Methods of treating subjects with CDC42-specific inhibitors

Abstract

Embodiments disclosed herein relate to methods for extending or modulating aspects of a subject's lifespan or for immunizing a subject. In particular, methods are provided for extending or modulating lifespan, survival time, lifespan, and healthspan, comprising administering an effective amount of at least one Cdc42-specific inhibitor to a subject in need of treatment. Additionally, methods are provided for immunizing a subject, comprising administering an effective amount of at least one Cdc42-specific inhibitor to a subject in need of immunization and administering one or more immunization doses to the subject. Methods are described that further include identifying a subject as one who would benefit from extended lifespan, extended survival time, extended lifespan, extended healthspan, or immunization. The subject may be identified based on the subject's age, the subject's current medical condition, the subject's current treatment, or the subject's Cdc42 activity.

Description

[Technical Field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with United States government support under HL076604 and DK077762 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0002] Methods are provided for extending aspects of a subject's lifespan, such as increasing lifespan, survival time, lifespan, and healthspan, by administering at least one inhibitor of a GTPase, such as Cdc42GTPase. Methods are also provided for immunizing a subject by administering at least one inhibitor of a GTPase, such as Cdc42GTPase, and one or more immunizations. [Background technology]

[0003] Rho family GTPases are molecular switches that control signaling pathways that regulate cytoskeletal rearrangements, gene expression, cell cycle progression, cell survival, and other cellular processes ( Etienne-Manneville, 2002 ).

[0004] Rho family proteins constitute one of the three major branches of the Ras superfamily. The development of inhibitors of Rho family GTPases may be a promising new avenue for novel therapeutic compounds. Summary of the Invention

[0005]

[0003] Embodiments disclosed herein relate to methods for extending aspects of a subject's lifespan or for immunizing a subject. In some embodiments, methods are provided for increasing lifespan, survival time, lifespan, and healthspan, comprising administering an effective amount of at least one Cdc42-specific inhibitor to a subject in need of treatment. In some embodiments, methods are provided for immunizing a subject, comprising administering an effective amount of at least one Cdc42-specific inhibitor to a subject in need of immunization and administering one or more immunization doses to the subject. In some embodiments, the method further comprises identifying the subject as one who would benefit from extended lifespan, extended survival time, extended lifespan, extended healthspan, or immunization. In some embodiments, the subject is identified based on the subject's age, the subject's current medical condition, the subject's current treatment, the subject's Cdc42 activity, and / or the subject's methylation status of CpG sites.

[0006] In some embodiments, the Cdc42-specific inhibitor is a small molecule. In some embodiments, the small molecule is a Cdc42 activity-specific inhibitor (CASIN). In embodiments described herein, the chemical structure of CASIN is: [ka]

[0007] In some embodiments, the small molecule comprises a compound of Formula (I) as a single enantiomer, a mixture of enantiomers, a pharmaceutically acceptable salt, solvate, or polymorph thereof; [ka] During the ceremony, Y is selected from the group consisting of -OR7, -NR8R9, and -NNR8R9; R7 is C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 alkoxy, wherein C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 Alkyl and phenyl are each optionally substituted with halo, -CN, -OH, C 1~6 Alkoxyl, heteroaryl, R 19 , and -OR 20 and substituted with one or more substituents each independently selected from the group consisting of: R8 and R9 are each independently hydrogen or R 20 or R8 and R9 optionally taken together with the nitrogen to which they are attached form indolinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, each of which may be selected from the group consisting of halo, cyano, nitro, hydroxy, C 1~6 Alkyl, (CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 or R and R are joined together as a ring; 1~3 It becomes alkyl, Each R 20 is C 1~6 Alkyl, C 3~7 cycloalkyl, and phenyl; 1~6 Alkyl, C 3~7 Cycloalkyl, and phenyl are each independently R 21 and R 22and optionally substituted with one or more substituents selected from the group consisting of Each R 21 are independently selected from the group consisting of halo, cyano, nitro, and hydroxy; Each R 22 separately, C 1~6 Alkyl, C 1~6 Alkoxy-(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, Hydroxy C 1~6 Alkyl, R 19 AND -OR 20 and wherein each is selected from the group consisting of halo, cyano, nitro, hydroxy, C 1~6 Alkyl, and C 1~6 and optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy; each u is independently 0, 1, 2, 3, or 4; R2 is hydrogen or C 1~6 Alkyl, C 3~7 cycloalkyl, and phenyl, wherein C 1~6 Alkyl, C 3~7 Cycloalkyl, and phenyl each independently represent halo, cyano, nitro, hydroxy, C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl, C substituted with up to 5 fluoro 1~6 Alkoxy and, each independently, halo, cyano, nitro, hydroxy, C 1~6 Alkyl, and C 1~6 -O(CH2) optionally substituted with one or more substituents selected from the group consisting of alkoxy u phenyl, or alkyl C, where R and R are taken together to link together as a ring; 1~3And R3, R4, R5 and R6 are each independently hydrogen, halo, cyano, nitro, hydroxy, C 1~6 Alkyl, (CH2) u C 3~7 Cycloalkyl, -O(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 alkoxy, wherein C 1~6 Alkyl, (CH2) u C 3~7 Cycloalkyl, -O(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 Alkyl, and phenyl each optionally contain one or more R 23 is replaced by Each R 23 are independently halo, cyano, nitro, hydroxy, C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 alkoxy, wherein the phenyl is selected from the group consisting of halo, cyano, nitro, hydroxy, C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 Alkyl, C substituted with up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6optionally substituted with one or more substituents independently selected from the group consisting of alkoxy; Each R 19 is C optionally substituted with halo, cyano, nitro, hydroxy, and up to five fluoro; 1~6 C optionally substituted with alkyl and up to five fluoro 1~6 aryl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy; Each R 20 are independently C optionally substituted with halo, cyano, nitro, hydroxy, and up to five fluoro; 1~6 C optionally substituted with alkyl and up to five fluoro 1~6 hydrogen or aryl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy; When Y is NR8R9, R8 and R2 are optionally joined together to form an alkyl C 1~3 However, However, R8 and R2 are linked together to form a ring. 1~3 provided that, when R4 is not substituted with hydroxyl.

[0008] Also provided herein are methods of immunizing a subject, wherein a Cdc42-specific inhibitor is administered to the subject before the subject receives one or more immunization doses. In some embodiments, the Cdc42-specific inhibitor is administered to the subject after the subject receives one or more immunization doses. In some embodiments, the Cdc42-specific inhibitor is administered to the subject both before and after the subject receives one or more immunization doses. In some embodiments, the subject is administered a Cdc42-specific inhibitor once or more than once. In some embodiments, the subject receives one or more immunization doses for the same disease or different diseases. In some embodiments, the subject's immune system is compromised. In some embodiments, Cdc42 activity in the subject is determined before administering a Cdc42-specific inhibitor to the subject.

[0009] Also provided herein are methods for increasing lifespan, survival time, lifespan, and healthspan, comprising administering to a subject in need of treatment an effective amount of at least one Cdc42-specific inhibitor, wherein the expected increase in lifespan, survival time, and / or healthspan survival time is about 1% to 100%, about 1% to 90%, about 1% to 80%, about 1% to 70%, about 1% to 60%, about 1% to 50%, about 1% to 40%, about 1% to 30%, about 1% to 20%, about 5% to 15%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, or a range bracketed by any of the foregoing values, wherein the percentage is relative to the subject's expected lifespan, survival time, lifespan, or healthspan. In some embodiments, the subject's expected lifespan, survival time, lifespan, or healthspan is the median or mean expected value for similarly situated subjects. In some embodiments, the expected increase is statistically significant. [Brief explanation of the drawings]

[0010] [Figure 1](a) Scheme showing the experimental setup for an in vivo model to determine the effects of a Cdc42-specific inhibitor (e.g., CASIN) on mammalian lifespan. (b) Mass spectrometry quantification of Cdc42-specific inhibitor (e.g., CASIN) concentrations in the serum of 75-week-old C57BL / 6 mice injected with an IP dose of 25 mg / kg CASIN every 24 h for four consecutive days. Blood was collected 3, 24, and 48 h after the final injection on day 4. n = 26 at 3 h, 12 at 24 h, and 7 at 48 h. (c) Representative images and quantification of Western blot / pulldown of total and active Cdc42 (Cdc42GTP) in bone marrow cells from young (10-week-old, labeled "Control Young") and aged (75-week-old, labeled "Control Old") control and CASIN-treated (labeled "CASIN Old") C57BL / 6 mice. n = 4 mice per group. *p < 0.05 vs. old controls by one-way ANOVA and Tukey's multiple comparison test. (d) Old control (labeled "old control" and represented by the black line bifurcating at approximately 75 weeks and terminating on the x-axis between 120 and 135 weeks) and CASIN (labeled "old CASIN" and represented by the gray line bifurcating at approximately 75 weeks and terminating on the x-axis between 150 and 165 weeks) treated mice. Treatments were performed according to the carton scheme shown in panel a. n = 18 for controls and 17 for CASIN. p < 0.0004 by Mantel-Cox test and p < 0.0032 by Gehan-Breslow-Wilcoxon test. Median survival time for controls was 123.5 weeks and for CASIN 136 weeks. (e)-(h) Cytokine array results. According to the scheme in panel a, serum was collected from the same mice on day 0 (old control) and day 7 (old control and old CASIN, indicated on the graph with the additional descriptor "d7"). Seven young (10-week-old) C57BL / 6 female mice were bled along with the old mice on day 0, and the serum was used as the young control sample for the cytokine array. The number of experimental mice included was n = 7 for young control (labeled "young control"), 27 for old control day 0 (labeled "old control"), 9 for old control day 7 (labeled "old control d7"), and 10 for old CASIN day 7 (labeled "old + CASIN d7").Serum samples were simultaneously loaded for all cytokines probed and into all experimental arms. Some samples did not produce signals above background due to technical reasons and were excluded from statistical analysis. Bars represent mean + / - SEM. *p<0.05, **p<0.01 by one-way ANOVA and Tukey's multiple comparison test. (i) Biological age prediction based on DNA methylation profiles of blood cells from aged control and aged CASIN-treated mice 8–9 weeks after treatment. Experiments were repeated twice with cohorts of 5–6 animals per group (n=12 for control and 11 for CASIN). Bars represent mean + / - SEM. *p<0.05 by unpaired t-test analysis. For aged controls ("Aged Control") and "AgedCASIN," bars are displayed on the left for "biological age" and on the right for "chronological age." There is a difference of approximately 9 weeks between the biological age and chronological age of the "Aged" group. [Figure 2](a) PK analysis of CASIN in mouse serum using LC / MS / MS ion chromatography of 100 μL serum samples from CASIN-injected mice. (b) Standard curve of CASIN in mouse serum by LC / MS / MS. CASIN was added to serum at concentrations of 0, 0.5, 1.0, and 5.0 μM and subsequently analyzed. (c) Body weight in grams of mice included in the lifespan study described in Figure 1(a). (d, e) White blood cell (WBC) and red blood cell (RBC) counts of mice included in the lifespan study described in Figure 1(a). (f, h) Flow cytometry analysis of peripheral blood (PB) from mice in the lifespan study described in Figure 1(a). Data are plotted as the percentage of B220+, Cd3+, and Gr1+, Mac1+, and Gr1+Mac1+ cells among all white blood cells (WBCs). (i, k) Lymphocyte (Ly), neutrophil (NE), and monocyte (Mo) cell counts from mice in the lifespan study described in Figure 1(a). Data from Figure 2(c) to (k) are shown as boxplots showing the minimum to maximum values; n = 18 for controls (labeled "control aged" and represented by white boxes) and n = 17 for CASIN-treated animals (labeled "CASINaged" and represented by gray boxes). Days of analysis according to the scheme in Figure 1a. [Figure 3] Data from cytokine array analysis. Serum was collected from mice on day 0 (old control) and day 7 (old control and old CASIN) according to the scheme shown in Figure 1(a). Blood from young (10-week-old) C57BL / 6 females was used for the young control samples in the cytokine array. The number of experimental mice included was 7 for young control ("young control"), 27 for old control day 0 ("old control"), 9 for old control day 7 ("old control d7"), and 10 for old CASIN day 7 ("old + CASIN d7"). Serum samples were loaded simultaneously for all cytokines probed and in all experimental arms. Some samples did not produce signals above background due to technical reasons and were excluded from statistical analysis. Bars represent mean + / - SEM. *p<0.05, **p<0.01 by one-way ANOVA and Tukey's multiple comparison test. [Figure 4] Abstract showing that short-term systemic treatment of aged mice with a Cdc42-specific inhibitor (e.g., CASIN) and its associated effects include an extension of median and maximum lifespan, a reduction in inflammatory cytokines (e.g., INFγ, IL-1α, IL-1β), and dialing back (e.g., resetting) the epigenetic clock of DNA methylation levels as an indication of chronological age compared to biological age. [Figure 5] (A) Scheme of the experimental setup for analyzing vaccination responses. (B) Quantification of interferon-gamma-positive CD3+CD8+ T cells according to the protocol in Figure 5(A). (C) Quantification of splenic Kb / C93-100-dimer+CD8+ T cell frequency determined by flow cytometry according to the protocol in Figure 3(A). (D) Antibody titers after viral vaccination showing the effect of age and Cdc42-specific inhibitor treatment according to the protocol in Figure 3(A). DETAILED DESCRIPTION OF THE INVENTION

[0011] Age-associated remodeling of the immune system impairs its functional integrity and contributes to increased morbidity and mortality in the elderly.

[0012] Age-related changes in immune system phenotype and function are primarily the result of age-related alterations in hematopoietic stem cell (HSC) function. HSC senescence is, in part, caused by elevated activity of the small RhoGTPase CDC42. Until recently, there was broad consensus that the phenotype of senescent HSCs was fixed and governed by cell-intrinsic regulatory mechanisms that could not be reversed by therapeutic intervention. However, new studies have identified Cdc42 activity as a pharmacological target for ameliorating HSC senescence, suggesting a novel and important mechanistic role for Cdc42 activity in HSC senescence. Therefore, pharmacological inhibition of elevated Cdc42 activity in senescent HSCs may rejuvenate senescent HSCs.

[0013] Cdc42 is involved in multiple diverse functions in eukaryotic cells, including actin cytoskeleton reorganization, cell polarity, and cell proliferation. Cdc42 cycles between an inactive GDP-bound state and an active GTP-bound state. The cycling between the GDP-bound and GTP-bound forms is tightly controlled by many different regulatory proteins, and its activity, and therefore Cdc42-GTP levels, are significantly elevated in the blood of aging animals (e.g., humans) and in several tissues of aging C57BL / 6 animals, including the heart, brain, lung, liver, bone marrow, spleen, and kidney. Cdc42 GTPase-activating protein (Cdc42GAP; also known as p50RhoGAP or ARHGAP1), is a ubiquitously expressed negative regulator of Cdc42 that catalyzes the hydrolysis of GTP bound to Cdc42. Genetic deletion of Cdc42GAP in mice (Cdc42GAP knockout) results in elevated levels of Cdc42-GTP in all tissues. This constitutive increase in Cdc42 activity in young mice affects several tissues and leads to a premature aging-like phenotype that shortens lifespan.

[0014] Presented herein is the surprising discovery that exposing a subject to a Cdc42-specific inhibitor can modulate or increase the subject's lifespan, survival time, lifespan, and / or healthspan, and can re-establish an immune system capable of mounting a strong immune response to vaccination. These and other novel aspects are described in further detail below.

[0015] As described herein, when a range of values is provided, it is intended that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is understood, unless the context clearly dictates otherwise. Any other stated or intervening value within that stated range is included in the embodiment. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, but are also included within the embodiment, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, a range excluding both of those included limits is also included in the embodiment. Furthermore, when a whole series of integers is reported for a particular value, e.g., 1, 2, 3, 4, 5, 6, etc., it is intended that the range may be recited from all of the aforementioned integers: 1 to 6, 2 to 6, 3 to 5, 1 to 4, etc.

[0016] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs when read in light of this disclosure.Although any method and material similar or equivalent to those described herein can also be used to carry out or test embodiments, preferred methods and materials are described herein.All publications mentioned herein are expressly incorporated by reference in their entirety.

[0017] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes a plurality of such methods, and reference to a "dosage" includes a reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0018] The terms "individual," "host," "subject," and "patient" are used interchangeably to refer to an animal that is the object of treatment, observation, and / or experimentation. "Animal" includes vertebrates and invertebrates, such as fish, crustaceans, reptiles, birds, and particularly mammals. "Mammals" include, but are not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates (monkeys, chimpanzees, apes, etc.), particularly humans.

[0019] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. When particular values are described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range of the particular value.

[0020] As used herein, the term "heterologous sequence or gene" means a nucleic acid (RNA or DNA) sequence that is not naturally found in association with the nucleic acid sequence of a particular molecule. The following sections detail several approaches that can be used to prepare inhibitors of Cdc42.

[0021] How to increase

[0003] Embodiments disclosed herein relate to administering an effective amount of at least one Cdc42-specific inhibitor to a subject in need of treatment. Certain methods described herein relate to methods for increasing longevity, survival time, lifespan, or healthspan in a subject, comprising administering an effective amount of at least one Cdc42-specific inhibitor to a subject in need of treatment.

[0022] "Lifespan" refers to the amount of time a subject is expected to live based on their year of birth and current age. It may further include demographic or determinant factors specific to the subject, such as gender, genetics, lifestyle (smoking, exercise, activities of daily living, alcohol consumption, diet, self-care practices, social contacts, work patterns, etc.), culture, politics, religion, socioeconomics, etc. See, e.g., "Men, Ageing and Health" (2001), 01 / WHO / NMH / NPH01.2.

[0023] "Survival time" refers to the expected time until a subject dies and may further include subject-specific demographics or determinants such as gender, genetics, lifestyle, culture, politics, religion, socioeconomics, etc.

[0024] "Lifespan" refers to the length of time a subject is expected to remain at a particular age and may further include subject-specific demographics or determinants such as gender, genetics, lifestyle, culture, politics, religion, socioeconomics, etc.

[0025] "Health span" refers to the expected length of time in a subject's life that the subject is reasonably healthy. In some embodiments, a subject's health considers one or more of physical, mental, and social well-being, absence of disease, and absence of infirmity.

[0026] The "increase" referred to in the disclosed embodiments refers to an "expected increase" in a subject, as opposed to the actual increase experienced by a particular subject. Therefore, it is not necessary to wait for a subject's lifespan, survival time, lifespan, or healthspan to expire in order to practice the disclosed embodiments. In preferred embodiments, the expected increase is statistically significant, although the expected increase may or may not be statistically significant. There are many known methods for calculating statistical significance, such as calculating a "p-value." In some embodiments, the statistical significance threshold is a p-value of ≦0.2, ≦0.15, ≦0.1, ≦0.05, ≦0.01, ≦0.005, about ≦0.2, about ≦0.15, about ≦0.1, about ≦0.05, about ≦0.01, or about ≦0.005. In some cases, a result may not be statistically significant, but the result may still be informative or suggest some given benefit. It is understood that the degree of significance to be attributed to a particular result is within the purview of an ordinary, skilled physician.

[0027] In some embodiments, the subject's expected lifespan, survival time, lifespan, or healthspan is the median expected value for similarly situated subjects. In other embodiments, the subject's expected lifespan, survival time, lifespan, or healthspan is the mean expected value for similarly situated subjects. Similarly situated subjects can be determined based on any one or more factors, including, but not limited to, age, health, family history, or Cdc42 activity level.

[0028] The expected increase in the subject's expected lifespan, survival time, lifespan, or healthspan is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or approximately any of the foregoing percentages. Boxed ranges (e.g., approximately 1% to 30%, approximately 5% to 25%, approximately 5% to 20%, approximately 5% to 15%, or 1% to 30%, 5% to 25%, 5% to 20%, 5% to 15%), 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 70%, 10% to 60%) 10%~50%, 10%~40%, 10%~30%, 10%~20%, 20%~100%, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, It may be 40% to 50%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 100%, 70% to 90%, 70% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, or approximately any of the aforementioned percentage ranges (e.g., about 10% to 70%, about 30% to 60%, or about 50% to 70%).

[0029] In some embodiments, the expected increase is in years relative to the subject's expected lifespan, survival time, lifespan, or healthspan, and is between 1 and 20 years, 1 and 19 years, 1 and 18 years, 1 and 17 years, 1 and 16 years, 1 and 15 years, 1 and 14 years, 1 and 13 years, 1 and 12 years, 1 and 11 years, 1 and 10 years, 1 and 9 years, 1 and 8 years, 1 and 7 years, 1 and 6 years, 1 and 5 years, 1 and 4 years, 1 and 3 years, 1 and 2 years, 1 year, at least the aforementioned years (e.g., at least 1 and 10 years), or about the aforementioned number of years (e.g., about 1 and 2 years or at least about 1 and 2 years).

[0030] In some embodiments, the expected increase is from days to months relative to expected lifespan, survival time, lifespan, or healthspan, which may be 1 day to 1 year, 1 day to 11 months, 1 day to 10 months, 1 day to 9 months, 1 day to 8 months, 1 day to 7 months, 1 day to 6 months, 1 day to 5 months, 1 day to 4 months, 1 day to 3 months, 1 day to 2 months, 1 day to 1 month, at least the aforementioned range of days to months (e.g., at least 1 day to 11 months), or approximately the aforementioned range of days to months (e.g., about 1 day to 6 months or at least about 1 day to 6 months).

[0031] In some embodiments, the expected increase is in weeks relative to the subject's expected lifespan, survival time, lifespan, or healthspan, and may be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, about any of the aforementioned weeks (e.g., about 15 weeks), at least any of the aforementioned weeks (e.g., at least about 15 weeks), or a range bracketed by 2 weeks within any of the aforementioned weeks (e.g., 2 to 30 weeks, or about 2 to 30 weeks, or at least about 2 to 30 weeks), 1 to 52 weeks, 2 to 50 weeks, 3 to 45 weeks, 4 to 40 weeks, 5 to 35 weeks, 6 to 30 weeks, 5 to 25 weeks, 6 to 20 weeks, 7 to 19 weeks, 8 to 18 weeks, 9 to 17 weeks, 10 to 16 weeks, 11 to 15 weeks, 12 to 14 weeks, at least in the aforementioned range of weeks (e.g., at least 6 to 20 weeks), or approximately in the aforementioned range of weeks (e.g., about 6 to 20 weeks or at least about 6 to 20 weeks).

[0032] In some embodiments, the expected increase is in days relative to the subject's expected lifespan, survival time, lifespan, or healthspan, which is 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, for any of the foregoing days (e.g., about 15 days), for at least about any of the foregoing weeks (e.g., at least about 15 days), or a range bracketed by any two of the foregoing weeks (e.g., 2-30 days, or about 2-30 days, or at least about 2-30 days).

[0033] In the disclosed embodiments, a "reduction" or "decrease" refers to an "actual" or "expected" reduction or decrease. An actual or expected reduction or decrease may or may not be statistically significant, although in preferred embodiments, any reduction or decrease is statistically significant. There are many known methods for calculating statistical significance, such as calculating a "p-value." In some embodiments, the threshold for statistical significance is a p-value ≦0.2, ≦0.15, ≦0.1, ≦0.05, ≦0.01, ≦0.005, about ≦0.2, about ≦0.15, about ≦0.1, about ≦0.05, about ≦0.01, or about ≦0.005. In some cases, a result may not be statistically significant, but the result may still be informative or suggest some given benefit. It is understood that the degree of significance to be attributed to a particular result is within the purview of an ordinarily skilled physician.

[0034] Immunization Method Embodiments disclosed herein relate to administering an effective amount of at least one Cdc42-specific inhibitor to a subject in need of immunization and administering one or more immunization doses to the subject. Surprisingly, direct pharmacological intervention of the Cdc42-specific inhibitor in the subject enables a potent immune response upon challenge with an immunogen. Such direct administration obviates the need to obtain and purify HSCs from a compatible donor, pretreat the HSCs with a Cdc42-specific inhibitor, and transplant the treated donor HSCs into the transplant recipient. Thus, under some embodiments disclosed herein, the Cdc42-specific inhibitor essentially acts as a type of adjuvant, enhancing the efficacy of immunization by modifying the subject's immune system and immune response. In some embodiments, the Cdc42-specific inhibitor is combined with the immunization dose prior to administration to the subject (e.g., the subject receives one or more immunization doses including both an immunogen and a Cdc42-specific inhibitor). In some embodiments, the immunization dose and the Cdc42-specific inhibitor are administered to the subject in separate doses.

[0035] In some embodiments, the Cdc42-specific inhibitor is administered to a subject before the subject receives one or more immunization doses. In other embodiments, the Cdc42-specific inhibitor is administered to a subject simultaneously with the subject receiving one or more immunization doses. Simultaneous administration can be achieved by multiple administrations within a limited period of time (e.g., a single office visit by a doctor), or simultaneous administration can be achieved simultaneously or nearly simultaneously.

[0036] The subject who needs to be administered a Cdc42-specific inhibitor to improve the immune response does not necessarily need to be identified before the first immunization. For example, the subject may be identified by showing an inadequate response to the first attempted immunization. According to some embodiments, the Cdc42-specific inhibitor is administered to the subject after the subject has received one or more immunizations.

[0037] In many cases, a subject does not receive sufficient immunity from a single immunization dose and must receive multiple immunization doses before sufficient immunity is achieved. A regimen can be easily and quickly designed, in which a subject receives a first Cdc42-specific inhibitor, the subject receives a first immunization dose after the first Cdc42-specific inhibitor administration, and the subject receives one or more subsequent immunization doses after the first immunization dose. Such a regimen can be continued, in which the subject receives a second immunization dose, followed by a third immunization dose. Thus, a subject may receive 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, 9-10, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 total immunization doses for immunization against individual diseases. The one or more Cdc42-specific inhibitors may be administered prior to the first immunization or prior to one or more subsequent immunizations.

[0038] In many cases, a subject requires immunization against one or more diseases (e.g., a subject is immunized against multiple diseases throughout childhood or adulthood). This may be due to the subject's health, upcoming travel, or the subject's lack of childhood immunization. One or more Cdc42-specific inhibitors can be administered prior to the first immunization dose, or, if the subject has been immunized against one or more diseases, prior to one or more subsequent immunization doses. Just as the first immunization dose and subsequent immunization doses can be the same or different (e.g., in terms of strength, concentration, or immunogen), the one or more Cdc42-specific inhibitors administered to the subject can be the same or different.

[0039] In some cases, a certain period of time elapses between administering immunization doses to the subject.In some embodiments, the period between immunization doses is about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year.In some embodiments, one or more Cdc42-specific inhibitors are administered to the subject during the period between administering the immunization doses to the subject.

[0040] A subject who would benefit from administration of a Cdc42-specific inhibitor during immunization may have a healthy immune system. Alternatively, in some embodiments, the subject does not have a healthy immune system. The subject's immune system may be compromised, particularly due to the subject's age, the subject's medical condition, or the subject's treatment for the subject's medical condition.

[0041] Unless otherwise specified, the full scope of the term "immunization" is intended to refer to the methods disclosed herein. Immunization can be passive or active. Passive immunity is when pre-synthesized elements of the immune system are transferred to a person, so that the body does not need to produce these elements itself. Antibodies can be used for passive immunization. Active immunity can occur naturally when a person comes into contact with a microorganism, such as an attenuated microorganism, or when a person comes into contact with a part of a microorganism. However, the immunogen need not be a microorganism; for example, in some embodiments, the immunogen is a small molecule (e.g., nicotine) or a macromolecule (e.g., a protein, such as an oncoprotein, or a hormone, such as ghrelin).

[0042] As discussed above, the immunization may also be a vaccine, although it is not necessarily so limited. Thus, in some embodiments, the immunization is not a vaccine. Many vaccines are recommended by the World Health Organization or the Centers for Disease Control and Prevention. In some embodiments, the immunization is a vaccine recommended by the medical community. In some embodiments, the vaccine is recommended by the Centers for Disease Control and Prevention. A vaccine may also be recommended by the World Health Organization, the Centers for Disease Control and Prevention, or the medical community for administration to adults. In some embodiments, the vaccine is for a disease that disproportionately affects adults compared to children. In some embodiments, the vaccine is for one or more of influenza, whooping cough, tetanus, diphtheria, shingles, pneumococcal disease, human papillomavirus, meningococcal disease, hepatitis A, hepatitis B, chickenpox, measles, mumps, and rubella.

[0043] Subject Identification Embodiments disclosed herein relate to administering an effective amount of at least one Cdc42-specific inhibitor, including a modulator of Cdc42-specific activity, to a subject in need of treatment. In some embodiments, not all subjects are candidates for such administration, and identifying a subject for treatment may be desirable. It is understood that patient selection depends on many factors within the laboratory of an ordinary skilled physician. Accordingly, some embodiments disclosed herein further include identifying a subject as one who would benefit from administering an effective amount of at least one Cdc42-specific inhibitor to extend lifespan, increase survival time, extend lifespan, or improve upon immunization. Subjects may be identified based on physiological factors specific to the subject, such as the subject's age, current medical condition, current treatment, prescribed treatment, the methylation status of CpG sites within any subject's Prima1, Hsf4, or Kcns1 genes, or any combination thereof, or, in preferred embodiments, based on the subject's Cdc42 activity. Assays for determining Cdc42 activity in a subject, particularly when measuring Cdc42 activity in a blood sample from the subject, are known in the art. See, e.g., Mizukawa et al., Blood (2017) 130:1336-46.

[0044] In some embodiments, physicians can rely on a combination of physiological factors of a given subject to identify the subject of treatment with an effective amount of at least one Cdc42-specific inhibitor.As mentioned above, subject age can be a factor in identifying the subject that needs treatment.For example, subject can be elderly (e.g., elderly human subject). An elderly human subject in some embodiments described herein is a subject aged 50 or greater, 51 or greater, 52 or greater, 53 or greater, 54 or greater, 55 or greater, 56 or greater, 57 or greater, 58 or greater, 59 or greater, 60 or greater, 61 or greater, 62 or greater, 63 or greater, 64 or greater, 65 or greater, 66 or greater, 67 or greater, 68 or greater, 69 or greater, 70 or greater, 71 or greater, 72 or greater, 73 or greater, 74 or greater, 75 or greater, 76 or greater, 77 or greater, 78 or greater, 79 or greater, 80 or greater, or a bracketed range of any of the foregoing ages (e.g., 50-80 or greater, 50-70 or greater, 50-60 or greater, 55-75 or greater, 55-75 or greater, 55-70 or greater, 55-65 or greater, 60-70 or greater, 52-71 or greater, 60-79 or greater, or 73-78 or greater).

[0045] In some embodiments, as discussed above, the subject is a human. However, this method is not limited to treating humans and is equally applicable to treating mammals. In such instances where a non-human mammal is treated, patient selection will depend on many factors within the laboratory of an ordinary skilled veterinarian or research scientist.

[0046] Cdc42-specific inhibitors Embodiments disclosed herein relate to compounds, compositions, pharmaceutical compositions, methods, uses, and kits comprising at least one Cdc42-specific inhibitor. In some embodiments, the Cdc42-specific inhibitor may be a chemical inhibitor, such as a small molecule (e.g., CAS1N). Small molecules include, for example, chemical molecules with low molecular weights (e.g., molecular weights less than 2,000 daltons). Furthermore, the Cdc42-specific inhibitor may be an siRNA molecule, an antisense molecule, a small RNA (e.g., microRNA) or modified nucleic acid, a ribozyme, an antibody (such as a neutralizing antibody), or a polypeptide (e.g., a dominant-negative peptide). Any type of inhibitor known to those skilled in the art may be used.

[0047] Another aspect of the embodiments relates to modulating biological pathways involving GTPases. Accordingly, some embodiments relate to all aspects of modulating Cdc42 GTPase activity, including an effective amount of an activator, an effective amount of a compound that specifically and / or selectively modulates Cdc42 GTPase activity, or a combination thereof. Modulated Cdc42 activity includes GTP binding, GDP binding, GEF binding, GTPase activity, integrin binding, and Cdc42 coupling or binding to receptors or effector-like molecules (integrins, growth factor receptors, tyrosine kinases, PI-3K, PIP-5K, etc.). Activity can be modulated by increasing, decreasing, antagonizing, or promoting Cdc42. Modulation of Cdc42 can be measured by assays such as GTP hydrolysis and GEF binding. An effective amount is any amount that modulates Cdc42 activity when administered. Activity can be modulated in cells, tissues, whole organisms, in situ, in vitro (such as in a test tube or on a solid support), in vivo, or in any desired environment. In some embodiments, an effective amount of a Cdc42-specific inhibitor is one that restores Cdc42 activity to normal levels in a subject. In some embodiments, an effective amount of a Cdc42-specific inhibitor is one that reverses tubulin apolarity in cells and inhibits Cdc42 activity in blood progenitor cells, such as hematopoietic cells, progenitor cells, or stem cells. In some embodiments, an effective amount of a Cdc42-specific inhibitor does not mobilize blood progenitor cells.

[0048] Other assays for signal transduction through Cdc42 can be accomplished according to procedures known in the art, for example, as described in U.S. Patent Nos. 5,141,851, 5,420,334, 5,436,128, and 5,482,954, all of which are incorporated herein by reference in their entireties where permitted. Additionally, peptides can be identified that inhibit the interaction, e.g., binding, between an activator and a G protein such as Cdc42.

[0049] Methods for detecting inhibition of Cdc42 activity are known in the art, as exemplified by the Active Cdc42 Pulldown and Detection Kit available from Thermo Fisher Scientific (Rockford, IL), as described in the Examples section below, and by the incorporated material in Asnaghi et al., Oncogene (2010) 29:2760-2771. Detecting inhibition can include comparing the inhibitory properties of the tested compound with those of one or more reference compounds. Such reference compounds can be, for example, CAS1N or other compounds described herein.

[0050] By "modulate," we mean that the addition of an agent affects activity or binding. Binding or activity modulation can be affected in a variety of ways, including inhibiting, blocking, preventing, increasing, enhancing, or promoting it. The binding or activity effect need not be achieved in a specific manner and can be competitive, noncompetitive, allosteric, sterically hindered, or via, for example, cross-linking between the agent and the GEF or GTPase. The agent can act on either the activator or the GTPase. The agent can be an agonist, antagonist, or partial agonist or antagonist. The presence or amount of binding can be determined in various ways, for example, by assaying the activity promoted or inhibited by the activator, such as guanine nucleotide exchange, GTP hydrolysis, or oncogenic transformation. Such assays are also described above or below and are known in the art. The agent can be obtained and / or prepared from a variety of sources, including natural and synthetic. It can include, for example, amino acids, lipids, carbohydrates, organic molecules, nucleic acids, inorganic molecules, or mixtures thereof.

[0051] As is understood in the art, detecting modulation can be performed in vitro or in vivo. Examples of in vitro and in vivo methods are provided herein. Results from evaluating the inhibitory properties of compounds provided herein can be measured, for example, by IC 50 , E.C.50 , K. i The results may be reported in art-understood terms, including, for example, , , or other standard terms known in the art. Thus, the evaluation provided herein can include evaluating the results, where evaluating the results includes determining the inhibitory properties of the test compound. In some cases, evaluating the results also includes comparing the inhibitory properties of the test compound to the inhibitory properties of one or more reference compounds. Such reference compounds can be, for example, CAS1N or other compounds described herein.

[0052] small molecule Small molecule inhibitors, as disclosed herein, can be used to specifically inhibit and / or modulate Cdc42. Any type of small molecule inhibitor known to those skilled in the art can be used. Many methods for identifying small molecule inhibitors are known, and commercial laboratories are available for screening small molecule inhibitors. For example, chemicals can be obtained from the compound collections of Merck® Research Laboratories (Rahway, NJ) or similar companies. Compounds can be screened for Cdc42 inhibition by automated robotic screening in a 96-well plate format. For example, compounds can be dissolved at an initial concentration of approximately 50 M in DMSO and dispensed into a 96-well plate. A 96-well plate assay may contain an appropriate number of units of Cdc42 and target (substrate). Compounds that cause greater than 50% inhibition of Cdc42 activity can be further diluted and tested to establish the concentration required for 50% inhibition of activity. In some embodiments, screening will involve Cdc42 protein and one or more of its binding proteins as well as candidate inhibitors. The inhibitory effect of screened compounds on disrupting Cdc42 target binding can be monitored, for example, using ELISA-type tests with Cdc42 or a surface-immobilized target, and residual binding can be detected, for example, using an antibody to the Cdc42 target (binding) molecule conjugated to a reporter (e.g., alkaline phosphatase). Binding assays can also be performed using surface plasmon resonance (SPR)-based interaction screens involving Cdc42 and its binding target and inhibitor, or other assays that screen for protein interactions (e.g., yeast two-hybrid systems, immunoprecipitation, immunocapture experiments combined with enzymatic or FACS detection, etc.). In some embodiments, candidate Cdc42 inhibitors can be tested for their ability to inhibit Cdc42 GTPase activity using assays known in the art.In other embodiments, Cdc42 inhibitors can be tested for their ability to decrease the amount of GTP-bound Cdc42, for example, relative to the amount of GDP-bound Cdc42, using assays known in the art.

[0053] In any of the embodiments described herein, the Cdc42 specific inhibitor, the inhibitor of Cdc42, the inhibitor of GTPase Cdc42, the GTPase Cdc42 inhibitor, the agent capable of inhibiting GTPase Cdc42, or the agent that specifically inhibits Cdc42 comprises a compound of Formula (I) as a single enantiomer, a mixture of enantiomers, a pharmaceutically acceptable salt, solvate, or polymorph thereof; [ka] During the ceremony, Y is selected from the group consisting of -OR7, -NR8R9, and -NNR8R9; R7 is C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 alkoxy, wherein C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 Alkoxy is optionally substituted with halo, -CN, -OH, C 1~6 Alkoxyl, heteroaryl, R 19 , and -OR 20and substituted with one or more substituents each independently selected from the group consisting of: R8 and R9 are each independently hydrogen or R 20 or R8 and R9 optionally taken together with the nitrogen to which they are attached form indolinyl, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl, each of which may be selected from the group consisting of halo, cyano, nitro, hydroxy, C 1~6 Alkyl, (CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl and up to 5 fluoro-substituted C 1~6 or alkyl C, optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy, or R and R are joined together as a ring; 1~3 And Each R 20 is C 1~6 Alkyl, C 3~7 cycloalkyl, and phenyl; 1~6 Alkyl, C 3~7 Cycloalkyl, and phenyl are each independently R 21 and R 22 and optionally substituted with one or more substituents selected from the group consisting of Each R 21 are independently selected from the group consisting of halo, cyano, nitro, and hydroxy; Each R 22 is separately C 1~6 Alkyl, C 1~6 Alkoxy-(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, Hydroxy C 1~6 Alkyl, R 19 AND -OR 20 and wherein each is selected from the group consisting of halo, cyano, nitro, hydroxy, C 1~6 Alkyl, and C1~6 and optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy; each u is independently 0, 1, 2, 3, or 4; R2 is hydrogen or C 1~6 Alkyl, C 3~7 cycloalkyl, and phenyl, wherein C 1~6 Alkyl, C 3~7 Cycloalkyl, and phenyl each independently represent halo, cyano, nitro, hydroxy, C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl, C substituted with up to 5 fluoro 1~6 Alkoxy and, each independently, halo, cyano, nitro, hydroxy, C 1~6 Alkyl, and C 1~6 -O(CH2) optionally substituted with one or more substituents selected from the group consisting of alkoxy u phenyl, or alkyl C, where R and R are taken together to link together as a ring; 1~3 And R3, R4, R5 and R6 are each independently hydrogen, halo, cyano, nitro, hydroxy, C 1~6 Alkyl, (CH2) u C 3~7 Cycloalkyl, -O(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 alkoxy, wherein C 1~6 Alkyl, (CH2)u C 3~7 Cycloalkyl, -O(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 Alkyl, and phenyl each optionally contain one or more R 23 is replaced by Each R 23 are independently halo, cyano, nitro, hydroxy, C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 alkoxy, wherein the phenyl is selected from the group consisting of halo, cyano, nitro, hydroxy, C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 Alkyl, substituted with up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 optionally substituted with one or more substituents independently selected from the group consisting of alkoxy; Each R 19 is C optionally substituted with halo, cyano, nitro, hydroxy, and up to five fluoro; 1~6 C optionally substituted with alkyl and up to five fluoro 1~6 aryl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy; Each R 20 are independently C optionally substituted with halo, cyano, nitro, hydroxy, and up to five fluoro; 1~6 C optionally substituted with alkyl and up to five fluoro 1~6hydrogen or aryl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy; When Y is NR8R9, R8 and R2 are optionally joined together to form an alkyl C 1~3 However, However, R8 and R2 are linked together to form a ring. 1~3 provided that, when R4 is not substituted with hydroxyl.

[0054] In some embodiments, one, two, or three of R3, R4, R5, and R6 are not hydrogen.

[0055] R4 is C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, -O(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 C substituted with alkoxy, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 alkoxy, wherein C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, -O(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy and phenyl are each optionally substituted with haloC 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, Hydroxy-C 1~6 C substituted with alkyl, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 and substituted with one or more substituents independently selected from the group consisting of alkoxy.

[0056] In some embodiments, Y is -NR8R9, where R8 is hydrogen and R9 is hydroxy, R 19 AND -OR 20 C optionally substituted with one or more substituents each independently selected from the group consisting of 1~6 alkyl, and each R 19 are independently substituted with halo, cyano, or up to five fluoro groups; 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 phenyl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy, 20 are independently C substituted with halo, cyano, nitro, hydroxy, and up to five fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 and oxygen or phenyl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy.

[0057] In some embodiments, each R 19 independently, halo, C 1~6 Alkyl, and C 1~6 phenyl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy, 20 independently, halo, C 1~6 Alkyl, and C 1~6 and phenyl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy.

[0058] In some embodiments, R2 and R8 are oxygen.

[0059] In some embodiments, Y is -NR8R9, and R8 and R2 together form a C 1~3 It becomes alkyl.

[0060] In some embodiments, R9 is hydrogen.

[0061] In some embodiments, R is hydroxy, R 19 -OR 20 C optionally substituted with one or more substituents each independently selected from the group consisting of 1~6 It is alkyl. Each R 19 are independently C optionally substituted with halo, cyano, or up to five fluoro; 1~6 C optionally substituted with alkyl and up to five fluoro 1~6 phenyl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy; Each R 20 are independently C optionally substituted with halo, cyano, nitro, hydroxy, and up to five fluoro; 1~6 C optionally substituted with alkyl and up to five fluoro 1~6 and hydrogen or phenyl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy.

[0062] In some embodiments, R9 is hydrogen or C 1~6 alkyl and hydroxyl, R 19 AND -OR 20 and optionally substituted with one or more substituents each independently selected from the group consisting of:

[0063] Each R 19 are independently C optionally substituted with halo, cyano, or up to five fluoro; 1~6 C optionally substituted with alkyl and up to five fluoro 1~6 phenyl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy; Each R 20 are independently C optionally substituted with halo, cyano, nitro, hydroxy, and up to five fluoro; 1~6 C optionally substituted with alkyl and up to five fluoro 1~6 and hydrogen or phenyl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy.

[0064] In some embodiments, R4 is C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, -O(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 C substituted with alkoxy, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 alkoxy, wherein C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, -O(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy and phenyl each optionally contain one or more R 23 Each R is replaced by 23 independently, halo, C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 C substituted with alkoxy, phenyl, and up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 alkoxy, wherein the phenyl is substituted with substituents independently selected from the group consisting of halo, C 1~6 Alkyl, -(CH2) u C 3~7 Cycloalkyl, C 2~6 Alkenyl, C 1~6 Alkoxy, C substituted with up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 and optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy.

[0065] In some embodiments, R4 is C 1~6 Alkyl, C 3~7Cycloalkyl, -OC 3~7 Cycloalkyl, phenyl, C substituted with up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 alkoxy, wherein the phenyl is selected from the group consisting of halo, C 1~6 Alkyl, C 1~6 Alkoxy, C substituted with up to 5 fluoro 1~6 Alkyl and C substituted with up to 5 fluoro 1~6 and optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy.

[0066] In some embodiments, Y can be -NR8R9, where R8 and R2 together are linked together as a ring. 1~3 It becomes alkyl.

[0067] In some embodiments, R2 is hydrogen or C 1~6 Alkyl, C 3~7 cycloalkyl, and phenyl, wherein C 1~6 The alkyl is optionally substituted with one or more halo.

[0068] In some embodiments, R2 is hydrogen.

[0069] In some embodiments, R9 is hydrogen or C 1~6 alkyl and hydroxyl, R 19 AND -OR 20 and optionally substituted with one or more substituents each independently selected from the group consisting of: Each R 19 are independently C optionally substituted with halo, cyano, or up to five fluoro; 1~6 C optionally substituted with alkyl and up to five fluoro 1~6 phenyl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy; Each R 20are independently C optionally substituted with halo, cyano, nitro, hydroxy, and up to five fluoro; 1~6 C optionally substituted with alkyl and up to five fluoro 1~6 and hydrogen or phenyl optionally substituted with one or more substituents each independently selected from the group consisting of alkoxy.

[0070] In some embodiments, the compound of formula (I) is selected from the group consisting of: [ka]

[0071] In some embodiments, the compound of formula (I) is CASIN: [ka] or a pharmaceutically acceptable salt thereof.

[0072] The term "ester" refers to an ester of the formula -(R) n refers to a chemical moiety having the formula -COOR', where R and R' are independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon), and heteroaryl (bonded through a ring carbon), and n is 0 or 1.

[0073] An "amide" is an amide of the formula -(R) n -C(O)NHR' or -(R) n A chemical moiety having the formula -NHC(O)R', where R and R' are independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon), and heteroalicyclic (bonded through a ring carbon), and n is 0 or 1. An amide can be an amino acid or peptide molecule attached to a molecule of the invention, thereby forming a prodrug.

[0074] Any amine, hydroxy, or carboxyl side chain on the compounds of the present invention can be esterified or amidated. The procedures and specific groups used to achieve this purpose are known to those of skill in the art and can be readily found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley & Sons, New York, NY, 1999, which is incorporated herein in its entirety.

[0075] As used herein, the terms "protecting group" and "protecting groups" refer to any atom or group of atoms added to a molecule to prevent an existing group in the molecule from undergoing an undesired chemical reaction. Examples of protecting group moieties are described in T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3. Ed. John Wiley & Sons, 1999, and J.F.W. McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973, both of which are incorporated herein by reference. Protecting group moieties can be selected to be stable to the reaction conditions applied and easily removed at a convenient stage using methodologies known in the art. A non-limiting list of protecting groups includes benzyl, substituted benzyl; alkylcarbonyl (e.g., t-butoxycarbonyl (BOC)); arylalkylcarbonyl (e.g., benzyloxycarbonyl, benzoyl); substituted methyl ethers (e.g., methoxymethyl ether); substituted ethyl ethers; substituted benzyl ethers; tetrahydropyranyl ethers; silyl ethers (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, or t-butyldiphenylsilyl); esters (e.g., benzoate esters); carbonates (e.g., methoxymethyl carbonate); sulfonates (e.g., tosylate, mesylate); acyclic ketals (e.g., dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane or 1,3-dioxolane); acyclic acetals; cyclic acetals; acyclic hemiacetals; cyclic hemiacetals; and cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane).

[0076] A "prodrug" refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may be bioavailable, for example, by oral administration, whereas the parent drug is not. A prodrug may also have improved solubility in pharmaceutical compositions compared to the parent drug. An example of a prodrug is, but is not limited to, a compound of the present invention administered as an ester ("prodrug") to facilitate transport across cell membranes where water solubility is detrimental to mobility, which is then metabolically hydrolyzed to the active carboxylic acid once inside cells where water solubility is beneficial. Another example of a prodrug is a short peptide (polyamino acid) bonded to an acid group, where the peptide is metabolized to reveal the active moiety.

[0077] The term "aromatic" refers to an aromatic group having at least one ring with a conjugated π-electron system, including both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (e.g., pyridine) groups. This term includes monocyclic and fused-ring polycyclic (i.e., rings that share adjacent pairs of carbon atoms) groups. The term "carbocyclic" refers to a ring structure that is closed by one or more covalent bonds, and in which the atoms forming the ring backbone are all carbon atoms. Thus, this term distinguishes carbocyclic from heterocyclic rings in which the ring backbone contains at least one atom other than carbon. The term "heteroaromatic ring" refers to an aromatic group that contains at least one heterocyclic ring.

[0078] As used herein, the term "alkyl" refers to an aliphatic hydrocarbon group. The alkyl moiety may be a "saturated alkyl" group, meaning that it does not contain an alkene or alkyne moiety. The alkyl moiety may also be an "unsaturated alkyl" moiety, meaning that it contains at least one alkene or alkyne moiety. An "alkene" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an "alkyne" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, straight-chain, or cyclic.

[0079] An alkyl group can have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as "1 to 20" refers to each integer within the given range. For example, "1 to 20 carbon atoms" means that the alkyl group consists of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms; however, the definition of this invention also covers appearances of the term "alkyl" without a specified numerical range). An alkyl group can also be a medium alkyl having 1 to 10 carbon atoms. An alkyl group can also be a lower alkyl having 1 to 5 carbon atoms. The alkyl group of the compounds of this invention can be designated as "C1-C4 alkyl" or similar designation. By way of example only, "C1-C4 alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl.

[0080] The alkyl group can be substituted or unsubstituted. When substituted, the substituents are one or more groups individually and independently selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino (including mono- and di-substituted amino groups), and protected derivatives thereof. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Whenever a substituent is described as being "optionally substituted," that substituent may be substituted with one of the above substituents.

[0081] The substituent "R" appearing alone without a number designation refers to a substituent selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon), and heteroalicyclic (bonded through a ring carbon).

[0082] An "O-carboxy" group refers to a RC(=O)O- group, where R is as defined herein.

[0083] A "C-carboxy" group refers to a -C(=O)OR group, where R is as defined herein.

[0084] An "acetyl" group refers to a -C(=O)CH3 group.

[0085] A "trihalomethanesulfonyl" group refers to a X3CS(=O)2- group where X is a halogen.

[0086] A "cyano" group refers to a -CN group.

[0087] An "isocyanate" group refers to an --NCO group.

[0088] A "thiocyanate" group refers to a -CNS group.

[0089] An "isothiocyanato" group refers to a -NCS group.

[0090] A "sulfinyl" group refers to a -S(=O)-R group, where R is defined herein.

[0091] An "S-sulfonamido" group refers to a -S(=O)NR, group, where R is defined herein.

[0092] An "N-sulfonamido" group refers to an RS(=O)2NH- group, where R is defined herein.

[0093] A "trihalomethanesulfonamide" group refers to a X3CS(=O)2NR- group, where X and R are defined herein.

[0094] An "O-carbamyl" group refers to an -OC(=O)-N(R)2, group, where R is defined herein.

[0095] An "N-carbamyl" group refers to an ROC(=O)NH- group, where R is defined herein.

[0096] An "O-thiocarbamyl" group refers to an -OC(=S)-N(R)2, group, where R is defined herein.

[0097] An "N-thiocarbamyl" group refers to an ROC(=S)NH- group, with R as defined herein.

[0098] A "C-amido" group refers to a -C(=O)-N(R) group, where R is defined herein.

[0099] An "N-amido" group refers to a RC(=O)NH- group, where R is defined herein.

[0100] The term "perhaloalkyl" refers to an alkyl group in which all hydrogen atoms have been replaced with halogen atoms.

[0101] The term "acylalkyl" refers to the group RC(=O)R'-, where R is as defined herein and R' is a diradical alkylene group. Examples of acylalkyl include, but are not limited to, CHC(=O)CH-, CHC(=O)CHCH-, CHCHC(=O)CHCH-, CHC(=O)CHCHCH-, and the like.

[0102] Unless otherwise specified, when a substituent is deemed "optionally substituted," it means that the substituent can be substituted with one or more groups individually and independently selected from cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, trihalomethanesulfonyl, and amino (including mono- and di-substituted amino groups), and protected derivatives thereof. Protecting groups that can form the protective derivatives of the above substituents are known to those of skill in the art and can be found in references such as Green and Wuts, above.

[0103] In the present context, the term "cycloalkyl" is intended to cover 3-, 4-, 5-, 6-, 7-, and 8- or more-membered rings containing only carbon atoms. Cycloalkyls can optionally contain one or more unsaturated bonds positioned in such a way that an aromatic pi-electron system does not result. Some examples of "cycloalkyl" are the carbocycles cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclopentadiene, cyclohexane, cyclohexene, 1,3-cyclohexadiene, 1,4-cyclohexadiene, cycloheptane, or cycloheptene.

[0104] As used herein, "heterocyclyl" refers to a cyclic ring system containing at least one heteroatom in the ring system backbone. The heteroatoms are independently selected from oxygen, sulfur, and nitrogen. A heterocyclyl can include multiple fused rings. A heterocyclyl can have any degree of saturation, as long as at least one ring in the ring system is aromatic. A heterocyclyl can be substituted or unsubstituted and is attached to other groups through any available valence, preferably any available carbon or nitrogen. Preferred monocyclic heterocycles are 5- or 6-membered. In a 6-membered monocyclic heterocycle, the heteroatoms are 1 to 3 selected from oxygen, sulfur, and nitrogen; if the heterocycle is 5-membered, it preferably has 1 or 2 heteroatoms selected from oxygen, sulfur, and nitrogen.

[0105] Heterocyclyl may further contain one or more carbonyl or thiocarbonyl functionalities, extending the definition to include oxo and thio systems such as lactams, lactones, cyclic imides, cyclic thioimides, cyclic carbamates, and the like.

[0106] Some examples of "heterocyclyl" include tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxane, 1,3-dioxane, 1,4-dioxane, 1,4-dioxane, piperazine, 1,3-oxathiane, 1,4-oxathiine, 1,4-oxathiane, tetrahydrofuran-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil. Heterocyclic radicals include, but are not limited to, morpholine, trioxane, hexahydro-1,3,5-triazine, tetrahydrofuran, pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline, pyrazolidine, imidazolidine, imidazolidine, 1,3-dioxane, 1,3-dioxane, 1,3-dithiol, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, and 1,3-oxathiolane. The point of attachment of the heterocyclic radical can be at the nitrogen heteroatom or through a carbon atom of the heterocycle.

[0107] In the present context, the term "aryl" is intended to mean a carbocyclic aromatic ring or ring system. Furthermore, the term "aryl" refers to a ring system that contains at least two aryl rings, or at least one aryl and at least one C 3~8 -Cycloalkyl includes fused ring systems in which at least one chemical bond is shared. Some examples of "aryl" rings include optionally substituted phenyl, naphthalenyl, phenanthrenyl, anthracenyl, tetralinyl, fluorenyl, indenyl, and indanyl. The term "aryl" refers to, for example, a ring connected through one of the ring-forming carbon atoms and optionally containing heterocyclyl, heteroaryl, halo, hydroxy, amino, cyano, nitro, alkylamido, acyl, C 1~6 Alkoxy, C 1~6 Alkyl, C 1~6 Hydroxyalkyl, C 1~6 Aminoalkyl, C 1~6The present invention relates to aromatic groups containing benzenoid groups having one or more substituents selected from alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl, or trifluoromethyl. The aryl group can be substituted at the para and / or meta positions. In other embodiments, the aryl group can be substituted at the ortho position. Representative examples of aryl groups include, but are not limited to, phenyl, 3-halophenyl, 4-halophenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, 3-aminophenyl, 4-aminophenyl, 3-methylphenyl, 4-methylphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 4-trifluoromethoxyphenyl, 3-cyanophenyl, 4-cyanophenyl, dimethylphenyl, naphthyl, hydroxynaphthyl, hydroxymethylphenyl, trifluoromethylphenyl, alkoxyphenyl, 4-morpholin-4-ylphenyl, 4-pyrrolidin-1-ylphenyl, 4-pyrazolylphenyl, 4-triazolylphenyl, and 4-(2-oxopyrrolidin-1-yl)phenyl.

[0108] As used herein, the term "heteroaryl" refers to an aromatic radical having one or more heteroatoms (e.g., oxygen, sulfur, or nitrogen) in the ring backbone, and may contain a single ring (e.g., pyridine) or multiple condensed rings (e.g., quinoline). Heteroaryl groups can have one or more substituents, each independently including halo, hydroxy, amino, cyano, nitro, cycloalkyl, haloalkyl, aryl, heterocyclyl, mercapto, alkylamido, acyl, C 1~6 -alkoxy, C 1~6 -Alkyl, C 1~6 -hydroxyalkyl, C 1~6 Aminoalkyl, C 1~6Heteroaryl groups are selected from alkylamino, alkylsulfenyl, alkylsulfinyl, alkylsulfonyl, sulfamoyl, and trifluoromethyl. Representative examples of heteroaryl groups include, but are not limited to, optionally substituted derivatives of furan, benzofuran, thiophene, benzothiophene, pyrrole, pyridine, indole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, isothiazole, imidazole, benzimidazole, pyrazole, indazole, tetrazole, quinoline, isoquinoline, pyridazine, pyrimidine, purine, and pyrazine, furazan, 1,2,3-oxadiazole, 1,2,3-thiazole, 1,2,4-thiazole, triazole, benzotriazole, pteridine, phenoxazole, oxadiazole, benzopyrazole, quinolizine, cinnoline, phthalazine, quinazoline, and quinoxaline. In some embodiments, the substituents are halo, hydroxy, cyano, OC 1~6 -Alkyl, C 1~6 -Alkyl, Hydroxy-C 1~6 -Alkyl, Amino-C 1~6 -alkyl.

[0109] antisense molecules In some embodiments, the Cdc42-specific inhibitor can be an antisense molecule. The term "antisense" (AS) or "antisense fragment" refers to a polynucleotide fragment (containing either deoxyribonucleotides, ribonucleotides, or a mixture of both) with inhibitory antisense activity, which causes a decrease in the expression of the endogenous genomic copy of the corresponding gene. An AS polynucleotide refers to a polynucleotide containing consecutive nucleotides of sufficient length and sequence homology to a sequence present in the sequence of a target gene to allow hybridization of the AS to the gene. Many reviews have covered the main aspects of antisense (AS) technology and its enormous therapeutic potential (see, e.g., Aboul-Fadl T., Curr Med. Chem. 2005; 12(19): 2193-214; Crooke ST, Curr Mol. Med. 2004 August; 4(5): 465-87; Crooke ST, Annu Rev Med. 2004; 55: 61-95; Vacek M et al., Cell Mol Life Sci. 2003 May; 60(5): 825-33; Cho-Chung YS, Arch Pharm Res. 2003 March; 26(3): 183-91; Moreira JN et al., Rev Recent Clin Trials 2006 September; 1(3): 217-35). Further reviews exist on the chemical (Crooke, 1995; Uhlmann et al., 1990), cellular (Wagner, 1994), and therapeutic (Hanania et al., 1995; Scanlon et al., 1995; Gewirtz, 1993) aspects of this technology. Antisense intervention in the expression of specific genes can be achieved by using synthetic AS oligonucleotide sequences (see, for example, Lefebvre-d'Hellencourt et al., 1995; Agrawal, 1996; Leflehman et al., 1997).

[0110] AS oligonucleotide sequences can be short sequences of DNA, typically 15- to 30-mers, but can be as small as 7-mers designed to complement a target mRNA of interest and form an RNA:AS duplex (Wagner et al., 1996). This duplex formation can interfere with the processing, splicing, transport, or translation of the associated mRNA. Furthermore, certain AS nucleotide sequences, upon hybridization with the target mRNA, can induce cellular RNase H activity, leading to mRNA degradation (Calabretta et al., 1996 Semin Oncol. 23(1):78-87). RNase H then cleaves the RNA component of the duplex, releasing the AS to further hybridize with additional molecules of the target RNA. An additional mechanism of action may be the interaction of AS with genomic DNA, forming a triple helix and rendering it transcriptionally inactive.

[0111] Sequence target segments of antisense oligonucleotides are selected so that they exhibit appropriate energy-related characteristics important for oligonucleotide duplex formation with their complementary templates, indicating a low likelihood of self-dimerization or self-complementarity (Anazodo et al., 1996). For example, the computer program OLIGO® (Primer Analysis Software, version 3.4) can be used to determine the melting temperature and free energy characteristics of antisense sequences and estimate potential self-dimerization and self-complementarity. This program allows for the determination of qualitative estimates of these two parameters (potential self-dimerization and self-complementarity) and provides indicators of "no potential," "some potential," or "essentially complete potential." Using this program, target segments that are predicted to have no potential for these parameters are typically selected. However, segments with "some potential" in one of the categories can be used. As is known in the art, a balance of parameters is used in the selection. Furthermore, oligonucleotides are also selected as needed to ensure that analog substitutions do not substantially affect function.

[0112] Phosphorothioate antisense oligonucleotides typically lack significant toxicity at concentrations effective in animals, exhibit sufficient pharmacodynamic half-lives (Agarwal et al., 1996), and are nuclease-resistant. Antisense-induced loss-of-function phenotypes related to cell development have been demonstrated for glial fibrillary acidic protein (GFAP), a protein responsible for establishing tectal plate formation in chicks (Galileo et al., 1991), and for the N-myc protein, which is responsible for maintaining cellular heterogeneity in neuroectodermal cultures (epithelial and neuroblastic cells, which differ in colony-forming, tumorigenic, and adhesive properties) (Rosolen et al., 1990; Whitesell et al., 1991). Antisense oligonucleotide inhibition of basic fibroblast growth factor (bFgF), which has mitogenic and angiogenic properties, suppressed glioma cell growth by 80% in a saturable and specific manner (Morrison, 1991). Due to their hydrophobicity, antisense oligonucleotides interact well with phospholipid membranes (Akhter et al., 1991). Following interaction with the cell plasma membrane, they are actively (or passively) transported into living cells (Loke et al., 1989) by a saturable mechanism predicted to involve specific receptors (Yakubov et al., 1989).

[0113] siRNA In other embodiments, the Cdc42-specific inhibitor may be a "small interfering RNA" (siRNA). siRNA refers to an RNA molecule that reduces or silences (prevents) the expression of its endogenous cellular counterpart gene / mRNA (e.g., Cdc42). This term is understood to include "RNA interference" (RNAi). RNA interference (RNAi) refers to the process of sequence-specific post-transcriptional gene silencing in mammals mediated by small interfering RNAs (siRNAs, e.g., short hairpin RNAs (shRNAs)) (Fire et al., 1998, Nature 391, 806). The corresponding process in plants is commonly referred to as specific post-transcriptional gene silencing or RNA silencing, and in fungi, it is also called silencing. The RNA interference response may be characterized by an endonuclease complex containing siRNA, commonly referred to as the RNA-induced silencing complex (RISC), which mediates the cleavage of single-stranded RNA with a sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA can occur in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al 2001, Genes Dev., 15, 188). For recent information on these terms and proposed mechanisms, see Bernstein E., Denli AM., Hannon GJ: The rest is silence. RNA. 2001 November;7(11):1509-21; and Nishikura K.: Ashortprimeron RNAi: RNA-directed RNA polymerase acts as a key catalyst. Cell. 2001 November 16;107(4):415-8.

[0114] RNAi is an efficient method for gene inactivation (Nature Reviews, 2002, v. 3, pp. 737-47; Nature, 2002, v. 418, pp. 244-51). Its method is based on the ability of dsRNA species to enter specific protein complexes, where they target and specifically degrade complementary cellular RNAs. More specifically, dsRNA is digested by type III RNases (DICER, Drosha, etc.) into short (17-29 bp) inhibitory RNAs (siRNAs) (Nature, 2001, v. 409, pp. 363-6; Nature, 2003, 425, pp. 415-9). These fragments, along with complementary mRNAs, are recognized by a specific RISC protein complex. The entire process is achieved by endonucleolytic cleavage of the target mRNA (Nature Reviews, 2002, v. 3, p. 737-47; Curr Opin Mol. Ther. 2003 June; 5(3): 217-24).

[0115] For disclosures regarding methods for designing and preparing siRNAs for known genes, see, e.g., Chalk AM, Wahlestedt C, Sonnhammer EL. 2004 Jun. 18; 319(1): 264-74; Sioud M, Leirdal M., Methods Mol. Biol. 2004; 252: 457-69; Levenkova N, Gu Q, Rux JJ. 2004 Feb. 12; 20(3): 430-2; and Ui-Tei K, Naito Y, Takahashi F, Haraguchi T, Ohki-Hamazaki H, Juni A, Ueda R, Saigo K., Nucleic Acids Res. 2004 Feb. 9; 32(3): 936-48. See PCT publications WO2004 / 015107 (Atugen) and WO02 / 44321 (Tuschl et al), also Chiu YL, Rana™. RNA 2003 September;9(9):1034-48 and US Pat. Nos. 5,898,031 and 6,107,094 (Crooke) for production of modified / more stable siRNAs.

[0116] DNA-based vectors capable of generating siRNAs in cells have been developed. This method generally involves the transcription of short hairpin RNAs that are efficiently processed to form siRNAs in cells (see, for example, Paddison et al. PNAS 2002, 99:1443-1448; Paddison et al. Genes & Dev 2002, 16:948-958; Sui et al. PNAS 2002, 8:5515-5520; and Brummelkamp et al. Science 2002, 296:550-553). These reports describe methods for generating siRNAs that can specifically target a large number of endogenously and exogenously expressed genes.

[0117] For methods related to the delivery of siRNA, see, for example, Shen et al. (FEBS letters 539:111-114 (2003)), Xia et al., Nature Biotechnology 20:1006-1010 (2002), Reich et al., Molecular Vision 9:210-216 (2003), Sorensen et al. (J. Mol. Biol. 327:761-766 (2003), Lewis et al., Nature Genetics 32:107-108 (2002) and Simeoni et al., Nucleic Acids Research 31,11:2717-2724 (2003)). siRNA has recently been successfully used for inhibition in primates. For further details, see, for example, Tolentino et al., Retina 24(1) February 2004 pp 132-138.

[0118] In some embodiments, the oligoribonucleotide according to the embodiments disclosed herein comprises a modified siRNA. In various embodiments, the siRNA comprises an RNA duplex comprising a first strand and a second strand, whereby the first strand comprises a ribonucleotide sequence at least partially complementary to about 18 to about 40 contiguous nucleotides of a target nucleic acid, the second strand comprises ribonucleotides at least partially complementary to the first strand, and the first strand and / or the second strand comprises multiple groups of modified ribonucleotides having a modification at the 2'-position of the sugar moiety, whereby within each strand, each group of modified ribonucleotides is adjacent to one or both sides of a group of adjacent ribonucleotides, whereby each ribonucleotide forming the group of adjacent ribonucleotides is selected from unmodified ribonucleotides or ribonucleotides having a modification that is different from the modification of the group of modified ribonucleotides.

[0119] Ribozymes In some embodiments, the Cdc42-specific inhibitor can be a ribozyme. The term "ribozyme" refers to an RNA molecule that has RNA catalytic ability and cleaves a specific site in a target RNA. According to embodiments disclosed herein, a ribozyme that cleaves mRNA (e.g., Cdc42 mRNA) can be used as an inhibitor. This may be necessary when antisense therapy is limited by stoichiometric considerations (Sarver et al., 1990, Gene Regulation and Aids, pp. 305-325). Ribozymes that target genes associated with myeloid diseases can then be used. The number of RNA molecules cleaved by the ribozyme is greater than that predicted by stochastic chemistry (Hampel and Tritz, 1989; Uhlenbeck, 1987).

[0120] Ribozymes catalyze the cleavage of phosphodiester bonds in RNA. Several structural families of ribozymes have been identified, including group I intron, RNase P, hepatitis delta virus ribozyme, hammerhead ribozyme, and hairpin ribozyme, originally derived from the negative strand of tobacco ringspot virus satellite RNA (sTRSV) (Sullivan, 1994; U.S. Patent No. 5,225,347). The latter two families are derived from viroids and virusoids, and ribozymes are thought to separate monomers from oligomers generated during rolling-circle replication (Symons, 1989 and 1992). Hammerhead and hairpin ribozyme motifs are most commonly adapted for trans-cleavage of mRNA for gene therapy (Sullivan, 1994). Ribozymes are generally approximately 30–100 nucleotides in length. Delivery of ribozymes is similar to that of AS fragments and / or siRNA molecules.

[0121] As used herein, the term "nucleic acid" may refer to DNA or RNA or modified versions thereof. Nucleic acids may also contain modified nucleotides that allow for correct readthrough by a polymerase and do not alter the expression of the polypeptide encoded by the nucleic acid. The terms "nucleic acid" and "oligonucleotide" are used interchangeably to refer to molecules containing multiple nucleotides. As used herein, these terms refer to oligoribonucleotides and oligodeoxyribonucleotides. The term also includes polynucleosides (e.g., polynucleotides minus the phosphate) and any other organic base-containing polymers. Nucleic acids include vectors, e.g., plasmids, as well as oligonucleotides. Nucleic acid molecules can be obtained from existing nucleic acid sources but are preferably synthetic (e.g., produced by oligonucleotide synthesis).

[0122] Polynucleotides used in accordance with the embodiments disclosed herein can be modified to have improved therapeutic properties. Nucleotide modifications or analogs can be introduced to improve the therapeutic properties of polynucleotides. Improved properties include increased nuclease resistance and / or increased ability to penetrate cell membranes. If necessary, nuclease resistance can be provided by any method known in the art that does not interfere with the biological activity of the AS polynucleotide, siRNA, cDNA, and / or ribozyme required for the method of use and delivery (Iyer et al., 1990; Eckstein, 1985; Spitzer and Eckstein, 1988; Woolf et al., 1990; Shaw et al., 1991). Modifications that can be made to oligonucleotides to enhance nuclease resistance include modification of the phosphorus or oxygen heteroatom of the phosphate backbone. These include methylphosphonates, phosphorothioates, phosphorodithioates, and the preparation of morpholino oligomers. In one embodiment, this is achieved by having phosphorothioate linkages linking the four to six 3'-terminal nucleotide bases. Alternatively, phosphorothioate linkages link all nucleotide bases. Other modifications known in the art can be used provided that biological activity is maintained but stability to nucleases is substantially increased.

[0123] Any analog or modification of a polynucleotide may be used with the embodiments disclosed herein, provided that the analog or modification does not substantially affect the function of the polynucleotide. Nucleotides may be selected from naturally occurring or synthetic modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine, and uracil. Modified nucleotide bases include inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl, and other alkyl adenines, 5-halouracil, 5-halocytosine, 6-azacytosine and 6-azathymine, pseudouracil, 4-thiuracil, 8-haloadenine, 8-aminoadenine, 8-thioladenine, 8-thiolalkyladenines, 8-hydroxyladenine and other 8-substituted adenines, 8-haloguanine, 8-aminoguanine, 8-thiolguanine, 8-thioalkylguanine, 8-hydroxylguanine and other substituted guanines, other aza and deazaadenines, other aza and deazaguanines, 5-trifluoromethyluracil, and 5-trifluorocytosine.

[0124] Furthermore, analogs of polynucleotides can be prepared in which the structure of the nucleotides is radically altered, making them more suitable as therapeutic or experimental reagents. An example of a nucleotide analog is peptide nucleic acid (PNA), in which the deoxyribose (or ribose) phosphate backbone in DNA (or RNA) is replaced with a polyamide backbone similar to that found in peptides. PNA analogs have been shown to be resistant to enzymatic degradation and to have extended life spans in vivo and in vitro. Furthermore, PNAs have been shown to bind more strongly to complementary DNA sequences than DNA molecules. This observation is attributed to the lack of charge repulsion between the PNA and DNA strands. Other modifications that can be made to oligonucleotides include polymer backbones, cyclic or acyclic backbones, and LNAs ("locked nucleic acids").

[0125] Embodiments disclosed herein also include nucleic acids (e.g., siRNAs) that can have the following degrees of homology or identity to a Cdc42-specific inhibitory nucleic acid: 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a range bracketed by any two of the foregoing percentages. Candidate Cdc42-specific inhibitory nucleic acids having 35% or greater homology or identity can be identified by methods known in the art and then tested using functional assays, such as those described herein and known in the art.

[0126] The term "homology" refers to the percent identity between two polynucleotide or two polypeptide moieties. The correspondence between sequences from one moiety to another can be determined by techniques known in the art. For example, homology can be determined by directly comparing the sequence information between two polynucleotide or polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridizing polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with a single-strand-specific nuclease and sizing the digested fragments. Two DNA or two polypeptide sequences are "substantially homologous" to each other if at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids match over a defined length of the molecule as determined using the methods described above.

[0127] Preparation of peptides and polypeptides In some embodiments, the Cdc42-specific inhibitor can be a polypeptide (e.g., a dominant-negative peptide, an antibody, or an affibody). The polypeptide can be produced, for example, through several methods known in the art (e.g., synthetically or through recombinant methods).

[0128] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are analogs or mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. Polypeptides can be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide," "peptide," and "protein" include glycoproteins as well as non-glycoproteins. Polypeptide products can be biochemically synthesized, such as using standard solid-phase techniques. Such methods include, but are not limited to, exclusive solid-phase synthesis, partial solid-phase synthesis, fragment condensation, and classical solution synthesis. These methods are preferably used when the peptide is relatively short (e.g., 10 kDa) and / or cannot be produced by recombinant technology (e.g., not encoded by a nucleic acid sequence) and therefore involve different chemistries. Solid-phase polypeptide synthesis procedures are well known in the art and are further described by John Morrow Stewart and Janis Dillaha Young, Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984). Synthetic polypeptides can be purified, if necessary, by preparative high performance liquid chromatography [Creighton T. (1983) Proteins, structures and molecular principles. W.H. Freeman and Co., NY], and their composition can then be confirmed by amino acid sequencing.When large amounts of polypeptide are desired, Bitter et al. (1987) Methods in Enzymol.153:516-544, Studier et al. (1990) Methods in Enzymol.185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. al.(1987)EMBOJ.6:307-311,Coruzzi et al.(1984)EMBOJ.3:1671-1680 and Brogli et al.,(1984)Science224:838-843,Gurley et al. al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

[0129] In some embodiments, a method for producing a polypeptide or fragment thereof is to clone a polypeptide containing the cDNA of a gene into an expression vector, culture cells containing the vector to express the encoded polypeptide, and then purify the resulting polypeptide, all of which are carried out using methods known in the art, e.g., as described in Marshak et al., "Strategies for Protein Purification and Characterization. A laboratory course manual." C.S.H.L. Press (1996) (and also, e.g., Bibl Hematol. 1965;23:1165-74; Appl Microbiol. 1967 July;15(4):851-6; CanJ.Biochem. 1968 May;46(5):441-4; Biochemistry. 1968 July;7(7):2574-80; ArchBiochemBiophys. 1968 Sep.10;126(3):746-72; BiochemBiophysResCommun. 1970 Feb.20;38(4):825-30).

[0130] The expression vector can contain a promoter to control the transcription of the heterologous material, and can be either a constitutive or inducible promoter to enable selective transcription. Optionally, an enhancer can be included to obtain the required transcription level. The expression vehicle can also contain a selection gene.

[0131] Vectors can be introduced into cells or tissues by any of a variety of methods known in the art, such as those described and generally found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Vega et al., Gene Targeting, CRC Press, Ann Arbor, Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988), and Gilboa et al. (1986).

[0132] Preparation of anti-Cdc42 antibody Antibodies that bind to Cdc42 or fragments derived therefrom can be prepared using the intact polypeptide or fragments containing smaller polypeptides as the immunizing antigen. For example, it may be desirable to generate antibodies that specifically bind to the N-terminus or C-terminus of Cdc42 or any other suitable domain. The polypeptide used to immunize animals can be derived from translated cDNA or chemically synthesized and, if necessary, conjugated to a carrier protein. Commonly used carriers that are chemically conjugated to polypeptides include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The conjugated polypeptide is then used to immunize animals.

[0133] If desired, polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or peptide to which the antibody was raised is bound. Those skilled in the art are aware of various techniques common in immunology for purifying and / or enriching polyclonal and monoclonal antibodies (Coligan et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1994).

[0134] Methods for producing all types of antibodies, including fragments, are known in the art (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988)). Immunization methods, including all the necessary steps of preparing the immunogen in an appropriate adjuvant, determining antibody binding, isolating the antibody, obtaining monoclonal antibodies, and humanizing the monoclonal antibodies, are all well known to those skilled in the art.

[0135] The antibody may be a humanized antibody or a human antibody. Antibodies can be humanized using various techniques known in the art, including CDR-grafting (EP 239,400, PCT publication WO 0.91 / 09967; US Pat. Nos. 5,225,539; 5,530,101, and 5,585,089, veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814(1994)); Roguska et al., PNAS 91:969-973(1994)), and chain shuffling (US Pat. No. 5,565,332).

[0136] Monoclonal antibodies as defined include antibodies derived from one species (mouse, rabbit, goat, rat, human, etc.) and antibodies derived from two (or more) species, such as chimeric and humanized antibodies.

[0137] Completely human antibodies are particularly desirable for the therapeutic treatment of human patients.Human antibodies can be produced by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences.See also U.S. Patent Nos. 4,444,887 and 4,716,111, and PCT applications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741, each of which is incorporated herein by reference in its entirety.

[0138] Additional information regarding antibodies of all types, including humanized antibodies, human antibodies and antibody fragments, can be found in WO01 / 05998, which is incorporated herein by reference in its entirety.

[0139] Neutralizing antibodies can be prepared by the methods described above, possibly with the additional step of screening for neutralizing activity, such as by a survival assay.

[0140] Embodiments disclosed herein also relate to the preparation and use of affibodies, which are binding proteins of non-Ig origin developed by combinatorial protein engineering principles, for example, as described in Nygren PA 2008 FEBS Journal 275:2668-2676.

[0141] The polypeptides used in the embodiments disclosed herein may also be modified, and in some cases chemically modified, to improve their therapeutic activity. "Chemically modified" refers to a polypeptide in which at least one of its amino acid residues has been modified by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques known in the art. Among the many well-known modifications, typical, but not exclusive, examples include acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of lipids or lipid derivatives, methylation, myristylation, PEGylation, prenylation, phosphorylation, ubiquitination, or similar processes.

[0142] Additional possible polypeptide modifications (such as those resulting from changes in the nucleic acid sequence) include substitutions, deletions, and insertions.

[0143] "Conservative substitution" refers to the substitution of amino acids of one class with amino acids of the same class, where the class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous polypeptides found in nature as determined, for example, by standard Dayhoff frequency exchange matrices or BLOSUM matrices.

[0144] A "non-conservative substitution" refers to the substitution of an amino acid of one class for an amino acid of another class, for example, the substitution of a class II residue, Ala, with a class III residue such as Asp, Asn, Glu, or Gln.

[0145] A "deletion" refers to a change in either the nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.

[0146] An "insertion" or "addition" refers to a change in a nucleotide or amino acid sequence that has resulted in the addition of one or more nucleotides or amino acid residues, respectively, compared to the naturally occurring sequence.

[0147] A "substitution" refers to the replacement of one or more nucleotides or amino acids with different nucleotides or amino acids, respectively. With respect to amino acid sequences, substitutions can be conservative or non-conservative.

[0148] Embodiments disclosed herein also include polypeptides (e.g., dominant-negative polypeptides or antibodies) that may have the following degrees of homology or identity to a Cdc42-specific inhibitory polypeptide: 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a range bracketed by any two of the foregoing percentages. Candidate Cdc42-specific inhibitory polypeptides having 35% or greater homology or identity can be identified by methods known in the art and then tested using functional assays, such as those described herein and known in the art.

[0149] Pharmaceutical Compositions and Administration Also provided herein are pharmaceutical compositions for the methods provided herein. In some embodiments, the pharmaceutical composition comprises a Cdc42-specific inhibitor and a pharmaceutically acceptable carrier.

[0150] The compounds or mixtures of compounds described herein can be synthetic, naturally occurring, or a combination thereof. The compounds or mixtures of compounds described herein can include amino acids, nucleotides, carbohydrates, lipids, polysaccharides, etc. The compounds or mixtures of compounds described herein preferably include a Cdc42-specific inhibitor (e.g., CASIN). The compounds or mixtures of compounds described herein can be formulated into pharmaceutical compositions containing pharmaceutically acceptable carriers and other excipients, as will be apparent to those skilled in the art. Such compositions can further include effective amounts of other compounds, particularly for the treatment of the conditions, diseases, and / or disorders described herein.

[0151] Some embodiments include administration of a pharmaceutically effective amount of an active agent or a pharmaceutically acceptable salt or ester thereof, an active agent analog or a pharmaceutically acceptable salt or ester thereof, or a combination thereof.

[0152] The compositions and preparations described preferably contain at least 0.1% of the active agent. Of course, the percentage of the compositions and preparations can vary and can comprise from about 2% to 60% by weight of the dosage. Preferably, the percentage of the compositions and preparations can comprise from about 2, 5, 10, or 15% to 30, 35, 40, 45, 50, 55, or 60% by weight of the dosage. The amount of active compound in such pharmaceutically useful compositions and preparations is such that a suitable dosage will be obtained.

[0153] Active agents can form salts, which are also within the scope of preferred embodiments. Reference to an active agent compound herein is understood to include reference to its salts, unless otherwise indicated. As used herein, the term "salt" refers to acidic and / or basic salts formed with inorganic and / or organic acids and bases. Furthermore, when an active agent contains both a basic moiety, such as, but not limited to, an amine, pyridine, or imidazole ring, and an acidic moiety, such as, but not limited to, a carboxylic acid, zwitterions ("inner salts") can be formed and are included in the term "salt" as used herein. While pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, other salts are also useful, for example, in isolation or purification steps that may be used during preparation. Salts of active agent compounds can be formed, for example, by reacting the active agent compound with an amount of acid or base, such as an equivalent amount, in a medium, such as a medium in which the salt precipitates, or in an aqueous medium, followed by lyophilization.

[0154] Active agents containing a basic moiety, such as, but not limited to, an amine, pyridine, or imidazole ring, can form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetate (such as those formed with acetic acid or trihaloacetic acid, e.g., trifluoroacetic acid), adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride (formed with hydrochloric acid), and hydrobromide (formed with hydrogen bromide). , hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate (formed with maleic acid), methanesulfonate (formed with methanesulfonic acid), 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate (such as formed with sulfuric acid), sulfonate (such as those described herein), tartrate, thiocyanate, toluenesulfonatosylate, undecanoate, and the like.

[0155] Active agents containing an acidic moiety, including but not limited to carboxylic acids, can form salts with a variety of organic and inorganic bases. Exemplary base salts include ammonium salts; alkali metal salts such as sodium, lithium, and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; salts with organic bases (e.g., organic amines) such as benzathine, dicyclohexylamine, and hydrabamine [formed with N,N-bis(dehydro-abietyl)ethylenediamine]; N-methyl-D-glucamine, N-methyl-D-glucamide, t-butylamine, and salts with amino acids such as arginine and lysine. Basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, butyl chloride, bromide, iodide), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, diamyl sulfate), linear halides (e.g., decyl, lauryl, myristyl, and stearyl chloride, bromide, and iodide), aralkyl halides (e.g., benzyl and phenethyl bromide), and the like.

[0156] Prodrugs and solvates of the compounds of preferred embodiments are also contemplated herein.As used herein, the term "prodrug" refers to a compound that, when administered to a subject, undergoes chemical conversion by metabolic or chemical processes to produce the compound of the active agent, and / or its salt and / or solvate.The solvate of the active agent is preferably a hydrate.

[0157] The active agents and their salts can exist in their tautomeric forms (e.g., as amides or imino ethers), and all such tautomeric forms are contemplated herein as part of the preferred embodiments.

[0158] All stereoisomers of the compounds of the present invention, including those that may exist due to asymmetric carbons on any of the substituents, including enantiomeric forms (which may exist even without asymmetric carbons) and diastereomeric forms, are contemplated and fall within the scope of the preferred embodiments. Individual stereoisomers of the compounds of the preferred embodiments can, for example, be substantially free of other isomers, or can be mixed with all or any other selected stereoisomers, for example, as racemates. The chiral centers of the preferred embodiments can have the S or R configuration as defined by the IUPAC 1974 Recommendations.

[0159] When the compound according to preferred embodiments is in the form of salt, it is preferably a pharmaceutically acceptable salt.Such salts include pharmaceutically acceptable acid addition salts, pharmaceutically acceptable base addition salts, pharmaceutically acceptable metal salts, ammonium and alkylated ammonium salts.Acid addition salts include the salts of inorganic acids and organic acids.Representative examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulfuric acid, nitric acid, etc. Representative examples of suitable organic acids include formic acid, acetic acid, trichloroacetic acid, trifluoroacetic acid, propionic acid, benzoic acid, cinnamic acid, citric acid, fumaric acid, glycolic acid, lactic acid, maleic acid, malic acid, malonic acid, mandelic acid, oxalic acid, picric acid, pyruvic acid, salicylic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, tartaric acid, ascorbic acid, pamoic acid, bismethylenesalicylic acid, ethanedisulfonic acid, gluconic acid, citric acid, aspartic acid, stearic acid, palmitic acid, EDTA, glycolic acid, p-aminobenzoic acid, glutamic acid, benzenesulfonic acid, p-toluenesulfonic acid, sulfates, nitrates, phosphates, perchlorates, borates, acetates, benzoates, hydroxynaphthoates, glycerophosphates, ketoglutarate, etc. Examples of metal salts include lithium, sodium, potassium, magnesium salts, etc. Examples of ammonium and alkylated ammonium salts include ammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, hydroxyethylammonium, diethylammonium, butylammonium, tetramethylammonium salts, etc. Examples of organic bases include lysine, arginine, guanidine, diethanolamine, choline, etc.

[0160] Pharmaceutically acceptable salts can be prepared by reacting the active agent with 1 to 4 equivalents of a base, such as sodium hydroxide, sodium methoxide, sodium hydride, potassium t-butoxide, calcium hydroxide, or magnesium hydroxide, in a solvent such as ether (THF), methanol, t-butanol, dioxane, isopropanol, or ethanol. Mixtures of solvents can be used. Organic bases, such as lysine, arginine, diethanolamine, choline, guanidine, and their derivatives, can also be used. Alternatively, acid addition salts, where applicable, can be prepared by treating with an acid, such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid, methanesulfonic acid, fonic acid, acetic acid, citric acid, maleic acid, salicylic acid, hydroxynaphthoic acid, ascorbic acid, palmitic acid, succinic acid, benzoic acid, benzenesulfonic acid, or tartaric acid, in a solvent such as ethyl acetate, ether, alcohol, acetone, THF, or dioxane. Mixtures of solvents can also be used.

[0161] The compounds can be formulated in a variety of forms, including solid and liquid forms such as tablets, gels, syrups, powders, aerosols, creams, lotions, tinctures, foams, and the like.

[0162] The compositions of preferred embodiments may contain physiologically acceptable diluents, fillers, lubricants, excipients, solvents, binders, stabilizers, etc. Diluents that can be used in the compositions include, but are not limited to, dicalcium phosphate, calcium sulfate, lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch, powdered sugar, and hydroxypropyl methylcellulose (HPMC) for sustained-release tablets. Binders that can be used in the compositions include, but are not limited to, starch, gelatin, and fillers such as sucrose, glucose, dextrose, and lactose.

[0163] Natural and synthetic gums that can be used in the compositions include, but are not limited to, sodium alginate, gum ghatti, carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone, and veegum. Excipients that can be used in the compositions include, but are not limited to, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, starch, magnesium stearate, lactose, and sucrose. Stabilizers that can be used include, but are not limited to, polysaccharides such as acacia, agar, alginic acid, guar gum, and tragacanth, amphiphiles such as gelatin, and synthetic and semi-synthetic polymers such as carbomer resins, cellulose ethers, and carboxymethylchitin.

[0164] Solvents that can be used include, but are not limited to, Ringer's solution, water, distilled water, up to 50% dimethyl sulfoxide in water, propylene glycol (neat or in water), phosphate buffered saline, balanced salt solutions, glycols, and other conventional fluids.

[0165] The dosage and regimen in which the compound is administered will vary depending on the dosage form, mode of administration, the condition being treated, and the particulars of the patient being treated. Thus, the optimal therapeutic concentration is best determined at that time and place through routine experimentation.

[0166] The compounds according to the preferred embodiments can also be administered enterally. Orally, the compounds according to the preferred embodiments are suitably administered at a rate of 100 μg to 100 mg per kg of body weight per day. Preferably, the compounds according to the preferred embodiments are suitably administered at a rate of about 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg to about 1, 5, 10, 25, 50, 75, or 100 mg per kg of body weight per day. The required dose can be administered in one or more portions. For oral administration, suitable dosage forms include, for example, tablets, gels, aerosols, pills, dragees, syrups, suspensions, emulsions, solutions, powders, and granules. A preferred method of administration involves using a suitable dosage form containing 1 mg to about 500 mg of the active substance. Preferably, the method of administration comprises using a suitable form containing from about 1, 2, 5, 10, 25, or 50 mg to about 100, 200, 300, 400, 500 mg of active agent.

[0167] The compounds according to the preferred embodiments can also be administered parenterally in the form of a solution or suspension for intravenous or intramuscular infusion or injection, in which case the compounds according to the preferred embodiments are generally administered at a rate of about 10 μg to 10 mg per kg of body weight per day, with the preferred method of administration being to use a solution or suspension containing about 0.01 mg to 1 mg of active substance per ml. Preferably, compounds according to preferred embodiments are generally administered at a rate of about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μg to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg per kg of body weight per day, with preferred methods of administration comprising using a solution or suspension containing about 0.01, 0.02, 0.03, 0.04, or 0.5 mg to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg of active agent per ml.

[0168] The active compounds and / or pharmaceutical compositions of the embodiments disclosed herein can be administered according to a variety of routes, typically by injection or oral administration, including local or systemic administration. Additionally, repeated administration can be given as needed.

[0169] For ex vivo administration, the active agent can be administered by any standard method that maintains cell viability, such as adding it to culture medium (appropriate for the target cells) and then adding this medium directly to the cells. As known in the art, any medium used in this method can be aqueous and non-toxic so as not to render the cells non-viable. Additionally, standard nutrients for maintaining cell viability can be included, if necessary. For in vivo administration, the complex can be added to a pharmaceutically acceptable carrier, such as saline and buffered saline, and administered by any of several means known in the art. Examples of administration include parenteral administration by subcutaneous or intramuscular injection, including, for example, by local perfusion through blood vessels supplying the tissue or organ bearing the target cells, or by inhalation of an aerosol; local administration into skin wounds and lesions; direct transfection into bone marrow cells prepared for transplantation and subsequent transplantation into a subject, and direct transfection into organs that are then transplanted into the subject. Additional administration methods include oral administration, particularly when the active agent is encapsulated, or rectal administration, particularly when the active agent is in the form of a suppository.

[0170] Such target cells may be located within a subject or human patient, in which case it is contemplated that a safe and effective amount of the active agent in a pharmacologically acceptable form is administered to the patient. Generally speaking, useful pharmaceutical compositions of preferred embodiments will comprise a convenient amount, e.g., about 0.001% to about 10% (w / w), of the selected active compound derivative, diluted in a pharmacologically or physiologically acceptable carrier, such as, for example, phosphate-buffered saline. The route of administration and the final amount of material administered to a subject under such circumstances will depend on the intended use and will be apparent to those skilled in the art in light of the following examples.

[0171] The composition selected should have low or no toxicity to cells. The toxicity of a particular compound may vary depending on the concentration of the compound used. It is also beneficial if the selected compound is metabolized or eliminated in the body in a manner that does not cause adverse toxicity.

[0172] The examples are illustrative of the types of compounds that may be used in the methods claimed herein, and the list is not intended to be exhaustive. Derivatives of the above compounds that meet the criteria of the claims are also preferably considered when selecting active compounds.

[0173] The compound is preferably administered so that a therapeutically effective concentration of the compound contacts affected cells of the body. In the context of preferred embodiments, the dose administered to a subject, particularly a human, is preferably sufficient to produce a therapeutic response in the subject over a reasonable period of time. The dose will be determined by the strength of the particular compound used, the condition of the subject, and the body weight of the subject being treated. The existence, nature, and extent of any adverse side effects that may accompany the administration of a particular compound will also determine the dose size and the particular route of administration employed for a particular patient. In general, the compounds of preferred embodiments are therapeutically effective at low doses. A generally useful dose range is from about 0.001 mM or less to about 100 mM or more. Preferably, the effective dose range is from about 0.01, 0.05, 0.1, 0.5, 0.6, 0.7, 0.8, or 0.9 mM to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM. Therefore, the compounds will generally be administered at low doses.

[0174] The compound can be administered in a pharmaceutically acceptable carrier.Pharmaceutical acceptable carriers are well known to those skilled in the art.The selection of carrier will be determined in part by the specific compound and the specific method used to administer the composition.Therefore, there are a wide variety of suitable formulations of the pharmaceutical composition of preferred embodiments.

[0175] The compound can be administered orally, topically, parenterally, by inhalation or spray, vaginally, rectally, or sublingually in dosage unit formulations. The term "administration by injection" includes, but is not limited to, intravenous, intraarticular, intramuscular, subcutaneous, and parenteral injections, as well as the use of infusion techniques. Dermal administration can include topical or transdermal administration. One or more compounds can be present in association with one or more non-toxic pharmaceutically acceptable carriers, and optionally other active ingredients.

[0176] Compositions intended for oral use can be prepared according to any suitable method known in the art for the manufacture of pharmaceutical compositions. Such compositions can contain one or more agents selected from the group consisting of diluents, sweeteners, flavoring agents, coloring agents, and preservatives to provide a palatable preparation. Tablets contain the active ingredient mixed with non-toxic pharmaceutically acceptable excipients suitable for the manufacture of tablets. These excipients can be, for example, inert diluents (such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate), granulating and disintegrating agents (such as corn starch or alginic acid), and binders (such as magnesium stearate, stearic acid, or talc). Tablets can be uncoated or coated by known techniques to delay disintegration and adsorption in the gastrointestinal tract, thereby providing a sustained action over a longer period. For example, a time-delay material, such as glyceryl monostearate or glyceryl distearate, can be used. These compounds can also be prepared in solid, rapid-release forms.

[0177] Formulations for oral use may also be presented as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

[0178] Aqueous suspensions containing the active material in admixture with excipients suitable for the manufacture of aqueous suspensions can also be used. Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, and dispersing or wetting agents such as naturally occurring phosphatides (e.g., lecithin), or condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, such as polyoxyethylene sorbitan monooleate, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides (e.g., polyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives, for example, ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0179] Dispersible powders and granules suitable for preparing an aqueous suspension by adding water provide the active ingredient mixed with a dispersing or wetting agent, a suspending agent, and one or more preservatives.Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above.Additional excipients, such as sweeteners, flavorings, and coloring agents, may also be present.

[0180] The compound can also be formulated as a non-aqueous liquid preparation, for example, an oily suspension, which can be formulated by suspending the active agent in vegetable oil (e.g., peanut oil, olive oil, sesame oil, or peanut oil) or mineral oil (such as liquid paraffin). Oily suspensions can contain thickening agents, such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners and flavoring agents, such as those mentioned above, can be added to provide a palatable oral preparation. These compositions can be preserved by adding antioxidants, such as ascorbic acid.

[0181] The compounds of the preferred embodiments can also be administered transdermally using methods known to those skilled in the art.For example, a solution or suspension of an active agent in a suitable volatile solvent, optionally containing a penetration enhancer, can be combined with additional additives known to those skilled in the art, such as matrix materials and bactericides.After sterilization, the resulting mixture can be formulated into a dosage form according to known procedures.Furthermore, when treated with an emulsifier and water, the solution or suspension of an active agent can be formulated into a lotion or ointment.

[0182] Suitable solvents for treating transdermal delivery systems are known to those skilled in the art and include lower alcohols such as ethanol or isopropyl alcohol, lower ketones such as acetone, lower carboxylic acid esters such as ethyl acetate, polar ethers such as tetrahydrofuran, lower hydrocarbons such as hexane, cyclohexane, benzene, or halogenated hydrocarbons such as dichloromethane, chloroform, trichlorotrifluoroethane, trichlorofluoroethane, etc. Suitable solvents may also include mixtures of one or more materials selected from lower alcohols, lower ketones, lower carboxylic acid esters, polar ethers, lower hydrocarbons, and halogenated hydrocarbons.

[0183] Suitable penetration enhancers for transdermal delivery systems are known to those skilled in the art and include, for example, monohydroxy or polyhydroxy alcohols (such as ethanol, propylene glycol, or benzyl alcohol), saturated or unsaturated C8-C18 fatty alcohols (such as lauryl alcohol or cetyl alcohol), saturated or unsaturated C8-C18 fatty acids (such as stearic acid), saturated or unsaturated fatty esters containing up to 24 carbons (such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl acetate, or monoglycerin esters of caproic, lauric, myristic, stearic, or palmitic acid), or diesters of saturated or unsaturated dicarboxylic acids containing up to about 24 carbons in total (such as diisopropyl adipate, diisobutyl adipate, diisopropyl sebacate, diisopropyl maleate, or diisopropyl fumarate). Additional penetration enhancers include phosphatidyl derivatives such as lecithin or cephalin, terpenes, amides, ketones, urea and their derivatives, and ethers such as dimethyl isosorbide and diethylene glycol monoethyl ether. Suitable penetration enhancer formulations may also include a mixture of one or more materials selected from monohydroxy or polyhydroxy alcohols, saturated or unsaturated C8-C18 fatty acid alcohols, saturated or unsaturated C8-C18 fatty acids, saturated or unsaturated fatty esters containing up to 24 carbons, diesters of saturated or unsaturated dicarboxylic acids containing up to 24 carbons in total, phosphatidyl derivatives, terpenes, amides, ketones, urea and their derivatives, and ethers.

[0184] Binder materials suitable for transdermal delivery systems are known to those skilled in the art and include polyacrylates, silicones, polyurethanes, block polymers, styrene-butadiene copolymers, and natural and synthetic rubbers. Cellulose ethers, derivatized polyethylenes, and silicates can also be used as matrix components. Additional additives, such as viscous resins and oils, can be added to increase the viscosity of the matrix.

[0185] In some embodiments, the composition can include, for example, a topical formulation. In some embodiments, the topical formulation is a non-transdermal composition formulated so as not to penetrate beyond the dermal layer. Non-transdermal formulations are known in the art and include matrix or micelle solutions, bandages, wound dressings, aerosol sprays, foams, non-transdermal topical patches, colorants, topical agents, and the like.

[0186] The pharmaceutical composition of a preferred embodiment can also be in the form of an oil-in-water emulsion. The oil phase can be a vegetable oil (e.g., olive oil or peanut oil) or a mineral oil (e.g., liquid paraffin) or a mixture thereof. Suitable emulsifiers can be naturally occurring gums (e.g., acacia gum or tragacanth gum), naturally occurring phosphatides (e.g., soybean, lecithin), and esters or partial esters derived from fatty acids and hexitol anhydrides (e.g., sorbitan monooleate) and condensation products of said partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). Emulsions can also contain sweeteners and flavorings. Syrups and elixirs can contain sweeteners such as glycerol, propylene glycol, sorbitol, or sucrose. Such formulations can also contain thickeners, preservatives, flavorings, and coloring agents.

[0187] The compound can also be administered in the form of a suppository for rectal or vaginal administration. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal or vaginal temperature, and therefore melts in the rectum or vagina to release the drug. Such materials include cocoa butter and polyethylene glycol.

[0188] In all use regimens disclosed herein for the active agents, the daily oral dosage regimen will preferably be from about 0.01 to about 200 mg / Kg of total body weight. Preferably, the daily oral dosage regimen will be from about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, or 5 to about 10, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / Kg of total body weight. The daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous, and parenteral injections, and for use with infusion techniques will preferably be from 0.01 to 200 mg / Kg of total body weight. Preferably, the daily dosage for administration by injection, including intravenous, intramuscular, subcutaneous, and parenteral injections, and use of infusion techniques, will be about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, or 5 to about 10, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / kg of total body weight. The daily vaginal administration regimen will preferably be 0.01 to 200 mg / kg of total body weight. The daily topical administration regimen will preferably be 0.01 to 200 mg administered one to four times daily. The concentration of the vaginal and topical administrations will preferably be 0.1 to 200 mg / kg, as needed to maintain the daily dose. Preferably, the daily oral dosage regimen will be from about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, or 5 to about 10, 50, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / Kg of total body weight. The daily inhalation dosage regimen will be from 0.01 to 10 mg / Kg of total body weight. Preferably, the daily inhalation dosage regimen will be from about 0.01, 0.05, 0.1, 0.5 to about 1, 2, 3, 4, 5, or 10 mg / Kg of total body weight.

[0189] It will be understood by those skilled in the art that specific administration method depends on various factors, all of which are routinely considered when administering therapeutic agents.However, it will also be understood that the specific dose level of a given patient depends on various factors, including the activity of the specific compound used, the age of the patient, the weight of the patient, the general health of the patient, the sex of the patient, the diet of the patient, the time of administration, the route of administration, excretion rate, drug combinations and the severity of the condition being treated.It will also be understood by those skilled in the art that the optimal course of treatment, i.e., the mode of treatment and the number of daily doses of the active agent or its pharmaceutically acceptable salt given over a defined period of time, can be determined by those skilled in the art using conventional treatment tests.

[0190] The active compounds can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human. Such compositions typically include the nucleic acid molecule, protein, modulator, or antibody and a pharmaceutically acceptable carrier.

[0191] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as a conventional media or agent is incompatible with the active compound, such media can be used in the compositions of preferred embodiments. Supplementary active compounds can also be incorporated into the compositions. The pharmaceutical compositions of preferred embodiments are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal, non-transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous administration can include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, and phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0192] In some embodiments, the active compounds are prepared with carriers that protect the compounds from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (containing liposomes) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

[0193] It is particularly advantageous to prepare oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, "dosage unit form" refers to a physically discrete unit suitable as a single dose for a subject to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the necessary pharmaceutical carrier. The specifications of the dosage unit form of preferred embodiments are determined and directly depend on the inherent characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technology for compounding such active compounds for individual treatment.

[0194] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the total amount of each active ingredient of a pharmaceutical composition or method sufficient to provide a meaningful patient benefit, e.g., a cure for a chronic condition or an increased cure rate for such a condition, or a decrease in an abnormal condition. This includes both therapeutic and prophylactic treatment. Thus, the compounds can be used at a very early stage of disease, before early onset, or after significant progression. When applied to an individual active ingredient administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to the combined amount of the active ingredients that results in the therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0195] When carrying out the method of treatment or use of the preferred embodiments, a therapeutically effective amount of one, two, or more active agents of the preferred embodiments is administered to a subject.The active agents of the preferred embodiments can be administered alone or in combination with other known therapies according to the method of the preferred embodiments.When co-administered with one or more other therapies, the active agents of the preferred embodiments can be administered simultaneously with the other therapies or sequentially.When administered sequentially, the attending physician will determine the appropriate order for administering the active agents of the preferred embodiments in combination with other therapies.

[0196] Generally, a therapeutically effective amount of active agent (i.e., an effective dosage) is in the range of about 0.001-5000 mg / kg body weight, more preferably about 0.01-1000 mg / kg body weight, more preferably about 0.01-500 mg / kg body weight, more preferably about 0.01-250 mg / kg body weight, more preferably about 0.01-100 mg / kg body weight, more preferably about 0.001-60 mg / kg body weight, more preferably about 0.01-25 mg / kg body weight, more preferably about 0.1-20 mg / kg body weight, and even more preferably about 1-10 mg / kg, 2-9 mg / kg, 3-8 mg / kg, 4-7 mg / kg, or 5-6 mg / kg body weight.

[0197] A skilled artisan will understand that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other diseases present, may affect the dosage required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount can include a single treatment or, preferably, a series of treatments. In a preferred example, a subject is treated with a dose in the range of about 0.1 to 20 mg / kg body weight once weekly for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably about 4, 5, or 6 weeks. It will also be understood that the effective amount used for treatment may increase or decrease over the course of a particular treatment. Variations in dosage may result from and be evident from the results of the diagnostic assays described herein.

[0198] Preferred embodiments include one or more additional agents that regulate the expression or activity of Cdc42 GTPase. The agent may be, for example, a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., heteroorganic and organometallic compounds) with a molecular weight of less than about 10,000 grams / mole, organic or inorganic compounds with a molecular weight of less than about 5,000 grams / mole, organic or inorganic compounds with a molecular weight of less than about 1,000 grams / mole, organic or inorganic compounds with a molecular weight of less than about 500 grams / mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0199] In one embodiment, the additional agent can be a prenylation inhibitor, such as those disclosed in U.S. Patent Nos. 6,649,638, 5,420,245, 5,574,025, 5,523,430, 5,602,098, 5,631,401, 5,705,686, 5,238,922, 5,470,832, and 6,191,147, all of which are incorporated by reference herein in their entireties.

[0200] In another embodiment, the additional agent is a compound described in U.S. Patent Nos. 6,572,850, 6,458,783, 6,423,751, 6,387,926, 6,242,433, 6,191,147, 6,166,067, 6,156,746, 6,083,979, 6,011,029, 5,929,077, 5,928,924, 5,843,941, 5,786,193, 5,629,302, 5,618,964, 5, No. 574,025, No. 5,567,841, No. 5,523,430, No. 5,510,510, No. 5,470,83 No. 2, No. 5,447,922, No. 6,596,735, No. 6,586,461, No. 6,586,447, No. 6, No. 579,887, No. 6,576,639, No. 6,545,020, No. 6,539,309, No. 6,535,82 No. 0, No. 6,528,523, No. 6,511,800, No. 6,500,841, No. 6,495,564, No. 6,49 No. 2,381, No. 6,458,935, No. 6,451,812, No. 6,441,017, No. 6,440,989 , No. 6,440,974, No. 6,432,959, No. 6,426,352, No. 6,410,541, No. 6,403, 581, 6,399,615, 6,387,948, 6,387,905, 6,387,903, No. 6,376,496, No. 6,372,747, No. 6,362,188, No. 6,358,968, No. 6,329, 376, 6,316,462, 6,294,552, 6,277,854, 6,268,394, No. 6,265,382, No. 6,262,110, No. 6,258,824, No. 6,248,756, No. 6,242, 458, 6,239,140, 6,228,865, 6,228,856, 6,225,322, 6,228,865, 6,228,856, 6,225,322, No. 6,218,401, No. 6,214,828, No. 6,214,827, No. 6,211,193, No. 6,194,and one or more inhibitors of farnesyl protein transferase (FPTase), prenyl protein transferase, or geranylgeranyl protein transferase described in US Pat. No. 4,338,438, which are specifically incorporated herein by reference in their entireties.

[0201] "Farnesyl protein transferase inhibitors" or "FPT inhibitors" or "FTIs" are defined herein as compounds that (i) potently inhibit FPT (but generally not geranylgeranyl protein transferase I) and (ii) block the intracellular farnesylation of Ras. FPT catalyzes the addition of an isoprenyl lipid moiety to a cysteine residue located near the carboxy terminus of the Ras protein. This is the first step in a post-translational processing pathway essential for both Ras membrane binding and Ras-induced oncogenic transformation. Many FPT inhibitors have been reported, including various peptidomimetic inhibitors and other small molecule inhibitors.

[0202] Farnesyltransferase inhibitors fall into two general classes: analogs of farnesyl diphosphate and protein substrates of farnesyltransferase. Farnesyltransferase inhibitors are described in U.S. Patent Nos. 5,756,528, 5,141,851, 5,817,678, 5,830,868, 5,834,434, and 5,773,455, all of which are incorporated herein by reference in their entireties. Among the farnesyltransferase inhibitors shown to be effective in inhibiting the transfer of farnesyl moieties to Ras-associated proteins are L-739,749 (a peptidomimetic analog of the CAAX sequence), L-744,832 (a peptidomimetic analog of the CAAX sequence), SCH, 44342 (1-(4-pyridylacetyl)-4-(8-chloro-5,6-dihydro-IIH-benzo[5,6]cyclohepta[1,2-b]pyridin-11-idene)piperidine), BZA-5B (a benzodiazepine peptidomimetic), FTI-276 (a CAAX peptidomimetic), and B1086 (a CAAX peptidomimetic). Administration of farnesyltransferase inhibitors (FTIs) is accomplished by standard methods known to those skilled in the art, most preferably by administration of tablets containing FTI, and is expected to be in the range of about 0.1 mg / kg body weight to about 20 mg / kg body weight per day.

[0203] In another embodiment, the additional agent comprises one or more inhibitors of geranylgeranyltransferase (GGT), as described in U.S. Patent No. 5,470,832 (Gibbs & Graham), which is incorporated herein by reference in its entirety. These compounds can be administered to an individual at a dosage of 0.5 mg / kg body weight to about 20 mg / kg body weight. Alternatively, one or more isoprenylation inhibitors, including farnesyltransferase (FT) inhibitors and / or geranylgeranyltransferase (GGT) inhibitors, are administered to the patient.

[0204] In another embodiment, the additional agent comprises one or more toxins, such as toxins A and B from Clostridium difficile and Clostridium sordellii lethal toxin (LT). Furthermore, Rho can inhibit Rac1 and Rac2 when specifically ADP-ribosylated by the C3 enzyme, a botulinum toxin, and the staphylococcal toxin EDIN (Narumiya, S. and Morii, S., Cell Signal, 5, 9-19, 1993; Sekine, A. et al., J. Biol. Chem., 264, 8602-8605, 1989, all of which are incorporated herein by reference in their entireties).

[0205] It is understood that the appropriate dose of a small molecule agent will depend on many factors within the laboratory of an ordinary skilled physician, veterinarian, or researcher. The dose of a small molecule will vary, for example, depending on the identity, size, and condition of the subject or sample being treated, as well as the route by which the composition is administered, if applicable, and the effect the practitioner desires the small molecule to have on the nucleic acid or polypeptide of the embodiment. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., from about 1 microgram per kilogram to about 500 milligrams per kilogram, from about 100 micrograms per kilogram to about 5 milligrams per kilogram, or from about 1 microgram per kilogram to about 50 micrograms per kilogram). Furthermore, it is understood that the appropriate dose of a small molecule will depend on the potency of the small molecule with respect to the expression or activity being modulated. Such appropriate doses can be determined using the assays described herein. When administering one or more of these small molecules to a subject (e.g., a human) to modulate the expression or activity of a polypeptide or nucleic acid of the preferred embodiments, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose initially, and then increase the dose until an appropriate response is obtained. Furthermore, it will be understood that the specific dose level for a particular subject will depend on various factors, such as the activity of the specific compound used, the age, weight, general health, sex, and diet of the subject, the time of administration, the route of administration, the excretion rate, any drug combinations, and the degree of expression or activity to be modulated.

[0206] While appropriate dosage ranges for active compounds may vary depending on these considerations, in general, compounds are administered in the range of about 0.1 μg / kg to 5 mg / kg body weight, preferably about 1 μg / kg to 300 μg / kg body weight, and more preferably about 10 μg / kg to 100 μg / kg body weight. Thus, for a typical 70 kg subject, the dosage range is about 0.7 μg to 350 mg, preferably about 700 μg to 21 mg, and most preferably about 700 μg to 7 mg. When compounds are administered orally or transdermally, dosages may be higher, for example, compared to intravenous administration. Compounds can be administered as a single bolus dose, as doses over time as in the case of intravenous or transdermal administration, or in multiple doses.

[0207] The amount of the active compound to be administered can be varied according to the discretion of those skilled in the art.The amount of the active compound to be administered to the recipient is within the range described herein.However, the administration of such amount will vary according to the criteria established by the clinician.

[0208] Dosing regimens for rejuvenating blood progenitor cells, skin epithelial progenitor cells, or intestinal epithelial progenitor cells, or for weight management with the active compounds, are based on a variety of factors, including the type of injury, age, weight, sex, individual medical condition, severity of the condition, route of administration, and the specific compound used. Thus, dosing regimens can vary widely but can be routinely determined by a physician using standard methods. Dosage levels of approximately 0.1 ng / kg to 10 mg / kg of body weight of the active compound are useful for all of the methods disclosed herein.

[0209] The treatment regimen will also depend on the condition being treated, based on a variety of factors including the type of injury, age, weight, sex, medical condition of the individual, the severity of the condition, the route of administration and the particular compound used.

[0210] In a preferred embodiment, the active compound is administered subcutaneously. A suitable subcutaneous dose of the active compound is preferably between about 0.1 ng / kg and about 10 mg / kg administered twice daily for a time sufficient to increase the rejuvenation of blood progenitor cells, skin epithelial progenitor cells, or intestinal epithelial progenitor cells. This dosing regimen maximizes the therapeutic benefit of treatment while minimizing the amount of drug required. Such administration minimizes costs and potentially adverse side effects.

[0211] For subcutaneous administration, the active ingredient can comprise 0.0001% to 10% w / w, e.g., 1% to 2% by weight of the formulation, and can even comprise 10% w / w, but preferably 5% w / w or less, more preferably 0.1% to 1% of the formulation. In a most preferred embodiment, subcutaneous administration of about 1 to 1000 μg / kg / day of active compound is initiated between one week before and one week after administration of a cancer treatment (e.g., a chemotherapeutic agent).

[0212] In all of these embodiments, the compound may be administered before, simultaneously with, or after any other therapeutic exposure.

[0213] The active compound can be administered by any suitable route, including orally, parenterally, by inhalation spray, rectally, or topically, in a dosage unit formulation containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. As used herein, the term parenteral includes subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques, or intraperitoneally. In some embodiments, the active compound is administered as a depot containing a biocompatible matrix formulated for continuous delivery of the drug in vivo. In some embodiments, the depot is formulated to degrade over time, thereby releasing the drug in a continuous or near-continuous manner. In some embodiments, the depot is formulated for drug release over a period ranging from about 1 day to about 1, 2, 3, 4, 5, 6 months, or longer. In some embodiments, the depot can be an injectable depot for local administration. In some embodiments, the injectable depot is formulated for subcutaneous, intravenous, intraarterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques, or intraperitoneal injection. In some embodiments, the injectable depot is formulated for local injection at or near the stroma of the intestinal tract.

[0214] The active compounds can be in solid form (including granules, powders, or suppositories) or liquid form (e.g., solutions, suspensions, or emulsions). These compounds can be applied to a variety of solutions. Solutions suitable for use in accordance with preferred embodiments are sterile, dissolve a sufficient amount of the peptide, and are not harmful for the proposed use. In this regard, the compounds disclosed herein are highly stable but are hydrolyzed by strong acids and strong bases. These compounds are soluble in organic solvents and aqueous solutions at pH 5-8.

[0215] The active compounds may be subjected to conventional pharmaceutical operations, such as sterilization, and / or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, and the like.

[0216] For administration, active compounds are usually combined with one or more adjuvants appropriate for the indicated route of administration. These compounds can be mixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphate and sulfate, acacia, gelatin, sodium alginate, polyvinylpyrrolidine, and / or polyvinyl alcohol, and can be tableted or encapsulated for conventional administration. Alternatively, the compounds disclosed herein can be dissolved in saline, water, polyethylene glycol, propylene glycol, carboxymethylcellulose colloidal solution, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and / or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical arts. Carriers or diluents can include time-delay materials, such as glyceryl monostearate or glyceryl distearate, alone or with waxes, or other materials known in the art.

[0217] In some embodiments, the pharmaceutical composition comprises a Cdc42-specific inhibitor in a dosage amount sufficient to reduce the level of GTP-bound Cdc42 in senescent progenitor cells to approximately the level of GTP-bound Cdc42 in normal, non-senescent progenitor cells, hi some embodiments, the pharmaceutical composition comprises a Cdc42-specific inhibitor in a dosage amount less than sufficient to mobilize hematopoietic stem and progenitor cells from the bone marrow to the peripheral blood.

[0218] Additional Doses and Regimen Some details regarding the administration of Cdc422-specific inhibitors are provided above. Additional information regarding administration and treatment regimens is provided herein.

[0219] In some embodiments, only a single administration of a Cdc42-specific inhibitor is required to treat a subject. As explained above, this may be determined by the subject's unique characteristics, such as age, health, and Cdc42 activity. However, often a subject may require multiple administrations of a Cdc42-specific inhibitor to achieve the desired therapeutic result. Thus, in some embodiments, the subject is administered a single Cdc42-specific inhibitor, while in other embodiments, the subject is administered two or more administrations of a Cdc42-specific inhibitor. Administration of a Cdc42-specific inhibitor can be performed as frequently as necessary for therapeutic effect, for example, in some embodiments, administration can be performed 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 1-10 times, 1-9 times, 1-8 times, 1-7 times, 1-6 times, 1-5 times, 1-4 times, 1-3 times, 2-10 times, 2-9 times, 2-8 times, 2-7 times, 2-6 times, 2-5 times, 2-4 times, 2-3 times, 3-10 times, 3-9 times, 3-8 times, 3-7 times, 3-6 times, 3-5 times, 3-4 times, 3-5 times, 3-6 times, 3-7 times, 3-8 times, 3-9 ...6 times, 3-6 times, 3-7 times, 3-8 times, 3-9 times, 3-4 times, 3-5 times, 3-4 times, 3-5 times, 3-6 times, 3-7 times, 3-6 times, 3-7 times, 3-8 times, 3-8 times, 3-9 times, 3-9 times, 3-8 times, 3-9 times, 3-9 times, 3-8 times, 3-9 times, 3-4 times, 3-5 times, 3-4 times, 3-5 times, 3-5 times, 3-6 times, 3 4-10 times, 4-9 times, 4-8 times, 4-7 times, 4-6 times, 4-5 times, 5-10 times, 5-9 times, 5-8 times, 5-7 times, 5-6 times, 6-10 times, 6-9 times, 6-8 times, 6-7 times, 7-10 times, 7-9 times, 7-8 times, 8-10 times, 8-9 times, 9-10 times, any of the aforementioned administration times (e.g., about 3 times or about 1-3 times), or at least any of the aforementioned administration times (e.g., at least 3 times, at least about 3 times, or at least about 1-3 times).

[0220] When two or more doses of a Cdc42-specific inhibitor are administered to a subject, each dose can be the same Cdc42-specific inhibitor, or the doses can be different Cdc42-specific inhibitors. For example, samples can be collected from the subject and screened to determine the best Cdc42-specific inhibitor at a given dose (e.g., the subject may respond better to a different Cdc42-specific inhibitor as treatment progresses). The dose of the Cdc42-specific inhibitor can be adjusted over the course of treatment, such that the subject receives the same or different doses of the same or different Cdc42-specific inhibitor during the course of treating the subject. Thus, in some embodiments, the principles of personalized medicine are utilized to determine which Cdc42-specific inhibitor to administer to a subject.

[0221] Administration of a Cdc42-specific inhibitor to a subject in need of treatment may be administered as a regimen. In some embodiments, administration is administered daily for 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, or 14 days, or a range bracketed by any of the aforementioned days (e.g., days 1-5 or 3-7), or approximately any of the aforementioned days (e.g., about 2 days, about 1-5 days, or about 3-7 days). In some embodiments, administration is administered on non-consecutive days. In some embodiments, administration occurs on non-consecutive days for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 10 days, 11 days, 12 days, 13 days, 14 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 5 years, 10 years, the remaining life of the subject, or a range enclosed by or about any of the aforementioned days, weeks, or months.

[0222] The subject may need multiple doses or even multiple dose regimens, as outlined above.Thus, in some embodiments, the daily regimen or the non-consecutive day regimen is repeated every day, every week, every month, every 6 months, every 9 months, every 12 months, every 2 years, every 5 years, or at the aforementioned intervals (for example, every day to every week, or every month to every 12 months), or at any of the aforementioned intervals (for example, about every day to every week, or about every month to every 12 months).

[0223] In some embodiments, the subject's Cdc42 activity is determined before the first administration of a Cdc42-specific inhibitor, or before a subsequent administration (e.g., determining activity before the first administration but not before the second administration, or determining activity before the first administration and before the second administration, or determining activity before the first administration but not before the second administration, and before the third administration). In some embodiments, it may be beneficial to determine the subject's Cdc42 activity before each administration of a Cdc42-specific inhibitor (e.g., determining activity before the first administration and before the second administration, or determining activity before the first administration, the second administration, and the third administration). It may be beneficial for a physician to utilize information gathered from determining a subject's Cdc42 activity to prepare a patient-specific regimen or provide a patient-specific treatment. Thus, in some embodiments, the regimen or administration of a Cdc42-specific inhibitor is determined or repeated administrations are performed based on the Cdc42 activity in the subject.

[0224] A threshold value for Cdc42 activity can be utilized to determine an appropriate regimen or administration of a Cdc42-specific inhibitor. In some embodiments, a regimen or administration of a Cdc42-specific inhibitor is initiated when the Cdc42 activity in a subject is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% of the Cdc42 activity in the subject prior to first administering the Cdc42-specific inhibitor to said subject. , 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, or 200%, approximately any of the aforementioned percentages, or a range enclosed within any of the aforementioned percentages (e.g., approximately 1% to 30%, approximately 5% to 25%, approximately 5% to 20%, approximately 5% to 15%, or 1% to 30%, 5% to 25%, 5% to 20%, 5% to 15%), 1% to 100%, 1% to 90%, 1% ~80%, 1%~70%, 1%~60%, 1%~50%, 1%~40%, 1%~30%, 1%~20%, 1%~10%, 10%~100%, 10%~90%, 10%~80%, 10%~70%, 10%~70%, 10%~60%, 10%~50%, 10%~40%, 10%~30%, 10%~20%, 20%~100%, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30 % to 60%, 30% to 50%, 30% to 40%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 100%, 70% to 90%, 70% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, approximately a percentage for any of the aforementioned ranges (e.g., approximately 10% to 70%,About 30% to 60%, or about 50% to 70%, or about 25%, about 30%, about 40%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 100%, about 105%, about 110%, about 115%, about 120%, or about 125%.

[0225] In addition to, or as an alternative to, relying on the subject's Cdc42 activity to determine the regimen or administration of a Cdc42-specific inhibitor, the regimen or administration can be determined by the ratio of Cdc42-GTP to total Cdc42 levels in the subject. In some embodiments, the ratio of Cdc42-GTP to total Cdc42 levels in the subject is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 before administration, or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0, or greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 prior to dosing, or greater than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 prior to dosing.In some embodiments, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%; , 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%; at least 10%-90%, 10%-80%, 10%-70%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10 %~30%, 10%~20%, 20%~100%, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, 50%~90%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the subject's blood progenitor cells comprise the aforementioned ratios of Cdc42-GTP to total Cdc42 levels before administration of a Cdc42-specific inhibitor. In some embodiments, the ratio of Cdc42-GTP to total Cdc42 levels in the subject's blood progenitor cells is decreased after administration of a Cdc42-specific inhibitor.In some embodiments, the ratio of Cdc42-GTP to total Cdc42 levels in the blood progenitor cells after administration is less than 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5, or less than about 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 after administration of a Cdc42-specific inhibitor. In some embodiments, the ratio of Cdc42-GTP to total Cdc42 levels in the blood progenitor cells is at least 0.8, 0.9, 1.0, 1.1, 1.2, or greater, or at least about 0.8, 0.9, 1.0, 1.1, 1.2, or greater after administration of a Cdc42-specific inhibitor.

[0226] Some subjects may benefit from repeated administration of a Cdc42-specific inhibitor over the remainder of their life to maintain the above levels and ratios of CDC42 activity. Other subjects may benefit from repeated administration of a Cdc42-specific inhibitor over the course of a treatment regimen to maintain the above levels and ratios of CDC42 activity. However, some subjects may not require continuous exposure to a Cdc42-specific inhibitor to maintain the above levels and ratios of CDC42 activity. Thus, in some embodiments, a subject is administered a Cdc42-specific inhibitor over the remainder of their life to maintain the above levels and ratios of CDC42 activity. In other embodiments, a subject is administered a Cdc42-specific inhibitor during the course of a treatment regimen to maintain the above levels and ratios of CDC42 activity. In yet other embodiments, a subject is discontinued from exposure to a Cdc42-specific inhibitor at the end of the course of a treatment regimen. In some embodiments, a subject is discontinued from exposure to a Cdc42-specific inhibitor, and the Cdc42-specific inhibitor-mediated changes in the subject are maintained after cessation of exposure.

[0227] In some embodiments, a subject is selected for treatment with a Cdc42-specific inhibitor based on one or more of the subject's circulating cytokine levels. In some embodiments, one or more of the subject's circulating cytokine levels are determined before the first administration of a Cdc42-specific inhibitor or before a subsequent administration (e.g., determining activity before the first administration but not before the second administration, or determining activity before the first administration and before the second administration, or determining activity before the first administration but not before the second administration, but before the third administration). In some embodiments, the determined circulating cytokine levels are interferon-γ, interleukin-1α, interleukin-1β, or interleukin-9, and any combination thereof. In some embodiments, one or more of the subject's circulating cytokine levels are used to select a subject for treatment with a Cdc42-specific inhibitor. In some embodiments, it may be beneficial to determine one or more of the subject's circulating cytokine levels before each administration of a Cdc42-specific inhibitor (e.g., determining circulating cytokine levels before the first administration and before the second administration, or determining circulating cytokine levels before the first administration, the second administration, and the third administration). It may be beneficial for a physician to utilize information gleaned from determining the levels of one or more circulating cytokines in a subject to prepare a patient-specific regimen or provide a patient-specific treatment. Thus, in some embodiments, the regimen or administration of a Cdc42-specific inhibitor is determined or repeated administration is performed based on the levels of one or more circulating cytokines in the subject.

[0228] In some embodiments, one or more of the subject's circulating inflammatory cytokine levels are determined before the first administration or before a subsequent administration of a Cdc42-specific inhibitor (e.g., determining activity before the first administration but not before the second administration, or determining activity before the first administration and before the second administration, or determining activity before the first administration but not before the second administration, but before the third administration). In some embodiments, the circulating inflammatory cytokine levels determined are interferon-γ, interleukin-1α, or interleukin-1β, and any combination thereof. In some embodiments, one or more of the subject's circulating inflammatory cytokine levels are used to select a subject for treatment with a Cdc42-specific inhibitor. In some embodiments, it may be beneficial to determine one or more of the subject's circulating inflammatory cytokine levels before each administration of a Cdc42-specific inhibitor (e.g., determining circulating inflammatory cytokine levels before the first administration and before the second administration, or determining circulating cytokine levels before the first administration, the second administration, and the third administration). It may be beneficial for a physician to utilize information gleaned from determining the levels of one or more circulating inflammatory cytokines in a subject to prepare a patient-specific regimen or provide a patient-specific treatment. Thus, in some embodiments, the regimen or administration of a Cdc42-specific inhibitor is determined or repeated administration is based on one or more circulating inflammatory cytokine levels in the subject.

[0229] Threshold circulating cytokine levels or circulating inflammatory cytokine levels can be utilized to identify subjects for treatment or to determine appropriate regimens or administration of Cdc42-specific inhibitors. In some embodiments, one or more of the determined circulating cytokine levels or circulating inflammatory cytokine levels in a subject are 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, or approximately any of the foregoing percentages, or a range bracketed by any of the foregoing percentages (e.g., about 1%-30%, about 5%-25%, about 5%-20%, about 5%-15% or 1%-30%) , 5%~25%, 5%~20%, 5%~15%), 1%~100%, 1%~90%, 1%~80%, 1%~70%, 1%~60%, 1%~50%, 1%~40%, 1%~30%, 1%~20%, 1%~10%, 10%~100%, 10%~90%, 10%~80%, 10%~70%, 10%~70%, 10%~60%, 10%~50%, 10%~40%, 10%~30%, 10%~20%, 20%~100%, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20% ~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, 50%~90%, 50%~80%, 50%~70%, 50%~60%, 60%~100%, 60%~90%, 60%~80%, 60%~70%, 70%~100%, 70%~90%, 70%~80%, 80%~100%,If the Cdc42-specific inhibitor IL-16 expression level is 80%-90%, 90%-100%, approximately any of the aforementioned percentage ranges (e.g., about 10%-70%, about 30%-60%, or about 50%-70%), or about 25%, about 30%, about 40%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 100%, about 105%, about 110%, about 115%, about 120%, or about 125%, the subject is selected for treatment with a Cdc42-specific inhibitor. In some embodiments, the control population is based on age. In some embodiments, the control population is 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 years old, or a range bracketed by any of the foregoing ages (e.g., 18-20, The age is equal to or greater than 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years of age, or greater than 18-20, 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years of age. In some embodiments, the circulating cytokine level determined is interferon-γ, interleukin-1α, interleukin-1β, or interleukin-9, as well as any combination thereof.

[0230] In some embodiments, a regimen or administration of a Cdc42-specific inhibitor is effective in reducing one or more of the circulating cytokine levels or circulating inflammatory cytokine levels determined in a subject by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 1 5%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, or approximately any of the foregoing percentages, or a range bracketed by any of the foregoing percentages (e.g., approximately 1%-30%, approximately 5%-25%, approximately 5%-20%, approximately 5%-15%, or 1%-30%, 5%-25%, 5%-20%, 5%-15%). %), 1%~100%, 1%~90%, 1%~80%, 1%~70%, 1%~60%, 1%~50%, 1%~40%, 1%~30%, 1%~20%, 1%~10%, 10%~100%, 10%~90%, 10%~80%, 10%~70%, 10%~70%, 10%~60%, 1 0%~50%, 10%~40%, 10%~30%, 10%~20%, 20%~100%, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30% ~70%, 30%-60%, 30%-50%, 30%-40%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, 90%-100%, approximately any of the aforementioned percentage ranges (e.g., approximately 10%-70%, approximately 30%-60%,or about 50%-70%, or about 25%, about 30%, about 40%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 100%, about 105%, about 110%, about 115%, about 120%, or about 125%. In some embodiments, the control population is based on age. In some embodiments, the control population is 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 years old, or a range bracketed by any of the foregoing ages (e.g., 18-20, Ages equal to or greater than 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years, or greater than 18-20, 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years.

[0231] In some embodiments, a regimen or administration of a Cdc42-specific inhibitor is effective to reduce or eliminate one or more of the circulating cytokine levels or circulating inflammatory cytokine levels determined in a subject by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 9%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, approximately any of the foregoing percentages, or a range bracketed by any of the foregoing percentages (e.g., about 1% to 30%, about 5% to 25%, about 5% to 20%, about 5% to 15%, or 1% to 30%) , 5%~25%, 5%~20%, 5%~15%), 1%~100%, 1%~90%, 1%~80%, 1%~70%, 1%~60%, 1%~50%, 1%~40%, 1%~30%, 1%~20%, 1%~10%, 10%~100%, 10%~90%, 10%~80%, 10%~7 0%, 10%~70%, 10%~60%, 10%~50%, 10%~40%, 10%~30%, 10%~20%, 20%~100%, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%-90%, 30%-80%, 30%-70%, 30%-60%, 30%-50%, 30%-40%, 40%-100%, 40%-90%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, 90%-100%, or approximately any of the aforementioned percentage ranges (e.g.,The repeat is about 10% to 70%, about 30% to 60%, or about 50% to 70%, or about 25%, about 30%, about 40%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 100%, about 105%, about 110%, about 115%, 120%, or about 125%. In some embodiments, the determined circulating cytokine level is interferon-γ, interleukin-1α, interleukin-1β, or interleukin-9, or any combination thereof.

[0232] In some embodiments, an effective amount of a Cdc42-specific inhibitor reduces or decreases the level of one or more circulating cytokine levels in a subject. In some embodiments, the reduction or decrease in one or more circulating cytokine levels or one or more circulating inflammatory cytokine levels is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 9%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, approximately any of the foregoing percentages, or a range bracketed by any of the foregoing percentages (e.g., approximately 1% to 30%, approximately 5% to 25%, approximately 5% to 20%). %, approximately 5%-15% or 1%-30%, 5%-25%, 5%-20%, 5%-15%), 1%-100%, 1%-90%, 1%-80%, 1%-70%, 1%-60%, 1%-50%, 1%-40%, 1%-30%, 1%-20%, 1%-10%, 10%-100%, 10%-90%, 10%-80%, 10%-70%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-6 0%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, 50%~90%, 50%~80%, 50%~70%, 50%~60%, 60%~100%, 60%~90%, 60%~80%, 60%~70%, 70%~100%, 70%~90%, 70%~80%,The percentage may be 80% to 100%, 80% to 90%, 90% to 100%, or approximately any of the aforementioned percentage ranges (e.g., about 10% to 70%, about 30% to 60%, or about 50% to 70%). In some embodiments, the circulating cytokine level determined is interferon-γ, interleukin-1α, or interleukin-1β, or any combination thereof.

[0233] In some embodiments, an effective amount of a Cdc42-specific inhibitor reduces or decreases the level of one or more circulating inflammatory cytokine levels in a subject. In some embodiments, the reduction or decrease in one or more circulating cytokine levels or one or more circulating inflammatory cytokine levels is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or more than ... , 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, approximately any of the aforementioned percentages, or a range bracketed by any of the aforementioned percentages (e.g., approximately 1% to 30%, approximately 5% to 25%, approximately 5% to 20%, Approximately 5% to 15% or 1% to 30%, 5% to 25%, 5% to 20%, 5% to 15%), 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%, 20% to 60% %, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, 50%~90%, 50%~80%, 50%~70%, 50%~60%, 60%~100%, 60%~90%, 60%~80%, 60%~70%, 70%~100%, 70%~90%, 70%~80%,The control population can be 80%-100%, 80%-90%, 90%-100%, or approximately any of the aforementioned percentage ranges (e.g., about 10%-70%, about 30%-60%, or about 50%-70%). In some embodiments, the control population is based on age. In some embodiments, the control population is 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 years old, or a range bracketed by any of the aforementioned ages (e.g., 18-20, The age is equal to or greater than 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years of age, or greater than 18-20, 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years of age. In some embodiments, the circulating cytokine level determined is interferon-γ, interleukin-1α, or interleukin-1β, as well as any combination thereof.

[0234] In some embodiments, an effective amount of a Cdc42-specific inhibitor increases or elevates the level of one or more circulating cytokine levels in a subject. In some embodiments, administration of an effective amount of a Cdc42-specific inhibitor increases or elevates the level of one or more circulating cytokine levels in a subject by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, approximately any of the foregoing percentages, or a range bracketed by any of the foregoing percentages (e.g., approximately 1% to 30%, approximately 5% to 25%, approximately 5% to 20%, approximately 5% to 15% or 1% to 30%, 5% to 25%, 5% to 20%, 5%-15%), 1%-100%, 1%-90%, 1%-80%, 1%-70%, 1%-60%, 1%-50%, 1%-40%, 1%-30%, 1%-20%, 1%-10%, 10%-100%, 10%-90%, 10%-80%, 10%-70%, 10%-70%, 10%-60%, 10%-50%, 10%-40%, 10%-30%, 10%-20%, 20%-100%, 20%-90%, 20%-80%, 20%-70%, 20%-60%, 20%-50%, 20%-40%, 20%-30%, 3 0%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, 50%~90%, 50%~80%, 50%~70%, 50%~60%, 60%~100%, 60%~90%, 60%~80%, 60%~70%, 70%~100%, 70%~90%, 70%~80%, 80%~100%, 80%~90%, 90%~100%,There is an increase or elevation in one or more circulating cytokine levels of about any of the aforementioned percentage ranges (e.g., about 10% to 70%, about 30% to 60%, or about 50% to 70%). In some embodiments, the increased or elevated circulating cytokine level is interleukin-9.

[0235] In some embodiments, administration of an effective amount of a Cdc42-specific inhibitor results in an increase in circulating cytokine levels or circulating inflammatory cytokine levels by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more of an increase in circulating cytokine levels or circulating inflammatory cytokine levels compared to a control population. , 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, approximately any of the foregoing percentages, or a range enclosed within any of the foregoing percentages (e.g., approximately 1% to 30%, approximately 5% to 25%, approximately 5% to 20%, approximately 5% to 15%, or 1% to 30%, 5% to 25%, 5% to 20%, 5% to 15%), 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50% %, 1%~40%, 1%~30%, 1%~20%, 1%~10%, 10%~100%, 10%~90%, 10%~80%, 10%~70%, 10%~70%, 10%~60%, 10%~50%, 10%~40%, 10%~30%, 10%~20%, 20%~100%, 20% ~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~9 There is an increase or elevation in one or more circulating cytokine levels of 0%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, 90%-100%, or about any of the aforementioned percentage ranges (e.g., about 10%-70%, about 30%-60%, or about 50%-70%).In some embodiments, the control population is based on age. In some embodiments, the control population is 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 years old, or a range bracketed by any of the foregoing ages (e.g., 18-20, In some embodiments, the increased or elevated circulating cytokine level is interleukin-9.

[0236] In addition to selecting a subject or determining a regimen or administration of a Cdc42-specific inhibitor based on a combination of one or more circulating cytokine levels, in some embodiments, the subject, regimen, or administration is determined based on a ratio of two or more circulating cytokine levels in the subject. In some embodiments, the circulating cytokines are one or more of interferon-γ, interleukin-1α, interleukin-1β, and / or interleukin-9. In some embodiments, the ratio is one or more of interferon-γ, interleukin-1α, or interleukin-1β to interleukin-9. In some embodiments, the ratio of circulating cytokine levels in the subject is 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 before administration, or about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 before administration. 0.8, 2.9, or 3.0, greater than 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 prior to dosing, or greater than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 prior to dosing.In some embodiments, the percentage of the ion exchange rate is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%; or at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 90%; , 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%; at least 10%-90%, 10%-80%, 10%-70%, 10%-70%, 10%-60%, 10%-50%, 10%-40% 0%, 10%~30%, 10%~20%, 20%~100%, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, Between 50% and 90%, between 50% and 80%, between 50% and 70%, between 50% and 60%, between 60% and 100%, between 60% and 90%, between 60% and 80%, between 60% and 70%, between 70% and 100%, between 70% and 90%, between 70% and 80%, between 80% and 100%, between 80% and 90%, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the subject's blood progenitor cells comprise the aforementioned circulating cytokine level ratios before administration of a Cdc42-specific inhibitor. In some embodiments, the circulating cytokine level ratios in the subject's blood progenitor cells are decreased after administration of a Cdc42-specific inhibitor. In some embodiments, the ratio of circulating cytokine levels in the blood progenitor cells is less than 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 after the administration, or less than about 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 after administration of a Cdc42-specific inhibitor.In some embodiments, the ratio of circulating cytokine levels in blood progenitor cells is at least or at least about 0.8, 0.9, 1.0, 1.1, 1.2 or more after administration of a Cdc42-specific inhibitor.

[0237] In some embodiments, subjects are selected for treatment with a Cdc42-specific inhibitor based on the methylation status of one or more CpG sites. A CpG site, or CG site, is a region of DNA containing a cytosine nucleotide followed by a guanine nucleotide in a linear sequence along the 5' to 3' direction. The cytosine in the CpG dinucleotide is methylated by DNA methyltransferase to form 5-methylcytosine. Methylation of cytosines within genes can alter their expression and is associated with epigenetics and gene regulation. In some embodiments, methylation of cytosines within CpG sites is used to select subjects for treatment with a Cdc42-specific inhibitor. Because age also has a strong influence on the DNA methylation level of cytosines within CpG sites, a highly precise biological clock (called the epigenetic clock or DNA methylation age) can be defined for each subject. In some embodiments, the "epigenetic clock" is used to select subjects for treatment with a Cdc42-specific inhibitor.

[0238] In some embodiments, the CpG site is within one or more of the Prima1, Hsf4, Kcns1 genes, and combinations thereof. In some embodiments, the methylation status of the CpG site within one or more of the Prima1, Hsf4, Kcns1 genes, and combinations thereof is determined before the first administration of a Cdc42-specific inhibitor, or before a subsequent administration (e.g., determining the level before the first administration but not before the second administration, or determining the level before the first administration and before the second administration, or determining the level before the first administration but not before the second administration, but determining the level before the third administration). In some embodiments, the methylation status of the CpG site in one or more of the Prima1, Hsf4, Kcns1 genes, and combinations thereof is used to select subjects for treatment with a Cdc42-specific inhibitor. In some embodiments, it may be beneficial to determine the methylation status of CpG sites in one or more of the Prima1, Hsf4, and Kcns1 genes, and combinations thereof, before each administration of a Cdc42-specific inhibitor (e.g., determining cytokine methylation levels before the first and second administrations, or determining cytokine methylation levels before the first, second, and third administrations). In some embodiments, it may be beneficial for a physician to utilize information gathered from determining the methylation status of CpG sites in one or more of the Prima1, Hsf4, and Kcns1 genes, and combinations thereof, to prepare a patient-specific regimen or provide a patient-specific treatment. Thus, in some embodiments, the regimen or administration of a Cdc42-specific inhibitor is determined or repeated based on the methylation status of CpG sites in one or more of the Prima1, Hsf4, and Kcns1 genes, and combinations thereof.

[0239] A threshold value of the methylation status of one or more CpG sites can be used to identify a subject for treatment or to determine an appropriate regimen or administration of a Cdc42-specific inhibitor. In some embodiments, the methylation status of one or more CpG sites in a subject's Prima1, Hsf4, Kcns1 genes, and combinations thereof, is used to identify a subject for treatment or to determine an appropriate regimen or administration of a Cdc42-specific inhibitor. In some embodiments, the methylation status of one or more CpG sites in a subject is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 0%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, or approximately any of the foregoing percentages, or a range bracketed by any of the foregoing percentages (e.g., approximately 1%-30%, approximately 5%-25%) , about 5% to 20%, about 5% to 15% or 1% to 30%, 5% to 25%, 5% to 20%, 5% to 15%), 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20 %, 20%~100%, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, 50%~90%, 50%~80%,If the Cdc42-specific inhibitor expression level is 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, 90%-100%, approximately any of the aforementioned percentage ranges (e.g., about 10%-70%, about 30%-60%, or about 50%-70%), or about 25%, about 30%, about 40%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 100%, about 105%, about 110%, about 115%, about 120%, or about 125%, then the subject is selected for treatment with a Cdc42-specific inhibitor. In some embodiments, the one or more CpG sites are within Prima1, Hsf4, Kcns1 genes, and combinations thereof. In some embodiments, the methylation status referred to is cytosine methylation. In some embodiments, the control population is based on age. In some embodiments, the control population is 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 years old, or a range bracketed by any of the foregoing ages (e.g., 18-20, Ages equal to or greater than 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years, or greater than 18-20, 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years.

[0240] In some embodiments, the methylation status of one or more CpG sites in a subject is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 116%, 117%, 118%, 119%, 220%, 221%, 222%, 230%, 240%, 250%, 265%, 270%, 275%, 280%, 285%, 291%, 302%, 303%, 304%, 305%, 306%, 307%, 308%, 309%, 3109%, 3110%, 3120%, 3121%, 3130%, 3131%, 3140%, 3142%, 3143%, 3144%, 3155%, 3156%, 3157%, 3158%, 3160%, 3161%, 3162%, 3163%, 3164%, 3165%, 3166%, 3167%, 3168%, 3169%, 3170%, 3171%, 3172%, 3173%, 3174%, 3175%, 3176%, 20%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, approximately any of the aforementioned percentages, or a range enclosed within any of the aforementioned percentages (e.g., approximately 1% to 30%, approximately 5% to 25%, approximately 5% to 20%, approximately 5% to 15%, or 1% to 30%, 5% to 25%, 5% to 20%, 5% to 15%), 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% ~20%, 1%~10%, 10%~100%, 10%~90%, 10%~80%, 10%~70%, 10%~70%, 10%~60%, 10%~50%, 10%~40%, 10%~30%, 10%~20%, 20%~100%, 20%~90%, 20%~80%, 20%~70% , 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40 % to 60%, 40% to 50%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 100%, 70% to 90%, 70% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, approximately any of the aforementioned percentage ranges (e.g., about 10% to 70%, about 30% to 60%, or about 50% to 70%), or about 25%, about 30%, about 40%, about 50%, about 55%, about 60%, about 70%, about 80%, about 90%, about 100%,If the Cdc42-specific inhibitor is about 105%, about 110%, about 115%, about 120%, or about 125%, the regimen or administration of the Cdc42-specific inhibitor is repeated. In some embodiments, the one or more CpG sites are within Prima1, Hsf4, Kcns1 genes, and combinations thereof. In some embodiments, the methylation status referred to is cytosine methylation. In some embodiments, the control population is based on age. In some embodiments, the control population is 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 years old, or a range bracketed by any of the foregoing ages (e.g., 18-20, Ages equal to or greater than 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years, or greater than 18-20, 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years.

[0241] In some embodiments, the methylation status of one or more CpG sites in the subject is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 115%, 120%, 130%, 140%, 155%, 160%, 170%, 180%, 195%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, 1000%, 1150%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1950%, 2000%, 2100%, 2200%, 2300%, 2400%, 2500%, 2600%, 2700%, 2800%, 2900%, 3000%, 3500%, 4000%, 4500%, 5000%, 5500%, 00%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, approximately any of the foregoing percentages, or a range enclosed within any of the foregoing percentages (e.g., approximately 1% to 30%, approximately 5% to 25%, approximately 5% to 20%, approximately 5% to 15% or 1% to 30%, 5% to 25%, 5% to 20%, 5% to 15%), 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1%~40%, 1%~30%, 1%~20%, 1%~10%, 10%~100%, 10%~90%, 10%~80%, 10%~70%, 10%~70%, 10%~60%, 10%~50%, 10%~40%, 10%~30%, 10%~20%, 20%~100%, 20 %~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~9 0%, 40%-80%, 40%-70%, 40%-60%, 40%-50%, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, 90%-100%, approximately any of the aforementioned percentage ranges (e.g., about 10%-70%, about 30%-60%, or about 50%-70%), or about 25%, about 30%, about 40%, about 50%, about 55%, about 60%,If the Cdc42-specific inhibitor is about 70%, about 80%, about 90%, about 100%, about 105%, about 110%, about 115%, about 120%, or about 125%, the regimen or administration of the Cdc42-specific inhibitor is repeated. In some embodiments, the one or more CpG sites are within Prima1, Hsf4, Kcns1 genes, and combinations thereof. In some embodiments, the methylation status referred to is cytosine methylation.

[0242] In some embodiments, an effective amount of a Cdc42-specific inhibitor reduces or decreases the methylation state of one or more CpG sites in a subject. In some embodiments, a regimen or administration of an effective amount of a Cdc42-specific inhibitor reduces or decreases the methylation state of one or more CpG sites in a subject by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or 30% or more compared to the methylation state of one or more CpG sites in the subject before the first administration of a Cdc42-specific inhibitor to the subject or the last administration of a Cdc42-specific inhibitor to the subject. 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, approximately any of the foregoing percentages, or a range bracketed by any of the foregoing percentages (e.g., approximately 1% to 30%, approximately 5% to 30%). 25%, approximately 5% to 20%, approximately 5% to 15% or 1% to 30%, 5% to 25%, 5% to 20%, 5% to 15%), 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 20% to 90%, 20% to 80%, 20% to 70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~40%, 40%~100%, 40%~90%, 40%~80%, 40%~70%, 40%~60%, 40%~50%, 50%~100%, 50%~90%, 50%~80%, 50%~70%, 50%~60%, 60%~100%, 60%~90%, 60%~80%, 60%~70%, 70%~100%, 70%~90%,The increase or elevation may be 70% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, or approximately any of the aforementioned percentage ranges (e.g., about 10% to 70%, about 30% to 60%, or about 50% to 70%). In some embodiments, the one or more CpG sites are within Prima1, Hsf4, Kcns1 genes, and combinations thereof. In some embodiments, the methylation state referred to is cytosine methylation.

[0243] In some embodiments, a regimen or administration of an effective amount of a Cdc42-specific inhibitor increases the methylation status of one or more CpG sites in a subject by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 14 5%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 225%, 250%, 300%, 350%, approximately any of the foregoing percentages, or a range bracketed by any of the foregoing percentages (e.g., approximately 1% to 30%, approximately 5% to 25%, approximately 5% to 20%, approximately 5% to 15%, or 1% to 30%, 5% to 25%, 5% to 20%, 5% to 15%), 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1%~60%, 1%~50%, 1%~40%, 1%~30%, 1%~20%, 1%~10%, 10%~100%, 10%~90%, 10%~80%, 10%~70%, 10%~70%, 10%~60%, 10%~50%, 10%~40%, 10%~30%, 10%~ 20%, 20%~100%, 20%~90%, 20%~80%, 20%~70%, 20%~60%, 20%~50%, 20%~40%, 20%~30%, 30%~100%, 30%~90%, 30%~80%, 30%~70%, 30%~60%, 30%~50%, 30%~4 A decrease or reduction of 0%, 40% to 100%, 40% to 90%, 40% to 80%, 40% to 70%, 40% to 60%, 40% to 50%, 50% to 100%, 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 60% to 90%, 60% to 80%, 60% to 70%, 70% to 100%, 70% to 90%, 70% to 80%, 80% to 100%, 80% to 90%, 90% to 100%, or about any of the aforementioned percentage ranges (e.g., about 10% to 70%, about 30% to 60%, or about 50% to 70%).In some embodiments, the control population is based on age. In some embodiments, the control population is 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 years old, or a range bracketed by any of the foregoing ages (e.g., 18-20, The age is equal to or greater than 25-35, 50-80, 50-70, 50-60, 55-75, 55-75, 55-70, 55-65, 60-70, 52-71, 60-79, or 73-78 years. In some embodiments, the one or more CpG sites are within Prima1, Hsf4, Kcns1 genes, and combinations thereof. In some embodiments, the methylation status referred to is cytosine methylation.

[0244] Procedural or other details supplementary to those set forth herein may be found in the cited references, which are specifically incorporated herein by reference.

[0245] The various methods and techniques described above provide several ways of implementing the present invention. Of course, it should be understood that not all of the described objectives or advantages can be achieved in accordance with the particular embodiments described herein. Thus, for example, one skilled in the art will recognize that a method can be implemented in a way that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other objectives or advantages that may be taught or suggested herein.

[0246] Moreover, one skilled in the art will recognize the interchangeability of various features from different embodiments. Similarly, the various features and steps discussed above, and other known equivalents of each such feature or step, can be mixed and matched by one of ordinary skill in the art to perform a method according to the principles described herein.

[0247] While the present invention has been disclosed in the context of particular embodiments and examples, it will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses, as well as obvious modifications and equivalents thereof. Accordingly, the present invention is not intended to be limited by the specific disclosures of preferred embodiments herein, but instead by reference to the claims appended hereto.

[0248] The following examples provide illustrations of some of the embodiments described herein, but are not intended to limit the invention. [Example]

[0249] General experimental procedure Unless otherwise stated, the following general procedures were applied:

[0250] Mice. Mice included in the study were female C57BL / 6 and were obtained from an internal split stock (C57BL / 6j mice obtained from both The Jackson Laboratory and NIA / Charles River, and C57BL / 6JRj mice obtained from Janvier). For the lifespan study described herein, 40 mice were randomly selected at 70 weeks of age. Mice were weighed and bled before treatment initiation (day 0) and on days 7 and 35. Mice that did not recover from blood collection and mice that died due to laboratory error were excluded. Mice that had to be euthanized because they were scored as "weak and dying" according to the approved animal license protocol for assessing mouse health remained part of the dataset. Allocation to control or treatment groups was random (20 mice per experimental group).

[0251] Median lifespan, 95% confidence intervals, and survival analyses were calculated using Prism GraphPad v7.0c. Notably, the median lifespan of the control group was consistent with data obtained for C57BL / 6j mice in a large set of lifespan studies of diverse inbred mouse strains. Young C57BL / 6 mice were 10 weeks old and housed in the same room and setting as the aged mice. Animals were maintained in accordance with the recommendations of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS123). Animals were housed in groups of up to four animals per cage in Macrolon Type II (long) cages provided with bedding and paper nesting material. Animals had free access to food (V1124-3, ssniff®) and water. Animals were maintained on a 12 / 12-h day / night rhythm throughout the experiment. Mouse experiments were performed in accordance with European and German laboratory animal welfare laws and according to approved protocols approved by the Institutional Review Board of Ulm University and the Regierungspraesidium Tuebingen, Baden-Württemberg.

[0252] CASIN solution and IP treatment. CASIN was freshly prepared prior to injection by dissolving the drug directly in beta-cyclodextrin solution (Sigma #H5784). IP injections were performed every 24 hours in the morning for 4 consecutive days during the 75-week age of the mice. Control mice were injected with an equal volume of vehicle. Serum for cytokine arrays was prepared from the day 7 bleed. Serum for mass spectrometry analysis of CASIN levels was prepared by bleeding equal cohorts of mice (sex, strain, origin, and age) 3, 24, and 48 hours after the end of treatment. The same CASIN stock was used throughout the study. CASIN was provided as a lyophilized powder.

[0253] Peripheral blood (PB) flow cytometry. Immunostaining of PB cells was performed according to standard procedures, and samples were analyzed on an LSRII flow cytometer (BD Biosciences). For PB lineage analysis, antibodies used were obtained from eBioscience: anti-CD3ε (clone 145-2C11), anti-B220 (clone RA3-6B2), anti-Mac-1 (clone M1 / 70), and anti-Gr-1 (clone RC57BL / 6-8C5). Lineage FACS analysis data were plotted as the percentage of B220+, CD3+, and myeloid (Gr-1+, Mac-1+, and Gr-1+Mac-1+) cells among total leukocytes. WBC (white blood cells), RBC (red blood cells), Ly (lymphocytes), NE (neutrophils), and Mo (monocytes) were counted using a hemocytometer Hemavet950, DREWSCIENTIFICInc, (FL33014), USA.

[0254] Liquid chromatography-mass spectrometry (LC-MS / MS) measurement of CASIN pharmacokinetics in mouse serum. Using an LC / MS / MS ion chromatography protocol, serum CASIN concentrations ranging from 0.1 to 10 μM were measured from mice injected i.p. at various times and compared with a standard curve obtained by spiking mouse serum in vitro with a defined dose of CASIN. A capped autosampler maximum recovery vial (Waters), a UPLC-MS system (Waters Quattro Premier XE Mass Spectrometer with ACQUITY UPLC System), an Acquity BEHC18 UPLC column (2.1 x 75 mm, 1.7 μm) (Waters), and an Acquity BEHC18 UPLC guard column (Waters) were used. For the LC gradient, solvent A contained acetonitrile / water (5 / 95) with 2 mM ammonium acetate, and solvent B contained acetonitrile / water (90 / 10). The gradient mobile phase was 60% solvent A to 57% solvent A in 2 min, 50% solvent A in 2.1 min, 46% solvent A in 9.9 min, and then changed to 30% solvent A in 12.1 min, followed by 1% solvent A over 5.9 min, then 60% solvent A in 18.1 min, which was held for 2 min. The column temperature was maintained at 25 °C. The electrospray negative (ES-) mode capillary voltage was 3 kV, the cone voltage was 45 V, the collision voltage was 30 V, and the desolvation temperature was 350 °C. The desolvation gas flow rate was 600 L / h, the source temperature was 120 °C, and the MRM transition m / z was 305.1 to 244.

[0255] Cytokine array. Lumimex method. Cytokine concentrations in sample supernatants were determined using the Milliplex™ Multiplex Kit (MilliporeSigma, Darmstadt, Germany) according to the manufacturer's protocol. Briefly, 25 μL of duplicate samples were incubated with 25 μL of antibody-coated beads in a 96-well black plate overnight at 4°C on a plate shaker. Next, the plate was washed twice using a BioTek 405TS (BioTek, Winooski, VT), and 25 μL of secondary antibody was added and incubated for 1 hour at room temperature with shaking. Finally, 25 μL of streptavidin-RPE was added directly to the secondary antibody and incubated for 30 minutes at room temperature with shaking. The plate was then washed two more times, and 150 μL of sheath fluid was added. The plate was shaken for 5 minutes before being read using luminex technology on a Milliplex Analyzer (MilliporeSigma, Darmstadt, Germany). Concentrations were calculated from a standard curve using recombinant proteins and are expressed in pg / ml.

[0256] Cdc42-GTPase effector domain pull-down assay. The relative levels of GTP-bound Cdc42 were determined by effector pull-down assay. Briefly, lineage-depleted BM cells (10 6 ) in 10% glycerol, 25 mM sodium fluoride, 1 mM sodium orthovanadate, and Mg containing protease inhibitor cocktail (Roche Diagnostics). 2+ Cells were lysed in lysis / wash buffer (Upstate Cell Signaling Solution). Samples were incubated with PAK-1 binding domain / agarose beads, and bound (activated) and unbound (non-activated) Cdc42 fractions were probed by immunoblotting with anti-Cdc42 antibody (Millipore, rabbit polyclonal). Activated protein was normalized to total protein and / or β-actin (Sigma), and relative amounts were quantified by densitometry.

[0257] Analysis of epigenetic signs of aging. CASIN (Xcessbio #M60040) was dissolved in DMSO at a concentration of 100 mM and freshly prepared before injection by diluting with cyclodextrin solution (Sigma #H5784). IP injections of 25 mg / kg were administered for four consecutive mornings starting at 77–86 weeks of age. Control mice were injected with an equal volume of vehicle. Mice were sacrificed 8–9 weeks after treatment, and blood was collected by cardiac puncture. DNA methylation levels were analyzed at three age-associated CpG dinucleotides (CpGs) as previously described. Briefly, genomic DNA was isolated from blood samples, bisulfite converted, and DNA methylation was analyzed within three genes (Prima1, Hsf4, and Kcns1) by pyrosequencing. DNA methylation results at these sites were integrated into a multivariate model for epigenetic age prediction in B6 mice and correlated with chronological age. Chronological and biological ages of CASIN-treated and control mice were compared using unpaired t-tests. Because the data were not normally distributed, all data were augmented to obtain a normal distribution before statistical analysis was performed.

[0258] Example 1 To determine whether short-term systemic treatment of aged animals with a Cdc42-specific inhibitor modulates lifespan, CASIN was administered intraperitoneally to 75-week-old female C57BL / 6 mice every 24 h for four consecutive days (Figure 1a). Serum CASIN levels were monitored by liquid chromatography-mass spectrometry (Figure 2a and Figure 2b). A single dose of 50 mg / kg initially resulted in serum concentrations of approximately 10 μM (data not shown). Therefore, a dose of 25 mg / kg was selected, and mass spectrometry data from the serum of aged mice showed CASIN levels in the expected range of 5 μM 3 h after injection (Figure 2b). CASIN concentrations decreased over the 48-h analysis window, with concentrations of approximately 2 μM detected after 3 h and approximately 1 μM after 2 h. By the end of the 48-h window, virtually no CASIN remained in serum.

[0259] Continuous injections over four days did not induce acute toxicity, and no treated mice died within four weeks of CASIN injection. Therefore, chronic toxicity due to the administration of the inhibitor is unlikely. Quantification of Cdc42 activity 24 hours after the final injection on day 5 showed that Cdc42-GTP in senescent bone marrow cells had decreased to levels seen in young mice (Figure 1c), confirming that CASIN indeed reduced Cdc42 activity after systemic in vivo treatment. As shown in Figure 1c, CASIN treatment reduced Cdc42 activity by approximately half, regardless of the age of the mice. Furthermore, CASIN treatment reversed Cdc42 activity in the aged mouse cohort to approximately the level seen in the young control cohort. Thus, CASIN treatment generally restored Cdc42 activity levels in aged mice to those present in young mice at approximately 60 weeks of age.

[0260] Notably, aged mice treated with CASIN for four consecutive days showed both mean and maximal lifespan extensions (Figure 1d). Thus, treatment with a Cdc42-specific inhibitor had a striking effect on lifespan extension, despite its removal from the bloodstream immediately after cessation of drug treatment (e.g., long-term effects of short-term treatment), thereby demonstrating a durable benefit from drug treatment.

[0261] Also noteworthy was that weight (Figure 2c), white blood cell (WBC) count (Figure 2d), red blood cell (RBC) count (Figure 2e), and the frequency and number of myeloid and lymphoid cells in peripheral blood (PB) remained unchanged in response to CASIN treatment, both short-term (e.g., day 7 after initiation of treatment) and long-term (e.g., day 35 after initiation of treatment) (Figure 2f-Figure 2k). Cytokine array analysis was performed to investigate the extent to which aging-associated inflammatory cytokines in the serum of aged mice were affected by CASIN treatment. The data showed a marked increase in the concentrations of INFγ, IL-1β, and IL-1α with age, and the concentrations of these cytokines were similar to those of young animals when aged animals were treated with CASIN (Figure 1e-Figure 1g). Therefore, CASIN treatment reduced the concentrations of these cytokines, and without being bound by theory, this decrease in inflammatory cytokines with age may contribute to the extended lifespan observed in these animals. Interestingly, IL-9 was the only cytokine whose serum concentration increased after CASIN treatment (Fig. 1h). The concentrations of IL-6, IL-2, GM-CSF, LIF, IL-4, and IL-7 tended not to increase significantly in aged mice. Although some (GM-CSF and IL-2) were decreased by CASIN, the concentrations of most cytokines were unchanged by aging or CASIN treatment (Fig. 3).

[0262] The DNA methylation status of CpG (5'-C-phosphate-G-3') sites within the Prima1, Hsf4, and Kcns1 genes may be a predictor of biological age in C57BL / 6 mice. Therefore, DNA methylation profiling of these CpGs was investigated as a biological epigenetic clock. This C57BL / 6-trained DNA methylation marker panel was applied to blood cells of aged CASIN-treated animals 9 weeks after treatment, and epigenetic age predictions were observed to no longer correlate with chronological age as in aged control animals. Instead, treatment with a Cdc42-specific inhibitor resulted in biological age predictions that were, on average, 9 weeks younger than the mice's chronological age (Figure 1i). Therefore, without being bound by any theory, mice treated with a Cdc42-specific inhibitor (e.g., CASIN) exhibit epigenetic changes that may influence the observed extended lifespan of aged CASIN-treated mice.

[0263] Furthermore, many age-related diseases, such as obesity, metabolic syndrome, diabetes, cardiovascular disease, cancer, depression, and Alzheimer's disease, share an inflammatory etiology, and activation of inflammatory pathways appears to be involved in the pathophysiology of sarcopenia and frailty. Without being bound by theory, the elevated levels of interferon-γ detected in these experiments may be a hallmark of youthfulness and functionally related to longevity. Similarly, both interleukin-1α and interleukin-1β are associated with many age-related phenotypes and diseases. Regarding IL-9, whose serum levels were elevated after CASIN treatment, IL-9 is a pleiotropic cytokine primarily produced by T helper 9 (Th9) cells and exerts well-documented effects on lymphocytes, mast cells, and resident lung cells. Without being bound by any theory, the data presented herein suggest a role for elevated levels of these cytokines in regulating longevity. Data further reveal that the methylation status of CpG sites within the Prima1, Hsf4, and Kcns1 genes in blood cells strongly correlates with biological age and serves as a validated biomarker of aging in C57BL / 6 mice.

[0264] As mentioned above, the data herein demonstrate that reducing Cdc42 activity in aged mice actually extends lifespan. Notably, inhibition of Cdc42 activity with CASIN and inhibition of mTOR with rapamycin are the only reported pharmacological treatments that significantly affect mouse lifespan when administered late in life (over 75 weeks of age) and only transiently (only 4 days for CASIN and 90 days for rapamycin). This indicates that the mechanism of action of CDC42-specific inhibitors differs from other longevity pharmacological interventions, which are typically administered early in life and / or continuously provided for extended periods to achieve significant effects.

[0265] Example 2 The experimental setup for the vaccination response experiment is shown in Figure 5(A). Briefly, 75-week-old C57BL / 6 mice (referred to as "old" in the results) and 12- to 16-week-old mice (referred to as "young" in the results) were intraperitoneally (IP) injected with the desired dose (e.g., 25 mg / kg) of a Cdc42-specific inhibitor (e.g., CASIN) or vehicle (referred to as "vehicle" in the results) as a control every 24 hours for 4 days. On day 5, the mice were treated with 5-fluorouracil (5-FU). Two methods of vaccination were investigated.

[0266] For DNA vaccination: 12 weeks after transplantation, recipient mice were immunized with pCI / C DNA encoding the Hepatitis B virus ("HBV") core antigen. Figure 5(B) shows the results of DNA vaccination with HBV core antigen 13 days after immunization, as well as the ovalbumin ("Ova")-specific immune response. Interferon-gamma-positive CD3 + CD8 + We analyzed the frequency of T cells, which is an established surrogate measure for the success of a DNA vaccination protocol. As Figure 5(B) shows, all mice showed interferon-gamma-positive CD3 T cells after immunization with HBV core antigen. + CD8 +Aged mice treated with CASIN showed a statistically significant increase in immune response. n = at least 3 per experiment. *p<0.05 Aged vehicle vs. Aged CASIN. In Figure 5(C), splenic K b / C 93-100 -Dimer + CD8 + T cell frequencies were determined by flow cytometry 13 days after immunization with HBV core antigen. These T cell frequencies are a direct measure of antigen-specific T cells. Aged mice treated with CASIN showed a statistically significant increase in antigen-specific T cells. n = at least 3 per experiment. *p<0.05 Aged vehicle vs. Aged CASIN.

[0267] For viral vaccination, animals were immunized twice with inactivated influenza virus (10 μg H3N2 + 10 μL Al(OH)3) via intramuscular injection 12 weeks after CASIN treatment, with a 4-week interval. Antibody titers were determined in serum 21 days after the second vaccination using ELISA techniques. n = at least 3 per experiment. These results are shown in Figure 5(D), which demonstrates increased antibody titers in CASIN-treated aged mice compared with vehicle-treated aged mice in the control cohort. This result suggests that CASIN-treated aged mice exhibited antibody titers approximately equivalent to those observed in young mice, regardless of CASIN treatment within that cohort.

Claims

1. 1. A composition comprising at least one Cdc42-specific inhibitor for use in a method of extending the lifespan of a subject, the method comprising administering to the subject an effective amount of at least one Cdc42-specific inhibitor, wherein the subject is selected based on the methylation status of a CpG site in a gene selected from the group consisting of Prima1, Hsf4, and Kcns1, and wherein the effective amount of the Cdc42-specific inhibitor reduces the amount of one or more circulating inflammatory cytokines selected from the group consisting of interferon-γ, interleukin-1α, and interleukin-1β in the subject, and wherein the at least one Cdc42-specific inhibitor is selected from the group consisting of: 【Chemical 1】 , composition.

2. The composition of claim 1 , wherein the administration of the Cdc42-specific inhibitor is performed two or more times.

3. 3. The composition of claim 1 or 2, wherein the Cdc42 activity in the subject is determined prior to administering the Cdc42-specific inhibitor.

4. The composition of any one of claims 1 to 3, wherein the effective amount of the Cdc42-specific inhibitor increases the amount of circulating interleukin-9 in the subject.

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