Metabolism and Disposition of a Novel B-Cell Lymphoma-2 Inhibitor Venetoclax in Humans and Characterization of Its Unusual Metabolites.

Venetoclax (ABT-199), a B-cell lymphoma-2 (Bcl-2) protein inhibitor, is currently in clinical development for the treatment of hematologic malignancies. We characterized the absorption, metabolism, and excretion of venetoclax in humans. After a single oral dose of [14C]venetoclax to healthy volunteers, the recovery of total radioactive dose was 100%, with feces being the major route of elimination of the administered dose, whereas urinary excretion was minimal (<0.1%). The extent of absorption was estimated to be at least 65%. Venetoclax was primarily cleared by hepatic metabolism (∼66% of the administered dose). ∼33% of the administered dose was recovered as the parent drug and its nitro reduction metabolite M30 [2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-amino-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)benzamide] (13%) in feces. Biotransformation of venetoclax in humans primarily involves enzymatic oxidation on the dimethyl cyclohexenyl moiety, followed by sulfation and/or nitro reduction. Nitro reduction metabolites were likely formed by gut bacteria. Unchanged venetoclax was the major drug-related material in circulation, representing 72.8% of total plasma radioactivity. M27 (oxidation at the 6 position of cyclohexenyl ring followed by cyclization at the α-carbon of piperazine ring; 4-[(10aR,11aS)-7-(4-chlorophenyl)-9,9-dimethyl-1,3,4,6,8,10,10a,11a-octahydropyrazino[2,1-b][1,3]benzoxazin-2-yl]-N-[3-nitro-4-(tetrahydropyran-4-ylmethylamino)phenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) was identified as a major metabolite, representing 12% of total drug-related material. M27 was primarily formed by cytochrome P450 isoform 3A4 (CYP3A4). Steady-state plasma concentrations of M27 in human and preclinical species used for safety testing suggested that M27 is a disproportionate human metabolite. M27 is not expected to have clinically relevant on- or off-target pharmacologic activities.

In Vitro and Clinical Evaluations of the Drug-Drug Interaction Potential of a Metabotropic Glutamate 2/3 Receptor Agonist Prodrug with Intestinal Peptide Transporter 1.

Despite peptide transporter 1 (PEPT1) being responsible for the bioavailability for a variety of drugs, there has been little study of its potential involvement in drug-drug interactions. Pomaglumetad methionil, a metabotropic glutamate 2/3 receptor agonist prodrug, utilizes PEPT1 to enhance absorption and bioavailability. In vitro studies were conducted to guide the decision to conduct a clinical drug interaction study and to inform the clinical study design. In vitro investigations determined the prodrug (LY2140023 monohydrate) is a substrate of PEPT1 with Km value of approximately 30 µM, whereas the active moiety (LY404039) is not a PEPT1 substrate. In addition, among the eight known PEPT1 substrates evaluated in vitro, valacyclovir was the most potent inhibitor (IC50 = 0.46 mM) of PEPT1-mediated uptake of the prodrug. Therefore, a clinical drug interaction study was conducted to evaluate the potential interaction between the prodrug and valacyclovir in healthy subjects. No effect of coadministration was observed on the pharmacokinetics of the prodrug, valacyclovir, or either of their active moieties. Although in vitro studies showed potential for the prodrug and valacyclovir interaction via PEPT1, an in vivo study showed no interaction between these two drugs. PEPT1 does not appear to easily saturate because of its high capacity and expression in the intestine. Thus, a clinical interaction at PEPT1 is unlikely even with a compound with high affinity for the transporter.

Investigation of metabolism and disposition of GSK1322322, a peptidase deformylase inhibitor, in healthy humans using the entero-test for biliary sampling.

GSK1322322 (N-((R)-2-(cyclopentylmethyl)-3-(2-(5-fluoro-6-((S)-hexahydropyrazino[2,1-c][1,4]oxazin-8(1H)-yl)-2-methylpyrimidin-4-yl)hydrazinyl)-3-oxopropyl)-N-hydroxy-formamide) is an antibiotic in development by GlaxoSmithKline. In this study, we investigated the metabolism and disposition of [(14)C]GSK1322322 in healthy humans and demonstrated the utility of the Entero-Test in a human radiolabel study. We successfully collected bile from five men using this easy-to-use device after single i.v. (1000 mg) or oral administration (1200 mg in a solution) of [(14)C]GSK1322322. GSK1322322 had low plasma clearance (23.6 liters/hour) with a terminal elimination half-life of ∼4 hours after i.v. administration. After oral administration, GSK1322322 was readily and almost completely absorbed (time of maximal concentration of 0.5 hour; bioavailability 97%). GSK1322322 predominated in the systemic circulation (>64% of total plasma radioactivity). An O-glucuronide of GSK1322322 (M9) circulated at levels between 10% and 15% of plasma radioactivity and was pharmacologically inactive. Humans eliminated the radioactive dose in urine and feces at equal proportions after both i.v. and oral doses (∼45%-48% each). Urine contained mostly unchanged GSK1322322, accounting for 30% of the dose. Bile contained mostly M9, indicating that glucuronidation was likely a major pathway in humans (up to 30% of total dose). In contrast, M9 was found in low amounts in feces, indicating its instability in the gastrointestinal tract. Therefore, without the Entero-Test bile data, the contribution of glucuronidation would have been notably underestimated. An unusual N-dehydroxylated metabolite (a secondary amide) of GSK1322322 was observed primarily in the feces and was most likely formed by gut microbes.

Pharmacokinetics, metabolism, and excretion of the antidiabetic agent ertugliflozin (PF-04971729) in healthy male subjects.

The disposition of ertugliflozin (PF-04971729), an orally active selective inhibitor of the sodium-dependent glucose cotransporter 2, was studied after a single 25-mg oral dose of [(14)C]-ertugliflozin to healthy human subjects. Mass balance was achieved with approximately 91% of the administered dose recovered in urine and feces. The total administered radioactivity excreted in feces and urine was 40.9% and 50.2%, respectively. The absorption of ertugliflozin in humans was rapid with a T(max) at ∼1.0 hour. Of the total radioactivity excreted in feces and urine, unchanged ertugliflozin collectively accounted for ∼35.3% of the dose, suggestive of moderate metabolic elimination in humans. The principal biotransformation pathway involved glucuronidation of the glycoside hydroxyl groups to yield three regioisomeric metabolites, M4a, M4b, and M4c (∼39.3% of the dose in urine), of which M4c was the major regioisomer (∼31.7% of the dose). The structure of M4a and M4c were confirmed to be ertugliflozin -4-O-ß- and -3-O-ß-glucuronide, respectively, via comparison of the HPLC retention time and mass spectra with authentic standards. A minor metabolic fate involved oxidation by cytochrome P450 to yield monohydroxylated metabolites M1 and M3 and des-ethyl ertugliflozin (M2), which accounted for ∼5.2% of the dose in excreta. In plasma, unchanged ertugliflozin and the corresponding 4-O-ß- (M4a) and 3-O-ß- (M4c) glucuronides were the principal components, which accounted for 49.9, 12.2, and 24.1% of the circulating radioactivity. Overall, these data suggest that ertugliflozin is well absorbed in humans, and eliminated largely via glucuronidation.

[LC-MSn analysis of metabolites of 1,2-[bis (1,2-benzisoselenazolone-3(2H)-ketone)]-ethane, a novel anti-cancer agent in rat].

This study is to elucidate the metabolic pathway of 1,2-[bis (1,2-benzisoselenazolone-3 (2H)-ketone)]-ethane (BBSKE) in rats. Rats were administrated with a single dose of BBSKE 200 mg x kg(-1). The metabolites in rat urine, feces, bile and plasma were identified by LC-MSn analysis. The characterization of fragment ions from LC-MSn chromatography and mass spectrometry was applied to the investigation of structures of metabolites. Three phase I metabolites were detected in rat urine and feces. Two of them were also found in plasma and one existed in bile. These products were derived from oxidized, methylated and S-methylated BBSKE, separately. One phase II glucuronide of BBSKE was also found in bile. Therefore, it is possible that BBSKE was metabolized by oxidization, methylation and glucuronidation.

Determination of bencycloquidium bromide in human urine using weak cation-exchange solid-phase extraction and LC-ESI-MS: Method validation and application to kinetic study of urinary excretion.

A sensitive LC-ESI-MS method has been developed and validated for the determination of bencycloquidium bromide (BCQB) in human urine samples. The method utilized a solid-phase extraction (SPE) procedure, choosing carboxy propyl phase (CBA) as the extracting sorbent for purification of BCQB, with better baseline and higher selectivity achieved. Sample preparation by this method yielded very good and consistent mean recovery of above 94.5%. Another major benefit of the present method was the high detectability, with a lower limit of quantification (LLOQ) of 0.02ng/ml. The developed method was successfully applied to determine BCQB in human urine, and was proved to be suitable for use in Phase I clinical pharmacokinetic study of BCQB.

Mice recognize recent urine scent marks by the molecular composition.

Male mice mark the territory with urine scent marks that are frequently renewed to maintain the territory ownership. We measured the response of male mice to small spots of urine deposed either 0, 5, 11, 22, 45, 90 min, or 24 h before testing and show that mice loose interest in sniffing scent marks as they become older and older. We asked what scent features tell a mouse how recent a scent mark is, and therefore, we studied the molecule-to-behavior relationship by correlating 6 behavioral variables--the number of sniffing acts, the latency to the first sniff, the number of urine marks, the latency to the first mark, the area of the marks, and the number of fecal pellets-to 2,4-dehydro-exo-brevicomin, linalool, 2-sec-butyl-4,5-dihydrothiazole, 2,4-dimethylphenol, 4-ethylphenol, and 6,10-dimethyl-5,9-undecadien-2-one released from urine spots over the time, identified, and quantified by gas chromatography and mass spectrometry. Canonical correlation between the molecular and the behavioral principal components was strong (R(1) = 0.96, P = 0.026). The principal component based on 2,4-dehydro-exo-brevicomin, linalool, and 2-sec-butyl-4,5-dihydrothiazole correlated negatively with countermarking and positively with the sniffing behavior, suggesting a semantic feature of fresh male mouse urine.

Pharmacokinetics, disposition, and metabolism of bicifadine in the mouse, rat, and monkey.

Bicifadine [DOV 220,075; (+/-)-1-(4-methylphenyl)-3-azabicyclo[3.1.0]-hexane HCl)] is a non-narcotic analgesic that is effective in animal models of acute and chronic pain. In this study, the pharmacokinetics, disposition, and metabolism of bicifadine were determined in male and female mice, rats, and cynomolgus monkeys following single oral and i.v. doses. [(14)C]Bicifadine was well absorbed in all three species. The oral bioavailability of bicifadine in mice and rats was 50 to 63% and 79 to 85%, respectively, and slightly lower in monkeys (33-42%). Based on the values of the area under the concentration-time curves, unchanged bicifadine comprised 7 to 12% of the plasma radioactivity after the oral dose and 14 to 26% after the i.v. dose in all three species. The major plasma metabolites were the lactam (M12), the lactam acid (M9), and the acid (M3) plus its glucuronide conjugate. At 0.5 h after the oral dose to rats, 63 to 64% of the radioactivity in the rat brain was bicifadine, and the remainder was the lactam. Most of the radioactivity after oral and i.v. dosing to the three species was recovered in the urine. The lactam acid was the major urinary metabolite in all species; bicifadine and the lactam were either not detected or were minor components in urine. Fecal radioactivity was due to the acid and lactam acid in the three species. Rat bile contained mainly the lactam acid and the acid plus its acyl glucuronide. Plasma protein binding of [(14)C]bicifadine was moderate in the mouse (80-86%) and higher in the rat and monkey (95-97%). In summary, bicifadine was well absorbed, extensively metabolized, and excreted via the urine and feces as metabolites.

Pharmacokinetics, disposition, and metabolism of bicifadine in humans.

Bicifadine [DOV 220,075; (+/-)-1-(4-methylphenyl)-3-azabicyclo-[3.1.0]hexane HCl)] is a non-narcotic analgesic that has proven to be effective for the treatment of acute pain in clinical studies. The pharmacokinetics, disposition, and metabolism of bicifadine were determined in eight healthy adult male subjects following a single oral dose of 200 mg of [(14)C]bicifadine in solution. The maximum concentration of total drug equivalents and bicifadine in plasma was at approximately 1 h; the elimination half-life was 2.6 and 1.6 h for radioactivity and bicifadine, respectively. Unchanged bicifadine represented 15% of the area under the concentration-time curve for total drug equivalents; the rest was due mainly to the lactam (M12), the acid (M3), and the lactam acid (M9). Total recovery of the dose was 92%, with most of the radioactivity recovered in the urine in the first 24 h; fecal excretion accounted for only 3.5% of the dose. Approximately 64% of the dose was metabolized to M9 and its acyl glucuronide; another 23% was recovered as M3 and its acyl glucuronide. Neither bicifadine nor M12 were detected in urine or feces. There were no reported serious or severe adverse events during the study.

[Pharmacokinetics and biodistribution of 3H-norcantharidin in mice].

A single dose of 3H-norcantharidin solution was intragastrically given, blood, tissues, urine and feces were collected as scheduled, and radioactivity in these samples was determined by tritium tracing method to investigate the pharmacokinetics, tissue distribution and excretion of norcantharidin in Kunming mice. The pharmacokinetic characteristics of norcantharidin were evaluated by DAS version 2.0. The blood concentration reached to maximum 0. 5 h after intragastric administration. The radioactivity in tissues was high in small intestine, gallbladder, stomach, adrenal gland, kidney, heart and uterus 15 minutes after administration, descending with time, and high in gallbladder, adrenal gland and uterus 3 hours post dosing. The 24 h accumulative excretion ratio of urine and feces were 65.40% and 1.33% respectively. 3H-norcantharidin was easily absorbed after orally given to mice, the radioactivity was high and existed for a long-time in gallbladder, adrenal gland and uterus, and low but also existed for a long-time in large intestine, thymus and fat tissue. 3H-norcantharidin was declined quickly in small intestine, stomach, kidney and heart, and occurred rarely in brain. Norcantharidin was excreted mainly by urinary route and seldom in feces, which may be the cause of the urinary stimulation side effects observed. Because the radioactivity measured were the sum of 3H labeled norcantharidin and its metabolites, further studies on the disposition of norcantharidin in mammal animals, on the separation or identification of metabolites and, if any, on their activities, are fairly needed.

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. Overall, 1 was well tolerated, with >94% of administered radioactivity excreted renally by 48 h postdose; lyophilization of all urine and plasma samples confirmed (3)H stability within [(3)H]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.

Metabolism, pharmacokinetics, and excretion of a nonpeptidic substance P receptor antagonist, ezlopitant, in normal healthy male volunteers: characterization of polar metabolites by chemical derivatization with dansyl chloride.

The excretion, biotransformation, and pharmacokinetics of ezlopitant [(2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl)-(5-isopropyl-2-methoxy-benzyl)-amine], a substance P receptor antagonist, were investigated in healthy male volunteers after oral administration of a single 200-mg (approximately 93 microCi/subject) dose of [(14)C]ezlopitant. The total recovery of administered radioactive dose was 82.8 +/- 5.1, with 32.0 +/- 4.2% in the urine and 50.8 +/- 1.4% in the feces. Mean observed maximal serum concentrations for ezlopitant and total radioactivity were achieved at approximately 2 h after oral administration; thus, ezlopitant was rapidly absorbed. Ezlopitant was extensively metabolized in humans, since no unchanged drug was detected in urine and feces. The major pathway of ezlopitant in humans was the result of the oxidation of the isopropyl side chain to form the omega-hydroxy and omega-1-hydroxy (M16) metabolites. M16 and omega,omega-1-dihydroxy (1,2-dihydroxy, M12) were identified as the major circulating metabolites accounting for 64.6 and 15.4% of total circulating radioactivity, respectively. In feces, the major metabolite M14 was characterized as the propionic acid metabolite and formed by further oxidation of the omega-hydroxy metabolite. The urinary metabolites were the result of cleaved metabolites caused by oxidative dealkylation of the 2-benzhydryl-1-aza-bicyclo[2.2.2]oct-3-yl moiety. The metabolites (M1A, M1B, and M4), approximately 34% of the total radioactivity in urine, were identified as benzyl amine derivatives. These were polar metabolites that were further characterized using the reaction with dansyl chloride to derivatize the primary amines and phenol moieties to less polar analytes. The other metabolites were the result of O-demethylation, dehydrogenation of the isopropyl group, and oxidation on the quinuclidine moiety.

Insect pheromones and precursors in female African elephant urine.

Using automated solid-phase dynamic extraction and gas chromatography-mass spectrometry, our search for urinary chemical signals from ovulatory female African elephants (Loxodonta africana) has revealed the bark beetle aggregation pheromones frontalin, exo-brevicomin, and endo-brevicomin, as well as their precursors and the aphid alarm pheromones (E,E)-alpha-farnesene and (E)-beta-farnesene. Enantiomeric ratios for brevicomins have been determined. Prior discovery of common insect/elephant pheromones in Asian elephants, namely, (Z)-7-dodecenyl acetate and frontalin, suggests that the present findings may yield valuable insights into chemical communication among African elephants.

Frontalin: a chemical message of musth in Asian elephants (Elephas maximus).

Musth is an important male phenomenon affecting many aspects of elephant society including reproduction. During musth, the temporal gland secretions (as well as the urine and breath) of adult male Asian elephants (Elephas maximus) discharge a variety of malodorous compounds together with the bicyclic ketal, frontalin. In contrast, teenage male elephants in musth release a sweet-smelling exudate from their facial temporal gland. We recently demonstrated that the concentration of frontalin becomes increasingly evident as male elephants mature. In the present study, we demonstrate that behaviors exhibited towards frontalin are consistent and dependent on the sex, developmental stage and physiological status of the responding conspecific individual. To examine whether frontalin functions as a chemical signal, perhaps even a pheromone, we bioassayed older and younger adult males, and luteal- and follicular-phase and pregnant females for their chemosensory and behavioral responses to frontalin. Adult males were mostly indifferent to frontalin, whereas subadult males were highly reactive, often exhibiting repulsion or avoidance. Female chemosensory responses to frontalin varied with hormonal state. Females in the luteal phase demonstrated low frequencies of responses, whereas pregnant females responded significantly more frequently, with varied types of responses including those to the palatal pits. Females in the follicular phase were the most responsive and often demonstrated mating-related behaviors subsequent to high chemosensory responses to frontalin. Our evidence strongly suggests that frontalin, a well-studied pheromone in insects, also functions as a pheromone in the Asian elephant: it exhibits all of the determinants that define a pheromone and evidently conveys some of the messages underlying the phenomenon of musth.

Disposition of radiolabeled ifetroban in rats, dogs, monkeys, and humans.

Ifetroban is a potent and selective thromboxane receptor antagonist. This study was conducted to characterize the pharmacokinetics, absolute bioavailability, and disposition of ifetroban after i.v. and oral administrations of [14C]ifetroban or [3H]ifetroban in rats (3 mg/kg), dogs (1 mg/kg), monkeys (1 mg/kg), and humans (50 mg). The drug was rapidly absorbed after oral administration, with peak plasma concentrations occurring between 5 and 20 min across species. Plasma terminal elimination half-life was approximately 8 h in rats, approximately 20 h in dogs, approximately 27 h in monkeys, and approximately 22 h in humans. Based on the steady-state volume of distribution, the drug was extensively distributed in tissues. Absolute bioavailability was 25, 35, 23, and 48% in rats, dogs, monkeys, and humans, respectively. Renal excretion was a minor route of elimination in all species, with the majority of the dose being excreted into the feces. After a single oral dose, urinary excretion accounted for 3% of the administered dose in rats and dogs, 14% in monkeys, and 27% in humans, with the remainder excreted in the feces. Extensive biliary excretion was observed in rats with the hydroxylated metabolite at the C-14 position being the major metabolite observed in rat bile. Ifetroban was extensively metabolized after oral administration. Approximately 40 to 50% of the radioactivity in rat and dog plasma was accounted for by parent drug whereas, in humans, approximately 60% of the plasma radioactivity was accounted for by ifetroban acylglucuronide.

Electrophysiological and biochemical responses of mouse vomeronasal receptor cells to urine-derived compounds: possible mechanism of action.

Receptor cells of the vomeronasal organ (VNO) are thought to detect pheromone-like molecules important for reproductive physiology. Several compounds derived from male mouse urine have been demonstrated to affect endocrine events in female mice. In the present study, the ability of these compounds to affect VNO activity was tested. In dissociated VNO cells held under voltage clamp conditions, application of dehydro-exo-brevicomin (DHB) evoked an outward current at negative holding potentials and an inward current at positive holding potentials. Under current clamp, DHB reduced action potential firing. Since DHB application caused a decrease in membrane conductance, this compound appeared to act by reducing inward current through closing an ion channel. Biochemical experiments tested the effects of DHB and 2-(sec-butyl)-4,5-dihydrothiazole (SBT) on cAMP levels in the VNO. A mixture of DHB and SBT decreased cAMP levels in VNO sensory tissue and had no effect on VNO non-sensory tissue. The results suggest that pheromones have an inhibitory influence on action potential generation and on cAMP levels in receptor cells of the VNO.