John A. Masucci

Allometric scaling of pharmacokinetic parameters in drug discovery: Can human CL, Vss and t1/2 be predicted fromin-vivo rat data?

SummaryIn a drug discovery environment, reasonable go/no-go humanin-vivo pharmacokinetic (PK) decisions must be made in a timely manner with a minimum amount of animalin-vivo orin-vitro data. We have investigated the accuracy of thein-vivo correlation between rat and human for the prediction of the total systemic clearance (CL), the volume of distribution at steady state (Vss), and the half-life (t1/2) using simple allometric scaling techniques. We have shown, using a large diverse set of drugs, that a fixed exponent allometric scaling approach can be used to predict humanin-vivo PK parameters CL, Vss and t1/2 solely from ratin-vivo PK data with acceptable accuracy for making go/no-go decisions in drug discovery. Humanin-vivo PK predictions can be obtained using the simple allometric scaling relationships CLHuman ≈ 40 CLRat (L/hr), Vss Human ≈ 200 Vss Rat (L), and t1/2 Human ≈ 4 t1/2 Rat (hr). The average fold error for human CL predictions for N=176 drugs was 2.25 with 79% of the drugs having a fold error less than 3. The average fold error for human Vss predictions for N=144 drugs was 1.85 with 84% of the drugs having a fold error less than 3. The average fold error for human t1/2 predictions for N=145 drugs was 2.05 with 76% of the drugs having a fold error less than 3. Using these simple allometric relationships, the sorting of drug candidates into a low/medium/high/very high human classification scheme was also possible from rat data. Since these simple allometric relationships between rat and human CL, Vss, and t1/2 are reasonably accurate, easy to remember and simple to calculate, these equations should be useful for making early go/no-goin-vivo human PK decisions for drug discovery candidates.

Rapidly distinguishing reversible and irreversible CYP450 inhibitors by using fluorometric kinetic analyses

SummaryIn this study we have evaluated the reliability of a fluorescence-based method used for rapid identification of irreversible CYP inhibitors (mechanism-based inhibitors). This was accomplished by comparing the time-dependence pattern of IC50 values from fluorometric kinetic measurements. For irreversible CYP inhibitors, IC50 values decreased as incubation proceeded. This was due to progressive inactivation of corresponding enzymes by reactive metabolites generated during the incubation. This change pattern was confirmed using a number of known irreversible CYP inhibitors, including furafylline, midazolam, erythromycin, clarithromycin, oleandomycin, 17α-ethynylestradiol and verapamil. The pattern was different in reversible inhibition, depending upon the compounds tested in the fluorometric kinetic assay. For some compounds, such as clotrimazole, IC50 values remained relatively stable, whereas other compounds, such as miconazole, terfenadine and ketoconazole showed a significant increase with incubation time. Monitoring tested compounds by LC-MS/MS during the incubation confirmed that increases of IC50 were probably caused by the loss of inhibitors, resulting from either metabolic degradation, or non-specific binding to microsomal proteins.

Metabolism and excretion of the antiepileptic/antimigraine drug, topiramate in animals and humans

SummaryThe metabolism and excretion of 2,3:4,5-bis-0-(l-methylethylidene)-β-D-fructopyranose sulfamate (TOPAMAX®, topiramate, TPM) have been investigated in animals and humans. Radiolabeled [14C] TPM was orally administered to mice, rats, rabbits, dogs and humans. Plasma, urine and fecal samples were collected and analyzed. TPM and a total of 12 metabolites were isolated and identified in these samples. Metabolites were formed by hydroxylation at the 7- or 8-methyl of an isopropylidene of TPM followed by rearrangement, hydroxylation at the 10-methyl of the other isopropylidene, hydrolysis at the 2,3-{ie151-1}-isopropylidene, hydrolysis at the 4,5-{ie151-2}-isopropylidene, cleavage at the sulfamate group, glucuronide conjugation and sulfate conjugation. A large percentage of unchanged TPM was recovered in animal and human urine. The most dominant metabolite of TPM in mice, male rats, rabbits and dogs appeared to be formed by the hydrolysis of the 2,3-{ie151-3}-isopropylidene group.

In vitro identification of metabolic pathways and cytochrome P450 enzymes involved in the metabolism of etoperidone

1. In vitro studies have been carried out to investigate the metabolic pathways and identify the hepatic cytochrome P450 (CYP) enzymes involved in etoperidone (Et) metabolism. 2. Ten in vitro metabolites were profiled, quantified and tentatively identified after incubation with human hepatic S9 fractions. Et was metabolized via three metabolic pathways: (A) alkyl hydroxylation to form OH-ethyl-Et (M1); (B) phenyl hydroxylation to form OH-phenyl-Et (M2); and (C) N-dealkylation to form 1-m-chlorophenylpiperazine (mCPP, M8) and triazole propyl aldehyde (M6). Six additional metabolites were formed by further metabolism of M1, M2, M6 and M8. 3. Kinetic studies revealed that all metabolic pathways were monophasic, and the pathway leading to the formation of OH-ethyl-Et was the most efficient at eliminating the drug. On incubation with microsomes expressing individual recombinant CYPs, formation rates of M1-3 and M8 were 10-100-fold greater for CYP3A4 than that for other CYP forms. The formation of these metabolites was markedly inhibited by the CYP3A4-specific inhibitor ketoconazole, whereas other CYP-specific inhibitors did not show significant effects. In addition, the production of M1-3 and M8 was strongly correlated with CYP3A4-mediated testosterone 6 β -hydroxylase activities in 13 different human liver microsome samples. 4. Dealkylation of the major metabolite M1 to form mCPP (M8) was also investigated using microsomes containing recombinant CYP enzymes. The rate of conversion of M1 to mCPP by CYP3A4 was 503.0 ± 3.1 pmole nmole−1 min−1. Metabolism of M1 to M8 by other CYP enzymes was insignificant. In addition, this metabolism in human liver microsomes was extensively inhibited by the CYP3A4 inhibitor ketoconazole, but not by other CYP-specific inhibitors. In addition, conversion of M1 to M8 was highly correlated with CYP3A4-mediated testosterone 6 β -hydroxylase activity. 5. The results strongly suggest that CYP3A4 is the predominant enzyme-metabolizing Et in humans.

The use of the suicide CYP450 inhibitor ABT for distinguishing absorption and metabolism processes in in-vivo pharmacokinetic screens

SummarySince drug candidates with low oral systemic exposure may be due to either or both absorption and metabolism factors, determining what factors limit the oral systemic exposure is not always obvious in a singlein-vivo pharmacokinetic (PK) assay. A rapid ratin-vivo PK screen where the oxidative drug metabolism has been attenuated using the suicide CYP450 inhibitor aminobenzotriazole (ABT) is described. We have shown that the roles of absorption and metabolism for drug candidates with low oral systemic exposure can be determined by comparing the PK parameters of drug candidates orally administered to non-treated and ABT-treated rats. Propranolol, metoprolol and climetidine are used as model drugs. Propranolol and metoprolol have low oral systemic exposures in rats primarily due to metabolism factors while the oral systemic exposure of climetidine is high in rats. For propranolol and metoprolol, large increases in the systemic exposure of these drugs were observed between non-treated and ABT-treated rats. ABT appeared not to increase or decrease significantly the rate and extent of absorption or metabolism of Cimetidine since that oral systemic exposure of non-treated and ABT-treated rats did not significantly change.These experiments suggest that for drug candidates with low systemic exposures in rats an observation of no change in the oral systemic exposure in ABT-treated rats when compared to the non-treated rats imply that absorption (or formulation) factors limit the systemic exposure of the drug while an increase in the systemic exposure in ABT-treated rats imply that metabolism factors limit the systemic exposure. Due to the ease of preparing and interpreting PK data from ABT-treated rats, is suggested that this assay could be used as an alternative toin vivo cannulation assays. Please send reprint requests to: Dr G. W. Caldwell,

Applied Pharmacokinetics in Drug Development

The process of discovering, developing, and marketing new drugs has changed considerably in the last decade; however, the cost associated with this process remains staggeringly high. Although there are many reasons for this high cost, one reason appears to be the continuing high attrition rates of drugs during costly early- and late-stage human clinical trials. To address this problem, drug discovery organizations are striving to rapidly identify high-potential drug candidates and eliminate as early as possible those with inferior potency, poor pharmacokinetic properties, and toxicity problems, so that these deficient drug candidates do not incur the high costs of clinical trials. During the last 5 years, a decision-making go/no-go strategy has been introduced into the drug discovery process, using pharmacokinetic principles to minimize the risks and maximize the benefits of selecting superior drug candidates.Pharmacological deficiencies are related in part to pharmacokinetic properties. To understand this process, a brief review of pharmacokinetic properties including oral bioavailability, half-life, absorption, clearance, and volume of distribution is presented. We examine in vitro — in vivo (human) and/or in vivo (animals) — in vivo (human) correlations for several of these pharmacokinetic properties, followed by a discussion of how this preclinical information is collected and used in drug discovery at the various stages to select drug candidates. Finally, we summarize how these methods are used to make go/no-go decisions in each step of the drug discovery process.

Evaluation of the absorption, excretion and metabolism of [14C] etoperidone in man

1. The absorption, excretion and metabolism of 2-{3-[4-(3-chlorophenyl)-1-piperazinyl]propyl}-4,5-diethyl-2,4-dihydro-3H-1,2,4 hydrochloride (etoperidone HCl) was investigated in six healthy men. Subjects were fasted overnight before receiving a single oral dose of a 100mg solution [14C] etoperidone HCl. 2. Plasma (0-48h), urine (0-120h) and faecal (0-120h) samples were collected. The terminal half-life of the total radioactivity from plasma was 21.7 ± 2.8 h with an apparent clearance of 1.01 ± 0.08 ml min-1. Recoveries of total radioactivity in urine and faeces were 78.8 ± 3.6% and 9.6 ± 4.1% of the dose, respectively. 3. Etoperidone and 21 metabolites were isolated and identified in the plasma, urine and faecal extracts. Unchanged etoperidone accounted for <0.01% of the dose in all excreta samples. Nine metabolites were identified in the plasma extracts and 21 urinary metabolites were identified. Seven faecal metabolites were identified. 4. Five proposed pathways were used to describe the formation of the metabolites: alkyl oxidation, piperazinyl oxidation, N -dealkylation, phenyl hydroxylation and conjugation. Alkyl oxidation of etoperidone resulted in the formation of 2-{3-[4-(3- chlorophenyl)-1-piperazinyl]propyl}-4-ethyl-2,4-dihydro-5-(1-hydroxyethyl)-3H-1 triazole-3-one. Piperazinyl oxidation of this metabolite leads to the formation of its N -oxide. N -dealkylation of the piperazinyl group led to the formation of 1-(3-chlorophenyl) piperazine and triazole propionic acid. Phenyl hydroxylation led to three important metabolites in the urine and faeces.

Evaluation of the Absorption, Excretion, and Metabolism of the Antihypertensive Agent RWJ-26899 in Male and Female CR Wistar Rats and Beagle Dogs

SummaryThe absorption, excretion and metabolism of N-(2,6-dichlorophenyl)-β-[[(1-methylcyclohexyl)methoxy]methyl]-N-(phenylmethyl)-1-pyrrolidineethanamine (RWJ-26899; McN-6497) has been investigated in male and female CR Wistar rats and beagle dogs. Radiolabeled [14C]RWJ-26899 was administered to rats as a single 24 mg/kg suspension dose while the dogs received 15 mg/kg capsules. Plasma (0–36 h; rat and 0–48 h; dog), urine (0–192 h; rat and dog) and fecal (0–192 h; rat and dog) samples were collected and analyzed. There were no significant gender differences observed in the data. The terminal half-life of the total radioactivity for rats from plasma was estimated to be 7.7±0.6 h while for dogs it was 22.9 ± 4.4 h. Recoveries of total radioactivity in urine and feces for rats were 8.7±2.9% and 88.3±10.4% of the dose, respectively. Recoveries of total radioactivity in urine and feces for dogs were 4.1±1.4% and 90.0 ±4.7% of the dose, respectively. RWJ-26899 and a total of nine metabolites were isolated and tentatively identified in rat urine, and fecal extracts. Unchanged RWJ-26899 accounted for approximately 1% of the dose in rat urine and 8% in rat feces. RWJ-26899 and a total of four metabolites were isolated and identified in dog urine, and fecal extracts. Unchanged RWJ-26899 accounted for approximately 1% of the dose in urine and 63% in feces in dog. Five proposed pathways were used to describe the metabolites found in rats: N-oxidation, oxidative N-debenzylation, pyrrolidinyl ring hydroxylation, phenylhydroxylation and methyl or cyclohexyl hydroxylation. Two biotransformation pathways in dogs are proposed: N-oxidation and methyl or cyclohexylring hydroxylation.

Evaluation of the excretion and metabolism of the new analgesic agent RWJ-22757 in male and female CR Wistar rats

The excretion and metabolism of (+/-)-trans-3-(2-bromophenyl)octahydroindolizine hydrochloride (RWJ-22757) have been investigated in male and female CR Wistar rats. Radiolabeled [14C] RWJ-22757 was administered orally to each of the rats as a single 60 mg/kg suspension dose. Plasma (0-48 h), urine (0-168 h) and fecal (0-168 h) samples were collected and analyzed. There were no significant gender differences observed in the data. The estimated elimination half-life of the total radioactivity from plasma was 19 h while the estimated elimination half-life of RWJ-22757 was 15 h. Recoveries of total radioactivity in urine and feces were 58.4+/-5.8 and 42.4+/-6.3%, respectively. RWJ-22757 and a total of 11 metabolites were isolated in rat plasma, urine, and fecal extracts. The structures of four of these metabolites were tentatively identified. Unchanged RWJ-22757 accounted for < 4% of the dose in plasma and urine and 28% in feces; thus, indicating the drug was extensively metabolized and either not absorbed well or biliary excreted. Identified metabolites accounted for > 80% of the total radioactivity contained in the samples. The following pathways were used to describe the formation of the metabolites identified in rats: octahydroindolizine ring oxidation, phenyl hydroxylation, octahydroindolizine ring oxidation followed by ring opening to a carboxylic acid function and octahydroindolizine ring oxidation followed by ring opening and N-methylation.

Evaluation of the excretion, and metabolism of the cardiotonic agent bemoradan in male rats and female beagle dogs

SummaryThe excretion and metabolism of (±) [6-(3,4-dihydro-3-oxo-1,4[2H]-benzoxazine-yl)-2,3,4,5-tetrahydro-5-methylpyridazin-3-one] (bemoradan; RWJ-22867) have been investigated in male Long-Evans rats and female beagle dogs. Radiolabeled [14C] bemoradan was administered to rats as a singkle 1 mg/kg suspension dose while the dogs received 0.1 mg/kg suspension dose. Plasma (0–24 h; rat and dog), urine (0–72 h; rat and dog) and fecal (0–72 h; rat and dog) samples were collected and analyzed. The terminal half-life of the total radioactivity for rats from plasma was estimate to be 4.3±0.1 h while for dogs it was 7.5±1.3 h. Recoveries of total radioactivity in urine and feces for rats were 49.1±2.4% and 51.1±4.9% of th dose, respectively. Recoveries of total radioactivity in urine and feces for dogs were 56.2±12.0% and 42.7 V 9.9% of the dose, respectively. Bemoradan and a total of nine metabolites were isolated and tentatively identified in rat and dog plasma, urine, and fecal extracts. Unchanged bemoradan accounted for approimately <2% of the dose in rat urine and 20% in rat feces. Unchanged bemoradan accounted for approximately 5% of the dose in urine and 16% in feces in dog. Six proposed pathways were used to describe the metabolites found in rats and dogs: pyridazinyl oxidations, methyl hydroxylation, hydration, N-oxidation, dehydration and phase II conjugations.