Mithat Gunduz

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. The present examination offers a comparison of 37 different methods for prediction of human clearance across compounds of diverse physicochemical properties. Furthermore, this work demonstrates the application of each prediction method to each charge class of the compounds, thus presenting an additional dimension to prediction of human pharmacokinetics. In general, the observations suggest that methods employing monkey clearance values and a method incorporating differences in plasma protein binding between rat and human yield the best overall predictions as suggested by approximately 60% compounds within 2‐fold geometric mean‐fold error. Other single‐species scaling or proportionality methods incorporating the fraction unbound in the corresponding preclinical species for prediction of free clearance in human were generally unsuccessful.

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 (VDss) 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. The authors also grouped the compounds according to their charge classes and show the applicability of each method considered to each class, offering further insight into the probability of a successful prediction. Furthermore, they found that the use of fraction unbound in plasma, to obtain unbound volume of distribution, is generally detrimental to accuracy of several methods, and they discuss possible reasons. Overall, the approach using dog and monkey data in the íie‐Tozer equation offers the highest probability of success, with an intrinsic diagnostic tool based on aberrant values (<0 or >1) for the calculated fraction unbound in tissue. Alternatively, methods based on dog data (single‐species scaling) and rat and dog data (íie‐Tozer equation with 2 species or multiple regression methods) may be considered reasonable approaches while not requiring data in nonhuman primates.

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. Metabolite M8 was structurally deciphered by liquid chromatographytandem mass spectrometry and NMR, and was readily generated in vitro upon incubation of [14C]1 with rat liver microsomes fortified with uridine 5′-diphosphoglucuronic acid trisodium salt and alamethicin under a CO2 atmosphere. Treatment of M8 with β-glucuronidase afforded 1 directly. The presence of M8 in bile and its notable absence from other matrices suggests the enterohepatic cycling of 1 via M8. Although the structure of M7 was not elucidated unequivocally due to its inability to be formed in vitro and its minimal absolute quantities in limited biological matrices, data herein clearly support its structural rationalization. Furthermore, since M7 is the precursor of M8, detection of M8 is indirect evidence of its existence. It is proposed that M7 arises from an equilibrium between 1 and dissolved CO2-equivalents both in vivo and in vitro, similar to carbamino bonds observed in hemoglobin and certain amino acids, respectively.

Metabolism and Disposition of a Selective α7 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 α7 nicotinic acetylcholinergic receptor agonist, were elucidated in humans (4 female, 4 male; all white) after an oral dose of [3H]1. Overall, 1 was well tolerated, with >94% of administered radioactivity excreted renally by 48 h postdose; lyophilization of all urine and plasma samples confirmed 3H stability within [3H]1. Across genders, 1 underwent low-to-moderate oral clearance comprising both renal (67%) and metabolic (33%) components, with the biotransformation of 1 occurring predominantly via oxidation of its furanopyridine moiety to carboxylic acid 2, and minimally by modification of its quinuclidine nitrogen to N-oxide 4 or N-glucuronide M5. Experiments using human in vitro systems were undertaken to better understand the enzyme(s) involved in the phase 1 biotransformation pathways. The formation of 2 was found to be mediated by CYP2D6, a polymorphically expressed enzyme absent in 5 to 10% of white people, whereas the generation of 4 was catalyzed by CYP2D6, FAD-containing monooxygenase 1 (FMO1), and FMO3. It is of interest that, although no overall gender-related differences in excretory routes, mass recoveries, pharmacokinetics, or metabolite profiles of 1 were evident, the observation of one of eight subjects (13%) showing disparate (relative to all other volunteers) systemic exposures to 1, and urinary and plasma quantitative profiles nearly devoid of 2 with the highest levels of 1, seem consistent with both the identification of CYP2D6 as the only major recombinant cytochrome P450 transforming 1 to 2 and the demographics of white CYP2D6 poor metabolizers. Data also reported herein suggest that 4 is generated predominantly by renal FMO1 in humans.

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. The objectives of the current investigation were to elucidate the structure of the novel N-carbamoyl glucuronide, to investigate the mechanism of N-carbamoyl glucuronide formation in vitro using stable labeled CO2, UDP glucuronosyltransferase (UGT) reaction phenotyping, and to assess whether M1 was formed to the same extent in vitro across species—mouse, rat, hamster, dog, monkey, and human. Structure elucidation was performed on a mass spectrometer with accurate mass measurement and MSn capabilities. 13C-labeled carbon dioxide was used for identification of the mechanism of N-carbamoyl glucuronidation. Mechanistic studies with 13C-labeled CO2 in rat liver microsomes revealed that CO2 from the bicarbonate buffer (in equilibrium with exogenous CO2) may be responsible for the formation of M1. M1 was formed in vitro in liver microsomes from multiple species, mainly rat and hamster, followed by similar formation in dog, monkey, mouse, and human. M1 could be detected in UGT1A1, UGT1A3, and UGT2B7 Supersomes in a CO2-rich environment. In conclusion, our study demonstrates that formation of M1 was observed in microsomal incubations across various species and strongly suggests incorporation of CO2 from the bicarbonate buffer, in equilibrium with exogenous CO2, into the carbamoyl moiety of the formed N-carbamoyl glucuronide.

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 α7 nicotinic acetylcholinergic receptor, were determined in both Sprague-Dawley rats and beagle dogs using [3H]1. Initially, 3-tritio-furanopyridine 1 ([3H]1a) was evaluated in pilot mass balance studies by determining total radioactivity recovery and pharmacokinetics in lyophilized excreta and nonlyophilized plasma, respectively. Lower mass balance and much greater circulatory radioactivity exposures were observed in rats than in dogs, with urinary tritiated water (HTO) only detected in rats. The 133-h half-life in rats, possibly due to very slowly eliminated metabolites, was more likely attributable to HTO formed from [3H]1a because of site-specific chemical and/or metabolic 3H instability, which was confirmed by urinary HTO. In contrast, dog data supported 3H stability within [3H]1a. Conflicting cross-species data with [3H]1a suggested species-specific metabolic fates for 1, requiring a 3H form of 1 resistant to 3H loss in rats. Therefore, tritiation of 1 at its furanopyridine C7, a site predicted to be both chemically and metabolically stable, yielded 7-tritio-N-(3R)-1-azabicyclo[2.2.2]oct-3-ylfuro[2,3-c]pyridine-5-carboxamide ditrifluoroacetate ([3H]1b), which allowed in both species the determination of all excretory pathways, total radioactivity pharmacokinetics, and major excretory and circulatory metabolites with complete radioactivity recovery without HTO generation. Definitive metabolite elucidation for 1 using [3H]1b confirmed the suspected species-dependent metabolic susceptibility for 3H loss from [3H]1a in rats, but not dogs, since the majority of rat metabolites resulted from furanopyridine biotransformation. The described studies explore the evaluation of tritium exchange risk from a mechanistic biotransformation perspective and highlight the need for careful deliberation when considering and designing 3H compounds for radiolabeled metabolism studies.

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. 11 of the 16 metabolites of levobunolol identified herein, including a direct acetyl conjugate of levobunolol observed in all ocular and liver fractions, have not been reported in the literature. The study documents the identification of six human ocular metabolites that have never been reported. The current investigation presents evidence for ocular and hepatic metabolism of levobunolol via non-cytochrome P450 pathways, which have not been comprehensively investigated to date. Our results indicated that rat liver S9 and human ocular S9 fractions formed the most metabolites. Furthermore, liver was a poor in vitro surrogate for eye, and rat and rabbit were poor surrogates for human in terms of the rate and extent of levobunolol metabolism.

Species Differences in the Biotransformation of an α4β2 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 α4β2 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[14C]-3- benzazepine hydrochloride ([14C]1) orally. Although both species chiefly (≥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. In monkeys, radioactivity recoveries were 50-fold greater in urine than feces and minimal (<5%) in bile. Both species metabolized 1 similarly: four-electron oxidation to one of four amino acids or two lactams (minor) and glucuronide formation (major). In rats, the latter pathway predominantly formed an N-carbamoyl glucuronide (M6), exclusively present in bile (69% of dose), whereas in monkeys it afforded an N-O-glucuronide (M5), a minor biliary component (4%) but the major plasma (62%) and urinary (42%) entity. In rats, first-pass hepatic conversion of 1 to M6, which was confirmed in rat hepatocytes, and its biliary secretion resulted in the indirect enterohepatic cycling of 1 via M6 and manifested in double-humped plasma concentration-time curves and long t1/2 for both 1 and total radioactivity. In monkeys, in which only M5 was formed, double-humped plasma concentration-time curves were absent, and moderate t1/2 for both 1 and total radioactivity were observed. A seemingly subtle, yet critical, difference in the chemical structures of these two glucuronide metabolites considerably affected the overall disposition of 1 in rats versus monkeys.

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. Cytochrome P450 (P450) phenotyping studies revealed that the formation of M1 and M2 were NADPH-dependent and primarily catalyzed by CYP3A4 among the studied P450 isoforms. In summary, an unusual and stable quinone methide metabolite of compound 1 was identified, and a mechanism was proposed for its formation via an oxidative ipso substitution.

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.