Enhanced brain delivery with lower hepatic exposure of lazaroid loaded nanostructured lipid carriers developed using a design of experiment approach.

The current study was designed to develop and optimize lazaroid loaded nano-structured lipid carriers (LAZ-NLCs) using design of experiment approach for enhancing lazaroid brain exposure. Response surface plots were used to determine the effects of independent variables (amount of PEGylating agent and liquid lipid) on dependent variables (particle size, zeta potential and encapsulation efficiency), while numerical optimization was used for optimizing LAZ-NLCs composition. The optimal LAZ-NLCs were spherical in shape with measured size of 172.3 ±â€¯3.54 nm, surface charge of -4.54 ±â€¯0.87 mV and encapsulation efficiency of 85.01 ±â€¯2.60%. The optimal LAZ-NLCs were also evaluated for hemolytic potential, storage stability and solid-state properties. The plasma pharmacokinetics along with brain and hepatic distributions of control lazaroid citrate solution and optimal LAZ-NLCs formulation were evaluated in Sprague-Dawley rats after the single bolus intravenous administration. The optimized LAZ-NLCs and the control lazaroid citrate solution had similar plasma pharmacokinetic profiles; however, differential organ bio-distributions were observed. The lazaroid exposure in brain was enhanced by two times with a decreased liver exposure by half for the NLCs group compared to the solution group.

UPLC-MS/MS assay of 21-aminosteroid (lazaroid U74389G) for application in pharmacokinetic study.

Lazaroids are potent inhibitors of lipid peroxidation, both in vitro and in vivo. Additionally, a member of the lazaroid family, U-74389G (LAZ) has been shown to have specific radio-protective and anti-proliferative effects. However, there is no quantitative analytical method developed for measuring the therapeutic levels of LAZ for the aforementioned effects. This article highlights the development and validation of a sensitive UPLC-MS/MS method for the quantification of LAZ, and its subsequent application in pharmacokinetic studies in rats with the lower limit of quantification (LLOQ) of 1.95 ng/mL. LAZ and internal standard diadzein (IS) were separated using ACQUITY UPLC(®) BEH C18 column. Gradient elution was used at a flow rate of 0.45 mL/min with mobile phases consisting of 0.1% formic acid in water and 0.1% formic in acetonitrile. LAZ (m/z 612→260) and IS (m/z 255→199) were detected by electrospray ionization (ESI) using multiple reaction monitoring (MRM) in a positive mode on QTRAP(®) 5500 System. The UPLC-MS/MS method was validated as per the US FDA Guidelines for Bio-analytical Validation. LAZ was extracted from rat plasma (100 µL) using protein precipitation by acetonitrile with mean recovery and matrix factor in range of 47.7-56.1%, and 85.6-89.4%, respectively. The calibration curve for LAZ was linear in the range of 1.95-250 ng/mL. The inter-day and intra-day accuracy and precision values for LLOQ, low, medium, high and very high concentration QC samples were within ±15%. LAZ was tested under different storage conditions, for short-term bench-top stability (1h and 3h at 25°C), long-term stability (1 month at -80°C), freeze-thaw cycle stability (1 cycle and 3 cycles) and stability of processed samples in auto-sampler (24h at 10°C) with stability values within ±15% range of nominal concentrations. The validated UPLC-MS/MS method was further applied to a pharmacokinetic study in rats after a single intravenous dose of LAZ at 5 mg/kg.

Venous irritation, pharmacokinetics, and tissue distribution of tirilazad in rats following intravenous administration of a novel supersaturated submicron lipid emulsion.

PURPOSE: To compare the venous irritation, pharmacokinetics, and tissue distribution of tirilazad in rats after intravenous administration of a submicron lipid emulsion with that of an aqueous solution. METHODS: Venous irritation was determined by microscopic evaluation of injury to the lateral tail veins of rats. Pharmacokinetic parameters were determined by following plasma concentrations of drug. Tissue distribution of [14C]-tirilazad was determined by quantitative whole body autoradiography. RESULTS: Single dose injections of tirilazad as an emulsion at doses ranging from 1.52 mg to 13.5 mg were non-irritating whereas the solution was irritating at a dose of 1.3 mg. The pharmacokinetic parameters were not statistically different between the emulsion and the solution (p > 0.2) at doses of 6 mg/kg/day and 20 mg/kg/day. However, at 65 mg/kg/day dose, a higher AUC(0,6) (4-fold) and lower V(ss), (18-fold) and CL(5-fold) were observed for the lipid emulsion as compared to the solution (p < 0.05). Tissue distribution showed higher initial concentrations (two fold or more) in most tissues for the solution. These values, however, equilibrated by 4 h and AUC(0,4) differences were less than two fold in most tissues. CONCLUSIONS: Formulating tirilazad in the lipid emulsion significantly reduces the venous irritation without changing the pharmacokinetics and tissue distribution at low doses.

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.

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.

Induction of tirilazad clearance by phenytoin.

Tirilazad is a membrane lipid peroxidation inhibitor being studied for the management of subarachnoid hemorrhage; phenytoin is used for seizure prophylaxis in the same disorder. The induction of tirilazad clearance by phenytoin was assessed in 12 volunteers (6 male, 6 female). Subjects received phenytoin orally every 8 h for 7 days (200 mg for nine doses and 100 mg for 13 doses) in one phase of a crossover study. In both study phases, 1.5 mg kg-1 tirilazad mesylate was administered by i.v. infusion every 6 h for 29 doses. Tirilazad mesylate and U-89678 (an active metabolite) in plasma were quantified by HPLC. After the final dose, tirilazad clearance was increased by 91.8% in subjects receiving phenytoin + tirilazad versus tirilazad alone. AUC0-6 for U-89678 after the last tirilazad dose was reduced by 93.1% by concomitant phenytoin. These effects were statistically significant. The time course of induction was consistent with that of phenytoin's effect on the ratio of urinary 6 beta-hydroxycortisol to cortisol, a measure of hepatic CYP3A activity. The results show that phenytoin induces metabolism of tirilazad and U-89678 in healthy subjects and that, under these conditions, tirilazad clearance approaches liver blood flow.

In vitro metabolic transformations of 2,4-dipyrrolidinylpyrimidine: a chemical probe for P450-mediated oxidation of tirilazad mesylate.

1. We have determined that 2,4-dipyrrolidinylpyrimidine (2,4-DPP), used as a model for studies of the metabolism of therapeutic agents containing this moiety, undergoes three characteristic hydroxylations when incubated with male rat liver microsomes. Analysis of microsomal incubates of stable isotope labelled analogues of 2,4-DPP by particle beam-liquid chromatography-mass spectrometry (LC-PB-MS) has shown that the three metabolites are 4-(3-hydroxypyrrolidinyl)-2-(pyrrolidinyl)-pyrimidine (M1), 4-(2-hydroxypyrrolidinyl)-2-(pyrrolidinyl)-pyrimidine (M2) and 2-(2-hydroxypyrrolidinyl)-4-(pyrrolidinyl)-pyrimidine (M3). 2. We determined that enzymes of the cytochrome P450 family are responsible for the in vitro hydroxylations of 2,4-DPP. 3. We observed that in microsomal incubations carried out in the presence of cyanide, a single cyanide adduct is formed implicating an iminium ion intermediate in the oxidation of the 2-pyrrolidine ring. 4. We also determined the intermolecular deuterium isotope effects for the formation of each of the three products. For M1, kH/kD = 14.55 +/- 0.54; for M2, kH/kD = 6.01 +/- 0.65; and for M3, kH/kD = 5.35 +/- 1.18. 5. We interpret these data as suggesting that M2 and M3 are formed by the same mechanism, probably including the formation of an iminium ion, and that M1 is formed by direct hydrogen abstraction.

Pharmacokinetics of tirilazad and U-89678, an active, reduced metabolite, following acute head trauma in adults.

The pharmacokinetics of tirilazad and U-89678 (an active metabolite) were evaluated in 55 adults (48 males, 7 females) with moderate or severe head injury who received 10.0 mg/kg/day tirilazad mesylate for 5 days. Trough plasma samples were obtained daily; serial plasma samples were obtained over one dosing interval on day 5. Plasma tirilazad and U-89678 were quantified by HPLC. Sixty-two percent of the subjects received concomitant anticonvulsants, of which 91% received phenytoin. Plasma tirilazad and U-89678 concentrations in head-injured patients were similar to or lower than those observed in healthy volunteers. Sufficient data were available to calculate pharmacokinetic parameters for day 5 in 26 patients; 11 received no anticonvulsants. The AUC0-6 (are under the concentration-time curve at 0-6 h) for tirilazad mesylate on day 5 in patients receiving anticonvulsants (median = 4972 ng h/mL) differed significantly from that in patients not receiving anticonvulsants (median = 9704 ng h/mL) (p = 0.0051). Similarly, the AUC0-6 of U-89678 in patients receiving anticonvulsants (median = 561 ng h/mL) was significantly different from that in patients who were not (median = 2494 ng h/mL) (p = 0.0016). Comparison of pharmacokinetic data from patients not receiving anticonvulsants to historical data in healthy volunteers suggests that head injury has little effect on tirilazad pharmacokinetics within 5 days of injury. These results suggest that the major factor affecting tirilazad pharmacokinetics in head-injured patients is concomitant use of enzyme-inducing anticonvulsants.

Pharmacokinetics of tirilazad in healthy male subjects at doses above 6 mg/kg/day.

The dose proportionality of tirilazad pharmacokinetics at dosages above 6.0 mg/kg/day were assessed in 18 healthy male volunteers between the ages of 19 and 46 years. Subjects were randomized to receive either 1.5 mg/kg, 3.0 mg/kg, or 4.0 mg/kg tirilazad mesylate every 6 hours for 29 doses (daily doses of 6.0, 12.0, and 16.0 mg/kg/day for 7 days). Each drug dose was administered intravenously over 10 minutes. Plasma tirilazad, U-89678, and U-87999 (active reduced metabolites) were quantified by HPLC. Two subjects in the high dose group withdrew before the end of the study. Following the first dose of tirilazad, dose-corrected pharmacokinetic parameters for all 3 compounds did not differ significantly among dose groups. After the final tirilazad the mean half-life of tirilazad was approximately 80 hours. Mean apparent tirilazad clearance did not differ significantly among groups. Mean U-89678 AUC0-6 following the last tirilazad dose did not differ significantly between the 6.0 and 12.0 mg/kg/day doses, but the value for the 16.0 mg/kg dose was higher than values from both lower doses (p = 0.044 and 0.056, respectively). Similar results were obtained for U-87999. The dose effects observed for the pharmacokinetics of these 2 metabolites may have been a function of intersubject variability. When combined with previous data concerning the dose proportionally of tirilazad pharmacokinetics at doses less than 6.0 mg/kg/day, the data from the present study suggest that the pharmacokinetics of tirilazad are approximately linear over a dosage range of 1.0-16.0 mg/kg/day. Due to the inability to assess the plasma protein binding of tirilazad and its reduced metabolites, the clinical significance of the departure from linearity of the pharmacokinetics of U-89678 and U-87999 cannot be directly assessed. Further study at higher doses will be needed to address this issue.

Pharmacokinetics of tirilazad and U-89678 in ischemic stroke patients receiving a loading regimen and maintenance regimen of 10 mg/kg/day of tirilazad.

The pharmacokinetics of tirilazad mesylate and its active reduced metabolite, U-89678, were evaluated in ischemic stroke patients receiving 2.5 mg/kg tirilazad every 3 hours for the first 12 hours of dosing followed by 2.5 mg/kg every 6 hours for a total of 22 doses (5 days). Trough and serial samples drawn during the 6 hours after administration of the last dose were analyzed for plasma levels of tirilazad and U-89678 by means of high-performance liquid chromatography. Complete concentration-time profiles were available for 20 patients, including 12 men (mean age, 68.0 years) and 8 women (mean age, 75.0 years). Trough concentrations of tirilazad and U-89678 were consistent with the loading regimen used. The mean area under the concentration-time curve from time 0 to 6 hours (AUC0-6) of tirilazad was 8181 +/- 2398 ng.hr/mL in men and 8135 +/- 3671 ng.hr/mL in women. The mean AUC0-6 of U-89678 was 2761 +/- 1834 ng.hr/mL in men and 1477 +/- 903 ng.hr/mL in women. These results show that gender has a modest effect on the pharmacokinetics of U-89678 but little effect on the pharmacokinetics of tirilazad in elderly ischemic stroke patients. These observations are consistent with previous findings in healthy young and elderly subjects.

Biotransformation of tirilazad in human: 1. Cytochrome P450 3A-mediated hydroxylation of tirilazad mesylate in human liver microsomes.

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. Characterization of three major microsomal metabolites of tirilazad by mass spectrometry indicated that hydroxylation had occurred in the pyrrolidine ring(s) and at the 6 beta-position of the steroid domain. A role for CYP3A4 in the formation of the three major metabolites (tirilazad hydroxylase activity) was established in human liver microsomal preparations: 1) Tirilazad hydroxylation was potently inhibited by troleandomycin and ketoconazole, specific inhibitors of CYP3A4. 2) The rates of tirilazad hydroxylation within a population of 14 human livers displayed a 9-fold interindividual variation and a significant correlation (r2 = .95) between tirilazad hydroxylation and testosterone 6 beta-hydroxylation. 3) Kinetic analysis of tirilazad hydroxylase activity in three human livers resulted in an apparent Km of 2.12, 1.68 and 1.66 microM, and Vmax = 0.85, 0.44 and 3.45 (nmol/mg protein/min) for HL14, HL17 and HL21, respectively. In addition, an apparent Km of 2.07 microM was established for tirilazad hydroxylation in a cDNA-expressed CYP3A4 microsomal system. Collectively, these data indicate that the metabolic clearance of tirilazad in humans is catalyzed primarily by CYP3A4 and provide an insight into factors (i.e., age, sex, drug-drug interactions) that modulate the metabolic clearance of tirilazad in vivo.

Biotransformation of tirilazad in human: 2. Effect of ketoconazole on tirilazad clearance and oral bioavailability.

The effect of ketoconazole, a CYP3A inhibitor, on the oral bioavailability of tirilazad mesylate was assessed in 12 healthy subjects, who received the following treatments in a crossover design: a) 10 mg/kg tirilazad mesylate solution orally on the fourth day of a 7-day regimen of 200 mg ketoconazole once daily, b) 10 mg/kg tirilazad mesylate solution orally, c) 2 mg/kg i.v. tirilazad mesylate solution on the fourth day of a 7-day regimen of 200 mg ketoconazole once daily and d) 2mg/kg i.v. tirilazad mesylate solution. Plasma concentrations of tirilazad mesylate and its active reduced metabolites (U-89678 and U-87999) were measured by high-performance liquid chromatography. Urinary ratios of 6 beta-hydroxycortisol to cortisol (6 beta-OHC/C) were measured as an index of hepatic CYP3A activity. Ketoconazole increased mean tirilazad mesylate area under the curve (AUC) values by 67% and 309% for i.v. and oral administration, respectively. Mean AUC values for U-89678 were increased 472% and 720% by ketoconazole coadministration with i.v. and oral tirilazad, respectively, whereas increases of > 10-fold in mean U-87999 AUC values were observed. These differences were statistically significant. These results indicate that ketoconazole inhibits the metabolism of these three compounds, which suggests that all of the compounds are substrates for CYP3A. Urinary 6 beta-OHC/C ratios did not reflect this level of effect of ketoconazole on CYP3A; this probe may not be useful for assessing the effect of CYP3A inhibitors. The absolute bioavailability of oral tirilazad was 8.7 +/- 4.8%; ketoconazole increased the bioavailability to 20.9 +/- 6.5%. Ketoconazole increased tirilazad mesylate bioavailability by decreasing the first-pass liver and gut wall metabolism of tirilazad mesylate to similar degrees.

Comparison of the pharmacokinetics of tirilazad mesylate in healthy volunteers and stable subjects with mild liver cirrhosis.

OBJECTIVE: The pharmacokinetics of tirilazad mesylate and an active reduced metabolite, U89678, were studied in 7 volunteers with mild cirrhosis of the liver, and seven age, sex, weight and smoking status matched healthy normal volunteers. Subjects received a single intravenous infusion of 2.0 mg.kg-1 tirilazad mesylate over 10 min. RESULTS: Mean tirilazad AUCzero-infinity was 8.83 mumol h.l-1 and 18.6 mumol h.l-1 in healthy volunteers and cirrhotic subjects, respectively. Mean tirilazad clearance in cirrhotics (12.7 l.h-1) was approximately 2.1 fold lower than in healthy volunteers (27.8 l.h-1). The differences were statistically significant. Mean U-89678 AUCzero-infinity in cirrhotic subjects (3.88 mumol h.l-1) was 2.5 fold higher than in healthy controls (1.53 mumol h.l-1), but the difference was marginally significant. CONCLUSION: These results indicate that clearance of both tirilazad mesylate and U89678 is decreased in subjects with hepatic impairment. This observation may be attributed either to decreases in liver blood flow and/or intrinsic clearance. The results of this study thus suggest that increased monitoring and or a reduction in tirilazad dosing may be necessary in patients with hepatic impairment.

Gender does not affect the degree of induction of tirilazad clearance by phenobarbital.

OBJECTIVE: Tirilazad mesylate is a membrane lipid peroxidation inhibitor being evaluated for the treatment of patients with subarachnoid haemorrhage (SAH); phenobarbital may be administered to these patients for seizure prophylaxis. Therefore, the effect of phenobarbital on tirilazad mesylate pharmacokinetics was assessed in 15 healthy volunteers (7M, 8F). METHODS: Subjects received 100 mg phenobarbital orally daily for 8 days in one phase of a two-way crossover study. In both phases, 1.5 mg.kg-1 tirilazed mesylate was administered (as a 10 minute IV infusion) every 6 hours for 29 doses. Three weeks separated study phases. Tirilazad mesylate and U-89678 (an active metabolite) in plasma were quantified by HPLC. RESULTS: Phenobarbital had no effect on the first dose pharmacokinetics of tirilazad or U-89678. After the final dose, clearance for tirilazad was increased 25% in males and 29% in females receiving phenobarbital + tirilazad versus tirilazad mesylate alone. These differences were statistically significant, and the degree of induction was not significantly different between genders. AUC(zero)-6 for U-89678 after the last tirilazad mesylate dose was reduced 51% in males and 69% in females. The decreases were statistically significant, and there was no gender by treatment interaction. CONCLUSION: The results show that phenobarbital induces metabolism of tirilazad and U-89678 similarly in both men and women. Lower levels of tirilazad and U-89678 in SAH patients receiving phenobarbital may adversely impact clinical response.

Lazaroids. CNS pharmacology and current research.

The 21-aminosteroids (lazaroids) are inhibitors of lipid membrane peroxidation and appear to function as oxygen free radical scavengers. The therapeutic potential of the lazaroid tirilazad mesylate has been extensively studied in several CNS disorders. Tirilazad and related compounds have been found to be highly beneficial in spinal cord trauma. Spinal cord injury studies utilising tirilazad are currently underway to determine the optimal combination of medications. Tirilazad has also been found to be beneficial in experimental head injury models, however current clinical studies have failed to confirm this efficacy, due in part to difficulties in obtaining therapeutic drug concentrations. Clinical studies using tirilazad in subarachnoid haemorrhage have been more promising. It has been shown to be beneficial in terms of reducing vasospasm and cerebral infarction associated with subarachnoid haemorrhage, and has now been approved in several European countries in this indication. Results from US studies are expected shortly. Finally, tirilazad has also been extensively tested in a variety of stroke models. Although it appears to be highly beneficial in experimental models, the clinical studies to date have failed to confirm this efficacy. Again, this failure appears to be due largely to inadequate drug concentrations having so far been tested.

In vitro metabolism of tirilazad mesylate in male and female rats. Contribution of cytochrome P4502C11 and delta 4-5 alpha-reductase.

Tirilazad mesylate, a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage and head injury. In rat, tirilazad seems to be highly extracted and is cleared almost exclusively via hepatic elimination. The biotransformation of tirilazad has been investigated in liver microsomal preparations from adult male and female Sprague-Dawley rats. Tirilazad metabolism in male rat liver microsomes resulted in the formation of two primary metabolites: M1 and M2. In incubations with female rat liver microsomes, M2 was the only primary metabolite detected. Structural characterization of M1 and M2 by mass spectrometry demonstrated that M2 was formed by reduction of the delta 4-double bond in the steroid A-ring, whereas M1 arose from oxidative desaturation of one pyrrolidine ring. Further structural analysis of M2 by proton NMR demonstrated that reduction at C-5 had occurred by addition of hydrogen in the alpha-configuration. Using metabolic probes and antibodies specific to individual hepatic microsomal enzymes, CYP2C11 and 3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase (5 alpha-reductase) were identified as responsible for the formation of M1 and M2, respectively. The formation of M1 was inhibited by testosterone, nicotine, cimetidine, and anti-CYP2C11 IgG. The formation of M2 was inhibited by finasteride, a potent inhibitor of 5 alpha-reductase. Kinetic analysis of CYP2C11-mediated M1 formation in male rat liver microsomal incubations revealed that M1 formation occurred through a low-affinity/low-capacity process (KM = 16.67 microM, Vmax = 0.978 nmol/mg microsomal protein/min); the formation of M2 was mediated by 5 alpha-reductase in a high-affinity/low-capacity process (KM = 3.07 microM, Vmax = 1.06 nmol/mg microsomal protein/min). In contrast, the formation of M2 in female rat liver microsomes was mediated by 5 alpha-reductase in a high-affinity/high-capacity process (KM = 2.72 microM, Vmax = 4.11 nmol/mg microsomal protein/min). Comparison of calculated intrinsic formation clearances (Vmax/KM) for M1 and M2 indicated that the female rat possessed a greater in vitro metabolic capacity for tirilazad biotransformation than the male rat. Therefore, the clearance of tirilazad mesylate in the rat is mediated primarily by rat liver 5 alpha-reductase, and the capacity in the female rat is 5-fold the capacity in the male. These observations correlate with documented differences in 5 alpha-reductase activity and predict a gender difference in tirilazad hepatic clearance in vivo.

The effect of phenytoin on the pharmacokinetics of tirilazad mesylate in healthy male volunteers.

The pharmacokinetic interaction between phenytoin and tirilazad was studied in 12 healthy men who received 200 mg phenytoin orally every 8 hours for 11 doses and 100 mg for the remaining 5 doses in one period of a two-way crossover study. In both periods, 1.5 mg/kg tirilazad mesylate was administered (as 10-minute intravenous infusions) every 6 hours for 21 doses (5 days). Plasma tirilazad mesylate and U-89678 (an active metabolite) were quantified by HPLC. After dose 21, area under the plasma concentration-time curve [AUC(0-6)] for tirilazad mesylate was significantly lower (p = 0.0061) after phenytoin treatment (3029 +/- 982 ng.hr/ml) than after tirilazad mesylate alone (4647 +/- 1562 ng.hr/ml). AUC(0-6) for U-89,678 after dose 21 was reduced from 1485 +/- 1173 ng.hr/ml after tirilazad mesylate alone to 195 +/- 223 ng.hr/ml after phenytoin coadministration. U-89678 normally accumulates during multiple dosing, but mean U-89678 trough concentrations decreased after 24 hours during tirilazad and phenytoin coadministration. No clinically significant interactions of tirilazad mesylate and phenytoin for medical events, vital signs, or laboratory parameters were identified. These results suggest that phenytoin rapidly induces tirilazad mesylate metabolism; it may also induce the metabolism of U-89678 or shunt tirilazad mesylate metabolism through other pathways.

Lack of a pharmacokinetic/pharmacodynamic interaction between nimodipine and tirilazad mesylate in healthy volunteers.

The potential interaction between tirilazad mesylate, a membrane lipid peroxidation inhibitor, and nimodipine, a calcium-channel antagonist, was assessed in 12 healthy male volunteers. Subjects received 60 mg nimodipine orally, 2.0 mg/kg tirilazad mesylate as a 10-minute intravenous infusion, and a combination of the two treatments according to a balanced 3-way crossover design. No significant effects of nimodipine on tirilazad mesylate pharmacokinetic parameters were observed (P > .05). Values for tirilazad mesylate clearance (34.9 +/- 8.96 L/hr) and half-life (29 +/- 7.83 hr) were consistent with previous studies. Nimodipine pharmacokinetic parameters exhibited substantial variability, and mean AUC was approximately 25% below the range of previously published values. However, no significant differences in nimodipine pharmacokinetics were observed between treatments. Nimodipine administration increased heart rate slightly without a change in blood pressure, which was not observed after tirilazad administration and was not altered when tirilazad and nimodipine were coadministered. Thus, no significant interaction between tirilazad mesylate and nimodipine is detectable after single-dose administration.