Topical Therapy of Skin Fibrosis and Melanoma

Abstract

Procedures for topical treatment of melanoma and skin fibrosis include administering to a mammal in need of treatment a compound including at least one BCL-2/BCL-xl inhibitor; and at least one ionic liquid. The compound can include Navitoclax dissolved in choline octanate in a concentration from approximately 150 ug/mL to approximately 160 ug/mL.

Claims

1. A composition of matter for topical treatment of disease, comprising: at least one BCL-2/BCL-xl inhibitor; and at least one ionic liquid.

2. The composition of claim 1, wherein the at least one BCL-2/BCL-xl inhibitor comprises at least one BCL-2 inhibitor selected from the group consisting of Navitoclax, ABT-737, Venetoclax or BM-1074.

3. The composition of claim 1, wherein the at least one BCL-2/BCL-xl inhibitor comprises at least one BCL-2 inhibitor selected from the group consisting of Navitoclax, ABT-737, Obatoclax Mesylate, TW-37, Venetoclax, BM-1074 or Gambogic Acid.

4. The composition of claim 1, wherein the at least one ionic liquid comprises choline octanate.

5. The composition of claim 4, wherein the at least one BCL-2/BCL-xl inhibitor comprises Navitoclax.

6. The composition of claim 5, wherein a concentration of Navitoclax with respect to choline octanate is from approximately 150 ug/mL to approximately 160 ug/mL.

7. The composition of claim 1, further comprising a transdermal patch.

8. A method of topical treatment of skin fibrosis, comprising: administering to a mammal in need thereof a compound comprising at least one BCL-2/BCL-xl inhibitor; and at least one ionic liquid.

9. The method of claim 8, wherein administering includes administering the compound wherein the at least one BCL-2/BCL-xl inhibitor comprises at least one BCL-2 inhibitor selected from the group consisting of Navitoclax, ABT-737, Venetoclax or BM-1074.

10. The method of claim 8, wherein administering includes administering the compound wherein the at least one BCL-2/BCL-xl inhibitor comprises at least one BCL-2 inhibitor selected from the group consisting of Navitoclax, ABT-737, Obatoclax Mesylate, TW-37, Venetoclax, BM-1074 or Gambogic Acid.

11. The method of claim 8, wherein administering includes administering the compound wherein the at least one ionic liquid comprises choline octanate.

12. The method of claim 11, further comprising producing the choline octanate by reacting choline bicarbonate and octanoic acid before administering.

13. The method of claim 12, further comprising dissolving Navitoclax in choline octanate in a concentration of from approximately 150 ug/mL to approximately 160 ug/mL.

14. The method of claim 11, further comprising producing the choline octanate by reacting at least two members selected from the group consisting of choline bicarbonate, octanoic acid, octenoic acid or geranic acid before administering.

15. A method of topical treatment of melanoma, comprising: administering to a mammal in need thereof a compound comprising at least one BCL-2/BCL-xl inhibitor; and at least one ionic liquid.

16. The method of claim 15, wherein administering includes administering the compound wherein the at least one BCL-2/BCL-xl inhibitor comprises at least one BCL-2 inhibitor selected from the group consisting of Navitoclax, ABT-737, Venetoclax or BM-1074.

17. The method of claim 15, wherein administering includes administering the compound wherein the at least one BCL-2/BCL-xl inhibitor comprises at least one BCL-2 inhibitor selected from the group consisting of Navitoclax, ABT-737, Obatoclax Mesylate, TW-37, Venetoclax, BM-1074 or Gambogic Acid.

18. The method of claim 15, wherein administering includes administering the compound wherein the at least one ionic liquid comprises choline octanate.

19. The method of claim 18, further comprising producing the choline octanate by reacting choline bicarbonate and octanoic acid before administering.

20. The method of claim 19, further comprising dissolving Navitoclax in choline octanate in a concentration of from approximately 150 ug/mL to approximately 160 ug/mL.

21. The method of claim 15, wherein administering includes administering the compound wherein the at least one ionic liquid comprises at least two members selected from the group consisting of choline bicarbonate, octanoic acid, octenoic acid or geranic acid.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0015] FIG. 1 illustrates oral gavage of NAVI and topical administration of COA/NAVI in accordance with an illustrative embodiment.

[0016] FIG. 2 illustrates blood quantification for NAVI PO and COA/NAVI in accordance with an illustrative embodiment.

[0017] FIG. 3 illustrates fibrotic area from beginning of treatment for 4 groups in accordance with an illustrative embodiment.

[0018] FIG. 4 illustrates body weight change for 4 groups in accordance with an illustrative embodiment.

[0019] FIG. 5 illustrates cell death % by apoptosis and necrosis for 8 groups in accordance with an illustrative embodiment.

[0020] FIG. 6 illustrates Western blot analysis for 5 groups in accordance with an illustrative embodiment.

[0021] FIG. 7 illustrates BCL-2 expression for 5 groups in accordance with an illustrative embodiment.

DETAILED DESCRIPTION

[0022] The disclosure of this application is technically related to co-pending U.S. Ser. No. ______ (attorney docket number UTEP2022-005-2), filed Nov. 21, 2022, the entire contents of which are hereby expressly incorporated by reference for all purposes.

[0023] Embodiments of this disclosure include methods and formulations that will ameliorate skin fibrosis. While not being bound by theory, the methods and formulations induce apoptosis of fibrosed cells.

[0024] A topical solution-based therapy combines an ionic liquid with a BCL-2 inhibitor. The ionic liquid based on choline bicarbonate and octanoic acid (COA) has been reintroduced as a highly effective method of transporting tiny hydrophobic molecules to the skin. By inhibiting the BCL-2 protein of a specific cell, the BCL-2 inhibitor Navitoclax (NAVI) was also very successful and safe chemotherapy for treating cancer. The COA/NAVI combination was created to promote skin penetration and drug retention in the target area for a more extended period, increasing therapeutic efficiency. Despite NAVI’s developing potential as a chemotherapeutic drug, its use is restricted because of the danger of severe thrombocytopenia. As a result, the regulatory body has limited their administration route to oral rather than intravenous or intramuscular.

[0025] The main challenges in treating fibrosis with chemotherapeutics are epidermal layer penetration, the appropriate concentration, and drug retention within the layer for a more extended period. The COA ionic liquid, as created, is intended to improve NAVI skin permeation, and keep the payload within the dermis and subcutaneous layer of the skin. NAVI is an FDA-approved hydrophobic anti-apoptotic protein B cell lymphoma/leukemia (BCL-2) inhibitor, making it a good candidate for COA enhanced negation of these variables. NAVI is a BH3 mimetic drug that binds directly to BCL-2, BCL-xL, and Bcl-w. BH3 mimetics are tiny molecules that inhibit anti-apoptotic BCL-2 family members. Individual anti-apoptotic factors like BCL-2, BCL-xL, BCL-W, and MCL are blocked. COA improves NAVI skin permeability and increases its solubility by up to 150 .Math.g/mL. With the development of COA/NAVI formulations, we overcame the clinical limitation of NAVI and facilitated its topical application to accelerate the treatment of local fibrosis. The COA/NAVI formulation delivers and maintains the drug at the desired fibrosis site for a more extended period, as confirmed by a pigskin in vitro permeation test (IVPT). In vitro studies performed on the human skin fibrotic skin fibroblast cell line Hs27 show its potential use as a chemotherapeutic agent for fibrosis treatment, and COA/NAVI shows significantly greater efficacy than free NAVI. In vivo studies were performed in a bleomycin-induced mouse model to evaluate the drugs’ effectiveness, safety, and bio distribution. The results of studies and general observations indicate that the COA/NAVI formulation can be considered an effective and safe treatment approach for treating cutaneous fibrosis.

[0026] A topical solution-based treatment comprises an ionic liquid and BCL-2 inhibitor. The Choline Octanoate (COA) ionic liquid was reported as a very effective agent for delivering small hydrophobic molecules through the skin. The BCL-2 inhibitor navitoclax (NAVI) was also a very effective and safe chemotherapeutic to treat cancer by inhibiting the BCL-2 protein of a specific cell. The COA/NAVI combination was specifically designed to improve skin penetration and enhance retention of the drug in the target area for an extended time to enhance therapeutic efficiency. Despite the emerging potential of NAVI as a chemotherapeutic agent, its applications are limited due to the risk of severe hematotoxicity. Therefore, the regulatory authority has restricted their route of administration to oral instead of intravenous and intramuscular.

[0027] The major challenges of melanoma treatment with chemotherapeutics are inadequate epidermal layer penetration, inadequate concentration, and poor drug retention within the layer for extended time periods. The as-developed ionic liquid COA is designed to enhance the skin permeation of NAVI and hold the payload within the dermis and subcutaneous layer of the skin. NAVI is a hydrophobic, anti-apoptotic protein B cell lymphoma/leukemia (BCL-2) inhibitor approved by the FDA for cancer treatment, making it an ideal candidate for COA enhancement negation of these factors. NAVI, a BH3 (BH3 mimetics are small compounds that antagonize anti-apoptotic BCL-2 family proteins) mimetic drug directly binds with BCL-2, Bcl-xL, and Bcl-w. It blocks individual anti-apoptotic factors like BCL-2, BCL-xL, BCL-W, and MCL. COA not only enhances skin permeation of NAVI but also increases solubility up to 150 .Math.g/mL. With the development of COA/NAVI formulation, we have overcome the clinical limitation of NAVI and facilitated their topical application to accelerate local melanoma treatment. COA/NAVI formulation delivers and holds the drug for an extended period at the targeted melanoma site, confirmed by in vitro permeation test (IVPT) using porcine skin. In vitro studies conducted on A375 human melanoma cells line demonstrate their potential use as a chemotherapeutic for treating melanoma, and COA/NAVI shows significantly higher efficacy than free NAVI. In vivo studies were conducted on an A375 xenograft mouse model to evaluate drug efficacy, safety, and biodistribution. The findings from comprehensive studies and observations reveal that the COA/NAVI formulation can be an effective and safer therapeutic approach for treating skin melanoma.

[0028] The components of the formulation compound can be substituted partially or wholly with other components that provide equivalent functionality. Examples of components that can be substituted include the following.

TABLE-US-00001 List of BCL-2 inhibitors Inhibitors Name CAS Navitoclax 923564-51-6 ABT-737 852808-04-9 Obatoclax Mesylate 803712-79-0 TW-37 877877-35-5 Venetoclax 1257044-40-8 BM-1074 1391108-10-3 Gambogic Acid 2752-65-0

List of Ionic Liquid Reagents

[0029] Choline Bicarbonate (CAS: 78-73-9), [0030] Octanoic Acid (CAS: 124-07-2), [0031] Octenoic Acid (CAS: 18719-24-9), [0032] and Geranic Acid (CAS: 459-80-3)

[0033] The choline octanate can be produced by reacting at least two members selected from the group consisting of choline bicarbonate, octanoic acid, octenoic acid or geranic acid. More specifically choline bicarbonate and at least one member selected from the group consisting of octanoic acid, octenoic acid or geranic acid.

EXAMPLES

[0034] Specific exemplary embodiments will now be further described by the following, nonlimiting examples which will serve to illustrate in some detail various features. The following examples are included to facilitate an understanding of ways in which embodiments of the present disclosure may be practiced. However, it should be appreciated that many changes can be made in the exemplary embodiments which are disclosed while still obtaining like or similar result without departing from the scope of embodiments of the present disclosure. Accordingly, the examples should not be construed as limiting the scope of the present disclosure.

Example 1

[0035] Mice were randomly distributed into four groups and treatments were given for four weeks. Our treatment groups were untreated, NAVI (50 .Math.M) (PO), topical administration of COA (50%), and COA/NAVI (50 .Math.M). (a)

[0036] Referring to FIG. 1, COA/NAVI 150 can be seen in a schematic representation of how NAVI transport via the topical and oral route of administration. Topical COA/NAVI delivery ensures a higher percentage of NAVI in the fibrotic area than NAVI PO administration. Also, topical COA/NAVI holds the drug longer in the fibrotic region and allows less NAVI transportation in the systemic circulation. Thus, we observe lower thrombocytopenia in COA/NAVI topical administration compared to NAVI PO.

[0037] Referring to FIG. 2, blood samples were quantified using a UV plate reader at 345 nm, and NAVI concentrations were quantified. The graph shows higher transport of NAVI (1.61 mg/mL) 250 via GI track than COA/NAVI (0.38 mg/mL) via skin fat layers.

[0038] Referring to FIG. 3, the fibrotic area was measured using slide calipers from the beginning of treatment. Data for COA/NAVI 350 shows fibrotic area was inhibited by 120% compared to the untreated group after four weeks of treatment. This data validates our initial claim of COA/NAVI anti-fibrotic therapeutic efficiency.

[0039] Referring to FIG. 4, body weight was measured during the treatments. We observe no sudden reduction of body weight, indicating our treatments were safe to administer to mice. The weight data for COA/NAVI 450 showed the smallest change. The data shown here is mean ± SD, the experiment was performed in N = 3. .sup.∗∗∗ Indicates statistical significance with p < 0.001; .sup.∗∗ indicates statistical significance with p < 0.01 and .sup.∗ indicates statistical significance with p < 0.05.

Example 2

[0040] Referring to FIG. 5, in the apoptosis-necrosis assay, the formulations COA/NAVI (20 .Math.M) 520 and COA/NAVI (50 .Math.M) 530 showed around 70% and 75% apoptosis, respectively, tested in A375 cells after 24 h of treatment compared to H.sub.2O.sub.2, as a positive control. However, both treatments COA/NAVI (20 .Math.M) 560 and COA/NAVI (50 .Math.M) 570 showed around 5% necrosis.

[0041] Referring to FIG. 6, Western blot analysis was done from Hs27 healthy skin fibroblast and A375 human melanoma cell lysate after treatment with the formulations. The experiment shows more inhibition of BCL-2 expression in COA/NAVI 640 and NAVI compared to COA or untreated melanoma and healthy cell. Housekeeping gene β-actin expression with COA/NAVI (50 .Math.M) 660 is about the same.

[0042] Referring to FIG. 7, the BCL-2 expression was quantified and represented compared to housekeeping gene β-actin. BCL-2 expression in COA/NAVI 750 is lower. Data represent mean ± SD, where n=6. .sup.∗ Indicates statistical significance with p < 0.05; .sup.∗∗∗ indicates statistical significance with p < 0.001.

[0043] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.