Biotransformation research advances - 2023 year in review.

This annual review marks the eighth in the series starting with Baillie et al. (2016) Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation. Its format is to highlight important aspects captured in synopsis followed by a commentary with relevant figure and references.

Bioactivation and reactivity research advances-2023 year in review.

Advances in the field of bioactivation have significantly contributed to our understanding and prediction of drug-induced liver injury (DILI). It has been established that many adverse drug reactions, including DILI, are associated with the formation and reactivity of metabolites. Modern methods allow us to detect and characterize these reactive metabolites in earlier stages of drug development, which helps anticipate and circumvent the potential for DILI. Improved in silico models and experimental techniques that better reflect in vivo environments are enhancing predictive capabilities for DILI risk. Further, studies on the mechanisms of bioactivation, including enzyme interactions and the role of individual genetic differences, have provided valuable insights for drug optimizations. Cumulatively, this progress is continually refining our approaches to drug safety evaluation and personalized medicine.Shuai Wang and Cyrus Khojasteh, on behalf of the authors.

Mechanisms of Nitrosamine Mutagenicity and Their Relationship to Rodent Carcinogenic Potency.

A thorough literature review was undertaken to understand how the pathways of N-nitrosamine transformation relate to mutagenic potential and carcinogenic potency in rodents. Empirical and computational evidence indicates that a common radical intermediate is created by CYP-mediated hydrogen abstraction at the α-carbon; it is responsible for both activation, leading to the formation of DNA-reactive diazonium species, and deactivation by denitrosation. There are competing sites of CYP metabolism (e.g., ß-carbon), and other reactive species can form following initial bioactivation, although these alternative pathways tend to decrease rather than enhance carcinogenic potency. The activation pathway, oxidative dealkylation, is a common reaction in drug metabolism and evidence indicates that the carbonyl byproduct, e.g., formaldehyde, does not contribute to the toxic properties of N-nitrosamines. Nitric oxide (NO), a side product of denitrosation, can similarly be discounted as an enhancer of N-nitrosamine toxicity based on carcinogenicity data for substances that act as NO-donors. However, not all N-nitrosamines are potent rodent carcinogens. In a significant number of cases, there is a potency overlap with non-N-nitrosamine carcinogens that are not in the Cohort of Concern (CoC; high-potency rodent carcinogens comprising aflatoxin-like-, N-nitroso-, and alkyl-azoxy compounds), while other N-nitrosamines are devoid of carcinogenic potential. In this context, mutagenicity is a useful surrogate for carcinogenicity, as proposed in the ICH M7 (R2) (2023) guidance. Thus, in the safety assessment and control of N-nitrosamines in medicines, it is important to understand those complementary attributes of mechanisms of mutagenicity and structure-activity relationships that translate to elevated potency versus those which are associated with a reduction in, or absence of, carcinogenic potency.

Bioactivation and reactivity research advances - 2022 year in review‡.

With the 50th year mark since the launch of Drug Metabolism and Disposition journal, the field of drug metabolism and bioactivation has advanced exponentially in the past decades (Guengerich 2023).This has, in a major part, been due to the continued advances across the whole spectrum of applied technologies in hardware, software, machine learning (ML), and artificial intelligence (AI). LC-MS platforms continue to evolve to support key applications in the field, and automation is also improving the accuracy, precision, and throughput of these supporting assays. In addition, sample generation and processing is being aided by increased diversity and quality of reagents and bio-matrices so that what is being analyzed is more relevant and translatable. The application of in silico platforms (applied software, ML, and AI) is also making great strides, and in tandem with the more traditional approaches mentioned previously, is significantly advancing our understanding of bioactivation pathways and how these play a role in toxicity. All of this continues to allow the area of bioactivation to evolve in parallel with associated fields to help bring novel or improved medicines to patients with urgent or unmet needs.Shuai Wang and Cyrus Khojasteh, on behalf of the authors.

Biotransformation research advances - 2022 year in review.

This annual review is the eighth of its kind since 2016 (Baillie et al. 2016, Khojasteh et al. 2017, Khojasteh et al. 2018, Khojasteh et al. 2019, Khojasteh et al. 2020, Khojasteh et al. 2021, Khojasteh et al. 2022). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation.

Mitigating a Bioactivation Liability with an Azetidine-Based Inhibitor of Diacylglycerol Acyltransferase 2 (DGAT2) En Route to the Discovery of the Clinical Candidate Ervogastat.

We recently disclosed SAR studies on systemically acting, amide-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) that addressed metabolic liabilities with the liver-targeted DGAT2 inhibitor PF-06427878. Despite strategic placement of a nitrogen atom in the dialkoxyaromatic ring in PF-06427878 to evade oxidative O-dearylation, metabolic intrinsic clearance remained high due to extensive piperidine ring oxidation as exemplified with compound 1. Piperidine ring modifications through alternate N-linked heterocyclic ring/spacer combination led to azetidine 2 that demonstrated lower intrinsic clearance. However, 2 underwent a facile cytochrome P450 (CYP)-mediated α-carbon oxidation followed by azetidine ring scission, resulting in the formation of ketone (M2) and aldehyde (M6) as stable metabolites in NADPH-supplemented human liver microsomes. Inclusion of GSH or semicarbazide in microsomal incubations led to the formation of Cys-Gly-thiazolidine (M3), Cys-thiazolidine (M5), and semicarbazone (M7) conjugates, which were derived from reaction of the nucleophilic trapping agents with aldehyde M6. Metabolites M2 and M5 were biosynthesized from NADPH- and l-cysteine-fortified human liver microsomal incubations with 2, and proposed metabolite structures were verified using one- and two-dimensional NMR spectroscopy. Replacement of the azetidine substituent with a pyridine ring furnished 8, which mitigated the formation of the electrophilic aldehyde metabolite, and was a more potent DGAT2 inhibitor than 2. Further structural refinements in 8, specifically introducing amide bond substituents with greater metabolic stability, led to the discovery of PF-06865571 (ervogastat) that is currently in phase 2 clinical trials for the treatment of nonalcoholic steatohepatitis.

Species differences in plasma protein binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease inhibitor nirmatrelvir.

Plasma protein binding (PPB) studies on the SARS-CoV-2 main protease inhibitor nirmatrelvir revealed considerable species differences primarily in dog and rabbit, which prompted further investigations into the biochemical basis for these differences.The unbound fraction (fu) of nirmatrelvir in dog and rabbit plasma was concentration (2-200 µM)-dependent (dog fu,p 0.024-0.69, rabbit fu,p 0.010-0.82). Concentration (0.1-100 µM)-dependent binding in serum albumin (SA) (fu,SA 0.040-0.82) and alpha-1-acid glycoprotein (AAG) (fu,AAG 0.050-0.64) was observed in dogs. Nirmatrelvir showed minimal binding to rabbit SA (1-100 µM: fu,SA 0.70-0.79), while binding to rabbit AAG was concentration-dependent (0.1-100 µM: fu,AAG 0.024-0.66). In contrast, nirmatrelvir (2 µM) revealed minimal binding (fu,AAG 0.79-0.88) to AAG from rat and monkeys. Nirmatrelvir showed minimal-to-moderate binding to SA (1-100 µM; fu,SA 0.70-1.0) and AAG (0.1-100 µM; fu,AAG 0.48-0.58) from humans across tested concentrations.Nirmatrelvir molecular docking studies using published crystal structures and homology models of human and preclinical species SA and AAG were used to rationalise the species differences to plasma proteins. This suggested that species differences in PPB are primarily driven by molecular differences in albumin and AAG resulting in differences in binding affinity.

Biotransformation novel advances - 2021 year in review.

Biotransformation field is constantly evolving with new molecular structures and discoveries of metabolic pathways that impact efficacy and safety. Recent review by Kramlinger et al. (2022) nicely captures the future (and the past) of highly impactful science of biotransformation (see the first article). Based on the selected articles, this review was categorized into three sections: (1) new modalities biotransformation, (2) drug discovery biotransformation, and (3) drug development biotransformation (Table 1).

Future of Biotransformation Science in the Pharmaceutical Industry.

Over the past decades, the number of scientists trained in departments dedicated to traditional medicinal chemistry, biotransformation and/or chemical toxicology have seemingly declined. Yet, there remains a strong demand for such specialized skills in the pharmaceutical industry, particularly within drug metabolism/pharmacokinetics (DMPK) departments. In this position paper, the members of the Biotransformation, Mechanisms, and Pathways Focus Group (BMPFG) steering committee reflect on the diverse roles and responsibilities of scientists trained in the biotransformation field in pharmaceutical companies and contract research organizations. The BMPFG is affiliated with the International Society for the Study of Xenobiotics (ISSX) and was specifically created to promote the exchange of ideas pertaining to topics of current and future interest involving the metabolism of xenobiotics (including drugs). The authors also delve into the relevant education and diverse training skills required to successfully nurture the future cohort of industry biotransformation scientists and guide them toward a rewarding career path. The ability of scientists with a background in biotransformation and organic chemistry to creatively solve complex drug metabolism problems encountered during research and development efforts on both small and large molecular modalities is exemplified in five relevant case studies. Finally, the authors stress the importance and continued commitment to training the next generation of biotransformation scientists who are not only experienced in the metabolism of conventional small molecule therapeutics, but are also equipped to tackle emerging challenges associated with new drug discovery modalities including peptides, protein degraders, and antibodies. SIGNIFICANCE STATEMENT: Biotransformation and mechanistic drug metabolism scientists are critical to advancing chemical entities through discovery and development, yet the number of scientists academically trained for this role is on the decline. This position paper highlights the continuing demand for biotransformation scientists and the necessity of nurturing creative ways to train them and guarantee the future growth of this field.

Transporter-Enzyme Interplay in the Pharmacokinetics of PF-06835919, A First-in-class Ketohexokinase Inhibitor for Metabolic Disorders and Non-alcoholic Fatty Liver Disease.

Excess dietary fructose consumption promotes metabolic dysfunction thereby increasing the risk of obesity, type 2 diabetes, non-alcoholic steatohepatitis (NASH), and related comorbidities. PF-06835919, a first-in-class ketohexokinase (KHK) inhibitor, showed reversal of such metabolic disorders in preclinical models and clinical studies, and is under clinical development for the potential treatment of NASH. In this study, we evaluated the transport and metabolic pathways of PF-06835919 disposition and assessed pharmacokinetics in preclinical models. PF-06835919 showed active uptake in cultured primary human hepatocytes, and substrate activity to organic anion transporter (OAT)2 and organic anion transporting-polypeptide (OATP)1B1 in transfected cells. "SLC-phenotyping" studies in human hepatocytes suggested contribution of passive uptake, OAT2- and OATP1B-mediated transport to the overall uptake to be about 15%, 60% and 25%, respectively. PF-06835919 showed low intrinsic metabolic clearance in vitro, and was found to be metabolized via both oxidative pathways (58%) and acyl glucuronidation (42%) by CYP3A, CYP2C8, CYP2C9 and UGT2B7. Following intravenous dosing, PF-06835919 showed low clearance (0.4-1.3 mL/min/kg) and volume of distribution (0.17-0.38 L/kg) in rat, dog and monkey. Human oral pharmacokinetics are predicted within 20% error when considering transporter-enzyme interplay in a PBPK model. Finally, unbound liver-to-plasma ratio (Kpuu) measured in vitro using rat, NHP and human hepatocytes was found to be approximately 4, 25 and 10, respectively. Similarly, liver Kpuu in rat and monkey following intravenous dosing of PF-06835919 was found to be 2.5 and 15, respectively, and notably higher than the muscle and brain Kpuu, consistent with the active uptake mechanisms observed in vitro. Significance Statement This work characterizes the transport/metabolic pathways in the hepatic disposition of PF-06835919, a first-in-class KHK inhibitor for the treatment of metabolic disorders and NASH. Phenotyping studies using transfected systems, human hepatocytes and liver microsomes signifies the role of OAT2 and OATP1B1 in the hepatic uptake and multiple enzymes in the metabolism of PF-06835919. Data presented suggest hepatic transporter-enzyme interplay in determining its systemic concentrations and potential enrichment in liver, a target site for KHK inhibition.

Disposition of Nirmatrelvir, an Orally Bioavailable Inhibitor of SARS-CoV-2 3C-Like Protease, across Animals and Humans.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease inhibitor PF-07321332 (nirmatrelvir), in combination with ritonavir (Paxlovid), was recently granted emergency use authorization by multiple regulatory agencies for the treatment of coronavirus disease 2019 (COVID-19) in adults and pediatric patients. Disposition studies on nirmatrelvir in animals and in human reagents, which were used to support clinical studies, are described herein. Plasma clearance was moderate in rats (27.2 ml/min per kg) and monkeys (17.1 ml/min per kg), resulting in half-lives of 5.1 and 0.8 hours, respectively. The corresponding oral bioavailability was moderate in rats (34%-50%) and low in monkeys (8.5%), primarily due to oxidative metabolism along the gastrointestinal tract in this species. Nirmatrelvir demonstrated moderate plasma protein binding in rats, monkeys, and humans with mean unbound fractions ranging from 0.310 to 0.478. The metabolism of nirmatrelvir was qualitatively similar in liver microsomes and hepatocytes from rats, monkeys, and humans; prominent metabolites arose via cytochrome P450 (CYP450)-mediated oxidations on the P1 pyrrolidinone ring, P2 6,6-dimethyl-3-azabicyclo[3.1.0]hexane, and the tertiary-butyl group at the P3 position. Reaction phenotyping studies in human liver microsomes revealed that CYP3A4 was primarily responsible (fraction metabolized = 0.99) for the oxidative metabolism of nirmatrelvir. Minor clearance mechanisms involving renal and biliary excretion of unchanged nirmatrelvir were also noted in animals and in sandwich-cultured human hepatocytes. Nirmatrelvir was a reversible and time-dependent inhibitor as well as inducer of CYP3A activity in vitro. First-in-human pharmacokinetic studies have demonstrated a considerable boost in the oral systemic exposure of nirmatrelvir upon coadministration with the CYP3A4 inhibitor ritonavir, consistent with the predominant role of CYP3A4 in nirmatrelvir metabolism. SIGNIFICANCE STATEMENT: The manuscript describes the preclinical disposition, metabolism, and drug-drug interaction potential of PF-07321332 (nirmatrelvir), an orally active peptidomimetic-based inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease, which has been granted emergency use authorization by multiple regulatory agencies around the globe for the treatment of coronavirus disease 2019 (COVID-19) in COVID-19-positive adults and pediatric patients who are at high risk for progression to severe COVID-19, including hospitalization or death.

Practical and Science-Based Strategy for Establishing Acceptable Intakes for Drug Product N-Nitrosamine Impurities.

The potential for N-nitrosamine impurities in pharmaceutical products presents a challenge for the quality management of medicinal products. N-Nitrosamines are considered cohort-of-concern compounds due to the potent carcinogenicity of many of the structurally simple chemicals within this structural class. In the past 2 years, a number of drug products containing certain active pharmaceutical ingredients have been withdrawn or recalled from the market due to the presence of carcinogenic low-molecular-weight N,N-dialkylnitrosamine impurities. Regulatory authorities have issued guidance to market authorization holders to review all commercial drug substances/products for the potential risk of N-nitrosamine impurities, and in cases where a significant risk of N-nitrosamine impurity is identified, analytical confirmatory testing is required. A key factor to consider prior to analytical testing is the estimation of the daily acceptable intake (AI) of the N-nitrosamine impurity. A significant proportion of N-nitrosamine drug product impurities are unique/complex structures for which the development of low-level analytical methods is challenging. Moreover, these unique/complex impurities may be less potent carcinogens compared to simple nitrosamines. In the present work, our objective was to derive AIs for a large number of complex N-nitrosamines without carcinogenicity data that were identified as potential low-level impurities. The impurities were first cataloged and grouped according to common structural features, with a total of 13 groups defined with distinct structural features. Subsequently, carcinogenicity data were reviewed for structurally related N-nitrosamines relevant to each of the 13 structural groups and group AIs were derived conservatively based on the most potent N-nitrosamine within each group. The 13 structural group AIs were used as the basis for assigning AIs to each of the structurally related complex N-nitrosamine impurities. The AIs of several N-nitrosamine groups were found to be considerably higher than those for the simple N,N-dialkylnitrosamines, which translates to commensurately higher analytical method detection limits.

Pharmacokinetics, mass balance, metabolism, and excretion of the liver-targeted acetyl-CoA carboxylase inhibitor PF-05221304 (clesacostat) in humans.

The disposition of the hepatoselective ACC inhibitor PF-05221304 (Clesacostat) was studied after a single 50-mg oral dose of [14C]-PF-05221304 to healthy human subjects.Mass balance was achieved with 89.9% of the administered dose recovered in urine and faeces, over the 11-day study period. The total administered radioactivity excreted in faeces and urine was 81.7 and 8.2%, respectively. Unchanged PF-05221304 accounted for 35.6% of the radioactive dose in faeces, suggesting ∼64% of the administered dose was absorbed.PF-05221304 was principally metabolised via oxidative and reductive pathways involving: (a) N-dealkylation, (b) isopropyl group monohydroxylation to yield enantiomeric metabolites (M2a and M2b), (c) hydroxylation on the 3-azaspiro[5.5]undecan-8-one moiety to metabolites M5 and 519c, and (d) carbonyl group reduction to enantiomeric alcohol metabolites M3, and M4. Secondary metabolites (521a, 521b, and 533), derived from a combination of oxidation and reduction of the primary metabolites accounted for ∼14.8% of the dose. In plasma, unchanged PF-05221304 represented 96.1% circulating radioactivity. Metabolites M1, M2b, and M2a represented 1.94, 1.76, and 0.18% of circulating radioactivity, respectively.Overall, these data suggest that PF-05221304 is well absorbed in humans and eliminated largely via phase I metabolism.

Comprehensive Nonclinical Safety Assessment of Nirmatrelvir Supporting Timely Development of the SARS-COV-2 Antiviral Therapeutic, Paxlovid™.

COVID-19 is a potentially fatal infection caused by the SARS-CoV-2 virus. The SARS-CoV-2 3CL protease (Mpro) is a viral enzyme essential for replication and is the target for nirmatrelvir. Paxlovid (nirmatrelvir co-administered with the pharmacokinetic enhancer ritonavir) showed efficacy in COVID-19 patients at high risk of progressing to hospitalization and/or death. Nonclinical safety studies with nirmatrelvir are essential in informing benefit-risk of Paxlovid and were conducted to support clinical development. In vivo safety pharmacology assessments included a nervous system/pulmonary study in rats and a cardiovascular study in telemetered monkeys. Potential toxicities were assessed in repeat dose studies of up to 1 month in rats and monkeys. Nirmatrelvir administration (1,000 mg/kg, p.o.) to male rats produced transient increases in locomotor activity and respiratory rate but did not affect behavioral endpoints in the functional observational battery. Cardiovascular effects in monkeys were limited to transient increases in blood pressure and decreases in heart rate, observed only at the highest dose tested (75 mg/kg per dose b.i.d; p.o.). Nirmatrelvir did not prolong QTc-interval or induce arrhythmias. There were no adverse findings in repeat dose toxicity studies up to 1 month in rats (up to 1,000 mg/kg daily, p.o.) or monkeys (up to 600 mg/kg daily, p.o.). Nonadverse, reversible clinical pathology findings without clinical or microscopic correlates included prolonged coagulation times at ≥60 mg/kg in rats and increases in transaminases at 600 mg/kg in monkeys. The safety pharmacology and nonclinical toxicity profiles of nirmatrelvir support clinical development and use of Paxlovid for treatment of COVID-19.

Novel advances in biotransformation and bioactivation research - 2020 year in review.

This annual review is the sixth of its kind since 2016 (see references). Our objective is to explore and share articles which we deem influential and significant in the field of biotransformation and bioactivation. These fields are constantly evolving with new molecular structures and discoveries of corresponding pathways for metabolism that impact relevant drug development with respect to efficacy and safety. Based on the selected articles, we created three sections: (1) drug design, (2) metabolites and drug metabolizing enzymes, and (3) bioactivation and safety (Table 1). Unlike in years past, more biotransformation experts have joined and contributed to this effort while striving to maintain a balance of authors from academic and industry settings.[Table: see text].

Organic Anion-Transporting Polypeptide 1B1/1B3-Mediated Hepatic Uptake Determines the Pharmacokinetics of Large Lipophilic Acids: In Vitro-In Vivo Evaluation in Cynomolgus Monkey.

It is generally presumed that uptake transport mechanisms are of limited significance in hepatic clearance for lipophilic or high passive-permeability drugs. In this study, we evaluated the mechanistic role of the hepato-selective organic anion-transporting polypeptides (OATPs) 1B1/1B3 in the pharmacokinetics of compounds representing large lipophilic acid space. Intravenous pharmacokinetics of 16 compounds with molecular mass ∼400-730 Da, logP ∼3.5-8, and acid pKa <6 were obtained in cynomolgus monkey after dosing without and with a single-dose rifampicin-OATP1B1/1B3 probe inhibitor. Rifampicin (30 mg/kg oral) significantly (P < 0.05) reduced monkey clearance and/or steady-state volume of distribution (VDss) for 15 of 16 acids evaluated. Additionally, clearance of danoprevir was reduced by about 35%, although statistical significance was not reached. A significant linear relationship was noted between the clearance ratio (i.e., ratio of control to treatment groups) and VDss ratio, suggesting hepatic uptake contributes to the systemic clearance and distribution simultaneously. In vitro transport studies using primary monkey and human hepatocytes showed uptake inhibition by rifampicin (100 µM) for compounds with logP ≤6.5 but not for the very lipophilic acids (logP > 6.5), which generally showed high nonspecific binding in hepatocyte incubations. In vitro uptake clearance and fraction transported by OATP1B1/1B3 (ft,OATP1B) were found to be similar in monkey and human hepatocytes. Finally, for compounds with logP ≤6.5, good agreement was noted between in vitro ft,OATP1B and clearance ratio (as well as VDss ratio) in cynomolgus monkey. In conclusion, this study provides mechanistic evidence for the pivotal role of OATP1B-mediated hepatic uptake in the pharmacokinetics across a wide, large lipophilic acid space. SIGNIFICANCE STATEMENT: This study provides mechanistic insight into the pharmacokinetics of a broad range of large lipophilic acids. Organic anion-transporting polypeptides 1B1/1B3-mediated hepatic uptake is of key importance in the pharmacokinetics and drug-drug interactions of almost all drugs and new molecular entities in this space. Diligent in vitro and in vivo transport characterization is needed to avoid the false negatives often noted because of general limitations in the in vitro assays while handling compounds with such physicochemical attributes.

Metabolism and excretion of (S)-6-(3-cyclopentyl-2-(4-trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid (PF-04991532), a hepatoselective glucokinase activator, in humans: confirmation of the MIST potential noted in first-in-Human metabolite scouting studies.

1. The absorption, metabolism, and excretion of a single oral 450-mg dose of [14C]-(S)-6-(3-cyclopentyl-2-(4-trifluoromethyl)-1H-imidazol-1-yl)propanamido)nicotinic acid (PF-04991532), a hepatoselective glucokinase activator, was investigated in humans. Mass balance was achieved with ∼94.6% of the administered dose recovered in urine and feces. The total administered radioactivity excreted in feces and urine was 70.6% and 24.1%, respectively. Unchanged PF-04991532 collectively accounted for ∼47.2% of the dose excreted in feces and urine, suggestive of moderate metabolic elimination in humans. 2. The biotransformation pathways involved acyl glucuronidation (M1), amide bond hydrolysis (M3), and CYP3A4-mediated oxidative metabolism on the cyclopentyl ring in PF-04991532 yielding monohydroxylated isomers (M2a-d). Unchanged PF-04991532 was the major circulating component (64.4% of total radioactivity) whereas M2a-d collectively represented 28.9% of the total plasma radioactivity. 3. Metabolites M2a-d were not detected systemically in rats and dogs, the preclinical species for the toxicological evaluation of PF-04991532. In contrast, cynomologus monkeys dosed orally with unlabeled PF-04991532 revealed M2a-d in circulation, whose UV abundance was comparable to the profile in humans. This observation suggested that monkeys could potentially serve as a non-rodent alternative for studying the toxicity of PF-04991532 and its metabolites M2a-d. 4. The present results are in excellent agreement with our previously generated metabolite scouting data, which provided preliminary evidence for the disproportionate metabolism of PF-04991532 in humans.

Predicting toxicities of reactive metabolite-positive drug candidates.

Because of the inability to predict and quantify the risk of idiosyncratic adverse drug reactions (IADRs) and because reactive metabolites (RMs) are thought to be responsible for the pathogenesis of some IADRs, the potential for RM formation within new chemical entities is routinely examined with the ultimate goal of eliminating or reducing the liability through iterative design. Likewise, avoidance of structural alerts is almost a standard practice in drug design. However, the perceived safety concerns associated with the use of structural alerts and/or RM screening tools as standalone predictors of toxicity risks may be overexaggerated. Numerous marketed drugs form RMs but do not cause idiosyncratic toxicity. In this review article, we present a critique of the structural alert/RM concept as applied in drug discovery and evaluate the evidence linking structural alerts and RMs to observed toxic effects. Pragmatic risk mitigation strategies to aid the advancement of drug candidates that carry a RM liability are also discussed.

Establishing Transcriptional Signatures to Differentiate PXR-, CAR-, and AhR-Mediated Regulation of Drug Metabolism and Transport Genes in Cryopreserved Human Hepatocytes.

The potential for drug-drug interactions (DDIs) arising from transcriptional regulation of drug-disposition genes via activation of nuclear receptors (NRs), such as pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR), remains largely unexplored, as highlighted in a recent guidance document from the European Medicines Agency. The goal of this research was to establish PXR-/CAR-/AhR-specific drug-metabolizing enzyme (DME) and transporter gene expression signatures in sandwich-cultured cryopreserved human hepatocytes using selective activators of PXR (rifampin), CAR (CITCO), and AhR (omeprazole). Dose response for ligand-induced changes to 38 major human DMEs and critical hepatobiliary transporters were assessed using a custom gene expression array card. We identified novel differentially expressed drug-disposition genes for PXR (↑ABCB1/MDR1, CYP2C9, CYP2C19, and EPHX1, ↓ABCB11), CAR [↑sulfotransferase (SULT) 1E1, uridine glucuronosyl transferase (UGT) 2B4], and AhR (↑SLC10A1/NTCP, SLCO1B1/OATP1B1], and coregulated genes (CYP1A1, CYP2B6, CYP2C8, CYP3A4, UGT1A1, UGT1A4). Subsequently, DME gene expression signatures were generated for known CYP3A4 inducers PF-06282999 and pazopanib. The former produced an induction signature almost identical to that of rifampin, suggesting activation of the PXR pathway, whereas the latter produced an expression signature distinct from those of PXR, CAR, or AhR, suggesting involvement of an alternate pathway(s). These results demonstrate that involvement of PXR/CAR/AhR can be identified via expression changes of signature DME/transporter genes. Inclusion of such signature genes could serve to simultaneously identify potential inducers and inhibitors, and the NRs involved in the transcriptional regulation, thus providing a more holistic and mechanism-based assessment of DDI risk for DMEs and transporters beyond conventional cytochrome P450 isoforms.

6-Chloro-5-[4-(1-Hydroxycyclobutyl)Phenyl]-1H-Indole-3-Carboxylic Acid is a Highly Selective Substrate for Glucuronidation by UGT1A1, Relative to ß-Estradiol.

6-Chloro-5-[4-(1-hydroxycyclobutyl)phenyl]-1H-indole-3-carboxylic acid (PF-06409577) is a direct activator of the human ß1-containing adenosine monophosphate-activated protein kinase (ΑMPK) isoforms. The clearance mechanism of PF-06409577 in animals and humans involves uridine diphosphoglucuronosyl transferase (UGT)-mediated glucuronidation to an acyl glucuronide metabolite of PF-06409577 [(2S,3S,4S,5R,6S)-6-((6-chloro-5-(4-(1-hydroxycyclobutyl)phenyl)-1H-indole-3-carbonyl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (M1)], which retains selective activation of human ß1-containing AMPK isoforms. This paper describes a detailed characterization of the human UGT isoform(s) responsible for glucuronidation of PF-06409577 to M1. Studies using a panel of 13 human recombinant UGT (hrUGT) enzymes indicated that PF-06409577 was converted to M1 in a highly selective fashion by UGT1A1, which was further verified in human liver microsomes treated with specific chemical inhibitors, and in different UGT1A1 expressers. Conversion of PF-06409577 to M1 by UGT1A1 occurred in a relatively selective fashion, compared with ß-estradiol (ES), a conventional probe substrate of UGT1A1. The Michaelis-Menten constant (K M) and V max values describing the formation of M1 from PF-06409577 in hrUGT1A1 and microsomal preparations from human intestine, liver, and kidney ranged from 131 to 212 µM (K M) and 107-3834 pmol/min per milligram (V max) in the presence of 2% bovine serum albumin. Relative activity factors (RAF) were determined for UGT1A1 using PF-06409577 and ES to enable estimation of intrinsic clearance from various tissues. RAF values from PF-06409577 and ES were generally comparable with the exception of intestinal microsomes, where ES overestimated the RAF of UGT1A1 due to glucuronidation by intestinal UGT1A8 and UGT1A10. Our results suggest the potential utility of PF-06409477 as a selective probe UGT1A1 substrate for UGT reaction phenotyping and inhibition studies in preclinical discovery/development.