Combination therapy comprising btk inhibitor and besudil

Abstract

Disclosed herein are methods of treating a disease selected from the group consisting of graft versus host disease (GVHD), systemic sclerosis (scleroderma), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), and obliterated bronchitis syndrome (BOS), using a combination of a BTK inhibitor, such as rezobrutinib, and besudil.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 513,952, filed July 17, 2023, and European Application No. 23214297.6, filed December 5, 2023, the disclosure of each of which is incorporated herein by reference in its entirety for any purpose. TECHNICAL FIELD

[0002] The present disclosure relates to methods of treating a disease selected from graft versus host disease (GVHD), systemic sclerosis (scleroderma), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), and bronchiolitis obliterans syndrome (BOS) using a combination of a BTK inhibitor, such as rilzabrutinib, and belumosudil. BACKGROUND

[0003] Bruton’s tyrosine kinase (BTK) is a cytoplasmic non-receptor tyrosine kinase belonging to the Tec kinase family. BTK acts downstream of the B-cell receptor (BCR) and is essential for B-cell lineage maturation and functional antibody production, and inhibition of BTK activity in cells produces phenotypic changes consistent with BCR blockade. BTK inhibition leads to downregulation of various B-cell activities, including cell proliferation, differentiation, maturation, and survival, as well as upregulation of apoptosis. Thus, BTK inhibitors have been investigated as therapeutic agents for treating various cancers. However, BTK also plays a critical role in signaling pathways associated with autoimmune and immune-mediated diseases. For example, BTK inhibition can suppress the production of autoantibodies that are thought to play an important role in the development of certain autoimmune diseases. In addition, BTK plays a role in the activation of innate immune cells such as macrophages and neutrophils, which are key players in inflammation. Thus, BTK inhibitors have the potential to target multiple pathways involved in inflammation and autoimmunity.

[0004] Belumosudil is an oral, selective Rho-associated coiled-coil containing protein kinase-2 (ROCK2) inhibitor. ROCK2 inhibition acts on a dysregulated adaptive immune system and fibrosis that occurs due to aberrant tissue repair. Studies have shown that belumosudil downregulates proinflammatory responses and also inhibits aberrant pro-fibrotic signaling. The FDA approved belumosudil (REZUROCK®) in 2021 for the treatment of cGVHD after failure of at least two prior systemic therapies. ®

[0005] ​Combination therapy is an attractive option for disease treatment, particularly with therapeutic agents that target different pathways. Accordingly, provided herein are methods of treating diseases such as graft versus host disease (GVHD), systemic sclerosis (scleroderma), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), and bronchiolitis obliterans syndrome (BOS) using a combination of a BTK inhibitor and bexarotene. SUMMARY

[0006] Described herein are methods of treating a disease selected from graft versus host disease (GVHD), systemic sclerosis (scleroderma), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), and bronchiolitis obliterans syndrome (BOS) using a combination of a BTK inhibitor and bexarotene.

[0007] Exemplary embodiments include the following.

[0008] Example 1. A method of treating a disease or condition selected from systemic sclerosis and transplant-related dysfunction in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

[0009] Example 2. A method of treating a disease or condition selected from graft versus host disease (GVHD), systemic sclerosis (scleroderma), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), and bronchiolitis obliterans syndrome (BOS) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

[0010] Example 3. A method of treating graft versus host disease (GVHD) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

[0011] Example 4. A method of treating systemic sclerosis (scleroderma) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

[0012] Example 5. A method of treating chronic lung allograft dysfunction (CLAD) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

[0013] Example 6. A method of treating restrictive allograft syndrome (RAS) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

[0014] Example 7. A method of treating bronchiolitis obliterans syndrome (BOS) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

[0015] Example 8. The method of any one of Examples 1-7, wherein the BTK inhibitor is a reversible BTK inhibitor.

[0016] Example 9. The method of any one of Examples 1-7, wherein the BTK inhibitor is an irreversible BTK inhibitor.

[0017] Embodiment 10. The method of any one of embodiments 1-9, wherein the BTK inhibitor is (i) (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2- enenitrile or a pharmaceutically acceptable salt thereof; (ii) 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1- piperidinyl]-2-propen-1-one or a pharmaceutically acceptable salt thereof; or (iii) (4-amino-3-(4-phenoxyphenyl)-1-[(3R)-1-(prop-2-enoyl)piperidin-3-yl]-1,3-dihydro-2H- imidazo[4,5-c]pyridin-2-one) or a pharmaceutically acceptable salt thereof.

[0018] Embodiment 11. The method of embodiment 10, wherein the BTK inhibitor is (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2- enenitrile or a pharmaceutically acceptable salt thereof.

[0019] Embodiment 12. A method of treating graft-versus-host disease (GVHD) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2- enenitrile or a pharmaceutically acceptable salt thereof, and (b) 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide or a pharmaceutically acceptable salt thereof.

[0020] Embodiment 13. The method of any one of embodiments 2, 3, and 12, wherein GVHD is chronic GVHD (cGVHD).

[0021] Embodiment 14. The method of any one of embodiments 1-13, wherein 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide or a pharmaceutically acceptable salt thereof is administered to the human patient at a daily dose of up to about 400 mg.

[0022] Embodiment 15. The method of embodiment 14, wherein 2-{3-[4-(lH-indazol-5- ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof, is administered to the human patient in a daily dose of about 50-400 mg.

[0023] Embodiment 16. The method of embodiment 14 or 15, wherein 2-{3-[4-(lH-indazol-5- ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof, is administered to the human patient in a dose of about 50 mg, 100 mg, 150 mg, or 200 mg.

[0024] Embodiment 17. The method of embodiment 16, wherein the dose is administered to the human patient once daily or twice daily.

[0025] Embodiment 18. The method of any one of embodiments 1-17, wherein 2-{3-[4-(lH-indazol-5- ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof, is administered orally.

[0026] Embodiment 19. The method of any one of embodiments 10-18, wherein (R)-2-[3-[4-amino-3-(2- fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient in a daily dose of up to about 800 mg.

[0027] Embodiment 20. The method of embodiment 19, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient in a daily dose of about 50-800 mg.

[0028] Embodiment 21. The method of embodiment 19 or 20, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient in a dose of about 100 mg, 200 mg, or 400 mg.

[0029] Embodiment 22. The method of embodiment 21, wherein a dose of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2- enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient once per day or twice per day.

[0030] Embodiment 23. The method of any one of embodiments 10-22, wherein a (E) isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2- enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient.

[0031] Embodiment 24. The method of any one of embodiments 10-22, wherein a (Z) isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2- enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient.

[0032] Embodiment 25. The method of any one of embodiments 10-22, wherein a mixture of (E) and (Z) isomers of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2- enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient.

[0033] Embodiment 26. The method of any one of embodiments 10-25, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2- enenitrile, or a pharmaceutically acceptable salt thereof, is administered orally.

[0034] Example 27. The method of any one of Examples 10-26, wherein (R)-2-[3-[4-amino-3- (2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4- methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, and 2-{3-[4-(1H-indol-5-ylamino)-2-quinolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof, are administered separately.

[0035] Example 28. The method of Example 27, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, and 2-{3-[4-(1H-indol-5-ylamino)-2-quinolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof, are administered sequentially.

[0036] Example 29. The method of any one of Examples 10-28, wherein (R)-2-[3-[4-amino-3- (2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4- methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, and 2-{3-[4-(1H-indol-5-ylamino)-2-quinolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof, are administered simultaneously. BRIEF DESCRIPTION OF DRAWINGS

[0037] The novel features of the application are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present application will be obtained by reference to the following detailed description that sets forth illustrative

[0038] Figure 1 shows flow cytometry analysis of the B cell function assay described in Example 1. Figure 1A Activated B cells stimulated with anti-CD79b only are shown. Figure 1B Activated B cells after anti-CD79b stimulation and treatment with 10 µM rizabrutinib are shown, and Figure 1C Activated B cells after anti-CD79b stimulation and treatment with 10 µM besutifin are shown.

[0039] Figure 2Dose-response plots for besuxibulin and rizabrutinib in the anti-CD79b-mediated B-cell activation assay described in Example 1 are shown.

[0040] Figure 3A Average GVHD scores in a GVHD mouse model measured daily from the start of treatment with rizabrutinib and ibrutinib at the indicated doses on day 21 after the start of disease until day 56 (start of dosing indicated by vertical dashed line) are shown, as described in Example 2. Figure 3B Total GVHD burden in vehicle, rizabrutinib, and ibrutinib treatment groups of the same sclerodermic GVHD model, expressed as AUC of daily scores per group, is shown, as described in Example 2.

[0041] Figure 4 Animal survival monitored daily over the duration of the study described in Example 4 is shown.

[0042] Figure 5A Body weight change of animals treated in the study described in Example 4 is shown. Animals were weighed daily and body weight change compared to day 0 was calculated. AUC was calculated from day 0 until day 56 using the trapezoidal rule and shown on the right. Statistical significance between groups was determined by one-way ANOVA and Dunnett’s multiple comparison test to compare all groups to the GVHD vehicle-control group. p < 0.01. Data presented as mean ± SEM. n = 6-12 per group.

[0043] Figure 5B Body weight change of animals treated in the study described in Example 4 - carried over to death weight. Animals were weighed daily and body weight change compared to day 0 was calculated. Data show the body weight at which the carried over animals were found dead or euthanized over the duration of the study. AUC was calculated from day 0 until day 56 using the trapezoidal rule and shown on the right. Statistical significance between groups was determined by one-way ANOVA and Dunnett’s multiple comparison test to compare all groups to the GVHD vehicle-control group. p < 0.05. Data presented as mean ± SEM. n = 6-12 per group.

[0044] Figure 6AGVHD score-standard score for animals treated in the study described in Example 4 are shown. Animals were scored daily according to the protocol shown in Table 4. AUC was calculated from Day 0 through the end of the study on Day 56 using the trapezoidal rule and is shown on the right. Statistical significance was determined by Kruskal-Wallis and Dunn’s post-test to compare all groups to the GVHD vehicle-control group. Data are presented as mean ± SEM. p < 0.01. n = 6-12 per group.

[0045] Figure 6B GVHD score-standard score-death score carryover for animals treated in the study described in Example 4 are shown. Animals were scored daily according to the protocol shown in Table 4. Data show the GVHD score at which carryover animals were found dead or euthanized over the duration of the study. AUC was calculated from Day 0 through the end of the study on Day 56 using the trapezoidal rule and is shown on the right. Statistical significance was determined by Kruskal-Wallis and Dunn’s post-test to compare all groups to the GVHD vehicle-control group. Data are presented as mean ± SEM. p < 0.001. n = 6-12 per group.

[0046] Figure 7A GVHD score-modified score for animals treated in the study described in Example 4 are shown. Animals were scored daily according to the protocol shown in Table 5. AUC was calculated from Day 0 through the end of the study on Day 56 using the trapezoidal rule to enable valid comparisons between groups by statistical tests. Statistical significance was determined by Kruskal-Wallis and Dunn’s post-test to compare all groups to the GVHD vehicle-control group. Data are presented as mean ± SEM. p < 0.01. n = 6-12 per group.

[0047] Figure 7B GVHD score-modified score-death score carryover for animals treated in the study described in Example 4 are shown. Animals were scored daily according to the protocol shown in Table 5. AUC was calculated from Day 0 through the end of the study on Day 56 to enable valid comparisons between groups by statistical tests. Statistical significance was determined by Kruskal-Wallis and Dunn’s post-test to compare all groups to the GVHD vehicle-control group. Data are presented as mean ± SEM. p < 0.001. n = 6-12 per group.

[0048] Figure 8A Progression-free survival (standard GVHD scale) tracked over the duration of the study described in Example 4 is shown and plotted as percentage of progression-free survival.

[0049] Figure 8B Progression-free survival (modified scGVHD scale) tracked over the duration of the study described in Example 4 is shown and plotted as percentage of progression-free survival.

[0050] Figure 9A Total histopathology scores for mouse lungs of animals treated in the study described in Example 4 are shown. Group means ± standard error of the mean (SEM). Cell migration is associated with significant disease induction in mouse lungs, with a slight decrease in total histopathology scores observed with ibrutinib or zanabrutinib treatment. Data were analyzed by non-parametric one-way ANOVA and post-hoc Dunn’s multiple comparison test. Indicates p < 0.01.

[0051] Figure 9B Sub-score histopathology scores for mouse lungs of animals treated in the study described in Example 4 are shown. Group means ± standard error of the mean (SEM). Cell migration is associated with significant disease induction in mouse lungs, with a slight decrease in total histopathology scores observed with ibrutinib or zanabrutinib treatment. Data were analyzed by non-parametric one-way ANOVA and post-hoc Dunn’s multiple comparison test. Indicates p < 0.01.

[0052] Figure 10A Total histopathology scores for mouse skin of animals treated in the study described in Example 4 are shown. Group means ± SEM. Total scores for skin were significantly different, with a small number of samples from each group exhibiting much more severe lesions compared to the group mean. The slight decrease in total scores observed with ibrutinib and zanabrutinib was associated with decreased scores for proliferative and fibrotic histopathology (see Figure 10B ). Data were analyzed by non-parametric one-way ANOVA and post-hoc Dunn’s multiple comparison test. Indicates p < 0.01.

[0053] Figure 10B Histopathology scores for mouse skin of animals treated in the study described in Example 4 are shown. Group means ± SEM. Data were analyzed by non-parametric one-way ANOVA and post-hoc Dunn’s multiple comparison test. p < 0.01.

[0054] Figure 11 Dermal thickness measurements of mouse skin of animals treated in the study described in Example 4 are shown. Group means ± SEM. Dermal thickness was measured at 5 sites and averaged for each animal. Although the dermis was thicker in animals that received cell transfer, statistical comparisons between groups were not significant. In animals treated with rezamulin, the slight decrease in dermal thickness corresponded to a decrease in dermal fibrosis score (p < 0.05). Figure 10B Data were analyzed by one-way ANOVA and post-hoc Tukey’s test.

[0055] Figure 12 Animal survival monitored daily over the duration of the study described in Example 5 is shown.

[0056] Figure 13 Body weight change of animals treated in the study described in Example 5 is shown. Animals were weighed daily and body weight change compared to day 0 was calculated. AUC was calculated using the trapezoidal rule transformation and shown on the right. Statistical significance between groups was determined by one-way ANOVA and Tukey’s multiple comparisons post-test to compare all groups. p < 0.0001. Data presented as mean ± SEM. n = 6-12 per group.

[0057] Figure 14 Body weight change of animals treated in the study described in Example 5 - death weight carryover is shown. Animals were weighed daily and body weight change compared to day 0 was calculated. Data show the body weight of carryover animals at the time they were found dead or euthanized over the duration of the study. AUC was calculated using the trapezoidal rule transformation and shown on the right. Statistical significance between groups was determined by one-way ANOVA and Tukey’s multiple comparisons post-test to compare all groups. p < 0.05; p < 0.001. Data presented as mean ± SEM. n = 6-12 per group.

[0058] Figure 15GVHD score-standard score for animals treated in the study described in Example 5 are shown. Animals were scored daily according to the protocol shown in Table 4. AUC was calculated from Day 0 through the end of the study on Day 56 using the trapezoidal rule and shown on the right. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison post-test to compare all groups. Data are presented as mean ± SEM. n = 6-12 per group. p < 0.05. n = 6-12 per group.

[0059] Figure 16 GVHD score-standard score-death score carryover for animals treated in the study described in Example 5 are shown. Animals were scored daily according to the protocol shown in Table 4. Data show the GVHD score at which the carryover animals were found dead or euthanized over the duration of the study. AUC was calculated from Day 0 through the end of the study on Day 56 using the trapezoidal rule and shown on the right. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison post-test to compare all groups. Data are presented as mean ± SEM. n = 6-12 per group.

[0060] Figure 17 GVHD score-modified score for animals treated in the study described in Example 5 are shown. Animals were scored daily according to the protocol shown in Table 5. AUC was calculated from Day 0 through the end of the study on Day 56 using the trapezoidal rule to enable effective comparison of groups by statistical tests. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison post-test to compare all groups. Data are presented as mean ± SEM. n = 6-12 per group.

[0061] Figure 18 GVHD score-modified score-death score carryover for animals treated in the study described in Example 5 are shown. Animals were scored daily according to the protocol shown in Table 5. AUC was calculated from Day 0 through the end of the study on Day 56 to enable effective comparison of groups by statistical tests. Statistical significance was determined by one-way ANOVA and Tukey’s multiple comparison post-test to compare all groups. Data are presented as mean ± SEM. n = 6-12 per group.

[0062] Figure 19 Progression free survival (standard GVHD scale) tracked over the duration of the study described in Example 5 is shown and plotted as percent progression free survival.

[0063] Figure 20 Progression free survival (modified scGVHD scale) tracked over the duration of the study described in Example 5 is shown and plotted as percent progression free survival. Detailed Implementation definition

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. It should be understood that both the general description above and the detailed description below are exemplary and illustrative only and do not limit any of the claimed subject matter. Section headings used herein are for organizational purposes only and should not be construed as limiting the described subject matter. If any material incorporated herein by reference is inconsistent with the express content of this disclosure, the express content shall prevail. In this application, the singular is used to include the plural unless otherwise expressly stated. It must be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used in this specification and the appended claims include plural indicators. In this application, the use of “or” means “and / or” unless otherwise stated. Furthermore, the use of the term “including” and other forms such as “include,” “includes,” and “included” is not restrictive.

[0065] The references to "some embodiments," "embodiments," "one embodiment," or "other embodiments" in the specification mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the invention, but not necessarily in all embodiments of the invention.

[0066] As used herein, ranges and quantities can be expressed as “about” a specific value or range. Thus, “about” includes the exact quantity modified by the term as well as the quantity expected to be within experimental error, such as, for example, within 15%, 10%, or 5%. For example, “about 200 mg” means “200 mg” plus a series of mg numbers within experimental error (e.g., adding or subtracting 15%, 10%, or 5% of 200 mg). As used herein, the term “about” can be used to modify a range or a specific value. It should be understood that using the term “about” before a list of values ​​or a range or value applies the term “about” to each listed value or across the entire range of values. For example, “about 50 mg, 100 mg, or 200 mg” should be understood to mean the same as “about 50 mg, about 100 mg, or about 200 mg”, and “about 50–200 mg” should be understood to mean the same as “about 50 mg to about 200 mg”.

[0067] As used herein, “administration” or “application to” (e.g., in relation to administering a drug, such as besudil or rizabrutinib, to a subject for treatment) means the act of prescribing a drug containing the drug for a subject to take during treatment, the act of dispensing the drug to a subject, and / or the act of the body receiving or ingesting the drug. Therefore, a drug (e.g., besudil or rizabrutinib) may be “administered” by: a physician or other medical professional who prescribes the drug; and / or a pharmacist who prepares the prescription and / or dispenses the drug to a subject; and / or a patient or subject ingesting the drug and / or his or her partner or caregiver.

[0068] As used herein, “besudil” refers to a compound having the chemical name 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propyl-2-yl)acetamide and the chemical structure shown below. Besudil is also known as KD025 and Slx-2119. Unless the context clearly indicates otherwise, "besudil" as used herein refers to any form of the compound and its pharmaceutically acceptable salts. The term "besudil" refers to the compound besudil (e.g., in free base form, amorphous form, and / or crystalline form), and pharmaceutically acceptable salts of besudil (e.g., REZUROCK). ® The mesylate form used in besudinil, as well as any form of besudinil that can be used in formulations or pharmaceutical compositions to administer the compound to a patient. Besudinil mesylate is currently marketed in the United States and other countries under the brand name REZUROCK. ® (Sold by Kadmon Corp. / Sanofi) for the treatment of patients with cGVHD, in some cases after failure of at least two lines of prior systemic therapy. REZUROCK ® The active pharmaceutical ingredient is besudil mesylate, with the molecular formula C. 27 H 28 N6O5S has a molecular weight of 548.62 g / mol and its chemical name is 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(prop-2-yl)acetamide methanesulfonate (1:1).

[0069] A “BTK inhibitor” or “inhibitor of BTK” refers to a compound that inhibits Bruton’s tyrosine kinase (BTK). “Irreversible BTK inhibitors” are characterized by a Michael acceptor moiety that is able to form a covalent bond with the conserved Cys481 residue in the ATP binding site. “Reversible BTK inhibitors” bind to a specific pocket in the SH3 domain through weak reversible interactions such as hydrogen bonds or hydrophobic interactions, resulting in an inactive conformation of the enzyme. In some embodiments, the BTK inhibitor is a hybrid BTK inhibitor, where the inhibitor binds to BTK in a reversible covalent manner, forming a reversible covalent bond with the Cys481 residue and temporarily inactivating the enzyme. An overview of BTK inhibitors is provided in Tasso et al., Molecules, 2021, 26, 7411, the disclosure of which is incorporated by reference herein in its entirety.

[0070] As used herein, “rezameterlinib” refers to the compound having the chemical name (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile and the chemical structure shown below. Rezameterlinib is also known as PRN1008. The compound has been disclosed in several patent publications, such as, for example, PCT Publication Nos. WO 2014 / 039899, WO 2015 / 127310, WO 2016 / 100914, WO 2016 / 105531, and WO 2018 / 005849, the respective disclosures of which are incorporated by reference herein in their entireties. Unless the context clearly indicates otherwise, references herein to “rezameterlinib” refer to the compound in any form and pharmaceutically acceptable salts thereof. The term “rezameterlinib” refers to the compound rezameterlinib (e.g., in free base form, amorphous form, and / or crystalline form), pharmaceutically acceptable salts of rezameterlinib, and any form of rezameterlinib that can be used in a formulation or pharmaceutical composition for administering the compound to a patient.

[0071] “Pharmaceutically acceptable salt” refers to a non-toxic, inorganic and organic acid addition salt of a compound, such as besuximod or rezameterlinib. In one embodiment, the pharmaceutically acceptable salt of besuximod is a mesylate salt.

[0072] As used herein, a “dose” refers to a specified amount of a drug, such as a compound described herein (e.g., bexagliflozin or rezameterlin), administered at one time. For example, a dose of 200 mg of bexagliflozin refers to the administration of 200 mg of bexagliflozin to a subject at one time, e.g., as a tablet or capsule. A “dose” refers to the specified amount, number, and frequency of doses over a specified period of time. For example, a daily dose of 400 mg of bexagliflozin refers to the administration of 400 mg of bexagliflozin to a subject in a single dose or multiple doses over a day (e.g., bexagliflozin administered twice daily in a dose of 200 mg).

[0073] As used herein, a “therapeutically effective amount” refers to an amount of a drug, such as bexagliflozin or rezameterlin, that provides the intended therapeutic effect. A “therapeutically effective amount” of a drug, such as bexagliflozin or rezameterlin, means an amount sufficient to effect a treatment for a disease state being treated, when administered to a human. When applied to any of the diseases disclosed herein (i.e., GVHD, scleroderma, CLAD, RAS, or BOS), “treating” or “treatment” includes (1) reducing the risk of developing a disease (GVHD, scleroderma, CLAD, RAS, or BOS) and / or inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; and (2) relieving the disease (GVHD, scleroderma, CLAD, RAS, or BOS), i.e., causing regression, reversal, or improvement of the disease or reducing the number, frequency, duration, or severity of its clinical symptoms. The therapeutically effective amount of a drug can vary depending on the health and physical condition of the subject to be treated, the severity of the disease progression, the assessment of the medical situation, and other relevant factors.

[0074] As used herein, the terms “individual,” “subject,” and “patient” mean any mammal and can be used interchangeably. In some embodiments, the mammal is a human. In some embodiments, the mammal is not a human. None of these terms requires or implies that a situation characterized by supervision (e.g., continuous or intermittent) by a health care worker (e.g., a physician, a registered nurse, a nurse practitioner, a physician’s assistant, a nursing attendant, or a hospice worker) is required or implied. In some embodiments, the patient is a human patient.

[0075] Although various features of the present application can be described in the context of individual embodiments, the features can also be provided alone or in any suitable combination. Conversely, although the present application can be described herein in the context of individual embodiments, the present application can also be implemented in a single embodiment. Bexagliflozin

[0076] The treatments described herein selected from the group consisting of graft versus host disease (GVHD), systemic sclerosis (scleroderma), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), and bronchiolitis obliterans syndrome (BOS) include administration of bardoxolone.

[0077] Bardoxolone is a ROCK2 inhibitor. Bardoxolone binds to and inhibits the serine / threonine kinase activity of ROCK2, and to a lesser extent, ROCK1. Bardoxolone inhibits ROCK2 and ROCK1 with IC 50 values of approximately 100 nM and 3 μM, respectively. Thus, bardoxolone is useful in the treatment of diseases, disorders, and conditions modulated by ROCK, including autoimmune and fibrotic disorders. Bardoxolone has been approved by the FDA for the treatment of chronic graft versus host disease (cGVHD).

[0078] Bardoxolone has the chemical name 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, and is represented by the chemical structure shown below. Bardoxolone

[0079] In some embodiments, bardoxolone is provided as a pharmaceutically acceptable salt. In some embodiments, bardoxolone is provided as a mesylate salt. In some embodiments, bardoxolone is provided as the free compound (i.e., not as a pharmaceutically acceptable salt).

[0080] In some embodiments, bardoxolone is provided as a capsule or tablet for oral administration. In some embodiments, bardoxolone is provided as a liquid formulation for oral administration.

[0081] In some embodiments, bardoxolone is provided at a dose of about 50 mg to about 400 mg of bardoxolone (measured as the equivalent of the free base). In some embodiments, bardoxolone is provided at a dose of about 50 mg, 100 mg, 200 mg, or 400 mg. In some embodiments, bardoxolone is provided at a dose of about 50 mg. In some embodiments, bardoxolone is provided at a dose of about 100 mg. In some embodiments, bardoxolone is provided at a dose of about 200 mg. In some embodiments, bardoxolone is provided at a dose of about 400 mg.

[0082] In some embodiments, bardoxolone is provided as a capsule or tablet. In some embodiments, bardoxolone is provided as a capsule or tablet comprising about 200 mg of bardoxolone. In some embodiments, bardoxolone is provided as a tablet comprising 200 mg of bardoxolone.

[0083] In some embodiments, the besuxibor is provided as a liquid formulation comprising about 50 mg, 100 mg, 200 mg, or 400 mg besuxibor. In some embodiments, the besuxibor is provided as a liquid formulation comprising about 50 mg besuxibor. In some embodiments, the besuxibor is provided as a liquid formulation comprising about 100 mg besuxibor. In some embodiments, the besuxibor is provided as a liquid formulation comprising about 200 mg besuxibor. In some embodiments, the besuxibor is provided as a liquid formulation comprising about 400 mg besuxibor. BTK inhibitor

[0084] The methods of treating a disease selected from graft versus host disease (GVHD), systemic sclerosis (scleroderma), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), and bronchiolitis obliterans syndrome (BOS) described herein comprise administering a BTK inhibitor (also referred to herein as “an inhibitor of BTK”).

[0085] In some embodiments, the BTK inhibitor is a reversible inhibitor of BTK. In some embodiments, the BTK inhibitor is an irreversible inhibitor of BTK. In some embodiments, the BTK inhibitor is a hybrid BTK inhibitor, wherein the inhibitor binds to BTK in a reversible covalent manner. In some embodiments, the BTK inhibitor is the BTK inhibitor described in Tasso et al., Molecules, 2021, 26, 7411, the disclosure of which is incorporated by reference herein in its entirety.

[0086] In some embodiments, the BTK inhibitor is (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile or a pharmaceutically acceptable salt thereof (rezamotamab). In some embodiments, the BTK inhibitor is 1-[(3R)-3-[4-amino-3-(4- phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one or a pharmaceutically acceptable salt thereof (ibrutinib). In some embodiments, the BTK inhibitor is (4-amino-3-(4-phenoxyphenyl)-1-[(3R)-1-(prop-2-enoyl)piperidin-3-yl]-1,3-dihydro-2H- imidazo[4,5-c]pyridin-2-one) or a pharmaceutically acceptable salt thereof (tolebrutinib).

[0087] In some embodiments, the BTK inhibitor is fenebrutinib. In some embodiments, the BTK inhibitor is evobrutinib. In some embodiments, the BTK inhibitor is orelabrutinib. In some embodiments, the BTK inhibitor is remibrutinib. In some embodiments, the BTK inhibitor is BIIB-091. In some embodiments, the BTK inhibitor is tirabrutinib. In some embodiments, the BTK inhibitor is acalabrutinib. In some embodiments, the BTK inhibitor is vecabrutinib. In some embodiments, the BTK inhibitor is zanubrutinib. In some embodiments, the BTK inhibitor is poseltinib. In some embodiments, the BTK inhibitor is pirtobrutinib. In some embodiments, the BTK inhibitor is spebrutinib. In some embodiments, the BTK inhibitor is olmutinib. In some embodiments, the BTK inhibitor is branebrutinib. In some embodiments, the BTK inhibitor is TAK-020. In some embodiments, the BTK inhibitor is elsubrutinib. In some embodiments, the BTK inhibitor is tolebrutinib.

[0088] In some embodiments, the BTK inhibitor is provided in Table 1. Table 1. Selected BTK inhibitors and chemical structures. Zanabrutinib

[0089] In some embodiments, the BTK inhibitor used in the methods described herein is zanabrutinib.

[0090] Zanabrutinib is a highly selective BTK inhibitor. Zanabrutinib functions as a reversible covalent BTK inhibitor and forms both non-covalent and covalent bonds with its target, thereby enhancing selectivity and prolonging inhibition at low systemic exposure. The reversible binding of zanabrutinib minimizes the potential for permanent modification peptides. Zanabrutinib is currently being developed for the treatment of immune-mediated diseases.

[0091] Rizabrutinib has the chemical name (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile and is represented by the chemical structure shown below. Rizabrutinib Rizabrutinib is also known as 2-[(3R)-2-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4[4-(oxetan-3-yl)piperazin-1-yl]-pent-2-enenitrile; 2-[(3R)-2-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4[4-(oxetan-3-yl)piperazin-1-yl]-(E and Z)-pent-2-enenitrile; (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile; (3R)-3-[4-amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-a-[2-methyl-2-[4-(3-oxetanyl)-1-piperazinyl]propylidene]-b-oxo-1-piperidinepropanenitrile; (EZ)-2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile; and the International Nonproprietary Names (INN) for pharmaceuticals (https: / / cdn.who.int / media / docs / default-source / international-nonproprietary-names-(inn) / pl121.pdf?sfvrsn=69617906_15&download=true) published by the World Health Organization with the following structure: .

[0092] Rizabrutinib exists as the (Z) isomer or the (E) isomer. A dose of rizabrutinib can contain less than about 2% by weight, such as less than about 1% by weight of the corresponding (Z) isomer as an impurity; a dose of rizabrutinib can contain less than about 2% by weight, such as less than about 1% by weight of the corresponding (E) isomer as an impurity. When rizabrutinib is referred to as a mixture of the (E) and (Z) isomers of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1- carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enonitrile, it is meant that the amount of (E) or (Z) isomer in the mixture is greater than about 2% by weight. In some embodiments, the molar ratio of the (E) isomer to the (Z) isomer is 8:2. In some embodiments, the molar ratio of the (E) isomer to the (Z) isomer is 9:1. In some embodiments, rizabrutinib is provided as the (E) isomer. In some embodiments, rizabrutinib is provided as the (Z) isomer. In some embodiments, rizabrutinib is provided as a mixture of the (E) and (Z) isomers.

[0093] In some embodiments, rizabrutinib is provided as a pharmaceutically acceptable salt. In some embodiments, rizabrutinib is provided as the free base (i.e., not as a pharmaceutically acceptable salt).

[0094] In some embodiments, rizabrutinib is formulated for oral administration. In some embodiments, rizabrutinib is provided as a capsule or tablet for oral administration. In some embodiments, rizabrutinib is provided as a liquid formulation for oral administration.

[0095] In some embodiments, rizabrutinib is provided at a dose of about 50 mg to about 800 mg of rizabrutinib. In some embodiments, rizabrutinib is provided at a dose of about 50 mg, 75 mg, 100 mg, 200 mg, 300 mg, or 400 mg. In some embodiments, rizabrutinib is provided at a dose of about 100 mg, 200 mg, or 400 mg. In some embodiments, rizabrutinib is provided at a dose of about 100 mg. In some embodiments, rizabrutinib is provided at a dose of about 200 mg. In some embodiments, rizabrutinib is provided at a dose of about 400 mg.

[0096] For oral administration, rizabrutinib can be provided in the form of tablets containing from about 1 to about 1000 mg of the active ingredient, particularly about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 600, 750, and 800 mg of the active ingredient. In some embodiments, rizabrutinib is provided as a capsule or tablet comprising about 100 mg or 300 mg rizabrutinib. In some embodiments, rizabrutinib is provided as a capsule or tablet comprising about 100 mg rizabrutinib. In some embodiments, rizabrutinib is provided as a capsule or tablet comprising about 300 mg rizabrutinib.

[0097] In some embodiments, rizabrutinib is provided as a liquid formulation comprising about 50 mg, 150 mg, 300 mg, 300 mg BID, 450 mg BID, or 600 mg rizabrutinib. In some embodiments, rizabrutinib is provided as a liquid formulation comprising about 300 mg rizabrutinib. In some embodiments, rizabrutinib is provided as a liquid formulation comprising about 300 mg BID rizabrutinib. In some embodiments, rizabrutinib is provided as a liquid formulation comprising about 450 mg BID rizabrutinib. In some embodiments, rizabrutinib is provided as a liquid formulation comprising about 600 mg rizabrutinib. Methods of treatment

[0098] In one aspect, provided herein are methods of treating a disease or condition selected from the group consisting of systemic sclerosis and a transplant-related dysfunction in a human patient in need thereof, comprising administering to the human patient a therapeutically effective amount of a combination comprising a BTK inhibitor and a besylate. In some embodiments, the disease or condition is systemic sclerosis. In some embodiments, the disease or condition is a transplant-related dysfunction. In some embodiments, the transplant-related dysfunction is graft-versus-host disease (GVHD), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), or bronchiolitis obliterans syndrome (BOS). In some embodiments, the transplant-related dysfunction is GVHD. In some embodiments, the transplant-related dysfunction is CLAD. In some embodiments, the transplant-related dysfunction is RAS. In some embodiments, the transplant-related dysfunction is BOS.

[0099] In further aspects, provided herein are methods of treating a disease or condition selected from graft versus host disease (GVHD), systemic sclerosis (scleroderma), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), and bronchiolitis obliterans syndrome (BOS) in a human patient in need thereof, comprising administering to the human patient a therapeutically effective amount of a combination comprising a BTK inhibitor and bexarotene. Also provided herein are methods of treating GVHD in a human patient in need thereof, comprising administering to the human patient a therapeutically effective amount of a combination comprising a BTK inhibitor and bexarotene. Further, provided herein are methods of treating CLAD in a human patient in need thereof, comprising administering to the human patient a therapeutically effective amount of a combination comprising a BTK inhibitor and bexarotene. Also provided herein are methods of treating RAS in a human patient in need thereof, comprising administering to the human patient a therapeutically effective amount of a combination comprising a BTK inhibitor and bexarotene. Also provided herein are methods of treating BOS in a human patient in need thereof, comprising administering to the human patient a therapeutically effective amount of a combination comprising a BTK inhibitor and bexarotene. Also provided herein are methods of treating systemic sclerosis (scleroderma) in a human patient in need thereof, comprising administering to the human patient a therapeutically effective amount of a combination comprising a BTK inhibitor and bexarotene.

[0100] Also provided herein are combinations of a BTK inhibitor and bexarotene for use in a method of treating a disease or condition selected from systemic sclerosis and transplant- related dysfunction in a human patient in need thereof. Further provided herein is the use of a combination of a BTK inhibitor and bexarotene for the manufacture of a medicament for treating a disease or condition selected from systemic sclerosis and transplant-related dysfunction in a human patient in need thereof.

[0101] Also provided herein are combinations of a BTK inhibitor and bexarotene for use in a method of treating a disease or condition selected from GVHD, scleroderma, CLAD, RAS, and BOS in a human patient in need thereof. Further provided herein is the use of a combination of a BTK inhibitor and bexarotene for the manufacture of a medicament for treating a disease or condition selected from GVHD, scleroderma, CLAD, RAS, and BOS in a human patient in need thereof.

[0102] In some embodiments, the disease or condition is graft versus host disease (GVHD). In some embodiments, the GVHD is chronic GVDH (cGVHD). In some embodiments, the GVHD is scleroderma-like chronic GVHD. In some embodiments, the GVHD is pulmonary GVHD. In some embodiments, the GVHD is pulmonary cGVHD.

[0103] In some embodiments, the disease or disorder is systemic sclerosis (scleroderma).

[0104] In some embodiments, the disease or disorder is chronic lung allograft dysfunction (CLAD).

[0105] In some embodiments, the disease or disorder is restrictive allograft syndrome (RAS).

[0106] In some embodiments, the disease or disorder is bronchiolitis obliterans syndrome (BOS) post-lung transplant.

[0107] In some embodiments, the disease or disorder is transplant-related dysfunction.

[0108] Diagnosis of any of the diseases or disorders disclosed herein can be made by a qualified healthcare worker, such as a physician, based on clinically accepted criteria. Subject

[0109] In some embodiments, the subject has graft versus host disease (GVHD). In some embodiments, the subject has chronic graft versus host disease (cGVHD).

[0110] In some embodiments, the subject has systemic sclerosis (scleroderma).

[0111] In some embodiments, the subject has chronic lung allograft dysfunction (CLAD).

[0112] In some embodiments, the subject has restrictive allograft syndrome (RAS).

[0113] In some embodiments, the subject has bronchiolitis obliterans syndrome (BOS) post-lung transplant.

[0114] In some embodiments, the subject has previously received an organ transplant. In some embodiments, the subject is a bone marrow transplant recipient. In some embodiments, the subject is a lung transplant recipient. In some embodiments, the subject is a single lung transplant recipient. In some embodiments, the subject is a double lung transplant recipient.

[0115] In some embodiments, the subject is an adult. In some embodiments, the adult is a male. In other embodiments, the adult is a female. In some embodiments, the adult is at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years of age. In some embodiments, the subject is a child or adolescent. In some embodiments, the subject is at least 12 years of age. In some embodiments, the subject is at least 12, 13, 14, 15, 16, 17, 18, or 19 years of age. administering

[0116] In some embodiments, the subject is administered besuxiborole orally. In some embodiments, the subject is administered a BTK inhibitor orally. In some embodiments, the subject is administered zanabrutinib orally. In some embodiments, the subject is administered a combination comprising a BTK inhibitor and besuxiborole orally. In some embodiments, the subject is administered a combination comprising zanabrutinib and besuxiborole orally.

[0117] In some embodiments, the combination disclosed herein (e.g., besuxiborole and a BTK inhibitor) is administered to the subject with food.

[0118] In some embodiments, the BTK inhibitor and besuxiborole are administered to the subject separately. In some embodiments, the BTK inhibitor and besuxiborole are administered to the subject sequentially. In some embodiments, the subject is administered besuxiborole prior to administration of the BTK inhibitor to the subject. In some embodiments, the subject is administered the BTK inhibitor prior to administration of besuxiborole to the subject. In some embodiments, the BTK inhibitor and besuxiborole are administered to the subject simultaneously. In some embodiments, the BTK inhibitor and besuxiborole are administered to the subject on the same day. In some embodiments, the BTK inhibitor and besuxiborole are administered to the subject at a time such that both the BTK inhibitor and besuxiborole are active (i.e., not fully metabolized) in the subject. In some embodiments, the BTK inhibitor and besuxiborole are administered to the subject on the same day and within 6 hours, such as within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, or 6 hours, of each other. In some embodiments, the BTK inhibitor and besuxiborole are administered to the subject on different days. In some embodiments, the BTK inhibitor and besuxiborole are administered to the subject on different days and within 3 weeks, such as within 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, or 21 days, of each other.

[0119] In some embodiments, the zanabrutinib and besuxiborole are administered to the subject separately. In some embodiments, the zanabrutinib and besuxiborole are administered to the subject sequentially. In some embodiments, the subject is administered besuxiborole prior to administration of zanabrutinib to the subject. In some embodiments, the subject is administered zanabrutinib prior to administration of besuxiborole to the subject. In some embodiments, the zanabrutinib and besuxiborole are administered to the subject simultaneously. In some embodiments, the zanabrutinib and besuxiborole are administered to the subject on the same day. In some embodiments, the zanabrutinib and besuxiborole are administered to the subject on different days.

[0120] In some embodiments, the besuxibulin is administered to the subject once daily or twice daily. In some embodiments, the besuxibulin is administered to the subject once daily. In some embodiments, the besuxibulin is administered to the subject twice daily.

[0121] In some embodiments, the BTK inhibitor is administered to the subject once daily, twice daily, or three times daily. In some embodiments, the BTK inhibitor is administered to the subject once daily. In some embodiments, the BTK inhibitor is administered to the subject twice daily. In some embodiments, the BTK inhibitor is administered to the subject three times daily.

[0122] In some embodiments, the revaplicin is administered to the subject once daily or twice daily. In some embodiments, the revaplicin is administered to the subject once daily. In some embodiments, the revaplicin is administered to the subject twice daily.

[0123] In some embodiments, the combination therapy (i.e., the BTK inhibitor and the besuxibulin) is continued based on patient tolerability until resolution or progression of active disease symptoms (GVHD, scleroderma, CLAD, RAS, or BOS). In some embodiments, the besuxibulin therapy is continued based on patient tolerability until resolution or progression of active disease symptoms. In some embodiments, the BTK inhibitor (such as revaplicin) therapy is continued based on patient tolerability until resolution or progression of active disease symptoms.

[0124] The duration of treatment depends on the patient. In some embodiments, the duration of treatment is 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, or 24 months. In some embodiments, the duration of treatment is further extended for an additional 24 months or more until disease progression.

[0125] In some embodiments, the methods of treating a disease described herein, such as GVHD, scleroderma, CLAD, RAS, or BOS, provide a method of improving FEVi in a subject having the disease, such as GVHD, scleroderma, CLAD, RAS, or BOS.

[0126] In some embodiments, the methods disclosed herein reduce the risk of retransplantation of an organ, such as a lung, in a subject.

[0127] In some embodiments, the methods disclosed herein improve the quality of life of a subject having a disease or condition selected from the group consisting of graft versus host disease (GVHD), systemic sclerosis (scleroderma), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), and bronchiolitis obliterans syndrome (BOS).

[0128] In some embodiments, the methods disclosed herein reduce mortality, reduce progressive bronchiectasis, reduce organ failure, reduce decline in pulmonary function, increase recovery and stabilization following organ transplantation, reduce hospitalization, reduce health care utilization, and / or reduce the risk of retransplantation.

[0129] In some embodiments, the methods disclosed herein comprise administering besuxiborole at a dose lower than besuxiborole administered as a single agent (i.e., monotherapy), as part of the combinations described herein. In some embodiments, the methods disclosed herein comprise administering besuxiborole at a daily dose lower than besuxiborole administered as a single agent (i.e., monotherapy), as part of the combinations described herein. Thus, in some cases, undesirable side effects caused by administration of besuxiborole can be reduced.

[0130] In some embodiments, the methods disclosed herein comprise administering a BTK inhibitor (such as zanabrutinib) at a dose lower than the BTK inhibitor administered as a single agent (i.e., monotherapy), as part of the combinations described herein. In some embodiments, the methods disclosed herein comprise administering a BTK inhibitor (such as zanabrutinib) at a daily dose lower than the BTK inhibitor administered as a single agent (i.e., monotherapy), as part of the combinations described herein. Thus, in some cases, undesirable side effects caused by administration of the BTK inhibitor can be reduced.

[0131] In some embodiments, the methods disclosed herein comprising administering a combination comprising besuxiborole and a BTK inhibitor (such as zanabrutinib) are more effective than administering besuxiborole or a BTK inhibitor alone (i.e., monotherapy). Dosing of besuxiborole

[0132] In any of the methods disclosed herein, besuxiborole is administered to a human patient at a daily dose of up to about 400 mg (measured as the equivalent of the free base). In some embodiments, besuxiborole is administered at a daily dose of about 50 mg, 100 mg, 200 mg, or 400 mg. In some embodiments, besuxiborole is administered at a daily dose of about 50 mg. In some embodiments, besuxiborole is administered at a daily dose of about 100 mg. In some embodiments, besuxiborole is administered at a daily dose of about 200 mg. In some embodiments, besuxiborole is administered at a daily dose of about 400 mg.

[0133] In some embodiments, bexagliflozin is administered at a dose of about 50 mg, 100 mg, 200 mg, or 400 mg. In some embodiments, bexagliflozin is administered at a dose of about 50 mg. In some embodiments, bexagliflozin is administered at a dose of about 100 mg. In some embodiments, bexagliflozin is administered at a dose of about 200 mg. In some embodiments, bexagliflozin is administered at a dose of about 400 mg.

[0134] In some cases, it can be necessary to adjust the dose and / or daily dose of bexagliflozin. For example, co-administration of bexagliflozin with a strong CYP3A inducer (an inducer of the CYP3A family of P-450 isozymes, including CYP3A4) can reduce the exposure of bexagliflozin, which can reduce the efficacy of bexagliflozin. In such cases, the dose and / or daily dose of bexagliflozin should be increased. In another example, co-administration of bexagliflozin with a proton pump inhibitor can reduce the exposure of bexagliflozin, which can reduce the efficacy of bexagliflozin. In such cases, the dose and / or daily dose of bexagliflozin should be increased.

[0135] In some embodiments, bexagliflozin is administered to a human patient once daily or twice daily. In some embodiments, bexagliflozin is administered to a human patient once daily. In some embodiments, bexagliflozin is administered to a human patient twice daily.

[0136] In some embodiments, bexagliflozin is administered to a human patient orally. In some embodiments, bexagliflozin is administered to a human patient as a capsule or tablet. In some embodiments, bexagliflozin is administered to a human patient as a liquid formulation. Dosing of rizabrutinib

[0137] In any of the methods disclosed herein, rizabrutinib is administered to a human patient at a daily dose of up to about 800 mg. In some embodiments, rizabrutinib is administered at a daily dose of about 100 mg, 300 mg, 400 mg, 600 mg, or 800 mg.

[0138] In some embodiments, rizabrutinib is administered to a human patient at a dose of about 50 mg, 75 mg, 100 mg, 200 mg, 300 mg, or 400 mg. In some embodiments, rizabrutinib is administered to a human patient at a dose of about 100 mg, 200 mg, or 400 mg. In some embodiments, rizabrutinib is administered to a human patient at a dose of about 100 mg. In some embodiments, rizabrutinib is administered to a human patient at a dose of about 200 mg. In some embodiments, rizabrutinib is administered to a human patient at a dose of about 400 mg.

[0139] In some embodiments, the human patient is administered ritazapimab once a day or twice a day. In some embodiments, the human patient is administered ritazapimab once a day. In some embodiments, the human patient is administered ritazapimab twice a day.

[0140] In some embodiments, ritazapimab is administered orally to the human patient. In some embodiments, ritazapimab is administered to the human patient as a capsule or tablet. In some embodiments, ritazapimab is administered to the human patient as a liquid formulation. Dosing of ibrutinib

[0141] In any of the methods disclosed herein, ibrutinib is administered to the human patient at a daily dose of up to about 420 mg. In some embodiments, ibrutinib is administered at a daily dose of about 140 mg to about 420 mg, such as about 150 mg to about 400 mg, about 150 mg to about 350 mg, about 150 mg to about 300 mg, about 150 mg to about 250 mg, about 150 mg to about 200 mg, about 200 mg to about 400 mg, about 200 mg to about 350 mg, about 200 mg to about 300 mg, about 200 mg to about 250 mg, about 250 mg to about 400 mg, about 250 mg to about 350 mg, about 250 mg to about 300 mg, about 300 mg to about 400 mg, and about 350 to about 400 mg. In some embodiments, ibrutinib is administered at a daily dose of about 140 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, or 420 mg.

[0142] In some embodiments, ibrutinib is administered at a daily dose of about 140 mg. In some embodiments, ibrutinib is administered at a daily dose of about 150 mg. In some embodiments, ibrutinib is administered at a daily dose of about 200 mg. In some embodiments, ibrutinib is administered at a daily dose of about 250 mg. In some embodiments, ibrutinib is administered at a daily dose of about 300 mg. In some embodiments, ibrutinib is administered at a daily dose of about 350 mg. In some embodiments, ibrutinib is administered at a daily dose of about 400 mg. In some embodiments, ibrutinib is administered at a daily dose of about 420 mg.

[0143] In some embodiments, ibrutinib is administered to a human patient at a dose of about 140 mg to about 420 mg, such as about 150 mg to about 400 mg, about 150 mg to about 350 mg, about 150 mg to about 300 mg, about 150 mg to about 250 mg, about 150 mg to about 200 mg, about 200 mg to about 400 mg, about 200 mg to about 350 mg, about 200 mg to about 300 mg, about 200 mg to about 250 mg, about 250 mg to about 400 mg, about 250 mg to about 350 mg, about 250 mg to about 300 mg, about 300 mg to about 400 mg, and about 350 to about 400 mg. In some embodiments, ibrutinib is administered at a dose of about 140 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, or 420 mg. In some embodiments, ibrutinib is administered to a human patient at a dose of about 140 mg. In some embodiments, ibrutinib is administered to a human patient at a dose of about 200 mg. In some embodiments, ibrutinib is administered to a human patient at a dose of about 250 mg. In some embodiments, ibrutinib is administered to a human patient at a dose of about 300 mg. In some embodiments, ibrutinib is administered to a human patient at a dose of about 400 mg. In some embodiments, ibrutinib is administered to a human patient at a dose of about 420 mg.

[0144] In some embodiments, ibrutinib is administered to a human patient once a day, twice a day, or three times a day. In some embodiments, ibrutinib is administered to a human patient once a day. In some embodiments, ibrutinib is administered to a human patient twice a day. In some embodiments, ibrutinib is administered to a human patient three times a day.

[0145] In some embodiments, ibrutinib is administered to a human patient orally. In some embodiments, ibrutinib is administered to a human patient as a capsule or tablet. In some embodiments, ibrutinib is administered to a human patient as a liquid formulation. Dosing of tolebrutinib

[0146] In any of the methods disclosed herein, tolebrutinib is administered to a human patient at a daily dose of about 60 mg to about 120 mg. In some embodiments, tolebrutinib is administered at a daily dose of about 60 mg. In some embodiments, tolebrutinib is administered at a daily dose of about 120 mg.

[0147] In some embodiments, tolebrutinib is administered to a human patient at a dose of about 60 mg to about 120 mg. In some embodiments, tolebrutinib is administered to a human patient at a dose of about 60 mg. In some embodiments, tolebrutinib is administered to a human patient at a dose of about 120 mg.

[0148] In some embodiments, tolebrutinib is administered to a human patient once a day.

[0149] In some embodiments, tolebrutinib is administered to a human patient orally. In some embodiments, tolebrutinib is administered to a human patient as a capsule or tablet. Kits / Articles of Manufacture

[0150] In certain embodiments, kits and articles of manufacture for use with one or more of the methods described herein are disclosed. Such kits include a carrier, package, or container, positioned to hold one or more containers, such as vials, tubes, and the like, each of which holds one of the separate elements to be used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic. In some embodiments, the kits include besuximilb and a BTK inhibitor. In some embodiments, the kits include besuximilb and rizabrutinib. In some embodiments, the kits include rizabrutinib.

[0151] Kits typically include a label indicating that the contents are to be used in a particular manner and / or for a particular treatment. Such kits also typically include a package insert explaining the use of the contents. In one embodiment, the label is on or associated with the container. In one embodiment, the label is on a container when letters, numbers or other characters forming the label are attached to the container itself, as by labeler, molding or etching into the container itself. In one embodiment, the label is associated with a container when letters, numbers or other characters forming the label are present on a tag or label which is carried with the container. For example, a tag or label is carried with the container when it is fastened to the container, e.g., by tacking with a glue or by a snap fit into an indentation on the container. In one embodiment, the label is associated with a container when it is present within a receptacle which also holds the container, e.g., as a package insert. In one embodiment, the label indicates that the contents are to be used for a particular treatment application. The label also indicates directions for using the contents, such as in the methods described herein.

[0152] Also disclosed herein are pharmaceutical compositions for use in the methods described herein. In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device containing one or more unit dosage forms containing the compounds provided herein. The pack for example contains metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the pharmaceutical for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for drugs or the approved product insert for a drug. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Examples

[0153] These examples are provided merely for illustrative purposes and do not limit the scope of the claims provided herein. Example 1. In vitro study of B cell activation in human whole blood by besuxibulin and rilzabrutinib. Background and objectives.

[0154] Besuxibulin exerts its effects in GVHD primarily through downregulation of effector T cells and STAT3 signaling (Flynn et al., Blood, 2016). The effects of besuxibulin on B cells, equally integral in aberrant inflammatory regulation, have not been fully characterized. The BTK inhibitor rilzabrutinib is known to block BCR-driven B cell activation. The goal of this study was to test the ability of besuxibulin to block BCR-driven human whole blood B cell activation in order to determine whether besuxibulin utilizes the same signaling pathway as rilzabrutinib. Methods.

[0155] To examine the effect of bexarotene and razakinutant on B cell activation, 95 pL of human whole blood per well was aliquoted and then different concentrations of bexarotene (ASTATECH, 42507) and razakinutant (Sanofi) ranging from 10 pM to 0.00017 pM were added in a 3-fold dilution series in PBS (Gibco, 20012-027). The whole blood was incubated with bexarotene or razakinutant for 1 h at 37 °C, 5% CO2, followed by the addition of 80 ng / mL anti-CD79b antibody (BD Biosciences, 557592, clone: 3A2-2E7) to activate B cells overnight at 37 °C, 5% CO2. The next day, the whole blood was lysed with BD PharmLyse (BD Biosciences, 555899) and stained with FITC mouse anti-human CD20 antibody (BD Biosciences, 555622, clone: 2H7), APC mouse anti-human CD69 antibody (BD Biosciences, 555533, clone: FN50), and fixable viability dye eFluor 780 (eBioscience, 65-0865-14) in staining buffer (0.5% BSA (Gibco, 15260-037) and 2 mM EDTA (Invitrogen, AM9261) in PBS). Flow cytometry analysis was performed using a BD LSRFortessa™ Cell Analyzer (BD Biosciences) and data were analyzed by FlowJo (BD Biosciences). B cell activation was determined by the increase of surface expression of the activation marker CD69 on B cells (CD20+) after anti-CD79b stimulation. The experiment was performed in duplicate. Results.

[0156] In line with the mechanism of action, flow cytometry analysis showed that 10 pM razakinutant inhibited anti-CD79b-mediated B cell activation in whole blood, reducing the number of activated cells from 73.4% to 8.03% ( Figure 1A -B). In contrast, flow cytometry analysis showed that bexarotene did not inhibit anti-CD79b-mediated B cell activation in whole blood ( Figure 1C ), with 82.4% of B cells showing positive staining for the activation marker CD69 after treatment with 10 pM bexarotene.

[0157] Figure 2 The dose-response plot of bexarotene and razakinutant provided in Figure 4 further demonstrates that bexarotene did not inhibit B cell activation in whole blood, while razakinutant inhibited B cell activation in whole blood in a dose-dependent manner, with an IC 50was 9.5 nM. Summary.

[0158] Ripretinib inhibits B cell activation, while no effect on B cell activation was observed with different concentrations of Blebbistatin. The ROCK2 pathway (used by Blebbistatin) is dispensable downstream of BCR signaling, which distinguishes it from the BTK pathway (used by ripretinib). Therefore, Blebbistatin and ripretinib utilize different signaling pathways, both of which have an indispensable role in the development of GVHD and CLAD. These data provide mechanistic rationale for the therapeutic combination of ROCK and BTK targeting agents by using a multi-pathway approach to treat certain diseases. Example 2. Efficacy of the BTK inhibitors ripretinib and ibrutinib in a sclerodermatous chronic graft versus host disease (cGVHD) mouse model. Table 2. Study design (single agent). Ibrutinib was administered for AM doses, then 0.5% MC vehicle was administered for PM doses.

[0159] The effect of a selective BTK inhibitor on disease progression was evaluated in a sclerodermatous chronic GVHD model. Sclerodermatous GVHD was initiated by exposing female C57B1 / 6 recipient mice to 8.5 Gy of total body irradiation on Day -1. Recipients: C57B1 / 6 (CD45.1), (8 weeks old) and donors: LP / J (000676), (6-10 weeks old) female mice were obtained from Jackson Labs and assigned to treatment and control groups. Animals were not replaced during the course of the study. Mice were handled following standard routine procedures. Animals were acclimated to the environment for at least 3 days prior to the start of the study. During this period, animals were observed daily for poor health and were culled. The study was conducted in an animal room that was supplied with filtered air, maintained at a temperature of 70 ± 5 °F, relative humidity of 50% ± 20%, and provided with an automatic timer for a light / dark cycle of 12 hours on and 12 hours off (without dusk).

[0160] On Day 0, a combination of bone marrow cells and splenocytes were transferred from donor LP / J mice to recipients as shown in Table 2. Specifically, C57B1 / 6 (CD45.1) recipients were injected intravenously (IV) with a combination of splenocytes and bone marrow cells from donor female LP / J (CD45.2) mice in sterile 1x PBS. Splenocytes were isolated using a Miltenyi GentleMACS Dissociator. BM cells were isolated using standard flushing practices. Following transplantation, all animals were housed under standard environmental conditions and had ad libitum access to appropriate irradiated sterile rodent chow and sterile water. Animals were dosed with vehicle, ibrutinib, or revmurabrutinib as indicated. All animals were monitored daily and weight, survival, and disease progression were recorded according to the scoring system described in Table 4.

[0161] On Day 20, animals were re-assigned to groups such that the average scGVHD score was similar across all groups. Treatment began on Day 21 and continued through Day 55. All dosing was performed BID to control for stress associated with oral gavage. Animals receiving ibrutinib were dosed with ibrutinib for AM doses and then vehicle for PM doses. On Days 33, 36, 39, 42, and 45, all animals were photographed under isoflurane anesthesia to assess disease severity.

[0162] 25 mg / kg ibrutinib treatment provided a numerical reduction in GVHD scores, similar to results previously reported in Dubovsky et al., JCI [Journal of Clinical Investigation] 2014, 124(11): 4867-4876 (see Figure 2 ). Data related to GVHD scores and total disease burden following treatment with ibrutinib and revmurabrutinib are shown in Figure 3A and Figure 3B The effects of revmurabrutinib were dose-dependent, with greater reductions in disease severity observed at the higher 40 mg / kg dose. The overall degree of disease reduction was similar to ibrutinib, which is approved for the treatment of steroid-refractory GVHD in adults.

[0163] These results demonstrate that BTK inhibitors can improve disease pathology in a model of GVHD and, specifically, revmurabrutinib, which has not been previously tested in this disease setting, has disease-modifying activity comparable to ibrutinib, which is approved for this indication. The results also establish safe and effective doses of revmurabrutinib, which can be used in combination with besuxoib for co-dosing studies in animals. Example 3. Efficacy of BTK inhibitors and besuxoib combination in a sclerodermatous chronic graft versus host disease (cGVHD) mouse model.

[0164] This study will evaluate the effects of besuxiborole and BTK inhibitors on a murine model of sclerodermatous chronic GVHD. Materials.

[0165] This study will be conducted under the same conditions as the single agent study described in Example 2.

[0166] Mice will be treated with the test articles described below.

[0167] Vehicle only. The vehicle contains 0.5% methylcellulose (MC) (Sigma Cat# M0262). From days 21 to 55, vehicle will be administered to mice at a dose of 0.1 mL / 20 g, twice daily, orally.

[0168] Besuxiborole. Besuxiborole will be formulated with vehicle (0.5% methylcellulose (MC)). Besuxiborole will be administered to mice in Groups 4, 7, and 8 at a dose of 125 mg / kg, orally, as follows. Animals will be treated twice daily from days 21-55.

[0169] Group 4. For the first / AM dose, only besuxiborole will be administered to the animals, and for the last / PM dose, 0.5% MC vehicle vehicle will be administered.

[0170] Group 7 For the first / AM dose, besuxiborole will be formulated in combination with ibrutinib, and for the last / PM dose, 0.5% MC vehicle vehicle will be administered. Testing formulation of the combination will be performed prior to the start of the study.

[0171] Group 8. For the first / AM dose, besuxiborole will be formulated in combination with zanabrutinib, and for the last / PM dose, 0.5% MC vehicle vehicle will be administered. Testing formulation of the combination will be performed prior to the start of the study.

[0172] Ibrutinib. Ibrutinib (PCI-32765; Selleckchem, Cat# S2680) will be formulated with vehicle (0.5% methylcellulose (MC)). Ibrutinib will be administered to mice in Groups 5 and 7 at a dose of 20 mg / kg, orally, as follows. Animals will be treated twice daily from days 21-55.

[0173] Group 5. For the first / AM dose, only ibrutinib will be administered to the animals, and for the last / PM dose, 0.5% MC vehicle vehicle will be administered.

[0174] Group 7.For the first / AM dose, ibrutinib will be formulated in combination with besuxibor, and for the last / PM dose, 0.5% MC vehicle will be administered. Testing formulation of the combination will be performed prior to study initiation.

[0175] Rizabrutinib. Rizabrutinib will be formulated with vehicle (0.5% methylcellulose (MC)). Rizabrutinib will be administered orally to mice in Groups 6 and 8 at a dose of 40 mg / kg as follows. Animals will be treated twice daily on Days 21-55. Rizabrutinib doses will be prepared as follows: weigh the amount of rizabrutinib required for 3 days of dosing and add to a mortar; add vehicle and grind with a pestle for 5 minutes; transfer to a clear glass vial and sonicate for 30 minutes or until a uniform suspension is formed; dilute as required for the dosing solution and prepare daily aliquots; and vortex / sonicate as required prior to dosing.

[0176] Group 6. For the first / AM dose, rizabrutinib will be formulated in combination with besuxibor, and for the last / PM dose, 0.5% MC vehicle will be administered. Testing formulation of the combination will be performed prior to study initiation.

[0177] Group 8. For the first / AM dose, ibrutinib will be formulated in combination with besuxibor, and for the last / PM dose, ibrutinib will be formulated alone. Testing formulation of the combination will be performed prior to study initiation. Experimental design.

[0178] A summary of the study design is provided in Table 3. Table 3. Study design (combination). Besuxibor and / or ibrutinib will be administered for the first / AM dose, and then 0.5% MC vehicle will be administered for the last / PM dose. Animals will be dosed with 125 mg / kg besuxibor from Day 21 to Day 32. Animals in Groups 4, 7, and 8 will be dosed with 100 mg / kg besuxibor from Day 33 to Day 55.

[0179] Upon arrival, mice will be randomized into one (1) group of six (6), one (1) group of twelve (12), and six (6) groups of ten (10) animals. Scleroderma-like chronic GVHD will be induced in C57B1 / 6 (CD45.1) mice using a single acute dose of total body irradiation (TBI) of 8.5 Gy on Day -1.

[0180] All animals will be monitored daily to record weight changes, survival, GVHD scores (Table 4), and incidence of diarrhea and bloody stool. In addition, a second scoring system for sclerodermatous chronic GVHD (scGVHD) will be used (Table 5). Table 4. Standard GVHD scoring regimen. Table 5. Modified scoring regimen for scGVHD.

[0181] On Day 20, animals in Groups 3-8 will be re-assigned to groups such that the average scGVHD score for Groups 3-8 is similar. Re-assignment will be based on the composite 5-point scGVHD score described in Table 5.

[0182] Animals will be dosed with vehicle or test article as detailed in Table 3. Dosing will begin on Day 21 and continue through Day 55. Animals receiving ibrutinib will be administered ibrutinib only for the first / AM dose, and vehicle for the last / PM dose.

[0183] On Days 33, 36, 39, 42, and 45, all animals will be photographed (top-down pictures, pure white background) under isoflurane anesthesia to assess disease severity. Each photograph will be labeled with the number of the corresponding animal to aid in identification.

[0184] Animals that develop pain will be administered buprenorphine, BID, as needed. Animals that are unable to care for themselves, feel cold to the touch, or are moribund will be euthanized. Animals requiring euthanasia will be euthanized by CO2 inhalation and will not be collected. Animals found dead will not be collected.

[0185] On Day 56, all surviving animals will be sacrificed by CO2 inhalation and the organs detailed in Table 2 will be collected. Experimental procedure.

[0186] GVHD induction. As detailed in Table 2, all recipient animals will receive a total body irradiation dose of 8.5 Gy on Day -1. On Day 0, Groups 3-8 animals will receive a bone marrow (BM) transplant containing a combination of bone marrow cells and splenocytes obtained from donor LP / J (CD45.2) mice via the tail vein (200 µL) according to Table 2. On Day 0, Group 2 animals will receive a bone marrow (BM) transplant containing only bone marrow cells obtained from donor LP / J (CD45.2) mice via the tail vein (200 µL) according to Table 2. Splenocytes will be isolated using a Miltenyi Gentlemacs dissociator. BM cells will be isolated using standard flushing practices (femur and tibia) and counted. Following transplantation, all recipient animals will be housed under standard environmental conditions and will have ad libitum access to appropriate rodent chow and sterile water.

[0187] Supportive care. Following TBI on Day -1 through Day 8, all animals will be given supplemental high caloric, highly palatable food in the pans at the bottom of the cages. Following transfer on Day 0, and then as needed upon transfer, all animals will be given 1 mL / animal / day of supplemental fluids (warmed Ringer’s solution) via subcutaneous injection. As needed, an additional 1 mL of fluids can be given pm. Any animal that loses greater than 15% of body weight will be given highly palatable soft food and 1 mL of supplemental fluids (warmed Ringer’s solution) via subcutaneous injection once daily. Any animal that loses greater than 20% of body weight will be given 1 mL of supplemental fluids (warmed Ringer’s solution) via subcutaneous injection twice daily. As needed, animals that appear in pain will be administered buprenorphine, BID.

[0188] Dosing. Throughout the study, animals in Group 1 will not be administered any treatment. Animals in Groups 2-8 will be administered vehicle or test article via oral gavage (PO), with dosing beginning on Day 21 and administered twice daily (BID) until Day 55, as follows. Animals in Groups 2, 3, and 6 will receive vehicle or rezameterlinib as shown in Table 2 for both BID doses. Animals in Groups 4, 5, and 7 will be administered ibrutinib and / or besutonimb for the first / AM dose and vehicle for the last / PM dose at each daily dosing. Animals in Group 8 will be administered rezameterlinib and besutonimb for the first / AM dose and rezameterlinib alone for the last / PM dose at each daily dosing.

[0189] In-life monitoring. Animals will be observed daily to assess for possible differences between treatment groups and / or possible toxicities resulting from treatment. Daily readings will be weight change, survival, standard GVHD scores (Table 3), modified GVHD scores (Table 4), incidence of diarrhea, and incidence of bloody stool.

[0190] Clinical scores for GVHD will be obtained daily throughout the duration of the study as assessed by the standard scoring system (Table 3) and the modified scoring system (Table 4). The standard GVHD score is based on 5 criteria: percent weight change, posture (hunched), activity, skin texture, and skin integrity (max index = 10). The modified GVHD score is based on 5 criteria: percent weight change, posture (hunched), activity, skin texture, and skin integrity (max index = 18). The overall score will be reported and the score for each individual parameter will be recorded.

[0191] On Days 33, 36, 39, 42, and 45, all animals will be photographed (top down picture, pure white background) under isoflurane anesthesia to assess disease severity. Each picture will be labeled with the number of the corresponding animal to aid in identification.

[0192] Findings of dead or moribund animals. Animals that are unable to self- care, feel cold to the touch, are moribund, or have lost more than 30% of their weight will be euthanized. Animals requiring euthanasia will be euthanized by CO2 inhalation and no organs will be collected.

[0193] Sacrifice. On Day 56, all surviving animals will be sacrificed by oral gavage of an overdose of metoclopramide and the organs detailed in Table 2 will be collected.

[0194] Sample collection. Blood, lung, skin, and spleen will be collected at the time of sacrifice.

[0195] Blood. Approximately 0.2 mL of blood will be collected from all animals by retro-orbital bleeds into K2EDTA tubes prior to sacrifice. The blood will be centrifuged and the plasma will be collected and stored at -80°C.

[0196] Lung. The lungs will be excised and the total lung and right lung will be weighed. The right lung will be ligated on the right bronchus, excised below the ligation point, snap frozen in liquid nitrogen and stored at -80°C. The left lung will be inflated with 10% NBF, ligated on the trachea to maintain inflation, fixed in 10% NBF for 24 hours, then moved to PBS prior to histopathology processing.

[0197] Skin.Prior to collection, the skin was shaved of hair. A 1 x 1 cm section of skin (from the back, between the shoulders, near the head) was excised, trimmed of any excess fat and / or connective tissue, sandwiched between foam in a tissue cassette, and placed in formalin. After 24 hours, the skin was transferred to PBS for storage until subsequent histological analysis. A second 1 cm x 1 cm section of skin (from the back, between the shoulders, directly below the first) was excised, trimmed of any excess fat and / or connective tissue, weighed, snap-frozen, and stored at -80 °C. If there were visible lesions, collection was performed in a manner that captured portions of the lesions in both the fixed and frozen sections.

[0198] Spleen. The spleen was excised and trimmed of any excess connective tissue. The spleen was then weighed, snap-frozen, and stored at -80 °C.

[0199] Histopathology. Fixed lung and skin samples were embedded in paraffin, sectioned at 5 microns, and slides were stained with hematoxylin and eosin (H&E). One slide per block was sectioned and stained with hematoxylin and eosin (H&E). All slides will be evaluated and scored using an optical microscope by a board-certified veterinary pathologist. Example 4. Activity of the BTK inhibitors, revzabrutinib and ibrutinib, in a sclerodermatous chronic graft versus host disease (cGVHD) mouse model.

[0200] The primary objective of this study was to evaluate the effect of selected BTK inhibitors on a murine model of sclerodermatous chronic graft versus host disease (GVHD). Animals.

[0201] Recipient female C57B1 / 6 (CD45.1) mice (n = 68; 8 weeks) with a mean starting body weight (± SEM) of 18.75 ± 0.12 g were obtained from Jackson Laboratories (Bar Harbor, ME). Donor female LP / J mice (n = 93; 6-10 weeks) were obtained from Jackson Laboratories (Bar Harbor, ME). Animals were acclimated for at least three days prior to study initiation. During this period, animals were observed daily to cull any animals that were not doing well.

[0202] The study was conducted in an animal room provided with HEPA filtered air, temperature of 70 ± 5 °F, and relative humidity of 50% ± 20%. Animals were housed in groups of 6-10 per cage. The animal room was set to maintain at least 12 to 15 air changes per hour. An automatic timer was used for a 12 hour light / dark cycle (no dusk). Alpha-dri® bedding was used. Cages, tops, and water bottles were washed with commercial cleaner and allowed to air dry. Floors were swept daily and mopped with commercial cleaner at least twice per week. Walls and cage racks were wiped with dilute bleach solution at least once per month. All cages were labeled with cage cards or labels with appropriate information identifying the study, dose, number of animals, and treatment group. Temperature and relative humidity were recorded during the study and retained.

[0203] Animals were fed LabDiet 5053 rodent diet and water ad libitum. Following total body irradiation (TBI) on Day -1 through Day 8, all animals were given supplemental high caloric, high palatability food.

[0204] At the start of the study, animals were randomized into seven (7) groups: one group of six (6) mice, one group of twelve (12) mice, and five (5) groups of ten (10) mice. Each animal was identified by ear punch corresponding to individual number. Each cage was identified using a cage card and the cage card was labeled with study number, treatment group number, and animal number. Study design.

[0205] Sclerodermatous chronic GVHD was induced in C57B1 / 6 (CD45.1) mice using a single acute dose of 8.5 Gy TBI on Day -1. On Day 0, C57B1 / 6 (CD45.1) recipients were intravenously (IV) injected with a combination of splenocytes and bone marrow cells in sterile lx PBS. Group 1 served as an untreated control group and did not receive TBI or cell transfer. Group 2 received allogeneic cell transfer from donor LP / J mice consisting only of bone marrow cells obtained from donor female LP / J (CD45.2) mice. Groups 3-7 received allogeneic cell transfer from donor LP / J mice consisting of both bone marrow and splenocytes obtained from donor female LP / J (CD45.2) mice. Splenocytes were isolated using a Miltenyi GentleMACS Dissociator. BM cells were isolated using standard flushing practices. Following transplantation, all animals were housed under standard environmental conditions and had ad libitum access to appropriate irradiated sterile rodent chow and sterile water. Additionally, all animals were given supplemental high caloric, high palatability food in the cage bottom pans after TBI on day -1 and through day 8. After the transfer on day 0, and then as needed after the transfer, all animals were given 1 mL / animal / day of supplemental fluids (warmed Ringer’s solution) via subcutaneous injection. Additional 1 mL of fluids were given as needed, possibly in the pm.

[0206] All animals were monitored daily to record weight changes, survival, and GVHD scores (Table 4 of Example 3), as well as the incidence of diarrhea and bloody stool. In addition, a second scoring system for sclerodermatous chronic GVHD was used (Table 5 of Example 3).

[0207] On day 20, animals in groups 3-7 were re-assigned to groups such that the average scGVHD scores for groups 3-7 were similar. The re-assignment was based on the composite 5-point scGVHD score described in Table 6. Animals were dosed with vehicle or test article as detailed in Table 6. Dosing began on day 21 and continued through day 55. Animals receiving ibrutinib were administered ibrutinib only for the AM dose, and then vehicle for the PM dose.

[0208] On days 33, 36, 39, 42, and 45, all animals were photographed (top-down pictures, pure white background) under isoflurane anesthesia to assess disease severity. Each picture was labeled with the number of the corresponding animal to aid in identification.

[0209] Animals that appeared to be in pain were administered buprenorphine, BID, as needed. Animals that were unable to self- care, felt cold to the touch, or were moribund were euthanized. Animals in need of euthanasia were euthanized by CO2 inhalation, and organs were not collected. Animals found dead were not collected. On day 56, all surviving animals were sacrificed by CO2 inhalation, and organs detailed in Table 6 were collected. Table 6. Study design. Total = 68 animals; Ibrutinib was administered for the AM dose, and then 0.5% MC vehicle was administered for the PM dose. Disease induction.

[0210] As detailed in Table 6, all recipient animals received a whole-body irradiation dose of 8.5 Gy on Day -1. On Day 0, Group 3-7 animals received bone marrow (BM) transplants containing a combination of bone marrow cells and splenocytes obtained from donor LP / J (CD45.2) mice via the tail vein (200 μL) according to Table 6. On Day 0, Group 2 animals received bone marrow (BM) transplants containing only bone marrow cells obtained from donor LP / J (CD45.2) mice via the tail vein (200 μL) according to Table 6. Splenocytes were isolated using a Miltenyi GentleMACS® Dissociator. BM cells were isolated using standard flushing practices (femur and tibia) and counted. Following transplantation, all recipient animals were housed under standard environmental conditions and had ad libitum access to appropriate rodent chow and sterile water. Dosing.

[0211] Vehicle. The vehicle was 0.5% methylcellulose (MC) administered at a dose of 0.1 mL / 20 g. Animals were treated twice daily on Days 21-55.

[0212] Ibrutinib. Ibrutinib was formulated with vehicle (0.5% MC). Ibrutinib was administered orally to mice in Group 4 at a dose of 12.5 mg / kg and to mice in Group 5 at a dose of 25 mg / kg. Animals were treated twice daily on Days 21-55 as follows: AM dose of Ibrutinib and PM dose of vehicle only.

[0213] Rizamatinib. Rizamatinib was formulated with vehicle (0.5% MC). Rizamatinib was administered orally to mice in Group 6 at a dose of 20 mg / kg and to mice in Group 7 at a dose of 40 mg / kg. Animals were treated twice daily on Days 21-55.

[0214] Throughout the study, animals in Group 1 were not administered any treatment. Animals in Groups 2-7 were administered vehicle or test article via oral gavage (PO), with dosing beginning on Day 21 and administered twice daily (BID) until Day 55. Animals in Groups 4 and 5 were administered Ibrutinib for the first / AM dose and vehicle for the last / PM dose at each daily dosing. Animals in Groups 2, 3, 6, and 7 received vehicle or test article as shown in Table 6 for both BID doses. In vivo monitoring and GVHD assessment.

[0215] Animals were observed daily to assess for possible differences between treatment groups and / or possible toxicities resulting from treatment. Daily readings were weight change, survival, standard GVHD score, modified GVHD score, incidence of diarrhea, and incidence of bloody stool.

[0216] Clinical scores for GVHD were obtained daily throughout the duration of the study as assessed by the standard scoring system (Table 4) and the modified scoring system for scGVHD (Table 5). The standard GVHD score was based on 5 criteria: percent weight change, posture (hunched), activity, coat texture, and skin integrity (max index = 10). The modified GVHD score for scGVHD was based on 5 criteria: percent weight change (0-4), posture (hunched) (0-3), activity (0-3), coat texture (0-4), and skin integrity (0-4) (max index = 18). The overall score as well as the score for each individual parameter was reported. Animals that exhibited pain were dosed with buprenorphine, BID, as needed.

[0217] On Days 33, 36, 39, 42, and 45, all animals were photographed (top down picture, pure white background) under isoflurane anesthesia to assess disease severity. Each picture was labeled with the number of the corresponding animal to aid in identification. Supportive care and euthanasia criteria

[0218] Following TBI on Day -1 through Day 8, all animals were given supplemental high caloric, highly palatable food in the tray at the bottom of the cage. Following transfer on Day 0, and then as needed following transfer, all animals were given 1 mL / animal / day of supplemental fluids (warmed Ringer’s solution) via subcutaneous injection. Additional 1 mL of fluids were given in the afternoon as needed. Any animal that lost > 15% of body weight was given highly palatable soft food and 1 mL of supplemental fluids (warmed Ringer’s solution) via subcutaneous injection once daily. Any animal that lost > 20% of body weight was given 1 mL of supplemental fluids (warmed Ringer’s solution) via subcutaneous injection twice daily. Animals that exhibited pain were dosed with buprenorphine, BID, as needed. Any animal that lost > 30% of body weight, was unable to eat, was unable to self-care, felt cold to the touch, or was moribund was euthanized. Animals that were found dead or euthanized prior to the scheduled termination day were not subjected to terminal collection. Sacrifice and sample collection.

[0219] At the end of the study (Day 56), all surviving animals were sacrificed by oral gavage of an overdose of metoclopramide and organs detailed in Table 6 were collected. Blood, lung, skin, and spleen were collected at the time of sacrifice.

[0220] Blood. Approximately 0.2 mL of blood was collected from all animals by retro-orbital bleeds into K2EDTA tubes prior to sacrifice. Blood was centrifuged and plasma was collected and stored at -80 °C.

[0221] Lung.Lungs were excised and whole lung and right lung were weighed. The right lung was ligated on the right bronchus, excised below the ligation point, snap frozen in liquid nitrogen and stored at -80°C. The left lung was insufflated with 10% neutral buffered formalin (NBF), ligated on the trachea to maintain insufflation, fixed in 10% NBF for 24 hours, then moved to PBS prior to histopathology processing.

[0222] Skin. Prior to collection, the skin was shaved of hair. A 1 x 1 cm section of skin (from the back of the animal, between the shoulders, near the head) was excised, trimmed of any excess fat and / or connective tissue, sandwiched between foam in a tissue cassette, and placed in formalin. After 24 hours, the skin was transferred to PBS for storage until subsequent histological analysis. A second 1 cm x 1 cm section of skin (from the back of the animal, between the shoulders, directly below the first section) was excised, trimmed of any excess fat and / or connective tissue, weighed, snap frozen, and stored at -80°C. If there were visible lesions, collection was performed in a manner that captured portions of the lesions in both the fixed and frozen sections.

[0223] Spleen. The spleen was excised and trimmed of any excess connective tissue. The spleen was then weighed, snap frozen, and stored at -80°C. Overall survival and progression-free survival.

[0224] Animal deaths were evaluated during the course of the study. In this model, animal deaths were generally attributed to severe illness. In this study, eight (8) animals were found dead, or were euthanized due to criteria for euthanasia or moribundity. Progression-free survival was defined as an increase in GVHD score of less than or equal to 2 compared to the Day 21 GVHD score (standard or modified). A difference in GVHD score of 3 or greater relative to Day 21 meant that the animal was no longer in progression-free survival and had begun to progress in disease. Skin and lung histopathology.

[0225] Fixed lung and skin samples were embedded in paraffin, sectioned at 5 microns, and slides were stained with hematoxylin and eosin (H&E). One slide per block was sectioned and stained with hematoxylin and eosin (H&E). All slides were evaluated and scored using a light microscope by a board-certified veterinary pathologist. Outcome evaluation.

[0226] Study endpoints were body weight change, survival, progression-free survival, standard GVHD score, modified scGVHD score, and histopathology. Statistical analysis.

[0227] Parametric data (weight change) were evaluated using one-way ANOVA and Dunnett’s multiple comparison test to compare all groups to the vehicle control group. Non-parametric data (GVHD scores) were analyzed with Kruskal-Wallis and Dunn’s post-test. All statistical analyses were performed using GraphPad Prism 8.4.3 software (La Jolla, CA). Statistical significance was reached when p < 0.05. Results.

[0228] Survival. Survival of all animals was tracked during the course of the study, and the percent survival is shown in Figure 4

[0229] All untreated animals (Group 1) survived to the end of the study. Two animals died prior to the start of treatment: one animal received BM only (Group 2), and one animal received BM with splenocytes. After the start of treatment on Day 21, all vehicle-treated animals that received BM only (Group 2) and diseased animals treated with 12.5 mg / kg ibrutinib (Group 4) survived to the end of the study. In the diseased animals, the survival rate was 90% for animals treated with 25 mg / kg ibrutinib (Group 5), 90% for animals treated with 40 mg / kg rezapolitib (Group 7), 82% for vehicle-treated animals (Group 3), and 80% for animals treated with 20 mg / kg rezapolitib (Group 6).

[0230] Body weight. GVHD disease induction (cell transfer of splenocytes and bone marrow cells) prevented normal weight gain in all diseased animals. Survival beyond Day 14 indicated successful engraftment of donor cells. Percent weight change is plotted in Figure 5A Figure 5B ​​​

[0231] To account for the mean weight change due to death in the study that resulted in survivor bias, death weights were performed until day 56 and presented in Figure 5B By AUC analysis from day 0, animals that did not receive treatment (group 1) showed significantly more weight gain compared to vehicle-treated animals that received BM only (group 2) (p < 0.05). After adjustment for survivor bias, vehicle-treated animals that received BM only (group 2) continued to show significantly more weight gain compared to diseased animals treated with vehicle (group 3). No significant difference in weight change was observed between diseased animals treated with test article (groups 4-7) and vehicle-treated (group 3) by AUC analysis. The trend of increased weight gain in diseased animals treated with 12.5 mg / kg ibrutinib (group 4) and 20 mg / kg rezanimod (group 6) compared to vehicle-treated (group 3) remained after adjustment for survivor bias.

[0232] GVHD score - standard scale. GVHD disease induction (cell transfer of splenocytes and bone marrow cells) induced disease in all animals in groups 3-7 as assessed using the multi-parameter GVHD scoring system shown in Table 4. Survival of animals beyond day 14 confirmed successful engraftment of transplanted cells. Standard GVHD scores are shown in Figure 6A By AUC analysis from day 0, animals that did not receive treatment (group 1) showed significantly lower standard GVHD scores compared to vehicle-treated diseased animals (group 3) (p < 0.01). Although not statistically significant, vehicle-treated animals that received BM only (group 2) showed lower standard GVHD scores compared to diseased animals treated with vehicle (group 3). No significant difference in standard GVHD scores was observed between diseased animals treated with test article (groups 4-7) and vehicle-treated (group 3). However, diseased animals treated with 25 mg / kg ibrutinib (group 5), 20 mg / kg rezanimod (group 6), and 40 mg / kg rezanimod (group 7) had significantly lower standard GVHD scores compared to vehicle-treated (group 3).

[0233] To account for the change in GVHD scores due to death in the study that resulted in survivor bias, death scores were performed until day 56 and presented in Figure 6BFigure 6 shows the mean modified GVHD score over time for each group. The animals that did not receive treatment (Group 1) showed a significantly lower modified GVHD score when compared to the diseased animals treated with vehicle (Group 3) by AUC analysis from Day 0 (p < 0.001). After adjustment for survivor bias, the animals that received BM only treated with vehicle (Group 2) continued to show a significantly lower modified GVHD score when compared to the diseased animals treated with vehicle (Group 3). No significant difference in modified GVHD score was observed between the diseased animals treated with the test articles (Groups 4-7) and the vehicle treated (Group 3). The trend of reduced modified GVHD score in the diseased animals treated with 25 mg / kg ibrutinib (Group 5), 20 mg / kg revmuratnib (Group 6), and 40 mg / kg revmuratnib (Group 7) was dampened after adjustment for survivor bias when compared to the vehicle treated (Group 3).

[0234] GVHD score - modified scale. For sclerodermic GVHD, as assessed using the modified multi-parameter GVHD scoring system shown in Table 5, GVHD disease induction (cell transfer of splenocytes and bone marrow cells) induced disease in all animals in Groups 3-7. Animal survival beyond Day 14 confirmed successful engraftment of the transplanted cells. The modified GVHD score is shown in Figure 7A Figure 7 shows the mean modified scGVHD score over time for each group. The animals that did not receive treatment (Group 1) showed a significantly lower modified scGVHD score when compared to the diseased animals treated with vehicle (Group 3) by AUC analysis from Day 0 (p < 0.01). Although not statistically significant, the animals that received BM only treated with vehicle (Group 2) showed a lower modified GVHD score when compared to the diseased animals treated with vehicle (Group 3). No significant difference in modified GVHD score was observed between the diseased animals treated with the test articles (Groups 4-7) and the vehicle treated (Group 3). However, the diseased animals treated with 25 mg / kg ibrutinib (Group 5), 20 mg / kg revmuratnib (Group 6), and 40 mg / kg revmuratnib (Group 7) had a significantly lower modified GVHD score when compared to the vehicle treated (Group 3).

[0235] To account for the average scGVHD score changes due to death in the study that caused survivor bias, a death score was performed until Day 56 and is presented in Figure 7Banimals (Group 1) showed significantly lower modified GVHD scores when mortality score resolution was analyzed (p < 0.001) compared to diseased animals treated with vehicle (Group 3). After adjustment for survivor bias, animals treated with vehicle that received BM only (Group 2) continued to show significantly lower modified GVHD scores compared to diseased animals treated with vehicle (Group 3). No significant differences in modified GVHD scores after mortality score resolution were observed between diseased animals treated with test article (Groups 4-7) and vehicle (Group 3). The trend of reduced modified GVHD scores in diseased animals treated with 25 mg / kg ibrutinib (Group 5), 20 mg / kg revmuratnib (Group 6), and 40 mg / kg revmuratnib (Group 7) compared to vehicle (Group 3) was dampened after adjustment for survivor bias.

[0236] Progression-free survival. Progression-free survival (PFS) was tracked for all animals during the course of the study and plotted as a percentage of animals that were progression-free, as shown in Figure 8A (standard GVHD scale) and Figure 8B (modified scGVHD scale). Progression-free survival was defined as an increase in GVHD score of less than or equal to 2 compared to Day 21 GVHD score (standard or modified). Disease progression was defined as an increase in GVHD score of greater than 2 compared to Day 21 GVHD score (standard or modified). Under both metrics, animals induced with GVHD showed reduced PFS compared to animals that did not receive treatment (Group 1), all of which did not experience disease progression. Analysis using the standard GVHD score scale indicated that 56% of animals treated with vehicle that received BM only (Group 2) did not show progressive disease by Day 56. Among the animals with GVHD, 40% of animals treated with 25 mg / kg ibrutinib (Group 5), 30% of animals treated with 20 mg / kg revmuratnib (Group 6), 10% of animals treated with 40 mg / kg revmuratnib (Group 7), 10% of animals treated with 12.5 mg / kg ibrutinib (Group 4), and 9% of animals treated with vehicle (Group 3) did not show progressive disease by Day 56. Due to the granularity of the modified score scale, analysis using the modified scGVHD scale showed that animals that received BM only (Group 2) showed progressive disease by Day 43, and animals that received BM with splenocytes (Groups 4-7) showed progressive disease by Day 32.

[0237] Skin and lung histopathology.Fixed skin and left lung samples from surviving animals (n = 60) were embedded in paraffin and sectioned at 5 microns. One slide per block was sectioned and stained with H&E. All slides were evaluated and scored by a board-certified veterinary pathologist using a light microscope. Lesions in both the lung and skin were scored for severity on an ordinal scale ranging from 0-5, where 0 = not present / within normal limits, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe, according to the extent found. Grade 0: within normal limits Grade 1 : minimal; < 10% of tissue compartment affected or minimal diffuse changes Grade 2: mild; 10-25% of tissue compartment affected or mild diffuse changes Grade 3: moderate; 26-50% of tissue compartment affected Grade 4: marked; 51-75% of tissue compartment affected Grade 5: severe; > 75% of tissue compartment affected In each organ, histological feature scores were added to obtain a total score. Lung range: 0-10; Skin range: 0-30.

[0238] Dermis thickness measurements. Using H&E-stained slides, the dermis was measured (pm) in five areas throughout the tissue, calculating the mean value for each sample. Measurements were taken from the epidermal basement membrane to the shallowest border of dermal collagen with subcutaneous fat. These measurements were only taken in non-tangential sectioned tissue areas that were free of histological artifacts and had an intact epidermis.

[0239] Statistical analysis. Data were presented as mean ± standard error of the mean (SEM). Semi-quantitative sum scores and individual histopathology scores were analyzed by non-parametric ANOVA (Kruskal-Wallis H test) and Dunn’s multiple comparison post-test (GraphPad Prism); skin measurements were analyzed by one-way ANOVA and post-hoc Tukey’s test (as appropriate). For all tests, significance was set at p < 0.05. Results and discussion. Histomorphology (H&E)

[0240] Lung.Histological findings in lung tissue were consistent with expected findings in a model of chronic GVHD. The primary finding was infiltration of mononuclear cells (predominantly lymphocytes, with fewer plasma cells and macrophages; rare neutrophils) in the perivascular region, and, less commonly, in the peribronchiolar region. Mononuclear cell infiltration was typically observed around the largest caliber vessels, with more severely affected samples exhibiting dense cuffs (up to 15 cells in thickness) from large to small caliber vessels. Extension into adjacent interstitium was rarely observed. Increased numbers of alveolar macrophages were also consistently visible, sometimes in interstitial infiltrate regions, but more commonly in the peripheral alveolar regions. Sporadically, untreated mice (Group 1) had minimal to mild perivascular / peribronchiolar mononuclear cell infiltration (consistent with bronchiolar-associated lymphoid tissue), with similar findings in mice receiving bone marrow cell transfer only (Group 2). A significant increase in total histopathology score was observed in animals receiving bone marrow and spleen cell transfer (Group 3) compared to animals receiving bone marrow transfer only (Group 2; p = 0.01) or no treatment (Group 1; p = 0.007) Figure 9A ). Both perivascular mononuclear cell and alveolar histiocyte proliferation scores were increased in GVHD mice Figure 9B . A small decrease in total and individual histopathology scores Figure 9A Figure 9B was observed with ibrutinib (Groups 4 and 5) or zanabrutinib (Groups 6 and 7) compared to vehicle, but none of the comparisons reached statistical significance.

[0241] Skin. ​There was a wide variation in the severity of histological findings in the skin among and within animals receiving cell transfer. Histological findings ranged from serum cell crusting, epidermal erosion or ulceration, epidermal and follicular hyperplasia, mononuclear inflammatory cell infiltration within the epidermis / hair follicle (interface dermatitis pattern), and dermal fibrosis. Note that the most common primary findings included minimal to mild epidermal hyperplasia, dermal mononuclear cell infiltration, and dermal fibrosis; crusting, significant necrosis with ulceration, interface-type epidermal inflammation, and significant epidermal and follicular hyperplasia were observed sporadically. Serum cell crusting was characterized by the accumulation of proteinaceous fluid, necrotic debris, and degenerative neutrophils on the surface of the epidermis, with or without epidermal necrosis, typically in short segments of partial thickness (erosion) or full thickness, sometimes extending into the subcutaneous tissue (ulceration). Epidermal hyperplasia was characterized by thickening of the epidermis, manifested as an increased layer of nucleated keratinocytes, 3-8 cells in thickness. Mononuclear cell (lymphocyte) infiltration in the superficial dermis rarely extended into the epidermis or hair follicle, with necrotic / apoptotic keratinocytes in the interface-type lesions, and follicular rupture was rarely observed; the latter finding was accompanied by perifollicular infiltration of neutrophils and macrophages. In most samples, mononuclear cell infiltration was primarily seen in the dermis, with inconsistent correlation to areas of hyperplastic epidermis and areas of dermal fibrosis, which was characterized by parallel deposition of collagen bundles or amphiphilic extracellular matrix, distorting the normal dermal collagen organization. Dermal fibrosis and inflammation resulted in slight thickening of the dermis. In untreated mice (Group 1), histological findings were minimal, with more severe findings observed with the addition of bone marrow (Group 2) compared to bone marrow + splenocyte transfer (Group 3); however, bone marrow + splenocyte transfer (Group 3) induced a significant decrease in total score compared to untreated (Group 1; p = 0.006) Figure 10A ]. Application of 25 mg / kg of ibrutinib (Group 5) or either dose of rezamolertinib (Groups 6 and 7) was associated with a decrease in total score compared to treatment with vehicle (Group 3) Figure 10A ]; these changes were driven by several features, but epidermal hyperplasia and dermal fibrosis scores Figure 10B ] were the most significant, including a significant decrease in dermal fibrosis score with ibrutinib (25 mg / kg; Group 5; p = 0.02) compared to vehicle (Group 3), and a statistical trend toward decreased score was observed with 40 mg / kg rezamolertinib (Group 7; p = 0.09). Dermal thickness measurements (H&E)

[0242] Skin from untreated mice (Group 1) was within the normal range, with minimal intra-group variability in dermal thickness measurements Figure 11). Cell transfer of bone marrow (Group 2) or bone marrow with splenocytes (Group 3) was associated with a slightly greater dermis thickness, which was associated with inflammatory cell infiltration and fibrosis. Test article treatment had minimal effect on dermis thickness, with a slight decrease in mean thickness observed in animals given 20 mg / kg of rezamolertinib (Group 6). In this study, dermis thickness measurements did not reach significance between groups (p = 0.3). Conclusions.

[0243] All untreated animals survived to the end of the study. After starting treatment on Day 21, all vehicle-treated animals that received only BM and diseased animals treated with 12.5 mg / kg of ibrutinib survived to the end of the study. In the diseased animals, the survival rate was 90% for animals treated with 25 mg / kg of ibrutinib, 90% for animals treated with 40 mg / kg of rezamolertinib, 82% for vehicle-treated animals, and 80% for animals treated with 20 mg / kg of rezamolertinib.

[0244] By AUC analysis, untreated animals showed significantly more weight gain when compared to vehicle-treated diseased animals. After adjustment for survivor bias, vehicle-treated animals that received only BM continued to show significantly more weight gain when compared to vehicle-treated diseased animals. No significant difference in weight change was observed between the diseased animals treated with test article and vehicle. The increasing trend in weight gain for diseased animals treated with 12.5 mg / kg of ibrutinib and 20 mg / kg of rezamolertinib compared to vehicle treatment persisted after adjustment for survivor bias.

[0245] By AUC analysis, untreated animals showed significantly lower post- mortality score adjusted standard GVHD scores when compared to vehicle-treated diseased animals. After adjustment for survivor bias, vehicle-treated animals that received only BM continued to show significantly lower standard GVHD scores when compared to vehicle-treated diseased animals. No significant difference in post-mortality score adjusted standard GVHD scores was observed between the diseased animals treated with test article and vehicle. The decreasing trend in standard GVHD scores for diseased animals treated with 25 mg / kg of ibrutinib, 20 mg / kg of rezamolertinib, and 40 mg / kg of rezamolertinib compared to vehicle treatment was suppressed after adjustment for survivor bias.

[0246] With AUC analysis, animals that did not receive treatment showed significantly lower modified GVHD scores when mortality score was censored compared to vehicle-treated diseased animals. After adjustment for survivor bias, animals that received BM only treated with vehicle continued to show significantly lower modified GVHD scores compared to vehicle-treated diseased animals. No significant difference in modified GVHD scores after mortality score was censored was observed between diseased animals treated with test article and vehicle. The trend of reduction in modified GVHD scores in diseased animals treated with 25 mg / kg ibrutinib, 20 mg / kg revmurafenib, and 40 mg / kg revmurafenib was dampened after adjustment for survivor bias compared to vehicle treatment.

[0247] Based on the PFS criteria and the modified GVHD score scale, animals that did not receive treatment did not show disease progression. Analysis using the standard GVHD score scale indicated that 56% of animals that received BM only treated with vehicle did not show progressive disease by day 56. Whereas in the GVHD animals, 40% of animals treated with 25 mg / kg ibrutinib, 30% of animals treated with 20 mg / kg revmurafenib, 10% of animals treated with 40 mg / kg revmurafenib, 10% of animals treated with 12.5 mg / kg ibrutinib, and 9% of animals treated with vehicle did not show progressive disease by day 56. Due to the granularity of the modified score scale, analysis using the modified scGVHD scale showed that animals that received BM only showed progressive disease by day 43, and all animals that received BM with splenocytes showed progressive disease by day 32.

[0248] Perivascular mononuclear cell infiltration was the predominant lung finding, with a range of findings in the skin, including epidermal and hair follicle hyperplasia, mononuclear cell infiltration, and dermal fibrosis, with sporadic development of more severe findings, including epidermal necrosis / ulceration, interface dermatitis-type mononuclear cell infiltration in diseased animals. Treatment with ibrutinib or revmurafenib produced minimal changes in lung and skin lesion severity scores compared to vehicle treatment; however, the reduction in dermal fibrosis score reached statistical significance in diseased animals treated with 25 mg / kg ibrutinib, or trended towards statistical significance in diseased animals treated with 40 mg / kg revmurafenib.

[0249] In the chronic GVHD model, administration of splenocyte + bone marrow cell transfer from LP / J donor mice to irradiated C57B1 / 6 mice was associated with the development of histologic lesions in the lung and skin. Perivascular mononuclear cell infiltration was the predominant lung finding, with a range of findings in the skin, including epidermal and hair follicle hyperplasia, mononuclear cell infiltration, and dermal fibrosis, with sporadic development of more severe findings, including epidermal necrosis / ulceration, interface dermatitis-type mononuclear cell infiltration.

[0250] Treatment with ibrutinib or zanabrutinib produced minimal changes in lung and skin lesion severity scores compared with vehicle treatment; however, in this study, the skin fibrosis score decreased to statistical significance with ibrutinib or tended toward statistical significance with zanabrutinib. Example 5. Activity of BTK inhibitors besuximab, zanabrutinib, and ibrutinib in a sclerodermatous chronic graft versus host disease (cGVHD) mouse model.

[0251] The primary goal of this study was to evaluate the impact of a combination of selected BTK inhibitors on a sclerodermatous chronic graft versus host disease (GVHD) murine model. However, as shown herein, the data were inconclusive due to multiple study limitations. Among the limitations, disease induction was not robust, and the positive control failed to show a significant difference. Therefore, treatments expected to show disease-modifying activity in the model did not perform as expected. Animals.

[0252] Recipient female C57B1 / 6 (CD45.1) mice (n = 78; 8 weeks) with a mean starting body weight (± SEM) of 18.55 ± 0.13 g were obtained from Jackson Laboratories (Bar Harbor, ME). Donor female LP / J mice (n = 108; 6-10 weeks) were obtained from Jackson Laboratories (Bar Harbor, ME). Animals were acclimated prior to study initiation. During this period, animals were observed daily to cull any animals that were not doing well.

[0253] The study was conducted in an animal room provided with HEPA filtered air, temperature of 70 ± 5 °F, and relative humidity of 50% ± 20%. Animals were housed in groups of 6-12 per cage. The animal room was set to maintain at least 12 to 15 air changes per hour. An automatic timer was used for a 12-hour light / dark cycle (no dusk). Alpha-dri® or equivalent bedding was used. Cages, tops, and water bottles were washed with commercial cleaner and allowed to air dry. Floors were swept daily and mopped with commercial cleaner at least twice per week. Walls and cage racks were wiped with a dilute bleach solution at least once per month. All cages were labeled with cage cards or tags with the appropriate information identifying the study, dose, number of animals, and treatment group. Temperature and relative humidity were recorded during the study and retained.

[0254] Animals were fed LabDiet 5053 rodent diet and water was provided ad libitum. At the start of the study, animals were randomized into eight (8) groups: one (1) group of six (6) animals, one (1) group of 12 animals, and six (6) groups of ten (10) animals each. Each animal was identified by ear punch corresponding to individual number. Each cage was identified using a cage card, and the cage card was labeled with study number, treatment group number, and animal number. Study design.

[0255] Sclerodermatous chronic GVHD was induced in C57B1 / 6 (CD45.1) mice using a single acute dose of 8.5 Gy of total body irradiation (TBI) on Day -1. On Day 0, C57B1 / 6 (CD45.1) recipients were intravenously (IV) injected with a combination of splenocytes and bone marrow cells in sterile lx PBS: Group 1 served as an untreated control group and did not receive TBI or cell transfer. Group 2 received allogeneic cell transfer from donor LP / J mice consisting only of bone marrow cells obtained from donor female LP / J (CD45.2) mice. Groups 3-8 received allogeneic cell transfer from donor LP / J mice consisting of both bone marrow and splenocytes obtained from donor female LP / J (CD45.2) mice. Splenocytes were isolated using a Miltenyi GentleMACS Dissociator. BM cells were isolated using standard flushing practices. Following transplantation, all animals were housed under standard environmental conditions and had ad libitum access to appropriate irradiated sterile rodent chow and sterile water.

[0256] Additionally, all animals were given supplemental high caloric, high palatability food in the tray at the bottom of the cage after TBI on day -1 and until day 8. After transfer on day 0, and then as needed after, all animals were given 1 mL / animal / day of supplemental fluids (warmed Ringer's solution) via subcutaneous injection. An additional 1 mL of fluids was given pm as needed.

[0257] All animals were monitored daily to record weight changes, survival, GVHD scores (Table 4), and incidence of diarrhea and bloody stool. In addition, a second scoring system for sclerodermatous chronic GVHD (scGVHD) was used (Table 5).

[0258] On day 20, animals in groups 3-8 were re-assigned to groups such that the average scGVHD score for groups 3-8 was similar. Re-assignment was based on the composite 5-point scGVHD score described in Table 5.

[0259] Animals were dosed with vehicle or test article as detailed below in Table 7. Dosing began on day 21 and continued through day 55. Animals receiving ibrutinib were dosed with ibrutinib only for the AM dose, and then with vehicle for the PM dose.

[0260] On days 33, 36, 39, 42, and 45, all animals were photographed (top-down pictures, pure white background) under isoflurane anesthesia to assess disease severity. Each photograph was labeled with the number of the corresponding animal to aid in identification.

[0261] Animals that appeared to be in pain were dosed with buprenorphine, BID, as needed. Animals that were unable to self- care, felt cold to the touch, or were moribund were euthanized. Animals that required euthanasia were euthanized by CO2 inhalation and were not collected. Animals found dead were not collected.

[0262] On day 56, all surviving animals were sacrificed by CO2 inhalation and organs were collected. Details of the study design are shown in Table 7. Table 7. Study design. Total = 78 animals; Bexagliflozin and / or ibrutinib were administered for the AM dose, and then 0.5% MC vehicle was administered for the PM dose; Animals were dosed with 125 mg / kg bexagliflozin from day 21 to day 32. Due to weight loss observed in bexagliflozin-treated animals (group 4), the bexagliflozin dose was reduced to 100 mg / kg for groups 4, 7, and 8. Animals in groups 4, 7, and 8 were dosed with 100 mg / kg bexagliflozin from day 33 to day 55. Disease induction.

[0263] As detailed in Table 7, all recipient animals received a whole-body irradiation dose of 8.5 Gy on Day -1. On Day 0, Group 3-8 animals received bone marrow (BM) transplants containing a combination of bone marrow cells and splenocytes obtained from donor LP / J (CD45.2) mice via the tail vein (200 μL) according to Table 7. On Day 0, Group 2 animals received bone marrow (BM) transplants containing only bone marrow cells obtained from donor LP / J (CD45.2) mice via the tail vein (200 μL) according to Table 7. Splenocytes were isolated using a Miltenyi GentleMACS® Dissociator. BM cells were isolated using standard flushing practices (femur and tibia) and counted. Following transplantation, all recipient animals were housed under standard environmental conditions and had ad libitum access to appropriate rodent chow and sterile water.

[0264] Vehicle. The vehicle was 0.5% methylcellulose (MC) administered at a dose of 0.1 mL / 20 g. Animals were treated twice daily (BID) on Days 21-55.

[0265] Bosutinib. Bosutinib was formulated with vehicle (0.5% MC). Bosutinib was administered orally to mice in Groups 4, 7, and 8 at a dose of 125 mg / kg on Days 21-32 and 100 mg / kg on Days 33-55. Animals were treated twice daily (BID) on Days 21-55 as follows: for Group 4, the AM dose was bosutinib and the PM dose was vehicle only; for Group 8, zanabrutinib was formulated in combination with bosutinib for the AM dose.

[0266] Ibrutinib. Ibrutinib was formulated with vehicle (0.5% MC). Ibrutinib was administered orally to mice in Group 5 and Group 7 at a dose of 20 mg / kg. Animals were treated twice daily (BID) on Days 21-55 as follows: for Group 5, the AM dose was i brutinib and the PM dose was vehicle only; for Group 7, i brutinib was formulated in combination with bosutinib for the AM dose.

[0267] Zanabrutinib. Zanabrutinib was formulated with vehicle (0.5% MC). Zanabrutinib was administered orally to mice in Group 6 and Group 8 at a dose of 40 mg / kg. Animals were treated twice daily (BID) on Days 21-55.

[0268] Animals in Group 1 were not administered any treatment throughout the study. Animals in Groups 2-8 were administered vehicle or test article via oral gavage (PO), with dosing starting on Day 21 and administered twice daily (BID) until Day 55. Animals in Groups 2, 3, and 6 received vehicle or rezameterlinib for both BID doses as shown in Table 7. Animals in Groups 4, 5, and 7 were administered ibrutinib and / or besuxoer at the AM dose and vehicle at the PM dose at each daily dosing. Animals in Group 8 were administered rezameterlinib and besuxoer at the AM dose and rezameterlinib alone at the PM dose at each daily dosing. On Day 33, the besuxoer dose was reduced from 125 mg / kg to 100 mg / kg for Groups 4, 7, and 8, In-life monitoring and GVHD assessment.

[0269] Animals were observed daily to assess for possible differences between treatment groups and / or possible toxicities resulting from treatment. Daily readings were weight change, survival, standard GVHD score, modified GVHD score, incidence of diarrhea, and incidence of bloody stool. The incidence of diarrhea and bloody stool was zero throughout the study.

[0270] Clinical scores for GVHD were obtained daily throughout the duration of the study as assessed by the standard scoring system (Table 4) and the modified scoring system for scGVHD (Table 5). The standard GVHD score was based on 5 criteria: percent weight change, posture (hunched), activity, coat texture, and skin integrity (maximum index = 10). The modified GVHD score was based on 5 criteria: percent weight change, posture (hunched), activity, coat texture, and skin integrity (maximum index = 18). The overall score as well as the score for each individual parameter was reported.

[0271] On Days 33, 36, 39, 42, and 45, all animals were photographed (top-down picture, pure white background) under isoflurane anesthesia to assess disease severity. Each picture was labeled with the number of the corresponding animal to aid in identification. Supportive care and euthanasia criteria.

[0272] Following TBI on Day -1 through Day 8, all animals were given supplemental high-calorie, high-palatability food in the cage floor pans. Following transfer on Day 0, and then as needed, all animals were given 1 mL / animal / day of supplemental fluids (warmed Ringer's Solution) via subcutaneous injection. Additional 1 mL of fluids were given in the afternoon as needed. Any animal that lost > 15% of body weight was given high-palatability chow and 1 mL of supplemental fluids (warmed Ringer's Solution) via subcutaneous injection once daily. Any animal that lost > 20% of body weight was given 1 mL of supplemental fluids (warmed Ringer's Solution) via subcutaneous injection twice daily. Animals that exhibited signs of pain were dosed with buprenorphine, BID, as needed. Any animal that lost > 30% of body weight, was unable to self- care, felt cold to the touch, or was moribund was euthanized. Animals requiring euthanasia were euthanized by CO2 inhalation, and no organs were collected. Euthanasia and sample collection.

[0273] On Day 56, all surviving animals were euthanized by overdosing with ketamine and xylazine, and organs detailed in Table 7 were collected. Blood, lung, skin, and spleen were collected at the time of euthanasia.

[0274] Blood. Approximately 0.2 mL of blood was collected from all animals by retro-orbital bleeds into K2EDTA tubes prior to euthanasia. Blood was centrifuged, and plasma was collected and stored at -80 °C.

[0275] Lung. The lungs were excised, and the whole lung and right lung were weighed. The right lung was ligated on the right bronchus, excised below the ligation point, snap-frozen in liquid nitrogen, and stored at -80 °C. The left lung was inflated with 10% neutral buffered formalin (NBF), ligated on the trachea to maintain inflation, fixed in 10% NBF for 24 hours, and then moved to PBS.

[0276] Skin. Prior to collection, the skin was shaved. A 1 x 1 cm section of skin (from the back of the animal, between the shoulders, near the head) was excised, excess fat and / or connective tissue was trimmed away, sandwiched between foam in a tissue cassette, and placed in formalin. After 24 hours, the skin was transferred to PBS for storage until subsequent histological analysis. A second 1 cm x 1 cm section of skin (from the back of the animal, between the shoulders, directly below the first section) was excised, excess fat and / or connective tissue was trimmed away, weighed, snap-frozen, and stored at -80 °C. If there were visible lesions, collection was performed in a manner that captured portions of the lesions in both fixed and frozen sections.

[0277] Spleen.The spleen was removed and trimmed of any excess connective tissue. The spleen was then weighed, snap-frozen, and stored at -80 °C. Survival.

[0278] Animal deaths were evaluated during the course of the study. In this model, animal deaths are generally attributed to severe illness. In this study, fifteen (15) animals were found dead, or euthanized for euthanasia criteria or moribundity. Progression-free survival was defined as an increase in GVHD score of less than or equal to 2 compared to the Day 21 GVHD score (standard or modified). A difference in GVHD score of 3 or greater relative to Day 21 meant that the animal was no longer in progression-free survival and had begun to progress in disease. Outcome evaluation.

[0279] Study endpoints were body weight change, survival, progression-free survival, standard GVHD score, and modified scGVHD score. Statistical analysis.

[0280] Data were evaluated for comparison of all groups using one-way ANOVA and Tukey’s multiple comparison post-test. All statistical analyses were performed using GraphPad Prism 9.5.1 software (La Jolla, CA). Statistical significance was achieved when p < 0.05. Results.

[0281] Survival. Survival of all animals was tracked during the course of the study, and the percent survival is shown in Figure 12 .

[0282] All untreated animals (Group 1) survived to the end of the study. Eight animals that received BM only (Group 2) died prior to the start of the study, and a 10% survival rate was observed in this group at the end of the study. All diseased animals that received rezameterlin (Group 6) and besuxorubin + rezameterlin (Group 8) therapy survived to the end of the study after starting treatment on Day 21. Among the diseased animals, the survival rate for the vehicle-treated animals (Group 3) was 91.7%, for the ibrutinib-treated animals (Group 5) was 90%, for the besuxorubin-treated animals (Group 4) was 80%, and for the besuxorubin + ibrutinib-treated animals (Group 7) was 80%.

[0283] Body weight. GVHD disease induction (cell transfer of spleen and bone marrow cells) prevented normal body weight gain in all diseased animals. Survival beyond Day 14 indicated successful engraftment of donor cells. Percent weight change is plotted in Figure 13 , and percent weight change after weight carryover of deaths is plotted in Figure 14 .

[0284] Untreated animals (Group 1) showed normal weight gain throughout the study. By AUC analysis, untreated animals (Group 1) showed significantly more weight gain compared to vehicle-treated diseased animals (Group 3) (p < 0.0001). No significant differences in weight change were observed between diseased animals treated with the test article (Groups 4-8) and vehicle-treated (Group 3).

[0285] To account for the mean weight change resulting from deaths in the study that caused survivor bias, death weights were taken until Day 56 and presented in Figure 14 By AUC analysis after death weight carryover, untreated animals (Group 1) showed significantly more weight gain compared to vehicle-treated diseased animals (Group 3) (p < 0.05 and p < 0.001).

[0286] After adjustment for survivor bias, only BM-only vehicle-treated animals (Group 2) showed significantly more weight loss compared to all other groups, however, this observation was not statistically significant. No significant differences in weight change were observed between diseased animals treated with the test article (Groups 4-8) and vehicle-treated (Group 3).

[0287] GVHD score - standard scale. GVHD disease induction (cell transfer of splenocytes and bone marrow cells) was assessed using the multi-parameter GVHD scoring system shown in Table 4. Animal survival beyond Day 14 confirmed successful engraftment of transplanted cells. Standard GVHD scores are shown in Figure 15

[0288] By AUC analysis from Day 0, vehicle-treated diseased animals (Group 3) showed significantly higher standard GVHD scores compared to untreated animals (Group 1) (p < 0.05). No significant differences in standard GVHD scores were observed between diseased animals treated with the test article (Groups 4-8) and vehicle-treated (Group 3). However, diseased animals treated with rezamolatinib (Group 6) had slightly lower standard GVHD scores compared to vehicle-treated (Group 3).

[0289] To account for the GVHD score changes resulting from deaths in the study that caused survivor bias, death scores were taken until Day 56 and presented in Figure 16 ​The animals receiving BM only (Group 2) had a significantly higher standard GVHD score relative to all groups by AUC analysis of the scores carried forward by death. However, this was not statistically significant. The trend of higher standard GVHD scores in the diseased animals treated with vehicle (Group 3) compared to the untreated animals (Group 1) was suppressed after adjustment for survivor bias and was no longer statistically significant. No significant difference in standard GVHD scores was observed between the diseased animals treated with the test article (Groups 4-8) and the vehicle treated (Group 3). However, the trend of lower standard GVHD scores in ruxabrutinib (Group 6) compared to vehicle treated (Group 3) remained.

[0290] GVHD score - modified scale. For sclerodermic GVHD, GVHD disease induction (cell transfer of splenocytes and bone marrow cells) was assessed using the modified multi-parameter GVHD scoring system shown in Table 5. Successful engraftment of the transplanted cells was confirmed by animal survival beyond Day 14. The modified GVHD scores are shown in Figure 17

[0291] By AUC analysis, no statistically significant differences in scGVHD scores were observed between the groups. However, the following trends were observed. The diseased animals treated with vehicle (Group 3) exhibited significantly higher scGVHD scores compared to the untreated animals (Group 1). The animals receiving BM only (Group 2) showed a mild lower modified GVHD score compared to the diseased animals treated with vehicle (Group 3).

[0292] To account for the average scGVHD score changes due to death in the study that caused survivor bias, the deaths were scored until Day 56 and are presented in Figure 18 By AUC analysis of the scores carried forward by death, no statistically significant differences in scGVHD scores were observed between the groups. However, the diseased animals treated with vehicle (Group 3) continued to exhibit significantly higher scGVHD scores compared to the untreated animals (Group 1). The animals receiving BM only (Group 2) showed a significantly higher modified GVHD score compared to the diseased animals treated with vehicle (Group 3). No trend of improved scGVHD scores was observed with the test article treatment compared to vehicle treatment.

[0293] Progression-free survival. Progression-free survival (PFS) was tracked for all animals during the course of the study and the percentage of progression-free survival was plotted as Figure 19 (the standard GVHD scale) and Figure 20 ​Progression-free survival was defined as an increase in GVHD score of less than or equal to 2 compared to Day 21 GVHD score (standard or modified). Disease progression was defined as an increase in GVHD score of greater than 2 compared to Day 21 GVHD score (standard or modified).

[0294] Progression-free survival analysis by standard GVHD scale indicated that approximately 80% of the untreated animals (Group 1) had a change in score of greater than 2. According to the scGVHD scale, approximately 60% of the untreated animals (Group 1) had a change in score of greater than 2. However, this was primarily due to grooming that occurred in the untreated group, which resulted in mild alopecia and resulted in higher than expected scores for coat texture.

[0295] Progression-free survival analysis using the standard GVHD score scale indicated that approximately 58% of the diseased animals treated with vehicle (Group 3) did not show progressive disease by Day 56. Analysis using the modified scGVHD scale indicated that approximately 75% of the diseased animals treated with vehicle (Group 3) did not show progressive disease by Day 56. These results are related to the lower disease severity and disease penetrance in this study. Conclusions.

[0296] All untreated animals and diseased animals treated with rezameterlinide and the combination of besuxib and rezameterlinide survived to the end of the study. A 10% survival rate was observed in animals that received BM only. In the diseased animals, the survival rate was 91.7% for animals treated with vehicle, 90% for animals treated with ibrutinib, 80% for animals treated with besuxib, and 80% for animals treated with the combination of besuxib and ibrutinib.

[0297] Analysis by AUC indicated that untreated animals showed significantly more weight gain compared to diseased animals treated with vehicle. After adjustment for survivor bias with carryover of death weight, untreated animals continued to show significantly more weight gain compared to diseased animals treated with vehicle. Animals that received BM only treated with vehicle showed significantly more weight loss compared to all other groups, however, this observation was not statistically significant. No significant differences in weight change were observed between diseased animals treated with test article and vehicle treated.

[0298] Analysis by AUC, diseased animals treated with vehicle showed significantly higher standard GVHD scores compared to animals that did not receive treatment. No significant difference in standard GVHD scores was observed between diseased animals treated with test article and vehicle. However, diseased animals treated with rezameterlinib had slightly lower standard GVHD scores compared to vehicle treatment. After mortality score carryover, animals that received BM only had significantly higher standard GVHD scores relative to all groups, however, this finding was not statistically significant. The trend of higher standard GVHD scores in diseased animals treated with vehicle compared to animals that did not receive treatment was suppressed after adjustment for survivor bias and was no longer statistically significant. The trend of lower standard GVHD scores with rezameterlinib compared to vehicle treatment remained when mortality score was carried over.

[0299] Analysis by AUC, no statistically significant difference in scGVHD scores was observed between groups. However, diseased animals treated with vehicle exhibited significantly higher scGVHD scores compared to animals that did not receive treatment. Animals that received BM only showed a mild lower modified GVHD score compared to diseased animals treated with vehicle. After mortality score carryover, diseased animals treated with vehicle continued to exhibit significantly higher scGVHD scores compared to animals that did not receive treatment. Although not statistically significant, when mortality score was carried over, significantly higher scGVHD scores were observed in animals that received BM only compared to all other groups. No trend of improved scGVHD scores was observed with test article compared to vehicle treatment.

[0300] Progression-free survival analysis using the standard GVHD score scale indicated that approximately 58% of diseased animals treated with vehicle did not show progressive disease by day 56.

[0301] Analysis using the modified scGVHD scale indicated that approximately 75% of diseased animals treated with vehicle did not show progressive disease by day 56. These results correlate with the lower disease severity and disease penetrance in this study.

[0302] As the data shows, there are limitations associated with this study. For example, disease induction was not robust. In addition, the positive control group did not show a statistically significant difference. Due to these limitations, the study outcomes are inconclusive.

[0303] While the foregoing application has been described in some detail for purposes of clarity and understanding, it will be appreciated that various changes, modifications and substitutions can be made by one skilled in the art without departing from the application. It is to be understood that various alternatives to the embodiments of the application described herein can be employed in practicing the application. The following claims are intended to define the scope of the application and are to be accorded the full scope of equivalents thereof. The disclosures of all patents and scientific articles cited herein are expressly incorporated herein by reference in their entirety for all purposes. To the extent that any material incorporated by reference contradicts or contradicts any aspect of the present disclosure, the present disclosure controls.

Claims

1. A method of treating a disease or condition selected from systemic sclerosis and a transplantation-related dysfunction in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

2. A method of treating a disease or condition selected from graft versus host disease (GVHD), systemic sclerosis (scleroderma), chronic lung allograft dysfunction (CLAD), restrictive allograft syndrome (RAS), and bronchiolitis obliterans syndrome (BOS) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

3. A method of treating graft versus host disease (GVHD) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

4. A method of treating systemic sclerosis (scleroderma) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

5. A method of treating chronic lung allograft dysfunction (CLAD) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

6. A method of treating restrictive allograft syndrome (RAS) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton’s tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof.

7. A method of treating bronchiolitis obliterans syndrome (BOS) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) a Bruton's tyrosine kinase (BTK) inhibitor, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide or a pharmaceutically acceptable salt thereof.

8. The method of any one of claims 1-7, wherein the BTK inhibitor is a reversible BTK inhibitor.

9. The method of any one of claims 1-7, wherein the BTK inhibitor is an irreversible BTK inhibitor.

10. The method of any one of claims 1-9, wherein the BTK inhibitor is (i) (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine- 1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile or a pharmaceutically acceptable salt thereof; (ii) l-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-lH-pyrazolo[3,4-d]pyrimidin-l-yl]-l- piperidinyl]-2-propen-l-one or a pharmaceutically acceptable salt thereof; or (iii) (4-amino-3-(4-phenoxyphenyl)-l-[(3R)-l-(prop-2-enoyl)piperidin-3-yl]-l,3-dihydro- 2H-imidazo[4,5-c]pyridin-2-one) or a pharmaceutically acceptable salt thereof.

11. The method of claim 10, wherein the BTK inhibitor is (R)-2-[3-[4-amino-3-(2-fluoro- 4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile or a pharmaceutically acceptable salt thereof.

12. A method of treating graft-versus-host disease (GVHD) in a human patient in need thereof, the method comprising administering to the human patient a therapeutically effective amount of a combination comprising: (a) (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine- 1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile or a pharmaceutically acceptable salt thereof, and (b) 2-{3-[4-(lH-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide or a pharmaceutically acceptable salt thereof.

13. The method of any one of claims 2, 3, and 12, wherein GVHD is chronic GVHD (cGVHD).

14. The method of any one of claims 1-13, wherein 2-{3-[4-(lH-indazol-5-ylamino)-2- quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof, is administered to the human patient at a daily dose of up to about 400 mg.

15. The method of claim 14, wherein 2-{3-[4-(lH-indazol-5-ylamino)-2- quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof, is administered to the human patient at a daily dose of about 50-400 mg.

16. The method of claim 14 or 15, wherein 2-{3-[4-(lH-indazol-5-ylamino)-2- quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof, is administered to the human patient at a dose of about 50 mg, 100 mg, 150 mg, or 200 mg.

17. The method of claim 16, wherein the dose is administered to the human patient once daily or twice daily.

18. The method of any one of claims 1-17, wherein 2-{3-[4-(lH-indazol-5-ylamino)-2- quinazolinyl]phenoxy}-N-(propan-2-yl)acetamide, or a pharmaceutically acceptable salt thereof, is administered orally.

19. The method of any one of claims 10-18, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient at a daily dose of up to about 800 mg.

20. The method of claim 19, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient at a daily dose of about 50-800 mg.

21. The method of claim 19 or 20, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient at a dose of about 100 mg, 200 mg, or 400 mg.

22. The method of claim 21, wherein a dose of (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient once per day or twice per day.

23. The method of any one of claims 10-22, wherein the (E) isomer (R)-2-[3-[4-amino-3- (2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4- methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient.

24. The method of any one of claims 10-22, wherein the (Z) isomer (R)-2-[3-[4-amino-3- (2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4- methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient.

25. The method of any one of claims 10-22, wherein a mixture of the (E) isomer and the (Z) isomer of (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1- yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered to the human patient.

26. The method of any one of claims 10-25, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, is administered orally.

27. The method of any one of claims 10-26, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile, or a pharmaceutically acceptable salt thereof, and 2-{3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy}-N-(propan-2- yl)acetamide, or a pharmaceutically acceptable salt thereof, are administered separately.

28. The method of claim 27, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin-1-yl]pent-2-enenitrile or a pharmaceutically acceptable salt thereof and 2-{3-[4-(1H-indazol-5-ylamino)-2-quinolinyl]phenoxy}-N-(propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof are administered sequentially.

29. The method of any one of claims 10-28, wherein (R)-2-[3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile or a pharmaceutically acceptable salt thereof and 2-{3-[4-(1H-indazol-5-ylamino)-2-quinolinyl]phenoxy}-N-(propan-2-yl)acetamide or a pharmaceutically acceptable salt thereof are administered simultaneously.

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