PALA local therapy for cancer

Abstract

Provided herein are compositions, systems, kits, and methods for topically applying a composition to a body area of ​​interest adjacent to and having precancerous and / or cancerous cells (e.g., skin cancer cells), wherein the composition comprises N-phosphonacetyl-L-aspartic acid (PALA) (also known as sparphosic acid) and water, and optionally further comprising at least one nonionic surfactant.

Description

Detailed Description of the Invention

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 492,381, filed Mar. 27, 2023, the entire disclosure of which is hereby incorporated by reference.

[0002] This invention was made with government support under W81XWH-16-1-0439 awarded by the Department of Defense. The government has certain rights in the invention.

[0003] [Technical Field] Provided herein are compositions, systems, kits, and methods for topically applying a composition to a body region of a subject having pre-cancerous cells and / or cancerous cells (e.g., skin cancer cells) and / or adjacent to the pre-cancerous cells and / or cancerous cells that are causative, wherein the composition comprises N-phosphonacetyl-L-aspartic acid (PALA) (also known as Sparfosic acid) and water, and optionally further comprises at least one non-ionic surfactant.

[0004] [Background Art] Surprisingly, 1 in 5 Americans will develop skin cancer in their lifetime 1 . Most skin cancers are caused by sun exposure and result in damage from ultraviolet light (UV) 2 . Skin cancers are classified as either melanoma or non-melanoma skin cancer (NMSC) 3 , and NMSC is the most common type of cancer overall and occurs in the basal cells, squamous cells, and Merkel cells of the skin 3 . Basal cell carcinoma is the most common, with approximately 5.4 million cases diagnosed annually in the United States 4 , followed by squamous cell carcinoma (SCC) as the second most common and the most metastatic 3 . SCC claims more than twice as many lives as melanoma, with an estimated 15,000 deaths annually 5Most SCCs arise from actinic keratosis (AK), a pre-cancerous lesion that affects over 58 million Americans, and AK is one of the most common skin diseases treated by dermatologists. 6 The direct cost of treating NMSC in the United States is estimated to be $4.8 billion annually, not including additional indirect costs due to wage loss. 7,8 。

[0005] Currently, there are several FDA-approved treatments for NMSC, each with its own advantages and limitations. The gold standard treatment is surgical removal (e.g., Mohs surgery). For pre-cancerous cells (AK), the gold standard is cryotherapy. These treatments can be effective in over 90% of individual lesions. However, a significant problem not addressed by current local destructive therapies (surgery or cryotherapy) is that most AK lesions occur within extensive areas of skin damaged by chronic sun exposure and are thus filled with pre-cancerous state cells, a phenomenon called "field carcinogenesis."

[0006] To address the statistically high risk of cancer occurring within these areas, several non-invasive topical treatments are currently approved, including fluorouracil (5-FU, an antimetabolite), imiquimod (IMQ, an immunomodulator), and tildrakizumab (a microtubule inhibitor). All three topical treatments are FDA-approved for AK, with response rates for AK of 40 - 80%, and 5-FU being the most effective. 95-FU and IMQ are also FDA approved for the treatment of superficial BCC and are often used off-label for the treatment of superficial SCC. Each of these topical agents can treat multiple diffuse lesions (thus addressing the "widespread carcinogenesis" problem) and often results in faster overall long-term clearance than cryotherapy, but each is also associated with strong topical skin reactions characterized by pain, burning, and skin erosion, and in some cases, hyperpigmentation or hypopigmentation of the skin. Therefore, there is still a need for improved topical NMSC therapies that are more effective and have fewer side effects than currently available options.

[0007] [Overview of the prefecture] Provided herein are compositions, systems, kits, and methods for topically applying a composition to a body area of ​​a subject having and / or causing precancerous and / or cancerous cells (e.g., skin cancer cells) and / or adjacent to such precancerous and / or cancerous cells, wherein the composition comprises N-phosphonacetyl-L-aspartic acid (PALA) (also known as sparphosic acid) and water, and optionally further comprising at least one nonionic surfactant. In certain 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, provided herein are methods for treating precancerous and / or cancerous cells of a subject, comprising topically applying the composition to a body area of ​​the subject having and / or causing precancerous and / or cancerous cells (e.g., skin cancer cells) and adjacent to the precancerous and / or cancerous cells, or providing a composition that allows the subject to apply the composition to a body area, wherein the composition comprises N-phosphonacetyl-L-aspartic acid (PALA) (also known as sparphosic acid) and water, and optionally further comprising at least one nonionic surfactant.

[0009] In certain embodiments, the area of ​​study includes skin (e.g., face, arms, legs, torso, etc.). In other embodiments, cancerous cells include melanoma and non-melanoma skin cancer cells. In further embodiments, non-melanoma skin cancer cells are basal cell carcinoma cells or squamous cell carcinoma cells. In certain embodiments, precancerous cells include actinic keratosis cells (e.g., as part of an actinic keratosis lesion). In certain embodiments, the subject is female, in which case the area of ​​study includes the surface of the subject's vagina, and precancerous cells and / or cancerous cells include vaginal precancerous cells and / or cancerous cells. In other embodiments, the area of ​​study includes the surface of the subject's oral cavity (e.g., gingiva), in which case precancerous cells and / or cancerous cells include oral precancerous cells and / or cancerous cells. In additional embodiments, the subject is female, in which case the area of ​​study includes the subject's cervix, and precancerous cells and / or cancerous cells include cervical precancerous cells and / or cancerous cells.

[0010] In some embodiments, applying or providing the composition is repeated 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, applying and providing is repeated at least 2 or 3 times per day for at least 7 days or at least 14 days. In certain embodiments, the method further includes administering an immune checkpoint inhibitor (e.g., one targeting CTLA4, PD-1, or PD-L1, e.g., pembrolizumab, ipilimumab, nivolumab, or atezolizumab) to a subject. In some embodiments, the subject is human, or dog, cat, cattle, pig, or horse. In certain embodiments, at least some or all of the cancerous or precancerous cells are reduced or shrinked by topical administration of the composition to a region or vicinity of a region of a subject having precancerous and / or cancerous cells. In certain embodiments, the composition is present in a skin patch or dispensing device. In certain embodiments, the nonionic surfactant is selected from the group consisting of polyglycerol alkyl ethers, glucosyl dialkyl ethers, crown ethers, ester-bonded surfactants, polyoxyethylene alkyl ethers, Brij, Span (sorbitan esters), and Tween (polysorbate). In certain embodiments, the nonionic surfactant is selected from the group consisting of capriol PGMC, polysorbate 80, polysorbate 20, sodium lauryl sulfate, poloxamer 188, polyoxyl 40 hydrogenated castor oil, monooleate mannide, and lauroyl polyoxyl-32 glyceride.

[0011] In some embodiments, systems and kits are provided herein, which include a) a composition comprising N-phosphonacetyl-L-aspartic acid (PALA) and water, and optionally at least one nonionic surfactant; and b) i) a dispensing container configured to distribute the composition topically to a body area of ​​interest, and / or ii) a topical patch configured to deliver the composition topically to a body area of ​​interest, and / or iii) at least one immune checkpoint inhibitor (e.g., those targeting CTLA4, PD-1, or PD-L1; e.g., pembrolizumab, ipilimumab, nivolumab, or atezolizumab).

[0012] In certain embodiments, the composition is present in a dispensing container or topical patch. In other embodiments, a dispensing container is present, which is a non-contact dispensing container that allows the composition to be distributed and rubbed onto a body area without other areas of the subject coming into contact with the composition. In certain embodiments, other areas of the subject include the subject's hand.

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

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

[0015] In some embodiments, at least one nonionic surfactant comprises a nonionic linear copolymer. In further embodiments, the nonionic linear copolymer comprises poloxamer 188. In other embodiments, the nonionic linear copolymer is present in the composition at a concentration of about 50% to 90%, about 60% to 80%, about 65% to 75%, or about 70%. In additional embodiments, at least one nonionic surfactant is present in the composition at a concentration of 10% to 30% or about 22%. In additional embodiments, at least one nonionic surfactant comprises caprylocaproyl polyoxyl-8 glyceride, or at least one nonionic surfactant excipient comprises a small amount of monoglycerides, diglycerides, and triglycerides of caprylic acid (C8) and capric acid and PEG-8 monoesters or diesters of (C10), or at least one nonionic surfactant comprises LABRASOL ALF.

[0016] In additional 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, optionally present in the composition at a concentration of about 0.5% to 7%, or about 1% to 5%, or about 3%. In other embodiments, the composition further comprises a solubilizer and / or emulsifier.

[0017] In certain embodiments, the composition further comprises macrogol glycerol ricinoleate, optionally being KOLLIPHOR EL, and optionally present in the composition at a concentration of about 2% to 10% or about 3% to 7%, or about 5%. In further embodiments, the composition further comprises at least one non-PALA anticancer agent, optionally comprising tilvanibrin, fluorouracil (5-FU), and / or imiquimod (IMQ). In certain embodiments, at least one nonionic surfactant comprises poloxamer 188 and / or caprylocaproyl polyoxyl-8 glyceride. In further embodiments, the composition is in the form of a cream, lotion, or gel (and also contains reagents necessary to be in such a form).

[0018] [Brief description of the drawing] [Figure 1] Topical PALA is non-toxic in mice up to 5% (w / v) and reduces tumor growth in a dose-dependent manner. (A) Percentage change in body weight during treatment. (B) Serum amyloid A (SAA) level at endpoint. (C) Colon length at endpoint. (D) Fecal lipocalin-(LCN2) level at endpoint. (E) Quantification of mean tumor number per mouse during treatment. (F) Change in tumor mass in each mouse during treatment as a percentage of the original tumor number. Mean ± SEM from three independent experiments, n=4-15 mice / group. Statistical significance determined by mixed-effects analysis and post-hoc Bonferroni multiple comparison test.

[0019] [Figure 2] Daily topical PALA application is better tolerated than topical treatment of standard NMSC therapy. (A) Percentage change in body weight during treatment. Initiation of supportive care is indicated by the arrow (↑). (B) Mean colon length on day 7. (C) Serum amyloid A level on day 7. (D) Mean spleen weight on day 7. (E) Histology of skin treated with topical drug for 7 days. Mean ± SEM, n=5-10 mice / group. Statistical significance determined by mixed-effects analysis and one-way ANOVA with post-hoc Bonferroni multiple comparison test or Dunnett multiple comparison test.

[0020] [Figure 3] Mice treated with topical PALA showed reduced tumor volume, tumor area, and tumor malignancy. (A) Percentage change in body weight during treatment. (B) Serum amyloid A level at endpoint. (C) Colon length at endpoint. (D) Lipocalin-2 (LCN2) level at endpoint. (E) Average number of tumors per mouse during treatment. (F) Percentage change in average number of tumors during treatment. (G) Quantification of tumor surface area from photographs during treatment. (H) Average tumor cross-sectional area measured from H&E stained tissue. Mean ± SEM, n=12 / group. Significance determined by mixed-effects model (REML) using Bonferroni multiple comparison test or unpaired t-test.

[0021] [Figure 4] Shows increased expression of the NOD2 target gene LL-37 by local PALA treatment. (A) Quantification of DefB14 stained area from multiplex immunofluorescence images of skin tumor sections. (B) Quantification of the percentage of LL-37+ cells within the tumor area 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 (vehicle) or 2% (w / v) PALA and stained for LL-37 (green), cytokeratin-14 (CK14, pink), or DNA (blue). Scale bar = 2 μm. High magnification of the box area shown as an insert, scale bar = 1 mm. Mean ± SEM, n = 12-19 tumors / group. Significance determined by unpaired t-test.

[0022] [Figure 5] Tumor-infiltrating cell populations are altered by local PALA treatment and exhibit immunomodulatory activity. Multiplex immunofluorescence images were used to quantify the proportion of tumor cells stained with specific markers. (A) Quantification of cells expressing the neutrophil cell marker myeloperoxidase (MPO, light blue). (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 T cell marker CD8 (red). All sections were co-stained with keratinocyte markers CK14 (gray) and DAPI (blue). (E) Representative multiplex immunofluorescence images highlighting differentially present cell types. Mean ± SEM, n=10-17 tumors / group. Scale bar = 2 μm. Significance determined by unpaired t-test.

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

[0024] [Figure 7] Local PALA treatment may induce cell cycle arrest. Representative images of immunostained skin from vehicle and 2% PALA-treated mice (left panel). Quantification of the percentage of positive cells within the tumor area is graphed (right panel). Mean ± SEM, n=12-16 tumors / group. Significance 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 indicated cell cycle markers (brown).

[0025] [Figure 8] Topical 2% PALA is nontoxic and shows a trend towards efficacy in reducing tumor load in a chemical carcinogen-induced skin cancer model. Skin cancer was induced in SKH1-Elite female mice by topical treatment with DMBA for 2 weeks, followed by topical treatment with TPA three times a week for 8 weeks. Mice were then randomized to treatment groups and topically treated with either vehicle (50% acetone / 10% glycerol) or 2% (w / v) PALA for 4 weeks. (A) Quantification of the mean change in tumor load per mouse during the treatment period as a percentage of tumor number at week 9. (B) Weight change during the treatment period as a percentage of baseline body weight. (C) Levels of hepatic and systemic inflammatory marker serum amyloid A (SAA) at endpoint. (D) Measurement of colon length at endpoint as a macroscopic measure of intestinal lesions. Mean ± SEM, n=8-9 / group. Significance determined by two-way ANOVA using Sidac's multiple comparison test or unpaired two-way t-test. All p-values ​​< 0.05 are shown in the graph.

[0026] [Figure 9] Topical 2% PALA formulated with Aquafor is effective in reducing tumor volume in a chemical carcinogen-induced skin cancer model. C57BL / 6 female mice were topically treated with DMBA for 2 weeks, followed by topical treatment with TPA three times a week for 12 weeks to induce skin cancer. Mice were then randomized to treatment groups and topically treated with either a vehicle (Aquafor) or 2% (w / v) PALA / Aquafor for 4 weeks. (A) Quantification of the mean change in tumor volume per mouse during the treatment period as a percentage of tumor numbers at week 13. (B) Quantification of the mean change in total tumor area per mouse during the treatment period as a percentage of measured values ​​at week 13. Mean ± SEM, n=5~6 / group. Significance determined by two-way ANOVA.

[0027] [Figure 10] Table 7 of Example 2 is shown. This table shows the appearance and HPLC assay of different hydrogels.

[0028] [Figure 11] Cumulative release profiles of various formulations with permeation enhancers (n=1). PALA aqueous solution is also reported for reference.

[0029] [Figure 12] Table 10 shows the appearance and assay of the formulation from Example 2.

[0030] [Figure 13] Cumulative release profile of a specific formulation (n=3).

[0031] [Figure 14] Skin tissue and plasma levels of PALA over 24 hours after a single topical application of 4% (w / v) PALA formulation AP0536 / 29 / 03 to mice. 10 μL of 4% PALA was applied to the dorsal skin of male CD-1 mice (n=3 / time point) (final applied dose = 0.4 mg / kg). Plasma and skin tissue samples were collected at 5, 15, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours post-administration, and PALA levels were determined by LC-MS. Graphs show mean values ​​± SEM.

[0032] [Figure 15] Reformulated topical 2% PALA is non-toxic and effective in reducing tumor volume and total tumor area. Skin cancer was induced in SKH1-Elite female mice by UV irradiation three times a week for 20 weeks. Mice were then randomized to treatment groups and treated topically daily for 6 weeks with either vehicle (AP0536 / 29 / 01), 2% (w / v) PALA (AP0536 / 29 / 02), or 4% (w / v) PALA (AP0536 / 29 / 03). (A) Change in body weight during the treatment period, expressed as a percentage of baseline body weight. (B) Levels of serum amyloid A (SAA), a hepatic and systemic inflammatory marker, at endpoint. (C) Measurement of colon length at endpoint as a macroscopic measure of intestinal lesions. (D) Levels of fecal lipocalin-2 (LCN2) at endpoint as a measure of intestinal inflammation. (E) Quantification of the mean number of tumors per mouse during the treatment period. (F) Mean change in tumor volume per mouse during the treatment period as a percentage of the original number of tumors. (G) Quantification of mean tumor surface area per mouse from photographs during the treatment period. Mean ± SEM, n=8~9 / group. Significance determined by one-way ANOVA using Bonferroni's multiple comparison test (REML) or Tukey's multiple comparison test. All p-values ​​< 0.05 are shown in the graph.

[0033] [Figure 16] Topical 2% PALA is better tolerated and more effective than topical treatment of two standard therapies for NMSC. Skin cancer was induced in SKH1-Elite female mice by UV irradiation three times a week for 20 weeks. Mice were then randomized to treatment groups and treated topically daily for 4 weeks with vehicle (AP0536 / 29 / 02), 2% (w / v) PALA (AP0536 / 29 / 02), 5% (w / w) 5-fluorouracil (5% 5-FU), or 5% (w / w) imiquimod (5% IMQ). (A) PALA-treated mice maintained better body weight than 5-FU or IMQ-treated mice. Body weight change from the start of treatment was graphed (mean ± SEM). Supportive care was provided to IMQ and 5-FU mice (started at the time indicated by †), and 5-FU-treated animals were discontinued after 2.5 weeks of treatment as directed by veterinary staff. (B) Levels of serum amyloid A (SAA), a hepatic and systemic inflammatory marker, at endpoint. (C) Levels of fecal lipocalin-2 (LCN2) at endpoint as a measure of intestinal inflammation. (D) Mice treated with 2% PALA had significantly smaller mean tumor sizes than mice treated with either vehicle or 5% IMQ. Quantification of mean tumor surface area per tumor measured from photographs using ImageJ software over the treatment period. (E) Growth profiles of individual tumors showing a reduced percentage of persistently growing tumors in 2% PALA-treated mice. (F) End-point tumor malignancy determined by evaluation of H&E-stained tumor sections, showing less squamous cell carcinoma (SCC) and SCC at tumor situ in mice treated with 2% PALA than in mice treated with vehicle or 5% IMQ. (G) Tumor levels of interleukin-1β were elevated in both 2% PALA-treated and 5% IMQ-treated mice, indicating induction of an anti-tumor immune response. (H) Tumor levels of interferon-α1 were elevated only in 2% PALA-treated mice, suggesting that the type of immune response induced was different from that stimulated by 5% IMQ.

[0034] [Figure 17] Local PALA treatment slows melanoma growth and increases the sensitivity of melanoma to anti-checkpoint inhibitor therapy in an orthotopic xenograft model of B16 melanoma. B16 melanoma cells were subcutaneously injected bilaterally into C57BL / 6 female mice (n=4-5 / group) (10 5 (Cells / flank, total of 8-10 injections). Tumor volume was measured with calipers every 2-3 days by a blinded operator for each treatment group. Data from palpable tumors were graphed as mean ± SEM. (A) Local treatment with 1% PALA daily for 12 days delays tumor growth. On day 12 after injection, mice were treated locally daily for 12 days with either vehicle (AP0536 / 29 / 02) or 1% (w / w) PALA (AP0536 / 29 / 02). Significant differences were determined by a mixed-effects model (REML) using Sidaq's multiple comparison test. **p<0.01B. Short-term local treatment of B16 melanoma with 2% PALA reduces the expression of the checkpoint inhibitor PD-L1. Seven days after injection, mice were treated locally daily for 5 days with either vehicle (AP0536 / 29 / 02) or 2% (w / w) PALA (AP0536 / 29 / 02). Fourteen days after injection, tumors were harvested, and PD-L1 protein levels were analyzed in tumor lysates by immunoblotting and quantified by image density measurement using Image J compared to tubulin levels (loading control). Graphs show mean values ​​± SEM. (C) Short-term local treatment with 2% PALA sensitizes B16 melanoma to anti-PD-L1 therapy. Seven days after injection, mice were treated locally daily for 5 days with either vehicle (AP0536 / 29 / 02) or 2% (w / w) PALA (AP0536 / 29 / 02). Mice were treated intraperitoneally every 48 hours from day 7 to day 19 with either an isotype or an anti-PD-L1 / atezolizumab antibody (100 μg / mouse). Statistical significance was determined by two-way ANOVA using Dunnett's multiple comparison test. ***p<0.001.

[0035] [Figure 18] Tumor growth of orthotopic xenografts of oral squamous cell carcinoma is delayed by local PALA treatment. MOC-1 squamous cell carcinoma cells were injected intratonally into the tongues of C57BL / 6 mice (male and female, n=6 / group). Once palpable tumors formed, the mouse tongues were treated locally five times a week with vehicle (AP0536 / 29 / 02) or 2% (w / w) PALA (AP0536 / 29 / 02). Tumor volume was measured every 2-3 days using calipers. Data were graphed as mean ± SEM. Statistical significance was determined by a mixed-effects model 'REML' using Sidac's multiple comparison test. *p<0.05, ****p<0.0001.

[0036] [Figure 19] PALA exhibits a U-shaped dose-response curve and stimulates biological effects independently of enzyme inhibition. (A) Bactericidal activity of human normal dermal fibroblasts stimulated by PALA treatment. Cells were stimulated with the indicated concentrations 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 viable bacteria were determined by seeding onto selective medium. Graphed mean ± SEM. (B) PALA-stimulated production of antimicrobial peptides in human normal dermal fibroblasts. Cells were stimulated in the same manner as in (A), and the supernatant was assayed by ELISA for human β-defensin 2 (HBD2) and human β-defensin 3 (HBD3). Graphed mean ± SEM. (C) The enzymatic activity of aspartate transcarbamylase, the enzyme target of PALA, decreases by only about 25% at the dose that induces maximum immunostimulation in human normal dermal fibroblasts. (A) Cell lysates from the cells shown were assayed for their ability to catalyze the formation of carbamyl aspartate (CA) in vitro. Graphed percentage change in CA levels; mean ± SD (D) and (E) similar U-shaped dose-response curves observed in response to topical PALA treatment of skin cancer in a UVB-induced NMSC mouse model. (D) Mean number of tumors per mouse at the end of the treatment period (graphed mean ± SEM). (D) Mice treated for 10 weeks with vehicle (Aquafor), 1%, 2%, or 5% PALA in Aquafor. (E) Mice treated for 4 weeks with vehicle (AP0536 / 29 / 02), 2% (w / w) PALA (AP0536 / 29 / 02), or 4% (w / w) PALA (AP0536 / 29 / 02).

[0037] [Modes for carrying out the invention] Provided herein are compositions, systems, kits, and methods for topically applying a composition to a body area of ​​interest (e.g., in the form of a lesion or tumor) adjacent to and having precancerous and / or cancerous cells (e.g., skin cancer cells), wherein the composition comprises N-phosphonacetyl-L-aspartic acid (PALA) (also known as sparphosic acid) and water, and optionally further comprising at least one nonionic surfactant. In certain 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%).

[0038] Phosphonoacetyl-L-aspartate (PALA) is a potent and highly specific inhibitor of carbamoyl phosphate synthetase II / aspartate transcarbamylase / dihydroorotase (CAD), a multi-enzyme protein that catalyzes the first three steps of novel pyrimidine nucleotide biosynthesis. PALA is designed as a transition state analog inhibitor of aspartate transcarbamylase and possesses nanomolar potency. 10 It is readily taken up by cells in culture, and aspartate transcarbamylase is required for this novel biosynthetic pathway, thus efficiently killing cells by starving them for pyrimidine nucleotides. PALA is highly specific because uridine, a salvage pyrimidine precursor, completely prevents its toxicity. Systemic administration of PALA showed high antitumor efficacy as a monotherapy in mouse B16 melanoma and Lewis lung cancer tumor models, but clinical trials of PALA as a monotherapy administered systemically in humans for colon cancer, breast cancer, malignant melanoma, or progressive soft tissue sarcoma have shown limited efficacy and have been disappointing. 11 However, this is thought to be at least partially due to the reversal of the inhibition of pyrimidine synthesis by dietary uridine.

[0039] In certain embodiments, the compositions herein (including PALA) are formulated in a form selected from the group consisting of creams, lotions, sprays, ointments, gels (e.g., hydrogels), pastes, and foundations. In other embodiments, such compositions exist in transdermal patches. In certain embodiments, the compositions may contain one or more additives selected from the group consisting of fragrances, colorants, thickeners, vegetable oils, emulsifiers, solvents, pH adjusters, disinfectants, preservatives, vitamins, sunscreens, surfactants, and combinations thereof. Various physical sunscreens such as titanium dioxide, silicone-treated titanium dioxide, zinc oxide, ferrous oxide, ferric chloride, talc, chromium oxide, or cobalt oxide may be included. Alternatively, or further, chemical sunscreens such as para-aminobenzoic acid, esters of para-aminobenzoic acid, salicylates, cinnamates, benzophenone, dihydroxyacetone, Persol 1789, or melanin may be included.

[0040] Compositions for such topical administration to a subject can be formulated as pharmaceutical compositions. In addition to containing PALA and water, such pharmaceutical compositions may contain additional agents. The present invention is not intended to be limited by the specific properties of the pharmaceutical preparations. For example, such compositions may be provided together with physiologically acceptable liquid, gel or solid carriers, diluents, adjuvants, and excipients. These therapeutic preparations can be applied topically to mammals for veterinary use in livestock and other animals, and for clinical use in humans.

[0041] [Examples] Example 1 Topical N-phosphonacetyl-L-aspartate is a dual-action candidate for the treatment of non-melanoma skin cancer (NMSC).

[0042] This example describes an investigation of topical application of PALA as an NMSC therapy by combining the chemotherapeutic and immunomodulatory properties of 5-fluorouracil and imiquimod. Daily topical application of PALA to mouse skin was well-tolerated and resulted in less irritation, fewer histopathological changes, and less inflammation compared to treatments caused by either 5-fluorouracil or imiquimod. In a UV-induced NMSC mouse model, topical PALA treatment significantly reduced tumor number, area, and malignancy compared to vehicle controls. This antitumor activity was attributed to increased expression of the antimicrobial peptide LL-37, as well as CD8 + T cells and F4 / 80 + It showed both immunomodulatory and antiproliferative effects, associated with increased recruitment of macrophages to tumors.

[0043] Materials and methods reagent PALA (NSC-224131) was obtained from the National Cancer Institute (NCI) / Division of Cancer Treatment and Diagnosis (DCTD) / Developmental Therapeutics Program (DTP) Open Chemical Repository. PALA was dissolved in 50% acetone / 10% glycerol (Fisher Scientific, Hampton, New Hampshire) at concentrations of 1%, 2%, or 5% (w / v). Aquafor 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).

[0044] UVB-induced NMSC mouse model The animal experiment procedures were approved by the Animal Experimentation Committee of the Cleveland Clinic (IACUC Protocol #2018-2096) and were carried out in accordance with relevant facility and national guidelines for the management and use of laboratory animals. Anesthetized female SKH1-Elite mice (Cr:SKH1-Hr hr (System code 477, Charles River Laboratories), previously listed 12 The subjects were exposed to UVB three times a week for 20 weeks. During UVB exposure, the head and tail regions were covered with felt to limit tumor induction to the dorsal skin only. The UVB exposure involved an average irradiance of 0.312–0.356 mW / cm². 2 A custom UV lamp unit (USHIO AmeriCA, Inc.) with 10 7.2W G8T5 bulbs (peak wavelength 305nm) was used. Before each exposure session, UVB irradiance was measured using a PMA2100 radiometer equipped with UVA and UVB detectors (Solar Light), and the time required to achieve the desired dose was calculated. The UVB dose was 80 mJ / cm². 2 Starting with [a dose], gradually increasing over the first 10 weeks (10% per week), with a final dose of 175 mJ / cm². 2 (The time range was increased to 8 minutes 11 seconds to 9 minutes 20 seconds, depending on the UVB irradiance measurement.)

[0045] After 20 weeks of UVB exposure, mice were divided into treatment groups based on their initial tumor count to balance initial tumor volume. Mice were treated daily with 200 μL of the drug, applied topically to the dorsal skin for 10 weeks. Mice were housed in small groups (n=5 / cage), and oral drug exposure was minimized by using a rapidly absorbed vehicle (50% acetone / 10% glycerol). Mouse body weight was measured weekly, and a score of physical condition was assessed. Tumor volume was determined weekly from human counts and digital photographs using ImageJ software. 13 Quantification was performed using the following method: Individual tumors were numbered and tracked during image analysis; if the tumor boundaries between individual tumors became dense (e.g., two tumors fused), they were still counted as two tumors and their areas were averaged.

[0046] At the time of collection, the length of the colon was measured, blood was collected in a K2-EDTA microtainer (Becton Dickinson), and then processed into plasma for serum amyloid A (SAA) measurement by ELISA. All skin lesions were counted at the time of collection, and tumors large enough to be excised were collected. Tumor tissue was excised, half was fixed with Histochoice and embedded in a paraffin block, and the other half was frozen for ELISA analysis. Tissue sections were stained with hematoxylin / eosin or subjected to multiplex immunofluorescence imaging for histopathological evaluation. Skin was carefully selected from mice with UV-induced tumors, and the largest tumor was bisected for staining. If a mouse had several tumors, it was not always possible to excise every tumor or lesion. Therefore, we prioritized larger and more prominent tumors in all mice and examined as many tumors as possible. Tissue examined by the resident dermatologist was not representative of the entire sample, and some even smaller precancerous lesions were excluded; however, this was done consistently across all treatment groups.

[0047] Skin irritation test Topical formulations of 2% PALA / 50% acetone / 10% glycerol, 5% imiquimod (IMQ), 5% 5-FU (5-FU), vehicle (50% acetone / 10% glycerol) as a control, and Aquafor (approximately 0.05 g / mouse) were applied daily to the dorsal skin of SKH1-ELite female mice (8 weeks old, n=5 / group) for 7 days. Since no significant differences were detected between these groups, the Aquafor and vehicle treatment groups were combined into a single group during the final analysis. Mice were monitored daily for weight changes, skin appearance, and signs of distress. In response to significant weight loss in the IMQ and 5-FU treatment groups, these mice were supplemented with Diet Gel76A (ClearH2O), starting on day 3 (IMQ) or day 5 (5-FU). Spleen weight and colon length were measured at the time of sampling. At the time of collection, blood was collected in a K2-EDTA microtainer (Becton Dickinson) and then processed into plasma for SAA measurement by ELISA. Skin tissue was collected, and one of the samples was fixed with Histochoice and embedded in a paraffin block, or frozen for ELISA analysis. Tissue sections were stained with hematoxylin / eosin for histopathological evaluation.

[0048] Enzyme-linked immunoassay Serum amyloid A levels were measured in plasma samples using the Mouse SAA ELISA Kit (E-90SAA, Immunology Consultants Laboratory, Inc.) according to the manufacturer's instructions. Lipocalin-2 (LCN2) was quantified from pre-weighed fecal samples homogenized in 0.5 mL of PBS using the Mouse Lipocalin-2 / NAL DuoSet ELISA (DY1857) and the DioSet Auxiliary Reagent Kit 2 (DY008, R&D Systems). Skin protein lysates were prepared by homogenizing frozen tissue with a pestle and scissors 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 cocktail) (A32965, ThermoFisher)). Cytokines and chemokines were quantified from 25 μg of lysate using a custom 10-plex U-PLEX panel (K15069L-2; Meso Scale Discovery) containing 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 the data were analyzed using MSD Discovery Workbench 4.0 software.

[0049] Multiplex immunofluorescence imaging Immunohistochemical staining was performed using the Discovery ULTRA automated staining system from Roche Diagnostics (Indianapolis, IN). Briefly, antigen retrieval was performed using proteinase K (IHCSelect, 21627; Millipore) and / or Tris / borate / EDTA buffer (Discovery C1, 06414575001; Roche), pH 8.0–8.5. The antigen was denatured using citrate buffer (Discovery CC2, 05424542001, Roche). The time, temperature, and dilution are listed in Tables 13–15.

[0050] [Table 1]

[0051] [Table 2]

[0052] [Table 3]

[0053] Antibodies used were OmniMap anti-rabbit HRP (05269679001; Roche) and UltraMap anti-rat HRP (05891884001; Roche), and were visualized in combination with opal fluorescent dyes from Akoya Biosciences (Marlborough, MA) (the dyes corresponding to each antibody are listed in Tables 1-3). The slides were counterstained with Spectral DAPI (FP1490; AKoya Biosciences).

[0054] For cell-secreted molecules (e.g., DefB14), the area of ​​positive staining within tumor tissue was measured using QPath software. First, the total tissue area (both tumor and non-tumor tissue) was assessed using the "Tissue Detection" command. Next, the amount of specific fluorescent dye molecules detected within the area was determined using the Thresholder command. The percentage of the area covered by the targeted fluorescent dye molecule was calculated for each sample.

[0055] Within the same annotation in the organizational domain, LL37 + cells, CD3 + T cells, CD8 + T cells, F4 / 80 + Macrophages and MPO + The proportion and density of cells were evaluated using a positive cell detection method on QuPath software. This allowed the use of a thresholding function to determine the detection threshold for cell radius and fluorescent dye molecules. The total number of cells in the tumor region was determined using DAPI or autofluorescence detection channels. The proportion of positive cells for each targeted fluorescent dye molecule was calculated for each detection channel.

[0056] Immunohistochemical staining Immunohistochemical staining was performed at the Cleveland Clinic's diagnostic imaging core facility using a Roche Diagnostics Discovery ULTRA automated staining system. Briefly, antigen retrieval was performed using Tris / borate / EDTA buffer (Discovery CC1, #06414575001; Roche), pH 8.0–8.5. Time, temperature, and dilution are found in Table 16.

[0057] [Table 4]

[0058] Antibodies were visualized using OmniMap anti-rabbit HRP (#05269679001, Roche) in combination with the ChromoMap DAB detection kit (#05266645001, Roche). Finally, the slides were counterstained with hematoxylin and blued.

[0059] statistical analysis All statistical analyses were performed using GraphPad Prism software (version 9.1). Significance of datasets with multiple variables was determined using one-way or two-way analysis of variance (ANOVA) with post-hoc Bonferroni or Tukey multiple comparison tests. Chi-squared tests were applied for classification-based variable comparisons. Details of the tests used in specific experiments are included in the figure legend.

[0060] result Topical PALA is non-toxic up to 5% (w / v) in mice and reduces tumor growth in a dose-dependent manner.

[0061] To determine the optimal dose of topical PALA for the treatment of NMSC, SKH1-Elite mice were UV-irradiated three times a week for 20 weeks to induce SCC, and then treated daily for 10 weeks with 0, 1, 2, or 5% (w / v) PALA in 50% acetone / 10% glycerol. Topical PALA treatment was well-tolerated over 10 weeks with no apparent skin irritation or weight loss (Figure 1A). Previous toxicity studies have identified the intestines, liver, and central nervous system as the primary targets of systemic PALA toxicity. 11Therefore, circulating serum amyloid A (SAA) levels were measured from plasma collected at the endpoint as a marker of hepatitis and systemic toxicity (Figure 1B). No significant differences were observed between treatment groups; however, mice treated with 1% and 5% PALA tended to have higher SAA levels (p=0.067 and p=0.066, respectively). The colon was examined for macroscopic pathology, and colonic length was measured at the time of collection as a quantitative measure of intestinal pathology. None of the mice showed obvious macroscopic colonic lesions or significant differences in colonic length between treatment groups (Figure 1C). Fecal lipocalin-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 vehicle (186.8±121.1 vs. 18.1±6.0, p=0.032, Figure 1D). Importantly, mice treated with 2% or 5% PALA had significantly fewer total tumors over a 10-week treatment period than mice treated with either the vehicle or 1% PALA (12.5±2.4 and 11.7±3.8 at the endpoint, respectively, p=0.029, Figure 1D) (3.83±0.6 and 6.20±1.8 at the endpoint, respectively). This was particularly evident when calculating the mean change from initial tumor volume; mean tumor volume steadily increased in the vehicle-treated and 1% PALA-treated groups (286.4% ± 62.6 and 275.0% ± 84.7 at endpoint compared to the initial size), but this was not observed in the 5% PALA group as early as 3 weeks after treatment (91.6% ± 13.4 at endpoint compared to the initial size), and not observed in the 2% PALA group until 8 weeks after the start of treatment (99.2% ± 20.5 at endpoint compared to the initial size, p=0.0018; Figure 1E). These results indicate that topical PALA is well-tolerated up to 5% (w / v) over 10 weeks of daily treatment and exhibits antitumor activity at concentrations of 2% or higher. Based on a significant antitumor effect (mean tumor volume change at the endpoint: 2% PALA dose was 99.2% ± 20.5% of the original size compared to 286.4% ± 62.6% in the vehicle, p = 0.0018), and the absence of elevations in inflammatory markers SAA and LCN2 at this dose, the 2% PALA dose was selected for further analysis.

[0062] Daily topical application of PALA is better tolerated than topical treatment of NMSC standard therapy. The tolerability of daily treatment with topical ointments of current standard therapy, 5% IMQ and 5% 5-FU, was compared over 7 days with 2% PALA and their vehicle controls in SKH1-Elite mice with no prior drug experience. Skin appearance, weight changes, and physical condition scores were assessed daily. In contrast to mice treated with vehicle and 2% PALA, mice treated topically with either IMQ or 5-FU showed significant weight loss, required interventional care, and one died (Figure 2A, arrow). Skin irritation was evident as early as day 2 in IMQ-treated mice and day 5 in the 5-FU group; no skin irritation was observed in either vehicle or 2% PALA-treated mice (Figure 6).

[0063] At the time of sampling on day 7, the macroscopic pathology of the spleen and intestines was evaluated, SAA levels were measured, and cutaneous histopathology and the expression of inflammatory mediators in the treated skin were assessed. No differences were observed between the groups in the macroscopic pathology of the intestinal tract or SAA levels (Figures 2B and 2C). Previous reports on local IMQ treatment in mice 14 Similarly, IMQ-treated mice showed splenomegaly (Figure 2D), significant epidermal thickening (thickening of the stratum spinosum; Figure 2E), and upregulation of IL-17 family cytokines in the treated skin tissue (Table I).

[0064] [Table 5]

[0065] Furthermore, skin levels of RANTES and GM-CSF were elevated in IMQ-treated animals (Table I). 5-FU-treated mice also exhibited toxicity characterized by a significant decrease in spleen weight (Figure 2D), epithelial erosion (Figure 2E), and increased expression of IL-6, IL-17C, KC, and GM-CSF in treated skin lysates (Table I). In contrast, PALA-treated mice showed no changes in spleen weight, colon length, SAA levels, dermatohistology, or expression of the inflammatory cytokine / chemokine panel in treated skin compared to vehicle controls. These findings suggest that daily topical application of PALA to mice is far more well-tolerated than topical treatment of current standard therapy for NMSC.

[0066] Mice treated with topical PALA showed reduced tumor volume, tumor area, and tumor malignancy. AK lesions and SCC tumors were induced in SKH1-Elite mice by exposure to UVB for 20 weeks. Tumor-bearing mice were treated daily for 10 weeks with either 2% PALA or a vehicle control, and the toxic side effects of the treatment, as well as the changes in the number, size, and malignancy of tumors induced by the treatment, were evaluated. Daily treatment of mice with topical PALA for 10 weeks was well-tolerated compared to vehicle-treated control mice, as reflected by no significant differences in body weight, SAA levels, macroscopic intestinal lesions, or LCN2 levels (Figures 3A-D). PALA-treated mice had significantly less tumor mass than vehicles-treated mice; the mean number of tumors in vehicles-treated mice increased during the 10-week treatment period (endpoint value of 252.3% ± 34.3, p = 0.00051), while the mean number of tumors per mouse in the PALA-treated group remained constant (endpoint value of 99.2% ± 20.5, p = 0.97) (Figures 3E and 3F). Tumor size also showed a decrease in mean tumor surface area (endpoint value 197.1 mm²). 2 30.3mm 2 (p<0.0001) and mean tumor cross-sectional area (endpoint value 4.4 mm) 2 2.4mm 2In both cases (p=0.003), tumors grew dramatically larger in the vehicle-treated group (Figures 3G and 3H). Analysis of individual tumor growth revealed that a larger proportion of tumors treated with PALA receded during the treatment period compared to the vehicle control (83.3% vs. 26.8%, p<0.0001, Table II).

[0067] [Table 6]

[0068] Furthermore, the growth phenotype differed between treatments, with most tumors treated with PALA shrinking after an initial growth phase or showing a continuous decrease in size (97.9%) (46.3%, p<0.0001; Table II), compared to the continuous growth observed in animals treated with the vehicle. These data support the use of topical PALA for the treatment of lesions in AK and SCC, for example.

[0069] The resected tumors were histologically classified into one of the following categories: benign / hypertrophic lesion, in-situ AK / SCC, well-differentiated SCC, or poorly differentiated SCC (Table III).

[0070] [Table 7]

[0071] Importantly, compared to controls treated with the vehicle, mice treated with PALA had a lower prevalence of well-differentiated and poorly differentiated SCC (38% vs. 69%, p<0.0001), a higher proportion of in-situ AK / SCC, and were characterized by benign lesions (62% vs. 31%, p<0.001), indicating that PALA treatment can block tumor progression.

[0072] The excised tumors were also stained for the expression of cell cycle markers to determine whether PALA could induce cell cycle arrest similarly to 5-FU. + Or Cyclin A2 +No differences were observed in the cell population (Figure 7). Compared to the vehicle control, phosphorylated histone H3 + A small increase in cell count was detected in PALA-treated samples (4.9% vs. 3.1%, p=0.0067), but the total number of positive cells was a very small proportion of the tissue (<5%), located at the base of the tumor, and it is unclear whether these cells are an integral component of the tumor and / or whether they are exposed to large amounts of the drug. However, tumors treated with PALA showed a higher Ki67 level per tumor than controls treated with the vehicle. + The average number of cells was higher (24.6% vs. 10.4%, p=0.005), and cyclin D1 per tumor was higher. + The lower percentage of cells (39.4% vs. 48.9%, p=0.0329, Figure 7) suggests that these tumor cells may have undergone cell cycle arrest in response to PALA treatment. 15 .

[0073] Activation of innate immunity and enhancement of cytotoxic T cell recruitment are stimulated by topical PALA treatment. Previous studies have demonstrated that topical PALA treatment of bacterial-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 these AMP mouse homologs (DefB14 and LL-37) were evaluated by multiplex immunofluorescence microscopy in tumor tissue of locally treated mice. DefB14 staining in the tumor was diffuse, indicating AMP secretion from cells, and the total area of ​​DefB14 staining did not differ between treatment groups (Figure 4A). In contrast, LL-37 staining was dispersed and co-localized with nuclear DAPI staining, indicating that the number of cells expressing LL-37 in the tumor tissue should be quantified. The results showed that LL-37 + We have demonstrated a significant increase in cells in tumor samples treated with PALA (Figure 4B).

[0074] Neutrophils and monocytes / macrophages are the major innate immune cell types that express both NOD2 and LL-37. 17,18Therefore, tumor sections were analyzed by multiplex immunofluorescence microscopy for neutrophil (myeloperoxidase, MPO) and macrophage (F4 / 80) markers to determine whether any of these immune cell populations were recruited to the tumor tissue in response to local PALA treatment. + The number of cells was the same between the treatment groups, indicating that neutrophils were not differentially recruited to the tumor tissue (Figure 5A). Instead, tumors treated with PALA showed a higher F4 / 80 ratio compared to controls treated with the vehicle. + The number of macrophages had increased (Figures 5B and 5E).

[0075] LL-37 has a controversial role in carcinogenesis. In some cancers, LL-37 is upregulated and stimulates the production of anti-inflammatory tumor-associated macrophages (TAMs) that promote tumorigenesis, while in other cancer types, LL-37 is anti-tumor CD8 + Promotes the activation and proliferation of cytotoxic T cells. 18 LL-37 in tumors treated with PALA + Cells and F4 / 80 + To better understand the role of macrophages, CD8 + The frequency of cytotoxic T cells was evaluated by multiplex immunofluorescence microscopy. CD3 in tumor tissue + The total number of T cells was the same between samples treated with the vehicle and samples treated with PALA (Figure 5C). However, tumors treated with PALA showed a higher CD8 count compared to controls treated with the vehicle. + The number of cytotoxic T cells was increased (Figures 5D and E). Taken together, these results suggest that local PALA treatment not only inhibits tumor growth but also activates an innate immune response that leads to an antitumor cytotoxic T cell response.

[0076] 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. 16,19~21The inventors have successfully demonstrated that daily topical treatment with 2% PALA not only reduces tumor growth but also prevents progression of tumors to more metastatic cell types. Multiplex immunofluorescence microscopy of treated tumors showed changes in cell cycle progression markers and increased immunomodulation, corresponding to what is known about the mechanism of action of this drug. 19 .

[0077] PALA had been widely investigated in the past as a systemic treatment for cancer. 22,23 Although the treatment was highly effective in two mouse models of colon cancer, this drug could not be used to treat human malignancies due to dose-limiting toxicity. 23 No evidence was found that PALA causes toxicity at low concentrations. The inventors found that topical treatment with PALA is the current standard treatment for NMSC, which is IMQ, 5-FU, and tilvanibrin; all of which cause severe skin irritation and inflammation. 24 We propose that PALA has advantages over other methods. When applied topically to mice for up to 10 weeks, PALA did not induce inflammatory responses or skin irritation. Because PALA is chemically non-reactive and can be stored at ambient temperature, it is a viable option for use in areas with limited economic resources. 20 .

[0078] This disclosure is not limited to any particular mechanism, and understanding the mechanism is not necessary to carry out the present invention. However, our results using topical PALA suggest that topical PALA can induce immunotherapeutic effects without requiring additional components for therapeutic action. Other studies investigating the potential of NOD2 activators as cancer immunotherapies have found that NOD2 activators primarily act as immune adjuvants. Derivatives of muramyl dipeptide (MDP), a NOD2 bacterial ligand bound to a lipid moiety, have shown promising antitumor effects in human clinical trials as well as in animal models. 25Direct injection of MDP lipid complexes into fibrosarcoma, hepatocellular carcinoma, and B16-F10 melanoma, or intravenous administration into UV-induced skin cancer models and multiple metastatic liver cancer models, suppressed tumor growth and metastasis, and / or improved the survival rate of tumor-bearing animals. 25,26 Although promising results were observed in early-stage clinical trials, only two MDP derivatives (Mifamurtide / Mepact® and ImmTher®) successfully completed Phase III trials and received approval in the European Union (Mifamurtide / Mepact®, or ImmTher®) as an orphan drug in combination with multi-agent chemotherapy for non-metastatic osteosarcoma after resection, and FDA approval. 27 The therapeutic effect includes the adjuvant effect of these MDP / lipid immunotherapies, which leads to enhanced tumor-killing activity of macrophages. 25,28 It is thought that this is involved, and this mechanism is potentially similar to our topical PALA formulation. Recently, it has been reported that muropeptides produced from Enterococcus faecium enhance cancer treatment with checkpoint inhibitors in a NOD2-dependent manner. 29 Similar to our findings in local PALA, this muropeptide is antitumor cytotoxic CD8 + It affected the macrophage population as well as increasing T cells.

[0079] Both IMQ and PALA are immunomodulatory agents that induce an antitumor immune response. IMQ is a nucleoside in the imidazoquinoline family and is often used in the treatment of NMSCs. 24,30 It activates anti-tumor immunity through the stimulation of TLR7 in macrophages and other immune cells. 24,30 IMQ is known to upregulate the expression of interferon-α and interleukin 1, 6, and 8, and similarly induce autophagy in macrophages. 24,30,31The inventors have shown that PALA treatment does not increase these cytokines and that macrophages are even more abundant in the treatment area, indicating that PALA acts through an immune mechanism different from that activated by IMQ. The present invention is not limited to any particular mechanism, and an understanding of the mechanism of the present invention is not necessary to carry it out, but PALA induces an antitumor response through the enhancement of cross-presentation of tumor antigens, and consequently CD8 + It is hypothesized that this enhances the tumor-specific cytolytic activity of T cells. This hypothesis explains the enhanced cross-presentation of NOD2-dependent MHC class I and MHC class II antigens in cells stimulated with MDP. 32~34 It is supported by [someone / something], but formal verification has not yet been carried out.

[0080] Immune cell infiltration is seen in most malignant tumors. 35 Macrophages are a major component of solid tumors and can promote tumorigenesis by stimulating angiogenesis, immunosuppression, invasion, and metastasis. 36,37 Tumor-associated macrophages (TAMs) are important regulators of the relationship between the immune system and cancer. 38 TAMs can promote rather than limit tumor progression, negatively impact the response to treatment, and suppress T cell recruitment. 38 However, activated macrophages are effective as cancer immunotherapy because they can kill cancer cells directly or indirectly through the recruitment of other immune cells such as cytotoxic T lymphocytes. 39 Within tumors, T cells often become dysfunctional. 40 Activated macrophages may be able to restore their cytolytic activity. 39 Recent research highlights how the relationship between TAM and the tumor microenvironment can lead to improvements in cancer treatment. 41,42Again, the present invention is not limited to any particular mechanism, and understanding the mechanism is not required to carry out the present invention, however, our findings suggest that PALA can recruit macrophages, activate them locally, and enhance the cytotoxic T cell response.

[0081] PALA inhibits pyrimidine nucleotide synthesis and inhibits cell proliferation through the depletion of dCTP and dTTP, preventing treated cells from completing DNA synthesis. 20 Ki-67 expression is commonly used as a marker for actively proliferating cells, but it may not be expressed only in quiescent cells in the G0 phase, and may be highly expressed in cells that have stopped at other stages of the cell cycle. 27 Cyclin D1 expression peaks at the end of G1 and is required for the cell to transition to the S phase. Known effects of PALA on the cellular pyrimidine pool. 20 Considering this, Ki-67 in PALA-treated tumors + Increase in the number of cells and cyclin D1 + The decrease in the percentage of cells supports the conclusion that cells may be undergoing arrest. These findings highlight the dual function of this drug as both a cell division inhibitor and an immunomodulator.

[0082] Example 2 PALA preparation This example describes the development of a topical formulation (CMI-34-1) for PALA. As a first approach, several hydrogels were prepared using various concentrations of natural and synthetic polymers: xanthan gum, methylcellulose, hydroxyethylcellulose, Carbopol 971P NF, poloxamer 188, and 407. Only poloxamer 188 20% w / w ultimately provided PALA permeability in in vitro diffusion tests (Franz cells). As a second approach, several permeability 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 labrasol ALF was included in the formulation. To further improve the formulation, isopropyl myristate and corifor EL were added for their emollient and tactile enhancement properties. Furthermore, they also play a role in promoting permeability. Even if they did not improve permeability in a test tube, they may still play a role in vivo. A formulation containing P188 20% w / w (70%), Labrasol ALF (21.6%), Corifor EL (5.4%), and isopropyl myristate (3%) was selected for formulation mouse testing. Stability tests conducted for 9 weeks under room temperature and refrigerated (2-8°C) conditions confirmed the stability of the formulation under both conditions.

[0083] HPLC method details A standard solution of PALA (CMI-34-1) was prepared at a concentration of 1 mg / mL in a diluent (100% type I water). 20 mg of PALA was accurately weighed into a 20 mL volumetric flask, and the volume was adjusted with 100% type I water. The sample was sonicated for 20 minutes until completely dissolved, and then injected into an HPLC for system suitability testing. To evaluate accuracy and reproducibility, standard A was injected five times and standard B twice, and the main peak area and % relative standard deviation (%RSD) were calculated. The %RSD of the main peak of the API in the standard solution should be less than 2.0% according to internal guidelines. Finally, the standard agreement was calculated as shown by the following formula:

[0084]

number

[0085] Here, area A and area B These are the areas under the peaks of standards A and B, respectively, and Conc A and Conc B These are the concentrations of standards A and B, respectively.

[0086] Preparation of placebo hydrogel Placebo hydrogels were prepared as shown in Table 4.

[0087] [Table 8] TIFF2026514385000011.tif47165

[0088] Preparation of formulations for in vitro testing (Franz cells) All formulations were prepared by accurately weighing 50 mg of PALA into a 4 mL glass vial. 950 mg of vehicle (listed in Table 5) was added to this. The vial was stirred at room temperature until a clear solution was obtained. With the exception of poloxamer P407 (20% w / w) and P188 (40% w / w), PALA solubilization was performed by stirring the vial in an ice bath (to prevent gelation that occurs at room temperature).

[0089] [Table 9]

[0090] C. Preparation of Bygel formulations xanthan gum-based baigel The Vigel consists 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 Lipophil WL1349 (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 to this. The vial was left to stand at room temperature with stirring until the API was completely solubilized. Next, 150 mg of the oil phase was slowly poured into the gelled aqueous phase and mixed in a homogenizer at 5000 rpm for 5 minutes at room temperature until a uniform emulsion was obtained.

[0091] Weigel based on Poloxamer 188 Poloxamer 188 (P188) 34% w / w (Table 5) was used as the aqueous phase. The oil phase was prepared by mixing Labrafac Lipophil WL1349 (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 to this. The vial was left to stand at room temperature with stirring until the API was completely solubilized. Next, 150 mg of the oil phase was slowly poured into the gelled aqueous phase and mixed in a homogenizer at 5000 rpm for 5 minutes at room temperature until a uniform emulsion was obtained.

[0092] [Table 10]

[0093] Hydrogel in vitro diffusion cell (Franz cell) An in vitro diffusion test (Franz cells, Copley) was performed to simulate the in vivo conditions for the transfer of PALA(CMI-34-1) from the formulation via the skin. The test was performed under sink conditions. A hydrogel or Bygel (containing 5 mg of API) loaded with 100 μL of PALA(CMI-34-1) was added to the donor chamber of the Franz cells. 7 mL of PBS pH 7.4 was added to the receptor chamber. The chambers were separated by a 25 mm diameter Strat-M membrane (transdermal diffusion test model, Merck). The Franz cells were incubated in a suitable heating block at 37°C with stirring at 450 rpm. At selected time points (15, 30, 60, 120, 180, and 1440 minutes), 100 μL of the sample was removed from the receptor chamber and analyzed by HPLC for drug release. The same volume was replaced with fresh PBS buffer preheated at 37°C.

[0094] HPLC assay of pharmaceutical formulations The formulation assay was performed in a two-step process using UV-HPLC analysis. Using a volumetric pipette, 300 μL of the formulation was collected, transferred to an Eppendorf pipette, 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 volumetric flask was then vortexed for 10 seconds, sonicated in an ultrasonic bath for 5 minutes, and analyzed by UV-HPLC.

[0095] 9-week stability test A 9-week stability test was performed on the lead formulation (AP0536 / 23 / 01) stored at room temperature and under refrigerated conditions (2-8°C). The formulation was stored in 4 mL clear glass vials. Samples were visually inspected (color and appearance) at specified time points (days 0, 7, 14, and 63) and analyzed by UV-HPLC. At each time point, 300 μL of the formulation was taken, transferred to an Eppendorf flask, 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 volumetric flask was then vortexed for 10 seconds, sonicated in an ultrasonic bath for 5 minutes, and analyzed by UV-HPLC. Each assay was performed in pairs.

[0096] Preparation for PK testing The lead formulation (AP0536 / 23 / 01) was newly prepared with three different drug loads (0, 2, and 4% w / w PALA). A 20% w / w aqueous solution of poloxamer 188 was prepared on a larger scale (30 g P188 + 120 g type I water) following the same procedure reported in Table 4. The placebo vehicle (AP0539 / 29 / 01) was prepared on a 150 g scale as follows: ·105.0g P188 20%w / w aqueous solution 32.4g Labrasol ALF 8.1g Corifer EL 4.5g Isopropyl myristate A 2% w / w PALA preparation (AP0539 / 29 / 02) was prepared on a 42g scale by accurately weighing 0.84g of PALA and adding 41.160g of vehicle (AP0539 / 29 / 01). A 4% w / w PALA preparation (AP0539 / 29 / 03) was prepared on a 42g scale by accurately weighing 1.68g of PALA and adding 40.32g of vehicle (AP0539 / 29 / 01).

[0097] Results and Discussion Hydrogel research This study investigated the development of suitable preclinical formulations for the compound PALA (CMI-34-1) after topical administration. Different polymer concentrations were investigated to prepare hydrogels, namely xanthan gum, methylcellulose, hydroxyethylcellulose, Carbopol 971P NF, and poloxamer 188 (P188) and poloxamer 407 (P407). As a control, PALA prepared in type I water was also examined in Franz cells. The control (water only) showed almost no detectable PALA permeation after 24 hours of HPLC analysis (approximately 0.3%) (Table 7, Figure 10). Hydrogels of xanthan gum, methylcellulose, hydroxyethylcellulose, and Carbopol 971P NF were first prepared with a polymer content of 2% w / w, resulting in very viscous gels. The hydrogels were loaded with a concentration of 50 mg / g PALA, and the diffusion of PALA from the hydrogels through the transdermal diffusion membrane was monitored in Franz cells. After 24 hours, no PALA permeation through the membrane was observed (Table 7, Figure 10). Based on these results, the polymer content of the hydrogel was reduced from 2% to 1-0.5% to decrease the viscosity of the formulation, ultimately promoting PALA permeation through the membrane. Unfortunately, with the exception of the poloxamer 188 hydrogel, which showed up to 60% PALA permeation after 24 hours (Table 7, Figure 10), no release was observed even at lower polymer concentrations (Table 7, Figure 10). As a result, the P188 hydrogel was selected for further investigation.

[0098] Since poloxamer 188 at 20% showed low viscosity, we considered increasing the concentration of P188 to provide a hydrogel with higher viscosity that would remain firmly on the skin after application. After several trials, an acceptable viscosity was provided at 34%, and thus, improved adhesion to the skin was confirmed (Table 8). Therefore, when 34% w / w P188 was prepared, 49.7% PALA was released after 24 hours.

[0099] [Table 11]

[0100] Further optimization of the 34% w / w P188 formulation was performed by incorporating different permeation enhancers, namely Labrasol ALF, Transktol HP, and isopropyl myristate, at 20% w / w. Details of the composition and visual observations are shown in Table 9. Isopropyl myristate was selected because it is a well-known emollient and texture enhancer in cosmetics. In addition to hydrogels, biogels were also investigated.

[0101] [Table 12]

[0102] The diffusion of PALA from the formulations listed in Table 9 was examined using Franz cells. Adding 20% ​​(w / w) Labrasol (AP0536 / 12 / 05) to poloxamer hydrogel resulted in a significant improvement in diffusion. The release profile (Figure 11) showed that approximately 50% of PALA was released within 90 minutes, and 76.6% was released after 3 hours. Using P188 + 20% w / w Transktol (AP0536 / 12 / 06), 6.1% of PALA was released within 90 minutes, reaching 52.5% release after 24 hours. No PALA release was detected within 180 minutes from P188 + 20% w / w isopropyl myristate (AP0536 / 12 / 07) and P188 Bygel (AP0536 / 12 / 04), but releases of 49.7% and 63.1%, respectively, were observed after 24 hours. No release was detected within 24 hours with xanthan gum Bygel (AP0536 / 7 / 02). Based on these results, an aqueous solution of P188 34% w / w (80% w / w) + Labrasol ALF (20% w / w) (AP0536 / 12 / 05) was considered the lead formulation and selected for further investigation (improvement of permeability and texture).

[0103] To further improve the permeability and texture of the formulation (AP0536 / 12 / 05), additional components such as corifor EL, isopropyl myristate, Teforce 63, and paraffin oil were added. To compensate for the increase in viscosity observed in initial tests in the presence of 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 their stability over 7 days. From the results, all formulations were stable after being stored at room temperature for 7 days (Table 10, Figure 12).

[0104] In vitro permeability tests were performed in triplicate with the selected formulations. Figure 13 shows that after 4 hours, approximately 50% and 70% of PALA were released from AP0536 / 16 / 01 and AP0536 / 17 / 01, respectively. The other two formulations, AP0536 / 17 / 02 and AP0536 / 17 / 03, released 20% and 0%, respectively, after 4 hours. Due to the high release profile and the presence of additional corifor EL and isopropyl myristate, which not only aid in the permeation of PALA in vivo but also provide a better texture of the formulation, the formulation consisting of 70% poloxamer 188 (20% aqueous solution), 21.6% labrasol ALF, 5.4% corifor EL, and 3% isopropyl myristate (AP0536 / 16 / 01) was ultimately selected as the lead to be examined in the PK test.

[0105] 9-week stability test A 9-week stability test was conducted. A lead formulation containing 70% P188 (20% aqueous solution), 21.6% Labrasol ALF, 5.4% Corifor EL, and 3% isopropyl myristate was newly prepared and stored under ambient and refrigerated conditions (2-8°C). As can be seen from Table 11, no changes in assay or purity were detected after 9 weeks under both storage conditions.

[0106] [Table 13]

[0107] Preparation of formulations for supply to support PK testing For PK testing, formulations containing 70% P188 (20% aqueous solution), 21.6% Labrasol ALF, 5.4% Corifor EL, and 3% isopropyl myristate were newly prepared with PALA content of 0% w / w (AP0536 / 29 / 01), 2% w / w (AP0536 / 29 / 02), and 4% w / w (AP0536 / 29 / 03). HPLC assays were performed, and the results are reported in Table 12.

[0108] [Table 14]

[0109] A hydrogel formulation containing poloxamer P188 20% w / w (70%), Labrasol ALF (21.6%), Corifor EL (5.4%), and isopropyl myristate (3%) was selected as the lead formulation for PK testing. This formulation, developed with a combination of poloxamer 188 hydrogel and Labrasol ALF (penetration enhancer), showed high PALA permeability in in vitro tests. The addition of Corifor EL and isopropyl myristate further enhanced the formulation's skin-softening properties and improved its texture. This formulation remained physically and chemically stable for at least 9 weeks.

[0110] In vivo results Figure 14. Skin tissue and plasma levels of PALA over 24 hours after a single topical application of the topical formulation AP0536 / 29 / 03 4% (w / v) PALA to mice. 10 μL of 4% PALA was applied to the dorsal skin of male CD-1 mice (n=3 / time point) (final applied dose = 0.4 mg / kg). Plasma and skin tissue samples were collected at 5, 15, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, and 24 hours after administration, and PALA levels were determined by LC-MS. Graphs show mean values ​​± SEM.

[0111] Figure 15 shows that the re-formulated topical 2% PALA is non-toxic and effective in reducing tumor volume and total tumor area. Skin cancer was induced in SKH1-Elite female mice by UV irradiation three times a week for 20 weeks. Mice were then randomized to treatment groups and treated topically daily for 6 weeks with either a vehicle (AP0536 / 29 / 01), 2% (w / v) PALA (AP0536 / 29 / 02), or 4% (w / v) PALA (AP0536 / 29 / 03). (A) Change in body weight during the treatment period, expressed as a percentage of baseline body weight. (B) Levels of serum amyloid A (SAA), a hepatic and systemic inflammatory marker, at endpoint. (C) Measurement of colon length at endpoint as a macroscopic measure of intestinal lesions. (D) Levels of fecal lipocalin-2 (LCN2) at endpoint as a measure of intestinal inflammation. (E) Quantification of the mean number of tumors per mouse during the treatment period. (F) Mean change in tumor volume per mouse during the treatment period as a percentage of the original number of tumors. (G) Quantification of mean tumor surface area per mouse from photographs during the treatment period. Mean ± SEM, n=8~9 / group. Significance determined by one-way ANOVA using Bonferroni's multiple comparison test (REML) or Tukey's multiple comparison test. All p-values ​​< 0.05 are shown in the graph.

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[0113] 36.Mantovani,A.& Sica,A.Macrophages,innate immunity and cancer:balance,tolerance,and diversity.Current opinion in immunology,22,231-237(2010). 37.Ruffell,B.& Coussens,L.M.Macrophages and therapeutic resistance in cancer.Cancer cell,27,462-472(2015). 38. Pathria, P., Louis, TL & Varner, JATargeting tumor-associated macrophages in cancer. Trends in immunology, 40, 310-327 (2019). 39. Wahl, LM & Kleinman, HK Tumor-associated macrophages as targets for cancer therapy. Vol. 90, 1583-1584 (Oxford University Press, 1998). 40. Andersen, MH, Schrama, D., thor Straten, P. & Becker, JCCytotoxic T cells. Journal of Investigative Dermatology, 126, 32-41 (2006). 41. Najafi, M., et al. Tumor microenvironment: Interactions and therapy. Journal of cellular physiology, 234, 5700-5721 (2019). 42.Reisfeld,RAThe tumor microenvironment:a target for combination therapy of breast cancer.Critical Reviews(trademark) in Oncogenesis,18(2013). All publications and patents referenced herein are incorporated herein by reference. Various modifications and changes to the methods and components of the present invention described above will be obvious to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention is described in relation to certain preferred embodiments, it will be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications to the described methods for carrying out the invention, which will be obvious to those skilled in the art, are intended to be within the scope of the following claims. [Brief explanation of the drawing]

[0114] [Figure 1] (A) Percentage change in body weight during treatment. (B) Serum amyloid A (SAA) level at endpoint. (C) Colon length at endpoint. (D) Fecal lipocalin-(LCN2) level at endpoint. (E) Quantification of the average number of tumors per mouse during treatment. (F) Change in tumor mass of each mouse during treatment as a percentage of the original number of tumors. [Figure 2] (A) Percentage change in body weight during treatment. Initiation of supportive therapy is indicated by the arrow (↑). (B) Mean length of the colon on day 7. (C) Serum amyloid A level on day 7. (D) Mean spleen weight on day 7. (E) Histology of skin treated with topical medication for 7 days. [Figure 3] (A) Percentage change in body weight during treatment. (B) Serum amyloid A level at endpoint. (C) Colon length at endpoint. (D) Lipocalin-2 (LCN2) level at endpoint. (E) Average number of tumors per mouse during treatment. (F) Percentage change in average number of tumors during treatment. (G) Quantification of tumor surface area from photographs taken during treatment. (H) Average tumor cross-sectional area measured from H&E stained tissue. [Figure 4] (A) Quantification of DefB14 stained area from multiplex immunofluorescence images of skin tumor sections. (B) Quantification of the percentage of LL-37+ cells within the tumor area 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 (vehicle) or 2% (w / v) PALA and stained for LL-37 (green), cytokeratin-14 (CK14, pink), or DNA (blue). [Figure 5] (A) Quantification of cells expressing the neutrophil cell marker myeloperoxidase (MPO, light blue). (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 T cell marker CD8 (red). All sections were simultaneously stained with keratinocyte markers CK14 (gray) and DAPI (blue). (E) Representative multiplex immunofluorescence images highlighting differentially present cell types. [Figure 6](A) Photographs of mice treated with vehicle, 2% PALA, and 5% IMQ 3 days after treatment. (B) Photographs of mice treated with vehicle, 2% PALA, and 5% 5-FU 7 days after treatment. [Figure 7] Representative images of immunostained skin from vehicle and 2% PALA-treated mice (left panel). Quantification of the percentage of positive cells within the tumor area is graphed (right panel). [Figure 8] (A) Quantification of the mean change in tumor mass per mouse during the treatment period, expressed as a percentage of tumor count at week 9. (B) Weight change during the treatment period, expressed as a percentage of baseline body weight. (C) Levels of hepatic and systemic inflammatory marker serum amyloid A (SAA) at the endpoint. (D) Measurement of colon length at the endpoint as a macroscopic measure of intestinal lesions. [Figure 9] (A) Quantification of the average change in tumor volume per mouse during the treatment period as a percentage of the number of tumors at week 13. (B) Quantification of the average change in total tumor area per mouse during the treatment period as a percentage of the measured values ​​at week 13. [Figure 10] Table 7 of Example 2 is shown. This table shows the appearance and HPLC assay of different hydrogels. [Figure 11] Cumulative release profiles (n=1) of various formulations with permeation enhancers. A PALA aqueous solution is also reported for reference. [Figure 12] Table 10 shows the appearance and assay of the formulation from Example 2. [Figure 13] Cumulative release profile of a specific formulation (n=3). [Figure 14] Levels of PALA in skin tissue and plasma over 24 hours after a single topical application of 4% (w / v) PALA formulation AP0536 / 29 / 03 to mice. [Figure 15](A) Change in body weight during the treatment period, expressed as a percentage of baseline body weight. (B) Levels of serum amyloid A (SAA), a hepatic and systemic inflammatory marker, at the endpoint. (C) Measurement of colon length at the endpoint as a macroscopic measure of intestinal lesions. (D) Fecal lipocalin-2 (LCN2) levels at the endpoint as a measure of intestinal inflammation. (E) Quantification of the mean number of tumors per mouse during the treatment period. (F) Mean change in tumor volume per mouse during the treatment period, expressed as a percentage of the original number of tumors. (G) Quantification of the mean tumor surface area per mouse from photographs during the treatment period. [Figure 16] (A) PALA-treated mice maintained better body weight than 5-FU or IMQ-treated mice. Body weight change from the start of treatment was graphed (mean ± SEM). Supportive care was provided to IMQ and 5-FU mice (started at the time indicated by †), and 5-FU-treated animals were discontinued after 2.5 weeks of treatment as directed by veterinary staff. (B) Levels of hepatic and systemic inflammatory marker serum amyloid A (SAA) at endpoint. (C) Fecal lipocalin-2 (LCN2) levels at endpoint as a measure of intestinal inflammation. (D) Mice treated with 2% PALA had significantly smaller mean tumor sizes than mice treated with either vehicle or 5% IMQ. Quantification of mean tumor surface area per tumor measured from photographs using ImageJ software over the treatment period. (E) Growth profiles of individual tumors showing a reduced percentage of persistently growing tumors in 2% PALA-treated mice. (F) Endpoint tumor malignancy, determined by evaluation of H&E-stained tumor sections, shows that mice treated with 2% PALA have less squamous cell carcinoma (SCC) and SCC at tumor site compared to mice treated with vehicle or 5% IMQ. (G) Tumor levels of interleukin-1β were elevated in both 2% PALA-treated and 5% IMQ-treated mice, indicating induction of an anti-tumor immune response. (H) Tumor levels of interferon-α1 were elevated only in 2% PALA-treated mice, suggesting that the type of immune response induced was different from that stimulated by 5% IMQ. [Figure 17](A) Local treatment with 1% PALA daily for 12 days slows tumor growth. Short-term local treatment of B16 melanoma with 2% PALA reduces the expression of the checkpoint inhibitor PD-L1. (C) Short-term local treatment with 2% PALA sensitizes B16 melanoma to anti-PD-L1 therapy. [Figure 18] Tumor growth of orthotopic xenografts of oral squamous cell carcinoma is delayed by local PALA treatment. [Figure 19] (A) Bactericidal activity of human normal dermal fibroblasts stimulated by PALA treatment. (B) PALA-stimulated production of antimicrobial peptides in human normal dermal fibroblasts. (C) Enzymatic activity of aspartate transcarbamylase, the enzymatic target of PALA, is reduced by only about 25% at the dose that induces maximum immune stimulation in human normal dermal fibroblasts. (D) and (E) Similar U-shaped dose-response curves observed in response to topical PALA treatment of skin cancer in a UVB-induced NMSC mouse model.

Claims

1. A method for treating target precancerous cells and / or cancerous cells, The method includes topically applying the composition to a body area of ​​a subject having and / or causing precancerous cells and / or cancerous cells, and / or adjacent to precancerous or cancerous cells, or providing the subject with the composition so that the subject can apply the composition to the body area. The method wherein the composition comprises N-phosphonacetyl-L-aspartic acid (PALA) and water, and optionally further comprises at least one nonionic surfactant.

2. The method according to 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 according to claim 1, wherein the area of ​​the target includes skin.

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

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

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

7. The method according to claim 1, wherein the subject is female, and the region of the subject includes the surface of the subject's vagina, and the precancerous cells and / or cancerous cells include precancerous cells and / or cancerous cells of the vagina.

8. The method according to claim 1, wherein the region of the target includes the surface of the oral cavity of the target, and the precancerous cells and / or cancerous cells include precancerous cells and / or cancerous cells of the oral cavity.

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

10. The method according to claim 1, wherein the composition further comprises a nonionic linear copolymer, optionally comprising poloxamer 188.

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

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

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

14. The method according to claim 13, wherein the nonionic surfactant comprises caprylocaproyl polyoxyl-8 glyceride, or the nonionic surfactant excipient comprises PEG-8 monoester or diester with small amounts of monoglycerides, diglycerides, and triglycerides of caprylic acid (C8) and capric acid (C10), or the nonionic surfactant comprises LABRASOL ALF.

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

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

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

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

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

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

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

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

23. The method according to any one of claims 1 to 22, wherein the composition further comprises the at least one nonionic surfactant, optionally wherein the nonionic surfactant comprises poloxamer 188 and / or caprylocaproyl polyoxyl-8 glyceride.

24. A composition, a) N-phosphonacetyl-L-aspartic acid (PALA) and; b) Water and; c) The composition comprising at least one nonionic surfactant.

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

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

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

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

30. The method according to claim 24, wherein the at least one nonionic surfactant comprises caprylocaproyl polyoxyl-8 glyceride, or the at least one nonionic surfactant excipient comprises PEG-8 monoester or diester with small amounts of monoglycerides, diglycerides, and triglycerides of caprylic acid (C8) and capric acid (C10), or the at least one nonionic surfactant comprises LABRASOL ALF.

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

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

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

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

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

36. The composition according to any one of claims 24 to 35, wherein the at least one nonionic surfactant comprises poloxamer 188 and / or caprylocaproyl polyoxyl-8 glyceride.

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

38. A system or kit: a) A composition comprising N-phosphoneacetyl-L-aspartic acid (PALA), water, and optionally at least one nonionic surfactant, b) Below: i) A dispensing container configured to distribute the composition locally to a target area of ​​the body, ii) A topical patch configured to deliver the composition locally to the body region of the target, or iii) The system or kit comprising at least one of the immune checkpoint inhibitors.

39. The kit system according to claim 38, wherein the composition is present in the dispensing container or the topical patch.

40. The system or kit according to claim 38, wherein a dispensing container is present, and the dispensing container is a non-contact type that enables the distribution and rubbing of the composition on the body area such that other areas of the object do not come into contact with the composition.

41. The system or kit according to claim 40, wherein the other area of ​​the target includes the hand of the target.

42. The system or kit according to claim 40, wherein the non-contact dispensing container includes a quantitative local applicator.

43. The system or kit according to 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 kit according to claim 40, wherein the aforementioned dispensing container includes a tube or sachet.

45. The system or kit according to 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 according to any one of claims 38 to 45, comprising at least one nonionic surfactant and a nonionic linear copolymer.

47. The system or kit according to claim 46, wherein the nonionic linear copolymer comprises poloxamer 188.

48. The system or kit according to claim 46, wherein the nonionic linear copolymer is present in the composition in an amount of about 50% to 90%, about 60% to 80%, about 65% to 75%, or about 70%.

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

50. The system or kit according to claim 38, wherein the at least one nonionic surfactant is present and comprises caprylocaproyl polyoxyl-8 glyceride, or the at least one nonionic surfactant excipient comprises PEG-8 monoester or diester with small amounts of monoglycerides, diglycerides, and triglycerides of caprylic acid (C8) and capric acid (C10), or the at least one nonionic surfactant comprises LABRASOL ALF.

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

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

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

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

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

56. The system or kit according to any one of claims 38 to 55, wherein the at least one nonionic surfactant comprises poloxamer 188 and / or caprylocaproyl polyoxyl-8 glyceride.

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

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