Synthetic structural modifications of neurosteroid pregnanolone sulfate: Assessment of neuroprotective effects in vivo.

Neuroactive steroid 20-oxo-5ß-pregnan-3α-yl L-glutamyl 1-ester (PA-Glu), a synthetic analogue of naturally occurring 20-oxo-5ß-pregnan-3α-yl sulfate (pregnanolone sulfate, PA-S), inhibits N-methyl-D-aspartate (NMDA) receptors and possesses neuroprotective properties and minimal adverse effects. Herein, we report in vivo effects of new structural modifications of the PA-S molecule: a nonpolar modification of the steroid D-ring (5ß-androstan-3α-yl L-glutamyl 1-ester, AND-Glu), attachment of a positively charged group to C3 (20-oxo-5ß-pregnan-3α-yl L-argininate dihydrochloride salt, PA-Arg) and their combination (5ß-androstan-3α-yl L-argininate dihydrochloride salt, AND-Arg). The first aim of this study was to determine the structure-activity relationship for neuroprotective effects in a model of excitotoxic hippocampal damage in rats, based on its behavioral correlate in Carousel maze. The second aim was to explore side effects of neuroprotective steroids on motor functions, anxiety (elevated plus maze) and locomotor activity (open field) and the effect of their high doses in mice. The neuroprotective properties of PA-Glu and AND-Glu were proven, with the effect of the latter appearing to be more pronounced. In contrast, neuroprotective efficacy failed when positively charged molecules (PA-Arg, AND-Arg) were used. AND-Glu and PA-Glu at the neuroprotective dose (1 mg/kg) did not unfavorably influence motor functions of intact mice. Moreover, anxiolytic effects of AND-Glu and PA-Glu were ascertained. These findings corroborate the value of research of steroidal inhibitors of NMDA receptors as potential neuroprotectants with slight anxiolytic effect and devoid of behavioral adverse effects. Taken together, the results suggest the benefit of the nonpolar D-ring modification, but not of the attachment of a positively charged group to C3.

Pregn-5-en-3ß-ol and androst-5-en-3ß-ol dicarboxylic acid esters as potential therapeutics for NMDA hypofunction: In vitro safety assessment and plasma stability.

Neurosteroids are endogenous steroidal compounds that can modulate neuronal receptors. N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that are of particular interest, as they participate in synaptic transmission and are implicated in various processes, such as learning, memory, or long-term neuronal potentiation. Positive allosteric modulators that increase the activity of NMDARs may provide a therapeutic aid for patients suffering from neuropsychiatric disorders where NMDAR hypofunction is thought to be involved, such as intellectual disability, autism spectrum disorder, or schizophrenia. We recently described a new class of pregn-5-ene and androst-5-ene 3ß-dicarboxylic acid hemiesters (2-24) as potent positive modulators of NMDARs. Considering the recommended guidelines for the early stage development of new, potent compounds, we conducted an in vitro safety assessment and plasma stability screening to evaluate their druglikeness. First, compounds were screened for their hepatotoxicity and mitochondrial toxicity in a HepG2 cell line. Second, toxicity in primary rat postnatal neurons was estimated. Next, the ability of compounds 2-24 to cross a Caco-2 monolayer was also studied. Finally, rat and human plasma stability screening revealed an unforeseen high stability of the C-3 hemiester moiety. In summary, by using potency/efficacy towards NMDARs data along with toxicity profile, Caco-2 permeability and plasma stability, compounds 14 and 15 were selected for further in vivo animal studies.