zebrafish danio rerio) no estudo do metabolismo de
TRANSCRIPT
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CARINA DE SOUZA ANSELMO
ZEBRAFISH (DANIO RERIO) NO ESTUDO DO METABOLISMO DE
SUBSTÂNCIAS DE INTERESSE NO CONTROLE ANTIDOPAGEM
Rio de Janeiro
2019
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CARINA DE SOUZA ANSELMO
ZEBRAFISH (DANIO RERIO) NO ESTUDO DO METABOLISMO DE
SUBSTÂNCIAS DE INTERESSE NO CONTROLE ANTIDOPAGEM
Tese de Doutorado apresentada ao Programa de Pós-Graduação em Ciências Farmacêuticas da Faculdade de Farmácia da Universidade Federal do Rio de Janeiro, como parte dos requisitos necessários à obtenção do título de Doutor em Ciências Farmacêuticas.
Orientadora: Profª.Drª. Valeria Pereira de Sousa
Coorientador: Profª. Dr. Henrique Marcelo Gualberto Pereira
Rio de Janeiro
2019
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FICHA CATALOGRÁFICA
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CARINA DE SOUZA ANSELMO
ZEBRAFISH (DANIO RERIO) NO ESTUDO DO METABOLISMO DE SUBSTÂNCIAS DE
INTERESSE NO CONTROLE ANTIDOPAGEM
Tese de Doutorado apresentada ao Programa de Pós-Graduação em Ciências Farmacêuticas
da Faculdade de Farmácia da Universidade Federal do Rio de Janeiro, como parte dos
requisitos necessários à obtenção do título de Doutor em Ciências Farmacêuticas.
Aprovada em 25 de janeiro de 2019.
Orientadora:
________________________________________________________________
Profª. Drª. Valéria Pereira de Sousa, Faculdade de Farmácia, UFRJ.
Coorientador:
_______________________________________________________________
Prof. Dr. Henrique Marcelo Gualberto Pereira, Instituto de Química, UFRJ.
Revisora:
______________________________________________________________
Profª. Drª Julia Helena Rosauro Clarke, Faculdade de Farmácia, UFRJ.
Banca examinadora:
_____________________________________________________________
Profª. Drª. Cássia Curan Turci, Instituto de Química, UFRJ.
_____________________________________________________________
Profª. Drª Claudia Moraes de Rezende, Instituto de Química, UFRJ.
_____________________________________________________________
Profª. Drª Cláudia Pinto Figueiredo, Faculdade de Farmácia, UFRJ
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À todas as mulheres que me precederam e
abriram caminhos, por vezes dolorosos, para
que hoje eu estivesse aqui. E em especial à
minha avó, Maria Aparecida, e à minha mãe,
Sonia Ligia, mulheres fortes e determinadas
que são minha inspiração.
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AGRADECIMENTOS
Primeiramente, devo agradecer a força Onipresente que me guia, me impulsiona para
frente e me faz crer que sempre haverá um amanhã com luz e coisas boas. Não foi uma
jornada fácil em muitos momentos. Conciliar trabalho, doutorado e uma Olimpíada por vezes
me esgotou e me deixou desacreditada da minha capacidade. Mas uma força maior, junto
com o amor de pessoas queridas, me conduziu. E por tudo isso, sou muito grata.
Aos meus pais, Sonia e Jorge, meus maiores incentivadores e apoiadores. Meu pai, que
com seu carinho, bom humor e afeto, sempre me apoiou em todas as decisões da minha vida.
Se escrevo essa tese hoje devo muito ao apoio dele que sempre teve muito orgulho de ter
uma filha estudante. E a minha mãe, que não exagero em dizer que foi meu chão em várias
ocasiões desta trajetória. Suas ligações, comidas, orações, apoio e amor foram fundamentais
e me conduziram até aqui. Por muitas vezes eu duvidei de mim, mas vocês nunca duvidaram.
Obrigada por tudo!
Ao meu irmão e cunhada, Vinicius e Thairony, que me deram meu maior presente, a
Sophia. Pequena, você não sabe, mas ajudou a tia a superar muitos perrengues durante essa
jornada. Sua alegria, gritinhos, risadas, brincadeiras, abraços e beijinhos tornaram meus dias
mais doces.
Aos meus cachorros Sherlock e Watson, meus peludos do coração. Eles não se
importam se tenho títulos, publicações ou dinheiro, e me recebem em casa sempre com todo
o amor e alegria independente de qualquer coisa. Vocês podem não compreender, mas me
ajudaram muito a tornar meus dias mais suaves sendo companheiros durante toda essa
jornada.
Aos meus amigos do Cefeteq e da vida, Ya, Tati e Vitinho. Vocês sempre estão lá seja
para beber, dançar, fazer churrascos e/ou ouvir os lamentos da vida. Estou aqui para vocês
como vocês sempre estiveram para mim.
As minhas amigas da faculdade e da vida, Lud, Bel, Lyne, Cyn e Lidia. Rimos, choramos,
nos apoiamos e nos divertimos. Meninas, vocês são uma ótima rede de apoio e moram no
meu coração.
A minha orientadora Valéria pelo apoio e confiança. Obrigada pelo incentivo em trocar
de projeto quando passei no concurso e passei a trabalhar no LBCD.
Ao meu orientador Henrique, que me aceitou de braços abertos no LBCD e como aluna
de doutorado, tornando esse trabalho possível. Obrigada por todos os ensinamentos,
confiança e incentivo durante essa jornada.
Ao meu ex-chefe, orientador do coração e amigo, Vinicius Sardela. Obrigada por todos
os ensinamentos sobre peixes, espectrômetro de massas, desenhos experimentais e
dopagem no esporte. Foi um período de troca maravilhoso o que tivemos no LBCD.
A professora Ana Ribeiro e ao funcionário Gabriel Reis pela colaboração e
ensinamentos sobre desenhos experimentais.
Aos meus amigos e companheiros de trabalho do LBCD: Amanda, Maria Elvira, Juliana,
Carol Duarte, Karina, Marina, Isabelle, Aline, Daniely, William, Felipe Alves, Mariani, Thamara,
Gustavo Ramalho, Gabriel e Nina. Alguns não estão trabalhando mais comigo, mas foram
partes fundamentais deste processo. Aprendi e aprendo muito com todos vocês. Obrigada
também por todas as risadas e descontração. Vocês fazem do ambiente de trabalho um lugar
muito agradável.
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Aos amigos que fui fazendo pela vida, Alinne, Cássio e Claudinha. Não nos vemos
muito, mas vocês sempre estão a postos para me ouvir. E quando nos vemos, parece que o
tempo não passou. Obrigada por todo o afeto.
A UFRJ, patrocinadores e clientes que fazem com que seja possível o funcionamento
do LBCD. Sou grata pelas instalações, equipamentos e reagentes a que tive acesso para
realizar esse trabalho.
A todos os funcionários do LBCD, incluindo, mas não se limitando à TI, ao
administrativo, a manutenção e limpeza, a garantia da qualidade e à segurança. Todos
contribuíram para que esse trabalho fosse realizado e têm minha gratidão.
A todos os funcionários do Programa de Pós-Graduação em Ciências Farmacêuticas da
UFRJ que sempre estiveram dispostos a auxiliar em diversas questões. E um agradecimento
especial ao funcionário Marcelo, pela sua simpatia e solicitude em atender os alunos.
A todos os professores membros da banca de acompanhamento pelas suas importantes
contribuições para a construção da tese. E as professoras membros da banca da defesa de
doutorado por aceitarem participar e contribuir para esse trabalho. E pela gentileza da
professora Júlia Clarke por ter feito a revisão da tese.
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“Todas as vitórias ocultam uma abdicação. ”
Simone de Beauvoir
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RESUMO
ANSELMO, Carina de Souza. Zebrafish (Danio rerio) no estudo do metabolismo de substâncias de interesse no controle antidopagem. Rio de Janeiro, 2019. Tese (Doutorado em Ciências Farmacêuticas), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 2019.
Um dos principais desafios do controle de antidopagem revela-se no grande
número de substâncias disponíveis e na dificuldade em encontrar os melhores alvos
analíticos para detectar seu uso indevido. Portanto, estudos de metabolismo
envolvendo substâncias proibidas são fundamentais. No entanto, estudos de
metabolismo em seres humanos nem sempre são viáveis do ponto de vista ético,
especialmente para substâncias não aprovadas para comercialização. O zebrafish
(Danio rerio) tornou-se um modelo popular em várias linhas de pesquisa biológicas
por compartilhar semelhanças fisiológicas, morfológicas e histológicas com
mamíferos. De fato, uma revisão da literatura mostrou que os ensaios em zebrafish
são realizados em diferentes fases do desenvolvimento do peixe, sendo tipicamente
realizados em animais adultos por meio de ensaios in vivo, seguido de fases larvais e
embriões jovens. Como em qualquer modelo não humano, o zebrafish apresenta
semelhanças e diferenças em relação ao perfil de metabólitos gerados quando
comparado ao observado em humanos. Assim, o objetivo deste trabalho é avaliar a
capacidade do zebrafish em produzir metabólitos que possam ser possíveis alvos
analíticos do controle antidopagem. Inicialmente, duas substâncias dopantes com
metabolismo bastante conhecido, a sibutramina e o estanozolol, foram testados para
verificar a aplicabilidade do modelo de zebrafish water tank (ZWT). Através da análise
por CL-EMAR (cromatografia líquida-espectrometria de massa de alta resolução)
pode-se observar que ZWT pode produzir vários metabólitos de sibutramina e
estanozolol, previamente detectados em urina humana. Outra parte do trabalho visou
o desenvolvimento de um método cromatográfico utilizando DoE (desenho
experimental) para separar os metabólitos urinários da sibutramina por CL-EMAR. De
acordo com a literatura, a sibutramina é extensamente metabolizada em N-desmetil-
sibutramina (M1), N-bisdesmetil-sibutramina (M2) e nos derivados mono-hidroxilados
M1 e M2. Além dos metabólitos hidroxilados previamente descritos em urina humana,
derivados di-hidroxilados de M1 e M2 também foram observados usando o método
desenvolvido pelo DoE. Esses metabólitos encontrados podem ser novos alvos
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analíticos do controle antidopagem. Finalmente, a sibutramina foi aplicada para a
otimização multivariada das condições do ZWT, e também para a comparação do
metabolismo entre ZWT, humanos e camundongos. Além disso, também foi avaliado
o metabolismo de outros agentes de dopagem no modelo de ZWT: selegilina, JWH-
073 e hexarelina. Após a otimização, 18 peixes com 168 horas de experimento é o
requisito mínimo para um painel relevante de produtos de biotransformação. Uma
comparação entre as espécies resultou na observação dos mesmos metabólitos
hidroxilados de sibutramina, porém com diferenças na razão de concentração dos
metabólitos. A estereoespecificidade do metabolismo no ZWT foi investigada usando
selegilina e nenhuma reação de racemização ou inversão foi observada. Além disso,
a investigação do metabolismo de JWH-073 foi realizada e os metabólitos observados
são os mesmos descritos para humanos, exceto para a hidroxilação no grupo indol.
Finalmente, a hexarelina foi utilizada como modelo para avaliar estudos no ZWT com
substâncias com baixa estabilidade. Como resultado, a hexarelina apresentou uma
metabolização muito rápida no ZWT e todos os metabólitos descritos para humanos
foram observados nesse modelo. Todos os fatos apresentados indicam que o ZWT é
um modelo valioso para a avaliação do metabolismo de xenobióticos com vários
propósitos, incluindo o controle antidopagem.
Palavras-chave: Zebrafish, espectrometria de massas de alta resolução, metabolismo,
metabólitos, controle antidopagem
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ABSTRACT
ANSELMO, Carina de Souza. Zebrafish (Danio rerio) in the study of the metabolism of substances of interest in anti-doping control. Rio de Janeiro, 2019. Tese (Doutorado em Ciências Farmacêuticas), Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 2019.
A major challenge in anti-doping control is the large number of substances available
and the difficulty in finding the best analytical targets to detect their misuse. Therefore,
metabolism studies involving prohibited substances are fundamental. However,
metabolism studies in humans are not always feasible from the ethical point of view,
especially for non-approved substances. Zebrafish (Danio rerio) has become a popular
model organism in several lines of biological research for sharing physiological,
morphological and histological similarities with mammals. In fact, a review of the
literature has shown that zebrafish screenings are performed at different life stages,
typically being carried out in adult fish through in vivo assays, followed by early larval
stages and embryos. As with any non-human model, the zebrafish presents similarities
and differences in relation to the profile of generated metabolites compared to that
observed in humans. Thus, the objective of this work is to evaluate zebrafish's ability
to produce metabolites to be possible anti-doping targets. Initially, two doping agents
with well known metabolism, sibutramine and stanozolol, were tested to verify the
applicability of the zebrafish water tank (ZWT) model. Through the analysis by LC-
HRMS (liquid chromatography-high resolution mass spectrometry) it can be seen that
ZWT could produce several sibutramine and stanozolol metabolites, all of which have
already been detected in human urine. Another part of the work aimed at the
development of a chromatographic method using DoE (experimental design) to
separate the urinary metabolites of sibutramine by LC-HRMS. According to the
literature, sibutramine is extensively metabolized in N-desmethyl-sibutramine (M1), N-
bisdesmethyl-sibutramine (M2) and monohydroxyl derivatives from M1 e M2. In
addition to the hydroxylated metabolites previously described in human urine,
dihydroxyl derivatives of M1 and M2 were also observed using the developed method
by DoE. These new metabolites that have been found may be new analytical targets
in anti-doping control. Finally, sibutramine was applied for the multivariate optimization
of ZWT conditions, also for the comparison of the metabolism among ZWT, humans
and mice. In addition, the metabolism of other doping agents in the developed ZWT
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model was also evaluated: selegiline, JWH-073 and hexarelin. After the optimization,
18 fish and 168 hours of experiments is the minimum requirement for a relevant panel
of biotransformation products. A comparison among the species resulted in the
observation of the same hydroxylated sibutramine metabolites, however with
differences in metabolites concentration ratio. The stereospecificity of the ZWT
metabolism was investigated using selegiline and no racemization or inversion
transformations were observed. Moreover, the investigation of JWH-073 metabolism
was performed and the metabolites observed are the same described for humans,
except for the hydroxylation at the indol group. Finally, hexarelin was used as a model
to evaluate studies by ZWT for drugs with low stability. As a result, hexarelin displays
a very fast metabolization in ZWT conditions and all the metabolites described for
human were also observed in ZWT. All the facts presented indicate that ZWT is a
valuable model for the evaluation of xenobiotic metabolism for various purposes,
including anti-doping control.
Keywords: Zebrafish, high resolution mass spectrometry, metabolism, metabolites,
doping control
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LISTA DE FIGURAS
Introdução
Fig 1. Number of publications over the years mentioning the terms “zebrafish” and “metabolism” or “metabolite” in the title, abstract and/or keywords…………..............................................…………………...........................................30
Fig 2. Overview of zebrafish experiments in relation to the type of experiment (a), life stage (b), exposure time (c) and use of organic solvents (d)............................................................................................................................................31
Capítulo I
Figure 1. Chemical structures of stanozolol, sibutramine, and both metabolites reported in the literature………………………………………………………………………….........………….….71
Figure 2. LC–HRMS extracted ion chromatograms at a mass tolerance of 6 ppm from the sibutramine metabolites with m/z 266.16700 (1), 252.15135 (2), 282.16192 (3), and 268.14627 (4) from (a) zebrafish samples collected before sibutramine administration and (b) zebrafish samples collected after 7 days of sibutramine administration.................................................73
Figure 3. LC–HRMS extracted ion chromatograms at a mass tolerance of 6 ppm from stanozolol metabolites with m/z 345.25365 (1), 361.24856 (2), 327.24309 (3), and 359.23291 (4) from (a) zebrafish samples collected before stanozolol administration and (b) zebrafish samples collected after 7 days of stanozolol administration…………………………………….75
Figure 4. LC–HRMS extracted ion chromatograms at a mass tolerance less than 15 ppm from stanozolol glucuronide metabolites with m/z 505.29083 (a), 521.28574 (b), 535.26501 (c), and 537.28066 (d) from zebrafish samples collected after stanozolol administration prepared by dilute and-shoot…………………………………………………………………...................……..78
Figure 5. ESI(+) MS/MS chromatograms and spectra of parent substance standards for N-desmethyl-sibutramine (a and 1) and 16β-hydroxy-stanozolol (c and 3) and from zebrafish samples after the administration of sibutramine (b and 2) and stanozolol (d and 4)…........………….......................................................................................................……….79
Capítulo II
Figure 1. Chemical structures of sibutramine and its metabolites………….........……………..88
Figure 2. Comparison between liquid-liquid extractions (LLE) at pH alkaline (A-LLE), neutral (N-LLE), acid (Ac-LLE) and solid phase extraction (SPE) in relation to yield values (recovery in % of N-desmethyl-sibutramine)………………………………………………...............……....98
Figure 3. LC-HRMS extracted ion chromatograms at a mass tolerance of 6 ppm from the sibutramine metabolites M5 with m/z 298.15683 from urine samples collected six hours after sibutramine ingestion under the chromatographic conditions obtained by the first experimental design (DoE) and final condition obtained by the second experimental design (DoE)………100
Figure 4. Surface response plots obtained by Box-Behnken design (DoE) showing the effects of mobile phase flow (X1), column temperature (X2) and ramp time (X3) on the resolutions R1-3 (A, B and C), R1-2 (D and E) and R5-6 (F)…………………………….........................…….101
Figure 5. LC-HRMS extracted ion chromatograms at a mass tolerance of 6 ppm from the urinary sibutramine metabolites M1 (A), M2 (B), M3 (C), M4 (D), M5 (E), M6 (F), M7 (G) and M8 (H) under the final chromatographic condition established by DoE………………………103
Figure 6. Proposal formation of the main fragments in product ion spectra of [M+H]+ of M3 (A), M4 (B), M5 (C) and M6 (D)……………………………………………………….............……….106
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Figure 7. LC-HRMS extracted ion chromatograms (at a mass tolerance of 6 ppm) and TIC from the urinary sibutramine metabolites……...............................................................................112
Capítulo III
Figure S.1 Main metabolites of SIB generated by demethylation and hydroxylation reactions mediated by CYP2B6…………………………………………………...............………………...130
Figure 1. Multivariate optimization results on surface plots for ZWT conditions….........……132
Figure 2. Comparison among the relative abundance (%) of SIB hydroxy isomers metabolites for human, mouse and ZWT…………………………………………..………......................…..135
Figure 3. Profile of the SIB metabolite M1-OH in humans (A), mice (B) and in zebrafish, under the three different conditions evaluated in the experimental design………............……….….136
Figure 4. In vivo metabolism percentage of SIB metabolites in ZWT (Control) and the percentage of the same SIB metabolites with introduction of selective inhibitor of CYP2B6 isoenzyme, ticlopidine, in the ZWT (+ ticlopidine)……………………………………………….139
Figure S.2. SIB metabolism by human, mice and zebrafish, over time……..........……….….137
Figure S.3. Metabolic route of selegiline observed in ZWT…………..……………..........…....140
Figure S.4. Ion chromatogram fragments formed from electron ionization and full-scan GC-MS for Mosher derivatives.……………………………………………………….……….............…..141
Figure 5. The metabolic profile of selegiline and its metabolites in ZWT…….........…...........142
Figure S.5. Metabolic route of the synthetic cannaminoid JWH-073 observed in ZWT......................................................................................................................................143
Figure 6. The metabolic profile of JWH-073 in ZWT evaluated by LC-HRMS......................144
Figure 7. The metabolic profile of hexarelin in a zebrafish model, evaluated by LC-HRMS..……….......................................................................................................................147
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LISTA DE TABELAS
Introdução
Table 1. Synteny comparison between zebrafish and the major human CYPs involved in the metabolism of xenobiotics.................................................................................27
Table 2. Publications involving the study of the metabolism of xenobiotics in zebrafish.....................................................................................................................33
Capítulo I
Table 1. Elemental composition and theoretical mass [M + H]+ of stanozolol, sibutramine and their metabolites reported in the literature........................................72
Table 2. Protonated molecules [M + H]+ of substances 19 (N-desmethyl-sibutramine) and 2 (16β-hydroxystanozolol) with resulting diagnostic product ions (using high resolution MS/MS experiments), elemental composition (experimental) and mass error (ppm)..........................................................................................................................80
Capítulo II
Table 1. Parameters evaluated in the chromatographic method adjustment..............93
Table 2. Box-Behnken design matrix and correspondent results for the second experimental design....................................................................................................93
Appendix A. Box-Behnken design matrix and correspondent results for the first experimental design....................................................................................................94
Table 3. Elemental composition and theoretical mass [M + H]+ of sibutramine and its metabolites.................................................................................................................95
Table 4. Protonated molecules [M + H]+ of sibutramine metabolites with resulting diagnostic product ions (using high resolution MS/MS experiments) and elemental composition (experimental).......................................................................................105
Table 5. Repeatability (RSD,%), matrix effect (RSD,%) and limit of detection (LOD, ng/mL) results for sibutramine metabolites...............................................................113
Capítulo III
Table 1. Experiments design for the three variables (number of fish, dosage of SIB and time) according to a Doehlert matrix and results obtained for each group evaluated..................................................................................................................126
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LISTA DE ABREVIATURAS
AAS Androgenic-Anabolic Steroids
AFB1 Aflatoxin B1
APAP Acetaminophen
BBD Box-Behnken design
bis-nor-sib N-bisdesmethyl-sibutramine
CB Chlorobenzene
C18 Octadecilsilano
CYP Cytocrome P450
COMT Catechol O-methyltransferases
dpf days post-fertilization
DAD Diode-array detection
DMSO Dimethyl sulfoxide
DoE Experimental design
EEC Experimental elemental composition
ESI Electrospray ionization
GC Gas chromatography
GST Glutathione S-transferases
HCB Hexachlorobenzene
HLM Human liver microsomes
hpf hours post-fertilization
HPLC High-performance liquid chromatography
HRMS High resolution mass spectrometry
FDA Food and Drug Administration
IS/ISTD Internal Standard
LC Liquid chromatography
LLE Liquid-liquid extraction
LOD Limit of detection
M1 N-desmethyl-sibutramine
M2 N-bisdesmethyl-sibutramine
m/z Mass-to-charge ratio
MRPL Minimum required performance level
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MS Mass spectrometry
NAPQI N-acetyl-p-benzoquinone imine
NAT N-acetyltransferase
nor-sib N-desmethyl-sibutramine
OECD Organization for Economic Cooperation and Development
OP Organophosphate
QqQ Triple quadrupole
RSD Relative standard deviation
SIB Sibutramine
SPE Solid-phase extraction
SRM Selected reaction monitoring
SULT Sulfotransferase
TBME Tert-butyl methyl ether
TH Thyroid hormones
TOF Time-of-flight
TPMT Thiopurine S-methyltransferases
UDP Uridine 5'-diphospho
UGT UDP-glucuronosyltransferase
UV Ultraviolet
WADA World Anti-Doping Agency
ZLM Zebrafish liver microsomes
ZWT Zebrafish water tank
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APRESENTAÇÃO DO TRABALHO
Este trabalho foi realizado no Laboratório Brasileiro de Controle de Dopagem
(LBCD) do Instituto de Química da Universidade Federal do Rio de Janeiro. A forma
de apresentação deste trabalho consiste da seguinte disposição:
▪ Introdução: Artigo de revisão publicado no periódico Comparative Biochemistry and
Physiology, Part C, contemplando o estado-da-arte sobre a utilização do zebrafish
para a avaliação do metabolismo de xenobióticos;
▪ Objetivos: gerais e específicos;
▪ Capítulo I: Artigo publicado no periódico Drug Testing and Analysis, contemplando a
avaliação da capacidade do zebrafish adulto para produzir metabólitos de sibutramina
e estanozolol, substâncias com um metabolismo bem conhecido que são amplamente
utilizados como agentes de dopagem no esporte;
▪ Capítulo II: Artigo submetido para publicação no periódico Journal of
Chromatography B, contemplando o desenvolvimento e validação de método de
cromatografia líquida acoplada a espectrômetro de massas de alta resolução para
detecção dos metabólitos urinários de sibutramina;
▪ Capítulo III: Artigo publicado no periódico Drug Testing and Analysis, contemplando
a comparação do metabolismo da sibutramina em humanos, camundongos e
zebrafish, além do metabolismo de outras substâncias utilizadas como dopagem no
esporte;
▪ Discussão geral;
▪ Conclusões gerais;
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SUMÁRIO
1 INTRODUÇÃO ............................................................................................................... 21
1.1 Zebrafish (Danio rerio): A valuable tool for predicting the metabolism of xenobiotics in
humans? ................................................................................................................................. 21
1.1.1 Abstract ......................................................................................................................... 21
1.1.2 Introduction.......................................................................................................................... 22
1.1.3 Development ........................................................................................................................ 24
1.1.4 Enzymes involved in the metabolism of xenobiotics ........................................................... 25
1.1.5 Metabolism of xenobiotics ................................................................................................... 29
1.1.6 Metabolites analysis ............................................................................................................. 46
1.1.7 Zebrafish considerations for metabolite screening ............................................................. 48
1.1.8 Conclusion ............................................................................................................................ 50
1.1.9 Conflict of interest statement ............................................................................................... 51
1.1.10 References .......................................................................................................................... 51
1.1.11 Justificativa do trabalho .................................................................................................. 57
2 OBJETIVOS .................................................................................................................... 62
2.1 Objetivos Específicos ......................................................................................................... 62
3. Capítulo I ..................................................................................................................... 64
3.1 Is zebrafish (Danio rerio) a tool for human-like metabolism study? ..................................... 64
3.1.1 Abstract ................................................................................................................................ 64
3.1.2 Introduction.......................................................................................................................... 65
3.1.3 Experimental ........................................................................................................................ 67
3.1.3.1 Chemicals and reagents .................................................................................................... 67
3.1.3.2 Zebrafish metabolism model ............................................................................................ 67
3.1.3.3 Samples ............................................................................................................................. 68
3.1.3.4 Instrumental conditions .................................................................................................... 68
3.1.3.4.1 Liquid chromatography .................................................................................................. 68
3.1.3.4.2 HRMS analysis ................................................................................................................ 69
3.1.3.4.3 Fragmentation analysis .................................................................................................. 69
3.1.4 Results and discussion .......................................................................................................... 70
3.1.4.1 Screening – Phase I metabolites of sibutramine and stanozolol ...................................... 70
detected in zebrafish samples by full-scan HRMS ........................................................................ 70
3.1.4.2 Screening – Phase II metabolites of stanozolol detected in ............................................. 77
zebrafish samples by full-scan HRMS ............................................................................................ 77
3.1.4.3 Comparison of product ion spectra of zebrafish samples and .......................................... 78
reference material solutions ......................................................................................................... 78
3.1.4 Conclusion ............................................................................................................................ 80
3.1.5 Acknowledgments ................................................................................................................ 81
3.1.6. References ........................................................................................................................... 81
4. Capítulo II .................................................................................................................... 87
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4.1 Development of liquid chromatographic Q-high resolution mass spectrometry method by
Box-Behnken design for investigation of sibutramine urinary metabolites ................................ 87
4.1.1 Abstract ................................................................................................................................ 87
4.1.2 Introduction.......................................................................................................................... 88
4.1.3 Materials and Methods ........................................................................................................ 90
4.1.3.1 Chemicals and reagents .................................................................................................... 90
4.1.3.2 Samples ............................................................................................................................. 91
4.1.3.3 Liquid-liquid extraction (LLE) ............................................................................................ 91
4.1.3.4 Solid-phase extraction (SPE) ............................................................................................. 91
4.1.3.5 Chromatographic adjustment by DoE ............................................................................... 92
4.1.3.6 Data analysis ...................................................................................................................... 94
4.1.3.7 HRMS analysis ................................................................................................................... 94
4.1.3.8 Fragmentation analysis ..................................................................................................... 95
4.1.3.9 Validation .......................................................................................................................... 96
4.1.3.9.1 Specificity ....................................................................................................................... 96
4.1.3.9.2 Repeatability .................................................................................................................. 96
4.1.3.9.3 Limit of Detection (LOD) ................................................................................................. 96
4.1.3.9.4. Carryover ....................................................................................................................... 97
4.1.3.9.5. Matrix interference ....................................................................................................... 97
4.1.3.9.6. Robustness .................................................................................................................... 97
4.1.4 Results and discussion .......................................................................................................... 97
4.1.4.1 Development of extraction method.................................................................................. 97
4.1.4.2 Chromatographic adjustment by DoE ............................................................................... 99
4.1.4.3 HRMS analysis of sibutramine metabolites in urine ....................................................... 102
4.1.4.4 Fragmentation analysis of sibutramine metabolites in urine ......................................... 104
4.1.4.4.1 M1 e M2 ....................................................................................................................... 104
4.1.4.4.2 M3 (M1-OH) ................................................................................................................. 106
4.1.4.4.3 M4 (M2-OH) ................................................................................................................. 107
4.1.4.4.4 M5 (M1-diOH) .............................................................................................................. 108
4.1.4.4.5 M6 (M2-diOH) .............................................................................................................. 109
4.1.4.4.6 M7 ................................................................................................................................ 109
4.1.4.4.7 M8 ................................................................................................................................ 110
4.1.4.5 Validation ........................................................................................................................ 110
4.1.4.5.1 Specificity ..................................................................................................................... 110
4.1.4.5.2 Repeatability ................................................................................................................ 111
4.1.4.5.3 Limit of Detection (LOD) ............................................................................................... 112
4.1.4.5.4 Carryover ...................................................................................................................... 113
4.1.4.5.5 Matrix interference ...................................................................................................... 113
4.1.4.5.6 Robustness ................................................................................................................... 114
4.1.5 Conclusion .......................................................................................................................... 114
4.1.6 References .......................................................................................................................... 115
5. Capítulo III ................................................................................................................. 119
5.1 Zebrafish (Danio rerio) Water Tank Model for the Investigation of Drug Metabolism:
Progress, Outlook, and Challenges .............................................................................................. 119
21
5.1.1 Abstract .............................................................................................................................. 119
5.1.2 Introduction........................................................................................................................ 120
5.1.3 Experimental Procedures ................................................................................................... 123
5.1.3.1 Chemicals and reagents .................................................................................................. 123
5.1.3.2 In vivo models.................................................................................................................. 123
5.1.3.2.1 Mice .............................................................................................................................. 123
5.1.3.2.2 Zebrafish ....................................................................................................................... 124
5.1.3.2.3 Humans ........................................................................................................................ 124
5.1.3.3 Experimental set up for ZWT ....................................................................................... 125
5.1.3.3.1 Optimization of the ZWT model for SIB metabolism .............................................. 125
5.1.3.3.2 Inhibition of sibutramine metabolism in zebrafish ...................................................... 126
5.1.3.3.3 ZWT drug metabolism: sibutramine, selegiline, JWH-073 and hexarelin .................... 126
5.1.3.4 Sample preparation ......................................................................................................... 127
5.1.3.4.1 For LC-HRMS analysis ................................................................................................... 127
5.1.3.4.2 For stereospecificity investigation ............................................................................... 127
5.1.3.4.3 For peptides analysis .................................................................................................... 128
5.1.3.5 Samples extract analysis ................................................................................................. 128
5.1.3.5.1 LC-HRMS conditions ..................................................................................................... 128
5.1.3.5.2 LC gradient condition optimized for metabolism investigation ................................... 129
5.1.3.5.3 GC-MS conditions ......................................................................................................... 129
5.1.4 Results and Discussion ....................................................................................................... 129
5.1.4.1 Optimization of the ZWT model for SIB metabolism ...................................................... 129
5.1.4.2 Comparative interspecies metabolism of sibutramine ................................................... 133
5.1.4.3 Characterization of the major CYP isoenzyme involved in the sibutramine ZWT metabolic
profile .......................................................................................................................................... 138
5.1.4.4 Stereospecificity investigation: selegiline as a model ..................................................... 139
5.1.4.5 Cannabimimetics biotransformation profile: JWH-073 study ........................................ 143
5.1.4.6 Peptides stability concept proof: hexarelin study ........................................................... 145
5.1.5 Conclusion .......................................................................................................................... 147
5.1.6 Conflict of interest .............................................................................................................. 149
5.1.7 Acknowledgments .............................................................................................................. 149
5.1.8 References .......................................................................................................................... 150
6. DISCUSSÃO GERAL ..................................................................................................... 156
7. CONCLUSÕES GERAIS ................................................................................................. 171
8. REFERÊNCIAS BIBLIOGRÁFICAS ............................................................................................... 175
ANEXOS ....................................................................................................................................... 179
21
1 INTRODUÇÃO
1.1 Zebrafish (Danio rerio): A valuable tool for predicting the metabolism of
xenobiotics in humans?
Carina de Souza Anselmo, Vinicius Figueiredo Sardela, Valeria Pereira de Sousa, Henrique Marcelo Gualberto Pereira.
Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46. https://doi.org/10.1016/j.cbpc.2018.06.005.
1.1.1 Abstract
Zebrafish has become a popular model organism in several lines of biological
research sharing physiological, morphological and histological similarities with
mammals. In fact, many human cytochrome P450 (CYP) enzymes have direct
orthologs in zebrafish, suggesting that zebrafish xenobiotic metabolic profiles
may be similar to those in mammals. The focus of the review is to analyse the
studies that have evaluated the metabolite production in zebrafish over the years,
either of the drugs themselves or xenobiotics in general (environmental
pollutants, natural products, etc.), bringing a vision of how these works were
performed and comparing, where possible, with human metabolism. Early studies
that observed metabolic production by zebrafish focused on environmental
toxicology, and in recent years the main focus has been on toxicity screening of
pharmaceuticals and drug candidates. Nevertheless, there is still a lack of
standardization of the model and the knowledge of the extent of similarity with
human metabolism. Zebrafish screenings are performed at different life stages,
typically being carried out in adult fish through in vivo assays, followed by early
larval stages and embryos. Studies comparing metabolism at the different
zebrafish life stages are also common. As with any non-human model, the
zebrafish presents similarities and differences in relation to the profile of
generated metabolites compared to that observed in humans. Although more
studies are still needed to assess the degree to which zebrafish metabolism can
be compared to human metabolism, the facts presented indicate that the
zebrafish is an excellent potential model for assessing xenobiotic metabolism.
© 2018 Elsevier Inc. All rights reserved
22
1.1.2 Introduction
Fish are the most numerous and phylogenetically diverse group of
vertebrates and are useful in the study of fundamental processes in
vertebrate evolution, development, toxicology and disease processes
(Garcia et al., 2016; Langheinrich, 2003; Spitsbergen and Kent, 2003).
In particular, zebrafish (Danio rerio), which is a tropical fish of the
Cyprinidae family, has been a prominent genetic model since 1981
(Streisinger et al., 1981), when its genetic amenability allowed the first
genetic screening for mutations that affect organ development in a
vertebrate (Kamel and Ninov, 2017). Since then, zebrafish has become
a popular model organism in several lines of biological research beyond
genetics, including developmental biology (Carten et al., 2011; Ho et al., 2003),
toxicology (Thompson et al., 2010; Weigt et al., 2011;
Zhang et al., 2016), drug discovery (Fleming et al., 2005; Kithcart and
MacRae, 2017), disease models (Brugman, 2016; Capiotti et al., 2014)
and neurobiology (Pinho et al., 2016; Shams et al., 2017). Therefore,
zebrafish represents a viable alternative to the classical mammalian
models currently used in biological research because it has some unique
properties: it is an intact organism with similar development, anatomy
and physiology to higher vertebrates (Otte et al., 2017); it is easier and
less expensive than popular rodent models; it is a genetically tractable
vertebrate model that can be easily injected with modified genes and
can absorb chemical mutagens through water; embryos are transparent
and develop externally, allowing the use of noninvasive imaging techniques;
embryos develop very rapidly compared to mammalian models, potentially
reducing the experimentation time (Garcia et al., 2016); and
it has a high reproduction capacity, where a pair of zebrafish can
generate hundreds of fertilized eggs, which develop rapidly into larvae
with functional metabolic organs (Kamel and Ninov, 2017).
Although there are some major differences resulting from adaptation to
aquatic life, most zebrafish organs perform the same functions as
their human counterparts and exhibit well-conserved physiology
23
(MacRae and Peterson, 2015). In fact, zebrafish share physiological,
morphological and histological similarities with mammals (Diekmann
and Hill, 2013) and its genome has been sequenced, revealing that
~70% of human genes have a zebrafish orthologue and approximately
82% of potential human disease-related genes have at least one obvious
zebrafish orthologue (Howe et al., 2013). Not surprisingly, in terms of
publication, zebrafish are one of the fastest growing model organisms
(Garcia et al., 2016). Furthermore, chemical-genetic screens in zebrafish have
already rendered the drug ProHema, which is undergoing
phase II clinical trials for improving hematopoietic stem cell engraftment and
expansion (Kamel and Ninov, 2017; North et al., 2007). Assessing the toxicity of
a compound is a critical step in the discovery of
new drugs. In forensic toxicology, it is also very important to assess the
toxicity of a compound, since the knowledge of its metabolism will be
fundamental for determining the analytical targets to be monitored as
the emergence of ever newer designer drugs is an ongoing challenge for
analytical toxicologists in forensic as well as clinical toxicology (Peters
and Martinez-Ramirez, 2010). To understand or predict the toxic potential of a
compound, knowledge of the absorption, distribution, metabolism, and
elimination of the compound is required (Garcia et al.,
2016).
Notably, extensive metabolism of a xenobiotic, an external and
foreign compound to the organism, could lead to reduced activity,
whereas creation of new active metabolites could lead to increased
toxicity (Diekmann and Hill, 2013). Zebrafish embryos represent an
attractive model for studies of developmental toxicity of xenobiotics
both for human and environmental risk assessment (Carlsson et al.,
2013). The reason for the preference in the use of embryos instead of
adult fish is because in Europe, zebrafish embryos are not considered
laboratory animals until the independent feeding stage (European
Commission, 2016), which makes them ideal candidates for several
chemical screenings. On the other hand, in the United States of America
fish are not protected for research use, while in Brazil, China and India,
24
all animals are protected for use in research (Sneddon et al., 2017). In
fact, the zebrafish embryo has already been accepted as a validated
alternative for the acute fish toxicity test (OECD TG236) (OECD, 2013).
In addition, toxicology may be the most prevalent use of zebrafish in
the industry, with the majority of large pharmaceutical companies reporting some
use of zebrafish for toxicology (MacRae and Peterson,
2015). The metabolism of the substance as a critical step in the toxicological
evaluation. Therefore, the purpose of this review is to analyse
publications involving xenobiotic metabolism studies at various stages
in the zebrafish's evolution over the years. Considerations will also be
made on the development of zebrafish, enzymes involved in the metabolism of
xenobiotics, and analytical methods for the detection of
metabolites, highlighting advantages and limitations of zebrafish as a
model for the evaluation of xenobiotic metabolism.
1.1.3 Development
The genetic signals and responses that drive early embryonic
development are fundamentally similar between zebrafish and mammalian
embryogenesis (Sipes et al., 2011). The formation of different
organ systems can be followed under the microscope during the developmental
period, offering many opportunities for the study of organogenesis (Dawid, 2004).
The zebrafish has a rapid development,
progressing from zygote to larvae in 72 h. During the early embryonic
stages, zebrafish are covered by a chorionic membrane (egg shell)
which will be lost between 3 or 4 dpf (days post-fertilization) (Kimmel et al., 1995).
The chorion is an acellular envelope containing pores that
are approximately 0.5 μm in diameter with 2 μm spacing (Lee et al.,
2009). A limitation to using the zebrafish embryo is its need to take up
substances through the chorion and other membranes, because the gills
of the adult fish are not yet developed (Vallverdú-Queralt et al., 2015).
Therefore, the chorion can significantly confound the early life zebrafish toxicity
assay by leading to false positives because of size-dependent limitations in the
25
uptake of large compounds (Langheinrich et al.,
2003; Mandrell et al., 2012). The chorion may be removed but not
without potentially affecting embryo integrity and behavior (Mandrell
et al., 2012; Sipes et al., 2011).
The morphogenesis of the liver, which is an organ that plays a key
role in the biotransformation of xenobiotics, starts at approximately
24 hpf (hours post-fertilization), and liver-specific markers occur even
earlier (16 hpf) (Tao and Peng, 2009). By 5 dpf, the heart, liver, brain,
pancreas, and other organs are developed (Garcia et al., 2016; Kimmel
et al., 1995). The zebrafish larvae begin independent feeding at approximately 6
dpf, growing rapidly and developing into juveniles at
approximately 30 dpf, and subsequently into adults at approximately
90 dpf (Kamel and Ninov, 2017). Similar to the mammalian liver, the
zebrafish liver is an essential organ in the body and performs several
vital activities, including metabolism, detoxification and homeostasis
(Menke et al., 2011; Tao and Peng, 2009). However, zebrafish have a
unique hepatic anatomy and cellular architecture, despite the high
conservation of cell types within the liver, compared with mammals
(Goessling and Sadler, 2015; Tao and Peng, 2009).
1.1.4 Enzymes involved in the metabolism of xenobiotics
Drug-metabolizing enzymes play central roles in the metabolism,
elimination and/or detoxification of xenobiotics introduced into the
organism (Xu et al., 2005). Zebrafish have the ability to perform both
phase I (oxidation, N-demethylation, O-demethylation and N-dealkylation) and
phase II (sulfation and glucuronidation) metabolism reactions, and the metabolic
enzymes responsible for these reactions are
highly conserved in relation to mammals (Brox et al., 2016; Chng et al.,
2012; Diekmann and Hill, 2013). Phase I enzymes consist primarily of
the cytochrome P450 (CYP) superfamily of microsomal enzymes, which
are found abundantly in the liver, gastrointestinal tract, lung and
kidney, catalyzing the oxidative and reductive metabolism of many
26
chemicals and endogenous compounds (Tseng et al., 2005; Xu et al.,
2005). In humans, it is believed that five CYP gene families, namely,
CYP1, CYP2, CYP3, CYP4 and CYP7, play crucial roles in hepatic as well
as extra-hepatic metabolism and elimination of xenobiotics and drugs,
as CYP1A2, CYP2C9, CYP2D6 and CYP3A4/5 are responsible for the
oxidative biotransformation of approximately 70% of most clinically
used drugs (Saad et al., 2017; Xu et al., 2005). Therefore, CYP families
1-3 are the main metabolizing enzymes responsible for xenobiotic
metabolism (Guengerich, 2008; Saad et al., 2016).
Many human CYP enzymes have direct orthologs in zebrafish
(Goldstone et al., 2010), suggesting that zebrafish metabolic profiles
may be similar to those of mammals. In addition, there are other CYPs
described in fish that lack orthologs in humans, such as CYP1C, CYP2AE
and CYP2X (Table 1) (Goldstone et al., 2010). Nevertheless, as CYP
activity is not necessarily correlated with its gene expression or even
protein levels, more focus is needed on CYP activity (Saad et al., 2017).
Like the human CYP3A genes, CYP3A65 transcription in the foregut
region was enhanced by treatment of the zebrafish larvae with dexamethasone
and rifampicin (Tseng et al., 2005). On the other hand, 7-
benzyloxy-4-(trifluoromethyl)coumarin, a vertebrate CYP3A substrate
not usually associated with CYP1 activity, was metabolized more efficiently by
CYP1A than CYP3A65 in zebrafish (Scornaienchi et al.,
2010). That is, a zebrafish CYP that has an ortholog in human will not
necessarily have the same behavior on a certain substrate. Another
interesting case is the metabolism of dextromethorphan in zebrafish;
although no ortholog of human CYP2D6 has been found in the fish (Table 1)
(Goldstone et al., 2010). Saad et al. (2017) showed that
zebrafish do metabolize dextromethorphan to dextrorphan, in addition
to 3-methoxymorphinan, but the ratio between both metabolite concentrations
was different than in man. This indicates that not having an
ortholog of human CYP in the zebrafish does not necessarily mean that
there will be no biotransformation of xenobiotics.
27
Table 1. Synteny comparison between zebrafish and the major human CYPs involved in the metabolism of xenobiotics. Adapted from Goldstone and co-workers, 2010.
Zebrafish Human
CYP1A CYP1A1/1A2 CYP1B1 CYP1B1 CYP1C1,2 - CYP1D1 CYP1D1P CYP2Ks CYP2W1 CYP2N13 CYP2J2 CYP2Ps CYP2J2 CYP2R1 CYP2R1 CYP2U1 CYP2U1 CYP2V1 CYP2J2 CYP2X1-10 - CYP2Y3,4 CYP2A/B/F/S CYP2AA1-12 - - CYP2D - CYP2C CYP2AD2,3,6 CYP2J2 CYP2AE1,2 - CYP3A65 CYP3A-se1,-se2a CYP3C1-4 CYP3A3,4,7 CYP4F43 CYP4F CYP4V7,8 CYP4V2 CYP4T8 - CYP5A1 CYP5A1 CYP7A1 CYP7A1 CYP7B1 CYP7B1 CYP7C1 -
In several mammalian species including man, CYP-related drug
metabolism tends to be significantly lower during early life stages, and
the same was observed in vitro in zebrafish (Saad et al., 2017), despite
several studies having already observed the production of metabolites
in the early larval stages (2 to 7 dpf) (Alderton et al., 2010; Chng et al.,
2012; Hu et al., 2012; Pardal et al., 2014) and embryos (fertilized eggs
exposed right after being collected) (Brox et al., 2016; Vallverdú-
Queralt et al., 2015; Wiegand et al., 2001). Regarding the embryos, the
biotransformation capability was five-fold higher in the older fish embryos (50 hpf)
than in the younger embryos (2 to 26 hpf) (Kühnert
et al., 2017). Concerning CYP3A65, the strength of transcription increased with
stages of development; CYP3A65 mRNA first appeared at
the 72 hpf stage, and in adult fish, it was intensively transcribed in liver
28
and intestine (Chang et al., 2013). The same is true for zebrafish
CYP1A, where expression is low during the early stages of development
and increases dramatically after hatching at approximately 72 hpf (Saad
et al., 2016). Overall, the expression of zebrafish CYPs appears to
broadly increase throughout normal embryonic development, with
higher expression observed in larvae posthatching (Jones et al., 2010).
During phase II drug metabolism, the drugs or metabolites from
phase I pathways are enzymatically conjugated with a hydrophilic endogenous
compound by the most common transferase enzymes: UDP-
glucuronosyltransferases (UGTs), sulfotransferases (SULTs), N-
acetyltransferases (NATs), glutathione S-transferases (GSTs), thiopurine S-
methyltransferases (TPMTs), and catechol O-methyltransferases
(COMTs) (Almazroo et al., 2017; Xu et al., 2005). Glucuronidation is
the major phase II drug metabolism pathway, with approximately 40%
to 70% of human endogenous and exogenous compounds conjugated to
glucuronidated forms to be excreted from the body (Almazroo et al.,
2017). In zebrafish, a total of 45 UGT genes were identified that can be
divided into three families consisting of Ugt1s, Ugt2s and Ugt5s (most abundant),
showing close similarities between mammalian and zebrafish Ugt1 and Ugt2
families (Huang and Wu, 2010; Christen and Fent,
2014). All the isoforms of UGTs have tissue-, sex- and developmental specific
expression patterns. UGTs expression level dropped at 2 dpf and
increased again from 3 to 5 dpf and features high expression of Ugt1a
and Ugt1b in the liver and intestine of female and male zebrafish
(Christen and Fent, 2014).
SULTs have also been identified in zebrafish being divided into five
families SULT 1, SULT 2, SULT 3 and SULT 6. Similar to the human
SULTs, the majority of the zebrafish SULTs that have been cloned belong to the
SULT1 gene family (Liu et al., 2010; Mohammed et al.,
2012). The amino acid sequence alignment demonstrates that the putative
zebrafish Naa10 (catalytic subunit Nα-acetyltransferase from
NAT) is highly similar to human Naa10 (Ree et al., 2015). Phylogenetic
analysis revealed a great diversity of fish GSTs, with 27 members found
29
in zebrafish (Glisic et al., 2015). Glisic et al. (2015) suggest involvement of GST
Pi class, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b in the
biotransformation of xenobiotics on their functional similarity to the
human orthologs in respect to the xenobiotic metabolism. Other work
showed further the considerable conservation with regard to the critical
amino acid residues between the zebrafish and mammalian COMTs
(Alazizi et al., 2011). In fact, many studies have revealed the presence
of phase II metabolites produced by zebrafish at different life stages
(Alderton et al., 2010; Anselmo et al., 2017; Brox et al., 2016; Shen
et al., 2017).
1.1.5 Metabolism of xenobiotics
The zebrafish is increasingly used as a vertebrate model for several
studies, including in vivo toxicological screenings, because it combines
the scale and throughput of in vitro systems with the physiological
complexity of a vertebrate whole animal (Garcia et al., 2016). Several
studies using zebrafish have observed the formation of metabolites of
the substances administered in the fish, with several goals in several
areas of interest, such as pollutants of the environment, drug discovery
and toxicological evaluation. However, there is still a lot to be understood about
the model and several particularities to be discussed.
Through an advanced search in the Science Direct database (https://
www.sciencedirect.com/science/search), searching for “zebrafish and
metabolism or metabolite” in the abstract, title and keywords fields, the
result presented in Fig. 1 was obtained. As can be observed, the subject
in question presented a considerable growth in the number of publications
overtime, especially after 2005. After evaluating the results of
the search and withdrawing the studies that did not focus on the observation of
the metabolism of exogenous substances by the fish, the
data presented in Table 2 were obtained, which included studies that,
with different aims, evaluated the production of xenobiotic metabolites
in zebrafish.
30
Fig 1. Number of publications over the years mentioning the terms “zebrafish” and “metabolism” or “metabolite” in the title, abstract and/or keywords. Source: https://www.sciencedirect.com/science/search.
Most of the studies evaluated the metabolism of xenobiotics through
in vivo assays, four used in vitro assays, and two compared in vitro with
in vivo (Table 2; Fig. 2a). Zebrafish screenings involving metabolite
analysis are performed at different life stages, typically being carried
out in adult fish, followed by early larvae stages and embryos (Table 2;
Fig. 2b). Studies comparing metabolism at the different zebrafish life
stages are also common (Chng et al., 2012; Hertl and Nagel, 1993; Saad
et al., 2017). In cases where adult fish were used, most studies used
both sexes, and some did not cite this type of information (Table 2). The
in vitro studies were performed using liver homogenate, liver microsomes or
whole zebrafish, whereas in vivo assays generally directly
exposed the fish to water or medium containing the substances, with
two exceptions in the routes of administration: spiked food (Wen et al.,
2015) and intraperitoneal injection (Troxel et al., 1997). The number of
animals used in the studies was widely variable, as generally when
using embryos or larvae the number tends to be higher (25 (Brox et al.,
2016), 60 (Vallverdú-Queralt et al., 2015) and 200 (Hertl and Nagel,
1993) embryos; 20 (Chng et al., 2012), 30 (Jones et al., 2012), 40 (Alderton et
al., 2010) and 100 (Hertl and Nagel, 1993) larvae) than
when using adult animals (6 (Shen et al., 2017), 12 (Anselmo et al.,
31
2017), 16 (Wen et al., 2015) and 20 animals (Kasokat et al., 1987,
1989)), but the number of animals used is not always provided.
Fig 2. Overview of zebrafish experiments in relation to the type of experiment (a), life stage (b),
exposure time (c) and use of organic solvents (d).
Another factor that has been shown to be quite variable is the exposure
time of the fish to the substances tested: in the in vitro assays the
exposure time tends to be shorter and less variable (from 45 min to
24 h) than in the in vivo tests (from 3 h to 20 days) (Fig. 2c). The mean
fish maintenance temperature, in the case of the in vivo experiments and
the in vitro assays, ranged from 23 to 28.5 °C, except for an in vitro study
that used a temperature of 35 °C (Wiegand et al., 2001) (Table 2).
Zebrafish are classified as eurythermal, as they exhibit a tolerance for
wide temperature ranges, and usually a temperature of 28.5 °C is considered
optimal and recommended (Kimmel et al., 1995; Lawrence,
2007), although OECD guidelines also suggest ranges between 21 and
25 °C (OECD, 1992) and 26 ± 1 °C (OECD, 2013). Overall, adult zebrafish were
cultured in a 12h:12h (Shen et al., 2017; G. Wang et al.,
32
2017; Zok et al., 1991) or 14 h:10 h (Saad et al., 2017; Troxel et al.,
1997) day/night cycle as recommended (12–16 h photoperiod) (OECD,
2013).
Concerning the use of organic solvents to increase the solubility
of tested compounds, several studies used DMSO at different concentrations
(0.01 to 1%, v/v); two used methanol (0.1 or 1%, v/v),
ethanol (0.01 or 0.0015%, v/v) or toluene (0.007%, v/v); and less than
half of the studies did not use any type of solvent (Fig. 2d). It is important to note
that even low concentrations of organic solvents may
influence the activity of zebrafish phase I and II enzymes (David et al.,
2012), and some studies have used high concentrations of DMSO and
methanol (1%, v/v) (Table 2).
33
Table 2. Publications involving the study of the metabolism of xenobiotics in zebrafish.
Author Zebrafish´s life stage
Brief Experimental design
Analytical technique
Results Observations
Kasokat, Nagel & Urich, 1987
Adult (female; age not informed)
The metabolism of phenol and substituted phenols in zebrafish was studied
In vivo. Exposure in water with ethanol 0.01% (v/v) for some compounds. Water analysed. ETa: 48h/35h.Tb: 26°C
HPLC-UV
Several phenol and substituted phenols metabolites have been identified, with phase II being more common than phase I metabolites.
Overall, the metabolites recovered were higher for substituted phenols than or phenol itself indicating that some substituted phenols are more rapidly metabolized
Kasokat, Nagel & Urich, 1989
Adult (female; age not informed)
The metabolism of chlorobenzene (CB) and hexachlorobenzene (HCB) in zebrafish was studied
In vivo. Exposure in water with ethanol 0.01/toluene 0.007% (v/v). Water and fish analysed. ETa: 48h Tb: 26°C
HPLC-UV
Hydroxylate and conjugate metabolites were detected for CB. Turning to HCB, no metabolites could be detected under the experimental conditions
Zebrafish is able to hydroxylate aromatic compounds. The finding was in contrast to published work concerning the metabolism of HCB in fish.
Gorge & Nagel, 1990
Larvae (28 dpf)
Study of the metabolism of two radiolabelled pesticides, lindane and atrazine
In vivo. Exposure in water. Water analysed. ETa: 40h / Tb: (26 ± 1)°C
HPLC-UV Presence of 12 metabolites of lindane and 4 metabolites of atrazine
Lindane more metabolized than atrazine.
Zok et al., 1991
Adult (both sexes; 12 weeks)
Biotransformation products of anilines in the zebrafish were analysed by HPLC-UV
In vivo. Exposure in water. Water and fish analysed. ETa: 24hTb: (26.5 ± 1)°C
HPLC-UV
Corresponding acetanilides were the only metabolites identified in water. No biotransformation for 2- and 4-nitroaniline.
Substitution in the ortho position of the aniline sterically hinders acetylation. No ring hydroxylation of the anilines was observed.
Hertl & Nagel, 1993
Embryos (4hpf), larvae (4dpf and 17 dpf) and adult (female; 24 weeks)
Metabolism of 3,4-dichloroaniline (3,4-DCA)
In vivo. Exposure in water. Fish analysed. ETa: 48h. Tb: 26°C
HPLC-UV All life stages of zebrafish possess the capability to generate the metabolite 3,4-dichloroacetanilide
There is evidence of hydroxylation product of the aniline.
34
Troxel et al., 1997
Adult (sexually mature, both sexes)
Characterize the in vivo metabolism and hepatic DNA adduction of the carcinogenic aflatoxin B1 (AFB1) in the zebrafish
In vivo/in vitro. Intraperitoneal injection.. Water analysed. ET: 24h / T: (26 ± 1)°C. Zebrafish liver homogenate. ETa: 45 min / Tb: 28°C
HPLC-UV
The major metabolites of AFB1 recovered in the water were identified as aflatoxicol and aflatoxicol-glucuronide
The results demonstrate that zebrafish have the capacity for both phase I and phase II metabolism of AFB1
Wiegand et al., 2001
Embryos (fertilized eggs; prim 6 stage embryos)
Characterize glutathione S-transferases (GST)-formed atrazine metabolites
In vitro. Enzyme assay with zebrafish’s embryos. ETa: 24h / Tb: 35°C
HPLC-UV-ESI-QqQ
Zebrafish embryos conjugate atrazine to glutathione by the GSTs in the same way that plants do
Phase I metabolism has not been evaluated, although in animals, hydroxylation and N-dealkylation are described as the main metabolism pathway.
Lindholst et al., 2003
Adult (both sexes; age not informed)
Metabolism of bisphenol A in zebrafish (Danio rerio)
In vivo. Exposure in water. Water and tissue. ETa: 7 days. Tb: (27 ± 1)°C
HPLC-ESI-MSD Zebrafish produced the metabolites bisphenol A glucuronic acid (BPAGA) and bisphenol A sulfate
The high concentration of BPAGA found in the bile indicates that biliary excretion into the intestines is a major route of elimination.
Li et al., 2009 Larvae (2dpf)
Zebrafish CYP3A4 and CYP2D6 functional activity assays for assessing drug metabolism
In vitro. Microplate whole zebrafish assay with 0.1% DMSO. ETa: 24h. Tb: Not informed
CYP’s specific chemiluminogenic substrate
CYP’s functional activity was up and downregulated in zebrafish similar to the response in humans.
These findings underscore the high degree of CYP conservation in zebrafish.
Alderton et al., 2010
Larvae (7 dpf)
Accumulation and metabolism of drugs commonly used as probes for human cytochrome P450s (CYPs) in zebrafish larvae
In vivo. Exposure in medium with DMSO 1% (v/v). Medium and larvae analysed. ETa: 4h Tb: (28.5 ± 0.5)°C
HPLC-ESI-MS/MS
Cisapride, chlorpromazine, verapamil, testosterone, and dextromethorphan showed that the zebrafish larvae catalyze a range of phase I and phase II reactions.
Metabolism of amodiaquine, midazolam, felodipine, and S-mephenytoin could not be detected. Both similarities and differences in the metabolism were observed in zebrafish when compared to mammals
Jones et al., 2010
Larvae (4 dpf)
Identify genes homologous to mammalian CYP’s and UDP-UGT genes and assess the ability of zebrafish to metabolize substrates for these enzymes
In vivo Exposure in water with 0.1% of DMSO. ETa: 10h. Tb: (28 ± 1)°C
Fluorescence ECODc, ERODd and OOMRe assay
Zebrafish larvae express genes similar to mammalian CYP and UGT isoforms throughout early development and have activities toward model CYP substrates
The continued use of these model organisms in toxicity testing is supported by this study.
35
Smith el., 2010
Adult (sexually mature, both sexes)
In vitro hepatic fluoxetine metabolism was determined in several model fish species: rainbow trout, goldfish, zebrafish and killifish.
In vitro. Liver microsomes. ETa: 1h. Tb: 25°C
HPLC-APCI-MS/MS
Fluoxetine metabolism in fish is much less than that seen in mammals. The major mammalian metabolite, norfluoxetine, is not the primary metabolite in fish
The low level of metabolism is likely the reason that fish bioaccumulate fluoxetine in vivo. After killifish, zebrafish was the fish model with higher rates of metabolism of fluoxetine.
Chen et al., 2012
Embryos (2hpf)
Determine if zebrafish larvae can be used to evaluate thyroid endocrine disruption of BDE-209 and if it bioaccumulates and is metabolized.
In vivo. Exposure in BDE solution with 0.01% of DMSO. Larvae analyse. ETa: 14 days. Tb: (28 ± 0.5)°C
GC-MS
Bioconcentration of BDE-209 in the larvae was evident, and BDE-209 could be metabolized. Both T4 and T3 levels were changed by BDE- 209 exposure in the larvae.
The limited metabolites of BDE-209 detected compared with previous studies suggest lower metabolic capability in larvae,
Chng et al., 2012
Larvae (5dpf) and adult (age not informed; female)
Investigation of the bioactivation potential and metabolism profile of zebrafish versus human using acetaminophen (APAP) and testosterone
In vitro/in vivo. Human (HLM) and adult zebrafish’s (ZLM) liver microsomes; larvae exposure in medium with methanol 0.1% (v/v) ETa (in vitro): 2h/Tb: 28.5°C. ETa (in vivo): 3h/Tb: 28.5°C.
HPLC-MS/MS/ HPLC-QTOF/MS/MS
NAPQI (APAP’s reactive metabolite) was generated by ZLM at lower levels than HLM. Several metabolites for testosterone were identified in ZLM and larvae.
The ZLM overall metabolite profile of testosterone had more hydroxylated metabolites compared with HLM. The zebrafish larvae produced only two hydroxilated metabolites
Hu et al., 2012
Larvae (2dpf) Characterize the metabolic profile of calycosin in a zebrafish model
In vivo. Exposure in medium with 0.1% DMSO. Medium and larvae analysed.ETa: 24h. Tb: 28.5°C.
HPLC-MS/MS
A total of ten calycosin metabolites formed from glucuronidation, glucosylation, sulfation, oxidation, demonstrating existence of both phase I and phase II metabolic activities.
The predominant phase II conjugation of calycosin observed in zebrafish larvae were similar to the metabolic profiles of other flavonoids in mammals
Jones et al., 2012
Larvae (3dpf)
Determine if zebrafish larvae could metabolise ibuprofen (CYP2C substrate) that has well characterised metabolism in mammals
In vivo. Exposure in water with ethanol 0.0015% (v/v). Water and fish analysed. ETa: 24h. Tb: (28 ± 1)°C
HPLC-LTQ-orbitrap -MS/MS
Hydroxy-ibuprofen was also identified in larvae extracts and water
Zebrafish larvae (72–96 hpf) can absorb, oxidize and excrete ibuprofen, similar to that identified in mammalian systems.
36
Carlsson et al., 2013
Embryos (fertilized eggs 15 min after collection)
Potential risk to fish embryos of veterinary pharmaceuticals were investigated and metabolism by the embryos was studied for albendazole (AL), febantel, fenbendazole and oxfendazole.
In vivo. Exposure in medium with 0.1% DMSO. Medium analysed. ETa: 6 days. Tb: (25.1 ± 0.4)°C
HPLC-ESI-MS/MS
AL was metabolized efficiently into albendazole sulfoxide, resulting in reduced toxicity. The prodrug febantel was metabolized to fenbendazole which is further metabolized to oxfendazole in various species.
The results also demonstrate the great value of including sublethal endpoints and chemical analysis to the zebrafish embryo test for hazard identification and risk assessment
Pardal et al., 2014
Larvae (3dpf)
Metabolism of resveratrol and its glucoside (piceid) in zebrafish
In vivo. Exposure in medium with 1% DMSO. Medium and larvae analysed. ETa: 48h. Tb: 27°C
HPLC-UV-MS/MS
The principal metabolites found were monoglucoronide and monosulfate forms of resveratrol. Other metabolites found in the literature were not detected in zebrafish larvae
Besides the metabolites found in this study for piceid, it’s already been identified glucuronidated and sulfated piceid in rat urine after oral administration.
Wen et al., 2014
Adults (20 weeks, both sexes) and eggs (collected every morning before feeding)
Evaluate potential risks posed by BDE-47 and their analogs in aquatic environment, distribution among tissues of adult fish and transfer from females to eggs
In vivo. Adult exposure by spiked food. Adults and eggs analysed. ETa: 20 days. Tb: (28 ± 1)°C
GC-MS/MS
Concentrations of compounds in the liver of males were higher than in females. Significant transfer of all compounds from adult females to eggs was observed. Several metabolites formed.
Transfer of residues from adult females to eggs was thought to be one of the main factors for the sex-dependent difference in concentrations in liver.
Vallverdú-Queralt et al., 2015
Embryos (fertilized eggs; 24hpf)
To provide accurate and comprehensive identification of the phenolic constituents found in wine extracts and zebrafish embryos
In vivo. Exposure in medium. Embryos analysed. ETa: 24h. Tb: (27 ± 1)°C
HPLC-LTQ-Orbitrap-MS
Quercetin-O-glucoronide and anthocyanin metabolites was detected in embryos.
This research constitutes the first comprehensive identification of phenolic compounds in zebrafish by HPLC-HRMS
Brox et al., 2016
Embryos (fertilized eggs; incubated until 72hpf before use)
Study of xenobiotic metabolism of zebrafish embryo (ZE) using clofibric acid and comparing it with the metabolism in other primarily higher organisms.
In vivo. Exposure in medium. Medium and embryos analysed. ETa: 24h. Tb: (26 ± 1)°C
HPLC-QTOF-MS
Eighteen metabolites was detected formed by phase I and phase II metabolism. Transformation of clofibric acid involves conjugation with sulfate or glucuronic acid, carnitine, taurine, and aminomethanesulfonic acid.
The results of this study outline the high metabolic potential of the ZE with respect to the transformation of xenobiotics. Similarities but also differences to other test systems were observed.
37
Anselmo et al., 2017
Adult (age and sex not informed)
The ability of adult zebrafish to produce metabolites of sibutramine and stanozolol, substances with a well-known metabolism that are widely used as doping agents in sports, was evaluated.
In vivo. Exposure in water. Water analysed. ETa: 7 days. Tb: (26 ± 2)°C
HPLC-QExactive -Orbitrap-MS
Adult zebrafish could produce several sibutramine and stanozolol metabolites, including demethylated, hydroxylated, dehydroxylated, and reduced derivatives, all of which have already been detected in human
This study demonstrates that adult zebrafish can absorb, oxidise, and excrete several metabolites in a manner similar to humans.
Shen et al., 2017
Adult (24-40 weeks; both sexes)
Metabolite profiles of ginsenosides Rk1 and Rg5 from red ginseng or red notoginseng in zebrafish were qualitatively analysed
In vivo. Exposure in water 0.5% DMSO. Medium and embryos analysed. ETa: 24h. Tb: (23 ± 1)°C
HPLC-QTOF-MS
Several metabolites of Rk1 (4) and Rg5 (7), were identified. There are glucuronidation, sulfation, dihydroxylation and dehydroxy-methylation products in metabolites of Rk1 and/or Rg5.
Desugarization, glucuronidation, sulfation, and dehydroxy-methylation was observed. Dehydroxylation and loss of C-17 residue were also metabolic pathways of ginsenoside Rg5
Saad et al., 2017
Different developmental stages (5 and 24hpf, 2, 3, 4 and 5 dpf) and adult (age not informed; both sexes)
Drug metabolism human CYP-specific substrates: dextromethorphan (DXM), diclofenac (DIC), testosterone (TST) and midazolam (MDZ) was investigated in adult (both sexes), embryos and larvae
In vitro. Liver microsomes with < 0.5% (v/v) DMSO. ETa: 2h. Tb: 28.5°C
HPLC-MS/MS
Adult zebrafish produced the two major human metabolites of DIC and DXM. For DIC the metabolite ratio was similar to that in man, whereas it was different for DXM. For TST, the major human metabolite could not be detected and MDZ was not metabolized.
No sex-related differences were detected, except for the higher TST depletion rate in adult females. Zebrafish embryos and larvae showed no or low biotransformation capacity.
Wang et al., 2017a
Adult (20 weeks; both sexes)
The metabolism of six organophosphate (OP) flame retardants was investigated in adult zebrafish
In vivo. Exposure in water 0.01% DMSO. Water and fish analysed. ETa: 19 days. Tb: (24 ± 1)°C
HPLC- QTOF-MS
Twenty main metabolites were detected in the liver of OPs-exposed zebrafish using high resolution mass spectrometry
The reaction pathways involving scission of the ester bond (hydrolysis), cleavage of the ether bond, oxidative hydroxylation, dechlorination, and coupling with glucuronic acid
Wang et al., 2017b
Larvae (7dpf)
Zebrafish as the biotransformation system for rapid identification of metabolites derived from herbal compounds
In vivo. Exposure in water 0.5% DMSO. Water analysed. ETa: 24h. Tb: 28.5°C
HPLC- QTOF-MS
46 metabolites screened from water samples of zebrafish treated with total Epimedium flavonoids (EFs) could be matched with their corresponding parent compounds.
37 of the metabolites were identified and validated. 75% of the identified EFs metabolites were successfully detected in urine samples of rats
a: Exposure time; b: temperature; dpf: days post fertilization; hpf: hours post fertilization. ECODc assay: The metabolism of 7-ethoxycoumarin to 7-hydroxycoumarin as a general marker for CYP2
activities in mammals; ERODd assay: The metabolism of 7-ethoxyresorufin to resorufin as a general marker for CYP1 activities in mammals; OOMRe assay: The conversion of
octyloxymethylresorufin to resorufin was used as a substrate for CYP3 activities in mammals.
38
The first publications that verified the generation of metabolites by
zebrafish were focused on environmental toxicology, since the knowledge of
biotransformation pathways of xenobiotics plays a fundamental
role in environmental monitoring programs. To the best of our knowledge, the
pioneering work that evaluated xenobiotics metabolites in
zebrafish was conducted in 1987 (Kasokat et al., 1987) (Table 2), in
which the metabolism of phenol and substituted phenols was studied in
adult zebrafish following in vivo exposure in the water. On this occasion, phenyl
glucuronide, phenyl sulfate, and quinol sulfate were
identified as metabolites of phenol; 2-cresyl glucuronide, 2-cresyl sulfate, and 2-
hydroxybenzoic acid were determined to be metabolites of
2-cresol; and only the glucuronide and sulfate conjugates were detected
as metabolites of 4-nitrophenol, 4-chlorophenol and pentachlorophenol. Overall,
no significant differences from related freshwater fish species were found, with
the exception of the considerably
lower oxidation rates which were observed in the case of phenol and 2-cresol
(Kasokat et al., 1987). Following this line of study, the metabolism of
chlorobenzene (CB) and hexachlorobenzene (HCB) was also
evaluated in adult zebrafish in a very similar way to the previous work
(Kasokat et al., 1989). The metabolites found for CB were isomeric
chlorophenols (phase I products) and the corresponding glucuronide
and sulfate conjugates (phase II products), and no metabolites were
detected for HCB. The results for CB were in agreement with those
observed in other freshwater fish species. However, other fish species
have already been shown to metabolize HCB, which may indicate that
failure to observe metabolites in this work may be due to the exposure
time, mode of exposure or use of the organic solvent toluene.
Continuing to use the zebrafish for xenobiotic analysis with impact
on the environment, Gorge and Nagel (1990) evaluated the hazards of
the pesticides lindane and atrazine to know whether if and to what
extent substances are incorporated, bioconcentrated and eliminated
when exposure is stopped. None of the metabolites were identified, but
it was possible to observe that atrazine was metabolized to a small
39
extent (4%) and that only three metabolites were formed. In contrast,
50% of lindane was converted to twelve more polar metabolites than
lindane, which must be dechlorination products of the compound. Zok
et al. (1991) conducted an experiment to evaluate the toxicokinetics of
nine anilines in adult zebrafish following in vivo exposure in water. The
corresponding acetanilides were the only metabolites identified in
water, and no biotransformation occurred at all for 2- and 4-nitroaniline,
suggesting that substitution in the ortho position of the aniline
sterically hinders acetylation. The acetylation reaction is catalyzed by
an acetyltransferase that depends on acetyl-CoA, which exists in zebrafish (Ree
et al., 2015). No hydroxylation of the aromatic ring was
observed for the anilines in zebrafish, which was not in agreement with
results observed in humans, where N-acetyl-4-aminophenol was the
predominant urinary aniline metabolite and acetanilides were found
only in minor amounts (Modick et al., 2016).
Hertl and Nagel (1993) evaluated the in vivo metabolism of 3,4-
dichloroaniline, which is an intermediate product of the degradation of
several herbicides, at different life stages of zebrafish. All life stages of
zebrafish possess the capability to metabolize 3,4-dichloroaniline to
3,4-dichloroacetanilide, and acetanilide formation appeared to be
higher in the more advanced stages of the fish. In this work, although
without confirmation, there is evidence of the presence of the hydroxylated
metabolite for 3,4-dichloroaniline. Other studies with environmental pollutants
were performed, such as the evaluation of the
metabolism of atrazine and bisphenol A in zebrafish. Atrazine is a
widely used herbicide, as the induction of GST provides an effective
resistance against atrazine in plants, and it was evaluated whether if the zebrafish
was capable of performing this reaction (Wiegand et al.,
2001). In vitro experiments were performed with enzyme extracts from
zebrafish embryos and atrazine being showed that atrazine-glutathione
conjugates products were formed. In this work, phase I metabolism had
not been evaluated, although in animals, hydroxylation and N-dealkylation are
described as the main metabolic pathways. A study of the
40
metabolism of bisphenol A, an endocrine disrupter which is used in the
production of plastics, has shown that the zebrafish produced the phase
II metabolites bisphenol A glucuronic acid and bisphenol A sulfate after
exposure of adult animals in water (Lindholst et al., 2003). Another
toxicological analysis performed on zebrafish was with carcinogenic
agents. Zebrafish were used as alternative in vivo and in vitro models for
chemical carcinogenesis studies of aflatoxin B1 (AFB1) (Troxel et al.,
1997). The major metabolites of AFB1 recovered in the water at various
time points after adult intraperitoneal injection were identified as
aflatoxicol (predominant metabolite, produced by a cytosolic reductase
reaction) and aflatoxicol-glucuronide. The in vitro assay assessing AFB1
metabolism again demonstrated the proficiency of AFL formation in
zebrafish and produced AFB1-8,9-epoxide, which is responsible for the
carcinogenic effects. Male zebrafish did appear to have lower activity
toward the formation of the AFB1–epoxide than females. This publication
demonstrated not only the ability of zebrafish to generate phase
I and II metabolites but also sex differences in relation to the metabolism of
xenobiotics.
Due to their low cost and high performance, polybrominated diphenyl
ethers (PBDEs) have been widely used for many years as flame
retardants in various commercial products and are an environmental
problem. As PBDEs have similar structures to those of thyroid hormones
(THs), they have the potential to disrupt thyroid endocrine activities;
thus, it is important to understand their in vivo metabolism (Chen et al.,
2012; Wen et al., 2015). After 14 days of exposure, the bioconcentration of BDE-
209 in the larvae was evident and BDE-209 could also be
metabolized, with the main metabolite being nona-BDE. Other BDE
metabolites have already been identified in zebrafish, including tri-BDE, tetra-
BDE, penta-BDE, hex-BDE, hepta-BDE and octa-BDE after
exposure for 5 months (He et al., 2011). The limited production of BDE-
209 metabolites in this study (Chen et al., 2012) compared with He
et al. (2011) can suggest lower metabolic capability in larvae, but may
also be due to the different exposure times and species used. Both T4
41
and T3 levels were changed by BDE-209 exposure in the larvae, suggesting
thyroid disruption (Chen et al., 2012). Other work evaluated
maternal transfer, distribution, and metabolism of BDE-47 and its related
hydroxylated, methoxylated analogs in zebrafish, because analogs
may be more toxic than PBDEs (Wen et al., 2015). Unlike the other
studies, adult fish were exposed by spiked food in this study. The results
indicated that the concentrations of all compounds in the liver of male
zebrafish were significantly greater than those in the liver of females,
suggesting transfer of the residues from adult females to eggs, with
several metabolites formed in adult fish. More recently, G. Wang et al.
(2017) evaluated the in vivo metabolism of six typical organophosphate
(OP) flame retardants using adult zebrafish. In this work, they found
several phase I and phase II metabolites, including OP diesters, hydroxylated OP
triesters, hydroxylated OP diesters, and glucuronic acid
conjugated metabolites, after hydroxylation.
In 2009, the first study to evaluate the zebrafish CYP response to
drugs used to treat humans was carried out, seeking an in vivo comparison
between the species. Li et al. (2009) developed a microplate
whole zebrafish larvae (2 dpf) assay to evaluate zebrafish CYP3A4 and
CYP2D6 functional activity with various specific substrates of these
enzymes. The overall prediction success rate was 86% for CYP3A4 inhibition and
induction in zebrafish, 100% for CYP3A4 inhibition and
71% for CYP3A4 induction (lovastatin and phenytoin did not induce
CYP3A4 in zebrafish as they do in humans); zebrafish CYP2D6 was
upregulated by treatment with dexamethasone, 2,4-dichlorophenoxyacetic acid
and ethanol. These results highlight the high
degree of CYP conservation in zebrafish. After this work, several others
have succeeded in evaluating drug metabolism in zebrafish.
Alderton et al. (2010) examined the accumulation and metabolism of a
number of drugs and commonly used probes for human CYPs in
zebrafish larvae (7 dpf) under conditions relevant to pharmacological
and toxicological assays. Metabolism studies with cisapride, chlorpromazine,
verapamil, testosterone, and dextromethorphan showed that
42
the zebrafish larvae catalyze several phase I (oxidation, N-demethylation, O-de-
ethylation, hydroxylation and N-dealkylation) and phase II
(sulfation and glucuronidation) reactions, where both similarities and
differences in the metabolic pathways were observed in zebrafish larvae
when compared to mammals. Additionally, the metabolism of phenacetin to
paracetamol and dextromethorphan to dextrorphan (metabolic
reactions catalyzed by CYP1A2 and CYP2D6 in humans, respectively)
was observed in the zebrafish larvae. Metabolism of amodiaquine,
midazolam, felodipine, and S-mephenytoin could not be detected under
the conditions used in these experiments. When zebrafish larvae were
exposed to cisapride, the only metabolite observed was cisapride Nsulfate, which
is a minor metabolite in mammalian species observed
only in the dog. Although some hydroxylation of testosterone (very
low) was observed in the current experiments, the predominant metabolic
pathway of testosterone observed in zebrafish larvae was glucuronidation. In
addition to that, the amount of any specific metabolite
formed was always much lower than that of the parent compound. All
these factors may indicate that the high amount of DMSO used (1%) in
this work may have influenced the results, since it has already been
demonstrated that low concentrations of organic solvents may influence
the activity of zebrafish phase I and II enzymes (David et al., 2012).
Jones et al. (2010) have assessed the expression of genes that have
been identified as similar to mammalian CYP1A, CYP2B6, CYP3A5, and
UGT1A1, which are considered key mammalian families responsible for
the oxidative metabolism of a variety of pharmaceuticals. Overall, this
study provided further evidence that zebrafish larvae express genes and
proteins with activities that are responsible for xenobiotic metabolism
with both oxidative and conjugative metabolism similar to those of
mammalian systems. Another study evaluated the in vitro hepatic
fluoxetine metabolism of adult zebrafish compared with several others
model fish: rainbow trout, goldfish and killifish (Smith et al., 2010).
After killifish, zebrafish was the fish model with the highest rates of
metabolism of fluoxetine. In humans, fluoxetine is primarily metabolized into
43
norfluoxetine (major metabolite) through demethylation by
CYP2D6 (has no ortholog in zebrafish, Table 1) (Goldstone et al., 2010)
and to a lesser extent, by CYPs 2C9, 3A4 and 2C19. In zebrafish, the loss
of fluoxetine is much greater than the production of the demethylated
metabolite, norfluoxetine, suggesting that there may be production of
multiple fluoxetine metabolites in fish and that norfluoxetine is unlikely
to be the primary fluoxetine metabolite, as it is in humans (Smith et al.,
2010).
Chng et al. (2012) progressed forward and investigated the bioactivation
potential and metabolism profile of zebrafish (in vitro and in
vivo) versus human (in vitro) using acetaminophen (APAP) and testosterone. The
hepatotoxicity of APAP is attributed to its reactive metabolite, NAPQI (N-acetyl-
p-benzoquinone imine), that was generated by
adult zebrafish liver microsomes (ZLM) (possibly by CYP3A65), albeit
at lower levels than by human liver microsomes (HLM). Several hydroxylated
metabolites of testosterone were identified, including 2α-,
6β-, and 16β hydroxytestosterone and three putative metabolites (M2,
M3, and M7) in the ZLM and only 6β-hydroxytestosterone, testosterone
glucuronide, and M8 in zebrafish larvae homogenate and media. Although the
main metabolite formed by zebrafish was 6β-hydroxytestosterone, the overall
metabolite profile was distributed across
more hydroxylated metabolites compared with that of HLM, and a
number of unique hydroxytestosterone metabolites were observed only
in zebrafish. This result suggests a difference in either the function or
the expression of the drug-metabolizing enzymes between zebrafish
larvae and adult zebrafish, in addition to demonstrating differences
between human and zebrafish in vitro testosterone metabolism.
Another study evaluated the metabolism of ibuprofen in zebrafish
larvae (3 dpf) after in vivo exposure in water with ethanol 0.0015% (v/v) (Jones
et al., 2012). Ibuprofen metabolism is well described in humans, involving
oxidation of the parent compound to hydroxy-ibuprofen and carboxy-ibuprofen by
CYP2C8/9, followed by conjugation
with glucuronic acid. Although no CYP2C-corresponding orthologs
44
have been identified in zebrafish, it has been able to generate hydroxyibuprofen
and other hydroxylated derivatives. However, the presence of
carboxy-ibuprofen and glycoconjugate derivatives was not observed,
revealing differences between zebrafish and human metabolism of
ibuprofen.
Several other studies have observed the in vivo production of phase I
and II metabolites during different life stages of zebrafish using the
following various substances, showing once again that the zebrafish is a
promising model that shares similarities and differences with mammals:
flavonoids (Hu et al., 2012), veterinary pharmaceuticals (albendazole,
febantel, fenbendazole and oxfendazole) (Carlsson et al., 2013), resveratrol
(Pardal et al., 2014), phenolic constituents found in wine
(Vallverdú-Queralt et al., 2015), clofibric acid (Brox et al., 2016) and
ginsenosides Rk1 and Rg5 (Shen et al., 2017). Pardal et al. (2014)
evaluated the metabolism of the phenolic compounds resveratrol and
its glucoside derivative (piceid) in vivo after exposure of zebrafish
larvae in 1% (v/v) DMSO medium. The principal metabolites found in
zebrafish larvae were monoglucuronide and monosulfate forms of resveratrol (the
most abundant metabolite), which is in agreement with
other mammals, including human. In the case of the piceid group, the
most abundant metabolite was the sulfated form of resveratrol, even if
it appeared later. In this work, it was not possible to identify glucuronidated or
sulfated piceid and other metabolites found in rat urine,
possibly for one of the following reasons: inability of the zebrafish
model to generate these metabolites; minor compounds that cannot be
detected under the experimental conditions; high amount of DMSO
used that may have interfered in the metabolism.
Brox et al. (2016) evaluated the metabolism of clofibric acid using
zebrafish embryos and compared it with the metabolism in other, primarily higher
organisms. A total of 18 metabolites of clofibric acid were
detected, including reactions of hydroxylation, demethylation, hydrolysis,
methylation, decarboxylation and conjugation with sulfate and
glucuronic acid. In addition, conjugation with taurine, aminomethanesulfonic acid,
45
and carnitine represents new phase II conjugates that
have not been described in zebrafish before this work. In humans, the
acyl glucuronide of clofibric acid (also generated by zebrafish) represents the
major metabolite, and taurine-conjugated clofibric acid
was previously detected in dogs, cats, and ferrets, and now in zebrafish
but not in humans.
Anselmo et al. (2017) evaluated the ability of adult zebrafish to
produce metabolites of sibutramine and stanozolol after in vivo exposure, with
the focus of using the model to search for analytical targets
for the detection of doping abuse. Several sibutramine and stanozolol
metabolites were identified, including demethylated, hydroxylated,
dehydroxylated, and reduced derivatives and stanozolol conjugates, all
of which have already been detected in human urine. This study suggests that
adult zebrafish can absorb, oxidize, and excrete several metabolites in a manner
similar to humans.
Saad et al. (2017) made an important contribution by comparing
the in vitro drug metabolism of human CYP-specific substrates at different life
stages of zebrafish with humans. Several CYP-related reactions in adult
zebrafish using human CYP2- and CYP3-specific substrates were detected. No
in vitro biotransformation of the tested
substrates was observed during zebrafish embryonic development until
96 hpf (end of organogenesis). Unlike other studies, the biotransformation rates
were predominantly higher in zebrafish compared
to humans, except for CYP2D6-like activity. Sex-related differences
were only present for testosterone. Diclofenac biotransformation in
zebrafish into 4- and 5-OH-diclofenac reveals similar biotransformation
pathways compared to humans by the production of similar major
metabolites with similar proportions in both species. However, dextromethorphan
was also biotransformed into similar main metabolites as in humans but with
altered ratios as shown (dextrorphan/3-methoxymorphinan). In this study, no
production of the main testosterone
metabolite was observed, which was already reported in another work
(Chng et al., 2012). For midazolam, neither consumption nor metabolites could
46
be detected in ZLM, whereas this substrate was largely metabolized into 1-OH-
midazolam and, to a much lesser extent, into 4-OHmidazolam in HLM. As a
result, midazolam, a typical substrate for
CYP3A in man, appeared to have no affinity for any of the zebrafish
CYPs. Therefore, the in vitro CYP-mediated drug metabolism can be
substantially different in zebrafish compared with man.
C. Wang et al. (2017) proposed a rapid identification of metabolites
derived from herbal compounds after in vivo exposure of zebrafish
larvae (7 dpf), compared with metabolism in rats, a representative
mammalian model. This work identified 46 potential metabolites that
could be matched with their corresponding parent compounds, and 37
of them were identified and validated by the known metabolic pathways and
fragmentation patterns. Among the 37 metabolites identified
in zebrafish, 28 were found in rat urine. This result encourages researchers to
use zebrafish larvae as the biotransformation system for
preliminary metabolism study.
1.1.6 Metabolites analysis
The first studies that carried out the detection of xenobiotic metabolites in
zebrafish used HPLC (high-performance liquid chromatography) with diode-array
detection (DAD) analysis (Gorge and Nagel,
1990; Hertl and Nagel, 1993; Kasokat et al., 1987, 1989; Troxel et al.,
1997; Zok et al., 1991). HPLC-DAD provides a means for analysing
various compounds, but is limited by the non-specific nature of UV
detection. On the other hand, gas chromatography–mass spectrometry
(GC–MS), together with detailed GC–MS spectral libraries, is a very
useful tool for toxicological analysis, but non-volatile, polar and thermally labile
compounds are difficult or impossible to analyse without a
long derivatization procedure (Couchman and Morgan, 2011).
An important contribution for analytical toxicology was the development of
the electrospray ionization (ESI) (Fenn, 2002), which was
responsible for the coupling of liquid chromatography (LC) and mass
47
spectrometry (MS), expanding the detection range of new metabolites,
and increasing the detection capacity of substances even at low concentrations.
Triple quadrupole (QqQ) instruments operating in selected
reaction monitoring (SRM) mode are the most common mass analysers
used in bioanalysis (Núñez et al., 2013), as can be observed in several
studies (Alderton et al., 2010; Carlsson et al., 2013; Chng et al., 2012;
Saad et al., 2017). However, for the detection by tandem-MS techniques, as the
triple quadrupole, the potential metabolites must be previously known or need to
share a high degree of structural similarity
with the parent compound to allow their determination by neutral loss
or precursor ion scan. However, these conditions are not necessarily
possible in metabolism studies (Brox et al., 2016).
An emerging approach for toxicological analysis is that of the
determination of exact mass with high-resolution (HRMS). Through a fullscan
HRMS experiment at high mass accuracy (mass errors below
6 ppm), it is possible to very precisely filter the full-scan data and extract analyte
chromatograms with very low background noise, which
allows the identification of metabolites by knowledge of the elemental
composition alone, without the need for reference material (Couchman
and Morgan, 2011). Time-of-flight (TOF) and Orbitrap-based technologies are
currently the most common analysers used in LC–HRMS
(Núñez et al., 2013). In fact, studies using LC-HRMS to search for xenobiotic
metabolites have been able to identify and sometimes characterize a greater
number of substances (Anselmo et al., 2017; Brox
et al., 2016; Shen et al., 2017; C. Wang et al., 2017) compared to other
techniques. The major challenge in the identification of metabolites by
LC-MS is the detection and structural elucidation of trace levels of
unknown metabolites in the presence of large amounts of complex interfering
ions of endogenous components, called matrix effect (Zhu et al., 2011). Thus, a
major advantage of using the in vivo zebrafish
model for predicting metabolism is that the matrix is cleaner than urine,
for example, widely used in other animal models, which reduces the
matrix effect.
48
1.1.7 Zebrafish considerations for metabolite screening
In general, the studies that evaluated the production of xenobiotic
metabolites in zebrafish observed several different phase I and II metabolites,
demonstrating the utility of the model in the prediction of the
metabolism of several substances. Therefore, there is further evidence
that zebrafish express genes and proteins with activities responsible for
xenobiotic metabolism with both oxidative and conjugative metabolism
similar to mammalian systems. Regarding the zebrafish CYP orthologs
in humans, it is important to highlight that three cases were observed:
no orthology with production of metabolites (fluoxetine (Smith et al.,
2010), CYP2D6; and ibuprofen (Jones et al., 2012), CYP2C); presence of
orthology without production of metabolites (midazolam (Saad et al.,
2017), CYP3A) and presence of orthology with production of metabolites
(sibutramine and stanozolol (Anselmo et al., 2017), CYP2B6 and
CYP3A4, respectively), which is the ideal case and the most often
found. Even in cases where human orthologs of CYP are present in
zebrafish, divergences may occur in relation to the major metabolites
generated or the production ratio of these metabolites in comparison
with humans (Alderton et al., 2010; Chng et al., 2012; Jones et al.,
2012). So, further studies are needed to understand to what extent the
zebrafish is able to reproduce the metabolism observed in humans.
Nevertheless, it is important to highlight that even the mouse, a classic
model for prediction of toxicity and evaluation of metabolites, presents
some differences in relation to human metabolism (Lootens et al.,
2009). Therefore, this issue does not prevent zebrafish from being
considered a model with great potential for studying the metabolism of
xenobiotics.
Zebrafish larvae represent a very interesting alternative model to
animal experimentation, although it is important to remember that
early life stages (3 to 5 dpf) are still undergoing embryogenic processes
and that their metabolic status is different from that of the young or
49
adult zebrafish (Pardal et al., 2014). In addition, when using zebrafish
embryos, there is a need for them to take up substances through the
chorion and other membranes (Vallverdú-Queralt et al., 2015), which
can generate results compromised by the differentiated absorption at
this life stage. Moreover, it has already been observed that the activity
of most CYPs involved in xenobiotic metabolism is increased dramatically after
hatching approximately 3 dpf (Saad et al., 2016) and that the
liver is fully formed at approximately 5 dpf (Kimmel et al., 1995). Although several
studies have observed the production of metabolites in
the embryonic and larval stages of zebrafish (Table 2), Saad et al.
(2017) observed no in vitro biotransformation during zebrafish embryonic
development until 96 hpf. Therefore, the age of the zebrafish is
a controversial issue and should be taken into account during experimental
design and when comparing the results with other mammals.
In zebrafish in vivo screening models, the main route of exposure is
dermal until approximately 5 dpf when the mouth is opened and both
dermal and enteral routes exist from 5 to 14 dpf while the embryo is
free-feeding (Garcia et al., 2016; Sipes et al., 2011). Usually, in conventional in
vivo experiments, a known and measurable amount of drug
is administered to the animal. However, in the zebrafish in vivo models,
the uptake from the water into the zebrafish is non-uniform and unpredictable,
which can make it difficult to interpret screening results
and to make comparisons between compounds (Fleming and Alderton,
2013).
Another important issue to be discussed in relation to zebrafish
screening is the use of organic solvents to increase the solubility of the
substances tested and their impact on metabolism enzymes. David et al.
(2012) observed that metabolism of ethoxyresorufin in zebrafish larvae
was significantly reduced by dimethylsulfoxide (DMSO) and methanol (0.1% and
0.05% v/v, respectively) after 24 h of exposure. The decreases in activity of
ethoxyresorufin were accompanied by decreased
expression of various genes coding for drug metabolizing enzymes
corresponding to CYP1, CYP2, CYP3 and UGT family enzymes (David
50
et al., 2012). Thus, relatively low concentrations of organic solvents
may impact the biotransformation of certain xenobiotics in zebrafish
larvae, and this deserves consideration when evaluating substances for
the study of metabolism and toxicity in this species. This fact, together
with the age of the fish, the amount of substance used in the experiment
and the unpredictable uptake from the exposure water, may be a determinant in
the metabolic profile that will be found and should be
taken into account when designing and interpreting results from metabolite
screening.
1.1.8 Conclusion
In the last 30 years, the zebrafish has been used to predict the metabolism
of xenobiotics, with different focuses. In the early years, studies focused on
environmental toxicology, and in the possible impact of
the generated metabolites on increasing the toxicity of the contaminants.
Currently, several studies have evaluated the metabolism of
drugs aiming to establish zebrafish as a model for toxicological
screening, either for the development of new drugs or for analytical
targets in anti-doping control. These studies have shown that the zebrafish
enzymes responsible for metabolism are analogous with those in
humans, performing several phase I and II reactions. Some factors need
to be considered for the use of the model, since they can directly influence the
results, such as: fish age, impact of the use of organic solvents and time of
exposure. In addition, a limitation of the in vivo model
of zebrafish is that it is not possible to control the amount of substance
administered by each fish and can generate non-reproducible results.
As with any non-human model, zebrafish model presents similarities
and differences in relation to the profile of metabolites generated
compared to that observed in humans. All the facts presented indicate
that zebrafish is a valuable potential model for the evaluation of xenobiotic
metabolism, but further studies are still needed to assess the
degree to which it can be compared to human metabolism.
51
1.1.9 Conflict of interest statement
The authors declare that there are no conflicts of interest.
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Cruchten, S., 2016. Xenobiotic metabolism in the zebrafish: a review of the spatiotemporal distribution, modulation and activity of Cytochrome P450 families 1 to 3. J. Toxicol. Sci. 41, 1–11. https://doi.org/10.2131/jts.41.1.
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in the cytochrome P450 1 family enzymes from Zebrafish (Danio rerio) using heterologously expressed proteins. Arch. Biochem. Biophys. 502, 17–22. https://doi. org/10.1016/j.abb.2010.06.018.
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1.1.11 Justificativa do trabalho
Um dos principais desafios da ciência antidopagem reside na gigantesca
diversidade de substâncias disponíveis e capazes de promover o aumento do
desempenho, sejam desenvolvidas na indústria formalmente constituída, sejam
moléculas desenvolvidas na clandestinidade e comercializadas no mercado
clandestino (KWIATKOWSKA et al., 2015; THEVIS & SCHÄNZER, 2014). A
maioria dos agentes de dopagem é convertida em metabólitos de fase I e II para
facilitar a excreção na urina. O metabolismo de fase I consiste em reações de
oxidação, redução ou hidroxilação catalisada pelo citocromo (CYP), localizado
principalmente no fígado. Por outro lado, a glicuronidação e a sulfonação são as
principais reações de fase II. Portanto, o perfil de excreção metabólico de cada
substância proibida pela WADA (World Anti-Doping Agency) deve ser
monitorado para assegurar a seleção adequada das substâncias-alvo no
controle antidopagem (BADOUD et al., 2011).
O uso de substâncias que melhoram o desempenho, particularmente
esteróides anabólicos androgênicos (EAA) e estimulantes, foi reconhecido como
uma prática comum, especialmente em esportes de elite (PEREIRA &
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SARDELA, 2014; ALQURAINI & AUCHUS, 2018). Os estimulantes são
substâncias capazes de reduzir o cansaço e aumentar a agilidade,
competitividade e agressividade. Esta classe farmacológica inclui os
estimulantes psicomotores, aminas simpatomiméticas e estimulantes do sistema
nervoso central (SNC), como a sibutramina. Já os EAA são uma classe de
substâncias sintéticas normalmente derivadas da testosterona, hormônio
anabólico e androgênio endógeno (WANG et al., 2017). Por esses efeitos, os
EAA são uma classe importante de substâncias com potencial para utilização
indevida no esporte. O estanozolol é um esteróide anabolizante exógeno
derivado da testosterona (HELFMAN & FALANGA, 1995; WANG et al., 2017) e,
como a sibutramina, é proibido pela WADA.
O estanozolol é metabolizado intensamente e seus metabólitos urinários
podem ser detectados por muito mais tempo do que a droga mãe, sendo
amplamente descritos na literatura (POZO et al., 2009; SCHÄNZER,
OPFERMANN & DONIKE, 1990; TUDELA, DEVENTER & VAN EENOO, 2013;
WANG et al., 2017). A sibutramina também é extensivamente metabolizada nos
seus dois metabólitos desmetilados farmacologicamente ativos, e
posteriormente, nos derivados seus correspondentes derivados hidroxilados
(STRANO-ROSSI, COLAMONICI & BOTRE, 2007; SARDELA et al., 2009). Por
serem substâncias extensamente metabolizadas apresentando metabólitos de
fase I e II já elucidados, além de serem amplamente utilizadas como dopagem
no esporte, a sibutramina e o estanozolol constituem alvos para o
desenvolvimento de novos modelos para avaliação do metabolismo de
xenobióticos.
Diferentes estratégias para a investigação do metabolismo de fármacos
estão disponíveis, sejam modelos in vivo ou in vitro (MARQUES et al., 2014;
PARDAL et al., 2014; ZHAO et al., 2014). Esposito e colaboradores (2015)
avaliaram o metabolismo de pequenos peptídeos hormonais através dos
modelos de microssomas e frações hepáticas e soro humano. Os autores
demonstraram a correlação entre os dois modelos, algo promissor por criar a
oportunidade da eliminação de estudo em humanos.
Höppner e colaboradores (2014) estudaram o metabolismo de fármacos
que ativam a enzima SRT1 (envolvida em processos de aumento da tolerância
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a exercícios) comparando-se os metabólitos produzidos in vivo, em urina e
sangue de rato, com os observados in vitro, com enzimas microssomais de ratos
e humanos. Neste estudo evidenciou-se que há uma correlação entre os
modelos in vivo e in vitro, em relação aos metabólitos gerados. No entanto,
alguns metabólitos hidroxilados observados in vitro não foram encontrados em
urina e sangue de ratos.
A constatação de que modelos in vitro por vezes apresentam desvios de
resultados quando comparados a estudos clínicos leva a busca por modelos
alternativos. Lootens e colaboradores (2011 e 2009) avaliaram o perfil
metabólico de esteróides anabolizantes utilizando camundongos transplantados
com hepatócito humano (uPA(+/+)-SCID) gerando animal quimérico,
denominado “rato humanizado”. O esteroide anabolizante metandienona foi
utilizado como modelo. Nos resultados, diversos esteroides hidroxilados foram
detectados como metabólitos minoritários, em contraste com trabalhos utilizando
apenas culturas de hepatócitos.
Outro modelo emergente no campo da avaliação in vivo de metabólitos de
xenobióticos envolve o peixe zebrafish (Danio rerio), devido à grande
similaridade genética que apresenta com os vertebrados, em especial com os
humanos (SANTORO, 2014; ZHOU et al., 2015). Algumas vantagens que fazem
do zebrafish uma alternativa viável em relação a outros modelos in vivo são: mais
fácil e econômico que os populares modelos de roedores; os embriões se
desenvolvem rapidamente em comparação com modelos de mamíferos,
reduzindo potencialmente o tempo de experimentação (GARCIA, NOYES &
TANGUAY, 2016); e os embriões possuem alta capacidade de reprodução
(KAMEL & NINOV, 2017). Além disso, diversas enzimas CYP humanas têm
ortólogas diretas no zebrafish (GOLDSTONE et al., 2010), sugerindo que os
perfis metabólicos desse peixe podem ser semelhantes aos dos mamíferos.
Em relação as técnicas analíticas utilizadas para análise de metabólitos
em matrizes biológicas, notavelmente, até o final do século XX, a técnica de
cromatografia gasosa acoplada à espectrometria de massas (CG-EM) era a
ferramenta analítica com especificidade e sensibilidade suficientes para essa
detecção (MATEUS-AVOIS, MANGIN & SAUGY, 2005). Entretanto, desde o
início do século XXI, a noção de quais metabólitos devem ser monitorados sofreu
60
o impacto da introdução de novas ferramentas analíticas. Entre essas, a técnica
de eletronebulização (electrospray ionization – ESI), responsável pelo
acoplamento da cromatografia líquida (CL) a espectrometria de massas (EM),
ampliou o leque de detecção de novos metabólitos, aumentando a capacidade
de detecção de substâncias em concentrações ainda mais baixas (COSTELLO,
1997; FENN et al., 1989 & FENN, 2002), sendo muito útil na busca de novos
metabólitos.
Considerando-se que muitos fármacos são extensivamente, ou até
mesmo completamente metabolizados antes de serem excretados, o estudo do
metabolismo é peça fundamental na ciência antidopagem, principalmente na
busca de marcadores (metabólitos de fase I e II) mais eficientes para o
diagnóstico do abuso de substâncias proibidas por atletas (BADOUD et al.,
2011). A análise de metabólitos contidos em amostras biológicas aumenta
consideravelmente o número de analitos potenciais que podem ser detectados
e identificados em caso de dopagem (BADOUD et al., 2011). Além disso, por
vezes, a peculiaridade das substâncias utilizadas no contexto da dopagem
esportiva – ausência de investigações de toxicidade típicas do desenvolvimento
de fármacos – torna oportuno o emprego de modelos não humanos (LOOTENS
et al., 2011; ESPOSITO et al., 2015). Sendo assim, neste trabalho o modelo in
vivo utilizado para predição dos metabólitos será o zebrafish devido a sua
relevância como modelo para predição do metabolismo de xenobióticos.
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62
2 OBJETIVOS
Os objetivos deste trabalho são o desenvolvimento e caracterização do
modelo de zebrafish para avaliação do perfil metabólico de substâncias
monitoradas no controle antidopagem.
2.1 OBJETIVOS ESPECÍFICOS
Avaliar a capacidade do zebrafish em produzir metabólitos de sibutramina
e estanozolol, substâncias com um metabolismo bem conhecido que são
amplamente utilizados como agentes de dopagem no esporte;
Desenvolver e validar método de cromatografia líquida acoplada a
espectrômetro de massas de alta resolução para detecção dos
metabólitos urinários de sibutramina;
Comparar o metabolismo da sibutramina em humanos, camundongos e
zebrafish;
Avaliar a enzima CYP responsável pelo metabolismo de sibutramina em
zebrafish;
Avaliar o metabolismo de outras substâncias utilizadas como dopagem no
esporte em zebrafish.
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64
3. Capítulo I
3.1 Is zebrafish (Danio rerio) a tool for human-like metabolism study?
Carina de Souza Anselmo, Vinicius Figueiredo Sardela, Bernardo Fonseca Matias, Amanda
Reis de Carvalho,Valeria Pereira de Sousa, Henrique Marcelo Gualberto Pereira, Francisco
Radler de Aquino Neto
Drug Test Anal. 2017 Nov;9(11-12):1685-1694. doi: 10.1002/dta.2318. Epub 2017 Nov 13.
3.1.1 Abstract
One of the greatest challenges in anti-doping science is the large number of
substances available and the difficulty in finding the best analytical targets to
detect their misuse. Therefore, metabolism studies involving prohibited
substances are fundamental. However, metabolism studies in humans could face
an important ethical bottleneck, especially for non-approved substances. An
emerging model for metabolism assessment is the zebrafish, due to its genetic
similarities with humans. In the present study, the ability of adult zebrafish to
produce metabolites of sibutramine and stanozolol, substances with a well-known
metabolism that are widely used as doping agents in sports, was evaluated. They
represent 2 of the most abused classes of doping agents, namely, stimulants and
anabolic steroids. These are classes that have been receiving attention because
of the upsurge of synthetic analogues, for which the side effects in humans have
not been assessed. The samples collected from the zebrafish tank water were
hydrolysed, extracted by solid-phase extraction, and analysed by liquid
chromatography with high resolution mass spectrometry (LC–HRMS). Adult
zebrafish could produce several sibutramine and stanozolol metabolites,
including demethylated, hydroxylated, dehydroxylated, and reduced derivatives,
all of which have already been detected in human urine. This study demonstrates
that adult zebrafish can absorb, oxidise, and excrete several metabolites in a
manner similar to humans. Therefore, adult zebrafish seem to be a very
promising tool to study human-like metabolism when aiming to find analytical
targets for doping control.
Copyright © 2017 John Wiley & Sons, Ltd.
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3.1.2 Introduction
One of the major challenges of anti-doping science lies in the huge
diversity of substances available and capable of promoting performance
enhancement in the formally established industry or molecules developed
underground and sold on the black market. [1,2] Considering that many drugs are
extensively or even completely metabolised before being excreted, the study of
metabolism is a key element in anti-doping science, especially in the search for
more efficient markers for the detection of prohibited substances used by
athletes. [3] Nevertheless, the investigation into the metabolism of new drugs
usually faces an ethical bottleneck in exposing humans to, sometimes, untested
drugs.
Thus, different strategies to investigate drug metabolism are available in
the literature, using either in vivo or in vitro models. Most in vitro techniques utilize
hepatic fractions, as microsomes and S9 [4–7] or culture of hepatocytes. [8–10]
In addition to the study of metabolism in humans, there are other in vivo models
that use rats and mice, [11–14] mice with humanized liver [15–17] and other
vertebrates. [18,19] Despite the tendency to perform in vitro tests for ethical
issues, it is notable that some studies have shown dissociations between
metabolites generated by in vitro models with those produced in vivo. [16,20,21]
Besides, previous studies have observed important differences between the
profile of metabolites produced in mice compared to human.[ 16,21] Therefore,
there is still a need for the identification of alternative in vivo models that
adequately reproduce human metabolism.
The zebrafish (Danio rerio) was introduced by Streisiger as an animal
model in genetic studies in the early 1980s; the zebrafish is a small teleost (3 to
4 cm) typically from sweet waters. [22] Some important advantages have been
associated with the zebrafish in terms of their use in research, such as the small
size, easy maintenance, low cost of breeding, and high reproductive rate. In
addition, its genome has already been sequenced, and it presents important
homology with mammals, besides the morphological and molecular basis of
tissues and organs similar to humans. [23–26] So, zebrafish is emerging as a
66
predictive vertebrate animal model for in vivo assessment of drug efficacy,
toxicity, and safety. [26–31] Besides, some studies have evaluated the ability of
zebrafish larvae at different stages of ripening to generate xenobiotic metabolites.
[32–35] However, studies have shown a difference either in the function or
expression of the drug metabolizing enzymes between zebrafish larvae and adult
zebrafish, suggesting that adults are more effective in generating xenobiotic
metabolites. [36,37] Therefore, to assess the zebrafish’s ability to mimic human
metabolism, it is interesting to use substances that are extensively metabolized
and whose metabolites are known, such as sibutramine and stanozolol.
The use of performance-enhancing substances, particularly androgenic-
anabolic steroids (AAS) and stimulants, has been recognized as a common
practice, especially in elite sports. [38,39] Sibutramine is a drug classified as
stimulant by the World Anti-Doping Agency (WADA) [40] that acts by inhibiting
the re-uptake of the neurotransmitters serotonin, norepinephrine and dopamine
and enhancing satiety. [41] Sibutramine undergoes primary and secondary
metabolism, and its elimination occurs after biotransformation to demethylated
metabolites, which are in turn hydroxylated and, finally, conjugated. [42,43] The
peripheral effects of sibutramine, due to the pharmacologically active
demethylated metabolites, commonly include elevated blood pressure, increased
pulse rate, and bronchodilatation, which is complemented by diminished fatigue
and improved alertness. These beneficial effects are considered the major
reasons that athletes abuse stimulants in sports, and numerous doping rule
violations have been recorded ever since systematic doping controls have been
conducted. [42,44,45]
AAS is a class of synthetic substances normally derived from testosterone,
which is an endogenous androgen and protein anabolic hormone. [46] Because
of this, AAS is an important class of performance-enhancing drugs with the
potential for misuse in sports. [47,48] Stanozolol is an exogenous anabolic steroid
designed from testosterone, [46,49] and like sibutramine, it is prohibited by
WADA. [40] Stanozolol is intensively metabolized and its urinary metabolites can
be detected much longer than the parent compound. The main phase-I metabolic
products that are important for human doping control purposes are 30-hydroxy-
stanozolol, 4β-hydroxystanozolol, 16β-hydroxy-stanozolol and 4,16-dihydroxy-
67
stanozolol, which are excreted in human urine as glucuronide conjugates.
[46,48,50,51] The aim of this work is to evaluate the similarities between the
metabolism of adult zebrafish and humans in relation to the production of phase
I and II metabolites of the xenobiotics sibutramine and stanozolol.
3.1.3 Experimental
3.1.3.1 Chemicals and reagents
Sibutramine and sibutramine metabolite (N-desmethyl-sibutramine) were
purchased from LGC (Teddington, Middlesex, UK). Stanozolol and stanozolol
metabolite (16β-hydroxystanozolol) were purchased from the National
Measurement Institute (Sydney, Australia). Buspirone (used as internal standard)
was supplied by SigmaAldrich (São Paulo, Brazil). All chemicals (ie, formic acid,
acetic acid, ammonium formate, sodium phosphate and methanol) were
analytical or high performance liquid chromatography (HPLC) grade (Tedia,
Fairfield, USA). The ultrapure water used was Milli-Q grade (Millipore, Billerica,
USA). The enzyme used for the enzymatic hydrolysis of the conjugates was β-
glucuronidase (from Escherichia coli) (Roche, Rio de Janeiro, Brazil).
3.1.3.2 Zebrafish metabolism model
Adult zebrafish (Danio rerio) were kept in tanks with 4 L of aerated water
at 26 ± 2°C. The fish were purchased at a fish store in Rio de Janeiro. The fish
were fed with Tetramin® ration (composed of fish derivatives, vitamins, and
minerals) once a day during all experiments. Powder standards of sibutramine
and stanozolol diluted in water were used to treat the tanks. Four tanks were
used: 2 negative controls (with fish/without substance addition and without
fish/with substance addition), a tank treated with sibutramine (15 mg), and a tank
treated with stanozolol (1 mg). For stanozolol, a poorly water-soluble substance,
a smaller amount of 1 mg was placed in water (500 mL) leaving in the sonicator
for 30 minutes, before adding to the tanks. Twelve fish were placed in all tanks.
The experiments were repeated 3 times. From all tanks, aliquots of 5 mL were
collected prior to the administration of sibutramine and stanozolol and at the
68
following times: 0, 3, 6, 24, 48, 72, 96, 120, 144, and 168 hours. This study was
approved by the Ethics Committee on the Use of Animals of the Federal
University of Rio de Janeiro through protocol number 022/17.
3.1.3.3 Samples
The procedure used was adapted from the laboratory routine. Aliquots of
1 mL from all tanks were hydrolysed and then extracted before injection into the
LC–HRMS system. Briefly, 5 μL of buspirone solution (internal standard, 50
ng/mL), 750 μL of pH 7.0 phosphate buffer and 50 μL of β-glucuronidase solution
were added to 1 mL of sample. The tubes were shaken and left in a water bath
at 50°C for 1 hour. The samples were extracted using a Strata-X-CW column
(Phenomenex, Torrance, CA, USA). The cartridges were conditioned with 1 mL
of methanol, followed by 1 mL of water. After this step, 1 mL of the sample was
applied. The cartridges were washed with 1 mL of water and then 1 mL of the
methanol:water (1:1) solution. The cartridges were allowed to dry, and the
analytes were recovered with 1 mL of methanol and 5% (v/v) formic acid. The
tubes were left under N2 flow at 40°C. The contents of the tubes were
reconstituted with 100 μL of a water:methanol (70:30) 0.1% formic acid/5 mM
ammonium formate solution. Standard solutions of N-desmethyl-sibutramine and
16β-hydroxystanozolol were also prepared in the same manner as the samples.
Additionally, 200 μL from all tanks were mixed (1:1) with a water:methanol (99:1)
solution with 0.1% formic acid and then directly injected into the LC–HRMS
system (dilute-and-shoot, DS).
3.1.3.4 Instrumental conditions
3.1.3.4.1 Liquid chromatography
All LC experiments were performed using a Thermo Scientific Dionex
Ultimate 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Reversed-phase
liquid chromatography was performed using a Syncronis C18 column (2.1 × 50
mm, 1.7 μm). The injection volume was 8 μL. The solvents used were: water
69
containing 0.1% formic acid and 5 mM ammonium formate (eluent A) and
methanol containing 0.1% formic acid (eluent B). The gradient programme
started at 5% B and increased to 10% B after 0.5 minutes, to 25% B after 0.5
minutes, to 90% B in 6 minutes, and to 100% B in 8 minutes. The column was
flushed for 2 minutes at 100% B and finally re-equilibrated at 5% B for 2 minutes.
The flow rate was set at 400 μL/min, and the column temperature, at 40°C.
3.1.3.4.2 HRMS analysis
Detection of substances was performed using a QExactive™ Hybrid
Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham,
MA, USA) using electrospray ionization (ESI). The mass calibration was
performed daily before starting the analysis using a calibration solution provided
by the manufacturer. The capillary temperature was set to 380°C, and the spray
voltage was 3.9 kV; other parameters for MS were sheath gas 60, aux gas 20
and sweep gas 0. An initial screening for metabolites was carried out in full scan
positive mode in a range of m/z 100–800. The chromatograms were evaluated
using TraceFinder 3.2.512.0 software (Thermo Fisher Scientific, Waltham, MA,
USA). Fragmentation analysis The detection of substances was performed using
a QExactive™ Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher
Scientific, Waltham, MA, USA) using ESI. For fragmentation studies, the product
ion spectra were recorded with a collision energy of 80 eV for 16β-
hydroxystanozolol (m/z 345.25) and 35 eV for N-desmethyl-sibutramine (m/z
266.17) from reference standard solutions and from samples of tanks treated with
stanozolol and sibutramine. The spectra and chromatograms were evaluated
using TraceFinder 3.2.512.0 software (Thermo Fisher Scientific, Waltham, MA,
USA).
3.1.3.4.3 Fragmentation analysis
The detection of substances was performed using a QExactive™ Hybrid
Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham,
MA, USA) using ESI. For fragmentation studies, the product ion spectra were
70
recorded with a collision energy of 80 eV for 16β-hydroxystanozolol (m/z 345.25)
and 35 eV for N-desmethyl-sibutramine (m/z 266.17) from reference standard
solutions and from samples of tanks treated with stanozolol and sibutramine. The
spectra and chromatograms were evaluated using TraceFinder 3.2.512.0
software (Thermo Fisher Scientific, Waltham, MA, USA).
3.1.4 Results and discussion
3.1.4.1 Screening – Phase I metabolites of sibutramine and stanozolol
detected in zebrafish samples by full-scan HRMS
The present work aimed to evaluate the ability of adult zebrafish to produce
metabolites of drugs, using substances abused as doping in sports as an
example. For this, water samples from the tanks with zebrafish treated with
sibutramine and stanozolol were analysed in full-scan mode (positive and
negative) by LC–HRMS and compared with the 2 negative controls (with
fish/without substance addition and without fish/with substance addition). Initial
metabolite identification was carried out using LC–HRMS and then by LC–
MS/MS to obtain more detailed structural information regarding the metabolites.
The use of analysers with accurate-mass capabilities, such as Orbitrap, increases
the sensitivity in full-scan mode. Additionally, the high mass accuracy allows for
extract-ion chromatograms with narrow m/z windows (normally, below 10 ppm),
which increases the sensitivity of the method by removing most of the chemical
interference signals in the sample analysis results.[52]
In humans, sibutramine undergoes biotransformation to at least 6 known
metabolites: the demethylated metabolites, N-desmethyl-sibutramine (nor-sib)
and N-bisdesmethyl-sibutramine (bis-nor-sib). These are in turn hydroxylated,
generating the known hydroxylated nor-sib and bis-nor-sib, with the hydroxyl
group linked to the cyclobutane ring (OH-nor-Sib1 and OH-bis-nor-Sib1) and to
the isopropyl chain (OH-nor-Sib2 and OH-bis-norSib2).[42,43] The chemical
structures for the metabolites of sibutramine are presented in Figure 1, and the
respective m/z values are in Table 1.
71
Figure 1. Chemical structures of stanozolol, sibutramine, and both metabolites reported in the literature. 1 = stanozolol; 2 = 16β-hydroxystanozolol; 3, 4, 5, 6, 7, 8 & 9 = hydroxyl-stanozolol metabolites; 10, 11, 12 & 13 = dihydroxyl-stanozolol metabolites; 14, 15, 16 & 17 = stanozolol metabolites; 18 = sibutramine; 19 = N-desmethyl-sibutramine; 20 = N-bisdesmethyl-sibutramine; 21 & 22 = hydroxyl N-bisdesmethyl-sibutramine metabolites; 23 & 24 = hydroxyl N-desmethyl-sibutramine metabolites.
72
Table 1. Elemental composition and theoretical mass [M + H]+ of stanozolol, sibutramine and their metabolites reported in the literature.
Substance Elemental
Composition
Theoretical mass
[M + H]+
1 C21H32N2O 329.25874
2, 3, 4, 5, 6, 7, 8 & 9 C21H33N2O2 345.25365
10, 11, 12 & 13 C21H33N2O3 361.24856
14 &15 C21H31N2O 327.24309
16 &17 C21H31N2O3 359.23291
18 C17H26ClN 280.18265
19 C16H24ClN 266.16700
20 C15H22ClN 252.15135
In Figure 2, the extracted ion chromatograms from the sibutramine
metabolites with m/z corresponding to nor- sib, [1], bis-nor-sib [2], OH-nor-
Sib1/OH-nor-Sib2 [3], and OH-bis-norSib1/OH-bis-nor-Sib2 [4] from (a) zebrafish
samples collected before sibutramine administration and (b) zebrafish samples
collected after 7 days of sibutramine administration are presented. There was no
production of sibutramine metabolites in tanks without fish with sibutramine (data
not shown). In the tanks in which sibutramine was administered, it was possible
to observe several peaks that indicate the production of demethyl and hydroxyl
metabolites of sibutramine, as is observed in human urine.
In fact, it was already possible to observe the production of these
metabolites in treated tanks (tanks with fish that received sibutramine) only 24
hours after the administration of sibutramine (data not shown). During the 7- day
experiment, a decrease in the sibutramine concentration occurred with the onset
of the demethyl and hydroxyl metabolites. The demethyl metabolites were
present in greater amounts than the hydroxyl ones throughout the experiment. In
the 2 negative control tanks, no peaks were observed, indicating the absence of
metabolite production, which was expected. Analysing the chromatograms for the
m/z of OH-nor-sib (Figure 2 (3,b)) and OH-bisnor-sib (Figure 2 (4,b)), it is possible
to observe 7 peaks, indicating that, similar to humans, zebrafish are capable of
hydroxylating the nor-sib and bis-nor-sib molecules in multiple positions.
73
Figure 2. LC–HRMS extracted ion chromatograms at a mass tolerance of 6 ppm from the sibutramine metabolites with m/z 266.16700 (1), 252.15135 (2), 282.16192 (3), and 268.14627 (4) from (a) zebrafish samples collected before sibutramine administration and (b) zebrafish samples collected after 7 days of sibutramine administration.
74
In addition to the urinary metabolites already described in the literature for
sibutramine, in the screening by full-scan positive MS, it was also possible to
observe dihydroxyl derivatives of nor-sib and bis-norsib present in tanks treated
with sibutramine. Therefore, zebrafish may be useful in investigating xenobiotic
metabolism even when identifying new metabolites, since tank water is a cleaner
matrix than urine.
Like sibutramine, stanozolol is a widely metabolized substance. Pozo et
al[51] found more than 16 metabolites of stanozolol present in human urine, most
of which were hydroxylation (mono and di), reduction, methylation, demethylation
and epimerization reaction products. The most interesting targets for doping
control analysis were found to be metabolites 2, 4, 9, 11, and 14 (Figure 1), which
could be detected in urine collected 120 hours after the administration.[51] The
chemical structures for the metabolites of stanozolol are presented in Figure 1
[2–17], and the respective m/z values, in Table 1. There was no production of
stanozolol metabolites in tanks without fish with stanozolol (data not shown).
In Figure 3, the extracted ion chromatograms for the stanozolol
metabolites with m/z corresponding to hydroxy metabolites 2, 3, 4, 5, 6, 7, 8, and
9 [1], dihydroxy metabolites 10, 11, 12, and 13 [2], 14, and 15 [3] and 16 and 17
[4] from (a) zebrafish samples collected before stanozolol administration and (b)
zebrafish samples collected after 7 days of stanozolol administration are
presented. In the tanks where stanozolol was administered, it is possible to
observe several peaks that indicate the production of the same metabolites of
stanozolol as observed in human urine. All m/z values described for the
stanozolol metabolites (Table 1) were found in the tanks treated with stanozolol,
including m/z 327.24309 and 361.24856, which have been indicated as targets
for anti-doping control because of their longer urinary excretion.[46] Like
sibutramine, it was already possible to observe the production of stanozolol
metabolites in the treated tanks after 24 hours of administration (data not shown),
and no peaks were observed in the negative controls.
75
Figure 3. LC–HRMS extracted ion chromatograms at a mass tolerance of 6 ppm from stanozolol metabolites with m/z 345.25365 (1), 361.24856 (2), 327.24309 (3), and 359.23291 (4) from (a) zebrafish samples collected before stanozolol administration and (b) zebrafish samples collected after 7 days of stanozolol administration.
Metabolite 2 was the most produced (2x higher concentration than the
other metabolites) throughout the experiment, followed by the 14/15, 16/17, and
76
10/11/12/13 metabolites. Several peaks were observed in the chromatograms for
the m/z of the stanozolol metabolites in Figure 3, indicating that zebrafish are
capable of performing various types of phase I reactions, generating different
metabolites of stanozolol. For example, the m/z 327.24309 corresponds to
metabolites 14 and 15 from a stanozolol molecule that underwent hydroxylation,
demethylation and methylation that are present in the tanks with zebrafish treated
with stanozolol. In addition to the demethylation and hydroxylation reactions
observed for sibutramine, using stanozolol revealed that adult zebrafish adults
are also capable of promoting the reduction and epimerization of xenobiotics.
The majority of xenobiotic biotransformation processes occur in the liver,
which contains a large number of metabolizing enzymes. The first step of the
biotransformation is called phase I metabolism and includes various reactions,
such as dehydrogenations, hydrolyses, reductions or oxidations, many of which
are catalysed by enzymes of the cytochrome P450 (CYP450) group.[53,54] In
humans, CYP2B6 is the major enzyme responsible for the sequential metabolism
of sibutramine into nor-sib and then bis-nor-sib.[55] On the other hand, CYP3A4
is responsible for the biotransformation of several steroids in humans.[56,57]
Interestingly, zebrafish CYP families that are analogous to CYP2B6 and CYP3A4
in humans include CYP2Y and CYP3A65, respectively. In fact, in this work, adult
zebrafish proved to be effective in generating metabolites for sibutramine and
stanozolol that have been described in humans, as the xenobiotics are possible
substrates for CYP2Y and CYP3A65.
An important issue to be evaluated in relation to drug administration in
zebrafish tanks is solubility in water. For this purpose, we use a water-soluble
substance (sibutramine) and a water insoluble substance (stanozolol).
Considering the liposolubility of stanozolol, a smaller amount (1 mg) than that
given for sibutramine (15 mg) was used. Using this amount of stanozolol in the
aquarium it was possible to observe phase I and II metabolites. Since the
aquarium has a large amount of water (4 L) and the amount of drug required is
very small, the lipophilicity issue is less critical. The question of solubility can be
critical for other drugs that are even more liposoluble than stanozolol and perhaps
it will be necessary to develop new administration forms in the tanks.
77
3.1.4.2 Screening – Phase II metabolites of stanozolol detected in
zebrafish samples by full-scan HRMS
In humans, xenobiotics are oxidized by CYP enzymes to inert or
pharmacologically active metabolites during first-pass metabolism. The
increased hydrophilicity of the substances facilitates clearance from the body,
either directly or following subsequent modification by phase II enzymes through
the addition or removal of polar and non-polar groups, respectively.[58]
Glucuronidation, which is catalysed by several UDP glucuronosyltransferase
isoforms, is an especially important route of phase II drug metabolism in humans,
in addition to sulfotransferases and glutathione S-transferases enzymes.[59,60]
The zebrafish possesses a total of 94 CYP genes in its genome, which fall into
18 CYP gene families that are also found in humans and other mammals,
especially the CYP families 5–51 that are involved in the metabolism of
endogenous substrates.[25] The zebrafish has been shown to express a number
of drug metabolizing enzymes, including glucuronyltransferases,
sulfotransferases and glutathione S-transferases.[36] A previous study noted that
adult zebrafish were able to produce phase II metabolites of xenobiotics.[33]
Stanozolol and sibutramine undergo phase II metabolism in
humans.[48,61] In Figure 4, the LC–HRMS extracted ion chromatograms from
the stanozolol glucuronide metabolites of m/z 505.29083 (a), 521.28574 (b),
535.26501 (c), and 537.28066 (d) derivatives of the metabolites of m/z
329.25874, 345.25365, 359.23291, and 361.24856, respectively, observed in
zebrafish samples collected after stanozolol administration and prepared by
dilute-and-shoot, without enzymatic hydrolysis, are presented. These stanozolol
glycoconjugate metabolites produced by zebrafish have already been observed
in human urine.[46,62] No peaks were observed in the samples from the negative
control tanks (data not shown). Under the analysed conditions, adult zebrafish
were able to generate phase II metabolites only for stanozolol; however, these
were observed in a much lower amount than the free metabolites (Figure 4). It is
possible that the conjugation process for the excretion of sibutramine and
stanozolol metabolites is more pronounced for humans than zebrafish. This
hypothesis should be checked in future studies.
78
Figure 4. LC–HRMS extracted ion chromatograms at a mass tolerance less than 15 ppm from stanozolol glucuronide metabolites with m/z 505.29083 (a), 521.28574 (b), 535.26501 (c), and 537.28066 (d) from zebrafish samples collected after stanozolol administration prepared by dilute and-shoot.
3.1.4.3 Comparison of product ion spectra of zebrafish samples and
reference material solutions
After the analysis in full-scan mode, the fragmentation spectra of the peaks
generated in the tanks treated with sibutramine and stanozolol were compared
with standard solutions of 16β-hydroxystanozolol and N-desmethyl-sibutramine.
In Figure 5, the chromatograms and their respective fragmentation spectra are
presented for N-desmethyl-sibutramine (a and 1) and 16β-hydroxystanozolol (c
and 3) standard solutions and the metabolites produced in the zebrafish tanks
treated with sibutramine (b and 2), and stanozolol (d and 4).
79
Figure 5. ESI(+) MS/MS chromatograms and spectra of parent substance standards for N-desmethyl-sibutramine (a and 1) and 16β-hydroxy-stanozolol (c and 3) and from zebrafish samples after the administration of sibutramine (b and 2) and stanozolol (d and 4).
The fragments m/z 81.04532, 95.08608, 107.08598, and 119.08586 were
the most abundant in the 16β-hydroxystanozolol spectra, being diagnostic ions
for the confirmation of this metabolite. [63] These fragments were also observed
in the spectrum relative to the 7.49-minute peak observed in the tanks treated
with stanozolol. The experimental elemental composition (EEC) and mass error
80
are presented in Table 2, and it is possible to observe the same EEC for
fragments derived from the 16β-hydroxystanozolol reference material solution
and from the metabolite produced by zebrafish. For N-desmethyl-sibutramine, the
most intense ions were m/z 125.01546, 139.03093, 97.10166, and 153.04662
(Figure 4). Like stanozolol, the same EEC for fragments obtained from the N-
desmethyl-sibutramine standard solution and the peak at 6.74 minutes produced
by zebrafish were observed, proving that they correspond to the same molecule.
Thus, adult zebrafish were able to produce metabolites that are identical to the
human metabolites for stanozolol and sibutramine.
Table 2. Protonated molecules [M + H]+ of substances 19 (N-desmethyl-sibutramine) and 2 (16β-hydroxystanozolol) with resulting diagnostic product ions (using high resolution MS/MS experiments), elemental composition (experimental) and mass error (ppm).
Substance Precursor ion
[M + H]+ m/z
CE*
(eV)
Product ion (m/z)a /
Product ion (m/z)b
Elemental
comp. (exp.)
Error (ppm)c /
Error (ppm)d
19 266.17 35
125.01549 / 125.01543 C7H6Cl 1.9 / 1.4
139.03094 / 139.03091 C8H8Cl 0.2 / 0.1
97.10168 / 97.10163 C7H13 5.2 / 4.7
153.04665 / 153.04659 C9H10Cl 0.6 / 0.2
2 345.25 80
81.04535 / 81.04530 C4H5N2 7.8 / 7.1
95.08612 / 95.08603 C7H11 6.2 / 5.2
107.08602 / 107.08593 C8H11 4.6 / 3.8
119.08591 / 119.08581 C9H11 3.2 / 2.4
3.1.4 Conclusion
This study demonstrates that adult zebrafish can absorb, oxidise, and
excrete several sibutramine and stanozolol phase I metabolites similar to those
identified in humans. Overall, this study provides further evidence that adult
zebrafish possess the machinery required for drug metabolism. Furthermore,
since the zebrafish model of metabolism is rather cheap and has a clean matrix,
it may be a good tool to study the production of metabolites. An important point
to consider is the possibility of isolating metabolites from the tank water aiming to
generate reference materials for analytical quality control purposes. This
81
perspective is particularly interesting since the quantity of drug that could be used
in the zebrafish model is considerably larger than for other metabolic models.
Therefore, there is the potential to generate a large enough mass of metabolites
to allow the required isolation and characterization. This promising approach is
corroborated by the fact that the matrix used in the experiment is much cleaner
than the biological matrices, even purified hepatocytes. Therefore, adult zebrafish
may be an important tool in the search for metabolites in metabolomics studies
in general and, more specifically, for doping agents that may be potential
analytical targets in doping control.
3.1.5 Acknowledgments
We thank Dr Manoel Luis Costa for his help with fish cultivation and Dr
Elisa Suzana Carneiro Poças for the exchange of ideas about the zebrafish. We
are grateful to American Journal Experts for their English review. We also
acknowledge financial support from CNPq, FAPERJ and the Ministry of Sport.
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4. Capítulo II
4.1 Development of liquid chromatographic Q-high resolution mass
spectrometry method by Box-Behnken design for investigation of
sibutramine urinary metabolites
4.1.1 Abstract
The knowledge of the metabolism of prohibited substances in sports is essential
for the determination of the most appropriate analytical targets in doping control.
Sibutramine is a substance listed by World Anti-Doping Agency as a stimulant
agent. According to the literature, sibutramine is extensively metabolized in N-
desmethyl-sibutramine (M1), N-bisdesmethyl-sibutramine (M2) and
monohydroxyl derivatives from M1 e M2. Previous works has evaluated the
metabolism of sibutramine through CG-MS (gas chromatography- mass
spectrometry) and LC-MS/MS (liquid chromatography–mass spectrometry).
Therefore, it is interesting to verify the existence of new metabolites of
sibutramine through current analytical methodologies, such as liquid
chromatography coupled to high resolution mass spectrometer (LC-HRMS).
Furthermore, the development of a comprehensive method to investigate the
metabolism of sibutramine allows the amplification of the window of detection of
the abuse of this stimulant and enable advances in pharmacology studies for
different fields. Experimental design (DoE), like Box-Behnken design (BBD), is a
systematic and scientific approach to study the interactions between independent
and dependent variables using minimum analytical runs. The objective of this
work was to develop and validate an LC-HRMS methodology using DoE for the
analysis of sibutramine metabolites in human urine. After optimization by DoE,
the final optimal chromatographic condition was based on reversed-phase
chromatography using a C18 column: ramp time of 25 minutes, flow rate of 0.17
mL min-1 and temperature of 50 °C; mobile phase A (water with 0.1% formic acid
and 5 mM ammonium formate) and B (methanol with 0.1% formic acid); initial
gradient percentage of 15% B and injection volume of 5μL. In addition to the
hydroxylated metabolites previously described in human urine, dihydroxyl
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derivatives of M1 and M2 were also observed using the developed method.
These new metabolites that have been found may be new analytical targets in
anti-doping control.
4.1.2 Introduction
Sibutramine is a halogenated amphetamine-like compound with
anorexiant action already commercialized with relative success in the treatment
of obesity and overweight in several countries. In humans, sibutramine is rapidly
metabolized to N-desmethyl and N-bisdesmethyl metabolites, and the in vivo
effects of sibutramine are predominantly due to the actions of these two active
metabolites (Figure 1) [1]. The pharmacological effects are based on the
serotonin-norepinephrine reuptake inhibition [2,3].
Figure 1. Chemical structures of sibutramine and its metabolites.
Initially, the drug was commercialized as a good alternative for the classical
anorexiant agents as fenproporex and amfepramone. More recently, the use of
sibutramine has been associated with various cardiovascular and psychiatric
symptoms. Increased incidence of nonfatal myocardial infarction, psychomotor
disturbances, and DNA damage were also reported [4–6]. However, its presence
in “natural” loss weight products as contaminant was already reported by different
authors. Sibutramine was also detected in counterfeit drugs or slimming products
[6–9]. Such presences were not stated on the label. The harmful side effects of
sibutramine create interest for forensic analysis. On the top of that, sibutramine
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is nominally cited in the World Anti-Doping Agency List of Prohibited Substances
as a specified stimulant [10]. In 2007, just after to be released on the Brazilian
marked, sibutramine figured as one of the most used stimulant doping agent used
by athletes in Brazil [11].
In this context, the evaluation of the urinary metabolites aiming at the
definition of the target compound is relevant. Sibutramine urinary metabolites
were firstly investigated by Thevis and co-workers (2006) [12] using liquid
chromatography–mass spectrometry (LC-MS/MS) where the mono and bis N-
desmethyl metabolites were established as target compound. Strano-Rossi and
coworkers (2007) [13] used gas chromatography (GC) approach based on the
formation of trimethylsylilderivates opening the range of possibilities through the
identification of hydroxylated metabolites in human urine. GC-MS approach was
also suggested by Sardela and co-workers [14] using double derivatization
strategy, with N-methyl-N-(trimethylsilyl)-trifluoroacetamide and N-methyl-bis-
(trifluoroacetamide) increasing the specificity of the analysis. The hydroxy-
cyclobutane-bis-nor-sibutramine, which becomes the more abundant metabolite
in the first 10h, and hydroxy-isopropyl-bis-nor-sibutramine, which becomes the
most abundant after 40h in human urine, were proposed for doping monitoring
by GC-MS.
Hakala and co-workers (2009) [15] identified in vitro using rat hepatocytes
nineteen sibutramine metabolites and several of their isomers formed via
demethylation, mono and dihydroxylation, dehydrogenation, acetylation,
attachment of CO2, and glucuronidation using LC-MS/MS experiments. Recently,
Anselmo and co-workers (2017)[16] used the in vivo model of zebrafish for the
evaluation of sibutramine metabolites, finding all the human phase I urinary
metabolites already described by LC-HRMS (high resolution mass spectrometry).
Progressively, LC-HRMS has become a key methodology in doping control
laboratories. Indeed, the technique is particularly powerful in metabolism studies,
increasing the perspective of discovery of new analytical targets. In particular, in
the Q-Exactive technology, different mass experiments could be performed.
Among then, full-scan HRMS experiment at high mass accuracy allows the
precisely filter the full-scan data and the extraction analyte chromatograms with
very low background noise, which allows the identification of unknown
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metabolites by knowledge of the elemental composition [17]. However, biological
samples could be very complex and challenge. Sometimes, despite of the high
accuracy observed in the mass data acquisition, the complexity of the matrix
could jeopardize the identification of the new analytical targets due to extensive
co-elution of isobaric compounds. In situation like this, optimization of the matrix
clean up and chromatographic conditions allows to reach the best selectivity –
specificity, ultimate target of the so called hyphenated approaches.
Considering the high number of parameters likely to impact in
chromatography resolution, experimental design approaches has the potential do
reduce the number of experiments, costs project and work overload, reaching the
desired results in less time. Experimental design (DoE) is a statistical technique
for planning, conducting, analyzing, and interpreting data from experiments [18].
Among the advantages of DoE that make it useful in analytical chemistry are
decreasing of experiments, lower consumption of samples and reagents,
obtaining of statistical models allowing interpretation of results [19]. Herein, it is
reported an urinary metabolic study of sibutramine in humans after optimization
of the analytical parameters by DoE and detection by LC-HRMS methodology
aiming the characterization of new analytical targets for the abuse of this doping
agent.
4.1.3 Materials and Methods
4.1.3.1 Chemicals and reagents
N-desmethyl-sibutramine (M1) and N-bisdesmethyl-sibutramine (M2)
were purchased from LGC (Teddington, Middlesex, UK). The internal standard
(IS) buspirone was supplied by Sigma-Aldrich (São Paulo, Brazil). For the study
of excretion in human, it was used tablets containing 15 mg of monohydrate
sibutramine hydrochloride (Biosintética, São Paulo, Brazil). All chemicals (i.e.,
formic acid, acetic acid, ammonium formate, sodium phosphate, tert-butyl methyl
ether (TBME), ethyl acetate and methanol) were analytical or HPLC grade (Tedia,
Fairfield, USA). The ultrapure water used was Milli-Q grade (Millipore, Billerica,
USA). The enzyme used for the enzymatic hydrolysis was β-glucuronidase (from
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Escherichia coli) (Roche, Rio de Janeiro, Brazil).
4.1.3.2 Samples
Human urine samples were collected after administration of a single dose
of 15 mg of monohydrated sibutramine chloridrate, to two healthy males and two
healthy females. Urine samples were also collected prior to administration of
sibutramine, used as a negative control of the experiments. The excretion study
was carried out according to International and Brazilian regulations and approved
by the University’s Ethics Committee (63965616.4.0000.5257).
4.1.3.3 Liquid-liquid extraction (LLE)
Positive control of N-desmethyl-sibutramine (M1) at 50 ng mL-1 were
prepared in urine. To verify the recovery of the extraction method a positive
control of M1 fortified after the extraction was used. A volume of 5 μL of buspirone
solution (internal standard, 50 ng mL-1), 750 μL of pH 7.0 phosphate buffer and
50 μL of β-glucuronidase solution were added to 1 mL of urine. The tubes were
shaken and left in a water bath at 50°C for 1h. The samples (1mL) were extracted
by liquid-liquid extraction in three different pH ranges: alkaline (adding carbonate
buffer pH 9.0), neutral (adding phosphate buffer pH 7.0) and acid (acetate buffer
pH 4.0). Four replicates were used for each pH range. The samples were
homogenized for 5 seconds. Liquid-extraction was performed by adding 2.5 mL
of 1:1 TBME/ethyl acetate. The samples were placed on a rotary shaker for 20
minutes and then centrifuged at 1,500 G-force for 10 minutes. The organic layer
was separated, and the solvent was evaporated. The dried residue was
resuspended in 100 μL of the mobile phase.
4.1.3.4 Solid-phase extraction (SPE)
Positive control of M1 at 50 ng mL-1 were prepared in urine. To verify the
recovery of the extraction method a positive control of M1 fortified after the
extraction was used. Aliquots of 1 mL of urine were hydrolyzed (or not) and then
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extracted before injection into the LC-HRMS system based on Sardela et. al
method developed [20]. Briefly, 5μL of buspirone solution (internal standard, 50
ng mL-1), 750 μL of pH 7.0 phosphate buffer and 50 μL of β-glucuronidase
solution were added to 1 mL of sample. Unhydrolyzed aliquots were also made.
The tubes were shaken and left in a water bath at 50°C for 1h. The samples were
extracted using a Strata-X-CW column (Phenomenex, Torrance, USA). The
experiment was carried out with four replicates. The cartridges were conditioned
with 1 mL of methanol, followed by 1 mL of water. After this step, 1 mL of the
sample had the pH adjusted to 9.0 with carbonate buffer, and it was applied to
the cartridges. The cartridges were washed with 1 mL of water and then 1 mL of
the methanol:water (1:1) solution. The cartridges were allowed to dry, and the
analytes were recovered with 1 mL of methanol and 5% (v/v) formic acid. The
tubes were left under N2 flow at 40°C. The contents of the tubes were
reconstituted with 100 μL of a water:methanol (70:30) 0.1% formic acid/5 mM
ammonium formate solution.
4.1.3.5 Chromatographic adjustment by DoE
Chromatographic preliminary experiments, described as conditions A and
B of Table 1, were performed to identify the critical factors and to set their levels
(maximum and minimum) for the experimental design. In this step the following
parameters were obtained: C18 Syncronis column (2.1 x 50 mm, 1.7 μm); mobile
phase A (water with 0.1% formic acid and 5 mM ammonium formate) and B
(methanol with 0.1% formic acid); initial gradient percentage of 15% B and
injection volume of 5μL were the fixed conditions. The critical conditions (factors)
that were identified in the preliminary experiments and adjusted by DoE were the
mobile phase flow, column temperature and ramp time (time required to reach
100% of the mobile phase B). A three-factor, three-level Box-Behnken design with
five replicates at the center point was used. The responses evaluated in the trials
were the resolutions between at least three peaks of the M1-diOH (M5)
metabolite. Two experimental designs were used to adjust the chromatographic
method. The first experimental design evaluated the flow levels 0.2, 0.3 and 0.4
mL min-1, temperatures of 40, 50 and 60°C and ramp time of 10, 15 and 20
93
minutes (Appendix A). The second experimental design is shown in Table 2,
where the levels of flow and ramp time were modified.
Table 1. Parameters evaluated in the chromatographic method adjustment.
Condition A B C D
Temperature
(°C)
((º( ((º (ºC)
40 50 54.1 50
Flow (mL/min) 0.4 0.3 0.2 0.17
% initial of Ba 5 15 15 15
Ramp time
(min)b
10 20 20 25
Total run (min) 14 25 25 30
a: Initial percentage of mobile phase B at the start of the chromatographic run.b: Time, in minutes, that the analysis took to reach 100% of mobile phase B. A: Inicial chromatographic condition; B: Chromatographic test condition performed to identify the critical factors; C: chromatographic condition obtained by the first experimental design and D: final condition obtained by the second experimental design.
Table 2. Box-Behnken design matrix and correspondent results for the second experimental design.
Assay Flow
(mL/min) T (ºC)
Ramp time (min)
Response (resolution) between peaks of M5
X1 X2 X3 R1-2 (Y3) R2-3 (Y4) R5-6 (Y5)
1 0.1 40 20 0.00 0.00 0.00 2 0.2 40 20 1.26 1.35 0.60
3 0.1 60 20 0.00 0.00 0.00
4 0.2 60 20 1.25 1.19 0.67
5 0.1 50 15 0.00 0.00 0.00
6 0.2 50 15 1.10 1.23 0.60
7 0.1 50 25 0.00 0.00 0.00
8 0.2 50 25 1.20 1.20 0.88
9 0.15 40 15 1.42 1.28 0.71
10 0.15 60 15 1.37 1.25 0.69
11 0.15 40 25 1.84 1.53 0.70
12 0.15 60 25 1.48 1.25 0.90
13 0.15 50 20 1.48 1.23 0.54
14 0.15 50 20 1.56 1.37 0.59
15 0.15 50 20 1.62 1.25 0.66
16 0.15 50 20 1.61 1.49 0.66
17 0.15 50 20 1.75 1.35 0.63
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Appendix A. Box-Behnken design matrix and correspondent results for the first experimental design.
Assay Flow
(mL/min) T (ºC)
Ramp time (min)
Response (resolution) between peaks of M5
X1 X2 X3 R1-3 (Y1) R3-4 (Y2)
1 0.2 40 15 1.08 0.74
2 0.4 40 15 0.00 0.00
3 0.2 60 15 0.98 0.91
4 0.4 60 15 0.00 0.00
5 0.2 50 10 0.00 0.00
6 0.4 50 10 0.00 0.00
7 0.2 50 20 1.22 0.93
8 0.4 50 20 0.89 0.89
9 0.3 40 10 0.00 0.00
10 0.3 60 10 0.00 0.00
11 0.3 40 20 0.94 0.87
12 0.3 60 20 0.99 0.96
13 0.3 50 15 0.00 0.00
14 0.3 50 15 0.00 0.00
15 0.3 50 15 0.00 0.00
16 0.3 50 15 0.00 0.00
17 0.3 50 15 0.00 0.00
4.1.3.6 Data analysis
The comparison between liquid-liquid extraction and solid-phase
extraction was performed by one-way ANOVA with Tukey's-GraphPad Prism. The
Box-Behnken design, as well as statistical analysis of the experiments, was
performed using version 10.1 Statistica® (Stat-Ease Inc., Minneapolis, USA).
Optimization was performed using a desirability function and the solver tool of
Microsoft Excel®2007 software (Microsoft, Redmond, USA), considering the
criterion of maximum resolution of 2.0 as the goal.
4.1.3.7 HRMS analysis
The detection of substances was performed using a QExactive™ Hybrid
Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham,
USA) with electrospray ionization (ESI). The mass calibration was performed
daily before starting the analysis using a calibration solution. The capillary
temperature was set to 250°C, and the spray voltage was 2.5 kV; other
parameters for MS were sheath gas 42, aux gas 10 and swept gas 2 in arbitrary
units. The mass spectrometer acquired full scan data in positive ionization mode
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at a resolution of 70 000. An initial screening for metabolites was carried out in
full scan positive mode in a range of m/z 100–800 [20]. The chromatograms were
evaluated using TraceFinder 3.2.512.0 software (Thermo Fisher Scientific,
Waltham, USA). The protonated exact mass values of the respective metabolites
are given in Table 3.
Table 3. Elemental composition and theoretical mass [M + H]+ of sibutramine and its metabolites.
Substance Elemental
Composition
Theoretical mass
[M + H]+
Sib C17H26ClN 280.18265
M1 C16H24ClN 266.16700
M2 C15H22ClN 252.15135
M3 (M1-OH) C16H24ClNO 282.16192
M4 (M2-OH) C15H22ClNO
268.14627
M5 (M1-
diOH)
C16H24ClNO2 298.15683
M6 (M2-
diOH)
C15H22ClNO2 284.14118
M7 C17H24ClNO2 310.15683
M8 C16H22ClNO2 296.14118
M1-Glu C23H32ClNO8 486.18892
M2-Glu C22H30ClNO8 472.17327
M3-Glu C22H32ClNO7 458.19401
M4-Glu C21H30ClNO7 444.17836
4.1.3.8 Fragmentation analysis
The detection of substances was performed using a QExactive™ Hybrid
Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific™, Waltham,
USA) using electrospray ionization (ESI). Sibutramine metabolites were analysed
in positive mode by a mass spectrometry experiment in which all ions within a
selected m/z range are fragmented and analysed in a second stage of tandem
mass spectrometry (Full‐MS/MS2). For fragmentation studies, the product ion
spectra were recorded with a collision energy of 20 eV and 30eV. The spectrum
was evaluated using Xcalibur Qual Browser 3.0.63 software (Thermo Fisher
Scientific™, Waltham, USA). The theoretical fragmentation of sibutramine
metabolites was verified by the software Mass Frontier 7.0 (Thermo Fisher
Scientific™, Waltham, USA).
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4.1.3.9 Validation
The repeatability, carryover and matrix interference parameters were
evaluated about the M1 and M2 metabolites. The other parameters specificity
and limit of detection were evaluated for all metabolites. Sibutramine metabolites
were identified by the previously described LC HRMS method. The identification
criteria were the protonated exact mass values of the respective metabolites
(Table 3) and the retention time. The experimental design used for the validation
experiments was based on WADA’s Technical Document International Standard
for Laboratories [21] and the Guidance for Bioanalytical Method Validation from
FDA (Food and Drug Administration) [22].
4.1.3.9.1 Specificity
The absence of interferences was verified through the analysis of ten
different blank urines. The exact m/z values of sibutramine metabolites were
investigated in these urine samples.
4.1.3.9.2 Repeatability
Seven replicates of blank urine fortified with M1 and M2 metabolites at 50
ng mL-1 were analysed. The repeatability was verified about the value of % RSD
(relative standard deviation) of the ratio between the analyte peak area and the
internal standard.
4.1.3.9.3 Limit of Detection (LOD)
Serial dilutions (1/2, 1/10, 1/25, 1/50, 1/100, 1/200, 1/500 e 1/1000) of
sibutramine excretion urine were performed to verify LOD values for the
metabolites. The criteria established were the lowest concentration that would be
detected with signal-to-noise > 3.
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4.1.3.9.4. Carryover
The carryover was evaluated by analysing two blank urines interspersed
with a fortified control of M1 and M2 at 100 ng mL-1.
4.1.3.9.5. Matrix interference
The matrix interference was verified through the analysis of ten different
blank urines fortified with M1 and M2 metabolites at 50 ng mL-1. This parameter
was verified about the value of % RSD of peak area and in the absence of
interfering with the retention times of the analytes of interest.
4.1.3.9.6. Robustness
Seven replicates of blank urine fortified with M1 and M2 metabolites at 50
ng mL-1 were analysed with the following variations in the chromatographic
method: column temperature change to 45ºC (three replicates) and flow change
to 0.16 mL min-1 (three replicates). The robustness was verified about the value
of % RSD of the ratio between the analyte peak area and the internal standard.
4.1.4 Results and discussion
4.1.4.1 Development of extraction method
Modern technologies bring new paradigm of detectability in metabolites
investigations. Nevertheless, sample preparation procedures should be
considered carefully aiming to complementary goals: i) eliminate as much as
possible the interferers; ii) include as much as metabolites on the analytical
instrumentation, allowing posterior detection. In addition, the choose of the
sample preparation protocol has an even important role when ESI ionization is
used, based on the well know suppression/enhancement ion effect [23,24].
Therefore, the primary goal of this work was to select a sample preparation
method to isolate the metabolites of sibutramine from the other components of
98
urine.
Liquid-liquid extractions were tested at three different pH values (acid,
neutral and basic), in addition to solid phase extraction. The metabolite M1 was
used for comparison purposes. According to Figure 2, it is possible to observe a
recovery around 70% in all procedures, with exception of the acid extraction
(bellow 60%). LLE at pH alkaline and SPE presented the higher yields. Significant
statistical differences were only observed between LLE at alkali pH x LLE at acidic
pH and SPE x LLE at acidic pH.
Figure 2. Comparison between liquid-liquid extractions (LLE) at pH alkaline (A-LLE), neutral (N-LLE), acid (Ac-LLE) and solid phase extraction (SPE) in relation to yield values (recovery in % of N-desmethyl-sibutramine). n = 3. *: P <0.05 (One-way ANOVA with Tukey's - GraphPad Prism).
Thus, the extraction being conducted in alkaline pH favors the partition of
the non-ionized moiety in the organic solvents, increasing the recovery.
Corroborating this rational, the recovery is poor in acid pH (Figure 2). Based on
the structural similarity, the same inference could be assumed for all sibutramine
metabolites. As in this work, Lu and co-workers (2010) [25] did not observe
differences between alkaline ELL and SPE in the extraction of sibutramine from
human urine. Sibutramine is a tertiary amine exhibiting pKa 10.2 [26]. The SPE
is one of the most used methods in the preparation of samples for injection in the
chromatographic systems coupled to the mass spectrometer [23,24,27]. Several
studies that analysed stimulants, including sibutramine, has used SPE as a
treatment of the samples [14,28].
Bylda and co-workers (2014) [24] demonstrated that studies comparing
different sample preparation methods in relation to matrix effects and analyte
recovery shows that mixed-mode strong anion exchange SPE was more effective
99
than LLE for polar and non-polar analytes in plasma. Considering the similarity of
the recovery rate observed between alkaline LLE and SPE, and the best clean
up result typically observed in the last one, SPE was chosen to be performed in
this project. As a result of the better clean up, it is possible to cite the decrease in
the matrix effect and lower use of organic solvents.
4.1.4.2 Chromatographic adjustment by DoE
Initially, a liquid chromatographic gradient was used to analyse the
metabolites of sibutramine in urine (A, Table 1). Clearly, this was not efficient in
separating the compounds, as can be seen in Figure 3A. Figure 3A shows that
the excretion of the M1-diOH (M5 metabolite, m/z 298.15683) results in multiple
chromatographic peaks between 5 and 7 minutes, opening the perspective of
several isobaric metabolites with very poor chromatographic resolution.
The selection of critical factors for the chromatographic methods is
essential for the efficiency of the optimization process [19]. Therefore, a
chromatographic test condition was performed to identify the critical factors and
to set their levels (maximum and minimum) for the experimental design for
optimization of the chromatographic method (B, Table 1). In this test condition, it
was chosen to increase the temperature of the chromatographic column, reduce
the mobile phase flow and increase the ramp time (required to reach 100% of the
mobile phase B) aiming to improve the resolution between the chromatographic
peaks.
In Figure 3B it can be seen that the test condition improved the resolution
among the chromatographic peaks, highlighting the relevance of the altered
parameters. Thus, the fixed conditions obtained in the test condition were: C18
Syncronis column (2.1 x 50 mm, 1.7 μm); mobile phase A (water with 0.1% formic
acid and 5 mM ammonium formate) and B (methanol with 0.1% formic acid); initial
gradient percentage of 15% B and injection volume of 5μL. On the other hand,
the critical factors that were identified in the test condition were the mobile phase
flow (X1), column temperature (X2) and ramp time (X3). Therefore, a three-factor,
three-level Box-Behnken (BB) design with five replicates at the center point was
used to adjust critical conditions to improve resolution between the M5
100
chromatographic peaks.
Figure 3. LC-HRMS extracted ion chromatograms at a mass tolerance of 6 ppm from the sibutramine metabolites M5 with m/z 298.15683 from urine samples collected six hours after sibutramine ingestion under the chromatographic conditions A: inicial chromatographic condition; B: chromatographic test condition performed to identify the critical factors; C: chromatographic condition obtained by the first experimental design (DoE) and D: final condition obtained by the second experimental design (DoE). The specific parameters evaluated in each A, B, C and D are present in Table 1.
Two BB designs were used to adjust the chromatographic method. The
first experimental design evaluated the flow levels 0.2, 0.3 and 0.4 mL min-1,
temperatures of 40, 50 and 60ºC and ramp time of 10, 15 and 20 minutes
(Appendix A). The responses were the resolutions R1-3 (Y1) and R3-4 (Y2) between
three peaks of the M5 metabolite, at 4.18 (1), 4.82 (3) and 5.62 (4) minutes
(Figure 3B). Statistical analysis of this design showed significant regression
models for the responses Y1 and Y2 (Equations 1 and 2), without lack of fit (R2 of
0.9085 and 0.9811, respectively), allowing the construction of response surface
plots to evaluate the influence of the factors on the responses of R1-3, which have
been identified as the most critical resolution (Figure 4A, B, and C).
Through these plots, it is possible to observe that the resolution between
the chromatographic peaks R1-3 increases with the ramp time and decreases with
increasing of mobile phase flow (Figure 4B). Optimization by Derringer and Suich
101
desirability function was performed, resulting in the final optimal condition C
presented in Table 1, which provided the chromatogram presented in Figure 3C.
It is possible to observe that this condition revealed the presence of another
chromatographic peak between 1 and 3, from now on named peak 2.
𝑌1 = 1.209 − 0.187𝑋1 + 0.23𝑋3 − 0.099𝑋1𝑋3 Eq.1
𝑌2 = 1.159 − 0.153𝑋1 + 0.135𝑋2 + 0.239𝑋3 − 0.05𝑋22 + 0.062𝑋3
2 + 0.084𝑋1𝑋2 −
0.067𝑋1𝑋3 + 0.05𝑋2𝑋3 + 0.072𝑋1𝑋22 − 0.062𝑋1
2𝑋3 Eq.2
Therefore, a second experimental design was performed, focused on the
resolution increase between peaks 1,2 and 2,3 (R1-2 and R2-3), using lower levels
for the mobile phase flow and higher levels for the ramp time. Thus, the second
BB design evaluated the flow levels 0.1, 0.15 and 0.2 mL min-1, temperatures of
40, 50 and 60ºC and ramp time of 15, 20 and 25 minutes (Table 2). Significant
mathematical models for the resolutions R1-2 (Y3) and R2-3 (Y4) (Equations 3 and
4), without lack of fit (R2 of 0.9707 and 0.9784, respectively), were used to
construct response surface plots (Figure 4D and E).
Figure 4. Surface response plots obtained by Box-Behnken design (DoE) showing the effects of mobile phase flow (X1), column temperature (X2) and ramp time (X3) on the resolutions R1-3 (A, B and C), R1-2 (D and E) and R5-6 (F). A, B and C represent the plots obtained in the first experimental design (Appendix A); D, E and F represent the graphs obtained in the second experimental design (Table 2).
102
Considering the experimental levels used in this new BB design, no
significant influence of ramp time and chromatographic column temperature on
the responses Y3 and Y4 was observed. In all conditions tested with the flow of
0.1 mL min-1, the chromatographic peaks appeared together at the beginning of
the run, taking the resolution to zero, as can be seen from the graphs D and E
(Figure 4). Therefore, the resolution between chromatographic peaks 1 and 2
tends to increase with the decrease of the flow to a certain extent, when the flow
near to 0.1 mL min-1 the peak resolution is reduced (Figure 4D and E).
As an evidence of the complexity of the metabolism pathway, during this
second experimental design, it was observed new chromatographic peaks (5 and
6) and a significant mathematical model (R2 of 0.9629, without lack of fit) for the
resolution between then (R5-6) was obtained (Y5) (Equation 5). The surface
response plot of R5-6 (Figure 4F) shows the ramp time, besides the flow rate,
affected this resolution, which was slightly increased with the increase in the ramp
time. Therefore, considering the improvement of the resolution R5-6 besides R1-2
and R2-3, these three responses were used to achieve a new optimal
chromatographic condition. After optimization by Derringer and Suich desirability
function, the final optimal chromatographic condition proposed was a ramp time
of 25 minutes, the flow rate of 0.17 mL min-1 and temperature of 50ºC (Table 1D).
The resolutions obtained were 1.28 (R1-2), 1.18 (R2-3) and 0.68 (R5-6). This
condition resulted in the chromatogram presented in Figure 3D, being validated
for analysis of sibutramine metabolites.
𝑌3 = 1.569 + 0.602𝑋1 − 0.968𝑋12 Eq.3
𝑌4 = 1.335 + 0.621𝑋1 − 0.713𝑋12 Eq.4
𝑌5 = 0.634 + 0.343𝑋1 − 0.339𝑋12 + 0.095𝑋3
2 + 0.069𝑋1𝑋3 Eq.5
4.1.4.3 HRMS analysis of sibutramine metabolites in urine
Sibutramine urinary metabolites were firstly investigated by Thevis and co-
workers (2006) [12] using liquid chromatography–mass spectrometry where the
mono and bis N-desmethyl metabolites were identified. In the following years, the
detection of sibutramine abuse was enhanced by the identification of
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hydroxylated derivatives from M1 and M2 in human urine [13,14]. In Figure 5 it is
possible to observe mono (M1) and bidesmethylated (M2) metabolites (Figure 5A
and B) and their respective monohydroxylated (M1-OH and M2-OH) (Figure 5C
and D) and dihydroxylated derivatives (M1-diOH and M2-diOH) (Figure 5E and
5F) observed in human urine after 6 hours of sibutramine ingestion.
Dihydroxyl derivatives of M1 and M2 have previously been described in
zebrafish [16] and rat hepatocytes (only M2-diOH) [15]. However, to the best of
our knowledge, as a result of the optimized chromatographic conditions allied to
the high accuracy from the HRMS, this is the first work to verify the presence of
dihydroxyl sibutramine metabolites in human urine. Multiple chromatographic
peaks for the same m/z value of mono or dihydroxylated metabolite indicate that
they are position isomers. For example, after M1 is demethylated and
hydroxylated, the second hydroxyl group can be added in several positions,
generating multiple dihydroxyl metabolites having the same value of m/z (M1-
diOH).
Figure 5. LC-HRMS extracted ion chromatograms at a mass tolerance of 6 ppm from the urinary sibutramine metabolites M1 (A), M2 (B), M3 (C), M4 (D), M5 (E), M6 (F), M7 (G) and M8 (H) under the final chromatographic condition established by DoE.
104
Metabolites of m/z 310.15683 (M7) and m/z 296.14118 (M8) previously
described by Hakala and co-workers in rat hepatocytes were also identified in
human urine, based on accurate mass. Glycoconjugate derivatives of M1, M2,
M1-OH, and M2-OH (Table 3) were also observed in the urine samples where no
hydrolysis was performed, indicating high excretion yields of the metabolites
without phase II metabolism (data not shown). Further information regarding the
structure of the hydroxyl metabolites will be revealed by the fragmentation
spectrum.
4.1.4.4 Fragmentation analysis of sibutramine metabolites in urine
For fragmentation studies, the product ion spectra were recorded with a
collision energy of 20 eV and 30eV using a QExactive™ Hybrid Quadrupole-
Orbitrap mass spectrometer. The appropriate collision energy was selected for
each metabolite to have the presence of the parent ion corresponding to at least
15% of the relative intensity of the fragmentation spectrum. The spectrum was
analysed using Xcalibur Qual Browser 3.0.63 software. Fragmentation spectrum
has fragments with exact mass and theoretical chemical formula with an error
less than 4 ppm. The theoretical fragmentation of sibutramine metabolites was
verified by the software Mass Frontier 7.0.
4.1.4.4.1 M1 e M2
The metabolites M1 and M2 correspond to the demethylated and
bisdemethylated metabolites of sibutramine, respectively. Fragmentation spectra
were obtained with a collision energy of 20eV for both metabolites. The fragments
m/z 125.01538, 139.03090, 153.04652, 97.10153 and 179.06221 are
characteristic of these metabolites (Table 4) and are present in Figure 6.
Fragmentation behavior of M1 e M2 in MS/MS experiments was similar to that
reported in the literature [12].
The fragment m/z 125.01538 is a marker of the presence of sibutramine
and its metabolites since it conserves the aromatic ring bound to the chlorine
atom characteristic of these substances, being present in all metabolites
105
analysed. The fragmentation spectra of M1 and M2 are quite similar since the
only difference between these metabolites is that M1 lost one methyl group in the
amine while M2 lost both methyl groups, becoming a primary amine.
Table 4. Protonated molecules [M + H]+ of sibutramine metabolites with resulting diagnostic product ions (using high resolution MS/MS experiments) and elemental composition (experimental).
Substance Precursor ion
[M + H]+ m/z
CE*
(eV)
Product ion
(m/z)
Elemental
comp. (exp.)
M1/M2 266.17/252.15 20
125.01538 C7H6Cl
139.03090 C8H8Cl
153.04652 C9H10Cl
97.10153 C7H13
179.06221 C11H12 Cl
M3 (M1-OH) 282.15 30
177.04660 C11H10Cl
191.06219 C12H12Cl
163.03093 C10H8Cl
233.10902 C15H18Cl
208.08867 C12H15NCl
M4 (M2-OH) 268.15 20
177.04657 C11H10Cl
233.10909 C15H18Cl
191.06213 C12H12Cl
163.03085 C10H8Cl
194.07314 C11H13NCl
M5 (M1-
diOH) 298.16 30
191.06216 C12H12Cl
231.09334 C15H16Cl
163.03079 C10H8Cl
224.08362 C12H15ONCl
206.07306 C12H13NCl
M6 (M2-
diOH) 284.14 30
191.06214 C12H12Cl
231.09329 C15H16Cl
203.06206 C13H12Cl
210.06795 C11H13ONCl
192.05742 C11H11NCl
All experimental molecular formulas were obtained with errors less than 4ppm mass error.
106
Figure 6. Proposal formation of the main fragments in product ion spectra of [M+H]+ of M3 (A), M4 (B), M5 (C) and M6 (D). Reactions were generated as an output from the software Mass Frontier 7.0 (Thermo Fisher Scientific™, Waltham, USA). +H+: protonation; i: inductive cleavages; rHR: charge-remote rearrangement; rHB: α,β-charge-site rearrangement; rH1,2: hydrogen shift to adjacent position; Lib: fragmentation library mechanisms and -H2O: inductive cleavages.
4.1.4.4.2 M3 (M1-OH)
The metabolites with m/z 282.16192 correspond to the hydroxylated
derivatives from M1. Fragmentation spectra were obtained with a collision energy
of 30eV. Through Figure 5C, it is possible to observe four chromatographic peaks
indicating positional isomers after hydroxylation. The fragments m/z 177.04660,
107
191.06219, 163.03093 and 233.10902 are characteristic of these metabolites
(Table 4) being found in the four retention times (12.9, 13.9, 14.8 and 15.6
minutes) in this order of intensity in all metabolites, except at the retention time
15.4 minutes. These fragments can be generated through fragmentation of the
M1-OH molecule having hydroxyls groups in the aliphatic chain or the
cyclobutane ring. For example, the m/z 233.10902 fragments may be derived
from the fragmentation of M1-OH with a hydroxyl on the cyclobutane ring,
generating the fragment or in the aliphatic chain (Figure 6A).
On the other hand, the fragment m/z 208.08867 when coming from an M1-
OH with the hydroxyl group in the aliphatic chain becomes more stable because
the positive charge is on the nitrogen atom generating the fragment, not on the
carbon atom (Figure 6A). This fragment is the most intense and present only at
the retention time of 15.4 minutes, suggesting that this metabolite possesses a
hydroxyl group in the aliphatic chain, and the others in the retention times of 13.9
and 14.8 in the cyclobutane ring. The metabolite in 12.9 minutes is present in a
much lower quantity than the others, and its fragmentation spectrum did not
reveal great information about its structure. These results are in agreement with
the literature which describes M1 derivatives hydroxylated in the aliphatic chain
and the cyclobutane ring [14].
4.1.4.4.3 M4 (M2-OH)
The metabolites with m/z 268.14627 correspond to the hydroxylated
derivatives of M2 (Figure 5D). Fragmentation spectra were obtained with a
collision energy of 20eV. As for M1-OH, it is possible to observe four
chromatographic peaks indicating that the hydroxyl group can enter different
positions (Figure 5d). The same diagnostic fragments for M1-OH are found for
M2-OH at the four retention times: m/z 177.04657, 233.10909, 191.06213 and
163.03085 (Table 4) in that order of intensity. All these fragments can be
generated through fragmentation of the M2-OH molecule having hydroxyls in the
aliphatic chain or the cyclobutane ring. However, the fragment m/z 194.07314
with the positive charge on the nitrogen atom generates the fragment which is
expected to be more stable than on the carbon atom (Figure 6B). This fragment
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is the most intense and presents only at the retention time of 16.1 minutes,
suggesting a hydroxyl group in the aliphatic chain, and the others in the retention
times of 13.0, 14.2 and 15.1 in the cyclobutane ring. These results are in
agreement with the literature which describes M2 derivatives hydroxylated in the
aliphatic chain and the cyclobutane ring [14].
4.1.4.4.4 M5 (M1-diOH)
The metabolites with m/z 298.15683 correspond to the dihydroxylated
derivatives of M1 (Figure 5E). Fragmentation spectra were obtained with a
collision energy of 30eV. To the best of our knowledge, this metabolite has not
yet been described in human urine, only in zebrafish [16]. Figure 5E shows that
are six more intense chromatographic peaks. The fragments m/z 191.06216,
231.09334 and 163.03079 are the most intense in the fragmentation spectra of
the metabolites at retention times 5.46, 7.69, 9.16 and 13.24 minutes. However,
for the metabolites at retention times 8.20 and 10.42 minutes, fragments m/z
224.08362, 206.07306 and 231.09334 are the most intense. The fragments m/z
191.06216 and 163.03079 are also observed in the monohydroxylated
derivatives, being characteristic of the loss of the hydroxyl groups, either in the
aliphatic chain or the cyclobutane ring. These fragments are present at all
retention times of the M1-diOH metabolites, albeit at low intensity in the
fragmentation spectra of the metabolites of the retention times 8.20 and 10.42.
The fragment m/z 231.09334 may be generated with the hydroxyl groups
on the cyclobutane ring or the aliphatic chain. The fragments m/z 224.08362 and
206.07306 (generated from loss of the hydroxyl group in the cyclobutane ring in
fragment m/z 224.08362) (Figure 6C) are indicative of the presence of one
hydroxyl group in the cyclobutane ring, with the other being in the aliphatic chain.
Therefore, this would be the possible structure of the metabolites at retention
times 8.20 and 10.42 minutes. The fragments with m/z 191.06216 (C12H12Cl) and
206.07306 (C12H13NCl) are not intended to be formed with two hydroxyls on the
cyclobutane ring according to the software Mass Frontier 7.0. So, for the other
metabolites (5.46, 7.69, 9.16 and 13.24 minutes), the two hydroxyls may be
together in the aliphatic chain, differing only in the positions in the aliphatic chain.
109
4.1.4.4.5 M6 (M2-diOH)
The metabolites with m/z 284.14118 correspond to the dihydroxylated
derivatives of M2 (Figure 5F). Fragmentation spectra were obtained with a
collision energy of 30eV. Through Figure 5F it is possible to observe six more
intense chromatographic peaks. The fragments m/z 191.06225, 231.09354 and
203.06206 are the most intense in the fragmentation spectra of the metabolites
at retention times 6.36, 7.93, 9.44, 9.89 and 13.75 minutes. However, only for the
metabolite at retention times 11.54 minutes, fragments m/z 192.05742,
210.06799 and 231.09354 are the most intense, and the fragments m/z
191.06225, 231.09354 and 203.06206 are present at lower intensities. The
fragments m/z 191.06225 and 231.09354 are the same as those identified in M1-
diOH.
The fragments m/z 210.06799 and 192.05742 (generated from loss of the
hydroxyl group in the cyclobutane ring in fragment m/z 210.06799) (Figure 6D)
are indicative of the presence of one hydroxyl group in the cyclobutane ring, with
the other being in the aliphatic chain. Thus, this would be the possible structure
of the metabolites at retention time 11.54 minutes. The fragment with m/z
192.05742 (C11H11NCl) is not intended to be formed with two hydroxyls on the
cyclobutane ring according to the software Mass Frontier 7.0. Therefore, for the
other metabolites (6.36, 7.93, 9.44, 9.89 and 13.75) the two hydroxyls may be
together in the aliphatic chain.
4.1.4.4.6 M7
The metabolites with m/z 310.15683 correspond to the molecular formula
C17H24ClNO2 (Table 3). Fragmentation spectra were obtained with a collision
energy of 20eV. Through Figure 5G it is possible to observe that there are two
distinct populations of M7 metabolites, one around 15.5 minutes and another
around 21 minutes. Chromatographic peaks in the 15.5 minutes region show the
more intense fragments m/z 144.08078 (C10H10N), 161.10736 (C9H18Cl) and
177.04662 (C11H10Cl) and those in the region of 21 minutes m/z 177.04661
(C11H10Cl), 233.10901 (C15H18Cl) and 191.06222 (C12H12Cl). Through the
110
molecular formula and the fragmentation spectrum, M7 may be
dihydroxylated/monodehydrogenated derivatives of sibutramine or acetylates
derived from M2-OH, but it is not possible to be sure about the molecular structure
without further investigation.
4.1.4.4.7 M8
The metabolites with m/z 296.14118 correspond to the molecular formula
C16H22ClNO2 (Table 3). Fragmentation spectra were obtained with a collision
energy of 30eV. Figure 5H shows that there are two distinct populations of M8
metabolites, some in the region around 16 minutes and another in 20 minutes.
Chromatographic peaks in the 16 minutes region show the more intense
fragments m/z 191.06219 (C12H12Cl), 219.09336 (C14H16Cl) and 247.08819
(C15H16OCl) and those in the region of 21 minutes m/z 177.04661 (C11H10Cl),
191.06222 (C12H12Cl) and 163.03082 (C10H8Cl). Through the molecular formula
and the fragmentation spectrum, M8 may be generated by carboxylation of M1
or hydroxylated/dehydrogenated derivatives of M1-OH, but it is not possible to be
sure about the molecular structure with the data available.
4.1.4.5 Validation
The validation of the method was performed for the main metabolites
identified in human urine: M1, M2 and their respective mono and dihydroxyl
derivatives. The repeatability, carryover and matrix interference parameters were
evaluated about the M1 and M2 metabolites, which had reference standards. The
other parameters specificity and limit of detection were evaluated for all
metabolites.
4.1.4.5.1 Specificity
The specificity was evaluated through the absence of interfering
chromatographic peaks at retention times for the sibutramine metabolites and the
internal standard (IS). The protonated exact mass m/z values (6 ppm error) of
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sibutramine metabolites were investigated in these urine samples. Blank urines
from ten different sources were analysed, and there was no endogenous
interference at the retention time of each metabolite (data not shown).
In Figure 7, A and B correspond to TIC (total ion current) and to
chromatograms extracted with the protonated exact mass values of the
sibutramine metabolites and IS buspirone (Table 3) in blank urine without the
addition of analytes and buspirone. Figure 7 (C and D) shows urine excretion of
sibutramine spiked with internal standard buspirone, where it is possible to
observe TIC and chromatographic peaks relative to the protonated exact mass
of the metabolites and buspirone.
The TIC corresponds to the sum of intensities of all mass spectral peaks
belonging to the same scan, in this case in the range of m/z 100-800, including
background noise as well as urine components. However, through the
chromatograms extracted with protonated exact mass values of the metabolites,
it is possible to observe the specificity of the method, since there are no
interferers co-eluting at the retention times of the analytes.
4.1.4.5.2 Repeatability
Repeatability (intra-assay precision) expresses the precision under the
same operating conditions over a short interval of time [21]. To measure this
parameter seven replicates of blank urine fortified with M1 and M2 metabolites at
50 ng mL-1 were analysed. This concentration (50 ng mL-1) was chosen because
it represents 50% of the MRPL (minimum required performance level) value
required by WADA for detection of sibutramine and its metabolites. The
repeatability was verified about the value of % RSD of the ratio between the
analyte peak area and the IS. The values obtained for repeatability about M1 and
M2 are less than 15% (Table 5), fulfilling the criteria established by the FDA [22].
112
Figure 7. LC-HRMS extracted ion chromatograms (at a mass tolerance of 6 ppm) and TIC from the urinary sibutramine metabolites M1 (m/z 266.16700), M2 (m/z 252.15135), M3 (m/z 282.16192), M4 (m/z 268.14627), M5 (m/z 298.15683) and M6 (m/z 284.14118) in blank urines (A and B) and excretion urine of sibutramine (C and D).
4.1.4.5.3 Limit of Detection (LOD)
Serial dilutions of sibutramine excretion urine were performed to verify
LOD values for the metabolites. The concentration of metabolites in urine was
estimated based on the ratio analyte/internal standard obtained for a positive
control with M1 fortified in urine at 50 ng mL-1. As the developed method is
qualitative, LOD was considered the lowest concentration that would be detected
with signal-to-noise > 3. The values obtained for the LOD of the sibutramine
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metabolites are below 0.5 ng mL-1 (Table 5). The sensitivity provided by LOD
values is sufficient for analysis of sibutramine metabolites in human urine.
Table 5. Repeatability (RSD,%), matrix effect (RSD,%) and limit of detection (LOD, ng/mL) results for sibutramine metabolites.
Substance Repeatability
(RSD%)
Matrix
effect
(RSD%)
Robustness
(RSD%)
LOD
(ng/mL)
M1 11.48 20.70 8.85 0.38
M2 8.97 11.35 10.25 0.28
M3 (M1-OH) - - - 0.41
M4 (M2-OH) - - - 0.33
M5 (M1-
diOH)
- - - 0.44
M6 (M2-
diOH)
- - - 0.42
4.1.4.5.4 Carryover
The carryover was evaluated by analysing two blank urines interspersed
with a fortified control with twice of the analyte concentration used in the
repeatability assay. There was no chromatographic peak at the same retention
time of M1 and M2 in the blank urines, indicating the absence of carryover effect
in the analytical method developed (data not shown).
4.1.4.5.5 Matrix interference
The molecules originating from the sample matrix that coelute with the
compounds of interest can interfere with the ionization in liquid chromatography
massspectrometry based on electrospray ionization [29]. The solid phase
extraction sample clean up, the low mobile phase flow, and the adequate
chromatographic resolution contribute to the reduction of the matrix effect. A part
of the matrix interference was assessed by analysing the chromatograms of the
blank urine extracted with the protonated exact mass values (m/z) of the
sibutramine metabolites. Through Figure 7 (A and B) it is possible to observe the
absence of urinary interferences coeluting in the retention times of sibutramine
metabolites (Figure 7C and D). Also, the influence of different matrices in the peak
area of the chromatographic peaks of M1 and M2 through the %RSD value was
114
evaluated. Through Table 4 it can be seen that the matrix effect was higher for
M1 than for M2. In the repeatability assay, the RSD% values for the peak area of
M1 and M2 were 10.69 and 6.87, respectively. Therefore, the values of matrix
effect are acceptable for the analysis.
4.1.4.5.6 Robustness
To be considered robust the method shall be determined to produce similar
results concerning minor variations in analytical conditions [21]. To make this
assessment, small variations in the chromatographic method were performed,
reducing the flow to 0.16 mL min-1 and changing the column temperature to 45°C.
The robustness was verified about the value of % RSD of the ratio between the
analyte peak area and the IS. The samples did not show significant changes
about the alterations made, based on the value of % RSD for M1 and M2 (Table
4).
4.1.5 Conclusion
The investigation of the metabolism of substances is a fundamental step
in the detection of doping in sport. Although different liquid-liquid extraction
conditions were evaluated, solid phase extraction was the most comprehensive
and suitable for different sibutramine target metabolites. The DoE design
approach proved to be a rapid, economical and efficient way to optimize the
experimental conditions for the liquid chromatography resolution of isomeric
sibutramine metabolites from human urine samples. The final optimal
chromatographic condition was based on reversed-phase chromatography with
ramp time of 25 minutes, flow rate of 0.17 mL min-1 and temperature of 50 °C;
mobile phase A (water with 0.1% formic acid and 5 mM ammonium formate) and
B (methanol with 0.1% formic acid); initial gradient percentage of 15% B and
injection volume of 5μL. As an outcome from the DoE, the chromatographic
resolution reached among the metabolites were 1.28 (R1-2), 1.18 (R2-3) and 0.68
(R5-6).
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The LC-HRMS method developed and validated proved to be effective in
the detection of sibutramine urinary metabolites. In addition to the previously
described urinary metabolites, new metabolites (M5, M6, M7, and M8) have been
identified. Moreover, it was shown that specific fragments observed on high
resolution could elucidate some sibutramine metabolites. The identified
metabolites may be new analytical targets for the detection of sibutramine abuse
in sport doping or general forensic cases.
4.1.6 References
[1] I.D. Hind, J.E. Mangham, S.P. Ghani, R.E. Haddock, C.J. Garratt, R.W. Jones, Sibutramine pharmacokinetics in young and elderly healthy subjects, Eur. J. Clin. Pharmacol. 54 (1999) 847–849. doi:10.1007/s002280050565.
[2] C.A. Luque, J.A. Rey, The discovery and status of sibutramine as an anti-obesity drug, Eur. J. Pharmacol. 440 (2002) 119–128. doi:10.1016/S0014-2999(02)01423-1.
[3] J.C.G. Halford, Pharmacotherapy for obesity, Appetite. 46 (2006) 6–10. doi:10.1016/j.appet.2005.07.010.
[4] P. Saha, U.K. Mazumder, P.K. Haldar, S.K. Sen, S. Naskar, Antihyperglycemic activity of Lagenaria siceraria aerial parts on streptozotocin induced diabetes in rats, Diabetol. Croat. 40 (2011) 49–60. doi:10.4158/EP161365.GL.
[5] A.J. Krentz, K. Fujioka, M. Hompesch, Evolution of pharmacological obesity treatments: focus on adverse side-effect profiles, Diabetes, Obes. Metab. 18 (2016) 558–570. doi:10.1111/dom.12657.
[6] B. Shapira, L. Goldstein, A. Reshef, A. Poperno, A Rare Case of Psychomotor Disturbances Linked to the Use of an Adulterated Dietary Supplement Containing Sibutramine, Clin. Neuropharmacol. 39 (2016) 154–156. doi:10.1097/WNF.0000000000000141.
[7] S. Strano-Rossi, S. Odoardi, E. Castrignanò, G. Serpelloni, M. Chiarotti, Liquid chromatography-high resolution mass spectrometry (LC-HRMS) determination of stimulants, anorectic drugs and phosphodiesterase 5 inhibitors (PDE5I) in food supplements, J. Pharm. Biomed. Anal. 106 (2015) 144–152. doi:10.1016/j.jpba.2014.06.011.
[8] Y. Zeng, Y. Xu, C.L. Kee, M.Y. Low, X. Ge, Analysis of 40 weight loss compounds adulterated in health supplements by liquid chromatography quadrupole linear ion trap mass spectrometry, Drug Test. Anal. 8 (2016) 351–356. doi:10.1002/dta.1846.
[9] K. Skalicka-Woźniak, M.I. Georgiev, I.E. Orhan, Adulteration of herbal sexual enhancers and slimmers: The wish for better sexual well-being and perfect body can be risky, Food Chem. Toxicol. (2016). doi:10.1016/j.fct.2016.06.018.
[10] T.H.E.W.A. Code, Standard Prohibited List, (2017) 1–9.
[11] H.M.G. Pereira, V.F. Sardela, Stimulant doping agents used in Brazil: prevalence, detectability, analytical implications, and challenges., Subst. Use Misuse. 49 (2014) 1098–114. doi:10.3109/10826084.2014.907653.
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[12] M. Thevis, G. Sigmund, A.-K. Schiffer, W. Schanzer, Determination of N-desmethyl- and N-bisdesmethyl metabolites of Sibutramine in doping control analysis using liquid chromatography-tandem mass spectrometry., Eur. J. Mass Spectrom. (Chichester, Eng). 12 (2006) 129–136. doi:10.1255/ejms.797.
[13] S. Strano-Rossi, C. Colamonici, F. Botre, Detection of sibutramine administration: a gas chromatography/mass spectrometry study of the main urinary metabolites, Rapid Commun. Mass Spectrom. 21 (2007) 79–88. doi:10.1002/rcm.
[14] V.F. Sardela, M.T.R. Motta, M.C. Padilha, H.M.G. Pereira, F.R.A. Neto, Analysis of sibutramine metabolites as N-trifluoroacetamide and O-trimethylsilyl derivatives by gas chromatography-mass spectrometry in urine, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 877 (2009) 3003–3011. doi:10.1016/j.jchromb.2009.07.013.
[15] K.S. Hakala, M. Link, B. Szotakova, L. Skalova, R. Kostiainen, R.A. Ketola, Characterization of metabolites of sibutramine in primary cultures of rat hepatocytes by liquid chromatography-ion trap mass spectrometry, Anal. Bioanal. Chem. 393 (2009) 1327–1336. doi:10.1007/s00216-008-2540-8.
[16] C.S. Anselmo, V.F. Sardela, B.F. Matias, A.R. de Carvalho, V.P. de Sousa, H.M.G. Pereira, F.R. de Aquino Neto, Is zebrafish (Danio rerio) a tool for human-like metabolism study?, Drug Test. Anal. (2017). doi:10.1002/dta.2318.
[17] L. Couchman, P.E. Morgan, LC-MS in analytical toxicology: Some practical considerations, Biomed. Chromatogr. 25 (2011) 100–123. doi:10.1002/bmc.1566.
[18] D.B. Hibbert, Experimental design in chromatography: A tutorial review, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 910 (2012) 2–13. doi:10.1016/j.jchromb.2012.01.020.
[19] S.L.C. Ferreira, V.A. Lemos, V.S. De Carvalho, E.G.P. Silva, A.F.S. Queiroz, C.S.A. Felix, L.F. Silva, G.B. Dourado, R. V Oliveira, Multivariate optimization techniques in analytical chemistry - an overview, Microchem. J. 140 (2018). doi:10.1016/j.microc.2018.04.002.
[20] V.F. Sardela, M.E.P. Martucci, A.L.D. de Araújo, E.S. Leal, D.S. Oliveira, G.R. Carneiro, K. Deventer, P. Van Eenoo, H.M.G. Pereira, F.R. Aquino Neto, Comprehensive analysis by liquid chromatography-Q-Orbitrap mass spectrometry: Fast screening of peptides and organic molecules, J. Mass Spectrom. (2018) 476–503. doi:10.1002/jms.4077.
[21] World Anti-Doping Agency, World Anti-Doping Code International Standard for Laboratories (ISL), (2016).
[22] U.S. Department of Health and Human Services Food and Drug Administration (FDA)., Bioanalytical Method Validation: Guidance for Industry, (2018).
[23] N.H. Snow, Solid-phase micro-extraction of drugs from biological matrices, J. Chromatogr. A. 885 (2000) 445–455. doi:10.1016/S0021-9673(00)00192-8.
[24] C. Bylda, R. Thiele, U. Kobold, D.A. Volmer, Recent advances in sample preparation techniques to overcome difficulties encountered during quantitative analysis of small molecules from biofluids using LC-MS/MS, Analyst. 139 (2014) 2265. doi:10.1039/c4an00094c.
[25] J. Lu, S. Wang, Y. Dong, X. Wang, S. Yang, J. Zhang, J. Deng, Y. Qin, Y. Xu, M. Wu, G. Ouyang, Simultaneous analysis of fourteen tertiary amine stimulants in human urine for doping control purposes by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry, Anal. Chim. Acta. 657 (2010) 45–52. doi:10.1016/j.aca.2009.10.016.
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[26] R.V.S. Nirogi, V. Kandikere, M. Shukla, K. Mudigonda, S. Maurya, Sensitive and reproducible liquid chromatography-tandem mass spectrometry method for quantification of sibutramine in human plasma, Forensic Toxicol. 25 (2007) June-. doi:10.1007/s11419-007-0024-8.
[27] M.-. C. Hennion, Solid-phase extraction: method development, sorbents, and coupling with liquid chromatography, J Chromatogr A. 856 (1999) 3–54. doi:10.1016/S0021-9673(99)00832-8.
[28] A. Musenga, D.A. Cowan, Use of ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry for fast screening in high throughput doping control, J. Chromatogr. A. 1288 (2013) 82–95. doi:10.1016/j.chroma.2013.03.006.
[29] D. Moreno-González, J. Alcántara-Durán, B. Gilbert-López, J.F. García-Reyes, A. Molina-Díaz, Matrix-effect free quantitative liquid chromatography mass spectrometry analysis in complex matrices using nanoflow liquid chromatography with integrated emitter tip and high dilution factors, J. Chromatogr. A. 1519 (2017) 110–120. doi:10.1016/j.chroma.2017.09.006.
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119
5. CAPÍTULO III
5.1 Zebrafish (Danio rerio) Water Tank Model for the Investigation of Drug
Metabolism: Progress, Outlook, and Challenges
Vinicius Figueiredo Sardela, Carina de Souza Anselmo, Isabelle Karine da Costa Nunes,
Gabriel Reis Alves Carneiro, Gustavo Ramalho Cardoso dos Santos, Aline Reis de Carvalho,
Bruna de Jesus Labanca, Daniely Silva Oliveira, William Dias Ribeiro, Amanda Lessa Dutra de
Araujo, Monica Costa Padilha, Cleverton Kleiton Freitas de Lima, Valeria Pereira de Sousa,
Francisco Radler de Aquino Neto, Henrique Marcelo Gualberto Pereira
Drug Test Anal. 2018 Nov;10(11-12):1657-1669. doi: 10.1002/dta.2523. Epub 2018 Nov 22.
5.1.1 Abstract
Zebrafish (Danio rerio) water tank (ZWT) approach was investigated as an alternative
model for metabolism studies based on six different experiments with four model
compounds. Sibutramine was applied for the multivariate optimization of ZWT
conditions, also for the comparison of the metabolism among ZWT, humans and mice,
beyond the role of CYP2B6 in ZWT. After the optimization, 18 fish and 168 hours of
experiments is the minimum requirement for a relevant panel of biotransformation
products. A comparison among the species resulted in the observation of the same
hydroxylated metabolites, with differences in metabolites concentration ratio. However,
the ZWT allowed tuning of the conditions in order to obtain a specific metabolic profile,
depending on the need. In addition, by utilizing CYP2B6 inhibition, a relevant ZWT
pathway for the demethylation of drugs was determined. The stereospecificity of the ZWT
metabolism was investigated using selegiline and no racemization or inversion
transformations were observed. Moreover, the investigation of metabolism of
cannabimimetics was performed using JWH-073 and the metabolites observed are the
same described for humans, except for the hydroxylation at the indol group, which was
explained by the absence of CYP2C9 orthologues in zebrafish. Finally, hexarelin was
used as a model to evaluate studies by ZWT for drugs with low stability. As a result,
hexarelin displays a very fast metabolization in ZWT conditions and all the metabolites
described for human were observed in ZWT. Therefore, the appropriate conditions,
merits and relevant limitations to conduct ZWT experiments for the investigation of drug
metabolism were described.
© 2018 John Wiley & Sons, Ltd.
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5.1.2 Introduction
Cytochrome P450 (CYP450) is a major source of variability in drug
pharmacokinetics, and it is responsible for the biotransformation of most foreign
substances in clinical use, based on phase I metabolism (among others,
oxidation, N-demethylation, O-demethylation and N-dealkylation).1 This step
increases the polarity of molecules by exposing or adding polar groups,
transforming them into polar, water-soluble, and excretable metabolites. The
primary CYP isoforms involved in drug metabolism, as part of the CYP450
system, are CYP2A9, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5.2
Additionally, other CYPs belong to drug P450 metabolizing families in the human
liver, such as CYP2B6. The expression of each CYP is influenced by a unique
combination of mechanisms and factors, including genetic polymorphisms,
induction by xenobiotics, regulation by cytokines, hormones, disease states, sex,
and age.1 Moreover, the final products of the biotransformation can differ in their
distribution, concentration, chemical structure and molecular properties across
different species, such as mouse, rat, dog, monkey, and human.3
The anti-doping tests involve analytical aspects based on the evolution of
separation techniques, such as gas chromatography and liquid chromatography.4
These approaches are improving in parallel with the requirements for increasing
sensitivity and selectivity for detecting prohibited substances in biological
samples from athletes.4 However, due to the increasing number of prohibited
substances in sports, being the major bottleneck of doping control, and the fact
that the rate of metabolism determines the duration and intensity of
pharmacological drug effects, more studies are required to identify analytical
targets to detect their misuse. In addition, to evaluate prohibited substances,
especially when they have not yet been approved for use and no drug is available
on the legal market, it is necessary to use alternative methods to study their
metabolism.
Zebrafish water tank (ZWT) is a new approach that was recently
introduced for the investigation of metabolism. Similar to the murine model, ZWT
also presents genetic similarities with humans; the sequencing of the zebrafish
genome has shown that 70% of human genes have a zebrafish orthologue.5
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Another advantage is that zebrafish embryos develop very rapidly compared to
mammalian study models.6 In addition, ZWT has a high reproductive capacity, as
a pair of zebrafish can generate hundreds of fertilized eggs, which develop rapidly
into larvae with metabolic capabilities.7
As described by Goldstone et al.,5 Zebrafish has a total of 94 CYP genes,
distributed among 18 gene families, as found also in mammals. There are 32
genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs.
The high degree of sequence similarity suggests conservation of enzyme
activities for CYP19, aromatase; CYP17, steroid 17a-hydroxylase, and the
CYP26 retinoic acid hydroxylases. Also, there are orthologous relationships for
some CYP1s and some CYP3s between zebrafish and human. Therefore,
because several CYP enzymes expressed by zebrafish are directly orthologous
with human CYPs, a possible similarity among the metabolic drugs profiles
generated by both species has been recently investigated.8 However, in contrast,
zebrafish have 47 CYP2 genes, compared to 16 in human, from which only two
(CYP2R1 and CYP2U1) are recognized as orthologous based on sequence.4 As
a result, investigations related to CYP2 metabolism pathways on ZWT could face
relevant limitations to detect similar metabolites with humans.
Anselmo et al., 2017 showed that ZWT might be able to absorb, oxidize
and excrete various metabolites, in a manner similar to humans, for sibutramine
(SIB) and the primary SIB metabolites: N-desmethyl-sibutramine (M1), N-bis-
desmethyl-sibutramine (M2), and their respective hydroxylated counterparts,
which were observed in a similar pathway as that described for human urine.9 In
humans, CYP2B6 is the most important drug metabolizing enzyme for the
sequential biotransformation of SIB into the active metabolites M1 and M2.
Moreover, CYP2B6 is involved in the metabolism of many drugs, such as
selegiline, bupropion, cyclophosphamide, efavirenz, ketamine, and methadone.10
In addition, CYP2B6 is one of the most polymorphic CYP genes in humans, and
variants have been shown to affect transcriptional regulation, splicing, mRNA and
protein expression, and catalytic activity.11
Although the cytochrome P450 CYP2B6 has been studied in depth in
humans, this isoform has not been well studied in zebrafish. Recent evidence
suggests an important role for CYP2B6 as a major determinant of the
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stereoselective metabolism of drugs, such as methadone elimination in
humans.12 The identity of the CYPs responsible for stereoselective metabolism
and clearance is incompletely understood in zebrafish. Insufficient understanding
of this relevant step in metabolism is a barrier to predicting the applicability of
ZWT for investigating analytical targets for doping control purposes.
Also described by Anselmo et al. in 2017, ZWT is rather cheap, has a clean
matrix and may be a good tool for studying the production of metabolites.9
However, similar to any other model, the ZWT model has limitations.8 In vivo and
in vitro models are currently employed for the evaluation of drug metabolism.
Some models rely on state-of-the-art technologies, such as humanized mice,13,14
whereas others rely on dynamic testing, such as microsomes.15,16 Each of these
methods has distinct advantages and limitations. Thus, the optimal choice and
employment of a specific method depends on the nature of the studies being
performed. Establishing the limitations of the ZWT model for drug metabolism
investigation is relatively complex. The instability of a drug over days in an
environment under 28 °C, underwater, microbiota, and in glass, may create
limitations for the study of peptide metabolism. A slightly less complex and
indirect limitation relies on the possible absence of some orthologous CYPs
between human and zebrafish. In this state of the art article, we highlight the
merits, limitations, and appropriate uses of the current in vivo ZWT model for the
study of drug metabolism.
Thus, the objective of this work is to stress the applications of the ZWT
model for doping control analysis and the study of metabolism, in general. During
this process, different relevant aspects were explored, including the possibility of
modulating the biotransformation of metabolites based on experimental designs,
comparisons of the results obtained through ZWT and those using classical
models (interspecies comparison), the characterization of the CYP isozymes
involved in the ZWT metabolic profile, and the evaluation of the outcomes of the
model in regards to stereospecific metabolic pathways. In addition, the
metabolism of the cannabimimetics and peptides, recently introduced by the
World Anti-Doping Agency (WADA) Prohibited List, were evaluated to examine
the challenges that the model might face.
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5.1.3 Experimental Procedures
5.1.3.1 Chemicals and reagents
SIB and N-desmethyl-sibutramine (M1) were purchased from LGC
(Teddington, Middlesex, UK). The internal standards (ISTD) for LC-HRMS
analysis, buspirone and 7-propylteophiline, and the inhibitor ticlopidine were
supplied by Sigma-Aldrich (São Paulo, Brazil). For the study of excretion in
humans, mice, and zebrafish, tablets containing 15 mg of SIB (Biosintética, São
Paulo, Brazil) were used. All chemicals (i.e., formic acid, acetic acid, ammonium
formate, sodium phosphate and methanol) were analytical or HPLC grade (Tedia,
Fairfield, USA). The ultrapure water used was Milli-Q grade (Millipore, Billerica,
USA). The enzyme used for the enzymatic hydrolysis was β-glucuronidase (from
Escherichia coli) (Roche, Rio de Janeiro, Brazil). (+/-)-amphetamine, (S)-
methamphetamine, (R)-selegiline (or (L)-(R)-deprenyl) and hexarelin were
purchased from Sigma Aldrich (São Paulo, Brazil), JWH-073, JWH-073-N-3-
hydroxybutyl, JWH-073-N-4-hydroxybutyl and the JWH073-carboxy metabolite
were purchased from Chiron AS (Trondheim, Norway). (R)-N-
desmethylselegiline and (R)-methamphetamine hydrochloride were purchased
from TRC (Toronto, Canada). The internal standard for enantiomeric
identification, phentermine, was supplied by NMI (Sydney, Australia). Mosher
reagent (R)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride, MTPA-Cl) was
obtained from Fluka analytical (Buchs, Switzerland). Hexane and ethyl acetate
were purchased from Tedia (Fairfield, USA).
5.1.3.2 In vivo models
5.1.3.2.1 Mice
Six male A/J mice (33-48 g, 6-months-old) were obtained from the Faculty of
Pharmacy-UFRJ breeding facility and maintained in the animal-housing facilities of
the central biotherium of the Faculty of Pharmacy at controlled room temperature
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(22 ºC - 25 ºC), with a 12-h light/dark cycle. All the procedures involving the care
and use of laboratory animals in this study were examined and approved by the
Animal Ethics Committee of Federal University of Rio de Janeiro-UFRJ (CEUA -
UFRJ - protocol number 050/18). The treatment with SIB was performed by means
of an oral gavage (5 mg.kg-1) in a 0.9% NaCl solution (saline), daily for 5 days. After
the oral administration of the drug, the mice were placed in metabolic cages for the
collection of urine at different times (0, 3, 6, 24, 72, and 120 h). Day zero
corresponds to the first urine collection, considered as the negative control for each
animal. After the collection of the biological material, it was stored in a freezer at -
30 °C for later extraction or analysis.
5.1.3.2.2 Zebrafish
Adult zebrafish (Danio rerio) of both sexes (3-5 months) were maintained
in tanks with 4 L of aerated water by an Atman Hf100 filter 160 L/H (Guangdong,
China), under a 12-h light/12-h dark cycle, at 28 ± 1 °C. The fish were
acclimatized for a period of 3 days prior to the experiments. The fish were fed
with Tetramin® ratio (composed of fish derivatives, vitamins, and minerals) twice
a day during all experiments. Powder standards of the drugs under evaluation
were previously diluted in water and then introduced in the ZWT. Five tanks for
each experiment were used: two negative controls (with fish/without substance
addition and without fish/with substance addition) and tanks treated with the
following substances (three replicates): experiment 1, 15 mg SIB; experiment 2,
10 mg Jumexil® (selegiline hydrochloride); experiment 3, 1 mg JWH-073; and
experiment 4, 1 mg hexarelin. Eighteen fish were placed in each tank. Aliquots of
5 mL were collected at the following times: 0, 3, 6, 24, 48, 72, 96, 120, 144 and
168 h. This study was approved by the Ethics Committee on the Use of Animals
of the Federal University of Rio de Janeiro through protocol number 022/17.
5.1.3.2.3 Humans
Urine samples were collected after administration of a single dose of 15
mg of monohydrated sibutramine chloridrate, to a healthy female with 30 years
125
old and 60 kg. The excretion study was carried out according to International and
Brazilian regulations and approved by the University’s Ethics Committee
(protocol 168/02). A total of 37 aliquots were collected, performing approximately
90 h of excretion study.
5.1.3.3 Experimental set up for ZWT
5.1.3.3.1 Optimization of the ZWT model for SIB metabolism
To establish the conditions for the ZWT experiments, a multivariate
optimization according to a Dohelert design was used.17 The variables included
were as follows: the number of fish, the SIB dosage, and the time course of the
experiment. The number of levels for these variables were established according
to the geometry of the design employed, resulting in a study with seven points for
time, five points for the number of fish and three points for SIB dosage, totaling
15 experiments (Table 1). The time was varied between 1 and 7 days, the number
of fishes between 6 and 22, and the SIB dosage between 1 and 15 mg for tanks
with 4 liters of water. Data obtained were modeled by using the software Statistica
for Windows version 12.0 (Statsoft, São Paulo, Brazil), employing the
Experimental Design module. Triplicates of experiment 7 described in Table 1
were performed to estimate the experimental variance.
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Table 1. Experiments design for the three variables (number of fish, dosage of SIB and time) according to a Doehlert matrix and results obtained for each group evaluated.
*Area of analite/ISTD Area Group 1: primary metabolites evaluation (demethylated) Group 2: secondary metabolites evaluation (hydroxylated)
5.1.3.3.2 Inhibition of sibutramine metabolism in zebrafish
Enzyme inhibition experiments were conducted by incubating 18 fish with
15 mg of the inhibitor ticlopidine for 1h at room temperature. After this period,
the fish were transferred to a tank containing 15 mg of SIB, and they remained
in that environment for 1 hour. Then, the fish were transferred to a third
aquarium containing clean water, and aliquots were collected at different times
(0, 3, 6, 24 and 48 h). Then, the aliquots were frozen for further extraction and
analysis.
5.1.3.3.3 ZWT drug metabolism: sibutramine, selegiline, JWH-073 and
hexarelin
The ZWT experiments were conducted using five tanks for each drug: two
negative controls (with fish/without substance addition and without fish/with
substance addition) and tanks treated with the following substances (three
ZWT Number of
fish Sib dosage
(mg) Time
(hours) Group 1
(Area Ratio*) Group 2
(Area Ratio*)
1 10 8 168 0.911 0.022
2 18 8 168 0.859 0.040
3 14 15 144 1.186 0.033
4 10 1 120 0.127 0.006
5 18 1 96 0.169 0.013
6 6 8 96 0.674 0.007
7.1 14 8 96 0.784 0.014
7.2 14 8 96 0.816 0.014
7.3 14 8 96 0.926 0.028
8 22 8 96 1.492 0.019
9 10 15 72 1.211 0.020
10 18 15 72 0.864 0.032
11 14 1 48 0.543 0.007
12 10 8 24 0.340 0.004
13 18 8 24 0.245 0.005
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replicates): 15 mg SIB, 10 mg Jumexil® (selegiline hydrochloride), 1 mg JWH-
073, and 1 mg hexarelin. Powder standards of the drugs under evaluation were
previously diluted in water and then introduced in the ZWT. Eighteen fish were
placed in each tank. Aliquots of 5 mL were collected at the following times: 0, 3,
6, 24, 48, 72, 96, 120, 144 and 168 h, except for hexarelin were aliquots of 2 mL
were collected every two hours.
5.1.3.4 Sample preparation
5.1.3.4.1 For LC-HRMS analysis
The extraction procedure for the LC-HRMS analysis of ZWT aliquots for
the study of SIB, selegiline, and JWH-073 was adapted from Sardela et. al.18
Briefly, 3 mL aliquots from all ZWTs were added to buspirone solution (internal
standard, 50 ng/mL) and 7-propylteophiline, 750 µL of pH 7.0 phosphate buffer
and 50 µL of β-glucuronidase solution from E. coli. The tubes were shaken and
placed in a water bath at 50 °C for 1 h. After this step, and only for SIB, the
samples were adjusted to a pH of 9.0 with carbonate buffer. The samples were
extracted using a Strata-X-CW column (Phenomenex, Torrance, USA). The
cartridges were conditioned with 1 mL of methanol, followed by 1 mL of water.
Then, 3 mL of the sample was applied. The cartridges were washed with 1 mL of
water and then 1 mL of a methanol:water (1:1) solution, and the analytes were
recovered with 1 mL of methanol and 5% (v/v) formic acid. The eluates were left
under N2 flow at 40 °C to dry. The contents of the tubes were reconstituted with
100 µL of water:methanol (70:30), 0.1% formic acid, and either 5 mM ammonium
formate, for SIB, or 2% acetic acid for selegiline and JWH-073. Standard
solutions of M1 and selegiline were prepared in the same manner as the samples.
The samples were then injected into the LC-HRMS system.
5.1.3.4.2 For stereospecificity investigation
For the stereospecificity investigation, the sample preparation was
performed as described by Sardela et al. (2013).19 Briefly, a liquid-liquid
128
extraction was conducted with a 2 mL aliquot from each tank using 5 mL of
hexane after the addition of phentermine (I.S., final concentration at 1 µg/mL).
Then, 60 µL of Mosher:hexane solution 2% (v/v) and 120 µL of aqueous 5M
potassium hydroxide were added. After centrifugation, all organic layers were
separated and dried under N2 flow. The dry extract was solubilized with 100 µL
of anhydrous ethyl acetate. The analysis was performed by gas chromatography
coupled to mass spectrometry (GC-MS).
5.1.3.4.3 For peptides analysis
For hexarelin and metabolites analysis the extraction procedure from ZWT
aliquots was adapted from Thomas et al.20 Briefly, for 2 mL of aliquots from all
ZWTs D4-GHRP-4 solution (internal standard, 2 ng/mL) was added. The samples
were extracted using a Strata-X-CW column (Phenomenex, Torrance, USA). The
cartridges were conditioned with 1 mL of methanol, followed by 1 mL of water.
Then, 2 mL of the sample was applied. The cartridges were washed with 1 mL of
water and then 1 mL of methanol, and the analytes were recovered with 1 mL of
methanol and 5% (v/v) formic acid. The eluates were dried in a speedvac at 45
°C for 45 min. The contents of the tubes were reconstituted with 50 µL of 2%
acetic acid. The samples were then injected into the LC-HRMS system.
5.1.3.5 Samples extract analysis
5.1.3.5.1 LC-HRMS conditions
For the analysis of JWH-073 and selegiline by LC-HRMS, the method used
was the same described by Sardela et al., 2018.18 The liquid chromatography
(LC) system was a Dionex (Thermo Fisher Scientific, Bremen, Germany) coupled
to a Q-Exactive Plus Orbitrap mass spectrometer (MS) (Thermo Fisher Scientific,
Bremen, Germany). The chromatographic separation was performed in a
reversed-phase column (Syncronis – Thermo, USA; C18, 1.7 µm, 50 mm × 2.1
mm) at 40 °C, and the mobile phases were composed of (A) H2O with 5.0 mM
ammonium formate and (B) MeOH; both phases containing 0.1% formic acid. The
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LC effluent was pumped to MS which was operated by switching between positive
and negative ionization modes and equipped with an Electrospray Ionization
(ESI) source. The MS acquired Full scan data in both ionization modes at a
resolution of 70.000 full widths at half maximum (FWHM). In addition, an all-ion
fragmentation Full scan was achieved in both ionization modes with normalized
collision energy (NCE) of 40 and a resolution of 17.500 FWHM. For the
identification of specific metabolites, mass spectra were obtained by an extra MS
experiment in which all ions within a selected m/z range are fragmented and
analyzed in a second stage of tandem mass spectrometry (FULL-MS/MS2).
5.1.3.5.2 LC gradient condition optimized for metabolism investigation
For the analysis of SIB and hexarelin, the same method was used, but
adapted in the gradient program. For SIB, the gradient program started with a
170 μL·min-1 flow at 15% B and increased to 100% B after 25 min; flushed for 2
min with 100% B and finally re-equilibrated with 15% B for 3 min. For hexarelin,
a mobile phase gradient of 5-60% methanol with 0.1% formic acid over 15 min at
a flow rate of 350 μL/min was performed
5.1.3.5.3 GC-MS conditions
For diastereomeric derivatives analysis, the GC-MS analysis was
performed with a quadrupole mass spectrophotometry ISQ (Thermo Scientific,
San Jose/United States) equipped with a TriPlus RSH autosampler. The injector,
column and mass spectrometry conditions were the same as described by
Carneiro et al.21 The GC temperature programming was set as follows: initial
column oven temperature 130 °C (held 1 min), then programmed to increase to
210 °C at 10 °C/min, then to 250 °C at 10 °C/min and to 300 °C at 30 °C/min.
5.1.4 Results and Discussion
5.1.4.1 Optimization of the ZWT model for SIB metabolism
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The results were analyzed in two groups of substances, with group 1
referring to the ratio of the sum of areas obtained for the chromatographic peak
related to the demethylated metabolites, M1, and bisdemethylated metabolites,
M2, and the I.S. standard, and group 2 referring to the ratio of the sum of areas
obtained for the chromatographic peaks related to the hydroxylated derivatives
of M1 and M2 (M1-OH and M2-OH, respectively) and the I.S. (Figure S.1).
Multivariate optimization is considered to be more efficient than univariate
processes because it allows the evaluation of the influences of several
parameters within a procedure simultaneously using a smaller number of
experiments.21 This division in groups was performed to analyze the different
phases of metabolism and the kinetics of the formation of metabolites, as those
belonging to group 1 were the precursors of the other SIB metabolites and those
belonging group 2 are the inactive and more water-soluble metabolites, according
to previous studies.22
Figure S.1 Main metabolites of SIB generated by demethylation and hydroxylation reactions mediated by CYP2B6.
The optimization of the ZWT conditions was performed by employing a
Response Surface Methodology based on a Doehlert matrix.21 The number of
fish was the principal variable for investigation because it is directly related to the
kinetics of the process, and the concentration of the drug in the ZWT was
evaluated because it is a relevant variable for other in vivo models. For the
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Doehlert design, when three variables are considered, the solid obtained
presents a cuboctahedron geometry, which is produced through the uniform
distribution of points over the whole experimental region.23 Therefore, the
duration of the experiment was set as a third variable, and the kinetics of the
biotransformation were evaluated along the time in hours.
As described, the experimental variables evaluated were the total number
of fish (Fish), the amount, in milligrams, of SIB added in each ZWT (Dosage), and
the duration of the experiment in hours (Time). The number of levels for these
variables were established according to the geometry of the design employed,
resulting in a study with seven levels for Fish, five levels for Dosage and three
levels for Time, totaling 13 experiments. The total number of fish varied among 6
and 22 specimens, the Dosage between 1 and 15 mg and the time for aliquot
collection (Time, in hours) between 24 and 168 hours. Table 1 shows the
numbered experiments designed for these variables according to Doehlert matrix
and the area ratio (metabolites/Internal Standard) obtained for groups 1 and 2.
The obtained data were modeled using the software Statistica for Windows
(Version 6) by employing the Experimental Design module.
The results of the multivariate optimization can be observed by the surface
plots in Figure 1, where the green area represents the lowest ratios, and the red
area represents the highest ratios obtained during the optimization. The number
of fish has a low impact on the production of primary metabolites (Group 1). As
can be observed, the peak concentration of these metabolites occurs 120 hours
after the start of the experiment, regardless of whether the ZWT system contained
4 or 22 specimens (Figure 1, Group 1.A). The concentrations of these metabolites
decrease, which can be explained by the continuity of biotransformation
pathways, leading to hydroxylated metabolites.
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Figure 1. Multivariate optimization results on surface plots for ZWT conditions: green area represents the lowest ratios and the red area represents the highest ratios. (A) Evaluation of Fish x Time, (B) Dosage x Time, and (C) Dosage x Fish.
Hydroxylated metabolites were more extensively formed when the number
of fish in the ZWT was higher than 18 specimens (Figure 1, Group 2.A). In
addition, the hydroxylated metabolites were observed in significant
concentrations after 120 hours, and their increase was continuously observed.
Therefore, 18 fish and 168 hours are the minimum experimental requirements to
obtain all the SIB metabolites in high concentrations in ZWT. Moreover, the
experiment could be modulated; for example, if the target metabolites were the
primary metabolites, the number of the fish should be lower than 18 and the time
less than 120 hours. This type of modulation is one advantage of the ZWT model
compared to other in vivo systems.
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The increase in the dose of SIB consistently improved the response in the
surface plots shown in Figure 1.C, for both groups. In addition, the asymmetry
observed in the curve of the formation of primary metabolites demonstrates the
classical biochemical relationship: substrate-enzyme, where a lower dosage (2
mg) of SIB (substrate) and a high number of specimens of fish (enzymes) results
in the total consumption of the substrate. In Figure 1.C, group 2, the substrate-
enzyme relationship becomes even more evident: the increase in the exponential
rate of the formation of the final metabolites is followed by the increase in the SIB
concentration and the number of fish. Moreover, in this experiment it was
observed that a low dose of the drug in the ZWT resulted in a SIB metabolite
profile that is consistent with those described in studies performed in humans
following a single dose administration, where the detectability of primary
metabolites decreases after some time and hydroxylated metabolites are
observed in higher abundance (Figure 1.C). However, with 15 mg of SIB in the
ZWT, the SIB metabolites profile observed is similar to that for chronic use by
humans.
The N-demethyl metabolites are observed for the duration of the
experiment, and these reach a stable concentration and can be detected together
with hydroxylated metabolites. In addition, should be considered that zebrafish
excrete the metabolites into the water and can subsequently ingest and
metabolize them again. Considering the aim of the present research, the
metabolite profile of the drugs was evaluated to collect the most comprehensive
data. Therefore, 18 fish, seven days and a high dose of the substances was used.
For SIB, 15 mg was the dose chosen as the best condition for a typical ZWT
experiment that allows for the detection of all metabolites simultaneously.
5.1.4.2 Comparative interspecies metabolism of sibutramine
The in vivo model that has been most widely used as an alternative for the
study of drug metabolism in humans is rodents.24 In recent years, the use of mice
has been highlighted to the detriment of other rodents, due to advantages, such
as small size, which allows a large population per cage and, consequently, a
reduction in costs. An average of 5 to 10 mouse pups can be born every 3 weeks.
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In addition, the murine genome is very similar to the human genome.25 It was
observed that the rodents feature 102 CYP genes, while humans have 57. In
addition, 36 pairs of these CYP genes are orthologous, with isolated or identical
functions in both species.26 However, due to interspecies variations in the
expression, activity, and induction of CYP enzymes, murine models present
limitations for comparison with humans.27
To compare the metabolic profiles of the drug SIB among different species
(human, mice, and zebrafish under ZWT condition), and to characterize and
validate zebrafish as an alternative model for studies of drug metabolism, the
metabolic profiles of SIB among these three species was evaluated. For this
purpose, the concentrations of SIB, its demethylated metabolites (M1 and M2)
and the hydroxylated derivatives of these demethylated metabolites (M1-OH and
M2-OH) were compared.
Although all three species produced metabolites corresponding to the
hydroxylated derivatives of M1 and M2, the ratio was different (Figure 2). For
example, the isomer of M1-OH metabolite, which appears at retention time
indicated by the number 1 (tR = 13.7 min), is more intense than the isomers
indicated by numbers 2 (tR = 14.6 min) and 3 (tR = 15.4 min), in humans (Figure
2.A) and ZWT (Figure 2.C), but not in mice (Figure 2.B). In case of the isomers
of hydroxylated metabolites from M2 (Figure 2.D-F), all species appear to have
specific hydroxylation patterns based on the intensity of formation of M2-OH
metabolites.
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Figure 2. Comparison among the relative abundance (%) of SIB hydroxy isomers metabolites for human, mouse and ZWT. The isomers of M1-OH are represented by the numbers (1), (2) and (3) in A for human, B for mouse and C for ZWT. The isomers for M2-OH are represented by the numbers (4), (5) and (6) in D for humans, E for mouse, and F for ZWT.
As described by Martignoni et al. in 2006, animal models are commonly
used in science to predict the metabolic behavior of substances in humans.3
However, the authors highlighted the importance of realizing that humans differ
from animals with regards to the isoform composition, expression and catalytic
activities of drug-metabolizing enzymes. Therefore, studies of the preclinical
development of new drugs must use the most relevant animal species for
extrapolating results to human metabolism. In the doping control field, this
perspective must be even more complex, as the results from a non-human
metabolism study could be the primary data used to define targets for drug
control. In doping control perspective, besides the presence of the metabolites,
an understanding of which metabolite is the most abundant, or presents a longer
detection window, is a critical goal for its applicability
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Although CYPs in zebrafish shows appreciable interspecies differences
regarding catalytic activity, the ZWT model allows tuning of the system, by
modifications of the ZWT conditions, including the number of fish and the time for
water collection. In Figures 3A and B, it is possible to compare the first samples
collected from the excretion of urine from humans and mice, respectively. The
differences in the intensities of the metabolites are again observed. However, in
ZWT, the ratio of this metabolite was modulated by the results obtained in the
Doelhert experiment. Figure 3, C1, C2, and C3, represent different conditions of
ZWT for SIB. To achieve the same metabolic profile observed in humans (Fig
3.A), an experiment with the conditions of 168 h, 10 fish and 8 mg of SIB was
used. Based on these results, the hypotheses that experimental conditions could
be modulated, starting from a simple statistical analysis, to enhance the formation
of a specific metabolite was demonstrated.
Figure 3. Profile of the SIB metabolite M1-OH in humans (A), mice (B) and in zebrafish, under the three different conditions evaluated in the experimental design (C1, C2 and C3). Condition 1: 120 hours, 10 fish and 1 mg of SIB (Table 1, ZWT→4); Condition 2: 8 hours, 14 fish and 8 mg of SIB (Table 1, ZWT→7); and Condition 3: 168 hours, 10 fish and 8 mg of SIB (Table 1, ZWT→1).
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Therefore, drugs with metabolic profiles well-known in humans can be
used as models to calibrate the conditions on ZWT through the same variables
presented, and using Doelhert matrix experiments. After that, new substances
(e.g., new designer drugs with similar structures) can be submitted for
metabolism evaluation under conditions more feasible to access a similar human
profile thoroughly characterized. Then, target metabolites for sports drug testing
could be properly selected or isolated from ZWT and applied as reference
material.
According to Figure S.2.A, it is possible to observe a large concentration
of SIB in the typical ZWT throughout the experiment, unlike what has been
observed in the urine from humans and mice. The large concentration of SIB is
explained by the fact that the drug was administered to ZWT by the dilution of 15
mg directly into the water tank, such that a fraction remains in the water that was
not absorbed by the fish. For humans, a single dose of SIB was administered. As
SIB is rapidly metabolized to M1 and M2 in humans,28 it is practically undetectable
in the unaltered drug urine (Figure S.2A). The same is observed for mice, even
though, for this specimen, the daily administration of SIB was performed (Figure
S.2A).
Figure S.2. SIB metabolism by human, mice and zebrafish, over time. A: SIB; B: M1; C: M2; D: M1-OH and E: M2-OH. The aliquots were obtained from the ZWT. For mice and humans, samples were obtained from urine. The data represent the averages of 3 independent experiments.
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In humans, there is a peak concentration of M1 and M2 within the first few
hours after ingestion of SIB, followed by a continuous decrease from 24 hours to
day 5 (Figure S.2 B-C). For mice and ZWT, the concentrations of M1 and M2 are
lower than in humans, increasing discretely until day 5, except for M1, which, in
ZWT, presents a considerable increase in concentration on the fifth day. The
hydroxylated derivatives of M1 and M2 have distinct behaviors among the three
species. In humans, the peak concentrations of M1-OH occur at 24 h and of M2-
OH occurs before 24 h; in mice and ZWT, the concentration grows slightly over
time, with ZWT presenting the lowest amount of these metabolites. It should be
noted that the same metabolites were observed for all three species, although in
different ratios. This result shows the feasibility of performing metabolic studies
using zebrafish as a model.
5.1.4.3 Characterization of the major CYP isoenzyme involved in the
sibutramine ZWT metabolic profile
Because the major metabolic pathways of SIB in zebrafish were identified
and these findings showed correspondence with the metabolites formed in
humans, the contribution of the CYP2B6 isoenzyme to the oxidative metabolism
of SIB in ZWT was studied, as the literature reports the involvement of this
isoenzyme in human metabolism.11 Thus, to identify if CYP2B6 is involved in the
formation of M1 and M2 metabolites, 15 mg ticlopidine, a CYP2B6 inhibitor drug,
was administered to a typical ZWT, also containing 15 mg of SIB.
The significant reduction in the production of the major SIB metabolites M1
from 57.7% to 23.4% and M2 from 56.3% to 29.1%, in the presence of 15 mg of
the inhibitor ticlopidine was observed (Figure 4). It is noteworthy that this inhibitor
was still able to reduce the production of the metabolites M1-OH from 43% to
21.4% and M2-OH from 41% to 22.8%. Based on these data, it was possible to
infer that there is an enzymatic correlation between the human and zebrafish
enzymes involved in SIB metabolism.
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Figure 4. In vivo metabolism percentage of SIB metabolites in ZWT (Control) and the percentage of the same SIB metabolites with introduction of selective inhibitor of CYP2B6 isoenzyme, ticlopidine, in the ZWT (+ ticlopidine).
According to the literature, the CYP2s constitute the largest CYP gene
family in both zebrafish and mammals, with 47 CYP2 genes in zebrafish, in
contrast to 16 in humans.5 In zebrafish, CYP2Y CYP2Y3 and CYP2Y4 present
the same order of conservation in the chromosomes, with a set of CYP2 genes
for human drug-metabolizing enzymes, including CYP2A6 and CYP2B6.5
5.1.4.4 Stereospecificity investigation: selegiline as a model
The relevance of stereospecificity in toxicology has a strong basis in the
literature29,30. Recently, enantiomeric analysis has contributed to demonstrating
that doping cases involving clenbuterol could be the result of unintentional
ingestion of contaminated meat in different countries.29,30 In forensic analytical
toxicology, a classic example of this relies on the possibility of discriminating the
context for the administration of amphetamine-like compounds from allowed
medicines to recreation abuse.
Selegiline (R(-)-N-methyl-N-(1-phenyl-2-propyl)-2-propylamine) is used to
treat Parkinson’s disease31. The metabolic pathway results in the formation of the
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very powerful stimulants amphetamine and methamphetamine. Consequently,
selegiline is listed by the World Anti-Doping Agency as a stimulant in the
prohibited list of substances in sport32. However, the selegiline metabolic outputs
are N-desmethylselegiline (in low concentration), (R)(-)-methamphetamine, (R)(-
)-amphetamine and their conjugated p-hydroxy derivatives (Figure S.3).33
Enantiomeric analysis has become necessary to discern between the clinical use
of selegiline and the abuse of the street drug “speed” (methamphetamine in
crystal form).34
Obviously, the abovementioned approach is possible due to the
preservation of the enantiomeric group, despite the biotransformation steps
involved, as demonstrated previously in humans and rats.33,35The highest
contribution to the hepatic clearance of selegiline in humans was calculated to be
exerted by CYP2B6 and CYP2C19, whereas CYP3A4 and CYP1A2 were of less
importance.36
Figure S.3. Metabolic route of selegiline observed in ZWT.
In ZWT, five urinary metabolites of selegiline and the unchanged drug were
identified by LC-HRMS: (R)-desmethylselegiline, (R)-methamphetamine, (R)-
amphetamine, (R)-p-hydroxy-amphetamine and (R)-p-hydroxy-
methamphetamine, being the last one detected in trace levels. A chiral
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derivatization reagent was used to transform the enantiomers into their
diastereomers before chromatography on a non-chiral system. The differentiation
between the enantiomers of methamphetamine (S and R) was possible by
comparing the retention time from the GC/MS of peaks with those of authentic
compounds, based on the Mosher reaction. For distinguishing amphetamine
enantiomers (S and R), analysis of retention time was performed, based on the
literature data, which showed that R enantiomers eluted before the corresponding
S-enantiomers (Figure S.4).37,38
Figure S.4. Ion chromatogram fragments formed from electron ionization and full-scan GC-MS for Mosher derivatives. (A) m/z 260, 162 and 228 for amphetamine ((R)-amphetamine, A1 and (S)-amphetamine, A2) and (B) m/z 189, 274 and 91 for methamphetamine ((R)-methamphetamine, B3 and (S)-methamphetamine, B4) enantiomers obtained through the Mosher reaction. Detection of (C) (R)-amphetamine and (D) (R)-methamphetamine at ZWT after 168 h of selegiline administration.
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The metabolite concentrations increased up to 9 hours after the
administration of 10 mg of selegiline hydrochloride to the ZWT, and the selegiline
concentration decreased to half after 168 h (Figure 5). It is possible to note, in
Figure 5, that the excretion profiles of methamphetamine and desmethylselegiline
were very similar and that both substances were excreted at higher
concentrations than the other metabolites. In contrast, the increase in the
amphetamine concentration was very slight in the ZWT.
Figure 5. The metabolic profile of selegiline and its metabolites in ZWT. Evaluation of the ratio between analite area and internal standard (7-propyltheophylline) area by LC-HRMS during 168 hours. The decrease of selegiline levels (see right axis) followed the increase of the selegiline metabolites desmethylselegiline, methamphetamine and amphetamine (see left axis).
Selegiline is primarily metabolized by three distinct pathways: N-
dealkylation, beta-carbon hydroxylation, and ring-hydroxylation, with the last two
being minor metabolic routes33. The products of beta-carbon hydroxylation, such
as ephedrine, pseudoephedrine, norephedrine, and norpseudoephedrine, were
not found in the ZWT. The major metabolite was (R)-methamphetamine, followed
by (R)-desmethylselegiline, which is similar to human urine excretion.33 As
observed in humans, during metabolism, the levorotatory (l) form is preserved.
No racemization or inversion transformation was observed, and N-dealkylation
showed stereoselectivity in the ZWT. This result raises the possibility that the
zebrafish model can be applied to metabolic investigations of other doping agents
with chiral centers and when the target definition requires a stereoselectivity
evaluation.
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5.1.4.5 Cannabimimetics biotransformation profile: JWH-073 study
Different studies have been published on the metabolism of
cannabimimetics: JWH-015 using rat liver microsomes in vitro,39 JWH-073 in
humans,40-42 and JWH-250 in humans and rats.43 All of these cannabimimetics
contain an aminoalkylindole group; JWH-073 also has a naphthyl group in
common with many other JHWs (Figure S.5). The in vitro metabolism of
cannabimimetics produces a large variety of metabolites not observed in human
metabolism studies39, such as for JWH-073, for which the diversity of products
generated by in vitro approaches greatly exceeds those typically reported from
urine.40-42
Figure S.5. Metabolic route of the synthetic cannaminoid JWH-073 observed in ZWT.
The human urinary metabolites ascribed to JWH-073 are
biotransformationally driven by the cytochrome P450 CYP2C9, which promotes
the monohydroxylation of the indole group, and CYP3A4 on the alkyl ω site,
followed by the ω-carboxylation of the alkyl chain by UDP-
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glucuronosyltransferase.40,41 All monohydroxylated forms are fully
glucuronidated, while only a fraction (<50%) of the carboxylated products are
glucuronidated.
In the ZWT, only two monohydroxylated JWH-073 metabolites were
observed in the first 24 hours, both on alkyl chains. The JWH-073-N-3-
hydroxybutyl showed the highest intensity after 24 hours of JWH administration
in the ZWT. This was followed by a decrease in concentration over the next 7
days, primarily because of the continuous reabsorption of this metabolite by the
fish, followed by an increase in the dihydrodiol metabolites concentrations. JWH-
073-N-4-hydroxybutyl was also observed in the first days of the study, and its
presence decreased, followed by the formation of an acid product. The ω-
carboxylation acid metabolite was the majority metabolite present in the ZWT at
the end of the experiment (Figure 6).
Figure 6. The metabolic profile of JWH-073 in ZWT evaluated by LC-HRMS. An increase of hydroxybutyl metabolites in the first hours are observed followed by a decrease after 72 hours. The acid metabolite increase continually after 24 hours from the beginning of the experiment.
Dihydrodiol metabolites were reported as major metabolites by
Wintermeyer et al. in a 2010 in vitro study,44 while they are not reported in
humans. Therefore, it is possible to assume that the model ZWT could be
modulated so that experiments end prior to 7 days, to obtain a profile similar to
that observed in human urine, and the most relevant target could be identified or
isolated from ZWT and used as reference material.
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In human urine, monohydroxylation of the indole group has also been
described for JWH-073. However, there are no known CYP2C9 orthologues in
zebrafish.5 Thus, the absence of a CYP orthologue between specific human and
zebrafish CYPs could be responsible for the absence of monohydroxylation on
the JWH-073 indole group, in ZWT. Therefore, the primary metabolites observed
in ZWT are the same as those observed in human urine: JWH-073-N-3-
hydroxybutyl, JWH-073-N-4-hydroxybutyl, and the respective carboxy
metabolites. The monohydroxy metabolites on the indole group were not
observed. In addition, it is reasonable to assume that other cannabinoids would
follow a similar pattern.
5.1.4.6 Peptides stability concept proof: hexarelin study
Hexarelin (His-D-2-methyl-Trp-Ala-Trp-D-Phe-Lys-NH2) is a potent,
synthetic, peptidic, nasally and orally active, centrally penetrant, and highly
selective agonist of the ghrelin/growth hormone secretagogue receptor (GHSR).
The biological activity of hexarelin is associated with the binding with the GH
secretagogue 1a receptor (GHS-R1a), a G protein-coupled receptor originally
identified in the hypothalamus and pituitary gland and recognized as the receptor
for ghrelin.45,46 Hexarelin is a nonapproved pharmaceutical drug that is used in
research and is misused for doping purposes. This compound has a low
detectability for the intact drug, when using traditional mass spectrometry
analyses, because it has a short half-life.47
In superior organisms, the metabolization of hexarelin is performed
systematically, through enzymes such as exopeptidase (amino- or carboxy-),
amidase, or endopeptidase, that act by cleaving the peptides to generate smaller
and smaller peptides46. As a result, hexarelin displays a very fast metabolization
with a half-life of approximately 2.5 h, similar to other GHRPs.48 A metabolite was
identified as hexarelin deamidated at the lysine residue.47
Semenistaya et al., described the stability of hexarelin and other peptides
in urine for 2 weeks at 4 °C, and 72 h in post-preparative QC samples at 8 °C.49
In addition, the authors highlighted that the metabolites identified differ regarding
their long-term storage stability at -20 °C. After 6 months of storage at -20 °C, a
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drastic decrease in the targeted peptides was observed. Moreover, the influence
of the non-specific adsorption of hexarelin and other peptides on plastic or glass
container surfaces, which are generally used in clinical and experimental
procedures, was also described.49,50 Therefore, the evaluation of hexarelin by the
ZWT model is a big challenge because of the temperature, the time of drug
exposure and the possibility of glass interaction in the tank. In the ZWT, all
hexarelin was metabolized in the first 12 hours of the experiment (Figure 7), which
was faster than expected, followed by the increase of all of the metabolites,
including hexarelin (1-3), hexarelin (4-6) free acid, and hexarelin (2-6) free acid
(Figures 7B, C and D).
The exopeptidase (amino- or carboxy-), amidase or endopeptidase
activities were observed in the water tank without fish, probably because an
environment constituted by unicellular organisms present in the tank was able to
promote such transformations. Only the metabolites hexarelin (4-6) free acid,
hexarelin (1-3) and hexarelin (2-6) free acid were found exclusively in a water
tank containing zebrafish. Therefore, ZWT can be used as a model for
investigating peptide metabolism. However, the drug instability in the tank
appears to be a challenge for peptide metabolism investigation, and additional
experiments are required to optimize the duration of the process and to determine
the pathways of the biotransformations.
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Figure 7. The metabolic profile of hexarelin in a zebrafish model evaluated by LC-HRMS. (A) shows the decrease in hexarelin levels, followed by an increase in the metabolites hexarelin (1-3), hexarelin (4-6) free acid and hexarelin (2-6) free acid (B, C and D respectively).
5.1.5 Conclusion
In this work, the merits, limitations and appropriate use of the current in
vivo ZWT model for the study of drug metabolism were highlighted. The
optimization of the ZWT conditions was performed by multivariate statistical
analysis using Doehlert matrix and SIB excretion metabolism profile. The number
of fish, the concentration of drug and the time were considered the principal
variables for ZWT conditions. The number of fish has a low impact on the
production of primary SIB metabolites, and hydroxylated metabolites were more
extensively formed under ZWT conditions with 18 specimens. In addition, the
hydroxylated metabolites were observed in significant concentrations after 120
148
hours. Therefore, 18 fish and 168 hours are the minimum experimental
requirements to obtain all the SIB metabolites in high concentrations in ZWT.
Using SIB as a study object, interspecies comparisons demonstrated the
differences regarding the metabolic patterns among the concentration of SIB
metabolites in humans, mice, and the ZWT models. All three species produced
the same hydroxylated metabolites, but with specific hydroxylation patterns.
However, the conditions of ZWT was easily modulated from Doehlert matrix data
to achieve the same metabolic pattern observed in human. For that, modification
on the time of the experiment to 168 h, the number of fish to 10 and the amount
of SIB to 8 mg in 4 L of water in the tank resulted in the observation of the same
concentration of OH isomers from M1. Therefore, this approach can be used to
calibrate the ZWT conditions to investigate new substances mimicking the human
results, which helps to determine target metabolite for methods applied on sports’
drug testing or increase the concentration of specific metabolites in order to
isolate them for future use as reference material.
For the first time, a characterization of the metabolic process for the major
CYP isozyme involved in the sibutramine ZWT metabolic profile was elucidated.
The contribution of the CYP2B6 isoenzyme to the oxidative metabolism of SIB in
ZWT was identified after the inhibition by ticlopidine and an enzymatic correlation
between the human and zebrafish enzymes involved in SIB metabolism was
demonstrated.
Also the stereospecificity investigation of ZWT metabolism was studied
using selegiline as a model. Five urinary metabolites of selegiline and the
unchanged drug were identified, and the excretion profiles of methamphetamine
and desmethylselegiline were very similar between human and ZWT. And, as
observed in humans, during metabolism under ZWT conditions, the levorotatory
(l) form is preserved. No racemization or inversion transformation was observed,
and N-dealkylation showed stereoselectivity. The results raise the possibility of
applying the ZWT model to investigations where the target definition requires a
stereoselectivity evaluation is required.
JWH-073 was applied as a model for cannabimimetics biotransformation
under ZWT condition. Different studies in humans have been published on the
metabolism of this class of substances, and among them the biotransformation
149
process is similar. In the ZWT, only two monohydroxylated JWH-073 metabolites
were observed, both on alkyl chains, and in the last days of the experiments, the
ω-carboxylation acid metabolite was the majority product present in ZWT. The
monohydroxylation in the indole group was not observed on ZWT results, as
described for humans. The absence of a specific CYP orthologue between
human and zebrafish could be responsible for the differences in the
biotransformation pathway observed.
The peptide hexarelin was used as a model to evaluate challenges to
conduct ZWT experiments for drugs with low stability in an oxidative environment.
As a result, hexarelin displayed a very fast metabolization in ZWT condition, with
a half-life lower than 12 hours. All metabolites described for humans were
observed in ZWT, including hexarelin (1-3), hexarelin (4-6) free acid, and
hexarelin (2-6) free acid. However, some of them also were observed in the water
tank without fish, probably a natural biora was able to promote such
transformations. Therefore, the ZWT can be used as a model for investigating
peptide metabolism, once a specific optimization is performed.
5.1.6 Conflict of interest
The authors declare they do not have any conflicts of interest associated
with the present work.
5.1.7 Acknowledgments
This work was supported by CNPq, FAPERJ and Ministério do Esporte.
The technical assistance of Geovana Maria de Lima Gomes was greatly
appreciated.
150
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6. DISCUSSÃO GERAL
O zebrafish tornou-se um modelo popular em várias linhas de pesquisa
biológicas pois compartilha semelhanças fisiológicas, morfológicas e histológicas
com os mamíferos (DIEKMANN & HILL, 2013; MACRAE & PETERSON, 2015).
De fato, muitas enzimas do citocromo P450 humano (CYP) têm ortólogos diretos
no zebrafish, sugerindo que os perfis metabólicos dos xenobióticos produzidos
pelo peixe podem ser semelhantes aos dos mamíferos (GOLDSTONE et al.,
2010). Anselmo e colaboradores (2018) relataram que os primeiros estudos que
observaram a produção de metabólitos no zebrafish focavam em toxicologia
ambiental, e somente nos últimos anos o foco tem sido a avaliação dos
metabólitos produzidos e da toxicidade das substâncias. No entanto, ainda há
uma falta de padronização do modelo de zebrafish, além da necessidade de se
conhecer a extensão da similaridade com o metabolismo humano. Como em
qualquer modelo não humano, o zebrafish apresenta semelhanças e diferenças
em relação ao perfil de metabólitos gerados quando comparado ao observado
em humanos (ANSELMO et al., 2018).
Um dos grandes desafios da ciência antidopagem consiste na diversidade
de substâncias disponíveis capazes de promover o aumento do desempenho
dos atletas, seja na indústria formalmente estabelecida ou no mercado
clandestino (KWIATKOWSKA et al., 2015; THEVIS & SCHÄNZER, 2014).
Levando-se em conta que muitas substâncias são extensivamente ou mesmo
completamente metabolizadas antes de serem excretadas, o estudo do
metabolismo é fundamental na ciência antidopagem, especialmente na busca
por alvos analíticos eficientes na detecção de substâncias proibidas (BADOUD
et al., 2011). No entanto, devido a questões éticas, geralmente não é possível
realizar a investigação do metabolismo em humanos.
A primeira etapa do trabalho visou avaliar a aplicação do modelo de
zebrafish (ZWT) para predição do metabolismo de sibutramina e estanozolol,
duas substâncias amplamente utilizadas como dopagem no esporte e com
metabolismo amplamente estudado. Para tal, amostras de água dos aquários
com zebrafish e tratados com sibutramina e estanozolol foram analisadas no
modo de varredura total (full-scan) positivo e negativo por LC-HRMS. Nos seres
157
humanos, a sibutramina sofre biotransformação em pelo menos seis metabólitos
conhecidos: os metabólitos desmetilados M1 e M2, que por sua vez, são
hidroxilados gerando os derivados M1-OH e M2-OH, com o grupamento hidroxila
ligado ao anel ciclobutano ou à cadeia isopropílica (STRANO-ROSSI,
COLAMONICI & BOTRÈ, 2007; SARDELA et al., 2009).
Assim como a sibutramina, o estanozolol é uma substância amplamente
metabolizada sendo encontrados mais de dezesseis metabólitos em urina
humana, a maioria dos quais são produtos de reação de hidroxilação (mono e
di), redução, metilação, desmetilação e epimerização (POZO et al., 2009;
SCHÄNZER, OPFERMANN & DONIKE, 1990; TUDELA, DEVENTER & VAN
EENOO, 2013; WANG et al., 2017). Todos os metabólitos de sibutramina e
estanozolol previamente identificados em urina humana também foram
produzidos pelo zebrafish (Capítulo I, p. 73-75). Para a sibutramina, além dos
metabólitos previamente descritos, foram identificados derivados dihidroxilados
de M1 e M2 (ANSELMO et al., 2017). No entanto, a resolução entre os sinais
cromatográficos dos derivados dihidroxilados de sibutramina não estava
adequada com o método analítico utilizado. Sendo assim, a próxima etapa do
trabalho foi desenvolver um método de LC-HRMS para detecção dos metabólitos
de sibutramina.
O método de extração dos metabólitos de sibutramina, bem como a
técnica de LC-HRMS para detecção dos metabólitos foram desenvolvidos em
urinas de excreção de sibutramina. Isto se deveu ao fato dos metabólitos
dihidroxilados, que apresentavam a pior resolução cromatográfica, serem
produzidos em baixas concentrações no modelo de ZWT. Primeiramente, foi
desenvolvido o método de extração dos metabólitos de sibutramina da urina. O
procedimento de preparo da amostra deve ser considerado cuidadosamente
visando eliminar, tanto quanto possível, os interferentes, ao mesmo tempo em
que se concentram os analitos. Além disso, a escolha do protocolo de preparo
da amostra tem um papel importante quando a ionização por ESI é utilizada,
para que se evite o efeito de supressão/aumento iônico (BYLDA et al., 2014;
SNOW, 2000).
No desenvolvimento do método de extração foram testadas extrações
líquido-líquido em três diferentes valores de pH (ácido, neutro e básico), além da
158
extração em fase sólida. Os resultados foram avaliados em relação a
recuperação do metabólito M1 para fins de comparação. Em todas as condições
foram observadas uma recuperação de M1 em torno de 70%, com exceção da
extração ácida (abaixo de 60%). As extrações líquido-líquido em pH alcalino e a
extração em fase sólida apresentaram os maiores rendimentos (Capítulo II, p.
98). Como a sibutramina possui pKa 10,2 (NIROGI et al., 2007), as extrações
realizadas em pH alcalino favorecem a partição da porção não ionizada nos
solventes orgânicos, aumentando a recuperação. A extração em fase sólida é
um dos métodos mais utilizados na preparação de amostras para injeção nos
sistemas cromatográficos acoplados ao espectrômetro de massas (BYLDA et al.,
2014; HENNION, 1999 & SNOW, 2000). Considerando a similaridade da taxa de
recuperação observada entre as extrações líquido-líquido em pH alcalino e a
extração em fase sólida, e o melhor resultado de limpeza da matriz tipicamente
observado com a fase sólida, além da economia de solventes orgânicos, este
método de extração foi selecionado para as extrações dos metabólitos de
sibutramina, seja da urina ou do modelo de ZWT.
Após a definição do método de extração, seguiu-se para o ajuste do
método cromatográfico. O metabólito de sibutramina dihidroxilado M1-diOH
resultava em múltiplos sinais cromatográficos, sem resolução adequada, no
método original de LC-HRMS, indicando a presença de vários isóbaros (Capítulo
II, p. 100). Considerando o alto número de parâmetros com probabilidade de
impactar a resolução cromatográfica, as abordagens de desenho experimental
(DoE) têm o potencial de reduzir o número de experimentos, custos e quantidade
de trabalho, alcançando os resultados desejados em menor tempo. No entanto,
a seleção de fatores críticos para os métodos cromatográficos é essencial para
a eficiência do processo de otimização por DoE (FERREIRA et al., 2018;
HIBBERT, 2012). Após ensaios preliminares as condições fixas selecionadas
foram coluna C18 Syncronis (2,1 x 50 mm, 1,7 μm); fase móvel A (água com
0,1% de ácido fórmico e 5 mM de formato de amónio) e B (metanol com 0,1% de
ácido fórmico); porcentagem inicial de gradiente de 15% B e volume de injeção
de 5μL. Por outro lado, os fatores críticos da cromatografia (que impactavam
diretamente na resolução) que foram identificados nos ensaios preliminares
foram o fluxo de fase móvel, a temperatura da coluna e o tempo do gradiente da
159
fase móvel (tempo de rampa), o que está de acordo com a literatura (BORGES
et al., 2013).
Em seguida, um DoE utilizando Box-Behnken (BB) de três fatores e três
níveis com cinco réplicas no ponto central foi utilizado para ajustar as condições
críticas visando melhorar a resolução entre os sinais cromatográficos de M1-
diOH. O BB já foi utilizado anteriormente para ajuste de métodos cromatográficos
(BORGES et al., 2013; HATAMBEYGI, ABEDI &TALEBI, 2011). Dois desenhos
BB foram utilizados para ajustar o método cromatográfico. O primeiro DoE
avaliou os níveis de fluxo 0,2, 0,3 e 0,4 mL min-1, temperaturas de 40, 50 e 60ºC
e tempos de rampa de 10, 15 e 20 minutos. A resposta avaliada foi em relação
a resolução cromatográfica entre os múltiplos sinais cromatográficos de M1-
diOH. A condição ótima obtida por esse DoE revelou a presença de outro sinal
cromatográfico que não havia sido identificado nos ensaios preliminares
(Capítulo II, p. 100). Sendo assim, foi necessário realizar um segundo DoE.
O segundo DoE foi realizado focando no aumento da resolução entre os
sinais cromatográficos recém identificados no primeiro DoE. Sendo assim, foram
utilizados níveis mais baixos para o fluxo da fase móvel (0,1, 0,15 e 0,2 mL min-
1) e maiores para o tempo de rampa (15, 20 e 25 minutos). Os níveis de
temperaturas de 40, 50 e 60ºC foram mantidos. A condição cromatográfica ótima
proposta, com a qual se conseguiu a melhor resolução entre os sinais
cromatográficos, foi obtida com um tempo de rampa de 25 minutos, vazão de
0,17 mL min-1 e temperatura de 50ºC (Capítulo II, p. 100). Essa condição foi
utilizada para a análise dos metabólitos de sibutramina em urina por LC-HRMS
e LC-MS/MS.
Os metabólitos urinários de sibutramina foram primeiramente investigados
por Thevis e colaboradores (2006) usando LC-MS onde os metabólitos mono
(M1) e bis N-desmetil (M2) foram identificados. Nos anos seguintes, a detecção
de abuso de sibutramina foi aperfeiçoada pela identificação de derivados
hidroxilados de M1 e M2 em urina humana (STRANO-ROSSI, COLAMONICI &
BOTRE, 2007; SARDELA et al., 2009). No presente trabalho foram identificados
em urina humana por LC-HRMS além dos metabólitos já descritos, derivados di-
hidroxilados (M1-diOH e M2-diOH) de M1 e M2, somente identificados
anteriormente em zebrafish (ANSELMO et al., 2017) e hepatócitos de ratos
160
(HAKALA et al., 2009). É interessante frisar que em hepatócitos de ratos apenas
M2-diOH foram observados. Metabólitos de m/z 310,15683 (M7) e m/z
296,14118 (M8) previamente descritos por Hakala e colaboradores (2009) em
hepatócitos de ratos também foram identificados em urina humana. Além disso,
derivados glicoconjugados de M1, M2, M1-OH e M2-OH também foram
observados nas amostras de urina aonde não foi realizada hidrólise. Para
elucidação estrutural dos metabólitos foi utilizada fragmentação por LC-MS/MS.
Os múltiplos sinais cromatográficos para o mesmo valor de m/z dos
metabólitos mono ou di-hidroxilados indicam que durante o metabolismo são
gerados múltiplos isômeros de posição, e que por isso, apresentam diferentes
tempos de retenção (Capítulo II, p. 103). Para os estudos de fragmentação, os
espectros de íons produto foram obtidos com energias de colisão de 20 eV e 30
eV. Para cada metabólito foi selecionada a energia de colisão apropriada para
se ter a presença do íon pai correspondendo a pelo menos 15% da intensidade
relativa do espectro de fragmentação. Os espectros de fragmentação possuem
fragmentos com massa exata e fórmula molecular teórica com erro menor que 4
ppm.
Os metabólitos desmetilados de sibutramina M1 e M2 apresentam os
fragmentos característicos m/z 125,01538, 139,03090, 153,04652, 97,10153 e
179,06221 (Capítulo II, p. 105), que estão de acordo com a literatura (THEVIS et
al., 2006). O fragmento m/z 125,01538 é um marcador da presença de
sibutramina e seus metabólitos, uma vez que conserva o anel aromático ligado
ao átomo de cloro característico dessas substâncias, estando presente em todos
os metabólitos analisados. Os metabólitos de m/z 282,16192 correspondem aos
derivados hidroxilados de M1 (M1-OH). Os fragmentos m/z 177,04660,
191,06219, 163,03093 e 233,10902 são característicos destes metabólitos
sendo encontrados nos quatro tempos de retenção nesta ordem de intensidade
e em todos os metabólitos (Capítulo II, p. 105), exceto no tempo de retenção
15,4 minutos. Por sua vez, o fragmento m/z 208,08867 quando vindo de um M1-
OH com o grupo hidroxila na cadeia alifática se torna mais estável porque a carga
positiva está no átomo de nitrogênio, e não no átomo de carbono. Este fragmento
é o mais intenso e presente apenas no tempo de retenção de 15,4 minutos,
sugerindo que este metabólito possui um grupo hidroxila na cadeia alifática, e os
161
demais no anel ciclobutano. Estes resultados estão de acordo com a literatura
que descreve os derivados M1 hidroxilados na cadeia alifática e no anel
ciclobutano (SARDELA et al., 2009).
Os metabólitos de m/z 268,14627 correspondem aos derivados
hidroxilados de M2 (M2-OH). Da mesma forma que para o M1-OH, são
observados quatro sinais cromatográficos indicando que o grupo hidroxila pode
entrar em diferentes posições. Os mesmos fragmentos de diagnóstico para M1-
OH são encontrados para M2-OH nos quatro tempos de retenção (Capítulo II, p.
105). Todos estes fragmentos podem ser gerados através da fragmentação da
molécula M2-OH possuindo hidroxilas na cadeia alifática ou no anel ciclobutano.
Contudo, o fragmento m/z 194,07314 com a carga positiva no átomo de
nitrogênio é o fragmento que se espera que seja mais estável do que no átomo
de carbono. Este fragmento é o mais intenso e apresenta-se apenas no tempo
de retenção de 16,1 minutos, sugerindo um grupo hidroxila na cadeia alifática, e
os demais no anel ciclobutano. Esses resultados também estão de acordo com
a literatura que descreve derivados de M2 hidroxilados na cadeia alifática e anel
de ciclobutano (SARDELA et al., 2009).
Os metabólitos com m/z 298,15683 correspondem aos derivados di-
hidroxilados de M1 (M1-diOH). Este metabólito foi descrito em urina humana pela
primeira vez nesse trabalho. Os cromatogramas indicam que são produzidos no
mínimo seis metabólitos com a massa exata de M1-diOH. Os fragmentos m/z
191,06216, 231,09334 e 163,03079 são os mais intensos nos espectros de
fragmentação dos metabólitos nos tempos de retenção 5,46; 7,69; 9,16 e 13,24
minutos (Capítulo II, p. 105). No entanto, para os metabólitos nos tempos de
retenção 8,20 e 10,42 minutos, os fragmentos m/z 224,08362, 206,07306 e
231,09334 são os mais intensos. Os fragmentos m/z 224,08362 e 206,07306
(gerado a partir da perda do grupo hidroxila no anel ciclobutano no fragmento
m/z 224,08362) são indicativos da presença de um grupo hidroxila no anel
ciclobutano, sendo o outro na cadeia alifática. Portanto, esta seria a possível
estrutura dos metabólitos nos tempos de retenção 8,20 e 10,42 minutos.
Segundo o software Mass Frontier 7.0, os fragmentos com m/z 191,06216 e
206,07306 não são formados com duas hidroxilas no anel ciclobutano. Assim,
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para os outros metabólitos (5,46; 7,69; 9,16 e 13,24 minutos), as duas hidroxilas
podem estar juntas na cadeia alifática.
Os metabólitos com m/z 284,14118 correspondem aos derivados di-
hidroxilados de M2 (M2-diOH). Assim como para M1-diOH, os cromatogramas
indicam que são produzidos no mínimo seis metabólitos com a massa exata de
M2-diOH. Os fragmentos m/z 191,06225, 231,09354 e 203,06206 são os mais
intensos nos espectros de fragmentação dos metabólitos nos tempos de
retenção 6,36, 7,93, 9,44, 9,89 e 13,75 minutos (Capítulo II, p. 105). Entretanto,
somente para o metabólito no tempo de retenção 11,54 minutos, os fragmentos
m/z 192,05742, 210,06799 e 231,09354 são os mais intensos, e os fragmentos
m/z 191,06225, 231,09354 e 203,06206 estão presentes em intensidades
menores. Os fragmentos m/z 191,06225 e 231,09354 são os mesmos
identificados em M1-diOH. Os fragmentos m/z 210,06799 e 192,05742 são
indicativos da presença de um grupo hidroxila no anel ciclobutano, sendo o outro
na cadeia alifática. Assim, esta seria a possível estrutura dos metabólitos no
tempo de retenção 11,54 minutos. O fragmento com m/z 192,05742 não é
formado com duas hidroxilas no anel ciclobutano de acordo com o software Mass
Frontier 7.0. Portanto, para os outros metabólitos (6,36, 7,93, 9,44, 9,89 e 13,75),
as duas hidroxilas podem estar juntas na cadeia alifática.
Além de serem descritos dois novos metabólitos de sibutramina em urina
humana, M1-diOH e M2-diOH, realizou-as a elucidação estrutural de suas
fórmulas estruturais baseada nos espectros de fragmentação. Metabólitos com
m/z 310,15683 (M7) e 296,14118 (M8) também foram identificados, seus
fragmentos descritos, porém não foi possível a elucidação inequívoca das suas
estruturas. No entanto, através das fórmulas moleculares e dos espectros de
fragmentação, acredita-se que M7 possam ser derivados di-
hidroxilados/monodesidrogenados de sibutramina, ou ainda derivados
acetilados de M2-OH. Da mesma forma, M8 pode ser gerado por carboxilação
de M1 ou através de derivados hidroxilados/desidrogenados de M1-OH.
A validação do método de LC-HRMS foi realizada para os principais
metabólitos identificados em urina humana: M1, M2 e seus respectivos derivados
mono e di-hidroxilados. Os parâmetros de repetibilidade, arraste e interferência
de matriz foram avaliados para os metabólitos M1 e M2, para os quais haviam
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padrões de referência. A especificidade e o limite de detecção foram avaliados
para todos os metabólitos. Todos os parâmetros avaliados no ensaio de
validação foram considerados adequados para o método desenvolvido, de
acordo com os parâmetros estabelecidos pela Norma Internacional para
Laboratórios Técnicos da WADA (ISL, 2016) e pelo Guia para a Validação de
Métodos Bioanalíticos do FDA (FDA, 2018) (Capítulo II, p. 110-114). Os
metabólitos descritos nesse trabalho podem ser úteis para a detecção da
dopagem de sibutramina no esporte.
Em seguida, o método desenvolvido de extração por fase sólida e de LC-
HRMS para analisar os metabólitos de sibutramina foi utilizado para avaliar o
metabolismo da sibutramina no ZWT. Para estabelecer as condições para os
experimentos do ZWT, foi utilizada uma otimização multivariada de acordo com
o desenho experimental de Dohelert (HIBBERT, 2012; FERREIRA et al., 2018).
O Dohelert foi escolhido para essa otimização por necessitar de menos
experimentos, em relação ao BB, e já ter sido utilizado anteriormente para
otimização de experimentos envolvendo outros organismos (SANTOUR et al.,
2001; SENA et al., 2012). As variáveis incluídas foram o número de peixes (6-
22), a dosagem de sibutramina (1-15 mg) e o tempo de duração do experimento
(1-7 dias). Os resultados foram analisados em dois grupos de substâncias, sendo
o grupo 1 referente aos metabólitos desmetilados, M1 e bis-dimetilados, M2, e o
grupo 2 referente aos derivados hidroxilados de M1 e M2 (M1-OH e M2-OH,
respectivamente). Essa divisão em grupos foi realizada para analisar as
diferentes fases do metabolismo e da cinética de formação dos metabólitos de
sibutramina.
Através do DoE foi observado que o número de peixes tem baixo impacto
na produção de metabólitos primários (Grupo 1), visto que o pico de
concentração desses metabólitos ocorre 120 horas após o início do experimento,
independentemente do sistema ZWT conter 4 ou 22 peixes (Capítulo III, p. 132).
As concentrações dos metabólitos do Grupo 1 diminuem ao longo do tempo, o
que pode ser explicado pela continuidade das vias de biotransformação, levando
aos metabólitos hidroxilados (SARDELA et al., 2009). Os metabólitos
hidroxilados foram mais extensivamente formados quando o número de peixes
no ZWT foi superior a 18. Além disso, os metabólitos hidroxilados foram
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observados em concentrações significativas após 120 horas, e seu aumento foi
continuamente observado após esse período. Portanto, 18 peixes e 168 horas
são os requisitos experimentais mínimos para obter todos os metabólitos da
sibutrmina em altas concentrações no ZWT. Uma vantagem do modelo de ZWT,
quando comparado a outros sistema in vivo, é a possibilidade da modulação do
experimento. Por exemplo, se os metabólitos alvo fossem os metabólitos
primários, o número de peixes deveria ser menor que 18 e o tempo menor que
120 horas para favorecer a geração deste antes de serem convertidos nos
derivados hidroxilados.
O aumento na dose de sibutramina aumentou a formação dos metabólitos
do grupo 1 e 2, ao longo do tempo (Capítulo III, p. 132). Além disso, neste
experimento observou-se que uma dose baixa do medicamento no ZWT resultou
em um perfil metabólico de sibutramina comparável aos humanos após
administração de dose única, onde a detectabilidade de metabólitos primários
diminui após alguns tempo e metabólitos hidroxilados são observados em maior
abundância (SARDELA et al., 2009). No entanto, com 15 mg de sibutramina no
ZWT, o perfil de metabólitos observado é semelhante ao de uso crônico por
humanos. Ou seja, os metabólitos N-desmetilados são observados durante toda
a duração do experimento, e estes atingem uma concentração estável e podem
ser detectados em conjunto com metabólitos hidroxilados. Além disso, deve-se
considerar que no modelo de ZWT o zebrafish excreta os metabólitos na água e
pode subsequentemente ingeri-los e metabolizá-los novamente. Para
sibutramina, 15 mg foi a dose escolhida como a melhor condição para um
experimento típico de ZWT que permite a detecção de todos os metabólitos
simultaneamente. Portanto, foram utilizados 18 peixes, 15 mg de sibutramina e
sete dias de experimento.
Em seguida, realizou-se a comparação do metabolismo de sibutramina
em humanos, camundongos e no zebrafish. O modelo in vivo mais utilizado como
alternativa para o estudo do metabolismo de xenobióticos em humanos é o
roedor (ANDES; CRAIG, 2002). No entanto, devido a variações interespécies na
expressão, atividade, e indução de enzimas CYP, modelos murinos apresentam
limitações para comparação com humanos (BOGAARDS et al., 2001). Sendo
assim, foram comparadas as concentrações de sibutramina, seus metabólitos
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desmetilados (M1 e M2) e os derivados hidroxilados desses metabólitos
desmetilados (M1-OH e M2-OH) nessas três espécies.
Embora todas as três espécies produzissem metabólitos correspondentes
aos derivados hidroxilados de M1 e M2, a razão entre os metabólitos foi diferente
(Capítulo III, p. 135). No entanto, na perspectiva do controle de dopagem, além
da presença dos metabólitos, a compreensão de qual metabólito é o mais
abundante, ou apresenta uma janela de detecção mais longa, é um objetivo
crítico para sua aplicabilidade (BADOUD et al., 2011). Embora as CYPs do
zebrafish mostrem diferenças interespécies em relação à atividade catalítica, o
modelo ZWT permite o ajuste do sistema, por modificações das condições do
modelo, como por exemplo o número de peixes e o tempo de coleta de água.
Por exemplo, para atingir o mesmo perfil metabólico observado em humanos
para M1-OH, foi utilizado um experimento com as condições de 168h, 10 peixes
e 8 mg de sibutramina (Capítulo III, p. 136). Esta capacidade de modular a
produção dos metabólitos através do DoE é uma possível vantagem do modelo
de ZWT em comparação com outros modelos in vivo (SARDELA et al., 2018).
Durante todo o experimento de ZWT observa-se uma grande
concentração de sibutramina nos aquários, ao contrário do que é observado em
urinas de humanos e camundongos (Capítulo III, p. 137). A alta concentração de
sibutramina é explicada pelo fato de que o fármaco foi administrado diretamente
ao ZWT, de tal forma que uma fração não absorvida pelo peixe permanece na
água. Para humanos, uma dose única de sibutramina foi administrada, sendo
esta rapidamente metabolizada em M1 e M2, e praticamente indetectável na
urina. O mesmo é observado para camundongos, embora, para esta espécie, foi
realizada a administração diária de sibutramina. Em humanos, há um pico de
concentração de M1 e M2 nas primeiras horas após a ingestão de sibutramina,
seguido por uma diminuição contínua após 24 horas até o dia 5. Em
camundongos e ZWT, as concentrações de M1 e M2 são menores que em
humanos, aumentando discretamente até o dia 5, com exceção do M1, que no
ZWT apresenta aumento considerável na concentração no quinto dia. Os
resultados obtidos demonstram que os derivados hidroxilados de M1 e M2
possuem comportamentos distintos entre as três espécies. Em humanos, as
concentrações máximas de M1-OH ocorrem às 24h e de M2-OH ocorrem antes
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das 24h; em camundongos e ZWT, a concentração cresce lentamente ao longo
do tempo, com o ZWT apresentando a menor quantidade desses metabólitos.
Estes resultados mostram a viabilidade de realizar estudos metabólicos usando
ZWT (SARDELA et al., 2018).
A próxima etapa do trabalho foi avaliar a correspondência entre a CYP2B6
humana e a CYP2Y do zebrafish. Em humanos, a CYP2B6 está diretamente
envolvida no metabolismo da sibutramina (BAE et al., 2008). O zebrafish
apresenta uma enzima ortóloga a CYP2B6, denominada CYP2Y (GOLDSTONE
et al., 2010). Assim, para identificar se a CYP2Y está envolvida na formação dos
metabólitos M1 e M2 em ZWT, utilizou-se a ticlopidina, um fármaco inibidor da
CYP2B6. Foi observada uma redução significativa na produção dos principais
metabólitos de sibutramina, tanto para os derivados demetilados quando para os
hidroxilados (Capítulo III, p. 139). Com base nesses dados é possível inferir que
há uma correlação enzimática entre a enzima humana e do zebrafish envolvidas
no metabolismo da sibutramina (SARDELA et al., 2018).
Após o estabelecimento do modelo de ZWT com a sibutramina, as
condições otimizadas foram aplicadas para outras substâncias utilizadas como
dopagem no esporte. As substâncias escolhidas visaram avaliar algumas
características específicas do modelo: estereoespecificidade (selegilina) e
metabolismo de substância com baixa estabilidade (hexarelina). Além disso,
avaliou-se o metabolismo do canabimimético JWH-073, substância que
apresentou divergências entre os metabólitos observado in vitro e em humanos.
A relevância da estereoespecificidade na toxicologia consiste no fato de
se permitir diferenciar entre a ingestão de substâncias a partir de medicamentos
do consumo de drogas de abuso (PARR et al., 2017; THEVIS et al., 2013). A
selegilina, em sua forma de enantiômero R é comercializada para o tratamento
de Parkinson (PALHAGEN et al., 2006). No entanto, a droga de abuso
denominada “speed” (metanfetamina na forma de cristal) é vendida no mercado
clandestino como mistura racêmica. Durante o metabolismo da selegilina, são
produzidos N-desmetil -selegilina (em baixa concentração), (R)(-)
metanfetamina, (R)(-) anfetamina e seus derivados p-hidroxi conjugados
(MAURER & KRAEMER, 1992; SHIN et al., 1997).
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No metabolismo da selegilina em humanos e ratos ocorre a preservação
do grupo enantiomérico (SHIN et al., 1997; SZOKO, KALÁSZ & MAGYAR, 1999).
No ZWT, cinco metabólitos urinários de selegilina e a droga inalterada foram
identificados por LC-HRMS: (R)-desmetilselegilina, (R)-metanfetamina, (R)-
anfetamina, (R)-p-hidroxi-anfetamina e (R)-p-hidroxi-metanfetamina, sendo a
última detectada em níveis de traços. Posteriormente, utilizou-se um reagente
de derivatização quiral (Mosher) para transformar os enantiômeros dos
metabólitos da selegilina nos seus diastereisomêros antes da análise por CG-
MS.
A selegilina é primariamente metabolizada por três vias distintas: N-
desalquilação, hidroxilação do carbono beta e hidroxilação do anel, sendo as
duas últimas as rotas metabólicas menores (SHIN et al., 1997). Os produtos de
hidroxilação de carbono beta, como efedrina, pseudoefedrina, norefedrina e
norpseudoefedrina, não foram encontrados no ZWT. O metabólito principal foi a
(R)-metanfetamina, seguida pela (R)-desmetil-selegilina, o que é semelhante à
excreção urinária humana (SHIN et al., 1997). Nenhuma racemização ou
transformação de inversão foi observada, e a N-desalquilação mostrou
estereosseletividade no ZWT (Capítulo III, p. 141). Este resultado revela a
possibilidade de se poder utilizar o zebrafish para análises de substâncias com
metabolismo estereoespecífico (SARDELA et al., 2018).
O metabolismo in vitro do JWH-073 produz uma grande variedade de
metabólitos não observados em estudos de metabolismo humano (GRIGORYEV
et al., 2011; ZHANG et al., 2006, MORAN et al., 2011; CHIMALAKONDA et al.,
2011). Os metabólitos urinários humanos atribuídos ao JWH-073 são
biotransformados pelo citocromo P450 CYP2C9 promovendo a mono-
hidroxilação do grupo indol, e pela CYP3A4 no sítio alquila, seguido pela co-
carboxilação da cadeia alquílica pela UDP-glucuronosiltransferase (MORAN et
al., 2011; CHIMALAKONDA et al., 2011). No ZWT, apenas dois metabólitos
JWH-073 mono-hidroxilados foram observados nas primeiras 24 horas, ambos
em cadeias alquílicas. O metabólito JWH-073-N-3-hidroxibutil apresentou a
maior intensidade após 24 horas de administração no ZWT, sendo seguido por
uma diminuição na concentração ao longo dos próximos 7 dias (Capítulo III, p.
144), principalmente devido à reabsorção contínua deste metabólito pelos
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peixes, seguida por um aumento na concentração dos metabólitos diidrodióis. O
metabolito ácido da co-carboxilação foi o metabolito majoritário presente no ZWT
no final do experimento.
Os derivados metabólicos diidrodióis foram relatados como metabólitos
principais em um estudo in vitro, enquanto não são relatados em seres humanos
(WINTERMEYER et al., 2010). Portanto, como uma vantagem do ZWT, os
experimentos podem ser modulados para que terminem antes de 7 dias, para
que se obtenha um perfil semelhante ao observado em urina humana. Outro fator
interessante a ser observar é que não há ortólogos da CYP2C9 em zebrafish
(GOLDSTONE et al., 2010), sendo que essa enzima é responsável pela mono-
hidroxilação do grupo indol em humanos. Assim, essa pode ser a explicação
para a ausência da mono-hidroxilação no grupo indol em ZWT. No entanto, os
metabólitos primários observados no ZWT são os mesmos que os observados
na urina humana: JWH-073-N-3-hidroxibutil, JWH-073-N-4-hidroxibutil e os
respectivos metabólitos carboxilados (SARDELA et al., 2018).
A hexarelina possui baixa detectabilidade para o fármaco intacto, quando
se utiliza análises tradicionais de espectrometria de massa, pois possui meia-
vida curta de apenas 2,5h (KOJIMA et al., 2001). Em organismos superiores, a
metabolização da hexarelina é realizada sistematicamente, por meio de enzimas
como exopeptidase (amino ou carboxi), amidase ou endopeptidase, que atuam
clivando os peptídeos e gerando peptídeos cada vez menores (HOWARD et al.,
1996). Um metabólito já descrito desse peptídeo foi identificado como sendo uma
hexarelina desamidada no resíduo de lisina (THOMAS et al., 2012).
Além da meia-vida curta, a hexarelina apresenta instabilidade quando
armazenada por seis meses a -20°C (SEMENISTAYA et al., 2015). Além disso,
também foi descrita a influência da adsorção inespecífica de hexarelina e outros
peptídeos sobre superfícies de recipientes de plástico ou vidro, que são
geralmente utilizados em procedimentos clínicos e experimentais (GOEBEL‐
STENGEL et al., 2011; SEMENISTAYA et al., 2015. Portanto, a avaliação da
hexarelina pelo modelo ZWT é um desafio por causa da temperatura, do tempo
de exposição à droga e a possibilidade de interação com o vidro do aquário. No
ZWT, toda a hexarelina foi metabolizada nas primeiras 12 horas do experimento,
que foi mais rápida do que o observado para as outras substâncias, seguido pelo
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aumento de todos os metabólitos, incluindo hexarelina (1-3), ácido livre de
hexarelina (4-6) e ácido livre de hexarelina (2-6) (Capítulo III, p. 147).
As atividades de exopeptidase (amino ou carboxi), amidase ou
endopeptidase foram observadas no aquário sem peixes e com hexarelina,
provavelmente porque um ambiente constituído por organismos unicelulares
presentes no aquário foi capaz de promover tais transformações. Apenas os
metabólitos ácido livre de hexarelina (4-6), hexarelina (1-3) e ácido livre de
hexarelina (2-6) foram encontrados exclusivamente no aquário contendo
zebrafish. Portanto, o ZWT pode ser usado como um modelo para investigar o
metabolismo peptídico. No entanto, a instabilidade do fármaco no aquário parece
ser um desafio para a investigação do metabolismo do peptídeo e são
necessárias experiências adicionais para otimizar, por DoE, a necessidade de
duração do processo (SARDELA et al., 2018).
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7. CONCLUSÕES GERAIS
Nos últimos 30 anos o zebrafish vem sendo utilizado para prever o
metabolismo de xenobióticos. Nos primeiros anos, o enfoque principal era na
toxicologia ambiental e no possível impacto dos metabólitos gerados no aumento
da toxicidade dos contaminantes. Atualmente, vários estudos avaliaram o
metabolismo de substâncias visando estabelecer o zebrafish como um modelo
para a avaliação de toxicidade e no desenvolvimento de novos medicamentos.
No melhor do nosso conhecimento, este é o primeiro trabalho a utilizar o
zebrafish na busca de alvos analíticos para o controle antidopagem.
Primeiramente, demonstrou-se que o zebrafish utilizado no modelo de
ZWT pode absorver, oxidar e excretar vários metabólitos de fase I e II da
sibutramina e do estanozolol semelhantes aos identificados em humanos. Além
disso, uma vantagem do modelo ZWT, quando comparado a outros modelos in
vivo, consiste no fato dos metabólitos poderem ser isolados da água do aquário
(matriz mais limpa que outras matrizes biológicas) com o objetivo de gerar
materiais de referência para o controle antidopagem.
Embora diferentes condições de extração líquido-líquido tenham sido
avaliadas para análise dos metabólitos de sibutramina em urina, a extração em
fase sólida foi a mais abrangente e adequada para os diferentes metabólitos
alvos da sibutramina. A abordagem por DoE provou ser uma maneira rápida,
econômica e eficiente de otimizar as condições experimentais para otimizar a
resolução por LC dos metabólitos isóbaros de sibutramina. O método de LC-
HRMS desenvolvido e validado mostrou-se eficaz na detecção dos metabólitos
urinários da sibutramina. Além dos metabólitos urinários previamente descritos,
novos metabólitos de sibutramina foram identificados. Além disso, fragmentos
específicos analisados por LC-HRMS auxiliaram na elucidação estrutural dos
metabólitos mono e diidroxilados da sibutramina. Os metabólitos identificados
podem ser novos alvos analíticos para a detecção de abuso de sibutramina no
esporte.
Neste trabalho, avaliou-se os méritos, limitações e uso apropriado do
modelo in vivo de ZWT para o estudo do metabolismo de xenobióticos. A
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otimização das condições do ZWT foi realizada por DoE para a sibutramina. O
número de peixes, a concentração de xenobiótico e o tempo de experimento
foram considerados as principais variáveis para as condições do ZWT. O número
de peixes teve um baixo impacto na produção dos metabólitos primários de
sibutramina (demetilados) e os metabólitos hidroxilados foram mais
extensivamente formada sob condições ZWT com 18 espécimes. Além disso, os
metabólitos hidroxilados foram observados em concentrações significativas
depois de 120 horas. Portanto, 18 peixes e 168 horas são os requisitos
experimentais mínimos para obter todos os metabolitos sibutramina em altas
concentrações no ZWT.
As comparações interespécies foram realizadas em relação ao
metabolismo de sibutramina em humanos, camundongos e ZWT. Todas as três
espécies produziram os mesmos metabólitos hidroxilados, mas com padrões
específicos de hidroxilação, demonstrada pela diferente razão entre os
metabólitos produzidos. No entanto, as condições de ZWT são facilmente
moduladas a partir dos dados de DoE, o que é outra vantagem do modelo
quando comparado a outros modelos in vivo. Portanto, essa abordagem pode
ser utilizada para calibrar as condições do ZWT para investigar novas
substâncias obtendo resultados mais semelhantes ao metabolismo humano.
A contribuição da isoenzima ortóloga a CYP2B6 em zebrafish (CYP2Y) foi
avaliada após a inibição pela ticlopidina. Sendo assim, demonstrou-se uma
correlação enzimática entre a enzima humana e do zebrafish envolvidas no
metabolismo da sibutramina. A investigação de estereoespecificidade do
metabolismo do ZWT utilizando a selegilina como modelo demonstrou que os
cinco metabolitos urinários de selegilina e a droga inalterada foram identificados,
e o perfil de excreção de metanfetamina e desmetilselegilina foi semelhante ao
humano. E, como observado em humanos, durante metabolismo em ZWT a
forma levógira (l) foi preservada, ou seja, nenhuma racemização ou
transformação de inversão foi observada, e a N-dealquilação mostrou
estereosseletividade. Os resultados levantam a possibilidade de aplicar o
modelo ZWT às investigações onde a definição de alvo requer
estereoespecificidade.
173
O JWH-073 foi aplicado como modelo para avaliar a biotransformação de
cannabimiméticos no ZWT. Apenas dois metabólitos JWH-073 mono-
hidroxilados foram observados, ambos em cadeias alquílicas, e nos últimos dias
dos experimentos, o metabólito ácido de carboxilação foi o produto presente
majoritariamente. A mono-hidroxilação no grupo indol descrita em humanos não
foi observada no ZWT, possivelmente em decorrência da ausência de CYP
ortóloga ao humano responsável por essa via metabólitca em zebrafish.
O peptídeo hexarelina foi utilizado como modelo para avaliar desafios
para realizar experimentos ZWT para substâncias com baixa estabilidade em um
ambiente oxidativo. Como resultado, a hexarelina exibiu uma metabolização
rápida em ZWT, com meia-vida inferior a 12 horas. Todos os metabólitos
descritos para humanos foram observados no ZWT, incluindo hexarelina (1–3),
ácido livre de hexarelina (4–6) e ácido livre de hexarelina (2-6) livre. No entanto,
alguns deles também foram observados no aquário com hexarelina, mas sem
peixes, e provavelmente uma microbiota natural foi capaz de promover tais
transformações. Portanto, o ZWT pode ser utilizado como um modelo para
investigar o metabolismo de diversas substâncias.
174
175
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ANEXOS
ANEXO A - Autorização da detentora dos direitos autorais do artigo
“Zebrafish (Danio rerio): A valuable tool for predicting the metabolism of
xenobiotics in humans?˜
180
ANEXO B - Autorização da detentora dos direitos autorais do artigo “Is
zebrafish (Danio rerio) a tool for human-like metabolism study?”
181
ANEXO C - Autorização da detentora dos direitos autorais do artigo “Zebrafish
(Danio rerio) water tank model for the investigation of drug metabolism: Progress,
outlook, and challenges.”