Polyglutamic acid compositions and methods of use thereof

Abstract

Disclosed herein are compositions comprising one or more polyglutamic acid compounds for treating, ameliorating, preventing or alleviating symptoms of vaginal dryness and symptoms associated with menopause. Suppositories of the compositions are also described.

Description

[Technical field]

[0001] Background technology Vaginal dryness is a common condition that can occur in women of all ages. However, vaginal dryness is most common in postmenopausal women. In postmenopausal women, vaginal dryness may be related to a decline in estrogen. Local estrogen therapy is the most common treatment for vaginal dryness, but this treatment has risks such as vaginal bleeding and increased risk of uterine cancer. For patients who do not want to consider or cannot undergo estrogen therapy, an alternative treatment is hyaluronic acid. However, hyaluronic acid has side effects such as vulvar candidiasis and bacterial vaginosis. Thus, conventional methods and compositions for treating vaginal dryness are inadequate, and this specification provides a solution that helps relieve vaginal dryness. Summary of the Invention

[0002] Aspects of this disclosure relate to novel compositions comprising one or more polyglutamic acid compounds and their use in treating and / or ameliorating symptoms of vaginal dryness and symptoms associated with menopause.

[0003] These and other features, aspects, and advantages of the present embodiments will become apparent with reference to the following description and claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] Detailed Description This specification describes compositions comprising one or more polyglutamic acid compounds and a pharma- ceutically acceptable carrier. Each polyglutamic acid compound can be prepared by reacting glutamic acid ester monomers with a suitable initiator. The molecular weight of each polyglutamic acid compound depends on the number of glutamic acid monomers attached, which corresponds to the n value shown below.

[0005] The structure of a polyglutamic acid compound is shown below:

[0006] [ka]

[0007] N refers to the chain length of the polyglutamic acid compound and can be from 300 to 30,000. The molecular weight of the polyglutamic acid compound of the present application can be from about 50 to 4,000 kDa. The compositions described herein can include one or more polyglutamic acid compounds with different polymer chain lengths (i.e., different n values).

[0008] For example, but not limited to, the composition may contain a first polyglutamic acid compound having an n value of about 300-500, a second polyglutamic acid compound having an n value of about 1,000-10,000, and a third polyglutamic acid compound having an n value of about 20,000-30,000.

[0009] The number of polyglutamic acid compounds with different n values ​​in the composition is not particularly limited. In some embodiments, the composition may contain 1, 2, 3, 4, 5, 6 or more polyglutamic acid compounds with different n values.

[0010] Without being bound by any particular theory, compositions comprising one or more polyglutamic acid compounds of the present disclosure produce unexpectedly superior results due to the ability of the compounds to penetrate various layers of vaginal epithelial tissues depending on the specific ratio of polyglutamic acid compounds with different n values. For example, the composition can comprise a first polyglutamic acid compound and a second polyglutamic acid compound, where the n value of the first polyglutamic acid compound is smaller than that of the second polyglutamic acid compound. It is believed that the polyglutamic acid compound with the smaller n value can penetrate deeper layers of the vaginal epithelium than the polyglutamic acid compound with the larger n value. It is also believed that the second polyglutamic acid compound with the larger n value than the first polyglutamic acid compound does not penetrate deeper epithelial layers, but rather the second polyglutamic acid compound remains closer to the surface than the first polyglutamic acid compound.

[0011] Particular embodiments contain a first polyglutamic acid compound, a second polyglutamic acid compound and a third polyglutamic acid compound. In particular embodiments, the first polyglutamic acid compound is a polyglutamic acid compound that contains more glutamic acid monomers than the second polyglutamic acid compound. The second polyglutamic acid compound contains more glutamic acid monomers than the third polyglutamic acid compound. In particular embodiments, the first polyglutamic acid compound is configured to penetrate the stratum corneum of the vaginal wall of the subject, the second polyglutamic acid compound is configured to penetrate the granular layer of the vaginal wall of the subject, and the third polyglutamic acid compound is configured to penetrate the spinous layer of the vaginal wall of the subject. In some embodiments, one or more of the polyglutamic acid compounds have a sufficiently large n value that they do not penetrate into the skin layer and remain on the skin surface and can act as a lubricant.

[0012] The amount of each polyglutamic acid compound in the composition described in this specification is not particularly limited, and may be about 10 μg to about 10 g. For example, the amount may be 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, 55 μg, 60 μg, 65 μg, 70 μg, 75 μg, 80 μg, 85 μg, 90 μg, 95 μg, 100 μg, 125 μg, 150 μg, 175 μg, 200 μg, 225 μg, 250 μg, 275 μg, 300 μg, 325 μg, 350 μg, 375 μg, 400 μg, 425 μg, 450 μg, 475 μg, 500 μg, 525 μg, 575 μg, , 600μg, 625μg, 650μg, 675μg, 700μg, 725μg, 750μg, 775μg, 800μg, 825μg, 850μg, 875μg, 900μg, 925μg, 950μg, 975μg, 1000μg, 5m g, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 125mg, 15 0mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 825mg, 850mg, 875mg, 900mg, 925mg, 950mg, 975mg, 1000mg, 1.25g, 1.5 g, 1.75 g, 2.0 g, 2.25 g, 2.5 g, 2.75 g, 3.0 g, 3.25 g, 3.5 g, 3.5 g, 3.75 g, 4.0 g, 4.25 g, 4.5 g, 4.75 g, 5.0 g, 5.25 g, 5.5 g, 5.75 g, 6.0 g, 6.25 g, 6.5 g, 6.75 g, 7.0 g, 7.25 g, 7.5 g, 7.75 g, 8.0 g, 8.25 g, 8.5 g, 8.75 g, 9.0 g, 8.25 g, 9.5 g, 9.75 g, 10 g, or any amount in between.

[0013] The compositions disclosed herein can be used to treat, prevent, or ameliorate vaginal dryness, vaginal itching, and / or vaginal burning. In some embodiments, the compositions described herein can be used to maintain healthy levels of vaginal moisture. In some embodiments, the compositions described herein can be used to speed wound healing, prevent urinary tract infections, and / or maintain the pH of vaginal tissue. In certain embodiments, the compositions described herein are administered to a subject to avoid painful intercourse. In certain embodiments, the compositions described herein are administered to a subject to rejuvenate vaginal tissue.

[0014] In certain embodiments, the composition can be administered to the vaginal tissue of a subject to treat, prevent, ameliorate, and / or reduce symptoms of vaginal dryness. In certain embodiments, the composition can be administered to the vaginal tissue of a subject to maintain a healthy level of vaginal moisture. In certain embodiments, the composition can be administered to the vaginal tissue of a subject to treat, prevent, and / or ameliorate symptoms of vaginal dryness due to menopause. In some embodiments, the composition can be administered to the vaginal tissue of a subject to treat, prevent, and / or ameliorate one or more symptoms of menopause. In certain embodiments, the composition can be administered to the vaginal tissue of a subject to reduce pain associated with sexual activity, including intercourse. In certain embodiments, the composition can be administered to the vaginal tissue of a subject to treat, prevent, and / or ameliorate vaginal itching. In certain embodiments, the composition can be administered to the vaginal tissue of a subject to treat, prevent, and / or ameliorate vaginal burning. In certain embodiments, the composition can be used for one or more of the purposes previously described.

[0015] In some embodiments, the compositions disclosed herein are in the form of pharma- ceutically effective salts. The term "pharmaceutically acceptable salts" is art-recognized and is used herein to include, but is not limited to, salts of acidic or basic groups that may be present in the compositions disclosed herein. Essentially basic compounds can form a wide variety of salts with various inorganic and organic acids. Acids that can be used to prepare pharma- ceutically acceptable acid addition salts of such basic compounds include non-toxic acid addition salts, i.e., sulfuric acid, citric acid, maleic acid, acetic acid, oxalic acid, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, hydrogen sulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, hydrogen tartrate, ascorbate. The acids that form salts with pharmacologically acceptable anions include, but are not limited to, succinate, maleate, gentisate, fumarate, gluconate, glycerate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylenebis-(2-hydroxy-3-naphthoic acid)) salts. Compounds containing an amino moiety in the compositions disclosed herein can also form pharma-ceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds that are acidic in nature in the compositions disclosed herein can form salts with various pharma-ceutically acceptable cations. Non-limiting examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, silicon, phosphorus, and iron salts.

[0016] The term "pharmaceutically acceptable carrier" is art-recognized and includes any pharma- ceutically acceptable material, composition, or vehicle suitable for administration of the compounds of the present invention to a mammal. Carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that are involved in the transport or transfer of the agent of interest from one organ or part of the body to another.

[0017] Each carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the formulation and not harmful to the patient. Examples of substances that can be used as pharmaceutically acceptable carriers include sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; fats and oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, glyceride, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer; and other non-toxic compatible substances used in pharmaceutical formulations. In one embodiment, the pharma- ceutically acceptable carrier is suitable for intravenous administration, hi another embodiment, the pharma- ceutically acceptable carrier is suitable for local injection.

[0018] The term "pharmaceutical acceptable esters" refers to the relatively non-toxic esterification products of the compounds of the invention. These esters can be prepared in situ during the final isolation and purification of the compounds, or by reacting the purified compounds in the form of the free acid or with a hydroxyl group with a suitable esterifying agent. Carboxylic acids can be converted to esters by treatment with an alcohol in the presence of a catalyst. Hydroxyl groups can be converted to esters by treatment with an esterifying agent such as an alkanoyl halide. The term also includes lower hydrocarbon groups that can be solvated under physiological conditions, e.g., alkyl esters, methyl esters, ethyl esters, and propyl esters.

[0019] The formulation of the composition described herein is not particularly limited. In certain embodiments, the composition is formulated as a topical preparation, such as a cream or ointment. In certain embodiments, the composition is formulated as a gel. In certain embodiments, the composition is formulated as a suppository.

[0020] In certain embodiments, the suppository comprises one or two polyglutamic acid compounds, a solubilizer, a humectant, a surfactant, and / or one or more suppository bases. The suppository may also comprise a softener. An example of a solubilizer is water. An example of a humectant is glycerin. An example of a surfactant is polysorbate 80. Examples of suppository bases are PEG4000, Gelucire 43 / 01, PEG3350, witepsol® W-35, and saturated fatty acid glyceride C. 12 -C 18An example of the softener is softisan 378. The molecular weight of one or two polyglutamic acid compounds is 50 kDa to 2000 kDa. For example, the molecular weight of the polyglutamic acid compound is 50 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1000 kDa, 105 The weight percent (% w / w) of each polyglutamic acid compound in the compositions described herein is not particularly limited and may be from about 0.01% to about 1%.For example, it is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%, 0.21%, 0.22%, 0.23%, 0.24%, 0.25%, 0.26%, 0.27%, 0.28%, 0.29%, 0.30%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.36%, 0.37%, 0.38%, 0.39%, 0.40%, 0.41%, 0.42%, 0.43%, 0.44%, 0.45%, 0.46%, 0.47%, 0.48%, 0.49%, 0.50%, 0.51%, 0. 52%, 0.53%, 0.54%, 0.55%, 0.56%, 0.57%, 0.58%, 0.59%, 0.60%, 0.61%, 0.62%, 0.63%, 0.64%, 0.65%, 0.66%, 0.67%, 0.68%, 0.69%, 0.70%, 0.71%, 0.72%, 0.73%, 0.74%, 0.75%, 0.76%, 0.77%, 0.78% %, 0.79%, 0.80%, 0.81%, 0.82%, 0.83%, 0.84%, 0.85%, 0.86%, 0.87%, 0.88%, 0.89%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, 1% or more, or any value therebetween. The weight percentage (%w / w) of the solubilizer, wetting agent, surfactant, one or more suppository bases, and softener in the suppository is also not particularly limited and may be 1% to 100%, about 0.1% to 11.4%, 0.1% to 1.4%, 0% to 100%, and 0% to 100%, respectively. The amounts of polyglutamic acid compound, solubilizer, wetting agent, surfactant, suppository base and / or softener are in a synergistic ratio. In this specification, "synergistic ratio" refers to a ratio that causes unexpectedly superior pharmacological and physiological effects in a subject. An example of a synergistic ratio of the amount of polyglutamic acid compound, the amount of solubilizer, the amount of wetting agent, the amount of surfactant and the amount of suppository base is 5:396:50:28:1521.

[0021] In certain embodiments, the suppository contains only one polyglutamic acid compound. In certain embodiments, the molecular weight of the compound is 100 kDa, 700 kDa, or 2000 kDa. In certain embodiments, the suppository contains a solubilizer, a wetting agent, a surfactant, and a suppository base. In certain embodiments, the solubilizer is water, the wetting agent is glycerin, the surfactant is polysorbate 80, and the suppository base is saturated fatty acid glyceride C. 12 -C 18 It is.

[0022] In certain embodiments, the suppository comprises two polyglutamic acid compounds, hi certain embodiments, the molecular weights of the compounds are 100 kDa and 700 kDa.

[0023] In certain embodiments, the suppository disintegrates in 5-10 minutes, 10-20 minutes, or 20-30 minutes. In certain embodiments, the suppository can be administered every 2 days, 3 days, 4 days, 5 days, 6 days, weekly, 8 days, 9 days, 10 days, biweekly, monthly, or more to provide rapid and long-lasting relief from symptoms of vaginal dryness and menopause-related symptoms.

[0024] Although the invention has been described in some detail for purposes of clarity and understanding, it will be understood that various changes in form and detail may be made without departing from the true scope of the invention. EXAMPLES

[0025] Example 1 As an example, polyglutamic acid (molecular weight: 100 kDa; 5 mg; 0.25% w / w) was mixed with water (396 mg; 19.8% w / w), glycerin (50 mg; 2.5% w / w), polysorbate 80 (28 mg; 1.4% w / w), and saturated fatty acid glyceride C 12 -C 18(1521 mg; 76.04% w / w) to obtain a white suppository. Dissolution experiments of the suppositories were carried out using phosphate buffer with a pH of about 4.2-4.5, which is within the range of normal vaginal pH (3.8-5.0). The percentage of polyglutamic acid in the buffer was measured after 0.5, 0.75, 1, 2, 3, 6, 24, 30 and 48 hours. The dissolution data are shown in Table 1.

[0026] [Table 1]

[0027] The suppository was observed to completely disintegrate within 30 minutes. The data shows that 67% of the polyglutamic acid compound was dissolved at 30 minutes and completely dissolved in the buffer at 3 hours. Unexpectedly, stable dissolution was observed from 0.5 to 3 hours. Even more unexpectedly, 100% of the polyglutamic acid compound remained in the buffer after 48 hours, indicating that the compound is stable in vaginal tissue and can moisturize vaginal tissue for more than 2 days after administration. These unexpected results indicate that the composition can provide rapid and long-term relief of symptoms of vaginal dryness.

[0028] Example 2 A study is conducted to evaluate the short-term stability of the vaginal suppository composition described in Example 1 compared to a placebo prototype composition. The placebo composition is identical to the vaginal suppository composition except that it does not contain a polyglutamic acid compound. The two compositions are subjected to a 3-month ICH stability study at 2-8°C and 25°C / 60%RH. At the end of the first and second month, the stability of the vaginal suppository composition is evaluated by appearance and the amount of polyglutamic acid compound remaining in the composition. At the end of the third month, the stability of the vaginal suppository composition is evaluated by appearance and the amount of polyglutamic acid compound dissolved in addition to the amount of polyglutamic acid remaining in the composition. For the placebo composition, stability is evaluated by appearance and placebo interference at the end of each month.

[0029] Example 3 An in vitro vaginal tissue test device is used to perform an assay to determine the activity and efficacy of polyglutamic acid compounds of various polymer lengths. Various (i.e., different n values) polyglutamic acid compounds are applied to the device, and their ability to penetrate various layers of vaginal wall tissue is correlated with the n value (i.e., the number of monomers in the polyglutamic acid compound) and their relative effect on vaginal moisture. Additionally, compositions having various concentrations of polyglutamic acid compounds with different polymer chain lengths (i.e., different n values) are tested to determine their effect on vaginal moisture and penetration into vaginal tissue layers.

[0030] Example 4 The effect of certain compositions described herein on the vaginal epithelium in a postmenopausal rat model is examined.

[0031] Thirty female Wistar / Sprague rats (8 weeks old) are purchased from the Firat University Laboratory Animal Center. All rats are housed individually in a clean environment with controlled temperature, humidity and light (12-h light / dark cycle) and are provided with standard commercial food and water ad libitum. All experimental procedures follow the guidelines for the use of laboratory animals and are approved by the Animal Care and Use Committee of Firat University.

[0032] Rats that showed a regular 4-day cycle are selected for the experiment. The rats are divided into 7 groups as shown in Table 2. After one acclimatization period, the rats are ovariectomized (OVX) or sham-operated (control) under anesthesia. The same weight of adipose tissue is removed from the rats in the control group. After surgery, vaginal smears are evaluated daily. Only rats whose vaginal smears showed a regular 4-day cycle (before and after sham / OVX) and whose white blood cells remained constant (after OVX) are used (10 rats per group). The remaining rats are administered placebo (vehicle) gel intravaginally or orally in the same amount as the rats administered the composition. The rats are weighed weekly.

[0033] [Table 2]

[0034] On day 15, the therapeutic effect of each composition on vaginal atrophy is determined by histological examination of vaginal tissue in vaginal smears obtained from each rat. The vaginal smears are fixed and explored to determine the effect of each composition on the vaginal microecosystem by expression of leukocyte esterase, β-glucuronidase, and coagulase. All rats are then sacrificed by cervical dislocation and the vagina and uterus are removed. After careful removal of adhering tissue, the wet weight of the uterus is measured. To minimize variability due to the incision, the same length of incision is made above the cervix for all uteruses. The vaginal tissue is then removed, cut into 1 mm x 1 mm x 2 mm sections, and stained with 2.5% glutaraldehyde. Some of the vaginal samples are taken and processed by standard procedures for histological examination (4-5 μm sections stained with hematoxylin + eosin).

[0035] The repair ability of each composition is detected by Western blot. Vaginal VEGF, p-AKT, COX2, ERα and ERβ are detected by Western blot. In Western blot, proteins are separated in 10% polyacrylamide gel, electrotransferred to polyvinylidene difluoride membrane, bands are blocked with 5% nonfat dry milk for 1 hour at room temperature and incubated with various primary antibodies (1:1000). Incubation is performed in PBS supplemented with 5% nonfat dry milk. After washing with Tris-buffered saline containing 0.1% Tween® 20 (TBS-T), the membrane is incubated with anti-rabbit IgG antibody conjugated with horseradish peroxidase for another hour at room temperature. After washing with TBS-T, antigen-antibody complexes are visualized using an enhanced chemiluminescence detection kit.

Claims

1. A composition for treating vaginal dryness comprising one or more polyglutamic acid compounds and a pharma- ceutically acceptable carrier.

2. The composition of claim 1 , wherein the one or more polyglutamic acid compounds comprise a first polyglutamic acid compound and a second polyglutamic acid compound.

3. The composition of claim 2 further comprising a third polyglutamic acid compound.

4. 3. The composition of claim 2, wherein the first polyglutamic acid compound has a smaller n value than the second polyglutamic acid compound, the n value being the number of glutamic acid monomers that combine to form each polyglutamic acid compound.

5. 4. The composition of claim 3, wherein the first polyglutamic acid compound has a smaller n value than the second polyglutamic acid compound and the third polyglutamic acid compound has a larger n value than the second polyglutamic acid compound, the n value being the number of glutamic acid monomers that combine to form each polyglutamic acid compound.

6. The composition of claim 1 , wherein the composition is a suppository.

7. The composition of claim 1 , wherein the composition is a cream.

8. The composition of claim 6, wherein the suppository comprises one or two polyglutamic acid compounds.

9. The composition of claim 8, further comprising a solubilizing agent, a wetting agent, a surfactant, and one or more suppository bases.

10. 10. The composition of claim 9, wherein the amounts of said polyglutamic acid compound, said solubilizing agent, said wetting agent, said surfactant and said suppository base are in a synergistic ratio.

11. The composition of claim 8, wherein the composition comprises one polyglutamic acid compound.

12. The composition of claim 11, wherein the molecular weight of the polyglutamic acid compound is 100 kDa.

13. The composition of claim 12, further comprising a solubilizing agent, a wetting agent, a surfactant, and a suppository base.

14. The solubilizing agent is water, the wetting agent is glycerin, the surfactant is polysorbate 80, and the suppository base is saturated fatty acid glyceride C １２ -C １８ The composition of claim 13, wherein

15. The composition of claim 4, wherein the first polyglutamic acid compound has a molecular weight of 100 kDa and the second polyglutamic acid compound has a molecular weight of 700 kDa.

16. The composition of claim 15, wherein the composition is a suppository.

17. 17. The composition of claim 16, further comprising a solubilizing agent, a wetting agent, a surfactant, and a suppository base.

18. The solubilizing agent is water, the wetting agent is glycerin, the surfactant is polysorbate 80, and the suppository base is saturated fatty acid glyceride C １２ -C １８ 18. The composition of claim 17, wherein

19. 13. A method for treating vaginal dryness, comprising administering the composition of claim 1 to the vaginal cavity of a subject.

20. 20. The method of claim 19, wherein the composition is a suppository.

21. 21. The method of claim 20, wherein the suppository contains only one polyglutamic acid compound.

22. 21. The method of claim 20, wherein the suppository comprises a first polyglutamic acid compound and a second polyglutamic acid compound.

23. 13. A method for maintaining healthy levels of vaginal moisture, comprising administering the composition of claim 1 to the vaginal tissue of a subject.

24. 13. A method for treating, preventing and / or ameliorating one or more symptoms of menopause, comprising administering the composition of claim 1 to the vaginal tissue of a subject.

25. 25. The method of claim 24, wherein the symptom is vaginal dryness, vaginal itching, vaginal burning and / or pain with sexual activity.

26. 26. The method of claim 25, wherein the pain is due to sexual intercourse.

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