CN108676061B

CN108676061B - Mannatide and purification method thereof

Info

Publication number: CN108676061B
Application number: CN201810502021.1A
Authority: CN
Inventors: 王淑娟; 徐昊; 康威
Original assignee: Sinopharm Yixin Pharmaceutical Co Ltd
Current assignee: Sinopharm Yixin Pharmaceutical Co Ltd
Priority date: 2018-05-23
Filing date: 2018-05-23
Publication date: 2019-12-10
Anticipated expiration: 2038-05-23
Also published as: CN108676061A

Abstract

The invention relates to the field of medicines, in particular to mannatide and a purification method thereof. The invention aims at the structural characteristics that mannan peptide is a macromolecular compound formed by mannan and peptide with different molecular weights and different charge conditions through glycosidic bonds. Separating the components with obvious difference in electric quantity by high performance liquid ion exchange chromatography and separating the components with obvious difference in molecular weight by high performance liquid size exclusion chromatography (HPSEC). On the basis of allergy investigation, a separation method combining high performance liquid ion exchange chromatography and high performance liquid size exclusion chromatography (HPSEC) is established, mannatide is separated and purified, and adverse reactions of mannatide in biological models such as guinea pigs can be effectively reduced.

Description

Mannatide and purification method thereof

Technical Field

The invention relates to the field of medicines, in particular to mannatide and a purification method thereof.

background

mannatide (Mannatide) is named mainly as "mannaide", which is called as "Mannatide" by the national drug administration in 2000, is white or yellowish amorphous powder, odorless, tasteless, easily soluble in water, insoluble in organic solvents, stable in chemical properties, uniform in certain degree, free of micromolecular saccharides such as monosaccharide and disaccharide and free amino acids, and completely composed of Mannatide molecules with different chain lengths. The content of total sugar in the mannatide is 87.7-90.3%, and the mannatide mainly contains mannose and a small amount of glucose residues; the total content of amino acids is 4.5% -6.2%, mainly including aspartic acid, threonine, serine, glutamic acid, alanine and leucine, wherein the sugar chain and the peptide chain are connected by N-glycosidic bond connection and O-glycosidic bond connection.

the drug product is reported to have a small number of adverse reactions during clinical use. The reported mean molecular weight of mannatide is 30000-60000. Macromolecular peptide substances enter the body to be used as antigen substances to stimulate the immune system of the body to cause congestion and edema of skin mucous membranes, which are manifested as flushing and rash of the skin; the drug macromolecules are combined with the blood purple protein to cause the reaction of foreign proteins, and the reaction can cause fever, chill, chest distress and dyspnea even anaphylactic shock in severe cases. At present, ultrafiltration and alcohol precipitation methods, enzymolysis processes, multi-stage ultrafiltration serial methods and ultrafiltration and gel column chromatography methods are mostly adopted. However, the purification effect is not ideal, and the medicament still has the phenomenon of allergy. Therefore, the mannan peptide is purified and separated by a more effective method, which is an effective way for reducing anaphylactic reaction.

Disclosure of Invention

In view of the above, the present invention provides mannatide and a purification method thereof. Can effectively reduce the adverse reaction of mannatide in biological models such as guinea pig and the like.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides a method for purifying mannatide, which comprises the following steps:

step 1: obtaining a crude product of the mannan peptide;

Step 2: purifying the crude product of the mannan peptide by adopting a high performance liquid ion exchange chromatography, collecting eluent, and filtering to obtain a first purified substance;

And step 3: and (3) purifying the first purified product by adopting high performance liquid phase size exclusion chromatography, and collecting eluent.

The invention aims at the structural characteristics that mannan peptide is a macromolecular compound formed by mannan and peptide with different molecular weights and different charge conditions through glycosidic bonds. Separating the components with obvious difference in electric quantity by high performance liquid ion exchange chromatography and separating the components with obvious difference in molecular weight by high performance liquid size exclusion chromatography (HPSEC). On the basis of allergy investigation, a separation method combining high performance liquid ion exchange chromatography and high performance liquid size exclusion chromatography (HPSEC) is established, mannatide is separated and purified, and adverse reactions of mannatide in biological models such as guinea pigs can be effectively reduced.

In some embodiments of the invention, the purification in step 2 is performed using water and 1mol/L sodium chloride solution as mobile phase, eluting according to the following gradient: sodium chloride solution ═ 10:90(1min) → water: sodium chloride solution ═ 40:60(3min) → water: sodium chloride solution 60:40(3min) → water: sodium chloride solution 90:10(1 min).

In some embodiments of the present invention, the collecting the eluent in step 2 is collecting the eluent for 0-0.5 min and 1.5-3 min.

in some embodiments of the invention, the filtering in step 2 is to remove impurities having a molecular weight of less than 8000.

in some embodiments of the invention, the purification in step 3 is performed using water as eluent at a flow rate of 0.5 ml/min.

In some embodiments of the invention, the eluent has a pH of 6.8.

In some embodiments of the present invention, the collecting the eluent in step 3 is collecting the eluent for 10-12 min, 13-15 min and 18-21 min.

In some embodiments of the present invention, the step of collecting the eluate in step 3 further comprises a step of ultrafiltration.

in some embodiments of the present invention, the obtaining of the crude mannatide in step 1 specifically comprises: the konjac flying powder is used as a raw material, and fermentation liquor is collected through biological fermentation.

The invention also provides mannatide prepared by the purification method.

The invention aims at the structural characteristics that mannan peptide is a macromolecular compound formed by mannan and peptide with different molecular weights and different charge conditions through glycosidic bonds. Separating the components with obvious difference in electric quantity by high performance liquid ion exchange chromatography and separating the components with obvious difference in molecular weight by high performance liquid size exclusion chromatography (HPSEC). On the basis of allergy investigation, a novel separation method combining high performance liquid ion exchange chromatography and high performance liquid size exclusion chromatography (HPSEC) is established to separate and purify mannatide.

The technical scheme provided by the invention effectively reduces the molecular weight range of the mannatide, and improves the physicochemical properties of the mannatide raw material drug, such as properties and the like. And the relationship between mannatide and adverse reaction is established by screening a biological model. The method for separating and purifying can effectively reduce the adverse reaction of mannatide in biological models such as guinea pig and the like.

Drawings

in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a high performance liquid phase ion exchange chromatogram of example 1;

FIG. 2 shows a high performance liquid size exclusion chromatogram of example 3;

FIG. 3 shows multinucleated giant cell-like myobud tissue and interstitial inflammatory cell infiltration 40 × (48 h after the last dose, animal No./group: M007/test article 1 group, section No.: 171758-35-1) at the injection site of control lateral quadriceps femoris muscle;

FIG. 4 shows myofibrotic necrosis at the site of injection of lateral quadriceps femoris with 40X infiltration of interstitial inflammatory cells (48 h after the last dose, animal No./group: F007/test article 1 group, section No.: 171757-35-2);

FIG. 5 shows that no significant histopathological changes were observed in the injection site of the lateral quadriceps femoris muscle 40

(48 hours after the last administration, animal No./group: M009/test sample 2 group, section No. 171760-35-1);

FIG. 6 shows myofibrotic necrosis at the site of lateral quadriceps injection with interstitial inflammatory cell infiltration 40 × (48 h after the last dose, animal No./group: M009/test article 2 group, section No.: 171760-35-2);

FIG. 7 shows that no significant histopathological changes were seen at the control lateral quadriceps injection site by 40 × (48 h post last dose, animal No./group: F011/test article 3 group, section No.: 171761-35-1);

FIG. 8 shows that no significant histopathological changes were seen at the site of injection into the lateral quadriceps femoris muscle (48 h after the last dose, animal No./group: F011/test article 3 group, section No.: 171761-35-2);

FIG. 9 shows that no significant histopathological changes were seen at the control lateral quadriceps injection site by 40 × (end of recovery period, animal No./group: F008/test article 1, section No.: 171852-35-1);

FIG. 10 shows that no significant histopathological changes were seen at the injection site of the quadriceps femoris muscle on the side of administration by 40 × (end of recovery period, animal No./group: F008/test article 1 group, section No. 171852-35-2);

FIG. 11 shows that no significant histopathological changes were seen at the control lateral quadriceps injection site by 40 × (end of recovery period, animal No./group: M010/test article 2 group, section No.: 171855-35-1);

FIG. 12 shows that no significant histopathological changes were seen at the injection site of the quadriceps femoris muscle on the side of administration by 40 × (end of recovery period, animal number/group: M010/test article 2 group, section number: 171855-35-2);

FIG. 13 shows that no significant histopathological changes were seen at the control lateral quadriceps injection site by 40 × (end of recovery period, animal No./group: F012/test article 3 group, section No.: 171856-35-1);

FIG. 14 shows that no significant histopathological changes were seen at the injection site of the quadriceps femoris muscle on the side of administration by 40 × (end of recovery period, animal No./group: F012/test article 3 group, section No.: 171856-35-2);

FIG. 15 shows perivascular edema at the control side margin intravenous injection site 40 × (72 h after the last administration, animal No./group: M001/test article 1 group, section No. 171752-43-1);

FIG. 16 shows perivascular hemorrhage, edema with inflammatory cell infiltration 40X at the site of intravenous injection at the ear margin on the administration side (72 h after the last administration, animal No./group: M001/test article 1 group, section No.: 171752-43-2);

FIG. 17 shows that no significant histopathological changes were seen 40X proximal to the control lateral marginal vein (72 h post last dose, animal No./group: M001/test article 1 group, section No.: 171752-43-1);

FIG. 18 shows that no significant histopathological changes were seen 40X proximal to the periauricular vein on the side of administration (72 h after the last administration, animal No./group: M001/test article 1 group, section No.: 171752-43-2);

FIG. 19 shows perivascular edema at the control lateral margin intravenous injection site 40 × (72 h after the last dose, animal No./group: F003/test article 2 group, section No.: 171753-43-1);

FIG. 20 shows the edema with inflammatory cell infiltration around the blood vessel at the site of intravenous injection of the administration side margin,

Perivascular organization was 40 × (72 h after the last dose, animal number/group: F003/test 2 group, section number:

171753-43-2)；

FIG. 21 shows that no significant histopathological changes were seen 40X in the proximal end of the control lateral marginal vein (72 h after the last dose, animal No./group: F003/test article 2 group, section No.: 171753-43-1);

FIG. 22 shows that no significant histopathological changes were seen 40X proximal to the periauricular vein on the side of administration (72 h after the last administration, animal No./group: F003/test article 2 group, section No.: 171753-43-2);

FIG. 23 shows that no significant histopathological changes were seen at the control side ear margin intravenous site by 40 × (72 h post last dose, animal No./group: F005/test article 3 group, section No.: 171755-43-1);

FIG. 24 shows organization of perivascular tissues at the site of intravenous injection at the ear margin on the administration side, inflammatory cell infiltration 40 × (72 h after the last administration, animal No./group: F005/test article 3 group, section No.: 171755-43-2);

FIG. 25 shows that no significant histopathological changes were seen 40X in the proximal control lateral ear vein (72 h after the last dose, animal No./group: F005/test article 3 group, section No.: 171755-43-1);

FIG. 26 shows that no significant histopathological changes were seen 40X proximal to the auricular vein on the side of administration (72 h after the last administration, animal No./group: F005/test article 3 group, section No.: 171755-43-2);

FIG. 27 shows that no significant histopathological changes were observed at the control lateral ear margin intravenous site by 40 × (end of recovery period, animal No./group: M002/test article 1 group, section No.: 171847-43-1);

FIG. 28 shows that no significant histopathological changes were observed at the site of intravenous injection at the ear margin on the administration side by 40 × (end of recovery period, animal No./group: M002/test article 1 group, section No. 171847-43-2);

FIG. 29 shows that no significant histopathological changes were seen 40X in the proximal control lateral ear vein (end of recovery period, animal No./group: M002/test article 1 group, section No.: 171847-43-1);

FIG. 30 shows that no significant histopathological changes were observed 40X at the proximal end of the periaural vein on the administration side (end of recovery period, animal No./group: M002/test article 1 group, section No. 171847-43-1);

FIG. 31 shows that no significant histopathological changes were seen at the control lateral ear margin intravenous site by 40 × (end of recovery period, animal No./group: F004/test article 2 group, section No.: 171848-43-1);

FIG. 32 shows that no significant histopathological changes were observed at the site of intravenous injection at the ear margin on the administration side by 40 × (end of recovery period, animal No./group: F004/test article 2 group, section No.: 171848-43-2);

FIG. 33 shows that no significant histopathological changes were seen 40X in the proximal end of the control lateral marginal vein (end of recovery period, animal No./group: F004/test article 2 group, section No.: 171848-43-1);

FIG. 34 shows that no significant histopathological changes were seen 40X at the proximal end of the periauricular vein on the side of administration (end of recovery period, animal No./group: F004/test article 2 group, section No.: 171848-43-2);

FIG. 35 shows that no significant histopathological changes were seen at the control side ear vein injection site by 40 × (end of recovery period, animal No./group: M006/test article 3 group, section No.: 171851-43-1);

FIG. 36 shows that no significant histopathological changes were observed at the site of intravenous injection at the ear margin on the administration side by 40 × (end of recovery period, animal No./group: M006/test article 3 group, section No.: 171851-43-2);

FIG. 37 shows that no significant histopathological changes were seen 40X in the proximal control lateral ear vein (end of recovery period, animal No./group: M006/test article 3 group, section No.: 171851-43-1);

FIG. 38 shows that no significant histopathological changes were seen 40X proximal to the peripheral vein on the administration side (end of recovery period, animal No./group: M006/test article 3 group, section No.: 171851-43-2);

FIG. 39 shows a negative control passively sensitized group F041;

Fig. 40 shows a negative control passively sensitized group F042;

Fig. 41 shows a negative control passively sensitized group F043;

FIG. 42 shows a negative control passively sensitized group M041;

Fig. 43 shows a negative control passively sensitized group M042;

Fig. 44 shows a negative control passively sensitized group M043;

Fig. 45 shows a positive control passively sensitized group F044;

Fig. 46 shows a positive control passively sensitized group F045;

Fig. 47 shows a positive control passively sensitized group F046;

Figure 48 shows a positive control passively sensitized group M044;

Fig. 49 shows a positive control passively sensitized group M045;

Figure 50 shows the positive control passively sensitized group M046;

fig. 51 shows test article 1 low-dose passively sensitized group F047;

figure 52 shows test article 1 low dose passively sensitized group F048;

Fig. 53 shows the test article 1 low-dose passively sensitized group F049;

Fig. 54 shows test article 1 low dose passively sensitized group M047;

Fig. 55 shows test article 1 low dose passively sensitized group M048;

figure 56 shows test article 1 low dose passively sensitized group M049;

Figure 57 shows test article 1 high dose passively sensitized group F050;

Fig. 58 shows the test article 1 high-dose passively sensitized group F051;

Figure 59 shows test article 1 high dose passively sensitized group F052;

figure 60 shows test article 1 high dose passively sensitized group M050;

Fig. 61 shows the test article 1 high dose passively sensitized group M051;

figure 62 shows test article 1 high dose passively sensitized group M052;

figure 63 shows the test article 2 low dose passively sensitized group F053;

Figure 64 shows the test article 2 low dose passively sensitized group F054;

figure 65 shows sample 2 low dose passively sensitized group F055;

fig. 66 shows the test article 2 low-dose passively sensitized group M053;

Figure 67 shows the test article 2 low dose passively sensitized group M054;

figure 68 shows sample 2 low dose passively sensitized group M055;

figure 69 shows the test article 2 high dose passively sensitized group F056;

figure 70 shows the test article 2 high dose passively sensitized group F057;

Figure 71 shows the test article 2 high dose passively sensitized group F058;

Figure 72 shows the test article 2 high dose passively sensitized group M056;

fig. 73 shows the test article 2 high dose passively sensitized group M057;

Figure 74 shows the test article 2 high dose passively sensitized group M058;

figure 75 shows the test article 3 low dose passively sensitized group F059;

figure 76 shows the low dose passively sensitized group F060 for test article 3;

Fig. 77 shows the test article 3 low dose passively sensitized group F061;

Figure 78 shows the test article 3 low dose passively sensitized group M059;

fig. 79 shows the test article 3 low dose passively sensitized group M060;

Fig. 80 shows the test article 3 low-dose passively sensitized group M061;

figure 81 shows the test article 3 high dose passively sensitized group F062;

FIG. 82 shows high dose passively sensitized group F063 for test article 3;

figure 83 shows sample 3 high dose passively sensitized group F064;

Figure 84 shows sample 3 high dose passively sensitized group M062;

FIG. 85 shows a high dose passively sensitized group M063 of test article 3;

Figure 86 shows sample 3 high dose passively sensitized group M064;

FIG. 87 shows sample 1 after sample application for 3 h;

FIG. 88 shows sample 2 after sample application for 3 hours;

FIG. 89 shows sample 3 after being applied for 3 hours.

Detailed Description

The invention discloses mannatide and a purification method thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The mannatide and the raw materials and reagents used in the purification method thereof provided by the invention can be purchased from the market.

the invention is further illustrated by the following examples:

example 1

The konjac flying powder is used as a raw material, and fermented liquid is collected through biological fermentation to prepare a mannan peptide crude product.

The mannatide was studied using BIA ion exchange column DEAE-0.1(CIMac ^TM). A typical spectrum is shown in FIG. 1:

Sample pretreatment: taking 200g of the crude product of the mannan peptide, dissolving the crude product in 1000mL of purified water, fully stirring and dissolving to obtain 1140mL of sample solution, filtering the sample solution with a 0.2um filter membrane for standby, wherein the sample concentration is as follows: 175 mg/mL.

a DEAE-0.1(CIMac ^TM) isotype preparative chromatography is used as a chromatographic column, water and sodium chloride solution are used as mobile phases, elution is carried out according to the following gradient that the sodium chloride solution is 10:90(1min) → water: the sodium chloride solution is 40:60(3min) → water: the sodium chloride solution is 60:40(3min) → water: the sodium chloride solution is 90:10(1min), samples of 0-0.5 min and 1.5-3 min are respectively received, the samples are respectively desalted and chlorine ions are respectively removed by a dialysis bag, impurities with the molecular weight being less than 8000, and the ultrafiltered samples are concentrated to 8g/L, and the two-component mannopeptide is freeze-dried to obtain the two-component mannopeptide, wherein the component of 0-0.5 min is 34.2g, the yield is 17.1%, the two-component is a dark brown solid (sample I), the average molecular weight is 40000-.

Example 2 systemic active hypersensitivity test (ASA)

TABLE 1 comparison of ASA test methods in the pharmacopoeia and guidelines

in this example, the evaluation method and standard of pharmacopoeia are adopted.

evaluation criteria for systemic sensitization:

"-" negative allergic reaction-normal

the + mild and positive anaphylaxis includes restlessness, hair erection, shivering and nasal pruritus

Positive allergic reaction, sneezing, cough, shortness of breath, urination, defecation, and lacrimation

"+ + + +" shows strong positive anaphylaxis, dyspnea, wheezing, purple addiction, gait instability, jumping, wheezing, spasm, rotation, and tidal breathing

The very positive allergic reaction, death, is "+ + + + +".

Hartley-Dunkin guinea pigs, SPF grade, 5/sex/group were used. Sensitization of the samples I and II was investigated by using the unseparated sample, sample I, and sample II (sensitization amount: 3mg/kg), and physiological saline as a negative control.

negative control group-saline: 0 animals had 2 or more of pili, nasal pruritus, dyspnea, purpura, tachypnea and tidal respiration, and no allergic reaction was observed.

control-not isolated sample: 5/10 animals have 2 or more of pili, nasal pruritus, dyspnea, purpura, tachypnea and tidal respiration, and are strong and positive in anaphylaxis.

Experimental group 1 — sample I: 6/10 animals have 2 or more of pili, nasal pruritus, dyspnea, purpura, tachypnea and tidal respiration, and are strong and positive in anaphylaxis.

experimental group 2 — sample II: 5/10 animals have 2 or more of pili, nasal pruritus, dyspnea, purpura, tachypnea and tidal respiration, and are strong and positive in anaphylaxis.

The separated samples have certain differences in properties and molecular weights, but have small differences in allergenicity.

Example 3

Mannatide was studied using Shodex Ohpak SB-805HQ molecular size exclusion chromatography column. A typical spectrum is shown in FIG. 2:

Sample pretreatment: taking 200g of sample II, dissolving in 1000mL of purified water, fully stirring and dissolving to obtain 1140mL of sample solution, filtering with a 0.2um filter membrane for later use, wherein the sample concentration is as follows: 175 mg/mL.

the pH was adjusted to 6.8 using Shodex Ohpak SB-805HQ molecular size exclusion chromatography column with purified water as eluent using sodium monohydrogen phosphate-disodium hydrogen phosphate buffer salt. The flow rate was 0.5 ml/min. Elution was carried out. Respectively receiving samples for 10-12 min, 13-15 min and 18-21 min. Ultrafiltering the sample with coiled membrane of model GM1812C-34D produced by GE, concentrating the ultrafiltered sample to 5g/L, and lyophilizing to obtain two components of mannatide. Wherein the component accounts for 120.6g in 10-12 min, the yield is 60.3%, the component is white powdery solid, and the average molecular weight is 45000-; 14.2g of 13-15 min component, yield 7.1%, brown solid, average molecular weight 50000-60000 (sample IV); 22.6g of 18-21 min fraction, yield 11.3%, light yellow solid, average molecular weight 30000-50000 (sample V).

Example 4 systemic active allergy test (ASA)

Hartley-Dunkin guinea pigs, SPF grade, 5/sex/group were used. Sensitization of the unseparated sample, sample III, sample IV and sample V (sensitization measurement: 3mg/kg) was investigated using physiological saline as a negative control.

experimental group 3 — sample III: there were 1/10 animals with restlessness and nasal pruritus, which were weakly positive for allergic reactions.

Experimental group 4 — sample IV: 5/10 animals have 2 or more of pili, nasal pruritus, dyspnea, purpura, tachypnea and tidal respiration, and are strong and positive in anaphylaxis.

Experimental group 5 — sample V: 5/10 animals have 2 or more of pili, nasal pruritus, dyspnea, purpura, tachypnea and tidal respiration, and 1 animal dies and has extremely strong positive anaphylaxis.

Research results show that the sample III (10-12 min component, average molecular weight of 45000-.

Example 5 histopathological examination of stimulation of mannatide administration by intramuscular injection to rabbits

experiment groups 1-3: three different lots of mannatide sample III from example 3.

1 presentation situation

The method comprises the steps of carrying out multiple intramuscular injection on New Zealand rabbits on 3 different batches of mannatide samples III prepared in example 3 with the same specification for a stimulation test, taking 12 healthy rabbits at 6 female/6, randomly dividing the rabbits into an experiment group 1, an experiment group 2 and an experiment group 3 according to sex and weight, and carrying out intramuscular injection on mannatide sample III liquid medicine (10 mg/ml ^-1) prepared in example 3 and sodium chloride injection with the same volume of 0.9% respectively at the shearing positions of the right side (R) and the left side (L) of each rabbit by using an aseptic operation method by adopting a homologous left-right side self-contrast method and an equivalent left-right side self-contrast method by shearing animals by using electric hair scissors to expose the quadriceps femoris muscles of the left side and the right side, wherein the administration volume is 0.5 ml/side, the administration is carried out for 1 time per day, and the administration is carried out for 7d continuously.

1/2 animals were sacrificed 48h after the last dose and continued to recover 14d each group, 1/1 male, the quadriceps femoris muscle was dissected out, cut longitudinally, visually observed the stimulation response status of the muscle tissue at the injection site and scored accordingly, fixed in 10% neutral formalin, and histopathological examination was performed on the routine tissue sections.

2 inspection method

grouping and group identification:

12 healthy rabbits are selected, 6 female/6 male rabbits are randomly divided into an experimental group 1, an experimental group 2 and an experimental group 3 according to sex and weight, and each group is 4 female/2 male rabbits. The experimental group is marked by blue cage cards, F for male animals and M for female animals, and F007-F012 for male animals and M007-M012 for male animals.

TABLE 2

The method comprises the steps of taking 12 healthy rabbits, wherein the rabbits are 6 female/6 male, randomly divided into an experimental group 1 group, an experimental group 2 group and an experimental group 3 group according to sex and body weight, each group is 4, 2 female/2 male, a left and right self-control method of the same body is adopted, the animals are sheared by electric hair clippers firstly, the quadriceps muscles on the left and right sides are exposed, YXH-1501 liquid medicine (10 mg/ml ^-1) and sodium chloride injection with the same volume of 0.9% are respectively injected into the shearing parts on the right (R) side and the left (L) side of each rabbit through muscles by an aseptic operation method, the administration volume is 0.5 ml/side, the administration is carried out for 1 time every day, and the administration is carried out for 7 days continuously.

dosing concentration and dosing volume:

the drug side was administered with a test solution (10 mg/ml ^-1), and the control side was administered with 0.9% sodium chloride injection, at a dose volume of 0.5 ml/side.

The dosage design basis is as follows:

Relevant contents in the guiding principle: the design of the irritation test dosage mainly takes the concentration of a test object and the total dosage into consideration, and generally adopts the same concentration as a clinical preparation;

the maximum concentration of the clinical muscle application of the product is 10 mg/ml ^-1;

10-20 mg of an adult, wherein the surface area of 60kg of the adult is 1.627m ², the surface area of 2kg of the rabbit is 0.1515m ², the dosage of the obtained rabbit is about 1.83 mg/rabbit, and the administration volume is about 0.18 ml/rabbit by adopting the maximum concentration of 10 mg/ml ^-1 planned for clinical muscle;

The rabbit muscle stimulation test generally adopts the administration volume of 0.5-1.0 ml/rabbit;

in conclusion, the subject was administered with a clinical intramuscular intended maximum concentration of 10 mg/ml ^-1, with a volume of 0.5 ml/side (in vivo or ex vivo self-control), which is about 2.8 times the clinically equivalent dose.

The administration frequency is as follows:

The administration was 1 time per day for 7 days.

Observing the administration site 48h before and after the last administration and at the end of convalescent period, observing whether the administration causes local reactions such as red swelling, congestion, exudation, degeneration or necrosis, and filling in local symptom observation table.

48H after the last administration, each group was euthanized by CO ₂, 1/2 animals were killed by euthanasia, 1/1 male, the quadriceps femoris muscles were dissected and taken out, longitudinally cut, the stimulation reaction conditions of the muscle tissues at the injection site were visually observed and scored according to Table 1, graded according to Table 2, and corresponding photographs were provided, fixed in 10% neutral formalin, sectioned after paraffin embedding, and examined by H-E staining under a microscope.

The remaining animals were further observed for 14d, euthanized with CO ₂ anesthesia after the end of the observation period, and the status of the stimulatory responses in the muscle tissue of the injection site was visually observed and scored according to Table 1, graded according to Table 2, and corresponding photographs were provided.

TABLE 3 grading Standard of muscle irritation response

Stimulation response	Reaction stage
		Without obvious change	0
Mild hyperemia in the range of 0.5 × 1.0cm	1
		Moderate congestion, in the range of 0.5 × 1.0cm or more	2
Severe hyperemia with muscular degeneration	3
		Necrosis with brown degeneration	4
generalized necrosis occurs	5

TABLE 4 mean scores and grades

Mean score	grade
		0.0～0.4	is free of
0.5～1.4	Light and slight
		1.5～2.4	mild degree of
2.5～3.4	Of moderate degree
		3.5～4.4	Severe degree
4.5 and above	Severe severity of disease

3 examination results and discussion

3.1 gross necropsy examination results (results are shown in Table 5):

3.1.1 irritation study 48h after last dose:

Experimental group 1 group

The muscle tissues of the injection sites of the biceps quadriceps of 2 animals have no obvious gross pathological changes;

Experimental group 2 groups

experimental group 3 groups

No gross pathological changes were evident in the muscle tissues of the bilateral quadriceps femoris injection sites of 2 animals.

3.1.2 recovery phase end irritation study:

Experimental group 1 group

experimental group 2 groups

Experimental group 3 groups

the animals generally were in good condition and did not die during the test period; no obvious abnormality is observed in the local part of the drug administration by visual observation before each drug administration.

At 48h after the last administration and at 14d after the continued recovery, all animals have no local reactions such as red swelling, hyperemia, exudation, degeneration or necrosis on the control side and the administration side by visual observation, and no obvious difference is seen in comparison of the three groups;

histopathological examination results: no drug-related vascular-stimulation histopathological changes were observed on the control side and the administration side of all animals 48h after the last administration and 14d after the continued recovery, and no significant differences were observed in the three group comparisons.

TABLE 5 individual table of examination results of the dissection

Animal breed/strain: Rabbit/New Zealand Rabbit

3.2 histopathological examination results (see tables 6 and 7 for results)

TABLE 6 microscopic examination of individual body surface (48 h after last dose)

Note 1: the content of the column of the name of the tissue organ can be adjusted according to different experimental requirements; the absence of histopathological changes in the "seen lesions" column is indicated by "NR" and the visible histopathological changes are described in their corresponding positions.

Note 2: in the "degree of pathology" column, "-" indicates no significant pathological change; "+/-" indicates a change in lightness; "+" indicates a moderate change; "B" indicates that no tissue or organ is found.

TABLE 7 microscopic examination of the individual body surface (end of recovery period)

3.2.1 irritation study 48h after last dose (see FIGS. 1-6)

Experimental group 1 group

No obvious histopathological changes were observed in the F007 animal control lateral quadriceps injection site; myofibrosis necrosis and interstitial inflammatory cell infiltration at the injection site of the lateral quadriceps femoris muscle are administered.

M007 animal contrast side quadriceps injection part has multinuclear giant cell-like myobud tissue, and infiltration of myofibrillar interstitial inflammatory cells; no significant histopathological changes were seen at the site of injection of the quadriceps femoris muscle on the side of administration.

Experimental group 2 groups

No significant histopathological changes were seen in F009 animals at the sites of bilateral quadriceps injection.

no significant histopathological changes were seen in the M009 animal control lateral quadriceps injection site; administration of myofibrotic necrosis, interstitial hemorrhage, inflammatory cell infiltration at the site of lateral quadriceps injection.

Experimental group 3 groups

No significant histopathological changes were seen in the bilateral quadriceps injection sites of 2 animals (F011, M011).

3.2.2 recovery phase end irritation study (see FIGS. 9-14)

Experimental group 1 group

no significant histopathological changes were seen in the bilateral quadriceps femoris injection sites of 2 animals (F008, M008);

Experimental group 2 groups

No significant histopathological changes were seen in the bilateral quadriceps injection sites of 2 animals (F010, M010);

Experimental group 3 groups

No significant histopathological changes were observed in the bilateral quadriceps femoris injection sites of 2 animals (F012, M012);

In summary, 48 hours after the last administration, histopathological changes of the injection site of quadriceps femoris muscle of the animals of the experimental group 1 and the experimental group 2 are observed, the pathological changes are extremely light, and compared with histopathological changes on two sides, no obvious difference is observed, and the histopathological changes are considered to be the needle track injury reaction caused by local injection administration.

after the recovery period is over, no obvious histopathological changes are found at the injection site of the quadriceps femoris muscle of the administration side of the animals in the experimental group 1, the experimental group 2 and the experimental group 3, and the histopathological changes can be recovered after the administration is stopped.

4 conclusion

Histopathological examination results indicate that under the test conditions, the mannan peptide sample III prepared in the same specification and 3 different batch numbers in example 3 is subjected to irritation test by multiple intramuscular injections of New Zealand rabbits, and no drug irritation histopathological change is found at the injection site of the quadriceps femoris muscle of the administration side of animals in the experimental group 1, the experimental group 2 and the experimental group 3.

EXAMPLE 6 Rabbit intravenous administration of mannatide stimulation test histopathological assay prepared according to the present invention

1 presentation situation

The method comprises the following steps of carrying out multiple times of intravenous administration on a mannan peptide sample III prepared in example 3 with the same specification and different batches for irritation tests on New Zealand rabbits, namely taking 12 healthy rabbits, 6 female/6, randomly dividing the rabbits into an experimental group 1, an experimental group 2 and an experimental group 3 according to sex and body weight, and carrying out 4 rabbits, 2 female/2 female on each group, adopting a homologous left-right self-control method, administering the mannan peptide sample III liquid medicine prepared in example 3 (0.08 mg. ml ^-1) to the right auricular vein of each animal, administering the sodium chloride injection with the same volume of 0.9% to the left auricular vein, administering the sodium chloride injection with the volume of 12 ml. kg. ^-1, administering the speed of 5 ml. min ^-1, administering for 1 time every day and continuously administering for 7 days.

1/2(1 male/1 female) animals are killed in each group 72h after the last administration and after the recovery of the animals is finished for 14d, ear marginal tissues are respectively cut from the injection part and two parts of the auricle vein close to the heart, the stimulation reaction condition of the injection part is observed by naked eyes, 10% neutral formalin is fixed, and the histopathology examination is carried out by a conventional tissue section.

2 inspection method

grouping and group identification: taking 12 healthy rabbits at 6/6 female parent. The test groups are divided into 1 group of test group, 2 groups of test group and 3 groups of test group according to sex and weight at random, and each group is 4 and 2/2 female parent. The experimental group is marked by red cage card, the female animals are marked by F, the numbers are F001-F006, the male animals are marked by M, and the numbers are M001-M006.

TABLE 8

Group of	Color of cage card	Female	Male sex
				Experimental group 1 group	Red colour	F001～F002	M001～M002
Experimental group 2 groups	red colour	F003～F004	M003～M004
				experimental group 3 groups	Red colour	F005～F006	M005～M006

the method comprises the steps of taking 12 healthy rabbits, 6/6 male parent, randomly dividing the rabbits into an experimental group 1 group, an experimental group 2 group and an experimental group 3 group according to gender and weight, 4 rabbits each group, 2/2 female parent, adopting a homobody left and right self-control method, administering the mannatide sample III liquid medicine (0.08 mg/ml ^-1) prepared in example 3 to the right ear vein of each animal, administering the sodium chloride injection with the same volume of 0.9% to the left ear vein, administering the sodium chloride injection with the volume of 12 ml/kg ^-1, administering the speed of 5ml/min ^-1, administering the mannatide sample III liquid medicine for 1 time each day, continuously administering the liquid medicine for 7 days, killing 1/2(1 female/1 male) animals each group after last administration for 72 hours and after continuing to recover for 14 days, respectively cutting ear marginal tissues from the injection positions and the near-center ends of the ear marginal veins, observing the stimulation reaction conditions of the injection positions by naked eyes, fixing 10% neutral formalin, and performing histopathological examination by a conventional tissue section.

Route of administration

a vein.

reasons for choosing the route of administration

the clinically planned administration route of the product is vein, and the clinical planned administration route of the product is vein according to the requirements of the technical guidance principle of research on drug irritation, anaphylaxis and hemolysis.

Method of administration

The same-body left and right self-contrast method is adopted.

the administration was carried out using a Senno SN-50C6 micro-syringe pump at a rate of 5.0 ml/min ^-1, and the mannatide sample III prepared in example 3 was administered to the right-hand auricular vein of each animal, and the same volume of 0.9% sodium chloride injection was administered to the left-hand auricular vein.

administration concentration and administration volume

The drug side was administered with the test solution (0.08 mg/ml ^-1), and the control side was administered with 0.9% sodium chloride injection, at a volume of 12 ml/kg ^-1.

Basis of dosage design

Relevant contents in the guiding principle: irritation test dosage design should primarily take into account subject concentrations and total dosages, generally using the same concentrations as in the clinic, and the administration volume should not be too small.

the maximum intended concentration of the clinical vein of the product is 0.08 mg/ml ^-1;

The dosage of the clinical planned usage is 10-20 mg for an adult, the surface area of 60kg for the adult is 1.627m ², the surface area of 2kg for a rabbit is 0.1515m ², the maximum clinical equivalent dosage of the rabbit is about 0.93 mg/kg ^-1, and the dosage volume is about 11.6 ml/kg ^-1 by adopting the clinical planned concentration of 0.08 mg/ml ^-1;

In conclusion, the subject uses the clinical intravenous maximum planned concentration of 0.08 mg/ml ^-1 for administration, and for the convenience of administration, the volume of administration is 12 ml/kg ^-1, and the administration is carried out 1 time per day for 7 days continuously.

the administration frequency is as follows:

The administration was 1 time per day for 7 days.

3 examination results and discussion

Gross necropsy examination results (see table 9):

TABLE 9 individual table of examination results of the dissection

Animal breed/strain: Rabbit/New Zealand Rabbit

Stimulation study 72h after the last dose:

Experimental group 1 group

No gross pathological changes were evident in bilateral ear margin tissues of 2 animals;

Experimental group 2 groups

Experimental group 3 groups

no gross pathological changes were evident in bilateral ear margin tissues of 2 animals.

3.1.2 recovery phase end irritation study:

Experimental group 1 group

Experimental group 2 groups

experimental group 3 groups

histopathological examination results (see table 10, table 11):

TABLE 10 microscopic examination of individual body surface (48 h after last dose)

Note 1: the contents of the columns of the name of the tissue organ and the tissue part can be adjusted according to different experimental requirements; the absence of histopathological changes in the "seen lesions" column is indicated by "NR" and the visible histopathological changes are described in their corresponding positions.

Note 2: in the "degree of pathology" column, "-" indicates no significant pathological change; "+/-" indicates a change in lightness; "+" indicates a slight change; "+ +" indicates a moderate change; "+ + + +" indicates a severe change; "B" indicates that no tissue or organ is found.

TABLE 11 microscopic examination of individual body surface (end of recovery period)

Stimulation study 72h after the last dose: (see FIGS. 15-26).

Experimental group 1 group

No obvious histopathological changes are observed at the control side ear edge intravenous injection part and the proximal end of the F001 animal; edema around the blood vessel at the intravenous injection site of the administration side ear edge, no significant histopathological changes were observed near the heart end.

The M001 animal has edema around the blood vessel of the control side ear edge intravenous injection part, and no obvious histopathological change is seen at the proximal end; peripheral bleeding and edema with inflammatory cell infiltration at the site of intravenous injection of the lateral margin of the drug administration.

experimental group 2 groups

The F003 animals control the perivascular edema of the side ear edge intravenous injection part, and no obvious histopathological change is seen at the proximal end; edema around the blood vessel of the intravenous injection site of the administration side ear edge is accompanied by infiltration of inflammatory cells and organization of the tissue around the blood vessel.

No significant histopathological changes were observed in M003 animals at the sites of bilateral auricular marginal intravenous injection and proximal to the heart.

experimental group 3 groups

No obvious histopathological changes are observed at the control side ear edge intravenous injection part and the proximal end of the F005 animal; perivascular organization, inflammatory cell infiltration, of the site of intravenous injection at the lateral margin of the drug administration.

no significant histopathological changes were observed in the M005 animals at the sites of bilateral auricular marginal intravenous injection and proximal to the heart.

3.2.2 recovery phase end irritation study: (see FIGS. 27-38)

Experimental group 1 group

No significant histopathological changes were observed in 2 animals (F002, M002) at the sites of bilateral auricular edge intravenous injection and proximal to the heart.

Experimental group 2 groups

No significant histopathological changes were observed in 2 animals (F004, M004) at the sites of bilateral auricular edge intravenous injection and proximal to the heart.

Experimental group 3 groups

no significant histopathological changes were observed in 2 animals (F006, M006) at the site of bilateral auricular vein injection and proximal to the heart.

in conclusion, the animals generally worked well during the test period and did not die; no obvious abnormality is observed in the local part of the drug administration by visual observation before each drug administration. 72h after the last administration, histopathological changes of the ear vein injection parts of the administration side and the control side of animals in the experimental group 1, the experimental group 2 and the experimental group 3 are visible, the pathological change degree is extremely light, and compared with histopathological changes on two sides, the histopathological changes are not obviously different, and the histopathological changes are considered to be the needle track injury reaction caused by local injection administration. After the recovery period is over, no obvious histopathological changes are found at the administration side ear edge intravenous injection part of the animals in the experimental group 1, the experimental group 2 and the experimental group 3, and the histopathological changes can be recovered after the administration is stopped.

4 conclusion

Histopathological examination results suggest that in the test condition, the mannan peptide sample III irritation test of the same specification and 3 different batch numbers prepared in example 3 is repeatedly intravenously administered to New Zealand rabbits, and no obvious drug irritation histopathological change is observed in the edge vein on the administration side of animals.

Example 7 Guinea pig administration of mannatide provided by the present invention Passive skin allergy test

1-3 of a sample: three different lots of mannatide sample III from example 3.

The method takes 64 healthy guinea pigs, and 32 male and female. The test sample is randomly divided into 16 groups of a negative control sensitization group, a positive control sensitization group, a sample 1 low-dose sensitization group, a sample 1 high-dose sensitization group, a sample 2 low-dose sensitization group, a sample 2 high-dose sensitization group, a sample 3 low-dose sensitization group, a sample 3 high-dose sensitization group, a negative control passive sensitization group, a positive control passive sensitization group, a sample 1 low-dose passive sensitization group, a sample 1 high-dose passive sensitization group, a sample 2 low-dose passive sensitization group, a sample 2 high-dose passive sensitization group, a sample 3 low-dose passive sensitization group and a sample 3 high-dose passive sensitization group according to gender and weight, wherein each group of sensitized animals is 2, 1 female/1, and each group of the passive sensitized animals is 6, 3 female/3 male.

The administration route is as follows:

the sensitization administration route is abdominal cavity, the passive sensitization administration route is intradermal, and the excitation administration route is vein.

Reasons for choosing the route of administration

according to the recommendation in the anaphylaxis test method of the research technical guidance principle of drug irritation, anaphylaxis and hemolysis, intravenous, abdominal and subcutaneous methods which are easy to generate antibodies can be adopted for administration in the sensitization stage, and rapid intravenous injection administration is required to be excited.

The administration method comprises the following steps:

The sensitization administration is intraperitoneal injection (ip), the passive sensitization administration is intradermal injection (id), and the excitation administration is rapid plantar intravenous injection (iv).

Dosing concentration and dosing volume:

Negative control sensitized group: priming phase ip 0.9% sodium chloride injection (0.5 ml/tube) was given.

Positive control sensitized group: sensitization phase ip 1% ovalbumin (0.5 ml/mouse) was administered.

Test article 1 low dose sensitized group: YXH-1501 solution (lot: 170901, 1.0 mg/ml-1, 0.5 ml/tube) was administered ip as the sensitization phase.

test article 1 high dose sensitized group: YXH-1501 solution (lot No. 170901, 10 mg/ml-1, 0.5 ml/tube) was administered ip as the sensitization phase.

Test article 2 low dose sensitized group: YXH-1501 solution (lot: 170902, 1.0 mg/ml-1, 0.5 ml/tube) was administered ip as the sensitization phase.

test article 2 high dose sensitized group: the sensitization stage ip was performed with the mannatide sample III solution (lot No.: 170902, 10 mg/ml-1, 0.5 ml/piece) prepared in example 3.

Test article 3 Low dose sensitization group A drug solution of mannatide sample III prepared in example 3 (lot No. 170903, 1.0 mg/ml ^-1, 0.5 ml/piece) was ip-administered in the sensitization phase.

Test article 3 high dose sensitization group the sensitization phase ip was administered with the mannatide sample III solution (lot No. 170903, 10 mg/ml ^-1, 0.5 ml/tube) obtained in example 3.

Negative control passive sensitized group: 0.1ml of serum is injected into each point of the back skin before the passive sensitization stage in an intradermal way according to the dilution sequence of the serum of the negative control sensitization group. The priming phase was injected intravenously with 1ml of the same priming antigen as the priming dose plus an equivalent amount of 0.5% Evans blue dye.

Positive control passive sensitized group: 0.1ml of serum is injected into each point of the back skin before the passive sensitization stage in turn according to the dilution sequence of the serum of the positive control sensitization group correspondingly. The priming phase was injected intravenously with 1ml of the same priming antigen as the priming dose plus an equivalent amount of 0.5% Evans blue dye.

Test article 1 low dose passive sensitization group: 0.1ml of serum is injected into each point of the back skin before the passive sensitization stage correspondingly in the intracutaneous injection according to the dilution sequence of the serum of the low-dose sensitization group of the test article 1. The priming phase was injected intravenously with 1ml of the same priming antigen as the priming dose plus an equivalent amount of 0.5% Evans blue dye.

Test article 1 high dose passive sensitization group: 0.1ml of serum is injected into each point of the back skin before the passive sensitization stage in turn according to the dilution sequence of the high-dose sensitization group serum of the test sample 1. The priming phase was injected intravenously with 1ml of the same priming antigen as the priming dose plus an equivalent amount of 0.5% Evans blue dye.

Test article 2 low dose passive sensitization group: before the passive sensitization stage, each point of the back skin is sequentially and correspondingly injected with 0.1ml of serum by intracutaneous injection according to the dilution sequence of the low-dose sensitization group serum of the test article 2. The priming phase was injected intravenously with 1ml of the same priming antigen as the priming dose plus an equivalent amount of 0.5% Evans blue dye.

Test article 2 high dose passive sensitization group: 0.1ml of serum is injected into each point of the back skin before the passive sensitization stage in turn according to the dilution sequence of the high-dose sensitization group serum of the test article 2. The priming phase was injected intravenously with 1ml of the same priming antigen as the priming dose plus an equivalent amount of 0.5% Evans blue dye.

Test article 3 low dose passive sensitization group: 0.1ml of serum is injected into each point of the back skin before the passive sensitization stage correspondingly in the intracutaneous way according to the dilution sequence of the low-dose sensitization group serum of the test sample 3. The priming phase was injected intravenously with 1ml of the same priming antigen as the priming dose plus an equivalent amount of 0.5% Evans blue dye.

Test article 3 high dose passive sensitization group: 0.1ml of serum is injected into each point of the back skin before the passive sensitization stage in turn according to the dilution sequence of the high-dose sensitization group serum of the test sample 3. The priming phase was injected intravenously with 1ml of the same priming antigen as the priming dose plus an equivalent amount of 0.5% Evans blue dye.

The dosage design basis is as follows:

Maximum concentration for clinical application 10 mg-ml ^-1 (intramuscular administration)

The clinical application dosage is 10-20 mg for an adult, the body surface area of the adult of 60kg is 1.627m ², the body surface area of a 300g guinea pig is 0.04m ², and the maximum equivalent dose of the guinea pig is calculated to be about 0.49 mg/pig;

Guidelines specify a passive skin allergy test, including at least the highest dose or concentration clinically planned, administered in a volume of 0.5 ml;

In conclusion, the subject high dose group was administered at the clinically intended concentration (10 mg. multidot.ml ^-1, administration volume: 0.5ml), and the low dose group was administered at 0.5 mg/dose (1.0 mg. multidot.ml ^-1, administration volume: 0.5ml, approximately the clinically equivalent dose).

3.6 dosing frequency

the sensitization stage comprises every other day of administration, 3 times of administration, killing sensitized animals at 14d after the last sensitization, passively sensitizing the animals the next day, and exciting the animals after 4h of passive sensitization.

And (3) sensitizing, namely, 0.5ml of 0.9% sodium chloride injection is given to a negative control sensitized animal, 0.5ml of 1% ovalbumin is given to a positive control sensitized animal, 0.5ml of mannatide sample III liquid medicine prepared in example 3 is given to a low-dose and high-dose sensitized animal to be tested (the medicine concentration is respectively 1.0 mg/ml ^-1 and 10 mg/ml ^-1), all groups of animals are subjected to intraperitoneal injection every other day for 3 times, 14d after the last sensitization, a guinea pig is subjected to intraperitoneal injection of pentobarbital sodium according to a 45 mg/kg ^-1 dose for anesthesia, the large abdominal vein is subjected to blood collection, centrifugation is carried out at 3000rpm for 15min after blood coagulation in a natural state, and serum is separated and is stored at minus 10 to minus 20 ℃ for later use.

And (3) passive sensitization, namely, depilating the front back of each passively sensitized animal (about 8 multiplied by 8cm ²), symmetrically marking two points at about 2cm positions on two sides of the midline of the back, wherein 4 points are provided, and diluting antiserum of each group into 1:2, 1:4, 1:8 and 1:16 by 0.9% sodium chloride injection at intervals of about 4 cm. between two adjacent points on the same side, wherein the guinea pigs of each group are sequentially injected with serum in the depilated positions in an intradermal mode, and each point is injected with 0.1ml, and the 4 points (two sides of the spinal column) are respectively serum diluent with each dilution degree.

Excitation: after 4h of passive sensitization, each passive sensitized animal was injected intravenously with 1ml of the same amount of challenge antigen as the sensitizing dose plus an equal amount of 0.5% Evans blue dye.

observing that CO ₂ is used for anaesthetizing each group of guinea pigs after being excited for 30min, cutting back skin, measuring the diameter of the blue spot on the inner layer of the skin, recording '0' if no spot exists, calculating the diameter of the irregular round blue spot according to (major diameter + minor diameter)/2, judging that the diameter of the blue spot is larger than 5mm to be positive, and evaluating that whether the test article has passive skin anaphylactic reaction and the intensity of anaphylactic reaction are judged according to the blue spot range.

The results are shown in Table 12 and FIGS. 39 to 86.

The general state of each group of animals during the test period is good, and no obvious abnormality is seen;

No obvious blue spot is seen at the position of the negative control passive sensitized guinea pig intracutaneously injected with antiserum, and the negative control passive sensitized guinea pig intracutaneously injected with antiserum is negative according to the passive skin anaphylaxis judgment standard;

obvious blue spots can be seen at the position of the positive control passive sensitized guinea pig injected with antiserum in an intradermal way, the diameter of the blue spots at the intradermal injection position with the antiserum dilution ratio of 1:2, 1:4, 1:8 and 1:16 is more than 5mm, the positive blue spots are determined according to the passive skin anaphylaxis judgment standard, and the incidence rate is 100%;

No obvious blue spots are found at the antiserum injection part of the low-dose and high-dose passively sensitized guinea pigs of the test sample, and the test sample is negative according to the judgment standard of passive skin anaphylactic reaction.

TABLE 12 animal blue Spot diameter Individual data (mm)

Conclusion under the test conditions, the evaluation results of the passive skin allergy test of the mannatide sample III prepared in example 3 given to the guinea pigs in 3 different batches are negative, and obvious differences are not found among the three.

Example 8 in vitro hemolytic assay of mannatide provided by the present invention

taking 17 test tubes, respectively adding 0.5, 0.4, 0.3, 0.2 and 0.1ml of mannatide sample III liquid medicine prepared in example 3 (test sample 1, batch number: 170901, 2.5 mg.ml ^-1) into the 1 st to 5 th tubes, respectively adding 0.9% sodium chloride injection 2.0, 2.1, 2.2, 2.3 and 2.4ml, respectively adding 0.9% sodium chloride injection 2.0, batch number: 170902, 2.5 mg.ml ^-1)0.5, 0.4, 0.3, 0.2 and 0.1ml into the 6 th to 10 th tubes, respectively adding 0.9% sodium chloride injection 2.0, 2.1, 2.2, 2.3 and 2.4ml, respectively adding 0.9% sodium chloride injection 2.0, 2.1, 2.2.2.3 and 2.4ml, respectively adding 0.9% sodium chloride injection 2.0, 2.1, 2.2.2, 2.3 and 2.4ml into the 11 th to 15 th tubes, respectively adding mannatide sample III liquid medicine prepared in example 3 (test sample 3, batch number: 365, batch number: 2.57, batch number: 2.2.2.2.2.5, 2.5ml, 2.5. 0.5. 0.4. 0.5. 0.1. 0.5. 0.1. 0.5. 0.4. 0.1. 0.4. 0.7. 0.1. 0.2. 0.4. 0.1. 0.9. 0.2. 0.1. 0.4. ml of haemolytic sodium chloride, respectively.

the results are shown in tables 13 to 17, FIGS. 87 to 89.

TABLE 13 YXH-1501 (batch No.: 170901) in vitro hemolytic assay

TABLE 14 YXH-1501 (batch No.: 170902) in vitro hemolytic assay

TABLE 15 YXH-1501 (batch No.: 170903) in vitro hemolytic assay

detection method

And (3) keeping the sample adding test tube in a water bath kettle at 37 +/-0.5 ℃ for heat preservation, observing once every 15min at the beginning, observing once every 1h after 1h, continuously observing for 3h, and observing the hemolysis and agglutination condition of each tube by naked eyes. The observation point was photographed for 3 h. (see Table 16 for criteria).

TABLE 16 criteria for determining hemolysis and agglutination of erythrocytes

After the tubes 1 to 5 (sample 1), 6 to 10 (sample 2) and 11 to 15 (sample 3) are added with the erythrocyte suspension, the solution is in a turbid state at the beginning stage, but the clear red or brown phenomenon does not appear, the erythrocyte at the bottom of the tube is deposited in a small amount, and the erythrocyte deposition at the bottom of the tube is gradually increased along with the time; water bath is carried out for 3h, the No. 1 to No. 15 tubes are in a layered state, the upper layer is a colorless transparent solution, a large amount of red blood cells at the bottom of the tubes are deposited and can be uniformly dispersed by shaking lightly, and no obvious difference is found compared with the No. 16 negative control tube, which indicates that hemolysis and agglutination phenomena do not occur; clear red appears in the positive control tube No. 17 within 15min after deionized ultrapure water is added, and no erythrocyte is deposited at the bottom of the tube, indicating that the tube is completely hemolyzed.

Conclusion under the test conditions, mannatide samples III prepared in example 3 with the same specification and different batches have no hemolysis and agglutination on rabbit blood erythrocytes, and no obvious difference is seen among the three.

Example 9 guinea pig administration of mannatide provided by the present invention to a human being

1-3 of a sample: three different lots of mannatide sample III from example 3.

The method takes 64 healthy guinea pigs, and 32 male and female. The test sample is randomly divided into 8 groups of a negative control group, a positive control group, a test sample 1 low dose group, a test sample 1 high dose group, a test sample 2 low dose group, a test sample 2 high dose group, a test sample 3 low dose group and a test sample 3 high dose group according to gender and weight, wherein each group comprises 8 animals and is 4/4 female parent.

Grouping and group identification

And according to sex and weight, dividing the test sample into 8 groups of a negative control group, a positive control group, a test sample 1 low-dose group, a test sample 1 high-dose group, a test sample 2 low-dose group, a test sample 2 high-dose group, a test sample 3 low-dose group and a test sample 3 high-dose group by using TOXSTAT2006 software, wherein each group comprises 8 female/4 male. After grouping, the negative control group is marked by a white cage card, the positive control group is marked by a yellow cage card, the low-dose group of the sample is marked by a blue cage card, the high-dose group of the sample is marked by a red cage card, animals are numbered, female animals are numbered from F, the numbers are F001-F032, and male animals are numbered from M, the numbers are M001-M032.

TABLE 17

Treatment of the remaining animals after grouping:

Grouping the rest 4 animals, 2 male parent/2 female parent, observing once a day, delivering CO ₂ at 2017.12.5 for anesthesia and euthanasia,

Route of administration, dose, frequency and duration of administration and reasons for selection:

The administration route is as follows:

the sensitization administration route is abdominal cavity, and the stimulation administration route is vein.

Reasons for the choice of route of administration:

According to the recommendation in the anaphylaxis test method of the research technical guidance principle of drug irritation, anaphylaxis and hemolysis, the method for easily generating antibodies such as vein, abdominal cavity and subcutaneous can be adopted in the sensitization stage for administration, the rapid intravenous injection administration is required to be excited, the abdominal cavity injection is adopted in the sensitization administration of the subject, and the intravenous injection is adopted in the excitation administration.

The administration method comprises the following steps:

The priming administration is intraperitoneal (ip) administration, and the priming administration is rapid plantar intravenous (iv) administration.

Dosing concentration and dosing volume:

Negative control group: the prime phase was given ip 0.9% sodium chloride injection (0.5 ml/tube) and at challenge iv 0.9% sodium chloride injection (1.0 ml/tube).

Positive control group: the prime phase was given ip 1% ovalbumin (0.5 ml/mouse) and the challenge was given iv 1% ovalbumin (1.0 ml/mouse).

Test article 1 Low dose group the mannosan peptide sample III solution (lot: 170901, 1.0 mg/ml ^-1, 0.5 ml/piece) prepared in example 3 was ip-administered in the priming stage, and the mannosan peptide sample III solution (lot: 170901, 1.0 mg/ml ^-1, 1.0 ml/piece) prepared in example 3 was iv-administered in the priming stage.

test article 1 high dose group the mannosan peptide sample III solution (lot No. 170901, 10 mg/ml ^-1, 0.5 ml/piece) prepared in example 3 was administered ip at the priming stage, and the mannosan peptide sample III solution (lot No. 170901, 10 mg/ml ^-1, 1.0 ml/piece) prepared in example 3 was administered iv at the priming stage.

test article 2 Low dose group the mannosan peptide sample III solution (lot: 170902, 1.0 mg/ml ^-1, 0.5 ml/piece) prepared in example 3 was ip-administered in the priming stage, and the mannosan peptide sample III solution (lot: 170902, 1.0 mg/ml ^-1, 1.0 ml/piece) prepared in example 3 was iv-administered in the priming stage.

Test article 2 high dose group the mannosan peptide sample III solution (lot # 170902, 10 mg/ml ^-1, 0.5 ml/piece) prepared in example 3 was administered ip at the priming stage, and the mannosan peptide sample III solution (lot # 170902, 10 mg/ml ^-1, 1.0 ml/piece) prepared in example 3 was administered iv at the priming stage.

Test article 3 Low dose group the mannosan peptide sample III solution (lot: 170903, 1.0 mg/ml ^-1, 0.5 ml/piece) prepared in example 3 was ip-administered in the priming stage, and the mannosan peptide sample III solution (lot: 170903, 1.0 mg/ml ^-1, 1.0 ml/piece) prepared in example 3 was iv-administered in the priming stage.

Test article 3 high dose group the mannosan peptide sample III solution (lot No. 170903, 10 mg/ml ^-1, 0.5 ml/piece) prepared in example 3 was administered ip at the priming stage, and the mannosan peptide sample III solution (lot No. 170903, 10 mg/ml ^-1, 1.0 ml/piece) prepared in example 3 was administered iv at the priming stage.

the dosage design basis is as follows:

Guidelines specify that a systemic active hypersensitivity test, including at least the highest dose or concentration clinically planned, the volume administered: sensitization stage 0.5ml, excitation stage 1.0 ml;

In summary, the high dose group of the present invention was administered at the clinically proposed concentrations (10 mg. multidot.ml ^-1, administration volume: sensitization phase 0.5ml, challenge phase 1.0ml), and the low dose group was administered at 0.5 mg/dose (1.0 mg. multidot.ml ^-1, administration volume: sensitization phase 0.5ml, challenge phase 1.0ml, approximately the clinically equivalent dose).

The administration frequency is as follows:

The sensitization stage comprises alternate daily administration for 3 times, wherein half of animals at 14 th day after the last sensitization are injected into plantar vein rapidly for excitation, and the rest animals at 21 st day after the last sensitization are excited.

sensitization, 0.5ml of 0.9% sodium chloride injection is given to animals in a negative control group, 0.5ml of 1% ovalbumin is given to animals in a positive control group, 0.5ml of the liquid medicine of the mannatide sample III prepared in the example 3 is given to animals in low and high dose groups of the test sample (the medicine concentration is respectively 1 mg/ml ^-1 and 10 mg/ml ^-1), and the animals in each group are subjected to intraperitoneal injection every other day for 3 times.

Excitation: half of animals (2 male parent/2 male parent) in each group are respectively selected to be injected into plantar veins of a test object with sensitization concentration of 2 times of volume 14d and 21d after last sensitization.

And (4) observation: symptoms of anaphylaxis were observed and recorded from immediately to 30min after challenge.

evaluation: and judging whether the test sample has anaphylactic reaction or not and the anaphylactic reaction intensity according to the anaphylactic reaction scoring standard.

test result judgment standard after excitation administration

immediately after challenge to about 30min after challenge, animals were observed for detailed responses and time to appearance and disappearance as indicated in table 18. And the allergic reaction of the product is judged according to the table 19.

TABLE 18 symptoms of anaphylaxis

0	Is normal	7	Shortness of breath	14	Gait instability
						1	medicine for treating restlessness	8	Urination apparatus	15	Jumping
2	Vertical hair	9	excrement discharging device	16	Wheezing
						3	Hair trembling	10	Lacrimation	17	spasm of muscle and tendon
4	Scratching nose	11	Dyspnea	18	Rotate
						5	Sneezing	12	wheeze sound	19	Tidal breathing
6	Cough with asthma	13	purpura	20	Death was caused by death

TABLE 19 evaluation criteria for systemic sensitization

0	－	negative allergic reaction
			1 to 4 symptoms	+	weak positive of anaphylaxis
5 to 10 symptoms	++	positive allergic reaction
			11 to 19 symptoms	+++	Strong positive of allergic reaction
20	++++	Very strong positive of allergic reaction

dead and moribund animal handling during the experiment:

Animals in none of the groups died except the positive control group at challenge.

treatment of surviving animals at the end of the experiment:

At the end of the experiment, all surviving animals were euthanized under CO ₂ anesthesia.

And (3) counting and processing data:

The guinea pig is heavy, the statistical influence of whether to keep decimal numbers is negligible, and the data are scattered, one third of the standard deviation is greater than 1, so that the individual data and the statistical data are rounded. Carrying out statistics on the animal body weight, calculating a MEAN value and a standard deviation by using EXCEL software, and expressing the MEAN value and the standard deviation in a report as MEAN +/-SD; analysis was performed using TOXSTAT 2006.

And (4) counting the clinical symptoms of the animals after the drug administration is stimulated, and judging the allergic reaction result.

results the general state of each group of animals during the test period is good, and no obvious abnormality is seen;

effect on animal body weight: the weights of animals in the positive control group and the test article low and high dose group are not statistically significant different from those in the negative control group;

TABLE 20 animal body weight Individual data (units: g)

TABLE 20 continuation

Note: at the 2 nd challenge dose, 4 animals were present per dose group.

TABLE 21 statistical data for animal body weights (mean. + -. SD, unit: g)

Note: n is 4 and the rest n is 8 in the 2 nd challenge administration.

the test result after the priming administration judges that no obvious allergic symptoms are found in the negative control group animals within 30min after the two times of priming administration, and the allergic reaction evaluation result is as follows: the anaphylactic reaction is negative;

The positive control group animals immediately appear symptoms such as restlessness, piloerection, nasal pruritus, tachypnea, urination, defecation, dyspnea, gait instability, jumping, spasm, rotation and the like after the first excitation administration, and die within 4min after the administration; immediately after the 2 nd excitation administration, the animals have symptoms of restlessness, nasal pruritus, tachypnea, urination, defecation, dyspnea, unstable gait, jumping, spasm, rotation and the like, die within 4min after the administration, and the result of anaphylactic reaction evaluation is as follows: the anaphylactic reaction is extremely strong and positive; no obvious allergic symptoms are found in 30min after the animals with low and high test sample doses are stimulated twice, and the allergic reaction evaluation result is as follows: the anaphylactic reaction is negative;

no obvious allergic symptoms are found in 30min after the animals with low and high test sample doses are stimulated twice, and the allergic reaction evaluation result is as follows: the allergic reaction is negative.

TABLE 22 animal symptom Individual data (first challenge dosing)

Note: "-" indicates a failure to meet this result, and "+" indicates a failure to meet this result.

TABLE 23 animal symptom Individual data (2 nd challenge dose)

TABLE 24 animal symptom List (first elicitation dose)

Note: n/m represents the number of animals presenting symptoms/total number of animals.

TABLE 25 animal Condition List (2 nd challenge administration)

TABLE 26 evaluation of systemic sensitization Individual data (first challenge dosing)

TABLE 27 evaluation of systemic sensitization Individual data (2 nd challenge dose)

TABLE 28 results of systemic sensitization evaluation (first challenge administration)

Note: n/m represents the number of animals present corresponding to the result/total number of animals.

TABLE 29 evaluation results of systemic sensitization (2 nd challenge administration)

Conclusion under the test conditions, the results of systemic sensitization evaluation of the mannatide sample III (product number: YXH-1501) obtained in example 3 given to guinea pigs in 3 different batches were all negative, and no significant difference was observed between the three.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A method for purifying mannatide, which is characterized by comprising the following steps:

step 1: obtaining a crude product of the mannan peptide;

and step 3: purifying the first purified product by high performance liquid size exclusion chromatography, and collecting eluate;

And (2) eluting the purified product by using water and 1mol/L sodium chloride solution as mobile phases according to the following gradient, wherein the water is as follows: sodium chloride solution ═ 10:90(1min) → water: sodium chloride solution ═ 40:60(3min) → water: sodium chloride solution 60:40(3min) → water: sodium chloride solution 90:10(1 min);

In the step 3, water is used as an eluent for purification, and elution is carried out at the flow rate of 0.5 ml/min;

in the step 3, the collected eluent is the eluent collected for 10-12 min;

the step 3 of collecting the eluent further comprises a step of ultrafiltration.

2. The purification method according to claim 1, wherein the collecting of the eluent in the step 2 is collecting the eluent for 0 to 0.5min and 1.5 to 3 min.

3. The purification process of claim 1 or 2, wherein the filtration in step 2 is to remove impurities having a molecular weight of less than 8000.

4. The purification process according to claim 3, wherein the eluent has a pH of 6.8.

5. The purification method according to any one of claims 1 to 4, wherein the step 1 of obtaining the crude mannatide product comprises: the konjac flying powder is used as a raw material, and fermentation liquor is collected through biological fermentation.

6. mannatide obtained by the purification method according to any of claims 1 to 5.