PHARMACOLOGY
Year : 2010  |  Volume : 2  |  Issue : 4  |  Page : 379-383 Table of Contents     

Modulation of the P-glycoproein-mediated intestinal secretion of glibenclamide: In Vitro and In Vivo assessments


Department of Pharmacology, Vaagdevi College of Pharmacy, Warangal, AP, India

Date of Web Publication14-Oct-2010

Correspondence Address:
P Srirangam
Department of Pharmacology, Vaagdevi College of Pharmacy, Warangal, AP
India
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DOI: 10.4103/0975-1483.71632

PMID: 21264098

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   Abstract 

The everted gut sac method was used to assess the role of the P-glycoprotein (P-gp) on the intestinal secretion of glibenclamide, a prototype of drug used to treat diabetic mellitus. The study included the evaluation of a P-gp modulator carbamazepine used at equimolar doses in the rat. Furthermore, the influence of carbamazepine on the disposition kinetics of glibenclamide in plasma was characterized. For the in vitro experiments, ileal sacs were incubated with glibenclamide in the presence or absence of carbamazepine. In the in vivo experiments, albino rats of either sex were randomly allocated to two groups (n = 6) and oral treatment with glibenclamide (3.6 mg/kg), alone and coadministration with carbamazepine (90 mg/kg). Blood samples were collected at an interval of 1, 2, 4, 6, and 8 h, respectively. Glibenclamide concentrations in both in vitro and in vivo samples were estimated by a sensitive RP-HPLC method. The rate of glibenclamide accumulation in the intestine wall of everted sacs was significantly lower after its incubation with carbamazepine when compared to glibenclamide alone treated. In the agreement with the in vivo and in vitro experiments, the presence of carbamazepine induced an enhancement in the concentrations of glibenclamide in plasma and gastrointestinal tract. The results obtained in this study, both under in vivo and in vitro conditions confirm the relevance of P-gp-mediated transport to the intestinal secretion of glibenclamide.

Keywords: P-glycoprotein, glibenclamide, carbamazepin


How to cite this article:
Srirangam P, Vidya SJ. Modulation of the P-glycoproein-mediated intestinal secretion of glibenclamide: In Vitro and In Vivo assessments. J Young Pharmacists 2010;2:379-83

How to cite this URL:
Srirangam P, Vidya SJ. Modulation of the P-glycoproein-mediated intestinal secretion of glibenclamide: In Vitro and In Vivo assessments. J Young Pharmacists [serial online] 2010 [cited 2012 Mar 20];2:379-83. Available from: /text.asp?2010/2/4/379/71632


   Introduction Top


P-glycoprotein (P-gp) is a transmembrane protein, associated with a phenotype of multidrug resistance to certain anticancer drugs in mammalian cancer cells, that is able to pump a broad range of structurally and functionally unrelated compounds out of the cell by an ATP-dependent process. New in vitro models have been developed to predict in vivo P-gp activity, in which Caco-2 cell monolayers are widely used to estimate transepithelial passage of different P-gp substrates. [1],[2] However, the correlation between this in vitro model and in vivo studies is rather poor. The use of everted gut sacs has been proposed as a new in vitro model for quantification of P-gp-mediated intestinal efflux for various drugs.

This study is planned to evaluate the safety of glibenclamide (an anti-diabetic drug) therapy in the presence of carbamazepine (a prototype drug used to treat painful diabetic neuropathy) in healthy rats. The interaction mechanisms in preclinical studies help to avoid adverse effects when this particular combination was administered to patients suffering with painful diabetic neuropathy.


   Materials and Methods Top


Drugs and chemicals

Glibenclamide and carbamazepine are the gift samples from Alka Pharmaceuticals (Hyderabad, India). D-PBS (Sigma-Aldrich, USA), the HPLC grade methanol and acetonitrile of Qualigens Fine Chemicals, Mumbai were procured from local chemical suppliers. All other chemicals used were of analytical grade.

Animals

Experiments were performed with albino rats procured from Mahaveera Enterprises (Hyderabad, AP, India), weighing between 180 and 210 g. The animals were housed in colony cages (four per cage) under conditions of standard lighting, temperature (22 ± 1°c) and humidity for at least 1 week before the beginning of experiment, to adjust to the new environment and to overcome stress possibly incurred during transit. During this period, they had free access to food and water. The experiments were planned after the approval of Institutional Animal Ethical Committee (IEAC), Vaagdevi College of Pharmacy, Warangal, AP, India.


   Methods Top


In vitro experiment

The experimental procedure was performed by modified method of Yumoto et al.[3] The whole small intestine was isolated and flushed with 50 mL of ice-cold saline after killing the animal by overdose of pentobarbital. The small intestine was divided into duodenum, jejunum, and ileum. Ileul segment was everted, and a 10-cm long sac was prepared (i.e., P-gp is more expressive in ileum). About 1 mL of glibenclamide solution (1 mg/mL) was introduced into the everted sac (serosal side) and both ends of the sac were ligated tightly. The sac-containing glibenclamide solution was immersed into 30 mL of Dulbecco's phosphate buffer solution (D-PBS) containing 25 mM of glucose, and the same concentration of carbamazepine in DMSO was introduced into the mucosal side and the solution was prewarmed and oxygenated throughout the experiment. The transport of glibenclamide from serosal to mucosal surface across the intestine was measured by collecting samples from the mucosal medium periodically at different intervals 0, 10, 20, 30, 60, 90, and 120 min and all samples were conserved at -20° C until analysis. Using this medium, the transport of glibenclamide in the absence (control) and presence (test) was measured.

In vivo experiment

Albino rats of either sex were randomly distributed into two groups of six animals in each group. Before experiment, all animals were fasted for 18 h and water ad libitum, water was withdrawn during experiment. After collection of initial blood samples, drugs were administered in the following order. Animals in Group A (GLB alone) received GLB at 3.6 mg/kg by oral route and rats in Group-B (GLB + CAR) received CAR (90 mg/kg) before 1 h of GLB (3.6 mg/kg) treatment by oral route. The blood samples were collected from orbital sinuses using heparinized capillaries into a microcentrifugation tubes contain anticoagulant at 1, 2, 4, 6, and 8 h after treatment. Plasma was separated by centrifugation and stored at −20°C until further analysis.

Bioanalytical method

Glibenclamide concentrations from in vitro and in vivo samples were determined with a validated high-performance liquid chromatography (HPLC) method. Briefly, the HPLC system consisted of a Waters 717 plus Autosampler (Waters Co., Milford, MA, USA), a Waters 501 pump (Waters Co), and a 785 UV Detector (Applied Biosystems, Foster City, CA, USA) operated at 253 nm. The stationary phase was a Waters Symmetry C18 column (250 mm × 4.6 mm, 5 μm, Waters Co.). The mobile phase used was 25 mM sodium phosphate/acetonitrile (65:35, v/v, pH = 3.5) at a flow rate of 1.0 mL/min. Glibenclamide and an internal standard (glipizide) were isolated from plasma by liquid-liquid extraction with methanol. The organic phase was separated and evaporated, and the remaining residue was reconstituted with 250 μL of mobile phase before applied to the HPLC system. The method was validated and found to be linear over the concentration range of 0.1-10 μg/mL. Using a linear-weighted least squares regression, the lower limit of quantitation (LLQ) was 0.1 μg/mL. The intra-assay and inter-assay coefficients of variation (%CV) for the three quality control standards (0.25, 2.00, and 8.00 μg/mL) were ≤10.2% and 4.9%, respectively.

Pharmacokinetic analysis

The pharmacokinetic parameters of glibenclamide were calculated using a sophisticated tool known as Win Nonlin (4.1) and the parameters includes half-life (t1/2 ), clearance (Cl), volume of distribution (Vd ), Cmax , Tmax , and area under the concentration-time curve (AUC).

Statistical analysis

Data were expressed as mean ± standard deviation (SD). The significance was determined by applying Student's paired t-test.


   Results Top


In vitro experiments

The time course of glibenclamide transport across the everted ileum of healthy rats was shown in [Figure 1]. The percentage of glibenclamide remain present in ileum are shown in [Figure 2], and results of carbamazepine inhibition at different time points are given in [Table 1].
Table 1 :Glibenclamide (μg/mL) transport in the everted ileum of healthy rat in the presence and absence of carbamazepine


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Figure 1 :Transport of glibenclamide from serosal to mucosal side in the everted ileum of the healthy rat in presence and absence of carbamazepine (n = 6)

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Figure 2 :Percentage of glibenclamide remaining in the everted ileum of healthy rat at different time points in the presence and absence of carbamazepine (n = 6)

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In vivo experiments

The plasma glibenclamide levels and pharmacokinetic parameters of glibenclamide such as AUC, T1/2, clearance, Vd , Cmax , and Tmax were altered significantly when comparing the results between GLB alone and combination group (GLB + CAR), and the results are shown in [Table 2], [Table 3] and [Figure 3], respectively.
Table 2 :Mean plasma glibenclamide concentration (μg/m) before and after oral administration of carbamazepine in healthy rats (n = 6)


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Table 3 :The comparison of pharmacokinetic parameters of glibenclamide (3.6 mg/mL) following retreatment with carbamazepine (90 mg/kg) by oral administration in healthy rats (n = 6)


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Figure 3 :Mean ± SD plasma concentration-time profile of glibenclamide (3.6 mg/mL) following pretreatment with carbamazepine (90 mg/kg) by oral administration in healthy rats (first day)

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   Discussion Top


The pharmacological effects of a given drug compound are highly influenced by its pharmacokinetic behavior. Physicochemical properties are critical to the absorption, distribution, metabolism, and excretion of different xenobiotic compounds. However, it has now become apparent that different transport proteins play an important role in regulating the kinetic disposition of several drugs. [4]

In addition, clinically relevant drug interactions may occur after the concomitant administration of various compounds. The goal of the interaction studies between P-gp substrate and modulator has been addressed to identify the pharmacokinetic consequences and to predict the clinical outcome.

Several approaches are available to assess in vitro role of the specific proteins involved in the intestinal drug transport processes. However, the inference from the quantitative information obtained in the in vitro models to the in vivo situation may be limited. Thus, the prediction of the absorption process for a given P-gp substrate from in vitro assays could not be correlated to the in vivo results obtained when coadministration with a P-gp modulator is performed. [5] This outcome could be due to differences in the length of drug exposure, drug concentrations, concomitant induction of metabolic pathways, and extent of P-gp inhibition. [6]

Previous in vitro studies reported that GLB was actively secreted by multidrug-resistant tumor cells and by cells transfected with the gene coding for P-gp in the mouse. In addition, Barthe et al. [7] described the everted gut sac as a simple method to study intestinal absorption of digoxin in the presence or absence of verapamil or quinidine, obtaining a high correlation with the in vitro Caco-2 cells [8] and in vivo methods using knockout mice. The results of the current trial confirm that the everted sac technique is a useful in vitro model system for studying the P-gp-mediated efflux of GLB. The satisfactory results obtained with the use of the everted sac model to assess the P-gp-mediated modulation of GLB transport may be relevant to achieve reproducible results between the in vitro and in vivo assays.

The GLB concentration profiles measured in the ileal wall was markedly modified by the presence of CAR. The GLB tissue accumulation at 60 min of incubation in the presence of CAR was 20.1-fold higher compared with the incubation of GLB alone. Therefore, the rate of GLB accumulation in the intestinal wall was significantly higher in the presence of CAR. Thus, these results demonstrated that the everted sac technique was useful to compare the P-gp inhibition by modulators like CAR from different generations used at equimolar doses.

Interestingly, the results reported in our in vitro experiments were directly related to the observed in vivo changes in the pharmacokinetic behavior of GLB after coadministration with CAR [Table 3]. This is a relevant issue, considering that in previous studies, it was not always possible to reproduce in vivo results observed in in vitro. For instance, where as ketoconazole seemed to be the most valuable tool for increasing the intracellular quantity of [14C] moxidectin in rat hepatocyte cell cultures, the compound had no effect on the in vivo pharmacokinetics of moxidectin in lambs. [9],[10],[11],[12],[13],[14]


   Conclusion Top


The results from the studies reported here indicate that the plasma disposition of GLB was strongly affected by P-gp activity. In addition, we have demonstrated that GLB disposition kinetics in the intestinal tissues was significantly modified by the presence of CAR, under both in vivo and in vitro conditions, which confirms the relevance of this cellular transporter on the intestinal secretion of GLB. The characterization of the intestinal elimination pathway for GLB would be of therapeutic significance and the high GLB concentrations could be available at the target tissues, and which improving its clinical efficacy.

 
   References Top

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2.Lin JH. Drug-drug interaction mediated by inhibition and induction of P-glycoprotein. Adv Drug Deliv Rev 2003;55:53-81.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]  
3.Yumoto R, Murakami T, Nakamoto Y, Hasegawa R, Nagai J, Takano M. Transport of rhodamine 123, a P-glycoprotein substrate, across rat intestine and Caco-2 cell monolayers in the presence of cytochrome P-450 3A-related compounds. J Pharmacol Exp Ther 1999;289:149-55.   Back to cited text no. 3  [PUBMED]  [FULLTEXT]  
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5.Yee S. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth. Pharm Res (NY) 1997;14:763-6.  Back to cited text no. 5      
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7.Barthe L, Bessouet M, Woodley J, Houin G. The inproved everted gut sac: a simple method to study intestinal P-glycoprotein. Int J Pharm 1998;173:255-8.  Back to cited text no. 7      
8.Cavet ME, West M, Simmons NL. Transport and epithelial secretion of the cardiac glycoside, digoxin, by human intestinal epithelial (Caco-2) cells. Br J Pharmacol 1996;118:1389-1396.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]  
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10.Fisher GA, Sikic BI. Clinical studies with modulators of multidrug resistance. Hematol Oncol Clin North Am 1995;9:363-82.  Back to cited text no. 10  [PUBMED]    
11.Fojo AT, Ueda K, Slamon DJ, Poplack DG, Gottesman MM, Pastan I. Expression of the multidrug-resistance gene in human tumors and tissues. Proc Natl Acad Sci USA 1987;84:265-9.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]  
12.Garrigos M, Mir LM, Orlowski S. Competitive and non-competitive inhibition of the multidrug-resistance-associated P-glycoprotein ATPase-further experimental evidence for a multisite model. Eur J Biochem 1997;244:664-73.  Back to cited text no. 12  [PUBMED]  [FULLTEXT]  
13.Stephens RH, O'Neill CA, Warhurst A, Carlson GL, Rowland M, Warhurst G. Kinetic profiling of P-glycoprotein-mediated drug efflux in rat and human intestinal epithelia. J Pharmacol Exp Ther 2001;296:584-91.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]  
14.Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. Cellular localization of the multidrug-resistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA 1987;84:7735-8.  Back to cited text no. 14  [PUBMED]  [FULLTEXT]  


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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