Ganaxolone for use in treating genetic epileptic disorders

Abstract

To provide agents for treating cyclin-dependent kinase-like 5 (CDKL5) deficiency.SOLUTION: Provided is a ganaxolone oral solution for treating CDKL5 deficiency, the ganaxolone being initially administered to a patient in an amount of 0.5 mg / kg / day to about 15 mg / kg / day, and over a period of four weeks, and a dose of ganaxolone being gradually increased to an amount of about 18 mg / kg / day to about 60 mg / kg / day, with a total dose of ganaxolone of up to about 1,800 mg / day for patients weighing more than 30 kg and a total dose of ganaxolone of about 63 mg / kg / day for patients weighing less than 30 kg.SELECTED DRAWING: None

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 584,403, filed November 10, 2017, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Infantile epileptic encephalopathies and rare childhood epilepsies represent a significant unmet medical need. These conditions include PCDH19-related epilepsy, CDKL5 deficiency (CDD), Dravet syndrome, Lennox-Gastaut syndrome (LGS), continuous sleep waves in sleep (CSWS), status epilepticus during sleep (ESES), and other intractable genetic epilepsy conditions that clinically resemble PCDH19-related epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES.

[0003] PCDH19-associated epilepsy is a severe and rare epilepsy syndrome that primarily affects women. The condition is caused by inherited mutations in the protocadherin 19 (PCDH19) gene, located on the X chromosome, and is characterized by early-onset and highly variable cluster seizures, cognitive and sensory impairments, and behavioral disturbances. Currently, there is no approved treatment or effective standard of care for PCDH19-associated epilepsy.

[0004] CDKL5 is a rare X-linked genetic disorder that results in early onset, difficult-to-control seizures, and severe neurodevelopmental disabilities. The most common feature of CDKL5 deficiency is early, drug-resistant epilepsy, usually beginning within the first few months of life. Seizures are generally highly polymorphic. Complex partial seizures, infantile spasms, myoclonic seizures, generalized tonic-clonic seizures, and tonic seizures have all been reported. Many different seizure types can also occur in the same patient, frequently varying over time. Patients treated with antiepileptic drugs ("AEDs") experience a short, seizure-free honeymoon period, followed by relapse (Kilstrup-Nielsen et al., 2012). CDKL5 deficiency is one of the most treatment-refractory epileptic encephalopathies.

[0005] The lack of meaningful treatment benefit from AEDs or any other intervention in CDKL5 is well summarized by the patient advocacy group CDKL5UK: "At present, we are not aware of any specific medications that would be beneficial for people with CDKL5 deficiency. Some people have had vagus nerve stimulators implanted; this has been beneficial for some. Some find that their children do not respond to any antiepileptic drugs, and specialists make the difficult decision to discontinue all antiepileptic drugs. Many parents have found that their children's seizures are significantly better when they fast, but the ketogenic diet has not worked for most people. We hope that a better understanding of the CDKL5 gene and its function will lead to new, more effective treatments."

[0006] No uniformly effective therapeutic agents have been found for the treatment of epileptic encephalopathies and rare childhood epilepsies, and multiple therapeutic agents (e.g., anticonvulsants) are often used in combination to treat PCDH19-related epilepsy, Dravet syndrome, Lennox-Gastaut syndrome (LGS), continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), and other intractable epileptic conditions and intractable genetic epileptic conditions that clinically resemble PCDH19-related epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES.

[0007] Additionally, there are no approved or licensed therapies for the treatment of patients with CDKL5 deficiency in the United States. There is no accepted standard of care or guidelines from authoritative scientific organizations for the treatment of such patients. However, most antiepileptic drugs ("AEDs"), including steroids / adrenocorticotropic hormone (ACTH), ketogenic diets, vagus nerve stimulation, and corpus callosotomy (to disrupt interhemispheric connections for the relief of secondary generalized seizures) have been attempted to treat the condition.

[0008] The effectiveness of multiple AEDs and ketogenic diets in patients with CDKL5 mutations is very low. Newer drugs tend to have less sedative effects, less adverse effects on memory and learning, and less likelihood of causing allergic reactions and serious side effects. However, some of the most commonly used AEDs to treat CDKL5 deficiency-related seizures are associated with the following serious side effects: Cognitive side effects are a major concern with topiramate. Felbamate may cause aplastic anemia or liver failure. Vigabatrin can permanently narrow a child's vision. Stevens-Johnson syndrome is a severe allergic drug reaction and remains a concern regarding lamotrigine.

[0009] Compared with drugs used before 1990, many of the newer drugs have a broader spectrum of action and are more likely to be effective against generalized seizures, although some, such as gabapentin, pregabalin, oxcarbazepine, and tiagabine, appear to be effective only against seizures with focal onset.

[0010] Seizures in PCDH19-related epilepsy, CDKL5 deficiency, Dravet syndrome, Lennox-Gastaut syndrome (LGS), continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), and other intractable genetic epilepsy conditions that share common seizure patterns and are clinically similar to PCDH19-related epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES are sometimes resistant to conventional antiepileptic and anticonvulsant medications.

[0011] More effective treatments, especially those with minimal side effects compared to existing treatments, are needed for children with refractory epileptic encephalopathies and rare childhood epilepsies.

[0012] The present invention fulfills this need by providing oral liquid, solid, and injectable neurosteroid formulations for the treatment of PCDH19-associated epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES, and such conditions; and methods of diagnosis and treatment of PCDH19-associated epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES, and such conditions. Summary of the Invention

[0013] The object of the present invention is to provide a treatment for early infantile epileptic encephalopathy.

[0014] Another object of the present invention is to utilize the gamma-aminobutyric acid (GABA)ergic mechanism of action of ganaxolone to provide therapeutic benefit for seizures, neuropsychological disorders, and sleep disorders associated with PCDH19-related epilepsy, CDKL5 epileptic encephalopathy, Dravet syndrome, Lennox-Gastaut syndrome (LGS), persistent sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), and other intractable genetic epileptic conditions that share common seizure patterns and are clinically similar to PCDH19-related epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES.

[0015] In furtherance of the above objectives and others, the present invention relates, in part, to an oral immediate release formulation comprising particles comprising: (i) a pregnenolone neurosteroid (e.g., ganaxolone) and (ii) one or more pharmaceutically acceptable excipients (e.g., oral suspension, tablet, or capsule), wherein the particles have a particle size that ensures the absence of aggregation after dispersion in simulated gastrointestinal fluid (SGF and / or SIF) and remains unchanged upon storage of the formulation for one month at 25°C / 60% RH. In a preferred embodiment, the formulation releases about 70% or about 80% or more of the pregnenolone neurosteroid in 500 ml of dissolution medium (e.g., 5% SLS in SGF (simulated gastric fluid) and / or 5% SLS in SIF (simulated intestinal fluid)) at 100 rpm, 37°C + 0.5°C, in 45 minutes in a USP Apparatus 1 (Basket) and achieves plasma levels of pregnenolone neurosteroid of about 55 ng / mL, about 60 ng / mL, or about 65 ng / mL, to plasma levels of pregnenolone neurosteroid of about 240 ng / mL to 400 ng / mL (e.g., 262 ng / mL) for at least about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, or about 12 hours after administration of a single dose and / or multiple doses. In some of these embodiments, the particles have a volume weighted median diameter of about 250 nm to about 450 nm (eg, about 332 nm). In some embodiments, the particles have a D(10) particle size of about 200 nm to about 220 nm, a D(50) particle size of about 250 nm to about 450 nm, and a D(90) particle size of about 480 nm to about 700 nm, the formulation is cyclodextrin-free, including sulfoalkyl ether cyclodextrins and modifications thereof, and is for treating in humans a disorder selected from the group consisting of PCDH19-associated epilepsy, CDKL5 epileptic encephalopathy, Dravet syndrome, Lennox-Gastaut syndrome (LGS), continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), and other intractable genetic epileptic conditions clinically similar to PCDH19-associated epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES.

[0016] The present invention also relates, in part, to an oral immediate release formulation comprising particles comprising: (i) ganaxolone, and (ii) one or more pharmaceutically acceptable excipients (e.g., an oral suspension, tablet, or capsule). wherein the particles have an average particle size of about 0.3 microns (i.e., a volume-weighted median diameter (D50) of about 0.3 microns); the particle size does not change upon storage of the formulation for one month at 25°C / 60% RH; the formulation releases about 70% or about 80% or more of the ganaxolone in 500 ml of dissolution medium (e.g., 5% SLS in SGF (simulated gastric fluid) and / or 5% SLS in SIF (simulated intestinal fluid)) at 100 rpm, 37°C + 0.5°C, and 45 minutes in a USP Apparatus 1 (Basket); and the formulation releases about 55 ng / mL, about 60 ng / ml, or about 65 ng / ml of ganaxolone in blood after single and / or multiple doses. The formulation is intended for use in treating disorders in humans, including or selected from the group consisting of PCDH19-associated epilepsy, CDKL5 epileptic encephalopathy, Dravet syndrome, Lennox-Gastaut syndrome (LGS), continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), and other intractable genetic epileptic conditions clinically similar to PCDH19-associated epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES. Plasma levels of ganaxolone of about 55 ng / mL, about 60 ng / mL, or about 65 ng / mL, to about 240 ng / mL to 400 ng / mL (e.g., 262 ng / mL), may be achieved following fasted and / or fed administration of the formulation.In some of these embodiments, an average particle size of about 0.3 microns is important to achieve about 70% or about 80% or greater dissolution of the pregnenolone neurosteroid in 45 minutes upon placement of the formulation in simulated gastrointestinal fluid (SGF and / or SIF) and to achieve plasma levels of the pregnenolone neurosteroid of about 55 ng / mL, about 60 ng / ml, or about 65 ng / ml, and plasma levels of the pregnenolone neurosteroid of about 240 ng / ml to 400 ng / ml (e.g., 262 ng / mL) for at least 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, or about 12 hours.

[0017] The present invention also relates, in part, to an immediate-release formulation comprising particles comprising: (i) ganaxolone, and (ii) one or more pharmaceutically acceptable excipients (e.g., oral suspension, tablet, or capsule), wherein the particles have an average particle size of about 0.3 microns; the average particle size does not change upon storage of the formulation at 25° C. / 60% RH for 2 months, and / or 3 months, and / or 4 months; the formulation releases 80% or more of the ganaxolone in 500 ml of dissolution medium (e.g., 5% SLS in SGF (simulated gastric fluid) and / or 5% SLS in SIF (simulated intestinal fluid)) in a USP Apparatus 1 (Basket) at 100 rpm, 37° C.+0.5° C., and 45 minutes; and the formulation maintains plasma levels of ganaxolone of about 55 ng / mL, about 60 ng / ml, or about 65 ng / ml for at least 6 hours after administration. and for a period of 12 hours to a plasma level of about 240 ng / ml to 400 ng / ml (e.g., 262 ng / mL), and for treating in humans a disorder selected from the group consisting of PCDH19-associated epilepsy, CDKL5 epileptic encephalopathy, Dravet syndrome, Lennox-Gastaut syndrome (LGS), continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), and other intractable genetic epileptic conditions clinically similar to PCDH19-associated epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES.

[0018] The present invention further relates to a pregnenolone neurosteroid and a composition comprising the pregnenolone neurosteroid for use in a method for treating an epileptic disorder in a mammal (e.g., a human), wherein the pregnenolone neurosteroid is administered orally or parenterally to the mammal after determining that the mammal has low plasma levels of the endogenous neurosteroid. The endogenous neurosteroid may be, for example, allopregnanolone or allopregnanolone sulfate. Low plasma levels of allopregnanolone sulfate are below 2500 pg mL -1 A low plasma level of allopregnanolone is 200 pg mL -1 Plasma levels of: 2500 pg mL -1 Plasma levels of allopregnanolone sulfate below 200 pg mL indicate that a mammal is likely to respond to treatment with pregnenolone neurosteroids (i.e., experience a 25% or greater reduction in seizure frequency). -1A plasma level of allopregnanolone below 100% indicates that the mammal is likely to respond to treatment with the pregnenolone neurosteroid (i.e., experience a 25% or greater reduction in seizure frequency). A low level of endogenous neurosteroid may be determined by obtaining a biological sample (e.g., plasma) from the mammal and performing an assay on the biological sample to determine the level of endogenous neurosteroid. The results of the assay may be communicated to the mammal or a healthcare provider before or after administration of the pregnenolone neurosteroid. The pregnenolone neurosteroid may be, for example, a compound of Formula IA, a compound of Formula IB, a compound of Formula II, or a compound of Formula III. In a preferred embodiment, the pregnenolone neurosteroid is selected from the group consisting of allopregnanolone, pregnanolone, 5-alpha DHP (5-alpha dihydroprogesterone), pregnanolone, dehydroepiandrosterone (DHEA), ganaxolone, 3α-hydroxy-3β-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5β-pregnan-20-one, pharmaceutically acceptable salts of any of the foregoing, and combinations of any of the foregoing. In a more preferred embodiment, the pregnenolone neurosteroid is ganaxolone. The pregnenolone neurosteroid can be administered in an amount of about 1 mg / day to about 5000 mg / day in one, two, three, or four divided doses. The pregnenolone neurosteroid may be administered for, for example, at least 1 day, at least 2 days, at least 3 days, 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks. When administered orally, the pregnenolone neurosteroid may be administered in an oral suspension or capsule as described herein. The oral suspension and capsule may contain particles containing the pregnenolone neurosteroid, the particles having an average particle size of about 0.3 microns (i.e., a volume-weighted median diameter (D50) of about 0.3 microns). Preferably, the average particle size does not change after the oral suspension or capsule is stored at 25°C / relative 60% RH for one month.The oral suspension may be administered three times daily, and the capsules may be administered twice daily. Administration of the pregnenolone neurosteroid results in a plasma level of the pregnenolone neurosteroid of about 55 ng / mL, about 60 ng / mL, or about 65 ng / mL, to about 240 ng / mL to about 400 ng / mL (e.g., 262 ng / mL) for at least about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, or about 12 hours. Preferably, the subject experiences a reduced frequency of symptoms (e.g., seizures) of the epileptic disorder. The epileptic disorder may be selected from the group consisting of, for example, CDKL5 deficiency, PCDH19-associated epilepsy, Lennox-Gastaut syndrome, Ohtahara syndrome, early myoclonic epileptic encephalopathy, West syndrome, Dravet syndrome, Angelman syndrome, continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), Rett syndrome, Fragile X syndrome, X-linked myoclonic seizures, spasticity and intellectual disability syndrome, idiopathic infantile epilepsy-dyskinetic encephalopathy, epilepsy and mental retardation limited to females, and severe infantile multifocal epilepsy. In a more preferred embodiment, the epileptic disorder is PCDH19-associated epilepsy, Dravet syndrome, Lennox-Gastaut syndrome (LGS), continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), or other intractable epileptic conditions and intractable genetic epileptic conditions clinically similar to PCDH19-associated epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES. The reduction in seizure frequency achieved by administration of a pregnenolone neurosteroid is generally 35% or greater, preferably about 40% or greater, more preferably about 45% or greater, and more preferably about 50% or greater, after 28 days of administration of the pregnenolone neurosteroid, compared to the seizure frequency during the 28 days prior to the first administration.

[0019] The present invention also relates to methods for treating epileptic disorders, comprising identifying a mammal (e.g., a human) with an epileptic disorder, determining whether the mammal has low levels of endogenous neurosteroids, and, if the mammal has low plasma levels of endogenous neurosteroids, administering to the mammal a dosing regimen of a pharmaceutically acceptable pregnenolone neurosteroid in an amount effective to reduce seizure frequency in the mammal, as well as the use of pregnenolone neurosteroids and compositions comprising pregnenolone neurosteroids in such methods. The endogenous neurosteroid may be, for example, allopregnanolone or allopregnanolone sulfate. Low plasma levels of allopregnanolone sulfate are defined as those below 2500 pg mL -1 Low levels of allopregnanolone are 200 pg mL -1The epileptic disorder may be selected from the group consisting of CDKL5 deficiency, PCDH19-associated epilepsy, Lennox-Gastaut syndrome, Ohtahara syndrome, early myoclonic epileptic encephalopathy, West syndrome, Dravet syndrome, Angelman syndrome, continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), Rett syndrome, fragile X syndrome, X-linked myoclonic seizures, spasticity and intellectual disability syndrome, idiopathic infantile epilepsy-dyskinetic encephalopathy, epilepsy and mental retardation limited to females, and severe infantile multifocal epilepsy. The dosing regimen may be administered orally or parenterally. The pregnenolone neurosteroid may be a compound of formula IA (e.g., ganaxolone). The pregnenolone neurosteroid may be administered in an amount of about 1 mg / day to about 5000 mg / day in 1, 2, 3, or 4 divided doses for at least 1 day, at least 2 days, at least 3 days, 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks. Administration of the pregnenolone neurosteroid results in a plasma level of the pregnenolone neurosteroid of about 55 ng / mL, about 60 ng / mL, or about 65 ng / mL to about 240 ng / mL to 400 ng / mL (e.g., 262 ng / mL) for at least about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, or about 12 hours. Preferably, the subject experiences a reduction in seizure frequency.

[0020] The present invention further relates to ganaxolone and compositions comprising ganaxolone for use in methods for treating epileptic disorders in mammals (e.g., humans), wherein the ganaxolone is administered orally or parenterally to the mammal after determining that the mammal has low plasma levels of endogenous neurosteroids. The endogenous neurosteroid may be, for example, allopregnanolone or allopregnanolone sulfate. Low plasma levels of allopregnanolone sulfate are below 2500 pg mL -1A low plasma level of allopregnanolone is 200 pg mL -1 Plasma levels of: 2500 pg mL -1 Plasma levels of allopregnanolone sulfate below 200 pg mL indicate that a mammal is likely to respond to treatment with ganaxolone (i.e., experience a 25% or greater reduction in seizure frequency). -1A plasma level of allopregnanolone below or equal to 0.05 indicates that the mammal is likely to respond to treatment with ganaxolone (i.e., experience a 25% or greater reduction in seizure frequency). Low levels of endogenous neurosteroids may be determined by obtaining a biological sample (e.g., plasma) from the mammal and performing an assay on the biological sample to determine the level of endogenous neurosteroids. The results of the assay may be communicated to the mammal or a healthcare provider before or after administration of ganaxolone. Ganaxolone can be administered in an amount of about 1 mg / day to about 5000 mg / day in one, two, three, or four divided doses. Ganaxolone may be administered, for example, for at least 1 day, at least 2 days, at least 3 days, 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks. When administered orally, ganaxolone may be administered in an oral suspension or capsule, as described herein. The oral suspension may be administered three times daily, and the capsule may be administered twice daily. The oral suspension or capsule may contain particles containing ganaxolone, the particles having an average particle size of about 0.3 microns (i.e., a volume-weighted median diameter (D50) of about 0.3 microns). Preferably, the average particle size does not change after the oral suspension or capsule is stored at 25°C / 60% relative humidity for one month. The epileptic disorder may be selected from the group consisting of, for example, CDKL5 deficiency, PCDH19-associated epilepsy, Lennox-Gastaut syndrome, Ohtahara syndrome, early myoclonic epileptic encephalopathy, West syndrome, Dravet syndrome, Angelman syndrome, continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), Rett syndrome, Fragile X syndrome, X-linked myoclonic seizures, spasticity and intellectual disability syndrome, idiopathic infantile epilepsy-dyskinetic encephalopathy, epilepsy and mental retardation limited to females, and severe infantile multifocal epilepsy.In a more preferred embodiment, the epileptic disorder is PCDH19-associated epilepsy, Dravet syndrome, Lennox-Gastaut syndrome (LGS), continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), or other intractable epileptic conditions and intractable genetic epileptic conditions clinically similar to PCDH19-associated epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES. For example, after 28 days of administration, seizure frequency may be reduced by 35% or more, preferably about 40% or more, more preferably about 45% or more, or more preferably about 50% or more, compared to the seizure frequency in the 28 days prior to the first administration. The method may further include establishing a baseline seizure frequency in the mammal; initially administering to the mammal an amount of ganaxolone from about 0.5 mg / kg / day to about 15 mg / kg / day; and gradually increasing the dose of ganaxolone over four weeks to an amount of about 18 mg / kg / day to about 65 mg / kg / day. The total dose of ganaxolone may be up to about 1800 mg / day. For mammals weighing 30 kg or less, the total daily dose of ganaxolone may be less (e.g., about 63 mg / day).

[0021] The present invention also relates to a pregnenolone neurosteroid or a composition comprising a pregnenolone neurosteroid for use in a method of treating a mammal with an epileptic disorder, the method comprising determining whether the mammal has low levels of endogenous neurosteroids; and, if the mammal has low levels of endogenous neurosteroids, chronically administering to the mammal a pharmaceutically acceptable pregnenolone neurosteroid. In a preferred embodiment, the mammal is a human; the epileptic disorder is selected from the group consisting of CDKL5 deficiency, PCDH19-associated epilepsy, Lennox-Gastaut syndrome, Rett syndrome, and Fragile X syndrome; the endogenous neurosteroid is allopregnanolone sulfate; and the low levels of endogenous neurosteroids are 2500 pg mL-1 or less. -1 and / or the endogenous neurosteroid is allopregnanolone, and low levels of endogenous neurosteroids are 200 pg mL-1 the pregnenolone neurosteroid is ganaxolone, and the ganaxolone is orally administered in an amount of about 1 mg / day to about 5000 mg / day in 1, 2, 3, or 4 divided doses for at least 1 day, at least 2 days, at least 3 days, 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks. Administration of ganaxolone preferably reduces seizure frequency by 35% or more, preferably about 40% or more, more preferably about 45% or more, or more preferably about 50% or more after 28 days of administration compared to the seizure frequency in the 28 days prior to the first administration.

[0022] The present invention further relates to methods for treating a mammal with a genetic epileptic disorder, comprising chronically administering a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) to the mammal with the genetic epileptic disorder in an amount effective to reduce seizure frequency in the mammal, and to the use of pregnenolone neurosteroids and compositions comprising pregnenolone neurosteroids in such methods. In certain preferred embodiments, the mammal is a human; and the epileptic disorder is a genetic epileptic disorder, such as early infantile epileptic encephalopathy. In certain preferred embodiments, the disorder is selected from, for example, cyclin-dependent kinase-like 5 ("CDKL5") deficiency, protocadherin 19 ("PCDH19") epilepsy, Lennox-Gastaut syndrome ("LGS"), Rett syndrome, fragile X syndrome, Ohtahara syndrome, early myoclonic epileptic encephalopathy, West syndrome, Dravet syndrome, Angelman syndrome, persistent spikes and waves during sleep (CSWS) epilepsy syndrome, and other diseases, such as X-linked myoclonic seizures, spasticity and intellectual disability syndrome, idiopathic infantile epilepsy-dyskinetic encephalopathy, epilepsy and mental retardation limited to females, and severe infantile multifocal epilepsy. In some of these embodiments, the human has low levels of endogenous neurosteroids (e.g., allopregnanolone sulfate (Allo-S)).

[0023] The present invention also relates to a method of treating a mammal having an epileptic encephalopathy, the method comprising orally administering to the mammal twice daily (e.g., every 10-13 hours) a solid oral immediate release formulation comprising a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone), wherein the half-life of the neurosteroid is about 18 to about 24 hours, the formulation releases about 70% or about 80% or more of the ganaxolone within 45 minutes of placement of the formulation in simulated gastrointestinal fluid (SGF and / or SIF), and wherein administration results in a reduction in seizure frequency per 28 days of at least about 35%, about 40%, about 45%, or about 50% compared to the seizure frequency in the 28 days prior to the first administration.

[0024] The present invention further relates to a method of treating a mammal having an epileptic encephalopathy, the method comprising orally administering to the mammal three times daily (e.g., every 6-8 hours) a liquid oral immediate release formulation comprising a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone), wherein the half-life of the neurosteroid is about 18 to about 24 hours, and the formulation releases about 70% or about 80% or more of the ganaxolone within 45 minutes of placement of the formulation in simulated gastrointestinal fluid (SGF and / or SIF), wherein administration results in a reduction in seizure frequency per 28 days of at least about 35%, about 40%, about 45%, or about 50% compared to the seizure frequency in the 28 days prior to the first administration.

[0025] The present invention also relates to a method for treating a patient with a pregnenolone neurosteroid, wherein the human is suffering from encephalopathy, the method comprising: By obtaining biological samples from humans; and by performing an assay on the biological sample to determine the level of endogenous neurosteroids; Determining whether a human has low levels of endogenous neurosteroids, comprising: 2500pg mL -1 Below, 2000pg mL -1 Below, 1500pg mL -1Below, 1000pg mL -1 Below, 900pg mL -1 Below, 800pg mL -1 Below, 700pg mL -1 Below, 600pg mL -1 Below, 500pg mL -1 Below, 400pg mL -1 Below, 300pg mL -1 Below, 200pg mL -1 Below, 100pg mL -1 Below, 75pg mL -1 Below, 50pg mL -1 or less than 25 pg mL -1 the following levels of endogenous neurosteroids indicate that the human has low levels of endogenous steroids: and if the person has low levels of endogenous steroids, orally administering to the patient a pregnenolone neurosteroid (e.g., ganaxolone) at a dose of 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day in two or three divided doses for at least one day. In some of these embodiments, the method comprises administering to a subject a subject in need thereof a method for treating a subject in need thereof, the method comprising ... -1 Below, 2000pg mL -1 Below, 1500pg mL -1 Below, 1000pg mL -1 Below, 900pg mL -1 Below, 800pg mL -1 Below, 700pg mL -1 Below, 600pg mL -1 Below, 500pg mL -1 Below, 400pg mL -1 Below, 300pg mL -1 Below, 200pg mL -1 Below, 100pg mL -1 Below, 75pg mL-1 Below, 50pg mL -1 or less than 25 pg mL -1 The presence of the following expression level indicates that administration of the ganaxolone is likely to reduce the patient's seizure frequency after 28 days of administration, for example, by 35% or more; about 40% or more; about 45% or more; or about 50% or more, compared to the seizure frequency during the 28 days prior to the first administration. The endogenous neurosteroid may be selected from the group including or consisting of pregnanolone, pregnanolone sulfate, 5-alpha DHP, allopregnanolone, allopregnanolone-S, pregnanolone, pregnanolone-S, DHEA, and combinations thereof. The pregnenolone neurosteroid may be selected from the group including or consisting of allopregnanolone, ganaxolone, alphaxalone, alphadolone, hydroxydione, minaxolone, pregnanolone, acebrocol, or tetrahydrocorticosterone, and pharmaceutically acceptable salts thereof. In some of these embodiments, the method further comprises communicating the results of the assay to the patient or healthcare provider before or after administration of the pregnenolone neurosteroid.

[0026] The present invention also provides a method of treating a human with ganaxolone, wherein the human is suffering from encephalopathy, the method comprising: Human allopregnanolone sulfate levels of 2500 pg mL -1 determining whether: Human allopregnanolone sulfate levels of 2500 pg mL -1 orally administering ganaxolone to a human in two or three divided doses for at least one day at a dose of 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day; In some of these embodiments, the method comprises administering 2500 pg mL -1The following allopregnanolone sulfate levels indicate that administration of the ganaxolone is likely to reduce seizure frequency in a human by, for example, at least about 35%, about 40%, about 45%, or about 50% after 28 days of administration compared to the seizure frequency in the 28 days prior to the first administration.

[0027] The present invention further provides a method of treating a human with ganaxolone, wherein the human is suffering from encephalopathy, the method comprising: Human allopregnanolone sulfate levels of 2500 pg mL -1 determining whether: Human allopregnanolone sulfate levels of 2500 pg mL -1 If the patient has: endogenous neurosteroids (e.g., allopregnanolone, pregnanolone) or synthetic neurosteroids (e.g., Co26749 / WAY-141839, Co134444, Co177843, Sage-217 (3α-hydroxy-3β-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5β-pregnan-20-one), ganaxolone) at 1 mg / orally administering to a human in two or three divided doses for at least one day at a dose of about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day; Human allopregnanolone sulfate levels of 2500 pg mL -1 and refraining from administering endogenous or synthetic neurosteroids to the human and / or administering different anticonvulsants if the neurosteroid concentration exceeds The different anticonvulsant drugs are, for example, selected from the group consisting of benzodiazepines (e.g., clobazam, diazepam, clonazepam, midazolam), clorazepic acid, levetiracetam, felbamate, lamotrigine, fatty acid derivatives (e.g., valproic acid), carboxamide derivatives (rufinamide, carbamazepine, oxcarbazepine, etc.), amino acid derivatives (e.g., levocarnitine), barbiturates (e.g., phenobarbital), or a combination of two or more of the foregoing agents.

[0028] The present invention also provides a method of treating a human with ganaxolone, wherein the human is suffering from encephalopathy, the method comprising: Human allopregnanolone sulfate levels of 2500 pg mL -1 determining whether: Human allopregnanolone sulfate levels of 2500 pg mL -1 orally administering ganaxolone to a human in two or three divided doses for at least one day at a dose of 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day; In some of these embodiments, the method comprises administering 2500 pg mL -1 The following allopregnanolone sulfate levels indicate that administration of the ganaxolone is likely to reduce seizure frequency in a human by, for example, at least about 35%, about 40%, about 45%, or about 50% after 28 days of administration compared to the seizure frequency in the 28 days prior to the first administration.

[0029] The present invention further provides a method of treating a human with ganaxolone, wherein the human is suffering from encephalopathy, the method comprising: Human allopregnanolone levels of 200 pg mL -1 determining whether: Human allopregnanolone levels of 200 pg mL -1 orally administering ganaxolone to a human at a dose of 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day in two or three divided doses for at least one day; Human allopregnanolone levels of 200 pg mL -1 and refraining from administering ganaxolone to a human if In some of these embodiments, 200 pg mL -1 The following allopregnanolone levels indicate that administration of the ganaxolone is likely to reduce seizure frequency in a human by, for example, at least about 35%, about 40%, about 45%, or about 50% after 28 days of administration compared to the seizure frequency in the 28 days prior to the first administration:

[0030] The present invention further provides a method of treating a human with ganaxolone, wherein the human is suffering from encephalopathy, the method comprising: Human allopregnanolone levels of 200 pg mL -1 determining whether: Human allopregnanolone levels of 200 pg mL -1 orally administering ganaxolone to a human at a dose of 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day in two or three divided doses for at least one day; Human allopregnanolone levels of 200 pg mL -1 and refraining from administering ganaxolone to a human if The present invention relates to a method comprising:

[0031] The present invention also provides a method of treating encephalopathy in a human, comprising administering a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) to the human at a dose of about 1800 mg or less per day for at least one day, wherein the human is also consuming a number of neurosteroids, including ALDH7A1, KCNQ2, KCNQ3, TBC1D24, PRRT2, SCN2A, SCN8A, ST3GAL5, CACNA1A, GABRA1, GABRB3, KCNT1, AARS, ARV1, DOCK7, FRRS1L, GUF1, ITPA, NECAP1, PLCB1, SLC12A5, SLC12A6, SLC12B7, SLC12B8, SLC12C9, SLC12C14, SLC12C15, SLC12C16, SLC12C17, SLC12C18, SLC12C20, SLC12C30, SLC12C40, SLC12C50, SLC12C60, SLC12C71, SLC12C82, SLC12C93, SLC12C14, SLC12C15, SLC12C16, SLC12C18, SLC12C19, SLC12C22, SLC12C23, SLC12C24, SLC12C25, SLC12C30, SLC12C44, SLC12C16, SLC12C25, SLC12C26, SLC12C31, SLC12C42, SLC12C51, SLC12C14, SLC12C27, SLC12C28, SLC12C32, SLC12C43, SLC12C52, SLC12C SLC13A5, SLC25A12, SLC25A22, ST3GAL3, SZT2, WWOX, CDKL5, ARHGEF9, ALG13, PCDH19, DNM1, EEF1A2, FGF12, GABRB1, GNAO1, GRIN2B, GRIN2D, HCN1, KC NA2, KCNB1, SIK1, SLC1A2, SPTAN1, STXBP1, UBA5, SCN1A, SCN9Ab, GPR98, SCN9A, CPA6, GABRD, GABRG2, SCN1B, STX1B, KCNMA1, SLC6A1, CHD2, GRIN2A, CA CNA1H, CLCN2a, EFHC1, CACNB4, SLC2A1, CASR, ADRA2B, CNTN2, GAL, GI1, KCNC1, CERS1, CSTB, EPM2A, GOSR2, KCTD7, LMNB2, NHLRC1, PRDM8, PRICKLE1, S CARB2, CHRNA2, CHRNA4, CHRNB2, DEPDC5, UBE3A, MeCP2, TSC1, TSC2, FOXG1, TPP1, ZEB2, ARX, CHRNA7, TCF4, POLG, SLC9A6, MEF2C, MBD5, CLN3, CLN5, CLN 6, ATP1A2, LG11, KANSL1, GAMT, CNTNAP2, KCNJ10, PNKP, PPT1, ADSL, MFSD8, SYN1, CLN8, ATP6AP2, CTSD, DNAJC5, FOLR1, GATM, GOSR2, LIAS, MAG12, NRXN1, SRPX2, and a combination of any two or more of the foregoing, and at least one of the symptoms experienced by the person is (i) uncontrolled cluster seizures (three or more seizures over a 12-hour period) for a period of 4 to 8 weeks (e.g., 6 weeks);(ii) intermittent seizures of status epilepticus; (iii) uncontrolled non-cluster seizures (focal cognitive impairment, focal convulsions, atypical absence, unilateral seizures, convulsions, or tonic seizures) with a seizure frequency of ≥ 4 over a 4-8 week (e.g., 4 week) period; (iv) ≥ 4 generalized convulsive seizures (tonic-clonic, tonic, clonic, or atonic) over a 4-8 week (e.g., 4 week) period; and (v) a combination of any two or more of the foregoing. In some of these embodiments, the pharmaceutically acceptable pregnenolone neurosteroid is ganaxolone and is administered orally in an amount of about 200 mg / day to about 1800 mg / day, about 300 mg / day to about 1800 mg / day, about 400 mg / day to about 1800 mg / day, about 450 mg / day to about 1800 mg / day, about 675 mg / day to about 1800 mg / day, about 900 mg / day to about 1800 mg / day, about 1125 mg / day to about 1800 mg / day, about 1350 mg / day to about 1800 mg / day, about 1575 mg / day to about 1800 mg / day, or about 1800 mg / day, administered orally in two or three divided doses. In some embodiments, administration of a pharmaceutically acceptable pregnenolone neurosteroid reduces the average seizure frequency per 28 days by 35% or more (e.g., about 40%, about 45%, about 50%, about 55%) compared to the seizure frequency in the 28 days prior to the first administration. In some embodiments, the improvement is 50% or more.

[0032] The present invention also relates to the treatment of human patients experiencing early-onset infantile epileptic encephalopathies, including, but not limited to, Ohtahara syndrome, early myoclonic epileptic encephalopathy, West syndrome, Dravet syndrome, PCDH19 (protocadherin 19) epilepsy, CDKL5 (cyclin-dependent kinase-like 5) epilepsy, Lennox-Gastaut syndrome (LGS), persistent spikes during sleep (CSWS), and other disorders such as X-linked myoclonic seizures, spasticity and intellectual disability syndrome, idiopathic infantile epilepsy-dyskinetic encephalopathy, epilepsy and mental retardation limited to females, and severe infantile multifocal epilepsy. The method involves administering a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) to a mammal at a dose of about 1 mg / kg / day to about 63 mg / kg / day. However, the total dose of ganaxolone should not exceed 1800 mg / day.

[0033] The present invention further provides a method for treating epilepsy in a patient with (i) uncontrolled cluster seizures (3 or more seizures over a 12-hour period) for a period of 4-8 weeks (e.g., 6 weeks), and / or (ii) intermittent bouts of status epilepticus, and / or (iii) uncontrolled non-cluster seizures (focal cognitive impairment, focal convulsions, atypical absence, unilateral seizures, convulsions, or tonic seizures) with a seizure frequency of ≥ 4 for a period of 4-8 weeks (e.g., 4 weeks), and / or (iv) ≥ 4 generalized convulsive seizures (tonic-clonic, tonic, clonic, atonic) for a period of 4-8 weeks (e.g., 4 weeks). A method of treating a mammal (e.g., a human) with a history of the disease, comprising administering a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) to the mammal at a dose of about 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day, provided that the total amount of ganaxolone administered does not exceed 1800 mg / day.

[0034] In an additional aspect, the present invention relates to a method of treating a mammal (e.g., a human) with subclinical CSWS syndrome, with or without EEG clinical events, comprising administering a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) to the mammal at a dose of about 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day, provided that the total amount of ganaxolone administered does not exceed 1800 mg / day.

[0035] The present invention relates, in part, to the use of pregnenolone neurosteroids, such as ganaxolone, in the treatment of genetically related early-onset infantile epileptic encephalopathies, such as PCDH19 female-predominant epilepsy and CDKL5 deficiency. Administration of pregnenolone neurosteroids in accordance with the present invention can help compensate for the effects of allopregnanolone deficiency.

[0036] The present invention also relates to a method of treating a mammal (e.g., a human) with a PCDH19 disorder, comprising administering a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) to the mammal at a dose of about 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day, provided that the total amount of ganaxolone administered does not exceed 1800 mg / day.

[0037] The present invention also relates to a method of treating a mammal (e.g., a human) with Dravet syndrome, comprising administering a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) to the mammal at a dose of about 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day, provided that the total amount of ganaxolone administered does not exceed 1800 mg / day.

[0038] The present invention also relates to a method of treating a mammal with LGS, comprising administering a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) to the mammal at a dose of about 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day, provided that the total amount of ganaxolone administered does not exceed 1800 mg / day.

[0039] The present invention also relates to a method of treating a mammal with CSWS, comprising administering a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) to the mammal at a dose of about 1 mg / kg / day to about 63 mg / kg / day, about 2 mg / kg / day to about 63 mg / kg / day, about 3 mg / kg / day to about 63 mg / kg / day, about 4 mg / kg / day to about 63 mg / kg / day, about 5 mg / kg / day to about 63 mg / kg / day, about 6 mg / kg / day to about 63 mg / kg / day, or about 7 mg / kg / day to about 63 mg / kg / day, provided that the total amount of ganaxolone administered does not exceed 1800 mg / day.

[0040] In certain embodiments, the methods of the present invention further include periodic measurement of plasma levels of the administered pharmaceutically acceptable pregnenolone neurosteroid, and / or concomitant AEDs, if present, and / or allopregnanolone (3α-hydroxy-5α-pregnan-20-one), and / or related endogenous CNS-active steroids. In some embodiments, plasma levels of liver enzymes (AST, ALT, and ALK Phos) are also measured before, during, or after initiation of treatment with the pharmaceutically acceptable pregnenolone neurosteroid. Plasma levels may be measured, for example, weekly, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, or every 12 weeks.

[0041] In certain embodiments, low endogenous levels of neurosteroids can be measured in humans as plasma allopregnanolone sulfate of about 2500 pg / ml or less. Thus, low endogenous levels of neurosteroids in humans can be measured in humans as, for example, 2400 pg / ml or less, 2300 pg / ml or less, 2200 pg / ml or less, 2100 pg / ml or less, 2000 pg / ml or less, 1900 pg / ml or less, 1800 pg / ml or less, 1700 pg / ml or less, 1600 pg / ml or less, 1500 pg / ml or less, 1400 pg / ml or less, 1300 pg / ml or less, 1200 pg / ml or less, 1100 pg / ml or less, 1000 pg / ml or less, 900 pg / ml or less, It may be 850pg / ml or less, 800pg / ml or less, 750pg / ml or less, 700pg / ml or less, 650pg / ml or less, 600pg / ml or less, 550pg / ml or less, 500pg / ml or less, 450pg / ml or less, 400pg / ml or less, 350pg / ml or less, 300pg / ml or less, 250pg / ml or less, 200pg / ml or less, 1500pg / ml or less, 100pg / ml or less, 50pg / ml or less, 25pg / ml or less, 10pg / ml or less, or 5pg / ml or less.

[0042] In certain embodiments, low endogenous levels of neurosteroids can be measured in humans as plasma allopregnanolone of about 200 pg / ml or less. Thus, low endogenous levels of neurosteroids in humans can be, for example, 200 pg / ml or less, 199 pg / ml or less, 198 pg / ml or less, 197 pg / ml or less, 196 pg / ml or less, 195 pg / ml or less, 194 pg / ml or less, 193 pg / ml or less, 192 pg / ml or less, 191 pg / ml or less, 190 pg / ml or less, 189 pg / ml or less, 188 pg / ml or less, 187 pg / ml or less, 186 pg / ml or less, 185 pg / ml or less, 184 pg / ml or less, 183 pg / ml or less, 182pg / ml or less, 181pg / ml or less, 180pg / ml or less, 179pg / ml or less, 178pg / ml or less, 177pg / ml or less, 176pg / ml or less, 175pg / ml or less, 174pg / ml or less, 172pg / ml or less, 171pg / ml or less, 170pg / ml or less, 169pg / ml or less, 168pg / ml or less, 167pg / ml or less, 166pg / ml or less, 165pg / ml or less, 164pg / ml or less, 163pg / ml or less, 162pg / ml or less, 161pg / ml or less, 160pg / ml or less, 159pg / ml or less, 158pg / ml or less, 157pg / ml or less, 156pg / ml or less, 155pg / ml or less, 154pg / ml or less, 153pg / ml or less, 152pg / ml or less, 151pg / ml or less, 150pg / ml or less, 149pg / ml or less, 148pg / ml or less, 147pg / ml or less, 146pg / ml or less, 145pg / ml or less, 144pg / ml or less, 143pg / ml or less, 142pg / ml or less, 141pg / ml or less, 140pg / ml or less, 139pg / ml or less, 138pg / ml or less, 137pg / ml or less, 136pg / ml or less, 135pg / ml or less, 134pg / ml or less, 133pg / ml or less, 132pg / ml or less, 131pg / ml or less, 130pg / ml or less, 129pg / ml or less, 128pg / ml or less, 127pg / ml or less, 126pg / ml or less, 125pg / ml or less, 124pg / ml or less, 123pg / ml or less, 122pg / ml or less, 121pg / ml or less, 120pg / ml or less, 119pg / ml or less,118pg / ml or less, 117pg / ml or less, 116pg / ml or less, 115pg / ml or less, 114pg / ml or less, 113pg / ml or less, 112pg / ml or less, 111pg / ml or less, 110pg / ml or less, 109pg / ml or less, 108pg / ml or less, 107pg / ml or less, 106pg / ml or less, 105pg / ml or less, 104pg / ml or less, 103pg / ml or less, 102pg / ml or less, 101pg / ml or less, 100pg / ml or less, 99pg / ml or less, 98pg / ml or less, 97pg / ml or less, 96pg / ml or less, 95pg / ml or less, 94pg / ml or less, 93pg / ml or less, 92pg / ml or less, 91pg / ml or less, 90pg / ml or less, 89pg / ml or less, 88pg / ml or less, 87pg / ml or less, 86pg / ml or less, 85pg / ml or less, 84pg / ml or less, 83pg / ml or less, 82pg / ml or less, 81pg / ml or less, 80pg / ml or less, 79pg / ml or less, 78pg / ml or less, 77pg / ml or less, 76pg / ml or less, 75pg / ml or less, 74pg / ml or less, 73pg / ml or less, 72pg / ml or less, 71pg / ml or less, 70pg / ml or less, 69pg / ml or less, 68pg / ml or less, 67pg / ml or less, 66pg / ml or less, 65pg / ml or less, 64pg / ml or less, 63pg / ml or less, 62pg / ml or less, 61pg / ml or less, 60pg / ml or less, 59pg / ml or less, 58pg / ml or less, 57pg / ml or less, 56pg / ml or less, 55pg / ml or less, 54pg / ml or less, 53pg / ml or less, 52pg / ml or less, 51pg / ml or less, 50pg / ml or less, 49pg / ml or less, 48pg / ml or less, 47pg / ml or less, 46pg / ml or less, 45pg / ml or less, 44pg / ml or less, 43pg / ml or less, 42pg / ml or less, 41pg / ml or less, 40pg / ml or less, 39pg / ml or less, 38pg / ml or less, 37pg / ml or less, 36pg / ml or less, 35pg / ml or less, 34pg / ml or less, 33pg / ml or less, 32pg / ml or less, 31pg / ml or less, 30pg / ml or less, 29pg / ml or less, 28pg / ml or less, 27pg / ml or less, 26pg / ml or less, 25pg / ml or less, 24pg / ml or less, 23pg / ml or less, 22pg / ml or less, 21pg / ml or less,It may be 20 pg / ml or less, 19 pg / ml or less, 18 pg / ml or less, 17 pg / ml or less, 16 pg / ml or less, 15 pg / ml or less, 14 pg / ml or less, 13 pg / ml or less, 12 pg / ml or less, 11 pg / ml or less, 10 pg / ml or less, 9 pg / ml or less, 8 pg / ml or less, 7 pg / ml or less, 6 pg / ml or less, 5 pg / ml or less, 4 pg / ml or less, 3 pg / ml or less, 2 pg / ml or less, 1 pg / ml or less, or 0 pg / ml.

[0043] Pregnenolone neurosteroids can be administered orally or parenterally. In certain preferred embodiments, the pregnenolone neurosteroid is ganaxolone, administered as an oral suspension or oral solid dosage form (e.g., oral capsule) at a total dose of up to 63 mg / kg / day, and ganaxolone is preferably administered in an amount of up to 1800 mg / day. Preferably, ganaxolone is administered chronically, for example, as long as the patient receives therapeutic benefit from the treatment without undesirable side effects that require discontinuation of treatment. In certain embodiments, ganaxolone is administered for at least 1 day, at least 2 days, at least 3 days, 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, or at least 12 weeks.

[0044] When administered as an oral suspension, the pregnenolone neurosteroid may be administered, for example, anywhere from 1 to about 3 times per day. In certain preferred embodiments, when administered orally, the pregnenolone neurosteroid (e.g., ganaxolone) may be administered with food (for better absorption) or without food. When administered as an oral tablet or capsule, the pregnenolone neurosteroid may be administered, for example, anywhere from 1 to about 4 times per day. When administered parenterally, the pregnenolone neurosteroid may be administered, for example, anywhere from 1 to about 3 times per day.

[0045] The present invention further provides a method for treating gene-related early-onset infantile epileptic encephalopathy, comprising identifying a human patient suffering from gene-related early-onset infantile epileptic encephalopathy, determining whether the human patient has low endogenous levels of a neurosteroid, and administering to the human patient a dosing regimen of a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) in an amount effective to reduce the frequency of seizures in the human patient. A low level of endogenous neurosteroid is, for example, below 2500 pg mL -1 Allopregnanolone sulfate levels of less than or equal to 200 mg mL -1 The level of allopregnanolone may be as follows: In certain embodiments, the gene-related early-onset infantile epileptic encephalopathy is selected from, for example, CDKL5 deficiency, PCDH19 epilepsy, Lennox-Gastaut syndrome, Rett syndrome, fragile X syndrome, Ohtahara syndrome, early myoclonic epileptic encephalopathy, West syndrome, Dravet syndrome, and other disorders, for example, X-linked myoclonic seizures, spasticity and intellectual disability syndrome, idiopathic infantile epilepsy-dyskinetic encephalopathy, epilepsy and mental retardation limited to females, and severe infantile multifocal epilepsy. In certain embodiments, the gene-related early-onset infantile epileptic encephalopathy is CDKL5, and the patient has a CDKL5 gene mutation.

[0046] The present invention also relates to a method for treating a symptom or syndrome of a genetic epileptic encephalopathy, comprising testing a subject for a PCDH19 gene mutation and / or a CDKL5 gene mutation and / or an SCN1A mutation, and, if the subject has a PCDH19 gene mutation and / or a CDKL5 gene mutation and / or an SCN1A mutation, administering a therapeutically effective amount of a pregnenolone neurosteroid (e.g., ganaxolone) to the subject over a long period of time, the method including the step of communicating the results of the genetic testing to the subject and / or a healthcare provider after the testing and / or before the administration.

[0047] The present invention also relates to a method for treating a symptom or syndrome of a genetic epileptic encephalopathy, comprising determining whether a subject has multiple types of generalized seizures, including, for example, a reduction in seizures (atonic, tonic, or myoclonic) for at least six months and an EEG pattern reporting diagnostic criteria for LGS (abnormal background activity with a slow spike pattern <2.5 Hz) at some point in the subject's medical history, and, if so, administering a therapeutically effective amount of a pregnenolone neurosteroid (e.g., ganaxolone) to the subject long-term, including the step of communicating the results of a genetic test to the subject and / or a healthcare provider after the test and before the administration.

[0048] The present invention also relates to a method for treating a symptom or syndrome of a genetic epileptic encephalopathy, comprising determining whether a subject's current or past EEG during sleep is consistent with a diagnosis of CSWS (e.g., continuous [85%-100%] predominantly bisynchronous 1.5-2 Hz [and 3-4 Hz] spikes during non-REM sleep), and, if so, subsequently administering a therapeutically effective amount of a pregnenolone neurosteroid (e.g., ganaxolone) to the subject over time, including the step of communicating the results of the genetic testing to the subject and / or a healthcare provider after the testing and prior to the administration.

[0049] The present invention also relates to a method of treating a symptom or syndrome of a genetic epileptic encephalopathy, comprising determining whether a subject has previously had a positive response to administration of steroids or ACTH, and, if the subject has, subsequently administering a therapeutically effective amount of a pregnenolone neurosteroid (e.g., ganaxolone) to the subject over an extended period of time, the method including communicating the results of a genetic test to the subject and / or a healthcare provider after said testing and prior to said administration.

[0050] definition The recitation of ranges of values is merely intended to serve as a shorthand method of referring individually to each separate value within the range, unless otherwise specified herein, and each separate value is incorporated herein as if it were individually set forth herein. The endpoints of all ranges are included within the range and are independently combinable. All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "etc."), is intended merely as illustrative and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0051] The terms "a" and "an" do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item.

[0052] The term "about" is used synonymously with the term "approximately." One of ordinary skill in the art will understand that the exact boundaries of "about" will depend on the components of the composition. Illustratively, the use of the term "about" indicates values slightly outside the cited value, i.e., plus or minus 0.1% to 10% outside, which are also effective and safe. Thus, compositions slightly outside the cited range are also encompassed by the claims.

[0053] An "active agent" is any compound, element, or mixture that, when administered to a patient alone or in combination with another agent, directly or indirectly exerts a physiological effect on the patient. When the active agent is a compound, salts, solvates (including hydrates) of the free compound or salt, crystalline and amorphous forms, and various polymorphs of the compound are included. Because compounds may contain one or more asymmetric elements, such as stereogenic centers and stereogenic axes, e.g., asymmetric carbon atoms, the compounds can exist in various stereoisomeric forms. These compounds can be, for example, racemic compounds or optically active forms. For compounds with two or more asymmetric elements, the compounds can also be mixtures of diastereomers. For compounds with asymmetric centers, it should be understood that pure optical isomers and mixtures thereof are all encompassed. Furthermore, compounds with carbon-carbon double bonds can exist in Z- and E-forms, and all isomeric forms of the compounds are encompassed by the present invention. In these situations, the single enantiomers, i.e., optically active forms, can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example, a chiral HPLC column.

[0054] The term "endogenous neurosteroids" refers to steroids that are produced in the brain and can regulate neuronal excitability by interacting with neuronal membrane receptors and ion channels, primarily GABA-A receptors, and includes, for example, pregnane neurosteroids (e.g., allopregnanolone, allotetrahydrodeoxycorticosterone), androstane neurosteroids (e.g., androstanediol, etiocholanone), and sulfated neurosteroids (e.g., pregnanolone sulfate, dehydroepiandrosterone sulfate (DHEAS)).

[0055] The term "pregnenolone neurosteroid" refers to endogenous or exogenous steroids that can modulate neuronal excitability by interacting with neuronal membrane receptors and ion channels, primarily GABA-A receptors, and includes, for example, endogenous neurosteroids and synthetic neurosteroids synthesized or derived from pregnenolone in vitro and in vivo.

[0056] The term "biomarker" refers to serum or plasma levels of neurosteroids that distinguish drug responders from non-responders.

[0057] As disclosed herein, the terms "serum" and "plasma" may be used interchangeably.

[0058] The terms "comprise," "include," and "containing" are non-limiting. Other unrecited elements may be present within embodiments claimed by these transitional phrases. When "comprise," "contain," or "include," is used as a transitional phrase, other elements may be included and still form embodiments within the scope of the claim. The open-ended transitional phrase "comprise" encompasses the intermediate transitional phrase "consisting essentially of" and the closed-ended phrase "consisting of."

[0059] A "bolus dose" is a relatively large amount of medication administered over a short period of time, for example, within 1 to 30 minutes.

[0060] "C max " is the concentration of the active agent in the plasma at the point of maximum concentration.

[0061] "Ganaxolone" is also known as 3α-hydroxy-5α-pregnan-20-one and is alternatively referred to as "GNX" in this document.

[0062] "Infusion" administration is parenteral administration, typically intravenous administration, although other parenteral routes such as epidural administration are included in some embodiments. Infusion administration occurs over a longer period than bolus administration, e.g., over a period of at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, or at least 4 hours.

[0063] A "patient" is a human or non-human animal in need of medical treatment. Medical treatment includes treatment of an existing condition, such as a disorder or injury. In certain embodiments, treatment also includes prophylactic or diagnostic treatment.

[0064] "Child" means a human being between the ages of 1 day and 18 years (eg, between the ages of 1 day and 15 years), inclusive.

[0065] "Adult" means a human being over the age of 18.

[0066] A "pharmaceutical composition" is a composition comprising at least one active agent, such as a compound of Formula (I), or a salt, solvate, or hydrate, and at least one other substance, such as a carrier. A pharmaceutical composition optionally contains one or more additional active agents. Where specified, the pharmaceutical composition meets U.S. FDA GMP (Good Manufacturing Practice) standards for human or non-human drugs. A "pharmaceutical combination" is a combination of at least two active agents, which may be combined in a single dosage form or provided together in separate dosage forms, with an indication that the active agents should be used together to treat a disorder, such as a seizure disorder.

[0067] "Povidone," also known as polyvidone and polyvinylpyrrolidone (PVP), is a water-soluble polymer formed from the monomer, N-vinylpyrrolidone. Plasdone C-12 and C-17 are pharmaceutical-grade homopolymers of N-vinylpyrrolidone. Plasdone C-12 has a K value of 10-2 to 13.8 and a nominal molecular weight of 4,000 d. Plasdone C-17 has a K value of 15.5 to 17.5 and a nominal molecular weight of 10,000 d.

[0068] "Sterilization" means the inactivation of substantially all biological contaminants in a sample, formulation, or product. A one million-fold reduction in bioburden is also considered "sterile" for most pharmaceutical applications.

[0069] The term "reducing" seizures or seizure activity refers to a detectable decrease in seizure frequency, severity, and / or duration. Reduction in seizure frequency, severity, and / or duration can be measured by self-assessment (e.g., patient report) or by a trained clinical observer. Determination of reduction in seizure frequency, severity, and / or duration can be made by comparing the patient's condition before and after treatment.

[0070] A "therapeutically effective amount" or "effective amount" is the amount of a pharmaceutical agent to achieve a pharmacological effect. The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. An "effective amount" of a neurosteroid is the amount needed to achieve the desired pharmacological effect or therapeutic improvement without undue adverse side effects. The effective amount of a neurosteroid will be selected by one of skill in the art depending on the particular patient and disease. It is understood that an "effective amount" or "therapeutically effective amount" may vary from subject to subject due to variations in neurosteroid metabolism, the subject's age, weight, general condition, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0071] "Treating" or "treatment" refers to any treatment of a disorder or disease, such as inhibiting the disorder or disease, for example, arresting the progression of the disorder or disease, relieving the disorder or disease, causing regression of the disorder or disease, relieving symptoms caused by the disease or disorder, or alleviating the symptoms of the disease or disorder.

[0072] "Alkyl" refers to a branched or straight-chain saturated aliphatic hydrocarbon group having a specified number of carbon atoms, typically from 1 to about 8 carbon atoms. As used herein, the term C1-C6-alkyl refers to an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms. Other embodiments include alkyl groups having 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 or 2 carbon atoms, such as C1-C8-alkyl, C1-C4-alkyl, and C1-C2-alkyl. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, t-butyl, n-pentyl, and sec-pentyl.

[0073] "Aryl" refers to an aromatic group containing only carbon atoms in the aromatic ring. Typical aryl groups contain 1 to 3 separate, fused, or pendant rings and 6 to about 18 ring atoms, with no heteroatoms as ring members. Where indicated, such aryl groups may be further substituted with carbon or non-carbon atoms or groups. Aryl groups include, for example, phenyl, naphthyl, including 1-naphthyl, 2-naphthyl, and biphenyl. An "arylalkyl" substituent is an aryl group, as defined herein, attached to the group it substitutes via an alkylene linker. Alkylene is an alkyl group as described herein, except that it is divalent.

[0074] "Cycloalkyl" is a saturated hydrocarbon ring group having a specified number of carbon atoms. Monocyclic cycloalkyl groups typically have 3 to about 8 carbon ring atoms, or 3 to 6 (3, 4, 5, or 6) carbon ring atoms. The cycloalkyl substituents may be pendant from a substituted nitrogen, oxygen, or carbon atom, or a substituted carbon atom that may have two substituents may have the cycloalkyl group attached as a spiro group. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0075] A "heteroalkyl" group is an alkyl group as described in which at least one carbon is replaced by a heteroatom, such as N, O, or S.

[0076] As used herein, the term "substituted" means that any one or more hydrogens on the designated atom or group have been replaced with a selection from the indicated group, provided that the replacement does not exceed the normal valence of the designated atom. When a substituent is oxo (i.e., =O), two hydrogens on the atom are replaced. When an oxo group replaces a heteroaromatic moiety, the resulting molecule may sometimes take tautomeric forms. For example, a pyridyl group substituted at the 2- or 4-position with oxo can sometimes be written as pyridine or hydroxypyridine. Combinations of substituents and / or variables are permissible only if such combinations result in stable compounds or useful synthetic intermediates. A stable compound or stable structure is defined to mean a compound that is sufficiently robust to survive isolation from a reaction mixture and subsequent formulation into an effective therapeutic agent. Unless otherwise specified, substituents are named in the core structure. For example, aminoalkyl should be understood to mean that the point of attachment of the substituent to the core structure is within the alkyl portion, and alkylamino means that the point of attachment is to the nitrogen of the amino group.

[0077] Suitable groups that may be present on a "substituted" or "optionally substituted" position include, but are not limited to, halogen; cyano; -OH; oxo; -NH; nitro; azido; alkanoyl (e.g., C2-C6 alkanoyl groups); C(O)NH; alkyl groups having 1 to about 8 carbon atoms, or 1 to about 6 carbon atoms, including cycloalkyl and (cycloalkyl)alkyl groups; alkenyl and alkynyl groups (including groups having one or more unsaturated bonds and 2 to about 8, or 2 to about 6 carbon atoms); alkoxy groups having one or more oxygen linkages and 1 to about 8, or 1 to about 6 carbon atoms; aryloxy, such as phenoxy; alkylthio groups (including groups having one or more thioether linkages and 1 to about 8, or 1 to about 6 carbon atoms). alkylsulfinyl groups (including those having one or more sulfinyl linkages and 1 to about 8 carbon atoms, or 1 to about 6 carbon atoms); alkylsulfonyl groups (including those having one or more sulfonyl linkages and 1 to about 8 carbon atoms, or 1 to about 6 carbon atoms); aminoalkyl groups (including groups having one or more N atoms and 1 to about 8, or 1 to about 6 carbon atoms); mono- or dialkylamino groups (including groups having an alkyl group of 1 to about 6 carbon atoms); mono- or dialkylaminocarbonyl groups having an alkyl group of about 1 to about 6 carbon atoms (i.e., alkylNHCO- or (alkyl1)(alkyl2)NCO-); and aryls having 6 or more carbons.

[0078] "AARS" means alanyl-tRNA synthetase.

[0079] "ADRA2B" means alpha-2B-adrenergic receptor.

[0080] "ALDH7A1" means aldehyde dehydrogenase 7 family, member A1.

[0081] "ALG13" means asparagine-linked glycosylation 13 (S. cerevisiae), a homolog thereof.

[0082] "ARHGEF9" means RHO guanine nucleotide exchange factor 9.

[0083] "ARV1" means ARV1 (S. cerevisiae), a homolog thereof.

[0084] "CACNA1A" means calcium channel, voltage-gated, P / Q type, alpha-1A subunit.

[0085] "CACNA1H" means calcium channel, voltage-gated, T-type, alpha-1H subunit.

[0086] "CACNB4" means calcium channel, voltage-gated, beta-4 subunit.

[0087] "CASR" means calcium-sensing receptor.

[0088] "CDKL5" means cyclin-dependent kinase-like 5.

[0089] "CERS1" means ceramide synthase 1.

[0090] "CHD2" means chromosomal domain helicase DNA-binding protein 2.

[0091] "CHRNA2" means cholinergic receptor, neuronal nicotinic, alpha polypeptide 2.

[0092] "CHRNA4" means cholinergic receptor, neuronal nicotinic, alpha polypeptide 4.

[0093] "CHRNB2" means cholinergic receptor, neuronal nicotinic, beta polypeptide 2.

[0094] "CLCN2" means chloride channel 2; CNTN2, contactin 2.

[0095] "CPA6" means carboxypeptidase A6; CSTB, cystatin B.

[0096] "DEPDC5" means DEP domain-containing protein 5.

[0097] "DNM1" means dynamin 1.

[0098] "DOCK7" means dedicator of cytokinesis 7.

[0099] "EEF1A2" means eukaryotic translation elongation factor 1, alpha-2.

[0100] "EFHC1" means EF-hand domain (C-terminus) containing protein 1.

[0101] "EPM2A" refers to the EPM2A gene that encodes laforin.

[0102] "FGF12" means fibroblast growth factor 12.

[0103] "FRRS1L" means iron chelate reductase 1-like.

[0104] "GABRA1" means gamma-aminobutyric acid receptor, alpha-1.

[0105] "GABRB1" means gamma-aminobutyric acid receptor, beta-1.

[0106] "GABRB3" means gamma-aminobutyric acid receptor, beta-3.

[0107] "GABRD" means gamma-aminobutyric acid receptor, delta.

[0108] "GABRG2" means gamma-aminobutyric acid receptor, gamma-2.

[0109] "GAL" means galanin; GNAO1, guanine nucleotide-binding protein, alpha-activating polypeptide O.

[0110] "GOSR2" means Golgi snap receptor complex member 2.

[0111] "GPR98" means G protein-coupled receptor 98.

[0112] "GRIN2A" means glutamate receptor, ionotropic, N-methyl-D-aspartate, subunit 2A.

[0113] "GRIN2B" means glutamate receptor, ionotropic, N-methyl-D-aspartate, subunit 2B.

[0114] "GRIN2D" means glutamate receptor, ionotropic, N-methyl-D-aspartate, subunit 2D.

[0115] "GUF1" refers to GUF1 GTPase (S. cerevisiae), a homolog thereof.

[0116] "HCN1" means hyperpolarization-activated cyclic nucleotide-gated potassium channel 1.

[0117] "ITPA" means inosine triphosphatase.

[0118] "KCNA2" means potassium channel, voltage-gated, shaker-related subfamily, member 2.

[0119] "KCNB1" means potassium channel, voltage-gated, shab-related subfamily, member 1.

[0120] "KCNC1" means potassium channel, voltage-gated, shaw-related subfamily, member 1.

[0121] "KCNMA1" means potassium channel, calcium-activated, large conductance, subfamily M, alpha member 1.

[0122] "KCNQ2" means potassium channel, voltage-gated, KQT-like subfamily, member 2.

[0123] "KCNQ3" means potassium channel, voltage-gated, KQT-like subfamily, member 3.

[0124] "KCNT1" means potassium channel, subfamily T, member 1.

[0125] "KCTD7" means potassium channel tetramerization domain-containing protein 7.

[0126] "LGI1" means leucine-rich gene, glioma inactivated, 1.

[0127] "LMNB2" means lamin B2.

[0128] "NECAP1" means NECAP endocytosis-associated protein 1.

[0129] "NHLRC1" means NHL repeat-containing 1 gene.

[0130] "PCDH19" means protocadherin 19.

[0131] "PLCB1" means phospholipase C, beta-1.

[0132] "PNPO" means pyridoxamine 5-prime-phosphate oxidase.

[0133] "PRDM8" means PR domain-containing protein 8.

[0134] "PRICKLE1" means prickle (Drosophila), its homolog, 1.

[0135] "PRRT2" means proline-rich transmembrane protein 2.

[0136] "SCARB2" means scavenger receptor class B, member 2.

[0137] "SCN1A" means sodium channel, neuronal type I, alpha subunit.

[0138] "SCN1B" means sodium channel, voltage-gated, type I, beta subunit.

[0139] "SCN2A" means sodium channel, voltage-gated, type II, alpha subunit.

[0140] "SCN8A" means sodium channel, voltage-gated, type VIII, alpha subunit.

[0141] "SCN9A" means sodium channel, voltage-gated, type IX, alpha subunit.

[0142] "SIK1" means salt-inducible kinase 1.

[0143] "SLC1A2" means solute carrier family 1 (glial high-affinity glutamate transporter), member 2.

[0144] "SLC12A5" means solute carrier family 12 (potassium / chloride transporter), member 5.

[0145] "SLC13A5" means solute carrier family 13 (sodium-dependent citrate transporter), member 5.

[0146] "SLC25A12" means solute carrier family 25 (mitochondrial carrier, aralar), member 12.

[0147] "SLC25A22" means solute carrier family 25 (mitochondrial carrier, glutamate), member 22.

[0148] "SLC2A1" means solute carrier family 2 (facilitated glucose transporter), member 1.

[0149] "SLC6A1" means solute carrier family 6 (neurotransmitter transporter, GABA), member 1.

[0150] "SPTAN1" means spectrin, alpha, non-erythroid 1.

[0151] "ST3GAL3" means ST3 beta-galactoside alpha-2,3-sialyltransferase 3.

[0152] "ST3GAL5" means ST3 beta-galactoside alpha-2,3-sialyltransferase 5.

[0153] "STX1B" means syntaxin 1B.

[0154] "STXBP1" means syntaxin binding protein 1.

[0155] "SZT2" means seizure threshold 2 (mouse), its homologue.

[0156] "TBC1D24" means Tre2-Bub2-Cdc16 / TBC1 domain family, member 24.

[0157] "UBA5" means ubiquitin-like modifier activating enzyme 5.

[0158] "WWOX" means WW domain-containing oxidoreductase. [Brief explanation of the drawings]

[0159] [Figure 1]

[0023] Figure 1 shows the efficacy of AEDs after 12 months of use in patients with PCDH19. Abbreviations can be found in Lotte et al. 2016, which is incorporated herein by reference.

[0160] [Figure 2] 1 is a graphical representation of particle size data from the manufacture of ganaxolone nanomill dispersion, bulk IR beads, and encapsulated IR beads. The typical decrease in particle size during milling is followed by a growth in particle size after the addition of stabilizers during curing, and a plateau reached at approximately 300 nm.

[0161] [Figure 3A]

[0023] Figure 1 shows an overview of the key steps in the manufacturing process to produce a 50 mg / ml suspension and 225 mg capsules containing IR-releasing ganaxolone particles. As shown, both products utilize a common stabilizing dispersion intermediate.

[0162] [Figure 3B] 1 is a summary of the major steps in the suspension manufacturing process used for the 50 mg / ml ganaxolone suspension of Example 1.

[0163] [Figure 3C] 1 is a summary of the major steps in the manufacturing process used for the 225 mg ganaxolone capsules of Example 2.

[0164] [Figure 3D] 1 is a graph of particle size stability of ganaxolone nanomill suspension and encapsulated IR beads.

[0165] [Figure 3E] Graph of the hardening curve of ganaxolone particles containing parabens. The stabilized 300 nm nanoparticles show good stability against particle growth in pediatric suspension and encapsulated drug product formats. The stabilization process is controlled by the precise addition and dissolution of the water-soluble stabilizer, paraben. The hardening process is controlled by adjusting the holding time and temperature of the stabilized dispersion prior to suspension dilution (for 50 mg / ml ganaxolone suspension) or fluidized bed bead coating (for 225 mg ganaxolone capsules).

[0166] [Figure 4] 1 shows cumulative responder curves for 28-day seizure frequency for individual seizures and cluster sums from Example 4.

[0167] [Figure 5] 1 is the mean ganaxolone plasma concentration profile following a single oral dose of ganaxolone 0.3 micron capsules of Example 2 in healthy volunteers after a high-fat meal (Example 5).

[0168] [Figure 6] 1 is the mean ganaxolone plasma concentration-time profile following single and multiple BID oral doses of the 0.3 micron ganaxolone capsules of Example 2 with a standard meal or snack in healthy volunteers (Example 5).

[0169] [Figure 7] 1 is a diagram showing the mean ganaxolone plasma concentration-time profiles after single and multiple BID oral doses of 0.3 micron ganaxolone capsules with a standard meal or snack in healthy volunteers.

[0170] [Figure 8] 1 is the mean ganaxolone plasma concentration-time profile following multiple BID oral doses of 0.3 micron ganaxolone capsules with a standard meal or snack in healthy volunteers.

[0171] [Figure 9] Mean ganaxolone plasma trough levels (semi-logarithmic scale) after multiple BID oral doses of 0.3 micron ganaxolone capsules with a standard meal or snack. Subjects received 600 mg ganaxolone BID on days 4-6; 800 mg ganaxolone BID on days 7-9; and 1000 mg ganaxolone BID on days 10-12. Values for days 6.5, 9.5, and 12.5 are from evening samples collected 12 hours after the last dose on the PK sampling day.

[0172] [Figure 10] 11. Plasma Allo-S concentrations (pg mL −1) in responders and non-responders of Example 11.

[0173] [Figure 11] Stratification of PCDH19 subjects by allopregnanolone sulfate (Allo-S) levels and concomitant seizure frequency response to ganaxolone in Example 11. A "-100 change" means complete seizure freedom, with the patient experiencing no seizures during the 26-week period. Anything from "0" to "-100%" indicates efficacy. Circles indicate "responders" (≥25% reduction in seizure frequency) and squares indicate "non-responders" (<25% reduction in seizure frequency).

[0174] [Figure 12] Stratification of CDKL5 subjects by allopregnanolone (Allo) levels and associated seizure frequency response to ganaxolone in Example 11. Each closed circle represents a unique subject in the trial.

[0175] [Figure 13]1 shows the relationship between dose and exposure (AUC) of ganaxolone in a 0.3 micron capsule formulation, showing saturation of exposure as dose approaches 2000 mg / day. DETAILED DESCRIPTION OF THE INVENTION

[0176] CDKL5 CDKL5 deficiency or CDKL5 stands for cyclin-dependent kinase-like 5.

[0177] The CDKL5 gene is located on the X chromosome and was previously called STK9.

[0178] Most CDKL5-affected children show perinatal irritability, early seizures, hand stereotypies, severe psychomotor developmental disorders, and severe hypotonia. In contrast to classic Rett syndrome, there may also be the absence of a classic regression period, poor eye contact, generally normal head circumference and other growth parameters, and a relative absence of autonomic dysfunction.

[0179] Other symptoms of CDKL5 deficiency often include: low muscle tone, hand wringing or hand to mouth, significant developmental delay, limited or absent speech, absent or poor eye contact, gastroesophageal reflux, constipation, small and cold feet, breathing abnormalities such as hyperventilation, teeth grinding, episodes of laughing or crying for no reason, low / weak muscle tone, very limited hand skills, some autistic-like tendencies, scoliosis, cerebral visual impairment (CVI), also known as "cortical blindness," apraxia, eating and drinking challenges, sleep disorders and sidelong glances, and leg crossing.

[0180] CDKL5 deficiency is a form of genetic epilepsy with encephalopathy that is virtually always refractory to treatment.

[0181] Seizures begin within days to months of birth and become increasingly difficult to treat in most patients. The best initial response to therapeutic agents other than neurosteroids is to valproic acid, but the 12-month responder rate is only 9% (Mueller et al.). AEDs commonly used to treat CDKL5 deficiency include vigabatrin, felbamate, and valproic acid. All three AEDs are associated with significant side effects. In addition to the risk of visual field loss with vigabatrin and aplastic anemia with felbamate, the tolerability of these three drugs is relatively poor, especially with long-term treatment. Patients may also be treated with high-dose pulsed steroids or ACTH, but neither can be administered long-term due to frequent and severe side effects. Vagus nerve stimulation and corpus callosotomy are attempted in extremely desperate cases, but both are invasive and generally ineffective. Corpus callosotomy is particularly invasive and provides only temporary relief from generalized seizures (and only in some cases). Unlike ganaxolone, which may improve cognitive and motor function, many available AEDs have side effects, including cognitive slowing, ataxia, liver toxicity, and serious weight management problems. None of these have been associated with ganaxolone use. In contrast to drugs with narrow therapeutic indices, such as sodium channel blockers, phenytoin, and carbamazepine, ganaxolone does not require frequent monitoring of blood levels.

[0182] CDKL5 was identified by an exon-trapping strategy designed to screen for candidate genes within Xp22, a region of the X chromosome to which several other genetic disorders have been mapped (Montini et al. 1998). CDKL5 is a member of the proline-directed kinase subfamily, which shares homology with both CDKL kinases and cell cycle-dependent kinases known as microtubule-associated proteins (MAPs) (Lin et al. 2005; Guerrini and Parrini 2012).

[0183] The human CDKL5 gene occupies approximately 240 kb of the Xp22 region and consists of 24 exons, the first three of which (exons 1, 1a, and 1b) are untranslated, while the coding sequence is contained within exons 2–21. Two splice variants with distinct 5' untranslated regions (5' UTRs) (also known as leader sequences or leader RNAs) have been identified: isoform I, containing exon 1, is transcribed in a wide range of tissues, whereas isoform II, containing exons 1a and 1b, is restricted to the testis and fetal brain. Alternative splicing events result in at least three distinct human protein isoforms. The original CDKL5 transcript generates a protein of 1,030 amino acids (CDKL5-115; 115 kDa). While CDKL5-115 is primarily expressed in the testis, a recently identified transcript characterized by alterations in the C-terminal region may be related to CDKL5 brain function. Such differential enrichment of CDKL5 splice variants by organ suggests the involvement of alternative splicing in regulating protein function.CDKL5 is a ubiquitous protein, but is primarily expressed in the brain (cerebral cortex, hippocampus, cerebellum, striatum, and brainstem), thymus, and testis (Lin et al. 2005).

[0184] CDKL5 is a protein whose gene is located on the X chromosome. The CDKL5 gene provides instructions for forming proteins essential for normal brain development, and mutations cause deletions at the protein level (LouLou Foundation Website; http: / / www.louloufoundation.org / about-cdkl5.html). CDKL5 deficiency syndrome is characterized by early-onset, intractable seizures, severe gross motor skill impairment, and global developmental delay accompanied by sleep disorders, abnormal muscle tone, teeth grinding, scoliosis, and gastrointestinal problems (Mangatt M, Wong K, Anderson B, Epstein A, Hodgetts S, Leonard H., Downs J. Prevalence and onset of comorbidities in the CDKL5 deficiency disorder differ from Rett syndrome. Orphanet Journal of Rare Diseases. 2016;11:39).

[0185] Kalscheuer et al. (2003) reported two unrelated girls who presented with infantile spasms (then diagnosed as West syndrome) and severe developmental delay. In both patients, the CDKL5 gene was disrupted by a breakpoint on the X chromosome due to a balanced translocation. The overlapping clinical similarities between these initial patients and atypical Rett syndrome raised the possibility of CDKL5 gene mutations as a possible underlying genetic etiology in patients diagnosed with either the classic or atypical variants of Rett syndrome, who exhibited early seizures and were negative for the methyl-CpG-binding protein 2 (MECP2) gene mutation typically associated with Rett syndrome (Tao et al. 2004; Weaving et al. 2004; Mari et al. 2005; Scala et al. 2005; Bahi-Buisson et al. 2008a). This underlying genetic mutation would later form the basis of a new clinical entity known as CDKL5 deficiency.

[0186] Clinical features commonly associated with CDKL5 mutations include early-onset seizures, severe intellectual and gross motor impairments, and certain dysmorphic features. Epilepsy manifests early in almost all patients with CDKL5 gene deletion mutations. Typical seizures are either infantile spasms (i.e., West syndrome) or multifocal myoclonic seizures (Archer et al. 2006; Bahi-Buisson et al. 2008b; Mei et al. 2010). Early severe seizure disorders are accompanied by very limited developmental progression and marked hypotonia. Patients with CDKL5 gene abnormalities have been reported to be normal for the first few days of life, then show early signs of poor developmental skills, including poor feeding and poor eye contact, even before the onset of seizures. Reduced fetal movement has been reported retrospectively in mothers (Archer et al. 2006). Subsequently, lack of purposeful hand use, severe developmental delay, and deficient language skills become evident (Archer et al. 2006; Bahi-Buisson et al. 2008b; Elia et al. 2008; Nemos et al. 2009; Mei et al. 2010; Neul et al. 2010; Melani et al. 2011). Approximately one-third of patients eventually walk (Bahi-Buisson et al. 2008b). Males are on the more severe side of the phenotypic spectrum, with virtually no motor acquisition (Van Esch et al. 2007; Sartori et al. 2009; Melani et al. 2011), whereas the rare female patient may achieve some independence and achieve better-than-expected language and motor milestones (Archer et al. 2006). Prior to identifying the link between the CDKL5 gene and Rett syndrome, many CDKL5 patients were classified as having atypical Rett syndrome with early onset seizures (Hanefeld variant).This is because the documented severe hypotonia, impaired psychomotor development, and stereotypic hand movements are within the range of typical Rett syndrome clinical symptoms (Artuso R et al. 2010; Stalpers XL et al. 2012; Nemos C et al. 2009). However, unlike Rett syndrome, CDKL5 epileptic encephalopathy patients typically do not regress later in life. Patients with CDKL5 epileptic encephalopathy exhibit similar sleep and respiratory symptoms to those of Rett syndrome: sleep disturbances (characterized by difficulty falling asleep, frequent awakenings, poor sleep efficiency, reduced rapid eye movement (REM) sleep, teeth grinding, daytime somnolence, and apnea (central or obstructive)). While sleep disturbances are likely related to the underlying neurological disorder, gastric reflux, seizures, and AEDs may contribute to some extent (Hagebeuck et al. 2012; Mangat et al. 2016). Gastrointestinal symptoms are highly common in patients with CDKL5 epileptic encephalopathy, with approximately 90% reporting constipation, gastroesophageal reflux, and / or aerophagia. The likelihood of experiencing constipation and reflux increases with age, especially after age 10. The dysmorphic features of CDKL5 epileptic encephalopathy have been reported to be subtle, with the exception of acquired microcephaly (delayed head growth associated with increased height and weight). The spectrum of features is similar overall in females and males. Frequently observed facial features include: a prominent and / or broad forehead; a high hairline; relative midface hypoplasia; deep-set, yet "large"-appearing eyes; and infraorbital shadowing. There are no approved or licensed therapies for the treatment of patients with CDKL5 deficiency in the United States.

[0187] Clinical features commonly associated with CDKL5 mutations include early-onset drug-refractory seizures, severe intellectual and gross motor disability, and severe sleep disturbances. Clinical manifestations of CDKL5 deficiency for which ganaxolone may show some therapeutic benefit are summarized as follows:

[0188] Intractable epilepsy Epilepsy appears early in almost all patients with CDKL5 gene deletion mutations. Typical seizures are either infantile spasms (i.e., West syndrome) or multifocal myoclonic seizures (Archer et al. 2006; Bahi-Buisson et al. 2008b; Mei et al. 2010). Some patients exhibit a unique seizure pattern consisting of prolonged generalized tonic-clonic events lasting 2–4 minutes (consisting of tonic oscillatory contractions followed by a clonic phase with a series of convulsions, gradually transforming into repetitive distal myoclonic jerks). It has also been noted that seizures are generally highly polymorphic, and many different seizure types can occur in the same patient and evolve over time.

[0189] In a cohort of 86 patients (77 females, 9 males) from the International Rett Syndrome Registry and Database (InterRett; Fehr et al. 2013), seizures were reported in all but one female. Seizures occurred in approximately 90% of patients by 3 months of age, with a mean age of onset of 7.3 weeks (range 0.3–34.8 weeks) in females and slightly earlier at 6.4 weeks (range 2.1–13 weeks) in males. Seizure control was generally poor, with 52 of 72 (72%) females and 8 of 9 (89%) males experiencing daily seizures (Fehr et al. 2013).

[0190] Data obtained primarily from the International CDKL5 Deficiency Disorder Database (ICDD, where "CDD" stands for CDKL5 deficiency) reported a similar lack of seizure control (Mangatt et al. 2016). Information on seizure frequency was available for 137 / 145 patients in the cohort study. Ninety-five (69.3%, 95 / 137) experienced seizures daily, with the mean daily seizure frequency ranging from 1 to 21. Of those who provided information on the number of daily seizures (n = 82), approximately one-third experienced at least five seizures daily.

[0191] severe developmental delay Early severe epileptic seizure disorder is associated with very limited developmental progression and marked hypotonia. Patients with CDKL5 gene defects are normal for the first few days of life but then, even before the onset of seizures, show early signs of developmental deficits, including poor feeding and poor eye contact. Fetal motor decline has been reported retrospectively by mothers (Archer et al. 2006). Subsequently, lack of purposeful hand use, severe developmental delay, and poor language skills become evident (Archer et al. 2006; Bahi-Buisson et al. 2008b; Elia et al. 2008; Nemos et al. 2009; Mei et al. 2010; Neul et al. 2010; Melani et al. 2011). Approximately one-third of patients eventually walk (Bahi-Buisson et al. 2008b). Males are at the more severe end of the phenotypic spectrum and have virtually no motor gains (Van Esch et al., 2007; Sartori et al., 2009; Melani et al., 2011), while rare affected females can achieve some independence and achieve better-than-expected language and motor milestones (Archer et al., 2006). Most children have severely impaired social interactions and lack gaze fixation (Guerrini, R and Parrini, E, 2012).

[0192] Sleep disorders Nearly all patients with CDKL5 deficiency exhibit sleep disturbances characterized by difficulty falling asleep, frequent awakenings, poor sleep efficiency, reduced rapid eye movement (REM) sleep, teeth grinding, daytime somnolence, and apnea (central or obstructive). Sleep disturbances are likely related to the underlying neurological disorder, while gastric reflux, seizures, and AEDs may contribute to some extent (Hagebeuck et al., 2012; Mangat et al., 2016).

[0193] Night awakenings are the most persistent sleep problem, experienced by more than half of patients. Night awakenings are particularly disturbing and disruptive to parents, as they are often accompanied by disturbing screams or loud laughing spells (Bahi-Buisson et al., 2008b; Mangat et al., 2016). A study by Mori et al. assessed the impact of caring for a child with CDKL5 deficiency on parental health and family quality of life. Data were sourced from the International CDKL5 Deficiency Database, where 192 families with children carrying pathogenic CDKL5 mutations provided data through January 2016. Significant impairment in mental health was observed in this caregiver population and was associated with increased severity of the child's sleep problems (Mori et al., 2017).

[0194] Severe gross motor skill disorder The ICDD was able to collect data from parents to provide statistics on gross motor skills. It is important to note that the sample size was relatively small and the data was parent-driven. Based on a sample size of 116 children (102 females and 14 males) from 17 different countries, ranging in age from 4 months to 29 years (median age 6 years) for females and from 2 years to 22 years 8 months (median age 9 years 2 months) for males, the findings for gross motor skills were as follows: Rolling over: About 84% of girls vs. 35% of boys Sitting without support: 55% of girls vs. 23% of boys Crawling: Nearly 21% of girls vs. 10% of boys · Independence: Nearly 20% of girls Walking without support: Nearly 18.8% of girls Running without support: 8% of girls

[0195] Most boys required maximal support to sit, stand, move, and walk; however, in this study, three boys learned to stand with support and two learned to walk with support. Due to the number of boys affected by CDKL5 deficiency, the sample size was very small. However, over the past two years, the International Foundation for CDKL5 Research (IFCR) has identified boys who were mildly affected compared to most boys, and some have been reported to be able to walk, run, and climb.

[0196] Likely reduced life expectancy Due to the rarity of CDKL5 deficiency, little is known about the long-term prognosis and life expectancy. Most confirmed patients are under 18 years of age, but identifying older children and adults is often difficult due to the frequent lack of complete growth records and genetic testing in this older population. However, there are several confirmed adults living with the disorder in their 20s, 30s, and even 40s. Identical twins believed to be in their 50s are living in Europe. However, it is important to note that, as with any condition affecting multiple organ systems, such as CDKL5 deficiency, there is a high likelihood of death due to other factors contributing to epilepsy syndromes, severe respiratory infections, and digestive problems / failure (http: / / www.curecdkl5.org / ).

[0197] Information from various social media sources involving the UK CDKL5 patient advocacy group indicates that several young children have died in the past few years, primarily due to either respiratory failure from pneumonia or complications related to gastrointestinal disorders. Several children have died unexpectedly, most likely due to sudden unexpected death in epilepsy (SUDEP). Patients with CDKL5 deficiency are at increased risk of SUDEP due to frequent generalized tonic-clonic seizures.

[0198] According to the American Academy of Neurology (Practice Guideline Summary: Sudden Unexpected Death in Epilepsy Incidence Rates and Risk Factors, April 2017): The occurrence of generalized tonic-clonic seizures (GTCS) (vs. the absence of GTCS) may increase the risk of SUDEP, based on moderate confidence in evidence from two Class II studies. GTCS frequency is very likely associated with an increased risk of SUDEP (based on two class II studies upgraded from moderate to high due to effect size). SUDEP risk increases threefold (>3 GTCS frequencies / year compared with 1-2 GTCS frequencies / year). Having a seizure within the past year, compared with no seizures, may increase the risk of SUDEP (moderate confidence in the evidence based on two Class II studies), as may having a seizure within the past five years (moderate confidence in the evidence based on one Class I study).

[0199] There are an estimated 1,200 patients identified at any one time as having one of the CDKL5 mutations. It is unknown how many of these patients have the pathological mutation, and only approximately 400 patients are currently included in various registries worldwide. Many patients in these registries are likely deceased based on social media reports, and others do not fall within the 2-17 year age range. Any research into patients with this disorder is significantly hampered by the difficulty of enrolling sufficient numbers of subjects to conduct adequately powered, randomized, controlled studies in which a reduction in seizure count or the proportion of subjects responding (classically defined as at least a 50% reduction from baseline seizure count) is the primary efficacy endpoint. These studies typically enroll 200-400 subjects, which would essentially represent the entire population of eligible subjects worldwide.

[0200] Future studies will develop a primary endpoint measuring the overall treatment effect in this specific population, such as the CGI-I, along with secondary endpoints capturing the most clinically meaningful endpoints, in addition to endpoints related to seizure frequency.

[0201] PCDH19 The PCDH19 gene encodes the protein protocadherin 19, which is part of a family of molecules that support communication between cells in the central nervous system. As a result of mutations, protocadherin 19 may be malformed, have reduced function, or not be produced at all.

[0202] Aberrant expression of protocadherin 19 is associated with highly variable and intractable seizures, cognitive impairment, and behavioral or social impairment with autistic traits.

[0203] PCDH19 female-predominant childhood epilepsy affects approximately 15,000 to 30,000 women in the U.S. This genetic disorder involves seizures that begin early in life, most often cluster-focal seizures that can last for several weeks.

[0204] Mutations in the PCDH19 gene have been associated with low levels of allopregnanolone.

[0205] Protocadherin 19 (PCDH19)-associated epilepsy is a severe epilepsy syndrome characterized by early-onset cluster seizures, varying degrees of cognitive and sensory impairment, and psychiatric and behavioral disorders (Depienne et al., 2012a). PCDH19-associated epilepsy is characterized as a rare disease by the National Institutes of Health Office of Rare Diseases Research (NIH-PCDH19-associated female-only epilepsy). The disorder is caused by mutations in the PCDH19 gene, which encodes protocadherin 19 on the X chromosome (Dibbens et al., 2008; Depienne and LeGuern, 2012b; Depienne et al., 2009). The mechanism by which this mutation contributes to the progression of epilepsy and intellectual disability is not fully understood. However, protocadherin 19 is a calcium-dependent cell-cell adhesion molecule transmembrane protein that is strongly expressed in neural tissues (e.g., hippocampus, cerebral cortex, thalamus, amygdala) and appears to be involved in synaptic transmission and the formation of synaptic connections during brain development (Depienne et al., 2014). PCDH19-associated epilepsy has an unusual X-linked pattern of genetic transmission, and symptoms are primarily limited to females (Depienne and LeGuern, 2012b).

[0206] People affected by this gene mutation were found to have reduced levels of endogenous allopregnanolone compared to age-matched controls.

[0207] The clinical features of PCDH19-associated epilepsy have been well characterized (Depienne and LeGuern, 2012b; Higurashi et al., 2013). The hallmark features of PCDH19-associated epilepsy are short clusters of seizures, beginning in infancy or early childhood (ranging from 4 to 60 months; mean age of onset = 12.9 months), and varying degrees of cognitive impairment (Depienne and LeGuern, 2012b; Higurashi et al., 2013; www.pcdh19info.org; Specchio et al., 2011). The onset of the initial cluster of seizures usually coincides with fever (i.e., febrile seizures) or immunization, and subsequent seizures can be febrile or afebrile. However, fever may exacerbate the seizures (Depienne and LeGuern, 2012b; Higurashi et al., 2013; Marini et al., 2010). Patients with PCDH19-FPE may experience individual attacks in addition to cluster and multiple attack types. In some patients, attacks improve upon reaching puberty, possibly due to increased endogenous levels of progesterone and allopregnanolone.

[0208] Seizure clusters are characterized by brief seizures lasting 1–5 minutes and are often preceded by a frightening cry (Depienne and LeGuern, 2012b; Higurashi et al., 2013; Marini et al., 2010). These clusters may occur more than 10 times per day for several days, with variable time between seizure clusters (Depienne and LeGuern, 2012b). Patients with PCDH19-related epilepsy may experience one or more seizure types over the course of the disorder, with generalized tonic-clonic, tonic-clonic, and / or focal seizures being the most common. Absence seizures, atonic seizures, and myoclonus may also occur, although less frequently (Depienne and LeGuern, 2012b; Marini et al., 2010; Scheffer et al., 2008). Status epilepticus may occur early in the course of the disorder. Furthermore, seizures are often refractory to treatment, especially in infancy and childhood. Notably, seizure frequency and tolerance to treatment tend to decrease over time, with some patients becoming seizure-free in adolescence or remaining seizure-free on monotherapy into adulthood (Depienne et al., 2012a; Specchio et al., 2011; Scheffer et al., 2008; Camacho et al., 2012).

[0209] PCDH19-related epilepsy is usually, but not always, associated with cognitive impairment. It is estimated that up to 75% of patients with PCDH19-related epilepsy have cognitive impairment ranging from borderline to severe (Depienne et al., 2009; www.pcdh19info.org; Specchio et al., 2011; Scheffer et al., 2008). Children's growth typically follows one of three courses: normal growth with regression following seizures, normal growth without regression, or delayed growth from birth through adulthood (www.pcdh19info.org). Cognitive impairment does not appear to be related to the frequency or severity of seizures (Depienne et al., 2012a; Specchio et al., 2011).

[0210] PCDH19-related epilepsy may also be associated with various psychiatric disorders, particularly autism or autistic features (up to 60% of patients), attention deficit hyperactivity disorder (ADHD), behavioral disorders, obsessive-compulsive disorder, or motor stereotypies, aggression, and anxiety (Depienne et al., 2013; Marini et al., 2010; www.pcdh19info.org; Scheffer et al., 2008). Other neurological abnormalities may also be present, including sleep disorders, ictal apnea, movement disorders, hypotonia, speech delay, sensory integration problems, and autonomic dysfunction (www.pcdh19info.org; Smith et al., 2018).

[0211] Mutations in the PCDH19 gene were first identified in 2008 in seven large families with female-only epilepsy and mental retardation (EFMR), and subsequently in individuals initially diagnosed with Dravet syndrome (DS) who did not display the characteristic gene mutation (SCN1A) associated with DS (Dibbens et al., 2008; Depienne LeGuern et al., 2012b). This disorder shares clinical features with other early-onset epileptic encephalopathies, such as DS, but is unique in its distinct progression and specific genetic mutations in the PCDH19 gene. Since the discovery of PCDH19-associated epilepsy, a significant number of patients have been diagnosed with this disorder, and mutations related to PCDH19 have become the second most frequently associated gene in the field of epilepsy (Depienne LeGuern et al., 2012b; Higurashi et al., 2013; Marini et al., 2010).

[0212] Prior to the discovery of the role of PCDH19 in childhood epilepsy, many patients were diagnosed with DS. There are also several differences between the two disorders. Males are overrepresented in the DS population (male-to-female ratio 2:1). Conversely, females with PCDH19 mutations are severely affected, while males with the mutations are usually phenotypically normal with respect to seizures and cognition (Depienne et al., 2009). Additional differences in the clinical manifestations of the two disorders exist, including differences in seizure type (e.g., fewer myoclonus and absence seizures in patients with PCDH19-related epilepsy). Patients with PCDH19 also exhibit an older mean age of seizure onset, a higher incidence of seizure clusters, and a lack of photosensitivity compared to patients with DS (Trivisano et al., 2016 and Steel, 2017).

[0213] Protocadherin 19 (PCDH19) is an adhesion molecule within the cadherin superfamily that is highly expressed in the central nervous system (CNS), particularly in the brain. The mechanism by which mutations in this gene contribute to the development of epilepsy and intellectual disability is not fully understood. However, protocadherin 19 is a calcium-dependent cell-cell adhesion molecule transmembrane protein that is highly expressed in neural tissues (e.g., hippocampus, cerebral cortex, thalamus, and amygdala) and appears to be involved in synaptic transmission and the formation of synaptic connections during brain development (Depienne et al., 2009). PCDH19-related epilepsy has an unusual X-linked pattern of genetic transmission, with the phenotype being primarily restricted to females, and carrier males generally unaffected (Depienne, LeGuern et al., 2012b). The role of this gene in childhood epilepsy was only discovered in 2008 (Dibbens et al., 2008). A large systematic review and meta-analysis of 271 PCDH19-mutated individuals reported in the literature was recently published, providing a comprehensive review of the disorders and typical phenotypic consequences caused by this mutation (Kolc et al., 2018).

[0214] The prevalence of PCDH19-associated epilepsy is largely unknown due to the recent discovery of the gene and its contribution to early-onset childhood epilepsy. A top-down population-based approach estimates that there are approximately 5,755 children with PCDH19-associated epilepsy in the United States. This number is derived from 470,000 children (<18 years of age) living in the United States with active epilepsy (Zack and Kobau 2017), of which approximately 24.5% are thought to have epilepsy of genetic etiology (Trump et al., 2016, Berg et al., 2017, and Lindy et al., 2018, unweighted average). Of the approximately 112,800 children living in the United States with genetic epilepsy, approximately 5% are thought to be associated with pathogenic PCDH19 gene mutations (Trump et al., 2016 and Lindy et al., 2018, unweighted average). Despite this methodological approach, the number of children formally diagnosed with PCDH19-associated epilepsy is believed to be significantly lower than the above estimates. The PCDH19 Alliance, a leading US-based patient advocacy organization, estimates that the number of formally diagnosed individuals with PCDH19-associated epilepsy is approximately 1,000 worldwide. It is hypothesized that many individuals are misdiagnosed due to limited awareness of PCDH19, or remain undiagnosed due to lack of access or reimbursement for genetic testing.

[0215] Clinical manifestations of PCDH19 gene mutations The phenotypic spectrum in individuals affected by mutations in the PCDH19 gene is large, and genotype-phenotype correlations have not yet been established. PCDH19 is largely characterized by early-onset (approximately 10 months of age) seizures that typically occur in clusters. Seizures are typically triggered by a febrile illness. Findings are variable, but seizures appear to offset during an age period that correlates with puberty (van Harssel et al., 2013 and Scheffer et al., 2008). In addition to the burden of seizures, affected individuals with PCDH19 mutations also experience severe intellectual disability (Depienne et al., 2009 and Marini et al., 2010) and behavioral dysregulation (Depienne et al., 2011 and Dibbens et al., 2008). While there is some phenotypic overlap with PCDH19-related epilepsies and Dravet syndrome (DS), numerous reports describe the unique clinical manifestations of each genetic epilepsy. Before the discovery of the PCDH19 gene, many patients were diagnosed with DS. In fact, it is thought that approximately 25% of SCN1A-negative patients diagnosed with DS may be PCDH19-positive (Jonghe 2011). This figure may change as awareness of PCDH19-related epilepsy increases.

[0216] Intractable epilepsy Seizures pose a significant clinical burden for individuals with PCDH19-related epilepsy, especially early in life. Seizure onset occurs approximately 8–12 months of age (Marini et al., 2010; Smith et al., 2018). Both generalized and focal seizures have been reported in this condition (Smith et al., 2018; Marini et al., 2010; Specchio et al., 2011). Absence seizures, atonic seizures, and myoclonus may also occur, although less frequently (Depienne and LeGuern 2012b; Marini et al., 2010; Scheffer et al., 2008). A hallmark of PCDH19 seizures is that they typically occur in clusters and are characterized by brief seizures lasting 1–5 minutes, often preceded by a frightening cry (Depienne and LeGuern, 2012b; Higurashi et al., 2013; Marini et al., 2010). These clusters may occur more than 10 times per day for several days, with variable time between seizure clusters (Depienne and LeGuern 2012b). Patients with PCDH19-related epilepsy may experience one or more seizure types throughout the course of the disorder. Status epilepticus may occur early in the course of the disorder. Furthermore, seizures are often refractory to treatment, particularly in infancy and childhood.

[0217] Intellectual disability Before the discovery of the PCDH19 gene, girls with this condition were diagnosed with "epilepsy in mentally retarded females" (EFMR). Autism spectrum disorder (ASD) and intellectual disability (ID) are present in 75-80% of individuals with PCDH19 mutations (Breuillard et al., 2016; Smith et al., 2018). Cognitive outcomes are highly heterogeneous, ranging from mild to severe impairment. ID has been diagnosed by low scores in all cognitive domains, with greater impairment in theory of mind. There was no correlation between epilepsy severity and level of ID (Specchio et al., 2011; Depienne et al., 2011).

[0218] behavioral dysregulation Behavioral and psychiatric comorbidity is well documented in affected individuals with PCDH19 gene mutations. These problems include aggression, depressed mood, and psychotic traits. A large meta-analysis of 271 individuals with PCDH19 variants reported that 60% of women, 80% of affected mosaic males, and 9 hemizygous males developed psychiatric features, with hyperactivity, autistic traits, and obsessive-compulsive behaviors being common (Kolc et al., 2018). Furthermore, behavioral and psychiatric disorders are commonly a primary concern for patients and caregivers. While seizure burden typically decreases with age, behavioral and psychiatric comorbidity remains relatively unchanged throughout life.

[0219] Sleep disorders Sleep dysregulation has also been reported as a common attribute of PCDH19-related epilepsy and is a major concern for families. The disorder has been described as trouble falling asleep and / or staying asleep. Sleep disturbances were reported in 53% (20 / 38) of probands and were primarily described as sleep maintenance insomnia, with many children waking too early and struggling to fall back asleep (Smith et al., 2018). It is unknown how seizure activity correlates with sleep dysfunction, or vice versa.

[0220] PCDH19 gene and protein The PCDH19 gene is located at position 22.1 on the long (q) arm of the X chromosome, and its coding sequence consists of six exons. This gene encodes the 1148-amino acid protein protocadherin 19, a member of the protocadherin family that plays an important role in cell-cell interactions. Protocadherins, including PCDH19, play important roles in axon guidance / sorting, neurite self-avoidance, and synapse formation (Garret and Weiner 2009; Lefebvre et al., 2012).

[0221] The majority of PCDH19-related epilepsy gene mutations were observed in the extracellular domain of the protein, encoded by exon 1. Missense variants were most common (approximately 45%), followed by frameshift (27%) and nonsense (20%) mutations (Kolc et al., 2018).

[0222] PCDH19-associated epilepsy is an X-linked disorder, and paradoxically, females with point mutations in the PCDH19 gene are severely affected, but transmitting males are not. In most X-linked dominant disorders, males are typically more severely affected than females, and intrauterine death is more common. In large case series in which inheritance was determined, half of the PCDH19 mutations occurred de novo, and half were inherited from healthy fathers with no evidence of seizures or cognitive impairment (Depienne et al., 2012a; Depienne et al., 2009). The expression of PCDH19 mutations is highly variable, with some individuals barely affected and others exhibiting severe disease. Even identical twins with mutations can vary in the frequency of seizures and the degree of cognitive impairment (Higurashi et al., 2013).

[0223] There are several hypothetical mechanisms for this abnormal transmission, including the presence of a compensatory protocadherin gene on the Y chromosome or cellular interference (Depienne et al., 2012a; Depienne et al., 2009). Regarding the latter, if a mutation is present, two cell populations may arise: one with the mutant PCDH19 gene and the other with the normal gene. This natural mosaicism could potentially harm normal brain function. Because males only have one X chromosome and one copy of the PCDH19 gene, a mutation event would result in a single, homogeneous cell population, which would likely not harm brain cells. The fact that non-mosaic hemizygous males exhibit PCDH19-associated epilepsy phenotypes suggests that the PCDH19 protein may not be essential in humans.

[0224] Unmet therapeutic need There remains a clear and significant unmet medical need for individuals affected by PCDH19-associated epilepsy. To date, no approved medications or treatments have been shown for this specific patient population. Individuals are currently treated with a variety of antiepileptic drugs (AEDs) without any established standard of care. Furthermore, some antiseizure medications have significant negative side effects and worsen other outcomes, such as behavioral changes. Therefore, there is a need for safe, durable medications that can effectively control seizures while potentially assisting with other neuropsychiatric disorders.

[0225] The need for improved seizure control Despite the availability of many AEDs, their therapeutic efficacy is limited and highly variable in this patient population. Lotte et al. retrospectively reviewed the efficacy of AEDs in 58 women with PCDH19-associated epilepsy. The results are shown in Figure 1. Despite the reported modest efficacy of clobazam, many individuals continue to experience seizures and remain inadequately treated.

[0226] Other reports have also documented that the majority of patients with PCDH19-related epilepsy experience uncontrollable, refractory seizures. 58 (58%) of probands in a cohort of 38 remained refractory to three or more seizure medications (Smith et al., 2018). Furthermore, a recent study documented that only 17 of 271 probands achieved seizure control (Kolc et al., 2018).

[0227] Currently, there are no antiepileptic drugs (AEDs) approved for PCDH19-associated epilepsy, leaving a significant unmet need in this patient population.

[0228] During the first few years of PCDH19-associated epilepsy, seizure clusters are frequent and severe, persist despite appropriate treatment, and may eventually become treatment-refractory (Higurashi et al., 2013). Despite the many available AEDs, none currently provide consistent seizure control in patients with PCDH19-associated epilepsy. Higurashi and colleagues investigated the efficacy of AEDs in patients with PCDH19-associated epilepsy (Higurashi et al., 2013). The authors noted that the efficacy of carbamazepine was very low, particularly in children experiencing severe cluster seizures. After reducing or discontinuing midazolam (which was able to control seizures in this patient), seizure recurrence and, occasionally, worsening of seizures were observed. Other AEDs, such as phenytoin / fosphenytoin or phenobarbital, showed only transient efficacy. Smith et al. also reported a cohort of 38 patients with PCDH19-associated epilepsy recruited through a patient registry. Of these patients, 30 (79%) demonstrated uncontrolled seizures, often despite receiving three or more AEDs (Smith et al., 2018). For these reasons, there is a need for new AEDs with novel mechanisms of action and improved side effect profiles that can maintain seizure control in patients with PCDH19-associated epilepsy.

[0229] Thus, there is an unmet medical need for PCDH19-associated epilepsy, a distinct generic form of epilepsy. The formulations and methods disclosed herein may meet this need.

[0230] In addition to the methods disclosed herein, ganaxolone may also have a positive effect on the neuropsychiatric, behavioral, and sleep disorders associated with PCDH19-associated epilepsy. Potential drug treatments that could provide multimodal effects relevant to the various symptoms faced by these individuals would be a therapeutic improvement over the current standard of care. Such treatments would be within the scope of the present invention.

[0231] Reduced steroidogenesis in patients with PCDH19-related epilepsy Endogenous neurosteroids play an important role in maintaining homeostasis of brain activity, and two recent reports provided compelling evidence that endogenous neurosteroid production is reduced in people affected by PCDH19 gene mutations.

[0232] Tan et al. were the first group to report this phenomenon. They performed gene expression analysis on primary skin fibroblasts from individuals affected with PCDH19-associated epilepsy and age-matched controls. They reported that the AKR1C1-3 gene was significantly dysregulated compared to controls. This gene is known to be important for the production of the steroid hormone metabolic enzyme responsible for the production of allopregnanolone. This gene expression result was further confirmed by analytical assessment of circulating allopregnanolone (Tan et al., 2015).

[0233] The findings of Tan et al. were further supported when Trivisano et al. reported on the blood levels of various neurosteroids, including allopregnanolone, in 12 PCDH19 patients and compared the levels with age-matched controls. Overall, reduced steroid synthesis was found in those affected by the gene mutation (Trivisano et al., 2017).

[0234] Therefore, administration of pregnenolone neurosteroids may help minimize the effects of allopregnanolone deficiency.

[0235] Dravet syndrome Dravet syndrome is a rare genetic epileptic encephalopathy described in 1978. It begins within the first year of life in otherwise healthy infants. Prior to 1989, the syndrome was known as epilepsy with polymorphic seizures, polymorphic epilepsy of infancy (PMEI), or severe myoclonic epilepsy of infancy (SMEI). The disorder begins in infancy but is lifelong.

[0236] Approximately 80% of people with the syndrome have a gene mutation (most commonly SCN1A) that causes problems in the pathway by which ion channels in the brain function. Approximately 95% of patients with Dravet syndrome have a de novo heterozygous mutation, which explains the unaffected status of many siblings and parents.

[0237] The first seizure is often accompanied by fever and may be a tonic-clonic or hemi-clonic seizure. The seizures are almost always intractable. Most children develop some degree of developmental disability and have other symptoms associated with the syndrome. Infants are developing normally at the time of seizure onset, and magnetic resonance imaging (MRI) and electroencephalography (EEG) studies are also normal in infancy.

[0238] Seizures early in life are often prolonged (lasting more than 2 minutes) or repetitive and may lead to status epilepticus. Children with Dravet syndrome can develop many different seizure types: myoclonic, tonic-clonic, absence or atypical absence seizures, atonic, focal seizures, and nonconvulsive status epilepticus. Myoclonic seizures appear between the ages of 1 and 5 years in 85% of children with Dravet syndrome.

[0239] Seizures occur without fever. However, these children are very susceptible to infections and often have seizures if they are sick or have a fever. Seizures can also be triggered by slight changes in body temperature that are not caused by infection, such as warm or hot bath water or hot weather. Many children have photosensitive seizures. Emotional stress or excitement can also trigger seizures in some children.

[0240] Children usually develop normally in the early years. After age 2, they may miss developmental milestones or not develop as quickly as they get older, and they experience an increase in seizures. There appears to be a correlation between the frequency of seizures, the frequency with which status epilepticus occurs, and the degree of developmental delay in children. Around age 6, cognitive problems may stabilize or begin to improve in some children. However, most children with Dravet syndrome have some degree of developmental disability that persists.

[0241] Other possible problems include: reduced motor tone (which may contribute to leg pain problems), unsteady gait (may result in a crouched gait), chronic infections, low humoral immunity, growth and nutrition problems (autonomic nervous system problems), and behavioral or developmental problems (such as autism spectrum disorders).

[0242] LGS Lennox-Gastaut syndrome (LGS) is a severe form of epilepsy. Seizures usually begin before age 4. Seizure types vary among patients but include tonic, atonic, atypical absence, and myoclonic. Periods of frequent seizures may be interspersed with brief, relatively seizure-free periods.

[0243] Most children with LGS experience some degree of impairment in intellectual function or information processing, along with developmental delay and behavioral problems. Lennox-Gastaut syndrome can be caused by brain malformations, perinatal asphyxia, severe head injury, central nervous system infections, and inherited degenerative or metabolic conditions. In 30–35% of cases, no cause can be found. Many cases of LGS have genetic mutations that are clinically relevant to the diagnosis. These may include known encephalopathy epilepsy genes in Rett syndrome, CNTNAP1, XP22.33, SCN2A, GABR3, Shank2, Shank3, and other genetic conditions associated with clinical epilepsy in LGS.

[0244] Patients with LSG, and those with other genetic conditions of intractable epilepsy that clinically resemble LGS symptoms, have occasionally been treated with and responded to classes of corticosteroids such as prednisone or adrenocorticotropic hormone (ACTH).

[0245] Non-degenerative genotypes or idiopathic refractive cases of LGS may respond to neurosteroid treatment as described herein.

[0246] CSWS Sustained spikes during sleep (CSWS) begin with seizures between the ages of 2 and 12; peaking at 4-5 years, EEG findings of continuous spikes during slow wave sleep usually occur 1-2 years after seizure onset (22). Males predominate (62%), and up to one-third of patients exhibit an abnormal mental status. Clinical manifestations include three evolutionary stages:

[0247] Stage 1, pre-CSWS: Infrequent nocturnal focal motor seizures, frequent hemiplegic status epilepticus, absence seizures, atonic seizures, complex focal seizures, and generalized tonic-clonic seizures occur.

[0248] Stage 2 of CSWS: Seizures become more frequent and complex, with typical or more frequent atypical absences, myoclonic absences, absence status epilepticus, rare atonic or clonic seizures, and focal simple cognitive impairment or partial complex cognitive impairment seizures (usually at night during CSWS on EEG), as well as some secondary generalized tonic-clonic or primary generalized tonic-clonic seizures. Tonic seizures do not occur. Marked psychomotor slowing and behavioral abnormalities, as well as Wernicke's or global language regression, occur, with localization of the Sylvian cortex on EEG and magnetoencephalography (MEG) studies.

[0249] The third stage (several months to usually 2-10 years later) involves remission of CSWS and seizures, as well as general improvement, normalization of the CSWS pattern, and residual language or other learning difficulties.

[0250] New genetic overlaps have been identified in the genetics of autism and epilepsy, primarily with Grin2A or Grin2B. Many may be idiopathic to testing.

[0251] Early infantile epileptic encephalopathy Early infantile epileptic encephalopathy is a genetic disease that affects newborns. It is characterized by seizures. Infants primarily have tonic seizures (which cause stiffening of the muscles of the body, typically the back, legs, and arms), but may also experience focal seizures and, rarely, myoclonic seizures (which cause jerks or spasms of the upper body, arms, or legs). Symptom episodes can occur more than 100 times a day.

[0252] Status epilepticus (SE) Status epilepticus (SE) is a severe seizure disorder in which a patient with epilepsy experiences a seizure lasting more than five minutes or multiple seizures within a five-minute period without recovery between seizures. In some cases, seizures can last for days or weeks. Status epilepticus is treated in the emergency room with traditional anticonvulsant medications. GABAergic drugs, such as benzodiazepines (BZ), are also effective. A Receptor modulators are the first-line treatment. Patients who do not respond to BZ alone are usually treated with anesthetics or barbiturates in combination with BZ. Approximately 23–43% of patients with status epilepticus treated with benzodiazepines and at least one additional antiepileptic drug do not respond to treatment and are considered refractory (Rossetti, AO and Lowenstein, DH, Lancet Neurol. (2011) 10(10):922–930). Currently, there are no good treatment options for these patients. Mortality rates are high for patients with refractory status epilepticus (RSE), and most RSE patients do not return to their pre-RSE clinical state. Approximately 15% of patients hospitalized with SE belong to a subgroup of RSE patients known as very refractory SE (SRSE), in which seizures persist or recur more than 24 hours after the initiation of anesthetic therapy. SRSE is associated with high mortality and morbidity (Shorvon, S., and Ferlisi, M., Brain, (2011) 134(10):2802-2818).

[0253] Early severe epileptic seizures Early severe epileptic seizure disorder is associated with very limited developmental progression and marked hypotonia.

[0254] Fragile X syndrome (FXS) Fragile X is a genetic condition characterized by a variety of developmental problems, including learning disabilities and cognitive impairments.

[0255] Neurosteroids Endogenous neurosteroids play an important role in maintaining homeostasis of brain activity. Neurosteroids have the ability to rapidly enact changes in the brain in response to changes in the brain environment. Neurosteroids do not interact with classical steroid hormone receptors that regulate gene transcription, but rather regulate brain excitability primarily through interactions with neuronal membrane receptors and ion channels.

[0256] Neurosteroids act in a variety of ways, depending on the chemical structure of the steroid molecule, such as GABA A GABA can be a positive or negative regulator of receptor function (Pinna and Rasmussen, 2014; Reddy, 2003). A Structurally, GABA receptors mediate the major role of synaptic inhibition in the CNS. A The receptor is a heteropentamer of five protein subunits that form a chloride ion channel. There are seven different classes of subunits, some of which have multiple homologous variants (α1-6, β1-3, γ1-3, σ1-3, δ, ε, θ), and most GABA receptors are A The receptor is composed of α, β, and γ or δ subunits. The neurotransmitter GABA activates the opening of chloride channels, allowing chloride influx and subsequent hyperpolarization. A The receptors prevent the generation of action potentials by preventing the depolarization caused by excitatory neurotransmission. There are two types of inhibitory neurotransmission mediated through GABA receptors: synaptic (transient) inhibition and extrasynaptic (sustained) inhibition. Neurosteroids inhibit both synaptic and extrasynaptic GABA. ABy modulating receptors, it enhances both transient and sustained currents. Transient inhibition results from the activation of γ2-containing receptors at the synapse by the intermittent release of millimolar concentrations of GABA from the axon terminals of presynaptic GABAergic neurons. In contrast, tonic inhibition is mediated by the continuous activation of δ-containing extrasynaptic receptors outside the synaptic cleft by low levels of ambient GABA that escape reuptake by GABA transporters. Tonic inhibition plays a unique role in controlling hippocampal excitability by setting a baseline of excitability (Reddy 2010).

[0257] Neurosteroids such as ganaxolone inhibit GABA A The first observation that neurosteroids potentiate GABA-induced responses mediated by GABA receptors was reported in 1984 for alphaxolone (Harrison and Simmonds, 1984). This modulatory effect of neurosteroids is due to the GABA receptors located within the transmembrane domains of the α- and β-subunits. A This occurs through binding to distinct sites on the receptor (Hosier et al, 2007; Hosier et al, 2009). The binding site for neurosteroids is distinct from that of GABA, benzodiazepines, and barbiturates. The exact location of the neurosteroid binding site is currently unknown, but a highly conserved glutamine at position 241 within the M1 domain of the α-subunit has been shown to play an important role in neurosteroid regulation (Hosier et al, 2009). In addition to the binding site, there is also a GABA-dependent interaction between neurosteroids and benzodiazepines. A Neurosteroids interact with most GABA receptors differently. A While benzodiazepines modulate GABA receptor isoforms containing the γ2 subunit and not the α4- or α6-subunits, AAlthough specific α-subunits may influence the efficacy of neurosteroids, γ-subunit types may act only on GABA receptors (Lambert et al., 2003; Reddy, 2010). A This can affect both the efficacy and potency of neurosteroid modulation of receptors (Lambert et al, 2003).

[0258] Recent research has shown that GABA A The existence of at least three neurosteroid binding sites on the receptor has been demonstrated: one for the allosteric potentiation of GABA-evoked currents by allopregnanolone, one for direct activation by allopregnanolone, and one for the antagonistic action of sulfated neurosteroids such as pregnanolone sulfate at low (nM) concentrations (Lambert et al., 2003; Hosie et al., 2007). A Neurosteroid potentiation of receptor chloride currents occurs through an increase in both channel open frequency and open duration (Reddy, 2010). Thus, neurosteroids enhance GABA receptor activity, allowing for the influx of large amounts of chloride ions. A By significantly increasing the likelihood of receptor chloride channel opening, they promote potentiation of inhibitory GABAergic transmission. These effects occur at physiological concentrations of neurosteroids. Thus, endogenous neurosteroid levels are associated with increased GABAergic activity. A It continuously regulates receptor function (Reddy, 2010).

[0259] Extrasynaptic δ-subunit-containing GABA A The receptor exhibits increased sensitivity to neurosteroids, suggesting a key regulatory role in tonic inhibition (Wohlfarth et al., 2002). GABA receptors containing the δ subunit AThe receptor is more sensitive to neurosteroid-induced enhancement of GABA responses (Stell et al., 2003). Mice lacking the δ subunit exhibit a dramatic reduction in sensitivity to neurosteroids (Mihalek et al., 1999). The δ-subunit does not contribute to the neurosteroid binding site, but appears to enhance the transduction of neurosteroid action after neurosteroids bind to the receptor. GABA containing the δ subunit A The receptor is poorly desensitized and is activated by ambient concentrations of GABA in the extracellular space. A Promotes mediation of receptor currents. A The receptor current causes stable inhibition of the neuron and reduces its excitability. GABA is a delta-containing GABA A Although they are receptor agonists, even if they bind with high affinity, their efficacy is relatively low (Glykys and Mody, 2007). Thus, neurosteroids do not inhibit the δ-containing GABA receptor, even in the presence of saturating GABA concentrations. A During neuronal activity, perisynaptic δ-subunit-containing GABA receptors and extrasynaptic δ-subunit-containing GABA receptors can significantly enhance the electrical current generated by the receptor. A It is expected that there will be substantial release of GABA from active GABAergic interneurons, which are able to interact with the receptors. Overall, the robust actions of neurosteroids are due to the synaptic and perisynaptic release of GABA. A Receptors / Extrasynaptic GABA A This is likely due to its effect on both receptors (Reddy, 2010).

[0260] Pregnane and pregnenolone neurosteroids are a class of compounds useful as anesthetics, sedatives, hypnotics, anxiolytics, antidepressants, antitremors, autistic behavior treatments, and anticonvulsants. These compounds are characterized by very low water solubility, which limits formulation options. The present invention provides nanoparticle formulations of pregnane and pregnenolone neurosteroids that are orally and parenterally bioavailable.

[0261] Injectable formulations of pregnane and pregnenolone neurosteroids are particularly desirable because the compounds are used for clinical indications where oral administration is excluded, such as anesthesia, and particularly for the emergency treatment of active seizures.

[0262] The present disclosure includes injectable nanoparticulate neurosteroid formulations.

[0263] The pregnane and pregnenolone neurosteroids of the present invention are, respectively, compounds of formula IA: [ka] or a pharmaceutically acceptable salt thereof, wherein: X is O, S, or NR 10 and; R 1 is hydrogen, hydroxyl, —CH2A, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl; A is hydroxyl, O, S, NR 11 or an optionally substituted nitrogen-containing 5-membered heteroaryl, or an optionally substituted nitrogen-containing bicyclic heteroaryl or bicyclic heterocyclyl; R 4is hydrogen, hydroxyl, oxo, optionally substituted alkyl, or optionally substituted heteroalkyl; R 2 , R 3 , R 5 , R 6 , and R 7 are each independently absent, hydrogen, hydroxyl, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxyl (e.g., methoxyl), or optionally substituted heteroalkyl; R 8 and R 9 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl (e.g., methyl), halogenated C1-C6 alkyl (e.g., trifluoromethyl), or C1-C6 alkoxyl (e.g., methoxyl), or R 8 and R 9 forms an oxo group; R 10 is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl, each alkyl being C1 to C6 10 alkyl, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)C1-C4 alkyl, and optionally, a single bond is replaced by a double bond or a triple bond; Each heteroalkyl group may be substituted with one or more methyl groups independently selected from —O—, —S—, —N(R 10 )-, -S(=O)-, or -S(=O)2-; R 10 is hydrogen, alkyl, or alkyl in which one or more methylene groups are replaced by -O-, -S-, -NH, or -N-alkyl; R 11 -H2 or -HR 12 and; R 12 is C1-C6 alkyl or C1-C6 alkoxy.

[0264] The pregnane and pregnenolone neurosteroids of the present invention may each be a compound of formula IA, wherein: X is O; R 1 are hydrogen, -CH3, -CH2OH, 1H-imidazol-1-yl, 1-oxidoquinolin-6-yloxyl, and 4-cyano-1H-pyrazol-1'-yl. R 4 is hydrogen, hydroxyl, oxo, optionally substituted alkyl, or optionally substituted heteroalkyl; R 2 , R 3 , R 5 , R 6 , and R 7 are each independently absent, hydrogen, hydroxyl, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxyl (e.g., methoxyl), or optionally substituted heteroalkyl; R 8 and R 9 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl (e.g., methyl), halogenated C1-C6 alkyl (e.g., trifluoromethyl), or C1-C6 alkoxyl (e.g., methoxyl), or R 8 and R 9 forms an oxo group; R 10 is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl, each alkyl being C1 to C6 10 alkyl, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)C1-C4 alkyl, and optionally, a single bond is replaced by a double bond or a triple bond; Each heteroalkyl group may be substituted with one or more methyl groups independently selected from —O—, —S—, —N(R10 )-, -S(=O)-, or -S(=O)2-; R 10 is hydrogen, alkyl, or alkyl in which one or more methylene groups are replaced by -O-, -S-, -NH, or -N-alkyl.

[0265] The pregnane and pregnenolone neurosteroids of the present invention are, respectively, compounds of formula IB: [ka] or a pharmaceutically acceptable salt thereof, wherein: X is O, S, or NR 10 and; R 1 is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl; R 4 is hydrogen, hydroxyl, oxo, optionally substituted alkyl, or optionally substituted heteroalkyl; R 2 , R 3 , R 5 , R 6 , and R 7 are each independently hydrogen, hydroxyl, halogen, optionally substituted alkyl, or optionally substituted heteroalkyl; R 8 is hydrogen or alkyl, and R 9 is hydroxyl; or R 8 and R 9 together form an oxo group; R 10is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl, each alkyl being C1 to C6 10 alkyl, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)C1-C4 alkyl, and optionally, a single bond is replaced by a double bond or a triple bond; Each heteroalkyl group may be substituted with one or more methyl groups independently selected from —O—, —S—, —N(R 10 )-, -S(=O)-, or -S(=O)2-; R 10 is hydrogen, alkyl, or alkyl in which one or more methylene groups are replaced by -O-, -S-, -NH, or -N-alkyl.

[0266] Compounds of formula IA and formula IB include, for example, allopregnanolone, ganaxolone, alphaxalone, alphadolone, hydroxydione, minaxolone, pregnanolone, acebrocol, or tetrahydrocorticosterone, and pharmaceutically acceptable salts thereof.

[0267] The pregnane and pregnenolone neurosteroids of the present invention also each have the formula II: [ka] or a pharmaceutically acceptable salt thereof, wherein: X is O, S, or NR 10 and; R 1 is hydrogen, hydroxyl, —CH2A, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl; A is hydroxyl, O, S, NR 11or an optionally substituted nitrogen-containing bicyclic heteroaryl or bicyclic heterocyclyl; R 4 is hydrogen, hydroxyl, oxo, optionally substituted alkyl, or optionally substituted heteroalkyl; R 2 , R 3 , R 5 , R 6 , and R 7 are each independently absent, hydrogen, hydroxyl, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxyl (e.g., methoxyl), or optionally substituted heteroalkyl; R 8 and R 9 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl (e.g., methyl), halogenated C1-C6 alkyl (e.g., trifluoromethyl), or C1-C6 alkoxyl (e.g., methoxyl), or R 8 and R 9 forms an oxo group; R 10 is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl, each alkyl being C1 to C6 10 alkyl, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)C1-C4 alkyl, and optionally, a single bond is replaced by a double bond or a triple bond; Each heteroalkyl group may be substituted with one or more methyl groups independently selected from —O—, —S—, —N(R 10 )-, -S(=O)-, or -S(=O)2-; R 10 is hydrogen, alkyl, or alkyl in which one or more methylene groups are replaced by -O-, -S-, -NH, or -N-alkyl; R 11 -H2 or -HR12 and; R 12 is C1-C6 alkyl or C1-C6 alkoxy.

[0268] The pregnane and pregnenolone neurosteroids of the present invention also each have the formula III: [ka] or a pharmaceutically acceptable salt thereof, wherein: X is O, S, or NR 10 and; R 1 is hydrogen, hydroxyl, —CH2A, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl; A is hydroxyl, O, S, NR 11 or an optionally substituted nitrogen-containing bicyclic heteroaryl or bicyclic heterocyclyl; R 4 is hydrogen, hydroxyl, oxo, optionally substituted alkyl, or optionally substituted heteroalkyl; R 2 , R 3 , R 5 , R 6 , and R 7 are each independently absent, hydrogen, hydroxyl, halogen, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkoxyl (e.g., methoxyl), or optionally substituted heteroalkyl; R 8 and R 9 are each independently selected from the group consisting of hydrogen, C1-C6 alkyl (e.g., methyl), halogenated C1-C6 alkyl (e.g., trifluoromethyl), or C1-C6 alkoxyl (e.g., methoxyl), or R 8 and R9 forms an oxo group; R 10 is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl, each alkyl being C1 to C6 10 alkyl, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)C1-C4 alkyl, and optionally, a single bond is replaced by a double bond or a triple bond; Each heteroalkyl group may be substituted with one or more methyl groups independently selected from —O—, —S—, —N(R 10 )-, -S(=O)-, or -S(=O)2-; R 10 is hydrogen, alkyl, or alkyl in which one or more methylene groups are replaced by -O-, -S-, -NH, or -N-alkyl; R 11 -H2 or -HR 12 and; R 12 is C1-C6 alkyl or C1-C6 alkoxy.

[0269] Ganaxolone Ganaxolone (CAS Registry Number 38398-32-2, 3α-hydroxy-3β-methyl-5α-pregnan-20-one) (GNX) is a new chemical entity under investigation as an antiepileptic drug (AED) for rare pediatric seizure disorders, such as protocadherin (PCDH)19 female-predominant epilepsy (also known as PCDH19 female-limited epilepsy), cyclin-dependent kinase-like 5 (CDKL5) mutation-associated epilepsy (CDKL5 deficiency), and Lennox-Gastaut syndrome, with additional potential utility in Dravet syndrome, Angelman syndrome, status epilepticus, and neuropsychiatric and behavioral disorders, such as fragile X syndrome (FXS), postpartum depression, premenstrual dysphoric disorder, and other mood or movement disorders.

[0270] The structural formula of ganaxolone is: [ka]

[0271] Ganaxolone inhibits gamma-aminobutyric acid type A (GABA) in the central nervous system (CNS). A Ganaxolone is a 3β-methylated synthetic analog of the endogenous neurosteroid allopregnanolone, an endogenous allosteric modulator of the steroid hormone receptor (SHR). Ganaxolone has the same core chemical structure as allopregnanolone, but with the addition of a 3β-methyl group designed to prevent conversion back to its active form at nuclear hormone receptors, eliminating the opportunity for undesired hormonal effects while increasing the bioavailability of the neurosteroid and maintaining its desired CNS activity.

[0272] Similar to allopregnanolone, ganaxolone (a neuroactive steroid) exhibits potent antiepileptic, anxiolytic, sedative, and hypnotic activity in animals by allosterically modulating gamma-aminobutyric acid type A (GABAA) receptors in the central nervous system (CNS). Ganaxolone has potency and activity comparable to allopregnanolone in activating synaptic and extrasynaptic GABAA receptors at sites distinct from benzodiazepine sites.

[0273] Ganaxolone binds both synaptic and extrasynaptic GABA receptors at class-specific binding sites. A It works by interacting with extrasynaptic GABA receptors. Outside the synapse, ganaxolone is absorbed and diffuses into the cell membrane, where it binds to extrasynaptic GABA receptors. A It activates receptors to achieve constant, or tonal, regulation of GABA inhibitory signals that calm overexcited neurons.

[0274] Ganaxolone has anticonvulsant activity and is useful, for example, in the treatment of epilepsy and other central nervous system disorders.

[0275] Ganaxolone is insoluble in water. Its solubilities in 95% alcohol, propylene glycol, and polyethylene glycol are 13 mg / mL, 3.5 mg / mL, and 3.1 mg / mL, respectively.

[0276] Ganaxolone is primarily metabolized by the CYP3A family of hepatic enzymes, but interactions based on hepatic metabolism are limited to interactions caused by induction or inhibition of CYP3A4 / 5 by other drugs, such as ketoconazole.

[0277] In vitro, ganaxolone clearance appears to be primarily driven by CYP3A4. In adult clinical studies, grapefruit administration increased ganaxolone exposure in healthy volunteers. Ganaxolone levels were decreased in patients treated concomitantly with enzyme-inducing AEDs. These data further support the hypothesis that CYP3A4 significantly contributes to ganaxolone clearance in humans.

[0278] In adults, plasma concentrations of ganaxolone after oral administration are characterized by high variability. Single-dose PK parameters were strongly influenced by the rate and extent of ganaxolone absorption and whether the subject was in the fed or fasted state.

[0279] In the pediatric population, levels of CYP3A4 expression approach adult levels by approximately 2 years of age, although there is a high degree of interindividual variability (de Wildt et al, 2003). Therefore, patients older than 2 years of age are expected to have clearance rates of ganaxolone similar to adults.

[0280] Ganaxolone has a relatively long half-life of approximately 20 hours in human plasma after oral administration (Nohria, V. and Giller, E., Neurotherapeutics, (2007) 4(1):102-105). Furthermore, ganaxolone has been shown to be effective against T maxThe onset time is short, which means that therapeutic blood levels are reached rapidly. Therefore, an initial bolus dose (loading dose) may not be required, which represents an advantage over other treatment methods. Ganaxolone is useful in treating seizures in adult and pediatric epileptic patients.

[0281] Ganaxolone affects GABAA receptors by interacting with a recognition site distinct from other allosteric GABAA receptor modulators, such as benzodiazepines. Ganaxolone binds to intrasynaptic and extrasynaptic receptors, mediating both transient and sustained modulation, respectively. Ganaxolone's unique binding to these two receptors does not lead to the tolerance seen with benzodiazepines. In contrast to allopregnanolone, ganaxolone is orally bioavailable, cannot be converted back in the body to intermediates such as progesterone, and has classical steroid hormone activity; therefore, it does not activate progesterone receptors, either directly or indirectly via metabolic conversion.

[0282] Intravenous ganaxolone has also been evaluated and shown to induce burst suppression-like electroencephalographic (EEG) patterns in otherwise normal rats and to block seizure responses in a model representative of clinical status epilepticus (SE). Ganaxolone produced a sedative, but not a full anesthetic, response.

[0283] In addition to its anticonvulsant activity, ganaxolone has been shown to have anxiolytic properties as well as improve autism-related behaviors. In a mouse model of posttraumatic stress disorder (PTSD), ganaxolone treatment reduced aggression and anxiety-like behaviors induced by social isolation (Pinna and Rasmussen, 2014). In another study, ganaxolone treatment improved socialization in the BTBR mouse model of autism (Kazdoba et al., 2016). In a clinical study of ganaxolone treatment in children and adolescents with fragile X syndrome (FXS), ganaxolone reduced anxiety and hyperactivity and improved attention in those with higher baseline anxiety (Ligsay et al., 2017).

[0284] Ganaxalone has been shown to exhibit potent antiseizure activity in multiple animal models and has been shown to be safe and effective in preliminary studies in children with intractable epilepsy (Nohria and Giller, 2007).

[0285] The anticonvulsant activity of ganaxalone has been established in multiple in vivo models of seizure activity. The results of these studies indicate that ganaxalone can block seizure propagation, increase the seizure threshold, and reverse status epilepticus with acute or delayed administration.

[0286] Safety pharmacology studies were conducted with ganaxolone.

[0287] Ganaxalone did not interact with the human ether-a-go-go-related gene (hERG) receptor at a measured concentration of 70 nM (n = 2). Ganaxalone did not affect cardiovascular parameters in dogs after single doses of up to 15 mg / kg (maximum concentration [Cmax] 1000 ng / mL and area under the concentration-time curve (AUC) (0-24) 10,000 ng·h / mL). In a toxicity study in 1-year-old dogs (Cmax > 1500 ng / mL), transient sinus tachycardia (> 190 beats per minute [bpm]) was observed after 3 months of dosing in four animals, accompanied by reductions in PR and QT intervals, but there was no treatment effect on QRS duration or corrected QT interval (QTc). No pulmonary effects were observed in female rats at doses up to 40 mg / kg.

[0288] There was physiologically normal shortening of the PR and QT intervals in response to higher heart rates. There were no effects on the QRS duration or QTc interval. No pulmonary effects were observed in female rats at doses up to 40 mg / kg.

[0289] Ganaxalone induces the major cytochrome P450 (CYP) isozymes 1A1 / 2 and 2B1 / 2 in female but not male rats, and autoinduction has been observed in mice and rats, but not in dogs.

[0290] Tissue distribution studies in mice and rats have shown that 14 C]-ganaxolone was rapidly distributed throughout the body into highly perfused organs, the intestine, and adipose tissue, demonstrating that brain ganaxolone concentrations were approximately 5-fold higher than in plasma.

[0291] In all species, most of the radioactivity excreted was via the feces (>70%), with the remainder excreted in the urine.

[0292] In toxicology studies, the most common effect after treatment with ganaxalone was GABAergic ADose-related sedation was observed, an expected pharmacological effect of a positive receptor modulator. There was little evidence of target organ or systemic toxicity associated with either single-dose or multiple-dose treatment with ganaxolone in both oral and IV programs. No functional or anatomical changes were observed in hematopoietic tissues or in any specific organ, such as the liver, kidney, or gastrointestinal (GI) system, in repeated-dose studies. In rats, ganaxalone induced liver enzymes, with the effect being more pronounced in females. This correlated with increased liver weight and dose-related hepatocyte hypertrophy in the 6-month study.

[0293] Long-term oral toxicity studies in dogs have shown that C > 1500 ng / mL (10 and 15 mg / kg / day) max Mean levels of were associated with increased body weight and total plasma cholesterol levels.

[0294] When administered IV to rats and dogs, the primary dose-limiting toxicity finding was sedation. In rats, after 14 days of IV dosing, the no-observed-adverse-effect level (NOAEL) was established at 42 mg / kg / day in males and 30 mg / kg / day in females. In dogs, after administration of ganaxolone by IV bolus followed by 28 days of continuous IV infusion, the NOAEL was 7.20 mg / kg / day, corresponding to steady-state concentrations of approximately 330 ng / mL and 333 ng / mL, respectively. In rabbits, there were no findings in local tolerance studies. Finally, in vitro, ganaxalone did not cause hemolysis and was compatible with human plasma.

[0295] Ganaxalone is not teratogenic in rats or mice and does not significantly affect the development of offspring. Ganaxalone did not affect fertility or early embryonic development in rats. No mutagenic potential was detected. Treatment of neonatal rats with ganaxalone resulted in the expected signs of sedation but did not affect development or show any post-mortem changes.

[0296] In oral dosing programs, the therapeutic index in dogs is approximately 2-3 fold from the non-human NOAEL level to adult dog partial seizure epilepsy and pharmacokinetic studies (sedation).

[0297] Ganaxolone has been shown to terminate generalized convulsive seizures in animal models of both epilepsy and status epilepticus.

[0298] In addition to seizure relief, ganaxolone may also be beneficial for sleep in subjects with genetic epilepsy as well as behavioral comorbidities.

[0299] In one embodiment of the invention, ganaxolone is used to treat rare childhood seizure disorders, such as protocadherin (PCDH)19 childhood epilepsy (also known as PCDH19-associated epilepsy), cyclin-dependent kinase-like 5 (CDKL5) deficiency (CDD), and Lennox-Gastaut syndrome (LGS), with additional potential utility in status epilepticus (SE), and neuropsychiatric and behavioral disorders, such as fragile X syndrome (FXS), postpartum depression, premenstrual dysphoric disorder, and other mood disorders.

[0300] Allopregnarone Allopregnanolone (CAS Registry Number 516-54-1, 3α,5α-tetrahydroprogesterone) is an endogenous progesterone derivative with anticonvulsant properties. [ka]

[0301] Allopregnanolone has a relatively short half-life of approximately 45 minutes in human plasma.

[0302] Allopregnanolone is a GABA A Due to its receptor-modulating activity, it exhibits potent antiepileptic, anxiolytic, sedative, and hypnotic activities in animals.

[0303] In addition to its effectiveness in treating seizures, allopregnanolone is being evaluated for use in treating neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, and for the treatment of lysosomal storage diseases characterized by abnormalities in cholesterol synthesis, such as Niemann-Pick A, B, and C, Gaucher disease, and Tay-Sachs disease (see U.S. Pat. No. 8,604,011, incorporated herein by reference, for teachings regarding the use of allopregnanolone to treat neurological disorders).

[0304] It has been hypothesized that disruption of certain neurosteroid hormones, such as allopregnanolone, may be involved in the molecular pathogenesis of PCDH19-associated epilepsy (Tan et al., 2015 and Trivisano et al., 2017). Allopregnanolone acts by inhibiting GABA A It is a neurosteroid known for its anticonvulsant and anxiolytic properties, acting as a positive allosteric modulator of the receptor. Gecz and colleagues have investigated various aspects of the molecular pathology of PCDH19-associated epilepsy (Tan et al., 2015). Expression analysis of skin fibroblasts from PCDH19-associated epilepsy patients suggests downregulation of specific sex-based genes in this disease. The AKR1C gene is the most consistently altered gene. When skin cell specimens from girls with PCDH19 mutations and controls were stimulated with progesterone, fibroblasts from PCDH19-mutated patients were poor metabolizers of progesterone to allopregnanolone. This suggests that impaired AKR1C mRNA, protein levels, and enzyme activity may contribute to allopregnanolone deficiency in patients with PCDH19-associated epilepsy. Gecz and colleagues are currently investigating further preclinical models to evaluate allopregnanolone deficiency in PCDH19-associated epilepsy (Tan et al., 2015).

[0305] The relationship between progesterone and its metabolite, allopregnanolone, and seizures has been extensively studied in women with catamenial epilepsy, a condition characterized by varying seizure frequency associated with different phases of the menstrual cycle. At times during the menstrual cycle when progesterone is lower (e.g., perimenopause), seizures tend to be more likely (French 2005). Circulating allopregnanolone levels parallel progesterone levels. The reproductive effects of progesterone are related to its interaction with intracellular progesterone receptors, whereas the anticonvulsant effects of progesterone are not (Reddy and Rogawski 2009). The antiseizure activity of progesterone is derived from its conversion to the neurosteroid allopregnanolone (Kokate et al., 1999). Allopregnanolone acts by inhibiting GABA receptors. A Due to its effects on the receptor, it has been shown to protect against seizure activity in several animal models (Reddy and Rogawski 2009). Ganaxolone, a synthetic analog of allopregnanolone that lacks progesterone-related effects, may be useful in treating seizures associated with PCDH19-related epilepsy.

[0306] Alfaxalone Alphaxalone, also known as alfaxalone (CAS Registry Number 23930-19-0, 3α-hydroxy-5α-pregnane-11,20-dione), is an anesthetically active neurosteroid. Alphaxalone is used as a general anesthetic in veterinary practice. The anesthetic is often administered in combination with an anticonvulsant for the treatment of refractory seizures. Injectable nanoparticulate neurosteroid formulations containing alphaxalone alone or in combination with either ganaxolone or allopregnanolone are within the scope of the present disclosure. [ka]

[0307] Afadlon Alphadolone, also known as alfadolone (CAS Registry Number 14107-37-0, 3α,21-dihydroxy-5α-pregnane-11,20-dione), is a neurosteroid with anesthetic properties. Its salt, alphadolone acetate, in combination with alphaxalone, is used as a veterinary anesthetic. [ka]

[0308] Additional neurosteroids Newly published data provide further evidence that pregnenolone, a neurosteroid related to ganaxolone, may specifically aid in the repair of neuronal damage caused by CDKL5 deficiency. The kinase CDKL5 is defective in patients with CDKL5 gene mutations, requiring the IQ motif-containing GTPase-activating protein 1 (IQGAP1) to form a functional complex with its effector Rac1 and the microtubule plus-end tracking protein CLIP170. This complex is required for target cell migration and polarity, both of which affect neuronal morphology. CDKL5 deficiency disrupts CLIP170's microtubule binding and thus disrupts its dynamics. CLIP170 is the cellular target of pregnenolone, a neurosteroid highly similar in structure and function to ganaxolone. Pregnenolone restores microtubule binding of CLIP170 in CDKL5-deficient cells by blocking CLIP170 in its active conformation, rescuing the morphological defects of CDKL5-deficient neurons (Barbiero I, Peroni D, Tramarin M, Chandola C, Rusconi L, Landsberger N, Kilstrup-Nielsen C. The neurosteroid pregnenolone reverts microtubule derangement induced by the loss of a functional CDKL5-IQGAP1 complex. Hum Mol Genet. 2017 Jun 21. doi:10.1093 / hmg / ddx237. [Epub ahead of print]). These findings provide novel insight into CDKL5 function and pave the way for target-specific therapeutic strategies, such as ganaxolone, for individuals affected by CDKL5 disorders.

[0309] Additional neurosteroids that may be used in the nanoparticulate neurosteroid formulations of the present disclosure and methods disclosed herein include hydroxydione (CAS Registry Number 303-01-5, (5β)-21-hydroxypregnane-3,20-dione), minaxolone (CAS Registry Number 62571-87-3, 2β,3α,5α,11α)-11-(dimethylamino)-2-ethoxy-3-hydroxypregnan-20-one), pregnanolone (CAS Registry Number 128-20-1, (3α,5β)-d-hydroxypreganan-20-one), lennanolone (CAS Registry Number 565-99-1, 3α-hydroxy-5β-pregnane-11,20-dione), or tetrahydrocorticosterone (CAS Registry Number 68-42-8, 3α,5α-pregnane-20-dione).

[0310] Additional neurosteroids that may be used in the nanoparticulate neurosteroid formulations of the present disclosure and the methods disclosed herein include Co26749 / WAY-141839, Co134444, Co177843, and Sage-217, Sage-324, and Sage-718. Co26749 / WAY-141839, Co134444, Co177843, and Sage-217 have the following structures: [Table A]

[0311] Additional neurosteroids that may be used in the nanoparticulate neurosteroid formulations of the present disclosure and the methods disclosed herein include compounds disclosed in U.S. Patent Application Publication No. 2016-0229887 (U.S. Patent Application No. 14 / 913,920, filed February 23, 2016), which is incorporated herein by reference in its entirety.

[0312] Dosage The pregnenolone neurosteroid in the methods of the present invention can be administered in an amount of about 1 mg / day to about 5000 mg / day in 1, 2, 3, or 4 divided doses. In certain embodiments, doses of 1600 mg / day and 2000 mg / day may be associated with somnolence, and the 1800 mg / day dose defines an optimal combination of drug exposure, dosing convenience, and tolerability.

[0313] When the pregnenolone neurosteroid is ganaxolone, the target and maximum dose of ganaxolone is about 1800 mg / day. In these embodiments, this dose provides the highest achievable exposure based on the nonlinear kinetics of ganaxolone. Thus, when the pregnenolone neurosteroid is ganaxolone, the amount of ganaxolone administered in the methods of the present invention is generally at a dose of 1 mg / kg / day to about 63 mg / kg / day, given in 1, 2, 3, or 4 divided doses, about 200 mg / day to about 1800 mg / day, about 300 mg / day to about 1800 mg / day, about 400 mg / day to about 1800 mg / day, about 450 mg / day to about 1800 mg / day, about 675 mg / day to about 1800 mg / day, about 900 mg / day to about 1800 mg / day, about 1125 mg / day to about 1800 mg / day, about 1350 mg / day to about 1800 mg / day, about 1575 mg / day to about 1800 mg / day, or about 1800 mg / day.

[0314] In certain embodiments, about 900 mg to about 1800 mg, about 950 mg to about 1800 mg, about 1000 mg to about 1800 mg, about 1100 mg to about 1800 mg, or about 1200 mg of ganaxolone is administered per day for two or more consecutive days. Ganaxolone may be administered orally or parenterally in one, two, three, or four doses per day.

[0315] Whether a person takes ganaxolone twice or three times daily depends on the formulation. For patients taking oral immediate-release capsules, ganaxolone is generally administered twice daily, with each dose separated by 8 to 12 hours from the next and / or previous dose. For patients taking oral suspension, ganaxolone is generally administered three times daily, with each dose separated by 4 to 8 hours from the next and / or previous dose.

[0316] When the pregnenolone neurosteroid is ganaxolone, the methods of the invention involve administration of ganaxolone at a dose of 1 mg / kg / day to about 63 mg / kg / day, provided that the total amount of ganaxolone administered does not exceed 1800 mg / day.

[0317] The pharmacokinetics of ganaxolone in formulations containing immediate-release 0.3 micron particles (e.g., the formulation of Example 2) is linear up to approximately 1200 mg / day (administered twice daily ("BID")), with a modest increase in exposure at a dose of 1600 mg / day and little or no further increase at a dose of 2000 mg / day. Therefore, a dose of 1800 mg is generally targeted to maintain the highest possible trough levels in all subjects. Dose levels higher than 1800 mg / day would not be medically advantageous because they would require dosing more than three times daily, which would not result in greater exposure and may further hinder patient compliance.

[0318] In certain embodiments, ganaxolone is administered at a dose of more than 5 mg / kg / day, e.g., about 6 mg / kg / day to about 63 mg / kg / day, provided that the total amount of ganaxolone administered does not exceed 1800 mg / day.

[0319] In certain embodiments, the dose of ganaxolone is adjusted from 15 mg / kg / day up to 63 mg / kg / day, with a maximum dose of 1800 mg per day during treatment.

[0320] In certain embodiments, the treatment method comprises administering at least 33 mg / kg / day of ganaxolone in 1, 2, 3, or 4 doses, with a maximum daily dose of about 1800 mg.

[0321] In certain embodiments, the human is about 0.6 to about 7 years old and is administered a ganaxolone dose of about 1.5 mg / kg BID (3 mg / kg / day) to 12 mg / kg three times daily ("TID") (36 mg / kg / day). In such embodiments, when the human receives a 12 mg / kg TID dosage regimen, a trough concentration of at least about 38.5±37.4 ng / mL is achieved.

[0322] In certain embodiments, ganaxolone is administered orally with food to humans aged 5 to 15 years at a dose of 6 mg / kg BID (12 mg / kg / day) to 12 mg / kg TID (36 mg / kg / day) in a β-cyclodextrin formulation, achieving plasma concentrations of ganaxolone of up to 22.1 ng / mL and 5.7 to 43.7 ng / mL at weeks 4 and 8, respectively, of administration.

[0323] In certain embodiments, ganaxolone is administered orally in the same formulation to epileptic patients aged 1 to 13 years at a dose of 1 to 12 mg / kg TID (3 to 36 mg / kg / day) with food to achieve a maximum ganaxolone plasma concentration of 5.78 ng / mL (1 mg / kg TID) to 10.3 to 16.1 ng / mL (12 mg / kg TID).

[0324] In certain embodiments, ganaxolone is administered orally to patients 4 to 41 months of age (0.33 to 3.42 years) at a dose of 3 to 18 mg / kg TID (9 to 54 mg / kg / day) in an oral suspension formulation to achieve a ganaxolone C of about 123 ng / mL. max , and a trough concentration of approximately 23 ng / mL is achieved.

[0325] In certain embodiments, the average ganaxolone C min(trough) was 55 ng / ml to about 100 ng / ml based on 1000 mg ganaxolone administered three times daily in a 0.3 μm ganaxolone suspension (i.e., the formulation of Example 1), and C max Levels are approximately 240ng / ml to 400ng / ml (e.g., 262ng / mL).

[0326] In certain embodiments, the method provides a mean C based on twice-daily administration of 1000 mg ganaxolone in a 0.3 micron ganaxolone capsule formulation (i.e., the formulation of Example 2). min (trough) and C max This results in levels of about 56.9 ng / ml and about 262 ng / mL, respectively.

[0327] In certain embodiments, administration of ganaxolone results in a C of greater than 3, 3.5, 4, 4.5, 5, or 6. min / C max This C min / C max The ratio may be achieved after administration of a single dose and / or after administration at steady state. In certain embodiments, regardless of the dose of ganaxolone administered, C min / C max The ratio remains the same.

[0328] In certain embodiments, the administered dose is determined from a pediatric pharmacokinetic model, which shows that, in various pediatric age ranges, C s are achieved similar to those achieved according to the effective dose determined in the adult epilepsy population. max and AUC exposure. The model can be constructed in a standard manner, for example, taking into account the pharmacokinetic data in this application.

[0329] In certain embodiments, pregnenolone neurosteroid may be administered to a patient using several titration steps until a therapeutically effective dosing regimen is achieved, for example, approximately 6-8 titration steps may be used depending on the patient's size.

[0330] In certain embodiments, the treatment method of the present invention comprises establishing a patient's baseline seizure frequency, initially administering to the patient a dose of ganaxolone in an amount of about 0.5 mg / kg / day to about 15 mg / kg / day, and gradually increasing the dose of ganaxolone to about 18 mg / kg / day to about 60 mg / kg / day over a four-week period, with a total dose of ganaxolone of up to about 1800 mg / day for patients weighing more than 30 kg and about 63 mg / day for patients weighing less than 30 kg. In certain preferred embodiments, the initial dose of ganaxolone is about 4.5 mg / kg / day. In certain preferred embodiments, the dose of ganaxolone is increased to about 36 mg / kg / day. In certain preferred embodiments, the dose of ganaxolone is reduced to its previous level if the patient experiences a dose-limiting adverse event.

[0331] In certain embodiments, for subjects weighing more than 30 kg, treatment is initiated at a dose of 900 mg / day in divided doses. Thereafter, the dose is increased by approximately 20-50% at intervals of not less than 3 days but not more than 2 weeks (e.g., an increase from 900 mg / day to 1200 mg / day is a 33% increase), provided that the current dose is reasonably tolerated until the desired efficacy is achieved or the maximum tolerated dose (MTD) level is reached. Subsequent dose adjustments may be made in increments of approximately 20-50%, with a minimum of 3 days between dose changes, unless required for safety. The maximum tolerated dose in this embodiment is 1800 mg / day.

[0332] In certain embodiments, for subjects weighing 30 kg or less, treatment is initiated at 18 mg / kg / day and may be increased in approximately 20% to 50% increments at intervals of at least 3 days but not more than 2 weeks, provided that the current dose is reasonably tolerated until the desired efficacy is achieved or the maximum tolerated dose (MTD) level is reached. Subsequent dose adjustments may be made in approximately 20% to 50% increments, with a minimum of 3 days between dose changes, unless required for safety. The maximum tolerated dose in this embodiment is 63 mg / kg / day.

[0333] For humans weighing ≥ 28 kg (62 lbs), ganaxolone may be initiated at a dose of about 300 mg / day to about 600 mg / day (e.g., 400 mg / day) in divided doses, with the dose increasing by 450 mg / day every 7 days until 1800 mg / day or the maximum tolerated dose is reached.

[0334] For humans weighing <28 kg (62 lbs), ganaxolone may be initiated at a dose of about 10 mg / kg / day to about 30 mg / kg / day (e.g., 18 mg / kg / day), increasing by about 15 mg / kg / day each week until 63 mg / kg / day is reached.

[0335] In certain embodiments, ganaxolone is administered as an oral suspension in increments of 10 mg / day to 20 mg / day (e.g., 15 mg / kg / day) up to 63 mg / kg / day (up to 1800 mg / day), or as an oral capsule in increments of 225 mg / day to 900 mg / day (e.g., 450 mg / day). In some such embodiments, ganaxolone may be dosed, for example, as follows: 6 mg / kg three times daily (TID) (18 mg / kg / day) (suspension) / 225 mg twice daily (BID) (450 mg / day) (capsules) - Days 1 to 7 11 mg / kg TID (33 mg / kg / day) (suspension) / 450 BID (900 mg / day) (capsules) - days 8 to 14; 16 mg / kg TID (48 mg / kg / day) (suspension) / 675 BID (1350 mg / day) (capsules) - days 15 to 21; 21 mg / kg TID (63 mg / kg / day, not to exceed 1800 mg / day) (suspension) / 900 BID (1800 mg / day) (capsules) - Days 22 to 28.

[0336] In certain embodiments, ganaxolone is administered in an oral suspension and the following titration schedule is used: [Table B]

[0337] In certain embodiments, ganaxolone is administered in capsules and the following titration schedule is used: [Table C]

[0338] In certain embodiments, the trough concentrations associated with maximum efficacy are within the range of about 55 ng / mL, about 60 ng / ml, or about 65 ng / ml (0.3 micron suspension; TID dosing), and an 1800 mg / day dose (0.3 micron capsule, BID dosing) achieves trough plasma concentrations within this range.

[0339] The methods of treatment disclosed herein include administration of a neurosteroid (e.g., ganaxolone) with or without food. In certain embodiments, ganaxolone is administered with food.

[0340] Treatment period Treatment periods according to the present invention may range from 1 day to over 2 years. For example, treatment periods may be 1 day to 80 years, 1 day to 70 years, 1 day to 60 years, 1 day to 50 years, 1 day to 45 years, 2 days to 45 years, 2 days to 40 years, 5 days to 35 years, 10 days to 30 years, 10 days to 30 years, or 15 days to 30 years. In some embodiments, the treatment period is as long as the subject continues to derive therapeutic benefit from administration of the neurosteroid. In some embodiments, the treatment period is 14 days, 28 days, 30 days, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 6 months, 1 year, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, or 10 years.

[0341] In certain embodiments, at the end of the treatment period, or immediately upon discontinuation of treatment, the dose is gradually reduced over a period of 1 to 4 weeks based on the subject's age, weight, dose, and duration of treatment.

[0342] formulation The formulations of the present invention comprise a pregnenolone neurosteroid (e.g., ganaxolone) and one or more pharmaceutically acceptable excipients. In certain embodiments, the formulations are free of cyclodextrins, including sulfoalkyl ether cyclodextrins and modified forms thereof.

[0343] In a preferred embodiment, the amount of pregnenolone neurosteroid in the formulation is therapeutically effective to treat symptoms of a disorder selected from the group consisting of PCDH19-associated epilepsy, CDKL5 epileptic encephalopathy, Dravet syndrome, Lennox-Gastaut syndrome (LGS), continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), and other refractory genetic epileptic conditions that share a common seizure type and that clinically resemble PCDH19-associated epilepsy, CDKL5 deficiency, Dravet syndrome, LGS, CSWS, and ESES immediately after administration of the formulation for 1 week and / or 2 weeks and / or 3 weeks and / or 4 weeks and / or 6 weeks and / or 7 weeks and / or 8 weeks and / or 9 weeks and / or 10 weeks and / or 11 weeks and / or 12 weeks. The symptom may be selected from the group consisting of intractable epilepsy, developmental delay, intellectual disability, sleep disorder, gross motor skill disorder, behavioral dysregulation, and a combination of two or more of the foregoing. In some such embodiments, the amount of pregnenolone neurosteroid is effective to reduce seizure frequency in a human after administration at the dosages and for periods described herein.

[0344] In a preferred embodiment of the present invention, a pregnenolone neurosteroid, such as ganaxolone, is incorporated into a pharmaceutically acceptable composition for oral administration. In certain preferred embodiments, such a formulation may be a liquid (e.g., an aqueous liquid, including a suspension, solution, etc.). In other preferred embodiments, the oral formulation may be an oral solid dosage form (e.g., an oral capsule or tablet). In a most preferred embodiment, the oral formulation is an oral suspension containing a pregnenolone neurosteroid. Preferably, a unit dose of the oral formulation contains a therapeutically effective amount of a pregnenolone neurosteroid that can be orally administered to a (e.g., human) patient (e.g., an infant, child, adolescent male or female, or adult). In certain embodiments, the oral suspension is administered to a patient via an oral syringe. For example, the oral suspension is intended for use in children weighing less than 30 kg. Alternatively, the oral suspension may be administered to patients who would have difficulty swallowing a solid oral dosage form. Children weighing more than 30 kg may take a solid dosage form, such as a ganaxolone capsule. Ganaxolone oral suspension may be administered via an oral dosing syringe, for example, three times daily. Ganaxolone capsules may be administered, for example, twice daily. Patients experience better absorption of ganaxolone with food (milk).

[0345] In certain preferred embodiments, the liquid formulations of the present invention may be those described and prepared in Applicant's prior U.S. Patent No. 8,022,054 (entitled "Liquid Ganaxolone Formulations and Methods for the Making and Use Thereof"), which is incorporated herein by reference in its entirety. However, oral liquid (e.g., suspension) formulations of pregnenolone neurosteroids may also be prepared according to other methods known to those skilled in the art.

[0346] As described in U.S. Patent No. 8,022,054, the liquid formulation may be an aqueous dispersion of stabilized pregnenolone neurosteroid (e.g., ganaxolone) particles, comprising ganaxolone, a hydrophilic polymer, a wetting agent, and an effective amount of a complexing agent to stabilize particle growth after initial particle growth and after reaching an endpoint, the complexing agent being selected from the group of small organic molecules having a molecular weight of less than 550 and containing a moiety selected from the group consisting of a phenolic moiety, an aromatic ester moiety, and an aromatic acid moiety, the stabilized particles having a particle volume-weighted median diameter (D50) of about 50 nm to about 500 nm, the complexing agent being present in an amount of about 0.05% to about 5 wt% based on the weight of the particles, and the particles being dispersed in an aqueous solution further containing at least two preservatives in an amount sufficient to inhibit microbial growth. The hydrophilic polymer may be present in an amount of about 3% to about 50 wt% based on the weight of the solid particles. The humectant may be present in an amount of about 0.01% to about 10% w / w based on the weight of the solid particles. The pregnenolone neurosteroid (e.g., ganaxolone) may be present in an amount of about 10% to about 80% (and in certain embodiments, about 50% to about 80%) based on the weight of the stabilized particles. The stabilized particles, when dispersed in simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) at a concentration of 0.5 to 1 mg ganaxolone / mL and placed in a water bath at 36°C to 38°C for 1 hour, may exhibit an increase in volume-weighted median diameter (D50) of up to about 150% compared to the volume-weighted median diameter (D50) of the stabilized particles dispersed in distilled water under the same conditions, and the volume-weighted median diameter (D50) of the stabilized particles dispersed in SGF or SIF is less than about 750 nm. The stabilized particles may exhibit an increase in volume-weighted median diameter (D50) of about 150% or less when the formulation is dispersed in 15 mL of SGF or SIF at a concentration of 0.5-1 mg ganaxolone / mL compared to the volume-weighted median diameter (D50) of the stabilized particles when the particles are dispersed in distilled water under the same conditions, and the volume-weighted median diameter (D50) of the stabilized particles dispersed in SGF or SIF is less than about 750 nm. The complexing agent may be a paraben, benzoic acid, phenol, sodium benzoate, methyl anthranilate, etc.The hydrophilic polymer may be a cellulose polymer, a vinyl polymer, or a mixture thereof. The cellulose polymer may be a cellulose ether, such as hydroxypropylmethylcellulose. The vinyl polymer may be a polyvinyl alcohol, such as vinylpyrrolidone / vinyl acetate copolymer (S630). The humectant may be sodium lauryl sulfate, a pharmaceutically acceptable salt of docusate, or a mixture thereof. The aqueous dispersion may further contain a sweetener, such as sucralose. The preservative may be selected from the group consisting of potassium sorbate, methylparaben, propylparaben, benzoic acid, butylparaben, ethyl alcohol, benzyl alcohol, phenol, benzalkonium chloride, and mixtures of any of the foregoing.

[0347] In some embodiments, a liquid pregnenolone neurosteroid (e.g., ganaxolone) formulation is provided, comprising the ganaxolone particles described herein and at least one dispersing or suspending agent for oral administration to a subject. The ganaxolone formulation may be a powder and / or granules for suspension, which upon mixing with water results in a substantially uniform suspension. As described herein, the aqueous dispersion may contain amorphous and non-amorphous ganaxolone particles of multiple effective particle sizes, such that ganaxolone particles with smaller effective particle sizes are absorbed more rapidly and ganaxolone particles with larger effective particle sizes are absorbed more slowly. In certain embodiments, the aqueous dispersion or suspension is an immediate-release formulation. In another embodiment, the aqueous dispersion containing amorphous ganaxolone particles is formulated so that about 50% of the ganaxolone particles are absorbed within about 3 hours after administration, and about 90% of the ganaxolone particles are absorbed within about 10 hours after administration. In another embodiment, the addition of a complexing agent to the aqueous dispersion increases the span of the ganaxolone-containing particles to extend the drug absorption phase, such that 50-80% of the particles are absorbed in the first 3 hours, and about 90% is absorbed in about 10 hours.

[0348] A suspension is "substantially uniform" if it is nearly homogeneous, i.e., if the suspension is comprised of approximately the same concentration of pregnenolone neurosteroid (e.g., ganaxolone) at every point throughout the suspension. Preferred embodiments provide essentially the same (within 15%) concentration when measured at various points in the ganaxolone aqueous oral formulation after shaking. Particularly preferred are aqueous suspensions and dispersions that maintain homogeneity (within a maximum of 15% variation) when measured two hours after shaking. Homogeneity should be determined by a consistent sampling method for determining the homogeneity of the entire composition. In one embodiment, the aqueous suspension can be resuspended into a homogeneous suspension by physical agitation lasting less than 1 minute. In another embodiment, the aqueous suspension can be resuspended into a homogeneous suspension by physical agitation lasting less than 45 seconds. In yet another embodiment, the aqueous suspension can be resuspended into a homogeneous suspension by physical agitation lasting less than 30 seconds. In yet another embodiment, no agitation is required to maintain a homogeneous aqueous dispersion.

[0349] In some embodiments, the pregnenolone neurosteroid (e.g., ganaxolone) powders for aqueous dispersion described herein contain stable ganaxolone particles having an effective particle size of less than 500 nm by weight, formulated with ganaxolone particles having an effective particle size of greater than 500 nm by weight. In such embodiments, the formulation has a particle size distribution in which about 10% to about 100% by weight of the ganaxolone particles are between about 75 nm and about 500 nm, about 0% to about 90% by weight of the ganaxolone particles are between about 150 nm and about 400 nm, and about 0% to about 30% by weight of the ganaxolone particles are greater than about 600 nm. The ganaxolone particles described herein can be amorphous, semi-amorphous, crystalline, semi-crystalline, or a mixture thereof.

[0350] In one embodiment, the aqueous suspensions or dispersions described herein contain ganaxolone particles or ganaxolone complexes at a concentration of about 20 mg / ml to about 150 mg / ml of suspension. In another embodiment, the aqueous oral dispersions described herein contain ganaxolone particles or ganaxolone complexes at a concentration of about 25 mg / ml to about 75 mg / ml of solution. In yet another embodiment, the aqueous oral dispersions described herein contain ganaxolone particles or ganaxolone complexes at a concentration of about 50 mg / ml of suspension. The aqueous dispersions described herein are particularly beneficial for administering ganaxolone to infants (under 2 years of age), children under 10 years of age, and any patient group unable to swallow or ingest solid oral dosage forms.

[0351] Liquid pregnenolone neurosteroid (e.g., ganaxolone) formulations for oral administration can be aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, and syrups. See, for example, Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002). In addition to ganaxolone particles, the liquid dosage form may contain additives such as: (a) disintegrants, (b) dispersants, (c) wetting agents, (d) at least one preservative, (e) viscosity enhancers, (f) at least one sweetener, (g) at least one flavoring agent, (h) complexing agents, and (i) ionic dispersion modulators. In some embodiments, the aqueous dispersion may further contain a crystallization inhibitor.

[0352] Examples of disintegrants used in aqueous suspensions and dispersions include, but are not limited to, starches, such as natural starches, such as corn starch or potato starch, pregelatinized starches, such as National 1551 or Amijele®, or sodium starch glycolate, such as Promogel® or Explotab®; celluloses, such as wood products, microcrystalline cellulose, such as Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, MingTia®, and Solka-Floc®, methylcellulose, croscarmellose, or crosslinked celluloses, such as crosslinked sodium carboxymethylcellulose (Ac-Di-Sol®). cross-linked starches, such as sodium starch glycolate; cross-linked polymers, such as crospovidone; cross-linked polyvinylpyrrolidone; alginates, such as alginic acid, or salts of alginic acid, such as sodium alginate; clays, such as Veegum® HV (magnesium aluminum silicate); gums, such as agar, guar, locust bean, karaya, pectin, or tragacanth; sodium starch glycolate; bentonite; natural sponge; surfactants; resins, such as cation exchange resins; citrus pulp; sodium lauryl sulfate; and sodium lauryl sulfate in combination with starch.

[0353] In some embodiments, dispersing agents suitable for the aqueous suspensions and dispersions described herein are known in the art and include, for example, hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and carbohydrate-based dispersing agents, such as hydroxypropyl cellulose and hydroxypropyl cellulose ethers (e.g., HPC, HPC-SL, and HPC-L), hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ethers (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100). K100M), sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate stearate, amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone / vinyl acetate copolymers (Plasdone®, e.g., S-630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g., Pluronics F68®, F88®, and F108®, which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic 9080, also known as Poloxamine 9080, which is a tetrafunctional block copolymer derived from the sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany, NJ)).In other embodiments, the dispersing agent is selected from the group that does not include one of the following agents: hydrophilic polymers; electrolytes; Tween® 60 or 80; PEG; polyvinylpyrrolidone (PVP); hydroxypropyl cellulose and hydroxypropyl cellulose ethers (e.g., HPC, HPC-SL, and HPC-L); hydroxypropyl methylcellulose and hydroxypropyl methylcellulose ethers (e.g., HPMC K100, HPMC K4M, HPMC K15M, HPMC K100M, and Pharmacoat® USP 2910 (Shin-Etsu Chemical Co., Ltd.); sodium carboxymethylcellulose; methylcellulose; hydroxyethylcellulose; hydroxypropylmethyl-cellulose phthalate; hydroxypropylmethyl-cellulose acetate stearate; amorphous cellulose; magnesium aluminum silicate; triethanolamine; polyvinyl alcohol (PVA); 4-(1,1,3,3-tetramethylbutyl)-phenol polymers with ethylene oxide and formaldehyde; poloxamers (e.g., Pluronics F68®, F88®, and F108®, which are block copolymers of ethylene oxide and propylene oxide); or poloxamines (e.g., Tetronic 908®, also known as Poloxamine 908%).

[0354] Suitable wetting agents (including surfactants) for the aqueous suspensions and dispersions described herein are known in the art and include, but are not limited to, acetyl alcohol, glycerol monostearate, polyoxyethylene sorbitan fatty acid esters (e.g., commercially available Tweens®, e.g., Tween 20® and Tween 80® (ICI Specialty Chemicals)), polyethylene glycols (e.g., Carbowaxs 3350® and 1450®, and Carpool 934® (Union Carbide)), oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate, docusate sodium, triacetin, vitamin E TPGS, sodium taurocholate, simethicone, and phosphotidylcholine.

[0355] Suitable preservatives for the aqueous suspensions or dispersions described herein include, for example, potassium sorbate, parabens (e.g., methylparaben and propylparaben) and their salts, benzoic acid and its salts, other esters of parahydroxybenzoic acid, such as butylparaben, alcohols, such as ethyl alcohol or benzyl alcohol, phenolic compounds, such as phenol, or quaternary compounds, such as benzalkonium chloride. Preservatives used herein are incorporated into dosage forms at concentrations sufficient to inhibit microbial growth. In one embodiment, the aqueous dispersion may contain methylparaben and propylparaben at concentrations ranging from about 0.01% to about 0.3% by weight of methylparaben based on the weight of the aqueous dispersion, and 0.005% to 0.03% by weight of propylparaben based on the weight of the total aqueous dispersion. In yet another embodiment, the aqueous dispersion may contain 0.05 to about 0.1% by weight of methylparaben and 0.01 to 0.02% by weight of propylparaben based on the weight of the aqueous dispersion.

[0356] Suitable thickening agents for the aqueous suspensions or dispersions described herein include, but are not limited to, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, Plasdone® S-630, carbomer, polyvinyl alcohol, alginate, gum arabic, chitosan, and combinations thereof. The concentration of the viscosity-enhancing agent will depend on the agent selected and the viscosity desired.

[0357] Examples of natural and artificial sweeteners suitable for the aqueous suspensions or dispersions described herein include, for example, acacia syrup, acesulfame K, alitame, anise, apple, aspartame, banana, bavarois, berry, blackcurrant, butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus fruits, citrus punch, citrus cream, cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate (cyclamate, cylamate), dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glycidinate (MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mint cream, and mixed berries. Flavoring ingredients include, for example, anise-menthol, cherry-anise, cinnamon orange, cherry-cinnamon, chocolate mint, chocolate mint, honey lemon, lemon-lime, lemon mint, menthol eucalyptus, orange cream, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol, or any combination of these flavoring ingredients, such as anise-menthol, cherry-anise, cinnamon orange, cherry-cinnamon, chocolate mint, honey lemon, lemon-lime, lemon mint, menthol eucalyptus, orange cream, vanilla mint, and mixtures thereof. In one embodiment, the aqueous dispersion may include a sweetening or flavoring agent at a concentration ranging from about 0.0001% to about 10.0% by weight of the aqueous dispersion, hi another embodiment, the aqueous dispersion may include a sweetening or flavoring agent at a concentration ranging from about 0.0005% to about 5.0% by weight of the aqueous dispersion.In yet another embodiment, the aqueous dispersion may include a sweetening or flavoring agent at a concentration ranging from about 0.0001% to 0.1% by weight, from about 0.001% to about 0.01% by weight, or from 0.0005% to 0.004% by weight of the aqueous dispersion.

[0358] In addition to the additives described above, the liquid pregnenolone neurosteroid (e.g., ganaxolone) formulation may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents.

[0359] In some embodiments, the pharmaceutical pregeneronone neurosteroid (e.g., ganaxolone) formulations described herein may be self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are generated by vigorous mechanical dispersion. In contrast to emulsions or microemulsions, SEDDS spontaneously form emulsions when added to excess water without any external mechanical dispersion or agitation. The advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. In addition, water or an aqueous phase can be added immediately before administration, ensuring the stability of unstable or hydrophobic active ingredients. Therefore, SEDDS provide an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. SEDDS may improve the bioavailability of hydrophobic active ingredients. Methods for producing self-emulsifying dosage forms are known in the art, including, but not limited to, U.S. Pat. No. 5,858,401, U.S. Pat. No. 6,667,048, and U.S. Pat. No. 6,960,563, each of which is specifically incorporated by reference.

[0360] Exemplary emulsifiers include ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, sodium lauryl sulfate, sodium docusenate, cholesterol, cholesterol esters, taurocholic acid, phosphotidylcholine, oils such as cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan, or mixtures of these substances.

[0361] In certain preferred embodiments, the liquid pharmaceutical formulation comprises ganaxolone, hydroxypropylmethylcellulose, polyvinyl alcohol, sodium lauryl sulfate, simethicone, methylparaben, propylparaben, sodium benzoate, citric acid, and sodium citrate at a pH of 3.8 to 4.2. The suspension may contain ganaxolone at a concentration of 50 mg / ml. The formulation may further comprise a pharmaceutically acceptable sweetener (e.g., sucralose) and / or a pharmaceutically acceptable flavoring (e.g., cherry). The formulation may be packaged in, for example, a 120 mL, 180 mL, 240 mL, or 480 mL bottle.

[0362] In certain preferred embodiments, the oral solid formulations of the present invention may be those described and prepared in Applicant's prior U.S. Patent No. 7,858,609 (entitled "Solid Ganaxolone Formulations and Methods for the Making and Use Thereof"), which is incorporated herein by reference in its entirety. However, oral solid dosage formulations (e.g., oral capsules or tablets) of pregnenolone neurosteroids may also be prepared according to other methods known to those skilled in the art.

[0363] For example, as disclosed in U.S. Patent No. 7,858,609, an oral solid formulation comprises stabilized particles comprising a pregenolone neurosteroid (e.g., ganaxolone), a hydrophilic polymer, a wetting agent, and an effective amount of a complexing agent to stabilize particle growth after initial particle growth and after reaching an endpoint. The complexing agent is a small organic molecule having a molecular weight of less than 550 and containing a moiety selected from the group consisting of a phenol moiety, an aromatic ester moiety, and an aromatic acid moiety. The stabilized particles have a particle volume-weighted median diameter (D50) of about 50 nm to about 500 nm, and the complexing agent is present in an amount of about 0.05% to about 5 wt.% based on the weight of the solid particles. The hydrophilic polymer may be present in an amount of about 3% to about 50 wt.% based on the weight of the solid particles. The wetting agent may be present in an amount of about 0.01% to about 10 wt.% based on the weight of the solid particles. The pregnenolone neurosteroid (e.g., ganaxolone) may be present in an amount of about 10% to about 80% (and in certain embodiments, about 50% to about 80%), based on the weight of the stabilized particles. The stabilized particles, when dispersed in simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) at a concentration of 0.5 to 1 mg ganaxolone / mL and placed in a water bath at 36°C to 38°C for 1 hour, may exhibit an increase in volume-weighted median diameter (D50) of no more than about 150% compared to the volume-weighted median diameter (D50) of the stabilized particles dispersed in distilled water under the same conditions, and the volume-weighted median diameter (D50) of the stabilized particles dispersed in SGF or SIF is less than about 750 nm. The stabilized particles may exhibit an increase in volume-weighted median diameter (D50) of about 150% or less when the formulation is dispersed in 15 mL of SGF or SIF at a concentration of 0.5 to 1 mg ganaxolone / mL compared to the volume-weighted median diameter (D50) of the stabilized particles when the particles are dispersed in distilled water under the same conditions, and the volume-weighted median diameter (D50) of the stabilized particles dispersed in SGF or SIF is less than about 750 nm. The stabilized solid particles may be combined with optional excipients and prepared for administration in powder form or incorporated into a dosage form selected from the group consisting of tablets or capsules. The complexing agent may be paraben, benzoic acid, phenol, sodium benzoate, methyl anthranilate, etc.The hydrophilic polymer may be a cellulose polymer, a vinyl polymer, or a mixture thereof. The cellulose polymer may be a cellulose ether, such as hydroxypropylmethylcellulose. The vinyl polymer may be polyvinyl alcohol, such as vinylpyrrolidone / vinyl acetate copolymer (S630). The wetting agent may be sodium lauryl sulfate, a pharmaceutically acceptable salt of docusate, or a mixture thereof. When the particles are incorporated into a solid dosage form, the solid dosage form may further comprise at least one pharmaceutically acceptable excipient, such as an ionic dispersion modulator, a water-soluble spacer, a disintegrant, a binder, a surfactant, a plasticizer, a lubricant, a diluent, or any combination or mixture thereof. The water-soluble spacer may be a sugar or an ammonium salt, such as fructose, sucrose, glucose, lactose, or mannitol. The surfactant may be, for example, a polysorbate. The plasticizer may be, for example, polyethylene glycol. The disintegrant may be, for example, cross-linked sodium carboxymethylcellulose, crospovidone, or a mixture thereof.

[0364] Capsules can be prepared, for example, by placing the bulk blend pregnenolone neurosteroid (e.g., ganaxolone) formulation described above inside the capsule. In some embodiments, the ganaxolone formulation (non-aqueous suspension and solution) is placed inside a soft gelatin capsule. In other embodiments, the ganaxolone formulation is placed inside a standard gelatin or non-gelatin capsule, such as a capsule containing HPMC. In other embodiments, the ganaxolone formulation is placed inside a sprinkle capsule, which may be swallowed whole or opened and the contents sprinkled on food before a meal. In some embodiments of the present invention, the therapeutic dose is divided into multiple (e.g., two, three, or four) capsules. In some embodiments, the entire dose of the ganaxolone formulation is delivered in capsule form.

[0365] In certain embodiments, each capsule contains 200 mg or 225 mg of ganaxolone, as well as hydroxypropyl methylcellulose, sucrose, polyethylene glycol 3350, polyethylene glycol 400, sodium lauryl sulfate, sodium benzoate, citric acid anhydrous, methylparaben sodium, microcrystalline cellulose, 30% simethicone emulsion, gelatin capsule, polysorbate 80, and sodium chloride. In some embodiments, the capsule size is 00.

[0366] Alternatively, the oral dosage forms of the present invention may be in the form of controlled release dosage forms, as described in US Pat. No. 7,858,609.

[0367] Pregnenolone neurosteroid (e.g., ganaxolone) formulations suitable for use in the present invention may be administered parenterally. In such embodiments, formulations are suitable for intramuscular, subcutaneous, or intravenous injection and may include physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol, and cremophor), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters, such as ethyl oleate. Additionally, ganaxolone can be dissolved at concentrations >1 mg / ml using water-soluble beta-cyclodextrins (e.g., beta-sulfobutyl-cyclodextrin and 2-hydroxypropyl beta-cyclodextrin). A particularly suitable cyclodextrin is the substituted beta-cyclodextrin Captisol®. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Ganaxolone formulations suitable for subcutaneous injection may contain additives such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, benzoic acid, benzyl alcohol, chlorobutanol, phenol, and sorbic acid. The inclusion of isotonic agents, such as sugars and sodium chloride, may also be desirable. Drug absorption from injectable pharmaceutical forms can be prolonged by using agents that delay absorption, such as aluminum monostearate and gelatin. Ganaxolone suspension formulations designed for sustained release via subcutaneous or intramuscular injection can avoid first-pass metabolism, and low dosages of ganaxolone would be required to maintain plasma levels of approximately 50 ng / ml. In such formulations, the size of the ganaxolone particles and the size spread of the ganaxolone particles can be used to control the release of the drug by controlling the rate of dissolution in fat or muscle.

[0368] Particularly useful injectable formulations are disclosed in Applicant's U.S. Patent Application Publication No. 2017 / 0258812 (U.S. Patent Application No. 15 / 294,135, filed October 14, 2016), which is incorporated herein by reference in its entirety. Other useful injectable formulations of pregnenolone neurosteroids known to those of skill in the art can also be used.

[0369] Combination treatment The present disclosure includes embodiments in which the neurosteroid is the only active agent, as well as embodiments in which the neurosteroid is administered in combination with one or more additional active agents. When used in combination with additional active agents, the neurosteroid and the additional active agents may be combined in the same formulation or may be administered separately. The neurosteroid may be administered at the same time as the additional active agent (coadministration), or may be administered before or after the additional active agent (sequential administration).

[0370] The present disclosure includes embodiments in which the additional active agent is an anticonvulsant. Anticonvulsants include GABA. A These include receptor modulators, sodium channel blockers, GAT-1 GABA transporter modulators, GABA transaminase modulators, voltage-gated calcium channel blockers, and peroxisome proliferator-activated alpha modulators.

[0371] The present disclosure includes embodiments in which the patient is administered an anesthetic or sedative in combination with the neurosteroid. The anesthetic or sedative may be administered at a concentration sufficient to cause the patient to lose consciousness, such as a concentration sufficient to medically induce a coma or a concentration effective to induce general anesthesia. The anesthetic or sedative may also be administered at a low dose that is effective for sedation but not sufficient to induce loss of consciousness.

[0372] Benzodiazepines are used as both anticonvulsants and anesthetics. Benzodiazepines useful as anesthetics include diazepam, flunitrazepam, lorazepam, and midazolam.

[0373] In certain embodiments, the neurosteroid is administered in combination with a benzodiazepine (e.g., clobazam, diazepam, clonazepam, midazolam), clorazepic acid, levetiracetam, felbamate, lamotrigine, a fatty acid derivative (e.g., valproic acid), a carboxamide derivative (e.g., rufinamide, carbamazepine, oxcarbazepine, etc.), an amino acid derivative (e.g., levocarnitine), a barbiturate (e.g., phenobarbital), or a combination of two or more of the foregoing agents.

[0374] The disclosed neurosteroid nanoparticle injectable formulations may be administered in conjunction with another anticonvulsant, which includes several drug classes and overlaps to some extent with coma-inducing drugs, anesthetics, and sedatives that may be used in combination with neurosteroids.Anticonvulsants that may be used in combination with the neurosteroid nanoparticle injectable formulations of the present disclosure include aldehydes, such as paraldehyde; aromatic allylic alcohols, such as stiripentol; barbiturates (including those mentioned above, as well as methylphenobarbital and barbexaclone); benzodiazepines (alprazolam, bretazenil, bromazepam, brotizolam, chloridazepoxide, cinolazepam, clonazepam, colazepate, clopazam, clotiazepam, cloxazolam, delorazepam, diazepam, estazolam, etizolam, ethyl loflazepate, flunitrazepam, flurazepam, flutoprazepam, halazepam, ketazolam, loprazolam, lorazepam, lormetazepam, medazepam, midazolam, nimetazepam, nitrazepam, nordazepam, oxazepam, phenenazepam, pinazepam, prazepam, premazepam, pyrazolam, quazepam, temazepam, tatrazepam, and triazolam); bromides such as potassium bromide; carboxamides such as carbamazepine, oxcarbazepine, and eslicarbazepine acetate; fatty acids, such as valproic acid, sodium valproate, and divalproex sodium; fructose derivatives, such as topiramate; GABA analogues, such as gabapentin and pregabalin, hydantoins, such as ethotoin, phenytoin, mephenytoin, and fosphenytoin; other neurosteroids, such as allopregnanolone, oxazolidinediones, such as paramethadione, trimethadione, and ethadione, propionates, such as beclamide; pyrimidinediones, such as For example, primidone, pyrrolidines such as brivaracetam, levetiracetam, and seletracetam, succinimides such as ethosuximide, pensuximide, and mesuximide; sulfonamides such as acetazolamide, sulthiame, methazolamide, and zonisamide; triazines such as lamotrigine, ureas such as feneturide and phenacemide; NMDA antagonists such as felbamate, and valproylamides such as valpromide and valnoctamide; and perampanel.

[0375] Biomarkers Predictive biomarkers are used to identify patient populations that are more homogeneous and more likely to respond to treatment.

[0376] Allopregnanolone, a metabolite of progesterone, is a GABAA receptor positive allosteric modulator (PAM) with known anticonvulsant properties. Deficiency of this endogenous GABAA modulator may result in hyperexcitable neuronal networks in the brain, leading to an increased risk of seizures.

[0377] It is believed that most / all people with PCDH19 mutations exhibit this allopregnanolone deficiency, supporting the hypothesis that treatment with ganaxolone may reduce the frequency of attacks and ameliorate the additional symptoms of PCDH19.

[0378] Individuals affected by PCDH19-associated epilepsy were found to exhibit endogenous allopregnanolone deficiency when compared with age-matched controls (Tan C et al. 2015). The mechanism for this observation was attributed to the downregulation of the AKR1C2 and AKR1C3 genes, which encode key enzymes responsible for steroid metabolism leading to allopregnanolone.

[0379] Unexpectedly, it has now been discovered that a method for identifying high responders to neurosteroid treatment is by measuring endogenous neurosteroid (e.g., allopregnanolone sulfate; Allo-S) levels in patients. It is hypothesized that Allo-S correlates with allopregnanolone and may be the dominant analyte between the two in plasma. Low levels of endogenous neurosteroids can be used to identify patient populations with potentially much higher response rates to ganaxolone treatment than patient populations with high levels of endogenous neurosteroids.

[0380] A post hoc review of baseline endogenous neurosteroid levels in 11 PCDH19 subjects described in Example 11 yielded additional important observations. Allopregnanolone sulfate (Allo-S) levels and 28-day seizure rates were assessed in these subjects. Ganaxolone responders were identified by a post hoc definition as those with at least a 25% reduction in 28-day seizure rate. In these 11 PCDH19 subjects, plasma Allo-S concentrations in responders (n = 6) and non-responders (n = 5) were 501 ± 430 pg mL-1 and 9,829 ± 6,638 pg mL-1 (mean ± SD), respectively. There appeared to be a bimodal distribution of Allo-S plasma levels, with one subset of subjects having dramatically elevated levels compared to the other (Figure 10). When comparing seizure frequency from baseline to 6 months, the biomarker-positive group significantly improved (p=0.02, Wilcoxon), while the biomarker-negative (high Allo-S) group did not improve but did not significantly worsen (p=0.25, Wilcoxon).

[0381] Furthermore, retrospective separation of the PCDH19 cohort according to Allo-S levels revealed that Allo-S levels were ≥ 2,500 pg mL -1 In seven subjects with Allo-S levels below 2,500 pg mL (G. Pinna Lab Method), there was a 53.9% reduction in seizure rate. -1 In four subjects with over 100, a 247% increase was found.

[0382] Thus, in certain embodiments of the invention, allopregnanolone sulfate (Allo-S) is used as a predictive biomarker for response to ganaxolone, an analog of allopregnanolone. In these embodiments, 2,500 pg mL -1 Allo-S plasma levels below 2,500 pg mL indicate that a subject is likely to respond to and benefit from ganaxolone therapy. -1Allo-S plasma levels above 2,500 pg mL indicate that the subject is unlikely to respond to ganaxolone therapy and that a different therapeutic agent should be used. -1 Administration of ganaxolone to the following subjects may restore normal neural networks and reduce the frequency of seizures in these subjects:

[0383] Detailed Description of the Preferred Embodiments The following examples of formulations according to the present invention should not be construed as limiting the invention in any way, but are merely samples of the various formulations described herein.

[0384] During the development of ganaxolone formulations, various formulations were evaluated to establish a formulation that exhibited appropriate pharmacokinetic ("PK") parameters and was suitable for development and commercialization. Other formulations of ganaxolone that have been used include ganaxolone mixed with sodium lauryl sulfate, hydroxypropyl-beta-cyclodextrin (HP-β-CD) in solution, and beta-cyclodextrin (β-CD) administered as various suspensions, as well as ganaxolone 0.5 micron particles in suspension, tablet, and controlled-release capsule formulations, and an IV solution using sulfobutyl ether cyclodextrin (Captisol®) for ganaxolone solubilization. Development efforts have resulted in an oral suspension containing 0.3 micron immediate-release ganaxolone particles, as described in Example 1, and an oral capsule formulation containing 0.3 micron immediate-release ganaxolone particles, as described in Example 2. The pharmacokinetics of these formulations of ganaxolone in humans have been investigated in several single-dose and multiple-dose studies in adults. The results of these studies are summarized in Examples 6 and 7.

[0385] Example 1 A 50 mg / ml ganaxolone suspension is prepared having the ingredients shown in Table 1 below: [Table 1]

[0386] Table 2 shows the function of the excipients used in the 50 mg / ml ganaxolone suspension. [Table 2]

[0387] The oral bioavailability of a 50 mg / ml ganaxolone suspension depends on the rate and extent of nanoparticle drug dissolution in a relevant physiological environment. The particle sizing method and specifications are intended to ensure that the ganaxolone drug product exhibits an absence of aggregation after dispersion in simulated gastrointestinal fluid.

[0388] FIG. 3B outlines the major steps in the suspension manufacturing process used for the 50 mg / ml ganaxolone suspension.

[0389] A dispersion nanomilling process is used to reduce the particle size of ganaxolone, resulting in stable ganaxolone nanoparticles. The nanomilling process involves the use of yttria-stabilized zirconia (YTZ) milling media under high-energy agitation in a nanomill. To ensure consistent slurry particle size prior to dispersion nanomilling, Marinus developed a high-energy rotor / stator premilling process using a VakuMix DHO-1. After nanomilling, the dispersion is diluted from 25% w / w ganaxolone to 20% w / w ganaxolone, filtered through a 20-micron filter, and stabilizers (methylparaben, sodium benzoate, and anhydrous citric acid) are added to promote controlled growth at room temperature for 5–10 days until the nanoparticles reach approximately 300 nm. Figure 9 shows a typical particle size growth profile. The stabilized 300 nm nanoparticles demonstrate good stability against particle growth in pediatric suspension and encapsulated drug product formats. The stabilization process is controlled by the precise addition and dissolution of the water-soluble stabilizer parabens. The hardening process is controlled by adjusting the holding time and temperature of the stabilized dispersion prior to suspension dilution (for the 50 mg / ml ganaxolone suspension) or fluidized bed bead coating (for the 225 mg ganaxolone capsules described in Example 2).

[0390] The three dispersion batches prepared in the dispersion nanomilling scale-up study were diluted and stabilized by the addition of sodium methylparaben, sodium benzoate, and anhydrous citric acid and allowed to cure for 7 days. After curing, the particle sizes were measured and are shown in Table 3. [Table 3] As shown, the D(50) particle size was stable within the specification of 250-450 nm.

[0391] Example 2 Ganaxolone capsules (225 mg) are prepared having the ingredients set forth in Tables 4 and 5 below. [Table 4]

[0392] Table 5 summarizes the functions of the excipients used in the 225 mg ganaxolone capsule formulation. [Table 5]

[0393] Figure 3C shows an overview of the major steps in the suspension manufacturing process used for the 225 mg ganaxolone capsules. The manufacturing process used to prepare the capsules utilizes the same drug product specifications and quantitative composition, as well as the same nanomilling dispersion dilution and dispersion stabilization processes. Thus, the product of Example 2 utilizes stabilized dispersion intermediates in common with the product of Example 1. Sodium methylparaben may be substituted for methylparaben.

[0394] Table 5A summarizes the results of the 36-month formal stability data for ganaxolone immediate release (IR) 225 mg capsules. [Table 5A]

[0395] Example 3 Example 3 describes a phase 2, multicenter, open-label, proof-of-concept trial of ganaxolone (GNX) in a cohort of children with genetic epilepsies (PCDH19, CDKL5 LGS, and CSWS) (ClinicalTrials.gov Identifier: NCT02358538). There were 11 girls aged 5 to 16 years with PCDH19 epilepsy who had confirmed genetic mutations. In the CDKL5 cohort, there were 6 girls and 1 boy with confirmed genetic mutations. In the Lennox-Gastaut syndrome cohort, there were 10 children. Two children with CSWS were enrolled in the study. The study was conducted with a 12-week baseline and 52 weeks of open-label treatment followed by up to 26 weeks of treatment. The primary efficacy outcome was the percentage change in seizure frequency per 28 days from baseline calculated using daily seizure diaries [time frame: 26 weeks]. Secondary outcome measures were: Clinician Global Impression of Change score assessed by questionnaire [time frame: 26 weeks]; Patient Global Impression of Change score assessed by questionnaire [time frame: 26 weeks]; assessment of the safety and tolerability (based on adverse event logs and other clinical safety assessments) of open-label ganaxolone as adjunctive treatment for uncontrolled seizures in children with rare genetic epilepsies [time frame: 26 weeks]; responder rate [time frame: 26 weeks]; and seizure-free days [time frame: 26 weeks].

[0396] As shown in Table 6, across multiple placebo-controlled studies of ganaxolone across multiple indications, including epilepsy, few side effects were reported, occurring at rates higher than those reported in placebo-treated subjects. The side effects were generally mild and always reversible. Compared to other available treatments, ganaxolone has been shown to be generally safe and well-tolerated, and a safe long-term option for children with well-controlled seizures. Four of the seven children enrolled in the study continue to receive ganaxolone. Adverse events in this study were similar to those reported for all previously completed placebo-controlled studies, as summarized in Table 6 below. [Table 6]

[0397] After screening and baseline assessments, consenting patients were enrolled in a 26-week study, during which investigators administered ganaxolone at variable doses up to 1,800 mg / day for patients weighing >30 kg or 63 mg / kg / day for patients weighing <30 kg. The primary efficacy measure was the percent change from baseline in the 28-day seizure frequency count. Safety and tolerability were within the secondary objectives of the study.

[0398] In this study, oral ganaxolone suspension or capsules were administered for 2 to 4 weeks, up to a total dose of 63 mg / kg / day (maximum 1800 mg / day). Approximately 6 to 8 titration steps were used depending on the patient's size. Children weighing over 30 kg may receive ganaxolone capsules. Ganaxolone oral suspension was administered via an oral dosing syringe three times daily. Ganaxolone capsules were administered twice daily. Patients experience better absorption of ganaxolone with food (milk).

[0399] Table 7 shows a suggested titration schedule for ganaxolone oral suspension by weight. [Table 7]

[0400] Table 8 shows a suggested titration schedule for ganaxolone oral capsules by weight. [Table 8]

[0401] Similar to patients with CDKL5 deficiency, signals of antiepileptic treatment efficacy of ganaxolone emerged in the PCDH19 cohort of this phase 2, open-label study of ganaxolone in children with rare genetic epilepsy with uncontrolled seizures despite multiple prior concurrent AED regimens. Preliminary data from 11 PCDH19 patients showed that 9 of 11 patients demonstrated some degree of seizure relief, with 4 achieving greater than 50% seizure relief that lasted for more than 6 months. Two patients completed 78 weeks of ganaxolone treatment and are now receiving ganaxolone under an investigator-sponsored IND. Although not shown, clinician- and parent / caregiver-assessed CGI-I scores demonstrated improvement consistent with seizure control.

[0402] Preliminary data are shown in Table 9. [Table 9]

[0403] Narratives from investigators describing the clinical status of their patients indicate that some children treated with ganaxolone appear to have meaningful improvement in non-seizure-related problems.

[0404] According to doctors who treated the five subjects with CDKL5 deficiency, all of the subjects benefited from treatment in some way, such as a decrease in seizure frequency, a decrease in seizure severity, and / or increased alertness with a calmer demeanor.

[0405] Based on its known mechanism of action, preclinical and clinical data, and narrative reports from investigators, ganaxolone has the potential to address seizure and non-seizure-related problems, including anxiety, poor social interaction, movement disorders, and poor sleep, all of which are common and severely disabling in children with CDKL5 deficiency.

[0406] Adverse events potentially associated with ganaxolone treatment are shown in Table 10 below. [Table 10]

[0407] Of the four CDKL5 patients who completed the study, three of four showed a >50% reduction in seizure frequency (52%, 59%, and 88%, respectively). Two of four showed a significant improvement in seizure-free days (78% and 368%). The investigator's (CGI-I) and parent's (CGI-P) Connor's Global Index showed improvement consistent with seizure control. One patient discontinued due to lack of seizure control, leading the investigator to question the caregiver's reliability. The safety and tolerability profile observed in these patients was consistent with previous studies.

[0408] Preliminary data from the first six CDKL5 patients showed improved seizure control, lasting up to six months, in three of the six patients. The seventh patient, recently added to the study, experienced a substantial reduction in seizures after the first 28 days of treatment. Four of the seven patients also experienced an increase in the number of seizure-free days. Although not shown, clinician- and parent / caregiver-assessed Clinical Global Impression-Improvement (CGI-I) scores showed improvements consistent with seizure control. All subjects benefited from treatment in some way, including reduced seizure frequency, decreased seizure severity, and / or increased attention accompanied by a calmer demeanor. Reports of reduced seizure severity and duration and increased attention, as well as increased social interaction, have been reported in children with PCDH19 and Lennox-Gastaut syndrome, further confirming the need to address these important endpoints in upcoming clinical studies of ganaxolone in CDKL5 deficiency. One child with a PCDH19 mutation was severely autistic and non-verbal before ganaxolone treatment. After initiating ganaxolone treatment, social interaction and speech improved significantly (documented by video for reference; such video can be an important way to document changes in the children's functional abilities during ganaxolone treatment).

[0409] Table 11 shows steroid and neurosteroid levels for the top three high-responders versus the bottom three non-responders. High-responders had a >70% reduction in seizures. Non-responders had a >100% increase in seizures. One high-responder and one non-responder only had baseline values, so baseline values were used for both baseline and 26-week time points. [Table 11]

[0410] These results indicate that, unlike other patients, patients who continue to show a very high response rate, with up to 100% seizure reduction, have significantly lower background plasma neurosteroids, with the exception of pregnanolone and pregnanolone sulfate, which may actually compete with allopregnanolone for GABAA binding sites. This particular pattern of high plasma neurosteroid levels persists up to 26 weeks of ganaxolone treatment. This means that patients with very high background levels of neurosteroids, particularly allopregnanolone, and especially allopregnanolone sulfate, can be predicted to respond poorly to allopregnanolone, ganaxolone, or other pregnanolone-based treatments. This finding allows the use of pregnanolone-based treatments, such as ganaxolone, to be selectively targeted to patients with low background levels of neurosteroids, particularly allopregnanolone and allopregnanolone sulfate, as these patients may be most likely to respond to these treatments with the highest degree of seizure reduction and overall epilepsy control.

[0411] In subjects with focal-onset seizure disorder, post-hoc analysis showed a statistically significant reduction in seizure frequency in subjects receiving ganaxolone who were taking three or more concomitant antiepileptic drugs (AEDs) compared with subjects receiving placebo (ClinicalTrials.gov Identifier: NCT02358538). Ganaxolone was associated with a 20% greater reduction in median seizure frequency than placebo (p=0.02) (Lappalainen J, Tsai J, Amerine W, Patroneva. A Multicenter, Double-Blind, Randomized, Placebo-Controlled Phase 3 Trial to Determine the Efficacy and Safety of Ganaxolone as Adjunctive Therapy for Adults with Drug-Resistant Focal-Onset Seizures. Neurology 2017:88,16 Supplement P5.237). Although numerically superior, there was no statistically significant benefit of ganaxolone compared with placebo for subjects taking fewer than three AEDs. These data demonstrate the efficacy of ganaxolone for treating the most refractory epilepsy patients requiring the most intensive drug regimens. Patients with CDKL5 deficiency are almost universally refractory to all available AEDs, despite treatment with multiple concomitant medications.

[0412] Based on the results obtained to date, ganaxolone has demonstrated a long-term safety and tolerability profile in children with severe, currently untreatable disorders. As shown in Table 12, the median percent seizure reduction of 43% and 34% for children with CDKL5 and PCDH19 disorders, respectively, indicates the potential for ganaxolone to be a significant improvement over existing therapies for severe, refractory pediatric genetic epileptic encephalopathies, particularly for children with CDKL5 deficiency. [Table 12]

[0413] These preliminary data compare favorably with the results cited in Muller A et al. (Muller A, Helbig I, Jansen C, Bast T, Guerrini R, Jahn J, Muhle H, Auvin S, Korenke GC, Philip S, Keimer R, Striano P, Wolf NI, Pust B, Thiels Ch, Fogarasi A, Waltz S, Kurlemann G, Kovacevic-Preradovic T, Ceulemans B, Schmitt B, Philippi H, Tarquinio D, Buerki S, von Stulpnagel C, Kluger G. Retrospective evaluation of low long-term efficacy of antiepileptic drugs and ketogenic diet in 39 patients with CDKL5-related epilepsy. Eur J Paediatr Neurol. 2016;Jan;20(1):147-51.l), the overall responder rate was 43% (3 / 7 subjects), with one additional subject nearly achieving responder status at 3 months and 33% (2 / 6 subjects) at 6 months. This compares to an overall response rate of less than 10% for most AEDs and steroids at 6 months. Five of the seven subjects improved their seizure-free days, which in some cases was significant.

[0414] conclusion Based on its known mechanism of action, preclinical and clinical data, and narrative reports from investigators, ganaxolone has the potential to address seizure and non-seizure-related problems, including anxiety, poor social interaction, movement disorders, and poor sleep, all of which are common and severely disabling in children with CDKL5 deficiency, PCDH19-associated epilepsy, and other genetic epilepsies.

[0415] Previous studies in PCDH19 patients have shown the following: (i) the median change from baseline in 28-day seizure frequency (primary endpoint) in the intention-to-treat (ITT) population was a 26% reduction (n=11, 4 patients had LOCF); (ii) the median change from baseline in seizure-free days (key secondary endpoint) in the ITT population was a 14% increase (n=11); (iii) investigator- (CGI-I) and caregiver- (CGI-P) assessed Clinical Global Impression scales were consistent with seizure control; (iv) 2 subjects completed the 52 extension and continue to receive ganaxolone through the investigator-initiated IND.

[0416] An additional cohort of subjects in this study had PCDH19 epilepsy. This cohort completed the study. PCDH19 pediatric epilepsy is a severe and rare epilepsy syndrome that primarily affects females. The condition is caused by inherited mutations in the protocadherin 19 (PCDH19) gene, located on the X chromosome, and is characterized by early-onset and highly variable cluster seizures, cognitive and sensory impairments, and behavioral disorders. The PCDH19 gene encodes the protein protocadherin 19, which is part of a family of molecules that support communication between cells in the CNS. As a result of the mutation, protocadherin 19 may be malformed, its function reduced, or it may not be produced at all. Abnormal expression of protocadherin 19 is associated with highly variable and intractable seizures, cognitive impairment, and behavioral or social impairments with autistic characteristics. Currently, there are no approved treatments for PCDH19 pediatric epilepsy.

[0417] A total of 11 female subjects aged 4 to 15 years with confirmed PCDH19 gene mutations and uncontrolled seizures despite antiepileptic medication were enrolled. Ganaxolone was studied as adjunctive treatment, administered as a PO suspension or capsules according to the titration schedule in Tables 7 and 8 for 26 weeks after establishing baseline seizure frequency for up to 12 weeks. Primary and secondary endpoints were the same as in the CDKL5 deficiency cohort.

[0418] Example 4 The study in Example 3 was designed to investigate whether ganaxolone provides anticonvulsant effects in children with uncontrolled seizures in PCDH19 epilepsy, CDD, LGS, and CSWS epilepsy in an open-label, proof-of-concept study (due to competing clinical trials, subjects with Dravet syndrome were not enrolled). This example provides additional details, results, and conclusions regarding the study in Example 3.

[0419] After establishing baseline seizure frequency, licensed subjects were enrolled in the study and treated with open-label ganaxolone oral suspension or ganaxolone capsules at doses up to 1800 mg / day for up to 6 months. Maximum study participation was 94 weeks (up to 12 weeks of screening to establish baseline seizure frequency, up to 26 weeks of treatment, a 52-week extension for subjects who benefited from ganaxolone treatment, and up to a 4-week down-titration period). Inclusion criteria included a PCDH19 or CDD gene mutation confirmed by genetic testing in a certified genetic laboratory and considered pathogenic or likely associated with an epilepsy syndrome (subjects with Dravet syndrome would have an SCN1A mutation confirmed by genetic testing in a certified genetic laboratory and considered pathogenic or likely associated with an epilepsy syndrome). Subjects enrolled in the CSWS cohort had to have a clinical diagnosis of CSWS as determined by a pediatric neurologist, and current or past EEG during sleep consistent with this diagnosis (e.g., continuous [85%-100%] predominantly bisynchronous 1.5-2 Hz [and 3-4 Hz] spikes during non-REM sleep). Refractive cases of LGS or CSWS that previously responded to steroids or ACTH could also be enrolled. Additionally, subject seizure criteria were: the subject had a) uncontrolled cluster seizures (≥3 seizures over 12 hours) or intermittent seizures of status epilepticus every 6 weeks or less during baseline, or b) uncontrolled non-cluster seizures (focal cognitive impairment, focal convulsions, atypical absence, unilateral seizures, convulsions, or tonic seizures) with a seizure frequency of ≥4 per 28 days during baseline, or c) ≥4 generalized convulsions (tonic-clonic, tonic, clonic, or atonic) per 28-day baseline period during baseline, or d) subclinical CSWS syndrome with or without clinical events on EEG.

[0420] Ganaxolone was provided as either an oral suspension or capsules and was taken with food. Grapefruit and grapefruit juice were prohibited during the study.

[0421] Ganaxolone oral suspension was administered three times daily (TID) by a parent or legal guardian via an oral medication syringe after morning, midday, and evening meals or snacks. Doses were spaced a minimum of 4 hours and a maximum of 8 hours apart. Missed doses of ganaxolone could be taken up to 4 hours before the next scheduled dose; otherwise, the missed dose was not given.

[0422] Ganaxolone capsules were administered twice daily (BID) with a glass of water or other liquid after the morning and evening meals or snacks. Ganaxolone was given as an oral suspension or capsules based on the subject's weight at the start of the study. Ganaxolone oral suspension was administered TID by a parent or guardian via an oral medication syringe after the morning, midday, and evening meals or snacks. Doses were administered a minimum of 4 hours and a maximum of 8 hours apart. Ganaxolone capsules were administered BID after the morning and evening meals or snacks. Doses were administered a minimum of 8 hours and a maximum of 12 hours apart. Missed doses of medication could be taken up to 8 hours before the next dose; otherwise, they were not administered. Capsules were to be swallowed whole; they were not opened, crushed, or chewed.

[0423] Ganaxolone suspension contains 50 mg ganaxolone / mL, hydroxypropyl methylcellulose, polyvinyl alcohol, sodium lauryl sulfate, simethicone, methylparaben, propylparaben, sodium benzoate, citric acid, and sodium citrate at a pH of 3.8-4.2, and is sweetened with sucralose and flavored with artificial cherry. The suspension has a milky white appearance and is packaged in a high-density polyethylene (HDPE) bottle with a child-resistant closure. Ganaxolone is supplied at a concentration of 50 mg / mL (ganaxolone equivalent) in a 120 mL bottle containing 110 mL of ganaxolone.

[0424] Ganaxolone capsules were placed in size 00 white / opaque gelatin capsules packaged in HDPE bottles with foil induction seals and child-resistant closures. Each capsule contained 200 mg or 225 mg of ganaxolone, as well as hydroxypropyl methylcellulose, sucrose, polyethylene glycol 3350, polyethylene glycol 400, sodium lauryl sulfate, sodium benzoate, anhydrous citric acid, sodium methylparaben, microcrystalline cellulose, 30% simethicone emulsion, gelatin capsule, polysorbate 80, and sodium chloride.

[0425] For subjects weighing over 30kg

[0426] Ganaxolone treatment was initiated at a dose of 900 mg / day in two or three doses. The dose was increased by approximately 20% to 50% at intervals of at least 3 days but not more than 2 weeks, provided the current dose was reasonably tolerated until the desired efficacy was achieved or the maximum tolerated dose (MTD) level (maximum 1800 mg / day) was reached. Subsequent dose adjustments were made in approximately 20% to 50% increments, with a minimum of 3 days between dose changes, unless required for safety. Each dose escalation above 1500 mg / day required a scheduled clinic visit 4 to 6 days after the dose increase to assess safety and tolerability. The maximum tolerated dose was 1800 mg / day.

[0427] For those weighing less than 30kg

[0428] For subjects weighing 30 kg (66 lbs) or less, dosing was initiated at 18 mg / kg / day in two or three divided doses. The dose was then increased by approximately 20% to 50% increments at intervals of at least 3 days but not more than 2 weeks, provided the current dose was reasonably tolerated until the desired efficacy was achieved or the MTD level was reached. Subsequent dose adjustments were made by approximately 20% to 50% increments, with a minimum of 3 days between dose changes, unless required for safety. Each dose escalation above 54 mg / kg / day required a scheduled clinic visit 4 to 6 days after the dose increase to assess safety and tolerability. The maximum tolerated dose was 63 mg / kg / day (maximum 1800 mg / day).

[0429] Efficacy evaluation The primary outcome measure was the percentage change in seizure frequency (both individual seizures and clusters) per 28 days relative to baseline.

[0430] Secondary efficacy outcome measures included the percent change from baseline in seizure frequency (individual seizures only) per 28 days; the percent change from baseline in cluster frequency per 28 days; the percent change from baseline in number of seizures per cluster; the percent change from baseline in seizure frequency (individual and cluster seizures) per 28 days per seizure subtype; the longest period (%) without a seizure or cluster; the change from baseline in the number of days without a cluster per 28 days; the change from baseline in the number of days without an individual seizure per 28 days; the change from baseline in the number of days without an individual seizure per 28 days; the proportion of subjects achieving a 28-day seizure frequency (individual and cluster seizures) reduction of ≥ 25%, 50%, or 75% compared to baseline; and Clinical Global Impression of Improvement: Clinician (CGII-C) and Clinical Global Impression of Improvement: Patient / Caregiver (CGII-P) ratings.

[0431] Post-baseline 28-day total seizure frequency was calculated by dividing the total number of individual seizures and clusters during the 26-week open-label treatment period by the number of days during the period with available seizure / cluster data and multiplying by 28. Baseline 28-day total seizure frequency was calculated by dividing the total number of individual seizures and clusters during the baseline period by the number of days during the period with available seizure / cluster data and multiplying by 28. Calculations for the percent change from baseline in 28-day total seizure frequency were performed for each subject as follows:

number

[0432] Baseline and post-baseline values, as well as arithmetic and percent change from baseline in 28-day total seizure frequency, were summarized by cohort, using descriptive statistics separately for the MITT and PP populations if they differed.

[0433] Secondary efficacy analyses included the following: percent change from baseline in individual seizure frequency per 28 days; percent change from baseline in cluster frequency per 28 days; percent change from baseline in mean number of seizures per cluster; percent change from baseline in total seizure frequency (individual and cluster seizures) per 28 days per seizure subtype; change from baseline in percentage of individual seizure- and cluster-free days; change from baseline in percentage of individual seizure-free days; change from baseline in percentage of cluster-free days; change (%) in longest individual seizure- and cluster-free period; proportion of subjects achieving a 25%, 50%, or 75% reduction in 28-day total seizure frequency (sum of individual seizures and clusters) compared to baseline; and frequency and percentage of response to the CGII-C (Clinician's Clinical Global Impression of Improvement) and CGII-P (Patient / Caregiver's Clinical Global Impression of Improvement). All secondary efficacy variables were summarized using descriptive statistics.

[0434] A total of 30 subjects were enrolled in the study: half (15 subjects) completed the 26-week open-label treatment period, and half (15 subjects) discontinued the study. Overall, the primary reasons for study discontinuation in the safety population were lack of efficacy (8 subjects [26.7%]) and AEs or SAEs (4 subjects [13.3%]). All 30 subjects (100.0%) were in the safety and MITT populations. Table 13 shows the subject disposition over the 26-week open-label period. [Table 13]

[0435] Demographic and other baseline characteristics of the MITT and PP cohorts were similar to those of the safety cohort. [Table 14]

[0436] Primary efficacy analysis Table 13 shows the percent change in 28-day total seizure frequency for individual seizures and cluster sums over the 26-week open-label treatment period relative to baseline for the MITT population. Through the first 3 months (Day 91), the mean percent change from baseline was 31.23% (SD=41.44%), 122.10% (SD=321.12%), and 52.83% (SD=234.08%) for the CDD, LGS, and PCDH19 cohorts, respectively. The median percent change at Day 91 was 47.34%, 10.22%, and 25.98% for the CDD, LGS, and PCDH19 cohorts, respectively.

[0437] At week 26, the mean percent change from baseline was 20.55% (SD=60.59%), 125.38% (SD=319.05%), and 46.36% (SD=235.66%) for the CDD, LGS, and PCDH19 cohorts, respectively. The median percent change from baseline to week 26 was 37.70%, 9.19%, and 24.59% for the CDD, LGS, and PCDH19 cohorts, respectively.

[0438] In the PP population, the mean percent change in 28-day total seizure frequency from baseline to week 26 was 20.55%, 18.43%, and 60.99% for the CDD, LGS, and PCDH19 cohorts, respectively. The median percent change from baseline to week 26 was 37.70%, 11.15%, and 22.11% for the CDD, LGS, and PCDH19 cohorts, respectively. Table 15: Summary of 28-day seizure frequency for individual seizures and clusters combined (MITT population) [Table 15]

[0439] Figure 4 shows cumulative responder curves for 28-day seizure frequency for individual seizures and clusters combined.

[0440] Table 16 summarizes the percent change in 28-day individual seizure frequency relative to baseline for the MITT population. The CDD and PCDH19 cohorts had fewer individual seizures at day 91 compared to baseline (mean percent change from baseline -30.33% [SD=39.83%] and -16.92% [SD=89.11%], respectively), while the LGS cohort had an increase in individual seizure frequency at day 91 compared to baseline (mean percent change from baseline 226.72% [SD=496.75%]). At week 26, the trend remained the same, with mean percent change from baseline of -21.06 (SD=59.25%) for the CDD cohort, 229.90% (SD=494.16%) for the LGS cohort, and -23.95% (SD=88.22%) for the PCDH19 cohort. [Table 16]

[0441] Regarding Clinical Global Impression of Improvement, at the end of week 26, in the CDD cohort, 3 subjects (42.9%) were greatly improved and 0 subjects were very improved; in the LGS cohort, 1 subject (14.3%) was greatly improved and 1 subject (14.3%) was very improved; in the PCDH19 cohort, 2 subjects (22.2%) were greatly improved and 2 subjects (22.2%) were very improved.

[0442] Ganaxolone was generally safe and well tolerated in subjects with epilepsy disorders. Overall, based on an evaluation of treatment-emergent adverse events ("TEAEs") in the safety population, treatment with ganaxolone was well tolerated across cohorts. In the CDD cohort, 6 subjects (85.7%) experienced a total of 45 TEAEs; in the CSWS cohort, 1 subject (50.0%) experienced 7 TEAEs; in the LGS cohort, 7 subjects (70.0%) experienced 24 TEAEs; and in the PCDH19 cohort, 11 subjects (100.0%) experienced 95 TEAEs.

[0443] A total of 83.3% of subjects experienced TEAEs overall: 23.3% mild, 46.7% moderate, and 13.3% severe. Six subjects (20.0%) experienced SAEs, 16 subjects (53.3%) had treatment-related TEAEs, and 4 subjects (13.3%) had TEAEs that led to discontinuation of study drug. No deaths were reported in this study.

[0444] Preliminary findings regarding the correlation between baseline endogenous allopregnanolone levels and seizure frequency change (efficacy) are shown in Figure 10, which is a plot of plasma allopregnanolone in each subject compared to the percentage change in seizure frequency with administration of ganaxolone according to the present example. In Figure 10, each closed circle represents a unique subject in the clinical trial. In Figure 10, a percentage change in seizure frequency of -100% means complete freedom from seizure activity, i.e., the subject did not experience any seizures during the 26-week study period, which would represent the best possible outcome. For the ganaxolone dosing regimen of this example, anywhere from 0 to -100% indicates efficacy. As can be seen from the results shown in Figure 10, subjects with plasma allopregnanolone levels below 200 pg / ml (below 100 pg / ml, below about 75 pg / ml, and in certain patients below about 50 pg / ml) responded best to the ganaxolone dosing regimen of this example.

[0445] Example 5 Single / Dose Fasting Feeding Study The 0.3 micron ganaxolone suspension of Example 1 was administered to healthy volunteers at 200 mg in a fasted study and at 400 mg in a fasted and high-fat study. A two- and three-fold fed / fasted effect was observed in AUC (0~∞) and C max The fasting effects of the 200 and 400 mg doses were dose-proportional.

[0446] Table 17 summarizes the ganaxolone pharmacokinetic parameters following a single dose of the 0.3 micron ganaxolone suspension in healthy volunteers in the fed and fasted states. [Table 17]

[0447] The 0.3 micron ganaxolone capsules of Example 2 were tested in healthy volunteers at single fed / fasted doses of 200, 400, and 600 mg, and multiple doses of 200, 400, and 600 mg BID (400 mg / day, 800 mg / day, and 1200 mg / day).

[0448] FIG. 5 shows the mean ganaxolone plasma concentration profile following a single oral dose of the ganaxolone 0.3 micron capsule of Example 2 in healthy volunteers after a high-fat meal.

[0449] FIG. 6 shows the mean ganaxolone plasma concentration-time profiles after single and multiple BID oral doses of the 0.3 micron ganaxolone capsules of Example 2 with a standard meal or snack in healthy volunteers.

[0450] After PO administration, the 0.3 micron ganaxolone capsules exhibited a rapid distribution phase followed by a longer elimination phase (Figure 5).

[0451] Single dosing in fasted and high-fat fed states resulted in fed / fasted geometric mean ratios (GMRs) of C for the 200, 400, and 600 mg doses, respectively. max 2.2, 3.2, and 4.9 for (0~∞) In the fed state, the AUC (0~∞) Value and C max The values were close to dose proportional. max Value and AUC (0~∞) The values were not significantly dose-proportional across the 200 mg, 400 mg, and 600 mg dose ranges, and C max is AUC(0~∞) In the fed state, the AUC values for 0.3 micron ganaxolone capsules at doses of 200, 400, and 600 mg were less than (0~t) The value is C max The GMR values were close to dose proportional (108% and 130% GMR, respectively) as were the fasted values (91% and 106%, respectively). A high CL of ganaxolone was observed in the fasted state. In the 0.3 micron capsule study, oral clearance CL / F values were not statistically different from the 200-600 mg doses, ranging from 586 to 433 L / h. Fasted and high-fat fed PK parameters for this study are shown in Figures 5 and 6, respectively.

[0452] Table 18 summarizes the ganaxolone pharmacokinetic parameters following a single oral dose of ganaxolone 0.3 micron capsules in healthy volunteers in the fasted state. [Table 18]

[0453] Table 19 provides a summary of ganaxolone pharmacokinetic parameters following a single oral dose of ganaxolone 0.3 micron capsules in healthy volunteers after a high-fat meal. [Table 19]

[0454] The 0.3 micron ganaxolone capsule formulation maximizes contact time in the stomach and small intestine, resulting in effective T-cell delivery when administered under BID multiple dosing conditions. 1 / 2 Upon acute dosing, the capsules produced greater PK variability compared to the 0.3 micron suspension, likely due to particle retention in the stomach and small intestine, resulting in more variable data 24 hours after dosing. The intra-subject variability of plasma concentrations from 24 to 72 hours after dosing was highest with the elimination T 1 / 2 T values were obtained from ganaxolone capsules at doses of 200 mg to 600 mg. 1 / 2The increase in T did not appear to be a saturation effect, as the AUC values from 200 mg to 600 mg were close to dose proportional, whereas the T 1 / 2 The T values were 3.46 hours and 18.7 hours, respectively. 1 / 2 The increase may be due to the fact that at the higher doses, the elimination phase for the formulation was more discernible in subjects due to higher drug loading in lipophilic tissues.

[0455] No GI site-specific absorption analysis of ganaxolone was performed. However, the time to maximum concentration (T max ) values were largely within the range of expected delivery to the colon (7-10 hours), suggesting that the majority of ganaxolone absorption likely occurs in the small intestine.

[0456] Table 20 summarizes the ganaxolone pharmacokinetic parameters (mean [SD]) following a single dose of the ganaxolone 0.3 micron formulation in healthy volunteers after a high-fat meal. [Table 20]

[0457] Example 6 Multiple-dose PK study Multiple-dose studies of oral ganaxolone formulations were conducted in healthy volunteers. 0.3 micron ganaxolone capsules were administered BID at increasing doses for 7 days with a standard meal or snack. Table 21 shows the PK data for these studies.

[0458] In a 7-day study with 0.3 micron ganaxolone capsules at doses of 200, 400, and 600 mg BID, steady state was achieved within 48 hours when administered with a standard meal or snack. At steady state, mean C max and AUC (0~12) was close to dose proportional. max and AUC (0~12)was similar between doses when comparing dosing with a high-fat or standard meal / snack, and the 600 mg dose had a mean AUC (0~12) However, with a standard meal / snack, the AUC was approximately 25% lower than with a high-fat meal. Trough levels after 7 days of BID dosing at 200, 400, and 600 mg were 14.3 ng / mL, 39.4, and 56.4 ng / mL. (0~12) The cumulative effect was approximately 43-81%, and effective T 1 / 2 The time-dependent plasma concentration curves are shown in Figure 7. The steady-state PK of the 0.3 micron ganaxolone capsules showed no significant circadian effect.

[0459] Healthy subjects received 600, 800, and 1000 mg of ganaxolone BID and achieved steady state within 3 days. The medium- and high-dose regimens were initiated 3 days after the low- and medium-dose regimens, respectively. In general, ganaxolone was rapidly absorbed after PO administration, with a mean C max was achieved within 2 hours after multiple dosing. max was independent of dose level. Mean C max were 224, 263, and 262 ng / mL for the three dose levels. This was not statistically proportional to dose, with the disproportionality being primarily caused by the lack of an increase in exposure from 800 mg to 1000 mg. min , C avg , and AUC τshowed similar trends, in subproportionality. Proportionality was preserved from 600 mg to 800 mg. Figure 8 shows the time-dependent plasma curves, and Figure 9 shows daily trough levels. The mean apparent systemic CL and mean variability at steady state ranged from 609 to 770 L / hr and 172% to 191%, respectively, across the ganaxolone BID dose range of 600 to 1000 mg. These data demonstrate that, under fed conditions, exposure to ganaxolone increases over the 600 to 1000 mg BID dose range, but not significantly with increasing dose; this imbalance was more pronounced at the upper end of the dose range. [Table 21] Values on days 6.5, 9.5, and 12.5 are from evening samples collected 12 hours after the last dose on the PK sampling day. Subjects received 600 mg ganaxolone BID on days 4-6; 800 mg ganaxolone BID on days 7-9; and 1000 mg ganaxolone BID on days 10-12.

[0460] Example 7 Mass Balance Mass balance was measured in healthy male volunteers at 300 mg. 14 C-GNX (with HP-β-CD) was evaluated after administration of a single PO dose. The achieved total plasma radioactivity concentrations were much higher than the GNX plasma levels in clinical studies with unlabeled GNX. This result suggests the presence of metabolites in plasma. The total radioactivity also appears to have a longer elimination half-life than intact GNX (230 hours vs. approximately 25 hours). Over 94% of the total radioactivity was excreted and collected in urine and feces over 30 days. This is consistent with the 30-day elimination half-life of all administered doses. 14 The results showed nearly complete recovery of C-GNX. Approximately 80% of the total radioactivity was excreted in feces and urine by day 14. The majority of the recovered radioactivity was in feces (68.95%), with the remainder in urine (25.34%). Table 22 shows the results of the 14-day excretion of C-GNX in healthy male volunteers. 14Summarize PK parameters (mean ± SD) of C-GNX-driven total radioactivity (study number CA042 9402.01 [n=5]). [Table 22]

[0461] Example 8 Food Effects All current and previous ganaxolone formulations have demonstrated higher levels and exposures in the fed versus fasted state.

[0462] The magnitude of the fed / fasted effect of the current formulation is C when compared to the previous ganaxolone βCD complex suspension. max about 3 times, and AUC (0~∞) The amount of oxidized cellulose in the cellulose was reduced by 7 to 8 times.

[0463] Fed / fasted studies with 0.3 micron ganaxolone capsules in healthy volunteers at doses of 200, 400, and 600 mg demonstrated an increasing food effect with increasing dose (Table 31).

[0464] Table 23 shows the geometric mean ratios (fed / fasted) of ganaxolone pharmacokinetic parameters following administration of 0.3 micron ganaxolone capsules to healthy volunteers. [Table 23]

[0465] The effect of various types of food on 0.3 ganaxolone capsules was indirectly measured. As shown in the table, C max and AUC (0~∞) In another study, using a 400 mg BID dosing regimen at steady state with a standard meal versus a liquid diet (8 oz. Ensure®), C max and AUC (0~12) The ratios were 1.2 and 1.3, respectively.

[0466] Table 24 summarizes the mean ratios (high fat / standard meal) of ganaxolone pharmacokinetic parameters following administration of 0.3 micron ganaxolone capsules to healthy volunteers. [Table 24]

[0467] These results show that ganaxolone absorption is enhanced in the presence of food with the 0.3 micron formulation, with a reduced high-fat to standard-fed or fasted ratio compared to previous formulations. The fed / fasted ratio also increases with increasing dose.

[0468] Example 9 Gender effect A repeated study using healthy volunteers showed no gender effect on PK parameters with ganaxolone dosing. Table 25 shows representative examples following dosing of ganaxolone β-CD suspension. [Table 25]

[0469] Example 10 Biomarkers Additionally, a critical retrospective review of baseline endogenous neurosteroid levels in the studies described in Examples 3 and 4 revealed preliminary evidence of a powerful predictive biomarker (allopregnanolone sulfate; Allo-S) and allopregnanolone (Allo), which can be used to identify patient populations with potentially much higher response rates to ganaxolone treatment than biomarker-negative patient populations. It is hypothesized that this sulfated version of allopregnanolone is more readily found in the circulation and may qualitatively represent allopregnanolone levels in the brain.

[0470] Methods: Individuals (n = 11) with confirmed PCDH19 mutations and minimal seizure burden were enrolled between May 2015 and November 2015 at six centers in the United States and Italy. Percent change in seizure frequency was assessed as the primary endpoint, revealing a 25% or greater reduction in seizure rate in responders. Plasma neurosteroid levels were quantified using a previously published GC / MS method (doi:10.1016 / S0028-3908(99)00149-5). In two cases, baseline neurosteroid levels were not measured. In these cases, 6-month values were used because neurosteroid levels were observed not to change significantly over time.

[0471] Results: The median change from baseline in 28-day seizure frequency (all seizure types) for all visitors (n = 11) was a 26% reduction. In this group, the mean plasma allopregnanolone sulfate (Allo-S) concentration was 4,741 pg mL -1 (median value = 433 pg mL -1 Responder analysis and correlation with Allo-S revealed two discrete populations. Responders (n = 6) (≥ 25% reduction in attack rate) and non-responders (n = 5) had plasma Allo-S concentrations of 501 ± 430 pg mL−1, respectively. -1 and 9,829 ± 6,638 pg mL -1 (mean ± SD, p = 0.05, Mann-Whitney) (Figure 10).

[0472] When comparing seizure frequency from baseline to 6 months, the biomarker-positive group significantly improved (p=0.02, Wilcoxon), while the biomarker-negative (high Allo-S) group did not improve but did not significantly worsen (p=0.25, Wilcoxon).

[0473] Biomarker positive (n=7, Allo-S<2,500pg mL -1 ) vs. biomarker-negative (n=4, Allo-S>2,500pg mL -1A retrospective analysis of biomarker+ and biomarker-negative subjects yielded a median % change in seizure rate of -53.9% and 247%, respectively (p=0.006, Mann-Whitney) (Figure 11). Furthermore, when comparing seizure frequency from baseline to 6 months, the biomarker-positive group significantly improved (p=0.02, Wilcoxon signed rank), and the biomarker-negative group did not significantly worsen (p=0.25, Wilcoxon signed rank). The % in Figure 11 shows the change in seizure frequency (primary efficacy endpoint) stratified by biomarker+ and biomarker- subjects.

[0474] Allopregnanolone can be used as a biomarker in subjects with CDKL5. Figure 12 shows the % change in seizure frequency in responders in the CDKL5 cohort. Each closed circle represents a unique subject in the trial. A "change of -100" means complete freedom from seizures; the patient has not experienced any seizures during the 26-week period. Anything from "0" to "-100%" indicates efficacy. Patient with increased seizures—their seizures worsened during the study. This patient had approximately 10x the level of allopregnanolone because other patients had positive responses (reduced seizures).

[0475] These results indicate that plasma neurosteroid (allopregnanolone sulfate (Allo-S) and / or allopregnanolone (Allo)) biomarkers can be used to predict seizure response when treated with ganaxolone, for example, in PCDH19, CDD, and other epileptic encephalopathies.

Claims

1. 1. A pregnenolone neurosteroid for use in a method for treating an epileptic disorder in a mammal, wherein the pregnenolone neurosteroid is administered orally or parenterally to the mammal after determining that the mammal has low levels of endogenous neurosteroids.

2. The use according to claim 1, wherein the endogenous neurosteroid is allopregnanolone or allopregnanolone sulfate.

3. The endogenous neurosteroid is allopregnanolone sulfate, and the low level of the endogenous neurosteroid is 2500 pg mL －１ The use according to claim 3, wherein the level is:

4. The endogenous neurosteroid is allopregnanolone, and the low level of the endogenous neurosteroid is 200 pg mL －１ The use according to claim 3, wherein the level is:

5. The use according to any one of claims 1 to 4, wherein the mammal is a human.

6. 6. The use of claim 5, wherein the epileptic disorder is selected from the group consisting of CDKL5 deficiency, PCDH19-associated epilepsy, Lennox-Gastaut syndrome, Ohtahara syndrome, early myoclonic epileptic encephalopathy, West syndrome, Dravet syndrome, Angelman syndrome, continuous sleep waves during sleep (CSWS), status epilepticus of sleep (ESES), Rett syndrome, Fragile X syndrome, X-linked myoclonic seizures, spasticity and intellectual disability syndrome, idiopathic infantile epilepsy-dyskinetic encephalopathy, epilepsy and mental retardation limited to females, and severe infantile multifocal epilepsy.

7. The pregnenolone neurosteroid has the compound of formula IA: 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein: X is O, S, or NR １０ and R １ is hydrogen, hydroxyl, -CH ２ A is optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl; A is hydroxyl, O, S, NR １１ , an optionally substituted nitrogen-containing 5-membered heteroaryl, or an optionally substituted nitrogen-containing bicyclic heteroaryl or bicyclic heterocyclyl; R ４ is hydrogen, hydroxyl, oxo, optionally substituted alkyl, or optionally substituted heteroalkyl; R ２ , R ３ , R ５ , R ６ , and R ７ are each independently absent, hydrogen, hydroxyl, halogen, or optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ alkoxyl (e.g., methoxyl), or optionally substituted heteroalkyl; R ８ and R ９ are each independently hydrogen, C １ ~C ６ Alkyl (e.g., methyl), halogenated C １ ~C ６ alkyl (e.g., trifluoromethyl), or C １ ~C ６ alkoxyl (e.g., methoxyl), or R ８ and R ９ forms an oxo group; R １０ is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl, and each alkyl is selected from the group consisting of C １ ~C １０ Alkyl, C ３ ~C ６ cycloalkyl, (C ３ ~C ６ Cycloalkyl)C １ ~C ４ alkyl, and optionally, a single bond is replaced by a double or triple bond; Each heteroalkyl group may be a heteroaryl group, where one or more methyl groups are independently selected from —O—, —S—, —N(R １０ )-, -S(=O)-, or -S(=O) ２ is an alkyl group substituted by -, and R １０ is hydrogen, alkyl, or alkyl in which one or more methylene groups are replaced by —O—, —S—, —NH, or —N-alkyl; R １１ is -H ２ or -HR １２ and R １２ is C １ ~C ６ Alkyl or C １ ~C ６ The use according to any one of claims 1 to 6, wherein the alkoxy is alkoxy.

8. 8. The use of claim 7, wherein the pregnenolone neurosteroid is selected from the group consisting of allopregnanolone, pregnanolone, 5-alpha DHP (5-alpha dihydroprogesterone), pregnanolone, dehydroepiandrosterone (DHEA), ganaxolone, 3α-hydroxy-3β-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5β-pregnan-20-one, pharmaceutically acceptable salts of any of the foregoing, and combinations of any of the foregoing.

9. 9. The use according to claim 8, wherein the pregnenolone neurosteroid is ganaxolone.

10. 10. The use according to claim 9, wherein ganaxolone is administered orally.

11. The determining the low level of the endogenous neurosteroid comprises: obtaining a biological sample from said mammal; performing an assay of said biological sample to determine the level of said endogenous neurosteroid; The use according to any one of claims 1 to 10, which is carried out by

12. 12. The use of claim 11, wherein the results of the assay are communicated to the mammal or a healthcare provider before or after the administration of the pregnenolone neurosteroid.

13. establishing a baseline seizure frequency in said mammal; initially administering to said mammal a dose of ganaxolone in an amount of about 0.5 mg / kg / day to about 15 mg / kg / day; gradually increasing the dose of ganaxolone to an amount of about 18 mg / kg / day to about 60 mg / kg / day over a period of four weeks; 13. The use of any one of claims 1 to 12, further comprising:

14. 1. A method of treating an epileptic disorder, comprising: Identifying mammals with epileptic disorders; determining whether the mammal has a low level of endogenous neurosteroids; if said mammal has a low level of endogenous neurosteroids, administering to said mammal a dosage regimen of a pharmaceutically acceptable pregnenolone neurosteroid in an amount effective to reduce the frequency of seizures in said mammal; A method comprising:

15. 15. The method of claim 14, wherein the endogenous neurosteroid is allopregnanolone or allopregnanolone sulfate.

16. The endogenous neurosteroid is allopregnanolone sulfate, and the low level of the endogenous neurosteroid is 2500 pg mL －１ The method of claim 15, wherein the level is:

17. The endogenous neurosteroid is allopregnanolone, and the low level of the endogenous neurosteroid is 200 pg mL －１ The method of claim 15, wherein the level is:

18. the mammal is a human; said epileptic disorder is selected from the group consisting of CDKL5 deficiency, PCDH19-associated epilepsy, Lennox-Gastaut syndrome, Ohtahara syndrome, early myoclonic epileptic encephalopathy, West syndrome, Dravet syndrome, Angelman syndrome, continuous sleep waves during sleep (CSWS), status epilepticus during sleep (ESES), Rett syndrome, fragile X syndrome, X-linked myoclonic seizures, spasticity and intellectual disability syndrome, idiopathic infantile epilepsy-dyskinetic encephalopathy, epilepsy and mental retardation limited to females, and severe infantile multifocal epilepsy; The dosing regimen is administered orally or parenterally; The pregnenolone neurosteroid has the compound of formula IA: 【Chemistry 2】 or a pharmaceutically acceptable salt thereof, wherein: X is O, S, or NR １０ and R １ is hydrogen, hydroxyl, -CH ２ A is optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl; A is hydroxyl, O, S, NR １１ , an optionally substituted nitrogen-containing 5-membered heteroaryl, or an optionally substituted nitrogen-containing bicyclic heteroaryl or bicyclic heterocyclyl; R ４ is hydrogen, hydroxyl, oxo, optionally substituted alkyl, or optionally substituted heteroalkyl; R ２ , R ３ , R ５ , R ６ , and R ７ are each independently absent, hydrogen, hydroxyl, halogen, or optionally substituted C １ ~C ６ Alkyl, optionally substituted C １ ~C ６ alkoxyl (e.g., methoxyl), or optionally substituted heteroalkyl; R ８ and R ９ are each independently hydrogen, C １ ~C ６ Alkyl (e.g., methyl), halogenated C １ ~C ６ alkyl (e.g., trifluoromethyl), or C １ ~C ６ alkoxyl (e.g., methoxyl), or R ８ and R ９ forms an oxo group, R １０ is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl, and each alkyl is selected from the group consisting of C １ ~C １０ Alkyl, C ３ ~C ６ cycloalkyl, (C ３ ~C ６ Cycloalkyl)C １ ~C ４ alkyl, and optionally, a single bond is replaced by a double or triple bond; Each heteroalkyl group may be a heteroaryl group, where one or more methyl groups are independently selected from —O—, —S—, —N(R １０ )-, -S(=O)-, or -S(=O) ２ is an alkyl group substituted by -, and R １０ is hydrogen, alkyl, or alkyl in which one or more methylene groups are replaced by —O—, —S—, —NH, or —N-alkyl; R １１ is -H ２ or -HR １２ and R １２ is C １ ~C ６ Alkyl or C １ ~C ６ is an alkoxy, The method according to any one of claims 14 to 16.

19. 19. The method of claim 18, wherein the pregnenolone neurosteroid is administered orally twice daily or three times daily.

20. 20. The method of claim 18 or 19, wherein the pregnenolone neurosteroid is ganaxolone.

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