CN100394981C - Human granulocyte-macrophage colony stimulating factor spray and its prepn process - Google Patents
Human granulocyte-macrophage colony stimulating factor spray and its prepn process Download PDFInfo
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- CN100394981C CN100394981C CNB2005101032015A CN200510103201A CN100394981C CN 100394981 C CN100394981 C CN 100394981C CN B2005101032015 A CNB2005101032015 A CN B2005101032015A CN 200510103201 A CN200510103201 A CN 200510103201A CN 100394981 C CN100394981 C CN 100394981C
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Abstract
The present invention provides a spraying agent of natural or recombinant human granulocyte macrophage colony stimulating factors, and a preparation method thereof. The spraying agent comprises 0.02 to 0.5 portion of human granulocyte macrophage colony stimulating factor, 10.0 to 100.0 portions of stabilizing agent and 0.05 to 0.5 portion of absorption enhancer by weight, wherein the stabilizing agent is selected from one or several of human serum albumin, lactose, trehalose, sucrose, mannitol, glycerine, propanediol, glycine and hyaluronic acid. The absorption enhancer is one or several of phosphatidyl choline, sodium cholate, sodium deoxycholate, oleic acid polysorbate 80 and brij. The spraying agent of human granulocyte-macrophage colony stimulating factors based on the present invention has the characteristics of high partial bioavailability, small side effect on the whole body, easy use, portability, etc.
Description
Technical field
The present invention relates to a kind of spray and preparation method thereof.Particularly, the present invention relates to a kind of human granulocyte-macrophage colony stimulating factor spray and preparation method thereof of natural or reorganization.
Background technology
Granulocyte macrophage colony stimulating factor (GM-CSF) is one of important Hemopoietic factor of discovered in recent years, is a kind of cytokine that can regulate the hemopoietic progenitor cell proliferation and differentiation.It is the glycoprotein of the single component of 23KD that people's ability in 1977 separation and purification from mouse lung organization condition culture fluid goes out to stimulate granulocyte and macrophage colony to form molecular weight, and therefore is named as GM-CSF (Burgess, et al., 1977); Nicola in 1978 etc. take the lead in having extracted human GM-CSF (Nicola et al from people's Placenta Hominis conditioned medium.，1979)。1985 human granulocyte macrophage colony stimulus factor (rhGM-CSF) gene cloned out, be encoded to 127 amino acid whose glycoproteins.At yeast with at the glycosyl and the non-glycosylated GM-CSF of expression in escherichia coli, still has the activity of natural GM-CSF.Drugs approved by FDA rhGM-CSF formally put on market in 1991, and the independently developed rhGM-CSF of China produced by the Ministry of Public Health approval is formal in 1998.In recent years along with the clinical extensive use of rhGM-CSF and at laboratory to its further further investigation, find that macrophage colony stimulating factor of recombinant human granulocyte can sequentially start neutrophilic granulocyte, the series of biologic of oxyphil cell and macrophage is learned function, make cell produce chemotaxis, make them concentrate on infection site then and bring into play phagocytic function, strengthen oxidative metabolism in the cell, to kill the pathogen of absorption, regulate body infection and antiulcer action thereby reach, under the immunocompetence and the infection and the ulcer that cause such as leukopenia have specific action.After reporting that infecting the leishmanial mice in the acute torrid zone gives GM-CSF, its intraperitoneal macrophage is through cultivating, and the infection rate of cell is reduced to below 10%, and matched group is 30% (Cheers et al., 1988; Jensen et al., 1988; Anaissie et al., 1989).According to clinical report GM-CSF the acute leukemia stomatitis there is certain preventive and therapeutic effect (modern combination of Chinese and Western medicine magazine 2005,14 (7)).When the part of human body such as oral ulcer, GM-CSF plays the effect of weak chemical inhibitor, and it can leave inflammation part by the inflammation-inhibiting cell, and can with the G-CSF synergism.Metcalf thinks that GM-CSF and other colony stimulating factor are worked in coordination with in inflammatory reaction and plays a role.It is remarkable that in vivo test proves that GM-CSF is applied to the sick effect of systemic Candida albicans, and effect is still remarkable when especially being applied to the immunologic hypofunction patient, and this point is even more important, because the Candida albicans patient immune function is most low.This shows that GM-CSF has the effect (2003 the 27th the 6th phases of volume of Chinese Medical Journal) of good anti-infection by Candida albicans.U.S. Berbudez report as the macrophage that then can activate infected with GM-CSF, and then suppresses or kills intracellular mycobacteria, if with GM-CSF and the coupling of anti-mycobacteria antibiotic, can further improve curative effect.
In order to illustrate that GM-CSF can strengthen the anti-infective effect, 32 routine patients have selected in University of Munich, wherein 22 examples were accepted the antibiotic therapy more than 3 kinds, all the other 10 examples were also used the antifungal drug of general, found that, numeration of leukocyte is elevated to normal person rapidly behind every use GM-CSF, and its required antibiotic therapy time obviously fails rapidly than numeration of leukocyte behind those uses GM-CSF that rising person lacks, and this result illustrates that effectively GM-CSF has certain anti-infection ability.Wandl has reported the skin ulcer for the treatment of the patient with breast cancer with GM-CSF, has also obtained gratifying effect.For the infection that a lot of complicated reasons cause, GM-CSF also has significant effect.In addition, there is report to use GM-CSF that obvious curative effects (Marques da, et al.Am J Surg1997,65 (7): 2876-82) are arranged by leg ulcer and the genital infection that Candida albicans causes.Report that in addition CM-CSF has certain curative effect (Balki B, et al.Eur Respir J 1977,10 (4): 846-50) to treatment chronic tracheitis, bronchitis, asthma etc.Jiang Li people such as rue thinks that in the viral morbific process of research GM-CSF plays an important role in the process with immunity of causing a disease of dengue virus.And we confirm further that by zoopery GM-CSF infects effective in cure to anti-herpes simplex virus and resisiting influenza virus.
Illustrate all that below macrophage colony stimulating factor of recombinant human granulocyte is a kind of anti-infectives safely and effectively.As topical, macrophage colony stimulating factor of recombinant human granulocyte gel as China Patent No. 02150093.2 and China Patent No. 0310044.X invention, the ulcer of main treatment skin burn, scald and skin or mucosa, this gel can only be located medication at skin and oral cavity, can not be in the medication of nasal cavity, throat and even trachea and pulmonary.And the macrophage colony stimulating factor of recombinant human granulocyte spray has overcome this shortcoming, especially use new prescription and new preparation method, make macrophage colony stimulating factor of recombinant human granulocyte stability better, the adjuvant that adds in the prescription more helps skin and the mucosa Transdermal absorption to GM-CSF.
And, because macrophage colony stimulating factor of recombinant human granulocyte is a kind of macromolecular polypeptides matter, because molecular structure and performance decision GM-CSF are compared with its less stable of peptide materials such as insulin, interferon, more be subject to the influence of external condition, it is comparatively stable to be made generally in lyophilized injectable powder.But because in the market macrophage colony stimulating factor of recombinant human granulocyte lyophilized injectable powder is a systemic administration, principal agent through the first pass effect of liver and and other anti-proteic material effect of body after arrive focus again, drug effect just obviously reduces; And if increase dosage, having to increase systemic toxic side effect.Therefore need provide can more effective administration dosage form.The patent No. is that 00114318.2 Chinese patent discloses a kind of insulin spray, thereby this dosage form is by oral cavity medicine treatment diabetes; Number of patent application is that 95102788.3 Chinese patent discloses a kind of leucocyte interferon spray; Number of patent application is that 03147580.9 Chinese patent discloses a kind of recombinant human interferon-alpha spray and treats respiratory virus infection and skin surface herpesvirus infection respectively; The Chinese patent application of number of patent application 01142682.9 discloses a kind of recombination human epidermal growth factor spray, and it is the regenerated biological preparation that promotes injured epidermis cell.But aspect treatment oral ulcer and anti-inflammatory, also do not have a peptide species class medicine spray to come out, more do not have macrophage colony stimulating factor of recombinant human granulocyte spray report.The blank of macrophage colony stimulating factor of recombinant human granulocyte aspect this dosage form filled up in the research and development that the present inventor carries out this.
Summary of the invention
One of purpose of the present invention provides a kind of human granulocyte-macrophage colony stimulating factor spray, and wherein human granulocyte macrophage colony stimulus factor comprises the natural human granulocyte macrophage colony stimulus factor and the human granulocyte macrophage colony stimulus factor of gene recombinaton.
A further object of the present invention provides the method for the above-mentioned spray of preparation.
According to a technical scheme of the present invention, the invention provides a kind of human granulocyte-macrophage colony stimulating factor spray, comprise the human granulocyte macrophage colony stimulus factor of 0.02～0.5 weight portion, the stabilizing agent of 10.0～100.0 weight portions and the absorption enhancer of 0.05～0.5 weight portion in per 1000 weight portion spray solutions, wherein said stabilizing agent is to be selected from two or more the adjuvant that comprises among the human albumin, lactose, trehalose, sucrose, mannitol, glycerol, propylene glycol, glycine, hyaluronic group; Described absorption enhancer comprises lecithin, sodium cholate, the adjuvant of one or more in the group of NaTDC, oleic acid, polyoxyethylene sorbitan monoleate, Brij for being selected from.
In spray according to the present invention, the content of preferred principal agent human granulocyte macrophage colony stimulus factor is 0.15 weight portion/1000 weight portion spray solutions.
According to " Pharmacopoeia of People's Republic of China) " three regulations of version in 2005, the ratio work of human granulocyte macrophage colony stimulus factor is 1.0 * 10
7IU/mg, the proportion of this spray solution is 1.00 to 1.02, promptly this solution of 1000 grams is equivalent to 1000 milliliters, so 0.15 weight portion/1000 weight portion spray solutions are equivalent to contain 1,500,000 IU/ml of principal agent active unit, the principal agent of same other weight portion, concerning those skilled in the art, be to know active unit's concentration that solution contains by inference.
Comprise selected especially stabilizing agent in the spray of the present invention, this medicine stabilizing agent has sufficient protective effect to principal agent.Preferred used stabilizing agent is human albumin, mannitol, trehalose and glycerol, and preferred four weight ratio 1: 4: 2: 1.And in spray according to the present invention, the content of preferred stabilizer is 40 weight portions/1000 weight portion spray solutions.This stabilizing agent can not only keep the effective biological activity of principal agent for a long time in spray solution, and has improved the viscosity of spray effectively, has prolonged the action time of medicine in focus.
Use Percutaneous absorption enhancer in the spray of the present invention.This absorption enhancer important feature is that it is taking into account stabilizing agent and absorption enhancer dual-use function, both principal agent is played the synergic stabilizer function of antioxygen, plays the effect that promotes that the principal agent transdermal absorbs again.Described absorption enhancer does not have any destruction to biomembrane or skin, can improve the part of principal agent body or the bioavailability of whole body fully, and it can be by the body biodegradation.Preferably use fabaceous lecithin and polyoxyethylene sorbitan monoleate as absorption enhancer, and when both weight ratios are 2: 3, promote the Transdermal absorption best results of principal agent.The content of preferred absorption enhancer is 0.2 weight portion/1000 weight portion spray solutions.
Can also contain antiseptic according to spray of the present invention, used antiseptic can be in one in methyl hydroxybenzoate, nipagin A and the nipasol or several, preferred nipagin A, the content of antiseptic is 1.0～1.5 weight portions/1000 weight portion spray solutions, preferred 1.2 weight portions/1000 weight portion spray solutions.
The used buffer of spray according to the present invention is phosphate buffer, acetate buffer or citrate buffer, its equivalent concentration be 0.02N to 0.2N, pH value is 5.0 to 7.5, wherein the phosphate buffer with 0.1N, pH7.2 serves as preferred.
According to the spray according to the present invention osmotic pressure regulator of sodium chloride as spray, the pH value of preferred spray solution is 5.5～7.5, and osmotic pressure is 280～310mOsm/L.Used adjuvant is medicinal rank, so this spray drug effect is clear and definite, drug safety, life-time service do not have any potential untoward reaction yet.
The invention provides a kind of method for preparing above-mentioned spray.Spray preparing process has many kinds, select suitable preparation method according to the contained adjuvant that medicine and medicine added.Because GM-CSF is a kind of macromolecular protein, easily be dissolved in water, and has certain biologic activity, so when preparation GM-CSF spray, at first to consider not destroy the structure of this protein molecular, secondly also will consider to reduce proteic biologic activity, this just requires us to select gentle preparation method.In preparation in accordance with the present invention, with phosphate buffer complex stabilizer is configured to mother solution I, composite absorption promoter is mixed with mother solution II, with mother solution I dilution GM-CSF stock solution, room temperature was placed 30 minutes, the consumption of mother solution I be the spray solution total amount that is made into 8～15%, preferred 10%, add mother solution II and antiseptic again, the consumption of mother solution II be the spray solution total amount that is made into 8～15%, preferred 10%, fixed molten and filter packing with phosphate buffer at last.Used GM-CSF contains macrophage colony stimulating factor of recombinant human granulocyte secretions by what the thalline fermentation expression went out, through slightly carry, essence puies forward the macrophage colony stimulating factor of recombinant human granulocyte stock solution that obtains meeting national quality testing standard.
Characteristics such as human granulocyte-macrophage colony stimulating factor spray according to the present invention has local bioavailability height, and systemic side effects is little, and is easy to use, portable.In liquid spray according to the present invention, added proper quantity of medicinal auxiliary material, not only made its principal agent stability be not less than lyophilized preparation, but also can promote the principal agent of macromole polypeptide class better to be absorbed, improved the bioavailability of principal agent widely by focus.This spray is applied to nasal cavity, oral cavity, bottleneck throat, body surface and phallic ulcer or inflammation, especially resists fungus-caused sex such as Candida albicans and grows device and infect and inflammation.Its principal agent not only can concentrate the efficacy of a drug in the focus wound surface after spraying, and makes the part that higher curative effect be arranged, and compares with the injection of this principal agent, can reduce the dosage that treatment is infected greatly, has reduced the whole body side reaction simultaneously.
The specific embodiment
Preparation embodiment
Preparation embodiment 1
1. fill a prescription:
GM-CSF 0.02g
Mannitol 20.0g
Lactose 10.0g
Oleic acid 0.1g
Tween-80 0.1g
(0.1N 7.2pH) adds to 1000ml to phosphate buffer
2. preparation method:
(1) be 7.2 with sodium hydrogen phosphate and sodium dihydrogen phosphate preparation pH, concentration is that the 0.1N phosphate buffer is molten;
(2) measure the molten 100ml of the phosphate buffer that has prepared with graduated cylinder, add by load weighted mannitol of prescription and lactose, and be stirred to preliminarily solubilised, make mother solution I;
(3) measure the molten 100ml of the phosphate buffer that has prepared with graduated cylinder, add by load weighted oleic acid of prescription and Tween-80, and be stirred to preliminarily solubilised, make mother solution II;
(4) the GM-CSF stock solution (containing GC-CSF albumen 0.02g) of adding 13ml in mother solution I;
(5) mix mother solution I, II, it is molten to add phosphate buffer again, is dissolved in 1000ml surely, full and uniform to dissolving fully.
(6) with packing after the aseptic microporous filter membrane aseptic filtration of 0.22 μ m.
Preparation embodiment 2
1. fill a prescription:
GM-CSF 0.15g
Human albumin 2.0g
Trehalose 20.0
Soybean phospholipid 0.1g
Tween-80 0.1g
Ethyl hydroxybenzoate 1.5g
(0.1N 7.2pH) adds to 1000ml to phosphate buffer
2. preparation method:
(1) be 7.2 with sodium hydrogen phosphate and sodium dihydrogen phosphate preparation pH, concentration is that the 0.1N phosphate buffer is molten.
(2) measure the molten 100ml of the phosphate buffer that has prepared with graduated cylinder, add by load weighted human albumin of prescription and trehalose, and be stirred to preliminarily solubilised, make mother solution I;
(3) measure the molten 100ml of the phosphate buffer that has prepared with graduated cylinder, add by load weighted soybean phospholipid of prescription and Tween-80, and be stirred to preliminarily solubilised, make mother solution II;
(4) the GM-CSF stock solution (containing GC-CSF albumen 0.15g) of adding 86ml in mother solution I;
(5) mother solution II is added among the mother solution I, and adds antiseptic, the reuse phosphate buffer is molten fixed molten to 1000ml, and fully dissolving.
(6) with packing after the aseptic microporous filter membrane aseptic filtration of 0.22 μ m.
Preparation embodiment 3
1. fill a prescription:
GM-CSF 0.15g
Human albumin 2.0g
Mannitol 20.0g
Hyaluronic acid 5.0
Glycerol 5.0
Soybean phospholipid 0.1g
Tween-80 0.1g
Ethyl hydroxybenzoate 1.3g
(0.1N 7.2pH) adds to 1000ml to phosphate buffer
2. preparation method:
(1) (0.1N, 7.2pH) dissolving are mixed with the mother solution that contains blood albumin 2%, mannitol 20%, hyaluronic acid 5% and glycerol 5% as preparation mother solution I with phosphate buffer as complex stabilizer with human albumin, mannitol, hyaluronic acid and glycerol.
(2) (0.1N, 7.2pH) dissolving is mixed with the mother solution II that contains 0.1% soybean phospholipid and 0.1% Tween-80 with phosphate buffer as composite absorption promoter with soybean phospholipid and Tween-80.
(3) get 100ml mother solution I, add the GM-CSF stock solution (2.24mg/ml) of 67ml then, room temperature was placed 30 minutes behind the mixing.
(4) add 100ml mother solution II, add the antiseptic ethyl hydroxybenzoate by prescription then, stirring and dissolving, the reuse phosphate buffer is fixed molten to 1000ml.
(5) with packing after the aseptic microporous filter membrane aseptic filtration of 0.22 μ m.
Preparation embodiment 4
1. fill a prescription:
GM-CSF 0.15g
Human albumin 5.0g
Mannitol 20.0g
Trehalose 10.0
Glycerol 5.0
Soybean phospholipid 0.08g
Tween-80 0.12g
Ethyl hydroxybenzoate 1.2g
(0.1N 7.2pH) adds to 1000ml to phosphate buffer
2. preparation method: (preferred manufacturing procedure)
(1) (0.1N, 7.2pH) dissolving are mixed with the mother solution that contains blood albumin 5%, mannitol 20%, trehalose 10% and glycerol 5% as preparation mother solution I with phosphate buffer as complex stabilizer with human albumin, mannitol, trehalose and glycerol.
(2) (0.1N, 7.2pH) dissolving is mixed with the mother solution II that contains 0.08% soybean phospholipid and 0.12% Tween-80 with phosphate buffer as composite absorption promoter with soybean phospholipid and Tween-80.
(3) get 100ml mother solution I, add the GM-CSF stock solution (2.24mg/ml) of 67ml then, room temperature was placed 30 minutes behind the mixing.
(4) get 100ml mother solution II and mix, add the antiseptic ethyl hydroxybenzoate by prescription then with above-mentioned solution, stirring and dissolving, the reuse phosphate buffer is fixed molten to 1000ml.
(5) with packing after the aseptic microporous filter membrane aseptic filtration of 0.22 μ m.
Preparation embodiment 5
1. fill a prescription:
GM-CSF 0.15g
Human albumin 3.0g
Mannitol 20.0g
Sucrose 10.0
Glycine 5.0
Soybean phospholipid 0.08g
Tween-80 0.12g
Propyl hydroxybenzoate 1.5g
(0.1N 7.2pH) adds to 1000ml to phosphate buffer
2. preparation method:
(1) (0.1N, 7.2pH) dissolving are mixed with the mother solution that contains blood albumin 5%, mannitol 20%, sucrose 10% and glycine 5% as preparation mother solution I with phosphate buffer as complex stabilizer with human albumin, mannitol, trehalose and glycerol.
(2) (0.1N, 7.2pH) dissolving is mixed with the mother solution II that contains 0.08% soybean phospholipid and 0.12% Tween-80 with phosphate buffer as composite absorption promoter with soybean phospholipid and Tween-80.
(3) get 100ml mother solution I, add the GM-CSF stock solution (2.24mg/ml) of 67ml then, room temperature was placed 30 minutes behind the mixing.
(4) add 100ml mother solution II, add the antiseptic propyl hydroxybenzoate by prescription then, stirring and dissolving, the reuse phosphate buffer is fixed molten to 1000ml.
(5) with packing after the aseptic microporous filter membrane aseptic filtration of 0.22 μ m.
Preparation embodiment 6
1. fill a prescription:
GM-CSF 0.05g
Human albumin 5.0g
Mannitol 20.0g
Trehalose 10.0
Glycerol 5.0
Soybean phospholipid 0.08g
Tween-80 0.12g
(0.1N 7.2pH) adds to 1000ml to phosphate buffer
2. preparation method:
(1) (0.1N, 7.2pH) dissolving are mixed with the mother solution that contains blood albumin 4%, mannitol 16%, trehalose 8% and glycerol 4% as preparation mother solution I with phosphate buffer as complex stabilizer with human albumin, mannitol, trehalose and glycerol.
(2) (0.1N, 7.2pH) dissolving is mixed with the mother solution II that contains 0.05% soybean phospholipid and 0.08% Tween-80 with phosphate buffer as composite absorption promoter with soybean phospholipid and Tween-80.
(3) get 125ml mother solution I, add the GM-CSF stock solution (2.24mg/ml) of 22.5ml then, room temperature was placed 30 minutes behind the mixing.
(4) get 150ml mother solution II and mix with above-mentioned solution, the reuse phosphate buffer is fixed molten to 1000ml.
(5) with packing after the aseptic microporous filter membrane aseptic filtration of 0.22 μ m.
Preparation embodiment 7
1. fill a prescription:
GM-CSF 0.15g
Sucrose 10.0g
Propylene glycol 10.0g
NaTDC 0.05g
(0.1N 6.8pH) adds to 1000ml to acetate buffer
2. preparation method:
(1) (0.1N, 5.6pH) dissolving are mixed with and contain sucrose 10%, propylene glycol 10% mother solution as preparation mother solution I with acetate buffer as complex stabilizer with sucrose and propylene glycol.
(2) (0.1N, 5.6pH) dissolving is mixed with the mother solution II that contains 0.5% NaTDC with acetate buffer as absorption enhancer with big NaTDC.
(3) get 100ml mother solution I, add the GM-CSF stock solution (2.24mg/ml) of 67ml then, room temperature was placed 30 minutes behind the mixing.
(4) add 100ml mother solution II, the reuse acetate buffer is fixed molten to 1000ml.
(5) with packing after the aseptic microporous filter membrane aseptic filtration of 0.22 μ m.
Preparation embodiment 8
1. fill a prescription:
GM-CSF 0.50g
Glycine 5.0g
Lactose 15.0g
Brij 0.15g
Oleic acid 0.1g
Methyl hydroxybenzoate 1.2g
(0.1N 7.2pH) adds to 1000ml to phosphate buffer
2. preparation method:
(1) with glycine and lactose as complex stabilizer with phosphate buffer (0.1N, 7.2pH) dissolving, be mixed with contain glycine 6.25%, lactose 18.75% mother solution as preparation mother solution I.
(2) (0.1N, 7.2pH) dissolving is mixed with the mother solution II that contains Brij 0.18% and oleic acid 0.12% with phosphate buffer as compound accelerant with Brij and oleic acid.
(3) get 80ml mother solution I, add the GM-CSF stock solution (2.51mg/ml) of 202ml then, room temperature was placed 30 minutes behind the mixing.
(4) add 85ml mother solution II, add the antiseptic methyl hydroxybenzoate by prescription then, stirring and dissolving, the reuse phosphate buffer is fixed molten to 1000ml.
(5) with packing after the aseptic microporous filter membrane aseptic filtration of 0.22 μ m.EXPERIMENTAL EXAMPLE
EXPERIMENTAL EXAMPLE 1: the stability test of spray
(1) experiment material:
According to the prepared GM-CSF spray sample of preparation embodiment 1～8,25 ℃ and 37 ℃ of constant temperature and humidity couveuses.
(2) experimental technique:
According to the method for three appendix XF of Pharmacopoeia of the People's Republic of China version in 2005, above sample is regularly carried out determination of activity.
(3) experimental result:
Under the situation of different protective agent protections, the comparative result of GM-CSF spray biologic activity stability.
(the biological activity unit of GM-CSF is * 1 to the steadiness of 25 ℃ of placements
6Iu/ml):
| 0 month | March | June | JIUYUE | December | |
| The prescription of preparation embodiment 1 | 0.21 | 0.20 | 0.19 | 0.16 | 0.12 |
| The prescription of preparation embodiment 2 | 1.52 | 1.52 | 1.50 | 1.49 | 1.35 |
| The prescription of preparation embodiment 3 | 1.51 | 1.53 | 1.51 | 1.42 | 1.26 |
| The prescription of preparation embodiment 4 | 1.51 | 1.51 | 1.53 | 1.50 | 1.49 |
| The prescription of preparation embodiment 5 | 1.50 | 1.51 | 1.52 | 1.51 | 1.44 |
| The prescription of preparation embodiment 6 | 0.52 | 0.50 | 0.51 | 0.47 | 0.44 |
| The prescription of preparation embodiment 7 | 1.50 | 1.47 | 1.32 | 1.08 | 0.87. |
| The prescription of preparation embodiment 8 | 5.02 | 4.98 | 4.85 | 4.57 | 4.16 |
(the biological activity unit of GM-CSF is 1.5 * 10 to the steadiness of 37 ℃ of placements
6Iu/ml):
| 0 month | January | February | March | June | |
| The prescription of preparation embodiment 1 | 0.21 | 0.20 | 0.19 | 0.15 | 0.10 |
| The prescription of preparation embodiment 2 | 1.52 | 1.51 | 1.48 | 1.42 | 1.19 |
| The prescription of preparation embodiment 3 | 1.51 | 1.49 | 1.50 | 1.38 | 1.14 |
| The prescription of preparation embodiment 4 | 1.51 | 1.52 | 1.49 | 1.50 | 1.41 |
| The prescription of preparation embodiment 5 | 1.50 | 1.51 | 1.51 | 1.49 | 1.33 |
| The prescription of preparation embodiment 6 | 0.52 | 0.51 | 0.48 | 0.43 | 0.29 |
| The prescription of preparation embodiment 7 | 1.50 | 1.49 | 1.27 | 0.87 | 0.43 |
| The prescription of preparation embodiment 8 | 5.02 | 4.92 | 4.84 | 4.15 | 3.76 |
According to the stability test result, decision is to prepare embodiment 4 as optimization formula.
EXPERIMENTAL EXAMPLE 2: nasal cavity synergistic corrosion virus test
(1) experiment material:
1. be subjected to the reagent product: according to GM-CSF spray 50ug/ml, 150ug/ml, the 500ug/ml of preparation embodiment 4 (removing the dosage difference of GM-CSF) preparation; Spray substrate (except that not containing the GM-CSF, other composition and purchase) all according to preparation embodiment 4, the positive control drug virazole.
2. animal: Kunming kind white mice, body weight 18～22g, male and female half and half.
3. viral: the influenza virus that known LD50 is provided.
(2) experimental technique:
A, get 90 of mices, be divided into normal control group, model group, positive drug control group, test group and comprise that GM-CSF spray 50ug/ only organizes, 150ug/ only organizes, 500ug/ only organizes.Every group of 15 mices, model group nasal cavity spray substrate liquid, all the other administration group nasal cavities spray corresponding medicine, divide 2 administrations every day, spray 3 times at every turn, wherein comprise the spray that 2 spray GM-CSF sprays and 1 spray virazole are made in each 3 sprays of test group.Administration in continuous 10 days, the 11st day, except that the normal control group; all the other respectively organize equal collunarium infection 10LD50 virus quantity, continue to be administered to 14 days after 2 hours, record mice death toll 14 days every days and average life day; observed for 2 weeks, calculate dead protective rate (%) and prolong vital rates (%).
(3) experimental result:
The record result who observes:
Above presentation of results, GM-CSF spray and antiviral agents are united use, can resist the infection of influenza virus.
EXPERIMENTAL EXAMPLE 3: anti-herpes simplex virus causes the skin infection effect
(1) experiment material:
1. be subjected to the reagent product: according to GM-CSF spray 50ug/ml, 150ug/ml, the 500ug/ml of embodiment four (removing the dosage difference of GM-CSF) preparation; Positive control drug is with normal saline acyclovir to be mixed with concentration 7% acyclovir spray solution.
2. animal: Cavia porcellus is commercially available animal.
3. viral: herpes simplex virus.
(2) experimental technique:
According to " new drug (Western medicine) clinical research guideline compilation ", bureau of drug administration of Ministry of Health of the People's Republic of China, in July, 1993,169 pages method experimentizes.
(3) experimental result:
1. behind the virus inoculation 6-8 days, in inoculation place one rigid pimple, bulla or irregular being dispersed in property vesicle take place, the model success.
2. after the administration 1-2 days, each administration group and model group relatively sb.'s illness took a favorable turn degree no significant difference.The GM-CSF spray has the obvious shortening monovesicle viral herpes course of disease, accelerates the effect of vesicle incrustation, healing.
3. compare with positive control drug, the GM-CSF spray can be stranded in disease damage position and induce various lymphocytes performance immunity and phagocytosis, thereby strengthens the effect that anti-herpes simplex virus infects, and the while, its toxic and side effects was more much smaller than chemical antiviral drugs.
EXPERIMENTAL EXAMPLE 4: anti-fungal infection test
(1) experiment material:
1. be subjected to the reagent product: according to GM-CSF spray 50ug/ml, 150ug/ml, the 500ug/ml of embodiment four (removing the dosage difference of GM-CSF) preparation; Spray substrate (except that not containing the GM-CSF, other composition and purchase) all according to embodiment four, cyclophosphamide.
2. animal: the Wistar rat, all female.
3. fungus: known Candida albicans is provided.
(2) experimental technique:
1. get 25 of Adult female rats, be divided into 5 groups, do the contrast except that 1 group, other 4 groups equal every day oral cyclophosphamide 1.0mg, take at twice, carry out 1 all immunosuppressive actions.
2. the Candida albicans that fresh Sha Shi chloramphenicol agar sugar culture-medium is cultivated is used Plondrel phthalate buffer (PBS) centrifuge washing 2 times, is mixed with every milliliter 1.0 * 10 then
5The bacterium liquid of individual cell.
3. get the genitals that the bacterium liquid for preparing splashes into 5 groups of female Wistar rats respectively with dropper, two every.
4. next day, except that matched group, other 4 groups respectively by spray matrix group, GM-CSF spray 50ug/ only organize, 150ug/ only organizes and 500ug/ only organizes administration.Divide 2 administrations every day, spray 3 times at every turn, administration in continuous 10 days, the result of record observation in the 11st day.
(3) experimental result:
Observed and recorded:
| Phenomenon | Matched group | Matrix group | The 50ug/ml group | The 150ug/ml group | The 500ug/ml group |
| The nympha redness | ± | ++++ | + | ± | ± |
| Exfoliation | - | +++ | ± | - | - |
| Scratch | + | +++ | ± | ± | - |
| Pustule | - | ++ | - | - | - |
| Vaginal secretions | + | ++++ | + | ± | ± |
| Secretions cheese sample → bean dregs sample | - | +++ | - | - | - |
| Stink | - | +++ | + | - | - |
(how much "+" number represents the phenomenon order of severity, and "-" representative does not have this phenomenon)
The anatomical slice observed result:
| Observation item | Matched group | Matrix group | The 50ug/ml group | The 150ug/ml group | The 500ug/ml group |
| Vaginal wall congestion and edema degree | ± | ++++ | ± | ± | ± |
| Vaginal wall has or not white folder film | - | ++ | ± | - | - |
| The erythema of vaginal wall and rotten to the corn face degree | - | ++ | - | - | - |
(how much "+" number represents the phenomenon order of severity, and "-" representative does not have this phenomenon)
Above observed result explanation, the GM-CSF spray has the effect of fungal infection such as anti-Candida albicans.
EXPERIMENTAL EXAMPLE 5: general pharmacology test
(1) experiment material
1, is subjected to the reagent product: according to GM-CSF spray 50ug/ml, 150ug/ml, the 500ug/ml of embodiment four (removing the dosage difference of GM-CSF) preparation.
2, animal: Kunming kind white mice, healthy cat.
3, instrument:
XZC-4 type toy autonomic activities analyzer
LMS-2R two a roads heat physiograph
The Cardiofax electrocardiograph
(2) experimental technique
1, to the neural influence of spirit (mice autonomic activities method)
On the basis of prerun mice is divided equally 4 groups: matched group, the basic, normal, high dosage group of GM-CSF spray, the different time mice of naming a person for a particular job is put into toy autonomic activities analyzer behind the nasal spray, surveys autonomic activities number of times in its 10min.Each administration group and matched group are organized a t check.
2, to the influence of cardiovascular system
24 cats are divided equally 4 groups: matched group, the basic, normal, high dosage group of GM-CSF spray, earlier with the conventional anesthesia of cat, tracheal intubation, the common carotid artery intubate is connected on the two roads heat physiograph recording blood pressure by pressure transducer, simultaneously with extremity two recording ecg that leads, nasal spray, the variation of measuring and writing down administration front and back blood pressure, electrocardio, heart rate, heart rate, each index of administration group and matched group are organized a t check.
3, to the influence of respiratory system
24 cats are divided equally 4 groups: matched group, the basic, normal, high dosage group of GM-CSF spray, earlier with the conventional anesthesia of cat, hook the position of cat breast abdominal respiration big rise and fall with hook, be connected on the two roads heat physiograph by tonotransducer, nasal spray, index is inhaled in the variation of the respiratory frequency and the degree of depth before and after the record administration, administration group calling and matched group is organized a t check.
(3) experimental result:
The index of the various dose administration group of three tests does not all have significant difference with matched group, illustrates that the GM-CSF spray of this prescription all has no adverse effects to nervous system, cardiovascular system and respiratory system.
EXPERIMENTAL EXAMPLE 6: acute toxicity test:
Be subjected to the reagent product: GM-CSF spray 15mg/ml is the high concentration spray of 100 times of clinical dosages.
Animal: Wistar rat.
3, method: prerun, investigate the GM-CSF spray and can measure LD50, if can measure then square routinely its LD50 of survey; Rat is divided equally 2 groups if can not survey then: matched group, GM-CSF spray group, the spray group is surveyed its maximum tolerated dose by maximum drug level, the administration of maximum administration volume, and active situation, the hair color, two of observing and write down each treated animal in 1～2 week just reach body weight change.
(3) experimental result:
Observe and find that after the GM-CSF spray medication of 15mg/ml, toxic reaction does not promptly appear in the just all no abnormal performance of the active situation of all test group and animals of control group, hair color, two.This dosage is 100 times of clinical dosages, illustrates that the clinical dosage medication of GM-CSF spray is as safe as a house.
EXPERIMENTAL EXAMPLE 7: long term toxicity test:
Be subjected to the reagent product: GM-CSF spray 50ug/ml, 150ug/ml, 500ug/ml; Blank substrate liquid is contrast liquid.
2, animal: Wistar rat.
3, method: according to The acute toxicity tests, divide 4 groups (matched group, the high, medium and low dosage groups of GM-CSF spray) 20 every group, administration every day 1 time, continuous 30 days with rat, weigh weekly during this time 1 time, and record dietary amount, amount of drinking water and two just, hair color, active situation.24h after the last administration, each group is lived and is killed 1/2 animal, get blood and survey each hematological indices and blood parameters, cut open core, internal organs such as liver, spleen, lung, kidney, adrenal gland, thyroid, gonad, brain are weighed the calculating organ index, be fixed then to do pathological examination and take a picture.1/2 animal was cooked above-mentioned same detection in 14 days in drug withdrawal in addition.
4, calculate: each detects index and organizes a t check.
(3) experimental result:
Medication group and animals of control group observed and recorded weekly shows, diet, drinking-water and two are just, hair color, active situation be all normal.
24 hours each the medication group and the t test value of matched group all do not have significant difference after the drug withdrawal, drug withdrawal after 14 days each medication group and the t test value of matched group do not have significant difference yet, illustrate that the clinical consumption of GM-CSF spray does not have long term toxicity reaction, clinical application is very safe.
EXPERIMENTAL EXAMPLE 8: hypersensitive test
(1) experiment material:
Be subjected to the reagent product: GM-CSF spray 150ug/ml, 10% fresh albumen.
2, animal: 20 of albino guinea-pigs, body weight 250～300g, male and female are not limit.
(2) experimental technique:
20 Cavia porcelluss are divided equally two groups: GM-CSF spray 50ug/ only organizes and positive control drug 10% Ovum Gallus domesticus album group, two groups respectively continuous every other day nasal spray 3 times, each group is divided equally 2 groups again after the sensitization, 2 groups respectively after the administration first time 14 days and 21 days intravenously administrables excite, excite dosage: GM-CSF injection 1.0ml/ only, positive control drug 10% Ovum Gallus domesticus album group 1.0ml/ only.Each Cavia porcellus symptoms of allergic when observation and record intravenously administrable excite.
(3) experimental result:
Observe and find, all occur in various degree erythema and edema before positive controls guinea pig skin or the nose, and do not find have erythema and edema to take place before skin or the nose in the Cavia porcellus of medication group.The GM-CSF spray of 150ug/ml is described, dosage is under 0.2ml/ time the dosage, and 3 spray noses do not have irritated reaction repeatedly.
EXPERIMENTAL EXAMPLE 9: irritation test
(1) experiment material:
1, be subjected to the reagent product: GM-CSF spray 150ug/ml, normal saline is contrast liquid.
2, animal: 16 of Wistar rats, body weight 180～200g, male and female half and half.
(2) experimental technique:
16 rats are divided equally two groups: matched group, GM-CSF spray 150IU/ only organize, nasal spray, administration every day 2 times, successive administration 7 days, observe and the record nasal membrane has or not phenomenons such as hyperemia, redness, after the last administration 2 hours, put to death animal and cut open and get nasal membrane and put in the 10% neutral formalin solution, pathological examination, photograph are done in section.
(3) experimental result:
Section is done pathologic finding and is found that hyperemia, redness and downright bad phenomenon all appear in the rat of GM-CSF spray and matched group, and the GM-CSF spray of 150ug/ml is described, dosage is the dosage spray nose of 3 times (0.2ml/ time), to the nasal cavity nonirritant.
Claims (15)
1. human granulocyte-macrophage colony stimulating factor spray, it is characterized in that, comprise the human granulocyte macrophage colony stimulus factor of 0.02～0.5 weight portion, the stabilizing agent of 10.0～100.0 weight portions and the absorption enhancer of 0.05～0.5 weight portion in the spray solution of per 1000 weight portions
Wherein said stabilizing agent is to be selected to comprise among the human albumin, lactose, trehalose, sucrose, mannitol, glycerol, propylene glycol, glycine, hyaluronic group two or more;
Described absorption enhancer comprises lecithin, sodium cholate, the adjuvant of one or more in the group in NaTDC, oleic acid, polyoxyethylene sorbitan monoleate, the Brij for being selected from.
2. spray as claimed in claim 1 is characterized in that, described human granulocyte macrophage colony stimulus factor comprises natural human granulocyte macrophage colony stimulating factor and gene recombinaton human granulocyte macrophage colony stimulus factor.
3. spray as claimed in claim 1 is characterized in that containing in per 1000 weight portion spray solutions the described human granulocyte macrophage colony stimulus factor of 0.15 weight portion.
4. spray as claimed in claim 1 is characterized in that, described stabilizing agent is human albumin, mannitol, trehalose and glycerol.
5. spray as claimed in claim 4 is characterized in that, the weight ratio of human albumin, mannitol, trehalose and glycerol 1: 4: 2: 1.
6. as claim 1,4 or 5 described sprays, it is characterized in that the spray solution of per 1000 weight portions contains the stabilizing agent of 40 weight portions.
7. spray as claimed in claim 1 is characterized in that, described absorption enhancer is fabaceous lecithin and polyoxyethylene sorbitan monoleate.
8. spray as claimed in claim 7 is characterized in that, the weight ratio of fabaceous lecithin and polyoxyethylene sorbitan monoleate is 2: 3.
9. as claim 1,7 or 8 described sprays, it is characterized in that, contain the absorption enhancer of 0.2 weight portion in the spray solution of per 1000 weight portions.
10. spray as claimed in claim 1, the pH value that it is characterized in that spray solution is 5.5～7.5, osmotic pressure is 280～310mOsm/L.
11. prepare the method for spray as claimed in claim 1, it is characterized in that this method may further comprise the steps:
1) stabilizing agent described in the claim 1 is dissolved in the inorganic salt buffer, thereby makes mother solution I;
2) absorption enhancer described in the claim 1 is dissolved in the inorganic salt buffer, thereby makes mother solution II;
3) with mother solution I dilution GM-CSF stock solution, the consumption of mother solution I is 8～15wt% of the spray solution total amount that is made into;
4) add mother solution II, the consumption of mother solution II is 8～15wt% of the spray solution total amount that is made into;
5) with fixed molten overanxious degerming in back of inorganic salt buffer and packing,
Wherein, step 1), 1) and 5) in described inorganic salt buffer be to be selected from phosphate buffer, acetate buffer or the citrate buffer one or more.
12. preparation method as claimed in claim 11 is characterized in that, the consumption of mother solution I is 10% of the spray solution total amount that is made into, and the consumption of mother solution II is 10% of the spray solution total amount that is made into.
13. preparation method as claimed in claim 11 is characterized in that, in step 1), 2) and 5) in the inorganic salt buffer that uses be phosphate buffer.
14., it is characterized in that the equivalent concentration of described inorganic salt buffer is 0.02N～0.2N as claim 11 or 13 described preparation methoies, pH value is 5.0～7.5.
15., it is characterized in that the equivalent concentration of described inorganic salt buffer is that 0.1N, pH value are 7.2 as claim 11 or 13 described preparation methoies.
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| CN100455324C (en) * | 2005-09-23 | 2009-01-28 | 华北制药金坦生物技术股份有限公司 | Externally applied liquid prepn of recombinant human granulocyte-macrophage colony stimulating factor |
| CN101085348B (en) * | 2007-06-18 | 2010-08-18 | 南京农业大学 | Nasal cavity immunity composite adjuvant for avian influenza inactivation antigen |
| CN101773453A (en) * | 2010-02-10 | 2010-07-14 | 冯来坤 | Application of granulocyte-macrophage colony stimulating factor in preparing beauty cosmetics |
| MX2022011498A (en) * | 2020-03-17 | 2022-10-07 | Drugrecure Aps | Liquid formulation of gm-csf for inhalation. |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1132098A (en) * | 1995-03-30 | 1996-10-02 | 北京兴华生物新技术开发中心 | Human leucocyte interferon spray |
| WO2002094342A2 (en) * | 2001-05-21 | 2002-11-28 | Vapotronics, Inc. | Compositions for protein delivery via the pulmonary route |
| WO2003035028A1 (en) * | 2001-10-19 | 2003-05-01 | Nektar Therapeutics | Modulating charge density to produce improvements in the characteristics of spray-dried proteins |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1132098A (en) * | 1995-03-30 | 1996-10-02 | 北京兴华生物新技术开发中心 | Human leucocyte interferon spray |
| WO2002094342A2 (en) * | 2001-05-21 | 2002-11-28 | Vapotronics, Inc. | Compositions for protein delivery via the pulmonary route |
| WO2003035028A1 (en) * | 2001-10-19 | 2003-05-01 | Nektar Therapeutics | Modulating charge density to produce improvements in the characteristics of spray-dried proteins |
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