RESUMEN

16-Dehydropregnenolone (16-DHP) is an active compound with an unsatisfied in vivo behavior and poor water-solubility, which limits its clinical application. To improve its in vivo behavior and water-solubility, a Hydroxypropyl-beta-Cyclodextrin (HP-ß-CD) inclusion complex of 16-DHP was prepared in this paper. Pharmacokinetic studies after oral administration of 16-DHP-HP-ß-CD at doses of 37.5, 75, 150 mg/kg were carried out to investigate its dose proportionality in rats. The relative bioavailability was researched by comparing the area under the plasma concentration-time curve of 16-DHP-HP-ß-CD and free 16-DHP after oral administration in rats at the dose of 75 mg/kg. At the same time, tissue distribution of 16-DHP-HP-ß-CD after oral administration at the dose of 240 mg/kg in mice was also investigated. Consequently, 16-DHP-HP-ß-CD appeared to be a linear pharmacokinetic character after peroral administration to the rat at the doses tested. Compared to free 16-DHP, inclusion complex could significantly improve the relative bioavailability (467%). Tissue distribution studies indicated that 16-DHP-HP-ß-CD tended to distribute into stomach, intestine, lung, brain and liver.

Asunto(s)

Portadores de Fármacos/química , Pregnenolona/análogos & derivados , beta-Ciclodextrinas/química , 2-Hidroxipropil-beta-Ciclodextrina , Administración Oral , Animales , Disponibilidad Biológica , Liberación de Fármacos , Femenino , Ratones , Pregnenolona/administración & dosificación , Pregnenolona/química , Pregnenolona/farmacocinética , Ratas , Ratas Sprague-Dawley , Solubilidad , Distribución Tisular

RESUMEN

16-dehydropregnenolone (DHP) is a promising novel antihyperlipidemic agent developed and patented by Central Drug Research Institute (CDRI), India. The purpose of the present study was to investigate whether DHP influences the activities and mRNA expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, CYP2D2, CYP2E1 and CYP3A1) in Sprague-Dawley (SD) rats. A cocktail suspension of CYP probe substrates which contained caffeine (CYP1A2), tolbutamide (CYP2C11), dextromethorphan (CYP2D2), chlorzoxazone (CYP2E1) and dapsone (CYP3A1) was administered orally on eighth- or fifteenth-day to rats pre-treated with DHP intragastrically at a dose of 36 and 72mg/kg for one week and two weeks. The concentrations of probe drugs in plasma were estimated by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Alongside, the effect of DHP on CYPs activity and mRNA expression levels were assayed in isolated rat liver microsomes and by real-time reverse transcription-polymerase chain reaction (RT-PCR), respectively. DHP had significant inducing effects on CYP1A2, 2C11, 2D2 and 2E1 with no effect on CYP3A1 in dose- and time-dependent manner, as revealed from the pharmacokinetic profiles of the probe drugs in rats. In-vitro microsomal activities and mRNA expression results were in good agreement with the in-vivo pharmacokinetic results. Collectively, the results unveiled that DHP is an inducer of rat hepatic CYP enzymes. Hence, intense attention should be paid when DHP is co-administered with drugs metabolized by CYP1A2, 2C11, 2D2 and 2E1, which might result in drug-drug interactions and therapeutic failure.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP3A/genética , Familia 2 del Citocromo P450/genética , Hipolipemiantes/farmacocinética , Pregnenolona/análogos & derivados , Esteroide 16-alfa-Hidroxilasa/genética , Administración Oral , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Cafeína/metabolismo , Cafeína/farmacología , Clorzoxazona/metabolismo , Clorzoxazona/farmacología , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Familia 2 del Citocromo P450/metabolismo , Dapsona/metabolismo , Dapsona/farmacología , Dextrometorfano/metabolismo , Dextrometorfano/farmacología , Regulación de la Expresión Génica , Hipolipemiantes/administración & dosificación , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Pregnenolona/administración & dosificación , Pregnenolona/farmacocinética , Ratas , Ratas Sprague-Dawley , Esteroide 16-alfa-Hidroxilasa/metabolismo , Tolbutamida/metabolismo , Tolbutamida/farmacología

RESUMEN

OBJECTIVE: 16-Dehydropregnenolone (16-DHP) is a potential antitumor compound with poor solubility. A liposome entrapped 16-DHP (16-DHP-LM) formulation was developed to surmount its solubility obstacle. The aim of this study is to investigate the pharmacokinetics of 16-DHP-LM and 16-DHP solution in female mice and tissue distribution of 16-DHP-LM in female tumor-bearing nude mice. METHODS: Rotary-evaporated film method was used to prepare 16-DHP-LM. The comparison of pharmacokinetics between 16-DHP-LM and 16-DHP solution in female mice was investigated after intravenous administration at a single dose of 15 mg/kg. The dose proportionality of 16-DHP-LM was also evaluated after intravenous administration of 16-DHP-LM at the doses of 7.5, 15.0 and 30.0 mg/kg. The tissue distribution of 16-DHP-LM in female tumor-bearing nude mice was evaluated after intravenous administration of 16-DHP-LM at a single dose of 30.0 mg/kg. RESULTS: The pharmacokinetic study indicated that the 16-DHP-LM group had higher area under the plasma concentration-time curve (AUC), lower apparent volume of distribution (Vz) and smaller systemic clearance (CL) than the 16-DHP solution group. For dose proportionality, good linearity of the pharmacokinetics of 16-DHP after intravenous administration of 16-DHP-LM was observed in the regression analysis of the AUC-dose plot (r = 0.99) and the Cmax-dose plot (r = 0.98). The tissue distribution study showed that the main tissue depots for 16-DHP in tumor-bearing nude mice were plasma, liver, spleen and tumor, which was benefit to anti-tumor effect. All these results provided a significant basis for the design of clinical trial of 16-DHP-LM.

Asunto(s)

Liposomas/farmacocinética , Pregnenolona/análogos & derivados , Distribución Tisular/fisiología , Administración Intravenosa/métodos , Animales , Área Bajo la Curva , Disponibilidad Biológica , Química Farmacéutica/métodos , Relación Dosis-Respuesta a Droga , Femenino , Infusiones Intravenosas/métodos , Inyecciones Intravenosas/métodos , Ratones , Ratones Desnudos , Soluciones Farmacéuticas/farmacocinética , Pregnenolona/farmacocinética

RESUMEN

Fast excitatory synaptic transmission that is contingent upon N-methyl d-aspartate receptor (NMDAR) function contributes to core information flow in the central nervous system and to the plasticity of neural circuits that underlie cognition. Hypoactivity of excitatory NMDAR-mediated neurotransmission is hypothesized to underlie the pathophysiology of schizophrenia, including the associated cognitive deficits. The neurosteroid pregnenolone (PREG) and its metabolites pregnenolone sulfate (PregS) and allopregnanolone in serum are inversely associated with cognitive improvements after oral PREG therapy, raising the possibility that brain neurosteroid levels may be modulated therapeutically. PregS is derived from PREG, the precursor of all neurosteroids, via a single sulfation step and is present at low nanomolar concentrations in the central nervous system. PregS, but not PREG, augments long-term potentiation and cognitive performance in animal models of learning and memory. In this report, we communicate the first observation that PregS, but not PREG, is a potent (EC50 ∼2 pM) enhancer of intracellular Ca(2+) that is contingent upon neuronal activity, NMDAR-mediated synaptic activity, and L-type Ca(2+) channel activity. Low picomolar PregS similarly activates cAMP response element-binding protein (CREB) phosphorylation (within 10 minutes), an essential memory molecule, via an extracellular-signal-regulated kinase/mitogen-activated protein kinase signal transduction pathway. Taken together, the results are consistent with a novel biologic role for the neurosteroid PregS that acts at picomolar concentrations to intensify the intracellular response to glutamatergic signaling at synaptic but not extrasynaptic, NMDARs by differentially augmenting CREB activation. This provides a genomic signal transduction mechanism by which PregS could participate in memory consolidation of relevance to cognitive function.

Asunto(s)

Señalización del Calcio , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Pregnenolona/farmacología , Potenciales Sinápticos , Animales , Canales de Calcio Tipo L/metabolismo , Células Cultivadas , Concentración 50 Inhibidora , Sistema de Señalización de MAP Quinasas , Masculino , Pregnenolona/farmacocinética , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/fisiología

RESUMEN

AIM: The present study was designed to investigate the activity of two glibenclamide derivatives on glucose concentration. An additional aim was to identify the biodistribution of glibenclamide derivatives in different organs in a diabetic animal model. METHODS: The effects of two glibenclamide derivatives on glucose concentration were evaluated in a diabetic animal model. In addition, glibenclamide derivatives were bound to Tc-99m using radioimmunoassay methods. To evaluate the pharmacokinetics of the glibenclamide derivatives over time (15, 30, 45 and 60 min) the Tc-99m-glibenclamide conjugates were used. RESULTS: The results showed that glibenclamide-pregnenolone had greater hypoglycemic activity than glibenclamide or glibenclamide-OH. The data also showed that the biodistribution of Tc-99m-glibenclamide-OH in all organs was less than that of the Tc-99m-glibenclamide-pregnenolone derivative. CONCLUSIONS: The glibenclamide-pregnenolone derivative had greater hypoglycemic effects and its biodistribution was wider than glibenclamide-OH. The data suggest that the steroid nucleus may be important to the hypoglycemic activity of the glibenclamide-pregnenolone derivative and this could be related to the degree of lipophilicity induced by the steroid nucleus in the chemical structure of glibenclamide-pregnenolone.

Asunto(s)

Glucemia/metabolismo , Diabetes Mellitus Experimental/sangre , Gliburida/uso terapéutico , Pregnenolona/uso terapéutico , Aloxano , Animales , Combinación de Medicamentos , Femenino , Gliburida/análogos & derivados , Gliburida/farmacocinética , Hipoglucemiantes/metabolismo , Hipoglucemiantes/farmacocinética , Metformina/uso terapéutico , Pregnenolona/farmacocinética , Ratas , Ratas Wistar , Distribución Tisular

RESUMEN

OBJECTIVES: This manuscript addresses key pharmacokinetic issues in support of the development of a potent candidate lipid-lowering drug molecule, 16-dehydropregnenolone (DHP). METHODS: Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay for simultaneous estimation of DHP and its metabolites, including 5-pregnene-3ß-ol-16, 17-epoxi-20-one (M(1) ) was validated in male and female Sprague-Dawley rat plasma and applied to different studies. Pharmacokinetic studies of DHP after intravenous and oral administration were carried out to assess any gender effect. Dose-proportionality after oral administration was assessed at three dose levels. Protein binding was estimated using the modified charcoal adsorption method. KEY FINDINGS: Rapid elimination of DHP from the systemic circulation resulted in a comparatively lesser systemic exposure in male compare to female rats. The area under the curve (AUC) after oral administration in males was significantly different to females. The large volume of distribution and low degree of protein binding suggest extensive distribution of DHP. An increase in the oral dose led to a disproportionate change in peak concentration (C(max) ) and AUC, indicating variable absorption. However, the dose-normalized AUC and C(max) at two dose levels were not found to be statistically different. CONCLUSIONS: The extent of conversion of DHP to M(1) was higher after oral administration in male rats but was insignificant in female rats. DHP showed low systemic oral bioavailability and exhibited dose-independent pharmacokinetics and gender differences.

Asunto(s)

Hipolipemiantes/farmacocinética , Pregnanolona/análogos & derivados , Pregnenolona/análogos & derivados , Administración Oral , Animales , Área Bajo la Curva , Disponibilidad Biológica , Cromatografía Liquida , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Hipolipemiantes/administración & dosificación , Inyecciones Intravenosas , Masculino , Pregnanolona/farmacocinética , Pregnenolona/administración & dosificación , Pregnenolona/farmacocinética , Unión Proteica , Ratas , Ratas Sprague-Dawley , Factores Sexuales , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Distribución Tisular

RESUMEN

Neurosteroids hold great promise for the treatment of diseases of the central nervous system (CNS). We compared the uptake by 11 brain regions and appearance in blood of tritium-labeled pregnenolone and progesterone after intranasal and intravenous (IV) injection. Both neurosteroids appeared in blood and brain after either method of administration, but with important differences in uptake. Bioavailability based on appearance in arterial serum showed that about 23% and 14% of the intranasal administered doses of pregnenolone and progesterone, respectively, entered the blood. Brain levels were about two fold lower after intranasal administration for the two neurosteroids. With intranasal administration, brain levels of the two steroids did not vary over time (2-120 min), whereas brain levels were higher early (10 min or less) after i.v. administration. With i.v. administration, uptake by brain regions did not vary, whereas the olfactory bulb, hippocampus, and hypothalamus had high uptake rates after intranasal administration. Intranasal administration of prenenolone improved memory, whereas progesterone decreased anxiety, thus demonstrating that therapeutic levels of neurosteroids can be delivered to the brain by intranasal administration. The neurosteroids were rapidly degraded after i.v. or intranasal delivery, but pregnenolone was more resistant to degradation in the brain after intranasal administration and in serum after i.v. administration. These results show that either the i.v. or intranasal routes of administration can deliver neurosteroids to blood and brain, but that the two routes have significant differences with intranasal administration favoring some brain regions.

Asunto(s)

Encéfalo/metabolismo , Pregnenolona/metabolismo , Pregnenolona/farmacocinética , Progesterona/metabolismo , Progesterona/farmacocinética , Administración Intranasal , Animales , Disponibilidad Biológica , Transporte Biológico , Encéfalo/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Hipocampo/metabolismo , Hipotálamo/metabolismo , Inyecciones Intravenosas , Masculino , Ratones , Neurotransmisores/metabolismo , Neurotransmisores/farmacología , Pregnenolona/administración & dosificación , Pregnenolona/sangre , Pregnenolona/farmacología , Progesterona/administración & dosificación , Progesterona/sangre , Progesterona/farmacología

RESUMEN

A sensitive, selective and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous quantification of 16-dehydropregnenolone (DHP) and its five metabolites 4,16-pregnadien-3, 20-dione (M(1)), 5-pregnene-3beta-ol-20-one (M(2)), 5-pregnene-3beta, 20-diol (M(3)), 5-pregnene-3beta-ol-16, 17-epoxi-20-one (M(4)) and 5,16-pregnadien-3beta, 11-diol-20-one (M(5)) in rabbit plasma using dexamethasone as internal standard (IS). The analytes were chromatographed on Spheri-5 RP-18 column (5 microm, 100 mm x 4.6 mm i.d.) coupled with guard column using acetonitrile:ammonium acetate buffer (90:10, v/v) as mobile phase at a flow rate of 0.65 ml/min. The quantitation of the analytes was carried out using API 4000 LC-MS-MS system in the multiple reaction monitoring (MRM) mode. The method was validated in terms of linearity, specificity, sensitivity, recovery, accuracy, precision (intra- and inter-assay variation), freeze-thaw, long-term, auto injector and dry residue stability. Linearity in plasma was observed over a concentration range of 1.56-400 ng/ml with a limit of detection (LOD) of 0.78 ng/ml for all analytes except M(3) and M(5) where linearity was over the 3.13-400 ng/ml with LOD of 1.56 ng/ml. The absolute recoveries from plasma were consistent and reproducible over the linearity range for all analytes. The intra- and inter-day accuracy and precision method were within the acceptable limits and the analytes were stable after three freeze-thaw cycles and their dry residues were stable at -60 degrees C for 15 days. The method was successfully applied to determine concentrations of DHP and its putative metabolites in plasma during a pilot pharmacokinetic study in rabbits.

Asunto(s)

Cromatografía Líquida de Alta Presión/métodos , Pregnenolona/análogos & derivados , Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Pregnenolona/sangre , Pregnenolona/farmacocinética , Conejos , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad

RESUMEN

We present a method for the analysis of urinary 16(5alpha)-androsten-3alpha-ol together with 5beta-pregnane-3alpha,20alpha-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol (5alphaA), 5beta-androstane-3alpha,17beta-diol (5betaA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6 per thousand, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual delta(13)C-values for 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol are less than 0.9 per thousand. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol, whereas the isotopic ratio values of 5beta-pregnane-3alpha,20alpha-diol vary by more 5 per thousand upon ingestion of pregnenolone. We have observed delta(13)C-value changes lower than 1 per thousand for 16(5alpha)-androsten-3alpha-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5beta-pregnane-3alpha,20alpha-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.

Asunto(s)

Androstenoles/uso terapéutico , Doping en los Deportes , Androstenoles/farmacología , Cromatografía de Gases , Deshidroepiandrosterona/farmacocinética , Humanos , Indicadores y Reactivos , Espectrometría de Masas , Pregnenolona/análisis , Pregnenolona/química , Pregnenolona/farmacocinética , Estándares de Referencia , Valores de Referencia , Reproducibilidad de los Resultados , Testosterona/análogos & derivados , Testosterona/orina

RESUMEN

An accurate and precise HPLC assay has been developed and validated for determination of dehydropregnenolone (DHP) in rat plasma, bile, urine and feces. Separation was achieved using a C-18 reversed phase column with a mobile phase comprising of acetonitrile and deionized water (55:45% v/v) using a UV detector, set at a wavelength of 248 nm. The method, applicable to 200-microl plasma, bile and urine, involved double extraction of the samples with n-hexane. The sample clean up for feces involved single extraction of 50 mg of sample with 3 ml of acetonitrile. The method was sensitive with a limit of quantitation of 20 ng/ml in all the matrices and absolute recovery >92%. Precision and accuracy were within the acceptable limits, as indicated by relative standard deviation varying from 4.7 to 11.2% and bias values ranging from 1.8 to 8.8%. Moreover, DHP was stable in plasma, bile and urine up to 90 days of storage at -60 degrees C and after being subjected to three freeze-thaw cycles. The method was applied to generate the pharmacokinetics of DHP in rats after oral and intravenous administration.

Asunto(s)

Hipolipemiantes/análisis , Hipolipemiantes/farmacocinética , Pregnenolona/análogos & derivados , Pregnenolona/análisis , Pregnenolona/farmacocinética , Administración Oral , Animales , Cromatografía Líquida de Alta Presión/métodos , Matriz Extracelular/metabolismo , Hipolipemiantes/administración & dosificación , Masculino , Pregnenolona/administración & dosificación , Ratas , Ratas Sprague-Dawley , Espectrofotometría Ultravioleta/métodos

RESUMEN

(-)-Verbenone, a monoterpene bicyclic ketone, is a component of the essential oil from rosemary species such as Rosmarinus officinalis L., Verbena triphylla, and Eucalyptus globulus and is used for an herb tea, a spice, and a perfume. In this study, (-)-verbenone was found to be converted to 10-hydroxyverbenone by rat and human liver microsomal cytochrome p450 (p450) enzymes. The product formation was determined by high-performance liquid chromatography with UV detection at 251 nm. There was a good correlation between activities of coumarin 7-hydroxylation and (-)-verbenone 10-hydroxylation catalyzed by liver microsomes of 16 human samples, indicating that CYP2A6 is a principal enzyme in (-)-verbenone 10-hydroxylation in humans. Human recombinant CYP2A6 and CYP2B6 catalyzed (-)verbenone 10-hydroxylation at Vmax values of 15 and 21 nmol/min/nmol p450 with apparent Km values of 16 and 91 microM, respectively. In contrast, rat CYP2A1 and 2A2 did not catalyze (-)-verbenone 10-hydroxylation at all, suggesting that there were species-related differences in the catalytic properties of human and rat CYP2A enzymes in the metabolism of (-)-verbenone. In the rat, recombinant CYP2C11, CYP2B1, and CYP3A2 catalyzed (-)-verbenone 10-hydroxylation with Vmax and Km ratios (ml/min/nmol p450) of 0.73, 0.20, and 0.03, respectively. Male-specific CYP2C11 was a major enzyme in (-)-verbenone 10-hydroxylation by untreated rat livers, and CYP2B1 catalyzed this reaction in liver microsomes of phenobarbital-treated rats. Rat CYP2C12, a female-specific enzyme, did not catalyze (-)verbenone 10-hydroxylation. These results suggest that human CYP2A6 and rat CYP2C11 are the major catalysts in the metabolism of (-)-verbenone by liver microsomes and that there are species-related differences in human and rat CYP2A enzymes and sex-related differences in male and female rats in the metabolism of (-)-verbenone.

Asunto(s)

Hidrocarburo de Aril Hidroxilasas/fisiología , Citocromo P-450 CYP2B1/fisiología , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/fisiología , Oxidorreductasas N-Desmetilantes/fisiología , Esteroide 16-alfa-Hidroxilasa/fisiología , Terpenos/metabolismo , Animales , Monoterpenos Bicíclicos , Línea Celular , Citocromo P-450 CYP2A6 , Citocromo P-450 CYP2B6 , Familia 2 del Citocromo P450 , Activación Enzimática , Inducción Enzimática , Femenino , Expresión Génica , Humanos , Hidroxilación , Masculino , NADP/biosíntesis , NADP/metabolismo , Fenobarbital/administración & dosificación , Fenobarbital/farmacocinética , Pregnenolona/administración & dosificación , Pregnenolona/farmacocinética , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Estereoisomerismo , Terpenos/farmacocinética , beta-naftoflavona/administración & dosificación , beta-naftoflavona/farmacocinética

RESUMEN

In peripheral nerves, progesterone synthesized by Schwann cells has been implicated in myelination. In spite of such an important function, little is known of the regulation of progesterone biosynthesis in the nervous system. We show here that in rat Schwann cells, expression of the 3 beta-hydroxysteroid dehydrogenase and formation of progesterone are dependent on neuronal signal. Levels of 3 beta-hydroxysteroid dehydrogenase mRNA and synthesis of [3H]progesterone from [3H]pregnenolone were low in purified Schwann cells prepared from neonatal rat sciatic nerves. However, when Schwann cells were cultured in contact with sensory neurons, both expression and activity of the 3 beta-hydroxysteroid dehydrogenase were induced. Regulation of 3 beta-hydroxysteroid dehydrogenase expression by neurons was also demonstrated in vivo in the rat sciatic nerve. 3 beta-hydroxysteroid dehydrogenase mRNA was present in the intact nerve, but could no longer be detected 3 or 6 days after cryolesion, when axons had degenerated. After 15 days, when Schwann cells made new contact with the regenerating axons, the enzyme was re-expressed. After nerve transection, which does not allow axonal regeneration, 3 beta-hydroxysteroid dehydrogenase mRNA remained undetectable. The regulation of 3 beta-hydroxysteroid dehydrogenase mRNA after lesion was similar to the regulation of myelin protein zero (P0) and peripheral myelin protein 22 (PMP22) mRNAs, supporting an important role of locally formed progesterone in myelination.

Asunto(s)

Comunicación Celular/fisiología , Ganglios Espinales/metabolismo , Neuronas Aferentes/metabolismo , Nervios Periféricos/metabolismo , Progesterona/biosíntesis , Células de Schwann/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Axones/metabolismo , Axones/ultraestructura , Células Cultivadas/citología , Células Cultivadas/metabolismo , Ganglios Espinales/citología , Masculino , Compresión Nerviosa/efectos adversos , Neuronas Aferentes/citología , Nervios Periféricos/citología , Pregnenolona/farmacocinética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Células de Schwann/citología , Nervio Ciático/metabolismo , Nervio Ciático/fisiopatología , Nervio Ciático/cirugía , Transducción de Señal/fisiología , Tritio/farmacocinética

RESUMEN

The course of the transformation of six 5-ene steroids with varying substituents at C-17 or/and C-3: dehydroepiandrosterone (DHEA), 5-androsten-3beta,17beta-diol, 17alpha-methyl-5-androsten-3beta,17beta-diol, 5-androsten-17-one, 5-androsten-3beta-ol and pregnenolone by Fusarium culmorum was investigated. Three substrates with oxygen functions at C-3 and C-17 i.e. DHEA, 5-androsten-3beta,17beta-diol and 17alpha-methyl-5-androsten-3beta,17beta-diol were hydroxylated entirely at 7alpha-axial, allylic position. The mixture of 7alpha-hydroxy- and 7alpha,15alpha-dihydroxyderivatives was formed during the transformation of pregnenolone and 5-androsten-17-one, from the latter 2alpha,7alpha-dihydroxyderivative was also obtained. 7alpha,15alpha- Dihydroxyderivative was the only product isolated from the 5-androsten-3beta-ol post-transformation mixture. The time-course of the DHEA transformation by F. culmorum shows that the substrate induces 7alpha-hydroxylase activity. DHEA was transformed by androstenedione induced F. culmorum cultures to a larger extent than by a noninduced microorganism; the selectivity of the transformation remained unchanged.

Asunto(s)

Fusarium/metabolismo , Esteroides/farmacocinética , Androstenodiona/metabolismo , Androstenoles/farmacocinética , Biotransformación , Cromatografía de Gases/métodos , Deshidroepiandrosterona/metabolismo , Deshidroepiandrosterona/farmacocinética , Hidroxilación , Espectroscopía de Resonancia Magnética , Pregnenolona/farmacocinética , Esteroides/metabolismo

RESUMEN

We have studied the distribution of the neurosteroids pregnenolone (Pe) and pregnenolone sulfate (PeS) in seven brain regions, and plasma and fat tissues in male adult rats following the intravenous infusion of 14 mg/kg Pe and 18 mg/kg PeS, respectively. After chromatographic separation of steroid sulfate esters and non-conjugated steroids by solid phase octadecyl C18 columns and celite column chromatographic separation of Pe from cross-reacted steroids, the concentrations of Pe and PeS were determined by radioimmunoassay. We found that both Pe and PeS concentrations were significantly increased in plasma, fat and brain compared to the vehicle controls after i.v. infusion of Pe and PeS. In the controls, Pe concentrations were highly correlated within brain regions and between fat and brain regions. Most correlations were lost after Pe and PeS infusions. The content of Pe and PeS was not uniformly distributed in the brain. The hypothalamus contained the highest level of Pe in controls, Pe-infused and PeS-infused rats (12 +/- 3.1, 3500 +/- 180 and 590 +/- 54 ng/g, respectively). The highest concentration of PeS was detected in the hypothalamus (26 +/- 8.2 ng/g) and striatum (17 +/- 4.1 ng/g) in controls, in the hypothalamus (200 +/- 24 ng/g) after PeS infusion as well as in the hypothalamus and medulla oblongata (57 +/- 9.6 and 55 +/- 7.6 ng/g, respectively) after Pe infusion. This study has yielded evidence that PeS injected i.v. can cross the blood-brain barrier without being hydrolysed to the more lipophilic Pe, and can thus be taken up by the brain.

Asunto(s)

Encéfalo/metabolismo , Hipotálamo/metabolismo , Pregnenolona/farmacocinética , Análisis de Varianza , Animales , Barrera Hematoencefálica , Encéfalo/efectos de los fármacos , Hipotálamo/efectos de los fármacos , Infusiones Intravenosas , Masculino , Pregnenolona/sangre , Ratas , Ratas Sprague-Dawley , Esteroides/metabolismo , Esteroides/farmacología , Distribución Tisular

RESUMEN

The mare possesses unique steroid hormone metabolic activity during pregnancy in that peripheral 4-pregnene-3,20-dione (progesterone; P4) is undetectable by 220 days gestation. This study examines in vivo metabolism of progestins by the pregnant mare and in vitro metabolic activity of maternal and fetal tissues. Pregnant mares (n = 3) received intravenous infusions of 3 beta-hydroxy-5-pregnen-20-one (pregnenolone; P5), P4, 5 alpha-pregnane-3,20-dione (5 alpha-DHP), 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-5 alpha), deuterium labeled (D4)-P5, D4-3 beta-5 alpha and vehicle. Anestrous mares (n = 2) were infused with P5, P4, and vehicle. Also, placenta, endometrium, fetal gonad, maternal and fetal liver, and adrenal from 4 animals were incubated in D4-P5, D4-5 alpha-DHP, or D4-3 beta-5 alpha. Pregnant mares (in vivo) converted infused P5 predominantly to 5-pregnene-3 beta,20 beta-diol (P5-beta beta), 5 alpha-DHP, 20 alpha-hydroxy-5 alpha-pregnan-3-one (20 alpha-5 alpha), and 3 beta-5 alpha while only minor concentrations of P4 were detected. Infused P4 was converted primarily to 5 alpha-DHP and 20 alpha-hydroxypregnanes and when 5 alpha-DHP served as a substrate, other 5 alpha-pregnanes were formed. Infused 3 beta-5 alpha was either reduced to beta alpha-diol or oxidized to 5 alpha-DHP. Regardless of treatment, anestrous mares were incapable of producing any 20 alpha-hydroxypregnanes but could convert P5 to P5-beta beta and P4 in quantities similar to that of pregnant mares. In vitro, placenta converted D4-P5 to D4-P4 while D4-3 beta-5 alpha was oxidized to D4-5 alpha-DHP. Endometrium converted substrate primarily to D4-20 alpha-hydroxypregnanes. Both maternal and fetal liver produced D4-20 beta-hydroxy compounds regardless of substrate. Maternal and fetal adrenal were capable of conversion of D4-P5 to D4-P4 while fetal gonad did not perform any significant metabolism of substrate, though it produced P5. These data explain the absence of P4 and presence of other progestin metabolites in maternal circulation during mid- and late pregnancy. Pregnenolone can be 5 alpha-reduced to 3 beta-5 alpha, and 3 beta-5 alpha could be 3-oxidized to 5 alpha-DHP. It is 5 alpha-DHP that may serve as substrate for other 5 alpha-pregnanes.

Asunto(s)

Caballos/metabolismo , Preñez/metabolismo , Progestinas/farmacocinética , 5-alfa-Dihidroprogesterona , Animales , Biotransformación , Deuterio , Endometrio/metabolismo , Femenino , Técnicas In Vitro , Infusiones Intravenosas , Hígado/embriología , Hígado/metabolismo , Especificidad de Órganos , Vehículos Farmacéuticos , Placenta/metabolismo , Embarazo , Pregnanodionas/farmacocinética , Pregnanolona/farmacocinética , Pregnenolona/farmacocinética , Progestinas/metabolismo

RESUMEN

We have studied the duration of the ACTH inhibition effect on the incorporation and transformation of [1-14 C] eicosatrienoic acid, in isolated adrenocortical cells of normal rats. The effect of esculetin, indomethacin and nordihydroguaiaretic acid alone, or in the presence of ACTH or diBucAMP on arachidonate biosynthesis was also investigated. ACTH and diBucAMP produced a significant inhibition in arachidonic acid biosynthesis. The inhibition produced by ACTH on delta 5 desaturating activity was considered to be a short-term effect. Nordihydroguaiaretic acid and esculetin provoked a depression in the uptake of 20:3 (n-6) acid and an inhibition in the formation of 20:4 (n-6) acid by adrenocortical cells. This effect was potentiated when the cells were currently treated with either ACTH or diBucAMP. Indomethacin produced no changes in the uptake of 20:3 (n-6) acid, while induced an increment on delta 5 desaturation activity. This effect would indicate that, normally, the metabolites produced by the cyclooxygenase pathway would operate by depressing arachidonic acid biosynthesis. Considering the negative regulation of the delta 5 desaturase activity system produced by ACTH through cAMP, and the positive modulation inferred by the results obtained in this work, it is possible to assume that there are, at least, two mechanisms that take place on 20:4 (n-6) acid formation. These mechanisms seem to work independently from one another and they probably interact when effecting a bi-directional control.

Asunto(s)

Corteza Suprarrenal/metabolismo , Ácido Araquidónico/biosíntesis , Cumarinas/metabolismo , Inhibidores de la Ciclooxigenasa/metabolismo , Glicósidos/metabolismo , Indometacina/metabolismo , Masoprocol/metabolismo , Pregnenolona/análogos & derivados , Corteza Suprarrenal/citología , Corteza Suprarrenal/efectos de los fármacos , Hormona Adrenocorticotrópica/metabolismo , Animales , Bucladesina/metabolismo , Cumarinas/farmacocinética , Inhibidores de la Ciclooxigenasa/farmacocinética , Femenino , Glicósidos/farmacocinética , Indometacina/farmacocinética , Masoprocol/farmacocinética , Pregnenolona/metabolismo , Pregnenolona/farmacocinética , Ratas , Ratas Wistar

RESUMEN

Unconjugated steroids were extracted with dichloromethane from the serum of pre-term neonates. Conjugated steroid fraction in the residue was separated and purified by high performance liquid chromatography. The purified steroid fraction was subjected to enzyme (arylsulfatase from Helix pomatia) or acid hydrolysis. Liberated steroid was analyzed by gas chromatography-mass spectrometry (GC/MS) and gas chromatography-Fourier transformation infrared spectroscopy (GC/FT-IR). All of the results obtained from GC/MS and GC/FT-IR analysis were identical with those of authentic 16-dehydropregnenolone (3 beta-hydroxy-5,16-pregnadien-20-one, 16-DHP). This is the first report to demonstrate the presence of 16-DHP in human blood.

Asunto(s)

Recien Nacido Prematuro/sangre , Pregnenolona/análogos & derivados , Arilsulfatasas/metabolismo , Análisis Químico de la Sangre , Cromatografía Líquida de Alta Presión , Cromatografía de Gases y Espectrometría de Masas , Edad Gestacional , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Recién Nacido , Cloruro de Metileno/química , Pregnenolona/sangre , Pregnenolona/aislamiento & purificación , Pregnenolona/farmacocinética , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

Human chorion and decidua use pregnenolone sulfate (P5S) and dehydroepiandrosterone sulfate (DHAS) as substrates for local estrogen and progesterone synthesis. We hypothesized that the local estrogen/progesterone ratio may influence contractility of the adjacent myometrium and hence effect the timing of parturition. Thus, we studied steroid sulfohydrolase activity for P5S in these tissues and investigated the potential interaction of other steroids on the rates of hydrolysis of P5S and DHAS. The enzyme was present in both tissues, predominantly in the microsomal fraction. With P5S as substrate, the Michaelis-Menten constant (Km) was similar in chorion (1.3 +/- 0.2 mumol/L, mean +/- SEM) and decidua (0.9 +/- 0.1 mumol/L) but the maximum velocity (Vmax) was significantly greater in chorion (2.6 +/- 0.4 vs. 1.1 +/- 0.3 nmol/mg protein/15 min, P less than 0.05). In both tissues there was a tendency towards greater activity in tissues obtained before labor compared to tissues obtained after spontaneous labor onset. Using either DHAS or P5S as substrate, there was significant inhibition of sulfohydrolase activity by other steroids at concentrations similar to those in late pregnancy fetal and maternal plasma. In microsomal preparations using DHAS as substrate, activity was inhibited by equimolar concentrations of estrone sulfate (E1S, by 38 +/- 2%), P5S (by 74 +/- 2%), and cholesterol sulfate (C27S, by 38 +/- 3%). With P5S as substrate, equimolar concentrations of E1S, DHAS, and C27S caused inhibition of sulfohydrolase activity by 19 +/- 5%, 16 +/- 4%, and 18 +/- 2%, respectively. These inhibitory effects also were observed using a tissue explant system with intact cells. In kinetic inhibition studies using DHAS as substrate, E1S and P5S were competitive inhibitors with inhibition constants (Ki) of 4.8 +/- 1.3 and 0.7 +/- 0.1 mumol/L, respectively. Using P5S as substrate, E1S and DHAS also were competitive inhibitors with Ki values of 8.2 +/- 2.1 and 9.6 +/- 1.2 mumol/L, respectively. For both substrates, the pattern of inhibition by C27S was complex. Preliminary experiments to distinguish, on the basis of differing physical-chemical properties, separate enzymes for different substrates were inconclusive. We conclude that human chorion and decidua can hydrolyze several steroid sulfoconjugates and this activity may regulate local estrogen and progesterone synthesis. There are significant interactions among steroid sulfoconjugates in regulating this activity. These activities may be important components of a paracrine system that determines myometrial contractility and the timing of parturition.

Asunto(s)

Arilsulfatasas/fisiología , Corion/enzimología , Decidua/enzimología , Deshidroepiandrosterona/metabolismo , Pregnenolona/metabolismo , Sulfatasas/fisiología , Técnicas de Cultivo , Deshidroepiandrosterona/farmacocinética , Estrógenos/biosíntesis , Estrona/análogos & derivados , Estrona/metabolismo , Femenino , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Trabajo de Parto/metabolismo , Microsomas/metabolismo , Embarazo , Pregnenolona/farmacocinética , Progesterona/biosíntesis , Esteril-Sulfatasa , Temperatura

RESUMEN

Radioiodinated benzoyl esters and amides of epimeric 20-hydroxy- and 20-aminopregn-5-en-3 beta-ols were synthesized in an effort to find an agent that would be rapidly and selectively taken up by adrenal cortical tissue. Achievement of such a goal would provide a basis for the development of adrenal imaging agents superior to those currently available for clinical use. The iodobenzoyl derivatives were obtained by treating the appropriate epimer with 2-iodobenzoic acid in the presence of dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine. The resulting esters and amides were readily labeled with radioiodine by isotope exchange with sodium iodide-125 in pivalic acid. Tissue distribution studies in female rats revealed that only the esters displayed appreciable adrenal specificity, and the ester having the same configuration at C-20 as cholesterol was significantly better than the corresponding C-20 epimer.

Asunto(s)

Radioisótopos de Yodo , Yodobenzoatos/síntesis química , Pregnenolona/análogos & derivados , Corteza Suprarrenal/diagnóstico por imagen , Amidas/síntesis química , Amidas/farmacocinética , Animales , Ésteres/síntesis química , Ésteres/farmacocinética , Femenino , Yodobenzoatos/farmacocinética , Marcaje Isotópico , Pregnenolona/síntesis química , Pregnenolona/farmacocinética , Cintigrafía , Ratas , Ratas Endogámicas , Relación Estructura-Actividad , Distribución Tisular

RESUMEN

A series of radioiodinated benzoate and carbamate esters of cholesterol and pregnenolone wherein the acyl moiety served as the carrier for radioiodine was synthesized and evaluated as potential imaging agents for the adrenal cortex. 2,6-Dimethyl-3-iodobenzoyl and N-(4-iodophenyl) carbamoyl groups were chosen as the acyl functionality in an attempt to provide esters resistant to in vivo hydrolysis. Tissue disposition studies in rats revealed that their biodistribution was determined by the attached sterol carrier-the cholesterol esters demonstrated significant uptake at 24 h in the adrenal whereas the corresponding pregnenolone derivatives showed only slight affinity for steroid-secreting tissues at this time.

Asunto(s)

Corteza Suprarrenal/diagnóstico por imagen , Ésteres del Colesterol/síntesis química , Pregnenolona/análogos & derivados , Animales , Ésteres del Colesterol/farmacocinética , Radioisótopos de Yodo , Pregnenolona/síntesis química , Pregnenolona/farmacocinética , Cintigrafía , Ratas