ABSTRACT:Carboxylic acid-containing nonsteroidal anti-inflammatory drugs (NSAIDs) can be metabolized to chemically reactive acyl glucuronide and/or S-acyl-CoA thioester metabolites capable of transacylating GSH. We investigated the metabolism of the NSAID mefenamic acid (MFA) to metabolites that transacylate GSH, leading to MFA-S-acyl-GSH thioester (MFA-SG) formation in incubations with rat and human hepatocytes and in vivo in rat bile. Thus, incubation of MFA (1-500 M) with rat hepatocytes led to the detection of MFA-1--O-acyl glucuronide (MFA-1--O-G), MFA-S-acylCoA (MFA-SCoA), and MFA-SG by liquid chromatography-tandem mass spectrometric analysis. The C max of MFA-SG (330 nM; 10-min incubation with 100 M MFA) was 120-to 1400-fold higher than the C max of drug S-acyl-GSH adducts detected from studies with other carboxylic acid drugs to date. MFA-SG was also detected in incubations with human hepatocytes, but at much lower concentrations. Inhibition of MFA acyl glucuronidation in rat hepatocytes had no effect on MFA-SG formation, whereas a 58 ؎ 1.7% inhibition of MFA-SCoA formation led to a corresponding 66 ؎ 3.5% inhibition of MFA-SG production. Reactivity comparisons with GSH in buffer showed MFA-SCoA to be 80-fold more reactive than MFA-1--O-G forming MFA-SG. MFA-SG was detected in MFAdosed (100 mg/kg) rat bile, where 17.4 g was excreted after administration. In summary, MFA exhibited bioactivation in rat and human hepatocytes and in vivo in rat, leading to reactive acylating derivatives that transacylate GSH. The formation of MFA-SG in hepatocytes was shown not to be mediated by reaction with MFA-1--O-G, and not solely by MFA-SCoA, but perhaps also by intermediary MFA-acyladenylate formation, which is currently under investigation.
BMN 250 is being developed as enzyme replacement therapy for Sanfilippo type B, a primarily neurological rare disease, in which patients have deficient lysosomal alpha-N-acetylglucosaminidase (NAGLU) enzyme activity. BMN 250 is taken up in target cells by the cation-independent mannose 6-phosphate receptor (CI-MPR, insulin-like growth factor 2 receptor), which then facilitates transit to the lysosome. BMN 250 is dosed directly into the central nervous system via the intracerebroventricular (ICV) route, and the objective of this work was to compare systemic intravenous (IV) and ICV delivery of BMN 250 to confirm the value of ICV dosing. We first assess the ability of enzyme to cross a potentially compromised blood–brain barrier in the Naglu−/− mouse model and then assess the potential for CI-MPR to be employed for receptor-mediated transport across the blood–brain barrier. In wild-type and Naglu−/− mice, CI-MPR expression in brain vasculature is high during the neonatal period but virtually absent by adolescence. In contrast, CI-MPR remains expressed through adolescence in non-affected non-human primate and human brain vasculature. Combined results from IV administration of BMN 250 in Naglu−/− mice and IV and ICV administration in healthy juvenile non-human primates suggest a limitation to therapeutic benefit from IV administration because enzyme distribution is restricted to brain vascular endothelial cells: enzyme does not reach target neuronal cells following IV administration, and pharmacological response following IV administration is likely restricted to clearance of substrate in endothelial cells. In contrast, ICV administration enables central nervous system enzyme replacement with biodistribution to target cells.
ABSTRACT:Flunoxaprofen (FLX) is a chiral nonsteroidal anti-inflammatory drug that was withdrawn from clinical use because of concerns of potential hepatotoxicity. FLX undergoes highly stereoselective chiral inversion mediated through the FLX-S-acyl-CoA thioester (FLX-CoA) in favor of the (R)-(؊)-isomer. Acyl-CoA thioester derivatives of acidic drugs are chemically reactive species that are known to transacylate protein nucleophiles and glutathione (GSH). In this study, we investigated the relationship between the stereoselective metabolism of (R)-(؊)-and (S)-(؉)-FLX to FLX-CoA and the subsequent transacylation of GSH forming FLX-S-acyl-glutathione (FLX-SG) in incubations with rat hepatocytes in suspension. Thus, when hepatocytes (2 million cells/ml) were treated with (R)-(؊)-or (S)-(؉)-FLX (100
b S Supporting Information P rostaglandin D 2 (PGD 2 ) is produced by mast cells in high concentrations during allergic responses and plays a key role in mediating allergic reactions seen in asthma, allergic rhinitis, atopic dermatitis, and allergic conjunctivitis. 1,2 PGD 2 exerts its activity through two G protein-coupled receptors (GPCRs), DP (prostanoid D receptor, DP 1 ) and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells, DP 2 ). These two GPCRs act in concert to promote a number of biological effects associated with the development and maintenance of the allergic responses. Numerous studies using DP and CRTH2 antagonists, combined with genetic analysis, support the view that these receptors play a pivotal role in mediating allergic diseases. [3][4][5][6] Therefore, there has been significant interest in the development of selective DP and CRTH2 antagonists. [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] We have pursued the thesis that blockade of both receptors may prove more beneficial in alleviating allergic diseases triggered by PGD 2 than inhibiting either one separately; therefore, we have been interested in identifying potent CRTH2/DP dual inhibitors. We previously reported on the discovery of AMG 009 (1; Figure 1) as a potent CRTH2 and DP dual antagonist. 23 In this article, we report our continued optimization efforts, which led to the discovery of AMG 853 (2), which possesses increased DP and CRTH2 potency as compared to 1.Compounds 1-25 were synthesized according to the chemistry depicted in Scheme 1. 4-Chloro-3-nitrobenzoyl chloride was reacted with amines to form the corresponding amides. Displacement of the chlorine adjacent to the nitro group with 4-hydroxyphenylacetic acids gave the bis-aryl ethers in good yields. Reduction of the nitro group followed by protection of the carboxylic acids as methyl esters provided the aniline methyl esters. The final two steps were carried out in one pot.The anilines were converted to the benzene sulfonamides by treating with sulfonyl chlorides, and then, hydrolysis of the methyl esters using sodium hydroxide afforded the final compounds (1-25).Compound 1 had high affinity toward the CRTH2 receptor, even in the presence of human plasma. It inhibited the binding of 3 H-PGD 2 to the CRTH2 receptors on HEK-293 cells with an IC 50 of 0.021 μM in the presence of human plasma. It also inhibited the binding of 3 H-PGD 2 to the DP receptors with a moderate IC 50 of 0.28 μM in the presence of plasma. Therefore, we set out to find compounds with improved activity on DP while maintaining the excellent activity on CRTH2 displayed by 1. In addition, in vitro metabolism studies with 1 identified the methoxy, amide, and sulfonamide phenyl groups as potential metabolic sites. Thus, we focused our efforts on modifying these regions.Compound 1 was one of the most potent dual CRTH2 and DP inhibitors identified from this series when it was selected for preclinical studies. Potency comparisons were mainly based on IC 50 values in th...
Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B) is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase activity, leading to increased levels of nondegraded heparan sulfate (HS). A mouse model has been useful to evaluate novel treatments for MPS IIIB, but has limitations. In this study, we evaluated the naturally occurring canine model of MPS IIIB for the onset and progression of biochemical and neuropathological changes during the preclinical stages (onset approximately 24–30 months of age) of canine MPS IIIB disease. Even by 1 month of age, MPS IIIB dogs had elevated HS levels in brain and cerebrospinal fluid. Analysis of histopathology of several disease-relevant regions of the forebrain demonstrated progressive lysosomal storage and microglial activation despite a lack of cerebrocortical atrophy in the oldest animals studied. More pronounced histopathology changes were detected in the cerebellum, where progressive lysosomal storage, astrocytosis and microglial activation were observed. Microglial activation was particularly prominent in cerebellar white matter and within the deep cerebellar nuclei, where neuron loss also occurred. The findings in this study will form the basis of future assessments of therapeutic efficacy in this large animal disease model.
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