Compositions and methods for use of CDC42 inhibitors in the treatment of skin disorders

Abstract

A pharmacological agent and method for treatment of cancer and vascular-related disorders of skin and colon are described. The pharmacological agent, which is a small molecule that targets and inhibits both RhoJ and CDC42 signaling, demonstrates potent anti-vascular effects in normal skin, colon, and tumors, combined with low toxicity, especially as compared to BRAF inhibitors (such as, vemurafenib used in melanoma treatments), selective CDC42 inhibitors (such as CASIN), and VEGF inhibitors (known as anti-vascular agents, such as, for example, linifanib). As such, the pharmacological agent is particularly suited for applications related to treating cancer, as well as disorders of the skin and colon that exhibit increased vasculature.

Description

COMPOSITIONS AND METHODS FOR USE OF CDC42 INHIBITORS IN THE TREATMENT OF SKIN DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 120 to U.S. Patent Application No. 63 / 631 ,887, filed on April 9, 2024, the disclosures of which are hereby incorporated by reference herein in their entireties.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under Grant No. CA244571 awarded by the National Cancer Institute; as well as by the Cancer Center Support Grant No. CA-62203 and UCI Skin Grant AR-075047, awarded by the University of California, Irvine. The Government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The invention is generally directed to a pharmacological agent, a pharmaceutical composition comprising thereof, and a method of treatment comprising the same, for treatment of cancer and disorders of the skin and colon that exhibit increased vasculature, and more particularly for the treatment of skin tumors, photoaging, rosacea, and skin inflammatory disorders.BACKGROUND OF THE INVENTION

[0004] .Tumors that arise in the epidermis must develop a vascular supply to grow beyond a millimeter in depth. In particular, vascular morphogenesis in tissues is a dynamic process, wherein endothelial cells modulate their cytoskeleton and cell matrix adhesions to properly respond to environmental cues (as described, for example, in Gerhardt H., et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol. 2003; 161 (6): 1163-77, the disclosure of which is incorporated herein by reference). As such, the dynamic rearrangement of endothelial cells’ cytoskeleton allows them to migrate both forward and backward within the growing blood vessel (see, forexample: Jakobsson L., et al. Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat Cell Biol. 2010;12(10):943-53; Lee H.W., et al. Flow goes forward and cells step backward: endothelial migration. Exp Mol Med. 2022;54(6):711-9; and Arima S., et al. Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement. Development. 2011 ;138(21 ):4763- 76; the disclosures of which are incorporated herein by reference). Furthermore, the generation of the capillary plexus depends on the regression of existing vessels during the maturation process, and similar types of vessel regression are observed in conditions where the adult vasculature needs to be remodeled (Kam C.Y. , et al. Mechanisms of skin vascular maturation and maintenance captured by longitudinal imaging of live mice. Cell. 2023; 186(11 ):2345-60 e16, the disclosure of which is incorporated herein by reference). Accordingly, all tumors, and particularly those that arise in the epidermis and lack their own vasculature, are dependent on the ability to induce the growth of vessels around them (Wu Z., et al. The role of angiogenesis in melanoma: Clinical treatments and future expectations. Front Pharmacol. 2022; 13: 1028647; Cho W.C., et al. Role of angiogenesis in melanoma progression: Update on key angiogenic mechanisms and other associated components. Semin Cancer Biol. 2019;59:175-86; and Bielenberg D.R., and Zetter B.R. The Contribution of Angiogenesis to the Process of Metastasis. Cancer J. 2015;21 (4):267-73; the disclosures of which are incorporated herein by reference). As such, tumors rapidly accelerate angiogenesis by producing a cornucopia of angiogenesispromoting factors, inducing a haphazard, tortuous vessel tree that is unable to deliver nutrients evenly to the tumor (Lugano R., et al. Tumor angiogenesis: causes, consequences, challenges and opportunities. Cell Mol Life Sci. 2020;77(9):1745-70; and Hanahan D., and Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996;86(3):353-64; the disclosures of which are incorporated herein by reference). Therefore, a thorough understanding of angiogenesis processes and agents is needed to develop potent strategies and therapies for fighting skin tumors.

[0005] Notably, combining tissue clearing with semi-automated tracing has provided great insight into how angiogenesis occurs in the skin and other tissues (Lee E.J., et al.Three-dimensional visualization of cerebral blood vessels and neural changes in thick ischemic rat brain slices using tissue clearing. Sci Rep. 2022; 12(1 ): 15897; Takahashi K., et al. An analysis modality for vascular structures combining tissue-clearing technology and topological data analysis. Nat Commun. 2022; 13(1 ):5239; and Kolesova H., et al. Tissue clearing and imaging methods for cardiovascular development. iScience. 2021 ;24(4): 102387; the disclosures of which are incorporated herein by reference). Furthermore, this approach has been recently applied to tumors (Frenkel N., et al. Tissue clearing and immunostaining to visualize the spatial organization of vasculature and tumor cells in mouse liver. Front Oncol. 2023; 13: 1062926; and Kostrikov S., et al. Optical tissue clearing and machine learning can precisely characterize extravasation and blood vessel architecture in brain tumors. Commun Biol. 2021 ;4(1 ):815; the disclosures of which are incorporated herein by reference), characterizing a tumor vasculature with large tortuous vessels, leaky basement membranes, and unperfused voids (Stolz B.J., et al. Multiscale topology characterizes dynamic tumor vascular networks. Sci Adv. 2022;8(23):eabm2456; and Dobosz M., et al. Multispectral fluorescence ultramicroscopy: three-dimensional visualization and automatic quantification of tumor morphology, drug penetration, and antiangiogenic treatment response. Neoplasia. 2014; 16(1 ): 1-13; the disclosures of which are incorporated herein by reference). Other studies have also utilized longitudinal imaging approaches to provide structural insight into how vessels develop or repair after injury (MacRitchie N., and Maffia P. Light sheet fluorescence microscopy for quantitative three-dimensional imaging of vascular remodelling. Cardiovasc Res. 2021 ;117(2):348-50; the disclosure of which is incorporated herein by reference). However, these approaches have not yet been applied to study skin conditions characterized by pathologic angiogenesis, such as inherited skin vascular anomalies (Ricci K. Medical Therapeutics for the Treatment of Vascular Anomalies: Part 3. Oral Maxillofac Surg Clin North Am. 2024;36(1 ): 125-36; and Gupta R. Propranolol for Vascular Anomalies: Efficacy and Complications in Pediatric Patients. J Indian Assoc Pediatr Surg. 2023;28(3): 194-205; the disclosures of which are incorporated herein by reference) or melanoma tumors, wherein existing anti-angiogenic therapies are many times toxic, or only minimally effective (Gacche R.N. Changing landscape of anti-angiogenic therapy: Novel approaches and clinical perspectives. Biochim Biophys Acta Rev Cancer. 2023; 1878(6): 189020, the disclosure of which is incorporated herein by reference).

[0006] Furthermore, part of the challenge in identifying potent anti-vascular agents lies in the fact that many intracellular signaling pathways can activate angiogenesis (Liu Z.L, et al. Angiogenic signaling pathways and anti-angiogenic therapy for cancer. Signal Transduct Target Ther. 2023;8(1 ):198, the disclosure of which is incorporated herein by reference). To this end, vascular endothelial growth factors, which activate intracellular signaling cascades, and, in turn, induce actin remodeling and focal adhesion assembly (Shibuya M. Vascular Endothelial Growth Factor (VEGF) and Its Receptor (VEGFR) Signaling in Angiogenesis: A Crucial Target for Anti- and Pro-Angiogenic Therapies. Genes Cancer. 2011 ;2(12):1097-105, the disclosure of which is incorporated herein by reference), are major chemo-attractants for migrating endothelial cells. For example, spatial activation of VEGFR2 signaling is critical in the maintenance of the adult vasculature. As another example, Semaphorins have also been identified as regulators of vasculogenesis - Semaphorin 3E binds to the PlexinDI receptor to induce receptor internalization, focal adhesion disassembly, and, subsequently, vessel regression (Oh W.J., and Gu C. The role and mechanism-of-action of Sema3E and Plexin-D1 in vascular and neural development. Semin Cell Dev Biol. 2013;24(3): 156-62; and Kim J, et al. Semaphorin 3E-Plexin-D1 signaling regulates VEGF function in developmental angiogenesis via a feedback mechanism. Genes Dev. 2011 ;25(13):1399-411 ; the disclosures of which are incorporated herein by reference). Moreover, two CDC42 GTPases - RhoJ and CDC42 - are critical mediators of both attractive and repulsive cues generated by activation of VEGF and Plexin receptors, respectively (Uemura A, and Fukushima Y. Rho GTPases in Retinal Vascular Diseases. Int J Mol Sci. 2021 ;22(7), the disclosure of which is incorporated herein by reference). However, despite the dependence of tumors on angiogenesis for growth and the current knowledge of various angiogenesis activation pathways, many tumors, including melanoma tumors, are minimally responsive to current anti-angiogenesis / anti-vascular agents, highlighting the need for more effective and safe drugs in this class. Accordingly, there exists a greatneed for more effective and safe pharmacologic agents with anti-vascular activity in both skin tumors and other skin conditions.SUMMARY OF THE INVENTION

[0007] Various embodiments are directed to a pharmacological agent for medical treatment of disorders and conditions characterized by an increased vasculature, wherein the pharmacological agent is a small molecule characterized by an ability to inhibit both RhoJ and CDC42 effector interactions.

[0008] In various such embodiments, the disorders and conditions are selected from the group consisting of: a cancer, an inherited vascular disorder, a vascular skin disease or condition, a vascular colon disease or condition, another condition characterized by increased or pathologic vascularity, and any combination thereof.

[0009] In still various such embodiments, the vascular skin disease or condition is a disorder selected from the group consisting of: photoaging, rosacea, port-wine birthmarks, atopic dermatitis, radiation dermatitis, psoriasis, skin cancer, another skin condition characterized by an increased or pathologic vascularity, and any combination thereof.

[0010] In still yet various embodiments, the vascular colon disease or condition is a disorder selected from the group consisting of: hereditary hemorrhagic telangiectasia, inflammatory bowel disease, colon cancer, another colon condition characterized by an increased or pathologic vascularity, and any combination thereof.

[0011] In yet still various such embodiments, the small molecule is a compound of Formula (I):or pharmaceutically acceptable salt or solvate thereof, wherein:Y is -N- or -CH-,ring A is selected from: pyrrolidine; phenyl; a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole; and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, NR2indazole; ring B is selected from: a six-membered saturated or partially unsaturated cycle, a six membered saturated or partially unsaturated heterocycle, and an aniline with one or more optional substituents; and ring C is selected from: a six-membered saturated or partially unsaturated cycle; a six membered saturated or partially unsaturated heterocycle, and an aniline with one or more optional substituents; wherein:R2is selected from: H, (C1 -C6)alkyl, halo(C1 -C6)alkyl, amino, amino(C1 - C6)alkyl, hydroxyl, hydroxy(C1 -C6)alkyl, alkoxy-(C1 -C6)-alkyl, carboxyl, ester, and C1-C6-acyloxy

[0012] In yet various such embodiments, the small molecule is a compound of Formula (H):or pharmaceutically acceptable salt or solvate thereof, wherein: dashed bond may be a single or a double bond,Y is -N- or -CH-, ring A is selected from: pyrrolidine; phenyl; a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole; and a bicyclic-heteroaromatic ring furtherselected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; with the proviso that when Y is -N-, A is different from phenyl including heteroaryl groups; ring A' is selected from: an aromatic ring and a heteroaromatic ring, further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole substituted by one or more substituents R2, and bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; each one of X1and X2are independently selected from: -N(H)-, -N(R2)- or -0-R1is selected from: H, (C 1 -C6)alkyl, halo(C1-C6)alkyl, amino(C1 -C6)alkyl, cycloalkyl, hydroxy(C1 -C6)alkyl, alkoxy-(C1-C6)-alkyl, C1 -C6 acyloxy;R2is selected from: H, (C1-C6)alkyl, halo(C1 -C6)alkyl, amino, amino(C1 -C6)alkyl, hydroxyl, hydroxy(C1 -C6)alkyl, alkoxy-(C1-C6)-alkyl, carboxyl, ester, C1-C6- acyloxy.

[0013] In still various such embodiments, the small molecule is a compound of Formula (III):or pharmaceutically acceptable salt or solvate thereof, wherein: each one of R1and R2is independently selected from: H, C1 -C4 alkyl, cycloalkyl; each one of X1and X2is independently selected from: -N(H)-, -N(R2)- or -O-, dashed bond is a single or double bond; ring D is optional; in the absence of ring D, -N(R2)- is -N(R2)(H);when ring D is present, -N(R2)- is embedded into ring D such as to form a five- or six-membered (hetero)aromatic, or nonaromatic ring selected from: an indole, indoline, quinoxaline, tetrahydroquinoxaline, quinoline, or tetrahydroquinoline, as depicted below:with the proviso that when ring D is absent, the dashed bond is a double bond.

[0014] In yet still various such embodiments, the small molecule is a compound of Formula (IV):or pharmaceutically acceptable salt or solvate thereof, wherein:Y1and Y2are independently selected from: -CH-, and -N-, and at least one of Y1and Y2is -N-; the dashed bond is a single or a double bond;R1is selected from: H, C1 -C4 alkyl, cycloalkyl;A is a non-substituted or a R3-substituted ring A selected from: phenyl; pyrrolidine; N-dimethylaminoaniline; a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole; and a bicyclic-heteroaromatic ring further selected from: benzofuran, N Flindole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole, with the proviso that when Y2is -N-, A is not phenyl; whereinR2is selected from: H, C1-C4 alkyl, and cycloalkyl; and whereinR3is selected from: C1-C4 alkyl, C1-C4 haloalkyl (preferably -CF3), and cycloalkyl;A' is a non-substituted or a R4-mono- or -di-substituted ring A' selected from: an aromatic ring and a heteroaromatic ring, further selected from: phenyl, pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole substituted by one or more substituents; and a bicyclic- heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole, wherein R2may be: H, C1-C4 alkyl, cycloalkyl; and wherein each R4is independently selected from: -OH, -COOH, -COOR2, C1-C4 alkyl, C1-C4 haloalkyl (preferably -CF3), C1-C4-haloalkyloxy (preferably -OCF3 or -OCHF2), C1 -C4-alkoxy (preferably -OCH3), cycloalkyl, -NR2, and -N(R2)2; with the proviso that when Y1is -N- and Y2is -C-, R1is H and A is phenyl, then the dashed bond of piperidine is present as a double bond.

[0015] In still yet various such embodiments, the small molecule is a compound of Formula (V):or pharmaceutically acceptable salt or solvate thereof, wherein:Y1and Y2are independently selected from: -CH- and -N-, wherein only one of Y1and Y2are -N-, the dashed bond is a single or a double bond;R1is selected from: H, C1 -C4 alkyl, cycloalkyl;A' is a non-substituted or a R4-mono- or -di-substituted ring A' selected from: an aromatic ring and a heteroaromatic ring, further selected from: phenyl, pyrazole, N-dimethylaminoaniline, N-R2pyrazole, pyrrole, N-R2pyrrole,pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole substituted by one or more substituents R2; and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; whereinR2is selected from: H, C1-C4 alkyl, cycloalkyl; wherein each R4is independently selected: -OH, -COOH, -COOR2, C1-C4 alkyl, C1 - C4 haloalky I (preferably -CF3), C1 -C4-haloalkyloxy (preferably -OCF3 or -OCHF2), C1-C4-alkoxy (preferably -OCH3), cycloalkyl, -N(H)R2’ and - N(R2)2; and wherein when Y1is -C- and Y2is -N-, R1is H and A' is phenyl, then the dashed bond of piperidine is present as a double bond.

[0016] In various such embodiments, the small molecule is a compound of Formula (VI):or pharmaceutically acceptable salt or solvate thereof, wherein:R1is selected from: H, C1 -C4 alkyl, cycloalkyl;A' is a non-substituted or a R4-mono- or -di-substituted ring A' selected from: an aromatic ring and a heteroaromatic ring, further selected from: phenyl, pyrazole, N-dimethylaminoaniline, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole substituted by one or more substituents R2, and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; whereinR2is selected from: H, C1-C4 alkyl, and cycloalkyl; and whereineach R4is independently selected from: -OH, -COOH, -COOR2, C1 -C4 alkyl, C1- C4 haloalkyl (preferably -CF3), C1 -C4-haloalkyloxy (preferably -OCHF2 ), C1-C4- alkoxy (preferably -OCH3), cycloalkyl, -N(H)R2, and -N(R2)2.

[0017] In still various such embodiments, the small molecule is a compound of Formula (VII):or pharmaceutically acceptable salt or solvate thereof, wherein:R5is selected from: H and C1 -C4 alkyl (preferably -CH3);R6is selected from: H, C1-C4-alkyl (preferably -CH3), and -C2H2- (i.e., -CH=CH-); wherein when R6is -C2H2-, it forms, together with the nitrogen atom to which R6is bound and the phenyl ring to which the nitrogen is bound, a 6-indolyl radical of general formula:the dashed C-C bond is optional, with the proviso that when R6is -C2H2- and forms the 6-indolyl radical, then the dashed bond is present as a double bond in the piperidine ring.

[0018] In yet still various such embodiments, the small molecule is a compound of Formula (VIII):or pharmaceutically acceptable salt or solvate thereof, wherein: the dashed bond is a single or a double bond; ring A is a R3-substituted phenyl or a non-substituted or a R3-substituted ring A selected from: pyrrolidine, and a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole, and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; whereinR3is selected from: C1 -C4 alkyl, C1-C4 haloalkyl (preferably -CF3 ), and cycloalkyl; whereinR2is selected from: H, C1-C4 alkyl, cycloalkyl; and whereinR4is selected from C1-C4 alkyl, C1-C4 haloalkyl (preferably -CF3), and cycloalkyl.

[0019] In still yet various such embodiments, the small molecule is a compound selected from the group consisting of:ARN25092

[0020] In yet various such embodiments, the small molecule is a compound of Formula(IX):or pharmaceutically acceptable salt or solvate thereof, wherein:Y is selected from the group consisting of CH and N;A and A’ are independently selected from the group consisting of: a 6-membered aromatic ring and a 6-membered heteroaromatic ring; wherein the 6-membered heteroaromatic ring contains 1 or 2 nitrogen atoms; and further wherein the 6- membered aromatic ring or the 6-membered heteroaromatic ring is optionally substituted at any position with a substituent selected from the group consisting of: Ci-ealkyl, halogen, Ci-ealkyl(halogen), OH, OCi ealkyl, OCi-ealkyl(alkoxy), NH2, NHCi-ealkyl, and N(Ci-6alkyl)2;X1and X2are independently selected from the group consisting of: CH2, NR2, and 0;Ri is selected from the group consisting of H and Ci-ealkyl;R2 is selected from the group consisting of H, Ci ealkyl, Ci-6alkyl(alkoxy), C(O)Ci- ealkyl, and C(O)Ci-ealkyl(alkoxy); provided that at least one of X1and X2is CH2.

[0021] In various such embodiments, the small molecule is a compound selected from the group consisting of:and pharmaceutically acceptable salts or solvates thereof.

[0022] In still various such embodiments, the small molecule is a compound selected from the group consisting of:ARN25375 ARN25499. and pharmaceutically acceptable salts or solvates thereof.

[0023] Various other embodiments are directed to a pharmaceutical composition including the pharmacological agent, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

[0024] Still various other embodiments are directed to a pharmaceutical composition including the pharmacological agent and one or more other therapeutic agent.

[0025] In various such embodiments, the pharmaceutical composition is administered separately, simultaneously, or sequentially.

[0026] Yet various other embodiments are directed to a medicament including the pharmacological agent, or the pharmaceutical composition, or pharmaceutically acceptable salts or solvates thereof.

[0027] Some embodiments of the disclosure are directed to a method for inhibiting angiogenesis in skin or colon by targeting and blocking both RhoJ and CDC42 signaling in a subject, wherein the method includes administering to the subject in need thereof a therapeutically effective amount of the pharmacological agent, or the pharmaceutical composition, or the medicament, or pharmaceutically acceptable salts or solvates thereof.

[0028] In various such embodiments, the subject has a disorder or conditions selected from the group consisting of: a cancer, an inherited vascular disorder, a vascular skin disease or condition, a vascular colon disease or condition, another condition characterized by increased or pathologic vascularity, and any combination thereof.

[0029] In still various such embodiments, the vascular skin disease or condition is a disorder selected from the group consisting of: photoaging, rosacea, port-wine birthmarks, atopic dermatitis, radiation dermatitis, psoriasis, skin cancer, another skin condition characterized by an increased or pathologic vascularity, and any combination thereof.

[0030] In still yet various embodiments, the vascular colon disease or condition is a disorder selected from the group consisting of: hereditary hemorrhagic telangiectasia, inflammatory bowel disease, colon cancer, another colon condition characterized by an increased or pathologic vascularity, and any combination thereof.

[0031] Additional embodiments and features are set forth in part in the description that follows, and in part will become apparent to those skilled in the art upon examination of the specification or may be learned by the practice of the disclosed subject matter. A further understanding of the nature and advantages of the present disclosure may be realized by reference to the remaining portions of the specification and the drawings, which form a part of this disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] These and other features and advantages of the present invention will be better understood by reference to the following detailed description when considered in conjunction with the accompanying data and figures, wherein:

[0033] FIGs. 1A through 1 E provide images and data illustrating experiments conducted to investigate RhoJ regulation of vessel arborization in skin, wherein FIG. 1A shows a diagram of optical tissue clearing and vessel analysis platform and method used to obtain the data, wherein each image (1.107 x 1.107 mm; 1024 x 1024 pixels) has >50 z-stacks (5 pm / stack), and wherein Z-stacks are compressed into 2D Tiff file, converted to grayscale, and analyzed with AngioTool, and also wherein the images are generated from three mice per group; FIG. 1B provides representative images of skin vasculature from RhoJ wild type (WT) and knockout (KO) mice, wherein fluorescence images of lectin labeled structures were obtained, converted to grayscale, and traced with AngioTool. (scale bar is 50 pm); FIG. 1C provides scatter bar plot of the frequency of vesselbranches, termini, and vessel diameters observed in FIG. 1B, wherein **“p < 0.05, 0.0005; dots on each plot correspond to the number of image stacks analyzed, n= 3 mice per genotype; FIG. 1D provides a diagram of optical tissue clearing and vessel analysis, wherein each image (1.107 x 1.107 mm; 1024 x 1024 pixels) has about 50-100 z-stacks (5 pm / stack), Z-stacks are compressed into 3D Tiff file, converted to grayscale, and analyzed with NeuTube, and images were generated from three mice per group; FIG. 1E provides representative images of skin vasculature from RhoJ wild type (WT) vs. knockout (KO) [vehicle vs 40 mg / kg twice daily a week] mice, wherein fluorescence images of lectin labeled structures were obtained, converted to grayscale, and traced with NeuTube ( scale bar is 100 pm); FIG. 1F provides scatter plot with bar graphs showing the statistical quantification of vessel parameters (number of branch / end points and branching, and tortuosity) after 3D vessel tracing, wherein each dot corresponds to an analyzed image, and wherein unpaired 2 -tail t-test was used to determine p-value, and further wherein *"”*p < 0.05, 0.01 , 0.0005, vehicle vs drug treated tumors, and > 3 mouse skins (WT or RhoJ knockout) were analyzed; FIG. 1G shows WG elastin and H and E staining of skin from RhoJ-WT and RhoJ-KO mice (notably showing the presence of an intact epidermis and hair follicles, indicating that RhoJ deletion did not induce skin necrosis; while FIG. 1 H provides scatter plot showing the percentage of Cd31+or Pdgfra+live cells in the skin and spleen from >4 mice per group (RhoJ-WT and RhoJ-KO), in accordance with embodiments of the application.

[0034] FIGs. 2A through 2D provide images and data illustrating experiments conducted to investigate the activity of CDC42 inhibitors (represented by ARN22089) towards altering vascular arborization patterns in tumors and compare it to that of vemurafenib (used in standard-of-care therapies for melanoma); wherein FIG. 2A provides plots of tumor volume as a function of time in mice treated with vehicle (circles), vemurafenib (small circles), or differently dosed ARN22089 (squares for [20 mg / kg] and triangles for [40 mg / kg], respectively), wherein the plots represent mean+SEM, *p-value < 0.05 (vehicle vs vemurafenib or 20 mg / kg), “p-value < 0.01 (vehicle vs ARN22089 40 mg / kg), and n=5 mice per group; FIG. 2B provides schematic diagram of tissue clearing, visualization, and vessel analysis of the tumors, wherein each image (1.107 x 1.107 mm;1024 x 1024 pixels) has >200 z-stacks (5 pm / stack); FIG 2C shows representative z- stacks of fluorescent images from tumors in mice treated with vehicle, vemurafenib (25 mg / kg twice daily), or ARN22089 (20 and 40 mg / kg twice daily) orally, wherein the bottom portion represents corresponding grayscale vessel tracing images (scale is 100 pm); FIG. 2D provides scatter plots with bar graphs showing the statistical quantification of vessel parameters (number of branch / end points and branching, number of vessels, length, and tortuosity) after 3D vessel tracing for >3 tumors per group, wherein dots correspond to the number of image stacks that were analyzed, and an equal number were analyzed in each tumor, < 0.05, 0.01 , 0.0005, and effects were compared between vehicle and drug treated mice; FIG. 2E provides additional representative 3D images of cleared tumors showing labeled vessels labeled from mice treated with the indicated inhibitors for two weeks, wherein the images were converted to grayscale and saved as a tiff file for 3D tracing with NeuTube (as shown in FIG. 2B) (scale bar = 100 pm), wherein each image (1.107 x 1.107 mm; 1024 x 1024 pixels) has about 207 z-stacks (5 pm / stack); FIG. 2F provides scatter bar plots showing additional vessel parameters, similar to those described in FIG. 2D, wherein the number of vessels in treated tumors that were < 20 microns in diameter, between 21 -50 microns in diameter, and >50 pm in diameter was plotted, and wherein each dot corresponds to an individual stack, 3 tumors were analyzed per group, and ‘ “ “‘p < 0.05, 0.01 , 0.0005, (vehicle vs treated tumor vessels); while FIG. 2G shows representative H&E and CD31 staining of vehicle and ARN22089-treated tumors, demonstrating that the imaging approach was detecting changes in blood vessel number, in accordance with embodiments of the application.

[0035] FIGs. 3A through 3D illustrate activity of ARN25062 towards disruption of tumor angiogenesis and compares it to that of ARN22089 and vemurafenib, wherein FIG. 3A provides growth curves of tumors treated with ARN25062 and ARN22089 by IV, as well as vemurafenib 10 mg / kg BID for two weeks once daily, wherein mean+SEM, two-way ANOVA test indicates significance between vehicle and treated mice for ARN22089, ARN25062 and Vemurafenib ***p < 0.0005; FIG. 3B provides representative 3D images of cleared tumor showing vessels labeled with lectin DyLight from mice treated with indicated inhibitors, wherein images in the middle were converted to grayscale and savedas a tiff file for 2D tracing with AngioTool or 3D tracing with NeuTube, while the bottom images show H&E staining of tumors (scale bar = 50 pm), and wherein each image (1 .107 x 1.107 mm; 1024 x 1024 pixels) has about 207 z-stacks (5 pm / stack); FIG. 3C provides scatter plots with bars showing the statistical quantifications from AngioTool tracing of vessel branching and endpoints; while FIG. 3D provides scatter plots with bars showing metrics obtained from NeuTube tracing of vessel branching and endpoints, wherein unpaired 2 -tail T-test applied in 2D and 3D analysis, ** ***p < 0.01 , 0.0005, and at least 3 tumors per condition were analyzed, in accordance with embodiments of the application.

[0036] FIGs. 4A through 4G provide images and data illustrating that CDC42 inhibitors disrupt vasculature in tumor organoids, wherein FIG. 4A provides a diagram showing a double chamber microfluidic designer device with two independently fed vascular chambers flanking a central 200 pm wide tumor chamber separated by 3 PDMS posts on each side used in the relevant experiments, wherein the left vascular chamber was treated with ARN22089 (2 pM) on day 6, while the right chamber received vehicle only; FIG. 4B shows representative fluorescent micrographs of double chamber VMT with A375 in the center and vasculature on either side; FIG. 4C provides quantification of A375 tumor-associated vasculature showing fold change in vessel length from baseline for left side (treated) vs. right side (control); FIG. 4D provides a schematic showing a single dualchamber microfluidic device used in the relevant experiments, wherein the vascular chamber connects to the tumor chamber (800 pm wide), separated by 6 PDMS posts spaced 50 pm apart that serve as burst valves to prevent the gel from traversing the chamber, and wherein EC and LF are introduced into loading port L1 , and cancer cells are introduced separately into loading port L2, while loading is facilitated by a pressure regulator (PR), and also wherein tissues are maintained via hydrostatic pressure generated across microfluidic channels connecting media reservoirs M1 -M2, while physiological flow rates are established by microfluidic resistors; FIG. 4E provides representative fluorescent micrographs of dual-chamber VMT containing A375 (top) or WM3248 (bottom) tumor and vasculature treated with either control (vehicle only), ARN25062 (2 pM), or vemurafenib (2 pM) for 48 hours starting on day 4, wherein media was refreshed on day 6, day 8, and day 10 (scale bar = 500 pm); FIG. 4F providesquantification of A375 tumor growth and tumor associated vasculature in the VMT showing fold change from baseline *p < 0.05, “p < 0.01 , while FIG. 4G provides quantification of WM3248 tumor growth and tumor-associated vasculature showing fold change in tumor vessel length from baseline, *p < 0.05, **p < 0.01 , in accordance with embodiments of the application.

[0037] FIGs. 5A through 5E provide images and data illustrating that CDC42 inhibitors alter vessel arborization in skin, wherein FIG. 5A shows skin of mice that were treated with vehicle, Vemurafenib, Linifanib, and ARN22089, wherein the skin was cleared and imaged to generate Z-stacks, with representative stacks shown, and wherein the bottom of each stack includes the 2D AngioTool tracing of pixels in grayscale (scale bar is 100 pm), and further wherein each image (1 .107 x 1.107 mm; 1024 x 1024 pixels) has >50 z- stacks (5 pm / stack); FIG. 5B shows skin of mice that were treated with vehicle, vemurafenib, and ARN22089, optically cleared, and imaged to generate Z-stacks, wherein the flattened images from additional samples to those presented in FIG. 5A are also shown, and further wherein images were converted to grayscale and vessels traced with AngioTool (scale bar is 100 pm), wherein each image (1.107 x 1.107 mm; 1024 x 1024 pixels) is from >50 flattened z-stacks (5 pm / stack); FIG. 5C provides scatter plots with bar graphs showing the frequency of branches, termini, and number of vessels with different thickness quantified from flattened images using the 2D AngioTool and Fiji software, with two-way ANOVA comparison, >3 mice per condition, and wherein dots on graphs correspond to each image stack, *““*p<0.05, 0.01 , 0.0005; and comparison to vehicle or indicated by the bar lines; FIG. 5D shows micrographs of skin harvested from mice treated with ARN22089 (with notable presence of observable vessels); while FIG. 5E provides images of VVG elastin and H and E staining of skin from WT and RhoJ KO mice, wherein, similarly to the RhoJ KO, the presence of an intact epidermis and hair follicles are observed in all samples, with a slight decrease in VVG highlighted areas in treated animals, in accordance with embodiments of the application.

[0038] FIGs. 6A through 6D provide images and data illustrating that ARN22089 disrupts vessels in skin and colon, but not the brain, wherein FIG. 6A illustrates that ARN22089 treatment does not affect mouse viability, wherein weights of mice treatedwith ARN22089 were measured after day and day 6 of treatment, mean+SD; FIG. 6B provides representative images of brain vessels labeled with lectin dylight (scale bar = 100 pm) from mice treated with ARN22089 or vehicle, wherein representative H&E staining of the brain tissue is shown; FIG. 6C shows PK profile of ARN22089 in the plasma and brain of mice after oral administration of 10 mg / kg, wherein the amount of compound detected in the brain was about 10 fold less than that in the plasma; while FIG. 6D provides representative images of cleared gastrointestinal tissues from mice treated with the indicated doses of ARN22089, wherein tissues were labeled with lectin dy Light, red fluorescence images were converted to grayscale using Imaged, decreased vessels were observed at the 12 and 40 mg / kg doses, and wherein representative H&E staining of the intestinal tissues is shown, highlighting the decrease of villi in mice treated with 40 mg / kg of ARN22089, in accordance with embodiments of the application.

[0039] FIGs. 7A and 7B provide images and data illustrating that ARN222089 inhibits angiogenesis in skin adjacent to tumors in NSG mice, wherein FIG. 7A provides representative grayscale images of adjacent skin vessels from mice bearing tumors that were treated with 20 or 40 mg / kg twice a day of inhibitor for two weeks (scale bar = 100 pm), wherein the bottom row shows 2D tracing with AngioTool; and FIG. 7B provides scatter plots showing number of branches and vessel termini in mice treated with 20 and 40 mg / kg of ARN22089, wherein each image (1.107 x 1.107 mm; 1024 x 1024 pixels) has >50 z-stacks (5 pm / stack), and further wherein each dot corresponds to an individual image stack, *p < 0.05, with >3 tumors per group included, in accordance with embodiments of the application.

[0040] FIGs. 8A through 8C provide images and data further illustrating effect of CDC42 inhibitors on vessel arborization in skin, wherein FIG. 8A provides a schematic showing a single dual-chamber microfluidic device, wherein the vascular chamber is 800 pm wide and is separated by 6 PDMS posts spaced 50 pm apart to serve as burst valves to prevent the gel from traversing the chamber, and wherein EC and LF are introduced into loading port L1 , while loading is facilitated by a pressure regulator (PR), tissues are maintained via hydrostatic pressure generated across microfluidic channels connecting media reservoirs M1 -M2, and physiological flow rates are established by microfluidicresistors; FIG. 8B provides representative fluorescent micrographs of dual chamber VMO showing vasculature (greyscale), treated with either control (vehicle only), ARN25062 (2 pM), ARN22089 (2 pM), or vemurafenib (2 pM) for 48 hours starting on day 4, wherein media was refreshed on day 6, day 8, and day 10; while FIG. 8C illustrates quantification of VMO-associated vasculature by showing fold change in vessel length and number of branchpoints compared to baseline, wherein *p < 0.05, “p < 0.01 , in accordance with embodiments of the application.

[0041] FIGs. 9A through 9F provide images and data illustrating that CDC42 inhibitors block skin angiogenesis in a RhoJ dependent manner, wherein FIG. 9A provides a table of genes summarizing experiments wherein four mice were treated with 12 mg / kg ARN22089 or vehicle twice daily for one week, after which their skin was harvested, and RNA was extracted from skin and subjected to bulk RNA sequencing (n=4 animals per group), and wherein differentially expressed genes (p< 01 ) were used to identify pathways in the STRING and PANTHER database that were downregulated in ARN22089 treated skin (pathways with a False Discovery Rate < 0.002 are shown); FIG. 9B provides heatmap showing significantly downregulated genes (at least 1.5-fold difference and p<.005) from the pathways identified in the table of FIG. 9A; FIG. 9C provides scatterplot showing percent live skin and spleen cells with Cd31+or Pdgfra+from 4 mice per group (RhoJ-WT: vehicle vs 12 mg / kg, n=4 per group); FIG. 9D summarizes experiments wherein skin from ARN22089 or vehicle treated mice was harvested to bulk RNAseq, and wherein differentially expressed genes between ARN22089 and treated mice that play a role in endothelial cell or fibroblast biology (as identified by the PANTHER database) were plotted on a heatmap; FIG. 9E provides representative grayscale and AngioTool tracing of RhoJ KO skin vessels (treated with vehicle or 40 mg / kg ARN22089); while FIG. 9F provides scatter plot showing the frequency of branches, termini, and vessel thickness as measured by the 2D AngioTool and Local Thickness (Fiji) software, wherein n.s. no significant difference (n>3 per condition), in accordance with embodiments of the application.

[0042] FIGs. 10A through 10H provide images and data illustrating that the selective CDC42 inhibitor CASIN does not affect vessel arborization in skin or vessel elongation invitro, wherein FIG. 10A shows representative stack images of skin vessels from wild type mice treated with vehicle or 40 mg / kg CASIN IP, wherein the bottom image of each stack includes the 2D AngioTool tracing of pixels in grayscale (scale bar is 100 pm); FIG. 10B provides scatter plots with bar graphs showing the frequency of branches, termini obtained from flattened images using the 2D AngioTool, wherein dots in graphs corresponds to analyzed image stacks, and unpaired t-test two tail was used to determine significance, with n=3 mice per condition; FIG. 10C provides representative stack images of skin vessels from mice treated with vehicle or CASIN 40 mg / kg IP daily for one week, wherein the bottom image of each stack includes the 2D AngioTool tracing of pixels in grayscale (scale bar is 100 pm); FIG. 10D provides scatter plots with bar graphs showing the frequency of branches, termini obtained from flattened images using the 2D AngioTool, wherein unpaired t-test two tail was used to determine significance, and also wherein each dot corresponds to a Z stack images, with n=2 animals per condition; FIG. 10E shows representative H&E staining of C57B6 skin obtained from CASIN or vehicle treated mice, wherein the thickness of the dermis was quantified and compared between vehicle and drug treated mice, with n=3 animals per group; FIG. 10F shows representative H&E staining of skin from wild type or knockout RhoJ mice treated with vehicle or CASIN at 40 mg / kg IP daily, wherein the dermal thickness was quantified and compared between drug and vehicle treated RhoJ knockout mice, and wherein scale bar is 100 pm, *pvalue < 0.05, and n=2-3 per condition, and also wherein unpaired t-test two tail was used to determine significance; FIG. 10G provides representative fluorescent micrographs of dual chamber VMO showing vasculature (greyscale), treated with either control (vehicle only), ARN22089 (2 pM), or CASIN (2 pM) for 48 hours starting on day 4, wherein media was refreshed on day 6, day 8, and day 10; FIG. 10H provides quantification of VMO-associated vasculature showing fold change in vessel length and number of branchpoints compared to baseline, wherein “p < 0.01 , in accordance with embodiments of the application.DETAILED DISCLOSURE

[0043] Turning now to the schemes, images, and data, a pharmacological agent for medical treatment of disorders and conditions that present with features of increased vasculature, as well as a pharmaceutical composition comprising thereof, and a method of using the same. In many embodiments, the pharmacological agent is a small molecule characterized by an ability to inhibit both RhoJ and CDC42 effector interactions. In many embodiments, the disorders and conditions are selected from the group comprising: a cancer, an inherited vascular disorder, a vascular skin disease or condition, a vascular colon disease or condition, another condition characterized by increased or pathologic vascularity, and any combination thereof. In many embodiments, the vascular skin disease or condition is a disorder selected from the group comprising: photoaging, rosacea, port-wine birthmarks, atopic dermatitis, radiation dermatitis, psoriasis, another skin condition characterized by an increased or pathologic vascularity, and any combination thereof. In many embodiments, the vascular colon disease or condition is a disorder selected from the group comprising: hereditary hemorrhagic telangiectasia, inflammatory bowel disease, another colon condition characterized by an increased or pathologic vascularity, and any combination thereof. In many embodiments, the small molecule is a compound exemplified by molecules selected from the group comprising:ARN25375 ARN25499 and pharmaceutically acceptable salts or solvates thereof. In addition, in many embodiments, the pharmaceutical composition comprises the pharmacological agent and at least one pharmaceutically acceptable excipient. In many embodiments, thepharmaceutical composition comprises the pharmacological agent and one or more other therapeutic agent. In some such embodiments, the pharmaceutical composition is administered separately, simultaneously, or sequentially. Some embodiments are directed to a medicament comprising the pharmacological agent, or the pharmaceutical composition, or pharmaceutically acceptable salts or solvates thereof.

[0044] In many embodiments, the method comprises inhibiting angiogenesis in skin by targeting and blocking both RhoJ and CDC42 signaling in a subject. In many such embodiments, the method comprises administering to the subject in need thereof a therapeutically effective amount of the pharmacological agent, or the pharmaceutical composition, or the medicament, or pharmaceutically acceptable salts or solvates thereof. In many embodiments, the subject has a vascular skin disorder or condition selected from the group comprising: photoaging, rosacea, radiation dermatitis, inherited vascular disorders, cancer, another disorder in skin characterized by increased or pathologic vascularity, and any combination thereof. In some embodiments, the subject has a vascular colon disorder or condition selected from the group comprising: inflammatory bowel disease, hereditary hemorrhagic telangiectasia, cancer, another disorder in colon characterized by increased or pathologic vascularity, and any combination thereof . It will be understood that the embodiments of the invention described herein are not intended to be exhaustive or to limit the invention to precise forms disclosed. Rather, the embodiments selected for description have been chosen to enable one skilled in the art to practice the invention.

[0045] Skin tumors need to develop a vascular supply to grow, yet, despite this known dependence on angiogenesis for growth, some tumors, such as, for example, melanoma tumors, are minimally responsive to current anti-angiogenesis agents, pointing to the need for more effective drugs in this class. In particular, one family of proteins - CDC42 GTPases, which includes CDC42, RhoJ, and RhoQ - has been shown to be critical to epidermal angiogenesis, wherein RhoJ and CDC42 are known to be critical mediators of both attractive and repulsive cues generated by activation of VEGF and Plexin receptors, respectively. More specifically, RhoJ, a protein with 55% homology to CDC42, is highly expressed in endothelial cells and induces actin depolymerization, focal adhesiondisassembly, and endothelial cell contraction (Shi T.T., et al. The Role of RhoJ in Endothelial Cell Biology and Tumor Pathology. Biomed Res Int. 2016;2016:6386412; and Fukushima Y., et al. RhoJ integrates attractive and repulsive cues in directional migration of endothelial cells. EMBO J. 2020;39(12):e102930; the disclosures of which are incorporated herein by reference). In contrast, CDC42 activates focal adhesion assembly and stimulates the migration of endothelial cells (Sundararaman A, and Mellor H. A functional antagonism between RhoJ and Cdc42 regulates fibronectin remodelling during angiogenesis. Small GTPases. 2021 ;12(4):241 -5; Wakayama Y., et al. Cdc42 mediates Bmp-induced sprouting angiogenesis through Fmnl3-driven assembly of endothelial filopodia in zebrafish. Dev Cell. 2015;32(1 ): 109-22; Ma J., et al. Role of activated Rac1 / Cdc42 in mediating endothelial cell proliferation and tumor angiogenesis in breast cancer. PLoS One. 2013;8(6):e66275; and Hu G.D., et al. The generation of the endothelial specific cdc42-deficient mice and the effect of cdc42 deletion on the angiogenesis and embryonic development. Chin Med J (Engl). 2011 ; 124(24):4155-9; the disclosures of which are incorporated herein by reference). As such, together, RhoJ and CDC42 mediate crosstalk between receptors that activate angiogenesis, particularly the VEGFR2 and Plexin receptors. Furthermore, RhoJ deficiency results in impaired VEGF- induced endothelial cell migration (Kaur S., et al. RhoJ / TCL regulates endothelial motility and tube formation and modulates actomyosin contractility and focal adhesion numbers. Arterioscler Thromb Vase Biol. 2011 ;31 (3):657-64, the disclosure of which is incorporated herein by reference), impaired retinal vascular angiogenesis early during development (Takase H., et al. Genome-wide identification of endothelial cell-enriched genes in the mouse embryo. Blood. 2012;120(4):914-23; and Kusuhara S., et al. Arhgef15 promotes retinal angiogenesis by mediating VEGF-induced Cdc42 activation and potentiating RhoJ inactivation in endothelial cells. PLoS One. 2012;7(9):e45858; the disclosures of which are incorporated herein by reference), and impaired tumor angiogenesis (Kim C., et al. Vascular RhoJ is an effective and selective target for tumor angiogenesis and vascular disruption. Cancer Cell. 2014;25(1 ): 102-17, the disclosure of which is incorporated herein by reference).

[0046] However, targeting RhoJ / CDC42 with small molecules has been problematic because of their globular structure and lack of readily apparent druggable pockets (Maldonado M.D.M., et al. Targeting Rac and Cdc42 GEFs in Metastatic Cancer. Front Cell Dev Biol. 2020;8:201 ; and Maldonado M.D.M., and Dharmawardhane S. Targeting Rac and Cdc42 GTPases in Cancer. Cancer Res. 2018;78(12):3101 -11 , the disclosures of which are incorporated herein by reference). Nevertheless, two approaches to target this family of GTPases have recently been developed, wherein one of the strategies involves blocking the ability of these GTPases to interact with guanine nucleotide exchange factors (Peterson J.R., et al. Biochemical suppression of small-molecule inhibitors: a strategy to identify inhibitor targets and signaling pathway components. Chem Biol. 2006;13(4):443-52, the disclosure of which is incorporated herein by reference); while the other approach involves blocking interactions between these GTPases and their downstream effectors (Brindani N., et al. Design, Synthesis, In Vitro and In Vivo Characterization of CDC42 GTPase Interaction Inhibitors for the Treatment of Cancer. J Med Chem. 2023;66(8):5981 -6001 ; and Jahid S., et al, Acquistapace IM, Hachey SJ, Flesher J L, et al. Structure-based design of CDC42 effector interaction inhibitors for the treatment of cancer. Cell Rep. 2022;39(4):110760, the disclosures of which are incorporated herein by reference). To this end, CASIN is a small molecule that specifically blocks CDC42 GTP exchange without affecting GTP exchange of other CDC42 family members. As such, in vivo administration of CASIN promotes hematopoietic stem cell mobilization (Liu W., et al. Rational identification of a Cdc42 inhibitor presents a new regimen for long-term hematopoietic stem cell mobilization. Leukemia. 2019;33(3):749- 61 , the disclosure of which is incorporated herein by reference), induces skin thickening (Zhang Y., et al. CASIN exerts anti-aging effects through RPL4 on the skin of naturally aging mice. Aging Cell. 2024;23(12):e14333, the disclosure of which is incorporated herein by reference), extends the lifespan of experimental animals (Florian M.C., et al. Inhibition of Cdc42 activity extends lifespan and decreases circulating inflammatory cytokines in aged female C57BL / 6 mice. Aging Cell. 2020; 19(9):e13208, the disclosure of which is incorporated herein by reference), and modulates T regulatory cell function in tumors (Kalim K.W., et al. Targeting of Cdc42 GTPase in regulatory T cells unleashesantitumor T-cell immunity. J Immunother Cancer. 2022; 10(11 ), the disclosure of which is incorporated herein by reference).

[0047] Even more recently, a structure-based drug design approach was used to discover a class of small molecules that block RhoJ / CDC42 downstream effector interactions. More specifically, the discovered agents were found to block S6 and ERK activation and inhibited angiogenesis in vascularized tumor organoids, and inhibited the growth of mouse- and patient-derived tumors in vivo. Notably, this in vivo activity was distinct from that reported for other CDC42 inhibitors.

[0048] However, while many anti-angiogenic agents have been identified and used to treat cancer and other conditions, these agents have significant and frequent side effects (Bodnar R. J. Anti-Angiogenic Drugs: Involvement in Cutaneous Side Effects and Wound- Healing Complication. Adv Wound Care (New Rochelle). 2014;3(10):635-46; and Elice F, and Rodeghiero F. Side effects of anti-angiogenic drugs. Thromb Res. 2012; 129 Suppl 1 :S50-3, the disclosures of which are incorporated herein by reference), which preclude their use in genetic conditions that may arise in early life, or those that would require chronic treatment, such as inflammatory skin disease. Moreover, the effects of VEGF inhibitors, the best characterized anti-vascular agents, are transient, with normal vessel structures returning even after one or two days of treatment cessation (Mancuso M.R., et al. Rapid vascular regrowth in tumors after reversal of VEGF inhibition. J Clin Invest. 2006; 116(10):2610-21 ; and Inai T., et al. Inhibition of vascular endothelial growth factor (VEGF) signaling in cancer causes loss of endothelial fenestrations, regression of tumor vessels, and appearance of basement membrane ghosts. Am J Pathol. 2004; 165(1 ):35- 52, the disclosures of which are incorporated herein by reference). For example, the drugs to inhibit skin vascularization have included alpha adrenergic agonists and laser therapies. As another example, the drugs have included MEK inhibitors. Furthermore, alpha adrenergic agents are largely not used anymore due to rebound and limited efficacy. Still, lasers have been used to treat skin conditions, but such treatments require repeated applications. Moreover, MEK inhibitors induce significant systemic toxicity, such as cardiotoxicity, which are problematic. Accordingly, a more thorough investigation, such that includes quantification and comparison of the effects of drugs on vasculature inthree dimensions, is needed to develop better anti-vascular and anti-tumor agents and therapies.

[0049] This application is directed to embodiments of a pharmacological agent with anti-vascular activity in tumors, skin, and colon, as well as a pharmaceutical composition comprising thereof, and a method of using the same for treating tumors and vascular- related conditions in skin. In particular, the application is directed to embodiments of a pharmaceutical composition comprising a pharmacological agent that is a CDC42 interaction inhibitor. In many such embodiments, the pharmacological agent blocks both RhoJ and CDC42 effector interactions and, as such, blocks angiogenesis and alters vessel arborization patterns in normal skin without apparent toxicity to endothelial or stromal cells in a RhoJ-dependent manner. Notably, in many embodiments, the pharmacological agent’s anti-vascular activity and efficacy is in stark contrast to either BRAF inhibitors (such as, for example, vemurafenib used in melanoma treatments), or selective CDC42 inhibitors (such as CASIN), both of which show no measurable effect on skin vessels; or VEGF inhibitors (known as anti-vascular agents, such as, for example, linifanib), which demonstrate less potent effects on capillary accumulation, without influencing vessel arborization. Accordingly, in many embodiments, the pharmacological agent and or the pharmaceutical composition comprising thereof is used in treatment of a tumor or a skin or a colon condition characterized by increased or abnormal / pathologic vascularity (angiogenesis). In many such embodiments, the pharmacological agent is used to treat the skin condition, wherein the skin condition is a condition selected from the group comprising: photoaging, rosacea, radiation dermatitis, inherited vascular disorders, cancer, including skin cancer and melanoma, as well as skin conditions that exhibit increased vascularization. However, in many other embodiments, the pharmacological agent is used to treat the colon condition, wherein the colon condition is a condition selected from the group comprising: inflammatory bowel disease, hereditary hemorrhagic telangiectasia, cancer, and other colon conditions that exhibit increased vascularization. It should be noted here, that no pharmacologic agent currently exists for treatment of photoaging and radiation dermatitis, while only limited therapeutic optionsare available for rosacea. In many embodiments, the pharmacological agent is a small molecule.

[0050] In many embodiments, the pharmacological agent of the instant application, which targets both RhoJ and CDC42 signaling, has a potent anti-vascular effect in normal skin and colon. In many such embodiments, the anti-vascular potency of the pharmacological agent is higher than that of any other small molecule or pharmacological agent that targets only CDC42 signaling (such as, for example CASIN), rather than both CDC42 and RhoJ. More specifically, the pharmacological agent of many embodiments demonstrates selective anti-vascular activity in skin and colon, wherein selective CDC42 inhibitor CASIN does not. Accordingly, in many embodiments, and in stark contrast to other CDC42 inhibitors, the pharmacological agents is particularly well-suited for use in the treatment of vascular disorders in skin and colon. As such, it should be noted and emphasized here, that identifying the pharmacological agent as having anti-vascular abilities distinct from those of other CDC42 inhibitors is non-intuitive and unobvious, as one might expect that all CDC42 inhibitors act the same.

[0051] In addition, in many embodiments, the pharmacological agent, characterized by an ability to inhibit CDC42 GTPases, including RhoJ, RhoQ, and CDC42, also inhibits tumor growth and vessel branching within tumors to a degree similar to that of BRAF inhibitors (which are included in standard-of-care therapies for melanoma, e.g., vemurafenib). However, in contrast to BRAF inhibitors, which only affect vessel arborization in tumors, in many embodiments, the pharmacological agent alters vessel arborization in normal skin, colon, and human skin organoids, notably, without tissue toxicity. In many embodiments, the pharmacological agent inhibits vessel formation in both mouse and human skin. In many embodiments, the pharmacological agent potently inhibits the vasculature in the skin without any sclerosis, without inducing necrosis, or any other obvious side effects. In many such embodiments, the anti-vascular potency of the pharmacological agent is dose-dependent, demonstrating a quantitative dose-dependent effect on skin vessels formation.

[0052] Furthermore, in many embodiments, the pharmacological agent demonstrates lesser systemic and CNS (central nervous system) toxicity than any other currently knownanti-vascular medicament, yet specifically affects skin and colon. Notably, in relevant experiments, the pharmacological agent have demonstrated no effect on brain vessels in mice, and a milder effect of colon vessels at only the highest dose. Accordingly, in some embodiments, the pharmacological agent, or the pharmaceutical composition comprising thereof, is used topically to prevent vascularization of skin tumors, or to inhibit vessel accumulation in the context of inherited vascular disorders or skin inflammatory diseases.

[0053] In many embodiments, the pharmacological agent and the pharmaceutical compositions comprising thereof inhibit tumor growth, and also inhibit skin and colon angiogenesis by inhibiting vessel formation and promoting vessel turnover. In many embodiments, the anti-tumor activity of the pharmacological agent is on par with MAP kinase inhibitors. Accordingly, in some embodiments, the pharmacological agent, or a pharmaceutical composition comprising thereof, is used as a systemic anti-cancer agent.

[0054] In many embodiments, the pharmacological agent, or the pharmaceutical compositions comprising thereof, is used in the context of rare vascular disorders wherein vessels accumulate in the skin, such as, for example, port wine birthmarks, or in the colon, such as, for example, in hereditary hemorrhagic telangiectasias. In many embodiments the pharmacological agent treats such conditions via a mechanism that directly disrupts vessels.Compositions

[0055] Regarding the compositions and molecular structures disclosed herein, the following paragraphs provide definitions of the various chemical moieties of the compounds according to the various embodiments and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0056] The term “alkyl”, as used herein by itself or as a part of another substituent, refers to aliphatic hydrocarbon groups. Such term includes linear (unbranched) chains or branched chains, which may be fully saturated, mono- or polyunsaturated.

[0057] The term “unsaturated” aliphatic hydrocarbon group encompasses alkenyl and alkynyl.

[0058] The term “alkenyl”, as used herein, refers to alkyl groups, preferably having from 2 to 6 carbon atoms and containing at least one carbon-carbon double bond.

[0059] The term “alkynyl”, as used herein, refers to alkyl groups, preferably having from 2 to 6 carbon atoms and containing at least one carbon-carbon triple bond.

[0060] Non-limiting examples of alkyl groups according to various embodiments are, for example, methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, ethenyl, 1 -propenyl, 2-propenyl, 1- or 2-butenyl, ethynyl, 1-propynyl, 2- propynyl, 1 - or 2-butynyl and the like.

[0061] The term “alkoxy”, as used herein, refers to an alkyl group that is linked to the remainder of the compound by an oxygen atom.

[0062] The term “halogen”, as used herein, refers to fluorine, chlorine, bromine and iodine.

[0063] The term “aromatic ring”, as used herein, refers to a moiety wherein the constituent carbon atoms make up an unsaturated ring system, all atoms in the ring system are sp2hybridized and the total number of TT-electrons is equal to 4n+2, wherein n is an integer.

[0064] The term “heteroaromatic ring”, as used herein, refers to an aromatic ring as defined above wherein one to four carbon atoms are independently replaced by heteroatoms chosen from the group consisting of nitrogen, oxygen and sulfur. Nonlimiting examples of heteroaromatic ring groups are, for example, pyrrolyl, furyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, indolyl, benzofuranyl, benzothiophenyl, benzimidazolyl, benzopyrazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, triazolyl, oxadiazolyl, tetrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl.

[0065] Unless otherwise indicated, the term “substituted”, as used herein, means that one or more hydrogen atoms of the above mentioned groups are covalently replaced with another non-hydrogen atom or functional group, provided that normal valencies are maintained and that the substitution results in a stable compound.

[0066] The term “pharmaceutically acceptable salts” refers to salts of the below identified compounds that retain the desired biological activity and are accepted by regulatory authorities.

[0067] As used herein, the term “salt” refers to any salt of a compound according to the various embodiments prepared from an inorganic or organic acid or base and internally formed salts. Typically, such salts have a physiologically acceptable anion or cation.

[0068] Furthermore, the compounds disclosed herein may form an acid addition salt or a salt with a base, depending on the kind of the substituents, and these salts are included in various embodiments, as long as they are pharmaceutically acceptable salts.

[0069] Examples of such salts include, but are not restricted to acid addition salts formed with inorganic acids (e. g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, trifluoroacetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, fumaric acid, maleic acid, ascorbic acid, benzoic acid, alginic acid, polyglutamic acid and naphthalene sulfonic acid.

[0070] Physiologically or pharmaceutically acceptable salts are particularly suitable for medical applications because of their greater aqueous solubility relative to the parent compound.

[0071] Pharmaceutically acceptable salts may also be prepared from other salts including other pharmaceutically acceptable salts of the compounds disclosed herein using conventional methods.

[0072] Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates”. For example, a complex with water is known as a “hydrate”. Solvates of various embodiments of the compounds are within the scope of the disclosure. The compounds disclosed herein may readily be isolated in association with solvent molecules by crystallization or evaporation of an appropriate solvent to give the corresponding solvates.

[0073] The compounds disclosed herein may be in crystalline form. In certain embodiments, the crystalline forms of the compounds are polymorphs.

[0074] Various embodiments are also directed to isotopically-labelled compounds, which are identical to those recited herein, but differ for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the various embodiments of the compounds and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine, iodine, and chlorine, such as2H,3H,11C,13C,14C,15N,17O, 18Q 31 p 32p 35g 18p 36Q| 123| 1251

[0075] Compounds according to various embodiments and pharmaceutically acceptable salts of said compounds that contain the aforementioned isotopes and / or other isotopes of other atoms are within the scope of the disclosure. Isotopically-labelled compounds according to various embodiments, for example those into which radioactive isotopes such as3H,14C are incorporated, are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e.3H, and carbon-14, i.e.14C, isotopes are particularly preferred for their ease of preparation and detectability.11C and18F isotopes are particularly useful in PET (Positron Emission Tomography), and125l isotopes are particularly useful in SPECT (Single Photon Emission Computerized Tomography), all useful in brain imaging. Furthermore, substitution with heavier isotopes such as deuterium, i.e.2H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements and, hence, may be preferred in some circumstances. Isotopically-labelled compounds according to various embodiments can generally be prepared by replacing a non- isotopically-labelled reagent with a readily available isotopically-labelled reagent.

[0076] Certain groups / substituents included according to various embodiments may be present as isomers or in one or more tautomeric forms. Accordingly, in certain embodiments, the compounds disclosed herein may exist in the form of other tautomers or geometrical isomers in some cases, depending on the kinds of the substituents. In the present disclosure, the compounds may be described in only one form of such isomers,but various embodiments include all such isomers, isolated forms of the isomers, or a mixture thereof. Furthermore, the compounds may have asymmetric carbon atoms or axial asymmetries in some cases and, correspondingly, they may exist in the form of optical isomers such as an (R)-form, an (S)-form, and the like. Various embodiments include all such isomers, including racemates, enantiomers and mixtures thereof.

[0077] In particular, various embodiments include all stereoisomeric forms, including enantiomers, diastereoisomers, and mixtures thereof, including racemates and the general reference to the compounds includes all the stereoisomeric forms, unless otherwise indicated.

[0078] In general, the compounds or salts according to various embodiments should be interpreted as excluding those compounds (if any) which are so chemically unstable, either per se or in water, that they are clearly unsuitable for pharmaceutical use through all administration routes, whether oral, parenteral, or otherwise. Such compounds are known to the skilled chemist.

[0079] To this end, in many embodiments, the small molecule characterized by the RhoJ and CDC42 inhibitory activity is a compound of general Formula (I):or pharmaceutically acceptable salt or solvate thereof, wherein:Y is -N- or -CH-, ring A is selected from: pyrrolidine; phenyl; a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole; and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, NR2indazole;ring B is selected from: a six-membered saturated or partially unsaturated cycle, a six membered saturated or partially unsaturated heterocycle, and an aniline with one or more optional substituents; and ring C is selected from: a six-membered saturated or partially unsaturated cycle; a six membered saturated or partially unsaturated heterocycle, and an aniline with one or more optional substituents; whereinR2is selected from: H, (C1 -C6)alkyl, halo(C1 -C6)alkyl, amino, amino(C1 - C6)alkyl, hydroxyl, hydroxy(C1 -C6)alkyl, alkoxy-(C1 -C6)-alkyl, carboxyl, ester, and C1-C6-acyloxy.

[0080] In many such embodiments, the small molecule is, more specifically, a compound of Formula (II):or pharmaceutically acceptable salt or solvate thereof, wherein: dashed bond may be a single or a double bond,Y is -N- or -CH-, ring A is selected from: pyrrolidine; phenyl; a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole; and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; with the proviso that when Y is -N-, A is different from phenyl including heteroaryl groups; ring A' is selected from: an aromatic ring and a heteroaromatic ring, further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene,thiazole, isothiazole, oxazole, isoxazole substituted by one or more substituents R2, and bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; each one of X1and X2are independently selected from: -N(H)-, -N(R2)- or -0-R1is selected from: H, (C 1 -C6)alkyl, halo(C1-C6)alkyl, amino(C1 -C6)alkyl, cycloalkyl, hydroxy(C1 -C6)alkyl, alkoxy-(C1-C6)-alkyl, C1 -C6 acyloxy;R2is selected from: H, (C1-C6)alkyl, halo(C1 -C6)alkyl, amino, amino(C1 -C6)alkyl, hydroxyl, hydroxy(C1-C6)alkyl, alkoxy-(C1-C6)-alkyl, carboxyl, ester, C1-C6- acyloxy.

[0081] However, in many other embodiments, the small molecule is, more specifically, a compound of Formula (III):or pharmaceutically acceptable salt or solvate thereof, wherein: each one of R1and R2is independently selected from: H, C1 -C4 alkyl, cycloalkyl; each one of X1and X2is independently selected from: -N(H)-, -N(R2)- or -O-, dashed bond is a single or double bond; ring D is optional; in the absence of ring D, -N(R2)- is -N(R2)(H); when ring D is present, -N(R2)- is embedded into ring D such as to form a five- or six-membered (hetero)aromatic, or nonaromatic ring selected from: an indole, indoline, quinoxaline, tetrahydroquinoxaline, quinoline, or tetrahydroquinoline, as depicted below:with the proviso that when ring D is absent, the dashed bond is a double bond.

[0082] Still, in many other embodiments, the small molecule is, more specifically, a compound of Formula (IV):or pharmaceutically acceptable salt or solvate thereof, wherein:Y1and Y2are independently selected from: -CH-, and -N-, and at least one of Y1and Y2is -N-; the dashed bond is a single or a double bond;R1is selected from: H, C1 -C4 alkyl, cycloalkyl;A is a non-substituted or a R3-substituted ring A selected from: phenyl; pyrrolidine; N-dimethylaminoaniline; a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole; and a bicyclic-heteroaromatic ring further selected from: benzofuran, N Flindole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole, with the proviso that when Y2is -N-, A is not phenyl; wherein R2is selected from: H, C1-C4 alkyl, and cycloalkyl; and wherein R3is selected from: C1-C4 alkyl, C1-C4 haloalkyl (preferably -CF3), and cycloalkyl;A is a non-substituted or a R4-mono- or -di-substituted ring A' selected from: an aromatic ring and a heteroaromatic ring, further selected from: phenyl, pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole,oxazole, isoxazole substituted by one or more substituents; and a bicyclic- heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole, wherein R2may be: H, C1-C4 alkyl, cycloalkyl; and wherein each R4is independently selected from: -OH, -COOH, -COOR2, C1-C4 alkyl, C1-C4 haloalkyl (preferably -CF3), C1-C4-haloalkyloxy (preferably -OCF3 or -OCHF2), C1 -C4-alkoxy (preferably -OCH3), cycloalkyl, -NR2, and -N(R2)2; with the proviso that when Y1is -N- and Y2is -C-, R1is H and A' is phenyl, then the dashed bond of piperidine is present as a double bond.

[0083] Still yet, in many other embodiments, the small molecule is, more specifically, a compound of Formula (V):or pharmaceutically acceptable salt or solvate thereof, wherein:Y1and Y2are independently selected from: -CH- and -N-, wherein only one of Y1and Y2are -N-, the dashed bond is a single or a double bond;R1is selected from: H, C1 -C4 alkyl, cycloalkyl;A' is a non-substituted or a R4-mono- or -di-substituted ring A' selected from: an aromatic ring and a heteroaromatic ring, further selected from: phenyl, pyrazole, N-dimethylaminoaniline, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole substituted by one or more substituents R2; and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; whereinR2is selected from: H, C1-C4 alkyl, cycloalkyl; wherein each R4is independently selected: -OH, -COOH, -COOR2, C1-C4 alkyl, C1 - C4 haloalkyl (preferably -CF3), C1 -C4-haloalkyloxy (preferably -OCF3 or -OCHF2), C1-C4-alkoxy (preferably -OCH3), cycloalkyl, -N(H)R2' and - N(R2)2; and wherein when Y1is -C- and Y2is -N-, R1is H and A1is phenyl, then the dashed bond of piperidine is present as a double bond.

[0084] In some embodiments, wherein the small molecule is the compound of Formula (V), still more specifically:R1 is H;R2is H or -CH3;A1is a non-substituted or a R4-mono- or -di-substituted ring A1selected from: an aromatic ring and a heteroaromatic ring, further selected from: phenyl, pyridine, NH-indole, N-R2indole, N-dimethylaminoaniline; and each R4is independently selected from: -OH, -COOH, -COOR2, -CH3, -CF3, -OCF3, -OCHF2, -OCH3, -N(H)R2, and -N (R2)2.

[0085] In some such embodiments, wherein the small molecule is the compound of Formula (V), even more specifically:Y1and Y2are independently selected from: -CH-and -N-; wherein only one of Y1and Y2is -N-; the dashed bond is a single or a double bond;R1is H;A1is a non-substituted or a R4-mono- or -di-substituted ring A selected from: phenyl, pyridine, N-dimethylaminoaniline, NH-indole, N-R2indole;R2is -CH3; and each R4is independently selected from: -OH, -COOH, -COOR2, -CH3, -CF3, - OCF3, -OCHF2, -OCH3, -N(H)R2, and -N(R2)2.

[0086] Still, in some other embodiments, the small molecule is, more specifically, a compound of Formula (VI):or pharmaceutically acceptable salt or solvate thereof, wherein:R1is selected from: H, C1 -C4 alkyl, cycloalkyl;A' is a non-substituted or a R4-mono- or -di-substituted ring A' selected from: an aromatic ring and a heteroaromatic ring, further selected from: phenyl, pyrazole, N-dimethylaminoaniline, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole substituted by one or more substituents R2, and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; whereinR2is selected from: H, C1-C4 alkyl, and cycloalkyl; and wherein each R4is independently selected from: -OH, -COOH, -COOR2, C1-C4 alkyl, C1-C4 haloalkyl (preferably -CF3), C1-C4-haloalkyloxy (preferably -OCHF2 ), C1 -C4-alkoxy (preferably -OCH3), cycloalkyl, -N(H)R2, and - N(R2)2.

[0087] In many such embodiments, wherein the small molecule is the compound of Formula (VI), even more specifically:R1is selected from: H, C1 -C4 alkyl, and cycloalkyl;A' is a non-substituted or a R4-mono- or -di-substituted ring A selected from: phenyl, pyridine, N-dimethylaminoaniline; wherein each R4is independently selected from: -OH, -COOH, -COOR2, -CH3, - CF3, -OCF3, -OCHF2, -OCH3, -NR2, and -N(R2)2; and whereinR2is -CH3.

[0088] Yet, in many other embodiments, the small molecule is, more specifically, a compound of Formula (VII):or pharmaceutically acceptable salt or solvate thereof, wherein:R5is selected from: H and C1 -C4 alkyl (preferably -CH3);R6is selected from: H, C1-C4-alkyl (preferably -CH3), and -C2H2- (i.e., -CH=CH-); wherein when R6is -C2H2-, it forms, together with the nitrogen atom to which R6is bound and the phenyl ring to which the nitrogen is bound, a 6-indolyl radical of general formula:the dashed C-C bond is optional, with the proviso that when R6is -C2H2- and forms the 6-indolyl radical, then the dashed bond is present as a double bond in the piperidine ring.

[0089] In some such embodiments, wherein the small molecule is the compound of Formula (VII), even more specifically, R5and R6are independently selected from: -H and -CH3.

[0090] Yet still, in many other embodiments, the small molecule is, more specifically, a compound of Formula (VIII):or pharmaceutically acceptable salt or solvate thereof, wherein: the dashed bond is a single or a double bond;ring A is a R3-substituted phenyl or a non-substituted or a R3-substituted ring A selected from: pyrrolidine, and a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole, and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; whereinR3is selected from: C1 -C4 alkyl, C1-C4 haloalkyl (preferably -CF3 ), and cycloalkyl; whereinR2is selected from: H, C1-C4 alkyl, cycloalkyl; and whereinR4is selected from C1-C4 alkyl, C1 -C4 haloalkyl (preferably -CF3), and cycloalkyl.

[0091] In some such embodiments, wherein the small molecule is the compound of Formula (VIII), even more specifically:A is a R3-substituted phenyl or a non-substituted or a R3-substituted ring A selected from: benzofuran, NH-indole, N-R2indole, pyrazole, pyrrolidine, isoquinoline, quinoline, pyrimidine, pyrrole, indazole, N-R2indazole, imidazole, N-R2imidazole, thiophene;R3is H or -CH3;R2is H or -CH3;R4is selected from: C1-C4 alkyl (preferably -CH3), C1-C4 haloalkyl (preferably - CF3), and cycloalkyl.

[0092] Accordingly, in many embodiments, the small molecule is selected from the group comprising molecules:ARN25092 and pharmaceutically acceptable salts or solvates thereof.

[0093] In many embodiments, the small molecule characterized by the RhoJ andCDC42 inhibitory activity is a compound of Formula (IX)or pharmaceutically acceptable salt or solvate thereof, wherein:Y is selected from the group comprising: CH and N;A and A’ are independently selected from the group comprising: a 6-membered aromatic ring and a 6-membered heteroaromatic ring; wherein the 6-membered heteroaromatic ring contains 1 or 2 nitrogen atoms; and further wherein the 6-membered aromatic ring or the 6-membered heteroaromatic ring is optionally substituted at any position with a substituent selected from the group comprising: Ci-ealkyl, halogen, Ci-6alkyl(halogen), OH, OCi-ealkyl, OCi- ealkyl(alkoxy), NH2, NHCi-ealkyl, and N(Ci-ealkyl)2;X1and X2are independently selected from the group comprising: CH2, NR2, and 0;R1is selected from the group comprising: H and Ci-ealkyl;R2is selected from the group comprising H, Ci-ealkyl, Ci-ealkyl(alkoxy), C(O)Ci- ealkyl, and C(O)Ci-ealkyl(alkoxy); provided that at least one of X1and X2is CH2.

[0094] In some such embodiments, wherein the small molecule is the compound of Formula (IX), more specifically: the 6-membered heteroaromatic ring contains 1 nitrogen atom, and the 6- membered aromatic ring is optionally substituted at any position with a substituent selected from the group comprising: halogen, OCi-ealkyl, NH2, NHCi-ealkyl, and N(Ci-ealkyl)2; andR1is hydrogen;R2is hydrogen.

[0095] Furthermore, in some such embodiments, wherein the small molecule is the compound of Formula (IX), even more specifically:Y is CH;A is selected from the group comprising: a 6-membered heteroaromatic ring that contains 1 nitrogen atom in position 2 or 3, and a 6-membered aromatic ring optionally substituted with a substituent selected from the group comprising: halogen and OCi-salkyl;A’ is selected from the group comprising: a 6-membered aromatic ring optionally substituted in meta or para positions with a substituent selected from the group comprising: halogen, OCi-ealkyl, and N(Ci-ealkyl)2;X1is selected from the group comprising: CH2, NH and 0;X2is selected from the group comprising: CH? and NH;R1is hydrogen; provided that at least one of X1and X2is CH2.

[0096] Still, in many embodiments, wherein the small molecule is the compound of Formula (IX), more specifically:A is selected from the group comprising: a heteroaromatic 6-membered ring that contains 1 nitrogen atom, and a 6-membered aromatic ring that is unsubstituted.

[0097] Yet still, in many embodiments, wherein the small molecule is the compound of Formula (IX), still more specifically:A’ is the 6-membered aromatic ring substituted in para or meta position with a substituent selected from the group comprising: OCialkyl and N(Ci-ealkyl)2

[0098] Accordingly, in many embodiments, the small molecule is selected from the group comprising molecules:ARN24928 29 and pharmaceutically acceptable salts or solvates thereof.

[0099] In many embodiments, the small molecule is selected from the group comprising molecules:ARN25375 ARN25499 and pharmaceutically acceptable salts or solvates thereof.

[0100] In many embodiments, the small molecule is selected from the group comprising molecules:ARN22089 ARN25062 and pharmaceutically acceptable salts or solvates thereof.

[0101] In many embodiments, the pharmaceutical composition is a composition comprising the small molecule described herein or a pharmaceutically acceptable salt thereof. In many such embodiments, the pharmaceutical composition also comprises atleast one pharmaceutically acceptable excipient. In many embodiments, the pharmaceutical composition is administered separately, simultaneously, or sequentially.EXAMPLARY EMBODIMENTS

[0102] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., s or sec, second(s); min, minute(s); h or hr, hour(s); and the like.

[0103] A series of experiments and analysis were conducted to investigate the activity and potency of the instant pharmacological agent in treatment of various skin conditions, as well as to compare the angiogenesis activity and toxicity of the instant pharmacological agent to those of other known anti-vascular agents. More specifically, experiments were conducted to elucidate the mechanism, although not to be bound by any theory, behind angiogenesis modulation in skin and tumors by the pharmacological agent of many embodiments in vivo, wherein the pharmacological agent is a CDC42-effector interactions inhibitor that, critically, also inhibits RhoJ To this end, a platform, which combines optical tissue clearing, vascular labeling with fluorescent antibodies / multiphoton imaging, and three-dimensional semi-automated vessel tracing, was utilized to quantify and compare the effects of various anti-vascular agents in vivo. More specifically, this platform (NeuTube), previously only used to trace neurons, offered a straightforward, 3D quantitative approach to studying vasculature, and provided more insight into examined vascular structure, than approaches relying on flattening 3D images into 2D, such as AngioTool. (which has also been used in the studies discussed herein).

[0104] Accordingly, first, it was demonstrated that RhoJ and CDC42 GTPases play non-overlapping roles in skin (and vessel) homeostasis, by comparing RhoJ knockout and CDC42 knockout mice, and showing that RhoJ-deficient mice had significantly altered vessel arborization / vascularization (i.e. , fewer small vessels / capillaries) in their adult skin, notably without other signs of overt toxicity, while RhoJ deletion did not grossly affect the skin structure, wherein the number of endothelial cells and fibroblasts was preserved, and the epidermis appeared intact with observable hair follicles. In contrast, the skin of CDC42-deficient mice was characterized by epidermal thickening without changes to the vasculature (Zhang M., et al. Cdc42 Deficiency Leads To Epidermal Barrier Dysfunction by Regulating Intercellular Junctions and Keratinization of Epidermal Cells during Mouse Skin Development. Theranostics. 2019;9(17):5065-84, the disclosure of which is incorporated herein by reference). This indicates that, contrary to previous assertions (but also not to be bound by any theory), RhoJ and CDC42 play nonoverlapping roles in angiogenesis in skin and colon, wherein RhoJ regulates angiogenesis in these tissues, while CDC42 does not impact angiogenesis in these tissues. Accordingly, in contrast to selective CDC42 inhibitors (such as CASIN), which have no effects on vascularization, agents that target both RhoJ and CDC42 have selective anti-vascular effects.

[0105] Furthermore, the platform / imaging approach described herein was applied to patient-derived melanoma xenografts treated with the instant pharmacological agent (which is CDC42 effector interaction inhibitor) and, for comparison, other standard-of-care melanoma therapies (e.g., BRAF inhibitors), to demonstrate that both types of drugs had similar tumor suppressive effects and could alter patterns of vessel arborization in tumors. However, it was also experimentally observed that the pharmacological agent of many embodiments could alter vessel arborization patterns in the skin to a greater extent than the VEGF inhibitor linifanib (a known anti-vascular agent), while vemurafenib had no effect on skin blood vessel arborization.

[0106] Furthermore, the effect of the pharmacological agent of many embodiments on human blood vessels was confirmed in vitro using humanized organotypic micro- physiological systems (VMO and VMT) that recapitulate in vivo neo-vascularization innormal tissues and tumors. Importantly, it was observed that the selective CDC42 inhibitor CASIN could not block skin vascularization in vivo, nor inhibit blood vessel elongation in human-derived vascularized micro-organs, which is in stark contrast to the activity of the pharmacological agent of many embodiments, wherein the pharmacological agent targets both CDC42 and RhoJ. Together, these observations illustrate, according to many embodiments, the activity and potency of the instant pharmacological agent as an anti-vascular agent with utility for treating cancer and various vascular conditions in skin.Example 1 - RhoJ deletion disrupts the vascular network in the skin of RhoJ knockout (KO) mice.

[0107] RhoJ is known to control vessel branching during development, as well as to selectively regulate tumor angiogenesis and retinal vascular angiogenesis during early development in mice, a phenotype that resolves during adulthood. Furthermore, although RhoJ knockout mice do have defective wound healing (Eelen G., et al. Role of glutamine synthetase in angiogenesis beyond glutamine synthesis. Nature. 2018;561 (7721 ):63-9, the disclosure of which is incorporated herein by reference), RhoJ is not known to play a role in adult tissue homeostasis, and its influence on vascular homeostasis in adult tissues has never been directly measured. Accordingly, it was sought to examine whether RhoJ KO mice had altered vascular architecture in the skin as compared to RhoJ wildtype mice. To this end, as illustrated in FIG. 1A, an existing protocol (Renier N., et al. iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell. 2014;159(4):896-910, the disclosure of which is incorporated herein by reference) was modified to clear skin tissue harvested from RhoJ KO and wild-type mice infused with Tomato-lectin prior to sacrifice to highlight the vasculature, as has been done previously in the brain (Loren M., et al. Optical clearing potential of immersion-based agents applied to thick mouse brain sections. PLoS One. 2019;14(5):e0216064; and Khouri K., et al. Simple methodology to visualize whole-brain microvasculature in three dimensions. Neurophotonics. 2021 ;8(2):025004, the disclosures of which are incorporated herein by reference). As such, the cleared tissues were imaged to generateZ-stack images of skin, and the differences in vessel architecture were measured using two different analytic pipelines. FIG. 1B illustrates a gross comparison of flattened images generated from RhoJ KO and wild-type mouse skin, which reveals that RhoJ KO mice has a decrease in the number of skin vessels. Furthermore, FIG. 1C illustrates the analysis of RhoJ KO (RhoJ-KO) and wild-type (RhoJ-WT) skin with AngioTool - a software package that takes 2D images, quantifies pixels in grayscale, and measures junctions and endpoints (Zudaire E., et al. A computational tool for quantitative analysis of vascular networks. PLoS One. 2011 ;6(11 ):e27385 the disclosure of which is incorporated herein by reference). To this end, FIG. 1C reveals that the skin of RhoJ-KO mice has fewer vessel junctions and endpoints as compared to RhoJ-WT skin. In addition, the same images were analyzed with NeuTube, which takes 3D z-stacks and quantifies the number of terminal nodes, branch nodes, and the total number of neurons (Feng L., et al. NeuTube 1 .0: A New Design for Efficient Neuron Reconstruction Software Based on the SWC Format. eNeuro. 2015;2(1 ), the disclosure of which is incorporated herein by reference) (or in this case vessels). As such, FIGs. 1D through 1F show that RhoJ-KO skin has fewer terminal nodes, branch nodes, vessel branches, and number of vessel segments as compared to RhoJ-WT skin. Furthermore, the distribution of vessel sizes in RhoJ-WT and RhoJ-KO skin were compared as provided in FIG. 10, which shows that RhoJ-KO skin had significantly fewer capillaries (0-10 pm diameter), and less arterioles / venules (between 11 and 45 pm diameter), although this change was not statistically significant.

[0108] Notably, despite the observed decrease in vessel density, RhoJ-KO skin showed no obvious pathological changes in the epidermis and dermis, as visualized by haematoxylin and eosin staining (H&E) shown in FIG. 1G (right). Nevertheless, a slight decrease in thickness of the superficial dermal layer of RhoJ-KO skin was observed, as highlighted by a Verhoeff-Van Gieson (WG) stain provided in FIG. 1G (left), which stains collagen and elastin. Moreover, flow cytometry analysis revealed that RhoJ-KO and RhoJ-WT mice had similar number of endothelial cells (CD31 + cells (Goncharov N.V., et al. Markers of Endothelial Cells in Normal and Pathological Conditions. Biochem (Mose) Suppl Ser A Membr Cell Biol. 2020; 14(3): 167-83, the disclosure of which is incorporatedherein by reference)) and fibroblasts (PDGRa+ cells (Yao L., et al. Temporal control of PDGFRalpha regulates the fibroblast-to-myofibroblast transition in wound healing. Cell Rep. 2022;40(7):111192, the disclosure of which is incorporated herein by reference)) in their skin (FIG. 1 H, top) and spleen (FIG. 1H, bottom). Accordingly, these experiments and data illustrate that, while RhoJ deletion affects vessel arborization in the skin and modulates skin vascular architecture, it does not grossly affect skin structure or the viability of fibroblasts or endothelial cells.Example 2 - CDC42 inhibitors alter vascular arborization patterns in patient-derived xenografts implanted into mice.

[0109] It is known that melanoma tumors are minimally responsive to antiangiogenesis agents, and that tumor vessels can rapidly regrow after the cessation of VEGF-targeted therapies (Lee C.G., et al. Vascular endothelial growth factor (VEGF) induces remodeling and enhances TH2-mediated sensitization and inflammation in the lung. Nat Med. 2004; 10(10): 1095-103, the disclosure of which is incorporated herein by reference), with VEGF inhibitors (such as, for example, linifanib) having proclivity for targeting vessels at the periphery of the tumor, rather than those at the center.

[0110] On the other hand, it has been previously shown that a certain class of CDC42 interaction inhibitors could inhibit tumor growth in vivo and block angiogenesis in vascularized microtumors in vitro. However, the instantly described experiments were conducted to examine whether the CDC42 interaction inhibitors, such as the pharmacological agent of many embodiments, represented here by a ARN22089 of formula:ARN22089 could inhibit the growth and vascularization of melanoma patient-derived xenografts. To this end, a melanoma patient-derived xenograft was implanted into the flanks of NSG mice and, when the tumors reached a volume of 200 mm3, mice were treated with dosesof ARN22089, vemurafenib (a BRAF inhibitor), or vehicle twice daily for two weeks by oral gavage to the result shown in FIG. 2A. As seen from FIG. 2A, ARN22089 inhibited the growth of tumors as effectively as vemurafenib.

[0111] Next, to compare how CDC42 interaction inhibitors (again, represented in these experiments by ARN22089) and vemurafenib affect arborization of vessels in tumors, the mice was sacrificed after the two week treatment, infused with Tomato-lectin, and cleared, and the tumors were imaged to quantify their vascular branching using NeuTube as shown in FIG. 2B. FIG. 2C illustrates the gross examination of stacked images from drug- treated mice, revealing a decrease in vasculature in treated mice as compared to controls (FIG. 2C, top). Moreover, NeuTube-based quantification of vessel architecture shown in FIG. 2C (bottom) reveals that ARN22089 affects vessel number and branching to a similar degree as vemurafenib. In addition, FIG. 2D presents NeuTube analysis of images, which reveals that ARN22089 affects the number of branches, the number of terminal nodes, vessel tortuosity, and vessel length. Still furthermore, FIGs 2E and 2F show that ARN22089 and vemurafenib demonstrate similar effects on vessels of all sizes (< 20, between 20 and 50, and >50 pm), with the greatest impact on the smaller vessels, wherein tumor vessels were larger and more tortuous than those seen in skin, consistent with studies published by others. In addition, FIG. 2G provides images showing stained ARN22089 treated tumors with CD31 , which reveal that ARN22089 treated tumors had fewer CD31 -stained vessels.

[0112] In addition, daily tail vein injection of another CDC42 inhibitor representative of the pharmacological agent, ARN25062 of formulaARN25062 , were performed to further investigate the anti-vascularization behavior of the pharmacological agent of many embodiments. To this end, FIGs. 3A though 3C provide the data obtained from these experiments, which illustrates that the pharmacological agent represented by CDC42 inhibitor ARN25062:1 ) stunted tumor growth (FIG. 3A);2) reduced vessel arborization in tumors grossly (FIG. 3B); and3) induced vessel arborization changes that were detectable using both AngioTool and NeuTube (FIGs. 3C and 3D).These results were on par with the results obtained with ARN22089, particularly when considering that the frequency and route of administration of the two agents were different (ARN22089 was administered orally, while ARN25062 was administered intravenously). Accordingly, in many embodiments, CDC42 inhibitors, such as the pharmacological agent of the instant application, disrupt tumor angiogenesis.Example 3 - CDC42 interaction inhibitors block angiogenesis in human-derived vascularized microtumors in vitro

[0113] It has been previously shown, using single-chamber Vascularized Micro Tumor (VMT) models (Jahid S., et al. Structure-based design of CDC42 effector interaction inhibitors for the treatment of cancer. Cell Rep. 2022;39(1 ):110641 , the disclosure of which is incorporated herein by reference), that CDC42 interaction inhibitors such as ARN22089 could inhibit vessel growth in human derived vascularized microtumors in vitro. More specifically, it has been shown that tumor cells in these models maintain: 1 ) in vivo gene expression profile; 2) physiologic cell-cell interactions; and, 3) responsiveness to anti-cancer drugs (Hachey S.J., et al. An in vitro vascularized microtumor model of human colorectal cancer recapitulates in vivo responses to standard-of- care therapy. Lab Chip. 2021 ;21 (7): 1333-51 ; and Hachey S.J., et al. Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research. J Vis Exp. 2023(199), the disclosures of which are incorporated herein by reference). However, it remained unclear to date whether CDC42 interaction inhibitors had direct effects on tumor vasculature. To investigate this aspect, a second VMT platform design with independently treated vascular chambers and an intervening tumor chamber (FIG. 4A) was used to measure the effects of drugs after infusion on one side of the chamber versus the infusion of the other with vehicle. To this end, it was demonstrated that CDC42 interaction inhibitors and the pharmacological agent, represented in these experimentsby ARN22089, disrupted the vasculature in drug-treated but not vehicle treated chambers with a concomitant effect on tumor growth (FIG. 4B), similarly to the previously observed effects. Notably, ARN22089-treated chambers had shorter vessels (FIG. 4C) as measured by REAVER (MATLAB) (Corliss B.A., et al. REAVER: A program for improved analysis of high-resolution vascular network images. Microcirculation. 2020;27(5):e12618, the disclosure of which is incorporated herein by reference).

[0114] Next, another representative of the pharmacological agent, ARN25062, was also tested, and its anti-vascular activity compared to vemurafenib. To this end, the ability of ARN25062 and vemurafenib to inhibit vessel growth was studied in a dual-chamber microfluidic device that consisted of a vascular network adjacent to a second chamber containing the tumor, modeling ingrowth / cooption of vessels by tumors, as shown in FIG. 4D. As such, it was observed that in the dual-chamber VMT, A375 tumors and associated vascular structures regressed significantly in response to treatment with ARN25062 and vemurafenib (FIG. 4E, top), as indicated by a reduction in tumor growth on days 8 and 10 and decreased vessel length at each time point (FIG. 4F). Notably, no significant difference was observed in response between ARN25062 and vemurafenib in the VMT. Furthermore, similar results (shown in FIGs. 4E (bottom) and FIG. 4G) were observed for the melanoma cell line WM3248; wherein a significant reduction in tumor growth and vessel length was observed for treatments with both ARN25062 and vemurafenib, while a significant decrease in vessel length was only observed in ARN25062 treated organoids. Accordingly, these results indicated that CDC42 inhibitors, such as the pharmacological agent of many embodiments, block vessel elongation in tumors in vitro, however, it remained unclear and unobvious whether the observed vascular-specific effects of the pharmacological agent could be maintained in the absence of tumors.Example 4 - CDC42 interaction inhibitors disrupt skin vasculature in mice in vivo and in human-derived vascularized microorgans in vitro.

[0115] As discussed above and illustrated in FIGs. 1A through 1 H, it has been observed that RhoJ KO mice had notable changes in the vascular arborization of skin compared to wild-type mice, suggesting that inhibiting RhoJ signaling could affect skinvasculogenesis. As such, next, FIGs. 5A through 5E provide images and data that compare the effects of ARN22089, vemurafenib, and linifanib treatments on the arborization of vessels in the skin of wild type mice. More specifically, in these experiments, C57B6 mice were treated with either ARN22089 (4, 12, or 40 mg / kg), vehicle, 25 mg / kg vemurafenib or linifanib (10 mg / kg) twice daily for one week, the mice were infused with Lectin, and cleared, and imaged, wherein vessel arborization was quantified with AngioTool. To this end, FIGs 5A and 5B show gross examination of image stacks, revealing that CDC42 interaction inhibitors, such as the pharmacological agent of many embodiments, represented here by ARN22089, alter skin vascular arborization patterns, which becomes even more obvious from the examination of the flattened images. Furthermore, ARN22089 had a dose-dependent effect on the number of observed junctions and endpoints (FIG. 5C). However, in stark contrast, vemurafenib and linifanib had minimal effect on the number of observed junctions, and no effect on the number of observed endpoints (FIG. 5C).

[0116] Next, the size distribution of skin vessels after treatments with the same agents were measured to the results provided in FIG. 5C. As seen from FIG. 5C, Vemurafenib did not have a significant effect on the distribution of capillaries (<10 pm) or arterioles / venules (10-45 pm) in the skin. Furthermore, Linifanib treated skin had fewer capillaries, while ARN22089 most significantly inhibited the accumulation of both capillaries and arterioles / venules in a dose dependent manner (FIG. 5C). Moreover, ARN22089 did not have any gross effects on skin appearance (FIG. 5D), however, it did have modest effects on the thickness of the superficial dermal layer as detected by WG and standard H&E staining (FIG. 5E). Notably, the observed effects on dermal thickness were on par with what was observed in RhoJ KO mice.

[0117] In summary, VEGF inhibitor (linifanib) did not affect the number of branches and endpoints in skin, even though it did have some effects on the accumulation of skin capillaries (FIG. 50). Furthermore, the relatively modest effect of this VEGF inhibitor on skin vascularization is consistent with the known lack of efficacy of such agents in treating skin tumors. In addition, a treatment with vemurafenib (one established melanoma therapy) was also tested for ability to inhibit tumor growth and angiogenesis, wherein itnormalized vessel tortuosity and decreased vessel numbers in tumors in vivo (FIG. 2C), as well as inhibited vessel elongation in vascularized microtumors in vitro (FIGs. 4E-G). Accordingly, in many embodiments, the pharmacological agent demonstrates anti- vascular activity on par with targeted therapies.

[0118] Furthermore, to investigate the tissue toxicity of the pharmacological agent of many embodiments, the effect of its representative agent, ARN22089, on the vascularization of organs other than skin was examined. To this end, it was observed that ARN22089 treatment had no effect on mouse weight (FIG. 6A), nor on blood vessel accumulation in the brain (FIG. 6B), although PK data did reveal that ARN22089 could cross the blood brain barrier to a small degree (FIG. 6C). Notably, treatment with ARN22089 did have an observable effect on the accumulation of vasculature in the colon, which was accompanied by loss of villi (FIG. 6D).

[0119] Moreover, FIGs. 7A and 7B illustrate experiments conducted to verify that the effect of the pharmacological agent (here, represented by ARN22089) on skin vascularization is independent of mouse strain background. To this end, skin from the NSG mice bearing tumors that were treated with CDC42 interaction inhibitor (here, ARN22089), vehicle, or vemurafenib at regions that were at a minimum 5 mm away from the implanted tumor was harvested and analyzed as shown in FIGs. 7A and 7B. As such, it was observed that ARN22089 treatment at either 20 or 40 mg / kg BID inhibited the accumulation of vessel junctions and endpoints as measured by AngioTool (FIGs. 7A and 7B). Notably, vemurafenib did not significantly alter either the accumulation of vessel junctions or endpoints (FIG. 7B), similarly to its effect in C57B6 skin. Accordingly, these results indicate that the anti-vascular effects of the pharmacological agent are independent of the presence of a functional immune system.

[0120] In addition, to further verify that the pharmacological agent of many embodiments has vascular-specific effects, its representatives ARN22089 and ARN25062, along with vemurafenib for comparison, were examined via Vascularized Micro-Organs (VMO) for their effect on vessels, wherein VMO is the tumor-free version of VMT micro-physiological system, as shown in FIG. 8A. As such, gross inhibition of vessel structure formation was observed in the VMOs that were treated with ARN22089orARN25062, but not, apparently, in those treated with vemurafenib or vehicle (FIG. 8B). Furthermore, REAVER quantification of the obtained VMO images revealed that treatment with ARN22089 and ARN25062 did, indeed, induce vascular disruption, as evidenced by a significant reduction in vessel length and the number of branch-points (FIG. 8C). In contrast, the vasculature was not significantly affected by vemurafenib treatment (FIG. 8C). Accordingly, these experimental results indicate that, according to many embodiments, the instant pharmacological agent, which is a CDC42 interaction inhibitor, specifically modulates vessel elongation.Example 5 - CDC42 interaction inhibitors affect the vascularization of the skin by similar mechanisms observed in RhoJ KO mice.

[0121] To better understand how the pharmacological agent of many embodiments modulates angiogenesis (although not to be bound by any theory), bulk RNA sequencing were performed on WT skin treated with either 12 mg / kg ARN22089 (representing the pharmacological agent) or vehicle twice daily for a week. As such, genes involved in CDC42 signaling and cell adhesion were identified (FIG. 9A) as genes downregulated after treatment with an agent. In addition, it was noted that marker genes of cells that are carried within vascular compartments were also downregulated after treatment with an agent. Also notably, the identified genes downregulated after treatment with an agent included several genes known to be involved in angiogenesis, including CDC42 (Yoshida Y., et al. Cdc42 has important roles in postnatal angiogenesis and vasculature formation. Dev Biol. 2021 ;477:64-9, the disclosure of which is incorporated herein by reference), RhoA (Eckenstaler R., et al. A current overview of RhoA, RhoB, and RhoC functions in vascular biology and pathology. Biochem Pharmacol. 2022;206:115321 , the disclosure of which is incorporated herein by reference), and CCL4 (Lu C.C., et al. The Chemokine CCL4 Stimulates Angiopoietin-2 Expression and Angiogenesis via the MEK / ERK / STAT3 Pathway in Oral Squamous Cell Carcinoma. Biomedicines. 2022; 10(7), the disclosure of which is incorporated herein by reference). Moreover, several genes involved in tumor angiogenesis / vasculogenesis, including leupaxin (Hou T., et al. Leupaxin Promotes Bladder Cancer Proliferation, Metastasis, and Angiogenesis Through the PI3K / AKTPathway. Cell Physiol Biochem. 2018;47(6):2250-60, the disclosure of which is incorporated herein by reference), CD177 (Jiang J., et al. Relationship between CD177 and the vasculogenic mimicry, clinicopathological parameters, and prognosis of epithelial ovarian cancer. Ann Palliat Med. 2020;9(6):3985-92, the disclosure of which is incorporated herein by reference), and CCL12 (Lala P.K., et al. Roles of prostaglandins in tumor-associated lymphangiogenesis with special reference to breast cancer. Cancer Metastasis Rev. 2018;37(2-3):369-84, the disclosure of which is incorporated herein by reference), were also downregulated in treated skin. In addition, markers of lymphatic vasculature (Lyvel ) (Jackson D.G., et al. LYVE-1 , the lymphatic system and tumor lymphangiogenesis. Trends Immunol. 2001 ;22(6):317-21 , the disclosure of which is incorporated herein by reference) and genes that mark cell types trafficked in lymphatics (lymphocytes, neutrophils, macrophages) were also observed to be downregulated in drug treated skin (FIG 9B). Furthermore, several components of PI3 kinase signaling were downregulated, consistent with earlier results that drug treatment affects S6 signaling. Importantly, no gross skin toxicity was observed in drug / agent treated animals, as evidenced by the preserved structure of the hair follicles (as seen in FIG. 5E), and as also evidenced by similar numbers of fibroblasts and endothelial cells in the skin of drug treated and vehicle treated mice (as seen in FIG. 9C).

[0122] In addition, while no difference in the number of endothelial cells (ECs) or fibroblasts that accumulated in treated or untreated skin was observed, changes in the expression of 16 genes involved in fibroblast and endothelial cell function were, nonetheless, identified, as illustrated in FIG.9D. To this end, one of the identified changes was a decreased expression of Col5a1 , a gene that, when mutated, affects the accumulation of collagen in the dermis of Ehler’s Danlos patients (Wenstrup R.J., et al. COL5A1 haploinsufficiency is a common molecular mechanism underlying the classical form of EDS. Am J Hum Genet. 2000;66(6): 1766-76, the disclosure of which is incorporated herein by reference). In addition, loss of expression of Fbnll , a gene involved in elastic fiber formation (Argraves W.S., et al. Fibulins: physiological and disease perspectives. EMBO Rep. 2003;4(12):1127-31 , the disclosure of which is incorporated herein by reference) was also observed. Accordingly, consistent with theseobservations, a decrease in the thickness of the WG stained layer (which highlights collagen) was identified in the superficial dermis in drug treated mice (FIG. 5E), as compared to control mice (FIG. 1 G), similarly to what was observed in RhoJ KO mice. Therefore, together, these findings validate the observations that the pharmacological agent of many embodiments, such as, for example represented by ARN22089, modulates the expression of genes involved in the generation of collagen ultrastructure (collagen, fibulin).

[0123] Previously, it has been demonstrated that ARN22089 and its analogues blocked the interactions between both RhoJ and CDC42 and their downstream effectors. Accordingly, the experiments described below were conducted to examine whether the effects of ARN22089 (as a representative of the pharmacological agent of many embodiments) on angiogenesis were RhoJ-dependent. To this end, wild-type and RhoJ KO mice were treated with ARN22089, and the approach illustrated in FIG. 1A was used to measure changes in vascularization. As such, analysis of stacked and flattened images obtained from the treated mice revealed no apparent differences in the vessel arborization patterns of RhoJ KO mice that were treated with ARN22089 as compared to baseline (FIG. 9E). Similarly, no differences in junctions or endpoints between RhoJ KO treated and untreated mice was observed as measured by AngioTool (FIG. 9F). Furthermore, no change in the vessel size was observed with ARN22089 treatment in the RhoJ-KO mice (FIG. 9F). Notably, 3D vessel analysis with NeuTube similarly showed no differences in the number of branch nodes and end nodes between RhoJ KO mice and RhoJ KO mice treated with ARN22089 (FIGs. 1E and 1 F).

[0124] Moreover, to further verify that the vascular specific effects of the pharmacological agent of many embodiments are achieved through modulation of RhoJ, rather than CDC42 function, the ability of the pharmacological agent (a CDC42 interaction inhibitor) to modulate skin vascularity was compared to that of CAS IN - a known selective CDC42 inhibitor. More specifically, CASIN is a small molecule that selectively inhibits CDC42 GTP exchange without affecting RhoJ activation. As such, wild-type and RhoJ KO mice were treated with CASIN, after which drug-induced changes in vascularization of the skin of mice were quantified. To this end, FIGs. 10A through 10D provide theanalysis of stacked and flattened images from such treated mice and reveal no apparent differences in vessel arborization patterns between CASIN-treated and vehicle-treated wild-type (FIGs. 10A and 10B) or RhoJ knockout mice (FIGs. 10C and 10D). Notably, it has been previously reported that CDC42 knockout mice and CASIN-treated mice had an increase in dermal thickness when compared to control animals, and the instant studies, indeed, confirmed observable dermal thickening induced by CASIN treatment in both wild-type (FIG. 10E) and RhoJ-knockout mice (FIG. 10F). This result is critically important, as it points out that the CDC42 inhibitors described herein (wherein the instant CDC42 inhibitors block both RhoJ and CDC42) have drastically different effects than those of the agents / drugs that block only CDC42. Furthermore, in contrast to earlier reports, which group CDC42 and RhoJ as having overlapping functions, the studies and experimental results described and discussed herein show that their functions are nonoverlapping, at least with regards to vascularization in tumor, skin, and colon. Accordingly, CDC42 interaction inhibitors represented by the pharmacological agent have activities that are distinct from those of CASIN and other CDC42 inhibitors.

[0125] In addition, the strategy and methods described herein and shown in FIG. 4D were used to examine whether CASIN could inhibit vessel elongation in human-derived vascular organoids. As such, it was observed that, while ARN22089 (as a representative of the pharmacological agent of many embodiments) inhibited vessel elongation in organoids devoid of tumors, CASIN did not (FIG. 10G and 10H). Accordingly, these results indicate that the anti-vascular effects observed for the pharmacological agent of many embodiments, which is a CDC42 interaction inhibitor, is RhoJ-dependent.

[0126] In summary, the experimentally obtained various data provided herein illustrates that, according to many embodiments, the pharmacological agent of many embodiments, which is a small molecule that targets and inhibits both RhoJ and CDC42 signaling, demonstrates potent anti-vascular effects in skin, especially, and surprisingly, as compared to those that target only CDC42. In particular, the pharmacological agent, which is a CDC42 interaction inhibitor, has been shown, according to many embodiments, to have vessel growth inhibitory effects on tumors in vivo that are similar to those of vemurafenib - a BRAF inhibitor used in standard-of-care melanoma therapies. However,the studies in skin and in vitro vascular organoids described herein have shown that, also according to many embodiments, the anti-angiogenesis properties of the pharmacological agent are profoundly different from those of vemurafenib. More specifically, in many embodiments, the pharmacological agent-treated skin has fewer capillaries and arterioles (as exemplified by data shown in FIG. 5C) as compared to vemurafenib, linifanib, or nontreated skin, which is consistent with the observed broad effects of the pharmacological agent of many embodiments on tumor vessels (FIG. 2C). In addition, in many embodiments, the pharmacological agent also affects the number of observed endpoints and vascular branches, suggesting, although not to be bound by any theory, that they inhibit vascularization by a different mechanism than either vemurafenib or linifanib (a VEGF inhibitor commonly used as an anti-vascular agent) in skin (as exemplified by data shown in FIG. 50). In contrast, neither CDC42-specific inhibitor CASIN (FIGs. 10A and 10B), nor vemurafenib (FIGs. 5A and 50), affects skin vascularization the way it is affected by the pharmacological agent. Furthermore, in many embodiments, only the pharmacological agent can inhibit vessel elongation in vitro in vascular organoids, while neither vemurafenib (FIGs. 8B and 80), nor CASIN (FIGs. 10G and 10H), show any effect on vessel elongation in these models. Accordingly, per the combination of the in vitro data, confirming an effect of the pharmacological agent on vessel elongation, and the in vivo data, confirming that the pharmacological agent does not affect the number of endothelial cells, and according to many embodiments, the pharmacological agent modifies vessel architecture without inducing gross endothelial cell cytotoxicity.

[0127] In addition, in many embodiments, the pharmacological agent described herein, which is a GTPase implicated in inhibiting CDC42 effector interactions, is a potent and efficient anti-angiogenesis agent in skin, especially, and unexpectedly, as compared to selective CDC42 inhibitors. To this end, in many embodiments, treatment with the pharmacological agent induces the downregulation of genes involved in cell migration and adhesion, as well as other genes known to play a role in angiogenesis (FIGs. 9A and 9B). Furthermore, in addition to the effects on angiogenesis, in many embodiments, treatment with the pharmacological agent downregulates the expression of genes, such as collagen and fibulin, which impacts dermal thickness; wherein so treated micedemonstrates decreased dermal thickness and collagen deposition, as measured by WG staining and exemplified by data and images shown in FIG. 5E. Notably, a similar to that of the pharmacological agent’s phenotype was also observed in RhoJ knockout mice (FIG. 1G). Also, notably, the anti-vascular activity of the pharmacological agent of many embodiments requires the presence of functional RhoJ, wherein the pharmacological agent does not inhibit angiogenesis in RhoJ-KO mouse skin, as exemplified by data and images shown in FIGs. 9E and 9F. In many embodiments, the pharmacological agent demonstrates broad anti-vascular activity, wherein, while it has no effect on brain vasculature or mouse weight, it does affect vasculature in the colon (as exemplified by data and images in FIGs. 6B and 6D).

[0128] In many embodiments, the pharmacological agent’s strong anti-angiogenesis performance is due to, although not to be bound by any theory, its ability to inhibit both CDC42 and RhoJ. To this end, in notable contrast, CASIN, which is a selective CDC42 inhibitor, shows no effects on skin vasculature (FIGs. 10A and 10B) and induces dermal thickening (FIG. 10E). Both of the phenotypes induced by CASIN are not RhoJ- dependent (FIG. 10C through 10F). Accordingly, the experimental results presented herein, indicate, although not to be bound by any theory, that RhoJ plays a specific role in skin vascularization, while CDC42 appears to control epidermal thickness. Therefore, while CASIN and other agents that target CDC42 GTPases have promising anti-aging properties, they would not be useful as anti-vascular agents. In contrast, the ability of the pharmacological agent, which is a CDC42 interaction inhibitor, to also target RhoJ is, in many embodiments, central to its anti-vascular activity. As such, in many embodiments, the pharmacological agent has a non-redundant role in angiogenesis in skin, but also, in some embodiments, in different other tissues. In many embodiments, the pharmacological agent downregulates CDC42 expression with possible secondary effects on CDC42 signaling.Example 6 - Materials and Methods

[0129] In vivo skin and tumor experiments. All animal experiments were approved by the UC Irvine Institutional Animal Care and Use Committee (IACUC) (AUP-20-161 ).C57BL6 mice and RhoJ KO mice in the C57B6 background were used in studies examining skin, colon, and brain vascularization while NOD. Cg-Prkdcscid IL2rgtm1WjlSzJ (NSG) mice were used for the patient-derived xenograft experiments. An equal number of male and female mice were used in treatment with inhibitors. For skin treatment, wild-type or RhoJ KO C57BL6 mice were treated with inhibitors by oral gavage twice daily for one week. For PDX tumor experiments, tumors were maintained by passaging into NSG mice before treatment. Inoculation of tumors in NSG mice was performed as previously described. When tumors reached 200 mm3, mice were treated by oral gavage twice daily with 20 or 40 mg / kg ARN22089 or with vemurafenib at 10 or 25 mg / kg, or with linifanib 10 mg / kg. Tail-vein injection was done once a day at 10 mg / kg ARN22089. Intraperitoneal injection of CASIN at 40 mg / kg was performed once a day for a week. All tumor experiments involved treating animals for two weeks while skin experiments involved treatment for 1 week.

[0130] Cardiac perfusion and tissue collection. At time of harvest, mice were infused with lectin-DyLight-649 (200 uL, 25% lectin-DyLight and 75% PBS) via tail vein injection to label endothelial cells. One hour later, mice were euthanized, and cardiac perfusion with 50 mL saline followed by 4% methanol-free paraformaldehyde (PFA) was performed as described previously. For skin, the fur was shaved and depilated and dorsal skin was removed from the back prior to perfusion and tissue collection. Tumors were removed from the flanks of the dorsal region. Brain, kidney, intestines, livers, hearts, and tissues were placed in 4% PFA for 24-48 h and then placed in 1xPBS at 4°C for subsequent tissue clearing.

[0131] Tissue clearing and imaging analysis. A modified iDISCO protocol was used to clear tissues. Tissues were dehydrated in graded series (20, 40, 60, 80, 100, 100%) of methanol in water for 48 h at RT. Samples were incubated with 66% dichloromethane (DCM) and 33% methanol for 48 h at RT. Next, samples were washed twice for 15 min each with DCM and placed in dibenzyl ether (DBE) for storage and used as a medium for imaging. The Leica TCS SP8 X instrument was used to image tissues and processed with a LAS X Navigator suite. The sample was placed on a makeshift holder attached to a microscope slide and filled with DBE; a coverslip was placed over the sample, makingsure the sample and liquid touches the coverslip. Images were captured using an HC PL FLOUTAR 10x / 0.30 objective. Image dimension: 1024 x 1024 pixel dimension and 1107.14 pm by 1107.14 pm, 5 pm per z-stack. Vascular fluorescence was detected by scanning for signals in the DyLight649 spectrum (670-708 nm). For image analysis, Z- stack images were opened with Fiji ImageJ, and color images were converted to black background and white pixels using color-split and saved as a tiff file either in three- dimenasional (3D) or compressed into 2D image using Z stack projection. 3D tiff files were analyzed using NeuTube software, while 2D images were analyzed using AngioTool software. Both automated and manual tracing were performed on Tiff images in NeuTube. Automated tracing was applied initially, with manual curation as needed in cases of low contrast between vessels and background and where large vessels cannot be correctly auto-traced. A custom MATLAB script was used to extract the number of branch and end points, vessel length, vessel diameter, and tortuosity in the neuTube SWC format files (individual nodes with x, y, z coordinates, radius and node connectivity). In the SWC format, vessel structures are simplified into spherical nodes with the radius of the vessel at a given location. Branch points (green nodes in images) appear where branches occur, and end points (yellow nodes in images) appear at the end of a vessel segment (all other nodes are red in images). A vessel segment is defined as a series of nodes that are bounded by two branch points or one branch point and one end point. The length of a vessel segment is defined as the distance between its pair of boundary points. The diameter of the vessel is calculated using the radius of the nodes. Tortuosity is calculated by dividing the chord (distance of two ends of a vessel) over the vessel length; value < 0.5 is highly tortuous. Parameters for AngioTool analysis were set the same for all images analyzed. Same 2D images, which were used in AngioTool, were used to determine the width of the vessel thickness using Local Thickness (masked, calibrated, and silenced) plugin on Fiji software; width < 10 pm represents veins and capillaries and between 11 and 45 pm represents arterioles in the skin (Liu Y.H., et al. Non-invasive longitudinal imaging of VEGF-induced microvascular alterations in skin wounds. Theranostics. 2022;12(2):558-73, the disclosure of which is incorporated herein by reference). For NSG skin stack images, the first and last few images in the stacks wereremoved to reduce background noise; all stacks from C57BL6 skin were analyzed. For image analysis >3 tumors or skin tissues were cleared, imaged and analyzed.

[0132] Statistics. GraphPad Prism software was used to generate graphs and perform statistical significance, using one- and two-way ANOVA test and unpaired T-test. One-way ANOVA and unpaired T-test were applied on vessel analysis and two-way ANOVA test was used to determine significance for tumor growth curves. Custom R script was used to convert pixel value to micrometer for diameter and distance; it was also used to calculate the frequency of branch nodes, end nodes, number of vessels and branching, and tortuosity.

[0133] Microfluidic device fabrication Microfluidic device fabrication and loading have been previously described. In summary, a custom polyurethane master mold was created using a two-part polyurethane liquid plastic (Smooth Cast 310, Smooth-On Inc.). Subsequently, a PDMS layer was replicated from this master mold, and holes were punched to create inlets and outlets. The platform was assembled in two stages: first, the PDMS layer was chemically glued and subjected to 2 minutes of oxygen plasma treatment to affix it to the bottom of a bottomless 96-well plate (Greiner). Following this, a 150 pm thin transparent membrane was bonded to the bottom of the PDMS device layer through an additional 2-minute treatment with oxygen plasma. The fully assembled platform was then placed in a 60°C oven overnight, covered with a standard 96-well plate polystyrene lid, and sterilized using UV light for 30 minutes before cell loading.

[0134] Cell culture and microfluidic device loading. To establish the vascular chamber, normal human lung fibroblasts and ECFC-ECs (endothelial colony forming cell endothelial cells) or HLIVECs (human umbilical vein endothelial cells) were harvested and resuspended in fibrinogen solution at a concentration of 3x106cells / mL and 7x106cells / mL, respectively. For VMT, A375 and WM3248 melanoma cells were introduced into the tumor chamber at a concentration of 1 x 1 Q5to 2 x 1 Q5cells / mL fibrinogen solution. Fibrinogen solution was prepared by dissolving 70% clottable bovine fibrinogen (Sigma- Aldrich) in EBM2 basal media (Lonza) to a final concentration of 5 mg / mL. The cell-matrix suspension was mixed with thrombin (50 U / mL, Sigma-Aldrich) at a concentration of 3 U / mL, quickly seeded into the microtissue chambers, and allowed topolymerize in a 37 °C incubator for 15 minutes. Laminin (1 mg / mL, LifeTechnologies) was then introduced into the microfluidic channels through medium inlets and incubated at 37 °C for an additional 15 minutes. After incubation, culture medium (EGM-2, Lonza) was introduced into the microfluidic channels and medium wells. The medium was changed every other day, and the hydrostatic pressure head re-established daily to maintain interstitial flow.

[0135] Drug treatment in the VMO and VMT. Following a culture period of 4-5 days to facilitate the development of a perfused vasculature within each VMO or VMT, the culture medium was replaced with a medium containing the specified drug concentrations. Drugs were administered to the microtissues through the newly formed vascular bed via gravity-driven flow. Specifically, ARN25062, ARN22089, and vemurafenib were used at a 2 pM dose. A375 VMT, WM3248 VMT, and VMO were randomly assigned to one of four conditions: control (vehicle only), 2 pM ARN25062, 2 pM ARN22089, 2 pM vemurafenib, or 2 pM CASIN. For experiments with two vascular side chambers, the left chamber received 2 pM ARN22089, while the right chamber served as control (vehicle only). Both VMO and VMT underwent a 48-hour treatment period, with complete medium replacement every 48 hours. Fluorescent micrographs of VMT were captured every 48 hours for 6 days post-treatment, and the quantification of tumor and vasculature growth was performed.

[0136] Fluorescence imaging and analyses of VMTA / MO. Fluorescence images were acquired with a Biotek Li- onheart fluorescent inverted microscope using automated acquisition and standard 10x air objective. To test vessel perfusion, 25 pg / mL FITC- or rhodamine- conjugated 70 kDa dextran was added to the medium inlet prior to treatment. For quantifying vessel length in VMOs and VMTs, REAVER software (MATLAB) was employed. Imaged software (National Institutes of Health) was utilized to determine the total fluorescence intensity (mean grey value) for each tumor image, providing a measure of tumor growth. Normalization to baseline was performed for each chamber. In VMTs, tumor growth was quantified by measuring the total fluorescence intensity in the color channel representing the tumor cells. This measurement accounted for both the area and depth of individual tumors, considering that thicker areas appear brighter. Any imageadjustments made were applied uniformly to ensure consistency across all images in the experimental group.

[0137] Tissue Histology and Immunohistochemistry Tissues were fixed in 4% formaldehyde and washed in 1xPBS. For embedding, tissues were incubated in 65% ethanol for 30 min at RT and kept in 70% ethanol before sending the samples to Experimental Tissue Resource (ETR) at IICI for embedding, WG (Piccinin M.A., and Schwartz J. StatPearls. Treasure Island (FL) ineligible companies. Disclosure: Janice Schwartz declares no relevant financial relationships with ineligible companies.; 2024, the disclosure of which is incorporated herein by reference) and H&E staining (Fischer A.H., et al. Hematoxylin and eosin staining of tissue and cell sections. CSH Protoc. 2008;2008:pdb prot4986, the disclosure of which is incorporated herein by reference).

[0138] Flow Cytometry. Back skin from mice was first shaved and depilated. A large surface area (6 cm x 3 cm) of the skin on the back was cut and fat removed. The skin was minced, and digest buffer added (RPMI without Ca / Mg (no FBS / EDTA), 23.2 mM HEPES and 2.32 mM sodium pyruvate, 0.25 mg / mL Liberase, 46.4 unit DNase) and incubated for 1.5 h at 37°C shaking. Spleen samples were removed from the same animals, minced and RPMI added to minced tissue. All samples were filtered with 70 micrometer cell strainer, and washed with FAC buffer (5% FBS, 2 mM EDTA, 1 % non- essential amino acids, 3.9x beta-mercapthoethanol (from 1000x)) twice and spun at 18.0 xg for 10 min 4°C. Skin samples were blocked with TruStain FcX PLUS and spleen samples with Fc block, both, at 1 :100 for 15 min at 4°C. All samples washed twice with FAC buffer and incubated in primary Cd31 (1 :50, brilliant violet 421 Invitrogen 404031182), PDGFRa (1 :100, APA5 Life Technology 12140181 ), viability (1 :200, eBioscience dyefluor 780 65086514) in FAC buffer for 30 min at 4°C nutating. Samples were then washed 2x and fixed with 1 % formaldehyde for 15 min and washed twice again. Samples were resuspended with 400 uL FAC buffer and ran on the BD Fortessa X20 Flow cytometer. Compensation and FMO for both skin and spleen were run before running the experimental samples. Samples were analyzed using FlowJo.

[0139] Quantification of brain levels of ARN22089 following oral administrationAnimals were treated with ARN22089 at 10 mg / Kg by oral administration, as describedelsewhere. Mouse brains were collected at 1 , 2, 4 and 8 hours after administration. The brains were homogenized and, following protein precipitation with acetonitrile, the amount of ARN22089 was quantified by LC-MS / MS as described.

[0140] Bulk RNA sequencing and pathway analysis A small piece (approximately 3 x 3 cm section) of the skin from a mouse was removed and placed in Qiagen buffer RLT (plus beta-mercaptoethanol). A Precellys Homogenizer was used to homogenize the skin sample in Precellys beads. RNeasy kit (Qiagen) was used to extract RNA, with DNase I digestion step. The RNA samples were sent to the Genomic Core Facility at UCI for library construction and sequencing. Paired-end sequencing reads were aligned and feature count to the mouse reference UCSC / mm10 (ERCC spike-in reference was concatenated with the mouse genomic and transcript reference) (Pine P.S., et al. Evaluation of the External RNA Controls Consortium (ERCC) reference material using a modified Latin square design. BMC Biotechnol. 2016;16(1 ):54, the disclosure of which is incorporated herein by reference) with HISAT2 v2.2.1 (Kim D., et al. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37(8):907-15, the disclosure of which is incorporated herein by reference) and samtools / 1.15.1 (Liao Y., et al. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923-30; and Li H., et al. The Sequence Alignment / Map format and SAMtools. Bioinformatics. 2009;25(16):2078-9, the disclosures of which are incorporated herein by reference). Count matrix was generated in R programming and RUVSeq (Risso D., et al. Normalization of RNA-seq data using factor analysis of control genes or samples. Nat Biotechnol. 2014;32(9):896-902, the disclosure of which is incorporated herein by reference) package was used to normalize (RUVr, residuals) RNA-seq data and determine differential expression. List of differential genes (vehicle vs ARN22089 (12 mg / kg)) was used to run functional processes on PANTHER (Thomas P.D., et al. H. PANTHER: Making genome-scale phylogenetics accessible to all. Protein Sci. 2022;31 (1 ):8-22; and Mi H., et al. Protocol Update for large-scale genome and gene function analysis with the PANTHER classification system (v.14.0). Nat Protoc. 2019; 14(3): 703-21 , the disclosures of which are incorporated herein by reference)(p<0.09) and STRINGv12 (Szklarczyk D., et al. The STRING database in 2023: proteinprotein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023;51 (D1 ):D638-D46, the disclosure of which is incorporated herein by reference) (p<0.009) to determine pathway analysis (Mi H., and Thomas P. PANTHER pathway: an ontology-based pathway database coupled with data analysis tools. Methods Mol Biol. 2009;563:123-40, the disclosure of which is incorporated herein by reference) and gene ontology (see Supplemental Tables). Heatmap was generated using R programming.

[0141] All the compounds underwent UPLC-MS quality control analysis using reversed phase chromatography. Analyses were conducted on a Waters Acquity UPLC- MS system consisting of a single quadrupole detector (SQD) mass spectrometer equipped with an electrospray ionization interface (ESI) and a photodiode array detector (PDA) from Waters Inc. (Milford, MA, USA). The scan range was set to 110-650 mlz for both polarities (ESI+ and ESI-). The PDA wavelength range was 210-400 nm (bottom image), and the UV purity was determined at the specific wavelength of 215 nm (upper image). The analyses were performed on an Acquity UPLC BEH Cis column (100x2.1mmlD, particle size 1.7pm) with a VanGuard BEH Cis pre-column (5x2.1 mmlD, particle size 1.7pm) using 10 mM NH4OAC in H2O at pH 5 adjusted with AcOH and 10 mM NH4OAC in MeCN-H2O (95:5) at pH 5 (B) as mobile phase. After an initial hold for 0.2 min at 10% B, a linear gradient was applied to 90% B in 6 min, then from 90 to 100% B in 0.1 min, followed by a hold at 100% B for 0.4 min. A 10 mM stock solution in dry DMSO-cfe was prepared for each test compound, and further diluted 20-fold in MeCN- H2O (1 :1) prior to analysis.DOCTRINE OF EQUIVALENTS

[0142] This description of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form described, and many modifications and variations are possible in light of the teaching above. The embodiments were chosen and described in order to best explain the principles of the invention and its practical applications. This description willenable others skilled in the art to best utilize and practice the invention in various embodiments and with various modifications as are suited to a particular use. The scope of the invention is defined by the following claims.

Claims

CLAIMS:

1. A pharmacological agent for medical treatment of disorders and conditions characterized by an increased vasculature, wherein the pharmacological agent is a small molecule characterized by an ability to inhibit both RhoJ and CDC42 effector interactions.

2. The pharmacological agent of claim 1 , wherein the disorders and conditions are selected from the group consisting of: a cancer, an inherited vascular disorder, a vascular skin disease or condition, a vascular colon disease or condition, another condition characterized by increased or pathologic vascularity, and any combination thereof.

3. The pharmacological agent of claim 2, wherein the vascular skin disease or condition is a disorder selected from the group consisting of: photoaging, rosacea, portwine birthmarks, atopic dermatitis, radiation dermatitis, psoriasis, skin cancer, another skin condition characterized by an increased or pathologic vascularity, and any combination thereof.

4. The pharmacological agent of claim 2, wherein the vascular colon disease or condition is a disorder selected from the group consisting of: hereditary hemorrhagic telangiectasia, inflammatory bowel disease, colon cancer, another colon condition characterized by an increased or pathologic vascularity, and any combination thereof.

5. The pharmacological agent of claim 1 , wherein the small molecule is a compound of Formula (I):or pharmaceutically acceptable salt or solvate thereof, wherein:Y is -N- or -CH-, ring A is selected from: pyrrolidine; phenyl; a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole; and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, NR2indazole; ring B is selected from: a six-membered saturated or partially unsaturated cycle, a six membered saturated or partially unsaturated heterocycle, and an aniline with one or more optional substituents; and ring C is selected from: a six-membered saturated or partially unsaturated cycle; a six membered saturated or partially unsaturated heterocycle, and an aniline with one or more optional substituents; wherein:R2is selected from: H, (C1 -C6)alkyl, halo(C1 -C6)alkyl, amino, amino(C1 - C6)alkyl, hydroxyl, hydroxy(C1 -C6)alkyl, alkoxy-(C1 -C6)-alkyl, carboxyl, ester, and C1-C6-acyloxy6. The pharmacological agent of claim 5, wherein the small molecule is a compound of Formula (II):or pharmaceutically acceptable salt or solvate thereof, wherein: dashed bond may be a single or a double bond,Y is -N- or -CH-, ring A is selected from: pyrrolidine; phenyl; a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole,isothiazole, oxazole, isoxazole; and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; with the proviso that when Y is -N-, A is different from phenyl including heteroaryl groups; ring A' is selected from: an aromatic ring and a heteroaromatic ring, further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole substituted by one or more substituents R2, and bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; each one of X1and X2are independently selected from: -N(H)-, -N(R2)- or -0-R1is selected from: H, (C 1 -C6)alkyl, halo(C1-C6)alkyl, amino(C1 -C6)alkyl, cycloalkyl, hydroxy(C1 -C6)alkyl, alkoxy-(C1-C6)-alkyl, C1 -C6 acyloxy;R2is selected from: H, (C1-C6)alkyl, halo(C1 -C6)alkyl, amino, amino(C1 -C6)alkyl, hydroxyl, hydroxy(C1 -C6)alkyl, alkoxy-(C1-C6)-alkyl, carboxyl, ester, C1-C6- acyloxy.

7. The pharmacological agent of claim 5, wherein the small molecule is a compound of Formula (III):or pharmaceutically acceptable salt or solvate thereof, wherein: each one of R1and R2is independently selected from: H, C1 -C4 alkyl, cycloalkyl; each one of X1and X2is independently selected from: -N(H)-, -N(R2)- or -0-, dashed bond is a single or double bond;ring D is optional; in the absence of ring D, -N(R2)- is -N(R2)(H); when ring D is present, -N(R2)- is embedded into ring D such as to form a five- or six-membered (hetero)aromatic, or nonaromatic ring selected from: an indole, indoline, quinoxaline, tetrahydroquinoxaline, quinoline, or tetrahydroquinoline, as depicted below:with the proviso that when ring D is absent, the dashed bond is a double bond.

8. The pharmacological agent of claim 5, wherein the small molecule is a compound of Formula (IV):or pharmaceutically acceptable salt or solvate thereof, wherein:Y1and Y2are independently selected from: -CH-, and -N-, and at least one of Y1and Y2is -N-; the dashed bond is a single or a double bond;R1is selected from: H, C1 -C4 alkyl, cycloalkyl;A is a non-substituted or a R3-substituted ring A selected from: phenyl; pyrrolidine; N-dimethylaminoaniline; a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole; and a bicyclic-heteroaromatic ring further selected from: benzofuran, N Flindole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole,with the proviso that when Y2is -N-, A is not phenyl; whereinR2is selected from: H, C1-C4 alkyl, and cycloalkyl; and whereinR3is selected from: C1-C4 alkyl, C1-C4 haloalkyl (preferably -CF3), and cycloalkyl;A' is a non-substituted or a R4-mono- or -di-substituted ring A' selected from: an aromatic ring and a heteroaromatic ring, further selected from: phenyl, pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole substituted by one or more substituents; and a bicyclic- heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole, wherein R2may be: H, C1-C4 alkyl, cycloalkyl; and wherein each R4is independently selected from: -OH, -COOH, -COOR2, C1-C4 alkyl, C1-C4 haloalkyl (preferably -CF3), C1-C4-haloalkyloxy (preferably -OCF3 or -OCHF2), C1 -C4-alkoxy (preferably -OCH3), cycloalkyl, -NR2, and -N(R2)2; with the proviso that when Y1is -N- and Y2is -C-, R1is H and A' is phenyl, then the dashed bond of piperidine is present as a double bond.

9. The pharmacological agent of claim 5, wherein the small molecule is a compound of Formula (V):or pharmaceutically acceptable salt or solvate thereof, wherein:Y1and Y2are independently selected from: -CH- and -N-, wherein only one of Y1and Y2are -N-, the dashed bond is a single or a double bond;R1is selected from: H, C1 -C4 alkyl, cycloalkyl;A' is a non-substituted or a R4-mono- or -di-substituted ring A' selected from: an aromatic ring and a heteroaromatic ring, further selected from: phenyl, pyrazole, N-dimethylaminoaniline, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole substituted by one or more substituents R2; and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; whereinR2is selected from: H, C1-C4 alkyl, cycloalkyl; wherein each R4is independently selected: -OH, -COOH, -COOR2, C1-C4 alkyl, C1 - C4 haloalky I (preferably -CF3), C1 -C4-haloalkyloxy (preferably -OCF3 or -OCHF2), C1-C4-alkoxy (preferably -OCH3), cycloalkyl, -N(H)R2' and - N(R2)2; and wherein when Y1is -C- and Y2is -N-, R1is H and A' is phenyl, then the dashed bond of piperidine is present as a double bond.

10. The pharmacological agent of claim 5, wherein the small molecule is a compound of Formula (VI):or pharmaceutically acceptable salt or solvate thereof, wherein:R1is selected from: H, C1 -C4 alkyl, cycloalkyl;A is a non-substituted or a R4-mono- or -di-substituted ring A' selected from: an aromatic ring and a heteroaromatic ring, further selected from: phenyl, pyrazole, N-dimethylaminoaniline, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole substituted by one or moresubstituents R2, and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; whereinR2is selected from: H, C1-C4 alkyl, and cycloalkyl; and wherein each R4is independently selected from: -OH, -COOH, -COOR2, C1 -C4 alkyl, C1- C4 haloalkyl (preferably -CF3), C1 -C4-haloalkyloxy (preferably -OCHF2 ), C1-C4- alkoxy (preferably -OCH3), cycloalkyl, -N(H)R2, and -N(R2)2.

11. The pharmacological agent of claim 5, wherein the small molecule is a compound of Formula (VII):or pharmaceutically acceptable salt or solvate thereof, wherein:R5is selected from: H and C1 -C4 alkyl (preferably -CH3);R6is selected from: H, C1-C4-alkyl (preferably -CH3), and -C2H2- (i.e., -CH=CH-); wherein when R6is -C2H2-, it forms, together with the nitrogen atom to which R6is bound and the phenyl ring to which the nitrogen is bound, a 6-indolyl radical of general formula:the dashed C-C bond is optional, with the proviso that when R6is -C2H2- and forms the 6-indolyl radical, then the dashed bond is present as a double bond in the piperidine ring.

12. The pharmacological agent of claim 5, wherein the small molecule is a compound of Formula (VIII):or pharmaceutically acceptable salt or solvate thereof, wherein: the dashed bond is a single or a double bond; ring A is a R3-substituted phenyl or a non-substituted or a R3-substituted ring A selected from: pyrrolidine, and a heteroaromatic ring further selected from: pyrazole, N-R2pyrazole, pyrrole, N-R2pyrrole, pyridine, pyrimidine, pyrazine, pyridazine, imidazole, N-R2imidazole, thiophene, thiazole, isothiazole, oxazole, isoxazole, and a bicyclic-heteroaromatic ring further selected from: benzofuran, NH-indole, N-R2indole, isoquinoline, quinoline, indazole, N-R2indazole; whereinR3is selected from: C1 -C4 alkyl, C1-C4 haloalkyl (preferably -CF3 ), and cycloalkyl; whereinR2is selected from: H, C1-C4 alkyl, cycloalkyl; and whereinR4is selected from C1-C4 alkyl, C1-C4 haloalkyl (preferably -CF3), and cycloalkyl.

13. The pharmacological agent according to any one of claims 6 to 12, wherein the small molecule is a compound selected from the group consisting of:ARN25092The pharmacological agent of claim 5, wherein the small molecule is a compound of Formula (IX):or pharmaceutically acceptable salt or solvate thereof, wherein:Y is selected from the group consisting of CH and N;A and A’ are independently selected from the group consisting of: a 6-membered aromatic ring and a 6-membered heteroaromatic ring; wherein the 6-membered heteroaromatic ring contains 1 or 2 nitrogen atoms; and further wherein the 6-membered aromatic ring or the 6-membered heteroaromatic ring is optionally substituted at any position with a substituent selected from the group consisting of: Ci-ealkyl, halogen, Ci-6alkyl(halogen), OH, OCi-ealkyl, OCi-ealkyl(alkoxy), NH2, NHCi-ealkyl, and N(Ci-ealkyl)2;X1and X2are independently selected from the group consisting of: CH2, NR2, and 0;Ri is selected from the group consisting of H and Ci-ealkyl;R2is selected from the group consisting of H, Ci-ealkyl, Ci-6alkyl(alkoxy), C(O)Ci- ealkyl, and C(O)Ci-ealkyl(alkoxy); provided that at least one of X1and X2is CH2.

15. The pharmacological agent of claim 14, wherein the small molecule is a compound selected from the group consisting of:ARN24928 29 and pharmaceutically acceptable salts or solvates thereof.

16. The pharmacological agent according to any one of claims 1 to 6, wherein the small molecule is a compound selected from the group consisting of:ARN25375 ARN25499. and pharmaceutically acceptable salts or solvates thereof.

17. A pharmaceutical composition comprising the pharmacological agent according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

18. A pharmaceutical composition comprising the pharmacological agent according to any one of claims 1 to 7, and one or more other therapeutic agent.

19. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is administered separately, simultaneously, or sequentially.

20. A medicament comprising the pharmacological agent according to any one of claims 1 to 16, or the pharmaceutical composition according to any one of claims 17 to 19, or pharmaceutically acceptable salts or solvates thereof.

21. A method for inhibiting angiogenesis in skin or colon by targeting and blocking both RhoJ and CDC42 signaling in a subject, wherein the method comprises administering to the subject in need thereof a therapeutically effective amount of the pharmacological agent according to any one of claims 1 to 16, or the pharmaceutical composition according to any one of claims 17 to 19, or the medicament according to claim 20, or pharmaceutically acceptable salts or solvates thereof.

22. The method according to claim 21 , wherein the subject has a disorder or conditions selected from the group consisting of: a cancer, an inherited vascular disorder, a vascular skin disease or condition, a vascular colon disease or condition, another condition characterized by increased or pathologic vascularity, and any combination thereof.

23. The method according to claim 22, wherein the vascular skin disease or condition is a disorder selected from the group consisting of: photoaging, rosacea, portwine birthmarks, atopic dermatitis, radiation dermatitis, psoriasis, skin cancer, another skin condition characterized by an increased or pathologic vascularity, and any combination thereof.

24. The method according to claim 22, wherein the vascular colon disease or condition is a disorder selected from the group consisting of: hereditary hemorrhagic telangiectasia, inflammatory bowel disease, colon cancer, another colon condition characterized by an increased or pathologic vascularity, and any combination thereof.

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