ABSTRACT
Mavacamten is a small molecule modulator of cardiac myosin designed as an orally administered drug for the treatment of patients with hypertrophic cardiomyopathy. The current study objectives were to assess the preclinical pharmacokinetics of mavacamten for the prediction of human dosing and to establish the potential need for clinical pharmacokinetic studies characterizing drug-drug interaction potential. Mavacamten does not inhibit CYP enzymes, but at high concentrations relative to anticipated therapeutic concentrations induces CYP2B6 and CYP3A4 enzymes in vitro. Mavacamten showed high permeability and low efflux transport across Caco-2 cell membranes. In human hepatocytes, mavacamten was not a substrate for drug transporters OATP, OCT and NTCP. Mavacamten was determined to have minimal drug-drug interaction risk. In vitro mavacamten metabolite profiles included phase I- and phase II-mediated metabolism cross-species. Major pathways included aromatic hydroxylation (M1), aliphatic hydroxylation (M2); N-dealkylation (M6), and glucuronidation of the M1-metabolite (M4). Reaction phenotyping revealed CYPs 2C19 and 3A4/3A5 predominating. Mavacamten demonstrated low clearance, high volume of distribution, long terminal elimination half-life and excellent oral bioavailability cross-species. Simple four-species allometric scaling led to predicted plasma clearance, volume of distribution and half-life of 0.51 mL/min/kg, 9.5 L/kg and 9 days, respectively, in human.
Subject(s)
Benzylamines/pharmacokinetics , Uracil/analogs & derivatives , Animals , Benzylamines/chemistry , Benzylamines/metabolism , Caco-2 Cells , Cardiac Myosins/metabolism , Cardiomyopathy, Hypertrophic/drug therapy , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Dogs , Drug Interactions , Hepatocytes/metabolism , Humans , Macaca fascicularis , Male , Metabolic Clearance Rate , Mice, Inbred ICR , Microsomes, Liver , Rats, Sprague-Dawley , Uracil/chemistry , Uracil/metabolism , Uracil/pharmacokineticsABSTRACT
To identify aldehyde oxidase (AO) substrates, an assay procedure was developed that leverages the capabilities of high-resolution mass spectrometry to simultaneously monitor parent loss and formation of hydroxylated metabolite over time in incubations with liver cytosol. By incorporating metabolite monitoring, false positives resulting from metabolism by other cytosolic enzymes or processes were decreased. A diverse set of 34 kinase inhibitors containing AO-substrate motifs was screened, and 35% of the compounds were identified as human AO substrates. Confirmation was obtained through determination of the site of metabolism. Human AO substrates identified contained unsubstituted diazanaphthalene moieties (A77-01, INCB 28060, ML-347, LDN-193189, and SB-525334), 4-aminoquinazoline cores (lapatinib, lapatinib M1, and CL-387785), and terminal pyridine and pyrimidine groups (imatinib, bafetinib, and AMG 900). Rat and cynomolgus monkey AO displayed substrate specificities that overlapped moderately with human; rates of metabolism were often higher and lower for cynomolgus monkey and rat, respectively, compared with human. A subset of novel AO substrates identified in this study was also subjected to two other methods for AO substrate determination: comparison of human liver microsome and hepatocyte stability, and the effect of hydralazine, an AO-specific inhibitor, on hepatocyte stability. These methods appeared to correlate and be capable of identifying AO substrates when more than one-third of metabolism in hepatocytes was AO-mediated; however, significant limitations exist. Considering the sensitivity, efficiency, and definitiveness of the cytosol assay with metabolite monitoring, its use is recommended as a primary screen for AO substrates.
Subject(s)
Aldehyde Oxidase/metabolism , Pharmaceutical Preparations/metabolism , Protein Kinase Inhibitors/metabolism , Animals , Cytosol/metabolism , Hepatocytes/metabolism , Humans , Macaca fascicularis , Male , Microsomes, Liver/metabolism , Rats , Substrate SpecificityABSTRACT
Chronic hepatitis B (CHB) virus infection afflicts hundreds of millions of people and causes nearly one million deaths annually. The high levels of circulating viral surface antigen (HBsAg) that characterize CHB may lead to T-cell exhaustion, resulting in an impaired antiviral immune response in the host. Agents that suppress HBsAg could help invigorate immunity toward infected hepatocytes and facilitate a functional cure. A series of dihydropyridoisoquinolizinone (DHQ) inhibitors of human poly(A) polymerases PAPD5/7 were reported to suppress HBsAg in vitro. An example from this class, RG7834, briefly entered the clinic. We set out to identify a potent, orally bioavailable, and safe PAPD5/7 inhibitor as a potential component of a functional cure regimen. Our efforts led to the identification of a dihydropyridophthalazinone (DPP) core with improved pharmacokinetic properties. A conformational restriction strategy and optimization of core substitution led to GS-8873, which was projected to provide deep HBsAg suppression with once-daily dosing.
ABSTRACT
Glycogen synthase 1 (GYS1), the rate-limiting enzyme in muscle glycogen synthesis, plays a central role in energy homeostasis and has been proposed as a therapeutic target in multiple glycogen storage diseases. Despite decades of investigation, there are no known potent, selective small-molecule inhibitors of this enzyme. Here, we report the preclinical characterization of MZ-101, a small molecule that potently inhibits GYS1 in vitro and in vivo without inhibiting GYS2, a related isoform essential for synthesizing liver glycogen. Chronic treatment with MZ-101 depleted muscle glycogen and was well tolerated in mice. Pompe disease, a glycogen storage disease caused by mutations in acid α glucosidase (GAA), results in pathological accumulation of glycogen and consequent autophagolysosomal abnormalities, metabolic dysregulation, and muscle atrophy. Enzyme replacement therapy (ERT) with recombinant GAA is the only approved treatment for Pompe disease, but it requires frequent infusions, and efficacy is limited by suboptimal skeletal muscle distribution. In a mouse model of Pompe disease, chronic oral administration of MZ-101 alone reduced glycogen buildup in skeletal muscle with comparable efficacy to ERT. In addition, treatment with MZ-101 in combination with ERT had an additive effect and could normalize muscle glycogen concentrations. Biochemical, metabolomic, and transcriptomic analyses of muscle tissue demonstrated that lowering of glycogen concentrations with MZ-101, alone or in combination with ERT, corrected the cellular pathology in this mouse model. These data suggest that substrate reduction therapy with GYS1 inhibition may be a promising therapeutic approach for Pompe disease and other glycogen storage diseases.
Subject(s)
Glycogen Storage Disease Type II , Mice , Animals , Glycogen Storage Disease Type II/drug therapy , Glycogen Synthase/metabolism , Glycogen Synthase/pharmacology , Mice, Knockout , Glycogen/metabolism , Muscle, Skeletal/metabolism , Enzyme Replacement Therapy/methodsABSTRACT
The nonclinical safety profile of GS-8873, a hepatitis B virus RNA transcript inhibitor was evaluated in rat and monkey 13-week toxicity studies with 8-week recovery phases. Vehicle or GS-8873 was dosed orally for 13 weeks at 2, 6, 20, and 60 mg/kg/day to Wistar Han rats and at 0.5, 1.5, 3, and 6 mg/kg/day to cynomolgus monkeys. In vitro and in vivo screening results from an analog discovered prior to GS-8873 informed the 13-week toxicology study designs. Neuroelectrophysiology and neurobehavioral evaluations were included at weeks 4 and 13 of the dosing and recovery phases for GS-8873. No adverse neurobehavioral effects were observed. Significant nerve conduction velocity (NCV) decreases and latency increases occurred at the high doses after 4 weeks of dosing. By week 13, dose-responsive NCV reductions and latency increases worsened across all dose groups compared with controls. Some reversal occurred 8 weeks after the last dose administered, but not to vehicle control levels. A minimal, axonal degeneration was observed in rat spinal and peripheral nerves across dose groups compared with controls. No monkey nervous system microscopic findings were observed. No-observed-adverse-effect-levels could not be determined for either species due to the neuroelectrophysiology findings and development was halted in the interest of safety. A retrospective risk assessment approach utilizing benchmark dose (BMD) modeling contributed 13-week NCV BMDL estimates (lower limits of the 95% confidence interval) in lieu of no-observed-adverse-effect-levels. The best-fitted models extrapolated NCV BMDLs for the rat caudal and monkey sural nerve at 0.3 and 0.1 mg/kg/day, respectively.
Subject(s)
Antiviral Agents , Hepatitis B virus , Administration, Oral , Animals , Antiviral Agents/pharmacology , Antiviral Agents/toxicity , Haplorhini , Hepatitis B virus/drug effects , Rats , Rats, Wistar , Retrospective StudiesABSTRACT
Chronic hepatitis B (CHB) is a global health care challenge and a major cause of liver disease. To find new therapeutic avenues with a potential to functionally cure chronic Hepatitis B virus (HBV) infection, we performed a focused screen of epigenetic modifiers to identify potential inhibitors of replication or gene expression. From this work we identified isonicotinic acid inhibitors of the histone lysine demethylase 5 (KDM5) with potent anti-HBV activity. To enhance the cellular permeability and liver accumulation of the most potent KDM5 inhibitor identified (GS-080) an ester prodrug was developed (GS-5801) that resulted in improved bioavailability and liver exposure as well as an increased H3K4me3:H3 ratio on chromatin. GS-5801 treatment of HBV-infected primary human hepatocytes reduced the levels of HBV RNA, DNA and antigen. Evaluation of GS-5801 antiviral activity in a humanized mouse model of HBV infection, however, did not result in antiviral efficacy, despite achieving pharmacodynamic levels of H3K4me3:H3 predicted to be efficacious from the in vitro model. Here we discuss potential reasons for the disconnect between in vitro and in vivo efficacy, which highlight the translational difficulties of epigenetic targets for viral diseases.
Subject(s)
Hepatitis B virus , Hepatitis B, Chronic , Humans , Animals , Mice , Antiviral Agents/pharmacology , Hepatitis B, Chronic/drug therapy , EpigenomicsABSTRACT
We describe here the anti-HBV activity of natural and synthetic retinoids in primary human hepatocytes (PHHs). The most potent compounds inhibited HBsAg, HBeAg, viral RNA and DNA production by HBV infected cells with EC50 values ranging from 0.4 to 2.6⯵M. The activity was independent of PHH donor and HBV genotype used in testing. 13-cis retinoic acid (Accutane) was selected for further evaluation in the PXB chimeric mouse model of HBV infection at doses allowing to achieve Accutane peak serum concentrations near its antiviral EC90 and exposures â¼5-fold higher than a typical clinical dose. While these supraclinical exposures of 100â¯mg/kg/day were well-tolerated by regular Balb/c mice, PXB mice were more sensitive and even a lower those of 60â¯mg/kg/day led to significant weight loss. Despite dosing at this maximal tolerated dose for 28 days, Accutane failed to show any anti-HBV activity. RAR target engagement was verified using transcriptome analysis of liver samples from treated versus vehicle groups. However, gene expression changes in PXB liver samples were vastly muted when compared to the in vitro PHH system. When comparing transcriptional changes associated with the conditioning of fresh hepatocytes toward enabling HBV infection, we also observed a large number of changes. Noticeably, a significant number of genes that were up- or down-regulated by the conditioning process were down- or up-regulated by HBV infected PHH treatment with Accutane, respectively. While the lack of efficacy in the PXB model may have many explanations, the observed, opposing transcriptional changes upon conditioning PHH and treating these cultured, HBV-infected PHH with Accutane allow for the possibility that the PHH system may yield artificial anti-HBV hits.
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B virus/drug effects , Hepatitis B/drug therapy , Hepatocytes/virology , Retinoids/pharmacology , Animals , Antiviral Agents/blood , Cell Survival/drug effects , DNA, Viral/metabolism , Disease Models, Animal , Down-Regulation , Gene Expression/drug effects , Hepatitis B/virology , Hepatitis B Surface Antigens/drug effects , Hepatitis B e Antigens/drug effects , Hepatitis B virus/genetics , Hepatocytes/metabolism , Humans , Isotretinoin/pharmacology , Male , Mice , Mice, Inbred BALB C , RNA, Viral/metabolism , Retinoids/blood , Up-Regulation , Virus Replication/drug effectsABSTRACT
Atropisomerism is a type of axial chirality in which enantiomers or diastereoisomers arise due to hindered rotation around a bond axis. In this manuscript, we report a case in which torsional scan studies guided the thoughtful creation of a restricted axis of rotation between two heteroaromatic systems of a phosphoinositide 3-kinase (PI3K) ß inhibitor, generating a pair of atropisomeric compounds with significantly different pharmacological and pharmacokinetic profiles. Emblematic of these differences, the metabolism of inactive ( M)-28 is primarily due to the cytosolic enzyme aldehyde oxidase, while active ( P)-28 has lower affinity for aldehyde oxidase, resulting in substantially better metabolic stability. Additionally, we report torsional scan and experimental studies used to determine the barriers of rotation of this novel PI3Kß inhibitor.
Subject(s)
Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Adenosine Triphosphate/metabolism , Animals , Enzyme Inhibitors/metabolism , Inhibitory Concentration 50 , Mice , Molecular Docking Simulation , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Protein Conformation , Quinazolines/chemistry , Quinazolines/metabolism , Quinazolines/pharmacology , Stereoisomerism , Substrate SpecificityABSTRACT
Illudins S and M are extremely cytotoxic products of the fungus Omphalotus illudens. They were evaluated as possible anticancer chemotherapeutic agents but displayed unfavorable therapeutic indices. Irofulven (6-hydroxymethylacylfulvene), a less toxic, synthetic derivative of illudin S, has proven very effective in many preclinical and clinical studies. It has been postulated that metabolism via hydrogenation of the 8,9-double bonds of these molecules would unmask the electrophilic, and thus, the toxic nature of their cyclopropyl moieties. Illudins S and M were found to be rapidly metabolized by NADPH-dependent alkenal/one oxidoreductase (AOR) with maximal rates of 115.9 and 44.1 micromol x min(-1) mg(-1), and K(m)s of 308 and 109 microM, respectively. Irofulven was reduced at a much slower rate: V(max) 275 nmol min(-1) mg(-1) and K(m) 145 microM. Human 293 cells transfected with an AOR overexpression vector were 100-fold more sensitive than control cells to irofulven, but displayed little differential sensitivity to illudin M. Addition of glutathione to the alpha,beta-unsaturated ketone moiety of illudin M, but not irofulven, occurred readily at physiological concentrations. Electrophilic intermediates of irofulven and illudin M that were activated by AOR were trapped with glutathione and identified by high performance liquid chromatography with tandem mass spectrometry. Samples of the 60 human tumor cell line panel used by the National Cancer Institute to evaluate potential chemotherapeutic compounds were assayed for AOR activity, which correlated positively with previously determined growth inhibitory measures for irofulven, but not illudin M or S. Collectively, these data indicate that bioactivation of irofulven by AOR plays a predominant role in its chemotherapeutic activity.
Subject(s)
Alcohol Oxidoreductases/metabolism , Alkylating Agents/pharmacology , Sesquiterpenes/pharmacology , Antibiotics, Antineoplastic/pharmacology , Antineoplastic Agents/pharmacology , Cell Line , Cell Line, Tumor , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Glutathione/metabolism , Humans , Ketones/chemistry , Kinetics , Mass Spectrometry , Models, Chemical , Polycyclic Sesquiterpenes , Sensitivity and Specificity , Time Factors , TransfectionABSTRACT
In this review we summarize recent data on the use of phase 2 enzyme inducers as cancer chemopreventive agents in preclinical and clinical studies. These agents elevate the expression of genes involved in the detoxication of electrophiles and free radicals that contribute to carcinogenesis. Their mechanisms of action, efficacy and limitations are discussed. Particular attention is paid to isothiocyante and dithiolethione classes of agents, as these are the most developed.
Subject(s)
Enzyme Activators/therapeutic use , Neoplasms/drug therapy , Neoplasms/prevention & control , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Antioxidants/metabolism , Clinical Trials as Topic , Enzyme Activation/drug effects , Humans , Isothiocyanates/pharmacology , Neoplasms/enzymology , Neoplasms/metabolism , Thiones/pharmacologyABSTRACT
Neonicotinoid insecticide metabolism involves considerable substrate specificity and regioselectivity of the relevant CYP450, aldehyde oxidase, and phase II enzymes. Human CYP450 recombinant enzymes carry out the following conversions: CYP3A4, 2C19, and 2B6 for thiamethoxam (TMX) to clothianidin (CLO); 3A4, 2C19, and 2A6 for CLO to desmethyl-CLO; 2C19 for TMX to desmethyl-TMX. Human liver aldehyde oxidase reduces the nitro substituent of CLO to nitroso much more rapidly than it does that of TMX. Imidacloprid (IMI), CLO, and several of their metabolites do not give detectable N-glucuronides but 5-hydroxy-IMI, 4,5-diol-IMI, and 4-hydroxythiacloprid are converted to O-glucuronides in vitro with mouse liver microsomes and UDP-glucuronic acid or in vivo in mice. Mouse liver cytosol with S-adenosylmethionine converts desmethyl-CLO to CLO but not desmethyl-TMX to TMX. Two organophosphorus CYP450 inhibitors partially block IMI, thiacloprid, and CLO metabolism in vivo in mice, elevating brain and liver levels of the parent compounds while reducing amounts of the hydroxylated metabolites.
Subject(s)
Aldehyde Oxidase/antagonists & inhibitors , Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Insecticides/metabolism , Nitro Compounds/metabolism , Aldehyde Oxidase/chemistry , Animals , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Guanidines/chemistry , Guanidines/metabolism , Humans , Imidazoles/chemistry , Imidazoles/metabolism , Insecticides/chemistry , Kinetics , Male , Microsomes, Liver/chemistry , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Neonicotinoids , Nitro Compounds/chemistry , Oxazines/chemistry , Oxazines/metabolism , Substrate Specificity , Thiamethoxam , Thiazoles/chemistry , Thiazoles/metabolismABSTRACT
The major neonicotinoid insecticide imidacloprid (IMI) is used worldwide for crop protection and pest control on pets. IMI is extensively metabolized, oxidatively by cytochromes P450 and via aerobic nitroreduction by the molybdo-flavoenzyme aldehyde oxidase (AOX). Rabbit liver AOX is capable of reducing IMI to both its nitrosoguanidine (IMI-NO) and aminoguanidine (IMI-NH2) derivatives; however, when IMI-NO is used as a substrate, less than stoichiometric amounts of IMI-NH2 are detected while IMI-NO is completely consumed. The disappearance of IMI-NO requires both a source of AOX and an AOX-specific electron donor substrate and is not inhibited by the addition of catalase and superoxide dismutase. Experiments to evaluate IMI-NO as a possible time-dependent inactivator of AOX reveal the following four characteristics: First, partially purified AOX (ppAOX) is inactivated at a moderate rate by the electron donor substrate N-methylnicotinamide (NMN); second, AOX is inactivated by IMI-NO in an NMN-dependent manner at a 10-fold greater rate; third, IMI does not inactivate AOX; and finally, GSH protects AOX from inactivation but not to a degree greater than IMI-NO-deficient incubations. Values for the kinetic constants of KI and kinact are measured to be 1.3 mM and 0.35 min(-1), respectively. Ultrafiltration is used to establish that IMI-NO inactivation is not reversible and to determine a partition ratio of 1.6. [3H]IMI-NO labeling shows that significant amounts (19%) of this molecule covalently bind to protein following reduction by ppAOX. The addition of 10 mM GSH attenuates this binding almost completely. These findings demonstrate that IMI-NO is metabolically activated by rabbit AOX to form both an irreversible inhibitor and a reactive intermediate that is capable of covalently binding to protein.
Subject(s)
Aldehyde Oxidase/antagonists & inhibitors , Enzyme Inhibitors/toxicity , Imidazoles/toxicity , Liver/enzymology , Nitro Compounds/toxicity , Nitroso Compounds/toxicity , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Imidazoles/chemistry , Imidazoles/metabolism , Neonicotinoids , Nitro Compounds/chemistry , Nitro Compounds/metabolism , Nitroso Compounds/chemistry , Nitroso Compounds/metabolism , Protein Binding , Rabbits , Time FactorsABSTRACT
The nitroguanidine or nitromethylene moiety of the newest major class of insecticides, the neonicotinoids, is important for potency at insect nicotinic receptors and selectivity relative to mammalian receptors. Aldehyde oxidase (AOX) was recently identified as the imidacloprid nitroreductase of mammalian liver, producing both nitrosoguanidine and aminoguanidine metabolites. The present study considers the ability of AOX, partially purified from rabbit liver, to reduce five commercial nitroguanidine (i.e., imidacloprid, thiamethoxam, clothianidin, and dinotefuran) and nitromethylene (i.e., nitenpyram) neonicotinoid insecticides and three derivatives thereof (i.e., the N-methyl and nitromethylene analogues of imidacloprid and desmethylthiamethoxam). LC/MS/MS was used to demonstrate that AOX reduces nitroguanidines to both nitroso- and aminoguanidines, while nitromethylenes are reduced only to the corresponding nitroso metabolites. Additionally, nitrosonitenpyram was found to spontaneously dehydrate to form a 2-cyanoamidine metabolite, mimicking a predominant photoreaction. The substrate specificity of AOX was characterized as follows: Neonicotinoids with a tertiary nitrogen (N-methylimidacloprid and thiamethoxam) are poor substrates; nitroguanidines are metabolized faster than nitromethylenes; and clothianidin is the most rapidly reduced. Kinetic constants were measured for reduction of three nitroguanidines at two concentrations of AOX. At 2 mg protein/mL, only nitroso metabolites were detected, with Km values of 1.03, 2.99, and 2.41 mM and Vmax values of 5.13, 2.54, and 0.98 nmol/min/mg protein measured for clothianidin, imidacloprid, and dinotefuran, respectively. At 5 mg protein/mL, both amino and nitroso metabolites were detected. However, with each nitroguanidine, the formation of nitroso metabolites did not saturate at substrate levels up to 4 mM, whereas amino metabolite formation exhibited Km values of 0.052, 0.16, and 0.084 mM with corresponding Vmax values of 0.80, 1.24, and 0.79 nmol/min/mg protein for clothianidin, imidacloprid, and dinotefuran, respectively. These in vitro observations show large structural differences in the rates of AOX-catalyzed reduction and help to interpret the extensive studies on in vivo metabolism of neonicotinoid insecticides.
Subject(s)
Aldehyde Oxidase/metabolism , Anabasine/analogs & derivatives , Anabasine/metabolism , Guanidines/metabolism , Insecticides/metabolism , Nitro Compounds/metabolism , Aldehyde Oxidase/chemistry , Animals , Guanidines/analysis , Guanidines/chemistry , Imidazoles/metabolism , Insecticides/chemistry , Kinetics , Liver/enzymology , Neonicotinoids , Nitro Compounds/chemistry , Nitroso Compounds/analysis , Nitroso Compounds/metabolism , Oxazines/metabolism , Oxidation-Reduction , Pyridines/metabolism , Rabbits , Substrate Specificity , Thiamethoxam , ThiazolesABSTRACT
Imidacloprid (IMI), the prototypical neonicotinoid insecticide, is used worldwide for crop protection and flea control on pets. It is both oxidatively metabolized by cytochrome P450 enzymes and reduced at the nitroguanidine moiety by a previously unidentified cytosolic "neonicotinoid nitroreductase", the subject of this investigation. Two major metabolites are detected on incubation of IMI with rabbit liver cytosol: the nitrosoguanidine (IMI-NO) and the aminoguanidine (IMI-NH2). Three lines of evidence identify the molybdo-flavoenzyme aldehyde oxidase (AOX, EC 1.2.3.1) as the neonicotinoid nitroreductase. First, classical AOX electron donor substrates (benzaldehyde, 2-hydroxypyrimidine, and N-methylnicotinamide) dramatically increase the rate of formation of IMI metabolites. Allopurinol and diquat are also effective electron donors, while NADPH and xanthine are not. Second, AOX inhibitors (potassium cyanide, menadione, and promethazine) inhibit metabolite formation when N-methylnicotinamide is utilized as an electron donor. Without the addition of an electron donor, rabbit liver cytosol reduces IMI only to IMI-NO at a slow rate. This reduction is also inhibited by potassium cyanide, menadione, and promethazine, as well as by additional AOX inhibitors, cimetidine and chlorpromazine. Finally, IMI nitroreduction by AOX is sensitive to an aerobic atmosphere, but to a much lesser extent than cytochrome P450 2D6. Large species differences are observed in the IMI nitroreductive activity of liver cytosol. While rabbit and monkey (Cynomolgus) give the highest levels of total metabolite formation, human, mouse, cow, and rat also metabolize IMI rapidly. In contrast, dog, cat, and chicken liver cytosols do not reduce IMI at appreciable rates. AOX, as a neonicotinoid nitroreductase, may limit the persistence of IMI, and possibly other neonicotinoids, in mammals.
Subject(s)
Aldehyde Oxidase/chemistry , Anabasine/metabolism , Imidazoles/pharmacology , Nitroreductases/chemistry , Aldehyde Oxidase/antagonists & inhibitors , Aldehyde Oxidase/metabolism , Anabasine/analogs & derivatives , Animals , Cytosol/drug effects , Cytosol/enzymology , Cytosol/metabolism , Enzyme Inhibitors/pharmacology , Guanidines/metabolism , Humans , Imidazoles/metabolism , Imidazoles/toxicity , Liver/drug effects , Liver/enzymology , Liver/metabolism , Molecular Conformation , Neonicotinoids , Nitro Compounds , Nitroreductases/antagonists & inhibitors , Nitroreductases/metabolism , Nitrosoguanidines/metabolism , Time FactorsABSTRACT
Four neonicotinoid nitroguanidine insecticides (imidacloprid, thiamethoxam, clothianidin, and dinotefuran) acting as nicotinic agonists account for 10-15% of worldwide insecticide sales. General methods are needed for synthesis of their guanidine and aminoguanidine metabolites so they may be used as analytical standards and for evaluation of nicotinic receptor potency. The guanidines are obtained by treating the parent nitroguanidines with Fe powder in aqueous C2H5OH containing NH4Cl and isolated by silica chromatography. The aminoguanidines are prepared as mixtures with the guanidines on reaction of the parent nitroguanidines and Zn powder in glacial acetic acid. The imidacloprid aminoguanidine is isolated as the acetone imine or trifluoroacetamide and the clothianidin and dinotefuran aminoguanidines as the acetone imines using silica chromatography. Deprotection under acidic conditions then leads to the aminoguanidine.HCl salts. Because of stability considerations, a pH partitioning method is used to separate thiamethoxam aminoguanidine and guanidine. An alternate procedure to the aminoguanidine of imidacloprid (but not thiamethoxam, clothianidin, or dinotefuran) is reaction with hydrazine hydrate and NH4Cl in anhydrous C2H5OH. Ambiguities in further biological reactions are clarified by synthesizing authentic standards of three purported metabolites formed via the imidacloprid aminoguanidine: the 1,2,4-triazol-3-one derivative with ethyl chloroformate or ethyl pyrocarbonate, the acetaldehyde imine with acetaldehyde, and the 3-methyl-1,2,4-triazin-4-one derivative with ethyl pyruvate in refluxing toluene. The purported triazolone metabolite is reassigned as the aminoguanidine acetaldehyde imine probably formed as an artifact from acetaldehyde present in the ethyl acetate used for metabolite extraction. Potency at the Drosophila nicotinic receptor is greatly decreased on converting a nitroguanidine to a guanidine or aminoguanidine. In sharp contrast, potency at the vertebrate alpha4beta2 nicotinic receptor is generally increased on conversion from the nitroguanidine to aminoguanidine and particularly guanidine derivatives.
Subject(s)
Guanidine/analogs & derivatives , Guanidine/metabolism , Insecticides/metabolism , Receptors, Nicotinic/metabolism , Amination , Animals , Drosophila melanogaster , Guanidine/chemical synthesis , Guanidine/chemistry , Inhibitory Concentration 50 , Insecticides/chemistry , Molecular StructureABSTRACT
NADPH-dependent alkenal/one oxidoreductase (AOR) from the rat is a phase 2/antioxidative enzyme that is known to catalyze the reduction of the carbon-carbon double bond of alpha,beta-unsaturated aldehydes and ketones. It is also known for its leukotriene B(4) 12-hydroxydehydrogenase activity. In order to begin to understand these dual catalytic activities and validate its classification as a reductase of the medium-chain dehydrogenase/reductase family, an investigation of the mechanism of its NADPH-dependent activity was undertaken. Recombinant AOR and a 3-nonen-2-one substrate were used to perform steady-state initial velocity, product inhibition, and dead end inhibition experiments, which elucidated an ordered Theorell-Chance kinetic mechanism with NADPH binding first and NADP(+) leaving last. A nearly 20-fold preference for NADPH over NADH was also observed. The dependence of kinetic parameters V and V/K on pH suggests the involvement of a general acid with a pK of 9.2. NADPH isomers stereospecifically labeled with deuterium at the 4-position were used to determine that AOR catalyzes the transfer of the pro-R hydride to the beta-carbon of an alpha,beta-unsaturated ketone, illudin M. Two-dimensional nuclear Overhauser effect NMR spectra demonstrate that this atom becomes the R-hydrogen at this position on the metabolite. Using [4R-(2)H]NADPH, small primary kinetic isotope effects of 1.16 and 1.73 for V and V/K, respectively, were observed and suggest that hydride transfer is not rate-limiting. Atomic absorption spectroscopy indicated an absence of Zn(2+) from active preparations of AOR. Thus, AOR fits predictions made for medium-chain reductases and bears similar characteristics to well known medium-chain alcohol dehydrogenases.