Comparison of mechanism-based inhibition of human cytochrome P450 2C19 by ticlopidine, clopidogrel, and prasugrel.

Mechanism-based inhibition of CYP2C19 in human liver microsomes by the thienopyridine antiplatelet agents clopidogrel, prasugrel and their thiolactone metabolites was investigated by determining the time- and concentration-dependent inhibition of the activity of S-mephenytoin 4'-hydroxylase as typical CYP2C19 activity and compared with ticlopidine and its metabolite. Clopidogrel was shown to be a mechanism-based inhibitor of CYP2C19 with the inactivation kinetic parameters, k(inact) and K(I), equal to 0.0557 min(-1) and 14.3 microM, respectively, as well as ticlopidine (0.0739 min(-1) and 3.32 microM, respectively). The thiolactone metabolite of ticlopidine and clopidogrel inhibited CYP2C19 only in a concentration-dependent manner. In contrast, neither prasugrel nor its thiolactone metabolite inhibited CYP2C19 at concentrations up to 100 microM. The oxidation of the thiophene moiety of clopidogrel to form their respective thiolactones was found to be the critical reaction that produces the chemically reactive metabolites which cause the mechanism-based inhibition of CYP2C19. Estimation of in vivo drug-drug interaction using in vitro parameters predicted clinically observed data. For clopidogrel, there was no increase in the area under the curve (AUC) at its clinical dose level as predicted by the in vitro parameters, and for ticlopidine the prediction agreed with the clinically observed AUC increase. In conclusion, clopidogrel is potent mechanism-based inhibitors of CYP2C19 as well as ticlopidine, whereas prasugrel did not inactivate CYP2C19. Administration of prasugrel would not cause a clinically relevant interaction with CYP2C19.

Pharmacokinetics and pharmacodynamics of prasugrel in subjects with moderate liver disease.

BACKGROUND AND OBJECTIVE: Prasugrel is a thienopyridine antiplatelet agent under investigation for the prevention of atherothrombotic events in patients with acute coronary syndrome who undergo percutaneous coronary intervention. Patients with chronic liver disease are among those in the target population for prasugrel. As hepatic enzymes play a key role in formation of prasugrel's active metabolite, hepatic impairment could affect the safety and/or efficacy of prasugrel in such patients. METHODS: This was a parallel-design, open-label, multiple dose study of 30 subjects, 10 with moderate hepatic impairment (Child-Pugh Class B) and 20 with normal hepatic function. Prasugrel was administered orally as a 60-mg loading dose (LD) and daily 10-mg maintenance doses (MDs) for 5 days. Pharmacokinetic parameters (AUC(0-t), C(max) and t(max)) and maximal platelet aggregation (MPA) by light transmission aggregometry were assessed after the LD and final MD. RESULTS AND DISCUSSION: Exposure to prasugrel's active metabolite was comparable between healthy subjects and those with moderate hepatic impairment. Point estimates for the ratios of geometric least square means for AUC(0-t) and C(max) after the LD and last MD ranged from 0.91 to 1.14. MPA to 20 microm ADP was similar between subjects with moderate hepatic impairment and healthy subjects for both the LD and MD. Prasugrel was well tolerated by all subjects, and adverse events were mild in severity. CONCLUSION: Moderate hepatic impairment appears to have no effect on exposure to prasugrel's active metabolite. Furthermore, MPA results suggest that moderate hepatic impairment has little or no effect on platelet aggregation relative to healthy controls. Overall, these results suggest that a dose adjustment would not be required in moderately hepatically impaired patients taking prasugrel.

Prasugrel pharmacokinetics and pharmacodynamics in subjects with moderate renal impairment and end-stage renal disease.

OBJECTIVE: The pharmacokinetic (PK) and pharmacodynamic (PD) responses to prasugrel were compared in three studies of healthy subjects vs. those with moderate or end-stage renal impairment. METHODS: Two of the three protocols were parallel-design, open-label, single dose (60-mg prasugrel) studies in subjects with end-stage renal disease (ESRD; n = 12) or moderate renal impairment (n = 10) and matched healthy subjects with normal renal function (n = 10). The third protocol was an open-label, single-dose escalation (5, 10, 30 and 60 mg prasugrel) study in subjects with ESRD (n = 16) and matched healthy subjects with normal renal function (n = 16). Plasma concentrations of prasugrel's active metabolite were determined and pharmacokinetic parameter estimates were derived. Maximum platelet aggregation (MPA) was measured by light transmission aggregometry using 20 mum adenosine diphosphate as agonist. RESULTS: Across all studies, prasugrel's C(max) and AUC(0-t) were 51% and 42% lower in subjects with ESRD than in healthy subjects. AUC(0-t) did not differ between healthy subjects and subjects with moderate renal impairment. The magnitude of change and time-course profiles of MPA was similar for healthy subjects compared with subjects with moderate renal impairment and those with ESRD. Prasugrel was well-tolerated in all subjects. CONCLUSION: There was no difference in pharmacokinetics or PD responses between subjects with moderate renal impairment and healthy subjects. Despite significantly lower exposure to prasugrel's active metabolite in subjects with ESRD, MPA did not differ between healthy subjects and those with ESRD.

Effect of rifampin on the pharmacokinetics and pharmacodynamics of prasugrel in healthy male subjects.

OBJECTIVE: Prasugrel is a thienopyridine antiplatelet agent for the prevention of atherothrombotic events in patients with acute coronary syndrome undergoing percutaneous coronary intervention. Since cytochrome P450 enzymes CYP3A4 and CYP2B6 play a major role in prasugrel's active metabolite formation, the effect of potent CYP induction by rifampin on the pharmacokinetics of prasugrel and on the pharmacodynamic response to prasugrel was evaluated in healthy male subjects. RESEARCH DESIGN AND METHODS: This was an open-label, two-period, fixed-sequence study conducted at a single clinical research center. In the first treatment period, subjects received prasugrel as an oral 60-mg loading dose (LD) on the first day followed by ten oral, 10-mg daily maintenance doses. After a 2-week washout period, subjects received oral rifampin alone (600 mg once daily) for 8 days, followed by coadministration of oral rifampin with prasugrel, given as a 60-mg LD on the first day followed by five daily 10-mg MDs. Blood collection for pharmacokinetic and pharmacodynamic analyses occurred after the LD and fifth MD of prasugrel in both periods. CLINICAL TRIAL SYNOPSIS: clinicalstudyresults.org ID #8976 RESULTS: Rifampin coadministration (600 mg daily) did not affect exposure to prasugrel's active metabolite (R-138727). However, at 2 and 4 h after the prasugrel loading dose (60 mg), rifampicin coadministration was associated with a 6-9 percentage point decrease (p < 0.01) in the magnitude of platelet inhibition; similarly, a 5-17 percentage point decrease (p < 0.05) was observed with rifampin coadministration during the prasugrel maintenance dose (10 mg) period. Post hoc in vitro experiments demonstrated a dose-dependent R-138727-rifampin interaction at the P2Y(12) level unrelated to enzyme induction. A limitation of this study is that while results of the in vitro post hoc study indicate a pharmacodynamic interaction with rifampin, the mechanism underlying this interaction has not been elucidated. CONCLUSIONS: Dose adjustment should not be necessary when prasugrel is administered with CYP inducers since formation of prasugrel's active metabolite is not affected by potent enzyme induction with rifampin.

Comparison of formation of thiolactones and active metabolites of prasugrel and clopidogrel in rats and dogs.

Prasugrel and clopidogrel are antiplatelet prodrugs that are converted to their respective active metabolites through thiolactone intermediates. Prasugrel is rapidly hydrolysed by esterases to its thiolactone intermediate, while clopidogrel is oxidized by cytochrome P450 (CYP) isoforms to its thiolactone. The conversion of both thiolactones to the active metabolites is CYP mediated. This study compared the efficiency, in vivo, of the formation of prasugrel and clopidogrel thiolactones and their active metabolites. The areas under the plasma concentration versus time curve (AUC) of the thiolactone intermediates in the portal vein plasma after an oral dose of prasugrel (1 mg kg(-1)) and clopidogrel (0.77 mg kg(-1)) were 15.8 +/- 15.9 ng h ml(-1) and 0.113 +/- 0.226 ng h ml(-1), respectively, in rats, and 454 +/- 104 ng h ml(-1) and 23.3 +/- 4.3 ng h ml(-1), respectively, in dogs, indicating efficient hydrolysis of prasugrel and little metabolism of clopidogrel to their thiolactones in the intestine. The relative bioavailability of the active metabolites of prasugrel and clopidogrel calculated by the ratio of active metabolite AUC (prodrug oral administration/active metabolite intravenous administration) were 25% and 7%, respectively, in rats, and 25% and 10%, respectively, in dogs. Single intraduodenal administration of prasugrel showed complete conversion of prasugrel, resulting in high concentrations of the thiolactone and active metabolite of prasugrel in rat portal vein plasma, which demonstrates that these products are generated in the intestine during the absorption process. In conclusion, the extent of in vivo formation of the thiolactone and the active metabolite of prasugrel was greater than for clopidogrel's thiolactone and active metabolite.

Common polymorphisms of CYP2C19 and CYP2C9 affect the pharmacokinetic and pharmacodynamic response to clopidogrel but not prasugrel.

BACKGROUND: Thienopyridines are metabolized to active metabolites that irreversibly inhibit the platelet P2Y(12) adenosine diphosphate receptor. The pharmacodynamic response to clopidogrel is more variable than the response to prasugrel, but the reasons for variation in response to clopidogrel are not well characterized. OBJECTIVE: To determine the relationship between genetic variation in cytochrome P450 (CYP) isoenzymes and the pharmacokinetic/pharmacodynamic response to prasugrel and clopidogrel. METHODS: Genotyping was performed for CYP1A2, CYP2B6, CYP2C19, CYP2C9, CYP3A4 and CYP3A5 on samples from healthy subjects participating in studies evaluating pharmacokinetic and pharmacodynamic responses to prasugrel (60 mg, n = 71) or clopidogrel (300 mg, n = 74). RESULTS: In subjects receiving clopidogrel, the presence of the CYP2C19*2 loss of function variant was significantly associated with lower exposure to clopidogrel active metabolite, as measured by the area under the concentration curve (AUC(0-24); P = 0.004) and maximal plasma concentration (C(max); P = 0.020), lower inhibition of platelet aggregation at 4 h (P = 0.003) and poor-responder status (P = 0.030). Similarly, CYP2C9 loss of function variants were significantly associated with lower AUC(0-24) (P = 0.043), lower C(max) (P = 0.006), lower IPA (P = 0.046) and poor-responder status (P = 0.024). For prasugrel, there was no relationship observed between CYP2C19 or CYP2C9 loss of function genotypes and exposure to the active metabolite of prasugrel or pharmacodynamic response. CONCLUSIONS: The common loss of function polymorphisms of CYP2C19 and CYP2C9 are associated with decreased exposure to the active metabolite of clopidogrel but not prasugrel. Decreased exposure to its active metabolite is associated with a diminished pharmacodynamic response to clopidogrel.

Disposition and metabolic fate of prasugrel in mice, rats, and dogs.

The disposition and metabolism of prasugrel, a thienopyridine prodrug and a potent inhibitor of platelet aggregation in vivo, were investigated in mice, rats, and dogs. Prasugrel was rapidly absorbed and extensively metabolized. In the mouse and dog, maximum plasma concentration of radioactivity was observed in less than 1 h after an oral [14C]prasugrel dose. Most of the administered prasugrel dose was recovered in the faeces of rats and dogs (72% and 52-73%, respectively), and in mice urine (54%). Prasugrel is hydrolysed by esterases to a thiolactone, which is subsequently metabolized to thiol-containing metabolites. The main circulating thiol-containing metabolite in the three animal species is the pharmacologically active metabolite, R-138727. The thiol-containing metabolites are further metabolized by S-methylation and conjugation with cysteine.

Absorption, distribution and excretion of the new thienopyridine agent prasugrel in rats.

Prasugrel is converted to the pharmacologically active metabolite after oral dosing in vivo. In this study, (14)C-prasugrel or prasugrel was administered to rats at a dose of 5 mg kg(-1). After oral and intravenous dosing, the values of AUC(0-infinity) of total radioactivity were 36.2 and 47.1 microg eqx h ml(-1), respectively. Oral dosing of unlabeled prasugrel showed the second highest AUC(0-8) of the active metabolite of six metabolites analyzed. Quantitative whole body autoradiography showed high radioactivity concentrations in tissues for absorption and excretion at 1 h after oral administration, and were low at 72 h. The excretion of radioactivity in the urine and feces were 20.2% and 78.7%, respectively, after oral dosing. Most radioactivity after oral dosing was excreted in bile (90.1%), which was reabsorbed moderately (62.4%). The results showed that orally administered prasugrel was rapidly and fully absorbed and efficiently converted to the active metabolite with no marked distribution in a particular tissue.

Cytochrome P450 3A inhibition by ketoconazole affects prasugrel and clopidogrel pharmacokinetics and pharmacodynamics differently.

Prasugrel and clopidogrel inhibit platelet aggregation through active metabolite formation. Prasugrel's active metabolite (R-138727) is formed primarily by cytochrome P450 (CYP) 3A and CYP2B6, with roles for CYP2C9 and CYP2C19. Clopidogrel's activation involves two sequential steps by CYP3A, CYP1A2, CYP2C9, CYP2C19, and/or CYP2B6. In a randomized crossover study, healthy subjects received a loading dose (LD) of prasugrel (60 mg) or clopidogrel (300 mg), followed by five daily maintenance doses (MDs) (15 and 75 mg, respectively) with or without the potent CYP3A inhibitor ketoconazole (400 mg/day). Subjects had a 2-week washout between periods. Ketoconazole decreased R-138727 and clopidogrel active metabolite Cmax (maximum plasma concentration) 34-61% after prasugrel and clopidogrel dosing. Ketoconazole did not affect R-138727 exposure or prasugrel's inhibition of platelet aggregation (IPA). Ketoconazole decreased clopidogrel's active metabolite AUC0-24 (area under the concentration-time curve to 24 h postdose) 22% (LD) to 29% (MD) and reduced IPA 28% (LD) to 33% (MD). We conclude that CYP3A4 and CYP3A5 inhibition by ketoconazole affects formation of clopidogrel's but not prasugrel's active metabolite. The decreased formation of clopidogrel's active metabolite is associated with reduced IPA.

Preclinical characterization of 2-[3-[3-[(5-ethyl-4'-fluoro-2-hydroxy[1,1'-biphenyl]-4-yl)oxy]propoxy]-2-propylphenoxy]benzoic acid metabolism: in vitro species comparison and in vivo disposition in rats.

Assessment of the pharmacokinetics of [14C]2-[3-[3-[(5-ethyl-4'-fluoro-2-hydroxy[1,1'-biphenyl]-4-yl)oxy]propoxy]-2-propylphenoxy-]benzoic acid ([14C]LY293111), an experimental anti-cancer agent, suggested long-lived circulating metabolites in rats. In vivo metabolites of LY293111 were examined in plasma, bile, urine, and feces of Fischer 344 (F344) rats after oral administration of [14C]LY293111. Metabolites were profiled by high-performance liquid chromatography-radiochromatography, and identified by liquid chromatography (LC)/mass spectrometry and LC/NMR. The major in vivo metabolites of LY293111 identified in rats were phenolic (ether), acyl, and bisglucuronides of LY293111. Measurement of radioactivity in rat plasma confirmed that a fraction of LY293111-derived material was irreversibly bound to plasma protein and that this bound fraction increased over time. This was consistent with the observed disparity in half-lives between LY293111 and total radioactivity in rats and monkeys, and is likely due to covalent modification of proteins by the acyl glucuronide. In vitro metabolism of [14C]LY293111 in liver slices from CD-1 mice, F344 rats, rhesus and cynomolgus monkeys, and humans indicates that glucuronidation was the primary metabolic pathway in all species. The acyl glucuronide was the most prevalent radioactive peak (16% of total 14C) produced by F344 rat slices, whereas the ether glucuronide was the major metabolite in all other species (26-36% of total 14C). Several minor hydroxylated metabolites were detected in F344 rat slice extracts but were not observed in other species. The data presented suggest that covalent modification of proteins by LY293111 acyl glucuronide is possible in multiple species, although the relative reactivity of this metabolite appears to be low compared with those known to cause adverse drug reactions.

Disposition of LY333531, a selective protein kinase C beta inhibitor, in the Fischer 344 rat and beagle dog.

1. Studies were conducted in the Fischer 344 rat and beagle dog to determine the disposition of LY333531 and its equipotent active des-methyl metabolite, LY338522, both potent and selective inhibitors of the beta-isozyme of protein kinase C. 2. Male Fischer 344 rats and female beagle dogs received a single 5-mg kg(-1) oral dose of (14)C-LY333531. Urine, faeces, bile and plasma were collected and analysed for (14)C, LY333531 and LY338522. 3. LY333531 was eliminated primarily in the faeces (91% by 120 h in rat, 90% by 96 h in dog). Bile contributed the majority of the radioactivity excreted in the faeces in rat (66% in the cannulated bile duct study) and a variable but significant proportion in dog. 4. Pharmacokinetics following a single 5 mg kg(-1) oral dose of (14)C-LY333531 to the male rat produced C(max) and AUC(0-infinity ) for LY333531 of 14.7 ng ml(-1) and 60.8 ng h ml(-1), respectively, with a half-life of 2.5 h. LY338522 and total radioactivity showed similar profiles. 5. In the female dog at the same dose, C(max) and AUC(0-infinity ) of LY333531 were higher, producing 245 +/- 94 ng ml(-1) and 1419 +/- 463 ng h ml(-1), respectively, with a half-life of 5.7 h. 6. The data indicate that the disposition of LY333531 is similar in rat and dog.

Salt form selection and characterization of LY333531 mesylate monohydrate.

LY333531 is a potent protein kinase C(beta) (PKC(beta)) inhibitor currently under development for the treatment of diabetic complications. Seven salts of LY333531 (hydrochloride, sulfate, mesylate, succinate, tartrate, acetate and phosphate) were evaluated during the early phase of development. Physical property screening techniques including microscopy, DSC, TGA, XRPD, hygroscopicity and solubility were utilized to narrow the selection to two salts: the mesylate and hydrochloride. Identification of the optimal salt form was based upon solubility, bioavailability, physical stability and purity. During the evaluation process three hydrated forms (anhydrate, monohydrate, and tetrahydrate) of the hydrochloride salt were identified. The mesylate salt was found to give only one, a monohydrate. Processing parameters (e.g. filtration rate, crystal form stability) demonstrated that the anhydrate was the preferred form of the hydrochloride salt. Bioavailability studies in dogs indicated that the C(max) and area under the plasma concentration vs. time curve (AUC) for LY333531 and its active metabolite, LY338522, following administration of the mesylate salt were approximately 2.6 times those obtained after the LY333531 HCl dose. This difference was presumed to be due primarily to the fact that the mesylate was five times more soluble than the hydrochloride salt in water. These factors led to selection and development of LY333531 mesylate monohydrate as the active pharmaceutical ingredient for clinical evaluation.

Confidence interval criteria for assessment of dose proportionality.

PURPOSE: The aim of this work was a pragmatic, statistically sound and clinically relevant approach to dose-proportionality analyses that is compatible with common study designs. METHODS: Statistical estimation is used to derive a (1-alpha)% confidence interval (CI) for the ratio of dose-normalized, geometric mean values (Rdnm) of a pharmacokinetic variable (PK). An acceptance interval for Rdnm defining the clinically relevant, dose-proportional region is established a priori. Proportionality is declared if the CI for Rdnm is completely contained within the critical region. The approach is illustrated with mixed-effects models based on a power function of the form PK = beta0 x Dose(beta1); however, the logic holds for other functional forms. RESULTS: It was observed that the dose-proportional region delineated by a power model depends only on the dose ratio. Furthermore, a dose ratio (rho1) can be calculated such that the CI lies entirely within the pre-specified critical region. A larger ratio (rho2) may exist such that the CI lies completely outside that region. The approach supports inferences about the PK response that are not constrained to the exact dose levels studied. CONCLUSION: The proposed method enhances the information from a clinical dose-proportionality study and helps to standardize decision rules.

High-performance liquid chromatographic determination of the enantiomers of the benzoquinolinone LY191704, a human type 1 5 alpha-reductase inhibitor, in plasma.

A stereoselective reversed-phase liquid chromatographic method for the determination of compounds LY300502 and LY300503 (enantiomers of LY191704) in rat and dog plasma was developed. The assay involved extraction of the compounds using a strong cation-exchange solid-phase extraction column, from which the compounds are eluted with 1% of 1 M HCI in methanol. The enantiomers were separated on a Daicel Chiralcel OD-R column. The mobile phase consisted of water-acetonitrile-methanol (50:40:10, v/v) at a flow-rate of 0.3 ml/min. UV detection was achieved at 220 nm. The disposition of the enantiomers of LY191704 in rats and dogs was found to be stereoselective and species specific.

Stereoselective disposition of the enantiomers of the benzoquinolinone LY191704, a human type I 5 alpha-reductase inhibitor. Differences between rats and dogs.

The benzoquinolinone LY191704, a potent and selective human type I 5 alpha-reductase inhibitor, is a racemic mixture of the compounds LY300502 and LY300503. Rats were treated orally with 10, 30, or 100 mg of LY191704/kg/day for 1 month. Plasma concentrations of LY191704 increased with dose. Both the AUC and maximal concentration values were reduced at 30 and 100 mg/kg on the last day, compared with the first day of treatment. In rat plasma the ratio of LY300502 to LY300503 ranged from 1.6 to 2.0 after the first dose and from 1.6 to 2.4 after the last dose. In dogs administered the same daily oral doses of LY191704 for 1 month, the plasma ratio of LY300502 to LY300503 was essentially unity at the beginning and end of the study. After daily oral administration of LY300502 or LY300503 to rats, at 30, 100, or 300 mg/kg for 14 days, the mean dose-normalized AUC value for LY300503 was 56% of that for LY300502 after the first dose and 38% after the last dose. The rate of metabolic oxidation for LY300502 in rat liver microsomes was approximately 32% of that for LY300503, whereas no differences were observed in the metabolism of the enantiomers in dog liver microsomes. The differences observed between LY300502 and LY300503 were attributed to preferential metabolism of LY300503 in rats. The data indicated that LY300503 was subject to a greater rate of metabolism than LY300502 and induced its own metabolism in rats and that the preferential metabolism of LY300503 was species-specific.

Liquid chromatographic control of the identity, purity and "potency" of biomolecules used as drugs.

The use of high-performance liquid chromatography (HPLC) in the control of rDNA-derived human insulin and human growth hormone is described. Powerful identity tests based upon reversed-phase HPLC separation of enzymatic digests have been developed. Size exclusion and reversed-phase assays are used to control higher molecular weight materials and monomeric derivatives, respectively, for both proteins. Finally, HPLC is used to control the relevant protein content, which in concert with other information controls the biopotency of the protein preparations.

Pressor responses to tyramine and norepinephrine after subchronic administration of fluoxetine to man.

The effects of subchronic, oral administration of fluoxetine (60 mg daily for 45 days) were studied in three healthy male volunteers. The pressor responses to intravenous bolus tyramine injections or norepinephrine infusions were assessed during the one-week placebo period, periodically after daily fluoxetine dosing, and then for 11 days post-fluoxetine dosing. The dose-pressor responses, determined from the incremental elevation of systolic blood pressure, were unchanged in each of the three dosing intervals. These results indicate that fluoxetine does not significantly impair the catecholamine uptake mechanism in the peripheral adrenergic neuron on acute or subchronic dosing, nor is any rebound-increased sensitivity evident after subchronic administration. Further, fluoxetine does not appear to demonstrate peripheral alpha-adrenolytic properties in man.

Single-dose and steady-state pharmacokinetics of tomoxetine in normal subjects.

A pharmacokinetic profile of tomoxetine, a selective norepinephrine uptake inhibitor, was developed in human volunteers following single and multiple oral administrations. Following the administration of a single 90-mg oral dose of tomoxetine to four normal volunteers, the plasma half-life was 4.3 +/- 0.5 hours. Mean plasma clearance was 0.60 +/- 0.14 L/Kg/hr, and the mean volume of distribution was 3.7 +/- 0.9 L/kg. Multiple doses of tomoxetine (20 mg bid and 40 mg bid) for seven days were administered to an additional seven subjects. The data appeared to have a bimodal distribution. The mean plasma half-life determined following the last dose was 4.6 +/- 0.5 hours in five subjects. The other two subjects, one at each dose level, demonstrated accumulation of tomoxetine occurring from the first to last dose where tomoxetine disappeared from plasma with a mean half-life of 19 hours.