Derivatisation of parthenolide to address chemoresistant chronic lymphocytic leukaemia.

Parthenolide is a natural product that exhibits anti-leukaemic activity, however, its clinical use is limited by its poor bioavailability. It may be extracted from and protocols for growing, extracting and derivatising it are reported A novel parthenolide derivative with good bioavailability and pharmacological properties was identified through a screening cascade based on anti-leukaemic activity and calculated "drug-likeness" properties, and pharmacokinetics studies and hERG liability testing. studies showed the most promising derivative to have comparable anti-leukaemic activity to DMAPT, a previously described parthenolide derivative.

Acute liver effects, disposition and metabolic fate of [C]-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 [C]-fenclozic acid showed acute centrilobular hepatocellular necrosis, but no other regions of the liver were affected.

In vitro exploration of potential mechanisms of toxicity of the human hepatotoxic drug fenclozic acid.

The carboxylic acid NSAID fenclozic acid exhibited an excellent preclinical safety profile and promising clinical efficacy, yet was withdrawn from clinical development in 1971 due to hepatotoxicity observed in clinical trials. A variety of modern in vitro approaches have been used to explore potential underlying mechanisms. Covalent binding studies were undertaken with [(14)C]-fenclozic acid to investigate the possible role of reactive metabolites.

In vitro models of xenobiotic metabolism in trout for use in environmental bioaccumulation studies.

1. In vitro screens are sought as informative, alternatives to the use of animals in vivo and to improve upon the current use of fish liver 9000 g supernatants (S9) in environmental risk assessment. 2.

Differential effect of troglitazone on the human bile acid transporters, MRP2 and BSEP, in the PXB hepatic chimeric mouse.

The aims of this study were to assess the utility of the PXB mouse model of a chimeric human/mouse liver in studying human-specific effects of an important human hepatotoxic drug, the PPARγ agonist, troglitazone. When given orally by gavage for 7 days, at dose levels of 300 and 600 ppm, troglitazone induced specific changes in the human hepatocytes of the chimeric liver without an effect on the murine hepatic portions. The human hepatocytes, in the vehicle-treated PXB mouse, showed an accumulation of electron-dense lipid droplets that appeared as clear vacuoles under the light microscope in H&E-stained sections.

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.

Evaluation of the pharmacokinetics, biotransformation and hepatic transporter effects of troglitazone in mice with humanized livers.

The pharmacokinetics, biotransformation and hepatic transporter effects of troglitazone were investigated following daily oral dosing, at 300 and 600 mg/kg, for 7 days to control (SCID) and chimeric (PXB) mice with humanized livers. Clinical chemistry revealed no consistent pattern of changes associated with troglitazone treatment in the PXB mouse. Human MRP2 but not mouse mrp2 was down-regulated following troglitazone treatment.

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 %).

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.

In vitro hepatic metabolism of cediranib, a potent vascular endothelial growth factor tyrosine kinase inhibitor: interspecies comparison and human enzymology.

The in vitro metabolism of cediranib (4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]quinazoline), a vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor (TKI) of all three VEGF receptors in late-stage development for the treatment of colorectal cancer and recurrent glioblastoma was investigated in hepatic proteins from preclinical species and humans using radiolabeled material. In human hepatocyte cultures, oxidative and conjugative metabolic pathways were identified, with pyrrolidine N(+)-glucuronidation being the major route. The primary oxidative pathways were di-and trioxidations and pyrrolidine N-oxidation.

Characterisation and identification of the human N+-glucuronide metabolite of cediranib.

Cediranib (4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxy-7-[3-(1-pyrrolidinyl)propoxy]quinazoline; RECENTIN), a vascular endothelial growth factor (VEGF) tyrosine kinase inhibitor (TKI) of all three VEGF receptors, is currently in Phase III clinical trials for the first-line treatment of colorectal cancer and the treatment of recurrent glioblastoma. During its clinical development a unique human metabolite, an N(+)-glucuronide, was identified as a major circulating metabolite and one of the major metabolites excreted into faeces. Given the possibility of four sites for the conjugation of the glucuronic acid moiety, determination of the location of the conjugation site on cediranib was warranted.

A quantitative high-throughput trapping assay as a measurement of potential for bioactivation.

Idiosyncratic adverse drug reactions (ADRs) are one of the most common causes of pharmaceutical withdrawals and labeling changes. Most ADRs are caused by drugs that form reactive species that can bind covalently to macromolecules such as proteins. The current methodology for the measurement of covalent binding relies on the use of radiolabeled material that requires an investment in time and resources not typically expended until later in the discovery process.

A semi-automated method for measuring the potential for protein covalent binding in drug discovery.

Introduction: Covalent protein binding of metabolically reactive intermediates of drugs has been implicated in drug toxicity including the occurrence of idiosyncratic drug toxicity. Investigators therefore would prefer to avoid developing compounds that produce significant amounts of reactive metabolites. By incubating the radiolabeled drug of interest with liver microsomes it is possible to evaluate the propensity of a drug candidate to covalently bind to proteins.

Assessment of P450 induction in the marmoset monkey using targeted anti-peptide antibodies.

The identity and expression of hepatic P450 enzymes in marmosets was investigated using a panel of anti-peptide antibodies originally targeted against human P450 enzymes. In immunoblotting, of 12 antibodies examined, 10 bound specifically to bands in marmoset liver microsomal fraction corresponding to P450 enzymes. It is proposed that these represent marmoset CYP1A1, CYP1A2, CYP2A, CYP2B, CYP2C forms (CYP2C-1 and CYP2C-2), CYP2D19, CYP3A21 and another CYP3A form (CYP3A-m).

Structure-function analysis of human CYP3A4 using a specific proinhibitory antipeptide antibody.

An anti-peptide antibody targeted against residues 253 to 269 of human CYP3A4 was produced that specifically and potently inhibited its activity in human hepatic microsomal fraction (>90%). The function of this region in P450 catalysis was investigated. Antibody binding to CYP3A4 was unable to affect the magnitude of the Type I spectrum on addition of testosterone.

Selective and potent inhibition of human CYP2C19 activity by a conformationally targeted antipeptide antibody.

A conformationally targeted anti-peptide antibody was produced by immunizing a rabbit with a cyclized peptide corresponding to a loop region of human CYP2C19 (residues 250-261). In an enzyme-linked immunosorbent assay, the antibody bound strongly to recombinant CYP2C19 and poorly to recombinant CYP2C8, CYP2C9, and CYP2C18. In immunoblotting studies, the antibody bound strongly to recombinant CYP2C19 and weakly to recombinant CYP2C8.

Affinity and potency of proinhibitory antipeptide antibodies against CYP2D6 is enhanced using cyclic peptides as immunogens.

A series of antipeptide antibodies directed against CYP2D6 were produced by immunizing rabbits with peptides that were sterically unrestrained (linear) or conformationally restricted by cyclization. A variety of sites within the region comprising residues 254 to 290 of CYP2D6 were targeted. In immunoblotting studies, each of the antibodies against the linear and cyclic peptides recognized only a single immunoreactive band of 54 kDa in human liver microsomal fraction and bound to recombinant CYP2D6, but not recombinant CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1, or CYP3A4.

Polymorphic debrisoquine 4-hydroxylase activity in the rat is due to differences in CYP2D2 expression.

The female Dark Agouti rat is widely used as an animal model for the CYP2D6 poor metabolizer phenotype, males of other strains such as Sprague Dawley or Wistar serving as models for the extensive metabolizer phenotype. To determine the relative level of expression of CYP2D enzymes in the liver of female and male Dark Agouti, Sprague Dawley and Wistar rats, anti-peptide antibodies were raised in rabbits against short synthetic peptides representing the C-termini of the rat P450 enzymes CYP2D1, CYP2D2, CYP2D3, CYP2D4 and CYP2D5. In immunoblotting studies, it was found that the hepatic expression of CYP2D1 was greater in Dark Agouti rats than Sprague Dawley or Wistar rats.

Expression and localisation of CYP2D enzymes in rat basal ganglia.

P450 enzymes in the CYP2D subfamily have been suggested to contribute to the susceptibility of individuals in developing Parkinson's disease. We have used specific anti-peptide antisera and peroxidase immunohistochemistry to investigate the expression of CYP2D enzymes in the rat brain and some possible factors that may affect their regulation. In male Wistar rats, CYP2D1 was not detected in the basal ganglia or in any other brain region.