CN107073077A

CN107073077A - CNTF nasal drug delivery system and its preparation method and application

Info

Publication number: CN107073077A
Application number: CN201480083218.6A
Authority: CN
Inventors: 解荷芝; 范蔷薇
Original assignee: Shaanxi Micot Technology Ltd
Current assignee: Shaanxi Micot Technology Ltd
Priority date: 2014-11-07
Filing date: 2014-11-07
Publication date: 2017-08-18
Anticipated expiration: 2034-11-07
Also published as: CN107073077B; WO2016070419A1

Abstract

A kind of fat CNTF nasal cavity administrated preparations for the treatment of for adding sorbefacient, improve the bioavilability of biopharmaceutical macromolecular drug nose administration, high molecular weight protein drug delivery can be entered blood, reach and exceed the effective blood drug concentration of injection, there is significant loss of weight to act on normal mouse, fat (DIO) mouse of diet induced, DIO rat body weights, better than subcutaneous administration mode and listing positive drug, and in dose dependent.Pharmacodynamic result shows that loss of weight stability is substantially better than subcutaneous administration mode, and antibody yield is substantially reduced, and does not cause weight gain and apositia to be reacted.The more common intranasal formulation of said preparation greatly improves bioavilability, and preparation technology is simple, stable and controllable for quality, is adapted to industrialized production.

Description

Ciliary neurotrophic factor nasal administration system and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines, and particularly relates to a ciliary neurotrophic factor (CNTF) nasal delivery system, and a preparation method and application thereof.

Background

The incidence of obesity is on the rise worldwide. Obese patients in the world currently have at least 12 hundred million, and the proportion of Chinese obese people in 2011 is about 33.4%. Obesity can increase the incidence of cardiovascular and cerebrovascular diseases, diabetes, fatty liver, tumor, endocrine, etc.

The medicine is one of main measures for reducing weight and treating obesity, and the common weight-reducing medicine sibutramine has been suspended or withdrawn from the market in 68 countries in the world in 2011 because of having serious cardiovascular risk; fenfluramine has been withdrawn from the market in 1997 for its potential to cause heart valve damage; orlistat (the only OTC antiobesity drug at present) can cause pancreatitis, oxalate nephropathy.

In the early 90 s, ciliary neurotrophic factor (CNTF) was used in experimental studies for the treatment of Amyotrophic Lateral Sclerosis (ALS). The study conducted clinical studies with a group of ALS patients, and soon after the study began, revealed a significant reduction in patient weight, with the results showing a significant reduction in weight in the drug group compared to the placebo group, and this result continued throughout the study. The company Regeneron then decided to conduct an experimental study of the weight loss of CNTF. The results show that the CNTF can be combined with a specific receptor of a hypothalamus in vivo, activate a key signal channel for suppressing appetite, suppress eating desire and prevent the body from generating hunger sensation, thereby reducing the body weight. Because CNTF is a polypeptide drug, it needs to be administered by injection. Therefore, Regeneron pharmaceutical company made it into injection to study the effect of losing weight and go to phase iii clinical trial.

Compared with the defects that the conventional weight-losing medicine has large side effect, high price, easy rebound and the like, the clinical research result of the CNTF shows that: the CNTF has good weight-reducing effect, is not easy to rebound, and is not easy to cause anorexia.

However, the CNTF injection has the defects of clinical application, such as the inability to penetrate the blood brain barrier and certain immunogenicity, and about 70% of patients generate antibodies in vivo after subcutaneous injection for a period of time, and many research units improve the defects at present, for example, CN200710198546.2(CN101185762A) discloses a method of ciliary neurotrophic factor modified by polyethylene glycol polymer, which adopts hydrophilic polyethylene glycol to covalently bind with CNTF to prolong the half-life of the CNTF in vivo, increase the stability of protein and reduce immunogenicity, but no research report of penetrating the blood brain barrier is found; CN201010552864.6(CN102462662A) discloses a method for encapsulating recombinant human ciliary neurotrophic factor by liposome, and after the liposome-encapsulated recombinant human CNTF is intravenously injected to a mouse, the amount of medicine reaching the brain of the mouse is obviously increased. However, the preparation process of the method is complex and the cost is high.

In order to enable the CNTF to be more convenient to use, higher in bioavailability, less in antibody generation, lower in cost and more suitable for industrial production, the inventor conducts a large amount of experimental researches to finally obtain the invention.

Disclosure of Invention

The invention aims to provide a novel nasal delivery system of CNTF (natural and mutant).

The invention provides a CNTF nasal administration preparation added with an absorption enhancer and used for treating obesity.

The nasal administration preparation is a nasal spray, a spray or a nasal drop.

The CNTF nasal administration preparation contains CNTF, an absorption enhancer, a stabilizer and a buffer system.

Specifically, the CNTF nasal administration preparation contains the following components: 0.01-10 parts of CNTF, 5-30 parts of absorption accelerator, 0.01-0.4 part of stabilizer and 100 parts of buffer system by volume.

Preferably, the CNTF nasal administration formulation contains the following ingredients: 0.01-5 parts of CNTF, 10-30 parts of absorption accelerator, 0.01-0.2 part of stabilizer and 100 parts of buffer system by volume.

Further preferably, the CNTF nasal administration preparation contains the following components: 0.01-1 part of CNTF, 10-28 parts of absorption accelerator, 0.01-0.1 part of stabilizer and 100 parts of buffer system by volume.

Still more preferably, the CNTF nasal delivery formulation contains the following ingredients: 0.015-0.030 part of CNTF, 14-25 parts of absorption accelerator, 0.05-0.1 part of stabilizer and 100 parts of buffer system by volume.

Most preferably, the CNTF formulation for nasal administration contains the following ingredients: 0.025 parts of CNTF, 20.41 parts of absorption accelerator, 0.05 parts of stabilizer and 100 parts of buffer system by volume.

In the above administration formulation:

the CNTF (ciliary neurotrophic factor) is a natural ciliary neurotrophic factor, preferably the CNTF is a natural CNTF mutant or an amino acid sequence which has at least 70% homology with natural CNTF, and further preferably the 17 th cysteine of the natural CNTF is changed into serine, and 16 amino acid sequences at the C end are removed.

Natural CNTF can be purchased commercially, CNTF mutants are provided by the beijing chinese drug biologicals assay, and methods for making CNTF mutants are also known in the art, for example: CN1750205A discloses the preparation of various mutants of CNTF.

The absorption enhancer is selected from one or more of chitosan, sodium polyphosphate, hydroxypropyl-beta-cyclodextrin, aminopeptidase inhibitors, polysorbate 20, polysorbate 60, polysorbate 80, polyoxyethylene castor oil, polyethylene glycol stearate 15(Solutol HS15), tocopherol polyethylene glycol succinate (TBGS), poloxamer F68, sodium cholate, sodium deoxycholate or phospholipid, and is preferably hydroxypropyl-beta-cyclodextrin or polyethylene glycol stearate 15.

The stabilizer is one or more selected from mannitol, lactose, sucrose, trehalose, glycine, arginine, aspartic acid, glutaminyl or albumin.

The buffer system is selected from a PBS (phosphate buffer system), an acetic acid-sodium acetate buffer system or citric acid-sodium citrate. The pH value is 5 to 9, preferably 6.5 to 7.5, and more preferably 7.2 to 7.4.

The parts by volume and parts by weight are international units, for example: parts by volume are ml, parts by weight are g, parts by volume are L, parts by weight are kg.

The invention also provides a preparation method of the nasal administration preparation, which comprises the following steps:

1) the buffer system configuration method comprises the following steps: judging the proportion relation of two substances in different types of buffer systems according to the pH value, dissolving the two substances in water respectively to prepare solution A and solution B, and adjusting the pH value of the solution B to 5-9 by using the solution A for later use;

2) processing of CNTF: taking the CNTF stock solution, carrying out ultrafiltration and centrifugation, collecting supernatant, then adding distilled water, carrying out centrifugation again, collecting supernatant, concentrating, and carrying out freeze drying for later use;

3) the preparation method of the nasal drop comprises the following steps: weighing each component according to the formula amount, dissolving the absorption enhancer and the stabilizer in the buffer solution, placing the buffer solution in a bottle of the transnasal administration device, placing the CNTF in a storage rack of the transnasal administration device, and covering a bottle cover.

The invention also provides application of the nasal cavity administration preparation in preparing a weight-losing medicine.

The weight loss is used for treating common obesity and diabetes type obesity, wherein the common obesity is constitutional obesity or nutritional obesity.

The CNTF nasal administration preparation provided by the invention has the following advantages:

1. the inventor considers that the CNTF is prepared into a nasal administration preparation by adding an absorption enhancer, and the absorbent with strong absorption enhancing capability is obtained through a large number of tests and screens, and the optimal formula is also obtained. Because the olfactory epithelium is the only tissue in which the central nervous system is in direct contact with the outside world, the axons of the olfactory sensory neurons, covered by the nasal cilia, form bundles that can pass through the lamina cribosa into the cranial cavity and form synaptic links with the mitral and plexus cells of the olfactory bulb in the brain, which is the olfactory mucosal epithelial pathway through which drugs are taken from the nasal cavity into the brain. After nasal administration, drug molecules are retained on olfactory patch mucosa and are easy to absorb into cerebrospinal fluid, so that the drug molecules are easy to bypass blood brain barrier and enter the central nervous system.

The research on the nasal mucosa administration system at present discovers that the preparation can stimulate the nasal mucosa, mainly has cilia toxicity effect, and comprises the effect of medicaments, accessory agents, penetration enhancers and preservatives on cilia activity, so that the nasal mucosa administration preparation has certain limitation in use; in addition, different types of absorption enhancers have different effects of promoting absorption.

The invention screens and obtains the accelerant with the optimal CNTF absorption effect and the optimal formula, provides a new drug delivery mode and overcomes the defects of the original drug delivery.

2. The nasal administration preparation provided by the invention improves the bioavailability of the biological macromolecular drug administered through the nose, can deliver the macromolecular protein drug into blood to reach and exceed the effective blood concentration of an injection, has obvious weight loss effect on the body weight of a normal mouse, a Diet Induced Obesity (DIO) mouse and a DIO rat, is superior to a subcutaneous administration mode and a positive drug on the market, and is dose-dependent. Pharmacodynamic results show that the weight loss stability is obviously superior to that of a subcutaneous administration mode, the antibody production is obviously reduced, and weight rebound and anorexia reaction are not caused. Compared with the common nasal drop preparation, the preparation provided by the invention greatly improves the bioavailability, has simple preparation process and stable and controllable quality, and is suitable for industrial production.

Description of the drawings:

FIG. 1: the influence of CNTF nasal drop on the body weight of DIO rats, wherein Model is a Model group, CNTF-SC-0.2mg/kg is a CNTF subcutaneous administration group, Fenfluramine-ig-5.6mg/kg is a positive control group (the positive drug is Fenfluramine), CNTF-1-IN-3mg/kg is a nasal drop administration group of example 1, CNTF-2-IN-3mg/kg is a nasal drop administration group of example 2, CNTF-3-IN-3mg/kg is a nasal drop administration group of example 3, CNTF-4-IN-3mg/kg is a nasal drop administration group of example 4, and CNTF-5-IN-3mg/kg is a nasal drop administration group of example 5;

FIG. 2: the effect of CNTF nasal drop on the Lee's index of DIO rats, wherein Model is a Model group, CNTF-SC-0.2mg/kg is a CNTF subcutaneous administration group, Fenfluramine-ig-5.6mg/kg is a positive control group, Fenfluramine is administered, CNTF-1-IN-3mg/kg is a nasal drop administration group of example 1, CNTF-2-IN-3mg/kg is a nasal drop administration group of example 2, CNTF-3-IN-3mg/kg is a nasal drop administration group of example 3, CNTF-4-IN-3mg/kg is a nasal drop administration group of example 4, and CNTF-5-IN-3mg/kg is a nasal drop administration group of example 5;

FIG. 3: the influence of the CNTF nasal drop on the weight change rate of normal mice is shown IN the specification, wherein Model is a Model group, Fenfluramine is a positive control group, CNTF-1-IN-20mg/kg is a nasal drop administration group IN example 1, CNTF-3-IN-20mg/kg is a nasal drop administration group IN example 3, and CNTF-5-IN-20mg/kg is a nasal drop administration group IN example 5;

FIG. 4: the effect of CNTF nasal drop on the conditioned anorexia of mice, wherein CNTF-IN-7.5mg/kg is the nasal drop small dose group of example 5, CNTF-IN-15mg/kg is the nasal drop medium dose group of example 5, and CNTF-IN-30mg/kg is the nasal drop large dose group of example 5.

FIG. 5: plasma concentration-time profiles for nasal administration of 5mg/kg, 2mg/kg and 0.8mg/kg of CNTF. group1 was the nasal 5mg/kg dose group of example 5, group2 was the nasal 2mg/kg dose group of example 5, group3 was the nasal 0.8mg/kg dose group of example 5, and blank group was the nasal drop group of CNTF in PBS buffer.

FIG. 6: CNTF was administered subcutaneously as a 100ug/kg blood concentration-time profile.

the specific embodiment is as follows:

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

Example 1: CNTF nose drops

1. Consists of the following components: 0.015g of CNTF, 25g of hydroxypropyl-beta-cyclodextrin, 0.1g of mannitol and 100ml of PBS buffer (pH 7.4).

2. The preparation method comprises the following steps:

1) the preparation method of the PBS buffer solution comprises the following steps: 2.457g of sodium dihydrogen phosphate is weighed and dissolved in 50ml of water to prepare 0.315M solution A; 5.6405g of disodium hydrogen phosphate is weighed and dissolved in 20ml of water to obtain solution B; adjusting the pH value of the solution B to 7.4 by using the solution A; the volume of the solution B is 50 ml. When in use, 10ml of the mixture is taken and added with 0.45g of sodium chloride to be 50ml, and the concentration is 0.063M for standby.

2) Processing of CNTF: taking 32ml of CNTF stock solution (provided by Beijing China pharmaceutical and biological product assay, the same below) and placing in a liquid storage tube of an ultrafiltration centrifugal device, centrifuging for 30min at 6500r at 4 ℃ for three times; adding 10ml of distilled water into the filtrate, centrifuging for 30min, and collecting the concentrated solution. And (3) filling the CNTF concentrated solution into a penicillin bottle, and freeze-drying for later use.

3) The preparation method of the nasal drop comprises the following steps: dissolving the hydroxypropyl-beta-cyclodextrin and the mannitol with the formula ratio in the PBS buffer solution, placing the solution in a bottle body of a transnasal administration device, then placing the CNTF in a storage rack of the transnasal administration device, and covering a bottle cap for later use.

Example 2: CNTF nose drops

1. Consists of the following components: CNTF0.015g, polysorbate 2010g, lactose 0.1g and PBS buffer (pH7.4)100 ml.

2. The preparation process comprises the following steps:

PBS buffer and CNTF treatment methods are the same as example 1;

dissolving polysorbate and lactose with formula amount in PBS buffer solution, placing in a bottle body of a transnasal administration device, then placing CNTF in a storage rack of the transnasal administration device, and covering a bottle cap for later use.

Example 3: CNTF nose drops

1. Consists of the following components: 0.030g of CNTF, 1514.29g of polyethylene glycol stearate, 0.1g of lactose and 100ml of PBS buffer (pH7.4).

2. The preparation process comprises the following steps:

PBS buffer and CNTF treatment methods are the same as example 1;

dissolving polyethylene glycol stearate 15 and lactose according to the formula amount in PBS buffer solution, placing the solution in a bottle body of a transnasal administration device, then placing CNTF in a storage rack of the transnasal administration device, and covering a bottle cap for later use.

Example 4: CNTF nose drops

1. Consists of the following components: 0.020g of CNTF, 1525g of polyethylene glycol stearate, 0.05g of lactose and 100ml of PBS buffer (pH7.4).

2. The preparation process comprises the following steps: the same as in example 3.

Example 5: CNTF nose drops

1. Consists of the following components: 0.025g of CNTF, 1520.41g of polyethylene glycol stearate, 0.05g of lactose and 100ml of PBS buffer (pH7.2).

Experimental example 1: weight-losing effect of CNTF nasal drops on obese DIO rats

1. Experimental animals: healthy and adult SPF SD rats, male, with the body mass of 200g-250g, purchased from the Western's university of transportation medical laboratory animal center, and having the animal production permit number SCXK (Shaan) 2012-003 and the animal qualification number 0014258. And (5) placing the chicken in an SPF animal house for breeding.

2. Grouping experiments:

SD rats 100 were fed with DIO feed for 1 month to become fat rats. Weighing body weight every week from 1 day before feeding high nutrition feed, eliminating rats with slow weight increase, and taking qualified body weight into the experiment. According to the criteria of obesity and overweight in humans. Obesity greater than 10% was considered overweight, greater than 20% was considered obese and the obesity model was considered successful. Control mice were fed with conventional feed for 4 weeks.

The obesity calculation formula is as follows: obesity (%) - (actual body weight of test group-average body weight of control group)/average body weight of control group x 100.

The obese rats which were successfully molded were 64 animals divided into 8 groups, each group was 8 animals, specifically, a model group, a CNTF subcutaneous injection group, a positive control group, and the nasal drip administration groups of examples 1, 2, 3, 4, and 5. Wherein:

the model group is dripped into a common auxiliary material (20 ul of PBS buffer solution, and the specific preparation method is shown in example 1);

CNTF subcutaneous administration group (CNTF-SC-0.2mg/kg, CNTF freeze-dried powder is directly dissolved in normal saline to prepare 0.2mg/mL solution);

a positive control group (positive drug Fenfluramine, Fenfluramine-ig-5.6 mg/kg);

example 1 nasal drop administration group (CNTF-1-IN-3 mg/kg);

example 2 nasal drop administration group (CNTF-2-IN-3 mg/kg);

example 3 nasal drop administration group (CNTF-3-IN-3 mg/kg);

example 4 nasal drop administration group (CNTF-4-IN-3 mg/kg);

example 5 nasal drop administration group (CNTF-5-IN-3 mg/kg).

3. The experimental method comprises the following steps:

the high fat diet was continued for each group except the model group for the regular diet.

Each group of rats was dosed once a day for 28 consecutive days, with daily regular recording of the body weight and the food intake for 24 h. On the last day, the rats are fasted for 12h and administered for the last time for 1h, blood is collected through a carotid artery cannula after anesthesia, the blood sample is placed for 2h at room temperature, centrifuged for 15min at 3000r/min, and serum is taken for freezing storage; fat around the testis and kidney on both sides is taken, the wet weight is weighed, and the fat index is calculated.

4. Results

4.1 weight change: see FIG. 1

As can be seen from fig. 1:

compared with the model group, the rats in the CNTF subcutaneous administration group have significant difference (P <0.05) from the 4 th day after the administration, and the rats in the example 5 nasal drop administration group, the example 4 nasal drop administration group, the example 3 nasal drop administration group, the example 2 nasal drop administration group, the example 1 nasal drop administration group and the positive control group have significant difference (P <0.05) from the 5 th, 8 th, 12 th, 15 th, 14 th and 9 th days respectively, which shows that the rats in the examples 1 to 5 nasal drop administration groups, the CNTF subcutaneous administration group and the positive control group have the effect of reducing the body weight of the DIO rats, the CNTF subcutaneous injection has the fastest effect, and the CNTF nasal drop has the lower effect speed than the CNTF subcutaneous injection. In the groups of the examples 4 and 5, the weight growth rate of the drug is superior to that of the positive control group, and in the group of the example 3, the weight growth rate of the drug is basically consistent with that of the positive control group and is far lower than that of the model group; the weight growth rate of the nasal drop administration groups of examples 1 and 2 was higher than that of the positive drug, but lower than that of the model group.

4.2 Lee's index: see FIG. 2

As can be seen from fig. 2: administration of each group had the effect of reducing the leo's index in DIO rats compared to the model group (P < 0.05).

4.3 Change in food intake: see Table 1

Table 1: effect of CNTF administration on the Rate of Change in food intake in DIO rats (X%)

As can be seen from Table 1, after administration, the food intake of rats in each group was decreased as compared with the model group, the decrease effect of CNTF subcutaneous injection group was most significant, the food intake of subcutaneous injection group reached the lowest value on day 8 after administration, and then the food intake began to rise, the food intake was equivalent to the model group on day 25 after administration, and nasal drip groups and positive control group were lower than the model group during administration and were relatively stable.

5. And (4) conclusion: the CNTF nasal drop has obvious weight loss effect and excellent stability compared with a subcutaneous injection group; the weight reducing effect is better than that of positive drug (fenfluramine), and weight rebound can not be caused after drug withdrawal.

Experimental example 2: CNTF nose drops for losing weight of normal mice

1. Experimental animals: adult and healthy ICR mice with male sex and weight of 18-22 g are selected and provided by an animal center of the medical college of Western-Ann university of transportation, an animal production license number SCXK (Shaan) 2012-003 and an animal qualification number 0014398, and are adapted to experiments in an experiment center after one week.

2. Grouping experiments: 60 mice, randomly divided into 6 groups of 10 mice each. Specifically, the test results showed that the test results were a normal control group, a positive control group, a CNTF subcutaneous administration group, and example 1, 3, and 5 nasal drops administration groups, wherein:

the normal control group was administered with an adjuvant solution (20 ul PBS buffer, see example 1 for specific formulation);

a positive control group (Fenfluramine-ig-8 mg/kg for Fenfluramine);

CNTF subcutaneous administration group (CNTF-SC-0.5 mg/kg);

example 1 nasal drop administration group (CNTF-1-IN-20 mg/kg);

example 3 nasal drop administration group (CNTF-3-IN-20 mg/kg);

example 5 nasal drop administration group (CNTF-5-IN-20 mg/kg).

3. And (3) experimental operation:

each group of mice was administered once a day (administration was as above) for 28 consecutive days, and the body weight and the food intake were recorded at regular intervals every day for 24 hours.

On the last day, after fasting for 12h and the last administration for 1h, collecting blood by eyeball, standing the blood sample at room temperature for 2h, centrifuging at 3000r/min for 15min, and taking serum to measure cholesterol, triglyceride and other indexes by using a kit; taking fat around testis and kidney at two sides, weighing wet weight, and calculating fat index; serum antibody levels were measured.

4. Results of the experiment

4.1 mouse weight change rate results: as can be seen in figure 3 of the drawings,

as can be seen from FIG. 3, compared with the adjuvant group, the CNTF nasal drop administration groups, the positive control group and the CNTF subcutaneous administration group all can reduce the weight gain rate of mice, the effect of the nasal drop administration groups of examples 3 and 5 is obvious, and the weight gain rate of the nasal drop administration group of example 1 is slightly lower than that of the positive control group.

4.2 effects on mouse perirenal and peritesticular fat weight: see table 2.

TABLE 2 Effect of CNTF on mouse perirenal and peritesticular fat weight

As can be seen from table 2: compared with a normal control group, the weight of fat around the kidney and the weight of fat around the testis of each group subjected to the nasal administration of the CNTF and the positive control group have statistical difference (P <0.05), which indicates that the CNTF nasal drop and the positive fenfluramine can reduce the fat content of mice; compared with the CNTF subcutaneous administration group, the weight of perirenal fat and the weight of peritesticular fat of the nasal drip administration group and the positive control group in example 5 have statistical difference (P <0.05), which shows that the fat content of mice is reduced better than that of the CNTF subcutaneous administration group in the nasal drip administration group and the positive control group in example 5. The fat content of the CNTF subcutaneous group was not statistically different from that of the adjuvant group.

Compared with the adjuvant group, after one month of continuous administration, the positive control group, the CNTF subcutaneous administration group and the nasal drip administration group have no significant influence on the blood sugar, cholesterol and triglyceride levels of the mice.

Antibody detection results: the amount of the antibody generated by each group of the CNTF nasal drop administration is obviously lower than that of the CNTF subcutaneous administration group, and the CNTF nasal drop administration group has good weight reduction effect at the dosage.

5. And (4) conclusion: the CNTF nasal drop has a weight-losing effect on normal mice, the weight-losing degree can reach and exceed the weight-losing effect of subcutaneous injection, the stability is good, the weight-losing effect is superior to that of a positive drug (fenfluramine), and weight rebound cannot be caused after drug withdrawal.

Experimental example 3: effect of CNTF nasal drops on body weight rebound in DIO mice

1. Experimental animals: adult and healthy C57 mice with male body weight of 18-22 g are selected, provided by the animal center of the medical college of Western-Ann university of transportation, and have the production license number SCXK (Shaan) 2012-003 and the animal qualification number 0014318. Feeding in SPF experimental animal room.

2. Grouping experiments: 50C 57 mice were fed DIO diet for 1 month to become obese. Weighing weekly starting 1 day before feeding high nutrition feed, eliminating mice with slow weight increase, and including qualified mice in the experiment (same standard as example 7)

30 obese mice successfully molded are divided into 5 groups, specifically, a model group, a positive control group, and the groups of small nasal drops, medium and large doses in example 5, and each group comprises 6 mice. Wherein:

the model group is dripped into the nasal cavity to be added with auxiliary materials (PBS buffer solution 20ul, the specific preparation method is shown in example 1);

a positive control group (Fenfluramine-ig-8 mg/kg);

example 5 nasal drop bolus (CNTF-5-IN-7.5 mg/kg);

example 5 nasal drop middle dose group (CNTF-5-IN-15 mg/kg);

example 5 nasal drops bolus (CNTF-5-IN-30 mg/kg).

3. And (3) experimental operation:

Each group of rats was dosed once a day for 14 consecutive days, and the body weight and the food intake of the rats were recorded regularly every day for 24 hours. The medication was stopped from day 15 and the body weight and food intake were continued for 14 days.

4. The results are shown in FIG. 4 and Table 3.

TABLE 3 weight changes

As can be seen from fig. 4 and table 3, at 14 days of administration, the nasal drops large dose group of example 5, the positive control group both had the effect of reducing the body weight of DIO mice (P < 0.05); after 14 days of withdrawal, the mice in the nasal drops bolus group of example 5 still had a significant difference (P <0.05) compared to the model group, but the positive control group had no difference.

5. And (4) conclusion: the Fenfluramine can cause weight rebound after stopping the medicine, and the CNTF nasal drops can not cause weight rebound after stopping the medicine.

Experimental example 4: effect of CNTF nasal drops on the conditioned anorexia response in mice

1. Experimental animals: adult, healthy and C57 mice with male weight of 18-22 g are selected, provided by the animal center of the medical college of Western-Ann university of transportation, and have the production license number SCXK (Shaan) 2012-003 and the animal qualification number 0014318. Feeding in SPF experimental animal room.

2. Grouping experiments: 30 mice were divided into 5 groups, specifically model group, positive control group, example 5CNTF nasal drops, medium and large dose groups, and 6 mice per group. Wherein:

a positive control group (lithium chloride, LiCl-i.p. -185 mg/kg);

example 5 nasal drop bolus group (CNTF-5-IN-10 mg/kg);

example 5 nasal drop middle dose group (CNTF-5-IN-20 mg/kg);

EXAMPLE 5 nasal drops bolus (CNTF-5-IN-40 mg/kg).

3. And (3) experimental operation: the 5 groups of mice were given 2 bottles of water (normal water) for 7 days, and then were trained on water deprivation, i.e. water deprivation was carried out for 23h and water supply lh was carried out for 3 days (times).

On day 4 (x) water deprivation for 23h, followed by 2 bottles of 0.1% saccharin water lh, followed immediately by dosing. After administration, free drinking water is recovered for 2 days (48h), then water is forbidden for 23h, 1 bottle of normal water and 1 bottle of saccharin water are given, the normal drinking water amount and the saccharin water amount of rats in lh are recorded, and the test of the conditioned anorexia response is carried out. The anorexia degree is expressed as the ratio of the normal drinking water amount to the total drinking water amount (sum of normal drinking water amount and saccharin drinking water amount) in the lh test period (drinking water selection ratio). In this experiment, 0.1% saccharin water was the taste conditioned stimulus, and lithium chloride or the test agent was the unconditional stimulus. The greater the drinking selection rate, the greater the anorexia. The drinking water selection ratio calculation formula is as follows:

and (4) judging a result standard: the greater the drinking selection rate, the greater the anorexia.

4. The experimental results are as follows: see table 4.

Table 4 mouse drinking water selection ratio (%, n ═ 6)

As can be seen from Table 4, the difference in the drinking water selection ratio among the mice of each nasal drop group of example 5 was not statistically significant compared to the model group.

5. And (4) conclusion: the CNTF has small side effect when being applied to nasal drops, and can not cause anorexia.

Experimental example 5: evaluation of intracerebral tropism of rat with CNTF nasal drops

1. Experimental animals: adult, healthy and ICR mice are selected, male mice with the weight of 18-22 g are selected, the mice are provided by an animal center of the medical college of the Western-Ann transportation university, the animal production license number SCXK (Shaan) 2012-003 and the animal qualification number 0014398, and the experiments are carried out after the experiment center is adapted for one week.

2. Grouping experiments: 48 mice were randomly divided into 4 groups of 12 mice each, and the CNTF subcutaneous administration group (10mg/kg) and the high, medium and low nasal drop dose groups of example 5 were administered via nasal administration, specifically:

CNTF subcutaneous administration group (CNTF-5-SC-10mg/kg)

EXAMPLE 5 nasal drops high dose group (CNTF-5-IN-10mg/kg)

EXAMPLE 5 nasal drop Medium dose group (CNTF-5-IN-5mg/kg)

EXAMPLE 5 nasal drip Low dose group (CNTF-5-IN-2.5mg/kg)

3. And (3) experimental operation: mice in each group were sacrificed 10min, 30min, 1h, 2h, and 3h after administration. Taking out brain tissue, separating olfactory bulb, washing with physiological saline to remove blood stain, and storing at-20 deg.C. The drug content in brain tissue was determined by double antibody sandwich ELISA.

4. The results are shown in Table 5

TABLE 5 comparison of the CNTF content in the brains of mice from the group of subcutaneous CNTF injections and from the group of CNTF nasal drops

Note: p <0.05, P <0.01

As can be seen from Table 5, the CNTF subcutaneous injection group had a small amount of entrance into the brain tissue, and the entrance of the nasal drops into the brain tissue was significantly improved.

5. And (4) conclusion: the CNTF nasal drop has obvious brain targeting effect

Experimental example 6: stimulation of CNTF nasal drops on epithelial cells of nasal mucosa of rat

The rats of example 1 were sacrificed 28 days after the administration, and nasal mucosa was taken, fixed with 10% formaldehyde, taken from the normal material, embedded in paraffin, sectioned, stained, and observed under an optical microscope.

The results show that: examples 1 and 4 nasal drop administration groups showed slight erosion of the phosphate epithelium coating the surface of the nasal mucosa tissue. The other nasal drop compositions have complete covering of the phosphorus epithelium on the nasal mucosa tissue, no erosion or ulcer is formed, and no edema or inflammatory cell infiltration is observed in the inherent layer.

And (4) conclusion: the CNTF nasal drop has small irritation to epithelial cells of nasal mucosa of a rat and is safe.

Example 7: immunogenicity Studies of CNTF nasal drops

1. Experimental animals: healthy and adult SPF SD rats, male, with the mass of 200g-250g, purchased from the Western's university of transportation medical laboratory animal center, with an animal production license number SCXK (Shaan) 2013 and an animal qualification number 0011711. Experiments were performed after one week of acclimation in the animal house.

2. Grouping experiments:

the obese rats (same as example 1)32 successfully molded were divided into 4 groups of 8 rats each, the CNTF subcutaneous injection group, and the CNTF nasal drip high, medium and low dose group of example 5. Wherein:

example 5 nasal drop high dose group (CNTF-5-IN-6 mg/kg);

example 5 nasal drop middle dose group (CNTF-5-IN-3 mg/kg);

example 5 nasal drop Low dose group (CNTF-5-IN-1.5 mg/kg);

3. the experimental method comprises the following steps:

each group of rats was dosed once daily for 28 consecutive days. On the last day, the rats are fasted for 12h and administered for the last time for 1h, blood is collected through a carotid artery cannula after anesthesia, the blood sample is placed for 2h at room temperature, centrifuged for 15min at 3000r/min, and serum is taken to measure the serum antibody level.

4. As a result: serum antibodies were measured after 5000-fold dilution of serum, and 100% of rat CNTF antibodies were positive in the nasal drip high dose group of example 5, 50% of rat CNTF antibodies were positive in the nasal drip medium dose group of example 5, 38% of rat CNTF antibodies were positive in the nasal drip low dose group of example 5, and 100% of rat CNTF antibodies were positive in the CNTF subcutaneous dose group. It can thus be seen that: the antibody production amount is increased along with the increase of the administration dosage. The amount of antibody produced by the nasal drops of the medium-and low-dose group of example 5 was significantly lower than that of the CNTF subcutaneous administration group, and the nasal drops of example 5 were 7.5 times higher than that of the subcutaneous administration group. The nasal drip high dose group produced an amount of antibody equivalent to that produced by the subcutaneous administration group, but the amount administered was 30 times that of the subcutaneous administration group.

5. And (4) conclusion: the production of CNTF nasal drops at equivalent blood concentrations is significantly lower than that of subcutaneous administration.

Example 8: pharmacokinetics study of CNTF nasal drops

2. Grouping experiments: 40 rats were randomly divided into 5 groups of 8 rats, each group was nasally administered with 5mg/kg, 2mg/kg, and 0.8mg/kg of CNTF of example 5, 100. mu.g/kg of CNTF by subcutaneous injection, and 2mg/kg of CNTF by nasal drip of the blank group, respectively.

Blank group (CNTF dissolved in PBS buffer solution, without absorption enhancer, 2mg/kg for nasal drop administration)

CNTF subcutaneous administration group (100. mu.g/kg)

EXAMPLE 5 nasal drip 5mg/kg dose group (CNTF-5-IN-5mg/kg)

EXAMPLE 5 nasal drip 2mg/kg dose group (CNTF-5-IN-2mg/kg)

EXAMPLE 5 nasal drip 0.8mg/kg dose group (CNTF-5-IN-0.8mg/kg)

3. And (3) experimental operation: the rats in each group are subjected to tail vein blood collection before administration (0h) and 10min, 30min, 45min, 1h, 2h, 3h, 4h, 5h and 6h after administration. The blood sample is centrifuged at 3000r for 15min and the separated serum is stored at-20 ℃. And detecting the melting of the front chamber temperature and diluting the sample diluent. The double antibody sandwich ELISA method is used for measuring the drug content in the serum.

4. The results are shown in Table 6, FIG. 5 and FIG. 6.

TABLE 6 pharmacokinetic parameters of CNTF nasal drops on rats

Table 6 shows that the pharmacokinetic characteristics of the rat nasal cavity for the 5mg/kg nasal drop dose group of example 5, 2mg/kg nasal drop dose group of example 5 and 0.8mg/kg nasal drop dose group of example 5 conform to a one-compartment model, Cmax is 78.53, 37.82 and 10.58ug/L, AUC0-t is 77.67, 48.22 and 11.64ug/L/h, AUC0- ∞ is 86.38, 53.22 and 12.28ug/L/h, Tmax is 0.23h, and both Cmax and AUC increase with the increase of the administration dose. The maximum blood concentration of the subcutaneous administration group is equivalent to that of the nasal drop 0.8mg/kg dose group in example 5, the peak time of each nasal drop dose group in example 5 is obviously shorter than that of the subcutaneous administration group, namely, the absorption rate of the nasal drop preparation in example 5 is faster.

Fig. 5 shows the plasma concentration-time curve of the rat nasal administration example 5 nasal drop 5mg/kg dose group, the example 5 nasal drop 2mg/kg dose group, the example 5 nasal drop 0.8mg/kg dose group, and fig. 6 shows the plasma concentration-time curve of the rat subcutaneous injection example 5 nasal drop 100ug/kg dose group, and the comparison of the two graphs shows that: a. the CNTF can reach the blood concentration when 0.8mg/kg is administrated to the nasal cavity of a rat to 100 mu g/kg of the subcutaneous CNTF, namely, the blood concentration of the effective dose can be reached when the nasal drop preparation of the embodiment 5 is administrated to 0.8 mg/kg.

Similarly, the results of the tests conducted on the other compositions and examples of the present invention were the same as those of example 5.

5. And (4) conclusion: the CNTF nasal drop has good absorption effect and can reach and exceed the blood concentration of a subcutaneous injection.

industrial applicability

The invention provides a nasal drop drug delivery system of ciliary neurotrophic factor (CNTF), the weight-reducing effect can reach or be superior to positive drugs and subcutaneous drug delivery, the weight-reducing stability is obviously superior to subcutaneous drug delivery, and the antibody production of CNTF is reduced; greatly improves the compliance and safety of medication (from injection to external use), and has adverse reaction: compared with the positive control medicament, the medicament does not cause weight rebound and anorexia reaction. Compared with the common nasal drop preparation, the preparation provided by the invention greatly improves the bioavailability, has simple preparation process and stable and controllable quality, and is suitable for industrial production.

Claims

A CNTF nasal administration preparation for treating obesity with addition of absorption enhancer is provided.
The formulation of claim 1, wherein the nasally administered formulation is a nasal spray, spray or nasal drops.
The formulation of claim 1, wherein the CNTF formulation for nasal administration comprises CNTF and absorption enhancers, stabilizers, buffers.
The formulation according to claim 3, characterized in that it contains, as components: 0.01-10 parts of CNTF, 5-30 parts of absorption accelerator, 0.01-0.4 part of stabilizer and 100 parts of buffer system by volume.
The formulation according to claim 3, characterized in that it contains, as components: 0.01-5 parts of CNTF, 10-30 parts of absorption accelerator, 0.01-0.2 part of stabilizer and 100 parts of buffer system by volume.
The formulation according to claim 3, characterized in that it contains, as components: 0.01-1 part of CNTF, 10-28 parts of absorption accelerator, 0.01-0.1 part of stabilizer and 100 parts of buffer system by volume.
The formulation according to claim 3, characterized in that it contains, as components: 0.015-0.030 part of CNTF, 14-25 parts of absorption accelerator, 0.05-0.1 part of stabilizer and 100 parts of buffer system by volume.
The formulation according to claim 3, characterized in that it contains, as components: 0.025 parts of CNTF, 20.41 parts of absorption accelerator, 0.05 parts of stabilizer and 100 parts of buffer system by volume.
Formulation according to any one of claims 1 to 8, characterized in that said CNTF is a natural ciliary neurotrophic factor, preferably said CNTF is a natural CNTF mutant or an amino acid sequence at least 70% homologous to natural CNTF, further preferably the natural CNTF has its 17 th cysteine changed to serine and the C-terminal 16 amino acid sequence removed.
The formulation according to any one of claims 1 to 8, wherein the absorption enhancer is selected from one or more of chitosan, sodium polyphosphate, hydroxypropyl- β -cyclodextrin, aminopeptidase inhibitor, polysorbate 20, polysorbate 60, polysorbate 80, polyoxyethylene castor oil, polyethylene glycol stearate 15(Solutol HS15), tocopherol polyethylene glycol succinate (TBGS), poloxamer F68, sodium cholate, sodium deoxycholate or phospholipids, preferably hydroxypropyl- β -cyclodextrin or polyethylene glycol stearate 15.
The formulation of any one of claims 1 to 8, wherein the stabilizer is selected from one or more of mannitol, lactose, sucrose, trehalose, glycine, arginine, aspartic acid, glutaminyl or albumin.
Formulation according to any one of claims 1 to 8, characterized in that the buffer system is selected from the group consisting of a PBS buffer system (phosphate buffer system), an acetic acid-sodium acetate buffer system or citric acid-sodium citrate.
Formulation according to claim 12, characterized in that the pH of the buffer system is 5-9, preferably 6.5-7.5, further preferably 7.2-7.4.
A method of preparing a formulation as claimed in any one of claims 3 to 14, comprising the steps of:

1) the buffer system configuration method comprises the following steps: judging the proportion relation of two substances in different types of buffer systems according to the pH value, dissolving the two substances in water respectively to prepare solution A and solution B, and adjusting the pH value of the solution B to 5-9 by using the solution A for later use;

2) processing of CNTF: taking the CNTF stock solution, carrying out ultrafiltration and centrifugation, collecting supernatant, then adding distilled water, carrying out centrifugation again, collecting supernatant, concentrating, and carrying out freeze drying for later use;

3) the preparation method of the nasal drop comprises the following steps: weighing each component according to the formula amount, dissolving the absorption enhancer and the stabilizer in the buffer solution, placing the buffer solution in a bottle of the transnasal administration device, placing the CNTF in a storage rack of the transnasal administration device, and covering a bottle cover.
Use of a formulation according to any one of claims 1 to 13 in the manufacture of a medicament for the treatment of obesity.
The use according to claim 15, wherein the weight loss is the treatment of obesity vulgaris, obesity of the diabetic type, said obesity vulgaris being constitutional or nutritional obesity.