Selective ROCK2 inhibition for the treatment of edema and related conditions

Abstract

The present disclosure provides methods and compositions for treating conditions associated with impaired lymphatic drainage.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 63 / 145,526, filed February 4, 2021, U.S. Provisional Application No. 63 / 209,324, filed June 10, 2021, and U.S. Provisional Application No. 63 / 228,404, filed August 2, 2021, each of which is incorporated by reference in its entirety.

[0002] Federally Sponsored Research This invention was made with government support under grants EB025765, EB008396, EB000262, and HL129733 awarded by the National Institutes of Health. The Government has certain rights in the invention. [Background technology]

[0003] background Conditions associated with impaired lymphatic drainage, such as lymphedema, are major pathological conditions with unknown mechanisms. Lymphedema is the excessive accumulation of interstitial fluid resulting from impaired drainage of fluid into lymphatic vasculature (1, 2) and affects more than 150 million people worldwide, but there is no effective treatment and standard care is mainly palliative (3). The lack of experimental models of normal and impaired lymphatic drainage is a major obstacle to better understanding and treatment of lymphedema. DISCLOSURE OF THEINVENTION

[0004] overview Provided herein, in some aspects, are methods and compositions for treating subjects with or at risk for conditions associated with impaired lymphatic drainage (e.g., lymphedema) with selective Rho-associated kinase 2 (ROCK2) inhibitors. Also provided herein is a biomimetic model of lymphatic vessels that was used to identify ROCK mechanisms in regulating lymphatic drainage. This microfluidic culture model of lymphatic vessels draining interstitial fluid was designed and used to demonstrate that inflammatory cytokines that disrupt vascular junctions rather tighten lymphatic vessel cell-cell junctions, preventing lymphatic drainage. Data herein show that inhibition of ROCK normalized fluid drainage under cytokine challenge by loosening lymphatic junctions in culture and tightening vascular junctions, and completely reversed lymphedema in a mouse model. Additionally, we identified the ROCK2 / JAM-A / ZO-1 complex, which is associated with distinct cytokine responses in lymphatic junctions.

[0005] Thus, some aspects of the present disclosure provide a method of administering a selective ROCK2 inhibitor to a subject (e.g., a human subject) having or at risk of a condition associated with impaired lymphatic drainage. In some embodiments, the subject has a condition associated with impaired lymphatic drainage. In some embodiments, the condition is edema. In some embodiments, the condition is lymphedema. The lymphedema can be primary lymphedema or secondary lymphedema. Some aspects of the present disclosure provide a method comprising administering a selective ROCK2 inhibitor to a subject in an amount effective to restore vascular barrier function.In some embodiments, the amount of the selective ROCK2 inhibitor is effective to reduce fluid leakage from the blood vessel of the subject compared to a control.In some embodiments, the amount of the selective ROCK2 inhibitor is effective to increase lymphatic drainage in the subject compared to a control.

[0006] Another aspect of the disclosure provides a method that includes administering to a subject a selective ROCK2 inhibitor in an amount effective to improve lymphatic drainage at a lymphatic junction in the subject compared to a control. A further aspect of the present disclosure provides a method comprising administering to a subject a selective ROCK2 inhibitor in an amount effective to prevent formation of tight lymphatic junctions in the subject. In some embodiments, the selective ROCK2 inhibitor is administered in an amount effective to reduce a symptom associated with impaired lymphatic drainage. In some embodiments, the symptom is swelling in a body part of the subject. In some embodiments, the selective ROCK2 inhibitor is administered orally. In some embodiments, the selective ROCK2 inhibitor is formulated as a tablet. In some embodiments, the selective ROCK2 inhibitor is administered at a dose of 200-400 mg.

[0007] Another aspect of the present disclosure provides a method comprising contacting a dermal lymphatic endothelial cell with a selective ROCK2 inhibitor in an amount effective to prevent formation of lymphatic-tite junctions. Yet another aspect of the present disclosure provides a method of contacting a lymphatic-tite junction with a selective ROCK2 inhibitor in an amount effective to improve lymphatic drainage at the lymphatic junction compared to a control. In some embodiments, the selective ROCK2 inhibitor binds to ROCK2 and inhibits ROCK2 serine / threonine kinase activity. In some embodiments, the selective ROCK2 inhibitor is selected from a polypeptide inhibitor, a polynucleotide inhibitor, and a small molecule inhibitor. In some embodiments, the selective ROCK2 inhibitor is a small molecule inhibitor.

[0008] In some embodiments, the small molecule inhibitor has an IC50 of 50 nM to 150 nM for ROCK2. 50 In some embodiments, the small molecule inhibitor binds to ROCK2 with an IC value of 50 nM to 150 nM without binding to PKA, PKG, PKC, or MRCK.50 In some embodiments, the small molecule inhibitor binds to ROCK1 with an IC value of 20,000 to 25,0000 nM. 50 Other ROCK2 inhibitors may be used in some embodiments, including SR-3677 (TOCHRIS®) and Chroman 1 (MCE®). In some embodiments, the selective ROCK2 small molecule inhibitor is 2-[3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy]-N-(1-methylethyl)-acetamide (KD025). In some embodiments, the selective ROCK2 small molecule inhibitor is selected from compounds 1-559, or a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, co-crystal, or prodrug thereof.

[0009] In some embodiments, the selective ROCK2 small molecule inhibitor is selected from a compound in Table 1, or a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, co-crystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is selected from a compound in Table 2, or a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, co-crystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is selected from a compound in Table 3, or a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, co-crystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is selected from a compound in Table 4, or a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, co-crystal, or prodrug thereof.

[0010] Yet another aspect of the present disclosure provides a biomimetic model of a lymphatic vessel, comprising a polymer-based housing comprising at least two parallel cylindrical channels within a three-dimensional gel, wherein human dermal cells are seeded into one of the cylindrical channels, and optionally, wherein the model further comprises lymphangiogenic growth factors. Yet another aspect of the present disclosure provides a method that includes: providing a biomimetic device comprising a polymer-based housing comprising at least two parallel cylindrical channels within a three-dimensional gel, where human dermal cells are seeded into one of the channels to generate modified lymphatic vessels expressing lymphatic markers; and stimulating the human dermal cells with lymphangiogenic growth factors to cause sprouting of lymphatic vessels. In some embodiments, the method further includes pressurizing the other channel with a surrogate lymph fluid composed of fluorescently labeled lymph molecules including fatty acids, phospholipids, albumin, and insoluble particles, and evaluating the lymph fluid drained by the modified lymphatic vessel.

[0011] In some embodiments, the polymer-based housing is selected from polydimethylsiloxane (PDMS), polypropylene, polystyrene, polyurethane, polycaprolactone, polymethylmethacrylate, and polyacetate. In some embodiments, the gel is selected from collagen gels, fibrin, materiel, synthetic hydrogels, and hydrogels made from other extracellular matrix (ECM) components. In some embodiments, the human dermal cells are human dermal lymphatic endothelial cells (LECs). In some embodiments, the LECs exhibit proper apical-basal orientation. In some embodiments, the lymphangiogenic growth factor is vascular endothelial growth factor C (VEGFC). In some embodiments, the modified lymphatic vessels express lymphatic endothelial markers. [Brief description of the drawings]

[0012] [Figure 1A-F] Figures 1A-1J. Lymphatics-on-chip recapitulates lymphatic structure, drainage, and dysfunction. (Figure 1A) Schematic of lymphatics-on-chip. (Figure 1B) Apical podoplanin (PDPN) expression at the luminal side of the vessels. (Figure 1C) Lymphatic sprouting in response to VEGFC stimulation. (Figure 1D) Immunostaining of LEC-generated lymphatic vessels and BEC-generated blood vessels with tight junction markers (JAM-A, ZO-1). (Figure 1E) Staining of adherens junctions. Red stars represent portal vein-like structures. (Figure 1F) F-actin staining confirming portal vein-like structures in lymphatic vessels. [Figure 1G-J] (Figure 1G) Percent drainage of lymphatic molecules through LEC and BEC lined channels compared to acellular channels (no cells). (Figure 1H) Representative images of BODIPY-C16 fatty acid drainage by LEC-generated lymphatic vessels and BEC-generated blood vessels. (Figure 1I) Percent lymphatic drainage after cytokine exposure. (Figure 1J) VE-cadherin staining of engineered lymphatic vessels after cytokine exposure. Scale bars (Figure 3A, 3B, 3C, 3D, 3E, 3H) = 100 μm; Scale bars (Figure 3F, 3J) = 50 μm. * (p < 0.05) and ** (p < 0.01) indicate statistical significance. [Figure 2A-E] Figures 2A-2J. ROCK inhibition normalizes LEC and BEC junctions and reverses lymphedema in vivo. (Figure 2A) Percent lymphatic drainage with Y27632. (Figure 2B) VE-cadherin staining of LECs exposed to Y27632. (Figure 2C) Percent lymphatic drainage with Y27632, fasudil, and ripasudil. (Figure 2D) Dye leakage-based permeability assay and VE-cadherin images of engineered blood vessels. (Figure 2E) Permeability coefficient of engineered blood vessels in inflammation with and without Y27632. [Figure 2F-J](Figure 2F) Effect of ROCK inhibition on LEC and BEC junctions. (Figure 2G) Changes in tail volume by Y27632 in a lymphedema model. (Figure 2H) Drainage to iliac lymph nodes 1 h after intradermal injection of Alexa647-conjugated albumin into the tails of mice treated with Y27632 or vehicle. (Figure 2I) Changes in tail volume with delayed exposure to Y27632. (Figure 2J) Representative images of tails before and after Y27632 treatment. Scale bar (Figure 2B and Figure 2D, lower panel) = 50 μm; Scale bar (Figure 2D, upper panel) = 200 μm; Scale bar (J) = 5 mm; * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) indicate statistical significance.

[0013] [Figure 3A-G] 3A-3L. ROCK2 forms a unique tight junction complex in inflamed LEC but not BEC. (FIG. 3A) Western blot showing basal levels of ROCK1 / 2 expression in LEC and BEC. (FIG. 3B) Western blot showing downregulation of ROCK1 in inflamed LEC. (FIG. 3C) siRNA-mediated knockdown of ROCK1 and ROCK2 in LEC. (FIG. 3D) Percent lymphatic drainage by LEC knockdown ROCK1 or ROCK2 in normal or IL-2 conditions. (FIG. 3E) VE-cadherin images of modified LV with or without ROCK1 / 2 knockdown in normal or IL-2 conditions. (FIG. 3F) Permeability coefficient of modified BV with or without ROCK1 knockdown in normal or IL-2 conditions. (FIG. 3G) VE-cadherin images of modified BV with or without ROCK1 knockdown in normal or IL-2 conditions. [Figure 3H-L](Fig. 3H) Immunoprecipitation data showing ROCK2 interaction with junctional adhesion molecule-A (JAM-A) and zonula occludens-1 (ZO-1) in inflamed LEC or ROCK1 kd LEC. (Fig. 3I) Immunofluorescence data showing ROCK2 localization to junctional regions and reinforced tight junctions (JAM-A) in inflamed LEC or ROCK1 kd LEC. (Fig. 3J) VE-cadherin images of modified LV with JAM-A knockdown in normal or IL-2 conditions. (Fig. 3K) Immunoprecipitation data showing that ROCK2 downregulated BEC do not form ROCK2-JAM-A complexes in normal or IL-2 conditions. (Fig. 3L) Percent lymphatic drainage by ROCK2 specific inhibitor KD025 (20 μM) in normal or IL-2 conditions. Scale bar 50 μm. *(p<0.05) indicates statistical significance.

[0014] [Figure 4] Figure 4A is a photograph showing that tail vein edema after cautery injury is visualized by swelling of the tail (left) and is reduced by treatment with KD025 (right). Figure 4B is a graph of the quantified tail volume over time. Figure 4C is a graph of data showing drainage to iliac lymph nodes 1 hour after intradermal injection of Alexa647-conjugated albumin into the tails of mice treated with KD025 or vehicle.

[0015] Detailed Description In some aspects, a method is provided herein for administering a selective Rho-associated kinase 2 (ROCK2) inhibitor to a subject having or at risk of a condition associated with impaired lymphatic drainage. Also provided herein is a method for treating a condition associated with impaired lymphatic drainage. It should be understood that "treating a condition" in a subject includes administering to the subject an agent that alleviates the symptom(s) associated with the condition. For example, treating lymphedema in a subject's extremities (e.g., legs or arms) can include administering to the subject a selective ROCK2 inhibitor to reduce or eliminate swelling in the extremities.

[0016] Another aspect of the present disclosure provides a method, comprising contacting dermal lymphatic endothelial cells with an effective amount of a selective Rho-associated kinase 2 (ROCK2) inhibitor to prevent the formation of lymphatic tight junctions.For example, FIG. 2G shows the preventive effect of ROCK2 inhibition in lymphedema.In some embodiments, the formation of tight junctions can be analyzed by staining VE-cadherin or JAM-A (junction marker) together with LYVE-1 or podoplanin (lymphatic marker). Yet another aspect of the disclosure provides a method comprising contacting a lymphatic-tite junction with a selective ROCK2 inhibitor in an amount effective to improve lymphatic drainage at the lymphatic junction compared to a control.

[0017] Lymphatic system The lymphatic system is an important part of the immune system. It helps maintain tissue homeostasis, including transport of interstitial proteins, tissue fluid balance, and the development of cell-mediated immunity. Lymph nodes, present throughout the lymphatic system, contain large numbers of lymphocytes, macrophages, and antigen-presenting cells that together initiate the primary immune response. Specialized lymphatic endothelial cells (LECs) aid in the primary immune response by recruiting intravascular lymphocytes into the circulation. In addition, LECs are responsible for facilitating the transmigration of intravascular lymphocytes into the reticular meshwork where they can interact with antigen-presenting cells. LECs have also been shown to be associated with chronic inflammation and cancer.

[0018] The data provided herein show that ROCK2 is selectively expressed in LECs (FIGS. 3A and 3B), and that loss of ROCK2 expression loosens adherens junctions (FIG. 3E). Taken together, these data suggest that inhibition of ROCK2 normalizes tight adherens junctions to support lymphatic drainage. Thus, some aspects of the disclosure provide methods that include contacting dermal lymphatic endothelial cells with a selective ROCK2 inhibitor in an amount effective to prevent lymphatic-tite junction formation. Further studies described herein show that KD025, a ROCK2-specific small molecule inhibitor, reverses the tightening of LEC junctions (data not shown) and impaired drainage (FIG. 3L) in response to IL-2 challenge. Taken together, the data show that ROCK2 tightens lymphatic junctions when inflammatory signals are present. Thus, some aspects of the present disclosure provide methods that include contacting lymphatic junctions with a selective ROCK2 inhibitor in an amount effective to improve lymphatic drainage at lymphatic junctions compared to a control. In some embodiments, the control is a measure of lymphatic drainage at lymphatic junctions before contacting with the selective ROCK2 inhibitor.

[0019] Impaired lymphatic drainage and edema Edema is the accumulation of fluid in the interstitial space that occurs when capillary filtration exceeds the limits of lymphatic drainage, causing prominent clinical signs and symptoms (Trayes KP et al. Am Fam Physician. 2013; 88(2):102-110). Chronic accumulation of edema in one or both lower limbs often indicates venous insufficiency, especially when dependent edema and hemosiderin deposition are present. Impaired lymphatic drainage occurs due to obstruction / blockage of lymphatic vessels that slows or prevents lymphatic circulation. A "condition associated with impaired lymphatic drainage" is a condition that develops as a result of impaired lymphatic drainage. Impaired lymphatic drainage can cause edema, such as, for example, lymphedema. Thus, edema (e.g., lymphedema) is a non-limiting example of a condition associated with impaired lymphatic drainage. Causes of impaired lymphatic drainage (e.g., lymphedema) include, for example, removal or injury of lymph nodes (e.g., during cancer treatment), obstruction due to benign or malignant tumor growth, physical trauma, surgery, obesity, infection of lymph nodes, chronic venous insufficiency, or malformation of the developing lymph nodes and lymphatic system. Edema and lymphedema include, by way of non-limiting examples, pulmonary, facial, mastectomy, extremity, cerebral, groin, and cardiac edema, whether due to surgical or postoperative sequelae, or other inflammatory, cardiovascular, or cancer-related processes.

[0020] Nearly every organ has lymphatic vessels present in it, which drain not only fluids but also lipids, immune cells, and metabolic waste products. Other examples of lymphatic drainage disorders therefore include lipedema (abnormal lipid / fat deposits), obesity and metabolic disease (intestinal lymphatic "mammary" dysfunction), CNS degeneration (cerebral lymphatic dysfunction and waste deposits), and cellulitis (frequent skin infections due to impaired lymphatic drainage of dendritic cells under infection). In some embodiments, the subject with lymphatic drainage disorder develops lymphedema.Lymphedema is abnormal swelling, for example, in arms, legs, breasts, or trunk, when lymphatic system is impaired (see, for example, Grada AA et al. Lymphedema 2017; 77(6): 1009-1020).Lymphedema can be either primary or secondary.Primary lymphedema occurs in the absence of disease or condition that causes it (e.g., may be hereditary), while secondary lymphedema is caused by another disease or condition. Causes of secondary lymphedema include, but are not limited to, surgery (e.g., removal or damage to lymph nodes and lymphatic vessels), radiation therapy (e.g., radiation can cause scarring and inflammation of lymph nodes or lymphatic vessels), cancer (e.g., lymphedema can occur when cancer cells block lymphatic vessels), and / or infection (e.g., lymph node infection or parasites can restrict lymphatic flow). In some embodiments, the subject has primary lymphedema. In other embodiments, the subject has secondary lymphedema.

[0021] Symptoms of impaired lymphatic drainage (e.g., lymphedema) include, for example, swelling, heaviness or pressure, limited range of motion, pain or discomfort, recurrent infections, and / or hardening and thickening of the skin (fibrosis) in part or all of an arm or leg, including fingers and toes. In some embodiments, the selective ROCK2 inhibitor is administered in an amount effective to reduce symptoms (e.g., reduce the severity of symptoms) associated with impaired lymphatic drainage. Reducing symptoms can be determined by the subject and / or a medical professional (e.g., a primary care physician). For example, the selective ROCK2 inhibitor can be administered in an amount effective to reduce (e.g., at least 10%, 25%, 50%, or 75%) or eliminate swelling in a body part (e.g., an arm or leg) of the subject compared to a control. In some embodiments, the control is the degree of swelling (e.g., volume of fluid) in a particular body part prior to administration of the selective ROCK2 inhibitor.

[0022] It should be understood that a subject having a condition associated with impaired lymphatic drainage is one who is exhibiting symptoms of impaired lymphatic drainage and / or who has been diagnosed (e.g., by a medical professional) as having a condition associated with impaired lymphatic drainage (e.g., lymphedema). Subjects at risk for having a condition associated with impaired lymphatic drainage are those who have undergone or will undergo (e.g., within 1 to 6 months) surgery and / or radiation therapy, or who have cancer or an infection (e.g., in or near a lymph node).

[0023] Selective ROCK2 inhibitors ROCK2 (ex: UniProtKB O75116) is a member of the Rho-associated protein kinase family. It is a key regulator of the actin cytoskeleton and cell polarity and is involved in the regulation of smooth muscle contraction, actin cytoskeleton organization, stress fiber and focal adhesion formation, neurite retraction, cell adhesion, and motility (e.g., Kawano Y. et al. J. Cell Biol. 1999; 147: 1023-1038;Sebbagh M. et al. J. Exp. Med. 2005; 201: 465-471;Tanaka T. et al. J. Biol. Chem. 2006; 281: 15320-15329;Ma Z. et al. Mol. Cell. Biol. 2006; 26: 9016-9034;Wang Y. et al. Circ. Res. 2009; 104: 531-540;Lock F. et al. PLoS ONE 2009; 4: E8190-E8190; Wang H. et al. Cancer Res. 2011; 71: 68-77; and Herskowitz J. et al. J. Biol. Chem. 2011; 286:6117-6127).

[0024] As used herein, a selective ROCK2 inhibitor has an IC2 activity that is at least 2-fold lower (e.g., 2-10-fold lower) for ROCK2 compared to ROCK1. 50In some embodiments, a selective ROCK2 inhibitor is an agent (e.g., a polypeptide, polynucleotide, or a compound (e.g., a small molecule)) having an IC 50 In some embodiments, the selective ROCK2 inhibitor can inhibit ROCK2 gene expression, ROCK2 protein production, and / or ROCK2 protein activity without substantially inhibiting the gene expression, protein production, and / or protein function of other ROCK family members, such as ROCK1 (e.g., UniProtKB Q13464). The use of selective ROCK2 inhibitors, in some embodiments, avoids the negative side effects associated with ROCK1 and / or pan-ROCK1 inhibition, such as hypotension.

[0025] Inhibition includes preventing gene expression and / or protein activity such that gene expression, protein production, and / or protein activity cannot be detected using standard gene expression, protein expression, and / or protein activity assays. Inhibition also includes reducing gene expression, protein expression, and / or protein activity such that gene expression, protein production, and / or protein activity can be detected using standard gene expression, protein production, and / or protein activity assays at a lower level than the control. For example, ROCK2 gene expression, ROCK2 protein production, and / or ROCK2 protein activity can be reduced by at least 10% in the presence of a selective ROCK2 inhibitor (e.g., a small molecule inhibitor) compared to a control. In some embodiments, ROCK2 gene expression, ROCK2 protein production, and / or ROCK2 protein activity is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% in the presence of a selective ROCK2 inhibitor (e.g., a small molecule inhibitor) compared to a control. In some embodiments, ROCK2 gene expression, ROCK2 protein production, and / or ROCK2 protein activity is reduced by 10%-100% in the presence of a selective ROCK2 inhibitor (e.g., a small molecule inhibitor) compared to a control. For example, ROCK2 gene expression, ROCK2 protein production, and / or ROCK2 protein activity may be reduced by 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 10%-90%, 20%-90%, 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%, 80%-90%, 10%-80%, 20%-80%, 30%-80%, 40%-80%, 50%-80%, 60%-80%, or 70%-80% in the presence of a selective ROCK2 inhibitor (e.g., a small molecule inhibitor) compared to a control. In some embodiments, the control is ROCK2 gene expression in the absence of a ROCK2 inhibitor (all other conditions being the same).In some embodiments, the control is ROCK2 protein production in the absence of a ROCK2 inhibitor (all other conditions are the same). In some embodiments, the control is ROCK2 protein activity in the absence of a ROCK2 inhibitor (all other conditions are the same).

[0026] Methods for measuring gene expression levels, protein expression levels, and / or protein expression levels are known. Non-limiting examples of gene expression assays include in situ hybridization and polymerase chain reaction. Non-limiting examples of protein production and / or activity assays include antibody-based assays such as enzyme-linked immunosorbent assay (ELISA), immunostaining, immunoblotting, and flow cytometry. In some embodiments, the assay for measuring gene expression level, protein expression level, and / or protein expression level is performed on a sample obtained from a subject (e.g., a subject administered a selective ROCK2 inhibitor). The sample can be a blood sample or a tissue sample. Other biological samples can also be used.

[0027] In some embodiments, the selective ROCK2 inhibitor binds to ROCK2 and inhibits ROCK2 serine / threonine kinase activity. In some embodiments, the selective ROCK2 inhibitor binds to ROCK2 and inhibits ROCK2 serine / threonine kinase activity by at least 10% compared to a control. For example, the selective ROCK2 inhibitor can bind to ROCK2 and inhibit ROCK2 serine / threonine kinase activity by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% compared to a control. In some embodiments, the selective ROCK2 inhibitor binds to ROCK2 and inhibits ROCK2 serine / threonine kinase activity by 10% to 100% compared to a control. For example, a selective ROCK2 inhibitor may bind to ROCK2 and inhibit ROCK2 serine / threonine kinase activity by 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 10%-90%, 20%-90%, 30%-90%, 40%-90%, 50%-90%, 60%-90%, 70%-90%, 80%-90%, 10%-80%, 20%-80%, 30%-80%, 40%-80%, 50%-80%, 60%-80%, or 70%-80% relative to a control. In some embodiments, the control is ROCK2 serine / threonine kinase activity in the absence of a ROCK2 inhibitor (all other conditions being the same).

[0028] Non-limiting examples of polynucleotides that selectively inhibit the expression of the ROCK2 gene and / or ROCK2 protein include antisense molecules and RNA interference (RNAi) molecules that specifically bind to the ROCK2 gene (e.g., the promoter, coding region, non-coding region, or other regulatory region of the ROCK2 gene). Examples of RNAi molecules include, but are not limited to, small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). Non-limiting examples of polypeptides that selectively inhibit ROCK2 protein activity include antibodies, aptamers, and other protein-binding molecules. The term "antibody" includes whole antibodies and antibody fragments (e.g., single-chain variable fragments (scFv)). In some embodiments, the selective ROCK2 inhibitor is an antibody (e.g., a monoclonal antibody) that specifically binds to ROCK2.

[0029] Non-limiting examples of compounds that selectively inhibit ROCK2 gene expression and / or ROCK2 protein activity include small molecules (e.g., less than 10 kDa). For example, small molecule inhibitors can inhibit ROCK2 with IC 50 In some embodiments, small molecule inhibitors bind with an IC value of 50 nM to 150 nM (e.g., 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM). 50 For example, a small molecule inhibitor binds to ROCK1 with an IC value of greater than 500 nM, greater than 1000 nM, or greater than 10,000 nM. 50 In some embodiments, the small molecule inhibitor binds to ROCK1 with an IC value of 20,000 nM to 25,000 nM. 50 Join by value. In some embodiments, the selective ROCK2 inhibitor is 2-[3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy]-N-(1-methylethyl)-acetamide (KD025). KD025 is also known as SLx-2119 (Boerma et al. Blood Coagul Fibrinolysis 2009; 19(7): 709-718). KD025 inhibits ROCK2 by binding to and suppressing its serine / threonine kinase activity. This inhibits the signal transduction pathway mediated by ROCK2. Surprisingly, as discussed herein, KD025 alone was sufficient to reduce lymphatic drainage in an in vitro model system (see FIG. 3L). Thus, in some embodiments, the present disclosure provides a method of administering KD025 to a subject having or at risk of a condition associated with impaired lymphatic drainage. Other non-limiting examples of ROCK2 inhibitors include SR-3677 (TOCHRIS®) and Chroman 1 (MCE®).

[0030] Other non-limiting examples of selective ROCK2 inhibitors are provided in Table 1 and International Publication No. WO 2019 / 145729 A1, the entirety of which is incorporated herein by reference. In some embodiments, the selective ROCK2 small molecule inhibitor is a compound of formula (I) (i.e., any subgenera or species thereof) disclosed in International Publication No. WO 2019 / 145729 A1 (the entirety of which is incorporated herein by reference); or a pharmaceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, cocrystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is a compound of formula (I) (i.e., any subgenera or species thereof) disclosed in International Publication No. WO 2019 / 145729 A1 (the entirety of which is incorporated herein by reference); or a pharmaceutically acceptable salt thereof.

[0031] For example, in some embodiments, the selective ROCK2 small molecule inhibitor is a compound of formula (I), or a pharma- ceutically acceptable salt thereof: [ka] During the ceremony: A 1 , A 2 and A 3 are each independently CH, CR 7 and N; B represents a 5-10 membered carbocyclic or 5-10 membered heterocyclic ring system; R 1 ,LR 2 where L is a bond or -L 1 -L 2 - and; L 1 is a bond, -(CR A R B ) 1~3 -, -O(CR A R B ) 1~3 -, -(CR A R B ) 0~3 O-, and -NR C (CR A R B ) 1~3 - selected from, and L 2 is a bond, -(CR A R B ) 1~3 -, -O-, -NR D -, -C(O)NR D -, -NR D C(O)-, -C(O)O-, -OC(O)-, -C(O)-, -S(O)2NR D -, -NR D S(O)2-, -S(O)2-, -S(O)(NR D )-, -NR D C(O)NR E -, -OC(O)NR D - and -C(O)NR D S(O)2-;

[0032] R 2-H, -CN, C 1~6 Alkyl, C 1~6 Haloalkyl, -OR F C replaced with 1~6 Alkyl, -NR F R G C replaced with 1~6 Alkyl, -OR F C replaced with 1~4 Haloalkyl, C substituted with -OH 3~8 Cycloalkyl, 3-8 membered heterocycloalkyl substituted C 1~4 C substituted with alkyl, 6-membered heteroaryl 1~4 Alkyl, -(CR H R I ) 1~3 OR F , -(CR H R I ) 1~3 NR F R G , -(CR N R O ) 1~3 C(O)OR F , -(CR N R O ) 1~3 C(O)NR F R G , C 3~10 a carbocyclic ring system, and a 3- to 10-membered heterocyclic ring system, wherein the carbocyclic or heterocyclic ring system is unsubstituted or is selected from the group consisting of ═O, —NR F R G , -C(O)R F , halogen, -CN, C 1~4 Alkyl, C 1~4 Haloalkyl or -OR F C replaced with 1~4 is substituted with alkyl; R 4 represents independently in each occurrence a halogen, C 1~4 Alkyl, C 1~6 Haloalkyl, -CN, -OR J , =O, -OR J C replaced with 1~4 Alkyl, -NR J R K , -NR J RK C replaced with 1~4 Alkyl, C 3~8 Cycloalkyl, C 3~8 Cycloalkyl-substituted C 1~4 Alkyl, 3- to 8-membered heterocycloalkyl, and C substituted with 3- to 8-membered heterocycloalkyl 1~4 alkyl;

[0033] R 5 -H, C 1~4 Alkyl, -OR L C replaced with 1~4 Alkyl, -NR L R L C replaced with 1~4 Alkyl, C 3~8 Cycloalkyl, substituted or unsubstituted phenyl, 3-8 membered heterocycloalkyl, C 3~8 Cycloalkyl-substituted C 1~4 C substituted with alkyl, 3-8 membered heterocycloalkyl 1~4 alkyl, and substituted or unsubstituted 5- or 6-membered heteroaryl, wherein the phenyl or heteroaryl group is selected from one or two R 9 may be substituted with; R 6 -H and C 1~4 alkyl; R 7 -H, halogen, -OR M , C 1~4 Alkyl, C 1~6 Haloalkyl, C 1~4 Alkenyl, -CN, and C 3~8 cycloalkyl; R 8 -H, halogen, C 1~4 Alkyl, C 1~6 Haloalkyl, -CN, and C 3~8 cycloalkyl; R 9 is halogen and C 1~4 alkyl; n is 0, 1, or 2;

[0034] R A and R B -H, C 1~4 Alkyl, and C 1~4 haloalkyl or R A and R B together with the atoms to which they are attached form a 3- to 6-membered cycloalkyl ring or a 3- to 6-membered heterocycloalkyl ring; R C , R D , R E , R F and R G are each independently H, C 1~4 Alkyl, and C 1~4 haloalkyl; R H and R I Each represents a pair of R H and R I is -H, except that, taken together with its carbon atoms, forms a 3- to 6-membered cycloalkyl ring or a 3- to 6-membered heterocycloalkyl ring; and R J , R K , R L , R M , R N and R O are each independently in each occurrence H and C 1~4 Additional embodiments, including subgenera and species of Formula (I), are provided in International Publication No. WO 2019 / 145729 A1, which is incorporated herein by reference in its entirety.

[0035] In some embodiments, the selective ROCK2 small molecule inhibitor is a compound of formula (I), or a pharma- ceutically acceptable salt thereof: [ka] During the ceremony: A 1 , A 2 and A 3 are each independently CH, CR 7 , and N; B represents a 5-10 membered carbocyclic or 5-10 membered heterocyclic ring system; R 1 ,LR 2 where L is -L 1 -L 2 - and; L 1 is a bond, -(CR A R B ) 1~3 -, -O(CR A R B ) 1~3 -, -(CR A R B ) 0~3 O-, and -NR C (CR A R B ) 1~3 - selected from, and L 2 is a bond, -(CR A R B ) 1~3 -, -O-, -NR D -, -C(O)NR D -, -NR D C(O)-, -C(O)O-, -OC(O)-, -C(O)-, -S(O)2NR D -, -NR D S(O)2-, -S(O)2-, -S(O)(NR D )-, -NR D C(O)NR E -, -OC(O)NR D - and -C(O)NR D S(O)2-;

[0036] R 2 -H, -CN, C 1~6 Alkyl, C 1~6 Haloalkyl, -OR F C replaced with 1~6 Alkyl, -NR F R G C replaced with 1~6 Alkyl, C substituted with -OH 3~8 Cycloalkyl, 3-8 membered heterocycloalkyl substituted C 1~4 C substituted with alkyl, 6-membered heteroaryl 1~4 Alkyl, -(CRH R I ) 1~3 OR F , -(CR H R I ) 1~3 NR F R G , C 3~10 Carbocyclic ring systems and 3- to 10-membered heterocyclic ring systems, where the carbocyclic or heterocyclic ring systems are unsubstituted or include ═O, —NR F R G , halogen, C 1~4 Alkyl or -OR F C replaced with 1~4 is substituted with alkyl; R 4 is a halogen, C 1~4 Alkyl, C 1~6 Haloalkyl, -CN, -OR J , =O, -OR J C replaced with 1~4 Alkyl, -NR J R K , -NR J R K C replaced with 1~4 Alkyl, C 3~8 Cycloalkyl, C 3~8 Cycloalkyl-substituted C 1~4 Alkyl, 3- to 8-membered heterocycloalkyl, and C substituted with 3- to 8-membered heterocycloalkyl 1~4 alkyl;

[0037] R 5 -H, C 1~4 Alkyl, -OR L C replaced with 1~4 Alkyl, C 3~8 Cycloalkyl, substituted or unsubstituted phenyl, 3-8 membered heterocycloalkyl, C 3~8 Cycloalkyl-substituted C 1~4 C substituted with alkyl, 3-8 membered heterocycloalkyl 1~4 alkyl, and substituted or unsubstituted 5- or 6-membered heteroaryl, wherein the phenyl or heteroaryl group is selected from one or two R 9 may be substituted with; R 6 -H and C 1~4 alkyl; R 7 -H, halogen, -OR M , C 1~4 Alkyl, C 1~6 Haloalkyl, -CN, and C 3~8 cycloalkyl; R 8 -H, halogen, C 1~4 Alkyl, C 1~6 Haloalkyl, -CN, and C 3~8 cycloalkyl; R 9 is halogen and C 1~4 alkyl; n is 0, 1, or 2;

[0038] R A and R B -H, C 1~4 Alkyl, and C 1~4 haloalkyl or R A and R B together with the atoms to which they are attached form a 3- to 6-membered cycloalkyl ring or a 3- to 6-membered heterocycloalkyl ring; R C , R D , R E , R F and R G are each independently -H and C 1~4 alkyl; R H and R I Each represents a pair of R H and R I is H, except for the carbon atom which together with the carbon atom forms a 3- to 6-membered cycloalkyl ring or a 3- to 6-membered heterocycloalkyl ring; and R J , R K , R L and R M is independently in each occurrence -H and C 1~4Additional embodiments, including subgenera and species of Formula (I), are provided in International Publication No. WO 2019 / 145729 A1, which is incorporated herein by reference in its entirety.

[0039] In some embodiments, the selective ROCK2 small molecule inhibitor comprises a compound selected from compounds 1-317 in Table 1. In some embodiments, the selective ROCK2 small molecule inhibitor is selected from compounds 1-317 in Table 1. In some embodiments, the selective ROCK2 small molecule inhibitor is any one of compounds 1-317 in Table 1, or a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, co-crystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is any one of compounds 1-317 in Table 1, or a pharma- ceutically acceptable salt thereof.

[0040] Table 1. Examples of selective ROCK2 inhibitors [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

[0041] In some embodiments, the selective ROCK2 inhibitor comprises compound 1. In some embodiments, the selective ROCK2 inhibitor comprises compound 2. In some embodiments, the selective ROCK2 inhibitor comprises compound 3. In some embodiments, the selective ROCK2 inhibitor comprises compound 4. In some embodiments, the selective ROCK2 inhibitor comprises compound 5. In some embodiments, the selective ROCK2 inhibitor comprises compound 6. In some embodiments, the selective ROCK2 inhibitor comprises compound 7. In some embodiments, the selective ROCK2 inhibitor comprises compound 8. In some embodiments, the selective ROCK2 inhibitor comprises compound 9. In some embodiments, the selective ROCK2 inhibitor comprises compound 10. In some embodiments, the selective ROCK2 inhibitor comprises compound 11. In some embodiments, the selective ROCK2 inhibitor comprises compound 12. In some embodiments, the selective ROCK2 inhibitor comprises compound 13. In some embodiments, the selective ROCK2 inhibitor comprises compound 14. In some embodiments, the selective ROCK2 inhibitor comprises compound 15.

[0042] In some embodiments, the selective ROCK2 inhibitor comprises compound 16. In some embodiments, the selective ROCK2 inhibitor comprises compound 17. In some embodiments, the selective ROCK2 inhibitor comprises compound 18. In some embodiments, the selective ROCK2 inhibitor comprises compound 19. In some embodiments, the selective ROCK2 inhibitor comprises compound 20. In some embodiments, the selective ROCK2 inhibitor comprises compound 21. In some embodiments, the selective ROCK2 inhibitor comprises compound 22. In some embodiments, the selective ROCK2 inhibitor comprises compound 23. In some embodiments, the selective ROCK2 inhibitor comprises compound 24. In some embodiments, the selective ROCK2 inhibitor comprises compound 25. In some embodiments, the selective ROCK2 inhibitor comprises compound 26. In some embodiments, the selective ROCK2 inhibitor comprises compound 27. In some embodiments, the selective ROCK2 inhibitor comprises compound 28. In some embodiments, the selective ROCK2 inhibitor comprises compound 29. In some embodiments, the selective ROCK2 inhibitor comprises compound 30. In some embodiments, the selective ROCK2 inhibitor comprises compound 31.

[0043] In some embodiments, the selective ROCK2 inhibitor comprises compound 32. In some embodiments, the selective ROCK2 inhibitor comprises compound 33. In some embodiments, the selective ROCK2 inhibitor comprises compound 34. In some embodiments, the selective ROCK2 inhibitor comprises compound 35. In some embodiments, the selective ROCK2 inhibitor comprises compound 36. In some embodiments, the selective ROCK2 inhibitor comprises compound 37. In some embodiments, the selective ROCK2 inhibitor comprises compound 38. In some embodiments, the selective ROCK2 inhibitor comprises compound 39. In some embodiments, the selective ROCK2 inhibitor comprises compound 40. In some embodiments, the selective ROCK2 inhibitor comprises compound 41. In some embodiments, the selective ROCK2 inhibitor comprises compound 42. In some embodiments, the selective ROCK2 inhibitor comprises compound 43. In some embodiments, the selective ROCK2 inhibitor comprises compound 44. In some embodiments, the selective ROCK2 inhibitor comprises compound 45. In some embodiments, the selective ROCK2 inhibitor comprises compound 46. In some embodiments, the selective ROCK2 inhibitor comprises compound 47.

[0044] In some embodiments, the selective ROCK2 inhibitor comprises compound 48. In some embodiments, the selective ROCK2 inhibitor comprises compound 49. In some embodiments, the selective ROCK2 inhibitor comprises compound 50. In some embodiments, the selective ROCK2 inhibitor comprises compound 51. In some embodiments, the selective ROCK2 inhibitor comprises compound 52. In some embodiments, the selective ROCK2 inhibitor comprises compound 53. In some embodiments, the selective ROCK2 inhibitor comprises compound 54. In some embodiments, the selective ROCK2 inhibitor comprises compound 55. In some embodiments, the selective ROCK2 inhibitor comprises compound 56. In some embodiments, the selective ROCK2 inhibitor comprises compound 57. In some embodiments, the selective ROCK2 inhibitor comprises compound 58. In some embodiments, the selective ROCK2 inhibitor comprises compound 59. In some embodiments, the selective ROCK2 inhibitor comprises compound 60. In some embodiments, the selective ROCK2 inhibitor comprises compound 61. In some embodiments, the selective ROCK2 inhibitor comprises compound 62. In some embodiments, the selective ROCK2 inhibitor comprises compound 63.

[0045] In some embodiments, the selective ROCK2 inhibitor comprises compound 64. In some embodiments, the selective ROCK2 inhibitor comprises compound 65. In some embodiments, the selective ROCK2 inhibitor comprises compound 66. In some embodiments, the selective ROCK2 inhibitor comprises compound 67. In some embodiments, the selective ROCK2 inhibitor comprises compound 68. In some embodiments, the selective ROCK2 inhibitor comprises compound 69. In some embodiments, the selective ROCK2 inhibitor comprises compound 70. In some embodiments, the selective ROCK2 inhibitor comprises compound 71. In some embodiments, the selective ROCK2 inhibitor comprises compound 72. In some embodiments, the selective ROCK2 inhibitor comprises compound 73. In some embodiments, the selective ROCK2 inhibitor comprises compound 74. In some embodiments, the selective ROCK2 inhibitor comprises compound 75. In some embodiments, the selective ROCK2 inhibitor comprises compound 76. In some embodiments, the selective ROCK2 inhibitor comprises compound 77. In some embodiments, the selective ROCK2 inhibitor comprises compound 78. In some embodiments, the selective ROCK2 inhibitor comprises compound 79.

[0046] In some embodiments, the selective ROCK2 inhibitor comprises compound 80. In some embodiments, the selective ROCK2 inhibitor comprises compound 81. In some embodiments, the selective ROCK2 inhibitor comprises compound 82. In some embodiments, the selective ROCK2 inhibitor comprises compound 83. In some embodiments, the selective ROCK2 inhibitor comprises compound 84. In some embodiments, the selective ROCK2 inhibitor comprises compound 85. In some embodiments, the selective ROCK2 inhibitor comprises compound 86. In some embodiments, the selective ROCK2 inhibitor comprises compound 87. In some embodiments, the selective ROCK2 inhibitor comprises compound 88. In some embodiments, the selective ROCK2 inhibitor comprises compound 89. In some embodiments, the selective ROCK2 inhibitor comprises compound 90. In some embodiments, the selective ROCK2 inhibitor comprises compound 91. In some embodiments, the selective ROCK2 inhibitor comprises compound 92. In some embodiments, the selective ROCK2 inhibitor comprises compound 93. In some embodiments, the selective ROCK2 inhibitor comprises compound 94. In some embodiments, the selective ROCK2 inhibitor comprises compound 95.

[0047] In some embodiments, the selective ROCK2 inhibitor comprises compound 96. In some embodiments, the selective ROCK2 inhibitor comprises compound 97. In some embodiments, the selective ROCK2 inhibitor comprises compound 98. In some embodiments, the selective ROCK2 inhibitor comprises compound 99. In some embodiments, the selective ROCK2 inhibitor comprises compound 100. In some embodiments, the selective ROCK2 inhibitor comprises compound 101. In some embodiments, the selective ROCK2 inhibitor comprises compound 102. In some embodiments, the selective ROCK2 inhibitor comprises compound 103. In some embodiments, the selective ROCK2 inhibitor comprises compound 104. In some embodiments, the selective ROCK2 inhibitor comprises compound 105. In some embodiments, the selective ROCK2 inhibitor comprises compound 106. In some embodiments, the selective ROCK2 inhibitor comprises compound 107. In some embodiments, the selective ROCK2 inhibitor comprises compound 108. In some embodiments, the selective ROCK2 inhibitor comprises compound 109. In some embodiments, the selective ROCK2 inhibitor comprises compound 110. In some embodiments, the selective ROCK2 inhibitor comprises compound 111.

[0048] In some embodiments, the selective ROCK2 inhibitor comprises compound 112. In some embodiments, the selective ROCK2 inhibitor comprises compound 113. In some embodiments, the selective ROCK2 inhibitor comprises compound 114. In some embodiments, the selective ROCK2 inhibitor comprises compound 115. In some embodiments, the selective ROCK2 inhibitor comprises compound 116. In some embodiments, the selective ROCK2 inhibitor comprises compound 117. In some embodiments, the selective ROCK2 inhibitor comprises compound 118. In some embodiments, the selective ROCK2 inhibitor comprises compound 119. In some embodiments, the selective ROCK2 inhibitor comprises compound 120. In some embodiments, the selective ROCK2 inhibitor comprises compound 121. In some embodiments, the selective ROCK2 inhibitor comprises compound 122. In some embodiments, the selective ROCK2 inhibitor comprises compound 123. In some embodiments, the selective ROCK2 inhibitor comprises compound 124. In some embodiments, the selective ROCK2 inhibitor comprises compound 125. In some embodiments, the selective ROCK2 inhibitor comprises compound 126. In some embodiments, the selective ROCK2 inhibitor comprises compound 127.

[0049] In some embodiments, the selective ROCK2 inhibitor comprises compound 128. In some embodiments, the selective ROCK2 inhibitor comprises compound 129. In some embodiments, the selective ROCK2 inhibitor comprises compound 130. In some embodiments, the selective ROCK2 inhibitor comprises compound 131. In some embodiments, the selective ROCK2 inhibitor comprises compound 132. In some embodiments, the selective ROCK2 inhibitor comprises compound 133. In some embodiments, the selective ROCK2 inhibitor comprises compound 134. In some embodiments, the selective ROCK2 inhibitor comprises compound 135. In some embodiments, the selective ROCK2 inhibitor comprises compound 136. In some embodiments, the selective ROCK2 inhibitor comprises compound 137. In some embodiments, the selective ROCK2 inhibitor comprises compound 138. In some embodiments, the selective ROCK2 inhibitor comprises compound 139. In some embodiments, the selective ROCK2 inhibitor comprises compound 140. In some embodiments, the selective ROCK2 inhibitor comprises compound 141. In some embodiments, the selective ROCK2 inhibitor comprises compound 142. In some embodiments, the selective ROCK2 inhibitor comprises compound 143.

[0050] In some embodiments, the selective ROCK2 inhibitor comprises compound 144. In some embodiments, the selective ROCK2 inhibitor comprises compound 145. In some embodiments, the selective ROCK2 inhibitor comprises compound 146. In some embodiments, the selective ROCK2 inhibitor comprises compound 147. In some embodiments, the selective ROCK2 inhibitor comprises compound 148. In some embodiments, the selective ROCK2 inhibitor comprises compound 149. In some embodiments, the selective ROCK2 inhibitor comprises compound 150. In some embodiments, the selective ROCK2 inhibitor comprises compound 151. In some embodiments, the selective ROCK2 inhibitor comprises compound 152. In some embodiments, the selective ROCK2 inhibitor comprises compound 153. In some embodiments, the selective ROCK2 inhibitor comprises compound 154. In some embodiments, the selective ROCK2 inhibitor comprises compound 155. In some embodiments, the selective ROCK2 inhibitor comprises compound 156. In some embodiments, the selective ROCK2 inhibitor comprises compound 157. In some embodiments, the selective ROCK2 inhibitor comprises compound 158. In some embodiments, the selective ROCK2 inhibitor comprises compound 159.

[0051] In some embodiments, the selective ROCK2 inhibitor comprises compound 160. In some embodiments, the selective ROCK2 inhibitor comprises compound 161. In some embodiments, the selective ROCK2 inhibitor comprises compound 162. In some embodiments, the selective ROCK2 inhibitor comprises compound 163. In some embodiments, the selective ROCK2 inhibitor comprises compound 164. In some embodiments, the selective ROCK2 inhibitor comprises compound 165. In some embodiments, the selective ROCK2 inhibitor comprises compound 166. In some embodiments, the selective ROCK2 inhibitor comprises compound 167. In some embodiments, the selective ROCK2 inhibitor comprises compound 168. In some embodiments, the selective ROCK2 inhibitor comprises compound 169. In some embodiments, the selective ROCK2 inhibitor comprises compound 170. In some embodiments, the selective ROCK2 inhibitor comprises compound 171. In some embodiments, the selective ROCK2 inhibitor comprises compound 172. In some embodiments, the selective ROCK2 inhibitor comprises compound 173. In some embodiments, the selective ROCK2 inhibitor comprises compound 174. In some embodiments, the selective ROCK2 inhibitor comprises compound 175.

[0052] In some embodiments, the selective ROCK2 inhibitor comprises compound 176. In some embodiments, the selective ROCK2 inhibitor comprises compound 177. In some embodiments, the selective ROCK2 inhibitor comprises compound 178. In some embodiments, the selective ROCK2 inhibitor comprises compound 179. In some embodiments, the selective ROCK2 inhibitor comprises compound 180. In some embodiments, the selective ROCK2 inhibitor comprises compound 181. In some embodiments, the selective ROCK2 inhibitor comprises compound 182. In some embodiments, the selective ROCK2 inhibitor comprises compound 183. In some embodiments, the selective ROCK2 inhibitor comprises compound 184. In some embodiments, the selective ROCK2 inhibitor comprises compound 185. In some embodiments, the selective ROCK2 inhibitor comprises compound 186. In some embodiments, the selective ROCK2 inhibitor comprises compound 187. In some embodiments, the selective ROCK2 inhibitor comprises compound 188. In some embodiments, the selective ROCK2 inhibitor comprises compound 189. In some embodiments, the selective ROCK2 inhibitor comprises compound 190. In some embodiments, the selective ROCK2 inhibitor comprises compound 191.

[0053] In some embodiments, the selective ROCK2 inhibitor comprises compound 192. In some embodiments, the selective ROCK2 inhibitor comprises compound 193. In some embodiments, the selective ROCK2 inhibitor comprises compound 194. In some embodiments, the selective ROCK2 inhibitor comprises compound 195. In some embodiments, the selective ROCK2 inhibitor comprises compound 196. In some embodiments, the selective ROCK2 inhibitor comprises compound 197. In some embodiments, the selective ROCK2 inhibitor comprises compound 198. In some embodiments, the selective ROCK2 inhibitor comprises compound 199. In some embodiments, the selective ROCK2 inhibitor comprises compound 200. In some embodiments, the selective ROCK2 inhibitor comprises compound 201. In some embodiments, the selective ROCK2 inhibitor comprises compound 202. In some embodiments, the selective ROCK2 inhibitor comprises compound 203. In some embodiments, the selective ROCK2 inhibitor comprises compound 204. In some embodiments, the selective ROCK2 inhibitor comprises compound 205. In some embodiments, the selective ROCK2 inhibitor comprises compound 206. In some embodiments, the selective ROCK2 inhibitor comprises compound 207.

[0054] In some embodiments, the selective ROCK2 inhibitor comprises compound 208. In some embodiments, the selective ROCK2 inhibitor comprises compound 209. In some embodiments, the selective ROCK2 inhibitor comprises compound 210. In some embodiments, the selective ROCK2 inhibitor comprises compound 211. In some embodiments, the selective ROCK2 inhibitor comprises compound 212. In some embodiments, the selective ROCK2 inhibitor comprises compound 213. In some embodiments, the selective ROCK2 inhibitor comprises compound 214. In some embodiments, the selective ROCK2 inhibitor comprises compound 215. In some embodiments, the selective ROCK2 inhibitor comprises compound 216. In some embodiments, the selective ROCK2 inhibitor comprises compound 217. In some embodiments, the selective ROCK2 inhibitor comprises compound 218. In some embodiments, the selective ROCK2 inhibitor comprises compound 219. In some embodiments, the selective ROCK2 inhibitor comprises compound 220. In some embodiments, the selective ROCK2 inhibitor comprises compound 221. In some embodiments, the selective ROCK2 inhibitor comprises compound 222. In some embodiments, the selective ROCK2 inhibitor comprises compound 223.

[0055] In some embodiments, the selective ROCK2 inhibitor comprises compound 224. In some embodiments, the selective ROCK2 inhibitor comprises compound 225. In some embodiments, the selective ROCK2 inhibitor comprises compound 226. In some embodiments, the selective ROCK2 inhibitor comprises compound 227. In some embodiments, the selective ROCK2 inhibitor comprises compound 228. In some embodiments, the selective ROCK2 inhibitor comprises compound 229. In some embodiments, the selective ROCK2 inhibitor comprises compound 230. In some embodiments, the selective ROCK2 inhibitor comprises compound 231. In some embodiments, the selective ROCK2 inhibitor comprises compound 232. In some embodiments, the selective ROCK2 inhibitor comprises compound 233. In some embodiments, the selective ROCK2 inhibitor comprises compound 234. In some embodiments, the selective ROCK2 inhibitor comprises compound 235. In some embodiments, the selective ROCK2 inhibitor comprises compound 236. In some embodiments, the selective ROCK2 inhibitor comprises compound 237. In some embodiments, the selective ROCK2 inhibitor comprises compound 238. In some embodiments, the selective ROCK2 inhibitor comprises compound 239.

[0056] In some embodiments, the selective ROCK2 inhibitor comprises compound 240. In some embodiments, the selective ROCK2 inhibitor comprises compound 241. In some embodiments, the selective ROCK2 inhibitor comprises compound 242. In some embodiments, the selective ROCK2 inhibitor comprises compound 243. In some embodiments, the selective ROCK2 inhibitor comprises compound 244. In some embodiments, the selective ROCK2 inhibitor comprises compound 245. In some embodiments, the selective ROCK2 inhibitor comprises compound 246. In some embodiments, the selective ROCK2 inhibitor comprises compound 247.

[0057] In some embodiments, the selective ROCK2 inhibitor comprises compound 248. In some embodiments, the selective ROCK2 inhibitor comprises compound 249. In some embodiments, the selective ROCK2 inhibitor comprises compound 250. In some embodiments, the selective ROCK2 inhibitor comprises compound 251. In some embodiments, the selective ROCK2 inhibitor comprises compound 252. In some embodiments, the selective ROCK2 inhibitor comprises compound 253. In some embodiments, the selective ROCK2 inhibitor comprises compound 254. In some embodiments, the selective ROCK2 inhibitor comprises compound 255. In some embodiments, the selective ROCK2 inhibitor comprises compound 256. In some embodiments, the selective ROCK2 inhibitor comprises compound 257. In some embodiments, the selective ROCK2 inhibitor comprises compound 258. In some embodiments, the selective ROCK2 inhibitor comprises compound 259. In some embodiments, the selective ROCK2 inhibitor comprises compound 260. In some embodiments, the selective ROCK2 inhibitor comprises compound 261. In some embodiments, the selective ROCK2 inhibitor comprises compound 262. In some embodiments, the selective ROCK2 inhibitor comprises compound 263.

[0058] In some embodiments, the selective ROCK2 inhibitor comprises compound 264. In some embodiments, the selective ROCK2 inhibitor comprises compound 265. In some embodiments, the selective ROCK2 inhibitor comprises compound 266. In some embodiments, the selective ROCK2 inhibitor comprises compound 267. In some embodiments, the selective ROCK2 inhibitor comprises compound 268. In some embodiments, the selective ROCK2 inhibitor comprises compound 269. In some embodiments, the selective ROCK2 inhibitor comprises compound 270. In some embodiments, the selective ROCK2 inhibitor comprises compound 271. In some embodiments, the selective ROCK2 inhibitor comprises compound 272. In some embodiments, the selective ROCK2 inhibitor comprises compound 273. In some embodiments, the selective ROCK2 inhibitor comprises compound 274. In some embodiments, the selective ROCK2 inhibitor comprises compound 275. In some embodiments, the selective ROCK2 inhibitor comprises compound 276. In some embodiments, the selective ROCK2 inhibitor comprises compound 277. In some embodiments, the selective ROCK2 inhibitor comprises compound 278. In some embodiments, the selective ROCK2 inhibitor comprises compound 279.

[0059] In some embodiments, the selective ROCK2 inhibitor comprises compound 280. In some embodiments, the selective ROCK2 inhibitor comprises compound 281. In some embodiments, the selective ROCK2 inhibitor comprises compound 282. In some embodiments, the selective ROCK2 inhibitor comprises compound 283. In some embodiments, the selective ROCK2 inhibitor comprises compound 284. In some embodiments, the selective ROCK2 inhibitor comprises compound 285. In some embodiments, the selective ROCK2 inhibitor comprises compound 286. In some embodiments, the selective ROCK2 inhibitor comprises compound 287.

[0060] In some embodiments, the selective ROCK2 inhibitor comprises compound 288. In some embodiments, the selective ROCK2 inhibitor comprises compound 289. In some embodiments, the selective ROCK2 inhibitor comprises compound 290. In some embodiments, the selective ROCK2 inhibitor comprises compound 291. In some embodiments, the selective ROCK2 inhibitor comprises compound 292. In some embodiments, the selective ROCK2 inhibitor comprises compound 293. In some embodiments, the selective ROCK2 inhibitor comprises compound 294. In some embodiments, the selective ROCK2 inhibitor comprises compound 295. In some embodiments, the selective ROCK2 inhibitor comprises compound 296. In some embodiments, the selective ROCK2 inhibitor comprises compound 297. In some embodiments, the selective ROCK2 inhibitor comprises compound 298. In some embodiments, the selective ROCK2 inhibitor comprises compound 299. In some embodiments, the selective ROCK2 inhibitor comprises compound 300. In some embodiments, the selective ROCK2 inhibitor comprises compound 301. In some embodiments, the selective ROCK2 inhibitor comprises compound 302. In some embodiments, the selective ROCK2 inhibitor comprises compound 303.

[0061] In some embodiments, the selective ROCK2 inhibitor comprises compound 304. In some embodiments, the selective ROCK2 inhibitor comprises compound 305. In some embodiments, the selective ROCK2 inhibitor comprises compound 306. In some embodiments, the selective ROCK2 inhibitor comprises compound 307. In some embodiments, the selective ROCK2 inhibitor comprises compound 308. In some embodiments, the selective ROCK2 inhibitor comprises compound 309. In some embodiments, the selective ROCK2 inhibitor comprises compound 310. In some embodiments, the selective ROCK2 inhibitor comprises compound 311. In some embodiments, the selective ROCK2 inhibitor comprises compound 312. In some embodiments, the selective ROCK2 inhibitor comprises compound 313. In some embodiments, the selective ROCK2 inhibitor comprises compound 314. In some embodiments, the selective ROCK2 inhibitor comprises compound 315. In some embodiments, the selective ROCK2 inhibitor comprises compound 316. In some embodiments, the selective ROCK2 inhibitor comprises compound 317.

[0062] In some embodiments, the selective ROCK2 inhibitor is compound 1. In some embodiments, the selective ROCK2 inhibitor is compound 2. In some embodiments, the selective ROCK2 inhibitor is compound 3. In some embodiments, the selective ROCK2 inhibitor is the hydrochloride salt of compound 3. In some embodiments, the selective ROCK2 inhibitor is the dihydrochloride salt of compound 3. In some embodiments, the selective ROCK2 inhibitor is compound 4. In some embodiments, the selective ROCK2 inhibitor is compound 5. In some embodiments, the selective ROCK2 inhibitor is compound 6. In some embodiments, the selective ROCK2 inhibitor is compound 7. In some embodiments, the selective ROCK2 inhibitor is compound 8. In some embodiments, the selective ROCK2 inhibitor is compound 9.

[0063] In some embodiments, the selective ROCK2 inhibitor is the hydrochloride salt of compound 9. In some embodiments, the selective ROCK2 inhibitor is compound 10. In some embodiments, the selective ROCK2 inhibitor is compound 11. In some embodiments, the selective ROCK2 inhibitor is compound 12. In some embodiments, the selective ROCK2 inhibitor is compound 13. In some embodiments, the selective ROCK2 inhibitor is compound 14. In some embodiments, the selective ROCK2 inhibitor is compound 15. In some embodiments, the selective ROCK2 inhibitor is compound 16. In some embodiments, the selective ROCK2 inhibitor is compound 17. In some embodiments, the selective ROCK2 inhibitor is compound 18. In some embodiments, the selective ROCK2 inhibitor is compound 19. In some embodiments, the selective ROCK2 inhibitor is compound 20. In some embodiments, the selective ROCK2 inhibitor is compound 21. In some embodiments, the selective ROCK2 inhibitor is compound 22. In some embodiments, the selective ROCK2 inhibitor is compound 23.

[0064] In some embodiments, the selective ROCK2 inhibitor is compound 24. In some embodiments, the selective ROCK2 inhibitor is compound 25. In some embodiments, the selective ROCK2 inhibitor is compound 26. In some embodiments, the selective ROCK2 inhibitor is compound 27. In some embodiments, the selective ROCK2 inhibitor is compound 28. In some embodiments, the selective ROCK2 inhibitor is compound 29. In some embodiments, the selective ROCK2 inhibitor is compound 30. In some embodiments, the selective ROCK2 inhibitor is compound 31. In some embodiments, the selective ROCK2 inhibitor is compound 32. In some embodiments, the selective ROCK2 inhibitor is compound 33. In some embodiments, the selective ROCK2 inhibitor is compound 34. In some embodiments, the selective ROCK2 inhibitor is compound 35. In some embodiments, the selective ROCK2 inhibitor is compound 36. In some embodiments, the selective ROCK2 inhibitor is compound 37. In some embodiments, the selective ROCK2 inhibitor is compound 38.

[0065] In some embodiments, the selective ROCK2 inhibitor is compound 39. In some embodiments, the selective ROCK2 inhibitor is compound 40. In some embodiments, the selective ROCK2 inhibitor is compound 41. In some embodiments, the selective ROCK2 inhibitor is compound 42. In some embodiments, the selective ROCK2 inhibitor is compound 43. In some embodiments, the selective ROCK2 inhibitor is compound 44. In some embodiments, the selective ROCK2 inhibitor is compound 45. In some embodiments, the selective ROCK2 inhibitor is compound 46. In some embodiments, the selective ROCK2 inhibitor is compound 47. In some embodiments, the selective ROCK2 inhibitor is compound 48. In some embodiments, the selective ROCK2 inhibitor is compound 49. In some embodiments, the selective ROCK2 inhibitor is compound 50. In some embodiments, the selective ROCK2 inhibitor is compound 51. In some embodiments, the selective ROCK2 inhibitor is compound 52. In some embodiments, the selective ROCK2 inhibitor is compound 53.

[0066] In some embodiments, the selective ROCK2 inhibitor is compound 54. In some embodiments, the selective ROCK2 inhibitor is compound 55. In some embodiments, the selective ROCK2 inhibitor is compound 56. In some embodiments, the selective ROCK2 inhibitor is compound 57. In some embodiments, the selective ROCK2 inhibitor is compound 58. In some embodiments, the selective ROCK2 inhibitor is compound 59. In some embodiments, the selective ROCK2 inhibitor is compound 60. In some embodiments, the selective ROCK2 inhibitor is compound 61. In some embodiments, the selective ROCK2 inhibitor is compound 62. In some embodiments, the selective ROCK2 inhibitor is compound 63. In some embodiments, the selective ROCK2 inhibitor is compound 64. In some embodiments, the selective ROCK2 inhibitor is compound 65. In some embodiments, the selective ROCK2 inhibitor is compound 66. In some embodiments, the selective ROCK2 inhibitor is compound 67. In some embodiments, the selective ROCK2 inhibitor is compound 68.

[0067] In some embodiments, the selective ROCK2 inhibitor is compound 69. In some embodiments, the selective ROCK2 inhibitor is compound 70. In some embodiments, the selective ROCK2 inhibitor is compound 71. In some embodiments, the selective ROCK2 inhibitor is compound 72. In some embodiments, the selective ROCK2 inhibitor is compound 73. In some embodiments, the selective ROCK2 inhibitor is the hydrochloride salt of compound 73. In some embodiments, the selective ROCK2 inhibitor is compound 74. In some embodiments, the selective ROCK2 inhibitor is compound 75. In some embodiments, the selective ROCK2 inhibitor is compound 76. In some embodiments, the selective ROCK2 inhibitor is compound 77. In some embodiments, the selective ROCK2 inhibitor is compound 78. In some embodiments, the selective ROCK2 inhibitor is compound 79. In some embodiments, the selective ROCK2 inhibitor is compound 80. In some embodiments, the selective ROCK2 inhibitor is compound 81. In some embodiments, the selective ROCK2 inhibitor is compound 82. In some embodiments, the selective ROCK2 inhibitor is compound 83.

[0068] In some embodiments, the selective ROCK2 inhibitor is compound 84. In some embodiments, the selective ROCK2 inhibitor is compound 85. In some embodiments, the selective ROCK2 inhibitor is compound 86. In some embodiments, the selective ROCK2 inhibitor is compound 87. In some embodiments, the selective ROCK2 inhibitor is compound 88. In some embodiments, the selective ROCK2 inhibitor is compound 89. In some embodiments, the selective ROCK2 inhibitor is compound 90. In some embodiments, the selective ROCK2 inhibitor is compound 91. In some embodiments, the selective ROCK2 inhibitor is compound 92. In some embodiments, the selective ROCK2 inhibitor is compound 93. In some embodiments, the selective ROCK2 inhibitor is compound 94. In some embodiments, the selective ROCK2 inhibitor is compound 95. In some embodiments, the selective ROCK2 inhibitor is compound 96. In some embodiments, the selective ROCK2 inhibitor is compound 97. In some embodiments, the selective ROCK2 inhibitor is compound 98.

[0069] In some embodiments, the selective ROCK2 inhibitor is compound 99. In some embodiments, the selective ROCK2 inhibitor is compound 100. In some embodiments, the selective ROCK2 inhibitor is compound 101. In some embodiments, the selective ROCK2 inhibitor is compound 102. In some embodiments, the selective ROCK2 inhibitor is compound 103. In some embodiments, the selective ROCK2 inhibitor is compound 104. In some embodiments, the selective ROCK2 inhibitor is compound 105. In some embodiments, the selective ROCK2 inhibitor is compound 106. In some embodiments, the selective ROCK2 inhibitor is compound 107. In some embodiments, the selective ROCK2 inhibitor is compound 108. In some embodiments, the selective ROCK2 inhibitor is compound 109. In some embodiments, the selective ROCK2 inhibitor is compound 110. In some embodiments, the selective ROCK2 inhibitor is compound 111. In some embodiments, the selective ROCK2 inhibitor is compound 112. In some embodiments, the selective ROCK2 inhibitor is compound 113.

[0070] In some embodiments, the selective ROCK2 inhibitor is compound 114. In some embodiments, the selective ROCK2 inhibitor is compound 115. In some embodiments, the selective ROCK2 inhibitor is compound 116. In some embodiments, the selective ROCK2 inhibitor is compound 117. In some embodiments, the selective ROCK2 inhibitor is compound 118. In some embodiments, the selective ROCK2 inhibitor is compound 119. In some embodiments, the selective ROCK2 inhibitor is compound 120. In some embodiments, the selective ROCK2 inhibitor is compound 121. In some embodiments, the selective ROCK2 inhibitor is compound 122. In some embodiments, the selective ROCK2 inhibitor is compound 123. In some embodiments, the selective ROCK2 inhibitor is compound 124. In some embodiments, the selective ROCK2 inhibitor is compound 125. In some embodiments, the selective ROCK2 inhibitor is compound 126. In some embodiments, the selective ROCK2 inhibitor is compound 127. In some embodiments, the selective ROCK2 inhibitor is compound 128.

[0071] In some embodiments, the selective ROCK2 inhibitor is the hydrochloride salt of compound 128. In some embodiments, the selective ROCK2 inhibitor is the dihydrochloride salt of compound 128. In some embodiments, the selective ROCK2 inhibitor is compound 129. In some embodiments, the selective ROCK2 inhibitor is compound 130. In some embodiments, the selective ROCK2 inhibitor is compound 131. In some embodiments, the selective ROCK2 inhibitor is compound 132. In some embodiments, the selective ROCK2 inhibitor is compound 133. In some embodiments, the selective ROCK2 inhibitor is compound 134. In some embodiments, the selective ROCK2 inhibitor is compound 135. In some embodiments, the selective ROCK2 inhibitor is compound 136. In some embodiments, the selective ROCK2 inhibitor is compound 137. In some embodiments, the selective ROCK2 inhibitor is compound 138. In some embodiments, the selective ROCK2 inhibitor is compound 139. In some embodiments, the selective ROCK2 inhibitor is compound 140. In some embodiments, the selective ROCK2 inhibitor is compound 141. In some embodiments, the selective ROCK2 inhibitor is compound 142. In some embodiments, the selective ROCK2 inhibitor is compound 143.

[0072] In some embodiments, the selective ROCK2 inhibitor is compound 144. In some embodiments, the selective ROCK2 inhibitor is compound 145. In some embodiments, the selective ROCK2 inhibitor is compound 146. In some embodiments, the selective ROCK2 inhibitor is compound 147. In some embodiments, the selective ROCK2 inhibitor is compound 148. In some embodiments, the selective ROCK2 inhibitor is compound 149. In some embodiments, the selective ROCK2 inhibitor is compound 150. In some embodiments, the selective ROCK2 inhibitor is compound 151. In some embodiments, the selective ROCK2 inhibitor is compound 152. In some embodiments, the selective ROCK2 inhibitor is compound 153. In some embodiments, the selective ROCK2 inhibitor is compound 154. In some embodiments, the selective ROCK2 inhibitor is compound 155. In some embodiments, the selective ROCK2 inhibitor is compound 156. In some embodiments, the selective ROCK2 inhibitor is compound 157. In some embodiments, the selective ROCK2 inhibitor is compound 158.

[0073] In some embodiments, the selective ROCK2 inhibitor is compound 159. In some embodiments, the selective ROCK2 inhibitor is compound 160. In some embodiments, the selective ROCK2 inhibitor is compound 161. In some embodiments, the selective ROCK2 inhibitor is compound 162. In some embodiments, the selective ROCK2 inhibitor is compound 163. In some embodiments, the selective ROCK2 inhibitor is compound 164. In some embodiments, the selective ROCK2 inhibitor is compound 165. In some embodiments, the selective ROCK2 inhibitor is compound 166. In some embodiments, the selective ROCK2 inhibitor is compound 167. In some embodiments, the selective ROCK2 inhibitor is compound 168. In some embodiments, the selective ROCK2 inhibitor is compound 169. In some embodiments, the selective ROCK2 inhibitor is compound 170. In some embodiments, the selective ROCK2 inhibitor is compound 171. In some embodiments, the selective ROCK2 inhibitor is compound 172. In some embodiments, the selective ROCK2 inhibitor is compound 173.

[0074] In some embodiments, the selective ROCK2 inhibitor is compound 174. In some embodiments, the selective ROCK2 inhibitor is compound 175. In some embodiments, the selective ROCK2 inhibitor is compound 176. In some embodiments, the selective ROCK2 inhibitor is compound 177. In some embodiments, the selective ROCK2 inhibitor is compound 178. In some embodiments, the selective ROCK2 inhibitor is compound 179. In some embodiments, the selective ROCK2 inhibitor is compound 180. In some embodiments, the selective ROCK2 inhibitor is compound 181. In some embodiments, the selective ROCK2 inhibitor is compound 182. In some embodiments, the selective ROCK2 inhibitor is compound 183. In some embodiments, the selective ROCK2 inhibitor is compound 184. In some embodiments, the selective ROCK2 inhibitor is compound 185. In some embodiments, the selective ROCK2 inhibitor is compound 186. In some embodiments, the selective ROCK2 inhibitor is compound 187. In some embodiments, the selective ROCK2 inhibitor is compound 188.

[0075] In some embodiments, the selective ROCK2 inhibitor is compound 189. In some embodiments, the selective ROCK2 inhibitor is compound 190. In some embodiments, the selective ROCK2 inhibitor is compound 191. In some embodiments, the selective ROCK2 inhibitor is compound 192. In some embodiments, the selective ROCK2 inhibitor is compound 193. In some embodiments, the selective ROCK2 inhibitor is compound 194. In some embodiments, the selective ROCK2 inhibitor is compound 195. In some embodiments, the selective ROCK2 inhibitor is compound 196. In some embodiments, the selective ROCK2 inhibitor is compound 197. In some embodiments, the selective ROCK2 inhibitor is compound 198. In some embodiments, the selective ROCK2 inhibitor is compound 199. In some embodiments, the selective ROCK2 inhibitor is compound 200. In some embodiments, the selective ROCK2 inhibitor is compound 201. In some embodiments, the selective ROCK2 inhibitor is compound 202. In some embodiments, the selective ROCK2 inhibitor is compound 203.

[0076] In some embodiments, the selective ROCK2 inhibitor is compound 204. In some embodiments, the selective ROCK2 inhibitor is compound 205. In some embodiments, the selective ROCK2 inhibitor is compound 206. In some embodiments, the selective ROCK2 inhibitor is compound 207. In some embodiments, the selective ROCK2 inhibitor is compound 208. In some embodiments, the selective ROCK2 inhibitor is compound 209. In some embodiments, the selective ROCK2 inhibitor is compound 210. In some embodiments, the selective ROCK2 inhibitor is compound 211. In some embodiments, the selective ROCK2 inhibitor is compound 212. In some embodiments, the selective ROCK2 inhibitor is compound 213. In some embodiments, the selective ROCK2 inhibitor is compound 214. In some embodiments, the selective ROCK2 inhibitor is compound 215. In some embodiments, the selective ROCK2 inhibitor is compound 216. In some embodiments, the selective ROCK2 inhibitor is compound 217. In some embodiments, the selective ROCK2 inhibitor is compound 218.

[0077] In some embodiments, the selective ROCK2 inhibitor is compound 219. In some embodiments, the selective ROCK2 inhibitor is compound 220. In some embodiments, the selective ROCK2 inhibitor is compound 221. In some embodiments, the selective ROCK2 inhibitor is compound 222. In some embodiments, the selective ROCK2 inhibitor is compound 223. In some embodiments, the selective ROCK2 inhibitor is compound 224. In some embodiments, the selective ROCK2 inhibitor is compound 225. In some embodiments, the selective ROCK2 inhibitor is compound 226. In some embodiments, the selective ROCK2 inhibitor is compound 227. In some embodiments, the selective ROCK2 inhibitor is compound 228. In some embodiments, the selective ROCK2 inhibitor is compound 229. In some embodiments, the selective ROCK2 inhibitor is compound 230. In some embodiments, the selective ROCK2 inhibitor is compound 231. In some embodiments, the selective ROCK2 inhibitor is compound 232. In some embodiments, the selective ROCK2 inhibitor is compound 233.

[0078] In some embodiments, the selective ROCK2 inhibitor is compound 234. In some embodiments, the selective ROCK2 inhibitor is compound 235. In some embodiments, the selective ROCK2 inhibitor is compound 236. In some embodiments, the selective ROCK2 inhibitor is compound 237. In some embodiments, the selective ROCK2 inhibitor is compound 238. In some embodiments, the selective ROCK2 inhibitor is compound 239. In some embodiments, the selective ROCK2 inhibitor is compound 240. In some embodiments, the selective ROCK2 inhibitor is compound 241. In some embodiments, the selective ROCK2 inhibitor is compound 242. In some embodiments, the selective ROCK2 inhibitor is compound 243. In some embodiments, the selective ROCK2 inhibitor is compound 244. In some embodiments, the selective ROCK2 inhibitor is compound 245. In some embodiments, the selective ROCK2 inhibitor is compound 246. In some embodiments, the selective ROCK2 inhibitor is compound 247. In some embodiments, the selective ROCK2 inhibitor is compound 248.

[0079] In some embodiments, the selective ROCK2 inhibitor is compound 249. In some embodiments, the selective ROCK2 inhibitor is compound 250. In some embodiments, the selective ROCK2 inhibitor is compound 251. In some embodiments, the selective ROCK2 inhibitor is compound 252. In some embodiments, the selective ROCK2 inhibitor is compound 253. In some embodiments, the selective ROCK2 inhibitor is compound 254. In some embodiments, the selective ROCK2 inhibitor is compound 255. In some embodiments, the selective ROCK2 inhibitor is compound 256. In some embodiments, the selective ROCK2 inhibitor is compound 257. In some embodiments, the selective ROCK2 inhibitor is compound 258.

[0080] In some embodiments, the selective ROCK2 inhibitor is compound 259. In some embodiments, the selective ROCK2 inhibitor is compound 260. In some embodiments, the selective ROCK2 inhibitor is compound 261. In some embodiments, the selective ROCK2 inhibitor is compound 262. In some embodiments, the selective ROCK2 inhibitor is compound 263. In some embodiments, the selective ROCK2 inhibitor is compound 264. In some embodiments, the selective ROCK2 inhibitor is compound 265. In some embodiments, the selective ROCK2 inhibitor is compound 266. In some embodiments, the selective ROCK2 inhibitor is compound 267. In some embodiments, the selective ROCK2 inhibitor is compound 268. In some embodiments, the selective ROCK2 inhibitor is compound 269. In some embodiments, the selective ROCK2 inhibitor is compound 270. In some embodiments, the selective ROCK2 inhibitor is compound 271. In some embodiments, the selective ROCK2 inhibitor is compound 272. In some embodiments, the selective ROCK2 inhibitor is compound 273.

[0081] In some embodiments, the selective ROCK2 inhibitor is compound 274. In some embodiments, the selective ROCK2 inhibitor is compound 275. In some embodiments, the selective ROCK2 inhibitor is compound 276. In some embodiments, the selective ROCK2 inhibitor is compound 277. In some embodiments, the selective ROCK2 inhibitor is compound 278. In some embodiments, the selective ROCK2 inhibitor is compound 279. In some embodiments, the selective ROCK2 inhibitor is compound 280. In some embodiments, the selective ROCK2 inhibitor is compound 281. In some embodiments, the selective ROCK2 inhibitor is compound 282. In some embodiments, the selective ROCK2 inhibitor is compound 283. In some embodiments, the selective ROCK2 inhibitor is compound 284. In some embodiments, the selective ROCK2 inhibitor is compound 285. In some embodiments, the selective ROCK2 inhibitor is compound 286. In some embodiments, the selective ROCK2 inhibitor is compound 287. In some embodiments, the selective ROCK2 inhibitor is compound 288.

[0082] In some embodiments, the selective ROCK2 inhibitor is compound 289. In some embodiments, the selective ROCK2 inhibitor is compound 290. In some embodiments, the selective ROCK2 inhibitor is compound 291. In some embodiments, the selective ROCK2 inhibitor is compound 292. In some embodiments, the selective ROCK2 inhibitor is compound 293. In some embodiments, the selective ROCK2 inhibitor is compound 294. In some embodiments, the selective ROCK2 inhibitor is compound 295. In some embodiments, the selective ROCK2 inhibitor is compound 296. In some embodiments, the selective ROCK2 inhibitor is compound 297. In some embodiments, the selective ROCK2 inhibitor is compound 298. In some embodiments, the selective ROCK2 inhibitor is compound 299. In some embodiments, the selective ROCK2 inhibitor is compound 300. In some embodiments, the selective ROCK2 inhibitor is compound 301. In some embodiments, the selective ROCK2 inhibitor is compound 302. In some embodiments, the selective ROCK2 inhibitor is compound 303.

[0083] In some embodiments, the selective ROCK2 inhibitor is compound 304. In some embodiments, the selective ROCK2 inhibitor is compound 305. In some embodiments, the selective ROCK2 inhibitor is compound 306. In some embodiments, the selective ROCK2 inhibitor is compound 307. In some embodiments, the selective ROCK2 inhibitor is compound 308. In some embodiments, the selective ROCK2 inhibitor is compound 309. In some embodiments, the selective ROCK2 inhibitor is compound 310. In some embodiments, the selective ROCK2 inhibitor is compound 311. In some embodiments, the selective ROCK2 inhibitor is compound 312. In some embodiments, the selective ROCK2 inhibitor is compound 313. In some embodiments, the selective ROCK2 inhibitor is compound 314. In some embodiments, the selective ROCK2 inhibitor is compound 315. In some embodiments, the selective ROCK2 inhibitor is compound 316. In some embodiments, the selective ROCK2 inhibitor is compound 317.

[0084] In some embodiments, the selective ROCK2 small molecule inhibitor is a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, co-crystal, or prodrug of any one of the aforementioned compounds (e.g., compounds 1-317 in Table 1). In some embodiments, the selective ROCK2 small molecule inhibitor is a pharma- ceutically acceptable salt of any one of the aforementioned compounds (e.g., compounds 1-317 in Table 1). In some embodiments, the selective ROCK2 small molecule inhibitor is a stereoisomer of any one of the aforementioned compounds (e.g., compounds 1-317 in Table 1). In some embodiments, the selective ROCK2 small molecule inhibitor is a tautomer of any one of the aforementioned compounds (e.g., compounds 1-317 in Table 1). In some embodiments, the selective ROCK2 small molecule inhibitor is an isotopically labeled derivative (e.g., deuterated analog) of any one of the aforementioned compounds (e.g., compounds 1-317 in Table 1). In some embodiments, the selective ROCK2 small molecule inhibitor is a solvate of any one of the aforementioned compounds (e.g., compounds 1-317 in Table 1). In some embodiments, the selective ROCK2 small molecule inhibitor is a hydrate of any one of the aforementioned compounds (e.g., compounds 1-317 in Table 1). In some embodiments, the selective ROCK2 small molecule inhibitor is a polymorph of any one of the aforementioned compounds (e.g., compounds 1-317 in Table 1). In some embodiments, the selective ROCK2 small molecule inhibitor is a co-crystal of any one of the aforementioned compounds (e.g., compounds 1-317 in Table 1). In some embodiments, the selective ROCK2 small molecule inhibitor is a prodrug of any one of the aforementioned compounds (e.g., compounds 1-317 in Table 1).

[0085] The term "salt" as used herein refers to any salt, including pharma- ceutically acceptable salts. Salts include ionic compounds resulting from the neutralization reaction of an acid with a base. A salt is composed of one or more cations (positively charged ions) and one or more anions (negative ions), so that the salt is electrically neutral (has no net charge). Salts of the compounds of the present disclosure include those derived from inorganic and organic acids and bases. Examples of acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed using other methods known in the art, such as ion exchange. Other salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobiotin, and tetrahydrofuran. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and the like. + (C 1~4Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further salts include ammonium, quaternary ammonium, and amine cations formed with counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0086] The term "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., within the scope of sound medical judgment, and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed using other methods known in the art, such as ion exchange. Other pharma- ceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, and 2-hydroxy-ethanesulfonate. Salts derived from appropriate bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N-butyl salts. + (C 1~4Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharma- ceutically acceptable salts include ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates.

[0087] Other non-limiting examples of selective ROCK2 inhibitors are provided in Table 2 and International Publication No. WO 2016 / 138335 A1, the entirety of which is incorporated herein by reference. In some embodiments, the selective ROCK2 small molecule inhibitor is a compound of formula (I) or (II) (i.e., including any subgenus or species thereof) disclosed in International Publication No. WO 2016 / 138335 A1 (the entirety of which is incorporated herein by reference); or a pharmaceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, cocrystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is a compound of formula (I) or (II) (i.e., including any subgenus or species thereof) disclosed in International Publication No. WO 2016 / 138335 A1 (the entirety of which is incorporated herein by reference); or a pharmaceutically acceptable salt thereof.

[0088] In some embodiments, the selective ROCK2 small molecule inhibitor comprises a compound selected from compounds 318-400 in Table 2. In some embodiments, the selective ROCK2 small molecule inhibitor is selected from compounds 318-400 in Table 2. In some embodiments, the selective ROCK2 small molecule inhibitor is any one of compounds 318-400 in Table 2, or a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, cocrystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is any one of compounds 318-400 in Table 2, or a pharma- ceutically acceptable salt thereof. Where a particular salt form of a compound (e.g., a trifluoroacetic acid (TFA) salt) is shown in Table 2, other pharma- ceutically acceptable salts of the compound are also provided as part of this disclosure.

[0089] Table 2. Examples of selective ROCK2 inhibitors [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] #=Compound

[0090] In some embodiments, the selective ROCK2 inhibitor comprises compound 318. In some embodiments, the selective ROCK2 inhibitor comprises compound 319. In some embodiments, the selective ROCK2 inhibitor comprises compound 320. In some embodiments, the selective ROCK2 inhibitor comprises compound 321. In some embodiments, the selective ROCK2 inhibitor comprises compound 322. In some embodiments, the selective ROCK2 inhibitor comprises compound 323. In some embodiments, the selective ROCK2 inhibitor comprises compound 324. In some embodiments, the selective ROCK2 inhibitor comprises compound 325. In some embodiments, the selective ROCK2 inhibitor comprises compound 326. In some embodiments, the selective ROCK2 inhibitor comprises compound 327. In some embodiments, the selective ROCK2 inhibitor comprises compound 328. In some embodiments, the selective ROCK2 inhibitor comprises compound 329. In some embodiments, the selective ROCK2 inhibitor comprises compound 330. In some embodiments, the selective ROCK2 inhibitor comprises compound 331. In some embodiments, the selective ROCK2 inhibitor comprises compound 332. In some embodiments, the selective ROCK2 inhibitor comprises compound 333.

[0091] In some embodiments, the selective ROCK2 inhibitor comprises compound 334. In some embodiments, the selective ROCK2 inhibitor comprises compound 335. In some embodiments, the selective ROCK2 inhibitor comprises compound 336. In some embodiments, the selective ROCK2 inhibitor comprises compound 337. In some embodiments, the selective ROCK2 inhibitor comprises compound 338. In some embodiments, the selective ROCK2 inhibitor comprises compound 339. In some embodiments, the selective ROCK2 inhibitor comprises compound 340. In some embodiments, the selective ROCK2 inhibitor comprises compound 341. In some embodiments, the selective ROCK2 inhibitor comprises compound 342. In some embodiments, the selective ROCK2 inhibitor comprises compound 343. In some embodiments, the selective ROCK2 inhibitor comprises compound 344. In some embodiments, the selective ROCK2 inhibitor comprises compound 345. In some embodiments, the selective ROCK2 inhibitor comprises compound 346. In some embodiments, the selective ROCK2 inhibitor comprises compound 347. In some embodiments, the selective ROCK2 inhibitor comprises compound 348.

[0092] In some embodiments, the selective ROCK2 inhibitor comprises compound 349. In some embodiments, the selective ROCK2 inhibitor comprises compound 350. In some embodiments, the selective ROCK2 inhibitor comprises compound 351. In some embodiments, the selective ROCK2 inhibitor comprises compound 352. In some embodiments, the selective ROCK2 inhibitor comprises compound 353. In some embodiments, the selective ROCK2 inhibitor comprises compound 354. In some embodiments, the selective ROCK2 inhibitor comprises compound 355. In some embodiments, the selective ROCK2 inhibitor comprises compound 356. In some embodiments, the selective ROCK2 inhibitor comprises compound 357. In some embodiments, the selective ROCK2 inhibitor comprises compound 358. In some embodiments, the selective ROCK2 inhibitor comprises compound 359. In some embodiments, the selective ROCK2 inhibitor comprises compound 360. In some embodiments, the selective ROCK2 inhibitor comprises compound 361. In some embodiments, the selective ROCK2 inhibitor comprises compound 362. In some embodiments, the selective ROCK2 inhibitor comprises compound 363. In some embodiments, the selective ROCK2 inhibitor comprises compound 364. In some embodiments, the selective ROCK2 inhibitor comprises compound 365.

[0093] In some embodiments, the selective ROCK2 inhibitor comprises compound 366. In some embodiments, the selective ROCK2 inhibitor comprises compound 367. In some embodiments, the selective ROCK2 inhibitor comprises compound 368. In some embodiments, the selective ROCK2 inhibitor comprises compound 369. In some embodiments, the selective ROCK2 inhibitor comprises compound 370. In some embodiments, the selective ROCK2 inhibitor comprises compound 371. In some embodiments, the selective ROCK2 inhibitor comprises compound 372. In some embodiments, the selective ROCK2 inhibitor comprises compound 373. In some embodiments, the selective ROCK2 inhibitor comprises compound 374. In some embodiments, the selective ROCK2 inhibitor comprises compound 375. In some embodiments, the selective ROCK2 inhibitor comprises compound 376. In some embodiments, the selective ROCK2 inhibitor comprises compound 377. In some embodiments, the selective ROCK2 inhibitor comprises compound 378. In some embodiments, the selective ROCK2 inhibitor comprises compound 379. In some embodiments, the selective ROCK2 inhibitor comprises compound 380. In some embodiments, the selective ROCK2 inhibitor comprises compound 381. In some embodiments, the selective ROCK2 inhibitor comprises compound 382.

[0094] In some embodiments, the selective ROCK2 inhibitor comprises compound 383. In some embodiments, the selective ROCK2 inhibitor comprises compound 384. In some embodiments, the selective ROCK2 inhibitor comprises compound 385. In some embodiments, the selective ROCK2 inhibitor comprises compound 386. In some embodiments, the selective ROCK2 inhibitor comprises compound 387. In some embodiments, the selective ROCK2 inhibitor comprises compound 388. In some embodiments, the selective ROCK2 inhibitor comprises compound 389. In some embodiments, the selective ROCK2 inhibitor comprises compound 390. In some embodiments, the selective ROCK2 inhibitor comprises compound 391. In some embodiments, the selective ROCK2 inhibitor comprises compound 392. In some embodiments, the selective ROCK2 inhibitor comprises compound 393. In some embodiments, the selective ROCK2 inhibitor comprises compound 394. In some embodiments, the selective ROCK2 inhibitor comprises compound 395. In some embodiments, the selective ROCK2 inhibitor comprises compound 396. In some embodiments, the selective ROCK2 inhibitor comprises compound 397. In some embodiments, the selective ROCK2 inhibitor comprises compound 398. In some embodiments, the selective ROCK2 inhibitor comprises compound 399. In some embodiments, the selective ROCK2 inhibitor comprises compound 400.

[0095] In some embodiments, the selective ROCK2 inhibitor is compound 318. In some embodiments, the selective ROCK2 inhibitor is compound 319. In some embodiments, the selective ROCK2 inhibitor is compound 320. In some embodiments, the selective ROCK2 inhibitor is compound 321. In some embodiments, the selective ROCK2 inhibitor is compound 322. In some embodiments, the selective ROCK2 inhibitor is compound 323. In some embodiments, the selective ROCK2 inhibitor is compound 324. In some embodiments, the selective ROCK2 inhibitor is compound 325. In some embodiments, the selective ROCK2 inhibitor is compound 326. In some embodiments, the selective ROCK2 inhibitor is compound 327. In some embodiments, the selective ROCK2 inhibitor is compound 328. In some embodiments, the selective ROCK2 inhibitor is compound 329. In some embodiments, the selective ROCK2 inhibitor is compound 330. In some embodiments, the selective ROCK2 inhibitor is compound 331. In some embodiments, the selective ROCK2 inhibitor is compound 332.

[0096] In some embodiments, the selective ROCK2 inhibitor is compound 333. In some embodiments, the selective ROCK2 inhibitor is compound 334. In some embodiments, the selective ROCK2 inhibitor is compound 335. In some embodiments, the selective ROCK2 inhibitor is compound 336. In some embodiments, the selective ROCK2 inhibitor is compound 337. In some embodiments, the selective ROCK2 inhibitor is compound 338. In some embodiments, the selective ROCK2 inhibitor is compound 339. In some embodiments, the selective ROCK2 inhibitor is compound 340. In some embodiments, the selective ROCK2 inhibitor is compound 341. In some embodiments, the selective ROCK2 inhibitor is compound 342. In some embodiments, the selective ROCK2 inhibitor is compound 343. In some embodiments, the selective ROCK2 inhibitor is compound 344. In some embodiments, the selective ROCK2 inhibitor is compound 345. In some embodiments, the selective ROCK2 inhibitor is compound 346. In some embodiments, the selective ROCK2 inhibitor is compound 347.

[0097] In some embodiments, the selective ROCK2 inhibitor is compound 348. In some embodiments, the selective ROCK2 inhibitor is compound 349. In some embodiments, the selective ROCK2 inhibitor is compound 350. In some embodiments, the selective ROCK2 inhibitor is compound 351. In some embodiments, the selective ROCK2 inhibitor is compound 352. In some embodiments, the selective ROCK2 inhibitor is compound 353. In some embodiments, the selective ROCK2 inhibitor is compound 354. In some embodiments, the selective ROCK2 inhibitor is compound 355. In some embodiments, the selective ROCK2 inhibitor is compound 356. In some embodiments, the selective ROCK2 inhibitor is compound 357. In some embodiments, the selective ROCK2 inhibitor is compound 358. In some embodiments, the selective ROCK2 inhibitor is compound 359. In some embodiments, the selective ROCK2 inhibitor is compound 360. In some embodiments, the selective ROCK2 inhibitor is compound 361. In some embodiments, the selective ROCK2 inhibitor is compound 362.

[0098] In some embodiments, the selective ROCK2 inhibitor is compound 363. In some embodiments, the selective ROCK2 inhibitor is compound 364. In some embodiments, the selective ROCK2 inhibitor is compound 365. In some embodiments, the selective ROCK2 inhibitor is compound 366. In some embodiments, the selective ROCK2 inhibitor is compound 367. In some embodiments, the selective ROCK2 inhibitor is compound 368. In some embodiments, the selective ROCK2 inhibitor is compound 369. In some embodiments, the selective ROCK2 inhibitor is compound 370. In some embodiments, the selective ROCK2 inhibitor is compound 371. In some embodiments, the selective ROCK2 inhibitor is compound 372. In some embodiments, the selective ROCK2 inhibitor is compound 373. In some embodiments, the selective ROCK2 inhibitor is compound 374. In some embodiments, the selective ROCK2 inhibitor is compound 375. In some embodiments, the selective ROCK2 inhibitor is compound 376. In some embodiments, the selective ROCK2 inhibitor is compound 377.

[0099] In some embodiments, the selective ROCK2 inhibitor is compound 378. In some embodiments, the selective ROCK2 inhibitor is compound 379. In some embodiments, the selective ROCK2 inhibitor is compound 380. In some embodiments, the selective ROCK2 inhibitor is compound 381. In some embodiments, the selective ROCK2 inhibitor is compound 382. In some embodiments, the selective ROCK2 inhibitor is compound 383. In some embodiments, the selective ROCK2 inhibitor is compound 384. In some embodiments, the selective ROCK2 inhibitor is compound 385. In some embodiments, the selective ROCK2 inhibitor is compound 386. In some embodiments, the selective ROCK2 inhibitor is compound 387.

[0100] In some embodiments, the selective ROCK2 inhibitor is compound 388. In some embodiments, the selective ROCK2 inhibitor is compound 389. In some embodiments, the selective ROCK2 inhibitor is compound 390. In some embodiments, the selective ROCK2 inhibitor is compound 391. In some embodiments, the selective ROCK2 inhibitor is compound 392. In some embodiments, the selective ROCK2 inhibitor is compound 393. In some embodiments, the selective ROCK2 inhibitor is compound 394. In some embodiments, the selective ROCK2 inhibitor is compound 395. In some embodiments, the selective ROCK2 inhibitor is compound 396. In some embodiments, the selective ROCK2 inhibitor is compound 397. In some embodiments, the selective ROCK2 inhibitor is compound 398. In some embodiments, the selective ROCK2 inhibitor is compound 399. In some embodiments, the selective ROCK2 inhibitor is compound 400.

[0101] Other non-limiting examples of selective ROCK2 inhibitors are provided in Table 3 and International Publication No. WO 2018 / 039539 A1, the entirety of which is incorporated herein by reference. In some embodiments, the selective ROCK2 small molecule inhibitor is a compound of formula (I) (i.e., any subgenus or species thereof) disclosed in International Publication No. WO 2018 / 039539 A1 (the entirety of which is incorporated herein by reference); or a pharmaceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, cocrystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is a compound of formula (I) (i.e., any subgenus or species thereof) disclosed in International Publication No. WO 2018 / 039539 A1 (the entirety of which is incorporated herein by reference); or a pharmaceutically acceptable salt thereof.

[0102] In some embodiments, the selective ROCK2 small molecule inhibitor comprises a compound selected from compounds 401-483 in Table 3. In some embodiments, the selective ROCK2 small molecule inhibitor is selected from compounds 401-483 in Table 3. In some embodiments, the selective ROCK2 small molecule inhibitor is any one of compounds 401-483 in Table 3, or a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, cocrystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is any one of compounds 401-483 in Table 3, or a pharma- ceutically acceptable salt thereof. Where a particular salt form of a compound (e.g., a trifluoroacetic acid (TFA) salt) is shown in Table 3, other pharma- ceutically acceptable salts of the compound are also provided as part of this disclosure.

[0103] Table 3. Examples of selective ROCK2 inhibitors [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] #=Compound

[0104] In some embodiments, the selective ROCK2 inhibitor comprises compound 401. In some embodiments, the selective ROCK2 inhibitor comprises compound 402. In some embodiments, the selective ROCK2 inhibitor comprises compound 403. In some embodiments, the selective ROCK2 inhibitor comprises compound 404. In some embodiments, the selective ROCK2 inhibitor comprises compound 405. In some embodiments, the selective ROCK2 inhibitor comprises compound 406. In some embodiments, the selective ROCK2 inhibitor comprises compound 407. In some embodiments, the selective ROCK2 inhibitor comprises compound 408. In some embodiments, the selective ROCK2 inhibitor comprises compound 409. In some embodiments, the selective ROCK2 inhibitor comprises compound 410. In some embodiments, the selective ROCK2 inhibitor comprises compound 411. In some embodiments, the selective ROCK2 inhibitor comprises compound 412. In some embodiments, the selective ROCK2 inhibitor comprises compound 413. In some embodiments, the selective ROCK2 inhibitor comprises compound 414. In some embodiments, the selective ROCK2 inhibitor comprises compound 415. In some embodiments, the selective ROCK2 inhibitor comprises compound 416.

[0105] In some embodiments, the selective ROCK2 inhibitor comprises compound 417. In some embodiments, the selective ROCK2 inhibitor comprises compound 418. In some embodiments, the selective ROCK2 inhibitor comprises compound 419. In some embodiments, the selective ROCK2 inhibitor comprises compound 420. In some embodiments, the selective ROCK2 inhibitor comprises compound 421. In some embodiments, the selective ROCK2 inhibitor comprises compound 422. In some embodiments, the selective ROCK2 inhibitor comprises compound 423. In some embodiments, the selective ROCK2 inhibitor comprises compound 424. In some embodiments, the selective ROCK2 inhibitor comprises compound 425. In some embodiments, the selective ROCK2 inhibitor comprises compound 426. In some embodiments, the selective ROCK2 inhibitor comprises compound 427. In some embodiments, the selective ROCK2 inhibitor comprises compound 428. In some embodiments, the selective ROCK2 inhibitor comprises compound 429. In some embodiments, the selective ROCK2 inhibitor comprises compound 430. In some embodiments, the selective ROCK2 inhibitor comprises compound 431. In some embodiments, the selective ROCK2 inhibitor comprises compound 432. In some embodiments, the selective ROCK2 inhibitor comprises compound 433.

[0106] In some embodiments, the selective ROCK2 inhibitor comprises compound 434. In some embodiments, the selective ROCK2 inhibitor comprises compound 435. In some embodiments, the selective ROCK2 inhibitor comprises compound 436. In some embodiments, the selective ROCK2 inhibitor comprises compound 437. In some embodiments, the selective ROCK2 inhibitor comprises compound 438. In some embodiments, the selective ROCK2 inhibitor comprises compound 439. In some embodiments, the selective ROCK2 inhibitor comprises compound 440. In some embodiments, the selective ROCK2 inhibitor comprises compound 441. In some embodiments, the selective ROCK2 inhibitor comprises compound 442. In some embodiments, the selective ROCK2 inhibitor comprises compound 443. In some embodiments, the selective ROCK2 inhibitor comprises compound 444. In some embodiments, the selective ROCK2 inhibitor comprises compound 445. In some embodiments, the selective ROCK2 inhibitor comprises compound 446. In some embodiments, the selective ROCK2 inhibitor comprises compound 447. In some embodiments, the selective ROCK2 inhibitor comprises compound 448. In some embodiments, the selective ROCK2 inhibitor comprises compound 449.

[0107] In some embodiments, the selective ROCK2 inhibitor comprises compound 450. In some embodiments, the selective ROCK2 inhibitor comprises compound 451. In some embodiments, the selective ROCK2 inhibitor comprises compound 452. In some embodiments, the selective ROCK2 inhibitor comprises compound 453. In some embodiments, the selective ROCK2 inhibitor comprises compound 454. In some embodiments, the selective ROCK2 inhibitor comprises compound 455. In some embodiments, the selective ROCK2 inhibitor comprises compound 456. In some embodiments, the selective ROCK2 inhibitor comprises compound 457. In some embodiments, the selective ROCK2 inhibitor comprises compound 458. In some embodiments, the selective ROCK2 inhibitor comprises compound 459. In some embodiments, the selective ROCK2 inhibitor comprises compound 460. In some embodiments, the selective ROCK2 inhibitor comprises compound 461. In some embodiments, the selective ROCK2 inhibitor comprises compound 462. In some embodiments, the selective ROCK2 inhibitor comprises compound 463. In some embodiments, the selective ROCK2 inhibitor comprises compound 464. In some embodiments, the selective ROCK2 inhibitor comprises compound 465.

[0108] In some embodiments, the selective ROCK2 inhibitor comprises compound 466. In some embodiments, the selective ROCK2 inhibitor comprises compound 467. In some embodiments, the selective ROCK2 inhibitor comprises compound 468. In some embodiments, the selective ROCK2 inhibitor comprises compound 469. In some embodiments, the selective ROCK2 inhibitor comprises compound 470. In some embodiments, the selective ROCK2 inhibitor comprises compound 471. In some embodiments, the selective ROCK2 inhibitor comprises compound 472. In some embodiments, the selective ROCK2 inhibitor comprises compound 473. In some embodiments, the selective ROCK2 inhibitor comprises compound 474. In some embodiments, the selective ROCK2 inhibitor comprises compound 475. In some embodiments, the selective ROCK2 inhibitor comprises compound 476. In some embodiments, the selective ROCK2 inhibitor comprises compound 477. In some embodiments, the selective ROCK2 inhibitor comprises compound 478. In some embodiments, the selective ROCK2 inhibitor comprises compound 479. In some embodiments, the selective ROCK2 inhibitor comprises compound 480. In some embodiments, the selective ROCK2 inhibitor comprises compound 481. In some embodiments, the selective ROCK2 inhibitor comprises compound 482. In some embodiments, the selective ROCK2 inhibitor comprises compound 483.

[0109] In some embodiments, the selective ROCK2 inhibitor is compound 401. In some embodiments, the selective ROCK2 inhibitor is compound 402. In some embodiments, the selective ROCK2 inhibitor is compound 403. In some embodiments, the selective ROCK2 inhibitor is compound 404. In some embodiments, the selective ROCK2 inhibitor is compound 405. In some embodiments, the selective ROCK2 inhibitor is compound 406. In some embodiments, the selective ROCK2 inhibitor is compound 407. In some embodiments, the selective ROCK2 inhibitor is compound 408. In some embodiments, the selective ROCK2 inhibitor is compound 409. In some embodiments, the selective ROCK2 inhibitor is compound 410. In some embodiments, the selective ROCK2 inhibitor is compound 411. In some embodiments, the selective ROCK2 inhibitor is compound 412. In some embodiments, the selective ROCK2 inhibitor is compound 413. In some embodiments, the selective ROCK2 inhibitor is compound 414. In some embodiments, the selective ROCK2 inhibitor is compound 415.

[0110] In some embodiments, the selective ROCK2 inhibitor is compound 416. In some embodiments, the selective ROCK2 inhibitor is compound 417. In some embodiments, the selective ROCK2 inhibitor is compound 418. In some embodiments, the selective ROCK2 inhibitor is compound 419. In some embodiments, the selective ROCK2 inhibitor is compound 420. In some embodiments, the selective ROCK2 inhibitor is compound 421. In some embodiments, the selective ROCK2 inhibitor is compound 422. In some embodiments, the selective ROCK2 inhibitor is compound 423. In some embodiments, the selective ROCK2 inhibitor is compound 424. In some embodiments, the selective ROCK2 inhibitor is compound 425. In some embodiments, the selective ROCK2 inhibitor is compound 426. In some embodiments, the selective ROCK2 inhibitor is compound 427. In some embodiments, the selective ROCK2 inhibitor is compound 428. In some embodiments, the selective ROCK2 inhibitor is compound 429. In some embodiments, the selective ROCK2 inhibitor is compound 430.

[0111] In some embodiments, the selective ROCK2 inhibitor is compound 431. In some embodiments, the selective ROCK2 inhibitor is compound 432. In some embodiments, the selective ROCK2 inhibitor is compound 433. In some embodiments, the selective ROCK2 inhibitor is compound 434. In some embodiments, the selective ROCK2 inhibitor is compound 435. In some embodiments, the selective ROCK2 inhibitor is compound 436. In some embodiments, the selective ROCK2 inhibitor is compound 437. In some embodiments, the selective ROCK2 inhibitor is compound 438. In some embodiments, the selective ROCK2 inhibitor is compound 439. In some embodiments, the selective ROCK2 inhibitor is compound 440. In some embodiments, the selective ROCK2 inhibitor is compound 441. In some embodiments, the selective ROCK2 inhibitor is compound 442. In some embodiments, the selective ROCK2 inhibitor is compound 443. In some embodiments, the selective ROCK2 inhibitor is compound 444. In some embodiments, the selective ROCK2 inhibitor is compound 445.

[0112] In some embodiments, the selective ROCK2 inhibitor is compound 446. In some embodiments, the selective ROCK2 inhibitor is compound 447. In some embodiments, the selective ROCK2 inhibitor is compound 448. In some embodiments, the selective ROCK2 inhibitor is compound 449. In some embodiments, the selective ROCK2 inhibitor is compound 450. In some embodiments, the selective ROCK2 inhibitor is compound 451. In some embodiments, the selective ROCK2 inhibitor is compound 452. In some embodiments, the selective ROCK2 inhibitor is compound 453. In some embodiments, the selective ROCK2 inhibitor is compound 454. In some embodiments, the selective ROCK2 inhibitor is compound 455. In some embodiments, the selective ROCK2 inhibitor is compound 456. In some embodiments, the selective ROCK2 inhibitor is compound 457. In some embodiments, the selective ROCK2 inhibitor is compound 458. In some embodiments, the selective ROCK2 inhibitor is compound 459. In some embodiments, the selective ROCK2 inhibitor is compound 460.

[0113] In some embodiments, the selective ROCK2 inhibitor is compound 461. In some embodiments, the selective ROCK2 inhibitor is compound 462. In some embodiments, the selective ROCK2 inhibitor is compound 463. In some embodiments, the selective ROCK2 inhibitor is compound 464. In some embodiments, the selective ROCK2 inhibitor is compound 465. In some embodiments, the selective ROCK2 inhibitor is compound 466. In some embodiments, the selective ROCK2 inhibitor is compound 467. In some embodiments, the selective ROCK2 inhibitor is compound 468. In some embodiments, the selective ROCK2 inhibitor is compound 469. In some embodiments, the selective ROCK2 inhibitor is compound 470. In some embodiments, the selective ROCK2 inhibitor is compound 471.

[0114] In some embodiments, the selective ROCK2 inhibitor is compound 472. In some embodiments, the selective ROCK2 inhibitor is compound 473. In some embodiments, the selective ROCK2 inhibitor is compound 474. In some embodiments, the selective ROCK2 inhibitor is compound 475. In some embodiments, the selective ROCK2 inhibitor is compound 476. In some embodiments, the selective ROCK2 inhibitor is compound 477. In some embodiments, the selective ROCK2 inhibitor is compound 478. In some embodiments, the selective ROCK2 inhibitor is compound 479. In some embodiments, the selective ROCK2 inhibitor is compound 480. In some embodiments, the selective ROCK2 inhibitor is compound 481. In some embodiments, the selective ROCK2 inhibitor is compound 482. In some embodiments, the selective ROCK2 inhibitor is compound 483.

[0115] In some embodiments, the selective ROCK2 small molecule inhibitor is selected from compounds 484 and 485 in Table 4. In some embodiments, the selective ROCK2 small molecule inhibitor is selected from compounds 484 and 485 in Table 4, and pharma- ceutically acceptable salts, stereoisomers, tautomers, cocrystals, polymorphs, solvates, hydrates, prodrugs, and topo-labeled derivatives thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is selected from compounds 484 and 485 in Table 4, and pharma-ceutically acceptable salts thereof.

[0116] Table 4. Examples of selective ROCK2 inhibitors [Table 4]

[0117] In some embodiments, the selective ROCK2 inhibitor comprises compound 484. In some embodiments, the selective ROCK2 inhibitor comprises compound 485. In some embodiments, the selective ROCK2 inhibitor is compound 484. In some embodiments, the selective ROCK2 inhibitor is compound 485. Other non-limiting examples of selective ROCK2 inhibitors are provided in Table 5 and US Patent Publication No. US20190276440A1, the entirety of which is incorporated herein by reference. In some embodiments, the selective ROCK2 small molecule inhibitor is a compound of formula (I) (i.e., any subgenus or species thereof) disclosed in US Patent Publication No. US20190276440A1 (the entirety of which is incorporated herein by reference); or a pharmaceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, cocrystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is a compound of formula (I) (i.e., any subgenus or species thereof) disclosed in US Patent Publication No. US20190276440A1 (the entirety of which is incorporated herein by reference); or a pharmaceutically acceptable salt thereof.

[0118] In some embodiments, the selective ROCK2 small molecule inhibitor comprises a compound selected from compounds 486-559 in Table 5. In some embodiments, the selective ROCK2 small molecule inhibitor is selected from compounds 486-559 in Table 5. In some embodiments, the selective ROCK2 small molecule inhibitor is any one of compounds 486-559 in Table 5, or a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, cocrystal, or prodrug thereof. In some embodiments, the selective ROCK2 small molecule inhibitor is any one of compounds 486-559 in Table 5, or a pharma- ceutically acceptable salt thereof. Where a particular salt form of a compound (e.g., a trifluoroacetic acid (TFA) salt) is shown in Table 5, other pharma- ceutically acceptable salts of the compound are also provided as part of this disclosure.

[0119] Table 5. Examples of selective ROCK2 inhibitors [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] #=Compound

[0120] In some embodiments, the selective ROCK2 inhibitor comprises compound 486. In some embodiments, the selective ROCK2 inhibitor comprises compound 487. In some embodiments, the selective ROCK2 inhibitor comprises compound 488. In some embodiments, the selective ROCK2 inhibitor comprises compound 489. In some embodiments, the selective ROCK2 inhibitor comprises compound 490. In some embodiments, the selective ROCK2 inhibitor comprises compound 491. In some embodiments, the selective ROCK2 inhibitor comprises compound 492. In some embodiments, the selective ROCK2 inhibitor comprises compound 493. In some embodiments, the selective ROCK2 inhibitor comprises compound 494. In some embodiments, the selective ROCK2 inhibitor comprises compound 495. In some embodiments, the selective ROCK2 inhibitor comprises compound 496. In some embodiments, the selective ROCK2 inhibitor comprises compound 497. In some embodiments, the selective ROCK2 inhibitor comprises compound 498. In some embodiments, the selective ROCK2 inhibitor comprises compound 499. In some embodiments, the selective ROCK2 inhibitor comprises compound 500.

[0121] In some embodiments, the selective ROCK2 inhibitor comprises compound 501. In some embodiments, the selective ROCK2 inhibitor comprises compound 502. In some embodiments, the selective ROCK2 inhibitor comprises compound 503. In some embodiments, the selective ROCK2 inhibitor comprises compound 504. In some embodiments, the selective ROCK2 inhibitor comprises compound 505. In some embodiments, the selective ROCK2 inhibitor comprises compound 506. In some embodiments, the selective ROCK2 inhibitor comprises compound 507. In some embodiments, the selective ROCK2 inhibitor comprises compound 508. In some embodiments, the selective ROCK2 inhibitor comprises compound 509. In some embodiments, the selective ROCK2 inhibitor comprises compound 510. In some embodiments, the selective ROCK2 inhibitor comprises compound 511. In some embodiments, the selective ROCK2 inhibitor comprises compound 512. In some embodiments, the selective ROCK2 inhibitor comprises compound 513. In some embodiments, the selective ROCK2 inhibitor comprises compound 514.

[0122] In some embodiments, the selective ROCK2 inhibitor comprises compound 515. In some embodiments, the selective ROCK2 inhibitor comprises compound 516. In some embodiments, the selective ROCK2 inhibitor comprises compound 517. In some embodiments, the selective ROCK2 inhibitor comprises compound 518. In some embodiments, the selective ROCK2 inhibitor comprises compound 519. In some embodiments, the selective ROCK2 inhibitor comprises compound 520. In some embodiments, the selective ROCK2 inhibitor comprises compound 521. In some embodiments, the selective ROCK2 inhibitor comprises compound 522. In some embodiments, the selective ROCK2 inhibitor comprises compound 523. In some embodiments, the selective ROCK2 inhibitor comprises compound 524. In some embodiments, the selective ROCK2 inhibitor comprises compound 525. In some embodiments, the selective ROCK2 inhibitor comprises compound 526. In some embodiments, the selective ROCK2 inhibitor comprises compound 527.

[0123] In some embodiments, the selective ROCK2 inhibitor comprises compound 528. In some embodiments, the selective ROCK2 inhibitor comprises compound 529. In some embodiments, the selective ROCK2 inhibitor comprises compound 530. In some embodiments, the selective ROCK2 inhibitor comprises compound 531. In some embodiments, the selective ROCK2 inhibitor comprises compound 532. In some embodiments, the selective ROCK2 inhibitor comprises compound 533. In some embodiments, the selective ROCK2 inhibitor comprises compound 534. In some embodiments, the selective ROCK2 inhibitor comprises compound 535. In some embodiments, the selective ROCK2 inhibitor comprises compound 536. In some embodiments, the selective ROCK2 inhibitor comprises compound 537. In some embodiments, the selective ROCK2 inhibitor comprises compound 538. In some embodiments, the selective ROCK2 inhibitor comprises compound 539. In some embodiments, the selective ROCK2 inhibitor comprises compound 540. In some embodiments, the selective ROCK2 inhibitor comprises compound 541. In some embodiments, the selective ROCK2 inhibitor comprises compound 542.

[0124] In some embodiments, the selective ROCK2 inhibitor comprises compound 543. In some embodiments, the selective ROCK2 inhibitor comprises compound 544. In some embodiments, the selective ROCK2 inhibitor comprises compound 545. In some embodiments, the selective ROCK2 inhibitor comprises compound 546. In some embodiments, the selective ROCK2 inhibitor comprises compound 547. In some embodiments, the selective ROCK2 inhibitor comprises compound 548. In some embodiments, the selective ROCK2 inhibitor comprises compound 549. In some embodiments, the selective ROCK2 inhibitor comprises compound 550. In some embodiments, the selective ROCK2 inhibitor comprises compound 551. In some embodiments, the selective ROCK2 inhibitor comprises compound 552. In some embodiments, the selective ROCK2 inhibitor comprises compound 553. In some embodiments, the selective ROCK2 inhibitor comprises compound 554. In some embodiments, the selective ROCK2 inhibitor comprises compound 555. In some embodiments, the selective ROCK2 inhibitor comprises compound 556. In some embodiments, the selective ROCK2 inhibitor comprises compound 557. In some embodiments, the selective ROCK2 inhibitor comprises compound 558. In some embodiments, the selective ROCK2 inhibitor comprises compound 559.

[0125] The selective ROCK2 inhibitor can be administered (delivered) to the subject by any route suitable for the dosage form of the inhibitor.For example, the selective ROCK2 inhibitor can be formulated as an intravenous solution and delivered to the subject via intravenous administration.As another example, the ROCK2 inhibitor can be formulated as a tablet (or other pill form) or liquid suspension and delivered to the subject via oral administration.Other dosage forms and delivery routes are contemplated herein, such as, but not limited to, intranasal and intramuscular. In general, formulation refers to the process of combining a drug, such as a selective ROCK2 inhibitor, with carriers, excipients, and / or other inactive drugs to produce a therapeutic composition.See, for example, Remington: The Science and Practice of Pharmacy, 19* Edition (1995) and / or Handbook of Pharmaceutical Granulation Technology, Chapter 7, "Drugs and the Pharmaceutical Sciences", vol. 81, 1997.

[0126] Biomimetic Models Also provided herein is a biomimetic model of lymphatic vessels and a method of use. In some embodiments, the model comprises a three-dimensional collagen hydrogel, comprising a polydimethylsiloxane housing and two parallel cylindrical channels (see, for example, Nguyen DT et al. PNAS 2013; 110(17): 6712-6717). In some embodiments, human dermal lymphatic endothelial cells are seeded into one of the cylindrical channels. In some embodiments, the model further comprises lymphangiogenic endothelial growth factor C or other growth factors. In some embodiments, the biomimetic model is used to evaluate lymphatic vessels or lymphatic drainage, for example, drainage of interstitial fluid. Thus, provided herein is a method of measuring drainage, such as lymphatic drainage, using a biomimetic model of lymphatic vessels. In some embodiments, the biomimetic model includes at least one (e.g., 1, 2, 3, 4, or more) channel, e.g., on a gel (e.g., collagen gel, fibrin, material, synthetic hydrogel, or hydrogel made from other ECM elements). In some embodiments, the at least one channel is contained within a material selected from polydimethylsiloxane (PDMS), polypropylene, polystyrene, polyurethane, polycaprolactone, polymethylmethacrylate, and polyacetate. EXAMPLES

[0127] example Identifying the pathophysiological mechanisms underlying this multifactorial disease using animal models of lymphedema is challenging because it is difficult to separate the relative contributions of biological and biophysical factors. In contrast, two-dimensional (2D) cell culture models are highly controllable but do not recapitulate the 3D organization of lymphatic vessels in vivo.

[0128] Example 1. Development of an in vitro lymphatic drainage model. To overcome the problems outlined above, we developed a microfluidic 3D culture model featuring engineered lymphatic vessels capable of draining interstitial fluid. The device contains a polydimethylsiloxane (PDMS) housing and two parallel cylindrical channels within a 3D collagen hydrogel (Figure 1A). Human dermal lymphatic endothelial cells (LECs) seeded in one channel generated engineered lymphatic vessels (Figure 1A) that expressed lymphatic endothelial markers with proper apical-basal orientation (Figure 1A and Figure 1B). Furthermore, stimulation with lymphangiogenic vascular endothelial growth factor C (VEGF) induced lymphatic vessel sprouting (Figure 1C). To characterize the integrity of lymphatic endothelium within the device, their junctional tissue was examined by immunofluorescence. Junctional adhesion molecule A (JAM-A) and zoneural occludin 1 (ZO-1) staining revealed weak tight junctions (TJs) in LEC-generated lymphatic vessels, in contrast to the strongly established TJs in blood vessels generated by human dermal microvascular endothelial cells (BECs) as a control (Figure 1D), similar to those described for native lymphatic vessels. Similarly, VE-cadherin staining to visualize adherens junctions (AJs) showed irregular wavy AJs in lymphatic vessels, in contrast to smooth continuous AJs in blood vessels (Figure 1E), and portal vein-like structures in lymphatic vessels only (Figures 1E and 1F), reminiscent of the first lymphatic “button-like junctions” observed in vivo (4, 5). Together, these data suggest structural features of rudimentary lymphatic vessels.

[0129] To functionally evaluate this rudimentary lymphatic vessel, measurements of lymphatic drainage were required. To accomplish this, a second cell-free channel was pressurized with a surrogate lymph fluid composed of fluorescently labeled lymphatic molecules, including fatty acids, phospholipids, albumin, and insoluble particles (data not shown). The pressure of the interstitial fluid triggered convective fluid transport into the modified lymphatic vessel. Lymphatic fluid draining from the lymphatic vessel was accumulated in a connected reservoir and collected for analysis. Human dermal microvascular endothelial cell-generated blood vessels or cell-free channels were used as controls. For all lymphatic molecules tested, the modified lymphatic vessels showed superior drainage capabilities compared to the modified blood vessels (Figure 1G). Time-lapse microscopy of the drainage further confirmed that fluid entered the lumen of the lymphatic vessels faster than the blood vessels (Figure 1H and data not shown). Surprisingly, while blood vessels appeared to be compressed by elevated interstitial fluid pressure, lymphatic vessel lumens expanded under the same conditions (data not shown); this is consistent with in vivo observations that lymphatic vessels expand in response to elevated interstitial fluid pressure. (6)(7, 8) Together, these findings suggest that our lymphatic vessel-on-a-chip recapitulates the structural and functional characteristics of native lymphatic vessels.

[0130] Example 2: Identification and use of proinflammatory cytokine inhibitors in vivo in lymphedema. Since inflammation is a major driver of lymphedema (2, 9, 10), in the next set of experiments we tested whether inflammatory cytokines impede drainage in this system. Indeed, IL-2, GM-CSF, and G-CSF each significantly reduced lymphatic drainage (Figure 1I) and tightened AJs (Figure 1J) and TJs (data not shown). To characterize what signaling pathways are involved in tightening lymphatic junctions, we identified inhibitors that could affect IL-2-induced junction assembly (data not shown): PP2, Src family of protein tyrosine kinase inhibitors; Wortmannin, a PI3K inhibitor; Y27632, a ROCK inhibitor; GGTI298, a Rap1 inhibitor; NSC23766, a Rac1 inhibitor; blebbistatin, a myosin II inhibitor; and Cpd22, an integrin-linked kinase inhibitor. Only the ROCK inhibitor Y27632 completely normalized the drainage impairment in IL-2-treated lymphatic vessels (data not shown). Y27632 also normalized GM-CSF- and G-CSF-treated conditions (Figure 2A and 2B; data not shown), and the clinically used ROCK inhibitors fasudil and ripasudil (11) similarly improved lymphatic drainage (Figure 2C), together suggesting a role for ROCK in regulating inflammation at lymphatic junctions and drainage.

[0131] Interestingly, inflammatory cytokines are known to have the opposite effect on capillaries, increasing vascular permeability by loosening cell-cell junctions (12), which may also contribute to lymphedema (2). Indeed, in engineered BEC-based vessels, IL-2, GM-CSF, and G-CSF all disrupted vascular barrier function (Figure 2D and Figure 2E). Here, treatment with Y27632 restored barrier function (Figure 2E, data not shown), suggesting that ROCK inhibition may ameliorate lymphedema by simultaneously reducing fluid leakage from blood vessels and increasing lymphatic drainage (Figure 2F).

[0132] To investigate the potential efficacy of ROCK inhibition in lymphedema, Y27632 was tested in an established mouse model of lymphedema (13, 14). Lymphedema was induced by surgical ligation of peripheral lymphatic vessels in the tails of mice (Figure 2G). Treatment with Y27632 completely prevented the lymphedema normally induced by this procedure (Figure 2G) and further led to a 10-fold higher drainage to proximal lymph nodes compared to untreated control mice (Figure 2H). Although these results suggest the possibility of preventing lymphedema, we also investigated whether ROCK inhibition could reverse existing lymphedema. Mice with injured tails were left for one month without any treatment. Although a few animals recovered spontaneously, several animals showed persistent tail swelling, indicating chronic lymphedema. When these mice were treated with Y27632, lymphedema gradually improved and was completely resolved by 2 weeks after treatment (Figures 2I and 2J; data not shown).

[0133] Example 3: Characterization of ROCK2 and ROCK2 inhibitors in LECs and BECs. Although these results suggested a promising avenue for the treatment of lymphedema, it remained unclear how ROCK activity could establish lymphatic endothelial cell (LEC) junctions while exerting the opposite effect on blood endothelial cell (BEC) junctions. Without wishing to be bound by theory, it was hypothesized that these differences could result from distinct functions of the two isoforms of ROCK, ROCK1 and ROCK2 (15). Under basal conditions, ROCK1 was detected at similar levels in LEC and BEC, whereas ROCK2 was barely detectable in BEC (Figure 3A). Interestingly, IL-2 in LEC substantially downregulated ROCK1, leaving ROCK2 as the predominant isoform (Figure 3B). To test whether alterations in ROCK1 / 2 levels alter LEC junctions and drainage, the effect of siRNA knockdown of ROCK1 / 2 was examined (Figure 3C). Indeed, knockdown of ROCK1 impaired drainage in LECs, phenocopying the effects of IL-2 exposure, whereas loss of ROCK2 phenocopied Y27632, maintaining drainage regardless of IL-2 (Figure 3D). These striking differences between ROCK1 and ROCK2 were also reflected in the phenotype of adherens junctions (Figure 3E). Loss of ROCK1 tightened the junctions, whereas loss of ROCK2 loosened them. Given the prominent expression of ROCK1 in BECs, the effect of knockdown of ROCK1 was also examined in these cells. As before, control BECs displayed high barrier function (low permeability) in basal conditions that was lost upon exposure to IL-2 (Figure 3F). Consistent with the effect in LECs, knockdown of ROCK1 allowed the cells to maintain high barrier function (Figure 3F) and intact junctions (Figure 3G). Together, these data suggest that ROCK2 is required for cytokine-induced junction tightening in LECs and that inhibition of ROCK2 can normalize these junctions to support lymphatic drainage.

[0134] Next, by isolating endogenous ROCK2 by immunoprecipitation, we identified its interaction with junctional adhesion molecule A (JAM-A) and zoneural occludin 1 (ZO-1), which appeared only in cytokine-exposed LECs (Fig. 3H). ROCK1 was not present in junction complexes, but knockdown of ROCK1 induced complex assembly that phenocopied the effects of IL-2 (Fig. 3H). Immunofluorescence staining confirmed that IL-2 or ROCK1 knockdown induced ROCK2 localization with tight junctions (Fig. 3I). To test whether JAM-A is functionally important for the ROCK pathway, we knocked down JAM-A; LECs did not tighten junctions in the presence of IL-2 (Fig. 3J). These ROCK2 / JAM-A / ZO-1 complexes were not observed in BECs (Fig. 3K). Finally, in a phase II clinical trial to treat chronic graft-versus-host disease, idiopathic pulmonary fibrosis, and psoriasis, a ROCK2-specific small molecule inhibitor, KD025, was administered under normal and IL-2 conditions in LECs (Figure 3L). KD025 (20 μM) reversed the tightening of LEC junctions (data not shown) and impaired drainage (Figure 3L) in response to IL-2 challenge. In summary, ROCK2 tightens lymphatic junctions by associating with JAM-A and ZO-1 when inflammatory signals are present.

[0135] Example 4: Characterization of selective ROCK2 inhibitors in vivo Administration of KD025 in mice reduced visible tail vein edema swelling after cautery injury (Figure 4A). Changes in tail volume (Figure 4B) and drainage to iliac lymph nodes were assessed 1 h after intradermal injection of Alexa647-conjugated albumin into the tails of mice treated with KD025 or vehicle (Figure 4C).

[0136] Here, using a 3D organotypic model of lymphatic vessels that emulates lymphatic junction structure and drainage function, the data show that inflammatory cytokines induce lymphatic endothelial cells to tighten cell-cell junctions via the ROCK2 / JAM-A / ZO-1 protein complex, which is not found in blood endothelium. Given that ROCK inhibitors have limited clinical utility because ROCK1-mediated relaxation of vascular smooth muscle leads to hypotension (11), our findings suggest that ROCK2-specific therapeutics, such as KD025, may be used to treat lymphedema with less toxicity. This study highlights the central role of cell-cell junctions in regulating the passage of fluid from blood vessels through the interstitial space back to the lymphatic circulation, and identifying the special mechanisms that cells use to dynamically regulate these adhesions will be central to normalizing perfusion. Furthermore, this work highlights how recapitulating appropriate architectures enables the emergence of multicellular functions previously only observed in vivo, and such advances in biomimetic cultures are likely to prove to play a key role in bridging traditional in vitro and in vivo models of physiology and disease.

[0137] References

number

number

[0138] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, and in some cases may be incorporated in their entirety. The indefinite articles "a" and "an," as used in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one." It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes multiple steps or acts, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are recited.

[0139] In the claims and the above specification, all transitional phrases such as "comprising," "including," "holding," "having," "containing," "involving," "holding," "comprising," and the like, are to be understood to mean open ended, i.e., including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as provided in the United States Patent Office's Patent Examining Manual Section 2111.03. The terms "about" and "substantially" preceding a numerical value mean ±10% of the stated numerical value. When a range of values ​​is provided, each value between the upper and lower limits of the range is specifically contemplated and described herein.

Claims

1. A composition comprising a selective Rho-associated kinase 2 (ROCK2) inhibitor for treating a subject having or at risk for a condition associated with impaired lymphatic drainage.

2. 2. The composition of claim 1, wherein the selective ROCK2 inhibitor is in an amount effective to reduce symptoms associated with impaired lymphatic drainage.

3. The composition of claim 2 , wherein the symptom is swelling in a body part of the subject.

4. A composition comprising a selective ROCK2 inhibitor in an amount effective to restore vascular barrier function.

5. 5. The composition of claim 4, wherein the amount of the selective ROCK2 inhibitor is effective to reduce fluid leakage from blood vessels in a subject receiving the composition compared to a control.

6. 6. The method of claim 4 or 5, wherein the amount of the selective ROCK2 inhibitor is effective to increase lymphatic drainage in a subject to which the composition is administered, as compared to a control.

7. A composition comprising a selective ROCK2 inhibitor in an amount effective to improve lymphatic drainage at lymphatic junctions in a subject to which the composition is administered, compared to a control.

8. A composition comprising a selective ROCK2 inhibitor in an amount effective to prevent the formation of lymphotite junctions in a subject to which the composition is administered.

9. The composition of any one of claims 1 to 8, wherein the subject has a condition associated with impaired lymphatic drainage.

10. 10. The composition of claim 9, wherein the condition is edema.

11. 10. The composition of claim 9, wherein the condition is lymphedema.

12. The composition of claim 11, wherein the lymphedema is primary lymphedema.

13. The composition of claim 11, wherein the lymphedema is secondary lymphedema.

14. The method of any one of claims 1 to 13, wherein the composition is for oral administration.

15. The composition of claim 14 formulated as a tablet.

16. The composition according to any one of claims 1 to 15, for administering the selective ROCK2 inhibitor at a dose of 200 to 400 mg.

17. 1. A composition comprising an effective amount of a selective Rho-associated kinase 2 (ROCK2) inhibitor to prevent the formation of lymphatic-tite junctions, the composition being for use in a method comprising contacting dermal lymphatic endothelial cells with the composition.

18. 1. A composition comprising a selective Rho-associated kinase 2 (ROCK2) inhibitor in an amount effective to improve lymphatic drainage at a lymphatic junction compared to a control, for use in a method comprising contacting a lymphatic-tite junction with the composition.

19. The composition of any one of claims 1 to 18, wherein the selective ROCK2 inhibitor binds to ROCK2 and inhibits ROCK2 serine / threonine kinase activity.

20. The composition of any one of claims 1 to 19, wherein the selective ROCK2 inhibitor is selected from a polypeptide inhibitor, a polynucleotide inhibitor, and a small molecule inhibitor.

21. The composition of claim 20 , wherein the selective ROCK2 inhibitor is a small molecule inhibitor.

22. Small molecule inhibitors have IC ５０ The composition of claim 21 , which binds to ROCK2 at a value

23. 23. The composition of claim 21 or 22, wherein the small molecule inhibitor binds to ROCK2 but does not bind to protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), or myotonic dystrophy kinase-related CDC42-binding kinase (MRCK).

24. Small molecule inhibitors with IC ５０ The composition of any one of claims 21 to 23, which binds to ROCK1 at a value

25. The composition of any one of claims 21 to 24, wherein the small molecule inhibitor is 2-[3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy]-N-(1-methylethyl)-acetamide (KD025).

26. The composition of any one of claims 21 to 24, wherein the small molecule inhibitor is selected from compounds 1-559, or a pharma- ceutically acceptable salt, stereoisomer, tautomer, isotopically labeled derivative, solvate, hydrate, polymorph, co-crystal, or prodrug thereof. 【Chemistry 1】 【Chemistry 2】 【Chemistry 3】 【Chemistry 4】 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 【Chemistry 10】 【Chemistry 11】 【Chemistry 12】 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 【Chemistry 17】 【Chemistry 18】 【Chemistry 19】 【Chemistry 20】 【Chemistry 21】 【Chemical 22】 【Chemistry 23】 【Chemistry 24】 【Chemistry 25】 【Chemistry 26】 【Chemical 27】 【Chemistry 28】 【Chemical Formula 29】 【Chemistry 30】 【Chemistry 31】 【Chemistry 32】 【Chemical Formula 33】 【Chemical 34】 【Chemistry 35】 【Chemical 36】 【Chemical 37】 【Chemical Formula 38】 【Chemical Formula 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 【Chemistry 46】 【Chemistry 47】 【Chemistry 48】 【Chemistry 49】 【Chemistry 50】 【Chemistry 51】 【Chemistry 52】 【Chemistry 53】 【Chemical 54】 【Chemistry 55】 【Chemistry 56】 【Chemistry 57】 【Chemistry 58】

27. The composition of claim 26, wherein the small molecule inhibitor is selected from compounds 1-559, or a pharma- ceutically acceptable salt thereof.

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