Acute liver effects disposition and metabolic fat

Authors: Kathryn Pickup Scott Martin Elizabeth A Partridge Huw B Jones Jonathan Wills Tim SchulzUtermoehl Alan McCarthy Alison Rodrigues Chris Page Kerry Ratcliffe Sunil Sarda Ian D Wilson

Publish Date: 2016/11/28

Volume: 91, Issue: 7, Pages: 2643-2653

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Abstract

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 ductcannulated 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 postdose Mice dosed with 14Cfenclozic acid showed acute centrilobular hepatocellular necrosis but no other regions of the liver were affected The majority of the 14Cfenclozic acidrelated 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 glutathionederived 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 proteinFenclozic acid 24chlorophenylthiazol4ylacetic acid Myalex was identified as a promising antirheumatic in the 1960’s that exhibited no adverse hepatic effects in preclinical animal tests or initial studies in man Chalmers et al 1969a b However whilst doses of 100 mg twice daily were well tolerated doses of 400 mg resulted in a relatively high incidence of jaundice 2 out of 12 subjects in one centre Hart et al 1970 As further cases of jaundice and abnormal hepatic function were detected in other trials where the 400 mg dose of fenclozic acid was administered clinical studies were halted and development was terminated Alcock 1970 Following the termination of development the preclinical data for fenclozic acid were reexamined and additional work in a wider range of preclinical species was undertaken in an effort to understand the toxicity of the compound but to no effect Alcock 1970 Fenclozic acid therefore stands as an example of a drug candidate with a good preclinical safety profile and good efficacy that nevertheless caused serious doserelated nonidiosyncratic adverse drug reactions ADR’s in man Whilst such examples of humanspecific ADRs are fortunately rare they raise questions regarding the effectiveness of preclinical safety studies that would benefit from a thorough mechanistic investigation of the causes of the toxicity Recent studies showed that the bioactivation of fenclozic acid in vitro resulted in high levels of covalent binding to microsomal preparations supplemented with NADPH Rodrigues et al 2013 but not UDPGA So whilst the reactive species responsible for the covalent binding proved refractory to identification in either microsomal or hepatocytebased systems it seemed likely that oxidative metabolism was responsible for the bioactivation of the drug Indeed although potentially reactive acyl glucuronides are also formed during the metabolism of fenclozic acid in vitro covalent binding in microsomal systems supplemented with UDPGA was lower than systems optimised for oxidative metabolism Further studies in the HRN™ hepatic reductase null mouse where hepatic CYP450s are inactive and the drug is essentially subject to biotransformation via acyl conjugation to glucuronic acid and amino acid conjugates showed only low levels of hepatic covalent binding Pickup et al 2014 More recently studies in bile ductcannulated rats showed the presence of metabolites that were clearly derived from reactive metabolites detoxified via reaction with glutathione Martin et al 2014 However as this study did not employ radiolabelled material it was not possible to determine the contribution of this route to the overall metabolic fate of the drug Here the metabolic fate and hepatic effects of 14Cfenclozic acid in normal and bile ductcannulated male C57BL/6J mice have been investigatedUnlabelled fenclozic acid 224chlorophenylthiazol4ylacetic acid was obtained from Compound Management AstraZeneca Alderley Park Cheshire UK batch number AZ10002189024 14Cfenclozic acid 224chlorophenyl214Cthiazol4ylacetic acid was synthesised and supplied by Isotope Chemistry AstraZeneca at 237 μCi/mg and a radiochemical purity 99 Ultima Gold scintillation cocktail was purchased from Packard Instruments Pangbourne UK CarboSorb oxidiser absorbant and Permafluor E+ scintillant were obtained from PerkinElmer Beaconsfield UK whilst FLUOTHANE™ was obtained from the AstraZeneca Group of Companies Pierce BCA protein assay kit was obtained from Perbio Science Cramlington UK Acetonitrile ethanol formic acid hexane tetrahydrofuran THF trifluoroacetic acid TFA and xylene were all of analytical grade and supplied by Fisher Scientific Loughborough UK Phosphate buffer pH 74 was supplied by AZ media AstraZeneca All other chemicals or solvents were purchased from commercial suppliers and were of analytical grade or the best equivalentFifteen wildtype mice strain C57BL/6J aged approximately 8 weeks and weighing between 228 and 272 g were supplied by the AstraZeneca Rodent Breeding Unit Alderley Park All animal procedures and treatments were carried out in accordance with approved animal licenses and guidelines issued by the British Home office Animals Scientific Procedures Act 1986All of the mice used in these studies were identified by unique tail markings and acclimatised for 3 days before dosing Mice were kept at room temperature and exposed to a 12 h dark/12 h artificial light cycle and an RM No 1 Modified Irradiated diet potable water was available ad libitum Mice were fasted overnight for 12 h prior to oral administration by gavage of either 14Cfenclozic acid 10 mg/kg or phosphate buffer dosing volume of 5 mL/kg the following morning Phosphate buffer pH 74 was used as the vehicle control doseStudy 1 Semiquantitative whole body autoradiography QWBA was performed in order to determine the distribution of 14Cfenclozic acid dose 10 mg/kg and 200 μCi/kg following oral administration to 6 male C57BL/6J mice 14CFenclozic acid specific activity of 237 μCi/mg was diluted with unlabelled fenclozic acid to give material with a specific activity of ca 110 μCi/mg Unlabelled fenclozic acid was dissolved in phosphate buffer pH 74 and an appropriate volume of this solution was added to the 14Cfenclozic acid to give a solution at the targetspecific activity 20 μCi/mg and a target concentration of 2 mg/mL After dosing the mice were housed individually in polycarbonate cages with stainless steel mesh inserts with two animals killed at each of the following time points 6 24 and 72 h postdose by FLUOTHANE™ inhalation Carcasses were immediately frozen in a mixture of hexane and dry ice at ca −80 °C Following freezing the hind legs and tail were removed and each carcass was then embedded left side uppermost in a block of carboxymethylcellulose ca 2 in water The blocks were mounted on the stage of a Leica CM3600 cryomicrotome Leica Microsystems Germany maintained at approximately −20 °C Sagittal sections 30 µm were prepared from each animal based on the work of Ullberg Ullberg 1954 to include as many tissues as possible Sections were then allowed to freezedry prior to exposure to FUJI phosphor imaging plates Raytek Sheffield UK Sections for imaging were enclosed in a lighttight cassette and were exposed for 7 days After exposure the imaging plates were removed from the cassettes under dark room conditions and the plates scanned using a FUJI FLA 5000 phosphor imager Images were obtained and quantified using AIDA version 422 Raytest UK Chesterfield UKStudy 2 A study was undertaken to profile the metabolites present in the excreta of male C57BL/6J mice n = 3 14Cfenclozic acid following oral administration dose 10 mg/kg and 1100 μCi/kg in pH 74 phosphate buffer with a dosing volume of 5 mL/kg 14CFenclozic acid specific activity 237 μCi/mg was diluted with unlabelled fenclozic acid to give material with a specific activity of ca 110 μCi/mg as described above A further 3 mice were administered phosphate buffer to provide a vehicle only control dose for histopathology see below Mice were housed individually in glass metabolism cages and urine faeces and aqueous cage wash were collected from individual animals for 72 h postdose urine and cage wash 0–6 6–12 and 12–24 h then every 24–72 h faeces were collected 0–24 24–48 and 48–72 h and samples were collected over dry ice to ensure sample stability after which animals were killed by FLUOTHANE™ inhalation Following death the mice were exsanguinated and the blood spun for 2 min at approximately 7800 g and ambient temperature to produce plasma The liver with gall bladder removed and kidneys from each animal were removed a small sample of each liver was taken and fixed for histopathology and the remainder of the liver and kidneys were snapfrozen in liquid nitrogen

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