WO2015062158A1

WO2015062158A1 - A composition via nasal administration of recombinant human ciliary neurotmphic factor and a preparation method thereof

Info

Publication number: WO2015062158A1
Application number: PCT/CN2014/000464
Authority: WO
Inventors: 吴闻哲; 侯惠民; 姚孝林; 罗世华; 徐晓寒
Original assignee: 上海现代药物制剂工程研究中心有限公司
Priority date: 2013-10-31
Filing date: 2014-05-05
Publication date: 2015-05-07
Also published as: CN103845727A; CN103845727B

Abstract

Provided in the present invention are a composition via nasal administration of recombinant human ciliary neurotmphic factor which contains 0-1% recombinant human ciliary neurotmphic factor, 0-20% of auxiliary material for protein permeation, 0-5% inorganic salts, 0-99% of water by weight percentage and a preparation method thereof. The effect on controlling the weight by the composition via nasal administration of the present invention is equivalent to that of administration by injcetion, and will not easily produce antibodies, can be uesd for losing weight.

Description

Nasal administration composition of recombinant human ciliary neurotrophic factor and preparation method thereof

The present invention relates to a recombinant human ciliary neurotrophic factor composition and a process for the preparation thereof. According to the latest epidemiological survey data, the overweight rate of Chinese residents is 17.16% and the obesity rate is 5.16%. Overweight and obesity have become important diseases affecting the health of residents. Nearly 40% of patients with hypertension due to overweight and obesity, nearly 35% of diabetes, and the direct economic burden of diseases caused by overweight and obesity is 36.90 billion yuan (2003 data). There is a huge market capacity for the treatment of obesity and its related diseases. Therefore, the development of drugs for the treatment of obesity has broad market prospects.

The human ciliary neurotmphic factor (hCNTF), named for its ability to promote the survival of chicken embryo ciliary ganglion neurons, is a neurotrophic factor other than the NGF family. It has a variety of biological activities in the body, mainly in the nervous system, and has a wide range of pro-survival effects on a variety of central and peripheral neurons. In recent years, when hCNTF was used as a therapeutic drug for the treatment of amyotrophic lateral sclerosis, it was found that CNTF can cause weight loss and loss of appetite. Subsequent studies have shown that CNTF has a similar leptin-like effect, which can suppress appetite, reduce body weight, regulate blood sugar and lipid metabolism, and have certain curative effects on certain chronic complications of diabetes.

Recombinant human ciliary neurotrophic factor (rhCNTF) is different from traditional chemical anti-obesity drugs. It is a protein macromolecular drug that is identical to the CNTF amino acid sequence secreted by the human body or changes (mutant) to a small part of amino acids. In a variety of rodent obesity or type 2 diabetes models, rhCNTF administration can correct or improve hyperinsulinemia, hyperphagia and hyperlipidemia and other obesity-related diseases, and can also regulate blood sugar and other metabolic indicators. The prevention and treatment of type II diabetes and its complications play an important role and have broad clinical application prospects. The completed animal pharmacodynamics and preclinical safety evaluation experiments confirmed that rhCNTF is a safe and effective drug for the treatment of obesity and diabetic obesity.

Regenron's recombinant human ciliary neurotrophic factor Axokine was genetically engineered for CNTF. The clinical trial results of Ι/Π/ΠΙ have shown that the drug has good safety and very little side effects. After 12 weeks of follow-up for 12 months, the body weight rebounded very slowly and the effect lasted for 12 months. However, antibodies produced by long-term injections are not yet on the market.

Since rhCNTF is a protein drug with a short half-life in vivo, it requires a daily subcutaneous bet as a weight-loss drug. Shot, about 3 months a course of treatment, inconvenient, there is no need to develop non-injectable preparations. Due to the poor stability of protein drugs, oral absorption, and easy degradation, there is no research report on the non-injection of rhCNTF.

Due to the absorption limitation of the nasal mucosa, drugs with a molecular weight greater than 1000 are considered to be difficult to enter the body and function through the nose. Therefore, recombinant human ciliary neurotrophic factor with a molecular weight of more than 20,000 is generally considered to be impossible to be administered from the nasal cavity. Therefore, no reports or patent applications concerning nasal administration of the drug have been observed. In fact, it has been confirmed by experiments that the solution of rhCNTF is directly administered to the nasal cavity of an animal, and no relevant pharmacological effects are observed.

Summary of the invention

It is an object of the present invention to provide a nasal administration composition for recombinant human ciliary neurotrophic factor and a preparation method thereof, which meet the needs of clinical applications.

Based on a large number of experiments, the present invention unexpectedly discovered a method for effectively overcoming technical obstacles, and successfully transporting a protein macromolecular drug into a living body through a nasal cavity to produce a weight loss effect comparable to injection.

The nasal administration composition of the recombinant human ciliary neurotrophic factor, in percentage by weight, comprises: recombinant human ciliary neurotrophic factor 0~1%

Protein penetration aid 0~20%

Buffer component (pH5~9) 0-5%

Inorganic salt 0~5%

Water 0~99%

The sum of the percentages of the components is 100%;

Preferably, in weight percent, contains:

Recombinant human ciliary neurotrophic factor 0.01~0.1%

Protein penetration aid 1~10%

Buffer component with pH=5~9 0~2%

Inorganic salt 0~2%

Water 86~97%

The sum of the percentages of the components is 100%;

The recombinant human ciliary neurotrophic factor (rhCNTF) has a molecular weight of 20,000~24, OOODa, which may be a protein sequence completely identical to the native human CNTF, or may be a genetically engineered variety of human CNTF mutants. , including changing 17 cysteine to alanine, 63 glutamic acid to arginine, and C-terminal deletion 14 to 16 amino acids Mutant.

The protein permeation auxiliary material is selected from the group consisting of chitosan, sodium polyphosphate, protamine, cyclodextrin or a derivative thereof, cholate and its derivative, aminopeptidase inhibitor, polyoxyethylene hydrogenated castor oil , polyoxyethylene lauryl ether, polyethylene glycol stearate, polyoxyethylene and polyoxypropylene copolymer, polysorbate, poloxamer, fusidic acid derivative, PLGA, phospholipid and its derivatives Or one or more of basic amino acid polymers;

among them:

The phospholipid is selected from the group consisting of lecithin, diacetyl phosphatidylcholine, lysophosphatidylglycerol, dipalmitoylphosphatidylcholine or dimyristoylphosphatidylglycerol;

The cholate is preferably sodium cholate, sodium glycocholate, ursodeoxycholic acid or taurocholate;

The basic amino acid polymer is preferably polyarginine (e.g., octaarginine), polylysine, transcriptional activator Tat (49-57) of the (HIV-1) viral genome;

The fusidic acid derivative is selected from the group consisting of sodium dihydrogenate (STDHF), sodium dihydrochloride, and the like;

The cyclodextrin or a derivative thereof is selected from the group consisting of α-, β-, γ-cyclodextrin, dimethyl-α- or β-cyclodextrin, hydroxypropyl-β- or γ-cyclodextrin, and the like;

The basic amino acid polymer is preferably octaarginine;

The buffer component of ρΗ=5~9 is selected from the group consisting of Tris-HCl, sodium acetate-sodium acetate, sodium citrate-sodium citrate or sodium dihydrogen phosphate-disodium hydrogen phosphate;

The inorganic salt is sodium chloride or potassium chloride;

Preferably, the nasal administration preparation of the recombinant human ciliary neurotrophic factor further comprises a preservative, and is selected from the group consisting of a common preservative such as phenol, benzalkonium chloride, chlorobutanol, thimerosal, and paraben;

Preferably, the nasal administration preparation of the recombinant human ciliary neurotrophic factor further comprises a bioadhesive material selected from the group consisting of gellan gum, aminated gelatin, cellulose or a derivative thereof, carbomer or hyaluronic acid, and the like. One or more of the above; preferably, the nasal administration composition of recombinant human ciliary neurotrophic factor, in weight percentage, contains: recombinant human ciliary neurotrophic factor 0.01 to 0.1%

Protein penetration aid 3.0~10.0%

Buffer component with pH=5~9 0.1 1.0%

Inorganic salt 0~0.6%

Preservative or / and bioadhesive material 0.0 ~ 5.0%

Water 83~96% The sum of the percentages of the components is 100%;

The preparation method of the above nasal composition containing recombinant human ciliary neurotrophic factor is as follows:

The protein infiltration auxiliary material and the appropriate amount of water are prepared into a nanosuspension by homogenization, spray drying, ion gel method, etc., and then uniformly mixed with rhCNTF and other protein permeation auxiliary materials, and added with a buffer of pH=5~9. a liquid component, an inorganic salt or the like to obtain the nasal administration preparation of the recombinant human ciliary neurotrophic factor;

Or:

The intranasal administration preparation of the recombinant human ciliary neurotrophic factor can be obtained by dissolving and mixing the protein permeation auxiliary material and rhCNTF in an appropriate amount of buffer, and then uniformly mixing with other components.

The nasal administration preparation containing the recombinant human ciliary neurotrophic factor can be administered through the nasal cavity, and the dose of rhCNTF is generally 0.5-lug/kg/day, which can be determined by the physician according to the patient's condition and age; The above nasal administration composition may be administered by nasal or nasal spray in the form of a solution, or may be administered by nebulizing or reconstituting the nasal cavity powder after nasal administration or nasal spray.

The invention has the advantages that: the developed recombinant human ciliary neurotrophic factor nasal administration composition has the same weight control effect as the injection, and is not easy to produce antibodies, and can be used for weight loss, and the effect is obvious.

DRAWINGS

Figure 1 shows the weight gain curve of mouse nasal instillation and intraperitoneal injection of CNTF. Wherein represents intranasal instillation and ip represents intraperitoneal administration.

Figure 2 is a graph showing the changes in body weight of nasal hypertrophy and subcutaneous injection of CNTF in a rat model of high nutritional obesity. Wherein i. n represents intranasal instillation and s. c represents subcutaneous injection.

Fig. 3 is a graph showing the weight gain curve of mouse nasal instillation and intraperitoneal injection of CNTF in Example 10. Wherein i. n represents intranasal instillation, and i. P represents intraperitoneal administration.

detailed description

Example 1

Recombinant human ciliary neurotrophic factor 0. 02%

Octa-arginine 10. 0 %

Phosphate buffer component (pH 6. 0) 0. 5 %

Sodium chloride 0. 5%

Water 88. 98%

Formulation of an aqueous solution of phosphate buffered salt according to the above prescription, recombinant human ciliary neurotrophic factor, octameric ammonia The acid is dissolved in an appropriate amount of buffered saline solution, mixed, and the other components are dissolved in the buffered saline solution, and uniformly mixed.

Example 2

Recombinant human ciliary neurotrophic factor 0. 02%

Protamine 3. 0 %

Polosham 188 0. 2 %

Phosphate buffer component (PH6) 0. 3 %

Sodium chloride 0. 5%

Water 95. 98% and remove the ethanol, and then uniformly mixed with the recombinant human ciliary neurotrophic factor, poloxamer 188 in the above ratio, and added to the buffer and other components.

Example 3

Recombinant human ciliary neurotrophic factor 0. 025%

Chitosan 2. 0 %

Sodium polyphosphate 0. 8%

Polyoxyethylene lauryl ether 3. 0 %

Acetate-sodium acetate buffer component (PH5) 0. 5 %

Sodium chloride 0. 5%

Water 93· 175%

Preparation method: adding sodium polyphosphate to chitosan solution to prepare nano suspension by rapid stirring, and uniformly mixing with recombinant human ciliary neurotrophic factor and polyoxyethylene lauryl ether according to the above ratio, adding appropriate buffer salt And other components, that is.

Example 4

Recombinant human ciliary neurotrophic factor 0.1%

Diacetyl phosphatidylcholine 6. 0 %

Polosham 407 1. 0 %

The citric acid-sodium citrate buffer component (pH 8.5) 0. 9 %

Potassium chloride 0. 3%

Carbomer 934 0. 5% Water 91· 2%

Preparation method: diacetyl phosphatidylcholine, poloxamer and appropriate amount of water are homogeneously emulsified into a nano suspension, and then uniformly mixed with the recombinant human ciliary neurotrophic factor according to the above ratio, adding carbomer solution, Buffer and other components, that is.

Example 5

Recombinant human ciliary neurotrophic factor 0. 02 %

PLGA 5. 0 %

Aminopeptidase inhibitor 4. 0 %

Tris-HCl buffer component (pH 7.5) 0. 1 %

Glycerin 0. 3 %

Sodium carboxymethyl cellulose 0. 5%

Water 90. 08%

Preparation method: PLGA is prepared into a nanosuspension by double emulsion solvent evaporation method, and then uniformly mixed with recombinant human ciliary neurotrophic factor, aminopeptidase inhibitor and glycerin according to the above ratio, and appropriate buffer salts and other components are added, ie Got it.

Example 6

Clean-grade Kunming mice weighing 14 to 18 grams were selected and weighed and randomized into groups of 6 animals each. A blank control group was given. 20 μl of normal saline was administered twice daily in the nasal cavity. In the intraperitoneal injection group, CNTF solution was intraperitoneally injected twice a day for 10 days. The experimental group was given nasal cavity of Examples 1, 2, 3 and 4 respectively. The composition was administered (in-l, in-2, in-3, in-4). The doses in the drug-administered group were 2 ug CNTF/day/day, and the test animals of each group were weighed once a day before administration. The results are shown in Figure 1.

The CNTF pharmaceutical compositions of Examples 2 and 4 were administered nasally, and the body weight gain rate of the mice was substantially the same as that of the intraperitoneal injection group, which was much lower than that of the blank control group. The CNTF pharmaceutical composition of Example 1 was administered nasally, and the body weight growth rate of the mice was significantly lower than that of the intraperitoneal injection group, which was much lower than that of the blank control group. The CNTF pharmaceutical composition of Example 3 was administered nasally, and the growth rate of the mice was higher than that of the intraperitoneal injection group, which was lower than that of the blank control group.

Example 7 Rat model of high-nutrition obesity: 10 out of 90 weaning 1-3 day rats were randomly selected as normal control group, fed with common vocabulary, and other rats were fed with high-nutrition vocabulary to establish diet induction. Type of obesity (DI0) model. Starting from the day of feeding high-nutrition vocabulary, weighed weekly, eliminated rats with slow weight gain, and included those who were qualified. In the DI0 group, the body weight of the rats was 20% higher than that of the normal control group, and the model was successfully established for 5 weeks. At the end of the 5th week, from Eight of the normal rats were selected as normal controls, and 40 obese rats were selected from the successful model rats, which were divided into model control group, positive control group (subcutaneous injection) and rhCNTF nasal administration test group. 8 in each group. Body weights were weighed before administration, one week, two weeks, three weeks, and four weeks after administration. The results are shown in Fig. 2.

The CNTF pharmaceutical compositions of Examples 2 and 4 were administered nasally. The weight gain rate of the rats was slightly higher than that of the subcutaneous injection group in the first two weeks, and the subcutaneous injection group was lower in the third week. The CNTF pharmaceutical composition of Example 5 was administered nasally, and the body weight for three weeks was higher than that of the subcutaneous injection group, which was lower than that of the model group. The CNTF pharmaceutical composition was administered intranasally and by injection, and the weight gain was alleviated as compared with the model group.

Grouping and administration:

Group 1: Normal control group, fed with common vocabulary, 8 rats

Group 2: Model control group, fed with high nutrition materials, 8 rats

Group 3: Positive control group, rhCNTF 50 μg/kg, sc, q. d, fed with high nutrition, 8 rats Group 4: in-1 group, rhCNTF 50 μg/kg, examples 2, in, bid, fed with high nutrition, 8 rats

Group 5: in- 2 group, rhCNTF 50 μg/kg, in, bid, Example 4, fed with high nutritional nutrition, 8 rats Group 6 in-3 group: rhCNTF 50 μg/kg i. n, qd, Example 5, fed a high nutritional word, 8 rats.

Example 8

Recombinant human ciliary neurotrophic factor 0. 05 %

Hydroxypropyl-β-cyclodextrin 8. 0 %

Tris-HCl buffer component (pH 8. 0 ) 0. 6 %

Knotted gelatin 0. 3 %

Water 91· 05 %

The aqueous solution of Tris-HCl buffer salt is prepared according to the above prescription, and the recombinant human ciliary neurotrophic factor, hydroxypropyl-β-cyclodextrin and gellan gum are separately dissolved in an appropriate amount of buffered saline solution, and uniformly mixed.

Example 9

Recombinant human ciliary neurotrophic factor 0. 01 %

Sodium dihydromomellate 0. 5 %

Acetic acid-sodium acetate buffer component (ρΗ5. 5 ) 0. 3 %

Glucose 3. 5 %

Benzalkonium chloride 0. 005 % Water 95· 685 %

According to the above prescription, an aqueous solution of acetic acid-sodium acetate buffer salt is prepared, and the recombinant human ciliary neurotrophic factor, sodium dihydrogen hyaluronate and glucose, and benzalkonium chloride are respectively dissolved in an appropriate amount of buffered saline solution, uniformly mixed, and freeze-dried, that is, Got it. Reconstitute with water before use.

Example 10

Clean-grade Kunming mice weighing 14 to 18 grams were selected and weighed and randomized into groups of 5 animals each. A blank control group was given. 20 μl of normal saline (i. n-normal saline) was administered twice daily in the nasal cavity. The intraperitoneal injection group (i. p) was intraperitoneally injected with rhCNTF solution twice a day for 10 days. Nasal administration compositions (i. nl, i. n-2) of Examples 8, 9 were administered nasally. The dose in the administration group was 2 g rhCNTF/day/day, and the test animals of each group were weighed once a day before the administration on the first day of administration. The results are shown in Figure 3.

The CNTF pharmaceutical compositions of Examples 8 and 9 were administered nasally, and the growth rate of the mice was lower than that of the blank control group. In the nasal cavity, the CNTF pharmaceutical composition of Example 8 was administered, and the body weight growth rate of the mice was significantly lower than that of the intraperitoneal injection group, which was much lower than the blank control group. The CNTF pharmaceutical composition of Example 9 was administered nasally, and the body weight growth rate of the mice was higher than that of the intraperitoneal injection group, which was significantly lower than that of the blank control group.

Example 11

ICR mice weighing about 20 grams were selected and weighed and randomized into groups of 6 animals each. A blank control group was given. 20 μl of normal saline was administered twice daily in the nasal cavity; intraperitoneal injection group (ip), intraperitoneal injection of rhCNTF solution once a day; nasal administration group (in), nasal administration of Example 2 twice daily. 4, 8 rhCNTF nasal administration composition. The dose in the administration group was 2 g rhCNTF/day/day, and the drug was stopped 20 days after administration. Blood samples were collected from the venous plexus of the eyes of the test animals on the 14th and 20th day after the administration and the 12th day after the drug was stopped. The serum rhCNTF antibody IgG was measured. The cutoff value ^ ). 055 was determined to be positive by more than 0.1. The results are shown in Schedule 1.

The results showed that: 12 days after discontinuation of the drug, 5 or 6 out of 6 mice in the i.p. group had antibody production, and the antibody positive rate was almost 100%. The rhCNTF compositions of Examples 2, 4, and 8 were administered to the nasal administration (i. n) group, and 1, 2, and 0 of the 6 mice respectively produced antibodies, and the positive rate was low. No antibody was found in the saline nasal drops. Table 1. Table of antibody positives after administration of rhCNTF in ICR mice:

Ip 1 group 2/6 3/6 5/6

2 groups 3/6 6/6 6/6 in-Example 2 3 groups 0/6 0/6 1/6 in-Example 4 4 groups 1/6 2/6 2/6 in-Example 8 Group 5 0/6 0/6 0/6 in-salt 6 groups 0/6 0/6 0/6 Note: 2/6 indicates that 2 antibodies were positive in 6 mice.

Claims

Claim

A nasal administration composition for recombinant human ciliary neurotrophic factor, characterized by comprising 0 to 1% by weight of recombinant human ciliary neurotrophic factor

Protein penetration aid 0~20%

Buffer component (pH5~9) 0-5%

Inorganic salt 0~5%

Water 0-99%

The sum of the percentages of the components is 100%.

2. A nasal administration composition for recombinant human ciliary neurotrophic factor, characterized by comprising, by weight percent, recombinant human ciliary neurotrophic factor 0.01 0.1%

Protein penetration aid 1~10%

Buffer component with pH=5~9 0~2%

Inorganic salt 0~2%

Water 86~97%

The sum of the percentages of the components is 100%.

The nasal administration composition of recombinant human ciliary neurotrophic factor according to claim 1, wherein the recombinant human ciliary neurotrophic factor (hCNTF) has a molecular weight of 20,000 to 24,000 Da.

The nasal administration composition of recombinant human ciliary neurotrophic factor according to claim 2, wherein the recombinant human ciliary neurotrophic factor (hCNTF) has a molecular weight of 20,000 to 24,000 Da.

The nasal administration composition of recombinant human ciliary neurotrophic factor according to claim 3 or 4, wherein the recombinant human ciliary neurotrophic factor is a protein sequence completely identical to native human CNTF, Or genetically engineered human CNTF mutants, including the change of 17-cysteine to alanine, 63-glutamic acid to arginine, and C-terminal deletion of 14 to 16 amino acids. body.

The nasal administration composition for recombinant human ciliary neurotrophic factor according to claim 3 or 4, wherein the protein permeation auxiliary material is selected from the group consisting of chitosan, sodium polyphosphate, and protamine , cyclodextrin or its derivatives, cholate and its derivatives, aminopeptidase inhibitors, polyoxyethylene hydrogenated castor oil, polyoxyethylene lauryl ether, Polyethylene glycol stearate, polyoxyethylene and polyoxypropylene copolymer, polysorbate, poloxamer, fusidic acid derivative, PLGA, phospholipid and its derivatives or basic amino acid polymer More than one;

among them:

The phospholipid and its derivative are selected from the group consisting of lecithin, diacetyl phosphatidylcholine, lysophosphatidylglycerol, dipalmitoylphosphatidylcholine or dimyristoylphosphatidylglycerol;

The inorganic salt is sodium chloride or potassium chloride.

The nasal administration composition for recombinant human ciliary neurotrophic factor according to claim 6, which further comprises one or more of a preservative or/and an adhesive material.

The nasal administration composition for recombinant human ciliary neurotrophic factor according to claim 7, characterized in that, by weight percentage, contains:

Recombinant human ciliary neurotrophic factor 0.01-0.1%

Protein penetration aid 3.0-10.0%

Buffer component with pH=5~9 0.1-1.0%

Inorganic salt 0-0.6%

Preservative or / and bioadhesive material 0.0 ~ 5.0%

Water 83-96%

The sum of the percentages of the components is 100%.

The method for preparing a nasal composition comprising a recombinant human ciliary neurotrophic factor according to any one of claims 1 to 8, comprising the steps of:

The protein infiltration auxiliary material and the appropriate amount of water are prepared into a nanosuspension by homogenization, spray drying, ion gel method, etc., and then uniformly mixed with rhCNTF and other protein permeation auxiliary materials, and added with a buffer of pH=5~9. Liquid a nasal administration preparation of the recombinant human ciliary neurotrophic factor can be obtained by using a component, an inorganic salt or the like; or:

The amount of the rhCNTF is generally 0.5-lug/kg/, and the dose of the rhCNTF is generally 0.5. day.