pH-Dependent Functional Activity of P-Glycoprotein in Limiting Intestinal Absorption of Protic Drugs: Kinetic Analysis of Quinidine Efflux In Situ MANTHENA V.S. VARMA, RAMESH PANCHAGNULA Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Phase X, SAS. Nagar, Punjab 160062, India

Received 3 March 2005; revised 29 June 2005; accepted 29 June 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20489

ABSTRACT: The purpose of this investigation is to evaluate the quantitative contribution of pH-dependent passive permeability on the functional activity of P-glycoprotein (P-gp) in limiting intestinal absorption of weakly basic drugs, in order to include this effect in prediction models. pH-dependent octanol/buffer partition coefficient, artificial membrane permeability and in situ rat intestinal permeability of quinidine were determined in the physiological pH range of gastrointestinal tract. In situ permeability, as a function of luminal pH, was also determined in the presence of P-gp inhibitor, verapamil (500 mM). Octanol/buffer partition coefficient, transport across artificial membrane, and rat in situ permeability showed high pH-dependency. Absorption quotient (AQ), calculated from in situ permeability to express the functional activity of Pgp, declined with increase in luminal pH or increase in luminal quinidine concentration because of the increased passive permeability or saturation of P-gp. AQ was 0.57
0.02 and 0.41
0.05, while passive permeability was 0.32
0.01  104 cm/sec and 0.43
0.02  104 cm/sec, in jejunum and ileum, respectively, at pH 7.4. Further, apparent Michaelis–Menten constants (KM, JP-gp,max) for the quinidine efflux in jejunum indicated that efflux activity was more at luminal pH 4.5 over pH 7.4. KM values for jejunum quinidine efflux at pH 4.5 and pH 7.4 were determined to be 77.63
10.90 and 22.86
5.22 mM, with JP-gp,max values of 1.47
0.08 and 0.62
0.04 nM/cm2/sec, respectively. AQ vs passive permeability showed significant relationship indicating dependency of P-gp-mediated efflux on pH-dependent passive permeability, which is dictated by ionization status for a protic or ampholytic drug. In conclusion, an orally administered drug is absorbed from various segments of intestine, which inherit difference in luminal pH, transcellular permeability and P-gp expression. In situ data suggests that pH-dependency and regional variability in passive permeability of protic substrates significantly influence their P-gp-mediated efflux and may have implications on predictions of the in vivo drug absorption. ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2632–2643, 2005

Keywords: pH dependency; passive permeability; P-glycoprotein efflux; prediction; oral absorption

INTRODUCTION Correspondence to: Ramesh Panchagnula (Telephone: 91172-214700; Fax: 91 172 214692; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 94, 2632–2643 (2005) ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association

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Absorption of drugs from the gastrointestinal tract is determined by fundamental processes, solubility and permeability.1 Permeability being complex, is still unpredictable determinant and it

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is informative to explore mechanisms contributing to permeability given the interest in development of highly reliable computational models of this property. P-glycoprotein (P-gp), an energydependent transmembrane drug efflux pump, is localized in wide range of tissues including enterocytes.2 It is believed that this multidrug transporter hinders absorption of various orally administered drugs by attenuating intestinal permeability and by potentially enhancing intestinal metabolism of substrates that are subjected to first pass gut metabolism.3 The contribution of P-gp in limiting intestinal absorption of drugs is determined by: (i) affinity of drug towards P-gpmediated efflux, (ii) expression levels of P-gp and variability in expression levels across the intestinal tract, and (iii) the passive permeability of the drug molecules across the enterocytes.4 In our previous studies, we showed that transport of P-gp substrates with moderate to less passive permeability is highly attenuated by P-gp, while passive permeability overrules the P-gpmediated efflux for high permeability molecules.5 Further, we proposed a quantitative model to predict influence of P-gp-mediated efflux on in vivo drug absorption, based on the P-gp activity and passive permeability of P-gp substrates.6 For protic or ampholytic drugs, charge state is an important factor associated with their passive permeability.7,8 The membrane permeability of uncharged species of a drug molecule is much higher than that of charged species. Thus, oral absorption of protic drugs depends on the epithelial permeability of unionized form of the drug, its pKa(s), and the relative difference in permeability between unionized form and the ionized form.9–11 As the protic drug moves down the GI tract, it experiences various pH conditions and exists with varying degree of ionization, which may determine the passive permeability. It should be appreciated that both passive transport and P-gp efflux processes operating in mutually opposite directions, contribute to overall drug permeability and thus influence the absorption process.3 With this background, it was hypothesized that the functional activity of P-gp to efflux protic drugs may alter as a function of luminal pH. This study was designed to investigate the pH-dependent functional role of P-gp in limiting protic drug permeability and its implications on predicting in vivo intestinal drug absorption. Permeability studies were carried with rat in situ single-pass intestinal perfusion model, as they are more reliable for predicting in vivo

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absorption in humans.12–14 Much of the information on drug interactions with P-gp has come from use of cultured epithelial models such as Caco-2 and MDRI-MDCKII.15,16 Although these models provide lots of mechanistic information, the relevance of these findings to drug permeability in normal intestine is doubtful, given that these monolayers are derived from colonic tumor cells (Caco-2) and often over-express efflux transporters and exhibit permeability characteristics different from normal epithelium, further with large lab-to-lab and batch-to-batch variability.17,18 In the present study, in situ rat permeability of a weakly basic P-gp substrate, quinidine, was determined along with permeability markers without and with P-gp inhibitor, in the pH range 4.5 to 8.0. Furthermore, the kinetic profiling at pH 4.5 and pH 7.4 was also investigated. Finally, quantitative relationship between passive permeability of quinidine and the functional activity of P-gp as a function of luminal pH was established to predict the contribution of pH-dependent passive transport to the P-gp-mediated efflux and the total permeability.

MATERIALS AND METHODS Chemicals Frusemide was obtained from Dr. Reddy’s Lab. (Hyderabad, India). Hydrochlorthiazide was received from Aristo Pharmaceutical Ltd. (Daman, India). Propranolol HCl was from Sun Pharmaceutical Industries Ltd. (Mumbai, India). Verapamil and quinidine were purchased from Sigma-Aldrich Co. (MO). Lecithin and dodecane were purchased from Himedia Lab. Pvt. Ltd., (Mumbai, India). n-octanol and DMSO were procured from Sigma-Aldrich Co. All the other solvents used were of HPLC grade (J.T. Baker, Mexico) and reagents and chemicals were of analytical grade. Measurement of Partition Coefficient (Log D) and Apparent Artificial Membrane Permeability (PPAMPA) pH-lipophilicity profile of quinidine in n-octanol/ buffer system was obtained at 37
58C by the shake-flask procedure and analyzed by HPLC. Artificial membrane permeability studies were performed in the same manner described previously.19–21 In brief, a 96-well microplate (acceptor compartment) was completely filled with phosphate buffer (pH 7.4) containing 5% DMSO.

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Each filter of the donor plate (Millipore Corp., Bedford, MA) was impregnated with 5 mL of 10% (w/v) lecithin in dodecane. A 200 mL of 100 mM quinidine solution in buffers in the pH range of 3 to 10 (n ¼ 4 for each pH) was added immediately and incubated at room temperature for 16 h. A reference solution defining equilibrium conditions was prepared at the same pH as the sample solution with no membrane barrier. The filter surface was wetted with 5 mL of a 60% (v/v) methanol/buffer solution for the reference. Quinidine concentration in the acceptor compartment was quantified by HPLC. Apparent artificial membrane permeability (PPAMPA) was calculated as previously reported.21 Permeability Studies: In Situ Perfusion Experiments Sprague–Dawley rats (270–350 g) were used to perform in situ single-pass perfusion. Anesthesia, surgical and perfusion procedures were justified in detail and were approved by the Institutional Animal Ethics Committee (IAEC, NIPER). The study complied with local and federal requirements for animal studies. Surgical procedure and the in situ single-pass perfusion experiments were performed according to the methods described previously.12,22,23 Rats were fasted for 16–18 h, prior to study, with tap water ad libidum. After anesthesia via intraperitoneal administration of thiopental sodium (50 mg/kg), rats were placed on a heating pad to maintain body temperature at 378C. Intestine was exposed by a midline abdominal incision and 8– 10 cm jejunum or ileum segment was cannulated at both ends with glass tubing. Isolated intestinal segment was rinsed with phosphate buffer saline (10 mL) and the perfusate solution maintained at 378C was pumped at a flow rate of 0.2 mL/min using syringe pump (Harvard Apparatus PHD 2000 pump, MA). Perfusion solution (pH 7.4) consisted of NaCl 48 mM, KCl 5.4 mM, Na2HPO4 2.8 mM, NaH2PO4 4 mM and D-glucose 1 g/L; and contained drugs with or without verapamil (500 mM) as P-gp inhibitor. Test solutions were prepared immediately before use by dissolving each component in the perfusion buffer. Final concentrations used in the studies were 50 mM, hydrochlorthiazide; 100 mM, propranolol; 50 mM, frusemide and 3–300 mM, quinidine. Concentrations were low enough to avoid precipitation in the lumen during the course of the study. After dissolving the drugs, the pH of the solution was readjusted when necessary.

A two-step perfusion procedure was followed to determine the permeability of compounds with and without P-gp inhibitor.23 This included sampling every 5 min for a 30 min perfusion period with perfusion solution (without P-gp inhibitor) after 20 min equilibrium, and then switching to second perfusion solution (with P-gp inhibitor) and similarly sampling every 5 min for 30 min perfusion period after 20 min equilibrium. Equilibrium of 20 min prior to sampling was found to be sufficient for both washout and to reach an initial steady-state.23 Water flux was quantified based on direct measurement of the volume at the outlet.20 To further keep a check on the intra- and interindividual variability and presence of steady state, permeability of propranolol (passive, highly permeable), frusemide and hydrochlorthiazide (passive, low permeable) were monitored, by coperfusing with quinidine.

Bioanalysis by RP-HPLC RP-HPLC with dual wavelength UV detector was used for simultaneous quantification of quinidine, permeability markers and P-gp inhibitor, verapamil.24 In brief, HPLC was conducted on Waters (Waters Corp., MA) equipped with 600 controller pumps, Waters 2487 UV–Vis dual l absorbance detector and configured to Millenium32 software. Perfusion sample was loaded onto the column by means of 717plus autosampler (Waters Corp.). Reversed-phase column used for chromatographic separations was 4.6 mm id, 250 mm length and 5 mm particle size C18 (Symmetry1, Waters) attached to a guard column of C18 (Waters). Chromatographic separations were accomplished using mobile phase consisting of acetate buffer (10 mM, adjusted to pH 5.0 with glacial acetic acid) and methanol (40:60 v/v), pumped in isocratic mode at a flow rate of 0.6 mL/min at ambient temperature. Samples of 20 mL were automatically injected after filtration through 0.45 mm filter (Millipore). Elution of quinidine, propranolol and verapamil was monitored at 230 nm and frusemide and hydrochlorthiazide was monitored at 275 nm, using a dual wavelength detector.

Data Treatment and Statistics Permeabilities (without and with P-gp inhibitor) are calculated after correcting the outlet concentration for water flux on the basis of the ratio of

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volume of perfusion solution collected and infused for each sampling point (5 min).20    Cin Peff;Control ðorÞ Peff ;Inh ¼ Q: 1 2Prl Cout ð1Þ Where Q is the flow rate, Cin and Cout are the respective inlet and outlet concentration, r is radius of intestine (0.21 cm), and l is length of intestine measured after completion of perfusion.12,25 In order to evaluate the quantitative contribution of P-gp to limit the intestinal absorption, we calculated the absorption quotient (AQ).26 The permeability obtained by in situ perfusion without and with P-gp inhibitor can be described by Equations (2) and (3) respectively. Peff;Control ¼ PPD  PPgp

ð2Þ

Peff;Inh ¼ PPD

ð3Þ

Where PP-gp is permeability due to P-gp efflux and PPD is passive permeability. Considering one-site Michaelis–Menten saturable kinetics for P-gp substrates,3,15,27 AQ can be obtained by: AQ ¼

Peff ;Control PPgp ¼1 PPD Peff;Inh

ð4Þ

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within the pH range of 2.0 and 10.0. Quinidine, a diprotic base with pKas 4.2 and 7.9, showed pHdependent n-octanol/buffer partition coefficient and PPAMPA in the GI pH range (Fig. 1). Log D of quinidine is 1.98
0.55 at pH 2.0, 0.98
0.00 at pH 4.5, and 1.37
0.05 at pH 7.4. The log PPAMPA profile as a function of donor pH ranged from 1.80 to 4.18 cm/s. Theoretical profiles were generated based on the pKas and the log P or log PPAMPA,O, where P and PPAMPA,O are octanol/ buffer partition coefficient and artificial membrane permeability when quinidine exists as completely unionized species.8 Theoretical and experimental data indicated that quinidine is a passively permeable molecule with partition coefficient and artificial membrane permeabilities exponentially increasing in the physiological pH range of 4.5 to 8.0. pH-Dependent In Situ Permeability and Regional Variability Quinidine (10 mM) exhibited marked difference in the in situ permeability when perfused in the absence and presence of P-gp inhibitor, verapamil (500 mM), indicative of efflux by P-gp. Further, permeability in the absence of P-gp-mediated transport is significantly higher than that in the

AQ quantifies the passive drug transport attenuation by P-gp across the intestinal enterocytes and provide information on the extent to which Pgp influences intestinal absorption of P-gp substrates across the enterocytes. The active transport flux rates were estimated by subtracting the control flux rate (under the influence of efflux transport) from the passive diffusion flux rate obtained from the same rat. Michaelis Menten kinetics (Km, JP-gp,max) and other nonlinear regression analysis were obtained using PRISM 2.01 (GraphPad Software Inc., San Diego, CA). Values were indicated as mean
SEM for permeability in four independent rats. Statistical difference between the permeabilities of the drugs without and with P-gp inhibitor, and kinetic parameters was evaluated by Student’s t-test (SigmaStat version 2.03, SPSS Inc., IL) at p < 0.05.

RESULTS Lipophilicity (log D) and Artificial Membrane Permeability (PPAMPA) Profiles of Quinidine Log D in n-octanol/buffer system and permeability across artificial membranes was investigated

Figure 1. pH-dependent octanol/buffer partition coefficient (^) and artificial membrane permeability (~) profiles of quinidine. Artificial membrane constituted of hydrophobic filter impregnated with 5 mL of 10% (w/v) lecithin in dodecane. Solid and dashed curves represent theoretical profiles generated based on the pKas and the log P or log PPAMPA,O, respectively.7,8 Values are mean
SEM (n ¼ 4).

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presence of P-gp-mediated transport (p < 0.01), in the investigated pH range of 4.5 to 8.0, in both jejunum and ileum (Fig. 2). Permeability in the absence and presence of verapamil was 0.03
0.00  104 cm/s and 0.20
0.00  104 cm/s, respectively, in jejunum at luminal pH 4.5. However, permeability increased significantly (p < 0.05) at pH 7.4 and pH 8.0. Both in the presence and absence of P-gp efflux, quinidine permeability was high in ileum over the jejunum. Functional activity of P-gp was quantified by AQ, which is the ratio of P-gp-mediated permeability (PP-gp) and complete passive permeability (PPD or Peff,Inh). AQ was found to decline as a function of luminal pH in both the intestinal regions. In jejunum, at pH 4.5, AQ was 0.82
0.03, indicating about 82% of passive permeability of quinidine is attenuated by P-gp-mediated efflux. However, AQ was significantly less (p < 0.05) than that at pH 4.5, when the luminal pH was above pH 6.5. Similar to jejunum, ileum also demonstrated large difference in AQ when pH was changed from pH 4.5 to pH 7.4, however, AQ was significantly less than that in jejunum at the corresponding pH. The permeability values of transcellular markers in the pH range without and with verapamil are given in Table 1. No significant change was observed for acidic markers, hydrochlorthiazide and frusemide, at different luminal pH or in the

presence of P-gp inhibitor. Weakly basic marker, propranolol, showed increased permeability with increase in pH, however, no significant change was observed between Peff,control and Peff,Inh at corresponding pH. Regional difference was also observed for propranolol with high permeability values in ileum.28 pH-Dependent Kinetic Profiling of P-gp-Mediated Efflux Figure 3 shows in situ jejunum permeability of quinidine in the absence and presence of verapamil (500 mM), as a function of drug concentration, at pH 4.5 and 7.4. Passive permeability (Peff,Inh) of quinidine (300 mM) was 0.20
0.02  104 cm/s and 0.32
0.03  104 cm/s at pH 4.5 and pH 7.4, respectively, and was found to be constant irrespective of quinidine concentration, at the particular pH. Peff,control showed concentrationdependency at both the pH investigated. At pH 4.5, Peff,control was increased from 0.03
0.01  104 cm/s to 0.15
0.02  104 cm/s, when the luminal concentration was increased from 3 to 300 mM. While at pH 7.4, increment in permeability as a function of concentration was relatively more, with Peff,control being 0.06
0.02  104 cm/s at luminal quinidine concentration 3 mM and 0.30
0.02  104 cm/s at 300 mM. No

Figure 2. Effect of luminal pH on the in situ permeability, in the absence and presence of P-gp inhibitor, and P-gp activity (AQ) of quinidine in jejunum and ileum. Quinidine (10 mM) along with permeability markers and without or with verapamil (500 mM) was perfused in situ. Permeability increased as a function of pH in jejunum and ileum, however, with regional variability in permeability and P-gp activity. Data is represented as mean
SEM (n ¼ 4). The significance of difference from the control at corresponding pH (*p < 0.05) and from corresponding permeability of pH 4.5 (#p < 0.05) were determined by Student’s t-test. Key: Ver, verapamil. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 12, DECEMBER 2005

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Table 1. The Effective Permeability Values of Transcellular Markers in the pH-Dependent In Situ Studies In situ Rat Jejunum Permeabilitya Compound Hydrochlorthiazide

Frusemide

Propranolol

Luminal pH Jejunum 4.5 5.5 6.5 7.4 8.0 Ileum 4.5 7.4 Jejunum 4.5 5.5 6.5 7.4 8.0 Ileum 4.5 7.4 Jejunum 4.5 5.5 6.5 7.4 8.0 Ileum 4.5 7.4

Peff,control (104 cm/s)

Peff,in (104 cm/s)b

0.072
0.015 0.061
0.029 0.047
0.013 0.052
0.013 0.026
0.011

0.080
0.012 0.047
0.030 0.028
0.025 0.051
0.006 0.024
0.015

0.095 0.304 0.689 0.024 0.113

0.082
0.015 0.075
0.024

0.081
0.009 0.069
0.018c

0.006 0.087

0.043
0.010 0.023
0.011 0.034
0.008 0.035
0.014 0.036
0.018

0.050
0.020 0.030
0.010 0.049
0.010b 0.041
0.020 0.043
0.030

0.146 0.227 0.178 0.155 0.169

0.061
0.021 0.046
0.019

0.046
0.014 0.042
0.019

0.326 0.095

0.239
0.057 0.328
0.089 0.425
0.023 0.483
0.035 0.597
0.059

0.254
0.015 0.310
0.054 0.428
0.028 0.563
0.033 0.632
0.045

0.059 0.056 0.007 0.141 0.056

0.349
0.035c 0.742
0.079c

0.368
0.042c 0.718
0.095

0.052 0.033

AQ

a In situ permeability values were measured in the absence (Peff,control) or presence (Peff,inh) of P-gp inhibitor, in jejunum and ileum at physiologically relevant pH range. Values are presented as mean
SEM (n ¼ 4). b Values of Peff,control and Peff,inh were not significantly different (p > 0.05), except where noted. c Significant difference (p < 0.05) from corresponding jejunum Peff,control or Peff,inh, at same luminal pH.

significant difference was observed between Peff, control and Peff,Inh of quinidine (300 mM) at pH 7.4, however, at pH 4.5, Peff,control is significantly less than Peff,Inh (p < 0.05). AQ for 3, 100, and 300 mM was 0.83, 0.43, and 0.26, respectively, at pH 4.5, while at pH 7.4, the values were 0.82, 0.18, and 0.06, respectively, at same concentrations. It is interesting to observe that although the functional activity of P-gp (AQ) is almost same at lowest concentration at both the luminal pH conditions, it reduced significantly with relatively small increments in quinidine concentration, at pH 7.4. Active transport flux of quinidine in situ was concentration dependent and saturable at both the pH 4.5 and pH 7.4 (Fig. 4). Table 2 shows the

apparent Michaelis–Menten constants (Km, JPgp,max) for quinidine transport by P-gp. It is evident from the results, that active transport was saturable with relatively lower concentration at pH 7.4 over pH 4.5. Verapamil permeability was also determined in each study by taking the advantage of simultaneous quantification HPLC method.24 Verapamil, a monoprotic base with pKa 6.5, showed pHdependent permeability (Fig. 5). Presence of quinidine in the concentration range of 3–300 mM showed no significant change in verapamil permeability, at both pH 4.5 and pH 7.4, indicating that verapamil at 500 mM concentration completely inhibited efflux transport irrespective of luminal pH.11

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Figure 3. Concentration dependency of the quinidine in situ permeability at luminal (A) pH 4.5 and (B) pH 7.4. Inset shows quinidine concentration-dependent functional activity of P-gp (AQ) at the corresponding pH. Quinidine permeability, in the presence and absence of verapamil (500 mM), was carried in the concentration range of 3–300 mM. Data is represented as mean
SEM (n ¼ 4). Key: Ver, verapamil.

Relationship Between Passive Permeability and AQ AQ values, obtained from in situ permeability studies in the luminal pH range of 4.5 to 8.0, were plotted against in situ passive permeability

(Peff,Inh), of the corresponding pH (Fig. 6A). It is obvious from the results that functional activity of P-gp in effluxing quinidine declined with increase in passive permeability, which for a basic drug is a function of degree of ionization as the pH increases (see Fig. 2). Data fitted to the relationship AQ ¼ PP-gp/Peff,Inh keeping the PP-gp constant, obtained from the mean of PP-gp at different pH in jejunum. Further, AQ showed linear relationship with log D and log PPAMPA (Fig. 6B and C).

DISCUSSION

Figure 4. Concentration dependency of the active transport flux rates of quinidine in jejunum at luminal pH 4.5 and pH 7.4. The passive diffusion flux rates of quinidine were determined over a concentration range of 3–300 mM in the presence of verapamil (500 mM) and the effective permeability flux rates were determined in the absence of any P-gp inhibitor. The active transport flux rates were determined by subtracting the effective permeability flux rates from passive diffusion flux rates, at the corresponding quinidine concentration (Eq. 4). Data points are mean
SEM (n ¼ 4). The significance of difference from the active transport flux rate at pH 4.5 (*p < 0.05) were determined by Student’s t-test. Key: ver, verapamil.

Currently there is immense interest in predicting the role of drug efflux in the poor and/or variable absorption of drugs administered via oral route.29 In this study, we have provided physiologicallyrelevant evidences and a quantitative relationship to describe and predict the influence of pH-dependent passive permeability on P-gpmediated efflux of a weakly basic drug, using single-pass rat intestinal perfusion model. The data presented here represent the first attempt to demonstrate quantitative contribution of pHdependent passive permeability on the P-gpmediated efflux and its kinetic profiling in rat in situ. Quinidine was chosen as the substrate for these studies because of various reasons that include: (i) its strong affinity to P-gp and

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Table 2. Apparent KM and Vmax Values for the Active Transport of Quinidine In Situ, at Luminal pH 4.5 and pH 7.4 Michaelis–Menten Constantsa KM (mM) JP-gp,max (nM/cm2/sec)

pH 4.5

pH 7.4

77.63
10.90 1.47
0.08

22.86
5.22b 0.62
0.04b

a KM and JP-gp,max reveals the affinity constant of quinidine for the efflux transport and the maximal active transport flux rate, respectively. Results are obtained by best-fit of data in Figure 4 to Michaelis–Menten kinetics and are expressed as mean
SEM (n ¼ 4). b Significant difference ( p < 0.05) when compared with the values at luminal pH 4.5.

susceptibility to drug efflux and also demonstrated pH-dependent P-gp-mediate drug interactions in Caco-2 monolayers,30 (ii) availability of a wealth of data demonstrating its interaction with P-gp in vitro, in situ, and in vivo,31 (iii) quinidine is a diprotic base and may demonstrate pH-dependent passive permeability as predicted from structure based theoretical log D profiles (data not shown; Pallas 2.0, CompuDrug Chemistry Ltd.), and (iv) being sufficiently permeable, it is expected to produce less variable results in the in situ model.31 Quinidine showed pH-dependent octanol/buffer partition coefficient and permeability across artificial membrane. Experimental results closely

followed the theoretical profiles based on pKas and log P or PPAMPA,O indicating that quinidine is transported by transcellular diffusion mechanism, and the degree of ionization dictated by the pH influences the passive permeability of quinidine. Based on these results, it is anticipated that passive permeability of the drug is highly influenced by the luminal pH in vivo.19,32 Moreover, pH-dependent in situ permeability of quinidine (10 mM) was demonstrated under both the presence and absence of P-gp-mediated efflux, in jejunum and ileum. AQ was 30.5% less in pH 7.4 than that of pH 4.5 in jejunum, suggestive of a significant decrease in P-gp efflux as a function of pH. Thus, quinidine may be considered as high

Figure 5. pH-dependent and quinidine concentration-dependent permeability of verapamil (500 mM). pH-dependent verapamil permeability values were obtained in the presence of 10 mM quinidine. Data was obtained from samples of quinidine permeability studies, using a simultaneous analytical methodology.24 Data points are mean
SEM (n ¼ 4). The significance of difference from the permeability at pH 4.5 (#p < 0.05); and difference from verapamil permeability in the presence of [quinidine] 3 mM, were determined by Student’s t-test. No significant difference was observed between verapamil permeabilities (p > 0.05), when compared at different quinidine concentrations. Key: Qui, quinidine; Ver, verapamil. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 12, DECEMBER 2005

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Figure 6. Relationship between AQ and (A) in situ passive permeability (Peff,Inh), (B) log D, and (C) log of artificial membrane permeability (PPAMPA). Dashed line represents AQ ¼ PP-gp/Peff,Inh, where PP-gp was kept constant (0.186  104 cm/s, mean of PP-gp obtained at various pH in jejunum). AQ showed significant correlation (p < 0.01) with Peff,Inh and linear correlation with log D and log PPAMPA.

responder to P-gp efflux at lower pH but tends towards features with low responder at high pH.26 Similar pH-dependent difference was also observed in ileum, however, permeability values in the absence and presence of P-gp inhibitor, at a particular pH, are higher in ileum. Passive permeability may show regional variation, as there are inherent differences in transcellular permeability in the gut.28,33 Regional variability in transcellular permeability was observed for quinidine, where the secretory transport was much greater in jejunum because of a higher passive diffusion component in ileum, throughout the pH range.31 In the present study, hydrochlorthiazide, frusemide and propranolol were used as permeability markers so as to check the intra- and interindividual variability and presence of steady state on different occasions and to obtain accurate permeability data of quinidine under different conditions (Viz. presence and absence of P-gpmediated efflux). Only insignificant difference was found between permeability in the absence and presence of P-gp inhibitor for transport markers, indicating that changes in quinidine permeabil-

ities obtained were result of P-gp inhibition. Similar to quinidine, propranolol demonstrated regional variability and showed high permeability values in ileum. Quinidine showed concentration-dependent permeability, at both pH 4.5 and pH 7.4, which was eliminated in the presence of P-gp inhibitor, consistent with transport by a saturable efflux mechanism. Results obtained in this study are consistent to concentration-dependent permeability of quinidine across Caco-2 monolayers.17 AQ, although was same at lowest concentration, declined with relatively little increment in quinidine concentrations at pH 7.4. Michaelis–Menten constants (Km, JP-gp,max) for efflux of quinidine were significantly different at luminal pH 4.5 and pH 7.4 (Tab. 2). Most often luminal pH determines passive permeability of basic drugs, however P-gp activity is not directly regulated by pH changes.30,34 Furthermore, the intracellular pH is independent of luminal pH, in the in situ model, and thus the change in luminal pH may not change the pH at P-gp interaction site. Thus, the difference in efflux kinetics is attributable to the

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differences in the transcellular flux rates. For the drug molecule to be effluxed, it should be passively permeable and available in the enterocytes, thus PP-gp is always less than passive permeability, and in an ideal situation, AQ value is always <1.26 However, P-gp-mediated efflux rate may be far higher depending on the substrate affinity and may be limited by passive permeability, especially when the passive permeability and/or the luminal substrate concentration are too low to maintain substrate concentration within the enterocytes. Thus, at low passive permeability high luminal concentration is required to maintain the saturable concentration at P-gp drug interaction site. By far the differences in efflux kinetics were attributed to the difference in P-gp expression and differences in transcellular transport characteristics between the models.35 Various reports showed evidence that efflux kinetics show regional variability because of the differences in level of P-gp expression and transcellular transport.28,36 However, this study shows only the influence of passive permeability, keeping the transporter affinity and P-gp expression levels constant. Our results clearly indicated that the differences in the transcellular flux rates contribute significantly to the differences in efflux kinetic constants. The dependence of efflux kinetics on luminal pH is a significant finding because an orally administered drug is absorbed from various segments of intestine, which exhibits a considerable pH gradient. Past the pyloric sphincter separating the stomach and the duodenum, the pH steeply rises to about 4.6.37 Between proximal jejunum and the distal ileum, the pH gradually rises from about 6.0 to 8.0, and can drop to as low as 5.0 in the colon.37 Moreover, the pH in the unstirred water layer is about 5.2–6.2 and might be regulated independently of the lumenal pH.38,39 Considering these physiological facts, cell culture models (especially using nongradient pH 7.4 medium in apical and basolateral compartment) may underestimate the involvement of P-gp in limiting intestinal absorption of basic P-gp substrates. Finally, P-gp functional activity was observed to decline with increase in Peff,Inh, when AQ values of quinidine, obtained at different pH conditions, are plotted against Peff,Inh at corresponding pH (Fig. 6). Experimentally determined AQ values significantly followed the rule AQ ¼ PP-gp/Peff,Inh, assuming the PP-gp as constant and passive permeability as variable due to pH change or regional differences. Functional activity of P-gp in ileum also followed the same relationship estab-

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lished with jejunum permeability data. P-gp expression was reported to be high in ileum,28 and the resultant AQ in ileum should be higher than that of jejunum at the corresponding passive permeability. However, the regional variability in P-gp expression is masked by the inherently high transcellular permeability in ileum. High propranolol permeability in ileum substantiates the regional passive permeability differences of quinidine. AQ showed a linear relationship with log D and log PPAMPA, which are physicochemical features of drugs that determine passive permeability.7 Thus, the functional activity of P-gp in drug efflux is directly related to its passive permeability, and simple AQ vs. Peff,Inh relationship may be used to predict how P-gp substrates would behave with respect to varying passive permeability with change in luminal pH. Overall, our results indicated that pH-dependent efflux kinetics and regional variability in functional activity of P-gp further adds complexity to the prediction models, however may provide basis for understanding the in vivo poor and variable absorption of protic drugs.

ACKNOWLEDGMENTS Authors are grateful to Sweta Modi and Kanwaljit Kaur for critically reading the manuscript and for helpful suggestions. A part of this work was presented at AAPS annual meeting and exposition 2004, Baltimore, USA with kind support from Department of Science and Technology, India and NIPER, India.

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