The disposition of veliparib (R)-2-(2-methylpyrrolidin-2-yl)-1H-benzodimidazole-4-carboxamide, ABT-888, a novel and potent inhibitor of poly(ADP-ribose) polymerase for the treatment of cancers, was ...investigated in rats and dogs after intravenous and oral administration of (3)Hveliparib and compared with that of humans. Veliparib absorption was high. Dosed radioactivity was widely distributed in rat tissues. The majority of drug-related material was excreted in urine as unchanged drug (approximately 54, 41, and 70% of the dose in rats, dogs, and humans, respectively). A lactam M8 and an amino acid M3 were two major excretory metabolites in animals. In the circulation of animals and humans, veliparib was the major drug-related component, and M8 was one of the major metabolites. Monooxygenated metabolite M2 was significant in the rat and dog, and M3 was also significant in the dog. Veliparib biotransformation occurred on the pyrrolidine moiety via formation of a lactam, an amino acid, and an N-carbamoyl glucuronide, in addition to oxidation on benzoimidazole carboxamide and sequential glucuronidation. In vitro experiments using recombinant human cytochrome P450 (P450) enzymes identified CYP2D6 as the major enzyme metabolizing veliparib with minor contributions from CYP1A2, 2C19, and 3A4. Veliparib did not inhibit or induce the activities of major human P450s. Veliparib was a weak P-glycoprotein (P-gp) substrate, showing no P-gp inhibition. Taken together, these studies indicate a low potential for veliparib to cause clinically significant P-gp or P450-mediated drug-drug interactions (DDIs). Overall, the favorable dispositional and DDI profiles of veliparib should be beneficial to its safety and efficacy.
The tobacco-specific nitrosamines N‘-nitrosonornicotine (NNN, 1) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2) are potent carcinogens in rodents. Bioactivation of NNN and NNK by ...cytochrome P450 enzymes generates a pyridyloxobutylating agent 6, which alkylates DNA to produce pyridyloxobutyl (POB)−DNA adducts. POB−DNA adduct formation plays a critical role in NNN and NNK carcinogenicity in rodents. To further investigate the significance of this pathway, we developed a high-performance liquid chromatography−electrospray ionization−tandem mass spectrometry (HPLC-ESI-MS/MS) method for quantitative analysis of four POB−DNA adducts with known structures. The corresponding deuterated analogues were synthesized and used as internal standards. DNA samples, spiked with internal standards, were subjected to neutral thermal hydrolysis followed by enzymatic hydrolysis. The hydrolysates were partially purified by solid phase extraction prior to HPLC-ESI-MS/MS analysis. The method was accurate and precise. Excellent sensitivity was achieved, especially for O 2-4-(3-pyridyl)-4-oxobut-1-ylthymidine (O2-POB-dThd, 11) with a detection limit of 100 amol per mg DNA. DNA samples treated with different concentrations of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3) were subjected to HPLC-ESI-MS/MS analysis. 7-4-(3-Pyridyl)-4-oxobut-1-ylguanine (7-POB-Gua, 12) was the most abundant adduct, followed by O 6-4-(3-pyridyl)-4-oxobut-1-yl-2‘-deoxyguanosine (O 6-POB-dGuo, 8), O 2-POB-dThd, and O 2-4-(3-pyridyl)-4-oxobut-1-ylcytosine (O 2-POB-Cyt, 13). Lung and liver DNA isolated from NNK-treated rats were analyzed. Consistent with the in vitro data, 7-POB-Gua was the major POB−DNA adduct formed in vivo. However, levels of O 6-POB-dGuo were the lowest of the four adducts analyzed, suggesting efficient repair of this adduct in vivo. In contrast to the other three adducts, O 6-POB-dGuo was more abundant in lung than in liver. O 2-POB-dThd appeared to be poorly repaired in vivo, and its levels were comparable to those of 7-POB-Gua. The results of this study provide a sensitive HPLC-ESI-MS/MS method for comprehensive quantitation of four POB−DNA adducts, support an important role of O 6-POB-dGuo in NNK lung tumorigenicity in rats, and suggest that O 2-POB-dThd may be a useful tobacco-specific DNA biomarker for future tobacco carcinogenesis studies.
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Aberrant activation of calpain has been observed in various pathophysiological disorders including neurodegenerative diseases such as Alzheimer’s Disease. Here we describe our efforts ...on ketoamide-based 1-benzyl-5-oxopyrrolidine-2-carboxamides as a novel series of highly selective calpain inhibitors mitigating the metabolic liability of carbonyl reduction. The most advanced compound from this new series, namely A-1212805 (ABT-957, Alicapistat) proceeded to clinical phase I studies.
At the time of writing, although siRNA therapeutics are approved for human use, no official regulatory guidance specific to this modality is available. In the absence of guidance, preclinical ...development for siRNA followed a hybrid of the small molecule and biologics guidance documents. However, siRNA differs significantly from small molecules and protein-based biologics in its physicochemical, absorption, distribution, metabolism and excretion properties, and its mechanism of action. Consequently, certain reports typically included in filing packages for small molecule or biologics may benefit from adaption, or even omission, from an siRNA filing. In this white paper, members of the 'siRNA working group' in the IQ Consortium compile a list of reports included in approved siRNA filing packages and discuss the relevance of two in vitro reports-the plasma protein binding evaluation and the drug-drug interaction risk assessment-to support siRNA regulatory filings. Publicly available siRNA approval packages and the literature were systematically reviewed to examine the role of siRNA plasma protein binding and drug-drug interactions in understanding pharmacokinetic/pharmacodynamic relationships, safety and translation. The findings are summarized into two decision trees to help guide industry decide when in vitro siRNA plasma protein binding and drug-drug interaction studies are warranted.
Calpain overactivation has been implicated in a variety of pathological disorders including ischemia/reperfusion injury, cataract formation, and neurodegenerative diseases such as Alzheimer’s disease ...(AD). Herein we describe our efforts leading to the identification of ketoamide-based 2-(3-phenyl-1H-pyrazol-1-yl)nicotinamides as potent and reversible inhibitors of calpain with high selectivity versus related cysteine protease cathepsins, other proteases, and receptors. Broad efficacy in a set of preclinical models relevant to AD suggests that inhibition of calpain represents an attractive approach with potential benefit for the treatment of AD.
Novel dual GIP and GLP-1 receptor agonist, tirzepatide (TZP), is being developed as a potential weekly treatment for type 2 diabetes (T2DM), weight management and nonalcoholic steatohepatitis. The ...absorption, metabolism and excretion of a single subcutaneous (SC) dose of 14C-tirzepatide was investigated in Sprague Dawley rat and cynomolgus monkey. In addition, tissue distribution of 14C-tirzepatide was assessed in quantitative whole-body autoradiography (QWBA) study in pigmented Long Evans rat following a single SC dose.
14C-Tirzepatide was prepared by incorporating four 14C’s in the mini-PEG linker between the peptide backbone and the di-acid chain to provide a specific activity of ∼ 40 µCi/mg. Following a single SC dose of 14C-tirzepatide in rat (3 mg/kg) and monkey (0.5 mg/kg), total radioactivity recovery was > 97% over the course of study (336 hours for rat and 672 hours for monkey). The dosed radioactivity was similarly excreted via urine and feces in rats and monkeys. Metabolism of 14C-tirzepatide was characterized in plasma and excreta. Parent drug was the major component in circulation accounting for approximately 87% of total radioactivity in rat and 84% in monkey. Tirzepatide was primarily metabolized via catabolism of the peptide backbone and β-oxidation of the di-acid chain.
Following a single SC dose of 14C-tirzepatide in rats (3 mg/kg), radioactivity was distributed to tissues as early as the first collection time point at 1-hour post dose. The tissues with the highest radioactivity concentrations were observed in the dose site, kidney, cecum, urinary bladder, intervertebral ligaments, arterial wall, lungs, and liver, generally at 12 to 48 hours post dose.
Disclosure
J. Martin: Employee; Self; Eli Lilly and Company. K. Cassidy: Employee; Self; Eli Lilly and Company. B. Czeskis: None. J. Alberts: Employee; Self; Eli Lilly and Company. Y. Lao: Employee; Spouse/Partner; Eli Lilly and Company. J. Gluff: None. A.M. Niedenthal: None.
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
Cvenetoclax 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-pyrrolo2,3-bpyridin-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-octahydropyrazino2,1-b1,3benzoxazin-2-yl-N-3-nitro-4-(tetrahydropyran-4-ylmethylamino)phenylsulfonyl-2-(1H-pyrrolo2,3-bpyridin-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.
Sodium caprate (C10) has been widely evaluated as an intestinal permeation enhancer for the oral delivery of macromolecules. However, the effect of C10 on the intestinal absorption of peptides with ...different physicochemical properties and its permeation-enhancing effect in vivo remains to be understood. Here, we evaluated the effects of C10 on intestinal absorption in rats with a glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GIP-GLP1) dual agonist peptide (LY) and semaglutide with different enzymatic stabilities and self-association behaviors as well as the oral exposure of the LY peptide in minipigs. Furthermore, we investigated the mechanism of action (MoA) of C10 for improving the intestinal absorption of the LY peptide in vivo via live imaging of the rat intestinal epithelium and tissue distribution of the LY peptide in minipigs. The LY peptide showed higher proteolytic stability in pancreatin and was a monomer in solution compared to that in semaglutide. C10 increased in vitro permeability in the minipig intestinal organoid monolayer to a greater extent for the LY peptide than for semaglutide. In the rat jejunal closed-loop model, C10 increased the absorption of LY peptide better than that of semaglutide, which might be attributed to higher in vitro proteolytic stability and permeability of the LY peptide. Using confocal live imaging, we observed that C10 enabled the rapid oral absorption of a model macromolecule (FD4) in the rat intestine. In the duodenum tissues of minipigs, C10 was found to qualitatively reduce the tight junction protein level and allow peptide uptake to the intestinal cells. C10 decreased the transition temperature of the artificial lipid membrane, indicating an increase in membrane fluidity, which is consistent with the above in vivo imaging results. These data indicated that the LY’s favorable physicochemical properties combined with the effects of C10 on the intestinal mucosa resulted in an ∼2% relative bioavailability in minipigs.
The development of glycine transporter 1 (GlyT1) inhibitors may offer putative treatments for schizophrenia and other disorders associated with hypofunction of the glutaminergic N-methyl-d-aspartate ...(NMDA) receptor. Herein, we describe the synthesis and biological evaluation of a series of 3,4-disubstituted pyrrolidine sulfonamides as competitive GlyT1 inhibitors that arose from de novo scaffold design. Relationship of chemical structure to drug–drug interaction (DDI) and bioactivation was mechanistically investigated. Murine studies were strategically incorporated into the screening funnel to provide early assessments of in vivo target occupancy (TO) by ex vivo binding studies. Advanced compounds derived from iterative structure–activity relationship (SAR) studies possessed high potency in ex vivo binding studies and good brain penetration, promising preliminary in vivo efficacy, acceptable preclinical pharmacokinetics, and manageable DDI and bioactivation liabilities.
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