Amino acid synthesis for fast antipruritic effect and its external skin preparation

Abstract

This invention discloses a long-acting antipruritic and antibacterial topical skin preparation, comprising N-ethyl-γ-L-glutamine, 1-1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-N-(L-γ-glutamyl-N-ethyl)-ethanolamine (POPE-N-Et-Gln-α), chlorhexidine acetate, and saffron extract as active ingredients, formulated into a hydrogel or cream with a pharmaceutically acceptable matrix. POPE-N-Et-Gln-α provides rapid transdermal action, while N-ethyl-γ-L-glutamine offers sustained-release and long-lasting effects; saffron extract provides anti-inflammatory and antioxidant effects, and chlorhexidine acetate provides antibacterial and antifungal effects. This invention is hormone-free and non-addictive, achieving both rapid and long-lasting antipruritic effects, and is suitable for eczema, urticaria, dry skin, neurodermatitis, psoriasis, microbial infections, and skin itching caused by insect bites.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical topical preparations, specifically to a compound topical preparation that simultaneously possesses rapid antipruritic, long-lasting sustained-release antipruritic, anti-inflammatory, antioxidant, and antibacterial effects. It can be prepared into hydrogels and creams for the treatment and care of skin itching caused by eczema, urticaria, dry skin, neurodermatitis, psoriasis, microbial infections, and insect bites. Background Technology

[0002] Skin itching is a common skin symptom that can be caused by a variety of factors, including allergic reactions, dry skin, fungal and bacterial infections, neurodermatitis, eczema, urticaria, infections, and insect bites. Traditional treatments for skin itching mainly include the use of antihistamines, corticosteroids, and local anesthetics. However, these methods have some limitations: (1) Antihistamines: Although they can relieve itching, they are not effective for certain types of itching (such as neuropruritus) and may cause side effects such as drowsiness.

[0003] (2) Glucocorticoids: Long-term use may lead to adverse reactions such as skin atrophy, pigmentation, and hormone-dependent dermatitis.

[0004] (3) Local anesthetics: Although they can quickly relieve itching, their effect is short-lived, requiring frequent use and may cause skin irritation.

[0005] Therefore, developing a safe, hormone-free, non-addictive topical formulation with long-lasting antipruritic effects is an important research direction in the field of dermatology.

[0006] N-ethyl-γ-L-glutamine (Gln) is an amino acid extracted from tea leaves and possesses various biological activities, including sedation, anti-anxiety, and anti-fatigue effects. Recent studies have shown that Gln also has a significant antipruritic effect. This mechanism may be related to Gln's ability to inhibit the transmission of neurogenic pruritus signals. Although topical formulations such as hydrogels and creams can prolong the release of Gln, thus extending its duration of action, theanine has a low transdermal absorption rate and is easily degraded by esterases in the skin, resulting in a short duration of its antipruritic effect. 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) is an important zwitterionic phospholipid widely distributed in biological membranes. Its unique chemical structure gives it a significant advantage in transdermal delivery systems. The zwitterionic nature and flexibility of POPE can protect drugs from degradation, prolonging their duration of action in the skin. Furthermore, the covalent linkage of the drug with phosphatidylethanolamine significantly improves its transdermal efficiency, enabling it to penetrate the skin barrier and reach deeper tissues. Therefore, this patent combines Gln and POPE via an amide reaction to generate a novel molecule capable of rapidly relieving itching.

[0007] Saffron (Crocus sativus L.) is a valuable medicinal plant, whose main active ingredients include crocin I, crocin II, and crocinin. These components have significant anti-inflammatory, antioxidant, and anti-tumor effects. In dermatology, saffron extract is used to treat various skin diseases, such as eczema and dermatitis. Its anti-inflammatory effect is mainly achieved by scavenging free radicals, inhibiting the release of inflammatory mediators, and reducing the infiltration of inflammatory cells.

[0008] Chlorhexidine acetate is a broad-spectrum antibacterial agent with strong antibacterial and antifungal activity.

[0009] It kills bacteria and fungi by disrupting the cell membranes of microorganisms, causing leakage of cell contents. In topical skin preparations, chlorhexidine acetate is commonly used for the prevention and treatment of skin infections caused by bacteria and fungi.

[0010] For the reasons mentioned above, we prepared two formulations: a hydrogel and a cream, with N-ethyl-γ-L-glutamine, POPE, chlorhexidine acetate and saffron extract as active ingredients. Summary of the Invention

[0011] This invention relates to a compound topical preparation that combines Glun, POPE-N-Et-Gln-α, chlorhexidine acetate, and saffron extract to prepare a formulation that simultaneously possesses rapid-acting antipruritic, long-acting sustained-release antipruritic, anti-inflammatory, antioxidant, and antibacterial effects. Currently, there are no related reports.

[0012] One object of this invention is to synthesize an active compound, POPE-N-Et-Gln-α, which is highly effective in relieving itching. Specifically, this compound is generated by reacting Gln with POPE via an amide reaction under conditions of adding an activator composition.

[0013] According to some embodiments, the activator composition is selected from several or one of the following: carbodiimide and N-hydroxysuccinimide, N,N'-diisopropylcarbodiimide and N-hydroxysuccinimide, and dicyclohexylcarbodiimide and N-hydroxysuccinimide, and the molar ratio of the activator composition to Gln is one of 1.2:1.2:1, 1.5:1.5:1, and 1:1:1.

[0014] According to some embodiments, the dosage form of the compound topical preparation is selected from hydrogels and creams.

[0015] According to some embodiments, the hydrogel has a matrix of sodium alginate, chitosan, hyaluronic acid, methacrylated hyaluronic acid, gelatin, methacrylated gelatin, silk fibroin, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, carbomer, polyvinyl alcohol, xanthan gum, sodium lauryl sulfate, triethanolamine, polysorbate 80, poloxamer, fatty alcohol polyoxyethylene ether, poloxamer 407, and poloxamer 188, with a preference for hydroxypropyl methylcellulose, triethanolamine, and carbomer.

[0016] According to some embodiments, the cream has a base of several or one of the following: isopropyl myristate, petrolatum, solid paraffin, glycerin, liquid paraffin, microcrystalline wax, lanolin, beeswax, cetyl alcohol, stearyl alcohol, cetyl alcohol, glyceryl monostearate, POPEG (molecular weight 400-8000), glycerin gelatin, methylcellulose, sodium carboxymethyl cellulose, xanthan gum, sodium lauryl sulfate, triethanolamine, polysorbate 80, poloxamer, fatty alcohol polyoxyethylene ether, glyceryl monostearate, Span 80, Span 60, lanolin, calcium soap, cholesterol, poloxamer 407, and poloxamer 188, with a preference for a combination of petrolatum, liquid paraffin, ceryl alcohol, polyethylene glycol 400, glycerin, sodium lauryl sulfate, and xanthan gum.

[0017] A second objective of this invention is to provide a method for synthesizing a rapid and long-lasting antipruritic compound. To facilitate understanding of the method for synthesizing this compound by those skilled in the art, the following synthetic process is provided exemplaryly, but not limitingly: Gln and POPE were dissolved in dimethylformamide (DMF) or dichloromethane (DCM) at a molar ratio of 1:1 to 1:2. An activator composition of 1.2 to 1.5 molar amounts was added, and the mixture was stirred at room temperature to 40°C for 2 to 6 hours. After the reaction was complete, the product was purified by silica gel / dextran gel column chromatography, eluting with a methanol / water mixture to obtain the target product. The reaction concentrations of Gln and POPE were 1-1000 mg. A third objective of this invention is to provide two methods for preparing compound topical preparations. To facilitate understanding of the methods for preparing these topical preparations by those skilled in the art, the following preparation processes are provided exemplarily, but not limitingly: (1) Preparation of long-lasting antipruritic and antibacterial hydrogel 1) Dissolve or disperse Gln, POPE-N-Et-Gln-α, chlorhexidine acetate, and saffron extract in water to obtain a mixed solution; 2) Dissolve the hydrogel matrix material in water to obtain a matrix solution; 3) Add the mixed solution obtained in step 1) to the matrix solution obtained in step 2) and stir until homogeneous; 4) Adjust the pH to 5.0-7.0 and continue stirring until homogeneous to obtain the hydrogel formulation.

[0018] The contents (w / v) of Gln, POPE-N-Et-Gln-α, chlorhexidine acetate, and saffron extract in the mixed solution were 0.1%–10%, 0.1%–30%, 0.05%–10%, and 0.01%–20%, respectively. The matrix content in the matrix solution was 0.1%–10%.

[0019] (2) Preparation of long-acting antipruritic and antibacterial cream 1) Dissolve or disperse Gln, POPE-N-Et-Gln-α, chlorhexidine acetate, saffron extract and water-soluble matrix material in an appropriate amount of water to obtain solution A; 2) Several fat-soluble matrix materials were mixed and melted at 65℃-80℃ to obtain mixture B; 3) Pour mixture B into solution A and stir for 10 minutes to obtain the initial cream.

[0020] 4) Grind the initial cream using a three-axis roller mill for 2-10 cycles to obtain the final long-acting antipruritic and antibacterial cream.

[0021] The contents (w / v) of Gln, POPE-N-Et-Gln-α, chlorhexidine acetate, and saffron extract in solution A are 0.1%–10%, 0.1%–30%, 0.05%–10%, and 0.01%–20%, respectively; the contents (w / v) of water-soluble matrix material are 0.2-30%; and the mass ratio of mixture B to solution A is 0.1-20.

[0022] Compared with the prior art, the technical advantages of the present invention are as follows: By combining N-ethyl-γ-L-glutamine (Gln) with phosphatidylethanolamine (POPE) to create a novel molecule, the transdermal efficiency and stability of the compound are significantly improved, prolonging the duration of its antipruritic effect. Simultaneously, the anti-inflammatory and antioxidant effects of saffron extract and the antibacterial and antifungal properties of chlorhexidine acetate provide comprehensive protection for the treatment of pruritus. The hydrogel and cream formulations of this invention not only achieve rapid itch relief and long-lasting sustained release but also avoid the side effects of traditional treatments, such as hormone dependence and skin irritation. They are safe, hormone-free, and non-addictive innovative topical formulations. Attached Figure Description

[0023] Figure 1 The synthetic route diagram for POPE-N-Et-Gln-α; Figure 2 The hydrogen spectrum of POPE-N-Et-Gln-α; Figure 3 The in vitro transdermal curves of POPE-N-Et-Gln-α cream; Figure 4 Draize score after using POPE-N-Et-Gln-α cream; Figure 5 HE image of skin after application of POPE-N-Et-Gln-α cream; Detailed Implementation The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0024] Example 1: Synthesis of POPE-N-Et-Gln-α 100 mg Gln and 771 mg POPE were placed in a 50 mL round-bottom flask, and 10 mL of anhydrous DMF was added. The mixture was sonicated at room temperature for 5 min under nitrogen protection to obtain a slightly milky white solution. 171 mg EDC·HCl and 17 mg DMAP were added sequentially, and after nitrogen purging, the mixture was stirred in an oil bath at 400 rpm for 4 h at 40 °C (TLC monitoring showed the disappearance of Gln spots). After cooling, the reaction solution was quenched with 0.5 mL of water, concentrated to 2 mL by rotary evaporation, and precipitated with 20 mL of cold diethyl ether. The byproduct was collected by centrifugation at 4 °C, and the upper ether phase was dried under nitrogen. The residue was dissolved in 0.5 mL of methanol / water (95:5), and eluted with a methanol / water gradient on a Sephadex LH-20 column. The 90:10 eluent containing POPE-N-Et-Gln-α was combined, rotary evaporated, and dried under vacuum for 12 h to obtain a white waxy solid. The specific synthetic route is as follows: Figure 1 .

[0025] Example 2: Preparation of POPE-N-Et-Gln-α hydrogel 430 mg of POPE-N-Et-Gln-α, 1.0 g of Gln, and 1.0 g of carbomer 974P were dispersed in 15 mL of deionized water and stirred magnetically overnight at 25 °C until fully swollen. The solution was then neutralized dropwise with triethanolamine to pH 6.8–7.2, increasing the viscosity to approximately 4500 cP. 0.2 g of glycerol and 0.02 g of methylparaben were added, and homogenization was continued for 15 min. The solution was then degassed under vacuum for 30 min to obtain a transparent, spreadable POPE-N-Et-Gln-α carbomer matrix hydrogel, which was stored at room temperature.

[0026] Example 3: Preparation of POPE-N-Et-Gln-α loaded cream 50 g glycerin, 50 g POPE-N-Et-Gln-α, 50 g Gln, 1 g saffron extract, 1 g chlorhexidine acetate, and 10 g sodium dodecyl sulfate were dissolved in 450 mL of 75 °C deionized water to form the aqueous phase. Separately, 50 g isopropyl myristate, 50 g white petrolatum, 60 g liquid paraffin, 1 g tea tree oil, 90 g stearyl alcohol, and 50 g polyethylene glycol 400 were mixed and melted at 80 °C until transparent to form the oil phase. The oil phase was slowly added dropwise to the aqueous phase at a rate of 10 mL·min⁻¹ under mechanical stirring at 800 rpm. After the addition was complete, the temperature was raised to 85 °C and homogenized for 10 min (10000 rpm), followed by gradient cooling to 40 °C and continued low-speed stirring to defoam for 30 min, yielding a pale yellow, fine O / W cream with a drug loading of 6g. % (Gln+POPE-N-Et-Gln-α), pH 6.2, viscosity 22000 cP, stable at room temperature.

[0027] Example 1: 1H N-Et-Gln-α Characterization 1. Experimental Methods ¹H NMR spectra of POPE-N-Et-Gln-α samples (approximately 5–10 mg) were performed at 25 °C using a Bruker 300 MHz NMR spectrometer. The samples were dissolved in 0.6 mL DMSO-d6 (containing 0.05% TMS as an internal standard), loaded into 5 mm standard NMR tubes, rotated at 20 Hz, scanned 16 times, with a spectral width of 20 ppm and a center frequency of 5 ppm. Data were processed using TopSpin software, with Fourier transforms performed using a 0.3 Hz exponential window function, and manual phase and baseline corrections were applied to obtain high-resolution proton NMR data.

[0028] 2. Experimental Results like Figure 2 The 1H NMR spectrum (¹H NMR, DMSO-d6, 300 MHz) of the sample shows that the target molecule is a complex lipid derivative containing amino groups, amides, phosphate esters, and long fatty chains. Characteristic signals include: ① Low field region δ 7.99–8.03 (1H, s, amide NH), 7.01–7.05 (2H, s, terminal amide NH2), 5.28–5.40 (2H, m, alkene CH=CH), and 4.18–4.22 (1H, m, CH-OP adjacent to phosphate ester); ② Medium-high field region δ 4.05–4.47 showing three sets of homozygous / non-equivalent CH2 (phosphate ester-glycerol region, J ≈ 8.5 Hz), δ 3.30–3.61 (2H, m, CH-N adjacent to amide / amine), δ 2.01–2.65 (multiple CH2, α-carbonyl / allyl / amine region), and δ 1.18–1.72 (approximately 30H, wide packing, long chain -(CH2)n-); ③ Terminal methyl group δ 0.84–0.92 (6H, t, J ≈ 7 Hz). The overall integral ratio is consistent with the POPE-N-Et-Gln-α structure, and the chemical shift and coupling mode are as expected, which can be used for structural confirmation.

[0029] Experimental Example 2: Evaluation of the slow transdermal delivery of POPE-N-Et-Gln-α-loaded cream 1. Experimental Methods A vertical Franz diffusion cell (effective diffusion area 1.13 cm², receiving cell volume 7 mL) was used, with 0.5 mm thick, integrity-verified (resistivity >15 kΩ·cm²) isolated abdominal skin of Bama miniature pigs as a barrier. The skin temperature was maintained by circulating water in a 32 ± 0.5 °C constant temperature bath. The receiving solution was pH 7.4 PBS (containing 20% ​​ethanol to improve solubility), and magnetic stirring at 500 rpm was used to avoid concentration polarization. Creams containing equal amounts of Gln (10 mg) and POPE-N-Et-Gln-α (10 mg) were accurately weighed and applied to the stratum corneum side of the skin in the supply cell. At 0.5, 1, 2, 4, 6, 8, 12, and 24 h, all receiving solutions were collected and immediately replenished with isothermal fresh receiving solution. After filtration through a 0.22 µm filter membrane, the samples were analyzed by HPLC (C18 column, mobile phase 0.05 mol·L⁻¹). - ¹ K₂HPO₄ / acetonitrile 90:10, λ=210 nm) was used to directly determine free Gln; for the POPE-N-Et-Gln-α group, an equal amount of 0.1 mol·L⁻¹ HCl was first added to hydrolyze at 60 °C for 30 min to break phospholipid bonds and release total Gln before injection, and quantification was performed using the external standard method. The results were expressed as cumulative permeate (µg·cm⁻¹). -2 Plot the in vitro transdermal curve using time (h).

[0030] 2. Experimental Results like Figure 3 In vitro transdermal experiments showed that the cumulative permeation of POPE-N-Et-Gln-α cream was significantly higher than that of free Gln cream at all time points within 24 hours, indicating that POPE modification can significantly promote the transdermal delivery of Gln. Its permeation advantage was particularly evident within 0.5–4 hours, suggesting that POPE-N-Et-Gln-α can rapidly penetrate the stratum corneum and release continuously, which may be related to its enhanced lipid solubility, compatibility with the skin lipid bilayer, and sustained-release characteristics of the phospholipid carrier system, thereby effectively improving the transdermal efficiency and bioavailability of Gln.

[0031] Experimental Example 3: Safety Evaluation of POPE-N-Et-Gln-α 1. Experimental Methods Healthy SD rats (half male and half female, weighing 180–220 g, n=12) were used for acute skin toxicity testing. Twenty-four hours before administration, hair was removed from both sides of the spine using an electric razor (area 3 cm × 3 cm). A self-controlled design was used; 0.5 g of POPE-N-Et-Gln-α cream (equivalent to 10 times the clinical dose) was applied to intact skin on the left side, while an equal volume of blank matrix was applied to the right side as a control. Twenty-four hours later, residual ointment was gently wiped off with warm water. Skin irritation reactions such as erythema, edema, desquamation, and crusting were observed and recorded daily for 14 consecutive days, quantified according to the Draize scoring system (0–4 points). Simultaneously, rat body weight, food intake, activity level, and blood biochemical indicators (ALT, AST, BUN, Cr) were monitored. Guinea pigs (n=10) were also used for active skin sensitization testing: 0.2 g of ointment was applied to the shaved area on days 0, 7, and 14 for induction; on day 28, rats were challenged with 0.1 g of ointment. After h, observe whether erythema, edema or papules appear, and take skin samples from the treatment area for HE staining to evaluate the integrity of the stratum corneum, inflammatory infiltration and changes in epidermal thickness, and comprehensively judge the local and systemic safety of POPE-N-Et-Gln-α cream.

[0032] 2. Experimental Results No deaths or significant behavioral abnormalities were observed in the rats in the POPE-N-Et-Gln-α cream group during the 14-day observation period, and their weight gain curves coincided with those of the blank control group. P >0.05), the Draize skin irritation index was consistently <0.5 (indicating no irritation), and only one rat developed mild, transient erythema (1 point) 2 hours after the first administration, which subsided spontaneously within 24 hours. Figure 4 As shown in Table 1, all blood biochemical indicators were within the normal reference range, and there was no statistically significant difference compared with the blank group. P >0.05). In the guinea pig sensitization test, no erythema, edema, or papules were observed 48 hours after challenge, and the sensitization rate was 0% (Table 2); skin histopathology showed that the structure of each layer of the epidermis was clear, the stratum corneum was continuous, there was no inflammatory cell infiltration in the dermis, and the epidermal thickness was comparable to that of the negative control group. P >0.05), indicating that POPE-N-Et-Gln-α cream is non-irritating and non-sensitizing to intact skin, and has good local and systemic safety.

[0033] Table 1. Body weight and blood biochemical indicators Compared with the blank matrix group, P>0.05, there was no statistically significant difference.

[0034] Table 2 Skin Reaction Scoring Table for Sensitization Test Sensitization rate = 0 / 10 = 0%, therefore it is judged as "non-sensitizing".

[0035] Experimental Example 4: Relief of itching in 2,4-dinitrofluorobenzene (DNFB)-induced allergic contact dermatitis by POPE-N-Et-Gln-α cream. 1. Experimental Methods (1) Model building SPF-grade female BALB / c mice (6–8 weeks, 18–22 g) were sensitized by applying 0.5% DNFB (acetone:olite = 4:1, 100 μL) to the abdomen after shaving on day 0. From day 5 to day 9, 0.2% DNFB 20 μL / ear was applied to the inner side of both ears daily to induce allergic contact dermatitis with itching.

[0036] (2) Grouping and administration Starting on day 5, participants were randomly divided into 5 groups (n=10): normal control group, model group, positive control group (oral diphenhydramine 10 mg / kg), low-dose POPE-N-Et-Gln-α cream group (POPE-N-Et-Gln-α+Gln, 10 mg / kg), and high-dose POPE-N-Et-Gln-α cream group (POPE-N-Et-Gln-α+Gln, 20 mg / kg). All groups received the medication once daily for 5 consecutive days.

[0037] (3) Indicator Measurement Within 30 minutes after each DNFB challenge, the number of times mice scratched their ears and the cumulative duration of scratching were recorded in a double-blind manner. Ear thickness was measured 24 hours after the last administration to calculate ear swelling, and skin inflammation was scored according to a 0–4 scale.

[0038] Experimental results: Table 3 shows that, compared with the normal control group, the model group had significantly higher scratching frequency (45.8±6.2 times / 30 min), scratching duration (128.6±15.3 s), ear swelling (0.58±0.08 mm), and inflammation score (3.2±0.4) (P<0.001). The positive control drug diphenhydramine significantly relieved itching and reduced inflammation (inhibition rate 60.1%). POPE-N-Et-Gln-α cream showed a dose-dependent effect: the inhibition rate was 37.8% in the low-dose group and 65.7% in the high-dose group. The number of scratches (15.7±3.8 times), scratching time (38.6±9.2 s), ear swelling (0.28±0.06 mm), and inflammation score (1.3±0.2) were all better than those in the positive control group (P<0.01), indicating that the new drug has better antipruritic and anti-inflammatory effects than traditional antihistamines at a dose of 20 mg / kg.

[0039] Table 3. Pruritus score of DNFB-induced allergic contact dermatitis Note: Data are expressed as mean ± standard deviation. Comparison with the model group: P <0.05, P <0.01, P <0.001; Compared with the positive drug group: # P <0.05, ## P <0.01.

[0040] Experimental Example 5: Relief of itching caused by psoriasis in mice by POPE-N-Et-Gln-α cream 1. Experimental Methods (1) Animals and adaptation SPF-grade female BALB / c mice (6–8 weeks, 18–22 g) were acclimatized for 7 days (22±2 ℃, 50–60 % humidity, 12 h light-dark cycle).

[0041] (2) Model induction On days 0 and 1, apply 62.5 mg of 5% imiquimod (IMQ) ointment after shaving the abdomen; from days 3 to 7, apply 25 mg of 0.5% IMQ evenly to the skin inside both ears and on the back daily for 5 consecutive days to induce psoriatic dermatitis with severe itching.

[0042] (3) Grouping and administration Starting on day 3, participants were randomly assigned to 5 groups (n=10): a normal control group (Vaseline), a model group (IMO), a positive control group (oral methotrexate 1 mg / kg), a low-dose group of POPE-N-Et-Gln-α cream (POPE-N-Et-Gln-α+Gln, 10 mg / kg), and a high-dose group of POPE-N-Et-Gln-α cream (POPE-N-Et-Gln-α+Gln, 20 mg / kg). Administered once daily for 5 consecutive days.

[0043] (4) Assessment of itching and skin lesions Within 30 minutes after daily application of the medication, the number of scratches and the cumulative scratching time of both ears were recorded in a double-blind manner. 24 hours after the last administration, the ear thickness was measured to calculate the degree of swelling, and erythema, scaling, and thickness were assessed according to a 0–4 scale (PASI score).

[0044] 2. Experimental Results Figure 4 shows that the IMQ model group mice exhibited significantly increased pruritus, with 52.3±7.8 scratches per 30 min and a duration of 145.6±18.4 s. Ear swelling increased to 0.62±0.09 mm, and the PASI total score reached 9.8±0.6. After methotrexate intervention at 1 mg / kg, the number of scratches decreased to 19.5±3.6, the duration shortened to 52.3±9.1 s, ear swelling decreased to 0.33±0.05 mm, PASI decreased to 4.7±0.4, and the pruritus inhibition rate was 62.7%. In the low-dose POPE-N-Et-Gln-α cream group, the number of scratches was 31.8±5.2, the duration was 89.4±12.7 s, ear swelling was 0.45±0.07 mm, and PASI was [missing data]. The POPE-N-Et-Gln-α cream high-dose group showed the best performance, with only 16.2±3.4 scratches lasting 43.1±8.6 s, ear swelling of 0.29±0.04 mm, PASI 3.9±0.3, and an inhibition rate of 69.0%, significantly better than the methotrexate group. P <0.01), showing an overall dose-dependent improvement, suggesting that the new drug has both potent antipruritic and anti-psoriasis effects.

[0045] Table 4. Itching score caused by psoriasis Note: Data are expressed as mean ± standard deviation. Comparison with the model group: P <0.05, P <0.01, P <0.001; Compared with the positive drug group: # P <0.05, ## P <0.01.

[0046] Experimental Example 6: Relief of itching caused by fungal infection in mice by POPE-N-Et-Gln-α cream 1. Experimental Methods (1) Animals and adaptation SPF-grade female C57BL / 6 mice (7–9 weeks, 18–22 g) were acclimatized for 5 days (22±2 ℃, 50–60 % humidity, 12 h light-dark cycle).

[0047] (2) Itching caused by fungal inoculation On day 0, the shaved area on the abdomen (2 cm × 2 cm) was lightly abraded with sandpaper until slightly red, and 100 μL containing 1×10 7CFU / mL Trichophyton rubrum spore suspension (0.1% Tween-80 saline), fixed with gauze for 4 h; thereafter, change the cage daily and maintain environmental humidity at 70% to promote infection, and establish a fungal dermatitis with pruritus model for 7 consecutive days.

[0048] (3) Grouping and administration Starting on day 3, participants were randomly assigned to 5 groups (n=10): a normal control group (uninfected), a model group (infected + solvent), a positive control group (terbinafine 20 mg / kg + hydroxyzine 10 mg / kg, combined with antifungal and antihistamine), a low-dose group of POPE-N-Et-Gln-α cream (POPE-N-Et-Gln-α + Gln, 10 mg / kg), and a high-dose group of POPE-N-Et-Gln-α cream (POPE-N-Et-Gln-α + Gln, 20 mg / kg). Administered once daily for 7 consecutive days.

[0049] (4) Assessment of itching and infection Two hours after the last administration, the affected area was placed in a transparent box to record the number of scratches (scratching of the inoculated area by the hind paws) and the cumulative scratching time over 30 minutes. Subsequently, the skin erythema score (0–4) and scaling score (0–4) were measured, and the affected skin was taken for fungal KOH microscopic examination to calculate the hyphal positivity rate.

[0050] 2. Experimental Results The results of the fungal infection pruritus model (Table 5) showed that Trichophyton rubrum infection caused a surge in scratching frequency in mice to 46.7±6.5 times / 30 min, with a cumulative scratching time of 132.4±16.3 s. Skin erythema scored 3.6 points, scaling 3.4 points, and the hyphae microscopic positivity rate was as high as 90%, indicating a high correlation between fungal load and pruritus severity. Terbinafine combined with hydroxyzine significantly reduced scratching frequency to 17.3±3.4 times and 48.6±8.9 s (inhibition rate 63.0%), and reduced the hyphae positivity rate to 20%, verifying that antifungal combined with antihistamines can simultaneously alleviate pruritus and skin lesions. The low-dose group of POPE-N-Et-Gln-α cream was only partially effective, with scratching inhibition at 34.0% and hyphae positivity still at 55%. The low-dose group of POPE-N-Et-Gln-α cream further reduced scratching to 14.5±3.0 times and a duration of 41.3±7.8 s, with an inhibition rate of 69.0% and a hyphal positivity rate of only 15%. The erythema and scaling scores were also superior to the positive combination group. P The value is <0.01, indicating that the new drug has both potent antifungal and antipruritic effects at high doses, and its anti-inflammatory effect is more thorough.

[0051] Table 5. Pruritus score caused by fungal infection Note: Data are expressed as mean ± standard deviation. Comparison with the model group: P <0.05, P <0.01, P <0.001; Compared with the positive drug group: # P <0.05, ## P <0.01.

[0052] Experimental Example 7: Relief of itching caused by mosquito bites in mice by POPE-N-Et-Gln-α cream 1. Experimental Methods (2) Animals and adaptation SPF-grade female ICR mice (6–8 weeks, 18–22 g) were acclimatized for 5 days (22±2 ℃, 50–60% humidity, 12h light-dark cycle), and the abdominal hair was shaved 2 cm × 2 cm 24 h before the experiment.

[0053] (3) Mosquito bite irritation On day 0, 30 female Aedes albopictus mosquitoes (starved for 24 hours) were placed in a mosquito cage and the mice were fixed to the abdominal wall with a net and exposed for 15 minutes, so that each mouse was bitten about 20–25 times. They were then immediately returned to the cage to establish an acute pruritus model induced by mosquito bites.

[0054] (4) Grouping and administration Thirty minutes after the bite, participants were randomly divided into 5 groups (n=10): normal control group (no bite), model group (bite + solvent), positive control group (chlorpheniramine 10 mg / kg), low-dose POPE-N-Et-Gln-α cream group (POPE-N-Et-Gln-α+Gln, 10 mg / kg), and high-dose POPE-N-Et-Gln-α cream group (POPE-N-Et-Gln-α+Gln, 20 mg / kg). Administered once daily for 5 consecutive days.

[0055] (5) Itching behavior measurement Starting 30 minutes after administration, mice were placed in a transparent observation box, and the number of scratches (hind paw scratches) and the cumulative scratching time of the bite area were recorded continuously over 30 minutes. At the same time, the degree of erythema and edema at the bite site was evaluated using a 0–4 point scale.

[0056] 2. Experimental Results Table 6 shows that the number of scratches in the mosquito bite model group of mice reached 38.5±5.9 times within 30 min, with a cumulative scratching time of 108.7±14.2 s. Erythema at the bite site scored 3.2 points and edema scored 2.8 points, indicating severe itching accompanied by significant local inflammation. Positive antihistamine 10 mg / kg reduced the scratching index to 15.2±3.3 times and 42.6±8.1 s, with an inhibition rate of 60.5%, while erythema and edema were halved, indicating that classic antihistamine treatment was effective but still had residual symptoms. The new drug showed a dose-dependent effect; low-dose POPE-N-Et-Gln-α cream only reduced scratching by 31.7%, while high-dose POPE-N-Et-Gln-α cream further reduced it to 12.8±2.9 times and 35.1±7.3 s, with an inhibition rate of 66.8%, significantly better than chlorpheniramine (…). P The scores for erythema and edema decreased to 1.1 and 1.0 respectively, almost returning to baseline levels, suggesting that the new drug has a faster and more comprehensive antipruritic and anti-inflammatory advantage than traditional antihistamines in an acute mosquito bite pruritus model.

[0057] Table 6. Itching scores caused by mosquito bites Note: Data are expressed as mean ± standard deviation. Comparison with the model group: P <0.05, P <0.01, P <0.001; Compared with the positive drug group: # P <0.05, ## P <0.01.

[0058] Experimental Example 8: POPE-N-Et-Gln-α cream relieved itching caused by neurodermatitis in mice. 1. Experimental Methods (1) Animals and adaptation SPF-grade female C57BL / 6 mice (7–9 weeks, 18–22 g) were acclimatized for 5 days (22±2 ℃, 50–60% humidity, 12 h light-dark cycle), and the back of the neck was shaved 2 cm × 2 cm 24 h before the experiment.

[0059] (2) Induction of chronic neurodermatitis The strategy of "repeated barrier disruption + local grouping" is adopted: Starting from day 0, use sandpaper (#600) to gently scrape the shaved area twice a day (9:00 and 17:00) in one direction 10 times until the skin is slightly red but does not bleed; Immediately afterwards, 0.3% mustard oil (allyl isothiocyanate, dissolved in 70% ethanol, 20 μL / time) was applied for 14 consecutive days to establish a neurodermatitis-like chronic pruritus model caused by persistent keratin damage and nerve sensitization.

[0060] (3) Grouping and administration On day 7 (when obvious scratching and erythema appeared), they were randomly divided into 5 groups (n=10): normal control group (no treatment); Model group (continued scraping + mustard oil, solvent gavage); positive control group (hydroxyzine 10 mg / kg + dexamethasone 1 mg / kg, combined with antihistamine + glucocorticoid); low-dose POPE-N-Et-Gln-α cream group (POPE-N-Et-Gln-α + Gln, 10 mg / kg), high-dose POPE-N-Et-Gln-α cream group (POPE-N-Et-Gln-α + Gln, 20 mg / kg); once daily for 7 days; throughout the experiment, daily scraping + mustard oil stimulation continued to maintain the chronic state.

[0061] (4) Behavioral and skin assessment Two hours after the last administration, the number of scratches (scratching of the shaved area by the hind paws) and the cumulative scratching time within 30 minutes were recorded; skin thickness was measured (micrometer) and swelling was calculated; erythema, scaling, and lichenification were assessed according to a 0–4 score (total score of 0–12 for neurodermatitis); IL-4 and IL-13 levels in the skin lesions were detected by ELISA.

[0062] 2. Experimental Results Table 7 shows that chronic sandpaper damage combined with mustard oil stimulation successfully induced typical neurodermatitis-like pruritus: the number of scratches in the model group mice increased dramatically to 49.6±6.8 times in 30 min, the cumulative scratching time was 138.4 s, the skin thickness increased to 0.59 mm, the total dermatitis score was 9.1, and IL-4 reached as high as 142 pg / mg, suggesting that continuous barrier disruption activates the Th2-IL-4 / 13 pathway and induces neurosensitization; the combination of hydroxyzine and dexamethasone reduced the number of scratches to 18.3 times, with an inhibition rate of 63.1%, and the inflammation and lichenification were significantly relieved but not completely reversed; the new drug showed dose-dependent effects, with high doses of 10 Further testing at mg / kg increased scratching intensity to 15.8 times, with an inhibition rate of 68.1%. Skin thickness and IL-4 levels were significantly better than the positive group (P<0.01), and the total dermatitis score was the lowest (3.7). This indicates that the dose has both potent antipruritic and anti-neurogenic inflammatory effects, and can significantly block the "barrier damage-neurosensitization-itch cycle," demonstrating potential therapeutic advantages for chronic neurodermatitis.

[0063] Table 7. Itching score caused by neurodermatitis

Claims

1. A long-acting topical antipruritic and antibacterial preparation, characterized in that, It contains the following active ingredients by weight percentage: a) N-ethyl-γ-L-glutamine 0.1%–10%; b) 1,1-Palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-N-(L-γ-glutamyl-N-ethyl)-ethanolamine 0.1%–15%; c) Chlorhexidine acetate 0.05%–2%; d) Saffron extract 0.01%–5%; And pharmaceutically acceptable matrices and / or transdermal excipients.

2. The topical preparation according to claim 1, characterized in that, The 1-1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-N-(L-γ-glutamyl-N-ethyl)-ethanolamine (POPE-N-Et-Gln-α) is generated by the amide reaction of N-ethyl-γ-L-glutamine (Gln) with phosphatidylethanolamine (POPE) under the condition of adding an activator composition.

3. The activator composition 1 according to claim 2, characterized in that, The activator composition is one or more of the following: carbodiimide and N-hydroxysuccinimide, N,N'-diisopropylcarbodiimide and N-hydroxysuccinimide, and dicyclohexylcarbodiimide and N-hydroxysuccinimide, wherein the molar ratio of the activator composition to Gln is one of 1.2:1.2:1, 1.5:1.5:1, or 1:1:

1.

4. The topical preparation according to claim 1, characterized in that, The dosage form of the topical preparation is selected from hydrogels and creams.

5. The topical preparation according to claim 4, characterized in that, The hydrogel has a matrix consisting of several or one of the following: sodium alginate, chitosan, hyaluronic acid, methacrylated hyaluronic acid, gelatin, methacrylated gelatin, silk fibroin, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, carbomer, polyvinyl alcohol, xanthan gum, sodium lauryl sulfate, triethanolamine, polysorbate 80, poloxamer, fatty alcohol polyoxyethylene ether, poloxamer 407, and poloxamer 188.

6. The topical preparation according to claim 4, characterized in that, The cream is characterized in that the base is one or more of the following: petrolatum, solid paraffin, liquid paraffin, microcrystalline wax, glycerin, lanolin, beeswax, cetyl alcohol, stearyl alcohol, octadecanol, glyceryl monostearate, PEG (molecular weight 400-8000), glycerin gelatin, methylcellulose, sodium carboxymethylcellulose, xanthan gum, sodium lauryl sulfate, triethanolamine, polysorbate 80, poloxamer, fatty alcohol polyoxyethylene ether, glyceryl monostearate, Span 80, Span 60, lanolin, calcium soap, cholesterol, poloxamer 407, and poloxamer 188.

7. The topical preparation according to claim 1, characterized in that, The topical preparation wherein the total content of crocin I, crocin II, crocin and crocinic acid in the saffron extract is ≥5 wt%.

8. The topical preparation according to any one of claims 1-7, for use in treating eczema, urticaria, dry skin, neurodermatitis, fungal or bacterial infections, and pruritus caused by insect bites.

9. A method for preparing the POPE-N-Et-Gln-α according to claim 3, comprising: Gln and POPE were dissolved in dimethylformamide (DMF) or dichloromethane (DCM) at a molar ratio of 1:1 to 1:

2. An activator composition of 1.2 to 1.5 times the molar amount was added, and the mixture was stirred at room temperature to 40°C for 2 to 6 hours. After the reaction was complete, or after purification by silica gel / dextran gel column chromatography, the product POPE-N-Et-Gln-α was obtained by elution with a methanol / water mixture. The reaction concentration of Gln was 1-1000 mg, and the reaction concentration of POPE was 1-1000 mg.

10. A method for preparing the topical formulation of claim 1, comprising: (1) Preparation of long-lasting antipruritic and antibacterial hydrogel 1) Dissolve or disperse Gln, POPE-N-Et-Gln-α, chlorhexidine acetate, and saffron extract in water to obtain a mixed solution; 2) Dissolve the hydrogel matrix material in water to obtain a matrix solution; 3) Add the mixed solution obtained in step 1) to the matrix solution obtained in step 2) and stir until homogeneous; 4) Adjust the pH to 5.0-7.0 and continue stirring until homogeneous to obtain the hydrogel formulation. (2) Preparation of long-acting antipruritic and antibacterial cream 1) Dissolve or disperse Gln, POPE-N-Et-Gln-α, chlorhexidine acetate, saffron extract and water-soluble matrix material in an appropriate amount of water to obtain solution A; 2) Several fat-soluble matrix materials were mixed and melted at 65℃-80℃ to obtain mixture B; 3) Pour mixture B into solution A and stir for 10 minutes to obtain the initial cream; 4) Grind the initial cream using a three-axis roller mill for 2-10 cycles to obtain the final long-acting antipruritic and antibacterial cream.

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