research article ˘ ˇ ˇˆ an impurity in tenofovir ... · potassium dihydrogen orthophosphate,...

C

urre

nt P

harm

aceu

tical

Ana

lysi

s��

��

��

��

Jinqi Zhenga,b

, Haihong Huc, Lin Liu

a, Yuefeng Rao

a, Yan Lou

a* and Su Zeng

c

aThe First Affiliated Hospital, College of Medicine, Zhejiang University, 79 QingChun Road, Hangzhou, Zhejiang

310000, People’s Republic of China; bZhejiang Institute for Food and Drug Control, Hangzhou, 310004, People’s Re-

public of China; cLaboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of

Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058,

People’s Republic of China

A R T I C L E H I S T O R Y

Received: December 11, 2015

Revised: March 16, 2016 Accepted: March 21, 2016

DOI:

10.2174/15734129126661605241218

37

Abstract: Background: Tenofovir disoproxil fumarate (TDF) is an oral prodrug and a nucleotide analog

used for treating human immunodeficiency virus (HIV) and hepatitis B virus (HBV). A growing subset of

TDF-treated HIV(+) individuals experienced drug-associated acute renal failure, suggesting drug-related

nephrotoxicity. Impurities in pharmaceutical drugs generally present a risk, and no obvious benefit.

Method: In an effort to determine whether the impurities also contribute to the nephrotoxicity, seven im-

purities were isolated and identified by using nuclear magnetic resonance (NMR) and mass spectra (MS).

Human renal organic anion transporter 1 (hOAT1) localized in the kidney was the major transporter of

tenofovir and presumably mediates its accumulation in the renal proximal tubules. On the basis of the

Guidance for Industry document published by the United States Food and Drug Administration (U. S.

FDA), we conducted an uptake assay and performed inhibition analysis using the Madin-Darby canine

kidney cells stably expressing human OAT1 (MDCK-hOAT1), before analyzing our data by liquid chro-

matography tandem mass spectrometry (LC-MS/MS).

Results: The uptake of the newly detected impurity RS5 was significantly higher than that of TDF. The 3-

(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that RS5 was more

cytotoxic than TDF. The structure-affinity relationships indicated that 9-methyl functioned as a detoxifi-

cation group.

Conclusion: Our findings provide useful information for understanding the structure and toxicity of TDF

and its impurities, and for assessing the clinical applications of TDF.

Keywords: Tenofovir disoproxil fumarate, impurity, NMR, cytotoxicity, MDCK-hOAT1, LC-MS/MS.

1. INTRODUCTION

TDF is fumaric acid salt of the bis-isopropoxy carbony-loxy methyl ester derivative of tenofovir. It is a nucleotide (nucleoside monophosphate) analogue exhibiting pharma-ceutical activity against retroviruses, including HIV-1, HIV-2, and hepadnaviruses. Numerous case reports have de-scribed severe renal tubular toxicity associated with TDF exposure [1-4]. However, the mechanism of TDF nephrotox-icity remains elusive. From the absorption and metabolism standpoint, nucleosides are regulated in biological systems via transport and metabolism in the cellular compartments. Transporter-mediated systems have been considered to play major roles in tubular drug uptake [5]. OAT1 plays an im-portant role in the transepithelial movement of small nega-tively charged molecules from the systemic circulation into the tubular lumen [6]. An active renal transport of acyclic

*Address correspondence to this author at The First Affiliated hospital,

College of Medicine, Zhejiang University, 79 QingChun Road, Hangzhou,

Zhejiang 310000, People’s Republic of China; Tel: +86 571 8726675; Fax: +86 571 87236675; E-mail: [email protected]

nucleotides via OAT1 could promote the dose-dependent accumulation of these molecules in the proximal tubule cells, according to previous studies [7]. The disruption of OAT1 activity prevents the renal proximal tubular toxicity of teno-fovir (the active form of TDF) in vivo [7].

Impurities are usually present in pharmaceutical drugs or drug products. Process-related impurities are not completely removed during the purification process. They are also formed owing to the degradation of the drug substances to-wards the end of the product’s shelf-life. Unlike the active therapeutic compound, the impurities often produce un-wanted side effects and present a risk, with no obvious bene-fits [8, 9]. The International Conference on Harmonisation (ICH) (documents Q3A(R2), Q3B(R2), and Q3C(R4)) pro-vides recommendations for the identification, toxicological qualification, and derivation of allowable limits for drug substance impurities, drug product degradants, and solvents, respectively [10-12]. Therefore, a structural elucidation and subsequent toxicological evaluation of these impurities is necessary for the quality assurance and safety assessment of various drugs.

1875-676X/17 $58.00+.00 © 2017 Bentham Science Publishers

Send Orders for Reprints to [email protected] 232

Current Pharmaceutical Analysis, 2017, 13, 232-240

RESEARCH ARTICLE

An Impurity in Tenofovir Disoproxil Fumarate Exhibits High Human Organic Anion Transporter 1 Dependent

Cytotoxicity

An Impurity Exhibits High OAT1 Dependent Cytotoxicity Current Pharmaceutical Analysis, 2017, Vol. 13, No. 3 233

A previously conducted specificity test showed that there was no interference from the excipients commonly found in the commercial pharmaceutical formulations of TDF [13]. A previous study reported that TDF was very sensitive to vari-ous stress conditions, including acidic, alkaline, and oxida-tive environments, and that it readily degrades into tenofovir monoester [14]. Although twenty-one impurities have been detected in the commercial TDF samples, only three have been already identified so far [15]. They have named as monoesters tenofovir and fumarate, respectively.

However, information pertaining to the cytotoxic effects

of the known impurities of TDF is limited, and very little

information is available on the newly detected impurity. In this study, we have investigated the interaction of TDF and

its impurities with hOAT1, to assess the potential for its ac-

tive accumulation in the proximal tubules. Firstly, the impu-rities were isolated and characterized with MS and NMR.

The nephro-cytotoxicity of these impurities was then as-

sessed using the MTT assay. Finally, in accordance with the guidelines stipulated by U.S. FDA’s Drug Interaction Stud-

ies [16], the OAT-mediated cytotoxicity of TDF and its im-

purities were evaluated by conducting uptake and inhibition studies with MDCK-hOAT1 in vitro.

2. MATERIAL AND METHODS

2.1. Materials

TDF sample (drug substance) and its impurities were

synthesized by the Heze Pharmaceutical Technology (Hangzhou, China). Probenecid (99.5%) was purchased from

National Institutes for Food and Drug Control (Beijing,

China). G418, penicillin, streptomycin, fetal bovine serum (FBS), trypsin, Dulbecco'smodified Eagle's medium

(DMEM) and RPMI 1640 medium were purchased from

GIBCO (Invitrogen Life Technologies, USA). Dimethylsul-phoxide-d6 (DMSO-d6), tetrahydrofuran and 6-

carboxyfluorescein (6-CFL) were obtained from Sigma–

Aldrich (St. Louis, MO, USA). MTT was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). BCA protein

assay kit was bought from Beyotime Institute of Biotechnol-

ogy (Jiangsu, China). HPLC grade acetonitrile and methanol, potassium dihydrogen orthophosphate, ammonium acetate,

hydrochloric acid, sodium hydroxide and hydrogen peroxide

were all of AR grade, procured from Merck (India) Ltd. A Milli-Q water system (Millipore, Bedford, MA, USA) was

used to prepare ultrapure water. The thermal degradation

study was performed using a constant temperature bath (Prima, England) capable of controlling the temperature with

in +2°C. Photo-degradation was carried out in a SHH-

100GD Drug Illumintion Test Chamber (Chongqing Yong-sheng Laboratory Instrument Works Co., China. MDCK-

MOCK and MDCK-hOAT1 cells grew in DMEM containing

10% FBS, antibiotics, and 350 μg/ml geneticin. All other chemicals were purchased from commercial sources and

were of analytical grade.

2.2. HPLC Instrumentation and Operating Conditions

An Agilent 1260 HPLC (Palo Alto, California, USA) equipped with 1260 VWD detector was used. The separation was performed using a Zorbax Eclipse Plus Phenyl-Hexyl,

4.6 100 mm, 3.5 μm analytical column (Palo Alto, Califor-nia, USA). The mobile phase was composed of an ammo-nium dihydrogen phosphate buffer (2.3g/L) filtered through a 0.45 μm polypropylene membrane (GHP, Pall Gelman) and ultrasonically degassed for 15 min. A gradient with am-monium dihydrogen phosphate buffer (A) and methanol-tetrahydrofuran-water (45:30:25) (B) was programmed. Ini-tially, mobile phase B maintains at 5%, increases to 40% in 10 min and hold at 40% for 5min, then progressing linearly to 70% over 15 min and hold at 70% for 2 min before falling back down to 5% in 5min. Total runtime was 37 min. Mobile phase was delivered at a flow rate of 1.0 mL/min and the injection volume was 50 L. The column temperature was maintained at 8°C and the detection was monitored at a wavelength of 260 nm.

2.3. LC–MS/MS Instrumentation and Chromatographic Conditions

The concentrations of TDF and its impurities in the up-take samples were determined by LC–MS/MS. An API4000 triple quadrupole mass spectrometer (AB Sciex, Ontario, Canada) with electrospray ionization (ESI) coupled to an Agilent 1200 series LC system (Agilent Technologies, Inc., USA) with Agilent eclips XDB C18 column (3.5 μm, 3.0

100 mm) was applied. The mobile phase consisted by 70% methanol and 30% water and the flow rate was set at 0.5 mL/min. The turbo ion spray interface was operated in nega-tive ion mode with ion spray voltage, -4500 V; temperature, 450°C; and dwell time, 200 ms. Collision gas (CAD), curtain gas (CUR), ion source gas1 (GS1) and ion source gas2 (GS2) were 8, 20, 70, and 40 psi, respectively.

2.4. Forced Degradation Studies

The drug substance was subjected to forced degradation

under acidic, basic and neutral conditions in 1 M hydrochlo-

ric acid, 0.03 M sodium hydroxide and aqueous 30% hydro-gen peroxide for 8 h, 12 h, and 24 h, respectively. Before

injection into the LC, the solutions were neutralized with

NaOH or HCl solutions, respectively. For thermal stress, sample of drug substance was placed in a controlled tem-

perature oven at 80°C for 12 h. For photolytic stress, the

sample was exposed to photolyte of 1.0 104 lx for 120 h.

After the exposure to the above stress conditions, solutions

of these samples were prepared by dissolving the samples in

diluent and then diluted to the desired concentration analysed by HPLC. For all degradation studies in solution, the drug

concentration used was 1.0 mg/mL.

2.5. Impurities Isolation

Impurities present in the crude samples at about 0.13–

0.5% level by area normalization. RS1 was isolated from degradation products and the other six impurities were iso-

lated during the process of synthesis of TDF. The reaction

scheme for the synthesis of TDF was as shown in the previ-ous report [17]. Fractions of >95% were pooled together,

concentrated in vacuum to remove acetonitrile. Chroma-

tographic purities of impurities had been determined by the HPLC method described and the purities were 94.4% (RS1),

94.8% (RS2), 93.9% (RS3), 97.0% (RS4), 96.5% (RS5),

96.4% (RS6) and 97.0% (RS7), respectively.

234 Current Pharmaceutical Analysis, 2017, Vol. 13, No. 3 Zheng et al.

2.6. Impurity Characterization

NMR experiments (1D and 2D) were performed using a Bruker ARX 300 spectrometer, using DMSO-d6 as solvent and tetramethylsilane (TMS) as internal standard.

1H NMR

measurements were carried out at 300 MHz, while 13

C NMR experiments were performed at 75 MHz.

2.7. Cell Culture

MDCK-hOAT1 cells stably expressed OAT1 were con-structed in our lab (18). MDCK cells were transfected with empty vector pcDNA3.1 (+) as mock cells. The activity of hOAT1 in the MDCK-hOAT1 cells was validated by model substrates, 6-CFL (18). Cells were grown at 37°C, 90% rela-tive humidity, 5% CO2 atmosphere in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum), 100 units/ml penicillin, 100 μg/mL streptomycin, and 350 μg/mL G418. The cells were sub-cultured after reaching 90% confluence.

2.8. Cytotoxicity by MTT Assay

The MTT assay was performed as described by Mos-mann [18] with some modifications. Briefly, after incuba-tion, MOCK-MDCK cells were washed once with 37°C PBS and then added 0.2 mL serum-free medium containing 0.5% MTT to each well. The plates were maintained for 4 h at 37°C in a humidified 5% CO2 atmosphere. After this time the medium was removed and 0.2 mL of DMSO was added to each well to solubilize the formazan formed. The plates were shaken gently for 10 min and the absorbance was measured at 490 nm. The absorbance of treated cells was compared with the absorbance of the controls, which cells were exposed only to the vehicle and were considered as 100% viability value. MTT experiments were performed at least three times using triplicates for each condition.

2.9. Cellular Uptake Assays

MDCK-hOAT1 and mock cells were seeded in 24-well plates coated with polydlysine (Costar Corning Inc., NY, USA) at a destiny of 1 10

5/well. After incubating for 48 h,

the uptake studies were performed as the method reported with minor modifications [19]. Briefly, Studies were con-ducted at 37°C, 50 rpm in an orbital shaker. The cells were pre-incubated with Hank’s balanced salt solution (HBSS, with 25 mM HEPES) for 10 min, and then HBSS containing impurities (10–500 μM) was added to initiate the uptake. The uptake of TDF and its impurities in hOAT1- and mock cells were measured in a time-dependent experiment, at the incubation times of 2, 5, 10 and 20 min; in a concentration-dependent experiment (uptake time: 10 min), at TDF and its impurities concentrations of 10, 50, 100, 200, 300 and 500 μM. At the designated times, uptake was terminated by washing the cells three times with chilled buffer after remov-ing the incubation buffer and lysed with 200 μL 0.1% so-dium dodecyl sulfonate (SDS). Then cell lysate was precipi-tated with 3-fold volume of acetonitrile (containing internal standard). The mixture was vortexed and centrifuged at 13,000 rpm for 10 min.

The impurities in the cells were quantified with LC–MS/MS and normalized to the total protein content in the

lysates using a bicinchoninic acid protein assay kit with bo-vine serum albumin as a standard. Uptake kinetics of TDF and its impurities were investigated at the concentration range from 10 to 500 μM in MDCK-hOAT1 cells compared with mock cells. All experiments were performed in tripli-cate.

2.10. Inhibition Assay

Concentration-dependent inhibition experiments were conducted by determining the inhibitory effect of TDF and its impurities to 6-CFL, a classic substrate, and uptake in MDCK-hOAT1 cells compared with mock cells. The cells were exposed to 6-CFL of 4 M in the absence or presence of TDF and its impurities or probenecid (1 mM, positive control) at 37°C for 4 min in the HBSS. After incubation, the cells were washed three times with ice-cold PBS, lysed and analyzed by SpectraMax M2 microplate reader (Molecular Devices Corporation Sunnyvale, CA, USA). The excitation and emission wavelength of 6-CFL were set at 480 nm and 525 nm, respectively. The uptake in presence of inhibitors was expressed as the percentage of the vehicle control (% of control).

2.11. Statistical Analysis

Data are expressed as mean + SD derived from at least three independent studies. Michaelis constants were esti-mated by fitting to the Michaelis–Menten equation V = Vmax·S/(Km+ S), where Vmax is the maximum uptake velocity, S is the substrate concentration and Km is the substrate con-centration resulting in half-maximal uptake rate (Graphpad Prism version 5, San Diego, CA, USA). Unpaired two-side Student’s t-test was applied for comparisons of two groups. P values < 0.05 were considered statistically significant.

3. RESULTS

3.1. Detection of Impurity

All peaks of the impurities were well resolved from TDF peak and the representative chromatogram was shown in Fig. (1).

3.2. Formation of Impurities

The degradation samples and a stability batch of TDF drug product were analyzed by HPLC. RS1 (0.5%), RS2 (0.07%), RS3 (0.02%), RS4 (0.04%), RS5 (0.06%), RS6 (0.02%), and RS7 (0.08%) were found in TDF drug sub-stance. TDF was very sensitive to various stress conditions and readily degraded into tenofovir monoester (RS1). Under photolysis, oxidation and thermal stress conditions, up to 0.9%, 2.6%, and 2.5% of RS1 were formed, respectively. Under base stressed conditions, RS1 increased to 10.9%, while acid stressed degradation up to 19.9%. Hence the acid stressed degradation route was chosen to enrich the impurity. RS1 was the degradation product and RS2 to RS7 were process impurities based on their trend Fig. (2). RS2 origi-nated from the condensation of pinacolyl methylphosphonic acid (PMPA) with chloromethyl n-propyl carbonate in the preparation of tenofovir disoproxil (TD), in which chloro-methyl n-propyl carbonate presented as an impurity in the raw material chloromethyl isopropyl carbonate. RS3


Fig. (1). Typical HPLC chromatogram of TDF drug substance spiked with impurities at specification level.

N

N

N

NH2N

OH

CH3H2O-

N

N

N

NH2N

CH3

RS3

N

N

NH2

N

N

O P

O

O O O

OO

O O CH3

O CH3

CH3

CH3

HOOCCOOH

1

23

45

6

78

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

.

TDF

9

degradation N

N

NH2

N

N

O

CH3

P

OH

O O O

OO CH3

CH3

1

2

6

3

45

78

9

10

16

11

12

13

14

15

RS1

6

2 13

4

57

8

9

N N

N

N

H2NO

H3C

P

HO

OH

O

Cl O O

OPMPA

Cl O

O

HOOCCOOH

NN

N

NH2N

O

CH3

P

O

OO

O

O

O

O

O

O

HOOCCOOH

.

RS2

12

6

3

4

5 7

8

9

1011

13 16

17

18

12

19

20

15

21

22

23

24

CH2O

RS7

1

21

62

3

4

579

8

1011

1214

16

19

20

13

15

17

18

NN

N

NNHO

H3C

P

O

OO

OO

O

O

O

O

NN

NN

NH

O

H3C P

O

O

O

O

O

O

O

O O

1'

2'

3'

4'

5'

6'

7'

8'

9' 10'11'

12'13'

14'15'

16'

17'

18'19'

20'

Cl O O

O

Cl O

O

HOOCCOOH

NN

N

NH2N

O

CH3

P

O

OO

O

O

O

O

O

O

HOOCCOOH

.

RS4

1

2

6

3

4

5 7

8

9

1011

13 16

17

12

19

20

15

21

22

23

24

Cl O O

O

Cl O O

O

HOOCCOOH

NN

N

NH2N

O

CH3

P

O

OO

O

O

CH3

O

O

O

O

HOOCCOOH

.

RS6

12

6

3

4

5 7

8

9

1011

13 16

12

19

20

15

21

22

23

24

N

NNH

N

NH2

N

N

N

NH2N

OH

Cl O O

O

Adenine

OH

HOTsO P

O

OEt

OEt

HOOCCOOH

NN

N

NH2N

O P

O

OO

O

O

O

O

O

O

HOOCCOOH

.

RS5

12

6

3

4

57

8

1011

13 16

17

12

19

20

15

21

22

23

24

18

condensation

Fig. (2). Formation of impurities during degradation or synthesis of TDF.

��

��

��

��

��

�

�

� ��

� ��

� ��

��

��

��

��

� ��

��

��

��

��

��

��

� ��

��


Table 1. NMR spectral parameters of RS5 (620a).

Position 1H

13 C HMBC

1 8.10(s,1H) 141.1 C-2; C-5

2 118.5

3 155.7

4 8.18(s,1H) 152.1 C3; C5

5 149.4

6 6.65(s,2H)

7 4.35(m,2H) 45.1 C1; C5; C8

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

3.92(m,2H)

4.03(m,2H)

5.57(m,2H)

5.57(m,2H)

4.84(m,1H)

4.84(m,1H)

1.24(d, 3H)

1.24(d, 3H)

1.24(d, 3H)

1.24(d, 3H)

7.32(s, 1H)

7.32(s, 1H)

70.5

64.6

84.1

84.2

152.5

152.5

72.8

72.8

21.2

21.2

21.2

21.2

166.0

134.0

134.0

166.0

C5; C7

C8

C13

C14

C13;C17;C18;

C14;C19;C20

C15

C15

C16

C16

C21

C24

Recorded in DMSO-d6. in ppm. HMBC connectivity between the proton indicated in the first column and carbons indicated in the last column. a: protonated molecular ion of RS5. originated from the dehydration of R-9-(2-hydroxypropyl) adenine during the synthesis. The formation of RS4 had the similar mechanism with those of RS2. Briefly, except chloromethyl n-propyl carbonate, chloromethyl ethyl car-bonate was also as the impurity existed in the raw material chloromethyl isopropyl carbonate. RS4 originated from the condensation of PMPA with chloromethyl ethyl carbonate in the preparation of TD. RS5 formed via the impurity ethylene alcohol presented in the R-propylene carbonate and con-densed with adenine, then the product R-9-hydroxyethyl adenine underwent the condensation with the tosylated hy-droxymethyl phosponate diester followed by hydrolysis. RS6 was formed via the similar pathways as RS2 and RS4, which all due to the other solvent impurities during the synthesis of chloromethyl isopropyl carbonate. The difference was that the formation of RS6 was due to the methanol, and the corre-sponding intermediate is chloromethyl methyl carbonate. RS7 originated from the condensation of formaldehyde with two molecules TD into dimers during the synthesis.

3.3. Structural Elucidation

Based on the mass values and NMR chemical shift data, the structures of the impurities were confirmed as given in

Fig. (2). The MS and NMR chemical shift values of the new impurity RS5 are shown in the Table 1. The values of TDF and the known impurities are presented in Table 1s and 2s (supporting information).

3.4. Quantitation of TDF and its Impurities via LC-

MS/MS

The MRM transitions (Q1 and Q3), declustering potential

(DP), collision energy (CE) and collision exit potential

(CXP) of analyties were as follows: m/z 634.3>115.1, -30

(DP), -17 (CE), and -10 (CXP) for TDF, m/z 620.5>115.1, -

40 (DP), -14 (CE), and -9 (CXP) for RS4, m/z 620.5>115.1,

-30 (DP), -13 (CE), and -6 (CXP) for RS5, and m/z

606.5>115.0, -40 (DP), -13 (CE), and -6 (CXP) for RS6, m/z

402.2>372.1, -100 (DP), -17 (CE), and -12 (CXP) for RS1 as the internal standard Fig. (3).

The method was fully validated with respect to linear

calibration, accuracy, precision and recovery. The limit of

quantifications (LOQ) and the linear calibration ranges were

2.3 ng/mL and 2.3-455.8 ng/mL for TDF, 2.9 ng/mL and

2.9-592.5 ng/mL for RS4, 2.8 ng/mL and 2.6-520 ng/mL for

RS6, respectively. The inter-day and intra-day RSDs of TDF


and its impurities were lower than 15%. The intra-day accu-

racy ranged from 95.8% to 105.1% for TDF, from 97.2% to

100.1% for RS4, from 98.4% to 103.1% for RS5, and from

97.9% to 106.1% for RS6, respectively. Inter-day accuracy

ranged from of 94.6% to 101.4% (TDF), 95.8% to 104.4%

(RS4), 96.6% to 99.4% (RS5), and 95.6% to 108.4% (RS6),

respectively. The absolute recovery ranged from 81.2% to

94.1% (for TDF), 85.2% to 97.1% (for RS4), 89.2% to

99.1% (for RS5), and 93.2% to 99.8% (for RS6). The abso-lute recovery of IS at 50 ng/mL was 104.5%.

3.5. MTT Assay

The toxicities of TDF and its impurities on the MDCK-MOCK cells were examined by MTT. After incubating for 48 h, the toxicities of RS1, RS2, RS3, and RS7 were lower than that of TDF. Therefore, the following toxic studies were

investigated with TDF, RS4, RS5 and RS6. The cells viabil-ities of TDF and RS4-RS6 were reduced in a concentration dependent manner. Moreover, RS5 markedly attenuated the viability reduction and the viability of cells was decreased to 65 + 7.3%, 60 + 5.7%, 51 + 3.3%, 13 + 0.8%, 7.9 + 0.7%, and 4.2 + 0.2% (n = 3) of cells treated with vehicle (0.25% DMSO), respectively Fig. (4). Above data indicated that RS5 was more cytotoxic than the drug TDF.

3.6. The Uptake of TDF and its Impurities

The uptake results revealed that, the uptake of TDF in MDCK-hOAT1 was markedly higher than that in mock cells in time and concentration dependent manners Fig. (5A). The MDCK-hOAT1 exhibited marked accumulation (about 1.8-fold for TDF, 1.3-fold for RS4, 6.2-fold for RS5, 4.6-fold for RS6) relative to empty vector-transfected cells. The kinetic

Fig. (3). MRM chromatograms of TDF, RS4, RS5, RS6 and IS.

� ��

��

��

� ��

� ��

� ��

� ��

��

� ��

��

� �

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

��

� ��

� ��

� ��

��

� � � � � � � � � � � � � ��

� �

��

��

� ��

� ��

��

��

��

��

��

��

��

��

��

� ��

��

� ��

� �

� ��

� ��

��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� � � � � �

� �

� � � � � � � �

� ��

��

��

� �

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� ��

� �

!� ��

��

"�#��!#$��

!

"�#��!#$��

!

"�#��!#$��

!

"�#��!#$��

!"�#��!#$��

!

�� !��"#�"��"#�""#�"��$�� %�� !��"#�"�&��"#�""#�"��$��"��

� ��

��

�� '��'�� !��"#�"�&��"#�""#�"��$��"(��

�� !��"#�"��"#�""#�"��$��

��

�� !��"#�"��"#�""#�"��$��


Fig. (4). The viability of MDCK-hOAT1 treated with TDF, RS4,

RS5, and RS6 for 48 h. Values were calculated as percentage of the

control group (0.25% DMSO). Data are expressed as mean +SD, n=

3. * P<0.05, ** P<0.01, *** P<0.001 vs control group. # #

P<0.01, #

# # P<0.001 vs TDF group.

study revealed that the uptake of TDF and its impurities in mock cells was linear in the concentration range of 10–500 μ . The OAT1-mediated uptake followed Michaelis–Menten kinetics, and the Km and Vmax were shown as Fig. (5B). The above results indicated that TDF and its impurities had the low affinity to hOAT1. And the affinity of RS5 was highest among 4 compounds tested Fig. (5B).

However, it is uncertain whether these impurities could be hOAT1 inhibitors. TDF and three impurities at 100 μM were assessed for inhibitory effects on 6-CFL uptake in MDCK-hOAT1 cells and probenecid (1 mM) was set as positive control. The positive inhibitor of OAT1 decreased the uptake ratio of 6-CFL significantly. Neither the TDF nor its impurities could influence the uptake ratio, which could be deduced that they were not the inhibitor of hOAT1 Fig. (6).

DISCUSSION

Controlling and minimizing the adverse effects of drugs are the key issues in ensuring a safe drug therapy. The strin-

Fig. (5). Time course of TDF uptake (100 μM) into MDCK-hOAT1 and mock cells for 2, 5, 10, 20 min at 37°C (A). Saturation curve of the

hOAT1-mediated uptake incubated with RS4, RS5, RS6 and TDF (10–500μM) for 10-min incubation (B). Data are expressed as mean + SD, n = 3.

�)�

��

��

��

��

��*��*+��+��*��

(�+�,�

� �!� -��+��*

�%&'%��

��

�

��

��

��*��*+��+��*��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

��

(�+�,�

� �!� -��+��*� �*

(�+�,�

� �!� -��+��*� �*

(�+�,�

� �!� -��+��*� �*

(�+�,�

� �!� -��+��*� �*

��

�

��

��

��*��*+��+��*�� "�.��*��*+��+��*��

�

��

��

��

��

��

'�

��

��

(�

��

��

��

��

��

��

��

��

��

�� !��!�"!��

######

######

$$$

###

######

######

###

###

####

##

###

$

##

##

##

$$


gent purity requirement [10] that all the individual impuri-ties, which are 0.1%, must be identified and characterized, and are 0.05%, should be reported. Although impurities RS1-RS7 add up to < 1% of the total, the reduced systemic exposure of impurities are expected to decrease the renal uptake and accumulation of toxics, which may potentially improve the long-term renal safety profile of TDF relative to TDF with impurities. Furthermore, this study will provide useful reference information for a large number of organic chemists from pharmaceutical companies and drug regula-tory authorities. To the best of our knowledge, RS5 was a newly detected impurity. RS1 was isolated as a major degra-dation product, and the other impurities were generated dur-ing the synthetic process, either as starting materials or in-termediates. RS1, RS3, and RS7 corresponded to tenofovir monoester, propylene adenine, and the TDF dimer, respec-tively. Unlike TDF, RS2, RS4, and RS6 exhibited variations in the diester moiety.

Tenofovir released from TDF undergoes active renal se-cretion via organic anion transporters (OAT1, OAT3), lead-ing to higher exposure of renal proximal tubules to tenofovir and a potential for renal adverse effects in a small subset of TDF-treated patients [20, 21]. The previous study suggested OAT1 and OAT3 expression increased the tenofovir cellular uptake by>70-fold and 8.2-fold, respectively. In addition, whereas tenofovir was significantly more cytotoxic in OAT1 than in OAT3-expressing cells [22]. Here we evaluate the interaction of TDF and its impurities with OAT1 to assess the potential for theirs active accumulation in proximal tu-bules. As compared to tenofovir, TDF showed a lower affin-ity for OAT1 [22], which indicated that the diester lead to the reduced affinity for OAT1. RS4 and RS6 are hydrolyzed to yield tenofovir, and RS5 is hydrolyzed to get demethy-lated tenofovir in vivo. These hydrolysis products demon-strate a higher affinity and a greater toxicity than their re-spective parent molecules. Even though the chemical struc-tures of TDF and RS5 are quite similar, the principal deter-minants of their corresponding affinities were not similar. As illustrated in Fig. (4 and 5A), RS5 was more cytotoxic and showed a higher affinity to OAT1 than TDF. This difference could be explained on the basis of structural variations. The major structural difference is presence of a methyl moiety on the 9-position. Lipophilicity has been reported to be an im-portant determinant of the affinities and inhibitory interac-tions of a variety of organic compounds towards transporters,

enzymes, and ion channels [23, 24]. The interaction with the lipophilic environment of the transporter protein as well as the direct interaction of certain amino acid residues in the substrate-binding pocket with various substrates and inhibi-tors determines the substrate recognition of OAT [25]. On the other hand, the main determinant of the affinity of xan-thine-related compounds for OAT1 is the position of the methyl group [23]. It is quite likely that TDF shows a lower potency than RS5 owing to steric hindrance. On the basis of these results, we conclude that 9-demethylated tenofovir shows an increased affinity and toxicity towards hOAT1. Thus, RS5 seems to negatively impact the renal proximal tubules by producing a more severe renal adverse effect than TDF, following hydrolysis in vivo.

As part of the Drug Interaction Studies conducted by the U.S. FDA, the decision trees were used to evaluate whether an investigational drug was a substrate or an inhibitor for OAT1 [17]

(Please refer to Fig. (6) in the Guidance for In-

dustry document published by the U.S. FDA). In this study, the values of the uptake kinetics parameters (Km and Vmax) suggested that RS5 showed a much more potent affinity for OAT1 than TDF. Results from the inhibition transport indi-cated that these impurities were not the inhibitors of OAT1, which implied that these impurities did not diminish the po-tency of TDF. We concluded that the impurities present in the TDF samples could be responsible for the toxic adverse effects.

CONCLUSION

In summary, seven prominent impurities in the bulk drug

of TDF were identified and characterized. The toxicological

evaluations of TDF and its impurities were conducted in

vitro using an MTT assay. The in vitro uptake studies with

hOAT1 clearly showed that the new impurity “RS5” exhib-

ited a higher affinity towards hOAT1 than TDF. The inhibi-

tion studies showed that the impurities were not the inhibi-

tors of hOAT1. An impurity with potency greater than that of

the test compound can cause the test compound to appear

more potent than it is in reality. These results highlight the

importance of biological safety studies that need to be con-

ducted in order to increase our knowledge on the potential

toxicity of drug-related substances. It is equally important to

conduct in vivo toxicity studies in order to draw meaningful conclusions regarding drug safety.

Fig. (6). Inhibitory effect of TDF, RS4, RS5 and RS6 on hOAT1-mediated 6-CFL transport. Probenecid was indicated as positive control. The results shown are means SD (n=3). *** P<0.001 vs control group.

��

��

��

&(��#)�#(��&

�*�+ #�,�

-.�(/��(

�#)(01

�(�#)(0��

��2)(3��4


CONFLICT OF INTEREST

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

ACKNOWLEDGEMENTS

This article was supported by the Natural Science Foun-dation of China (#81230080), Zhejiang Medicine and Health Platform Backbone Personnel Fund Scheme (2013RCA015) and Funds of department of science and technology for analysis test (No. 2014C37059).

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publisher's website along with the published article.

REFERENCES

[1] Fux, C.A.; Christen, A.; Zgraggen, S.; Mohaupt, M.G.; Furrer, H.

Effect of tenofovir on renal glomerular and tubular function. AIDS, 2007, 21, 1483-1485.

[2] Mocroft, A.; Kirk, O.; Gatell, J.; Reiss, P.; Gargalianos, P.; Zilmer, K.; Beniowski, M.; Viard, J.P.; Staszewski, S.; Lundgren, J.D.

Chronic renal failure among HIV-1-infected patients. AIDS, 2007, 21, 1119-1127.

[3] Côté, H.C.; Magil, A.B.; Harris, M.; Scarth, B.J.; Gadawski, I.; Wang, N.; Yu, E.; Yip, B.; Zalunardo, N.; Werb, R.; Hogg, R.;

Harrigan, P.R.; Montaner, J.S. Exploring mitochondrial nephrotoxicity as a potential mechanism of kidney dysfunction

among HIV-infected patients on highly active antiretroviral therapy. Antivir. Ther., 2006, 11, 79-86.

[4] Vidal, F.; Domingo, J.C.; Guallar, J.; Saumoy, M.; Cordobilla, B.; Sánchez de la Rosa R.; Giralt, M.; Alvarez, M.L.; López-Dupla,

M.; Torres, F.; Villarroya, F.; Cihlar, T.; Domingo, P. In vitro cytotoxicity and mitochondrial toxicity of tenofovir alone and in

combination with other antiretrovirals in human renal proximal tubule cells. Antimicrob. Agents Chemother., 2006, 50, 3824-3832.

[5] Itagaki, S.; Sugawara, M.; Kobayashi, M.; Nishimura, S.; Fujimoto, M.; Miyazaki, K.; Iseki, K. Major role of organic anion transporters

in the uptake of phenolsulfonphthalein in the kidney. Eur. J Pharmacol., 2003, 475, 85-92.

[6] Hosoyamada, M.; Sekine, T.; Kanai, Y.; Endou, H. Molecular cloning and functional expression of a multispecific organic anion

transporter from human kidney. Am. J Physiol., 1999, 276, F122-F128.

[7] Kohler, J.J.; Hosseini, S.H.; Green, E.; Abuin, A.; Ludaway, T.; Russ, R.; Santoianni, R.; Lewis, W. Tenofovir renal proximal

tubular toxicity is regulated by OAT1 and MRP4 transporters. Lab. Invest., 2011, 91, 852-858.

[8] Kogawa A.C.; Salgado H.RN. Impurities and Forced Degradation Studies: A Review. Curr. Pharm. Anal., 2016, 12, 18-24.

[9] Verbeken, M.; Wynendaele, E.; Lefebvre, R.A.; Goossens, E.; Spiegeleer, B.D. The influence of peptide impurity profiles on

functional tissue-organ bath response: the 11-mer peptide INSL6[151-161] case. Anal. Biochem., 2012, 421, 547-555.

[10] International Conference on Harmonization of Technical Require-ments for Registration of Pharmaceuticals for Human Use Steering

Committee. ICH Q3A(R2) Impurities in new drug substances, 2006.

[11] International Conference on Harmonization of Technical Require-

ments for Registration of Pharmaceuticals for Human Use Steering Committee. ICH Q3B(R2) Impurities in new drug products, 2006.

[12] International Conference on Harmonization of Technical Require-ments for Registration of Pharmaceuticals for Human Use Steering

Committee. ICH Q3C(R4) Impurities: guideline for residual sol-vents, 2009.

[13] Karunakaran, A.; Kamarajan, K.; Thangarasu, V. Development and Validation of First-Derivative Spectrophotometric Method for the

Simultaneous Estimation of Lamivudine and Tenofovir disoproxil fumerate in Pure and in Tablet Formulation. Der. Pharmacia.

Lettre., 2010, 2, 221-228. [14] Rama Prasad, L.A.; Rao, J.V.L.N.S.; Pamidi, S.; Vara, Prasad J.;

Hemalatha, J. New stability indicating HPLC method for simultaneous estimation of lamivudine, tenofovir df and nevirapine

in extended release tablets. Int. J. Pharm., 2013, 3, 136-144. [15] Ashenafi, D.; Chintam, V.; van Veghel, D.; Dragovic, S.;

Hoogmartens, J.; Adams, E. Development of a validated liquid chromatographic method for the determination of related

substances and assay of tenofovir disoproxil fumarate. J. Sep. Sci., 2010, 33, 1708-16.

[16] U.S. Food and Drug Administration (FDA). Guidance for Industry: Drug Interaction Studies — Study Design, Data Analysis,

Implications for Dosing, and Labeling Recommendations (Draft Guidance). 2012; pp. 69-70.

[17] Brown Ripin, D.H.; Teager, D.S.; Fortunak, J.; Basha, S.M.; Bivins, N.; Boddy, C.N.; Byrn, S.; Catlin, K. K.; Houghton, S.R.;

Jagadeesh, S.T.; Kumar, K.A.; Melton, J.; Muneer, S.; Rao, L.N.; Rao, R. V.; Ray, P.C.; Reddy, N.G.; Reddy, R.M.; Shekar, K.C.;

Silverton, T.; Smith, D.T.; Stringham, R.W.; Subbaraju, G.V.; Talley, F.; Williams, A. Process Improvements for the Manufacture

of Tenofovir Disoproxil Fumarate at Commercial Scale. Org. Process Res. Dev., 2010, 14, 1194-201.

[18] Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J.

Immunol. Methods., 1983, 65, 55-63. [19] Ma, L.P.; Zhao, L.; Hu, H.H.; Qin, Y.H.; Bian, Y.C.; Jiang, H.D.;

Zhou, H.; Yu, L.S.; Zeng, S. Interaction of five anthraquinones from rhubarb with human organic anion transporter 1 (SLC22A6)

and 3 (SLC22A8) and drug-drug interaction in rats. J Ethnopharmacol., 2014, 153, 864-871.

[20] Truong, D.M.; Kaler, G.; Khandelwal, A.; Swaan, P.W.; Nigam, S.K. Multi-level analysis of organic anion transporters 1, 3, and 6

reveals major differences in structural determinants of antiviral discrimination. J. Biol. Chem., 2008, 283, 8654-863.

[21] Kearney, B.P.; Flaherty, J.F.; Shah, J. Tenofovir disoproxil fumarate: clinical pharmacology and pharmacokinetics. Clin.

Pharmacokinet., 2004, 43, 595–612. [22] Bam, R.A.; Yant, S.R.; Cihlar, T. Tenofovir alafenamide is not a

substrate for renal organic anion transporters (OATs) and does not exhibit OAT-dependent cytotoxicity. Antivir. Ther., 2014, 19, 687-

692. [23] Sugawara, M.; Mochizuki, T.; Takekuma, Y.; Miyazaki, K.

Structure-affinity relationship in the interactions of human organic anion transporter 1 with caffeine, theophylline, theobromine and

their metabolites. Biochim. Biophys. Acta., 2005, 1714, 85-92. [24] Sugawara, M.; Kato, M.; Kitakubo, M.; Takekuma, Y.; Ganapathy,

V.; Miyazaki, K. Inhibitory effects of basic drugs on the sodium-dependent transport of L-alanine via system B0 in the small

intestine. Drug Metab. Pharmacokinet., 2003, 18, 186-193. [25] Feng, B.; Shu, Y.; Giacomini, K.M. Role of aromatic

transmembrane residues of the organic anion transporter, rOAT3, in substrate recognition. Biochem., 2002, 41, 8941-8947.

research article ˘ ˇ ˇˆ an impurity in tenofovir ... · potassium dihydrogen orthophosphate,...

Documents