Pharmacokinetics of 16-dehydropregnenolone hydroxypropyl-ß-cyclodextrin inclusion complex following peroral administration.

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.

Promnestic effects of intranasally applied pregnenolone in rats.

The neurosteroid pregnenolone (PREG) has been shown to have memory-enhancing and anti-depressant action. The present study addresses the question of whether intranasally applied pregnenolone (IN-PREG) also has promnestic properties in the rat. We examined the effects of IN-PREG at doses of 0.187 and 0.373mg/kg on memory for objects and their location on learning and retention of escape in a water maze, and on behavior on the elevated plus maze. The main findings were: (a) Pre-trial, but not post-trial, administration of IN-PREG facilitated long-term memory in a novel object-preference test and a novel object-location preference test when tested 48h after dosing. (b) Over the duration of 5days of extinction trials, after learning to escape onto a hidden platform in a water maze, the animals treated with IN-PREG spent more time in searching for the absent platform, indicating either, or both, superior memory for the former position of the escape platform, or a higher resistance to extinction. (c) Administration of the anticholinergic, scopolamine, disrupted learning to escape from the water maze in the vehicle-treated group. The IN-PREG treated groups exhibited superior escape learning in comparison with vehicle controls, indicating that the treatment countered the scopolamine effect. IN-PREG treatment had no influence on behaviors on the elevated plus maze. Our results demonstrate that IN-PREG is behaviorally active with cognitive enhancing properties comparable to those known from studies employing systemic PREG administration.

Pregnenolone co-treatment partially restores steroidogenesis, but does not prevent growth inhibition and increased atresia in mouse ovarian antral follicles treated with mono-hydroxy methoxychlor.

Mono-hydroxy methoxychlor (mono-OH MXC) is a metabolite of the pesticide, methoxychlor (MXC). Although MXC is known to decrease antral follicle numbers, and increase follicle death in rodents, not much is known about the ovarian effects of mono-OH MXC. Previous studies indicate that mono-OH MXC inhibits mouse antral follicle growth, increases follicle death, and inhibits steroidogenesis in vitro. Further, previous studies indicate that CYP11A1 expression and production of progesterone (P4) may be the early targets of mono-OH MXC in the steroidogenic pathway. Thus, this study tested whether supplementing pregnenolone, the precursor of progesterone and the substrate for HSD3B, would prevent decreased steroidogenesis, inhibited follicle growth, and increased follicle atresia in mono-OH MXC-treated follicles. Mouse antral follicles were exposed to vehicle (dimethylsulfoxide), mono-OH MXC (10 µg/mL), pregnenolone (1 µg/mL), or mono-OH MXC and pregnenolone together for 96 h. Levels of P4, androstenedione (A), testosterone (T), estrone (E1), and 17ß-estradiol (E2) in media were determined, and follicles were processed for histological evaluation of atresia. Pregnenolone treatment alone stimulated production of all steroid hormones except E2. Mono-OH MXC-treated follicles had decreased sex steroids, but when given pregnenolone, produced levels of P4, A, T, and E1 that were comparable to those in vehicle-treated follicles. Pregnenolone treatment did not prevent growth inhibition and increased atresia in mono-OH MXC-treated follicles. Collectively, these data support the idea that the most upstream effect of mono-OH MXC on steroidogenesis is by reducing the availability of pregnenolone, and that adding pregnenolone may not be sufficient to prevent inhibited follicle growth and survival.

Improvement of Remyelination in Demyelinated Corpus Callosum Using Human Adipose-Derived Stem Cells (hADSCs) and Pregnenolone in the Cuprizone Rat Model of Multiple Sclerosis.

Adipose-derived stem cells (ASCs) have neuroprotective effects, and their repair ability has been approved in neurodegenerative studies. Pregnenolone as a neurosteroid plays significant roles in neurogenesis. We aimed to consider the effect of ADSCs and pregnenolone injection on the multiple sclerosis (MS) model created by cuprizone. Male Wistar rats (n = 36) were fed with an ordinary diet or a diet with cuprizone (0.6%) for 3 weeks. H-ADSCs were taken from patients with lipoaspirate surgery. The rats were divided into six groups (n = 6): healthy, MS, sham, pregnenolone injection, ADSCs injection, pregnenolone and ADSCs injection. Behavioral test, histological examination and TEM were conducted. The specific markers for myelin and cell differentiation were assessed using immunohistochemistry staining. Additionally, the measure of MBP and MOG gene expression and the amount of related proteins were determined using real-time RT-PCR and ELISA techniques, respectively. Histologic results showed that induced demyelination in corpus callosum fibers. TEM revealed an increased thickness of myelin in fibers in the treated groups (P < 0.05). Injection of hADSC and pregnenolone significantly increased the expression levels of MBP and MOG (P < 0.001). The mean percentage of MOG and MBP markers were significantly increased in the treated groups compared to MS and sham groups (P < 0.05). Moreover, the OD level of MBP and MOG proteins showed that their values in the ADSCs/pregnenolone group were close to those of the control group without a significant difference. Our data indicated the remyelination potency and cell differentiation can improve with ADSCs and pregnenolone treatments in the multiple sclerosis model which created by cuprizone in rats.

Positive allosteric modulators that target NMDA receptors rectify loss-of-function GRIN variants associated with neurological and neuropsychiatric disorders.

N-methyl-d-aspartate receptors (NMDARs) mediate a slow component of excitatory synaptic transmission that plays important roles in normal brain function and development. A large number of disease-associated variants in the GRIN gene family encoding NMDAR GluN subunits have been identified in patients with various neurological and neuropsychiatric disorders. Many of these variants reduce the function of NMDARs by a range of different mechanisms, including reduced glutamate potency, reduced glycine potency, accelerated deactivation time course, decreased surface expression, and/or reduced open probability. We have evaluated whether three positive allosteric modulators of NMDAR receptor function (24(S)-hydroxycholesterol, pregnenolone sulfate, tobramycin) and three co-agonists (d-serine, l-serine, and d-cycloserine) can mitigate the diminished function of NMDARs harboring GRIN variants. We examined the effects of these modulators on NMDARs that contained 21 different loss-of-function variants in GRIN1, GRIN2A, or GRIN2B, identified in patients with epilepsy, intellectual disability, autism, and/or movement disorders. For all variants, some aspect of the reduced function was partially restored. Moreover, some variants showed enhanced sensitivity to positive allosteric modulators compared to wild type receptors. These results raise the possibility that enhancement of NMDAR function by positive allosteric modulators may be a useful therapeutic strategy.

Metabolomics approach to anabolic steroid urine profiling of bovines treated with prohormones.

In livestock production, illegal use of natural steroids is hard to prove because metabolites are either unknown or not significantly above highly fluctuating endogenous levels. In this work we outlined for the first time a metabolomics based strategy for anabolic steroid urine profiling. Urine profiles of controls and bovines treated with the prohormones dehydroepiandrosterone (DHEA) and pregnenolone were analyzed with ultraperformance liquid chromatography in combination with time-of-flight accurate mass spectrometry (UPLC-TOFMS). The obtained full scan urinary profiles were compared using sophisticated preprocessing and alignment software (MetAlign) and multivariate statistics, revealing hundreds of mass signals which were differential between untreated control and prohormone-treated animals. Moreover, statistical testing of the individual accurate mass signals showed that several mass peak loadings could be used as biomarkers for DHEA and pregnenolone abuse. In addition, accurate mass derived elemental composition analysis and verification by standards or Orbitrap mass spectrometry demonstrated that the observed differential masses are most likely steroid phase I and glucuronide metabolites excreted as a direct result from the DHEA and pregnenolone administration, thus underlining the relevance of the findings from this untargeted metabolomics approach. It is envisaged that this approach can be used as a holistic screening tool for anabolic steroid abuse in bovines and possibly in sports doping as well.

Simultaneous quantification of GABAergic 3alpha,5alpha/3alpha,5beta neuroactive steroids in human and rat serum.

The 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone enhance GABAergic neurotransmission and produce inhibitory neurobehavioral and anti-inflammatory effects. Despite substantial information on the progesterone derivative (3alpha,5alpha)-3-hydroxypregnan-20-one (3alpha,5alpha-THP, allopregnanolone), the physiological significance of the other endogenous GABAergic neuroactive steroids has remained elusive. Here, we describe the validation of a method using gas chromatography-mass spectrometry to simultaneously identify serum levels of the eight 3alpha,5alpha- and 3alpha,5beta-reduced derivatives of progesterone, deoxycorticosterone, dehydroepiandrosterone and testosterone. The method shows specificity, sensitivity and enhanced throughput compared to other methods already available for neuroactive steroid quantification. Administration of pregnenolone to rats and progesterone to women produced selective effects on the 3alpha,5alpha- and 3alpha,5beta-reduced neuroactive steroids, indicating differential regulation of their biosynthetic pathways. Pregnenolone administration increased serum levels of 3alpha,5alpha-THP (+1488%, p<0.001), (3alpha,5alpha)-3,21-dihydroxypregnan-20-one (3alpha,5alpha-THDOC, +205%, p<0.01), (3alpha,5alpha)-3-hydroxyandrostan-17-one (3alpha,5alpha-A, +216%, p<0.001), (3alpha,5alpha,17beta)-androstane-3,17-diol (3alpha,5alpha-A-diol, +190%, p<0.01). (3alpha,5beta)-3-hydroxypregnan-20-one (3alpha,5beta-THP) and (3alpha,5beta)-3-hydroxyandrostan-17-one (3alpha,5beta-A) were not altered, while (3alpha,5beta)-3,21-dihydroxypregnan-20-one (3alpha,5beta-THDOC) and (3alpha,5beta,17beta)-androstane-3,17-diol (3alpha,5beta-A-diol) were increased from undetectable levels to 271+/-100 and 2.4+/-0.9 pg+/-SEM, respectively (5/8 rats). Progesterone administration increased serum levels of 3alpha,5alpha-THP (+1806%, p<0.0001), 3alpha,5beta-THP (+575%, p<0.001), 3alpha,5alpha-THDOC (+309%, p<0.001). 3alpha,5beta-THDOC levels were increased by 307%, although this increase was not significant because this steroid was detected only in 3/16 control subjects. Levels of 3alpha,5alpha-A, 3alpha,5beta-A and pregnenolone were not altered. This method can be used to investigate the physiological and pathological role of neuroactive steroids and to develop biomarkers and new therapeutics for neurological and psychiatric disorders.

Steady-state activation and modulation of the synaptic-type α1ß2γ2L GABAA receptor by combinations of physiological and clinical ligands.

The synaptic α1ß2γ2 GABAA receptor is activated phasically by presynaptically released GABA. The receptor is considered to be inactive between synaptic events when exposed to ambient GABA because of its low resting affinity to the transmitter. We tested the hypothesis that a combination of physiological and/or clinical positive allosteric modulators of the GABAA receptor with ambient GABA generates measurable steady-state activity. Recombinant α1ß2γ2L GABAA receptors were expressed in Xenopus oocytes and activated by combinations of low concentrations of orthosteric (GABA, taurine) and allosteric (the steroid allopregnanolone, the anesthetic propofol) agonists, in the absence and presence of the inhibitory steroid pregnenolone sulfate. Steady-state activity was analyzed using the three-state cyclic Resting-Active-Desensitized model. We estimate that the steady-state open probability of the synaptic α1ß2γ2L GABAA receptor in the presence of ambient GABA (1 µmol/L), taurine (10 µmol/L), and physiological levels of allopregnanolone (0.01 µmol/L) and pregnenolone sulfate (0.1 µmol/L) is 0.008. Coapplication of a clinical concentration of propofol (1 µmol/L) increases the steady-state open probability to 0.03. Comparison of total charge transfer for phasic and tonic activity indicates that steady-state activity can contribute strongly (~20 to >99%) to integrated activity from the synaptic GABAA receptor.

Inhibition of Hedgehog Signaling in Fibroblasts, Pancreatic, and Lung Tumor Cells by Oxy186, an Oxysterol Analogue with Drug-Like Properties.

The widespread involvement of the Hedgehog (Hh) signaling pathway in human malignancies has motivated the clinical development of Smoothened (Smo) antagonists, such as vismodegib and sonidegib. However, Smo antagonists have failed to benefit patients suffering from Hh pathway-dependent solid tumors, such as pancreatic, colorectal, or ovarian cancer. Hh-dependent cancers are often driven by activating mutations that occur downstream of Smo and directly activate the transcription factors known as glioma-associated oncogenes (Gli1-3). Hence, the direct targeting of Gli could be a more effective strategy for achieving disease modification compared to Smo antagonism. In this study, we report on the biological and pharmacological evaluation of Oxy186, a semisynthetic oxysterol analogue, as a novel inhibitor of Hh signaling acting downstream of Smo, with encouraging drug-like properties. Oxy186 exhibits strong inhibition of ligand-induced Hh signaling in NIH3T3-E1 fibroblasts, as well as in constitutively activated Hh signaling in Suppressor of Fused (Sufu) null mouse embryonic fibroblast (MEF) cells. Oxy186 also inhibits Gli1 transcriptional activity in NIH3T3-E1 cells expressing exogenous Gli1 and Gli-dependent reporter constructs. Furthermore, Oxy186 suppresses Hh signaling in PANC-1 cells, a human pancreatic ductal adenocarcinoma (PDAC) tumor cell line, as well as PANC-1 cell proliferation in vitro, and in human lung cancer cell lines, A549 and H2039.

Nanomolar concentrations of pregnenolone sulfate enhance striatal dopamine overflow in vivo.

The balance between GABA-mediated inhibitory and glutamate-mediated excitatory synaptic transmission represents a fundamental mechanism for controlling nervous system function, and modulators that can alter this balance may participate in the pathophysiology of neuropsychiatric disorders. Pregnenolone sulfate (PS) is a neuroactive steroid that can modulate the activity of ionotropic glutamate and GABA(A) receptors either positively or negatively, depending upon the particular receptor subtype, and modulates synaptic transmission in a variety of experimental systems. To evaluate the modulatory effect of PS in vivo, we infused PS into rat striatum for 20 min via a microdialysis probe while monitoring local extracellular dopamine (DA) levels. The results demonstrate that PS at low nanomolar concentrations significantly increases extracellular DA levels. The PS-induced increase in extracellular DA is antagonized by the N-methyl-d-aspartate (NMDA) receptor antagonist, d-AP5 [d-(-)-2-amino-5-phosphonopentanoic acid], but not by the sigma receptor antagonist, BD 1063 [1(-)[2-(3,4-dichlorophenyl)-ethyl]-4-methylpiperazine]. The results demonstrate that exogenous PS, at nanomolar concentrations, is able to increase DA overflow in the striatum through an NMDA receptor-mediated pathway.

Modulatory effect of neurosteroids in haloperidol-induced vacuous chewing movements and related behaviors.

RATIONALE: Tardive dyskinesia is a syndrome of abnormal and involuntary movements which occurs as a complication of long-term neuroleptic therapy especially classical neuroleptics such as haloperidol and chlorpromazine. Dysfunction of GABA receptor mediated inhibition, and increased glutamatergic neurotransmission has been implicated in the development of orofacial dyskinesia in rats and tardive dyskinesia in humans. Neurosteroids modulate both GABAergic as well as glutamatergic neurotransmission in various brain areas. OBJECTIVE: The objective of the present study was to elucidate the role of various neurosteroids in neuroleptic-induced vacuous chewing movements and related behaviors in rats by using behavioral, biochemical, and neurochemical parameters. MATERIALS AND METHODS: Animals chronically treated with haloperidol (1 mg/kg i.p.) for a period of 21 days exhibited marked increase in vacuous chewing movements, tongue protrusions, and facial jerkings as compared to vehicle-treated controls. It also resulted in increased superoxide anion levels and lipid peroxidation, whereas decreased levels of endogenous antioxidant enzymes (catalase and superoxide dismutase) in rat brain striatum homogenates. Neurochemical studies revealed that chronic administration of haloperidol resulted in significant decrease in the levels of dopamine, serotonin, and norepinephrine in rat brain striatum homogenates, whereas urine biogenic amines metabolite levels were increased. In a series of experiments, rats co-administered with allopregnanolone (0.5, 1, and 2 mg/kg i.p.) and progesterone (5, 10, and 20 mg/kg i.p.), both positive GABA-modulating [negative N-methyl-D-aspartate (NMDA)-modulating] neurosteroids prevented, whereas pregnenolone (0.5, 1, and 2 mg/kg i.p.) and dihydroxyepiandrosterone sulfate (0.5, 1, and 2 mg/kg i.p.) both negative GABA-modulating (positive NMDA-modulating) neurosteroids aggravated all the behavioral, biochemical, and neurochemical parameters. CONCLUSIONS: These results suggest that neurosteroids may play a significant role in the pathophysiology of vacuous chewing movements and related behaviors by virtue of their action on either the GABA or NMDA modulation. Furthermore, neurosteroids showing selectivity for positive GABA modulation and/or negative NMDA modulation may be particularly efficacious as novel therapeutic agents for the treatment of tardive dyskinesia and deserve further evaluation.

Add-On Pregnenolone with L-Theanine to Antipsychotic Therapy Relieves Negative and Anxiety Symptoms of Schizophrenia: An 8-Week, Randomized, Double-Blind, Placebo-Controlled Trial.

AIMS: Pregnenolone (PREG) and L-theanine (LT) have shown ameliorative effects on various schizophrenia symptoms. This is the first study to evaluate the efficacy and safety of augmentation of antipsychotic treatment among patients with chronic schizophrenia or schizoaffective disorder with PREG-LT. METHODS: Double-blind, placebo-controlled trial of PREG-LT or placebo augmentation was conducted for eight weeks with 40 chronic DSM-IV schizophrenia and schizoaffective disorder patients with suboptimal response to antipsychotics. Oral PREG (50 mg/day) with LT (400 mg/day) or placebo were added to a stable regimen of antipsychotic medication from March 2011 to October 2013. The participants were rated using the Scale for the Assessment of Negative Symptoms (SANS), the Hamilton Scale for Anxiety (HAM-A), and the Positive and Negative Syndrome Scale (PANSS) scales bi-weekly. The decrease of SANS and HAM-A scores were the co-primary outcomes. Secondary outcomes included assessments of general functioning and side effects. RESULTS: Negative symptoms such as blunted affect, alogia, and anhedonia (SANS) were found to be significantly improved with moderate effect sizes among patients who received PREG-LT, in comparison with the placebo group. Add-on PREG-LT also significantly associated with a reduction of anxiety scores such as anxious mood, tension, and cardiovascular symptoms (HAM-A), and elevation of general functioning (GAF). Positive symptoms, antipsychotic agents, concomitant drugs, and illness duration did not associate significantly with effect of PREG-LT augmentation. PREG-LT was well-tolerated. CONCLUSIONS: Pregnenolone with L-theanine augmentation may offer a new therapeutic strategy for treatment of negative and anxiety symptoms in schizophrenia and schizoaffective disorder. Further studies are warranted. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01831986.

Neuroactive steroids modulate HPA axis activity and cerebral brain-derived neurotrophic factor (BDNF) protein levels in adult male rats.

Depression is characterized by hypothalamo-pituitary-adrenocortical (HPA) axis hyperactivity. In this major mood disorder, neurosteroids and neurotrophins, particularly brain-derived neurotrophic factor (BDNF), seem to be implicated and have some antidepressant effects. BDNF is highly involved in regulation of the HPA axis, whereas neurosteroids effects have never been clearly established. In this systematic in vivo study, we showed that the principal neuroactive steroids, namely dehydroepiandrosterone (DHEA), pregnenolone (PREG) and their sulfate esters (DHEA-S and PREG-S), along with allopregnanolone (ALLO), stimulated HPA axis activity, while also modulating central BDNF contents. In detail, DHEA, DHEA-S, PREG, PREG-S and ALLO induced corticotropin-releasing hormone (CRH) and/or arginine vasopressin (AVP) synthesis and release at the hypothalamic level, thus enhancing plasma adrenocorticotropin hormone (ACTH) and corticosterone (CORT) concentrations. This stimulation of the HPA axis occurred concomitantly with BDNF modifications at the hippocampus, amygdala and hypothalamus levels. We showed that these neurosteroids induced rapid effects, probably via neurotransmitter receptors and delayed effects perhaps after metabolization in other neuroactive steroids. We highlighted that they had peripheral effects directly at the adrenal level by inducing CORT release, certainly after estrogenic metabolization. In addition, we showed that, at the dose used, only DHEA, DHEA-S and PREG-S had antidepressant effects. In conclusion, these results highly suggest that part of the HPA axis and antidepressant effects of neuroactive steroids could be mediated by BDNF, particularly at the amygdala level. They also suggest that neurosteroids effects on central BDNF could partially explain the trophic properties of these molecules.

Intrahippocampal allopregnanolone decreases voluntary chronic alcohol consumption in non-selected rats.

We have recently shown that 0.2 microg of the neurosteroid allopregnanolone (AlloP) administered to the hippocampus induced an anxiolytic-like profile and also reduced alcohol withdrawal symptoms in voluntary and chronic alcohol-drinking rats. The aim of the present work was to study whether the administration of this dose of AlloP could affect alcohol consumption in non-selected rats that have been voluntarily ingesting high doses of alcohol for long periods of time in a limited access procedure. We used a free-choice drinking procedure that involved providing the rats with an alcoholic solution (10% ethanol) at an early age. Alcohol and control rats were assigned randomly to three groups that received an intrahippocampal (dorsal CA1) injection before the period of alcohol consumption after a long history of chronic alcohol intake. The injection groups were AlloP (0.2 microg, 1.26 microM), pregnenolone sulfate (PregS) (5 ng, 24 microM) or vehicle. Blood alcohol concentrations (BAC) were assessed before testing the effects of injections on alcohol consumption. Although AlloP did not eliminate alcohol ingestion, it significantly decreased alcohol consumption. The intrahippocampal administration of PregS, at the dose tested, did not effectively modify alcohol consumption levels. These results indicate that the positive modulation of hippocampal GABA(A) receptors induced by neurosteroids can be an important neurobiological target for reducing chronic alcohol consumption.

Pregnenolone as an adjunct to risperidone for treatment of women with schizophrenia: A randomized double-blind placebo-controlled clinical trial.

There have been few studies of pregnenolone therapy in schizophrenia and those that exist have been subject to several critical limitations, thus yielding inconsistent results. We attempted to assess the therapeutic effect of pregnenolone in a patient sample as homogeneous as possible. In this randomized double-blind clinical trial, 82 female inpatients with chronic schizophrenia, who had discontinued their antipsychotic medications for at least one week in case of any oral antipsychotic medication or a month for any depot antipsychotic medication, received risperidone plus either pregnenolone (50 mg/day) or placebo for 8 weeks. Inclusion criteria were acute illness with a baseline Positive and Negative Syndrome Scale (PANSS) negative subscale score of ≥20. Exclusion criteria were the presence of severe depression or other concomitant psychiatric disorders. Primary outcome was defined as the difference in the PANSS total score change from baseline to week 8 in the pregnenolone group compared to the placebo group. No significant difference was found in the PANSS total score changes between the two arms (mean difference (CI 95%) = -9.41 (-20.24 to 1.41); p = 0.087). Significant differences were initially found for PANSS negative change scores (mean difference (CI 95%) = -2.61 (-5.03 to -0.19); p = 0.035) and general psychopathology change scores (mean difference (CI 95%) = -5.93 (-11.37 to -0.48); p = 0.033). However, these findings did not survive Bonferroni correction for multiple testing. While this trial may suggest a potential effect of pregnenolone on schizophrenia symptoms, further studies are warranted.

ATP Maintenance via Two Types of ATP Regulators Mitigates Pathological Phenotypes in Mouse Models of Parkinson's Disease.

Parkinson's disease is assumed to be caused by mitochondrial dysfunction in the affected dopaminergic neurons in the brain. We have recently created small chemicals, KUSs (Kyoto University Substances), which can reduce cellular ATP consumption. By contrast, agonistic ligands of ERRs (estrogen receptor-related receptors) are expected to raise cellular ATP levels via enhancing ATP production. Here, we show that esculetin functions as an ERR agonist, and its addition to culture media enhances glycolysis and mitochondrial respiration, leading to elevated cellular ATP levels. Subsequently, we show the neuroprotective efficacies of KUSs, esculetin, and GSK4716 (an ERRγ agonist) against cell death in Parkinson's disease models. In the surviving neurons, ATP levels and expression levels of α-synuclein and CHOP (an ER stress-mediated cell death executor) were all rectified. We propose that maintenance of ATP levels, by inhibiting ATP consumption or enhancing ATP production, or both, would be a promising therapeutic strategy for Parkinson's disease.

The stress response factor daf-16/FOXO is required for multiple compound families to prolong the function of neurons with Huntington's disease.

Helping neurons to compensate for proteotoxic stress and maintain function over time (neuronal compensation) has therapeutic potential in aging and neurodegenerative disease. The stress response factor FOXO3 is neuroprotective in models of Huntington's disease (HD), Parkinson's disease and motor-neuron diseases. Neuroprotective compounds acting in a FOXO-dependent manner could thus constitute bona fide drugs for promoting neuronal compensation. However, whether FOXO-dependent neuroprotection is a common feature of several compound families remains unknown. Using drug screening in C. elegans nematodes with neuronal expression of human exon-1 huntingtin (128Q), we found that 3ß-Methoxy-Pregnenolone (MAP4343), 17ß-oestradiol (17ßE2) and 12 flavonoids including isoquercitrin promote neuronal function in 128Q nematodes. MAP4343, 17ßE2 and isoquercitrin also promote stress resistance in mutant Htt striatal cells derived from knock-in HD mice. Interestingly, daf-16/FOXO is required for MAP4343, 17ßE2 and isoquercitrin to sustain neuronal function in 128Q nematodes. This similarly applies to the GSK3 inhibitor lithium chloride (LiCl) and, as previously described, to resveratrol and the AMPK activator metformin. Daf-16/FOXO and the targets engaged by these compounds define a sub-network enriched for stress-response and neuronally-active pathways. Collectively, these data highlights the dependence on a daf-16/FOXO-interaction network as a common feature of several compound families for prolonging neuronal function in HD.

Effects of some neurosteroids injected into some brain areas of WAG/Rij rats, an animal model of generalized absence epilepsy.

Neurosteroids are synthesized in the brain and have been demonstrated to modulate various cerebral functions. Allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), a naturally occurring neurosteroid, and ganaxolone (3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a synthetic derivative, are two neurosteroids acting as positive allosteric modulators of the GABA(A) receptor complex acting on a specific steroid recognition site. Both agents antagonize generalized tonic-clonic seizures in various animal models of epilepsy. Pregnenolone sulphate (3beta-hydroxy-5alpha-pregnen-20-one 3-sulphate; PS) is a negative allosteric modulator of GABA(A) receptors and a positive modulator of the NMDA receptors. We have evaluated the effects of such compounds in a genetic animal model of absence epilepsy, the WAG/Rij rat. Animals were chronically implanted with five frontoparietal cortical electrodes for electrocorticogram (EEG) recordings and bilateral guide cannulae into specific brain areas of the cortico-thalamic circuit in order to evaluate the effects of these compounds on the number and duration of epileptic spike-wave discharges (SWDs). The focal and bilateral microinjection of the two GABA(A) positive modulators into some thalamic nuclei (nucleus ventralis posteromedialis, nucleus reticularis thalami, nucleus ventralis posterolateralis was usually able to significantly worsen the occurrence of SWDs in WAG/Rij rats. Whereas both compounds were able to reduce the number and duration of SWDs when microinjected into the peri-oral region of the primary somatosensory cortex. The effects of PS were more complex depending on both the dose and the site of administration, generally, at low doses in thalamic nuclei and cortex, PS induced an increase of absence activity and a reduction at higher doses. These findings suggest that neurosteroids might play a role in absence epilepsies and that it might depend on the involvement of specific neuronal areas.

Evaluation of the impact of 16-dehydropregnenolone on the activity and expression of rat hepatic cytochrome P450 enzymes.

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.