Methods of treating neurofibromatosis using farnesyl dibenzodiazepinones

Abstract

Methods for treating Neurofibromatosis (NF) in subjects having the disorder or in subjects predisposed to develop the disorder via administration of farnesyl dibenzodiazepinone compounds are provided.

Description

METHODS OF TREATING NEUROFIBROMATOSIS USING FARNESYL DIBENZODIAZEPINONES BACKGROUND OF INVENTION

[0001] Neurofibromatosis is a neurocutaneous disorder first described by Friedrich Daniel von Recklinghausen in 1882. Neurofibromatosis has three forms. NF1 (neurofibromatosis type 1), which affects 1 in 3,164 people, is caused by mutations of the gene located at 17q11.2 coding for the protein neurofibromin. NF2 (neurofibromatosis type 2), which has a birth incidence of 1 in 50,000, was discovered to be caused by mutation of the NF2 gene identified by Rouleau et al. (1990) and Wolf et al. (1992) located 22q12.2 which codes for the protein neurofibromin 2, also called merlin or schwannomin. The third type is called schwannomatosis, for which candidate gene is SMARCB1. The three genes associated with neurofibromatosis are all considered tumor suppressors.

[0002] The clinical features of NF1 in humans are highly diverse, with characteristic features including café-au-lait spots, neurofibromas (tumors on nerves), freckling, Lisch nodules (benign iris hamartomas), and skeletal abnormalities. Individuals with NF1 may also experience a variety of symptoms such as learning disabilities, cognitive impairments, attention deficits, and autism spectrum social difficulties. About 60 percent of children with NF1 have mild to moderate difficulties in school. Furthermore, NF1 is associated with an increased risk of certain tumours, including benign neurofibromas and malignant peripheral nerve sheath tumours. The NF1 gene encodes multiple biochemical domains, including a Ras-GAP domain, and it has four splice variants, two of which are expressed in the CNS. The type I isoform of the NF1 gene encodes a version of Neurofibromin with efficient Ras-GAP activity. This variant is predominantly expressed in neurons.

[0003] NF1 gene expression occurs across multiple brain systems that participate in a broad variety of behaviors. Within the CNS, NF1 transcription is seen in cortex, striatum, substantia nigra, brainstem, hippocampus, and cerebellum. In cortex and hippocampus, NF1 is expressed in pyramidal neurons, interneurons, and glia. It is also highly expressed in the Purkinje neurons of the cerebellum. This broad range of brain systems in which NF1 is expressed is also likely to contribute to the range of cognitive symptoms associated with its loss of function.

[0004] People with neurofibromatosis type 2 can exhibit the same type of skin symptoms as type 1, but not necessarily in every case. Symptoms may include pain due to pressure on nerves, tinnitus, weakness in fingers, numbness, headaches. The symptom most characteristic of NF2 is hearing loss. The hearing loss occurs due to the pressure of tumors on the acoustic nerve. The same pressure can cause headaches, dizziness, and nausea.

[0005] The main symptom of schwannomatosis is localized pain. This pain is due to tissues and nerves experiencing more pressure because of nearby tumors.

[0006] There has been relatively little clinical study of pharmacological interventions in Neurofibromatosis, particularly in relation to the cognitive and neurological deficits present in NF1. One medication is approved for the treatment of NF1. The MEK inhibitor selumetinib is approved for the treatment of children 2 years of age and older with neurofibromatosis type 1 (NF1) who have plexiform neurofibromas (PN) that cannot be completely removed by surgery. Selumetinib is not brain penetrant and is associated with serious side effects related to cardiac, muscle, eye and skin tissues. As of July 2024 there are four ongoing clinical studies specifically focused on neurofibromatosis with industry sponsors listed on ClinicalTrials.gov (NCT05331105, NCT05913037, NCT05130866 and NCT04374305). There is therefore an ongoing need for novel medicines for neurofibromatosis that are clinically safe and well tolerated, particularly ones that are brain penetrant and have potential to treat CNS mediated symptoms such as learning and cognitive deficits. BRIEF SUMMARY OF INVENTION

[0007] The present invention relates to methods for treating neurofibromatosis (NF) in subjects having the disorder or in subjects predisposed to develop the disorder. As discussed more fully below, it has been found that farnesyl dibenzodiazepinone compounds can reduce symptoms of the disorder in NF individuals to which the compounds are administered.

[0008] Thus, and in a first embodiment, the invention is drawn to a method of treating a subject having NF, wherein the method comprises administering 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 to a subject having NF.

[0009] In a second embodiment, the invention is drawn to a method of inhibiting development of NF in a subject predisposed to develop NF, wherein the method comprises administering 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 to a subject predisposed to develop NF.

[0010] In a third embodiment, the invention is drawn 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 having NF or in a method of treating a subject having NF.

[0011] In a fourth embodiment, the invention is drawn 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 inhibition of development of NF in a subject predisposed to develop NF or in a method of inhibiting development of NF in a subject predisposed to develop NF.

[0012] In a fifth embodiment, the invention is drawn 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 the treatment of a subject having NF or in a method of treating a subject having NF.

[0013] In a sixth embodiment, the invention is drawn 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 the inhibition of development of NF in a subject predisposed to develop NF or in a method of inhibiting development of NF in a subject predisposed to develop NF.

[0014] In a seventh embodiment, the invention is drawn to the use of at least one farnesyl dibenzodiazepinone compound for the manufacture of a medicament for treating a subject having NF.

[0015] In an eighth embodiment, the invention is drawn to the use of at least one farnesyl dibenzodiazepinone compound for the manufacture of a medicament for inhibiting development of NF in a subject predisposed to develop NF.

[0016] In each of the embodiments and aspects of the invention, the at least one farnesyl dibenzodiazepinone compound is selected from compounds encompassed by Formula I or pharmaceutically acceptable salts thereof: O W2CH3Formula Ieach of W1, W2and W3is independently CH3H CH3CH3CH3H or the chain from thebeing either –CH=O or –CH2OH; A is –NH–, –NCH2R1– or –NC(O)R1–, where R1is C1-6 alkyl, C2-6 alkene, aryl or heteroaryl; each of R2, R3, and R4is independently H, R5, or –C(O)R6, where each R5is independently C1-6alkyl, C2-7 alkalene, aryl or heteroaryl, and where each R6is independently H, C1-6 alkyl, C2-7 alkalene, aryl or heteroaryl.

[0017] In each of the embodiments and aspects of the invention, the at least one farnesyl dibenzodiazepinone compound may be the compound AMO-01 or a pharmaceutically acceptable salt thereof.O 01

[0018] todevelop NF a a or

[0019] In each of the embodiments and aspects of the invention, the therapeutically effective amount of the at least one farnesyl dibenzodiazepinone compound or the pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is between about 0.1 ug to about 200 mg per kg of the agent per body weight of the subject.

[0020] In each of the embodiments and aspects of the invention, the at least one farnesyl dibenzodiazepinone compound or the pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is administered to the subject via intravenous, subcutaneous, or other acceptable means.

[0021] 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 herein may be readily utilized as a basis for modifying or designing other means for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent means 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 is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1. The effect of AMO-0130 mg / kg i.p. in heterozygous Nf1tm1Fcrmice on the Novel object recognition test of learning and memory. This test was used because it is sensitive to lesions of the hippocampus and regions where neurofibromin is highly expressed. Wild type (WT) controls spent more time exploring the novel object (N) than Nf1tm1Fcrmice (p <0.0001). AMO-01 reversed this phenotype in the Nf1tm1Fcrmice (p <0.0001) whilst having no effect on learning and memory performance in Wild Type mice (NS). Data are mean ± sem of time spent with novel object (seconds) in groups of N = 10 mice.

[0023] Figure 2. The effect of AMO-0130 mg / kg i.p. in heterozygous Nf1tm1Fcrmice on the T-maze spontaneous alternation test of spatial working memory. Wild type (WT) controls alternated more than chance and significantly more than Nf1tm1Fcrmice (p <0.0001) who alternated at chance. AMO-01 reversed this phenotype in the Nf1tm1Fcrmice (p <0.0001) whilst having no effect on learning and memory in Wild Type control mice. Data are mean ± sem of percentage alternation in groups of N = 10 mice. DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0024] As used herein, “a” or “an” may mean one or more. As used herein when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0025] As used herein, “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., + / - 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.

[0026] As used herein, “treat” and all its forms and tenses (including, for example, treat, treating, treated, and treatment) refer to both therapeutic treatment and prophylactic orpreventative treatment. Subjects in need of treatment include those already diagnosed with NF as well as those predisposed to develop NF but not yet diagnosed as having NF. II. The Present Invention

[0027] As indicated above, the present invention is based on the discovery by the inventors that certain farnesyl dibenzodiazepinone compounds can reduce symptoms of neurofibromatosis (NF) in individuals having the disorder and to whom the compounds are administered. Thus, the present invention is directed to methods for treating NF in subjects having the disorder or in subjects predisposed to develop the disorder.

[0028] A subject having NF or predisposed to develop NF is a subject having a chromosomal deletion, duplication, insertion, substitution or mutation at 17q11.2 or 22q12.2 and / or in whom expression of neurofibromin or neurofibromin 2 proteins is reduced and / or in whom a dysfunctional neurofibromin or neurofibromin 2 proteins are produced.

[0029] The neurofibromin or neurofibromin 2 proteins are tumor suppressor proteins. Neurofibromin acts as a repressor of activation of Ras and subsequent activation of the Ras- MEK-ERK MAP kinase pathway that is in turn responsible for activating gene transcription related to cell division in dividing tissues and synapse generation in mature non-dividing neurons (refs). Neurofibromin 2, otherwise known as merlin or Schwannomin also regulates the Ras- MEK-ERK MAP kinase pathway (ref). Loss of function changes in NF1 and NF2 lead to aberrant activation of the Ras-MEK-ERK pathway and consequently abnormal cellular or synaptic proliferation (refs). A desirable goal for a curative therapeutic for neurofibromatosis is therefore inhibition of aberrant activation of the Ras-MEK-ERK pathway. Inhibition of MEK or ERK may be associated with side effects since these kinases are critical regulators. Selective MEK inhibitors are associated with pyrexia, fatigue, diarrhea, rash, hypertension, transaminase elevation and ocular damage (Garutti et al., 2023; Han and Chen, 2023), and cardiovascular events such as pulmonary embolism or decreases in left ventricular ejection fraction (Mincu et al., 2019). Chin et al. (2020) report dose limiting side effects of selective ERK inhibitors include diarrhea, nausea, fatigue, rash, transaminase elevation, hypertension, neurotoxicity and myocardial infarction. Ocular and neurological toxicities seen in early development of MEK and ERK inhibitors has led to the design of inhibitors of these targets that do not penetrate the bloodbrain barrier (Livingstone et al., 2014) and do not therefore have the potential to treat learning and memory difficulties due to compromised neuronal function in NF.

[0030] On the basis of this over-activity, and as described below, a brain-penetrant inhibitor of the Ras-ERK pathway, termed AMO-01 herein, was assessed in a knockout mouse model of NF. A single i.p. dose of AMO-01 administered to test animals was found to significantly improve aberrant neurobehavioral features associated with the mice, thus establishing the basis for the present invention. Farnesyl Dibenzodiazepinones

[0031] AMO-01 (10-farnesyl-4,6,8-trihydroxy-dibenzodiazepin-11-one) is a farnesyl dibenzodiazepinone and a member of a class of dibenzodiazepinone compounds containing a farnesyl moiety. The structure of AMO-01 is as follows: O 01 Each of theof AMO-01, or a pharmaceutically acceptable salt thereof, to a subject having NF or a subject predisposed to develop the NF.

[0032] Farnesyl dibenzodiazepinone compounds are produced by strains of Micromonospora, a genus of bacteria of the family Micromonosporaceae that are gram-positive, spore-forming, generally aerobic, and that form a branched mycelium. Members of the genus also commonly produce aminoglycoside antibiotics.

[0033] AMO-01 is produced by Micromonospora sp. strain 046-ECO11. Strain 046-ECO11 was deposited on March 7, 2003, with the International Depositary Authority of Canada (IDAC), Bureau of Microbiology, Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2, under Accession No.070303-01. More details on strain 046-ECO11 and means for producing AMO-01 may be found in international patent publication WO 2004 / 065591, published August 5, 2004, the contents of which are incorporated herein by reference.

[0034] In addition to AMO-01, and pharmaceutically acceptable salts thereof, each of the methods of the present invention may be practiced via administration to a subject having NF or a subject predisposed to develop NF at least one farnesyl dibenzodiazepinone compound selected from compounds encompassed by Formula I or a pharmaceutically acceptable salt thereof: O W2CH3Formula Ieach of W1, W2and W3is independently CH3H CH3CH3CH3H or the chain from thebeing either –CH=O or –CH2OH; A is –NH–, –NCH2R1– or –NC(O)R1–, where R1is C1-6alkyl, C2-6alkene, aryl or heteroaryl; each of R2, R3, and R4is independently H, R5, or –C(O)R6, where each R5is independently C1-6 alkyl, C2-7 alkalene, aryl or heteroaryl, and where each R6is independently H, C1-6 alkyl, C2-7alkalene, aryl or heteroaryl.

[0035] In certain embodiments, the invention provides compounds of Formula I, wherein A is selected from the group consisting of NH, NCH2R1, and NC(O)R1; wherein R2is H; R3is H; and R4is H. In another embodiment, R2, R3and R4are each H; and all other groups are as previously defined. In a further embodiment, R2, R3and R4are each H; and W1 is -CH=CH- and all other groups are as previously defined. In a further embodiment, R2, R3and R4are each H, and W2is –CH=CH- and all other groups are as previously defined. In a further embodiment, R2, R3and R4are each H; and W3is –CH=CH- ; and all other groups are as previously defined. In a further embodiment, A is NH; R2, R3and R4are each H; and all other groups are as previously defined. In a further embodiment, A is NH; each of W1, W2, and W3is –CH=CH-; and all othergroups are as previously defined. The invention encompasses all pharmaceutically acceptable salts of the foregoing compounds.

[0036] The following are additional exemplary compounds of Formula I and of the farnesyl dibenzodiazepinone compounds of the invention: O CH CH CH O CH CH CH N CH ; CH ;O CH CH CH O CH CH CH N CH N CH ; CH ;O CH CH CH O CH CH CH CH ;Formula XI Formula XII O CH CH CH O CH CH CH N CH N CH O O O O ; ;O CH CH CH O CH CH CH N CH N CH ; ;O CH CH CH O CH CH CH ;O CH CH CH O CH CH CH N CH N CH O O CH ;O CH CH CH O CH CH CH N CH N CH CH ;ıIJCH CH CH O CH CH CH N CH N CH ;CH CH CH O CH CH CH ;O CH CH CH O CH CH CH N CH N CH ; ;O CH CH O CH CH O OH N N ;O O O OH N N ;ı4O CH CH CH O CH CH CH N CH N CH OHOH OHO CH CH CH N CH .“alkyl” refers to linear or branched hydrocarbon groups. Examples of alkyl groups include, without limitation, methyl, ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl, heptyl, cyclopentyl, cyclohexyl, cyclohexymethyl, and the like. Alkyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, oxo, guanidino and formyl.

[0038] The term “alkenyl” refers to linear, branched or cyclic hydrocarbon groups containing at least one carbon-carbon double bond. Examples of alkenyl groups include, without limitation, vinyl, 1-propen-2-yl, 1-buten-4-yl, 2-buten-4-yl, 1-penten-5-yl and the like. Alkenyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, formyl, oxo and guanidino. The double bond portion(s) of the unsaturated hydrocarbon chain may be either in the cis or trans configuration.

[0039] The terms “cycloalkyl” and “cycloalkyl ring” refer to a saturated or partially unsaturated carbocyclic ring in a single or fused carbocyclic ring system having from three to fifteen ring members. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl. Cycloalkyl may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

[0040] The terms “heterocyclyl” and “heterocyclic” refer to a saturated or partially unsaturated ring containing one to four hetero atoms or hetero groups selected from O, N, NH, NRx, PO2, S, SO or SO in a single or fused heterocyclic ring system having from three to fifteen ring members. Examples of a heterocyclyl or heterocyclic ring include, without limitation, morpholinyl, piperidinyl, and pyrrolidinyl. Heterocyclyl, heterocyclic or heterocyclyl ring may optionally be substituted with substituents selected from acyl, amino, acylamino, acyloxy, oxo, thiocarbonyl, imino, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

[0041] The term “amino acid” refers to any natural amino acid; natural amino acids are well known to a person skilled in the art.

[0042] The term “halo” refers to a halogen atom, e.g., bromine, chlorine, fluorine and iodine.

[0043] The terms “aryl” and “aryl ring” refer to aromatic groups in a single or fused ring system, having from five to fifteen ring members. Examples of aryl include, without limitation, phenyl, naphthyl, biphenyl, terphenyl. Aryl may optionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, azido, alkythio, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and formyl.

[0044] The terms “heteroaryl” and “heteroaryl ring” refer to aromatic groups in a single or fused ring system, having from five to fifteen ring members and containing at least one hetero atom such as O, N, S, SO and SO2. Examples of heteroaryl groups include, without limitation, pyridinyl, thiazolyl, thiadiazoyl, isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl, triazolyl, and pyrrolyl groups. Heteroaryl groups may optionally be substituted with one or more substituent group selected from acyl, amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido,cyano, halo, hydroxyl, nitro, thio, thiocarbonyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl, and formyl.

[0045] The terms “aralkyl” and “heteroaralkyl” refer to an aryl group or a heteroaryl group, respectively bonded directly through an alkyl group, such as benzyl. Aralkyl and heteroaralkyl may be optionally substituted as the aryl and heteroaryl groups.

[0046] Similarly, the terms “aralkenyl” and “heteroaralkenyl” refer to an aryl group or a heteroaryl group, respectively bonded directly through an alkene group, such as benzyl. Aralkenyl and heteroaralkenyl may be optionally substituted as the aryl and heteroaryl groups.

[0047] The compounds of the present invention can possess one or more asymmetric carbon atoms and can exist as optical isomers forming mixtures of racemic or non-racemic compounds. The compounds of the present invention are useful as single isomers or as a mixture of stereochemical isomeric forms. Diastereoisomers, i.e., nonsuperimposable stereochemical isomers, can be separated by conventional means such as chromatography, distillation, crystallization or sublimation. The optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes.

[0048] As used herein, reference to a subject having NF is a subject identified as having a chromosomal deletion at 17q11.2 or 22q12.2 having at least two of the following symptoms: hypotonia (low tone), normal or accelerated growth, absent or severely delayed speech, and global developmental delay.

[0049] As used herein, reference to a subject predisposed to develop NF is a subject identified as having a chromosomal deletion at 17q11.2 or 22q12.2 and / or a subject having at least two of the following symptoms: learning difficulties, autism features, hyperactivity disorder, neurofibromas and schwannomatosis.

[0050] As will be apparent, the identity of the gene mutations(s) in a subject having NF or predisposed to develop NF can vary in size, number and specific location within the loci. Further, because NF is thought to be related to haploinsufficiency of the NF1 or NF2 genes, the chromosomal deletion at 17q11.2 or 22q12.2 may be in one or both of the alleles. When in both alleles, the deletion can be different between the alleles.III. Methods of Treatment

[0051] The present invention is drawn to methods of treating a subject having NF or a subject predisposed to develop NF. Each of the methods of the invention comprise administering 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 to a subject, such as a subject having NF or a subject predisposed to develop NF. In certain embodiments, the at least one farnesyl dibenzodiazepinone compound is AMO-01.

[0052] The invention is also drawn 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) treating a subject having NF, (b) methods of treating a subject having NF, (c) treating a subject predisposed to develop NF or (d) methods of treating a subject predisposed to develop NF. In certain embodiments, the at least one farnesyl dibenzodiazepinone compound is AMO-01.

[0053] The invention is also drawn 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 having NF, (b) a method of treating a subject having NF, (c) treating a subject predisposed to develop NF, or (d) methods of treating a subject predisposed to develop NF. In certain embodiments, the at least one farnesyl dibenzodiazepinone compound is AMO-01.

[0054] The invention is further drawn to the use of at least one farnesyl dibenzodiazepinone compound for the manufacture of a medicament for treating a subject having NF, and to the use of at least one farnesyl dibenzodiazepinone compound for the manufacture of a medicament for inhibiting development of NF in a subject predisposed to develop NF. In certain embodiments, the at least one farnesyl dibenzodiazepinone compound is AMO-01.

[0055] As used herein, the terms “treat”, “treating” and “treatment” have their ordinary and customary meanings, and include one or more of the following: ameliorating a symptom of NF in a subject having NF or a subject predisposed to develop NF; blocking a symptom of NF in such a subject; blocking or ameliorating recurrence of a symptom of NF in such a subject; decreasing in severity and / or frequency a symptom of NF in such a subject; blocking progressionof NF in such a subject; and curing or resolving NF in such a subject. Treatment means ameliorating, etc. by about 1% to about 100% versus a subject to whom the treatment has not been administered. Preferably, the ameliorating, 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% versus a subject to whom the treatment has not been administered. The treatment may begin prior to, concurrent with, or after the onset of clinical symptoms of the disorder. Thus, the subject may be showing symptoms of NF, or merely diagnosed as predisposed to develop NF but so far free of symptoms of the disorder. The results of the treatment may be permanent or may continue for a period of days (such as 1, 2, 3, 4, 5, 6 or 7 days), weeks (such as 1, 2, 3 or 4 weeks) or months (such as 1, 2, 3, 4, 5, 6 or more months).

[0056] The amount of the at least one farnesyl dibenzodiazepinone administered to a subject when the methods of the invention are practiced will vary by such factors as the age and weight of the subject, and the identity and severity of the symptoms of the disorder. However, the amount administered to a subject will be a therapeutically effective amount, i.e. an amount sufficient to effect treatment. As an example, a therapeutically-effective amount of the at least one farnesyl dibenzodiazepinone is between about 0.1 ug to about 200 mg per kg of body weight of the subject. Additional ranges of therapeutically-effective amounts include, but are not limited to, about 10 ug / kg to 200 mg / kg; about 100 ug / kg to 200 mg / kg; about 1 mg / kg to 200 mg / kg; about 10 mg / kg to 200 mg / kg; about 10 ug / kg to 100 mg / kg; about 100 ug / kg to 100 mg / kg; about 1 mg / kg to 100 mg / kg; about 10 mg / kg to 100 mg / kg; about 0.1 ug / kg to 50 mg / kg; about 1 ug / kg to 50 mg / kg; about 10 ug / kg to 50 mg / kg, about 100 ug / kg to 50 mg / kg, about 1 mg / kg to 50 mg / kg, about 10 mg / kg to 50 mg / kg, and about 20 ug / kg to 40 mg / kg of body weight of the subject. Specific examples of therapeutically-effective amounts include, but are not limited to, about 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, 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 60 mg / kg of body weight of the subject.

[0057] The at least one farnesyl dibenzodiazepinone compound may be administered to a subject once, or twice, three times, four times, five times, six times or more, over a course of treatment. The timing between each dose in a dosing schedule may range between days, weeks, months, or years, an includes administered 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 quantity ofthe at least one farnesyl dibenzodiazepinone may be administered in each dose of the dosing schedule, or the amounts in each dose may vary. Appropriate doses and dosing schedules can be readily determined by techniques well known to those of ordinary skill in the art without undue experimentation. Such a determination will be based, in part, on the tolerability and efficacy of a particular dose.

[0058] Administration frequencies include 4, 3, 2 or once daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once weekly, every eight days, every nine days, every ten days, bi-weekly, monthly and bi-monthly. The duration of treatment will be based on the symptoms and severity of the disorder in the particular subject and will be best determined by the attending physician. However, continuation of treatment is contemplated to last for a number of days, weeks, months or years. Indeed, in some instances, treatment may continue for the entire life of the subject.

[0059] Depending on the means of administration, the dosage may be administered all at once, such as with an oral formulation in a capsule or liquid, or slowly over a period of time, such as with an intramuscular or intravenous administration.

[0060] As used herein, “subject” includes, but is not limited, to a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal. IV. Formulations and Doses

[0061] In each of the methods of treatment of the invention, the at least one farnesyl dibenzodiazepinone compound and pharmaceutical formulations are administered in a pharmaceutically acceptable form and in substantially non-toxic quantities.

[0062] The pharmaceutical formulations of the invention will comprise at least one farnesyl dibenzodiazepinone compound as described herein. The pharmaceutical formulations will also comprise one or more pharmaceutically acceptable carrier or diluent.

[0063] 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™), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g. Cremophor EL), poloxamer 407 and 188, hydrophilic and hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fatemulsions, lipids, PEGylated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes. The terms specifically exclude cell culture medium. The formulations may further comprise stabilizing agents, buffers, antioxidants and preservatives, tonicity agents, bulking agents, emulsifiers, suspending or viscosity agents, inert diluents, fillers, and combinations thereof.

[0064] The identity of the carriers and diluents will depend on the manner in which the pharmaceutical formulations are being used and the means used to administer the pharmaceutical formulations to a subject. For example, pharmaceutical formulations for intramuscular preparations can be prepared where the carrier is water-for-injection, 0.9% saline, or 5% glucose solution.

[0065] Additionally, the pharmaceutical formulations may be administered in a liquid form. The liquid can be for oral dosage, for ophthalmic or nasal dosage as drops, or for use as an enema or douche. When the pharmaceutical formulation is formulated as a liquid, the liquid can be either a solution or a suspension of the pharmaceutical formulation. There is a variety of suitable formulations for the solution or suspension of the pharmaceutical formulations that are well known to those of skill in the art, depending on the intended use thereof. Liquid formulations for oral administration prepared in water or other aqueous vehicles may contain various suspending agents such as methylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone, and polyvinyl alcohol. The liquid formulations may also include solutions, emulsions, syrups and elixirs containing, together with the active compound(s), wetting agents, sweeteners, and coloring and flavoring agents.

[0066] The farnesyl dibenzodiazepinone compounds and pharmaceutical formulations may be formulated for and administered via any of the means commonly known, including, but not limited to oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, pulmonary, topical or parenteral administration. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of drug formulations can be used to effect such administration.V. Examples Mice

[0067] Nf1tm1Fcrheterozygous mice, initially obtained from the Jackson Laboratory, and wild type (WT) littermates were generated on a C57BL / 6J background. The Nf1tm1Fcrmouse is a long standing and recognized model of NF1 neurofibromatosis having construct validity and being suitable for identifying potential novel therapeutics for neurofibromatosis (Brannan et al., 1994). Of note, this model replicates the learning and memory and cognitive deficits of NF1 (Silva et al., 1997). These mice have a heterozygous knock out of the Nf1 gene, which reflects the heterozygosity of tumors in NF1 patients (Jacks et al., 1994). The resulting Nf1tm1Fcrmice were housed in groups of the same genotype in a temperature and humidity controlled room with a 12- h light-dark cycle (lights on 7 am to 7 pm). Room temperature and humidity were recorded continuously in the holding room. Testing was conducted during the light phase. Food and water were available ad libitum. Testing was conducted on Nf1tm1Fcrmice and their wild type littermates, with N = 10 mice per treatment group, tested at two months of age during behavioral experiments. Mice were housed in commercial plastic cages and experiments were conducted in line with the requirements of the UK Animals (Scientific Procedures) Act, 1986. All experiments were conducted with experimenters blind to genotype and drug treatment. Animals were allowed a minimum acclimatization period of one week prior to performing any experiment. Only healthy animals were used. No prophylactic or therapeutic treatment was to be administered during the acclimatization period. Behavioral Testing

[0068] AMO-01 from AMO Pharma Ltd was administered to mice as follows: A. AMO-01 at a single dose of 30 mg / kg intraperitoneally in a vehicle of ethanol, PEG 400 and polysorbate 80 and 5% dextrose B. Vehicle dosed intraperitoneally as 15 ml / kg Groups of N = 10 mice were treated 4 hours before testing on the first day of testing.

[0069] The following groups were studied: 1. Nf1tm1Fcrmice + vehicle, N = 10 2. Nf1tm1Fcrmice + AMO-0130 mg / kg i.p., N = 10 3. Wild Type litter mates + vehicle, N = 10 4. Wild Type litter mates + AMO-0130 mg / kg i.p., N = 10The following tests were administered: Novel Object Recognition (NOR): This test assesses recognition memory. Mice were presented with a familiar and a novel object, and memory is measured by the preference for exploring the novel object. Impaired recognition memory in NF1 mice may indicate cognitive deficits. T-Maze: The T-maze memory test is a widely used behavioral assay in mice to assess spatial working memory and learning abilities. In this test, a T-shaped maze is typically employed, where one arm of the T is designated as the "start arm," and the other two arms are the "choice arms." The test relies on the natural tendency of mice to explore new environments and relies on their spatial memory to make choices. Alterations in spontaneous alternation behavior may indicate deficits in working memory in NF1 mice.

[0070] Results from novel object recognition testing are shown in Figure 1 where time spent exploring a novel object (N) and a familiar object (F) (seconds) is shown. Wild Type mice, whether treated with vehicle or AMO-01 showed a difference in time spent exploring the two objects, with significantly more time spent exploring the novel object following both treatments (and no effect of treatment). In Nf1tm1Fcrmice the time spent exploring novel and familiar objects was identical and therefore not significantly different, reflecting the fact that the Nf1tm1Fcrmice did not distinguish between a familiar (remembered) object and a novel object. Treatment of Nf1tm1Fcrmice with AMO-01 restored the increase in time spent exploring the novel object, such that there was no difference between the behaviors of AMO-01 treated Nf1tm1Fcrmice and Wild Type mice. This reflected normal behavior following acute treatment with AMO-01.

[0071] Results from the T maze alternation test are provided in Figure 2 which shows rescue of spontaneous alternation behavior by AMO-01 in Nf1tm1Fcrmice. Working memory was assessed by alternation behavior of Nf1tm1Fcror Wild Type mice after a single dose 30 mg / kg ip AMO-01 or vehicle administered 4 hours before testing in groups of N = 10 mice. Wild Type mice treated with vehicle showed alternation of entry in arms of the T maze between 60 and 80 percent of test occasions. Administration of AMO-01 had no significant effect on performance in Wild Type mice. These data showed Wild Type mice exhibited a working memory of the arm they first visited, which was unaffected by AMO-01. Nf1tm1Fcrmice alternated an average of 50 percent of the time when treated with vehicle. This showed their responding was at chance levels reflecting a working memory deficit. Treatment with AMO-01 in Nf1tm1Fcrmice resulted in alternation levels indistinguishable from Wild Type mice, reflecting a normalization of working memory.* * * *

[0072] While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications 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.REFERENCES

[0073] All patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. Each cited patent and publication is incorporated herein by reference in its entirety. All of the following references have been cited in this application: Brannan CI, Perkins AS, Vogel KS, Ratner N, Nordlund ML, Reid SW, Buchberg AM, Jenkins NA, Parada LF, Copeland NG. Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues. Genes Dev.1994 May 1;8(9):1019-29. Jacks T, Shih TS, Schmitt EM, Bronson RT, Bernards A, Weinberg RA. Tumour predisposition in mice heterozygous for a targeted mutation in Nf1. Nat Genet.1994 Jul;7(3):353-61. doi: 10.1038 / ng0794-353. PMID: 7920653. Rouleau GA, Seizinger BR, Wertelecki W, Haines JL, Superneau DW, Martuza RL, Gusella JF. Flanking markers bracket the neurofibromatosis type 2 (NF2) gene on chromosome 22. Am J Hum Genet.1990 Feb;46(2):323-8 Silva AJ, Frankland PW, Marowitz Z, Friedman E, Laszlo GS, Cioffi D, Jacks T, Bourtchuladze R. A mouse model for the learning and memory deficits associated with neurofibromatosis type I. Nat Genet.1997 Mar;15(3):281-4. doi: 10.1038 / ng0397-281. Erratum in: Nat Genet 2002 Aug;31(4):439. Lazlo G [corrected to Laszlo GS]. PMID: 9054942. Wolf RK, Frazer KA, Jacker RK, Lanser MJ, Pitts LH, Cox DR. Analysis of chromosome22 deletions in neurofibromatosis type 2-related tumors. Am J Hum Genet.1992;51:478– 485

Claims

WHAT IS CLAIMED IS:

1. A method of treating a subject having Neurofibromatosis (NF), comprising administering 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 to a subject having NF.

2. A method of inhibiting development of NF in a subject predisposed to develop NF, comprising administering 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 to a subject predisposed to develop NF.

3. The method of claim 1 or 2, wherein the at least one farnesyl dibenzodiazepinone compound is selected from compounds encompassed by Formula I or pharmaceutically acceptable salts thereof: O 3 Formula Ieach of W1, W2and W3is independently CH3H CH3or the chain from thebeing either –CH=O or –CH2OH; A is –NH–, –NCH2R1– or –NC(O)R1–, where R1is C1-6alkyl, C2-6alkene, aryl or heteroaryl;each of R2, R3, and R4is independently H, R5, or –C(O)R6, where each R5is independently C1-6 alkyl, C2-7 alkalene, aryl or heteroaryl, and where each R6is independently H, C1-6 alkyl, C2-7 alkalene, aryl or heteroaryl.

4. The method of claim 1 or 2, wherein the at least one farnesyl dibenzodiazepinone compound is AMO-01 or a pharmaceutically acceptable salt thereof. O 015. The method of any one of claims 1-4, wherein the subject having NF or the subject predisposed to develop NF is a subject having a chromosomal deletion at 17q11.2 or 22q12.

2.

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

7. The method of any one of claims 1-6, wherein the at least one farnesyl dibenzodiazepinone compound or the pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is administered to the subject via intravenous or subcutaneous administration.

8. 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 having NF or in a method of treating a subject having NF.

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

10. The use of claim 8 or 9, wherein the at least one farnesyl dibenzodiazepinone compound is selected from compounds encompassed by Formula I or pharmaceutically acceptable salts thereof: O W2Formula Ieach of W1, W2and W3is independently CH3H CH3CH3CH3H or the chain from thebeing either –CH=O or –CH2OH; A is –NH–, –NCH2R1– or –NC(O)R1–, where R1is C1-6 alkyl, C2-6 alkene, aryl or heteroaryl; each of R2, R3, and R4is independently H, R5, or –C(O)R6, where each R5is independently C1-6alkyl, C2-7 alkalene, aryl or heteroaryl, and where each R6is independently H, C1-6 alkyl, C2-7 alkalene, aryl or heteroaryl.

11. The use of claim 8 or 9, wherein the at least one farnesyl dibenzodiazepinone compound is AMO-01 or a pharmaceutically acceptable salt thereof.O 0112. use any one or the subject predisposed to develop NF is a subject having a chromosomal deletion at 17q11.2 or 22q12.

2.

13. The use of any one of claims 8-12, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepinone compound is between 0.1 ug / kg and 200 mg / kg of the farnesyl dibenzodiazepinone compound per body weight of the subject.

14. The use of any one of claims 8-13, wherein the at least one farnesyl dibenzodiazepinone compound or the pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is administered to the subject via intravenous or subcutaneous administration.

15. 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 treating a subject having NF or a method of treating a subject having NF.

16. 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 inhibition of development of NF in a subject predisposed to develop NF or in a method of inhibiting development of NF in a subject predisposed to develop NF.

17. The therapeutically effective amount of claim 15 or 16, wherein the at least one farnesyl dibenzodiazepinone compound is selected from compounds encompassed by Formula I or pharmaceutically acceptable salts thereof:O W21CH3Ieach of CH3H CH3CH3CH3H or the chain from the being either–CH=O or –CH2OH; A is –NH–, –NCH2R1– or –NC(O)R1–, where R1is C1-6alkyl, C2-6alkene, aryl or heteroaryl; each of R2, R3, and R4is independently H, R5, or –C(O)R6, where each R5is independently C1-6alkyl, C2-7 alkalene, aryl or heteroaryl, and where each R6is independently H, C1-6 alkyl, C2-7 alkalene, aryl or heteroaryl.

18. The therapeutically effective amount of claim 15 or 16, wherein the at least one farnesyl dibenzodiazepinone compound is AMO-01 or a pharmaceutically acceptable salt thereof. 0119. The therapeutically effective amount of any one of claims 15-18, wherein the subject having NF or the subject predisposed to develop NF is a subject having a chromosomal deletion at 17q11.2 or 22q13.3.

20. The therapeutically effective amount of any one of claims 15-19, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepinone compound is between 0.1 ug / kg and 200 mg / kg of the farnesyl dibenzodiazepinone compound per body weight of the subject.

21. The therapeutically effective amount of any one of claims 15-20, wherein the at least one farnesyl dibenzodiazepinone compound or the a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is administered to the subject via intravenous or subcutaneous administration.

22. Use of at least one farnesyl dibenzodiazepinone compound for the manufacture of a medicament for treating a subject having NF.

23. Use of at least one farnesyl dibenzodiazepinone compound for the manufacture of a medicament for inhibiting development of NF in a subject predisposed to develop NF.

24. The use of claim 22 or 23, wherein the at least one farnesyl dibenzodiazepinone compound is selected from compounds encompassed by Formula I or pharmaceutically acceptable salts thereof: O 3 Formula Ieach of W1, W2and W3is independently CH3H CH3the–CH=O or –CH2OH;A is –NH–, –NCH2R1– or –NC(O)R1–, where R1is C1-6 alkyl, C2-6 alkene, aryl or heteroaryl; each of R2, R3, and R4is independently H, R5, or –C(O)R6, where each R5is independently C1-6 alkyl, C2-7alkalene, aryl or heteroaryl, and where each R6is independently H, C1-6alkyl, C2-7alkalene, aryl or heteroaryl.

25. The use of claim 22 or 23, wherein the at least one farnesyl dibenzodiazepinone compound is AMO-01 or a pharmaceutically acceptable salt thereof. O 0126. The use of any one of claims 22-25, wherein the subject having NF or the subject predisposed to develop NF is a subject having a chromosomal deletion at 17q11.2 or 22q12.

2.

27. The use of any one of claims 22-26, wherein the therapeutically effective amount of the at least one farnesyl dibenzodiazepinone compound is between 0.1 ug / kg and 200 mg / kg of the farnesyl dibenzodiazepinone compound per body weight of the subject.

28. The use of any one of claims 22-27, wherein the at least one farnesyl dibenzodiazepinone compound or the a pharmaceutical formulation comprising at least one farnesyl dibenzodiazepinone compound and a pharmaceutically acceptable carrier or diluent is administered to the subject via intravenous or subcutaneous administration.

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