Post-finasteride syndrome : an emerging clinical problem

The presence of side effects during pharmacological treatment is unfortunately a quite common problem. In this review, we focused our attention on adverse events related to 5 alpha-reductase (5α-R) inhibitors (i.e., finasteride and dutasteride), approved for the treatment of benign prostatic hyperplasia and androgenetic alopecia (AGA). Although these drugs are generally well tolerated, many reports described adverse effects in men during treatment, such as sexual dysfunction and mood alteration. In addition, it has been also reported that persistent side effects may occur in some AGA patients. This condition, termed post-finasteride syndrome (PFS) is characterized by sexual side effects (i.e., low libido, erectile dysfunction, decreased arousal and difficulty in achieving orgasm), depression, anxiety and cognitive complaints that are still present despite drug withdrawal. Indeed, some national agencies (e.g., Swedish Medical Products Agency, the Medicines and Healthcare Products Regulatory Agency of UK and the U.S. Food and Drug Administration) required to include multiple persistent side effects within the finasteride labels. As here reported, these observations are mainly based on self-reporting of the symptomatology by the patients and few clinical studies have been performed so far. In addition, molecular mechanisms and/or genetic determinants behind such adverse effects have been poorly explored both in patients and animal models. Therefore, results here discussed indicate that PFS is an emerging clinical problem that needs to be further elucidated

L\ubfATTIVAZIONE DI TSPO E DI LXR STIMOLA LA NEUROSTEROIDOGENESI ED E\ubf PROTETTIVA IN UN MODELLO DI NEUROPATIA PERIFERICA DI RATTO DIABETICO

Diabetes mellitus (DM) is one of the first leading cause of death in most high-income countries and there is evidence that it is epidemic in many economically developing and newly industrialized nations. Beside these considerations, there are epidemiologic evaluations that show a dramatic increase of the number of diabetic and with impaired glucose tollerance patients in the next years. It is also important to underline that complications from diabetes, such as coronary artery and peripheral vascular disease, stroke, diabetic neuropathy, renal failure and blindness result in increasing disability, reduced life expectancy and enormous health costs for virtually every society. In particular, diabetic peripheral neuropathy can account for the 50-75% of non traumatic amputations; diabetes is therefore undoubtedly one of the most challenging health problems of our century. Recently, in an animal model of diabetic neuropathy (i.e. the rat raised diabetic with the injection of streptozotocin - STZ), it has been demonstrated that, in plasma and in sciatic nerve, the levels of neuroactive steroids are dramatically decreased. Promising results have been obtained in the same experimental model after treatments with progesterone, testosterone and their derivatives. The use of molecules able to increase the neuroactive steroid levels directly in the nervous system could be an interesting therapeutic approach because it may avoid the endocrine side effects usually observed after systemic treatment with steroids. In order to evaluate the possible neuroprotective effects mediated by an increase of neuroactive steroid levels in the peripheral nervous system, using the model of the STZ rat we here analyzed the effects of two different pharmacological approaches. In the first part of this thesis, we analyzed the effect of Ro5-4864, a ligand of the translocator protein of 18 kDa (TSPO). TSPO is a key regulator of neuroactive steroid synthesis and it is involved in the rate limitig step of this process (i.e. the entrance of cholesterol into the mitochondria). Indeed, it has been recently demonstrated that injection of TSPO ligands, like for instance Ro5-4864, was able to increase the capability of steroidogenic machinery in adrenal cortex, placenta, testis, ovary and glial cells. In the second part of this thesis, the effect of GW3965, a ligand of the Liver X Receptor (LXR), was analyzed. The rationale of this approach was based on the finding that cholesterol homeostasis is partially controlled by the regulation of different genes activated by LXR. Moreover, LXR activation is able to increase the steroidogenesis in the adrenal glands. At 2 months after STZ injection, two different protocols of TSPO ligand administration were assessed; in particular, animals were injected every 2 days or once a week whit the ligand (i.e., they received 16 or 4 injections). In the case of GW3965 treatment, at 2 months, diabetic animals were treated once a week (i.e., they received 4 injections). Functional and behavioural parameters, like for instance hot plate test and the recording of nerve conduction velocity, as well as biochemical parameters, like Na+,K+-ATPase activity and myelin protein gene expression, were analyzed in order to assess the neuroprotective effects of treatments. Moreover, in the case of Ro5-4864 treatment, the intra-epidermal nerve fiber density was quantified in the skin of the hindpaw footpad. Furthermore, in the experiment with GW3965 treatment, the levels of gene expression of LXR targets and of steroidogenic enzimes were determined in the sciatic nerve of diabetic animals. Data obtained showed that activation of TSPO and of LXR using their ligands once a week resulted in an increased levels of neuroactive steroids and protection of the sciatic nerve from neuropathy induced by diabetes. Conversely, 16 treatments with Ro5-4864 did not produced any protective effects. The increase of neuroactive steroid levels observed in the sciatic nerve after the treatment with the two molecules was in agreement with the improve of functional and biochimical parameters observed in diabetic rats, like for istance the thermal nociceptive activity, nerve conduction velocity, and Na+,K+-ATPase activity. Moreover, a recover of the intra-epidermal nerve fiber density after Ro5-4864 injection and a significant increase of the gene expression of some enzymes of steroidogenesis after GW3965 administration were detected. In contrast to what observed after TSPO activation, treatment with GW3965 did not consistently countered the diabetes-induced decrease in expression of the myelin protein zero observed in the sciatic nerve of STZ-treated animals. This observation suggests somewhat different mechanisms of action for LXR and TSPO ligands, an issue that it should be explored in the future. In conclusion the data here reported show that Ro5-4864 and GW3965 are neuroprotective in an animal model of diabetic peripheral neuropathy. Since a pharmacological treatment for this pathology is not yet available, data here provide suggest that these two molecules may represent promising candidates for the treatment of diabetic neuropathy

Physiopathological role of neuroactive steroids in the peripheral nervous system

Peripheral neuropathy (PN) refers to many conditions involving damage to the peripheral nervous system (PNS). Usually, PN causes weakness, numbness and pain and is the result of traumatic injuries, infections, metabolic problems, inherited causes, or exposure to chemicals. Despite the high prevalence of PN, available treatments are still unsatisfactory. Neuroactive steroids (i.e., steroid hormones synthesized by peripheral glands as well as steroids directly synthesized in the nervous system) represent important physiological regulators of PNS functionality. Data obtained so far and here discussed, indeed show that in several experimental models of PN the levels of neuroactive steroids are affected by the pathology and that treatment with these molecules is able to exert protective effects on several PN features, including neuropathic pain. Of note, the observations that neuroactive steroid levels are sexually dimorphic not only in physiological status but also in PN, associated with the finding that PN show sex dimorphic manifestations, may suggest the possibility of a sex specific therapy based on neuroactive steroids

Analysis of the finasteride treatment and its withdrawal in the rat hypothalamus and hippocampus at whole-transcriptome level

Purpose: As reported in patients treated for androgenetic alopecia with finasteride (i.e., a blocker of the enzyme 5 alpha-reductase) and in an animal model, side effects affecting sexual, psychiatric, neurological, and physical domains, may occur during the treatment and persist with drug suspension. The etiopathogenesis of these side effects has been poorly explored. Therefore, we performed a genome-wide analysis of finasteride effects in the brain of adult male rat. Methods: Animals were treated (i.e., for 20 days) with finasteride (1mg/rat/day). 24 h after the last treatment and 1 month after drug suspension, RNA sequencing analysis was performed in hypothalamus and hippocampus. Data were analyzed by differential expression analysis and Gene-Set Enrichment Analyses (GSEA). Results: Data obtained after finasteride treatment showed that 186 genes (i.e., 171 up- and 15 downregulated) and 19 (i.e., 17 up- and 2 downregulated) were differentially expressed in the hypothalamus and hippocampus, respectively. Differential expression analysis at the drug withdrawal failed to identify dysregulated genes. Several gene-sets were enriched in these brain areas at both time points. Conclusion: Some of the genes reported to be differentially expressed (i.e., TTR, DIO2, CLDN1, CLDN2, SLC4A5, KCNE2, CROT, HCRT, MARCKSL1, VGF, IRF2BPL) and GSEA, suggest a potential link with specific side effects previously observed in patients and in the animal model, such as depression, anxiety, disturbance in memory and attention, and sleep disturbance. These data may provide an important background for future experiments aimed at confirming the pathological role of these genes

Physiopathological role of the enzymatic complex 5α-reductase and 3α/β-hydroxysteroid oxidoreductase in the generation of progesterone and testosterone neuroactive metabolites

The enzymatic complex 5\u3b1-reductase (5\u3b1-R) and 3\u3b1/3\u3b2-hydroxysteroid oxidoreductase (HSOR) is expressed in the nervous system, where it transforms progesterone (PROG) and testosterone (T) into neuroactive metabolites. These metabolites regulate myelination, brain maturation, neurotransmission, reproductive behavior and the stress response. The expression of 5\u3b1-R and 3\u3b1-HSOR and the levels of PROG and T reduced metabolites show regional and sex differences in the nervous system and are affected by changing physiological conditions as well as by neurodegenerative and psychiatric disorders. A decrease in their nervous tissue levels may negatively impact the course and outcome of some pathological events. However, in other pathological conditions their increased levels may have a negative impact. Thus, the use of synthetic analogues of these steroids or 5\u3b1-R modulation have been proposed as therapeutic approaches for several nervous system pathologies. However, further research is needed to fully understand the consequences of these manipulations, in particular with 5\u3b1-R inhibitors

Allopregnanolone: An overview on its synthesis and effects

Allopregnanolone, a 3α,5α-progesterone metabolite, acts as a potent allosteric modulator of the γ-aminobutyric acid type A receptor. In the present review, the synthesis of this neuroactive steroid occurring in the nervous system is discussed with respect to physiological and pathological conditions. In addition, its physiological and neuroprotective effects are also reported. Interestingly, the levels of this neuroactive steroid, as well as its effects, are sex-dimorphic, suggesting a possible gender medicine based on this neuroactive steroid for neurological disorders. However, allopregnanolone presents low bioavailability and extensive hepatic metabolism, limiting its use as a drug. Therefore, synthetic analogues or a different therapeutic strategy able to increase allopregnanolone levels have been proposed to overcome any pharmacokinetic issues

Sex differences in steroid levels and steroidogenesis in the nervous system : Physiopathological role

The nervous system, in addition to be a target for steroid hormones, is the source of a variety of neuroactive steroids, which are synthesized and metabolized by neurons and glial cells. Recent evidence indicates that the expression of neurosteroidogenic proteins and enzymes and the levels of neuroactive steroids are different in the nervous system of males and females. We here summarized the state of the art of neuroactive steroids, particularly taking in consideration sex differences occurring in the synthesis and levels of these molecules. In addition, we discuss the consequences of sex differences in neurosteroidogenesis for the function of the nervous system under healthy and pathological conditions and the implications of neuroactive steroids and neurosteroidogenesis for the development of sex-specific therapeutic interventions

Altered methylation pattern of the SRD5A2 gene in the cerebrospinal fluid of post-finasteride patients: A pilot study

Context: Post-finasteride syndrome (PFS) occurs in patients with androgenic alopecia after suspension of the finasteride treatment, leading to a large variety of persistent side effects. Despite the severity of the clinical picture, the mechanism underlying the PFS symptoms onset and persistence is still unclear. Objective: To study whether epigenetic modifications occur in PFS patients. Methods: Retrospective analysis of a multicentric, prospective, longitudinal, case\u2013control clinical trial, enrolling 16 PFS patients, compared to 20 age-matched healthy men. Main outcomes were methylation pattern of SRD5A1 and SRD5A2 promoters and concentration of 11 neuroactive steroids, measured by liquid chromatography-tandem mass spectrometry, in blood and cerebrospinal fluid (CSF) samples. Results: SRD5A1 and SRD5A2 methylation analysis was performed in all blood samples (n = 16 PFS patients and n = 20 controls), in 16 CSF samples from PFS patients and in 13 CSF samples from controls. The SRD5A2 promoter was more frequently methylated in CSF of PFS patients compared to controls (56.3 vs 7.7%). No promoter methylation was detected in blood samples in both groups. No methylation occurred in the SRD5A1 promoter of both groups. Unmethylated controls compared to unmethylated SRD5A2 patients showed higher pregnenolone, dihydrotestosterone and dihydroprogesterone, together with lower testosterone CSF levels. Andrological and neurological assessments did not differ between methylated and unmethylated subjects. Conclusions: For the first time, we demonstrate a tissue-specific methylation pattern of SRD5A2 promoter in PFS patients. Although we cannot conclude whether this pattern is prenatally established or induced by finasteride treatment, it could represent an important mechanism of neuroactive steroid levels and behavioural disturbances previously described in PFS

Evaluation of neuroactive steroid levels by liquid chromatography-tandem mass spectrometry in central and peripheral nervous system : effect of diabetes

The nervous system is a target for physiological and protective effects of neuroactive steroids. Consequently, the assessment of their levels in nervous structures under physiological and pathological conditions is a top priority. To this aim, identification and quantification of pregnenolone (PREG), progesterone (PROG), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), dihydrotestosterone (DHT), 5aandrostan- 3a, 17b-diol (3a-diol), 17a- and 17b-estradiol (17a-E and 17b-E) by liquid chromatography and tandem mass spectrometry (LC\u2013MS/ MS) has been set up. After validation, this method was applied to determine the levels of neuroactive steroids in central (i.e., cerebral cortex, cerebellum and spinal cord) and peripheral (i.e., brachial nerve) nervous system of control and diabetic rats. In controls only the brachial nerve had detectable levels of all these neuroactive steroids. In contrast, 17a-E in cerebellum, 17a-E, 17b-E, DHP and THP in cerebral cortex, and 17a-E, 17b-E and DHP in spinal cord were under the detection limit. Diabetes, induced by injection with streptozotocin, strongly affected the levels of some neuroactive steroids. In particular, the levels of PREG, PROG and T in cerebellum, of PROG, T and 3a-diol in cerebral cortex, of PROG, DHTand 3a-diol in spinal cord and of PREG, DHP, THP, T, DHTand 3a-diol in brachial nerve were significantly decreased. In conclusion, the data here reported demonstrate that the LC\u2013MS/MS method allows the assessment of neuroactive steroids in the nervous system with high sensitivity and specificity and that diabetes strongly affects their levels, providing a further basis for new therapeutic tools based on neuroactive steroids aimed at counteracting diabetic neuropathy