An antipruritic gel containing a 3-hydroxydecanoic acid microemulsion and a method of making the same

Abstract

This invention discloses an antipruritic gel containing 3-hydroxydecanoic acid microemulsion and its preparation method. The gel combines the natural antipruritic ingredient 3-hydroxydecanoic acid and peppermint oil. By preparing it into a microemulsion form, nano-sized droplets are combined with skin absorption enhancers and gel sustained-release agents, thereby improving transdermal absorption efficiency and prolonging the residence time in the skin to achieve a long-lasting antipruritic effect. 3-hydroxydecanoic acid exerts its antipruritic effect through anti-inflammatory action, blocking pruritus signaling pathways, and reducing mast cell degranulation. This antipruritic gel is stable at room temperature, easy to use, and suitable for relieving skin itching caused by mosquito bites, histamine, or non-histamine substances.

Description

Technical Field

[0001] This invention belongs to the technical field of antipruritic drugs and their preparations, specifically relating to an antipruritic gel containing 3-hydroxydecanoic acid microemulsion and its preparation method. Background Technology

[0002] As the body's natural barrier, the skin effectively prevents exogenous substances from entering the body, protecting internal organs and blood vessels from damage. However, the skin barrier function also presents challenges for transdermal drug delivery, making it difficult for topical medications to effectively penetrate the skin and exert their effects. Because the skin is composed of multiple cell layers, smaller compounds with a certain degree of lipophilicity are more likely to cross the skin barrier. Currently, various methods exist to promote transdermal absorption, such as adding penetration enhancers, preparing drugs as nanoparticles or emulsions, and using physical methods such as ultrasound or microneedles. Microemulsions, with particle sizes less than 20 nanometers, are transparent, have good thermal and cold stability, and can be stored for long periods without delamination, thus being considered an ideal transdermal drug delivery method. However, establishing methodologies for preparing microemulsions is complex, requiring experimental screening of suitable surfactants, co-surfactants, and their ratios.

[0003] Itching is an uncomfortable sensation that triggers the urge to scratch, and chronic itching is often accompanied by dermatitis or systemic diseases, severely impacting quality of life. The occurrence of itching is closely related to various cells and factors, such as keratinocytes, nerve cells, immune cells, as well as interleukins and chemokines (Du et al., Neuroscience 495, 2022:74-85). Currently, purely natural antipruritic ingredients used for mosquito bites, such as comfrey, menthol, eugenol, and camphor oil, have very limited antipruritic effects. Many antipruritic products for mosquito bites still require the addition of chemically synthesized antipruritic components such as dexamethasone and methyl salicylate. Clinically commonly used oral antipruritic drugs include antihistamines (such as loratadine and diphenhydramine), while topical medications are mostly corticosteroids (such as dexamethasone and hydrocortisone). Long-term use of these can lead to serious side effects and is ineffective against nonhistamine-mediated itching.

[0004] 3-Hydroxydecanoic acid (HDC) is a natural compound widely found in bacterial glycolipids and polyhydroxyalkanoates (PHAs) (Applied Microbiology and Biotechnology, 2000, 53:167-172), making it abundant and safe. While there are no reports on the antipruritic effects of HDC, we have incidentally observed its strong anti-inflammatory activity in cell experiments, suggesting that it may exert its antipruritic effect by inhibiting inflammation. Furthermore, as a lipophilic compound, HDC may possess transdermal absorption capabilities. However, HDC is poorly soluble in water, making direct application as a solid inconvenient; therefore, it needs to be prepared into easily usable formulations. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an antipruritic gel containing 3-hydroxydecanoic acid microemulsion and its preparation method.

[0006] The core principle of this invention lies in the fact that 3-hydroxydecanoic acid (3-hydroxydecanoic acid) alleviates skin itching caused by inflammation by inhibiting the production of inflammatory factors from immune cells such as macrophages and mast cell degranulation. Furthermore, 3-hydroxydecanoic acid can also inhibit the pruritus-related TRP neural pathway. Compared with conventional chemically synthesized antipruritic agents, 3-hydroxydecanoic acid has a milder effect and exhibits both specific and non-specific inhibitory effects, thus being effective not only for histamine-mediated allergic pruritus but also for relieving non-histamine-induced pruritus.

[0007] The technical solution of the present invention is as follows:

[0008] An antipruritic gel containing 3-hydroxydecanoic acid microemulsion, comprising 20-50% 3-hydroxydecanoic acid microemulsion and 50-80% carbomer gel;

[0009] The formulation of the 3-hydroxydecanoic acid microemulsion is as follows: 5-10% 3-hydroxydecanoic acid, 1-2% peppermint oil, 15-30% surfactant, 15-30% co-surfactant, and the balance being pure water; wherein, the surfactant is preferably an alkyl glycoside, and the co-surfactant is preferably glycerol;

[0010] The carbomer gel was obtained by mixing carbomer 940 and pure water at a mass ratio of 2:98, and then adjusting the pH to 7.4 with triethanolamine.

[0011] The method for preparing the antipruritic gel containing 3-hydroxydecanoic acid microemulsion according to the present invention is as follows:

[0012] (1) Preparation of 3-hydroxydecanoic acid microemulsion

[0013] 3-Hydroxydecanoic acid was mixed with peppermint essential oil, and surfactant, co-surfactant and pure water were added in sequence. The mixture was heated to 40-50°C and stirred at 5000-10000 rpm for 5-30 minutes to obtain 3-hydroxydecanoic acid microemulsion.

[0014] (2) Preparation of carbomer gel

[0015] Carbomer 940 was mixed with pure water, and the pH was adjusted to 7.4 with triethanolamine while stirring to obtain a transparent carbomer gel.

[0016] (3) Preparation of antipruritic gel

[0017] Add the carbomer gel obtained in step (2) to the 3-hydroxydecanoic acid microemulsion obtained in step (1) and stir for 5 to 30 minutes to obtain the antipruritic gel.

[0018] The antipruritic gel containing 3-hydroxydecanoic acid microemulsion described in this invention can be used to prepare drugs for treating mosquito bites and skin itching caused by histamine or non-histamine substances (such as quinoa pod hairs).

[0019] The beneficial effects of this invention are as follows:

[0020] This invention is the first to use 3-hydroxydecanoic acid as an antipruritic agent. Its small molecular weight and lipophilic properties allow it to effectively penetrate the skin, inhibit various inflammatory factors, and block itch signaling pathways, thus achieving a good antipruritic effect. Furthermore, 3-hydroxydecanoic acid is derived from microbial fermentation, ensuring high safety and minimal side effects, making it suitable for long-term use. Through microemulsion and gel formulations, the droplet size of 3-hydroxydecanoic acid is reduced to below 10 nm, and its synergistic effect with penetration-enhancing surfactants improves transdermal absorption and prolongs its residence time in the skin, thereby enhancing the antipruritic effect. Attached Figure Description

[0021] Figure 1 Appearance and stability of the antipruritic gel containing 3-hydroxydecanoic acid microemulsion after centrifugation, high temperature and low temperature treatment.

[0022] Figure 2 The condition of the skin surface of volunteers who were bitten by mosquitoes 1 hour after applying anti-itch gel.

[0023] Figure 3 The study investigated the secretion of three inflammatory factors in mouse macrophages RAW264.7 after stimulation with lipopolysaccharide (LPS), and found that the secretion of inflammatory factors decreased after treatment with 3-hydroxydecanoic acid (3-HDA).

[0024] Figure 4 TRPV1 protein immunofluorescence staining (red fluorescence), cell nuclei stained with DAPI (blue fluorescence). Capsaicin activates the TRPV1 pathway, while 3-hydroxydecanoic acid inhibits its activation.

[0025] Figure 5 Mast cells (RBL-2H3) degranulate; the arrow indicates the degranulated mast cells. Detailed Implementation

[0026] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0027] In the following embodiments,

[0028] 3-Hydroxydecanoic acid was provided by Huzhou Zijin Biotechnology Co., Ltd., with a purity of 90% and the main impurity being 3-hydroxylauric acid.

[0029] The peppermint essential oil is purchased from the Taihe peppermint base in Anhui Province and is 100% natural peppermint extract.

[0030] The surfactant alkyl glycoside was provided by Jiangsu Wanqi Biotechnology Co., Ltd.

[0031] Carbomer 940 was purchased from Wuhan Desheng Biochemical Technology Co., Ltd.

[0032] Example 1: Formulation of 3-hydroxydecanoic acid microemulsion, carbomer gel and antipruritic gel

[0033] 3-Hydroxydecanoic acid is almost insoluble in water. When mixed with peppermint essential oil, it forms an oily liquid. After adding surfactants, co-surfactants, and water, and heating to 40-50°C with thorough stirring, a light yellow and transparent microemulsion is formed.

[0034] Table 1 Formulation of 3-hydroxydecanoic acid microemulsion

[0035]

[0036] Carbomer 940 and pure water were mixed at a mass ratio of 2:98. Triethanolamine was then added while stirring to adjust the pH to 7.4, resulting in a transparent gel. The carbomer gel was added to the hydroxydecanoic acid microemulsion (number 2 in the table above) while stirring continuously. After the addition was complete, stirring continued for 5-30 minutes. The formulation parameters are shown in Table 2. A homogeneous and transparent gel was ultimately obtained. Its morphology is as follows... Figure 1 As shown.

[0037] Table 2. Mixing ratio of 3-hydroxydecanoic acid microemulsion to carbomer gel

[0038]

[0039] Example 2: Particle size and stability analysis of the antipruritic gel

[0040] The particle size of the microemulsions in the gels numbered 1-3# in Table 2 was determined using a nanoparticle size analyzer. Their stability under centrifugation (1000 rpm for 10 minutes), low temperature (stored at -5℃ for 7 days), and high temperature (stored at 45℃ for 7 days) was also measured. The results are shown in Table 3.

[0041] Table 3. Particle size and stability analysis of microemulsion gels

[0042]

[0043] The results showed that the microemulsion particles of the antipruritic gel were all less than 10 nm in size, and the gel exhibited good stability, tolerating both high and low temperature storage environments without stratification (appearance as...). Figure 1 (As shown).

[0044] Example 3: Evaluation of the transdermal effect of 3-hydroxydecanoic acid in antipruritic gel

[0045] Fresh pigskin (skin near the nipples) was obtained from a local slaughterhouse, placed in PBS buffer, refrigerated, and transported back to the laboratory within 1 hour. The pigskin was cut into 1cm × 1cm pieces, and 0.1g of each was applied to the surface of an antipruritic gel (product #2 prepared in Example 1) and a 3-hydroxydecanoic acid microemulsion (formulation #2 in Table 1). Transdermal permeability was then measured every hour. Three replicates were set up at each time point. Transdermal permeability % = total residual hydroxydecanoic acid on the pigskin surface (mg) / initial amount of hydroxydecanoic acid (mg) × 100%. The results are shown in Table 4.

[0046] Table 4. Comparison of transdermal penetration rates of antipruritic gel (2#), 3-hydroxydecanoic acid solid, and 3-hydroxydecanoic acid microemulsion on fresh pigskin.

[0047]

[0048] As shown in the table above, 3-hydroxydecanoic acid, as a waxy solid, is difficult to apply to the skin surface and has almost no transdermal penetration. Preparing it as a microemulsion significantly improves the transdermal penetration rate of 3-hydroxydecanoic acid, thereby increasing its utilization rate; transdermal penetration reaches over 70% after 2 hours. This is likely due to the nanoscale nature of the microemulsion and the presence of a large amount of surfactants that aid in transdermal absorption, thus the anti-itch gel formulation exhibits good transdermal effects. However, as a liquid formulation, the microemulsion is prone to runoff from the skin surface and has a poor skin feel. While the anti-itch gel slows down the transdermal penetration rate of 3-hydroxydecanoic acid to some extent, the gel form facilitates its retention on the skin surface, resulting in a better skin feel and a longer-lasting anti-itch effect.

[0049] Example 4: Experiment on the inhibition of histamine-induced skin itching in mice by microemulsion gel

[0050] Experimental Methods (Reference: Yang et al. Journal of Ethnopharmacology 298, 2022, 115543): Twenty mice, half male and half female, were randomly divided into a blank control group, a model group, a positive control group, and an antipruritic gel treatment group, with five mice in each group. One day before the experiment, the hair on the back of the neck of the mice was removed with a shaver, covering an area of ​​approximately 2cm × 2cm. The mice were housed individually for 24 hours to acclimatize to the environment. Topical medication was applied to each group: sterile saline was applied to the blank control group and the model group, a commercially available antipruritic solution was applied to the control group, and the antipruritic gel (numbered 2# in Example 1) was applied to the treatment group. Each group was treated twice.

[0051] Following medication, except for the control group, mice in all other groups received an intradermal injection of 100 μL of histamine into the hairless area on the back of the neck. Immediately afterward, the mice were placed in experimental observation cages. Under quiet conditions, the number of times the mice scratched the injection site with their hind paws within 30 minutes was recorded. The formula for calculating the pruritus inhibition rate is:

[0052] Itching suppression rate (%) = (average number of scratches in the model group - average number of scratches in the treatment group) / average number of scratches in the model group × 100%.

[0053] The average number of scratches within 30 minutes after histamine injection in each group of mice is shown in Table 5. Histamine injection induced an itching response in the mice. Compared with the model group, the number of scratches in the treatment group was significantly reduced, indicating that the antipruritic gel in this invention has a good inhibitory effect on histamine-induced skin itching in mice.

[0054] Table 5. Itching status of animals in each group during the histamine-induced mouse pruritus model experiment.

[0055]

[0056] Example 5: Experiment on the inhibition of nonhistamine-induced skin itching in mice by antipruritic gel

[0057] Experimental Methods (Reference: W.-J. Lee et al. J Ginseng Res 42(2018)470-475): Following Example 4, only the subcutaneous injection of histamine was replaced with 4 ng of *Chenopodium album* pod extract (Reference: The Journal of Neuroscience, 2008, 28:4331-4335), with all other experimental procedures remaining the same. The treatment group was treated with the antipruritic gel numbered 2# in Example 1. The average number of scratches by mice in each group within 30 minutes after *Chenopodium album* pod injection was recorded (see Table 6). Compared with the model group, the number of scratches by mice in the treatment group was significantly reduced, indicating that the antipruritic gel of this invention has a good inhibitory and antipruritic effect on non-histamine-induced pruritus induced by *Chenopodium album* pods.

[0058] Table 6. Itching status of animals in each group in the mouse nonhistamine pruritus pathway model experiment.

[0059]

[0060] Example 6: Volunteer test of the anti-itch gel's effect on mosquito bites.

[0061] Subjects and Grouping: This study included 60 volunteers who had been bitten by mosquitoes. Clinical manifestations included red papules, swelling, and bumps at the bite site, accompanied by local itching and burning pain. The subjects were randomly divided into a treatment group and a control group. The treatment group consisted of 30 patients (10 males and 20 females, aged 25 to 60 years); the control group consisted of 30 patients (12 males and 18 females, aged 22 to 55 years). There were no statistically significant differences in age and gender between the two groups, making them comparable.

[0062] Experimental method (refer to CN106138241B): The treatment group used the anti-itch gel numbered 2# in Example 1, and the control group used a certain brand of anti-itch floral water. Both were applied to the mosquito bite sites 2 to 4 times a day.

[0063] Efficacy evaluation criteria:

[0064] -Significant effect: Clinical symptoms completely disappear within 30 minutes, red spots and swelling completely subside, and itching and burning sensations disappear.

[0065] - Effective: Symptoms basically disappear or are significantly relieved within 60 minutes, red spots basically disappear, swelling and bumps are significantly reduced, and itching and burning pain are significantly reduced.

[0066] - Ineffective: Symptoms did not improve significantly within 4 hours, red spots did not disappear, redness and swelling remained unchanged, and itching and burning sensations did not improve.

[0067] Experimental results showed that the antipruritic gel prepared in this invention had a total effective rate of 97% for mosquito bites, with itching disappearing within an average of 10 minutes and redness and swelling subsiding within 30 minutes. The total effective rate of the control group's floral water was 77%. The results indicate that the antipruritic gel of this invention is significantly effective in treating red spots, swelling, bumps, itching, and burning pain caused by mosquito bites, and is superior to commercially available floral water. The treatment effects of the two groups are shown in Table 7. Typical volunteer bite sites are shown in the images below. Figure 2 As shown, the itching decreased in about 3 minutes after applying the anti-itch gel, and the redness and swelling subsided significantly within 1 hour.

[0068] Table 7. Comparison of clinical efficacy of antipruritic gel in treating mosquito bites.

[0069]

[0070] Example 7: Analysis of the antipruritic mechanism of 3-hydroxydecanoic acid-containing microemulsion gel

[0071] The antipruritic gel of this invention uses 3-hydroxydecanoic acid as the main active ingredient, and its antipruritic effect is a first-time discovery. Therefore, it is necessary to conduct an in-depth analysis of its antipruritic mechanism. In vitro cell experiments were conducted to investigate its effects on three aspects: inhibiting the secretion of inflammatory factors, blocking pruritus signaling pathways, and inhibiting mast cell degranulation.

[0072] 1. Inhibition of Inflammatory Factor Secretion Assay (Reference: Xu et al. Molecules 2022, 27, 5037): Mouse RAW264.7 cells were used as a model and divided into control, model, and experimental groups. The control group was cultured in normal medium for 12 hours, the model group was cultured with 1 mg / L LPS (lipopolysaccharide), and the experimental group was cultured with 10 mg / L 3-hydroxydecanoic acid in addition to LPS for 12 hours. Enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of inflammatory factors such as TNF-α in the cell supernatant. Figure 3 As shown, compared with the control group, the inflammatory factor TNF-α in the model group was significantly increased; the 3-hydroxydecanoic acid in the experimental group significantly inhibited the increase of inflammatory factors, indicating that it may exert its antipruritic effect by inhibiting the secretion of inflammatory factors.

[0073] 2. Effects on Signaling Pathways (Reference: Yang et al. International Immunopharmacology 96, 2021, 107620): RAW264.7 cells were seeded in 96-well plates and divided into a blank control group, a model group, and an experimental group. The blank control group and model group were treated with HBSS buffer, while the experimental group was treated with HBSS buffer containing 10 mg / L 3-hydroxydecanoic acid. After 2 hours of pretreatment, the model group and experimental group were stimulated with 100 μM capsaicin. After 24 hours of incubation, TRPV1 protein was immunofluorescently stained, and cell nuclei were labeled with DAPI. Observation under a laser confocal microscope showed that 3-hydroxydecanoic acid significantly reduced TRPV1 protein expression, indicating that it reduces the transduction of pruritus signals by inhibiting the TRPV1 channel. Figure 4 ).

[0074] 3. Mast cell degranulation experiment (refer to WO2018115295A1): Using mast cells (RBL-2H3) as a model, they were divided into a control group, a model group, and an experimental group. The control group was cultured in normal culture medium for 2 hours, the model group was stimulated with 10 μM capsaicin, and the experimental group was cultured with 10 mg / L 3-hydroxydecanoic acid in addition to capsaicin for 2 hours. After toluidine blue staining, the changes in purple-red granules in the cytoplasm were observed under a light microscope. The results showed that compared with the model group, 3-hydroxydecanoic acid in the experimental group significantly inhibited the degranulation of mast cells, and its effect was comparable to that of dexamethasone. Figure 5 Inhibiting mast cell degranulation can reduce the subsequent production of histamine, thereby relieving itching.

[0075] Based on the above mechanistic studies, the antipruritic mechanism of 3-hydroxydecanoic acid is mainly achieved through three aspects: inhibiting the secretion of inflammatory factors, blocking the TRPV1 signaling pathway, and reducing mast cell degranulation. These findings provide a theoretical basis for the antipruritic gel of this invention and verify the scientific validity and rationality of its antipruritic effect.

[0076] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The use of an antipruritic gel containing 3-hydroxydecanoic acid microemulsion in the preparation of a drug for treating mosquito bites and skin itching caused by histamine or nonhistamine substances; The antipruritic gel containing 3-hydroxydecanoic acid microemulsion is composed of 20-50% 3-hydroxydecanoic acid microemulsion and 50-80% carbomer gel; all percentages are mass fractions.

2. Use according to claim 1, wherein The formulation of the 3-hydroxydecanoic acid microemulsion is as follows: 5-10% 3-hydroxydecanoic acid, 1-2% peppermint oil, 15-30% surfactant, 15-30% co-surfactant, and the balance being pure water; all percentages are by mass.

3. Use according to claim 2, wherein the compound is ###0002### The surfactant is an alkyl glycoside.

4. The use according to claim 2, wherein the compound is ###0002### The co-surfactant is glycerol.

5. The use according to claim 1, wherein Carbomer gel is prepared by mixing carbomer 940 and pure water at a mass ratio of 2:98, and then adjusting the pH to 7.4 with triethanolamine.

6. The use according to claim 1, wherein The method for preparing the antipruritic gel containing 3-hydroxydecanoic acid microemulsion is as follows: (1) Preparation of 3-hydroxydecanoic acid microemulsion 3-Hydroxydecanoic acid was mixed with peppermint essential oil, and surfactant, co-surfactant and pure water were added in sequence. The mixture was heated to 40-50°C and stirred at 5000-10000 rpm for 5-30 minutes to obtain 3-hydroxydecanoic acid microemulsion. (2) Preparation of carbomer gel Carbomer 940 was mixed with pure water, and the pH was adjusted to 7.4 with triethanolamine while stirring to obtain a transparent carbomer gel. (3) Preparation of antipruritic gel Add the carbomer gel obtained in step (2) to the 3-hydroxydecanoic acid microemulsion obtained in step (1) and stir for 5~30 minutes to obtain the antipruritic gel.

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