Isolation of aldose reductase inhibitors from the flowers ofChrysanthemum boreale

The methanol extract from the whole parts of the flowers ofChrysanthemum boreale was found to exhibit a significant inhibition of rat lens aldose reductase (RLAR) activityin vitro. Bioassay guided systematic fractionation of the methanol extract led to the isolation of four flavonoids which were identified as acacetin (I), apigenin (II), luteolin (III) and linarin (IV). Compounds I–III were demonstrated to exhibit a significant inhibition of RLAR. Luteolin (III) was found to be the most potent AR inhibitor with IC₅₀ value of 5×10^?7M.     

Synthesis and biological activity of novel Schiff bases derived from metronidazole

A number of novel Schiff bases (5a–i) and (7a–d) derived from metronidazole were synthesized. Reaction of 2-(2-methyl-5-nitro-imidazol-1-yl)-ethyl ester toluene-4-sulfonate with 4-hydroxybenzaldehyde and with 3-hydroxybenzaldehyde in the presence of a base afforded 4-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)benzaldehyde (5) and 3-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethoxy)benzaldehyde (7), respectively. The reaction of aldehydes 5 and 7 with a number of primary aromatic amines produced Schiff bases 5a–i and 7a–d, respectively. Structures of these compounds were confirmed through different spectroscopic methods such as ¹H-NMR, ¹³C-NMR, mass spectrometry, and also by elemental analyses. The prepared compounds were evaluated in vitro for their antigiardial, anti-trichomonal, antibacterial, and antifungal activities. Compounds 5e, 5g, 5i, 7a, 7b, 7c, and 7d exhibited remarkable antigiardial activity and were found to be more active than metronidazole with IC₅₀ of 7.2, 3.3, 1.5, 5.8, 4.5, 2.9, and 3.8 µg/mL, respectively. Compounds 5a and 5c also exhibited antigiradial activity with similar IC₅₀ values compared to the reference drug metronidazole with IC₅₀ of 7.2 µg/mL. The other compounds 5b, 5d, 5f, and 5 h also showed antigiardial activity but with higher IC₅₀ compared to the reference drug. Compounds were also tested for their anti-trichomonal activity, they, however, exhibited higher IC₅₀ compared to the reference drug metronidazole (7.4 µg/mL), except for compound 5a which exhibited anti-trichomonal activity with an IC₅₀ of 6.3 µg/mL. On the bases of preliminary screening, the newly synthesized compounds exhibited moderate to potent antimicrobial activities. Compound 5e inhibited the growth of Methicillin resistant Staphylococcus aureus (MRSA) and Bacillus cereus and compound 5c inhibited Candida Pathogenic fungus at 50 μg/mL compared with the positive control (Nystatin) which inhibits Candida at 25 μg/mL.     

Arsenic release from shallow aquifers of the Hetao basin,Inner Mongolia: evidence from bacterial community in aquifer sediments and groundwater

Indigenous microbes play crucial roles in arsenic mobilization in high arsenic groundwater systems. Databases concerning the presence and the activity of microbial communities are very useful in evaluating the potential of microbe-mediated arsenic mobilization in shallow aquifers hosting high arsenic groundwater. This study characterized microbial communities in groundwaters at different depths with different arsenic concentrations by DGGE and one sediment by 16S rRNA gene clone library, and evaluated arsenic mobilization in microcosm batches with the presence of indigenous bacteria. DGGE fingerprints revealed that the community structure changed substantially with depth at the same location. It indicated that a relatively higher bacterial diversity was present in the groundwater sample with lower arsenic concentration. Sequence analysis of 16S rRNA gene demonstrated that the sediment bacteria mainly belonged to Pseudomonas, Dietzia and Rhodococcus, which have been widely found in aquifer systems. Additionally, NO₃ ^?-reducing bacteria Pseudomonas sp. was the largest group, followed by Fe(III)-reducing, SO₄ ^2?-reducing and As(V)-reducing bacteria in the sediment sample. These anaerobic bacteria used the specific oxyanions as electron acceptor and played a significant role in reductive dissolution of Fe oxide minerals, reduction of As(V), and release of arsenic from sediments into groundwater. Microcosm experiments, using intact aquifer sediments, showed that arsenic release and Fe(III) reduction were microbially mediated in the presence of indigenous bacteria. High arsenic concentration was also observed in the batch without amendment of organic carbon, demonstrating that the natural organic matter in sediments was the potential electron donor for microbially mediated arsenic release from these aquifer sediments.     

The interaction of escitalopram and R-citalopram at the human serotonin transporter investigated in the mouse

Rationale

Escitalopram appears to be a superior antidepressant to racemic citalopram. It has been hypothesized that binding of R-citalopram to the serotonin transporter (SERT) antagonizes escitalopram binding to and inhibition of the SERT, thereby curtailing the elevation of extracellular 5-hydroxytryptamine (5-HT_Ext), and hence antidepressant efficacy. Further, it has been suggested that a putative allosteric binding site is important for binding of escitalopram to the primary, orthosteric, site, and for R-citalopram’s inhibition hereof.

Objectives

Primary: Investigate at the human (h)SERT, at clinical relevant doses, whether R-citalopram antagonizes escitalopram-induced 5-HT_Ext elevation. Secondary: Investigate whether abolishing the putative allosteric site affects escitalopram-induced 5-HT_Ext elevation and/or modulates the effect of R-citalopram.

Methods

Recombinant generation of hSERT transgenic mice; in vivo microdialysis; SERT binding; pharmacokinetics; 5-HT sensitive behaviors (tail suspension, marble burying).

Results

We generated mice expressing either the wild-type human SERT (hSERT^WT) or hSERT carrying amino acid substitutions (A505V, L506F, I507L, S574T and I575T) collectively abolishing the putative allosteric site (hSERT^{ALI/VFL+SI/TT}). One mg/kg escitalopram yielded clinical relevant plasma levels and brain levels consistent with therapeutic SERT occupancy. The hSERT mice showed normal basal 5-HT_Ext levels. Escitalopram-induced 5-HT_Ext elevation was not decreased by R-citalopram co-treatment and was unaffected by loss of the allosteric site_. The behavioral effects of the clinically relevant escitalopram dose were small and tended to be enhanced by R-citalopram co-administration.

Conclusions

We find no evidence that R-citalopram directly antagonizes escitalopram or that the putative allosteric site is important for hSERT inhibition by escitalopram.     

Determinants of bumetanide response in the dog: Effect of probenecid

The pharmacokinetics and pharmacodynamics of intravenous bumetanide (0.250 mg/kg), alone (treatment I) and after probenecid pretreatment (treatment II), were studied in four mongrel dogs. Lactated Ringer's solution was administered by vein throughout both treatments at a flow rate of 2 ml/min to avoid fluid and electrolyte depletion. Bumetanide and probenecid concentrations were analyzed by HPLC, sodium by flame photometry, and creatinine by colorimetry. Although the probenecid markedly reduced the plasma and renal clearances of bumetanide, as well as the fraction excreted unchanged in the urine, there was no significant difference between treatments I and II in the 4-hr natriuretic and diuretic responses. However, analysis of the dose-response curves between treatments I and II showed that sodium, excretion was better correlated with bumetanide urinary excretion rate than with plasma concentration. The reasons for a poor correlation between treatments during the early time periods are discussed.     

Role of ACE I/D gene polymorphisms on the effect of ramipril in inflammatory response and myocardial injury in patients undergoing coronary artery bypass grafts

Background

Angiotensin-converting enzyme (ACE) inhibitors block angiotensin II formation and release bradykinin, which is effective in the regulation of oxidoinflammatory injury. Some reports denote alterations in the effectiveness of ACE inhibitors in association with ACE insertion/deletion (I/D) gene polymorphisms. This study investigates the effects of ramipril on the oxidoinflammatory cytokines (IL-6, IL-8, TNF-alpha) and TnT (myocardial injury marker) and their alteration in association with ACE I/D gene polymorphisms.

Methods

The study group (n?=?51) patients received ramipril before coronary artery bypass grafting (CABG), while patients not receiving ramipril (n?=?51) constituted the controls. TNFα, IL-6, and IL-8 were evaluated using ELISA and TnT by electrochemiluminescence methods before the induction of anesthesia (t1), at the 20th minute following cross-clamping (t2), at the end of the operation (t3), and at the 24th hour from the commencement of anesthesia (t4). Genotyping was performed by PCR.

Results

Differences between the groups were significant at t4 for the TNFα and at t3 for IL-6 (p?p?p?>?0.05). The IL-6, IL-8, TNFα, and TnT serum levels had no correlation with the ACE I/D gene polymorphism.

Conclusion

Low cytokine and TnT levels in the study group, especially after cross-clamping, may indicate the protective effect of ramipril from oxidoinflammatory injury. This effect did not appear to be associated with the ACE I/D gene polymorphism.     

Prefrontal dopamine D1 receptors and working memory in schizotypal personality disorder: a PET study with [11C]NNC112

Rationale

Schizotypal personality disorder (SPD) is associated with working memory (WM) impairments that are similar to those observed in schizophrenia. Imaging studies have suggested that schizophrenia is associated with alterations in dopamine D1 receptor availability in the prefrontal cortex (PFC) that may be related to the WM impairments that characterize this disorder.

Objectives

The aim of this study was to characterize prefrontal D1 receptor availability and its relation to WM performance in SPD.

Methods

We used positron emission tomography (PET) and the radiotracer [¹¹C]NNC112 with 18 unmedicated SPD and 21 healthy control participants; as an index of D1 receptor availability, binding potential (BP) measures (BP_F, BP_ND, and BP_P) were calculated for prefrontal and striatal subregions. To assess WM, SPD participants completed the 2-back and Paced Auditory Serial Addition Test (PASAT).

Results

There were no significant group differences in PFC BP. BP_F and BP_P in the medial PFC were significantly negatively related to PASAT performance (r _s ?=??0.551, p?=?.022 and r _s ?=??0.488, p?=?.047, respectively), but BP was not related to 2-back performance.

Conclusions

In contrast to what has been found in schizophrenia, SPD was not associated with significant alterations in prefrontal D1 receptor availability. Similar to previous schizophrenia findings, however, higher prefrontal D1 receptor availability was associated with poorer WM performance (as measured by the PASAT) in SPD. These findings suggest that schizophrenia and SPD may share a common pathophysiological feature related to prefrontal dopamine functioning that contributes to WM dysfunction, but that in SPD, alterations in D1 may occur only in a subset of individuals and/or to an extent that is minor relative to what occurs in schizophrenia.     

Jungia sellowii suppresses the carrageenan-induced inflammatory response in the mouse model of pleurisy

This study was conducted to explore the anti-inflammatory effect of Jungia sellowii (Asteraceae) using a murine model of pleurisy induced by carrageenan (Cg). This plant is used in southern Brazil to treat inflammatory diseases. J. sellowii leaves were extracted with ethanol/water to obtain the crude extract (CE), which was fractionated with different solvents, yielding n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH) fractions, and aqueous fraction (Aq). The major compounds succinic acid (SA) and lactic acid (LA) were isolated from Aq fraction, and their structures were determined by ¹H and ¹³C NMR. Pleurisy was induced by Cg (Saleh et al. 1996). The leukocytes, exudation, myeloperoxidase (MPO) and adenosine–deaminase (ADA) activities, metabolites of nitric oxide (NO _x ) levels, protein levels and mRNA expression for interleukin 1 beta (IL-1β), tumour necrosis factor alpha (TNF-α), interleukin 17A (IL17A) and inducible of nitric oxide synthase (iNOs), and p65 protein phosphorylation (NF-κB) were analysed 4 h after pleurisy induction. Animals pre-treated with CE, BuOH, Aq, SA, or LA inhibited leukocytes, exudation, MPO and ADA activities, NO _x , IL-1β, TNF-α, and IL-17A levels, and the mRNA expression for IL-1β, TNF-α, IL-17A, iNOS, and p65 protein phosphorylation (NF-κB) (p < 0.05). Our study demonstrated that J. sellowii can protect against inflammation induced by Cg by decreasing the leukocytes and exudation. Its effects are related to the decrease of either proinflammatory cytokines and/or NO _x . The isolated compounds SA and LA may play an important role in this anti-inflammatory action by inhibiting all the studied parameters. The anti-inflammatory properties of these compounds are due to the downregulation of NF-κB.     

Synthesis of two nitro analogs of tranylcypromine: Relations of aromatic substitution of nitro groups to MAO-Inhibitory activity

Two new nitro analogs of tranylcypromine, (E)-2-(p-nitrophenyl)cyclopropylamine ((E)-p-NTCP) and (E)-2-(m-nitrophenyl)cyclopropylamine ((E)-m-NTCP) were synthesized in order to examine the effect of aromatic nitro substitution on the MAO-inhibitory activity of 2-phenylcyclopropylamines. The compounds were obtained by treatingt-butyl (E)-2-(p-nitrophenyl) cyclopropanecarbamate andt-butyl (E)-2-(m-nitrophenyl)cyclopropanecarbamate withp-toluenesulfonic acid in CH₃CN. Inhibitions of rat brain mitochondrial MAO-A and B by the compounds were examined using serotonin and benzylamine as the substrate at bothin vitro andex vivo levels. It was found fromin vitro measurements that(E)-p-NTCP at 6.0×10^?5M elicited merely 22.5% inhibition against MAO-B without any effect on MAO-A. In contrast,(E)-m-NTCP showed fair degrees of inhibitions of MAO-A and B with IC₅₀ values, 2.5×10^?7M and 1.4×10^?6M, respectively. It was also noted from(E)-m-NTCP thatm-nitro substitution caused a shift of selectivity of the inhibition toward MAO-A. According toex vivo measurements at 1.5, 3, 6, and 12 hr following the administration of a dose of 0.015 mmol/kg, i.p. to the rats, the inhibition percents of MAO-A by(E)-m-NTCP were 58.6, 63.7 63.6, and 46.6%, slightly lower than those observed by tranylcypromine. Whereas,(E)-p-NTCP at the same dose level did not show significant inhibitions against both MAO-A and MAO-B. Possible reasons for the difference in potencies between(E)-m-NTCP and(E)-p-NTCP were sought in relation to differing electron withdrawing effects ofm-andp-substituents which will influence electron density of the side chain amino functions and the partitions.     

Potential anti-tubercular and in vitro anti-inflammatory agents: 9-substituted 1,8-dioxo-octahydroxanthenes through cascade/domino reaction by citric fruit juices

A series of 9-substituted 1,8-dioxo-octahydroxanthenes, 3a–k were synthesized and evaluated for their anti-tubercular and in vitro anti-inflammatory potential. Compounds 3b, 3d, 3e, 3f, 3g, and 3k demonstrated their potential anti-tubercular activity at 0.4 μg/ml comparable to streptomycin and pyrazinamide (standard drugs) screened by Microplate Alamar Blue assay against Mycobacterium tuberculosis H37 Ra. Compounds 3j, 3e, 3f, and 3c showed good in vitro anti-inflammatory activity by the inhibition of enzyme MMP-2 75, 65, 70, and 60 % and inhibition of enzyme MMP-9 by 90, 70, 60, and 60 %, respectively by gelatin zymography on gel electrophoresis. Standard drug tetracycline (300 μg/ml) is used as positive control which showed 90 % inhibition. The percentage inhibition was determined by a gel documentation system. DMSO was used as solvent control which did not show any inhibition.     

The molecular structure of (+)-6-methoxy-α-methy1-2-naphthaleneacetic acid determined by X-ray method

The molecular structure of (+)-6-Methoxy-α-methyl-2-naphthaleneacetic acid (Naproxen), C₁₄H₁₄O₃, was determined by X-Ray diffraction technique. Naproxen crystallized in P2₁ with two molecules in the unit cell of dimensionsa=7.855,b=5.783,c=13.347Å and β=93.9°. The structure was solved with the SHELX program system. The present R-value is 0.099. The molecules are connected by the intermolecular OH…O hydrogen bonds.     

Curcumin,demethoxycurcumin and bisdemethoxycurcumin differentially inhibit morphine's rewarding effect in rats

Rationale

Recent animal studies reported that curcumin, the active constituent of Curcuma longa, has several central actions and may attenuate morphine tolerance.

Objectives

In the present study, we utilized the intracranial self-stimulation (ICSS) paradigm to examine the effects of the commercially available curcuminoid mixture and each one of its components, individually, on brain stimulation reward and on the reward-facilitating effect of morphine.

Methods

Male Sprague-Dawley rats were implanted with an electrode into the medial forebrain bundle and trained to respond for electrical stimulation using a rate-frequency paradigm. In the first study, rats were injected with graded doses either of the curcuminoid mixture, or curcumin I, or II, or III. In the second study, we examined whether a low dose of the curcuminoid mixture or each individual curcumin analogue composing it could counteract the reward-facilitating effect of morphine.

Results

At low doses, both the curcuminoid mixture and curcumin I did not affect brain stimulation reward, whereas, higher doses increased ICSS thresholds. Curcumin II and curcumin III did not affect brain stimulation reward at any doses. Subthreshold doses of the curcuminoid mixture and curcumin I inhibited the reward-facilitating effect of morphine.

Conclusion

Both the curcuminoid mixture and curcumin I lack hedonic properties and moderate the reward-facilitating effect of morphine. Our data suggest that curcumin interferes with brain reward mechanisms responsible for the expression of the acute reinforcing properties of opioids and provide evidence that curcumin may be a promising adjuvant for attenuating morphine’s rewarding effects in patients who are under long-term opioid therapy.     

Synthesis and anti-HIV-1 screening of novel N′-(1-(aryl)ethylidene)-2-(5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-4(1H)-yl)acetohydrazides

A novel series of N′-(1-(aryl)ethylidene)-2-(5,5-dioxido-3-phenylbenzo[e]pyrazolo[4,3-c][1,2]thiazin-4(1H)-yl)acetohydrazides was synthesized. The synthesis was carried out by thermal method as well as ultrasonic bath to reduce reaction time and to enhance product yields. The synthesized compounds were characterized by spectroscopic techniques like NMR, infrared and EIMS. The structure of compound 5w was elucidated by X-ray crystallography. The titled compounds were evaluated for anti-human immunodeficiency virus type 1 (anti-HIV-1) and cytotoxic activities. Biological studies indicated that amongst these compounds, 5a, b, j, h and i showed the activity with median effective concentration (EC₅₀) values less than 20 μM. Compound 5i exhibited the most potent anti-HIV-1 activity (EC₅₀ = 3.2 μM) while 5h showed anti-HIV-1 activity (EC₅₀ = 3.8 μM) with no toxicity at all in primary human lymphocytes, CEM and VERO cells.     

One-pot synthesis and cytotoxic evaluation of amide-linked 1,4-disubstituted 1,2,3-bistriazoles

A series of amide-linked 1,4-disubstituted 1,2,3-bistriazoles have been synthesized employing copper(I)-catalyzed azide–alkyne cycloaddition reaction. All the newly synthesized compounds were screened for in vitro cytotoxicity against a panel of five human cancer cell lines; Fibrosarcoma (HT-1080), Colon (colo205, HCT-116), and Lung (A549, NCIH322). Some of the bistriazoles exhibited moderate to good activity. Compounds 3n and 3o were found to be the more active and displayed broad spectrum activity against all the cancer cell lines under investigation. Further, to study the binding modes for the two more potent compounds 3n and 3o against Human topoisomerase II, docking simulations have been carried out.     

Oral Delivery of Glucagon Like Peptide-1 by a Recombinant Lactococcus lactis

Purpose

To develop a live oral delivery system of Glucagon like peptide-1 (GLP-1), for the treatment of Type-2 Diabetes.

Methods

LL-pUBGLP-1, a recombinant Lactococcus lactis (L. lactis)) transformed with a plasmid vector encoding GLP-1 cDNA was constructed and was used as a delivery system. Secretion of rGLP-1 from LL-pUBGLP-1 was characterized by ELISA. The bioactivity of the rGLP-1 was examined for its insulinotropic activity on HIT-T15 cells. Transport of rGLP-1 across MDCK cell monolayer when delivered by LL-pUBGLP-1 was studied. The therapeutic effect of LL-pUBGLP-1 after oral administration was investigated in ZDF rats.

Results

DNA sequencing and ELISA confirmed the successful construction of the LL-pUBGLP-1 and secretion of the active form of rGLP-1. In vitro insulinotropic studies demonstrated that LL-pUBGLP-1 could significantly (p?GLP-1 transport rate across the MDCK cell monolayer was increased by eight times (p?p?0-11h (2.5 times, p?Conclusion The present study demonstrates that L. lactis when genetically modified with a recombinant plasmid can be used for the oral delivery of GLP-1.     

Effect of two-linked mutations of the FMO3 gene on itopride metabolism in Chinese healthy volunteers

Purpose

Itopride is an effective gastroprokinetic agent mainly used for the treatment of functional dyspepsia. Flavin-containing monooxygenase 3 (FMO3) has been confirmed to be the key enzyme involved in the main itopride metabolic pathway. We investigated whether the FMO3 genotypes can affect itopride metabolism in Chinese healthy volunteers.

Methods

Twelve healthy volunteers who had been genotyped for FMO3 gene were selected to participate in our study. Volunteers were given 50 mg itopride orally and then blood samples were collected from 0 to 24 h. The plasma concentrations of itopride and itopride N-oxide were determined by HPLC-MS/MS method.

Results

Itopride and itopride N-oxide both exhibit FMO3 genotype-dependent pharmacokinetic profiles. The area under the plasma concentration–time curve (AUC) of itopride increased by 127.82?±?41.99 % (P?P?FMO3 hhdd subjects (n?=?6) compared with the HHDD group (n?=?6). The CL/F value was lower in the hhdd group than that in the HHDD group (36.60?±?7.06 vs. 80.20?±?15.34 L/h, P?1/2 value and t_max of itopride and itopride N-oxide were observed between these two genotypes.

Conclusion

The FMO3 allele can significantly affect the metabolism of itopride. The pharmacokinetic parameters of both itopride and itopride N-oxide were significantly different between these two genotypes.     

5α-reductase type I expression is downregulated in the prefrontal cortex/Brodmann’s area 9 (BA9) of depressed patients

Rationale

The implications of the neurosteroid 3α-hydroxy-5α-pregnan-20-one [allopregnanolone (Allo)] in neuropsychiatric disorders have been highlighted in several recent clinical investigations. For instance, Allo levels are decreased in the cerebrospinal fluid (CSF) of patients with posttraumatic stress disorder (PTSD) and major unipolar depression. Neurosteroidogenic antidepressants [i.e., selective brain steroidogenic stimulants (SBSSs)], including fluoxetine and analogs, correct this decrease in a manner that correlates with improved depressive symptoms. Allo positively and allosterically modulates GABA action at postsynaptic and extrasynaptic GABA_A receptors. It is synthesized in both the human and rodent brain cortices by principal glutamatergic pyramidal neurons from progesterone by the sequential action of 5α-reductase type I (5α-RI), which is the rate-limiting step enzyme in Allo biosynthesis, and 3α-hydroxysteroid dehydrogenase (3α-HSD), which converts 5α-dehydroprogesterone into Allo.

Hypothesis

We thus hypothesized that decreased CSF levels of Allo in depressed patients could reflect a brain dysfunction of 5α-RI.

Methods

In a pilot study of samples from six patients per group [six depressed patients and six nonpsychiatric subjects (NPS)], we studied the expression of 5α-RI messenger RNA (mRNA) in prefrontal cortex Brodmann’s area 9 (BA9) and cerebellum from depressed patients obtained from the Maryland Brain Collection at the Maryland Psychiatric Research Center (Baltimore, MD) that were age-matched with NPS.

Results

The levels of 5α-RI mRNA were decreased from 25?±?5.8 in NPS to 9.1?±?3.1 fmol/pmol neuronal specific enolase (NSE) (t_1,10?=?2.7, P?=?0.02) in depressed patients. These differences are absent in the cerebellum of the same patients. The levels of neurosteroids were determined in the prefrontal cortex BA9 of depressed patients obtained from the Stanley Foundation Brain Bank Neuropathology Consortium, Bethesda (MD). The BA9 levels of Allo in male depressed patients failed to reach statistical difference from the levels of NPS (1.63?±?1.01 pg/mg, n?=?8, in NPS and 0.82?±?0.33 pg/mg, n?=?5, in nontreated depressed patients). However, depressed patients who had received antidepressant treatment (three patients SSRI and one TCA) exhibited increased BA9 Allo levels (6.16?±?2.5 pg/mg, n?=?4, t_1,9?=?2.4, P?=?0.047) when compared with nontreated depressed patients.

Conclusions

Although in a small number of patients, this finding is in-line with previous reports in the field that have observed an increase of Allo levels in CSF and plasma of depressed patients following antidepressant treatment. Hence, the molecular mechanisms underlying major depression may include a GABAergic neurotransmission deficit caused by a brain Allo biosynthesis downregulation, which can be normalized by SBSSs.     

Proof-of-concept randomized controlled trial of pregnenolone in schizophrenia

Rationale

Preclinical and clinical data suggest that pregnenolone may be a promising therapeutic in schizophrenia. Pregnenolone is neuroprotective and enhances learning and memory, myelination, and microtubule polymerization. Treatment with pregnenolone elevates allopregnanolone (a neurosteroid that enhances GABA_A receptor responses) and pregnenolone sulfate (a positive NMDA receptor modulator). Pregnenolone could thus potentially mitigate GABA dysregulation and/or NMDA receptor hypofunction in schizophrenia via metabolism to other neurosteroids.

Objective

The objective of this study is to conduct a randomized controlled trial of adjunctive pregnenolone in schizophrenia.

Methods

Following a placebo lead-in, 120 participants were randomized to pregnenolone or placebo for 8 weeks (Institute for Mental Health, Singapore). Primary endpoints were changes in MATRICS Consensus Cognitive Battery (MCCB) composite scores (cognitive symptoms), UCSD Performance-based Skills Assessment—Brief (UPSA-B) composite scores (functional capacity), and Scale for Assessment of Negative Symptoms (SANS) total scores (negative symptoms). A modified intent-to-treat analysis approach was utilized.

Results

No significant changes compared to placebo were demonstrated in composite MCCB scores. In contrast, participants randomized to pregnenolone (n?=?56) demonstrated greater improvements in functional capacity (UPSA-B composite changes) compared to placebo (n?=?55), p?=?0.03. Pregnenolone was also superior to placebo in the communication subscale of the UPSA-B (p?r _s?=?0.497, p?n?=?17) but not in males. Mean total SANS scores were very low at baseline and did not improve further post-treatment. Pregnenolone was well-tolerated.

Conclusions

Pregnenolone improved functional capacity in participants with schizophrenia, but did not improve cognitive symptoms over an 8-week treatment period. Neurosteroid changes correlated with functional improvements in female participants. Neurosteroid interventions may exhibit promise as new therapeutic leads for schizophrenia.     

11-trifluoromethyl-phenyldiazirinyl neurosteroid analogues: potent general anesthetics and photolabeling reagents for GABAA receptors

Rationale

While neurosteroids are well-described positive allosteric modulators of gamma-aminobutyric acid type A (GABA_A) receptors, the binding sites that mediate these actions have not been definitively identified.

Objectives

This study was conducted to synthesize neurosteroid analogue photolabeling reagents that closely mimic the biological effects of endogenous neurosteroids and have photochemical properties that will facilitate their use as tools for identifying the binding sites for neurosteroids on GABA_A receptors.

Results

Two neurosteroid analogues containing a trifluromethyl-phenyldiazirine group linked to the steroid C11 position were synthesized. These reagents, CW12 and CW14, are analogues of allopregnanolone (5α-reduced steroid) and pregnanolone (5β-reduced steroid), respectively. Both reagents were shown to have favorable photochemical properties with efficient insertion into the C–H bonds of cyclohexane. They also effectively replicated the actions of allopregnanolone and pregnanolone on GABA_A receptor functions: they potentiated GABA-induced currents in Xenopus laevis oocytes transfected with α₁β₂γ_2L subunits, modulated [³⁵S]t-butylbicyclophosphorothionate binding in rat brain membranes, and were effective anesthetics in Xenopus tadpoles. Studies using [³H]CW12 and [³H]CW14 showed that these reagents covalently label GABA_A receptors in both rat brain membranes and in a transformed human embryonal kidney (TSA) cells expressing either α₁ and β₂ subunits or β₃ subunits of the GABA_A receptor. Photolabeling of rat brain GABA_A receptors was shown to be both concentration-dependent and stereospecific.

Conclusions

CW12 and CW14 have the appropriate photochemical and pharmacological properties for use as photolabeling reagents to identify specific neurosteroid-binding sites on GABA_A receptors.     

Effect of para halogen modification of S-3-(phenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethyl-phenyl)-propionamides on metabolism and clearance

The purpose of this  study was to better understand why para-halogen modifications of S-3-(4-halophenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethylphenyl) propionamide selective androgen receptor modulators (SARMs) had the opposite of expected effects on total clearance, in which electron-withdrawing groups generally protect benzene ring from hydroxylation. We determined the plasma protein binding of this series of halogen substituted SARMs and characterized the qualitative effects of B-ring halogen substitution on in vivo metabolism. In vivo metabolism of S-9, S-10, and S-11 were determined in rats using LC-MSⁿ analysis. Intrinsic clearance was measured by in vitro metabolism using rat liver microsomes. Rat plasma protein binding was measured by equilibrium dialysis and drug concentrations after dialysis were analyzed by LC-MS. The major metabolic pathways of the halogen-substituted SARMs examined were very similar and included three major phase I pathways; (1) hydrolysis of the amide bond, (2) B-ring hydroxylation, and (3) A-ring nitro reduction to an aromatic amine. In plasma protein binding studies, S-1 (F, fu = 0.78 ± 0.17 %) showed the greatest unbound fraction, followed by S-9 (Cl, fu = 0.10 ± 0.04 %), S-10 (Br, fu = 0.03 ± 0.01 %), and S-11 (I, fu = 0.008 ± 0.001 %). The CL_int values of S-1, S-9, S-10, and S-11 were 2.4, 2.5, 2.8, and 4.6 μL/min/mg, respectively. These findings suggest that as lipophilicity increased the free fraction was reduced thus compensating for metabolic liability and resulting in the apparent discrepancy between CL_int and CL total of halogen-substituted SARMs series.