Challenges and Opportunities with Non-CYP Enzymes Aldehyde Oxidase, Carboxylesterase, and UDP-Glucuronosyltransferase: Focus on Reaction Phenotyping and Prediction of Human Clearance.

Over the years, significant progress has been made in reducing metabolic instability due to cytochrome P450-mediated oxidation. High-throughput metabolic stability screening has enabled the advancement of compounds with little to no oxidative metabolism. Furthermore, high lipophilicity and low aqueous solubility of presently pursued chemotypes reduces the probability of renal excretion. As such, these low microsomal turnover compounds are often substrates for non-CYP-mediated metabolism. UGTs, esterases, and aldehyde oxidase are major enzymes involved in catalyzing such metabolism. Hepatocytes provide an excellent tool to identify such pathways including elucidation of major metabolites. To predict human PK parameters for P450-mediated metabolism, in vitro-in vivo extrapolation using hepatic microsomes, hepatocytes, and intestinal microsomes has been actively investigated. However, such methods have not been sufficiently evaluated for non-P450 enzymes. In addition to the involvement of the liver, extrahepatic enzymes (intestine, kidney, lung) are also likely to contribute to these pathways. While there has been considerable progress in predicting metabolic pathways and clearance primarily mediated by the liver, progress in characterizing extrahepatic metabolism and prediction of clearance has been slow. Well-characterized in vitro systems or in vivo animal models to assess drug-drug interaction potential and intersubject variability due to polymorphism are not available. Here we focus on the utility of appropriate in vitro studies to characterize non-CYP-mediated metabolism and to understand the enzymes involved followed by pharmacokinetic studies in the appropriately characterized surrogate species. The review will highlight progress made in establishing in vitro-in vivo correlation, predicting human clearance and avoiding costly clinical failures when non-CYP-mediated metabolic pathways are predominant.

Integrated Pharmacokinetic/Pharmacodynamic Analysis for Determining the Minimal Anticipated Biological Effect Level of a Novel Anti-CD28 Receptor Antagonist BMS-931699.

BMS-931699 (lulizumab pegol), a domain antibody (dAb) conjugated with 40-kDa branched polyethylene glycol, is a human anti-CD28 receptor antagonist under development for the treatment of inflammatory and autoimmune diseases. In the present work, the minimal anticipated biologic effect level (MABEL) was determined for BMS-931699 by integrating all the available preclinical data. The relevance of the in vitro mixed lymphocyte reaction (MLR) assay to a whole blood CD28 receptor occupancy (RO) assessment, as well as the relationship between the CD28 RO and the inhibition of T-cell-dependent antibody response to keyhole limpet hemocyanin in vivo, was demonstrated through an integrated pharmacokinetic/pharmacodynamic analysis using anti-hCD28 dAb-001 (differing from BMS-931699 by two additional amino acids at the N-terminus) and a mouse surrogate. Based on this analysis, the EC10 value (0.32 nM) from the human MLR assay and the human plasma volume (0.04 l/kg) were employed to calculate the MABEL (0.01 mg) of BMS-931699 in humans, with a CD28 RO predicted to be ≤10%. The estimated MABEL dose was threefold higher than the value derived from the binding constant and twofold less than the MABEL converted from animal efficacy studies based on the body surface area. Furthermore, it was 2900-fold lower than the human equivalent dose derived from the no observed adverse effect level in monkeys (15 mg/kg/week for 5 doses, intravenous dosing) with a 10-fold safety factor applied. Therefore, the MABEL dose represented a sound approach to mitigate any potential risk in targeting CD28 and was successfully used as the first-in-human starting dose for BMS-931699.

Discovery of ((4-(5-(Cyclopropylcarbamoyl)-2-methylphenylamino)-5-methylpyrrolo[1,2-f][1,2,4]triazine-6-carbonyl)(propyl)carbamoyloxy)methyl-2-(4-(phosphonooxy)phenyl)acetate (BMS-751324), a Clinical Prodrug of p38α MAP Kinase Inhibitor.

In search for prodrugs to address the issue of pH-dependent solubility and exposure associated with 1 (BMS-582949), a previously disclosed phase II clinical p38α MAP kinase inhibitor, a structurally novel clinical prodrug, 2 (BMS-751324), featuring a carbamoylmethylene linked promoiety containing hydroxyphenyl acetic acid (HPA) derived ester and phosphate functionalities, was identified. Prodrug 2 was not only stable but also water-soluble under both acidic and neutral conditions. It was effectively bioconverted into parent drug 1 in vivo by alkaline phosphatase and esterase in a stepwise manner, providing higher exposure of 1 compared to its direct administration, especially within higher dose ranges. In a rat LPS-induced TNFα pharmacodynamic model and a rat adjuvant arthritis model, 2 demonstrated similar efficacy to 1. Most importantly, it was shown in clinical studies that prodrug 2 was indeed effective in addressing the pH-dependent absorption issue associated with 1.

Role of hepatic blood flow and metabolism in the pharmacokinetics of ten drugs in lean, aged and obese rats.

1. The effect of age and obesity on the pharmacokinetics (PK), hepatic blood flow (HBF) and liver metabolism of 10 compounds was determined in rats. The animals fed a high-fat diet were defined as the diet-induced obese (DIO) group, while the animals that were aged similar to the DIO rats but not fed with high-fat diet were called the age-matched (AM) group. 2. The clearance (CL) values of high CL compounds (CL > 50 mL/min/kg, namely propranolol, diazepam, phenytoin, ethinylestradiol, lorcaserin and fenfluramine) decreased significantly (1.5- to 6-fold) in DIO and AM rats as compared to lean rats, while there was no clear trend for change in CL for the low-to-moderate CL compounds (CL < 50 mL/min/kg, namely atenolol, chlorzoxazone, vancomycin and sibutramine). Hepatocytes incubations revealed a change in half life (t1/2) only for phenytoin. The body weight normalized liver weights and HBF of AM and DIO rats were found to be 2- to 3-fold lower than in lean rats. 3. Our findings suggest that age, and diet to a lesser extent, can reduce HBF and body normalized liver weights and, hence, also reduce CL values for high CL compounds in rats.

Estimation of the unbound brain concentration of P-glycoprotein substrates or nonsubstrates by a serial cerebrospinal fluid sampling technique in rats.

The unbound concentration in plasma drives the transport of the drug into the brain, and the unbound drug concentration in the central nervous system (CNS) drives the interaction with the target eliciting the pharmacological effect. Delivery of the drug to the CNS is a challenge because of the unique neurovascular unit, which restricts the passage of drugs into the brain. The efflux transporters [especially P-glycoprotein (P-gp)] present at the blood-brain barrier (BBB) act as one of the major detractors for keeping drugs outside the CNS. The cerebrospinal fluid (CSF) drug concentration has been used as a surrogate for unbound brain concentrations and has proven to be a good indicator to relate to CNS activity. Herein, we have established a serial CSF sampling technique in rats, which allowed CSF sampling from a single animal and reduced the number of animals required, as well as the interanimal variance associated with a composite/terminal study design. Concentrations in the CSF sampled from the cisterna magna serially from the same rat were compared with the concentrations obtained from discrete CSF sampling and with brain concentrations. The serial CSF sampling technique was also authenticated by ensuring no change in the barrier without any indication of damage caused by the repeated puncture of cisterna magna. This technique was corroborated using three passively permeable compounds (carbamazepine, theophylline, and propranolol), three P-gp substrates (quinidine, verapamil, and digoxin), and one l-amino acid uptake transporter substrate (gabapentin). The P-gp substrates were also used in separate studies with the P-gp inhibitor elacridar to assess the effect on CSF concentration versus brain concentration on P-gp inhibition. The CSF concentration and unbound brain concentration were comparable (within 3-fold) for all compounds, including P-gp substrates even in the presence of elacridar. Therefore, this technique can prove to be beneficial for predicting the unbound drug concentrations in the brain from the CSF concentrations and reduce the cost incurred in preclinical animal models. Chemical inhibition by elacridar and prediction of the brain unbound concentrations from the serial CSF sampling of P-gp substrates in the rat may be an attractive alternative to the use of genetically knocked out rodents.

Synthesis and evaluation of carbamoylmethylene linked prodrugs of BMS-582949, a clinical p38α inhibitor.

A series of carbamoylmethylene linked prodrugs of 1 (BMS-582949), a clinical p38α inhibitor, were synthesized and evaluated. Though the phosphoryloxymethylene carbamates (3, 4, and 5) and α-aminoacyloxymethylene carbamates (22, 23, and 26) were found unstable at neutral pH values, fumaric acid derived acyloxymethylene carbamates (2, 28, and 31) were highly stable under both acidic and neutral conditions. Prodrugs 2 and 31 were also highly soluble at both acidic and neutral pH values. At a solution dose of 14.2mpk (equivalent to 10mpk of 1), 2 gave essentially the same exposure of 1 compared to dosing 10mpk of 1 itself. At a suspension dose of 142mpk (equivalent to 100mpk of 1), 2 demonstrated that it could overcome the solubility issue associated with 1 and provide a much higher exposure of 1. To our knowledge, the unique type of prodrugs like 2, 28, and 31 was not reported in the past and could represent a novel prodrug approach for secondary amides, a class of molecules frequently identified as drug candidates.

Impact of nonlinear midazolam pharmacokinetics on the magnitude of the midazolam-ketoconazole interaction in rats.

Numerous groups have described the rat as an in vivo model for the assessment and prediction of drug-drug interactions (DDIs) in humans involving the inhibition of cytochrome P450 3A forms. Even for a well-established substrate-inhibitor pair like midazolam-ketoconazole, however, the magnitude of the DDI in rats (e.g. 1.5- to 5-fold) does not relate to what is observed clinically (e.g. 5- to 16-fold). Because nonlinear substrate pharmacokinetics (PK) may result in a weaker interaction, it was hypothesized that the lower magnitude of interaction observed in rats was due to the saturation of metabolic pathway(s) of midazolam at the doses used (10-20 mg/kg). Therefore, the inhibitory effects of ketoconazole were reevaluated at lower oral (1 and 5 mg/kg) and intravenous (IV) (1 mg/kg) doses of midazolam. In support of the hypothesis, oral exposure at 5 mg/kg dose of midazolam was 18-fold higher compared to that at 1 mg/kg. Furthermore, when the interaction was investigated at the lower midazolam dose (1 mg/kg), ketoconazole increased the IV and oral exposure of midazolam by 7-fold and 11-fold, respectively. A weaker DDI (1.5- to 1.8-fold) was observed at the higher oral midazolam dose. Collectively, these results suggest that the lower reported interaction in rats is likely due to saturation of midazolam clearance at the doses used. Therefore, when the rat is used as a DDI model to screen and differentiate compounds, or predict CYP3A inhibition in humans, it is important to use low doses of midazolam and ensure linear PK.

Discovery of pyrrolo[2,1-f][1,2,4]triazine C6-ketones as potent, orally active p38α MAP kinase inhibitors.

Pyrrolo[2,1-f][1,2,4]triazine based inhibitors of p38α have been prepared exploring functional group modifications at the C6 position. Incorporation of aryl and heteroaryl ketones at this position led to potent inhibitors with efficacy in in vivo models of acute and chronic inflammation.

Discovery of 4-(5-(cyclopropylcarbamoyl)-2-methylphenylamino)-5-methyl-N-propylpyrrolo[1,2-f][1,2,4]triazine-6-carboxamide (BMS-582949), a clinical p38α MAP kinase inhibitor for the treatment of inflammatory diseases.

The discovery and characterization of 7k (BMS-582949), a highly selective p38α MAP kinase inhibitor that is currently in phase II clinical trials for the treatment of rheumatoid arthritis, is described. A key to the discovery was the rational substitution of N-cyclopropyl for N-methoxy in 1a, a previously reported clinical candidate p38α inhibitor. Unlike alkyl and other cycloalkyls, the sp(2) character of the cyclopropyl group can confer improved H-bonding characteristics to the directly substituted amide NH. Inhibitor 7k is slightly less active than 1a in the p38α enzymatic assay but displays a superior pharmacokinetic profile and, as such, was more effective in both the acute murine model of inflammation and pseudoestablished rat AA model. The binding mode of 7k with p38α was confirmed by X-ray crystallographic analysis.

Examination of CYP3A and P-glycoprotein-mediated drug-drug interactions using animal models.

With the advent of polytherapy for cancer treatment it has become prudent to minimize, as much as possible, the potential for drug-drug interactions (DDI). Toward this end, the metabolic and transporter pathways involved in the disposition of a drug candidate (phenotyping) and potential for inhibition and induction of drug-metabolizing enzymes and transporters are evaluated in vitro. Such in vitro human data can be made available prior to human dosing and enable in vitro to in vivo-based predictions of clinical outcomes. Despite some success, however, in vitro systems are not dynamic and sometimes fail to predict drug-drug interactions for a variety of reasons. In comparison, relatively less effort has been made to evaluate predictions based on data derived from in vivo animal models. This chapter will attempt to summarize different examples from the literature where animal models have been used to predict cytochrome P450 3A (CYP3A)- and P-glycoprotein-based DDI. When employing data from animal models one needs to be aware of species differences in enzyme- and transporter-activity leading to differences in pharmacokinetics, clearance pathways as well as species differences in selectivity and affinity of probe substrates and inhibitors. Because of these differences, in vivo animal studies alone, cannot be predictive of human DDI. Despite these caveats, the information obtained from validated in vivo animal models may prove useful when used in conjunction with in vitro-in vivo extrapolation methods. Such an integrated data set can be used to select drug candidates with a reduced DDI potential.

Preclinical pharmacokinetics and in vitro metabolism of brivanib (BMS-540215), a potent VEGFR2 inhibitor and its alanine ester prodrug brivanib alaninate.

PURPOSE: Brivanib alaninate is a prodrug of brivanib (BMS-540215), a potent oral VEGFR-2 inhibitor and is currently in development for the treatment of hepatocellular and colon carcinomas. In vitro and in vivo studies were conducted to characterize the preclinical pharmacokinetics and disposition of brivanib and brivanib alaninate, and antitumor efficacy in mice bearing human xenografts. METHODS: In vitro studies were conducted in liver and intestinal fractions, plasma and Caco-2 cells to assess the metabolic stability. Pharmacokinetics of brivanib were determined in preclinical species after administration of single intravenous or oral doses of both brivanib and brivanib alaninate. The antitumor efficacy was assessed at equimolar doses in nude mice bearing human tumor xenografts. Human efficacious dose was predicted based on projected human pharmacokinetic parameters and exposure at efficacious doses in the mouse efficacy models. RESULTS: In vitro and in vivo studies indicated that brivanib alaninate was efficiently converted to brivanib. Brivanib showed good brain penetration in rats consistent with its high intrinsic permeability and lack of active efflux in Caco-2 cells. The oral bioavailability of brivanib varied among species (22-88%) and showed dissolution rate-limited absorption even when combined with organic co-solvents. Administration of brivanib as brivanib alaninate allowed completely aqueous vehicles, and an improvement in the oral bioavailability (55-97%) was observed. The clearance of brivanib in humans is anticipated to be low to intermediate (hepatic extraction ratio < 0.7), while its volume of distribution is expected to be high. The minimum efficacious dose of brivanib alaninate was determined to be 60 mg/kg per day. CONCLUSIONS: Brivanib alaninate is rapidly and efficiently converted to the parent, brivanib, as demonstrated both in vitro and in vivo and offers an excellent mode to deliver brivanib orally.

Novel tricyclic inhibitors of IkappaB kinase.

The design and synthesis of a novel series of oxazole-, thiazole-, and imidazole-based inhibitors of IkappaB kinase (IKK) are reported. Biological activity was improved compared to the pyrazolopurine lead, and the expedient synthesis of the new tricyclic systems allowed for efficient exploration of structure-activity relationships. This, combined with an iterative rat cassette dosing strategy, was used to identify compounds with improved pharmacokinetic (PK) profiles to advance for in vivo evaluation.

The discovery of (R)-2-(sec-butylamino)-N-(2-methyl-5-(methylcarbamoyl)phenyl) thiazole-5-carboxamide (BMS-640994)-A potent and efficacious p38alpha MAP kinase inhibitor.

A novel structural class of p38alpha MAP kinase inhibitors has been identified via iterative SAR studies of a focused deck screen hit. Optimization of the lead series generated 6e, BMS-640994, a potent and selective p38alpha inhibitor that is orally efficacious in rodent models of acute and chronic inflammation.

Benzothiazole based inhibitors of p38alpha MAP kinase.

Rational design, synthesis, and SAR studies of a novel class of benzothiazole based inhibitors of p38alpha MAP kinase are described. The issue of metabolic instability associated with vicinal phenyl, benzo[d]thiazol-6-yl oxazoles/imidazoles was addressed by the replacement of the central oxazole or imidazole ring with an aminopyrazole system. The proposed binding mode of this new class of p38alpha inhibitors was confirmed by X-ray crystallographic studies of a representative inhibitor (6a) bound to the p38alpha enzyme.

Design, synthesis, and anti-inflammatory properties of orally active 4-(phenylamino)-pyrrolo[2,1-f][1,2,4]triazine p38alpha mitogen-activated protein kinase inhibitors.

A novel structural class of p38 mitogen-activated protein (MAP) kinase inhibitors consisting of substituted 4-(phenylamino)-pyrrolo[2,1- f][1,2,4]triazines has been discovered. An initial subdeck screen revealed that the oxindole-pyrrolo[2,1- f][1,2,4]triazine lead 2a displayed potent enzyme inhibition (IC 50 60 nM) and was active in a cell-based TNFalpha biosynthesis inhibition assay (IC 50 210 nM). Replacement of the C4 oxindole with 2-methyl-5- N-methoxybenzamide aniline 9 gave a compound with superior p38 kinase inhibition (IC 50 10 nM) and moderately improved functional inhibition in THP-1 cells. Further replacement of the C6 ester of the pyrrolo[2,1- f][1,2,4]triazine with amides afforded compounds with increased potency, excellent oral bioavailability, and robust efficacy in a murine model of acute inflammation (murine LPS-TNFalpha). In rodent disease models of chronic inflammation, multiple compounds demonstrated significant inhibition of disease progression leading to the advancement of 2 compounds 11b and 11j into further preclinical and toxicological studies.

Preclinical pharmacokinetics and in vitro metabolism of dasatinib (BMS-354825): a potent oral multi-targeted kinase inhibitor against SRC and BCR-ABL.

PURPOSE: Dasatinib (BMS-354825), a potent oral multi-targeted kinase inhibitor against SRC and BCR-ABL, has recently been approved for the treatment of chronic myelogenous leukaemia (CML) in imatinib-acquired resistance and intolerance. In vitro and in vivo studies were conducted to characterize the pharmacokinetics and metabolism of dasatinib in mouse, rat, dog, and monkey. Possible mechanisms contributing to the incomplete oral bioavailability of dasatinib in animals were investigated. METHODS: Metabolic stability of dasatinib was measured after incubation with liver microsomes (either NADPH- or UDPGA-fortified) and isolated hepatocytes obtained from mouse, rat, dog, monkey, and human. In all cases, substrate depletion over time was measured, and appropriate scaling factors were used to predict in vivo clearance. Pharmacokinetics of dasatinib were determined in mice, rats, dogs, and monkeys after administration of single intravenous or oral doses. In addition, the routes of excretion were investigated after administration of dasatinib to bile duct cannulated (BDC) rats. Absorption and first-pass metabolism were evaluated as possible reasons for the incomplete oral bioavailability using various in vitro and in vivo models like Caco-2 cells, P-glycoprotein (P-gp) knockout mice, and intra-portal dosing in rats. RESULTS: In vivo systemic plasma clearance values of dasatinib were 62, 26, 25, and 34 ml/min/kg in mouse, rat, dog, and monkey, respectively. Scaling of in vitro hepatocyte and liver microsomal data gave reasonably good predictions of in vivo clearances across all species. Percent distribution in blood cells ranged from 43% in mouse to 57% in dog. Dasatinib showed high volumes of distribution (>3 l/kg) and high serum protein binding values (>90%) in all four species tested. Oral bioavailability of dasatinib ranged from 14% in the mouse to 34% in the dog. In rats, bioavailability after an intraportal dose was comparable to that after intra-arterial administration. In BDC rats, less than 15% of an intravenous dose was excreted unchanged in urine, bile, and the gastrointestinal tract, suggesting that dasatinib is cleared primarily via metabolism. Dasatinib has high intrinsic permeability in Caco-2 cells, however, the efflux ratio was approximately two-fold indicating that it may be a substrate for an intestinal efflux transporter. However, in vivo studies in P-gp knockout mice versus wild-type mice showed no difference in the amount of dasatinib remaining unabsorbed in the gastrointestinal tract, suggesting that P-gp may not be responsible for the incomplete bioavailability. CONCLUSIONS: Dasatinib shows intermediate clearance in mouse, rat, dog, and monkey, and distributes extensively in those species. Oxidative metabolism appears to be the predominant clearance pathway. The incomplete oral bioavailability may be due to both incomplete absorption and high first-pass metabolism. However, the efflux transporter, P-glycoprotein does not appear to be limiting oral absorption.

Effect of commonly used organic solvents on the kinetics of cytochrome P450 2B6- and 2C8-dependent activity in human liver microsomes.

The effect of common organic solvents on the activities of various human cytochromes P450 has been reported. However, very little is known about their influence on CYP2B6 and CYP2C8 enzymes. The purpose of this study was to investigate the effect of solvents on the kinetics of representative CYP2B6 (bupropion hydroxylase) and CYP2C8 (paclitaxel hydroxylase) reactions in human liver microsomes. Methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied at increasing volumes (v/v). Acetonitrile, DMSO, and ethanol were shown to increase the Km and decrease the intrinsic clearance (CLint) of CYP2B6-mediated bupropion hydroxylation in a concentration-dependent manner. These solvents did not noticeably alter the Vmax at concentrations of < or =1% (v/v). Unlike the other solvents studied, the effect of methanol (< or =0.5%, v/v) on CYP2B6 kinetics was negligible. Both DMSO and ethanol increased the Km and decreased the CL(int) of CYP2C8-mediated paclitaxel hydroxylation in a concentration-dependent manner. Acetonitrile had minimal influence on CYP2C8 enzyme kinetics at concentrations of < or =1% (v/v). Methanol decreased the Km of paclitaxel at low concentrations followed by an increase at concentrations of > or =2% (v/v). This differential influence on Km resulted in an increased CLint at low concentrations followed by a decrease at high concentrations. The studied solvents had minimal influence on Vmax of paclitaxel. Collectively, DMSO and ethanol were not suitable for characterizing CYP2B6- and CYP2C8-mediated reactions because they showed concentration-dependent inhibition. Methanol and acetonitrile at concentrations of < or =0.5% and < or =1% (v/v) appeared to be suitable for the measurement of CYP2B6- and CYP2C8-mediated activities, respectively.

Development and validation of a preclinical food effect model.

A preclinical canine model capable of predicting a compound's potential for a human food effect was developed. The beagle dog was chosen as the in vivo model. A validation set of compounds with known propensities for human food effect was studied. Several diets were considered including high-fat dog food and various quantities of the human FDA meal. The effect of pentagastrin pretreatment was also investigated. The high-fat dog food did not predict human food effect and was discontinued from further evaluation. The amount of FDA meal in the dog was important in the overall prediction of the magnitude of human food effect. Fed/fasted Cmax and AUC ratios using a 50-g aliquot of the FDA meal in the dog were in the closest qualitative agreement to human data. Pentagastrin pretreatment did not affect the AUC in the fed state, but increased the fasted AUC for weakly basic compounds. Pentagastrin pretreatment and a 50-g aliquot of the FDA meal in the dog predicted the human food effect for a validation set of compounds. This model, which is intended for compound screening, will be helpful for determining food effect as a liability when compounds progress from discovery to clinical development.

In vivo animal models for investigating potential CYP3A- and Pgp-mediated drug-drug interactions.

With the advent of polytherapy it has become prudent to minimize, as much as possible, the potential for drug-drug interactions. Towards this end, the metabolic and transporter pathways involved in the disposition of a drug candidate (phenotyping) are evaluated in vitro employing available human tissue and specific reagents. Likewise, in vitro screening for inhibition and induction of drug-metabolizing enzymes and transporters is conducted also. Such in vitro human data can be made available prior to human dosing and enable in vitro to in vivo-based predictions of clinical outcomes. Despite some success, however, in vitro systems are not dynamic and sometimes fail to predict drug-drug interactions for a variety of reasons. In comparison, relatively less effort has been made to evaluate predictions based on data derived from in vivo animal models. This review will attempt to summarize different examples from the literature where animal models have been used to predict cytochrome P450 3A (CYP3A)- and P-glycoprotein (Pgp)-based drug-drug interactions. When employing data from animal models one needs to be aware of species differences in pharmacokinetics, clearance pathways and selectivity and affinity of probe substrates and inhibitors. Because of these differences, in vivo animal studies alone, cannot be predictive of human drug-drug interactions. Despite these caveats, the information obtained from validated in vivo animal models may prove useful when used in conjunction with in vitro-in vivo extrapolation methods. Such an integrated data set can be used to select drug candidates with a reduced drug interaction potential.

Novel fluoro-substituted camptothecins: in vivo antitumor activity, reduced gastrointestinal toxicity and pharmacokinetic characterization.

PURPOSE: The novel fluoro-substituted camptothecin analog, BMS-286309, and its prodrug, BMS-422461, were evaluated for their pharmacologic, toxicologic, metabolic and pharmacokinetic developmental potential. METHODS: In vitro and in vivo assays were used to assess the compounds for topoisomerase I activity, antitumor activity, gastrointestinal (GI) toxicity, and pharmacokinetic parameters. RESULTS: BMS-286309-induced topoisomerase I-mediated DNA breaks in vitro and was similar in potency to camptothecin. Both BMS-286309 and -422461 were comparable to irinotecan regarding preclinical antitumor activity assessed in mice bearing distal site murine and human tumors. BMS-422461 was also found to be orally active. Both analogs were >100-fold more potent in vivo than irinotecan and both were superior to irinotecan with respect to toxicological assessment of GI injury in mice. The generation of parent compound from BMS-422461 was qualitatively similar in mouse, rat and human blood and liver S9 fractions. The percentage of BMS-286309 remaining as the active lactone form at equilibrium was comparable in mouse and human plasma. The pharmacokinetic profile in rat blood demonstrated that BMS-422461 was rapidly cleaved to BMS-286309. CONCLUSIONS: The favorable in vivo metabolic activation of BMS-422461, and the pharmacokinetic characteristics of BMS-286309, suggest that the good efficacy of BMS-422461 is derived from robust in vivo release of BMS-286309 in rodents and the likelihood that this biotransformation will be preserved in humans. The comparable antitumor activity of BMS-422461 to irinotecan, as well as reduced preclinical GI toxicity, make this novel camptothecin analog attractive for clinical development.