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Progesterone, 5a-dihydropogesterone and allopregnanolone's effects on seizures: A review of animal and clinical studies

Open ArchivePublished:October 28, 2018DOI:https://doi.org/10.1016/j.seizure.2018.10.012

      Highlights

      • Progesterone's anti-seizure effects are mediated by its metabolites: DHP and ALLO.
      • Progesterone, DHP and ALLO suppress non-absence seizures in animals.
      • Progesterone, ALLO and an ALLO analog suppress seizures in clinical trials.
      • Their anti-seizure effects are mostly independent of its genomic receptors.
      • Rational drug design may mitigate progesterone's hormonal side effects.

      Abstract

      The anti-seizure effects of progesterone family compounds have long been known. Over the years, however, most studies have focused on progesterone and on its secondary metabolite allopregnanolone (ALLO), with less attention being paid to its primary metabolite 5a-dihydroprogesterone (DHP).
      Here we review animal and clinical studies related to the anti-seizure effects of progesterone and its 5a neuroactive metabolites, including DHP and ALLO.
      Progesterone and its reduced metabolites all have demonstrated seizure-suppression effects in animal models – except in models of absence seizures – with the common side effects of sedation and ataxia. Progesterone and ALLO have also shown anti-seizure effects in clinical trials. A large Phase III trial has revealed that female patients with premenstrual exacerbations of seizures benefit most from progesterone therapy. A liquid suspension of ALLO has also been tested in patients with supra-refractory status epilepticus with some success in a small phase II trial. ALLO's C3 methyl analog ganaxolone is under development as an anti-seizure drug.
      Progesterone's anti-seizure effects are mostly independent of its genomic receptors and are, in large part, due to its active metabolites. ALLO is a potent allosteric modulator of GABA receptors. Other membrane receptors are thought to be involved in the DHP's anti-seizure actions, but their exact nature is not yet known.
      Potential drawbacks to the development of progesterone family compounds as anti-seizure drug are their endocrine effects. These compounds might form a basis for the future development of novel anti-seizure drugs, however, with hormonal side effects being mitigated through rational drug design.

      Keywords

      1. Progesterone and seizures

      Epilepsy, characterized by spontaneous and recurrent seizures, currently affects 1% of the world population [
      • Banerjee P.N.
      • Filippi D.
      • Hauser W.A.
      The descriptive epidemiology of epilepsy – a review.
      ]. Despite many available therapeutic options, 1/3 of patients do not achieve seizure control [
      • Schmidt D.
      • Löscher W.
      Drug resistance in epilepsy: putative neurobiologic and clinical mechanisms.
      ]. Thus, searching for new and effective anti-epileptic drugs remains a central theme of epilepsy research. Among the many different candidates for drug development, the progesterone family emerges as a potential therapeutic option.
      Progesterone (pregn-4-ene-3,20-dione, P4) is a neurosteroid hormone synthesized in both males and females. In females, there is a clear link between progesterone, the female fertility cycle and seizures. Generally speaking, seizure thresholds are high when blood progesterone levels are high, and low when blood progesterone is low or falling [
      • Edwards H.E.
      • Burnham W.M.
      • Mendonca A.
      • Bowlby D.A.
      • MacLusky N.J.
      Steroid hormones affect limbic afterdischarge thresholds and kindling rates in adult female rats.
      ]. This has suggested the development of progesterone-like compounds for epilepsy therapy.
      The present review will discuss progesterone and its 5a metabolites in animal and clinical trials related to seizure therapy.

      2. Synthesis and metabolism

      The synthesis of progesterone synthesis occurs in the mitochondrial membranes of body cells and in the brain [
      • Corpéchot C.
      • Young J.
      • Calvel M.
      • Wehrey C.
      • Veltz J.N.
      • Touyer G.
      • et al.
      Neurosteroids: 3 alpha-hydroxy-5 alpha-pregnan-20-one and its precursors in the brain, plasma, and steroidogenic glands of male and female rats.
      ]. As the first step, cholesterol is converted to pregnenolone by CYP11A1 though C20 carbon side-chain cleavage. Pregnenolone is then further reduced at the C3 position by 3b-hydroxysteroid dehydrogenase to produce progesterone [
      • Hill M.
      • Vrbíková J.
      • Zárubová J.
      • Kancheva R.
      • Velíková M.
      • Kancheva L.
      • et al.
      The steroid metabolome in lamotrigine-treated women with epilepsy.
      ].
      The further metabolism of progesterone takes place via multiple pathways, which are different in the peripheral and central compartments. In the peripheral compartment, the 5 beta reduction pathway predominates [
      • Atherden L.M.
      Progesterone metabolism; investigation of the products of metabolism with human liver in vitro.
      ,
      • Havlíková H.
      • Hill M.
      • Kancheva L.
      • Vrbíková J.
      • Pouzar V.
      • Cerny I.
      • et al.
      Serum profiles of free and conjugated neuroactive pregnanolone isomers in nonpregnant women of fertile age.
      ], while in the central compartment the 5 alpha pathway is predominant in rats [
      • Hanukoglu I.
      • Karavolas H.J.
      • Goy R.W.
      Progesterone metabolism in the pineal, brain stem, thalamus and corpus callosum of the female rat.
      ,
      • Karavolas H.J.
      • Hodges D.
      • O’Brien D.
      Uptake of (3H)progesterone and (3H)5alpha-dihydroprogesterone by rat tissues in vivo and analysis of accumulated radioactivity: accumulation of 5alpha-dihydroprogesterone by pituitary and hypothalamic tissues.
      ], monkeys [
      • Billiar R.B.
      • Little B.
      • Kline I.
      • Reier P.
      • Takaoka Y.
      • White R.J.
      The metabolic clearance rate, head and brain extractions, and brain distribution and metabolism of progesterone in the anesthetized, female monkey (Macaca mulatta).
      ], and humans [
      • Bixo M.
      • Andersson A.
      • Winblad B.
      • Purdy R.H.
      • Bäckström T.
      Progesterone, 5alpha-pregnane-3,20-dione and 3alpha-hydroxy-5alpha-pregnane-20-one in specific regions of the human female brain in different endocrine states.
      ].
      In the 5a reduction pathway, progesterone is first converted to 5a-dihydroprogesterone (DHP) by 5a-reductase which irreversibly reduces the double bond between C4 and C5. 5a-dihydroprogesterone is then converted by 3a-hydroxysteroid dehydrogenase to allopregnanolone (3a,5a-tetrahydroprogesterone, ALLO) by changing the ketone group on C3 to an alcohol group. This step is reversible.
      In the rat brain, most progesterone is metabolized to DHP within 10 minutes and DHP is metabolized to ALLO within 30 minutes [
      • Karavolas H.J.
      • Hodges D.
      • O’Brien D.
      Uptake of (3H)progesterone and (3H)5alpha-dihydroprogesterone by rat tissues in vivo and analysis of accumulated radioactivity: accumulation of 5alpha-dihydroprogesterone by pituitary and hypothalamic tissues.
      ]. Finasteride inhibits 5a-reductase [
      • Kokate T.G.
      • Banks M.K.
      • Magee T.
      • Yamaguchi S.
      • Rogawski M.A.
      Finasteride, a 5alpha-reductase inhibitor, blocks the anticonvulsant activity of progesterone in mice.
      ], whereas. Indomethacin and medroxyprogesterone acetate inhibit of3a-HSD [
      • Penning T.M.
      • Carlson K.E.
      • Sharp R.B.
      Affinity-labelling of the anti-inflammatory drug and prostaglandin-binding site of 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol with 17 beta- and 21-bromoacetoxysteroids.
      ]. These enzyme inhibitors play important roles in studies of the progesterone family (Fig. 1).
      Fig. 1
      Fig. 1The metabolism pathway of progesterone. Enzyme inhibitors are shown in red. CYP11A4: cytochrome P45011A4 enzyme, 3beta-HSD: 3beta-hydroxysteroid dehydrogenase, 3alpha-HSD: 3alpha-hydroxysteroid dehydrogenase, MPA: medroxyprogesterone acetate.

      3. Concentrations in body and brain

      Concentrations of progesterone in the peripheral compartment vary widely in females during the estrous cycle, with lower levels and less variation being seen in males [
      • Van der Molen H.J.
      • Groen D.
      Determination of progesterone in human peripheral blood using gas-liquid chromatography with electron capture detection.
      ]. In the central compartment, progesterone and its metabolites fluctuate according to circadian rhythms [
      • Corpéchot C.
      • Collins B.E.
      • Carey M.P.
      • Tsouros A.
      • Robel P.
      • Fry J.P.
      Brain neurosteroids during the mouse oestrous cycle.
      ], as well as (in females) varying with the estrous cycle. These variations have profound effects on cognition and behavior [
      • Corpéchot C.
      • Collins B.E.
      • Carey M.P.
      • Tsouros A.
      • Robel P.
      • Fry J.P.
      Brain neurosteroids during the mouse oestrous cycle.
      ,
      • McEwen B.S.
      • Woolley C.S.
      Estradiol and progesterone regulate neuronal structure and synaptic connectivity in adult as well as developing brain.
      ]. Endogenous progesterone and its 5a metabolites tend to accumulate in the brain [
      • Karavolas H.J.
      • Hodges D.
      • O’Brien D.
      Uptake of (3H)progesterone and (3H)5alpha-dihydroprogesterone by rat tissues in vivo and analysis of accumulated radioactivity: accumulation of 5alpha-dihydroprogesterone by pituitary and hypothalamic tissues.
      ,
      • Irwin R.W.
      • Solinsky C.M.
      • Loya C.M.
      • Salituro F.G.
      • Rodgers K.E.
      • Bauer G.
      • et al.
      Allopregnanolone preclinical acute pharmacokinetic and pharmacodynamic studies to predict tolerability and efficacy for Alzheimer's disease.
      ], with the peak concentrations being much higher than concentrations in the periphery [
      • Corpéchot C.
      • Collins B.E.
      • Carey M.P.
      • Tsouros A.
      • Robel P.
      • Fry J.P.
      Brain neurosteroids during the mouse oestrous cycle.
      ].
      Following exogenous administration, brain concentrations of progesterone are 3 times higher than concentrations in the periphery. After intravenous (IV) injections of progesterone and 5a-DHP in rats, for instance, both compounds are found to accumulate to high levels in the central department. The highest levels being found in the hypothalamus and anterior pituitary region, with very little being found in the cerebral cortex [
      • Karavolas H.J.
      • Hodges D.
      • O’Brien D.
      Uptake of (3H)progesterone and (3H)5alpha-dihydroprogesterone by rat tissues in vivo and analysis of accumulated radioactivity: accumulation of 5alpha-dihydroprogesterone by pituitary and hypothalamic tissues.
      ].
      A comprehensive review of progesterone's fate in the body may be found in Taraborrelli [
      • Taraborrelli S.
      Physiology, production and action of progesterone.
      ] and a comprehensive pharmacokinetic study is presented by Irwin et al. [
      • Irwin R.W.
      • Solinsky C.M.
      • Loya C.M.
      • Salituro F.G.
      • Rodgers K.E.
      • Bauer G.
      • et al.
      Allopregnanolone preclinical acute pharmacokinetic and pharmacodynamic studies to predict tolerability and efficacy for Alzheimer's disease.
      ].

      4. Anti-seizure effects of progesterone

      4.1 Animal studies

      Table 1 summarizes the studies done on progesterone in different animal seizure models. Seyle was perhaps the first to report the anti-seizure effects of progesterone, administering progesterone to pentylenetetrazole (PTZ)-treated immature male rats in 1942 [
      • Selye H.
      The antagonism between anesthetic steroid hormones and pentamethylenetetrazol (Metrazol).
      ]. Later, these effects were confirmed by others, using both male and female animals, and in a variety of models, including the amygdala-kindling model [
      • Lonsdale D.
      • Nylen K.
      • McIntyre Burnham W.
      The anticonvulsant effects of progesterone and its metabolites on amygdala-kindled seizures in male rats.
      ,
      • Lonsdale D.
      • Burnham W.M.
      The anticonvulsant effects of progesterone and 5alpha-dihydroprogesterone on amygdala-kindled seizures in rats.
      ,
      • Mohammad S.
      • Abolhassan A.
      • Pourgholami M.H.
      Evaluation of the anticonvulsant profile of progesterone in male amygdala-kindled rats.
      ], the hippocampal-kindling model [
      • Jeffrey M.
      • Lang M.
      • Gane J.
      • Chow E.
      • Wu C.
      • Zhang L.
      Novel anticonvulsive effects of progesterone in a mouse model of hippocampal electrical kindling.
      ], the PTZ model in mature rats [
      • Kokate T.G.
      • Banks M.K.
      • Magee T.
      • Yamaguchi S.
      • Rogawski M.A.
      Finasteride, a 5alpha-reductase inhibitor, blocks the anticonvulsant activity of progesterone in mice.
      ,
      • Akula K.K.
      • Dhir A.
      • Kulkarni S.K.
      Effect of various antiepileptic drugs in a pentylenetetrazol-induced seizure model in mice.
      ,
      • Craig C.R.
      Anticonvulsant activity of steroids: separability of anticonvulsant from hormonal effects.
      ,
      • Reddy D.S.
      • Castaneda D.C.
      • O’Malley B.W.
      • Rogawski M.A.
      Anticonvulsant activity of progesterone and neurosteroids in progesterone receptor knockout mice.
      ], and the kainic acid model [
      • Frye C.A.
      • Scalise T.J.
      Anti-seizure effects of progesterone and 3alpha, 5alpha-THP in kainic acid and perforant pathway models of epilepsy.
      ]. Most studies have reported an ED50 higher than 50 mg/kg, and have also reported sedation as a common side effect. In the kindling model, progesterone suppresses the generalized convulsions, but only partially suppresses focal discharge even at high doses [
      • Jeffrey M.
      • Lang M.
      • Gane J.
      • Chow E.
      • Wu C.
      • Zhang L.
      Novel anticonvulsive effects of progesterone in a mouse model of hippocampal electrical kindling.
      ] [
      • Albright P.S.
      • Burnham W.M.
      Development of a new pharmacological seizure model: effects of anticonvulsants on cortical- and amygdala-kindled seizures in the rat.
      ].
      Table 1Progesterone as an anti-seizure drug in animal studies. PTZ: pentylenetetrazole, I.P.: intraperitoneal, S.C.: subcutaneous, P.O.: oral route, ED50: effective dose 50.
      AuthorSubjectsSeizure ModelTreatmentResults
      Selye (1942)White rats, male, immature, 45 gPTZ 2 doses of ∼70 mg/kg each, S.C.Progesterone (110 mg/kg, I.P.)Increase seizure threshold

      Adverse effects: sedation, anesthesia
      Craig (1966)Swiss-Webster mice, male and female, 20–30 gPTZ 85 mg/kg, S.C.Progesterone (0–1000 mg/kg, P.O.)Suppress seizures ED50 = 200 mg/kg

      Adverse effects: sedation, motor impairment
      Mohammad et al (1998)White rats, male, 260–300 gAmygdala kindlingProgesterone (0, 10, 30, 60, 75, 90 mg/kg, I.P.)Suppress generalized seizures at 75 mg/kg

      Adverse effects: sedation
      Kokate et al (1999)NIH Swiss mice, male, 25–30 gPTZ, 85 mg/kg, S.C.Progesterone (50–200 mg/kg, I.P.)Suppress clonic seizures ED50 = 94 mg/kg

      Adverse effect: sedation
      Frye & Saclise, (2000)Ovariectomized Long-Evans rats, 55 days old upon arrivalKainic acid, 32 mg/kg, S.C.Progesterone (0, 4, 8 mg/kg, S.C.)Decrease the duration of partial and full seizures at 4 and 8 mg/kg
      Van Luijtelaar et al. (2001)WAG/Rij mice, femaleWAG/Rij mice, genetic absence modelProgesterone (20 mg/kg, 30 mg/kg, I.P.)Increase the number and duration of spike-wave discharge
      Lonsdale & Burnhan (2003)Wistar rats, female, 210–260 gAmygdala kindlingProgesterone dose-response (0, 10, 20, 40, 120, 160 mg/kg, S.C.)

      Progesterone time-response (2.5, 5, 10, 15, 20, 40, 80, or 160 min S.C.)
      Suppression of generalize seizures (ED50= 103 mg/kg) and focal seizures (Rmax = 20%).

      Suppression of generalized seizures reached 100% 20 min post-treatment

      Adverse effects: sedation
      Reddy et al (2004)Adult C57BL6/129SvEv mice, male and female, 25–30 gPTZ, 85 mg/kg, S.C.Progesterone (10–150 mg/kg, I.P.)Suppression of clonic seizures male:

      ED50 = 106 mg/kg female:

      ED50= 70 mg/kg

      Adverse effects: sedation, motor impairment
      Lonsdale et al (2006)Wistar rats, male, 300–400 gAmygdala kindlingProgesterone dose-response (0, 40, 80, 120, 160 mg/kg, S.C.)

      Progesterone time-response (10, 20, 40, 80, or 160 min S.C.)
      Suppress generalize seizures (ED50= 65.3 mg/kg) and focal seizures (ED50 = 114 mg/kg).

      Suppression of generalized seizures at 100% and suppression of focal seizures at 37.5% at 40 min post-treatment

      Adverse effects: sedation
      Akula et al (2009)Albino mice, Laka strain, mice, 22–30 gPTZ I.V. timed infusionProgesterone (20–80 mg/kg, S.C.)Increase threshold for tonic extensor seizure by 50% at 30 mg/kg
      Jeffrey et al. (2014)C57 black mice, male, 6–10 monthsHippocampal kindlingProgesterone (10, 35, 100, 160 mg/kg)Suppress generalized seizures and reduce the duration of focal seizures in a dose-dependent manner
      Zhong (2015)Wistar rats, female, 50 days oldAmygdala kindlingProgesterone (0–160 mg/kg, S.C.)Only suppress generalized seizures in 20% subjects at 100 mg/kg and above, adverse effect: ataxia, respiratory depression
      Compared to the classical anti-seizure drugs, progesterone is not very potent. Akula et al. compared progesterone with other anti-seizure drugs in the intravenous PTZ model in mice and concluded that progesterone is more potent than tiagabine, GABA, adenosine, gabapentin and ethanol, but less potent than triazolam, clonazepam, diazepam, phenobarbital, carbamazepine and phenytoin [
      • Akula K.K.
      • Dhir A.
      • Kulkarni S.K.
      Effect of various antiepileptic drugs in a pentylenetetrazol-induced seizure model in mice.
      ]. Progesterone is also less potent than (+)MK-801 and diazepam in protecting against picrotoxin-, bicuculline- and NMDA-induced lethality [
      • Członkowska A.I.
      • Krzaścik P.
      • Sienkiewicz-Jarosz H.
      • Siemiatkowski M.
      • Szyndler J.
      • Bidziński A.
      • et al.
      The effects of neurosteroids on picrotoxin-, bicuculline- and NMDA-induced seizures, and a hypnotic effect of ethanol.
      ].
      Progesterone has also shown pro-seizure effects in one animal model, the WAG/Rij model of absence seizures. In this model, 20 mg/kg of progesterone increased the number and duration of the spike-wave discharges [
      • van Luijtelaar G.
      • Budziszewska B.
      • Jaworska-Feil L.
      • Ellis J.
      • Coenen A.
      • Lasoń W.
      The ovarian hormones and absence epilepsy: a long-term EEG study and pharmacological effects in a genetic absence epilepsy model.
      ]. The pro-seizure action was probably due to a progesterone metabolite, not to progesterone itself, since the effect was attenuated by blocking the 5a hydroxylation pathway with finasteride [
      • van Luijtelaar G.
      • Budziszewska B.
      • Tetich M.
      • Lasoń W.
      Finasteride inhibits the progesterone-induced spike-wave discharges in a genetic model of absence epilepsy.
      ]. Progesterone's secondary metabolite, ALLO is a known GABA agonist [
      • Hogenkamp D.J.
      • Tahir S.H.
      • Hawkinson J.E.
      • Upasani R.B.
      • Alauddin M.
      • Kimbrough C.L.
      • et al.
      Synthesis and in vitro activity of 3 beta-substituted-3 alpha-hydroxypregnan-20-ones: allosteric modulators of the GABAA receptor.
      ], and most GABA agonists exacerbate absence seizures [
      • Snead O.C.
      Basic mechanisms of generalized absence seizures.
      ].

      4.2 Clinical studies

      Clinical studies with progesterone have usually been done in women with catamenial epilepsy. In “catamenial” epilepsy, seizures tend to cluster during specific phases of the menstrual cycles, such as ovulation and menstruation [
      • Zimmerman A.W.
      • Holden K.R.
      • Reiter E.O.
      • Dekaban A.S.
      Medroxprogesterone acetate in the treatment of seizures associated with menstruation.
      ]. Herzog has proposed a scheme that classifies these seizures into 3 categories: 1) C1 or the “perimenstural” pattern involves seizures that occur around day 0 of menstrual cycle, a period that is characterized by low progesterone levels; 2) C2 or the “periovulatory” pattern involves seizures that occur around Day10–14 of the cycle, a period that corresponds to a physiological surge in estrogen and low progesterone; and 3) C3 or the “inadequate luteal” pattern involves seizures that occur in the luteal phase. Normally progesterone is high during this phase, but women with this pattern do not experience the normal progesterone surge, making their luteal phases “inadequate” [
      • Herzog A.G.
      • Klein P.
      • Ransil B.J.
      Three patterns of catamenial epilepsy.
      ].
      Table 2 summarizes clinical studies on progesterone and its analogs as anti-seizure therapy. As an established contraceptive, progesterone has a number of commercially available analogs and formulations – each with a slightly different spectrum of activity and potency – which has greatly facilitated the clinical studies of progesterone.
      Table 2Progesterone and its analogs as anti-seizure drugs in clinical studies. RCT: randomized clinical trials, P.O.: oral route, I.M.: intramuscular injection, I.V.: intravenous injection, BID: twice daily, TID: three times daily.
      AuthorPatientSeizure Type(s)MedicationsResults
      Dana-Haeri & Richens (1983)Double-blind, placebo-controlled RCT

      N = 9, 20–30 y/o,
      Tonic-clonic seizures and/or partial seizures with catemenial exercerbationOn day 5–21 of each menstrual cycle, the patient will receive either

      1. 5 mg Noresthisterone, TID

      2. 350ug Noresthisterone, TID

      Placebo
      No decrease in seizure frequency
      Mattson et al (1984a)Open clinical trial

      N = 14, adult women
      13 patients with complex partial seizures

      1 patient with absence seizures
      Prior medications plus

      1. Provera®(in 8 patients, 10 mg q2-4d, P.O.)

      Depo-Provera (in 6 patients, 120–150 mg I.M.)
      39% reduction in seizure frequency (3 patients withdrew from study) Adverse effects: amenorrhea, spotting
      Backstrom. et al (1984)Open clinical study

      N = 7, women, 22–43 y/o
      Complex partial seizures with one distinct focus, selected for greater than 1 epileptic discharge per 5 min of EEG recordingPrior medications plus

      Progesterone, I.V. 0.5–3.0 mg bolus plus 4–12 mg/hr infusion to achieve luteal phase progesterone levels (29 ng/ml)
      Significant reduction in frequency of epileptic discharges in 4 out of 7 patients
      Herzog (1986)Open clinical trial

      N = 8, women, 16–41 y/o
      Complex partial seizures (catamenial pattern)Prior medications plus

      50–400 mg progesterone BID during periods of high seizure frequency – achieved luteal phase levels of 5–25 ng/ml
      68% reduction in seizure frequency Adverse effects: transient fatigue, depression in 50% of patients
      Herzog (1995)Open clinical trial

      N = 25, women, 18–40 y/o
      Complex partial seizures-13/25 patients also experienced secondarily generalized seizures (catamenial pattern)Prior medications plus

      200 mg progesterone lozenges TID during periods of high seizure frequency – achieved luteal phase levels of 5–25 ng/ml
      55% reduction in seizure frequency Adverse effects: asthenia, depression (2/25 patients)
      Herzog (1999)Open clinical trial

      N= 15, women from 1995 trial who remained on cyclic progesterone therapy
      Complex partial seizures (catamenial pattern)Prior medications plus

      200 mg progesterone lozenges TID during periods of high seizure frequency – achieved luteal phase levels of 5–25 ng/ml
      62% reduction in seizure frequency
      Herzog et al (2012)Double-blind, placebo-controlled, phase III, multicenter RCT,

      N= 294, women, 13–45 y/o
      Intractable partial seizures (catamenial and non-catamenial pattern)Prior medication plus 200 mg progesterone lozenges TID on day 14–21 of menstrual cycleNo significant difference in responding rates between treatment and control groups, post hoc analysis revealed that the level of perimenstrual exacerbation is a significant predictor of responding rate
      Najafi et al. (2013)Double-blind RCT,

      N = 38 women, 18–45 y/o
      Complex partial seizures, secondarily generalized seizures, or primary generalized seizures (catamenial pattern)Prior medications plus 40 mg progesterone tablets (Mejestrol) BID on day 15–25 of menstrual cycle81% reduction in seizure frequency
      Motta et al (2013)RCT, 36 women, 20–40 y/oPrimary generalized tonic-clonic seizures, complex/simple partial seizures with and without generalized seizures, myoclonic seizures with primary generalized tonic-clonic seizures (catamenial pattern)Prior medications plus 25 mg progesterone sublingual tablets BID on day 16–25 of menstrual cycle26/36 patients experienced a reduction of seizures
      One of the first attempts to treat drug intractable epilepsy with a form of progesterone occurred in an 8 year old girl, who had frequent seizures prior to her menstrual period [
      • Zimmerman A.W.
      • Holden K.R.
      • Reiter E.O.
      • Dekaban A.S.
      Medroxprogesterone acetate in the treatment of seizures associated with menstruation.
      ]. Zimmerman et al. reasoned that if it were possible to stop her menstrual cycle and it might also be possible stop her seizures. Medroxyprogesterone acetate (MPA), a potent contraceptive with the brand name Provera, was prescribed in both oral form and intramuscular injection. The girl remained seizure free during her 4-month treatment period [
      • Zimmerman A.W.
      • Holden K.R.
      • Reiter E.O.
      • Dekaban A.S.
      Medroxprogesterone acetate in the treatment of seizures associated with menstruation.
      ].
      In the years after Zimmerman's first case report, many other open-label add-on clinical trials have been conducted in women with catamenial seizures (Table 2). Most clinical trials have reported a reduction in seizure frequency, with one notable exception. This was a clinical trial conducted by Dana-Haeri and Richens [
      • Dana-Haeri J.
      • Richens A.
      Effect of norethisterone on seizures associated with menstruation.
      ]. In the Dana-Haeri & Richens study, noresthisterone was prescribed as the progesterone-like compound. Noresthisterone is more potent than progesterone itself in terms of activating the progesterone nuclear receptors. Noresthisterone, however, was not effective. This failure may suggest that the progesterone nuclear receptor is not involved in producing progesterone's anti-seizure effects.
      Following these open label studies, a full-scale, double-blind, placebo-controlled randomized clinical trial (RCT)–a “gold standard” trial – was finally done in 2012. In that trial, Herzog et al. recruited 294 female patients with intractable partial seizures, with or without catamenial exacerbation [
      • Herzog A.G.
      • Fowler K.M.
      • Smithson S.D.
      • Kalayjian L.A.
      • Heck C.N.
      • Sperling M.R.
      • et al.
      Progesterone vs placebo therapy for women with epilepsy: a randomized clinical trial.
      ]. In this fully controlled trial, there was no overall difference in responding between the treatment and control groups. A post-hoc analysis, however, revealed that patients those with catamenial epilepsy and severe perimenstrual exacerbations (C1) did benefit from progesterone therapy [
      • Najafi M.
      • Sadeghi M.M.
      • Mehvari J.
      • Zare M.
      • Akbari M.
      Progesterone therapy in women with intractable catamenial epilepsy.
      ].
      Subsequently, a second double-blind, placebo-controlled clinical study was conducted by Najafi's at al. This trial, which involved only women with catamential seizures, also showed a statistically significant reduction in seizure frequency in the progesterone treated group [
      • Najafi M.
      • Sadeghi M.M.
      • Mehvari J.
      • Zare M.
      • Akbari M.
      Progesterone therapy in women with intractable catamenial epilepsy.
      ]. In clinical studies, therefore, it appears that progesterone benefits women with catamenial seizures, but not necessarily women with other types of epilepsy.

      5. Anti-seizure effects of allopregnanolone

      It has been known for some time that the anti-seizure effects of progesterone are largely mediated by its metabolites. In animal studies, when the 5a metabolism of progesterone is blocked by finasteride, the anti-seizure effect of progesterone is nearly abolished [
      • Kokate T.G.
      • Banks M.K.
      • Magee T.
      • Yamaguchi S.
      • Rogawski M.A.
      Finasteride, a 5alpha-reductase inhibitor, blocks the anticonvulsant activity of progesterone in mice.
      ,
      • Frye C.A.
      • Scalise T.J.
      • Bayon L.E.
      Finasteride blocks the reduction in ictal activity produced by exogenous estrous cyclicity.
      ]. There is also a clinical report where a woman who had good seizure control on progesterone relapsed when the doctor prescribed her finasteride. Finasteride was later removed from her treatment and she achieved good seizure control on progesterone again [
      • Herzog A.G.
      • Frye C.A.
      Seizure exacerbation associated with inhibition of progesterone metabolism.
      ].
      The metabolite that has been most extensively studied is allopregnanolone (ALLO), progesterone's secondary metabolite

      5.1 Animal studies

      Table 3 summarizes the animal studies that have examined the anti-seizure effects of, ALLO in animal seizure models. As indicated, ALLO suppresses generalized convulsive seizures in the PTZ model [
      • Reddy D.S.
      • Castaneda D.C.
      • O’Malley B.W.
      • Rogawski M.A.
      Anticonvulsant activity of progesterone and neurosteroids in progesterone receptor knockout mice.
      ,
      • Kokate T.G.
      • Svensson B.E.
      • Rogawski M.A.
      Anticonvulsant activity of neurosteroids: correlation with gamma-aminobutyric acid-evoked chloride current potentiation.
      ], the kainic acid model [
      • Frye C.A.
      • Scalise T.J.
      Anti-seizure effects of progesterone and 3alpha, 5alpha-THP in kainic acid and perforant pathway models of epilepsy.
      ] and the 6 Hz model [
      • Kaminski R.M.
      • Livingood M.R.
      • Rogawski M.A.
      Allopregnanolone analogs that positively modulate GABA receptors protect against partial seizures induced by 6-Hz electrical stimulation in mice.
      ]. It also alleviates kainic acid and pilocarpine induced-status epilepticus in mice [
      • Kokate T.G.
      • Cohen A.L.
      • Karp E.
      • Rogawski M.A.
      Neuroactive steroids protect against pilocarpine- and kainic acid-induced limbic seizures and status epilepticus in mice.
      ] and suppresses the secondarily generalized convulsive seizures in the rat amygdala kindling model [
      • Lonsdale D.
      • Nylen K.
      • McIntyre Burnham W.
      The anticonvulsant effects of progesterone and its metabolites on amygdala-kindled seizures in male rats.
      ,
      • Lonsdale D.
      • Burnham W.M.
      The anticonvulsant effects of allopregnanolone against amygdala-kindled seizures in female rats.
      ] and the mouse hippocampus kindling model [
      • Jeffrey M.
      • Lang M.
      • Gane J.
      • Chow E.
      • Wu C.
      • Zhang L.
      Novel anticonvulsive effects of progesterone in a mouse model of hippocampal electrical kindling.
      ]. (Focal kindled seizures are discussed below.) The reported ED50s have varied, but most studies have reported ED50s below 20 mg/kg – considerably lower than the ED50s reported for progesterone in animal studies, Side effects, especially sedation, have been reported even at these lower doses.
      Table 3Allopregnanolone as an anti-seizure drug in animal studies. PTZ: pentylenetetrazole, I.P.: intraperitoneal, S.C.: subcutaneous, P.O.: oral route.
      AuthorSubjectsSeizure ModelTreatmentResults
      Kokate et al. (1994)

      7932175
      NIH Swiss mice, male, 25–30 gPTZ, 80 mg/kg, S.C.

      MES
      THP (-up to 100 mg/kg, I.P.)ED50 = 13.7 mg/kg in PTZ model,

      Not effective against MES-induced seizures at any dose

      Adverse effect: sedation, motor toxicity (TD50 = 42.0 mg/kg)
      Kokate et al. (1996)NIH Swiss mice, male, 25–30 gpilocarpine, 416 mg/kg, S.C., limbic motor seizures and status epilepticsTHP (-up to 20 mg/kg), I.P.ED50 = 7.0 mg/kg
      Kokate et al. (1996)NIH Swiss mice, male, 25–30 gKainic acid, 32 mg/kg, S.C.THP (-up to 40 mg/kg), I.P., one injection or two injection spaced one hourprotected 40% subjects that received one injection and 100% subjects that received two injections against limbic seizures and status epilepticus

      Side effect: sedation
      Budziszewska et al. (1999)WAG/Rij rats

      10022363
      WAG/Rij, genetic absence modelTHP (5–20 mg/kg, I.P.)Increase number and duration of spike wave discharges
      Frye & Scalise, (2000)Ovariectomized Long-Evans rats, 55 days old upon arrivalKainic acid, 32 mg/kg, S.C.THP (0, 4, 8 mg/kg, S.C.)Increase latency to initial partial seizures, decrease number and durations of full and partial seizures
      Kaminski et al. (2004)NIH Swiss mice, male, 25–30 g6 Hz corneal stimulationTHP (2–100 mg/kg, I.P.)ED50= 14.2 mg/kg
      Reddy et al. (2004)Adult C57BL6/129SvEv mice, male and female, 25–30 gPTZ, 85 mg/kg, S.C.THP (0–100 mg/kg, I.P.)Suppress of clonic seizures,

      male: ED50 = 15 mg/kg,

      female: ED50 = 7 mg/kg
      Lonsdale et al, (2006)Wistar rats, male, 300–400 gAmygdala kindlingTHP dose-response (0, 1.25, 5, 10, 20, or 30 mg/kg, I.P.)Suppress generalized seizures (ED50 = 15.2 mg/kg),

      Adverse effect: ataxia (TD50 = 26.6 mg/kg)
      Lonsale et al, (2007)Wistar rats, female, 240–265 gAmygdala kindlingTHP dose-response (0, 0.625, 1.25, 2.5, 5.0, 7.7 10, or 20 mg/kg, S.C.)

      THP time-response (10, 20, 30, 60, 120, 240 min, S.C.)
      Suppress generalized seizures (ED50= 1.1 mg/kg),

      Suppression of generalized seizures reached 75% from 15 to 60 min post-treatment

      Adverse effect: ataxia (TD50 = 8.6 mg/kg)
      Rogawski et al. (2013)Pediatric P9 ratsKainic acid, 2 mg/kg, I.P., drug resistant status epileptus modelTHP dose 30 mg/kg I.P.Terminates electrographic seizure discharges, the effects last longer than 5 mg/kg diazepam treated control groups
      Jeffrey et al. (2014)C57 black mice, male, 6–10 months oldHippocampal kindlingTHP (1, 3.5, 10, 30 mg/kg, I.P.)Suppress generalized seizures and reduce the length of focal seizures, completely suppress generalized seizure at 10 and 30 mg/kg
      Zhong (2015)Wistar rats, female, 50 days oldAmygdala kindlingTHP (0–20 mg/kg, S.C.)Suppress generalized seizures (ED50 = 24 mg/kg),

      Adverse effect: ataxia (TD50 = 27 mg/kg)
      ALLO is relatively potent as compared to other anti-seizure drugs. Taubøll & Gjerstad compared ALLO's and phenobarbital's effects on recurrent inhibition in rat hippocampal slices, and concluded that ALLO was the most potent of the three [
      • Taubøll E.
      • Gjerstad L.
      Comparison of 5 alpha-pregnan-3 alpha-ol-20-one and phenobarbital on cortical synaptic activation and inhibition studied in vitro.
      ]. ALLO is also more potent than 5β-tetrahydrodeoxycortisone and (+)MK-801 against picrotoxin- and bicuculline-induced seizures in male albino mice [
      • Członkowska A.I.
      • Krzaścik P.
      • Sienkiewicz-Jarosz H.
      • Siemiatkowski M.
      • Szyndler J.
      • Bidziński A.
      • et al.
      The effects of neurosteroids on picrotoxin-, bicuculline- and NMDA-induced seizures, and a hypnotic effect of ethanol.
      ].
      ALLO, however, has not proven to be effective in all models of generalized seizures. It has failed to suppress tonic hindlimb extension in the maximal electroshock model’ [
      • Kokate T.G.
      • Svensson B.E.
      • Rogawski M.A.
      Anticonvulsant activity of neurosteroids: correlation with gamma-aminobutyric acid-evoked chloride current potentiation.
      ]. It also exacerbates the absence seizures modeled by the WAG/Rij rats [
      • van Luijtelaar G.
      • Budziszewska B.
      • Tetich M.
      • Lasoń W.
      Finasteride inhibits the progesterone-induced spike-wave discharges in a genetic model of absence epilepsy.
      ].
      ALLO has produced apparently conflicting results when tested against limbic focal seizures in kindled subjects. Limbic focal seizures in kindled animals are thought to model drug-resistant complex-partial seizures in humans [
      • Albright P.S.
      • Burnham W.M.
      Development of a new pharmacological seizure model: effects of anticonvulsants on cortical- and amygdala-kindled seizures in the rat.
      ] and, therefore, the suppression of limbic focal seizures in kindled animals is of great interest to drug development. Jeffrey et al. reported that ALLO suppressed focal hippocampal afterdischarges in mice [
      • Jeffrey M.
      • Lang M.
      • Gane J.
      • Chow E.
      • Wu C.
      • Zhang L.
      Novel anticonvulsive effects of progesterone in a mouse model of hippocampal electrical kindling.
      ]. Jeffrey et al., however, were measuring afterdischarge duration rather than the complete suppression of afterdischarge. Lonsdale, who used a stricter criterion – the complete suppression of the focus rather than the shortening of afterdischarge – reported no focal seizure suppression in the amygdala-kindled rats – or less than 40% of suppression at a very high [
      • Lonsdale D.
      • Nylen K.
      • McIntyre Burnham W.
      The anticonvulsant effects of progesterone and its metabolites on amygdala-kindled seizures in male rats.
      ,
      • Lonsdale D.
      • Burnham W.M.
      The anticonvulsant effects of allopregnanolone against amygdala-kindled seizures in female rats.
      ]. These differing results clearly relate to the criteria used. Jeffery et al. would have reported no suppression of focal seizures if she had used Lonsdale's criterion [
      • Jeffrey A.M.
      Progesterone and its metabolites: anticonvulsant and behavioral studies. Thesis.
      ].

      5.2 Clinical studies

      Table 4 presents a summary of the clinical trials using ALLO and its commercial analog ganaxolone. Clinically, ALLO seems to have been tested for only one particular indication, super-refractory status epilepticus (SRSE). SRSE occurs when status epilepticus has resisted all of the standard medications, and has even resisted general anesthesia. It is diagnosed when status is uncontrolled 24 hours after initiating anesthetic treatment [
      • Delaj L.
      • Novy J.
      • Ryvlin P.
      • Marchi N.A.
      • Rossetti A.O.
      Refractory and super-refractory status epilepticus in adults: a 9-year cohort study.
      ]. Case reports indicate that intravenous ALLO effectively suppresses SRSE in both adults [
      • Vaitkevicius H.
      • Husain A.M.
      • Rosenthal E.S.
      • Rosand J.
      • Bobb W.
      • Reddy K.
      • et al.
      First-in-man allopregnanolone use in super-refractory status epilepticus.
      ] and paediatric patients [
      • Broomall E.
      • Natale J.E.
      • Grimason M.
      • Goldstein J.
      • Smith C.M.
      • Chang C.
      • et al.
      Pediatric super-refractory status epilepticus treated with allopregnanolone.
      ]. Recently, a commercial aqueous formulation of ALLO – brexanolone – has been tested in an open-label add-on clinical trial in SRSE patients, and has proved successful in 77% of patients [
      • Rosenthal E.S.
      • Claassen J.
      • Wainwright M.S.
      • Husain A.M.
      • Vaitkevicius H.
      • Raines S.
      • et al.
      Brexanolone as adjunctive therapy in super-refractory status epilepticus.
      ]. However, due to the small sample size in this open trial, some caution must be applied. It will be better to wait for the results from the randomized double-blind placebo-controlled Phase III trial which is currently underway (NCT02477618, N = 132) before drawing any firm conclusions.
      Table 4Allopreganolone and its analog ganaxolone as anti-seizure drugs in clinical studies, listed in chronological order.
      AuthorPatientSeizure Type(s)MedicationsResults
      Laxer et al.

      (2000)

      10999558
      Double blind RCT

      N = 52,18 to 65 y/o
      Complex partial seizures with and without secondary generalizationGanaxolone

      500 mg TID on day1 with food

      625 mg TID on day2–8 with food

      Medication given with other AEDs blood concentration below 25% therapeutic level
      Less people discontinued study because of seizures (37.5% in treatment group compared to 70.8% in control group)
      Kerrigan et al. (2000)

      11074186
      Open label, add-on trial

      N = 20, 7 months to 7 y/o,
      Infantile spasmGanaxolone,

      titrated to 36 mg/kg/d or max. tolerated dose over 4 weeks,

      Maintained for 8 weeks, then discontinued by tapering
      Reduced spasm frequency by 50–33%
      Pieribone et al. (2007)Pilot, open label, dose-esclation study

      N = 15, 5–15 y/o
      Seizures not controlled by at least 2 conventional AEDsGanaxolone oral suspension, 1 mg/kg BID to 12 mg/kg TID, maintained over 8 weeks25% subjects experienced >50% seizure reduction, 13% subjects experienced moderate (>25%, but <50%) seizure reduction

      Adverse Effects: Somnolence
      Sperling et al. (2017)Double blind, placebo controlled RCT,

      N = 147,18–49 y/o
      Uncontrolled partial-onset seizuresGanaxolone 1500 mg/day,

      Treatment period = 10 weeks (2 weeks of titration + 8 weeks of maintenance)
      Reduce average numbers of weekly seizures, mean percentage change in seizure frequency is -17.6% in the treatment group

      Adverse effects: dizziness, fatigue, somnolence
      Rosenthal et al. (2017)Open label, add-on trial

      N = 22, 10–76 y/o
      Supre-refractory status epilepticus, uncontrolled by first-line and second-line anesthetic agents and cannot wean off third-line anestheticsBrexanolone I.V. infusion, loading dose 296.6ug/kg for 1 hour, and 4 day maintenance infusion 86 or 156ug/kg/h77% patients successfully weaned off third-line anesthetics before tapering brexanolone

      Well tolerated in patiuents
      ALLO also has a commercially available analog, ganaxolone, whose chemical structure differs by only one methyl group on C3 [
      • Hogenkamp D.J.
      • Tahir S.H.
      • Hawkinson J.E.
      • Upasani R.B.
      • Alauddin M.
      • Kimbrough C.L.
      • et al.
      Synthesis and in vitro activity of 3 beta-substituted-3 alpha-hydroxypregnan-20-ones: allosteric modulators of the GABAA receptor.
      ]. This methyl substitution prolongs ALLO's half-life without diminishing its potency. Most clinical trials, therefore, have been conducted with ganaxolone rather than with ALLO. All of the findings of these trials have been positive, indicating that ganaxolone has good anti-seizure effects in people with epilepsy. Since ganaxolone – like ALLO – is a potent GABA-A agonist [
      • Hogenkamp D.J.
      • Tahir S.H.
      • Hawkinson J.E.
      • Upasani R.B.
      • Alauddin M.
      • Kimbrough C.L.
      • et al.
      Synthesis and in vitro activity of 3 beta-substituted-3 alpha-hydroxypregnan-20-ones: allosteric modulators of the GABAA receptor.
      ], it is not surprising that the side effects have included fatigue and sedation.
      It is worth noting that both male and female subjects have been recruited in ganaxolone trials, whereas only females have been recruited in progesterone trials. ALLO, unlike progesterone, has no hormonal effects [
      • Rapkin A.J.
      • Morgan M.
      • Goldman L.
      • Brann D.W.
      • Simone D.
      • Mahesh V.B.
      Progesterone metabolite allopregnanolone in women with premenstrual syndrome.
      ].

      6. Anti-seizure effects of 5a-dihydroprogesterone

      Following the early finasteride experiments, attention centered on progesterone's secondary metabolite ALLO – a known GABA agonist. Finasteride, however, blocks the synthesis of both DHP and A LLO. It was possible, therefore, that DHP might also be contributing to progesterone's anti-seizure effects. In fact, progesterone still has strong anti-seizure effects when co-administered with indomethacin, which blocks the formation of ALLO [
      • Shahzamani A.
      Progesterone as an anti-convulsant. Thesis.
      ]. This suggested that DHP itself had anti-seizure effects and led to the further study of DHP.
      DHP has been far less studied than progesterone or ALLO. No clinical studies have as yet been done with DHP, so only animal studies will be discussed here.

      6.1 Animal studies

      Table 5 summarizes the animal studies on DHP. Landgren et al. actually first described the anti-seizure effects of DHP in penicillin-induced seizures in ovariectomized cats in the 1980s [
      • Landgren S.
      • Aasly J.
      • Bäckström T.
      • Dubrovsky B.
      • Danielsson E.
      The effect of progesterone and its metabolites on the interictal epileptiform discharge in the cat's cerebral cortex.
      ].
      Table 55a-dihydroprogesterone (DHP) as an anti-seizure drug in animal studies. (s.c.: subcutaneous; i.v.: intravenous, ED50: effective dose 50, TD50: toxic dose 50).
      AuthorSubjectsSeizure ModelTreatmentResults
      Landgren et al., 1987Ovariectomized adult catsPenicillin-induced seizuresDHP (0.5 mg/mL, 1.1, 3.4, 6.3 mL/min I.V. infusion via lingual artery for 20s)Reduction of epileptiform interictal spiking, by 9%
      Lonsdale & Burnham, 2003Wistar rats, female, 240–265 gAmygdala-kindlingDHP (0, 2.5, 5.0 mg/kg, S.C.)Suppresses generalized seizures (ED50 = 2.9 mg/kg) and focal seizures (ED50= 4.3 mg/kg)

      No sedation was observed
      Lonsdale et al., 2006Wistar rats, male, 300–400 gAmygdala-kindlingDHP (0, 2.5, 5.0, 7.5 mg/kg, S.C.)Suppresses generalized seizures (ED50 = 6.2 mg/kg) and focal seizures (ED50= 6.2 mg/kg)

      No sedation was observed
      Jeffrey, 2014C75 black mice, male, 6–10 months oldHippocampal-kindlingDHP (0, 5, 10 mg/kg, I.P.)No effect on generalized motor seizures or focal seizures
      Wu & Burnham, 2018Wistar rats, female, 60 days oldAmygdala-kindlingDHP dose-response (0, 1.25, 2.5, 5.0, 7.5 mg/kg, I.V.)

      DHP time-response (5, 15, 30, 45, 60, or 80 min, I.V.)
      Suppresses generalized seizures (ED50= 1.69 mg/kg) and focal seizures (ED50= 3.48 mg/kg)

      Adverse effect: ataxia (TD50 = 3.57 mg/kg)

      Maximum effect was observed immediately after injection
      There was little subsequent research in this field, however, until Lonsdale and collaborators returned to this area in 2003. They reported that subcutaneous DHP suppressed both focal seizures and secondarily generalized motor seizures in amygdala-kindled rats. Suppression was found at non-sedating doses and it occurred in both males [
      • Lonsdale D.
      • Nylen K.
      • McIntyre Burnham W.
      The anticonvulsant effects of progesterone and its metabolites on amygdala-kindled seizures in male rats.
      ] and females [
      • Lonsdale D.
      • Burnham W.M.
      The anticonvulsant effects of progesterone and 5alpha-dihydroprogesterone on amygdala-kindled seizures in rats.
      ]. Of particular interest was the suppression of the amygdala focus at non-toxic doses.
      Not all subsequent studies, however, have supported Lonsdale's work. Jeffrey, for instance, reported that intraperitoneal DHP had no effect of focal or generalized seizures in hippocampal-kindled mice [
      • Jeffrey A.M.
      Progesterone and its metabolites: anticonvulsant and behavioral studies. Thesis.
      ]. It is hard to compare Lonsdale's and Jeffrey's studies, however, since they differed in the animal species, the kindling site and the route of administration. There is also the question of solubility, since DHP has a high partition coefficient and is very hard to keep in solution.
      A recent study by Wu & Burnham has switched to the intravenous route of injection, which allows for the use of lower doses which are easier to keep in solution [
      • Wu Y.V.
      • Burnham W.M.
      The anti-seizure effects of IV 5α-dihydroprogesterone on amygdala-kindled seizures in rats.
      ]. Female Wistar rats were implanted with an electrode in the right basolateral amygdala. They were then kindled to 15 stage 5 seizures, stability tested, and cannulated in the jugular vein. Multiple doses of IV DHP were tested against focal electrographic seizures and secondarily generalized convulsions. A dose-dependent suppression of both generalized and focal seizures was found, with ED50s of 1.69 mg/kg for the generalized convulsive seizures and of 3.48 mg/kg for the focal electrographic seizures. Ataxia, as rated by the Löscher ataxia scale, was also seen, and had a TD50 of 3.57 mg/kg. These effects were seen almost immediately, and lasted to about 60 minutes post-injection [
      • Wu Y.V.
      • Burnham W.M.
      The anti-seizure effects of IV 5α-dihydroprogesterone on amygdala-kindled seizures in rats.
      ]. These data confirm the ability of DHP to suppress the drug-resistant amygdala focus in kindled animals. Future studies will have to address the question of why ataxia was seen in the Wu et al. studies but not in Lonsdale's previous intraperitoneal studies. The different routes of injection may offer an explanation.

      7. Possible mechanisms: progesterone, ALLO and DHP

      7.1 Progesterone

      Progesterone itself seems to have some anti-seizure actions, in addition to the anti-seizure actions of its metabolites. These may be seen in the presence of finastreride pretreatment, but they occur at high and sedating doses [
      • Jeffrey M.
      • Lang M.
      • Gane J.
      • Chow E.
      • Wu C.
      • Zhang L.
      Novel anticonvulsive effects of progesterone in a mouse model of hippocampal electrical kindling.
      ]
      Progesterone has several molecular targets in the central nervous system which might mediate these actions. These include both genomic progesterone receptors [
      • Brinton R.D.
      • Thompson R.F.
      • Foy M.R.
      • Baudry M.
      • Wang J.
      • Finch C.E.
      • et al.
      Progesterone receptors: form and function in brain.
      ] and non-genomic receptors [
      • Singh M.
      • Su C.
      • Ng S.
      Non-genomic mechanisms of progesterone action in the brain.
      ]. The non-genomic receptors include: 1) the membrane progesterone receptor (mPRs) – the G-protein coupled receptors; 2) progesterone membrane receptor components (PGMRCs); and 3) sigma receptors.
      Since progesterone's anti-seizure effects can be seen shortly after administration, they presumably relate to its binding to non-genomic receptors. In fact, progesterone's anti-seizure actions are preserved even when nuclear progesterone receptor is antagonized by RU486 [
      • Kokate T.G.
      • Banks M.K.
      • Magee T.
      • Yamaguchi S.
      • Rogawski M.A.
      Finasteride, a 5alpha-reductase inhibitor, blocks the anticonvulsant activity of progesterone in mice.
      ] or in mice where the nuclear receptors have been knocked-out [
      • Reddy D.S.
      • Castaneda D.C.
      • O’Malley B.W.
      • Rogawski M.A.
      Anticonvulsant activity of progesterone and neurosteroids in progesterone receptor knockout mice.
      ].
      The fact that progesterone's ED50 in animals is quite high (over 50 mg/kg), and that it loses much of its potency in the presence of finasteride [
      • Frye C.A.
      • Scalise T.J.
      • Bayon L.E.
      Finasteride blocks the reduction in ictal activity produced by exogenous estrous cyclicity.
      ] suggests that progesterone's low-dose anti-seizure effects are largely mediated by its active metabolites.

      7.2 ALLO

      ALLO is considerably more potent than progesterone, with an ED50 below 20 mg/kg in animal seizure models. Since its anti-seizure actions appear shortly after administration (Table 3), it also presumably works on non-genomic receptors. Similar to barbiturate, ALLO is also a positive allosteric modulator of the cell-surface GABA-A receptor [
      • Majewska M.D.
      • Harrison N.L.
      • Schwartz R.D.
      • Barker J.L.
      • Paul S.M.
      Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor.
      ]. At nM concentrations, ALLO enhances GABA's ability to open chloride channels [
      • Lambert J.J.
      • Belelli D.
      • Harney S.C.
      • Peters J.A.
      • Frenguelli B.G.
      Modulation of native and recombinant GABA(A) receptors by endogenous and synthetic neuroactive steroids.
      ], and at higher concentrations, ALLO opens the GABA-A related chloride channel even in the absence of GABA. As expected, sedation and ataxia are common side effects of ALLO – and its analog ganaxolone – as they are with other GABA-enhancing drugs [
      • Schousboe A.
      • Madsen K.K.
      • Barker-Haliski M.L.
      • White H.S.
      The GABA synapse as a target for antiepileptic drugs: a historical overview focused on GABA transporters.
      ].
      ALLO is also an agonist at cell-surface mPRs in GT17 neuronal cell lines [
      • Thomas P.
      • Pang Y.
      Membrane progesterone receptors: evidence for neuroprotective, neurosteroid signaling and neuroendocrine functions in neuronal cells.
      ] which may partially contribute to its anti-seizures effects. It does not bind to nuclear progesterone receptors (nPRs) [
      • Mellon S.H.
      Neurosteroid regulation of central nervous system development.
      ].

      7.3 DHP

      DHP is also more potent than progesterone, with an ED50 in intravenous studies below 5 mg/kg. Once again its anti-seizure effects are seen shortly after administration suggesting that they are mediated by non-genomic receptors.
      The mechanisms of DHP's actions have been little studied as yet. Binding studies, however, have revealed that DHP binds to both mPRs and nPRs. Its affinity for mPRs is stronger than its affinity for nPRs [
      • Grazzini E.
      • Guillon G.
      • Mouillac B.
      • Zingg H.H.
      Inhibition of oxytocin receptor function by direct binding of progesterone.
      ,
      • Smith J.L.
      • Kupchak B.R.
      • Garitaonandia I.
      • Hoang L.K.
      • Maina A.S.
      • Regalla L.M.
      • et al.
      Heterologous expression of human mPRalpha, mPRbeta and mPRgamma in yeast confirms their ability to function as membrane progesterone receptors.
      ]. It does bind to nPRs, however, with an affinity that is weaker than progesterone, but stronger than ALLO [
      • Iswari S.
      • Colas A.E.
      • Karavolas H.J.
      Binding of 5 alpha-dihydroprogesterone and other progestins to female rat anterior pituitary nuclear extracts.
      ,
      • Rupprecht R.
      • Berning B.
      • Hauser C.A.
      • Holsboer F.
      • Reul J.M.
      Steroid receptor-mediated effects of neuroactive steroids: characterization of structure-activity relationship.
      ]. Binding to nPRs probably doesn’t contribute to its (very rapid) anti-seizure effects, but might be a source of hormonal side effects if DHP were developed as an anti-seizure drug.
      DHP also binds to PGMRCs [
      • Singh M.
      • Su C.
      • Ng S.
      Non-genomic mechanisms of progesterone action in the brain.
      ,
      • Tischkau S.A.
      • Ramirez V.D.
      A specific membrane binding protein for progesterone in rat brain: sex differences and induction by estrogen.
      ]. Little is understood of the function of PGMRC receptors [
      • Cahill M.A.
      The evolutionary appearance of signaling motifs in PGRMC1.
      ]. They are hypothesized to participate in cholesterol synthesis, to modulate cell cycling, and to direct axonal migration during brain development. It seem unlikely that any of these actions would contribute to DHP's rapid anti-seizure effects, but, once again, they might be a source of side effects if DHP were developed as a drug.

      8. Possible mechanisms: progesterone family compounds as anti-inflammatory drugs

      In addition to their acute anti-seizure actions, compounds in the progesterone family might also alleviate difficult-to-treat seizures by modulating neuroinflammation. Both systemic and CNS inflammatory markers are elevated in drug-resistant seizure disorders such as West syndrome (infantile spasms), viral encephalitis-induced epilepsy [
      • Barker-Haliski M.L.
      • Löscher W.
      • White H.S.
      • Galanopoulou A.S.
      Neuroinflammation in epileptogenesis: Insights and translational perspectives from new models of epilepsy.
      ], drug-resistant temporal lobe epilepsy [
      • Aronica E.
      • Crino P.B.
      Inflammation in epilepsy: clinical observations.
      ], and status epilepticus [
      • Janigro D.
      • Iffland P.H.
      • Marchi N.
      • Granata T.
      A role for inflammation in status epilepticus is revealed by a review of current therapeutic approaches.
      ]. These include markers such as IL-1B, IL-6, TNFa [
      • Ho Y.-H.
      • Lin Y.-T.
      • Wu C-WJ
      • Chao Y.-M.
      • Chang A.Y.W.
      • Chan J.Y.H.
      Peripheral inflammation increases seizure susceptibility via the induction of neuroinflammation and oxidative stress in the hippocampus.
      ] and NF-kB [
      • Shi Y.
      • Zhang L.
      • Teng J.
      • Miao W.
      HMGB1 mediates microglia activation via the TLR4/NF-κB pathway in coriaria lactone induced epilepsy.
      ].
      There is some evidence that progesterone, DHP and ALLO may all attenuate inflammatory responses. The evidence is strongest for progesterone. In a model of early brain injury, for instance, progesterone ameliorates the elevation of NF-kB pathway and other inflammatory markers. It has also shown anti-inflammatory effects in animal models of traumatic brain injury and stroke [
      • Deutsch E.R.
      • Espinoza T.R.
      • Atif F.
      • Woodall E.
      • Kaylor J.
      • Wright D.W.
      Progesterone's role in neuroprotection, a review of the evidence.
      ]. In a recent study, progesterone decreased both systemic and central inflammation cytokines – such as IL-1B, IL-6, and TNFa – in a model of neonatal arterial ischemic stroke. It also decreased stroke-induced seizure occurrence and severity [
      • Ho Y.-H.
      • Lin Y.-T.
      • Wu C-WJ
      • Chao Y.-M.
      • Chang A.Y.W.
      • Chan J.Y.H.
      Peripheral inflammation increases seizure susceptibility via the induction of neuroinflammation and oxidative stress in the hippocampus.
      ].
      ALLO has similarly been shown to reduce the expression of cytokines such as TNF-a, IL-8, and IL-1b in an in vitro model [
      • Noorbakhsh F.
      • Ellestad K.K.
      • Maingat F.
      • Warren K.G.
      • Han M.H.
      • Steinman L.
      • et al.
      Impaired neurosteroid synthesis in multiple sclerosis.
      ], and both progesterone and ALLO reduce IL-1b and TNF-a in the acute phase of traumatic brain injury in rats [
      • He J.
      • Evans C.-O.
      • Hoffman S.W.
      • Oyesiku N.M.
      • Stein D.G.
      Progesterone and allopregnanolone reduce inflammatory cytokines after traumatic brain injury.
      ,
      • VanLandingham J.W.
      • Cekic M.
      • Cutler S.
      • Hoffman S.W.
      • Stein D.G.
      Neurosteroids reduce inflammation after TBI through CD55 induction.
      ].
      There is less evidence for DHP. DHP, however, at 0.25–0.5 mg/kg has been shown to reduce hilar neuron loss and vimentin expression in reactive astrocytes in the kainic-acid model [
      • Ciriza I.
      • Carrero P.
      • Frye C.A.
      • Garcia-Segura L.M.
      Reduced metabolites mediate neuroprotective effects of progesterone in the adult rat hippocampus. The synthetic progestin medroxyprogesterone acetate (Provera) is not neuroprotective.
      ].
      As with progesterone's anti-seizure effects, progesterone's anti-inflammatory and neuroprotective effects may mainly relate to its metabolites. When 5a-reductase is blocked by finasteride, progesterone's neuroprotective effects are abolished [
      • Ciriza I.
      • Azcoitia I.
      • Garcia-Segura L.M.
      Reduced progesterone metabolites protect rat hippocampal neurones from kainic acid excitotoxicity in vivo.
      ]. Interestingly, when indomethacin is used to block the interconversion between DHP and ALLO, both DHP and ALLO's neuroprotective effects are equally diminished. A possible explanation is that both DHP and ALLO are required to provide neuroprotection [
      • Ciriza I.
      • Azcoitia I.
      • Garcia-Segura L.M.
      Reduced progesterone metabolites protect rat hippocampal neurones from kainic acid excitotoxicity in vivo.
      ].
      The anti-inflammatory actions of the progesterone family, and the possible anti-seizure effects of anti-inflammatory compounds, warrant further investigation.

      9. Directions for future drug development

      Compounds in the progesterone family have proven their anti-seizure effects in both animal and clinical studies. Some are anti-seizure at non-sedating doses, and appear to suppress even pharmacoresistant limbic seizures. ALLO has already been developed as the anti-seizure compound ganaxolone [
      • Hogenkamp D.J.
      • Tahir S.H.
      • Hawkinson J.E.
      • Upasani R.B.
      • Alauddin M.
      • Kimbrough C.L.
      • et al.
      Synthesis and in vitro activity of 3 beta-substituted-3 alpha-hydroxypregnan-20-ones: allosteric modulators of the GABAA receptor.
      ], but DHP has not yet been exploited.
      Progesterone and its 5a metabolites, however, have some side effects that would need to be reduced through rational drug design – especially for long-term use. First, progesterone itself suppresses the fertility cycle in females, which is why progesterone-related compounds are widely used as contraceptives [
      • Chabbert-Buffet N.
      • Kolanska K.
      • Daraï E.
      • Bouchard P.
      Selective progesterone receptor modulators: current applications and perspectives.
      ]. In male sex offenders, the progesterone analog medroxyprogesterone acetate has been used to reduce sexual arousal. In this population, MPA lowered testosterone and luteinizing hormone levels, as well as decreased testicular size [
      • Murray J.B.
      Psychopharmacological therapy of deviant sexual behaviour.
      ]. These hormone effects, mediated via intracellular receptors, would need to be reduced in drugs directly based on progesterone.
      DHP, like progesterone, might also affect the female reproductive cycle. Direct evidence is lacking, but indirect evidence suggests that DHP affects the secretion of luteinizing hormone [
      • Mahesh V.B.
      • Murphy L.L.
      • O’Conner J.L.
      Selective modulation of FSH and LH secretion by steroids.
      ] and follicle stimulating hormone (FSH) [
      • Sanyal M.K.
      • Todd R.B.
      5-Dihydroprogesterone influence on ovulation of prepuberal rats.
      ]. In addition, DHP induces inducing lordosis in female Sprague-Dawley rats [
      • Whalen R.E.
      • Gorzalka B.B.
      The effects of progesterone and its metabolites on the induction of sexual receptivity in rats.
      ]. Krebs et al. have suggested that this effect may be regulated by PGMRC1 [
      • Krebs C.J.
      • Jarvis E.D.
      • Chan J.
      • Lydon J.P.
      • Ogawa S.
      • Pfaff D.W.
      A membrane-associated progesterone-binding protein, 25-Dx, is regulated by progesterone in brain regions involved in female reproductive behaviors.
      ]. Similarly, chronic administration of DHP at 500ug to estrogen-primed, ovariectomized female mice increases receptively in a strain-dependent manner [
      • Gorzalka B.B.
      • Whalen R.E.
      Genetic regulation of hormone action: selective effects of progesterone and dihydroprogesterone (5alpha-pregnane-3,20-dione) on sexual receptivity in mice.
      ]. It has been found that this DHP-induced receptivity in a dominant trait in mice [
      • Gorzalka B.B.
      • Whalen R.E.
      Effects of genotype on differential behavioral responsiveness to progesterone and 5alpha-dihdroprogesterone in mice.
      ].
      ALLO, on the other hand, has negligible affinity to the nPR receptor under physiological conditions [
      • Rupprecht R.
      • Berning B.
      • Hauser C.A.
      • Holsboer F.
      • Reul J.M.
      Steroid receptor-mediated effects of neuroactive steroids: characterization of structure-activity relationship.
      ]. These results must be interpreted with caution, however, ALLO's possible effects on the reproductive and other systems should not be overlooked [
      • Herbison A.E.
      Physiological roles for the neurosteroid allopregnanolone in the modulation of brain function during pregnancy and parturition.
      ]. ALLO suppresses the stress response, for instance, by modulating the hypothalamus-pituitary-adrenal axis [
      • Biggio G.
      • Pisu M.G.
      • Biggio F.
      • Serra M.
      Allopregnanolone modulation of HPA axis function in the adult rat.
      ]. Upregulated ALLO during late pregnancy is crucial in maintaining the hypo-responsiveness to stress, a mechanism independent of progesterone and DHP [
      • Brunton P.J.
      Neuroactive steroids and stress axis regulation: pregnancy and beyond.
      ]. Acute, intracerebroventricular administration of 6uM ALLO also alters luteinizing hormone, progesterone and prolactin levels[
      • Laconi M.R.
      • Chavez C.
      • Cavicchia J.C.
      • Fóscolo M.
      • Sosa Z.
      • Yunes R.F.
      • et al.
      Allopregnanolone alters the luteinizing hormone, prolactin, and progesterone serum levels interfering with the regression and apoptosis in rat corpus luteum.
      ] and inhibits lordosis, therefore decreasing sexual receptivity[
      • Laconi M.R.
      • Cabrera R.J.
      Effect of centrally injected allopregnanolone on sexual receptivity, luteinizing hormone release, hypothalamic dopamine turnover, and release in female rats.
      ]. ALLO also affects feeding behavior via central mechanisms. Repeated exposure to ALLO at 10–20 mg/kg daily increases food intake and promotes weight gain in rats [
      • Holmberg E.
      • Bäckström T.
      • Johansson M.
      • Löfgren M.
      • Haage D.
      Allopregnanolone induces a diurnally dependent hyperphagic effect and alters feeding latency and duration in male Wistar rats.
      ,
      • Holmberg E.
      • Johansson M.
      • Bäckström T.
      • Löfgren M.
      • Haage D.
      Repeated allopregnanolone exposure induces weight gain in schedule fed rats on high fat diet.
      ]. In humans, high levels of ALLO are correlated with weight gain in pregnancy and in those with polycystic ovarian disease [
      • Holmberg E.
      • Sjöstedt J.
      • Malinina E.
      • Johansson M.
      • Turkmen S.
      • Ragagnin G.
      • et al.
      Allopregnanolone involvement in feeding regulation, overeating and obesity.
      ].
      Tumorigenesis in mammary tissues is another possible effect of both progesterone and DHP. Wiebe's group has proposed that DHP in particular promotes cellular proliferation and decreases cell adhesion based on in vitro studies [
      • Wiebe J.P.
      • Pawlak K.J.
      • Kwok A.
      Mechanism of action of the breast cancer-promoter hormone, 5α-dihydroprogesterone (5(P), involves plasma membrane-associated receptors and MAPK activation.
      ,
      • Wiebe J.P.
      • Muzia D.
      The endogenous progesterone metabolite, 5a-pregnane-3,20-dione, decreases cell-substrate attachment, adhesion plaques, vinculin expression, and polymerized F-actin in MCF-7 breast cancer cells.
      ]. DHP has also been shown to promote the neoplastic transformation of non-tumor C4HD murine mammary cells in vivo [
      • Wiebe J.P.
      • Rivas M.A.
      • Mercogliano M.F.
      • Elizalde P.V.
      • Schillaci R.
      Progesterone-induced stimulation of mammary tumorigenesis is due to the progesterone metabolite, 5α-dihydroprogesterone (5αP) and can be suppressed by the 5α-reductase inhibitor, finasteride.
      ].
      It seems, however, that DHP stimulates tumorigenesis via mitogen-activated protein kinase (MAPK) pathway which is activated by mPRs. MPRs seem to have little roles in suppressing limbic seizures [
      • Wiebe J.P.
      • Rivas M.A.
      • Mercogliano M.F.
      • Elizalde P.V.
      • Schillaci R.
      Progesterone-induced stimulation of mammary tumorigenesis is due to the progesterone metabolite, 5α-dihydroprogesterone (5αP) and can be suppressed by the 5α-reductase inhibitor, finasteride.
      ]. Thus, it might be possible to develop an analog that lack mPR activity while maintaining anti-seizure effects.
      If DHP were to be developed as an anti-seizure agent, both its hormonal and turmorigenic effects would have to be reduced through rational drug design.

      Conflict of interest declaration

      Yinhao Violet Wu and W. McIntyre Burnham declare that they have no conflict of interest.

      Acknowledgement

      This work was partly supported by research grant from the Ontario Brain Institute (grant number: 496399 ).

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