draft · 2019. 10. 17. · draft investigation of intestinal elimination and biliary excretion of...

Draft

Investigation of intestinal elimination and biliary excretion of ibuprofen in control and hyperglycemic rats

Journal: Canadian Journal of Physiology and Pharmacology

Manuscript ID cjpp-2019-0164.R1

Manuscript Type: Article

Date Submitted by the Author: 09-Jun-2019

Complete List of Authors: Kovacs, Noemi-Piroska; 1SC. Salix Pharm SRLAlmási, Attila; University of Pecs, Institute of Pharmaceutical ChemistryGarai, Kitti; University of Pécs, Institute of Pharmaceutical BiotechnologyKuzma, Mónika; University of Pécs, Institute of Pharmaceutical ChemistryVancea, Szende; University of Medicine, Pharmacy, Science and Technology of Târgu-MureşFischer, Emil; University of Pecs, Institute of Pharmacology and PharmacotherapyPerjesi, Pal; University of Pecs, Institute of Pharmaceutical chemistry

Is the invited manuscript for consideration in a Special

Issue:Not applicable (regular submission)

Keyword: Ibuprofen, STZ-induced hyperglycemia, Intestinal elimination, Biliary excretion, HPLC

https://mc06.manuscriptcentral.com/cjpp-pubs

Canadian Journal of Physiology and Pharmacology

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Investigation of intestinal elimination and biliary excretion of ibuprofen in

control and hyperglycemic rats

Noémi-Piroska Kovács1,2, Attila Almási2, Kitti Garai3, Mónika Kuzma2, Szende Vancea4, Emil

Fischer5, Pál Perjési2*

1SC. Salix Pharm SRL, Pandurilor str.113, RO-540501, Târgu-Mureş, Romania2Institute of Pharmaceutical Chemistry, University of Pécs, Rókus str.2, H-7624 Pécs, Hungary3Institute of Pharmaceutical Biotechnology, University of Pécs, Rókus str.2, H-7624 Pécs,

Hungary4Department of Physical Chemistry, University of Medicine, Pharmacy, Science and

Technology of Târgu-Mureş, Gheorghe Marinescu str. 38, RO-540139 Târgu-Mureş, Romania5Institute of Pharmacology and Pharmacotherapy, University of Pécs, Szigeti str. 12, H-7624

Pécs, Hungary

*Corresponding authorPál Perjési, PhD

E-mail: [email protected]

Postal address: H-7624 Pécs, Rókus str. 2.

Hungary

Phone: +36 72 503 626

Fax: +36 72 503 627

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Abstract

An in vivo intestinal perfusion model was used to investigate how experimental hyperglycemia

affects intestinal elimination and biliary excretion in the rat. Experimental diabetes was induced

by administration of streptozotocin (65mg/kg i.v.). The intestinal perfusion medium contained

250 µM (±)-ibuprofen. An isocratic HPLC method with UV-Vis detection was developed to

quantitate ibuprofen in the intestinal perfusate and a gradient method was applied to quantitate

ibuprofen and ibuprofen-β-D-glucuronide in the bile. The limit of quantitation of ibuprofen was

found to be 0.51 µM in the small intestinal perfusate. In the bile, the limit of quantitation of

ibuprofen and ibuprofen-β-D-glucuronide was 4.42 µM and 10.3 µM, respectively.

Unconjugated ibuprofen and ibuprofen-β-D-glucuronide were detected in the bile, however, no

β-D-glucuronide of ibuprofen could be detected in the intestinal perfusate. The results indicate

that experimental diabetes can cause a decrease in the disappearance of ibuprofen from the

small intestine. Excretion of both ibuprofen and ibuprofen-β-D-glucuronide decreased to the

bile in experimental diabetes. The results can be explained by the results of molecular biological

studies indicating STZ-initiated alterations in the intestinal and hepatic transport processes.

KeywordsIbuprofen, STZ-induced hyperglycemia, Glucuronidation, Intestinal elimination, Biliary

excretion, HPLC-UV-Vis

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Graphical Abstracts

Investigation of intestinal elimination and biliary excretion of ibuprofen in control and

hyperglycemic rats



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IntroductionIbuprofen (± 2-(4-isobutylphenyl)propionic acid; IBP) is a widely used non-steroidal

anti-inflammatory drug (NSAID) of the 2-arylpropionic acid (2-APA) class (Adams et al.

1969a; 1969b; Mazaleuskaya et al. 2015). At low doses (800-1200 mg/day) it is mainly used to

relieve minor pain and inflammation. In higher doses (1800-2400 mg/day) it is used for the

long-term treatment of rheumatoid arthritis, osteoarthritis and other chronic conditions

(Rainsford 2009). Ibuprofen is the most commonly administered orally. It is rapidly and

completely absorbed after oral administration (Davies 1998). It is able to penetrate to the central

nervous system and can accumulate at peripheral sites where analgesia or anti-inflammatory

effect is required (Rainsford 2009; Davies 1998).

Like most NSAIDs, IBP is also administered as a racemic mixture, being the (S)-

enantiomer largely responsible for its pharmacological activity (Davies 1998; Caldwell et al.

1998). Following administration, about 50-60% of the (R) enantiomer undergoes an (R)-(S)

inversion mainly in the liver (Davies 1998; Caldwell et al. 1998; Hall et al. 1993), but it may

occur in the gut as well (Jamali et al. 1992; Rudy et al. 1991). It is a relatively weak acid (pKa

4.4) and its solubility in water or at acidic pH is very low. This results in a relatively long

residence time in the acidic environment of the stomach, which slows down absorption of the

substance (Chiarini et al. 1984). Most absorption is likely to occur in the small intestine, a

smaller but significant part of it is taken up by the stomach (Adams et al. 1969b; Kepp et al.

1997).

Ibuprofen is reported to be almost completely metabolized in humans, with little or no

parent compound found in the urine (Mazaleuskaya et al. 2015; Rudy et al. 1991; Kepp et al.

1997; Mills et al. 1973). It undergoes conjugation with glucuronic acid forming ibuprofen-acyl-

glucuronide (Mazaleuskaya et al. 2015; Rudy et al. 1991) (Fig 1.). Formation of oxidative

metabolites (mainly 2-hydroxyibuprofen and 3-carboxyibuprofen) represents the major

metabolic pathways. The primary oxidative metabolites (hydroxylated derivatives) can also be

converted to and excreted as the respective acyl-glucuronides. The main place of the reactions

is the liver but they can occur in other tissues where the corresponding metabolizing enzymes

are expressed (Mazaleuskaya et al. 2015; Davies 1998; Rudy et al. 1991; Kepp et al. 1997;

Mills et al. 1973).

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OCOOH

HO

OH

OH

O C CHO

CH3

CH2 CH(CH3)2CH

H3C

H3CCH2 CH COOH

CH3

I II

Fig. 1. The structures of ibuprofen (I) and ibuprofen-β-D glucuronide (II)

Several factors, including pathophysiological circumstances, diseases, e.g. diabetes, can

influence biotransformation and excretion of xenobiotics (Toda et al. 2005; Ritter 2007; Hao et

al. 2011). There are only a few data, however, about the change of metabolic profile of the small

intestine in diabetes or in the hyperglycemic state. One of the animal models used to investigate

intestinal absorption of xenobiotics is the present rat ex vivo model (Toda et al. 2005; Fischer

et al. 2015; Almási et al. 2018). The relevance of the model in absorption is supported by the

results of investigations of oral absorption of a series of 43 drugs, of which absorption was

found to be close to that of humans (Chiu et al. 2000). Furthermore, STZ-induced

hyperglycemia is a well-characterized and frequently used animal model to study the

physiological consequences of hyperglycaemic conditions (Sakata et al.2012; Wang-Fisher and

Garyantes, 2018). Such condition has been reported to affect several metabolic enzymes and

transporters both in rats and humans (Anger et al. 2009; Nawa et al. 2010; Dostalek et al. 2011;

Vahabzadeh and Mohammadpou 2015; Tran and Elbarbry 2016).

In our earlier studies, in vivo intestinal absorption and Phase II metabolism of 4-

nitrophenol (PNP), as a model compound, was investigated in control and hyperglycemic rats.

It was found that experimental hyperglycemia can cause changes in intestinal absorption and

metabolism. It increased intestinal glucuronidation of PNP but did not influence sulfate

conjugation (Fischer et al. 2015). On the other hand, biliary excretion of both conjugates was

depressed in the hyperglycemic rats (Almási et al. 2018).

As a continuation of our earlier studies on this field, the present experiments were

planned to investigate how experimental diabetes affects the intestinal disappearance and

conjugative metabolism of ibuprofen. To receive information about the possible role of the

hyperglycemia-induced changes, jejunal perfusion of streptozotocin (STZ)-treated rats was

performed to compare these results with data obtained in the control rats. During the

experiments, perfusate and bile samples were collected and analyzed by a newly developed,

validated HPLC-UV-Vis method for ibuprofen and ibuprofen-β-D glucuronide.

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Materials and methodsChemicals and reagents

Ibuprofen (IBP), ammonium acetate (NH4OOCCH3) and glacial acetic acid was

purchased from Molar Chemicals (Budapest, Hungary); acetonitrile (ACN), potassium chloride

(KCl), calcium chloride (CaCl2x2H2O), disodium hydrogen phosphate (Na2HPO4

x2H2O), and

TRIS from Reanal (Budapest, Hungary); sodium chloride (NaCl), diclofenac sodium (DICL),

sodium citrate dehydrate, citric acid and streptozotocin (STZ) from Sigma- Aldrich (Budapest,

Hungary); magnesium sulfate (MgSO4) and sodium dihydrogen phosphate (NaH2PO4) from

Spektrum-3D (Hungary); and ibuprofen-β-D-glucuronide from Toronto Research Chemicals

(Toronto, Canada). The solvents were HPLC grade. The standard isotonic perfusion medium

had the following composition (mmol/l): NaCl 96.4, KCl 7.0, CaCl2 3.0, MgSO4 1.0, sodium

phosphate buffer (pH 7.4) 0.9, TRIS buffer (pH 7.4) 29.5, glucose 14.0, mannitol 14.0. UV-Vis

measurements were performed on a Jasco V650 spectrophotometer at ambient temperature.

Distilled water was purified in the Department of Pharmaceutical Chemistry, University of Pécs

by use of a Purelab Option Q7 Water System. A Mettler Toledo MP 220 pH meter and a Mettler

Toledo Inlab 413 electrode were used to adjusting the pH. Blood glucose level was controlled

with an AccuChek blood glucose meter (Roche).

Animal experiments

A similar procedure was used as described before (Fischer et al. 2015; Almási et al.

2018). Male Wistar rats (weighing 220-280 g) were anesthetized with urethane (1.2 g/kg i.p.).

The abdomen was opened by a mid–line incision and a jejunal loop (length about 10 cm) was

isolated and cannulated. The lumen of the jejunal loop was gently flushed with a warmed

isotonic solution to remove digesta and food residues and then blown empty with 4-5 ml of air.

The intestinal perfusion was carried out by a peristaltic pump (flow rate: 13 ml/min) with 250

µM ibuprofen dissolved in isotonic perfusion medium. During the 90 minute period of the

perfusion, 250 µl samples were collected in previously set time points. While the biliary

excretion was investigated, the bile duct was cannulated with PE-10 tubing and the bile outflow

was collected in 15-min periods. The samples were stored in a refrigerator (- 20 °C) until

analysis.

Experimental diabetes was induced by i.v. administration of streptozotocin (freshly

dissolved in 0.1 M citrate buffer pH 4.0) in a dose of 65 mg/kg (2ml/kg)). Control animals were

treated with the vehicle. Experiments were performed after one week of the streptozotocin

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treatment. Blood glucose levels were tested before the STZ-treatment and before starting the

experiments. The experimental animals were provided standard chaw (Innovo, Gödöllő,

Hungary) and water ad libitum, The standard chaw was withdrawn 12 hours before the glucose

level testings. Perfusate and bile samples were obtained from the same rats of two different

groups (control and STZ-pretreated). The number of rats in each group was five. The values

represent the mean ± S.E. of five independent experiments.

Sample preparations

“The perfusion samples (250 µl) were allowed to rise to ambient temperature and

vortex-mixed. To 200 µl of each sample, 50 µl of 1 mM diclofenac sodium solution (as internal

standard) was added (200 µM final concentration), vortex-mixed and centrifuged at 900 g for

10 min.

The bile samples were allowed to rise to ambient temperature and vortex-mixed. To 50

µl of the samples, 200 µl of 0.1 M perchloric acid solution (containing 31.25 µM diclofenac

sodium as internal standard; 25 M final concentration) was added, vortex-mixed and

centrifuged at 10.000 g for 10 minutes.”

Instrumentation and chromatographic conditions

Analysis of the perfusates and the bile samples was performed by an Agilent 1100 HPLC

system equipped with a quaternary HPLC pump (G1311A), degasser (G1379A), an

autosampler (G1313A), a thermostated column compartment (G1316A) and a diode-array

detector (G1315B). Data were recorded and evaluated by Agilent ChemStation software

(Rev.B.03.02-SR2). Reversed-phase Zorbax SB C18 (4.6 mm x 150 mm, 5µm particle size) and

Teknokroma TR-C-160K1 guard columns were used for analytical separations.

HPLC-UV-Vis analysis of the intestinal perfusate samples (Method I)

The mobile phase consisted of acetonitrile and 5 mM acetate buffer (55:45, v/v %)

adjusted to pH 3 with concentrated acetic acid. The flow rate of the eluent was 1 ml/min. The

volume of the samples was 20 µl and the column was thermostated at 40 °C. The detection was

performed at 220 nm. Standard solutions were prepared by the addition of known concentration

of substance - prepared in phosphate buffer and added to the drug-free perfusate.

The standard solutions contained 200 M DICL as an internal standard. The constructed

calibration curves were based on the ratio of the integrated peak areas of the ibuprofen standards

and the internal standard. Five replicate injections of the standard solutions were made. The

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relative standard deviation (RSD) of peak areas and retention times was calculated to assess

intra-day precision. The same solutions stored at 4°C were used to assess inter-day assay

variations.

HPLC-UV-Vis analysis of the bile samples (Method II)

Gradient separation using acetonitrile (A) and 5 mM ammonium acetate buffer (pH 3.0)

(B) of ibuprofen, ibuprofen-β-D-glucuronide and diclofenac sodium was performed. The used

gradient was as follows: 65 % B was descended to 45 % in six minutes, kept for 5.5 minutes

and increased to the initial conditions in six minutes. The HPLC system was equilibrated for

fifteen minutes (65 % B) before injecting the next sample. The flow rate of the eluent was set

to 1.0 ml/min and the run time was 10 min. The wavelength of the detection was 240 nm. The

volume of the samples was 20 µl, the column was thermostated at 40 °C.

The standard solutions contained 25 M DICL as an internal standard. The calibration

curve derived from the ratio of area under the curve of ibuprofen, ibuprofen-β-D-glucuronide,

and diclofenac-sodium. The relative standard deviations (RSD) of peak areas and retention

times were calculated for intra-day and day-to-day relations.

Validation of the chromatographic methods

Specificity

Specificity was defined as the ability of the methods to differentiate and quantify the

analytes in the presence of endogenous constituents of the perfusate and the samples. Figure 2

depicts the representative HPLC chromatograms of the blank perfusate (Figure 2A), the

standards prepared with the blank perfusate, and the perfusate generated in rat small intestine

luminal perfusion experiments at the beginning (Figure 3A) and the 90th minute of the

perfusion period (Figure 3B). There were no endogenous peaks in the regions of the retention

times of ibuprofen, ibuprofen--D-glucuronide and diclofenac sodium. Similar results were

obtained by comparison of the HPLC chromatograms of the blank bile (Figure 4A), that of the

standards prepared in blank bile (Figure 4B) and of the 90th-minute bile sample of the perfusion

experiments (Figure 5).

Linearity

Linearity of Method I was studied by preparing series of ibuprofen solutions of drug-

free small intestinal perfusate (containing 200 µM diclofenac sodium) in the range of 5-250 µM

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(5, 10, 50, 100, 250 M). Data were obtained from five parallel injections of two independent

weighings (10 injections altogether) applied at each concentration level. Calibration curves

were created by plotting the theoretical concentrations against the relative peak areas. Linearity

was determined by least-squares regression. The regression equation for ibuprofen was as

follows: y= 0.0092x+0.1131 (R²=0.9942).

The linearity of Method II was studied by preparing series of ibuprofen, ibuprofen--D-

glucuronide and diclofenac sodium (as internal standard) solutions of drug-free bile in the range

of 7.5-150 µM (7.5, 15, 30, 75, 150 M). Data were obtained from five parallel injections at

each concentration level. Calibration curves were created by plotting the theoretical

concentrations against the relative peak areas. Linearity was determined by least-squares

regression. The obtained regression equations for ibuprofen and ibuprofen-β-D-glucuronide

were y= 0.0062x + 0.0013 (R2=1) and y= 0.0049x – 0.0008 (R2 = 0.9999), respectively.

System suitability

System suitability data were extracted from chromatograms of the standard solutions of

ibuprofen and ibuprofen--D-glucuronide in drug-free perfusate and bile. Results were

obtained from five parallel injections. The percent RSD values found for intra-day retention

times and integrated peak areas of the standard solutions are shown in Table 1 (Method I) and

Tables 2 and 3 (Method II).

Precision

The precision of the chromatographic methods was investigated by determination of the

intra-day precision (repeatability) and inter-day precision (reproducibility). Repeatability of the

retention times and the integrated peak areas were determined by analysis of five concentration

levels of ibuprofen (in drug-free small intestinal perfusate) (Method I), and of ibuprofen and

ibuprofen-β-D-glucuronide (in drug-free bile) (Method II). Each concentration was analyzed

five times. The percent RSD values found for intra-day retention times and integrated peak

areas of the standard solutions are shown in Tables 1-3.

For determination of inter-day precision (reproducibility) of retention times and

integrated peak areas, one set of calibration with different ibuprofen (in small intestinal

perfusate and bile) and ibuprofen-β-D-glucuronide (in bile) contents was prepared and

measured on the same working days over five consecutive weeks. The percent RSD values for

retention time and integration areas obtained from the measurements are summarized in Tables

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1-3. The percent RSDs of the inter-day precision of the retention times of ibuprofen (Method I)

were RSD = 1.83-2.17; and those of the integrated peak areas RSD = 1.58–10.40. In the case

of Method II, the corresponding values were RSD = 0.22-1.08 and RSD = 3.79-8.76,

respectively. Fort the ibuprofen-β-D-glucuronide (Method II), the inter-day precision of the

retention times were RSD = 1.05-2.45; and those of the integrated peak areas RSD = 4.68 –

8.32 (Tables 1-3).

Determination of LOD and LOQ

Limit of detection (LOD) was determined experimentally, and taken as the

concentration producing a detector signal that could be clearly distinguished from the baseline

noise (3 times baseline noise) (ICH 2005). The LOD of ibuprofen was found to be 0.15 µM in

the small intestinal perfusate. In the bile, the limit of detection of ibuprofen and ibuprofen-β-

D-glucuronide was 1.33 µM and 3.09 µM, respectively.

The percentage of relative standard errors for the calibration curves obtained for

ibuprofen in Method I was 3.98, while 7.49 and 4.48 for ibuprofen and ibuprofen-β-D-

glucuronide, respectively, in Method II. Based on the calculation of the root mean square error

(RMSE) over the concentration range of 5.0-50.0 µM in Method I and over the 7.5-75.0 µM

range (ibuprofen and ibuprofen-β-D-glucuronide) in Method II, the limit of quantification

(LOQ) (calculated as (10xRMSE)/m) where m was the slope of the calibration curve). of

ibuprofen was found to be 0.51 µM in the small intestinal perfusate. In the bile, the limit of

quantitation of ibuprofen and ibuprofen-β-D-glucuronide was 4.42 µM and 10.3 µM,

respectively.

Calculations and statistical analysis

The luminal appearance of ibuprofen was calculated on the base of its luminal

concentrations and the volume of perfusion solution. The biliary excretion was calculated on

the base of the volume of bile flow in 15 minutes periods. Data show the mean ± SE of five

independent experiments. The difference among groups was determined by the Student’s t-test.

Significant differences from the control value: * p<0.05 and ** p < 0.01.

Ethical approval

All animal procedures were performed according to the Hungarian Animal Protection

Act Scientific Procedures (Government Regulation 243/1998), and the study was approved by

the Institutional Animal Care and Use Committee of the University of Pécs.

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Results Diabetes can be induced with many different STZ dosing regimens in rodents (Wang-

Fischer and Garyantes 2018). With rats, a single dose of 65 mg/kg STZ i.p. or i.v. is used most

frequently (Sakata et al. 2012; Wang-Fisher and Garyantes 2018). Hyperglycaemia was

confirmed after 1 weak of the STZ-treatment. Using i.v. treatment, the hyperglycemia is

stabilized by that time (Bojcsev et al. 1996). The average blood glucose level of the control

animals was 7.85±1.02 mM, while that of the STZ-treated rats was 23.32 ± 2.06 mM.

The concentration of ibuprofen in the small intestine perfusates was determined by

HPLC-UV-Vis analysis (Method I). The disappearance of ibuprofen from the intestinal

perfusion solution indicates the absorption rate and the intestinal elimination (excretion and

metabolism) of the drug. There were no endogenous peaks in the regions of the retention times

of ibuprofen and diclofenac sodium. Presence of other ibuprofen-derived compounds could not

be detected (=220 nm) under the experimental conditions (Figures 2 and 3).

Fig. 2. HPLC chromatogram (Method I) of the blank small intestinal perfusate (Fig. 2.A) and the ibuprofen (2) and diclofenac sodium (internal standard) standards (1) (Fig. 2.B). The retention times in the sample of small intestinal perfusate for ibuprofen (2) and diclofenac sodium (1) were 6.11 and 5.21 respectively.

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Fig. 3. HPLC chromatograms (Method I) of samples generated in rat small intestine luminal perfusion experiment at the beginning (Fig. 3.A.) and at the 90th minute (Fig. 3.B) of the perfusion period. The retention times of diclofenac sodium (1) and ibuprofen (2) were 5.21 and 6.11 min, respectively.

The gradient HPLC-UV-Vis method (Method II) using acetonitrile (A) and 5 mM

ammonium acetate buffer pH 3.0 (B) as mobile phase (linear gradient from 35% to 55% A in 6

minutes; 55% A for 5.5 minutes; linear gradient from 55% to 35% A in 3.5 minutes; 35%

acetonitrile for 10 minutes) provided a baseline separation of ibuprofen--D-glucuronide (1),

diclofenac (2) and ibuprofen (3) (Figure 4B). The detection wavelength was 240 nm. There

were no endogenous peaks in the regions of the retention times of ibuprofen, ibuprofen--D-

glucuronide and diclofenac sodium (Figure 4A). In Method I, the retention time of ibuprofen

was shorter than that of in a similar RP-HPLC method [19], while Method II provided a reliable

separation and determination of ibuprofen-β-D-glucuronide and ibuprofen (Figure 5.).

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Fig. 4. HPLC chromatograms (Method II) of the blank bile (Fig. 4.A) and the system suitability solution containing ibuprofen-β-D-glucuronide, diclofenac sodium as internal standard and ibuprofen standards (Fig. 4.B.). The retention times of ibuprofen-β-D-glucuronide (1), diclofenac sodium (2) and ibuprofen (3) were 4.74, 10.08 and 10.92 min, respectively.

Fig.5. HPLC chromatogram (Method II) of a bile sample generated in an STZ pretreated rat at the 90th minute of the perfusion period. The retention times of ibuprofen-β-D-glucuronide (1), diclofenac sodium (2) and ibuprofen (3) were 4.74, 10.08 and 10.92 min, respectively.

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System suitability and validation (intra-day precision and inter-day precision) data are

summarized in Tables 1-3.

Table 1. Intra-day and inter-day precision of retention time and peak areas of ibuprofen (IBP) standard solutions in perfused buffer medium (Method I). The precisions of the peak areas were calculated from the ratios of the peak areas of ibuprofen and diclofenac sodium.

Concentration of standard IBP solution (µM)

Intra-day precision of

retention time(RSD %) (n=5)

Intra-day precision of peak areas

(RSD %) (n=5)

Inter-day precision of

retention time( RSD %) (n=5)

Inter-day precision ofpeak areas

(RSD %) (n=5)5 0.53 12.91 2.17 10.40

10 0.55 5.71 2.00 3.41

50 0.64 3.59 1.97 3.17

100 0.67 5.99 1.83 3.46

250 0.57 3.20 2.10 1.58

*IBP: Ibuprofen.

Table 2. Intra-day and inter-day precision of retention time and peak areas of ibuprofen (IBP) in standard solutions in bile (Method II). The precisions of the peak areas were calculated from the ratios of the peak areas of ibuprofen and diclofenac sodium.

Concentration of standard

IBP* solution (µM)




(RSD %) (n=5)




(RSD %) (n=5)7.5 0.10 7.80 1.08 5.56

15 0.10 7.33 0.26 3.79

30 0.14 4.58 0.22 4.35

75 0.11 4.59 0.63 8.76

150 0.09 2.76 0.26 6.19

*IBP: Ibuprofen.

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Table 3. Intra-day and inter-day precision of retention time and peak areas of ibuprofen-β-D-glucuronide in standard solutions in bile (Method II). The precisions of the peak areas were calculated from the ratios of the peak areas of ibuprofen-β-D-glucuronide and diclofenac sodium.

Concentration of standard IBP-

β-D-glucuronide* solution (µM)




(RSD %) (n=5)




(RSD %) (n=5)

7.5 0.54 5.12 1.34 8.32

15 0.25 5.20 1.19 6.49

30 0.05 4.83 1.05 8.33

75 0.25 3.45 2.45 4.68

150 0.09 5.27 1.78 6.82

* IBP-β-D-glucuronide: Ibuprofen-β-D-glucuronide

Figure 6. shows the time-course of the disappearance of ibuprofen from the perfused rat

jejunal segment of the small intestine under the control and hyperglycemic conditions. Based

on the HPLC-UV-Vis studies, a continuous decrease of the disappearance of ibuprofen could

be detected in the small intestinal perfusates during the experiments. The disappearance of the

drug was consequently lower in the hyperglycemic animals, and the differences became

statistically significant in the last 30 minutes of the experiments.

Control+IBP

STZ+IBP

**

0 50 100

time (min)

0

500

1000

1500

2000

2500

3000

3500

4000

*

amou

nt o

f ibu

prof

en (n

mol

)

Control+IBP

STZ+IBP

**

0 50 100

time (min)

0

500

1000

1500

2000

2500

3000

3500

4000

*

Control+IBP

STZ+IBP

**

0 50 100

time (min)

0

500

1000

1500

2000

2500

3000

3500

4000Control+IBP

STZ+IBP

**

0 50 100

time (min)

0

500

1000

1500

2000

2500

3000

3500

4000

*

amou

nt o

f ibu

prof

en (n

mol

)

Fig.6. Time-course of the disappearance of ibuprofen in the rat small intestinal perfusate during perfusion of the jejunal loop with isotonic medium containing 250 µM ibuprofen (IBP). Each value represents the average of five independent experiments ± standard error. Significant differences from the control value: * p<0.05 and ** p < 0.01. (STZ: streptozoticin.)

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Figure 7. shows the cumulative biliary excretion (sum of excreted amounts in 90

minutes) of non-metabolized ibuprofen in the control and the STZ-treated rats. Significantly

lower amount of ibuprofen was excreted into the bile in the diabetic rats.

Fig. 7. Changes in the excreted amount of ibuprofen (IBP) into the bile during perfusion of the jejunal loop of rate with isotonic medium containing 250 µM ibuprofen. Each value represents the average of five independent experiments ± standard error. Significant difference from the control value: ** p < 0.01. (STZ: streptozotocin.)

Figure 8. demonstrates the cumulative biliary excretion (sum of excreted amounts in 90

minutes) of ibuprofen-β-D-glucuronide. It can be seen that STZ- pretreatment significantly

decreased the biliary excretion of ibuprofen-β-D-glucuronide in comparison to the control

values.

Fig.8. Change in the excreted amount of ibuprofen-β-D-glucuronide in the bile of the rat during perfusion of the jejunal loop with isotonic medium containing 250 µM ibuprofen (IBP). Each value represents the average of five independent experiments ± standard error. Significant difference from the control value: * p<0.05. (STZ: streptozotocin.)

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DiscussionThe developed isocratic RP-HPLC method for the quantitation of ibuprofen (IBP) from

small intestinal perfusate samples provided rapid and well repeatable series of measurements

with a retention time less than 8 minutes (Fig.2). Other papers have already described similar

methods for the analysis of ibuprofen in urine and plasma (Wang et al. 2005; Ge et al. 2011;

Jung et al. 1993; Berner et al. 1993) with similar validation parameters. For the unstable

metabolite, ibuprofen-β-D-glucuronide, previous methods suggested RP-HPLC determination

after solid phase extraction from plasma (Castillo and Smith 1993) and UPLC-MS detection

and identification in urine (Plumb et al. 2007).

The validated HPLC-UV-Vis method was applied for monitoring the jejunal absorption

of ibuprofen in the rat small intestine luminal perfusion experiments. Figure 6 shows the

amounts of ibuprofen, which is continuously decreased until the end of the perfusion

experiment. At the beginning of the experiments, the time course of disappearance rate of IBP

was similar in the STZ-treated and the control rats, however, the amount of IBP in the intestinal

perfusate of the diabetic rats was statistically higher in the last 30 minutes of the experiments.

This finding is in contrary to our previous results with 4-nitrophenol (Almási et al. 2006) and

salicylic acid (Nyúl et al. 2018), which showed these two compounds to be disappeared more

effectively from the small intestine in the hyperglycemic rats.

Zhang et al. have demonstrated that long term (8 weeks) experimental diabetes may

decrease P-gp function and expression in the brain and intestines, while it also may induce

increases in the liver or kidney (Zhang et al. 2011). Some other studies reported shorter STZ-

induced hyperglycemic period (9 days) to observe reduced expression of P-gp (Bojcsev et al.

1996, Anger et al. 2009; Nawa et al. 2010) Depressed hepatic expression of the efflux

transporters MDR1B (P-gp), MRP2 and BCRP was observed under our experimental

conditions as well (Garai et al. 2018). Transport studies on in vitro BBB models showed that

administration of verapamil, as a competitive inhibitor of P-gp (abcb1) (Römermann et al.,

2013), increased the permeability of ibuprofen and diclofenac (Novakova et al. 2014).

Furthermore, sulindac and ibuprofen were reported to inhibit P-gp activity at clinically

achievable doses (Angelini et al. 2008). Accordingly, the observed alteration can be explained

by the reduced activity of P-gp due to the experimental hyperglycemia and the accumulating

ibuprofen in the endothelial cells. The progressively increasing cellular ibuprofen level

attenuates the concentration gradient, which is considered to be the most important factor of the

entrance of ibuprofen (Mazaleuskaya et al. 2015).

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Analysis of the perfusate samples by the HPLC Method II (LOD: 0.15 M) did not show

the presence of the IBP-glucuronide. The low detection level of the IBP-glucuronide conjugate

indicates only a negligible level of the metabolite in the intestinal perfusate. In vitro

experiments indicate that several UGT isoforms can catalyze formation of glucuronide

derivative of the parent ibuprofen, including UGT1A3, UGT1A9, UGT2B4, UGT2B7,

UGT2B17 and UGT1A10 (Kuehl et al. 2005; Sakaguchi et al. 2004; Turgeon et al. 2003; Basu

et al. 2004). The main UGT isoforms expressed in the rat liver and intestine are UGT1A1,

UGT1A6, UGT1A7 and UGT1A8 (Miles et al. 2006; Grams et al. 2000). Previous studies

revealed that conjugation reaction on the carboxyl moiety with glucuronic acid is mediated by

the UGT2B1 in the rat (King et al. 2001) – UGT2B7 in human (Ji et al. 2018) - which is

expressed in an insignificant level in the rat small intestine. On the contrary, the phenol-

glucuronide conjugate of 4-nitrophenol (Almási et al. 2006; Fisher et al. 2015) and capsaicin

(Kozma et al. 2015) could be detected in the intestinal perfusates of similar experiments. These

observations are presumably associated with the preference of the UGT1A1, UGT1A6, and

UGT1A8 isoforms, which catalyze ether-O-glucuronidation reactions (Baojian et al. 2011; Dong

et al. 2012). Our earlier study showed that intestinal UGT activity almost doubled and the beta-

glucuronidase activity increased by more than four times in the STZ-treated rats (Fischer et al.

2015). Taking into consideration that O-acyl glucuronides are reported to be reactive

metabolites, which can easily undergo hydrolysis, intramolecular O-acyl migration, and

nucleophilic displacement reactions (Kuehl et al. 2005), the increased glucuronidase activity

can effectively reduce the intracellular (thus, the excreted) level of the IBP-glucuronide. On the

other hand, O-ether glucuronides are reported to be stable, non-toxic metabolites of alcohols

and phenols (Parkinson et al. 2013).

The gradient HPLC method was successfully applied for the detection of ibuprofen and

its -D-glucuronide in the bile. Besides the oxidative metabolites 2-hydoxyibuprofen and

carboxyibuprofen (Lockwood and Wagner 1982; Geisslinger and Dietzel 1989), ibuprofen-β-

D-glucuronide represents a significant part of the ibuprofen metabolites. Its tendency for

hydrolysis and acyl migration, however, makes it rather unstable; what ensures a complicated

quantitation (Kuehl et al. 2005).

HPLC analysis of the bile samples demonstrated a significantly lower biliary excretion

of IBP and IBP-glucuronide in the STZ-treated rats (Figures 7. and 8.). Similar observation was

made while biliary excretion of 4-nitrophenol, as well as its glucuronide and sulfate conjugates,

were investigated under the same experimental conditions (Toda et al. 2005; Almási et al. 2011,

Almási et al. 2018). These results correspond with the findings that hyperglycemia reduces the

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activity of the UGT2B7 (Dostalek et al. 2011). A similar result was observed in the STZ-treated

rats: UGT activity halved in the hyperglycaemic rats in comparison to the controls (Almási et

al. 2018). Furthermore, expression of the efflux transporter P-glycoprotein (Pgp) has been

reported to be reduced in hyperglycemia (Anger et al. 2009; Nawa et al. 2010; Zhang et al.

2011). The obtained results are also in agreement with our molecular biology studies on liver

samples of the experimental animals. The results showed depressed expression of the efflux

transporters MDR1B (P-gp), MRP2 and BCRP, which are involved in the efflux of organic

anions from the hepatocytes into the bile canaliculus. Furthermore, it was found that expression

of OATP1 (organic-anion-transporting polypeptide-1) – the transporter that participates in

hepatic uptake of organic anions – is also depressed in the STZ-treated hyperglycemic rats

(Garai et al. 2018). The sum of these changes can result in depressed excretion of IBP and IBP

glucuronide in the hyperglycaemic rats.

ConclusionsThe present results show that ibuprofen being a weak organic acid is effectively

absorbed from the small intestine as it was expected. Experimental hyperglycemia caused a

decrease in its intestinal disappearance rate. Contrary to 4-nitrophenol (Almási et al. 2006) and

capsaicin (Kuzma et al. 2015) – both are phenolic derivatives - the glucuronide conjugate of

ibuprofen could not be detected in the small intestinal perfusates. On the other hand, ibuprofen

and ibuprofen-β-D-glucuronide overwhelmingly excreted by the liver, and the biliary excretion

was statistically depressed in hyperglycemic animals. A similar observation was made while

biliary excretion of 4-nitrophenol and its glucuronide conjugate was investigated under the

same experimental conditions (Almási et al. 2011). Our present results demonstrate that

hyperglycemia is able to influence both the intestinal and the biliary excretory processes and

the metabolic reactions as well. Although noticeable similarities between the change of

activities of the intestinal and hepatic metabolic enzymes and transporters in humans and the

experimental animals could be observed, further research on the field is needed for better

understanding of the diabetes-induced pharmacokinetic changes with clinical relevance.

Acknowledgment

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The present contribution is dedicated to the 650th anniversary of the Foundation of University

of Pécs, Pécs, Hungary. This study was supported by the European Union, co-financed by the

European Social Fund (EFOP-3.6.1.-16-2016-00004), the University of Pécs, Pharmaceutical

Talent Center program, and the Transylvanian Museum Society (Romania).

Conflict of interestThe authors declare no conflict of interest. All authors have completed the Unified Competing

Interest form and declare that there was no support from any organization for the submitted

work, no financial relationships with any organizations that may have an interest in the

submitted work in the previous 3 years, and no other relationships or activities that may have

influenced the submitted work.

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Vahabzadeh. M., and Mohammadpour. H.A. 2015. Effect of diabetes mellitus on the metabolism of drugs and toxins . J. Clin. Toxicol. 5:233. doi: 10.4172/2161-0495.1000233

Wang, P., Qi, M., Liu, L., and Fang, L. 2005. Determination of ibuprofen in dog plasma by liquid chromatography and application in pharmacokinetic studies of an ibuprofen prodrug in dogs. J. Pharm. Biomed. Anal. 38: 714-719. doi:10.1016/j.jpba.2005.02.013. PMID:15967300.

Wang-Fischer, Y., and Garyantes, T. 2018. Improving the reliability and utility of streptozotocin-induced rat diabetic model. J. Diabet. Res. 2018: 8054073. doi: 10.1155/2018/8054073.

Wu, B., Basu, S., Meng, S., Wang, X., Zhang, S., and Hu, M. 2011. Regioselective sulfation and glucuronidation of phenolics: Insights into the structural basis of conjugation. Curr. Drug Metab. 12: 900-916. PMID:21933112.

Zhang, L., Lu, L., Jin, S., Jing, X.J., Yao, D., Hu, N., Liu, L., Duan, R., Liu, X.D., Wang, G.J., and Xie, L.2011. Tissue-specific alterations in expression and function of P-glycoprotein in streptozotocin-induced diabetic rats. Acta Pharmacol. Sin. 32: 956-966. doi:10.1038/aps.2011.33. PMID:21685928.

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https://doi.org/10.15406/mojbb.2016.02.00016

http://dx.doi.org/10.1124/dmd.31.5.670

https://doi.org/10.1016/j.jpba.2005.02.013

https://www.ncbi.nlm.nih.gov/pubmed/?term=Wu%20B%5BAuthor%5D&cauthor=true&cauthor_uid=21933112

https://www.ncbi.nlm.nih.gov/pubmed/?term=Basu%20S%5BAuthor%5D&cauthor=true&cauthor_uid=21933112

https://www.ncbi.nlm.nih.gov/pubmed/?term=Meng%20S%5BAuthor%5D&cauthor=true&cauthor_uid=21933112

https://www.ncbi.nlm.nih.gov/pubmed/?term=Wang%20X%5BAuthor%5D&cauthor=true&cauthor_uid=21933112

https://www.ncbi.nlm.nih.gov/pubmed/?term=Zhang%20S%5BAuthor%5D&cauthor=true&cauthor_uid=21933112

https://www.ncbi.nlm.nih.gov/pubmed/?term=Hu%20M%5BAuthor%5D&cauthor=true&cauthor_uid=21933112

https://doi.org/10.1038/aps.2011.33

Draft

Graphical Abstracts

Investigation of intestinal elimination and biliary excretion of ibuprofen in control and

hyperglycemic rats



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Draft

OCOOH

HO

OH

OH

O C CHO

CH3

CH2 CH(CH3)2CH

H3C

H3CCH2 CH COOH

CH3

I II

Fig. 1. The structures of ibuprofen (I) and ibuprofen-β-D glucuronide (II)

Fig. 2. HPLC chromatogram (Method I) of the blank small intestinal perfusate (Fig. 2.A) and the ibuprofen (2) and diclofenac sodium (internal standard) standards (1) (Fig. 2.B). The retention times in the sample of small intestinal perfusate for ibuprofen (2) and diclofenac sodium (1) were 6.11 and 5.21 respectively.

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Draft

Fig. 3. HPLC chromatograms (Method I) of samples generated in rat small intestine luminal perfusion experiment at the beginning (Fig. 3.A.) and at the 90th minute (Fig. 3.B) of the perfusion period. The retention times of diclofenac sodium (1) and ibuprofen (2) were 5.21 and 6.11 min, respectively.

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DraftFig. 4. HPLC chromatograms (Method II) of the blank bile (Fig. 4.A) and the system suitability solution containing ibuprofen-β-D-glucuronide, diclofenac sodium as internal standard and ibuprofen standards (Fig. 4.B.). The retention times of ibuprofen-β-D-glucuronide (1), diclofenac sodium (2) and ibuprofen (3) were 4.74, 10.08 and 10.92 min, respectively.

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DraftFig.5. HPLC chromatogram (Method II) of a bile sample generated in an STZ pretreated rat at the 90th minute of the perfusion period. The retention times of ibuprofen-β-D-glucuronide (1), diclofenac sodium (2) and ibuprofen (3) were 4.74, 10.08 and 10.92 min, respectively.

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Draft

Control+IBP

STZ+IBP

**

0 50 100

time (min)

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*

amou

nt o

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en (n

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time (min)

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500

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Control+IBP

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)

Fig.6. Time-course of the disappearance of ibuprofen in the rat small intestinal perfusate during perfusion of the jejunal loop with isotonic medium containing 250 µM ibuprofen (IBP). Each value represents the average of five independent experiments ± standard error. Significant differences from the control value: * p<0.05 and ** p < 0.01. (STZ: streptozotocin.)

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DraftFig. 7. Changes in the excreted amount of ibuprofen (IBP) into the bile during perfusion of the jejunal loop of rate with isotonic medium containing 250 µM ibuprofen. Each value represents the average of five independent experiments ± standard error. Significant difference from the control value: ** p < 0.01. (STZ: streptozotocin.)

Fig.8. Change in the excreted amount of ibuprofen-β-D-glucuronide in the bile of the rat during perfusion of the jejunal loop with isotonic medium containing 250 µM ibuprofen (IBP). Each value represents the average of five independent experiments ± standard error. Significant difference from the control value: * p<0.05. (STZ: streptozotocin.)

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Draft

Table 1. Intra-day and inter-day precision of retention time and peak areas of ibuprofen (IBP) standard solutions in perfused buffer medium (Method I). The precisions of the peak areas were calculated from the ratios of the peak areas of ibuprofen and diclofenac sodium.

Concentration of standard IBP solution (µM)




(RSD %) (n=5)


retention time( RSD %) (n=5)


(RSD %) (n=5)5 0.53 12.91 2.17 10.40

10 0.55 5.71 2.00 3.41

50 0.64 3.59 1.97 3.17

100 0.67 5.99 1.83 3.46

250 0.57 3.20 2.10 1.58

*IBP: Ibuprofen.

Table 2. Intra-day and inter-day precision of retention time and peak areas of ibuprofen (IBP) in standard solutions in bile (Method II). The precisions of the peak areas were calculated from the ratios of the peak areas of ibuprofen and diclofenac sodium.

Concentration of standard

IBP* solution (µM)




(RSD %) (n=5)




(RSD %) (n=5)7.5 0.10 7.80 1.08 5.56

15 0.10 7.33 0.26 3.79

30 0.14 4.58 0.22 4.35

75 0.11 4.59 0.63 8.76

150 0.09 2.76 0.26 6.19

*IBP: Ibuprofen.

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Draft

Table 3. Intra-day and inter-day precision of retention time and peak areas of ibuprofen-β-D-glucuronide in standard solutions in bile (Method II). The precisions of the peak areas were calculated from the ratios of the peak areas of ibuprofen-β-D-glucuronide and diclofenac sodium.

Concentration of standard IBP-

β-D-glucuronide* solution (µM)




(RSD %) (n=5)




(RSD %) (n=5)

7.5 0.54 5.12 1.34 8.32

15 0.25 5.20 1.19 6.49

30 0.05 4.83 1.05 8.33

75 0.25 3.45 2.45 4.68

150 0.09 5.27 1.78 6.82

* IBP-β-D-glucuronide: Ibuprofen-β-D-glucuronide

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