Application of beta-guanidinopropionic acid in preparation of medicine for chronic inflammatory dermatosis

Abstract

The invention belongs to the technical field of biological medicines, and relates to an application of beta-guanidinopropionic acid (3-Guanidinopropionic acid, beta-GPA) in preparation of a medicine for treating chronic inflammatory dermatosis. The invention discovers that the creatine transporter SLC6A8 is remarkably and highly expressed in skin lesion tissues of psoriasis for the first time, and the protein is strongly associated with occurrence and development of chronic inflammatory skin diseases such as psoriasis. The SLC6A8 transporter inhibitor beta-guanidino propionic acid can obviously inhibit the expression level of keratinocyte inflammatory factors, and meanwhile, the imiquimod-induced psoriasis model mouse skin lesion symptom is reversed, so that the beta-guanidino propionic acid has the effect of resisting psoriasis. The invention can provide new therapeutic targets and new drug choices for psoriasis and other chronic inflammatory skin diseases.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the use of β-guanidinopropionic acid (3-Guanidinopropionic acid, β-GPA) in the preparation of drugs for treating chronic inflammatory skin diseases, and especially to the use of β-GPA and a pharmaceutical composition thereof in the treatment of psoriasis and / or atopic dermatitis. Background Art

[0002] Chronic inflammatory skin diseases are a group of diseases characterized by persistent inflammatory responses of the skin. Common types include psoriasis and atopic dermatitis. Their typical characteristics are skin erythema, itching, desquamation and recurrent attacks. Some patients also have skin barrier dysfunction. These diseases can occur at any age, but the predisposition populations for different diseases vary. About 20-30% of the world's population is affected by these diseases, and some severe patients may have systemic diseases such as metabolic syndrome or arthritis.

[0003] Traditional topical treatments for psoriasis and atopic dermatitis are mainly topical glucocorticoids, but long-term use of such drugs is prone to hormone resistance, and side effects such as skin atrophy, capillary dilation, and pigmentation may occur. In recent years, monoclonal antibody drugs for cytokines such as tumor necrosis factor α (TNF-α), interleukin 12 (IL-12), interleukin 17 (IL-17), and interleukin 23 (IL-23) for the treatment of psoriasis, and monoclonal antibody drugs for cytokines such as interleukin 4 (IL-4), interleukin 13 (IL-13), and interleukin 31 (IL-31) for the treatment of atopic dermatitis have been launched one after another, and have been widely used in the treatment of chronic inflammatory skin diseases. However, such preparations can lead to adverse reactions such as adverse reactions to local injections, increased risk of infection due to immunosuppression, and immune dysfunction caused by long-term use. At the same time, the high cost of medication and strict storage conditions are not conducive to long-term use by patients. In addition, small molecule inhibitors targeting Janus tyrosine kinase (JAK) and phosphodiesterase 4 (PDE4) also severely limit the willingness of patients with psoriasis and atopic dermatitis to use them due to problems such as systemic immunosuppression, increased risk of infection, and gastrointestinal side effects.

[0004] In view of the above situation, the development of new safe and effective therapeutic drugs is a key issue facing the clinical treatment of chronic inflammatory skin diseases. The current scientific research field pays great attention to the application potential of small molecule compounds in the treatment of chronic inflammatory skin diseases. With their economic advantages, diverse administration routes and the ability to target specific pathological mechanisms, these drugs are promoting the development of new chronic inflammatory skin disease therapeutic drugs.

[0005] Creatine transporter SLC6A8 is a transmembrane protein through which creatine enters cells. Creatine then generates phosphocreatine under the catalysis of creatine kinase, rapidly storing and releasing high-energy phosphate groups, maintaining the stability of intracellular ATP levels, and providing immediate energy support for cells. Studies have found that β-guanidinopropionic acid (3-Guanidinopropionicacid, β-GPA) is a small molecule inhibitor of SLC6A8. This drug plays a key regulatory role in skeletal muscle endurance regulation, brain tissue biosynthesis metabolism, and tumor cell proliferation by competitively inhibiting cell uptake of creatine. However, there are currently no reports on the expression of SLC6A8 in chronic inflammatory skin diseases (including psoriasis and atopic dermatitis, etc.) and its association with the occurrence and development of such diseases. There are also no reports on the role of SLC6A8 inhibitor β-GPA in such disease models and its clinical application value. Summary of the invention

[0006] In order to achieve the above-mentioned invention object, the relevant technical solutions are as follows:

[0007] The present invention provides an application of a SLC6A8 transporter inhibitor β-guanidinopropionic acid (3-Guanidinopropionicacid, β-GPA) in the preparation of a drug for treating chronic inflammatory skin diseases.

[0008] The molecular formula of the β-GPA is C 4 H 9 N 3 O 2 , the structural formula is as follows:

[0009]

[0010] Preferably, the chronic inflammatory skin disease is psoriasis and / or atopic dermatitis.

[0011] More preferably, the psoriasis is psoriasis vulgaris.

[0012] Further preferably, the drug consists of β-GPA and a pharmaceutically acceptable carrier, including but not limited to common excipients (such as starch, water, etc.), lubricants (such as magnesium stearate, etc.), disintegrants (such as microcrystalline cellulose, etc.), etc.

[0013] Further preferably, the drug is in the form of an external preparation, an oral preparation or an injection preparation.

[0014] Further preferably, the drug is in the form of a gel, spray, aerosol, patch, tincture, paste, lotion, cream, ointment, granules, capsule, pill, tablet, powder, oral liquid, syrup, or injection.

[0015] Preferably, the β-GPA content in the β-GPA external preparation is 0.5% to 10%, and more preferably, the β-GPA content is 5%.

[0016] Preferably, the effective therapeutic dose of β-GPA oral preparation or injection preparation (calculated as β-GPA used per kilogram of human body weight) is 10 mg / Kg / day to 100 mg / Kg / day, and more preferably, the effective therapeutic dose is 50 mg / Kg / day.

[0017] The "effective therapeutic dose" mentioned in the present invention refers to the dynamic dosing range determined by medical professionals based on comprehensive variables such as drug delivery methods, dosage form characteristics, individual physiological parameters of patients, and clinical manifestations of target indications. This dosage parameter has the characteristics of individualized administration and complies with the principles of precision medicine treatment. Its numerical range will be adaptively adjusted with the patient's metabolic characteristics, degree of pathological progression, and pharmacokinetic characteristics.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] Chronic inflammatory skin diseases have complex causes, long course of disease, and symptoms are prone to recurrence, which seriously affects the quality of life of patients. The present invention combines clinical sample analysis and in vitro and in vivo efficacy evaluation to find for the first time that the creatine transporter SLC6A8 is strongly associated with the progression of chronic inflammatory skin diseases such as psoriasis, and its inhibitor β-GPA can exert significant anti-inflammatory effects and reverse psoriasis symptoms. Our discovery provides new therapeutic targets and new drug options for psoriasis and other chronic inflammatory skin diseases.

[0020] The present invention provides an application of a creatine SLC6A8 transporter inhibitor in the preparation of a drug for treating chronic inflammatory skin diseases, mainly involving the use of the SLC6A8 transporter inhibitor β-GPA for treating psoriasis and / or atopic dermatitis. The present invention first discovered that the creatine transporter SLC6A8 is highly expressed in psoriasis lesion tissues, and β-GPA can exert significant anti-inflammatory and therapeutic effects in both an in vitro inflammatory cell model and an IMQ-induced psoriasis mouse model. It can be seen that SLC6A8 is an important target for the treatment of chronic inflammatory skin diseases such as psoriasis, and its inhibitor β-GPA can provide a new therapeutic drug option for the treatment of chronic inflammatory skin diseases such as clinical psoriasis, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The expression of SLC6A8 in healthy controls and psoriasis lesion tissues was investigated by H&E staining and immunohistochemistry.

[0022] Figure 2The RT-qPCR method was used to investigate the regulatory effect of SLC6A8 transporter inhibitor β-guanidinopropionic acid (β-GPA) on the expression levels of inflammatory factors (IL-6, IL-8 and IL-1β) in human keratinocytes HaCaT induced by TNF-α / IFN-γ.

[0023] Figure 3 The experimental therapeutic effect of β-GPA on the IMQ-induced mouse psoriasis model was investigated by constructing the model. DETAILED DESCRIPTION

[0024] The following examples are intended to enable those skilled in the art to more fully understand the technical solutions and implementation effects of the present invention, but the protection scope of the present invention is not limited thereto. This section further describes the present invention in detail in conjunction with specific implementation cases, and its technical features and advantages will be clearly reflected in the description. It should be pointed out that the embodiments are only exemplary descriptions and are not limitations on the scope of the claims of the present invention. Any detail adjustment, equivalent replacement or adaptive improvement based on the core principles of the present invention falls within the substantive protection scope of the present invention.

[0025] Example 1 The expression level of SLC6A8 in the skin lesions of patients with psoriasis is significantly higher than that in the skin tissues of healthy controls 1.1 Experimental methods

[0026] Skin lesion tissue samples (n=3) from patients with clinically confirmed psoriasis were selected as the experimental group, and healthy skin tissue samples (n=3) were selected as the control group, and H&E staining and immunohistochemical staining were performed. HE staining steps: tissue samples were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced ​​(thickness 4μm); hematoxylin staining (5 minutes) and eosin staining (2 minutes) were performed in sequence, and the histopathological characteristics were observed under an optical microscope after dehydration and sealing. Immunohistochemical (IHC) detection of SLC6A8 expression: after the sections were dewaxed and hydrated, antigen heat repair (citrate buffer, pH 6.0, 95℃ for 20 minutes) was performed; endogenous peroxidase was blocked (3% H 2 O 2 , 10 minutes), incubated with primary antibody (anti-SLC6A8 antibody, 1:200 dilution, 4°C overnight); added HRP-labeled secondary antibody (room temperature for 1 hour), DAB color development, hematoxylin counterstaining, and neutral gum sealing.

[0027] 1.2 Experimental Results

[0028] like Figure 1As shown in the figure, HE staining results showed that the psoriasis group showed epidermal hyperkeratosis, incomplete keratinization, thickened stratum spinosum, and dilated capillaries in the dermal papilla, which were consistent with the typical pathological characteristics of psoriasis, while the skin tissue structure of the control group was normal. Immunohistochemistry results showed that SLC6A8 was mainly located in the epidermal stratum corneum in psoriasis lesions and was significantly enriched in the cell membrane of keratinocytes, with a staining intensity significantly higher than that of the control skin tissue.

[0029] 1.3 Experimental Conclusion

[0030] This example confirms through HE staining and immunohistochemistry that the expression level of SLC6A8 in psoriasis lesion tissue is significantly higher than that in healthy control skin tissue. Its high expression is closely related to the pathological characteristics of psoriasis (such as epidermal dysplasia and inflammatory response), suggesting that SLC6A8 may be used as a biomarker or therapeutic target for the diagnosis of psoriasis, and this discovery provides an experimental basis for the development of inhibitors against SLC6A8, which has important clinical application potential.

[0031] Example 2 β-Guanidin propionic acid (β-GPA) significantly inhibits the expression of inflammatory factors in the HaCaT cell inflammation model induced by TNF-α / IFN-γ dual factors

[0032] 2.1 Experimental methods

[0033] Human keratinocytes HaCaT with good growth status were selected. When the cells grew to more than 80%, the cells were collected by trypsin digestion and plated in 6-well plates. After the cells were completely attached to the wall on the second day of plating, the cells were divided into 5 groups: Ctrl, TNF-α / IFN-γ (all 10ng / ml), TNF-α / IFN-γ+0.5mMβ-GPA, TNF-α / IFN-γ+1mMβ-GPA, and TNF-α / IFN-γ+2mMβ-GPA. In the experiment, the cells were first pretreated with different concentrations of β-GPA for 10h, and then TNF-α / IFN-γ (final concentration of 10ng / ml) was directly added to stimulate the cells for 2h, and then the cell samples were uniformly collected and RNA was extracted for real-time fluorescence quantitative reverse transcription PCR (RT-qPCR) detection.

[0034] 2.2 Experimental Results

[0035] like Figure 2 As shown in the figure, after TNF-α / IFN-γ stimulated HaCaT, the mRNA expression of inflammatory factors IL-6, IL-8 and IL-1β increased significantly. On this basis, after intervention with different concentrations (0.5mM, 1mM, 2mM) of β-GPA, the expression levels of IL-6, IL-8 and IL-1β decreased significantly, and the trend was concentration-dependent ( #Represents the comparison between TNF-α / IFN-γ group and blank control, ### P<0.001; * represents the comparison between the β-GPA group and the TNF-α / IFN-γ group, NS indicates no significant difference, *P<0.05, **P<0.01, ***P<0.001).

[0036] 2.3 Experimental Conclusion

[0037] In the TNF-α / IFN-γ-induced HaCaT cell inflammation model, β-GPA can significantly inhibit the release of related inflammatory cytokines, indicating that β-GPA can exhibit significant anti-inflammatory activity in the in vitro model.

[0038] Example 3 Preparation of β-GPA external gel

[0039] 3.1 Preparation of β-GPA (1%, 5%) external gel

[0040] To prepare 20 g of β-GPA topical gel, first weigh the relevant raw materials according to the following weights: β-GPA (0.2 g for 1% and 1.0 g for 5%), carbomer 940 (0.158 g), triethanolamine (0.1736 g), low molecular weight sodium hyaluronate (0.0242 g), glycerol (2.422 g), azone (0.4 g), and ultrapure water (1%: 16.6224 g; 5%: 15.8224 g).

[0041] After weighing the raw materials, prepare the gel according to the following steps: ① Preparation of compound moisturizer: weigh 0.0242g of low molecular weight sodium hyaluronate and 2.422g of glycerol, mix well and set aside. ② Preparation of phase A (solution containing β-GPA): weigh β-GPA according to the target concentration (0.2g for 1% and 1.0g for 5%), first add 10mL of ultrapure water (about 10g), stir until completely dissolved; add 1mL of compound moisturizer (about 1g, the remaining compound moisturizer is reserved for phase B) to the solution, mix well; add 0.4g of azone and stir until homogeneous. ③ Preparation of phase B (carbomer swelling solution): weigh 0.158g of carbomer 940 and mix with the remaining compound moisturizer (about 1.446g); add the remaining ultrapure water (1%: 6.6224g; 5%: 5.8224g), and continue stirring for 20-30 minutes to allow the carbomer to fully swell and disperse. ④ Mixing and gelation: slowly pour phase B into phase A, stirring while adding, and mix evenly; add 0.1736g of triethanolamine and continue stirring for 5-6 minutes until a transparent and uniform gel is formed.

[0042] 3.2 Preparation of placebo gel (without β-GPA)

[0043] The method is the same as above, but β-GPA is not added to the gel preparation, and the total amount of ultrapure water used is 16.8224 g.

[0044] Example 4 β-Guanidine propionic acid (β-GPA) can significantly alleviate IMQ-induced psoriasis-like symptoms in mouse skin 4.1 Experimental methods

[0045] Twenty-eight 6-week-old female BALB / c mice were randomly divided into 4 groups, 6 mice in each group, and the backs were shaved (area 2cmх3cm). They were raised normally, and different treatments were given to the shaved areas of the mice on the second day. Imiquimod (IMQ) was used to induce psoriasis skin lesions in mice in the experiment. The specific grouping and operation are as follows: ① Blank control group (Control): no treatment; ② Model group (IMQ+placebo gel): placebo gel once in the morning and evening, IMQ once a day. That is, IMQ 62.5mg / mouse / day was applied to the back skin of mice at 2 pm every day for 6 days; placebo gel 62.5mg / mouse / day was applied at 8 am and 8 pm respectively, for 6 days. ③ Low-concentration treatment group (IMQ+1% β-GPA gel): 1% β-GPA gel was applied once in the morning and evening, IMQ was applied once a day. That is, IMQ 62.5 mg / mouse / day was applied to the back skin of mice at 2 pm every day for 6 days; 1% β-GPA gel 62.5 mg / mouse / day was applied at 8 am and 8 pm respectively for 6 days. ④ High concentration treatment group (IMQ + 5% β-GPA gel): 5% β-GPA gel was applied once in the morning and evening, and IMQ was applied once a day. That is, IMQ 62.5 mg / mouse / day was applied to the back skin of mice at 2 pm every day for 6 days; 5% β-GPA gel 62.5 mg / mouse / day was applied at 8 am and 8 pm respectively for 6 days.

[0046] The mice in the above groups were fed and drank water normally, and the skin performance of the mice was observed every day and given PASI scores. On the 7th day, the skin lesions of the mice were recorded, and the mice were dislocated and killed to end the experiment. Among them, the PASI score sets 3 evaluation indicators, and 0 to 4 points are given according to the severity of the skin lesions on the back of the mice. The three scores are summarized as the total PASI score. The specific scores are as follows: ① Evaluation of erythema: The following scores are used for the scoring of erythema on the back of mice: no erythema is 0; light red is 1; red is 2; dark red is 3; very deep red is 4. ② Evaluation of thickening: The following scores are used for the scoring of thickening on the back of mice: flush with normal skin is 0; slightly raised compared to normal skin is 1; moderately raised with rounded or sloped edges is 2; obvious raised with thickened edges is 3; extremely obvious raised and highly thickened is 4. ③ Evaluation of scaling: The scaling on the back of mice was scored as follows: smooth surface and no scaling was assigned as 0; local lesions were covered with scaling, mainly fine scaling was assigned as 1; most lesions were covered with scaling, the scaling was thicker, and the scaling was mainly flaky was assigned as 2; almost all lesions were covered with scaling, the scaling was thicker, and the scaling was distributed in layers was assigned as 3; all lesions were covered with scaling, the scaling was very thick, and the scaling was overlapping in layers was assigned as 4.

[0047] 4.2 Experimental Results

[0048] The changes in the back skin of each group of mice on the 7th day are as follows Figure 3 .Depend on Figure 3 A shows that compared with the blank control group (Control) mice, obvious erythema and scaling were observed on the back skin of the modeling group (IMQ+placebo gel) mice. The erythema and scaling of the low-concentration treatment group (IMQ+1% β-GPA gel) and the high-concentration treatment group (IMQ+5% β-GPA gel) were significantly alleviated after topical drug treatment, and the high-concentration treatment group had better treatment effect.

[0049] Figure 3 BE is the PASI score of mice. Compared with the model group, the erythema in the 5% β-GPA gel treatment group was significantly improved ( Figure 3 B); Compared with the model group, the thickening and scaling of the back skin of mice in the 1% β-GPA gel treatment group and the 5% β-GPA gel treatment group were significantly improved ( Figure 3 CD); compared with the model group, the PASI total scores in the 1% β-GPA gel treatment group and the 5% β-GPA gel treatment group were significantly reduced ( Figure 3 E) (NS means no significant difference, *P<0.05, **P<0.01).

[0050] 4.3 Experimental Conclusion

[0051] In the IMQ-induced psoriasis mouse model, topical β-GPA can significantly alleviate the IMQ-induced psoriasis-like skin lesions.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. Application of β-guanidinopropionic acid in the preparation of drugs for treating chronic inflammatory skin diseases.

2. The use according to claim 1, characterized in that: The chronic inflammatory skin disease is psoriasis and / or atopic dermatitis.

3. The use according to claim 1 or 2, characterized in that: The medicine consists of β-GPA and a pharmaceutically acceptable carrier.

4. The use according to any one of claims 1 to 3, characterized in that: The medicine is in the form of an external preparation, an oral preparation or an injection preparation.

5. The use according to claim 4, wherein the drug is in the form of a gel, spray, aerosol, patch, tincture, paste, lotion, cream, ointment, granule, capsule, pill, tablet, powder, oral liquid, syrup, or injection.

6. The use according to claim 4 or 5, characterized in that: The content of β-GPA in the external preparation is 0.5% to 10%, and the effective therapeutic dose of the oral preparation and the injection preparation is 10 mg / kg / day to 100 mg / kg / day based on the amount of β-GPA used per kilogram of human body weight.

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