RESUMEN
1. 4-Oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxylic acid (4-methylaminomethyl-phenyl)-amide (1), developed for general anxiety disorder, was discontinued from clinical development due to unsuitable oral pharmacokinetics. 2. In humans, (1) demonstrated an unacceptable high apparent oral clearance (Cl(p)/F) that also demonstrated a supraproportional dose-exposure relationship. Secondary peaks in the plasma concentration-time profile suggested possible enterohepatic recirculation of (1). A combination of in vitro mechanistic tools was applied to better understand the processes underlying these complex clinical pharmacokinetic profiles of (1). 3. In metabolism experiments, (1) was shown to be a substrate of monoamine oxidase A (MAO-A) as well as being metabolized by cytochrome P450. The former appeared to be a high K(M) process with a high capacity, while the latter showed saturation between 1 and 10 microM, consistent with the supraproportional dose-exposure relationship. 4. In a sandwich-cultured hepatocyte model, (1) was shown to be a substrate for both uptake and efflux into the canicular space, which is consistent with the observation of pharmacokinetics suggestive of enterohepatic recirculation. Finally, in human epithelial colon adenocarcinoma cell line (Caco-2) and Madin-Darby canine kidney cells transwell flux experiments, (1) was shown to have relatively low permeability and a basolateral-to-apical flux ratio consistent with the activity of P-glycoprotein. 5. In combination, a compounding of the contributions of MAO-A, hepatic uptake and efflux transporters, and P-glycoprotein to the disposition of (1) may underlie the low oral exposure, saturable clearance, and aberrant concentration versus time profiles observed for this compound in humans.
Asunto(s)
Anilidas/metabolismo , Anilidas/farmacocinética , Ansiolíticos/metabolismo , Ansiolíticos/farmacocinética , Agonistas de Receptores de GABA-A , Indoles/metabolismo , Indoles/farmacocinética , Anilidas/química , Animales , Ansiolíticos/química , Línea Celular Tumoral , Células Cultivadas , Sistema Enzimático del Citocromo P-450/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Indoles/química , Hígado/metabolismo , Estructura Molecular , Monoaminooxidasa/metabolismoRESUMEN
CYP2D15 is the canine ortholog of human CYP2D6, the human CYP2D isoform involved in the metabolism of drugs such as antiarhythmics, adrenoceptor antagonists, and tricyclic antidepressants. Similar to human, canine CYP2D15 is expressed in the liver, with detectable levels in several other tissues. Three different CYP2D15 cDNA clones were obtained by RT-PCR from dog liver RNA. Two clones corresponded to variant full-length CYP2D15 cDNAs (termed CYP2D15 WT2 and CYP2D15 V1); the third was identified as a splicing variant missing exon 3 (termed CYP2D15 V2). Recombinant baculoviruses were constructed containing full-length cDNAs and used to express CYP2D15 WT2 and CYP2D15 V1 in Spodoptera frugiperda (Sf9) cells with expression levels of up to 0.14 nmol/mg cell protein. As with human CYP2D6, the recombinant CYP2D15 enzymes exhibited bufuralol 1'-hydroxylaseand dextromethorphan O-demethylase activities whencoexpressed with rabbit NADPH:P450 oxidoreductase. For bufuralol 1'-hydroxylase, apparent Km values were 4.9, 3.7, and 2.5 microM and the Vmax values were 0.14, 0.034, and 0.60 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. For dextromethorphan O-demethylase, apparent Km values were 0.6, 0.6, and 2.0 microM and the Vmax values were 0.18, 0.034, and 0.057 nmol/min/mg protein for dog liver microsomes, CYP2D15 WT2, and the variant CYP2D15 V1, respectively. The human CYP2D6-specific inhibitor quinidine and the rat CYP2D1-specific inhibitor quinine were both shown to be inhibitors of bufuralol 1'-hydroxylase activity for dog liver microsomes, CYP2D15 WT2, and the CYP2D15 V1 variant with nearly equal potency. Thus, the dog expresses a CYP2D ortholog possessing enzymatic activities similar to human CYP2D6, but is affected by the inhibitors quinine and quinidine in a manner closer to that of rat CYP2D1.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/química , Animales , Línea Celular , Clonación Molecular , Sistema Enzimático del Citocromo P-450/genética , Perros , Activación Enzimática , Femenino , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas/biosíntesis , Isoenzimas/química , Isoenzimas/genética , Cinética , Masculino , Microsomas Hepáticos/enzimología , NADPH-Ferrihemoproteína Reductasa/biosíntesis , NADPH-Ferrihemoproteína Reductasa/genética , Especificidad de Órganos , Conejos , SpodopteraRESUMEN
A series of novel, potent and selective pyrido[1,2-a]pyrazine dopamine D4 receptor antagonists are reported including CP-293,019 (D4 Ki = 3.4 nM, D2 Ki > 3,310 nM), which also inhibits apomorphine-induced hyperlocomotion in rats after oral dosing.
Asunto(s)
Antagonistas de Dopamina/síntesis química , Antagonistas de los Receptores de Dopamina D2 , Actividad Motora/efectos de los fármacos , Pirazinas/síntesis química , Pirimidinas/síntesis química , Animales , Apomorfina/farmacología , Antagonistas de Dopamina/química , Antagonistas de Dopamina/farmacología , Diseño de Fármacos , Haloperidol/farmacología , Indicadores y Reactivos , Estructura Molecular , Pirazinas/química , Pirazinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Ratas , Receptores de Dopamina D4 , Relación Estructura-ActividadRESUMEN
We have investigated the metabolism of polychlorinated biphenyls and endogenous steroids by the major phenobarbital (PB)-inducible hepatic cytochromes P450 in dogs and rats, PBD-2 and PB-B, respectively. Previous results from our laboratory indicate that dog PBD-2 purified from microsomes of PB-treated animals is similar to rat PB-B with respect to structure and the regioselective metabolism of warfarin and androstenedione. The results also strongly suggest that PBD-2 is the P450 form responsible for metabolizing 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) in liver microsomes from untreated dogs. In the present study, a cytochrome P450 with similar chromatographic behavior to that of PBD-2 has been purified from liver microsomes of untreated dogs. This protein is identical to PBD-2 based on (i) mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) reactivity with anti-PBD-2 IgG, (iii) amino-terminal sequence, and (iv) 245-HCB metabolite profile. Induction and antibody-inhibition data suggest that PBD-2 is responsible for the metabolism of 2,2',3,3',6,6'-hexachlorobiphenyl (236-HCB) in microsomes obtained from both untreated and PB-treated dogs. In contrast, metabolism of 4,4'-dichlorobiphenyl (4-DCB) by dog microsomes is poor, and does not appear to be catalyzed to a significant extent by PBD-2. Antibody-inhibition studies with intact microsomes corroborate previous results that androstenedione is metabolized by purified PBD-2 to the same major metabolite (16 beta-OH androstenedione) produced by rat PB-B. Dog PBD-2 metabolizes progesterone primarily to the 21-OH metabolite, while metabolism by rat PB-B leads to the formation of the 16 alpha-OH product. On the other hand, upon Ouchterlony double-immunodiffusion analysis, anti-PBD-2 IgG reacts strongly with PB-B but not PB-C, the major rat liver progesterone 21-hydroxylase. The data suggest that dog PBD-2 is a constitutive P450 important in the metabolism of various PCBs and endogenous steroids. Dog PBD-2 and rat PB-B appear to be similar enzymes, yet they differ in their regioselective metabolism of progesterone.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Bifenilos Policlorados/metabolismo , Esteroides/metabolismo , Secuencia de Aminoácidos , Androstenodiona/metabolismo , Animales , Catálisis , Perros , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Progesterona/metabolismo , RatasRESUMEN
Chloramphenicol (CAP) is a potent and effective mechanism-based inactivator of the major phenobarbital (PB)-inducible isozyme of dog liver cytochrome P-450 (PBD-2) in vitro. In a reconstituted system containing PBD-2, CAP causes a time- and NADPH-dependent irreversible loss of 7-ethoxycoumarin deethylase activity, with no loss of spectrally detectable cytochrome P-450. Inactivation is enhanced by cytochrome b5, and, in the presence of cytochrome b5, the concentration of CAP at which the rate constant for inactivation is half-maximal (Kl) and the maximal rate constant for inactivation (Kinact) are 5 microM and 1.2 min-1, respectively. CAP binds covalently to PBD-2 with a stoichiometry of 1 nmol of [14C]CAP bound/nmol of cytochrome P-450 inactivated. In addition, CAP is a selective inactivator of PBD-2. In intact liver microsomes from PB-treated dogs, CAP irreversibly inhibits androstenedione 16 alpha and 16 beta, but not 6 beta hydroxylation. Covalent binding of [14C]CAP to dog liver microsomes in vitro is increased 5.5 times by PB induction. This increase correlates well with the increased levels of immunochemically determined PBD-2 (5.8-fold) and 16 alpha and 16 beta hydroxylation of androstenedione (5.7- and 5.8-fold) in microsomes from PB-treated compared to control animals. Anti-PBD-2 IgG specifically inhibits by greater than 80% the covalent binding of [14C]CAP to microsomes from control and PB-treated dogs. Finally, in liver microsomes from PB-treated and control dogs, CAP appears to bind covalently to a single protein with the same molecular weight as PBD-2 as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography.
Asunto(s)
Cloranfenicol/farmacología , Inhibidores Enzimáticos del Citocromo P-450 , Hígado/enzimología , Fenobarbital/farmacología , Animales , Autorradiografía , Sistema Enzimático del Citocromo P-450/biosíntesis , Grupo Citocromo b/metabolismo , Citocromos b5 , Perros , Electroforesis , Inducción Enzimática/efectos de los fármacos , Técnicas In Vitro , Isoenzimas/metabolismo , Cinética , Hígado/efectos de los fármacos , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , NADP/metabolismo , Proteínas/metabolismoRESUMEN
The biochemical basis for the marked difference in the rate of the hepatic metabolism of 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB) by Beagle dogs and Sprague-Dawley rats has been investigated. Control dog liver microsomes metabolize this substrate 15 times faster than control rat liver microsomes. Upon treatment with phenobarbital (PB), at least two cytochrome P-450 isozymes are induced in the dog, and the hepatic microsomal metabolism of 245-HCB is increased on both a per nanomole P-450 basis (twofold) and a per milligram protein basis (fivefold). One of the PB-induced isozymes, PBD-2, has been purified to a specific content of 17-19 nmol/mg protein and to less than 95% homogeneity, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a reconstituted system containing cytochrome b5, this isozyme shows an activity toward 245-HCB which is greater than threefold that seen in intact liver microsomes from PB-induced dogs. A reconstituted system containing the major isozyme induced by PB in the rat (PB-B) metabolizes 245-HCB at 1/10 the rate observed with purified PBD-2. Antibody inhibition studies have shown that PBD-2 accounts for greater than 90% of the hepatic microsomal metabolism of 245-HCB in control and PB-induced dogs, while PB-B only accounts for about half of the metabolism of this compound by microsomes obtained from PB-treated rats. Immunoblot analysis has revealed that the level of PBD-2 in dog liver microsomes increases nearly sixfold with PB treatment, and this increase correlates well with the fivefold increase in the rate of hepatic microsomal metabolism of 245-HCB by dogs. Together these data support a primary role for isozyme PBD-2 in the hepatic metabolism of 245-HCB in control and PB-induced dogs. In addition, these results suggest that, in contrast to rats, dogs can readily metabolize 245-HCB as a result of the presence of a cytochrome P-450 isozyme with efficient 245-HCB metabolizing activity.