WO2022001784A1 - 谷氨酰胺酶抑制剂在制备治疗银屑病的药物中的应用 - Google Patents

谷氨酰胺酶抑制剂在制备治疗银屑病的药物中的应用 Download PDF

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WO2022001784A1
WO2022001784A1 PCT/CN2021/101850 CN2021101850W WO2022001784A1 WO 2022001784 A1 WO2022001784 A1 WO 2022001784A1 CN 2021101850 W CN2021101850 W CN 2021101850W WO 2022001784 A1 WO2022001784 A1 WO 2022001784A1
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mice
psoriasis
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卢传坚
徐永跃
王茂杰
黄闰月
包德威·伯格林
焦琳
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广东省中医院
广州中医药大学第二附属医院
广州中医药大学第二临床医学院
广东省中医药科学院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/433—Thidiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/473—Quinolines; Isoquinolines ortho- or peri-condensed with carbocyclic ring systems, e.g. acridines, phenanthridines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/501—Pyridazines; Hydrogenated pyridazines not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics

Definitions

  • the invention belongs to the technical field of medicine and relates to the use of glutaminase inhibitors, in particular to the application of glutaminase inhibitors in the preparation of medicines for treating psoriasis.
  • Psoriasis is a chronic inflammatory skin disease with skin erythema, papules, and scales as the main clinical manifestations. The global incidence is about 1-3%. Psoriasis is characterized by excessive proliferation of skin lesions and infiltration of local inflammatory cells, and changes in the metabolic spectrum of local skin lesions. The pathogenesis is complex, involving multiple aspects such as genetics, immune-inflammation, and vascular abnormalities. Procrastination is hard to heal.
  • the drugs currently used for the treatment of psoriasis mainly include glucocorticoids, tretinoin drugs, vitamin D3 derivatives, dithranol, cytotoxic drugs, etc.
  • tazarotene, moderate and strong glucocorticoids Hormones and calcipotriol are currently the first-line drugs for topical treatment of psoriasis.
  • the therapeutic effect thereof is still not completely satisfactory, and there is still a lack of a drug for the treatment of psoriasis with significant curative effect and small side effects in the prior art.
  • CB-839 (CAS: 1439399-58-2) is a glutaminase 1 (GLS1) inhibitor, which can regulate cellular metabolic processes by targeting the inhibition of GLS1 enzyme activity. Its molecular weight is 571.574, and its molecular formula is C 26 H 24 F 3 N 7 O 3 S, the structural formula is as follows:
  • the purpose of the present invention is to provide the application of glutaminase inhibitor in the preparation of medicine for treating psoriasis.
  • a glutaminase inhibitor in the preparation of a medicament for the treatment of psoriasis.
  • the glutaminase inhibitor may be selected from the group consisting of Acivicin (CAS: 42228-92-2), Compound 968 (CAS: 311795-38-7), BPTES (CAS: 314045-39) -1), one or more of CB-839.
  • the glutaminase inhibitor can be CB-839.
  • CB-839 is a GLS inhibitor, which can block the production of basic substances such as energy and amino acids by inhibiting the conversion of glutamine into glutamate.
  • the weight percentage of CB-839 may be 2%-8%.
  • CB-839 can be used as a single active ingredient or together with other pharmaceutically acceptable ingredients in the manufacture of a medicament for the treatment of psoriasis.
  • the pharmaceutically acceptable ingredient may include a pharmaceutically active ingredient that has no antagonistic effect with CB-839 and/or one or more pharmaceutically acceptable excipients.
  • the drug for the treatment of psoriasis may be administered by injection, oral or transdermal administration.
  • the drug for the treatment of psoriasis may be administered by topical transdermal administration.
  • the drug for treating psoriasis may be in the form of an ointment, gel, patch, plaster, lotion, film, paste, aerosol, or spray.
  • glutaminase is an important target of targeted metabolism in the treatment of psoriasis
  • glutaminase inhibitors including CB-839 and BPTES, etc.
  • CB-839 can: 1) significantly inhibit keratinocyte metabolism of glutamine; 2) significantly inhibit miR-31-induced up-regulated keratinocyte mitochondrial respiration; 3) significantly inhibit miR-31-induced mTOR 4) Significantly reduce cell activity and promote apoptosis, especially in keratinocytes with high expression of miR-31; 5) Downregulate the expression of a series of miR-31 and/or TNF- ⁇ -induced pro-inflammatory factors, Especially GM-CSF and TGF- ⁇ 1.
  • Figure 1 shows the total uptake of glutamine by keratinocytes during 12h of CB-839 intervention.
  • *** indicates that compared with the marked group, P ⁇ 0.001;
  • Figure 2 shows the total amount of glutamate secreted by keratinocytes during CB-839 intervention for 12h.
  • *** indicates that compared with the marked group, P ⁇ 0.001;
  • Figures 3-9 show the changes in the metabolic levels of glutamine, aspartic acid, ⁇ -ketoglutaric acid, succinic acid, fumaric acid and malic acid with time in keratinocytes after CB-839 intervention;
  • Figure 10 shows the changes in the oxygen consumption rate of keratinocytes induced by miR-31 in different treatment groups.
  • *** indicates the statistical difference between the miR-31 group and the miR-31+CB839 group at the same time point P ⁇ 0.001, ns means there is no significant statistical difference between the two;
  • Figure 11 shows the phosphorylation levels of mTOR pathway downstream molecules 4EBP1, S6 and p70S in cells of different treatment groups
  • Figure 12 shows the growth curves of keratinocytes in different treatment groups.
  • * means compared with the Ctrl group, P ⁇ 0.05;
  • *** means compared with the Ctrl group, P ⁇ 0.001;
  • Figure 13 shows the clone formation of keratinocytes intervened by different concentrations of CB-839;
  • Figures 14-15 sequentially show the inhibitory effect of CB-839 on the expression of pro-inflammatory factors in keratinocytes induced by miR-31 and TNF- ⁇ ;
  • Figure 16 shows the changes in body weight of mice in each group during administration
  • Figure 17 shows the skin lesions of mice in each group on the 8th day
  • Figure 18 shows the results of H&E staining of mouse skin lesions in each group
  • Figure 19 shows the Baker score results of the skin of the skin lesions of the mice in each group, in the figure, ** means compared with the marked group, P ⁇ 0.01; *** means compared with the marked group, P ⁇ 0.001;
  • Figure 20 shows the epidermal thickness of the skin lesions of mice in each group, in the figure, * means compared with the marked group, P ⁇ 0.05; *** means compared with the marked group, P ⁇ 0.001; **** means compared with the marked group Compared with the marked group, P ⁇ 0.0001;
  • Figure 21 shows the counting results of the subcutaneous microvessels in the skin lesions of the mice in each group, in the figure, * means compared with the marked group, P ⁇ 0.05; *** means compared with the marked group, P ⁇ 0.001;
  • Figures 22-23 show the proliferation of Ki67-positive cells in the epidermis of mouse skin lesions in each group.
  • ** means compared with the marked group, P ⁇ 0.01;
  • *** means compared with the marked group , P ⁇ 0.001;
  • Figures 24-25 show the expression and distribution of GLS enzymes in the skin lesions of mice in each group.
  • * indicates that compared with the marked group, P ⁇ 0.05;
  • *** indicates that compared with the marked group, P ⁇ 0.05 ⁇ 0.001;
  • Figure 26 shows the effect of different administration modes of CB-839 on the skin lesions of IMQ mice
  • Figure 27 shows the results of H&E staining of mouse skin lesions in each group
  • Figure 28 shows the comparison results of the spleen index of mice in each group; in Figure 27, * means compared with the marked group, * P ⁇ 0.05; ** means compared with the marked group, * P ⁇ 0.01; **** means **** P ⁇ 0.0001 compared to the marked group;
  • Fig. 29 shows the epidermal thickness ( ⁇ m) of the skin lesions of mice in each group; in Fig. 27, * indicates that compared with the marked group, * P ⁇ 0.05; ** indicates that compared with the marked group, * P ⁇ 0.01; *** means *** P ⁇ 0.001 compared to the marked group ; **** means **** P ⁇ 0.0001 compared to the marked group;
  • Figure 30 shows the expression profile of cell metabolism genes specifically expressed in the skin lesions of psoriasis patients
  • Figures 31-32 show the staining results and quantitative analysis results of miR-31 in psoriatic skin lesions and normal human skin.
  • Figure 32 *** indicates that compared with the marked group, P ⁇ 0.001;
  • Figure 33 shows the results of detecting the levels of metabolites using metabolomics technology after overexpression of miR-31;
  • Figure 34 shows the expression levels of cell metabolism-related gene proteins on keratinocytes and non-keratinocytes after expressing miR-31;
  • Figure 35 is a summary graph of the results of Figures 31-34.
  • the pathological process of psoriasis is inseparable from the metabolic reorganization of keratinocytes induced by miR-31.
  • the specific manifestation is that the affected basal keratinocytes first overexpress glucose transporter 1 (GLUT1) and glutaminase (GLS) to increase glycolysis and glutamine metabolic pathways, thereby providing sufficient energy and basic substances for its rapid proliferation and differentiation.
  • GLUT1 glucose transporter 1
  • GLS glutaminase
  • the mTOR pathway maintains cell survival and participates in other pathological changes.
  • Example 1 CB-839 inhibits the uptake and metabolism of glutamine in keratinocytes
  • CB-839 can significantly inhibit the uptake of glutamine by keratinocytes (P ⁇ 0.001), and reduce the amount of glutamate secreted into the culture medium by keratinocytes (P ⁇ 0.001).
  • CB-839 can significantly inhibit glutamate, aspartate, alpha-ketoglutarate (alpha-KG), succinic acid in keratinocytes.
  • Example 2 CB-839 inhibits miR-31-induced mitochondrial respiration in keratinocytes
  • Oxygen consumption rate is the oxygen consumption rate of cells, which is an indicator of mitochondrial respiration capacity.
  • Oxygen consumption detection principle The value displayed before adding oligo represents the basal oxygen consumption of the cell, including mitochondrial oxidative phosphorylation and proton leakage oxygen consumption, that is, after protons form potential energy in the mitochondrial membrane through the respiratory chain, part of Proton reflux can form ATP by ATP synthase, converting potential energy into energy in ATP. Part of it passes through the mitochondrial membrane but is only oxidized, the potential energy is converted to heat, but not used to synthesize ATP. Oligo is an ATP synthase inhibitor.
  • FCCP is an uncoupling agent. As a proton carrier, it makes a large number of protons return and consume a lot of oxygen, but this proton return cannot form ATP.
  • the increase in oxygen consumption after FCCP represents the maximum oxygen consumption capacity of mitochondria, which indirectly shows the largest oxygen consumption capacity. breathing capacity, and its high value relative to the base value represents its additional breathing potential.
  • the last additions are antimycin A and oligomycin (R+A), both of which are respiratory chain inhibitors and completely prevent mitochondrial oxygen consumption.
  • HaCaT cells were transfected with control miRNA mimic (ThermoFisher Scientific, 4464059) or miR-31 mimic (ThermoFisher Scientific, 4464067) after 24 hours of intervention with DMSO and 10 ⁇ M CB-839 respectively; among them, DMSO intervention was given 24 hours after transfection of control miRNA mimic 24 hours after transfection of miR-31 mimic was recorded as Ctrl group, 24 hours after transfection of miR-31 mimic was recorded as miR-31 group, and 24 hours after transfection of control miRNA mimic was recorded as CB839 group, and 10 ⁇ M CB-839 intervention was recorded as CB839 group, transfected with miR- After 31 mimic 24 hours, the 10 ⁇ M CB-839 intervention group was designated as miR-31+CB839 group. The level of OCR was detected 24h after the intervention.
  • the basal medium was low sugar (1 mM) glutamine (2 mM) medium. The results are shown in Figure 10.
  • Western Blotting assay can detect the expression of target protein.
  • HaCaT cells transfected with control miRNA mimic or miR-31 mimic were given DMSO and 10 ⁇ M CB-839 for 24 hours after transfection; among them, those transfected with control miRNA mimic and given DMSO for 24 hours were recorded as the Ctrl-mimic group, transfected with miR- 31 mimic treated with DMSO after 24 hours was recorded as miR-31 mimic group, transfected with control miRNA mimic 24 hours later and given 10 ⁇ M CB-839 intervention as CB839 group, transfected with miR-31 mimic 24 hours later and given 10 ⁇ M CB-839 group. 839 intervention group was denoted as miR-31+CB839 group.
  • HaCaT cells were transfected with control siRNA (ThermoFisher Scientific, AM4635) and GS-siRNA (ThermoFisher Scientific, AM16708) 24 hours later and each was given DMSO intervention as a control, which were recorded as the Ctrl-siRNA group and the GS-siRNA group, respectively. After 24 hours of intervention, the cells were collected to extract total cell protein for Western Blotting experiment, and the phosphorylation levels of 4EBP1, S6 and p70S downstream molecules of mTOR pathway were detected. The results are shown in Figure 11.
  • Figure 11 shows that overexpression of miR-31 and inhibition of GS (glutamine synthase) gene expression levels can significantly increase the phosphorylation levels of 4EBP1, S6, and p70S, while CB839 can significantly inhibit miR-31-induced mTOR pathway Increased phosphorylation levels of downstream molecules 4EBP1, S6, p70S.
  • MTS (CAS: 138169-43-4) is a tetrazolium compound, which can be reduced to colored formazan products by various dehydrogenases in the mitochondria of living cells. related.
  • HaCaT cells were transfected with control miRNA mimic, miR-31 mimic or miR-31 inhibitor (ThermoFisher Scientific, 4464084) for 24 hours, and each was given different concentrations of CB-839 to intervene for 16 hours; marked as Ctrl group, miR-31+ group and miR-31-group;
  • Inoculation of cells trypsinize various cells, then prepare a single cell suspension with a culture medium containing 10% fetal calf serum, inoculate 1000 cells per well into a 96-well plate, and the volume of each well is 100 ⁇ L;
  • CB-839 has a significant inhibitory effect on the activity of keratinocytes, and the inhibitory effect on keratinocytes overexpressing miR-31 is the most obvious.
  • the keratinocyte Hacat cells were cultured in 5% FBS MEM medium, 80% of the culture dish was confluent, and the seed plate was trypsinized, 800 cells per well/6-well plate. The next day, the cells were adhered and the medium was changed, and different concentrations of CB-839 were used for stimulation for 7 days. Then, the culture medium was removed for crystal violet staining, and the cells were counted and photographed under a microscope to collect data. The results are shown in Figure 13.
  • Example 5 CB-839 can down-regulate the expression of a series of miR-31 or TNF- ⁇ -induced pro-inflammatory factors
  • the inflammation model of keratinocytes was induced by transfection of miR-31 mimic for 48h (miR-31 group) or by adding TNF- ⁇ 20ng/mL and stimulated for 16h (TNF- ⁇ group), and then treated with 10 ⁇ M CB-839 overnight.
  • the control group was transfected with miRNA mimic or added DMSO without CB-839 intervention. Finally, the cell culture medium was collected, and the expression of a series of cytokines was detected by Luminex.
  • Example 6 The treatment experiment of glutaminase inhibitor CB-839 on IMQ-induced psoriasis model mice
  • Drug preparation Take an appropriate amount of CB-839 powder, dissolve it in DMSO solvent, and prepare 2mg/mL, 6mg/mL, and 18mg/mL CB-839 solutions for use.
  • the blank group (Ctrl group) was not given imiquimod external coating, but was given Vaseline external coating in the morning, and 100 ⁇ L DMSO solvent was externally coated in the afternoon.
  • the model group (IMQ group) was given 50 mg of imiquimod (imiquimod cream, produced by Sichuan Mingxin Pharmaceutical Co., Ltd.) daily in the morning, and 100 ⁇ L of DMSO solvent was given externally in the afternoon.
  • CB-839 low-dose group (CB-839L group) was given 50 mg imiquimod in the morning and 2 mg/mL CB-839 100 ⁇ L in the afternoon.
  • CB-839 middle-dose group (CB-839M group) was given 50 mg imiquimod in the morning and 6 mg/mL CB-839 100 ⁇ L in the afternoon.
  • CB-839H group The CB-839 high-dose group (CB-839H group) was given 50 mg imiquimod in the morning and 18 mg/mL CB-839 100 ⁇ L in the afternoon.
  • Calcipotriol betamethasone gel group (Xamiol group) was given 50 mg of imiquimod in the morning, and 40 ⁇ L of calcipotriol betamethasone gel (Samel) was given externally in the afternoon.
  • mice Before administration, the skin of about 2 ⁇ 3 cm on the back of all mice was sheared and depilated to expose the back skin. Drugs were administered on days 1-7, and the experiment was completed on day 8 and materials were collected. The body weight of the mice was recorded on days 0, 2, 4, 6 and 8, and the back skin of the mice was photographed every day.
  • Fig. 17 shows the back skin of the mouse on the 8th day of the experiment. From the results of Fig. 17, it can be seen that erythema, papules and scales appeared on the skin of the mouse after induction by daily administration of 50 mg of imiquimod. Compared with the model group, the skin lesions of the mice in each CB-839 dose group were improved at different levels, erythema was reduced, papules and scales were reduced, and the improvement effect was better than that of the calcipotriol betamethasone gel group. . This indicated that CB-839 could significantly improve IMQ-induced skin psoriasis-like changes in mice.
  • mice in the model group had obvious hyperplasia of the epidermis, local inflammatory cell infiltration, increased angiogenesis, hyperkeratosis of the epidermis stratum corneum with parakeratosis, which was similar to the pathology of psoriatic skin lesions.
  • CB-8396mg/mL After external application of CB-8396mg/mL, the skin lesions of the mice were significantly improved, the acanthosis was reduced, the inflammatory infiltration and angiogenesis were reduced. This indicates that CB-839 has therapeutic effect on psoriasis mouse model.
  • Epidermal thickness reflects the speed of basal cell proliferation and is also one of the pathological features of psoriasis.
  • the microvessels in the skin lesions of the mice in each group were counted, and the results are shown in Figure 21 . It can be seen from the results in Fig. 21 that the number of microvessels in the skin lesions of the mice in the model group was significantly increased. Compared with the model group, the number of microvessels in the mice in the CB-839 treatment group was significantly reduced, and the number of microvessels in the middle-dose CB-839 administration group decreased most significantly (P ⁇ 0.001).
  • Drug preparation Take an appropriate amount of CB-839 powder, dissolve the drug in the external coating group in DMSO solvent, and prepare a 6 mg/mL CB-839 solution for later use.
  • the drug solvent of CB-839 oral group was 0.9%NaCl, the final concentration was 20mg/mL; the drug solvent of intraperitoneal injection group and subcutaneous injection group was 20%DMSO+1%Tween20+0.9%NaCl, and the final concentration was 6mg/mL.
  • the blank group (Ctrl group) was not given imiquimod external coating, and was given Vaseline external coating every morning.
  • the model group (IMQ group) was given 50 mg of imiquimod (imiquimod cream, produced by Sichuan Mingxin Pharmaceutical Co., Ltd.) every morning.
  • the CB-839 external coating group (Topical group) was given 50 mg of imiquimod in the morning and 100 ⁇ L of CB-839 6 mg/mL in the afternoon.
  • the CB-839 oral group (Oral group) was given 50 mg of imiquimod in the morning, and 20 mg/mL of CB-839 100 ⁇ L in the afternoon.
  • CB-839 intraperitoneal injection group (Intraperitoneal group) was given 50 mg of imiquimod in the morning, and 6 mg/mL CB-839 100 ⁇ L intraperitoneal injection was given in the afternoon.
  • the CB-839 subcutaneous injection group (Subcutaneous group) was given 50 mg of imiquimod in the morning and 100 ⁇ L of CB-839 100 ⁇ L subcutaneously in the afternoon.
  • mice Before administration, the skin of about 2 ⁇ 3 cm on the back of all mice was sheared and depilated to expose the back skin. Drugs were administered on days 1-4, and the experiment was completed on day 5 and materials were collected. The body weight of mice was recorded on days 1, 3, and 5.
  • Fig. 26 shows the back skin of the mouse on the fifth day of the experiment. From the results of Fig. 26, it can be seen that erythema, papules and scales appeared on the skin of the mice after being induced by daily administration of 50 mg of imiquimod. Compared with the model group, the skin lesions of the mice in the CB-839 external coating group were significantly improved, the erythema was reduced, the papules and scales were reduced, and the improvement effect was better than the oral group, the intraperitoneal injection group, and the subcutaneous injection group. It shows that the administration mode of CB-839 external coating has the most obvious improvement on IMQ-induced skin psoriasis in mice.
  • the skin of the skin lesions of the mice in each group was taken, fixed and then prepared into paraffin sections and stained with H&E.
  • the results are shown in Figure 27. From the results in Figure 27, compared with the blank group, the epidermis of the mice in the model group was significantly proliferated, local inflammatory cells infiltrated, angiogenesis increased, and the epidermal stratum corneum was hyperkeratinized with parakeratosis, which was similar to the pathology of psoriatic skin lesions. After external application of CB-8396mg/mL, the skin lesions of the mice were significantly improved, the acanthosis was reduced, the inflammatory infiltration and angiogenesis were reduced. The degree of improvement was better than that of other administration route groups, suggesting that external application of CB-839 was the optimal administration method.
  • mice in each group were taken and weighed.
  • Spleen index spleen weight (mg) ⁇ body weight (g) ⁇ 10 ⁇ 100%. It can be seen from the results in Fig. 28 that, compared with the blank group, the spleen index of the mice in the model group was significantly increased. Compared with the model group, the spleen index of the mice in the external application CB-839 group was significantly reduced, and it was better than the oral group, the intraperitoneal injection group and the subcutaneous injection group, suggesting that the external application of CB-839 inhibited the hyperactivity of immune cells induced by IMQ in mice The most obvious effect.
  • Epidermal thickness reflects the speed of basal cell proliferation and is also one of the pathological features of psoriasis.
  • the results of the epidermal thickness of the skin lesions of the mice in each group are shown in Figure 29. It can be seen from the results in Figure 29 that the average thickness of the skin lesions of the mice in each group increased after induction with imiquimod. Compared with the model group, the epidermal thickness of the mice in the CB-839 external coating group was significantly improved compared with the model group, and the difference was statistically significant. The improvement of the epidermal thickness of the mice in the CB-839 external coating group was better than that in the intraperitoneal injection group. There was no statistical difference in the improvement of epidermal thickness between the oral group and the subcutaneous injection group compared with the model group.
  • GSE13355 and GSE30999 were downloaded from the Pubmed database through bioinformatics technology and data mining methods. Both studies used the same gene chip technology to detect gene expression in psoriasis patient skin lesions (PL), non-lesion (PN) and healthy human skin (HC) tissues.
  • PL psoriasis patient skin lesions
  • PN non-lesion
  • HC healthy human skin
  • a total of 164 gene expression data involved in glycolysis pathway, glutamine metabolism pathway, tricarboxylic acid cycle, pentose phosphate metabolism pathway, fatty acid metabolism, and metabolite transporters were extracted, and PL specimens were found by principal component analysis (Fig. 30a). Aggregated together and separated from HC and/or PN specimens, indicating that the gene expression profiles of metabolic enzymes in PL specimens were significantly different from those in HC and PN.
  • a series of genes specifically expressed in PL tissue were identified, including 12 up-regulated genes and 6 down-regulated genes.
  • up-regulated genes GLUT1, HK2, PGM2 and SLC16A1 involved in glycolysis were included; GOT1, GOT2 and GS involved in glutamine metabolic pathway were also included (Fig. 30b).
  • Glycolytic pathway and glutamine metabolism pathway are important sources for rapidly proliferating cells to synthesize energy and substances. The above results suggest that the rapid proliferation of psoriatic skin lesions is related to hyperglycolytic and glutamine metabolism.
  • Example 8 miR-31 is specifically highly expressed in psoriatic skin lesions and participates in the metabolic reorganization of skin lesions keratinocytes
  • miR-31 The expression of miR-31 in the skin of psoriasis patients or healthy volunteers was detected by in situ hybridization; the expression of key metabolic enzymes was detected by cell experiments and metabolomics experiments.
  • Figures 31-32 show the staining results and quantitative analysis results of miR-31 in psoriatic skin lesions (Ps group) and normal human skin (HC group) in turn.
  • the results show that: (1) miR-31 is highly expressed in psoriasis (2) miR-31 was mainly distributed in the spinous layer and granular layer, but not in the basal layer.
  • Figure 33 shows the metabolite levels detected by metabolomic technology after overexpression of miR-31. It was found that miR-31 significantly affects metabolites including glucose metabolism and amino acid metabolism.
  • FIG. 34 shows the results of amino acid marker proteomics.
  • miR-31 was overexpressed in a keratinocyte cell line (HaCaT) and a non-keratinocyte cell line (HEK 293T), and then metabolomic detection was used in both cells.
  • HaCaT keratinocyte cell line
  • HEK 293T non-keratinocyte cell line
  • Figure 35 is a summary graph illustrating that miR-31 has a role in mediating cellular metabolic reorganization.

Abstract

本发明公开谷氨酰胺酶抑制剂在制备治疗银屑病的药物中的应用。本发明通过体外细胞实验证实了谷氨酰胺酶抑制剂可以抑制角质细胞代谢谷氨酰胺、抑制miR-31诱导上调的角质细胞线粒体呼吸、抑制miR-31诱导的mTOR通路、降低细胞活性并促进细胞凋亡以及具有抗炎作用；以及通过动物实验证实了谷氨酰胺酶抑制剂可以有效治疗咪喹莫特诱导的小鼠银屑病样病理改变，包括：显著降低皮损的Baker评分、有效减轻表皮肥厚程度、显著减少表皮下微血管数量、显著减少表皮层Ki67阳性细胞数和降低表皮GLS的表达。因此，谷氨酰胺酶抑制剂可用于制备治疗银屑病的药物。

Description

谷氨酰胺酶抑制剂在制备治疗银屑病的药物中的应用 技术领域
本发明属于医药技术领域，涉及谷氨酰胺酶抑制剂的用途，具体涉及谷氨酰胺酶抑制剂在制备治疗银屑病的药物中的应用。
背景技术
银屑病是以皮肤红斑、丘疹、鳞屑为主要临床表现的慢性炎症性皮肤病，全球发病率约为1-3％。银屑病以皮损表皮过度增殖、局部炎症细胞浸润为主要病理特征，并且局部皮损代谢谱发生改变，发病机制复杂，涉及遗传、免疫-炎症、血管异常等多个方面，容易反复发作，迁延难愈。目前用于治疗银屑病的药物主要包括糖皮质激素、维A酸类药物、维生素D3衍生物、地蒽酚、细胞毒性药物等，其中，他扎罗汀、中效与强效的糖皮质激素、卡泊三醇为目前银屑病局部治疗的一线药物。虽然目前银屑病的药物治疗方案较多，但其治疗效果仍未能完全令人满意，现有技术中仍缺少一种疗效显著、副作用小的治疗银屑病的药物。
CB-839(CAS:1439399-58-2)是一种谷氨酰胺酶1(GLS1)抑制剂，可通过靶向抑制GLS1酶活性调控细胞代谢过程，其分子量为571.574，分子式为C 26H 24F 3N 7O 3S，结构式如下：
Figure PCTCN2021101850-appb-000001
廖英等发表的论文表明CB-839对三阴性乳腺癌、神经胶质瘤和多发性骨髓等多种肿瘤细胞均具有抗增殖活性；对耐药小细胞肺癌细胞在极低浓度作用下即存在强杀死作用；此外，CB-839的抗肿瘤作用在肾上皮细胞、软组织肉瘤等中也得到了证实(详见：廖英，冀林华，崔森.谷氨酰胺酶新型抑制剂及其抗肿瘤活性研究进展[J].中国肿瘤临床,2019,46(7):366-369.)
发明内容
本发明的目的在于提供谷氨酰胺酶抑制剂在制备治疗银屑病的药物中的应用。
根据本发明的一个方面，提供了谷氨酰胺酶抑制剂在制备治疗银屑病 的药物中的应用。
在一些实施方式中，谷氨酰胺酶抑制剂可以选自阿西维辛(Acivicin，CAS:42228-92-2)、化合物968(CAS:311795-38-7)、BPTES(CAS:314045-39-1)、CB-839中的一种或多种。
在一些实施方式中，谷氨酰胺酶抑制剂可以为CB-839。CB-839为GLS抑制剂，可以通过抑制谷氨酰胺转化为谷氨酸的生化过程，从而阻断细胞以谷氨酰胺作为原料生产能量和氨基酸等基本物质。
在一些实施方式中，在治疗银屑病的药物中，CB-839的重量百分含量可以为2％-8％。
在一些实施方式中，CB-839可以作为单一活性成分或与其他药学上可接受的成分一起用于制备治疗银屑病的药物。
在一些实施方式中，药学上可接受的成分可以包括与CB-839没有拮抗作用的药物活性成分和/或药学上可接受的一种或多种辅料。
在一些实施方式中，治疗银屑病的药物的给药方式可以为注射、口服或透皮给药。
在一些实施方式中，治疗银屑病的药物的给药方式可以为局部透皮给药。
在一些实施方式中，治疗银屑病的药物的剂型可以为软膏剂、凝胶剂、贴膏剂、膏药、洗剂、涂膜剂、糊剂、气雾剂或喷雾剂。
本发明的有益效果在于：
(1)发现了谷氨酰胺酶(glutaminase,GLS)是靶向代谢治疗银屑病的重要靶标，提供了谷氨酰胺酶抑制剂(包括CB-839和BPTES等)在制备治疗银屑病药物中的用途。
(2)通过体外细胞实验证实了CB-839可以：1)显著抑制角质细胞代谢谷氨酰胺；2)显著抑制miR-31诱导上调的角质细胞线粒体呼吸；3)显著抑制miR-31诱导的mTOR通路；4)显著降低细胞活性并促进细胞凋亡，特别是促进高表达miR-31的角质细胞凋亡；5)下调一系列miR-31和/或TNF-α诱导的促炎因子的表达，特别是GM-CSF和TGF-β1。
(3)进一步通过动物实验证明了外涂CB-839可有效治疗咪喹莫特(IMQ)诱导的小鼠银屑病样病理改变，包括：1)显著降低皮损的Baker评分；2)有效减轻表皮肥厚程度；3)显著减少表皮下微血管数量；4)显著减少表皮层Ki67阳性细胞数；5)显著降低表皮GLS的表达。
(4)通过动物实验证明了外涂CB-839给药对IMQ诱导的银屑病小鼠模型的治疗作用优于口服给药、腹腔给药和皮下注射给药，包括：1)外涂给药能够显著减轻IMQ小鼠表皮皮损；2)显著减轻IMQ小鼠脾脏指数； 3)显著降低皮损肥厚程度。
附图说明
图1表示CB-839干预12h过程中角质细胞对谷氨酰胺的总摄取量，图中， ***表示与所标记组相比，P<0.001；
图2表示CB-839干预12h过程中角质细胞分泌的谷氨酸的总分泌量，图中， ***表示与所标记组相比，P<0.001；
图3-9依次表示CB-839干预后角质细胞中谷氨酰胺、天冬氨酸、α-酮戊二酸、琥珀酸、延胡索酸和苹果酸的代谢水平随时间的变化情况；
图10表示不同处理组，miR-31诱导的角质细胞细胞氧消耗率的变化情况，图中， ***表示同一时间点miR-31组与miR-31+CB839组之间的统计学差异P<0.001，ns表示两者无显著统计学差异；
图11表示不同处理组细胞中mTOR通路下游分子4EBP1、S6、p70S的磷酸化水平；
图12表示不同处理组角质细胞的生长曲线，图中，同一时间点， *表示与Ctrl组相比，P<0.05； ***表示与Ctrl组相比，P<0.001；
图13表示不同浓度CB-839干预的角质细胞的克隆形成情况；
图14-15依次表示CB-839对miR-31、TNF-α两种方法诱导的角质细胞的促炎因子的表达的抑制作用；
图16表示给药期间各组小鼠的体重变化；
图17表示各组小鼠第8天的皮损情况；
图18表示各组小鼠皮损组织H&E染色结果；
图19表示各组小鼠皮损部位皮肤的Baker评分结果，图中， **表示与所标记组相比，P<0.01； ***表示与所标记组相比，P<0.001；
图20表示各组小鼠皮损组织表皮厚度，图中， *表示与所标记组相比，P<0.05； ***表示与所标记组相比，P<0.001； ****表示与所标记组相比，P<0.0001；
图21表示各组小鼠皮损组织的皮下微血管的计数结果，图中， *表示与所标记组相比，P<0.05； ***表示与所标记组相比，P<0.001；
图22-23表示各组小鼠皮损组织的表皮层Ki67阳性细胞的增殖情况，图24中， **表示与所标记组相比，P<0.01； ***表示与所标记组相比，P<0.001；
图24-25表示各组小鼠皮损组织中GLS酶的表达与分布情况，图26中， *表示与所标记组相比，P<0.05； ***表示与所标记组相比，P<0.001；
图26表示CB-839不同给药方式对IMQ小鼠皮损的影响；
图27表示各组小鼠皮损组织H&E染色结果；
图28表示各组小鼠脾脏指数比较结果；图27中， *表示与所标记组相比， *P<0.05； **表示与所标记组相比， *P<0.01； ****表示与所标记组相比， ****P<0.0001；
图29表示各组小鼠皮损组织的表皮厚度(μm)；图27中， *表示与所标记组相比， *P<0.05； **表示与所标记组相比， *P<0.01； ***表示与所标记组相比， ***P<0.001； ****表示与所标记组相比， ****P<0.0001；
图30表示银屑病患者皮损组织中特异表达的细胞代谢基因表达谱；
图31-32表示miR-31在银屑病皮损,和正常人皮肤中的染色结果和定量分析结果，图32中， ***表示与所标记组相比，P<0.001；
图33表示过表达miR-31后利用代谢组学技术检测代谢物的水平的结果；
图34表示表达miR-31后，角质细胞和非角质细胞上细胞代谢相关基因蛋白的表达水平；
图35为图31-34的结果的总结图。
具体实施方式
下面结合具体实施例和附图对本发明作进一步详细的说明。如无特殊说明，实施例中所用试剂均为市售，所用的技术手段为本领域技术人员所熟知的常规手段。
本发明发明人在长期的银屑病的作用及机制研究过程中，通过相关研究发现：
银屑病病理进程与miR-31参与诱导的角质细胞代谢重组密不可分，具体表现为受累基底层角质细胞首先过表达葡萄糖转运蛋白1(GLUT1)和谷氨酰胺酶(GLS)而提高糖酵解和谷氨酰胺代谢途径，从而为其快速增殖和分化提供足够的能量和基础物质。随后，基底层角质细胞向表层分化，而分化的角质细胞通过上调miR-31而抑制GLUT1并维持细胞高表达GLS，最终致使棘层和颗粒层的角质细胞依旧可以依赖谷氨酰胺代谢途径和激活的mTOR通路维持细胞生存并参与其他病理改变。由此可见GLS在银屑病皮损角质细胞代谢重组中的扮演着关键角色。基于上述发现，完成本发明。
实施例1 CB-839抑制角质细胞谷氨酰胺的摄取和代谢实验
将10μM的CB-839(CB-839组)和等体积的DMSO(Ctrl组)分别添加到角质细胞HaCaT细胞的培养液中，干预12h后，收集细胞培养液，利用靶向代谢组学的方法检测收集到的细胞培养液中谷氨酰胺(glutamine)和谷氨酸(glutamate)的含量，并通过与新鲜的培养液对比，计算出不同干预 条件下细胞摄取的谷氨酰胺的量(即谷氨酰胺的消耗量)和分泌到细胞培养液中的谷氨酸的量(即谷氨酸的分泌量)，结果如图1-2所示。由图1-2的结果可知，CB-839可以显著抑制角质细胞摄取谷氨酰胺(P<0.001)，以及减少角质细胞分泌到培养液中的谷氨酸的量(P<0.001)。
收集培养液后，往细胞中添加新鲜的培养液继续对细胞进行培养，并每隔一段时间收集细胞，同样采用靶向代谢组学的方法检测细胞样品中多个谷氨酰胺代谢途径下游的代谢物的含量。结果如图3-9所示。
由图3-9的结果可知，除谷氨酰胺外，CB-839可以明显抑制角质细胞内包括谷氨酸、天冬氨酸(Aspartate)、α-酮戊二酸(alpha-KG)、琥珀酸(succinate)、延胡索酸(fumarate)和苹果酸(malate)的水平，说明CB-839能阻断角质细胞对谷氨酰胺的代谢，降低包括多个三羧酸循环途径中代谢物的水平，从而对角质细胞的物质和能量代谢产生影响。
实施例2 CB-839抑制miR-31诱导的角质细胞线粒体呼吸能力
Oxygen consumption rate(OCR)是细胞氧消耗率，为线粒体呼吸能力指标。耗氧量的检测原理：在加oligo之前显示的数值，代表的是细胞的基础耗氧量，包括线粒体氧化磷酸化及质子漏耗氧，即质子在线粒体膜通过呼吸链形成电势能后，一部分质子回流可以通过ATP合酶形成ATP，将势能转化为ATP中的能量。一部分通过线粒体膜但是只是发生氧化，势能转化为热量，但是没有用于合成ATP。oligo是ATP合酶抑制剂，加入此药后减少的耗氧代表的是机体用于ATP合成的耗氧量，间接显示此时细胞的ATP产量。FCCP是一种解偶联剂，作为一种质子载体使得大量质子回流，大量耗氧，但是这种质子回流不能形成ATP，FCCP后耗氧的增加，代表线粒体的最大耗氧能力，间接显示最大的呼吸能力，而其相对与基础值的高值代表其还具有的呼吸潜力。最后加入是抗霉素A和寡霉素(R+A)，二者是呼吸链抑制剂，完全阻止线粒体耗氧。
HaCaT细胞转染对照miRNA mimic(ThermoFisher Scientific,4464059)或miR-31 mimic(ThermoFisher Scientific,4464067)24小时后各给予DMSO和10μM CB-839干预；其中，转染对照miRNA mimic 24小时后给予DMSO干预的记为Ctrl组，转染miR-31 mimic 24小时后给予DMSO干预的记为miR-31组，转染对照miRNA mimic 24小时后给予10μM CB-839干预的记为CB839组，转染miR-31 mimic 24小时后给予10μM CB-839干预的记为miR-31+CB839组。干预24h后检测OCR的水平。基础培养基为低糖(1mM)含谷氨酰胺(2mM)培养基。结果如图10所示。
由图10的结果可知，miR-31组细胞基础水平OCR和激发的最高OCR 水平都显著高于miR-31+CB839组(P<0.001)，说明CB839可显著抑制miR-31诱导的角质细胞高水平的OCR。
实施例3 CB-839抑制miR-31诱导的mTOR通路的Western Blotting实验
Western Blotting实验可以检测目的蛋白的表达。HaCaT细胞转染对照miRNA mimic或miR-31 mimic 24小时后各给予DMSO和10μM CB-839干预；其中，转染对照miRNA mimic 24小时后给予DMSO干预的记为Ctrl-mimic组，转染miR-31 mimic 24小时后给予DMSO干预的记为miR-31 mimic组，转染对照miRNA mimic 24小时后给予10μM CB-839干预的记为CB839组，转染miR-31 mimic 24小时后给予10μM CB-839干预的记为miR-31+CB839组。另取HaCaT细胞转染对照siRNA(ThermoFisher Scientific,AM4635)和GS-siRNA(ThermoFisher Scientific,AM16708)24小时后各给予DMSO干预作为对照，分别记为Ctrl-siRNA组和GS-siRNA组。干预24h后收集细胞提取细胞总蛋白进行Western Blotting实验，检测mTOR通路下游分子4EBP1、S6、p70S的磷酸化水平。结果如图11所示。
图11结果表明，过表达miR-31和抑制GS(谷氨酰胺合成酶)基因的表达水平均可以显著提高4EBP1、S6、p70S的磷酸化水平，而CB839可以显著抑制miR-31诱导的mTOR通路下游分子4EBP1、S6、p70S的磷酸化水平的提高。
实施例4 CB-839降低角质细胞活性并促进细胞凋亡的实验
1、MTS实验检测细胞活性
MTS(CAS：138169-43-4)是一种四唑类化合物，可被活细胞线粒体中的多种脱氢酶还原成有色的甲瓒产物，其颜色深浅与活细胞数在一定范围内高度相关。
具体步骤包括：
(1)HaCaT细胞转染对照miRNA mimic、miR-31 mimic或miR-31 inhibitor(ThermoFisher Scientific,4464084)24小时后各给予不同浓度的CB-839干预16h；分别记为Ctrl组、miR-31+组和miR-31-组；
(2)接种细胞：用胰蛋白酶消化各种细胞，然后用含10％胎小牛血清的培养液配成单个细胞悬液，以每孔1000个细胞接种到96孔板，每孔体积100μL；
(3)呈色：每孔加MTS溶液20μL继续孵育2～4h；
(4)比色：选择490nm波长，在酶联免疫监测仪上测定各孔光吸收值， 记录结果，以时间为横坐标，吸光值为纵坐标绘制细胞生长曲线。结果如图12所示。
由图12的结果可知，CB-839对角质细胞活性具有显著抑制作用，对miR-31过表达角质细胞的抑制作用最明显。
2、Colony Forming(细胞集落形成)实验
将角质形成细胞Hacat细胞培养于5％FBS MEM培养基，80％铺满培养皿后用胰蛋白酶消化种板，每孔800细胞/6孔板。次日细胞贴壁后换液，应用不同浓度的CB-839干预刺激7天，然后去培养基行结晶紫染色液染色，镜下计数拍照，采集数据。结果如图13所示。
由图13的结果可知，随着CB-839浓度的增加，其对细胞克隆形成的抑制作用越明显。
实施例5 CB-839可下调一系列miR-31或TNF-α诱导的促炎因子的表达
分别通过转染miR-31 mimic 48h(miR-31组)或加入TNF-α20ng/mL刺激16h(TNF-α组)两种方法诱导角质细胞的炎症模型，然后给予10μM CB-839治疗过夜，转染对照miRNA mimic或加入DMSO、不给予CB-839干预为Ctrl组，最后收集细胞培养液，通过Luminex检测一系列细胞因子的表达。结果如图14-15所示，其中，miR-31-、CB-839-表示转染对照miRNA mimic但不给予CB-839干预，miR-31+、CB-839-表示转染miR-31 mimic但不给予CB-839干预，miR-31+、CB-839+表示转染miR-31 mimic且给予CB-839干预；TNF-α-、CB-839-表示加入DMSO但不给予CB-839干预，TNF-α+、CB-839-表示加入TNF-α但不给予CB-839干预，TNF-α+、CB-839+表示加入TNF-α且给予CB-839干预。
图14-15结果表明两种不同造模方式均可以诱导不同促炎因子的产生，即造模成功，而CB-839在不同的模型上表现出对不同的抑制作用，其中对GM-CSF、IL-1β、sCD163和TGF-β的抑制作用最稳定，说明CB-839具有一定的抗炎作用。
实施例6 谷氨酰胺酶抑制剂CB-839对IMQ诱导的银屑病模型小鼠的治疗实验
1、药物准备：取适量的CB-839粉末，溶于DMSO溶剂中，分别制备2mg/mL、6mg/mL、18mg/mL的CB-839溶液，备用。
2、实验过程：采用SPF级雄性BALB/c小鼠，随机分配成6组，每组4-5只。组别及相应处理分别是：
空白组(Ctrl组)，不给予咪喹莫特外涂，每日上午给予凡士林外涂，下午给予DMSO溶剂100μL外涂。
模型组(IMQ组)，每日上午给予咪喹莫特(咪喹莫特乳膏，四川明欣药业有限责任公司生产)50mg外涂，下午给予DMSO溶剂100μL外涂。
CB-839低剂量组(CB-839L组)，每日上午给予咪喹莫特50mg外涂，下午给予2mg/mL CB-839100μL外涂。
CB-839中剂量组(CB-839M组)，每日上午给予咪喹莫特50mg外涂，下午给予6mg/mL CB-839100μL外涂。
CB-839高剂量组(CB-839H组)，每日上午给予咪喹莫特50mg外涂，下午给予18mg/mL CB-839100μL外涂。
卡泊三醇倍他米松凝胶组(Xamiol组)，每日上午给予咪喹莫特50mg外涂，下午给予卡泊三醇倍他米松凝胶(赛美尔)40μL外涂。
给药前先对所有小鼠后背约2×3cm大小皮肤进行剪毛、脱毛，使其后背皮肤暴露。第1-7天给药，第8天实验结束并取材。第0、2、4、6、8天记录小鼠体重，每天对小鼠后背皮肤拍照。
3、实验结果：
3.1、CB-839治疗对IMQ诱导的银屑病模型小鼠的体重的影响
结果如图16所示。由图16的结果可知，给予IMQ诱导第二天可使小鼠体重明显减轻(体重损失约10％)，第4天体重开始回升，而CB-839低、中、高剂量组的小鼠体重变化，在IMQ给药期间均较为平稳。
3.2、CB-839治疗对IMQ诱导的银屑病模型小鼠皮损的影响
图17表示的是实验第8天的小鼠的后背皮肤，由图17的结果可知，每天给予50mg咪喹莫特诱导之后小鼠皮肤出现红斑、丘疹和鳞屑。相对于模型组，各CB-839剂量组的小鼠皮损组织均出现不同层度的改善，红斑减少，丘疹和鳞屑有所减少，且改善效果优于卡泊三醇倍他米松凝胶组。说明CB-839可显著改善IMQ诱导的小鼠皮肤银屑病样改变。
3.3、CB-839治疗后IMQ诱导的银屑病模型小鼠的皮损组织病理评价(1)H&E染色
取各组小鼠皮损部位的皮肤，固定后制备石蜡切片，并进行H&E染色，结果如图18所示。由图18的结果可知，相对于空白组，模型组小鼠表皮明显增生，局部炎症细胞浸润，血管生成增多，表皮角质层过度角化伴角化不全，与银屑病皮损病理相类似。而给予CB-8396mg/mL浓度药物外涂后，小鼠皮损明显好转，棘层肥厚减轻，炎症浸润减少，血管生成减少。 说明CB-839对银屑病小鼠模型具有治疗作用。
(2)Baker评分
银屑病的组织病理观察主要采用Baker评分，具体标准如表1所示：
表1 银屑病的Baker评分标准
Figure PCTCN2021101850-appb-000002
利用显微镜观察H&E染色后的各组小鼠皮损部位的皮肤的结构，并进行Baker评分，结果如图19所示。由图19的结果可知，相对于对照组，咪喹莫特诱导模型组小鼠Baker评分显著增加(P＜0.0001)。CB-839治疗组Baker评分有所改善，其中，中剂量(6mg/mL)治疗组Baker改善具有统计学意义(P＜0.01)。说明CB-839治疗对咪喹莫特诱导银屑病模型小鼠皮损组织的炎症具有一定的抑制作用。
(3)皮损组织表皮厚度
表皮厚度反应基底层细胞增殖的快慢，也是银屑病病理特征之一。
各组小鼠皮损组织表皮厚度的结果如图20所示。由图20的结果可知，给予咪喹莫特诱导后各组小鼠皮损组织表皮平均厚度均有所增加。相对于模型组，CB-839不同剂量组治疗小鼠的表皮厚度都有所改善，差异具有统计学意义。
(4)微血管分布
对各组小鼠皮损组织的微血管进行计数，结果如图21所示。由图21的结果可知，模型组小鼠皮损组织的微血管数量显著增多。相对于模型组，CB-839治疗组小鼠的微血管数量均显著减少，其中以中剂量的CB-839给药组微血管数量减少最为明显(P＜0.001)。
3.4、CB-839对IMQ诱导的银屑病模型小鼠皮损组织角质细胞增殖的影响
通过免疫组化法分析各组小鼠皮损组织中Ki67阳性细胞的增殖情况，结果如图22和图23所示。由图22和图23的结果可知，模型组小鼠表皮基底层Ki67阳性细胞数显著高于空白组小鼠(P＜0.001)。而经过CB-839中剂量(6mg/mL)治疗后，小鼠Ki67阳性细胞数显著降低(P＜0.01)，说明CB-839对IMQ诱导的银屑病模型小鼠具有抗角质细胞过度增殖的作 用。
3.5、CB-839对IMQ诱导的银屑病模型小鼠皮损组织GLS酶表达的影响
通过免疫组化法分析各组小鼠皮损组织中GLS酶的表达与分布，结果如图24和图25所示。由图24和图25的结果可知，相对于空白组小鼠，模型组小鼠皮损组织GLS表达水平显著升高(P＜0.001)。CB-839中剂量(6mg/mL)治疗组小鼠GLS酶表达水平显著降低(P＜0.05)，说明CB-839对IMQ诱导的银屑病模型小鼠皮损组织GLS酶的表达具有抑制作用。
4、CB-839不同给药方式(外涂、口服、腹腔注射、皮下注射)对IMQ诱导的银屑病模型小鼠皮损的影响
4.1、药物准备：取适量的CB-839粉末，外涂组药物溶于DMSO溶剂中，制备6mg/mL的CB-839溶液，备用。CB-839口服组药物溶剂为0.9％NaCl，终浓度为20mg/mL；腹腔注射组、皮下注射组药物溶剂为20％DMSO+1％Tween20+0.9％NaCl，终浓度均为6mg/mL。
4.2、实验过程：采用SPF级雄性BALB/c小鼠，随机分配成6组，每组5只。组别及相应处理分别是：
空白组(Ctrl组)，不给予咪喹莫特外涂，每日上午给予凡士林外涂。
模型组(IMQ组)，每日上午给予咪喹莫特(咪喹莫特乳膏，四川明欣药业有限责任公司生产)50mg外涂。
CB-839外涂组(Topical组)，每日上午给予咪喹莫特50mg外涂，下午给予6mg/mL CB-839100μL外涂。
CB-839口服组(Oral组)，每日上午给予咪喹莫特50mg外涂，下午给予20mg/mL CB-839100μL灌胃。
CB-839腹腔注射组(Intraperitoneal组)，每日上午给予咪喹莫特50mg外涂，下午给予6mg/mL CB-839100μL腹腔注射。
CB-839皮下注射组(Subcutaneous组)，每日上午给予咪喹莫特50mg外涂，下午给予6mg/mL CB-839100μL皮下注射。
给药前先对所有小鼠后背约2×3cm大小皮肤进行剪毛、脱毛，使其后背皮肤暴露。第1-4天给药，第5天实验结束并取材。第1、3、5天记录小鼠体重。
4.3、实验结果：
(1)CB-839治疗对IMQ诱导的银屑病模型小鼠皮损的影响
图26表示的是实验第5天的小鼠的后背皮肤，由图26的结果可知， 每天给予50mg咪喹莫特诱导之后小鼠皮肤出现红斑、丘疹和鳞屑。相对于模型组，CB-839外涂组的小鼠皮损组织明显改善，红斑减少，丘疹和鳞屑有所减少，且改善效果优于口服组、腹腔注射组、皮下注射组。说明CB-839外涂的给药方式对IMQ诱导的小鼠皮肤银屑病样的改善最为明显。
(2)H&E染色
取各组小鼠皮损部位的皮肤，固定后制备石蜡切片，并进行H&E染色，结果如图27所示。由图27的结果可知，相对于空白组，模型组小鼠表皮明显增生，局部炎症细胞浸润，血管生成增多，表皮角质层过度角化伴角化不全，与银屑病皮损病理相类似。而给予CB-8396mg/mL浓度药物外涂后，小鼠皮损明显好转，棘层肥厚减轻，炎症浸润减少，血管生成减少。改善程度优于其他给药途径组，提示外涂CB-839为最优给药方式。
(3)脾脏指数
取各组小鼠的脾脏，称重。脾脏指数＝脾脏重量(mg)÷体重(g)×10×100％。由图28的结果可知，相对于空白组，模型组小鼠脾脏指数显著升高。相较于模型组，外涂CB-839组小鼠脾脏指数显著降低，并且优于口服组、腹腔注射组和皮下注射组，提示外涂CB-839对IMQ诱导小鼠免疫细胞反应亢进的抑制作用最明显。
(4)皮损组织表皮厚度
表皮厚度反应基底层细胞增殖的快慢，也是银屑病病理特征之一。
各组小鼠皮损组织表皮厚度的结果如图29所示。由图29的结果可知，给予咪喹莫特诱导后各组小鼠皮损组织表皮平均厚度均有所增加。相对于模型组，CB-839外涂给药组治疗小鼠的表皮厚度较模型组显著改善，差异具有统计学意义。CB-839外涂组小鼠表皮厚度改善优于腹腔注射组。口服组和皮下注射组小鼠表皮厚度改善较模型组无统计学差异。
实施例7 生信手段分析证实银屑病患者皮损组织具有特异的细胞代谢基因表达谱
通过生物信息学技术与数据挖掘方法，从Pubmed数据库下载GSE13355和GSE30999研究数据。这两项研究均使用相同基因芯片技术检测银屑病患者皮损(PL)、非皮损(PN)和健康人皮肤(HC)组织中基因的表达。共提取164个参与糖酵解通路、谷氨酰胺代谢通路、三羧酸循环、磷酸戊糖代谢途径、脂肪酸代谢以及代谢物转运蛋白的基因表达数据，通过主成分分析(图30a)发现PL标本聚集一起并与HC和(或)PN标本分离，说明PL标本代谢酶的基因表达谱明显与HC和PN不同。
通过比较PL与HC各代谢基因的表达，鉴定了在PL组织中特异表达 的一系列基因，其中上调基因12个，下调基因6个。在上调基因中，包括了参与糖酵解的GLUT1、HK2,、PGM2和SLC16A1；也包括了参与谷氨酰胺代谢途径的GOT1、GOT2和GS(图30b)。糖酵解途径和谷氨酰胺代谢途径是快速增殖细胞合成能量和物质的重要来源，以上结果提示在银屑病皮损细胞的快速增殖与其亢进的糖酵解和谷氨酰胺代谢相关。
实施例8 miR-31在银屑病皮损特异高表达且参与皮损角质细胞的代谢重组
通过原位杂交技术检测银屑病患者或健康志愿者皮肤中的miR-31表达情况；通过细胞实验与代谢组学实验检测关键代谢酶的表达。
图31-32依次为miR-31在银屑病皮损(Ps组)和正常人皮肤(HC组)中的染色结果和定量分析结果，结果表明：(1)miR-31高表达于银屑病皮损组织；(2)miR-31主要分布于棘层和颗粒层，而非基底层。
图33为过表达miR-31后利用代谢组学技术检测代谢物的水平，结果发现miR-31显著影响包括糖代谢和氨基酸代谢相关的代谢物。
下图34为氨基酸标记蛋白质组学的结果，在该实验中，通过在角质细胞株(HaCaT)和非角质细胞株(HEK 293T)中过表达miR-31，然后利用代谢组检测在两种细胞上细胞代谢相关基因蛋白的表达水平，结果显示miR-31参与多个代谢酶的表达调控。
图35为总结图，说明了miR-31具有介导细胞代谢重组的作用。
以上所述的仅是本发明的一些实施方式。对于本领域的普通技术人员来说，在不脱离本发明创造构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。

Claims (9)

  1. 谷氨酰胺酶抑制剂在制备治疗银屑病的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述谷氨酰胺酶抑制剂选自阿西维辛、化合物968、BPTES、CB-839中的一种或多种。
  3. 根据权利要求2所述的应用，其特征在于，所述谷氨酰胺酶抑制剂为CB-839。
  4. 根据权利要求3所述的应用，其特征在于，所述治疗银屑病的药物中，CB-839的重量百分含量为2％-18％。
  5. 根据权利要求3或4所述的应用，其特征在于，所述CB-839作为单一活性成分或与其他药学上可接受的成分一起用于制备治疗银屑病的药物。
  6. 根据权利要求5所述的应用，其特征在于，所述药学上可接受的成分包括与CB-839没有拮抗作用的药物活性成分和/或药学上可接受的一种或多种辅料。
  7. 根据权利要求6所述的应用，其特征在于，所述治疗银屑病的药物的给药方式为注射、口服或透皮给药。
  8. 根据权利要求7所述的应用，其特征在于，所述治疗银屑病的药物的给药方式为局部透皮给药。
  9. 根据权利要求8所述的应用，其特征在于，所述治疗银屑病的药物的剂型为软膏剂、凝胶剂、贴膏剂、膏药、洗剂、涂膜剂、糊剂、气雾剂或喷雾剂。
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