Metabolism Of Saquinavir Research Articles

INFLUENCE OF POMEGRANATE JUICE ON THE CYP3A4-MEDIATED METABOLISM AND P-GLYCOPROTEIN MEDIATED TRANSPORT OF SAQUINAVIR IN VIVO AND EX VIVO MODELS

Cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) play an important role in the first pass metabolism thereby limits the oral bioavailability of many clinically important and frequently prescribed drugs. The absolute oral bioavailability of saquinavir is very low (i. e. 4%) due to its extensive first pass metabolism by the major metabolizing isozyme CYP3A4 and it is also a substrate of P-gp. Pomegranate juice (PGJ) was known to be a modulator of CYP3A4 and P-gp. Therefore, the aim of this study was to evaluate the influence of PGJ on the pharmacokinetics (PK) of saquinavir in wistar rats and on the P-gp mediated intestinal transport of saquinavir in everted gut sacs ex vivo. Rats were treated orally with saquinavir (100 mg/kg) alone and in combination with PGJ (0.5, 1.0 and 2.0 mL/200g, body weight) for 15 consecutive days. Blood samples were collected on 1 st day in single dose pharmacokinetic study (SDS) and on 15 th day in multiple dose pharmacokinetic study (MDS). The peak plasma concentration (C max )and area under the plasma concentration-time curve (AUC 0-24 ) of saquinavir was increased with PGJ in SDS (p<0.001) may be due to inhibition of CYP3A4 and P-gp. But interestingly, the C max and AUC 0-24 of saquinavir was decreased significantly with PGJ in MDS. This is may be due to induction of CYP3A4. The transport of saquinavir was increased in presence of PGJ and known P-gp inhibitors (Verapamil, Ketoconazole and Quinindine) across the rat everted gut sacs ex vivo. The present study results suggested that PGJ has both effects (inhibition, in SDS and induction, in MDS) on CYP3A4-mediated saquinavir metabolism in vivo and inhibitory effect on the P-gp mediated intestinal transport of saquinavir ex vivo. Further studies are needed to confirm this interaction at cellular level using cell lines and in humans.

CPY3A4-Mediated α-Hydroxyaldehyde Formation in Saquinavir Metabolism

Saquinavir (SQV) is a protease inhibitor widely used for the treatment of human immunodeficiency virus (HIV) infection. We profiled SQV metabolism in mice using a metabolomic approach. Thirty SQV metabolites were identified in mouse feces and urine, of which 20 are novel. Most metabolites observed in mice were recapitulated in human liver microsomes. Among these novel metabolites, one α-hydroxyaldehyde produced from SQV N-dealkylation was noted and verified for the first time. Meanwhile, the corresponding product (3S)-N-tert-butyldecahydro-isoquinoline-3-carboxamide and its further metabolites were identified in mouse urine. The α-hydroxyaldehyde pathway was confirmed by using semicarbazide as a trapping reagent as well. Using recombinant cytochrome P450 (CYP450) isoenzymes and Cyp3a-null mice, CYP3A was identified as the dominant enzyme contributing to the formation of α-hydroxyaldehyde. This study enhances our knowledge of SQV metabolism, which can be used for predicting drug-drug interactions and further understanding the mechanism of adverse effects associated with SQV.

Analysis of the Pharmacokinetic Boosting Effects of Ritonavir on Oral Bioavailability of Drugs in Mice

Ritonavir dramatically increases the bioavailability of a variety of concurrently administered drugs by inhibition of metabolic enzymes and drug transporters. The purpose of this study was to investigate the extent to which ritonavir's inhibition of drug transporters and/or CYP3A contributes to the increased oral bioavailability in mice. The area under the plasma concentration-time curves (AUC) for orally administered saquinavir after coadministration with 50 mg/kg ritonavir dramatically increased (325-fold). As a result, the bioavailability, Fa·Fg and Fh increased 75-, 38- and twofold, respectively. In addition, the increase in the AUC predicted from the in vitro Ki value was ninefold, which was derived from the inhibition of metabolic enzymes by ritonavir in the liver. The remaining 36-fold increase in the AUC was considered to be derived from the inhibition in the small intestine. The AUCinf for probe substrate midazolam, fexofenadine, and pravastatin increased after the oral administration of ritonavir by only five-, 13-, and sevenfold, respectively. Moreover, the AUC0-12 for saquinavir was affected negligibly by itraconazole. These results indicate ritonavir mainly affects the first-pass effect of saquinavir in the small intestine, increasing the bioavailability of orally administered saquinavir. Furthermore, cyp isoforms other than CYP3A, which contribute to the metabolism of saquinavir in humans, are involved in the metabolism of saquinavir in mice.

Docosahexaenoic acid (DHA) inhibits saquinavir metabolism in-vitro and enhances its bioavailability in rats

This study investigated the effect of docosahexaenoic acid (DHA) on the metabolism of saquinavir by cytochrome P450 3A (CYP3A) in-vitro using rat liver microsomes and in-vivo using rats. DHA showed a concentration-dependent inhibition of in-vitro saquinavir metabolism with Km, Vmax and Ki values of 2.21 microM, 0.054 micromol h(-1) (mg protein)(-1) and 149.6 microM, respectively. After oral co-administration with 250 microg kg(-1) DHA, the bioavailability of saquinavir significantly increased approximately 4 fold (P < 0.01) without affecting the elimination half-life, as compared with the control. In contrast, oral administration of DHA did not affect the kinetic parameters of saquinavir administered intravenously. These results suggest that the inhibitory effect of DHA on saquinavir metabolism predominantly takes place in the gut and imply that DHA impairs the function of enteric, but not of hepatic, CYP3A. The pharmacokinetic interaction occurred only when DHA was taken simultaneously with oral administration of saquinavir. These results considered together with the lack of time-dependent saquinavir metabolism inactivation effects in-vitro, imply that the inhibitory effect of DHA is primarily reversible. It is concluded that DHA inhibited saquinavir metabolism in-vitro and enhanced the oral bioavailability of saquinavir in rats.

Mechanistic simulations explain paradoxical saquinavir metabolism during in vitro vectorial transport study

We were confronted by an unexpected observation: in a Transwell study of confluent, Cyp3A4 and P-gp-expressing Caco-2 cells, higher intracellular saquinavir levels, yet less first metabolite (M7) formation were observed following apical dosing, compared to basal dosing [1]. To test two seemingly plausible hypothesized explanations, we constructed an in silico working analogue using the synthetic method. Neither mechanism alone was sufficient, but when combined and tuned within the analogue, the results generated were a semi-quantitative match to the experimental data. After 60 cycles, more of the simulated dose was present within analogue cells as parent drug after apical dosing. Furthermore, less M7 was present after apical dose. The paradox disappeared by having simulated drugs equilibrate among separate intracellular zones. Building, studying, and exploring mechanistic explanations for complex wet-lab phenomena using the new methods improved insight into the referent system, while providing a straightforward, scientific means of testing the plausibility of mechanistic hypotheses.

Clearance and clearance inhibition of the HIV-1 protease inhibitors ritonavir and saquinavir in sandwich-cultured rat hepatocytes and rat microsomes

The metabolism and active transport of ritonavir and saquinavir were studied using sandwich-cultured rat hepatoyctes and rat liver microsomes. For ritonavir four comparable metabolites were observed in the sandwich-culture and in microsomes. For saquinavir eight metabolites were observed in sandwich-culture and 14 different metabolites in microsomes. Ketoconazole did not affect the metabolism of ritonavir in sandwich-culture or microsomes and slightly inhibited the metabolism of saquinavir in sandwich-culture. This inhibition resulted in a different metabolite profile for saquinavir in microsomes. Ritonavir had a pronounced inhibiting effect on the metabolism of saquinavir and affected the hydroxylation of 6beta-testosterone negatively. In the active transport studies, cyclosporin A and PSC833 enhanced the metabolism of ritonavir, suggesting that ritonavir is normally excreted into the bile canaliculi. Verapamil, showed no effect on the metabolism of ritonavir. The intrinsic clearance was estimated at 1.65 and 67.5 microl/min/1 x 10(6) cells and the hepatic metabolism clearance at 0.017 and 6.83ml/min/SRW for ritonavir and saquinavir respectively. In conclusion, for saquinavir the metabolism rate and the amount of metabolites produced was higher than for ritonavir. Ritonavir had a strong inhibitory effect on the metabolism of saquinavir and seemed to be excreted into the bile.

Rate-Limiting Steps in Hepatic Drug Clearance: Comparison of Hepatocellular Uptake and Metabolism with Microsomal Metabolism of Saquinavir, Nelfinavir, and Ritonavir

The intrinsic metabolic clearance of saquinavir, nelfinavir, and ritonavir was determined over a range of concentrations (0.02-20 microM) in both rat liver microsomes and fresh isolated rat hepatocytes in suspension. Clearance values were found to be concentration dependent for both systems, and at low concentrations, microsomal clearance was much greater (7-14-fold) than in hepatocytes. Kinetic parameters showed substantially lower microsomal K(m) values (5-42 nM) compared with suspended rat hepatocytes (34-270 nM) but similar scaled V(max) values (2-26 nmol/min/g liver). In the absence of metabolism (achieved by pretreating hepatocytes with a mechanism-based inhibitor of cytochrome P450), saquinavir, nelfinavir, and ritonavir were actively and rapidly taken up into hepatocytes (cell/medium concentration ratios of 306-3352), and intracellular unbound drug concentrations between 5- and 12-fold higher than extracellular unbound concentrations were achieved. Comparison of the rate of uptake into hepatocytes with the rate of metabolism in hepatocytes and microsomes indicates that the former is the rate-limiting step at low concentrations. The rate of metabolism saturates at lower concentrations (100-400-fold) than the rate of uptake; hence, at the high concentrations metabolic rate-limited clearance occurs. In conclusion, the clearance of saquinavir, nelfinavir, and ritonavir is extremely rapid, and it is proposed that in the case of hepatocytes and by inference in vivo, the rate of uptake limits the metabolic clearance of these three drugs.

Gender: A Possible Determinant in Dosing of Dermatologic Drugs—an overview

The role of gender in pharmacokinetics could play a significant role in tailoring HIV and other drug regimens. Here we investigate sex as a factor in saquinavir pharmacokinetics. The HIV-positive women who use saquinavir in combination with other protease inhibitors frequently demonstrate higher saquinavir concentrations than their male counterparts. The majority of the data indicates that the root of women's higher saquinavir concentrations is not inherent to their gender. Rather, extenuating factors associated with HIV play a prominent role in the observed difference between men and women's pharmacokinetic parameters. The source of observed differences in saquinavir pharmacokinetics could not be definitively traced to CYP3A, the enzyme responsible for saquinavir's metabolism or drug transporters. Hormonal changes associated with HIV infection together with concurrent use of other protease inhibitors could help explain development of sex differences. These findings can be used to further investigate when and why gender-based differences in saquinavir pharmacokinetics exist and possible future dosage and therapy considerations. Our knowledge of gender-related pharmacokinetics should be extended for other systemic drugs in dermatology.

Inhibitory effect of stiripentol on carbamazepine and saquinavir metabolism in human.

To characterize the in vitro and in vivo inhibitory effect of stiripentol, a new anticonvulsant, on the metabolism of carbamazepine and saquinavir, which are substrates of CYP3A4. Human liver microsomes and cDNA-expressed CYP enzymes were used for the in vitro experiments. Pharmacokinetic data from epileptic children and healthy adults were used for the carbamazepine and saquinavir in vivo studies, respectively. Carbamazepine biotransformation to its 10,11-epoxide by human liver microsomes (Vmax = 10.3 nmol min(-1) nmol(-1) P450, apparent Km = 362 microm), cDNA-expressed CYP3A4 (Vmax = 1.17 nmol min(-1) nmol(-1) P450, apparent Km = 119 microm) and CYP2C8 (Vmax = 0.669 nmol min(-1) nmol(-1) P450, apparent Km = 757 microm) was inhibited by stiripentol (IC50 14, 5.1, 37 microM and apparent Ki 3.7, 2.5, 35 microm, respectively). Saquinavir biotransformation to its major metabolite M7 by human liver microsomes (Vmax = 5.7 nmol min(-1) nmol(-1) P450, apparent Km = 0.79 microm) was inhibited by stiripentol (IC50 163 microM, apparent Ki 86 microm). In epileptic children treated with carbamazepine and stiripentol, the plasma concentration ratio of carbamazepine epoxide/carbamazepine was decreased by 65%. The in vivo apparent Ki for stiripentol ranged from 10.5 to 41.4 microm. The pharmacokinetics of saquinavir was not modified by stiripentol in healthy adults. The 95% confidence intervals for the difference for Cmax and AUC of saquinavir between the placebo and stiripentol phase were (-39.8, 39.8) and (-33.2, 112), respectively. These results showed that stiripentol was a weak inhibitor of saquinavir metabolism both in vitro and in vivo. In contrast, stiripentol is a potent inhibitor of carbamazepine 10,11-epoxide formation in vitro and in vivo in epileptic patients.

Pharmacokinetics and pharmacodynamics of saquinavir in pediatric patients with human immunodeficiency virus infection.

Our objective was to investigate the clinical pharmacologic characteristics of saquinavir given as a soft gelatin capsule, either alone or in combination with nelfinavir, to children and adolescents with human immunodeficiency virus infection. The pharmacokinetics of 50 mg/kg saquinavir 3 times a day (tid) alone versus 33 mg/kg saquinavir tid plus 30 mg/kg nelfinavir tid was assessed after single-dose administration and after short- and long-term administration. The single-dose pharmacokinetics of fixed (1200 mg) versus unrestricted weight-adjusted dosing (50 mg/kg) was also investigated. Saquinavir as the sole protease inhibitor resulted in lower saquinavir exposure in children (steady-state geometric mean area under the concentration-time curve from time zero to 24 hours [AUC (0-24 h)], 5790 ng x h/ml; steady-state concentration 8 hours after drug administration [C(8h,SS)], 65 ng/ml) and adolescents [steady-state geometric mean AUC(0-24 h), 5914 ng x h/ml] than that reported in adults treated with 1200 mg tid [steady-state geometric mean AUC(0-24 h), 21,700 ng x h/ml; C(8h,SS), 223 ng/ml]. This finding appeared to be attributable to markedly higher apparent oral clearance, potentially as a result of increased systemic clearance and reduced oral bioavailability. Nelfinavir combined with saquinavir reduced apparent oral clearance, increasing saquinavir exposure in children [steady-state geometric mean AUC(0-24 h), 11,070 ng x h/ml; C(8h,SS), 380 ng/ml] to levels that approach those observed in adults. A significant correlation between average trough concentration and sustained viral load suppression was observed in children. The apparent threshold for maintaining viral load suppression was a mean trough saquinavir concentration above 200 ng/ml. The pharmacokinetics of saquinavir in children is different from that of adults, and administration of saquinavir alone will not give consistently efficacious plasma levels. The best way of improving saquinavir exposure in children is through combination therapy with other protease inhibitors that inhibit saquinavir metabolism.

CYP3A4-mediated hepatic metabolism of the HIV-1 protease inhibitor saquinavir in vitro

1. The aim was to identify the major metabolites of saquinavir (SQV) from human hepatic microsomal incubations and the CYP isoform(s) responsible.2. Ten fractions containing various metabolites were separated by isocratic reversed- phase HPLC and characterized by HPLC, mass spectrometry and NMR.3. Metabolites were either mono- or di-hydroxylated derivatives of SQV. Fast-atom bombardment and electrospray MS showed that hydroxylation was predominantly situated on the decahydroisoquinoline ring. A major metabolite (M4) was rigorously identified as 6-equatorial-hydroxy SQV.4. Metabolism of saquinavir to all metabolites was inhibited by the CYP3A4-selective inhibitor ketoconazole (IC50 = 0.55 ± 0.12 µM). Other isoform-selective inhibitors were non-inhibitory. The protease inhibitors ritonavir, indinavir and nelfinavir potently inhibited SQV metabolism in hepatic microsomes with IC50 = 0.025 ± 0.004, 0.82 ± 0.26 and 0.58 ± 0:14 µM, respectively.5. Saquinavir metabolism correlated with immunochemically determined CYP3A4 levels and testosterone 6(β-hydroxylation, but it failed to correlate with either immunochemically determined CYP1A2 levels or marker activities for CYP1A2, 2C9 or 2E1.6. Heterologously expressed CYP3A4 metabolized saquinavir with a similar metabolic profile to that of human liver microsomes.7. Km and Vmax for total SQV metabolism were 0.61±0.19µM and 1.82 ± 1.13 nmolmg−1 min−1, respectively.8 The extensive involvement of hepatic CYP3A4 in the metabolism of saquinavir predicts high intrinsic clearance of saquinavir. Inhibitors of CYP3A4 such as other protease inhibitors will substantially increase the bioavailability of saquinavir.

P-glycoprotein limits oral availability, brain, and fetal penetration of saquinavir even with high doses of ritonavir.

The low oral bioavailability of the HIV protease inhibitor (HPI) saquinavir is dramatically increased by coadministration of the HPI ritonavir. Because saquinavir and ritonavir are substrates and inhibitors of both the drug transporter P-glycoprotein (P-gp) and of the metabolizing enzyme CYP3A4, we wanted to sort out whether the ritonavir effect is primarily mediated by inhibition of CYP3A4 or P-gp or both. P-gp is known to limit the bioavailability, brain, testis, and fetal penetration of its substrates, so effective inhibition of P-gp by ritonavir in vivo might open up pharmacological sanctuary sites for saquinavir, with the potential of beneficial effects on therapy, but also of increased toxicity. In vitro, P-gp-mediated transport of saquinavir and ritonavir was only moderately inhibited by both HPIs compared with the potent P-gp inhibitor PSC833. When [(14)C]saquinavir was orally coadministered with a maximum tolerated dose of ritonavir to wild-type and P-gp-deficient mice, saquinavir bioavailability was dramatically increased in both strains, but P-gp still limited the oral bioavailability of saquinavir, and its penetration into brain and fetus. These data indicate that in vivo, ritonavir is a relatively poor P-gp inhibitor. The highly increased bioavailability of saquinavir because of ritonavir coadministration most likely results from reduced saquinavir metabolism. Importantly, our data indicate that it is unlikely that ritonavir coadministration will substantially affect the contribution of P-gp to pharmacological sanctuary sites such as brain, testis, and fetus. Thus, if one wanted to effectively open these sites for therapeutic purposes, more efficient P-gp inhibitors should be applied.

Inhibition of the CYP3A4-mediated metabolism and P-glycoprotein-mediated transport of the HIV-1 protease inhibitor saquinavir by grapefruit juice components.

Cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (P-gp) are both expressed in the intestinal mucosa and present a barrier to oral drug delivery. CYP3A4 and P-gp share both overlapping tissue distribution and substrate specificity. Grapefruit juice interactions with CYP3A4 substrates are well documented and occur as a consequence of down regulation of intestinal CYP3A4. The aim of the present study was to screen grapefruit juice components against the CYP3A4-mediated metabolism and P-gp mediated transport of the HIV-1 protease inhibitor saquinavir. Five grapefruit juice components: quercetin, naringin, naringenin, 6', 7'-dihydroxybergamottin and bergamottin were screened as potential inhibitors of the metabolism of saquinavir by human liver microsomes. The known CYP3A4 inhibitor ketoconazole was also screened for inhibitory potential. These compounds were also screened as modulators of P-gp activity by assessing the directional transport of saquinavir across Caco-2 cell monolayers which express P-gp. The effect of verapamil, a known modulator of P-gp function, was also determined in these cell lines. On preincubation, 6', 7'-dihydroxybergamottin and bergamottin inhibited the metabolism of saquinavir, with IC50 values of 0.33+/-0.23 muM and 0.74+/-0.13 muM, respectively (n=3). Ketoconazole achieved an IC50 of 0. 55+/-0.12 muM (n=4). The other compounds studied failed to reach IC50 at concentrations of up to 100 muM. The transport of saquinavir in the basolateral-->apical (BL-->AP) direction exceeded that in the apical -->basolateral direction (AP-->BL), with apparent permeability coefficients of 199.2+/-15.8x10-7 cm s-1 and 8.00+/-1. 13x10-7 cm s-1, respectively (n=3) which is indicative of a polarized efflux mechanism. The ratio of BL-->AP/AP-->BL for saquinavir was 25, but in the presence of verapamil and ketoconazole this ratio was reduced to 3.6 and 4.0, respectively (n=3), indicating extensive inhibition of P-gp mediated saquinavir efflux. Of the grapefruit juice components studied only naringin and 6', 7'-dihydroxybergamottin had any appreciable effect, reducing the ratio to 7.6 and 7.1, respectively (n=3); but this was due solely to increased AP-->BL transport. Grapefruit juice components inhibit CYP3A4-mediated saquinavir metabolism and also modulate, to a limited extent, P-gp mediated saquinavir transport in Caco-2 cell monolayers. The in vivo effects of grapefruit juice coadministration are most likely the result of effects on CYP3A4 (inhibition and down regulation) and only to a minor extent on modulation of P-gp function.

Pharmacokinetic enhancement of inhibitors of the human immunodeficiency virus protease by coadministration with ritonavir.

Coadministration with the human immunodeficiency virus (HIV) protease inhibitor ritonavir was investigated as a method for enhancing the levels of other peptidomimetic HIV protease inhibitors in plasma. In rat and human liver microsomes, ritonavir potently inhibited the cytochrome P450 (CYP)-mediated metabolism of saquinavir, indinavir, nelfinavir, and VX-478. The structural features of ritonavir responsible for CYP binding and inhibition were examined. Coadministration of other protease inhibitors with ritonavir in rats and dogs produced elevated and sustained plasma drug levels 8 to 12 h after a single dose. Drug exposure in rats was elevated by 8- to 46-fold. A > 50-fold enhancement of the concentrations of saquinavir in plasma was observed in humans following a single codose of ritonavir (600 mg) and saquinavir (200 mg). These results indicate that ritonavir can favorably alter the pharmacokinetic profiles of other protease inhibitors. Combination regimens of ritonavir and other protease inhibitors may thus play a role in the treatment of HIV infection. Because of potentially substantial drug level increases, however, such combinations require further investigation to establish safe regimens for clinical use.