Potential volunteers were selected from the records of FOCUS Clinical Drug Development GmbH, Neuss, Germany. 27 nonsmoking healthy volunteers of Caucasian descent (22 men and 5 women; age range, 22 to 45 years; weight range, 53 to 105 kg) participated in the study after giving written informed consent that was approved by the responsible Independent Ethics Committee of the trial center: Ethikkommission der, Düsseldorf, Germany. All subjects were determined to be in good health (on the basis of medical history, physical examination, electrocardiogram, and routine laboratory blood and urine tests) before they were enrolled in the study. None of the subjects received any medication, including any known enzyme inducer or inhibitor, for 1 month prior to first dosing with study drug. They were not allowed to use grapefruit-containing products or St. John's wort for 10 days prior to and throughout the study. All female subjects had a negative pregnancy test and were advised to use a medically acceptable birth control method other than an oral contraceptive.

The study involved an open-label, randomized, multiple-oral-dose, two-way crossover design with a washout duration of at least 4 weeks between the 2 study periods. The subjects ingested a 20-mg extended-release formulation of gepirone (20 mg daily for 2 days, followed by 40 mg daily for 5 days), alone (study period A) or in combination with rifampin 600 mg once daily from days 1 to 8 (study period B). For each study period, all subjects were hospitalized in the research unit from 24 hours prior until 2 hours after dosing of study drug on day 1, as well as from the evening of day 6 through completion of pharmacokinetic sampling on day 9. Outpatient visits for study drug dosing and safety evaluation occurred on days 2 to 6.

On day 7 of each study period, each subject received the last 40-mg dose of gepirone ER after a 10-hour overnight fast. Lunch was provided 4 hours after dosing. Timed blood samples of 10 mL each were drawn from the contralateral arm of each subject before and at 0.5, 1, 3, 4, 5, 6, 7, 8, 10, 12, 18, 24, 30, 36, and 48 hours after the day 7 dose of gepirone. All blood samples were processed within 30 minutes to harvest plasma, which was then divided into aliquots and stored at –20ºC until analysis of drug concentrations. To assess attainment of steady-state concentrations in the subjects, predose blood samples of 10 mL each were also obtained on days 5 and 6. These two predose concentration values were compared with the predose concentration value on day 7 and the 24-hour sample on day 8.

To assess CYP3A4 induction using the endogenous 6β-hydroxycortisol to free cortisol ratio (6β-OHC/FC), 24-hour urine samples were collected prior to first dosing on day 1 and after the last dosing on day 7 of each study period. The urine samples were stored at 4ºC during each collection period. The total urine volume was recorded, and a 20 mL aliquot was stored at –20ºC until analysis.

Plasma samples were analyzed for concentrations of gepirone, 1-PP, and 3´-OH-gepirone by liquid chromatographic (LC)-electrospray mass spectrometry, with buspirone as the internal standard for the analysis of gepirone and 3´-OH-gepirone, and 2-aminonorbornane as the internal standard for the analysis of 1-PP. Briefly, for analysis of gepirone and 3´-OH-gepirone, 0.5 mL of plasma was mixed with saturated sodiumtetraborate solution and the internal standard. The samples were then extracted twice with a mixture of n-hexane/chloroform (2:1 v/v). The final organic extract was allowed to dry by evaporation under a gentle stream of nitrogen. The dry residue was reconstituted with 100 µL of a 50:50 acetonitrile/0.05 M ammonium acetate solution, and an aliquot was injected into the LC system. For analysis of 1-PP, the procedure was the same, except that a different internal standard was used and the organic extract was back-extracted with 100 µL of 1% formic acid solution. An aliquot of the aqueous solution was then injected into the LC system.

A Luna^® C₁₈ (Phenomenex Inc.) column and a gradient system with flow rate of 600 μl/min were used for chromatography. The samples were analyzed by electrospray mass spectrometry. The product ions of the [M+H]⁺ion were m/z 121.8 for gepirone, m/z 122.0 for 3´-OH-gepirone, m/z 122.0 for buspirone, m/z 122.2 for 1-PP, and m/z 95 for aminonorbornane. The limits of quantification were 0.1 ng/mL for gepirone and 0.2 ng/mL for both metabolites. Low-, medium-, and high-concentration quality-control (QC) samples for all three analytes were prepared and analyzed with each subject's samples. Coefficients of variation of the different QC samples were in the range of 4.5% to 13.2%. No interfering peaks were observed in chromatograms after rifampin administration.

Urine samples were analyzed for determination of cortisol and 6β-hydroxycortisol concentrations by gradient HPLC. Briefly, 1 mL of urine samples were mixed with the internal standard, 6β-hydroxycortisone, before solid-phase extraction. Following evaporation of the organic phase, the residues were reconstituted in 200 µL of water:ethanol (1:1) mixture and 50 µL was injected into the HPLC system. The analytes were separated by a µBondapak^® (Waters Corporation) C₁₈ column and detected at 243 nm. The low-, medium-, and high-concentration QC samples, respectively, were 52.2, 209, and 522 ng/mL for cortisol and 40.8, 204, and 815 ng/mL for 6β-hydroxycortisol. The lower limits of detection were 10.4 ng/mL for free cortisol and 15 ng/mL for 6β-hydroxycortisol. Coefficients of variation of low-, medium-, and high-concentration QC samples were in the range of 1.1% to 7.1% for 6β-hydroxycortisol and 1.3% to 17.8% for free cortisol.

Pharmacokinetic parameters of gepirone, 3´-OH-gepirone, and 1-PP, including area under the plasma concentration-time curve from 0 to 24 hours (AUC_0-24), apparent clearance (Cl/F), and terminal elimination half-life (t_1/2), were calculated using standard noncompartmental methods with the SAS program, version 6.12. The peak plasma concentration (C_max) and time to peak concentration (T_max) were determined by examining the concentration-versus-time curves for each subject. The terminal segment of the natural log concentration-versus-time curve for all individual subjects was visually identified. The elimination rate constant, β, was determined by simple linear regression of each subject’s data. Elimination half-life was calculated as 0.693/β. The AUC_0-24 was estimated with the trapezoidal rule to the measured concentration at 24 hours. Apparent oral clearance was calculated as administered dose/AUC. Percentage chan-ges in pharmacokinetic parameters for gepirone treatment with or without rifampin coadministration were assessed for each subject.

Repeated-measures analysis of variance (ANOVA) was used to assess whether steady state was achieved for gepirone and the two metabolites after multiple dosing of gepirone for 7 days. All pharmacokinetic parameters in both study periods were expressed as mean ± SD in Table 1. The pharmacokinetic parameters, as well as the AUC ratios (for treatment with/without rifampin) of gepirone and the two metabolites were compared by repeated-measures ANOVA. A P value < 0.05 was considered to be statistically significant.

Table 1

Steady-State Pharmacokinetic Parameters of Gepirone, 3´-OH-Gepirone, and 1-PP in 24 Healthy Subjects on Day 7 of Gepirone Treatment, with or without Concurrent Rifampin 600 mg Daily

Of the 27 subjects who enrolled in the study, 3 discontinued from the study (2 subjects due to occurrence of adverse events and 1 subject due to a protocol violation). For the 24 subjects completing the study, the mean ± standard deviation values were as follows: age, 33.4 ± 5.4 years; weight 76.6 ± 11.5 kg; height, 179 ± 8.1 cm; and body mass index 23.9 ± 2.3 kg/m².

For each study period, steady state for gepirone and the two metabolites was achieved in all 24 subjects after 2 days of administration of 40 mg of gepirone ER. The pharmacokinetic parameters of gepirone, 3´-OH-gepirone, and 1-PP after gepirone treatment with and without rifampin coadministration are summarized in Table 1.

Rifampin significantly reduced the plasma concentrations of both gepirone and 3´-OH-gepirone (Fig. 2). The mean C_max and AUC_0-24 of gepirone were reduced by 92.1% ± 9.9% and 94.9% ± 5.2%, respectively. The corresponding reduction in mean C_max and AUC_0-24 for 3´-OH-gepirone was 58.0% ± 11.7% and 64.6% ± 10.5%, respectively. Reductions in C_max and AUC_0-24 were observed for both gepirone and 3´-OH-gepirone in each individual subject. Also, the AUC_0-24 for gepirone was decreased by a greater percentage than was the AUC_0-24 for 3´-OH-gepirone in all subjects. The AUC_0-24 ratio (with/without rifampin) was significantly higher for 3´-OH-gepirone (0.35 ± 0.11) than for gepirone (0.05 ± 0.05).

Fig. (2) Arithmetic mean plasma concentrations of gepirone, 1-PP and 3’-OH-gepirone in 24 healthy subjects after multiple dosing of gepirone (40 mg/day), with or without rifampin therapy (600 mg/day).

Rifampin also resulted in a 37% reduction in the elimination half-life of 3´-OH-gepirone. For gepirone, the terminal phase was not visually identifiable in 4 subjects during rifampin treatment. During the gepirone-only treatment, 1 sub-ject had an elimination half-life that was more than 15 times longer than that in the other subjects; no clinical and/or analytical reasons were present that could account for the significant discrepancy. Comparison of gepirone elimination half-life values was therefore based only on 23 subjects for the gepirone-only treatment and 18 subjects for gepirone and rifampin co-administration and showed a doubling of the elimination half-life during rifampin treatment. Rifampin shortened the mean T_max of 3´-OH-gepirone by about 2 hours. There was no significant difference in the T_max of gepirone with concurrent use of rifampin.

In contrast, concurrent use of rifampin produced only minimal changes in mean C_max (2.3% ± 23.1% reduction) and AUC (7.1% ± 30.7% reduction) for 1-PP (Fig. 2). There was also much more variability in the direction and magnitude of change in the two pharmacokinetic parameters of 1-PP: 12 subjects had 1.9% to 37.4% increases in C_max and the other 12 subjects had 0.6% to 61.9% decreases in C_max. The AUC of 1-PP was reduced in 15 subjects (by 4.2% to 65.5%) and increased in 9 subjects (by 1.9% to 54.7%). In addition, the AUC ratio (with/without rifampin) for 1-PP (0.93 ± 0.31) were significantly higher than that for gepirone and 3´-OH-gepirone. Rifampin had no significant effect on the T_max or elimination half-life of 1-PP.

The baseline total amount of 24-hour urinary 6β-hydroxycortisol prior to first dosing of gepirone was similar in both study periods: 197 ± 117 µg versus 181 ± 75.6 µg, with approximately 8-fold and 4.5-fold interindividual variability, respectively; these findings are consistent with those reported in the literature. With concurrent use of rifampin, the subjects had a more than 4-fold increase in the 24-hour excretion of 6β-hydroxycortisol (Table 2). Similarly, there was more than 4-fold increase in the mean urinary 6β-OHC/FC ratio from baseline. The extent of this increase ranged from 135% to 1100%. On the other hand, dosing of gepirone alone for 7 days only resulted in a 3.1% ± 47.5% mean increase in the 6β-OHC/FC ratio. The extent of change in the ratio is more variable, with a range of 61% decrease to 133% increase. There is also no consistent pattern of incr-ease in urinary excretion of 6β-hydroxycortisol as that demonstrated with rifampin administration in all 24 subjects.

Table 2

Comparison of 6β -Hydroxycortisol and 6β-Hydroxycortisol-to-Free-Cortisol Ratio in all 24 Subjects During Gepirone Treatment, with or without Concurrent Rifampin (600 mg/day)

Headache, dizziness, and fatigue were the most common adverse effects reported by the subjects. Their occurrence was most frequent during the first couple of days of each study period. Similar frequencies of these adverse effects were observed with and without rifampin treatment. However, four subjects reported moderate (3 cases) to severe (1 case) dizziness on day 1 or day 3 during the gepirone-only phase, as compared to no subjects during coadministration of rifampin.