Species-specific Bioactivation of Morpholines as a Causative of Drug Induced Liver Injury Observed in Monkeys.

Background: Everolimus, an allosteric mechanistic target of rapamycin (mTOR) inhibitor, recently demonstrated the therapeutic value of mTOR inhibitors for Central Nervous System (CNS) indications driven by hyperactivation of mTOR. A newer, potent brain-penetrant analog of everolimus, referred to as (1) in this manuscript [(S)-3-methyl-4-(7-((R)-3-methylmorpholino)-2- (thiazol-4-yl)-3H-imidazo[4,5-b]pyridin-5-yl)morpholine,(1)] catalytically inhibits mTOR function in the brain and increases the lifespan of mice with neuronal mTOR hyperactivation.

Introduction: Early evaluation of the safety of 1 was conducted in cynomolgus monkeys in which oral doses were administered to three animals in a rising-dose fashion (from 2 to 30 mg/kg/day).

Metabolism and Excretion of Therapeutic Peptides: Current Industry Practices, Perspectives, and Recommendations.

Therapeutic peptides (TPeps) have expanded from the initial endogenous peptides to complex modified peptides through medicinal chemistry efforts for almost a century. Different from small molecules and large proteins, the diverse submodalities of TPeps have distinct structures and carry different absorption, distribution, metabolism, and excretion (ADME) properties. There is no distinct regulatory guidance for the industry on conducting ADME studies (what, how, and when) for TPeps.

Species-dependent hepatic and intestinal metabolism of selective oestrogen receptor degrader LSZ102 by sulphation and glucuronidation.

LSZ102 is an orally bioavailable selective oestrogen receptor degrader in clinical development for the treatment of breast cancer. Preclinical studies showed efficacy in xenograft models on oral dosing. However, oral bioavailability was relatively low in several preclinical species (7-33%), and was associated with first-pass metabolism, particularly intestinal first-pass.

Models and Approaches Describing the Metabolism, Transport, and Toxicity of Drugs Administered by the Ocular Route.

The eye is a complex organ with a series of anatomic barriers that provide protection from physical and chemical injury while maintaining homeostasis and function. The physiology of the eye is multifaceted, with dynamic flows and clearance mechanisms. This review highlights that in vitro ocular transport and metabolism models are confined by the availability of clinically relevant absorption, distribution, metabolism, and excretion (ADME) data.

New Perspectives on Acyl Glucuronide Risk Assessment in Drug Discovery: Investigation of In vitro Stability, In situ Reactivity, and Bioactivation.

Background: Acyl glucuronides of xenobiotics have been a subject of wide interest from the pharmaceutical industry with respect to biochemical reactivity, hepatic disposition, and enterohepatic circulation. The reactivity and lack of stability of an acyl glucuronide for a clinical candidate could pose major developability concerns. To date, multiple in vitro assays have been published to assess the risk associated with acyl glucuronides.

Genotoxicity of 4-(piperazin-1-yl)-8-(trifluoromethyl)pyrido[2,3-e][1,2,4] triazolo[4,3-a]pyrazine, a Potent H4 Receptor Antagonist for the Treatment of Allergy: Evidence of Glyoxal Intermediate Involvement.

Background: 4-(piperazin-1-yl)-8-(trifluoromethyl)pyrido[2,3-e][1,2,4]triazolo[4,3-a]pyrazine (1) is a small-molecule which demonstrated a sub-nM inhibitory potency toward the histamine H4 receptor (H4R). However, it was found to be mutagenic in an in vitro Ames assay. Metabolic bioactivation of 1 could potentially arise from the piperazine moiety by forming reactive intermediates such as glyoxal, aldehyde-imine and/or iminium ion, which could all lead to genotoxicity.

Investigation of Ocular Bioactivation Potential and the Role of Cytochrome P450 2D Enzymes in Rat.

Background: Timolol is clinically administered topically (ocular) to reduce intraocular pressure and treat open-angle glaucoma. Ocular administration of timolol in low doses (0.5% w/v in the form of eye drops) has led to challenges for in vivo metabolite identification.

Do We Need to Study Metabolism and Distribution in the Eye: Why, When, and Are We There Yet?

The liver is known to be the principal site of drug metabolism. Depending on the route of administration, especially in cases of topical and local delivery, evaluation of local drug metabolism in extrahepatic tissues is vital to assess fraction of the drug metabolized. This parameter becomes important from the point of view of drug availability or the contribution to overall clearance.

In vitro ocular metabolism and bioactivation of ketoconazole in rat, rabbit and human.

Oral ketoconazole is clinically administered for treatment of severe cases for fungal keratitis. Pharmacodynamics and efficacy of oral and topical (ocular) ketoconazole have been explored in rabbit. However, metabolism of ketoconazole in the eye in any species is not well explored in any preclinical species or human.

Ocular Metabolism of Levobunolol: Historic and Emerging Metabolic Pathways.

Although ocular transport and delivery have been well studied, metabolism in the eye is not well documented, even for clinically available medications such as levobunolol, a potent and nonselective β-adrenergic receptor antagonist. Recently, we reported an in vitro methodology that could be used to evaluate ocular metabolism across preclinical species and humans. The current investigation provides detailed in vitro ocular and liver metabolism of levobunolol in rat, rabbit, and human S9 fractions, including the formation of equipotent active metabolite, dihydrolevobunolol, with the help of high-resolution mass spectrometry.

Metabolic Enzyme Sulfotransferase 1A1 Is the Trigger for N-Benzyl Indole Carbinol Tumor Growth Suppression.

In an attempt to identify novel therapeutics and mechanisms to differentially kill tumor cells using phenotypic screening, we identified N-benzyl indole carbinols (N-BICs), synthetic analogs of the natural product indole-3-carbinol (I3C). To understand the mode of action for the molecules we employed Cancer Cell Line Encyclopedia viability profiling and correlative informatics analysis to identify and ultimately confirm the phase II metabolic enzyme sulfotransferase 1A1 (SULT1A1) as the essential factor for compound selectivity. Further studies demonstrate that SULT1A1 activates the N-BICs by rendering the compounds strong electrophiles which can alkylate cellular proteins and thereby induce cell death.

Comprehensive assessment of human pharmacokinetic prediction based on in vivo animal pharmacokinetic data, part 2: clearance.

A comprehensive analysis on the prediction of human clearance based on intravenous pharmacokinetic data from rat, dog, and monkey for approximately 400 compounds was undertaken. This data set has been carefully compiled from literature reports and expanded with some in-house determinations for plasma protein binding and rat clearance. To the authors- knowledge, this is the largest publicly available data set.

Comprehensive assessment of human pharmacokinetic prediction based on in vivo animal pharmacokinetic data, part 1: volume of distribution at steady state.

The authors present a comprehensive analysis on the estimation of volume of distribution at steady state (VD(ss) ) in human based on rat, dog, and monkey data on nearly 400 compounds for which there are also associated human data. This data set, to the authors- knowledge, is the largest publicly available, has been carefully compiled from literature reports, and was expanded with some in-house determinations such as plasma protein binding data. This work offers a good statistical basis for the evaluation of applicable prediction methods, their accuracy, and some methods-dependent diagnostic tools.

Oxidative ipso substitution of 2,4-difluoro-benzylphthalazines: identification of a rare stable quinone methide and subsequent GSH conjugate.

In vitro metabolite identification and GSH trapping studies in human liver microsomes were conducted to understand the bioactivation potential of compound 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol], an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a GSH adduct (M2). The stability of this metabolite arises from extensive resonance-stabilized conjugation of the substituted benzylphthalazine moiety.

In vitro-in vivo correlation for intrinsic clearance for CP-409,092 and sumatriptan: a case study to predict the in vivo clearance for compounds metabolized by monoamine oxidase.

Oxidative deamination of the GABA(A) partial agonist CP-409,092 and sumatriptan represents a major metabolic pathway and seems to play an important role for the clearance of these two compounds. Similar to sumatriptan, human mitochondrial incubations with deprenyl and clorgyline, probe inhibitors of monoamine oxidase B and monoamine oxidase A (MAO-B and MAO-A), respectively, showed that CP-409,092 was metabolized to a large extent by the enzyme MAO-A. The metabolism of CP-409,092 and sumatriptan was therefore studied in human liver mitochondria and in vitro intrinsic clearance (CL(int)) values were determined and compared to the corresponding in vivo oral clearance (CL(PO)) values.

A new structural alert for benzimidazoles: 2,6-dimethylphenyl substituents increase mutagenic potential and time-dependent CYP3A4 inhibition risk.

A series of 2-[(2,6)-dimethylphenyl]benzimidazole analogs displayed strong potential for mutagenicity following metabolic activation in either TA98 or TA100 Salmonella typhimurium strains. The number of revertants was significantly reduced by replacing the 2,6-dimethylphenyl group with a 2,6-dichlorophenyl moiety. Time-dependent CYP3A4 inhibition was also observed with a compound containing a 2-[(2,6)-dimethylphenyl] benzimidazole ring, implying risk for this scaffold to generate reactive metabolites.

Identification of a novel N-carbamoyl glucuronide: in vitro, in vivo, and mechanistic studies.

1-[4-Aminomethyl-4-(3-chlorophenyl)-cyclohexyl]-tetrahydro-pyrimidin- 2-one, 1, was developed as an inhibitor of dipeptidyl peptidase-4 enzyme. Biotransformation studies with 1 revealed the presence of an N-carbamoyl glucuronide metabolite (M1) in rat bile and urine. N-Carbamoyl glucuronides are rarely observed, and little is understood regarding the mechanism of N-carbamoyl glucuronidation.

Species differences in the biotransformation of an alpha 4 beta 2 nicotinic acetylcholine receptor partial agonist: the effects of distinct glucuronide metabolites on overall compound disposition.

The metabolism and disposition of (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-3-benzazepine (1), an alpha(4)beta(2) nicotinic acetylcholine receptor partial agonist, was investigated in Sprague-Dawley rats and cynomolgus monkeys receiving (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-4[(14)C]-3- benzazepine hydrochloride ([(14)C]1) orally. Although both species chiefly (>or=62%) cleared 1 metabolically, species-specific dispositional profiles were observed for both 1 and total radioactivity. Radioactivity was excreted equally in the urine and feces of intact rats but largely (72%) in bile in bile duct-cannulated animals.

Metabolism and disposition of a gamma-aminobutyric acid type A receptor partial agonist in humans.

The metabolism and disposition of N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-indole-3-carboxamide (1), a potent subtype-selective partial agonist at the gamma-aminobutyric acid type A receptor complex, were elucidated in humans following a p.o. dose of N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-[3-(14)C]indole-3-carboxamide monomethane-sulfonate ([(14)C]1).

Metabolism and disposition of a selective alpha(7) nicotinic acetylcholine receptor agonist in humans.

The metabolism and disposition of N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide (1), an alpha(7) nicotinic acetylcholinergic receptor agonist, were elucidated in humans (4 female, 4 male; all white) after an oral dose of [(3)H]1.

Using a tritiated compound to elucidate its preclinical metabolic and excretory pathways in vivo: exploring tritium exchange risk.

The metabolism and excretion of N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide (1), an agonist of the alpha7 nicotinic acetylcholinergic receptor, were determined in both Sprague-Dawley rats and beagle dogs using [3H]1.

Biotransformation of a GABAA receptor partial agonist in sprague-dawley rats and cynomolgus monkeys: identification of two unique N-carbamoyl metabolites.

The absorption, metabolism, and excretion of N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-indole-3-carboxamide monomethanesulfonate (1), a GABAA receptor partial agonist potentially useful in treating generalized anxiety disorder, have been evaluated in both Sprague-Dawley rats and cynomolgus monkeys using [14C]1. In both species, mass balance was achieved within 48 h postdose, with the majority of drug-related material excreted within the feces; the clearance of 1 in each species had both metabolic and renal components. In addition to the metabolites produced by aliphatic hydroxylation and/or N-dealkylation of 1, two unique metabolites were detected: a putative carbamic acid (M7) in rat plasma and monkey bile, and an N-carbamoyl glucuronide (M8) in both rat and monkey bile.

Hypothalamic-pituitary-adrenocortical (HPA) axis response and biotransformation of oral naltrexone: preliminary examination of relationship to family history of alcoholism.

We examined HPA axis response to 50 mg oral naltrexone compared with placebo in 17 healthy male and female nonalcoholic subjects, approximately half of whom had a positive family history of alcoholism (FH+) and half of whom who did not (FH-). Mood response and naltrexone biotransformation were also examined at various intervals. Subjects participated in two morning test sessions (50 mg naltrexone or identical placebo pill) after an overnight stay in the Rockefeller University GCRC.