Compositions and methods for treating vascular ehlers-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. TIFF2022504990000037.tif80128

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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 which are incorporated herein by reference in their entireties.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 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.

[0003] Field The present invention relates to connective tissue disorders. [Background technology]

[0004] background 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 bleeding, distinctive facial features, and develop spontaneous dissections of medium to large blood vessels as well as organ (uterus, colon) rupture, which results in premature death and a median survival of approximately 45 years. 1 .

[0005] Many features of vEDS are distinct from other inherited vasculopathies, such as Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS), which are associated with excessive TGF-β activity. These features include aortic dissection and rupture without prior aneurysms in medium-sized or large vessels and hollow organs. These features make disease prediction and monitoring difficult or impossible, and in the majority of adults diagnosed with vEDS, the presenting symptom is vascular dissection or organ rupture, with 25% of patients experiencing major complications by age 20. 1,2 Thus, there is an unmet need for treating vEDS and related connective tissue disorders. Summary of the Invention

[0006] overview Provided herein are compositions, formulations, and methods for inhibiting, treating, preventing, and / or reducing the severity of symptoms of connective tissue disorders, such as vasculopathy. Aspects of the subject matter relate to the use of agents for the treatment of a wide range of connective tissue disorders. In certain embodiments, the connective tissue disorder comprises vasculopathy, and in some embodiments, the vasculopathy comprises vascular Ehlers-Danlos syndrome (vEDS).

[0007] Included herein is a method for treating a vasculopathy (e.g., vEDS) in a subject, the method including 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).

[0008] 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), thereby inhibiting the activity of ERK, PLC, IP3, or PKC.

[0009] In certain embodiments, the agent 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 kinase.

[0010] In embodiments, the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof. In certain embodiments, the agent comprises a small molecule.

[0011] In some cases, the agent comprises a small molecule.Small molecule is a compound with a mass of less than 2000 Daltons.The molecular mass of 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.

[0012] Small molecules are organic or inorganic. Exemplary organic small molecules include, but are not limited to, aliphatic hydrocarbons, alcohols, aldehydes, ketones, organic acids, esters, mono- and disaccharides, aromatic hydrocarbons, amino acids, and lipids. Exemplary inorganic small molecules include trace elements, ions, free radicals, and metabolites. Alternatively, small molecules can be synthetically engineered to consist of fragments, or small parts, or longer amino acid chains to fill the binding pocket of an enzyme. Typically, small molecules are less than 1 kilodalton.

[0013] In some cases, the agent comprises a nucleic acid molecule. For example, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) 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), thereby inhibiting the activity of ERK, PLC, IP3 or PKC. In some cases, the nucleic acid comprises small interfering RNA (siRNA), RNA interference (RNAi), messenger RNA (mRNA), small or short hairpin RNA (shRNA), double-stranded ribonucleic acid (dsRNA), antisense RNA or microRNA, or any part thereof. However, those skilled in the art can easily identify additional nucleic acids that inhibit / antagonize or activate / agonist ERK or PKC, or IP3 or PLC.

[0014] Polynucleotides, polypeptides, or other agents, as used herein, are purified and / or isolated. Specifically, as used herein, "isolated" or "purified" nucleic acid molecules, polynucleotides, polypeptides, or proteins are substantially free of other cellular material, culture medium if produced by recombinant technology, or chemical precursors or other chemicals if chemically synthesized. A purified compound is at least 60% by weight (dry weight) of the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99% by weight of the compound of interest. For example, a purified compound is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w / w) of the desired compound by weight. Purity is measured by any suitable standard method, for example, 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 and sequences which flank it in its native state. A purified or isolated polypeptide is free of the amino acids and sequences which flank it in its native state. "Purified" also defines a degree of sterility that is safe for administration to a human subject, e.g., devoid of infectious or toxic agents.

[0015] In embodiments, the agent comprises cobimetinib, or a pharma- ceutically acceptable salt thereof. In other embodiments, the agent comprises ruboxistaurin, or a pharma- ceutically acceptable salt thereof. In other embodiments, the agent comprises enzastaurin, or a pharma- ceutically acceptable salt thereof. In other contemplated embodiments, the agent comprises sotrastaurin, or a pharma- ceutically acceptable salt thereof.

[0016] In an alternative embodiment, the method further comprises administering an agent that decreases the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3).

[0017] In some embodiments, the method comprises administering an effective amount of the agent. The effective amount of the agent is about 0.001 mg / kg to about 250 mg / kg body weight, for example, 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. The doctor or veterinarian in charge will ultimately decide the appropriate amount and dosage regimen.

[0018] 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 can be suitably administered for a period of one day, one week, one month, two months, three months, six months, nine months, or one year.In some cases, the agent is administered every day, for example, every 24 hours.Or, the agent is administered continuously or several times a day, for example, every 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.

[0019] Additionally, the methods described herein reduce or prevent the severity of vasculopathy (vEDS) by at least about 1%, e.g., 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%.

[0020] A variety of administration routes are available, for example, agents may be administered topically, orally, by inhalation, or by injection.

[0021] The subject is preferably a mammal in need of such treatment or prevention, for example, a subject diagnosed with or predisposed to vasculopathy.The mammal can be any mammal, for example, human, primate, mouse, rat, dog, cat, horse, and livestock or animals raised for food consumption, for example, cow, sheep, pig, chicken, and goat.In a preferred embodiment, the mammal is a human.

[0022] In some aspects, a subject having or at risk of suffering from a connective tissue disorder, such as angiopathy (in some embodiments, vEDS), has a level of ERK protein or PKC protein or mRNA that is different from that of a normal control. In some embodiments, a test sample obtained from a subject contains a level of ERK protein or PKC protein or mRNA that is different from that of a normal control. For example, a test sample can contain 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 higher level of ERK protein or PKC protein or mRNA compared to a normal control.

[0023] In certain embodiments, the test sample may contain 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 levels of ERK activity or PKC activity compared to a normal control.

[0024] A "control" sample or value refers to a sample that serves 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 that has or needs to be diagnosed with a connective tissue disorder, such as angiopathy (e.g., vEDS), and compared with a sample from a known condition, for example, a subject (or subjects) that does not have a disease (negative or normal control), or a subject (or subjects) that has a disease (positive control). A control can also represent an average value collected from multiple tests or results. Those skilled in the art will recognize that controls can be designed for the evaluation of any number of parameters. Those skilled in the art will understand which controls are useful in a given situation and can analyze data based on comparison with the control value. Controls are also useful for determining the significance of data. For example, if the value for a given parameter is variable in the control, the variation in the test sample is not considered significant.

[0025] With respect to a compound (e.g., protein or mRNA), the term "normal amount" refers to the normal amount of the compound in an individual who does not have a connective tissue disorder such as angiopathy (e.g., vEDS) or in a healthy or general population. The amount of the compound can be measured in a test sample and compared to a "normal control" level using techniques such as reference limits, discrimination limits, or risk-defined thresholds to define cutoff points and outliers (e.g., for a particular vEDS or its symptoms). Normal control levels refer to the levels of one or more compounds or compound compounds that are typically found in subjects known not to be affected by vEDS. Such normal control levels and cutoff points can vary based on whether the compound is used alone or in combination with other compounds in the formula to make the index. Alternatively, the normal control level can be a database of compound patterns from previously tested subjects who did not develop vEDS or its specific symptoms over a clinically relevant time range (e.g., when testing subjects who will develop or already have vEDS).

[0026] The determined level may be the same as the control level or cut-off level or threshold level, or may be increased or decreased compared to the control level or cut-off level or threshold level. In some aspects, the control subject is a matched control of the same species, sex, ethnicity, age group, smoking status, body mass index (BMI), current treatment regimen status, medical history, or a combination thereof, but differs from the subject to be diagnosed in that the control does not suffer from or is not at risk for the disease (or symptoms thereof) in question.

[0027] Compared to a control level, the determined level can be an increased level. As used herein, the term "increased" with respect to a level (e.g., a protein or mRNA level) refers to any % increase above a control level. In various embodiments, an increased level can be at least or about a 5% increase, at least or about a 10% increase, at least or about a 15% increase, at least or about a 15% increase, at least or about a 20% increase, at least or about a 20% increase, at least a 25% or about a 25% increase, at least a 30% or about a 30% increase, at least a 35% or about a 35% increase, at least a 40% or about a 40% increase, at least a 45% or about a 45% increase, at least a 50% or about a 50% increase, at least a 55% or about a 55% increase, at least a 60% or about a 60% increase, at least a 65% or about a 65% increase, at least a 70% or about a 70% increase, at least a 75% or about a 75% increase, at least a 80% or about a 80% increase, at least a 85% or about a 85% increase, at least a 90% or about a 90% increase, or at least a 95% or about a 95% increase compared to a control level.

[0028] Compared to a control level, the determined level can be a decreased level. As used herein, the term "decreased" with respect to a level (e.g., a protein or mRNA level) refers to any % decrease below a control level. In various embodiments, a decreased level can be at least or about a 5% decrease, at least or about a 10% decrease, at least or about a 15% decrease, at least or about a 15% decrease, at least or about a 20% decrease, at least or about a 20% decrease, at least a 25% or about a 25% decrease, at least a 30% or about a 30% decrease, at least a 35% or about a 35% decrease, at least a 40% or about a 40% decrease, at least a 45% or about a 45% decrease, at least a 50% or about a 50% decrease, at least a 55% or about a 55% decrease, at least a 60% or about a 60% decrease, at least a 65% or about a 65% decrease, at least a 70% or about a 70% decrease, at least a 75% or about a 75% decrease, at least a 80% or about a 80% decrease, at least a 85% or about a 85% decrease, at least a 90% or about a 90% decrease, or at least a 95% or about a 95% decrease compared to a control level.

[0029] In aspects, the test sample obtained from the subject includes blood, serum, plasma, saliva, tears, vitreous, cerebrospinal fluid, sweat, cerebrospinal fluid, or urine.

[0030] Further provided herein is a method for treating a connective tissue disorder in a subject. The method includes administering an effective amount of an agent, wherein the agent reduces the activity or expression of extracellular signal-related kinase (ERK) or protein kinase C (PKC). In some embodiments, the connective tissue disorder is selected from the group consisting of vasculopathy (e.g., vEDS), Marfan syndrome, Loeys-Dietz syndrome, and familial thoracic aortic aneurysm. Other examples include, but are not limited to, non-myopathic dermatomyositis; bizzare parosteal osteochondromatous proliferation; classic Ehlers-Danlos syndrome; fragile cutaneous Ehlers-Danlos syndrome; Eolers-Danlos syndrome; eosinophilic fasciitis; epidermolysis bullosa (EB); Legg-Calvé-Perthes disease; Marfan syndrome; melorheostosis; melorheostosis with bone mottling; mixed connective tissue disease; rheumatoid factor negative polyarthritis; osteosclerosis with ichthyosis and premature ovarian failure; pacman dysplasia; Paget's disease of bone; familial Paget's disease of bone; polymyositis; progressive hearing loss with stapes fixation; Ribbing's disease; scleroderma; temporomandibular joint ankylosis ankyloses); 20-onychodystrophy; vascular Ehlers-Danlos syndrome; Weill-Marchesani syndrome; and Worth autosomal dominant osteosclerosis. In some embodiments, the connective tissue disorder comprises angiopathy.

[0031] In addition to treating Marfan syndrome itself, the present invention also provides methods of treating various Marfan-related disorders, in some embodiments, the Marfan-related disorder is selected from the group consisting of Loeys-Dietz syndrome, familial aortic aneurysm, bicuspid aortic valve with aortic dilatation, familial ectopia lentis (dislocated lens), mitral valve prolapse syndrome, Marfan habitus, congenital contracture arachnodactyly (Beales syndrome), Stickler syndrome, Shprintzen-Goldberg syndrome, Weill-Marchesani syndrome, and Ehlers-Danlos syndrome.

[0032] 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.

[0033] In embodiments, the agent comprises cobimetinib, or a pharma- ceutically acceptable salt thereof. In other embodiments, the agent comprises ruboxistaurin, or a pharma- ceutically acceptable salt thereof. In other embodiments, the agent comprises enzastaurin, or a pharma- ceutically acceptable salt thereof. In other contemplated embodiments, the agent comprises sotrastaurin, or a pharma- ceutically acceptable salt thereof.

[0034] In an alternative embodiment, the method further comprises administering an agent that decreases the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3).

[0035] Further provided herein is a pharmaceutical composition for treating vasculopathy.In some 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 its fragment, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof.

[0036] The compositions described herein are administered via oral, intravenous, topical, parenteral, intraperitoneal, intramuscular, intrathecal, intralesional, intracranial, intranasal, intraocular, intracardiac, intravitreal, intraosseous, intracerebral, intraarterial, intraarticular, intradermal, transdermal, transmucosal, sublingual, enteral, sublabial, insufflation, suppository, inhalation, or subcutaneous administration.

[0037] Further provided herein is a kit for treating vasculopathy. In some embodiments, the kit includes (1) a pharmaceutical composition of any of the compositions described herein, and (2) instructions for treating vasculopathy.

[0038] In another aspect, a method for treating connective tissue disorders (e.g., Marfan syndrome) is contemplated. In some embodiments, the method comprises administering an effective amount of an agent, wherein the agent comprises an antibody or a fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof. In some embodiments, a therapeutically effective amount of one or more agents is co-administered to the subject.

[0039] In some embodiments, a method for treating a connective tissue disorder (e.g., Marfan syndrome) comprises administering an agent that reduces the activity or expression of protein kinase C (PKC). In some embodiments, a method for treating a connective tissue disorder (e.g., Marfan syndrome) comprises administering an agent that reduces the activity or expression of extracellular signal-regulated kinase (ERK). In some embodiments, a method for treating a connective tissue disorder (e.g., Marfan syndrome) comprises administering an agent that reduces the activity or expression of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), or a combination thereof.

[0040] In some embodiments, a method for treating a connective tissue disorder (e.g., Marfan syndrome) comprises administering an agent that reduces or inhibits 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), thereby inhibiting activity of ERK, PLC, IP3, or PKC.

[0041] In certain embodiments, a method for treating a connective tissue disorder (e.g., Marfan syndrome) comprises 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 kinase.

[0042] In some embodiments, the patient is a human patient. In some embodiments, the patient is 15 years of age or older. In some embodiments, the patient is an adult patient. In some embodiments, the patient is a pediatric patient.

[0043] In some embodiments, the method is initiated upon (or shortly after) vEDS is diagnosed, hi some embodiments, the method is initiated when the patient is 15 years of age or when first diagnosed.

[0044] In some embodiments, the patient is diagnosed based on a vEDS phenotype or based on a molecular test vEDS (e.g., the patient is determined to have vEDS based on one or more genetic tests, such as a test that determines that the patient has a glycine substitution in the triple helix or a splice site variant).

[0045] In some embodiments, the patient has a COL3A1 mutation. In some embodiments, the patient has a glycine substitution or a splice site variant within the triple helix. In some embodiments, the patient has a missense substitution for glycine in the collagen triple helix repeat (Gly-XY)n sequence, and / or a splice site variant leading to in-phase exon skipping. In some embodiments, the patient has a glycine substitution within the triple helix (Group I). In some embodiments, the patient has a splice site variant, an in-frame insertion-deletion or a duplication (Group II). In some embodiments, the patient has a variant leading to haploinsufficiency (Group III).

[0046] In some embodiments, the patient has previously had an acute vEDS-related event (e.g., arterial event, rupture or dissection, intestinal or uterine rupture) prior to the first dose of one or more agents embodied herein, or a pharma- ceutically acceptable salt thereof.

[0047] In certain embodiments, the agent reduces levels of PKC protein or mRNA 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 / 2, 3 / 4, or 5 / 5 compared to a normal control. In other embodiments, the agent reduces the level of PKC activity 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%, or 5-fold compared to a normal control.

[0048] In some embodiments, the agent reduces levels of extracellular signal-regulated kinase (ERK) protein or mRNA 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 / 2, 1 / 3, 1 / 4, or 1 / 5 compared to a normal control. In other embodiments, the agent reduces the level of ERK activity 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%, or 5-fold compared to a normal control.

[0049] In some 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 examples, the agent for treating a connective tissue disorder (e.g., Marfan syndrome) comprises ruboxistaurin, or a pharmaceutically acceptable salt thereof.

[0050] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure belongs.The following references provide those skilled in the art with the general definitions 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 given to them below, unless otherwise specified.

[0051] The term "pharmaceutical composition" refers to any composition that contains at least one therapeutically or biologically active agent and is suitable for administration to a patient. Any of these formulations can be prepared by methods well known and accepted in the art. For example, see Remington: The Science and Practice of Pharmacy, 20th edition, (ed. AR Gennaro), Mack Publishing Co., Easton, Pa., 2000.

[0052] "G protein-coupled receptor (GPCR)" refers to a protein receptor that senses molecules outside the cell and activates a signaling pathway and ultimately a cellular response inside the cell. GPCRs are called seven-transmembrane receptors because they pass through the cell membrane seven times.

[0053] "Agonist" refers to a chemical that binds to a receptor and activates the receptor to produce a biological response. An agonist causes an action, whereas an "antagonist" blocks the action of an agonist, and an inverse agonist causes an action opposite to that of an agonist. As used herein, the terms "antagonist" and "inhibitor" are used interchangeably to refer to any molecule that counteracts or inhibits, reduces, or suppresses the biological activity of its target molecule. In some embodiments, an agonist is a "superagonist" if it induces or increases the biological activity of its target molecule. In some embodiments, an antagonist is a "superantagonist" if it counteracts or inhibits, reduces, or suppresses the biological activity of its target molecule. Suitable inhibitors, antagonists, and agonists include soluble receptors, peptide inhibitors, small molecule inhibitors, ligand fusions, and antibodies.

[0054] As used herein, the term "salt" refers to the acid salt or base salt of the agent used herein.Illustrative but non-limiting examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, etc.) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid, etc.) salts, and quaternary ammonium (methyl iodide, ethyl iodide, etc.) salts.

[0055] As used herein, "antagonist" can refer to an antibody or fragment thereof, a peptide, a polypeptide or fragment thereof, a small molecule, and an inhibitory nucleic acid or fragment thereof that interferes with the activity or binding of another, for example, by competing for one or more binding sites of an agonist, but does not induce an activity response.

[0056] "Wild type" or "WT" refers to the typical phenotype of a species as it exists in nature. Alternatively, wild type may be conceptualized as the product of a standard "normal" allele at a genetic locus, as opposed to one produced by a nonstandard "mutant" allele.

[0057] The term "administering" as used herein refers to any manner of transferring, delivering, introducing, or transporting an agent to a subject in need of treatment, for example, for a disease or condition. Such manners include, but are not limited to, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

[0058] "Co-administer" means that the compositions described herein are administered simultaneously, immediately before, or immediately after the administration of additional therapy. The agents or compositions of the present disclosure can be administered to a patient alone or simultaneously. Co-administration means simultaneous or sequential administration of the compounds (two or more compounds or agents) individually or in combination. The preparation can also be combined with other active substances as needed.

[0059] As used herein, "sequential administration" includes administration of two agents (e.g., agents or compositions described herein) occurring separately on the same day or not on the same day (e.g., occurring on consecutive days).

[0060] As used herein, "concurrent administration" includes at least partial overlap in duration. For example, when two agents (e.g., any of the agents described herein having biological activity) are administered in parallel, their administration occurs within a certain desired time. The administration of the agents can begin and end on the same day. The administration of one agent can also precede the administration of the second agent by days, as long as both agents are taken at least once on the same day. Similarly, the administration of one agent can extend beyond the administration of the second agent, as long as both agents are taken at least once on the same day. The biologically active agents / agents do not need to be taken at the same time every day to include concurrent administration.

[0061] As used herein, "intermittent administration" includes administration of an agent for a period of time (which may be considered a "first administration period"), followed by a time during which the agent is not taken or is taken at a lower maintenance dose (which may be considered an "off period"), followed by a period during which the agent is again administered (which may be considered a "second administration period"). Generally, during the second administration phase, dosage levels of the agent will be consistent with those administered during the first administration period, but may be increased or decreased as medically necessary.

[0062] As used herein, "alteration" also includes a two-fold or more, e.g., five-fold, ten-fold, twenty-fold, thirty-fold, forty-fold, fifty-fold, one hundred-fold, five hundred-fold, one thousand-fold, or more, change in expression level or activity of a gene or polypeptide.

[0063] As defined herein, the terms "inhibit", "inhibit", "inhibiting" and the like with respect to protein inhibitor (e.g., ERK or PKC inhibitor, or PLC or IP3 inhibitor) interaction refer to adversely affecting (e.g., decreasing) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor (e.g., decreasing the activity or amount of ERK or PKC, or PLC or IP3, decreasing the ability of ERK or PKC, or PLC or IP3 to bind to a receptor, decreasing the ability of a receptor to bind to ERK or PKC, or decreasing ERK or PKC signaling upon ERK or PKC, or PLC or IP3 binding to a receptor). In some embodiments, inhibition refers to a reduction in the symptoms of a disease or 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, decreasing, preventing, delaying, inactivating, desensitizing, or downregulating activity.

[0064] By "ameliorate" is meant to decrease, inhibit, alleviate, ameliorate, arrest, or stabilize the onset or progression of a disease, such as, for example, a pseudoallergic-type reaction.

[0065] "Amplify" means to increase the number of copies of a molecule. In one example, the polymerase chain reaction (PCR) is used to amplify nucleic acids.

[0066] "Binding" means having a physicochemical affinity for a molecule. Binding is measured by any of the methods disclosed herein, for example, by a drug / compound with a receptor expressed on a cell.

[0067] In this disclosure, "comprises," "including," "containing," "having," and the like can have the meaning given them in U.S. patent law and can mean "include," "comprise," and the like; the terms "consisting essentially of" or "consisting essentially of" likewise have the meaning given them in U.S. patent law, and these terms are open-ended, permitting the presence of more than is recited, but excluding prior art aspects, so long as the basic or novel characteristics of what is recited are not altered by the presence of more than is recited.

[0068] "Effective amount" refers to the amount required to improve the symptoms of a disease compared to untreated patients. The effective amount of the active compound used to carry out the present invention for therapeutic treatment of a disease varies 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 dosage regimen. Such an amount is referred to as an "effective" amount.

[0069] The dosage and frequency (single or multiple doses) administered to a mammal may vary depending on a variety of factors, such as whether the mammal is suffering from another disease and its route of administration; the size, age, sex, health, weight, body mass index, and diet of the recipient; the nature and extent of symptoms of the disease being treated, the type of concomitant treatment, complications from the disease being treated, or other health-related issues. Other therapeutic regimens or agents may be used in conjunction with the methods and agents of the present disclosure. Adjustment and manipulation of established dosages (e.g., frequency and duration) are well within the capabilities of those skilled in the art.

[0070] For any agent described herein, the therapeutically effective amount (e.g., effective dose or amount) can be initially determined from cell culture assays. The target concentration is the concentration of the therapeutic agent that can achieve the methods described herein, as measured using methods described herein or known in the art.

[0071] As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models.For example, dosages for humans can be formulated to achieve concentrations found to be effective in animals.Dosages in humans can be adjusted by monitoring the effectiveness of the agent and adjusting dosage upwards or downwards, as described above.It is well within the capabilities of those skilled in the art to adjust dosages to achieve maximum efficacy in humans based on the above and other methods.

[0072] Dosage may vary depending on the patient's requirements and the therapeutic agent used. The dose administered to the patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dose is also determined by the existence, nature, and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with a smaller dosage that is less than the optimal dose of the agent. Thereafter, the dosage is increased by small increments until the optimal effect is achieved according to the situation. Dosage and interval can be adjusted individually to provide an effective level of the administered agent for the particular clinical indication being treated. This provides a treatment regimen that is commensurate with the severity of the individual's disease state.

[0073] Effective doses of agents (e.g., pharmacological inhibitors) of the disclosure for treating vEDS, treating Marfan syndrome, and / or altering expression or activity of the PLC / IP3 / PKC / ERK signaling pathway include from about 0.001 mg / kg to about 0.01 mg / kg of agent, from about 0.01 mg / kg to about 0.1 mg / kg of agent, from about 0.1 mg / kg to about 1.0 mg / kg of agent, from about 1.0 mg / kg to about 5.0 mg / kg of agent, from about 5.0 mg / kg to about 10 mg / kg of agent, from about 10 mg / kg to about 15 mg / kg of agent, from about 15 mg / kg to about 20 mg / kg of agent, from about 20 mg / kg to about 25 mg / kg of agent, from about 25 mg / kg to about 30 mg / kg of agent, from about 30 mg / kg to about 35 mg / kg of agent, from about 35 mg / kg to about 40 mg / kg of agent, The active ingredient may be about 40 mg / kg to about 45 mg / kg of active ingredient, about 45 mg / kg to about 50 mg / kg of active ingredient, about 50 mg / kg to about 55 mg / kg of active ingredient, about 55 mg / kg to about 60 mg / kg of active ingredient, about 60 mg / kg to about 65 mg / kg of active ingredient, about 65 mg / kg to about 70 mg / kg of active ingredient, about 70 mg / kg to about 75 mg / kg of active ingredient, about 75 mg / kg to about 80 mg / kg of active ingredient, about 80 mg / kg to about 85 mg / kg of active ingredient, about 85 mg / kg to about 90 mg / kg of active ingredient, about 90 mg / kg to about 95 mg / kg of active ingredient, or about 95 mg / kg to about 100 mg / kg of active ingredient.

[0074] In some aspects, the disclosure includes compositions comprising an effective dose of an agent of the disclosure, where the agent can be from about 0.1% to about 20% w / v of the composition. Weight percentages of ingredients are based on the total weight of the formulation or composition in which the ingredient is included, unless specifically specified to the contrary.

[0075] For example, an effective dose of an agent disclosed herein may be from about 0.001% to about 0.01%, from about 0.01% to about 0.1%, from about 0.1% to about 1.0%, from about 1.0% to about 2.0%, from about 2.0% to about 3.0%, from about 3.0% to about 4.0%, from about 4.0% to about 5.0%, from about 5.0% to about 6.0%, from about 6.0% to about 7.0%, from about 7.0% to about 8.0%, from about 8.0% to about 9.0%, from about 9.0% to about 10.0%, from about 10.0% to about 11.0%, from about 10.0% to about 12.0%, from about 10.0% to about 13.0%, from about 10.0% to about 14.0%, from about 10.0% to about 15.0%, from about 10.0% to about 16.0%, from about 10.0% to about 17.0%, from about 10.0% to about 18.0%, from about 10.0% to about 19.0%, from about 10.0% to about 20.0%, from about 10.0% to about 21.0%, from about 10.0% to about 22.0%, from about 10.0% to about 23.0%, from about 10.0% to about 24.0%, from about 10.0% to about 25.0%, from about 10.0% to about 26.0%, from about 10.0% to about 27.0%, from about 10.0% to about 28.0%, from about 10.0% to about 29.0%, from about 10.0% to about 30.0%, from about 10 %, about 7.0% to about 8.0%, about 8.0% to about 9.0%, about 9.0% to about 10%, about 10% to about 11%, about 11% to about 12%, about 12% to about 13%, about 13% to about 14%, about 14% to about 15%, about 15% to about 16%, about 16% to about 17%, about 17% to about 18%, about 18% to about 19%, or about 19% to about 20% w / v.

[0076] The terms "treating" and "treatment", as used herein, refer to the administration of an agent or formulation to a clinically symptomatic individual suffering from an adverse condition, disorder, or disease to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or their underlying causes, and / or promote amelioration or repair of damage.

[0077] The terms "subject", "patient", "individual", and the like, as used herein, are not intended to be limiting and are generally interchangeable. An individual described as a "subject", "patient", "individual", and the like, does not necessarily have a given disease, but may simply be seeking medical advice. The terms "subject", "patient", "individual", and the like, as used herein, include all members of the animal kingdom that may be afflicted with the indicated disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is a human.

[0078] The term "sample" as used herein refers to a biological sample obtained for the purpose of in vitro evaluation. In several embodiments, the sample may include bodily fluids. In some embodiments, bodily fluids include, but are not limited to, whole blood, plasma, serum, lymph, breast milk, saliva, mucus, semen, cell extracts, inflammatory fluid, cerebrospinal fluid, vitreous humor, tears, vitreous, aqueous humor, or urine obtained from a subject. In some aspects, the sample is a composite panel of two or more bodily fluids. In exemplary aspects, the sample includes blood or a fraction thereof (e.g., plasma, serum, or a fraction obtained by leukapheresis).

[0079] It is understood that the ranges provided herein are shorthand for all values ​​within the range.For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group 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 intervening decimal values ​​between the aforementioned integers, such as 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 endpoint of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0080] "Recombinant" means a nucleic acid molecule formed by laboratory methods of genetic recombination (e.g., molecular cloning) to combine genetic material from multiple sources to create sequences not otherwise found in an organism.

[0081] The word "expression" or "expressing" as used herein with respect to a DNA nucleic acid sequence (e.g., a gene) refers to the transcription and / or translation product of that sequence. The level of expression of a DNA molecule in a cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.7-18.88). When used with respect to a polypeptide, expression includes any step involved in the production of a polypeptide, 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.).

[0082] By "reduce" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0083] "Reference" means a standard or control condition.

[0084] Unless otherwise specified or clear from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural. Unless otherwise specified or clear from context, as used herein, the term "or" is understood to be inclusive.

[0085] Unless otherwise specified or clear from the context, the term "about" as used herein is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean. About can be understood to be 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".

[0086] As used herein, the term "derivative" refers to a compound having a structure derived from that of a parent compound (e.g., a compound disclosed herein) that is sufficiently similar to that disclosed herein that it would be expected by one of skill in the art to exhibit the same or similar activity and utility as the claimed compound based on that similarity, or to induce, as a precursor, 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.

[0087] Each aspect disclosed herein is contemplated as being applicable to each of the other disclosed aspects, and thus, all combinations of the various elements described herein are within the scope of the invention.

[0088] Other features and advantages of the present invention will be apparent from the following description of its preferred embodiment and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated by reference. Genbank and NCBI submissions referenced by accession numbers cited herein are incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference. In case of conflict, the present specification, including definitions, shall control. In addition, the materials, methods, and examples are only illustrative and are not intended to be limiting. [The present invention 1001] administering an effective amount of an agent that decreases the activity or expression of extracellular signal-regulated kinase (ERK) or protein kinase C (PKC), thereby treating the vasculopathy; 23. A method of treating vasculopathy in a subject, comprising: [The present invention 1002] The method of the present invention 1001, wherein the vasculopathy includes vascular Ehlers-Danlos syndrome (vEDS). [The present invention 1003] The method of any one of claims 1 to 10, wherein said agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof. [The present invention 1004] The method of any one of claims 10 to 3, wherein the agent comprises a small molecule. [The present invention 1005] The method of claim 10, wherein said agent comprises cobimetinib or a pharma- ceutically acceptable salt thereof. [The present invention 1006] The method of claim 10, wherein said agent comprises ruboxistaurin or a pharma- ceutically acceptable salt thereof. [The present invention 1007] The method of claim 10, wherein said agent comprises enzastaurin or a pharma- ceutically acceptable salt thereof. [The present invention 1008] The method of claim 10, wherein said agent comprises sotrastaurin or a pharma- ceutically acceptable salt thereof. [The present invention 1009] The method of claim 1001, further comprising administering an agent that decreases the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3). [The present invention 1010] The method of claim 1001, wherein the effective amount of said agent is about 0.001 mg / kg to 250 mg / kg of body weight. [The present invention 1011] The method of claim 1001, wherein said subject comprises a level of ERK protein or PKC protein or mRNA that differs from a normal control. [The present invention 1012] The method of the present invention, wherein the subject comprises 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 higher levels of ERK or PKC protein or mRNA compared to a normal control. [The present invention 1013] The method of the present invention, wherein the subject comprises 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 higher level of ERK activity or PKC activity compared to a normal control. [The present invention 1014] The method of any one of claims 1 to 10, wherein said level is the level in a test sample obtained from said subject. [The present invention 1015] The method of any one of claims 10 to 14, wherein the test sample comprises blood, serum, plasma, saliva, tears, vitreous, cerebrospinal fluid, sweat, cerebrospinal fluid, or urine. [The present invention 1016] administering an effective amount of an agent that decreases the activity or expression of extracellular signal-regulated kinase (ERK) or protein kinase C (PKC), thereby treating a connective tissue disorder; 23. A method for treating a connective tissue disorder in a subject, comprising: [The present invention 1017] The method of claim 1016, wherein said connective tissue disorder is selected from the group consisting of vasculopathy, Marfan syndrome, Loeys-Dietz syndrome, and familial thoracic aortic aneurysm. [The present invention 1018] The method of claim 1016, wherein the vasculopathy comprises vascular Ehlers-Danlos syndrome (vEDS). [The present invention 1019] The method of claim 1016, wherein said agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof. [The present invention 1020] The method of any one of claims 10 to 19, wherein the agent comprises a small molecule. [The present invention 1021] The method of claim 1019, wherein said agent comprises cobimetinib or a pharma- ceutically acceptable salt thereof. [The present invention 1022] The method of claim 1019, wherein the agent comprises enzastaurin or a pharma- ceutically acceptable salt thereof. [The present invention 1023] The method of claim 1019, wherein the agent comprises sotrastaurin or a pharma- ceutically acceptable salt thereof. [The present invention 1024] The method of claim 1019, wherein the agent comprises ruboxistaurin or a pharma- ceutically acceptable salt thereof. [The present invention 1025] The method of claim 1016, further comprising administering an agent that decreases the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3). [The present invention 1026] A pharmaceutical composition for the treatment of vasculopathy comprising an effective amount of an agent that decreases the activity or expression of extracellular signal-regulated kinase (ERK) or protein kinase C (PKC), thereby treating the vasculopathy. [The present invention 1027] The pharmaceutical composition of the present invention 1026, wherein the agent comprises an antibody or a fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof. [The present invention 1028] A kit comprising: (1) a pharmaceutical composition of the present invention 1026 or 1027; and (2) instructions for treating a vasculopathy. [The present invention 1029] administering an effective amount of an agent comprising an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof, thereby treating the connective tissue disorder. 23. A method for treating a connective tissue disorder comprising: [The present invention 1030] The method of claim 1029, wherein said connective tissue disorder comprises Marfan syndrome. [The present invention 1031] The method of claim 1029, wherein said agent decreases the activity or expression of protein kinase C (PKC). [The present invention 1032] The method of the present invention 1031, wherein the agent reduces the level of the PKC protein or mRNA 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 / 2, 1 / 3, 1 / 4, or 1 / 5 compared to a normal control. [The present invention 1033] The method of the present invention 1031, wherein the agent reduces the level of PKC activity 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%, or 1 / 5 compared to a normal control. [The present invention 1034] The method of claim 1029, wherein the agent comprises enzastaurin or a pharma- ceutically acceptable salt thereof. [The present invention 1035] The method of claim 1029, wherein the agent comprises sotrastaurin or a pharma- ceutically acceptable salt thereof. [The present invention 1036] The method of claim 1029, wherein the agent comprises ruboxistaurin or a pharma- ceutically acceptable salt thereof. [Brief description of the drawings]

[0089] [Figure 1A] Graph demonstrating that the G209S / + mouse model recapitulates the vascular Ehlers-Danlos syndrome (vEDS) phenotype. % survival versus age is shown. Median survival was 400 days; p<0.0001. [Figure 1B] FIG. 1 is a graph demonstrating that the G938D / + mouse model recapitulated the vEDS phenotype. Percent survival versus age is shown. Median survival was 45 days; p<0.0001. [Figure 1C] Images of the heart and aorta of a vEDS mouse. The arrow (left) indicates the ascending aorta, and the red arrow (right) indicates the site of aortic dissection in the descending aorta. [Figure 1D] FIG. 13 is a graph demonstrating that vEDS samples clustered separately from controls, indicating significant differences in the transcriptome of vEDS aortas. [Figure 1E]FIG. 13 demonstrates network analysis showing that vEDS aortas exhibited gene expression signatures for elevated mitogen-activated protein kinase (MAPK) activity (P38, JNK, AKT, ERK, ERK1 / 2). [Figure 1F] 1 is a table showing that upstream analysis predicted that transcriptional differences in vEDS aortas were driven by G protein-coupled receptor (GPCR) activation, signaling through the related PLC / IP3 / PKC / ERK axis. [Figure 1G] Immunoblots and graphs demonstrating a signature for elevated 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. [Figure 2A] 1 is a graph depicting survival of mice treated with cobimetinib, an FDA-approved MEK inhibitor. A 94% survival rate was observed after 45 days of treatment, compared to only 55% survival rate without treatment. [Figure 2B] 1 is a graph depicting survival 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 without treatment. [Figure 2C] 1 is a graph depicting survival of mice treated with hydralazine, which blocks the PLC / IP3 / PKC / ERK axis. Protection was observed with a 98% survival rate at age 45 days, the median survival time for untreated vEDS mice. [Figure 3-1] Figure 3A is a graph depicting that in the vEDS mouse model described herein, pregnancy and lactation are associated with a 60% mortality rate due to arterial dissection in the first 30 days after birth in vEDS mice. Figure 3B is a graph depicting that prevention of lactation by removal of pups after birth can prevent dissection and death in vEDS mice, with 100% survival observed. [Figure 3-2] Figure 3C is a graph depicting that nearly complete survival (95%) was achieved with treatment with hydralazine (16 mg / kg / day), which blocks the oxytocin-activated PLC / IP3 / PKC / ERK axis. Figure 3D depicts that protection (95% survival rate) was observed with treatment with trametinib (GSK-1120212) 1 mg / kg / day, an FDA-approved inhibitor of MEK, a kinase that activates ERK. [Figure 3-3] Figure 3E is a bar graph depicting that increased risk of death correlated with increased ERK activation, as measured by immunoblotting, while protection from aortic dissection correlated with decreased ERK activation. Figure 3F is a bar graph depicting that increased risk of death correlated with increased ERK activation, as measured by ERK target gene expression, while protection from aortic dissection correlated with decreased ERK activation. [Figure 4] Figure 4A is an image of an immunoblot showing that pharmacological inhibition of PKCβ prevented autophosphorylation of PKC as well as phosphorylation of ERK in the aortic wall as assessed by immunoblot of aortic lysates, and that pharmacological inhibition of MEK not only correlated with the expected reduction in phosphorylation of ERK, a downstream substrate of MEK, but also, curiously, reduced PKC phosphorylation, suggesting the presence of a positive feedback loop. Figures 4B and 4C are graphs depicting quantification of immunoblot images depicting that pharmacological inhibition of PKCβ prevented autophosphorylation of PKC as well as phosphorylation of ERK in the aortic wall as assessed by immunoblot of aortic lysates, and that pharmacological inhibition of MEK not only correlated with the expected reduction in phosphorylation of ERK, a downstream substrate of MEK, but also, curiously, reduced PKC phosphorylation, suggesting the presence of a positive feedback loop (*p<0.05, **p<0.01, ***p<0.001). Neither cobimetinib nor ruboxistaurin had any effect on blood pressure. [Diagram 5]Figures 5A and 5B are graphs depicting that blocking the PLC / IP3 / PKC / ERK axis, hydralazine (32 mg / kg / d) mice provided significant protection with a 98% survival rate at 45 days of age, the median survival time for untreated, vEDS mice, and that survival was affected at puberty, with risk seen almost exclusively in male mice (Figure 5A male mice and Figure 5B female mice). [Figure 6] 1 is a graph depicting that the combination of hydralazine (32 mg / kg / d) and bicalutamide (50 mg / kg / d) results in a 90% survival rate in male mice compared to only 24% survival rate in male mice treated with hydralazine alone. Male mice continued to survive after bicalutamide was removed after puberty, suggesting that there was a time dependency on androgen sensitivity in this mouse model. [Figure 7] FIG. 13 depicts male mice treated with bicalutamide alone led to a moderate survival of approximately 80%, and no males continued to survive after removal of bicalutamide after puberty, suggesting that inhibition of androgen signaling alone was not sufficient to prevent aortic disease in our mouse model of vEDS. [Figure 8] 8A and 8B are graphs depicting that mice treated with spironolactone alone (100 mg / kg / d) showed moderate survival of about 80%, similar to bicalutamide alone, but 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. [Figure 9] 1 is a graph depicting that mice treated with higher doses of hydralazine had an increased dose of hydralazine (50 mg / kg / d) and their survival was not improved any more than the 32 mg / kg / d dose did. [Figure 10]FIG. 10A is a graph depicting that the addition of a specific oxytocin receptor antagonist 2 to mouse treatment resulted in a 95% survival rate in the first 30 days after parturition in still lactating vEDS mice, demonstrating that the significantly elevated risk of death due to aortic dissection in pregnancy was specifically driven by activation of the oxytocin receptor during lactation. FIG. 10B is a graph depicting that propranolol, a non-specific β antagonist that lowers blood pressure in vEDS mice, does not improve survival in the first 30 days after parturition in still lactating vEDS mice, demonstrating that the significantly elevated risk of death due to aortic dissection in pregnancy is not improved by reducing blood pressure. [Figure 11] Figure 11A is a graph showing that inhibition of angiotensin-II signaling by treating mice with the angiotensin receptor antagonist, losartan (60 mg / kg / d), showed no effect on survival (Figure 11A). Treatment of mice with the thrombin receptor antagonist, vorapraxar (1 mg / kg / d), was also shown to have no effect on survival (Figure 11B). [Figure 12] FIG. 13 is a graph depicting that treatment of mice with the nonspecific tyrosine kinase receptor antagonist, nintedanib (50 mg / kg / d), had no effect on survival, suggesting that tyrosine kinase receptor activation did not drive activation of the PLC / IP3 / PKC / ERK signaling pathway in vEDS mice. [Figure 13] 13A-13C are graphs depicting treatment of mice with the nonspecific β antagonist, propranolol (80 mg / kg / d) (FIG. 13A), the specific β antagonist, atenolol (120 mg / kg / d) (FIG. 13B), and the β antagonist / β agonist, celiprolol (200 mg / kg / d) (FIG. 13C), showing that none of these manipulations resulted in improved survival in our vEDS mouse model, despite a reduction in blood pressure. [Figure 14] FIG. 13 is a graph depicting celiprolol accelerating the risk of aortic dissection in a mouse model; vEDS mutants with Col3a1G209S / + mutations also demonstrated increased risk of aortic dissection in celiprolol. [Figure 15] Graph depicting that amlodipine (12 mg / kg / d) also increased the risk of aortic dissection in a mouse model, which was shown to be consistent with MFS mice as well. [Figure 16] FIG. 16A is a graph demonstrating pharmacological inhibition of PKCβ using a second specific PKCβ inhibitor, enzastaurin. FIG. 16A: Enzastaurin (60 mg / kg / d) also rescued 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). FIG. 16B is a graph demonstrating that treatment of mice with the endothelin receptor antagonist, bosentan, improved survival. FIG. 16B: Treatment with bosentan resulted in 80% survival after 40 days after treatment compared to only 50% survival in untreated vEDS mice (p=0.0298). [Figure 17] Immunofluorescence staining demonstrating that signaling pathways are elevated in vascular tissue samples from human patients with vEDS. Figure 17 demonstrates PKC phosphorylation in two tissue samples (iliac artery and descending thoracic aorta) from a patient with vEDS. [Figure 18] Immunofluorescence staining demonstrating that signaling pathways are elevated in vascular tissue samples from human patients with vEDS. Figure 18 demonstrates ERK1 / 2 phosphorylation in two tissue samples (iliac artery and descending thoracic aorta) from a patient with vEDS. [Figure 19] 1 is a graph demonstrating that ruboxistaurin treatment reduces aortic root growth in 129 MFS mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0090] Detailed Description The present invention is based at least in part on the identification of novel targetable signaling abnormalities that contribute to the pathogenesis of vEDS. In particular, it has been observed that pharmacological agents that inhibit ERK1 / 2 activation or PKC activation can successfully prevent death caused by aortic dissection. Furthermore, agents that inhibit the activation of the PLC / IP3 / PKC / ERK signaling pathway also prevent death caused by aortic dissection. Taken together, these novel findings provide the first evidence for targetable signaling abnormalities that contribute to the pathogenesis of vEDS and potentially other vasculopathies, as well as other connective tissue disorders.

[0091] vasculopathy Vasculopathy is a term used to describe diseases that affect blood vessels. It often includes vascular abnormalities caused by degenerative, metabolic and inflammatory conditions, embolic diseases, coagulation disorders, and functional disorders, such as reversible occipital leukoencephalopathy. The etiology of vasculopathy is generally unknown, and the condition is often not pathologically proven. Vasculitis, on the other hand, is a more specific term and is defined as inflammation of the blood vessel wall.

[0092] As used herein, "vasculitis (angiitis or angitis)" refers to inflammation of blood vessels, e.g., arteritis, phlebitis, or inflammation of lymphatic vessels, e.g., lymphangitis. Vasculitis can take various forms, such as cutaneous vasculitis, urticarial vasculitis, leukocytoclastic vasculitis, livedotype vasculitis, and nodular vasculitis. Small vasculitis can refer to inflammation of small or medium-sized blood or lymphatic vessels, e.g., capillaries, venules, arterioles, and arteries.

[0093] Vascular Ehlers-Danlos Syndrome (vEDS) Vascular Ehlers-Danlos syndrome (vEDS) is an inherited connective tissue disorder caused by heterozygous mutations in the type III collagen α1 chain (COL3A1) gene. The leading cause of death in vEDS is arterial dissection and / or rupture, but little is known about the pathogenesis of this disease. No effective treatment strategies exist for this devastating condition. The current belief is that a reduction in the amount of collagen III directly leads to the signs and symptoms of vEDS due to an inherent decrease in the structural integrity of the tissue. However, early pathogenic models of Marfan syndrome (MFS) also cause significant tissue fragility conferred by failed elastic fiber formation, however, subsequent studies have demonstrated that the defective gene product in MFS is the culprit. 3,4 , clearly demonstrated enhanced transforming growth factor beta (TGF-β) signaling in a mouse model lacking fibrillin-1. Follow-up studies continued to show that TGF-β and downstream cell signaling molecules were the main 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 inhibitors of ERK1 / 2 activation RDEA119 / trametinib, could suppress aortic disease in MFS mice. 3-6 .

[0094] However, similar to other inherited vasculopathies such as Marfan syndrome and Loeys-Dietz syndrome, provided herein are signaling abnormalities that are major mediators of disease pathology in vEDS. RNA-seq profiling on aortas of mice carrying patient-derived Col3a1 mutations demonstrated 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.

[0095] In one embodiment, COL3A1 has the following amino acid sequence (NCBI Accession No: AAH28178.1 (SEQ ID NO: 1), which is incorporated by reference in its entirety): Contains TIFF0007678748000001.tif79148.

[0096] In one embodiment, COL3A1 is represented by the following nucleic acid sequence (NCBI Accession No: NM_000090.3 (SEQ ID NO: 2), which is incorporated by reference in its entirety): TIFF0007678748000002.tif143149TIFF0007678748000003.tif223148, with the start and stop codons in bold and underlined.

[0097] MAP / ERK pathway The MAPK / ERK pathway (also known as the Ras-Raf-MEK-ERK pathway) is a chain of proteins in cells that transmit signals from receptors on the surface of the cell to DNA in the nucleus of the cell.

[0098] A signal begins when a signaling molecule binds to a receptor on the cell surface and ends when DNA in the nucleus expresses a protein that results in some change in the cell, such as cell division. Pathways involve many proteins, including MAPKs (mitogen-activated protein kinases, formerly called ERKs, extracellular signal-regulated kinases), which communicate by adding phosphate groups to adjacent proteins, acting as "on" or "off" switches.

[0099] 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, MAPK 1, p44-ERK1, ERT2, p44-MAPK, or microtubule-associated protein 2 kinase.

[0100] 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.

[0101] In one embodiment, ERK1 has the following amino acid sequence (NCBI Accession No: P27361.4 (SEQ ID NO: 3), which is incorporated by reference in its entirety): Contains TIFF0007678748000004.tif27146.

[0102] In one embodiment, ERK1 is represented by the following nucleotide sequence (NCBI Accession No: X60188.1 (SEQ ID NO: 4), which is incorporated by reference in its entirety): Contains TIFF0007678748000005.tif126148, coding regions are bold and underlined.

[0103] In one embodiment, ERK2 has the following amino acid sequence (NCBI Accession No: P28482.3 (SEQ ID NO: 5), incorporated herein by reference in its entirety): Contains TIFF0007678748000006.tif23146.

[0104] In one embodiment, ERK2 is represented by the following nucleotide sequence (NCBI Accession No: NM_138957.3 (SEQ ID NO: 6), which is incorporated by reference in its entirety): TIFF0007678748000007.tif102148, coding sequence is bold and underlined.

[0105] In some embodiments, the present disclosure provides a method for treating vasculopathy (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 ERK activity or expression.Examples of ERK inhibitors include ASN007 (Asana BioSciences, Bridgewater, New Jersey), ulixertinib (BVD-523) (BioMed Valley Discoveries, Kansas City, MO), CC-90003 (Celgene Corporation, Summit, New Jersey), GDC-0994 (Array BioPharma, Boulder, CO), KO-947 (Kura Oncology, San Diego, California), LTT462 (Novartis, Basel, Switzerland), LY3214996 (Eli Lilly and Company, Indianapolis, IN), MK-8353 (Merck Sharp and Dohme Corp, Kenilworth, NJ).

[0106] MEK Inhibitors In some embodiments, the present disclosure provides a method for treating vasculopathy (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 some embodiments, the Ras pathway inhibitor is 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 (binimetinib), AS-703026 (pimasertib), SL-327, BIX02188, AZD8330, TAK-733, cobimetinib or PD318088, and ERK inhibitors, such as LY3214996, BVD-523, or GDC-0994.

[0107] In some embodiments, the MEK inhibitor is selected from the group consisting of trametinib, refametinib, cobimetinib, TAK-733, PD0325901, PD184352 (CI-10-40), R05126766, RO-4987655; E6201; GDC-0623; CH5126766; G-573; WX-554; selumetinib, binimetinib, and pimasertib. In some embodiments, the MEK inhibitor comprises cobimetinib. In some embodiments, the MEK inhibitor comprises trametinib. In some embodiments, the MEK inhibitor comprises trametinib and cobimetinib. In some embodiments, the MEK inhibitor, or a pharmaceutically acceptable salt thereof, is also contemplated herein.

[0108] In certain embodiments, MEK inhibitors may be used 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.

[0109] Protein kinase C (PKC) inhibitors Protein kinase C, commonly abbreviated as PKC, is a family of protein kinase enzymes, or members of this family, involved in the regulation of the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues on these proteins. The PKC enzymes then phosphorylate either diacylglycerol (DAG) or calcium ions (Ca 2+ PKC enzymes are activated by signals such as an increase in the concentration of PKC kinase inhibitors (PKC kinase inhibitors). Thus, PKC enzymes play important roles in several signal transduction cascades. The PKC family consists of 15 isozymes in humans, which are divided into three subfamilies based on their second messenger requirements: conventional (or classical), novel, and atypical. Conventional PKCs are the isoforms α, β I , β II , and γ. These require Ca for activation. 2+ The new (n)PKCs, which include the δ, ε, η, and θ isoforms, require DAG for activation but do not require Ca. 2+ Thus, conventional and novel PKCs are activated through the same signaling pathways as phospholipase C. On the other hand, atypical (a) PKCs (including protein kinase Mζ and ι / λ isoforms) do not require Ca for activation. 2+Neither diacylglycerol nor diacylglycerol are required. The term "protein kinase C" as used herein generally refers to the entire family of isoforms.

[0110] Exemplary PKC agonists include, but are not limited to, ruboxistaurin, chelerythrine, myabenol C, myricitrin, gossypol, verbascoside, BIM-1, or bryostatin 1. In some embodiments, the PKC inhibitor comprises enzastaurin. In some embodiments, the PKC inhibitor comprises ruboxistaurin. In some embodiments, the PKC agonist, or a pharma- ceutically acceptable salt thereof, is also contemplated herein.

[0111] In some embodiments, the PKC inhibitor is TIFF0007678748000008.tif34128, or a salt thereof, During the ceremony, Ring A is a substituted or unsubstituted C5-C6 cycloalkyl, a substituted or unsubstituted 5-6 membered heterocycloalkyl (having one or more N, O or S ring members), a substituted or unsubstituted phenyl, or a substituted or unsubstituted 5-6 membered heteroaryl (having one or more N, O or S ring members); 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 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered, having 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 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered, having 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 membered, 5-9 membered, or 5-6 membered, having one or more N, O, or S ring members); or R 1 and R 2 is a substituted or unsubstituted alkylene (e.g., C1-C2, C1-C4, C1-C8, C1-C 10 ), or substituted or unsubstituted heteroalkylene (e.g., 2-8 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered with 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 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered with one or more N, O, or S ring members), substituted or unsubstituted arylene (e.g., C6-C 10 or phenylene), or 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) linked together to form a linker; Here, each R A , R B , R C , and R Dare independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl (e.g., 2-8 membered having 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 having 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 having one or more N, O or S ring members).

[0112] In some embodiments, Ring A is a 5-6 membered heteroaryl which may contain one or more nitrogens as ring members.

[0113] In some embodiments, the PKC inhibitor is TIFF0007678748000009.tif34128, R 1 and R 2 is as described above.

[0114] In some embodiments, the PKC inhibitor is TIFF0007678748000010.tif34128, R 1 and R 2 is as described above.

[0115] In some embodiments, the PKC inhibitor is TIFF0007678748000011.tif34128, R 1 and R 2 is as described above.

[0116] In some embodiments, for a PKC inhibitor of formula (I), (II), (III), (IV-A), or (IV-B), each R 1 and R 2is 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 some 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. 1 and R 2 is independently hydrogen, or unsubstituted C1-C4 alkyl.

[0117] In some embodiments, for the PKC inhibitor of formula (II), R 1 is a substituted or unsubstituted piperidinyl, piperazinyl, pyridyl, or pyrimidyl. For example, R 1 teeth, The file is TIFF0007678748000012.tif32128.

[0118] In some embodiments, R 2 is hydrogen or unsubstituted C1-C4 alkyl. In some embodiments, R 2 is hydrogen. In some embodiments, R 2 is methyl. In some embodiments, R 2 is ethyl. In some embodiments, R 2 is propyl. In embodiments, R 2 is butyl. In some embodiments, R 2 is t-butyl.

[0119] In some embodiments, for a PKC inhibitor of formula (I), (II), (III), (IV-A), or (IV-B), R 1 and R 2are linked together 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 together to form a substituted or unsubstituted C1-C8 alkylene linker. 1 and R 2 are linked together to form a substituted C1-C8 alkylene linker. 1 and R 2 are linked together to form an unsubstituted C1-C8 alkylene linker. 1 and R 2 are linked together 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 together to form an unsubstituted 2-8 membered heteroalkylene linker having one or more N, O or S atoms in the heteroalkylene linker chain.

[0120] In some embodiments, R 1 and R 2 teeth, are concatenated together to form TIFF0007678748000013.tif30153, where R 3 are 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 membered, 2-6 membered, 4-6 membered, 2-3 membered, or 4-5 membered, having one or more N, O, or S members); substituted or unsubstituted cycloalkyl (e.g., C3-C8, C3-C6, C4-C6, or C5-C6); substituted or unsubstituted heterocycloalkyl (e.g., 3-8 membered, 3-6 membered, 4-6 membered, 4-5 membered, or 5-6 membered, and having one or more N, O, or S ring members); substituted or unsubstituted aryl (e.g., C6-C8, 10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., having 5-10, 5-9, or 5-6 members and one or more N, O, or S ring members). A , R B , R C , and R D is written as above.

[0121] In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF0007678748000014.tif20128, and these are 3 may be substituted with one or more of:

[0122] In some embodiments, R 1 and R 2 teeth, are concatenated together to form TIFF0007678748000015.tif14128, and R 3 is written as above.

[0123] In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF0007678748000016.tif9128, and these are 3 may be substituted with one or more of:

[0124] In some embodiments, for a PKC inhibitor of formula (IV-A) or (IV-B), R1 and R 2 are linked together to form a substituted or unsubstituted C5-C6 cycloalkylene linker. 1 and R 2 are linked together to form a substituted C-C cycloalkylene linker, e.g., which may be one or more of the R 3 In some embodiments, R 1 and R 2 are linked together to form an unsubstituted C5-C6 cycloalkylene linker.

[0125] In some embodiments, R 1 and R 2 are linked together to form a substituted or unsubstituted 5-6 membered heterocycloalkylene linker (e.g., having one or more N, O or S atoms in the linker chain). 1 and R 2 are linked together to form a substituted 5-6 membered heterocycloalkylene linker having one or more N, O or S atoms in the linker chain, e.g., R 3 In some embodiments, R 1 and R 2 are linked together to form an unsubstituted 5-6 membered heterocycloalkylene linker having one or more N, O or S atoms in the linker chain.

[0126] In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF0007678748000017.tif19128, and these are 3 In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF0007678748000018.tif19128, and these are 3In some embodiments, R 1 and R 2 teeth, These are then concatenated together to form TIFF0007678748000019.tif17128, which are then combined into the R 3 may be substituted with one or more of:

[0127] In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF0007678748000020.tif20128, and these are 3 In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF0007678748000021.tif19128, and these are 3 In some embodiments, R 1 and R 2 teeth, These are then concatenated together to form TIFF0007678748000022.tif19128, which are then combined into the R 3 may be substituted with one or more of:

[0128] In some embodiments, for a PKC inhibitor of formula (III), each R 1 and R 2 is independently hydrogen, substituted or unsubstituted C1-C4 alkyl, or substituted or unsubstituted 5-6 membered heterocycloalkyl (having one or more N, O, or S ring members). 1 is unsubstituted C1-C4 alkyl. In some embodiments, R 1 is a substituted 5-6 membered heterocycloalkyl having one or more N, O or S ring members, which can be defined as R 3 In some embodiments, R 1is a substituted 5-6 membered heterocycloalkyl having one or more N, O or S ring members, which can be defined as R 3 In some embodiments, R 1 is substituted or unsubstituted piperidinyl or piperazinyl. In some embodiments, R 1 is a substituted piperidinyl or piperazinyl, which are defined above as R 3 In some embodiments, R 1 is unsubstituted piperidinyl or piperazinyl. In some embodiments, R 1 teeth, TIFF0007678748000023.tif17128, and these are the R files mentioned above. 3 In some embodiments, R 1 teeth, The file is TIFF0007678748000024.tif17140.

[0129] In some embodiments, R 2 is hydrogen. In embodiments, R 2 is methyl.

[0130] As discussed above, a group that is "substituted" is one that is substituted at one or more available positions, typically 1, 2, 3, 4 or 5 positions, by other than hydrogen, by one or more suitable groups (which may be the same or different), such as, 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 hydroxy, alkyl), aminoalkyl (e.g., C1-C6 aminoalkyl), haloalkoxy (e.g., C1-C6 haloalkoxy), alkanoyl (e.g., C1-C8 alkanoyl), alkanone (e.g., C1-C8 alkanone), alkanoyloxy (e.g., C1-C8 alkanoyloxy), alkoxycarbonyl (e.g., C1-C8 alkoxycarbonyl), mono- and di-(C1-C8 alkyl)amino, mono- and di-(C1-C8 alkyl)aminoC1-C8 alkyl, mono- and di-(C1-C8 alkyl)carboxamide, mono- and di-(C1-C8 alkyl)sulfonamide, alkylsulfinyl (e.g., C1-C8 alkylsulfinyl), alkylsulfonyl (e.g., C1-C8 alkylsulfonyl), aryl (e.g., phenyl), arylalkyl (e.g., (C6-C 18 aryl) C1-C8 alkyl, e.g., benzyl and phenethyl), aryloxy (e.g., C6-C 18 Aryloxy (e.g., phenoxy), arylalkoxy (e.g., (C6-C 18 aryl), C1-C8 alkoxy), and / or 3-8 membered heterocyclic groups having one or more N, O or S ring members.

[0131] As referred to herein, a group having a designated number of "membered" or "membered" refers to the designated number of atoms in the group.

[0132] Exemplary PKC inhibitors include: TIFF0007678748000025.tif172167 may be mentioned.

[0133] In certain embodiments, a protein kinase inhibitor or monoclonal antibody that inhibits a protein kinase or a receptor involved in a growth factor signaling pathway, such as an EGFR, VEGFR, AKT, Erb1, Erb2, ErbB, Syk, Bcr-Abl, JAK, Src, GSK-3, PI3K, Ras, Raf, MAPK, MAPKK, mTOR, c-Kit, eph receptor or BRAF inhibitor, is administered to the subject. Non-limiting examples of inhibitors of protein kinase or growth factor signaling pathways include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ranibizumab, ruxolitinib, saracatinib, sorafenib, sunitinib, trastuzumab, and vandetanib. , AP23451, vemurafenib, MK-2206, GSK690693, A-443654, VQD-002, miltefosine, perifosine, CAL101, PX-866, LY294002, rapamycin, temsirolimus, everolimus, ridaforolimus, alvocidib, genistein, selumetinib, AZD-6244, vatalanib, P1446A-05, AG-024322, ZD1839, P276-00, GW572016 or mixtures thereof.

[0134] In certain embodiments, an agent that decreases PKC activity or expression 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.

[0135] In one embodiment, PKC has the following amino acid sequence (NCBI Accession No: NP_002728.1 (SEQ ID NO: 7), which is incorporated by reference in its entirety): Contains TIFF0007678748000026.tif47146.

[0136] In one embodiment, PKC comprises the following nucleotide sequence (NCBI Accession No: NM_002737.2 (SEQ ID NO: 8), which is incorporated by reference in its entirety): TIFF0007678748000027.tif31146TIFF0007678748000028.tif243148TIFF0007678748000029.tif243148TIFF0007678748000030.tif67148 sequence, coding sequence is bold and underlined.

[0137] Agents that decrease the expression or activity of phospholipase C (PLC) or inositol triphosphate (IP3) In some instances, an agent that acts as a vasodilator is used to block the activity or expression of PLC or IP3. In some embodiments, the PLC inhibitor comprises U-73122, U73343, and ET-18-OCH3. In some embodiments, the IP3 inhibitor comprises 2-APB and xestospondin C. In some embodiments, the agent comprises hydralazine. It is contemplated that the inhibitor can be used alone or in any combination. In some embodiments, the PLC or IP3 inhibitor, or its pharmaceutically acceptable salt, is also contemplated herein.

[0138] In certain embodiments, agents that decrease PLC or IP3 activity or expression may be administered at concentrations 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. In other embodiments, it is contemplated that agents are administered at higher concentrations, e.g., up to 1000 mg / kg body weight.

[0139] In some embodiments, combination therapy with androgen antagonists is contemplated (e.g., antiandrogens).Antiandrogens are a class of drugs that prevent androgens, such as testosterone and dihydrotestosterone (DHT), from mediating their biological effects in the body.They act by blocking the androgen receptor (AR) and / or inhibiting or suppressing androgen production.Exemplary antiandrogens include:

[0140] Androgen receptor antagonist: a drug that directly binds to AR and blocks AR. These drugs include steroidal antiandrogens: cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendolone, and osaterone acetate (veterinary medicine), and nonsteroidal antiandrogens: flutamide, bicalutamide, nilutamide, topirutamide, enzalutamide, and apalutamide. Apart from cyproterone acetate and chlormadinone acetate, a few other progestins used in oral contraceptives and / or in menopausal HRT also have various degrees of AR antagonist activity, including dienogest, drospirenone, medrogestone, nomegestrol acetate, promegestone, and trimegestone.

[0141] 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 ceviteronel, the CYP11A1 (P450scc) inhibitor aminoglutethimide, and the 5α-reductase inhibitors finasteride, dutasteride, epristeride, alphatradiol, and saw palmetto extract (Serenoa repens). Several other antiandrogens are also known to weakly inhibit androgen synthesis, including cyproterone acetate, spironolactone, medrogestone, flutamide, nilutamide, and bifluranol.

[0142] Antigonadotropin: A drug that suppresses gonadotropin-releasing hormone (GnRH)-induced release of gonadotropins and the resulting activation of gonadal androgen production. Examples include GnRH regulators, such as leuprorelin (GnRH agonist) and cetrorelix (GnRH antagonist), progestogens, such as allylestrenol, chlormadinone acetate, cyproterone acetate, gestorone caproate, hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrol acetate, osaterone acetate (veterinary medicine), and oxendolone, and estrogens, such as estradiol, estradiol esters, ethinylestradiol, conjugated estrogens, and diethylstilbestrol.

[0143] Additional examples: Drugs that counteract the effects of androgens by other means than those mentioned above. Examples include estrogens, especially oral and synthetic (e.g., ethinylestradiol, diethylstilbestrol), which stimulate sex hormone binding globulin (SHBG) production in the liver, thereby reducing the release and thus bioactive levels of testosterone and DHT; anticorticotropic drugs such as glucocorticoids, which suppress adrenocorticotropic hormone (ACTH)-induced production of adrenal androgens; and immunogens and vaccines against androstenedione, such as obandrotone albumin and androstenedione albumin, which reduce levels of androgens via the production of antibodies against androgens and the androgen precursor androstenedione (used only in veterinary medicine).

[0144] Connective tissue disorders 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 tissues are fat, bone, and cartilage. These disorders often involve joints, muscles, and skin, but they can also involve other organs and organ systems, including the eyes, heart, lungs, kidneys, gastrointestinal tract, and blood vessels. There are over 200 disorders that affect connective tissue. Causes and specific symptoms vary for different types.

[0145] Examples of tissue diseases (e.g., epithelial, connective, muscle and nervous tissue) potentially treatable with the compositions and methods include, but are not limited to, autoimmune, degenerative, inflammatory, infectious, cancerous, viral, fungal, injury or traumatic. Diseases of these tissues and / or organs may be primary diseases or may be caused by pre-existing diseases and / or conditions. Examples include amyloidosis, atrial fibrillation, convulsion, cramp, 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 arachnodactyly), 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 dystrophies, disuse atrophy, denervation atrophy, Duchenne muscular dystrophy, facioscapulohumoral muscular dystrophy), liver disease, hepatic disease myasthenia gravis gravis, myopathy, myositis, myositis ossificans, cancer, fibromyalgia, muscle fatigue, spasm, spasticity, sprains, contusions, brain injury, spinal cord injury, glioma, neuroeptheliomatous, hypertension, cardiovascular disease, diabetes, Alzheimer's disease, cystitis, AIDS, rickets, and nerve sheath tumors.Examples of tissues, organs and / or body systems that may be affected by disease and treated with the compositions and methods described herein include, but are not limited to, the immune system, the sensory organs (e.g., organs of taste, smell, vision, hearing), the digestive system (e.g., mouth, isthmus, pharynx, esophagus, abdomen, stomach, small intestine, large intestine, liver, pancreas), genitourinary system, endocrine system, metabolism, cardiovascular system (e.g., heart, blood pressure, arteries), hematology (e.g., blood chemistry), urinary system (e.g., kidneys, ureters, bladder, male urethra, female urethra, male reproductive organs (e.g., testes and their capsules, vas deferens, vesiculae seminales, ejaculatory duct, penis, prostate, bulbourethral glands), female reproductive organs (e.g., ovaries, fallopian tubes, uterus, vagina, clitoris, Bartholin's gland, external genitalia, breasts)), endocrine glands (e.g., thyroid gland, parathyroid gland, thymus, pituitary gland, pineal gland, chromaffin and cortical systems, and the like). corticol system, spleen), reproductive, respiratory (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 (e.g., loose junctions (e.g., loose, adipose, reticular), and dense junctions (e.g., parallel, interwoven)), cartilage (e.g., hyaline, elastic, fibrous), muscle (e.g., skeletal muscle (e.g., types I, II, IIa, IIx, IIb), cardiac muscle, smooth muscle), nerve (e.g., neurons (e.g., motor neurons, interneurons, sensory neurons), glia, spinal cord, nerves, brain).

[0146] In some embodiments, the connective tissue disorder comprises a vasculopathy (e.g., vascular Ehlers-Danlos syndrome), Marfan syndrome, Loeys-Dietz syndrome, or familial thoracic aortic aneurysm.

[0147] Ehlers-Danlos Syndrome (EDS) Ehlers-Danlos syndrome (EDS) is a group of genetic connective tissue disorders. Symptoms may include loose joints, elastic skin, and abnormal scarring. These may be noticed at birth or in early childhood. Complications may include aortic dissection, joint dislocation, scoliosis, chronic pain, or early osteoarthritis.

[0148] EDS is caused by a mutation in one of over a dozen different genes. The particular gene affected determines the particular EDS. Some cases result from a de novo mutation that occurs during early development, while others are inherited in an autosomal dominant or recessive manner. This causes a defect in the structure or processing of collagen. The diagnosis may be confirmed with genetic testing or a skin biopsy. People may be misdiagnosed as having hypochondria, depression, or chronic fatigue syndrome.

[0149] To date, there is no known cure, but physical therapy and bracing can help strengthen muscles and support joints. Some disorders result in a normal life expectancy, while disorders that affect the blood vessels generally result in a shorter life expectancy. EDS affects approximately 1 in every 5,000 people worldwide, and prognosis depends on the specific disorder.

[0150] EDS classification Hypermobility type EDS (type 3 hEDS) is primarily characterized by joint hypermobility affecting both large and small joints, which can result in recurrent joint dislocations and subluxations (incomplete dislocations). People with this type generally have soft, smooth, velvety skin with easy bleeding and chronic pain in muscles and / or bones. The mutations that cause this type of EDS are unknown. Less skin involvement is seen than in other types. No genetic testing is available for this type.

[0151] Classical EDS (type 1 cEDS) is associated with very elastic (stretchy), smooth skin that is fragile and bleeds easily; extensive, atrophic scarring (flat or depressed scars); and joint hypermobility. Molar pseudotumors (calcified hematomas at pressure sites such as the elbow) and spheroids (fat-containing cysts on the forearms and shins) are also frequently seen. Hypotonia and delayed motor development may occur. Mutations that cause this type of EDS are in the genes COL5A1, COL5A2, and COL1A1. It involves the skin more than hEDS.

[0152] Vascular EDS (type 4 vEDS) is characterized by thin, translucent skin that is extremely fragile and prone to bleeding. Arteries and certain organs, such as the intestines and uterus, are also fragile and prone to rupture. People with this type are typically short and have thin hair. It also has characteristic facial features, including large eyes, small chin, sunken cheeks, thin nose and lips, and ears without earlobes. Joint hypermobility is present, but is generally limited to the small joints (fingers, toes). Other common features include club feet, tendon and / or muscle rupture, acroprogeria (premature aging of the skin of the hands and feet), early onset varicose veins, pneumothorax (collapsed lungs), gum recession, and reduced subcutaneous fat mass. It can be caused by a mutation in the COL3A1 gene.

[0153] Kyphoscoliotic EDS (type 6 kEDS) is associated with severe hypotonia at birth, delayed motor development, progressive scoliosis (present from birth), and scleral fragility. Affected people may also have fragile arteries that rupture and bleed easily, usually small corneas, and osteopenia (low bone density). Other common features include a "Marfanoid habitus" (arachnodactyly) characterized by long, thin fingers, usually long limbs, and a sunken (pectus excavatum) or protruding (pectus carinatum) chest. It may be caused by a mutation in the gene PLOD1.

[0154] Multiple joint laxity type EDS (types 7A and B aEDS) is characterized by severe joint hypermobility and congenital hip dislocation. Other common features include fragile rubbery skin with easy bleeding, hypotonia, kyphoscoliosis (backward and scoliotic curvature), and mild osteopenia. Type I collagen is usually affected. It is very rare, with about 30 cases reported. It is more severe than the hypermobility type. Mutations in the genes COL1A1 and COL1A2 cause it.

[0155] Cutaneous fragile EDS (type 7C dEDS) is associated with extremely fragile skin that leads to severe bleeding and scarring; drooping, excess skin, especially on the face; and hernias. It is extremely rare, with approximately 10 reported cases.

[0156] Brittle Cornea Syndrome is characterized by thin corneas, early onset progressive bulbous or conus keratoconus, and blue sclera. Classic symptoms such as hypermobile joints and hyperelastic skin are also often present.

[0157] Classical EDS (type 1 cEDS) is characterized by skin hyperextensibility with a velvety feel but without atrophic scarring, generalized joint hypermobility with or without recurrent dislocations (most commonly of the shoulders and ankles), and skin that bruises easily or has spontaneous ecchymosis (discoloration of the skin due to bleeding under the skin).

[0158] Spondylodysplastic EDS (spEDS) is characterized by short stature (progressive during childhood), hypotonia (ranging from congenital severe to late-onset mild), and bowed limbs.

[0159] Muscle contracture-type EDS (mcEDS) is characterized by congenital multiple joint contractures, characteristically adduction-flexion contractures and / or equinovarus (clubfoot), evident at birth or in early infancy, characteristic craniofacial features, and cutaneous features such as skin hyperextensibility, easy bleeding, fragile skin with atrophic scarring, and excessive palmar wrinkling.

[0160] Myopathic EDS (mEDS) is characterized by congenital hypotonia and / or muscle atrophy that improves with age, proximal joint contractures (knee, hip and elbow joints), and distal joint hypermobility (ankle, wrist, foot and hand joints).

[0161] Periodontal EDS (pEDS) is characterized by early-onset (childhood or adolescence) severe and refractory periodontitis, loss of attached gingiva, anterior tibial spots, and a family history of first-degree relatives who meet the clinical diagnostic criteria.

[0162] Valvular EDS (cvEDS) is characterized by severe, progressive heart valve problems (aortic, mitral), skin problems (hyperextensibility, atrophic scarring, thin skin, easy bleeding), and joint hypermobility (generalized or limited to small joints).

[0163] Treatment The present disclosure provides a method for the treatment of vasopathy (e.g., vEDS) or connective tissue disorder in a subject in need thereof by administering to the subject a therapeutically effective amount of an agent that 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, for the preparation of a medicament useful for the treatment of vasopathy (e.g., vEDS) or connective tissue disorder.

[0164] The present disclosure also provides a method comprising combination therapy.As used herein, "combination therapy" or "co-therapy" includes the administration of a therapeutically effective amount of an agent (e.g., an agent that reduces the activity or expression of ERK, PKC, and / or PLC or IP3) or a pharma- ceutically acceptable salt thereof with at least one additional active agent, also referred to herein as "active pharmaceutical ingredient" ("API"), as part of a treatment regimen intended to provide beneficial effects from the co-action of the agent (e.g., agonist, antagonist, or inhibitor) and the additional active agent.

[0165] According to the embodiments described below, "additional API" is understood to refer to 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 is understood that two or more of the additional APIs described below can be used in the regimen. The term "combination therapy" or "combination therapy regimen" is not intended to encompass the administration of two or more therapeutic compounds as part of a separate monotherapy regimen that fortuitously and arbitrarily results in an unintended or unanticipated beneficial effect.

[0166] Preferably, administration of a composition comprising an agent (e.g., an agent that reduces the activity or expression of, for example, ERK, PKC, and / or PLC or IP3) in combination with one or more additional APIs as discussed herein provides a synergistic response in the treated subject. In this context, the term "synergistic" refers to the efficacy of the combination being more effective than the additive effect of either monotherapy alone.

[0167] The present disclosure also provides methods that include combination therapy for the treatment of vasopathies (e.g., vEDS) or connective tissue disorders. As used herein, "combination therapy" or "co-therapy" includes the administration of the compounds described herein with at least one additional agent as disclosed herein as part of a specific treatment regimen intended to provide beneficial effects from the co-action of these therapeutic compounds. The at least one additional agent can be a therapeutic or non-therapeutic agent. The beneficial effects of the combination include, but are not limited to, pharmacokinetic or pharmacodynamic co-actions resulting from the combination of therapeutic compounds. The beneficial effects of the combination can also relate to the reduction of toxicity, side effects, or adverse events associated with another agent in the combination. "Combination therapy" may, but generally is not intended to, encompass the administration of two or more of these therapeutic compounds as part of separate monotherapy regimens that coincidentally and arbitrarily result in the combination of the present disclosure.

[0168] Thus, in some embodiments, a subject in need thereof is administered one or more agents 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.

[0169] In certain embodiments, a subject in need thereof is administered one or more agents that inhibit 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 kinase.

[0170] In the context of combination therapy, the administration of the antagonist may be simultaneous with the administration of one or more additional agents, or may be consecutive with the administration of one or more additional agents.In another aspect, the administration of different components of combination therapy may be at different frequencies.The one or more additional agents may be administered before (e.g., 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 before), simultaneously with, or after (e.g., 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 after) the administration of the compound of the present disclosure.

[0171] One or more additional active substances can be formulated for co-administration with the active substance of the present disclosure in a single dosage form, as described in more detail herein.One or more additional active substances can be administered separately from the dosage form that contains the compound of the present disclosure.When the additional active substance is administered separately from the compound of the present disclosure, it can be by the same or different administration route as the compound of the present disclosure.

[0172] Preferably, the administration of the composition comprising the agent of the present disclosure in combination with one or more additional agents provides a synergistic response in subjects with the disorder, disease, or condition of the present disclosure.In this context, the term "synergistic" refers to the efficacy of the combination being more effective than the additive effect of either monotherapy alone.The synergistic effect of the combination therapy according to the present disclosure may allow the use of a lower dosage and / or less frequent administration of at least one agent in the combination compared to its dose and / or frequency outside the combination.The synergistic effect may manifest itself in the avoidance or reduction of adverse or undesirable side effects associated with the use of either therapy alone in the combination.

[0173] "Combination therapy" also includes administration of the compound of the present disclosure in further combination with non-drug therapy (e.g., surgery or radiation therapy).When combination therapy further includes non-drug treatment, the non-drug treatment can be performed at any suitable time, as long as the beneficial effect from the combination of therapeutic compound and non-drug treatment is achieved.For example, in appropriate cases, when non-drug treatment is separated in time from administration of therapeutic compound, perhaps by days or even weeks, beneficial effect is still achieved.

[0174] In the embodiments of the methods described herein, the agent (e.g., the 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 vasopathy (e.g., vEDS) or connective tissue disorder. In some embodiments, the agent and at least one additional agent are administered in a single dosage form. In another aspect, the agent and at least one additional agent are administered in separate dosage forms. In some embodiments, the at least one additional agent is a therapeutic agent. In some embodiments, the therapeutic agent is adapted for the treatment of vasopathy (e.g., vEDS) or connective tissue disorder. In another aspect, the agent is administered in combination with at least one additional agent that is not for the treatment of vasopathy (e.g., vEDS) or connective tissue disorder, for example, a second agent that serves to reduce toxicity or adverse events associated with another active agent administered in combination therapy.

[0175] In several embodiments, the at least one additional agent is directed to a targeted therapy, where the treatment targets a vasopathy (e.g., vEDS) or connective tissue disorder, protein, or tissue environment that contributes to vasopathy (e.g., vEDS) or connective tissue disorder progression.

[0176] In some embodiments, combination therapy with androgen antagonists is contemplated (e.g., antiandrogens).Antiandrogens are a class of drugs that prevent androgens such as testosterone and dihydrotestosterone (DHT) from mediating their biological effects in the body.They act by blocking androgen receptor (AR) and / or inhibiting or suppressing androgen production.

[0177] In embodiments, exemplary combinations include an androgen antagonist in addition to a PLC, IP3, PKC, or ERK inhibitor.

[0178] The term "therapeutically effective amount" refers to an amount sufficient to treat, ameliorate, reduce the severity or duration of a disease, disorder or condition, or enhance or improve the therapeutic effect of another therapy, or prevent a specified disease, disorder or condition, or exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The exact 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.

[0179] An effective amount of the agent can be administered once a day, two to five times a day, up to two or up to three times a day, or up to eight times a day. In embodiments, the agent is administered three times a day, twice a day, once a day, 14 days on (four times a day, three times a day or two times a day, or once a day) and 7 days off in a three week cycle, up to 5 or 7 days on (four times a day, three times a day or two times a day, or once a day) and 14 to 16 days off in a three week cycle, or once every two days, or once a week, or once every two weeks, or once every three weeks.

[0180] An effective amount of an agent (e.g., an agent that reduces the activity or expression of ERK, PKC, and / or PLC or IP3) may range from about 0.001 mg / kg to about 1000 mg / kg, from about 0.01 mg / kg to about 100 mg / kg, from about 0.1 mg / kg to about 10 mg / kg; or any range with the lower end of the range being any amount from 0.001 mg / kg and 900 mg / kg and the upper end of the range being any amount from 0.1 mg / kg and 1000 mg / kg (e.g., 0.005 mg / kg and 200 mg / kg, 0.5 mg / kg and 20 mg / kg). Effective doses will also vary, as will be recognized by those skilled in the art, depending on the disease being treated, the route of administration, excipient usage, and the possibility of co-use with other therapeutic treatments, such as the use of other agents.

[0181] In more specific aspects, the disclosed agents (e.g., agents that decrease ERK, PKC, and / or PLC or IP3 activity or expression) are administered at a dosage regimen 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 weeks, or more). In some embodiments, the compounds embodied herein are administered at a dosage regimen of 100-300 mg / day for 4 or 16 weeks. Alternatively, or thereafter, an agent embodied herein is administered at a dosage regimen of 100 mg twice daily for 8 weeks, or, optionally, for 52 weeks.

[0182] As used herein, "subject in need thereof" refers to a subject with a disease, disorder, or condition, or a subject with an increased risk of developing a disease, disorder, or condition compared to the entire population.In a preferred aspect, the subject in need thereof is a subject with vasopathy (e.g., vEDS) or connective tissue disorder, or a subject with an increased risk of developing a vasopathy (e.g., vEDS) or connective tissue disorder compared to the entire population.The subject in need thereof may be a subject that is "non-responsive" or "refractory" to currently available methods for disease or disorder.In this context, the terms "non-responsive" and "refractory" refer to a subject's response to a therapy that is not clinically adequate to alleviate one or more symptoms associated with disease or disorder.

[0183] "Subject" includes mammals. The mammal can be, for example, any mammal, such as a human, a primate, a vertebrate, a bird, a mouse, a rat, a 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.

[0184] The present disclosure provides a monotherapy for the treatment of diseases, disorders, or conditions as described herein. As used herein, "monotherapy" refers to the administration of a single active or therapeutic compound to a subject in need thereof. Preferably, monotherapy involves the administration of a therapeutically effective amount of an active compound. For example, monotherapy using the agent 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, where a combination of multiple active compounds is administered, and preferably each component of the combination is present in a therapeutically effective amount. In one aspect, monotherapy using the agent of the present disclosure is more effective than combination therapy in inducing a desired biological effect.

[0185] As used herein, "treatment," "treating," or "treat" 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 ameliorate the symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder.

[0186] As used herein, "prevention," "preventing," or "prevent" describes reducing or eliminating the onset of symptoms or complications of a disease, condition, or disorder, and includes the administration of an agent of the present disclosure to reduce the onset, onset, or recurrence of symptoms of a disease, condition, or disorder.

[0187] As used herein, the term "alleviate" is intended to describe the process by which the severity of a sign or symptom of a disorder is reduced. 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, although elimination is not necessary. An effective dosage is expected to reduce the severity of a sign or symptom.

[0188] As used herein, the term "symptom" is defined as an indication of disease, illness, injury, or something in the body that is not normal. Symptoms are felt or noticed by the individual experiencing the symptoms, but may not be readily noticeable by others, who are defined as non-medical personnel.

[0189] Treating a disorder, disease or condition according to the methods described herein can result in a decrease in vasopathy (e.g., vEDS) or connective tissue disorder progression rate.Preferably, after treatment, vasopathy (e.g., vEDS) or connective tissue disorder progression rate is reduced by at least 5% compared to the number before treatment; more preferably, vasopathy (e.g., vEDS) or connective tissue disorder progression rate is reduced by at least 10%; more preferably, at least 20%; more preferably, at least 30%; more preferably, at least 40%; more preferably, at least 50%; even more preferably, at least 50%; most preferably, at least 75%.Vasopathy (e.g., vEDS) or connective tissue disorder progression rate can be measured by any reproducible measurement means.

[0190] As used herein, the term "selectively" means that it tends to occur more frequently in one population than in another. The populations compared may be cell populations. Preferably, the agent of the present disclosure acts selectively on hyperproliferative cells but does not act on normal cells. The agent of the present disclosure acts selectively to regulate one molecular target but does not significantly regulate another molecular target.

[0191] Combination therapy In some embodiments, the present disclosure also provides a method comprising combination therapy of hydralazine and at least one additional active agent. In some embodiments, the at least one additional active agent is a therapeutic agent, such as an anti-androgen compound, such as bicalutamide or spironolactone.

[0192] As used herein, "combination therapy" or "co-therapy" includes the administration of a therapeutically effective amount of the agent described herein with at least one additional active agent as part of a specific treatment regimen intended to provide beneficial effects from the co-action of the agent and the additional active agent.The beneficial effects of the combination include, but are not limited to, pharmacokinetic or pharmacodynamic co-actions 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 that accidentally and arbitrarily result in beneficial effects that were not intended or expected.

[0193] Preferably, the combination therapy provides synergistic response in the treated subject.In this context, the term "synergistic" refers to the efficacy of the combination being more effective than the additive effect of either monotherapy alone.The synergistic effect of the combination therapy according to the present invention can allow the use of a lower dosage and / or less frequent administration of at least one active substance in the combination compared to its dosage and / or frequency outside the combination.The additional beneficial effect of the combination can be manifested in the avoidance or reduction of the harmful or undesirable side effects associated with the use of either therapy alone (also called monotherapy) in the combination.

[0194] "Combination therapy" also includes the administration of an agent that inhibits or reduces the biological activity and / or expression of the signal transduction pathway of the present invention (e.g., PLC / IP3 / PKC / ERK) in further combination with a non-drug therapy (e.g., surgery or radiation therapy). When the combination therapy further includes a non-drug treatment, the non-drug treatment can be performed at any suitable time, as long as the beneficial effect from the synergy of the combination of the therapeutic compound and the non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is separated in time from the administration of the therapeutic compound, perhaps by days or even weeks. The non-drug treatment can be selected from chemotherapy, radiation therapy, hormone therapy, anti-estrogen therapy, gene therapy, and surgery.

[0195] 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 sufficient to treat, improve, reduce the severity or duration of the disease being treated (e.g., vEDS), or to enhance or improve the therapeutic effect of another therapy, or to show a detectable therapeutic effect in the subject. In one embodiment, the therapeutically effective amount of the agent is an amount effective to reduce a signal transduction pathway (e.g., PLC / IP3 / PKC / ERK).

[0196] In some embodiments, administration of hydralazine and an antiandrogen according to the methods described herein results in elimination of the 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.

[0197] Pharmaceutical Compositions In some embodiments, the present invention provides pharmaceutical compositions comprising an agent used in the present invention (e.g., an agent that reduces the activity or expression of ERK, PKC and / or PLC or IP3). The agent may be appropriately formulated and introduced into a subject or cellular environment by any means recognized for such delivery.

[0198] A "pharmaceutical composition" is a formulation containing the agent described herein in a pharma- ceutically acceptable form suitable for administration to a subject. As used herein, the phrase "pharmaceutical acceptable" refers to compounds, materials, compositions, carriers, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals, within the bounds of sound medical judgment, without excessive toxicity, irritation, allergic reaction, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0199] Such compositions typically include the active agent and a pharma- ceutically acceptable carrier. As used herein, the term "pharma- ceutically 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 can also be incorporated into the composition.

[0200] As used herein, the term "pharmaceutically acceptable salts" refers to salts formed, for example, from acidic and basic groups of the agents described herein. Illustrative 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, besylate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate salts (e.g., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)).

[0201] A pharmaceutical composition is formulated to suit its 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 may contain the following components: a sterile diluent, e.g., water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, e.g., benzyl alcohol or methylparabens; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, or phosphates, and tonicity adjusters, e.g., sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0202] Pharmaceutical compositions suitable for injectable use include sterile 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, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy flow from a syringe 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, including, for example, water, ethanol, polyol (for example, 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 a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption, such as aluminum monostearate and gelatin in the composition.

[0203] Sterile injectable solutions can be prepared by mixing active compound in the required amount in selected solvent with one or combination of the above-listed components, and then sterilizing by filtration if necessary.Generally, dispersion is prepared by mixing active compound into a sterile vehicle that contains basic dispersion medium and other necessary components from above-listed.For the sterile powder for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces powder from the solution of active ingredient and any additional desired components that are previously sterilized by filtration.

[0204] Oral compositions generally include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, troches, or capsules, for example, gelatin capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash. Pharmaceutically compatible binders, and / or auxiliary substances can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0205] The compositions of the invention can also be formulated as nanoparticle formulations. The compounds of the invention can be administered for immediate, delayed, modified, sustained, pulsed and / or controlled release applications. The pharmaceutical compositions of the invention can contain 0.01-99% by weight of the active material. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a nebulizer or pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide. Such methods include those described in U.S. Pat. No. 6,468,798.

[0206] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams as generally known in the art. The compound can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0207] In one embodiment, the active compound is prepared with a carrier that protects the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid. Such formulations can be prepared using standard techniques. These materials can also be purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes that target infected cells with monoclonal antibodies 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. Pat. No. 4,522,811.

[0208] The toxicity and therapeutic efficacy of such compounds are measured, for example, by the LD50 (the dose lethal to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of the population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the ratio LD 50 / ED 50The therapeutic index can be expressed as: Compounds that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects can be used, but care should be taken in designing delivery systems that target such compounds to the affected tissue site in order to minimize potential damage to non-infected cells, thereby reducing side effects.

[0209] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. Such dosages of compounds can be administered with little or no toxicity to ED. 50 It is preferred that the circulating concentration range includes the IC50 / IC10 concentration range. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Dosages are based on the IC50 / IC10 concentration range determined in animal models and in cell culture. 50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

[0210] As defined herein, the therapeutically effective amount (i.e., effective dosage) of an agent depends on the agent selected. For example, a single dose of an agent within the range of approximately 1 pg to 1000 mg can be administered; in some 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 some embodiments, 1 to 5 g of the composition can be administered.

[0211] The therapeutically effective amount of the compound of the present invention can be determined by methods known in the art. In addition to depending on the selected agent and / or pharmaceutical formulation used, the therapeutically effective amount of the pharmaceutical composition of the present invention depends on the age and overall physiological condition of the patient and the route of administration. In some embodiments, the therapeutic dose is generally from about 10 and 2000 mg / day, preferably from about 30 and 1500 mg / day. Other ranges may also be used, including, for example, 50-500 mg / day, 50-300 mg / day, 100-200 mg / day.

[0212] Administration can be once a day, twice a day, or more frequently, and may be reduced during the maintenance phase of disease or disorder; for example, every other day or once every two days instead of every day or twice a day. The dose and frequency of administration depend on the clinical signs that confirm the maintenance of the remission phase, with the reduction or disappearance of at least one, or more preferably two or more, clinical signs of the acute phase known to those skilled in the art. Those skilled in the art will recognize that certain factors, including but not limited to the severity of disease or disorder, previous treatments, the overall physical health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutically effective amount of an agent can include a single treatment, or optionally, can include a series of treatments.

[0213] It can be appreciated that the method of introducing an agent into a cellular environment depends on the type of cell and the composition of its environment. An appropriate amount of agent must be introduced, and these amounts can be determined empirically using standard methods. Exemplary effective concentrations of individual agents in the cellular environment can be 500 millimolar or less, 50 millimolar or less, 10 millimolar or less, 1 millimolar or less, 500 nanomolar or less, 50 nanomolar or less, 10 nanomolar or less, or even compositions that are 1 nanomolar or less can be used.

[0214] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. EXAMPLES

[0215] 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 present invention and are not intended to limit the scope of what the inventors regard as their invention.

[0216] Example 1: Generation of knockin glycine-substituted vEDS mice Novel knockin glycine substitution (Col3a1 G209S / + and Col3a1 G938D / +) vEDS mice were generated using CRISPR / Cas9. The mouse models recapitulated human vascular phenotypes, and the molecular basis for the eventual failure of the vascular wall was elucidated through the exploitation of environmental influences on the disease phenotype.

[0217] To introduce each mutation, three guide RNAs (tracrRNA + crRNA) were designed to target DNA regions flanking the intended mutation site. A DNA oligo repair template (ssDNA oligo) was also designed, containing homologous sequences upstream and downstream of the target region, as well as the intended mutation. Pronuclear injection of one-cell C57BL / 6J embryos was performed by the JHU Transgenic Core using standard microinjection techniques with a mix of Cas9 protein, tracrRNA, crRNA, and ssDNA oligos diluted in RNase-free injection buffer. Injected embryos were transferred into the oviducts of pseudopregnant ICR females using established techniques. Sanger sequencing of the site was used to confirm introduction of the mutation, and mice were backcrossed for at least four generations to exclude off-target effects. For G209S mice, the introduced mutation was c.625_626GG>TC, which corresponds to p.Gly210Ser in humans. For G938D mice, the introduced mutation was c.2813G>A, which corresponds to p.Gly939Ser in humans. Wild-type human Col3a1 mRNA NCBI accession number: NM_000090.3; wild-type mouse Col3a1 mRNA NCBI accession number: NM_009930.2.

[0218] Example 2: Col3a1 G209S / + and Col3a1 G938D / + mouse models recapitulated the vEDS phenotype Both Col3a1 G209S / + and Col3a1 G938D / + mouse models recapitulated the vEDS phenotype. Mice with vEDS died suddenly due to aortic rupture, aortic dissection or organ rupture, and in most cases showed hemothorax or intraperitoneal bleeding at necropsy. Mice with the Col3a1 G938D / + mutation exhibit a more severe phenotype (median survival = 45 days versus 400 days for the Col3a1 G209S / + model, p<0.0001, Figure 1A-1C). Figure 1A is a graph demonstrating that the G209S / + mouse model recapitulated the vEDS phenotype, with a median survival of 400 days; p<0.0001. In Figure 1B, survival of the G938D / + mouse model is shown, recapitulating vEDS, with a median survival of 45 days; p<0.0001.

[0219] No echocardiographic evidence of aortic root aneurysms was observed in either mouse model.

[0220] Example 3: Signal transduction abnormalities were mediators of disease pathology in vEDS We hypothesized that signaling abnormalities may be a primary mediator of disease pathology in vEDS. To test this hypothesis, considering 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 aorta 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.

[0221] Network analysis showed that vEDS aortas displayed a gene expression signature for elevated mitogen-activated protein kinase (MAPK) activity [P38, JNK, AKT, ERK, ERK1 / 2] ( Figure 1 E).

[0222] Furthermore, upstream analysis predicted that transcriptional differences in vEDS aortas were driven by G protein-coupled receptor (GPCR) activation, signaling through the associated PLC / IP3 / PKC / ERK axis ( Figure 1F ).

[0223] The signature of elevated GPCR and MAPK signaling was confirmed by immunoblotting for markers of active signaling through this pathway, and ERK1 / 2 phosphorylation and PKC phosphorylation were found to be significantly higher in vEDS aortas (FIG. 1G).

[0224] In contrast to Marfan syndrome (MFS) or Loeys-Dietz syndrome (LDS), expression profiling of vEDS aortas did not show the synthetic repertoire typical of high TGFβ signaling, however, evidence of increased ERK activation (similar to MFS and LDS) was observed. Small human studies suggest that the β-blocker celiprolol has the potential to delay adverse events in patients with vEDS, while angiotensin receptor blockers such as losartan confer dramatic protection in mouse models of MFS or LDS.

[0225] 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. While initial data indicate that hydralazine, which inhibits the PLC / IP3 / PKC / ERK axis, confers some protection in vEDS mice, these data highlight the need for discovery-based methods to uncover unexpected therapeutic strategies.

[0226] To evaluate the modulation of phenotype severity, vEDS mutations were introduced onto the pure 129 and BL6 backgrounds. Both mutations are associated with early death due to aortic dissection on the BL6 background. Most notably, the 129 background provides complete protection from dissection for both vEDS genotypes, with a completely normal life span. Rescue is associated with normalization of gene expression profiles for the aortic wall. These data provide the rationale and motivation to conduct genetic studies to identify the source and mechanism of modification in vEDS mice, with the hope and intention of mimicking nature's successful strategy using pharmacological agents.

[0227] Example 4: Pharmacological inhibition of the PLC / IP3 / PKC / ERK axis reduced the risk of aortic rupture in vEDS mice Activation of the PLC / IP3 / PKC / ERK axis has been shown to be pathogenic in MFS. 7 , we hypothesized that pharmacological inhibition of this axis would reduce the risk of aortic rupture in vEDS mice.

[0228] Pharmacological ERK antagonists increased survival Pharmacological ERK antagonists were tested to support a role for ERK activation in the pathogenesis of vEDS and as a method to identify therapeutic strategies for vEDS. Pharmacological ERK antagonists are FDA-approved inhibitors of MEK, the kinase that activates ERK. 8 We then treated mice with cobimetinib [GDC-0973, RO5514041] (2 mg / kg / day). We hypothesized that if ERK activation truly drives disease risk, then ERK inhibition would rescue mortality risk from aortic dissection.

[0229] In line with this hypothesis, a 94% survival rate was observed 45 days after treatment, compared with only 55% survival rate without treatment (Figure 2A).

[0230] Pharmacological PKC inhibition increased survival Further studies were then conducted to test agents that inhibit PKC activation. 9 We then treated mice with ruboxistaurin [LY 333531] (10 mg / kg / day). We hypothesized that if ERK activation and therefore disease risk are driven by PLC / IP3 / PKC activation, then a pharmacological PKC inhibitor would also rescue mortality risk from aortic dissection.

[0231] In line with this hypothesis, 100% survival was observed 39 days after treatment, compared with only 55% survival without treatment (Figure 2B).

[0232] Agents that inhibit the PLC / IP3 / PKC / ERK signaling cascade increased survival Further studies were performed 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 therefore confers similar protection. 10 A striking protection was observed with a 98% survival rate at 45 days of age, the median survival time for untreated vEDS mice (Figure 2C).

[0233] While survival was affected at puberty, this risk was seen almost exclusively in male mice, and therefore treatment with androgen antagonists may be advantageous for these mice. Furthermore, the dose of hydralazine may be insufficient to completely inhibit this pathway in this mouse model, and higher doses of hydralazine may prove advantageous.

[0234] Mice are treated with a combination therapy of hydralazine and an androgen antagonist.

[0235] Additionally, mice are treated with higher doses of hydralazine.

[0236] These results suggest that inhibition of excessive PLC / IP3 / PKC / ERK signaling in the aorta rescues the risk of mortality due to aortic dissection in a mouse model of vEDS.

[0237] Example 5: Oxytocin-induced ERK signaling activated the PLC / IP3 / OKC / ERK axis and aggravated the risk of aortic dissection Patients with vascular Ehlers-Danlos syndrome (vEDS) experience dissection of the medium to large aorta. Many features of vEDS are distinct from other inherited vasculopathies, such as Marfan syndrome (MFS) and Loeys-Dietz syndrome (LDS), which are associated with excessive TGFβ activity. These do not include a specific predisposition for dissection without prior vasodilation and involvement of the aortic root. Vascular rupture in patients with vEDS is difficult to predict or prevent. Pregnancy specifically increases the risk of dissection, with complications occurring in over 50% of pregnancies and death occurring in approximately 12-25% of pregnancies. The prominent postpartum occurrence of vascular dissection is not consistent with a mechanism that significantly induces hemodynamic stress. Instead, we hypothesized that oxytocin, the hormone that initiates uterine contractions and is maintained after parturition and throughout lactation, may contribute to pregnancy-associated risk. Oxytocin receptor expression is induced in the aorta during pregnancy and the hormone stimulates peripheral tissues through activation of ERK, a signaling cascade previously implicated in the pathogenesis of MFS and LDS.

[0238] Previously, we showed 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, we used oxytocin-induced ERK signaling as a method to test whether activation of the PLC / IP3 / PKC / ERK axis exacerbates the risk of aortic dissection.

[0239] In the vEDS mouse model, we identified that pregnancy and lactation were associated with 60% mortality due to arterial dissection in the first 30 days after parturition in vEDS mice (Figure 3A). Also, prevention of lactation by removal of pups after birth was able to prevent dissection and death in vEDS mice (100% survival, Figure 3B). Furthermore, nearly complete survival (95%) was achieved with treatment with hydralazine (16 mg / kg / day), which blocks the oxytocin-activated PLC / IP3 / PKC / ERK axis (Figure 3C). Similar protection (95% survival) was observed with treatment with trametinib [GSK-1120212] (1 mg / kg / day), an FDA-approved inhibitor of MEK, the kinase that activates ERK (Figure 3D).

[0240] This increased risk of death correlated with increased ERK activation, whereas protection from aortic dissection correlated with decreased ERK activation, as measured by immunoblotting and ERK target gene expression (Figure 3E and Figure 3F). This data further supports that increasing activation of the PLC / IP3 / PKC / ERK signaling pathway resulted in a significantly elevated risk of death due to aortic dissection in the vEDS mouse model, and furthermore, inhibiting the PLC / IP3 / PKC / ERK signaling pathway ameliorated this risk.

[0241] Example 6: Compositions and methods for treating vascular Ehlers-Danlos syndrome and related disorders Use of both pharmacological MEK / ERK and PKC antagonists rescued mortality risk from aortic dissection, demonstrating that PKC-dependent ERK activation is a critical component of aortic disease in vEDS and that targeting this signaling pathway is advantageous in reducing aortic disease in vEDS mouse models.

[0242] Here, it was demonstrated that pharmacological inhibition of PKCβ using a second specific PKCβ inhibitor, enzastaurin (60 mg / kg / d), also rescued 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, FIG. 16A), providing further evidence that PKCβ phosphorylation is a critical component of aortic disease in vEDS mice.

[0243] Since activation of the PLC / IP3 / PKC / ERK signaling pathway was identified herein as pathogenic in vEDS, the identity of the receptors that may activate this abnormal signaling pathway was investigated. GPCRs (Gq) signal through this pathway - Common Gq receptors in the aorta include angiotensin II receptors, thrombin receptors, endothelin-1 receptors, vasopressin receptor 1, sphingosine-1-phosphate receptors, alpha-1 adrenergic receptors, and serotonin receptors. However, there are also orphan GPCRs, such as GPR56, which has been shown to interact with collagen3,4, that are also expressed in the aorta.

[0244] Without wishing to be bound by theory, we hypothesized that inhibiting the abnormally activated receptor would allow us to pinpoint exactly how the signaling pathways are activated. Additionally, we treated mice with bosentan (100 mg / kg / d), an orally bioavailable nonspecific endothelin receptor antagonist. Treatment with bosentan resulted in 80% survival after 40 days of treatment compared to only 50% survival in untreated vEDS mice (p=0.0298, FIG. 16B). This suggests that endothelin receptor signaling contributes to vEDS pathogenesis.

[0245] The use of both pharmacological MEK / ERK antagonists and PKC antagonists rescued the risk of death from aortic dissection, demonstrating that PKC-dependent ERK activation is a key component of aortic disease in vEDS and that targeting this signaling pathway is advantageous in 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 patients with vEDS. Both PKC phosphorylation (Figure 17) as well as ERK1 / 2 phosphorylation (Figure 18) were identified in two tissue samples (iliac artery and descending thoracic aorta) from patients with vEDS. None of these phosphorylated proteins were found in tissue samples taken from the ascending aorta of individuals without vEDS.

[0246] conclusion Collectively, the results provide the first evidence for a targetable signaling abnormality that contributes 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 mortality due to aortic dissection in a vEDS mouse model.

[0247] Inhibition of ERK activation through activators of ERK, pharmacological inhibition of MEK, 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 may offer therapeutic benefit for vascular Ehlers-Danlos syndrome and potentially other connective tissue disorders.

[0248] Furthermore, PLC / IP3 / PKC / ERK activators may show comparable upregulation in vEDS aortas, which will be tested through analysis of candidates emerging from RNA-Seq profiles.

[0249] References TIFF0007678748000031.tif232159

[0250] Example 7: Androgens played an important role in aortic dissection risk and combination therapy with hydralazine Pharmacological inhibition of PKCβ prevented PKC autophosphorylation as well as ERK phosphorylation in the aortic wall as assessed by immunoblotting of aortic lysates (Figures 4A-4C). Pharmacological inhibition of MEK not only correlated with the expected reduction in phosphorylation of ERK, a downstream substrate of MEK, but curiously also reduced PKC phosphorylation, suggesting the existence of a positive feedback loop (Figure 1, *p<0.05, **p<0.01, ***p<0.001). Neither cobimetinib nor ruboxistaurin had any effect on blood pressure.

[0251] Combination therapy of hydralazine and bicalutamide for the treatment of vEDS Treatment of mice with hydralazine (32 mg / kg / d), which blocks the PLC / IP3 / PKC / ERK axis, is remarkably protective, conferring a 98% survival rate at 45 days of age, the median survival time for untreated vEDS mice. While survival was affected at puberty, this risk was seen almost exclusively in male mice (Figures 2A and 2B), and therefore we hypothesized that treatment with an androgen antagonist might be advantageous for 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 a 24% survival rate in male mice treated with hydralazine alone (Figure 6).

[0252] Curiously, when bicalutamide was removed postpuberty (at 90 days of age), the same proportion of male mice continued to survive while remaining on hydralazine (Figure 6), suggesting that there was a time dependency for androgen sensitivity in this mouse model. Informatively, male mice treated with bicalutamide alone achieved a moderate survival of about 80%, and males did not continue to survive after removal of bicalutamide postpuberty (Figure 7), suggesting that inhibition of androgen signaling alone was not sufficient to prevent aortic disease in the mouse model of vEDS.

[0253] Combination therapy of hydralazine and spironolactone for the treatment of vEDS These new observations and understanding that androgens play an important role in aortic dissection risk in our vEDS mouse model led to the testing of another FDA approved medication in combination with hydralazine. Spironolactone is an FDA approved diuretic that has direct androgen antagonism as a side effect. Spironolactone is used off-label to specifically treat acne, hirsutism, and other androgen dependent disorders. 1 It was hypothesized that spironolactone might similarly be used as a direct androgen antagonist in this disorder.

[0254] Mice were treated with spironolactone alone (100 mg / kg / d) (Figure 8A), and a moderate survival of about 80% was observed, similar to 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 (Figure 8B).

[0255] Combination therapy of hydralazine and ruboxistaurin for the treatment of vEDS In an example, combination therapy is contemplated that includes administration of hydralazine and ruboxistaurin. Ruboxistaurin is a protein kinase C-β (PKC-β) inhibitor and a macrocyclic bisindolylmaleimide compound under development by Eli Lilly that has potential as a therapy for diabetic macular edema and other diabetic vascular disorders, such as diabetic retinopathy, diabetic peripheral neuropathy and diabetic nephropathy. Additional ruboxistaurin names include Arxxant (proposed trade name), IUPAC: (9S)-9-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9H,18H-5,21:12,17-di(metheno)dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecyne-18,20-dione, and CAS number: 169939-94-0.

[0256] The structure of ruboxistaurin is provided below. TIFF0007678748000032.tif39128

[0257] Combination therapy of hydralazine and enzastaurin for the treatment of vEDS In an example, a combination therapy is contemplated that includes administration of hydralazine and enzastaurin. Enzastaurin is a synthetic bisindolylmaleimide with potential antineoplastic activity. By binding to the ATP binding site, enzastaurin selectively inhibits protein kinase C beta (PKC-β), an enzyme involved in the induction of vascular endothelial growth factor (VEGF)-stimulated angiogenesis. This agent can reduce tumor blood supply and prevent growth. Additional 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.

[0258] The structure of enzastaurin is provided below. TIFF0007678748000033.tif50128

[0259] Combination therapy of hydralazine and sotrastaurin for the treatment of vEDS In an example, a combination therapy is contemplated that includes administration of hydralazine and sotrastuarin (EAB071), an investigational immunosuppressant that blocks T-lymphocyte activation through protein kinase C inhibition.

[0260] The structure of sotrastaurin is provided below. TIFF0007678748000034.tif49128

[0261] Combination therapy of hydralazine and antiandrogens for the treatment of vEDS Test additional combinations of hydralazine and different antiandrogens.For example, contemplate hydralazine and androgen receptor antagonists, including steroidal antiandrogens (cyproterone acetate, megestrol acetate, chlormadinone acetate, oxendolone acetate and osaterone acetate).Furthermore, contemplate hydralazine in combination with nonsteroidal antiandrogens, such as flutamide, nilutamide, topirutamide, enzalutamide, drospirenone or medrogestone.

[0262] In other examples, combination therapy is contemplated that includes hydralazine and androgen synthesis inhibitor or antigonadotropin.Exemplary androgen synthesis inhibitors include ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, epristeride, and alphatradiol.Exemplary antigonadotropins include leuprorelin and cetrorelix.Other contemplated antiandrogens include ethinylestradiol and diethylstilbestrol.

[0263] Increased doses of hydralazine Furthermore, the dose of hydralazine may be insufficient to completely inhibit this pathway in this mouse model, and higher doses of hydralazine may prove advantageous. Thus, these mice were treated with higher doses of hydralazine, and it was found that increasing doses of hydralazine (50 mg / kg / d) did not improve survival any more than the 32 mg / kg / d dose did (Figure 9).

[0264] Example 8: Oxytocin-induced signaling Oxytocin-induced ERK signaling was used as a method to test the hypothesis that activation of the PLC / IP3 / PKC / ERK axis exacerbates the risk of aortic dissection. In the vEDS mouse model described herein, pregnancy and lactation were found to be associated with a 60% mortality rate due to arterial dissection in the first 30 days after parturition in vEDS mice, and prevention of lactation by removal of pups after birth, treatment with hydralazine (16 mg / kg / d), or treatment with trametinib could prevent dissection and death in vEDS mice. This increased risk of death correlated with increased ERK activation, while protection from aortic dissection correlated with decreased ERK activation, as measured by immunoblotting and ERK target gene expression.

[0265] Specific oxytocin receptor antagonists 2 Addition of treatment of mice with oxytocin receptor antagonist desGly-NH2,d(CH2)5[d-Tyr2,Thr4]OVT (the selective oxytocin receptor antagonist used was desGly-NH2,d(CH2)5[d-Tyr2,Thr4]OVT) resulted in a 95% survival rate in the first 30 days postpartum in vEDS mice that were still lactating, demonstrating that the significantly elevated risk of mortality due to aortic dissection in pregnancy was specifically driven by activation of oxytocin receptors during lactation (Figures 10A and 10B).

[0266] Furthermore, it was demonstrated that treatment with propranolol (FIG. 10B), which is the standard of care in this population and reduces blood pressure without affecting PLC / IP3 / PKC / ERK signaling, did not affect survival in this pregnancy / lactation model (46% survival rate vs. 50% survival rate 30 days after delivery). This data further supports that increasing activation of the PLC / IP3 / PKC / ERK signaling pathway resulted in a significantly elevated risk of death due to aortic dissection in our vEDS mouse model, and furthermore, inhibiting the PLC / IP3 / PKC / ERK signaling pathway ameliorated this risk.

[0267] GPCR activated the PLC / IP3 / PKC / ERK signaling pathway Having identified activation of the PLC / IP3 / PKC / ERK signaling pathway as pathogenic in vEDS, we identified receptors that may activate this abnormal signaling pathway. GPCR (Gq) signals through this pathway - Common Gq receptors in the aorta include angiotensin II receptors, thrombin receptors, endothelin-1 receptors, vasopressin receptor 1, sphingosine-1-phosphate receptors, alpha-1 adrenergic receptors, and serotonin receptors. However, Gq receptors that interact with collagen 3, which are also expressed in the aorta, are not. 3,4 There are also orphan GPCRs, such as GPR56, which has been shown to mediate

[0268] We hypothesized that we would identify how signaling pathways could be activated when receptor activity was inhibited. Angiotensin-II signaling was first inhibited by treating mice with the angiotensin receptor antagonist, losartan (60 mg / kg / d) (Figure 11A), but it was found that this had no effect on survival. Next, mice were treated with the thrombin receptor antagonist, vorapraxal (1 mg / kg / d), but it was also found that this had no effect on survival (Figure 11B).

[0269] Additional specific Gq receptor inhibitors are tested.Exemplary Gq receptor inhibitors that are tested include endothelin-1 receptor, vasopressin receptor 1, sphingosine-1-phosphate receptor, alpha-1 adrenergic receptor, serotonin receptor, and orphan GPCRs, such as GPR56.

[0270] Tyrosine kinase receptors and the PLC / IP3 / PKC / ERK signaling pathway 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, leading to elevated PLC / IP3 / PKC / ERK signaling pathway, treated were treated with a nonspecific tyrosine kinase receptor antagonist, nintedanib (50 mg / kg / d), but it was found that it also had no effect on survival (Figure 12). This suggested that tyrosine kinase receptor activation was not driving the activation of the PLC / IP3 / PKC / ERK signaling pathway in vEDS mice.

[0271] Beta-Adrenergic Blockers and the Risk of Aortic Rupture Others have proposed that the vEDS phenotype is the result of chronically "weak" tissue. 5-8 We therefore propose that reducing blood pressure with β-adrenergic receptor blockers should reduce the risk of aortic rupture in these patients. To address this hypothesis, we used a nonspecific β antagonist, propranolol (80 mg / kg / d), a specific β antagonist, atenolol (120 mg / kg / d), and a β antagonist / β agonist, celiprolol (200 mg / kg / d), in mice, but found that none of these manipulations resulted in improved survival in our vEDS mouse model, despite the reduction in blood pressure (Figures 13A-13C). Celiprolol further accelerated aortic dissection in the mouse model, downregulating Col3a1. G209S / +vEDS patients with the mutation also demonstrated an increased risk of aortic dissection on celiprolol (Figure 14). This observation could be driven by the β2 agonism activity of celiprolol, as this was not common to all β antagonists.

[0272] Calcium channel blockers, which lower blood pressure by a different mechanism, were also tested. Amlodipine (12 mg / kg / d) was also shown to increase the risk of aortic dissection in a mouse model, which was also consistent with MFS mice. 9 (Figure 15).

[0273] Example 9: Treatment of Marfan Syndrome with Protein Kinase C-β (PKC-β) Inhibitors In an example, different PKC-β inhibitors are used for the treatment of Marfan syndrome. For example, the treatment of Marfan syndrome can include the administration of ruboxistaurin. Here, it is demonstrated that the pharmacological inhibition of PKCβ using ruboxistaurin (10 mg / kg / d) rescues aortic root growth in Marfan syndrome mice (p=2E-4, FIG. 19). In another example, the treatment of Marfan syndrome includes the administration of enzastaurin or sotrastaurin.

[0274] Combination therapy of hydralazine and antiandrogens for the treatment of vEDS Test additional combinations of hydralazine and different antiandrogens.For example, contemplate hydralazine and androgen receptor antagonists, including steroidal antiandrogens (cyproterone acetate, megestrol acetate, chlormadinone acetate, oxendolone acetate and osaterone acetate).Furthermore, contemplate hydralazine in combination with nonsteroidal antiandrogens, such as flutamide, nilutamide, topirutamide, enzalutamide, drospirenone or medrogestone.

[0275] In other examples, combination therapy is contemplated that includes hydralazine and androgen synthesis inhibitor or antigonadotropin.Exemplary androgen synthesis inhibitors include ketoconazole, abiraterone acetate, ceviteronel, aminoglutethimide, finasteride, dutasteride, epristeride, and alphatradiol.Exemplary antigonadotropins include leuprorelin and cetrorelix.Other contemplated antiandrogens include ethinylestradiol and diethylstilbestrol.

[0276] Example 10: Pharmacological inhibition increases survival in vEDS mice Pharmacological PKC inhibition with enzastaurin increases survival in vEDS mice Both Col3a1 G209S / + and Col3a1 G938D / + mouse models recapitulate the vEDS phenotype and are used to test agents for the treatment of vEDS. Mice are treated with the well-tolerated orally administered agent, enzastaurin, at approximately 30 mg / kg / day. By binding to the ATP binding site, enzastaurin selectively inhibits protein kinase C beta (PKC-β), and thus, it is hypothesized that a pharmacological PKC inhibitor rescues the risk of death from aortic dissection. Approximately 100% survival is observed after approximately 30 days (1 month) of treatment, compared to a significantly reduced survival rate in mice without treatment (e.g., statistically significant difference compared to control mice).

[0277] Mice are also treated for at least about 45 days and the survival rate of enzastaurin-treated mice versus control mice is assessed.

[0278] Mice are also treated with higher doses of enzastaurin, for example, about 40 mg / kg / day, 50 mg / kg / day, or 100 mg / kg / day. In other examples, mice are treated with enzastaurin once a day or twice a day.

[0279] These results suggest that inhibition of excessive PLC / IP3 / PKC / ERK signaling in the aorta with enzastaurin rescues the risk of mortality due to aortic dissection in a mouse model of vEDS.

[0280] Pharmacological PKC inhibition with sotrastaurin increases survival in vEDS mice Additional studies using the mouse models described herein (e.g., Col3a1 G209S / + and Col3a1 G938D / + mouse models) are used to test additional agents for the treatment of vEDS. Mice treated with the well-tolerated orally administered agent, sotrastaurin, at about 30 mg / kg / day are evaluated. Sotrastaurin (AEB071) is an immunosuppressant that blocks T-lymphocyte activation through protein kinase C inhibition. Similar to enzastaurin, we hypothesize that PKC inhibitors (e.g., sotrastaurin) also rescue the risk of death from aortic dissection. Approximately 100% survival is observed after about 30 days (1 month) of treatment, compared to significantly reduced survival in mice without treatment (e.g., statistically significant difference compared to control mice). Mice are also treated for at least about 45 days, and survival rates of sotrastaurin-treated mice versus control mice are evaluated.

[0281] The mice are 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 examples, the mice are treated with sotrastaurin once a day or twice a day.

[0282] These results provide evidence that inhibition of excessive PLC / IP3 / PKC / ERK signaling in the aorta with sotrastaurin rescues the risk of mortality due to aortic dissection in a mouse model of vEDS.

[0283] References TIFF0007678748000035.tif158160

[0284] Other Aspects Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0285] The patents and scientific literature referred to herein establish knowledge that is 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 published foreign patents and patent applications cited herein are incorporated by reference.Genbank and NCBI submissions indicated by accession numbers cited herein are incorporated by reference.All other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference.

[0286] While the present invention 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 invention as encompassed by the appended claims.

Claims

1. A pharmaceutical composition for the treatment of vascular Ehlers-Danlos syndrome (vEDS), comprising an effective amount of an active agent selected from the group consisting of ruboxistaurin, enzastaurin, and sotrastaurin, or a pharma- ceutical acceptable salt thereof.

2. 2. The pharmaceutical composition of claim 1, comprising an effective amount of enzastaurin.

3. The pharmaceutical composition of claim 1, comprising an effective amount of ruboxistaurin.

4. The pharmaceutical composition of claim 1, comprising an effective amount of sotrastaurin.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the effective amount of the agent or a pharma- ceutically acceptable salt thereof is from about 0.001 mg / kg to 250 mg / kg body weight, or from about 0.05 mg / kg to about 50 mg / kg body weight.

6. 6. The pharmaceutical composition of claim 5, wherein the effective amount of the agent or a pharma- ceutically acceptable salt thereof is from about 1 mg / kg to about 15 mg / kg, or from about 1 mg / kg to about 30 mg / kg.

7. A pharmaceutical composition for reducing the risk of aortic dissection or rupture, comprising an effective amount of enzastaurin or a pharma- ceutical acceptable salt thereof.

8. The pharmaceutical composition of claim 7, wherein the effective amount of enzastaurin or a pharma- ceutically acceptable salt thereof is from about 0.001 mg / kg to 250 mg / kg of body weight, or from about 0.05 mg / kg to about 50 mg / kg of body weight.

9. The pharmaceutical composition of claim 8, wherein the effective amount of enzastaurin or a pharma- ceutically acceptable salt thereof is from about 1 mg / kg to about 15 mg / kg, or from about 1 mg / kg to about 30 mg / kg.

10. A kit comprising: (1) the pharmaceutical composition of any one of claims 1 to 6; and (2) instructions for treating vEDS.

11. A kit comprising: (1) a pharmaceutical composition described in any one of claims 7 to 9; and (2) instructions for reducing the risk of aortic dissection or rupture.

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