阅读说明：本技术 使用法呢基二苯并二氮杂酮治疗Phelan McDermid综合征的方法 (Methods of treating Phelan McDermid syndrome using farnesyl dibenzodiazepinone ) 是由 迈克尔·斯内普 帕特里夏·科格拉姆 罗伯特·迪肯 于 2018-10-25 设计创作，主要内容包括：提供了通过给予法呢基二苯并二氮杂酮化合物来治疗患有Phelan McDermid综合征(PMS)的受试者或易患该疾病的受试者的Phelan McDermid综合征(PMS)的方法。还提供了法呢基二苯并二氮杂酮化合物在制备用于治疗患有PMS的受试者的药物中的用途。(Methods of treating Phelan McDermid Syndrome (PMS) in a subject suffering from, or susceptible to, Phelan McDermid Syndrome (PMS) by administering a farnesyl dibenzodiazepinone compound are provided. Also provided is the use of a farnesyl dibenzodiazepinone compound in the manufacture of a medicament for treating a subject suffering from PMS.)

1. a method of treating a subject having Phelan McDermid Syndrome (PMS), comprising administering to a subject having PMS a therapeutically effective amount of (i) at least one farnesyl dibenzodiazepine compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepine compound and a pharmaceutically acceptable carrier or diluent.

2. A method of inhibiting the development of PMS in a subject predisposed to PMS comprising administering to the subject predisposed to PMS a therapeutically effective amount of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent.

3. The method of claim 1 or 2, wherein at least one farnesyl dibenzodiazepine compound is selected from a compound encompassed by formula I:

wherein the content of the first and second substances,

W¹、W²and W³Each independently is

Or the tricyclic chain may be at W³、W²Or W¹Is denoted by W³、W²Or W¹Are each-CH ═ O or-CH₂OH termination;

a is-NH-, -NCH₂R¹-or-NC (O) R¹-, wherein R¹Is C_1-6Alkyl radical, C_2-6Alkenyl, aryl or heteroaryl;

R²、R³and R⁴Each independently is H, R⁵or-C (O) R⁶Wherein each R is⁵Independently is C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl, and wherein each R is⁶Independently H, C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl.

4. The method of claim 1 or 2, wherein the at least one farnesyl dibenzodiazepine compound is AMO-01 or a pharmaceutically acceptable salt thereof,

5. the method of any one of claims 1-4, wherein the subject with PMS or a subject predisposed to PMS is a subject having a chromosome deletion at 22q 13.3.

6. The method of any one of claims 1-5, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepinone compound is from 0.1 μ g/kg to 200mg/kg of farnesyl dibenzodiazepinone compound per body weight of the subject.

7. The method of any one of claims 1-5, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepine compound is 80-160mg/m²And is delivered to the subject within 4-8 h.

8. The method of any one of claims 1-5, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepine compound is 120mg/m²And delivered to the subject within 6 h.

9. The method of any one of claims 1-8, wherein the at least one farnesyl dibenzodiazepinone compound or the pharmaceutical formulation comprising the at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is administered to the subject by intravenous or subcutaneous administration.

Use of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent in the treatment of a subject with PMS or in a method of treating a subject with PMS.

Use of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent in or in a method of inhibiting the development of PMS in a subject predisposed to PMS.

12. The use according to claim 10 or 11, wherein at least one farnesyl dibenzodiazepine compound is selected from the group consisting of the compounds covered by formula I:

wherein the content of the first and second substances,

W¹、W²and W³Each independently is

a is-NH-, -NCH₂R¹-or-NC (O) R¹-, wherein R¹Is C_1-6Alkyl radical, C_2-6Alkenyl, aryl or heteroaryl;

R²、R³and R⁴Each independently is H, R⁵or-C (O) R⁶Wherein each R is⁵Independently is C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl, and wherein each R is⁶Independently H, C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl.

13. The use of claim 10 or 11, wherein the at least one farnesyl dibenzodiazepine compound is AMO-01 or a pharmaceutically acceptable salt thereof,

14. the use of any one of claims 10-13, wherein the subject with PMS or a subject predisposed to PMS is a subject with a chromosome deletion at 22q 13.3.

15. The use of any one of claims 10-14, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepinone compound is from 0.1 μ g/kg to 200mg/kg of farnesyl dibenzodiazepinone compound per body weight of the subject.

16. The use of any one of claims 10-14, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepine compound is 80-160mg/m²And is delivered to the subject within 4-8 h.

17. The use of any one of claims 10-14, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepine compound is 120mg/m²And delivered to the subject within 6 h.

18. The use of any one of claims 10-17, wherein the at least one farnesyl dibenzodiazepinone compound or the pharmaceutical formulation comprising the at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is administered to the subject by intravenous or subcutaneous administration.

19. A therapeutically effective amount of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent, for use in the treatment of a subject having PMS or in a method of treating a subject having PMS.

20. A therapeutically effective amount of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent, for use in inhibiting the development of PMS in a subject susceptible to PMS or a method of inhibiting the development of PMS in a subject susceptible to PMS.

21. A therapeutically effective amount according to claim 19 or 20, wherein at least one farnesyl dibenzodiazepine compound is selected from the group consisting of those encompassed by formula I:

wherein the content of the first and second substances,

W¹、W²and W³Each independently is

Or the tricyclic chain may be at W³、W²Or W¹Is denoted by W³、W²Or W¹Are each-CH ═ O or-CH₂OH termination;

a is-NH-, -NCH₂R¹-or-NC (O) R¹-, wherein R¹Is C_1-6Alkyl radical, C_2-6Alkenyl, aryl or heteroaryl;

R²、R³and R⁴Each independently is H, R⁵or-C (O) R⁶Wherein each R is⁵Independently is C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl, and wherein each R is⁶Independently H, C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl.

22. The therapeutically effective amount of claim 19 or 20, wherein the at least one farnesyl dibenzodiazepine compound is AMO-01 or a pharmaceutically acceptable salt thereof,

23. a therapeutically effective amount according to any one of claims 19 to 22, wherein the subject suffering from PMS or a subject susceptible to PMS is a subject having a chromosome deletion at 22q 13.3.

24. The therapeutically effective amount of any one of claims 19-23, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepinone compound is from 0.1 μ g/kg to 200mg/kg of the farnesyl dibenzodiazepinone compound per body weight of the subject.

25. The therapeutically effective amount of any one of claims 19-23, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepine compound is 80-160mg/m²。

26. The therapeutically effective amount of any one of claims 19-23, wherein the therapeutically effective amount of at least one farnesyl dibenzodiazepine compound is 120mg/m²。

27. The therapeutically effective amount of any one of claims 19-26, wherein the at least one farnesyl dibenzodiazepine compound or a pharmaceutical formulation comprising the at least one farnesyl dibenzodiazepine compound and a pharmaceutically acceptable carrier or diluent is administered to the subject by intravenous or subcutaneous administration.

28. Use of at least one farnesyl dibenzodiazepinone compound for the preparation of a medicament for treating a subject suffering from PMS.

29. Use of at least one farnesyl dibenzodiazepinone compound for the preparation of a medicament for inhibiting the development of PMS in a subject predisposed to PMS.

30. The use of claim 28 or 29, wherein at least one farnesyl dibenzodiazepine compound is selected from a compound encompassed by formula I:

wherein the content of the first and second substances,

W¹、W²and W³Each independently is

a is-NH-, -NCH₂R¹-or-NC (O) R¹-, wherein R¹Is C_1-6Alkyl radical, C_2-6Alkenyl, aryl or heteroaryl;

R²、R³and R⁴Each independently is H, R⁵or-C (O) R⁶Wherein each R is⁵Independently is C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl, and wherein each R is⁶Independently H, C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl.

31. The use of claim 28 or 29, wherein at least one farnesyl dibenzodiazepine compound is AMO-01 or a pharmaceutically acceptable salt thereof,

32. the use of any one of claims 28-31, wherein the subject with PMS or a subject predisposed to PMS is a subject having a chromosome deletion at 22q 13.3.

33. The use of any one of claims 28-32, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepinone compound is from 0.1 μ g/kg to 200mg/kg of farnesyl dibenzodiazepinone compound per body weight of the subject.

34. The use of any one of claims 28-32, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepine compound is 80-160mg/m²。

35. The use of any one of claims 28-32, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepine compound is 120mg/m²。

36. The use of any one of claims 28-35, wherein the at least one farnesyl dibenzodiazepinone compound or the pharmaceutical formulation comprising the at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is administered to the subject by intravenous or subcutaneous administration.

Background

Phelan McDermid Syndrome (PMS) is a neurodevelopmental disorder that was first mentioned in Watt et al (1985) and later described in more detail by Phelan et al (2001).

Clinical features of PMS in humans include severe neonatal hypotonia (> 97% of individuals), overall developmental delay (> 98%), normal to accelerated growth (95%), loss to severe language delay (> 98%) and mild teratogenic features (Phelan 2008). Behaviorally, individuals with pheranmcdermid often present with low sensitivity to pain, poor communication with the eye, stereotypy, decreased social functioning, hyperactivity, altered sensorimotor function, and aggressive behavior. Behavioral phenotypes may degenerate with age, showing a decline in social, motor or mental function (Soorya et al, 2013). Physically, PMS may be associated with mild teratogenic features and lymphedema.

A key complication of PMS is epilepsy (epilepsy). As many as 41% of affected individuals develop seizures (fever and/or non-fever) (soraya et al, 2013). Holder and Quach (2016) also describe that 46% of phelan mcdermid individuals develop seizures at some point in their life and present all seizure types. The most common seizures are atypical absence seizures (90%), with other types including tonic (54%), hypotonia (18%), tonic-clonic (9%) and myoclonic (9%). 20% of individuals characterized by Holder and Quach have status epilepticus, while 8% are diagnosed with Lennox Gastaut syndrome. Seizures may be pharmacologically controlled or drug resistant. With age, there is an increasing tendency for susceptibility to seizures, and seizures are associated with a deterioration in functional skills (Soorya et al, 2013). Various anticonvulsants have been used, and multiple medications for seizures (poly-pharmacy) are common.

The disease is characterized in humans by a chromosomal deletion at 22q13.3 containing the SHANK3/ProSAP2 gene (Bonaglia et al, 2001; Anderlid et al, 2002; Wilson et al, 2003). Consensus suggests that signs and symptoms of PMS are related to the haploinsufficiency of the SHANK3 protein.

Shcheglovitov et al (2013) studies have shown that Induced Pluripotent Stem (IPS) cell lines extracted from PMS patients have impaired postsynaptic AMPA and NMDA receptor responses, which reflect less excitatory synapses. These defects were associated with a decrease in the levels of the longest isoform of the SHANK3 protein (Shcheglovitov et al, (2013)) and were remedied by administration of insulin-like growth factor 1 (IGF-1). The authors suggested that SHANK3 is essential in the early stages of synapse formation, while IGF-1 promotes synaptic maturation by triggering deletion of SHANK3 and recruitment of PSD95 protein. Bozdagi et al (2013) also showed that the phenotype of the Shank3 knockout mouse was rescued using IGF-1. Kolevzon et al (2014) reported that IGF-1 was administered under placebo-controlled conditions, resulting in improved social, repetitive and abnormal behavior in Shank3 knockout mice over the course of 12 weeks of treatment.

Although the united states is conducting studies on the role of intranasal oxytocin in PMS, clinical studies on pharmacological intervention in PMS are relatively rare (clinical trials. gov identification nct 02710084). There is no approved PMS treatment. Therefore, there is a need for methods for treating PMS; the invention herein presented is directed to such objects and other important ends.

Disclosure of Invention

The present invention relates to a method for treating Phelan McDermid Syndrome (PMS) in a subject suffering from, or susceptible to, Phelan McDermid Syndrome (PMS). As discussed more fully below, farnesyl dibenzodiazepinone (farnesyl dibenzodiazepinone) compounds have been found to alleviate the symptoms of disease in PMS subjects administered with the compounds.

Thus, in a first embodiment, the invention relates to a method of treating a subject with PMS, wherein the method comprises administering to a subject with PMS a therapeutically effective amount of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent.

In a second embodiment, the invention relates to a method of inhibiting the development of PMS in a subject predisposed to PMS, wherein the method comprises administering to the subject predisposed to PMS a therapeutically effective amount of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent.

In a third embodiment, the present invention relates to the use of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent in the treatment of a subject with PMS or in a method of treating a subject with PMS.

In a fourth embodiment, the present invention relates to the use of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent in a method of inhibiting the development of PMS in a subject susceptible to PMS or in a method of inhibiting the development of PMS in a subject susceptible to PMS.

In a fifth embodiment, the invention relates to a therapeutically effective amount of (i) at least one farnesyl dibenzodiazanone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazanone compound and a pharmaceutically acceptable carrier or diluent for use in a method of treating a subject having PMS or treating a subject having PMS.

In a sixth embodiment, the invention relates to a therapeutically effective amount of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent for use in a method of inhibiting the development of PMS in a subject susceptible to PMS or inhibiting the development of PMS in a subject susceptible to PMS.

In a seventh embodiment, the present invention relates to the use of at least one farnesyl dibenzodiazepine compound for the preparation of a medicament for treating a subject suffering from PMS.

In an eighth embodiment, the present invention relates to the use of at least one farnesyl dibenzodiazepinone compound for the preparation of a medicament for inhibiting the development of PMS in a subject predisposed to PMS.

In various embodiments and aspects of the invention, the at least one farnesyl dibenzodiazepinone is selected from a compound encompassed by formula I:

wherein the content of the first and second substances,

W¹、W²and W³Each independently is

OrOr the tricyclic chain may be at W³、W²Or W¹Is denoted by W³、W²Or W¹Are each-CH ═ O or-CH₂OH termination;

a is-NH-, -NCH₂R¹-or-NC (O) R¹-, wherein R¹Is C_1-6Alkyl radical, C_2-6Alkenyl (alkene), aryl or heteroaryl;

R²、R³and R⁴Each independently is H, R⁵or-C (O) R⁶Wherein each R is⁵Independently is C_1-6Alkyl radical, C_2-7Alkenylene (alkalene), aryl or heteroaryl, and wherein each R is⁶Independently H, C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl.

In various embodiments and aspects of the invention, the at least one farnesyl diphenyldiazepine compound can be compound AMO-01 or a pharmaceutically acceptable salt thereof.

In a related embodiment or aspect of the invention, the PMS-prone subject is a subject having a chromosomal deletion at 22q 13.3.

In various embodiments and aspects of the invention, the therapeutically effective amount of the at least one farnesyl dibenzodiazepinone compound or the pharmaceutical formulation comprising the at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is from about 0.1 μ g to about 200mg per kg agent per body weight of the subject.

In various embodiments and aspects of the invention, the at least one farnesyl dibenzodiazepinone compound or a pharmaceutical formulation comprising the at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is administered to the subject intravenously, subcutaneously, or otherwise acceptable.

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other processes for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent methods do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It should be expressly understood, however, that any of the descriptions, figures, embodiments, etc. are provided for the purpose of illustration and description only and are not intended as a definition of the limits of the present invention in any way.

Drawings

FIG. 1 shows the results obtained in Wild Type (WT) mice and Shank3b^-/-Activation of the Ras-ERK pathway in Knockout (KO) transgenic mice is indicated by pERK levels in hippocampus and cortex. "ns" is not significant; is p<0.0001。

Figure 2 anxiety was assessed by the light-dark box (light-dark box) test in the Shank3b gene knockout transgenic model of PMS 4 hours prior to testing in groups of n-10 mice given a single dose of 30mg/kg intraperitoneal injection of AMO-01 or vehicle (vehicle). For analysis ofThe overall analysis of variance for this experiment was significant (F ═ 31.19, p < 0.0001). Vehicle treated Wild Type (WT) mice and Shank3b^-/-The differences between knockout mice (KO) were significant (p < 0.001). AMO-01 had no significant effect in wild type mice. In contrast, AMO-01 significantly reduced Shank3b^-/-Defects in mice (p < 0.001), but this reversal was not complete because of AMO-01 treated wild-type mice and Shank3b^-/-There were still significant differences between mice (p < 0.05). "ns" is not significant; is p<0.05; is p<0.0001。

FIG. 3 self-injurious behaviour expressed by excessive self-combing (self-grooming) in the Shank3b KO transgenic model of PMS after a single 30mg/kg intraperitoneal injection of AMO-01. The overall analysis of variance used to analyze the experiment was significant (F ═ 29.14, p<0.0001). Vehicle treated Wild Type (WT) mice and Shank3b^-/-The difference between knockout mice was significant (p < 0.001). AMO-01 had no significant effect in wild type mice. In contrast, AMO-01 significantly reduced Shank3b^-/-Defects in mice (p < 0.0001). This reversal was complete because AMO-01 treated wild-type mice and post-treatment Shank3b^-/-There was no significant difference between mice. "ns" is not significant; is p<0.0001。

FIG. 4 response to social novelty (socinovelty) in Shank3b KO transgenic model of PMS after a single 30mg/kg intraperitoneal injection of AMO-01, expressed by the time it took to interact with familiar or new mice. Wild-type animals (WT) took more time to interact closely with strange (not seen before) mice than with familiar mice (seen before), and this difference was not affected by AMO-01 treatment. No such difference was observed for vehicle treated Shank3b KO mice, indicating that the process of social novelty was disrupted. Treatment with AMO-01 normalized the differential response of Shank3b KO mice to social novelty. The overall analysis of variance used to analyze this experiment was significant (F52.75, p < 0.0001). "ns" is not significant; is p < 0.0001.

FIG. 5 Shank3b KO of PMS after a single dose of 30mg/kg of AMO-01 injected intraperitoneallyThe balance beam walking behavior of the transgenic model (beam walking behavbior). The overall analysis of variance used to analyze the experiment was significant (F ═ 29.76, p<0.0001). Vehicle treated wild type mice and Shank3b^-/-The differences between knockout mice were significant (p)<0.001). AMO-01 had no significant effect on wild type mice. In contrast, AMO-01 significantly reduced Shank3b^-/-Defects expressed in mice. This reversal was complete because AMO-01 treated wild-type mice and post-treatment Shank3b^-/-There was no significant difference between mice. "ns" is not significant; is p<0.001。

FIG. 6 Marble burying behavior in Shank3b KO transgenic model of PMS (marble burying behavior) following intraperitoneal injection of AMO-01 at a single dose of 30 mg/kg. The overall analysis of variance used to analyze this experiment was significant (F-42.15, p)<0.0001). Vehicle treated wild type mice and Shank3b^-/-The difference of the knockout mice is obvious (p is less than 0.001). AMO-01 had no significant effect on wild type mice. In contrast, AMO-01 significantly reduced Shank3b^-/-Mice exhibited defects (p < 0.0001). This reversal was complete because of wild-type mice treated with AMO-01 and post-treatment Shank3b^-/-There was no significant difference between mice. "ns" is not significant; is p<0.001; is p<0.0001。

FIG. 7 Audio Seizure Threshold (Audio Seizure Threshold) of Shank3b KO transgenic model of PMS after intraperitoneal injection of AMO-01 at a single dose of 30 mg/kg.

FIG. 8. schedule of AMO-01 administration to Phelan McDermid syndrome subjects.

FIG. 9 Total dose injected over 6 hours was 120mg/m²Before and after, visual evoked potentials recorded from the visual cortex of 23 year old men with genetically confirmed PhelanMcDermid syndrome.

Detailed Description

I. Definition of

As used herein, "a" or "an" can mean one or more. As used herein, the terms "a" or "an" when used in conjunction with the word "comprising" may mean one or more than one. As used herein, "another" may mean at least a second or more. Furthermore, unless the context requires otherwise, singular terms include the plural, and plural terms include the singular.

As used herein, "about" refers to a numerical value, whether or not explicitly indicated, including, for example, integers, fractions, and percentages. The term "about" generally refers to a range of values (e.g., +/-5-10% of the stated value) that one of ordinary skill in the art would consider equivalent to the stated value (e.g., having the same function or result). In some instances, the term "about" may include numbers that are rounded to the nearest significant figure.

As used herein, "treatment" and all forms and tenses thereof, including, for example, treatment (treatment, treating, treated, and treatment), refer to both therapeutic treatment (therapeutic treatment) and prophylactic (therapeutic) or preventative (preventative) treatment. Subjects in need of treatment include those already diagnosed with PMS as well as those susceptible to PMS but not yet diagnosed with PMS.

II. the invention

As noted above, the present invention is based on the inventors' discovery that a particular farnesyl dibenzodiazepine compound can alleviate the symptoms of Phelan McDermid Syndrome (PMS) in a subject suffering from the disease and to whom the compound is administered. Accordingly, the present invention relates to a method of treating PMS in a subject suffering from, or susceptible to, such a disease.

A subject suffering from PMS or a subject susceptible to PMS is a subject having a chromosomal deletion at 22q13.3 and/or a subject having reduced expression of the SHANK3 protein thereof and/or a subject having produced a dysfunctional SHANK3 protein thereof. Chromosomal site 22q13.3 included the SHANK3/ProSAP2 gene encoding SHANK3 protein.

SHANK3 ("SH 3 and multiple ankyrin repeat domains 3"; also known as proline-rich synapse-associated protein 2(ProSAP2)) is a scaffold protein found in dendrites. It is a member of a family of scaffold proteins present in postsynaptic density involved in synapse formation and may be linked to actin cytoskeleton (actin cytoskeleton) and G-protein coupled signaling pathways via neurotransmitter receptors, ion channels and other membrane proteins (Boeckers et al, 2002). The SHANK3 message is expressed in heart, brain and spleen (Lim et al, 1999). In the brain, SHANK3 is mainly expressed in neurons.

Studies have shown that different deletions of SHANK3 have different (although similar) effects on the activity of the SHANK3 protein isoform and on changes in specific receptors. Various murine knockout mice have been developed that shorten the Shank3 gene. Different exons of the gene have been addressed, as have different results associated with the expression of isoforms of the SHANK3 protein. Bozdagi et al (2010) created a mouse with the Shank3 gene deleted between exons 4 and 9, including the ankyrin repeat domain. Expression of this modified heterozygote showed a 50% reduction in shank3 mRNA levels. Basal glutamatergic AMPA-mediated transmission was reduced in young heterozygotes (3 to 4 weeks of age) by presynaptic and postsynaptic mechanisms. In this connection, maintenance of high frequency and theta burst induced hippocampal long-term potentiation (LTP; a form of synaptic plasticity) is reduced, but long-term inhibition (LTD) is not. These electrophysiologically determined changes are paralleled by failure to maintain synaptic volume with LTP increase and decrease AMPA receptor passage. These mice show a social interaction deficiency. Bozdagi et al (2013) showed that the defects in this model were reversed by IGF-1.

Krouser et al (2013) reported homozygous deletion of Shank3 exon 21 in studied 2 to 6 month old mice. This deletion reduced the major isoform of the SHANK3 protein in the hippocampus, but had the complex effect of the appearance of the shorter isoform. The glutamate receptors of this mouse show no major changes, except for the mGluR5 subtype of metabotropic glutamate receptors. Accordingly, hippocampal LTP is deficient, reflecting a decrease in NMDA/AMPA ratio. In these mice, there was no mGluR dependent LTD deficiency. These mice did not differ in dendritic spine density and complexity.

Heterozygote mice with deletion of the c-terminus (exon 21) of Shank3 were created and reported by Duffney et al (2015), indicating a 50 to 70% reduction in Shank3 protein. Teenagers show impaired social interaction testing behavior. NMDA-mediated EPSPs were reduced in the prefrontal cortex of these mice, while AMPA-mediated responses were intact. This finding was associated with a decrease in membrane NMDA receptor subunits. These mice also showed a decrease in rac-dependent silk-cutting protein (cofilin) signaling and, therefore, a decrease in synaptic actin-however, there was no change in dendritic spine density and, therefore, in synaptic number in these mice. The mice described by Bozdagi et al (2010) have N-terminal deletions, which result in the loss of the longest isomer, and also have a silk-cutting protein deficiency. Uppal et al (2015) investigated heterozygote N-terminal gene knockouts at 5 weeks and 3 months of age, and found no difference in synaptic density between these two ages. However, detailed examination showed that the number of punctured synapses increased at 5 weeks of age, but not at 3 months of age. At both ages, there was no difference in spine, head size, PSD length and PSD area at any age. Jaramilo et al (2016) reported the deletion of heterozygote exons 4 to 9N-terminal in mice 3 to 5 months of age at the time of study. This procedure results in a change in the complex pattern of the isoforms of the SHANK3 protein. These mice showed a decrease in hippocampal LTP, while LTD dependent on mGluR5 was intact. In the hippocampal region, synaptic transmission and the NDMA/AMPA ratio are unaffected, but in the striatum, the NMDA/AMPA ratio decreases due to a decrease in NMDA receptor function.

Peca et al (2011) generated knockout mice in which exons 13-16 of the Shank3 gene were targeted, deleting the PDZ domain. This gene knockout resulted in complete elimination of the SHANK3 α (longest isomer) and SHANK β isomers, and a significant reduction in the putative SHANK3 γ isomer. Homozygote forms of this mouse show reduced levels of SAPAP3, Homer-1b/c, and PSD93 in the striatum, as well as reduced glutamate receptor subunits GluR2, NR2A, and NR 2B. With the complexity of dendritic trees and the increase in total dendritic length in the striatum, this is accompanied by a decrease in dendritic spine density. In the absence of changes in the NMDA/AMPA ratio, population spikes (population spikes) in the striatum were reduced due to a decrease in post-synaptic AMPA responses. The hippocampal electrophysiological phenomena of these mice were not altered.

Thus, the loss of synaptic plasticity in the hippocampal between models is large in terms of synaptic impact, but different Shank3 deletions have different effects in terms of Shank3 protein isoforms and different specific receptor changes.

As reported by Shcheglovitov et al, the effect of knocking out the Shank3 gene in mice can be compared to an IPS cell line extracted from a PhellanMcDermid syndrome patient (2013). These authors report the following: postsynaptic AMPA and NMDA responses in PhelanMcDermid syndrome IPS cells were significantly impaired without differences in gabaergic inhibition. These changes reflect less excitatory synapses and are associated with reduced levels of the longest isoform of SHANK3 protein.

By referring to IPCs studies with increased input resistance compared to control neurons, which may be associated with decreased subunit expression of hyperpolarized activated cyclic nucleotide gated (HCN) channels, Holder and Quach (2016) cite data from Shcheglovitov et al (2013) related to increased epileptic sensitivity seen in phelan mcdermid syndrome. Holder and Quach proposed that "there is a tendency to increase excitability, potentially indicating an increased prevalence of epilepsy in individuals with a mutation in the SHANK3 gene compared to the general population".

The key molecular aberration of PMS is the overactivation of the Ras-ERK enzymatic pathway, which plays an important role in synaptic plasticity and neuronal survival (Grewal et al, 1999). The Ras-ERK pathway is also associated with the occurrence of seizures, based on evidence of a series of activations of this pathway in different experimental models and in the use of Ras-ERK pathway inhibitors. Ras-ERK pathway activation is indicated by the level of phosphorylated form of ERK (pERK). This activation is seen in various adaptations of brain excitability-based synaptic mechanisms. Davis et al (2000) showed that induction of hippocampal LTP in rats was accompanied by a rapid but transient increase in pERK.

Upon such over-activation, and as described below, at C57BL/6 of PMS^-/-Brain penetration inhibitors of the Ras-ERK pathway (referred to herein as AMO-01) were evaluated in a gene knockout mouse model. It was found that the administration of a single intraperitoneal dose of AMO-01 to two months old test animals significantly improved all abnormal neurobehavioral characteristics associated with the mice, thus establishing the basis for the present invention.

Farnesyl dibenzodiazepinone

AMO-01 (10-farnesyl-4,6,8-trihydroxy-dibenzodiazepin-11-one) is a farnesyl-dibenzodiazepin and is a member of a class of dibenzodiazepin compounds that contain a farnesyl group.

The structure of AMO-01 is as follows:

the methods of the invention can be practiced by administering AMO-01 or a pharmaceutically acceptable salt thereof to a subject having PMS or a subject susceptible to PMS.

Farnesyl dibenzodiazepinone compounds are prepared from strains of the genus Micromonospora (Micromonospora) which is a gram-positive spore, usually aerobic, of the family Micromonosporaceae and forms a branched mycelium. Members of this genus also commonly produce aminoglycoside antibiotics.

AMO-01 was prepared from Micromonospora strain 046-ECO 11. Strain 046-ECO11 was deposited at International Depositary Authority of Canada (IDAC), Bureau of microbiology, Health Canada,1015Arlington Street, Winnipeg, Manitoba, CanadaR3E 3R2) on 7.3.2003 with accession number 070303-01. More details on strain 046-ECO11 and methods of making AMO-01 can be found in international patent publication WO 2004/065591, published 8/5 2004, the contents of which are incorporated herein by reference.

In addition to AMO-01 and pharmaceutically acceptable salts thereof, each of the methods of the invention can be practiced by administering to a subject suffering from PMS or susceptible to PMS at least one farnesyl dibenzodiazepinone compound selected from a compound encompassed by formula I:

wherein the content of the first and second substances,

W¹、W²and W³Each independently is

OrOr the tricyclic chain may be at W³、W²Or W¹Is denoted by W³、W²Or W¹Are each-CH ═ O or-CH₂OH termination;

a is-NH-, -NCH₂R¹-or-NC (O) R¹-, wherein R¹Is C_1-6Alkyl radical, C_2-6Alkenyl, aryl or heteroaryl;

R²、R³and R⁴Each independently is H, R⁵or-C (O) R⁶Wherein each R⁵Independently is C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl, and wherein each R⁶Independently H, C_1-6Alkyl radical, C_2-7Alkenylene, aryl or heteroaryl.

In a particular embodiment, the present invention provides a compound of formula I wherein a is selected from NH, NCH₂R¹And NC (O) R¹(ii) a Wherein R is²Is H; r³Is H; and R is⁴Is H. In another embodiment, R²、R³And R⁴Each is H; and all other groups are as previously defined. In another embodiment, R²、R³And R⁴Each is H; and W¹is-CH ═ CH-, all other groups are as previously defined. In another embodiment, R²、R³And R⁴Each is H, and W²is-CH ═ CH-, all other groups are as previously defined. In another embodiment, R²、R³And R⁴Each is H; and W³is-CH ═ CH-; and all other groups are as previously defined. In another embodiment, a is NH; r²、R³And R⁴Each is H; and all other groups are as previously defined. In another embodiment, a is NH; w¹、W²And W³Each is-CH ═ CH-; and all other groups are as previously defined. The present invention includes all pharmaceutically acceptable salts of the above compounds.

The following are additional exemplary compounds of formula I and the farnesyl dibenzodiazepinone compounds of the invention:

as used herein, the term "alkyl" refers to a straight or branched chain hydrocarbon group. Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl, heptyl, cyclopentyl, cyclohexyl, cyclohexylmethyl (cyclohexexymethyl), and the like. Alkyl may be optionally substituted with a substituent selected from: acyl, amino, amido, acyloxy, carboalkoxy (carboalkoxy), carboxy, carboxyamino (carboxyamidio), cyano, halogen, hydroxy, nitro, thio (thio), alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, carbonyl (oxo), guanidino, and formyl.

The term "alkenyl" refers to a straight, branched, or cyclic hydrocarbon group containing at least one carbon-carbon double bond. Examples of alkenyl groups include, but are not limited to, vinyl, 1-propen-2-yl, 1-buten-4-yl, 2-buten-4-yl, l-penten-5-yl, and the like. The alkenyl group may be optionally substituted with a substituent selected from: acyl, amino, amido, acyloxy, carboalkoxy, carboxy, carboxyamino, cyano, halogen, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, formyl, carbonyl, and guanidino. The double bond portion of the unsaturated hydrocarbon chain may be in either the cis or trans configuration.

The terms "cycloalkyl" and "cycloalkyl ring" refer to saturated or partially unsaturated carbocyclic rings in a single or fused carbocyclic ring system having three to fifteen ring members. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl. Cycloalkyl groups may be optionally substituted with the following substituents: acyl, amino, amido, acyloxy, carboalkoxy, carboxy, carboxyamino, cyano, halogen, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

The terms "heterocyclyl" and "heterocycle" refer to monocyclic or fused ring systems containing from one to four heteroatoms or containing atoms selected from O, N, NH, NRx, PO in a monocyclic or fused ring system having from three to fifteen ring members₂A saturated or partially unsaturated ring of a hetero group of S, SO or SO. Examples of heterocyclyl or heterocyclic rings include, but are not limited to, morpholinyl, piperidinyl, and pyrrolidinyl. Heterocyclyl, heterocyclic or heterocyclic ring optionally selectedSubstituted with a substituent selected from: acyl, amino, amido, acyloxy, carbonyl, thiocarbonyl, imino, carboalkoxy, carboxyl, carboxyamino, cyano, halogen, hydroxy, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

The term "amino acid" refers to any natural amino acid; natural amino acids are well known to those skilled in the art.

The term "halogen" refers to halogen atoms such as bromine, chlorine, fluorine and iodine.

The terms "aryl" and "aryl ring" refer to an aromatic group in a monocyclic or fused ring system having five to fifteen ring members. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, biphenyl, terphenyl. Aryl groups may be optionally substituted with one or more substituents selected from: acyl, amino, amido, acyloxy, azido, alkylthio, carboalkoxy, carboxyl, carboxyamino, cyano, halogen, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, and formyl.

The terms "heteroaryl" and "heteroaryl ring" refer to rings in a single or fused ring system having five to fifteen ring members and containing at least one heteroatom such as O, N, S, SO and SO₂An aromatic group of (2). Examples of heteroaryl groups include, but are not limited to, pyridyl, thiazolyl, thiadiazolyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazolyl, triazolyl, and pyrrolyl. The heteroaryl group may be optionally substituted with one or more substituents selected from: acyl, amino, amido, acyloxy, carboalkoxy, carboxy, carboxyamino, cyano, halogen, hydroxy, nitro, thio, thiocarbonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

The terms "aralkyl" and "heteroaralkyl" refer to an aryl group or heteroaryl group, respectively, bonded directly through an alkyl group (e.g., benzyl). Aralkyl and heteroaralkyl groups may be optionally substituted with aryl and heteroaryl groups.

Similarly, the terms "arylalkenyl" and "heteroarylalkenyl" refer to an aryl or heteroaryl group, respectively, bonded directly through an alkenyl group (e.g., benzyl). Aralkenyl and heteroaralkenyl may be optionally substituted with aryl and heteroaryl.

The compounds of the present invention may have one or more asymmetric carbon atoms and may exist as optical isomers forming mixtures of racemic or non-racemic compounds. The compounds of the present invention may be used as a single isomer or as a mixture of stereochemically isomeric forms. Diastereomers, i.e. stereoisomers which are not superimposable, may be separated by conventional methods (e.g. chromatography, distillation, crystallization or sublimation). The optical isomers can be obtained by resolution of the racemic mixture according to conventional methods.

As used herein, reference to a subject with PMS is a subject identified as having a chromosomal deletion at 22q13.3 with at least two of the following symptoms: hypotonia (low muscle tone), normal or accelerated growth, lack or severe speech retardation, and overall developmental delay.

As used herein, reference to a subject susceptible to PMS is a subject identified as having a chromosomal deletion at 22q13.3 and/or having at least two of the following symptoms: hypotonia (low muscle tone), normal or accelerated growth, lack or severe speech retardation, and overall developmental delay.

Obviously, the identification of chromosomal deletions in subjects with PMS or susceptible to PMS can vary in size, number and at specific positions in the locus. Furthermore, since PMS is thought to be associated with haploid insufficiency of SHANK3, the chromosomal deletion at 22q13.3 can be in one or both alleles. When in two alleles, the deletions between alleles may be different.

As used herein, a reduction in the expression of SHANK3 protein in a subject is at least a 30% reduction in the expression of SHANK3 protein relative to a subject without a chromosomal deletion at 22q 13.3. The reduction in expression of SHANK3 protein may be at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more relative to a subject without a chromosomal deletion at 22q 13.3.

As used herein, a malfunctioning SHANK3 protein is a SHANK3 protein that does not support normal synaptic function.

Methods of treatment

The present invention relates to a method of treating a subject suffering from PMS or a subject susceptible to PMS. The methods of the invention comprise administering to a subject (e.g., a subject having PMS or a subject susceptible to PMS) a therapeutically effective amount of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent. In certain embodiments, the at least one farnesyl dibenzodiazepinone compound is AMO-01.

The invention also relates to the use of (i) at least one farnesyl dibenzodiazanone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazanone compound and a pharmaceutically acceptable carrier or diluent in (a) a method of treating a subject suffering from PMS, (b) a method of treating a subject suffering from PMS, (c) a subject susceptible to PMS, or (d) a method of treating a subject susceptible to PMS. In certain embodiments, the at least one farnesyl dibenzodiazepinone compound is AMO-01.

The present invention also relates to a therapeutically effective amount of (i) at least one farnesyl dibenzodiazepinone compound or (ii) a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent for use in (a) treating a subject with PMS, (b) a method of treating a subject with PMS, (c) treating a subject susceptible to PMS, or (d) treating a subject susceptible to PMS. In certain embodiments, the at least one farnesyl dibenzodiazepinone compound is AMO-01.

The invention also relates to the use of at least one farnesyl dibenzodiazepinone compound for the manufacture of a medicament for treating a subject suffering from PMS, and the use of at least one farnesyl dibenzodiazepinone compound for the manufacture of a medicament for inhibiting the development of PMS in a subject predisposed to PMS. In certain embodiments, the at least one farnesyl dibenzodiazepinone compound is AMO-01.

As used herein, the terms "treatment (treating, and treating)" have ordinary and customary meanings and include one or more of the following: alleviating symptoms of PMS in a subject suffering from PMS or a subject susceptible to PMS; preventing PMS symptoms in such a subject; preventing or reducing PMS symptom recurrence in such subject; reducing the severity and/or frequency of PMS symptoms in such a subject; preventing the development of PMS symptoms in such a subject; and curing or addressing PMS symptoms in such subjects. Treatment refers to a reduction in the degree of, etc., from about 1% to about 100% relative to an untreated subject. Preferably, the degree of remission, etc., is about 100%, about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, or about 30% relative to an untreated subject. Treatment may begin before, at the same time, or after the onset of clinical symptoms of the disease. Thus, a subject may be exhibiting symptoms of PMS or simply diagnosed as susceptible to PMS but not currently exhibiting symptoms of the disease. The outcome of the treatment may be permanent or may last for days (e.g., 1, 2, 3, 4, 5, or 7 days), weeks (e.g., 1, 2, 3, or 4 weeks), or months (e.g., 1, 2, 3, 4, 5, 6, or more months).

When practicing the methods of the invention, the amount of at least one farnesyl dibenzodiazepinone administered to a subject will vary depending on factors such as the age and weight of the subject, and the characteristics (identity) and severity of the symptoms of the disorder. However, the amount administered to the subject will be a therapeutically effective amount, i.e., an amount sufficient for treatment. As one example, a therapeutically effective amount of at least one farnesyl dibenzodiazepinone is about 0.1 μ g to 200mg per kg of body weight of the subject. Additional ranges of therapeutically effective amounts include, but are not limited to, about 10 μ g/kg to 200mg/kg, about 100 to 200mg/kg, about 1 to 200mg/kg, about 10 to 100mg/kg, about 100 to 100mg/kg, about 1 to 100mg/kg, about 10 to 100mg/kg, about 0.1 to 50mg/kg, about 1 to 50mg/kg, about 10 to 50mg/kg, about 100 to 50mg/kg, about 1 to 40mg/kg, about 1 to 30mg/kg, about 1 to 20mg/kg, about 1 to 10mg/kg, about 1 to 5mg/kg, About 0.1mg/kg to 10mg/kg, about 0.1mg/kg to 5mg/kg, about 10mg/kg to 50mg/kg and about 20 μ g/kg to 40mg/kg of the subject's body weight. Specific examples of therapeutically effective amounts include, but are not limited to, about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60mg/kg of body weight of the subject. In other words, the therapeutically effective amount of the at least one farnesyl dibenzodiazepinone is about 1mg/m²To about 1000mg/m². Additional ranges of therapeutically effective amounts include, but are not limited to: about 1mg/m²To about 1000mg/m²About 1mg/m²To about 500mg/m²About 1mg/m²To about 400mg/m²About 1mg/m²To about 300mg/m²About 1mg/m²To about 250mg/m²About 1mg/m²To about 200mg/m²About 1mg/m²To about 150mg/m²About 1mg/m²To about 100mg/m²About 50mg/m²To about 1000mg/m²About 50mg/m²To about 500mg/m²About 50mg/m²To about 400mg/m²About 50mg/m²To about 300mg/m²About 50mg/m²To about 200mg/m²About 50mg/m²To about 150mg/m²About 50mg/m²To about 100mg/m²About 100mg/m²To about 1000mg/m²About 100mg/m²To about 500mg/m²About 100mg/m²To about 400mg/m²About 100mg/m²To about 300mg/m²About 100mg/m²To about 200mg/m²About 100mg/m²To about 150mg/m². Specific examples of a therapeutically effective amount include, but are not limited to, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200mg/m²Or more.

The at least one farnesyl dibenzodiazepinone compound can be administered to the subject one or two, three, four, five, six or more times during a course of treatment. The time interval between doses in a dosage regimen may be days, weeks, months or years, including once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more weeks. The same amount of at least one farnesyl dibenzodiazepine is administered in each dose of the dosing regimen, or the amount in each dose can vary. Appropriate dosages and dosing regimens can be readily determined by techniques well known to those of ordinary skill in the art without undue experimentation. This manner of determination will be based in part on the tolerability and efficacy of the particular dose.

The frequency of administration includes 4, 3, 2, or 1 time per day, every other day, every third day, every fourth day, every fifth day, every sixth day, once per week, every eighth day, every ninth day, every tenth day, every second week, every month, and every second month. The duration of treatment will be based on the symptoms and severity of the disease in the particular subject and will be determined by the attending physician. However, the duration of treatment is expected to last days, weeks, months or years. Indeed, in certain instances, treatment may be continued throughout the subject's life.

Depending on the mode of administration, the dose may be administered all at once, for example as an oral formulation in capsule or liquid form, or slowly over a period of time, for example intramuscularly or intravenously. When administered over a period of time, the course of administration may be, for example, 10, 20, 30, 40, 50, or 60min, or 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10h, or longer. In one embodiment of the invention, 6 is smallThe dose delivered to the subject over time is 120mg/m²。

As used herein, a "subject" includes, but is not limited to, a human, a non-human primate, a bird, a horse, a cow, a goat, a sheep, a companion animal (e.g., a dog, cat, or rodent), or other mammal.

Formulation and dosage

In each of the methods of treatment of the present invention, at least one farnesyl dibenzodiazepinone compound and the pharmaceutical formulation are administered in a pharmaceutically acceptable form and in a substantially non-toxic amount.

Pharmaceutical formulations of the invention will comprise at least one farnesyl dibenzodiazepine compound as described herein. The pharmaceutical formulation will also comprise one or more pharmaceutically acceptable carriers or diluents.

Suitable examples of carriers and diluents are well known to those skilled in the art and include water, water for injection, saline, buffered saline, dextrose, glycerol, ethanol, propylene glycol, polysorbate 80 (Tween-80)^TM) Polyethylene glycols 300 and 400(PEG300 and 400) pegylated castor oil (e.g., cremophor el), poloxamers 407 and 188, hydrophilic and hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, pegylated phospholipids, polymer matrices, biocompatible polymers, lipid spheres, vesicles, particles, and liposomes. The term specifically excludes cell culture media. The formulations may additionally include stabilizers, buffers, antioxidants and preservatives, tonicity agents (tonicityagents), bulking agents (bulking agents), emulsifiers, suspending or viscosity agents, inert diluents, fillers (filler), and combinations thereof.

The nature of the carrier and diluent will depend on the manner in which the pharmaceutical formulation is used and the manner in which the pharmaceutical formulation is administered to a subject. For example, a pharmaceutical formulation may be prepared in the form of a formulation for intramuscular injection, wherein the carrier is water for injection, 0.9% saline or 5% dextrose solution.

In addition, the pharmaceutical formulation may be administered in liquid form. The liquid may be an oral, eye or nasal drop, or used as an enema or irrigation fluid. When the pharmaceutical formulation is formulated as a liquid, the liquid can be a solution or a suspension of the pharmaceutical formulation. There are a variety of suitable formulations for solutions or suspensions of pharmaceutical formulations well known to those skilled in the art, depending on their intended use. Liquid formulations for oral administration prepared in water or other aqueous vehicles may contain various suspending agents, for example, methylcellulose, alginates, tragacanth (tragacanth), pectin, sodium alginate, carrageenan, acacia, polyvinylpyrrolidone, and polyvinyl alcohol. Liquid preparations may also include solutions, emulsions, syrups and elixirs containing the active compound, wetting agents, sweetening agents and coloring and flavoring agents.

Farnesyl dibenzodiazepinone compounds and pharmaceutical formulations can be formulated and administered by any known conventional method, including but not limited to oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, pulmonary, topical, or parenteral administration. Parenteral administration includes, but is not limited to, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), intraarterial, intramedullary (intramedulary), intracardiac, intraarticular (joints), intrasynovial (synovial area), intracranial, intraspinal, and intrathecal (spinal fluid). Any known device for parenteral injection or infusion of pharmaceutical formulations may be used for such administration.

V. example 1

Mouse

Exon 13-16 deleted Shank3b originally obtained from Jackson laboratories^-/-Homozygous mice, and Wild Type (WT) littermates generated in a C57BL/6J background, and backcrossed repeatedly over eight generations on a C57BL/6J background. The resulting Shank3 knock-out (KO) mice were placed in a temperature and humidity controlled room in groups of the same genotype and subjected to a 12 hour light-dark cycle (lit at 7 am to 7 pm). Room temperature and humidity were continuously recorded in the holding chamber. The test was performed during the light phase. Food and water are available ad libitum. Shank3 knockout mice and their wild-type littermates were tested, N-10 mice per treatment group, at two months of age in the behavioral experiments. Mice were placed in commercial plastic cages, and tested 198Experiments were performed in the United kingdom animal (science procedures) Act (UK Animals (scientific procedures) Act) in 6 years. All experiments were performed on those with unknown genotype and drug therapy. Animals were allowed at least one week of acclimation prior to any experiment. Only healthy animals were used. No prophylactic or therapeutic treatment was performed during the acclimation period.

Ras-ERK Activity

In the initial experiment, the activity of the Ras-ERK pathway in brain and lymphocytes relative to untreated animals was determined. Comparison N-group of 10 mice. To determine the levels of brain biomarkers, the following methods and reagents were used. 20. mu.g of protein lysate (lysate) were separated from each sample on an 8-16% sodium dodecyl sulfate polyacrylamide electrophoresis gel (Bio-Rad, 161-0111222) and transferred to nitrocellulose (Bio-Rad, 162-0115). Membranes were treated after ECL immunoblotting substrate (Pierce, 32106) with (ERK)1/2(1/2000) (Cell Signaling). Total ERK1/2 protein content (phosphorylation) was assessed by blotting membranes with an anti-phospho (antiphospho) -ERK antibody (1/2000) (Cell Signaling). ERK phosphorylation was normalized to the protein content in the same sample and expressed as% change from basal conditions, with basal levels taken as 100%. Protein loading was assessed by stripping and re-blotting membranes with b-actin antibody (1/1000) (sigma chemical Co.). Phosphorylation-ERK 1/2(Thr-202/Tyr-204) (Cell Signaling)#4370) Total ERK, p44/42MAPK (Erk1/2) antibody #9102 (CellSignaling)). For the loaded control samples, the membranes were peeled off and re-probed with anti-tubulin antibody (Sigma, a 3853). For the determination of lymphocyte biomarkers, FACSTArplus (Becton Dickinson) was used, in which the excitation laser was tuned at 488nm and the green fluorescence from FITC (GST) was collected through a 515-545nm band pass filter. The mean FITC fluorescence intensity relative to the fluorescence of the reference cells was calculated. Mean cell fluorescenceLight intensity (MFI) is proportional to the average number of Ab molecules bound per cell.

Behavioral testing

AMO-01 from AMOPharma Ltd was administered to mice as follows:

A. intraperitoneal injection of AMO-01 in a single dose of 30mg/kg in an excipient of ethanol, PEG400, polysorbate 80 and 5% dextrose

B. The dosage of the excipient for intraperitoneal injection is 15mL/kg

On the first day of testing, groups of 10 mice were treated 4h prior to testing. Following a single intraperitoneal administration on the first day, a 5 day test was performed.

The following groups were studied:

1.Shank3b^-/-knockout mice + vehicle, N ═ 10

2.Shank3b^-/-Knockout mice + AMO-0130mg/kg intraperitoneal injection, N10

3. Wild type littermate + excipient, N10

4. Wild type littermate + AMO-0130mg/kg for intraperitoneal injection, wherein N is 10

The following tests were performed.

Day 1: increased anxiety in the light and dark box experiments. The test was performed in a two chamber tester (Med Associates, st. albans, VT), with one "dark chamber" always kept in the dark and the other chamber illuminated during the test as a "bright chamber". When a mouse is placed in the dark chamber, the light chamber is illuminated and the door between the two chambers is opened. The mice were allowed to explore the device freely for 5 min. The waiting time to enter the light cavity from the dark cavity and the percentage of time spent in the light cavity are used as indicators of anxiety-like behavior.

Day 2: excessive combing results in skin damage. Shank3b^-/-Knockout mice develop distinct skin lesions of varying degrees in the general population under storage. During this test, the time spent combing the following across all sequences was scored by an observer who did not understand the genotype during the scoring process: facial wipes, combing/rubbing of the head and ears, and whole body combing.

Day 3: social recognition abnormalities and social novelty reactions. The test consisted of three phases that placed various stimuli in the bilateral chambers, respectively. Stage 1 includes two identical non-social stimuli (NS1& NS1), stage 2 includes one non-social stimulus (NS1) and one social stimulus (Soc1), and stage 3 includes one known social stimulus (Soc1) and one new social stimulus (Soc 2). The preference index of one stimulus versus the other in each phase is compared.

Day 4: sensory motor function was evaluated by the balanced wood walking test. A standard paradigm was used to evaluate the balance beam walking performance (drop delay).

Day 5: activities of daily living/species typical behavior was assessed by marble burial. Marble burial behavior was evaluated using a standard paradigm.

Seizure thresholds were assessed by vocal source seizures in separate cohorts of N-10 each. Seizures were induced using a sustained stimulus of 60s (124 db). Blinded observers scored motor response rate and seizure severity after auditory stimulation according to classification: racing, seizure and respiratory arrest.

The results of the Ras-ERK pathway functional assay are shown in FIG. 1, where activation of the Ras-ERK pathway in Wild Type (WT) and Shank3 knock-out (KO) mice is indicated by the indicated levels of pERK in hippocampus and cortex. No change was observed in the hippocampus, similar to the lack of effect observed in this region using electrophysiology in studies by Peca et al (2011). In contrast, an almost three-fold increase was observed in pERK in the cortex (changes in striated cortex of this mouse were reported in Peca et al 2011). A significant increase was also observed in peripheral circulating lymphocytes (data not shown).

Figure 2 provides results of the light and dark box test showing rescue of light and dark box anxiety-mediated behavior by AMO-01 to Shank3b KO mice. In the group of N-10 mice, anxiety was assessed by the light and dark box test in the Shank3b KO transgenic model of PMS with a single 30mg/kg dose of vehicle administered 4h after peritoneal injection of AMO-01 or prior to testing. Gross analysis of variance significance for analysis of the experiment(F＝31.19，p<0.0001). Vehicle treated wild type mice and Shank3b^-/-The differences between Knockout (KO) mice were significant (p < 0.001). AMO-01 had no significant effect on wild type mice. AMO-01 significantly reduced the number of peaks in Shank3b^-/-Defects were shown in mice, but this reversal was not complete because of wild-type mice treated with AMO-01 and Shank3b^-/-The difference between KO mice was significant (P < 0.05).

Figure 3 provides the results of the combing test showing the rescue of excessive combing behavior by AMO-01 in Shank3b KO mice. Self-injurious behaviour as indicated by excessive self-combing in the Shank3b KO transgenic model of PMS following a single peritoneal injection of AMO-01 at 30 mg/kg. The overall analysis of variance used to analyze this experiment was significant (F ═ 29.14, p < 0.0001). Vehicle treated wild type mice and Shank3b^-/-The difference between knockout mice was significant (p < 0.001). AMO-01 had no significant effect on wild type mice. AMO-01 significantly reduces Shank3b^-/-Defects shown in mice (p < 0.0001). This reversal was complete because of wild type mice treated with AMO-01 and post-treatment Shank3b^-/-There was no significant difference between KO mice.

Figure 4 provides the results of the social approval and test for social novelty response showing rescue of social novelty response by AMO-01 in Shank3b KO mice. Social novelty response was expressed by the time it took to interact with a familiar or new mouse in the Shank3b KO transgenic model of PMS after a single 30mg/kg intraperitoneal injection of AMO-01. The overall analysis of variance used to analyze this experiment was significant (F52.75, p < 0.0001).

Figure 5 provides the results of the balance beam walk test showing AMO-01 rescue of balance beam walk behavior in Shank3b KO mice. Balance beam walking behavior of the Shank3b KO transgenic model of PMS after intraperitoneal injection of AMO-01 at a single dose of 30 mg/kg. The overall analysis of variance used to analyze this experiment was significant (F ═ 29.76, p < 0.0001). Vehicle treated wild type mice and Shank3b^-/-The difference between knockout mice was significant (p < 0.001). AMO-01 on wild type miceThere was no significant effect. AMO-01 significantly reduces Shank3b^-/-Mice showed defects (p < 0.0001). This reversal was complete because AMO-01 treated wild-type mice and treated Shank3b^-/-There was no significant difference between KO mice.

Fig. 6 provides the results of the marble burial test, which shows the rescue of AMO-01 to marble burial behavior in Shank3b KO mice. Marble burying behavior in the Shank3b KO transgenic model of PMS after intraperitoneal injection of AMO-01 at a single dose of 30 mg/kg. The overall analysis of variance used to analyze this experiment was significant (F ═ 42.15, p < 0.0001). Wild type mice and Shank3b treated in vehicle^-/-The difference between knockout mice was significant (p < 0.001). AMO-01 had no significant effect on wild type mice. AMO-01 significantly reduces Shank3b^-/-KO mice showed defects. This reversal was complete because AMO-01 treated wild-type mice and post-treatment Shank3b^-/-There was no significant difference between KO mice.

Figure 7 provides the results of the seizure threshold test showing rescue of reduced phonogenic seizure threshold by AMO-01 in Shank3b KO mice. Threshold value of endogenous seizure in Shank3b KO transgenic model of PMS after intraperitoneal injection of AMO-01 at a single dose of 30 mg/kg.

Thus, it can be seen that the experiments provided herein demonstrate Shank3b in PhelanMcDermid syndrome^-/-In a homozygous knockout mouse model, the Ras-ERK pathway is activated in the cortex (by a significant fold) and is unchanged in the hippocampus. These data are consistent with those reported by Peca et al (2011), which shows that in this model, the plasticity of the striatal cortical pathway is altered in the absence of hippocampal changes. The increase in Ras-ERK pathway activation is consistent with a decrease in the seizure threshold in the cortex as in Nateri et al (2007).

The peritoneal injection of AMO-01 administered at 30mg/kg had no effect on anxiety, grooming behavior, social ability, sensory motor function or species-typical behavior or cognition in wild-type mice. These data are consistent with the behavioral testing results of wild type mice previously using this compound. In contrast, this dose reversed the defects of all these regions in the Shank3 gene knockout transgenic mouse model of Phelan McDermid syndrome. This reversal indicates a significant normalization of function in the areas of anxiety and phonogenic seizures, and a complete normalization of function in grooming, social interaction, sensorimotor and species-typical behavior. After a single dose of AMO-01, the phenotype of phelan mcdermid syndrome can be widely cured and lasted for at least 5 days.

VI. example 2

AMO-01 was administered to 23 year old men who were genetically confirmed to be phelan mcdermid syndrome.

The drug substance under investigation was prepared from a sterile bulk formulation containing 32.8mg/mL (3.15% w/w) of the active drug substance dissolved in a sterile 0.9% saline solution to make an isotonic final dosage formulation. Each vial contained 70mg amo-01 drug substance in 2.134mL of the formulation (plus 2% excess). This corresponds to a total drug content of 71.4mg of active drug substance per 2.176mL of formulation. AMO-01 was administered by intravenous infusion in a single dose over 6 hours, with a total dose of 120mg/m²。

Seizure frequency and Visual Evoked Potentials (VEPs) were evaluated along with clinical testing. VEPs provide a non-invasive technique for assessing the functional integrity of the cerebral visual pathway from the retina to the visual cortex through optic nerve/light irradiation. VEPs are recorded from the head surface on the visual cortex and extracted from the ongoing EEG by signal averaging. VEP reflects the sum of excitatory and inhibitory postsynaptic potentials occurring on apical dendrites (Zemon et al, 1986), which regulate the excitatory and inhibitory signals received by pyramidal cells. The major positive and negative peaks and troughs in the VEP waveform reflect different cellular events. The contrast-reversing chequerboard stimuli (contrast-reversing chequerboards stimulus) used in this study produced a positive peak at about 60ms (P0 or P60), reflecting activation of the major visual cortex from the lateral geniculate nucleus. A negative peak at about 75ms (N0 or N75) reflects depolarization of superficial layers spreading into the primary visual cortex and glutamatergic postsynaptic activity, while a positive peak at about 100ms (P1 or P100) reflects surface hyperpolarization and GABAergic activity (Zemon et al, 1980). VEPs have been used in clinical trials; for example, antiepileptic drugs (gabapentin, Conte et al, 2009) and infant formulas (O' Connor et al, 2009). Both were FDA approved, based in part on positive results from the VEP study. Data from ongoing clinical trials in Mount Sinai provide support for VEPs as a measure of therapeutic response. According to the schematic of fig. 8, clinical response assessments were performed before treatment, 6 hours after treatment, and one, two and four weeks after treatment.

Results

Subjects had a full history of mixed seizures, which occurred 3 to 4 times a week (weekly) "like a clock work" for 4 years since the age of 19. His seizures included tonic-clonic tics with reduced oxygen saturation, and postural blood pressure changes. These seizures are of a severe, severe nature and may be life threatening.

The subject will develop one of these severe seizures within about one hour prior to infusion of the study drug-his pre-seizure phase can be detected due to the accompanying classical character and behavioral changes. Depending on the clinician of the study, the infused study drug appears to completely stop the impending seizure. It is noteworthy that the seizure completely ceased within the next two weeks (and according to the study clinician, this was the first round without seizures within two weeks, as the subject was in the middle and young age period at least five years ago. in this seizure free period, the young was "awake" (awakened) at several different levels, he became more social, more focused (e.g., better eye exchange), more cooperative, less restless, and his motor skills improved.

VEPs were recorded before and 6 hours after AMO-01 infusion. Figure 9 shows how the VEPs were reduced in this subject by treatment.

Together, these data demonstrate that administration of AMO-01 to a human subject with loss of function of the SHANK3 gene can produce clinical benefits in a variety of features of PhelanMcDermid syndrome over exposure to the compound, as observed in vivo in a SHANK3b gene knockout mouse model of the disease.

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While the invention has been described with reference to certain specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.

Reference to the literature

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