JP7023026B1 - JAK inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a naturally occurring JAK inhibitor useful for prevention and treatment of diseases related to the onset and exacerbation of JAK such as rheumatoid arthritis and atopic dermatitis. The active ingredient is an extract of at least one plant selected from Sanguisorba officinalis and Geranium thunbergii. [Selection diagram] Fig. 1

Description

The present invention relates to naturally occurring JAK inhibitors. More specifically, it relates to a JAK inhibitor containing a plant extract as an active ingredient.

JAK (Janus kinase) is one of the tyrosine kinases, plays an important role in intracellular immune activation signal transduction, and is involved in the onset and exacerbation of various diseases. The signal transduction involving JAK is that JAK, which is bound to the intracellular region of gp130, which is a receptor component of IL (interleukin) -6, which is an inflammatory cytokine, binds to the receptor by IL-6. It is activated and induces STAT (signal transduction and transcriptional activator) phosphorylation via phosphorylation of gp130, which is carried out by translocation of the phosphorylated STAT into the nucleus (JAK / STAT pathway). In recent years, a method of suppressing the onset and exacerbation of a disease by inhibiting the enzyme activity of JAK has attracted attention, and various JAK inhibitors have been reported so far. There are also JAK inhibitors already on the market, for example, tofacitinib is used as a therapeutic agent for rheumatoid arthritis, delgocitinib is used as a therapeutic agent for atopic dermatitis, and ruxolitinib is used as a therapeutic agent for myelofibrosis. In addition, research and development are underway on the effectiveness of JAK inhibitors for diseases such as malignant lymphoma, pancreatic cancer, psoriasis, and alopecia areata, and further, JAK inhibitors are effective for androgenetic alopecia (AGA). There is also a report such as. However, almost all of the JAK inhibitors reported so far are chemically synthesized substances, and the naturally occurring JAK inhibitors are, for example, furanocoumarin derivatives contained in Kyokatsu (Notopterygium), which is a raw drug derived from Umbelliferae. Although there is a report (Non-Patent Document 1) that notopterygium, which is known as one, has a JAK inhibitory effect, little is known. Therefore, the search for naturally occurring JAK inhibitors is still in a significant position.

Qiong Wang et al. , The Natural Compound Notorol Bindings and Targets JAK2 / 3 to Ameliorate Inflammation and Arthritis, Cell Reports 32,108158, September 15, 2020

Therefore, an object of the present invention is to provide a naturally occurring JAK inhibitor useful for the prevention and treatment of diseases related to the onset and exacerbation of JAK such as rheumatoid arthritis and atopic dermatitis.

As a result of diligent studies on the JAK inhibitory effect of plant extracts in view of the above points, the present inventors have found that the extract of Sanguisorba officinalis and the extract of Geranium thunbergii have a JAK inhibitory effect. Found to have.

As described in claim 1, the JAK 1 inhibitor of the present invention made based on the above findings contains an extract of Sanguisorba officinalis as an active ingredient (provided that it is used as an anti-inflammatory agent, an anti-pruritic agent, and an anti-allergic agent). Excludes) .

According to the present invention, it is possible to provide a naturally occurring JAK inhibitor useful for the prevention and treatment of diseases related to the onset and exacerbation of JAK such as rheumatoid arthritis and atopic dermatitis.

It is a graph which shows the JAK inhibitory action of the Sanguisorba extract (Ju extract) and the Geranium thunbergii extract in the test example.

The JAK inhibitor of the present invention contains an extract of at least one plant selected from Sanguisorba officinalis and Geranium thunbergii as an active ingredient.

Sanguisorba officinalis used in the present invention is a plant of the Rosaceae family, also called Jiyu, and dried roots and rhizomes are known by the crude drug name of Chiyu. Geranium thunbergii is a plant of the Geraniaceae family, and is a medicinal herb whose above-ground part is used for the purpose of antidiarrheal and intestinal regulation. However, there has been no report that any of them has a JAK inhibitory effect. Both Sanguisorba officinalis and Geranium thunbergii may be naturally grown or cultivated by the producer.

In the present invention, the extract of Waremokou is, for example, on roots and rhizomes (which may be shredded, dried or crushed), water as an extraction solvent or various organic solvents such as methanol, ethanol and isopropyl. Add monoalcohol such as alcohol, polyhydric alcohol such as 1,3-butylene glycol, ether, hexane, ethyl acetate, acetonitrile, etc. (the organic solvent may contain water), and let stand at room temperature for 1 hour to 1 week. It can be obtained as a filtrate by extracting the components by extracting the components or extracting the components by refluxing and then filtering. As the extraction solvent, a single solvent may be used, or a plurality of solvents may be mixed and used. The extract of Geranium thunbergii can be obtained as a filtrate by the same method as described above using, for example, the above-ground part (flowers, leaves and stems). The obtained filtrate may be used as it is as a liquid extract, or may be concentrated under reduced pressure or freeze-dried to form a solid or powder. The filtrate may be further purified by ammonium sulfate fractionation, gel filtration, ion chromatography, or the like, and the obtained fraction may be used as a liquid extract. In addition, for example, the extraction operation is first performed using hexane, and then the extraction operation is performed by gradually increasing the polarity of the solvent to be used, such as ethyl acetate, acetone, and water, and any of the solvents is used. The filtrate obtained in 1) may be used as a liquid extract.

The JAK inhibitor in the present invention can be taken or ingested as a drug or health food by formulating it into various pharmaceutical forms, and can also be ingested by blending it with various foods and drinks. .. The JAK inhibitor in the present invention can also be applied to the skin in the form of an external preparation such as an ointment, cream or lotion. The dose, ingestion, and application amount can be appropriately determined based on the age, sex, weight, degree of symptoms, etc. of the target person, and the JAK inhibition can be achieved by taking, ingesting, or applying an appropriate amount. Based on the action, it can be expected to have a preventive effect and a therapeutic effect on diseases related to the onset and exacerbation of JAK such as rheumatoid arthritis and atopic dermatitis.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not construed as being limited to the following description.

Test example: JAK inhibitory action of Sanguisorba officinalis extract and Geranium thunbergii extract 1. Inhibitory effect on phosphorylation of STAT3 induced by IL-6 Michael Forester et al. , 2016, Cell Chemical Biology 23,1335-1340, using the inhibitory effect on IL-6-induced phosphorylation of STAT3 as an index, a plant extract that is a candidate for a JAK inhibitor. A screening was performed. Specifically, about 2500 human epidermal keratinized cell lines HaCaT per well were inoculated on a 96-well plate in 100 μL of medium, cultured at 37 ° C. for 16 to 24 hours, and then into 100 μL of FBS-free medium. Stabilization was performed by changing and culturing for 6 hours. After Stalvation, 1 μL (final concentration 1%) of each of the various plant extracts was added, pretreated for 1 hour, then IL-6 with a final concentration of 20 ng / mL was added, and the cells were cultured at 37 ° C. for 15 minutes. After the culture was completed, the cells were washed twice with DPBS, and 50 μL of x4 Sample buffer (200 mM Tris-HCl [pH 6.8], 8% SDS, 400 mM DTT, 0.2% BPB, 40% glycerol) was added. The cells were lysed. The cytolysate was transferred to a 96-well PCR plate and heated at 95 ° C. for 20 minutes to decompose genomic DNA and extract intracellular proteins. Then, using a 10% TGX gel (Bio-Rad, USA), 5 μL of cell extract per well was electrophoresed. The gel was transferred to a PVDF membrane (Merk, Germany) and phosphorylated with STAT3 and non-phosphorylated STAT3 using anti-phospho-STAT3 antibody (Santa cruz, USA) and anti-STAT3 antibody (Santa cruz, USA). Bands were detected and narrowed down to plant extracts showing results comparable to the positive control results of 100 μM tofacitinib (absence of phosphorylated STAT3 band and presence of non-phosphorylated STAT3 band). is. As a result, Sanguisorba officinalis extract and Geranium thunbergii extract could be selected as plant extracts having an inhibitory effect on STAT3 phosphorylation induced by IL-6, which showed similar results to the results of tofacitinib. Both the cracked sardine extract and the geranium thunbergii extract used as samples are commercially available products, and the composition (% by weight) of the cracked sardine extract is root and rhizome extract: 0.8, 1,3-butylene glycol: 49. It was 6.6 and water: 49.6. The composition (% by weight) of the geranium thunbergii extract was flower, leaf and stem extract: 1.8, 1,3-butylene glycol: 49.1 and water: 49.1.

2. 2. Inhibition of JAK1 on enzyme activity (experimental method)
The inhibitory effect of the above-mentioned commercially available Geranium thunbergii extract and Geranium thunbergii extract on the enzyme activity of JAK1 was evaluated by ADP-Glo Kinase Assay (Promega, USA). Specifically, 50 ng / μL of JAK1 kinase (Promega, USA) 2.5 μL, 0.125 μL of extract diluted to each concentration (0.3%, 1%) with sterile purified water, x1 kinase buffer 4. An enzyme reaction solution consisting of 875 μL was prepared and incubated at 25 ° C. for 30 minutes. After completion of the incubation, 2.5 μL of 1 μg / μL substrate solution and 2.5 μL of 250 μM ATP were added and incubated at 25 ° C. for 60 minutes. Then, 12.5 μL of ADP-Glo Reagent was added and incubated at 25 ° C. for 40 minutes. Subsequently, 25 μL of Kinase Detection Buffer was added and incubated at 25 ° C. for 30 minutes. After the incubation was completed, the activity of JAK1 kinase was detected by measuring the fluorescence intensity with a fluorescent plate reader.

(Experimental result)
FIG. 1 shows a relative value of 0.5% 1,3-butylene glycol (0.5% BG) as a control with the fluorescence intensity as 100. FIG. 1 shows the results of 100 μM tofacitinib as a positive control and the results of 0.5% 1,3-butylene glycol and 0.1% DMSO as a negative control. As is clear from FIG. 1, both the crackle extract (ju extract) and the ginger extract exert an inhibitory effect on the enzyme activity of JAK1 in a concentration-dependent manner, and the inhibitory effect of 1% crackle extract is 100 μM. It was comparable to the inhibitory effect of tofacitinib.

Formulation Example 1: Tablet A tablet having the following component composition and having a JAK inhibitory action was produced by a method known per se (unit:% by weight).
Powder of Sanguisorba officinalis extract used in the test example 1
Lactose 80
Magnesium stearate 19

Pharmaceutical Example 2: Biscuit A biscuit having the following composition and having a JAK inhibitory effect was produced by a method known per se (unit:% by weight).
Sanguisorba extract used in the test example 1
Cake flour 32
Whole egg 16
Butter 16
Sugar 24
Water 10
Baking powder 1

Pharmaceutical Example 3: Jelly A jelly having the following component composition and having a JAK inhibitory action was produced by a method known per se (unit:% by weight).
Geranium thunbergii extract used in the test example 0.01
Gelling agent 1.3
Sugar 20
Acidity regulator 2.5
Water 76.19

Pharmaceutical Example 4: Ointment An ointment having the following composition and having a JAK inhibitory effect was produced by a method known per se (unit:% by weight).
Powder of Sanguisorba officinalis extract used in the test example 5
White petrolatum 95

Formulation Example 5: Cream A cream having the following component composition and having a JAK inhibitory action was produced by a method known per se (unit: weight%).
Geranium thunbergii extract powder used in the test example 5
White petrolatum 30
Cetanol 15
Sodium lauryl sulfate 1
Ethyl paraoxybenzoate 0.1
Butyl paraoxybenzoate 0.1
Water 48.8

Pharmaceutical Example 6: Lotion A lotion having a JAK-inhibiting action having the following composition was produced by a method known per se (unit:% by weight).
Geranium thunbergii extract used in the test example 10
Polyoxyethylene (40) Hardened Castor Oil 0.5
Ethanol 5
Paraben 0.2
Water 84.3

INDUSTRIAL APPLICABILITY The present invention is industrial in that it can provide a naturally occurring JAK inhibitor useful for the prevention and treatment of diseases related to the onset and exacerbation of JAK such as rheumatoid arthritis and atopic dermatitis. Has availability.

Claims (1)

A JAK 1 inhibitor containing an extract of Sanguisorba officinalis as an active ingredient (excluding uses for anti-inflammatory agents, antipruritic agents, and antiallergic agents) .

Images