Compositions and methods for treating vascular ehlers danlos syndrome and associated disorders

Abstract

To provide compositions and methods for treating vascular Ehlers Danlos Syndrome and associated connective tissue disorders.SOLUTION: A method comprises administering an effective amount of an agent that decreases the activity or expression of extracellular signal-regulated kinase (ERK) or protein kinase C (PKC). Preferably, the agent comprises an antibody or a fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof. More preferably, the agent comprises cobimetinib or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 838,049, filed April 24, 2019, U.S. Provisional Patent Application No. 62 / 747,587, filed October 18, 2018, and U.S. Provisional Patent Application No. 62 / 746,524, filed October 16, 2018, the entire contents of which are incorporated herein by reference in their entirety.

[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 vEDS have thin, translucent skin, easy bleeding, distinctive facial features, and spontaneous dissection of medium- to large-sized blood vessels and 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; the presenting symptom in the majority of adults diagnosed with vEDS is vascular dissection or organ rupture, and 25% of patients experience major complications by the age of 20. 1,2 Thus, there is an unmet need for treatments for vEDS and related connective tissue disorders. Summary of the Invention

[0006] overview In particular, 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 present 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 includes vasculopathy, and in some embodiments, the vasculopathy includes vascular Ehlers-Danlos syndrome (vEDS).

[0007] Included herein is a method for treating a vasculopathy (e.g., vEDS) in a subject, the method comprising 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 some 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, for example, 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 active substance comprises a small molecule.Small molecule is a compound whose mass is less than 2000 Dalton.The molecular mass of small molecule is preferably less than 1000 Dalton, more preferably less than 600 Dalton, for example, the compound is less than 500 Dalton, less than 400 Dalton, less than 300 Dalton, less than 200 Dalton or less than 100 Dalton.

[0012] Small molecules can be organic or inorganic. Exemplary organic small molecules include, but are not limited to, aliphatic hydrocarbons, alcohols, aldehydes, ketones, organic acids, esters, monosaccharides and disaccharides, aromatic hydrocarbons, amino acids, and lipids. Exemplary inorganic small molecules include trace elements, ions, free radicals, and metabolites. Alternatively, small molecules can be synthetically engineered to consist of fragments, 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 active substance 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 hairpin RNA 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 other nucleic acids that inhibit / antagonize or activate / agonize ERK or PKC, or IP3 or PLC.

[0014] As used herein, a polynucleotide, polypeptide, or other agent is purified and / or isolated. Specifically, as used herein, an "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein is 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. Purity is measured by any appropriate 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 that flank it in its native state. A purified or isolated polypeptide is free of the amino acids and sequences that flank it in its native state. "Purified" also defines a degree of sterility that is safe for administration to human subjects, e.g., lacking infectious or toxic agents.

[0015] In embodiments, the agent comprises cobimetinib or a pharmaceutically acceptable salt thereof. In other embodiments, the agent comprises ruboxistaurin or a pharmaceutically acceptable salt thereof. In other embodiments, the agent comprises enzastaurin or a pharmaceutically acceptable salt thereof. In other contemplated embodiments, the agent comprises sotrastaurin or a pharmaceutically 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 includes 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. Ultimately, the appropriate amount and dosage regimen will be determined by the attending physician or veterinarian.

[0018] In some cases, the active substance is administered at least once a day, at least once a week, or at least once a month.The active substance can be suitably administered for a period of 1 day, 1 week, 1 month, 2 months, 3 months, 6 months, 9 months, or 1 year.In some cases, the active substance is administered every day, for example, every 24 hours.Alternatively, the active substance is administered continuously or several times a day, for example, every 1 hour, every 2 hours, every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 7 hours, every 8 hours, every 9 hours, every 10 hours, every 11 hours, or every 12 hours.

[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 routes of administration are available, for example, agents are administered topically, orally, by inhalation, or by injection.

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

[0022] In some aspects, a subject having or at risk of developing a connective tissue disorder, such as an vasculopathy (e.g., vEDS in some embodiments), has a different level of ERK or PKC protein or mRNA than a normal control. In some embodiments, a test sample obtained from a subject contains a different level of ERK or PKC protein or mRNA than a normal control. For example, 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%, 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 than 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 higher 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 for comparison with a test sample, usually a known reference. For example, a test sample can be collected from a test subject, for example, a subject with or in need of diagnosis of 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) without a disease (negative or normal control), or a subject (or subjects) with 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 control values. 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., a protein or mRNA), the term "normal amount" refers to the normal amount of the compound in individuals without a connective tissue disorder such as a vasculopathy (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-defining thresholds to define cutoff points and outliers (e.g., for a particular vEDS or its symptoms). Normal control levels refer to levels of one or more compounds or composite compounds typically found in subjects known not to suffer from 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 an indexing formula. 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, cutoff level, or threshold level, or may be increased or decreased compared to the control level, cutoff 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 the control level. In various embodiments, the increased level can be at least or about a 5% increase, at least or about a 10% 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 an 80% or about a 80% increase, at least an 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 the 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 percent decrease below the 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 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 or about 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 an 80% or about a 80% decrease, at least an 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 comprises blood, serum, plasma, saliva, tears, vitreous, cerebrospinal fluid, sweat, cerebrospinal fluid, or urine.

[0030] Also provided herein are methods for treating a connective tissue disorder in a subject. The method includes administering an effective amount of an agent that reduces the activity or expression of extracellular signal-regulated 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 Ehlers-Danlos syndrome; Ehlers-Danlos syndrome; eosinophilic fasciitis; epidermolysis bullosa (EB); Legg-Calvé-Perthes disease; Marfan syndrome; melorheostosis; melorheostosis with bone poikilosis; 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 stapedial fixation; Ribbing's disease; scleroderma; temporomandibular joint ankylosis ankyloses); 20-nail dystrophy; vascular Ehlers-Danlos syndrome; Weill-Marchesani syndrome; and Worth autosomal dominant osteosclerosis. In some embodiments, the connective tissue disorder comprises an angiopathy.

[0031] In addition to treating Marfan syndrome itself, the present invention also provides methods for 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 contractural 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 pharmaceutically acceptable salt thereof. In other embodiments, the agent comprises ruboxistaurin or a pharmaceutically acceptable salt thereof. In other embodiments, the agent comprises enzastaurin or a pharmaceutically acceptable salt thereof. In other contemplated embodiments, the agent comprises sotrastaurin or a pharmaceutically 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 several 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 a fragment thereof, 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] Also 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 a connective tissue disorder (e.g., Marfan syndrome) is contemplated. In several embodiments, the method comprises administering an effective amount of an agent, wherein the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof. In some embodiments, therapeutically effective amounts of one or more agents are simultaneously 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 decreases 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 decreases 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 decreases 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 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.

[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 old 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 that leads 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 that leads 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 pharmaceutically acceptable salt thereof.

[0047] In certain embodiments, the agent reduces PKC protein or mRNA levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 5-50%, 50-75%, 75-100%, a 1 / 2 fold reduction, a 3 / 4 fold reduction, or a 5 / 5 fold reduction 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 extracellular signal-regulated kinase (ERK) protein or mRNA levels by at least about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99%, 100%, 5-50%, 50-75%, 75-100%, a 1 / 2 fold reduction, a 3 / 4 fold reduction, or a 5 / 5 fold reduction 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 several embodiments, the agent for treating connective tissue disorders (e.g., Marfan syndrome) comprises enzastaurin, or a pharmaceutically acceptable salt thereof. In other embodiments, the agent for treating connective tissue disorders (e.g., Marfan syndrome) comprises sotrastaurin, or a pharmaceutically acceptable salt thereof. In other contemplated examples, the agent for treating connective tissue disorders (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 that are commonly understood by those skilled in the art to which this disclosure belongs.The following references provide those skilled in the art with 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 HarperCollins 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 containing at least one therapeutically or biologically active agent and suitable for administration to a patient. Any of these formulations can be prepared by methods well known and accepted in the art. See, for example, 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 extracellular molecules and activates signal transduction pathways and ultimately cellular responses within the cell. GPCRs are called seven-transmembrane receptors because they cross the cell membrane seven times.

[0053] "Agonist" refers to a chemical substance that binds to a receptor and activates the receptor, resulting in 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, 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, 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 an acid or base salt of the agent used herein. Illustrative but non-limiting examples of acceptable salts include mineral acid salts (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, etc.), organic acid salts (acetic acid, propionic acid, glutamic acid, citric acid, etc.), and quaternary ammonium salts (methyl iodide, ethyl iodide, etc.).

[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 active response.

[0056] "Wild-type" or "WT" refers to the phenotype typical 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 non-standard "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 modes include, but are not limited to, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

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

[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 occurring 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 active substances (e.g., any of the active substances described herein having biological activity) are administered in parallel, their administration occurs within a desired time period. The administration of the active substances can begin and end on the same day. The administration of one active substance can also precede the administration of the second active substance by days, as long as both active substances are taken at least once on the same day. Similarly, the administration of one active substance can extend beyond the administration of the second active substance, as long as both active substances are taken at least once on the same day. The biologically active agents / active substances 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, the dosage level of the agent will correspond to that administered during the first administration period, but may be increased or decreased as medically necessary.

[0062] As used herein, "alteration" also includes a 2-fold or more, e.g., 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more, change in the expression level or activity of a gene or polypeptide.

[0063] As defined herein, the terms "inhibition," "inhibit," "inhibiting," and the like, with respect to a protein inhibitor (e.g., an ERK or PKC inhibitor, or a 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 disorder (e.g., a connective tissue disorder). Similarly, an "inhibitor" is a compound or protein that inhibits a target by binding, partially or completely blocking, reducing, preventing, delaying, inactivating, desensitizing, or downregulating activity.

[0064] By "ameliorate" is meant to decrease, inhibit, alleviate, relieve, 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 binding to a receptor expressed on a cell.

[0067] In this disclosure, the terms "comprises," "including," "containing," "having," and the like can have the meanings 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 meanings 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 basic or novel characteristics of what is recited are not altered by the presence of more than is recited.

[0068] "Effective amount" means the amount required to improve the symptoms of a disease compared to untreated patients. The effective amount of the active compound used to practice the present invention for the 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 various factors, such as whether the mammal is suffering from another disease and its route of administration; the recipient's size, age, sex, health, weight, body mass index, and diet; the nature and extent of symptoms of the disease being treated, the type of concurrent treatment, complications from the disease being treated, or other health-related problems. 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 effective 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, the dosage for humans can be formulated to achieve a concentration that has been found to be effective in animals.The dosage in humans can be adjusted by monitoring the effectiveness of the active substance and adjusting the dosage upward or downward, as described above.It is well within the ability of those skilled in the art to adjust the dosage to achieve maximum efficacy in humans based on the above-mentioned methods and other methods.

[0072] Dosage can vary depending on the patient's requirements and the therapeutic agent used. The dose administered to a 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 a practitioner. Generally, treatment is initiated with a smaller dosage that is less than the optimal dose of the active substance. Thereafter, the dosage is increased by small increments until the optimal effect is achieved according to the situation. Dosage and interval can be individually adjusted to provide an effective level of the administered active substance for the specific 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 about 0.001 mg / kg to about 0.01 mg / kg of agent, about 0.01 mg / kg to about 0.1 mg / kg of agent, about 0.1 mg / kg to about 1.0 mg / kg of agent, about 1.0 mg / kg to about 5.0 mg / kg of agent, about 5.0 mg / kg to about 10 mg / kg of agent, about 10 mg / kg to about 15 mg / kg of agent, about 15 mg / kg to about 20 mg / kg of agent, about 20 mg / kg to about 25 mg / kg of agent, about 25 mg / kg to about 30 mg / kg of agent, about 30 mg / kg to about 35 mg / kg of agent, about 35 mg / kg to about 40 mg / kg of agent, mg / kg, 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 present disclosure includes compositions comprising an effective dose of an agent of the present disclosure, wherein the agent may 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 otherwise 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%, or from about 7.0% to about 8.0% of the composition. %, 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 can be affected by the indicated disorder. In some aspects, the subject is a mammal, and in some aspects, the subject is 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 comprise a bodily fluid. In some embodiments, the bodily fluid includes, but is not limited to, whole blood, plasma, serum, lymph, breast milk, saliva, mucus, semen, cell extract, 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 comprises blood or a fraction thereof (e.g., plasma, serum, or a fraction obtained by leukapheresis).

[0079] Ranges provided herein are understood to be shorthand for all values within that range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange 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, as well as all intervening decimal values between the aforementioned integers, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from either 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 the laboratory method of genetic recombination (e.g., molecular cloning) to combine genetic material from multiple sources to create a sequence not otherwise found in an organism.

[0081] As used herein, the term "expression" or "expressing" in reference 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 in reference 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 value. 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 the structure of a parent compound (e.g., a compound disclosed herein), whose structure is sufficiently similar to that disclosed herein that it would be expected by one of ordinary skill in the art to exhibit the same or similar activity and utility as the claimed compound based on that similarity, or to induce the same or similar activity and utility as the claimed compound as a precursor. 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 accordingly, 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 the preferred embodiments thereof, and from the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated by reference. Genbank and NCBI submissions identified 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, will control. Additionally, the materials, methods, and examples are illustrative only 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; 10. A method of treating vasculopathy in a subject, comprising: [The present invention 1002] 1001. The method of claim 1001, wherein the vasculopathy comprises vascular Ehlers-Danlos syndrome (vEDS). [The present invention 1003] 1001. The method of claim 1001, 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 claim 1003, wherein said agent comprises a small molecule. [The present invention 1005] 1001. The method of claim 1001, wherein said agent comprises cobimetinib or a pharmaceutically acceptable salt thereof. [The present invention 1006] 1004. The method of claim 1003, wherein said agent comprises ruboxistaurin or a pharmaceutically acceptable salt thereof. [The present invention 1007] 1004. The method of claim 1003, wherein said agent comprises enzastaurin or a pharmaceutically acceptable salt thereof. [The present invention 1008] 1004. The method of claim 1003, wherein said agent comprises sotrastaurin or a pharmaceutically acceptable salt thereof. [The present invention 1009] 1001. The method of claim 1001, further comprising the step of administering an agent that decreases the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3). [The present invention 1010] 1001. 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] 1001. The method of claim 1001, wherein said subject comprises a level of ERK or PKC protein or mRNA that differs from a normal control. [The present invention 1012] 1011. 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 1011, 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 activity or PKC activity compared to a normal control. [The present invention 1014] The method of claim 1011, wherein said level is the level in a test sample obtained from said subject. [The present invention 1015] 1015. The method of claim 1014, wherein the test sample comprises blood, serum, plasma, saliva, tears, vitreous, cerebrospinal fluid, sweat, 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; 10. A method of treating a connective tissue disorder in a subject, comprising: [The present invention 1017] 1016. 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 claim 1019, wherein said agent comprises a small molecule. [The present invention 1021] The method of claim 1019, wherein said agent comprises cobimetinib or a pharmaceutically acceptable salt thereof. [The present invention 1022] 1019. The method of claim 1019, wherein said agent comprises enzastaurin or a pharmaceutically acceptable salt thereof. [The present invention 1023] 1019. The method of claim 1019, wherein said agent comprises sotrastaurin or a pharmaceutically acceptable salt thereof. [The present invention 1024] 1019. The method of claim 1019, wherein said agent comprises ruboxistaurin or a pharmaceutically 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] 1026. The pharmaceutical composition of claim 1026, 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 1028] A kit comprising (1) the pharmaceutical composition of the present invention 1026 or 1027, and (2) instructions for treating 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. 10. A method for treating a connective tissue disorder, comprising: [The present invention 1030] 1029. The method of claim 1029, wherein said connective tissue disorder comprises Marfan syndrome. [The present invention 1031] 1029. 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 claim 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, 3 / 4, or 5 / 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 5-fold compared to a normal control. [The present invention 1034] 1029. The method of claim 1029, wherein said agent comprises enzastaurin or a pharmaceutically acceptable salt thereof. [This invention 1035] 1029. The method of claim 1029, wherein said agent comprises sotrastaurin or a pharmaceutically acceptable salt thereof. [The present invention 1036] 1029. The method of claim 1029, wherein said agent comprises ruboxistaurin or a pharmaceutically acceptable salt thereof. [Brief explanation of the drawings]

[0089] [Figure 1A]Figure 11 is a graph demonstrating that the G209S / + mouse model recapitulates the vascular Ehlers-Danlos syndrome (vEDS) phenotype. % survival is shown relative to age in days. Median survival was 400 days; p<0.0001. [Figure 1B]

[0023] Figure 1 is a graph demonstrating that the G938D / + mouse model recapitulated the vEDS phenotype. % survival is shown relative to age in days. Median survival was 45 days; p<0.0001. [Figure 1C] 1 shows an image 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. 10 is a graph demonstrating that vEDS samples clustered separately from controls, indicating significant differences in the transcriptome of vEDS aortas. [Figure 1E] FIG. 10 demonstrates network analysis showing that vEDS aortas displayed a gene expression signature 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] Immunoblotting for markers of active signaling through this pathway demonstrated a signature for elevated GPCR and MAPK signaling. 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 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 98% survival at 45 days of age, 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) was observed with treatment with trametinib (GSK-1120212) 1 mg / kg / day, an FDA-approved inhibitor of MEK, the 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 the autophosphorylation of PKC and ERK in the aortic wall, as assessed by immunoblot of aortic lysates, and that pharmacological inhibition of MEK not only correlated with the expected decrease in phosphorylation of ERK, a downstream substrate of MEK, but also, curiously, reduced PKC phosphorylation, suggesting the existence of a positive feedback loop. Figures 4B and 4C are graphs depicting the quantification of immunoblot images showing that pharmacological inhibition of PKCβ prevented the autophosphorylation of PKC and ERK in the aortic wall, and that pharmacological inhibition of MEK not only correlated with the expected decrease in phosphorylation of ERK, a downstream substrate of MEK, but also, curiously, reduced PKC phosphorylation, suggesting the existence of a positive feedback loop (*p<0.05, **p<0.01, ***p<0.001). Neither cobimetinib nor ruboxistaurin had any effect on blood pressure. [Figure 5] Figures 5A and 5B are graphs depicting that treating mice with hydralazine (32 mg / kg / d), which blocks the PLC / IP3 / PKC / ERK axis, confers a significant protection of 98% survival at 45 days of age, the median survival time for untreated vEDS mice, and that survival is 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) resulted in a 90% survival rate in male mice compared to only a 24% survival rate in male mice treated with hydralazine alone. Male mice continued to survive after bicalutamide was removed after puberty, suggesting a time-dependent change in androgen sensitivity in this mouse model. [Figure 7]FIG. 10 is a graph depicting that male mice treated with bicalutamide alone achieved a moderate survival of approximately 80%, and no males continued to survive after removing 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] Figures 8A and 8B are graphs depicting that mice treated with spironolactone alone (100 mg / kg / d) showed a moderate survival of approximately 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] Figure 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 delivery in vEDS mice that were still lactating, 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. Figure 10B is a graph depicting that propranolol, a nonspecific β antagonist that lowers blood pressure in vEDS mice, does not improve survival in the first 30 days after delivery in vEDS mice that were still lactating, demonstrating that the significantly elevated risk of death due to aortic dissection in pregnancy is not ameliorated by reducing blood pressure. [Figure 11]Figure 11A is a graph showing that inhibiting angiotensin II signaling by treating mice with the angiotensin receptor antagonist, losartan (60 mg / kg / d), had no effect on survival (Figure 11A). Treatment of mice with the thrombin receptor antagonist, vorapraxar (1 mg / kg / d), was also found to have no effect on survival (Figure 11B). [Figure 12] FIG. 10 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] Figures 13A-13C are graphs depicting treatment of mice with the nonspecific β antagonist, propranolol (80 mg / kg / d) (Figure 13A), the specific β antagonist, atenolol (120 mg / kg / d) (Figure 13B), and the β antagonist / β agonist, celiprolol (200 mg / kg / d) (Figure 13C), and showed that none of these treatments resulted in improved survival in our vEDS mouse model, despite a reduction in blood pressure. [Figure 14] 1 is a graph depicting celiprolol accelerating the risk of aortic dissection in a mouse model; vEDS mutants with Col3a1 G209S / + mutation also demonstrated an increased risk of aortic dissection in celiprolol. [Figure 15] 1 is a graph depicting that amlodipine (12 mg / kg / d) also increased the risk of aortic dissection in a mouse model, which was also shown to be consistent with MFS mice. [Figure 16]Figure 16A is a graph demonstrating pharmacological inhibition of PKCβ using a second specific PKCβ inhibitor, enzastaurin. Figure 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). Figure 16B is a graph demonstrating that treatment of mice with the endothelin receptor antagonist, bosentan, improved survival. Figure 16B: Treatment with bosentan resulted in an 80% survival rate 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] 10 is a graph demonstrating that ruboxistaurin treatment reduces aortic root growth in 129 MFS mice. DETAILED DESCRIPTION OF THE INVENTION

[0090] Detailed Description The present invention is based, at least in part, on the identification of novel, targetable signal transduction 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 due to aortic dissection. Furthermore, agents that inhibit activation of the PLC / IP3 / PKC / ERK signaling pathway also prevent death due to aortic dissection. Together, these novel findings provide the first evidence for targetable signal transduction abnormalities that contribute to the pathogenesis of vEDS and potentially other vasculopathies and other connective tissue disorders.

[0091] vasculopathy Vasculopathy is a term used to describe diseases affecting 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, livedo-like 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 primary cause of death in vEDS is arterial dissection and / or rupture, yet little is known about the pathogenesis of this disease. No effective treatment strategies exist for this devastating condition. The current belief is that a decrease in collagen III levels directly leads to the signs and symptoms of vEDS due to an inherent decrease in the structural integrity of tissues. However, early pathogenic models of Marfan syndrome (MFS) also cause significant tissue fragility conferred by failed elastic fiber formation, but subsequent studies have demonstrated that the defective gene product in MFS is responsible for the loss of elastic fibers. 3,4 clearly demonstrated enhanced transforming growth factor-β (TGF-β) signaling in a mouse model lacking fibrillin-1. Follow-up studies continued to demonstrate that TGF-β and downstream cell signaling molecules were key mediators of disease pathology. Furthermore, therapies that attenuate TGF-β signaling and related pathways, such as TGF-β neutralizing antibodies (NAbs), the angiotensin-II (Ang-II) type 1 receptor blocker (ARB) losartan, or the inhibitor of ERK1 / 2 activation RDEA119 / trametinib, can suppress aortic disease in MFS mice. 3-6 .

[0094] However, similar to other inherited vasculopathies such as Marfan syndrome and Loeys-Dietz syndrome, signaling abnormalities are identified as key mediators of disease pathology in vEDS. RNA-seq profiling of aortas from 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): Includes TIFF2025118759000002.tif79148.

[0096] In one embodiment, COL3A1 is identified as having the following nucleic acid sequence (NCBI Accession No: NM_000090.3 (SEQ ID NO: 2), which is incorporated by reference in its entirety): TIFF2025118759000003.tif143149TIFF2025118759000004.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 the DNA in the cell's nucleus.

[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 leads to 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 neighboring 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): Includes TIFF2025118759000005.tif27146.

[0102] In one embodiment, ERK1 is represented by the following nucleotide sequence (NCBI Accession No: X60188.1 (SEQ ID NO: 4), herein incorporated by reference in its entirety): Contains TIFF2025118759000006.tif126148, coding region is 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): Includes TIFF2025118759000007.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): TIFF2025118759000008.tif102148, with the coding sequence in 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, the method 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), and 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, the method 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, or a MEK inhibitor such as AZD6244 (selumetinib), PD0325901, GSK1120212 (trametinib), U0126-EtOH, PD184352, RDEA119 (rafametinib), PD98059, BIX 02189, MEK162 (binimetinib), or 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, MEK inhibitors, or pharmaceutically acceptable salts thereof, are 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 regulating the function of other proteins through phosphorylation of the hydroxyl groups of serine and threonine amino acid residues on these proteins. PKC enzymes then phosphorylate the hydroxyl groups of diacylglycerol (DAG) or calcium ions (Ca 2+ PKC enzymes are activated by signals such as an increase in the concentration of PKC-like proteins. 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 novel (n)PKCs, including the δ, ε, η, and θ isoforms, require DAG for activation but do not require Ca. 2+ Therefore, conventional and novel PKCs are activated through the same signaling pathways as phospholipase C. On the other hand, atypical (α) PKCs (including protein kinase Mζ and ι / λ isoforms) 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 embodiments, the PKC inhibitor comprises enzastaurin. In embodiments, the PKC inhibitor comprises ruboxistaurin. In embodiments, PKC agonists, or pharmaceutically acceptable salts thereof, are also contemplated herein.

[0111] In some embodiments, the PKC inhibitor is TIFF2025118759000009.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, having 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, having 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; where each R at each occurrence 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 nitrogen ring members.

[0113] In some embodiments, the PKC inhibitor is TIFF2025118759000010.tif34128, R 1 and R 2 is as described above.

[0114] In some embodiments, the PKC inhibitor is TIFF2025118759000011.tif34128, R 1 and R 2 is as described above.

[0115] In some embodiments, the PKC inhibitor is TIFF2025118759000012.tif34128, and 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 are 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 TIFF2025118759000013.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. In some embodiments, R 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 with 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, TIFF2025118759000014.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-C 10 or phenyl), or substituted or unsubstituted heteroaryl (e.g., 5-10, 5-9, or 5-6 members and having one or more N, O, or S ring members). A , R B , R C , and R D is written as above.

[0121] In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF2025118759000015.tif20128, and these are then combined into the R 3 may be substituted with one or more of:

[0122] In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF2025118759000016.tif14128, and R 3 is written as above.

[0123] In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF2025118759000017.tif9128, and these are then combined into the R 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. In some embodiments, R 1 and R 2 are linked together to form a substituted C5-C6 cycloalkylene linker, e.g., which is one or more 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., which can be represented by the 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 TIFF2025118759000018.tif19128, and these are then combined into the R 3 In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF2025118759000019.tif19128, and these are then combined into the R 3In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF2025118759000020.tif17128, and these 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 TIFF2025118759000021.tif20128, and these are then combined into the R 3 In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF2025118759000022.tif19128, and these are then combined into the R 3 In some embodiments, R 1 and R 2 teeth, These are concatenated together to form TIFF2025118759000023.tif19128, and these 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 are 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, TIFF2025118759000024.tif17128, and these are the R files mentioned above. 3 In some embodiments, R 1 teeth, The file is TIFF2025118759000025.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, with other than hydrogen, with one or more suitable groups (which may be the same or different), 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)carboxamido, mono- and di-(C1-C8 alkyl)sulfonamido, alkylsulfinyl (e.g., C1-C8 alkylsulfinyl), alkylsulfonyl (e.g., C1-C8 alkylsulfonyl), aryl (e.g., phenyl), arylalkyl (e.g., (C6-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: TIFF2025118759000026.tif172167 can be cited.

[0133] In certain embodiments, a protein kinase inhibitor or monoclonal antibody that inhibits a protein kinase or 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 a mixture 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, the 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): Includes TIFF2025118759000027.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): TIFF2025118759000028.tif31146TIFF2025118759000029.tif243148TIFF2025118759000030.tif243148TIFF2025118759000031.tif67148 sequences, with the coding sequence in 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 embodiments, the PLC inhibitor includes U-73122, U73343, and ET-18-OCH3. In embodiments, the IP3 inhibitor includes 2-APB and xestospondin C. In embodiments, the agent includes hydralazine. It is contemplated that the inhibitors can be used alone or in any combination. In embodiments, PLC or IP3 inhibitors, or pharmaceutically acceptable salts thereof, are also contemplated herein.

[0138] In certain embodiments, an agent that decreases PLC or IP3 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. In other embodiments, it is contemplated that the agent will be administered at a higher concentration, 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 androgen receptors (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 such as cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendolone, and osaterone acetate (veterinary medicine), and non-steroidal antiandrogens such as flutamide, bicalutamide, nilutamide, topirutamide, enzalutamide, and apalutamide. In addition to cyproterone acetate and chlormadinone acetate, a few other progestins used in oral contraceptives and / or menopausal HRT, including dienogest, drospirenone, medrogestone, nomegestrol acetate, promegestone, and trimegestone, also have varying degrees of AR antagonist activity.

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

[0142] Antigonadotropin: A drug that inhibits 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), 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. Examples include estrogens, especially oral and synthetic (e.g., ethinylestradiol, diethylstilbestrol), which stimulate hepatic sex hormone-binding globulin (SHBG) production, 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 androgen levels 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 tissue 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. These tissue and / or organ diseases may be primary diseases or may be caused by pre-existing diseases and / or illnesses. Examples include amyloidosis, atrial fibrillation, convulsion, cramp, dermatomyositis, enchondroma, fibroma, lumbar pain, 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 dystrophy, disuse atrophy, denervation atrophy, Duchenne muscular dystrophy, facioscapulohumoral muscular dystrophy), liver disease, and 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, sensory organs (e.g., organs of taste, smell, sight, hearing), 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, seminal vesicles, ejaculatory ducts, penis, prostate, bulbourethral glands), female reproductive organs (e.g., ovaries, fallopian tubes, uterus, vagina, clitoris, Bartholin's glands, external genitalia, breasts)), endocrine glands (e.g., thyroid gland, parathyroid gland, thymus, pituitary gland, pineal gland, chromaffin and cortical systems), and 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), nervous (e.g., neurons (e.g., motor neurons, interneurons, sensory neurons), glia, spinal cord, nerves, brain).

[0146] In 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 specific gene affected determines the specific 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 defects in collagen structure or processing. The diagnosis can be confirmed with genetic testing or a skin biopsy. People may be misdiagnosed with 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. While some disorders result in a normal life expectancy, disorders that affect the blood vessels generally result in a shorter life expectancy. EDS affects approximately 1 in 5,000 people worldwide, and the 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 muscle and / or bone pain. The mutations that cause this type of EDS are unknown. Less skin involvement is seen than in other types. Genetic testing is not available for this type.

[0151] Classic EDS (type 1 cEDS) is associated with very elastic (stretchy), smooth skin that is fragile and bleeds easily; extensive, atrophic scars (flat or depressed scars); and joint hypermobility. Molluscum pseudotumors (calcified hematomas in pressure areas such as the elbows) and spheroid bodies (fat-containing cysts on the forearms and shins) are also frequently seen. Hypotonia and motor developmental delays may occur. Mutations that cause this type of EDS are in the genes COL5A1, COL5A2, and COL1A1. It involves the skin more often 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 thinning hair. They also have distinctive facial features, including large eyes, a small chin, sunken cheeks, a thin nose and lips, and ears without earlobes. Joint hypermobility is present but is generally limited to the small joints (fingers, toes). Other common features include clubfoot, tendon and / or muscle rupture, acroprogeria (premature aging of the skin on the hands and feet), early-onset varicose veins, pneumothorax (collapsed lungs), gum recession, and reduced subcutaneous fat mass. It may 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 individuals 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, slender 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 arthrochastic 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 curvature and scoliosis), and mild osteopenia. Type I collagen is usually affected. It is very rare, with approximately 30 reported cases. 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; loose, 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 keratoconus or keratoconus, and blue sclera. Classical symptoms, such as hypermobile joints and hyperelastic skin, are also often present.

[0157] Classical EDS (cEDS type 1) is characterized by hyperextensible skin 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] Myocontractile EDS (mcEDS) is characterized by congenital multiple joint contractures, characteristically adduction-flexion contractures and / or equinovarus (clubfoot), evident at birth or early infancy, characteristic craniofacial features, and cutaneous features such as skin hyperextensibility, easy bleeding, skin fragility 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, attached gingival defects, anterior tibial plaques, 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 method The present disclosure provides methods for treating vasopathy (e.g., vEDS) or connective tissue disorders in a subject in need thereof by administering a therapeutically effective amount of an agent to the subject, wherein the agent reduces the activity or expression of extracellular signal-regulated kinase (ERK) or protein kinase C (PKC). The method further comprises administering an agent that reduces the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3). In embodiments, the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination, for the preparation of a medicament useful for the treatment of vasopathy (e.g., vEDS) or connective tissue disorders.

[0164] The present disclosure also provides methods that include 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 pharmaceutically acceptable salt thereof with at least one additional active agent, also referred to herein as an "active pharmaceutical ingredient" ("API"), as part of a treatment regimen intended to provide beneficial effects from the co-action of the agent (e.g., an agonist, antagonist, or inhibitor) and the additional active agent.

[0165] According to the embodiments described below, an "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 decreases 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 a 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 separate monotherapy regimens that fortuitously and arbitrarily result in an unintended or unexpected beneficial effect.

[0166] Preferably, administration of a composition comprising an agent (e.g., an agent that decreases 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 involving combination therapy for the treatment of vasopathies (e.g., vEDS) or connective tissue disorders. As used herein, "combination therapy" or "cotherapy" includes the administration of a compound described herein with at least one additional agent, as disclosed herein, as part of a specific treatment regimen intended to provide a beneficial effect from the synergistic action of these therapeutic compounds. The at least one additional agent may be a therapeutic or non-therapeutic agent. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic coaction resulting from the combination of therapeutic compounds. The beneficial effect of the combination may 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 some 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 active substances, or may be sequential to the administration of one or more additional active substances.In another aspect, the administration of different components of combination therapy may be at different frequencies.One or more additional active substances 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 simultaneous 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 containing the compound of the present disclosure.When the additional active substance is administered separately from the compound of the present disclosure, it can be administered by the same or different administration route as the compound of the present disclosure.

[0172] Preferably, administration of a composition comprising the active substance of the present disclosure in combination with one or more additional active substances provides a synergistic response in subjects with the disorders, diseases, or conditions 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 for the use of a lower dosage and / or less frequent administration of at least one active substance in the combination compared to its dose and / or frequency outside the combination. The synergistic effect may be manifested in the avoidance or reduction of adverse or unwanted side effects associated with the use of either therapy alone in the combination.

[0173] "Combination therapy" also encompasses the administration of a compound of the present disclosure 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 administered at any suitable time, as long as the beneficial effect from the synergistic action of the combination of the therapeutic compound and the non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect can still be achieved when the non-drug treatment is separated in time from the administration of the therapeutic compound, perhaps by several days or even weeks.

[0174] In embodiments of the methods described herein, an agent (e.g., an agent that reduces the activity or expression of ERK, PKC, and / or PLC or IP3) can be administered alone or in combination with at least one additional agent in a method for treating vasopathy (e.g., vEDS) or connective tissue disorders. In several 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 several embodiments, the at least one additional agent is a therapeutic agent. In several embodiments, the therapeutic agent is indicated for the treatment of vasopathy (e.g., vEDS) or connective tissue disorders. In another aspect, the agent is administered in combination with at least one additional agent that is not intended for the treatment of vasopathy (e.g., vEDS) or connective tissue disorders, 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, wherein 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 (for example, 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 a disease, disorder, or condition, ameliorate its symptoms, reduce its severity, or reduce its duration, or enhance or improve the therapeutic effect of another therapy, or prevent the identified 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 precise effective amount for a subject will depend on the subject's weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration.

[0179] An effective amount of the agent can be administered once daily, two to five times daily, up to two or up to three times daily, or up to eight times daily. In embodiments, the agent is administered three times daily, twice daily, once daily, 14 days on (four times daily, three times daily, or twice daily, or once daily) and 7 days off in a three-week cycle, up to five or seven days on (four times daily, three times daily, or twice daily, or once daily) 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 decreases the activity or expression of ERK, PKC, and / or PLC or IP3) can be in the range of about 0.001 mg / kg to about 1000 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.1 mg / kg to about 10 mg / kg; or any range 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 depending on the disease being treated, the route of administration, excipient use, and the possibility of co-use with other therapeutic treatments, such as the use of other agents, as will be recognized by those skilled in the art.

[0181] In more specific aspects, an agent of the disclosure (e.g., an agent that decreases the activity or expression of ERK, PKC, and / or PLC or IP3) is 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, 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 who has a disease, disorder, or condition, or a subject who has 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 who has vasopathy (e.g., vEDS) or connective tissue disorder, or a subject who has an increased risk of developing vasopathy (e.g., vEDS) or connective tissue disorder compared to the entire population.The subject in need thereof may be a subject who 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 therapy that is not clinically appropriate 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 fowl, 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 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 an 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, in which a combination of multiple active compounds is administered, preferably with each component of the combination present in a therapeutically effective amount. In one aspect, monotherapy using an 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 agents 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 a process in 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 preferred embodiments, 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 that something in the body is not normal. Symptoms are felt or noticed by the individual experiencing the symptoms, but may not be readily apparent to others, which are defined as non-healthcare professionals.

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

[0190] As used herein, the term "selectively" means that a phenomenon tends to occur more frequently in one population than in another. The populations being compared may be cell populations. Preferably, the agent of the present disclosure selectively acts on hyperproliferative cells but does not act on normal cells. The agent of the present disclosure selectively acts 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 the 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 antiandrogen compound, for example, bicalutamide or spironolactone.

[0192] As used herein, "combination therapy" or "cotherapy" includes the administration of a therapeutically effective amount of an 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-action resulting from the combination of therapeutically active compounds. "Combination therapy" is not intended to encompass the administration of two or more therapeutic compounds as part of separate monotherapy regimens that accidentally and arbitrarily result in an unintended or unexpected beneficial effect.

[0193] Preferably, the combination therapy 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.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 dose 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 encompasses 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 administered at any suitable time, as long as the beneficial effect from the synergistic action of the combination of the therapeutic compound and the non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect can still be achieved when the non-drug treatment is separated in time from the administration of the therapeutic compound, perhaps by several 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 an agent administered to a subject is a therapeutically effective amount. The term "therapeutically effective amount" refers to an amount sufficient to treat the disease being treated (e.g., vEDS), ameliorate the symptoms, reduce the severity, or reduce the duration thereof, or to enhance or improve the therapeutic effect of another therapy, or to exhibit a detectable therapeutic effect in the subject. In one embodiment, a therapeutically effective amount of an 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 certain embodiments, the present invention provides pharmaceutical compositions comprising an agent used in the present invention (e.g., an agent that decreases 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 an agent described herein in a pharmaceutically acceptable form suitable for administration to a subject. As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, carriers, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals, within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0199] Such compositions typically include an active ingredient and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the composition.

[0200] As used herein, the term "pharmaceutically acceptable salt" refers to a salt formed, for example, from acidic and basic groups of an agent 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, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate salts (e.g., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)).

[0201] Pharmaceutical compositions are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent, e.g., water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, e.g., benzyl alcohol or methylparaben; an antioxidant, e.g., ascorbic acid or sodium bisulfite; a chelating agent, e.g., ethylenediaminetetraacetic acid; a buffer, e.g., acetate, citrate, or phosphate, and a tonicity adjuster, 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 syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and 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, for example, sugars, polyalcohols such as mannitol and sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0203] Sterile injectable solution can be prepared by mixing active compound in the required amount in selected solvent with one or combination of the components listed above, and then optionally sterilize by filtration.Generally, dispersion is prepared by mixing active compound into sterile vehicle that contains basic dispersion medium and other components required from the components listed above.For the sterile powder that is used to prepare sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying, which produces powder from the solution that active compound and any other desired components are added to sterilized by filtration before.

[0204] Oral compositions generally contain an inert diluent or an edible carrier. For oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, troches, or capsules, such as 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 present invention can also be formulated as nanoparticle formulations. The compounds of the present invention can be administered for immediate-release, delayed-release, modified-release, sustained-release, pulsed-release, and / or controlled-release applications. Pharmaceutical compositions of the present invention can contain 0.01 to 99% by weight of the active ingredient. 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 via transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, surfactants, 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 effectiveness of such compounds can be 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 a 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 uninfected cells and thereby reduce 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. The dosage of such compounds can be administered with little or no toxicity. 50 Preferably, the circulating concentration range includes the IC50 / IC100 / IC200 / IC100 / IC200 / IC100 / IC200 / IC200 / IC200 / IC300 / IC400 / IC500 / IC600 / IC700 / IC800 / IC9 ... 50 The test compound may be formulated to achieve a circulating plasma concentration range that includes the target dose (i.e., the concentration of the test compound that achieves a 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 the 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 also depends on the age and overall physiological condition of the patient and the route of administration. In some embodiments, the therapeutic dose is generally between about 10 and 2000 mg / day, preferably between about 30 and 1500 mg / day. Other ranges, including, for example, 50-500 mg / day, 50-300 mg / day, and 100-200 mg / day, can also be used.

[0212] Administration can be once a day, twice a day, or more frequently, and can 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.Dose and administration frequency depend on the clinical signs that confirm the maintenance of remission, which are accompanied by 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 treatment, the overall health and / or age of the subject, and other existing diseases, can affect the dosage and timing required to effectively treat the subject.In addition, treatment of a subject with a therapeutically effective amount of an active substance 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. Appropriate amounts of the 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 at 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. [Example]

[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 model recapitulates the human vascular phenotype, and the molecular basis for the eventual failure of the vascular wall was elucidated through the use of environmental influences on the disease phenotype.

[0217] To introduce each mutation, three guide RNAs (tracrRNA + crRNA) were designed to target the DNA region flanking the intended mutation site. DNA oligo repair templates (ssDNA oligos) were also designed, containing homologous sequences upstream and downstream of the target region and the intended mutation. Pronuclear injection of single-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 mutation site was used to confirm introduction of the mutation, and mice were backcrossed for at least four generations to rule out 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 the 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 most often exhibited hemothorax or intraperitoneal bleeding at autopsy. Mice with the Col3a1 G938D / + mutation exhibited a more severe phenotype (median survival = 45 days versus 400 days for the Col3a1 G209S / + model, p<0.0001; Figures 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. Figure 1B shows the survival of the G938D / + mouse model, which recapitulated 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 signal transduction abnormalities may be a primary mediator of disease pathology in vEDS. To test this hypothesis, given that the proximal descending aorta is the most common site of aortic dissection in this model, we performed RNA-seq 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 1E ).

[0222] Furthermore, upstream analysis predicted that transcriptional differences in vEDS aorta 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 (Figure 1G).

[0224] In contrast to Marfan syndrome (MFS) or Loeys-Dietz syndrome (LDS), expression profiling of vEDS aortas did not reveal the synthetic repertoire typical of elevated TGFβ signaling, but evidence of increased ERK activation (similar to MFS and LDS) was observed. Small human studies suggest that the beta-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 assess modulation of phenotypic severity, the vEDS mutation was introduced onto the inbred 129 and BL6 backgrounds. Both mutations are associated with early death due to aortic dissection on the BL6 background. Remarkably, the 129 background provides complete protection from dissection for both vEDS genotypes, with a completely normal lifespan. Rescue is associated with normalization of the gene expression profile of the aortic wall. These data provide the rationale and motivation for conducting genetic studies to identify the source and mechanism of alterations in vEDS mice, with the hope and intention of mimicking nature's successful strategies 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 the role of ERK activation in the pathogenesis of vEDS and as a way to identify therapeutic strategies for vEDS. Pharmacological ERK antagonists are FDA-approved inhibitors of MEK, the kinase that activates ERK. 8 Mice were treated with cobimetinib [GDC-0973, RO5514041] (2 mg / kg / day). We hypothesized that if ERK activation truly drives disease risk, 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 a 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 Mice were treated with ruboxistaurin [LY 333531] (10 mg / kg / day). We hypothesized that if ERK activation and consequent disease risk are driven by PLC / IP3 / PKC activation, a pharmacological PKC inhibitor would also rescue mortality risk from aortic dissection.

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

[0232] Agents that inhibit the PLC / IP3 / PKC / ERK signaling cascade increased survival Further studies were conducted to test other drugs that inhibit this signaling cascade. Mice were treated with hydralazine (32 mg / kg / d), which blocks the PLC / IP3 / PKC / ERK axis, hypothesizing that this drug targets the same pathway and thereby provides similar protection. 10 A significant 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 beneficial 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 beneficial.

[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 caused by 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 medium- to large-arteries. 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 aortic root involvement. Vascular rupture in patients with vEDS is difficult to predict or prevent. Pregnancy particularly increases the risk of dissection, resulting in complications in over 50% of pregnancies and death in approximately 12–25% of pregnancies. The prominent postpartum occurrence of vascular dissection is not consistent with a mechanism that significantly increases hemodynamic stress. Instead, we hypothesized that oxytocin, the hormone that initiates uterine contractions and is maintained postpartum and throughout lactation, may contribute to pregnancy-related 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 a vEDS mouse model, we identified that pregnancy and lactation were associated with a 60% mortality rate due to arterial dissection during the first 30 days after birth in vEDS mice (Figure 3A). Furthermore, preventing lactation by removing the pups after birth prevented dissection and death in vEDS mice (100% survival rate, 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 rate) 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] As measured by immunoblotting and ERK target gene expression (Figures 3E and 3F), this increased risk of death correlated with increased ERK activation, whereas protection from aortic dissection correlated with decreased ERK activation. These data further support 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 that 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 The use of both pharmacological MEK / ERK and PKC antagonists rescued the risk of mortality 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 a vEDS mouse model.

[0242] Here, we demonstrated that pharmacological inhibition of PKCβ using a second specific PKCβ inhibitor, enzastaurin (60 mg / kg / d), also rescued the risk of mortality from aortic dissection, with 80% of enzastaurin-treated vEDS mice surviving 40 days after treatment compared with 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] Because activation of the PLC / IP3 / PKC / ERK signaling pathway was identified herein as pathogenic in vEDS, we investigated the identity of the receptors that may activate this abnormal signaling pathway. GPCR (Gq) signals through this pathway. Common Gq receptors in the aorta include angiotensin II receptor, thrombin receptor, endothelin-1 receptor, vasopressin receptor 1, sphingosine-1-phosphate receptor, alpha-1 adrenergic receptor, and serotonin receptor. However, there are also orphan GPCRs, such as GPR56, which has been shown to interact with collagen3,4, which are also expressed in the aorta.

[0244] Without wishing to be bound by theory, we hypothesized that inhibiting abnormally activated receptors would allow us to pinpoint exactly how signaling pathways are activated. Furthermore, we treated mice with bosentan (100 mg / kg / d), an orally bioavailable, nonspecific endothelin receptor antagonist. Treatment with bosentan resulted in an 80% survival rate after 40 days of treatment, compared with only a 50% survival rate in untreated vEDS mice (p = 0.0298, Figure 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 beneficial in reducing aortic disease in a vEDS mouse model. Next, we sought to identify evidence that this signaling pathway is elevated in vascular tissue samples from human patients with vEDS. Both PKC phosphorylation (Figure 17) and ERK1 / 2 phosphorylation (Figure 18) were identified in two tissue samples (iliac artery and descending thoracic aorta) from patients with vEDS. Neither of these phosphorylated proteins was observed 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 pharmacological inhibition of ERK activators, MEK, or PKC, or through 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 TIFF2025118759000032.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 and 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 decrease in ERK phosphorylation, a downstream substrate of MEK, but also, curiously, 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, was significantly 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 almost exclusively seen in male mice (Figures 2A and 2B), and we therefore hypothesized that treatment with an androgen antagonist might be beneficial 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 with only a 24% survival rate in male mice treated with hydralazine alone (Figure 6).

[0252] Curiously, when bicalutamide was removed after puberty (at 90 days of age), male mice continued to survive at the same rate while remaining on hydralazine (Figure 6), suggesting a time-dependence of androgen sensitivity in this mouse model. Interestingly, male mice treated with bicalutamide alone achieved a moderate survival of approximately 80%, and no males continued to survive after bicalutamide was removed after puberty (Figure 7), suggesting that inhibition of androgen signaling alone was not sufficient to prevent aortic disease in this 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 the risk of aortic dissection 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 could similarly be used as a direct androgen antagonist in this disorder.

[0254] Mice treated with spironolactone alone (100 mg / kg / d) (Figure 8A) showed a moderate survival of approximately 80%, 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 one example, a combination therapy involving the administration of hydralazine and ruboxistaurin is contemplated. Ruboxistaurin is a protein kinase C-β (PKC-β) inhibitor and a macrocyclic bisindolylmaleimide compound under development by Eli Lilly, with 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 trademark 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. TIFF2025118759000033.tif39128

[0257] Combination therapy of hydralazine and enzastaurin for the treatment of vEDS In an example, a combination therapy involving the administration of hydralazine and enzastaurin is contemplated. 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 designations include 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. TIFF2025118759000034.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: TIFF2025118759000035.tif49128

[0261] Combination therapy of hydralazine and antiandrogens for the treatment of vEDS Additional combinations of hydralazine and different antiandrogens are tested.For example, hydralazine and androgen receptor antagonists are contemplated, including steroidal antiandrogens (cyproterone acetate, megestrol acetate, chlormadinone acetate, oxendolone and osaterone acetate).In addition, hydralazine is contemplated in combination with nonsteroidal antiandrogens, such as flutamide, nilutamide, topilutamide, enzalutamide, drospirenone or medrogestone.

[0262] In other examples, the combination therapy including hydralazine and androgen synthesis inhibitor or antigonadotropin is contemplated.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 beneficial. Accordingly, 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 more than did the 32 mg / kg / d dose (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 aortic dissection in the first 30 days after delivery in vEDS mice. Prevention of lactation by removal of pups after birth, treatment with hydralazine (16 mg / kg / d), or treatment with trametinib prevented 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 Additional treatment of mice with oxytocin receptor antagonist 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 death due to aortic dissection in pregnancy was specifically driven by activation of oxytocin receptors during lactation (Figures 10A and 10B).

[0266] Furthermore, we demonstrated that treatment with propranolol (Figure 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% vs. 50% survival at 30 days postpartum). This data further supports that increasing activation of the PLC / IP3 / PKC / ERK signaling pathway resulted in a significantly increased risk of death due to aortic dissection in our vEDS mouse model, and that inhibiting the PLC / IP3 / PKC / ERK signaling pathway ameliorated this risk.

[0267] GPCR activated the PLC / IP3 / PKC / ERK signaling pathway Since activation of the PLC / IP3 / PKC / ERK signaling pathway was identified 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, which are also expressed in the aorta and interact with collagen 3, are also expressed in the aorta. 3,4 There are also orphan GPCRs, such as GPR56, which has been shown to

[0268] We hypothesized that we could identify how signaling pathways might be activated when receptor activity is inhibited. Angiotensin II signaling was first inhibited by treating mice with the angiotensin receptor antagonist, losartan (60 mg / kg / d) (Figure 11A), but this was found to have no effect on survival. Next, we treated mice with the thrombin receptor antagonist, vorapraxal (1 mg / kg / d), but this was also found to have 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 activity, we treated the vEDS mice with the nonspecific tyrosine kinase receptor antagonist, nintedanib (50 mg / kg / d), but found that it also had no effect on survival (Figure 12). This suggests that tyrosine kinase receptor activation did not drive 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 Therefore, we 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 administered the nonspecific β-antagonist, propranolol (80 mg / kg / d), the specific β-antagonist, atenolol (120 mg / kg / d), and the β-antagonist / β-agonist, celiprolol (200 mg / kg / d) to mice. However, we found that none of these treatments, despite reducing blood pressure, resulted in improved survival in our vEDS mouse model (Figures 13A-13C). Celiprolol further accelerated aortic dissection in the mouse model, leading to increased Col3a1 expression. G209S / +vEDS patients with the mutation also demonstrated an increased risk of aortic dissection with celiprolol (Figure 14). Because this was not the case for all beta-antagonists, this observation could be driven by the beta-agonist activity of celiprolol.

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

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

[0274] Combination therapy of hydralazine and antiandrogens for the treatment of vEDS Additional combinations of hydralazine and different antiandrogens are tested.For example, hydralazine and androgen receptor antagonists are contemplated, including steroidal antiandrogens (cyproterone acetate, megestrol acetate, chlormadinone acetate, oxendolone and osaterone acetate).In addition, hydralazine is contemplated in combination with nonsteroidal antiandrogens, such as flutamide, nilutamide, topilutamide, enzalutamide, drospirenone or medrogestone.

[0275] In other examples, the combination therapy including hydralazine and androgen synthesis inhibitor or antigonadotropin is contemplated.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 the 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 therefore, we hypothesize that pharmacological PKC inhibitors rescue the risk of death from aortic dissection. Approximately 100% survival is observed after approximately 30 days (1 month) of treatment, compared with significantly reduced survival in mice without treatment (e.g., a 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 relative to 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 daily or twice daily.

[0279] These results suggest that inhibition of excessive PLC / IP3 / PKC / ERK signaling in the aorta with enzastaurin rescues the risk of mortality caused by 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) will be used to test additional agents for the treatment of vEDS. Mice treated with the well-tolerated orally administered agent, sotrastaurin, at approximately 30 mg / kg / day will be 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 will be observed after approximately 30 days (1 month) of treatment, compared with significantly reduced survival in mice without treatment (e.g., a statistically significant difference compared to control mice). Mice will also be treated for at least approximately 45 days, and the survival rate of sotrastaurin-treated mice relative to control mice will be 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 TIFF2025118759000036.tif158160

[0284] Other Aspects While 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.

[0287] Sequence information SEQUENCE LISTING <110> THE JOHNS HOPKINS UNIVERSITY <120> COMPOSITIONS AND METHODS FOR TREATING VASCULAR EHLERS DANLOS SYNDROME AND ASSOCIATED DISORDERS <150> US 62 / 838,049 <151> 2019-04-24 <150> US 62 / 747,587 <151> 2018-10-18 <150> US 62 / 746,524 <151> 2018-10-16 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 1163 <212> PRT <213> Homo sapiens <400> 1 Met Met Ser Phe Val Gln Lys Gly Ser Trp Leu Leu Leu Ala Leu Leu 1 5 10 15 His Pro Thr Ile Ile Leu Ala Gln Gln Glu Ala Val Glu Gly Gly Cys 20 25 30 Ser His Leu Gly Gln Ser Tyr Ala Asp Arg Asp Val Trp Lys Pro Glu 35 40 45 Pro Cys Gln Ile Cys Val Cys Asp Ser Gly Ser Val Leu Cys Asp Asp 50 55 60 Ile Ile Cys Asp Asp Gln Glu Leu Asp Cys Pro Asn Pro Glu Ile Pro 65 70 75 80 Phe Gly Glu Cys Cys Ala Val Cys Pro Gln Pro Pro Thr Ala Pro Thr 85 90 95 Arg Pro Pro Asn Gly Gln Gly Pro Gln Gly Pro Lys Gly Asp Pro Gly 100 105 110 Pro Pro Gly Ile Pro Gly Arg Asn Gly Asp Pro Gly Ile Pro Gly Gln 115 120 125 Pro Gly Ser Pro Gly Ser Pro Gly Pro Pro Gly Ile Cys Glu Ser Cys 130 135 140 Pro Thr Gly Pro Gln Asn Tyr Ser Pro Gln Tyr Asp Ser Tyr Asp Val 145 150 155 160 Lys Ser Gly Val Ala Val Gly Gly Leu Ala Gly Tyr Pro Gly Pro Ala 165 170 175 Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Thr Ser Gly His Pro Gly 180 185 190 Ser Pro Gly Ser Pro Gly Tyr Gln Gly Pro Pro Gly Glu Pro Gly Gln 195 200 205 Ala Gly Pro Ser Gly Pro Pro Gly Pro Pro Gly Ala Ile Gly Pro Ser 210 215 220 Gly Pro Ala Gly Lys Asp Gly Glu Ser Gly Arg Pro Gly Arg Pro Gly 225 230 235 240 Glu Arg Gly Leu Pro Gly Pro Pro Gly Ile Lys Gly Pro Ala Gly Ile 245 250 255 Pro Gly Phe Pro Gly Met Lys Gly His Arg Gly Phe Asp Gly Arg Asn 260 265 270 Gly Glu Lys Gly Glu Thr Gly Ala Pro Gly Leu Lys Gly Glu Asn Gly 275 280 285 Leu Pro Gly Glu Asn Gly Ala Pro Gly Pro Met Gly Pro Arg Gly Ala 290 295 300 Pro Gly Glu Arg Gly Arg Pro Gly Leu Pro Gly Ala Ala Gly Ala Arg 305 310 315 320 Gly Asn Asp Gly Ala Arg Gly Ser Asp Gly Gln Pro Gly Pro Pro Gly 325 330 335 Pro Pro Gly Thr Ala Gly Phe Pro Gly Ser Pro Gly Ala Lys Gly Glu 340 345 350 Val Gly Pro Ala Gly Ser Pro Gly Ser Asn Gly Ala Pro Gly Gln Arg 355 360 365 Gly Glu Pro Gly Pro Gln Gly His Ala Gly Ala Gln Gly Pro Pro Gly 370 375 380 Pro Pro Gly Ile Asn Gly Ser Pro Gly Gly Lys Gly Glu Met Gly Pro 385 390 395 400 Ala Gly Ile Pro Gly Ala Pro Gly Leu Met Gly Ala Arg Gly Pro Pro 405 410 415 Gly Pro Ala Gly Ala Asn Gly Ala Pro Gly Leu Arg Gly Gly Ala Gly 420 425 430 Glu Pro Gly Lys Asn Gly Ala Lys Gly Glu Pro Gly Pro Arg Gly Glu 435 440 445 Arg Gly Glu Ala Gly Ile Pro Gly Val Pro Gly Ala Lys Gly Glu Asp 450 455 460 Gly Lys Asp Gly Ser Pro Gly Glu Pro Gly Ala Asn Gly Leu Pro Gly 465 470 475 480 Ala Ala Gly Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro 485 490 495 Asn Gly Ile Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro 500 505 510 Gly Pro Ala Gly Pro Arg Gly Ala Ala Gly Glu Pro Gly Arg Asp Gly 515 520 525 Val Pro Gly Gly Pro Gly Met Arg Gly Met Pro Gly Ser Pro Gly Gly 530 535 540 Pro Gly Ser Asp Gly Lys Pro Gly Pro Pro Gly Ser Gln Gly Glu Ser 545 550 555 560 Gly Arg Pro Gly Pro Pro Gly Pro Ser Gly Pro Arg Gly Gln Pro Gly 565 570 575 Val Met Gly Phe Pro Gly Pro Lys Gly Asn Asp Gly Ala Pro Gly Lys 580 585 590 Asn Gly Glu Arg Gly Gly Pro Gly Gly Pro Gly Pro Gln Gly Pro Pro 595 600 605 Gly Lys Asn Gly Glu Thr Gly Pro Gln Gly Pro Pro Gly Pro Thr Gly 610 615 620 Pro Gly Gly Asp Lys Gly Asp Thr Gly Pro Pro Gly Pro Gln Gly Leu 625 630 635 640 Gln Gly Leu Pro Gly Thr Gly Gly Pro Pro Gly Glu Asn Gly Lys Pro 645 650 655 Gly Glu Pro Gly Pro Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly 660 665 670 Gly Lys Gly Asp Ala Gly Ala Pro Gly Glu Arg Gly Pro Pro Gly Leu 675 680 685 Ala Gly Ala Pro Gly Leu Arg Gly Gly Ala Gly Pro Pro Gly Pro Glu 690 695 700 Gly Gly Lys Gly Ala Ala Gly Pro Pro Gly Pro Pro Gly Ala Ala Gly 705 710 715 720 Thr Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Gly Leu Gly Ser 725 730 735 Pro Gly Pro Lys Gly Asp Lys Gly Glu Pro Gly Gly Pro Gly Ala Asp 740 745 750 Gly Val Pro Gly Lys Asp Gly Pro Arg Gly Pro Thr Gly Pro Ile Gly 755 760 765 Pro Pro Gly Pro Ala Gly Gln Pro Gly Asp Lys Gly Glu Gly Gly Ala 770 775 780 Pro Gly Leu Pro Gly Ile Ala Gly Pro Arg Gly Ser Pro Gly Glu Arg 785 790 795 800 Gly Glu Thr Gly Pro Pro Gly Pro Ala Gly Phe Pro Gly Ala Pro Gly 805 810 815 Gln Asn Gly Glu Pro Gly Gly Lys Gly Glu Arg Gly Ala Pro Gly Glu 820 825 830 Lys Gly Glu Gly Gly Pro Pro Gly Val Ala Gly Pro Pro Gly Lys Asp 835 840 845 Gly Thr Ser Gly His Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg Gly 850 855 860 Asn Arg Gly Glu Arg Gly Ser Glu Gly Ser Pro Gly His Pro Gly Gln 865 870 875 880 Pro Gly Pro Pro Gly Pro Pro Gly Ala Pro Gly Pro Cys Cys Gly Gly 885 890 895 Val Gly Ala Ala Ala Ile Ala Gly Ile Gly Gly Glu Lys Ala Gly Gly 900 905 910 Phe Ala Pro Tyr Tyr Gly Asp Glu Pro Met Asp Phe Lys Ile Asn Thr 915 920 925 Asp Glu Ile Met Thr Ser Leu Lys Ser Val Asn Gly Gln Ile Glu Ser 930 935 940 Leu Ile Ser Pro Asp Gly Ser Arg Lys Asn Pro Ala Arg Asn Cys Arg 945 950 955 960 Asp Leu Lys Phe Cys His Pro Glu Leu Lys Ser Gly Glu Tyr Trp Val 965 970 975 Asp Pro Asn Gln Gly Cys Lys Leu Asp Ala Ile Lys Val Phe Cys Asn 980 985 990 Met Glu Thr Gly Glu Thr Cys Ile Ser Ala Asn Pro Leu Asn Val Pro 995 1000 1005 Arg Lys His Trp Trp Thr Asp Ser Ser Ala Glu Lys Lys His Val 1010 1015 1020 Trp Phe Gly Glu Ser Met Asp Gly Gly Phe Gln Phe Ser Tyr Gly 1025 1030 1035 Asn Pro Glu Leu Pro Glu Asp Val Leu Asp Val Gln Leu Ala Phe 1040 1045 1050 Leu Arg Leu Leu Ser Ser Arg Ala Ser Gln Asn Ile Thr Tyr His 1055 1060 1065 Cys Lys Asn Ser Ile Ala Tyr Met Asp Gln Ala Ser Gly Asn Val 1070 1075 1080 Lys Lys Ala Leu Lys Leu Met Gly Ser Asn Glu Gly Glu Phe Lys 1085 1090 1095 Ala Glu Gly Asn Ser Lys Phe Thr Tyr Thr Val Leu Glu Asp Gly 1100 1105 1110 Cys Thr Lys His Thr Gly Glu Trp Ser Lys Thr Val Phe Glu Tyr 1115 1120 1125 Arg Thr Arg Lys Ala Val Arg Leu Pro Ile Val Asp Ile Ala Pro 1130 1135 1140 Tyr Asp Ile Gly Gly Pro Asp Gln Glu Phe Gly Val Asp Val Gly 1145 1150 1155 Pro Val Cys Phe Leu 1160 <210> 2 <211> 5490 <212> DNA <213> Homo sapiens <400> 2 ggctgagttt tatgacgggc ccggtgctga agggcaggga acaacttgat ggtgctactt 60 tgaactgctt ttcttttctc ctttttgcac aaagagtctc atgtctgata tttagacatg 120 atgagctttg tgcaaaaggg gagctggcta cttctcgctc tgcttcatcc cactattatt 180 ttggcacaac aggaagctgt tgaaggaga tgttcccatc ttggtcagtc ctatgcggat 240 agagatgtct ggaagccaga accatgccaa atatgtgtct gtgactcagg atccgttctc 300 tgcgatgaca taatatgtga cgatcaagaa ttagactgcc ccaacccaga aattccattt 360 ggagaatgtt gtgcagtttg cccacagcct ccaactgctc ctactcgccc tcctaatggt 420 caaggacctc aaggccccaa gggagatcca ggccctcctg gtattcctgg gagaaatggt 480 gaccctggta ttccaggaca accagggtcc cctggttctc ctggcccccc tggaatctgt 540 gaatcatgcc ctactggtcc tcagaactat tctccccagt atgattcata tgatgtcaag 600 tctggagtag footggagg actcgcaggc tatcctggac cagctggccc cccaggccct 660 cccggtcccc ctggtacatc tggtcatcct ggttcccctg gatctccagg ataccaagga 720 ccccctggtg aacctgggca agctggtcct tcaggccctc caggacctcc tggtgctata 780 ggtccatctg gtcctgctgg aaaagatgga gaatcaggta gacccggacg acctggagag 840 cgaggattgc ctggacctcc aggtatcaaa ggtccagctg ggatacctgg attccctggt 900 atgaaaggac acagaggctt cgatggacga aatggagaaa agggtgaaac aggtgctcct ggattaaagg gtgaaaatgg tcttccaggc gaaaatggag ctcctggacc catgggtcca agaggggctc ctggtgagcg aggacggcca ggacttcctg gggctgcagg tgctcggggt 1080 aatgacggtg ctcgaggcag tgatggtcaa ccaggccctc ctggtcctcc tggaactgcc 1140 ggattccctg gatcccctgg tgctaagggt gaagttggac ctgcagggtc tcctggttca 1200 aatggtgccc ctggacaaag aggagaacct ggacctcagg gacacgctgg tgctcaaggt cctcctggcc ctcctgggat taatggtagt cctggtggta aaggcgaaat gggtcccgct ggcattcctg gagctcctgg actgatggga gcccggggtc ctccaggacc agccggtgct 1380 aatggtgctc ctggactgcg aggtggtgca ggtgagcctg gtaagaatgg tgccaaagga gagcccggac cacgtggtga acgcggtgag gctggtattc caggtgttcc aggagctaaa ggcgaagatg gcaaggatgg atcacctgga gaacctggtg caaatgggct tccaggagct 1560 gcaggagaaa ggggtgcccc tgggttccga ggacctgctg gaccaaatgg catcccagga 1620 gaaaagggtc ctgctggaga gcgtggtgct ccaggccctg cagggcccag aggagctgct 1680 ggagaacctg gcagagatgg cgtccctgga ggtccaggaa tgaggggcat gcccggaagt 1740 ccaggaggac caggaagtga tgggaaacca gggcctcccg gaagtcaagg agaaagtggt 1800 cgaccaggtc ctcctgggcc atctggtccc cgaggtcagc ctggtgtcat gggcttcccc 1860 ggtcctaaag gaaatgatgg tgctcctggt aagaatggag aacgaggtgg ccctggagga 1920 cctggccctc agggtcctcc tggaaagaat ggtgaaactg gacctcaggg acccccaggg 1980 cctactgggc ctggtggtga caaggagac acaggacccc ctggtccaca aggattacaa 2040 ggcttgcctg gtacaggtgg tcctccagga gaaaatggaa aacctgggga accaggtcca 2100 aagggtgatg ccggtgcacc tggagctcca ggaggcaagg gtgatgctgg tgcccctggt 2160 gaacgtggac ctcctggatt ggcaggggcc ccaggactta gaggtggagc tggtccccct 2220 ggtcccgaag gaaggaaggg tgctctctggt cctcctgggc cacctggtgc tgctggtact 2280 cctggtctgc aaaggaatgcc tggagaaaga ggaggtcttg gaagtcctgg tccaaagggt 2340 gacaagggtg aaccaggcgg tccaggtgct gatggtgtcc cagggaaaga tggcccaagg 2400 ggtcctactg gtcctattgg tcctcctggc ccagctggcc agcctggaga taagggtgaa 2460 ggtggtgccc ccggacttcc aggtatagct ggacctcgtg gtagccctgg tgagagaggt 2520 gaaactggcc ctccaggacc tgctggtttc cctggtgctc ctggacagaa tggtgaacct 2580 ggtggtaaag gagaagagg ggctccgggt gagaaaggtg aaagggccc tcctggagtt 2640 gcaggacccc ctggaggttc tggacctgct ggtcctcctg gtccccaaagg tgtcaaaggt 2700 gaacgtggca gtcctggtgg acctggtgct gctggcttcc ctggtgctcg tggtcttcct 2760 ggtcctcctg gtagtaatgg taacccagga cccccaggtc ccagcggttc tccaggcaag 2820 gatgggcccc caggtcctgc gggtaacact ggtgctcctg gcagccctgg agtgtctgga 2880 ccaaaaggtg atgctggcca accaggagag aagggatcgc ctggtgccca gggcccacca 2940 ggagctccag gcccacttgg gattgctggg atcactggag cacggggtct tgcaggacca 3000 ccaggcatgc caggtcctag gggaagccct ggccctcagg gtgtcaaggg tgaaagtggg 3060 aaaccaggag ctaacggtct cagtggagaa cgtggtcccc ctggacccca gggtcttcct 3120 ggtctggctg gtacagctgg tgaacctgga agagatggaa accctggatc agatggtctt 3180 ccaggccgag atggatctcc tggtggcaag ggtgatcgtg gtgaaaatgg ctctcctggt 3240 gcccctggcg ctcctggtca tccaggccca cctggtcctg tcggtccagc tggaaagagt 3300 ggtgacagag gagaaagtgg ccctgctggc cctgctggtg ctcccggtcc tgctggttcc 3360 cgaggtgctc ctggtcctca aggcccacgt ggtgacaaag gtgaaacagg tgaacgtgga 3420 gctgctggca tcaaaggaca tcgaggattc cctggtaatc caggtgcccc aggttctcca 3480 ggccctgctg gtcagcaggg tgcaatcggc agtccaggac ctgcaggccc cagaggacct 3540 gttggaccca gtggacctcc tggcaaagat ggaaccagtg gacatccagg tcccattgga 3600 ccaccagggc ctcgaggtaa cagaggtgaa agaggatctg agggctcccc aggccaccca 3660 gggcaaccag gccctcctgg acctcctggt gcccctggtc cttgctgtgg tggtgttgga 3720 gccgctgcca ttgctgggat tggaggtgaa aaagctggcg gttttgcccc gtattatgga 3780 gatgaaccaa tggatttcaa aatcaacacc gatgagatta tgacttcact caagtctgtt 3840 aatggacaaa tagaaagcct cattagtcct gatggttctc gtaaaaaccc cgctagaaac 3900 tgcagagacc tgaaattctg ccatcctgaa ctcaagagtg gagaatactg ggttgaccct 3960 aaccaaggat gcaaattgga tgctatcaag gtattctgta atatggaaac tggggaaaca 4020 tgcataagtg ccaatccttt gaatgttcca cggaaacact ggtggacaga ttctagtgct 4080 gagaagaaac acgtttggtt tggagagtcc atggatggtg gttttcagtt tagctacggc 4140 aatcctgaac ttcctgaaga tgtccttgat gtgcagctgg cattccttcg acttctctcc 4200 agccgagctt cccagaacat alatatcac tgcaaaaata gcattgcata catggatcag 4260 gccagtggaa atgtaaagaa ggccctgaag ctgatggggt caaatgaagg tgaattcaag 4320 gctgaaggaa atagcaaatt cacctacaca gttctggagg atggttgcac gaaacacact 4380 ggggaatgga gcaaaacagt ctttgaatat cgaacacgca aggctgtgag actacctatt 4440 gtagatattg caccctatga cattggtggt cctgatcaag aatttggtgt ggacgttggc 4500 cctgtttgct ttttataaac caaactctat ctgaaatccc aaaaaaaa atttaactcc 4560 atatgtgttc ctcttgttct aatcttgtca accagtgcaa gtgaccgaca aaattccagt 4620 tatttattc caaaatgttt ggaaacagta taatttgaca aagaaaaatg atacttctct 4680 ttttttgctg ttccaccaaa tacaattcaa atgctttttg ttttatttt ttaccaattc 4740 caatttcaaa atgtctcaat ggtgctataa taaataaact tcaactct ttatgataac 4800 aacactgtgt tatattcttt gaatcctagc ccatctgcag agcaatgact gtgctcacca 4860 gtaaaagata acctttcttt ctgaaatagt caaatacgaa attagaaaag ccctccctat 4920 tttaactacc tcaactggtc agaaacacag attgtattct atgagtccca gaagatgaaa 4980 aaaattttat acgttgataa aacttataaa tttcattgat taatctcctg gaagattggt 5040 ttaaaaagaa aagtgtaatg caagaattta aagaaatatt tttaaagcca caattatttt 5100 aatattggat atcaactgct tgtaaaggtg ctcctcttttt ttcttgtcat tgctggtcaa 5160 gattactaat atttgggaag gctttaaaga cgcatgttat ggtgctaatg tactttcact 5220 tttaaactct agatcagaat tgttgacttg cattcagaac ataaatgcac aaaatctgta 5280 catgtctccc atcagaaaga ttcattggca tgccacaggg gattctcctc cttcatcctg 5340 taaaggtcaa caataaaaac caaattatgg ggctgctttt gtcacactag catagagaat 5400 gtgttgaaat ttaactttgt aagcttgtat gtggttgttg atcttttttt tccttacaga 5460 cacccataat aaaatatcat attaaaattc 5490 <210> 3 <211> 379 <212> PRT <213> Homo sapiens <400> 3 Put Ala Ala Ala Ala Ala Ala Gln Gly Gly Gly Gly Gly Glu Pro Arg Arg 1 5 10 15 Thr Glu Gly Val Gly Pro Gly Val Pro Gly Glu Val Glu Met Val Lys 20 25 30 Gly Gln Pro Phe Asp Val Gly Pro Arg Tyr Thr Gln Leu Gln Tyr Ile 35 40 45 Gly Glu Gly Ala Tyr Gly Met Val Ser Ser Ala Tyr Asp His Val Arg 50 55 60 Lys Thr Arg Val Ala Ile Lys Lys Ile Ser Pro Phe Glu His Gln Thr 65 70 75 80 Tyr Cys Gln Arg Thr Leu Arg Glu Ile Gln Ile Leu Leu Arg Phe Arg 85 90 95 His Glu Asn Val Ile Gly Ile Arg Asp Ile Leu Arg Ala Ser Thr Leu 100 105 110 Glu Ala Met Arg Asp Val Tyr Ile Val Gln Asp Leu Met Glu Thr Asp 115 120 125 Leu Tyr Lys Leu Leu Lys Ser Gln Gln Leu Ser Asn Asp His Ile Cys 130 135 140 Tyr Phe Leu Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala 145 150 155 160 Asn Val Leu His Arg Asp Leu Lys Pro Ser Asn Leu Leu Ile Asn Thr 165 170 175 Thr Cys Asp Leu Lys Ile Cys Asp Phe Gly Leu Ala Arg Ile Ala Asp 180 185 190 Pro Glu His Asp His Thr Gly Phe Leu Thr Glu Tyr Val Ala Thr Arg 195 200 205 Trp Tyr Arg Ala Pro Glu Ile Met Leu Asn Ser Lys Gly Tyr Thr Lys 210 215 220 Ser Ile Asp Ile Trp Ser Val Gly Cys Ile Leu Ala Glu Met Leu Ser 225 230 235 240 Asn Arg Pro Ile Phe Pro Gly Lys His Tyr Leu Asp Gln Leu Asn His 245 250 255 Ile Leu Gly Ile Leu Gly Ser Pro Ser Gln Glu Asp Leu Asn Cys Ile 260 265 270 Ile Asn Met Lys Ala Arg Asn Tyr Leu Gln Ser Leu Pro Ser Lys Thr 275 280 285 Lys Val Ala Trp Ala Lys Leu Phe Pro Lys Ser Asp Ser Lys Ala Leu 290 295 300 Asp Leu Leu Asp Arg Met Leu Thr Phe Asn Pro Asn Lys Arg Ile Thr 305 310 315 320 Val Glu Glu Ala Leu Ala His Pro Tyr Leu Glu Gln Tyr Tyr Asp Pro 325 330 335 Thr Asp Glu Pro Val Ala Glu Glu Pro Phe Thr Phe Ala Met Glu Leu 340 345 350 Asp Asp Leu Pro Lys Glu Arg Leu Lys Glu Leu Ile Phe Gln Glu Thr 355 360 365 Ala Arg Phe Gln Pro Gly Val Leu Glu Ala Pro 370 375 <210> 4 <211> 1866 <212> DNA <213> Homo sapiens <400> 4 cgttcctcgg cgccgccggg gccccagagg gcagcggcag caacagcagc agcagcagca 60 gcgggagtgg agatggcggc ggcggcggct caggggggcg ggggcgggga gccccgtaga 120 accgaggggg tcggcccggg ggtcccgggg gaggtggaga tggtgaaggg gcagccgttc 180 gacgtgggcc cgcgctacac gcagttgcag tacatcggcg agggcgcgta cggcatggtc 240 agctcggcct atgaccacgt gcgcaagact cgcgtggcca tcaagaagat cagccccttc 300 gaacatcaga cctactgcca gcgcacgctc cgggagatcc agatcctgct gcgcttcgc 360 catgagaatg tcatcggcat ccgagacatt ctgcgggcgt ccaccctgga agccatgaga 420 gatgtctaca ttgtgcagga cctgatggag actgacctgt acaagttgct gaaaagccag 480 cagctgagca atgaccatat ctgctacttc ctctaccaga tctgcgggg cctcaagtac 540 atccactccg ccaacgtgct ccaccgagat ctaaagccct ccaacctgct cagcaacacc 600 acctgcgacc ttaagatttg tgatttcggc ctggcccgga ttgccgatcc tgagcatgac 660 cacaccggct tcctgacgga gtatgtggct acgcgctggt accgggcccc agagatcatg 720 ctgaactcca agggctatac caagtccatc gacatctggt ctgtgggctg cattctggct 780 gagatgctct ctaaccggcc catcttccct ggcaagcact acctggatca gctcaaccac 840 attctgggca tcctgggctc cccatcccag gaggacctga attgtatcat caacatgaag 900 gcccgaaact acctacagtc tctgccctcc aagaccaagg tggcttgggc caagcttttc 960 cccaagtcag actccaaagc ccttgacctg ctggaccgga tgttaacctt taaccccaat 1020 aaacggatca cagtggagga agcgctggct cacccctacc tggagcagta ctatgacccg 1080 acggatgagc cagtggccga ggagcccttc accttcgcca tggagctgga tgacctacct 1140 aaggagcgg tgaaggagct catcttccag gagacagcac gcttccagcc cggagtgctg 1200 gaggccccct agcccagaca gacatctctg caccctgggg cctggacctg cctcctgcct 1260 gccctctcc cgccagactg ttagaaaatg gacactgtgc ccagcccgga ccttggcagc 1320 ccaggccggg gtggagcatg ggcctggcca cctctctcct ttgctgaggc ctccagcttc 1380 aggcaggcca aggccttctc ctccccaccc gccctcccca cggggcctcg ggagctcagg 1440 tggccccagt tcaatctccc gctgctgctg ctgctgcgcc cttaccttcc ccagcgtccc 1500 agtctctggc agttctggaa tggaagggtt ctggctgccc caacctgctg aagggcagag 1560 gtggagggtg gggggcgctg agtagggact cagggccatg cctgcccccc tcatctcatt 1620 caaaccccac cctagtttcc ctgaaggaac attccttagt ctcaagggct agcatccctg 1680 aggagccagg ccgggccgaa tccctccct gtcaaagctg tcacttcgcg tgccctcgct 1740 gcttctgtgt gtggtgagca gaagtggagc tggggggcgt ggagagcccg gcgcccctgc 1800 caccctcctg acccgtctaa tatataaata tagagatgtg tctatggctg aaaaaaaaa 1860 aaaaaa 1866 <210> 5 <211> 360 <212> PRT <213> Homo sapiens <400> 5 Met Ala Ala Ala Ala Ala Ala Gly Ala Gly Pro Glu Met Val Arg Gly 1 5 10 15 Gln Val Phe Asp Val Gly Pro Arg Tyr Thr Asn Leu Ser Tyr Ile Gly 20 25 30 Glu Gly Ala Tyr Gly Met Val Cys Ser Ala Tyr Asp Asn Val Asn Lys 35 40 45 Val Arg Val Ala Ile Lys Lys Ile Ser Pro Phe Glu His Gln Thr Tyr 50 55 60 Cys Gln Arg Thr Leu Arg Glu Ile Lys Ile Leu Leu Arg Phe Arg His 65 70 75 80 Glu Asn Ile Ile Gly Ile Asn Asp Ile Ile Arg Ala Pro Thr Ile Glu 85 90 95 Gln Met Lys Asp Val Tyr Ile Val Gln Asp Leu Met Glu Thr Asp Leu 100 105 110 Tyr Lys Leu Leu Lys Thr Gln His Leu Ser Asn Asp His Ile Cys Tyr 115 120 125 Phe Leu Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala Asn 130 135 140 Val Leu His Arg Asp Leu Lys Pro Ser Asn Leu Leu Leu Asn Thr Thr 145 150 155 160 Cys Asp Leu Lys Ile Cys Asp Phe Gly Leu Ala Arg Val Ala Asp Pro 165 170 175 Asp His Asp His Thr Gly Phe Leu Thr Glu Tyr Val Ala Thr Arg Trp 180 185 190 Tyr Arg Ala Pro Glu Ile Met Leu Asn Ser Lys Gly Tyr Thr Lys Ser 195 200 205 Ile Asp Ile Trp Ser Val Gly Cys Ile Leu Ala Glu Met Leu Ser Asn 210 215 220 Arg Pro Ile Phe Pro Gly Lys His Tyr Leu Asp Gln Leu Asn His Ile 225 230 235 240 Leu Gly Ile Leu Gly Ser Pro Ser Gln Glu Asp Leu Asn Cys Ile Ile 245 250 255 Asn Leu Lys Ala Arg Asn Tyr Leu Leu Ser Leu Pro His Lys Asn Lys 260 265 270 Val Pro Trp Asn Arg Leu Phe Pro Asn Ala Asp Ser Lys Ala Leu Asp 275 280 285 Leu Leu Asp Lys Met Leu Thr Phe Asn Pro His Lys Arg Ile Glu Val 290 295 300 Glu Gln Ala Leu Ala His Pro Tyr Leu Glu Gln Tyr Tyr Asp Pro Ser 305 310 315 320 Asp Glu Pro Ile Ala Glu Ala Pro Phe Lys Phe Asp Met Glu Leu Asp 325 330 335 Asp Leu Pro Lys Glu Lys Leu Lys Glu Leu Ile Phe Glu Glu Thr Ala 340 345 350 Arg Phe Gln Pro Gly Tyr Arg Ser 355 360 <210> 6 <211> 1514 <212> DNA <213> Homo sapiens <400> 6 gccctccct ccgcccgccc gcggcccgc ccgtcagtct ggcaggcagg caggcaatcg 60 gtccgagtgg ctgtcggctc ttcagctctc ccgctcggcg tcttccttc tctcccggt 120 cagcgtcggc ggctgcaccg gcggcggcgc agtccctgcg ggaggggcga caagagctga 180 gcggcggccg ccgagcgtcg agctcagcgc ggcggaggcg gcggcggccc ggcagccaac 240 atggcggcgg cggcggcggc gggcgcggc ccggagatgg tccgcgggca ggtgttcgac 300 gtggggccgc gctacaccaa cctctcgtac atcggcgagg gcgcctacgg catggtgtgc 360 tctgcttatg ataatgtcaa caaagttcga gtagctatca agaaaatcag cccctttgag 420 caccagacct actgccagag aaccctgagg gagataaaa tcttactgcg cttcagacat 480 gagaacatca ttggaatca tgacattatt cgagcaccaa ccatcgagca atgaagat 540 gtatatatag tacaggacct catggaaca gatctttaca agctcttgaa cacacac 600 ctcagcaatg accatatctg ctattctc taccagatcc tcagaggtt aaatatatc 660 cattcagcta acgttctgca ccgtgaccctc aagccttcca acctgctgct caacaccacc 720 tgtgatctca agatctgtga ctttggctg gcccgtgttg cagatccaga ccatgatcac 780 acagggttcc tgacagaata tgtggccaca cgttggtaca gggctccaga aattatgttg 840 aattccaagg gctacaccaa gtccattgat atttgtctg taggctgcat tctggcagaa 900 atgctttcta acaggccatt ctttccaggg aagcattatc ttgaccagct gaaccacatt 960 ttgggtattc ttggatcccc atcacaagaa gacctgaatt gtataataaa tttaaaagct 1020 aggaactatt tgctttctct tccacacaa aaaggtgc catggacag gctgttccca 1080 aatgctgact ccaagctct ggacttattg gataaatgt tgacattca cccacacaag 1140 aggattgaag tagacaggc tctgcccac ccatatctgg agcagtatta cgacccgagt 1200 gacgagccca tcgccgaagc accattcaag ttcgacatgg aattggatga cttgcctaag 1260 gaaaagctca aagaactaat tttgagag actgctagat tccagccagg atacagatct 1320 taaatttgtc aggtacctgg agtttaac agtgagctct agcaagggag gcgctgcctt 1380 ttgttttctag atattatgt tcctcaggt ccattatttt gtattcttt ccaagctcct 1440 tattggagg tatttttta aatttagaat taaaaattat tattgaagtt acatataaaaa 1500 AAAAAAAAAAAAAAAA 1514 <210> 7 <211> 672 <212> PRT <213> Homo sapiens <400> 7 Met Ala Asp Val Phe Pro Gly Asn Asp Ser Thr Ala Ser Gln Asp Val 1 5 10 15 Ala Asn Arg Phe Ala Arg Lys Gly Ala Leu Arg Gln Lys Asn Val His 20 25 30 Glu Val Lys Asp His Lys Phe Ile Ala Arg Phe Phe Lys Gln Pro Thr 35 40 45 Phe Cys Ser His Cys Thr Asp Phe Ile Trp Gly Phe Gly Lys Gln Gly 50 55 60 Phe Gln Cys Gln Val Cys Cys Phe Val Val His Lys Arg Cys His Glu 65 70 75 80 Phe Val Thr Phe Ser Cys Pro Gly Ala Asp Lys Gly Pro Asp Thr Asp 85 90 95 Asp Pro Arg Ser Lys His Lys Phe Lys Ile His Thr Tyr Gly Ser Pro 100 105 110 Thr Phe Cys Asp His Cys Gly Ser Leu Leu Tyr Gly Leu Ile His Gln 115 120 125 Gly Met Lys Cys Asp Thr Cys Asp Met Asn Val His Lys Gln Cys Val 130 135 140 Ile Asn Val Pro Ser Leu Cys Gly Met Asp His Thr Glu Lys Arg Gly 145 150 155 160 Arg Ile Tyr Leu Lys Ala Glu Val Ala Asp Glu Lys Leu His Val Thr 165 170 175 Val Arg Asp Ala Lys Asn Leu Ile Pro Met Asp Pro Asn Gly Leu Ser 180 185 190 Asp Pro Tyr Val Lys Leu Lys Leu Ile Pro Asp Pro Lys Asn Glu Ser 195 200 205 Lys Gln Lys Thr Lys Thr Ile Arg Ser Thr Leu Asn Pro Gln Trp Asn 210 215 220 Glu Ser Phe Thr Phe Lys Leu Lys Pro Ser Asp Lys Asp Arg Arg Leu 225 230 235 240 Ser Val Glu Ile Trp Asp Trp Asp Arg Thr Thr Arg Asn Asp Phe Met 245 250 255 Gly Ser Leu Ser Phe Gly Val Ser Glu Leu Met Lys Met Pro Ala Ser 260 265 270 Gly Trp Tyr Lys Leu Leu Asn Gln Glu Glu Gly Glu Tyr Tyr Asn Val 275 280 285 Pro Ile Pro Glu Gly Asp Glu Glu Gly Asn Met Glu Leu Arg Gln Lys 290 295 300 Phe Glu Lys Ala Lys Leu Gly Pro Ala Gly Asn Lys Val Ile Ser Pro 305 310 315 320 Ser Glu Asp Arg Lys Gln Pro Ser Asn Asn Leu Asp Arg Val Lys Leu 325 330 335 Thr Asp Phe Asn Phe Leu Met Val Leu Gly Lys Gly Ser Phe Gly Lys 340 345 350 Val Met Leu Ala Asp Arg Lys Gly Thr Glu Glu Leu Tyr Ala Ile Lys 355 360 365 Ile Leu Lys Lys Asp Val Val Ile Gln Asp Asp Asp Val Glu Cys Thr 370 375 380 Met Val Glu Lys Arg Val Leu Ala Leu Leu Asp Lys Pro Pro Phe Leu 385 390 395 400 Thr Gln Leu His Ser Cys Phe Gln Thr Val Asp Arg Leu Tyr Phe Val 405 410 415 Met Glu Tyr Val Asn Gly Gly Asp Leu Met Tyr His Ile Gln Gln Val 420 425 430 Gly Lys Phe Lys Glu Pro Gln Ala Val Phe Tyr Ala Ala Glu Ile Ser 435 440 445 Ile Gly Leu Phe Phe Leu His Lys Arg Gly Ile Ile Tyr Arg Asp Leu 450 455 460 Lys Leu Asp Asn Val Met Leu Asp Ser Glu Gly His Ile Lys Ile Ala 465 470 475 480 Asp Phe Gly Met Cys Lys Glu His Met Met Asp Gly Val Thr Thr Arg 485 490 495 Thr Phe Cys Gly Thr Pro Asp Tyr Ile Ala Pro Glu Ile Ile Ala Tyr 500 505 510 Gln Pro Tyr Gly Lys Ser Val Asp Trp Trp Ala Tyr Gly Val Leu Leu 515 520 525 Tyr Glu Met Leu Ala Gly Gln Pro Pro Phe Asp Gly Glu Asp Glu Asp 530 535 540 Glu Leu Phe Gln Ser Ile Met Glu His Asn Val Ser Tyr Pro Lys Ser 545 550 555 560 Leu Ser Lys Glu Ala Val Ser Ile Cys Lys Gly Leu Met Thr Lys His 565 570 575 Pro Ala Lys Arg Leu Gly Cys Gly Pro Glu Gly Glu Arg Asp Val Arg 580 585 590 Glu His Ala Phe Phe Arg Arg Ile Asp Trp Glu Lys Leu Glu Asn Arg 595 600 605 Glu Ile Gln Pro Pro Phe Lys Pro Lys Val Cys Gly Lys Gly Ala Glu 610 615 620 Asn Phe Asp Lys Phe Phe Thr Arg Gly Gln Pro Val Leu Thr Pro Pro 625 630 635 640 Asp Gln Leu Val Ile Ala Asn Ile Asp Gln Ser Asp Phe Glu Gly Phe 645 650 655 Ser Tyr Val Asn Pro Gln Phe Val His Pro Ile Leu Gln Ser Ala Val 660 665 670 <210> 8 <211> 8787 <212> DNA <213> Homo sapiens <400> 8 ggccgcagct ccccggcgga ggcaagaggt ggttgggggg gaccatggct gacgttttcc 60 cgggcaacga ctccacggcg tctcaggacg tggccaaccg cttcgcccgc aaaggggcgc 120 tgaggcagaa gaacgtgcac gaggtgaagg accacaaatt catcgcgcgc ttcttcaagc 180 agcccacctt ctgcagccac tgcaccgact tcatctgggg gtttgggaaa caaggcttcc 240 agtgccaagt ttgctgtttt gtggtccaca agaggtgcca tgaatttgtt actttttctt 300 gtccgggtgc ggataaggga cccgacactg atgaccccag gagcaagcac aagttcaaaa 360 tccacactta cggaagcccc accttctgcg atcactgtgg gtcactgctc tatggactta 420 tccatcaagg gatgaaatgt gacacctgcg atatgaacgt tcacaagcaa tgcgtcatca 480 atgtccccag cctctgcgga atggatcaca ctgagaagag ggggcggatt tacctaaagg 540 ctgaggttgc tgatgaaaag ctccatgtca cagtacgaga tgcaaaaaat ctaatcccta 600 tggatccaaa cgggctttca gatccttatg tgaagctgaa acttattcct gatcccaaga 660 atgaaagcaa gcaaaaaacc aaaaccatcc gctccacact aaatccgcag tggaatgagt 720 cctttacatt caaattgaaa ccttcagaca aagaccgacg actgtctgta gaaatctggg 780 actgggatcg aacaacaagg aatgacttca tgggatccct ttcctttgga gtttcggagc 840 tgatgaagat gccggccagt ggatggtaca agttgcttaa ccaagaagaa ggtgagtact 900 acaacgtacc cattccggaa ggggacgagg aaaggaaacat ggaactcagg cagaaattcg 960 agaaagccaa acttggccct gctggcaaca aagtcatcag tccctctgaa gacgggaaac 1020 aaccttccaa caaccttgac cgagtgaaac tcacggactt caatttcctc atggtgttgg 1080 gaaagggag ttttggaaag gtgatgcttg ccgacaggaa gggcacagaa gaactgtatg 1140 1200 tagaaaagcg agtcttggcc ctgcttgaca aacccccgtt cttgacgcag ctgcactcct 1260 gcttccagac agtggatcgg ctgtacttcg tcatggata tgtcaacggt ggggacctca 1320 1380 agatttccat cggattgttc tttcttcata aaagaggaat catttatagg gatctgaagt 1440 tagataacgt catgttggat tcagaaggac atatcaaaat tgctgacttt gggatgtgca 1500 aggaacacat gatggatgga gtcacgacca ggaccttctg tgggactcca gattatatcg 1560 ccccagagat aatcgcttat cagccgtatg gaaaatctgt ggactggtgg gcctatggcg 1620 tcctgttgta tgaaatgctt gccgggcagc ctccatttga tggtgaagat gaagacgagc 1680 tatttcagtc tatcatggag cacaacgttt cctatccaaa atccttgtcc aaggaggctg 1740 tttctatctg caaaggactg atgaccaaac acccagccaa gcggctgggc tgtgggcctg 1800 agggggagag ggacgtgaga gagcatgcct tcttccggag gatcgactgg gaaaaactgg 1860 agaacaggga gatccagcca ccattcaagc ccaaagtgtg tggcaaagga gcagagaact 1920 ttgacaagtt cttcacacga ggacagcccg tcttaacacc acctgatcag ctggttattg 1980 ctaacataga ccagtctgat tttgaagggt tctcgtatgt caacccccag tttgtgcacc 2040 ccatcttaca gagtgcagta tgaaactcac cagcgagaac aaacacctcc ccagccccca 2100 gccctccccg cagtgggaag tgaatcctta accctaaaat tttaaggcca cggccttgtg 2160 tctgattcca tatggaggcc tgaaaattgt agggttatta gtccaaatgt gatcaactgt 2220 tcagggtctc tctcttacaa ccaagaacat tatcttagtg gaagatggta cgtcatgctc 2280 agtgtccagt ttaattctgt agaagttacg tctggctcta ggttaaccct tcctagaaag 2340 caagcagact gttgccccat tttgggtaca atttgatata ctttccatac cctccatctg 2400 tggatttttc agcattggaa tcccccaacc agagatgtta aagtgagcct gtcccaggaa 2460 acatctccac ccaagacgtc tttggaatcc aagaacagga agccaagaga gtgagcaggg 2520 agggattggg ggtgggggag gcctcaaaat accgactgcg tccattctct gcctccatgg 2580 aaacagcccc tagaatctga aaggccggga taaacctaat cactgttccc aaacattgac 2640 aaatcctaac ccaaccatgg tccagcagtt accagtttaa acaaaaaaac ctcagatgag 2700 tgttgggtga atctgtcatc tggtaccctc cttggttgat aactgtcttg atacttttca 2760 ttctttgtaa gaggccaaat cgtctaagga cgttgctgaa caagcgtgtg aaatcatttc 2820 agatcagga taagccagtg tgtacatatg ttcattttaa tctctgggag attatttttc 2880 catccagggt gccatcagta atcatgccac tactcaccag tgttgttcgc cacacccac 2940 ccccacacac accacatt tgctgcctac cttgttatcc ttctcagaa gctgaagtgt 3000 acgccctctc cccttttgtg cttattatt taataggctg cagtgtcgct tatgaaagta 3060 cgatgtacag taacttaatg gaagtgctga cttagcatc agcctctacc gattgattt 3120 cctcccttct ctagccctgg atgtccactt agggataaaa agaatatggt ttggttccc 3180 atttctagtt cacgttgaat ggaggcctg gagctgtaga atcaggaac ccggatgcct 3240 aacagctcaa agatgttttg ttatagaag gattttaata cgttttgcaa atgcatcatg 3300 caatgaattt tgcatgttta taataacct taataacag tgaatctata ttattgatat 3360 aatcgtatca agtataaaga gagtattata ataattttat aagacacaat tgtgctctat 3420 ttgtgcaggt tctgtttct aatcctctt tctattaag tttagctga atcccttgct 3480 tctgtgcttt ccctccctgc acatgggcac tgtatcagat agattacttt ttaatgtag 3540 ataaaatttc aaaaatgaat ggctagttta cgtgatagat taggctctta ctacatatgt 3600 gtgtgtatat atatgtattt gattctacct gcaaacaaat ttttattggt gaggactatt 3660 tttgagctga cactccctct tagtttcttc atgtcacctt tcgtcctggt tcctccgcca 3720 ctcttctct tggggacaac aggaagtgtc tgattccagt ctgcctagta cgttggtaca 3780 cacgtggcat tgccgcagca cctgggctga cctttgtgtg tgcgtgtgtg tgtgtttcct 3840 tcttcccttc agcctgtgac tgttgctgac tccaggggtg ggagggatgg ggagactccc 3900 ctcttgctgt gtgtactgga cacgcaggaa gcatgctgtc ttgctgcctc tgcaacgacc 3960 tgtcgtttgc tccagcatgc acaaacttcg tgagaccaac acagccgtgc cctgcaggca 4020 ccagcacgtg cttttcagag gctgcggact ttcttccagc cattgtggca ttggcctttc 4080 cagtcttggg aggagcgcgc tgctttggtg agacacccc atgcaaggtc ctcagagtag 4140 ccgggttcta ccacaaacag aaacagaatg aaagtagctg tcagtcctttagagagccg 4200 ctctgtttcc tcccagaagc atctcccagc taagctcgca ttattttct cctctggctg 4260 ttgcctgaa gttcacagaa cacacaacca tgaaaggctt tttgaggtga gaggcccagg 4320 tggtcctggc aaccctgagt agaggagag acggggtagg gaacggggccc ggccagaaaa 4380 gaaccattc ttctgccatc tttgcac catagacatc gagactccag ggggtcctgg 4440 ctcccctgtc cctgcagccc tgcaggtcag tgcatgatct gggttcgt cctgaccagg 4500 tgctcctcct ttgatccgag gggaaaggga ctggttatta gAAagagcct aggagacaaa 4560 agggccagtc cccctgccca gatggagca gcagcaggac agacccccac gaggcccccc 4620 aggaggagg aagatcccac ggaggacac atgaggttag ggaccttgt tcagcacccc 4680 aaacagcctg cctgtttaaa gcaggcagca ggcttaggcc tccctgcaa cccaacc 4740 cacaagtttg ttctctagg aaacacattc actgtctcag ctggctgtta ctctctcaga 4800 ccatatggca aagttttcca agaaaatgcc ccgacagggg tgcccagcac actgcctgag 4860 ggacacaga catcagaca aacccccaga gagaacagt caaatcagg gcccggtgca 4920 gtgttgtcat gtggaacctg ctttatccat tgctgagtgt tgaatgtggg taatggttag 4980 ggctttccag atctcagcag ccaaagacag ttattgttgg aagactgtca tgtagataac 5040 catgagcaat ggctcgcctc agaatcagtt cataaaattc tatggtactg gccccttcgt 5100 gggtattgtg tgaaatgaga tggtggcgag gggtgcgctg tggaactgcc gcagccacgc 5160 aggaggtccc tgggggatgc tttgggaagt cttgcccct gagcactgcc tgattgccag 5220 ggcctgtgga ggtctaggcc gcctggcaga atctagcacc gtccgaatcc ccgcaggacc 5280 catggagcta tgaccacacc aggccattca aatggctctg cattatcttc ccttggaagg 5340 tggccactcc tcggtggcag ggcctttccc tgaggctgca ggccgtgggc tggcagcccg 5400 tctcttggca tttcaattga aggtcaccag gtgctgggtt tgaaaggaag tcactggagt 5460 gctgccaggg gccgccctcc aaggttaatg agaggcccac atccaggcaa gaactaattc 5520 aaaaggcaga tcagaaacca caggagtcaa aattattgct ccggcagtgc ttcccttcct 5580 ttcatccact ggcctcgtgt ggtccatgca gggccactgt ctgccctttc tgatgccacg 5640 tattaggctt tcttactcag aattttgata gaaaaccatg gggccaagag ctctggaagc 5700 ctggccggaa agaccaaggt tcatgcagcc aacaaatga ttgttgagca cctctcggag 5760 ccaaagtcct taggcgagtg tggtgacttc ctggaaggag gatgcagact tccagagagc 5820 ccccccaacg gacgtgctga gaagggagag ggaggcgggg gctgtagtca ggaaggagcc 5880 agagaagaac agggtttggg tgcatccaga aatatgcctg cataggagg gagaggaagg 5940 ggtgccaccg tcaacggctt cccatcggag gtggttggtg cagatggaag tttctgtctg 6000 ctggccctca agagagtgtt ttgccaggga cacagtctgt tcctcctcag aaaacacccc 6060 ccaaatgcta acaacatccc caccagctgc tagaagcccc tttcccctcc ccaccttgaa 6120 gtagctcata gttctctggg cagagccaga ccatccagtg taccccagag gccagtaggt 6180 tcctgcccat tttcctctct ggcttcctgc caagaattat ggcagctgag gatgaatgga 6240 gaagtaaaaa caactaacac cgcacaacta acaactaaca ccgcagttcc cacctgggtt 6300 ccacttagca ggagacattt cggagggttt tttttgtttt tgttcctgtt tttttttttt 6360 ttgctggaat ttgttttctc agtactgaaa agaaaaag tgacaatctt gtatttttaa 6420 aagcctcgga aaggtgatac catctgacag tcattttctc acgttggtct tctaaagtca 6480 cctatttctt gtgtgtgcac atcacaccat ttcctgtttc tttataaccc gacaagggta 6540 ggagtgcctg tttcccctgc tgggcacacc agacaatcgt aatcacaaaa cagacaactga 6600 gccaggggcc caaagggtgt gatcatgaga gttaccggga cagcagtagg catgacagtc 6660 accaggaagg acaagggtgc tctgttgtta gtggccacac accaatttga caaggagtgt 6720 tgcgaaattt ttattattt atttatttt tttgagatgg agtttcactc ttgttgccca 6780 ggctggagtg cggtggtaca atctcggctc actgcaacct ccacctccca ggttcaagcg 6840 attctcctgc ctcagcctcc caagtacctg ggactacagg tgcgtgccac cacaccagc 6900 taaattttgt gtttttagta gagatggggt ttcaccatgt tggccaggat ggtcttgaac 6960 ccctgacctc atgatctgcc tgcctcggcc tcccaaagtg ctgggattac aggcatgagc 7020 caccacgcc agccaaaata tttttttaaa gtcattttcc ttaagctgct tgggctacat 7080 gtgaaataca ctggacggtc aacattcctg tctcctccca tttgggctga tgcagcagat 7140 ccagggaatg ttacctgttt ctgctgctag aagatccagg aaattgggaa ggttacctga 7200 cgcacacatg gatgaaggcc atcatctaga aatggggtca accacaattg tgttaattcc 7260 gtagtgtcag ggattcttcg ggaaggtcaa gattgaag gattctgacc cctgtgcctc 7320 ccatttatgt gatcaggtga cagttaataa ccgtggaggt cacactcagc catccaacag 7380 ccttacagtg accctacaca aaagccccca aattccaaag actttttctt aacctaaagg 7440 aagaaattat ttgttaattc fòagagagca actgaatata ctgggctatt tgtacttttt 7500 tatagagaac tttaataata attctttaaa aatgagtttt tagaacaaag caactgacga 7560 tttcctaaga ttccaatgcc ctggagcttg taggaggact tagcctgggt cagctggagc 7620 acccccgacc tgatctccca ctgccagatt ttcccatgct cctagggtat ggagtccacg 7680 tgggaatgac tgcaagttca ggtggaactt ggccgactga tgctctgcga gtttttaata 7740 gacactgggg acaactgctt aaggtttaga aacttccaaa ccacaggaaa gacattttta 7800 gtgtccccca tccagaggca gccctggaat aggattccca ggggtttctg ggaccccttt 7860 ccttgctccg tgaggctctg tggccatctt ttggcaggag gaggatgctt ccttggctct 7920 gtgcccagac ccgcctggtc cccaggtctc tcaccttggg tgaagattca gagatgccct 7980 gtaaggattt tgcccactgg gcaactcaga aatacttcga tctcccaaga tataagaggc 8040 agcagcaaac gtgcctattg acgtctgttt catagttacc acttacgcga gtagacagaa 8100 ctcggctttt cagaaaatag gtgtcaagtc cactttataa gaaccttttt ttctaaaata 8160 agataaaagg tggctttgca ttttctgatt aaacgactgt gtctttgtca cctctgctta 8220 actttaggag tatccattcc tgtgattgta gacttttgtt gatattcttc ctggaagaat 8280 atcattcttt tcttgaaggg ttggtttact agaatattca aaatcaatca tgaaggcagt 8340 tactattttg agtctaaagg ttttctaaaa attaacctca catcccttct gttagggtct 8400 ttcagaatat cttttataaa cagaagcatt tgaagtcatt gcttttgcta catgatttgt 8460 gtgtgtgaag gacataccac gtttaaatca ttaattgaaa aacatcatat aagccccaac 8520 tttgtttgga ggaagagacg gaggttgagg tttttccttc tgtataagca cctactgaca 8580 aaatgtagag gccattcaac cgtcaaacac catttggtta tatcgcagag gagacggatg 8640 tgtaaattac tgcattgctt tttttttcag tttgtataac ctctaatctc cgtttgcatg 8700 atacgctttg ttagaaacat taattgtagt ttggaagcaa gtgtgtatga ataaagataa 8760 tgatcattcc aaaaaaaaaa aaaaaaa 8787

Claims

1. 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; 10. A method of treating a vasculopathy in a subject, comprising:

2. 2. The method of claim 1, wherein the vasculopathy comprises vascular Ehlers-Danlos syndrome (vEDS).

3. 10. The method of claim 1, wherein the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof.

4. The method of claim 3 , wherein the agent comprises a small molecule.

5. 10. The method of claim 1, wherein the agent comprises cobimetinib or a pharmaceutically acceptable salt thereof.

6. 4. The method of claim 3, wherein the agent comprises ruboxistaurin or a pharmaceutically acceptable salt thereof.

7. 4. The method of claim 3, wherein the agent comprises enzastaurin or a pharmaceutically acceptable salt thereof.

8. 4. The method of claim 3, wherein the agent comprises sotrastaurin or a pharmaceutically acceptable salt thereof.

9. 10. The method of claim 1, further comprising administering an agent that decreases the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3).

10. 10. The method of claim 1, wherein the effective amount of the agent is about 0.001 mg / kg to 250 mg / kg of body weight.

11. The method of claim 1, wherein the subject comprises a level of ERK or PKC protein or mRNA that differs from a normal control.

12. 12. The method of claim 11, 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.

13. 12. The method of claim 11, 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 greater level of ERK activity or PKC activity compared to a normal control.

14. 12. The method of claim 11, wherein the level is the level in a test sample obtained from the subject.

15. 15. The method of claim 14, wherein the test sample comprises blood, serum, plasma, saliva, tears, vitreous, cerebrospinal fluid, sweat, cerebrospinal fluid, or urine.

16. 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; 10. A method of treating a connective tissue disorder in a subject, comprising:

17. 17. The method of claim 16, wherein the connective tissue disorder is selected from the group consisting of vasculopathy, Marfan syndrome, Loeys-Dietz syndrome, and familial thoracic aortic aneurysm.

18. 17. The method of claim 16, wherein the vasculopathy comprises vascular Ehlers-Danlos syndrome (vEDS).

19. 17. The method of claim 16, wherein the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof.

20. 20. The method of claim 19, wherein the agent comprises a small molecule.

21. 20. The method of claim 19, wherein the agent comprises cobimetinib or a pharmaceutically acceptable salt thereof.

22. 20. The method of claim 19, wherein the agent comprises enzastaurin or a pharmaceutically acceptable salt thereof.

23. 20. The method of claim 19, wherein the agent comprises sotrastaurin or a pharmaceutically acceptable salt thereof.

24. 20. The method of claim 19, wherein the agent comprises ruboxistaurin or a pharmaceutically acceptable salt thereof.

25. 17. The method of claim 16, further comprising administering an agent that decreases the activity or expression of phospholipase C (PLC) or inositol triphosphate (IP3).

26. 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.

27. 27. The pharmaceutical composition of claim 26, wherein the agent comprises an antibody or fragment thereof, a polypeptide, a small molecule, a nucleic acid molecule, or any combination thereof.

28. A kit comprising: (1) the pharmaceutical composition of claim 26 or 27; and (2) instructions for treating a vasculopathy.

29. 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.

10. A method for treating a connective tissue disorder, comprising:

30. 30. The method of claim 29, wherein the connective tissue disorder comprises Marfan syndrome.

31. 30. The method of claim 29, wherein the agent decreases the activity or expression of protein kinase C (PKC).

32. 32. The method of claim 31, 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%, a half-fold reduction, a third-fold reduction, a quarter-fold reduction, or a fifth-fold reduction compared to a normal control.

33. 32. The method of claim 31, 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 5-fold compared to a normal control.

34. 30. The method of claim 29, wherein the agent comprises enzastaurin or a pharmaceutically acceptable salt thereof.

35. 30. The method of claim 29, wherein the agent comprises sotrastaurin or a pharmaceutically acceptable salt thereof.

36. 30. The method of claim 29, wherein the agent comprises ruboxistaurin or a pharmaceutically acceptable salt thereof.

Images