RESUMEN
Benzphetamine is a Schedule III anorectic agent that is a prodrug for d-amphetamine and d-methamphetamine and may have utility as an "agonist" medication for cocaine use disorder treatment. This study evaluated the pharmacokinetic-pharmacodynamic profile of benzphetamine using a drug discrimination procedure in rhesus monkeys. The potency and time course of cocaine-like discriminative stimulus effects were compared for benzphetamine (10-18mg/kg, intramuscular (IM)) and d-amphetamine (0.032-0.32mg/kg, IM) in monkeys (n=3-4) trained to discriminate IM cocaine (0.32mg/kg) from saline in a two-key food-reinforced discrimination procedure. Parallel pharmacokinetic studies in the same monkeys determined plasma benzphetamine, d-methamphetamine and/or d-amphetamine levels for correlation with behavioral effects. d-Amphetamine produced dose-dependent, time-dependent, and full cocaine-like effects, i.e. ≥90% cocaine-appropriate responding, in all monkeys without altering response rates. The time course of d-amphetamine's cocaine-like discriminative stimulus effects correlated with plasma d-amphetamine levels. Benzphetamine was 180-fold less potent than d-amphetamine and produced full cocaine-like effects in only 2 of 4 monkeys while significantly decreasing response rates. Benzphetamine administration increased plasma d-methamphetamine (peak at 100min) and d-amphetamine (peak at 24h) levels, but the time course of behavioral effects did not correlate with increased levels of benzphetamine, d-methamphetamine or d-amphetamine. These results suggest that benzphetamine yields d-amphetamine and d-methamphetamine as active metabolites in rhesus monkeys, but generation of these metabolites is not sufficient to account for benzphetamine behavioral effects. The incomplete cocaine substitution profile and protracted d-amphetamine plasma levels suggest that benzphetamine may still warrant further evaluation as a candidate pharmacotherapy for cocaine use disorder treatment.
Asunto(s)
Benzfetamina/farmacología , Dextroanfetamina/farmacología , Metanfetamina/farmacología , Animales , Conducta Animal/efectos de los fármacos , Benzfetamina/farmacocinética , Dextroanfetamina/farmacocinética , Macaca mulatta , Masculino , Metanfetamina/farmacocinéticaRESUMEN
Expression of dynorphin, an endogenous opioid peptide, increases with age and has been associated with memory impairments in rats. In human, prodynorphin (Pdyn) gene polymorphisms might be linked to cognitive function in the elderly. Moreover, elevated dynorphin levels have been reported in postmortem samples from Alzheimer's disease patients. However, the cellular and molecular processes affected by higher dynorphin levels during aging remain unknown. Using Pdyn(-/-) mice, we observed significant changes in the function and expression of Group 1 metabotropic glutamate receptor (mGluR). Compared with age-matched wild-type (WT) littermates, we found increased expression of mGluR1α and mGluR5 in the hippocampus and cortex of old, but not young, Pdyn(-/-) mice. Increased Group 1 mGluR expression in aged Pdyn(-/-) mice was associated with enhanced mGluR-mediated long-term depression, a form of synaptic plasticity. Notably, whereas aged WT mice developed spatial and recognition memory deficits, aged Pdyn(-/-) mice performed similarly as young mice. Pharmacological treatments with 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide, a positive modulator of mGlu5 receptors, or norbinaltorphimine, an antagonist for dynorphin-targeted κ-opioid receptor, rescued memory in old WT mice. Conversely, mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride impaired spatial memory of old Pdyn(-/-) mice. Intact cognition in aged Pdyn(-/-) mice paralleled with increased expression of Group 1 mGluR-related genes Homer 1a and Arc. Finally, aged Pdyn(-/-) mice displayed less anxiety-related behaviors than age-matched WT mice. Together, our results suggest that elevated Pdyn expression during normal aging reduces mGluR expression and signaling, which in turn impairs cognitive functions and increases anxiety.
Asunto(s)
Envejecimiento/fisiología , Ansiedad/metabolismo , Encefalinas/deficiencia , Regulación de la Expresión Génica/genética , Precursores de Proteínas/deficiencia , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Ansiedad/tratamiento farmacológico , Benzamidas/farmacología , Benzamidas/uso terapéutico , Benzfetamina/análogos & derivados , Benzfetamina/farmacología , Estimulantes del Sistema Nervioso Central/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Trastornos del Conocimiento/tratamiento farmacológico , Modelos Animales de Enfermedad , Antagonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/uso terapéutico , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Técnicas In Vitro , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/genética , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pirazoles/farmacología , Pirazoles/uso terapéutico , Piridinas/farmacología , Piridinas/uso terapéutico , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Reconocimiento en Psicología/efectos de los fármacos , Reconocimiento en Psicología/fisiologíaRESUMEN
We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a K(I) of 0.44 microM and k(inact) of 0.12 min(-1). The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 A away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by approximately 20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4.
Asunto(s)
Acetileno/análogos & derivados , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas/metabolismo , Acetileno/farmacología , Benzfetamina/farmacología , Catálisis/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Familia 2 del Citocromo P450 , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estereoisomerismo , Especificidad por Sustrato/efectos de los fármacosRESUMEN
A series of naphthylisopropylamine and N-benzyl-4-methylthioamphetamine derivatives were evaluated as monoamine oxidase inhibitors. Their potencies were compared with those of a series of amphetamine derivatives, to test if the increase of electron richness of the aromatic ring and overall size of the molecule might improve their potency as enzyme inhibitors. Molecular dockings were performed to gain insight regarding the binding mode of these inhibitors and rationalize their different potencies. In the case of naphthylisopropylamine derivatives, the increased electron-donating capacity and size of the aromatic moiety resulting from replacement of the phenyl ring of amphetamine derivatives by a naphthalene system resulted in more potent compounds. In the other case, extension of the arylisopropylamine molecule by N-benzylation of the amino group led to a decrease in potency as monoamine oxidase inhibitors.
Asunto(s)
Benzfetamina/análogos & derivados , Inhibidores de la Monoaminooxidasa/farmacología , Naftalenos/farmacología , Propilaminas/farmacología , Animales , Benzfetamina/química , Benzfetamina/farmacología , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Inhibidores de la Monoaminooxidasa/química , Naftalenos/química , Propilaminas/química , RatasRESUMEN
Structural plasticity of mammalian cytochromes P450 (CYP) has recently been explored in our laboratory and elsewhere to understand the ligand-binding promiscuity. CYP2B4 exhibits very different conformations and thermodynamic signatures in binding the small inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) versus the large bifonazole. Using four key active-site mutants (F296A, T302A, I363A, and V367L) that are involved in binding one or both inhibitors, we dissected the thermodynamic basis for the ability of CYP2B4 to bind substrates and inhibitors of different sizes and chemistry. In all cases, 1:1 binding stoichiometry was observed. The inhibitors 4-CPI, 1-(4-chlorophenyl)imidazole, and 1-(2-(benzyloxy)ethyl)imidazole bind to the mutants with a free energy difference (Delta Delta G) of approximately 0.5 to 1 kcal/mol compared with the wild type but with a large entropy-enthalpy compensation of up to 50 kcal/mol. The substrate testosterone binds to all four mutants with a Delta Delta G of approximately 0.5 kcal/mol but with as much as 40 kcal/mol of entropy-enthalpy compensation. In contrast, benzphetamine binding to V367L and F296A is accompanied by a Delta Delta G of approximately 1.5 and 3 kcal/mol, respectively. F296A, I363A, and V367L exhibit very different benzphetamine metabolite profiles, indicating the different substrate-binding orientations in the active site of each mutant. Overall, the findings indicate that malleability of the active site allows mammalian P450s to exhibit a high degree of thermodynamic fidelity in ligand binding.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/metabolismo , Inhibidores Enzimáticos/farmacología , Animales , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Benzfetamina/química , Benzfetamina/farmacología , Sitios de Unión , Calorimetría , Familia 2 del Citocromo P450 , Inhibidores Enzimáticos/química , Ligandos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato/efectos de los fármacos , Termodinámica , Agua/químicaRESUMEN
Extracts of the adult worms of both Schistosoma mansoni and Schistosoma haematobium can metabolise some typical P450 substrates but to differing degrees. S. mansoni worm extracts displayed a approximately 12-fold higher specific activity for an aminopyrine substrate than rat liver microsomes. At 4 mM substrate concentration the demethylation reaction with N-nitrosodimethylamine (NDMA) (5 nmol HCHO/mg protein/min) was only half that of rat liver microsomes, whereas in extracts of S. haematobium, no detectable activity was found towards NDMA. Using ethylmorphine as substrate the demethylation activity of S. mansoni extracts (1.82 nmol HCHO/mg protein/min) was 5.5-fold lower than that of rat liver microsomes. Benzphetamine demethylase activity was also readily detectable in S. mansoni worm extracts at 6.79 nmol HCHO/mg protein/min compared with 10.20 nmol HCHO/mg protein/min in the case of rat liver microsomes. When aniline was used as substrate, surprisingly, no activity was found in worm extracts of either S. mansoni or S. haematobium, whereas rat liver microsomes showed high activity towards this amine. The anti-P450 2E1 and 2B1/2 cross-reacted with both worm homogenates and gave a specific band corresponding to a protein of molecular weight of approximately 50.0 kDa. A study with anti-P450 IVA antibody revealed that while this protein was strongly expressed in S. haematobium worm extracts, no immunoreactivity was observed with extracts of S. mansoni. Immunoblotting analyses with anti-P450 IIIA and P450 1A1 did not detect immunoreactive protein in either S. mansoni or S. haematobium.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Schistosoma haematobium/enzimología , Schistosoma mansoni/enzimología , Aminopirina/farmacología , Compuestos de Anilina/farmacología , Animales , Benzfetamina/farmacología , Cricetinae , Sistema Enzimático del Citocromo P-450/química , Dimetilnitrosamina/farmacología , Activación Enzimática/efectos de los fármacos , Etilmorfina/farmacología , Femenino , Formaldehído/análisis , Formaldehído/metabolismo , Immunoblotting , Masculino , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxazinas/farmacología , Ratas , Schistosoma haematobium/química , Schistosoma mansoni/química , Especificidad por SustratoAsunto(s)
Depresores del Apetito/efectos adversos , Atención Dental para Enfermos Crónicos , Xerostomía/etiología , Adulto , Depresores del Apetito/farmacología , Benzfetamina/efectos adversos , Benzfetamina/farmacología , Caries Dental/etiología , Dietilpropión/efectos adversos , Dietilpropión/farmacología , Interacciones Farmacológicas , Femenino , Fenfluramina/efectos adversos , Fenfluramina/farmacología , Humanos , Masculino , Mazindol/efectos adversos , Mazindol/farmacología , Morfolinas/efectos adversos , Morfolinas/farmacología , Obesidad/tratamiento farmacológico , Enfermedades Periodontales/etiología , Fentermina/efectos adversos , Fentermina/farmacología , Fenilpropanolamina/efectos adversos , Fenilpropanolamina/farmacología , EmbarazoRESUMEN
We have studied the effect of propofol on the cytochrome P450-dependent mono-oxygenase system in human liver microsomes by assaying mono-oxygenase activities toward specific cytochrome P450 isoform test substrates, aniline, 7-ethoxycoumarin, benzphetamine and benzo(a) pyrene. Propofol inhibited benzo(a)pyrene hydroxylation to a greater extent than the oxidative metabolism of the other test substrates, even at 0.05 mmol litre-1. The degrees of inhibition of benzphetamine N-demethylation and 7-ethoxy-coumarin O-de-ethylation were similar, while aniline hydroxylation was least affected by propofol. Spectral analysis showed that propofol competed with carbon monoxide for binding to the haem moiety of haemoprotein in the P450 enzyme. The variable inhibition observed may be caused by the differential binding of propofol to P450 isoforms. Propofol 0.05-1.0 mmol litre-1 exhibited a concentration-dependent inhibitory effect on human cytochrome P450 2E1, 2B1 and 1A1. These inhibitory actions of propofol on human liver microsomal enzymes in vitro suggest that potential drug interactions may exist between propofol and other drugs administered clinically.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Oxigenasas/antagonistas & inhibidores , Propofol/farmacología , Compuestos de Anilina/metabolismo , Benzo(a)pireno/farmacología , Benzfetamina/farmacología , Cumarinas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Microsomas Hepáticos/enzimologíaRESUMEN
The suppression by cannabidiol (CBD) of the liver microsomal drug-metabolizing enzyme activities in female rats was demonstrated and its mechanism was examined. Pretreatment of rats with CBD (10 mg/kg, i.p.) caused temporary decreases in contents of cytochrome P450 (P450) and b5 and NADPH-cytochrome c (P450) reductase activity compared with values from the vehicle control group. p-Nitroanisole O-demethylase, aniline hydroxylase, d-benzphetamine N-demethylase and delta 9-tetrahydrocannabinol 11-hydroxylase were also decreased by the CBD pretreatment. The latter two activities took a longer time to return to control levels than the former two. However, the CBD pretreatment, which reduced the protein level of P450 UT-2 (CYP2C11) in adult male rats, did not decrease the protein level of P450 F-1 (CYP2C6) or F-2 (CYP2C12) in liver microsomes from female rats. These results suggest that the mechanisms by which CBD suppresses liver microsomal drug-metabolizing enzyme activities are different in male and female rats.
Asunto(s)
Cannabidiol/farmacología , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/antagonistas & inhibidores , Animales , Benzfetamina/farmacología , Dronabinol/farmacología , Femenino , Microsomas Hepáticos/efectos de los fármacos , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Caracteres SexualesRESUMEN
The effects of propofol on cytochrome P450 activity in rat hepatic microsomes were evaluated to determine the potential influence of this anesthetic on the metabolism of coadministered agents. In microsomes from untreated and isoniazid-treated rats, propofol was a weak inhibitor of enflurane metabolism, inhibiting activity only at 0.35 mM propofol. In contrast, toluene, a related compound, effectively impaired enflurane defluorination in microsomes from untreated, and isoniazid- and phenobarbital-treated rats at concentrations as low as 0.025 mM. Propofol, in contrast to toluene, was an effective inhibitor of benzphetamine demethylation where it inhibited this activity at propofol concentrations as low as 0.025 mM in microsomes from phenobarbital-treated rats. In microsomes from phenobarbital-treated rats, propofol potently inhibited the metabolism of aniline. Sixty-four percent inhibition was achieved at 0.03 mM propofol, whereas toluene had no effect at 1 mM. These data demonstrate that propofol does not effectively inhibit enflurane metabolism performed by the isoniazid-inducible cytochrome P450IIE1 but effectively impairs activities of the phenobarbital-inducible cytochrome P450 isozymes.
Asunto(s)
Inhibidores Enzimáticos del Citocromo P-450 , Microsomas Hepáticos/enzimología , Propofol/farmacología , Compuestos de Anilina/farmacología , Animales , Benzfetamina/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Enflurano/metabolismo , Hidroxilación , Isoniazida/farmacología , Masculino , Metilación , Microsomas Hepáticos/efectos de los fármacos , Fenobarbital/farmacología , Ratas , Ratas Sprague-Dawley , Tolueno/farmacologíaRESUMEN
The kinetics of NADPH-dependent reduction of cytochrome P450 LM2 in the soluble monomeric reconstituted system in the absence of any substrate is shown to be monophasic. We show that ferrous cytochrome c acts as a competitive inhibitor of the reduction. In the presence of 1 mM benzphetamine an additional extremely fast phase was observed. Under these conditions ferrous cytochrome c was found to be a competitive inhibitor of the slow phase of the reduction process, which accounted for 80% of the total reduction amplitude. Inhibition experiments yield a dissociation constant for the LM2-reductase complex of 3.0 +/- 1.5 microM. This constant was the same both in the presence and in the absence of benzphetamine. Based on these data we conclude that cytochromes P450 and c bind to the same center on the NADPH-cytochrome P450 reductase molecule. Comparative analysis of the amino acid sequences reveals a detectable similarity between cytochrome c and cytochrome P450 LM2 at positions 68-87 and 121-145, respectively. In addition, a substantial similarity was shown for sequence fragments 204-224 of NADPH-cytochrome P450 reductase and 40-60 of cytochrome b5. Based on these findings a hypothesis for the location of the centers of intermolecular interactions on the molecules of cytochrome P450 LM2 and NADPH-cytochrome P450 reductase is proposed.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Grupo Citocromo c/farmacología , Microsomas Hepáticos/enzimología , NADP/metabolismo , Secuencia de Aminoácidos , Animales , Benzfetamina/farmacología , Sitios de Unión , Unión Competitiva , Citocromos b5/metabolismo , Humanos , Cinética , Matemática , Datos de Secuencia Molecular , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxidación-Reducción , Conejos , Homología de Secuencia de Ácido NucleicoRESUMEN
The induction of lipid peroxidation in hepatic microsomes of rodents treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is well documented. The potential mechanisms involved in TCDD-induced microsomal lipid peroxidation were investigated, using selected inhibitors and free radical scavengers in vitro. Rats were treated with 40 micrograms TCDD/kg orally as a single dose. Inhibitors of phospholipase A2, including a variety of phenothiazines, dibucaine, imipramine, and verapamil, inhibited in vitro microsomal lipid peroxidation in response to TCDD administration. In addition, the lipoxygenase inhibitor quercetin, and the hydrogen peroxide scavenger aminopyrine inhibited lipid peroxidation with microsomes from TCDD-treated rats. The singlet oxygen scavenger beta-carotene, the cytochrome P-450 substrate benzphetamine, and the cyclooxygenase inhibitor indomethacin produced moderate enhancement of hepatic microsomal lipid peroxidation. The results suggest that activation of phospholipase A2 may play a critical role in the metabolic events associated with hepatotoxicity and ultimate cell death produced by TCDD. The results also support the involvement of hydrogen peroxide in TCDD-induced microsomal lipid peroxidation.
Asunto(s)
Peroxidación de Lípido/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Fosfolipasas A/fisiología , Dibenzodioxinas Policloradas/toxicidad , Aminopirina/farmacología , Animales , Benzfetamina/farmacología , Carotenoides/farmacología , Femenino , Indometacina/farmacología , Microsomas Hepáticos/enzimología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Fosforilasas/antagonistas & inhibidores , Fosforilasas/metabolismo , Quercetina/farmacología , Quinacrina/farmacología , Ratas , Ratas Endogámicas , beta CarotenoRESUMEN
In hepatic microsomes one or more isozymes of cytochrome P-450 inhibits the ATP-dependent Ca2+ pump. This inhibition is reversible by GSH and appears to be due to a direct oxidation of the pump proteins by the oxygenated cytochrome. To determine which isozyme mediates this inhibition, we have examined the effect of various substrates and inhibitors on the NADPH inhibition of Ca2+ uptake. We find that aminopyrine, benzphetamine and SKF-525A reverse this inhibition while a number of other substrates do not. This pattern suggests that a previously unreported isozyme of cytochrome P-450 mediates the Ca2+ pump inhibition.
Asunto(s)
ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/metabolismo , NADP/metabolismo , Aminopirina/farmacología , Animales , Benzfetamina/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Imidazoles/farmacología , Masculino , Metirapona/farmacología , Microsomas Hepáticos/efectos de los fármacos , Oxidación-Reducción , Proadifeno/farmacología , Ratas , Ratas EndogámicasAsunto(s)
Carbón Orgánico , Microsomas Hepáticos/efectos de los fármacos , NADPH-Ferrihemoproteína Reductasa/metabolismo , Fenobarbital/farmacología , Animales , Benzfetamina/farmacología , Sitios de Unión , Inducción Enzimática/efectos de los fármacos , Etilmorfina/farmacología , Masculino , Microsomas Hepáticos/enzimología , Oxidación-Reducción , Fenobarbital/aislamiento & purificación , Ratas , Factores de Tiempo , Conservación de TejidoRESUMEN
The effect of substrate on LM2 reduction was examined using a reconstituted system containing dilauroylphosphatidylcholine, NADPH-cytochrome P-450 reductase, and cytochrome P-450 LM2 in a 160:1.5:1 molar ratio. In general, most substrates increased the rate constants of both the first and second phases of reduction as well as the fraction of LM2 reduced in the first phase. The correlation between the high spin content of the cytochrome and each of these kinetic parameters was weaker than expected if spin state controlled LM2 reduction. Further, substrate was shown to exert a rapid effect on both the high spin content and stimulation of reduction indicating that the low spin to high spin shift cannot be responsible for the slow phase of reduction for this particular isoform. Cytochrome P-450 reduction was also examined in both phospholipid-containing and soluble systems where the LM2 and reductase were not present as a preformed complex. In these systems the reactions were substantially slower than with the standard reconstituted system. Addition of substrate enhanced the rate of reduction, indicating that the rate of association between LM2 and the reductase was increased by substrate addition. The strong correlation between the rate of LM2 reduction in a preformed complex and the logarithm of the rate of LM2 and reductase association implicates the rate of functional complex formation as the factor controlling the slow phase of reduction.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Animales , Benzfetamina/farmacología , Cinética , Microsomas Hepáticos/enzimología , Oxidación-Reducción , Conejos , Relación Estructura-ActividadRESUMEN
Rabbit cytochrome P450 isozyme 2 requires cytochrome b5 to metabolize the volatile anesthetic methoxyflurane but not the substrate benzphetamine [E. Canova-Davis and L. Waskell (1984) J. Biol. Chem. 259, 2541-2546]. To determine whether the requirement for cytochrome b5 for methoxyflurane oxidation is mediated by an allosteric effect on cytochrome P450 LM2 or cytochrome P450 reductase, we have investigated whether this anesthetic can induce a role for cytochrome b5 in benzphetamine metabolism. Using rabbit liver microsomes and antibodies raised in guinea pigs against rabbit cytochrome b5, we found that methoxyflurane did not create a cytochrome b5 requirement for benzphetamine metabolism. Methoxyflurane also failed to induce a role for cytochrome b5 in benzphetamine metabolism in the purified, reconstituted mixed function oxidase system. Studies of the reaction kinetics established that in the absence of cytochrome b5, methoxyflurane and benzphetamine are competitive inhibitors, and that in the presence of cytochrome b5, benzphetamine and methoxyflurane are two alternate substrates in competition for a single site on the same enzyme. These results all indicate that the methoxyflurane-induced cytochrome b5 dependence of the mixed function oxidase cytochrome P450 LM2 system is a direct result of the interaction between methoxyflurane and the substrate binding site of cytochrome P450 LM2 and suggest the focus of future studies of this question.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Grupo Citocromo b/metabolismo , Isoenzimas/metabolismo , Metoxiflurano/metabolismo , Microsomas Hepáticos/metabolismo , Animales , Benzfetamina/farmacología , Sitios de Unión , Unión Competitiva , Citocromos b5 , Cinética , Metoxiflurano/farmacología , Conejos , Especificidad por SustratoRESUMEN
The discriminative stimulus (DS) and subjective effects of caffeine (100 and 300 mg, PO) and benzphetamine (12.5 and 50 mg, PO) were studied in 18 normal human volunteers trained to discriminate between d-amphetamine (10 mg) and placebo. d-Amphetamine increased ratings of drug liking and activity level and produced a profile of subjective effects characteristic of amphetamine and related psychomotor stimulants. The DS effects of d-amphetamine generalized only partially to caffeine and benzphetamine; mean percent d-amphetamine-appropriate responding was 42 and 58 after 100 and 300 mg caffeine, respectively, and 17 and 56 after 12.5 and 50 mg benzphetamine, respectively. Neither dose of caffeine affected ratings of drug liking or activity level, but 300 mg caffeine did produce a profile of subjective effects that partially overlapped with that produced by d-amphetamine. Benzphetamine 50 mg, but not 12.5 mg, increased ratings of drug liking and activity level and produced a profile of subjective effects qualitatively similar to, but weaker than, that produced by d-amphetamine. For both caffeine and benzphetamine, a close relationship was observed between their subjective effects and their ability to substitute for the DS effects of d-amphetamine. These results correspond well with findings obtained from similar studies conducted with laboratory animals, providing further support for the reliability and validity of human drug discrimination paradigms.
Asunto(s)
Benzfetamina/farmacología , Cafeína/farmacología , Dextroanfetamina/farmacología , Discriminación en Psicología/efectos de los fármacos , Fenetilaminas/farmacología , Adulto , Aprendizaje Discriminativo/efectos de los fármacos , Emociones/efectos de los fármacos , Femenino , Humanos , Masculino , Factores de TiempoRESUMEN
The mechanism of the inactivation of the major phenobarbital-inducible isozyme of rat liver cytochrome P-450 (P-450 PB-B2) by chloramphenicol has been investigated. Preparations of the enzyme from animals treated in vivo with chloramphenicol (CAP PB-B2) have been isolated, and their catalytic, spectral, and physical properties have been compared with those of the native PB-B2. The CAP PB-B2 exhibited: 1) a 60-70% loss in the rate of NADPH-supported monooxygenase activity with the substrates benzphetamine, 7-ethoxycoumarin, and p-nitroanisole; 2) a 60% decrease in the extent of enzymatic P-450 reduction catalyzed by NADPH-cytochrome P-450 reductase under both aerobic and anaerobic conditions; 3) a 60% decrease in the steady-state level of the ferrous dioxygen complex in the presence of substrates; 4) a 60% decrease in the magnitude of the type I spectral change induced by benzphetamine; and 5) a shift in the wavelength maximum for the chemically reduced ferrous-carbonyl complex from 450 to 451.5 nm. On the other hand, the ability of the CAP PB-B2 to catalyze the iodosobenzene-supported metabolism of 7-ethoxycoumarin and p-nitroanisole was unaltered. The results are consistent with a scheme whereby the binding of metabolites of chloramphenicol to amino acid residues in the PB-B2 close to the heme moiety blocks electron transport from NADPH-cytochrome P-450 reductase, thereby leading to a loss of monooxygenase activity.
Asunto(s)
Cloranfenicol/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Isoenzimas/antagonistas & inhibidores , Fenobarbital/farmacología , 7-Alcoxicumarina O-Dealquilasa , Animales , Benzfetamina/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Inducción Enzimática , Isoenzimas/biosíntesis , Cinética , Masculino , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxigenasas/metabolismo , Ratas , Ratas Endogámicas , Espectrofotometría , Factores de TiempoRESUMEN
Reconstituted liposomal cytochrome P-450 LM2 was reacted with a series of benzphetamine analogues as substrates. Based on the thermodynamical model of Ristau et al. (Biochim. Biophys. Acta, 536 (1978) 226-234) the free enthalpy of substrate binding to the high spin form of the enzyme was shown to correlate with the total high spin content of the respective enzyme substrate complex. Reduction and substrate N-demethylation rates as well have been evidenced to linearly correlate with the substrate-induced spin shift delta alpha and moreover with the spin content alpha. The data obtained provide further experimental support for the spin state regulation of the reduction and conversion rate of cytochrome P-450 LM2.