Fenclozic Acid Research Articles

A retrospect on antimicrobial potential of thiazole scaffold

AbstractDue to its appreciable diversity in biological actions, thiazole and its substituted components, a significant class of heterocyclic compounds has developed as an influential scaffold in the field of chemical sciences. The variability of thiazole core has been expressed through the effective instigation of its anticancer (Dasatinib, Tiazofurin), antiretroviral (Ritonavir, Brecanavir), antimicrobial (Sulfathiazole, Ravuconazole) and anti‐inflammatory (Fenclozic acid, Meloxicam) derivatives. This reasonable diversity in the physiological reaction pattern led many scientists to refine and develop new structural alternatives with much more efficient pharmacological action. This review is crucial for previous studies and projects to survey the antimicrobial activity of thiazole and thiazole related compounds to the mid of 2019.

The metabolic fate of fenclozic acid in chimeric mice with a humanized liver

The metabolic fate of the human hepatotoxin fenclozic acid ([2-(4-chlorophenyl)-1,3-thiazol-4-yl]acetic acid) (Myalex) was studied in normal and bile-cannulated chimeric mice with a humanized liver, following oral administration of 10 mg/kg. This in vivo animal model was investigated to assess its utility to study “human” metabolism of fenclozic acid, and in particular to explore the formation of electrophilic reactive metabolites (RMs), potentially unique to humans. Metabolism was extensive, particularly involving the carboxylic acid-containing side chain. Metabolism resulted in the formation of a large number of metabolites and involved biotransformation via both oxidative and conjugative routes. The oxidative metabolites detected included a variety of hydroxylations as well as cysteinyl-, N-acetylcysteinyl-, and cysteinylglycine metabolites. The latter resulted from the formation of glutathione adducts/conjugates providing evidence for the production of RMs. The production of other classes of RMs included acyl-glucuronides, and the biosynthesis of acyl carnitine, taurine, glutamine, and glycine conjugates via potentially reactive acyl-CoA intermediates was also demonstrated. A number of unique “human” metabolites, e.g., those providing evidence for side-chain extension, were detected in the plasma and excreta of the chimeric liver-humanized mice that were not previously characterised in, e.g., the excreta of rat and C57BL/6 mice. The different pattern of metabolism seen in these chimeric mice with a humanized liver compared to the conventional rodents may offer clues to the factors that contributed to the drug-induced liver injury seen in humans.

Improved hepatic physiology in hepatic cytochrome P450 reductase null (HRN™) mice dosed orally with fenclozic acid.

Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit no functional expression of hepatic cytochrome P450 (P450) when compared to wild type (WT) mice, but have normal hepatic and extrahepatic expression of other biotransformation enzymes. We have assessed the utility of HRN™ mice for investigation of the role of metabolic bioactivation in liver toxicity caused by the nonsteroidal anti-inflammatory drug (NSAID) fenclozic acid. In vitro studies revealed significant NADPH-dependent (i.e. P450-mediated) covalent binding of [14C]-fenclozic acid to liver microsomes from WT mice and HRN™ mice, whereas no in vitro covalent binding was observed in the presence of the UDP-glucuronyltransferase cofactor UDPGA. Oral fenclozic acid administration did not alter the liver histopathology or elevate the plasma liver enzyme activities of WT mice, or affect their hepatic miRNA contents. Livers from HRN™ mice exhibited abnormal liver histopathology (enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration, necrosis, inflammatory cell infiltration) and plasma clinical chemistry (elevated alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities). Modest apparent improvements in these abnormalities were observed when HRN™ mice were dosed orally with fenclozic acid for 7 days at 100 mg kg-1 day-1. Previously we observed more marked effects on liver histopathology and integrity in HRN™ mice dosed orally with the NSAID diclofenac for 7 days at 30 mg kg-1 day-1. We conclude that HRN™ mice are valuable for assessing P450-related hepatic drug biotransformation, but not for drug toxicity studies due to underlying liver dysfunction. Nonetheless, HRN™ mice may provide novel insights into the role of inflammation in liver injury, thereby aiding its treatment.

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).

Significantly Different Covalent Binding of Oxidative Metabolites, Acyl Glucuronides, and S-Acyl CoA Conjugates Formed from Xenobiotic Carboxylic Acids in Human Liver Microsomes.

Xenobiotic carboxylic acids may be metabolized to oxidative metabolites, acyl glucuronides, and/or S-acyl-CoA thioesters (CoA conjugates) in vitro, e.g., in hepatocytes, and in vivo. These metabolites can potentially be reactive species and bind covalently to tissue proteins and are generally considered to mediate adverse drug reactions in humans. Acyl glucuronide metabolites have been the focus of reactive metabolite research for decades, whereas drug-CoA conjugates, which have been shown to be up to 40-70 times more reactive, have been given much less attention. In an attempt to dissect the contribution of different pathways to covalent binding, we utilized human liver microsomes supplemented with NADPH, uridine 5'-diphosphoglucuronic acid (UDPGA), or CoA to evaluate the reactivity of each metabolite separately. Seven carboxylic acid drugs were included in this study. While ibuprofen and tolmetin are still on the market, ibufenac, fenclozic acid, tienilic acid, suprofen, and zomepirac were stopped before their launch or withdrawn. The reactivities of the CoA conjugates of ibuprofen, ibufenac, fenclozic acid, and tolmetin were higher compared to those of their corresponding oxidative metabolites and acyl glucuronides, as measured by the level of covalent binding to human liver microsomal proteins. The highest covalent binding was observed for ibuprofenyl-CoA and ibufenacyl-CoA, to levels of 1000 and 8600 pmol drug eq/mg protein, respectively. In contrast and in agreement with the proposed P450-mediated toxicity for these drug molecules, the reactivities of oxidative metabolites of suprofen and tienilic acid were higher compared to the reactivities of their conjugated metabolites, with NADPH-dependent covalent binding of 250 pmol drug eq/mg protein for both drugs. The seven drugs all formed UDPGA-dependent acyl glucuronides, but none of these resulted in covalent binding. This study shows that, unlike studies with hepatocytes or in vivo, human liver microsomes provide an opportunity to investigate the reactivity of individual metabolites.

Identification of the reactive metabolites of fenclozic acid in bile duct cannulated rats.

Fenclozic acid (Myalex) was developed by ICI pharmaceuticals in the 1960s for the treatment of rheumatoid arthritis and was a promising compound with a good preclinical safety profile and efficacy. While it did not show adverse hepatic effects in preclinical animal tests or initial studies in man [ Chalmers et al. Ann. Rheum. Dis. 1969 , 28 , 595 and Chalmers et al. Ann. Rheum. Dis. 1969 , 28 , 590 ], it was later withdrawn from clinical development. Hepatotoxicity was observed in humans at daily doses of 400 mg but was not replicated in any of the animal species tested. Rodrigues et al. [ Arch. Toxicol. 2013 , 87 , 1569 ] published a mechanistic investigation using modern in vitro assays/techniques in order to investigate the hepatotoxicity; however, only the covalent binding in rat, dog, and human microsomes was identified as a potential indicator for hepatoxicity. Metabolites associated with or responsible for covalent binding could not be detected, likely due to the low in vitro metabolic turnover of fenclozic acid in microsomes. Foulkes [ J. Pharmacol. Exp. Ther. 1970 , 172 , 115 ] investigated the in vivo metabolism of fenclozic acid which included a rat bile duct cannulated (BDC) study characterizing the biliary and urinary metabolites; however, no reactive metabolites were identified. This study aimed to reinvestigate the in vivo metabolism of fenclozic acid in rat, with a focus on identifying any reactive metabolites that could explain the in vitro covalent binding in microsomes observed across the species. Using modern analytical techniques, we were successful in identifying an epoxide reactive metabolite, which upon conjugation with glutathione (GSH), formed up to 16 GSH-related products including positional and diastereoisomers. Not including the GSH related conjugates, 7 additional metabolites were identified compared to these previous metabolism studies.

Utility of the HRN™ (hepatic cytochrome P450 reductase null) mice for investigating mechanisms of liver toxicity of carboxylic-acid-containing drugs

Many carboxylic-acid-containing drugs cause liver injury in humans. Examples include fenclozic acid (FA), which was withdrawn due to jaundice observed in clinical trials, and diclofenac (DFC) which remains widely prescribed despite being associated with liver damage. To explore whether these toxicities could be due to metabolic bioactivation mediated by cytochrome P450 (CYP) or conjugative enzymes, covalent binding (CVB) assays were done using liver microsomal incubations from wild-type and hepatic cytochrome P450 reductase null (HRN™) mice, which are deficient in CYP activity. High levels of CYP-mediated CVB of [14C]-FA and [14C]-DFC were observed in wild-type microsomes, but not in HRN™ microsomes. No UDPGAmediated CVB was detected in microsomes incubated with [14C]-FA. Wild-type and HRN™ mice were orally administered DFC or FA orally for 7days. At 100 mg/kg, FA caused a significant (p<0.05) time-dependent increase in plasma alanine amino transferase (ALT) in wild-type but not HRN™ mice. Aberrant liver histopathology and liver clinical chemistry were evident in HRNTM mice and treatment with DFC and FA “normalised” the elevated ALT levels. These data demonstrate that FA undergoes CYP mediated bioactivation and that HRNTM mice are well suited to investigations of metabolism, but not of liver toxicity, due to impaired liver function.

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 [14C]-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.

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. Time-dependent covalent binding to protein was observed in NADPH-supplemented liver microsomes, although no metabolites were detected in these incubations or in reactive metabolite trapping experiments. In human hepatocytes, covalent binding was observed at lower levels than in microsomes and a minor uncharacterizable metabolite was also observed. In addition, covalent binding was observed in incubations undertaken with dog and rat hepatocytes, where a taurine conjugate of the drug was detected. Although an acyl glucuronide metabolite was detected when liver microsomes from human, rat and dog were supplemented with UDPGA, there was no detectable UDPGA-dependent covalent binding. No effects were observed when fenclozic acid was assessed for P450-dependent and P450-independent cytotoxicity to THLE cell lines, time-dependent inhibition of five major human cytochrome P450 enzymes, inhibition of the biliary efflux transporters BSEP and MRP2 or mitochondrial toxicity to THLE or HepG2 cells. These data suggest that Phase 1 bioactivation plays a role in the hepatotoxicity of fenclozic acid and highlight the unique insight into mechanisms of human drug toxicity that can be provided by investigations of biotransformation and covalent binding to proteins.

A modern approach to the synthesis of 2‐(4‐chlorophenyl)[2‐ 14C]thiazol‐4‐ylacetic acid ([14C] fenclozic acid) and its acyl glucuronide metabolite

An updated approach to the 1960s synthesis of [(14)C] fenclozic acid from labelled potassium cyanide is presented. By employing modern synthetic methodology and purification techniques, many of the inherent hazards in the original synthesis are avoided or significantly reduced. The concomitant labelled stereoselective synthesis of the key acyl glucuronide metabolite (the 1-β-O-acyl glucuronide) is also described.

The fate of fenclozic acid in the gut and its effect on some aspects of gut metabolism.

In the rat [14C]fenclozic acid is not metabolized in the gut and passes into the portal blood unchanged. After intraduodenal administration of [14C]fenclozic acid, a small proportion of the dose binds to high molecular weight substances in the gut wall. The incorporation of L-[U-14C]leucine and N-[3H]acetyl-D-glucosamine into acid-precipitable materials by isolated mucosal cells and homogenates of gut mucosal cells was inhibited by fenclozic acid in a dose-dependent manner. Other non-steroidal anti-inflammatory drugs (indomethacin, phenylbutazone, prednisolone, salicylic acid and paracetamol) were tested for their potency as inhibitors of glycoprotein production by whole cell preparations and by homogenized gut cell preparations. Marked differences were observed in the inhibitory potency of indomethacin, paracetamol and salicylic acid in the two experimental systems. Fenclozic acid had no major effect on the rate of total glycoprotein production by the isolated perfused rat liver or by the duodenal mucosa in situ. Fenclozic acid displaces albumin-bound [3H]tryptophan and increases the level of hepatic tryptophan pyrrolase approx. threefold. The inhibition of gut glycoprotein production by fenclozic acid was not prevented by free tryptophan.

Michaelis-Menten absorption kinetics in drugs: - examples and implications.

It is shown that the absorption of phenylbutazone, naproxen, and chlorthiazide is governed by a dose limiting mechanism. This dose dependency may be explained by a saturation absorption process mathematically obeying Michaelis-Menten type kinetics. Observed dose relationships for tetracycline, fenclozic acid and related compounds, phenytoin, and possibly digoxin and digitoxin may be explained if a saturable process in absorption is postulated. This behavior may be produced by insolubility of the drug compound, a limited "window of absorption" in the gastrointestinal tract, or a capacity limited absorption because of the carrier or the transport mechanism involved. The need for suitably designed dose response studies with new drug compounds is discussed.

The enhanced biliary secretion of a taurine conjugate in the rat after intraduodenal administration of high doses of fenclozic acid.

1. The metabolic fate of [14C]fenclozic acid (ICI 54450) in rat was determined after intraduodenal administration at different doses. 2. Increasing the dose from 2 to 100 mg/kg resulted in a five-fold increase in drug-related material secreted in bile. 3. At a dose of 2 mg/kg the taurine conjugate was a relatively minor metabolite, whereas at 100 mg/kg this conjugate was the major metabolite in bile and urine. 4. Enhanced biliary secretion of the taurine conjugate in rats receiving multiple doses of fenclozic acid results in exposure of the intestinal cells to much greater concn. of drug-related metabolites.

The effects of fenclozic acid on fertility of male rats

Fenclozic acid, a prostaglandin synthesis inhibitor, was administered to male rats by stomach intubation. A second group of rats received the vehicle (1% methylcellulose) alone. The dose schedule used was 75 mg/kg on day 1, 50 mg/kg on day 2 and 25 mg/kg on days 3, 4 and 5. The dose was progressively reduced to eliminate the toxic effects seen in the initial stages of the treatment. All the animals had normal fertility and libido before treatment and during administration of the vehicle. However, sterility or sub-fertility was seen in 4 of the 6 animals treated with fenclozic acid. Sperm were always present in the vaginal smears. It was concluded that the reduction in fertility was probably caused by an effect of the drug on maturation or metabolism of the sperm.

Enhancement of natural and antibody-dependent lymphocyte cytotoxicity by drugs which inhibit prostaglandin production by tumor target cells

Our previous observations suggested that the production of prostaglandins by tumor cells exposed to lymphocytes might constitute a mechanism by which the tumor cells Could subvert the effects of a cellular immune response directed against them. The present experiments tested this hypothesis by determining whether inhibition of prostaglandin production permitted enhanced expression of natural and antibody-dependent lymphocyte cytotoxicity against the target cells. Cell lines T 24 and HCV 29 were labelled with 51Chromium and incubated with purified lymphocytes obtained from venous blood of normal donors. Antiserum to T 24 and varying concentrations of inhibitors of prostaglandin synthetase (indomethacin, fenclozic acid, acetylsalicylic acid, and 2,6-xylenol) were added at the onset of incubation and assay tubes were incubated for varying times at 37 °C. In some experiments, lymphocytes or labeled target cells were preincubated with inhibitors and then washed prior to their addition to the assay tubes. Cytotoxicity was determined by measuring 51Chromium release and assessing any differences that might reflect the presence of the various drugs. Each prostaglandin synthetase inhibitor significantly enhanced both natural and antibody-dependent lymphocyte cytotoxicity. Enhancement appeared to reflect an effect on the target cells, presumeably by an inhibition of prostaglandin production. No increase in spontaneous 51Chromium release was apparent. The inhibitors did not appear to activate lymphocytes. This evidence supports the suggestion of a mechanism in which tumor cells may prevent the effect of a cellular immune response by producing inhibitory levels of prostaglandins. These results also suggest that manipulation of this mechanism can enhance the effectiveness of the lymphocyte response and may be a consideration in assessing lymphocyte/tumor cell interaction in vitro and in vivo.

The effect of adjuvant arthritis and drugs on the ability of rat plasma to inhibit the triton X-100 induced lysis of rabbit polymorphonuclear leucocyte granules

Plasma from normal rats has the ability to inhibit the Triton X-100 lysis of rabbits polymorphonuclear leucocyte granules. During the development of adjuvant-induced arthritis this activity decreases but no change occurs when rats are given injections of E. coli in oil into the foot pad. A study has been made of the levels of stabilizing activity present in the plasma of both normal and arthritic rats treated with either anti-inflammatory drugs or drugs alleged to affect the stability of lysosomal membranes. Paramethasone, prednisolone and menadione prevented the fall in levels during the onset of the adjuvant-induced arthritis but only paramethasone caused an increase in the protective effect when given to normal rats. Chloroquine, cortisone (lysosomal stabilizers) testosterone, oestradiol (lysosomal labilizers) progesterone and fenclozic acid failed to correct the fall in stabilizing activity in arthritic rats but progesterone, oestradiol and chloroquine caused a decrease in stabilizing activity when given to normal rats.

Metabolism of 2-(4-chlorophenyl)thiazol-4-ylacetic acid (fenclozic acid) and related compounds by microorganisms.

Metabolism of 2-(4-chlorophenyl)thiazol-4-ylacetic acid (fenclozic acid) and related compounds by microorganisms.

The pharmacology of fenclozic acid (2-(4-chlorophenyl)-thiazol-4-ylacetic acid; I.C.I. 54,450; 'Myalex'); a new compound with anti-inflammatory, analgesic and antipyretic activityl.

1. Fenclozic acid (2-(4-chlorophenyl)thiazol-4-ylacetic acid; I.C.I. 54,450; "Myalex") is one representative of a new class of compounds with antiinflammatory, analgesic and antipyretic properties as evidenced by its activity in a variety of tests in rats, mice and guinea-pigs.2. In tests of short duration the potency of fenclozic acid is similar to that of phenylbutazone.3. In tests of longer duration fenclozic acid is more potent than phenylbutazone.4. The activity of fenclozic acid is not mediated by stimulation of the adrenals and the compound is devoid of corticosteroid-like activity.