Ganaxolone for use in the treatment of tuberous sclerosis complex

Abstract

The present disclosure relates to methods for treating tuberous sclerosis or tuberous sclerosis-associated epilepsy, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutically acceptable pregnenolone neurosteroid, e.g., ganaxolone, to alleviate one or more symptoms of tuberous sclerosis or tuberous sclerosis-associated epilepsy.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 944,549, filed December 6, 2019, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] 1. Background Tuberous sclerosis complex (TSC), also known as tuberous sclerosis complex (TSC), is a rare, multisystem genetic disorder characterized by the growth of numerous noncancerous (benign) tumors found in numerous parts of the body (Northrup et al., (2013), Pediatr Neurol., 49(4):243-254). These tumors occur in the skin, brain, kidneys, and other organs and often cause serious health problems (ibid.).

[0003] Epilepsy is the most common neurological symptom in individuals with TSC ("TSC-associated epilepsy") and is a significant cause of morbidity and mortality. Julich and Sahin (2014), Pediatric Neurol., 50:290-296. Infantile spasms are the most common type of seizure occurring in infancy and are the initial manifestation of epilepsy in 50% of individuals (ibid.). In older children and adults, focal impaired consciousness seizures (formerly called complex partial seizures) are the most common (ibid.). Other focal and generalized seizures can also occur (ibid.).

[0004] TSC-associated epilepsy affects up to 90% of patients, with approximately 70% of these cases being treatment-resistant (Portocarrero et al., (2018), An Bras Dermatol., 93(3):323-331). Furthermore, patients with intractable epilepsy have a higher prevalence of intellectual disabilities, including autism, mental retardation, and mood disorders, compared with control patients, and the disease manifests throughout the affected individual's life (ibid.). Due to the detrimental impact of epilepsy on cognitive development and quality of life, seizure prevention and management are important goals in the treatment of TSC (Vergeer et al., (2019), Epilepsia Open, 4:581-592). There is no cure for this disorder. The first-line treatment for TSC-associated infantile spasms is vigabatrin (Uliel-Sibony et al., (2020), Child's Nervous System, 36:2511-2517). However, vigabatrin is associated with serious side effects (ibid.). For example, in approximately 21-34% of patients, vigabatrin is associated with irreversible retinal damage. Other possible side effects include brain abnormalities in the thalamus, basal ganglia, brainstem tegmentum, and cerebellar dentate nucleus, hyperkinetic movement disorders, and acute encephalopathy (ibid.). Furthermore, no other antiepileptic medications significantly reduce the severity or incidence of TSC-associated epilepsy. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Northrup et al.,(2013),Pediatr Neurol.,49(4):243-254 [Non-patent document 2] Julich and Sahin(2014),Pediatric Neurol.,50:290-296 [Non-patent document 3] Portocarrero et al.,(2018),An Bras Dermatol.,93(3):323-331 [Non-patent document 4] Vergeer et al.,(2019), Epilepsia Open, 4:581-592 [Non-Patent Document 5] Uliel-Sibony et al., (2020), Child's Nervous System, 36:2511-2517 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a great need for effective therapies to treat TSC. [Means for solving the problem]

[0007] 2. Overview The present disclosure relates to methods for treating TSC and / or TSC-associated epilepsy. As noted above, TSC and / or TSC-associated epilepsy is a highly complex and challenging condition to treat. In 70% of cases, patients are resistant to treatment and the epilepsy is refractory. Refractory epilepsy is usually associated with significant behavioral and developmental consequences. As described and exemplified herein, the inventors believe that ganaxolone can provide an effective treatment for TSC and / or TSC-associated epilepsy.

[0008] Given the complexity and difficulty of treating TSC and TSC-associated epilepsy, the inventors believed that effective treatment requires high doses of drugs (e.g., ganaxolone). For example, in a clinical trial involving adults with focal seizures, ganaxolone was administered at 1800 mg / day (900 mg twice daily), but clinical endpoints were not achieved. The inventors surprisingly discovered that administering ganaxolone at lower and more frequent daily doses can provide an effective treatment for TSC and TSC-associated epilepsy. While not wishing to be bound by a particular theory or mechanism, it is believed that administering ganaxolone more frequently (e.g., three times daily) but at lower doses than previously used may improve drug exposure by maintaining ganaxolone serum levels above a threshold level (e.g., trough concentration) for an extended period of time, resulting in effective treatment. For example, oral ganaxolone administered three (or more) times daily at a total daily dose of 1800 mg or less, 1700 mg or less, 1600 mg or less, 1500 mg or less, or 63 mg / kg / day can effectively reduce seizures by about 70% or more, achieving a ganaxolone plasma concentration of at least about 100 ng / ml over a 24-hour period. Furthermore, dosing three (or more) times daily generally requires less ganaxolone to achieve a trough ganaxolone concentration, which is beneficial to the treated subject. In fact, trough ganaxolone levels with twice-daily dosing generally remained below 100 ng / ml over the 24-hour treatment period.

[0009] The inventors also surprisingly discovered that a subpopulation of patients with TSC-associated epilepsy have low plasma concentrations of allopregnanolone sulfate (Allo-S) (e.g., less than 2,500 pg / ml) and may be more responsive to treatment with ganaxolone.

[0010] Accordingly, the present disclosure relates to methods for effectively treating TSC and / or TSC-associated epilepsy. The methods disclosed herein comprise administering to a subject in need thereof a therapeutically effective amount of a neurosteroid, preferably ganaxolone, or a pharmaceutically acceptable salt thereof. The ganaxolone is preferably administered in an amount that provides a trough ganaxolone level (e.g., ganaxolone plasma concentration) of about 100 ng / ml or greater over a 24-hour period by about 70% or greater.

[0011] To provide a plasma concentration of ganaxolone of about 100 ng / ml or more over a 24-hour period of about 70% or more, ganaxolone can be administered three times a day (or more) at a maximum of about 1,800 mg / day. For subjects weighing less than 40 kg, ganaxolone can be administered three times a day (or more) at a maximum dose of 63 mg / kg / day. Generally, a maximum of about 1,500 mg / day of ganaxolone administered three times a day produces a plasma concentration of about 100 ng / ml or more over a 24-hour period of about 70% or more. Ganaxolone can be administered three times a day at a dose of about 500 mg. It is understood that a skilled clinician can adjust the amount of ganaxolone administered three times a day (e.g., orally) to achieve the desired ganaxolone trough concentration, as long as the total amount does not exceed the maximum daily dose of ganaxolone.

[0012] Preferably, ganaxolone is administered orally (e.g., via oral suspension or oral capsules). Without being bound by theory, the inventors believe that triple dosing results in improved anti-seizure activity (i.e., reduced seizure frequency and / or seizure severity) due to increased plasma ganaxolone exposure. This is contrary to previous treatment protocols that taught administering high doses (e.g., daily doses) of ganaxolone to achieve a therapeutic effect. For example, administering ganaxolone twice daily at high doses.

[0013] Administration of an amount of ganaxolone that achieves a ganaxolone plasma concentration of at least about 100 ng / ml or more over a 24-hour period by at least about 70% reduces the subject's seizure frequency and / or seizure severity compared to baseline. Typically, a reduction in seizure frequency of at least about 20% is achieved compared to baseline seizure frequency. The subject's ganaxolone plasma concentration can be monitored during treatment, and / or the subject can be monitored for seizure activity using EEG. If the subject exhibits signs of seizures (e.g., seizure recurrence), the amount of ganaxolone administered can be adjusted accordingly.

[0014] The methods disclosed herein are suitable for treating any form of seizure associated with TSC or TSC-related epilepsy, including, but not limited to, infantile spasms, complex partial seizures, focal seizures, or generalized seizures.

[0015] One object of the present disclosure is to provide a treatment for tuberous sclerosis complex. One object of the present disclosure is to provide a treatment for tuberous sclerosis complex (TSC)-associated epilepsy. A further object of the present disclosure is to provide a treatment for seizures associated with TSC-associated epilepsy. Another object of the present disclosure is to utilize the gamma-aminobutyric acid (GABA)ergic mechanism of action of ganaxolone to provide a therapeutic benefit to humans with TSC and TSC-associated epilepsy.

[0016] In furtherance of the above and other objectives, the present disclosure relates, in part, to a method of treating a human suffering from tuberous sclerosis, comprising administering to the human a therapeutically effective amount of a pharmaceutically acceptable pregnenolone neurosteroid in an amount effective to reduce or inhibit one or more symptoms of tuberous sclerosis(s) in the human. The pharmaceutically acceptable pregnenolone neurosteroid may be administered parenterally and / or orally in an amount ranging from about 1 mg / day to about 5000 mg / day. Humans who may benefit therapeutically from administration of a pharmaceutically acceptable pregnenolone neurosteroid include those with low levels of allopregnanolone sulfate.

[0017] The plasma level of allopregnanolone sulfate appears to be positively correlated with the plasma level of allopregnanolone and may qualitatively represent the level of allopregnanolone in the brain. Therefore, low levels of allopregnanolone sulfate may indicate a deficiency of allopregnanolone in the brain. Plasma allopregnanolone sulfate levels of about 2500 pg / ml or less are considered low levels and may indicate a deficiency of endogenous neurosteroids in humans. Low levels of allopregnanolone sulfate are: 2400pg / ml or less, 2300pg / ml or less, 2200pg / ml or less, 2100pg / ml or less, 2000pg / ml or less, 1900pg / ml or less, 1800pg / ml or less, 1700pg / ml or less, 1600pg / ml or less, 1500pg / ml or less, 1400pg / ml or less, 1300pg / ml or less, 1200pg / ml or less, 1100pg / ml or less, 1000pg / ml or less, 900pg / ml or less, 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 The concentration may be below 500pg / ml, 450pg / ml, 400pg / ml, 350pg / ml, 300pg / ml, 250pg / ml, 200pg / ml, 150pg / ml, 100pg / ml, 90pg / ml, 80pg / ml, 70pg / ml, 60pg / ml, 50pg / ml, 40pg / ml, 30pg / ml, 20pg / ml, 15pg / ml, 10pg / ml, 9pg / ml, 8pg / ml, 7pg / ml, 6pg / ml, 5pg / ml, 4pg / ml, 3pg / ml, 2pg / ml, or 1pg / ml.

[0018] Because a deficiency of allopregnanolone in the brain can cause one or more symptoms of tuberous sclerosis, administration of a pharmaceutically acceptable pregnenolone neurosteroid according to the methods disclosed herein can correct this deficiency, thereby alleviating and / or reducing the severity and frequency of one or more symptom(s) of tuberous sclerosis according to the methods disclosed herein. Symptoms of tuberous sclerosis that may be alleviated or reduced by administration of a pharmaceutically acceptable pregnenolone neurosteroid include, but are not limited to, seizures, intellectual disability, developmental delay, abnormal behavior, skin abnormalities, pulmonary disease, and renal disease. Seizures include, for example, focal motor seizures without impaired consciousness or awareness, focal seizures with impaired consciousness or awareness, focal seizures resulting in bilateral generalized convulsive seizures, tonic-clonic seizures, and generalized seizures with a countable motor component, such as tonic-clonic, bilateral tonic, bilateral clonic, or atonic / absence seizures.

[0019] The present disclosure also relates to a method of treating a human suffering from tuberous sclerosis, comprising administering to the human a therapeutically effective amount of ganaxolone in an amount effective to alleviate or inhibit one or more symptoms of tuberous sclerosis in the human. Ganaxolone may be administered parenterally and / or orally. Humans who may benefit therapeutically from the administration of ganaxolone include those with low levels of allopregnanolone sulfate.

[0020] When ganaxolone is administered orally, a therapeutically effective amount of ganaxolone may be, for example, about 600 mg / day to about 2000 mg / day. In certain embodiments, this dose may be increased to about 2100 mg / day, 2200 mg / day, 2300 mg / day, or more to provide an improved response compared to lower doses, the limiting factor being the side effects caused by high doses. Generally, about 1,500 mg / day or 1,800 mg / day of ganaxolone is administered. In certain embodiments, to alleviate or reduce the severity of side effects(s) observed in humans at high doses, the dose may be reduced to about 550 mg / day, 500 mg / day, 450 mg / day, 300 mg / day, or less than 300 mg / day. Symptoms of tuberous sclerosis that may be alleviated and / or their frequency and / or severity reduced by administering ganaxolone in accordance with the methods disclosed herein include, but are not limited to, seizures, intellectual disability, developmental delay, abnormal behavior, skin abnormalities, pulmonary disease, and renal disease. Seizures include, for example, focal motor seizures without impaired consciousness or awareness, focal seizures with impaired consciousness or awareness, focal seizures that result in bilateral generalized convulsive seizures, tonic-clonic seizures, and generalized seizures with a countable motor component, such as tonic, bilateral tonic, bilateral, or atonic / absence seizures.

[0021] The present disclosure also relates to a method of treating a human with TSC or TSC-associated epilepsy, comprising chronically administering to the human a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) in an amount effective to reduce seizure frequency in the human, wherein the human has low plasma levels of endogenous neurosteroid(s) (e.g., allopregnanolone sulfate (Allo-S), as described above).

[0022] The present disclosure also relates to a method of treating a human having TSC or TSC-associated epilepsy, comprising orally administering to the human 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 neurosteroid has a half-life of about 18 to about 24 hours, and wherein the formulation releases at least about 70% or about 80% of the ganaxolone after 45 minutes in simulated gastrointestinal fluid (simulated gastric fluid (SGF) and / or simulated intestinal fluid (SIF)), resulting in at least about a 35%, about a 40%, about a 45%, or about a 50% reduction in seizure frequency per 28 days in the human compared to the seizure frequency 28 days prior to the initial administration.

[0023] Additionally, the present disclosure relates to a method of treating a human having TSC or TSC-associated epilepsy, comprising orally administering to the human 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 neurosteroid has a half-life of about 18 hours to about 24 hours, and wherein the formulation releases at least about 70% or about 80% of the ganaxolone after 45 minutes in simulated gastrointestinal fluid (simulated gastric fluid (SGF) and / or simulated intestinal fluid (SIF)), resulting in at least about a 35%, about a 40%, about a 45%, or about a 50% reduction in seizure frequency per 28 days in the human compared to the seizure frequency 28 days prior to the initial administration.

[0024] The present disclosure also relates to a method of treating a human having a pregnenolone neurosteroid, the human having TSC or TSC-related epilepsy, the method comprising: collecting or recovering a biological sample (e.g., a blood sample) from the human to determine whether the human has low levels of endogenous neurosteroids (e.g., Allo-S); and performing or subjecting the biological sample to an assay to determine the plasma level of the endogenous neurosteroid(s) in the biological sample. -1 Below, 2000pg mL -1Below, 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 following endogenous neurosteroid levels indicate low levels of endogenous steroids in humans: Subjects (e.g., humans) with low levels of endogenous steroids (e.g., Allo-S) can be orally administered 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, given in two or three divided doses at least once daily.

[0025] In some of these embodiments, 2500 pg mL -1 Below, 2000pg mL -1 Below, 1500pg mL -1 Below, 1000pg mL -1 Below, 900pg mL -1 , 800pg mL -1 Below, 700pg mL -1 Below, 600pg mL -1 Below, 500pg mL-1 or below, 400pg mL -1 Below, 300pg mL -1 Below, 200pg mL-1 or below, 100pg mL -1 Below, 75pg mL -1Below, 50pg mL -1 or less than 25 pg mL -1 The following endogenous neurosteroid levels indicate that administration of the above-described ganaxolone is likely to reduce a patient's seizure frequency by, for example, 35% or more, about 40% or more, about 45% or more, or about 50% or more for 28 days after administration compared to the seizure frequency during the 28-day period prior to the first administration: The endogenous neurosteroid may be selected from the group including or consisting of pregnanolone, pregnanolone sulfate, 5-αDHP, allopregnanolone, allopregnanolone-S, pregnanolone, pregnanolone-S, DHEA, and combinations thereof; for example, the pregnenolone neurosteroid may be selected from the group including or consisting of allopregnanolone, ganaxolone, alphaxolone, alphadolone, hydroxydione, minaxolone, pregnanolone, acetrocol, 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 and after administration of the pregnenolone neurosteroid. Ganaxolone is a preferred pregnenolone neurosteroid.

[0026] The present disclosure also relates to a method of treating a human with ganaxolone, wherein the human is afflicted with TSC or TSC-associated epilepsy, the method comprising administering to the human a dose of ganaxolone above 2500 pg mL -1 determining whether the human has a level of allopregnanolone sulfate (Allo-S) below 2500 pg mL; and -1 In some of these embodiments, the method comprises orally administering 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. -1The following allopregnanolone sulfate salts show that administration of the ganaxolone is likely to reduce seizure frequency in humans by, for example, at least about 35%, about 40%, about 45%, or about 50% for 28 days after administration compared to the seizure frequency during the 28-day period prior to the first administration.

[0027] Additionally, the disclosure relates to a method of treating a human suffering from TSC or TSC-associated epilepsy, the method comprising administering to the human a dose of 2500 pg mL -1 determining whether the human has a level of allopregnanolone sulfate below 2500 pg mL -1 If the condition is below this, endogenous neurosteroids (allopregnanolone, pregnanolone, etc.) or synthetic neurosteroids (e.g., Co26749 / WAY-141839, Col34444, Co177843, Sage-217 (3α-hydroxy-3β-methyl-21-(4-cyano-1H-pyrazol-1'-yl)-19-nor-5β-pregnan-20-one, ganaxolone, etc.) are administered at a dose of 1 mg / kg / day to approximately 200 mg. / kg / day, about 2 mg / kg / day to about 150 mg / kg / day, about 3 mg / kg / day to about 100 mg / kg / day, about 4 mg / kg / day to about 90 mg / kg / day, about 5 mg / kg / day to about 80 mg / kg / day, about 6 mg / kg / day to about 70 mg / kg / day, or about 7 mg / kg / day to about 65 mg / kg / day, administered orally in two or three divided doses for at least one day to achieve a human allopregnanolone sulfate level of 2500 pg mL -1If the level of the neurosteroid exceeds 100 mg / kg / day, the method includes refraining from administering endogenous or synthetic neurosteroids to the human and / or administering another anticonvulsant. The other anticonvulsant may be selected from the group consisting of, for example, benzodiazepines (e.g., clobazam, diazepam, clonazepam, midazolam, etc.), clorazepic acid, levetiracetam, felbamate, lamotrigine, fatty acid derivatives (e.g., valproic acid), carboxamide derivatives (e.g., rufinamide, carbamazepine, oxcarbazepine, etc.), amino acid derivatives (e.g., levocamitine), barbiturates (e.g., phenobarbital), or a combination of two or more of these agents. Any number of other anticonvulsants may be administered. Those skilled in the art are familiar with anticonvulsants.

[0028] The present disclosure also relates to a method of treating a human having TSC or TSC-associated epilepsy, the method comprising administering to the human a dose of 2500 pg mL -1 determining whether the human has a level of allopregnanolone sulfate below 2500 pg mL -1 In some of these embodiments, the method comprises orally administering 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 65 mg / kg / day in two or three divided doses for at least one day. -1 The following allopregnanolone sulfate salts show that administration of the ganaxolone is likely to reduce seizure frequency in humans by, for example, at least about 35%, about 40%, about 45%, or about 50% for 28 days after administration compared to the seizure frequency during the 28-day period prior to the first administration.

[0029] Additionally, the disclosure relates to a method of treating a human suffering from TSC or TSC-associated epilepsy, the method comprising administering to the human a dose of 200 pg mL -1determining whether the patient has a level of allopregnanolone below 200 pg mL; -1 If you have the following levels of allopregnanolone, administer orally in two or three divided doses for at least one day at a dose of 1 mg / kg / day to about 80 mg / kg / day, about 2 mg / kg / day to about 75 mg / kg / day, about 3 mg / kg / day to about 70 mg / kg / day, about 4 mg / kg / day to about 65 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, and receive a dose of 200 pg mL -1 In some of these embodiments, the method includes the step of withholding administration of ganaxolone to the human if the human has a level of allopregnanolone above 200 pg mL -1 The following allopregnanolone shows that administration of the ganaxolone is likely to reduce seizure frequency in humans by, for example, at least about 35%, about 40%, about 45%, or about 50% for 28 days after administration compared to the seizure frequency in the 28-day period prior to the first administration.

[0030] The methods disclosed herein may further include measuring plasma levels of allopregnanolone in a human with TSC or TSC-associated epilepsy. An allopregnanolone plasma level of about 200 pg / ml or less is a low level and may indicate that the human may have a deficiency of endogenous neurosteroids. Thus, in some embodiments, a low endogenous level of neurosteroids in a human may 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, or 185 pg / ml or less. , 184pg / ml or less, 183pg / 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 Below, 174pg / ml or less, 173pg / 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 / m l 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, 145 pg / 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, 1 35pg / 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,Below 125 pg / ml, below 124 pg / ml, below 123 pg / ml, below 122 pg / ml, below 121 pg / ml, below 120 pg / ml, below 119 pg / ml, below 118 pg / ml, below 117 pg / ml, below 116 pg / ml, below 115 pg / ml, below 114 pg / ml, below 113 pg / ml, below 112 pg / ml, below 111 pg / ml, below 110 pg / ml, below 109 pg / ml, below 108 pg / ml, below 107 pg / ml, below 106 pg / ml, below 105 pg / ml, below 104 pg / ml, below 103 pg / ml Below 1, below 102 pg / ml, below 101 pg / ml, below 100 pg / ml, below 99 pg / ml, below 98 pg / ml, below 97 pg / ml, below 96 pg / ml, below 95 pg / ml, below 94 pg / ml, below 93 pg / ml, below 92 pg / ml, below 91 pg / ml, below 90 pg / ml, below 89 pg / ml, below 88 pg / ml, below 87 pg / ml, below 86 pg / ml, below 85 pg / ml, below 84 pg / ml, below 83 pg / ml, below 82 pg / ml, below 81 pg / ml, below 80 pg / ml, below 79 pg / ml, below 78 Below pg / ml, below 77 pg / ml, below 76 pg / ml, below 75 pg / ml, below 74 pg / ml, below 73 pg / ml, below 72 pg / ml, below 71 pg / ml, below 70 pg / ml, below 69 pg / ml, below 68 pg / ml, below 67 pg / ml, below 66 pg / ml, below 65 pg / ml, below 64 pg / ml, below 63 pg / ml, below 62 pg / ml, below 61 pg / ml, below 60 pg / ml, below 59 pg / ml, below 58 pg / ml, below 57 pg / ml, below 56 pg / ml, below 55 pg / ml, below 54 pg / ml, 5 Below 3 pg / ml, below 52 pg / ml, below 51 pg / ml, below 50 pg / ml, below 49 pg / ml, below 48 pg / ml, below 47 pg / ml, below 46 pg / ml, below 45 pg / ml, below 44 pg / ml, below 43 pg / ml, below 42 pg / ml, below 41 pg / ml, below 40 pg / ml, below 39 pg / ml, below 38 pg / ml, below 37 pg / ml, below 36 pg / ml, below 35 pg / ml, below 34 pg / ml, below 33 pg / ml, below 32 pg / ml, below 31 pg / ml, below 30 pg / ml, below 29 pg / ml.It may be 28 pg / ml or less, 27 pg / ml or less, 26 pg / ml or less, 25 pg / ml or less, 24 pg / ml or less, 23 pg / ml or less, 22 pg / ml or less, 21 pg / ml or less, 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.

[0031] The present disclosure further relates to a method of treating TSC or TSC-associated epilepsy, the method comprising administering to a human a dose of 200 pg mL -1 determining whether the patient has a level of allopregnanolone below 200 pg mL; -1 If the patient has the following levels of allopregnanolone, the method includes orally administering ganaxolone at a dose of 1 mg / kg / day to about 100 mg / kg / day, about 2 mg / kg / day to about 80 mg / kg / day, about 3 mg / kg / day to about 70 mg / kg / day, about 4 mg / kg / day to about 65 mg / kg / day, about 5 mg / kg / day to about 65 mg / kg / day, about 6 mg / kg / day to about 65 mg / kg / day, or about 7 mg / kg / day to about 65 mg / kg / day for at least one day.

[0032] The present disclosure also relates to a method of treating endogenous neurosteroid deficiency in a human in need thereof, comprising administering a pharmaceutically acceptable pregnenolone neurosteroid (e.g., ganaxolone) at a dose of about 1800 mg or less for at least one day to the human, wherein the human has a genetic mutation in the TSC1 gene located on chromosome 9q34 and / or the TSC2 gene located on chromosome 16p13.3 and has hypopigmentation (≧3, at least Patients with one or more of the following conditions are present: angiofibroma (≥ 3) or forehead plaques, periungual fibromas (≥ 2), Charlene's patches, multiple retinal hamartomas, cerebral cortical atypia, subependymal nodules, subependymal giant cell astrocytomas, cardiac rhabdomyoma, lymphangioleiomyomatosis (LAM) angiomyolipomas (≥ 2), "scattered" small white spots, multiple small pits in dental enamel (≥ 3), oral fibromas (≥ 2), retinal apigmented macules, multiple renal cysts, and non-renal hamartomas.

[0033] In some of these embodiments, the pharmaceutically acceptable pregnenolone neurosteroid is ganaxolone and is orally administered in an amount of about 200 mg / day to about 2500 mg / day, about 200 mg / day to about 2250 mg / day, about 200 mg / day to about 2000 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, given in two or three divided doses. In some embodiments, a person experiencing seizures and receiving a pharmaceutically acceptable pregnenolone neurosteroid reduces the average frequency of seizures per 28 day period by 35% or more (e.g., about 40%, about 45%, about 50%, about 55%) compared to the frequency of seizures in the 28 day period prior to the first administration, hi some embodiments, the improvement is 50% or more.

[0034] The methods disclosed herein further include periodic measurement of plasma levels of the administered pharmaceutically acceptable pregnenolone neurosteroid(s) (e.g., ganaxolone), and / or concomitant AED(s), 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 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.

[0035] Pregnenolone neurosteroids (e.g., ganaxolone) can be administered chronically orally or parenterally using the methods described herein. 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, with ganaxolone 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 without undesirable side effects requiring 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. In some embodiments, ganaxolone can be administered for a period of 2 weeks to 100 years, or for the duration of a human lifetime.

[0036] When administered as an oral suspension, the pregnenolone neurosteroid may be administered, for example, from one to about three times per day. In certain preferred embodiments, oral administration of 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, from one to about four times per day. When administered parenterally, the pregnenolone neurosteroid may be administered, for example, from one to about three times per day.

[0037] The present disclosure relates to immediate-release formulations (e.g., oral suspensions, tablets, or capsules) comprising particles comprising (i) a pregnenolone neurosteroid (e.g., ganaxolone) and (ii) one or more pharmaceutically acceptable excipient(s), wherein the particles have a particle size that ensures no aggregation after dispersion in simulated gastrointestinal fluid (SGF and / or SIF) for use in TSC and / or TSC-associated epilepsy, and the formulations do not deteriorate upon storage at 25°C / 60% relative humidity for one month. 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., SGF (simulated gastric fluid) with 5% SLS and / or SIF (simulated intestinal fluid) with 5% SLS) at 37°C for 45 minutes and at 0.5°C at 100 rpm using USP Apparatus 1 (Basket), resulting in a plasma level of 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 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 (e.g., about 332 nm). In some embodiments, the particles have a D(10) diameter of about 200 nm to about 220 nm, a D(50) diameter of about 250 nm to about 450 nm, and a D(90) diameter of about 480 nm to about 700 nm, and the formulation does not include a sulfoalkyl ether cyclodextrin, including modified versions thereof, and is for treating TSC-associated epilepsy.

[0038] The present disclosure also relates, in part, to an oral immediate-release formulation (e.g., an oral suspension, tablet, or capsule) comprising particles comprising (i) ganaxolone and (ii) one or more pharmaceutically acceptable excipient(s) for use in treating TSC and / or TSC-associated seizures, 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), and the particles do not deteriorate upon storage of the formulation for one month at 25°C / 60% relative humidity. The formulations release about 70% or about 80% or more of the ganaxolone in 500 ml of dissolution medium (e.g., SGF (simulated gastric fluid) with 5% SLS and / or SIF (simulated intestinal fluid) with 5% SLS) at 37°C for 45 minutes and at 0.5°C at 100 rpm using USP Apparatus 1 (Basket), and achieve 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) for at least 6 to 12 hours after administration, following single and / or multiple doses of these formulations, for the treatment of TSC or TSC-related epilepsy. 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) can be provided following administration of the formulation in a fasted and / or postprandial state.

[0039] In some of these embodiments, an average particle size of about 0.3 microns is important for enabling the formulation to achieve about 70% or about 80% or greater pregnenolone neurosteroid dissolution in 45 minutes in simulated gastrointestinal fluid (SGF and / or SIF) and provide a plasma level of 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) over at least 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, or about 12 hours.

[0040] The present disclosure also relates, in part, to an immediate release formulation (e.g., oral suspension, tablet, or capsule) comprising particles comprising (i) ganaxolone and (ii) one or more pharmaceutically acceptable excipient(s) for use in TSC and / or TSC-associated epilepsy, wherein the particles have an average particle size of about 0.3 microns, and the particle size does not deteriorate upon storage at 25°C / 60% relative humidity 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., SGF (simulated gastric fluid) containing 5% SLS and / or SIF (simulated intestinal fluid) containing 5% SLS) at 37°C for 45 minutes and at 0.5°C using USP Apparatus 1 (Basket) at 100 rpm, and the formulation provides 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) for at least 6 to 12 hours after administration. 3. Brief description of the drawings [Brief explanation of the drawings]

[0041] [Figure 1] Baseline endogenous allopregnanolone sulfate ("Allo-S") levels are shown in female patients with PCDH19-associated epilepsy stratified into responders and non-responders. Patients with allopregnanolone sulfate (Allo-S) levels <2.5 ng mL-1 experienced a 50% median reduction in seizure frequency compared to baseline (n=7). This analysis was performed retrospectively on a small, open-label cohort. Nevertheless, the 1.5- to 2-order difference in allopregnanolone sulfate levels between responders and non-responders suggests that plasma allopregnanolone sulfate levels may be used to predict the efficacy of pharmaceutically acceptable pregnenolone neurosteroids. These data provide preliminary evidence that plasma allopregnanolone sulfate levels may be used as a predictive biomarker to proactively identify patients who may experience improved therapeutic benefit from ganaxolone. [Figure 2]We present allopregnanolone sulfate (Allo-S) levels between TSC patients and controls based on plasma samples from the Biosample Repository. The objective was to quantify endogenous neurosteroid levels using a proprietary, validated analytical method (LC / MS / MS) and compare these levels with healthy (unaffected) age-matched control samples. Plasma samples from epileptic TSC patients showed a trend toward decreased Allo-S levels (n = 47, median 1.8 ng mL-1) compared with controls (n = 60, median 4.1 ng mL-1). This finding was more pronounced when restricting the sample to patients / subjects aged 1-14 years (n = 28 TSC, n = 28 controls). Patients / subjects comprised 29 females and 18 males. The median age of women was 15 years (range 2-27 years). The median age of men was 10.5 years (range 2-33 years). Fluctuations in neurosteroid levels after puberty may confound the analysis of all patients. [Figure 3] Allo-S distribution in TSC patients and unaffected subjects (all individuals) is shown. [Figure 4] We present a comparison of allopregnanolone-sulfate (Allo-S) in PCDH19 and TSC, and potential opportunities to expand the scope of Allo-S biomarker investigation in TSC. [Figure 5] 1 shows a positive correlation between allopregnanolone (Allo) and allopregnanolone sulfate (Allo-S). DETAILED DESCRIPTION OF THE INVENTION

[0042] 4. Detailed Description Effective treatment of TSC and / or TSC-associated epilepsy is challenging, and conventional treatment protocols are ineffective for many patients. In fact, approximately 70% of patients are resistant to treatment and have intractable epilepsy. Most seizures begin within the first 12 months of life, resulting in a high prevalence of intellectual disability. Controlling seizures can reduce these developmental disabilities. Therefore, improved methods of treating TSC and / or TSC-associated epilepsy are desperately needed.

[0043] The present disclosure relates to novel methods for treating SE. As exemplified and described herein, treatment with these methods reduces seizure frequency and / or suppresses seizures in TSC-associated epilepsy. These methods can be used to treat all forms of TSC-associated epilepsy, including, but not limited to, infantile spasms, focal motor seizures without impaired consciousness or awareness, focal seizures with impaired consciousness or awareness, focal seizures that can become bilateral, generalized seizures such as tonic-clonic, bilateral tonic-clonic, bilateral clonic, countable atonic / astatic, myoclonic, or epileptic seizures, and motor seizures.

[0044] The methods described herein include administering to a subject a therapeutically effective amount of a neurosteroid. Ganaxolone is a preferred neurosteroid.

[0045] The methods described herein can further include administering ganaxolone in a therapeutically effective amount to achieve a ganaxolone plasma concentration of 100 ng / ml or greater for at least about 70% of 24 hours. This can be achieved by administering ganaxolone at least three times daily. Three times daily is preferred, but in some cases, it may be appropriate to administer ganaxolone more than three times daily to achieve the desired ganaxolone trough concentration. Achieving a plasma concentration of at least about 100 ng / ml or greater for at least about 70% of 24 hours daily improves seizure relief and / or seizure suppression. For example, a seizure reduction of at least 20% or greater compared to baseline seizure frequency can be achieved. Administering ganaxolone three times daily at a lower maximum dose can achieve the desired ganaxolone trough concentration. Typically, a maximum daily dose of about 1,800 mg, and preferably 1,500 mg, of ganaxolone is administered. The maximum daily dose of ganaxolone is administered in the same or different doses at least three times apart within a 24-hour period.

[0046] The methods disclosed herein can further include determining whether a patient with TSC or TSC-associated epilepsy will benefit from treatment with a neurosteroid (e.g., ganaxolone). A subpopulation of patients with TSC-associated epilepsy may have low plasma concentrations of Allo-S (e.g., less than 2,500 pg / ml) and may respond better to treatment with ganaxolone. The methods described herein can include measuring the subject's endogenous neurosteroid levels before initiating treatment with a neurosteroid (e.g., ganaxolone). A low level of endogenous neurosteroid indicates that the subject will respond to treatment with a neurosteroid. Once it is determined that the subject has low endogenous neurosteroid levels, the subject can be administered a therapeutically effective amount of a neurosteroid.

[0047] Further description of the method and guidance for carrying out the method are provided herein. For simplicity of exposition, further details and guidance are provided with respect to a preferred embodiment using ganaxolone. The further details and guidance are also intended to be relevant to treatment with other neurosteroids.

[0048] I. Definition The recitation of ranges of numerical values ​​is merely intended to serve as a shorthand method of referring individually to each separate value within the range, unless otherwise stated herein, and each separate value is incorporated herein by reference as if each separate value were individually set forth herein. All range endpoints are included within the range and are independently combinable. All methods described herein can be performed in any suitable order unless otherwise stated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as"), is intended solely for illustration and does not limit the scope of the invention unless a claim is specifically asserted. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0049] 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.

[0050] The term "about" is used synonymously with the term "approximately." As one of ordinary skill in the art would understand, the exact boundaries of "about" 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%, which are also effective and safe. Thus, compositions slightly outside the cited ranges are also within the scope of the claims.

[0051] An "active ingredient" is any compound, element, or mixture that, when administered to a patient alone or in combination with another agent, directly or indirectly imparts a physiological effect to the patient. When the active ingredient is a compound, it includes salts, free compounds, or solvates (including hydrates) of salts, crystalline and amorphous forms, and various polymorphs of the compound. Because compounds may contain one or more asymmetric atoms, such as stereogenic centers and stereogenic axes, e.g., asymmetric carbon atoms, the compounds can exist in various stereoisomeric forms. These compounds may be, for example, racemates or optically active forms. For compounds with two or more asymmetric atoms, these compounds may also be mixtures of diastereomers. For compounds with asymmetric centers, it should be understood that pure optical isomers and mixtures thereof are all included. Furthermore, compounds with carbon-carbon double bonds may exist in Z- and E-forms, and all isomeric forms of the compounds are included in 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.

[0052] The term "endogenous neurosteroids" refers to steroids produced in the brain that can modulate neuronal excitability by interacting with neuronal membrane receptors and ion channels, primarily GABA-A receptors, such as pregnane neurosteroids (e.g., allopregnanolone, allotetrahydrodeoxycorticosterone, etc.), androstane neurosteroids (e.g., androstanediol, etiocholanone, etc.), and sulfated neurosteroids (e.g., pregnanolone sulfate, dehydroepiandrosterone sulfate (DHEAS)).

[0053] 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.

[0054] The term "biomarker" refers to serum or plasma levels of neurosteroids that distinguish drug responders from non-responders.

[0055] As disclosed herein, the terms "serum" and "plasma" may be used interchangeably.

[0056] The terms "comprising," "including," and "containing" are open-ended. Other unrecited elements may be present within embodiments claimed by these transitional phrases. When "comprising," "including," and "containing" are used as transitional phrases, other elements may be included and still form embodiments within the scope of the claims. The open-ended transitional phrase "comprising" includes the intermediate transitional phrase "consisting essentially of" and the closed-ended phrase "consisting of."

[0057] A "bolus dose" is a relatively large amount of medication administered over a short period of time, eg, within 1 to 30 minutes.

[0058] "C max " is the concentration of the active ingredient in the plasma at the point of maximum concentration.

[0059] "Ganaxolone" is also known as 3α-hydroxy-5α-pregnan-20-one and is alternatively referred to herein as "GNX."

[0060] "Infusion" administration is parenteral administration, typically intravenous administration, but in some embodiments includes other parenteral routes such as epidural administration. 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.

[0061] 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 trauma, and in certain embodiments, treatment includes prophylactic or diagnostic treatment.

[0062] "Child" means a human between the ages of 1 day and 18 years (eg, between the ages of 1 day and 15 years), inclusive.

[0063] "Adult" means a human being over the age of 18.

[0064] A "pharmaceutical composition" is a composition containing at least one active ingredient, 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 ingredients. Specifically, the pharmaceutical composition meets the US FDA's GMP (Good Manufacturing Practice) standards for human or non-human drugs. A "pharmaceutical combination" is a combination of at least two active ingredients, which can be combined in a single dosage form or provided together in separate dosage forms with instructions for the combined use of the active ingredients, for treating a disorder, such as a seizure disorder.

[0065] "Povidone," also known as polyvidone and polyvinylpyrrolidone (PVP), is a water-soluble polymer made 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 10.2-13.8 and a nominal molecular weight of 4000 d. Plasdone C-17 has a K value of 15.5-17.5 and a nominal molecular weight of 10,000 d.

[0066] "Sterilization" means the inactivation of substantially all biological contaminants in a sample, preparation, or product. A one million-fold reduction in bioburden is also considered "sterile" for most medical uses.

[0067] The term "suppressing" seizures or seizure activity refers to a detectable decrease in seizure frequency, severity, and / or duration. Reductions 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 reductions in seizure frequency, severity, and / or duration can be made by comparing the patient's condition before and after treatment.

[0068] A "therapeutically effective amount" or "effective amount" is an amount of a pharmaceutical agent that achieves a pharmacological effect. The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. An "effective amount" of a neurosteroid is the amount necessary 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 will be understood that an "effective amount" or "therapeutically effective amount" may vary from subject to subject based on changes 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.

[0069] "Treat" or "treatment" refers to any treatment of a disorder or disease, such as inhibiting the disorder or disease, e.g., arresting the progression of the disorder or disease, alleviating the disorder or disease, regressing the disorder or disease, alleviating the symptoms caused by the disease or disorder, or alleviating the symptoms of the disease or disorder.

[0070] "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. In other embodiments, alkyl groups have 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.

[0071] "Aryl" refers to an aromatic group containing only carbon within the aromatic ring. Typical aryl groups contain 1 to 3 separate, fused, or pendant rings and 6 to about 18 ring atoms, but do not contain heteroatoms as ring members. Where indicated, such aryl groups may be further substituted with carbon or non-carbon atoms or groups. Examples of aryl groups include phenyl and naphthyl, such as 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.

[0072] "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 can be pendant from a substituted nitrogen, oxygen, or carbon atom, or a substituted carbon atom that can bear two substituents can have the cycloalkyl group attached as a spiro group. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0073] A "heteroalkyl" group is an alkyl group as described herein in which at least one carbon is replaced with a heteroatom, such as N, O, or S.

[0074] As used herein, the term "substituted" means that one or more hydrogens on the specified atom or group are replaced with a group of the options listed, provided that the normal valence of the specified atom is not exceeded. When a substituent is oxo (i.e., =0), two hydrogens on the atom are replaced. When an oxo group substitutes a heteroaromatic moiety, the resulting molecule may exist in tautomeric forms. For example, a pyridyl group substituted with oxo at the 2- or 4-position may be described as a pyridine or a 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 intended to mean a compound that is sufficiently stable to permit isolation from a reaction mixture and subsequent formulation into an effective therapeutic agent. Unless otherwise specified, substituents are named relative to the core structure. For example, aminoalkyl means that the point of attachment of the substituent to the core structure is within the alkyl moiety, and alkylamino means that the point of attachment is at the bond between the amino group and the nitrogen.

[0075] Suitable groups that may be present in the "substituted" or "optionally substituted" position include, for example, 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 (such as cycloalkyl and (cycloalkyl)alkyl groups); alkenyl and alkynyl groups having one or more unsaturated bonds and 2 to about 8, or 2 to about 6 carbon atoms, or the like; alkoxy groups having one or more oxygen bonds and 1 to about 8, or 1 to about 6 carbon atoms, or the like; aryloxy groups such as phenoxy; alkyl groups having one or more thioether bonds and 1 to about 8, or 1 to about 6 carbon atoms, or the like. alkylsulfinyl groups having one or more sulfonyl bonds and 1 to about 8 carbon atoms, or 1 to about 6 carbon atoms, etc.; alkylsulfonyl groups having one or more sulfonyl bonds and 1 to about 8 carbon atoms, or 1 to about 6 carbon atoms, etc.; aminoalkyl groups having one or more N atoms and 1 to about 8, or 1 to about 6 carbon atoms, etc.; mono- or dialkylamino groups having an alkyl group of 1 to about 6 carbon atoms, etc.; mono- or dialkylaminocarbonyl groups having an alkyl group of about 1 to about 6 carbon atoms (i.e., alkylNHCO- or (alkyl1)(alkyl2)NCO-); and aryl groups having 6 or more carbons, etc.

[0076] II. Tuberous sclerosis Tuberous sclerosis complex (TSC) is a multisystem disorder of embryonic cortical development that affects numerous organs through the abnormal growth of benign tumors known as hamartomas. Symptoms can include a combination of seizures, intellectual disability, developmental delay, abnormal behavior, skin abnormalities, pulmonary disease, and kidney disease. While the TSC phenotype is highly variable, neurological symptoms, such as epilepsy, are present in up to 90% of TSC patients (Krueger et al., 2013). The condition is caused by inherited mutations in either the TSC1 gene located on chromosome 9q34 or the TSC2 gene located on chromosome 16p13.3. TSC occurs at a frequency of 1:6,000, with mutations present in 85% of patients (Julich & Sahin, 2014). The gene products hamartin (TSC1) and tuberin (TSC2) form a regulatory complex that inhibits the small GTPase Ras homolog abundant in the brain (Rheb) to limit the activity of mammalian target of rapamycin complex 1 (mTORCl), a key intercellular regulator of growth and metabolism (Krueger et al., 2013). The mTOR inhibitor everolimus has been shown to reduce seizures (French et al., 2016; Mizuguchi et al., 2019).

[0077] TSC is one of the most common genetic causes of epilepsy, and seizure symptoms vary depending on the age of onset (Mich & Sahin, 2014). An effective treatment option is the use of adrenocorticotropic hormone (ACTH), with a potential mechanism consistent with the use of ganaxolone. ACTH has been shown to have a stimulatory effect on deoxycorticosterone (DOC), which is further synthesized into various neurosteroids. Specifically, ACTH has been shown to rapidly increase endogenous plasma and brain levels of allopregnanolone, which may further explain its beneficial effects on IS.

[0078] Infantile spasms ("IS") are the most common type of seizure presenting in infancy and represent the first symptom of epilepsy in 50% of patients. In older children and adults, focal impaired consciousness seizures (formerly known as complex partial seizures) are most common. Other focal and generalized seizures may occur, and more than 30% of patients develop treatment-resistant epilepsy (Julich & Sahin, 2014). While seizures are typically attributed to the nodules and surrounding cortex, seizures can arise in other brain regions or in TSC patients without nodules, and epilepsy in TSC can be considered multifactorial (Julich & Sahin, 2014).

[0079] A definite clinical diagnosis of TSC includes two major symptoms or one major symptom and two or more minor symptoms, as shown in Table 7 below. [Table 7]

[0080] γ-aminobutyric acid (GABA) is an endogenous GABA A Altered expression of receptor modulators appears to play a central role in the development of TSC-associated epilepsy ( di Michele et al., 2003 ).

[0081] Evidence supports a deficiency of the neuroactive steroids 3α,5α-tetrahydroprogesterone (THP) or allopregnanolone as a cause of epileptogenesis in TSC. Allopregnanolone is a positive modulator of GABAA receptors and has been shown to have antiepileptic effects in experimental animals and humans. Allopregnanolone is reduced relative to the functional GABAA antagonist 3β-THR in patients with TSC-associated epilepsy, but not in non-epileptic TSC patients or controls (di Michele et al., 2003). This reduced ratio may alter GABAA receptor-mediated neuronal excitability, potentially leading to epilepsy development. The role of GABAA receptor mediation is supported by the greater efficacy of vigabatrin, a specific and irreversible inhibitor of GABA aminotransferase, in seizures due to TSC compared with other epilepsies (di Michele et al., 2003).

[0082] TSC patients with epilepsy have reduced levels of endogenous neurosteroids, particularly Alio, similar to previous reports of PCDH19. Approximately 25% of all TSC patients have plasma levels of allopregnenalone sulfate below 6 ng / ml.

[0083] Anticonvulsant neuroactive steroids may be useful in the treatment of TSC and TSC-associated epilepsy. Anticonvulsant neuroactive steroids enhance GABAA-mediated signaling and may improve seizure control as well as behavioral abnormalities in individuals with TSC and TSC-associated epilepsy.

[0084] There is evidence that upregulation of the TSC-mTOR pathway enhances inflammatory responses and increases pro-inflammatory TOR, such as TLR4 signaling. Neurosteroids, including Allo, have been shown to positively modulate GABAA receptors and act as inhibitors of various neuroinflammatory pathways, including TLR4, broadening the mechanism of action of these compounds.

[0085] III. Neurosteroids Endogenous neurosteroids play an important role in maintaining homeostasis of brain activity. Neurosteroids have the ability to rapidly induce 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.

[0086] Neurosteroids act in concert with GABA, depending on the chemical structure of the steroid molecule. 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 receptors are heteropentamers of five protein subunits that form chloride ion channels. 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). Most GABA receptors 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 GABA receptors prevent the depolarization caused by excitatory neurotransmission, thereby preventing the generation of action potentials. A There are two types of inhibitory neurotransmission mediated by receptors: synaptic (transient) inhibition and extrasynaptic (sustained) inhibition. Neurosteroids inhibit both synaptic and extrasynaptic GABA receptors. AIt modulates receptors, thereby enhancing 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 regulating hippocampal excitability by setting a baseline of excitability (Reddy, 2010).

[0087] Neurosteroids such as ganaxolone inhibit GABA A Neurosteroids are potent positive allosteric modulators of GABA receptors (Akk et al, 2009). A The first observation that GABA receptors enhance GABA-induced responses was reported for alphaxolone in 1984 (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. Although the exact location of the neurosteroid binding site is currently unknown, 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 are also differences between neurosteroids and benzodiazepines, such as GABA A There are also differences regarding their interactions with receptors. Neurosteroids interact with most GABA receptors. A Although benzodiazepines modulate GABA receptor isoforms containing the γ2 subunit and not the α4- or α6-subunits, ASpecific α subunits may influence the efficacy of neurosteroids, whereas γ subunit types may affect the efficacy of 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).

[0088] Recent research has shown that GABA A The existence of at least three neurosteroid binding sites on the receptor has been demonstrated: allosteric enhancement of GABA-induced currents by allopregnanolone, direct activation by allopregnanolone, and antagonism by low (nM) concentrations of sulfated neurosteroids, such as pregnenolone sulfate (Lambert et al., 2003; Hosie et al., 2007). A Neurosteroid potentiation of GABA receptor chloride currents occurs through an increase in both channel open frequency and open duration (Reddy, 2010). Thus, neurosteroids inhibit GABA receptors, allowing for the influx of large amounts of chloride ions. A These effects occur at physiological concentrations of neurosteroids. Therefore, endogenous neurosteroid levels are associated with increased GABAergic activity. A It continuously regulates receptor function (Reddy, 2010).

[0089] Extrasynaptic δ-subunit-containing GABA A The GABA receptor exhibits increased sensitivity to neurosteroids, suggesting a key regulatory role in tonic inhibition (Wohlfarth et al., 2002). 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 Long-lasting GABA receptors with low receptor desensitization and activation by ambient concentrations of GABA in the extracellular space A Helps mediate receptor currents. Tonic GABA A The receptor current induces stable inhibition of the neuron, reducing its excitability. GABA binds with high affinity but relatively low efficacy to δ-containing GABA receptors. A Therefore, neurosteroids inhibit the production of δ-containing GABA receptors, even in the presence of saturating GABA concentrations. A During neural activity, perisynaptic δ-subunit-containing GABA receptors and extrasynaptic δ-subunit-containing GABA receptors can significantly enhance the electrical current generated by the receptors. A It is thought that there is a significant release of GABA from active GABAergic interneurons, which can interact with the receptors. Overall, the robust actions of neurosteroids are due to the release of GABA from synaptic and perisynaptic / extrasynaptic GABA receptors. A This is likely due to its effect on both receptors (Reddy, 2010).

[0090] Pregnane and pregnenolone neurosteroids are a class of compounds useful as anesthetics, sedatives, hypnotics, anxiolytics, antidepressants, antitremors, autistic behavioral treatments, and anticonvulsants. These compounds are characterized by very poor water solubility, which limits formulation options. Nanoparticle formulations of pregnane and pregnenolone neurosteroids are available that are bioavailable both orally and parenterally.

[0091] Injectable formulations of pregnane and pregnenolone neurosteroids are particularly desirable because these compounds are used for clinical indications where oral administration is not possible, such as anesthesia, particularly for the emergency treatment of active seizures.

[0092] The present disclosure includes injectable nanoparticulate neurosteroid formulations.

[0093] The pregnane neurosteroids and pregnenolone neurosteroids of the present invention each have the formula IA: [ka] or a pharmaceutically acceptable salt thereof, wherein X is O, S, or NR 10 and R 1 is hydrogen, hydroxyl, —CHA, 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 a 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 C-C alkyl, optionally substituted C-C alkoxyl (e.g., methoxyl), or optionally substituted heteroalkyl; R 8 and R 9are each independently selected from the group consisting of hydrogen, C-C alkyl (e.g., methyl), halogenated C-C alkyl (e.g., trifluoromethyl), or C-C 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, where each alkyl is C-C 10 alkyl, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)C1-C4 alkyl, and optionally containing a single bond substituted with a double or triple bond; Each heteroalkyl group may be substituted by one or more methyl groups, independently selected from -O-, -S-, -N(R 10 )-, -S(=O)- or -S(=O)2-substituted alkyl group, 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 -H2 or -HR 12 and R 12 is C1-C6 alkyl, or C1-C6 alkoxy.

[0094] The pregnane neurosteroids and pregnenolone neurosteroids of the present invention can each be a compound of formula IA, wherein: X is O, R 1 are hydrogen, —CH, —CHOH, 1H-imidazol-1-yl, 1-oxidoquinolin-6-yloxy, and 4-cyano-1H-pyrazol-1′-yl; R 4 is hydrogen, oxo, optionally substituted alkyl, or optionally substituted heteroalkyl; R 2 , R 3 , R 5 , R6 , and R 7 are each independently absent, hydrogen, hydroxyl, halogen, optionally substituted C-C alkyl, optionally substituted C-C alkoxyl (e.g., methoxyl), or optionally substituted heteroalkyl; R 8 and R 9 are each independently selected from the group consisting of hydrogen, C-C alkyl (e.g., methyl), halogenated C-C alkyl (e.g., trifluoromethyl), or C-C 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, where each alkyl is C-C 10 alkyl, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)C1-C4 alkyl, and optionally containing a single bond substituted with a double or triple bond; Each heteroalkyl group may be substituted by one or more methyl groups, independently selected from -O-, -S-, -N(R 10 )-, -S(=O)- or -S(=O)2-substituted alkyl group, and R 10 is hydrogen, alkyl, or alkyl in which one or more methylene groups are replaced by -O-, -S-, -NH, or -N-alkyl.

[0095] The pregnane neurosteroids and pregnenolone neurosteroids of the present invention each have the formula IB: [ka] or a pharmaceutically acceptable salt thereof, wherein X is O, S, or NR 10 and R 1is 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 are bonded to each other to form an oxo group, R 10 is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl, where each alkyl is C-C 10 alkyl, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)C1-C4 alkyl, and optionally containing a single bond substituted with a double or 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.

[0096] 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.

[0097] The pregnane neurosteroids and pregnenolone neurosteroids of the present invention 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, —CHA, 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 C-C alkyl, optionally substituted C-C alkoxyl (e.g., methoxyl), or optionally substituted heteroalkyl; R 8 and R 9 are each independently selected from the group consisting of hydrogen, C-C alkyl (e.g., methyl), halogenated C-C alkyl (e.g., trifluoromethyl), or C-C 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, where each alkyl is C-C 10 alkyl, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)C1-C4 alkyl, and optionally containing a single bond replaced with a double or 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.

[0098] The pregnane neurosteroids and pregnenolone neurosteroids of the present invention 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, —CHA, 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 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 C-C alkyl, optionally substituted C-C alkoxyl (e.g., methoxyl), or optionally substituted heteroalkyl; R 8 and R 9 are each independently selected from the group consisting of hydrogen, C-C alkyl (e.g., methyl), halogenated C-C alkyl (e.g., trifluoromethyl), or C-C alkoxyl (e.g., methoxyl), or R 8 and R 9 are bonded to each other to form an oxo group, R 10 is hydrogen, hydroxyl, optionally substituted alkyl, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted arylalkyl, where each alkyl is C-C 10 alkyl, C3-C6 cycloalkyl, (C3-C6 cycloalkyl)C1-C4 alkyl, optionally containing a single bond replaced with a double or 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.

[0099] a) Ganaxolone Ganaxolone (CAS Reg. No. 38398-32-2, 3α-hydroxy-3β-methyl-5α-pregnan-20-one) is a 3β-methylated synthetic analog of allopregnanolone, an endogenous allosteric modulator of CNS GABAA receptors. The structural formula of ganaxolone is: [ka]

[0100] Ganaxolone exhibits efficacy and potency comparable to allopregnanolone in activating synaptic and extrasynaptic GABAA receptors at sites distinct from those of benzodiazepines and barbiturates (Carter 1997). Ganaxolone also exhibits protective effects in various rodent seizure models (Reddy 2012, Bialer 2010). Clinical trials have demonstrated that ganaxolone possesses anticonvulsant properties with an acceptable safety and tolerability profile in adults and children at doses ranging from 900 to 1800 mg (Sperling 2017, Laxer 2000, Kerrigan 2000, Pieribone 2007). Furthermore, ganaxolone reduces seizures in ischemic stroke and refractory pediatric epilepsy. In an open-label (OL) study, pediatric patients aged 2 to 60 months with refractory seizures and a history of IS were treated with ganaxolone at doses up to 36 mg / kg for up to 3 months (Kerrigan, 2000). Sixteen of 20 patients completed treatment, 15 of whom had a history of IS. Five of the 15 patients experienced a 50% or greater reduction in seizure count from baseline, five experienced a 25-50% reduction, and five experienced a <25% reduction. One patient was seizure-free, and one non-responder (<25% reduction) experienced seizure resolution by weeks 2 to 7.

[0101] In addition to its anticonvulsant effects, ganaxolone has been shown to reduce anxiety, hyperactivity, and attention in children with fragile X syndrome (Ligsay, 2016). Similar behavioral problems are also observed in individuals with TSC, with rates of ADHD and autism at approximately 50% (Mich & Sahin, 2014). Therefore, ganaxolone treatment may enhance GABAA-mediated signaling to improve seizure control as well as behavioral abnormalities in individuals with TSC and TSC-associated epilepsy.

[0102] Ganaxolone has the same core chemical structure as allopregnanolone, but with the addition of a 3β-methyl group designed to prevent conversion back to the active entity at nuclear hormone receptors, thereby increasing the bioavailability of the neurosteroid and maintaining desired CNS activity while eliminating the opportunity for undesired hormonal effects.

[0103] Similar to allopregnanolone, ganaxolone (a neuroactive steroid) exhibits potent antiepileptic, anxiolytic, sedative, and hypnotic activity in animals through allosteric modulation of gamma-aminobutyric acid type A (GABAA) receptors in the central nervous system (CNS). Ganaxolone exhibits potency and efficacy comparable to allopregnanolone in activating synaptic and extrasynaptic GABAA receptors at sites distinct from benzodiazepine sites.

[0104] Ganaxolone binds both synaptic and extrasynaptic GABA receptors at binding sites unique to this class. A Outside the synapse, ganaxolone is absorbed and can diffuse across the cell membrane, blocking extrasynaptic GABA receptors. A It activates receptors to achieve stable or sustained regulation of GABA inhibitory signals that calm overexcited neurons.

[0105] Ganaxolone is insoluble in water, with solubilities of 13 mg / mL, 3.5 mg / mL, and 3.1 mg / mL in 95% alcohol, propylene glycol, and polyethylene glycol, respectively.

[0106] Ganaxolone is primarily metabolized by the CYP3A family of hepatic enzymes, but interactions based on hepatic metabolism are limited to interactions with other agents, such as ketoconazole, that result in induction or inhibition of CYP3A4 / 5.

[0107] In vitro, ganaxolone clearance appears to be primarily driven by CYP3A4. In adult clinical trials, grapefruit administration increased ganaxolone exposure in healthy volunteers. Ganaxolone levels were decreased in patients concomitantly treated with enzyme-inducing AEDs. These data further support the hypothesis that CYP3A4 significantly contributes to ganaxolone clearance in humans.

[0108] 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 a fed or fasted state.

[0109] In the pediatric population, levels of CYP3A4 expression, although highly variable between individuals, approach adult levels by approximately 2 years of age (de Wildt et al, 2003). Therefore, it is likely that patients older than 2 years of age have a clearance rate of ganaxolone similar to that of adults.

[0110] 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 a T max Ganaxolone has a short onset time, meaning that therapeutic blood levels are reached rapidly. Therefore, an initial bolus dose (loading dose) may not be required, which represents an advantage over other treatments. Ganaxolone is useful in treating seizures in adult and pediatric patients with epilepsy.

[0111] 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 result in the tolerance observed 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, so it does not activate progesterone receptors, either directly or indirectly via metabolic conversion.

[0112] Intravenous ganaxolone has also been evaluated 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.

[0113] In addition to its anticonvulsant activity, ganaxolone has demonstrated anxiolytic properties and improved behaviors associated with autism. In a mouse model of posttraumatic stress disorder (PTSD), treatment with ganaxolone 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 trial 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).

[0114] Ganaxalone did not interact with the human delayed rectifier potassium channel 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.

[0115] Physiologically normal shortening of the PR and QT intervals was observed in response to elevated heart rates. No effects on the QRS duration or QTc interval were observed. No pulmonary effects were observed in female rats at doses up to 40 mg / kg.

[0116] Ganaxalone induces the major cytochrome P450 (CYP) isozymes 1A1 / 2 and 2B1 / 2 in female rats but not in male rats, and autoinduction has been observed in mice and rats, but not in dogs.

[0117] Tissue distribution studies in mice and rats have shown that 14 C]-ganaxolone was rapidly distributed throughout the body to highly perfused organs, the intestine, and adipose tissue, and brain ganaxolone concentrations were approximately 5-fold higher than those in plasma.

[0118] In all species, the majority of radioactivity excreted is via the feces (>70%), with the remainder excreted in the urine.

[0119] In toxicology studies, the most common effect after treatment with ganaxalone was an increase in GABA ADose-related sedation was observed, a pharmacological effect expected for 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.

[0120] Long-term oral toxicity studies in dogs have shown that C > 1500 ng / mL (10 and 15 mg / kg / day) max Mean levels of α-glucan were associated with increased body weight and total plasma cholesterol levels.

[0121] Following intravenous administration to rats and dogs, the primary dose-limiting toxicity finding was sedation. In rats, the no-observed-adverse-effect level (NOAEL) was established at 42 mg / kg / day in males and 30 mg / kg / day in females after 14 days of intravenous administration. In dogs, the NOAEL after ganaxolone administration by IV bolus followed by 28 days of continuous IV infusion 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.

[0122] Ganaxalone was not teratogenic in rats or mice and did not significantly affect the development of the offspring. Ganaxalone did not affect fertility or early embryonic development in rats. No mutagenic potential was detected. Treatment of neonatal rats with ganaxalone produced the expected signs of sedation but did not affect development or reveal any postmortem changes.

[0123] b) Allopregnarone Allopregnanolone (CAS Registry Number 516-54-1, 3α,5α-tetrahydroprogesterone) is an endogenous progesterone derivative with anticonvulsant properties. [ka]

[0124] Allopregnanolone has a relatively short half-life of approximately 45 minutes in human plasma.

[0125] Allopregnanolone exhibits potent antiepileptic, anxiolytic, sedative, and hypnotic activities in animals due to its GABAA receptor modulating activity.

[0126] In addition to its effectiveness in treating stroke, allopregnanolone is being evaluated for use in the treatment of neurodegenerative diseases such as 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).

[0127] The relationship between progesterone, its metabolite allopregnanolone, and seizures has been extensively studied in women with catamenial epilepsy, a condition characterized by changes in seizure frequency associated with different phases of the menstrual cycle. Seizures tend to be more likely at times during the menstrual cycle when progesterone levels are low (e.g., perimenopause) (French, 2005). Circulating allopregnanolone levels correspond to progesterone levels. Progesterone's effects on reproduction are related to its interaction with intracellular progesterone receptors, whereas its anticonvulsant effects are not (Reddy and Rogawski, 2009). The antiseizure activity of progesterone derives from its conversion to the neurosteroid allopregnanolone (Kokate et al., 1999). Allopregnanolone has been shown to protect against seizure activity in several animal models due to its effects on GABAA receptors (Reddy and Rogawski, 2009). Ganaxolone, a synthetic analog of allopregnanolone that lacks progesterone-related effects, may be useful in treating seizures associated with PCDH19-associated epilepsy.

[0128] c) alfaxalone Alphaxalone (also known as alfaxalone) (CAS Reg. No. 23930-19-0, 3α-hydroxy-5α-pregnane-11,20-dione) is an anesthetically active neurosteroid. Alphaxalone is used in veterinary medicine as a general anesthetic. 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]

[0129] d) Afadlon Alphadolone (also known as alfadolone) (CAS Reg. No. 14107-37-0, 3α,21-dihydroxy-5α-pregnane-11,20-dione) is a neurosteroid with anesthetic properties. Its salt, alphadolone acetate, is used in combination with alphaxalone as a veterinary anesthetic. [ka]

[0130] e) Additional neurosteroids Additional neurosteroids that may be used in the nanoparticulate neurosteroid formulations of the present disclosure and in the methods disclosed herein include hydroxydione (CAS Registry Number 303-01-5, (5β)-21-hydroxypregnane-3,20-dione), minaxolone (CAS Reg. No. 62571-87-3, 2β,3α,5α,11α)-11-(dimethylamino)-2-ethoxy-3-hydroxypregnan-20-one), pregnanolone (CAS Reg. No. 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).

[0131] Additional neurosteroids that may be used in the nanoparticulate neurosteroid formulations and methods disclosed herein include Co26749 / WAY-141839, Co134444, Co177843, as well as Sage-217, Sage-324, and Sage-718. Co26749 / WAY-141839, Co134444, Co177843, and Sage-217 have the following structures: [Table 10]

[0132] Additional neurosteroids that may be used in the nanoparticulate neurosteroid formulations of the present disclosure and in the methods disclosed herein include the compounds disclosed in U.S. Patent Application Publication No. 2016-0229887 (U.S. Patent Application No. 14 / 913,920, filed February 23, 2016), the entire contents of which are incorporated herein by reference.

[0133] IV. Dosage The pregnenolone neurosteroid used in the methods disclosed herein 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 can lead to somnolence, and the 1800 mg / day dose defines an optimal combination of drug exposure, dosing convenience, and tolerability.

[0134] When the pregnenolone neurosteroid is ganaxolone, the target and maximum dose of ganaxolone is about 1800 mg / day, which in these embodiments provides the highest achievable exposure based on the nonlinear kinetics of ganaxolone. Therefore, when the pregnenolone neurosteroid is ganaxolone, the amount of ganaxolone administered in the methods of the present invention is generally 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, or 1 mg / kg / day to about 80 mg / kg / day, administered in one, two, three, or four divided doses. In certain embodiments, the target and maximum dose of ganaxolone may be limited, if necessary, depending on the side effect(s) (e.g., somnolence) and higher doses to achieve improved therapeutic efficacy.

[0135] In certain embodiments, about 300 mg to about 2000 mg, 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 orally administered per day for two or more consecutive days (e.g., for a period of one week to 50 years, or for the patient's lifetime). Ganaxolone may be administered orally or parenterally in one, two, three, or four doses per day.

[0136] Whether a person takes ganaxolone twice or three times daily depends on the formulation. For patients receiving oral immediate-release capsules, ganaxolone is typically administered twice daily, with each dose separated by 8 to 12 hours from the next and / or the previous dose. For patients receiving oral suspension, ganaxolone is typically administered three times daily, with each dose separated by 4 to 8 hours from the next and / or the previous dose.

[0137] When the pregnenolone neurosteroid is ganaxolone, the methods of the present invention involve administering ganaxolone at a dose of about 1 mg / kg / day to about 80 mg / kg / day, provided that the total amount of ganaxolone administered does not exceed 2000 mg / day.

[0138] The methods described herein can further include administering a therapeutically effective amount of ganaxolone to achieve a plasma concentration of ganaxolone of about 70% or more greater than 100 ng / ml at 24 hours. For example, the plasma concentration of ganaxolone can be at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more greater than 100 ng / ml at 24 hours.

[0139] To achieve a plasma concentration of 100 ng / ml or greater in about 70% or greater over a 24-hour period, ganaxolone can be administered three or more times per day. Three times per day is preferred, and ganaxolone can be administered three or more times per day as needed or desired to achieve the desired trough concentration of ganaxolone. For example, ganaxolone can be administered three, four, five, six, seven, eight, or more times per day.

[0140] The ganaxolone administered according to the methods disclosed herein can be administered at the same or lower daily doses as those used in clinical trials, and can increase drug exposure by maintaining ganaxolone serum concentrations of at least about 100 ng / ml for at least about 70% of a 24-hour period, for example. A total daily dose of about 1800 mg, about 1700 mg, about 1600 mg, about 1500 mg, or 63 mg / kg / day of ganaxolone can be administered, provided that the total daily dose is administered in three or more doses (preferably containing the same dose of ganaxolone) to produce a serum ganaxolone concentration of at least about 100 ng / ml for at least about 70% of a 24-hour period. Generally, a total daily dose of 1500 mg of ganaxolone can produce a plasma concentration of at least about 100 ng / ml for at least about 70% of a 24-hour period when administered three times a day.

[0141] For example, if ganaxolone is administered at 1500 mg per day, approximately 500 mg can be administered three times per day. For example, if a total of 1800 mg of ganaxolone is administered per day, approximately 600 mg can be administered three times per day. The maximum daily dose of ganaxolone is administered at least three times within a 24-hour period, either in the same dose or in different doses. A skilled clinician will understand that the amount of ganaxolone administered at least three times per day can be adjusted to achieve the desired ganaxolone trough level, as long as the total amount does not exceed the maximum daily dose of ganaxolone.

[0142] A plasma concentration of at least about 100 ng / ml is preferred, although some variation may occur based on, for example, differences in subject weight, metabolism, age, duration of attack, and severity of attack.

[0143] Ganaxolone can be administered orally (e.g., as an oral suspension or oral capsule) or in an intravenous formulation. Preferably, ganaxolone is administered orally. Oral administration includes, but is not limited to, oral suspension formulations and oral capsules.

[0144] A plasma concentration of at least about 100 ng / ml for at least about 70% of a 24-hour period improves seizure reduction and / or seizure suppression. For example, a seizure reduction of at least 20% compared to baseline seizure frequency can be achieved. For example, a seizure reduction of at least 35% compared to baseline seizure frequency can be achieved. Seizure burden and / or frequency can be monitored using EEG.

[0145] 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 doses of 1600 mg / day and little or no increase at doses of 2000 mg / day. Thus, to maintain the highest possible trough levels in all subjects, a dose of 1800 mg is generally targeted, but may be adjusted on an individual basis to provide optimal therapeutic effect.

[0146] In certain embodiments, ganaxolone is administered at a dose of more than 5 mg / kg / day, for example, from about 6 mg / kg / day to about 80 mg / kg / day, provided that the dose does not exceed 1800 mg / day.

[0147] In certain embodiments, the dose of ganaxolone is adjusted during treatment from 15 mg / kg / day to 100 mg / kg / day, up to a maximum dose of 1800 mg / day.

[0148] In certain embodiments, the method of treatment involves administering at least 33 mg / kg / day of ganaxolone in one, two, three, or four doses, with a maximum daily dose of about 1800 mg.

[0149] In certain embodiments, the human is about 0.6 to about 7 years old and is administered between about 1.5 mg / kg twice daily ("BID") (3 mg / kg / day) and 12 mg / kg three times daily ("TID") (36 mg / kg / day). In this embodiment, the human receives a 12 mg / kg TID dosing regimen and achieves a trough concentration of at least about 38.5±37.4 ng / mL.

[0150] In certain embodiments, ganaxolone is administered orally with food in a beta-cyclodextrin formulation at a dose of 6 mg / kg BID (12 mg / kg / day) to 12 mg / kg TID (36 mg / kg / day) to achieve maximum plasma concentrations of ganaxolone of 22.1 ng / mL and 5.7 to 43.7 ng / mL at weeks 4 and 8 of administration, respectively.

[0151] In certain embodiments, ganaxolone is administered orally with food at a dose of 1-12 mg / kg TID (3-36 mg / kg / day) to achieve a maximum ganaxolone plasma concentration of 5.78 ng / mL (1 mg / kg TID) to 10.3-16.1 ng / mL (12 mg / kg TID).

[0152] In certain embodiments, ganaxolone is administered orally in an oral suspension formulation at a dose of 3-18 mg / kg TID (9-54 mg / kg / day) to provide a ganaxolone C of about 123 ng / mL. max and achieve a trough concentration of approximately 23 ng / mL.

[0153] In certain embodiments, the average ganaxolone C min (trough) is 55ng / ml to approximately 100ng / ml, C maxLevels are approximately 240 ng / ml to 400 ng / ml (e.g., 262 ng / mL) based on an oral dose of 1000 mg of ganaxolone three times per day.

[0154] In certain embodiments, these methods provide a mean C based on twice-daily administration of 1000 mg ganaxolone. min (trough) and C max This results in levels of approximately 56.9 ng / ml and approximately 262 ng / mL, respectively.

[0155] In certain embodiments, administration of ganaxolone provides a C of greater than 3, 3.5, 4, 4.5, 5, or 6. min / C max This C provides the ratio min / C max The ratio can be provided after a single dose and / or after steady-state administration. In certain embodiments, regardless of the dose of ganaxolone administered, the C min / C max The ratio remains the same.

[0156] In certain embodiments, the dose administered is determined from a pediatric pharmacokinetic model that achieves similar C values ​​in various pediatric age ranges as those achieved according to effective doses determined in the adult epilepsy population. max and AUC exposure. This model can be constructed in a standard manner, for example, taking into account the pharmacokinetic data in this application.

[0157] 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.

[0158] In certain embodiments, the methods disclosed herein involve establishing a patient's baseline seizure frequency and initially administering a dose of ganaxolone to the patient in an amount of about 0.5 mg / kg / day to about 15 mg / kg / day, 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. For patients weighing 30 kg or less, the total daily dose of ganaxolone may be lower (e.g., about 63 mg / day). 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.

[0159] In certain embodiments, treatment of subjects weighing more than 30 kg begins with a dose of 900 mg / day in divided doses. Thereafter, if reasonably tolerated, the dose is increased by approximately 20-50% at intervals of at least 3 days but not more than 2 weeks (e.g., an increase from 900 mg / day to 1200 mg / day is a 33% increase) until the desired efficacy or maximum tolerated dose (MTD) level is achieved. Subsequent dose adjustments may be made in increments of approximately 20-50%, with a minimum of 3 days between dose changes, unless safety requires. The maximum tolerated dose in these embodiments is 1800 mg / day.

[0160] In certain embodiments, treatment of subjects weighing 30 kg or less begins at 18 mg / kg / day. If the current dose is reasonably tolerated and achieves the desired efficacy or reaches the maximum tolerated dose (MTD) level, the dose may be increased by approximately 20% to 50% at intervals of at least 3 days but not more than 2 weeks. Subsequent dose adjustments may be made by approximately 20% to 50% increments, with a minimum of 3 days between dose changes, unless safety requires. The maximum tolerated dose in these embodiments is 63 mg / day.

[0161] For humans weighing 28 kg (62 lbs) or more, ganaxolone can 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.

[0162] For humans weighing less than 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), and increased by about 15 mg / kg / day each week until 63 mg / kg / day is reached.

[0163] In certain embodiments, ganaxolone is administered as an oral suspension at 10 mg / day to 20 mg / day (e.g., 15 mg / kg / day), increasing to a maximum of 63 mg / kg / day (maximum of 1800 mg / day), or as an oral capsule at 225 mg / day to 900 mg / day (e.g., 450 mg / day). In some of these embodiments, ganaxolone may be administered, for example, as follows: 6 mg / kg three times daily (TID) (18 mg / kg / day) (suspension) / 225 twice daily (BID) (450 mg / day) (capsules) on days 1-7; 11 mg / kg TID (33 mg / kg / day) (suspension) / 450 BID (900 mg / day) (capsules) on days 8-14; 16 mg / kg TID (48 mg / kg / day) (suspension) / 675 BID (1350 mg / day) (capsules) on days 15-21; 21 mg / kg TID (63 mg / kg / day, not to exceed 1800 mg / day) (suspension) / 900 BID (1800 mg / day) (capsules) on days 22-28.

[0164] In certain embodiments, ganaxolone is administered as an oral suspension and the following titration schedule is used: [Table 11]

[0165] In certain embodiments, ganaxolone is administered in capsules and the following titration schedule is used: [Table 12]

[0166] In certain embodiments, trough concentrations associated with maximal efficacy are within the range of about 55 ng / mL, about 60 ng / ml, or about 65 ng / ml (0.3 micron suspension, TID administration), and an 1800 mg / day dose (0.3 micron capsule, BID administration) achieves trough plasma concentrations within this range.

[0167] The methods of treatment disclosed herein include administering a neurosteroid (e.g., ganaxolone) with or without food. In certain embodiments, ganaxolone is administered with food.

[0168] V. Treatment period Treatment periods according to the present invention can range from 1 day to more than 2 years. For example, treatment periods can range from about 1 day to about 80 years, about 1 day to about 70 years, about 1 day to about 60 years, about 1 day to about 50 years, about 1 day to about 45 years, about 2 days to about 45 years, about 2 days to about 40 years, about 5 days to about 35 years, about 10 days to about 30 years, about 10 days to about 30 years, or about 15 days to about 30 years. In some embodiments, treatment periods are as long as the subject continues to derive therapeutic benefit from administration of the neurosteroid (e.g., ganaxolone). 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.

[0169] In certain embodiments, at the end of the treatment period or once treatment is discontinued, the dose is gradually tapered over a period of 1 to 4 weeks based on the subject's age, weight, dose, and duration of treatment.

[0170] VI. Formulations Any desired formulation containing a pregnenolone neurosteroid (e.g., ganaxolone) and one or more pharmaceutically acceptable excipient(s) can be administered according to the methods disclosed herein. The pregnenolone neurosteroid is included in a therapeutically effective amount to treat one or more symptoms of TSC or TSC-associated epilepsy. In certain embodiments, the formulation is free of cyclodextrins, including sulfoalkyl ether cyclodextrins and modified forms thereof.

[0171] In a preferred embodiment, the amount of pregnenolone neurosteroid in the formulation is such that the formulation effectively treats symptoms of TSC-associated epilepsy for a period of, for example, 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 or more.

[0172] In preferred embodiments, the pregnenolone neurosteroid (e.g., 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 most preferred embodiments, the oral formulation is an oral suspension containing the pregnenolone neurosteroid or an oral capsule containing the pregnenolone neurosteroid. Preferably, a unit dose of the oral formulation contains a therapeutically effective amount of the 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 the patient via an oral syringe. For example, the oral suspension is contemplated for use in children weighing less than 30 kg (e.g., about 28 kg). Alternatively, the oral suspension may be administered to patients who would have difficulty swallowing a solid oral dosage form. Children over 30 kg may take a solid dosage form, such as ganaxolone capsules. 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).

[0173] As described in U.S. Patent No. 8,022,054, the liquid formulation can be an aqueous dispersion of stable pregnenolone neurosteroid (e.g., ganaxolone) particles, the particles comprising ganaxolone, a hydrophilic polymer, a wetting agent, and an effective amount of a complexing agent to stabilize initial particle growth and particle growth after an endpoint is reached, 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 volume-weighted median particle 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% (w / w) 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 can be in an amount of about 3% to about 50% (w / w) 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% (in certain embodiments, about 50% to about 80%) based on the weight of the stabilized particles. When the stabilized particles are 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 heat bath at 36°C to 38°C for 1 hour, the volume-weighted mean diameter (D50) does not increase by more than about 150% compared to the volume-weighted mean diameter (D50) of the stabilized particles dispersed in distilled water under the same conditions. In this case, the volume-weighted mean diameter (D50) of the stabilized particles dispersed in SGF or SIF is less than about 750 nm. The stabilized particles, when dispersed in 15 mL of SGF or SIF at a concentration of 0.5 to 1 mg ganaxolone / mL, do not exhibit an increase in volume-weighted median diameter (D50) of more than about 150% compared to the volume-weighted median diameter (D50) of the stabilized particles when dispersed in distilled water under the same conditions. In this case, the volume-weighted median diameter (D50) of the stabilized particles dispersed in SGF or SIF is less than about 750 nm. The complexing agent is selected from the group consisting of parabens, benzoic acid, phenol, sodium benzoate, methyl anthranilate, and the like. The hydrophilic polymer can be a cellulosic polymer, a vinyl polymer, or a mixture thereof.The cellulose-based 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 include 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.

[0174] In some embodiments, a liquid ganaxolone formulation is provided, comprising a pregnenolone neurosteroid (e.g., ganaxolone) formulation 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, when mixed with water, provides 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 rapidly absorbed and ganaxolone particles with larger effective particle sizes are slowly absorbed. 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 distance between ganaxolone-containing particles, extending the drug absorption phase so that 50-80% of the particles are absorbed in the first 3 hours and approximately 90% are absorbed by approximately 10 hours.

[0175] A suspension is "substantially uniform" if it is generally uniform, i.e., if it comprises approximately the same concentration of pregnenolone neurosteroid (e.g., ganaxolone) at any point in the suspension. Preferred embodiments are those that exhibit essentially the same concentration (within 15%) when measured at various points in the aqueous ganaxolone oral formulation after shaking. Particularly preferred are aqueous suspensions and dispersions that maintain uniformity (within a maximum of 15% variation) when measured two hours after shaking. For determining the uniformity of the overall composition, uniformity should be determined by a consistent sampling method. In one embodiment, the aqueous suspension can be resuspended into a uniform suspension by physical agitation lasting less than 1 minute. In another embodiment, the aqueous suspension can be resuspended into a uniform suspension by physical agitation lasting less than 45 seconds. In yet another embodiment, the aqueous suspension can be resuspended into a uniform suspension by physical agitation lasting less than 30 seconds. In yet another embodiment, no agitation is required to maintain a uniform aqueous dispersion.

[0176] 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.

[0177] 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 useful for administering ganaxolone to infants (under 2 years of age), children under 10 years of age, and any patient population unable to swallow or ingest solid oral dosage forms.

[0178] The pregnenolone neurosteroid (e.g., ganaxolone) formulation for oral administration can be an aqueous suspension selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, and syrups. See, e.g., Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002). In addition to ganaxolone particles, the liquid dosage form can include 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 modifiers. In some embodiments, the aqueous dispersion can further include a crystallization inhibitor.

[0179] Examples of disintegrants for use in aqueous suspensions and dispersions include starches, for example 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, for example wood products, microcrystalline cellulose (e.g. Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Examples of suitable crosslinking agents include, but are not limited to, crosslinked starches such as sodium starch glycolate; crosslinked polymers such as crospovidone; crosslinked 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, carob, karaya, pectin, or tragacanth; sodium starch glycolate; bentonite; natural sponges; surfactants; resins such as cation exchange resins; citrus pulp; sodium lauryl sulfate; sodium lauryl sulfate in mixed starches.

[0180] 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; trade name Plasdone®), 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 K100M), sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate stearate, amorphous cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone / vinyl acetate copolymer (Plasdone®, e.g., S-630), ethylene oxide and formaldehyde-containing 4-(1,1,3,3-tetramethylbutyl)-phenol polymer (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 are tetrafunctional block copolymers derived by 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 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® USP2910 (Shin-Etsu)); sodium carboxymethylcellulose; methylcellulose; hydroxyethylcellulose; hydroxypropylmethyl-cellulose phthalate; hydroxypropylmethyl-cellulose acetate stearate; amorphous cellulose; magnesium aluminum silicate; triethanolamine; polyvinyl alcohol (PVA); ethylene oxide and formaldehyde-containing 4-(1,1,3,3-tetramethylbutyl)-phenol polymers; poloxamers (e.g., Pluronics F68®, F88®, and F108®, which are block copolymers of ethylene oxide and propylene oxide; or poloxamine (e.g., Tetronic 908®, also known as poloxamine 908).

[0181] 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., those sold under the trade name Tweens®, e.g., Tween 20® and Tween 80® (ICI Specialty Chemicals)), and 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, sodium docusate, triacetin, vitamin E TPGS, sodium taurocholate, simethicone, phosphotidylcholine, and the like.

[0182] 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 the formulation at concentrations sufficient to inhibit microbial growth. In one embodiment, the aqueous liquid 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 from 0.005% to 0.03% by weight of propylparaben based on the total weight of the aqueous dispersion. In yet another embodiment, the aqueous liquid 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.

[0183] Suitable viscosity enhancing agents for the aqueous suspensions or dispersions described herein include, but are not limited to, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, Plasdone.RTM.S-630, carbomer, polyvinyl alcohol, alginate, acacia, chitosan, and combinations thereof. The concentration of the viscosity enhancing agent will vary depending on the agent selected and the viscosity desired.

[0184] 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, citrus punch, citrus cream, cotton candy, cocoa, cola, cool cherries, cocoa ... Citrus, cyclamate, silamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger, glycyrrhetinic acid salt, licorice (liquorice) syrup, grapes, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glycyrrhizate (MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mint cream, mixed berry, neohesperidin DC, neote Flavoring ingredients include, but are not limited to, raspberry, orange, pear, peach, peppermint, peppermint cream, Prosweet® Powder, raspberry, root beer, rum, saccharin, safrole, sorbitol, spearmint, spearmint cream, strawberry, strawberry cream, stevia, sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfame potassium, mannitol, talc, sucralose, sorbitol, Swiss cream, tagatose, tangerine, thaumatin, tutti frutti, vanilla, walnut, watermelon, wild cherry, wintergreen, xylitol, or any combination of these flavoring ingredients, such as anise and menthol, cherry and anise, cinnamon and orange, cherry and cinnamon, chocolate and mint, honey and lemon, lemon and lime, lemon and mint, menthol and eucalyptus, orange and cream, vanilla and mint, and mixtures thereof. In one embodiment, the aqueous liquid 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 liquid 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 liquid dispersion may contain a sweetening or flavoring agent at a concentration ranging from about 0.0001% to 0.1%, from about 0.001% to about 0.01%, or from 0.0005% to 0.004% by weight of the aqueous dispersion.

[0185] In addition to the additives listed above, the liquid pregnenolone neurosteroid (e.g., ganaxolone) may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers.

[0186] In some embodiments, the pharmaceutical pregnenolone neurosteroid (e.g., ganaxolone) formulations described herein may be self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase within another, usually in the form of droplets. Emulsions are generally created by strong mechanical dispersion. In contrast to emulsions or microemulsions, SEDDSs spontaneously form emulsions when added to excess water without any external mechanical dispersion or agitation. An advantage of SEDDSs is that only gentle mixing is required to disperse the droplets throughout the solution. Additionally, the addition of water or an aqueous phase immediately prior to administration ensures the stability of unstable or hydrophobic active ingredients. Therefore, SEDDSs provide an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. SEDDSs can improve the bioavailability of hydrophobic active ingredients. Methods for making self-emulsifying dosage forms are known in the art, for example, but not limited to, U.S. Pat. Nos. 5,858,401, 6,667,048, and 6,960,563, each of which is incorporated herein by reference.

[0187] 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 docusate, 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.

[0188] In certain preferred embodiments, the liquid pharmaceutical formulation contains ganaxolone, hydroxypropylmethylcellulose, polyvinyl alcohol, sodium lauryl sulfate, simethicone, methylparaben, propylparaben, sodium benzoate, citric acid, and sodium citrate, and has a pH of 3.8 to 4.2. The suspension may contain ganaxolone at a concentration of 50 mg / ml. The formulation may further contain a pharmaceutically acceptable sweetener (e.g., sucralose) and / or a pharmaceutically acceptable flavoring agent (e.g., cherry). The formulation may be packaged in, for example, 120 mL, 180 mL, 240 mL, or 480 mL bottles.

[0189] In certain preferred embodiments, oral solid formulations are prepared using those described in applicant's prior patent, U.S. Patent No. 7,858,609, entitled "Solid Ganaxolone Formulations and Methods for the Making and Use Thereof," the entire contents of which are incorporated by reference. Oral solid dosage forms (e.g., oral capsules or tablets) of pregnenolone neurosteroids may be prepared according to any suitable method.

[0190] For example, as described in U.S. Patent No. 7,858,609, an oral solid formulation can include stabilized particles containing a pregnenolone neurosteroid (e.g., ganaxolone), a hydrophilic polymer, a wetting agent, and an effective amount of a complexing agent to stabilize initial particle growth and particle growth after an endpoint is reached, the complexing agent being a small organic molecule 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, and the complexing agent being present in an amount of about 0.05% to about 5% (w / w) based on the weight of the solid particles. The hydrophilic polymer can be present in an amount of about 3% to about 50% (w / w) based on the weight of the solid particles. The wetting agent can 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 comprise about 10% to about 80% (in certain embodiments, about 50% to about 80%) of the stabilized particles by weight. When the stabilized particles are 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 heat bath at 36°C to 38°C for 1 hour, the volume-weighted mean diameter (D50) of the stabilized particles does not increase by more than about 150% compared to the D50 of the stabilized particles dispersed in distilled water under the same conditions. In this case, the volume-weighted mean diameter (D50) of the stabilized particles dispersed in SGF or SIF is less than about 750 nm. The stabilized particles, when dispersed in 15 mL of SGF or SIF at a concentration of 0.5 to 1 mg ganaxolone / mL, do not increase the volume-weighted median diameter (D50) of the stabilized particles by more than about 150% compared to the volume-weighted median diameter (D50) of the stabilized particles when dispersed in distilled water under the same conditions. In this case, the volume-weighted median diameter (D50) of the stabilized particles dispersed in SGF or SIF is less than about 750 nm. The solid stabilized particles can be combined with any excipient 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 can be paraben, benzoic acid, phenol, sodium benzoate, methyl anthranilate, etc. The hydrophilic polymer can be a cellulosic polymer, a vinyl polymer, or a mixture thereof.The cellulose-based polymer can be a cellulose ether, such as hydroxypropylmethylcellulose. The vinyl polymer can be polyvinyl alcohol, such as vinylpyrrolidone / vinyl acetate copolymer (S630). The wetting agent can be sodium lauryl sulfate, a pharmaceutically acceptable salt of docusate, and mixtures thereof. When the particles are incorporated into a solid dosage form, the solid dosage form can further comprise at least one pharmaceutically acceptable excipient, such as an ionic dispersion modifier, a water-soluble spacer, a disintegrant, a binder, a surface-active ingredient, a plasticizer, a lubricant, a diluent, and any combination or mixture thereof. The water-soluble spacer can be a sugar or an ammonium salt, such as fructose, sucrose, glucose, lactose, or mannitol. The surface-active ingredient can be, for example, a polysorbate. The plasticizer can be, for example, polyethylene glycol. The disintegrant can be cross-linked sodium carboxymethylcellulose, crospovidone, a mixture thereof, or the like.

[0191] Capsules can be prepared, for example, by placing the bulk blended pregnenolone neurosteroid (e.g., ganaxolone) formulations described herein 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 non-gelatin capsule, such as a standard gelatin capsule or an HPMC-containing capsule. In other embodiments, the ganaxolone formulation is placed inside a sprinkle capsule, where the capsule can be swallowed whole or opened and the contents sprinkled on food before eating. The therapeutic dose can be 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.

[0192] Preferably, each capsule contains about 200 to about 600 mg of ganaxolone, about 300 to about 600 mg of ganaxolone, about 400 to about 600 mg of ganaxolone, about 500 to about 600 mg of ganaxolone, about 200 mg of ganaxolone, about 250 mg of ganaxolone, about 300 mg of ganaxolone, about 500 mg of ganaxolone, or about 600 mg of ganaxolone.

[0193] In certain embodiments, each capsule contains either 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. In some embodiments, the capsule is size 00.

[0194] Alternatively, the oral dosage forms of the present invention may be in the form of controlled release dosage forms such as those described in US Pat. No. 7,858,609.

[0195] Pregnenolone neurosteroid (e.g., ganaxolone) formulations suitable for use in the present invention may also be administered parenterally. In such embodiments, formulations are suitable for intramuscular, subcutaneous, or intravenous injection and may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous 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 may be dissolved at a concentration of less than 1 mg / ml using water-soluble beta-cyclodextrins (e.g., beta-sulfobutyl-cyclodextrin and 2-hydroxypropyl beta-cyclodextrin). Particularly suitable cyclodextrins are substituted β-cyclodextrins, such as 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 also 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. It may be desirable to include isotonic agents such as sugars and sodium chloride. The use of agents that delay absorption, such as aluminum monostearate and gelatin, can prolong drug absorption from injectable pharmaceutical forms. Ganaxolone suspension formulations designed for sustained release via subcutaneous or intramuscular injection can avoid first-pass metabolism, and low doses of ganaxolone are 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 formulation and the rate of dissolution in fat or muscle.

[0196] Particularly useful injectable formulations are disclosed in commonly-assigned U.S. Patent Application Publication No. 2017 / 0258812 (U.S. Patent Application No. 15 / 294,135, filed October 14, 2016), the entire contents of which are incorporated herein by reference. Other useful injectable formulations of pregnenolone neurosteroids known to those of skill in the art can also be used.

[0197] VII. Combination Therapy The present disclosure includes embodiments in which the neurosteroid is the only active ingredient, as well as embodiments in which the neurosteroid is administered in combination with one or more additional active ingredients. When used in combination with additional active ingredients, the neurosteroid and the additional active ingredients 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 ingredient (co-administration), or may be administered before or after the additional active ingredient (sequential administration).

[0198] The present disclosure includes embodiments in which the additional active ingredient is an anticonvulsant, including GABA receptor modulators, sodium channel blockers, GAT-1 GABA transporter modulators, GABA transaminase modulators, voltage-dependent calcium channel blockers, and peroxisome proliferator-activated alpha modulators.

[0199] The present disclosure includes embodiments in which a patient is administered an anesthetic or sedative in combination with a 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 induce medical coma or a concentration effective to induce general anesthesia. The anesthetic or sedative may be administered at a low dose that is effective for sedation but not sufficient to induce loss of consciousness.

[0200] Benzodiazepines are used as both anticonvulsants and anesthetics. Benzodiazepines useful as anesthetics include diazepam, flunitrazepam, lorazepam, and midazolam.

[0201] 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 (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.

[0202] The injectable formulations of neurosteroid nanoparticles of the present disclosure may be administered in conjunction with another anticonvulsant, which comprises several drug classes and overlaps to some extent with the coma-inducing drugs, anesthetics, and sedatives that may be used in combination with neurosteroids.Anticonvulsants that may be used in combination with the injectable formulations of neurosteroid nanoparticles of the present disclosure include aldehydes, such as paraldehyde; aromatic allylic alcohols, such as stiripentol; barbiturates (including those listed above, as well as methylphenobarbital and barbexaclone); benzodiazepines (alprazolam, bretazenil, bromazepam, brotizolam, chloridazepoxide, cinolazepam, clonazepam, colazepate, clopazam, clotiazepam, cloxazolam, delorazepam, diazepam, enezolan, phenazepam ... stazolam, 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 acetic acid eslicarbazepine; 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.

[0203] VIII. Biomarkers Predictive biomarkers are used to identify patient populations that are more homogeneous and more likely to respond to treatment.

[0204] Allopregnanolone, a metabolite of progesterone, is a positive allosteric modulator (PAM) of the GABA receptor. Humans with TSC-associated epilepsy exhibit this allopregnanolone deficiency, which may support the hypothesis that treatment with a pharmaceutically acceptable pregnenolone (such as ganaxolone) may reduce seizure frequency and possibly ameliorate further symptoms of TSC-associated epilepsy.

[0205] 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. -1 Allo-S plasma levels below 2,500 pg mL indicate that a subject is likely to respond to and benefit from ganaxolone therapy. -1 An Allo-S plasma level above 0.05 indicates that the subject is unlikely to respond to ganaxolone therapy and that a different therapeutic agent should be used. [Example]

[0206] 5. Working Example The formulations in the following examples of the present invention should not be construed as limiting the invention in any way, but rather as merely samples of the various formulations described herein.

[0207] During the development of ganaxolone formulations, various formulations were evaluated to establish one that exhibited adequate pharmacokinetic ("PK") parameters and was suitable for development and commercialization. Other formulations of ganaxolone used included ganaxolone mixed with sodium lauryl sulfate and 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 sulfobutylether cyclodextrin (Captisol®) to solubilize ganaxolone. Development efforts 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.

[0208] Example 1 A 50 mg / ml ganaxolone suspension was prepared having the ingredients set forth in Table 1 below. [Table 1]

[0209] Table 2 shows the function of the excipients used in the 50 mg / ml ganaxolone suspension. [Table 2]

[0210] 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.

[0211] A dispersion nanomilling process was used to reduce the particle size of ganaxolone to obtain stable ganaxolone nanoparticles. The nanomilling process involved the use of yttria-stabilized zirconia (YTZ) milling media under high-energy agitation in the 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 was 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) were added to promote controlled growth at room temperature for a 5-10 day curing period to approximately 300 nm. The stabilized 300 nm nanoparticles exhibited good stability against particle growth in pediatric suspension and encapsulated pharmaceutical composition formats. The stabilization process was controlled by precisely adding and dissolving the water-soluble stabilizer paraben. The hardening process was controlled by adjusting the holding time and temperature of the stabilized dispersion prior to suspension dilution (in the case of the 50 mg / ml ganaxolone suspension) or fluidized bed bead coating (in the case of the 225 mg ganaxolone capsules described in Example 2).

[0212] The three dispersion batches prepared in the dispersion nanomilling scale-up study were diluted and stabilized with the addition of sodium methylparaben, sodium benzoate, and anhydrous citric acid, and then cured for 7 days. After curing, the particle sizes were measured and are shown in Table 3. [Table 3]

[0213] As shown here, the D(50) particle size was stabilized within the range of 250–450 nm.

[0214] Example 2: Ganaxolone capsules (225 mg) were prepared having the ingredients set forth in Tables 4 and 5 below. [Table 4]

[0215] Table 5 summarizes the functions of the excipients used in the 225 mg ganaxolone capsule formulation. [Table 5]

[0216] The manufacturing process used to prepare these capsules utilizes the same pharmaceutical composition specifications and quantitative composition, as well as the same nanomilling dispersion dilution and dispersion stabilization processes. Thus, the product of Example 2 utilizes the same stabilized dispersion intermediate as the product of Example 1. Methylparaben sodium can be substituted for methylparaben.

[0217] Table 6 summarizes the results of formal stability data for ganaxolone immediate-release (IR) 225 mg capsules over a 36-month period. [Table 6]

[0218] Example 3 Summary of previous epilepsy clinical trials in TSC-related epilepsy [Table 13]

[0219] Example 4 A Phase 2 clinical trial of ganaxolone for TSC-associated epilepsy will be conducted. Approximately 30 male and / or female patients, ages 2 to 65 years (inclusive), with TSC-associated epilepsy will be screened and enrolled. These patients will have a confirmed clinical diagnosis of TSC and a mutation in either the TSC1 or TSC2 gene. Patients will keep a daily diary of the effect of ganaxolone on their seizures.

[0220] The treatment phase includes two phases: Part A and Part B. In Part A, patients receive ganaxolone for a total of 12 weeks (4 weeks of titration, 8 weeks of maintenance) in addition to standard antiepileptic treatment. Patients are titrated to 63 mg / kg / day (maximum 1800 mg / day) over 4 weeks, then maintained at that dose for an additional 8 weeks. Ganaxolone is administered as an oral suspension with food in 15 mg / kg / day increments up to 63 mg / kg / day. Patients weighing 28 kg or less are administered on a mg / kg basis. Patients weighing more than 28 kg are administered a fixed regimen in 450 mg / day increments up to 1800 mg / day. Ganaxolone is administered as follows over the 4-week titration period: [Table 14]

[0221] Patients who do not tolerate the next dose step remain at the lower dose step for an additional number of days before moving on to the next dose. If the next dose is still not tolerated, patients can return to the next lower dose step. After the escalation period between maintenance periods, a minimum dose of 33 mg / kg / day or 900 mg / day is generally required unless the sponsor agrees to the lower dose due to tolerability issues, such as somnolence.

[0222] Dose modifications, including alternative dosing paradigms (e.g., lower doses during the day and higher doses in the evening), will be discussed with the sponsor's medical monitoring within 48 hours before and after making the modifications. Patients who discontinue ganaxolone treatment before completing the maintenance period in Part A will continue to be followed up per protocol and will be encouraged to continue keeping a daily seizure diary at least until the completion of Part A. These patients will also be seen 2 weeks after completing the diary for a safety follow-up assessment.

[0223] Patients who continue treatment with ganaxolone in the OLE phase (Part B) of the study who achieve a 35% or greater reduction in seizure frequency during the 12-week treatment period in Part A (e.g., a 4-week baseline period) compared to baseline and who have no other contraindications to continued treatment may continue into Part B ("OLE eligible"). Part B is an open-label extension study lasting approximately 24 weeks. Therefore, Part B is open to patients who respond to ganaxolone as defined by the protocol. The main differences between Parts A and B are the duration of treatment, less frequent assessments, and the ability to modify medication dosage (both ganaxolone and other AED treatment, including initiation and discontinuation of other medications) based on evaluation of the patient's clinical progress.

[0224] In Part B, ganaxolone patients will continue ganaxolone treatment at the dose they received at the completion of Part A. During Part B, patients weighing more than 28 kg may have their ganaxolone dose adjusted to a maximum of 600 mg TID, and patients weighing 28 kg or less may have their ganaxolone dose adjusted to a maximum dose of 21 mg / kg TID. Doses of other antiepileptic drugs may be adjusted during Part B (including tapering and treatment initiation) based on the investigator's discretion.

[0225] Patients who complete Part A and do not continue in Part B, or who complete Part B or discontinue GNX treatment, will undergo a 2-week drug de-escalation period and return to the study site for a safety follow-up visit after 2 weeks, unless medically contraindicated.

[0226] Patients who complete Part A and are deemed eligible for Part B will continue to receive ganaxolone at the same dose as when they completed Part A for an additional 24 weeks of treatment.

[0227] Patients may receive a lower dose during the day and a higher dose in the evening.

[0228] Those discontinuing ganaxolone should taper gradually over 2 weeks unless there is a medical contraindication, such as a drug-induced rash.

[0229] Patients who discontinue ganaxolone before completing the maintenance treatment period will be followed according to the protocol, and patients are encouraged to continue keeping a daily seizure diary until at least the completion of the treatment period in Part A. These patients will return to the study site for a follow-up safety assessment 2 weeks after tapering.

[0230] Patients may return for a 2-week follow-up safety check (e.g., due to early discontinuation of Part A or B, not participating in Part B, or completing Part B).

[0231] The primary efficacy endpoint is to assess the potential of ganaxolone in TSC-associated epilepsy over 12 weeks and / or the percent change in primary seizure frequency by the end of the 12-week treatment (titration and maintenance) period in Part A. Cardinal seizure types include focal motor seizures without impairment of consciousness or awareness, focal seizures with impairment of consciousness or awareness, focal seizures resulting in bilateral generalized convulsive seizures, and generalized seizures with countable motor elements.

[0232] Secondary objectives are to evaluate the safety and tolerability of ganaxolone as adjunctive therapy through the 12-week treatment (titration and maintenance) endpoint in Part A; to evaluate pharmacokinetic (PK) parameters in patients receiving ganaxolone doses up to 63 mg / kg / day (or up to 1800 mg / day) throughout the study; to evaluate pharmacokinetic (PK) parameters in patients receiving ganaxolone doses up to 63 mg / kg / day (or up to 1800 mg / day) throughout the study; and to evaluate the long-term safety and tolerability of GNX when administered as adjunctive therapy throughout Part B.

[0233] Exploratory objectives are to assess changes in quality of life, to assess behavioral / neuropsychiatric changes in patients receiving ganaxolone as adjunctive therapy through the endpoint of the 12-week treatment (titration and maintenance) period in Part A, to evaluate the relationship between efficacy response to ganaxolone and biomarker levels (e.g., neurosteroids), to evaluate the potential effect of ganaxolone on EEG activity, to evaluate changes in other types of seizures (non-primary) in TSC, to evaluate the effect of ganaxolone on primary seizure-free days, to evaluate the effect of ganaxolone on infantile / infantile spasms-free days, and to evaluate the effect of ganaxolone on infantile / infantile spasms-free days.

[0234] A. Pharmacokinetic Assessment: The PK population includes all patients who received at least one dose of GNX, had at least one sample collected, and had valid bioanalytical results. These samples were collected 1-5 hours or 4-8 hours after the last dose between Part A and Part B. Pharmacokinetic analysis is limited to a list of concentrations because sufficient concentration-time data are not available for noncompartmental analysis, such as Cmax, AUC, or tmax. Pharmacokinetic data from this study may be used in a population PK analysis, which will be conducted and reported separately from this study.

[0235] B. Neurosteroid serum and concomitant AED levels: Blood samples will be collected at the screening visit, week 12 of Part A, and the final visit of Part B to measure levels of neurosteroids (allopregnanolone and related endogenous CNS-active steroids and sulfated metabolites, such as allopregnanolone sulfate).

[0236] Example 5. Biomarkers Individuals (n = 11) with confirmed PCDH19 mutations and minimal seizure burden were enrolled at six centers in the United States and Italy between May 2015 and November 2015. Percent change in seizure frequency was assessed as the primary endpoint, with responders defined as a ≥25% reduction in seizure rate. 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 not observed to change significantly over time.

[0237] 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 = 433 pg mL -1 Responder analysis and correlation with Allo-S revealed two distinct populations. Plasma Allo-S concentrations in responders (n = 6) (≥ 25% reduction in attack rate) and non-responders (n = 5) were 501 ± 430 pg mL−1, respectively. -1 , and 9,829 ± 6,638 pg mL -1 (mean ± SD, p=0.05, Mann-Whitney).

[0238] Comparing seizure frequency between baseline and 6 months, the biomarker-positive group showed significant improvement (p = 0.02, Wilcoxon), whereas the biomarker-negative (high Allo-S) group showed no improvement or significant worsening (p = 0.25, Wilcoxon). -1 ) and biomarker-negative (n=4, Allo-S>2,500pg mL -1A retrospective analysis of the 6-month and 6-month controls showed a median change in seizure rate of -53.9% and 247%, respectively (p=0.006, Mann-Whitney). Furthermore, when comparing seizure frequency between baseline and 6 months, the biomarker-positive group significantly improved (p=0.02, Wilcoxon signed rank), while the biomarker-negative group did not significantly worsen (p=0.25, Wilcoxon signed rank).

[0239] Example 6. Case Report 1 of a Subject with TSC Enrolled in Part A of an Open-Label Phase 2 Study A subject with tuberous sclerosis complex (Subject 001) was enrolled in Part A of an open-label, Phase 2 study of adjunctive ganaxolone (GNX) treatment in tuberous sclerosis complex-associated epilepsy, according to the study protocol described in Example 4. The subject's baseline seizure burden was 132.41 per 28-day period. Ganaxolone was administered orally three times daily at a maximum daily dose of 1800 mg for 11 weeks (78 days). The subject completed the protocol and experienced a 64% reduction in seizures compared to baseline. This is the first subject to complete the protocol. Additional subjects are currently being enrolled but have not completed the study. [Table 8]

[0240] Example 7. Preliminary Pharmacokinetic and Pharmacodynamic (PK / PD) Analysis A preliminary PK / PD analysis was performed to investigate the presence or absence of a relationship between ganaxolone levels and the percentage change in grand motor seizure frequency.

[0241] a) Study design This global, randomized, double-blind, placebo-controlled phase 3 clinical trial evaluated the safety and efficacy of adjunctive ganaxolone for the treatment of seizures associated with CDD. Patients aged 2-21 years with a pathogenic or likely pathogenic mutation in the CDKL5 gene, a neurodevelopmental disorder, and at least 16 seizures per 28 days during the 2 months prior to screening and at least two refractory seizures to prior antiepileptic drug treatment were eligible for enrollment. The study consisted of a 6-week baseline period followed by a 17-week double-blind phase (ganaxolone or placebo, 1:1). The dose of ganaxolone 50 mg / mL suspension was titrated over 4 weeks to 63 mg / kg / day (21 mg / kg TID), not to exceed 1800 mg / day (600 mg TID), or to the maximum tolerated dose. Blood samples for PK analysis were scheduled to be taken at Visit 3 (Week 5), Visit 4 (Week 9), and Visit 5 (Week 17).

[0242] b) Method The mean ganaxolone concentration was calculated for each subject using available results from up to three laboratory measurements during the double-blind phase. Linear regression was performed on the arithmetic and natural log-transformed percent reduction (log e Regression diagnostics were performed using the mean ganaxolone concentration ([percent reduction + 100]) as the dependent variable and the natural log-transformed mean ganaxolone concentration as the single explanatory variable. Regression diagnostics included inspection of residual plots and normal probability plots, and determination of outliers and influence values. Cases with standardized residuals greater than 2 or less than -2 were removed from the model, and the regression was repeated.

[0243] The resulting samples were used to determine the Pearson correlation coefficient (using log-transformed values). Additionally, the Kruskal-Wallis test was used to compare the percentage reduction in seizures across three tertiles of mean ganaxolone concentrations per subject: low (N=13), medium (N=13), and high (N=12).

[0244] The number of CNS-related adverse events suggestive of potential dose-related toxicity (somnolence, sedation, lethargy, impaired attention, drooling, and hypotonia) in ganaxolone-treated participants, as well as the onset and duration of the events, were tabulated, and the number of participants who had CNS adverse events during each week of the double-blind phase was calculated.

[0245] c) Result Forty-four participants with seizure reduction data had at least one plasma ganaxolone level measurement (mean + standard deviation = 103.5 + 79.2 ng / mL). In a linear regression using the rate of seizure reduction as the dependent variable and the mean plasma ganaxolone concentration as the independent variable, six cases were determined to be outliers due to adjusted residuals >2 or <-2. When the linear regression was repeated excluding these cases (N = 38), the adjusted R 2 The correlation coefficient between mean plasma ganaxolone concentrations and percent reduction in grand mal motor seizures was 0.227 (F(1,36) = 1.89), p = 0.001). Using the same samples, the correlation coefficient between mean plasma ganaxolone concentrations and percent reduction in grand mal motor seizures was -0.499 (p = 0.001). A robust regression was performed including all observations (N = 44) that replicated the results of this analysis.

[0246] The mean and median percentage reductions in grand mal motor seizures were calculated for the low, middle, and high tertiles of ganaxolone concentrations (Table 9). A statistically significant difference was found between the groups in terms of the percentage reduction in grand mal motor seizure frequency (H(2) = 9.087, p = 0.011) (Figure 2). Post-hoc pairwise comparisons of the sample distributions of the three groups revealed a statistically significant difference between the low GNX level group and the high GNX level group, but not between the other groups. [Table 9]

[0247] In summary, a negative correlation was observed between the logarithm of plasma ganaxolone levels and the percentage change in grand motor seizure frequency. Increased plasma GNX levels were associated with a significant reduction in seizure frequency in patients with CDKL5 deficiency disorder (CDD), ranging from 27 to 333 ng / mL. Back-transformation of the logarithm predicted that a plasma concentration of approximately 100 ng / mL (mean for the CDD population) would reduce seizures by approximately 40% in participants in this study.

[0248] Modeled PK curves based on previous Phase 1 studies demonstrate that TID administration can increase trough GNX levels compared with BID administration. Preliminary PK / PD analysis results suggest that increased plasma ganaxolone concentrations are associated with improved seizure reduction, with concentrations of approximately 100 ng / mL associated with significant changes in seizure frequency. Based on the modeled PK curves, TID administration can achieve plasma ganaxolone levels greater than 100 ng / mL for approximately 78% of 24 hours compared with 53% achieved using BID administration. While these analyses do not maintain randomization and therefore may not demonstrate a causal role of GNX on changes in seizure frequency, they suggest that TID administration may result in increased antiepileptic effects. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for treating tuberous sclerosis or tuberous sclerosis-associated epilepsy, comprising administering to a subject in need thereof a pharmaceutically acceptable therapeutically effective amount of pregnenolone neurosteroid, or a pharmaceutically acceptable salt thereof. (Item 2) 2. The method of claim 1, wherein the pregnenolone neurosteroid is ganaxolone. (Item 3) 10. The method of any one of the preceding items, wherein the pregnenolone neurosteroid is administered three times daily. (Item 4) 3. The method according to any one of items 1 to 2, wherein the pregnenolone neurosteroid is administered twice daily. (Item 5) The method of any one of the preceding items, wherein the subject is administered about 200 mg / day to about 1,800 mg / day of ganaxolone. (Item 6) 5. The method of any one of items 1 to 4, wherein the subject is administered up to about 1,800 mg / day of ganaxolone. (Item 7) The method of any one of the preceding items, wherein the ganaxolone is administered at about 1,500 mg / day of ganaxolone. (Item 8) The method of any one of the preceding items, wherein ganaxolone is administered in an amount of up to 63 mg / kg / day. (Item 9) 10. The method of any one of the preceding items, wherein the pregnenolone neurosteroid is administered orally. (Item 10) 10. The method of any one of the preceding items, wherein the pregnenolone neurosteroid is administered as an oral suspension. (Item 11) 10. The method of any one of the preceding items, wherein the pregnenolone neurosteroid is administered in an oral capsule. (Item 12) 10. The method of any one of the preceding items, wherein the tuberous sclerosis-associated epilepsy is infantile spasms. (Item 13) 10. The method of any one of the preceding items, wherein the tuberous sclerosis-associated epilepsy is a focal impaired consciousness seizure. (Item 14) 10. The method of any one of the preceding items, wherein the tuberous sclerosis-associated epilepsy is focal seizures. (Item 15) 10. The method of any one of the preceding items, wherein the tuberous sclerosis-associated epilepsy is a generalized seizure. (Item 16) 10. The method of any one of the preceding items, wherein administering the pregnenolone neurosteroid reduces seizure frequency and / or seizure severity in the subject compared to baseline. (Item 17) 10. The method of any one of the preceding items, wherein administering the pregnenolone neurosteroid reduces seizure frequency by about 20% or more compared to baseline seizure frequency. (Item 18) 10. The method of any one of the preceding items, wherein administering the pregnenolone neurosteroid reduces seizure frequency by at least about 35% or more compared to baseline seizure frequency. (Item 19) 10. The method of any one of the preceding items, wherein the subject is monitored by electroencephalogram (EEG). (Item 20) 10. The method of any one of the preceding items, wherein seizure activity in the subject is monitored by electroencephalography (EEG). (Item 21) The method of any one of the preceding items, wherein ganaxolone is administered to the subject in an amount sufficient to provide the subject with a ganaxolone plasma concentration of about 100 ng / mL for at least about 70% of a 24-hour day. (Item 22) 22. The method of claim 21, wherein the ganaxolone is administered three times a day. (Item 23) measuring the level of endogenous neurosteroid in the subject prior to administering the pregnenolone neurosteroid, wherein a low level of endogenous neurosteroid in the subject indicates that the subject will respond to the pregnenolone neurosteroid; and 10. The method of any one of the preceding items, further comprising administering to a subject having low endogenous neurosteroid levels a therapeutically effective amount of the pregnenolone neurosteroid. (Item 24) 24. The method of claim 23, wherein the endogenous neurosteroid is allopregnanolone sulfate. (Item 25) The low level of endogenous neurosteroids is 2500 pg mL -1 25. The method of any one of items 23 or 24, wherein the amount is: (Item 26) 26. The method of claim 25, wherein the pregnenolone neurosteroid is ganaxolone. (Item 27) A method for treating tuberous sclerosis or tuberous sclerosis-associated epilepsy, comprising administering ganaxolone to a subject in need thereof in an amount sufficient to provide the subject with a ganaxolone plasma concentration of at least 100 ng / ml for at least about 70% of 24 hours per day. (Item 28) 28. The method of item 27, wherein ganaxolone is administered three times a day. (Item 29) 29. The method of any one of items 27 or 28, wherein ganaxolone is administered orally. (Item 30) 30. The method of any one of items 27 to 29, wherein ganaxolone is administered as an oral suspension. (Item 31) 30. The method of any one of items 27 to 29, wherein the ganaxolone is administered as an oral capsule. (Item 32) 32. The method according to any one of items 27 to 31, wherein ganaxolone is administered in an amount of up to 63 mg / kg / day. (Item 33) 33. The method according to any one of items 27 to 32, wherein ganaxolone is administered in an amount of up to 1,800 mg / day. (Item 34) 33. The method according to any one of items 27 to 32, wherein ganaxolone is administered in an amount of up to 1,500 mg / day. (Item 35) 35. The method according to any one of items 27 to 34, wherein the tuberous sclerosis-associated epilepsy is infantile spasms, focal impaired consciousness seizures, focal seizures, or generalized seizures. (Item 36) 36. The method of any one of items 27 to 35, wherein administering ganaxolone reduces seizure frequency and / or seizure severity in the subject compared to baseline. (Item 37) 36. The method of any one of items 27 to 35, wherein administration of ganaxolone reduces macromotor frequency in the subject compared to baseline. (Item 38) 36. The method of any one of items 27 to 35, wherein administration of ganaxolone reduces seizure frequency by about 20% compared to baseline seizure frequency. (Item 39) 36. The method of any one of items 27 to 35, wherein administering ganaxolone reduces seizure frequency by at least about 35% compared to baseline seizure frequency. (Item 40) 40. The method according to any one of items 27 to 39, wherein the subject is monitored by electroencephalogram (EEG). (Item 41) 40. The method of any one of items 27 to 39, wherein the subject is monitored for seizure activity by electroencephalography (EEG). (Item 42) 1. A method of treating a subject having or suspected of having epilepsy associated with tuberous sclerosis complex, comprising: determining whether the subject has a low level of endogenous neurosteroids; and The method comprises administering to the subject a therapeutically effective amount of a pharmaceutically acceptable pregnenolone neurosteroid, or a pharmaceutically acceptable salt thereof, if the subject has low levels of endogenous neurosteroids. (Item 43) 43. The method of claim 42, wherein the endogenous neurosteroid is allopregnanolone sulfate and the pregnenolone neurosteroid is ganaxolone. (Item 44) the endogenous neurosteroid is allopregnanolone sulfate, and the low level of endogenous steroid is 2500 pg mL -1 44. The method of any one of items 42 or 43, wherein the level is: (Item 45) The pregnenolone neurosteroid has formula IA:

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Claims

1. A composition for treating tuberous sclerosis or tuberous sclerosis-associated epilepsy in a subject, comprising ganaxolone, or a pharmaceutically acceptable salt thereof, wherein the subject is administered a dose of ganaxolone in an amount of 0.5 mg / kg / day to 15 mg / kg / day, and the dose of ganaxolone is gradually increased over four weeks to an amount of 18 mg / kg / day to 63 mg / kg / day, the total dose of ganaxolone being up to 1800 mg / day for patients weighing more than 30 kg.

2. 10. The composition of claim 1, wherein the composition is administered three times daily.

3. The composition according to any one of claims 1 to 2, characterized in that the composition is administered twice a day.

4. The composition of any one of claims 1 to 3, wherein the subject is administered about 200 mg / day to about 1,800 mg / day of ganaxolone.

5. A composition described in any one of claims 1 to 4, characterized in that the subject is administered approximately 1,500 mg / day of ganaxolone.

6. The composition according to any one of claims 1 to 5, characterized in that the composition is administered orally.

7. The composition according to any one of claims 1 to 6, characterized in that the composition is administered as an oral suspension.

8. The composition according to any one of claims 1 to 6, characterized in that the composition is administered as an oral capsule.

9. 9. The composition of any one of claims 1 to 8, wherein administration of the composition reduces the frequency of seizures and / or the severity of seizures in a subject compared to baseline.

10. 10. The composition of any one of claims 1 to 9, wherein administration of the composition reduces seizure frequency by about 20% or more compared to baseline seizure frequency.

11. The composition of any one of claims 1 to 10, wherein administration of the composition reduces seizure frequency by at least about 35% or more compared to baseline seizure frequency.

12. The composition according to any one of claims 1 to 11, characterized in that the subject is monitored by electroencephalography (EEG).

13. The composition according to any one of claims 1 to 12, characterized in that the seizure activity in the subject is monitored by electroencephalography (EEG).

14. 14. The composition of any one of claims 1 to 13, wherein the ganaxolone is administered to the subject in an amount sufficient to provide the subject with a ganaxolone plasma concentration of about 100 ng / mL for at least about 70% of 24 hours per day.

15. 15. The composition of claim 14, wherein the composition is administered three times daily.

16. 1. A composition for treating tuberous sclerosis or tuberous sclerosis-associated epilepsy in a subject in need thereof, the composition comprising ganaxolone, administered in a therapeutically effective amount to provide a ganaxolone plasma concentration of at least about 100 ng / ml for at least about 70% or more of a 24-hour period per day, wherein the subject is administered a dose of ganaxolone in an amount of 0.5 mg / kg / day to 15 mg / kg / day and the dose of ganaxolone is gradually increased over four weeks to an amount of 18 mg / kg / day to 63 mg / kg / day, the total dose of ganaxolone being up to 1800 mg / day for patients weighing more than 30 kg.

17. 17. The composition of claim 16, wherein the composition is administered three times daily.

18. 18. The composition according to claim 16 or 17, characterized in that the composition is administered orally.

19. The composition according to any one of claims 16 to 18, characterized in that the composition is administered as an oral suspension.

20. The composition according to any one of claims 16 to 18, characterized in that the composition is administered as an oral capsule.

21. Composition according to any one of claims 16 to 20, characterized in that the composition is administered in an amount of up to 1,500 mg / day of ganaxolone.

22. 22. The composition of any one of claims 16 to 21, wherein administration of the composition reduces the frequency of seizures and / or the severity of seizures in a subject compared to baseline.

23. The composition of any one of claims 16 to 21, wherein administration of the composition reduces the frequency of grand motor seizures in a subject compared to baseline.

24. 22. The composition of any one of claims 16 to 21, wherein administration of the composition reduces seizure frequency by about 20% compared to baseline seizure frequency.

25. The composition of any one of claims 16 to 21, wherein administration of the composition reduces seizure frequency by at least about 35% or more compared to baseline seizure frequency.

26. The composition according to any one of claims 16 to 21, characterized in that the subject is monitored by electroencephalography (EEG).

27. The composition according to any one of claims 16 to 21, characterized in that the seizure activity in the subject is monitored by electroencephalography (EEG).

28. The subject has a blood glucose level of 2500 pg mL －１ 22. The composition of any one of claims 1 to 21, having low levels of endogenous neurosteroids:

29. 22. The composition of any one of claims 1 to 21, wherein the subject exhibits at least two major features selected from the group consisting of: at least three hypopigmented macules at least 5 mm in diameter; at least three angiofibromas or at least one forehead plaque; at least two periungual fibromas; at least one charlene patch; multiple retinal hamartomas; cortical atypia including nodules and lines radiating from the white matter; subependymal giant cell astrocytoma; cardiac rhabdomyoma; and lymphangioleiomyomatosis.

30. 29. The composition of claim 28, wherein the endogenous neurosteroid is allopregnenalone sulfate and the level of the allopregnenalone sulfate in the subject is less than 6 ng / ml.

31. The composition of any one of claims 1 to 21, wherein the subject suffers from TSC treatment-resistant epilepsy.

32. 22. The composition of any one of claims 1 to 21, wherein the subject has an additional behavioral condition selected from the group consisting of anxiety, hyperactivity, attention-deficit / hyperactivity disorder (ADHD) and autism, and any combination thereof.

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