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.

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.

In vitro characterization of sarizotan metabolism: hepatic clearance, identification and characterization of metabolites, drug-metabolizing enzyme identification, and evaluation of cytochrome p450 inhibition.

In vitro biotransformation studies of sarizotan using human liver microsomes (HLM) showed aromatic and aliphatic monohydroxylation and dealkylation. Recombinant cytochromes P450 (P450) together with P450-selective inhibitors in HLM/hepatocyte cultures were used to evaluate the relative contribution of different P450s and revealed major involvement of CYP3A4, CYP2C9, CYP2C8, and CYP1A2 in sarizotan metabolism. The apparent K(m, u) and V(max) of sarizotan clearance, as investigated in HLM, were 9 microM and 3280 pmol/mg/min, predicting in vivo hepatic clearance of 0.94 l/h, which indicates that sarizotan is a low-clearance compound in humans and suggests nonsaturable metabolism at the targeted plasma concentration (< or =1 microM). This finding is confirmed by the reported human clearance (CL/F of 3.6-4.4 l/h) and by the dose-linear area under the curve increase observed with doses up to 25 mg. The inhibitory effect of sarizotan toward six major P450s was evaluated using P450-specific marker reactions in pooled HLM. K(i, u) values of sarizotan against CYP2C8, CYP2C19, and CYP3A4 were >10 microM, whereas those against CYP2D6 and CYP1A2 were 0.43 and 8.7 microM, respectively. Based on the estimates of sarizotan concentrations at the enzyme active sites, no clinically significant drug-drug interactions (DDIs) due to P450 inhibition are expected. This result has been confirmed in human DDI studies in which no inhibition of five major P450s was observed in terms of marker metabolite formation.

Species differences in the response of liver drug-metabolizing enzymes to (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949) in vivo and in vitro.

Induction of drug-metabolizing enzymes (DMEs) is highly species-specific and can lead to drug-drug interaction and toxicities. In this series of studies we tested the species specificity of the antidiabetic drug development candidate and mixed peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist (S)-4-O-tolylsulfanyl-2-(4-trifluormethyl-phenoxy)-butyric acid (EMD 392949, EMD) with regard to the induction of gene expression and activities of DMEs, their regulators, and typical PPAR target genes. EMD clearly induced PPARalpha target genes in rats in vivo and in rat hepatocytes but lacked significant induction of DMEs, except for cytochrome P450 (P450) 4A. CYP2C and CYP3A were consistently induced in livers of EMD-treated monkeys. Interestingly, classic rodent peroxisomal proliferation markers were induced in monkeys after 17 weeks but not after a 4-week treatment, a fact also observed in human hepatocytes after 72 h but not 24 h of EMD treatment. In human hepatocyte cultures, EMD showed similar gene expression profiles and induction of P450 activities as in monkeys, indicating that the monkey is predictive for human P450 induction by EMD. In addition, EMD induced a similar gene expression pattern as the PPARalpha agonist fenofibrate in primary rat and human hepatocyte cultures. In conclusion, these data showed an excellent correlation of in vivo data on DME gene expression and activity levels with results generated in hepatocyte monolayer cultures, enabling a solid estimation of human P450 induction. This study also clearly highlighted major differences between primates and rodents in the regulation of major inducible P450s, with evidence of CYP3A and CYP2C inducibility by PPARalpha agonists in monkeys and humans.

Simple-to-use, reference criteria for revealing drug-induced QT interval prolongation in conscious dogs.

Electrocardiogram (ECG) QT interval prolongation produced by drugs in certain animal models is currently believed to be predictive of cardiac proarrhythmic effects in humans. For this reason, nonclinical assessment of the effects of novel drugs on cardiac repolarization is a regulatory prerequisite for progressing such agents to clinical evaluation. The present investigation was carried out to develop reliable, simple-to-use reference criteria for identifying individual animals as responders to drugs that prolong the QT interval. ECG were recorded for 30 s at 0 (8 am), 2, 4, 6 and 24 h in 6 trained, conscious, beagle dogs during 5 control experimental sessions. QT intervals were measured and corrected for heart rate by applying the Van de Water algorithm (QTc). The maximal (QTc(max)) and minimal (QTc(min)) values of QTc observed in each of the five control recording sessions were noted. Two reference (R) criteria were used to designate an individual animal as a responder to drug treatment: 1) QTc(maxR) which was obtained by adding 10 ms to the largest value of QTc(max) observed during the five control recording sessions and 2) (QTc(max)-QTc(min))(maxR) which was obtained by increasing by 50% the largest of the (QTc(max)-QTc(min)) values [(QTc(max)-QTc(min))(max)] observed in the 5 control recording sessions. The sensitivity and reliability of these criteria were tested by determining QTc intervals before and 2, 4, 6 and 24 h after placebo or quinidine (200, 400 and 800 mg p.o. per animal). The reference values of QTc(maxR) and (QTc(max)-QTc(min))(maxR) for the various dogs ranged from 246 to 270 ms and from 15 to 19.5 ms, respectively. The number of dogs responding to treatment (T: quinidine at 200, 400 and 800 mg, p.o. per animal) with a QTc(maxT) and/or a (QTc(max)-QTc(min))(maxT) equal to or greater than the respective reference values was, respectively, 1/6, 3/6 and 5/6 dogs. Additionally, the number of responders correlated well with the concentration of free quinidine in the plasma. In conclusion, this investigation succeeded in establishing reliable, reference criteria for individual dogs despite the intrinsic daily variation of QTc interval. The application of these criteria allowed identifying individual animals responding to quinidine with delayed cardiac repolarization.

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.