CN111481657B - JZY-17 and use of compounds for the preparation of a medicament for the treatment of psoriasis - Google Patents

Abstract

The invention relates to JZY-17 and application of a compound in preparing a medicament for treating psoriasis, wherein the JZY-17 and the compound are combined to synergistically inhibit the proliferation of keratinocytes, so that the medicament has a better effect, and a mouse model also proves that the medicament combination has the effect of treating psoriasis and has a better market prospect.

Description

JZY-17 and use of compounds for the preparation of a medicament for the treatment of psoriasis

Technical Field

The invention relates to the field of medicines, in particular to JZY-17 and application of a compound in preparing a medicament for treating psoriasis.

Background

Psoriasis, commonly called psoriasis, has histopathological manifestations mainly including hyperkeratosis with parakeratosis of epidermal basal layer keratinocytes, capillary vessel hyperplasia and dilation of dermal layer, inflammatory cell infiltration, and the most common clinical manifestations of red or pink plaques, scales, etc. Psoriasis affects approximately 3% of the world population. The prevalence rate of psoriasis in Europe is higher and is 0.75% -2.9%, the average is about 2%, the prevalence rate in the United states is more than 2%, most countries in Asia is less than 1%, the national survey result in China except 1984 is 0.123%, the prevalence rate of psoriasis in Zhang Jiang is equal to that in (2007-2008) in six urban residents in the continent, and the result is 0.47%. The prevalence rates of mainland china and taiwan are increasing with time, and attention should be paid.

There is no current theory about the pathogenesis of psoriasis, and cellular immune system abnormality and keratinocyte abnormality are two main characteristic manifestations of psoriasis skin lesion tissues and are also main entry points for researching the pathogenesis of psoriasis. Among them, helper T cell (Th) 17 lymphocytes and cytokines thereof, signal transduction pathways of keratinocytes, and expression levels of microRNAs (miRNAs) in keratinocytes and the like are hot research points in recent years. In addition, Vascular Endothelial Growth Factor (VEGF) also plays an important role in the pathogenesis of psoriasis. Relevant cytokines and miRNAs are taken as treatment targets, and regulation and control of gene expression in an immune system and keratinocytes are important strategies for treating psoriasis.

Cytokines in keratinocytes can perform intracellular signal transduction through a variety of signal transduction pathways, including Ca2+ -dependent protein kinase C pathway, mitogen-activated protein kinase pathway, etc., wherein Janus kinase-signal transduction and transcriptional activator (JAKSTAT) signal transduction pathway is the research hotspot of psoriasis at present. In vitro experiments, keratinocytes in psoriatic patients partially completed the differentiation and proliferation defects of the cells under the catalysis of TRPC6 activator, while the extracellular myelin TRPV1 promoted an increase in partial cytokine expression, suggesting a bidirectional interference between TRP channels and inflammatory responses. In skin tissue biopsies of psoriatic patients, the expression of messenger RNA and related proteins of TRPC channels in keratinocytes is reduced, the level of differentiation markers is reduced, suggesting that the differentiation mechanism is impaired, and such defects are present in both skin damaged tissues and non-skin damaged tissues of psoriatic patients. In addition, TRP channels (TRPC5, TRPC5, TRPM4, TRPM7 channels) are involved in the development of relevant inflammatory responses by modulating cytokines released by T cells. Researches show that baicalein can promote the expression of keratin 1 and keratin 10 by activating TRPV4 receptor to induce calcium ion, and has the functions of promoting keratinocyte differentiation and inhibiting proliferation. At present, the TRP channel is taken as an important way for regulating the calcium ion level in tissues, and is expected to become a new target for relieving psoriasis symptoms and treating psoriasis.

There are also a number of methods for treating psoriasis in the prior art, such as huoyang, which exploits the effects of camptothecin on the autophagy and apoptosis of HaCaT cells, human primary Keratinocytes (KCs) to investigate the mechanism of camptothecin for treating psoriasis. The camptothecin has certain proliferation inhibition rate on primary keratinocytes after 24h, and is expected to be used for treating psoriasis.

However, there are currently a few studies on keratinocytes, and the provision of methods for treating psoriasis is not sufficient, and there is room for continued improvement and research.

Disclosure of Invention

When the inventors studied the skin disease-related compound, they unexpectedly found that a triterpene compound (see CN200680020518.5) in the prior art has an effect of inhibiting the proliferation of keratinocytes in addition to an antioxidant effect, an anti-inflammatory effect and a melanin production-inhibiting effect, and thus is expected to be useful for the treatment of psoriasis.

The invention provides a triterpene compound, which has a specific structure shown as a formula I.

The compounds of formula I are typically used in the form of pharmaceutical compositions comprising one or more compounds of formula I and a pharmaceutically acceptable carrier. Preferably, these compositions are in unit dosage forms, such as tablets, pills, capsules, powders, granules, suspensions, metered aerosol or liquid sprays, drops, ampoules, transdermal patches; for painting or coating or otherwise administering the drug. The principal active ingredients are typically mixed in a pharmaceutically acceptable carrier such as conventional tableting ingredients, for example corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate and dicalcium phosphate, or gums, dispersants, suspending agents or surfactants such as sorbitan monooleate and polyethylene glycol, and other pharmaceutically acceptable diluents such as water, to form a homogeneous preformulation composition containing the compound of the invention or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. .

In addition, the present inventors obtained multiple sets of polypeptides that specifically bind and inhibit proliferation of keratinocytes by polypeptide library screening.

In one aspect, the invention provides a polypeptide JZY-17 that specifically binds to and inhibits keratinocyte proliferation, the sequence of which is set forth in SEQ ID NO: 1 is shown. In preliminary analysis, the polypeptide of the invention is expected to be possibly used for treating psoriasis through model experiments.

In a further aspect, the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, a compound of formula I, and a polypeptide that inhibits keratinocyte proliferation, JZY-17. Preferably, the pharmaceutical composition is in a unit dosage form suitable for topical administration, such as an ointment or spray.

Further, the application of the compound shown in the formula I and the polypeptide JZY-17 for inhibiting the keratinocyte proliferation in the preparation of a composition for inhibiting the keratinocyte proliferation is also provided.

Further, the use of a compound of formula I and of the polypeptide JZY-17 inhibiting the proliferation of keratinocytes for the preparation of a composition for the treatment of psoriasis is also provided.

Advantageous effects

The research shows that the triterpene compound has the effects of resisting oxidation, resisting inflammation, inhibiting melanin generation and inhibiting keratinocyte proliferation; in addition, the polypeptide capable of combining and inhibiting the proliferation of the keratinocyte is obtained by screening a polypeptide library, the polypeptide and a compound are combined to synergistically inhibit the proliferation of the keratinocyte, so that the polypeptide has a good effect, and a mouse model also proves that the medicine composition has the effect of treating psoriasis and has a good market prospect.

Drawings

FIG. 1 is a graph showing the results of ELISA for binding activity of keratinocytes to polypeptides

FIG. 2 caspase-3 protein expression Effect map

Detailed Description

The present invention is described in detail below by way of examples, it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adaptations of the present invention based on the above-described disclosure.

Example 1 isolation and culture of keratinocytes

The foreskin tissue after the male circumcision is taken and immediately placed into PBS (phosphate buffer solution) added with gentamicin and amphotericin after the operation, and the tissue is cleaned for 3 times by the PBS to remove blood stains. Preparing a keratinocyte culture solution added with HKGS, gentamicin/amphotericin (500X), putting tissues into the culture solution, removing subcutaneous tissues, cutting skin tissues into small pieces, wherein the pieces are 5mmX5mm, adding dispaseII enzyme (1.2U/ml) and keeping out of the sun at 4 ℃ for overnight; separating epidermis and dermis, digesting epidermis with 0.5% pancreatin at 37 deg.C for about 5-lOmin, terminating digestion with 10% serum-containing DMEM medium, grinding on 200 mesh sieve, filtering, centrifuging, and washing with PBS twiceSuspending cells by epidermal cell culture medium with added antibiotics, and plating; cells can adhere to the wall in 3-4 days, fibroblasts do not grow in the epidermal culture medium, when the epidermal cells grow in clusters and the cell fusion degree reaches about 80 percent, trypLE is used^TMExpress enzyme digestion cells, passage, gradual reduction of antibiotic use until the use is stopped, and impurities in the cells are basically removed after the cells are passed to the second generation and liquid change. Under a microscope, the cells are observed to be in an oval or polygonal shape and a paving stone shape, the cell shapes are consistent, the cell nucleus is larger, the cell outline is clear, the edge is transparent, the cell membrane boundary is clear, the number of cells in the division period is large, and black secretory granules are arranged around part of the cells. This property is known in the art as keratinocyte cell property, and 3 rd generation cells were used for the experiment.

EXAMPLE 2 preparation of keratinocyte inhibitor Compounds

A mixture of ursolic acid (48.1g, 0.105mol), dimethyl-N, N-diethylaminophosphate (34.82g, 0.211mol), and dry tetrahydrofuran (1250ml) was heated to 35 ℃ to prepare a clear solution, and 1H-tetrazole (44.25g, 0.632mol) was added in one portion at an internal temperature of 27 ℃ and then stirred at room temperature (22 ℃) for 1 hour. After confirming the production of dimethyl phosphite by TLC, the reaction solution was cooled with acetone-dry ice, and an aqueous solution of 70% t-butyl hydroperoxide (84mL, 0.607mol) was added dropwise thereto at-20 ℃. The ice bath was removed, the temperature was gradually raised to room temperature, TLC was performed to confirm the disappearance of dimethyl phosphite and the production of dimethyl phosphate, and then the reaction was terminated with a 10% aqueous solution (300ml) of sodium hydrogen sulfite at 0 ℃. Ethyl acetate (1250mL) was added to the reaction solution, and the organic layer was separated. The organic layer was washed successively with a 10% aqueous solution of sodium hydrogen sulfite (100 ml. times.3), a 5% aqueous solution of sodium hydrogen carbonate (200 ml. times.3) and a saturated aqueous solution of sodium chloride, and dried over anhydrous magnesium sulfate. Subsequently, silica gel (400mL) was added to the organic layer, which was then concentrated to dryness under reduced pressure. The column was packed with silica gel adsorbing the organic layer, and then filled with hexane. Then, the unadsorbed material was further eluted by a hexane-packed column, and development with hexane/ethyl acetate (2: 1) was performed. Among the eluted fractions, fractions having a single component were concentrated to give 34.6g of a slightly impurity-containing compound as a gel powder. This was dissolved in dry dichloromethane (350mL), and bromotrimethylsilane (25mL, 186mmol) was added to a stream of argon gas, followed by reaction at room temperature for 1 hour. After confirmation by TLC, the reaction solution was concentrated under reduced pressure, and the residue was dissolved in dry toluene and concentrated (200ml × 2) to completely remove excess bromotrimethylsilane. The concentrate was dissolved in 95% methanol (300mL), and the solution was stirred at room temperature for 1 hour, whereby crystals were precipitated. The solution was concentrated under reduced pressure and dried with phosphoric anhydride at 50 ℃ overnight under reduced pressure, thereby yielding 23.1g of a purified compound.

Example 3 keratinocyte inhibitor polypeptide screening

Phage random 12 Peptide Library Kit (Ph.D. TM-12 Phage Display Peptide Library Kit) was purchased from New England Biolabs, USA, and the host bacterium is E.coli ER2738.

Random dodecapeptide phage display library bioscreening: pretreatment of microplates with 0.1mol/L polylysine, 1) coating 1X 10 keratinocytes prepared in example 1⁶Coated microwell plates per mL, wet box overnight at 4 ℃, then add 200 μ Ι/well fixation solution (glutaraldehyde) followed by 3 washes; 2) binding blocking buffer [ 0.1mol/LNaHCO₃(pH8.6),5mg/mL BSA,0.02％NaN₃Blocking, 6 washes of TBST, 10. mu.L of the original library diluted with 90. mu.L of TBST and added to the microwell with gentle shaking at room temperature for 60 min. 3) Eluting, namely pouring the liquid, washing the plate for 10 times, adding 100 mu L/hole of 0.2mol/L glycine-hydrochloric acid buffer solution (pH2.2), shaking for 15min at room temperature, and adding 15 mu L of 1mol/L Tris-HCl (pH9.1) to neutralize the eluent. 4) And (3) amplification, namely diluting the overnight culture solution of the Escherichia coli ER2738 by using an LB culture medium at a ratio of 1: 20, inoculating phage liquid, and carrying out vigorous shaking culture at 37 ℃ for 5 hours. The mixture was transferred to a 50mL centrifuge tube, centrifuged at 10000r/min for 10min at 4 ℃ and 80% of the supernatant was transferred, and 1/6 volumes of polyethylene glycol (PEG)/NaCl (20% PEG 8000,2.5mol/LNaCl) solution were added and precipitated overnight at 4 ℃. Suspending the precipitate in 1mL TBS after low-temperature high-speed centrifugation, adding 1/6-volume PEG/NaCl into the supernatant after short-time centrifugation, mixing uniformly, and re-precipitating on ice for 60 min; the pellet was finally resuspended in 200. mu.L TBS (containing 0.02% NaN3), and the number of phages in the eluate was titrated on LB/IPTG/X-gal plates at 4 DEG CThe supernatant was stored. The above screening process was repeated for 3 rounds. And (4) picking 30 phage blue spots in the 3 rd round, amplifying and preparing a purified phage suspension. enzyme-Linked immunosorbent assay (ELISA) assay 96-well plates were pretreated with 0.1mol/L polylysine and used with keratinocytes prepared in example 1 at 1X 10⁶Coated microwell plates per mL, wet box overnight at 4 ℃, then add 200 μ Ι/well fixation solution (glutaraldehyde) followed by 3 washes; blocking at 4 deg.C for 2H, washing the plate for 6 times, adding 100 μ L of a third round of prepared 30 phage suspension, shaking at room temperature for 2H, washing the plate for 6 times, adding HRP-labeled anti-phage secondary antibody, washing the plate for 6 times at room temperature for 1H, adding 100 μ L of TMB chromogenic substrate, adding 0.5mol/L H at 37 deg.C for 30min₂SO₄And measuring the value of A450 by using a microplate reader.

The ELISA results in fig. 1 show that 21 out of 30 clones bound well to keratinocytes and were determined to be positive. Numbered 1,2,4,5,7,8,9,10,11,13,14,15,16,17,19,21,22,23,24,25 and 27 respectively (fig. 1).

Extraction and sequencing of DNA: taking 500 mu L of phage suspension positive to ELISA experiment, adding 200 mu L of PEG/NaCl, reversing and mixing evenly, and standing for 10min at room temperature. Centrifuging at 4 deg.C and 10000r/min for 10min, centrifuging at radius of 6cm, and discarding supernatant. Resuspend the pellet in 100. mu.L iodide solution, add 250. mu.L ethanol, incubate for 10min at room temperature. Centrifuging at 4 deg.C and 10000r/min for 10min, centrifuging at radius of 6cm, discarding supernatant, washing precipitate with 70% ethanol, and resuspending the precipitate in 30 μ LTE (10 mmol/L Tris-HCl (pH8.0),1mmol/L EDTA). Sending to Shanghai bioengineering Co., Ltd for sequencing.

DNA sequencing was performed on 21 positive phage clones and the amino acid sequence of the fusion 12 peptide encoded by them was deduced from the determined DNA sequences, and it was found that the 12 peptide displayed by 4 positive clones had a highly repetitive sequence. The polypeptide sequences are respectively JZY-13: RRHVRCRWDNQH, JZY-14: MMSYVNDPWERR, JZY-17: SHRDYHWWRRSS, JZY-27: LQNQMATGHRAK are provided.

EXAMPLE 4 inhibition of cell proliferation by Polypeptides and Compounds

Taking cells in logarithmic growth phase to prepare 1 × 10⁵Cell suspension/mL, seeded into 96-well plates at 200. mu.L per well. Placing in an incubator for culturing. After the cells adhere to the wall for 2 hours, the old culture solution is discarded, 1mmol/L of each polypeptide is added for continuous culture for 48 hours, and the polypeptides are selectedThe 2-pore polypeptide with the smallest cell density, i.e., the polypeptide has the property of binding to cells and inhibiting cell proliferation. This species was selected as candidate polypeptide for subsequent experiments, respectively: JZY-13 and JZY-17. The next experiment was carried out for JZY-17 as follows.

Example 5 Crohn-Forming Rate assay to examine the Effect of Polypeptides and Compounds on keratinocyte cell proliferation

200 keratinocytes in the logarithmic growth phase were seeded in 12-well plates at a culture volume of 1 mL/well. And adding compounds and polypeptides with different concentrations for 24h, wherein the specific treatment groups are as follows:

experimental group 1: a compound of formula (I) at a final concentration of 2. mu. mol/L;

experimental group 2: polypeptide with final concentration of 2 mu mol/L;

experimental group 3: polypeptide at a final concentration of 2. mu. mol/L + compound of formula (I) at a final concentration of 2. mu. mol/L;

blank control group: drug free control

After further cultivation for 7 days, the number of clones was counted. The culture medium was discarded and washed 2 times with PBS. Fixing with methanol for 10min, discarding methanol, adding 0.02% amino black to dye for 10min, and washing off the dye solution. The number of clones was counted under an inverted microscope, and clones with more than 50 cells were defined as one valid clone, and the experiment was repeated 3 times. Then, the clone formation rate (%) was calculated as (number of clones/number of control clones) × 100% according to the formula. The specific results of the inhibition of cell proliferation by each of the groups of test drugs detected using the clonality method are shown in Table 1 below. The combination of the polypeptide and the compound can inhibit the clone formation efficiency to 97% at most, and the effect is very obvious.

TABLE 1 Effect of polypeptides and Compounds on keratinocyte cell proliferation

Group of	Clone number of formation	Clone formation efficiency (%)
			Blank control group	96.7±4.9	48.4±2.5
Experimental group 1	12.6±1.0	6.3±0.5
			Experimental group 2	22.4±1.4	11.2±0.7
Experimental group 3	5.9±0.8	3.0±0.4

EXAMPLE 6 Effect of Polypeptides and Compounds on Caspase-3 Activity

Detection of Caspase-3 activity was performed according to the Caspase-3 kit instructions. After 48 hours of reaction in each experimental group (set as in example 5), the cells of each group were collected to prepare cell suspensions, centrifuged at 4 ℃ for 10min at 1500r/min, the supernatant was discarded, the cells were adjusted to 1X 108/L after washing with cold PBS, and the cells were resuspended in lysis buffer after centrifugation at 1500r/min for 20 min. Repeatedly freezing and thawing the lysed cells, carrying out ice bath for 15min, centrifuging at the temperature of 4 ℃ for 20min at the speed of 15000r/min, carrying out protein quantification on the supernatant by using a Coomassie brilliant blue assay method, and determining the activity of caspase-3. The absorbance value was measured at 405nm with a microplate reader, and the enzyme activity was calculated according to the formula attached to the specification.

As shown in FIG. 2, the concentration of caspase-3 protein in the blank group was 18.22. + -. 1.00pmol/L, the concentration of caspase-3 protein in the compound group after reaction was 49.00. + -. 2.51pmol/L, the concentration of caspase-3 protein in the polypeptide group after reaction was 30.11. + -. 3.47pmol/L, and the concentration of caspase-3 protein in the polypeptide group after co-treatment was 75.16. + -. 2.31pmol/L, which showed that the polypeptide and the compound together had a better synergistic effect.

Example 6 experiment on psoriasis model

1. Production of animal model

The skin area of the back of BALB/c mice was cleaned to about 3cm by 3cm, and the exposed skin was topically treated with 5% imiquimod cream 42mg daily for 6 days. The skin of the mouse shows erythema, scales, scabbing and the like, which is regarded as successful model building.

2. The experiments were divided into 4 groups, blank control, model and experimental groups and positive control, 15 per group. The blank control group was not modeled, and the experimental groups were: polypeptide at a final concentration of 2. mu. mol/L + compound of formula (I) at a final concentration of 2. mu. mol/L; positive control group: the rapamycin ointment was administered continuously at a rate of 3 times per day for 14 days with a dose of 0.1% rapamycin applied continuously for 14 days. The other two groups are administered with normal saline to moisten skin 3 times per day for 14 days. And 5.0ml of abdominal aortic blood and skin of the skin lesion area were collected on day 15.

3. And (3) detecting the IFN-gamma content of the plasma of each group of mice: on the 15 th day, 5.0ml of abdominal aortic blood of each group of mice is taken, the abdominal aortic blood is centrifuged at the temperature of 4 ℃ for 3cm, the IFN-gamma content of the plasma of the mice is detected according to an ELISA method after the abdominal aortic blood is centrifuged at 2000g for 30min, the relative expression content of the p-JAK2, p-STAT1 and p-STAT3 proteins of the skin in the skin lesion area is detected by adopting a western blot technology, and the specific result is shown in Table 2.

TABLE 2 results of IFN-. gamma.and p-JAK2, p-STAT1, and p-STAT3 expression levels in mouse plasma

Compared with a control group, the serum cytokine IFN-gamma content of the model group is up-regulated, the serum IFN-gamma content of the experimental group is reduced after the medicine is taken, Western blotting shows that compared with a blank control group, the expressions of the model group skin lesion areas p-JAK2, p-STAT1 and p-STAT3 are obviously increased, and the expressions of the experimental groups p-JAK2, p-STAT1 and p-STAT3 are obviously lower than those of the model group and are also reduced more than those of a positive control group. This indicates that the effect of the experimental group is better than that of the positive control group. This demonstrates that a pharmaceutical combination of a compound and a polypeptide of the invention can protect psoriatic mice by reducing the expression of inflammatory factors in psoriatic mice.

It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of components set forth in the following description and/or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

Sequence listing

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Claims (5)

1, SEQ ID NO: 1 and the use of a compound of formula I for the preparation of a medicament for inhibiting keratinocyte proliferation

SEQ ID NO: 1 and the use of a compound of formula I for the preparation of a medicament for the treatment of psoriasis-related diseases

3. A pharmaceutical composition comprising SEQ ID NO: 1 and compounds of formula I

4. The composition of claim 3, further comprising a pharmaceutically acceptable carrier.

5. The composition according to claim 4, which is in the form of any one of spray, drop and transdermal patch.

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