European Journal of Pharmaceutical Sciences 25 (2005) 445–453

Enhanced oral paclitaxel absorption with vitamin E-TPGS: Effect on solubility and permeability in vitro, in situ and in vivo Manthena V.S. Varma, Ramesh Panchagnula ∗ Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research (NIPER), Phase X, SAS. Nagar, Mohali, Punjab 160062, India Received 21 February 2005; received in revised form 7 April 2005; accepted 11 April 2005 Available online 10 May 2005

Abstract Solubility and permeability being important determinants of oral drug absorption, this study was aimed to investigate the effect of d-␣tocopheryl polyethylene glycol 1000 succinate (TPGS) on the solubility and intestinal permeability of paclitaxel in vitro, in situ and in vivo, in order to estimate the absorption enhancement ability of TPGS. Aqueous solubility of paclitaxel is significantly enhanced by TPGS, where a linear increase was demonstrated above a TPGS concentration of 0.1 mg/ml. Paclitaxel demonstrated asymmetric transport across rat ileum with significantly greater (26-fold) basolateral-to-apical (B–A) permeability than that in apical-to-basolateral (A–B) direction. Presence of Pglycoprotein (P-gp) inhibitor, verapamil (200 ␮M), diminished asymmetric transport of paclitaxel suggesting the role of P-gp-mediated efflux. TPGS showed a concentration-dependent increase in A–B permeability and decreased B–A permeability. The maximum efflux inhibition activity was found at a minimum TPGS concentration of 0.1 mg/ml, however, further increase in TPGS concentration resulted in decreased A–B permeability with no change in B–A permeability. Thus, the maximum paclitaxel permeability attained with 0.1 mg/ml TPGS was attributed to the interplay between TPGS concentration dependent P-gp inhibition activity and miceller formation. In situ permeability studies in rats also demonstrated the role of efflux in limiting permeability of paclitaxel and inhibitory efficiency of TPGS. The plasma concentration of [14 C]paclitaxel following oral administration (25 mg/kg) was significantly increased by coadministration of TPGS at a dose of 50 mg/kg in rats. Bioavailability is enhanced about 4.2- and 6.3-fold when [14 C]paclitaxel was administrated with verapamil (25 mg/kg) and TPGS, respectively, as compared to [14 C]paclitaxel administered alone. The effect of verapamil on oral bioavailability of [14 C]paclitaxel was limited relative to the TPGS, consistent with the in vitro solubility and permeability enhancement ability of TPGS. In conclusion, the current data suggests that the coadministration of TPGS may improve the bioavailability of BCS class II–IV drugs with low solubility and/or less permeable as a result of significant P-gp-mediated efflux. © 2005 Elsevier B.V. All rights reserved. Keywords: Vitamin E-TPGS; P-glycoprotein; Oral absorption; Pharmacokinetics

1. Introduction Paclitaxel, an antimicrotubule anticancer drug used in wide variety of human cancers, is currently formulated with cremophor EL (polyethoxylated castor oil derivative) and dehydrated alcohol (1:1), is administered through intravenous

∗

Corresponding author. Tel.: +91 172 214 682; fax: +91 172 214 692. E-mail address: [email protected] (R. Panchagnula).

0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2005.04.003

infusion (Panchagnula, 1998). Ethanol-cremophor EL vehicle required to solubilize paclitaxel in this formulation is toxic and also produces vasodilation, labored breathing, lethargy, and hypotension. In order to develop safer clinical formulations, many studies have been directed to novel oral formulations (Dhanikula and Panchagnula, 1999; Mu and Feng, 2003; Feng et al., 2004; Yang et al., 2004; Win and Feng, 2005). Paclitaxel is very poorly absorbed on peroral administration because of its low solubility and low permeability. Apart from its unfavorable physicochemical features for

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passive permeability, it is also believed that P-glycoprotein (P-gp) hinders the transport of paclitaxel from the gut (Varma et al., 2005a). An increasing number of drugs, including HIV protease inhibitors like indinavir, ritonavir, saquinavir and anti-cancer drugs like docetaxel, vinblastine, etc have been reported to be substrates for P-gp (Varma et al., 2003). In vivo studies confirmed that P-gp significantly limits oral bioavailability of several drugs, where intestinal permeability showed dose dependence with increased permeability as lumen concentration increases (Williams and Sinko, 1999; Malingre et al., 2001). Studies using mdr1a(−/−) mice showed direct evidences that P-gp strictly limits the uptake of orally administered paclitaxel (Sparreboom et al., 1997). Woo et al. (2003) demonstrated that about half of paclitaxel dose administered is extruded to the gut lumen by P-gp and only small amount of drug is lost by gut wall and liver metabolism. Thus, the oral bioavailability of paclitaxel can be significantly enhanced by effectively inhibiting P-gp-mediated efflux. Solubility and permeability of a drug are the fundamental determinants of its oral bioavailability (Varma et al., 2004). Surfactants are extensively used to increase the absorption of drugs from the intestine; as they show increased solubility of hydrophobic macromolecules, increased membrane fluidity or disruption of tight junctions, interaction with metabolic enzymes and inhibition of efflux transporters (Nerurkar et al., 1997; Rege et al., 2002). Vitamin E-TPGS (TPGS) is non-ionic water soluble derivative of Vitamin E found to enhance the bioavailability of cyclosporin and amprenavir by enhancing solubility and/or permeability, or reducing intestinal metabolism (Sokol et al., 1991; Chang et al., 1996; Yu et al., 1999; Joshi et al., 2003). TPGS form micelles above the critical miceller concentration (CMC) and improve solubility of lipophilic compounds. Previous reports suggested that coadministration of TPGS enhanced oral absorption of cyclosporin A due to improved solubilization by micelle formation (Sokol et al., 1991; Boudreaux et al., 1993). Chang et al. (1996) evaluated the effect of TPGS on the oral pharmacokinetics of cyclosporin A in healthy volunteers, and suggested that enhanced absorption, decreased counter transport by P-gp, or some unknown mechanism is responsible for the observed decrease in apparent oral clearance. Later on, it was demonstrated that TPGS enhanced the cytotoxicity of doxorubicin, vinblastine, paclitaxel and colchicine in the G185 cells, by acting as reversing agent for P-gpmediated multidrug resistance (Dintaman and Silverman, 1999). In the light of above discussion, the present work investigated the effect of TPGS on the solubility and permeability of a biopharmaceutic classification system (BCS) class IV drug, paclitaxel in vitro, in situ and in vivo. The functional role of Pgp in limiting the permeability of paclitaxel was determined along with the influence of miceller drug concentration on the effective permeability. Furthermore, we also studied the influence of TPGS on the oral bioavailability of paclitaxel in rats.

2. Materials and methods 2.1. Chemicals Paclitaxel was gifted by Dabur India Ltd. (New Delhi, India), and [14 C]paclitaxel was purchased from Sigma–Aldrich Co. (MO, USA). [3 H]Imipramine was purchased from NEN Bio (Boston, MA). Hydrochlorthiazide was received from Aristo Pharmaceuticals Ltd. (Daman, India), and propranolol HCl was from Sun Pharmaceutical Industries Ltd. (Mumbai, India). Frusemide was gifted by Dr. Reddys’ Lab. (Hyderabad, India). l-Phenylalanine was purchased from Sisco Lab. (Mumbai, India). Other compounds verapamil, imipramine and d-glucose were purchased from Sigma Chemicals Co. (MO, USA). Lecithin and dodecane were purchased from Himedia Lab. Pvt. Ltd. (Mumbai, India). Vitamin E-TPGS was from Eastman, and DMSO was procured from Sigma–Aldrich Co. (MO, USA). Solvents used for quantification were of HPLC grade (JT Baker, Mexico) and all other chemicals and reagents were of analytical grade. 2.2. Animals and legal prerequisites Sprague–Dawley rats (270–350 g) were used for in vitro transport, in situ single-pass perfusion and in vivo pharmacokinetic studies. Anesthesia, surgical and disposal procedures were justified in detail and were approved by the Institutional Animal Ethics Committee (IAEC, NIPER). The studies complied with local and federal requirements for animal studies. 2.3. Solubility studies Equilibrium solubility of paclitaxel in 0–5 mg/ml concentration of TPGS was determined by shake-flask method (n = 3). Excess amount of drug was added in TPGS solution in water and equilibrated at 37 ± 0.2 ◦ C with vigorous shaking in shaker water bath (Julabo, Germany) for 48 h. Samples were filtered using 0.22 ␮m filter (Millipore, USA). Aliquots of each filtrate were diluted appropriately and analyzed by RP-HPLC method. 2.4. Measurement of apparent artificial membrane permeability (PPAMPA ) Artificial membrane permeability studies were performed in the same manner described previously (Kansy et al., 1998; Bermejo et al., 2004). In brief, a 96-well microplate (acceptor compartment) was completely filled with phosphate buffer (pH 7.4) containing 5% DMSO. Each filter of the donor plate (Millipore Corp., Bedford, MA) was impregnated with 5 ␮l of 10% (w/v) lecithin in dodecane. A 200 ␮l of 10 ␮M [14 C]paclitaxel (0.4 ␮Ci/ml) in different concentrations of TPGS solution (n = 4 for each TPGS concentration studied) was added immediately and incubated

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at room temperature for 16 h. A reference solution defining equilibrium conditions was prepared as the sample solution with no membrane barrier. The filter surface was wetted with 5 ␮l of 60% (v/v) methanol/buffer solution for the reference. [14 C]paclitaxel was quantified in the receiver compartment by liquid scintillation counter (Wallac, Finland). Apparent artificial membrane permeability (PPAMPA ) was calculated as previously reported (Ano et al., 2004).

hancing secretory transport (SQ) of its substrates, respectively, and PPD,AB and PPD,BA are passive permeability in the absorption and secretory directions (equal to A–B and B–A permeabilities in the presence of verapamil (200 ␮M)). AQ quantifies the passive drug transport attenuation by P-gp across the intestinal enterocytes and provide information on the extent to which P-gp influences intestinal absorption of P-gp substrates across the enterocytes.

2.5. Bi-directional transport studies

2.6. In situ permeability studies

Paclitaxel transport across rat ileum tissue was measured by methods previously reported (Collett et al., 1999; Stephens et al., 2002). Non-fasting male Sprague–Dawley rats were sacrificed by cervical dislocation after a mild anesthesia with thiopental (40 mg/kg). Ileum segment was immediately removed, washed and mounted intact in modified Ussing chambers. Intestinal mucosa was bathed with 6 ml each (donor and receptor chambers) of bicarbonatebuffered Ringer, pH 7.4 at 37 ◦ C, under continuous oxygenation. After an equilibration period of 30 min, bathing medium was replaced with TPGS solution (0–1 mg/ml) containing labeled (0.2 ␮Ci/ml) and unlabelled paclitaxel (5 ␮M) and imipramine (10 ␮M), from either the (A)pical or (B)asolateral chamber. P-gp inhibitor, verapamil (200 ␮M) was added to the apical chamber (inhibition studies). Samples (500 ␮l) from the receiver chamber were withdrawn every 30 min, for 2 h, and replaced with bicarbonate-buffered Ringer. Samples were analyzed by liquid scintillation counting. Imipramine, which is a passive permeable compound, was used to assess the inherent passive transcellular permeability and thus the intestinal tissue integrity. Cumulative amount permeated was plotted against time to calculate apparent permeability (Papp ) in cm/s, by

The surgical procedure and the in situ single-pass perfusion experiments were performed according to the methods described previously (Fagerholm et al., 1996; Hanafy et al., 2001; Varma et al., 2005b; Varma and Panchagnula, 2005) 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 37 ◦ C. Ileum segment of 8–10 cm was isolated and cannulated with glass tubing. The segment was rinsed with phosphate buffer saline (10 ml) and the perfusate solution maintained at 37 ◦ C was pumped at a flow rate of 0.1 ml/min using syringe pump (Harvard Apparatus PHD 2000 pump, MA, USA). The perfusion solution (pH 7.4) consisted of NaCl 48 mM, KCl 5.4 mM, Na2 HPO4 2.8 mM, NaH2 PO4 4 mM and d-glucose 1 g/l; contained paclitaxel (2 ␮M) with TPGS (0–1 mg/ml) or verapamil (200 ␮M). Paclitaxel concentration was low enough to avoid precipitation in the lumen during the course of the study. Single-pass perfusion procedure was followed to determine the permeability (Hanafy et al., 2001). Sampling was made every 5 min for a 60 min perfusion period with perfusion solution after 20 min equilibration. Equilibration of 20 min prior to sampling was found to be sufficient for both washout and to reach an initial steady state (Hanafy et al., 2001). Water flux was quantified based on direct measurement of the volume at the outlet (Bermejo et al., 2004; Varma and Panchagnula, 2005). To further keep a check on the intra- and inter-individual variability and presence of steady state, permeability of lphenylalanine and propranolol (passive, highly permeable), frusemide and hydrochlorthiazide (passive, low permeable), and d-glucose were monitored, by co-perfusing along with drug solution without and with P-gp inhibitor. D-glucose was estimated using GOD-POD based assay kit (Autozyme, ACCURex Biomedical Pvt. Ltd., Mumbai, India). RP-HPLC with dual wavelength UV detector was used for simultaneous quantification of paclitaxel and other permeability markers (l-phenylalanine, propranolol, frusemide and hydrochlorthiazide). In situ permeabilities without (Peff,control ) and with (Peff,inh ) P-gp inhibitor are calculated after correcting the outlet concentration for water flux on the basis of the ratio of volume of perfusion solution collected and infused for each sampling point (5 min) (Bermejo et al., 2004; Varma

Papp =

dQ/dt CA

(1)

where dQ/dt is the rate of appearance of compound in the receiver chamber, C the substrate concentration in donor chamber, and A is the cross-section area (0.44 cm2 ). Papp values are reported as mean ± S.D. of four tissues taken from independent rats. In order to evaluate the quantitative contribution of P-gp to limit the intestinal absorption, we calculated the absorption quotient (AQ) and secretory quotient (SQ) (Troutman and Thakker, 2003; Varma et al., 2005a): AQ =

PP-gp,AB PPD,BA − Papp,AB = PPD,AB PPD,BA

(2)

SQ =

PP-gp,BA Papp,BA − PPD,BA = PPD,BA PPD,BA

(3)

where PP-gp,AB and PP-gp,BA express the effect P-gp would have in attenuating absorption transport (AQ) and en-

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and Panchagnula, 2005): Peff,control (or)Peff,inh


 Q (Cin /Cout ) − 1 = 2Πrl

by (4)

where Q is the flow rate, Cin and Cout the respective inlet and outlet concentration, r the radius of intestine (0.21 cm) and l is the length of intestine measured after completion of perfusion.

F=

AUCoral DoseIV AUCIV Doseoral

(5)

2.9. Statistics Difference in pharmacokinetic parameters and difference in permeabilities of paclitaxel was evaluated by Student’s ttest (SigmaStat version 2.03, SPSS Inc., IL, USA) at P < 0.05 and <0.01.

2.7. Bioanalysis by RP-HPLC HPLC was conducted on Waters (Waters Corp., MA, USA) equipped with 600 controller pumps, Waters 2487 UV–vis dual λ absorbance detector and configured to Millenium32 software. Perfusion sample was loaded onto the column by means of 717plus autosampler (Waters Corp., MA, USA). The column used for chromatographic separations was 4.6 mm i.d., 250 mm length and 5 ␮m particle size C18 (Symmetry® , Waters, USA) attached to a guard column of C18 (Waters, USA). For paclitaxel, propranolol and frusemide, the mobile phase composed of 70% methanol, 26% pH 5.0 (20 mM) acetate buffer and 4% isopropyl alcohol was pumped at a flow rate of 0.6 ml/min and chromatograms were recorded at 220 and 230 nm. In case of l-phenylalanine and hydrochlorthiazide, mobile phase consisting of 52% methanol, 41.7% pH 5.0 (20 mM) acetate buffer and 6.3% isopropyl alcohol were pumped at a flow rate of 0.6 ml/min and chromatograms were recorded at 230 and 275 nm. 2.8. Pharmacokinetic study Male Sprague-Dawley rats were fasted, with water ad libitum, for 16 h before the oral administration. [14 C]paclitaxel was dissolved in cremophor EL and ethanol (1:1, v/v) and diluted fourfold with saline, and the final solution of 6.25 mg/ml (0.2 ␮Ci/mg [14 C]paclitaxel) was administered orally (25 mg/kg of rat) with a blunt needle via the esophagus into stomach; or 2 mg/ml paclitaxel was injected intravenously (2 mg/kg of rat), after cannulating femoral vein. The oral solution containing P-gp inhibitor included either verapamil (25 mg/kg) or TPGS (50 mg/kg). Subsequently, blood samples (200 ␮l) were collected under diethylether anesthesia, into heparinized micro-centrifuge tubes (100 IU/ml blood), from retro-orbital vein (Zhang et al., 2000). An equivalent volume of dextrose saline is administered intra-peritoneally to maintain central compartment (blood) volume. Blood samples were centrifuged for 5 min at 5000 × g and the [14 C]paclitaxel concentrations in the plasma were determined by liquid scintillation counting. Area under the paclitaxel concentration-time curve (AUC) were calculated by trapezoidal rule. The apparent bioavailability (Foral ) of orally administered paclitaxel was calculated

3. Results 3.1. Effect of TPGS on solubility and artificial membrane permeability (PPAMPA ) of paclitaxel Paclitaxel is a non-ionic molecule and is practically insoluble in water at 37 ◦ C. Equilibrium solubility is as low as 1.34 ± 0.18 ␮g/ml and the presence of TPGS at concentration up to 0.1 mg/ml showed no significant change in its solubility. However, paclitaxel solubility is directly proportional to the TPGS concentration, above 0.2 mg/ml (Fig. 1). At a TPGS concentration of 5 mg/ml, the solubility of paclitaxel in water was increased by about 38-fold. The linear increase in paclitaxel solubility above TPGS CMC (0.2 mg/ml) (Yu et al., 1999), indicate the distribution of paclitaxel in the TPGS micelles. The total paclitaxel concentration (Stotal ) above CMC of TPGS is given by Eq. (6): Stotal = Sfree [1 + Ka (SAA)m ]

(6)

where (SAA)m is the concentration of TPGS in micelle form, which is equal to the difference in the apparent TPGS concentration and the CMC. Free drug concentration (Sfree ) and the equilibrium distribution coefficient (Ka ) are calculated by fitting paclitaxel solubility and the TPGS miceller concentration in Eq. (6). Ka was found to be 0.86 mM−1 . Aqueous solubility and Ka being small, appreciable miceller solubilization

Fig. 1. TPGS concentration-dependent solubility of paclitaxel. Inset shows total solubility of paclitaxel vs miceller concentration of TPGS. Linear regression was obtained with Eq. (6) to estimate association constant (Ka ). Each bar represents mean ± S.D. (n = 3) of equilibrium solubility at 48 h.

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Fig. 2. Effect of TPGS concentration on artificial membrane permeability of paclitaxel (10 ␮M). Open points () depict intrinsic permeability calculated using Eq. (7). Apparent permeability of paclitaxel decreased above CMC of TPGS, but the intrinsic permeability did not change. Values represent mean ± S.D. (n = 4). Artificial membrane constituted of hydrophobic filter impregnated with 5 ␮l of 10% (w/v) lecithin in dodecane.

of paclitaxel occurred at relatively large TPGS concentrations. Fig. 2 shows the effect of TPGS on the PPAMPA of paclitaxel. PPAMPA was found to be 0.86 ± 0.1 × 10−6 cm/s across artificial membrane formed with 10% lecithin in dodecane. Presence of TPGS showed no effect on PPAMPA up to a concentration of 0.1 mg/ml, above which, gradual decrease was observed. The decline in PPAMPA is attributable to the reduction in availability of free drug due to TPGS micellization, above 0.2 mg/ml. To normalize for the free drug concentration, the intrinsic permeability PPAMPA,int was calculated using distribution constant (Ka ) and the (SAA)m by PPAMPA,int =

PPAMPA = PPAMPA [1 + Ka (SAA)m ] Ffree

(7)

Intrinsic permeability values, in the presence of TPGS above 0.2 mg/ml, were found to be similar to the permeability of paclitaxel in the absence of TPGS (Fig. 2). 3.2. Effect of TPGS on bi-directional transport of paclitaxel Paclitaxel demonstrated polarized permeability in bidirectional transport studies across excised rat ileum. B–A permeability was found to be 26-fold more then A–B permeability. P-gp inhibition by verapamil (200 ␮M) significantly increased A–B permeability (P < 0.05), and decreased B–A permeability (P < 0.05). AQ and SQ (calculated using A–B and B–A permeabilities in the presence of verapamil as passive permeability of paclitaxel in the absorptive and secretory directions, respectively) indicated that about 89% of passive permeability of paclitaxel is attenuated by P-gp-mediated efflux in A–B direction, and P-gp enhanced secretory permeability by 1.18-fold. The secretory permeability decreased to 66% in the presence of 0.002 mg/ml TPGS (from (9.24 ± 0.59 to

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Fig. 3. Paclitaxel bi-directional transport across rat ileum, in the presence and absence of P-gp inhibitor verapamil (200 ␮M) or different concentrations of TPGS. The apparent permeability coefficient (Papp ) in A–B direction increased and the Papp in B–A decreased as the TPGS concentration increased up to its CMC. AQ and SQ values are negligible when TPGS concentration is 0.1–0.2 mg/ml, indicating that TPGS completely inhibited P-gp efflux at a minimum concentration of 0.1 mg/ml. No change in B–A Papp was found above CMC of TPGS (0.2 mg/ml) while A–B Papp reduced, consistent with TPGS micelle formation above its CMC. Each bar indicates mean ± S.D. (n = 4) (Ver, verapamil).

Table 1 Bi-directional permeability of imipramine (10 ␮M), across rat ileum, in the presence of verapamil (200 ␮M) or different concentrations of TPGS Efflux ratiob

Papp (×106 cm/s)a A–B Control +Verapamil (200 ␮M) +TPGS (0.002 mg/ml) +TPGS (0.02 mg/ml) +TPGS (0.1 mg/ml) +TPGS (0.2 mg/ml) +TPGS (0.5 mg/ml) +TPGS (1.0 mg/ml)

10.2 11.3 10.9 12.1 12.4 9.9 10.1 12.9

B–A ± ± ± ± ± ± ± ±

1.2 0.9 1.5 1.2 2.1 1.6 1.8 2.0

11.4 11.8 9.2 8.4 13.6 12.4 9.1 10.7

± ± ± ± ± ± ± ±

1.9 0.9 1.1 1.6 1.0 1.8 1.2 0.8

1.1 1.0 0.8 0.7 1.1 1.2 0.9 0.8

a Data are presented as mean ± S.D. of four ileum segments. No statistical significance was found between permeabilities in absorptive and secretive directions. b Efflux ratio is the ratio of B–A permeability to A–B permeability.

6.12 ± 0.41) × 10−6 cm/s), whereas absorptive permeability increased 4.7-fold (Fig. 3). Increase in TPGS concentration up to 0.2 mg/ml diminished polarized transport by further increasing the absorptive transport and reducing the secretory transport. AQ and SQ decreased as a function of TPGS concentration and was found to be negligible at 0.1 and 0.2 mg/ml indicating that, similar to verapamil, TPGS inhibit P-gp and the maximum inhibition was achieved at 0.1 mg/ml concentration. Further increase in TPGS concentration to 0.5 and 1 mg/ml significantly decreased paclitaxel A–B permeability (P < 0.05). AQ increased to 0.45 at TPGS concentration 1 mg/ml, however, no change in secretory permeability was observed. Bi-directional transport of a passive permeability marker, imipramine, which was used to check the integrity of the biomembrane, showed no efflux and difference in its permeability in the presence of various concentrations of TPGS (Table 1).

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Fig. 4. Influence of luminal TPGS concentration on P-gp-mediated efflux and intestinal permeability of paclitaxel in rat ileum in situ. Open points () depict intrinsic permeability calculated using Eq. (7). Values represent mean ± S.D. (n = 4). ∗ P < 0.05 and ** P < 0.01, significantly different when compared to control. Permeability was measured in ileum by single-pass perfusion of drug solution in aqueous solutions of TPGS with and without verapamil (200 ␮M) (Ver, verapamil).

Fig. 5. Effect of TPGS on the rat in situ ileum permeability of high- and low-permeable passive and carrier-mediated transport markers. Except for propranolol and hydrochlorthiazide, which showed reduced permeability in the presence of 1 mg/ml TPGS, no significant difference was found with the markers in the presence of verapamil (200 ␮M) and/or TPGS (P > 0.05). Data points represent the mean ± S.D. (n = 4) (l-Phy, l-phenylalanine; Ppn, propranolol; Hct, hydrochlorthiazide; Fru, frusemide; Ver, verapamil).

3.3. Effect of TPGS on in situ permeability of paclitaxel In situ single-pass perfusion in rat ileum also demonstrated functional role of P-gp in limiting paclitaxel oral absorption (Fig. 4). Inhibition of P-gp by verapamil (200 ␮M), co-perfused with paclitaxel (2 ␮M), increased in situ permeability by 2.3-fold (from (0.08 ± 0.01 to 0.25 ± 0.02) × 10−4 cm/s). Similarly, 0.1 mg/ml TPGS also increased in situ permeability, but to a lesser extent. Combination of verapamil (200 ␮M) and 0.1 mg/ml TPGS exhibited permeability similar to that observed in the presence of only verapamil. As observed with bi-directional transport studies, 1 mg/ml TPGS demonstrated increased permeability over control, however, was significantly less than that of 0.1 mg/ml TPGS, consistent to micelle formation reduces the free drug and free TPGS concentration above CMC, leading to observed effects. Coperfusion of verapamil (200 ␮M) with paclitaxel and TPGS (1 mg/ml) also did not significantly increase the permeability, substantiating the effect of micelles on permeability. Intrinsic permeability of paclitaxel in the presence of 1 mg/ml TPGS was found to be (0.20 and 0.22) × 10−4 cm/s. TPGS did not show significant change in the transport of passive and carrier-mediated permeability markers in situ (Fig. 5). 3.4. Effect of TPGS on in vivo permeability of paclitaxel Plasma concentration-time profiles of [14 C]paclitaxel administered intravenously (2 mg/kg); given alone or in combination with verapamil (25 mg/kg) or in combination with TPGS (50 mg/kg), orally (25 mg/kg) in Sprague–Dawley rats, are given in Fig. 6. [14 C]paclitaxel is rapidly distributed after intravenous administration and is very poorly absorbed after oral administration with apparent bioavailability of 4.7 ± 0.5%. Coadministration of verapamil with [14 C]paclitaxel resulted in a significant increase

Fig. 6. Plasma concentration–time profile of [14 C]paclitaxel in rats after (a) intravenous administration (2 mg/kg); (b) after oral administration (25 mg/ml) of [14 C]paclitaxel alone and in combination with verapamil or TPGS. Data points represent mean and error bars show S.E.M. (n = 4). ∗ P < 0.05 and ** P < 0.01, significantly different when compared to oral paclitaxel alone. # P < 0.05 and ## P < 0.01, significantly different when compared to oral paclitaxel in combination with verapamil (Pcl, [14 C]paclitaxel; Ver, verapamil).

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Table 2 Pharmacokinetic parameters of [14 C]paclitaxel after intravenous (2 mg/kg) or oral (25 mg/kg) administration in rats Administrations

Cmax (ng/ml)

AUC0–24 (ng h/ml)

Foral a (%)

i.v. [14 C]paclitaxel Oral [14 C]paclitaxel only Oral [14 C]paclitaxel + verapamil (25 mg) Oral [14 C]paclitaxel + TPGS (50 mg)

82.1 ± 7.5 210.7 ± 24.6** 254.9 ± 18.9**

1782.3 ± 62.8 1051.7 ± 123.3 4433.9 ± 804.4** 6664.2 ± 626.3**

4.7 ± 0.5 19.9 ± 3.6** 29.9 ± 2.8**

a ∗∗

Data are presented as mean ± S.E.M. of four rats for each group. P < 0.01, in comparison to [14 C]paclitaxel administered alone orally. Statistical significance was assessed by Student’s t-test.

in plasma concentration of [14 C]paclitaxel. The maximum plasma concentration (Cmax ) and apparent bioavailability of [14 C]paclitaxel were 2.6- and 4.2-fold higher, when coadministrated with verapamil (Table 2). Further increase in [14 C]paclitaxel plasma concentration was observed after oral administration of [14 C]paclitaxel with TPGS. Cmax and apparent bioavailability of [14 C]paclitaxel administered with TPGS were 3.1- and 6.3-fold higher when [14 C]paclitaxel was administrated alone; 1.2- and 1.5-fold higher when administrated with verapamil.

4. Discussion Paclitaxel, belonging to BCS class IV, has low solubility and permeability as a result of which this clinically potent molecule is orally inactive (Varma et al., 2005a). Presence of TPGS significantly improved the solubility by miceller solubilization and enhanced permeability by P-gp efflux modulation. Below CMC, TPGS has no effect on the solubility of paclitaxel, while above the CMC, solubility linearly increased with TPGS concentration, which is in agreement with the previously reported results for cyclosporin (Ismailos et al., 1994) and amprenavir (Yu et al., 1999). To examine the effect of micelle formation on the passive permeability of paclitaxel, transport studies were carried out with artificial membranes (PAMPA) formed with lecithin in dodecane. Permeability was found to decline significantly when TPGS concentration in donor chamber is above its CMC. Intrinsic permeability was found to be similar to paclitaxel permeability below CMC and thus the decline in passive permeability is attributed to the presence of paclitaxel in TPGS micelles (Amidon et al., 1982). Paclitaxel demonstrated polarized transport across ileum segment, which is diminished in the presence of verapamil. AQ in the presence of P-gp-mediated efflux suggested that about 89% of paclitaxel passive transport is attenuated by P-gp and thus may have a major effect on the efficiency of intestinal paclitaxel absorption in vivo (Varma and Panchagnula, 2005). Below CMC, TPGS showed concentrationdependent increase in the net absorption of paclitaxel by decreasing efflux. Further increase in TPGS concentration (above CMC) lead to decrease in absorptive transport, which resulted in increased AQ. Decrease in absorptive permeabil-

ity, which, however, is higher than that of control, could be due to micelle formation where the availability of free drug and free TPGS is low. In situ permeability studies also demonstrated the role of P-gp in limiting intestinal permeability of paclitaxel, effect of TPGS on P-gp-mediated efflux and the influence of micelle formation on the overall permeability of paclitaxel. A distinct difference in paclitaxel permeability was observed when coperfused with TPGS below and above its CMC. Transport markers were used in bi-directional and in situ permeability studies so as to check the integrity of the intestinal tissue in the presence of TPGS. Only insignificant difference was found between permeability values in the absence and presence of TPGS for transport markers, indicating that changes in paclitaxel permeability in the presence of varying concentrations of TPGS are not due to compromise in membrane integrity. Because of the solubility and/or permeability limitations the absorption site for drugs belonging to BCS classes II–IV is shifted more towards the ileum, which has a transit time of ∼140 min (Kaus et al., 1999). The high P-gp expression levels in the lower intestine make the moderately absorbed P-gp substrates more susceptible to Pgp-mediated efflux (Mouly and Paine, 2003; Siegmund et al., 2003). Rat being the best Fa,human predictor animal model for passive permeability and further there exists a similar level of P-gp expression (Stephens et al., 2001) and overlapped substrate specificity with quantitatively same affinity for a large number of P-gp substrates in rat mdr1a and human MDR1 (Yamazaki et al., 2001), permeability studies in rat ileum provides more meaningful forecast on in vivo absorption of P-gp substrates (Varma and Panchagnula, 2005). A striking increase in the AUC of [14 C]paclitaxel was observed in rats when the drug was administered orally in combination with TPGS. Absorption enhancement is significant given the reasons for poor pharmacokinetics of [14 C]paclitaxel in vivo. It has been previously reported that systemic exposure to orally administered paclitaxel is substantially enhanced by coadministration of P-gp inhibitor such as cyclosporin A, PSC 833, verapamil, KR-30031 and MS-209 (Sokol et al., 1991; van Asperen et al., 1997, 1998; Kimura et al., 2002; Woo et al., 2003). Combining with these P-gp inhibitors enhanced bioavailability of orally administered paclitaxel in normal mice to levels similar to those obtained in mdr1a(−/−) mice given paclitaxel only (van Asperen et al., 1997 and 1998). Thus the increase in

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[14 C]paclitaxel AUC with TPGS is attributable in part to the P-gp modulating ability of TPGS, which in this study has been substantiated with bi-directional and in situ permeability data. TPGS dose of 50 mg/kg results in micelle formation in vivo, where maximum amount of TPGS exists in micelles form. However, in vitro studies indicated that free TPGS concentration (i.e. concentration equal to CMC) available in the gut completely inhibit P-gp-mediated efflux. The AUC of [14 C]paclitaxel was 1.5-fold higher when coadministered with TPGS, than when administrated with verapamil. Verapamil at the dose (25 mg/kg) administered in the present study is available at a far higher concentration than 200 ␮M, which completely eliminated P-gp-mediated efflux in vitro and in situ. Woo et al. reported the ability of KR-30031, a verapamil derivative, to reduce the active P-gp efflux is equipotent with verapamil, and further KR-30031 showed a dose-dependent increase in AUC of paclitaxel with no further increase above 20 mg/kg dose in Sprague–Dawley rats (Woo et al., 2003). Thus, it is anticipated that verapamil completely inhibited P-gp efflux in vivo, in the present study. Cyclosporin A enhances the plasma concentration of paclitaxel following oral administration not only by inhibiting the P-gp-mediated efflux, but also by modulating other mechanisms involving drug metabolism in gut and liver (van Asperen et al., 1998). In contrast to cyclosporin A, TPGS seems to show little or no effect on the metabolism of paclitaxel because, our pharmacokinetic study in rats showed about 1.5-fold higher AUC of paclitaxel when coadministered with TPGS over paclitaxel coadministered with verapamil, while intestinal and liver metabolism contributes relatively less to paclitaxel absorption (Woo et al., 2003). Previous reports indicated that only 3.5% of paclitaxel dose is eliminated by intestinal and first-pass liver metabolism and inhibition of both P-gp and CYP3A increased bioavailability in rats by only 20% in comparison to inhibition of only Pgp. Further, Wacher et al. (2002) reported that the sirolimus oral absorption in rats is not affected by TPGS at a dose of 50 mg/kg, but increased about 4.7-fold when coadministered with the CYP3A inhibitor, ketoconazole. Thus, modulation of intestinal and liver metabolism play a little or no role. The striking increase in bioavailability of paclitaxel after coadministration with TPGS, in comparison to paclitaxel administered with verapamil, is attributable to the solubility enhancing ability of TPGS. Previous studies also demonstrated that TPGS miceller solubilization enhanced the bioavailability of cyclosporin A in liver transplant patients with significantly improved absorption (Sokol et al., 1991), and increased cyclosporin A absorption in pediatric transplant recipients (Boudreaux et al., 1993). Solubility data from the present study showed that TPGS functioned as surfactant and solubilize paclitaxel through micelle formation. This dissolution enhancement ability of TPGS provides higher concentration of paclitaxel at the GI absorption site resulting to observed difference in the in vivo pharmacokinetics of paclitaxel when administered with verapamil and TPGS.

5. Conclusions In cancer chemotherapy, oral treatment with paclitaxel is to be preferred as oral drug administration is convenient to patients, reduces administration costs and facilitates the use of more chronic treatment regimens. In addition, circumvention of systemic exposure to the co-solvent cremophor EL is another advantage of oral therapy. In the present study, based on the preclinical data, we have shown the feasibility of oral administration of paclitaxel in cancer patients by concomitant administration of TPGS. Miceller solubilization ability and P-gp inhibitory activity of TPGS resulted in significant improvement in pharmacokinetic profile of paclitaxel. Data clearly indicated that TPGS effect in vivo is due to combination of increased solubility and inhibition of P-gpmediated efflux, and suggest that TPGS can serve to improve the bioavailability not only of orally administered paclitaxel but also of other BCS class II–IV drugs, which have either low solubility or limited permeability due to their efflux by P-gp or both.

Acknowledgements Authors are grateful to Vishwanand Bhoomi for his help in pharmacokinetic studies. Authors also like to thank Kanwaljit Kaur, Sweta Modi, Namita Kapoor and Mahua Sarkar for critically reading the manuscript and for helpful suggestions.

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