Topical PALA therapy for cancer

Abstract

Provided herein are compositions, systems, kits, and methods for topically applying a composition to a subject having pre-cancerous cells and / or cancerous cells (e.g., skin cancer cells) and / or a body region adjacent potential pre-cancerous cells and / or cancerous cells, wherein the composition comprises N-phosphonoacetyl-L-aspartic acid (PALA) (also known as phosphine aspartic acid) and water, and optionally further comprises at least one non-ionic surfactant.

Description

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 492,381, filed March 27, 2023, which is incorporated herein by reference in its entirety.

[0002] This invention was carried out with government support under license number W81XWH-16-1-0439 granted by the U.S. Department of Defense. The government holds certain rights to this invention.

[0003] Invention Field This document provides compositions, systems, kits, and methods for topically applying a composition to a body region of a subject having precancerous and / or cancerous cells (e.g., skin cancer cells) and / or adjacent to potential precancerous and / or cancerous cells, wherein the composition comprises N-phosphonoacetyl-L-aspartic acid (PALA) (also known as phosphonoaspartic acid) and water, and optionally further comprises at least one nonionic surfactant. Background Technology

[0004] Shockingly, one in five Americans will develop skin cancer in their lifetime. 1 Most skin cancers are caused by sun exposure, resulting in damage from ultraviolet (UV) radiation. 2 Skin cancer is classified as melanoma or non-melanoma (NMSC). 3 These are the most common types of cancer, and they originate from basal cells, squamous cells, and Merkel cells in the skin. 3 Basal cell carcinoma is the most common type, with an estimated 5.4 million cases diagnosed annually in the United States. 4 Secondly, squamous cell carcinoma (SCC) is the second most common and most metastatic type. 3 SCC causes more than twice as many deaths as melanoma, with an estimated 15,000 people dying from SCC each year. 5 Most SCCs originate from actinic keratosis (AK), a precancerous lesion affecting more than 58 million Americans, making AK one of the most commonly treated skin conditions by dermatologists. 6 The direct treatment cost of NMSC in the United States is estimated at $4.8 billion annually, excluding additional indirect costs such as lost wages. 7，8 .

[0005] Currently, there are many FDA-approved therapies for NMSCs, each with its own advantages and limitations. The gold standard treatment is surgical excision (e.g., Mohs surgery). For precancerous lesions (AK), the gold standard is cryotherapy. These treatments are effective in more than 90% of individual lesions. However, a major problem that locally destructive methods (surgery or cryosurgery) fail to address is that most AK lesions occur within large areas of skin that have been damaged by prolonged sun exposure and are therefore filled with pretumor cells, a phenomenon known as "regional carcinogenesis."

[0006] To address the high statistical risk of cancer development in these areas, several non-invasive local treatments have been approved, including fluorouracil (5-FU; an antimetabolite), imiquimod (IMQ; an immunomodulator), and tebanibulin (a microtubule inhibitor). All three local treatments are FDA-approved for AK, and reported AK response rates range from 40% to 80%, with 5-FU being the most effective. 9 5-FU and IMQ are also FDA-approved for the treatment of superficial BCC and are frequently used off-label for the treatment of superficial SCC. While each of these topical agents can treat multiple diffuse lesions (thus addressing the "regional cancer" problem) and often produces higher long-term clearance rates than cryotherapy overall, each agent is also associated with severe local skin reactions characterized by pain, burning, skin erosion, and, in some cases, hyperpigmentation or hypopigmentation. Therefore, there remains a need for improved topical NMSC therapies that are highly effective but have fewer side effects compared to currently available options. Summary of the Invention

[0007] This document provides compositions, systems, kits, and methods for topically applying a composition to a body region of a subject having precancerous and / or cancerous cells (e.g., skin cancer cells) and / or adjacent to potentially precancerous and / or cancerous cells, wherein the composition comprises N-phosphonoacetyl-L-aspartic acid (PALA) (also known as phosphonoaspartic acid) and water, and optionally further comprises at least one nonionic surfactant. In some embodiments, PALA is present in the composition at a concentration of about 0.1% to about 5.0% or 1.0% to 8.0%.

[0008] In some embodiments, this document provides a method for treating precancerous and / or cancerous cells in a subject, the method comprising: applying a composition topically to a body region of the subject having precancerous and / or cancerous cells and / or adjacent potential precancerous and / or cancerous cells, or providing the composition to the subject such that the subject is able to apply the composition to the body region, wherein the composition comprises N-phosphonoacetyl-L-aspartic acid (PALA) and water, and optionally further comprises at least one nonionic surfactant.

[0009] In some embodiments, the subject's area includes skin (e.g., face, arms, legs, torso, etc.). In other embodiments, cancerous cells include melanoma and non-melanoma skin cancer cells. In a further embodiment, non-melanoma skin cancer cells are basal cell carcinoma cells or squamous cell carcinoma cells. In a particular embodiment, precancerous cells include actinic keratosis cells (e.g., as part of an actinic keratosis lesion). In a particular embodiment, the subject is female, wherein the subject's area includes the surface of the subject's vagina, and wherein the precancerous and / or cancerous cells include vaginal precancerous cells and / or cancerous cells. In other embodiments, the subject's area includes the surface of the subject's oral cavity (e.g., gums), and wherein the precancerous and / or cancerous cells include oral precancerous cells or cancerous cells. In yet another embodiment, the subject is female, wherein the subject's area includes the subject's cervix, and wherein the precancerous and / or cancerous cells include cervical precancerous cells and / or cancerous cells.

[0010] In some embodiments, the composition is repeatedly applied or delivered daily for at least 7 days (e.g., 7…10…20… or 30 days), at least 14 days, at least 21 days, or at least 7 weeks (e.g., 7, 8, 9, 10…20…30… or 40 weeks). In certain embodiments, application and delivery are repeated at least two or three times daily for at least 7 days or at least 14 days. In some embodiments, the method further includes administering an immune checkpoint inhibitor (e.g., targeting CTLA4, PD-1, or PD-L1; such as pembrolizumab, ipilimumab, nivolumab, or atezolizumab) to the subject. In some embodiments, the subject is a human or a dog, cat, cow, pig, or horse. In certain embodiments, the composition is topically applied to an area of ​​the subject containing precancerous cells and / or cancerous cells, reducing or eliminating at least some or all of the cancerous or precancerous cells at or near said area. In certain embodiments, the composition is present in a skin patch or dispensing device. In some embodiments, the nonionic surfactant is selected from the group consisting of: polyglycerol alkyl ethers, glucosyl dialkyl ethers, crown ethers, ester-linked surfactants, polyoxyethylene alkyl ethers, Brij, Spans (sorbitol esters), and Tweens (polysorbates). In specific embodiments, the nonionic surfactant is selected from the group consisting of: Capryol PGMC, polysorbate 80, polysorbate 20, sodium lauryl sulfate, poloxamer 188, polyhydroxy40 hydrogenated castor oil, dimannitol monooleate, and lauroyl polyhydroxy-32 glycerol ester.

[0011] In some embodiments, systems and kits are provided herein comprising: a) a composition comprising N-phosphonoacetyl-L-aspartic acid (PALA) and water, and optionally at least one nonionic surfactant; and b) at least one of the following: i) a dispensing container configured to locally dispense the composition to a body region of a subject, and / or ii) a topical patch configured to locally deliver the composition to the body region of the subject, and / or iii) an immune checkpoint inhibitor (e.g., targeting CTLA4, PD-1, or PD-L1; such as pembrolizumab, ipilimumab, nivolumab, or atezolizumab).

[0012] In some embodiments, the composition is present in a dispensing container or a topical patch. In other embodiments, a dispensing container is present, and said dispensing container is a non-contact dispensing container that allows the composition to be dispensed and rubbed onto a body area such that other areas of the subject do not come into contact with the composition. In a particular embodiment, other areas of the subject include the subject's hands.

[0013] In some embodiments, the non-contact dispensing container includes a metered-dose topical applicator. In other embodiments, the non-contact dispensing container is selected from the group consisting of: CLICK metered-dose topical applicator, TOPI-CLICK applicator, TAPEMARK unit-dose semi-solid drug delivery system, MICROBRISTLE applicator (MBA™), BACK Easy lotion applicator, and LIQUIBAND XL skin closure system. In a further embodiment, the dispensing container is present and includes a tube or pouch.

[0014] In some embodiments, compositions are provided herein comprising: a) N-phosphonoacetyl-L-aspartic acid (PALA); b) water; and optionally c) at least one nonionic surfactant. In some embodiments, at least one nonionic surfactant is present. In some embodiments, PALA is present in the composition at a concentration of about 0.1% to about 8.0% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 8%). In some embodiments, the composition comprises about 20%-50% or 10%-75% water.

[0015] In some embodiments, at least one nonionic surfactant comprises a nonionic linear copolymer. In a further embodiment, the nonionic linear copolymer comprises poloxamer 188. In other embodiments, the nonionic linear copolymer is present in the composition at about 50%-90%, about 60%-80%, about 65%-75%, or about 70%. In yet another embodiment, at least one nonionic surfactant is present in the composition at 10%-30% or about 22%. In yet another embodiment, at least one nonionic surfactant comprises octanoyl hexanoyl polyhydroxy-8 glycerol ester, or wherein at least one nonionic surfactant excipient comprises PEG-8 monoesters and diesters of octanoic acid (C8) and decanoic acid (C10) with small amounts of monoglycerides, diglycerides, and triglycerides, or wherein at least one nonionic surfactant comprises LABRASOL ALF.

[0016] In other embodiments, the composition further comprises a hydrogel agent, and the composition is in the form of a hydrogel. In some embodiments, the composition further comprises isopropyl myristate, and optionally said isopropyl myristate is present in the composition at a concentration of about 0.5%-7%, about 1%-5%, or about 3%. In other embodiments, the composition further comprises a solubilizer and / or an emulsifier.

[0017] In some embodiments, the composition further comprises polyethylene glycol glycerol ricinoleate, and optionally said polyethylene glycol glycerol ricinoleate is KOLLIPHOR EL, and optionally said polyethylene glycol glycerol ricinoleate is present in the composition at a concentration of about 2%-10%, about 3%-7%, or about 5%. In further embodiments, the composition further comprises at least one non-PALA anticancer agent, and optionally said at least one non-PALA anticancer agent includes: tebanibulin, fluorouracil (5-FU), and / or imiquimod (IMQ). In particular embodiments, at least one nonionic surfactant includes poloxamer 188 and / or capryloyl hexanoyl polyhydroxy-8 glyceride. In further embodiments, the composition is in the form of a cream, lotion, or gel (and contains the agents necessary for this form). Attached Figure Description

[0018] Figure 1: Topical PALA was nontoxic at up to 5% (w / v) in mice and reduced tumor growth in a dose-dependent manner. A. Percentage change in body weight during treatment. B. Serum amyloid A (SAA) level at the endpoint. C. Colon length at the endpoint. D. Fecal lipocalin-2 (LCN2) level at the endpoint. E. Quantification of the mean number of tumors per mouse during treatment. F. Change in tumor burden per mouse during treatment, expressed as a percentage of the original number of tumors. Mean ± SEM, n = 4–15 mice / group, from 3 independent experiments. Statistical significance was determined by mixed-effects analysis and post-hoc Bonferroni's multiple comparison test.

[0019] Figure 2: Daily topical PALA application was better tolerated than standard NMSC topical treatment. A. Percentage change in body weight during treatment. Arrow (↑) indicates the start of supportive care. B. Mean colon length on day 7. C. Serum amyloid A level on day 7. D. Mean spleen weight on day 7. E. Histological examination of skin treated with topical medication for 7 days. Mean ± SEM, n = 5–10 mice / group. Statistical significance was determined by mixed-effects analysis using Bonfroni's multiple comparison test or by one-way ANOVA using Dunnett's multiple comparison test.

[0020] Figure 3: Reduction in tumor burden, tumor area, and tumor grade in mice treated with local PALA. A. Percentage change in body weight during treatment. B. Serum amyloid A level at the endpoint. C. Colon length at the endpoint. D. Lipocarrier protein-2 (LCN2) level at the endpoint. E. Mean number of tumors per mouse during treatment. F. Percentage change in mean number of tumors during treatment. G. Quantification of surface tumor area from photographs taken during treatment. H. Mean cross-sectional tumor area, measured by H&E-stained tissue. Mean ± SEM, n = 12 / group. Significance was determined by mixed-effects model (REML) using Bonfroni multiple comparison test or unpaired t-test.

[0021] Figure 4: Expression of the NOD2 target gene LL-37 increased by topical PALA treatment. A. Quantification of DefB14 staining area from multiplex immunofluorescence images of skin tumor sections. B. Quantification of the percentage of LL-37+ cells in the tumor region from multiplex immunofluorescence images of skin tumor sections. C. Representative multiplex immunofluorescence images of skin tumor sections, highlighting differential expression of LL-37 from mice treated with 50% acetone / 10% glycerol (mediator) or 2% (w / v) PALA, stained against LL-37 (green), cytokeratin-14 (CK14, pink), or DNA (blue). Scale bar = 2 mm. Higher magnification of the boxed areas is shown as insets, scale bar = 1 mm. Mean ± SEM, n = 12–19 tumors / group. Significance was determined by unpaired t-test.

[0022] Figure 5: Tumor-infiltrating cell populations altered by local PALA treatment, demonstrating immunomodulatory activity. Multiplex immunofluorescence images were quantified based on the percentage of tumor cells stained with specific markers. A. Quantification of cells expressing the neutrophil cell marker myeloperoxidase (MPO; aqua). B. Quantification of cells expressing the macrophage cell marker F4 / 80 (pink). C. Quantification of cells expressing the T cell marker CD3 (red). D. Quantification of cells expressing the cytotoxic T cell marker CD8 (green). All sections were co-stained with the keratinocyte marker CK14 (grey) and DAPI (blue). E. Representative multiplex immunofluorescence images highlighting differentially expressed cell types. Mean ± SEM, n = 10–17 tumors / group. Scale bar = 2 mm. Significance was determined by unpaired t-test.

[0023] Figure 6: Representative photographs of peak skin irritation observed during the local irritation test. A. Photographs of mice treated with the medium, 2% PALA, and 5% IMQ 3 days after treatment. B. Photographs of mice treated with the medium, 2% PALA, and 5% 5-FU 7 days after treatment.

[0024] Figure 7: Topical PALA treatment induces cell cycle arrest. Representative images of immunostained skin from mice treated with the medium and 2% PALA (left panel). Percentage of positive cells in tumor regions is quantified graphically (right panel); mean ± SEM, n = 12–16 tumors / group. Significance was determined by unpaired t-test. Multiplex immunofluorescence images of skin tumors stained with Ki67 (green) and cytokeratin-14 (CK14; red) are shown in the top row. Immunohistochemical images show the expression of the indicated cell cycle markers (brown).

[0025] Figure 8: Topical 2% PALA was non-toxic and showed a trend toward reducing tumor burden in a chemically carcinogenic skin cancer model. SKH1-Elite female mice were induced to develop skin cancer by topical treatment with DMBA for 2 weeks, followed by topical TPA three times weekly for 8 weeks. Mice were then randomly assigned to treatment groups and treated topically with either the medium (50% acetone / 10% glycerol) or 2% (w / v) PALA for 4 weeks. A. Quantitative change in mean tumor burden per mouse during the treatment period, expressed as a percentage of tumor number at week 9. B. Change in body weight during the treatment period, expressed as a percentage of baseline body weight. C. Levels of serum amyloid A (SAA), a liver and systemic inflammatory marker, at the endpoint. D. Measurement of colon length at the endpoint as a general measure of intestinal pathology. Mean ± SEM, n = 8–9 / group. Significance was determined by two-way ANOVA using Sidak's multiple comparison test or unpaired two-way t-test. All p-values ​​< 0.05 are shown on the chart.

[0026] Figure 9: Topical 2% PALA formulated in Aquaphor effectively reduced tumor burden in a chemically induced skin cancer model. C57BL / 6 female mice were induced to develop skin cancer by topical treatment with DMBA for 2 weeks, followed by topical TPA three times weekly for 12 weeks. Mice were then randomly assigned to either the treatment group or treated topically with either the medium (Aquaphor) or 2% (w / v) PALA / Aquaphor for 4 weeks. A. Quantitative change in mean tumor burden per mouse during the treatment period, expressed as a percentage of tumor number at week 13. B. Quantitative change in mean total tumor area per mouse during the treatment period, expressed as a percentage of the measurement at week 13. Mean ± SEM, n = 5–6 / group. Significance was determined by two-way ANOVA. Figure 10 shows Table 7 from Example 2, which illustrates the appearance and HPLC determination of different hydrogels.

[0027] Figure 11. Cumulative release curves of different formulations containing penetration enhancers (n=1). PALA aqueous solution is also reported as a reference.

[0028] Figure 12 shows Table 10, which displays the appearance and assays of the formulation from Example 2.

[0029] Figure 13. Cumulative release curves of a specific formulation (n=3).

[0030] Figure 14. Skin and plasma PALA levels in mice 24 hours after a single topical application of formulation AP0536 / 29 / 03 4% (w / v) PALA. 10 μL of 4% PALA (final dose = 0.4 mg / kg) was applied to the dorsal skin of male CD1 mice (n=3 / time point). Plasma and skin tissue samples were collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h after administration, and PALA levels were determined by LC-MS. A graph showing mean ± SEM values ​​is plotted.

[0031] Figure 15. The reformulated topical 2% PALA was non-toxic and effectively reduced tumor burden and total tumor area. SKH1-Elite female mice were irradiated with UV light three times a week for 20 weeks to induce skin cancer. Mice were then randomly assigned to treatment groups and treated topically once daily with a medium (AP0536 / 29 / 01), 2% (w / v) PALA (AP0536 / 29 / 02), or 4% (w / v) PALA (AP0536 / 29 / 03) for 6 weeks. A. Change in body weight during treatment, expressed as a percentage of baseline body weight. B. Levels of serum amyloid A (SAA), a marker of liver and systemic inflammation, at the endpoint. C. Measurement of colon length at the endpoint as a general measure of intestinal pathology. D. Levels of fecal lipid carrier protein-2 (LCN2) at the endpoint as a measure of intestinal inflammation. E. Quantification of mean tumor burden per mouse during treatment. F. Mean change in tumor burden per mouse during treatment, expressed as a percentage of the original tumor number. G. Mean tumor surface area per mouse was quantified from photographs taken during the treatment period. Mean ± SEM, n = 8–9 / group. Significance was determined by mixed-effects model using Bonfroni's multiple comparison test (REML) or one-way ANOVA using Tukey's multiple comparison test. All p-values ​​< 0.05 are shown on graphs.

[0032] Figure 16. Topical 2% PALA was better tolerated and more effective than two standard-care topical treatments for NMSC. SKH1-Elite female mice were irradiated with UV light three times a week for 20 weeks to induce skin cancer. Mice were then randomly assigned to treatment groups (n=5 / group) and treated topically once daily with a medium (AP0536 / 29 / 02), 2% (w / w) PALA (AP0536 / 29 / 02), 5% (w / w) 5-fluorouracil (5% 5-FU), or 5% (w / w) imiquimod (5% IMQ) for 4 weeks. A. Mice treated with PALA maintained their body weight better than mice treated with 5-FU or IMQ. Weight changes from the start of treatment are plotted (mean ± SEM). Supportive care was provided to IMQ and 5-FU mice (from... A. The time indicated (starting point), and animals treated with 5-FU were discontinued after 2.5 weeks of treatment, as requested by veterinary staff. B. The level of serum amyloid A (SAA), a marker of liver and systemic inflammation, at the endpoint. C. The level of fecal lipid transporter-2 (LCN2) at the endpoint as a measure of intestinal inflammation. D. Mice treated with 2% PALA had significantly smaller mean tumor size than mice treated with the vector or 5% IMQ. Quantification of the mean tumor surface area per tumor measured from photographs using ImageJ software during the treatment period. E. Growth curves of individual tumors show a lower percentage of persistently growing tumors in mice treated with 2% PALA. F. Endpoint tumor grading determined by evaluating tumor cross-sections stained with H&E showed fewer squamous cell carcinomas (SCC) and SCC in situ tumors in mice treated with 2% PALA compared to mice treated with the vector or 5% IMQ. G. Tumor levels of interleukin-1β were elevated in mice treated with both 2% PALA and 5% IMQ, indicating the induction of an antitumor immune response. Tumor levels of H. interferon-α1 were elevated only in mice treated with 2% PALA, suggesting that the type of immune response elicited was different from that induced by stimulation with 5% IMQ.

[0033] Figure 17: In a B16 melanoma orthotopic xenograft model, local PALA treatment delayed melanoma growth and sensitized melanoma against checkpoint inhibitor therapy. B16 melanoma cells (10T) were bilaterally subcutaneously injected into female C57Bl / 6 mice (n=4-5 / group). 5Tumor volume was measured by calipers every 2-3 days by an uninformed staff member. Data from palpable tumors were plotted as mean ± SEM. A. Tumor growth was delayed by daily topical treatment with 1% PALA for 12 days. On day 12 post-injection, mice were treated daily with either the mediator (AP0536 / 29 / 02) or 1% (w / w) PALA (AP0536 / 29 / 02) for 12 days. Significant differences were determined using a mixed-effects model (REML) with multiple comparisons by Chidak. **p<0.01 B. Short-term topical treatment of B16 melanoma with 2% PALA reduced the expression of the checkpoint inhibitor PD-L1. On day 7 post-injection, mice were treated daily with either the mediator (AP0536 / 29 / 02) or 2% (w / w) PALA (AP0536 / 29 / 02) for 5 days. Tumors were harvested on day 14 post-injection, and PD-L1 protein levels in tumor lysates were analyzed by immunoblotting and quantified relative to tubulin levels (loaded control) using ImageJ image density assay. Mean ± SEM plots were generated. C. Short-term local treatment with 2% PALA sensitized B16 melanoma to anti-PD-L1 therapy. Mice were treated daily with either the carrier (AP0536 / 29 / 02) or 2% (w / w) PALA (AP0536 / 29 / 02) for 5 days starting on day 7 post-injection. From day 7 to day 19, mice were also treated intraperitoneally every 48 hours with either allotype or anti-PD-L1 / atezolizumab antibody (100 µg / mouse). Significant differences were determined by two-way ANOVA using Dunnett's multiple comparison test. ***p<0.001 Figure 18: Tumor growth of oral squamous cell carcinoma orthotopic xenografts was delayed by local PALA treatment. C57Bl / 6 mice (male and female; n=6 / group) were injected with MOC-1 squamous cell carcinoma cells into their tongues. Once palpable tumors formed, the mice were locally treated with either the medium (AP0536 / 29 / 02) or 2% (w / w) PALA (AP0536 / 29 / 02) 5x / week. Tumor volume was measured every 2–3 days via calipers. Data are plotted as mean ± SEM. Significant differences were determined by a mixed-effects model (REML) using the Westdak multiple comparison test. *p<0.05, ****p<0.0001.

[0034] Figure 19: PALA exhibits a U-shaped dose-response curve and stimulates biological effects independent of enzyme inhibition. A. Bactericidal activity of normal human dermal fibroblasts stimulated by PALA treatment. Cells were stimulated with the indicated concentration of PALA for 16 hours. The conditioned supernatant was added to a logarithmic phase culture of methicillin-resistant Staphylococcus aureus (MRSA) for 2 hours, and viability was determined by selective plating. Graphs were plotted with mean ± SD. B. PALA-stimulated production of antimicrobial peptides in normal human dermal fibroblasts. Cells were stimulated as in A, and human β-defensin 2 (HBD2) and human β-defensin 3 (HBD3) in the supernatant were measured by ELISA. Graphs were plotted with mean ± SD. C. At the dose that induces maximal immunostimulation in normal human dermal fibroblasts, the enzyme activity of aspartate transcarbamate (the enzyme target of PALA) decreased by only about 25%. The ability of cell lysates from the cells shown in A to catalyze the formation of carbamoyl aspartate (CA) in vitro was determined. Plot the percentage change in CA levels; mean ± SD. D. and E. Similar U-shaped dose-response curves observed in response to topical PALA treatment for skin cancer in a UVB-induced NMSC mouse model. D. Mean number of tumors per mouse at the end of treatment (plotted as mean ± SEM). D. Mice treated with a medium (Aquaphor), 1%, 2%, or 5% PALA in Aquaphor for 10 weeks. E. Mice treated with a medium (AP0536 / 29 / 02), 2% (w / w) PALA (AP0536 / 29 / 02), or 4% (w / w) PALA (AP0536 / 29 / 02) for 4 weeks. Detailed Implementation

[0035] This document provides compositions, systems, kits, and methods for topically applying compositions to a subject's body regions having precancerous and / or cancerous cells (e.g., skin cancer cells) and / or adjacent to potentially precancerous and / or cancerous cells (e.g., in the form of lesions or tumors), wherein the compositions comprise N-phosphonoacetyl-L-aspartic acid (PALA) (also known as phosphonoaspartic acid) and water, and optionally further comprise at least one nonionic surfactant. In some embodiments, PALA is present in the composition at a concentration of about 0.1% to about 5.0% or about 1% to about 8% (e.g., about 2%, 4%, 6%, or 8%).

[0036] Phosphonylacetyl-L-aspartate (PALA) is a potent and highly specific inhibitor of the multi-enzyme protein carbamoyl phosphate synthase II / aspartate transcarbamoylase / dihydroorotase (CAD), which catalyzes the first three steps of de novo pyrimidine nucleotide biosynthesis. PALA was designed as a transition state analog inhibitor of aspartate transcarbamoylase and possesses nanomolar potency.10 It is readily absorbed by cultured cells and effectively kills cells by depriving them of pyrimidine nucleotides, as aspartate transcarbamate is essential for this de novo biosynthetic pathway. PALA is highly specific because rescuing the pyrimidine precursor uridine completely prevents its toxicity. In murine B16 melanoma and Lewis lung cancer tumor models, systemic administration of PALA showed good antitumor efficacy as a single agent, but clinical trials of PALA as a single agent administered systemically in humans for colon cancer, breast cancer, malignant melanoma, or advanced soft tissue sarcoma have been disappointing due to limited efficacy. 11 This may be at least partly due to the reversal of the inhibition of pyrimidine synthesis by dietary uridine.

[0037] In some embodiments, the compositions described herein (containing PALA) are formulated in the form of creams, lotions, sprays, ointments, gels (e.g., hydrogels), pastes, and foundations. In other embodiments, such compositions are present in patches. In specific embodiments, the compositions may contain one or more additives selected from the group consisting of fragrances, colorants, thickeners, vegetable oils, emulsifiers, solvents, pH adjusters, antibacterial agents, preservatives, vitamins, sunscreens, surfactants, and combinations thereof. Various physical sunscreens may be included, such as titanium dioxide, silicone-treated titanium dioxide, zinc oxide, ferrous oxide, ferric chloride, talc, chromium oxide, or cobalt oxide. Alternatively or additionally, chemical sunscreens may be included, such as para-aminobenzoic acid, esters of para-aminobenzoic acid, salicylates, cinnamic acid esters, benzophenone, dihydroxyacetone, Parsol 1789, or melanin.

[0038] Compositions intended for topical application to subjects can be formulated into pharmaceutical compositions. Such pharmaceutical compositions may contain additional agents besides PALA and water. This invention is not intended to be limited by the specific properties of pharmaceutical formulations. For example, such compositions may be provided with physiologically tolerable liquid, gel, or solid carriers, diluents, adjuvants, and excipients. These therapeutic formulations may be applied topically to mammals for veterinary use, such as for livestock, and for clinical use in humans.

[0039] Example Example 1 Topical N-phosphonylacetyl-L-aspartate is a dual-action candidate for the treatment of non-melanoma skin cancer (NMSC). This embodiment describes a study of topical PALA as a therapy for NMSCs by combining the chemotherapy and immunomodulatory properties of 5-fluorouracil and imiquimod. Daily topical application of PALA to the skin of mice was well tolerated and produced less irritation, fewer histopathological changes, and less inflammation compared to those induced by 5-fluorouracil or imiquimod. In a UV-induced NMSC mouse model, topical PALA treatment significantly reduced the number, size, and grade of tumors compared to the mediator control. This antitumor activity was associated with increased expression of the antimicrobial peptide LL-37 and CD8. + T cells and F4 / 80 + Increased recruitment of macrophages to tumors was associated with immunomodulatory and antiproliferative effects.

[0040] Materials and methods reagents PALA (NSC-224131) was obtained from the National Cancer Institute (NCI) / Division of Cancer Treatment and Diagnostics (DCTD) / Developmental Treatment Program (DTP) Open Chemistry Bank. PALA was dissolved in 50% acetone / 10% glycerin (Fisher Scientific, Hampton, New Hampshire) at concentrations of 1%, 2%, or 5% (w / v). Aquaphor Healing Ointment was purchased from Beiersdorf USA (Wilton, Connecticut). Imiquimod Cream (Aldara, 5%; NDC# 45802-368-62) was purchased from Perrigo Company (Allegan, Michigan). Fluorouracil Cream USP (Efudex, 5%; NDC# 51672-4118-6) was purchased from Taro Pharmaceutical Industries Ltd (Hawthorne, New York).

[0041] UVB-induced NMSC mouse model Animal procedures were approved by the Institutional Animal Care and Use Committee of the Cleveland Clinic (IACUC Protocol #2018-2096) and performed in accordance with relevant institutional and national guidelines for the care and use of laboratory animals. Anesthetized female SKH1-Elite mice (Cr1:SKH1- Hr hr ， strain code 477 (Charles River Laboratories) was exposed to UVB three times a week for 20 weeks, as previously described. 12During UVB exposure, the head and tail areas were covered with felt to confine tumor induction to the skin only on the back. The average irradiance was 0.312–0.356 mW / cm². 2 A custom UV lamp unit (USHIO America, Inc.) equipped with ten 7.2W G8T5 bulbs (305 nm peak wavelength) was used for UVB exposure. Before each exposure period, UVB irradiance was measured using a PMA2100 radiometer equipped with UVA and UVB detectors (Solar Light), and the time required to reach the desired dose was calculated. Over the first 10 weeks, the UVB dose was gradually increased (10% per week), from 80 mJ / cm². 2 The dose was started at 175 mJ / cm 2 The final dose (time range of 8 minutes 11 seconds to 9 minutes 20 seconds, depending on the UVB irradiance measurement).

[0042] After 20 weeks of UVB exposure, mice were divided into treatment groups based on the initial tumor number to balance the initial tumor burden. Mice were treated daily with 200 μL of the drug, applied topically to the dorsal skin for 10 weeks. Mice were housed in groups (n=5 / cage) and oral exposure to the drug was minimized by using a rapidly absorbed medium (50% acetone / 10% glycerol). Every other week, mice were weighed and their physical condition was assessed. Tumor burden was determined weekly based on human counts and digital photographs quantified using ImageJ software. 13 Individual tumors are numbered and tracked during image analysis; if the tumor boundaries between individual tumors become merged (e.g., two tumors merge), they are still counted as two tumors, and the area average is taken.

[0043] At harvest, colon length was measured and blood was collected in K2-EDTA micro-blood collection tubes (Becton Dickinson) and processed into plasma for serum amyloid A (SAA) measurement via ELISA. All skin lesions were counted at harvest, and tumors large enough to be bisected were collected. The tumor tissue was bisected, with one half fixed in a Histochoice and embedded in a paraffin block, and the other half frozen for ELISA analysis. Tissue sections were stained with hematoxylin / eosin for histopathological evaluation or multiplex immunofluorescence imaging. Skin collected from mice with UV-induced tumors was carefully selected to bisect the largest tumors for staining. In mice with multiple tumors, not every tumor or lesion could be excised. Therefore, we prioritized larger, more prominent tumors across all mice and examined as many tumors as possible. The tissue examined by the inpatient dermatologist was not representative of the entire sample and excluded some smaller precancerous lesions; however, this was consistent across all treatment groups.

[0044] Skin irritation test Topical formulations of 2% PALA / 50% acetone / 10% glycerol, 5% imiquimod (IMQ), 5% 5-FU (5-FU), a carrier-mediated treatment (50% acetone / 10% glycerol) control, and Aquaphor were applied daily (approximately 0.05 g / mouse) to the dorsal skin of SKH1-Elite female mice (8 weeks old; n=5 / group) for 7 days. During the final analysis, the Aquaphor and carrier-mediated treatment groups were merged into single groups as no differences were detected between them. Mouse weight changes, skin appearance, and signs of distress were monitored daily. In response to significant weight loss in the IMQ and 5-FU treatment groups, these mice were supplemented with dietary gel 76A (ClearH2O) starting on day 3 (IMQ) or day 5 (5-FU). At harvest, spleen weight and colon length were measured. Blood was collected in K2-EDTA micro-blood collection tubes (Becton Dickinson) and processed into plasma for SAA measurement by ELISA. Skin tissue was harvested and fixed in Histochoice and embedded in paraffin blocks, or frozen for ELISA analysis. Tissue sections were stained with hematoxylin / eosin for histopathological evaluation.

[0045] Enzyme-linked immunosorbent assay Serum amyloid A levels in plasma samples were measured using the Mouse SAA ELISA Kit (E-90SAA, Immunology Consultants Laboratory, Inc.) according to the manufacturer's instructions. Lipocarrier-2 (LCN2) levels were quantified from pre-weighed fecal samples homogenized in 0.5 mL PBS using the Mouse Lipocarrier-2 / NGAL DuoSet ELISA (DY1857) and DuoSet Auxiliary Reagent Kit 2 (DY008, R&D Systems). Skin protein lysates were prepared by homogenizing frozen tissue in RIPA buffer (50 mM Tris (pH 8), 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulfate, 1x Pierce protease inhibitor mixture (A32965, ThermoFisher)) using a pestle and scissors. The following cytokines and chemokines were quantified from 25 µg of lysate using a custom 10-plex U-PLEX panel (K15069L-2; Meso Scale Discovery): GM-CSF, KC / CXCL1, IFNα, IFNβ, IFNγ, IL-1β, IL-6, IL-12p70, IL-17A, IL-17C, IL-17F, IP-10 / CXCL10, MCP-1 / CCL2, RANTES / CCL5, and TNFα. U-Plex data were collected using a MESO SECTOR S 600 plate reader (IC0AA-0; Meso Scale Discovery) and analyzed using MSD Discovery Workbench 4.0 software.

[0046] Multiplex Immunofluorescence Imaging Immunohistochemical staining was performed using the Discovery ULTRA automated stainer from Roche Diagnostics (Indianapolis, IN). Briefly, antigen retrieval was performed using proteinase K (IHC Select, 21627; Millipore) and / or Tris / borate / EDTA buffer (Discovery CC1, 06414575001; Roche) (pH 8.0 to 8.5). Antigen denaturation was performed using citrate buffer (Discovery CC2, 05424542001; Roche). Time, temperature, and dilution are listed in Tables 13-15.

[0047] Table 13: Panel conditions for multiple staining of antimicrobial peptides Table 14: Panel conditions for multiplex staining of immune cells Table 15: Panel conditions for multiple staining of cell proliferation Antibodies were visualized using OmniMap anti-rabbit HRP (05269679001; Roche) and UltraMap anti-rat HRP (05891884001; Roche) combined with Akoya Biosciences (Marlborough, MA) opal fluorophores. The respective antibodies are listed in Tables 1-3. Slides were counterstained with Spectral DAPI (FP1490; Akoya Biosciences).

[0048] For cell-secreted molecules (e.g., DefB14), QPath software was used to measure the positively stained areas within tumor tissue. First, the total tissue area (both tumor and non-tumor tissue) was assessed using the "Tissue Detection" command. Next, the threshold command was used to determine the number of specific fluorophores detected within the region. For each sample, the percentage of area covered by the targeted fluorophore was calculated.

[0049] Within the same annotated tissue region, LL37 was assessed using the positive cell detection method on QuPath software. + Cells, CD3 + T cells, CD8 + T cells, F4 / 80 + Macrophages and MPO + Cell proportion and density. This allows the use of threshold functions to determine cell radius and fluorophore detection thresholds. The total number of cells in the tumor region is determined using either DAPI or autofluorescence detection channels. For each detection channel, the percentage of positive cells for each targeted fluorophore is calculated.

[0050] Immunohistochemical staining Immunohistochemical staining was performed by the Cleveland Clinic Imaging Core using the Discovery ULTRA automated stainer from Roche Diagnostics. Briefly, antigen retrieval was performed using Tris / borate / EDTA buffer (Discovery CC1, #06414575001, Roche) (pH 8.0 to 8.5). Time, temperature, and dilution are shown in Table 16.

[0051] Table 16: Staining conditions for cell cycle markers Antibodies were visualized using OmniMap anti-rabbit HRP (#05269679001, Roche) combined with the ChromoMap DAB assay kit (#05266645001, Roche). Finally, the slides were counterstained with hematoxylin and bluing.

[0052] Statistical analysis Statistical analysis was performed using GraphPad Prism software (version 9.1). One-way or two-way ANOVA with post-hoc Bonfroni or Duque multiple comparison tests was used to determine the significance of datasets with multiple variables. Chi-square tests were applied for comparisons of categorical variables. Test details for specific experiments are included in the legend.

[0053] result Topical PALA at concentrations up to 5% (w / v) was non-toxic and reduced tumor growth in a dose-dependent manner. To determine the optimal dose of topical PALA for the treatment of NMSCs, SKH1-Elite mice were irradiated with UV light three times weekly for 20 weeks to induce SCC, followed by daily treatment with 0%, 1%, 2%, or 5% (w / v) PALA in 50% acetone / 10% glycerol for 10 weeks. Topical PALA treatment was well tolerated over 10 weeks, with no significant skin irritation or weight loss (Figure 1A). Previous toxicology studies have identified the intestines, liver, and central nervous system as primary targets for systemic PALA toxicity. 11Therefore, circulating serum amyloid A (SAA) levels were measured from plasma collected at the endpoint as a marker of liver inflammation and systemic toxicity (Fig. 1B). No significant differences were found between treatment groups; however, there was a trend toward higher SAA levels in mice treated with 1% and 5% PALA (p = 0.067 and p = 0.066, respectively). Gross colon pathology was examined, and colon length was measured at harvest as a quantitative measure of intestinal pathology. No significant differences in gross colon pathology or colon length were observed between treatment groups in any of the mice (Fig. 1C). Fecal lipid transporter-2 (LCN2) levels were also quantified as a marker of intestinal inflammation. Only mice treated with 5% PALA had significantly higher LCN2 levels compared to mice treated with the vector (186.8 ± 121.1 vs. 18.1 ± 6.0, p = 0.032; Fig. 1D). Importantly, during the 10-week treatment period, the total tumor number in mice treated with 2% or 5% PALA (3.83 ± 0.6 and 6.20 ± 1.8 at the endpoint, respectively) was significantly less than that in mice treated with the mediator or 1% PALA (12.5 ± 2.4 and 11.7 ± 3.8 at the endpoint, respectively, p = 0.029; Figure 1D). This was particularly evident when calculating the mean change relative to the initial tumor burden; the mediator and 1% PALA treatment groups showed a steady increase in mean tumor burden (286.4% ± 62.6% and 275.0% ± 84.7% of the original size at the endpoint, respectively), which was not observed as early as week 3 in the 5% PALA group (91.6% ± 13.4% of the original size at the endpoint) or as late as week 8 in the 2% PALA group (99.2% ± 20.5% of the original size at the endpoint, p = 0.0018; Figure 1E). These results indicate that up to 5% (w / v) of topical PALA was well tolerated during 10 weeks of daily treatment and showed antitumor activity at concentrations of 2% and higher. Based on the significant antitumor effect (mean tumor burden change of 286.4% ± 62.6% in the mediator at endpoint compared to 99.2% ± 20.5% of the original size for 2% PALA, p = 0.0018) and the absence of any increase in the inflammatory markers SAA and LCN2 at this dose, the 2% PALA dose was selected for further analysis.

[0054] Daily application of topical PALA was better tolerated than standard care topical treatment for NMSCs. Tolerance to daily treatment with the current standard care topical ointment, 5% IMQ, and 5% 5-FU was compared to 2% PALA over 7 days in newly tested SKH1-Elite mice and their vector control. Skin appearance, weight change, and body condition scores were assessed daily. Significant weight loss was observed in mice treated with IMQ or 5-FU topical treatment compared to mice treated with the vector and 2% PALA, requiring interventional care and resulting in the death of one mouse (Fig. 2A, arrow). In the 5-FU group, skin irritation was evident in mice treated with IMQ as early as day 2 and day 5; no skin irritation was observed in mice treated with the vector or 2% PALA (Fig. 6).

[0055] Gross pathology of the spleen and intestines was assessed on day 7 after harvest, SAA levels were measured, and skin histopathology was performed to evaluate the expression of inflammatory mediators in the treated skin. No differences were observed between groups in gross intestinal pathology or SAA levels (Figures 2B and 2C). This is similar to previous reports of topical IMQ treatment in mice. 14 Mice treated with IMQ exhibited splenomegaly (Fig. 2D), significant epidermal thickening (acanthosis; Fig. 2E), and upregulation of IL-17 family cytokines in the treated skin tissue (Table I). Furthermore, elevated levels of skin RANTES and GM-CSF were observed in IMQ-treated animals (Table I). Mice treated with 5-FU also exhibited toxicity, characterized by a significant decrease in spleen weight (Fig. 2D), epithelial erosion (Fig. 2E), and increased expression of IL-6, IL-17C, KC, and GM-CSF in the lysates of treated skin (Table I). In contrast, mice treated with PALA showed no changes in spleen weight, colon length, SAA levels, skin histology, or expression of inflammatory cytokines / chemokine groups in the treated skin compared to the mediator control. These findings suggest that daily application of topical PALA to mice is significantly better tolerated than current standard-of-care topical treatments for NMSCs.

[0056] In mice treated with topical PALA, tumor burden, tumor area, and tumor grade were all reduced. AK lesions and SCC tumors were induced in SKH1-Elite mice by UVB exposure for more than 20 weeks. Tumor-bearing mice were treated daily with 2% PALA or a vector control for 10 weeks, and the toxicity of the treatment and changes in the number, size, and grade of treatment-induced tumors were evaluated. Daily topical PALA treatment for 10 weeks was well tolerated in mice, as reflected by no significant differences in body weight, SAA levels, gross intestinal pathology, or LCN2 levels compared to vector-treated control mice (Figs. 3A-D). The tumor burden in PALA-treated mice was significantly lower than that in vector-treated mice; the mean number of tumors in vector-treated mice increased over the 10-week treatment period (endpoint value 252.3% ± 34.3%, p = 0.00051), while the mean number of tumors per mouse remained unchanged in the PALA-treated group (endpoint value 99.2% ± 20.5%, p = 0.97) (Figs. 3E and 3F). Tumor size was also significantly larger in the mediator treatment group, with a mean surface tumor area (endpoint value of 197.1 mm). 2 Compared to 30.3 mm 2 (p<0.0001) and mean tumor cross-sectional area (endpoint value 4.4 mm). 2 Comparison 2.4 mm 2 (p=0.003) This was true for both (Figures 3G and 3H). When analyzing the growth of individual tumors, the proportion of tumor regression in PALA treatment was greater than that in the mediator control during the treatment period (83.3% vs. 26.8%, p<0.0001; Table II). Furthermore, the growth phenotypes differed between treatments, with most PALA-treated tumors shrinking or continuing to decrease in size after the initial growth phase (97.9%), compared to continued growth observed in animals treated with vectors (46.3%; p<0.0001; Table II). These data support the use of topical PALA for the treatment of lesions such as AK and SCC.

[0057] The excised tumors were histologically graded as benign / proliferative lesions, in situ AK / SCC, well-differentiated SCC, or poorly differentiated SCC (Table III). Importantly, compared with the control group treated with the vector, mice treated with PALA had a lower prevalence of well-differentiated and poorly differentiated SCC (38% vs. 69%, p<0.0001) and an increased proportion of in situ AK / SCC and benign lesions (62% vs. 31%, p<0.001), indicating that PALA treatment can block tumor progression.

[0058] The resected tumor was also stained to target the expression of cell cycle markers to determine whether PALA could induce cell cycle arrest similarly to 5-FU. This was also observed in cyclin B1. + Or cyclin A2 + No differences were observed in the cell population (Figure 7). Phosphorylated histone H3 was detected in PALA-treated samples compared to the mediator control. + A slight increase in cell number was observed (4.9% vs. 3.1%, p = 0.0067), but due to the very small percentage of positive cells in the tissue (<5%) and their location at the tumor base, it remains unclear whether these cells are part of the tumor and / or whether they have been exposed to significant amounts of the drug. However, compared to the control group treated with the drug, tumors treated with PALA contained a higher mean Ki67 per tumor. + Cell number (24.6% vs. 10.4%; p=0.005) and lower cyclin D1 levels per tumor were also observed. + The percentage of cells (39.4% vs. 48.9%, p=0.0329; Figure 7) indicates that these tumor cells may have undergone cell cycle arrest in response to PALA treatment. 15 .

[0059] Topical PALA treatment stimulates innate immune activation and enhanced recruitment of cytotoxic T cells. Previous work has shown that topical PALA treatment of bacterially infected human skin explants stimulates the expression of antimicrobial peptides such as human β-defensin 2 (HBD2) and cathelicidin (LL-37) in a NOD2-dependent manner. 16 Therefore, the levels of mouse homologs of these AMPs (DefB14 and LL-37) in tumor tissues of locally treated mice were assessed using multiplex immunofluorescence microscopy. DefB14 staining within the tumors was diffuse, indicating AMP secretion from cells, and the total area of ​​DefB14 staining did not differ between treatment groups (Fig. 4A). In contrast, LL-37 staining was discrete and co-localized with nuclear DAPI staining, suggesting that the number of cells expressing LL-37 should be quantified in the tumor tissue. These results indicate that LL-37 is present in PALA-treated tumor samples. + The number of cells increased significantly (Figure 4B).

[0060] Neutrophils and monocytes / macrophages are the main innate immune cell types that express NOD2 and LL-37. 17，18Therefore, multiplex immunofluorescence microscopy was used to analyze markers of neutrophils (myeloperoxidase; MPO) and macrophages (F4 / 80) in tumor sections to determine whether any of these immune cell populations were recruited to the tumor tissue in response to local PALA treatment. MPO levels were compared between treatment groups. + The number of cells was the same, indicating that neutrophils were not differentially recruited to the tumor tissue (Figure 5A). Conversely, tumors treated with PALA showed an increased number of F4 / 80 cells compared to the control group treated with the vector. + Macrophages (Fig. 5B and Fig. 5E).

[0061] The role of LL-37 in carcinogenesis is controversial. In some cancers, it upregulates and stimulates the production of anti-inflammatory tumor-associated macrophages (TAMs) that promote tumorigenesis, while in other cancer types, LL-37 promotes the production of anti-tumor CD8 cells. + Activation and expansion of cytotoxic T cells 18 To better understand LL-37 + Cells and F4 / 80 + The role of macrophages in tumors treated with PALA was evaluated using multiplex immunofluorescence microscopy to assess CD8 levels. + Prevalence of cytotoxic T cells. CD3 levels in tumor tissue between samples treated with the vector and those treated with PALA. + The total number of T cells was the same (Figure 5C). However, compared with the control group treated with the vector, tumors treated with PALA had an increased number of CD8 cells. + Cytotoxic T cells (Fig. 5D and Fig. 5E). In summary, the results indicate that local PALA treatment not only inhibits tumor proliferation but also activates the innate immune response, thereby generating an anti-tumor cytotoxic T cell response.

[0062] This embodiment demonstrates that topically applied PALA is a novel treatment for actinic keratosis and squamous cell carcinoma of the skin. PALA is a simple molecule that can treat cancer through two different mechanisms. 16，19-21 We have successfully demonstrated that daily topical treatment with 2% PALA not only reduces tumor growth but also inhibits tumor progression to more metastatic cell types. Multiplex immunofluorescence microscopy of the treated tumors showed changes in cell cycle progression markers and increased immune regulation, corresponding to the known mechanism of action of this drug. 19 .

[0063] PALA has previously been extensively tested as a systemic treatment for cancer. 22，23 Although the treatment was quite effective in two mouse models of colon cancer, the drug cannot be used to treat human malignancies due to dose-limiting toxicity. 23At low concentrations, we observed no evidence of toxicity from PALA. We believe that topical treatment with PALA will be more advantageous than IMQ, 5-FU, and tebanibulin; these are the current standard of care for NMSC and all cause severe skin irritation and inflammation. 24 PALA did not induce inflammation or skin irritation when applied topically to mice for up to 10 weeks. PALA is also chemically non-reactive and can be stored at ambient temperature, making it a viable option for use in resource-poor regions. 20 .

[0064] While this disclosure is not limited to any particular mechanism, and an understanding of such mechanism is not essential for practicing the invention, our results of local PALA demonstrate that it can induce an immunotherapeutic effect without the need for additional components to exert a therapeutic effect. Other studies have investigated the potential of NOD2 activators as cancer immunotherapies and found that they primarily act as immune adjuvants. Derivatives of the lipid-conjugated NOD2 bacterial ligand muramyl dipeptide (MDP) have demonstrated promising antitumor effects in animal models and human clinical trials. 25 Direct injection of lipid conjugates of MDP into fibrosarcoma, hepatocellular carcinoma, B16-F10 melanoma, or intravenous administration in UV-induced skin cancer models or multiple metastatic liver cancer models, prevented tumor growth, metastasis, and / or improved survival rates in cancer-bearing animals. 25，26 Although promising results were observed in early clinical trials, only two MDP derivatives (mifamin / Mepact) were used. ® and ImmTher ® The Phase III trial was successfully completed and approval was obtained for use in the European Union (Mepact). ® ), or has been approved by the FDA as an orphan drug (Immotherapeutic agent). ® In combination with multi-drug chemotherapy after resection for non-metastatic osteosarcoma 27 It is believed that the therapeutic effect is primarily mediated by the adjuvant effect of these MDP / lipid immunotherapies, which leads to enhanced tumor-killing activity of macrophages. 25，28 This is a mechanism that may be similar to our local PALA formulation. Recently, cell wall peptides produced from Enterococcus faecalis have been described as enhancing checkpoint inhibitor cancer therapy in a NOD2-dependent manner. 29 Similar to our findings with localized PALA, this cell wall peptide increased the antitumor cytotoxicity of CD8. + T cells were involved, and the macrophage population was also affected.

[0065] Both IMQ and PALA are immunomodulators that induce anti-tumor immune responses. IMQ is a nucleoside in the imidazoquinoline family and is frequently used to treat non-MSCs. 24，30 It activates anti-tumor immunity by stimulating TLR7 in macrophages and other immune cells. 24，30 IMQ is known to upregulate the expression of interferon-α, as well as interleukins 1, 6, and 8, and to trigger autophagic cell death in macrophages. 24，30，31 We have shown that PALA treatment does not increase these cytokines and that macrophages are more abundant in the treated area, thus indicating that PALA works through an immune mechanism different from that activated by IMQ. While the invention is not limited to any particular mechanism, and an understanding of the mechanism of the invention is not essential for its practice, it is hypothesized that PALA may induce an antitumor response through enhanced cross-presentation of tumor antigens, thereby leading to CD8... + Increased tumor-specific cytolytic activity of T cells. This hypothesis is supported by studies describing enhanced NOD2-dependent cross-presentation of MHC class I and MHC class II antigens in cells stimulated with MDP. 32-34 However, it still needs to be formally tested.

[0066] Immune cell infiltration is observed in most malignant tumors. 35 Macrophages are a major component of solid tumors and can promote tumorigenesis through stimulation of angiogenesis, immunosuppression, invasion, and metastasis. 36，37 Tumor-associated macrophages (TAMs) are key regulators of the link between the immune system and cancer. 38 TAM can promote, rather than limit, tumor progression and may negatively impact treatment response and inhibit T cell recruitment. 38 However, activated macrophages are effective as a cancer immunotherapy because they can kill cancer cells directly or indirectly by recruiting other immune cells such as cytotoxic T lymphocytes. 39 Within tumors, T cells often exhibit dysfunction. 40 Furthermore, activated macrophages may be able to restore their cytolytic activity. 39 Recent research has highlighted how the relationship between TAM and the tumor microenvironment can lead to improved cancer therapies. 41,42 Furthermore, while the invention is not limited to any particular mechanism, and an understanding of that mechanism is not essential for practicing the invention, our findings suggest that PALA can recruit and locally activate macrophages to enhance cytotoxic T cell responses.

[0067] PALA inhibits pyrimidine nucleotide synthesis and suppresses cell proliferation by depleting dCTP and dTTP, thereby preventing treated cells from completing DNA synthesis. 20 Although Ki-67 expression is frequently used as a marker for active cell cycles, it is only absent in G0 quiescent cells and may be highly expressed in cells arrested at other stages of the cell cycle. 27 Cyclin D1 expression peaks at the end of G1 phase and is essential for the transition of cells to S phase. Given the known effects of PALA on the cytosine pool... 20 Ki-67 in tumors treated with PALA + Increased cell number and cyclin D1 + The decreased percentage of cells supports the conclusion that the cells may be undergoing cellular stasis. These findings highlight the drug's dual function as both a cell inhibitor and an immunomodulator.

[0068] Example 2 PALA formulations This embodiment describes the development of a topical formulation of PALA (CMI-34-1). As a first approach, several hydrogels were prepared using different concentrations of natural and synthetic polymers: xanthan gum, methylcellulose, hydroxyethylcellulose, Carbopol 971P NF, poloxamer 188, and 407. Poloxamer 188 at 20% w / w resulted in the only hydrogel providing PALA permeability in in vitro diffusion tests (Franz pool). As a second approach, several penetration enhancers (transcutol HP, labrasol ALF, and isopropyl myristate) were added to the poloxamer 188 hydrogel to improve PALA permeability. In vitro diffusion tests confirmed that labrasol ALF significantly improved PALA permeability, and therefore it was incorporated into the formulation. To further refine the formulation, isopropyl myristate and kolliphor EL were added to obtain their emollient and texture-enhancing properties. Furthermore, they also have penetration-enhancing effects. Although they did not improve permeability in vitro, they can be effective in vivo. The formulation containing P188 20% w / w (70%), Labrasol ALF (21.6%), Kolliphor EL (5.4%), and isopropyl myristate (3%) was selected as the formulation for mouse studies. Stability studies conducted for 9 weeks at room temperature and under refrigeration (2℃–8℃) confirmed the stability of the formulation under both conditions.

[0069] Details of the HPLC method Prepare a 1 mg / mL PALA standard solution (CMI-34-1) in diluent (100% Type I water). Accurately weigh 20 mg of PALA into a 20 mL volumetric flask and adjust the volume with 100% Type I water. Sonicate the sample for 20 minutes until completely dissolved and inject it into an HPLC system for suitability testing. To assess precision and repeatability, inject standard A five times and standard B twice, calculating the peak area and relative standard deviation (RSD%). According to internal guidelines, the RSD% of the main peak of the API in the standard solution must be <2.0%. Finally, calculate the consistency of the standards as described by the following equation: Area A and area B These are the peak areas under the standard samples A and B, respectively, and their concentrations. A and concentration B These are the concentrations of standards A and B, respectively.

[0070] Preparation of placebo hydrogels The placebo hydrogels were prepared as shown in Table 4.

[0071] Table 4 - List of placebo hydrogels Preparation of formulations for in vitro testing (Franz pool) All formulations were prepared by accurately weighing 50 mg of PALA into 4 mL glass vials. 950 mg of the mediator (listed in Table 5) was added. The vials were stirred at room temperature until a clear solution was obtained. Except for poloxamer P407 20% w / w and P188 40% w / w, where PALA was solubilized by stirring the vials in an ice bath (to prevent gelation at room temperature).

[0072] Table 5 lists the formulations tested in the Franz pool. Formulation code Composition of media PALA concentration in the formulation AP0536 / 4 / 01 • 100% P188 (20% w / w) aqueous solution 50 mg / g AP0536 / 7 / 04 • 100% P188 (40% w / w) aqueous solution 50 mg / g AP0536 / 13 / 01 • 100% P188 (34% w / w) aqueous solution 50 mg / g AP0536 / 4 / 05 • 100% P407 (20% w / w) aqueous solution 50 mg / g AP0536 / 7 / 03 • 100% P407 + P188 (15% w / w + 10% w / w) aqueous solution 50 mg / g AP0536 / 4 / 02 • 100% Carbopol 971P NF (2% w / w) aqueous solution 50 mg / g AP0536 / 4 / 03 • 100% hydroxyethyl cellulose (2% w / v) aqueous solution 50 mg / g AP0536 / 7 / 05 • 100% hydroxyethyl cellulose (0.5% w / v) aqueous solution 50 mg / g AP0536 / 4 / 04 • 100% xanthan gum (2% w / v) aqueous solution 50 mg / g AP0536 / 7 / 01 • 100% xanthan gum (1% w / v) aqueous solution 50 mg / g AP0536 / 4 / 06 • 100% methylcellulose (2% w / v) aqueous solution 50 mg / g AP0536 / 7 / 06 • 100% methylcellulose (1% w / v) aqueous solution 50 mg / g AP0536 / 12 / 05 • 80% w / w P188 (34% w / w) aqueous solution • 20% w / w Labrasol ALF 50 mg / g AP0536 / 12 / 06 • 80% w / w P188 (34% w / w) aqueous solution • 20% w / w transcutol HP 50 mg / g AP0536 / 12 / 07 • 80% w / w P188 (34% w / w) aqueous solution • 20% w / w isopropyl myristate 50 mg / g AP0536 / 16 / 01 • 70% w / w P188 (20% w / w) aqueous solution • 21.6% w / w Labrasol ALF • 5.4% w / w Kolliphor EL • 3% w / w Isopropyl Myristate 50 mg / g AP0536 / 17 / 01 • 80% w / w P188 (30% w / w) aqueous solution • 20% w / w Labrasol ALF 50 mg / g AP0536 / 17 / 02 • 72% w / w P188 (20% w / w) aqueous solution • 10% w / w Tefose 63 • 10% w / w paraffin oil • 8% w / w Labrasol ALF 50 mg / g AP0536 / 17 / 03 • 82% w / w P188 (20% w / w) aqueous solution • 15% w / w Labrasol ALF • 2% w / w Tefose 63 • 1% w / w Paraffin oil 50 mg / g Preparation of bigel formulations xanthan gum-based bigel The bigel consisted of an aqueous phase and an oil phase. Xanthan gum 1% w / v (prepared as described in Table 5) was used as the aqueous phase. The oil phase was prepared by mixing labrafac lipophile WL 1349 (95% w / w) and emulfree duo (5% w / w) at room temperature. 50 mg of PALA was accurately weighed into a 4 mL vial. 800 mg of the aqueous phase was added. The vial was stirred at room temperature until the API was completely dissolved. Then, 150 mg of the oil phase was slowly poured into the gelled aqueous phase and homogenized at 5000 rpm for 5 minutes at room temperature until a homogeneous emulsion was obtained.

[0073] Poloxamer 188-based dual gel Poloxamer 188 (P188) 34% w / w (Table 5) was used as the aqueous phase. The oil phase was prepared by mixing labrafac lipophile WL 1349 (95% w / w) and emulfree duo (5% w / w) at room temperature. 50 mg of PALA was accurately weighed into a 4 mL vial. 800 mg of the aqueous phase was added. The vial was stirred at room temperature until the API was completely dissolved. Then, 150 mg of the oil phase was slowly poured into the gelled aqueous phase and homogenized at 5000 rpm for 5 minutes at room temperature until a homogeneous emulsion was obtained.

[0074] Table 6 - List of dual-gel formulations tested in the Franz pool In vitro diffusion cell for hydrogels (Franz cell) An in vitro diffusion assay (Franz pool, Copley) was performed to simulate in vivo conditions for transdermal transfer of PALA (CMI-34-1) from the formulation. The assay was conducted in a water bath. 100 μL of a hydrogel or duplex loaded with PALA (CMI-34-1) (containing 5 mg of API) was added to the donor chamber of the Franz pool. 7 mL of PBS pH 7.4 was added to the recipient chamber. The chambers were separated by Strat-M membranes (transdermal diffusion assay model, Merck) with a diameter of 25 mm. The Franz pool was incubated at 37 °C in a suitable heating block with stirring at 450 rpm. At selected time points (15, 30, 60, 120, 180, and 1440 min), 100 μL of sample was removed from the recipient chamber and drug release was analyzed by HPLC. The same volume was replaced with fresh PBS buffer preheated at 37 °C.

[0075] HPLC determination of the formulation The formulation was analyzed in duplicate by UV-HPLC. Using a positive displacement pipette, 300 µL of the formulation was transferred to an Eppendorf tube and centrifuged at 13,000 rpm for 5 minutes. 100 µL of the supernatant was accurately weighed into a 5 mL volumetric flask containing 100% type I water. The flask was then vortexed for 10 seconds and sonicated in an ultrasonic bath for 5 minutes before analysis by UV-HPLC.

[0076] 9-week stability study A 9-week stability study was conducted on the lead formulation (AP0536 / 23 / 01) stored at room temperature and under refrigeration (2°C–8°C). The formulation was stored in 4 mL clear glass vials. Samples were visually inspected (color and appearance) at specified time points (0, 7, 14, and 63 days) and analyzed by UV-HPLC. At each time point, 300 µL of the formulation was sampled and transferred to an Eppendorf tube and centrifuged at 13,000 rpm for 5 min. 100 µL of the supernatant was accurately weighed into a 5 mL volumetric flask containing 100% type I water. The flask was then vortexed for 10 seconds and sonicated in an ultrasonic bath for 5 min before analysis by UV-HPLC. The determination was performed in duplicate.

[0077] Formulations used in PK studies The lead formulation (AP0536 / 23 / 01) was freshly reconstituted at three different drug loadings (0%, 2%, and 4% w / w PALA). Poloxamer 188 20% w / w aqueous solution was prepared on a larger scale (30 g P188 + 120 g type I water) following the same procedure reported in Table 4. The placebo mediator (AP0539 / 29 / 01) was prepared on a 150 g scale as follows: 105.0 g P188 20% w / w aqueous solution 32.4 g Labrasol ALF 8.1 g kolliphor EL 4.5 g isopropyl myristate A 2% w / w PALA formulation (AP0539 / 29 / 02) was prepared in a 42 g scale by accurately weighing 0.84 g of PALA and adding 41.160 g of mediator (AP0539 / 29 / 01). A 4% w / w PALA formulation (AP0539 / 29 / 02) was prepared in a 42 g scale by accurately weighing 1.68 g of PALA and adding 40.32 g of mediator (AP0539 / 29 / 01).

[0078] Results and discussion Research on hydrogels This work investigated the development of suitable preclinical formulations of the compound PALA (CMI-34-1) for topical application. Different polymers at varying concentrations were investigated to prepare hydrogels: xanthan gum, methylcellulose, hydroxyethylcellulose, Carbopol 971P Nf, poloxamer 188 (P188), and poloxamer 407 (P407). As a control, PALA prepared in type I water was also tested in a Franz cell. The control (water only) showed almost no PALA permeation (approximately 0.3%) in HPLC analysis after 24 hours (Table 7, Figure 10). The xanthan gum, methylcellulose, hydroxyethylcellulose, and Carbopol 971P NF hydrogels were initially prepared with a 2% w / w polymer content, resulting in a very viscous gel. The hydrogels were loaded with PALA at a concentration of 50 mg / g, and the diffusion of PALA from the hydrogel through the transdermal diffusion membrane was monitored in a Franz cell.

[0079] No PALA permeation through the membrane was observed after 24 hours (Table 7, Figure 10). Based on these results, the polymer content of the hydrogel was reduced from 2% to 1%–0.5% to reduce the viscosity of the formulation and ultimately promote PALA permeation through the membrane. Unfortunately, no release was observed even at lower polymer concentrations (Table 7, Figure 10), except for the poloxamer 188 hydrogel, which showed up to 60% PALA permeation after 24 hours (Table 7, Figure 10). Therefore, the P188 hydrogel was selected for further investigation.

[0080] Since 20% poloxamer 188 exhibits low viscosity, increasing the concentration of P188 was investigated to provide a hydrogel with a higher viscosity that would remain firmly attached to the skin after application. After several trials, 34% was identified as providing an acceptable viscosity, thus improving adhesion to the skin (Table 8). Therefore, a 34% w / w P188 solution was prepared, and it was shown that 49.7% of PALA was released after 24 hours.

[0081] Table 8. Appearance and determination of P188 at different polymer concentrations The 34% w / w P188 formulation was further optimized by incorporating different penetration enhancers, namely 20% w / w Labrasol ALF, Transcutol Hp, and isopropyl myristate. Details regarding composition and visual observation are provided in Table 9. Isopropyl myristate was also selected as a well-known emollient and texture enhancer in cosmetics. In addition to hydrogels, bigels were also investigated.

[0082] Table 9 - Appearance and determination of hydrogel formulations and dipels containing penetration enhancers The diffusion of PALA from the formulations listed in Table 9 was tested using the Franz pool. A significant improvement in diffusion was observed upon the addition of 20% w / w labrasol (AP0536 / 12 / 05) to the poloxamer hydrogel. The release curves (Figure 11) show that approximately 50% of PALA was released within 90 minutes, reaching 76.6% release at 3 hours. With P188 + 20% w / w transcutol (AP0536 / 12 / 06), 6.1% of PALA was released within 90 minutes, reaching 52.5% release at 24 hours. No PALA release was detected within 180 minutes from P188 + 20% w / w isopropyl myristate (AP0536 / 12 / 07) and P188 dual gel (AP0536 / 12 / 04), but 49.7% and 63.1% releases were observed at 24 hours, respectively. No release was detected in the xanthan gum bigel (AP0536 / 7 / 02) within 24 hours. Based on these results, P188 34% w / w aqueous solution (80% w / w) + labrasol ALF (20% w / w) (AP0536 / 12 / 05) was selected as a lead formulation for further investigation (improved permeability and texture).

[0083] To further improve the permeability and texture of the formulation (AP0536 / 12 / 05), additional components such as kolliphor EL, isopropyl myristate, Tefose 63, and paraffin oil were added. To compensate for the viscosity increase observed in the initial tests with the additional excipients, the concentration of poloxamer 188 was reduced from 34% w / w to 20% w / w. Furthermore, to fine-tune the viscosity of AP0536 / 12 / 05, the concentration of P188 was reduced from 34% to 30% (AP0536 / 17 / 01). Table 10 (Figure 12) lists the composition of the optimized formulation and its stability over 7 days. The results show that all formulations remained stable after 7 days of storage at room temperature (Table 10; Figure 12).

[0084] In vitro permeation studies were conducted on triplicate of the selected formulations. Figure 13 shows that approximately 50% and 70% of PALA were released from AP0536 / 16 / 01 and AP0536 / 17 / 01, respectively, after 4 hours. The other two formulations, AP0536 / 17 / 02 and AP0536 / 17 / 03, released 20% and 0%, respectively, after 4 hours. The formulation (AP0536 / 16 / 01), composed of 70% poloxamer 188 (20% aqueous solution), 21.6% labrasol ALF, 5.4% kolliphor EL, and 3% isopropyl myristate, was ultimately selected as the lead formulation for the PK study because of its favorable release profile and the presence of kolliphor EL and isopropyl myristate, which may contribute to PALA permeation in vivo and provide a better texture for the formulation.

[0085] 9-week stability study A 9-week stability study was conducted. A lead formulation containing 70% P188 (20% aqueous solution), 21.6% Labrasol ALF, 5.4% Kolliphor EL, and 3% isopropyl myristate was freshly reconstituted and stored under both ambient and refrigerated conditions (2°C–8°C). As shown in Table 11, no changes in assays or purity were detected after 9 weeks under either storage condition.

[0086] Table 11. 4-week stability study of the lead formulation AP0536 / 23 / 01 Supply of formulations to support PK research Formulations containing 70% P188 (20% aqueous solution), 21.6% labrasol ALF, 5.4% kolliphor EL, and 3% isopropyl myristate were freshly reconstituted for PK studies at 0% w / w (AP0536 / 29 / 01), 2% w / w (AP0536 / 29 / 02), and 4% w / w (AP0536 / 29 / 03) PALA concentrations. HPLC determinations were performed, and the results are reported in Table 12.

[0087] Table 12 - Determination of T0 time for transport of formulations prior to PK studies A hydrogel formulation comprising poloxamer 188 20% w / w (70%), labrasol ALF (21.6%), kolliphor EL (5.4%), and isopropyl myristate (3%) was selected as a lead formulation for PK studies. This formulation, developed using a combination of poloxamer 188 hydrogel and labrasol ALF (penetration enhancer), demonstrated high permeability of PALA in in vitro studies. Further addition of kolliphor EL and isopropyl myristate increased the emollient properties and improved the texture of the formulation. The formulation was physically and chemically stable for at least 9 weeks.

[0088] In vivo results Figure 14. Skin and plasma PALA levels in mice 24 hours after a single topical application of formulation AP0536 / 29 / 03 4% (w / v) PALA. 10 μL of 4% PALA (final dose = 0.4 mg / kg) was applied to the dorsal skin of male CD1 mice (n=3 / time point). Plasma and skin tissue samples were collected at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h after administration, and PALA levels were determined by LC-MS. A graph showing mean ± SEM values ​​is plotted.

[0089] Figure 15. The reformulated topical 2% PALA was non-toxic and effectively reduced tumor burden and total tumor area. SKH1-Elite female mice were irradiated with UV light three times a week for 20 weeks to induce skin cancer. Mice were then randomly assigned to treatment groups and treated topically once daily with a medium (AP0536 / 29 / 01), 2% (w / v) PALA (AP0536 / 29 / 02), or 4% (w / v) PALA (AP0536 / 29 / 03) for 6 weeks. A. Change in body weight during treatment, expressed as a percentage of baseline body weight. B. Levels of serum amyloid A (SAA), a marker of liver and systemic inflammation, at the endpoint. C. Measurement of colon length at the endpoint as a general measure of intestinal pathology. D. Levels of fecal lipid carrier protein-2 (LCN2) at the endpoint as a measure of intestinal inflammation. E. Quantification of mean tumor burden per mouse during treatment. F. Mean change in tumor burden per mouse during treatment, expressed as a percentage of the original tumor number. G. Mean tumor surface area per mouse was quantified from photographs taken during the treatment period. Mean ± SEM, n = 8–9 / group. Significance was determined by mixed-effects model using Bonfroni multiple comparison test (REML) or one-way ANOVA using Duker's multiple comparison test. All p-values ​​< 0.05 are shown on graphs.

[0090] References All publications and patents mentioned in this application are incorporated herein by reference. Various modifications and variations of the methods and compositions described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications intended to carry out the described modes of the invention will be apparent to those skilled in the art and are within the scope of the following claims.

Claims

1. A method for treating precancerous cells and / or cancerous cells of a subject, the method comprising: The composition is applied topically to a body area of ​​the subject that has precancerous cells and / or cancerous cells and / or adjacent to potential precancerous or cancerous cells, or The composition is provided to the subject so that the subject can apply the composition to the body area. The composition comprises N-phosphonoacetyl-L-aspartic acid (PALA) and water, and optionally also comprises at least one nonionic surfactant.

2. The method of claim 1, wherein the PALA is present in the composition at a concentration of about 0.1% to about 5.0% or about 1.0% to 8.0%.

3. The method of claim 1, wherein the area of ​​the subject includes skin.

4. The method of claim 1, wherein the precancerous cells include actinic keratosis cells.

5. The method of claim 3, wherein the cancerous cells include melanoma or non-melanoma skin cancer cells.

6. The method of claim 4, wherein the non-melanoma skin cancer cells are basal cell carcinoma cells or squamous cell carcinoma cells.

7. The method of claim 1, wherein the subject is a woman, wherein the region of the subject includes the surface of the subject's vagina, and wherein the precancerous cells and / or cancerous cells include vaginal precancerous cells and / or cancer cells.

8. The method of claim 1, wherein the region of the subject includes the surface of the subject's oral cavity, and wherein the precancerous cells and / or cancerous cells include oral precancerous cells and / or cancer cells.

9. The method of claim 1, wherein the subject is female, wherein the region of the subject includes the cervix of the subject, and wherein the precancerous cells and / or cancerous cells include cervical precancerous cells or cancerous cells.

10. The method of claim 1, wherein the composition further comprises a nonionic linear copolymer, and optionally wherein the nonionic linear copolymer comprises poloxamer 188.

11. The method of claim 1, wherein the application of the composition or the provision of the composition is repeated daily for at least 7 days, or for at least 14 days, or for at least 21 days.

12. The method of claim 9, wherein the nonionic linear copolymer is present in the composition at about 50%-90%, about 60%-80%, about 65%-75%, or about 70%.

13. The method of any one of claims 1-12, wherein the composition further comprises a nonionic surfactant, and optionally wherein the nonionic surfactant is present in the composition at 10%-30% or about 22%.

14. The method of claim 13, wherein the nonionic surfactant comprises octanoyl hexanoyl polyhydroxy-8 glycerol ester, or wherein the nonionic surfactant excipient comprises PEG-8 monoesters and diesters of octanoic acid (C8) and decanoic acid (C10) with small amounts of glycerol monoesters, diesters and triesters, or wherein at least one nonionic surfactant comprises LABRASOL ALF.

15. The method of any one of claims 1-14, wherein the composition further comprises a hydrogel agent, and the composition is in the form of a hydrogel.

16. The method of any one of claims 1-15, wherein the composition further comprises isopropyl myristate, and optionally wherein the isopropyl myristate is present in the composition at a concentration of about 0.5%-7%, about 1%-5%, or about 3%.

17. The method of any one of claims 1-16, wherein the composition further comprises a solubilizer and / or an emulsifier.

18. The method of any one of claims 1-17, wherein the composition further comprises polyethylene glycol glycerol ricinoleate, and optionally wherein the polyethylene glycol glycerol ricinoleate is KOLLIPHOR EL, and optionally wherein the polyethylene glycol glycerol ricinoleate is present in the composition at a concentration of about 2%-10%, about 3%-7%, or about 5%.

19. The method of any one of claims 1-18, further comprising administering an immune checkpoint inhibitor to the subject.

20. The method of any one of claims 1-19, wherein the subject is a human, dog, or cat.

21. The method of any one of claims 1-20, wherein the composition further comprises at least one non-PALA anticancer agent, and optionally wherein the at least one non-PALA anticancer agent comprises: Tibanibulin, fluorouracil (5-FU), and / or imiquimod (IMQ).

22. The method of any one of claims 1-21, wherein the composition is in the form of a cream, emulsion, skin lotion, gel, ointment, spray, or is present in a skin patch.

23. The method of any one of claims 1-22, wherein the composition further comprises the at least one nonionic surfactant, and optionally wherein the nonionic surfactant comprises poloxamer 188 and / or capryloyl hexanoyl polyhydroxy-8 glycerol ester.

24. A composition comprising: a) N-phosphonoacetyl-L-aspartic acid (PALA); b) Water; and c) At least one nonionic surfactant.

25. The composition of claim 24, wherein the PALA is present in the composition at a concentration of about 0.1% to about 5.0% or about 1.0% to 8.0%.

26. The composition of any one of claims 24-25, wherein the at least one nonionic surfactant comprises a nonionic linear copolymer.

27. The composition of claim 26, wherein the nonionic linear copolymer comprises poloxamer 188.

28. The composition of claim 26, wherein the nonionic linear copolymer is present in the composition at about 50%-90%, about 60%-80%, about 65%-75%, or about 70%.

29. The composition of claim 24, wherein the at least one nonionic surfactant is present in the composition at 10%-30% or about 22%.

30. The composition of claim 24, wherein the at least one nonionic surfactant comprises octanoyl hexanoyl polyhydroxy-8 glycerol ester, or wherein the at least one nonionic surfactant excipient comprises PEG-8 monoester and diester of caprylic acid (C8) and decanoic acid (C10) with small amounts of glycerol monoester, diester and triester, or wherein the at least one nonionic surfactant comprises LABRASOL ALF.

31. The composition of any one of claims 24-30, wherein the composition further comprises a hydrogel agent, and the composition is in the form of a hydrogel.

32. The composition of any one of claims 24-31, wherein the composition further comprises isopropyl myristate, and optionally wherein the isopropyl myristate is present in the composition at a concentration of about 0.5%-7%, about 1%-5%, or about 3%.

33. The composition of any one of claims 24-32, wherein the composition further comprises a solubilizer and / or an emulsifier.

34. The composition of any one of claims 24-33, wherein the composition further comprises polyethylene glycol glycerol ricinoleate, and optionally wherein the polyethylene glycol glycerol ricinoleate is KOLLIPHOR EL, and optionally wherein the polyethylene glycol glycerol ricinoleate is present in the composition at a concentration of about 2%-10%, about 3%-7%, or about 5%.

35. The composition of any one of claims 24-34, wherein the composition further comprises at least one non-PALA anticancer agent, and optionally wherein the at least one non-PALA anticancer agent comprises: Tibanibulin, fluorouracil (5-FU), and / or imiquimod (IMQ).

36. The composition of any one of claims 24-35, wherein the at least one nonionic surfactant comprises poloxamer 188 and / or capryloyl hexanoyl polyhydroxy-8 glycerol ester.

37. The composition of any one of claims 24-36, wherein the composition is in the form of a cream, lotion, or gel.

38. A system or pillbox, said system or pillbox comprising: a) A composition comprising N-phosphonoacetyl-L-aspartic acid (PALA) and water, and optionally at least one nonionic surfactant; and b) At least one of the following: i) A dispensing container configured to locally dispense the composition to a body region of a subject. ii) A topical patch configured to locally deliver the composition to the body area of ​​the subject, or iii) Immune checkpoint inhibitors.

39. The system or cassette of claim 38, wherein the composition is present in the dispensing container or the topical patch.

40. The system or kit of claim 38, wherein a dispensing container is present, and wherein the dispensing container is a non-contact dispensing container that allows the composition to be dispensed and rubbed onto the body area such that other areas of the subject do not come into contact with the composition.

41. The system or cassette of claim 40, wherein the other area of ​​the subject includes the subject's hand.

42. The system or cartridge of claim 40, wherein the non-contact dispensing container comprises a metered-dose topical applicator.

43. The system or kit of claim 40, wherein the non-contact dispensing container may be selected from the group consisting of: CLICK metering topical applicator, TOPI-CLICK applicator, TAPEMARK unit-dose semi-solid drug delivery system, MICROBRISTLE applicator (MBA™), BACK Easy lotion applicator, and LIQUIBAND XL skin closure system.

44. The system or cassette of claim 40, wherein the dispensing container is present and includes a tube or pouch.

45. The system or kit of claim 38, wherein the PALA is present in the composition at a concentration of about 0.1% to about 5.0% or about 1.0% to 8.0%.

46. ​​The system or kit of any one of claims 38-45, wherein the at least one nonionic surfactant is present and comprises a nonionic linear copolymer.

47. The system or cartridge of claim 46, wherein the nonionic linear copolymer comprises poloxamer 188.

48. The system or cartridge of claim 46, wherein the nonionic linear copolymer is present in the composition at about 50%-90%, about 60%-80%, about 65%-75%, or about 70%.

49. The system or kit of claim 38, wherein the at least one nonionic surfactant is present in the composition at 10%-30% or about 22%.

50. The system or kit of claim 38, wherein the at least one nonionic surfactant is present and comprises octanoyl hexanoyl polyhydroxy-8 glycerol ester, or wherein the at least one nonionic surfactant excipient comprises PEG-8 monoesters and diesters of caprylic acid (C8) and decanoic acid (C10) with small amounts of glycerol monoesters, diesters and triesters, or wherein the at least one nonionic surfactant comprises LABRASOL ALF.

51. The system or kit of any one of claims 38-50, wherein the composition further comprises a hydrogel agent, and the composition is in the form of a hydrogel.

52. The system or kit of any one of claims 38-51, wherein the composition further comprises isopropyl myristate, and optionally wherein the isopropyl myristate is present in the composition at a concentration of about 0.5%-7%, about 1%-5%, or about 3%.

53. The system or cartridge of any one of claims 38-51, wherein the composition further comprises a solubilizer and / or an emulsifier.

54. The system or kit of any one of claims 38-53, wherein the composition further comprises polyethylene glycol glycerol ricinoleate, and optionally wherein the polyethylene glycol glycerol ricinoleate is KOLLIPHOR EL, and optionally wherein the polyethylene glycol glycerol ricinoleate is present in the composition at a concentration of about 2%-10%, about 3%-7%, or about 5%.

55. The system or kit of any one of claims 38-53, wherein the composition further comprises at least one non-PALA anticancer agent, and optionally wherein the at least one non-PALA anticancer agent comprises: Tibanibulin, fluorouracil (5-FU), and / or imiquimod (IMQ).

56. The system or kit of any one of claims 38-55, wherein the at least one nonionic surfactant comprises poloxamer 188 and / or capryloyl hexanoyl polyhydroxy-8 glycerol ester.

57. The system or kit of any one of claims 38-56, wherein the composition is in the form of a cream, lotion, or gel.

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