CN106668832B

CN106668832B - Application of polypeptide in preparation of medicine for treating enterovirus infection

Info

Publication number: CN106668832B
Application number: CN201710201536.3A
Authority: CN
Inventors: 凌建群; 章殷捷
Original assignee: Zhejiang Yingkang Biological Medicine Co ltd
Current assignee: Zhejiang Yingkang Biological Medicine Co.,Ltd.
Priority date: 2017-03-30
Filing date: 2017-03-30
Publication date: 2020-08-14
Anticipated expiration: 2037-03-30
Also published as: CN106668832A

Abstract

The invention discloses an application of polypeptide in preparing a medicament for treating enterovirus infection, belongs to the field of infectious disease treatment, and simultaneously provides a medicinal composition for treating enterovirus infection. The polypeptide can be used for treating and preventing enterovirus infection, particularly hand-foot-mouth disease and herpetic pharyngolaryngitis, and has the advantages of remarkable effect and good safety.

Description

Application of polypeptide in preparation of medicine for treating enterovirus infection

Technical Field

The invention relates to the field of treatment of enterovirus infection, in particular to application of a polypeptide in preparing a medicament for treating enterovirus infection.

Background

Enteroviruses (EVs)) are a class of pathogens that invade and multiply in intestinal cells, which induce a variety of inflammations in human intestinal epithelial cells. Enteroviruses belong to the genus enterovirus of the picornaviridae family and include Poliovirus (PV), Echovirus (Echovirus), coxsackievirus (CVA, CVB), novel enteroviruses, for a total of 106 serotypes. They were divided into 4 groups (species) according to biological and genetic characteristics (Kenowles NJ, Hovi T, Hypypia T, et al. Classification and nomenclature of viruses: n ü n th report of the international committee on taxonomy of viruses. san Diego: Elsevier, 2011): enterovirus group A (EV-A) included 21 serotypes, Enterovirus group B (EV-B) included 60 serotypes, Enterovirus group C (EV-C) included 21 serotypes, PV3 serotypes were included, and Enterovirus group D (EV-D) included 4 serotypes.

Enteroviruses cause Hand-Foot-and-Mouth disease (HFMD) and herpes-type pharyngolaryngitis (HA) worldwide (National institutional report of acquired and weaning of acquired diseases).http://www.moh.gov.cn/publicfiles//business/ htmlfiles/wsb/pyqxx/list.htm.) and particularly frequently occurs in infants and young children. HFMD is mainly clinically characterized by fever and rashes or herpes in the hands, feet, oral cavity, etc., and enteroviruses causing HFMD include enterovirus type 71 (EV71) and coxsackie group a virus (CoxA), some serotypes of echovirus (Echo). HA is mainly manifested by repeated high fever, angina, crying, antifeedant, salivation, dysphoria, vomiting, etc., and clinically caused by infection of Coxsackie group A virus, and part of EV 71. There are several complications of HFMD/HA in the prior reports, including meningesInflammation, encephalitis, acute delayed paralysis, acute cardiopulmonary failure, respiratory tract infections, myocardial damage and diarrhea (Hsiung GD, Wang JR. Enterovirus infections with specific reponsence to enterovirus 71.J Microbiol Immunol Infect.2000; 33: 1-8. Stalkup JR, Chilukuri. Enterovirus infections: a review of clinical presentation, diagnosis, and anddtreatment. DermatolClin.2002; 20: 217-23.)

Human is the only host of enterovirus, and both patients and recessive infectors are the sources of infection. The enterovirus is mainly spread through feces-mouth and respiratory droplets, and can also be infected by herpes fluid contacting the skin and mucous membranes of patients. The human is generally susceptible to enterovirus, and can obtain specific immunity after dominant infection and recessive infection, and the duration is not clear. There was no cross-immunity between the virus types. All age groups can be infected and attack, but the disease is frequently encountered in the age group less than or equal to 3 years old.

At present, no specific medicine exists for treating HFMD/HA, western medicine mainly comprises antiviral treatment and symptomatic treatment, clinical medicine mainly comprises antiviral drugs, traditional Chinese medicines, antibacterial drugs and the like, the antiviral treatment HAs large side effect and poor taste of the traditional Chinese medicines, the antibacterial drugs cannot kill viruses, the treatment effects are not ideal, the side effect is generated, the health of children is influenced, and the treatment and prevention of HFMD/HA infection become a medical problem.

The antibacterial peptide is a basic polypeptide, generally consists of about 12-50 amino acids, and has broad-spectrum antibacterial property. Currently, the antibacterial peptide database (APD, http:// APs. unmac. edu/AP /) contains 2817 antibacterial peptides from 6 classes of organisms, wherein 288 are from bacteria, 4 are from archaea, 8 are from protists, 13 are from fungi, 341 are from plants, and 2109 are from animals. In addition to antibacterial effects, different antibacterial peptides also have different effects, as Guangshun Wang reviews 80 antibacterial peptides for HIV antiviral effects (Guangshun Wan, Natural antimicrobial peptides improving anti-HIV vaccines, Curr Top peptide Protein Res.2012; 13: 93-110.). Melitin has inhibiting or killing effect on U937 human mononuclear leukosis cells, Du145 prostate cancer cells, SKOV3 ovarian cancer cells, B16 mouse melanoma cells and BEL-7402 human liver cancer cells (S.Saini, et al, Melitinactivations endogenesis p)hosphitase D during catalysis of human monocetylecium cells, Toxicon, vol.37, No.11, pp.1605-1619, 1999, P.J.Russell, et al, Cytoxic properties of microcontrogens stabilizing peptide101against promoter, Cancer Immunology, immunotherpay, vol.53, No.5, pp.411-421,2004.); LL-37is used for topical treatment to promote healing of venous leg ulcers (

A.et al. (2014). Treatment with LL-37is safe and effective healing chemicals of hard-to-magnetic rear capsules, while the mechanism of LL-37 promoting wound healing is not clear, but may involve wound healing components of epidermal regeneration, angiogenesis and inflammation, bovine Lactoferrin may regulate the immune system in THP-1 cells by inhibiting the induction of TNF- α and IL-6 by LPS (TLR4) (matrix-Balter, et al. L.A. (1996) Lactoferrin or fragment of therapeutic tissue of human Lactoferrin-tissue Repair, for the prevention of hyaluronic acid derivative PX-absorbed-tissue-6, cement-protein of hyaluronic acid condensate, for example, hyaluronic acid, for example, hyaluronic acid, sodium sulfate, sodium.

The applicant finds the polypeptide of the invention in research, the polypeptide is an antibacterial peptide, and a plurality of patents are applied for the characteristics of the antimicrobial peptide, and the like, and the prior art only reports the antibacterial performance of the polypeptide and does not report that the polypeptide has the effect of treating enterovirus infection.

Disclosure of Invention

The applicant finds that the polypeptide has a good effect on enterovirus infection, particularly hand-foot-and-mouth diseases and herpetic pharyngolaryngitis, and aims at the defects of the prior art, the primary object of the invention is to provide an application of the polypeptide in preparing a medicament for treating the enterovirus infection, particularly the hand-foot-and-mouth diseases and the herpetic pharyngolaryngitis, and the secondary object of the invention is to provide a polypeptide preparation for treating the enterovirus infection, particularly the hand-foot-and-mouth diseases and the herpetic pharyngolaryngitis, which is characterized in that: the preparation is prepared by taking effective dose of the polypeptide as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients. It is a third object of the present invention to provide a method for treating enteroviral infections, in particular hand-foot-and-mouth diseases, herpetic pharyngolaryngitis.

The amino acid sequence of the polypeptide is SEQ ID NO. 1.

The contents in the present invention are mass percentages unless otherwise specified.

The enterovirus infection of the invention is preferably hand-foot-and-mouth disease (HFMD)/and herpetic pharyngolaryngitis (HA).

The meanings of the present invention for treating enteroviral infections include, but are not limited to: ameliorating or alleviating symptoms associated with an enterovirus infection, reducing duration of symptoms, not worsening disease state, delaying or slowing disease progression, such as for HFMD/HA, lowering fever temperature or returning to normal body temperature, reducing rash, number of herpes, reducing number of vomiting, relieving or eliminating sore throat, congestion and ulceration, reducing or alleviating complications, and alleviating or curing other symptoms associated with HFMD/HA.

In one embodiment, the present invention provides the use of a polypeptide at a concentration of 0.01% to 1% for the manufacture of a medicament for the treatment of an enteroviral infection.

In one embodiment, the present invention provides the use of a polypeptide at a concentration of 0.02% to 0.2% for the manufacture of a medicament for the treatment of an enteroviral infection.

In one embodiment, the present invention provides a pharmaceutical composition for treating an enteroviral infection, characterized by: the composition is a polypeptide preparation prepared by taking effective dose of the polypeptide as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients. The polypeptide preparation is preferably a liquid preparation, preferably a spray and a lotion, and the liquid preparation can also be made into an inhalant when applicable. Meanwhile, when applicable, the polypeptide preparation can also be an external solid preparation and a semisolid preparation, the preferable solid preparation is freeze-dried powder, the preferable semisolid preparation is paste or gel,

in one embodiment, the present invention provides a pharmaceutical composition for treating enterovirus infection, wherein the concentration of the polypeptide is 0.01% -1%.

In one embodiment, the present invention provides a pharmaceutical composition for treating an enteroviral infection, said polypeptide being present at a concentration of 0.02% to 0.2%.

In one embodiment, the present invention relates to a method for treating an enteroviral infection, in particular a hand-foot-and-mouth disease, herpetic pharyngolaryngitis, comprising the steps of: the polypeptide preparation is applied to a part affected by the enterovirus infection, and the amino acid sequence of the polypeptide is SEQ ID NO. 1. When the polypeptide preparation is a spray, the spray bottle is shaken uniformly before use, and the spray is sprayed on the oral cavity, the throat and other infected parts for 4 times a day; when the polypeptide preparation is lotion, the preparation can be gargled orally or dipped in sterile gauze to wash the affected part for 4 times a day; when the polypeptide is an inhalation preparation, atomizing the polypeptide liquid preparation into fog particles by an ultrasonic medical atomizer, and delivering the fog particles to the oral mucosa lesion of a patient 2 times a day; when the polypeptide is paste or gel, it is dipped with sterilized cotton swab and applied to the affected part 4 times per day.

The site affected by an enteroviral infection may comprise a site adjacent to a site exhibiting symptoms of an enteroviral infection. Symptoms of enteroviral infection include, but are not limited to: fever, rash, herpes, ulceration of the skin and mucous membranes, vomiting, sore throat and congestion, with frequent pain in the neck, abdomen and extremities, and with severe symptoms, serious complications can occur, such as complicated aseptic meningitis, encephalitis, paralysis, nervous system damage, pulmonary edema, myocardial damage, heart failure, and the like.

The invention provides a new application of a known polypeptide in treating enterovirus infection, which not only has an antimicrobial effect, but also has the effects of resisting viruses, regulating an immune system and promoting ulceration healing. Compared with the prior art, the polypeptide preparation has better effect of treating enterovirus infection; the polypeptide has good water retention property, can prevent skin damage parts from being dried and promote ulceration healing; the polypeptide of the invention has high safety, and the final degradation product is amino acid without adverse reaction; in the process of regulating an immune system, the polypeptide also has the effects of preventing and treating secondary bacterial infection, and the polypeptide has the antibacterial effect through the action with a microbial cell membrane, so that the drug resistance of bacteria is not easy to generate.

Detailed Description

The invention provides an application of polypeptide in treating enterovirus infection, in particular hand-foot-mouth disease and herpes pharyngolaryngitis, wherein the amino acid sequence of the polypeptide is SEQ ID No.1 without special indication.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the respective raw materials added in the examples are commercially available unless otherwise specified.

The polypeptide of the invention is an antibacterial peptide, and the existing data only disclose that the polypeptide has antibacterial efficacy. In the research process of the applicant, the fact that the polypeptide has the effect of treating enterovirus infection, particularly hand-foot-and-mouth disease and herpetic pharyngolaryngitis is unexpectedly found, the specific treatment mechanism is not clear, but based on the existing documents of other antibacterial peptides, the applicant speculates that the polypeptide disclosed by the invention may have multiple unknown functions, the polypeptide may have an inhibitory effect on viruses, and on the basis of regulating the immune system of a body, microorganisms in the area complicated with the enterovirus infection are eliminated, the healing of the ulcer is promoted, and the effect of treating the enterovirus infection is finally achieved.

The invention provides a pharmaceutical composition for treating enterovirus infection, in particular hand-foot-mouth disease and herpetic pharyngolaryngitis, which is characterized in that: the composition is a preparation prepared by taking effective dose of the polypeptide as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

The auxiliary materials can be one or a plurality of combinations of conventional auxiliary materials in medicinal preparations such as sucrose, starch, dextrin, magnesium stearate, sorbitol, mannitol, lactose, microcrystalline cellulose, aerosil, carboxymethyl cellulose, water and the like, and the specific selection of which one or more auxiliary materials is determined according to the required preparation formulation. The auxiliary components refer to medicines capable of improving the curative effect of the polypeptide or treating enterovirus infection.

The polypeptide of the invention can be prepared into various dosage forms when preparing medicines for treating enterovirus infection, particularly hand-foot-and-mouth diseases and herpetic pharyngolaryngitis, the dosage form is pharmaceutically acceptable, and generally refers to a substance or a composition which is compatible with other components included in the dosage form and/or a patient to be treated by the dosage form chemically and/or toxicologically. The dosage form may include a spray, emulsion, suspension, paste, dressing, lotion, gel, powder, or other solid, semi-solid, or liquid composition. Such compositions may comprise: humectants, hydrating agents, penetrants, preservatives, emulsifiers, natural or synthetic oils, surfactants, gelling agents, thickeners, waxes, odor absorbers, colorants, powders, viscosity modifiers, and water, as well as minerals, polyphenols, silicones, vitamins, and the like.

Preferably, the polypeptide preparation is preferably a liquid preparation, preferably a spray and a lotion, and the liquid preparation can also be made into inhalants when applicable. Meanwhile, when applicable, the polypeptide preparation can also be an external solid preparation and a semisolid preparation, the preferable solid preparation is freeze-dried powder, the preferable semisolid preparation is paste or gel,

the polypeptide of the present invention can be prepared according to the method of the present invention as described in the previous patent application (application No. 201310245373.0, title: a method for preparing antibacterial peptide and application) of the applicant. The polypeptide of the present invention can also be obtained by chemical synthesis, or by expression, separation and purification by genetic engineering techniques (see Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

This section of the examples further illustrates the content of the invention but should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

The invention discloses the application of the polypeptide as a medicinal active ingredient in preparing medicaments for treating and preventing enterovirus infection, particularly hand-foot-and-mouth diseases and herpetic pharyngolaryngitis for the first time, so that the polypeptide alone or in combination with other substances as the medicinal active ingredient is combined with auxiliary materials to prepare a medicament, and the medicament belongs to the protection range of the invention as long as the medicament is used for treating and preventing the enterovirus infection, particularly the hand-foot-and-mouth diseases and the herpetic pharyngolaryngitis.

The polypeptide of the invention has the functions of treating and preventing enterovirus infection, especially hand-foot-mouth diseases and herpetic pharyngolaryngitis when being prepared into any dosage form. Any medicament, if the components of which contain the polypeptide and are prepared into a medicament, is marked on the package, the instruction book and the like or any other propaganda product, as long as the medicament is noted or suggested to have the effect of treating and preventing the enterovirus infection, particularly the hand-foot-and-mouth disease and the herpetic pharyngolaryngitis, and falls into the protection scope of the invention.

Example 1 preparation of lyophilized formulations according to the invention

Dissolving auxiliary materials of mannitol and arginine, mixing the dissolved auxiliary materials with the polypeptide, adjusting the pH value to 7.2 to form a liquid containing 1mg of the polypeptide, 32mg of mannitol and 15mg of arginine per milliliter, filtering the liquid to remove bacteria, and freeze-drying the liquid to obtain the freeze-dried preparation.

EXAMPLE 2 preparation of sprays according to the invention

For convenient transportation and storage, the polypeptide of the invention is prepared into a freeze-dried preparation, and can be prepared into a spray preparation according to requirements when in use: dissolving the lyophilized preparation with physiological saline or medical common solvent to obtain a solution containing the polypeptide with concentration of 0.01%, and spraying in a sprayer.

Example 3 preparation of lotions according to the invention

And (2) uniformly stirring 8% of glycerol in purified water, adding 0.02% of the polypeptide, 0.5% of arginine and 10% of lysine, mixing, uniformly stirring to a thick degree, and adjusting the pH to 7.2 to obtain the polypeptide lotion.

EXAMPLE 4 preparation of the gels of the invention

And (2) fully and uniformly stirring 1.5% of carbomer, adding 2.3% of hyaluronic acid, 0.08% of glycerol and 0.2% of the polypeptide, and continuously and uniformly stirring to obtain the gel.

EXAMPLE 5 therapeutic Effect of the polypeptide preparation of the present invention on hand-foot-mouth disease

1. Case data

The patients in this group had 82 cases, wherein 43 cases were male, 39 cases were female, and the disease course was l-2 days for infants aged 0.5-5.2 years. All the medical diagnosis standards accord with the diagnosis and treatment guideline for hand-foot-and-mouth disease 2010 edition (the sanitary department of the people's republic of China, the diagnosis and treatment guideline for hand-foot-and-mouth disease 2010 edition [ J ]. the international journal of respiration, 2010, 30 (24): 1473-: dysfunction of the heart, brain, lung and other distention organs; no treatment was performed before admission. The patients were divided into observation group and control group according to random number comparison table, wherein each group comprises 41 patients, and the age, sex and course of disease of the patients between the two groups have no obvious difference (P > 0.05).

2. Method of producing a composite material

Ribavirin (10-15 mg/kg/day) was administered to the control group (SishuiErkang pharmaceutical Co., Ltd.) and diluted with 5% glucose injection and administered by intravenous drip 1 time a day. The observation group uses the polypeptide spray of the embodiment 2 to spray and administer the medicine through oral cavity and throat, 4 times a day, and serious patients use the lotion of the embodiment 3 externally 4 times a day, and dip and wash the red rash, blister and erosion part of the foot and mouth of the hand and the foot with sterile gauze. 5 days is 1 course of treatment, and the two groups are both administered conventional liquid therapy and symptomatic treatment.

3. Evaluation criteria

The effect is shown: after 72h of treatment, the endosome temperature returns to normal, the herps in the angina are reduced, no ulcer is formed, and the rash of hands and feet is reduced; the method has the following advantages: the internal body temperature returns to normal after 5d of treatment, the herps of the angina part is reduced, no ulcer is formed, and the rash of hands and feet is reduced or eliminated; and (4) invalidation: the fever is still caused after 5 days of treatment, the general condition is not improved, and the herpes of the oral cavity and the herpes of the hands, the feet and the buttocks are not obviously reduced or increased.

Total effective rate (number of effective cases + number of effective cases)/total number of cases × 100%.

4. Statistical treatment

The statistical method comprises the following steps: using SPSS10.0 statistical analysis software, using t test for metering data and X for counting data²Inspection, P<0.05 indicates that the difference is statistically significant.

5. Results

The treatment effect of the control group and the observation group on the hand-foot-and-mouth disease patients is shown in table 1.

TABLE 1 comparison of the therapeutic effects of two groups of infants (n,%)

Therapeutic effect	Show effect	Is effective	Invalidation	Total effective rate (%)
					Control group (41)	15(36.5％)	22(53.7％)	4(9.8％)	90.2
Observation group (41)	23(56.1％)	16(39.0％)	2(4.9％)	95.1

Table 1 the results show that: the control group and the observation group have obvious total effective rate in treating hand-foot-and-mouth diseases, but the treatment effect of the polypeptide preparation is better than that of ribavirin.

EXAMPLE 6 therapeutic Effect of the polypeptide preparation of the present invention on herpetic pharyngolaryngitis

1 case data

116 children with herpangina are selected. Inclusion criteria were: (1) the symptoms and signs accord with the diagnosis standard of herpangina (Wangwaiping, Erscience, 8 th edition, M, national public health Press, 2013,3, 207-; (3) the disease onset time is within 2 days; (4) CRP > 10 mg/L. Exclusion criteria: (1) those with lung infections; (2) other studies have been undertaken. 60 male children patients and 56 female children patients are divided into an observation group and a control group according to a random number comparison table, wherein 58 patients in each group have no obvious difference in age, sex and disease course (P > 0.05).

2 method

All children patients take the children fermented soya beans and fructus forsythiae heat-clearing granules (manufacturer: Jichuan pharmaceutical industry group Co., Ltd.) three times a day, 3 g each time, 1 week of treatment course, the observation group is combined with the polypeptide preparation described in the example 2 and sprayed into oral cavity four times a day, 1 week of treatment course, the control group is combined with the stomatitis spray (manufacturer: Jiangxi Zhenming pharmaceutical industry) and sprayed into oral cavity four times a day, and 1 week of treatment course.

3 observation index

After one week, the body temperature recovery time, oral mucosa herpes healing time and cure rate of the children patients in the course of disease are collected in both groups, and the treatment effects are compared. The side reactions (rash, nausea, vomiting) occurred after the children patients applied the two spray groups were compared with the incidence rate of anaphylaxis.

4 statistical methods

Using SPSS10.0 statistical analysis software, using t test for metering data and X for counting data²Inspection, P<0.05 indicates that the difference is statistically significant.

5 results

The recovery time of two groups of children patients after treatment is different, the body temperature recovery time and the herpes healing time of the groups are observed to be obviously shorter than those of a control group (see table 2, P is less than 0.05, and P is less than 0.01), the effective rate is obviously higher than that of the control group (see table 3, P is less than 0.05), and the allergy incidence rate is obviously lower than that of the control group (see table 4, P is less than 0.05). Herpes t is 3.67, P <0.01.

Comparison of treatment time for two groups of infants (Table 2)

Body temperature t 2.66, P <0.05, compared to control group

Two groups of children patients curative effect comparison (Table 3)

Comparison with control group, χ₂＝6.85，P<0.05.

Comparison of allergic reactions in two groups of infants (Table 4)

Comparison with control group, χ₂＝6.92，P<0.05.

Because enterovirus infection, particularly hand-foot-mouth disease and herpetic pharyngolaryngitis frequently occur in infants, which is related to immature intestinal mucosa, high permeability, low immunity and the like of the infants, the polypeptide preparation can effectively treat the enterovirus infection, particularly the hand-foot-mouth disease and the herpetic pharyngolaryngitis, has obvious curative effect on treating and preventing the enterovirus infection, particularly the hand-foot-mouth disease and the herpetic pharyngolaryngitis, and selected cases are the infants and preschool children, but the treatment of enterovirus infection of children or adults before the preschool age, particularly the hand-foot-mouth disease and the herpetic pharyngolaryngitis can be expected to be effective. Meanwhile, according to the curative effect in the embodiment, when the outbreak of the enterovirus infection can be easily expected, the polypeptide preparation can prevent the enterovirus infection and reduce the morbidity.

SEQUENCE LISTING

<110> Gill Biotechnology Ltd of Jiangsu

<160>1

<170>PatentIn version 3.3

<210>1

<211>18

<212>PRT

<213> Artificial sequence

<400>1

Gly Arg Phe Lys Arg Phe Arg Lys Lys Phe Lys Lys Leu Phe Lys Lys

1 5 10 15

Leu Ser

Claims

1. The application of the polypeptide in preparing the medicine for treating the enterovirus infection is characterized in that the amino acid sequence of the polypeptide is SEQ ID NO.1, and the diseases caused by the enterovirus infection are hand-foot-and-mouth diseases and herpetic pharyngolaryngitis.

2. The use of claim 1, wherein the polypeptide is present at a concentration of 0.01% to 1% by weight.

3. The use according to claim 2, wherein the concentration of the polypeptide is 0.02 to 0.2% by mass.

4. The use of any one of claims 1-3, wherein the medicament is a liquid formulation.

5. Use according to claim 4, characterized in that: the liquid preparation is spray and lotion.