WO2022174378A1

WO2022174378A1 - Use of extract from rabbit skin inflamed by vaccinia virus in treatment of demyelinating disease of nervous system

Info

Publication number: WO2022174378A1
Application number: PCT/CN2021/076788
Authority: WO
Inventors: 刘承鑫
Original assignee: 骏运投资有限公司
Priority date: 2021-02-19
Filing date: 2021-02-19
Publication date: 2022-08-25
Also published as: TWI828061B; TW202233212A; CN117355317A; WO2022174743A1

Abstract

The present invention relates to a use of an extract from rabbit skin inflamed by a vaccinia virus in the preparation of a drug, and more specifically to a use in the preparation of a drug for treating a demyelinating disease of a nervous system. In addition, the present invention relates to a use of an extract from rabbit skin inflamed by a vaccinia virus in the preparation of a drug for restoring a neural function of a patient suffering from a demyelinating disease of a nervous system. The demyelinating disease may be multiple sclerosis, acute disseminated encephalomyelitis or neuromyelitis optica. In addition, the extract from rabbit skin inflamed by the vaccinia virus may be lepalvir.

Description

Use of vaccinia virus-induced inflammatory rabbit skin extract in the treatment of demyelinating diseases of the nervous system

Field of Invention

The present invention belongs to the field of medicine. In particular, the present invention relates to novel therapeutic uses of vaccinia virus-inflamed rabbit skin extracts. More specifically, the present invention relates to the therapeutic use of vaccinia virus-inflamed rabbit skin extracts. More specifically, the present invention relates to the medicinal use of vaccinia virus-inflamed rabbit skin extracts for the treatment of demyelinating diseases of the nervous system, especially demyelinating diseases of the central nervous system. The demyelinating disease of the nervous system can be multiple sclerosis, acute disseminated encephalomyelitis, or neuromyelitis optica.

Background technique

Nerve fibers are divided into unmyelinated nerve fibers and myelinated nerve fibers. Myelinated nerve fibers such as autonomic preganglionic fibers and larger somatic nerve fibers have an outer sheath on their axons called myelin. Myelin is made up of the cell membrane of myelin cells. The myelinating cells of the central nervous system are oligodendritic cells. The myelin sheath of peripheral nerve fibers consists of the cell membrane of Schwann cells. Myelin sheath is composed of lipids and proteins, which protect the axon and insulate nerve impulses and accelerate the conduction of nerve impulses. Fibers with thick myelin sheaths also conduct faster impulses. When the myelin sheath is destroyed, conduction slows down. Nerve conduction is also affected by temperature, and an increase in body temperature during demyelination can cause conduction block.

Demyelinating diseases can include demyelinating diseases of the central nervous system and demyelinating diseases of the peripheral nervous system. Common clinical demyelinating diseases of the central nervous system include multiple sclerosis, acute disseminated encephalomyelitis, neuromyelitis optica, etc. It has serious impact and poses a serious threat to the physical and mental health of patients.

Multiple sclerosis (MS) is a disease characterized by inflammatory demyelinating lesions of the central nervous system. The most frequently involved parts of the disease are the white matter, optic nerve, spinal cord, brain stem and cerebellum. The main clinical features are the scattered distribution of white matter in the central nervous system and the remission and recurrence in the course of the disease, and the spatial multiple and course of symptoms and signs. temporal multiplicity. Multiple sclerosis was included in China's "First List of Rare Diseases" in 2018.

Acute disseminated encephalomyelitis (ADEM) is a type of idiopathic central nervous system demyelinating disease that is more common in children but can occur at any age. According to the definition of the International Pediatric MS Study Group (IPMSSG), ADEM is an acute or subacute onset with encephalopathy (abnormal behavior or disturbance of consciousness) that affects multiple regions of the central nervous system. The first occurrence of demyelinating disease.

Neuromyelitis optica (NMO) is an acute or subacute demyelinating disease with simultaneous or sequential involvement of the optic nerve and spinal cord. The disease, first described by Devic (1894), is clinically characterized by acute or subacute onset blindness in one or both eyes, preceded or followed by transverse or ascending myelitis for days or weeks, and later this disease It's called Devic's disease or Devic's syndrome. Neuromyelitis optica was also included in China's "First List of Rare Diseases" in 2018.

There has always been a need in the art to develop drugs for alleviating, alleviating or treating demyelinating diseases of the nervous system.

Glucocorticoids have been used in the treatment of multiple sclerosis and acute disseminated encephalomyelitis. Representative glucocorticoids include methylprednisolone and prednisone (see Myhr KM et al., Corticosteroids in the treatment of multiple sclerosis, Acta Neurol Scand 2009: 120 (Suppl. 189): 73-80). However, as hormonal drugs, glucocorticoids can cause allergic reactions when used in large quantities in a short period of time; while long-term use of glucocorticoids has greater side effects, including muscle atrophy, obesity, hypertension, hyperlipidemia, elevated urine sugar, and osteoporosis. Therefore, there is still a need to develop other drugs instead of glucocorticoids for the treatment of central nervous system demyelinating diseases that do not produce the side effects of glucocorticoids.

Vaccinia virus-inflamed rabbit skin extract is an active substance extracted from the skin of rabbits inflamed by vaccinia virus. This vaccinia virus-inflamed rabbit skin extract is commercially available under the trade name Lepalvir for pain treatment. Vaccinia virus-inflamed rabbit skin extract has been shown to be clinically safe and does not produce the aforementioned side effects. In addition, WO2020/211009 discloses the use of vaccinia virus-induced inflammatory rabbit skin extracts for the treatment of hematopoietic system damage; WO 2020/248240 discloses vaccinia virus-induced inflammatory rabbit skin extracts for use in the treatment of cancer. Reference is incorporated herein.

Experimental autoimmune encephalitis (EAE) is an effective and commonly used model for the study of multiple sclerosis, and has been widely used in the study of the pathogenesis of multiple sclerosis and the exploration of therapeutic drugs (H. Levy et al., Characterization of brain lesions in a mouse model of progressive multiple sclerosis, Experimental Neurology 226 (2010) 148-158).

SUMMARY OF THE INVENTION

Objects of the present invention include providing medicaments for preventing, alleviating or treating demyelinating diseases of the nervous system. More specifically, the object of the present invention includes providing a medicament for preventing, alleviating or treating multiple sclerosis or acute disseminated encephalomyelitis.

The technical problem of the present invention is solved by providing an extract of vaccinia virus-inflamed rabbit skin, preferably immediately.

Overall, the inventors found that vaccinia virus-inflamed rabbit skin extract can effectively prevent, treat or alleviate demyelinating diseases of the nervous system, especially central nervous system demyelinating diseases, such as multiple sclerosis or acute disseminated disease. Encephalomyelitis. Furthermore, the inventors unexpectedly found that the vaccinia virus-inflamed rabbit skin extract was more effective than the approved prednisone in treating the disease. Such an effect is surprising since prednisone therapy is already widely used in the clinical treatment of multiple sclerosis.

In one aspect, the present invention relates to the use of a vaccinia virus inflamed rabbit skin extract in the manufacture of a medicament for the prevention or treatment of demyelinating diseases of the nervous system in a patient. In one aspect, the present invention relates to a vaccinia virus-inflamed rabbit skin extract for use in the prevention or treatment of a demyelinating disease of the nervous system in a patient. In one aspect, the present invention relates to a method of preventing or treating demyelinating disease of the nervous system in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a vaccinia virus inflamed rabbit skin extract.

In one aspect, the present invention relates to the use of a vaccinia virus-inflamed rabbit skin extract in the manufacture of a medicament for restoring nerve function in a patient suffering from a demyelinating disease of the nervous system. In one aspect, the present invention relates to a vaccinia virus-inflamed rabbit skin extract for use in restoring neurological function in a patient suffering from a demyelinating disease of the nervous system. In one aspect, the present invention relates to a method of restoring neural function in a patient suffering from a demyelinating disease of the nervous system, the method comprising administering to the patient a therapeutically effective amount of a vaccinia virus inflamed rabbit skin extract.

In one aspect, the demyelinating disease of the nervous system comprises demyelinating disease of the central nervous system or demyelinating disease of the peripheral nervous system, preferably demyelinating disease of the central nervous system.

In one aspect, the demyelinating disease of the nervous system comprises multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, diffuse sclerosis (Schilder disease) or concentric sclerosis (Balo disease) ), preferably multiple sclerosis or acute disseminated encephalomyelitis, more preferably multiple sclerosis. In one aspect, the disease is multiple sclerosis. In another aspect, the disease is acute disseminated encephalomyelitis.

In one aspect, the site of lesions in a demyelinating disease of the nervous system includes the white matter, optic nerve, spinal cord, brain stem, or cerebellum.

In one aspect, restoring nerve function includes ameliorating, alleviating, or eliminating the following symptoms: loss or abnormality of sensation, muscle weakness, blurred vision, increased reflexes, spasticity, dyskinesia, ataxia, limb tremor, dysphonia, dysphagia, nystagmus , ophthalmoplegia, optic neuritis, fatigue, diplopia, incontinence, impaired thinking or cognitive impairment.

In one aspect, the demyelinating disease of the nervous system is multiple sclerosis selected from the group consisting of clinically single syndrome (CIS), relapsing remitting (RRMS), secondary progressive (SPMS), primary progressive ( PPMS) or progressive relapsing (PRMS).

In one aspect, vaccinia virus inflamed rabbit skin extract can be used as the sole active ingredient for the treatment of demyelinating diseases of the nervous system. In this aspect, the vaccinia virus inflamed rabbit skin extract can be used as the sole active ingredient in the preparation of a medicament for the treatment of, or in a patient with, a demyelinating disease of the nervous system Restore nerve function. In a method of treating a demyelinating disease of the nervous system in a patient or in a method of restoring nerve function in a patient having a demyelinating disease of the nervous system, the vaccinia virus inflamed rabbit skin extract can be administered as the sole active ingredient. patient. "Sole Active Ingredient" means that the Vaccinia Virus Inflammatory Rabbit Skin Extract is not to be used, administered to a patient, or prepared in combination with other drugs for the treatment of demyelinating diseases of the nervous system. The other drug for treating demyelinating diseases of the nervous system may be glucocorticoid drugs, such as methylprednisolone or prednisone. Those skilled in the art understand that the combined administration of two drugs to a patient and the improvement of the therapeutic effect does not mean that the two drugs are administered separately to achieve the therapeutic effect. For example, the use of the extract of the present invention together with glucocorticoid drugs may lead to an increase in the efficacy of glucocorticoid drugs, but this does not mean that the extract of the present invention itself will be expected to have therapeutic effects by those skilled in the art. In addition, the combined use of the two drugs still cannot solve the side effects of glucocorticoid drugs themselves. In fact, the inventors have surprisingly found that the single use of the vaccinia virus-inflamed rabbit skin extract can achieve better efficacy than the single use of glucocorticoid drugs, while avoiding the significant side effects of glucocorticoid drugs.

In one aspect, the present invention relates to a pharmaceutical composition comprising a vaccinia virus inflamed rabbit skin extract and an optional pharmaceutically acceptable carrier, adjuvant or excipient. In one aspect of the present invention, the pharmaceutically acceptable carriers, adjuvants or excipients are those for formulating the drug into oral preparations or injections. In one aspect, the vaccinia virus inflammatory rabbit skin extract is formulated into an oral preparation or an injection, preferably an intramuscular injection or an intravenous injection. In one aspect, the vaccinia virus-inflamed rabbit skin extract is Lizaizhi. Accordingly, the present invention also relates to the use of the pharmaceutical composition for treating demyelinating diseases of the nervous system, or restoring nerve function in patients suffering from demyelinating diseases of the nervous system. The present invention also relates to the use of the vaccinia virus-induced inflammatory rabbit skin extract in preparing the pharmaceutical composition.

In one aspect, the vaccinia virus-inflamed rabbit skin extract is Lizaizhi.

In one aspect, the vaccinia virus-inflamed rabbit skin extract is formulated into oral preparations or injections, preferably intramuscular injections or intravenous injections.

In one aspect, the patient is a mammal, preferably a human.

In one aspect, the vaccinia virus inflamed rabbit skin extract is administered to the patient in an amount of 0.05 U/kg to 50 U/kg, preferably 0.1 U/kg to 10 U/kg, more preferably 0.5 U/kg to 5 U/kg, e.g. people.

In one aspect, the prepared medicament comprises 3U to 3000U, preferably 6U to 600U, more preferably 30U to 300U of the vaccinia virus inflamed rabbit skin extract.

In one aspect, the vaccinia virus inflamed rabbit skin extract (preferably Lizaizhi) is 0.05U/kg to 50U/kg, preferably 0.1U/kg to 10U/kg, more preferably 0.5U/kg to 5U/kg is administered to a patient, preferably a human. For example, the vaccinia virus-inflamed rabbit skin extract is administered to a patient, preferably a human, in an amount selected from: 0.05 U/kg, 0.06 U/kg, 0.07 U/kg, 0.08 U/kg, 0.09 U/kg, 0.1 U/kg U/kg, 0.2U/kg, 0.3U/kg, 0.4U/kg, 0.5U/kg, 0.6U/kg, 0.7U/kg, 0.8U/kg, 0.9U/kg, 1U/kg, 1.5U /kg, 2U/kg, 2.5U/kg, 3U/kg, 3.1U/kg, 3.2U/kg, 3.3U/kg, 3.4U/kg, 3.5U/kg, 3.6U/kg, 3.7U/kg , 3.8U/kg, 3.9U/kg, 4U/kg, 4.5U/kg, 5U/kg, 5.5U/kg, 6U/kg, 6.5U/kg, 7U/kg, 7.5U/kg, 8U/kg , 8.5U/kg, 9U/kg, 9.5U/kg, 10U/kg, 11U/kg, 12U/kg, 13U/kg, 14U/kg, 15U/kg, 16U/kg, 17U/kg, 18U/kg , 19U/kg, 20U/kg, 25U/kg, 30U/kg, 35U/kg, 40U/kg, 45U/kg, 50U/kg and ranges bounded by these figures. Those skilled in the art know that for the dosage to be administered, the human dose (U/kg or mg/kg) = the mouse dose (U/kg or mg/kg)/12.3; or the human dose (U/kg) or mg/kg) = dose in mice (U/kg or mg/kg) x 0.08. The above doses may be effective amounts to treat the above-mentioned diseases in a patient. In one aspect, the vaccinia virus-inflamed rabbit skin extract is administered by injection, such as intramuscularly or intravenously, at the above doses.

In one aspect of the present invention, the prepared medicament or pharmaceutical composition comprises 3U to 3000U, preferably 6U to 600U, more preferably 30U to 300U of the vaccinia virus inflamed rabbit skin extract. The medicament or pharmaceutical composition is for administration to a human, eg, an adult. The average weight of an adult is, for example, 60 kg. Correspondingly, the amount of the vaccinia virus inflammatory rabbit skin extract contained in the medicine prepared by the present invention is, for example, 3U, 4U, 5U, 6U, 7U, 8U, 9U, 10U, 15U, 20U, 25U, 30U, 35U, 40U, 42U, 44U, 45U, 46U, 47U, 48U, 49U, 50U, 55U, 60U, 65U, 70U, 80U, 90U, 100U, 120U, 150U, 160U, 170U, 180U, 190U, 192U, 194U, 195U, 196U, 198U, 200U, 220U, 240U, 260U, 280U, 300U, 350U, 400U, 500U, 600U, 700U, 800U, 900U, 1000U, 1500U, 2000U, 2500U, 3000U and ranges bounded by these numbers. In one aspect, the drug or pharmaceutical composition is prepared as an injection, such as an intramuscular injection or an intravenous injection. In one aspect, the medicament or injection is an indivisible fixed dose. In one aspect, the drug or injection cannot be divided into smaller doses within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days. In one aspect, the drug or injection is administered only once within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days.

In one aspect, the vaccinia virus inflamed rabbit skin extract is administered to the patient every 6-72 hours, preferably 12-48 hours, more preferably 24-36 hours, more preferably 24 hours.

In one aspect, the dosing regimen to the patient is three times a day, twice a day, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks, once every three weeks Once, once a month. For example, the extract of the present invention can be administered to a patient once a day.

In one aspect, the extract of the invention is administered to a patient for at least 24 months, at least 12 months, at least 6 months, at least 2 months, at least 1 month, at least 3 weeks, at least 2 weeks, at least 10 days, At least 7 days, at least 5 days, at least 2 days, or at least 1 day.

The "vaccinia virus-inflamed rabbit skin extract (extract from rabbit skin inflamed by vaccinia virus)" described herein refers to a kind of extract from the inflamed rabbit skin inoculated with vaccinia virus, such as through extraction, purification, purification and other procedures. Extracts of active substances. This extract is usually a yellow or pale yellow liquid, but can also be made into a solid by drying. The injection of the vaccinia virus-inflamed rabbit skin extract is commercially available under the trade name Lepalvir. The vaccinia virus inflamed rabbit skin extract of the present invention may contain peptides. For example, it has been found that the extract may contain naturally occurring peptides (ie not additionally added peptides). The peptides may be short peptides. WO2013173941 describes short peptides isolated from vaccinia virus inflamed rabbit skin extracts. Therefore, in this aspect, the vaccinia virus inflamed rabbit skin extract of the present invention may not be a non-protein extract.

In the preparation of the extract of the present invention, the preparation method includes extracting the inflamed rabbit skin fragments after vaccinia virus inoculation with an aqueous solution of phenol, wherein preferably the concentration of phenol is about 1%-10%, preferably about 2%-5%, more preferably about The 2% or about 3% phenol in water is carried out below about 12°C, eg, about 0-10°C, preferably about 2-8°C, more preferably about 3-6°C, more preferably about 4°C. In addition, the preparation method also includes adsorbing the extract treated with the aqueous phenol solution with an adsorbent (eg, activated carbon), and performing desorption under alkaline conditions, wherein the adsorption is preferably performed under acidic conditions (eg, about pH 3-6, more preferably about pH 3-6). pH 4-5, more preferably about pH 4.5), and the basic conditions for desorption are about pH 9-12, preferably about pH 10 or pH 11.

In one aspect, a vaccinia virus-inflamed rabbit skin extract or anisotropy can be prepared by a method comprising the steps of:

(1) collecting the inflamed rabbit skin after inoculation with vaccinia virus, fragmenting the rabbit skin, and extracting it with an extraction solvent to obtain solution A;

(2) acid and heat treatment are carried out to solution A to obtain solution B;

(3) alkali and heat treatment are carried out to solution B to obtain solution C;

(4) carry out adsorption and filtration to solution C under acidic condition, carry out desorption under alkaline condition, obtain solution D;

(5) neutralization and heat treatment are carried out to solution D to obtain solution E;

(6) Concentrating solution E to obtain the extract; and

(7) Optionally, the extract is mixed with a pharmaceutically acceptable carrier, adjuvant or excipient.

In one aspect, in step (1), a rabbit is inoculated with vaccinia virus, the skin of the pox is collected, the skin is fragmented, an aqueous solution of phenol is added, and the temperature is lower than about 12°C (eg, about 0-10°C, Preferably about 2-8°C, more preferably about 3-6°C, more preferably about 4°C) for at least about 12 hours (eg about 24-90 hours, preferably about 48-72 hours, more preferably about 70 or about 72 hours) ), centrifuged to obtain supernatant, and filtered to obtain solution A. The phenol concentration in the phenol aqueous solution is about 1%-10%, preferably about 2%-5%, more preferably about 2% or about 3%.

In one aspect, in step (2), solution A is made acidic (eg, about pH 4-6, more preferably about pH 4.5-5.5, more preferably about pH 5) with an acid (eg, hydrochloric acid), heated (eg, at about pH 5). 90-100°C, preferably about 95°C for at least about 10 minutes, such as about 20-50 minutes, preferably about 30-40 minutes), optionally at a lower temperature (eg, to below about 50°C, preferably below about 30°C) , followed by centrifugation to obtain the supernatant, which was filtered to obtain solution B. The step (2) can be carried out in a nitrogen atmosphere.

In one aspect, in step (3), solution B is adjusted to alkaline (eg, about pH 8-10, more preferably about pH 8.5-9.5, more preferably about pH 9 or about pH 9.5) with a base (eg, sodium hydroxide). 2), heating (for example at about 90-100°C, preferably about 95°C for at least 10 minutes, for example about 30-50 minutes, preferably about 30-40 minutes), optionally cooling (for example to below about 50°C, Preferably below about 30°C), solution C is obtained after filtration. The step (3) can be carried out in a nitrogen atmosphere.

In one aspect, in step (4), solution C is made acidic (eg, about pH 3-6, more preferably about pH 4-5, more preferably about pH 4.5) with an acid (eg, hydrochloric acid), and the adsorbent is added thereto (eg activated carbon) is soaked (eg, with agitation for at least about 1 hour, preferably about 2-10 hours, more preferably about 4 hours), after which the solution is removed and the adsorbent containing the active ingredient is collected. Subsequently, the above adsorbent is added to the eluent (eg, water), and the pH is adjusted to basic (eg, about pH 9-12, preferably about pH 10 or pH 11) with a base (eg, sodium hydroxide) to remove the active ingredient from the adsorbent. Separation (eg, stirring for at least about 1 hour, preferably 2-10 hours, more preferably 4 hours, followed by filtration, and washing of the adsorbent with water) yields solution D. The step (4) can be carried out in a nitrogen atmosphere.

In one aspect, in step (5), solution D is neutralized to weakly acidic (eg, about pH 5.5-6.6, preferably about pH 6) with an acid (eg, hydrochloric acid) to obtain solution E. Preferably, the step (5) can be performed under sterile conditions.

In one aspect, in step (6), solution E is concentrated (eg, concentrated under reduced pressure, preferably concentrated by evaporation under reduced pressure, eg, at about 50°C-70°C, preferably about 54°C-56°C), followed by filtration, An extract containing the active ingredient is obtained. The step (6) can be carried out in a nitrogen atmosphere.

Those skilled in the art understand that the extracts of the present invention can also be obtained by inoculating other animal tissues with vaccinia virus. For example, in the present invention, extracts of inflamed tissues inoculated with vaccinia virus can be used. The tissue may be derived from mammalian tissue, which may include companion animals, laboratory animals, livestock animals, such as rabbits, cows, horses, sheep, goats, monkeys, mice, pigs. The tissue can be skin.

Detailed ways

Unless otherwise defined, all scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Example methods and materials are described below, equivalents of which may be used. All publications and other references mentioned herein are incorporated by reference in their entirety.

The following examples are provided to further illustrate the present invention. The following examples are not intended to limit the scope of the invention for any reason.

Example

Example 1 - Therapeutic effect of Lizaixi on multiple sclerosis (EAE)

1. Purpose of the test

To study the therapeutic effect of Lizaixi on multiple sclerosis (EAE)

2. Test method

SPF grade C57BL/6 mice, 18-22g, 250, male. After the quarantine, except for 16 mice in the negative control group, all the other mice were injected with 0.2 mL of Mog35-55 emulsifier antigen at 3 points on the back skin (the injection volume of MOG35-55 was 200ug/mice), and 1/3 of each site was injected. . On the 1st and 3rd day, the pertussis toxin was injected intraperitoneally at 200 ng per animal. Mice with a neurological function score of about 2 were randomly divided into the model control group, the prednisone acetate group, and the "Lizaishi" low, medium and high dose groups, 16 mice/group. After grouping, the corresponding test drugs were given intraperitoneally every day, and the model control group was given the same amount of normal saline, once a day. On the 21st and 28th day of administration, half of the mice in each group were randomly selected for testing.

3. Experimental materials

3.1 Test drugs

Rabbit skin extract caused by vaccinia virus ("Lizaixi" stock solution, referred to as LZS).

3.2 Positive control drug

Prednisone acetate injection (referred to as PA).

4. Grouping of experimental animals

Mice with a neurological function score of about 2 were screened and randomly divided into model control group, prednisone acetate group, "Lizaixi" low (10U/Kg), medium (20U/Kg), and high-dose group (40U/Kg) /Kg), 16/group.

5. Drug preparation

5.1 Emulsifier preparation:

Dissolve 5 mg of MOG35-55 dry powder in PBS to 2.5 mL to obtain a solution with a concentration of 2 mg/mL. The MOG35-55 solution was mixed with the same volume of complete Freund's immune adjuvant (1:1), and the adjuvant added Mycobacterium tuberculosis 10 mg/mL to form an emulsifier.

5.2 High-dose LZS (4U/mL):

Take the LZS stock solution and dilute it with normal saline to 4U/ml.

5.3 Medium dose LZS (2U/mL):

Take high-dose LZS diluted 2 times with normal saline to make 2U/ml.

5.4 Low-dose LZS (1U/mL):

Take high-dose LZS diluted 4 times with normal saline to make 1U/ml.

5.5 Prednisone acetate (PA):

Take prednisone acetate injection and dilute it with normal saline to 0.7 mg/mL.

5.6 Pertussis Toxin (PTX):

PTX was dissolved in physiological saline, and the concentration was adjusted to 2 μg/mL.

6. Modeling method:

Except for the negative control group, all mice were injected with 0.2 mL of emulsifier (ie, the injection volume of MOG35-55 was 200 ug/mice). On day 1, 0.2 mL of the antigen formulation was injected subcutaneously at 3 points on the back of the mice, one point on the midline of the shoulder, and the other two points on both sides of the midline of the lower back. Inject 1/3 of each site. On the 1st and 3rd day, 200ng/pertussis toxin was injected intraperitoneally. On the 14th day of modeling, mice with an animal neurological function score of about 2 were screened and randomly divided into model control group, prednisone acetate group, "Li Zaishi" low (10U/Kg), medium (20U/Kg) ), high dose group (40U/Kg), 16 animals/group.

7. Administration method:

After grouping, the corresponding test drugs were given intraperitoneally every day, and the model control group was given the same amount of normal saline, once a day. On the 21st and 28th days of administration, 8 mice were selected from each group for testing:

Table 1 Groups, doses and dosing schedules

7. Detection indicators

The general clinical manifestations of the animals were observed daily, and the body weights of the animals were measured once a week. The neurological function score was performed once a week, and the degree of improvement was calculated.

Improvement (%) = (neural function score after administration - neurological function score before administration) / neurological function score before administration × 100%

Table 2 Neurological function score (Kono 5 scale)

8. Data statistics method:

All data use

SPSS 21.0 software was used for statistical analysis; if the variance of measurement data was homogeneous, or the variance was homogeneous after transformation, the one-way ANOVA method for pairwise comparison between groups was used; if the variance was still unequal after transformation, the rank and test for statistical analysis. Test level α=0.05.

9. Experimental results

9.1 General observations and body weight (see Table 3):

There was no abnormal performance in the daily observation of the animals in each group. The body weight of the animals in each group increased normally, and there was no statistical difference (P>0.05). No animal died during the experiment.

9.2 Neurological function score (see Table 4):

Compared with the model control group, the mean scores of neurological function in LZS low (10U/Kg), medium (20U/Kg), high-dose (40U/Kg) and prednisone acetate groups at 7-28 days of administration have a downward trend. After 14 days of administration, the functional scores of LZS middle-dose group and high-dose group were significantly decreased (P<0.05), and the improvement degrees were 35.3% and 55.6%, respectively. After 21 days of administration, the functional scores of LZS middle-dose group and high-dose group were significantly decreased (P<0.05), and the improvement degrees were 41.2% and 55.6%, respectively. After 28 days of administration, the functional scores of LZS low-dose group, middle-dose group and high-dose group were significantly decreased (P<0.05), and the improvement degrees were 38.9%, 52.9%, and 55.6%, respectively, which were all better than 27.8% of the prednisone acetate group.

Claims

Use of a vaccinia virus-inflamed rabbit skin extract in the manufacture of a medicament for preventing or treating demyelinating diseases of the nervous system in a patient.
The use of claim 1, wherein the medicament is for use in restoring nerve function in a patient suffering from a demyelinating disease of the nervous system.
The use of claim 1 or 2, wherein the demyelinating disease of the nervous system comprises a demyelinating disease of the central or peripheral nervous system.
The use of any one of claims 1-3, wherein the demyelinating disease of the nervous system comprises multiple sclerosis, neuromyelitis optica, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, diffuse sclerosis ( Schilder disease) or concentric sclerosis (Balo disease), preferably multiple sclerosis or acute disseminated encephalomyelitis.
5. The use of any one of claims 1-5, wherein the site of disease in a demyelinating disease of the nervous system comprises the white matter, optic nerve, spinal cord, brain stem or cerebellum.
The use of claim 2, wherein restoring nerve function comprises ameliorating, alleviating or eliminating the following symptoms: loss or abnormality of sensation, muscle weakness, blurred vision, increased reflexes, spasticity, dyskinesia, ataxia, limb tremor, dysphonia, dysphagia , nystagmus, ophthalmoplegia, optic neuritis, fatigue, diplopia, incontinence, impaired thinking or cognitive impairment.
The purposes of any one of claims 1-6, wherein the demyelinating disease of the nervous system is multiple sclerosis, the type of which is selected from clinical single syndrome (CIS), relapsing remitting (RRMS), secondary progressive (SPMS), primary progressive (PPMS) or progressive relapsing (PRMS).
7. The use of any one of claims 1-7, wherein the vaccinia virus-inflamed rabbit skin extract is Lizai.
The use according to any one of claims 1 to 8, wherein the vaccinia virus-inflamed rabbit skin extract is formulated into oral preparations or injections, preferably intramuscular injections or intravenous injections.
9. The use of any one of claims 1-9, wherein the patient is a human.
The use according to any one of claims 1-10, wherein the vaccinia virus-inflamed rabbit skin extract is in a dosage of 0.05 U/kg to 50 U/kg, preferably 0.1 U/kg to 10 U/kg, more preferably 0.5 U/kg to 0.5 U/kg An amount of 5U/kg was administered to the patient.
The use of any one of claims 1-11, wherein the medicament comprises 3U to 3000U, preferably 6U to 600U, more preferably 30U to 300U of the vaccinia virus inflamed rabbit skin extract.