CN105541947A - Drug molecule for antagonizing TLR7/8 and TLR9 activation and application - Google Patents

Abstract

The invention relates to a drug molecule for antagonizing TLR7/8 and TLR9 activation. The drug molecule is of a phosphorothioate modified molecule structure containing a sequence shown as SEQ ID NO: 1. The drug molecule is capable of antagonizing TLR7/8 and TLR9 activation and restraining release of proinflammatory factors such as TNF and IL to achieve the inflammation restraining effect; the drug molecule and normal saline are prepared into injection, treatment is conducted in a subcutaneous injection mode, and pathological injury of psoriasis can be remarkably relieved.

Description

Drug molecule and application for antagonizing TLR7/8 and TLR9 activation

technical field

The invention belongs to the field of biotechnology, and specifically relates to a drug molecule for antagonizing the activation of TLR7/8 and TLR9 and its use.

Background technique

Psoriasis, commonly known as psoriasis, is a common chronic inflammatory skin disease in clinical practice. It occurs more frequently in young and middle-aged people, with a longer course of disease and easy recurrence. It presents progressive skin lesions and thickened keratin. The clinical manifestations are mainly erythema and scales, and the whole body can be affected.

Although it was early recognized that the disease is an autoimmune disease related to heredity and infection. However, the specific pathogenic mechanism is not clear, so there is no specific drug at present, and non-specific drug treatment is mainly used to soften the stratum corneum as a symptomatic strategy, or a treatment plan that uses a small amount of hormones as an auxiliary treatment. Larger side effects, and unsatisfactory curative effect.

In recent years, many reports have gradually shown that the pathogenic mechanism of psoriasis is mainly due to the disorder of autoimmunity, TLR7/8, TLR7/8 in the Toll-like receptor (TLRs) family on immune cells such as skin macrophages and T lymphocytes. 9. After recognizing nucleic acid fragments in self-damaged cells and microorganisms, excessive inflammatory reactions are triggered, resulting in further damage to self-epidermal cells, which in turn promotes lymphocyte aggregation and epidermal keratinization, thus, repeated cycles make it worse.

Recently, foreign countries have used special antibodies to fight against inflammatory factors, such as antibodies against interleukin (IL) and tumor necrosis factor (TNF), and achieved preliminary curative effect. However, the preparation process is relatively complicated and expensive.

How to block the occurrence and development of psoriasis from the source of TLRs activation is a focus of attention of those skilled in the art.

Contents of the invention

The object of the present invention is to provide a drug molecule and its use for antagonizing the activation of TLR7/8 and TLR9. The drug molecule can antagonize the activation of TLR7/8 and TLR9, inhibit the release of pro-inflammatory factors such as TNF and IL, and achieve an anti-inflammatory effect; the drug molecule is prepared into an injection with normal saline or phosphate buffer, and is treated by subcutaneous injection , can significantly alleviate the pathological damage of psoriasis.

Technical scheme of the present invention is:

A drug molecule that antagonizes the activation of TLR7/8 and TLR9, the drug molecule is a sulfur-modified molecular structure including the sequence shown in SEQ ID NO:1.

The molecular structure is T*G*G*C*G*C*G*C*A*C*C*C*A*C*G*G*C*C*T*G, where * is thio grooming.

The use of the above-mentioned drug molecule in the preparation of a drug for treating psoriasis, the drug is an injection, the injection is composed of the above-mentioned drug molecule and normal saline, and the weight:volume ratio of the drug molecule and normal saline is 1~ 2:3~5.

The preparation process of the injection drug according to the present invention: according to the above nucleotide sequence, it is obtained by ordinary chemical synthesis, thio modification and purification, and the dosage form is (water-soluble) powder, which can be expanded alone or supplemented with mannitol After mixing with other agents, vacuum pack and store at room temperature or in a refrigerator at 4°C.

When in use, it is diluted with a common injection liquid (such as: physiological saline, etc.), and injected subcutaneously in a patient through a syringe. It can be injected once a day, and can be injected at multiple points, each point is 10-30μg/300μl, every two weeks is a course of treatment, and the actual number of courses of treatment can be determined according to the specific clinical situation.

Due to the advantages of clear action target and treatment site, significant effect and good safety, the injection of the present invention can significantly inhibit the release of pro-inflammatory factors and improve the pathological damage of psoriasis, and can be used as a medicine for treating psoriasis. It has urgent social health needs and broad application prospects.

The applicant has long been engaged in the research of Toll-like family-related innate immunity, in-depth research on the mechanism of action and indications of the TLR9 blocker c41 molecule. Through experimental verification, the molecule can not only block TLR9-mediated inflammatory activation, but also antagonize TLR7 /8-mediated activation; and in animal models of psoriasis, significant curative effects have been obtained. Moreover, the c41 molecule can metabolize itself in the cell without affecting the ability of the cell to respond again, which enables the c41 molecule to not only inhibit the active phase of the disease, but also not destroy the normal immunity of the body. After preliminary safety evaluation and pharmaceutical research, it is confirmed that the molecule has good stability and no toxic or side effects. Thus, the c41 molecule, that is, the drug molecule described in the present invention, is a drug molecule that blocks the inflammatory response from the initial stage of psoriasis pathogenesis, thereby achieving the therapeutic effect, and it is bound to bring hope to the majority of psoriasis patients , actively promote the development of clinical treatment.

In addition, psoriasis pathological damage mainly occurs in the epidermis, dermis and subcutaneous. By subcutaneous injection, the drug can quickly enter the affected area, preventing the activation of the accumulated immune cells, and the drug effect is better.

Weight in the present invention: volume, when the unit of weight is μg, the unit of volume is corresponding to μl; when the unit of weight is mg, the unit of volume is corresponding to ml; when the unit of weight is Kg, the unit of volume is corresponding to l.

Description of drawings

Figure 1 shows that the drug inhibits macrophage TLR7/8, 9 activation;

Figure 2 shows that the drug improves the pathological damage of psoriasis (X20, HE). Wherein a is the placebo group, and b is the c41 treatment group.

detailed description

The following examples can illustrate the present invention in detail, but do not limit the present invention thereto.

Embodiment 1: preparation of injection

Drug name: c41 molecule

Sequence structure: TGGCGCGCACCCACGGCCTG

Modification structure:

(* is thio modification)

Preparation method: chemical synthesis, entrusted to Shanghai Shenggong Company. Yield, purified by HPLC, vacuumed, stored in glass bottles at room temperature, and packed at 330 μg/bottle (dry powder).

Usage configuration: Take a small bottle, inject 600μl of normal saline, dilute and mix well, set aside.

Example 2: Identification of c41 molecule inhibiting the activation of TLR7/8 and 9

2.1 Main materials, reagents and equipment:

1) Cell line: RAW264.7 (ATCC, USA.).

2) Cell culture medium: DMEM (Gibco, Inc., USA).

3) Elisa kit: anti-mouse TNF-a (eBioscience, Inc., USA).

4) Cell incubator: FORMA3111.

5) Microplate reader: full-wavelength multifunctional microplate reader (ThermoScientific, USA).

2.2 Experimental method: RAW264.7 cells were diluted to 1×10 ⁶ /mL, added to a 96-well plate (200 μL/well), placed in a 37°C, 5% CO ₂ incubator for 2 hours, and then added TLR7/8 agonist R848 ( 5 μl, final concentration 100ng/ml) was the experimental group; TLR9 agonist CpG-ODN18265 μl (5 μl, final concentration 4 μM) was added as the second experimental group, each of the above groups had 8 replicate wells. Then, each component is divided into 4 multiple wells, and 5 μl of culture solution is added, respectively as a placebo group and two groups; the remaining 4 multiple wells of each group are respectively added with 5 μl of drug c41 molecule (final concentration 4 μM) of the present invention as treatment One group, two groups. After continuing to culture for 24 hours, the supernatant was collected and tested by Elisa to obtain the concentration of TNF-a.

2.3 Experimental results: In the placebo group, TLR7/8 is normally activated, stimulating RAW264.7 cells to release a large amount of TNF-a; in the placebo group two, TLR9 is normally activated, stimulating RAW264.7 cells to release a large amount of TNF-a; the treatment group and In the second group, under the action of the drug c41 molecule of the present invention, the level of TNF-a released by RAW264.7 cells was significantly inhibited (P<0.01), so the drug can effectively inhibit the key target of psoriasis and curb the excessive inflammatory response . As shown in Figure 1.

Example 3: Subcutaneous injection in the treatment of psoriasis and its curative effect.

3.1 Main materials, reagents and equipment:

1) Animal: Balb/c (Huafukang Biotechnology Co., Ltd., China).

2) Modeling drug: imiquimod (Mingxin Pharmaceutical, China).

3) Therapeutic drug: injection according to the present invention (see Example 1 for details)

3.2 Experimental method:

1) After the mice were shaved, the psoriasis model was established on the back of the mice after shaving, 20mg/cm2, once a day, for 6 days.

2) Grouping: placebo group and drug treatment group, each with 5 rats.

3) Administration: placebo group: subcutaneous injection of normal saline (NS), 100 μl/20g body weight; treatment group: C41 drug of the present invention diluted to 550 μg/ml with NS and subcutaneous injection of C41 molecule 100 μl/20g body weight.

4) Each group starts treatment from the first day and lasts for 6 days.

5) After 6 days, the skin tissues of each group were taken, stained with HE, and observed pathologically.

3.3 Experimental results:

In the placebo group, subcutaneous inflammatory cell infiltration, thickening of the epidermis, and obvious keratinization; while in the treatment group, there was no obvious inflammatory cell infiltration, and no keratinization of the epidermis, indicating that the injection of the present invention can effectively curb the pathology of psoriasis damage. As shown in Figure 2.

Claims (6)

1. a drug molecule for antagonism TLR7/8 and TLR9 activation, is characterized in that: the sequence thio-modification molecular structure as shown in SEQIDNO:1 of this drug molecule for comprising.

2. drug molecule according to claim 1, is characterized in that: the structure of described molecule is T*G*G*C*G*C*G*C*A*C*C*C*A*C*G*G*C*C*T*G, and wherein * is thio-modification.

3. the arbitrary described drug molecule of claim 1-2 is for the preparation of the purposes in treatment psoriasis.

4. purposes according to claim 3, is characterized in that: use medicine for injection.

5. purposes according to claim 4, is characterized in that: described injection is made up of the drug molecule described in claim 1 or 2 and physiological saline or phosphoric acid buffer.

6. purposes according to claim 5, is characterized in that: described drug molecule and the weight of physiological saline: volume ratio is 1 ~ 2:3 ~ 5.