The debate on animal ADME studies in drug development: an update.

The preparation and release of the International Conference on Harmonisation guideline on safety evaluation of human metabolites and the technical progresses in bioanalysis have triggered an intense debate on the value of absorption, distribution, metabolism and excretion radiolabelled studies in animals. Some authors have radically challenged the traditional approach whereas others, while accepting the need of significant changes, argue that these studies remain an irreplaceable component of the preclinical registration dossier. This paper reviews some of the representative positions and describes the potential evolution.

Dogs and monkeys in preclinical drug development: the challenge of reducing and replacing.

INTRODUCTION: Animal experimentation is a very contentious issue affecting reputation of drug industry. There are several reasons to forecast an increase in the number of dogs and monkeys used in safety and pharmacokinetic studies. This increase may trigger a strong reaction of the public opinion. There have been many proposals and initiatives to change the present approach to safety and metabolic studies. Tests based on new technologies, in vitro cell assays, stem cells, imaging, and computational systems, have the potential to anticipate effects in humans. Unfortunately, all these efforts and ideas have not changed standard approaches and regulatory expectations. AREAS COVERED: This review looks at opportunities to reduce the number of dogs and monkeys currently used in pharmaceutical research. It also discusses present efforts and approaches, their strengths and potentials and the reasons why they may not fulfill expectations. EXPERT OPINION: Unless the pharmaceutical industry gets more involved, an alternative paradigm of preclinical drug development is unlikely to be established in the foreseeable future. One can imagine a scenario where the political pressure against the use of dogs and monkeys in biomedical research becomes irresistible while alternative methods are not yet established. To avoid this situation, the pharmaceutical industry should take the lead and preclinical scientists at all levels need to influence decision makers and help develop new innovative approaches in drug safety evaluation.

Preclinical in vivo ADME studies in drug development: a critical review.

INTRODUCTION: The last two decades have brought many fundamental changes to the drug development process. One such change is the importance of preclinical pharmacokinetics, which has become an essential part of early drug discovery. Furthermore, bioanalytical methods have become more sensitive and the identification and quantitation of metabolites can now be carried out on limited amount of biological material. There has also been a change in regulatory expectations, which are now particularly focused on the safety of human metabolites. AREAS COVERED: The focus of this paper is on some 'traditional' in vivo ADME studies: excretion balance, metabolic profile and WBA in the toxicological species. These studies, performed with radiolabeled material, have a long history: and are a regular presence in submission dossiers. This paper reviews their value in the perspective of the contemporary drug development process. EXPERT OPINION: These experiments may sometimes still be relevant to explain toxicological findings or for other special purposes but should not be considered required pieces of the registration dossiers. An appropriate investigation of samples coming from safety evaluation and human Phase I studies and the knowledge generated during the lead optimization phase provide, in most instances, all the DMPK information needed to take decisions in the drug development process.

Plasma protein binding and blood-free concentrations: which studies are needed to develop a drug?

INTRODUCTION: The plasma protein binding of drugs and metabolites is known to influence their pharmacokinetics and, therefore, their effects. Evaluating the extent and the linearity of protein binding is an essential piece of information that has to be generated during drug development. Blood cell partitioning has a similar relevance. AREAS COVERED: This paper summarizes the regulatory requirements and focuses particularly on two questions pertaining to the drug development process. The first of these questions asks when is it necessary to perform detailed clinical studies on protein binding while the second asks whether the in vitro studies presently performed in plasma produce biased information. EXPERT OPINION: The authors propose that clinical ex vivo protein-binding studies should be performed on highly bound compounds (a definition of highly bound is suggested as > 95%). They also propose that in vitro studies, to measure the free drug, should be performed in whole blood, rather than in plasma, particularly if binding to proteins or blood cells is nonlinear.

Drug and metabolite concentrations in tissues in relationship to tissue adverse findings: a review.

INTRODUCTION: Drug blood (or plasma) levels measured during safety preclinical investigations do not always correlate with toxicological findings. Concentrations in target tissues or, even better, at target receptors would probably be more relevant. In addition, toxicity may be caused by drug metabolites which, in turn, can be tissue specific. Tissue concentrations and tissue metabolism may be crucial for interpreting tissue toxicity. AREAS COVERED: This paper, starting from the authors' direct experience, focuses on distribution of the parent compound and metabolites in target toxicity tissues and presents a review of several examples where organ or tissue concentrations have been either useful or not relevant for interpreting safety findings. Regulatory aspects and technological progresses are also mentioned. EXPERT OPINION: The authors advocate directing more attention and efforts toward investigating tissue distribution: this approach might reduce late stage attrition. When unexpected tissue toxicity is found, measuring drug concentrations in the target tissue and characterising and measuring tissue metabolites could bring relevant information for interpreting the adverse finding. Evidence of slow accumulation of a long lasting metabolite in a tissue should be considered as an alert: this evidence can be obtained during short-term toxicity studies.

Tissue distribution and characterization of drug-related material in rats and dogs after repeated oral administration of casopitant.

Casopitant [1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-N-((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)-N-methyl-(2R,4S)] has been shown to be a potent and selective antagonist of the human neurokinin 1 receptor, the primary receptor for substance P. During long-term toxicity studies conducted in rat and dog, evidence of cardiomyopathy and increased cardiac weight were observed. The distribution and metabolism of casopitant were studied in both species evaluating the accumulation of drug-related material (DRM) after repeat dosing and its potential relationship with pathological findings observed in myocardium. After repeat oral administration of [(14)C]casopitant to rats (20 days) and dogs (14 days), DRM was quantifiable in all of the tissues examined with lung and liver containing the highest level of radioactivity. The concentration of radioactivity was significantly higher in tissues than in plasma, declining slowly and still quantifiable after a recovery period of 20 days. The principal circulating components identified in both species were casopitant, M12 (oxidized deacetylated), M13 (hydroxylated piperazine), and M31 and M134 (two N-dealkylated piperazines). In tissues, a similar metabolic pattern was observed, in which casopitant, M31, M134, M76 (N-deacetylated), and M200 (N-deacetylated N,N-deethylated) were the major components quantified. After a 26-week repeat dose study in dog, casopitant and M13 were the major circulating components, whereas in myocardium, M200 and M134 were the major ones and their levels increased over time, reaching considerable concentrations (millimolar magnitude). After a washout period, all circulating derivatives decreased to undetectable levels, whereas M200 was still the major component in myocardium. Overall DRM in plasma did not correlate with the respective concentrations in tissues.

Casopitant: in vitro data and SimCyp simulation to predict in vivo metabolic interactions involving cytochrome P450 3A4.

Casopitant [1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-N-((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)-N-methyl-(2R,4S), GW679769] has previously been shown to be a potent and selective antagonist of the human neurokinin-1 receptor, the primary receptor of substance P, both in vitro and in vivo, with good brain penetration properties. On the basis of this mode of action it was evaluated for the prevention of chemotherapy-induced and postoperative nausea and vomiting, and for the chronic treatment of anxiety, depression, insomnia, and overactive bladder. Casopitant is shown to be a substrate, an inhibitor, and an inducer of CYP3A4, and, because of this complex behavior, it was difficult to identify the primary mechanism by which it may give rise to drug-drug interactions (DDIs) of clinical relevance. Moreover, the major circulating metabolite is itself an inhibitor of CYP3A4 in vitro. On the basis of the different clinical indications and the various potential comedications of casopitant, a relevant part of the clinical development plan was focused on the assessment of the importance of clinical DDIs. The present study provides an overview of the DDI potential profile of casopitant, based on in vitro data and clinical evidence of its interaction with CYP3A4 probe substrates midazolam and nifedipine, the strong inhibitor ketoconazole, and the inducer rifampin. Overall, the clinical data confirm the ability of casopitant to interact with CYP3A4 substrates, inhibitors, or inducers. The in vitro data are accurate and robust enough to build a reliable SimCyp population-based model to estimate the potential DDIs of casopitant and to minimize the clinical studies recommended.

Disposition and metabolism of [14C]SB-649868, an orexin 1 and 2 receptor antagonist, in humans.

N-[[(2S)-1-[[5-(4-fluorophenyl)-2-methyl-4-thiazolyl]carbonyl]-2-piperidinyl]methyl]-4-benzofurancarboxamide (SB-649868) is a novel orexin 1 and 2 receptor antagonist under development for insomnia treatment. The disposition of [(14)C]SB-649868 was determined in eight healthy male subjects using an open-label study design after a single oral dose of 30 mg. Blood, urine, and feces were collected at frequent intervals after dosing, and samples were analyzed by high-performance liquid chromatography-mass spectrometry coupled with off-line radiodetection for metabolite profiling and characterization. NMR spectroscopy was also used to further characterize certain metabolites. Elimination of drug-related material was almost complete over a 9-day period, occurring principally via the feces (79%), whereas urinary excretion accounted only for 12% of total radioactivity. Mean apparent half-life (t(1/2)) of plasma radioactivity was notably longer (39.3 h), with respect to that of unchanged SB-649868 (4.8 h), suggesting the presence of more slowly cleared metabolites. SB-649868 and an unusual hemiaminal metabolite, M98 (2-[((2S)-1-{[5-(4-fluorophenyl)-2-methyl-1,3-thiazol-4-yl]carbonyl}-2-piperidinyl)methyl]-3,5-dihydroxy-3,4-dihydro-1(2H)-isoquinolinone; GSK2329163), resulting from oxidation of the benzofuran ring and subsequent rearrangement, were the principal circulating components in plasma extracts. Two additional minor metabolites were also observed: a benzofuran ring-opened carboxylic acid M25 ([2-({[((2S)-1-{[5-(4-fluorophenyl)-2-methyl-1,3-thiazol-4-yl]carbonyl}-2-piperidinyl)methyl]amino}carbonyl)-6-hydroxyphenyl]acetic acid; GSK2329158) and an amine metabolite (M8). SB-649868 was extensively metabolized, and only negligible amounts were excreted unchanged. The principal route of metabolism was via oxidation of the benzofuran ring with the resultant M25 being the principal metabolite in excreta, representing at least 12% of the administered dose across urine and feces.

Metabolic disposition of casopitant, a potent neurokinin-1 receptor antagonist, in mice, rats, and dogs.

Casopitant [1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-N-((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)-N-methyl-(2R,4S)] is a potent and selective antagonist of the neurokinin-1 (NK1) receptor, developed for the prevention of chemotherapy-induced nausea and vomiting and postoperative nausea and vomiting. Absorption, distribution, metabolism, and elimination of [(14)C]casopitant have been investigated in the mouse, rat, and dog after single oral administration and compared with those in humans. [(14)C]Casopitant was rapidly absorbed in all three species: the maximum plasma concentration of radioactivity was generally observed 0.5 to 2 h after a single oral dose. In dog and female rat, as observed for humans, the principal circulating radiolabeled components were unchanged casopitant and its hydroxylated derivative M13. In rats, there was an evident sex-related difference in the rate of elimination of drug-related material with elimination being more rapid in males than in females. In dogs and mice, no notable sex differences were observed in the pattern of excretion. The elimination of drug-related radioactivity was largely by metabolism, with metabolites excreted primarily in the feces. The predominant route of metabolism was the oxidation of the parent molecule, observed together with loss of the N-acetyl group, N-demethylation, and modification of piperazine with consequent opening and cleavage of the ring, giving a complex pattern of metabolites. Conjugation of some of those oxidized products with glucuronic acid was observed. Urinary excretion in all three species was a minor route of elimination, accounting for between 2 and 7% of the dose, with unchanged parent drug never quantifiable.

Expression and distribution of CYP3A genes, CYP2B22, and MDR1, MRP1, MRP2, LRP efflux transporters in brain of control and rifampicin-treated pigs.

The in vivo effect of rifampicin, a potent ligand of PXR, on gene expression of CYP2B22, 3A22, 3A29, 3A46, CAR, PXR and MDR1, MRP1, MRP2, LRP transporters in liver and cortex, cerebellum, midbrain, hippocampus, meninges and brain capillaries of pig was investigated. Animals were treated i.p. with four daily doses of rifampicin (40 mg/kg). The basal mRNA expressions of the individual CYP3As, CYP2B22, CAR, and PXR in various brain regions, except meninges, were about or below 10% of the corresponding hepatic mRNA values, whereas the mRNAs of brain transporters were closer or comparable to those in liver. After pig treatment with rifampicin, the mRNA expression of CYPs and transporters from brain regions did not appear to change, except CYP3A22 and 3A29 in cortex and hippocampus, CYP2B22 in meninges. An enzymatic analysis for CYP3As and CYP2B, in microsomes and mitochondria from liver and brain tissues using the marker activities 7-benzyloxyquinoline O-debenzylase and the anthraldehyde oxidase, showed the lack of rifampicin induction in all the brain regions, unlike liver. Taken together, our results demonstrate that CYP2B22, CYP3As, and MDR1, MRP1, MRP2, and LRP transporters are all expressed, although at different extent, in the brain regions but, despite the presence of PXR and CAR, are resistant to induction indicating that the regulation of these proteins is more complex in brain than in liver. These data obtained in vivo in the brain regions and liver of pig may be of interest to human metabolism in CNS.

Disposition and metabolism of radiolabeled casopitant in humans.

Casopitant [1-piperidinecarboxamide,4-(4-acetyl-1-piperazinyl)-N-((1R)-1-(3,5-bis(trifluoromethyl)phenyl)-ethyl)-2-(4-fluoro-2-methylphenyl)-N-methyl-(2R,4S)-(GW679769)] is a novel neurokinin-1 receptor antagonist being developed for the prevention of chemotherapy-induced and postoperative nausea and vomiting. The disposition of [(14)C]casopitant was determined in a single-sequence study in six healthy male subjects after single-dose 90-mg i.v. and 150-mg oral administration. Blood, urine, and feces were collected at frequent intervals after dosing. Plasma, urine, and fecal samples were analyzed by high-performance liquid chromatography/mass spectrometry coupled with off-line radiodetection for metabolite profiling. Moreover, urine was also analyzed with (1)H-NMR to further characterize metabolites. Plasma pharmacokinetic parameters for casopitant, a major metabolite (M13, coded as GSK525060), and total radioactivity were determined. Absorption of radioactivity after oral administration appeared to be nearly complete; elimination was principally via the feces both after oral and intravenous administration. Urinary elimination accounted for only <8% of total radioactivity. The main circulating metabolites were a hydroxylated derivative, M13 (coded as GSK525060), and, after oral administration, a deacetylated and oxidized metabolite, M12 (coded as GSK631832). In addition, many other metabolites were identified in plasma and excreta: the principal route of metabolism included multiple oxidations, loss of the N-acetyl group, modifications or loss of the piperazine group, and cleavage of the molecule. Casopitant was extensively metabolized, and only negligible amounts were excreted as unchanged compound. Some phase II metabolites were also observed, particularly in urine.