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A metabolic mechanism-based characterization of antiepileptic drug-drug interactions (DDIs) with clobazam in patients with Lennox-Gastaut syndrome (LGS) was performed using a population pharmacokinetic (PPK) approach. To characterize potential DDIs with clobazam, pharmacokinetic (PK) data from 153 patients with LGS in study OV-1012 (NCT00518713) and 18 healthy participants in bioavailability study OV-1017 were pooled. Antiepileptic drugs (AEDs) were grouped based on their effects on the cytochrome P450 (CYP) isozymes responsible for the metabolism of clobazam and its metabolite, N-desmethylclobazam (N-CLB): CYP3A inducers (phenobarbital, phenytoin, and carbamazepine), CYP2C19 inducers (valproic acid, phenobarbital, phenytoin, and carbamazepine), or CYP2C19 inhibitors (felbamate, oxcarbazepine). CYP3A4 inducers-which did not affect the oral clearance of clobazam-significantly increased the formation of N-CLB by 9.4%, while CYP2C19 inducers significantly increased the apparent elimination rate of N-CLB by 10.5%, resulting in a negligible net change in the PK of the active metabolite. CYP2C19 inhibitors did not affect N-CLB elimination. Because concomitant use of AEDs that are either CYP450 inhibitors or inducers with clobazam in the treatment of LGS patients had negligible to no effect on clobazam PK in this study, dosage adjustments may not be required for clobazam in the presence of the AEDs investigated here.

An integrative population pharmacokinetics (PPK)-based approach was used to characterize the effect of hepatic impairment on clobazam PK and its major metabolite in systemic circulation, N-desmethylclobazam (N-CLB). At therapeutic clobazam dosages, N-CLB plasma concentrations are 3-5 times greater than the parent compound. PK data from clinical trials in patients with Lennox-Gastaut syndrome (LGS; OV-1002 and OV-1012), healthy participants (OV-1016), and participants with and without renal impairment (OV-1032), as well as those from a publication describing the effects of hepatic impairment on clobazam PK, were merged to create the PPK model. Individual patient clobazam PK parameters from the publication were used to generate patient plasma-concentration data. Clobazam PK was linear and the formation of N-CLB was elimination-rate limited. Hepatic impairment did not affect the total apparent clearance of clobazam but may affect the PK of N-CLB. Because the formation of N-CLB is elimination-rate limited and the total apparent clearance of clobazam is unaffected by hepatic impairment, the PPK model suggests that patients with LGS and hepatic impairment may not require clobazam dosage modification.

Purpose A thorough QT study was conducted to assess the proarrhythmic potential of clobazam and its active metabolite, N-desmethylclobazam (N-CLB). Methods In this Phase I, single-site, randomized, double-blind, double-dummy, parallel-group study, healthy participants were randomized to 1 of 4 treatment groups: clobazam 40 mg/d (maximum therapeutic dosage), clobazam 160 mg/d (supratherapeutic dosage), placebo, or moxifloxacin 400 mg (active control). Findings Of 280 enrolled participants (n = 70 per treatment arm), 250 (92%) completed the study; 194 were included in the pharmacokinetics population (clobazam 40 mg/d, n = 66; clobazam 160 mg/d, n = 62; and moxifloxacin, n = 66). Mean changes from baseline in QT interval placebo-corrected for heart rate using the Fridericia formula (primary end point), Bazett formula, and individual correction method (QTcF, QTcB, and QTcI, respectively) with clobazam 40 and 160 mg/d revealed no effect on QTc. No clinically relevant or treatment-related arrhythmias were observed, and there were no instances of second- or third-degree atrioventricular block. Given that clobazam is primarily demethylated to N-CLB by cytochrome P450 (CYP) enzyme, CYP3A4, the mean plasma time–concentration profile of clobazam was unchanged with the exclusion of CYP2C19 poor metabolizers. As N-CLB is metabolized by CYP2C19, the exclusion of CYP2C19 poor metabolizers resulted in slightly decreased mean plasma time–concentration profiles of N-CLB. Using a linear mixed-effects model, the effects of the clobazam and N-CLB Cmax values on the placebo-corrected changes from baseline in QTcF, QTcI, and QTcB were near zero or slightly negative, and are not considered clinically important. The incidence of treatment-emergent adverse events was greatest in the clobazam groups (number of moderate AEs experienced by patients: PBO, 3/70; MOXI, 5/70; CLB 40 mg/d, 18/70; CLB 160 mg/d, 21/70; severe AEs: PBO, MOXI, & CLB 160 mg/d, 0; CLB 40 mg/d, 2/70); there were no serious AEs in any treatment group. A total of 10% of participants experienced benzodiazepine-withdrawal symptoms (16%, 23%, and 3% in the clobazam 40 and 160 mg/d groups and the moxifloxacin group, respectively). Implications The findings from this thorough QT study are consistent with existing clinical data and support the lack of proarrhythmic potential with clobazam.

To assess withdrawal-related adverse event (AE) rates following abrupt clobazam discontinuation in Phase I trials and gradual clobazam tapering (2-3 weeks) following discontinuation from III trials met the criteria for potential/III trials, we evaluated AE data from four multiple-dosage Phase I trials (duration: 8-34 days). Therapeutic (20 and 40 mg/day) and supratherapeutic clobazam dosages (120 and 160 mg/day) were administered. Adverse events (AEs) were also assessed for patients with Lennox-Gastaut syndrome enrolled in Phase II (OV-1002) and Phase III (OV-1012) studies (duration ≤15 weeks) and in the open-label extension (OLE) trial OV-1004 (≤5 years). Potential withdrawal-related AEs were identified by preferred terms, provided that the AEs occurred ≥1 day following and ≤30 days after the last clobazam doses, or were deemed withdrawal symptoms by investigators. Clinical Institute Withdrawal Assessment for Benzodiazepines (CIWA-B) scale was used to evaluate withdrawal intensity in three of the four Phase I trials. A total of 207 participants in Phase I trials received steady-state clobazam dosages of 20-160 mg/day, 182 received clobazam dosages of ≥40 mg/day, and 94 received clobazam dosages of ≥120 mg/day. Abrupt clobazam discontinuation led to 193 withdrawal-related AEs for 68 Phase I participants. Nearly 50% of AEs occurred after discontinuation of clobazam dosages of ≥120 mg/day. Adverse events were mild or moderate and included headache (14% of Phase I participants), insomnia (12.6%), tremor (10.1%), and anxiety (8.7%). The CIWA-B scores varied (range: 0-59). Most scores were

Study objective To investigate potential drug-drug interactions between clobazam and cytochrome P450 (CYP) isoenzyme substrates, inhibitors, and inducers. Design Two, prospective, open-label, single-center, drug-drug interaction (DDI) studies and a population pharmacokinetics analysis of seven multicenter phase II-III trials. Setting Clinical research unit. Participants Fifty-four healthy adult volunteers were enrolled in the two drug-drug interaction studies; 53 completed the studies. The population pharmacokinetics analysis evaluated data from 171 participants from five studies with healthy volunteers and two studies with patients with Lennox-Gastaut syndrome. Participants in these studies received clobazam and stable dosages of the following antiepileptic drugs: phenobarbital, phenytoin, carbamazepine, valproic acid, lamotrigine, felbamate, or oxcarbazepine. Intervention In the first drug-drug interaction study, 36 participants received a single oral dose of clobazam 10 mg on day 1, followed by either ketoconazole 400 mg once/day or omeprazole 40 mg once/day on days 17-22, with a single dose of clobazam 10 mg coadministered on day 22, to study the effects of CYP3A4 or CYP2C19 inhibition, respectively, on clobazam and its active metabolite N-desmethylclobazam (N-CLB). In the second study, 18 participants received a drug cocktail consisting of caffeine 200 mg, tolbutamide 500 mg, dextromethorphan 30 mg, and midazolam 4 mg on days 1 and 19, and clobazam 40 mg/day on days 4-19, to study clobazam's effects on CYP1A2, CYP2C9, CYP2D6, and CYP3A4. Measurements and main results In the first DDI study, coadministration of ketoconazole (a CYP3A4 inhibitor) and clobazam increased clobazam's area under the concentration time curve from time zero extrapolated to infinity (AUC(0-∞) ) 54% and decreased clobazam's maximum plasma concentration (C(max) ) by 15% versus administration of clobazam alone, but the combination affected these pharmacokinetic parameters for N-CLB to a lesser degree. The CYP2C19 inhibitor omeprazole increased AUC(0-∞) and C(max) of N-CLB by 36% and 15%, respectively, but did not significantly affect the pharmacokinetics of clobazam. At steady state, N-CLB has 3-4 times greater exposure than clobazam. In the second DDI study, no clinically significant drug-drug interactions were observed between clobazam 40 mg and the CYP probe substrates caffeine or tolbutamide. Exposure to midazolam and its 1-hydroxymidazolam metabolite, however, decreased by 27% and increased 4-fold, respectively. Clobazam increased dextromethorphan (CYP2D6) AUC(0-∞) by 95% and C(max) by 59%. In the population pharmacokinetics analysis, stable dosages of common antiepileptic drugs that induce CYP3A4 or CYP2C19, or inhibit CYP2C19, had negligible effects on clobazam or N-CLB. Clobazam did not affect valproic acid or lamotrigine exposures. Conclusion These findings suggest no clinically meaningful drug-drug interactions between clobazam and drugs metabolized by CYP3A4, CYP2C19, CYP1A2, or CYP2C9. Concomitant use of drugs metabolized by CYP2D6 may require dosage adjustment. Clobazam may be administered safely as adjunctive therapy in patients with Lennox-Gastaut syndrome, without meaningful changes in clobazam pharmacokinetics that would require dosage adjustment.

Two toxicologic studies of vigabatrin were conducted with immature Sprague Dawley rats to characterize intramyelinic edema (IME) formation and assess potential impact on behavioral measures. Study 1 was a dosage-ranging characterization of overall toxicity of vigabatrin in young, developing rats. Study 2 evaluated vacuolar brain lesions found in Study 1.During Study 1, immature Sprague Dawley rats were orally administered deionized water (vehicle control), or vigabatrin at 5, 15, or 50mg/kg/day for ≤ 9 weeks, beginning at postnatal day 4 (PND 4) and followed by a recovery period. Toxicologic observations were collected, including adverse clinical signs, body weight gains, food consumption, ophthalmoloscopy, electroretinograms, sexual maturation, motor activity, memory, and learning behaviors. At sacrifice, CNS tissues were examined by light microscopy for evidence of IME. In Study 2, immature Sprague Dawley rats were again orally administered vigabatrin (50mg/kg/day for ≤ 9 weeks, beginning at PND 4). At sacrifice, CNS tissues were examined by both light and transmission electron microscopy for evidence of IME.At 5-50mg/kg/day, dosage-related reduced food consumption, decreased body weight, and delayed sexual maturation were found. Persisting through recovery, effects were more pronounced in males. Increased degrees of vacuolation were observed on PND 67 only after a dosage of 50mg/kg/day, and were attenuated during recovery. Vacuolar-change morphology was characteristic of IME, with no evidence of cellular or neuritic degeneration. Ultrastructural analyses revealed brain vacuoles initiated as splits of myelin sheaths along intra-period lines. These splits expanded, evolving into large membrane-rich vacuoles, and were more prominent at later stages of myelin development. Hypomyelination and gliopathy were noted from PNDs 4-15, and were likely related to vigabatrin exposure during active myelination. A lesser degree of hypomyelination was observed from PNDs 4-46 and 4-65. Vacuolation was markedly attenuated in post-recovery-period rats.The present studies indicated toxicities in young rats at vigabatrin dosages lower than those reported for toxicities in older rats. Dosages50mg/kg/day did not affect CNS, behavior, and reproductive development. However, at the greatest dosage, some retardation of physical growth, delay in sexual maturation, reduction in physical strength, and induction of CNS stimulation (handling-induced spasms) occurred. The key pathologic finding was vacuolar brain lesions in the white and gray matter, which generally reversed upon drug discontinuation. Vacuoles were confined to myelin sheaths, consistent with observations in adult rats. Vigabatrin delayed but did not eliminate myelination despite continued dosing, an effect greatest during active myelination.

An integrative population pharmacokinetics (PPK)-based approach was used to characterize the effect of hepatic impairment on clobazam PK and its major metabolite in systemic circulation, N-desmethylclobazam (N-CLB). At therapeutic clobazam dosages, N-CLB plasma concentrations are 3-5 times greater than the parent compound. PK data from clinical trials in patients with Lennox-Gastaut syndrome (LGS; OV-1002 and OV-1012), healthy participants (OV-1016), and participants with and without renal impairment (OV-1032), as well as those from a publication describing the effects of hepatic impairment on clobazam PK, were merged to create the PPK model. Individual patient clobazam PK parameters from the publication were used to generate patient plasma-concentration data. Clobazam PK was linear and the formation of N-CLB was elimination-rate limited. Hepatic impairment did not affect the total apparent clearance of clobazam but may affect the PK of N-CLB. Because the formation of N-CLB is elimination-rate limited and the total apparent clearance of clobazam is unaffected by hepatic impairment, the PPK model suggests that patients with LGS and hepatic impairment may not require clobazam dosage modification.

Summary Objective To evaluate the safety, tolerability, and comparative pharmacokinetics (PK) of intravenous and oral carbamazepine. Methods In this phase 1, open-label study, adult patients with epilepsy on a stable oral carbamazepine dosage (400–2,000 mg/day) were converted to intravenous carbamazepine (administered at 70% of the oral dosage). A 28-day outpatient period preceded an up to 10-day inpatient period and a 30-day follow-up period. Intravenous carbamazepine was administered over 15 or 30 min every 6 h on days 1–7; some patients in the 15-min group were eligible to receive four 2- to 5-min (rapid) infusions on day 8. Patients underwent blood sampling to determine the area under the concentration–time curve (AUC) for carbamazepine and metabolite carbamazepine-10,11-epoxide following oral (day 0) and intravenous carbamazepine administration (days 1, 7, and 8). Bioequivalence was evaluated in patients with normal renal function (creatinine clearance >80 ml/min). Safety assessments were conducted through day 38. Results Ninety-eight patients enrolled and 77 completed the PK component. The mean daily oral and intravenous carbamazepine dosage for 64 PK-evaluable patients with normal renal function was 962.5 and 675.1 mg (70% of oral dosage), respectively. Steady-state minimum concentration (Cmin) and overall exposure (AUC0–24) for intravenous carbamazepine infused over 30, 15, or 2–5 min were similar to oral carbamazepine. The 90% confidence intervals (CIs) for the ratios of the adjusted means for AUC0–24, maximum concentration (Cmax), and Cmin were within the 80%–125% bioequivalence range for 30-min intravenous infusions versus oral administration, but exceeded the upper limit for Cmax for the 15-min and rapid infusions. All intravenous carbamazepine infusions were well tolerated. Significance Intravenous carbamazepine infusions (70% of oral daily dose) of 30-, 15-, and 2- to 5-min duration, given every 6 h, maintained patients’ plasma carbamazepine concentrations. Intravenous carbamazepine 30-min infusions were bioequivalent to oral carbamazepine in patients with normal renal function; rapid infusions were well-tolerated in this study.

SummaryObjective To report tolerability findings and maintenance of seizure control from a pooled analysis of phase I open-label trial OV-1015 (NCT01079351) and phase III study 13181A (NCT01128959). Methods Patients receiving a stable oral dosage of carbamazepine were switched to an intravenous (IV) carbamazepine formulation solubilized in a cyclodextrin matrix (at a 70% dosage conversion) for either a 15- or a 30-min infusion every 6 h for up to 7 days and then switched back. A subset of patients who tolerated 15-min infusions also received 2- to 5-min (rapid) infusions. Assessments included physical and laboratory evaluations, electrocardiography (ECG) studies, as well as adverse event (AE) monitoring for tolerability. Convulsion/seizure AE terms and data from seizure diaries were used as proxies for the assessment of consistency of seizure control between formulations. Results Of the 203 patients exposed to IV carbamazepine (30 min, n = 43; 15 min, n = 160), 113 received 149 rapid infusions. During infusion, the most commonly reported AEs (≥5%) were dizziness (19%), somnolence (6%), headache (6%), and blurred vision (5%). IV carbamazepine was not associated with clinically relevant cardiac AEs. The tolerability profile appeared similar between patients who received

Micafungin is an antifungal agent metabolized by arylsulfatase with secondary metabolism by catechol-O-methyltransferase. The objectives of this study were to estimate the pharmacokinetic parameters and plasma protein binding of micafungin in volunteers with moderate hepatic dysfunction (n = 8), volunteers with creatinine clearance < 30 mL/min (n = 9), and matched controls (n = 8 and n = 9, respectively). Single-dose micafungin pharmacokinetics were estimated using noncompartmental techniques. There was a statistically lower area under the observed micafungin concentration-time curve (AUC) from time 0 to infinity for subjects with moderate hepatic dysfunction as compared to control subjects (97.5 +/- 19 microg.h/mL vs 125.9 +/- 26.4 microg.h/mL, P = .03), although there was no difference in micafungin weight-adjusted clearance (10.9 +/- 1.7 mL/h/kg vs 9.8 +/- 1.8 mL/h/kg, P = .2). The difference in area under the concentration-time curve may be explained by the differences in body weight between subjects and controls. Renal dysfunction did not alter micafungin pharmacokinetics.

Background The effects of tacrolimus (FK506) and malononitrilamides (MNA) (FK778 and FK779) monotherapy and combination therapy were examined in prevention of acute heart and kidney allograft rejection and reversal of ongoing acute heart allograft rejection in the rat. Methods. Brown Norway (RT1 n )-to-Lewis (RT1 1 ) and ACI (RT1 a )-to-Lewis (RT1 1 ) combinations were used, respectively, for heart and kidney transplantation models. Immunosuppressants were administered orally from day 1 to day 14 for preventing acute rejection and from day 4 to day 34 after transplantation for the reversal of ongoing acute rejection. Results. In the prevention of acute heart rejection model, recipient rats treated with monotherapy of tacrolimus or MNA (FK778, FK779) showed a dose-related prolongation of mean survival time (MST) compared with naive control rats (P

Background. Malononitrilamide FK778, an analogue of leflunomide's active metabolite, is a promising novel small molecule with immunosuppressive and immunomodulatory properties. In this study, we evaluated the ability of combination therapy of FK778 with tacrolimus to inhibit lymphocyte proliferation and to prevent acute allograft rejection. Methods. Proliferation assay was used to evaluate the effect of FK778 plus tacrolimus on murine splenocytes, monkey lymphocytes, and human peripheral blood mononuclear cells, after activation with T or B cell-specific mitogens. A rat kidney transplantation model was used to evaluate the ability of FK778 combined with tacrolimus to prolong allograft survival. Median-effect principle and combination index (CI) were used to determine synergism, summation, or antagonism. Results. A total of 58 combinations of FK778 plus tacrolimus were evaluated. Of the combinations tested, 82.8% (24/29) produced additive to synergistic effects in B cells, whereas 79.3% (23/29) produced moderate antagonistic effects in T cells. A concomitant 14-day therapy of FK778 (10 mg/kg/day) and tacrolimus (1 mg/kg/day) synergistically prolonged renal allograft survival to 25.5±5.9 days (CI=0.458). However, when addition of FK778 to tacrolimus therapy was delayed to day 7 after transplantation, a strong synergism was obtained (mean survival time=74.9±14.8 days, CI

BACKGROUND: Malononitrilamide 715 (FK778) is a new class of low-molecular-weight immunosuppressant that is a derivative of the active metabolite of leflunomide, A77 1726. In this study, the authors evaluated the combined effect of FK778 with tacrolimus in prevention of renal allograft rejection in Vervet monkeys. METHODS: Male Vervet monkeys were obtained from Caribbean Primates Ltd. Donor and recipient monkeys were from different breeding colonies. Eleven groups (n>or=4 per group) were involved in this study. FK778 and tacrolimus were administered orally for 60 days according to protocol. RESULTS: Naive controls rejected renal grafts, with a median survival time (MST) of 8.0 days in group 1. When recipient monkeys were treated with tacrolimus 1.0 mg/kg/day in group 2 or FK778 2.5 mg/kg/day in group 3, the MST was 16.0 days (P=0.001) and 11.0 days (P=0.266), respectively. Combination therapy of these two agents at the same doses immediately after transplantation resulted in an MST of 25.0 days (P=0.016) in group 4. When tacrolimus was initiated immediately after transplantation and FK778 treatment was delayed until day 7 after surgery in group 5, recipient survivals were significantly prolonged to 38.0 days (P=0.02). These results were repeatable when FK778 5.0 mg/kg/day (9.0 days, P=0.544 in group 6) was combined with tacrolimus 1.0 mg/kg/day immediately after transplantation (8.0 days, P=0.339) in group 7, or when FK778 was delayed 7 days (60.0 days, P=0.002) in group 8. Furthermore, it was also repeatable when FK778 10 mg/kg/day was combined with tacrolimus 1.0 mg/kg/day with a 7-day delay. CONCLUSIONS: A significant prolongation of renal allograft survival was produced when FK778 administration was delayed by 7 days combined with tacrolimus in Vervet monkeys.

Micafungin (FK463) is an echinocandin that demonstrates potent in vitro antifungal activities against Candida and Aspergillus species. However, little is known about its comparative antifungal activities in persistently neutropenic hosts. We therefore investigated the plasma micafungin pharmacokinetics and antifungal activities of micafungin against experimental disseminated candidiasis and invasive pulmonary aspergillosis in persistently neutropenic rabbits. The groups with disseminated candidiasis studied consisted of untreated controls (UCs); rabbits treated with desoxycholate amphotericin B (DAMB) at 1 mg/kg of body weight/day; or rabbits treated with micafungin at 0.25, 0.5, 1, and 2 mg/kg/day intravenously. Compared with the UCs, rabbits treated with micafungin or DAMB showed significant dosage-dependent clearance of Candida albicans from the liver, spleen, kidney, brain, eye, lung, and vena cava. These in vivo findings correlated with the results of in vitro time-kill assays that demonstrated that micafungin has concentration-dependent fungicidal activity. The groups with invasive pulmonary aspergillosis studied consisted of UCs; rabbits treated with DAMB; rabbits treated with liposomal amphotericin B (LAMB) at 5 mg/kg/day; and rabbits treated with micafungin at 0.5, 1, and 2 mg/kg/day. In comparison to the significant micafungin dosage-dependent reduction of the residual burden (in log CFU per gram) of C. albicans in tissue, micafungin-treated rabbits with invasive pulmonary aspergillosis had no reduction in the concentration of Aspergillus fumigatus in tissue. DAMB and LAMB significantly reduced the burdens of C. albicans and A. fumigatus in tissues ( P < 0.01). Persistent galactomannan antigenemia in micafungin-treated rabbits correlated with the presence of an elevated burden of A. fumigatus in pulmonary tissue. By comparison, DAMB- and LAMB-treated animals had significantly reduced circulating galactomannan antigen levels. Despite a lack of clearance of A. fumigatus from the lungs, there was a significant improvement in the rate of survival ( P < 0.001) and a reduction in the level of pulmonary infarction ( P < 0.05) in micafungin-treated rabbits. In summary, micafungin demonstrated concentration-dependent and dosage-dependent clearance of C. albicans from persistently neutropenic rabbits with disseminated candidiasis but not of A. fumigatus from persistently neutropenic rabbits with invasive pulmonary aspergillosis.

Unilamellar liposomal amphotericin B (AmBisome) (liposomal AMB) reduces the toxicity of this antifungal drug. The unique composition of liposomal AMB stabilizes the liposomes, producing higher sustained drug levels in plasma and reducing renal and hepatic excretion. When liposomes release their drug payload, unbound, protein-bound, and liposomal drug pools may exist simultaneously in the body. To determine the amounts of drug in these pools, we developed a procedure to measure unbound AMB in human plasma by ultrafiltration and then used it to characterize AMB binding in vitro and to assess the pharmacokinetics of nonliposomal pools of AMB in a phase IV study of liposomal AMB and AMB deoxycholate in healthy subjects. We confirmed that AMB is highly bound (>95%) in human plasma and showed that both human serum albumin and α 1 -acid glycoprotein contribute to this binding. AMB binding exhibited an unusual concentration dependence in plasma: the percentage of bound drug increased as the AMB concentration increased. This was attributed to the low solubility of AMB in plasma, which limits the unbound drug concentration to −1 kg −1 ) for both formulations. Unbound drug urinary clearances were equal to the glomerular filtration rate, and tubular transit rates were

The plasma pharmacokinetics and tissue distribution of the novel antifungal echinocandin-like lipopeptide micafungin (FK463) were investigated in healthy rabbits. Cohorts of three animals each received micafungin at 0.5, 1, and 2 mg/kg of body weight intravenously once daily for a total of 8 days. Serial plasma samples were collected on days 1 and 7, and tissue samples were obtained 30 min after the eighth dose. Drug concentrations were determined by validated high-performance liquid chromatographic methods. Plasma drug concentration data were fit to a two-compartment pharmacokinetic model, and pharmacokinetic parameters were estimated using weighted nonlinear least-square regression analysis. Micafungin demonstrated linear plasma pharmacokinetics without changes in total clearance and dose-normalized area under the concentration-time curve from 0 h to infinity. After administration of single doses to the rabbits, mean peak plasma drug concentrations ranged from 7.62 μg/ml at 0.5 mg/kg to 16.8 μg/ml at 2 mg/kg, the area under the concentration-time curve from 0 to 24 h ranged from 5.66 to 21.79 μg · h/ml, the apparent volume of distribution at steady state ranged from 0.296 to 0.343 liter/kg, and the elimination half-life ranged from 2.97 to 3.20 h, respectively. No significant changes in pharmacokinetic parameters and no accumulation was noted after multiple dosing. Mean tissue micafungin concentrations 30 min after the last of eight daily doses were highest in the lung (2.26 to 11.76 μg/g), liver (2.05 to 8.82 μg/g), spleen (1.87 to 9.05 μg/g), and kidney (1.40 to 6.12 μg/g). While micafungin was not detectable in cerebrospinal fluid, the concentration in brain tissue ranged from 0.08 to 0.18 μg/g. These findings indicate linear disposition of micafungin at dosages of 0.5 to 2 mg/kg and achievement of potentially therapeutic drug concentrations in plasma and tissues that are common sites of invasive fungal infections.

Amphotericin B (AmB) in small unilamellar liposomes (AmBisome) provides higher plasma concentrations and greater safety than the conventional deoxycholate formulation. The authors compared the disposition of the liposome's drug and cholesterol components by measuring AmB and radioactivity in plasma, urine, and feces for 1 week after a single 2-hour infusion of 14C-cholesterol-labeled AmBisome (2 mg/kg, 1 microgCi/kg) in healthy adults (4 males, 1 female). The plasma profile of 14C-cholesterol differed from that of AmB, lacking an initial rapid disappearance phase, having a lower total clearance, and having a volume of distribution (0.13 L/kg) close to that of the plasma compartment. The biphasic disappearance and long plasma half-life (147 h) of 14C-cholesterol were similar to those of other low-clearance liposomes. This and the low clearance of 14C-cholesterol from the plasma compartment suggest that it served as a liposome marker. The plasma drug-lipid ratio fell during the study, showing that AmB was cleared from plasma more rapidly than cholesterol or liposomes and suggesting that the composition of the liposomes changed over time. 14C-radioactivity was recovered mainly in the feces (9.5% of dose), consistent with the catabolism of cholesterol to bile salts. Combined fecal and renal clearances were < 18% of total clearance, suggesting that most of the liposomal drug and lipid remained in the body 1 week after dosing. Thus, AmBisome remains in the circulation for an extended period of time while releasing AmB, resulting in its markedly altered pharmacokinetic and safety profiles.

Amphotericin B remains the standard of care for the treatment of invasive and disseminated fungal infections. Various lipid-based formulations of amphotericin B have been developed to improve its therapeutic index by decreasing toxicity. Previous bioanalytic methods using microbial inhibition or high-pressure liquid chromatography quantified total amphotericin B (free, plasma protein-bound, and lipid-complexed). Sensitivity of this method with a low limit of quantitation of 0.05 microg/mL was inadequate to determine free (unbound) amphotericin B. A sensitive LC/MS/MS method was developed to determine the total amphotericin B value in human plasma and other biologic matrices and the free amphotericin B concentration in plasma. For determination of total plasma amphotericin B concentrations, the sample was diluted and injected onto the LC/MS/MS. For total amphotericin B in other matrices and free amphotericin B in plasma, solid-phase extraction was used. Natamycin served as an internal standard. A PE Sciex API 3000 (Sciex; Concord, Ontario, Canada) was used to assess free amphotericin B in plasma ultrafiltrate determination and an API 3+ for the other matrices, with electrospray interfaced to a C18 analytic column. The low limit of quantitation was 1 ng/mL for ultrafiltrate. For total amphotericin B, the low limits were 2 microg/mL for plasma, 0.05 microg/mL for urine, and 0.4 microg/mL for fecal homogenate. The methods were validated to show the standard range linearity, sensitivity, selectivity, accuracy, precision, and stability of amphotericin B in the matrices tested.

Tacrolimus (FK506, Prograf), marketed for the prophylaxis of organ rejection following allogenic liver or kidney transplantation, is virtually completely metabolized. The major metabolic pathways are P450 3A4-mediated hydroxylation and demethylation. Since P450 hepatic drug-metabolizing enzymes may be impaired in hepatic dysfunction, a study was conducted to characterize oral and intravenous tacrolimus pharmacokinetics in 6 patients with mild hepatic dysfunction and compared with parameters to those from normal subjects obtained in a separate study. Patients received two treatments: a single 0.020 mg/kg ideal body weight (IBW) i.v. dose infused over 4 hours and approximately 0.12 mg/kg IBW orally; normal subjects were dosed at 0.02 mg/kg 4-hour i.v. and 5 mg (0.065 mg/kg) p.o. Mean blood pharmacokinetic parameters with mild hepatic dysfunction were as follows: clearance = 0.035 L/h/kg, terminal exponential volume of distribution = 2.59 L/kg, terminal exponential half-life = 60.6 hours (i.v.), p.o. maximum blood concentration = 48.2 ng/mL, time of p.o. maximum blood concentration = 1.5 hours, and absolute bioavailability = 22.3%. The respective parameters in normal subjects were as follows: 0.040 L/h/kg, 1.91 L/kg, 34.2 hours (i.v.), 29.7 ng/mL, 1.6 hours, and 17.8%. Inasmuch as clearance and bioavailability were not substantially different from that in normal subjects, patients with mild hepatic impairment may initially be treated with conventional tacrolimus doses, with subsequent dosage adjustments based on response, toxicity, and therapeutic drug monitoring.

Tacrolimus (FK506, Prograf) is marketed for the prophylaxis of organ rejection following allogenic liver or kidney transplantation. This study investigated the effect of timing of a standardized breakfast meal on both the rate and extent of tacrolimus absorption following a single 5 mg oral dose. The protocol used a randomized, open-label, four-period, four-treatment, four-sequence crossover design in 16 healthy, nonsmoking, drug-free male subjects between the ages of 22 and 45 years who were within 15% of their ideal body weight. The four treatments were the following: (A) fasting for 10 hours, (B) ingestion 1 hour before breakfast, (C) ingestion immediately following consumption of the breakfast, and (D) ingestion 1.5 hours after beginning consumption of the breakfast. The breakfast, which was consumed over 15 minutes, contained 848 kcal, with 30%, 16%, and 54% of calories derived from fat, protein, and carbohydrate, respectively. Tacrolimus absorption in the fasting state provided the greatest relative bioavailability (p < 0.05 compared with all other three treatments). AUC(0-infinity)) averaged 312, 276, 205, and 203 ng x h/mL for treatments A, B, C and D, respectively. In contradistinction to taking the drug 1 hour prior to a meal, which had a relatively minor impact on the relative extent of absorption (approximately 12%) compared to the fasting state, ingestion of tacrolimus immediately after a meal (treatment C) or 1.5 hours subsequent to a meal (treatment D) had a more pronounced influence. Mean AUC(0-infinity) ratios (fasting to either postmeal treatments) were approximately 1.5, indicating that absorption extent was considerably reduced by ingesting tacrolimus capsules immediately after eating or 1.5 hours thereafter. Absorption was also prolonged following drug ingestion after a meal, as indicated by a mean tmax value in the fasting state of 1.84 hours, relative to 3.41 hours (immediately aftermeal, p = 0.0035) and 3.22 hours (1.5 hours postmeal, p = 0.0094). The only discernable difference in parameters between treatments C and D was with Cmax, with values of 7.19 and 9.04 ng/mL, respectively, but was not statistically significantly different (p = 0.231). Based on these results and those from a prior study, it is recommended that under therapeutic conditions, oral tacrolimus be administered in a consistent manner, both with respect to the type of meal as well as timing of ingestion relative to consumption of the meal.

Objective Our objective was to compare the intravenous and oral pharmacokinetics of tacrolimus among subjects of three different ethnic backgrounds, African American, white, and Latin American. Methods Ten African American, 12 white, and 12 Latin American subjects received intravenous and oral tacrolimus in an open-label, two-period, parallel group study. All of the subjects received intravenous tacrolimus (0.015 mg/kg) as a constant infusion over 4 hours and oral tacrolimus capsules (5 mg) as single doses in randomized order. Concentrations of tacrolimus and its metabolites were measured in whole blood with the use of a validated HPLC-mass spectrometry assay. Results There were no significant differences in pharmacokinetic parameters among the three study groups after intravenous administration of the drugs. After oral administration, the tacrolimus maximum concentration was significantly lower (P < .01) in the African American subjects (20.8 μg/L) than in the white subjects (37.8 μg/L) and Latin American subjects (33.0 μg/L). Absolute bioavailability was significantly lower (P = .01) in the African American subjects (11.9%) and in the Latin American subjects (14.4%) than in the white subjects (18.8%). After the oral dose, the area under the plasma concentration-time curve was lower in the African American subjects (179 μg/L · h, geometric mean) than in the white (293 μg/L · h) and Latin American subjects (239 μg/L · h, differences not statistically significant). Maximum concentration (P < .02) and area under the plasma concentration-time curve (not statistically significant) of the main tacrolimus metabolite 13-O-desmethyl tacrolimus was lower in the African American subjects than in the white and Latin American subjects. Conclusions Significant differences in tacrolimus pharmacokinetics exist among the three different ethnic groups. Our results indicate that this may result from differences in intestinal CYP3A or P-glycoprotein activities. Clinical Pharmacology & Therapeutics (2001) 69, 24–31; doi: 10.1067/mcp.2001.113183

Tacrolimus ointment, formulated for the treatment of atopic dermatitis, is the first in a class of topical immunomodulators. Its mechanism of action is based on calcineurin inhibition, which results in suppression of antigen-specific T-cell activation and inhibition of inflammatory cytokine release. Animal and human studies have shown that topically applied tacrolimus is minimally absorbed into the systemic circulation, the fraction that is absorbed is extensively distributed, and tacrolimus does not accumulate in tissues following repeated topical application. In addition, tacrolimus ointment is not inherently irritating, sensitizing, phototoxic, or photoallergenic when applied to intact skin. Unlike some topical corticosteroids, tacrolimus ointment does not cause a decrease in collagen synthesis or skin thickness, nor does it produce skin abnormalities or depigmentation. In animal studies, repeated daily application of tacrolimus ointment up to 1 year is associated with dermal findings similar to those following vehicle application (mild to moderate dermal irritation and microscopic findings of acanthosis, hyperkeratosis, and superficial inflammation). In a 52-week study with Yucatan micropigs, no noteworthy macroscopic or microscopic changes (either dermal or systemic) related to the application of tacrolimus ointment (0.03% to 0.3% concentrations) were observed. Tacrolimus ointment was shown to be safe and effective in phase 2 and early phase 3 studies. Significant improvements in atopic dermatitis were observed in the majority of patients treated with tacrolimus ointment. The most common adverse events associated with its use were a transient burning sensation and pruritus at the site of application. Blood tacrolimus concentrations were below the limit of quantitation in most patients. (J Am Acad Dermatol 2001;44:S17-27.)

Background. In nonprimates, organ allografts are often not rejected after withdrawal of immunosuppression.In this study, we examined whether such a phenomenon also occurs in primates. Methods. Vervet monkeys were transplanted with renal allografts and treated for 60 days with tacrolimus, or tacrolimus plus sirolimus. The drugs were totally withdrawn on day 61. The survival of the monkeys was monitored, and their response to donor- or third party-derived alloantigens was examined in vivo and in vitro. Results. The majority (80–100%) of the grafts survived for at least additional 30 days with no signs of acute rejection. The compromised rejection is donor-specific, because recipient monkeys failed to reject a donor-derived skin graft, but a third-party skin graft was rejected. In vitro mixed lymphocyte reaction and interleukin-2 production in the mixed lymphocyte reaction between the recipients and their donors or between the recipients and a third party had no discernable patterns, and thus did not reflect the in vivo status of the immune system. Although the recipients could not reject the graft acutely after drug withdrawal, the kidney grafts and the donor-derived skin grafts had pathological findings of chronic rejection. Conclusions. The rejection response of the monkeys to an established graft after withdrawal of immunosuppression is compromised. The compromised rejection is specific and is not due to a permanent alteration of the immune system by the initial drug treatment. The allografts are not inert but have low levels of interaction with the recipient immune system.

Background. Our previous studies confirmed that tacrolimus (FK506) and sirolimus [rapamycin (RAPA)], in combination, are not antagonistic but are synergistic in the prolongation of heart and small bowel grafts in the rodent. The aim of this study was to confirm further the synergistic effect of combined FK506 and RAPA in the more clinically relevant model, kidney transplantation in monkeys. Methods. A total of 60 male Vervet monkeys were randomly assigned to 10 groups (n≥5). Monkeys with renal allografts were treated with different doses of FK506 and/or RAPA orally for 60 days. Graft survival, body weight, clinical biochemistry determinations, oral glucose tolerance test, trough levels of the two drugs, and histopathology were investigated. Results. Low doses of FK506 (1 or 4 mg/kg) combined with RAPA (0.5 mg/kg) produced synergistic effect in the prolongation of renal graft survival [combination index (CI)=0.292, 0.565]. There were no additive or synergistic drug-associated toxicities such as hyperglycemia, nephrotoxicity, and hyperlipidemia. There also was no pharmacological antagonism. Conclusion. Concomitant therapy of low-dose (drug-optimal) FK506 and RAPA produced a synergistic effect in the prolongation of kidney allograft survival in Vervet monkeys without additive drug-associated toxicities.

Purpose. Amphotericin B in small, unilamellar liposomes (AmBisome) is safer and produces higher plasma concentrations than other formulations. Because liposomes may increase and prolong tissue exposures, the potential for drug accumulation or delayed toxicity after chronic AmBisome was investigated.

Tacrolimus (FK506, Prograf) is marketed for the prophylaxis of organ rejection following allogenic liver or kidney transplantation. A previously conducted, randomized, 24-subject, crossover bioavailability study of 1 and 5 mg capsules (one period each) failed to demonstrate bioequivalence. A single-dose, four-period, four-sequence, randomized, crossover, replicate study (N = 32) was therefore used to evaluate the bioequivalence of the marketed 1 and 5 mg capsules in healthy volunteers. Tacrolimus blood concentrations were measured serially over 72 hours using a commercially available ELISA assay. Noncompartmental pharmacokinetic parameters were determined. Ninety percent CIs of log-transformed parameter ratios were 90.5-101.9, 87.1-101.7, and 89.7-103.8 for Cmax, AUC0-t, and AUC0-infinity, respectively. Since all values were within 80% to 125%, the capsules are bioequivalent. Based on %CVs, intersubject variability was approximately two to three times greater than intrasubject variability. The safety of single 5 mg oral tacrolimus doses administered to healthy volunteers at 7-day intervals was also ascertained.

Amphotericin B has been the mainstay of systemic antifungal therapy for over 30 years, despite its serious side-effects, and, although numerous alternative antifungal agents have been developed, none to date has matched the efficacy of amphotericin B. However, modern drug delivery technology has improved the safety of amphotericin B by incorporating it into lipid-based delivery systems, including liposomes. Three such formulations, based on the natural affinity of amphotericin B for lipids, are currently marketed. All increase the therapeutic index of amphotericin B, thereby allowing more aggressive treatment than is possible with the conventional product. However, they differ in structure, side-effect profiles and evidence of proven efficacy as discussed in this review.

Tacrolimus is a marketed immunosuppressant used in liver and kidney transplantation. It is subject to extensive metabolism by CYP3A4 and is a substrate for P-glycoprotein-mediated transport. A pharmacokinetic interaction with rifampin, an antituberculosis agent and potent inducer of CYP3A4 and P-glycoprotein, and tacrolimus was evaluated in six healthy male volunteers. Tacrolimus was administered at doses of 0.1 mg/kg orally and 0.025 mg/kg/4 hours intravenously. The pharmacokinetics of tacrolimus were obtained from serial blood samples collected over 96 hours, after single oral and intravenous administration prior to and during an 18-day concomitant rifampin dosing phase. Coadministration of rifampin significantly increased tacrolimus clearance (36.0 +/- 8.1 ml/hr/kg vs. 52.8 +/- 9.6 ml/hr/kg; p = 0.03) and decreased tacrolimus bioavailability (14.4% +/- 5.7% vs. 7.0% +/- 2.7%; p = 0.03). Rifampin appears to induce both intestinal and hepatic metabolism of tacrolimus, most likely through induction of CYP3A and P-glycoprotein in the liver and small bowel.

Purpose. Amphotericin B (AmB) in small, unilamellar liposomes (AmBisome ®) has an improved therapeutic index, and altered pharmacokinetics. The repeat-dose safety and toxicokinetic profiles of AmBisome were studied at clinically relevant doses.

Tacrolimus is a potent immunosuppressant marketed for the prevention of rejection in liver and kidney transplantation. Topical tacrolimus has been shown to be effective in the treatment of atopic dermatitis, a disease with an immunologic basis, and is currently being developed for this indication. The objective of the current study was to determine whether repeated topical application of tacrolimus ointment could result in hypopigmentation at the application site(s). Each of 10 Yucatan miniature pigs received topical application of 0.03%, 0.1%, and 0.3% tacrolimus ointment, positive control (a combination of 2% 4-hydroxyanisole and 0.01% all-trans-retinoic acid, 4-HAArRA), and a negative control (vehicle placebo) to five test sites (approximately 12.5 cm2) along either side of the dorsal midline (10 sites per animal). Tacrolimus and controls were randomly assigned to the test site on each animal and were applied unoccluded twice daily for 8 weeks at a dose volume of 0.1 ml per site for tacrolimus and vehicle control and 0.025 ml per site for the positive control. Topical application of tacrolimus ointment in concentrations of up to 0.3% for 8 weeks had no effect on the cutaneous pigmentation of Yucatan miniature swine. In contrast, the application of 4-HAArRA produced a statistically significant (P

The acute phototoxic potential of tacrolimus (FK506) ointment was investigated in Crl:SKH1-hr hairless mice. Response toa series of ultravioletradiation (UVR) site exposures in males (five group) following a single topical administration of tacrolimus ointment (0%[vehicle], 0.03%, 0.1%, 0.3%, 1%, or 3%) indicated that tacrolimus ointment was neither photoprotective or phototoxic. However, a slight but dose-related decrease in body weight relative to the vehicle control was found at the end of the 4-day observation period in the tacrolimus ointment-treated groups. The same concentrations of tacrolimus ointment were applied to the back and sides of mice (five sex group; ∼40% of total body surface area) 5 days week in a 13-week range-finding study to assess the tolerance to the ointment and UVR exposure. Untreated animals served as a control group. Mice were exposed to 72 minutes of UVR from a 6.5-kW xenon lamp. Mortality occurred in the 0.3%, 1%, and 3% ointment groups. Application of 3% tacrolimus ointment was associated with reduced body weight. Mild to moderate erythema and/or edema were noted in all tacrolimus ointment-treated groups. Histopathologi-cal examination indicated a mild increase in the prominence of the stratum granulosum and mild acanthosis of the epidermis in both vehicle-treated and tacrolimus ointment-treated mice. Stomach, esophageal, and/ or abdominal cavity lesions, indicative of systemic toxicity and possibly associated with the in gestion of ointment vehicle during preening, were observed during necropsy. There was minimal photo-mediated dermal toxicity of tacrolimus ointment in the 13 weeks of exposure in the present study.

Objective To quantitate the effect of ketoconazole, an azole antifungal agent and potent inhibitor of CYP3A4 and P-glycoprotein, on the bioavailability of tacrolimus, a substrate of the CYP3A system and of P-glycoprotein. Subjects and Methods The pharmacokinetics of tacrolimus were studied in six healthy volunteers (two women and four men) in a four-dose study after each received single doses of tacrolimus alone (0.1 mg/kg orally and 0.025 mg/kg intravenously) and with coadministered ketoconazole (200 mg orally at bedtime for 12 days). The dose of tacrolimus was reduced during the ketoconazole phase (0.04 mg/kg orally; 0.01 mg/kg intravenously). Ketoconazole and tacrolimus doses were separated by approximately 10 hours. Whole blood tacrolimus concentrations were determined by enzyme-linked immunosorbent assay. Estimated pharmacokinetic parameters in whole blood (mean ± SD) before and with ketoconazole were calculated with noncompartmental techniques. Results Coadministration of ketoconazole did not consistently affect tacrolimus clearance (55.6 ± 16.7 ml/hr/kg versus 42.5 ± 7.6 ml/hr/kg), and steady-state volume of distribution was unchanged (0.99 ± 0.26 L/kg versus 0.93 ± 0.25 L/kg). However, a significant increase in tacrolimus bioavailability (14% ± 5% versus 30% ± 8%; p < 0.01) was observed with coadministered ketoconazole. Hepatic bioavailability was unchanged by the presence of ketoconazole (96% ± 1% versus 97% ± 1%). Conclusions Because ketoconazole did not alter hepatic bioavailability and because 10 hours separated administration times of the drugs, it appears that the marked increase in tacrolimus bioavailability can be explained by ketoconazole having a local inhibitory effect on tacrolimus gut metabolism or on intestinal P-glycoprotein activity. Clinical Pharmacology & Therapeutics (1997) 62, 41–49; doi

Tacrolimus (FK506) is a macrolide immunosuppressant approved for the prophylaxis of organ rejection in liver transplant. Immunoassays of low intra- and interday variability and high sensitivity are necessary to adequately characterize terminal elimination phase concentrations in pharmacokinetic studies. A new ELISA kit for the quantitation of tacrolimus in human whole blood has been validated for use in pharmacokinetic studies. Methanol sample extracts were dried and reconstituted in a horseradish peroxidase (HPR)-FK506 conjugate solution. The reconstituted samples and mouse anti-FK506 were added to a microplate, precoated with secondary antibody, and incubated, FK506 and the HPR-FK506 conjugate competed to bind with anti-FK506, which was immobilized by binding to the secondary antibody. Unbound FK506 was washed away, and substrate was added for color development. Once the reaction was stopped with 2 N H2SO4, the plate was read at 450 nm. The linear range was 0.5-60 ng/ml, with a limit of quantitation of 0.5 ng/ml. Interday precision and accuracy were < or = 10.4% C.V. and < or = 3% R.E. for quality control samples. The lack of interference from endogenous compounds was established by parallelism and recoveries of FK506 from six lots of control matrix. Cross-reactivity against the metabolites and analogs were not performed because the kit monoclonal antibody was from the same source as Kobayashi et al (1). The utility and sensitivity of the kit present a good method for the quantitation of tacrolimus in blood from pharmacokinetic studies. The method is robust and has been used to assay tacrolimus in several thousand whole blood samples by multiple analysts.

A highly sensitive enzyme-linked immunosorbent assay method has been developed for the determination of tacrolimus in human blood samples. The assay is a modification of the previously published assay with improved sensitivity. Following extraction of tacrolimus with methanol and sulfosalicylic acid, the samples are incubated for 2 h at room temperature on a Nunc Maxisorb plate that has been treated for nonspecific binding by precoating with polyclonal antibody. The analysis of human blood following the standard addition of tacrolimus (0.02-5.0 ng/ml) demonstrated excellent precision and accuracy over a 6-day period. The interday and intraday co-efficients of variation were 6.0-28.9 and 3.9-15.2%, respectively. The limit of quantitation was 0.05 ng/ml. The method was used to quantitate blood concentration of tacrolimus in patients following the administration of tacrolimus ointment.

Background. In an outbred pig model of total bowel transplantation, we previously showed that simultaneous donor-specific bone marrow infusion (DSBMI), rather than promoting engraftment, sensitizes recipients and causes rejection ; it also aggravates the risk of generalized graft-versus-host disease (GVHD) and infection, and tends to reduce recipient and graft survival. Small and large animal models of bone marrow-induced transplant tolerance suggest that some form of recipient preconditioning (RPC) may facilitate engraftment of co-transplanted bone marrow cells and fully expose their tolerogenic potential. Methods. In a preclinical model, we prospectively studied the effect of RPC on simultaneous DSBMI and total (i.e., small and large) bowel transplantation. RPC consisted of whole body irradiation with 400 R (day 0) ; some recipients additionally received horse anti-pig antithymocyte globulin (days -2, -1, and 0). We studied six groups of outbred pigs, all of which underwent at least a total bowel transplant : group 1, nonimmunosuppressed control pigs (n=5) ; group 2, nonimmunosuppressed DSBMI pigs (n=13) ; group 3, tacrolimus pigs (n=7) ; group 4, DSBMI+tacrolimus pigs (n=15) ; group 5, RPC+nonimmunosuppressed DSBMI pigs (n=11) ; and group 6, RPC+DSBMI+tacrolimus pigs (n=14). Results. RPC did not prolong overall survival at 7, 14, 21, and 28 days after transplant. Survival rates were 100%, 100%, 86%, and 71% in group 3 ; 71%, 43%, 29%, and 29% in group 6 ; 55%, 9%, 0%, and 0% in group 5 ; and 60%, 0%, 0%, and 0% in Group 1. Moreover, RPC (groups 5 and 6) increased the incidence of death from rejection, GVHD, and infection when compared with group 3. Survival was significantly higher for RPC+DSBMI+tacrolimus pigs (group 6), compared with RPC+nonimmunosuppressed DSBMI pigs (group 5). Survival greater than 28 days was noted only in pigs that received tacrolimus after transplant : 71% in group 3 versus 29% in group 6. With both RPC and DSBMI (groups 5 and 6), rejection, GVHD, and infection were not mutually exclusive events. In groups 5 and 6, at autopsy, the incidence of rejection and GVHD was 17% ; rejection and infection, 17% ; and GVHD and infection, 45%. A combination of all three immunologic events was noted in 14%. Conclusions. RPC, combined with DSBMI, and with or without posttransplant immunosuppression, does not prolong survival after total bowel transplantation. Rather, it increases the incidence of death from rejection, GVHD, infection, or a combination of these three immunologic events. According to this preclinical study, RPC and unmodified DSBMI do not improve patient and graft outcome after total bowel transplantation and need to be refined before being applied clinically.

Rejection remains a major barrier to successful bowel transplantation, and immunosuppressive protocols are far from standardized. In 88 nonrelated outbred pigs, we compared the effects of two immunosuppressive regimens-one with FK506, the other with cyclosporine (CsA) and pig antithymocyte globulin (ATG)-on incidence and severity of rejection in the early, critical posttransplant period. Group A (n=14) was nonimmunosuppressed (controls). Group B (n=17) received pig ATG (10 mg/kg/day ×10 days), CsA (3 mg/kg/day), prednisolone (2 mg/kg/day), and azathioprine (2.5 mg/kg/day); predisolone and azathioprine were each reduced by 50% at 8 and 15 days posttransplant. Trough CsA whole-blood concentrations were ≥400 ng/ml for the first 7 days, ≥200 ng/ml thereafter. Group C (n=13) received FK506 (0.2 mg/kg/day) and prednisolone (2 mg/kg/day); prednisolone was reduced by 50% at 8 and 15 days. FK506 whole-blood concentrations were ≥20 ng/ml. All immunosuppression in groups B and C was given intravenously. We performed orthotopic small bowel transplants with systemic venous drainage. Recipient bowel was resected distal to the second portion of the duodenum and proximal to the rectum at transplant; bowel continuity was restored by duodenojejunostomy; ileostomy was created distally to allow access for daily biopsies. We graded interstitial and vascular rejection separately, according to a scoring system (no, mild, moderate, and severe rejection). Rejection-free graft survivals at 7, 14, and 21 days posttransplant were 38%, 19%, and 0% n group A; 93%, 93%, and 62% in group B; and 100%, 91%, and 82% in group C (P

Drug development is a process that has no “short cuts” in spite of a detailed road map for developing safe and efficacious drugs. In this context, efficient drug development does not mean “quicker” or “less costly” but is a process that must maintain sight of safety and efficacy, produces information/data that is needed for “Go-No Go” decisions using rational study designs and utilizes flexible study sequences-not linear, ritualistic design. It is important to achieve the first goal: of an open IND, by use Exploratory Guidance as a tool to facilitate entry into humans when possible (IND-enabling development). It should be kept in mind that phase 1/1a studies, whether in healthy subjects or patients, provides the learning phase of drug development, whereas, as development progresses to patients, the safe and effective use are confirmed and thus provide the basis for marketing approval. All aspects of drug development are not addressed here. The present report primarily addresses early development (phase 1)as in many cases the “Go-NoGo” decisions are defined. An overview of the drug development process , regulatory aspects, and the important IND-enabling development, is presented. Considering that all development begins with evaluations of animal safety/toxicity, specifics germain to the transition from animals to humans are presented in some detail. The importance of clinical pharmacology, pharmacokinetics, and biopharmaceutics receive a more in depth treatment; collectively as these are the pivotal “learning” areas for successful confirmation as development moves to patients. In this context, decision making studies such as proof of concept and several phase 1 scenarios are presented as examples, drug metabolism and drug–drug interactions studies (in vitro and in vivo) are discussed in some detail. As was stated earlier, an all-encompassing review for drug development is not presented. However the text does provide a learning tool for those interested in the studies and decisions obtained to successful early drug development as a prelude to a more comprehensive development culminating with a safe and efficacious drug. Keywords: effective; early; study designs; drug–drug interactions; IND-enabling

Toxicology/safety studies in animals are an integral part of any drug development program, the results of which have a direct impact on both the IND and the NDA. Both bioavailability and bioequivalence concepts are applied to animal toxicology studies to determine and confirm the exposure needs in order to calculate the animal to human safety margins. This article focuses on bioavailability/bioequivalence (BA/BE) studies, IND-targeted nonclinical development, and NDA-targeted nonclinical development. Keywords: biopharmaceutics; safety studies; toxicology studies; preclinical development

Cyclosporine is a marketed immunosuppressive agent and a known substrate for CYP3A. Micafungin is an antifungal agent and a mild inhibitor of CYP3A-mediated metabolism in vitro. The objectives of this study were to evaluate the pharmacokinetics of cyclosporine and micafungin before and with concomitant administration. The pharmacokinetics of single-dose oral cyclosporine (5 mg/kg) were estimated on days 1, 9, and 15 (n = 27). Subjects received micafungin (100 mg/d over 1 hour) on days 7, 9, and 11 through 15. Micafungin pharmacokinetics were estimated on days 7, 9, and 15. Mean apparent oral cyclosporine clearances were estimated to be 645 ± 236 mL/h/kg, 546 ± 101 mL/h/kg (P = .01), and 540 ± 104 mL/h/kg (P = .02) for days 1, 9, and 15, respectively. Micafungin appears to be a mild inhibitor of cyclosporine metabolism.

The effects of tacrolimus (FK506) and malononitrilamides (MNA) (FK778 and FK779) monotherapy and combination therapy were examined in prevention of acute heart and kidney allograft rejection and reversal of ongoing acute heart allograft rejection in the rat.Brown Norway (RT1n)-to-Lewis (RT11) and ACI (RT1a)-to-Lewis (RT11) combinations were used, respectively, for heart and kidney transplantation models. Immunosuppressants were administered orally from day 1 to day 14 for preventing acute rejection and from day 4 to day 34 after transplantation for the reversal of ongoing acute rejection.In the prevention of acute heart rejection model, recipient rats treated with monotherapy of tacrolimus or MNA (FK778, FK779) showed a dose-related prolongation of mean survival time (MST) compared with naive control rats (P0.01). The mean survival time in combination therapy of tacrolimus (FK506) and FK778 indicated that an additive to synergistic interaction was produced when compared with the respective monotherapies (combination index [CI]=0.631-1.022). These results were reproducible with tacrolimus and FK779 combination therapy (CI=0.572-0.846). Furthermore, similar results were also found in the prevention of acute kidney allograft rejection in the rat (CI=0.137-0.516). In the reversal of ongoing acute heart allograft rejection, combination therapy of tacrolimus and FK778 demonstrated a strong synergistic interaction (CI=0.166-0.970) compared with monotherapy of tacrolimus or FK778.Combination therapy of tacrolimus and MNA (FK778, FK779) produces synergistic effects in prevention of acute heart and kidney rejection and reversal of ongoing heart allograft rejection in the rat.

Malononitrilamide FK778, an analogue of leflunomide's active metabolite, is a promising novel small molecule with immunosuppressive and immunomodulatory properties. In this study, we evaluated the ability of combination therapy of FK778 with tacrolimus to inhibit lymphocyte proliferation and to prevent acute allograft rejection.Proliferation assay was used to evaluate the effect of FK778 plus tacrolimus on murine splenocytes, monkey lymphocytes, and human peripheral blood mononuclear cells, after activation with T or B cell-specific mitogens. A rat kidney transplantation model was used to evaluate the ability of FK778 combined with tacrolimus to prolong allograft survival. Median-effect principle and combination index (CI) were used to determine synergism, summation, or antagonism.A total of 58 combinations of FK778 plus tacrolimus were evaluated. Of the combinations tested, 82.8% (24/29) produced additive to synergistic effects in B cells, whereas 79.3% (23/29) produced moderate antagonistic effects in T cells. A concomitant 14-day therapy of FK778 (10 mg/kg/day) and tacrolimus (1 mg/kg/day) synergistically prolonged renal allograft survival to 25.5+/-5.9 days (CI=0.458). However, when addition of FK778 to tacrolimus therapy was delayed to day 7 after transplantation, a strong synergism was obtained (mean survival time=74.9+/-14.8 days, CI0.001).This study demonstrates that the combination of FK778 with tacrolimus in vitro produces synergistic inhibition on B-cell proliferation but not on T cell proliferation in mice, nonhuman primates, and humans. When the addition of FK778 treatment was delayed to day 7 after transplantation, a strong synergism was produced in prolongation of renal allograft survival in the rat.

Malononitrilamide 715 (FK778) is a new class of low-molecular-weight immunosuppressant that is a derivative of the active metabolite of leflunomide, A77 1726. In this study, the authors evaluated the combined effect of FK778 with tacrolimus in prevention of renal allograft rejection in Vervet monkeys.Male Vervet monkeys were obtained from Caribbean Primates Ltd. Donor and recipient monkeys were from different breeding colonies. Eleven groups (nor=4 per group) were involved in this study. FK778 and tacrolimus were administered orally for 60 days according to protocol.Naive controls rejected renal grafts, with a median survival time (MST) of 8.0 days in group 1. When recipient monkeys were treated with tacrolimus 1.0 mg/kg/day in group 2 or FK778 2.5 mg/kg/day in group 3, the MST was 16.0 days (P=0.001) and 11.0 days (P=0.266), respectively. Combination therapy of these two agents at the same doses immediately after transplantation resulted in an MST of 25.0 days (P=0.016) in group 4. When tacrolimus was initiated immediately after transplantation and FK778 treatment was delayed until day 7 after surgery in group 5, recipient survivals were significantly prolonged to 38.0 days (P=0.02). These results were repeatable when FK778 5.0 mg/kg/day (9.0 days, P=0.544 in group 6) was combined with tacrolimus 1.0 mg/kg/day immediately after transplantation (8.0 days, P=0.339) in group 7, or when FK778 was delayed 7 days (60.0 days, P=0.002) in group 8. Furthermore, it was also repeatable when FK778 10 mg/kg/day was combined with tacrolimus 1.0 mg/kg/day with a 7-day delay.A significant prolongation of renal allograft survival was produced when FK778 administration was delayed by 7 days combined with tacrolimus in Vervet monkeys.

Tacrolimus (FK506, Prograf) is a macrolide lactone antibiotic widely used by the oral route for the prophylaxis of organ rejection in patients who have received allogenic liver or kidney transplants. This study investigated the influence of a high- versus a low-fat meal, relative to the fasting state (three treatments total), on the rate and extent of tacrolimus absorption following a single 5 mg oral dose. The protocol employed a three-period, randomized, crossover design employing 5 x 1 mg capsules in 15 healthy male nonsmoking, drug-free volunteers, 20 to 45 years of age, who were within 15% of their ideal body weight. Food had a clinically significant effect in reducing relative bioavailability, as well as slowing absorption, but did not affect terminal exponential half-life (approximately 34 hours). Mean maximum tacrolimus blood concentration (Cmax) values were 25.6, 5.88, and 9.03 ng/mL for the fasting, high-fat, and low-fat treatments, respectively; mean area under the blood concentration-time curve (AUC(0-infinity) values were 272, 181, and 201 (ng/mL)-h, respectively; and mean time of Cmax (tmax) values were 1.37, 6.47, and 3.20 hours, respectively. Differences in parameters between the fasting and each fed treatment were statistically significantly different (p < 0.05). Statistically significant differences also existed in tmax between the two meals. Results also indicated the safety of single 5 mg oral tacrolimus doses administered to healthy volunteers.

Tacrolimus is an immunosuppressant drug used for the prophylaxis of organ rejection in patients who receive allogenic liver or kidney transplants. This study investigated the relationship between tacrolimus blood concentration-time profiles after 3-, 7-, and 10-mg single oral doses were given to 18 healthy, drug-free, nonsmoking, institutionalized male volunteers. The protocol used a single-dose, 3-period, 3-treatment, nonmasked, randomized-block, complete crossover design. The 90% CIs of the ratios of dose-adjusted, mean, logtransformed values of maximum blood concentration, area under the tacrolimus blood concentration-time curve (0 to the last measurable concentration; lower limit of quantitation = 0.5 ng/mL), and area under the blood concentration-time curve (0 to ∞) fell within the range of 125%, indicating dose proportionality for these parameters under experimental conditions. Power to detect a 20% difference for the 3 doses tested was 82.2%, 66.7%, and 72.8%, respectively.

The safety, tolerance, and pharmacokinetics of a small unilamellar liposomal formulation of amphotericin B (AmBisome) administered for empirical antifungal therapy were evaluated for 36 persistently febrile neutropenic adults receiving cancer chemotherapy and bone marrow transplantation. The protocol was an open-label, sequential-dose-escalation, multidose pharmacokinetic study which enrolled a total of 8 to 12 patients in each of the four dosage cohorts. Each cohort received daily doses of either 1.0, 2.5, 5.0, or 7.5 mg of amphotericin B in the form of AmBisome/kg of body weight. The study population consisted of patients between the ages of 13 and 80 years with neutropenia (absolute neutrophil count, 3 ) who were eligible to receive empirical antifungal therapy. Patients were monitored for safety and tolerance by frequent laboratory examinations and the monitoring of infusion-related reactions. Efficacy was assessed by monitoring for the development of invasive fungal infection. The pharmacokinetic parameters of AmBisome were measured as those of amphotericin B by high-performance liquid chromatography. Noncompartmental methods were used to calculate pharmacokinetic parameters. AmBisome administered as a 1-h infusion in this population was well tolerated and was seldom associated with infusion-related toxicity. Infusion-related side effects occurred in 15 (5%) of all 331 infusions, and only two patients (5%) required premedication. Serum creatinine, potassium, and magnesium levels were not significantly changed from baseline in any of the dosage cohorts, and there was no net increase in serum transaminase levels. AmBisome followed a nonlinear dosage relationship that was consistent with reticuloendothelial uptake and redistribution. There were no breakthrough fungal infections during empirical therapy with AmBisome. AmBisome administered to febrile neutropenic patients in this study was well tolerated, was seldom associated with infusion-related toxicity, was characterized by nonlinear saturation kinetics, and was effective in preventing breakthrough fungal infections.

Background: Tacrolimus is a potent immunosuppressant used in organ transplant recipients; an ointment formulation is being developed as a therapeutic agent for atopic dermatitis. Objective: Our purpose was to define the pharmacokinetics and evaluate tacrolimus 0.3% ointment as therapy for moderate to severe atopic dermatitis. Methods: Thirty-nine patients, 5 to 75 years of age, received 14 applications over 8 days. Serial blood samples were collected on days 1 and 8, with predose samples collected on days 2 through 7. Overall response and signs/symptoms were rated daily on days 1 through 11. Incidence of adverse events and laboratory profile were determined. Results: Mean area under the curve (0.9 to 42.5 ng ·hr/ml) was highly variable and appeared to be related to size of application area. No systemic accumulation of tacrolimus was observed. Comparison to historical intravenous data indicates that absolute bioavailability of topical tacrolimus was less than 0.5%. Ninety-five percent of patients showed at least good improvement. All adverse events were transient. Burning was the most common application site adverse event and vasodilatation ("flushing/warmth") was the most common nonapplication site adverse event. No drug-related changes in laboratory profile were observed. Conclusion: The results of this study suggest that tacrolimus 0.3% ointment may be a safe and effective therapy for atopic dermatitis. (J Am Acad Dermatol 1998;38:69-76.)

Epidural administration of clonidine, a partial alpha 2-adrenergic agonist, provides effective relief for patients with intractable cancer pain. However, clonidine's long plasma elimination half-life suggests a potential for plasma accumulation following repeated doses or constant infusion. Adult male and female cancer patients experiencing severe, intractable pain were administered a continuous epidural infusion (30 micrograms/h) of clonidine hydrochloride for 14 days. Plasma clonidine concentrations were determined for 31 patients using a radioimmunoassay with a limit of quantitation of 0.062 ng/mL. Mean (+/- SD) plasma clonidine concentration and calculated total body clearance after 7 days of infusion (respectively, 2.19 +/- 1.17 ng/mL, n = 24; 279 +/- 184 mL/min, n = 27) were comparable to those following 14 days of infusion (2.50 +/- 1.51 ng/mL, n = 19; 272 +/- 163 mL/min, n = 21). Clonidine does not appear to accumulate in the plasma compartment during prolonged (14-day) continuous epidural infusion in cancer patients.

Objective: We assessed rates of withdrawal-related events following abrupt discontinuation and gradual tapering of clobazam during Phase I–III studies. Background Clobazam, a 1,5-benzodiazepine, is under FDA review for LGS. Design/Methods: Potential withdrawal-related AEs following abrupt CLB discontinuation were assessed in 4 multiple-dosage Phase I trials (dosing 8−34 days). These studies employed therapeutic (20 and 40 mg/day) and supratherapeutic dosages (120 and 160 mg/day). These events were also assessed in Phase II/III studies (dosing ≤18 weeks) and their open-label extension (OLE) (≤5 years). In these studies, gradual tapering over 2–3 weeks was conducted. Withdrawal-related AEs included sedation, somnolence, insomnia, irritability, anxiety, and seizures. AEs must have occurred ≥1 day following last clobazam dose and within 30 days after last dose, or must have been deemed withdrawal symptoms by investigators. Results: Phase I participants received steady-state dosages of 20–160 mg/day, with the vast majority receiving ≥40 mg/day and ∼50% receiving ≥120 mg/day (≤4-times maximum daily dosage in Phase II/III). In contrast, LGS patients received ≤40 mg/day during Phase II/III and ≤80 mg/day in the OLE. 87 patients discontinued clobazam during Phase II/III and tapered drug. None reported withdrawal-related AEs. Abrupt discontinuation led to AEs consistent with withdrawal. 93 AEs were reported for 68 of 207 in Phase I studies. Nearly all AEs occurred after abrupt discontinuation of clobazam ≥40 mg/day, with ∼50% occurring at ≥120 mg/day. The majority of AEs following abrupt discontinuation were mild, and included headache, insomnia, anxiety, and tremor. No status epilepticus occurred during withdrawal/tapering. Conclusions: Withdrawal-related AEs did not occur during 2- to 3-week tapering at the conclusion of Phase II/III studies of clobazam. No withdrawal-related AEs have been observed with long-term therapy through 5 years In Phase I, abrupt withdrawal led to generally mild AEs at rates comparable to those observed for other benzodiazepines. Supported by: Lundbeck Inc. Disclosure: Dr. Tolbert has received personal compensation for activities with Lundbeck Research USA, Inc as an employee. Dr. Harris has received research support from Lundbeck Research USA, Inc. Dr. Bekersky has received personal compensation for activities with Lundbeck Research USA, Inc. as an employee. Dr. Owen has received personal compensation for activities with Lundbeck Research USA, Inc.

A sensitive and selective high-performance liquid chromatographic (HPLC) method was developed for the determination of 1,3-di(4-imidazolino-2-methoxyphenoxy)propane (DMP) in rat, dog and human plasma (50-5000 ng/ml) and urine (0.1-10 micrograms/ml). DMP and DMPent (dimethoxyimidizolinopentamidine, the internal standard), are extracted from alkanized plasma with n-butyl chloride-n-butanol (9:1, v/v). The organic phase is dried under nitrogen, reconstituted in mobile phase, and washed with hexane. Separation is achieved by ion-pair chromatography on a Zorbax Rx C8 column with fluorescence detection. The analysis of pooled plasma (80, 400, and 4000 ng/ml) and urine controls (0.3, 1.6, and 8 micrograms/ml) demonstrated excellent precision and accuracy over a three-day period. The recovery of DMP is90% from rat, dog, and human plasma and85% from rat and human urine, and 60-70% from dog urine. The limit of quantitation (LOQ) of the assay is 50 ng/ml in rat, dog and human plasma. Using the high-sensitivity assay, the limit of quantitation was decreased to 5, 2 and 0.6 ng/ml in rat, dog and human plasma, respectively. The LOQ of the assay is 0.1 microgram/ml in rat, dog and human urine. The assay was used to determine plasma and urine concentrations of DMP in pharmacokinetic studies in rat and dog.