A composition for treating cancer, methods and use thereof

Abstract

The present disclosure relates to a composition comprising a peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent. It also relates to a process of preparing the composition and a method / use of the peptide alone or in combination with at least one anti-cancer agent in preventing or managing or treating cancer / precancerous condition.

Description

[0001] “A COMPOSITION FOR TREATING CANCER, METHODS AND USE THEREOF”

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to the field of compositions and therapeutics. Particularly, the present disclosure relates to a composition comprising a peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent. It also relates to a process of preparing the composition, use of the peptide having at least 90% homology to a peptide of SEQ ID No. 1 or use of the composition comprising a peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent in treating cancer.

[0004] BACKGROUND AND PRIOR ART

[0005] Skin cancer instigates in skin cells and causes abnormal growth or changes in the skin. There is a global trend of increase in the number of skin malignancies, especially in countries with large Caucasian populations (e.g. USA, UK and Australia). Based on the GLOBOCAN 2020 database, it was recently reported that melanoma is not that rare in India. Also, based on the Cancer Stat Facts by National Cancer Institute 2021, the incidence rates of skin cancer are increasing worldwide with an approximate 5 years of survival rate of nearly 90%.

[0006] Skin cancer comprises of several forms where basal cell carcinoma is commonly found. The other different forms include - squamous cell carcinoma, merkel cell carcinoma and melanoma. Melanoma is the most aggressive form of the skin cancer and is accountable for nearly 75% of all skin cancer associated deaths. Melanoma prevalence and the mortality rate of patients (advanced stage melanoma) in India and other countries in the world has been alarmingly increasing in recent years (Deo et al. 2005). Melanoma can affect people irrespective of their age and race. Among basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and malignant skin melanoma (MSM), SCC and MSM are more aggressive, invasive and can metastasize to other parts of body leading to severity of the condition. In 2019, it has been reported that there were around 4 million cases of BCC, 2.4 million cases of SCC and 0.3 million cases of MSM worldwide (Zhang et al., 2021). Interestingly, most invasive SCCs start off as premalignant skin lesions known as Actinic keratoses (Aks) which can occur anywhere on the body (Siegel et al., 2017). It has been estimated that around 40 million Americans develop these lesions each year (Neugebauer et al, 2018) and nearly 10% of AKs can progress into invasive SCCs (Fuchs and Marmur, 2007).

[0007] Ultraviolet radiation due to exposure to sun is the major cause for the skin cancer occurrence. Nevertheless, other factors such as human papillomavirus infections, smoking tobacco, genetics, environmental carcinogens, genetic factors, chronic non-healing wounds, light skin color and aging also are some of the causative factors for skin cancer condition.

[0008] As regards treatment, Melanoma treatment is one of the greatest challenges in cancer treatment and the survival rates are low during metastasis condition. Even though using latest treatment strategies by means of targeted inhibitors, survival rates in some patients has not improved and most of them do not respond to treatment due to heterogeneous complexity of tumor microenvironment.

[0009] Many of the treatment strategies for skin cancer include surgery, radiation therapy, topical chemotherapy and immunotherapy. However, some of these treatments have severe drawbacks such as fatigue, flu-like symptoms, DNA damage, hair-loss, skin irritation, development of secondary tumors and tumor cell migration into blood.

[0010] Commonly used topical treatment for skin lesions is 5% 5 -fluorouracil (5FU) cream since 5FU is a highly cytotoxic agent that inhibits DNA synthesis leading to tumor cell death. However, 5FU cannot be targeted to tumor tissue specifically as it can produce several side effects in patients such as skin irritation, erythema, pain, inflammation, pruritis etc around the site of application that can severely affect the patient’s quality of life. Topical treatment with 5FU is reported to have severe side effects resulting in adverse drug reaction that leads to multiple actinic keratosis of forearms, neck and face (Kishi P and Price CJ, 2018). In some extreme cases, the treatment with the 5FU creams can lead to infections and ulceration.

[0011] Topical therapeutic applications are cost-effective in comparison to the other treatment strategies and also allow for better patient amenability since the therapy can be dispensed from the household by the patient. However, topical treatment using miltefosine or Imiquimod compositions towards cutaneous melanoma condition has shown low response rates and the patients have shown only better response only after combining with other skin-directed therapies (Spratt et. Al, 2014). Thus, there is requirement for the therapeutic molecule / combination that can address the following:

[0012] • Reduce / halt the tumor growth in the patients

[0013] • Reduce the adverse side effects of 5FU / other cytotoxic drugs while maintaining the anticancer efficacy

[0014] • Non-irritant on the skin

[0015] • Enhancing the penetration of the formulation with the novel biologic / combination into the skin tumors

[0016] • Decrease the amount of the chemotherapeutic agent needed to achieve the desired prognosis.

[0017] The present disclosure achieves this objective by providing a therapeutic molecule and a composition, processes, methods and use of the molecule / the composition for treating cancer.

[0018] STATEMENT OF THE INVENTION

[0019] The present disclosure relates to a composition comprising a peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent, a process of preparing the composition and its use in preventing or managing or treating cancer / precancerous condition. The present disclosure also relates to a method of preventing or managing or treating cancer / precancerous condition, said method comprising administering / applying to a subject in need thereof an effective amount of a peptide having at least 90% homology to a peptide of SEQ ID No. 1 or a composition comprising the peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent. It also relates to use of a peptide having at least 90% homology to a peptide of SEQ ID No. 1 or a composition comprising the peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent in preventing or managing or treating cancer / precancerous condition.

[0020] BRIEF DESCRIPTION OF ACCOMPANYING FIGURES

[0021] Figure 1: Cell viability assay for B16F10 mouse melanoma cells treated with the peptide of SEQ ID No.l. B16F10 mouse melanoma cells were treated with the peptide at different concentrations (from 0 to 200 pM) for 24h, 48h and 72h. Using MTS reagent (Promega), absorbance values were measured. Peptide treated cells showed growth inhibition at the concentration of 25 M and further higher concentrations. Tumor cells showed IC50 value to nearly 60 M of peptide concentration.

[0022] Figure 2: Peptide + 5FU treatment induces B16F10 cell cytotoxicity at 24h. Tumor cells were treated with the peptide and 5FU at a concentration of 60 pM and 20 pM respectively for 24h individually and in combination. Calcein AM and Propidium Iodide was used to stain live and dead cells. Green (calcein AM stain) indicates live cells and Red (propidium iodide) indicates dead cells. lOOx magnification.

[0023] Figure 3: Peptide + 5FU treatment induces B16F10 cell cytotoxicity at 48 h. Tumor cells were treated with the peptide and 5FU at a concentration of 60 pM and 20 pM respectively for 48h individually and in combination. Calcein AM and Propidium Iodide was used to stain live and dead cells. Green (calcein AM stain) indicates live cells and Red (propidium iodide) indicates dead cells. lOOx magnification.

[0024] Figure 4: Peptide + 5FU treatment does not cause cytotoxicity on Mouse embryonic fibroblasts. The Mouse embryonic fibroblasts (MEFs) were treated with combination treatment of the peptide and 5FU at a concentration of 60 pM and 20 pM for 48h. Calcein AM and Propidium Iodide was used to stain live and dead cells. Green (calcein AM stain) indicates live cells and Red (propidium iodide) indicates dead cells. lOOx magnification.

[0025] Figure 5: Cell cycle analysis by flow cytometer. B16F10 cells were treated with the peptide (60 pM) for 48 h. Cells were fixed with 70% ethanol. Further, the DNA content of the cells was examined by staining with 50 pg / ml propidium iodide and RNase A (lOOpg / ml) mixture. Later, the cells were analyzed using flow cytometer under PI channel. The cells were gated in different populations as Gl, S, G2 / M phases.

[0026] Figure 6: Quantitative PCR analysis of Ki67 and Top2a genes. B16F10 cells were treated with the peptide for 24h. Later, RNA was isolated, reverse transcribed and mRNA levels were examined by amplifying using gene specific primers with the help of Sybr green PCR mix. 18sRNA was used as normalized control.

[0027] Figure 7: Penetration of the peptide into mouse melanoma B16F10 cells. Cells were treated with peptide at a concentration of 60 pM for 16h. Post-treatment, cells were fixed and treated with peptide specific antibody followed by FITC-tagged anti-mouse antibody. Cells were examined under the microscope. lOOx magnification. Figure 8 [A & B]: Topical application of the peptide in formulation alone or peptide in combination with 5FU (Peptide+5FU) in formulation reduces tumor growth in syngeneic mouse melanoma model. Tumors were induced in C57BL / 6 mice using B16F10 melanoma cells. When tumor reached nearly 75 mm , mice were treated topically with either different combinations of peptide and 5FU. Mice were divided into 5 groups for different combinations of treatments. Group - 1. Includes control mice with in-house formulation treatment without any active component, Group - 2. 5FU (IH) mice includes 5FU (100 mM) treatment in IH [In-house] formulation, Group - 3. 5FU (Comm) mice includes treatment with commercially available 5-FU cream (5%) (-380 mM), Group - 4. Includes mice treated with peptide (Img peptide / 1 gm IH formulation base), Group - 5. Includes mice treated with peptide (Img peptide / gm IH formulation base) and 5FU (100 mM) in IH formulation (5FU+ Peptide (IH). The treatment was carried out for all the groups weekly thrice for the duration of three weeks. 5FU commercially available cream 5FU (Comm) was used as a positive control. Control animals were treated with in-house (IH) formulation base. After three weeks, animals were sacrificed and tumors were collected to measure tumor volume and tumor weights.

[0028] Figure 9: Histo-morphological examination of tumor sections in treated and control mice. Histological tumor sections of control and differentially treated mice groups were collected after 3 weeks post treatment. Tissues were fixed, sections were made, stained with hematoxylin and eosin following standard procedures. Sections were observed under microscope for tumor morphology. Magnification, 100X.

[0029] Figure 10: Histo-morphological examination of skin sections. Histological dorsal skin sections of mice subjected to different treatments with the in house prepared formulation base [Control] was checked for any side effects on the skin. Skin sections were stained with hematoxylin and eosin post treatment following standard procedures. Epidermis in the skin sections of the animal groups treated with commercially available 5FU cream was affected largely whereas in all other treatment groups, epidermis was intact and unaffected. Magnification, 40X.

[0030] Figure 11: Comparison of activity of different concentrations of 5-Fluorouracil on xenograft tumor progression. Animals were treated with commercial 5-FU cream [-380 mM] and in-house 5-FU formulation [lOOmM] and commercial Imiquimod cream (5% Imiquimod). The formulations were applied topically to tumor bearing animals for 25 days and the tumor progression was monitored. NC represents negative control (animals treated with formulation without any compounds). Error bars represent mean + / - SEM.

[0031] Figure 12: Effect of the peptide on xenograft tumor progression in animals. Formulations containing different concentrations of the peptide were topically applied to tumor -bearing animals for 25 days and tumor progression was studied. NC represents negative control (animals treated with formulation without any peptide). Peptide alone showed a concentration dependent anti-tumor activity without any skin irritation to animals. Error bars represent mean + / - SEM

[0032] Figure 13A: Comparison of anti-tumor activity of commercial 5FU vs 5FU + peptide. Animals were treated with commercial 5-FU cream (-380 mM) or a combination of commercial 5-FU with 1 mg / g of peptide. The formulations were applied topically to tumor bearing animals for 25 days and the tumor progression was monitored. NC represents negative control (animals treated with formulation without any compounds). Error bars represent mean + / - SEM.

[0033] Figure 13B: Bottom panel: Comparison of dermal side effects caused by commercial 5- Fluorouracil and reduction in side effects by the peptide and the combination of peptide + commercial 5-FU. NC represents negative control (animals treated with formulation without any compounds).

[0034] Figure 14: Comparison of weights of excised tumors at the end of the study under each group (A) along with the images (B). Animals were treated with commercial 5-FU cream or a combination of commercial 5-FU with 1 mg / g of peptide. The formulations were applied topically to tumor bearing animals for 25 days. At the 25thday, animals were sacrificed, and the tumors were excised out and weighed. Panel B shows the images of the excised tumors. Error bars in the graph A represent mean + / - SEM.

[0035] Figures 15: Relative expression levels of Ki-67 mRNA in mice treated with different combinations of 5FU and peptide. Mice groups (Untreated control, commercial 5-FU cream, inhouse 5-FU formulation, peptide alone, in-house 5-FU formulation + peptide) were sacrificed after 3 weeks of treatment. RNA was isolated from excised tumors, cDNA was transcribed and quantitative PCR was performed using primers for Ki-67 genes. Quantification of mRNA levels was done using 18sRNA as internal control. Data are presented as mean ± SEM.

[0036] Figures 16: Relative expression levels of TOP2A mRNA in mice treated with different combinations of 5FU and peptide. Mice groups (Untreated control, commercial 5-FU cream, inhouse 5-FU formulation, peptide alone, in-house 5-FU formulation + peptide) were sacrificed after 3 weeks of treatment. RNA was isolated from excised tumors, cDNA was transcribed and quantitative PCR was performed using primers for T0P2A genes. Quantification of mRNA levels was done using 18sRNA as internal control. Data are presented as mean ± SEM.

[0037] Figure 17: Immunohistochemical analysis for Ki-67. Mice groups (Untreated control, commercial 5-FU cream, in-house 5-FU formulation, peptide alone, in-house 5-FU formulation + peptide) were sacrificed after 3 weeks of treatment. Tumors were excised and the sections were fixed in formalin and embedded in paraffin using standard procedures. Sections were prepared (4-5 pm) for all the embedded tumors and standard procedures were followed to rehydrate, retrieve antigen and further immune -histochemical analysis. Sections were incubated with anti- Ki-67 antibody followed by secondary antibody conjugated with HRP. Using DAB substrate and counterstaining with hematoxylin, the staining was observed. Slides were mounted, dried and analyzed under microscope. Magnification lOOx.

[0038] Figure 18: Anti-tumorigenic effects of peptide through intraperitoneal administration route in a syngeneic melanoma mouse model. Tumors were induced in C57BE6 mice using B16F10 melanoma cells. When tumor reached 75mm , mice were treated with either PBS [control] or 8mg / Kg of peptide every alternate day for three weeks. Animals were sacrificed after 3 weeks and tumors were collected. Tumor morphology was shown in all the mice (A) and tumor volumes were calculated (B) and weighed (c) and shown as Mean ± SEM of 3 mice per group.

[0039] DETAILED DESCRIPTION OF THE DISCLOSURE

[0040] The foregoing has broadly outlined the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter which forms the subject of the description of the disclosure. It should also be realized by those skilled in the art that such equivalent product / methods do not depart from the scope of the disclosure.

[0041] Definitions:

[0042] Unless otherwise defined, all terms used in the disclosure, including technical and scientific terms, have meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the singular forms ‘a’, ‘an’ and ‘the’ include both singular and plural referents unless the context clearly dictates otherwise.

[0043] The term ‘comprising’, ‘comprises’ or ‘comprised of as used herein are synonymous with ‘including’, ‘includes’, ‘containing’ or ‘contains’ and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. Likewise, certain terms may be interchangeably used throughout the specification and thus have the same meaning even when they are referred interchangeably. For example: “Peptide of SEQ ID No. 1” is interchangeably referred throughout the specification as “peptide”, “the peptide”, “protein”, “molecule” etc; “Cancer” is interchangeably referred throughout the specification as “tumor”; “In-house” as “HI”; “5FU”, “5FU (Comm)”, “peptide”, “5FU+ Peptide (IH)” as “compounds”.

[0044] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0045] The term ‘about’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of ±10% or less, preferably ±5% or less, more preferably ±1% or less and still more preferably ±0.1% or less of and from the specified value, insofar such variations are appropriate to perform the present disclosure. It is to be understood that the value to which the modifier ‘about’ refers is itself also specifically, and preferably disclosed.

[0046] The term “cancer” throughout the specification refers to a disease or group of diseases or a condition where cells grow abnormally and has the ability to spread or invade parts of the body. They can be a benign tumor to a malignant cancer.

[0047] The term “anti-cancer agent” throughout the specification refers to any agent / molecule / compound and their different forms not limiting to salts, esters, ethers, polymorphs, metabolites, pure form, particle size, isomers, mixtures of isomers, complexes, combinations and other derivatives.

[0048] The present disclosure relates to a composition comprising a peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent. In an embodiment of the present disclosure, the composition comprises a peptide having at least 95% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent.

[0049] In an embodiment of the present disclosure, the composition comprises a peptide having at least 96% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent.

[0050] In an embodiment of the present disclosure, the composition comprises a peptide having at least 97% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent.

[0051] In an embodiment of the present disclosure, the composition comprises a peptide having at least 98% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent.

[0052] In an embodiment of the present disclosure, the composition comprises a peptide having at least 99% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent.

[0053] In a preferred embodiment of the present disclosure, the composition comprises a peptide of SEQ ID No. 1 (KTLLEQTLALLPAALPLLAAPLAPLTLRRRRPKIKQ) and at least one anticancer agent.

[0054] In a preferred embodiment of the present disclosure, the anti-cancer agent is selected from a group comprising but not limiting to 5 -Fluorouracil [5FU], Imiquimod or combinations thereof.

[0055] In an embodiment of the present disclosure, concentration of the peptide is in the range of about 0.001% - 2% and concentration of and the anti-cancer agent is in the range of about 0.001% - 5%.

[0056] In an embodiment of the present disclosure, concentration of the peptide is about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 1.5% and about 2% or any other concentration within the range of 0.001% - 2%. In an embodiment of the present disclosure, concentration of the anti-cancer agent is about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5% and about 5% or any other concentration within the range of 0.001% - 5%.

[0057] In an embodiment of the present disclosure, the composition optionally comprises excipients.

[0058] In an embodiment of the present disclosure, the excipients are in the range of about 93% - 99.998%.

[0059] In an embodiment of the present disclosure, the excipients are at a concentration of about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, about 99.992%, %, about 99.994%, about 99.996%, about 99.998% and any other concentration within the range of 93% - 99.998%.

[0060] In an embodiment of the present disclosure, the excipients are selected from a group comprising but not limited to preservatives, buffering agents, stabilizers, binders, lubricants, compaction agents, disintegration agents, dispersion agents, coloring agents, additive, solvent, surfactant, anti-oxidant, suspending agent, emulsifying agent or combinations thereof.

[0061] In an embodiment of the present disclosure, the composition is administered through different modes such as but not limiting to oral, intratumoral, intravenous, intraperitoneal, intramuscular, topical / cosmetic, sub-cutaneous or combinations thereof.

[0062] In a preferred embodiment of the present disclosure, the composition is administered as a topical / cosmetic composition.

[0063] In yet another preferred embodiment of the present disclosure, the composition is administered through intraperitoneal route. In an embodiment of the present invention, the composition is formulated into various forms selected from a group comprising but not limiting to tablet, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion in hard or soft gel capsules, syrups, elixirs, cream, paste, spray, lotion, powder, ointment or combinations thereof.

[0064] In a preferred embodiment of the present disclosure, the composition is formulated into forms selected from a group comprising but not limited to a gel, cream, paste, spray, lotion, powder, ointment or combinations thereof.

[0065] The present disclosure also relates to a composition comprising a peptide having at least 90% homology to a peptide of SEQ ID No. 1 at a concentration ranging from about 0.001% - 2%, at least one anti-cancer agent at a concentration ranging from about 0.001% - 5% and excipients at a concentration ranging from about 93 % - 99.998%.

[0066] The present disclosure also relates to a process of preparing the composition, said method comprising steps of combining a peptide having at least 90% homology to a peptide of SEQ ID No. 1 with at least one anti-cancer agent.

[0067] In a preferred embodiment of the present disclosure, the anti-cancer agent is selected from a group comprising but not limiting to 5 -Fluorouracil [5FU], Imiquimod or combinations thereof.

[0068] In a preferred embodiment, the present disclosure relates to a process of preparing the composition, said method comprising steps of combining a peptide of SEQ ID No. 1 with 5-FU.

[0069] In an embodiment of the present disclosure, the peptide is synthesized before combining with anti-cancer agent.

[0070] In an embodiment of the present disclosure, concentration of the peptide is in the range of about 0.001% - 2% and the concentration of anti-cancer agent is in the range of about 0.001% - 5%. In an embodiment of the present disclosure, concentration of the peptide is about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 1.5% and about 2% or any other concentration within the range of 0.001% - 2%.

[0071] In an embodiment of the present disclosure, concentration of the anti-cancer agent is about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5% and about 5% or any other concentration within the range of 0.001% - 5%.

[0072] In an embodiment of the present disclosure, the process optionally comprises inclusion of excipients in the composition.

[0073] In an embodiment of the present disclosure, the excipients are in the range of about 93% - 99.998%.

[0074] In an embodiment of the present disclosure, the excipients are at a concentration of about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, about 99.992%, %, about 99.994%, about 99.996%, about 99.998% and any other concentration within the range of 93% - 99.998%.

[0075] The present disclosure also relates to a method of inhibiting cancer cells / tumor cells, said method comprising treating the cancer cells / tumor cells with a peptide having at least 90% homology to a peptide of SEQ ID No. 1 or an effective amount of a composition comprising the peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent.

[0076] The present disclosure also relates to a method of preventing or managing or treating cancer or a precancerous condition, said method comprising administering / applying to a subject in need thereof, an effective amount of a peptide having at least 90% homology to a peptide of SEQ ID No. 1 or an effective amount of a composition comprising the peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent.

[0077] In a preferred embodiment of the present disclosure, the anti-cancer agent is selected from a group comprising but not limiting to 5 -Fluorouracil [5FU], Imiquimod or combinations thereof.

[0078] In a preferred embodiment, the present disclosure relates to a method of preventing or managing or treating cancer / precancerous condition, said method comprising administering / applying to a subject in need thereof, an effective amount of a peptide of SEQ ID No. 1 or an effective amount of a composition comprising the peptide of SEQ ID No. 1 and 5FU.

[0079] In an embodiment of the present disclosure, the precancerous condition is selected from a group comprising but not limiting to actinic keratosis, epidermolysis bullosa, submucous fibrosis, lichen planus and discoid lupus erythematous.

[0080] In an embodiment of the present disclosure, the cancer is such as but not limiting to skin cancer, breast cancer, colon cancer, liver cancer, esophageal cancer, gastric cancer, renal cancer, head and neck cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer and glioma.

[0081] In an embodiment of the present disclosure, the skin cancer is such as but not limiting to basal cell carcinoma, squamous cell carcinoma, merkel cell carcinoma and melanoma.

[0082] In a preferred embodiment of the present disclosure, the skin cancer is melanoma.

[0083] The present disclosure also relates to a method of preventing or managing or treating skin cancer, said method comprising administering / applying to a subject in need thereof, an effective amount of a peptide of SEQ ID No. 1 or an effective amount of a composition comprising the peptide of SEQ ID No. 1 and at least one anti-cancer agent.

[0084] The present disclosure also relates to a method of preventing or managing or treating melanoma, said method comprising administering / applying to a subject in need thereof, an effective amount of a peptide of SEQ ID No. 1 or an effective amount of a composition comprising the peptide of SEQ ID No. 1 and at least one anti-cancer agent.

[0085] The present disclosure also relates to use of a peptide having at least 90% homology to a peptide of SEQ ID No. 1 or a composition comprising the peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent in manufacturing a medicament for preventing or managing or treating cancer / precancerous condition.

[0086] In a preferred embodiment of the present disclosure, the anti-cancer agent is selected from a group comprising but not limiting to 5 -Fluorouracil [5-FU], Imiquimod or combinations thereof.

[0087] In a preferred embodiment, the present disclosure relates to use of a peptide of SEQ ID No. 1 or a composition comprising the peptide of SEQ ID No. 1 and 5-FU in manufacturing a medicament for preventing or managing or treating cancer / precancerous condition.

[0088] In an embodiment of the present disclosure, the precancerous condition is selected from a group comprising but not limiting to epidermolysis bullosa, submucous fibrosis, lichen planus and discoid lupus erythematous.

[0089] In an embodiment of the present disclosure, the cancer is such as but not limiting to skin cancer, breast cancer, colon cancer, liver cancer, esophageal cancer, gastric cancer, renal cancer, head and neck cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer and glioma.

[0090] In an embodiment of the present disclosure, the skin cancer is such as but not limiting to basal cell carcinoma, squamous cell carcinoma, merkel cell carcinoma and melanoma.

[0091] In a preferred embodiment of the present disclosure, the skin cancer is melanoma.

[0092] The present disclosure also relates to use of a peptide of SEQ ID No. 1 or a composition comprising the peptide of SEQ ID No. 1 and at least one anti-cancer agent in manufacturing a medicament for preventing or managing or treating cancer / precancerous condition. The present disclosure also relates to use of a peptide of SEQ ID No. 1 or a composition comprising the peptide of SEQ ID No. 1 and at least one anti-cancer agent in manufacturing a medicament for preventing or managing or treating skin cancer.

[0093] The present disclosure also relates to use of a peptide of SEQ ID No. 1 or a composition comprising the peptide of SEQ ID No. 1 and at least one anti-cancer agent in manufacturing a medicament for preventing or managing or treating melanoma.

[0094] In a preferred embodiment of the present disclosure, the anti-cancer agent is selected from a group comprising but not limiting to 5 -Fluorouracil [5FU], Imiquimod or combinations thereof.

[0095] In an embodiment of the present disclosure, the subject is a mammal, including human being.

[0096] In an embodiment of the present disclosure, the composition is administered through different modes such as but not limiting to oral, intratumoral, intravenous, intraperitoneal, intramuscular, topical / cosmetic, sub-cutaneous and combinations thereof.

[0097] In a preferred embodiment of the present disclosure, the composition is administered as a topical / cosmetic composition.

[0098] In yet another preferred embodiment of the present disclosure, the composition is administered through intraperitoneal route.

[0099] In an embodiment of the present invention, the composition is formulated into various forms selected from a group comprising but not limiting to tablet, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion in hard or soft gel capsules, syrups, elixirs, cream, paste, spray, lotion, powder, ointment or combinations thereof. In a preferred embodiment of the present disclosure, the composition is formulated into forms selected from a group comprising but not limited to a gel, cream, paste, spray, lotion, powder, ointment or combinations thereof.

[0100] The present invention discloses use of a synthetic small peptide as a therapeutic molecule for treating cancer / precancerous condition either alone or in combination with at least one anticancer agent such as but not limiting to 5-FU, Imiquimod, etc. The peptide is 36 amino acids long and its sequence is as follows:

[0101] KTLLEQTLALLPAALPLLAAPLAPLTLRRRRPKIKQ - SEQ ID No.l

[0102] The peptide is shown to be an immuno-modulatory and anti-cancerous in nature. The peptide alone inhibited the growth of melanoma tumor in a syngeneic mouse model. C57BL / 6 mice injected with B16F10 melanoma cells subcutaneously and treated with 8 mg / kg body weight of the peptide for three weeks resulted in significantly reduced tumor volumes and tumor weight compared to those of the control mice. It was also observed that the treatment of Bl 6F 10 cells with the peptide led to downregulation of important genes like Ki67 and Top2a that are known to play important roles in tumor progression through cell cycle progression and proliferation. In addition to the stand-alone activity, when the peptide was combined with 5 -fluorouracil (an already approved chemotherapeutic agent for early-stage skin cancer and precancerous lesions) and applied topically to mice bearing induced melanoma tumors, it was observed that the combination showed a much better anti-tumor effect than the commercially available 5FU cream or was able to bring down the side effects associated with 5-FU. The combination led to reduced side effects like skin irritation, redness and swelling, which are the major side effects generally associated with 5FU treatment in skin cancer patients. The topical route of administration of the peptide in combination with 5FU (peptide+5FU) led to significant reduction in the tumor burden in animals. Overall, the results suggest that topical application of peptide as standalone or peptide+5FU reduce the melanoma tumor burden in mice and acts as potential anti-melanoma agent when used either alone or in combination with 5FU treatment. Thus, the peptide finds use in combination with existing molecule (like 5FU) for intervention in skin cancers via different modes of application. It is to be understood that the foregoing description is illustrative not a limitation. While considerable emphasis has been placed herein on particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein. Similarly, additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein.

[0103] Descriptions of well-known / conventional methods / steps and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above-described embodiments, and in order to illustrate the embodiments of the present disclosure, certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments may be practiced and to further enable those of skill in the art to practice the embodiments. Accordingly, following examples should not be construed as limiting the scope of the embodiments herein.

[0104] EXAMPLES

[0105] Example 1: Preparation of the peptide and formulations

[0106] The present study was designed to evaluate the anti-cancer activity of the composition comprising the peptide of SEQ ID No. 1 and 5-FU.

[0107] Peptide of SEQ ID No.l was synthesized, directly added to 5-FU at different concentrations with remaining being the excipients and mixed properly to obtain the composition. Similarly, peptide alone and 5-FU alone were prepared along with suitable excipients for conducting experiments.

[0108] Concentrations of the components for preparation are as follows:

[0109] Formulation comprising only the excipients is used as control [formulation base]

[0110] Commercially available 5-FU cream at 5% or ~380mM In-house 5-FU formulation at -1.43% or lOOmM

[0111] Peptide at different concentrations 12.5 uM (0.00498%), 25 uM (0.00996%), 50 uM (0.0199%), 100 uM (0.039%), 200 uM (0.079%), 0.1 mg / g of the formulation [0.01%], 0.5 mg / g of the formulation [0.05%], 1 mg / g of the formulation [0.1%]

[0112] Peptide was added to the commercially available 5-FU cream and in-house 5-FU formulation at the above concentration to test the effect of the combination

[0113] The excipients used in the formulations are as follows:

[0114] Sodium carboxymethyl cellulose

[0115] Sodium Chloride

[0116] Sodium Acetate

[0117] Glacial acetic acid

[0118] Methyl Paraben

[0119] Propyl Paraben m-Cresol

[0120] Lysine hydrochloride

[0121] Deionized water

[0122] All the formulations were stored at 4°C throughout the duration of the study.

[0123] The process steps for preparing the formulations are as follows:

[0124] 1) All the ingredients [excipients] were weighed / measured, water was added slowly and mixed with spatula up and down till the mixture becomes transparent with a semi solid texture to obtain the formulation base;

[0125] 2) Peptide and / or 5-FU at the above concentrations was added to the formulation base to obtain the peptide formulation, 5-FU formulation and 5-FU formulation + peptide; and

[0126] 3) All the formulations were stored at 4°C throughout the duration of the study. Example 2: Peptide exhibits in vitro anti-tumorigenic activity for melanoma

[0127] Cell viability assay was performed using mouse B16F10 tumor cells treated with different concentrations of peptide at different time intervals using MTS reagent (Promega, USA). In brief, B16F10 tumor cells were treated with the peptide at different concentrations [from 0, 12.5 uM (0.00498%), 25 uM (0.00996%), 50 uM (0.0199%), 100 uM (0.039%) and 200 uM (0.079%) for 24 h, 48 h and 72 h). Treated cells showed growth inhibition from the concentration of 25 pM and further higher concentrations of peptide at all different time intervals (Figure 1). Significant reduction in the cell viability was observed in the cells treated with > 25 pM of the peptide.

[0128] The peptide and 5-Fluorouracil (5FU) combination treatment (Peptide+5FU) inhibited cell proliferation in B16F10 cells and the IC50 values were found to be nearly 60 pM and 20 pM respectively. Hence, the cells were treated with the same concentration independently and in combination to find the effect on the cell viability. Peptide treatment has induced apoptosis in the B16F10 melanoma cells which were incubated for 24h whereas 5FU stalled proliferation process without much inducing apoptosis (Figure 2). The combination treatment of peptide and 5FU has less viable cells than independent treatments. When the treatment continued for 48 h, the same effect can be observed in the cells (Figure 3). Cytotoxicity was also tested in normal cells (mouse embryonic fibroblasts) by treating them with combination of 60 pM peptide and 20 pM 5FU. It was observed that these normal mouse embryonic fibroblast cells were unaffected by this combination treatment (Figure 4). From Table 1 below showing IC50 Values for the cell lines treated with peptide, it is evident that the peptide inhibits cell proliferation in human melanoma cell lines like A375 and Sk-Mel-28 but do not affect other normal cell lines like HEK293T, HaCaT.

[0129] Table 1: IC50 Values for the cell lines treated with peptide

[0130] Example 3: Peptide affects B16F10 cell cycle progression

[0131] In order to assess if the peptide affects the cell cycle progression, peptide-treated B16F10 cells were fixed with ethanol, stained with PI and analyzed through flow cytometer. The arrow in the treatment category clearly indicates accumulation in the cell population in G2 / M phase. The increase in this phase clearly indicates that B16F10 cells were not entering into cytokinesis (Figure 5). Further, markers for cell proliferation and cytokinesis (Ki67 and Top2a) were quantified using Q-PCR and the results showed that mRNA levels of Ki67 and Top2a genes were significantly downregulated in the peptide-treated cells (Figure 6).

[0132] Example 4: Peptide could penetrate into B16F10 cells

[0133] Penetration studies were carried out after treating the B16F10 tumor cells with the peptide. Peptide (60 pM) was treated to cells for 16h. Further, the cells were fixed in formaldehyde and processed for staining using in house raised peptide specific antibody. Using secondary mouse FITC-tagged antibody, the peptide was observed to be localized inside the cells. Microscopic observations revealed that peptide enters into the cytoplasm and nucleus of the cell after the treatment (Figure 7).

[0134] Example 5: Peptide treatment does not cause skin irritation in animals

[0135] It is known that 5 -Fluorouracil is recommended as topical therapeutic agent for skin cancer. However, it is associated with side effects as mentioned in the above sections. Although 5- Fluorouracil and Imiquimod is able to show reduction in the growth of the xenograft tumor in animal models (Figure 11), they produce skin irritation. In another set of experiment, different concentration of the peptide [O.lmg / g or 0.01%, 0.5 mg / g or 0.05% & Img / g or 0.1%] was tested on xenograft tumor progression. The peptide alone showed a concentration dependent anti-tumor activity at all concentrations with 1 mg / g of the peptide showing the best effect. While the peptide showed anti-tumor activity even at 0.1 mg / g and 0.5 mg / g, none of the concentrations showed any skin irritation to the animals establishing the point that the peptide is safe and nontoxic bearing no side effects (Figures 10 & 12). Example 6: In vivo anti-tumorigenic activity of the peptide applied topically

[0136] In vivo anti-tumorigenic studies in case of melanoma cancer revealed that the peptide and combination treatment with 5FU (peptide+5FU) reduces tumor growth in syngeneic mouse model (C57BL / 6). Melanoma Tumors were induced by injecting 3 x 106cells under the right flank of the mice. When tumors reached 75mm , mice were topically applied with only in-house formulation base as per Example 1. Peptide was applied topically (1 mg / g) using in-house (IH) formulation base every day for 3 weeks for peptide-treated groups. 5FU was applied topically (100 mM in the in-house formulation base [5FU (IH)]) every day for 3 weeks. A combination treatment of peptide and 5FU in the IH formulation [100 mM] was also performed to see the effect of peptide as part of adjunct therapy [peptide+5FU (IH)]. As positive control, commercially available 5% 5FU cream [-380 mM] was applied to a group of mice. Melanoma Tumor volumes and weights were monitored every week for these mice which were significantly reduced in the mice which received topical application of peptide in formulation or combination treatment of peptide+5FU in formulation [peptide+5FU (IH)] in comparison to those mice that received either commercial 5FU [5FU Comm)] or 5FU in IH formulation base [5FU (IH)] (Figures 8A and B). Thus, topical application of the peptide in formulation or the peptide in combination with 5FU in formulation showed promising result in reducing melanoma tumor growth.

[0137] Example 6: In vivo anti-tumorigenic activity of the peptide applied intraperitoneally

[0138] Tumors were induced in C57BL6 mice using B16F10 melanoma cells. When tumor reached 75mm , mice were treated with either PBS [control] or 8mg / Kg of peptide every alternate days for three weeks. Animals were sacrificed after 3 weeks and tumors were collected. Tumor morphology was shown in all the mice (A) and tumor volumes were calculated (B) and weighed (c) and shown as Mean ± SEM of 3 mice per group. Measurement of tumor volume and weight in all tested animals revealed that the peptide administration reduces overall tumor burden in B16F10 melanoma model in mice [Figure 18] as compared to the control group [PBS].

[0139] Example 7: Differential mRNA expression of Ki-67 and TOP2A in tumor samples following therapeutic interventions

[0140] Tumors were induced in C57BL / 6 mice using B16F10 melanoma cells. When tumor reached nearly 75 mm , the mice were divided into 5 groups. All the 5 groups were divided into Untreated (topical application of only formulation without any drug or peptide treatment), 5-FU (mice treated only with 5-FU mixed with in-house formulation), 5-FU comm (commercially available 5-FU cream), Peptide mixed with in-house formulation, combination treatment of peptide and 5-FU (combination of 5-FU and peptide in in-house formulation mixture). The treatment was given thrice a week for 3 weeks. After three weeks, animals were sacrificed and tumors were collected to measure tumor volume and tumor weights. Tumors were harvested post-treatment from mice and preserved. Total RNA was extracted, followed by cDNA synthesis. Quantitative PCR (qPCR) was performed to assess mRNA levels of proliferation markers Ki-67 and T0P2A. Expression levels were normalized to 18S rRNA.

[0141] A significant reduction in Ki-67 and T0P2A mRNA expression was observed in tumors treated with the peptide, with the most pronounced decrease seen in the combination treatment group (5- FU + peptide). Data are presented as mean ± SEM [Figures 15-16].

[0142] Example 8: Immunohistochemical analysis for Ki-67

[0143] For immunohistochemical analysis of tumor sections of mice, tumors from mice subjected to different treatments like Control (Untreated), Peptide (Peptide mixed with the in-house (IH) prepared formulation base), 5-FU (5-flourouracil mixed with the in-house formulation), commercially (comm) available 5-FU cream and combination treatment of 5-FU and peptide with the in-house formulation were collected and sections were made. Tumor sections were next stained for Ki-67 using immunohistochemical analysis procedure. Immunohistochemical analysis for Ki67 (Figure 17) expression showed that the untreated (control) and commercial 5- FU treated tumor sections have more Ki67 staining than those of the peptide treated samples. In the combination treatment (5-FU and Peptide) tumor sections, Ki67 staining was significantly lowered than those of the other tumor sections [Figure 17].

[0144] Example 7: Morphological examination of Melanoma tumor tissues

[0145] In this experiment, tumor tissues from all the groups were fixed, sections were made and stained with hematoxylin and eosin stain. Sections were observed under microscope. In untreated group (Control), tumor sections showed poorly differentiated cells with maximum of irregularly shaped cells with different nuclear sizes. In contrast, tumor sections in mice treated with peptide in formulation [peptide] or peptide in combination with 5FU formulation [peptide +5FU (IH)] had well differentiated cells with prominent nucleus (Figure 9). In the groups treated with either 5FU (Comm) or 5FU (IH) in formulation, it was observed that infiltration of immune cells was higher in untreated in comparison to the peptide-treated groups. Together, both Figures 8 and 9 suggest that the peptide treatment alone or in combination treatment showed significant regression in tumor burden as compared to untreated or 5FU-treated mice group.

[0146] Example 8: Reduction in side effects by 5FU in animals

[0147] Comparison of anti-tumor activity of commercial 5FU vs 5FU combined with peptide were further carried out in different set of experiments. Formulations were applied to animals bearing xenograft tumors [animals injected with A375 melanoma cells] once a day every day for 25 days and tumor volumes were measured at the indicated time points. It was noted that the treatment with peptide alone [0.5 mg / g & 0.1 mg / g], combination of 5FU commercial (~380mM) + Peptide [0.5 mg / g & 0.1 mg / g] showed better efficacy with a reduction in side effects as compared to 5FU commercial alone (Figures 10, 13 and Figure 14).

[0148] Table 2 below shows the weight of excised tumors at the end of the study under each group. The values clearly indicate that the peptide alone or in combination treatment [5FU commercial + Peptide] showed reduction in the tumor weights, thus confirming evidences that the peptide alone or in combination with 5FU plays a potential role in anti-cancer therapy.

[0149] Table 2: Excised tumor weights after various treatments

[0150] Also, peptide when combined with 5FU is able to reduce its side effects as evident from Figure 13B and Table 3 below, while still being able to retain anti-cancer activity as compared to 5FU alone.

[0151] Table 3: Side effects after treatment

Claims

We Claim:

1. A composition comprising a peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent.

2. The composition as claimed in claim 1 , wherein the anti-cancer agent is selected from a group comprising 5 -Fluorouracil [5FU], Imiquimod or combinations thereof.

3. The composition as claimed in claim 1, wherein concentration of the peptide is in the range of about 0.001% - 2% and concentration of the anti-cancer agent is in the range of about 0.001% - 5%.

4. The composition as claimed in claim 1, wherein the composition optionally comprises excipients present in the range of about 93% - 99.998%.

5. The composition as claimed in claim 4, wherein the excipients are selected from a group comprising preservatives, buffering agents, stabilizers, binders, lubricants, compaction agents, disintegration agents, dispersion agents, coloring agents, additive, solvent, surfactant, anti-oxidant, suspending agent, emulsifying agent or combinations thereof.

6. The composition as claimed in claim 1 , wherein the composition is administered through different modes selected from a group comprising oral, intratumoral, intravenous, intraperitoneal, intramuscular, topical / cosmetic, sub-cutaneous or combinations thereof.

7. The composition as claimed in claim 1, wherein the composition is formulated into various forms selected from a group comprising tablet, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion in hard or soft gel capsules, syrups, elixirs, cream, paste, spray, lotion, powder, ointment or combinations thereof.

8. A composition comprising a peptide having at least 90% homology to a peptide of SEQ ID No. 1 at a concentration ranging from about 0.001% - 2%, at least one anti-cancer agent at a concentration ranging from about 0.001% - 5% and excipients at a concentration ranging from about 93 % - 99.998%.

9. A process of preparing the composition claimed in claim 1, said process comprising steps of combining a peptide having at least 90% homology to a peptide of SEQ ID No. 1 with at least one anti-cancer agent.

10. The process as claimed in claim 9, wherein the anti-cancer agent is selected from a group comprising 5-Fluorouracil [5FU], Imiquimod or combinations thereof.

11. The process as claimed in claim 9, wherein the peptide is synthesized before combining with anti-cancer agent.

12. The process as claimed in claim 9, wherein concentration of the peptide is in the range of about 0.001% - 2% and concentration of the anti-cancer agent is in the range of about 0.001% - 5%.

13. The process as claimed in claim 9, wherein the process optionally comprise inclusion of excipients in the composition.

14. A method of inhibiting cancer cells / tumor cells, said method comprising treating the cancer cells / tumor cells with a peptide having at least 90% homology to a peptide of SEQ ID No. 1 or an effective amount of a composition comprising the peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent.

15. A method of preventing or managing or treating cancer or a precancerous condition, said method comprising administering / applying to a subject in need thereof, an effective amount of a peptide having at least 90% homology to a peptide of SEQ ID No. 1 or an effective amount of a composition comprising the peptide having at least 90% homology to a peptide of SEQ ID No. 1 and at least one anti-cancer agent.

16. The method as claimed in claim 15, wherein the anti-cancer agent is selected from a group comprising 5 -Fluorouracil [5FU], Imiquimod or combinations thereof.

17. The method as claimed in claim 15, wherein the precancerous condition is selected from a group comprising actinic keratosis, epidermolysis bullosa, submucous fibrosis, lichen planus and discoid lupus erythematous.

18. The method as claimed in claim 15, wherein the cancer is selected from a group comprising skin cancer, breast cancer, colon cancer, liver cancer, esophageal cancer, gastric cancer, renal cancer, head and neck cancer, lung cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer and glioma.

19. The method as claimed in claim 18, wherein the skin cancer is selected from a group comprising basal cell carcinoma, squamous cell carcinoma, merkel cell carcinoma and melanoma.

20. A peptide having at least 90% homology to a peptide of SEQ ID No. 1 or a composition comprising the peptide having at least 90% homology to a peptide of SEQ ID No. 1 andat least one anti-cancer agent in manufacturing a medicament for preventing or managing or treating cancer / precancerous condition.

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