WO2024030399A2 - Use of a gaba-a pam for reduction of tactile hypersensitivity - Google Patents

Abstract

The present disclosure provides methods of use of a particular GABA-A modulator compound, including in particular patient populations and/or for particular indications (e.g., diseases, disorders, or conditions).

Description

USE OF A GABA-A PAM FOR REDUCTION OF TACTILE HYPERSENSITIVITY

BACKGROUND

[1] Autism spectrum disorder (ASD) is a highly prevalent class of neurodevelopmental disorders characterized by impairments in social communication and interactions, as well as restricted and repetitive behaviors. Rates of ASD diagnoses are increasing, and the CDC identifies one in every 59 children in the United States as having ASD. In the United States alone, it is estimated that the ASD related healthcare costs exceed 230 billion dollars per year, or 1.4 million per individual with ASD over their lifetime.

SUMMARY

[2] 8-chloro-6-(2-fluorophenyl)-4H-benzo[f]pyrazolo[l,5-a][l,4]diazepine-2-carboxylic acid (Compound 1) is a recently discovered GABA-A PAM “Positive Allosteric Modulator” with low CNS exposure after oral or intravenous dosing.

Compound 1

[3] The present disclosure demonstrates that Compound l is a particularly useful agent for reducing tactile dysfunction. [4] In some embodiments, the present disclosure provides uses of Compound 1 (and/or of compositions that comprise and/or deliver Compound 1) in the treatment of subjects (e.g., human subjects) suffering from or susceptible to a tactile dysfunction condition and/or from a disease, disorder or condition that may be associated with tactile dysfunction.

[5] One aspect of the present disclosure, for example features a method of reducing tactile dysfunction in a subject (e.g., a human) by administering to the subject Compound 1, or a pharmaceutically acceptable salt thereof, for example according to a regimen (e.g., in an amount and for a duration - e.g., by administration of one or more doses) sufficient to reduce the tactile dysfunction. In some embodiments, such human subject has been diagnosed with Autism Spectrum Disorder (ASD), Rett syndrome (RTT), Phelan McDermid syndrome (PMS), or Fragile X syndrome.

[6] In another aspect, the present disclosure, for example features a method of reducing anxiety or social impairment in a subject (e.g., a human) by administering to the subject Compound 1, or a pharmaceutically acceptable salt thereof, for example according to a regimen (e.g., in an amount and for a duration - e.g., by administration of one or more doses) sufficient to reduce the anxiety or social impairment. In some embodiments, such human subject has been diagnosed with ASD, RTT, PMS, or Fragile X syndrome

[7] In another aspect, the present disclosure, for example features a method of reducing touch over-reactivity and/or pain and/or mechanical allodynia in a subject (e.g., a human) by administering to the subject Compound 1, or a pharmaceutically acceptable salt thereof, for example according to a regimen (e.g., in an amount and for a duration - e.g., by administration of one or more doses) sufficient to reduce the touch over-reactivity and/or pain and/or mechanical allodynia. In some embodiments, such human subject has been diagnosed with ASD, RTT, PMS, or Fragile X syndrome.

[8] In another aspect, the present disclosure, for example features a method of reducing tactile dysfunction in a subject (e.g., a human) diagnosed with Autism Spectrum Disorder (ASD), Rett syndrome (RTT), Phelan McDermid syndrome (PMS), or Fragile X syndrome by administering to the subject Compound 1, or a pharmaceutically acceptable salt thereof, for example according to a regimen (e.g., in an amount and for a duration - e g., by administration of one or more doses) sufficient to reduce the tactile dysfunction. [9] In another aspect, the present disclosure, for example features a method of reducing anxiety or social impairment in a subject (e.g., a human) diagnosed with ASD, RTT, PMS, or Fragile X syndrome by administering to the subject Compound 1, or a pharmaceutically acceptable salt thereof, for example according to a regimen (e.g., in an amount and for a duration - e.g., by administration of one or more doses) sufficient to reduce the anxiety or social impairment.

[10] In another aspect, the present disclosure, for example features a method of reducing touch over-reactivity and/or pain and/or mechanical allodynia in a subject (e.g., a human) diagnosed with ASD, RTT, PMS, or Fragile X syndrome by administering to the subject Compound 1, or a pharmaceutically acceptable salt thereof, for example according to a regimen (e g., in an amount and for a duration - e.g., by administration of one or more doses) sufficient to reduce the touch over-reactivity and/or pain and/or mechanical allodynia. In some embodiments, the touch overreactivity and/or pain is associated with a disease states selected from Sensory Processing Disorder (SPD) and fibromyalgia. In some embodiments, the mechanical allodynia is associated with nerve injury, shingles, diabetic neuropathy, chemotherapy-induced neuropathy, or a neuropathic pain state.

DEFINITIONS

[11] As used herein, the terms “Autism Spectrum Disorder” or “ASD” refer to a heterogeneous group of neurodevelopmental disorders as classified in the fifth revision of the American Psychiatric Association’s Diagnostic and Statistical Manual of Mental Disorders 5th edition (DSM-5). The DSM-5 redefined the autism spectrum to encompass the prior (DSM-IV- TR) diagnosis of autism, Asperger syndrome, pervasive developmental disorder not otherwise specified, childhood disintegrative disorder, and Rett syndrome. Autism spectrum disorders are typically characterized by social deficits and communication difficulties, stereotyped or repetitive behaviors and interests, and in some cases, cognitive delays. For example, an ASD is defined in the DSM-5 as exhibiting (i) deficits in social communication and interaction not caused by general developmental delays (must exhibit three criteria including deficits in social- emotional reciprocity, deficits in nonverbal communication, and deficits in creating and maintaining relationships appropriate to developmental level), (ii) demonstration of restricted and repetitive patterns of behavior, interest or activities (must exhibit two of the following four criteria: repetitive speech, repetitive motor movements or repetitive use of objects, adherence to routines, ritualized patterns of verbal or nonverbal, or strong resistance to change, fixated interests that are abnormally intense of focus, and over or under reactivity to sensory input or abnormal interest in sensory aspects of environment), (iii) symptoms must be present in early childhood, and (iv) symptoms collectively limit and hinder everyday functioning. In some embodiments, as will be clear from context to those skilled in the art reading the complete present disclosure, term “ASD” may be used herein to refer to or encompass Dravet’s syndrome and/or autistic-like behavior in non-human animals.

[12] As used herein, the terms “Rett syndrome” or “RTT” refer to an X-linked disorder that affects approximately one in ten-thousand girls. Patients with Rett syndrome typically go through four stages: Stage I) Following a period of apparently normal development from birth, the child begins to display social and communication deficits, similar to those seen in other autism spectrum disorders, between six and eighteen months of age. The child shows delays in their developmental milestones, particularly for motor ability, such as sitting and crawling. Stage II) Beginning between one and four years of age, the child goes through a period of regression in which they lose speech and motor abilities, developing stereotypical midline hand movements and gait impairments. Breathing irregularities, including apnea and hyperventilation also develop during this stage. Autistic symptoms are still prevalent at this stage. Stage III) Between age two and ten, the period of regression ends and symptoms plateau. Social and communication skills may show small improvements during this plateau period, which may last for most of the patients' lives. Stage IV) Motor ability and muscle deterioration continues. Many girls develop severe scoliosis and lose the ability to walk.

[13] As used herein, the terms “Phelan McDermid syndrome” or “PMS” refer to rare genetic condition caused by a deletion or other structural change of the terminal end of chromosome 22 in the 22ql3 region or a disease-causing mutation of the Shank3 gene. Although the range and severity of symptoms may vary, PMS is generally thought to be characterized by neonatal hypotonia (low muscle tone in the newborn), normal growth, absent to severely delayed speech, moderate to profound developmental delay, and minor dysmorphic features. People who have PMS often show symptoms in very early childhood, sometimes at birth and within the first six months of life. [14] As used herein, the term “Fragile X syndrome” refers to an X chromosome-linked condition that is characterized by a visible constriction near the end of the X chromosome, at locus q27.3 that causes intellectual disability, behavioral and learning challenges and various physical characteristics. Fragile X syndrome is the most common inherited form of mental retardation and developmental disability. Males with Fragile X syndrome usually have mental retardation and often exhibit characteristic physical features and behavior. Fragile X syndrome is characterized by behavior similar to autism and attention deficit disorder, obsessive-compulsive tendencies, hyperactivity, slow development of motor skills and anxiety fear disorder. When these disabilities are severe and occur simultaneously, the condition is sometimes described as autism, and may be associated with any degree of intelligence. Other characteristics are a likable, happy, friendly personality with a limited number of autistic-like features such as hand-flapping, finding direct eye contact unpleasant, and some speech and language problems. Physical features may include large ears, long face, soft skin and large testicles (called “macroorchidism”) in post pubertal males. Connective tissue problems may include ear infections, flat feet, high arched palate, double-jointed fingers and hyper-flexible joints.

[15] As used herein, the term “tactile dysfunction” refers to exhibiting symptoms such as withdrawing when being touched, refusing to eat certain “textured” foods and/or to wear certain types of clothing, complaining about having hair or face washed, avoiding getting hands dirty (e.g. , glue, sand, mud, finger-paint), and using finger tips rather than whole hands to manipulate objects. Tactile dysfunction may lead to a misperception of touch and/or pain (hyper- or hyposensitive) and may lead to self-imposed isolation, general irritability, distractibility, and hyperactivity.

[16] As used herein, the term “pain” has its art-understood meaning. In some embodiments, pain is acute pain. In some embodiments, pain is chronic pain. In some embodiments, pain may be or comprise nociceptive pain (i.e., caused by tissue damage). In some embodiments, pain can be neuropathic pain (i.e., caused by nerve damage).

[17] As used herein, the term “mechanical allodynia” refers to a painful sensation resulting from exposure to an ordinarily innocuous stimulus (e g., light touch) - i.e., one that does not normally provoke pain. As is known in the art, mechanical allodynia can result, for example, from physical trauma, nerve injury, shingles, diabetic neuropathy, chemotherapy-induced neuropathy, a neuropathic pain state, etc.

[18] As used herein, the term “anxiety” refers to emotions characterized by feelings of tension, worried thoughts and physical changes like increased blood pressure. In some cases, anxiety can be characterized by having recurring intrusive thoughts or concerns, avoiding certain situations (e.g., social situations) out of worry, and physical symptoms such as sweating, trembling, dizziness, or a rapid heartbeat.

[19] As used herein, the term “social impairment” refers to a distinct dissociation from and lack of involvement in relations with other people. It can occur with various mental and developmental disorders, such as autism. In some cases, social impairment may occur when an individual acts in a less positive way or performs worse when they are around others as compared to when alone. Nonverbal behaviors associated with social impairment can include deficits in eye contact, facial expression, and gestures that are used to help regulate social interaction. Often there is a failure to develop age appropriate friendships. Alternatively or additionally, in some cases, social impairment can include a lack of spontaneous seeking to share achievements or interests with other individuals. A person with social impairment may exhibit a deficit in social reciprocity with individuals, decreased awareness of others, lack of empathy, and/or lack of awareness of the needs of others.

[20] As used herein, the terms “blood brain barrier” and “BBB” refer to a transvascular permeability barrier that tightly controls entry of substances into the brain. Capillaries that perfuse the brain are lined with special endothelial cells that lack fenestrations and are sealed by endothelial tight junctions; this tight endothelium provides a physical barrier that together with metabolic barriers forms the basis of the BBB.

[21] As used herein, the terms “reduced CNS exposure” and/or “reduced CNS penetration” are used to describe peripherally acting compositions relative to an appropriate reference - e.g., based on an assessment performed in an appropriate model indicative of CNS exposure and/or penetration. In some embodiments, a relevant model is a blood brain barrier penetration model. In some embodiments, an appropriate reference is a GABAA PAM compound whose performance in the relevant model is already known. In some embodiments, a relevant reference is a known, BBB-permeant compound such as for example a brain-penetrating GABAA PAM. See, for example, Groeneveld et al Drug Discov Today Technol 20:27, 2016. In some embodiments, permeability may be assessed, for example, using NeuroCArt as described by Goreneveld et al. In some embodiments, a composition or compound is considered to have “reduced permeability” (or “low permeability”) if it is determined to have reduced CNS exposure, e.g., as described herein. In some embodiments, a compound is considered to have “reduced” CNS exposure and or penetration if it is determined to have decreased (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) performance in a relevant assay (e.g., that assesses ability to cross the blood brain barrier) relative to an appropriate control.

[22] As used herein, the term “reducing” refers to decreasing (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or about 100%) one or more side effects and/or symptoms (e.g. , tactile sensitivity, social impairment, or anxiety) of patients diagnosed with ASD, RTT, PMS, or Fragile X syndrome.

[23] As used herein, the terms “treatment” or “treating” refer to one or more of reducing or decreasing frequency and/or intensity of one or more characteristics or side effects (e.g., one or more of tactile dysfunction, anxiety, and social impairment) of a particular disease, disorder, condition, or state, (e.g., of ASD, RTT, PMS, and/or Fragile X syndrome), to delaying onset thereof, and/or to decreasing risk of progression thereto or thereof (e.g. , by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or about 100%) a particular disease or condition. Tn some embodiments, “treatment” may achieve such reduction relative to an appropriate reference or control (e.g., absence of treatment, presence of a reference treatment with known outcome) in a particular individual or test system, or in a population thereof. In some embodiments, “treatment” involves administration according to a dosing regimen that has been established (e.g., by statistically significant correlation) to achieve beneficial effect in a relevant population or system.

[24] As used herein, the terms “effective amount” or “therapeutically effective amount” refers to an amount sufficient to produce a desired result, for example, reducing (e.g. , by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) tactile dysfunction, social impairment, or anxiety in a subject upon administration of a composition containing a compound described herein. In some embodiments, a desired result may be assessed with respect to levels, symptoms, side effects, etc., as applicable, in a subject or population that has not received an administration, or has received a reference (e g., control) administration, or has received a prior administration, and/or may be assessed relative to a control or baseline average. In some embodiments, an “effective amount” may be included in a single dose; more commonly, however, an “effective amount” is achieved through administration of two or more doses, e.g., according to a dosing regimen (e.g., that has been established, for example by statistically significant correlation, to achieve the relevant desired result in a relevant subject or population).

125] As used herein, the term “subject,” refers to an animal which may, in some embodiments, be a mammal. In many embodiments, a subject is a human. In some embodiments, a subject may display, or have been determined to be at risk of displaying, one or more symptoms or side effects (e g , one or more of tactile dysfunction, anxiety, and social impairment) of a particular disease, disorder, condition, or state, (e.g., of ASD, RTT, PMS, and/or Fragile X syndrome). In some embodiments, a subject may have been diagnosed with a particular disease, disorder, condition, or state, (e.g., of ASD, RTT, PMS, and/or Fragile X syndrome). In some embodiments, a subject may have been determined to be at risk of (e.g., via genetic assessment, observation of certain symptoms or side effects, administration of one or more standard tests or assessments, etc.) a particular disease, disorder, condition, or state, (e.g., of ASD, RTT, PMS, and/or Fragile X syndrome). In some embodiments, a subject may be or have been diagnosed, or determined to be at risk of, a relevant disease, disorder, or condition, for example, by virtue of having been subjected to one or more standard tests, or may have been identified, without examination, as one at high risk due to the presence of one or more risk factors. In certain embodiments, a subject is a human. In certain embodiments, a subject is an adult. In certain embodiments, a subject is an adolescent. In some embodiments, a subject is a child. In certain embodiments, a child is less than 12 years of age. In certain embodiments, a child is less than 10 years of age. In certain embodiments, a child is less than 8 years of age. In certain embodiments, a child is less than 6 years of age. In certain embodiments, a child is less than 4 years of age. In certain embodiments, a child is less than 2 years of age. In certain embodiments, a child is 2-4 years of age. In certain embodiments, a child is 4-6 years of age. In certain embodiments, a child is 6-8 years of age. In certain embodiments, a child is 8-10 years of age. In certain embodiments, a child is greater than 12 years of age. In certain embodiments, a subject is elderly. In certain embodiments, a human subject is considered to be elderly if aged more than about 60, 65, 70, 75, 80, or 85 years. [26] As used herein, the term “pharmaceutical composition,” refers to a composition that comprises and/or delivers a compound described herein (and/or, in some embodiments, a therapeutically active metabolite thereof) formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal; often a pharmaceutical composition described herein comprises its active agent (e.g., the compound) in a particular salt form (e g., a pharmaceutically acceptable salt form). Those skilled in the art will appreciate that, in some embodiments, a pharmaceutical composition may be formulated (e.g., may have a form and/or may include particular excipient(s) appropriate for) administration by a particular route - for example for oral administration (e.g. , a tablet, capsule, caplet, gelcap, syrup or other drinkable liquid); for topical administration (e.g., as a cream, gel, lotion, or ointment); for parenteral (e.g., intravenous) administration (e g., as a sterile solution free of particulate emboli and in a solvent system suitable for parenteral use); or otherwise as described herein and/or understood by those skilled in the art.

[27] As used herein, the terms “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier,” refer to an ingredient in a pharmaceutical composition that is understood or considered not to be pharmaceutically “active”. For example, a pharmaceutically acceptable excipient or carrier is or comprises one or more ingredients (e.g., a vehicle capable of suspending or dissolving the active agent) other than a compound described herein and having the properties of being nontoxic and non-inflammatory in a patient. In some embodiments, excipients may include one or more of, for example: anti adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, or waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene, calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (com), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[28] As used herein, the term “pharmaceutically acceptable salt,” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66: 1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Typically, salts (e.g., salt forms) can be prepared in situ during the final isolation and purification of the compounds. Salts of a carboxylate may include representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[29] Those skilled in the art will appreciate that, in some embodiments, isotopes of compounds described herein may be prepared and/or utilized in accordance with the present invention. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. In some embodiments, an isotopic substitution (e.g., substitution of hydrogen with deuterium) may alter the physicochemical properties of the molecules, such as metabolism and/or the rate of racemization of a chiral center.

[30] Those skilled in the art will appreciate that pharmaceutically active compounds are often utilized (e.g., administered) in a prodrug form - e.g., in a form that converts to the active form after administration to a subject. In some embodiments of the present disclosure, pharmaceutically active compounds (e.g., Compound 1) are utilized in a prodrug form. For example, in some embodiments, a pharmaceutical composition that is administered to a subject includes a prodrug form of a relevant compound (e.g., Compound 1). Those skilled in the art are aware of various moieties and/or bonds that are commonly used to generate a prodrug of a compound of interest. Typically, a prodrug form of a compound of interest includes at least one moiety attached to the compound of interest by a bond that is relatively labile under physiological conditions. Commonly, for example, a prodrug moiety is attached by an ester linkage. In some embodiments, a prodrug of Compound 1 may have one of the following structures:

[31] Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[32] As is known in the art, many chemical entities (in particular many organic molecules and/or many small molecules) can adopt a variety of different solid forms such as, for example, amorphous forms and/or crystalline forms (e.g., polymorphs, hydrates, solvates, etc). In some embodiments, such entities may be utilized in any form, including in any solid form. In some embodiments, such entities are utilized in a particular form, for example in a particular solid form, or particular combination thereof.

[33] In some embodiments, Compound 1 may be provided and/or utilized in salt form.

BRIEF DESCRIPTION OF THE DRAWING

[34] Figure 1 depicts effects of 4 hours pre-treatment with 3 mg/kg Compound 1 on tactile PPI in Phelan McDermid syndrome model in Male Shank3 heterozygous mice.

[35] Figure 2 depicts effects of 0.5 hour pre-treatment with 10 mg/kg Compound 1 on tactile PPI in Phelan McDermid syndrome model in Male Shank3 heterozygous mice.

[36] Figure 3 depicts effects of 0.5 hour pre-treatment with 10 mg/kg Compound 1 on tactile PPI in Phelan McDermid syndrome model in Male Shank3 heterozygous mice. [37] Figure 4 depicts effects of 4 hours pre-treatment with 3 mg/kg Compound 1 on tactile PPI in Phelan McDermid syndrome model in Male Shank3 heterozygous mice.

[38] Figure 5 depicts effects of 4 hours pre-treatment with 10 mg/kg Compound 1 on tactile PPI in Phelan McDermid syndrome model in Male Shank3 heterozygous mice.

[39] Figure 6 depicts effects of 0.5 hour pre-treatment with 3 mg/kg Compound 1 on tactile PPI in Phelan McDermid syndrome model in Male Shank3 heterozygous mice.

[40] Figure 7 depicts effects of 0.5 hour pre-treatment with 3 mg/kg Compound 1 on tactile PPI in C57B1/6 wild type mice.

[41] Figure 8 depicts effects of 0.5 hour pre-treatment with 10 mg/kg Compound 1 on tactile PPI in C57B1/6 wild type mice.

[42] Figure 9 depicts effects of 0.5 hour pre-treatment with 30 mg/kg Compound 1 on tactile PPI in C57B1/6 wild type mice.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Diseases, Disorders and Conditions

[43] The present disclosure documents the surprising particular usefulness of Compound 1 in relieving touch hypersensitivity, and teaches the particular use of this compound in a variety of contexts including, for example, to treat one or more diseases, disorders or conditions (or features thereof) that may be associated with tactile dysfunction.

[44] As described herein, tactile dysfunction refers to exhibiting symptoms such as withdrawing when being touched, refusing to eat certain “textured” foods and/or to wear certain types of clothing, complaining about having hair or face washed, avoiding getting hands dirty (e.g. , glue, sand, mud, finger-paint), and using finger tips rather than whole hands to manipulate objects. Those skilled in the art, reading the present disclosure, will appreciate that its teachings may be applicable to tactile dysfunction (e.g., to misperception of touch and/or pain (hyper- or hyposensitive)) in various contexts - for example, in subjects diagnosed with one or more diseases, disorders or conditions as described herein and/or in subjects suffering from idiopathic tactile dysfunction, and will appreciate the applicability of provided technologies to contexts when reduction of tactile sensitivity (e.g., hyperreactivity) may be desirable. [45] For example, a majority of ASD patients (60.9%) report altered tactile sensitivity in both glabrous (smooth) and hairy skin, and altered sensitivity to vibration and thermal pain. As with idiopathic or non-syndromic ASD, pervasive developmental disorders that cause syndromic forms of ASD are also associated with disrupted somatosensation. For example, abnormalities in tactile perception are observed in patients with Phelan McDermid Syndrome (PMS) and Fragile X syndrome, which are both highly associated with ASD and are caused by mutations in Shank3 and Fmrl, respectively. Similarly, tactile hypersensitivity is common in patients with Rett syndrome (RTT), which is caused by mutations in the X-linked methyl-CpG-binding protein 2 (Mecp2) gene. There is an inverse correlation between the presence of ASD traits in human subjects and their neural responses to C-low threshold mechanoreceptor (LTMR)-targeted affective touch. Currently, there are no FDA approved treatments for ASD. Thus, a critical need exists for novel therapeutic approaches to treat ASD and related disorders such as Rett syndrome, Phelan McDermid Syndrome, and Fragile X syndrome.

[46] A range of mouse genetic models of Autism Spectrum Disorder (ASD), combined with behavioral testing, synaptic analyses, and electrophysiology have been used to define both the etiology of aberrant tactile sensitivity in ASD and the contribution of somatosensory dysfunction to the expression of ASD-like traits. Literature reports that mutations in genes associated with both syndromic and nonsyndromic forms of ASD cause tactile dysfunction, and that the Rett Syndrome (RTT)-, Phelan McDermid syndrome (PMS)-, and ASD-associated genes Mecp2, Shcmk3, and Gabrb3 function autonomously in peripheral somatosensory neurons for normal tactile behaviors. Abnormalities in tactile perception are observed in patients with Phelan McDermid Syndrome (PMS) and Fragile X syndrome, which are both highly associated with ASD and are caused by mutations in Shank3 and Fmrl, respectively. Similarly, tactile hypersensitivity is common in patients with Rett syndrome (RTT), which is caused by mutations in the X-linked methyl-CpG-binding protein 2 (Mecp2) gene. Tactile dysfunction associated with Mecp2 and Gabrb3 ASD models is believed to be caused by a deficiency of the b3 subunit of the GABAA receptor (GABRB3) and GABAA receptor-mediated presynaptic inhibition (PSI) of somatosensory inputs to the CNS. ShankS mutant DRG neurons, which are associated with PMS, on the other hand, exhibit hyperexcitability. Such somatosensory deficits during development may contribute to aberrant social behaviors as well as anxiety -like behaviors in adulthood. Thus, it may be that somatosensory neuron dysfunction underlies aberrant tactile perception in various disorders including, for example, ASD, RTT, PMS, and Fragile X syndrome, and/or that functional insufficiency of GABAA receptors, or hyperactivity of peripheral sensory neurons, cause tactile processing deficiency during development, which leads to anxiety-like behavior and social interaction deficits in adult mice. Thus, the present disclosure appreciates that peripheral sensory neurons represent exciting therapeutic targets for ASD, RTT, PMS, and Fragile X syndrome.

[47] The present disclosure appreciates that deficits in peripheral sensory neurons, and not neurons in the brain, account for touch hypersensitivity in mouse models of ASD. Moreover, the present disclosure appreciates that touch hypersensitivity during development causes anxiety and social interaction deficits in adulthood. The present disclosure specifically appreciates that Orefice et al (Cell 178:867, 2019-08-08) have proposed that “GABAAR agonists, GABA reuptake inhibitors, or GABAAR PAMS that are peripherally-restricted may reduce tactile overreactivity and improve brain microcircuit function and related ASD behaviors observed in certain patients with ASD, while minimizing or avoiding entirely potentially detrimental effects on brain development observed in clinical use of classical, FCA-approved GABAA drugs. . .” and that “Peripherally restricted methods for augmenting GABAAR signaling may also have applicability in other diseases and disorders in which touch over-reactivity is present, such as mechanical allodynia in neuropathic pain states, sensory processing disorder, and schizophrenia”.

[48] However, though the possibility that modulation of the GABAA receptors, which attenuate the activity of peripheral mechanosensory neurons, may be useful for treating tactile hypersensitivity and thus anxiety and social impairments in ASD and other patients is exciting, a need has remained to demonstrate that particular compounds can in fact achieve such benefits, without untoward side effects. In fact, treating young children with compounds that modulate GABAA receptors has traditionally been avoided because of undesirable side effects of these drugs in children. Indeed, there is great reluctance on the part of physicians to use FDA- approved compounds that modulate GABAA receptors because of undesirable side effects, including sedation, and serious complications associated with interference with brain development.

[49] The present disclosure addresses these needs and provides a particularly useful compound for uses as described herein. Compound 1 and Compositions Thereof

[50] Compounds that modulate GABAA receptors that have low CNS exposure have proven to be difficult to discover; those skilled in the art are aware that most or all FDA-approved GABAA drugs penetrate the blood brain barrier. See Groeneveld et al, 2016. Furthermore, there are numerous subtypes of GABAA receptors and a given modulator may or may not have desired and/or undesired effects based on subtype selectivity and pharmacokinetics. Thus, the demonstrations of in vitro and in vivo activities provided herein are surprising and unexpected.

[51] 8-chloro-6-(2-fluorophenyl)-4H-benzo[f]pyrazolo[l,5-a][l,4]diazepine-2-carboxylic acid (Compound 1) is a recently discovered GABA-A positive allosteric modulator with low CNS exposure after oral or intravenous dosing. The present disclosure provides an insight that Compound 1 might be both sufficiently peripherally restricted (and specific) and sufficiently effective in its GABAA modulation to usefully treat tactile dysfunction (e.g., touch hypersensitivity) and/or other disorders or conditions as discussed herein (e.g., as may be characterized by and/or arise from tactile dysfunction).

[52] The present disclosure furthermore documents that Compound 1 displayed robust efficacy in a mouse model of touch hypersensitivity, and teaches that Compound 1 is an excellent compound for reducing tactile dysfunction in a human subject diagnosed with Autism Spectrum Disorder (ASD), Rett syndrome (RTT), Phelan McDermid syndrome (PMS), or Fragile X syndrome, and/or reducing anxiety or social impairment in a subject (e g., a human) diagnosed with ASD, RTT, PMS, or Fragile X syndrome and/or treating touch over-reactivity and/or pain and/or mechanical allodynia in a human subject.

Compound 1

[53] In some embodiments, Compound 1 is administered to an appropriate subject in accordance with the present disclosure by administration of a pharmaceutical composition as described herein. In some embodiments, Compound 1 is administered according to a dosing regimen, e.g., involving administration of one or more unit doses (e g., unit dosage forms).

[54] In some embodiments, Compound 1 is utilized, and/or a pharmaceutical composition comprises and/or delivers Compound 1, in a particular form (e.g., salt form, solid form, etc.).

[55] In some embodiments, administration is oral and/or a pharmaceutical composition is formulated for oral administration (e.g., including one or more orally-acceptable carriers or excipients).

[56] In some embodiments, administration is via an immediate release formulation (e.g., an immediate release oral formulation). In some embodiments, administration is via an extended release formulation.

[57] In some embodiments, administration is via a pediatrically acceptable formulation such as, for example, a flavored liquid, or a solid or gel formulation that is or comprises small-sized particles (e.g., sprinkles).

[58] In some embodiments, a composition that comprises or delivers Compound 1 in accordance with the present disclosure is characterized in that it shows efficacy in a mouse tactile PPI model as described herein (with dosing and/or route of administration adjusted appropriately to the mouse). Alternatively or additionally, a composition that comprises or delivers Compound 1 in accordance with the present disclosure is characterized by low CNS exposure of Compound 1 upon administration of the composition to a subject (e.g., a human subject).

[59] In some embodiments, the present disclosure provides use of Compound 1 in (e.g., in the manufacture of a medicament for) reducing tactile dysfunction in a subject. In some embodiments, the present disclosure provides a use of Compound 1 (e.g., in the manufacture of a medicament for) reducing anxiety or social impairment in a subject. In some embodiments, the present disclosure provides use of Compound 1 in (e g., in the manufacture of a medicament for) treating touch over-reactivity and/or pain and/or mechanical allodynia in a human subject in need thereof.

[60] In some embodiments, the present disclosure provides use of Compound 1 as a reference for comparison of other GABAA modulators (e.g., GABAA PAMs), for example to establish a useful degree of modulation of touch hypersensitivity or other tactile dysfunction, a useful extent of efficacy in treatment of one or more diseases, disorders or conditions, a useful (limited) extent of CNS penetration or exposure, etc.

[61] Alternatively or additionally, in some embodiments, the present disclosure provides use of Compound 1 for assessing peripheral restriction of one or more GABAA-associated phenomena or events. For example, pain that responds to treatment with Compound 1 is peripheral pain; tactile dysfunction that responds to exposure to Compound 1 is peripheral tactile dysfunction. In some embodiments, it may be useful to assess a subject to determine whether one or more features of the subject’s state (e.g., of a disease, disorder or condition from which the subject suffers) can be treated or addressed by peripheral intervention, or instead might require central intervention. To give but one example, a subject suffering pain that does not respond to Compound 1 may require treatment with a CNS-active pain reliever. In some embodiments of the present disclosure, Compound 1 may be used to characterize (e.g., diagnose) one or more aspects of a subject’s state, and appropriate treatment (e.g., central and/or peripheral, which may be or comprise treatment with Compound 1) may be administered accordingly. Subjects

[62] In some embodiments, a subject (or population of subjects) to whom Compound 1 (e.g., a composition that comprises or delivers Compound 1) is administered in accordance with the present disclosure is a subject (or population thereof) who is displaying or has displayed one or more features of tactile dysfunction (e.g., touch over-reactivity, pain, mechanical allodynia). In some embodiments, such tactile hypersensitivity is associated with known genetic conditions. In some embodiments, such tactile hypersensitivity is idiopathic. In some embodiments, a subject (or population of subjects) to whom Compound 1 (e.g., a composition that comprises or delivers Compound 1) is administered in accordance with the present disclosure is a subject (or population thereof) who suffers from mechanical allodynia associated with physical trauma, nerve injury, shingles, diabetic neuropathy, chemotherapy-induced neuropathy or a neuropathic pain state.

[63] In some embodiments, a subject (or population of subjects) to whom Compound 1 (e.g., a composition that comprises or delivers Compound 1) is administered in accordance with the present disclosure is a subject (or population thereof) who suffers from or has been determined to be susceptible to (e.g., has been diagnosed with) one or more of Autism Spectrum Disorder (ASD), Rett syndrome (RTT), Phelan McDermid syndrome (PMS), or Fragile X syndrome.

[64] In some embodiments, a subject (or population of subjects) to whom Compound 1 (e.g., a composition that comprises or delivers Compound 1) is administered in accordance with the present disclosure is receiving or has recently received pain management therapy; in some embodiments, such pain management therapy may be or comprises administration of one or more of Acetaminophen, Alfentanil, Alprazolam, Amitriptyline, Amoxapine, Aspirin, Baclofen, Buprenorphine, Bupropion, Butorphanol, Carbamazepine, Celecoxib, Citalopram, Clomipramine, Clonazepam, Codeine, Desipramine, Desvenlafaxine, Diclofenac, Diflunisal, Doxepin, Duloxetine, Escital opram, Etodolac, Etoricoxib, Fenoprofen, Fentanyl, Fluoxetine, Flurbiprofen, Fluvoxamine, Gabapentin, Hydrocodone, Hydromorphone, Ibuprofen, Imipramine, Indomethacin, Ketoprofen, Ketorolac, Lacosamide, Lamotrigine, Levetiracetam, Levorphanol, Lorazpam, Mefenamic acid, Meloxicam, Meperidine, Methadone, Milnacipran, Mirtazepine, Morphine, Nabumetone, Nalbuphine, Naproxen, Nefazodone, Nortriptyline, Oliceridine, Opium, Oxaprozin, Oxcarbazepine, Oxycodone, Oxymorphone, Paroxetine, Pentazocine, Phenytoin, Piroxicam, Pregabalin, Propoxyphene, Protriptyline, Remifentanil, Sertraline, Sodium valproate, Sulfentanil, Sulindac, Tapentadol, Tizanidine, Tolmetin, Topiramate, Tramadol, Trazodone, Trimipramine, Valproic acid, Venlafaxine. In some embodiments, a subject (or population of subjects) to whom Compound 1 (e.g., a composition that comprises or delivers Compound 1) is administered in accordance with the present disclosure is receiving pain management therapy at a lower level than that required for comparable pain reduction absent administration of Compound 1.

Administration

[65] In some embodiments, administration in accordance with the present disclosure (e.g., of Compound 1 or a composition that comprises or delivers Compound 1, for example to a subject suffering from a disease, disorder or condition as described herein) is or comprises oral administration.

[66] In some embodiments, an amount administered (e.g., in a single dose or in a daily dose or via a dosing regimen) may be or comprise an amount of about 3 mg to about 1000 mg. In some embodiments, an amount administered (e.g., in a single dose or in a daily dose or via a dosing regimen) may be or comprise an amount of about 3 mg to about 15 mg, about 10 mg to about 25 mg, about 15 mg to about 50 mg, about 25 mg to about 75 mg, about 50 mg to about 100 mg, about 75 mg to about 125 mg, about 100 mg to about 150 mg, or about 125 mg to about 200 mg. In some embodiments, the present invention provides methods of reducing tactile dysfunction, wherein the method comprises administering to a subject in need thereof about 0.1 mg to about 10,000 mg of Compound 1. In some embodiments, provided methods comprise an amount administered (e.g., in a single dose or in a daily dose or via a dosing regimen) of about 0.1 mg to about 9000 mg, about 0.1 mg to about 8000 mg, about 0.1 to about 7000 mg, about 0.1 mg to about 6000 mg, about 0.1 mg to about 5000 mg, about 0.1 mg to about 4000 mg, about 0.1 mg to about 3000 mg, about 0.1 mg to about 2000, about 0.1 mg to about 1000 mg, about 0.1 mg to about 900 mg, about 0.1 mg to about 800 mg, about 0.1 mg to about 700 mg , about 0.1 mg to about 600 mg, about 0.1 mg to about 500 mg, about 0.1 mg to about 400 mg, about 0.1 mg to about 300 mg, about 0.1 mg to about 200 mg, about 0.1 mg to about 100 mg, about 0.1 mg to about 75 mg, about 0.1 mg to about 50 mg, about 0.1 mg to about 25 mg, about 0.1 mg to about 15 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 3 mg, or about 0.1 mg to about 1 mg.

[67] In some embodiments, provided methods comprise administering to a patient in need thereof Compound 1 or a composition that comprises or delivers Compound 1 in an amount that is equivalent to about 3 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, or about 30 mg/kg in a mouse. In some embodiments, provided methods comprise administering to a patient in need thereof Compound 1 in an amount that is equivalent to about 100 mg/kg in a mouse. In some such embodiments, the amount of Compound 1 is about 15 mg, about 25 mg, about 50 mg, about 75 mg, about 150 mg, or about 500 mg.

[68] In some embodiments, subjects receiving treatment with Compound 1 as described herein are monitored, for example, for changes (e.g., improvement) in one or more aspects of tactile dysfunction (e.g., touch hypersensitivity) and/or in one or more physical or behavioral symptoms, and/or for increase or decrease in experienced pain. In some embodiments, additional therapy is administered, and/or Compound 1 therapy is adjusted (e.g., increased, decreased, or terminated), responsive to such monitoring.

[69] In some embodiments, treatment with Compound 1 is initiated during a subject’s youth (e.g., when a subject is a baby or a child). In some embodiments, Compound 1 treatment is administered to a subject over a period of years, optionally with one or more breaks or rest periods during which Compound 1 therapy is not administered.

EXAMPLES

[70] Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention. Example 1: Synthesis of Compound 1 (8-chloro-6-(2-fluorophenyl)-4H- benzo[f]pyrazolo[l,5-a] [l,4]diazepine-2-carboxylic acid)

Compound 1 Experimental Protocol:

Step 1: ethyl l-(4-chlorophenyl)-5-methyl-lH-pyrazole-3-carboxylate

[71] A round bottom flask was charged with 4-chlorophenyl hydrazine HC1 (31.47 g, 0.176 mol), MeOH (500 mL), ethyl 2,4-dioxopentanoate (24.69 mL, 1 eq.) and DIPEA (30.62 mL, 1 eq.). The mixture was heated to reflux for 1.5hour. After completion, reaction mixture was cooled down to room temperature and silica gel (~70 g) was added. Solvent was evaporated and the silica deposit was subjected to flash chromatography on silica, eluting with 90% hexane: 10% ethyl acetate. 26.4 g was obtained (56% yield). LC-MS ESI+: 265.10 (M+l). Step 2: ethyl l-(4-chloro-2-(2-fluorobenzoyl)phenyl)-5-methyl-lH-pyrazole-3- carb oxy late

[72] Ethyl l-(4-chlorophenyl)-5-methyl-lH-pyrazole-3-carboxylate (5.27 g, 0.02 mol) and 2-fluoro-benzaldehyde (4.19 mL, 2 eq.) were dissolved in 1,2-di chloroethane (73 mL) and placed in a screwed vial. To the resulting solution was added Pd(TFA)2 (662 mg, 0.1 eq.) and the mixture was stirred for 45 min at room temperature. TBHP (5.5 M in nonane, 9.05 mL, 2.5 eq.) was added in one portion. Reaction mixture was heated to 100 °C for 24 h. Then it was cooled down to room temperature and washed with 0.5 M NaOH (30 mL), dried over anhydrous MgSO4 and evaporated under reduced pressure. The product was purified using silica gel flash chromatography eluting with a gradient from 100% hexane to 80% hexane : 20% ethyl acetate. 2.2 g was obtained (28%). LC-MS ESI+: 387.00 (M+l).

Step 3: ethyl 5-(bromomethyl)-l-(4-chloro-2-(2-fluorobenzoyl)phenyl)-lHpyrazole- 3 -carboxylate

[73] Ethyl 1 -(4-chloro-2-(2-fluorobenzoyl)phenyl)-5-methyl- lH-pyrazole-3 - carboxylate (6.61 g, 0.017 mol) was dissolved in DCE (375 mb). Then NBS (3.65 g, 1.2 eq.) was added and the mixture was heated to 90 °C (oil bath temperature). After 5 minutes, Luperox A75 (0.55 g, 0.1 eq.) was added, and heating was continued. After 2 hours progress of the reaction was checked by LC-MS and approximately 40% of starting material was still present in the reaction mixture. An additional portion of NBS (1.82g, 0.6 eq.) and benzoyl peroxide (0.27g, 0.05 eq.) were added. Heating was continued for 1 hour. After that time TLC analysis showed almost full conversion of the starting material. The reaction mixture was cooled to room temperature and silica gel (~60 g) was added. Solvent was evaporated under reduced pressure. Product was purified using flash chromatography on silica, eluting with a gradient from 100% hexane to 80% hexane:20% ethyl acetate. 4.93 g was obtained (62% yield). LCMS 464.95, 466.95 (M+l).

Step 4: ethyl 8-chloro-6-(2-fluorophenyl)-4H-benzo[f]pyrazolo[l,5- a] [1 ,4]diazepine-2-carboxylate

[74] Ethyl 5-(bromomethyl)-l-(4-chloro-2-(2-fluorobenzoyl)phenyl)-lH-pyrazole-3- carboxylate (4.93 g, 0.106 mol) was dissolved in IPA (100 mL), together with ammonium acetate (3.59 g, 3 eq.) and urotropine (4.45 g, 3 eq.). RM was heated to reflux for 1.5 hours. Then RM was cooled to room temperature and silica gel (~50 g) was added. Solvent was evaporated under reduced pressure. Product was purified using flash chromatography on silica, eluting with a gradient from 80%hexane : 20% ethyl acetate to 50% hexane : 50%ethyl acetate. 3.25 g was obtained (80%). LC-MS 384.05 (M+l).

Step 5: 8-chloro-6-(2-fluorophenyl)-4H-benzo[f]pyrazolo[l,5-a][l,4]diazepine-2- carboxylic acid

[75] Ethyl 8-chloro-6-(2-fluorophenyl)-4H-benzo[f]pyrazolo[l,5-a][l,4]diazepine-2- carboxylate (3.25 g, 0.0085 mol) was dissolved in a THF:water mixture (v:v, 1: 1, 84 mL), iOH H2O(1.93 g, 1.06 g, 3 eq.)was added in one portion and the reaction mixture was stirred at 60 °C temperature until completion, approximately 1 to 2 hours (indicated by TLC analysis, 100% ethyl acetate as eluent, SM Rf=0.6 and desired product DP Rf=0.0). After completion, THF was evaporated under reduced pressure. The water solution was acidified with formic acid to pH ~ 5. The resulting precipitate was collected and washed with distilled water (100 mL). NOTE: if a very pure product is obtained in the previous step, no purification is required after saponification and acidification. Product was purified using Cl 8 reversed phase chromatography, eluting with a gradient from 90% water : 10% MeOH to 100% MeOH.

2.2 g was obtained (73%). LC-MS 355.90 (M+l). 1H NMR (400 MHz, DMSO-d6) 5 13.35 -12.91 (s, 1H), 8.00 (d, J= 8.8 Hz, 1H), 7.84 (dd, J= 8.8, 2.4 Hz, 1H), 7.65 -7.52 (m, 2H), 7.37 -7.17 (m, 3H), 6.93 (s, 1H), 4.76 (s, 2H).

Example 2: In vitro Binding and Functional GABAA Activity of Compound 1

[76] Binding of Compound 1 to native GABAA receptors was determined at Eurofins Discovery using a rat brain tissue preparation, measuring the displacement of the benzodiazepine ³H-flunitrazepam (See: Damm, et.al. [3H] flunitrazepam: Its advantages as a ligand for the identification of benzodiazepine receptors in rat brain membranes. Res Commum Chem Pathol Pharmacol. 1978, 22(3):597-600, and Speth,, et.al., Benzodiazepine receptors: temperature dependence of [3H]flunitrazepam binding. Life Sci.1979, 24(4):351-357). Wistar rat brain (minus cerebellum) was used, with an incubation time of 60 minutes at 25°C and 50 mM phosphate buffer (pH 7.4) was used as incubation buffer. IOUM of diazepam was used as the non-specific ligand, and 1 nM ³H-flunitrazepam was used as the radio-ligand. The binding Ki of Compound 1 in this assay was found to be 170 nM.

[77] Functional activity of Compound 1 against various GABAA receptor subtypes was determined by B’SYS GmbH, measured by manual patch-clamp technique in CHO or Ltk cells stably expressing either aip2y3 (CHO), a3p2y3 (Ltk) or a5p2y3 (Ltk) receptors (Source: B’SYS GmbH, 4108 Witterswil, Switzerland).

[78] Confluent clusters of cells are electrically coupled. Because responses in distant cells are not adequately voltage clamped and because of uncertainties about the extent of coupling, all cells were cultivated at a density that enables single cells (without visible connections to neighboring cells) to be measured.

[79] For electrophysiological measurements, cells were seeded into 35 mm culture dishes without antibiotics or antimycotics.

[80] Equipment: Amplifier; EPC- 10, HEKA Electronics;

[81] Headstage: Preamplifier EPC- 10, HEKA Electronics;

[82] Software: PatchMaster, HEKA Electronics

[83] Conditions: Cells were incubated at 37°C in a humidified atmosphere with 5% CO2 (rel. humidity about 95%).

[84] Culture media: The cells were continuously maintained in and passaged in sterile culture flasks containing a 1: 1 mixture of Dulbecco’s modified eagle medium and nutrient mixture D- MEM/F-12 (lx, liquid, with L-Glutamine) for Ltk cells or HAM/F-12 (lx, liquid, with L Glutamine) for CHO cells, supplemented with 10% fetal bovine serum and 1.0% Penicillin/Streptomycin solution.

[85] Antibiotics: The complete medium as indicated above was supplemented with selected antibiotics:

GABAA (alB2y2): Hygromycin, Puromycin, Zeocin

GABAA (ot3B2y2) and GABAA (a5B2y2): Neomycin

[86] Each concentration of Compound 1 was analyzed in at least n=2 isolated cells on each GABA subtype. The stock solution of GABA was prepared prior to the experimental start of the present study, stored frozen (-10°C to -30°C) until the day of experimentation. Shortly prior to the electrophysiological experiments frozen stock solution was thawed and diluted. Negative control: Two additional cells were treated with vehicle (0.1% DMSO) only instead of test item to show current run-down during the course of the experiment (time-matched). Positive control: 1.0 pM Diazepam was used as a positive control to show accuracy of the experiment (n=2).

[87] Experimental procedure: The 35 mm culture dishes upon which cells were seeded at a density allowing single cells to be recorded, were placed on the dish holder of the microscope and continuously perfused (at approximately 1 mL/min) with the bath solution described in section 0. All solutions applied to cells, including the pipette solution, were maintained at room temperature (19°C to 30°C). After formation of a Gigaohm seal between the patch electrodes and an individual cell (pipette resistance range: 2.5 M to 6.0 MQ; seal resistance range: >1 GQ) the cell membrane across the pipette tip was ruptured to assure electrical access to the cell interior (whole-cell patch-clamp configuration). In case the quality of the seal was poor, the process of seal formation was repeated with a different cell and a new pipette. GABA inward currents were measured upon application of submaximal GABA concentration to patch-clamped cells. The cells were voltage-clamped at a holding potential of -80 mV. If current density was judged to be too low for measurement, another cell was recorded. Only data from cells treated with the test item were documented. All GABAAR subtypes were stimulated by GABA (2 pM) and cumulative, increasing concentrations of test item/GABA. Between two GABA applications bath solution (without or with test item) was perfused for at least 60 s. For control experiments, the test item was replaced by 0.1% DMSO and a single concentration of the positive control/GABA was applied at the end of the experiment.

[88] Concentration-response curves were generated by applying increasing, cumulative concentrations of Compound 1 in the presence of an ECso concentration of GABA.

[89] The ECso values for Compound 1 were:

127 nM for aip2y3 119 nM for ot3p2y3 36 nM for a p2y3

[90] The maximal response elicited by Compound 1 relative to GABA was found to be:

281% for alp2y3 140% for a3p2y3 210% for Ot5p2y3.

[91] These results indicate that Compound 1 strongly potentiates effect(s) of GABA across the receptors examined. The degree of potentiation observed was similar to the positive control, diazepam. Example 3: CNS Exposure After Administration to Rats of Compound 1

[92] Rat plasma protein binding (93.5 %), and rat brain homogenate binding (86.5 %) were determined by equilibrium dialysis.

[93] To male Sprague-Dawley Rats, Compound 1 was administered in a single IV dose (1 mg/kg) as a solution (5% DMSO in pH 8 phosphate buffer). Compound 1 did not readily cross the blood brain barrier following a single IV (1 mg/kg) dose with brain:plasma ratio of 0.018 observed one hour post administration. The ratio of unbound drug (brain: plasma) was 0.037.

[94] To male Sprague-Dawley Rats, Compound 1 was administered in single oral doses of 10 mg/kg and 100 mg/kg as solutions (5% DMSO in pH 8 phosphate buffer). Total brain levels of Compound 1 were measured 1 hour post administration as 176 ng/g at lOmg/kg and 2053 ng/g at 100 mg/kg. This results in a braimplasma ratio of 0.014 from the 10 mg/kg oral dose and 0.023 from the 100 mg/kg oral dose. The ratio of unbound drug (brain:plasma) was 0.029 from the 10 mg/kg oral dose and .048 from the 100 mg/kg oral dose. Four hours post oral administration, the plasma:brain ratio of 0.0135 and 0.0162 for the 10 mg/kg and 100 mg/kg dose respectively was noted. The ratio of unbound drug (braimplasma) at the 4 hr timepoint was 0.028 from the 10 mg/kg oral dose and .034 from the 100 mg/kg oral dose. Plasma to brain ratios were measured 24 hours post administration with the 100 mg/kg dose and was 0.0172, giving a braimplasma unbound drug ratio of 0.035. The change in braimplasma ratios of total and unbound drug changed little over the course of the study; 1, 4 and 24 hours post oral administration at either 10 mg/kg or 100 mg/kg.

[95] These data demonstrate low brain partitioning in rat with concentrations in brain ranging from 1-5% those in plasma.

Example 4: Activity in Mouse Touch Hypersensitivity Model (Tactile PPI)

[96] The tactile prepulse inhibition (PPI) assay was adapted from methods published by Orefice et al., 2016 (Cell 166, 299-313). The response of mice to tactile stimulus was measured using San Diego Instruments startle reflex system (SR-LAB™ Startle Response System). Tactile sensorimotor gating deficits were measured using a PPI assay where the pre-stimulus was an air puff (0.9 PSI or 0.6 PSI as specified below, 50 ms), administered to the back of the mouse to assess hairy skin sensitivity, followed by an acoustic startle stimulus (125 dB, 20 ms).

[97] Typical experimental procedure:

[98] Male Shank3 heterozygous mice were bred at PsychoGenics (as mentioned, the shank3 gene is mutated in the human condition known as Phelan McDermid syndrome).

[99] Upon weaning, animals were group-housed in OPTIMice ventilated cages for the remainder of the study. Testing commenced at 8 weeks of age. All animals were examined and weighed prior to initiation of the study to assure adequate health and suitability and to minimize non-specific stress associated with manipulation. Mice were maintained on a 12/12 light/dark cycle. The room temperature was maintained between 20 and 23°C with a relative humidity maintained at approximately 50%. Chow and water were provided ad libitum for the duration of the study. Animals were randomly assigned across treatment groups and balanced by test chamber. The test was performed during the animal’s light cycle.

[100] Compound 1 was evaluated at 3 and 10 mg/kg. Compound 1 was dissolved in 5% DMSO in Phosphate Buffer (pH 8) and administered p.o. at a dose volume of 10 ml/kg. Tactile PPI testing began at 30 minutes or 4 hours post-dose as indicated below.

[101] In each experiment described below (i.e., in Experiments 1-6), fourteen mice were included in the treatment group, and 14 were in the control group, so that total N=28. Each experiment consisted of two sessions separated by 72 hours. A 1-week washout separated the start of each experiment. A crossover design was used within each experiment. Experiments 1&2 used the same mice. Across the six (6) experiments, the following regimens were assessed:

Experiments 1 & 2

[102] Experiment 1 : 3 mg/kg Compound 1 with 4-hour pre-treatment Session la (e.g., 0.6 PSI); Session lb (e.g., 0.9 PSI) Group 1: Vehicle (PO), n = 14 Group 2: Compound 1 3 mg/kg (PO), n = 14 Session 2a (e.g., 0.6 PSI); Session 2b (e.g., 0.9 PSI) Group 1 : Compound 1 3 mg/kg (PO), n = 14 Group 2: Vehicle (PO), n = 14

[103] Experiment 2: 10 mg/kg Compound 1 with 30-minute pre-treatment Session la (e.g., 0.6 PSI); Session lb (e.g., 0.9 PSI) Group 1 : Vehicle (PO), n = 14 Group 2: Compound 1 10 mg/kg (PO), n = 14 Session 2a (e.g., 0.6 PSI); Session 2b (e.g., 0.9 PSI) Group 1 : Compound 1 10 mg/kg (PO), n = 14 Group 2: Vehicle (PO), n = 14 Mice were placed into a ventilated, cylindrical holder on a platform within a soundproof chamber. The session was run twice per day per animal (Session a and Session b) as detailed below. Session a consisted of an acclimation phase followed by 2 blocks of trials. The acclimation phase consisted of a 5-minute period during which constant background white noise (78 dB) was presented. Block I consisted of 5 prepulse stimuli alone trials(i.e., air puff trials all of which were presented at either 0.6 PSI or 0.9 PSI, depending on the session). Block II consisted of 5 pulse alone trials (i.e., acoustic startle stimuli trials, 125 dB, 20 ms), 10 prepul se+pulse trials, and 5 no stimulation trials which were presented in a pseudorandom order. Among the 10 prepul se+pulse trials, the prepulse intensity remained constant at either 0.6 or 0.9 PSI (20 ms) and the interstimulus interval (1ST) between prepulse and pulse varied from 250 ms (5 trials) to 500 ms (5 trials) in duration. Throughout the session, the intertrial intervals ranged between 10 to 50 seconds. Whole body flinch, or startle reflex, was quantified using an accelerometer sensor which measured the amplitude of movement of the animal within the cylindrical holder. Immediately upon completion of the session, the air puff pressure was adjusted from 0.6 PSI to 0.9 PSI (or vice versa). The pressure of the air puff delivery in each test chamber was then checked for accuracy using a manometer. Animals remained in the cylindrical holder within the test chamber while the air puff adjustments were made and checked. Once the pressure was successfully changed and checked, Session b was run in the same manner described above. In all experiments, the following measures were determined from the data obtained from the tactile PPI sessions: 1) response to the prepulse (air puff) alone as a percentage of the startle response); 2) response to the pulse (acoustic startle stimuli alone and 3) % pre-pulse inhibition. The response to the prepulse alone was calculated using the formula: ((average startle response to prepulse alone trials in Block Vaverage startle response to pulse alone trials in Block II) - (average startle response to no stim trials in Block Il/average startle response to pulse alone trials in Block II))*100. For each ISI trial type (250 ms and 500 ms), % pre-pulse inhibition was calculated using the formula: (l-(average startle response to the prepul se+pulse trials in Block Il/average startle response to pulse alone trials in Block II))* 100.

[104] The data sets generated from each experiment were analyzed independently in the following manner: The startle response to air puff alone (i.e., the Block I trials) and the startle response to the pulse alone (in the Block II trials) were analyzed using a paired t-test. Prepulse inhibition of the startle response (i.e., Block II prepulse/pulse trials relative to Block II pulse alone trials) was evaluated using a two-way repeated measures ANOVA with factors of Treatment and ISI as the within subject measures. Bonferroni’s post-hoc comparisons were performed if appropriate. An effect was considered significant if p < 0.05. The following exclusion criteria were applied to all data sets: 1) Startle amplitude less than 1000 A.U, 2) Negative % PPI at 250 and/or 500 ms ISI trial types in the vehicle group. Note that, due to the crossover design, data within an experiment were excluded in the event that an exclusion criterion was met. Data are represented as the mean and standard error to the mean (s.e.m).

Results for Experiments 1-2 are shown in Figures 1-2.

Experiments 3-6:

[105] Experiments 3-6 utilized a separate cohort of mice than was used in experiments 1&2, and in a different set of runs on a different month, again with 3mg/kg and 10 mg/kg of Compound 1 tested in the tactile PPI model in Male Shank3 heterozygous mice, with 0.6PSI and 0.9PSI air puffs, and with both 250ms and 500ms ISI. The results of Experiments 3-6, are shown in Figures 3-6.

[106] Overall, 5 of the 6 experiments had conditions that showed a statistically significant main effect of treatment with a p value of <0.05, with the other one experiments having conditions that showed trends of a main effect of treatment, but did not reach statistical significance. Evaluating across all six experiments in Male Shank3 heterozygous mice there is evidence of efficacy at 3mg/kg and 10 mg/kg of Compound 1 in the tactile PPI model. Example 5: Activity in Wild Type Mouse

Experiments 7-9:

[107] Experiments 7-9 tested 30-minutes pretreatment with 3 mg/kg, 10 mg/kg and 30 mg/kg of Compound 1 in C57B1/6 wild type mice, with 0.6 PSI and 0.9 PSI air puffs, and with both 250 ms and 500 ms ISI. The results of Experiments 7-9, are shown in Figures 7-9.

Overall, statistical significance with a p value of <0.05 was achieved in the wild type mice treated with 30 mg/kg of Compound 1 after a 30 minute pre-treatment whereas a trend towards efficacy was observed with 3 and 10 mg/kg of Compound 1 in the 0.6 PSI experiments.

Claims

CLAIMS A method of reducing tactile dysfunction in a human subject diagnosed with Autism Spectrum Disorder (ASD), Rett syndrome (RTT), Phelan McDermid syndrome (PMS), or Fragile X syndrome by administering to the subject a composition that comprises or delivers Compound 1 (8-chloro-6-(2-fluorophenyl)-4H-benzo[f]pyrazolo[l,5- a][l,4]diazepine-2-carboxylic acid), or a pharmaceutically acceptable salt thereof. A method of reducing anxiety or social impairment in a human subject diagnosed with ASD, RTT, PMS, or Fragile X syndrome by administering to the subject Compound 1 (8- chloro-6-(2-fluorophenyl)-4H-benzo[f]pyrazolo[l,5-a][l,4]diazepine-2-carboxylic acid), or a pharmaceutically acceptable salt thereof, in an amount and for a duration sufficient to reduce the anxiety or social impairment. A method of treating touch over-reactivity and/or pain and/or mechanical allodynia in a human subject in need thereof, comprising administering to the subject Compound 1 (8- chloro-6-(2-fluorophenyl)-4H-benzo[f]pyrazolo[l,5-a][l,4]diazepine-2-carboxylic acid), or a pharmaceutically acceptable salt thereof, in an amount and for a duration sufficient to reduce the touch over-reactivity and/or pain and/or mechanical allodynia. The method of claim 3, wherein the touch over-reactivity and/or pain is associated with a disease states selected from Sensory Processing Disorder (SPD) and fibromyalgia. The method of claim 3, wherein the mechanical allodynia is associated with nerve injury, shingles, diabetic neuropathy, chemotherapy-induced neuropathy, or a neuropathic pain state. The method of any one of claims 1-5, wherein the subject is a child.