Unmasking the dynamic interplay between intestinal P-glycoprotein and CYP3A4

Drug efflux by intestinal P-glycoprotein (P-gp) is known to decrease the oral bioavailability of many CYP3A4 substrates. We hypothesized that the interplay occurring between P-gp and CYP3A4 at the apical membrane would increase the opportunity for drug metabolism. To define the roles of P-glycoprotein (P-gp) and CYP3A4 in controlling the extent of intestinal absorption and metabolism, two substrates were tested. The transport, metabolism, and intracellular levels of N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K77, a cysteine protease inhibitor; P-gp and CYP3A4 substrate) and felodipine (CYP3A4 substrate only) were measured across CYP3A4-transfected Caco-2 cells in the presence of an inhibitor of CYP3A4 and P-gp, cyclosporine (CsA), or an inhibitor of P-gp and not CYP3A4, GG918 (N-[4-[2-(1,2,3,4-tetrahydro-6,7- dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine). The extent of metabolism was measured by calculating the extraction ratio (ER) across the cells, while accounting for intracellular changes occurring with P-gp inhibition. The (A)pical to (B)asolateral and B-->A ERs for K77 were 0.33 and 0.06, respectively. These changed with GG918 to 0.14 and 0.12 and with CsA to 0.06 and 0.04. Felodipine ERs were similar in both directions, 0.26 and 0.24 (A-->B and B-->A), and were unchanged in the presence of GG918 but decreased with CsA (0.14 and 0.11). The K77 absorption rate was increased 5 and 4.2-fold in the presence of CsA and GG918, respectively, whereas no change was observed for felodipine absorption. The decreased A-->B ER and increased absorption of K77 with GG918 suggest that P-gp influences the extent of drug metabolism in the intestine via prolonging the access of drugs to CYP3A4 near the apical membrane and decreasing transport across the cells.     

Differentiation of gut and hepatic first pass metabolism and secretion of saquinavir in ported rabbits

The current study was performed in intestinal and vascular access ported rabbits to quantify and differentiate the components of intestinal and hepatic first pass extraction (i.e., metabolism and secretion) of saquinavir (SQV) mediated by P-glycoprotein (P-gp) and CYP3A. SQV was administered i.v. (1-5 mg/kg) or into the upper small intestine (USI) (5 mg/kg). The roles of intestinal and hepatic secretion by means of P-gp and/or metabolism by CYP3A on the first pass gastrointestinal extraction of SQV were differentiated by using N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) (a P-gp inhibitor), midazolam (an inhibitor of CYP3A), or cyclosporine A (an inhibitor of P-gp and CYP3A). The bioavailability (BA) of SQV after USI dosing was 4%. In the presence of CYP3A and P-gp inhibitors, the BA of SQV increased 2- to 11-fold. Based on a relatively unchanged Cmax but prolonged Tmax and t(1/2), P-gp and CYP3A inhibition appeared to alter SQV disposition (i.e., enhanced oral bioavailability by diminishing SQV elimination and by increasing its net intestinal absorption). In conclusion, the current results substantiate the role of the liver and, for the first time, experimentally establish an important role for the intestine in the net absorption and disposition of SQV. The results also demonstrate that changes in SQV disposition due to the modulation of metabolism and secretion were important and may potentially have considerable implications on multiple drug therapeutic regimens used in the treatment of AIDS.     

Pharmacokinetics and interactions of a novel antagonist of chemokine receptor 5 (CCR5) with ritonavir in rats and monkeys: role of CYP3A and P-glycoprotein

The mechanisms of pharmacokinetic interactions of a novel anti-human immunodeficiency virus (anti-HIV-1) antagonist of chemokine receptor 5 (CCR5) [2-(R)-[N-methyl-N-(1-(R)-3-(S)-((4-(3-benzyl-1-ethyl-(1H)-pyrazol-5-yl)piperidin-1-yl)methyl)-4-(S)-(3-fluorophenyl)cyclopent-1-yl)amino]-3-methylbutanoic acid (MRK-1)] with ritonavir were evaluated in rats and monkeys. MRK-1 was a good substrate for the human (MDR1) and mouse (Mdr1a) multidrug resistance protein transporters and was metabolized by CYP3A isozymes in rat, monkey, and human liver microsomes. Both the in vitro MDR1-mediated transport and oxidative metabolism of MRK-1 were inhibited by ritonavir. Although the systemic pharmacokinetics of MRK-1 in rats and monkeys were linear, the oral bioavailability increased with an increase in dose from 2 to 10 mg/kg. The area under the plasma concentration-time curve (AUC) of MRK-1 was increased 4- to 6-fold when a 2 or 10 mg/kg dose was orally coadministered with 10 mg/kg ritonavir. Further pharmacokinetic studies in rats indicated that P-glycoprotein (P-gp) inhibition by ritonavir increased the intestinal absorption of 2 mg/kg MRK-1 maximally by approximately 30 to 40%, and a major component of the interaction likely resulted from its reduced systemic clearance via the inhibition of CYP3A isozymes. Oral coadministration of quinidine (10 and 30 mg/kg) increased both the extent and the first-order rate of absorption of MRK-1 (2 mg/kg) by approximately 40 to 50% and approximately 100 to 300%, respectively, in rats, thus further substantiating the role of P-gp in modulating the intestinal absorption of MRK-1 in this species. At the 10 mg/kg MRK-1 dose, however, the entire increase in its AUC upon coadministration with ritonavir or quinidine could be attributed to a reduced systemic clearance, and no effects on intestinal absorption were apparent. In contrast to rats, the effects of P-gp in determining the intestinal absorption of MRK-1 appeared less significant in rhesus monkeys at either dose.     

Contribution of down-regulation of intestinal and hepatic cytochrome P450 3A to increased absorption of cyclosporine A in a rat nephrosis model

This study examined the contribution of changes in regulation of intestinal and hepatic cytochrome P450 3A (CYP3A) and multidrug resistance transporter 1 (Mdr1) to absorption of cyclosporine A (CsA) in a rat nephrosis model. Interleukin (IL)-6 was also measured. Puromycin aminonucleoside at a dose of 20 mg/100 g was administered intravenously. Tissue samples were dissected out from the upper and middle intestines and liver after development of nephrosis to measure the expression levels of mRNA and protein. CsA at a dose of 0.5 mg/100 g was administered into a closed loop of the upper and middle intestines. Blood from the inferior vena cava (IVC) and portal vein was taken until 30 min after administration. The expression levels of CYP3A decreased markedly, whereas those of Mdr1 showed large interindividual variations for all of the tissues in the nephrotic rats. Plasma concentrations of CsA reached higher levels in the nephrotic than in the control rats and were higher when administered from the upper than the middle intestine in both the portal vein and IVC. IL-6 increased in urine in the nephrotic rats. In summary, intestinal and hepatic CYP3A were down-regulated in the nephrosis model accompanying the increased levels of IL-6. Consistent results were not obtained for the regulation of Mdr1. In conclusion, these findings suggest that the down-regulation of CYP3A in the upper intestine and liver predominantly contributes to the increase in CsA absorption, and Mdr1 showed less contribution in this rat nephrosis model.     

Gut instincts: CYP3A4 and intestinal drug metabolism

First-pass metabolism is a common cause of incomplete and variable absolute bioavailability for an orally dosed drug. The drug-metabolizing enzyme CYP3A4 is often implicated in this process, resulting, in some cases, in systemic exposures of less than 15% of the administered dose. By creating an elegant CYP3A4-transgenic mouse model, van Herwaarden et al. show in this issue of the JCI that first-pass metabolism of the anticancer agent docetaxel by the gut wall, and not the liver, is likely to be the major cause of its low oral bioavailability in humans (see the related article beginning on page 3583). This study helps explain interpatient differences in efficacy and safety following oral therapy with approved CYP3A4 substrates and provides a powerful new tool for preclinical predictions of first-pass metabolism for new drugs in development.     

Effects of clarithromycin on the pharmacokinetics of tacrolimus and expression of CYP3A4 and P-glycoprotein in rats

The objective of this study was to investigate the effect of clarithromycin on the pharmacokinetics of tacrolimus in rats and better understand its mechanism. In the control group (n = 6), rats received a single oral dose of 1 mg tacrolimus on day 6. In the experimental group (n = 6), rats received 0.25 g of clarithromycin daily for five consecutive days and then a single oral dose of 1 mg tacrolimus on day 6. Orbital venous blood (250 μL) was collected at 0, 0.25, 0.50, 0.75, 1, 2, 4, 8, 12, and 24 h before and after tacrolimus administration. Blood drug concentrations were detected via mass spectrometry. Small intestine and liver tissue samples were collected after rats were euthanized via dislocation, and CYP3A4 and P-glycoprotein (P-gp) protein expression was determined using western blotting. Clarithromycin increased the blood tacrolimus concentration and affected its pharmacokinetic properties in rats. Compared with those in the control group, the AUC_0–24, AUC_0–∞, AUMC_(0–t), and AUMC_(0–∞) of tacrolimus in the experimental group were significantly increased, whereas the CLz/F was significantly lower (P < 0.01). Simultaneously, clarithromycin significantly inhibited CYP3A4 and P-gp expression in the liver and intestine. Protein expression of CYP3A4 and P-gp in the liver and the intestinal tract was significantly downregulated in the intervention group compared with that in the control group. Clarithromycin significantly inhibited the protein expression of CYP3A4 and P-gp in the liver and intestine, thereby increasing the mean blood concentration and significantly increasing the AUC of tacrolimus.     

Investigation of the Functional Role of P-Glycoprotein in Limiting the Oral Bioavailability of Lumefantrine

In the quest to explore the reason for the low and variable bioavailability of lumefantrine, we investigated the possible role of P-glycoprotein (P-gp) in lumefantrine intestinal absorption. An in situ single-pass intestinal perfusion study in rats with the P-gp inhibitor verapamil or quinidine and an ATPase assay with human P-gp membranes indicated that lumefantrine is a substrate of P-gp which limits its intestinal absorption. To confirm these findings, an in vivo pharmacokinetic study was performed in rats. The oral administration of verapamil (10 mg/kg of body weight) along with lumefantrine caused a significant increase in its bioavailability with a concomitant decrease in clearance. The increase in bioavailability of lumefantrine could be due to inhibition of P-gp and/or cytochrome P450 3A in the intestine/liver by verapamil. However, in a rat intestinal microsomal stability study, lumefantrine was found to be resistant to oxidative metabolism. Further, an in situ permeation study clearly showed a significant role of P-gp in limiting the oral absorption of lumefantrine. Thus, the increase in lumefantrine bioavailability with verapamil is attributed in part to the P-gp-inhibitory ability of verapamil. In conclusion, lumefantrine is a substrate of P-gp, and active efflux by P-gp across the intestine partly contributed to the low/variable bioavailability of lumefantrine.     

Identification of a novel route of extraction of sirolimus in human small intestine: roles of metabolism and secretion

Using Caco-2 cell monolayers expressing CYP3A4, we investigated the interplay between metabolism and transport on the first-pass intestinal extraction of the immunosuppressant sirolimus, a CYP3A4/P-glycoprotein (P-gp) substrate. Modified Caco-2 cells metabolized [(14)C]sirolimus to the predicted amounts of CYP3A4-mediated products based on CYP3A4 content, which was approximately 20% of that measured in human small intestinal mucosal homogenate. [(14)C]Sirolimus also degraded to the known ring-opened product, seco-rapamycin. Unexpectedly, a ring-opened dihydro metabolite (M2) was the major product detected in cells at all sirolimus concentrations examined (2-100 microM). Greater M2 formation after apical versus basolateral dosing (1.6-fold) was explained by higher intracellular content of sirolimus after apical dosing. M2 was not detected in incubations with human liver and intestinal microsomes but was readily detected with corresponding homogenates. M2 formation was NADPH-dependent but unaffected by the CYP3A4 inhibitors ketoconazole and troleandomycin. Although M2 was formed from purified seco-rapamycin (20 microM) in the homogenates, it was not detected in cells when seco-rapamycin was added to the apical compartment, because seco-rapamycin was essentially impermeable to the apical membrane. Sirolimus, seco-rapamycin (basolaterally dosed), and M2 were all actively secreted across the apical membrane, and secretion of each was inhibited by the P-gp inhibitor LY335979 [(2R)-anti-5-[3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy]quinoline trihydrochloride]. Along with CYP3A4-mediated metabolism and P-gp-mediated secretion, we conclude that the following novel pathway, which occurs at least in the intestine, may contribute significantly to the first-pass extraction of sirolimus in humans: intracellular degradation of sirolimus to seco-rapamycin, metabolism of seco-rapamycin to M2 by an unidentified non-microsomal enzyme, and P-gp-mediated secretion of M2 and seco-rapamycin.     

Low bioavailability of amoxicillin in rats as a consequence of presystemic degradation in the intestine.

Several studies have been carried out to elucidate the causes of the low oral bioavailability of amoxicillin in rats. The hepatic first-pass effect of the antibiotic was estimated by comparing the area under the plasma drug concentration-versus-time curve from time zero to infinity (AUC0-infinity) obtained after injecting the drug into a mesenteric vein with the AUC0-infinity value obtained after injecting the drug into the jugular vein of conscious rats. No hepatic first-pass effect was detected. The bioavailability of amoxicillin after intraduodenal administration was only 51%, and the fraction of the dose remaining in the intestine at the end of the experiment was 4.5%. This was far less than the fraction that did not reach systemic circulation, which indicates a presystemic loss of drug, probably at the intestine. In vitro studies corroborated the fact that amoxicillin is subjected to presystemic degradation by intestinal juices and intestinal tissues. The greatest loss of drug occurred in the complete intestine (45% of the initial amount), and it was mainly due to the action of intestinal tissues (28% of the initial amount) but was also due to the action of intestinal juices (15% of the initial amount). The absorption of amoxicillin in three parts of the intestine (upper, middle, and lower) was also evaluated. The largest AUC0-infinity value and the highest plasma drug levels were obtained when amoxicillin absorption took place in the middle intestine. The smallest AUC0-infinity value and the lowest plasma drug levels corresponded to absorption from the upper intestine.     

Intestinal MDR transport proteins and P-450 enzymes as barriers to oral drug delivery.

Cytochrome P-450 3A4 (CYP3A4), the major phase I drug metabolizing enzyme in humans, and the multidrug efflux pump, MDR or P-glycoprotein (P-gp), are present at high levels in the villus tip enterocytes of the small intestine, the primary site of absorption for orally administered drugs. These proteins are induced or inhibited by many of the same compounds and demonstrate a broad overlap in substrate and inhibitor specificities, suggesting that they act as a concerted barrier to drug absorption. A series of studies from our laboratory of cyclosporine and tacrolimus in humans and a novel cysteine protease inhibitor in rats, dosed concomitantly with inhibitors and inducers of CYP3A4 and P-gp, suggest that gut extraction can be modeled using measures of intestinal metabolism and absorption rate, the latter reflecting changes in P-gp. Results evaluating a preliminary model applied to the CYP3A substrate drugs midazolam, indinavir, saquinavir, and rifabutin suggest that the model may be useful for predicting in vivo intestinal metabolism from in vitro data.     

CYP3A4-transfected Caco-2 cells as a tool for understanding biochemical absorption barriers: studies with sirolimus and midazolam

CYP3A4-transfected Caco-2 cells were used as an in vitro system to predict the importance of drug metabolism and transport on overall drug absorption. We examined the transport and metabolism of two drugs; midazolam, an anesthetic agent and CYP3A4 substrate, and sirolimus, an immunosuppressant and a dual CYP3A4/P-glycoprotein (P-gp) substrate, in the presence of cyclosporine (CsA, a CYP3A4/P-gp inhibitor) or N-[4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl]-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine (GG918) (an inhibitor of P-gp and not CYP3A4). All major CYP3A4 metabolites were formed in the cells (1-OH > 4-OH midazolam and 39-O-desmethyl > 12-OH > 11-OH sirolimus), consistent with results from human liver microsomes. There was no bidirectional transport of midazolam across CYP3A4-transfected Caco-2 cells, whereas there was a 2.5-fold net efflux of sirolimus (1 microM) that disappeared in the presence of CsA or GG918. No change in the absorption rate or extraction ratio (ER) for midazolam was observed when P-gp was inhibited with GG918. Addition of GG918 had a modest impact on the absorption rate and ER for sirolimus (increased 58% and decreased 25%, respectively), whereas a 6.1-fold increase in the absorption rate and a 75% decrease in the ER were found when sirolimus was combined with CsA. Although both midazolam and sirolimus metabolites were preferentially excreted to the apical compartment, only sirolimus metabolites were transported by P-gp as determined from inhibition studies with GG918. Using CYP3A4-transfected Caco-2 cells we determined that, in contrast to P-gp, CYP3A4 is the major factor limiting sirolimus absorption. The integration of CYP3A4 and P-gp into a combined in vitro system was critical to unveil the relative importance of each biochemical barrier.     

No influence of the MDR-1 C3435T polymorphism or a CYP3A4 promoter polymorphism (CYP3A4-V allele) on dose-adjusted cyclosporin A trough concentrations or rejection incidence in stable renal transplant recipients

BACKGROUND: A substantial proportion of the variability in the absorption and clearance of cyclosporin A (CsA) after oral administration has been attributed to variability in liver cytochrome P-450 3A4 (CYP3A4) activity and intestinal P-glycoprotein (P-gp) concentration. A polymorphism in the CYP3A4 promoter region, termed "variant" allele CYP3A4-V, was postulated to be associated with altered CYP3A4 enzyme activity. A polymorphism in exon 26 (C3435T) of the multidrug resistance-1 (MDR-1) gene was correlated with intestinal expression and in vivo activity of P-gp. METHODS: We investigated the occurrence of both polymorphisms in 124 stable Caucasian renal transplant recipients (>6 months after transplantation) on CsA as the primary immunosuppressant. Real-time, rapid-cycle PCR methods were developed and used for genotyping. RESULTS: The estimated allele frequencies for the MDR-1 C3435T allele (54%) and the CYP3A4-V allele (4.8%) were similar to those reported for Caucasian populations. No significant differences were found for the CsA doses needed to maintain similar CsA trough concentrations in patients with and without the CYP3A4-V allele or in patients with different MDR-1 C3435T genotypes. Furthermore, neither of the polymorphisms investigated was associated with renal function as assessed by creatinine plasma concentration or, in a retrospective analysis, the incidence of acute rejection. CONCLUSIONS: These findings suggest that the MDR-1 C3435T mutation and the CYP3A4-V variant are not major determinants of CsA efficacy in renal transplant recipients.     

Inhibition of cytochrome P450 2A6 increases nicotine's oral bioavailability and decreases smoking

BACKGROUND: Nicotine establishes and maintains tobacco dependence. Individuals with genetically deficient CYP2A6 nicotine metabolism are at lower risk to become smokers and, if dependent, will smoke fewer cigarettes. Hepatic CYP2A6 accounts for nicotine's low systemic bioavailability, precluding oral nicotine replacement to treat dependence. OBJECTIVE: We sought to determine whether CYP2A6 inhibition via oral methoxsalen decreases nicotine clearance, increases nicotine bioavailability, and decreases smoking. METHODS: Two within-subject designs in healthy tobacco-dependent volunteers were conducted: a singleblind kinetic study (n = 17) of methoxsalen 30, 10, or 3.5 mg or placebo given with nicotine 4 mg orally to abstinent smokers; and a double-blind randomized crossover study (n = 11) of methoxsalen 30 mg or placebo crossed with nicotine 4 mg given orally or placebo before 60 minutes' abstinence and 90 minutes' free smoking. RESULTS: Placebo plus nicotine 4 mg orally increased the mean 3-hour plasma nicotine level by 4 ng/mL over residual baseline nicotine level, whereas methoxsalen 10 or 30 mg plus nicotine increased it by 9 ng/mL (P<.01), demonstrating in vivo inhibition of CYP2A6 nicotine metabolism. Methoxsalen 30 mg plus nicotine 4 mg given orally decreased breath carbon monoxide concentration at the end of free smoking by 47% (4.6 versus 8.7 ppm; P<.01) and cigarettes smoked by 24% (3.1 versus 4.1, P<.01) compared with placebo plus placebo. CONCLUSIONS: Methoxsalen inhibits nicotine first-pass metabolism of orally administered nicotine, and the combination directly reduces smoking in a laboratory setting. CYP2A6 inhibitors may have an important role in smoking cessation and tobacco exposure reduction.     

Ex situ inhibition of hepatic uptake and efflux significantly changes metabolism: hepatic enzyme-transporter interplay

The disposition of digoxin and the influence of the organic anion transporting polypeptide (Oatp)2 inhibitor rifampicin and the P-glycoprotein (P-gp) inhibitor quinidine on its hepatic disposition were examined in the isolated perfused rat liver. Livers from groups of rats were perfused in a recirculatory manner after a bolus dose of digoxin (10 microg), a dual substrate for Oatp2 and P-gp as well as CYP3A. Perfusions of digoxin were also examined in groups of rats in the presence of the inhibitors: rifampicin (100 microM) or quinidine (10 microM). In all experiments, perfusate samples were collected for 60 min. Digoxin and its primary metabolite were determined in perfusate and liver by liquid chromatography/mass spectrometry. The area under the curve (AUC) from 0 to 60 min was determined. The AUC +/- S.D. of digoxin was increased from control (3880 +/- 210 nM x min) by rifampicin (5200 +/- 240 nM x min; p < 0.01) and decreased by quinidine (3220 +/- 340 nM x min; P < 0.05). It is concluded that rifampicin limits the hepatic entrance of digoxin and reduced the hepatic exposure of digoxin to CYP3A by inhibiting the basolateral Oatp2 uptake transport, whereas quinidine increased the hepatic exposure of digoxin to CYP3A by inhibiting the canalicular P-gp transport. These data emphasize the importance of uptake and efflux transporters on hepatic drug metabolism.     

Nonlinear intestinal first-pass metabolism of salicylamide in dogs after portacaval transposition

The dose-dependent first-pass metabolism and pharmacokinetics of salicylamide (SAM) were studied at four dose levels in dogs before and after portacaval transposition. Four minutes after each p.o. dose, a tracer dose of [14C]SAM was given i.v. to determine clearance and bioavailability. Over the dosage range studied pretransposition, 5 to 40 mg/kg, bioavailability increased from 0.24 +/- 0.14 (mean +/- S.D.) to 0.76 +/- 0.20 (P less than .05). Clearance decreased from 3.4 +/- 1.0 to 0.6 +/- 0.11 liter/min (P less than .01) and half-life increased from 5.0 +/- 1.2 to 23.5 +/- 6.1 min (P less than .01). Over the dosage range studied post-transposition, 1.5 to 20 mg/kg, bioavailability increased from 0.31 +/- 0.09 to 0.99 +/- 0.08. Clearance and half-life had the same values and showed the same dose-dependence as in the normal dogs. The amount of SAM removed by the intestine during first-pass remained constant at about 1 mg/kg over the dose range given to the post-transposition animals. Therefore, although more easily saturable than the liver, the intestine plays an important role in first-pass metabolism of low p.o. doses of SAM. In contrast to previous results in the normal dog, the p.o. coadministration of sodium sulfate did not reduce the bioavailability of SAM in transposed dogs. This indicates that the nonlinear intestinal first-pass metabolism of SAM is not due to the depletion of the cosubstrate precursor, inorganic sulfate.     

Effect of interleukin-2 on intestinal P-glycoprotein expression and functionality in mice

P-glycoprotein (Pgp), an active drug transporter expressed in enterocytes, can reduce intestinal absorption of drugs. Until now, interleukin-2 (IL2) has been reported as a Pgp modulator only in vitro. The present study examines the effects in vivo of IL2 after chronic treatment on intestinal Pgp protein expression and activity. This work also describes the effects of IL2 on the oral bioavailability of a Pgp substrate (digoxin) and of a Pgp/CYP3A cosubstrate (saquinavir). Human recombinant interleukin-2 (rIL2), administered to mice at 9 million international units/kg by intraperitoneal route twice daily for 4 days, led to a decrease in intestinal Pgp protein expression evaluated by Western blot with C219 antibody. In an in vitro everted gut sac model, rIL2 pretreatment decreased the Pgp-mediated transport of rhodamine 123 across mouse intestine by 37%. Moreover, rIL2 pretreatment markedly raised the area under the curve of orally administered digoxin from 3.5 +/- 0.5 to 9.7 +/- 1.5 mg min l(-1) as a consequence of the reduction in intestinal Pgp activity. rIL2 treatment increased saquinavir bioavailability from 2.5 to 4.5%, showing that first-pass metabolism is not affected and that Pgp by itself has only a moderate effect on saquinavir oral bioavailability. In conclusion, rIL2 pretreatment reduces intestinal Pgp protein expression and activity in mice. However, the effect of such a treatment on drug bioavailability depends on the extent of their metabolism by CYP3A.     

Cytochrome P450 3A4 and P-glycoprotein expression in human small intestinal enterocytes and hepatocytes: a comparative analysis in paired tissue specimens

OBJECTIVES: Our objectives were to determine the content of cytochrome P450 (CYP) 3A4, CYP3A5, and P-glycoprotein and to measure CYP3A4-dependent catalytic activity in paired human small intestinal and liver specimens. METHODS: Samples of duodenum or proximal jejunum and liver wedge biopsy specimens were obtained from 15 patients undergoing a gastrointestinal operation. Enterocytes were isolated from the intestinal samples. The contents of CYP3A4, CYP3A5, and P-glycoprotein and CYP3A4-mediated catalytic activities were determined in homogenized enterocyte and liver samples. RESULTS: The CYP3A4 protein content was about 3 times (P <.01) and the P-glycoprotein content about 7 times (P <.0001) higher in the enterocyte homogenates than in the liver homogenates. CYP3A5 protein was detected in all samples, but the levels were too low in most cases to allow quantification. The 2 cases with a quantifiable hepatic CYP3A5 content had the CYP3A5*1/*3 genotype; all other cases were homozygous for the CYP3A5*3 allele. No intraindividual correlations between the intestine and liver with respect to CYP3A4 content, P-glycoprotein content, or the measured catalytic activities were present. Values for the maximum rate of metabolism (V(max)) of verapamil N-dealkylation (formation of D-617) and N-demethylation (formation of norverapamil) activities correlated with the CYP3A4 protein content in both organs. CONCLUSIONS: This work demonstrated a much higher content of both CYP3A4 protein and P-glycoprotein in enterocytes isolated from human duodenal or jejunal mucosa than in paired specimens of liver tissue. These results lend support to the view that biotransformation in the gut wall substantially contributes to the overall first-pass metabolism of many CYP3A4 substrates. Furthermore, the high content of P-glycoprotein on the apical surface of enterocytes supports the theory that this efflux transporter may act in concert with CYP3A4 to limit oral drug bioavailability. Finally, these results indicate that neither CYP3A4 nor MDR1 (P-glycoprotein) is coordinately regulated in the liver and intestine.     

Modulation of Biotransformation Systems and ABC Transporters by Benznidazole in Rats

The effect of antichagasic benznidazole (BZL; 100 mg/kg body weight/day, 3 consecutive days, intraperitoneally) on biotransformation systems and ABC transporters was evaluated in rats. Expression of cytochrome P-450 (CYP3A), UDP-glucuronosyltransferase (UGT1A), glutathione S-transferases (alpha glutathione S-transferase [GST-α], GST-μ, and GST-π), multidrug-resistance-associated protein 2 (Mrp2), and P glycoprotein (P-gp) in liver, small intestine, and kidney was estimated by Western blotting. Increases in hepatic CYP3A (30%) and GST-μ (40%) and in intestinal GST-α (72% in jejunum and 136% in ileum) were detected. Significant increases in Mrp2 (300%) and P-gp (500%) proteins in liver from BZL-treated rats were observed without changes in kidney. P-gp and Mrp2 were also increased by BZL in jejunum (170% and 120%, respectively). In ileum, only P-gp was increased by BZL (50%). The activities of GST, P-gp, and Mrp2 correlated well with the upregulation of proteins in liver and jejunum. Plasma decay of a test dose of BZL (5 mg/kg body weight) administered intraduodenally was faster (295%) and the area under the concentration-time curve (AUC) was lower (41%) for BZL-pretreated rats than for controls. The biliary excretion of BZL was higher (60%) in the BZL group, and urinary excretion of BZL did not show differences between groups. The amount of absorbed BZL in intestinal sacs was lower (25%) in pretreated rats than in controls. In conclusion, induction of biotransformation enzymes and/or transporters by BZL could increase the clearance and/or decrease the intestinal absorption of coadministered drugs that are substrates of these systems, including BZL itself.     

The effect of age on P-glycoprotein expression and function in the Fischer-344 rat

We investigated the effect of age on P-glycoprotein (P-gp) expression and function in rat liver, intestine, kidney, and endothelial cells of the blood-brain barrier (BBB) and lymphocytes. Flow cytometric analysis was used to examine P-gp expression in lymphocytes from male Fischer-344 rats from three age groups (young at 3-4 months, intermediate at 13-14 months, and old at 25-26 months). In addition, P-gp function in lymphocytes was assessed by measuring the ability of the P-gp inhibitor verapamil to limit the efflux of the fluorescent P-gp substrate rhodamine 123. P-gp expression was evaluated in the remaining four tissues by Western blot analysis. The effect of age on P-gp expression was tissue-specific. Although lymphocytic and hepatic P-gp expression increased with age, renal P-gp content was lower in the old kidneys. No statistical difference was observed in P-gp expression in intestinal microsomes or in BBB cell lysates among the three age groups. P-gp function was also increased by 6- to 8-fold in lymphocytes from the old rats. When P-gp expression was compared with CYP3A expression in these rats (reported elsewhere in this journal), we found that P-gp expression increased with age, whereas CYP3A expression and activity declined in the old livers. The converse pattern was observed in the kidney. Thus, age-related changes in P-gp expression and function are likely to be tissue-specific, and these changes may be inversely related to differences in CYP3A expression.     

Targeting myocardial reperfusion injuries with cyclosporine in the CIRCUS Trial – pharmacological reasons for failure

The mitochondrial permeability transition (mPTP) is a key feature of cardiac cell death in ischaemia‐reperfusion injury (I/R). The mPTP blocker, cyclosporine A (CsA), has been shown to give protection against reperfusion‐induced myocardial necrosis and troubles generated by acute coronary artery repermeabilization. Nevertheless, the results of the CIRCUS trial (Does Cyclosporine Improve Clinical Outcome in ST‐Elevation Myocardial Infarction Patients) seem to go against this hypothesis. Pharmacological reasons linked to CsA pharmacokinetics and pharmacodynamics could be suggested. First, it could be explained by a limited diffusion of the drug in the area at risk, due to the only inclusion of patients with a TIMI 0 or 1 coronary blood flow in the anterior territory and the absence of collateral perfusion. Second, to explain a low tissue diffusion of the compound, blood cell capture and high metabolism could be suggested. Moreover, CsA is highly metabolized by cytochrome P450 3A4 (CYP3A4), a polymorphic enzyme leading to variations of C_max and AUC between 10–20% in patients using CsA. Finally, CsA blocks calcineurin, a protein implied in I/R damage but calcineurin inhibition could contribute to protection towards I/R damage only when Rcan1, a calcineurin natural inhibitor, expression is low. The results of the CIRCUS trial are disappointing and could contribute to the withdrawal of the mPTP blockade pharmacological strategy as a way to protect the myocardium from I/R lesions. Nevertheless, many pharmacological insights could have contributed to an increased variability and, as a consequence, an important reduction of the pharmacological power of the study.