Compositions and methods for treating vascular ellis- danlos syndrome and related disorders

Abstract

The present invention relates to compositions and methods for treating vascular Ehlers-Danlos syndrome and related connective tissue disorders.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 838,049, filed April 24, 2019, U.S. Provisional Application No. 62 / 747,587, filed October 18, 2018, and U.S. Provisional Application No. 62 / 746,524, filed October 16, 2018. The entire contents of these applications are incorporated herein by reference in their entirety.

[0003] Statement Regarding Federally Funded Research

[0004] This invention was made with government support under Grant No. 5T32GM007309-44 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0005] The present invention relates to connective tissue disorders. Background Art

[0006] Vascular Ehlers-Danlos Syndrome (vEDS) is an inherited connective tissue disorder caused by heterozygous mutations in the COL3A1 gene. Patients with vascular EDS have thin, translucent skin, easy bruising, a characteristic facial appearance, and spontaneous dissection of large and medium-sized blood vessels and organs (uterus, colon) when they rupture, which leads to premature death and a median survival of approximately 45 years. 1 .

[0007] Many features of vEDS distinguish it from other inherited vascular disorders, such as Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS), which have been associated with excessive TGF-β activity. These features include the development of aortic dissection in any medium-sized or large blood vessel and in the absence of previous aneurysms or ruptures in hollow organs. These features make disease prediction and monitoring difficult or impossible, and the presenting sign in most adults diagnosed with vEDS is a vessel dissection or organ rupture, with 25% of patients experiencing severe complications before the age of 20. 1,2 Thus, there is an unmet need for treatments for vEDS and related connective tissue disorders. Summary of the Invention

[0008] In particular, provided herein are compositions, formulations, and methods for inhibiting, treating, preventing, and / or alleviating symptoms of the severity of connective tissue disorders (e.g., vascular lesions). Various aspects of this subject matter relate to the use of agents for treating a variety of connective tissue disorders. In certain embodiments, the connective tissue disorder comprises a vascular lesion, and in certain embodiments, the vascular lesion comprises vascular Ehlers-Danlos syndrome (vEDS).

[0009] Included herein are methods for treating a vascular disorder (e.g., vEDS) in a subject. The methods include administering to the subject an effective amount of an agent such that the agent reduces the activity or expression of extracellular signal-regulated kinase (ERK) or protein kinase C (PKC).

[0010] In certain embodiments, the agent inhibits the expression of mitogen-activated protein kinase / extracellular signal-regulated kinase (MEK), extracellular signal-regulated kinase (ERK), phospholipase C (PLC), inositol triphosphate (IP3), or protein kinase C (PKC), and thereby inhibits the activity of ERK, PLC, IP3, or PKC.

[0011] In certain embodiments, the agent inhibits the activity or expression of one or more molecules involved in the mitogen-activated protein kinase (MAPK) pathway, such as RAS-RAF / MEK / extracellular signal-regulated kinase (ERK) protein kinase.

[0012] In an embodiment, the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof. In a specific embodiment, the agent comprises a small molecule.

[0013] In some cases, the pharmaceutical agent comprises a small molecule. A small molecule is a compound having a mass of less than 2000 daltons. The molecular mass of the small molecule is preferably less than 1000 daltons, more preferably less than 600 daltons, for example, the compound is less than 500 daltons, less than 400 daltons, less than 300 daltons, less than 200 daltons, or less than 100 daltons.

[0014] Small molecules are organic or inorganic. Exemplary organic small molecules include, but are not limited to, aliphatic hydrocarbons, alcohols, aldehydes, ketones, organic acids, esters, monosaccharides and disaccharides, aromatic hydrocarbons, amino acids, and lipids. Exemplary inorganic small molecules include trace minerals, ions, free radicals, and metabolites. Alternatively, small molecules can be synthetically engineered to consist of fragments or small portions or longer amino acid chains to fill the binding pocket of an enzyme. Typically, small molecules are less than one thousand daltons.

[0015] In certain instances, the agent comprises a nucleic acid molecule. For example, a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) inhibits the expression of mitogen-activated protein kinase / cellular extracellular signal-regulated kinase (MEK), extracellular signal-regulated kinase (ERK), phospholipase C (PLC), inositol triphosphate (IP3), or protein kinase C (pKC), and thereby inhibits the activity of ERK, PLC, IP3, or PKC. In certain instances, the nucleic acid comprises a small interfering RNA (siRNA), RNA interference (RNAi), messenger RNA (mRNA), small hairpin RNA or short hairpin RNA (shRNA), double-stranded ribonucleic acid (dsRNA), antisense RNA, or microRNA (microRNA), or any portion thereof. However, the skilled artisan can readily identify other nucleic acids that inhibit / antagonize or activate / agonize ERK or PKC or IP3 or PLC.

[0016] A polynucleotide, polypeptide, or other agent used herein is purified and / or isolated. Specifically, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein used herein is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. The purified compound is at least 60% (dry weight) of the compound of interest by weight. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% of the compound of interest by weight. For example, a purified compound is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the compound of interest by weight. Purity is measured by any appropriate standard method, such as by column chromatography, thin layer chromatography, or high performance liquid chromatography (HPLC) analysis. A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences flanking it in its naturally occurring state. A purified or isolated polypeptide is free of the amino acids or sequences flanking it in its naturally occurring state. Purified also defines the degree of sterility, which is safe for administration to a human subject, such as the lack of infectious or toxic agents.

[0017] In embodiments, the agent comprises cobimetinib or a pharmaceutically acceptable salt thereof. In other embodiments, the agent comprises Ubenimex or a pharmaceutically acceptable salt thereof. In other embodiments, the agent comprises Enzastaurin or a pharmaceutically acceptable salt thereof. In other contemplated embodiments, the agent comprises Sotrastaurin or a pharmaceutically acceptable salt thereof.

[0018] In alternative embodiments, the method further comprises administering an agent that reduces the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3).

[0019] In embodiments, the method includes administering an effective amount of a medicament. The effective amount of the medicament is from about 0.001 mg / kg to about 250 mg / kg body weight, for example, from about 0.001 mg / kg, about 0.05 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 25 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, about 125 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg or about 250 mg / kg body weight. Ultimately, the attending physician or veterinarian determines the appropriate amount and dosage regimen.

[0020] In some cases, the agent is administered at least once a day, at least once a week, or at least once a month. The agent may be suitably administered continuously for a period of 1 day, 1 week, 1 month, 2 months, 3 months, 6 months, 9 months, or 1 year. In some cases, the agent is administered daily, for example, once every 24 hours. Alternatively, the agent is administered continuously or several times a day, for example, once every 1 hour, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, every 10 hours, every 11 hours, or every 12 hours.

[0021] In addition, the methods described herein prevent or reduce the severity of vEDS by at least about 1%, for example, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%.

[0022] A variety of administration routes are available. For example, the agent can be administered topically, orally, via inhalation, or via injection.

[0023] The subject is preferably a mammal in need of such treatment or prevention, for example, a subject who has been diagnosed with a vascular disease or a predisposition thereto. The mammal is any mammal, for example, a human, a primate, a mouse, a rat, a dog, a cat, a horse, and livestock or animals raised for food consumption, for example, cattle, sheep, pigs, chickens, and goats. In a preferred embodiment, the mammal is a human.

[0024] In some aspects, there is a connective tissue disorder such as vasculopathy (and vEDS in certain embodiments) or a subject in the risk of suffering a connective tissue disorder such as vasculopathy (and vEDS in certain embodiments), there is an ERK or PKC protein or mRNA level different from the normal control. In certain embodiments, the test sample obtained from the subject comprises an ERK or PKC protein or mRNA level different from the normal control. For example, the test sample can comprise an ERK or PKC protein or mRNA level at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 5-50%, 50-75%, 75-100%, 1 times, 2 times, 3 times, 4 times or 5 times higher than the normal control.

[0025] In certain embodiments, the test sample can comprise a level of ERK or PKC activity that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, about 5 to about 50%, about 50 to about 75%, about 75 to about 100%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold greater than a normal control.

[0026] "Control" sample or numerical value represents a sample used as a reference (usually a known reference) for comparison with a test sample. For example, a test sample can be taken from a test subject, for example, a subject suffering from a connective tissue disorder such as a vascular lesion (for example, vEDS) or a subject in need of diagnosis, and compared with a sample from a known condition, for example, a subject (or subjects) without the disease (negative or normal control), or a subject (or subjects) with the disease (positive control). The control can also represent a mean value collected from many tests or results. Those skilled in the art will recognize that controls can be designed for evaluating any number of parameters. Those skilled in the art will understand which controls are valuable in a given situation and can analyze data based on comparison with control values. Controls are also valuable for determining the significance of data. For example, if the numerical value of a given parameter is variable in the control, the change in the test sample will not be considered significant.

[0027] The term "normal amount" with respect to a compound (e.g., protein or mRNA) means the normal amount of the compound in an individual without a connective tissue disorder such as a vascular lesion (e.g., vEDS), or in a healthy or general population. The amount of the compound in the test sample can be measured and cutoff points and abnormal values ​​(e.g., for a specific vEDS or its symptoms) can be defined using techniques such as reference limits, discrimination limits, or risk-defining thresholds to compare with "normal control" levels. Normal control levels refer to the levels of one or more compounds or combinations of compounds typically found in subjects known not to have vEDS. Such normal control levels and cutoff points can vary depending on whether the compound is used alone or in a formulation combined with other compounds to form an index. Alternatively, the normal control level can be a database of compound patterns from subjects previously tested who did not develop vEDS or its specific symptoms (e.g., in the case of vEDS development or testing of subjects who already have vEDS) within clinically relevant time limits.

[0028] The level determined can be the same as the control level or cutoff level or threshold level, or can be increased or decreased relative to the control level or cutoff level or threshold level. In some aspects, the control subject is a matched control with the same species, sex, ethnicity, age group, smoking status, body mass index (BMI), current treatment regimen status, medical history, or a combination thereof, but unlike the diagnosed subject, the control has not suffered from the disease in question (or its symptoms) or is not at risk for the disease.

[0029] As used herein, the term "increase" refers to an increase in the level of at least one of the control levels. For example, the level of at least one of the control levels (e.g., protein or mRNA level) can be increased by at least one of the following methods: at least one of the control levels (e.g., protein or mRNA level) and at least one of the control levels (e.g., mRNA or protein level). In some embodiments, the level of increase can be at least or approximately 5% increase, at least or approximately 10% increase, at least or approximately 15% increase, at least or approximately 20% increase, at least or approximately 25% increase, at least or approximately 30% increase, at least or approximately 35% increase, at least or approximately 40% increase, at least or approximately 45% increase, at least or approximately 50% increase, at least or approximately 55% increase, at least or approximately 60% increase, at least or approximately 65% ​​increase, at least or approximately 70% increase, at least or approximately 75% increase, at least or approximately 80% increase, at least or approximately 85% increase, at least or approximately 90% increase, at least or approximately 95% increase.

[0030] The determined level can be a reduced level relative to the control level. As used herein, the term "reduced" with respect to a level (e.g., a protein or mRNA level) means any % reduction below the control level. In various embodiments, the reduced level can be at least or about a 5% reduction, at least or about a 10% reduction, at least or about a 15% reduction, at least or about a 20% reduction, at least or about a 25% reduction, at least or about a 30% reduction, at least or about a 35% reduction, at least or about a 40% reduction, at least or about a 45% reduction, at least or about a 50% reduction, at least or about a 55% reduction, at least or about a 60% reduction, at least or about a 65% reduction, at least or about a 70% reduction, at least or about a 75% reduction, at least or about a 80% reduction, at least or about a 85% reduction, at least or about a 90% reduction, at least or about a 95% reduction relative to the control level.

[0031] In certain aspects, the test sample from the subject comprises blood, serum, plasma, saliva, tears, vitreous, cerebrospinal fluid, sweat, cerebrospinal fluid, or urine.

[0032] Also provided herein are methods of treating a connective tissue disorder in a subject. The methods comprise administering an effective amount of an agent, wherein the agent reduces the activity or expression of extracellular signal-regulated kinase (ERK) or protein kinase C (PKC). In embodiments, the connective tissue disorder is selected from vasculopathy (e.g., vEDS), Marfan syndrome, Loeys-Dietz syndrome, and familial thoracic aortic aneurysm. Other examples include, but are not limited to, myopathic dermatomyositis; bizzare parosteal osteochondromatous proliferation; classic Ehlers-Danlos syndrome; Ehlers-Danlos syndrome cutis hyperelastica; Ehlers-Danlos syndrome; eosinophilic fasciitis; epidermolysis bullosa (EB) junctional epidermolysis bullosa; Marfan syndrome; melorheostosis; melorheostosis with bone enostosis; mixed connective tissue disease; rheumatoid factor negative polyarthritis; sclerosteosis with ichthyosis and premature ovarian failure; pacman developmental anomaly; Paget's disease of bone; familial Paget's disease of bone; polymyositis; progressive deafness with fixation of the stapes; Ribbing's disease; scleroderma; temporomandibular joint ankylosis; 20-methylretinoid malnutrition; vascular Ehlers-Danlos syndrome; Weber-Christian syndrome; and Worth type autosomal dominant sclerosteosis. In certain embodiments, the connective tissue disorder comprises vasculopathy.

[0033] In addition to treatment of Marfan syndrome itself, the present application also provides methods of treating various Marfan-related disorders. In certain embodiments, the Marfan-related disorder is selected from the group consisting of Loeys-Dietz syndrome, familial aortic aneurysm, bicuspid aortic valve with aortic dilation, familial spherophakia (luxated lens), mitral valve prolapse syndrome, Marfanoid habitus, congenital contractural arachnodactyly (Beals syndrome), Stickler syndrome, Shprintzen-Goldberg syndrome, Wiedemann-Maestrini syndrome, and Ehlers-Danlos syndrome.

[0034] In embodiments, the method for treating a connective tissue disorder comprises administering an agent, wherein the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof. In preferred embodiments, the agent comprises a small molecule.

[0035] In embodiments, the agent comprises cobimetinib or a pharmaceutically acceptable salt thereof. In other embodiments, the agent comprises ruboxistaurin or a pharmaceutically acceptable salt thereof. In other embodiments, the agent comprises enzastaurin or a pharmaceutically acceptable salt thereof. In other contemplated embodiments, the agent comprises sorafenib or a pharmaceutically acceptable salt thereof.

[0036] In alternative embodiments, the method further comprises administering an agent that reduces the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3).

[0037] Also provided herein are pharmaceutical compositions for treating a vascular disorder. In embodiments, the composition comprises an effective amount of an agent that reduces the activity or expression of extracellular signal-regulated kinase (ERK) or protein kinase C (PKC). The pharmaceutical composition comprises an agent, wherein the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof.

[0038] The compositions described herein are administered by oral administration, intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intraarterial administration, intraarticular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation, suppository administration, inhalation, or subcutaneous administration.

[0039] Also provided herein are kits for treating a vascular disorder. In embodiments, the kit comprises 1) a pharmaceutical composition of any of the compositions described herein and 2) written instructions for treating a vascular disorder.

[0040] In other aspects, methods for treating a connective tissue disorder (e.g., Marfan syndrome) are contemplated. In embodiments, the methods comprise administering an effective amount of an agent, wherein the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof. In certain embodiments, a therapeutically effective amount of one or more agents are co-administered to the subject.

[0041] In certain embodiments, the methods for treating a connective tissue disorder (e.g., Marfan syndrome) comprise administering an agent that reduces the activity or expression of protein kinase C (PKC). In certain embodiments, the methods for treating a connective tissue disorder (e.g., Marfan syndrome) comprise administering an agent that reduces the activity or expression of extracellular signal-regulated kinase (ERK). In certain embodiments, the methods for treating a connective tissue disorder (e.g., Marfan syndrome) comprise administering an agent that reduces the activity or expression of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), or a combination thereof.

[0042] In certain embodiments, the methods for treating a connective tissue disorder (e.g., Marfan syndrome) comprise administering an agent that reduces or inhibits the expression of mitogen-activated protein kinase / extracellular signal-regulated kinase (MEK), extracellular signal-regulated kinase (ERK), phospholipase C (PLC), inositol triphosphate (IP3), or protein kinase C (pKC), and thereby inhibits the activity of ERK, PLC, IP3, or PKC.

[0043] In certain embodiments, the methods for treating a connective tissue disorder (e.g., Marfan syndrome) comprise administering an agent that reduces or inhibits the activity or expression of one or more molecules associated with the mitogen-activated protein kinase (MAPK) pathway (e.g., RAS-RAF / MEK / extracellular signal-regulated kinase (ERK) protein kinases).

[0044] In certain embodiments, the patient is a human patient. In certain embodiments, the patient is 15 years of age or older. In certain embodiments, the patient is an adult patient. In certain embodiments, the patient is a pediatric patient.

[0045] In certain embodiments, the methods begin at or shortly after diagnosis of vEDS. In certain embodiments, the methods begin when the patient is 15 years of age or at first diagnosis.

[0046] In certain embodiments, the patient is diagnosed based on a phenotype of vEDS, or based on a molecular test vEDS (e.g., the patient is determined to have vEDS based on one or more genetic tests, e.g., a test that determines the patient has a glycine substitution within a triple helix or a splice site variant).

[0047] In certain embodiments, the patient has a COL3A1 mutation. In certain embodiments, the patient has a glycine substitution within a triple helix or a splice site variant. In certain embodiments, the patient has a missense substitution of glycine in the repeating (Gly-X-Y)n sequence of the collagen triple helix, and or a splice site variant that causes phase-wise exon skipping. In certain embodiments, the patient has a glycine substitution within a triple helix (Group I). In certain embodiments, the patient has a splice site variant, in-frame insertion-deletion, or duplication (Group II). In certain embodiments, the patient has a variant that causes haploinsufficiency (Group III).

[0048] In certain embodiments, the patient has had an acute vEDS-related event (e.g., an arterial event such as a rupture or dissection, a bowel or uterine rupture) prior to the initial dose of one or more agents described herein, or a pharmaceutically acceptable salt thereof.

[0049] In certain embodiments, the agent reduces PKC protein or mRNA levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 5-50%, 50-75%, 75-100%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to a normal control. In other embodiments, the agent reduces PKC activity levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 5-50%, 5-fold compared to a normal control.

[0050] In certain embodiments, the agent reduces extracellular signal-regulated kinase (ERK) protein or mRNA levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 5-50%, 50-75%, 75-100%, 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold compared to a normal control. In other embodiments, the agent reduces ERK activity levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 5-50%, 5-fold compared to a normal control.

[0051] In embodiments, the agent for treating a connective tissue disorder (e.g., Marfan syndrome) comprises Enzastaurin or a pharmaceutically acceptable salt thereof. In other embodiments, the agent for treating a connective tissue disorder (e.g., Marfan syndrome) comprises Sotrastaurin or a pharmaceutically acceptable salt thereof. In other contemplated embodiments, the agent for treating a connective tissue disorder (e.g., Marfan syndrome) comprises Ruboxistaurin or a pharmaceutically acceptable salt thereof.

[0052] Definitions

[0053] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide one of ordinary skill with a general definition of many of the terms used in this disclosure: The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0054] The term "pharmaceutical composition" refers to any composition comprising at least one therapeutic or biologically active agent and being suitable for administration to a patient. Any of these formulations can be prepared by methods well known and accepted in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 20th Ed., (A. R. Gennaro, ed.), Mack Publishing Co., Easton, Pa., 2000.

[0055] A "G protein-coupled receptor (GPCR)" refers to a protein receptor that senses molecules outside the cell and activates intracellular signal transduction pathways and ultimately activates a cellular response. GPCRs are called seven-transmembrane receptors because they cross the cell membrane seven times.

[0056] An "agonist" refers to a chemical that binds to a receptor and activates the receptor to produce a biological response. An agonist causes an effect, whereas an "antagonist" blocks the effect of an agonist, and an inverse agonist causes an effect opposite to that of an agonist. As used herein, the terms "antagonist" and "inhibitor" are used interchangeably to mean any molecule that counteracts or inhibits, reduces or suppresses the biological activity of its target molecule. In certain embodiments, an agonist is a "super-agonist" when it induces or increases the biological activity of its target molecule. In certain embodiments, an antagonist is a "super-antagonist" when it counteracts or inhibits, reduces or suppresses the biological activity of its target molecule. Suitable inhibitors, antagonists, agonists include soluble receptors, peptide inhibitors, small molecule inhibitors, ligand fusions, and antibodies.

[0057] The term "salt" as used herein refers to acid or base salts of the agents used herein. Illustrative, but non-limiting examples of acceptable salts are mineral acid (hydrochloric, hydrobromic, phosphoric, sulfuric, etc.) salts, organic acid (acetic, propionic, glutamic, citric, etc.) salts, and quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts.

[0058] An "antagonist" as used herein can mean an antibody or fragment thereof, a peptide, a polypeptide or fragment thereof, a small molecule, and an inhibitory nucleic acid or fragment thereof, which interferes with the activity or binding of another, for example, by competing for one or more of the binding sites of the agonist, but does not induce a positive response.

[0059] "Wild type" or "WT" refers to the phenotype of the typical form of a species as it exists in nature. Alternatively, wild type is conceptualized as the product of the standard "normal" allele at a locus, as compared to the product of a non-standard "mutant" allele.

[0060] The term "administering" as used herein means any mode of transferring, delivering, introducing or transporting a pharmaceutical agent to a subject in need of treatment for a disease or disorder. Such modes include, but are not limited to, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

[0061] "Co-administration" means that the compositions described herein are administered at the same time as, shortly before, or shortly after the administration of other therapies. The agents or compositions of the present disclosure can be administered to a patient alone or can be co-administered. Co-administration is intended to encompass simultaneous or sequential administration of the compounds, either separately or in combination (more than one compound or agent). If desired, the above-mentioned formulations can also be combined with other active substances.

[0062] "Sequential administration" as used herein includes administration of two agents (e.g., the agents or compositions described herein) separately on the same day or not on the same day (e.g., over consecutive days).

[0063] "Simultaneous administration" as used herein includes at least partial overlap in duration. For example, when two agents (e.g., any of the biologically active agents described herein) are administered simultaneously, their administration occurs within some desired time. Administration of the agents can begin and end on the same day. Administration of one agent can also precede administration of the second agent by several days, so long as both agents are administered at least once on the same day. Similarly, administration of one agent can extend beyond administration of the second agent, so long as both agents are administered at least once on the same day. One or more biologically active agents need not be administered every day to include simultaneous administration.

[0064] "Intermittent administration" as used herein includes administration of an agent for a period of time (which can be considered a "first administration phase"), followed by a period of time in which the agent is not administered or is administered at a lower maintenance dose (which can be considered a "disengagement phase"), followed by a period of time in which the agent is administered again (which can be considered a "second administration phase"). Typically, during the second administration phase, the dose level of the agent will match the dose level administered during the first administration phase, but can be increased or decreased as medically indicated.

[0065] "Alteration" as used herein also includes a change of 2-fold or more, e.g., 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold or more, in the expression level or activity of a gene or polypeptide.

[0066] As defined herein, the terms "inhibition," "inhibit," "inhibiting," and the like with respect to protein inhibitors (e.g., ERK or PKC inhibitors, or PLC or IP3 inhibitors) interactions mean that, relative to the activity or function of the protein in the absence of the inhibitor, a negative impact (e.g., reduction) on the activity or function of the protein (e.g., reducing the activity or amount of ERK or PKC, or PLC or IP3, reducing the ability of ERK or PKC, or PLC or IP3 to bind to a receptor, reducing the ability of a receptor to bind to ERK or PKC, or reducing ERK or PKC, or PLC or IP3 after binding to a receptor or PKC signaling). In embodiments, inhibition represents the alleviation of a disease or a symptom of a disease (e.g., a connective tissue disorder). Similarly, an "inhibitor" is a compound or protein that inhibits a target by binding, partially or completely blocking, reducing, preventing, delaying, inactivating, desensitizing, or downregulating activity.

[0067] "Ameliorate" means to reduce, inhibit, attenuate, diminish, arrest, or stabilize the development or progression of a disease, such as pseudoallergic reaction.

[0068] "Amplification" refers to increasing the number of copies of a molecule. In one embodiment, the nucleic acid is amplified using the polymerase chain reaction (PCR).

[0069] "Binding" refers to having a physicochemical affinity for a molecule. Binding is measured by any of the methods disclosed herein, for example, a drug / compound having a receptor expressed on a cell.

[0070] In this disclosure, "including", "comprising", "containing", "having", etc. may have the meanings assigned to them by U.S. patent law and may mean "including", "comprising", etc.; the term "essentially consisting of..." or "consisting of..." also has the meaning assigned to it by U.S. patent law, and these terms are open-ended, allowing for more content than the listed content, as long as the basic or novel characteristics of the listed content are not changed by the existence of more content than the listed content, but excludes implementations of the prior art.

[0071] An "effective amount" is an amount required to improve the symptoms of a disease relative to an untreated patient. The effective amount of the active compound used in practicing the present invention to therapeutically treat a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosing regimen. Such an amount is referred to as an "effective" amount.

[0072] The dosage and frequency of administration (single or multiple doses) to a mammal can vary depending on factors such as whether the mammal is suffering from another disease, and its route of administration; the size, age, sex, health, body mass index and diet of the recipient; the nature and extent of the symptoms of the disease being treated, the kind of concurrent treatment, complications from the disease being treated, or other health-related problems. Other treatment regimens or agents can be used in conjunction with the methods and agents of the present disclosure. Adjustments and manipulations of the determined dosages (e.g., frequency and duration) are well within the capabilities of those skilled in the art.

[0073] For any agent described herein, the therapeutically effective amount (e.g., effective dose or amount) can be determined initially from cell culture assays. The target concentration will be that concentration or dose which achieves the concentration of the therapeutic agent in the methods described herein as measured using the methods described herein or known in the art.

[0074] As is well known in the art, therapeutically effective amounts for humans can also be determined from animal models. For example, a dose for humans can be formulated according to concentration that has been found to be effective in animals. The dosage in humans can be adjusted by monitoring the effectiveness of the agent and adjusting the dosage up or down, as described above. Adjusting the dosage in humans to achieve maximum efficacy based on the methods described above and other methods is well within the capabilities of the ordinarily skilled artisan.

[0075] The dosage can vary according to the requirements of the patient and the therapeutic agent used. The dosage administered to a patient should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose will also depend on the nature of any adverse side effects. The determination of optimum doses for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages that are less than the optimum dose, followed by an increase in dosage, until the optimum effect under circumstances is reached. Both the dose and interval between doses can be adjusted as required to

[0076] An effective dose of a pharmacological inhibitor of the present disclosure for treating vEDS, treating Marfan syndrome, and / or altering expression or activity of the PLC / IP3 / PKC / ERK signaling pathway can be about 0.001 mg / kg to about 0.01 mg / kg of the pharmacological inhibitor, about 0.01 mg / kg to about 0.1 mg / kg of the pharmacological inhibitor, about 0.1 mg / kg to about 1.0 mg / kg of the pharmacological inhibitor, about 1.0 mg / kg to about 5.0 mg / kg of the pharmacological inhibitor, about 5.0 mg / kg to about 10 mg / kg of the pharmacological inhibitor, about 10 mg / kg to about 15 mg / kg of the pharmacological inhibitor, about 15 mg / kg to about 20 mg / kg of the pharmacological inhibitor, about 20 mg / kg to about 25 mg / kg of the pharmacological inhibitor, about 25 mg / kg to about 30 mg / kg of the pharmacological inhibitor, about 30 mg / kg to about 35 mg / kg of the pharmacological inhibitor, about 35 mg / kg to about 40 mg / kg of the pharmacological inhibitor, about 40 mg / kg to about 45 mg / kg of the pharmacological inhibitor, about 45 mg / kg to about 50 mg / kg of the pharmacological inhibitor, about 50 mg / kg to about 55 mg / kg of the pharmacological inhibitor, about 55 mg / kg to about 60 mg / kg of the pharmacological inhibitor, about 60 mg / kg to about 65 mg / kg of the pharmacological inhibitor, about 65 mg / kg to about 70 mg / kg of the pharmacological inhibitor, about 70 mg / kg to about 75 mg / kg of the pharmacological inhibitor, about 75 mg / kg to about 80 mg / kg of the pharmacological inhibitor, about 80 mg / kg to about 85 mg / kg of the pharmacological inhibitor, about 85 mg / kg to about 90 mg / kg of the pharmacological inhibitor, about 90 mg / kg to about 95 mg / kg of the pharmacological inhibitor, or about 95 mg / kg to about 100 mg / kg of the pharmacological inhibitor.

[0077] In certain aspects, the present disclosure includes a composition having an effective dose of one or more pharmacological inhibitors of the present disclosure, wherein the pharmacological inhibitor can be about 0.1% to about 20% w / v of the composition. Unless there is an explicit indication to the contrary, the weight % of a component is based on the total weight of the formulation or composition in which the component is included.

[0078] For example, an effective dose of a pharmacological inhibitor of the present disclosure can be about 0.001%- about 0.01%, about 0.01%- about 0.1%, about 0.1%- about 1.0%, about 1.0%- about 2.0%, about 2.0%- about 3.0%, about 3.0%- about 4.0%, about 4.0%- about 5.0%, about 5.0%- about 6.0%, about 6.0%- about 7.0%, about 7.0%- about 8.0%, about 8.0%- about 9.0%, about 9.0%- about 10%, about 10%- about 11%, about 11%- about 12%, about 12%- about 13%, about 13%- about 14%, about 14%- about 15%, about 15%- about 16%, about 16%- about 17%, about 17%- about 18%, about 18%- about 19%, or about 19%- about 20% w / v of the composition.

[0079] As used herein, the terms "treating" and "treatment" refer to the administration of an agent or formulation to an individual suffering from clinical symptoms of an undesirable condition, disorder, or disease, thereby achieving a decrease in the severity and / or frequency of the symptoms, eliminating the symptoms and / or their underlying causes, and / or promoting improvement or remediation of the damage.

[0080] As used herein, the terms "subject," "patient," "individual," and the like are not intended to be limiting and are generally interchangeable. An individual described as a "subject," "patient," "individual," and the like does not necessarily suffer from a given disease, but may simply be seeking medical advice. As used herein, the terms "subject," "patient," "individual," and the like include all members of the animal kingdom that may suffer from a specified disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is a human.

[0081] The term "sample" as used herein refers to a biological sample obtained for in vitro evaluation purposes. In embodiments, the sample may comprise a body fluid. In certain embodiments, the body fluid includes, but is not limited to, whole blood, plasma, serum, lymph, breast milk, saliva, mucus, semen, cell extracts, inflammatory fluids, cerebrospinal fluid, vitreous humor, tears, vitreous body, aqueous humor, or urine obtained from a subject. In some aspects, the sample is a composite panel of two or more body fluids. In exemplary aspects, the sample comprises blood or a fraction thereof (e.g., plasma, serum, or a fraction obtained by leukapheresis).

[0082] The scope provided herein should be understood as a shorthand for all values ​​within the scope. For example, the scope of 1-50 should be understood to include any number, combination of numbers or subranges from a set consisting of 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 or 50, and all insertion decimal values ​​between the aforementioned integers, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8 and 1.9. With respect to subranges, "nested subranges" extending from either end of the scope are particularly encompassed. For example, nested sub-ranges of the exemplary range of 1-50 may include 1-10, 1-20, 1-30, and 1-40 in one direction, or 50-40, 50-30, 50-20, and 50-10 in the other direction.

[0083] "Recombinant" refers to a nucleic acid molecule formed by bringing together genetic material from multiple sources through laboratory methods of genetic recombination (eg, molecular cloning) to produce a sequence not otherwise found in a biological organism.

[0084] As used herein, the term "expression" or "expressed" in connection with a DNA nucleic acid sequence (e.g., a gene) refers to the transcription and / or translation product of the sequence. Based on the amount of the corresponding mRNA present in the cell or the amount of the protein encoded by the DNA produced by the cell, the expression level of the DNA molecule in the cell can be determined (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual (Molecular Cloning: Experimental Guide), 18.7-18.88). When used with respect to a polypeptide, expression is included in any step involved in polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting proteins (e.g., ELISA, western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0085] "Reduction" refers to a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0086] "Reference" refers to a standard or control condition.

[0087] Unless otherwise specified or obvious from the context, the terms "a", "an" and "the" used herein should be understood to be singular or plural. Unless otherwise specified or obvious from the context, the term "or" used herein should be understood to be inclusive.

[0088] Unless otherwise specified or apparent from the context, the term "about" as used herein is understood to mean within a range generally tolerated in the art, for example, within 2 standard deviations of the mean. Approximately can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0089] As used herein, the term "derivative" refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein), and whose structure is sufficiently similar to those disclosed herein, and based on this similarity, one skilled in the art would foresee that it would exhibit the same or similar activity and utility as the claimed compound, or act as a precursor to induce the same or similar activity and utility as the claimed compound. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of the parent compound.

[0090] It is contemplated that each of the embodiments disclosed herein can apply to each of the other disclosed embodiments. Thus, all combinations of various elements described herein are within the scope of the application.

[0091] Other features and advantages of the present application will be apparent from the following detailed description of the preferred embodiments, and from the claims. Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the application. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. Genbank and NCBI submission files indicated by accession numbers cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF DRAWINGS

[0092] Figure 1A is a graph demonstrating that the G209S / + mouse model recapitulates the phenotype of vascular Ehlers-Danlos syndrome (vEDS). The percent survival is shown relative to age. The median survival was 400 days, p < 0.0001.

[0093] Figure 1B is a graph demonstrating that the G938D / + mouse model recapitulates the phenotype of vEDS. The percent survival is shown relative to age. The median survival was 45 days, p < 0.0001.

[0094] Figure 1C is an image depicting the heart and aorta of a vEDS mouse. The arrow (left) indicates the ascending aorta, and the red arrow (right) indicates the location of the aortic dissection in the descending aorta.

[0095] Figure 1D is a graph demonstrating that vEDS samples cluster separately from controls, indicating significant differences in the transcriptome of vEDS aortas.

[0096] Figure 1E is an illustrative graph demonstrating network analysis indicating that vEDS aortas show a gene expression profile of elevated mitogen-activated protein kinase (MAPK) activity (P38, JNK, AKT, ERK, ERK1 / 2).

[0097] Figure 1FIs a table illustrating that upstream analysis predicts that transcriptional differences in vEDS aortas are driven by G protein-coupled receptor (GPCR) activation, which signals through the associated PLC / IP3 / PKC / ERK axis.

[0098] Figure 1G Figures 2 and 3 are immunoblots and graphs demonstrating elevated markers of GPCR and MAPK signaling by immunoblotting for markers of active signaling through this pathway. ERK1 / 2 phosphorylation and PKC phosphorylation were found to be significantly higher in vEDS aortas.

[0099] Figure 2A The graph depicts the survival rate of mice treated with cobimetinib (FDA-approved MEK inhibitor). A 94% survival rate was observed after 45 days of treatment compared to only 55% survival in untreated mice.

[0100] Figure 2B The graph depicts the survival rate of mice treated with ruboxistaurin, a well-tolerated, orally administered pharmacological agent that specifically inhibits PKCβ. 100% survival was observed after 39 days of treatment, compared to only 55% survival in untreated mice.

[0101] Figure 2C The graph depicts the survival of mice treated with hydralazine (which blocks the PLC / IP3 / PKC / ERK axis). Protection of 98% survival was observed at 45 days of age (median survival of untreated vEDS mice).

[0102] Figure 3A The graph depicts that in the vEDS mouse model described herein, pregnancy and lactation are associated with a 60% lethality rate in vEDS mice due to arterial dissection within the first 30 days postpartum.

[0103] Figure 3B The graphic depicts that arresting lactation by removing pups after birth was able to prevent dissection and mortality in vEDS mice, with 100% survival observed.

[0104] Figure 3C The graph depicts that near complete survival (95%) was achieved following treatment with hydralazine (16 mg / kg / day), which blocks the PLC / IP3 / PKC / ERK axis activated by oxytocin.

[0105] Figure 3DThe graph depicts the protection (95% survival) observed following treatment with 1 mg / kg / day of trametinib (GSK-1120212), an FDA-approved inhibitor of MEK, a kinase that activates ERK.

[0106] Figure 3E The bar graph depicts that increased risk of death is associated with increased ERK activation, while protection from aortic dissection is associated with decreased ERK activation, as measured by immunoblotting.

[0107] Figure 3F The bar graph depicts that increased risk of death is associated with increased ERK activation, while protection from aortic dissection is associated with decreased ERK activation, as measured by ERK target gene expression.

[0108] Figure 4A are immunoblot images demonstrating that pharmacological inhibition of PKCβ prevents PKC autophosphorylation and phosphorylation of ERK in the aortic wall, as assessed by immunoblotting of aortic lysates, and indicating that pharmacological inhibition of MEK is associated with the expected reduction in phosphorylation of ERK, a downstream substrate of MEK, and surprisingly also with reduced PKC phosphorylation, suggesting the existence of a positive feedback loop.

[0109] Figure 4B and Figure 4C The figure depicts quantification of immunoblot images, which shows that pharmacological inhibition of PKCβ prevented PKC autophosphorylation and ERK phosphorylation in the aortic wall, while pharmacological inhibition of MEK was associated with the expected reduction in phosphorylation of ERK (a downstream substrate of MEK) and, surprisingly, also with reduced PKC phosphorylation, suggesting the existence of a positive feedback loop (*p<0.05, **p<0.01, ***p<0.001). Neither cobimetinib nor rubutrazol had an effect on blood pressure.

[0110] Figure 5A and Figure 5B The graph depicts that treatment of mice with hydralazine (32 mg / kg / d), which blocks the PLC / IP3 / PKC / ERK axis, provided significant protection: 98% survival at 45 days of age (median survival for untreated vEDS mice), with survival being affected during puberty, and this risk was seen almost exclusively in male mice ( Figure 5A Male mice and Figure 5B female mice).

[0111] Figure 6The graph depicts that the combination of hydralazine (32 mg / kg / d) and bicalutamide (50 mg / kg / d) resulted in a 90% survival rate in male mice, compared to only a 24% survival rate in male mice treated with hydralazine alone. Male mice continued to survive after the removal of bicalutamide after puberty, suggesting a time-dependent androgen sensitivity in this mouse model.

[0112] Figure 7 The graph depicts that treatment of male mice with bicalutamide alone resulted in an intermediate survival rate of approximately 80%, and males failed to survive after removal of bicalutamide after puberty, suggesting that inhibition of androgen signaling alone is not sufficient to prevent aortic disease in our vEDS mouse model.

[0113] Figure 8A and Figure 8B The graph depicts that mice treated with spironolactone (100 mg / kg / d) alone exhibited an intermediate survival rate of approximately 80%, similar to bicalutamide alone, but the combination of spironolactone (100 mg / kg / d) and hydralazine (32 mg / kg / d) resulted in 100% survival after 50 days of treatment, similar to the combination of hydralazine and bicalutamide.

[0114] Figure 9 The graph depicts that mice treated with higher doses of hydralazine had increasing doses of hydralazine (50 mg / kg / d) and their survival was not improved above the 32 mg / kg / d dose.

[0115] Figure 10A The graphic depicts that in vEDS mice, adding a specific oxytocin receptor antagonist 2 to the treatment of mice while still lactating resulted in 95% survival during the first 30 days postpartum, indicating that the significantly elevated risk of mortality due to aortic dissection and pregnancy is specifically driven by activation of the oxytocin receptor during breastfeeding.

[0116] Figure 10B The graph depicts that propranolol, a nonspecific beta antagonist that lowers blood pressure in vEDS mice, did not improve survival during the first 30 days postpartum in vEDS mice while still lactating, suggesting that the significantly elevated risk of death due to aortic dissection and pregnancy is not ameliorated by lowering blood pressure.

[0117] Figure 11A The graph indicates that inhibition of angiotensin-II signaling by treating mice with the angiotensin receptor antagonist losartan (60 mg / kg / d) did not show an effect on survival ( Figure 11A). Treatment of mice with the thrombin receptor antagonist vorapraxar (1 mg / kg / d) was found to have no effect on survival Figure 11B ) either.

[0118] Figure 12 The graph of Figure 4 depicts that treatment of mice with the non-specific tyrosine kinase receptor antagonist nintedanib (50 mg / kg / d) had no effect on survival, suggesting that tyrosine kinase receptor activation does not drive activation of the PLC / IP3 / PKC / ERK signaling pathway in vEDS mice.

[0119] Figures 13A-13C The graph of Figure 5 depicts that treatment of mice with the non-specific beta antagonist propranolol (80 mg / kg / d) Figure 13A ), the specific beta 1 antagonist atenolol (120 mg / kg / d) Figure 13B ) and the beta 1 antagonist / beta 2 agonist celiprolol (200 mg / kg / d) Figure 13C ) showed that these manipulations, although lowering blood pressure, did not result in an improvement in survival in our vEDS mouse model.

[0120] Figure 14 The graph of Figure 6 depicts that celiprolol, which accelerates the risk of aortic dissection in the mouse model, and vEDS mutations with Col3a1 G209S / + mutations also exhibit an increased risk of aortic dissection due to celiprolol.

[0121] Figure 15 The graph of Figure 7 depicts that amlodipine (12 mg / kg / d) also increases the risk of aortic dissection in the mouse model, and this has been shown to be consistent with MFS mice as well.

[0122] Figure 16A The graph of Figure 8 confirms pharmacological inhibition of PKC beta using a second specific PKC beta inhibitor, enzastaurin. Figure 16A : Enzastaurin (60 mg / kg / d) also rescues the risk of death from aortic dissection, with 80% of vEDS mice treated with enzastaurin surviving after 40 days of treatment, compared to only 50% of untreated vEDS mice (p = 0.0305).

[0123] Figure 16B The graph of Figure 9 confirms that treatment of mice with the endothelin-constrictor peptide-receptor antagonist bosentan improves survival. Figure 16BBosentan treatment resulted in 80% survival after 40 days of treatment, compared to only 50% survival in untreated vEDS mice (p=0.0298).

[0124] Figure 17 is an immunofluorescence staining demonstrating elevated signaling pathways in vascular tissue samples from human patients with vEDS. Figure 17 PKC phosphorylation was demonstrated in two tissue samples (iliac arteries and descending thoracic aorta) from vEDS patients.

[0125] Figure 18 is an immunofluorescence staining demonstrating elevated signaling pathways in vascular tissue samples from human patients with vEDS. Figure 18 ERK1 / 2 phosphorylation was demonstrated in two tissue samples (iliac arteries and descending thoracic aorta) from vEDS patients.

[0126] Figure 19 The graph demonstrates that lubutaurin treatment reduces aortic root growth in 129MFS mice. DETAILED DESCRIPTION

[0127] The present invention is based, at least in part, on the identification of novel, targetable signaling abnormalities that contribute to the pathogenesis of vEDS. Specifically, it was observed that pharmacological agents that inhibit ERK1 / 2 activation or PKC activation were able to successfully prevent mortality due to aortic dissection. Furthermore, agents that inhibit activation of the PLC / IP3 / PKC / ERK signaling pathway also prevented mortality due to aortic dissection. Together, these novel findings provide the first evidence for a targetable signaling abnormality that contributes to the pathogenesis of vEDS and potentially other vascular and connective tissue disorders.

[0128] Vascular pathologies

[0129] Vasculopathy is a term used to describe diseases that affect blood vessels. It often includes vascular abnormalities caused by degenerative, metabolic, and inflammatory diseases, embolic diseases, coagulopathy, and functional disorders such as posterior reversible encephalopathy syndrome. The cause of vasculopathy is often unknown, and the disease is often not pathologically confirmed. On the other hand, vasculitis is a more specific term and is defined as inflammation of the blood vessel wall.

[0130] As used herein, "vasculitis (angiitis)" refers to inflammation of blood vessels, e.g., arteritis, phlebitis, or lymphatic vessels, e.g., lymphangitis. Vasculitis can take many forms, such as cutaneous vasculitis, urticarial vasculitis, leukocytoclastic vasculitis, livedo vasculitis, and nodular vasculitis. Small vessel vasculitis can refer to inflammation of small or medium-sized blood vessels or lymphatic vessels, e.g., capillaries, venules, arterioles, and arteries.

[0131] Vascular Ehlers-Danlos Syndrome (vEDS)

[0132] Vascular Ehlers-Danlos syndrome (vEDS) is an inherited connective tissue disorder caused by heterozygous mutations in the collagen type III alpha 1 chain (COL3A1) gene. The primary cause of death in vEDS is arterial dissection and / or rupture, but the pathogenesis of the disease is poorly understood. There are no effective treatment strategies for this devastating condition. Current thinking is that reduced amounts of collagen III lead directly to the signs and symptoms of vEDS due to an inherent loss of the structural integrity of the tissue. However, early pathogenic models of Marfan syndrome (MFS) also abnormally induced tissue weakness resulting from failed elastogenesis, but subsequent work clearly demonstrated that in the absence of fibrillin-1, the gene product missing in MFS 3,4 ) in a mouse model of MFS. Subsequent work continues to demonstrate that TGF-β and downstream cell signaling molecules are major mediators of disease pathology. Furthermore, therapies that attenuate TGF-β signaling and related pathways, such as TGF-β neutralizing antibodies (Nabs), the angiotensin-II (Ang-II) type 1 receptor blocker (ARB) losartan, or the ERK1 / 2 activation inhibitor RDEA119 / trametinib, can inhibit aortic disease in MFS mice. 3-6 .

[0133] However, similar to other inherited vascular disorders such as Marfan syndrome and Lois-Dietz syndrome, signaling abnormalities presented herein are the primary mediators of disease pathology in vEDS. RNA-seq profiling of mouse aortas harboring patient-derived Col3a1 mutations revealed elevated PLC / IP3 / PKC / ERK signaling compared to wild-type aortas. Immunoblotting of the proximal descending thoracic aorta confirmed elevated PKC and ERK1 / 2 activation.

[0134] In certain embodiments, COL3A1 comprises the following amino acid sequence (NCBI Accession No.: AAH28178.1 (SEQ ID NO: 1), herein incorporated by reference in its entirety):

[0135]

[0136] In certain embodiments, COL3A1 comprises the following nucleic acid sequence, with the start and stop codons indicated in bold and underlined (NCBI Accession No.: NM_000090.3 (SEQ ID NO: 2), herein incorporated by reference in its entirety):

[0137]

[0138]

[0139]

[0140] MAP / ERK pathway

[0141] The MAPK / ERK pathway (also known as the Ras-Raf-MEK-ERK pathway) is a chain of proteins in cells that transmits signals from receptors on the cell surface to DNA in the cell nucleus.

[0142] Signaling begins when a signaling molecule binds to a receptor on the cell surface and ends when the DNA in the cell nucleus expresses the protein and produces some change in the cell, such as cell division. This pathway involves many proteins, including MAPK (mitogen-activated protein kinase, originally called ERK, extracellular signal-regulated kinase), which communicate by adding phosphate groups to neighboring proteins, which act as "on" or "off" switches.

[0143] The term "ERK" refers to any human ERK1 or ERK2 gene or protein. ERK1 is known by several names, including, for example, mitogen-activated protein kinase 3, extracellular signal-regulated kinase 1, insulin-stimulated MAP2 kinase, MAP kinase 1, MAPK1, p44-ERK1, ERT2, p44-MAPK, or microtubule-associated protein 2 kinase.

[0144] ERK2 is known by several names including, for example, mitogen-activated protein kinase 1, extracellular signal-regulated kinase 2, mitogen-activated protein kinase 2, MAP kinase 2, MAPK 2, p42-MAPK, or ERT1.

[0145] In certain embodiments, ERK1 comprises the following amino acid sequence (NCBI Accession No.: P27361.4 (SEQ ID NO: 3), herein incorporated by reference in its entirety):

[0146]

[0147] In certain embodiments, the ERK1 comprises the following nucleotide sequence, with the coding region indicated in bold and underlined (NCBI Accession No.: X60188.1 (SEQ ID NO: 4), herein incorporated by reference in its entirety):

[0148]

[0149]

[0150] In certain embodiments, ERK2 comprises the following amino acid sequence (NCBI Accession No.: P28482.3 (SEQ ID NO: 5), herein incorporated by reference in its entirety):

[0151]

[0152] In certain embodiments, the ERK2 comprises the following nucleotide sequence, with the coding sequence indicated in bold and underlined (NCBI Accession No.: NM_138957.3 (SEQ ID NO: 6), herein incorporated by reference in its entirety):

[0153]

[0154]

[0155] In certain embodiments, the present disclosure provides methods for treating a vascular disorder (eg, vEDS) in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an agent that reduces the activity or expression of ERK. Examples of ERK inhibitors include ASN007 (Asana BioSciences, Bridgewater, NJ), Ulixertinib (BVD-523) (BioMed Valley Discoveries, Kansas City, MO), CC-90003 (Celgene Corporation, Summit, NJ), GDC-0994 (Array BioPharma, Boulder, CO), KO-947 (Kura Oncology, San Diego, CA), LTT462 (Novartis, Basel, Switzerland), LY3214996 (Eli Lilly and Company, Indianapolis, IN), MK-8353 (Merck Sharp and Dohme Corp), Kenilworth, NJ.

[0156] MEK inhibitors

[0157] In embodiments, the present disclosure provides methods for treating vascular diseases (e.g., vEDS) in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an agent that reduces the activity or expression of ERK or PKC, for example, a Ras / Raf / MEK / ERK pathway inhibitor. In an embodiment, the Ras pathway inhibitor is selected from a Raf inhibitor such as vemurafenib, sorafenib or dabrafenib, a MEK inhibitor such as AZD6244 (selumetinib), PD0325901, GSK1120212 (trametinib), U0126-EtOH, PD184352, RDEA119 (rafametinib), PD98059, BIX 02189, MEK162 (bimetinib), AS-703026 (pimasertib), SL-327, BIX02188, AZD8330, TAK-733, cobimetinib or PD318088, and an ERK inhibitor such as LY3214996, BVD-523 or GDC-0994.

[0158] In an embodiment, the MEK inhibitor is selected from trametinib, lafatinib, cobimetinib, TAK-733, PD0325901, PD184352 (CI-10-40), R05126766, RO-4987655; E6201; GDC-0623; CH5126766; G-573; WX-554; selumetinib, bimetinib, and pimacitinib. In an embodiment, the MEK inhibitor comprises cobimetinib. In an embodiment, the MEK inhibitor comprises trametinib. In an embodiment, the MEK inhibitor comprises trametinib and cobimetinib. In an embodiment, a MEK inhibitor or a pharmaceutically acceptable salt thereof is also contemplated.

[0159] In certain embodiments, the MEK inhibitor may be administered at a concentration of about 0.001 mg / kg to about 250 mg / kg body weight, e.g., 0.001 mg / kg, 0.05 mg / kg, 0.01 mg / kg, 0.05 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, or 250 mg / kg body weight.

[0160] Protein kinase C (PKC) inhibitors

[0161] Protein kinase C (often abbreviated as PKC) is a family of protein kinases or members of this family that participate in controlling the function of other proteins by phosphorylating the hydroxyl groups of serine and threonine residues on these proteins. PKC enzymes are in turn activated by signals such as diacylglycerol (DAG) or calcium ions (Ca 2+ ) is activated by an increase in the concentration of PKC. Thus, PKC enzymes play an important role in several signal transduction cascades. The PKC family consists of 15 isoenzymes in humans and is divided into three subfamilies based on their second messenger requirements: conventional (or classical), novel, and atypical. Conventional PKC contains isoforms α, β I , β II and γ. These require Ca 2+ The novel (n)PKC includes δ, ε, η and θ isoforms and requires DAG but not Ca for activation. 2+ Thus, conventional and novel PKCs are activated through the same signal transduction pathway as phospholipase C. On the other hand, the activation of atypical (a)PKCs (including protein kinase Mζ and ι / λ isoforms) requires neither Ca nor phospholipase C. 2+Diacylglycerol is also not required.The term "protein kinase C" as used herein refers generally to the entire family of isoforms.

[0162] Exemplary PKC agents include, but are not limited to, lubutaurin, chelerythrine, miyabenol C, myricetin, gossypol, verbascoside, BIM-1, or bryostatin 1. In embodiments, the PKC inhibitor comprises enzastaurin. In embodiments, the PKC inhibitor comprises lubutaurin. In embodiments, PKC agents or pharmaceutically acceptable salts thereof are also contemplated.

[0163] In certain embodiments, the PKC inhibitor may have the following structure:

[0164] or a salt thereof,

[0165] in:

[0166] Ring A is substituted or unsubstituted C5-C6 cycloalkyl, substituted or unsubstituted 5-6 membered heterocycloalkyl having one or more N, O or S ring members, substituted or unsubstituted phenyl, or substituted or unsubstituted 5-6 membered heteroaryl having one or more N, O or S ring members;

[0167] Each R 1 and R 2 are independently hydrogen, halogen, -N3, -CN, -NO2, -NR A R B 、-C(O)R C 、-C(O)-OR C 、-C(O)NR A R B 、-OR D 、-NR A C(O)R C 、-NR A C(O)OR C , substituted or unsubstituted alkyl (e.g., C1-C2, C1-C4, C1-C8 or C1-C 10 ), substituted or unsubstituted heteroalkyl (e.g., 2-8, 2-6, 4-6, 2-3, or 4-5 membered ring members having one or more N, O, or S), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8, 3-6, 4-6, 4-5, or 5-6 membered ring members having one or more N, O, or S), substituted or unsubstituted aryl (e.g., C6-C 10or phenyl) or substituted or unsubstituted heteroaryl (e.g., 5-10, 5-9, or 5-6 membered having one or more N, O, or S ring members); or

[0168] R 1 and R 2 are linked together to form substituted or unsubstituted alkylene groups (e.g., C1-C2, C1-C4, C1-C8, C1-C 10 ), or substituted or unsubstituted heteroalkylene (e.g., 2-8, 2-6, 4-6, 2-3, or 4-5 membered with one or more N, O, or S in the heteroalkylene chain), substituted or unsubstituted cycloalkylene (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkylene (e.g., 3-8, 3-6, 4-6, 4-5, or 5-6 membered with one or more N, O, or S ring members), substituted or unsubstituted arylene (e.g., C6-C8, C3-C6, C4-C6, or C5-C6), 10 or phenylene) or a substituted or unsubstituted heteroarylene (e.g., 5-10 membered, 5-9 membered, or 5-6 membered having one or more N, O, or S ring members)) linker;

[0169] Each R A 、R B 、R C and R D is independently at each occurrence hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl (e.g., 2-8 membered with one or more N, O, or S atoms), substituted or unsubstituted cycloalkyl (e.g., 2-3 membered), substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered with one or more N, O, or S ring members), substituted or unsubstituted aryl such as phenyl, or substituted or unsubstituted heteroaryl (e.g., 5-10 membered with one or more N, O, or S ring members).

[0170] In certain embodiments, the ring A is a 5-6 membered heteroaryl group which may include one or more nitrogen as ring members.

[0171] In certain embodiments, the PKC inhibitor may have the following structure:

[0172] R 1 and R 2 is as described above.

[0173] In certain embodiments, the PKC inhibitor may have the following structure:

[0174] R1 and R 2 is as described above.

[0175] In certain embodiments, the PKC inhibitor may have the following structure:

[0176] R 1 and R 2 is as described above.

[0177] In certain embodiments, with respect to the PKC inhibitors of Formula (I), (II), (III), (IV-A), or (IV-B), each R 1 and R 2 is hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted 2-6 membered heteroalkyl (e.g., having one or more N, O, or S ring members), substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 5-10 membered heterocycloalkyl (e.g., having one or more N, O, or S ring members), substituted or unsubstituted phenyl, or substituted or unsubstituted 5-10 membered heteroaryl (e.g., having one or more N, O, or S ring members). In certain embodiments, each R 1 and R 2 is independently hydrogen, substituted or unsubstituted C1-C4 alkyl, or substituted heterocycloalkyl having one or more N, O, or S ring members. In certain embodiments, each R 1 and R 2 are independently hydrogen or unsubstituted C1-C4 alkyl.

[0178] In certain embodiments, with respect to the PKC inhibitors of formula (II), R 1 is a substituted or unsubstituted piperidinyl, piperazinyl, pyridinyl or pyrimidinyl. For example, R 1 yes

[0179] In certain embodiments, R 2 is hydrogen or unsubstituted C1-C4 alkyl. 2 is hydrogen. In certain embodiments, R 2 In certain embodiments, R 2 In certain embodiments, R 2 In an embodiment, R 2 In certain embodiments, R 2 It is tert-butyl.

[0180] In certain embodiments, with respect to the PKC inhibitors of Formula (I), (II), (III), (IV-A), or (IV-B), R1 and R 2 are connected to each other to form a substituted or unsubstituted C1-C8 alkylene, or a substituted or unsubstituted 2-8 membered heteroalkylene linker having one or more N, O or S atoms in the heteroalkylene chain. 1 and R 2 are linked to each other to form a substituted or unsubstituted C1-C8 alkylene linker. 1 and R 2 are connected to each other to form a substituted C1-C8 alkylene linker. 1 and R 2 are linked to each other to form an unsubstituted C1-C8 alkylene linker. 1 and R 2 are linked to each other to form a 2-8 membered heteroalkylene linker having one or more N, O, or S atoms in the heteroalkylene chain. 1 and R 2 are linked to each other to form an unsubstituted 2-8 membered heteroalkylene linker having one or more N, O or S atoms in the heteroalkylene linker chain.

[0181] In certain embodiments, R 1 and R 2 Connect to each other to form where R 3 It is hydrogen, halogen, -N3, -CN, -NO2, -NR A R B 、-C(O)R C 、-C(O)-OR C 、-C(O)NR A R B 、-OR D 、-NR A C(O)R C 、-NR A C(O)OR C , substituted or unsubstituted alkyl (e.g., C1-C2, C1-C4, C1-C8 or C1-C 10), substituted or unsubstituted heteroalkyl (e.g., 2-8, 2-6, 4-6, 2-3, or 4-5 membered with one or more N, O, or S ring members), substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6), substituted or unsubstituted heterocycloalkyl (e.g., 3-8, 3-6, 4-6, 4-5, or 5-6 membered with one or more N, O, or S ring members), substituted or unsubstituted aryl (e.g., C6-C 10 or phenyl) or substituted or unsubstituted heteroaryl (e.g., 5-10, 5-9, or 5-6 members and having one or more N, O, or S ring members). A 、R B 、R C and R D is as described above.

[0182] In certain embodiments, R 1 and R 2 Connect to each other to form It can be one or more of the above R 3 replace.

[0183] In certain embodiments, R 1 and R 2 Connect to each other to form R 3 is as described above.

[0184] In certain embodiments, R 1 and R 2 Connect to each other to form It can be one or more of the above R 3 replace.

[0185] In certain embodiments, with respect to the PKC inhibitors of Formula (IV-A) or (IV-B), R 1 and R 2 are linked to each other to form a substituted or unsubstituted C5-C6 cycloalkylene linker. 1 and R 2 are linked to each other to form a substituted C5-C6 cycloalkylene linker, for example, which may be replaced by one or more of the above-mentioned R 3 In certain embodiments, R 1 and R 2 are linked to each other to form an unsubstituted C5-C6 cycloalkylene linker.

[0186] In certain embodiments, R1 and R 2 are connected to each other to form a substituted or unsubstituted 5-6 membered heterocycloalkylene linker having one or more N, O, or S atoms in the linker chain. In certain embodiments, R 1 and R 2 are connected to each other to form a substituted 5-6 membered heterocycloalkylene linker having one or more N, O, or S atoms in the linker chain, which can be substituted with one or more R 3 as described above. In certain embodiments, R 1 and R 2 are connected to each other to form an unsubstituted 5-6 membered heterocycloalkylene linker having one or more N, O, or S atoms in the linker chain.

[0187] In certain embodiments, with respect to the PKC inhibitors of Formula (III), each R 1 and R 2 are connected to each other to form which can be substituted with one or more R 3 as described above. In certain embodiments, R 1 and R 2 are connected to each other to form which can be substituted with one or more R 3 as described above. In certain embodiments, R 1 and R 2 are connected to each other to form which can be substituted with one or more R 3 as described above.

[0188] In certain embodiments, with respect to the PKC inhibitors of Formula (III), each R 1 and R 2 are connected to each other to form which can be substituted with one or more R 3 as described above. In certain embodiments, R 1 and R 2 are connected to each other to form which can be substituted with one or more R 3 as described above. In certain embodiments, R 1 and R 2 are connected to each other to form which can be substituted with one or more R 3 as described above.

[0189] In certain embodiments, with respect to the PKC inhibitors of Formula (III), each R 1 and R 2is independently hydrogen, substituted or unsubstituted C1-C4 alkyl, or substituted or unsubstituted 5-6 membered heterocycloalkyl and having one or more N, O or S ring members. In certain embodiments, R 1 is unsubstituted C1-C4 alkyl. In certain embodiments, R 1 is a substituted 5-6 membered heterocycloalkyl having one or more N, O or S ring members, which may be replaced by one or more of the above R 3 In certain embodiments, R 1 is a substituted 5-6 membered heterocycloalkyl having one or more N, O or S ring members, which may be replaced by one or more of the above R 3 In certain embodiments, R 1 is substituted or unsubstituted piperidinyl or piperazinyl. In certain embodiments, R 1 is a substituted piperidinyl or piperazinyl group, which may be replaced by one or more of the above R 3 In certain embodiments, R 1 is unsubstituted piperidinyl or piperazinyl. In certain embodiments, R 1 yes It can be one or more of the above R 3 In certain embodiments, R 1 yes

[0190] In certain embodiments, R 2 is hydrogen. In an embodiment, R 2 It's methyl.

[0191] As discussed above, a "substituted" group is substituted at one or more available positions (typically 1, 2, 3, 4, or 5 positions) with one or more suitable groups (which may be the same or different) other than hydrogen. For example, halogen, cyano, amino, hydroxy, nitro, azido, carboxamido, -COOH, SO2NH2, alkyl (e.g., C1-C8 alkyl), alkenyl (e.g., C2-C8 alkenyl), alkynyl (e.g., C2-C8 alkynyl), alkoxy (e.g., C1-C8 alkoxy), alkyl ether (e.g., C2-C8 alkyl ether), alkylthio (e.g., C1-C8 alkylthio), mono- or di-(C1-C8 alkyl)amino, haloalkyl (e.g., C1-C6 haloalkyl), hydroxyalkyl (e.g., C1-C6 hydroxyalkyl), aminoalkyl (e.g., C1-C6 aminoalkyl), haloalkoxy (e.g., C1-C6 C1-C8 alkyl)amino, mono- and di-(C1-C8 alkyl)aminoC1-C8 alkyl, mono- and di-(C1-C8 alkyl)carboxamido, mono- and di-(C1-C8 alkyl)sulfonamido, alkylsulfinyl (e.g., C1-C8 alkylsulfinyl), alkylsulfonyl (e.g., C1-C8 alkylsulfonyl), aryl (e.g., phenyl), arylalkyl (e.g., (C6-C8 18 Aryl) C1-C8 alkyl, such as benzyl and phenethyl), aryloxy (e.g., C6-C 18 aryloxy such as phenoxy), arylalkoxy (e.g., (C6-C 18 aryl) C1-C8 alkoxy) and / or a 3-8 membered heterocyclic group having one or more N, O or S ring members.

[0192] As referred to herein, a group having a specified number of "members" or "members" means a group having the specified number of atoms.

[0193] Exemplary PKC inhibitors may include

[0194]

[0195]

[0196] In certain embodiments, the subject is administered a protein kinase inhibitor, or a monoclonal antibody that inhibits a receptor involved in a protein kinase or growth factor signaling pathway, such as EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptors, or a BRAF inhibitor. Non-limiting examples of protein kinase or growth factor signaling pathway inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, muritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saratinib, sorafenib, sunitinib, trastuzumab, fentanyl, AP23451, vemurafenib, and fentanyl. , MK-2206, GSK690693, A-443654, VQD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, rifolimus, avosidixide, genistein, selumetinib, AZD-6244, varanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016, or a mixture thereof.

[0197] In certain embodiments, the agent that reduces the activity or expression of PKC can be administered at a concentration of about 0.001 mg / kg to about 250 mg / kg body weight, e.g., 0.001 mg / kg, 0.05 mg / kg, 0.01 mg / kg, 0.05 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 125 mg / kg, 150 mg / kg, 175 mg / kg, 200 mg / kg, 225 mg / kg, or 250 mg / kg body weight.

[0198] In certain embodiments, the PKC comprises the following amino acid sequence (NCBI Accession No.: NP_002728.1 (SEQ ID NO: 7), herein incorporated by reference in its entirety):

[0199]

[0200]

[0201] In certain embodiments, the PKC comprises the following nucleotide sequence, with the coding sequence indicated in bold and underlined (NCBI Accession No.: NM_002737.2 (SEQ ID NO: 8), herein incorporated by reference in its entirety):

[0202]

[0203]

[0204]

[0205] Agents that reduce the expression or activity of phospholipase C (PLC) or inositol triphosphate (IP3)

[0206] In certain embodiments, certain agents that act as vasodilators are used to block the activity or expression of PLC or IP3. In embodiments, the PLC inhibitor comprises U-73122, U73343, and ET-18-OCH3. In embodiments, the IP3 inhibitor comprises 2-APB and Xestospongin C. In embodiments, the agent comprises hydralazine. It is contemplated that the inhibitor may be used alone or in any combination. In embodiments, PLC or IP3 inhibitors or pharmaceutically acceptable salts thereof are also contemplated.

[0207] In certain embodiments, with the concentration of about 0.001mg / kg to about 250mg / kg body weight, for example, the concentration of 0.001mg / kg, 0.05mg / kg, 0.01mg / kg, 0.05mg / kg, 1mg / kg, 5mg / kg, 10mg / kg, 25mg / kg, 50mg / kg, 75mg / kg, 100mg / kg, 125mg / kg, 150mg / kg, 175mg / kg, 200mg / kg, 225mg / kg or 250mg / kg body weight, use the activity reducing PLC or IP3 or the medicament of expression.In other embodiments, expection can be with higher concentration, for example, up to the concentration of 1000mg / kg body weight, use described medicament.

[0208] In certain embodiments, combination therapy with androgen antagonists (e.g., antiandrogens) is contemplated. Antiandrogens are a class of drugs that prevent androgens such as testosterone and dihydrotestosterone (DHT) from mediating their biological effects in the body. They work by blocking androgen receptors (AR) and / or inhibiting or curbing androgen production. Exemplary antiandrogens include:

[0209] Androgen receptor antagonists: Drugs that directly bind to and block the AR. These include the steroidal antiandrogens cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, ocantolone, and osatetron acetate (veterinary), and the nonsteroidal antiandrogens flutamide, bicalutamide, nilutamide, topilutamide, enzalutamide, and apalutamide. In addition to cyproterone acetate and chlormadinone acetate, several other progestins used in oral contraceptives and / or menopausal HRT (including dienogest, drospirenone, medrogestrel, nomegestrol acetate, promegestrol, and trimegestone) also have varying degrees of AR antagonist activity.

[0210] Androgen synthesis inhibitors: Drugs that directly inhibit the enzymatic biosynthesis of androgens (such as testosterone and / or DHT). Examples include the CYP17A1 inhibitors ketoconazole, abiraterone acetate, and sevitronel, the CYP11A1 (P450scc) inhibitor aminoglutethimide, and the 5α-reductase inhibitors finasteride, dutasteride, epristeride, α-estradiol, and saw palmetto extract (Serenoa repens). Many other antiandrogens, including cyproterone acetate, spironolactone, medrogestrel, flutamide, nilutamide, and pentofluphenazine, are also known to weakly inhibit androgen synthesis.

[0211] Antigonadotropins: Drugs that inhibit gonadotropin-releasing hormone (GnRH)-induced gonadotropin release and subsequent activation of gonadal androgen production. Examples include GnRH modulators such as leuprolide (a GnRH agonist) and cetrorelix (a GnRH antagonist), progestogens such as allylestradiol, chlormadinone acetate, cyproterone acetate, progesterone caproate, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, osatron acetate (veterinary) and orsendolone, and estrogens such as estradiol, estradiol esters, ethinylestradiol, conjugated estrogens, and diethylstilbestrol.

[0212] Other examples: drugs that antagonize the effects of androgens by means other than those described above. Examples include estrogens, especially oral and synthetic estrogens (e.g., ethinylestradiol, diethylstilbestrol), which stimulate the production of sex hormone-binding globulin (SHBG) in the liver, thereby reducing the free levels of testosterone and DHT, and thus reducing their biologically active levels; anticorticotropins such as glucocorticoids, which inhibit adrenocorticotropic hormone (ACTH)-induced adrenocorticotropic hormone production; and immunogens and vaccines against androstenedione, such as ovoandrostenedione albumin and androstenedione albumin, which reduce androgen levels by producing antibodies against androgens and the androgen precursor androstenedione (used only in veterinary medicine).

[0213] Connective tissue disorders

[0214] Connective tissue diseases refer to a group of disorders involving the protein-rich tissues that support organs and other parts of the body. Examples of connective tissue are fat, bone, and cartilage. These disorders often involve the joints, muscles, and skin, but they can also affect other organs and organ systems, including the eyes, heart, lungs, kidneys, gastrointestinal tract, and blood vessels. There are over 200 disorders affecting connective tissue. Causes and specific symptoms vary depending on the type.

[0215] Examples of tissue diseases (e.g., epithelial tissue, connective tissue, muscle tissue, and neural tissue) that may be treated with the compositions and methods include, but are not limited to, the following: autoimmune diseases, degenerative diseases, inflammatory diseases, infectious diseases, cancerous diseases, viral diseases, fungal diseases, injuries, or trauma-derived diseases. These tissue and / or organ diseases may be primary diseases or may be caused by existing diseases and / or conditions. Examples include amyloidosis, atrial fibrillation, convulsions, angina, dermatomyositis, enchondroma, fibroma, lumbao, hereditary connective tissue disorders (e.g., Marfan syndrome, Peyronie's disease, Ehlers-Danlos syndrome, osteogenesis imperfecta, Stickler syndrome, Alport syndrome, congenital contracture of elongated fingers (toes)), autoimmune connective tissue disorders (e.g., systemic lupus erythematosus (SLE), rheumatoid arthritis, scleroderma, Sjögren's syndrome, mixed connective tissue disease , psoriatic arthritis), scurvy, muscle diseases (e.g., muscle tumors, muscular dystrophy, disuse atrophy, denervation atrophy, Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy), liver disease, myasthenia gravis, myopathy, myositis, myositis ossificans, cancer, fibromyalgia, muscle fatigue, cramps, spasticity, sprains, strains, brain injury, spinal cord injury, glioma, neuroepithelioma, hypertension, cardiovascular disease, diabetes, Alzheimer's disease, cystitis, AIDS, rickets, and nerve sheath tumors.Examples of tissues, organs, and / or body systems that are affected by disease and that can be treated with the compositions and methods described herein include, but are not limited to, the following: the immune system, sensory organs (e.g., organs of taste, smell, vision, hearing), the digestive system (e.g., mouth, throat, pharynx, esophagus, abdomen, stomach, small intestine, large intestine, liver, pancreas), genitourinary organs, the endocrine system, the metabolic system, the cardiovascular system (e.g., heart, blood pressure, arteries), the blood system (e.g., blood chemistry), urinary organs (e.g., kidneys, ureters, bladder, male urethra, female urethra), male reproductive organs (e.g., testicles and their coverings, vas deferens, seminal vesicles, ejaculatory ducts, penis, prostate, bulbourethral glands), female reproductive organs (e.g., ovaries, uterine tubes, uterus, vagina, clitoris, Bartholin's glands, external organs, breasts), ductless glands (e.g., thyroid, parathyroid, thymus, pituitary, pineal, chromaffin and cortical system, spleen), reproductive system, respiratory system (e.g., larynx, trachea, bronchi, pleura, mediastinum, lungs), central nervous system (e.g., nerves, nerve fibers), skin, epithelium (e.g., simple, stratified, pseudostratified, columnar, glandular), connective tissue (e.g., loose connective tissue (e.g., lacunar, adipose, reticular) and dense connective tissue (e.g., dense regular, dense irregular), cartilage (e.g., hyaline, elastic, fibrous), muscle (e.g., skeletal muscle (e.g., type I, II, IIa, IIx, IIb), cardiac muscle, smooth muscle), nerves (e.g., neurons (e.g., motor neurons, interneurons, sensory neurons), glia, spinal cord, nerves, brain).

[0216] In embodiments, the connective tissue disorder comprises a vascular disorder (eg, vascular Ehlers-Danlos syndrome), Marfan syndrome, Loes-Dietz syndrome, or familial thoracic aortic aneurysm.

[0217] Ehlers-Danlos Syndrome (EDS)

[0218] Ehlers-Danlos syndrome (EDS) is a group of inherited connective tissue disorders. Symptoms may include loose joints, excessive skin elasticity, and abnormal scarring. These symptoms may be noticed at birth or in early childhood. Complications may include aortic dissection, joint dislocations, scoliosis, chronic pain, or early osteoarthritis.

[0219] EDS is caused by mutations in one of more than a dozen different genes. The specific gene affected determines the specific form of EDS. Some cases arise from new mutations that occur during early development, while others are inherited in an autosomal dominant or recessive manner. This results in defects in collagen structure or processing. The diagnosis can be confirmed with genetic testing or a skin biopsy. People may be misdiagnosed with hypochondriasis, depression, or chronic fatigue syndrome.

[0220] To date, there is no cure, but physical therapy and bracing may help strengthen muscles and support joints. While some disorders result in a normal life expectancy, those affecting blood vessels generally result in a shorter life expectancy. EDS affects approximately one in every 5,000 people worldwide, and the prognosis depends on the specific disorder.

[0221] EDS classification

[0222] The primary characteristic of hypermobility EDS (type 3 hEDS) is joint hypermobility affecting both large and small joints, which may lead to recurrent joint dislocations and subluxations (partial dislocations). Generally, people with this type have soft, smooth, tender skin, easy bruising, and chronic muscle and / or bone pain. The mutation that causes this type of EDS is unknown. Skin involvement is less common than in other types. There are no genetic tests for this type.

[0223] Classic EDS (type 1 cEDS) features smooth, extremely elastic (stretchy) skin that is fragile and bruises easily; extensive atrophic scars (flat or depressed scars); and joint hypermobility. Molluscum pseudotumors (calcified hematomas at pressure points such as the elbows) and globules (fat-filled cysts on the forearms and shins) are also common. Hypotonia and delayed motor development may occur. Mutations causing this type of EDS are in the genes COL5A1, COL5A2, and COL1A1. It involves more skin than hEDS.

[0224] Vascular EDS (type 4 vEDS) is characterized by thin, translucent skin that is very fragile and bruises easily. Arteries and certain organs (such as the intestines and uterus) are also fragile and easily ruptured. People with this type of EDS typically have a short stature and sparse hair. It also has characteristic facial features, including large eyes, a small chin, sunken cheeks, a thin nose and lips, and ears without earlobes. Joint hypermobility is present, but is usually limited to small joints (fingers and toes). Other common features include clubfoot, tendon and / or muscle rupture, acrodermatitis (premature aging of the skin on the hands and feet), early-onset varicose veins, pneumothorax (collapsed lung), gum recession, and a decreased amount of subcutaneous fat. It can be caused by mutations in the COL3A1 gene.

[0225] Kyphoscoliosis EDS (type 6 kEDS) is associated with severe hypotonia at birth, delayed motor development, progressive scoliosis (present from birth), and scleral fragility. Affected individuals may also have easy bruising, fragile arteries that rupture easily, abnormally small corneas, and osteopenia (low bone density). Other common features include the "Marfan syndrome body type," characterized by long, thin fingers (arachnodactyly), abnormally long limbs, and either a sunken chest (pectus excavatum) or a protruding chest (pectus carinatum). It can be caused by mutations in the gene PLOD1.

[0226] Arthrohysterectomy (aEDS type 7A&B) is characterized by severe joint hypermobility and congenital hip dislocation. Other common features include fragile, elastic skin that bruises easily, hypotonia, kyphosis (kyphosis and scoliosis), and mild osteopenia. Type I collagen is typically affected. It is very rare, with approximately 30 cases reported. It is more severe than the hypermobility type. Mutations in the genes COL1A1 and COL1A2 cause it.

[0227] EDS (type 7C dEDS) is characterized by very fragile skin that causes severe bruising and scarring; sagging, excess skin, especially on the face; and hernias. It is extremely rare, with about 10 cases reported.

[0228] Fragile corneal syndrome is characterized by thin corneas, early-onset progressive sphericity or keratoconus, and blue sclerae. Classic symptoms, such as hypermobile joints and hyperelastic skin, are also often seen.

[0229] Classic EDS (type 1 cEDS) is characterized by hyperextensibility of the skin, a soft texture, the absence of atrophic scarring, generalized joint hypermobility with or without recurrent dislocations (most commonly of the shoulders and ankles), and easy bruising or spontaneous ecchymoses (discoloration of the skin caused by underlying bleeding).

[0230] Spinal dysplasia EDS (spEDS) is characterized by short stature (progressing during childhood), hypotonia (ranging from severe congenital to milder late-onset forms), and bowing of the limbs.

[0231] Muscular dystrophy EDS (mcEDS) is characterized by multiple congenital contractures, characteristic adduction-flexion contractures and / or clubfoot (clubfoot), distinctive craniofacial features evident at birth or in early infancy, and cutaneous features such as skin hyperextensibility, bruising, skin fragility, atrophic scarring, and increased palmar wrinkling.

[0232] Myopathic EDS (mEDS) is characterized by congenital muscle hypotonia and / or muscle atrophy that improves with age, proximal joint contractures (knees, hips, and elbows), and distal joint hypermobility (ankles, wrists, feet, and hands).

[0233] Periodontal EDS (pEDS) is characterized by severe and persistent early-onset periodontitis (childhood or adolescence), absence of attached gingiva, pretibial plaque, and a family history in first-degree relatives meeting clinical criteria.

[0234] Cardiac valvular EDS (cvEDS) is characterized by severe, progressive heart valve problems (aortic, mitral), skin problems (hyperextensibility, atrophic scarring, thin skin, easy bruising), and joint hypermobility (generalized or limited to small joints).

[0235] Therapeutic methods

[0236] The present disclosure provides a method for treating a vascular lesion (e.g., vEDS) or a connective tissue disorder in a subject in need thereof by administering to the subject a therapeutically effective amount of an agent, wherein the agent reduces the activity or expression of extracellular signal-regulated kinase (ERK) or protein kinase C (PKC). The method further comprises administering an agent that reduces the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3). In embodiments, the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof for the preparation of a medicament that can be used to treat a vascular lesion (e.g., vEDS) or a connective tissue disorder.

[0237] The present disclosure also provides methods comprising conjoint therapy. "Conjoint therapy" or "co-therapy" as used herein includes administering a therapeutically effective amount of a medicament (e.g., an agent that reduces the activity or expression of ERK, PKC and / or PLC or IP3) or a pharmaceutically acceptable salt thereof, together with at least one additional active agent (also referred to herein as an "active pharmaceutical ingredient" ("API")), as part of a treatment regimen, to provide the beneficial effects of the combined action of the medicament (e.g., an agonist, antagonist or inhibitor) and the additional active agent.

[0238] According to the embodiments described below, "additional API" is understood to mean at least one additional API administered in a combination therapy regimen with an agent (e.g., an agent that reduces the activity or expression of ERK, PKC and / or PLC or IP3). In addition, it should be understood that more than one additional API described below may be used in a regimen. The terms "combination therapy" or "combination therapy regimen" are not intended to encompass the administration of two or more therapeutic compounds as part of an independent monotherapy regimen, where the monotherapy regimen incidentally and arbitrarily results in an unexpected or unpredicted beneficial effect.

[0239] Preferably, administration of a composition comprising an agent in combination with one or more of the additional APIs discussed herein (e.g., an agent that reduces the activity or expression of ERK, PKC and / or PLC or IP3) provides a synergistic response in the treated subject. In this context, the term "synergistic" means that the effect of the combination is more effective than the additive effect of any of the monotherapies alone.

[0240] The present disclosure also provides methods comprising combination therapies for treating vascular lesions (e.g., vEDS) or connective tissue disorders. As used herein, "combination therapy" or "co-therapy" includes administering the compounds described herein as part of a specific treatment regimen, along with at least one additional agent disclosed herein, with the intention of providing a beneficial effect from the combined action of these therapeutic compounds. The at least one additional agent can be a therapeutic agent or a non-therapeutic agent. The beneficial effects of the combination include, but are not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic compounds. The beneficial effects of the combination can also involve mitigating toxicities, side effects, or adverse events associated with another agent in the combination. "Combination therapy" may (but typically is not) intended to encompass the administration of two or more of these therapeutic compounds as part of separate monotherapy regimens, where the monotherapy regimens incidentally and arbitrarily lead to the combination of the present disclosure.

[0241] Thus, in certain embodiments, one or more agents are administered to a subject in need thereof that inhibit the expression or activity of mitogen-activated protein kinase / extracellular signal-regulated kinase (MEK), extracellular signal-regulated kinase (ERK), phospholipase C (PLC), inositol triphosphate (IP3), or protein kinase C (PKC), thereby inhibiting the activity of ERK, PLC, IP3, or PKC.

[0242] In certain embodiments, one or more agents that inhibit the activity or expression of one or more molecules involved in the mitogen-activated protein kinase (MAPK) pathway (e.g., RAS-RAF / MEK / extracellular signal-regulated kinase (ERK) protein kinase) are administered to a subject in need thereof.

[0243] In the context of conjoint therapy, the administration of antagonist can be carried out simultaneously or successively with the administration of one or more other medicaments. In another aspect, the administration of the different components of conjoint therapy can be carried out with different frequencies. Can be before administering compound of the present disclosure (for example, before 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks), concomitantly or afterwards (for example, afterwards 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks), administer one or more other medicaments.

[0244] As described in more detail herein, one or more additional agents can be formulated for co-administration with an agent of the present disclosure in a single dosage form. One or more additional agents can be administered separately from a dosage form comprising a compound of the present disclosure. When an additional agent is administered separately from a compound of the present disclosure, it can be by the same or a different route of administration as the compound of the present disclosure.

[0245] Preferably, the composition comprising the medicament of the present invention in combination with one or more additional medicaments is administered to provide a synergistic response in a subject suffering from an obstacle, disease or condition of the present invention. In this context, the term "synergistic" means that the efficacy of the combination is more effective than the additive effect of any single therapy alone. According to the synergistic effect of the combined therapy of the present invention, it is possible to allow the use of a lower dose and / or lower frequency than that outside the combination to administer at least one medicament in the combination. A synergistic effect can be manifested as avoiding or reducing adverse or undesirable side effects associated with using only any one of the therapies in the combination.

[0246] "Combination therapy" also encompasses the administration of a compound of the present disclosure in combination with a non-drug therapy (e.g., surgery or radiation therapy). Where the combination therapy further includes a non-drug therapy, the non-drug therapy may be administered at any appropriate time, as long as the beneficial effects of the combined effects of the therapeutic compound and the non-drug therapy are achieved. For example, in appropriate circumstances, when the non-drug therapy is temporarily removed from the administration of the therapeutic compound, a beneficial effect may still be achieved, which may persist for days or even weeks.

[0247] In embodiments of the methods described herein, an agent (e.g., an agent that reduces the activity or expression of ERK, PKC and / or PLC or IP3) can be administered alone or in combination with at least one additional agent in a method for treating a vascular lesion (e.g., vEDS) or a connective tissue disorder. In embodiments, the agent and the at least one additional agent are administered in a single dosage form. In another aspect, the agent and the at least one additional agent are administered in separate dosage forms. In embodiments, the at least one additional agent is a therapeutic agent. In embodiments, the therapeutic agent is indicated for treating a vascular lesion (e.g., vEDS) or a connective tissue disorder. In another aspect, the agent is co-administered with at least one additional agent that is not used to treat a vascular lesion (e.g., vEDS) or a connective tissue disorder, such as a second agent for alleviating the toxicity or adverse events associated with another active agent administered in a combination therapy.

[0248] In embodiments, the at least one additional agent is directed to a targeted therapy, wherein the treatment targets a vascular lesion (e.g., vEDS) or a connective tissue disorder, a protein that promotes progression of a vascular lesion (e.g., vEDS) or a connective tissue disorder, or the tissue environment.

[0249] In certain embodiments, conjoint therapy with androgen antagonists (e.g., antiandrogens) is encompassed. Antiandrogens are a class of drugs that prevent androgens such as testosterone and dihydrotestosterone (DHT) from mediating their biological effects in vivo. They work by blocking androgen receptors (ARs), and / or inhibiting or curbing androgen production.

[0250] In embodiments, exemplary combinations include an androgen antagonist plus a PLC, IP3, PKC, or ERK inhibitor.

[0251] The term "therapeutically effective amount" means an amount sufficient to treat a disease, disorder, or condition, ameliorate its symptoms, lessen its severity, or reduce its duration, or to enhance or improve the therapeutic effect of another therapy, or to prevent an identified disease, disorder, or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay known in the art. The precise effective amount for a subject will depend on the subject's weight, size, and health; the nature and extent of the condition; and the therapeutic agent or combination of therapeutic agents selected for administration.

[0252] An effective amount of the agent can be administered once a day, 2-5 times a day, up to 2 times a day or up to 3 times a day, or up to 8 times a day. In embodiments, the agent is administered 3 times a day, 2 times a day, once a day, 14 days on (4 times a day, 3 times a day, 2 times a day, or once a day) and 7 days off in a 3-week cycle, up to 5 or 7 days on (4 times a day, 3 times a day, 2 times a day, or once a day) and 14-16 days off in a 3-week cycle, or once every 2 days, or once a week, or once every 2 weeks, or once every 3 weeks.

[0253] The effective amount of an agent (e.g., an agent that reduces the activity or expression of ERK, PKC and / or PLC or IP3) can be in the range of about 0.001 mg / kg to about 1000 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 10 mg / kg; or any range below: wherein the lower end of the scope is any amount from 0.001 mg / kg to 900 mg / kg, and the upper end of the scope is any amount from 0.1 mg / kg to 1000 mg / kg (e.g., 0.005 mg / kg to 200 mg / kg, 0.5 mg / kg to 20 mg / kg). As known to those skilled in the art, effective doses will also vary, depending on the disease being treated, route of administration, excipient use, and the possibility of being used together with other therapeutic treatments (such as the use of other agents).

[0254] In more specific aspects, an agent of the present disclosure (e.g., an agent that reduces the activity or expression of ERK, PKC and / or PLC or IP3) is administered at a dosage of 30-300 mg / day (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg / day) for at least 1 week (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 36, 48, or more weeks). In certain embodiments, a compound described herein is administered at a dosage of 100-300 mg / day for 4 or 16 weeks. Alternatively or subsequently, an agent described herein is administered at a dosage of 100 mg twice daily for 8 weeks, or optionally, for 52 weeks.

[0255] As used herein, a "subject in need thereof" is a subject having a disease, disorder or condition, or a subject at increased risk of developing a disease, disorder or condition relative to the population as a whole. In a preferred aspect, a subject in need thereof is a subject having a vascular lesion (e.g., vEDS) or a connective tissue disorder, or a subject at increased risk of developing a vascular lesion (e.g., vEDS) or a connective tissue disorder relative to the population as a whole. A subject in need thereof may be a "non-responsive" or "refractory" subject to currently available therapies for the disease or disorder. In this context, the terms "non-responsive" and "refractory" mean that the subject's response to therapy is clinically insufficient to alleviate one or more symptoms associated with the disease or disorder.

[0256] "Subject" includes mammals. The mammal can be, for example, any mammal, e.g., a human, a primate, a vertebrate, a bird, a mouse, a rat, poultry, a dog, a cat, a cow, a horse, a goat, a camel, a sheep, or a pig. Preferably, the mammal is a human. The terms "subject" and "patient" are used interchangeably herein.

[0257] The present disclosure provides monotherapy for treating a disease, disorder or condition as described herein. As used herein, "monotherapy" means administering a single active or therapeutic compound to a subject in need thereof. Preferably, monotherapy will involve administering a therapeutically effective amount of the active compound. For example, a monotherapy using a medicament of the present disclosure can be administered to a subject in need of treatment in a therapeutically effective amount. Monotherapy can be contrasted with combination therapy, in which a combination of multiple active compounds is administered, preferably each component of the combination is present in a therapeutically effective amount. In one aspect, monotherapy with a medicament of the present disclosure is more effective than combination therapy in inducing a desired biological effect.

[0258] As used herein, "treatment," "treat," or "treating" describes the management and care of a patient for the purpose of combating a disease, condition, or disorder, and includes the administration of an agent of the present disclosure to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder.

[0259] As used herein, "prevention," "preventing," or "inhibit" describes the alleviation or elimination of the onset of symptoms or complications of a disease, condition, or disorder, and includes the administration of an agent of the disclosure to reduce the onset, development, or recurrence of symptoms of a disease, condition, or disorder.

[0260] As used herein, the term "relieve" is intended to describe the process of reducing the severity of a sign or symptom of a disorder. Importantly, a sign or symptom can be alleviated without being eliminated. In a preferred embodiment, administration of an agent of the present disclosure results in the elimination of a sign or symptom, however, elimination is not required. An effective dose is expected to reduce the severity of a sign or symptom.

[0261] As used herein, the term "symptom" is defined as an indication of a disease, condition, injury, or some discomfort in the body. Symptoms are felt or noticed by the individual experiencing the symptom, but may not be readily apparent to others. Others are defined as non-healthcare professionals.

[0262] Treatment of a disorder, disease, or condition according to the methods described herein can result in a decrease in the rate of progression of a vascular lesion (e.g., vEDS) or a connective tissue disorder. Preferably, after treatment, the rate of progression of the vascular lesion (e.g., vEDS) or a connective tissue disorder decreases by at least 5% relative to the pre-treatment figure; more preferably, the rate of progression of the vascular lesion (e.g., vEDS) or a connective tissue disorder decreases by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 50%; and most preferably, by at least 75%. The rate of progression of a vascular lesion (e.g., vEDS) or a connective tissue disorder can be measured by any reproducible means of measurement.

[0263] As used herein, the term "selectively" refers to a tendency to occur at a higher frequency in one population than in another. The populations compared may be cell populations. Preferably, the agents of the present disclosure act selectively on hyperproliferative cells while not acting on normal cells. The agents of the present disclosure act selectively to modulate one molecular target but do not significantly modulate another molecular target.

[0264] Combination therapy

[0265] In embodiments, the present disclosure also provides methods of combination therapy comprising hydralazine and at least one additional active agent. In embodiments, the at least one additional active agent is a therapeutic agent, for example, an anti-androgen compound such as bicalutamide or spironolactone.

[0266] As used herein, "combination therapy" or "co-therapy" includes the administration of a therapeutically effective amount of an agent described herein, together with at least one additional active agent, as part of a specific treatment regimen intended to provide a beneficial effect of the agent and the additional active agent acting together. The beneficial effects of the combination include, but are not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutically active compounds. "Combination therapy" is not intended to encompass the administration of two or more therapeutic compounds as part of separate monotherapy regimens, where the monotherapy regimens incidentally and arbitrarily result in unintended or unpredicted beneficial effects.

[0267] Preferably, conjoint therapy provides synergistic response in treated subject.In this context, term " synergistic " represents that the effectiveness of combination is more effective than the cumulative effect of any single monotherapy alone.According to the synergistic effect of conjoint therapy of the present invention, it is possible to allow the use of dosage and / or frequency lower than that outside the combination and / or lower frequency to administer at least one medicament in the combination.The other beneficial effect of the combination can be manifested as, avoids or reduces the adverse or undesirable side effect relevant to any therapy (also referred to as monotherapy) in the combination used alone.

[0268] "Combination therapy" also includes the administration of a medicament further combined with a non-drug therapy (e.g., surgery or radiation therapy), which inhibits or reduces the biological activity and / or expression of the signal transduction pathway (e.g., PLC / IP3 / PKC / ERK) of the present invention. In the case where combination therapy further includes non-drug therapy, non-drug therapy can be performed at any appropriate time, as long as the beneficial effect of the combined action of the therapeutic compound and the non-drug therapy is achieved. For example, in appropriate cases, when non-drug therapy is temporarily removed from the administration of the therapeutic compound, a beneficial effect is still achieved, and the beneficial effect may last for several days or even weeks. Non-drug therapy can be selected from chemotherapy, radiation therapy, hormone therapy, anti-estrogen therapy, gene therapy and surgery.

[0269] In the context of the methods described herein, the amount of the agent administered to the subject is a therapeutically effective amount. The term "therapeutically effective amount" refers to an amount that is sufficient to treat the disease being treated (e.g., vEDS), improve its symptoms, alleviate its severity, or reduce its duration, or enhance or improve the therapeutic effect of another therapy, or is sufficient to show a detectable therapeutic effect in the subject. In one embodiment, the therapeutically effective amount of an agent is an amount that effectively reduces a signaling pathway (e.g., PLC / IP3 / PKC / ERK).

[0270] In embodiments, administration of hydralazine and an antiandrogen according to the methods described herein results in elimination of symptoms or complications of the disease being treated (e.g., vEDS); however, elimination is not required. In one embodiment, the severity of the symptoms or complications is reduced.

[0271] Pharmaceutical compositions

[0272] In certain embodiments, the invention provides pharmaceutical compositions, which are included in the medicaments used in the present invention (for example, medicaments that reduce the activity or expression of ERK, PKC and / or PLC or IP3). By any means generally recognized for this delivery, the medicament can be suitably prepared and introduced into a subject or a cellular environment.

[0273] A "pharmaceutical composition" is a formulation containing an agent described herein in a pharmaceutically acceptable form suitable for administration to a subject. As used herein, the phrase "pharmaceutically acceptable" refers to compounds, substances, compositions, carriers, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0274] Such compositions generally include a pharmaceutical agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Supplementary active compounds may also be incorporated into the compositions.

[0275] As used herein, the term "pharmaceutically acceptable salt" is a salt formed, for example, by an acid and a basic group of the agents described herein. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, benzenesulfonate, gentisinate, fumarate, gluconate, glucaronate, sucrose, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (e.g., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)).

[0276] The pharmaceutical compositions are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0277] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating agents such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin. TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating agents such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0278] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound into a selected solvent along with one or a combination of ingredients listed above as needed, followed by sterilization filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other ingredients listed above as needed. With respect to sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying to produce a powder of the active ingredient and any other desired ingredients from a previously sterile-filtered solution thereof.

[0279] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be blended with an excipient and used in the form of tablets, lozenges or capsules (e.g., gelatin capsules). Oral compositions can also be prepared using a fluid carrier to be used as a mouthwash. Pharmaceutically suitable binders and / or excipients can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties: binders, such as microcrystalline cellulose, gum tragacanth or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, sodium carboxymethyl starch (Primogel) or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as mint, methyl salicylate or orange flavoring.

[0280] The compositions of the present invention can also be formulated into nanoparticle formulations. The compounds of the present invention can be used for immediate release, delayed release, regulated release, sustained release, pulsed release and / or controlled release applications. The pharmaceutical composition of the present invention can contain 0.01-99% by weight of active substance per volume. With regard to inhalation administration, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser (which contains a suitable propellant, for example, a gas such as carbon dioxide) or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0281] Systemic administration can also be by transmucosal or transdermal mode.About transmucosal or transdermal administration, use the penetrant that is suitable for the barrier that will penetrate in the preparation.This penetrant is generally known in the art, and for transmucosal administration, comprises for example detergent, bile salt and fusidic acid derivative.Transmucosal administration can be completed by using nasal spray or suppository.For transdermal administration, active compound is mixed with ointment, salves, gel or cream generally known in the art.Also can prepare described compound with the form of suppository (for example, with conventional suppository base, such as cocoa butter and other glycerides) or retention enema for rectal delivery.

[0282] In one embodiment, the active compound is prepared together with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Standard techniques can be used to prepare such preparations. The material can also be commercially available from Alza Corporation and Nova Pharmaceuticals. Liposomal suspensions (including liposomes that target infected cells with monoclonal antibodies directed against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent number 4,522,811.

[0283] The toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell culture or experimental animals, for example, by determining the LD 50 (a dose that is lethal to 50% of a population) and ED 50 (The dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 / ED 50 Compounds that exhibit high therapeutic indices are preferred. Although compounds with toxic side effects may be used, care should be taken to design delivery systems that target such compounds to the affected tissue site in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0284] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Such dosages of the compound are preferably such that circulating concentrations include the ED 50 The dosage may vary within this range depending on the dosage form employed and the route of administration utilized. For compounds used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC values ​​determined in cell culture. 50 (ie, the concentration of the test compound which achieves a half-maximal inhibition of symptoms.) Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0285] A therapeutically effective amount of an agent (i.e., an effective dose) as defined herein will depend on the agent selected. For example, a single dose of an agent can be administered in the range of about 1 pg to 1000 mg; in certain embodiments, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or 1000 mg can be administered. In certain embodiments, 1-5 g of a composition can be administered.

[0286] A therapeutically effective amount of a compound of the present application can be determined by methods known in the art. In addition to depending on the agent and the selected / pharmaceutical formulation used, the therapeutically effective amount of a pharmaceutical composition of the present application will also depend on the age and general physical condition of the patient and the route of administration. In certain embodiments, a therapeutic dose is typically about 10 to 2000 mg / day, and preferably about 30 to 1500 mg / day. Other ranges can be used, including, for example, 50-500 mg / day, 50-300 mg / day, 100-200 mg / day.

[0287] Administration can be once per day, twice per day, or more frequently, and can be reduced in a maintenance phase of the disease or disorder, e.g., once every two days or once every three days, rather than once per day or twice per day. The dose and frequency of administration will depend on clinical signs that confirm maintenance of the remission phase, and the reduction or absence of at least one or more, more preferably more than one, of the clinical signs of the acute phase known to the skilled artisan. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and the presence of other diseases. Moreover, treatment of a subject with a therapeutically effective amount of an agent can include a single treatment or, optionally, a series of treatments.

[0288] It is understood that the method of introducing an agent into a cellular environment will depend on the type of cell and the composition of its environment. Suitable amounts of the agent must be introduced, and these amounts can be determined empirically using standard methods. An exemplary effective concentration of a single agent in a cellular environment can be 500 micromolar or less, 50 micromolar or less, 10 micromolar or less, 1 micromolar or less, 500 nanomolar or less, 50 nanomolar or less, 10 nanomolar or less, or even a concentration of 1 nanomolar or less can be used.

[0289] The pharmaceutical composition can be included in a kit, container, pack, or dispenser together with instructions for administration.

[0290] Examples

[0291] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and treatment methods of the application, and are not intended to limit the scope of what the inventors regard as their application.

[0292] Example 1: Generation of vEDS mice with knock-in glycine substitutions.

[0293] New knock-in glycine substitution (Col3a1 G209S / + and Col3a1 G938D / +) vEDS mice were generated using CRISPR / Cas9. The mouse model recapitulates the human vascular phenotype and leverages the environmental impact on the disease phenotype to reveal the molecular basis of the ultimate failure of the blood vessel wall.

[0294] To introduce each mutation, three guide RNAs (tracrRNA + crRNA) were designed to target DNA regions flanking the intended mutation site. DNA oligo repair templates (ssDNA oligos) were also designed that included homology sequences upstream and downstream of the target region and the intended mutation. Single cell C57BL / 6J embryos were pronuclear injected by the JHU Transgenic Core using standard microinjection techniques using a mixture of Cas9 protein, tracrRNA, crRNA, and ssDNA oligo diluted in RNase-free injection buffer. Injected embryos were transferred into the oviducts of pseudo-pregnant ICR females using established techniques. Introduction of the mutation was confirmed by Sanger sequencing of the site, and mice were backcrossed for at least 4 generations to eliminate off-target effects. For G209S mice, the introduced mutation was c.625_626GG>TC, corresponding to p.Gly210Ser in humans. For G938D mice, the introduced mutation was c.2813G>A, corresponding to p.Gly939Ser in humans. NCBI Accession Number for wild-type human Col3a1 mRNA: NM_000090.3; NCBI Accession Number for wild-type mouse Col3a1 mRNA: NM_009930.2.

[0295] Example 2: Col3a1 G209S / + and Col3a1 G938D / + mouse models recapitulate vEDS phenotypes.

[0296] Col3a1 G209S / + and Col3a1 G938D / + mouse models recapitulate the vEDS phenotype. Mice with vEDS die suddenly due to aortic rupture, aortic dissection, or organ rupture, most commonly with hemothorax or hemoperitoneum at necropsy. Mice with the Col3a1 G938D / + mutation exhibit a more severe phenotype (median survival = 45 days vs. 400 days for the Col3a1 G209S / + model, p < 0.0001, Figures 1A-1C ).Figure 1A Figure 2 demonstrates that the G209S / + mouse model recapitulates the vEDS phenotype with a median survival of 400 days, p < 0.0001. In Figure 1B Figure 3 shows the survival of G938D / + mouse model, recapitulating vEDS with a median survival of 45 days, p < 0.0001.

[0297] In neither mouse model was evidence of aortic root aneurysm observed by echocardiography.

[0298] Example 3: Abnormal signaling is a mediator of disease pathology in vEDS.

[0299] It was hypothesized that aberrant signaling could be a major factor mediating disease pathology in vEDS. To test this hypothesis, given that the proximal descending aorta is the most common site of aortic dissection in this model, RNA-seq was performed on the proximal descending thoracic aortas of three Col3a1 G209S / + mice, three Col3a1 G938D / + mice, and three Col3a1+ / + (wild-type) mice. Unsupervised hierarchical clustering was performed using the most differentially expressed genes (FDR < 0.10). vEDS samples clustered separately from controls Figure 1D ), indicating significant differences in the transcriptome of vEDS aortas.

[0300] Network analysis indicated that vEDS aortas exhibited a gene expression profile of elevated mitogen-activated protein kinase (MAPK) activity [P38, JNK, AKT, ERK, ERK1 / 2] Figure 1E ).

[0301] Furthermore, upstream analysis predicted that transcriptional differences in vEDS aortas were driven by activation of G-protein coupled receptors (GPCRs), which signal through the associated PLC / IP3 / PKC / ERK axis Figure 1F ).

[0302] The signature of elevated GPCR and MAPK signaling was confirmed by immunoblotting for markers of active signaling through this pathway, and it was found that ERK1 / 2 phosphorylation and PKC phosphorylation were significantly higher in vEDS aortas Figure 1G ).

[0303] Compared with Marfan syndrome (MFS) or Lois-Dietz syndrome (LDS), the expression profile of vEDS aortas did not show the synthetic repertoire typical of high TGFβ signaling, but evidence of increased ERK activation was observed (similar to MFS and LDS). A small human study suggested that the beta-blocker celiprolol could potentially delay adverse events in vEDS patients, while angiotensin receptor blockers such as losartan provided striking protection in mouse models of MFS or LDS.

[0304] Furthermore, there was no evidence of protection from dissection or death in losartan-treated vEDS mice, whereas celiprolol was associated with a significant acceleration of dissection and death in both vEDS models; both drugs achieved the expected reduction in hemodynamic stress. Although early data suggest that hydralazine (which inhibits the PLC / IP3 / PKC / ERK axis) provides some protection in vEDS mice, these data highlight the need for a discovery-based approach to uncover unexpected therapeutic strategies.

[0305] vEDS mutations were introduced into pure 129 and BL6 backgrounds to assess modulation of phenotypic severity. Both mutations were associated with early mortality from aortic dissection on the BL6 background. Remarkably, the 129 background resulted in complete protection of both vEDS genotypes from dissection and a completely normal lifespan. Rescue was associated with normalization of the gene expression profile of the aortic wall. These data provide the rationale and motivation for genetic studies to identify the source and mechanism of the modifications in vEDS mice, with the hope and intent of mimicking the successful natural strategy using pharmacological agents.

[0306] Example 4: Pharmacological inhibition of the PLC / IP3 / PKC / ERK axis reduces the risk of aortic rupture in vEDS mice. Pharmacological ERK antagonists increase survival

[0307] Since activation of the PLC / IP3 / PKC / ERK axis has been shown to be pathogenic in MFS 7 , we therefore hypothesized that pharmacological inhibition of this axis would reduce the risk of aortic rupture in vEDS mice.

[0308] Figure 2A

[0309] Pharmacological ERK antagonists were tested to support a role for ERK activation in the pathogenesis of vEDS and as an approach to identify therapeutic strategies for vEDS. Mice were treated with cobimetinib [GDC-0973, RO5514041] (2 mg / kg / day), an FDA-approved inhibitor of MEK, a kinase that activates ERK. 8 We hypothesized that if ERK activation indeed drives disease risk, then ERK inhibition would rescue the risk of death from aortic dissection.

[0310] Consistent with this hypothesis, a 94% survival rate was observed after 45 days of treatment, compared with only 55% survival in the untreated group ( Pharmacological PKC inhibition increases survival ).

[0311] Figure 2B

[0312] Next, further studies were conducted to test agents that inhibit PKC activation. Mice were treated with lubutrone [LY 333531] (10 mg / kg / day), a well-tolerated, orally administered pharmacological agent that specifically inhibits PKCβ. 9 We hypothesized that if PLC / IP3 / PKC activation drives ERK activation and thereby disease risk, then pharmacological PKC inhibitors would also rescue the risk of death from aortic dissection.

[0313] Consistent with this hypothesis, 100% survival was observed after 39 days of treatment, compared with only 55% survival in the untreated group ( Agents that inhibit the PLC / IP3 / PKC / ERK signaling cascade increase survival ).

[0314] Figure 2C

[0315] Further studies were conducted to test other drugs that inhibit this signaling cascade. Mice were treated with hydralazine (32 mg / kg / d), which blocks the PLC / IP3 / PKC / ERK axis, hypothesizing that this drug targets the same pathway and thus provides similar protection. 10 Significant protection was observed, with 98% survival at 45 days of age (median survival of untreated vEDS mice). Example 5: Oxytocin-induced ERK signaling activates the PLC / IP3 / OKC / ERK axis and worsens the risk of aortic dissection ).

[0316] Although survival was affected during puberty, this risk was seen almost exclusively in male mice, so treatment with androgen antagonists may be beneficial in these mice. Furthermore, the dose of hydralazine may not be sufficient to completely inhibit this pathway in this mouse model; higher doses of hydralazine may prove beneficial.

[0317] Mice were treated with a combination of hydralazine and an androgen antagonist.

[0318] In addition, mice were treated with a higher dose of hydralazine.

[0319] These results suggest that inhibition of excessive PLC / IP3 / PKC / ERK signaling in the aorta rescues the risk of death from aortic dissection in a mouse model of vEDS.

[0320] Figure 3A Figure 3B

[0321] Patients with vascular Ehlers-Danlos syndrome (vEDS) experience dissections of medium- to large-sized arteries. Many features of vEDS differ significantly from other inherited vascular disorders, such as Marfan syndrome (MFS) and Lois-Dietz syndrome (LDS), which have been associated with excessive TGFβ activity. These include involvement of the aortic root and no particular predisposition to dissection in the absence of preexisting vasodilation. Vascular rupture in vEDS patients is difficult to predict or prevent. Pregnancy particularly increases the risk of dissection, with complications occurring in over 50% of pregnancies and approximately 12-25% of pregnancies resulting in mortality. Vascular dissections primarily occur postpartum, inconsistent with a mechanism solely induced by hemodynamic stress. Instead, it has been hypothesized that oxytocin, a hormone that triggers uterine contractions and persists during postpartum lactation, may contribute to pregnancy-associated risks. Oxytocin receptor expression is induced in the aorta during pregnancy, and this hormone stimulates surrounding tissues through activation of ERK, a signaling cascade previously implicated in the pathogenesis of MFS and LDS.

[0322] We have previously demonstrated that pregnancy-associated aortic dissection is largely driven by lactation-associated oxytocin release and oxytocin-induced ERK signaling in the aorta of MFS mice. 11 Therefore, oxytocin-induced ERK signaling was used as a method to test whether activation of the PLC / IP3 / PKC / ERK axis exacerbates the risk of aortic dissection.

[0323] In the vEDS mouse model, pregnancy and lactation were identified to be associated with a 60% lethality rate in vEDS mice caused by arterial dissection within the first 30 days postpartum ( Figure 3C Furthermore, blocking lactation by removing the pups after birth prevented dissection and mortality in vEDS mice (100% survival, Figure 3D Furthermore, treatment with hydralazine (16 mg / kg / day), which blocks the PLC / IP3 / PKC / ERK axis activated by oxytocin, resulted in near-complete survival (95%) ( Figure 3E Similar protection (95% survival) was observed after treatment with trametinib [GSK-1120212] (1 mg / kg / day), an FDA-approved inhibitor of MEK, a kinase that activates ERK ( Figure 3F ).

[0324] This increased risk of death was associated with increased ERK activation, as measured by immunoblotting and by ERK target gene expression, whereas protection from aortic dissection was associated with decreased ERK activation ( Example 6: Compositions and methods for treating vascular Ehlers-Danlos syndrome and related disorders and Figure 16AThese data further support that increased activation of the PLC / IP3 / PKC / ERK signaling pathway leads to a significantly increased risk of death from aortic dissection in the vEDS mouse model and that inhibition of the PLC / IP3 / PKC / ERK signaling pathway ameliorates this risk.

[0325] Figure 16B

[0326] Administration of pharmacological MEK / ERK antagonists and PKC antagonists rescued the risk of death from aortic dissection, suggesting that PKC-dependent ERK activation is a key component of aortic disease in vEDS and that targeting this signaling pathway could be beneficial in reducing aortic disease in vEDS mouse models.

[0327] Here we demonstrate that pharmacological inhibition of PKCβ using a second specific PKCβ inhibitor, enzastaurin (60 mg / kg / d), also rescues the risk of death from aortic dissection, with 80% of enzastaurin-treated vEDS mice surviving 40 days after treatment compared to only 50% of untreated vEDS mice (p = 0.0305, Figure 17 This provides further evidence that PKCβ phosphorylation is a key component of aortic disease in vEDS mice.

[0328] Since activation of the PLC / IP3 / PKC / ERK signaling pathway was identified herein as pathogenic in vEDS, the identity of receptors that might activate this aberrant signaling pathway was examined. GPCRs (Gq) signal through this pathway—Gq receptors commonly found in the aorta include angiotensin II receptor, thrombin receptor, endothelin-1 receptor, vasopressin receptor 1, sphingosine-1-phosphate receptor, alpha-1 adrenergic receptor, and serotonin receptor. However, there are also orphan GPCRs that are also expressed in the aorta, such as GPR56, which has been shown to interact with collagen 3. 3,4 .

[0329] Without wishing to be bound by theory, it is hypothesized that if the abnormally activated receptor is inhibited, one will be able to accurately identify how the signaling pathway is activated. Additionally, mice were treated with bosentan (100 mg / kg / d), an orally bioavailable, nonspecific endothelin receptor antagonist. Bosentan treatment resulted in an 80% survival rate after 40 days of treatment, compared to only 50% survival in untreated vEDS mice (p = 0.0298, Figure 18 ). This suggests that endothelin receptor signaling contributes to the pathogenesis of vEDS.

[0330] The use of pharmacological MEK / ERK antagonists and PKC antagonists rescued the risk of death from aortic dissection, suggesting that PKC-dependent ERK activation is a key component of aortic disease in vEDS and that targeting this signaling pathway is beneficial for reducing aortic disease in vEDS mouse models. We next sought to identify evidence that this signaling pathway is elevated in vascular tissue samples from human vEDS patients. PKC phosphorylation was identified in two tissue samples from vEDS patients: the iliac artery and the descending thoracic aorta. Example 7: Androgens have a significant role in aortic dissection risk and combination therapy with hydralazine. ) and ERK1 / 2 phosphorylation ( Figures 4A-4C These phosphorylated proteins were not seen in tissue samples taken from the ascending aorta of people without vEDS.

[0331] in conclusion

[0332] Together, these results provide the first evidence for targetable signaling abnormalities that contribute to the pathogenesis of vEDS. These data support the hypothesis that increased PLC / IP3 / PKC signaling drives increased MAPK / ERK activation, which in turn increases the risk of death from aortic dissection in a mouse model of vEDS.

[0333] Inhibition of ERK activation by pharmacological inhibition of MEK (an activator of ERK), or pharmacological inhibition of PKC, or pharmacological inhibition of the PLC / IP3 / PKC / ERK axis was shown to be sufficient to rescue death from aortic dissection. Agents that inhibit this pathway would provide therapeutic benefit for vascular Ehlers-Danlos syndrome and potentially other connective tissue disorders.

[0334] Furthermore, the possibility that PLC / IP3 / PKC / ERK activators might show coordinated upregulation in vEDS aortas was tested by analyzing candidates emerging from RNA-Seq profiles.

[0335] References

[0336] 1. Pepin M, Schwarze U, Superti-Furga A, Byers PH. Clinical and Genetic Features of Ehlers-Danlos Syndrome Type IV, the Vascular Type. N Engl JMed.2000; 342(10):673-680.doi:10.1056 / NEJM200003093421001.

[0337] 2.Pepin MG,Schwarze U,Rice KM,Liu M,Leistritz Dru,Byers PH.Survivalis affected by mutation type and molecular mechanism in vascular Ehlers-Danlos syndrome(EDS type IV).Genet Med.2014;16(12):881-888.doi:10.1038 / gim.2014.72.

[0338] 3.Habashi JP,Judge DP,Holm TM,等人Losartan,an AT1 Antagonist,PreventsAortic Aneurysm in a Mouse Model of Marfan Syndrome.Science(80-).2006;312(5770):117-121.doi:10.1126 / science.1124287.

[0339] 4.Holm TM,Habashi JP,Doyle JJ,等人Noncanonical TGFβsignalingcontributes to aortic aneurysm progression in Marfan syndromemice.Science.2011;332(6027):358-361.doi:10.1126 / science.1192149.

[0340] 5.Habashi JP,Doyle JJ,Holm TM,等人Angiotensin II Type 2ReceptorSignaling Attenuates Aortic Aneurysm in Mice Through ERK Antagonism.Science(80-).2011;332(6027):361-365.doi:10.1126 / science.1192152.

[0341] 6.Judge DP,Biery NJ,Keene DR,等人Evidence for a critical contributionof haploinsufficiency in the complex pathogenesis of Marfan syndrome.J ClinInvest.2004;114(2).doi:10.1172 / JCI200420641.

[0342] 7.Doyle JJ,Doyle AJ,Wilson NK,等人A deleterious gene-by-environmentinteraction imposed by calcium channel blockers in Marfansyndrome.Elife.2015;4.doi:10.7554 / eLife.08648.

[0343] 8.Larkin J,Ascierto PA,Dréno B,等人Combined Vemurafenib andCobimetinib in BRAF-Mutated Melanoma.N Engl J Med.2014;371(20):1867-1876.doi:10.1056 / NEJMoa1408868.

[0344] 9.PKC-DRS2 Group L,Aiello LP,Davis MD,等人Effect of ruboxistaurin onvisual loss in patients with diabetic retinopathy.Ophthalmology.2006;113(12):2221-2230.doi:10.1016 / j.ophtha.2006.07.032.

[0345] 10. Gurney AM, Allam M. Inhibition of calcium release from thesarcoplasmic reticulum of rabbit aorta by hydralazine. Br J Pharmacol. 1995; 114(1): 238-244. http: / / www.ncbi.nlm.nih.gov / pubmed / 7712024. Accessed September 24, 2018.

[0346] 11.Habashi JP, Gallo EM, Bagirzadeh R, et al. Oxytocin Antagonism PreventsPregnancy-Associated Aortic Dissection in a Mouse Model of Marfan Syndrome.; 2018.

[0347] Combination therapy of hydralazine and bicalutamide for treating vEDS

[0348] Pharmacological inhibition of PKCβ prevented PKC autophosphorylation and ERK phosphorylation in the aortic wall as assessed by immunoblotting of aortic lysates ( Figure 2A Pharmacological inhibition of MEK was associated not only with the expected reduction in ERK (a downstream substrate of MEK) phosphorylation but also, surprisingly, with a reduction in PKC phosphorylation, suggesting a positive feedback loop (Figure 1, *p < 0.05, **p < 0.01, ***p < 0.001). Neither cobimetinib nor rubutrazol had an effect on blood pressure.

[0349] Figure 6

[0350] Treatment of mice with hydralazine (32 mg / kg / d), which blocks the PLC / IP3 / PKC / ERK axis, provided significant protection: 98% survival at 45 days of age (the median survival of untreated vEDS mice). Although survival was affected during puberty, this risk was seen almost exclusively in male mice ( Figure 6 and 2B ), it was hypothesized that treatment with an androgen antagonist might be beneficial in these mice. Indeed, the combination of hydralazine (32 mg / kg / d) and bicalutamide (50 mg / kg / d) resulted in a 90% survival rate in male mice, compared to only 24% survival in male mice treated with hydralazine alone ( Figure 7 ).

[0351] Curiously, if bicalutamide was removed after puberty (90 days of life) while hydralazine continued to be administered, male mice continued to survive at the same rate ( Combination therapy of hydralazine and spironolactone for treating vEDS ), suggesting that there is a time-dependent androgen sensitivity in this mouse model. Informatively, treatment of male mice with bicalutamide alone resulted in an intermediate survival rate of approximately 80%, whereas males did not survive after bicalutamide removal after puberty ( Figure 8A ), suggesting that inhibition of androgen signaling alone is insufficient to prevent aortic disease in the vEDS mouse model.

[0352] Figure 8B

[0353] These new observations and insights into the significant role androgens play in the risk of aortic dissection in our vEDS mouse model led to testing another FDA-approved drug in combination with hydralazine. Spironolactone is an FDA-approved diuretic that has a direct androgen antagonist effect as a side effect. Spironolactone is used off-label specifically to treat acne, hirsutism, and other androgen-dependent disorders. 1 It is hypothesized that spironolactone may also act as a direct androgen antagonist in this disorder.

[0354] Mice treated with spironolactone (100 mg / kg / d) alone ( Combination therapy of hydralazine and ruboxylidine for treating vEDS ), an intermediate survival rate of approximately 80% was observed, similar to the results of treatment with bicalutamide alone. However, the combination of spironolactone (100 mg / kg / d) and hydralazine (32 mg / kg / d) resulted in a 100% survival rate after 50 days of treatment, similar to the combination of hydralazine and bicalutamide ( Combination therapy of hydralazine and enzalutamide for treating vEDS ).

[0355] Combination therapy of hydralazine and sotolcizumab for treating vEDS

[0356] In the embodiments, combination therapy comprising administration of hydralazine and lubustatin is contemplated. Lubustatin is a protein kinase C-β (PKC-β) inhibitor and a macrocyclic bisindolylmaleimide compound being developed by Eli Lilly and Company as a promising treatment for diabetic macular edema and other diabetic vascular diseases, including diabetic retinopathy, diabetic peripheral neuropathy, and diabetic nephropathy. Alternative lubustatin nomenclature includes Arxxant (proposed trademark name), IUPAC: (9S)-9-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9H,18H-5,21:12,17-di(methyl)dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecyne-18,20-dione, and CAS number: 169939-94-0.

[0357] The structure of lubutolone is provided below:

[0358]

[0359] Combination therapy of hydralazine and antiandrogens for treating vEDS

[0360] In the embodiments, combination therapies comprising administration of hydralazine and enzastaurin are contemplated. Enzastaurin is a synthetic bisindolylmaleimide with potential anti-tumor activity. By binding to the ATP-binding site, enzastaurin selectively inhibits protein kinase C beta (PKC-β), an enzyme involved in inducing vascular endothelial growth factor (VEGF)-stimulated angiogenesis. This agent may reduce tumor blood supply, thereby preventing growth. Alternative enzastaurin nomenclature includes: LY-317615, IUPAC: 3-(1-methylindol-3-yl)-4-[1-[1-(pyridin-2-ylmethyl)piperidin-4-yl]indol-3-yl]pyrrole-2,5-dione, and CAS number: 170364-57.

[0361] The structure of Enzastaurin is provided below:

[0362]

[0363] Increased doses of hydralazine

[0364] In the Examples, combination therapies comprising the administration of hydralazine and sotrastourin are contemplated. Sotrastourin (EAB071) is an investigational immunosuppressant that blocks the activation of T-lymphocytes through protein kinase C inhibition.

[0365] The structure of sotrastolin is provided below:

[0366]

[0367] Figure 9

[0368] Other combinations of hydralazine and different antiandrogens have been tested. For example, hydralazine and androgen receptor antagonists, including steroidal antiandrogens (cyproterone acetate, megestrol acetate, chlormadinone acetate, oxendolone and osatetron acetate), have been considered. In addition, combinations of hydralazine with nonsteroidal antiandrogens such as flutamide, nilutamide, toprotamide, enzalutamide, drospirenone or medrogestrel have been considered.

[0369] In other embodiments, combination therapies comprising hydralazine and androgen synthesis inhibitors or anti-gonadotropins are contemplated. Exemplary androgen synthesis inhibitors include ketoconazole, abiraterone acetate, aminoglutethimide (Cytadren), aminoglutethimide (Orimeten), finasteride, dutasteride, epristeride, and alpha-estradiol. Exemplary anti-gonadotropins include leuprolide and cetrorelix. Other contemplated anti-androgens include ethinyl estradiol and diethylstilbestrol.

[0370] Example 8: Oxytocin-induced signaling

[0371] Furthermore, the dose of hydralazine can not be sufficient in this mouse model to completely inhibit the pathway, and a higher dose of hydralazine can prove to be beneficial. Thus, these mice were treated with a higher dose of hydralazine, and it was found that increasing the dose of hydralazine (50 mg / kg / d) did not improve survival over the 32 mg / kg / d dose ( Figure 10A ).

[0372] Figure 10B

[0373] Oxytocin-induced ERK signaling was used as a means to test the hypothesis that activation of the PLC / IP3 / PKC / ERK axis worsens the risk of aortic dissection. In the vEDS mouse model described herein, it was found that pregnancy and lactation were associated with a 60% lethality in vEDS mice due to arterial dissection in the first 30 days postpartum, and that preventing lactation by removing pups after birth, treating with hydralazine (16 mg / kg / d), or treating with trametinib, was able to prevent dissection and death in vEDS mice. This increased risk of death was associated with an increase in ERK activation, as measured by immunoblotting and by ERK target gene expression, and protection from aortic dissection was associated with a decrease in ERK activation.

[0374] Treatment of mice with a specific oxytocin receptor antagonist was added 2 (using the selective oxytocin receptor antagonist des Gly-NH2, d(CH2)5[D-Tyr2,Thr4]OVT), which resulted in a 95% survival in vEDS mice that were still lactating in the first 30 days postpartum, indicating that the significantly elevated risk of death due to aortic dissection that accompanies pregnancy is specifically driven by activation of the oxytocin receptor during breast feeding GPCR activation of the PLC / IP3 / PKC / ERK signaling pathway and 10B ).

[0375] Furthermore, it was confirmed that treatment with propranolol ( Figure 11A) did not affect survival in this pregnancy / breastfeeding model (46% survival vs. 50% survival after 30 days postpartum). Propranolol is the standard of care in this population and reduces blood pressure without affecting PLC / IP3 / PKC / ERK signaling. This data further supports that increased activation of the PLC / IP3 / PKC / ERK signaling pathway leads to a significantly increased risk of death from aortic dissection in our vEDS mouse model and that inhibition of the PLC / IP3 / PKC / ERK signaling pathway ameliorates this risk.

[0376] Figure 11B

[0377] Since activation of the PLC / IP3 / PKC / ERK signaling pathway was identified as pathogenic in vEDS, receptors that may be activating this aberrant signaling pathway have been identified. GPCRs (Gq) signal through this pathway—Gq receptors commonly found in the aorta include angiotensin II receptor, thrombin receptor, endothelin-1 receptor, vasopressin receptor 1, sphingosine-1-phosphate receptor, alpha-1 adrenergic receptor, and serotonin receptor. However, there are also orphan GPCRs that are also expressed in the aorta, such as GPR56, which has been shown to interact with collagen 3. 3,4 .

[0378] It was hypothesized that if the activity of the receptor was inhibited, it would be possible to identify how the signaling pathway was activated. First, angiotensin-II signaling was inhibited by treating mice with the angiotensin receptor antagonist losartan (60 mg / kg / d). Tyrosine kinase receptors and the PLC / IP3 / PKC / ERK signaling pathway ), but it was found that it had no effect on survival. Then, mice were treated with the thrombin receptor antagonist vorapaxar (1 mg / kg / d), but it was found that it also had no effect on survival ( Figure 12 ).

[0379] Other specific Gq receptor inhibitors were tested. Exemplary Gq receptor inhibitors tested included endothelin-1 receptor, vasopressin receptor 1, sphingosine-1-phosphate receptor, alpha-1 adrenergic receptor, serotonin receptor, and orphan GPCRs such as GPR56.

[0380] Beta-adrenergic receptor blockers and the risk of aortic rupture

[0381] The PLC / IP3 / PKC / ERK signaling pathway can also be transactivated by tyrosine kinase receptors expressed in the aorta. These include EGFR, VEGFR, FGFR, and PDGFR. To test the hypothesis that tyrosine kinase receptors are abnormally activated and lead to increased PLC / IP3 / PKC / ERK signaling, treatment with the nonspecific tyrosine kinase receptor antagonist nintedanib (50 mg / kg / d) was conducted, but it was found that it also had no effect on survival ( Figures 13A-13C This suggests that tyrosine kinase receptor activation does not drive activation of the PLC / IP3 / PKC / ERK signaling pathway in vEDS mice.

[0382] Figure 14

[0383] Others have proposed 5-8 , the vEDS phenotype is the result of chronic "weak" tissue, and it has been proposed that lowering blood pressure with β-adrenergic receptor blockers would reduce the risk of aortic rupture in these patients. To address this hypothesis, mice were treated with the nonspecific β-antagonist propranolol (80 mg / kg / d), the specific β1-antagonist atenolol (120 mg / kg / d), and the β1-antagonist / β2-agonist celiprolol (200 mg / kg / d), but found that despite a reduction in blood pressure, none of these manipulations resulted in improved survival in our vEDS mouse model ( Figure 15 Celiprolol even accelerated the risk of aortic dissection in a mouse model and had a Col3a1 G209S / + The vEDS mutation also demonstrated an increased risk of aortic dissection with celiprolol ( Example 9: Treatment of Marfan Syndrome with a protein kinase C-beta (PKC-beta) inhibitor Since this is not common to all beta-antagonists, this observation may be driven by the beta2 agonist activity of celiprolol.

[0384] Calcium channel blockers, which lower blood pressure through another mechanism, were also tested. Amlodipine (12 mg / kg / d) was found to also increase the risk of aortic dissection in the mouse model, and this has been shown to be consistent with MFS (Marfan syndrome) mice. 9 ( Figure 19 ).

[0385] Combination therapy of hydralazine and antiandrogens for treating vEDS

[0386] In an embodiment, various PKC-β inhibitors are used to treat Marfan syndrome. For example, treatment of Marfan syndrome can include administration of lubutaurin. It is demonstrated herein that pharmacological inhibition of PKCβ using lubutaurin (10 mg / kg / d) rescues aortic root growth in Marfan syndrome mice (p=2E-4, Example 10: Pharmacological inhibition increases survival in vEDS mice.). In other embodiments, treatment of Marfan syndrome comprises administration of Enzastar or Sotar.

[0387] ​

[0388] Other combinations of hydralazine and different antiandrogens were tested. For example, hydralazine and androgen receptor antagonists, including steroidal antiandrogens (cyproterone acetate, megestrol acetate, chlormadinone acetate, ostarabine, and oxendolone) were considered. In addition, combinations of hydralazine with non-steroidal antiandrogens such as flutamide, nilutamide, toprilumide, enzalutamide, drospirenone, or medroxyprogesterone were considered.

[0389] In other embodiments, combination therapies comprising hydralazine and an androgen synthesis inhibitor or an anti-gonadotropin were considered. Exemplary androgen synthesis inhibitors include ketoconazole, abiraterone acetate, seletalisib, aminoglutethimide, finasteride, dutasteride, epristeride, and alpha-estradiol. Exemplary anti-gonadotropins include leuprolide and cetrorelix. Other antiandrogens considered include ethinyl estradiol and diethylstilbestrol.

[0390] ​

[0391] Pharmacological PKC inhibition with enzastaurin increases survival in vEDS mice

[0392] The Col3al G209S / + and Col3al G938D / + mouse models recapitulate the vEDS phenotype and were used to test agents to treat vEDS. The mice were treated with the well-tolerated orally administered agent enzastar at about 30 mg / kg / day. By binding to the ATP-binding site, enzastar selectively inhibits protein kinase C beta (PKC-beta), and thus pharmacological PKC inhibitors were hypothesized to rescue the risk of dying from aortic dissection. About 100% survival was observed after about 30 days (1 month) of treatment, in contrast to the significantly reduced survival in untreated mice (e.g., statistically significant difference compared to control mice).

[0393] The mice were also treated for at least about 45 days, and the survival of the enzastar-treated mice was evaluated relative to control mice.

[0394] The mice were also treated with higher doses of enzastar, e.g., about 40 mg / kg / day, 50 mg / kg / day, or 100 mg / kg / day. In other embodiments, the mice were treated with enzastar once per day or twice per day.

[0395] These results suggest that the use of enzastar in the aorta to inhibit excessive PLC / IP3 / PKC / ERK signaling rescues the risk of death from aortic dissection in the vEDS mouse model.

[0396] Pharmacological PKC inhibition with soratinib increases survival in vEDS mice

[0397] Other studies using the mouse models described herein (e.g., Col3a1 G209S / + and Col3a1 G938D / + mouse models) will be used to test other agents for the treatment of vEDS. Mice treated with the well-tolerated, orally administered agent, sotrastolin, at approximately 30 mg / kg / day, were evaluated. Sotrastolin (AEB071) is an immunosuppressant that blocks T-lymphocyte activation through protein kinase C inhibition. Similar to enzastaurin, it was hypothesized that PKC inhibitors (e.g., sotrastolin) would also rescue the risk of death from aortic dissection. Approximately 100% survival was observed after approximately 30 days (1 month) of treatment, compared to a significantly reduced survival rate in untreated mice (e.g., a statistically significant difference compared to control mice). Mice were also treated for at least about 45 days, and the survival rate of sotrastolin-treated mice relative to control mice was evaluated.

[0398] Mice were also treated with higher doses of sotrastaurin, for example, about 40 mg / kg / day, 50 mg / kg / day, or 100 mg / kg / day. In other embodiments, mice were treated with sotrastaurin once a day or twice a day.

[0399] These results demonstrate that inhibition of excessive PLC / IP3 / PKC / ERK signaling in the aorta using sotrastuzumab rescues the risk of death due to aortic dissection in the vEDS mouse model.

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[0410] Other implementation plans

[0411] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the following claims. Other aspects, advantages and modifications are within the scope of the following claims.

[0412] The patents and scientific literature mentioned herein establish the knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All disclosed foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by the accession numbers cited herein are hereby incorporated by reference. All other disclosed references, documents, manuscripts, and scientific literature cited herein are hereby incorporated by reference.

[0413] While the application has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the application encompassed by the appended claims.

Claims

1. Use of an agent in the preparation of a pharmaceutical composition for treating vascular Ehlers-Danlos syndrome (vEDS) in a subject, wherein the agent comprises a small molecule that reduces the activity or expression of extracellular signal-regulated kinase (ERK) or protein kinase C (PKC), wherein the small molecule is selected from cobimetinib, enzastaurin, sorafenib, lububurinib, or a pharmaceutically acceptable salt thereof.

2. The method according to claim 1, wherein the dosage of the drug is 0.001 mg / kg to 250 mg / kg body weight.

3. The method of claim 1, wherein the agent reduces the activity or expression of protein kinase C (PKC).

4. The use according to claim 3, wherein the agent reduces the level of PKC protein or mRNA by at least 5% compared to a normal control, and / or wherein the agent reduces the level of PKC activity by at least 5% compared to a normal control.

5. The method of claim 1, wherein the subject comprises a level of ERK or PKC protein or mRNA that is different from a normal control, and / or wherein the subject comprises a level of ERK or PKC protein or mRNA that is at least 5% higher than a normal control, and / or wherein the subject comprises a level of ERK or PKC activity that is at least 5% higher than a normal control.

6. The use of claim 5, wherein the level is in a test sample obtained from the subject, and wherein the test sample comprises blood, serum, plasma, saliva, tears, vitreous humor, sweat, cerebrospinal fluid, or urine.

7. The use according to any one of claims 1 to 6, wherein the use further comprises co-administration of an agent that reduces the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3).

8. The method according to claim 1, wherein the pharmaceutical agent is selected from lubutrone, enzastaurin, sotrastaurin or a pharmaceutically acceptable salt thereof.

9. The use according to claim 1, wherein the pharmaceutical agent comprises enzastaurin.

10. The use according to claim 8 or 9, wherein the dosage of the agent is 0.001 mg / kg to 250 mg / kg body weight.

11. The method according to claim 10, wherein the dosage of the medicament is 1 mg / kg to 15 mg / kg body weight.

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