Acute liver effects, disposition and metabolic fate of [14C]-fenclozic acid following oral administration to normal and bile-cannulated male C57BL/6J mice.

The distribution, metabolism, excretion and hepatic effects of the human hepatotoxin fenclozic acid were investigated following single oral doses of 10 mg/kg to normal and bile duct-cannulated male C57BL/6J mice. Whole body autoradiography showed distribution into all tissues except the brain, with radioactivity still detectable in blood, kidney and liver at 72 h post-dose. Mice dosed with [14C]-fenclozic acid showed acute centrilobular hepatocellular necrosis, but no other regions of the liver were affected. The majority of the [14C]-fenclozic acid-related material recovered was found in the urine/aqueous cage wash, (49%) whilst a smaller portion (13%) was eliminated via the faeces. Metabolic profiles for urine, bile and faecal extracts, obtained using liquid chromatography and a combination of mass spectrometric and radioactivity detection, revealed extensive metabolism of fenclozic acid in mice that involved biotransformations via both oxidation and conjugation. These profiling studies also revealed the presence of glutathione-derived metabolites providing evidence for the production of reactive species by mice administered fenclozic acid. Covalent binding to proteins from liver, kidney and plasma was also demonstrated, although this binding was relatively low (less than 50 pmol eq./mg protein).

Preclinical Comparison of Osimertinib with Other EGFR-TKIs in EGFR-Mutant NSCLC Brain Metastases Models, and Early Evidence of Clinical Brain Metastases Activity.

PURPOSE: Approximately one-third of patients with non-small cell lung cancer (NSCLC) harboring tumors with EGFR-tyrosine kinase inhibitor (TKI)-sensitizing mutations (EGFRm) experience disease progression during treatment due to brain metastases. Despite anecdotal reports of EGFR-TKIs providing benefit in some patients with EGFRm NSCLC brain metastases, there is a clinical need for novel EGFR-TKIs with improved efficacy against brain lesions. EXPERIMENTAL DESIGN: We performed preclinical assessments of brain penetration and activity of osimertinib (AZD9291), an oral, potent, irreversible EGFR-TKI selective for EGFRm and T790M resistance mutations, and other EGFR-TKIs in various animal models of EGFR-mutant NSCLC brain metastases. We also present case reports of previously treated patients with EGFRm-advanced NSCLC and brain metastases who received osimertinib in the phase I/II AURA study (NCT01802632). RESULTS: Osimertinib demonstrated greater penetration of the mouse blood-brain barrier than gefitinib, rociletinib (CO-1686), or afatinib, and at clinically relevant doses induced sustained tumor regression in an EGFRm PC9 mouse brain metastases model; rociletinib did not achieve tumor regression. Under positron emission tomography micro-dosing conditions, [11C]osimertinib showed markedly greater exposure in the cynomolgus monkey brain than [11C]rociletinib and [11C]gefitinib. Early clinical evidence of osimertinib activity in previously treated patients with EGFRm-advanced NSCLC and brain metastases is also reported. CONCLUSIONS: Osimertinib may represent a clinically significant treatment option for patients with EGFRm NSCLC and brain metastases. Further investigation of osimertinib in this patient population is ongoing. Clin Cancer Res; 22(20); 5130-40. ©2016 AACR.

Metabolic Disposition of Osimertinib in Rats, Dogs, and Humans: Insights into a Drug Designed to Bind Covalently to a Cysteine Residue of Epidermal Growth Factor Receptor.

Preclinical and clinical studies were conducted to determine the metabolism and pharmacokinetics of osimertinib and key metabolites AZ5104 and AZ7550. Osimertinib was designed to covalently bind to epidermal growth factor receptors, allowing it to achieve nanomolar cellular potency (Finlay et al., 2014). Covalent binding was observed in incubations of radiolabeled osimertinib with human and rat hepatocytes, human and rat plasma, and human serum albumin. Osimertinib, AZ5104, and AZ7550 were predominantly metabolized by CYP3A. Seven metabolites were detected in human hepatocytes, also observed in rat or dog hepatocytes at similar or higher levels. After oral administration of radiolabeled osimertinib to rats, drug-related material was widely distributed, with the highest radioactivity concentrations measured at 6 hours postdose in most tissues; radioactivity was detectable in 42% of tissues 60 days postdose. Concentrations of [(14)C]-radioactivity in blood were lower than in most tissues. After the administration of a single oral dose of 20 mg of radiolabeled osimertinib to healthy male volunteers, ∼19% of the dose was recovered by 3 days postdose. At 84 days postdose, mean total radioactivity recovery was 14.2% and 67.8% of the dose in urine and feces. The most abundant metabolite identified in feces was AZ5104 (∼6% of dose). Osimertinib accounted for ∼1% of total radioactivity in the plasma of non-small cell lung cancer patients after 22 days of 80-mg osimertinib once-daily treatment; the most abundant circulatory metabolites were AZ7550 and AZ5104 (<10% of total osimertinib-related material). Osimertinib is extensively distributed and metabolized in humans and is eliminated primarily via the fecal route.

A comparison of the metabolism of midazolam in C57BL/6J and hepatic reductase null (HRN) mice.

The hepatic cytochrome P450 reductase null (HRN) mouse, which has no functional hepatic Cyp P450s, may represent a useful model for examining extra-hepatic P450-related oxidative metabolism. Here the pharmacokinetics and metabolic fate of midazolam, a drug known to undergo such extra-hepatic metabolism, have been investigated in the HRN mouse and compared with a phenotypically normal strain (C57BL/6J). In addition, the effects of co-administration of the pan-P450 inhibitor 1'-aminobenzotriazole (ABT) on the metabolic profile have been compared in both strains. Significant pharmacokinetic differences for midazolam were observed between the two strains of mice with the HRN mice showing lower circulating concentrations of 1'-hydroxymidazolam but higher concentrations of 1'-hydroxymidazolam-O-glucuronide. A significant increase in midazolam exposure was seen upon ABT exposure for both strains of mice, but no differences in the area under the concentration time curves (AUC) for the monitored metabolites were observed. Although oxidative metabolism of midazolam was not abolished, significant decreases in 1'-hydroxymidazolam formation ratios were observed for both strains of mice exposed to ABT. Metabolite profiling of blood and bile showed a number of qualitative and quantitative differences between HRN and normal mice. These differences in midazolam metabolism between the two strains of mice clearly demonstrate the role that liver P450 enzymes play in the murine metabolism of midazolam. The fate of the compound in the HRN mice shows the importance of extrahepatic metabolism and also showed that these mice appear to be more capable of forming circulating phase II glucuronides than the normal strain.

Troglitazone metabolism and transporter effects in chimeric mice: a comparison between chimeric humanized and chimeric murinized FRG mice.

1. The biotransformation, hepatic transporter and blood chemistry effects of troglitazone were investigated following 7 days of dosing at 600 mg/kg/day to chimeric murinized or humanized FRG mice, Mo-FRG and Hu-FRG mice, respectively. 2. Clinical chemistry and histopathology analysis revealed a significant drop in humanization over the time course of the study for the Hu-FRG mice but no significant changes associated with troglitazone treatment in either the Mo-FRG or the Hu-FRG models. No changes in transporter expression in livers of these mice were observed. Oxidative and conjugative metabolic pathways were identified with a 15- to 18-fold increase in formation of troglitazone sulfate in the Hu-FRG mice compared with the Mo-FRG mice in blood and bile, respectively. This resembles the troglitazone metabolism in human and these data are comparable with the formation of this metabolite in the chimeric uPA(+/+)/SCID mice. 3. However, larger amounts of troglitazone glucuronide were also observed in the Hu-FRG mouse compared with the Mo-FRG mouse which may be an effect of the drop in humanization of the Hu-FRG mouse during the study. 4. Highly humanized mice have a considerable potential in providing a useful first insight into circulating human metabolites of candidate drugs metabolized in the liver.

The metabolic fate of [14C]-fenclozic acid in the hepatic reductase null (HRN) mouse.

1. The distribution, metabolism, excretion and hepatic effects of fenclozic acid were investigated following a single oral dose of 10 mg/kg to hepatic reductase null (HRN) mice. 2. The majority of the [(14)C]-fenclozic acid was eliminated via the urine/aqueous cage wash, (55%) with a smaller portion excreted in the faeces, (5%). The total recovery of radioactivity in the excreta over the 72 h period studied was ca. 60%. 3. Metabolism of fenclozic acid in the HRN mice was entirely to the carboxylic acid function and was dominated by amino acid conjugation to glycine and taurine, with lesser amounts of an acyl glucuronide. 4. Whole body autoradiography of mice showed general distribution into all tissues except the brain. Radioactivity was still detectable in the kidney and liver of the HRN mice at 72 h post-dose. Covalent binding studies showed evidence of binding to kidney, liver and plasma proteins however, the degree of binding was less than 50 pmol equiv/mg protein for all tissues. 5. The HRN mouse appears to be a useful in vivo model for the study of the Phase II conjugation metabolism of fenclozic acid in the absence of hepatic cytochrome P450-related oxidative metabolism.

Drug-drug interactions and metabolism in cytochrome P450 2C knockout mice: application to troleandomycin and midazolam.

Drug-drug interactions (DDIs) may cause serious drug toxicity and delay development of candidate drugs. Screening using human liver microsomes and hepatocytes can help predict DDIs but do not always provide the degree of certainty required for confident progression of a candidate drug. Thus a suitable in vivo test system could be of great value. Here a Cyp2c knockout (KO) mouse was investigated for studying DDIs using midazolam (MDZ) a standard human CYP3A4 substrate and troleandomycin (TAO) a potent human CYP3A4 inhibitor. Pharmacokinetics (PK) and biotransformation of MDZ were investigated following dosing to Cyp2c KO and wild type mice before and after TAO treatment. The noteworthy differences in the metabolism of MDZ in Cyp2c KO compared to wild type mice confirms the important role that Cyp2c enzymes play in the murine metabolism of MDZ in vivo. The impact of Cyp3a inhibition produced a further increase in circulating MDZ concentrations in all individuals from both strains of mice though the impact of the elimination of the Cyp2c pathway in the KO mice on the AUC was less than perhaps expected. We have shown that TAO produces an increase in the MDZ concentration and a reduction in the 1'hydroxymidazolam/midazolam formation ratio but the expected difference in the magnitude of this effect between the wild type and the Cyp2c KO mice was not seen. The magnitude of the TAO effect was also smaller than is reported in humans. Hence further work is required before this animal model could be used to predict clinical interactions.

Pharmacokinetics and metabolism of midazolam in chimeric mice with humanised livers.

The pharmacokinetics and biotransformation of midazolam were investigated following single oral doses of 0.1, 1 and 10 mg/kg to chimeric mice with humanised livers (PXB mice) and to severe combined immunodeficient (SCID) mice used as controls. Pharmacokinetic analysis, on whole blood, revealed rapid absorption of the administered midazolam with a higher C(max) in PXB compared to SCID. The exposure to 1'-hydroxymidazolam was approximately 14-fold greater than to midazolam in the SCID mice and close to equivalent in the PXB mice. The metabolism of midazolam in SCID mice was faster than in the PXB mice such that pharmacokinetic data for midazolam in SCID mice could not be generated from the lowest dose in these animals. Both oxidative and conjugative metabolic pathways were identified in the PXB mice. All the major circulating metabolites observed in humans; 1'-hydroxymidazolam, 4'-hydroxymidazolam, 1',4'-dihydroxymidazolam and 1'-hydroxymidazolam glucuronide, were detected in the blood of PXB mice. However, 4'-hydroxymidazolam and the 1'-hydroxymidazolam glucuronide were not detected in blood samples obtained from SCID mice. The midazolam metabolite profile in the PXB mouse was similar to that previously reported for human suggesting that the PXB mouse model can provide a model system for predicting circulating human metabolites.

The hepatic reductase null mouse as a model for exploring hepatic conjugation of xenobiotics: application to the metabolism of diclofenac.

The distribution, metabolism, excretion and hepatic effects of diclofenac were investigated following a single oral dose of 10 mg/kg to wild type and hepatic reductase null (HRN) mice. For the HRN strain the bulk of the [(14)C]-diclofenac-related material was excreted in the urine/aqueous cagewash within 12 h of administration (~82%) with only small amounts eliminated via the faeces (~2% in 24 h). Wild type mice excreted the radiolabel more slowly with ca. 52 and 15% of the dose recovered excreted in urine and faeces, respectively, by 24 h post dose. The metabolic profiles of the HRN mice were dominated by acyl conjugation to either taurine or glucuronic acid. Wild type mice produced relatively small amounts of the acyl glucuronide. Whole Body Autoradiography (WBA) of mice sacrificed at 24 h post dose indicated increased retention of radioactivity in the livers of HRN mice compared to wild type mice. Covalent binding studies showed no differences between the two strains. Metabolism of diclofenac in HRN mice involved mainly acyl glucuronide formation and taurine amide conjugation. This mouse model may find utility in understanding the impact of reactive metabolite formation via routes that involve the production of acyl-CoA or acyl glucuronides of acidic drugs.

Diclofenac metabolism in the mouse: novel in vivo metabolites identified by high performance liquid chromatography coupled to linear ion trap mass spectrometry.

The metabolism of [(14)C]-diclofenac in mice was investigated following a single oral dose of 10 mg/kg. The majority of the drug-related material was excreted in the urine within 24 h of administration (49.7 %). Liquid chromatographic analyses of urine and faecal extracts revealed extensive metabolism to at least 37 components, with little unchanged diclofenac excreted. Metabolites were identified using a hybrid linear ion-trap mass spectrometer via exact mass determinations of molecular ions and subsequent multi-stage fragmentation. The major routes of metabolism identified included: 1) conjugation with taurine; and 2) hydroxylation (probably at the 4'-and 5-arene positions) followed by conjugation to taurine, glucuronic acid or glucose. Ether, rather than acyl glucuronidation, predominated. There was no evidence for p-benzoquinone-imine formation (i.e. no glutathione or mercapturic acid conjugates were detected). A myriad of novel minor drug-related metabolites were also detected, including ribose, glucose, sulfate and glucuronide ether-linked conjugates of hydroxylated diclofenac derivatives. Combinations of these hydroxylated derivatives with acyl conjugates (glucose, glucuronide and taurine) or N-linked sulfation or glucosidation were also observed. Acyl- or amide-linked-conjugates of benzoic acid metabolites and several indolinone derivatives with further hydroxylated and conjugated moieties were also evident. The mechanisms involved in the generation of benzoic acid and indolinone products indicate the formation reactive intermediates in vivo that may possibly contribute to hepatotoxicity.

Utility of spatially-resolved atmospheric pressure surface sampling and ionization techniques as alternatives to mass spectrometric imaging (MSI) in drug metabolism.

Tissue distribution studies of drug molecules play an essential role in the pharmaceutical industry and are commonly undertaken using quantitative whole body autoradiography (QWBA) methods. The growing need for complementary methods to address some scientific gaps around radiography methods has led to increased use of mass spectrometric imaging (MSI) technology over the last 5 to 10 years. More recently, the development of novel mass spectrometric techniques for ambient surface sampling has redefined what can be regarded as "fit-for-purpose" for MSI in a drug metabolism and disposition arena. Together with a review of these novel alternatives, this paper details the use of two liquid microjunction (LMJ)-based mass spectrometric surface sampling technologies. These approaches are used to provide qualitative determination of parent drug in rat liver tissue slices using liquid extraction surface analysis (LESA) and to assess the performance of a LMJ surface sampling probe (LMJ-SSP) interface for quantitative assessment of parent drug in brain, liver and muscle tissue slices. An assessment of the utility of these spatially-resolved sampling methods is given, showing interdependence between mass spectrometric and QWBA methods, in particular there emerges a reason to question typical MSI workflows for drug metabolism; suggesting the expedient use of profile or region analysis may be more appropriate, rather than generating time-intensive molecular images of the entire tissue section.