Evobrutinib pathway to its major metabolite M463-2 and insights from a biotransformation and DDI perspective.

Evobrutinib is a highly selective, covalent, central nervous system-penetrant Bruton's tyrosine kinase (BTK) inhibitor, currently in Phase III trials for the treatment of relapsing multiple sclerosis. One major circulating metabolite of evobrutinib has been previously identified as the racemic dihydro-diol M463-2 (MSC2430422) in a Phase I human mass balance study.Phenotyping experiments were conducted to confirm the metabolic pathway of evobrutinib to M463-2. Ratio of the enantiomers was determined by enantioselective liquid chromatography with tandem mass spectrometry analysis of plasma samples from humans and preclinical species. Drug-drug interaction (DDI) characterisation, evaluation of pharmacological activity on BTK, and off-target screening experiments followed assessing safety of the metabolite.The biotransformation of evobrutinib to M463-2 was determined to be a two-step process with a CYP-mediated oxidation acting to form an epoxide intermediate, which was further hydrolysed by soluble and mitochondrial epoxide hydrolase. Only the (S)-enantiomer was determined to be a major metabolite, the (R)-enantiomer was minor. In vitro studies demonstrated the (S)-enantiomer lacked clinically relevant pharmacological activity, off-target effects and DDIs.The biotransformation of evobrutinib to its major metabolite has been elucidated, with the major (S)-enantiomer being shown to pose no on/off target or DDI risks.

Evobrutinib, a covalent Bruton's tyrosine kinase inhibitor: Mass balance, elimination route, and metabolism in healthy participants.

The highly selective, covalent Bruton's tyrosine kinase inhibitor evobrutinib is under investigation for treatment of patients with multiple sclerosis (MS). Early clinical studies in healthy participants and patients with relapsing MS indicated that evobrutinib is well-tolerated and effective. We undertook a mass balance study in six men who received a single 75-mg oral dose of evobrutinib containing ~ 3.6 MBq (100 µCi) 14 C-evobrutinib, to determine the absorption, metabolic pathways, and routes of excretion of evobrutinib. The primary objectives of this phase I study (NCT03725072) were to (1) determine the rates and routes of total radioactivity excretion, including the mass balance of total drug-related radioactivity in urine and feces, (2) assess the pharmacokinetics (PKs) of total radioactivity in blood and plasma, and (3) characterize the plasma PKs of evobrutinib. Exploratory end points included identifying and quantifying evobrutinib and its metabolites in plasma and excreta (urine and feces) and exploring key biotransformation pathways and clearance mechanisms. Evobrutinib was primarily eliminated in feces (arithmetic mean percentage, SD, 71.0, 2.1) and, to a lesser extent, in urine (20.6, 2.0), with most of the total radioactivity (85.3%) excreted in the first 72 h after administration. No unchanged evobrutinib was detected in excreta. Evobrutinib was rapidly absorbed and substantially metabolized upon absorption. Only one major metabolite M463-2 (MSC2430422) was identified in plasma above the 10% of total drug exposure threshold, which classifies M463-2 (MSC2430422) as a major metabolite according to the US Food and Drug Administration (FDA; metabolites in safety testing [MIST]) and the European Medicines Agency (EMA; International Conference on Harmonization [ICH] M3). These results support further development of evobrutinib and may help inform subsequent investigations.

Insights into Praziquantel Metabolism and Potential Enantiomeric Cytochrome P450-Mediated Drug-Drug Interaction.

The active enantiomer R-Praziquantel (PZQ) shows a clinically lower relative exposure when administered enantiomerically pure compared with a racemic form. We investigated the hypothesis that enantiomer-enantiomer interactions on cytochrome P450 (P450) enzymes could explain this observation and aimed to further deepen the understanding of PZQ metabolism. First, in an in vitro metabolite profiling study, the formation of multiple metabolites per P450, together with an observed interconversion of cis-4'-OH-PZQ to trans-4'-OH-PZQ in human hepatocytes, pointed out the inadequacy of measuring metabolite formation in kinetic studies. Thus, a substrate depletion approach to study PZQ enantiomeric interactions was applied. Second, an abundant CYP3A4 metabolite found in previous studies was structurally characterized. Third, substrate depletion methodologies were applied to determine P450 enzyme kinetics of PZQ and to further estimate enantiomer-enantiomer inhibitory parameters. A competitive inhibition between PZQ enantiomers for CYP2C9, 2C19, 3A4, and 3A5 was revealed. Analyses considering the clearance of only one or both enantiomers provided comparable enantiomer-enantiomer inhibition estimates. To conclude, this paper provides new insights into PZQ metabolic profile to enable a better understanding of enantioselective pharmacokinetics using substrate depletion-based methods. SIGNIFICANCE STATEMENT: In this study, enantiomer-enantiomer interactions of praziquantel on cytochrome P450 metabolizing enzymes are investigated via substrate depletion measurement using modeling methods. Together with new insights into the praziquantel metabolism, this work provides a novel data set to understand its pharmacokinetics.

Open-label, single-center, phase I trial to investigate the mass balance and absolute bioavailability of the highly selective oral MET inhibitor tepotinib in healthy volunteers.

Tepotinib (MSC2156119J) is an oral, potent, highly selective MET inhibitor. This open-label, phase I study in healthy volunteers (EudraCT 2013-003226-86) investigated its mass balance (part A) and absolute bioavailability (part B). In part A, six participants received tepotinib orally (498 mg spiked with 2.67 MBq [14C]-tepotinib). Blood, plasma, urine, and feces were collected up to day 25 or until excretion of radioactivity was <1% of the administered dose. In part B, six participants received 500 mg tepotinib orally as a film-coated tablet, followed by an intravenous [14C]-tepotinib tracer dose (53-54 kBq) 4 h later. Blood samples were collected until day 14. In part A, a median of 92.5% (range, 87.1-96.9%) of the [14C]-tepotinib dose was recovered in excreta. Radioactivity was mainly excreted via feces (median, 78.7%; range, 69.4-82.5%). Urinary excretion was a minor route of elimination (median, 14.4% [8.8-17.7%]). Parent compound was the main constituent in excreta (45% [feces] and 7% [urine] of the radioactive dose). M506 was the only major metabolite. In part B, absolute bioavailability was 72% (range, 62-81%) after oral administration of 500 mg tablets (the dose and formulation used in phase II trials). In conclusion, tepotinib and its metabolites are mainly excreted via feces; parent drug is the major eliminated constituent. Oral bioavailability of tepotinib is high, supporting the use of the current tablet formulation in clinical trials. Tepotinib was well tolerated in this study with healthy volunteers.

A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development under the "Metabolites in Safety Testing" Regulatory Guidance.

Since the introduction of metabolites in safety testing (MIST) guidance by the Food and Drug Administration in 2008, major changes have occurred in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and nonclinical studies to generate this information. In this cross-industry review, we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radio chromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated, which has led to more frequent determination of human metabolite profiles from multiple ascending-dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. Together with technological advances, these events have led to a general shift of focus toward earlier human metabolism studies using high-resolution mass spectrometry and to a reduction in animal radiolabel absorption/distribution/metabolism/excretion studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industry.

Metabolism of the MEK1/2 Inhibitor Pimasertib Involves a Novel Conjugation with Phosphoethanolamine in Patients with Solid Tumors.

Pimasertib (AS703026 or MSC1936369B) is a selective inhibitor of MEK1/2, the mitogen-activated protein kinase (MAPK) signaling pathway, which is often dysregulated in cancer cells. Pimasertib has shown potent preclinical antitumor activity and its clinical activity is being investigated in various tumor types. In this phase I study, the disposition and biotransformation of 14C-radiolabeled pimasertib was investigated in six patients with locally advanced or metastatic solid tumors (NCT01713036). Ultra-performance liquid chromatography-mass spectrometry and radiodetection techniques were used to investigate the profiles and structures of metabolites in plasma, urine, and feces after a single oral dose of 14C-pimasertib. A total of 14 different phase I and II metabolites of 14C-pimasertib were detected, which were principally generated through oxidations and conjugations (direct and indirect); but other reactions included isomerization, N-dealkylation, deamination, and deiodination to form minor metabolites. Two major metabolites (>10% of total drug-related material), M554 and M445, were identified in plasma and urine. In feces, M445 was the primary metabolite with only trace amounts of M554 excreted. All other metabolites, including enantiomers of M445 and pimasertib, were detected to a lesser extent (<5%) in these matrices. M445 was identified as a carboxylic acid of pimasertib. M554 was identified as a novel phosphoethanolamine conjugate on the propanediol moiety of pimasertib by high-resolution mass spectrometry and multiple nuclear magnetic resonance spectroscopy techniques. To our knowledge, a phosphoethanolamine conjugate is a novel metabolite not previously described for a pharmaceutical agent and requires detailed further investigations to understand any implications.

Pimasertib, a selective oral MEK1/2 inhibitor: absolute bioavailability, mass balance, elimination route, and metabolite profile in cancer patients.

AIM: This trial (NCT: 01713036) investigated the absolute bioavailability, mass balance and metabolite profile of pimasertib in a new design combining these investigations in a single group of patients. METHODS: Six male patients with pathologically confirmed, locally advanced or metastatic solid tumours were enrolled. Exclusion criteria included Eastern Cooperative Oncology Group performance status >1. In Part A of the trial, patients received a 60 mg oral dose of unlabelled pimasertib followed by an intravenous (i.v.) tracer dose of [14 C]pimasertib 2 µg (equalling 9 kBq) as a bolus injection, one hour after the oral dose, on Day 1. On Day 8, all patients received 60 mg pimasertib capsules spiked with 2.6 MBq of [14 C]pimasertib. Patients received 60 mg oral unlabelled pimasertib twice daily from Day 3 to Day 21 of Part A and in subsequent 21-day cycles in Part B. RESULTS: Following i.v. administration, [14 C]pimasertib exhibited a geometric mean total body clearance of 45.7 l h-1 (geometric coefficient of variation [geometric CV]: 47.2%) and a volume of distribution of 229 l (geometric CV: 42.0%). Absolute bioavailability was 73%. The majority of the oral [14 C] dose (85.1%) was recovered in excreta. Total radioactivity was mainly excreted into urine (52.8%) and faeces (30.7%) with 78.9% of the [14 C] dose recovered as metabolites. Two major circulating metabolites were identified in plasma: a carboxylic acid (M445) and a phosphoethanolamine conjugate (M554). The safety profile was in line with the published pimasertib trials. CONCLUSION: Pimasertib showed a favourable pharmacokinetic profile with high absolute bioavailability and a unique metabolic pathway (conjugation with phosphoethanolamine).

In vitro and in vivo drug disposition of cilengitide in animals and human.

Cilengitide is very low permeable (1.0 nm/sec) stable cyclic pentapeptide containing an Arg-Gly-Asp motif responsible for selective binding to αvß3 and αvß5 integrins administered intravenously (i.v.). In vivo studies in the mouse and Cynomolgus monkeys showed the major component in plasma was unchanged drug (>85%). These results, together with the absence of metabolism in vitro and in animals, indicate minimal metabolism in both species. The excretion of [(14)C]-cilengitide showed profound species differences, with a high renal excretion of the parent drug observed in Cynomolgus monkey (50% dose), but not in mouse (7 and 28%: m/f). Consistently fecal (biliary) secretion was high in mouse (87 and 66% dose: m/f) but low in Cynomolgus monkey (36.5%). Human volunteers administrated with [(14)C]-cilengitide showed that most of the dose was recovered in urine as unchanged drug (77.5%, referred to Becker et al. 2015), indicating that the Cynomolgus monkey was the closer species to human. In order to better understand the species difference between human and mouse, the hepatobiliary disposition of [(14)C]-cilengitide was determined in sandwich-cultured hepatocytes. Cilengitide exhibited modest biliary efflux (30-40%) in mouse, while in human hepatocytes this was negligible. Furthermore, it was confirmed that the uptake of cilengitide into human hepatocytes was minor and appeared to be passive. In summary, the extent of renal and biliary secretion of cilengitide appears to be highly species specific and is qualitatively well explained using sandwich hepatocyte culture models.

Metabolism and disposition of the αv-integrin ß3/ß5 receptor antagonist cilengitide, a cyclic polypeptide, in humans.

Cilengitide (EMD 121974, manufactured by Merck KGaA, Darmstadt, Germany) is an αv-integrin receptor antagonist showing high affinity for αvß3 and αvß5.This study determined the mass balance of cilengitide in healthy volunteers receiving a single intravenous infusion of 2.1 MBq (14) C-cilengitide spiked into 250 mL of 2000 mg of cilengitide. Blood, urine, and feces were collected up to day 15 or until excretion of radioactivity was below 1% of the administered dose. Total radioactivity derived from the administration of (14) C-cilengitide and unlabeled cilengitide levels were determined and used for calculation of pharmacokinetic parameters.(14) C-cilengitide-related radioactivity was completely recovered (94.5%; 87.4%-100.6%) and was mainly excreted into urine (mean, 79.0%; range, 70.3%-88.2%) and to a lesser extent into feces (mean, 15.5%; range, 9.3%-20.3%). Of the administered dose, 77.5% was recovered as unchanged cilengitide in urine. The concentration profiles of cilengitide and total radioactivity in plasma were comparable. No circulating metabolites were identified in plasma and urine. Two metabolites,M606-1 and M606-2, were identified in feces considered to be formed by intestinal peptidases or by peptidases from fecal bacteria. In conclusion, the data show that following intravenous administration, (14) C-cilengitide was completely recovered, was excreted mainly via renal elimination, and was not metabolized systemically.

Comparison of the in vitro and in vivo metabolism of Cladribine (Leustatin, Movectro) in animals and human.

New insight into the in vitro and in vivo metabolism of Cladribine (2-chloro-2'-deoxyadenosine, [2-CdA]) are presented. Following incubation of [(14)C]-2-CdA in mouse, rat, rabbit, dog, monkey and human hepatocyte cultures, variable turnover was observed with oxidations and direct glucuronidation pathways. The oxidative cleavage to 2-chloroadenine (2-CA, M1) was only observed in rabbit and rat. Following incubation of [(14)C]-2-CdA in whole blood from mouse, monkey and human, a significant turnover was observed. The main metabolites in monkey and human were 2-chlorodeoxyinosine (M11, 16% of total radioactivity) and 2-chlorodeoxyinosine (M12, 43%). In mouse, 2-CA was the major metabolite (2-CA; M1, 73%). After single intravenous and oral administration of [(14)C]-2-CdA to mice, 2-chlorodeoxyinosine (M11) was confirmed in plasma, while 2-chlorohypoxanthine (M12) and 2-CA (M1) were found in urine. Overall, the use of [(14)C]-2-CdA both in vitro (incubations in mouse, monkey and human whole blood) and in vivo (mouse) has confirmed the existence of an additional metabolism pathway leading to the formation of 2-chlorodeoxyinosine (M11) and 2-chlorohypoxanthine (M12). Formation of these two metabolites demonstrates that Cladribine as free form is not fully resistant to adenosine deaminase as suggested earlier, an enzyme involved in its mode of action.

Pharmacokinetics of ML3403 ({4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine), a 4-Pyridinylimidazole-type p38 mitogen-activated protein kinase inhibitor.

The p38 mitogen-activated protein kinase (MAPK) is a key mediator in cytokine-induced signaling events that are activated in response to a variety of extracellular stimuli such as stress factors, apoptosis, and proliferation. Therefore, the MAPK family plays an integral role in disease states including oncogenesis, autoimmune diseases, and inflammatory processes. Inhibition of these protein kinases represents an attractive strategy for therapeutic intervention. In particular, one class of p38 MAP kinase inhibitors, the pyridinyl imidazole derivatives, is intensely investigated by several industrial groups, but so far no studies concerning the metabolism of these structurally related substances seem to be available. The objective of our examinations was the preclinical characterization of ML3403, {4-[5-(4-fluorophenyl)-2-methylsulfanyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine, a potent inhibitor of p38 MAP kinase, comprising the basic pyridinyl imidazole structure. In human hepatic microsomal incubations, the sulfoxidation to ML3603 ({4-[5-(4-fluorophenyl)-2-methylsulfinyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine) and M-sulfone ({4-[5-(4-fluorophenyl)-2-methylsulfonyl-3H-imidazol-4-yl]-pyridin-2-yl}-(1-phenylethyl)-amine) was found to be the predominant metabolic transformation. In addition, oxidative removal of the phenylethyl moiety, pyridine N-oxidation, and hydroxylation reactions were observed. Incubations were carried out with hepatic microsomes from various species and with recombinant human cytochrome P450 isoenzymes, showing that CYP1A2, CYP2C19, CYP2D6, and CYP3A4 are the prominent enzymes in the metabolism of ML3403. Michaelis-Menten kinetics of ML3603 formation by these recombinant isoenzymes showed that CYP3A4 plays a pivotal role in the sulfoxidation reaction. In addition, pharmacokinetics of ML3403 were evaluated in male and female Wistar rats after oral gavage, showing a fast and high conversion to its active sulfoxide metabolite ML3603. A remarkable gender-specific difference in the systemic exposure to ML3403 and ML3603 was found in rats. No gender-specific difference was detected in incubations with human liver microsomes.

Overexpression, purification and characterization of SimL, an amide synthetase involved in simocyclinone biosynthesis.

Simocyclinone D8 is a potent inhibitor of bacterial gyrase, produced by Streptomyces antibioticus Tu 6040. It contains an aminocoumarin moiety, similar to that of novobiocin, which is linked by an amide bond to a structurally complex acyl moiety, consisting of an aromatic angucycline polyketide nucleus, the deoxysugar olivose and a tetraene dicarboxylic acid. We have now investigated the enzyme SimL, responsible for the formation of the amide bond of simocyclinone. The gene was cloned, expressed in S. lividans T7, and the protein was purified to near homogeneity, and characterized. The 60 kDa protein catalyzed both the ATP-dependent activation of the acyl component as well as its transfer to the amino group of the aminocoumarin ring, with no requirement for a 4'-phosphopantetheinyl cofactor. Besides its natural substrate, simocyclinone C4, SimL also accepted a range of cinnamic and benzoic acid derivatives and several other, structurally very diverse acids. These findings make SimL a possible tool for the creation of new aminocoumarin antibiotics.