Proof of concept: a PhRMA position paper with recommendations for best practice.

Proof of concept (POC) may be defined as the earliest point in the drug development process at which the weight of evidence suggests that it is "reasonably likely" that the key attributes for success are present and the key causes of failure are absent. POC is multidimensional but is focused on attributes that, if not addressed, represent a threat to the success of the project in crucial areas such as safety, efficacy, pharmaceutics, and commercial and regulatory issues. The appropriate weight of evidence is assessed through the use of mathematical models and by evaluating the consequences of advancing a candidate drug that is not safe, effective, or commercially viable, vs. failing to advance a candidate that possesses these attributes. Tools for POC include biomarkers, targeted populations, pharmacokinetic (PK)/pharmacodynamic (PD) modeling, simulation, and adaptive study designs. Challenges to the success of POCs include a shortage of skilled personnel, failure to integrate multiple disciplines and information, and the demand made by organizations for certainty.

A comparison of the pharmacokinetics and tolerability of the anti-migraine compound almotriptan in healthy adolescents and adults.

This study was designed to assess and compare the pharmacokinetics and tolerability of almotriptan, a 5-HT1B/1D agonist used to treat migraine attacks, in adolescents and adults. Healthy adolescents (n = 18) and adults (n = 18) received a single 12.5-mg dose of almotriptan after fasting overnight. Plasma and urinary almotriptan concentrations were measured by high-performance liquid chromatography. Pharmacokinetic parameters of almotriptan were determined by non-compartment analysis. The 90% confidence interval (CI) approach was employed to assess age effects. Mean Cmax, tmax, area under the curve (AUC0- infinity ), half-life, and percentage excreted in urine were nearly identical for the two populations. Mean oral (CLPO) and renal (CLR) clearances were similar between the age groups; however, weight-corrected CLPO was approximately 32% higher (90% CI 16, 51) in adolescents compared with adults. The higher weight-corrected CLPO appeared to offset increases in exposure expected on the basis of lower body weight in adolescents. The findings were the same when a subgroup (n = 9) of 12-14-year-old children was compared with adults. The type, incidence and severity of adverse events were similar between the two age groups and were consistent with those reported previously during adult clinical trials. Based on these pharmacokinetic and tolerability findings, no dose adjustment for almotriptan would be required when treating patients as young as 12 years old.

Lack of effect of reboxetine on cardiac repolarization.

OBJECTIVE: The effect of reboxetine on electrocardiographic parameters, particularly the QTc interval, was assessed in 20 healthy subjects (15 male, 5 female). METHODS: In a 5-way crossover study, subjects received placebo, 2 mg, 4 mg, or 6 mg reboxetine, or 6 mg reboxetine and 200 mg ketoconazole twice daily for 7 days. Plasma samples, vital signs, and 12-lead electrocardiograms (ECGs) were obtained during one dosing interval of days 1, 4, and 7. Additional ECGs were recorded immediately after an exercise paradigm, so that the RR versus QT relationship might be used in calculating QTc. Plasma concentrations of R,R (-)reboxetine and the more active S,S (+)reboxetine were measured by HPLC-dual mass spectrometry. RESULTS: No statistically significant differences among treatments in mean dose-corrected pharmacokinetic parameters were observed, except that the dose-corrected area under the concentration-time curve from time zero to 12 hours and the peak plasma concentration were significantly increased on days 4 and 7 in the presence of ketoconazole. As expected, heart rate increased from baseline (approximately 8-11 beats/min) at > or =8 mg reboxetine daily. No statistically significant prolongation of QTc (Fridericia correction) occurred after any of the treatments. No relationships between DeltaQTc and plasma concentrations of reboxetine enantiomers were apparent. Similar results were obtained with Bazett's correction and two linear corrections that relied on exercise data generated before drug administration. CONCLUSIONS: Reboxetine, at systemic exposures approximately twice the recommended dose, did not significantly affect cardiac repolarization in healthy subjects. Use of QT versus RR relationship in the drug-free state to correct QT for heart rate in the drug-treated state may provide an acceptable alternative to classic correction equations.

Effect of MAO-A inhibition on the pharmacokinetics of almotriptan, an antimigraine agent in humans.

AIMS: To assess the effect of a reversible MAO-A inhibitor, moclobemide, on the single-dose pharmacokinetics of almotriptan and assess the clinical consequences of any interaction. METHODS: Twelve healthy volunteers received the following treatments in a randomized, open-label, two-way crossover design (with a 1 week washout between treatments): (A) one 150 mg moclobemide tablet every 12 h for 8 days and one 12.5 mg almotriptan tablet on the morning of day 8; and (B) one 12.5 mg almotriptan tablet on day 8. Plasma almotriptan was quantified by h.p.l.c.-MS-MS, while urinary concentrations were measured by h.p.l.c.-u.v. Vital signs, ECGs, and adverse events were evaluated after almotriptan administration. Treatment effects on pharmacokinetics and vital signs were assessed by analysis of variance. RESULTS: Mean almotriptan AUC was higher (483 +/- 99.9 vs 352 +/- 75.4 ng ml-1 h, P = 0.0001) and oral clearance was lower (26.6 +/- 4.00 vs 36.6 +/- 5.89 l h-1, P = 0.0001) when almotriptan was administered with moclobemide. Mean half-life was longer (4.22 +/- 0.78 vs 3.41 +/- 0.45 h, P = 0.0002) after coadministration with moclobemide. Renal clearance of almotriptan was unaffected by moclobemide. No serious adverse events occurred and no clinically significant vital sign changes were observed. CONCLUSIONS: Moclobemide increased plasma concentrations of almotriptan on average by 37%, but the combined administration of these two compounds was well tolerated. The degree of interaction was much less than that seen previously for sumatriptan or zolmitriptan given with moclobemide.

Pharmacokinetics of reboxetine in healthy volunteers with different ethnic descents.

RATIONALE: Ethnicity can affect the pharmacokinetics and pharmacodynamics of psychopharmacologic drugs. OBJECTIVES: Reboxetine disposition differences among Asians, blacks, and Caucasians were examined. METHODS: Healthy subjects (12 Asians, 12 blacks, 12 Caucasians) received a single oral dose of one 4-mg reboxetine tablet in an open label, parallel study design. Plasma concentrations of reboxetine enantiomers [R,R(-) reboxetine and predominantly active S,S(+) reboxetine] were quantified using HPLC-MS-MS. Plasma unbound fractions of reboxetine enantiomers were evaluated by equilibrium dialysis. Ethnic group effects on pharmacokinetic parameters were assessed by ANOVA. RESULTS: Mean S,S(+) reboxetine CLPO for blacks was significantly greater, compared to Asians and Caucasians (154+/-82 ml/min, 101+/-19 ml/min and 101+/-18 ml/min, respectively). Mean S,S(+) reboxetine free fractions (fu) were significantly greater for Asians and blacks, compared to Caucasians (3.04+/-1.28%, 2.89+/-0.69%, and 1.99+/-0.58%, respectively). S,S(+) Reboxetine unbound clearance (CLu) was significantly less for Asians, compared to blacks and Caucasians (3742+/-1468 ml/min, 5187+/-2027 ml/min, and 5294+/-1163 ml/min, respectively). S,S(+) Reboxetine mean unbound AUC (AUCu) in these groups were 20.2+/-7.1 ng.h/ml, 14.6+/-5.1 ng.h/ml, and 13.2+/-3.2 ng.h/ml, respectively. AUCu was significantly greater for Asians. CLu and AUCu did not differ significantly between blacks and Caucasians. Ethnic effects of R,R(-) reboxetine were similar to those observed for S,S(+) reboxetine. CONCLUSIONS: The AUCu difference between Asian and black and Caucasian subjects was modest. Tolerability differences among groups were not observed. No dosage adjustment is necessary for Asians or blacks.

Lack of pharmacokinetic interaction between the antimigraine compound, almotriptan, and propranolol in healthy volunteers.

This study was designed to assess the pharmacokinetics of almotriptan, a 5-HT1B/1D agonist, when administered in the presence and absence of propranolol. Healthy male (n = 10) and female (n = 2) volunteers received (i) 80 mg propranolol twice daily for 7 days and 12.5 mg almotriptan on day 7, and (ii) 12.5 mg almotriptan on day 7, according to a two-way crossover design. Plasma and urinary almotriptan concentrations were measured by high performance liquid chromatography (HPLC) methods. Treatment effects on pharmacokinetic parameters were assessed by analysis of variance (ANOVA). Statistically significant differences between treatments in area under the curve (AUC), clearance, and half-life were observed (P < 0.03), but these differences were < 7%. Ninety percent confidence interval analysis of log-transformed pharmacokinetic parameters showed that the treatments were equivalent. Adverse events were mild to moderate in intensity, and no treatment effects on vital signs were observed. The results show that propranolol has no effect on the pharmacokinetics of almotriptan. Concomitant administration of the two drugs is well tolerated.

Evaluation of the potential pharmacokinetic interaction between almotriptan and fluoxetine in healthy volunteers.

This study was designed to assess the pharmacokinetics of almotriptan, a 5HT1B/1D agonist used to treat migraine attacks, when administered in the presence and absence of fluoxetine. Healthy male (n = 3) and female (n = 11) volunteers received (1) 60 mg fluoxetine daily for 8 days and 12.5 mg almotriptan on Day 8 and (2) 12.5 mg almotriptan on Day 8, according to a two-way crossover design. Plasma and urinary almotriptan concentrations were measured by HPLC methods. Treatment effects on pharmacokinetic parameters were assessed by analysis of variance. Mean almotriptan Cmax was significantly higher following combination treatment with fluoxetine (52.5 +/- 11.9 ng/ml vs. 44.3 +/- 10.9 ng/ml, p = 0.023). Mean AUC0-infinity was not significantly affected by fluoxetine coadministration (353 +/- 55.7 ng.h/ml vs. 333 +/- 33.6 ng.h/ml, p = 0.059). Confidence interval analysis (90%) of log-transformed pharmacokinetic parameters showed that the confidence interval for AUC0-infinity was within the 80% to 125% limit for equivalence, but Cmax was not (90% CI 106%-134% of the reference mean). Adverse events were mild to moderate in intensity, and no clinically significant treatment effects on vital signs or ECGs were observed. The results show that fluoxetine has only a modest effect on almotriptan Cmax. Concomitant administration of the two drugs is well tolerated, and no adjustment of the almotriptan dose is warranted.

Pharmacokinetics of reboxetine in elderly patients with depressive disorders.

OBJECTIVES: To examine the pharmacokinetic characteristics of the selective norepinephrine reuptake inhibitor, reboxetine, in elderly patients with depression. PATIENTS: Twelve female inpatients (mean age 80 +/- 4 years) with major depressive or dysthymic disorder were enrolled in a 4-week uncontrolled study of oral reboxetine 2-8 mg/day. METHODS: After a one-week washout period, patients were randomized into two groups (groups A and B, n = 6/group). Reboxetine was given twice daily, starting with 2 mg/day during week 1 and increasing by 2 mg/day each week to 8 mg/day in week 4. Pharmacokinetic evaluations were carried out at two dosage levels in each group: at the end of weeks 1 and 3 in group A (2 and 6 mg/day), and at the end of weeks 2 and 4 in group B (4 and 8 mg/day). Blood and urine samples were taken for determination of reboxetine pharmacokinetics. RESULTS: Reboxetine displayed linear pharmacokinetics, with dose-proportional changes, in elderly depressed patients. Mean total urinary recovery ranged from 4.06 to 6.17%. The mean area under the plasma concentration-time curve (AUCtau) and the maximum plasma drug concentration (Cmax) showed considerable variation between patients; at a dosage of 4 mg/day, AUCtau was 1,466-6,866 ngxh/ml and Cmax ranged from 169 to 663 ng/ml. CONCLUSIONS: The pharmacokinetics of reboxetine are linear across the dosage range of 2-8 mg/day in elderly depressed patients, although Cmax and AUCtau values are higher (and more variable) than in young adults. These results support the use of a lower starting dose (4 mg/day) of reboxetine in the elderly.

Pharmacokinetic interaction between verapamil and almotriptan in healthy volunteers.

OBJECTIVE: To assess the interaction between almotriptan, a 5-HT1B/1D-receptor agonist used to treat migraine, and verapamil, an agent for migraine prophylaxis. METHODS: Twelve healthy volunteers received the following treatments in a crossover design: (1) 120-mg sustained-release verapamil tablet twice daily for 7 days and one 12.5-mg almotriptan tablet on day 7 and (2) one 12.5-mg almotriptan tablet alone on day 7. Serial plasma and urine samples were obtained on day 7. Almotriptan plasma concentrations were determined by liquid chromatography-tandem mass spectrometry; urine samples were analyzed by ultraviolet HPLC. Safety measures included blood pressure and pulse measurements, electrocardiography, and adverse event monitoring. Statistical comparisons of pharmacokinetic parameters and vital sign data were made by ANOVA. RESULTS: Mean almotriptan peak concentration and area under the plasma concentration-time curve were significantly higher and volume of distribution and oral clearance were significantly lower after coadministration of almotriptan and verapamil compared with administration of almotriptan alone. The magnitudes of these differences were approximately 20%. Renal clearance was unaffected by verapamil coadministration. No significant effects of treatment on blood pressure or pulse were detected, with the exception of sitting systolic blood pressure at 2 hours after administration. However, the difference in mean change from baseline at this time point was only 8 mm Hg. CONCLUSIONS: Verapamil modestly inhibited almotriptan clearance to a degree consistent with the modest contribution of CYP3A4 to almotriptan metabolism. This observation and the lack of effect of verapamil on the tolerability to almotriptan administration suggest that no reduction of the almotriptan dose is warranted.

Pharmacokinetics of single-dose reboxetine in volunteers with renal insufficiency.

Reboxetine is a new selective norepinephrine reuptake inhibitor (selective NRI) for the short- and long-term treatment of depression that is effective and well tolerated at a dose of 8 to 10 mg/day. This study assessed the pharmacokinetics of reboxetine in volunteers with renal impairment. A single 4 mg dose of reboxetine was administered to a total of 18 volunteers with mild (n = 6), moderate (n = 6), or severe (n = 6) renal impairment (creatinine clearance: 56-64, 26-51, and 9-19 ml/min, respectively), and reboxetine concentrations were measured in plasma by HPLC. Mean AUC infinity increased by 43% (mild vs. severe; p < 0.01) as renal function declined, while renal clearance and total urinary excretion of unchanged reboxetine decreased by 67% and 62%, respectively (mild vs. severe; p < 0.01 for both parameters). tmax and t1/2 were not significantly different between groups. In comparison with historical data from young healthy volunteers, AUC infinity and t1/2 are at least doubled in volunteers with renal impairment, while CLr is halved. This pharmacokinetic study has shown that increasing renal dysfunction leads to increasing systemic exposure to reboxetine, particularly in severe renal insufficiency, although reboxetine was well tolerated by all volunteers. Thus, a reduction of the starting dose of reboxetine to 2 mg twice daily would be prudent in patients with renal dysfunction.

Clinical pharmacokinetics of reboxetine, a selective norepinephrine reuptake inhibitor for the treatment of patients with depression.

Reboxetine is a novel selective norepinephrine inhibitor that has been evaluated in the treatment of patients with depression. Reboxetine is a racemic mixture, and the (S,S)-(+)-enantiomer appears to be the more potent inhibitor. However, the ratio of the areas under the concentration-time curves of the (S,S)-(+)- and (R,R)-(-)-enantiomers in vivo is approximately 0.5. There is no evidence for chiral inversion. Differences in the clearances of the 2 enantiomers may be explained by differences in protein binding. The pharmacokinetics of reboxetine are linear following both single and multiple oral doses up to a dosage of 12 mg/day. The plasma concentration-time profile following oral administration is best described by a 1-compartment model, and the mean half-life (approximately 12 hours) is consistent with the recommendation to administer the drug twice daily. Reboxetine is well absorbed after oral administration. The absolute bioavailability is 94.5%, and maximal concentrations are generally achieved within 2 to 4 hours. Food affects the rate, but not the extent, of absorption. The distribution of reboxetine appears to be limited to a fraction of the total body water due to its extensive (>97%) binding to plasma proteins. The primary route of reboxetine elimination appears to be through hepatic metabolism. Less than 10% of the dose is cleared renally. A number of metabolites formed through hepatic oxidation have been identified, but reboxetine is the major circulating species in plasma. In vitro studies show that reboxetine is predominantly metabolised by cytochrome P450 (CYP) 3A4; CYP2D6 is not involved. Reboxetine plasma concentrations are increased in elderly individuals and in those with hepatic or renal dysfunction, probably because of reduced metabolic clearance. In these populations, reboxetine should be used with caution, and a dosage reduction is indicated. Ketoconazole decreases the clearance of reboxetine, so that the dosage of reboxetine may need to be reduced when potent inhibitors of CYP3A4 are coadministered. Quinidine does not affect the in vivo clearance of reboxetine, confirming the lack of involvement of CYP2D6. There is no pharmacokinetic interaction between reboxetine and lorazepam or fluoxetine. Reboxetine at therapeutic concentrations has no effect on the in vitro activity of CYP1A2, 2C9, 2D6, 2E1 or 3A4. The lack of effect of reboxetine on CYP2D6 and CYP3A4 was confirmed by the lack of effect on the metabolism of dextromethorphan and alprazolam in healthy volunteers. Thus, reboxetine is not likely to affect the clearance of other drugs metabolised by CYP isozymes.

Pharmacokinetics of reboxetine in healthy, elderly volunteers.

The pharmacokinetic characteristics of reboxetine, a unique selective noradrenaline reuptake inhibitor (selective NRI) for the treatment of depression, were studied in 12 healthy, elderly volunteers (mean age 81 years +/- 9 years). All subjects received a single 4-mg dose of reboxetine, and plasma reboxetine concentrations were measured by HPLC. Reboxetine was well tolerated by all subjects. Exposure to reboxetine was higher in this group of very elderly subjects, compared with data obtained in a similar study of young, healthy volunteers. Cmax in the elderly was 271 +/- 86 ng/ml, compared with 111 +/- 28 ng/ml in the young subjects after a single 4-mg dose, although in both groups Cmax was observed after 2 h. The AUC infinity was nearly four times that in the younger subjects (8345 +/- 3107 ng.h/ml vs. 2106 +/- 881 ng.h/ml) and the t1/2 was twice as long (24 +/- 6 h vs. 12 +/- 3 h). Renal clearance was also reduced. Reboxetine 8-10 mg/day has been effective and well tolerated in clinical trials in non-elderly depressed patients. The increased exposure to reboxetine observed in our very elderly subjects supports a reduction of the starting dose to 4 mg/day (in two divided doses) in the elderly.

Ketoconazole inhibits the clearance of the enantiomers of the antidepressant reboxetine in humans.

BACKGROUND: Ketoconazole is a potent inhibitor of the cytochrome P450 3A4 enzyme. Reboxetine, a selective norepinephrine reuptake inhibitor, is metabolized by cytochrome P450 3A4. The potential interaction of reboxetine with this representative from the azole derivative class was examined. METHODS: Eleven healthy volunteers received (1) 4 mg reboxetine orally on the second day of a 5-day regimen of 200 mg ketoconazole once daily and (2) 4 mg reboxetine orally in a crossover design. Plasma concentrations of reboxetine enantiomers [R,R(-)-reboxetine and the more active S,S(+)-reboxetine] were measured by high-performance liquid chromatography-tandem mass spectrometry. Effects of ketoconazole on enantiomer pharmacokinetics were assessed by ANOVA. RESULTS: Ketoconazole increased R,R(-)-reboxetine and S,S(+)-reboxetine mean area under the plasma concentration-time curves (AUC) by 58% and 43%, respectively (P < .02). Oral clearance of both enantiomers was consequently decreased 34% and 24%, respectively, by ketoconazole (P < .005). Ketoconazole did not significantly affect maximal plasma concentrations (P > .1). Mean terminal half-life after administration of ketoconazole (21.5 hours and 18.9 hours) was significantly longer than after reboxetine alone (14.8 hours and 14.4 hours; P < or = .005). The AUC ratio for R,R(-)-reboxetine to S,S(+)-reboxetine was reduced by ketoconazole administration (2.76 after ketoconazole versus 2.39; P < .003). CONCLUSION: Ketoconazole decreases clearance of both reboxetine enantiomers. Although the adverse effect profile for reboxetine was not altered by ketoconazole, the results of this study suggest that caution should be used and that a reduction in reboxetine dose should be considered when the two are coadministered.

Pharmacokinetics and tolerability of a novel 5-HT1D agonist, PNU-142633F.

OBJECTIVES: The objectives of this study were to characterize the safety, tolerability and pharmacokinetics of a single, oral dose of PNU-142633F escalating over the range of 1.0 mg to 100 mg (free base equivalents). METHODS: This was a randomized, double-blind, single-dose, placebo-controlled, dose-escalation trial, with each dose group (1.0, 3.0, 10, 30, 50, 75 and 100 mg) having eight volunteers (six PNU-142633F and two placebo). Clinical laboratory tests, electrocardiogram, Holter monitoring, and assessment of adverse events were used to gauge the tolerability of PNU-142633. Serial blood samples and urine collections were obtained and plasma and urine PNU-142633 concentrations were determined by a validated HPLC fluorescence method. RESULTS: PNU-142633 was well tolerated after oral administration. There were no reports of serious or unexpected adverse events. The most common adverse event that was possibly medication-related was transient dizziness. There were no clinically significant or dose-related effects of PNU-142633 on any vital sign parameters (aural temperature, systolic and diastolic blood pressure, pulse rate or respiratory rate), at any study time or dose. There were no clinically significant ECG changes. Only sporadic abnormalities in clinical chemistry values and hematology were noted. After the 1.0 mg and 3.0 mg doses, plasma concentrations of PNU-142633 were either below or only slightly above the lower limit of quantitation (2 ng/ml). At higher doses (30-100 mg) the terminal half-life was relatively constant at approximately 11 hours. Neither Cmax nor AUC(0-infinity) increased proportionally with the administered dose. The mean percentage of the dose excreted in the urine as intact PNU-142633 increased from 14.3% after the 1 mg dose to 49.3% after the 100 mg dose. CONCLUSIONS: The clinical safety and pharmacokinetic data support the study of this agent as a potential treatment for migraine attacks.

Population pharmacokinetics of tirilazad: effects of weight, gender, concomitant phenytoin, and subarachnoid hemorrhage.

PURPOSE: Data collected during Phase I and II in the development of tirilazad were pooled and analyzed using nonlinear mixed effects models to assess covariates which might affect tirilazad pharmacokinetics. METHODS: Four single dose and five multiple dose studies in normal volunteers were combined with two multiple dose studies performed in patients with subarachnoid hemorrhage (SAH) to identify factors related to intersubject variability in clearance (CL) and central compartment volume (Vc). Data from 253 subjects, which consisted of 7,219 tirilazad concentrations, were analyzed. The effects of weight, gender, patient versus volunteer status, and phenytoin use were evaluated. RESULTS: Relative to male volunteers not receiving concomitant phenytoin, significant effects on clearance included: a 46% increase in volunteers receiving phenytoin, and an 82% increase in clearance associated with SAH patients (all of whom received phenytoin). Significant effects on Vc were: a 26% increase for female volunteers not receiving phenytoin, a 12% decrease for volunteers receiving concomitant phenytoin, a 152% increase for male SAH patients, and a 270% increase for female SAH patients. Incorporating patient covariate effects substantially reduced the interindividual variability (from 27.9% to 24.7% for clearance and from 48.2% to 37.5% for Vc). Residual variability was estimated at 66% coefficient of variation (CV) in SAH patients and at 22-48% CV over the range of predicted concentrations in normal volunteers. CONCLUSIONS: The most important factors affecting tirilazad pharmacokinetics are the administration of phenytoin (increased CL) and SAH (increased Vc and residual variability). The effect of gender on tirilazad pharmacokinetics was modest.

Absolute bioavailability of reboxetine enantiomers and effect of gender on pharmacokinetics.

The absolute bioavailability of reboxetine enantiomers was assessed in six male and six female volunteers. In a two-way crossover study, subjects received 1.0 mg reboxetine orally and 0.3 mg reboxetine as an intravenous bolus. The R,R(-) and S,S(+) enantiomers in serial plasma and urine samples were determined by a validated LC-MS-MS method. There were no significant differences between treatments for clearance or dose-corrected AUC(0-infinity) values. The absolute bioavailability was 0.919 and 1.02 for R,R(-) reboxetine and S,S(+) reboxetine, respectively. A secondary objective of the study was to assess gender effects on pharmacokinetics of the enantiomers. Significant differences in volume of distribution between genders were observed, but differences in weight-corrected volumes were not significant. Weight-corrected systemic clearance and oral clearance tended to be lower in males, but this difference reached statistical significance only for weight-corrected oral clearance of R,R(-) reboxetine. C(max) after oral administration was 40 and 48% higher in women than men for R,R(-) reboxetine and S,S(+) reboxetine, respectively. These results indicate that reboxetine enantiomers are well absorbed after oral administration and that little first-pass metabolism occurs. There are no clinically significant effects of gender on the pharmacokinetics of reboxetine enantiomers.

Hormonal effects on tirilazad clearance in women: assessment of the role of CYP3A.

This study assessed whether the previously reported difference in tirilazad clearance between pre- and postmenopausal women is reversed by hormone replacement and whether this observation can be explained by differences in CYP3A4 activity. Ten healthy women from each group were enrolled: premenopausal (ages 18-35), postmenopausal (ages 50-70), postmenopausal receiving estrogen, and postmenopausal women receiving estrogen and progestin. Volunteers received 0.0145 mg/kg midazolam and 3.0 mg/kg tirilazad mesylate intravenously on separate days. Plasma tirilazad and midazolam were measured by HPLC/dual mass spectrophotometry (MS/MS) assays. Tirilazad clearance was significantly higher in premenopausal women (0.51 +/- 0.09 L/hr/kg) than in postmenopausal groups (0.34 +/- 0.07, 0.32 +/- 0.06, and 0.36 +/- 0.08 L/hr/kg, respectively) (p = 0.0001). Midazolam clearance (0.64 +/- 0.12 L/hr/kg) was significantly higher in premenopausal women compared to postmenopausal groups (0.47 +/- 0.11, 0.49 +/- 0.11, and 0.53 +/- 0.19 L/hr/kg, respectively) (p = 0.037). Tirilazad clearance was weakly correlated with midazolam clearance (r2 = 0.129, p = 0.02). Tirilazad clearance is faster in premenopausal women than in postmenopausal women, but the effect of menopause on clearance is not reversed by hormone replacement. Tirilazad clearance in these women is weakly related to midazolam clearance, a marker of CYP3A activity.

Biotransformation of tirilazad in human: 3. tirilazad A-ring reduction by human liver microsomal 5alpha-reductase type 1 and type 2.

Tirilazad mesylate (FREEDOX), a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage. In humans, tirilazad is cleared almost exclusively via hepatic elimination with a medium-to-high extraction ratio. In human liver microsomal preparations, tirilazad is biotransformed to multiple oxidative products and one reduced, pharmacologically active metabolite, U-89678. Characterization of the reduced metabolite by mass spectrometry and cochromatography with an authentic standard demonstrated that U-89678 was formed via stereoselective reduction of the Delta4 bond in the steroid A-ring. Kinetic analysis of tirilazad reduction in human liver microsomes revealed that kinetically distinct type 1 and type 2 5alpha-reductase enzymes were responsible for U-89678 formation; the apparent KM values for type 2 and type 1 were approximately 15 and approximately 0.5 microM, respectively. Based on pH dependence and finasteride inhibition studies, it was inferred that 5alpha-reductase type 1 was the high affinity/low capacity microsomal reductase that contributed to tirilazad clearance in vivo. In addition, a role for CYP3A4 in the metabolism of U-89678 was established using cDNA expressed CYP3A4 and correlation studies comparing U-89678 consumption with cytochrome P450 activities across a population of human liver microsomes. Collectively, these data suggest that formation of U-89678, a circulating pharmacologically active metabolite, contributes to the total metabolic elimination of tirilazad in humans and that clearance of U-89678 is mediated primarily via CYP3A4 metabolism. Therefore, concurrent administration of therapeutic agents that modulate 5alpha-reductase type 1 or CYP3A activity are anticipated to affect the pharmacokinetics of PNU-89678.

Biotransformation of tirilazad in human: 4. effect of finasteride on tirilazad clearance and reduced metabolite formation.

The effect of oral finasteride, an inhibitor of 5alpha-reductase, on the clearance of tirilazad, a membrane lipid peroxidation inhibitor, was assessed in eight healthy men who received: 1) 10 mg/kg tirilazad mesylate solution orally on the 7th day of a 10-day regimen of 5 mg finasteride once daily, 2) 10 mg/kg tirilazad mesylate orally, 3) 2 mg/kg tirilazad mesylate i.v. on the 7th day of a 10-day regimen of 5 mg finasteride once daily and 4) 2 mg/kg tirilazad mesylate i.v., in a four-way cross-over design. Plasma concentrations of tirilazad and its active reduced metabolites (U-89678 and U-87999) were measured by liquid chromatography with tandem mass spectrometry (LC-MS-MS). Finasteride increased mean tirilazad areas under the curve by 21 and 29% for i.v. and p.o. tirilazad, respectively. Mean U-89678 areas under the curve were decreased 92 and 75% by finasteride administration with i.v. and p.o. tirilazad, respectively, and decreases of 94 and 85% in mean U-87999 area under the curve values were observed. These differences were statistically significant. These results indicate that finasteride inhibits the metabolism of tirilazad to U-89678. However, this inhibition has only a moderate effect on the overall clearance of tirilazad. These results thus confirm earlier in vitro work that showed that tirilazad is predominantly metabolized by CYP3A4. Although the major circulating metabolites of tirilazad are formed via reduction, this represents a minor route of tirilazad elimination in man.

Kinetic characterization and identification of the enzymes responsible for the hepatic biotransformation of adinazolam and N-desmethyladinazolam in man.

The kinetics of the N-demethylation of adinazolam to N-desmethyladinazolam (NDMAD), and of NDMAD to didesmethyladinazolam (DDMAD), were studied with human liver microsomes using substrate concentrations in the range 10-1000 microM. The specific cytochrome P450 (CYP) isoforms mediating the biotransformations were identified using microsomes containing specific recombinant CYP isozymes expressed in human lymphoblastoid cells, and by the use of CYP isoform-selective chemical inhibitors. Adinazolam was demethylated by human liver microsomes to NDMAD, and NDMAD was demethylated to DDMAD; the substrate concentrations, Km, at which the reaction velocities were 50% of the maximum were 92 and 259 microM, respectively. Another metabolite of yet undetermined identity (U) was also formed from NDMAD (Km 498 microM). Adinazolam was demethylated by cDNA-expressed CYP 2C19 (Km 39 microM) and CYP 3A4 (Km 83 microM); no detectable activity was observed for CYPs 1A2, 2C9, 2D6 and 2E1. Ketoconazole, a relatively specific CYP 3A4 inhibitor, inhibited the reaction; the concentration resulting in 50% of maximum inhibition, IC50, was 0.15 microM and the inhibition constant, Ki, was < 0.04 microM in five of six livers tested. Troleandomycin, a specific inhibitor of CYP 3A4, inhibited adinazolam N-demethylation with an IC50 of 1.96 microM. The CYP 2C19-inhibitor omeprazole resulted in only partial inhibition (IC50 21 microM) and sulphaphenazole, alpha-naphthoflavone, quinidine and diethyldithiocarbamate did not inhibit the reaction. NDMAD was demethylated by cDNA-expressed CYP 3A4 (Km 220 microM, Hill number A 1.21), CYP 2C19 (Km 187 microM, Hill number A 1.29) and CYP 2C9 (Km 1068 microM). Formation of U was catalysed by CYP 3A4 alone. Ketoconazole strongly inhibited NDMAD demethylation (IC50 0.14 microM) and formation of U (IC50 < 0.1 microM) whereas omeprazole and sulphaphenazole had no effect on reaction rates. These results show that CYP 3A4 is the primary hepatic CYP isoform mediating the N-demethylation of adinazolam and NDMAD. Co-administration of adinazolam with CYP 3A4 inhibitors such as ketoconazole or erythromycin might lead to reduced efficacy, since adinazolam by itself has relatively weak benzodiazepine agonist activity, with much of the pharmacological activity of adinazolam being attributable to its active metabolite NDMAD.