Caprospinol: discovery of a steroid drug candidate to treat Alzheimer's disease based on 22R-hydroxycholesterol structure and properties.

The overall ability of the brain to synthesise neuroactive steroids led us to the identification of compounds that would reproduce aspects of neurosteroid pharmacology. The rate-determining step in neurosteroid biosynthesis is the import of the substrate cholesterol into the mitochondria, where it is metabolised into pregnenolone via the intermediate 22R-hydroxycholesterol. The levels of translocator protein 18-kDa, mediating the import of cholesterol into mitochondria, correlated with increased pregnenolone formation and reduced levels of 22R-hydroxycholesterol in biopsies from Alzheimer's disease (AD), but not age-matched control, brains. 22R-hydroxycholesterol was shown to protect against ß-amyloid (Aß(42) )-induced neurotoxicity. In search of 22R-hydroxycholesterol stable analogues, we identified the naturally occurring heterospirostenol, (22R,25R)-20α-spirost-5-en-3ß-yl hexanoate (caprospinol) and derivatives that protect neuronal cells against Aß(1-42) neurotoxicity. The neuroprotective effect of caprospinol is the result of a combination of overlapping properties, including: (i) the ability to bind to Aß(42) and reduce plaque formation in the brain in vivo; (ii) interaction with components of the mitochondria respiratory chain resulting in an anti-uncoupling effect; (iii) the capacity to scavenge Aß(42) monomers present in mitochondria; and (iv) the property of being a sigma-1 receptor ligand. In vivo, caprospinol crosses the blood-brain barrier, accumulates in the brain, and restores cognitive impairment in a pharmacological rat model of AD. Caprospinol is stable, does not bind to known steroid receptors, is devoid of mutagenic and genotoxic properties, and is devoid of acute toxicity in rodents. The pharmacokinetics and pharmacodynamics of caprospinol were studied, and long-term toxicity studies are under investigation, aiming to develop this compound as a disease-modifying drug for the treatment of AD.

The naturally occurring steroid solasodine induces neurogenesis in vitro and in vivo.

In this study, we explored the capacity of the naturally occurring compound solasodine to promote neurogenesis in vitro and in vivo. Mouse embryonic teratocarcinoma P19 cells exposed to solasodine for 2 days followed by a 5-day washout differentiated into cholinergic neurons that expressed specific neuronal markers and displayed important axonal formation that continued growing even 30 days after treatment. In vivo, a 2-week infusion of solasodine into the left ventricle of the rat brain followed by a 3-week washout resulted in a significant increase in bromodeoxyuridine uptake by cells of the ependymal layer, subventricular zone, and cortex that co-localized with doublecortin immunostaining, demonstrating the proliferative and differentiating properties of solasodine on neuronal progenitors. In addition, these data demonstrate that under our experimental conditions adult ependymal cells retrieved their proliferative and differentiating abilities. The GAP-43/HuD pathway was activated both in vitro and in vivo, suggesting a role in the differentiating process triggered by solasodine. Solasodine treatment in rats resulted in a dramatic increase in expression of the cholesterol- and drug-binding translocator protein in ependymal cells, suggesting a possible role played by neurosteroid production in solasodine-induced neurogenesis. In GAD65-GFP mice that express the green fluorescent protein under the control of the glutamic acid decarboxylase 65-kDa promoter, solasodine treatment increased the number of GABAergic progenitors and neuroblasts generated in the subventricular zone and present in the olfactory migratory tract. Taken together, these results suggest that solasodine offers an interesting approach to stimulate in situ neurogenesis from resident neuronal progenitors as part of neuron replacement therapy.

Caprospinol reduces amyloid deposits and improves cognitive function in a rat model of Alzheimer's disease.

Alzheimer's disease (AD), the most prominent form of dementia in elderly, is a yet incurable degenerative neurological illness characterized by memory loss. Here, we used an AD rat model to investigate the in vivo efficacy of caprospinol, a disease-modifying steroid developed on the concept that reduced synthesis of 22R-hydroxycholesterol in the AD brain increases beta-amyloid neurotoxicity. Caprospinol treatment of diseased rats attenuated memory impairment, as assessed using Morris watermaze tests. This recovery of cognitive function was accompanied by a reduction in hippocampal amyloid deposits, astrogliosis, neurodegeneration and Tau protein phopshorylation. In parallel studies, caprospinol bioavailability in normal rat forebrain was found to be dependent on the dose and duration of the treatment, demonstrating the ability of the compound to cross the blood-brain barrier. These results position caprospinol as a promising drug candidate for AD treatment.

22R-Hydroxycholesterol induces differentiation of human NT2 precursor (Ntera2/D1 teratocarcinoma) cells.

Recently, we have shown that 22R-hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, is present at lower levels in Alzheimer's disease (AD) hippocampus and frontal cortex tissue specimens than in age-matched controls, and that this substance protects against cell death induced by amyloid beta-peptide in both rat sympathetic nerve pheochromocytoma (PC12) and differentiated human Ntera2/D1 teratocarcinoma neurons. Herein we report that 22R-hydroxycholesterol inhibits the proliferation of human Ntera2/D1 teratocarcinoma precursor cells (NT2) and induces these cells to differentiate into "neuron-like" or "astrocyte-like" cells. 22R-Hydroxycholesterol-induced differentiation of NT2 cells is associated with increases in the expression of neurofilament protein NF200, the cytoskeletal proteins microtubule-associated protein type II (MAP2) a and MAP2b, glial fibrillary acidic protein (GFAP) and glial cell line-derived neurotrophic factor receptor-alpha 2 (GFRalpha2). These effects of 22R-hydroxycholesterol are considered to be stereospecific because its enantiomer 22S-hydroxycholesterol and other steroids failed to induce differentiation of NT2 cells. 22R-Hydroxycholesterol was found to lack specific binding for numerous receptors, including all steroid receptors tested. However, using a cholesterol protein binding blot assay we demonstrated the presence of a 22R-hydroxycholesterol-binding protein in NT2 cells distinct from the human oxysterol receptors liver X receptor LXRalpha and beta.

Peripheral-type benzodiazepine receptor in neurosteroid biosynthesis, neuropathology and neurological disorders.

The peripheral-type benzodiazepine receptor is a mitochondrial protein expressed at high levels in steroid synthesizing tissues, including the glial cells of the brain. Peripheral-type benzodiazepine receptor binds cholesterol with high affinity and is a key element of the cholesterol mitochondrial import machinery responsible for supplying the substrate cholesterol to the first steroidogenic enzyme, thus initiating and maintaining neurosteroid biosynthesis. Neurosteroid formation and metabolism of steroid intermediates are critical components of normal brain function. Peripheral-type benzodiazepine receptor also binds with high affinity various classes of compounds. Upon ligand activation peripheral-type benzodiazepine receptor-dependent cholesterol transport into mitochondria is accelerated leading in increased formation of neuroactive steroids. These steroids, such as allopregnanolone, have been shown to be involved in various neurological disorders, such as anxiety and mood disorders. Thus, peripheral-type benzodiazepine receptor drug ligand-induced neuroactive steroid formation offers a means to regulate brain dysfunction. Peripheral-type benzodiazepine receptor basal expression is upregulated in a number of neuropathologies, including gliomas and neurodegenerative disorders, as well as in various forms of brain injury and inflammation. In Alzheimer's disease pathology neurosteroid biosynthesis is altered and a decrease in the intermediate 22R-hydroxycholesterol levels is observed. This steroid was found to exert neuroprotective properties against beta-amyloid neurotoxicity. Based on this observation, a stable spirostenol derivative showing to display neuroprotective properties was identified, suggesting that compounds developed based on critical intermediates of neurosteroid biosynthesis could offer novel means for neuroprotection. In conclusion, changes in peripheral-type benzodiazepine receptor and neurosteroid levels are part of the phenotype seen in neuropathology and neurological disorders and offer potential targets for new therapies.

A capillary gas chromatography/mass spectrometric method for the quantification of hydroxysteroids in human plasma.

A specific and sensitive methodology for the quantitative determination of hydroxysteroids dehydroepiandrosterone and pregnenolone and their main metabolites in human plasma is described. Hydroxysteroids were extracted using methanol and steroids were further separated by reverse-phase high-performance liquid chromatography, allowing for minimization of the possible chromatographic interferences. Eluted fractions were collected, pooled, and analyzed by gas chromatography-mass spectrometry as trimethylsilyl ether derivatives. The quantification was performed with single-ion monitoring of the highly abundant m/z 129 or m/z 358 fragments. The combination of the chromatographic characteristics to the specific fragments ensured the selectivity and specificity of the method. Under these conditions the method was linear (typical R2 is superior to 0.98 for all hydroxysteroids studied) over the concentration range of 2 x 10(-9) to 10(-6)M with good precision and accuracy.

Acetylcholine release in the hippocampus of the urethane anaesthetised rat positively correlates with both peak theta frequency and relative power in the theta band.

The need to achieve a clearer understanding of relations between hippocampal theta characteristics and cholinergic septohippocampal neuron activity, prompted us to re-examine, in the urethane-anaesthetised rat, the statistical relationships between the electrophysiological and neurochemical variables using a procedure which is believed to enhance significantly the degree of confidence with which parameters of theta recorded with classic macroelectrodes can be related to concomitant acetylcholine output measured by high-performance liquid chromatography with electrochemical detection. Firstly, the theta rhythm and the acetylcholine content were derived from the same hippocampus. Secondly, the hippocampal electroencephalogram was quantified using spectral analysis which permits the more objective quantitative evaluation of selected electroencephalogram samples. Thirdly, a larger number of rats than in our previous study was used here, thus enhancing the validity of statistical results. This procedure yielded, in our time-course determination, two main findings. The first finding is that acetylcholine release was positively correlated with frequency at the peak power of the theta band which reflects the frequency of the theta signal. This finding had not been reported yet. The second finding is that hippocampal acetylcholine outflow also covaried with relative power of the theta band which reflects the amplitude of the theta signal. This finding is consistent with our previous study in which EEG was quantified by means of a traditional method. These findings suggest that the cholinergic component of the septohippocampal system, which is the main source of hippocampal acetylcholine, and neurophysiological mechanisms involved in the modulation of both the amplitude and the frequency of theta are functionally related. The possibility that, at least in the urethane-anaesthetised rat, hippocampal acetylcholine is involved in these modulator mechanisms is discussed.

iNOS contribution to the NMDA-induced excitotoxic lesion in the rat striatum.

1. The aim of this study was to assess whether an excitotoxic insult induced by NMDA may induce an iNOS activity which contributes to the lesion in the rat striatum. 2. For this purpose, rats were perfused with 10 mM NMDA through a microdialysis probe implanted in the left striatum. Microdialysate nitrite content, striatal Ca-independent nitric oxide synthase activity and lesion volume were measured 48 h after NMDA exposure in rats treated with dexamethasone (DXM) (3 mg kg(-1) x 4) or aminoguanidine (AG) (100 mg kg(-1) x 4). 3. A significant increase in microdialysate nitrite content and in the Ca-independent NOS activity was observed 48 h after NMDA infusion. Both these increases were reduced by DXM and AG. The NMDA-induced striatal lesion was also reduced by both treatments. 4. Our results demonstrate that NMDA excitotoxic injury induces a delayed, sustained activation of a Ca-independent NOS activity. This activity is blocked by DXM and AG, strongly suggesting the involvement of iNOS. The fact that AG and DXM reduce the NMDA-elicited lesion suggests that iNOS contributes to the brain damage induced by excitotoxic insult.

Glutamate induces hydroxyl radical formation in vivo via activation of nitric oxide synthase in Sprague-Dawley rats.

It was recently reported that neuronal nitric oxide synthase (NOS) generates oxygen-derived free radicals in vitro at low concentrations of L-arginine. Using the microdialysis technique, we monitored both hydroxyl radical (.OH) and nitric oxide (.NO) formation in rat striatum perfused with glutamate (500 mM). .OH and .NO were quantitated in microdialysates by measuring the amounts of the non-enzymatic hydroxylation product of salicylate (2,3-dihydroxybenzoic acid) and the metabolites of .NO (nitrite + nitrate), respectively. .OH levels were dramatically increased during glutamate perfusion, while .NO generation was virtually abolished. .OH production was inhibited by the specific NOS blocker, NG-nitro-L-arginine methyl ester. This effect was not reversed but potentiated by L-arginine. Thus, it is likely that NOS generates oxygen-derived free radicals instead of .NO in brain subjected to highly excitotoxic conditions.

Deleterious Ca-independent NOS activity after oxidative stress in rat striatum.

The aim of this study was to assess whether oxidative stress induces deleterious NOS activity in the central nervous system (CNS). For this purpose, the mitochondrial toxin malonate, which promotes free radical production, was infused into the left striatum of rats. Forty-eight hours after injection, an increase in Ca-independent NOS activity was observed in the injected striatum. This increase was blocked by alpha-phenyl-tert-butyl-nitrone, a free radical scavenger, and by aminoguanidine, an inhibitor of NOS 2. Both these drugs reduced the malonate-induced striatal necrotic volume. These results suggest that in the CNS oxidative stress can induce a Ca-independent NOS, probably of type 2, which contributes to the lesion.