Finasteride Inhibits the Disease-Modifying Activity of Progesterone in the Hippocampus Kindling Model of Epileptogenesis

1. Introduction

Acquired limbic epilepsy or temporal lobe epilepsy (TLE) is caused by diverse precipitating factors such as brain injury, stroke, infections, or prolonged seizures. The mechanisms underlying the development of acquired TLE are not very well understood. The term ‘epileptogenesis’ is used to describe the complex plastic changes in the brain that, following a precipitating injury, convert a normal brain into a brain debilitated by recurrent seizures [1,2]. There is an unmet need for drugs that truly prevent the development of epilepsy (antiepileptogenic agents) or alter its natural course, delaying the appearance or severity of epileptic seizures (disease-modifying agents) in people at risk. Progesterone (P) plays an important role in women with epilepsy [3,4]. P has long been known to have antiseizure activity in animal models [5–7] and in clinical studies [8–11]. Therefore, it has been suggested that women with epilepsy are prone to seizures in response to decreased levels of P during perimenstrual periods [12,13]. The antiseizure properties of P are due to its conversion in the brain to allopregnanolone (3α-hydroxy-5α-pregnane-20-one) and other structurally-related neurosteroids [6,7,14,15]. Recent studies show that neurosteroids are mainly present in principal neurons in many brain regions that are relevant to acquired TLE including the hippocampus [16]. Neurosteroids rapidly alter neuronal excitability through direct interaction with synaptic and extrasynaptic GABA-A receptors in the hippocampus [17]. Although they act on all GABA-A-receptor subtypes, they have pronounced effects on the extrasynaptic δ-subunit containing GABA-A receptors that mediate tonic inhibition [18]. Therefore, allopregnanolone and related 5α-reduced neurosteroids are powerful anticonvulsants in animal seizure models, and their synthetic analogs have been proposed for epilepsy therapy [19].

Neurosteroids are broad-spectrum anticonvulsants that confer seizure protection in various experimental models of evoked seizures in normal or healthy rats and mice [7,20–22]. They protect against seizures induced by GABA-A receptor antagonists, 6-Hz model, pilocarpine-induced limbic seizures, and seizures in kindled animals. Unlike benzodiazepines, tolerance to their actions does not occur during chronic administration in rats [19,23]. In addition to anticonvulsant activity, there is emerging evidence that neurosteroids play a role in regulating acquired epileptogenesis. Although the precise mechanisms underlying epileptogenesis remain unclear, epileptogenesis may involve an interaction of acute and delayed anatomic, molecular, and physiological events that are both complex and multifaceted. The initial precipitating factor activates diverse signaling events, such as inflammation, oxidation, apoptosis, neurogenesis and synaptic plasticity, which eventually lead to structural and functional changes in neurons [1,2]. These changes are eventually manifested as abnormal hyperexcitability and spontaneous seizures. In the rodent brain, the 5α-reductase type I and II isozymes convert P to dihydroprogesterone, ultimately leading to the production of allopregnanolone and related neurosteroids, which may retard epileptogenesis by the interruption of one or more of the above events. In addition to catalyzing the rate-limiting step in the reduction of P, both isoforms of the 5α-reductase enzyme are responsible for the conversion of testosterone and deoxycorticosterone to dihydroprogesterone and dihydrodeoxycorticosterone, respectively [13]. Finasteride, an irreversible inhibitor of 5α-reductase, blocks the synthesis of all 5α-reduced neurosteroids, including testosterone-derived androstanediol [24] and deoxycorticosterone-derived allotetrahydrodeoxycorticosterone (THDOC) [25] and thereby may affect epileptogenesis.

As a precursor of neurosteroids, P is an appealing agent for the intervention of epileptogenesis. Three observations support this contention. First, P’s inhibitory effect on kindling was observed in rats [26] and mice [27]. Second, following pilocarpine-induced status epilepticus, the neurosteroidogenic enzyme cytochrome P450 side chain cleavage (P450scc) was increased for several weeks, suggesting that it may be associated with the promotion of neurosteroidogenesis [28]. P450scc is a critical enzyme involved in the conversion of cholesterol into pregnenolone, a precursor for several neurosteroids. Third, inhibiting neurosteroid synthesis with finasteride accelerated the onset of spontaneous recurrent seizures [29], suggesting that endogenous neurosteroids play a role in restraining epileptogenesis. However, P has not been investigated widely for its potential disease-modifying activity in epileptogenic models. Using the mouse kindling model, we previously demonstrated that P inhibits the development of limbic epileptogenesis at doses that do not affect seizure expression and motor performance [27], indicating the disease-modifying activity of P. Kindling is a model of functional epileptogenesis, since seizures develop after repeated subthreshold stimulations applied in the hippocampus, amygdala or other brain regions, without induction of evident brain lesions [30]. However, the mechanisms underlying the protective effect of P against limbic epileptogenesis remain unclear.

In the present study, we sought to investigate the role of neurosteroids in the disease-modifying effect of P by using finasteride, a 5α-reductase inhibitor that blocks the synthesis of P-derived and 5α-reduced neurosteroids in the brain. Our results show for the first time that finasteride prevents P’s inhibition of hippocampal kindling epileptogenesis, indicating that P-derived neurosteroids such as allopregnanolone may inhibit epileptogenesis, thus confirming the new role of neurosteroids in limbic epileptogenesis and TLE.

2. Materials and Methods

3. Results

4. Discussion

The key observation of this study is that the disease-modifying effect of P in the kindling model of epileptogenesis is blocked by a pretreatment of finasteride, which is consistent with a protective role of 5α-reduced neurosteroids in limbic epileptogenesis. Finasteride inhibition of P’s kindling-retarding effect has not been reported previously. Finasteride is a 5α-reductase inhibitor that blocks the biosynthesis of endogenous neurosteroids such as allopregnanolone and related 5α-reduced steroids [32,33]. Consistent with animal studies, patients treated with finasteride had significantly reduced levels of neurosteroids [39]. Thus, it is suggested that inhibition of neurosteroids may accelerate the putative mechanisms that promote epileptogenesis in acquired TLE [2]. These results support the emerging notion that exogenous treatment with P delays the progression of acquired epileptogenesis [24,27].

However, there are certain caveats that must be considered in regards to the interpretation of these findings. First, the results apply only to the kindling model and protocol used in this study. Second, this study supports the disease-modifying potential of P to the extent that kindling development represents a model of limbic epileptogenesis. However, the relationship of this model to human TLE is not well validated. Third, the relevance of the dosages of P that were used in this study in relation to those required for true antiepileptogenic effect is uncertain. Fourth, we did not test P during earlier stages of kindling (stage 3) to ascertain whether this effect of P is because of antiseizure activity. Finally, it is likely that the effect of P on epileptogenesis may be model specific and false positive results are likely in the kindling model (e.g. levetiracetam) [40]. Therefore, further studies using spontaneous seizure models following kainic acid or pilocarpine-induced SE are needed to confirm antiepileptogenic efficacy of P. Nevertheless, our findings from multiple studies provide compelling evidence that P has disease-modifying or antiepileptogenic effect in the kindling model. To date, we tested P in three distinct kindling epileptogenesis protocols: (a) chronic P therapy in the conventional kindling [27]; (b) acute P therapy in the rapid kindling [2]; and (c) P therapy 30-min prior to kindling sessions (current study). In these studies, P showed kindling-retarding effect, especially at low doses that do not affect electrographic AD expression (Fig.1B), which is consistent with disease-modifying activity. Despite concerns on validity and generalization to other models, kindling is a very useful model for rapid identification of disease-modifying drugs.

P targets multiple molecular and cellular mechanisms relevant to epileptogenesis, including signaling cascades of inflammation, apoptosis, neurogenesis, and synaptic plasticity [41]. Therefore, P may be a natural disease-modifying agent. In the present study, P significantly suppressed the rate of development of kindled seizure activity. P’s effects on kindling epileptogenesis are sensitive to blockade by finasteride, which inhibits both type I and type II 5α-reductase isoenzymes that convert P into neurosteroids [32–36], predominantly allopregnanolone, that may mediate P’s antiepileptogenic effects. Given during kindling development, finasteride accelerated the kindling acquisition, supporting previous studies that indicate the significant role of endogenous neurosteroids in epileptogenesis in post-status epilepticus models [28,29]. Thus, it is likely that endogenous neurosteroids modulate the rate of epileptogenesis. The potential mechanisms of neurosteroid interruption of epileptogenesis in the hippocampus remain unclear. Neurosteroids such as allopregnanolone are potent positive allosteric modulators of synaptic and extrasynaptic GABA-A receptors that mediate phasic and tonic inhibition in the hippocampus [17,19]. Thus, neurosteroid augmentation of tonic inhibition in the hippocampus could inhibit the spread of the seizure discharge from the hippocampus focus and thereby suppress the development rate of behavioral kindled seizure activity without affecting the focal electrographic discharges (Fig.5). Increased tonic inhibition by allopregnanolone is shown to impair the N-methyl-D-asparate receptor-mediated excitability in the hippocampus [42]. It is likely that such a mechanism may underlie the neurosteroid’s disease-modifying effects in the kindling model.

In addition, there are other limitations of the kindling and finasteride protocol employed in this study. Although the estrous cycle has minimal effects on kindling seizures [43], there is evidence that kindling produces acyclicity and an increase in testosterone levels, seemingly due to polycystic ovaries [44]. Both conditions could affect seizures. It is possible that finasteride influences on this because it can block all 5α-reduced neurosteroids, including testosterone-derived androstanediol [24]. However, it is highly unlikely that seizure protection is independent of an effect on allopregnanolone biosynthesis because we utilized an exogenous P treatment protocol, which results in a massive increase in allopregnanolone in the brain. Apart from blocking the conversion of P to allopregnanolone, finasteride also inhibits the production of other 5α-reduced, anticonvulsant neurosteroids, including androstanediol and THDOC, which are derived from testosterone and deoxycorticosterone, respectively [24,25, 32,33]. The finasteride treatment protocol employed in this study offers little specificity regarding its inhibition of specific neurosteroid synthesis or the brain region involved in neurosteroidogenesis. Therefore, it is likely that the action of finasteride may be due to its ability to reduce all 5α-reduced neurosteroids in the brain.

Neurosteroid biosynthesis is regulated by various endogenous signals. Recent evidence indicates that neurosteroids are mainly present in principal neurons in the hippocampus and neocortex [16]. The biosynthesis of neurosteroids is controlled by the translocator protein (TSPO), formerly called peripheral or mitochondrial benzodiazepine receptor [45]. Activation of TSPO by endogenous signals and ligands facilitates the intramitochondrial flux of cholesterol and thereby promotes neurosteroid synthesis. Thus, dysregulation of neurosteroid synthesis may play a role in epileptogenesis. This premise has been demonstrated in a post-status epilepticus model of epileptogenesis [28,29]. In this study, the rate of kindling was accelerated by finasteride alone, which may reflect independent effects of finasteride on epileptogenesis due to inhibition of endogenous neurosteroid synthesis. Additional studies are needed to investigate whether augmentation of endogenous neurosteroid synthesis represents a unique strategy for preventing epileptogenesis.

The ovarian cycle regulates the seizure sensitivity in females by the modulation of GABA-A receptor plasticity [46,47]. In the present study, animals were not checked for estrous cycle stages due to a chronic experimental design that precluded grouping of animals based on their ovarian cycle. Nevertheless, the results of this study suggest that the finasteride blockade of the disease-modifying effect of P is due to the inhibition of brain neurosteroid synthesis and also neurosteroids derived from the conversion of ovarian P to allopregnanolone. There is pilot evidence of gender differences in neurosteroid sensitivity [48,49]. Clearly, more needs to be learned about the impact of estrous cycle and ovarian neurosteroids (gonadectomy) on kindling measurements using a large group size. These studies are underway, including the gender differences in P and neurosteroid’s effects on kindling epileptogenesis.

In this study, finasteride has little influence on acute seizure susceptibility in fully-kindled mice at the low dosage used in the experiments. This is very interesting observation given the effect of neurosteroids on acute seizures in epilepsy models. In the absence of exogenous P treatment, it is likely that finasteride inhibition of neurosteroid biosynthesis lead to little changes in the seizure expression in fully-kindled mice exhibiting maximal stage 5 seizures. Alternately, the time course of evaluation at 1 h post finasteride administration may be another factor in this paradoxical finding. However, finasteride is able to alter the rate of epileptogenesis when given during kindling development. Finasteride inhibits 5α-reductase enzyme that exists in two isoforms, designated as types I and II, which convert P into 5α-dihydroprogesterone and ultimately lead to the production of allopregnanolone. The type I isoenzyme is more prominent in the brain, whereas type II exists mainly in androgen-sensitive glandular tissues such as the prostate [33–35]. In human tissues, finasteride inhibits both isoforms but is more potent on the type II isoenzyme than the type I, thus conferring prostatic selectivity. However, in the rodent models, finasteride is also a highly potent inhibitor of the type I enzyme. Because finasteride does not select between the rodent isoenzymes, our studies do not provide an answer on the relative role of the two isoenzymes in neurosteroid biosynthesis, especially in rodents.

Finasteride treatment has led to exacerbation of seizures in epilepsy rats [50] and accelerated the rate of epileptogenesis in mice (Fig.3). However, there is little evidence that finasteride causes seizures in humans who do not have epilepsy or exacerbates epileptogenesis in persons at high risk for developing epilepsy. Finasteride is used clinically for the treatment of benign prostatic hypertrophy and male pattern baldness. There is published evidence of seizure exacerbation as an adverse event of finasteride treatment [51]. Finasteride provoked seizures in a woman with epilepsy who experienced an increase in seizure frequency and severity during finasteride use. Therefore, in the clinical setting, finasteride should be used with great caution in patients with epilepsy because of its potential to exacerbate seizures or epileptogenesis in patients at risk for developing epilepsy.

In conclusion, our results demonstrate for the first time that pretreatment with finasteride blocks the protective ability of P to retard kindling-induced limbic epileptogenesis. Thus, the disease-modifying or antiepileptogenic effect of P may be mediated through a mechanism involving neurosteroids, which are emerging endogenous agents that can retard acquired epileptogenesis [2,26]. Thus, neurosteroids such as allopregnanolone may possess antiepileptogenic properties. Finasteride inhibition of neurosteroidogenesis may influence seizure susceptibility in persons with TLE or related epileptogenic risk factors.