Peptide compositions for treating neoplastic lesions

Abstract

The present invention provides a pharmaceutical composition comprising the therapeutic peptide CyPep-1 for treating neoplastic lesions, particularly warts.

Description

Technical Field

[0001] This invention provides a pharmaceutical composition for treating warts or other neoplastic lesions, the pharmaceutical composition comprising, as an active ingredient, a therapeutic peptide based on the sequence of peptide CyPep-1, the therapeutic peptide having the amino acid sequence of SEQ ID NO:1. This invention also provides a method for treating warts or other neoplastic lesions using the composition of this invention. Background Technology

[0002] Warts are benign skin tumors (lesions) caused by human papillomavirus (HPV) infection. Common warts (verrucavulgaris) often appear on the skin, most commonly on the hands, as small, firm, rough growths. If present on the soles of the feet, warts are generally called plantar warts (verruca plantaris). Warts often grow on the genitals or anus; in this case, they are called anogenital warts (condylomata acuminata). Other less common types of warts include flat warts (verruca plana), which most commonly occur on the face or neck, hands, wrists, and knees; filiform warts, which often form on the face; and periungual warts, which appear in clusters around the fingernails or toenails.

[0003] It is generally believed that warts arise when damaged skin is exposed to HPV, which allows the virus to enter basal keratinocytes, the primary target of HPV infection of the skin. Both direct and indirect human-to-human transmission of warts occur through HPV shed from the wart (Lipke, Clinical Medicine and Research 4(4): 273-293, 2006). Different HPV serotypes are associated with different types of warts: for example, common warts and skin tags are most commonly caused by HPV types 27, 57, 2, and 1 (Bruggink et al., Journal of Clinical Virology 55(3): 250-255, 2012), while genital warts are most commonly caused by HPV types 6 and 11 (Lowy and Schiller, Journal of Clinical Investigation 116(5): 1167-1173, 2006).

[0004] Warts are very common, and it is believed that most people will develop them at some point in their lives. While warts may be considered harmless in some cases, most patients find them uncomfortable, and many also find their warts embarrassing, impacting their quality of life (Lipke, ibid.). However, effective treatments for warts are lacking—while several treatments exist (e.g., salicylic acid, cryotherapy, laser therapy, 5-fluorouracil, etc.), most lack evidence of their effectiveness (Sterling et al., British Journal of Dermatology 171:696-712, 2014). Without successful treatment, warts are generally self-limiting, but can take up to two years to heal completely. Even with seemingly successful treatment, recurrence is common. Therefore, new treatments for warts are needed. New treatments would also be beneficial for other neoplastic lesions.

[0005] WO 2011 / 092347 discloses an antitumor peptide, including CyPep-1, which consists of the amino acid sequence shown in SEQ ID NO:1. As shown in WO 2011 / 092347, CyPep-1 exhibits selective cytotoxicity against tumor cells, while co-pending application PCT / EP2019 / 066295 demonstrates that CyPep-1, when used in combination with checkpoint inhibitors, is highly effective in treating cancer. CyPep-1 has also been found to be particularly effective in treating warts.

[0006] CyPep-1 is a fusion peptide based on a fragment of the tumor suppressor protein Conductin / Axin2 coupled to the C-terminus of the HIV-TAT cell-penetrating peptide. The HIV-TAT cell-penetrating peptide is a cationic peptide, and unconstrained by theory, the selective cytotoxicity of CyPep-1 against tumor cells is thought to be due to the negative charge on the tumor cell membrane (conversely, the membranes of healthy mammalian cells tend to carry a more neutral charge). Advantageously, CyPep-1 promotes target cell lysis, leading to the release of cellular contents upon cell death. In the case of HPV-infected wart cells, this includes the release of HPV antigens. The immune system is exposed to the released HPV antigens, promoting a T-cell response that not only leads to the destruction of the wart but also results in durable immunity against HPV (or at least the infected HPV strain). Therefore, treating warts with CyPep-1 not only destroys existing warts but also prevents or reduces the likelihood of recurrence.

[0007] CyPep-1 also has antibacterial properties (possibly due to the negative charge on the cell membranes of many bacteria) and has been shown to have potent bactericidal activity against medically relevant species of Gram-positive and Gram-negative bacteria (see WO2011 / 092347). Summary of the Invention

[0008] This invention provides a CyPep-1 composition suitable for treating warts or even other tumors. This composition has been shown to have particularly advantageous properties regarding the solubility and stability of CyPep-1, and the slow release of CyPep-1 limits the frequency of CyPep-1 administration required for successful wart treatment. The use of this CyPep-1 formulation has been found to be effective in the treatment of warts.

[0009] Therefore, in a first aspect, the present invention provides an aqueous pharmaceutical composition comprising:

[0010] (i) 0.1-5% therapeutic oligopeptide, wherein the therapeutic oligopeptide contains the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity with it;

[0011] (ii) 0.1-5% preservative;

[0012] (iii) 0.1-5% organosilicon;

[0013] (iv) 10-20% di(ethylene glycol) ether;

[0014] (v) 0.005-0.1% butylated hydroxyanisole;

[0015] (vi) 1-10% liquid paraffin;

[0016] (vii) 1-10% soft paraffin;

[0017] (viii) 1-10% cetearyl alcohol; and

[0018] (ix) 0.1-5% cetearyl alcohol polyoxyethylene ether 20.

[0019] The compositions of the present invention are suitable for the treatment of warts, but may also be used for other purposes, as described below.

[0020] In another aspect, the present invention provides a composition as described above for treatment.

[0021] In another aspect, the present invention provides a composition as described above for treating tumors on the body surface (e.g., skin tumors). Specifically, the composition can be used for the treatment of warts. In another embodiment, it can be used for the treatment of skin cancer.

[0022] In a related aspect, the present invention provides a method for treating a tumor (e.g., warts) in a subject, comprising administering the composition described above.

[0023] In another related aspect, the present invention provides the use of the composition described above in the manufacture of a medicament for treating tumors (e.g., warts).

[0024] In the compositions shown above, the amount of each component is indicated as a percentage of that component within the compositions of the present invention. All percentage values ​​in the compositions shown above, and throughout this specification, are expressed as weight percentages (%w / w).

[0025] Therefore, the present invention provides a pharmaceutically acceptable composition for delivering therapeutic peptides. This may be particularly effective in the treatment of warts. That is, the compositions provided herein are suitable for treating warts. As used herein, “pharmaceutical” or “pharmaceutical acceptable” means that the composition is physiologically acceptable to the recipient to whom the composition is applied. The compositions of the present invention are creams suitable for topical application (i.e., application to the skin). Therefore, the compositions of the present invention are pharmaceutically acceptable creams. As creams, the compositions of the present invention are semi-solid. Pharmaceutically acceptable creams are non-toxic and preferably non-irritating, and provide a suitable carrier for delivering (discussed below) the active ingredient, particularly to the target wart. As pharmaceutical creams, the compositions of the present invention are emulsions comprising an aqueous phase and an oil phase. The compositions of the present invention are particularly likely to be oil-in-water emulsions. The compositions of the present invention are preferably sterile.

[0026] The compositions of the present invention are aqueous compositions. As mentioned herein, an aqueous composition is a composition containing or composed of water. In other words, the compositions of the present invention comprise the surfactants and various excipients described below, as well as water. Although the composition is aqueous, this does not mean that all components of the composition are soluble in water—on the contrary, the composition is an emulsion containing both an aqueous phase and an oil phase, as detailed above. However, as an aqueous composition, water generally forms the continuous phase of the emulsion, meaning that the emulsion is an oil-in-water emulsion as specified above. In the case where the compositions of the present invention are described herein, and the percentage content of each excipient does not (or may not) add up to 100%, the balance (i.e., the remainder) of the composition is water.

[0027] As mentioned above, the compositions of the present invention are suitable for treating tumors. "Tumor" is broadly defined to include any malignant, pre-malignant, or non-malignant tumor condition. Tumors may be characterized by uncontrolled or unwanted cell proliferation. Tumors can be tumors of the skin, including skin cancer or non-malignant skin tumors, or tumors occurring on the body surface or area where a cream can be applied. More specifically, the compositions are suitable for treating warts. The term "wart" as defined herein covers all types of warts, as further discussed below. Treatment of warts or other tumors using the compositions of the present invention is achieved through their active ingredient. The term "active ingredient" is used to refer to the component in the composition that has a therapeutic effect (i.e., anti-tumor or anti-wart effect). The active ingredient in the compositions of the present invention is a therapeutic oligopeptide. Upon application of the compositions of the present invention to warts or other tumors on a subject, the therapeutic oligopeptide comes into contact with the warts or other tumors and destroys them. Therefore, the therapeutic oligopeptides used in the present invention have anti-wart activity, or more generally, anti-tumor cell activity.

[0028] The therapeutic oligopeptide used in the compositions of the present invention comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:1. As detailed above, the therapeutic oligopeptide (CyPep-1) of SEQ ID NO:1 is disclosed in WO 2011 / 092347. SEQ ID NO:1 consists of a fragment of the tumor suppressor protein Conductin / Axin2 conjugated to the C-terminus of the HIV-TAT cell membrane-penetrating peptide. The aforementioned Conductin / Axin2 fragment has the amino acid sequence shown in SEQ ID NO:2 (corresponding to amino acids 13-27 of SEQ ID NO:1), while the HIV-TAT cell membrane-penetrating peptide has the amino acid sequence shown in SEQ ID NO:3 (corresponding to amino acids 1-12 of SEQ ID NO:1).

[0029] Oligopeptides are polymers formed by amino acids linked together by peptide bonds. As defined herein, an oligopeptide contains at least three amino acids, although therapeutic oligopeptides, as used herein, obviously contain more than three amino acids. As defined herein, an oligopeptide has no specific maximum length; for example, it can contain up to 30, 40, 50, or 100 amino acids or more, but the prefix "oligosaccharide" is typically used to indicate a relatively small number of amino acid subunits, i.e., less than 200, preferably less than 100, 90, 80, 70, 60, or 50 amino acids. The therapeutic oligopeptide used in the compositions of the present invention can therefore contain at least 23 and no more than 200 amino acids. In embodiments, it contains at least 24, 25, 26, or 27 amino acids. Alternatively, it can be defined as containing no more than 50, 45, 40, 35, 30, 29, 28, or 27 amino acids. Thus, the therapeutic oligopeptide can contain a plurality of amino acids within any integer range shown above for the minimum or maximum number of subunits. Therefore, the representative subunit ranges include 23-150, 23-100, 23-80, 23-50, 23-40, 23-30, 25-150, 25-100, 25-80, 25-50, 25-40, 25-30, 26-150, 26-100, 26-80, 26-50, 26-40, 26-30, 27-150, 27-100, 27-80, 27-50, 27-40, 27-30, 27-29, and 27-28.

[0030] The therapeutic oligopeptides used in the compositions of the present invention comprise the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity with it. In one particular embodiment, the therapeutic oligopeptide comprises the amino acid sequence shown in SEQ ID NO:1. In another embodiment, the therapeutic oligopeptide consists of the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity with it. In yet another embodiment, the therapeutic oligopeptide consists of the amino acid sequence shown in SEQ ID NO:1.

[0031] The level of sequence identity between two sequences (e.g., the target oligopeptide sequence and the sequence shown in SEQ ID NO:1) can be determined by sequence alignment. Sequence alignment can be performed using any suitable method, such as computer programs like the EMBOSS Needle or EMBOSS stretcher (both Rice P. et al., Trends Genet. 16(6): 276-277, 2000) for double sequence alignment, and Clustal Omega (Sievers F. et al., Mol. Syst. Biol. 7: 539, 2011) or MUSCLE (Edgar RC, Nucleic Acids Res. 32(5): 1792-1797, 2004) for multiple sequence alignment. Such a computer program can be used with standard input parameters, such as the standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1; or the standard EMBOSSNeedle parameters: matrix BLOSUM62, gap opening penalty 10, gap extension penalty 0.5. Alternatively, any other suitable parameters can be used. Sequence alignment must be performed globally, that is, across the entire length of the sequences being compared.

[0032] The therapeutic oligopeptides used in this invention may comprise only protein-derived amino acids (i.e., L-amino acids encoded by the standard genetic code). Alternatively, the therapeutic oligopeptides used in this invention may contain one or more non-protein-derived amino acids. For example, the therapeutic oligopeptides used in this invention may contain one or more D-amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 or more D-amino acids), human-engineered amino acids, or natural non-protein-derived amino acids, such as amino acids formed through metabolic processes. Examples of non-protein-derived amino acids that may be used include ornithine (a product of the urea cycle) and artificially modified amino acids, such as amino acids protected by 9H-fluorene-9-ylmethoxycarbonyl (Fmoc)-, tert-butoxycarbonyl (Boc)-, and 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), as well as amino acids having a carboxybenzyl (Z) group.

[0033] The in vitro and / or in vivo stability of therapeutic oligopeptides can be improved or enhanced by using stabilizing or protecting methods known in the art, such as adding protecting or stabilizing groups, incorporating amino acid derivatives or analogs, or chemically modifying amino acids. Such protecting or stabilizing groups can, for example, be added to the N-terminus and / or C-terminus. Examples of such groups are acetyl groups and other protecting groups or groups known in the art that can potentially stabilize peptides.

[0034] Peptides composed entirely of L-amino acids are referred to in the art as L-peptides, while peptides composed entirely of D-amino acids are referred to in the art as D-peptides. The term "reverse peptide" is used to refer to a peptide that has the same amino acid sequence as an L-peptide but is composed entirely of D-amino acids (i.e., a D-peptide with the same sequence as its corresponding L-peptide). Reverse peptides have a mirror structure to their corresponding L-peptides (i.e., L-peptides with the same amino acid sequence). Reverse peptides may be advantageous in a clinical setting (relative to L-peptides) because they are generally not easily degraded by serum proteases (due to their non-native conformation, reverse peptides may not be recognized by proteases). In a particular embodiment, the therapeutic oligopeptide used according to the invention is a reverse compound in which each amino acid is a D-amino acid. That is, the therapeutic oligopeptide may be a D-peptide. The oligopeptide compound may in particular comprise or consist of a D-peptide composed of or composed of the amino acid sequence shown in SEQ ID NO:1.

[0035] The therapeutic oligopeptides used in this article can be synthesized by a technician using standard biochemical techniques. If the oligopeptide is an L-peptide containing only protein-derived amino acids, it can be synthesized using recombinant DNA techniques. That is, the DNA sequence encoding the oligopeptide can be cloned and introduced into an expression vector. The DNA sequence encoding the therapeutic oligopeptide used in this article comprises or consists of a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 90%, or 95% sequence identity with it. Such a nucleotide sequence can be generated and synthesized effortlessly by a technician.

[0036] The DNA sequence encoding the therapeutic oligopeptide used herein can be amplified from a template using standard methods known in the art, such as by PCR generation or by artificial gene synthesis. The DNA sequence encoding the oligopeptide can then be introduced into an expression vector using standard molecular cloning techniques such as restriction endonucleases or Gibson assembly. Suitable expression vectors are known in the art. This expression vector can then be introduced into a cellular expression system using standard techniques. Suitable expression systems may include bacterial cells and / or eukaryotic cells, such as yeast cells, insect cells, or mammalian cells. Given that the therapeutic oligopeptides used herein may be toxic to bacterial cells (as discussed above), eukaryotic cells may be a more suitable cellular expression system for producing the oligopeptide compound.

[0037] Cell-free in vitro protein expression systems can be used instead of cell expression systems to synthesize the L-peptide compounds used in this paper. In such a system, the nucleotide sequence encoding the oligopeptide is transcribed into mRNA in vitro, and the mRNA is translated into protein. Cell-free expression system kits are widely available on the market and can be purchased from, for example, Thermo Fisher Scientific (USA).

[0038] The oligopeptides used in this invention can be optionally chemically synthesized in non-biological systems. Oligopeptides containing D-amino acids or other non-protein-derived amino acids are particularly suitable for chemical synthesis, as biosynthesis is generally not feasible in such cases. Liquid-phase or solid-phase protein synthesis can be used to generate polypeptides that can be formed within or contained in the oligopeptide compounds used in this invention. Such methods are well known to those skilled in the art, who can readily produce oligopeptides using appropriate methods common in the field.

[0039] As noted above, the therapeutic oligopeptides used in the compositions of this invention have anti-wart activity. This means that when the therapeutic oligopeptide is applied to warts on a subject, the warts heal over a period of time (e.g., at least 4 weeks). For example, the warts may shrink or fall off, and / or the skin that formed the warts may return to normal. To determine whether an oligopeptide has anti-wart activity, a composition containing the target oligopeptide can be applied to warts on several subjects, and the results can be compared with those obtained by subjects treated with a placebo (i.e., a composition or cream that is otherwise identical but lacks the target oligopeptide). If the results obtained using the composition containing the target oligopeptide are superior to those obtained using the placebo, the target oligopeptide can be considered to have anti-wart activity, thus constituting a therapeutic oligopeptide as defined herein.

[0040] The compositions of the present invention comprise from 0.1% to 5% of a therapeutic oligopeptide. In a specific embodiment, the composition comprises from 0.5% to 2%, 2.5%, or 3% of a therapeutic oligopeptide. In a preferred embodiment, the composition comprises 1% of a therapeutic oligopeptide. Most preferably, the composition comprises 1% CyPep-1, i.e., the composition comprises 1% of a therapeutic oligopeptide, and the therapeutic oligopeptide is a D-peptide consisting of the amino acid sequence shown in SEQ ID NO:1.

[0041] The compositions of the present invention further comprise a preservative. The preservative serves to maintain the sterility of the composition (i.e., it has antimicrobial activity). In particular, the preservative may have biocidal properties, enabling it to kill or inhibit the growth of bacteria and fungi such as yeast. Therefore, the preservative may have both antimicrobial and antifungal activity. A particularly preferred preservative is phenoxyethanol.

[0042] Phenoxyethanol:

[0043] Other suitable preservatives are known in the art and include parabens (including methylparaben, ethylparaben, propylparaben and butylparaben); sorbic acid (or 2,4-hexadienoic acid) and its salts, including sodium sorbate and potassium sorbate; and benzoic acid and its salts, including sodium benzoate and potassium benzoate.

[0044] The compositions of the present invention contain 0.1% to 5% of a preservative. In a specific embodiment, the composition contains 0.5% to 2%, 2.5%, or 3% of a preservative. In a preferred embodiment, the composition contains 1% of a preservative. Preferably, the compositions of the present invention contain 0.1% to 5% phenoxyethanol. In particular, the composition may contain 0.5% to 2%, 2.5%, or 3% phenoxyethanol. In a particularly preferred embodiment, the composition contains 1% phenoxyethanol.

[0045] The compositions of the present invention further comprise organosilicon (polysiloxane). Organosilicon serves to provide a pleasant texture to the composition. Generally, the organosilicon used in the compositions of the present invention is dimethyl silicone oil (dimethyl polysiloxane). Dimethyl silicone oils with different average polymer lengths are distinguished by their different kinematic viscosities (the longer the average polymer length, the higher the viscosity, and vice versa). In the case of dimethyl silicone oil, kinematic viscosity is measured using centistokes (cSt). The SI unit for kinematic viscosity is m³ / s. 2 / s. 1m 2 The kinematic viscosity of / s is equivalent to 10 6 The kinematic viscosity of cSt; conversely, the kinematic viscosity of 1 cSt is equivalent to 10 -6 m 2 Kinematic viscosity / s. Viscosity is measured at 25°C. Dimethicone with a wide viscosity range can be used as an excipient in pharmaceutical compositions. For example, a viscosity between 20 cSt and 1000 cSt may be suitable, depending on the concentration of dimethicone in the composition and the desired viscosity of the composition as a whole. Particularly preferred is that the composition contains dimethicone with a viscosity of 350 cSt at 25°C (commonly referred to as dimethicone 350 cSt).

[0046] The compositions of the present invention comprise from 0.1% to 5% organosilicon (preferably dimethicone). In a specific embodiment, the composition comprises from 0.5% to 2%, 2.5%, or 3% organosilicon (preferably dimethicone). In a preferred embodiment, the composition comprises 1% dimethicone. Preferably, the compositions of the present invention comprise from 0.1% to 5% dimethicone 350cSt. In particular, the composition may comprise from 0.5% to 2%, 2.5%, or 3% dimethicone 350cSt. In a particularly preferred embodiment, the composition comprises 1% dimethicone 350cSt.

[0047] The compositions of the present invention further comprise di(ethylene glycol) ethyl ether (DEGEE), commonly known by its trade name Transcutol-P.

[0048] DEGEE:

[0049] DEGEE exhibits activity as both a solvent and a penetration enhancer. As a "penetration enhancer," it refers to DEGEE that improves the absorption of the active ingredient through the skin when the compositions of the present invention are applied to a subject. The compositions of the present invention contain 10% to 20% DEGEE. In specific embodiments, the composition contains 10% to 18%, 10% to 16%, 12% to 20%, 12% to 18%, 12% to 16%, 14% to 20%, 14% to 18%, or 14% to 16% DEGEE, preferably 14% to 16%. Most preferably, the composition contains 15% DEGEE.

[0050] The compositions of the present invention further comprise butyl hydroxyanisole (BHA). BHA is a mixture of the isomers 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole.

[0051]

[0052] 2-tert-butyl-4-hydroxyanisole (left) and 3-tert-butyl-4-hydroxyanisole (right).

[0053] The BHA used in the compositions of the present invention may contain any proportion of 2-tert-butyl-isomer and 3-tert-butyl-isomer. In a specific embodiment, the BHA contains at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of 3-tert-butyl-4-hydroxyanisole.

[0054] BHA is an antioxidant because it can isolate free radicals in the composition within its aromatic ring. This prevents oxidation and degradation of other components of the composition, improving its stability. The compositions of the present invention contain from 0.005% to 0.1% BHA. In specific embodiments, the composition contains 0.01% to 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, or 0.05% BHA, preferably from 0.01% to 0.05%. In other embodiments, the composition contains 0.01% to 0.04% or 0.03% BHA. Most preferably, the composition contains 0.02% BHA.

[0055] The compositions of the present invention further comprise liquid paraffin (also known as mineral oil). Liquid paraffin acts as an emollient (or moisturizer), maintaining the hydration of the skin to which the composition is applied. The compositions of the present invention comprise from 1% to 10% liquid paraffin. In specific embodiments, the composition comprises 2% to 10%, 2% to 8%, 2% to 6%, 4% to 10%, 4% to 8%, or 4% to 6% liquid paraffin, preferably 4% to 6%. Most preferably, the composition comprises 4.8% liquid paraffin.

[0056] The compositions of the present invention further comprise molasses (also known as petrolatum or petrolatum). Both white and yellow molasses (white or yellow petrolatum) can be used in the compositions of the present invention. Preferably, white molasses (white petrolatum) is used (white molasses is petrolatum that has been highly refined to substantially decolorize it). Molasses (including white molasses) also act as a moisturizer.

[0057] The compositions of the present invention comprise from 1% to 10% pyrolytic wax (preferably white pyrolytic wax). In specific embodiments, the compositions comprise 1% to 9%, 1% to 7%, 3% to 10%, 3% to 9%, 3% to 7%, 5% to 10%, 5% to 9%, or 5% to 7% pyrolytic wax (preferably white pyrolytic wax). Preferably, the compositions comprise 5% to 7% pyrolytic wax (preferably white pyrolytic wax). Most preferably, the compositions comprise 6.3% pyrolytic wax, particularly 6.3% white pyrolytic wax.

[0058] The compositions of the present invention further comprise cetearyl alcohol. Cetearyl alcohol is a mixture of the solid alcohol cetyl alcohol (1-hexadecyl alcohol) and stearyl alcohol (1-octadecyl alcohol). Cetearyl alcohol acts as an emulsifier, thus stabilizing the emulsion of the compositions of the present invention and preventing phase separation.

[0059] The ratio of cetyl alcohol to stearyl alcohol in cetearyl alcohol is variable. Generally, the cetearyl alcohol used herein may contain 30-70% of both cetyl alcohol and stearyl alcohol. Cetearyl alcohol may not be a pure mixture of cetyl alcohol and stearyl alcohol, and may also contain small amounts of other alcohols, such as myristyl alcohol. Cetearyl alcohol may be a mixture of cetyl alcohol and stearyl alcohol in a ratio of (approximately) 70:30, 60:40, 50:50, 40:60, or 30:70. Most preferably, cetearyl alcohol is a 50:50 mixture of cetyl alcohol and stearyl alcohol. The 50:50 mixture of cetyl alcohol and stearyl alcohol in cetearyl alcohol is referred to herein as cetearyl alcohol 50:50.

[0060] The compositions of the present invention comprise from 1% to 10% cetearyl alcohol (preferably cetearyl alcohol 50:50). In specific embodiments, the compositions comprise 2% to 10%, 2% to 8%, 2% to 6.5%, 2% to 6%, 4% to 10%, 4% to 8%, 4% to 6.5%, 4% to 6%, 4.5% to 8%, 4.5% to 6.5%, 4.5% to 6% or 5% to 6% cetearyl alcohol (preferably cetearyl alcohol 50:50). In a preferred embodiment, the compositions comprise 4.5% to 6.5% cetearyl alcohol (preferably cetearyl alcohol 50:50). In another preferred embodiment, the compositions comprise 5% to 6% cetearyl alcohol (preferably cetearyl alcohol 50:50). Most preferably, the compositions comprise 5.5% cetearyl alcohol, particularly 5.5% cetearyl alcohol 50:50.

[0061] The compositions of the present invention further comprise cetearyl alcohol polyoxyethylene ether 20 (also known as cetearyl alcohol polyether-20 or polyethylene glycol cetearyl ether). Cetearyl alcohol polyoxyethylene ether 20 is a mixture of ethoxylated cetyl alcohol and stearyl alcohol, each having an average number of ethoxy groups between 17.2 and 25. Preferably, the ethoxylated cetyl alcohol and stearyl alcohol each have an average of 20 ethoxy groups. In a specific embodiment, both the ethoxylated cetyl alcohol and stearyl alcohol contain 20 ethoxy groups. Cetearyl alcohol polyoxyethylene ether 20 acts as both an emulsifier and a nonionic surfactant, promoting the formation and stability of the emulsion in the compositions of the present invention.

[0062] The compositions of the present invention comprise from 0.1% to 5% cetearyl alcohol polyoxyethylene ether 20. In specific embodiments, the composition comprises 0.1% to 4%, 0.1% to 3%, 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, 1% to 5%, 1% to 4%, 1% to 3%, 2% to 5%, 2% to 4%, or 2% to 3% cetearyl alcohol polyoxyethylene ether 20. In a preferred embodiment, the composition comprises 1% to 3% cetearyl alcohol polyoxyethylene ether 20. In another preferred embodiment, the composition comprises 2% to 3% cetearyl alcohol polyoxyethylene ether 20. Most preferably, the composition comprises 2.4% cetearyl alcohol polyoxyethylene ether 20.

[0063] The excipients used in the compositions of this invention are generally described in the 6th edition of the Handbook of Pharmaceutical Excipients (edited by Raymond Crowe, Paul J Sheskey, and Marian E Quinn, published by Pharmaceutical Publishers (UK) and the American Pharmacists Association, 2009). All excipients used are common in the art and are widely available from commercial suppliers. As noted above, the compositions of this invention are aqueous compositions; therefore, if the total amount of the active ingredient and excipients does not constitute 100% of the composition, the remainder may consist of water.

[0064] The water used in the compositions of this invention is sterile. Preferably, the water is also deionized. In a preferred embodiment, rinsing water is used. Rinsing water is well known in the art. The rinsing water is hypotonic, with an osmotic pressure of 0 Osm / L.

[0065] In a specific embodiment, the composition of the present invention comprises:

[0066] (i) 0.1-5% therapeutic oligopeptide, wherein the therapeutic oligopeptide contains the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity with it;

[0067] (ii) 0.1-5% preservative;

[0068] (iii) 0.1-5% organosilicon;

[0069] (iv) 10-20% di(ethylene glycol) ether;

[0070] (v) 0.005-0.1% butylated hydroxyanisole;

[0071] (vi) 1-10% liquid paraffin;

[0072] (vii) 1-10% soft paraffin;

[0073] (viii) 1-10% cetearyl alcohol; and

[0074] (ix) 0.1-5% cetearyl alcohol polyoxyethylene ether 20.

[0075] In another embodiment, the composition of the present invention comprises:

[0076] (i) 0.1-5% therapeutic oligopeptide, wherein the therapeutic oligopeptide consists of the amino acid sequence shown in SEQ ID NO:1, and the therapeutic oligopeptide is a reverse compound, wherein each amino acid is a D-amino acid;

[0077] (ii) 0.1-5% phenoxyethanol;

[0078] (iii) 0.1-5% dimethicone 350cSt;

[0079] (iv) 10-20% di(ethylene glycol) ether;

[0080] (v) 0.005-0.1% butylated hydroxyanisole;

[0081] (vi) 1-10% liquid paraffin;

[0082] (vii) 1-10% white soft paraffin;

[0083] (viii) 1-10% cetearyl alcohol 50:50; and

[0084] (ix) 0.1-5% cetearyl alcohol polyoxyethylene ether 20.

[0085] In another embodiment, the composition of the present invention comprises:

[0086] (i) 0.5-2% therapeutic oligopeptide, wherein the therapeutic oligopeptide contains the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity with it;

[0087] (ii) 0.5-2% preservative;

[0088] (iii) 0.5-2% organosilicon;

[0089] (iv) 14-16% di(ethylene glycol) ethyl ether;

[0090] (v) 0.01-0.05% butylated hydroxyanisole;

[0091] (vi) 4-6% liquid paraffin;

[0092] (vii) 5-7% soft paraffin;

[0093] (viii) 4.5-6.5% cetearyl alcohol; and

[0094] (ix) 1-3% cetearyl alcohol polyoxyethylene ether 20.

[0095] In another embodiment, the composition of the present invention comprises:

[0096] (i) 0.5-2% therapeutic oligopeptide, wherein the therapeutic oligopeptide consists of the amino acid sequence shown in SEQ ID NO:1, and the therapeutic oligopeptide is a reverse compound, wherein each amino acid is a D-amino acid;

[0097] (ii) 0.5-2% phenoxyethanol;

[0098] (iii) 0.5-2% dimethicone 350cSt;

[0099] (iv) 14-16% di(ethylene glycol) ethyl ether;

[0100] (v) 0.01-0.05% butylated hydroxyanisole;

[0101] (vi) 4-6% liquid paraffin;

[0102] (vii) 5-7% white soft paraffin;

[0103] (viii) 4.5-6.5% cetearyl alcohol 50:50; and

[0104] (ix) 1-3% cetearyl alcohol polyoxyethylene ether 20.

[0105] In another embodiment, the composition of the present invention comprises the following:

[0106] (i) 1% therapeutic oligopeptide, wherein the therapeutic oligopeptide contains the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85% sequence identity with it;

[0107] (ii) 1% preservative;

[0108] (iii) 1% organosilicon;

[0109] (iv) 15% di(ethylene glycol) ethyl ether;

[0110] (v) 0.02% butylated hydroxyanisole;

[0111] (vi) 4.8% liquid paraffin;

[0112] (vii) 6.3% soft paraffin;

[0113] (viii) 5.5% cetearyl alcohol;

[0114] (ix) 2.4% cetearyl alcohol polyoxyethylene ether 20; and

[0115] (x) 62.98% water.

[0116] In another embodiment, the composition of the present invention comprises the following:

[0117] (i) 1% therapeutic oligopeptide, wherein the therapeutic oligopeptide consists of the amino acid sequence shown in SEQ ID NO:1, and the therapeutic oligopeptide is a reverse compound, wherein each amino acid is a D-amino acid;

[0118] (ii) 1% phenoxyethanol;

[0119] (iii) 1% dimethicone oil 350cSt;

[0120] (iv) 15% di(ethylene glycol) ethyl ether;

[0121] (v) 0.02% butylated hydroxyanisole;

[0122] (vi) 4.8% liquid paraffin;

[0123] (vii) 6.3% white soft paraffin;

[0124] (viii) 5.5% cetearyl alcohol 50:50;

[0125] (ix) 2.4% cetearyl alcohol polyoxyethylene ether 20; and

[0126] (x) 62.98% water.

[0127] The compositions of the present invention, as creams, may be provided in tubes (e.g., made of plastic), barrels or cans (e.g., made of plastic, metal or glass), or any other suitable container.

[0128] The present invention further provides a composition as described above for treatment. Treatment refers to the administration of medicine to a subject suffering from a disease or physical condition. Generally, herein, treatment is used to refer to treatment applied to a subject in an attempt to cure a condition. The subject, as defined herein, refers to any mammal, such as farm animals like cattle, horses, sheep, pigs, or goats; pet animals like rabbits, cats, or dogs; or primates such as monkeys, chimpanzees, gorillas, or humans. Most preferably, and in most cases, the subject is a human.

[0129] The present invention also provides a composition as described above for treating tumors. In one embodiment, the present invention provides a composition as described above for treating skin tumors (i.e., skin neoplastic conditions). More specifically, the present invention provides a composition as described above for treating warts. Similarly, the present invention provides a method of treating a tumor in a subject, comprising administering the composition of the present invention to the subject (or more specifically, to the tumor). In one embodiment, the present invention provides a method of treating a skin tumor in a subject, comprising administering the composition of the present invention to the subject (or more specifically, to the tumor). In particular, the present invention provides a method of treating warts in a subject, comprising administering the composition of the present invention to the subject (or more specifically, to the wart). Similarly, the present invention provides the use of the compositions of the present invention in the manufacture of medicaments for treating tumors. In one embodiment, the present invention provides the use of the compositions of the present invention in the manufacture of medicaments for treating skin tumors. In particular, the present invention provides the use of the compositions of the present invention in the manufacture of medicaments for treating warts. That is, the compositions of the present invention are used to treat a subject suffering from a skin tumor (e.g., warts) (i.e., a subject requiring treatment of a tumor (e.g., warts)) to destroy or shrink the tumor (e.g., warts) and / or prevent the spread of the tumor (e.g., warts).

[0130] The compositions of this invention can be used to treat non-malignant skin conditions, including warts (as described above), actinic keratosis, seborrheic keratosis, and Bowen's disease (squamous cell carcinoma in situ). Actinic keratosis (also known as solar keratosis) is a non-malignant, rough patch of skin caused by sun exposure. Seborrheic keratosis is a waxy or wart-like growth of unknown cause. Normally, neither actinic keratosis nor seborrheic keratosis requires treatment. However, if the skin lesions caused by these conditions become irritating, treatment may be necessary. Actinic keratosis can develop into skin cancer, therefore providing treatment options for this condition is important for skin cancer prevention. Without treatment, Bowen's disease can transform into malignant squamous cell carcinoma. The compositions of this invention provide important new treatment options for these conditions.

[0131] The compositions of the present invention can also be used to treat malignant skin conditions (i.e., skin cancer). For example, the compositions of the present invention can be used to treat squamous cell carcinoma and basal cell carcinoma.

[0132] For all neoplastic skin conditions that can be treated with the compositions of the present invention, the compositions can be applied directly (locally) to the skin lesions.

[0133] As detailed above, the subjects treated with the compositions of the present invention can be any mammal, such as the farm animals or pet animals exemplified above, but preferably humans. The compositions of the present invention can be used to treat any human suffering from tumors (e.g., warts), including humans of any sex (male or female) and any age (adult or child).

[0134] As mentioned above, the term "wart" as used herein refers to any and all types of warts as described above. In a preferred embodiment, the composition is used to treat cutaneous warts. Cutaneous warts include common warts (verruca vulgaris), plantar warts (plantar warts, verruca plantaris), flat warts (verruca plana), intermediate warts (characterized by both common and flat warts), filiform warts, and periungual warts. Essentially, all warts except anogenital warts are defined herein as cutaneous warts. For example, the composition of the present invention can be used to treat common warts; the composition of the present invention can be used to treat plantar warts; the composition of the present invention can be used to treat flat or intermediate warts; the composition of the present invention can be used to treat filiform warts; and the composition of the present invention can be used to treat periungual warts. In another embodiment, the composition is used to treat anogenital warts.

[0135] This composition can be used to treat any number of warts on a subject in parallel. The composition can be used to treat only a single wart, but can also be optionally used to treat multiple warts simultaneously. For example, the composition of the present invention can be used to treat two, three, four, or five or more warts on a single subject simultaneously. Similarly, similar considerations can be applied to the treatment of other neoplastic lesions.

[0136] The compositions of the present invention are administered to a subject requiring treatment (e.g., for warts). A “therapeuticly effective amount” means an amount sufficient to demonstrate benefit to the subject’s condition. Whether an amount is sufficient to demonstrate benefit to the subject’s condition can be determined by the subject himself or by a physician or veterinarian. In particular, sufficient composition is applied to shrink or destroy the warts to be treated.

[0137] Generally, the invented composition is applied topically to the subject to be treated. The composition cream is applied directly to the surface of a tumor, such as a wart. The cream can be applied to the tumor (e.g., wart) by the subject himself or her, a doctor, or a third party (e.g., a relative of the subject, such as a parent). The cream can be easily applied directly from a container (e.g., from a tube) or by finger. Generally, the amount of the composition applied each time will be sufficient to cover the lesion or wart to be treated. After application of the cream, the lesion or wart can be covered with a patch or dressing (preferably a closed patch or dressing) to retain the cream at the site of the lesion or wart. In cases where the composition of the invention is used to treat multiple lesions or warts in parallel, the cream is applied separately to each lesion or wart to be treated.

[0138] The compositions of the present invention can be applied daily to the tumor (e.g., wart) to be treated, for example, once daily, twice daily, or three times daily. The compositions of the present invention can optionally be applied every other day or every three days, or weekly. In a preferred embodiment, the compositions of the present invention are applied to the tumor or wart to be treated once daily. In another preferred embodiment, the compositions are applied to the tumor or wart to be treated twice daily. The duration of treatment with the compositions of the present invention for a tumor or wart may be indeterminate, for example, until the tumor or wart is destroyed, that is, until treatment is successful. Alternatively, treatment with the compositions of the present invention for warts can be sustained for a defined duration, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer, or about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks or longer. The treatment can last for 1 month, 2 months, or 3 months or longer, or about 1 month, 2 months, or 3 months or longer. For example, treatment can last 2-8 weeks, such as 4-8 weeks or 6-8 weeks, 2-4 weeks or 2-6 weeks. In a preferred embodiment, treatment lasts approximately 4 weeks.

[0139] In a particularly preferred embodiment, treatment includes applying a cream to the warts to be treated once daily for 4 weeks or about 4 weeks. In other preferred embodiments, treatment includes applying a cream to the warts to be treated once daily for 2-8 weeks (e.g., 4-8 weeks), or applying a cream to the warts to be treated twice daily for 2-8 weeks (e.g., 4-8 weeks).

[0140] The invention can be further understood by referring to the following non-limiting embodiments. Attached Figure Description

[0141] Figure 1The results of a calibration experiment for determining CyPep-1 concentration by HPLC (using the method of Example 1) are shown. A shows a representative calibration curve for CyPep-1 standards prepared in deionized water in the concentration range of 2.5–500 μg / ml. The points shown in the figure are the average of three HPLC runs. B shows a chromatogram of a sample representing the CyPep-1 calibration standard (in this case, the CyPep-1 concentration is 100 μg / ml); and

[0142] Figure 2 The cumulative amount (μg / cm²) of CyPep-1 released per unit area from five test drug formulations through isoporous membranes into receptor solution (deionized water) between 1 and 7 hours (expressed as the square root of time) is shown. 2 The data are expressed as mean ± standard deviation (n = 6). Detailed Implementation

[0143] Example

[0144] Example 1 - Development of Analytical Methods

[0145] To measure the stability of CyPep-1 in test drug formulations, the following analytical method for the quantification of CyPep-1 by HPLC was developed. Various conditions (temperature, flow rate, etc.) were tested (data not shown), and the following procedure was found to be optimal.

[0146] Using Phenomenex (USA) Kinetex C18 5μm HPLC was performed using a 4.6×250 mm column and a Phenomenex SecurityGuard C18 5 μm guard column, as follows:

[0147]

[0148]

[0149] This HPLC procedure determined the limit of quantitation (LOQ) for CyPep-1 to be 2.5 μg / ml. Representative calibration curves for CyPep-1 standards and chromatograms of representative calibration standards obtained using the above HPLC method are shown in [the figures]. Figure 1 A and Figure 1 As shown in B.

[0150] Example 2 - Formulation Development

[0151] Preliminary experiments showed that CyPep-1 exhibits high solubility in deionized water (pH 4, pH 5, pH 6, or pH 7), ethanol, phenoxyethanol, and benzyl alcohol (the saturated solubility of CyPep-1 in each exceeds 10% w / w). The stability of CyPep-1 in these liquids was tested. Good recovery (at least 95%) of CyPep-1 was observed after 4 weeks of storage in deionized water (pH 4–7) at 40°C and 50°C, and in a mixture of ethanol and phenoxyethanol at 40°C.

[0152] Solubility Test - Method

[0153] The saturated solubility of CyPep-1 in various liquid excipients was evaluated as follows:

[0154] (i) Weigh CyPep-1 (approximately 20.0 mg) into a glass vial of suitable size.

[0155] (ii) Add each excipient (approximately 500 mg) to the respective glass vials from step (i).

[0156] (iii) Stir CyPep-1 and the excipient system in a pre-calibrated water bath at 25°C for 24 h (once saturation is observed).

[0157] (iv) During the 24 hours of stirring, visually inspect the solution every hour (if possible) to see if CyPep-1 has dissolved in the excipients.

[0158] (v) If CyPep-1 dissolution is observed, add approximately 20.0 mg increments of CyPep-1 to the vial until saturation is reached or until a total of 60 mg has been added at the point where solubility is reported as ≥10% w / w.

[0159] (vi) For the saturated system, remove any undissolved CyPep-1 from the saturated solution by centrifugation at approximately 16,000 x g for 10 minutes. Examine the sample of the saturated supernatant using an optical microscope with magnification of 200-1000x using a Leica (Germany) DME optical microscope. Place the saturated supernatant on a microscope slide and immediately examine for the presence of CyPep-1 crystals using both polarized and unpolarized light. Dilution (step (vii)) is performed immediately after the centrifugation step.

[0160] (vii) Since the solubility of CyPep-1 in these excipients is unknown, appropriate dilution should be performed before analysis to ensure that the concentration of CyPep-1 in the sample diluent (deionized water) is above the quantification limit for the HPLC method. For excipients saturated after adding approximately 20 mg of CyPep-1, the following dilutions should be performed:

[0161] (a) Weigh approximately 50 mg of the saturated solution into a 10 mL volumetric flask.

[0162] (b) Dilute the volumetric flask from step (a) to volume with sample diluent (deionized water) and vortex mix for 30 seconds.

[0163] (c) Transfer the aliquots of the diluted sample from step (b) to an HPLC vial and analyze them by HPLC as described above.

[0164] (viii) For excipients saturated after the addition of approximately 40 mg CyPep-1, dilute as detailed in step (vii), but using a 20 mL volumetric flask.

[0165] Stability Testing - Methods

[0166] The stability of CyPep-1 in liquid excipients was evaluated, and the evaluation showed that the saturated solubility of CyPep-1 is above 10% (w / w). The stability assessment of CyPep-1 in excipients is as follows:

[0167] (i) Weigh CyPep-1 (40.0±0.1mg) into a glass vial of suitable size.

[0168] (ii) Add each excipient (39.96 ± 0.2 g) to each suitable-sized glass vial from step (i).

[0169] (iii) Add the PTFE magnetic stirrer to the mixture from step (ii) and stir for about 16 hours.

[0170] (iv) Transfer the solution from step (iii) to a 3 mL borosilicate glass vial with a PTFE-lined cap. Prepare sufficient vials for testing at additional time points t = 0 and 2 at 25°C, 40°C, and 50°C; n = one aliquot prepared at each time point.

[0171] (v) A placebo solution (without CyPep-1) was also prepared and placed on stability according to step (iv).

[0172] (vi) After storage at 40°C and 50°C, the evaluation time points were t = 0 weeks, 2 weeks, and 4 weeks. The CyPep-1 content and %a / a analysis in the solution at each time point are as follows:

[0173] a) Weigh each CyPep-1 solution (500±10 mg) into three separate 10 mL volumetric flasks.

[0174] b) Dilute the volumetric flask to volume with sample diluent (deionized water), and after inverting it 5 times, mix thoroughly by vortexing for 30 seconds.

[0175] c) If the excipient is observed to be immiscible with the sample dilution (step (b)), stir the extract at ambient temperature for about 2 hours, and then centrifuge at about 16,000 x g for 10 minutes.

[0176] d) Transfer the aliquots of each sample (from step (b) or (c)) to an amber HPLC vial and analyze them using the HPLC described in Example 1.

[0177] e) For placebo solutions, follow steps (a) through (d), but prepare and analyze only one copy.

[0178] Based on these initial solubility and stability experiments, several CyPep-1 formulations were synthesized for further testing, as follows:

[0179] Cream 01:

[0180] Element Concentration (% w / w) CyPep-1 1 Deionized water 24 PEG 400 42 Transcutol-P 12 benzyl alcohol 1 Cetearyl alcohol 5 Crodamol GTCC 10 Dimethicone 1 Span 60 1.4 Twain 60 2.6

[0181] Cream 02:

[0182] Element Concentration (% w / w) CyPep-1 1 Deionized water 24 PEG 400 42 Transcutol-P 12 benzyl alcohol 1 Cetearyl alcohol 5 Crodamol GTCC 10 Dimethicone 1 Brij-S2 1.5 Brij-S20 2.5

[0183] Cream 03:

[0184] Element Concentration (% w / w) CyPep-1 1 Deionized water 24 PEG 400 42 Transcutol-P 12 benzyl alcohol 1 Cetearyl alcohol 5.5 Liquid paraffin 4.8 White paraffin 6.3 Dimethicone 1 Cetearyl alcohol polyether 20 2.4

[0185] Cream 04:

[0186]

[0187]

[0188] Cream 05:

[0189] Element Concentration (% w / w) CyPep-1 1 Deionized water 78 Phenoxyethanol 1 Liquid paraffin 11.17 Cetearyl alcohol 3.83 Dimethicone 1 Brij-S2 1.4 Brij-S20 2.6

[0190] Cream 06:

[0191] Element Concentration (% w / w) CyPep-1 1 Deionized water 78 Phenoxyethanol 1 Cetearyl alcohol 5 Crodamol GTCC 10 Dimethicone 1 Brij-S2 1.5 Brij-S20 2.5

[0192] Cream 07:

[0193] Element Concentration (% w / w) CyPep-1 1 Deionized water 77 benzyl alcohol 2 Cetearyl alcohol 5 Crodamol GTCC 10 Dimethicone 1 Brij-S2 1.5 Brij-S20 2.5

[0194] Cream 08:

[0195] Element Concentration (% w / w) CyPep-1 1 Deionized water 78 Phenoxyethanol 1 Cetearyl alcohol 5.5 Liquid paraffin 4.8 White paraffin 6.3 Cetearyl alcohol polyether 20 2.4 Dimethicone 1

[0196] Cream 09:

[0197] Element Concentration (% w / w) CyPep-1 1 Deionized water 62.98 Phenoxyethanol 1 Transcutol-P 15 Cetearyl alcohol 5.5 Liquid paraffin 4.8 White paraffin 6.3 BHA 0.02 Cetearyl alcohol polyether 20 2.4 Dimethicone 1

[0198] Cream 10:

[0199] Element Concentration (% w / w) CyPep-1 1 Deionized water 41.98 PEG 400 35 benzyl alcohol 2 BHA 0.02 Cetearyl alcohol 5.5 Liquid paraffin 4.8 White paraffin 6.3 Cetearyl alcohol polyether 20 2.4 Dimethicone 1

[0200] Ointment 1:

[0201] Element Concentration (% w / w) CyPep-1 1 PEG 400 58 Transcutol-P 15 benzyl alcohol 1 PEG 3350 25

[0202] Ointment 2:

[0203] Element Concentration (% w / w) CyPep-1 1 PEG 400 49 Transcutol-P 15 Deionized water 10 PEG 4000 25

[0204] Ointment 3:

[0205] Element Concentration (% w / w) CyPep-1 1 PEG 400 62.98 Phenoxyethanol 1 BHA 0.02 Deionized water 10 PEG 4000 25

[0206] Aqueous Gel 1:

[0207] Element Concentration (% w / w) CyPep-1 1 Deionized water 30 PEG 400 52 Transcutol-P 15 benzyl alcohol 1 Carbomer 974 1

[0208] Aqueous Gel 2:

[0209] Element Concentration (% w / w) CyPep-1 1 Deionized water 96 benzyl alcohol 2 Carbomer 974 1

[0210] Aqueous Gel 3:

[0211] Element Concentration (% w / w) CyPep-1 1 Deionized water 96 benzyl alcohol 2 Hydroxyethyl cellulose 1

[0212] Aqueous Gel 4:

[0213] Element Concentration (% w / w) CyPep-1 1 Deionized water 85.98 BHA 0.02 ethanol 10 Phenoxyethanol 1 Hydroxyethyl cellulose 2

[0214] Aqueous Gel 5:

[0215] Element Concentration (% w / w) CyPep-1 1 Deionized water 70.98 BHA 0.02 ethanol 10 Transcutol-P 15 Phenoxyethanol 1 Hydroxyethyl cellulose 2

[0216] Non-aqueous gel 1:

[0217] Element Concentration (% w / w) CyPep-1 1 PEG 400 68.1 ethanol 4.9 Transcutol-P 25 Hydroxypropyl cellulose 1

[0218] Example 3 - Formulation Testing

[0219] Ointment 1 and non-aqueous gel 1 were immediately discarded because CyPep-1 was shown to be insoluble in these formulations. The formulations were then centrifuged at 16 minutes. Formulations separated during that period were considered unstable and discarded. The following formulations were discarded because they were considered unstable upon separation during centrifugation: Cream 01 and Ointment 02. Aqueous gels 01 and 02 were discarded due to turbidity, indicating that the gelling agent (Carbomer 974) was not fully hydrated. This was determined to be due to the incompatibility of Carbomer 974 with CyPep-1. Creams 2 and 3, as well as aqueous gel 3, were also discarded. The stability of CyPep-1 in the various formulations was then investigated.

[0220] CyPep-1 Extraction Method

[0221] Before stability testing, a CyPep-1 extraction method was generated. Several methods were tested (data not shown). The optimal extraction method developed is as follows:

[0222] (i) Weigh 150 mg of the preparation into a 10 mL volumetric flask.

[0223] (ii) Dilute the flask to volume with a 50:50 v / v water:ethanol mixture.

[0224] (iii) Vortex the volumetric flask for 30 seconds to disperse the formulation, resulting in an extract containing CyPep-1 at a concentration of 150 μg / ml.

[0225] (iv) Add the PTFE magnetic stirrer to the volumetric flask from step (iii) and stir the contents of the volumetric flask at 500 rpm for 2 hours.

[0226] (v) Transfer the extraction solution from step (iv) to a centrifuge tube and centrifuge the sample at 16,000 x g for 10 min at 25 °C.

[0227] (vi) Filter the supernatant using a 0.2 μm PTFE filter.

[0228] The obtained filtered solution was used for HPLC analysis. This method was found to recover an estimated 96% of CyPep-1 from the formulation.

[0229] Short-term stability test

[0230] Ten of the above-mentioned non-discarded formulations were included in the stability test. Formulation samples were stored at 2–8°C, 25°C, and 40°C. Samples stored at 25°C and 40°C were evaluated after 2 weeks; samples stored at 2–8°C and 25°C were evaluated after 4 and 11 weeks.

[0231] At any point in time, no change was observed in the macroscopic (visual) or microscopic (400x magnification) appearance of any formulation.

[0232]

[0233] At t=0, CyPep-1 percentage recoveries were observed for all formulations prepared for short-term stability, ranging from 94.14% to 103.15% of the theoretical concentration. An exception was formulation aqueous gel 5, which showed a CyPep-1 recovery of 74.26% of the theoretical concentration at t=0. However, results at subsequent time points ranged between 96.68% and 106.26% of the theoretical CyPep-1 concentration, suggesting that the result obtained at t=0 was likely an anomaly.

[0234] After storage at 40°C for t=2 weeks, a decrease in CyPep-1 recovery was observed in all evaluated cream and ointment formulations (66.45%–78.11%). Based on these results, the remaining stability assessments were performed at 2–8°C and 25°C, as CyPep-1 is considered unstable at 40°C. No substantial changes in the percentage recovery of CyPep-1 were observed after storage at 25°C for t=2, 4, and 11 weeks, and after storage at 2–8°C for t=4 and 11 weeks (<10%), indicating that CyPep-1 is stable in these formulations for up to 11 weeks under the tested storage conditions. An exception was Ointment 3, which showed a decreasing trend in CyPep-1 recovery over time when stored at 25°C.

[0235] Based on the combination of these results and the aesthetics of each formulation, the following formulations were selected for further study:

[0236] Cream 05

[0237] Cream 09

[0238] Cream 10

[0239] ·Aqueous Gel 4

[0240] ·Aqueous gel 5.

[0241] The following formulations should be discontinued:

[0242] • Cream 04: This formulation exhibits relatively poor stability and is found to be aesthetically unappealing (grayish-white and greasy).

[0243] Cream 06: This formulation exhibits relatively poor stability.

[0244] Cream 07: This formulation exhibits relatively poor stability.

[0245] • Cream 08: Although this formulation exhibits equivalent stability to the selected formulation, it does not contain antioxidants. Oxidation has been identified as a potential pathway for CyPep-1 oxidation (data not shown), therefore this formulation has been discontinued.

[0246] • Cream 3: This formulation showed relatively poor stability and was found to be aesthetically unappealing (grayish-white and greasy).

[0247] Example 4 - In vitro drug release test

[0248] CyPep-1 exhibited minimal nonspecific binding to glass and plastic, meaning that no detergent was needed in the acceptor solution to prevent nonspecific binding. Therefore, deionized water was chosen as the acceptor solution for the study.

[0249] The back diffusion of the formulation to five synthetic membranes was then evaluated. The tested membranes were Supor, nitrocellulose, polycarbonate, cellulose, and nylon membranes. Only the cellulose membrane exhibited back diffusion after the formulation was applied with deionized water as the acceptor solution, which may alter the composition of the applied formulation. No back diffusion was observed with any of the other tested membranes.

[0250] All identical membranes were evaluated for CyPep-1 binding. The recoveries of CyPep-1 after incubation with the membranes are shown below (average recovery, n=3, compared to the control sample without membrane, incubation at 32°C):

[0251]

[0252]

[0253] Polycarbonate membranes were chosen for drug release studies because they showed the highest CyPep-1 recovery and no back diffusion was observed.

[0254] In vitro drug release assay - methods

[0255] This method uses individually calibrated static Franz cells, each with an average surface area and volume of approximately 2 cm². 2 And 10 mL. The static Franz cell was fitted with a polycarbonate membrane and filled with deionized water. The static Franz cell was then equilibrated to 32°C in a water bath, and an "unlimited dose" (300 mg / cm³) was administered after 30 minutes. 2 The test formulation was prepared in a Franz cell equipped with a polycarbonate membrane but without the formulation, serving as a blank.

[0256] At each time point (0, 1, 2, 3, 4, 5, 6, and 7 hours), an aliquot of 1 mL of receiver fluid (deionized water) was extracted from the Franz cell using a syringe via the sampling arm. Each 1 mL aliquot was replaced with 1 mL of preheated deionized water to maintain a constant volume and ensure no air bubbles were generated in the cell. Each aliquot was analyzed by HPLC using the method described above, and the cumulative amount of CyPep-1 released through the polycarbonate membrane was calculated. During the experiment, the Franz cell was shielded from light by sealing it with a paraffin membrane.

[0257] In vitro drug release experiment - results

[0258] The amount of CyPep-1 released into the receptor solution through the polycarbonate membrane during the 7-hour experiment for each formulation was as follows: Figure 2As shown in the figure, the test formulations aqueous gel 4 and aqueous gel 5 exhibited the highest CyPep-1 release levels. These formulations were observed to release CyPep-1 rapidly during the initial 1-hour experimental period, followed by minimal additional release. Compared to the gel formulations, the cream formulations cream 05, cream 09, and cream 10 initially released lower amounts of CyPep-1 at the 1-hour time point, but continued to release CyPep-1 between the 1-hour and 7-hour time points.

[0259] These experiments demonstrate that Cream 09 clearly possesses the most advantageous properties for use as a medicine. As shown in Example 3 above, CyPep-1 is very stable in this cream. The results shown in Example 4 indicate that CyPep-1 is released from this cream at a sustained high level. Sustained release of CyPep-1 is desirable because it means that after a single application of the formulation to the wart, CyPep-1 is continuously released onto the wart, implying a sustained therapeutic effect with each application of the cream. This, in turn, means that the cream requires a lower application frequency than formulations such as the tested aqueous gels, which release their CyPep-1 content essentially instantaneously, thus providing only a relatively short-lived therapeutic effect on the treated wart. Cream 09 readily releases the highest level of CyPep-1 among the tested creams (each cream exhibiting sustained release of the peptide), thus possessing the most advantageous properties among all tested formulations and potentially demonstrating the highest level of efficacy in wart treatment.

[0260] Example 5 - Manufacturing of Cream 09

[0261] Two containers, A and B, were used in the preparation process. Phenoxyethanol and Transcutol-P were added to container A, followed by BHA. The contents of the container were stirred until the BHA dissolved. Water was then added to container A, followed by CyPep-1. The mixture was stirred until a clear solution (free of particles or crystals) was obtained.

[0262] Liquid paraffin, soft white paraffin, cetearyl alcohol, and cetearyl alcohol polyoxyethylene ether 20 were added to container B. The contents of container B were heated to 65°C and melted. The contents of container A were also heated to 65°C. The contents of container B were then added to container A and homogenized at 10,000 rpm for several minutes. Dimethicone was added during homogenization. The formulation was then cooled to room temperature while continuously stirred.

[0263] Example 6 - Treatment of warts with cream 09

[0264] A clinical trial was conducted to test the effect of CyPep-1 on skin warts in the presence of cream 09. Patients were divided into two groups: one group received cream 09, and the other group received a placebo (placebo 09, equivalent to cream 09 except that CyPep-1 was replaced with water).

[0265] Apply the cream to the patient once daily, directly to the warts for 28 days. After application, cover the warts with a transparent film for 8-12 hours to prevent the cream from running off. Follow up with the patient weekly.

Claims

1. A cream comprising: (i) 0.5-2% w / w therapeutic oligopeptides, wherein the therapeutic oligopeptides consist of the amino acid sequence shown in SEQ ID NO:1; (ii) 0.5-2% w / w preservative; (iii) 0.5-2% w / w dimethicone; (iv) 14-16% w / w di(ethylene glycol) ether; (v) 0.01-0.05% w / w butylated hydroxyanisole; (vi) 4-6% w / w liquid paraffin; (vii) 5-7% w / w soft paraffin; (viii) 4.5-6.5% w / w cetearyl alcohol; and (ix) 1-3% w / w cetearyl alcohol polyoxyethylene ether 20.

2. The cream according to claim 1, wherein each amino acid of the therapeutic oligopeptide is a D-amino acid.

3. The cream according to claim 1, wherein the preservative is phenoxyethanol.

4. The cream according to claim 1, wherein the cream comprises 0.01-0.03% w / w butyl hydroxyanisole; 5-6% w / w cetearyl alcohol; and 2-3% w / w cetearyl alcohol polyoxyethylene ether 20.

5. The cream according to claim 4, wherein the dimethicone is a dimethicone with a viscosity of 350 cSt at 25°C.

6. The cream according to claim 1, wherein the cetearyl alcohol is a mixture of cetearyl alcohol and stearyl alcohol in a 50:50 ratio.

7. The cream according to claim 1, wherein the paraffin is white paraffin.

8. The cream according to claim 1, wherein the cream comprises: (i) 1% w / w therapeutic oligopeptide, wherein the therapeutic oligopeptide consists of the amino acid sequence shown in SEQ ID NO:1, and each amino acid thereon is a D-amino acid; (ii) 1% w / w phenoxyethanol; (iii) 1% w / w dimethicone oil with a viscosity of 350 cSt at 25°C; (iv) 15% w / w di(ethylene glycol) ether; (v) 0.02% w / w butylated hydroxyanisole; (vi) 4.8% w / w liquid paraffin; (vii) 6.3% w / w white soft paraffin; (viii) 5.5% w / w cetearyl alcohol, which is a mixture of cetearyl alcohol and stearyl alcohol in a 50:50 ratio; (ix) 2.4% w / w cetearyl alcohol polyoxyethylene ether 20; and (x) 62.98% w / w water.

9. Use of the cream as defined in any one of claims 1 to 8 in the manufacture of a medicament for treating warts.

10. The use according to claim 9, wherein the treatment comprises applying the cream topically to the wart.

11. The use according to claim 10, wherein the treatment comprises applying the cream to the wart daily for 4 weeks.

12. The use according to claim 9, wherein the wart is a skin wart.

Images