WO2016110173A1 - New inhibitor for influenza virus neuraminidase and uses thereof - Google Patents

Abstract

A new inhibitor for influenza virus neuraminidase and uses thereof in the preparation for medicines, food, and health care products for the prevention and/or treatment of human or animal diseases caused by influenza viruses. The neuraminidase inhibitor for is icariin and derivatives thereof.

Description

Novel inhibitor of influenza virus neuraminidase and use thereof Technical field

The present invention relates to a novel inhibitor of influenza virus neuraminidase, which is specifically icariin and a derivative thereof. The present invention also relates to the use of the novel inhibitor for the preparation of a medicament, a food, a health supplement for preventing and/or treating a disease caused by an influenza virus. It belongs to the fields of pharmacy and pharmacology.

Background technique

The following description is intended to help the reader understand. Neither the information provided nor the references cited are considered to be prior art to the present invention.

Influenza virus, or influenza virus, is an RNA virus that causes influenza in humans, dogs, horses, pigs, mammals, and poultry. Taxonomically, the influenza virus belongs to the Orthomyxoviridae family, which causes acute upper respiratory tract infections and can spread rapidly through the air, causing periodic pandemics around the world. The virus was first known in 1933 by the Englishman Wilson Smith, known as H1N1. H stands for hemagglutinin; N stands for neuraminidase. The numbers represent different types. Human influenza viruses are classified into three types: A (A), B (B), and C (C), which are the pathogens of influenza (flu). Among them, the influenza A virus antigen is susceptible to mutation and has caused a worldwide pandemic many times.

The influenza virus is spherical, and the newly isolated strain is mostly filamentous, with a diameter between 80 and 120 nanometers, and a filamentous influenza virus with a length of up to 400 nanometers. Influenza virus structure can be divided into envelope, matrix protein and core three parts from the outside. The core of the virus contains the genetic material that stores the virus information and the enzymes necessary to replicate the information. The genetic material of influenza virus is single-stranded negative-strand RNA, abbreviated as ss-RNA. Ss-RNA is combined with nuclear protein (NP) and entangled into ribonucleoprotein body (RNP), which exists in extremely high density. In addition to ribonucleoproteins, there are RNA polymerases responsible for RNA transcription. The RNA of influenza A virus consists of 8 segments. The influenza virus gene consists of eight single-stranded RNA fragments, the NA, HA, NP, M, NS, PB1, PB2, and PA genes, respectively. They encode 10 proteins: Hemagglutinin HA, Neuraminidase NA, Matrix protein 1 M1, Nucleoprotein NP, and RNA-dependent RNA polymerase PB1 , PB2 and PA) Ion channel protein M2 and non-structural proteins NS1 and NS2.

The matrix protein constitutes the outer shell skeleton of the virus, and in fact, in addition to the matrix protein (M1), the membrane protein (M2). The M2 protein has an ion (mainly Na+) channel and regulates the pH in the membrane, but in a small amount. The matrix protein binds tightly to the outermost envelope of the virus, protecting the core of the virus and maintaining the spatial structure of the virus. After the influenza virus completes its reproduction in the host cell, the matrix protein is distributed on the inner wall of the host cell membrane, and the shaped viral nucleocapsid recognizes the site containing the matrix protein on the host cell membrane, binds to form a viral structure, and is budding. The form of the prominent release of mature virus particles (Virion). The envelope is a layer of phospholipid bilayer membrane wrapped outside the matrix protein. This membrane is derived from the cell membrane of the host. The mature influenza virus sprouts from the host cell, and the host cell membrane is wrapped in the body and then detached from the cell to be infected. Next target. In addition to phospholipid molecules, there are two very important glycoproteins in the envelope: hemagglutinin and neuraminic acid. Enzyme. These two types of proteins protrude from the virus in vitro and are about 10 to 40 nanometers in length and are called spikes. Generally, an influenza virus surface is distributed with 500 hemagglutinin spikes and 100 neuraminidase spikes. In the influenza A virus, the antigens of hemagglutinin and neuraminidase mutate, which is the basis for distinguishing virus strain subtypes.

Hemagglutinin (HA) is columnar and can bind to receptors on the surface of red blood cells of animals such as humans, birds, and pig guinea pigs to cause blood clotting, so it is called hemagglutinin. After hemagglutinin proteolysis, it is divided into two parts: light chain and heavy chain. The latter can be combined with the sialic acid receptor on the host cell membrane. The former can assist the fusion of the viral envelope and the host cell membrane. Hemagglutinin plays an important role in the introduction of the virus into host cells. Hemagglutinin is immunogenic and anti-hemagglutinin antibodies neutralize influenza viruses. Neuraminidase (NA) is a mushroom-like tetrameric glycoprotein with activity in hydrolyzing sialic acid. After the mature influenza virus leaves the host cell in a budding manner, the hemagglutinin on the surface of the virus is kept in contact with the host cell membrane via the sialic acid receptor, and the sialic acid needs to be hydrolyzed by the neuraminidase to cut off the last connection between the virus and the host cell. So that the virus can be released from the host cell smoothly, and then infect the next host cell. Therefore, neuraminidase has also become a target of influenza treatment drugs, and oseltamivir (Duffy) designed for this enzyme is one of the most famous anti-influenza drugs. H can be divided into 17 subtypes (H1 to H17), and N has 10 subtypes (N1 to N10). Among them, only H1N1, H2N2, and H3N2 mainly infect humans, and many other subtypes of natural hosts are a variety of birds and animals. Among them, H5, H7 and H9 subtype strains are the most harmful to birds. Under normal circumstances, the avian flu virus does not infect animals other than birds and pigs. However, in 1997, Hong Kong reported for the first time 18 cases of H5N1 human infection with avian influenza, of which 6 died, causing widespread concern worldwide. Since 1997, there have been several incidents of avian influenza virus infection in the world. The highly pathogenic avian influenza viruses such as H5N1, H7N7, H7N9, and H9N2, once mutated and have human-to-human transmission ability, will lead to human avian influenza epidemic, indicating that the avian influenza virus has great potential for humans. Threat.

Because influenza virus antigens are highly variable, conventional vaccines are not yet effective in preventing influenza outbreaks and epidemics, so anti-influenza drug research is of great significance in influenza treatment. Anti-influenza viruses that have been and are being studied include amantadines, influenza virus neuraminidase inhibitors, influenza virus receptor blockers, and anti-influenza antisense oligonucleotides. Amantadine and rimantadine are commonly used clinical treatment drugs, but they are ineffective against influenza B, easily resistant and neurotoxic; influenza virus neuraminidase inhibitors such as oseltamivir are currently the most effective. First-line drugs against influenza virus; influenza virus receptor blockers have obvious antiviral effects, anti-influenza virus antisense oligonucleotides have been shown to specifically inhibit viral replication in vitro, and may become a new generation of anti-influenza specific drugs, but They are still in the research stage.

Icariin, Chinese name is Epimedium, English name is Icariin, Icariln, Ieariline, English chemical name is 3-[(6-Deoxy-alpha-L-mannopyranosyl)oxy]-7-(beta-D- Glucpyranosy-loxy)-5-hydroxy-2-(4-methoxyphenyl)-8-(3-methyl-2-buten-1-yl)-4H-1-benzopyran-4-one, chemical formula: C ₃₃ H ₄₀ O ₁₅ . Natural icariin is found in the genus E. pimedium, mainly distributed in the north temperate zone. There are 23 species of genus Epimedium in the world, and 13 species in China, such as: kinky E. brevicornum, E. sagitatum, E. icoroanum, etc. Chinese Patent Application CN1535976A describes in detail a method for extracting and purifying Epimedium from Epimedium. Extracted from the dried stems and leaves of Epimedium, the purified icariin is a pale yellow needle-like crystalline powder. Epimedium and its extract, icariin, have been used in Chinese patent medicines and health care products for many years. Even for thousands of years, no toxic or side effects have been found. It is safe for clinical use in Europe for the treatment of acute and chronic hepatitis. .

Based on the knowledge of the inventors, there is no report showing the correlation of the activity of neuraminidase of the influenza virus, especially icariin and its derivatives in the preparation of influenza drugs for the prevention and/or treatment of influenza viruses. The use of health products.

Unexpectedly, Applicants have found that drugs and health supplements containing icariin and its derivatives can effectively inhibit the activity of influenza virus neuraminidase, thereby achieving the effect of inhibiting the proliferation of influenza virus, and can be used for prevention and/or prevention. The treatment of influenza diseases achieves the object of the present invention.

Summary of the invention

In one aspect of the invention, there is provided an inhibitor of influenza virus neuraminidase, in particular icariin and a derivative thereof. It can be used for the purpose of preventing and/or treating drugs, foods, health products or cosmetics for pathological symptoms or diseases of human or animals caused by influenza viruses.

In still another aspect of the present invention, the present invention provides a pharmaceutical composition comprising icariin and a derivative thereof, wherein the pharmaceutical composition contains at least one active ingredient of icariin and a derivative thereof. And the composition may also contain at least one other compound or a herbal ingredient.

In another aspect of the invention, the present invention also provides a preparation comprising icariin and a derivative thereof or a pharmaceutical composition thereof, and a process for producing the same.

A specific object of the present invention is to provide an inhibitor of influenza virus neuraminidase, a composition containing the same, for use in the preparation of a cold medicine or a health supplement for preventing and/or treating influenza virus. The influenza diseases include influenza A, B and C viruses and their respective subtypes and various serotypes thereof, various gene mutants, and serotype variants. That is, H1 to H17, N1 to N10 of influenza A virus, and various gene mutants and serotype variants of M1, M2, PA, PB1, PB2, NS1, NS2; type B and C viruses and their respective Various subtypes and their various serotypes, various gene mutants, serotype variants.

Internationally, the inventors used an influenza virus neuraminidase inhibitor, icariin and its derivatives, which were screened from natural Chinese herbal medicines without any side effects, to effectively prevent and/or treat diseases caused by influenza virus for the first time. , proved the mechanism of action of the drug. To achieve scientific use of medicine, there is a targeted. It is also the first time that this function of icariin has been discovered, filling the domestic and international gaps in the modernization of Chinese herbal medicine, which is conducive to the rehabilitation of patients.

DRAWINGS

Figure 1. Icariin inhibits influenza virus neuraminidase activity assay.

Figure 2. Icariin inhibits influenza virus staining in MDCK cells by fluorescent antibody staining. A, MDCK cells infected with influenza virus H5N1, no icariin, all cells are infected with virus; B, MDCK cells infected with influenza virus H5N1, medium containing 5 μmol of icariin, only a small number of cells Infected with virus; C, MDCK cells infected with influenza virus H9N2, no icariin, all cells infected with virus; D, MDCK cells infected with influenza virus H9N2, medium containing 5 μmol of icariin, only A small number of cells are infected with the virus.

Figure 3. Icariin inhibits influenza virus proliferation in MDCK cells by flow cytometry analysis. A, MDCK cells infected with influenza virus H5N1, no icariin, almost all cells are infected with the virus; B, MDCK cells infected with influenza virus H5N1, the medium contains 5μmol of icariin, only a small amount The cells are infected with the virus; C, MDCK cells infected with influenza virus H9N2, no icariin, almost all cells are infected with the virus; D, MDCK cells infected with influenza virus H9N2, the medium contains 5 μmol of icariin Only a small number of cells are infected with the virus.

Figure 4. Icariin inhibits the replication of influenza virus in MDCK cells in real time with unlabeled cell function. A, MDCK cells infected with influenza virus H5N1, the line with triangles is the result of the culture plate wells of virus-infected cells without icariin; the line with dots is the control wells without virus infection; no dots The lines of the triangles and the triangles were plate wells with a gradient of concentration of 1 μmol, 5 μmol, 10 μmol, 20 μmol, 50 μmol, 100 μmol, and 200 μmol of icariin. B, MDCK cells infected with influenza virus H9N2, the line with triangles is the result of the culture plate wells of virus-infected cells without icariin; the line with dots is the control wells without virus infection; no dots The lines of the triangles and the triangles were plate wells with a gradient of concentration of 1 μmol, 5 μmol, 10 μmol, 20 μmol, 50 μmol, 100 μmol, and 200 μmol of icariin.

Figure 5. Animal experiments in which icariin inhibits influenza virus. A, the average weight gain and decrease of each experimental group after inoculation of H5N1 in mice, that is, the total weight of 10 mice - the total weight of the mice in the first 10 experiments was ÷ 10, and the solid line represents the infected mice and used 30 mg / The icariin was used to prevent changes in body weight of the experimental group mice (experimental group 2); the dotted line represents the change in body weight of the experimental group mice that did not use icariin after infection with the virus (experimental group 1). B, the survival rate of the mouse, the solid line represents the condition of the mice in the experimental group infected with the infected mice and using 30 mg / icariin (experimental group 2); the dotted line represents the mice infected with the virus without using Epimedium Survival of mice in the experimental group of glycosides (experimental group 1).

Figure 6. Analysis of pathological tissue sections. A, normal mouse lung tissue section (experimental group 4). B. Normal mice were administered 30 mg/time of icariin in lung tissue sections (experimental group 3), and there were increased immune cells such as macrophages. C, lung tissue sections of experimental mice without icariin after infection with mice (experimental group 1), thrombosis in small blood vessels of the lungs, and a small amount of neutrophil degeneration and necrosis in the widened alveolar septum There is a small amount of lymphocyte infiltration around the bronchi. D, mice were infected and 30 mg/time of icariin was used to prevent lung tissue sections of experimental mice (experimental group 2), and there was a widening of alveolar septum with a small amount of macrophage infiltration.

Figure 7. Comparison of dissolution profiles of sustained release tablets prepared in Example 7 of the present invention at different pH values.

detailed description

In order to provide a substantial understanding of the present invention, certain aspects, modes, embodiments, variations and features of the present invention are described in various detail. In the practice of the present invention, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology, and recombinant DNA have been used. These techniques are well known.

In one embodiment, the icariin is defined by the following formula (I):

In another embodiment, the icariin derivative defined by the following formula (II) is prepared:

among them,

Xa, Xb, Xc, Xd, Xe, Xf, Xg, Xh, Xi, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X _{7 are} independently an oxygen atom (O) or a sulfur atom (S );

Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh and Ri are independently a hydrogen atom (H), a 1-5Ry-substituted alkyl group (C ₁ -C ₁₀ ) or (C ₁ -C ₁₀ )-O- (C ₁ -C ₁₀ ), each alkyl group (C ₁ -C ₁₀ ) is substituted with 1-5 Ry, each Ry is Rq, or (C ₂ -C ₁₀ )alkyl, (C ₃ -C ₁₀ An alkyl group, (C ₃ -C ₁₀ )cycloalkylalkyl group, (C ₈ -C ₁₄ )bicycloalkylalkyl group, (C ₈ -C ₁₄ )tricycloalkylalkyl group, —(C ₅ - C ₁₀ ) cycloalkylalkyl, (C ₃ -C ₁₀ )cycloalkyl, (C ₈ -C ₁₄ )bicycloalkylalkyl, (C ₈ -C ₁₄ )tricycloalkylalkyl, phenyl , naphthyl, (C ₁₄ ) aromatic groups, each of which may be substituted with one or more Rz, each Rz being (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, (C ₁ -C ₆ )alkynyl, (C ₃ -C ₈ )cycloalkyl, (C ₆ -C ₈ )cycloalkenyl, phenyl, 3- to 5-membered heterocyclic ring, C(halogen) ₃ Or CH(halogen) ₂ ; R ₁ , R ₂ , R ₂ , R ₄ , R ₅ , R ₆ , R ₇ and R _{8 are} independently a hydrogen atom (H), or a (C ₁ -C ₁₀ )alkyl group, ( C ₂ -C ₁₀ )alkenyl, (C ₂ -C ₁₀ )alkynyl, (C ₃ -C ₁₀ )cycloalkyl, (C ₈ -C ₁₄ )bicycloalkylalkyl, (C ₈ -C ₁₄ ) tricyclo alkyl, phenyl, naphthyl, (C ₁₄₎ Fragrance groups, each of which may be unsubstituted, substituted by one or more Rz or Rq, one or more Rq substitutions with more than one Rz substitution, each Rq may be CN, OH, halogen , N ₃ , NO ₂ , N(Rz) ₂ , ═NR z, CH═NRz, NRz OH, ORz, CORz, C(O)ORz, OC(O)Rz, OC(O)ORz, SRz, S( O) Rz or S(O) ₂ Rz; Y ₁ , Y ₂ and Y _{3 are} independently CH ₃ , (C ₁ -C ₁₂ )alkyl, or may be a (C ₁ -C ₁₂ ) alkane substituted with a combination of Ry Each of the (C ₁ -C ₁₂ )alkyl groups may be substituted with from 1 to 5 R y or with one counter ion.

In a preferred aspect, Xa, Xb, Xb, Xd, Xe, Xf, Xg, Xh, Xi and X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X _{7 are} independently oxygen atoms (O And Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh, Ri, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R _{8 are} independently a hydrogen atom (H) , Y ₁ , Y ₂ and Y _{3 are} independently CH ₃ .

In a specific embodiment, Xa, Xb, Xb, Xd, Xe, Xf, Xg, Xh, Xi and X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X _{7 are} independently oxygen atoms ( O), Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh, Ri, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R _{8 are} independently a hydrogen atom (H) ), Y ₁ , Y ₂ and Y _{3 are} independently CH ₃ .

It will be understood by those skilled in the art that the icariin and its derivatives include their individual isomers, racemic or non-racemic mixtures of isomers, oxides, or pharmaceutically acceptable salts thereof And hydrates.

In a specific embodiment of the present invention, the icariin compound can be modified by a group such as amination, carboxylation, aldehyde formation, benzylation or the like to prepare epidemis with different activities and physical and chemical properties. A glycoside compound derivative.

In a specific embodiment, the icariin and its icariin derivative used in the present invention may be a natural or chemically synthesized and modified compound having the above formula (I) or (II). The compound can be produced by a known method. Solvents, temperatures, pressures, and other reaction conditions can be readily selected by those skilled in the art. Starting materials are commercially available or can be readily prepared by one skilled in the art by known methods.

In a preferred embodiment, the natural icariin used in the present invention is extracted and purified from Epimedium plants.

Alternatively, the natural icariin plant is extracted and purified from the Epimedium plant of the Berberidaceae family. The Epimedium plants include E. davidii, E. baojingense, E. icoroanum, and E. icoroanum. (E. fargesii), E. elongatum, E. acuminatum, E. simplicifolium, E. brachyrrhizum, multi-flower Epimedium (E. multiflorum), E. enshiense, E. reticulatum, E. sagittatum, E. latisepalum ), E.chlorandrum, E.platypetalum, E. truncatum, E. rhizomatosum, three-leaf clover (E.sagittatum), E. pauciflorum, E. myrianthum, E. ecalcaratum, E. glandulosopilosum, One or more mixtures of the group consisting of E. parvifolium, E. stellulatum, and E. ilicifolium. The definitions of some terms used in the specification, unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Prophylactic and therapeutic uses of icariin and its derivatives

In one embodiment of the invention, one skilled in the art screens to inhibit influenza virus neuraminidase activity by inhibiting influenza virus replication by performing enzymatic kinetic experiments using potential candidate compounds. In particular, it has been determined by enzymatic kinetic screening experiments of candidate compounds that icariin and its derivatives are capable of completely inhibiting influenza virus neuraminidase activity for the prevention and/or treatment of influenza virus diseases.

In a specific embodiment of the diseases caused by various influenza viruses, the ratio of the addition of the influenza virus to the icariin may be 1:0.00001-10000, preferably 1:0.0001-1000; more preferably 1 by weight. 0.001-500; most preferably 1:0.01-100 to achieve complete inhibition of influenza virus neuraminidase, thereby inhibiting replication of influenza virus.

In another embodiment of the invention, the prepared icariin and its derivatives treat or prevent pathological symptoms or diseases caused by influenza virus. The influenza virus includes influenza virus A, B and C and their various subtypes, serotypes, genotypes, gene mutant viruses; the pathological symptoms or diseases are caused by various viruses of the above viruses Type one or more diseases caused by influenza syndrome. Preferred are cold syndrome, cough, fever, sneezing, nasal congestion, runny nose, chills, headache, fatigue, body aches, pneumonia, tonsillitis, pharyngitis, laryngitis, tearing, hoarseness, respiratory syndrome, Cardiopulmonary disease, cold limbs, nausea and vomiting, dry cough, dry mouth, drowsiness, diarrhea, cold abortion, myocarditis, hypertension, myocardial infarction, stroke, nasal mucosal congestion, edema, secretions, pharyngeal light Degree of congestion, submandibular lymphadenopathy, pharyngeal congestion and edema.

The term "treating" as used herein, or refers to a therapeutic treatment and a preventive or preventive measure, wherein the subject's pathological symptoms or disorders are prevented or slowed down (weakened). If a therapeutic amount of icariin and its derivatives according to the methods described herein is accepted, the subject's symptoms are successfully "treated" and the subject shows that symptoms can be observed and/or determined One or more signs and symptoms are reduced and disappeared. It should also be understood that the various modes of treating or preventing a medical condition described herein are intended to mean "significant", which includes all treatment or prevention and less than all treatment or prevention, wherein certain biologically relevant or medically relevant results are achieved.

In various embodiments, suitable in vitro assays or in vivo assays are performed to determine the effect of a therapeutic agent based on a particular icariin and its derivatives and whether its administration is suitable for treatment. In various embodiments, in vitro assays can be performed on representative cells involved in a subject's condition to determine whether a given therapeutic drug based on icariin and its derivatives has an expectation of the virus type. Effect. If a therapeutic amount of icariin and its derivatives according to the methods described herein is accepted, the subject's symptoms are successfully treated, ie, the subject shows that the above pathological symptoms can be observed and/or determined Or one or more signs and symptoms of the disease are reduced and disappeared. It should also be understood that the various modes of treating or preventing a medical condition described herein are intended to mean "significant", which includes all treatment or prevention and less than all treatment or prevention, wherein certain biologically relevant or medically relevant results are achieved.

In a specific embodiment of the invention, the subject is administered a prophylactically and/or therapeutically effective amount of a medicament comprising icariin and a derivative thereof.

The candidate compounds used in the treatment can be tested in a suitable animal model system including, but not limited to, non-human primates (eg, baboons, orangutans, prior to testing the human subject). Monkeys; pet animals such as cats, dogs, snakes, etc; farm animals of pigs, horses, cows, goats, etc.; any animal of laboratory animals such as rats, mice, monkeys, rabbits, and the like. In one embodiment, icariin and a derivative thereof are administered to a subject suffering from or at risk of the above-mentioned pathological symptoms or diseases (a state in which the symptoms are readily available) in an attempt to improve the pathological symptoms Or one or more factors of the disease.

The term "effective amount" as used herein refers to an amount sufficient to achieve the desired therapeutic and/or prophylactic effect, for example, to cause prevention or alleviation of symptoms associated with pathological symptoms or diseases. The amount of the composition administered to the subject will depend on the type and severity of the disease as well as the nature of the individual, such as the usual health conditions, age, sex, weight, and tolerance to the drug. The amount will also depend on the severity, severity and type of disease. Those skilled in the art will be able to determine the appropriate dosage based on these and other factors. The composition may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the compounds of the invention can be administered to a subject having one or more signs or pathological symptoms of pathological symptoms. For example, "therapeutically effective amount" refers to an average level that minimizes the physiological effects of symptoms.

Generally, the dosage will be capable of preventing or reducing the severity or spread of the condition or indication being treated. The correct dosage will depend on the circumstances, such as the condition being treated, the schedule of administration, whether the compound is administered alone or in combination with another therapeutic agent, the plasma half-life of the compound, and the overall health of the subject.

The subject can be administered by oral, topical, inhalation, nasal, rectal, transdermal or injection administration.

In a specific embodiment of the invention, a daily dosage is prepared containing the compound disclosed herein. The regimen is from about 0.1 mg/kg to about 10000 mg/kg body weight, preferably from about 0.1 mg/kg to about 1000 mg/kg body weight, more preferably from 0.1 mg/kg to about 500 mg/kg, and most preferably from 0.5 mg/kg to About 200 mg/kg body weight. It can be administered from 1 to 6 times a day, preferably 2 or 3 times a day. The spacing may also be irregular, and those skilled in the art recognize that the optimal amount of the compound or its pharmaceutically acceptable salt and the interval between single administrations will depend on the nature and extent of the condition to be treated, the form, route and location of administration. The point and the particular condition of the subject being treated are determined and the most preferred protocol can be determined using conventional techniques. It will also be understood by those skilled in the art that the optimal course of treatment, i.e., the amount of the compound administered daily or a pharmaceutically acceptable salt thereof administered over a given number of days, can be utilized by those skilled in the art using conventional therapeutic test methods. determine. In therapeutic applications, relatively high doses in relatively short time intervals are sometimes required until the course of the disease slows or terminates, and preferably until the subject shows a partial or complete improvement in the disease or pathology symptom. Therefore, the patient can perform a prophylactic administration method to the patient. One of skill in the art will recognize that certain factors can affect the dosage and timing of effective treatment of a subject, including but not limited to the severity of the disease or disorder, prior treatment, health conditions, and/or subject's Age and other diseases that exist. Moreover, treating a subject with a therapeutically effective amount of a therapeutic control composition described herein can include a single treatment or a series of treatments.

The compounds of the invention may be administered with at least one other active ingredient other compound and/or a Chinese medicinal ingredient. The compounds can be administered simultaneously as a separate formulation or in a unit dosage form, or sequentially. The compound is ribavirin (1), interferon, thymosin alpha _, transfer factor, zidovudine (ZDV), didanosine, Lamivudine, stavudine, zalcitabine, abacavir, Ribavirin, (Virazole), nevirapine, diarrhife Delavirdine, efavirenz, saquinavir, ritonavir, nelfinavir, indinavir, amprenavir, Lopinavir, King Kong Amantadine, Rimantadine, Zanamivir, Acyclovir, Valacyclovir, Idoxuridine, Vidarabine , Lamivudine, Famciclovir, Penciclovir, oseltamivir and zanamivir, wherein at least one other active ingredient of the traditional Chinese medicine is Radix, Folium, Silver Aster, Forsythia, Nepeta, Honeysuckle, Houttuynia, Wild Chrysanthemum, Summer Sangju, Chai One or more combinations of traditional Chinese medicine ingredients such as andrographis paniculata. In any case, the plurality of therapeutic agents can be administered in any order or even simultaneously. If at the same time, the plurality of therapeutic agents can be provided in a single, uniform form. Or in multiple forms (for example, as a single tablet or as separate tablets or capsules, several different formulations of pills). One of the therapeutic agents may be provided in a multi-dose form, or Several may be provided as a multi-dose type. If not simultaneously, the time interval between multiple doses may range from greater than 0 weeks to less than about 1 week, less than about 2 weeks, less than about 4 weeks, less than about 2 months, A change in the range of less than about 4 months or even less than about half a year.

The term "unit dosage form" as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit comprising a separate predetermined amount of compound or in combination with other agents, the amount being calculated Sufficient to combine with a pharmaceutically acceptable diluent, carrier or vehicle to produce the desired effect.

In a specific embodiment of the present invention, a pharmaceutical composition containing icariin or a derivative thereof for treating and/or preventing a pathological symptom or disease associated with an improvement of a cold disease caused by an influenza virus is prepared. It is administered to a subject by oral administration, injection, inhalation, or the like. Therefore, the pharmaceutical composition of the present invention can be prepared into various dosage forms. For example, the composition for oral administration can be a capsule, a dropping pill, a gel, a tablet, a powder, a granule, a lozenge, an effervescent tablet, or a syrup. , emulsion, controlled release preparation (including sustained release tablets or sustained release capsules, etc.), fast-dissolving preparations, oral liquid preparations The composition for external use may be a smear-type drug, a cream, an external application, an ointment, a topical syrup, a liquid spray, an air spray, or the like. The composition for administration by inhalation may be a solution, a dispersion, a dry powder or the like; or a high-purity icariin compound may be extracted from the Chinese herbal medicine Epimedium, and a liposome-encapsulated nano icariin is prepared and prepared. Injectable preparations and sustained release pharmaceutical dosage forms. In the pharmaceutical composition, the proportion of icariin or a derivative thereof is from 0.0% to 99.0%, preferably from 0.0% to 90%, more preferably 10% by weight based on the total weight of the entire preparation. -80%, most preferably 25-70%.

The above preparation may be a controlled preparation, including a sustained release tablet or a sustained release capsule. The controlled release tablet comprises a core and a controlled release layer that encapsulates the core. The core contains at least two active ingredients, at least one of which is icariin and a derivative thereof. The core may further comprise at least one other active ingredient of Interferon, Thymosin alpha _, Ribavirin, Amantadine, Rimantadine oseltamivir and Zanamivir. The core may also contain at least one other traditional Chinese medicine active ingredient which is one or more combinations of traditional Chinese medicine ingredients such as Radix, Eucalyptus, Forsythia, Honeysuckle, Bupleurum, Andrographis paniculata. The controlled release layer of the controlled release tablet comprises a copolymer of methacrylic acid and ethyl acetate, a copolymer of methacrylic acid and methyl methacrylate, ethyl cellulose, and triethyl citrate. Wherein, according to the weight ratio, the ratio of the core and the controlled release layer of the package core is 1:0.01-100.

In a specific embodiment of the invention, a method of preparing a sustained release tablet is provided. The controlled release formulations produced provide pH independent release characteristics in the range of pH 1 to 10.0.

In the pharmaceutical preparation of the present invention, the oral preparation is exemplified, and the pharmaceutically acceptable excipients used include, but are not limited to, lubricants and co-solvents such as magnesium stearate, calcium stearate, zinc stearate, stearic acid. , behenic acid glyceride, octadecyl fumarate, talc, anhydrous colloidal silica, silica gel; disintegrating agents such as starch, cyclodextrin, maltodextrin, carboxymethyl cellulose, methyl Acrylic acid and ethyl acetate copolymer, vinyl acetate copolymer, methacrylic acid and methyl methacrylate copolymer, ethyl cellulose, triethyl citrate, croscarmellose, crosslinked polyethylene Pyrrolidone, gelatin, microcrystalline cellulose; diluent or compressive agent, such as maltodextrin glucose, lactose, galactose, sucrose, mannitol xylitol, erythritol, sorbitol, microcrystalline cellulose; coating , dry paint, N-vinyl butyrolactam, titanium dioxide; seasonings or other ingredients, such as strawberries, oranges, bananas, mint, honey; cosolvents, such as alcohol (beer, fruit wine, rice wine, white wine), vegetable oil, Animal oil, ethanol, isopropanol, glycerin , mannitol, propylene glycol, benzyl alcohol, dimethyl sulfite, polyethylene glycol, gum arabic, lecithin, pyrrolidone, oleic acid, azone; colorant.

The carrier particles may be crystals or spheres of lactose or sucrose; or composite spheres or granules, such as spheres of sugar formed by granulating sucrose with starch as a binder, calcium carbonate spheres or malt paste formed by starch as a binder fine. The carrier particles can also be particles of any other pharmaceutically acceptable excipient such as hydroxypropylcellulose granules, guar gum granules, xanthan gum granules. The carrier can take a wide variety of forms, and all such selections and amounts of adjustments are clearly within the abilities of those skilled in the art.

By the oral route, the compound of icariin or a derivative thereof according to the present invention or a composition thereof may comprise a liquid or a water-alcohol which is dissolved in a food or a health care product liquid, such as optionally dissolved in a flavoring. Class solution. It can be incorporated into a swallowable solid excipient, for example in the form of granules, pills, tablets, enteric coated tablets. It can also be placed in a liquid in a food or health supplement, optionally in the swallow capsule. For swallowing, a variety of oral composition embodiments can be employed, and in particular, can be oral composition embodiments of food addenda. The enteric coated tablets, colloidal capsules, gels, emulsions, tablets, capsules or solutions are prepared by conventional methods. In particular, the active agent according to the invention may be incorporated into a food supplement or any other form of fortified food or health supplement, such as a food or health care product stick, or compressed or uncompressed Among the powders. These powders can be diluted with water, diluted with water in soda, cheese products or soy derivatives, or can be incorporated into food or health care bars.

The present invention will be better understood by the following examples, but is not limited to the examples.

Example

Example 1. Preparation of influenza virus neuraminidase and analysis of its effect on inhibition of influenza virus neuraminidase activity by icariin

Take 104TCID50 of influenza virus H5N1 (A/chicken/Guangdong/174/04 (H5N1)), H9N2 (A/chicken/Guangdong/SS/94 (H9N2), in order to try to determine whether icariin is related to other subtypes of neuraminic acid The inhibitory effect of the enzyme was also selected from the H9N2 virus strain to prepare a neuraminidase solution, and Triflurane X-100 (Sigma) was added to a concentration of 1% to prepare an influenza virus neuraminidase solution for assay of inhibitor activity. The 98% pure icariin standard (ChromaDex, USA) was dissolved in 30% dimethyl nitrous oxide (Sigma) at a concentration of 10 mmol/L; Methylumbelliferyl-aDN-acetylneuramimic acid (MUNANA, sigma) was formulated into 20 Micromol/L concentration; oseltamivir (Oseltamivir, ChromaDex, USA) was formulated with water to a concentration of 10 mmol/L; 10 times buffer (325 mmol MES (Sigma), 40 mmol CaCl (Sigma), pH 6.5). The enzyme activity assay reaction system was 100 microliters and was carried out in a black 96-well plate. Influenza virus neuraminidase solution was pre-charged with different concentrations of icariin, and then added 3 μL of reaction buffer, 27 μL of water, which contained 1 μmol, 5 μmol, 50 μmol, 100 μmol, 200 μmol of icariin concentration gradient (final reaction system) concentration). At the same time, oseltamivir was set as a control experiment according to the above method, and the final reaction system concentration gradient was 1 μmol, 5 μmol, 50 μmol, 100 μmol, and 200 μmol. In addition, it is not necessary to add influenza virus neuraminidase solution (other components are added at the above concentration, the influenza virus neuraminidase solution is replaced with water), no icariin and oseltamivir are added (other ingredients are added according to the above concentration, Replenish the reaction volume with water), add no icariin and oseltami but add 1% DMSO (addition of other components at the above concentration, 1% DMSO is the final reaction system concentration). Add the reaction before MUNANA. The system is 40 μL, and each reaction system is shown in Table 1. After reacting at 37 ° C for 1 hour, all the reaction systems were added with 60 μl of 20 μmol/L MUNANA, reacted at 37 ° C for 1 hour, and terminated with 0.002 mol/L sodium hydroxide. reaction. Fluorescence intensity was measured using a super-microplate reader (Synergy HT, blo-tek, USA) at 360 excitation light and 460 emission light.

Table 1. Composition of each reaction system before adding MUNANA

Inhibition of influenza virus neuraminidase activity by icariin (Fig. 1) revealed that icariin inhibits influenza virus neuraminidase activity, which is nearly 40 times higher than oseltamivir, suggesting epimedium Glycoside can be used as a lead compound for the prevention and/or treatment of influenza virus diseases, and it is expected to develop a better drug for oseltamivir. In addition, this experiment also confirmed that icariin can also inhibit the neuraminidase activity of different subtypes, which can inhibit the activity of N1 (H5N1) and N2 (H9N2) enzymes, suggesting that icariin may inhibit Neuraminidase activity of various subtypes.

Example 2. Inhibition of influenza virus by icariin at the in vitro cell level

MDCK (Madin-Darby Canine Kidney) cells (provided by Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences) were selected as influenza virus proliferating cells, and DMEM (Gibco) was used as the base medium for MDCK cells. MDCK according to the method of Xie et al. (Yi Xie, James A. Schafer. Inhibition of ENaC by intracellular Cl ^- in an MDCK clone with high ENaC expression. Am J Physiol Renal Physiol. 2004 Oct; 287(4): F722-31.) Cell culture: 190 mL of DMEM medium was added to 20 mL of fetal bovine serum, 2 mL of green chain double antibody, 7.5% NaHCO _{3 to} adjust the pH to 7.4. The above components were mixed into MDCK complete medium, 15 passages of cell bottles were added, and the amount of digestive juice was added. It is advisable to cover the cells just before, and gently put them in a 37 ° C incubator for about 10 minutes. When the cells are rounded and do not associate with each other, gently digest the digestive juice, add the culture solution, and repeatedly suck and blow until the cells are The cells were dissociated, and the cells were counted. 1.0 × 10 ⁵ /mL cells were seeded in a new 25 mL culture flask, and cultured at 37 ° C in a 5% CO ₂ atmosphere. The influenza virus was inoculated into the 9-day-old chicken embryoal allantoic cavity, incubated at 35 ° C for 48-72 h, then the chicken embryo was placed in a refrigerator at 4 ° C overnight, the allantoic fluid was harvested the next day, centrifuged at 3000 rpm for 10 min, and the precipitate was discarded. The serum was subjected to a blood coagulation test, and it was confirmed that the titer was greater than 1:64, and the supernatant was harvested and stored at -80 °C. Influenza virus-infected cells were determined for virus titer: cultured cells were passaged and counted, transferred to a 6-well culture plate, and the cell density per well was adjusted to 1.0 × 10 ⁵ /mL. Take H5N1 subtype, H9N2 (in order to try to inhibit the effect of icariin on other subtypes of neuraminidase, we also selected H9N2 strain) subtype influenza virus 1mL was added to the A and B cells, The same amount of physiological saline was added to the C well as a negative control. The final volume of each well culture system was adjusted to 5 mL, and A and B cells were incubated for 72 h for a total of 3 groups. Each group was repeated 5 times. Cytopathic effect (CPE) was observed under a microscope to determine the virus titer. Refer to the method of Hatakeyama et al. (Shuji Hatakeyama, Yuko Sakai-Tagawa, Maki Kiso, Hideo Goto, Chiharu Kawakami, Keiko Mitamura, Norio Sugaya, Yasuo Suzuki and Yoshihiro Kawaoka. Enhanced Expression of an α2, 6-Linked Sialic Acid on MDCK Cells Improves Isolation Of Human Influenza Viruses and Evaluation of Their Sensitivity to a Neuraminidase Inhibitor.J Clin Microbiol.Aug 2005;43(8):4139–4146.), Icariin for Influenza Virus Inhibition Experiment: Icariin with 30% The dimethyl sigma was dissolved at a concentration of 1 mmol/mL and added to the DMEM cell culture medium. The experimental results of Example 1 were used to determine the concentration of 5 μmol for the detection of icariin inhibition virus replication assay. Icariin was replaced with PBS as a negative control to inhibit viral replication. Using a 96-well culture plate, set up 5 parallel experiments, 1.0 × 10 ⁵ /mL MDCK cells 100 μL, add 100 μL of virus containing 200 TCID ₅₀ titer and various concentrations of icariin, set at 37 ° C, 5% CO 2 environment The culture was allowed to stand for 3 days, and the virus value added was observed. Flow cytometry (Beckman Coulter, USA), refer to the method of Liu et al. (Liu H, Zhang GL, Shen L, Zeng Z, Zhou BL, Liu CH, Nie G. Application and evaluation of a pseudotyped virus assay for screening herbs for anti-H5N1 avian influenza virus.Zhong Xi Yi Jie He Xue Bao.2010Nov;8(11):1036-40.), fluorescent antibody (Anti-Influenza A H5N1 Hemagglutinin Antibody and Anti-Influenza A H9N2 Hemagglutinin Antibody, purchased from Sino Biotlogical Inc., with reference to the method of Ebrahimi et al. (Ebrahimi SM, Tebianian M, Aghaiypour K, Nili H, Mirjalili A. Prokaryotic expression and characterization of avian influenza A virus M2 gene as a candidate for universal Recombinant vaccine against influenza A subtypes;specially H5N1 and H9N2.Mol Biol Rep.2010 Jul;37(6):2909-14.), the virus proliferation was analyzed by staining and detected by fluorescence microscopy (Olympus, Germany). Real-time label-free cell function analyzer (xCELLigence RTCA MP, ACEA Biosciences Inc) analysis: 200 TCID50/mL influenza virus H5N1 and H9N2 were mixed with diluted inhibitors in a 1:1 ratio and placed in a 37 ° C incubator for incubation. One hour, a mixture of virus and inhibitor was then added to a 96-well cell electronic assay plate in which MDCK cells had been grown, and placed in a 37 ° C incubator for 190-200 hours. During the experiment, there were 7 icariin concentration gradients, ie, the medium containing 1 μmol, 5 μmol, 10 μmol, 20 μmol, 50 μmol, 100 μmol, 200 μmol, and a virus-negative control and icariin-free inoculated virus control . Culture and detection were performed using a real-time label-free cell function meter, and data acquisition was performed every 15 minutes.

Inhibition of influenza virus by icariin at the cellular level in vitro (Example 2), protection of MDCK cells from influenza virus destruction by viral pathogenicity and icariin of MDCK cells, including detection of infection using fluorescent antibodies Influenza-infected cells (Fig. 2), flow cytometry analysis (Fig. 3), and real-time unlabeled cell function analyzer analysis (Fig. 4) are all well demonstrated that icariin can inhibit influenza virus proliferation and protect it. Cells are protected from viruses. In this experiment, we used two strains of influenza virus neuraminidase, namely H5N1 (N1) and H9N2 (N2), and icariin showed these two different neuraminidase strains. Almost the same inhibitory effect, suggesting that the neuraminidase virus strain may be a broad-spectrum inhibitor of influenza virus subtypes, serotypes, genotypes, and icariin can be used as a preventive and/or therapeutic agent. The flu virus causes pathological symptoms or diseases of drugs, foods, and health products.

Example 3. Animal experiment of icariin inhibiting influenza virus

3.1 Experimental animal species and characteristics

BALB/C mice, the Experimental Animal Center of Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, were reproduced. The experimental animal quality license number was Heilongjiang-SYXK 2007-167.

Age 9 weeks to 10 weeks, weight 20g, male and female.

3.2 feeding conditions

The animal laboratory air is regularly ventilated and well lit, keeping the room temperature at the implementation room. Five animals were kept per cage, and the puffed feed specially made for mice was fed freely. Before the start of the experiment, observe the animals' feeding, activity and feces for a week. Only healthy mice were selected for the next experiment.

3.3 Animal safety evaluation of icariin

First, the acute toxicity test was used to evaluate the safety of icariin in order to provide a basis for the application of icariin. In this experiment, the maximum tolerance method for acute toxicity test was used. First, 20 healthy BALB/C mice weighing about 20 g were selected, male and female. The clinically recommended route of administration is oral, and the equity experiment is administered by intragastric administration. Icariin was prepared into a mushy paste with distilled water and administered to mice once a day (about 200 mg of icariin per mouse). The animals were fasted overnight and given free access to water before gavage. After the stomach was given a normal diet, the symptoms of poisoning and death were observed and weighed once a week for 2 weeks. Under the same conditions, the same batch of mice were fed with normal food and observed as the control group. The results of the experiment showed that all mice had any symptoms of poisoning, which proved that icariin is a non-toxic substance.

Icariin for the prevention of influenza virus infection in animals: 6-week-old male BALB/c mice were selected, each group was inoculated with 50 μL of 102 LD50 H5N1 virus (since H9N2 is pathogenic to mice, so this H9N2) was not selected in the experiment (experimental group 1 and experimental group 2), PBS was used as a negative control (experimental group 4), and a control group (experimental group 3) administered with icariin alone was infected with the virus-infected mouse. Four hours later, four experimental animal groups were set up, each group of 10 mice, each of which was orally administered with 1 mg, which was equivalent to oral administration of icariin 25 mg/kg body weight per day, twice a day for 14 days. The mice were weighed once a day, and the changes in body weight and survival rate were observed. Histopathological examination: Selecting the diseased, treatment group, oral administration of icariin and normal mouse lung tissue with 10% neutral-buffered formalin, then correcting and using different concentrations Dehydrated ethanol, soaked in xylene (Sima), embedded in paraffin (Paraffin, Sigma), cut into 3 micron thick slices, placed on glass slides, dewaxed continuously with alcohol, with hematoxylin (Hematoxylin) and Eosin (Sigma) staining were observed under light microscopy.

Table 2, animal experiment group experiment

Group	Vaccination strain	Icariin	PBS
Experimental group 1	infection	no	no
Experimental group 2	infection	Yes	no
Experimental group 3	Uninfected	Yes	no
Experimental group 4	Uninfected	no	Yes

An animal experiment in which icariin inhibits influenza virus (Example 3) showed that icariin was able to protect against infection with highly pathogenic avian influenza virus (H5N1) (Fig. 5). In addition, histopathological sections showed that icariin can also promote immune cell infiltration in lung tissue and improve the immune protection of lung tissue (Fig. 6). Icariin can be used in the preparation of icariin and its derivatives for the treatment or prevention of drugs, foods, and health products for pathological symptoms or diseases caused by influenza virus infection.

Example 4: Capsule preparation

The icariin 0-side composition used in the examples of the present invention may be icariin for purchasing a drug, 200 g of icariin, and pulverized into a fine powder. The component was passed through a 120 mesh sieve, weighed, and the amount of starch and magnesium stearate added in the above table were mixed and poured into a lower hopper. Open granulation coating machine, inlet air pressure O.6bar, inlet air temperature 30 °C, spray gun pressure (CYL) 3b cylinder, atomization pressure (CAP1) 0.8 bar, pour 250g blank pellet core, granulation, granulation speed 4 rpm, 12% creep pump, 145 rpm rotary speed, spray 200 g of 7% PVP solution (solvent is 90% ethanol). After the granulation is finished, it is dried at 50 ° C and discharged.

The prepared pellets were filled with a capsule containing amyl saponin by using a hard capsule drug filling machine to fill 200 mg of icariin per two capsules, respectively.

Example 5 Tablet Preparation a

Active agent	Add amount (g)
Icariin	300
Excipient
Cassava starch	30
Galactose	20
Microcrystalline cellulose	30

Preparation process: weigh 200g of icariin, pass 100 mesh sieve, tapioca starch, galactose, microcrystalline cellulose over 80 mesh sieve, mix evenly, add 100g of 6% PVP absolute ethanol solution to prepare granules, 60 °C Dry, 18 mesh sieve dry pellets, 2 g of magnesium stearate was added to the dry pellets.

c. The above a is punched with a double laminator to obtain 200 mg of icariin per tablet.

Example 6. Mixed capsule controlled release preparation

Designing a controlled release formulation for oral administration in accordance with the present invention requires selection and evaluation of the in vitro release profile and timing of the formulation, making it optimal for obtaining the desired in vivo plasma profile, preferably once daily. Thus, several different formulation principles for single unit matrix tablets have been investigated, namely providing formulations with different release profiles.

a, preparation of active drug core

Active agent	Add amount (g)
Icariin	200
Excipient
starch	200
Magnesium stearate	3
Adhesive
glucose	2
Maltodextrin	3
Vinyl acetate copolymer	1.5

The icariin component or icariin used in the examples of the present invention may be a purchased drug, and 200 g of icariin is weighed and pulverized into a fine powder. The component was passed through a 120 mesh sieve, weighed, and the amount of starch, magnesium stearate, glucose, maltodextrin and vinyl acetate copolymer added in the above table was mixed, passed through a 250 mesh sieve, and then poured into a lower hopper. in. Open granulation coating machine, inlet air pressure O.6bar, inlet air temperature 30 °C, spray gun pressure (CYL) 3b cylinder, atomization pressure (CAP1) 0.8 bar, pour 250g blank pellet core, granulation, granulation speed 4 rpm, 12% creep pump, 145 rpm rotary speed, spray 200 g of 7% PVP solution (solvent is 90% ethanol). After the granulation is finished, it is dried at 50 ° C and discharged.

b, preparation of coating

Coating composition	Add amount (g)
Methacrylic acid and ethyl acetate copolymer	90
Methyl methacrylate and methyl methacrylate copolymer	135
Ethyl cellulose	180
Triethyl citrate	450

90 g of methacrylic acid and ethyl acetate copolymer, 135 g of methacrylic acid and methyl methacrylate were dissolved in a fluidized bed at room temperature with an acetone/isopropanol/water mixture (54/36/10, weight percent) The copolymer, 180 g of ethyl cellulose and 45 g of triethyl citrate were dissolved while using the mixture to coat 450 g of the fine particles obtained in a. The coated particles are obtained after spraying. The average diameter is 270 μm.

c. The pellets prepared by a and b were filled with a hard capsule drug filling machine according to the coated fine particles to prepare a capsule, that is, a mixed controlled release capsule preparation of the present invention containing 0.5 g of icariin was obtained.

Example 7 Preparation of Controlled Release Tablets of the Present Invention

a,

Active agent	Add amount (g)
Icariin	200
Excipient
Cassava starch	30
Galactose	20
Microcrystalline cellulose	30

Preparation process: weigh icariin 200g over 100 mesh sieve, tapioca starch, galactose, microcrystalline cellulose over 80 mesh sieve, mix well, add 100g 6% PVP absolute ethanol solution to granulate, dry at 60 ° C 18 mesh sieved dry granules, and 2 g of magnesium stearate was added to the dry granules.

b, preparation of coating

Dissolve in a fluidized bed at room temperature with an acetone/isopropanol/water mixture (54/36/10, weight percent) to 90 g of methacrylic acid and ethyl acetate copolymer, 135 g of methacrylic acid and methacrylic acid The ester copolymer, 180 g of ethyl cellulose and 45 g of triethyl citrate were dissolved while using this mixture to coat 450 g of the fine particles obtained in a. The coated particles are obtained after spraying. The average diameter is 270 μm. In order to obtain a multi-layer coating, the spray is again carried out on the basis of the original coating, and the above steps are repeated to obtain the desired multilayer controlled release layer.

c. The above two components a and b are punched by a double laminating machine to obtain a two-layer tablet, each of which is 0.5 g of icariin.

The extended release tablets of the present invention utilize a swellable and partially aggressive polymeric matrix. Based on the hypothetical matrix, the release profile can describe the in vitro release profile of the sample approximately at the square root of time. Obtaining the in vitro release profile that is not affected by the pH value is not affected by the pH value of the gastrointestinal tract when absorbed in the body, and is beneficial to avoid the difference in individual absorption due to the difference in pH of the gastrointestinal tract. The polymer of the present invention constitutes an extended release matrix, and slowly releases icariin as a active ingredient and derivatives thereof and combinations thereof. After administration, after contact with the aqueous liquid, the polymer swells to form a viscous gel layer to regulate drug release. The viscosity of the polymer preferably ranges from 180 to 110,000 mPa.s (apparent viscosity of a 2% aqueous solution at 20 ° C).

The controlled release samples prepared in Example 6 or 7 of the present invention were simultaneously subjected to a release test at different pH values, and a release test curve was drawn based on the release test data. As shown in FIG. 7, the preparation of Example 7 can be seen. The sample has a consistent release profile in each pH medium and its release is virtually unaffected by the pH of the medium.

Example 8 Preparation of Injectable Formulations of the Compositions of the Invention

Formula: 10g icariin

20g mannitol

Weigh 10g of icariin, prepare 1000ml solution with water for injection, sterilely filter, fill with 1ml each, freeze-dry, tampon, and cover to obtain the finished injection.

Example 9: Preparation of coated tablets

The beneficial effects of the present invention are: icariin can inhibit the activity of influenza virus neuraminidase, thereby inhibiting the replication of influenza virus, and can be used for the prevention and treatment of various diseases caused by influenza virus infection, food, health care products. Development.

The invention is not limited to the specific embodiments described in the application, and is used as a single description of the individual aspects of the invention. A person skilled in the art will appreciate that various modifications and changes can be made without departing from the spirit and scope of the application. Functionally equivalent uses within the scope of the present disclosure will be apparent to those skilled in the art from this disclosure. Such modifications and variations are intended to fall within the scope of the appended claims. The present disclosure is only limited by the scope of the appended claims and the scope of the claims. It should be understood that the present disclosure is not limited to specific methods, reagents, compositions, and biological systems, of course, the methods, reagents, compositions, and biological systems may vary. It is also understood that the terminology used herein is for the purpose of describing the particular embodiments

Moreover, where features or aspects of the disclosure are described in the form of a Markush group, those skilled in the art will recognize that the present disclosure is also a single member or subgroup member of the Markush group. The way it is described.

All of the patents, patent applications, prior applications and publications are hereby incorporated by reference in their entirety in their entirety in their entireties in the entireties in Other embodiments are proposed within the scope of the claims.

Claims (54)

1. A novel inhibitor of influenza virus neuraminidase characterized in that the inhibitor is icariin and a derivative thereof, and the icariin and its derivatives are respectively of the following formula (I) and II) as defined, including its individual isomers, racemic or non-racemic mixtures of isomers, oxides, or pharmaceutically acceptable salts and hydrates thereof,

   

   Wherein Xa, Xb, Xc, Xd, Xe, Xf, Xg, Xh, Xi, X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ and X _{7 are} independently an oxygen atom (O) or a sulfur atom. (S);

   Ra, Rb, Rc, Rd, Re, Rf, Rg, Rh and Ri are independently a hydrogen atom (H), a 1-5Ry-substituted alkyl group (C ₁ -C ₁₀ ) or (C ₁ -C ₁₀ )-O- (C ₁ -C ₁₀ ), each alkyl group (C ₁ -C ₁₀ ) is substituted with 1-5 Ry, each Ry is Rq, or (C ₂ -C ₁₀ )alkyl, (C ₃ -C ₁₀ An alkyl group, (C ₃ -C ₁₀ )cycloalkylalkyl group, (C ₈ -C ₁₄ )bicycloalkylalkyl group, (C ₈ -C ₁₄ )tricycloalkylalkyl group, —(C ₅ - C ₁₀ ) cycloalkylalkyl, (C ₃ -C ₁₀ )cycloalkyl, (C ₈ -C ₁₄ )bicycloalkylalkyl, (C ₈ -C ₁₄ )tricycloalkylalkyl, phenyl , naphthyl, (C ₁₄ ) aromatic groups, each of which may be substituted with one or more Rz, each Rz being (C ₁ -C ₆ )alkyl, (C ₂ -C ₆ )alkenyl, (C ₁ -C ₆ )alkynyl, (C ₃ -C ₈ )cycloalkyl, (C ₆ -C ₈ )cycloalkenyl, phenyl, 3- to 5-membered heterocyclic ring, C(halogen) ₃ Or CH(halogen) ₂ ; R ₁ , R ₂ , R ₂ , R ₄ , R ₅ , R ₆ , R ₇ and R _{8 are} independently a hydrogen atom (H), or a (C ₁ -C ₁₀ )alkyl group, ( C ₂ -C ₁₀ )alkenyl, (C ₂ -C ₁₀ )alkynyl, (C ₃ -C ₁₀ )cycloalkyl, (C ₈ -C ₁₄ )bicycloalkylalkyl, (C ₈ -C ₁₄ ) tricyclo alkyl, phenyl, naphthyl, (C ₁₄₎ Fragrance groups, each of which may be unsubstituted, substituted by one or more Rz or Rq, one or more Rq substitutions with more than one Rz substitution, each Rq may be CN, OH, halogen , N ₃ , NO ₂ , N(Rz) ₂ , ═NRz, CH═NRz, NRz OH, ORz, CORz, C(O)ORz, OC(O)Rz, OC(O)ORz, SRz, S(O Rz or S(O) ₂ Rz; Y ₁ , Y ₂ and Y _{3 are} independently CH ₃ , (C ₁ -C ₁₂ )alkyl, or (C ₁ -C ₁₂ )alkyl substituted with a combination of Ry Each (C ₁ -C ₁₂ )alkyl group may be substituted with 1-5 Ry or a counter ion.
2. The novel inhibitor according to claim 1, characterized in that the influenza virus comprises influenza virus A, B and C and various subtypes thereof, serotypes, genotypes, gene mutant viruses.
3. The inhibitor according to claim 2, wherein the influenza virus is an influenza A virus.
4. The inhibitor according to claim 3, characterized in that the influenza virus is an H5N1 or H9N2 virus.
5. The inhibitor according to claim 1, characterized in that icariin and its derivative have a nearly 40-fold inhibitory effect on influenza virus neuraminidase activity compared to oseltamivir.
6. A method for screening novel inhibitors of influenza virus neuraminidase, characterized in that the method comprises adding icariin and a derivative thereof as an inhibitor of influenza virus neuraminidase to a cell or an animal.
7. The method of claim 6 wherein said cells are MDCK cells.
8. The method of claim 6 wherein said animal is a chicken or a mouse.
9. Use of the inhibitor according to any one of claims 1 to 5 for the preparation of a medicament, food, health supplement or cosmetic for preventing and/or treating a human or animal pathological condition or disease caused by influenza virus.
10. The use according to claim 9, wherein the icariin is derived from a natural icariin plant and is obtained by extracting and purifying a plant of the genus Epimedium of the genus Berberidaceae. The genus Epimedium includes E. davidii, E. baojingense, E. icoroanum, and E. icoroum. E. fargesii), E. elongatum, E. acuminatum, E. simplicifolium, E. brachyrrhizum, and more E. multiflorum, E. enshiense, E. reticulatum, E. sagittatum, E. latisepalum E.chlorandrum, E.platypetalum, E. truncatum, E. rhizomatosum, and three-leaf clover (E.rhizomatosum) E.sagittatum), E. pauciflorum, E. myrianthum, E. ecalcaratum, E. glandulosopilosum, leaflets Epimedium (E.parvifolium), star flower Epimedium (E.stellulatum), one of the group of Epimedium Zhenping (E.ilicifolium) consisting of a mixture of one or more.
11. The use according to claim 9 or 10, characterized in that the influenza virus comprises influenza virus A, B and C and various subtypes thereof, serotypes, genotypes, gene mutant viruses.
12. The use according to claim 11, characterized in that the influenza virus is an influenza A virus.
13. The use according to claim 12, characterized in that the influenza virus is an H5N1 or H9N2 virus.
14. The use according to claim 9 or 10, characterized in that the pathological symptoms or diseases include cough, fever, sneezing, nasal congestion, runny nose, chills, headache, fatigue, body aches, pneumonia, tonsillitis, Pharyngitis, laryngitis, tearing, hoarseness, respiratory syndrome, cardiopulmonary disease, cold limbs, nausea and vomiting, dry cough, dry mouth, drowsiness, diarrhea, cold abortion, myocarditis, hypertension, myocardial infarction, stroke One or more diseases of the group consisting of nasal mucosa congestion, edema, secretion, mild hyperemia of the pharynx, enlarged submandibular lymph nodes, pharyngeal congestion and edema, and cold syndrome.
15. The use according to claim 14, characterized in that the pathological symptoms or diseases include cough, fever, stuffy nose, chills, headache, fatigue, body aches, tears, cold limbs, nausea and vomiting, drowsiness, edema One or more diseases of the group consisting of cold syndrome.
16. The use according to claim 9 or 10, characterized in that the icariin and its derivative are administered to a subject by oral, topical, inhalation, nasal, rectal, transdermal or injection routes.
17. The use according to claim 16, wherein the icariin and a derivative thereof are administered to a subject by an oral or injection route.
18. The use according to claim 16 or 17, wherein the subject comprises an animal such as a human, a farm animal, a pet animal, or a laboratory animal.
19. The use according to claim 18, characterized in that the subject is a human.
20. The use according to any of claims 16-19, characterized in that the subject already has or is at risk of having or being at a pathological condition or disease of an influenza virus infection.
21. The use according to claim 20, characterized in that the human has already suffered from a pathological symptom or disease of influenza virus infection.
22. The use according to any one of claims 9 to 21, wherein the subject is administered an effective amount of the icariin of claim 1 and a derivative thereof according to the prophylactic and/or therapeutic effect.
23. The use according to claim 22, wherein the effective amount comprises administering from about 0.1 mg/kg to about 10000 mg/kg of body weight of icariin and a derivative thereof per day to a human.
24. The use according to claim 23, wherein the effective amount comprises administering icariin and a derivative thereof to the human daily from about 0.5 mg/kg to about 200 mg/kg of body weight.
25. The use according to claim 24, wherein the effective amount comprises administering 25 mg/kg body weight of icariin and a derivative thereof to a human daily.
26. Use according to any of claims 9-25, characterized in that icariin and its derivatives are administered 1 to 6 times to the subject.
27. The use according to claim 26, characterized in that icariin and a derivative thereof are administered twice or three times to said subject.
28. A pharmaceutical composition comprising the novel inhibitor of claim 1, characterized in that the pharmaceutical composition contains at least two active ingredients, at least one of which is icariin and a derivative thereof.
29. The pharmaceutical composition according to claim 28, wherein said at least one other active ingredient is Ribavirin 1 , Interferon, Thymosin α, Transfer factor, Qiduo Zidovudine ZDV), didanosine, lamivudine, stavudine, zalcitabine, abacavir, ribavirin, ribavirin (Virazole), nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, nelfinavir, indinavir, Amprenavir, Lopinavir, Amantadine, Rimantadine, Zanamivir, Acyclovir, Valacyclovir, Iodine (Idoxuridine), Vidarabine, Lamivudine, Famciclovir, Penciclovir, oseltamivir and zanamivir.
30. The pharmaceutical composition according to claim 28, wherein the at least one other traditional Chinese medicine active ingredient is Radix Isatidis, Folium Isatidis, Yinqiao, Forsythia, Nepeta, Honeysuckle, Houttuynia, Wild Chrysanthemum, Summer Sangju One or more combinations of traditional Chinese medicine ingredients such as Bupleurum and Andrographis paniculata.
31. Use of a composition according to any one of claims 28 to 30 for the manufacture of a medicament, food, nutraceutical or cosmetic for the prevention and/or treatment of pathological conditions or diseases of human or animal caused by influenza virus.
32. The use according to claim 31, characterized in that the influenza virus comprises influenza virus A, B and C and various subtypes thereof, serotypes, genotypes, gene mutant viruses.
33. The use according to claim 32, characterized in that the influenza virus is an influenza A virus.
34. A capsule, a dropping pill, a gel, a tablet, a powder, a granule, a lozenge, an effervescent tablet, a syrup, or the like, comprising the novel inhibitor of claim 1 or the pharmaceutical composition according to claims 28-30, Emulsions, controlled release formulations, fast-dissolving preparations, oral liquid formulations.
35. The formulation of claim 34, wherein said formulation further comprises adjuvants including, but not limited to, lubricants and co-solvents such as magnesium stearate, calcium stearate, zinc stearate, hard Fatty acid, glyceryl behenate, sodium stearyl fumarate, talc, silica gel; disintegrants such as starch, cyclodextrin, carboxymethylcellulose, croscarmellose, cross-linked poly Vinyl pyrrolidone, gelatin; diluent or compressive agent such as lactose, sucrose, mannitol xylitol, erythritol, sorbitol, microcrystalline cellulose; seasonings or other ingredients such as strawberries, oranges, bananas, mint , honey; cosolvents, such as alcohol (beer, fruit wine, rice wine, white wine), vegetable oil, animal oil, ethanol, isopropanol, glycerin, propylene glycol, benzyl alcohol, dimethyl sulfite, polyethylene glycol, gum arabic, Lecithin, pyrrolidone, oleic acid, azone.
36. A formulation according to claim 34 or 35, wherein the dosage form is a controlled release formulation.
37. The formulation of claim 36 wherein said controlled release formulation comprises a core and a controlled release layer that encapsulates the core.
38. A formulation according to any one of claims 34 to 37, wherein the core comprises at least two active ingredients, at least one of which is icariin and a derivative thereof.
39. The preparation according to any one of claims 34 to 38, wherein the core may further comprise at least one other active ingredient: Interferon, Thymosin alpha, Ribavirin, Amantadine (Amantadine), Rimantadine oseltamivir and zanamivir.
40. The preparation according to any one of claims 34 to 38, wherein the core may further comprise at least one other traditional Chinese medicine active ingredient which is a traditional Chinese medicine ingredient such as Radix, Radix, Forsythia, Honeysuckle, Bupleurum, Andrographis paniculata One or more combinations.
41. The controlled release preparation according to any one of claims 34 to 40, wherein said controlled release layer contains a copolymer of methacrylic acid and ethyl acetate, a copolymer of methacrylic acid and methyl methacrylate, and Cellulose, triethyl citrate.
42. The controlled release preparation according to any one of claims 1 to 4, wherein the controlled release preparation according to claim 1, wherein the core and the coated core are controlled release according to a weight ratio The ratio of the layers is 1:0.01-100.
43. A controlled release formulation according to any one of claims 34 to 42 wherein the formulation provides a pH independent release in vitro in the range of pH 1 to 10.0.
44. Use of a formulation according to any one of claims 34-43 for the manufacture of a medicament, food, nutraceutical or cosmetic for the prevention and/or treatment of pathological conditions or diseases of human or animal caused by influenza virus.
45. The use according to claim 44, characterized in that the influenza virus comprises influenza virus A, B and C and various subtypes thereof, serotypes, genotypes, gene mutant viruses.
46. The use according to claim 45, characterized in that the influenza virus is an influenza A virus.
47. The use according to any one of claims 44 to 46, wherein the pathological symptom or disease comprises cough, fever, sneezing, nasal congestion, runny nose, chills, headache, fatigue, body aches, pneumonia, Tonsillitis, pharyngitis, laryngitis, tearing, hoarseness, respiratory syndrome, cardiopulmonary disease, cold limbs, nausea and vomiting, dry cough, dry mouth, drowsiness, diarrhea, cold abortion, myocarditis, hypertension, myocardial infarction One or more diseases of the group consisting of brain stroke, nasal mucosal congestion, edema, secretion, mild hyperemia of the pharynx, enlarged submandibular lymph nodes, pharyngeal congestion and edema, and cold syndrome.
48. The use according to claim 47, characterized in that the pathological symptoms or diseases include cough, fever, stuffy nose, chills, headache, fatigue, body aches, tears, cold limbs, nausea and vomiting, drowsiness, edema One or more diseases of the group consisting of cold syndrome.
49. The use according to any one of claims 44 to 48, wherein said controlled release preparation is administered to said subject once a day.
50. The use according to claim 49, wherein the subject comprises an animal such as a human, a farm animal, a pet animal, or a laboratory animal.
51. The use according to claim 50, characterized in that the subject is a human.
52. Use according to any of claims 44-51, characterized in that the subject already has or is at risk of having or being at a pathological condition or disease of influenza virus infection.
53. The use according to claim 52, characterized in that the human has already suffered from a pathological symptom or disease of influenza virus infection.
54. A method of preparing a controlled release preparation, characterized in that the preparation method comprises at least one of the steps of: preparing a core step and preparing a sustained release layer of at least one layer of coating.

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