CN102178939B - Muramidase hydrolase external sterile cream for treating skin ulcer and preparation method thereof - Google Patents
Muramidase hydrolase external sterile cream for treating skin ulcer and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a muramidase hydrolase external sterile cream for treating skin ulcer and a preparation method thereof. The muramidase hydrolase external sterile cream is prepared from the following raw materials of: muramidase hydrolase, Span, poloxamer, Tween, sorbierite, sodium dihydrogen phosphate, ethylparaben, liquid paraffin, glycerin monostearate, cetyl alcohol, stearyl alcohol, albolene and sterile water. The invention also provides a preparation method for the muramidase hydrolase external sterile cream. The preparation method comprises the following steps of: dissolving; performing high temperature sterilization; filtering and removing bacteria by using a filter membrane; homogenizing at a low speed; homogenizing at a high speed; stirring; reducing temperature; and performing sterile filling. The muramidase hydrolase external sterile cream is used for treating the skin ulcer including bedsore, burn ulcer, calf ulcer, post-zoster ulcer, diabetic ulcer and postoperative ulcer in a transdermal administration form.
Description
Technical field
The present invention relates to a kind of lisozima external use sterile emulsifiable paste for the treatment of skin ulcer (ulcer, diabetic ulcer, postoperative ulcer behind decubital ulcer, scald ulcer, calf ulcer, the herpes zoster) and preparation method thereof, belong to medical technical field.
Background technology
Skin ulcer comprises decubitus ulcer, scalds ulcer, ulcer, diabetic ulcer, postoperative ulcer after the calf ulcer, herpes zoster, refers to local skin or sticking downright bad, and disintegrate, the formed limitation that comes off are damaged.
In China, skin ulcer and decubital ulcer disease are commonly encountered diseases, frequently-occurring disease.The skin ulcer decubital ulcer cause of disease is because of neurotrophy disorder and disturbance of blood circulation, local organization continuous ischemia, malnutrition and the soft tissue necrosis that occurs such as untimely treatment, can bring out other organ complication.According to related documents, skin ulcer decubital ulcer disease is 3%～14% at China hospital sickness rate, and according to incompletely statistics: have approximately every year and die from skin ulcer decubital ulcer complication more than the tens of thousands of people, global skin ulcer affects about 18,000,000 people in the whole world.
At present treatment skin ulcer decubital ulcer disease is divided into because of processing, topical therapeutic, operative treatment, Supporting Therapy, drug treatment etc.
To because processing: will cut off anaphylactogen such as anaphylaxis (drug eruption); The diabetes foot disease will be noted the control of blood glucose; Physicochemical damage will be noted away from etiology; Occupation disease, the condition that the elimination of trying one's best is caused a disease etc.Topical therapeutic: the ulcer debridement is changed dressings; Remove slough, cicatrix, outmoded granulation tissue, foreign body etc.Local physical therapy: the local irradiations such as He-Ne Lasers, ultrashort wave, infrared ray.Operative treatment: change dressings more greatly, for a long time for ulcer area and more should not consider operative treatment.Supporting Therapy: comprise the braking of good part, raise affected part, good nursing etc., note the balance of Water-Electrolyte, give the diet of high protein high-energy and vitamin, improve patient's immunity.The treatment of medicine: Claritin, the medicine of anti-parasite medicine, antibiotic and promotion ulcer healing etc.
In a word, all there is defective in various degree in above Therapeutic Method, and clinical practice is very limited.For satisfying the clinical demand of extensive patients, those skilled in the art are seeking to develop safety, efficient, the external used medicine for the treatment of skin ulcer quality controllable, easy to use for many years always.
At present at home the lisozima preparation mainly with oral solid formulation (buccal tablet, enteric coatel tablets), buccal tablet is clinical in acute and chronic pharyngitis, stomatocace and dys-expectoration, enteric coatel tablets clinical for chronic rhinitis, acute and chronic pharyngitis, oral ulcer, etc.Up to now, be used for the treatment of the aseptic external used medicine of lisozima for the treatment of skin ulcer (ulcer, diabetic ulcer, postoperative ulcer behind decubital ulcer, scald ulcer, calf ulcer, the herpes zoster) listing is not yet arranged.
Chinese patent file 200410020816.7 discloses the new purposes of a kind of human lysozyme in the dermopathic medicine of preparation treatment, relate to dermatosis that the preparation treatment is caused by antibacterial, fungus, virus, allergy, occupational, physical property, delayed ischemic neurological deficits, immunity, infect, fester disease medicine in application.Chinese patent file 200410020736.1 discloses the new purposes of human lysozyme in the medicine that preparation treatment wound infection does not more fester, the new purposes in the medicine that the infected wound that burn and diabetes and acquired immune deficiency syndrome (AIDS) cause non-healing wounds to cause festers.
Being used for ointment, ointment, the gel of burn or severe trauma according to Chinese Pharmacopoeia 2010 editions and European Pharmacopoeia, American Pharmacopeia, should be sterile preparation.Unsterilised or the sterilizing methods of used crude drug, adjuvant and the working condition of preparation is talked about improperly, produces easily clinically the secondary microbiological contamination, causes the more difficult healing of wound face, and therapeutic effect is very undesirable or ulcer surface produced larger harm.In addition, be exclusively used in the aseptic external used medicine of lisozima for the treatment of skin ulcer (ulcer, diabetic ulcer, postoperative ulcer behind decubital ulcer, scald ulcer, calf ulcer, the herpes zoster), particularly lisozima sterile milk paste formulation there is not yet report.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of lisozima external use sterile emulsifiable paste for the treatment of skin ulcer (ulcer, diabetic ulcer, postoperative ulcer behind decubital ulcer, scald ulcer, calf ulcer, the herpes zoster) and preparation method thereof is provided.
The term explanation:
Skin ulcer of the present invention refers to decubitus ulcer, scalds ulcer, ulcer, diabetic ulcer, postoperative ulcer after the calf ulcer, herpes zoster.
Lisozima, English name Lysozyme hydrochloride, CAS accession number 9066-59-5.
Detailed Description Of The Invention
A kind of lisozima external use sterile emulsifiable paste for the treatment of skin ulcer, raw materials quality part comprises following component:
A: 50～80 parts of lisozimas, enzymatic activity 6200～20000 units/mg;
B: 50～80 parts of span, 30～60 parts of poloxamers, 30～50 parts of tweens, 20～40 parts of sorbitol, 10～20 parts of sodium dihydrogen phosphate, 1～10 part of ethyl hydroxybenzoate;
C: 220～260 parts of liquid paraffin, 30～50 parts of glyceryl monostearates, 10～30 parts of hexadecanol, 10～30 parts of octadecanol, 70～100 parts of white vaseline;
D: 449～500 parts of sterilized water.
According to the present invention, preferred, a kind of lisozima external use sterile emulsifiable paste for the treatment of skin ulcer, raw materials quality part comprises following component:
A: 50～60 parts of lisozimas, enzymatic activity 12000～16000 units/mg;
B: 50～60 parts of Arlacel-40s, 30～50 parts of F68s, 30～40 parts of tween 80s, 20～30 parts of sorbitol, 10～15 parts of sodium dihydrogen phosphate, 1～5 part of ethyl hydroxybenzoate;
C: 220～240 parts of liquid paraffin, 30～40 parts of glyceryl monostearates, 10～20 parts of hexadecanol, 10～20 parts of octadecanol, 70～80 parts of white vaseline;
D: 450～470 parts of sterilized water.
The above-described technical scheme according to the present invention, B organizes each component and adds the sterilized water dissolving, as water; C organizes each component melts, and as oil phase, water and oil phase be 121～125 ℃ of high temperature sterilizes respectively; A component lisozima adds sterilized water dissolving, membrane filtration degerming; Water after the sterilization, lisozima and oil phase mix, shear, stir, and make aseptic emulsifiable paste.
According to the present invention, the preparation method of described lisozima sterile milk paste formulation comprises the steps:
(1) in 100,000 grades of clean areas, take by weighing B by proportioning and organize each component, add an amount of sterilized water dissolving, as water;
(2) in 100,000 grades of clean areas, take by weighing C by proportioning and organize each component, melting in 65～70 ℃ of water-baths is as oil phase;
(3) in 100,000 grades of clean areas, take by weighing A component lisozima by proportioning, add the sterilized water dissolving of surplus, after 0.22 μ m membrane filtration degerming, gained lisozima feed liquid is delivered in hundred grades of sterilized tanks of sterile workshop, room temperature is for subsequent use;
(4) oil phase that the water that step (1) is made and step (2) make was sterilized 30 minutes under 121～125 ℃ of conditions respectively, was delivered to respectively in hundred grades of sterilized tanks of sterile workshop and deposited, and is for subsequent use 65～70 ℃ of insulations;
(5) under hundred grades of aseptic conditions, the lisozima feed liquid that step (3) is made and step (4) insulation water for subsequent use mixes, and stirs, and it is for subsequent use to be warming up to 65～70 ℃ of insulations, gets the lisozima aqueous phase liquid;
(6) under hundred grades of aseptic conditions, under stirring step (4) insulation oil phase for subsequent use is joined in the lisozima aqueous phase liquid of step (5), under 65～70 ℃ of conditions of temperature, 1000～1750r/min low velocity shear is 8～10 minutes first, and then 2500～2880r/min high speed shear is 4～5 minutes; Stop to shear, open and stir, rotating speed is 60～90r/min, continues to stir 5～10 minutes, and then is that 30～50r/min slowly stirs at speed of agitator, begins cooling, stops when being cooled to 30～35 ℃ stirring, and makes lisozima sterile milk paste formulation product.
The said goods is carried out aseptic packaging, check, warehouse-in.
The operation of above-mentioned steps (6) is to carry out in shearing tank, and shearing tank is this area conventional equipment.
Need to prove that in above preparation method, the pipeline of the mass transport that is useful on must pass through sterilization treatment in advance.
With regard to preparation method, more than be the preferred method of the present invention, but be not limited to this.Those skilled in the art can suitably be adjusted according to its knowledge of knowing.Be not particularly limited in the above lisozima sterile milk paste formulation preparation method, can be with reference to prior art.
Characteristics of the present invention:
1, the lisozima emulsifiable paste is local skin external use sterile preparation, and most critical is exactly sterilization technology among the preparation technology.The physicochemical property of each composition of pharmaceutical adjunct according to the present invention, sticky because of oil phase substrate such as Filtration if adopt non-heat sterilization method, be difficult to filter.Through considering the characteristics of this preparation, the present invention adopts high-temperature heat sterilization technique that water, the oil phase of pharmaceutical adjunct are sterilized especially.
2, degeneration easily occuring, is unsuitable for adopting high temperature sterilization under the formulation products effective ingredient lisozima hot conditions of the present invention.Consider that lisozima dissolubility in water is better, in this product production process, the present invention will join aqueous phase after the lisozima dissolving first again, selects the Filtration sterilization, has both met the state-promulgated pharmacopoeia requirement to guarantee this preparation, can reach again the purpose of sterilization.
Lisozima emulsifiable paste sterile preparation quality standard is following but be not limited to this in the preparation method of the present invention.
Lisozima sterile milk paste formulation quality standard
The hydrochloric lysozyme of this product must not be less than 90% of labelled amount.
[shape] this product is the milky ointment
[discriminating]
(1) gets this product an amount of (being equivalent to the 2mg lisozima), add water 5ml and make dissolving, add 1 of 5 of 10% sodium hydroxide solutions and 10% copper-bath, displaing amaranth behind the mixing.
(2) it is an amount of to get this product, and (get anhydrous sodium acetate 6.7g, add the about 900ml of water, jolting makes dissolving, regulates pH value to 5.4 with acetic acid, and thin up shakes up to 1000ml to add acetic acid-sodium-acetate buffer.) make the solution of hydrochloric lysozyme 0.4mg among every 1ml, measure according to spectrophotography " two appendix IV of Chinese Pharmacopoeia version in 2010 A ", at the wavelength place of 280nm absorption maximum is arranged, trap should be 0.39～0.49.
[inspection]
Dissolubility is got this product 1g, adds water 20ml and stirs, and should be able to dissolve fully.
PH value is got this product 1g and is dissolved in the 20ml distilled water, is measuring pH value, should be at 3.5～6.5.
Viscosity is according to " two appendix VI of Chinese Pharmacopoeia version in 2010 G the second method "
Granularity checks according to granularity and particle size distribution method " Chinese Pharmacopoeia version appendix in 2010 IX E first method ", all must not detect the particle greater than 180um.
Aseptic according to Sterility Test " Chinese Pharmacopoeia version appendix in 2010 XI H " inspection, should be up to specification.
Other this product should meet other requirements under " Chinese Pharmacopoeia version appendix in 2010 I F " emulsifiable paste item.
[lysozyme content]
The preparation of need testing solution takes by weighing emulsifiable paste (5%) an amount of (being equivalent to lisozima 25mg), puts in the test tube, adds chloroform 5ml, after breakdown of emulsion is complete, with the extraction of PH6.2 phosphate buffered solution, puts in the separatory funnel, gets the upper strata.Extract 3 times, each 20ml with 20mlPH6.2 phosphate buffered solution flushing test tube and separatory funnel, is incorporated in 100 milliliters of measuring bottles again, decides scale with the PH6.2 phosphate buffered solution.Precision pipettes 2mL and puts in the 50ml volumetric flask, adds the pH6.2 phosphate buffer to scale, and is for subsequent use as need testing solution.
Standard solution prepares precision and takes by weighing 25 milligrams of lisozima standard substance, puts in the 100ml volumetric flask, adds the dissolving of phosphate buffer solution, is settled to scale.Precision pipettes 1mL respectively, 2mL solution is put in the 50ml volumetric flask, adds the PH6.2 phosphate buffered solution to scale, as standard solution (1) and standard solution (2).Sample solution, standard solution (1) standard solution (2) is put in the ice bath and is preserved
The preparation of substrate suspension takes by weighing hydrochloric acid lyase micrococcus 15～20mg, add phosphate buffer (pH6.2) 0.5～1ml, in mortar, ground 3 minutes, it is an amount of to add phosphate buffer (pH6.2) again, make cumulative volume be about 50ml, make suspension in the time of 25 ± 0.1 ℃, the trap that records at the wavelength place of 450nm is 0.70 ± 0.05 (before use preparation).
The algoscopy precision is measured lisozima substrate test solution 4ml, heating is 5 minutes in 35 ℃ of water-baths, precision pipettes the sample solution 100 μ l that heated in advance in water-bath 3 minutes, add in the substrate test solution, and in 35 ℃ of water-baths, heat after 10 minutes, accurate hydrochloric acid solution 0.5mL of measuring 1mol/L adds the end wherein again, shakes up.With water as blank, at wavelength 640nm place, the absorption value Ast of UV-VIS spectrophotometry sample solution.Standard solution (1), standard solution (2) are measured absorbance A s by above method
1, As
2
The unit of every g lysozyme=
Ws: the lysozyme standard substance amount of taking by weighing
Wt: the sample amount of taking by weighing * specification
As
1: standard solution (1) trap
As
2: standard solution (2) trap
The lisozima emulsifiable paste sterile preparation of the external used medicine for the treatment of skin ulcer of the present invention (ulcer, diabetic ulcer, postoperative ulcer behind decubital ulcer, scald ulcer, calf ulcer, the herpes zoster), adopting lisozima is the effective ingredient of medicine of the present invention, with selected pharmaceutic adjuvant, make medicament by techniques such as dissolving, high temperature sterilize, membrane filtration degerming, low speed homogenizing, high speed homogenization, stirring, cooling, sterile fillings.Through the external preparation for skin drug administration, the skin ulcer of different times (decubital ulcer, scald ulcer behind ulcer, calf ulcer, the herpes zoster, diabetic ulcer, postoperative ulcer) has been shown that all it has preferably clinical effect.More detailed effect experiment will be explained in an embodiment.
Description of drawings
Fig. 1-Fig. 8 is the rabbit skin irritation result of the test figure that makes as test sample of the aseptic emulsifiable paste of the lisozima of embodiment 3.Fig. 1-8 shows that all epidermis is complete, without hyperemia and inflammatory cell infiltration.Wherein,
Fig. 1 is rabbit head/intact skin medication side figure, pathology numbering: T429-10-1, and HE5 *;
Fig. 2 is rabbit head/intact skin control sides figure, pathology numbering: T429-10-2, and HE5 *;
Fig. 3 is rabbit tail/intact skin medication side figure, pathology numbering: T430-10-1, and HE5 *;
Fig. 4 is rabbit tail/intact skin control sides figure, pathology numbering: T430-10-2, and HE5 *;
Fig. 5 be rabbit left front/damaged skin medication side figure, pathology numbering: T431-10-1, HE5 *;
Fig. 6 be rabbit left front/damaged skin control sides figure, pathology numbering: T431-10-2, HE5 *;
Fig. 7 is before the rabbit right/damaged skin medication side figure, pathology numbering: T432-10-1, and HE5 *;
Fig. 8 is before the rabbit right/damaged skin control sides figure, pathology numbering: T432-10-2, and HE5 *.
The specific embodiment
The present invention is described further below in conjunction with embodiment, but be not limited to this.
The explanation of embodiment 1-3 supplementary material:
Embodiment 1, lisozima sterile milk paste formulation and preparation
1, raw and auxiliary material inventory proportioning (mass ratio):
Lisozima: span: poloxamer: tween: sorbitol: sodium dihydrogen phosphate: ethyl hydroxybenzoate: liquid paraffin: glyceryl monostearate: hexadecanol: octadecanol: white vaseline: sterilized water=50: 50: 30: 30: 20: 10: 1: 220: 30: 10: 10: 70: 450.
2, preparation
(1) in 100,000 grades of clean area; Get Arlacel-80 5000g, poloxamer-683000g, Tween-40 3000g, sodium dihydrogen phosphate 1000g, ethyl hydroxybenzoate 100g and sorbitol 2000g, add sterilized water 30000g dissolving, as water;
(2) in 100,000 grades of clean areas; Get liquid paraffin 22000g, hexadecanol 1000g, octadecanol 1000g, white vaseline 7000g, glyceryl monostearate 3000g, melting in 65～70 ℃ of water-baths is as oil phase;
(3) in 100,000 grades of clean areas; Get lisozima 5000g, add among the sterilized water 15000g, by sterilized material conveying pipe with 0.22 μ m membrane filtration degerming after; In hundred grades of sterile workshop of suction, room temperature is for subsequent use;
What the water that (4) step (1) is made and step (2) made sterilized 30 minutes under 121～125 ℃ of conditions respectively, by sterilized material conveying pipe, feed liquid is delivered to respectively placement in hundred grades of sterilized tanks of sterile workshop, for subsequent use 65～70 ℃ of insulations;
(5) in hundred grades of sterile workshop, the lisozima feed liquid that step (3) makes is incubated water for subsequent use with step (4) mixes, and stirs, and it is for subsequent use to be warming up to 65～70 ℃ of insulations, gets the lisozima aqueous phase liquid;
(6) in hundred grades of sterile workshop; Continuous the stirring step (4) insulation oil phase for subsequent use is joined in the lisozima aqueous phase liquid of step (5), the suction of gained mixture is sheared in the tank; Under 65～70 ℃ of conditions of temperature, open cutter low speed 1000～1650r/min and sheared 10 minutes, then 2500～2780r/min sheared 5 minutes at a high speed; Close cutter, open and stir; Rotating speed is 60～90r/min, continue to stir 5～10 minutes, and then is that 30～50r/min slowly stirs at speed of agitator, begins cooling, stops when being cooled to 33 ℃ stirring.Make lisozima sterile milk paste formulation product.
(7) product aseptic packaging, check, warehouse-in.
Embodiment 2, lisozima sterile milk paste formulation and preparation
1, raw and auxiliary material inventory proportioning (mass ratio):
Lisozima: span: poloxamer: tween: sorbitol: sodium dihydrogen phosphate: ethyl hydroxybenzoate: liquid paraffin: glyceryl monostearate: hexadecanol: octadecanol: white vaseline: sterilized water=60: 60: 50: 40: 30: 15: 5: 240: 40: 20: 20: 80: 470.
2, preparation
(1) in 100,000 grades of clean area; Take by weighing sorbester p18 00g, polysorbate40 00g, poloxamer 5000g, sodium dihydrogen phosphate 1500g, ethyl hydroxybenzoate 500g and sorbitol 3000g, add sterilized water 30000g, and dissolving, as water;
(2) in 100,000 grades of clean areas; Take by weighing liquid paraffin 24000g, hexadecanol 2000g, octadecanol 2000g, white vaseline 8000g, glyceryl monostearate 4000g, melting in 65～70 ℃ of water-baths is as oil phase;
(3) in 100,000 grades of clean areas; Take by weighing lisozima 6000g, add among the sterilized water 17000g, by sterilized material conveying pipe with 0.22 μ m membrane filtration degerming after; In hundred grades of sterile workshop of suction, room temperature is for subsequent use;
Step (4), (5) are identical with the corresponding steps of embodiment 1.
(6) in hundred grades of sterile workshop; Continuous the stirring step (4) insulation oil phase for subsequent use is joined in the lysozyme aqueous phase liquid of step (5), the suction of gained mixture is sheared in the tank; Under 67～70 ℃ of conditions of temperature, open cutter low speed 1200～1750r/min and sheared 9 minutes, then 2600～2880r/min sheared 5 minutes at a high speed; Close cutter, open and stir; Rotating speed is 70～90r/min, continue to stir 5～10 minutes, and then is that 40～50r/min slowly stirs at speed of agitator, begins cooling, stops when being cooled to 32 ℃ stirring.Make lisozima sterile milk paste formulation product.
(7) product aseptic packaging, check, warehouse-in.
Embodiment 3, lisozima sterile milk paste formulation and preparation
1, raw and auxiliary material inventory proportioning (mass ratio):
Lisozima: span: poloxamer: tween: sorbitol: sodium dihydrogen phosphate: ethyl hydroxybenzoate: liquid paraffin: glyceryl monostearate: hexadecanol: octadecanol: white vaseline: sterilized water=55: 55: 40: 35: 25: 12.5: 3: 230: 35: 15: 15: 75: 460.
2, preparation
(1) in 100,000 grades of clean areas, take by weighing span 5500g, poloxamer 4000g, tween 3500g, sodium dihydrogen phosphate 1250g, ethyl hydroxybenzoate 300g and sorbitol 2500g, add sterilized water 30000g dissolving, as water;
(2) in 100,000 grades of clean areas, take by weighing liquid paraffin 23000g, hexadecanol 1500g, octadecanol 1500g, white vaseline 7500g, glyceryl monostearate 3500g, melting in 65～70 ℃ of water-baths is as oil phase;
(3) in 100,000 grades of clean areas, take by weighing lisozima 5500g, add in the 16000g sterilized water, by sterilized material conveying pipe with 0.22 μ m membrane filtration degerming after; In hundred grades of sterile workshop of suction, room temperature is for subsequent use;
Step (4), (5) are identical with the corresponding steps of embodiment 1.
(6) in hundred grades of sterile workshop; Continuous the stirring step (4) insulation oil phase for subsequent use is joined in the lisozima aqueous phase liquid of step (5), the suction of gained mixture is sheared in the tank; Under 66～70 ℃ of conditions of temperature, open cutter low speed 1300～1750r/min and sheared 9 minutes, then 2700～2880r/min sheared 4 minutes at a high speed; Close cutter, open and stir; Rotating speed is 80～90r/min, continue to stir 5～10 minutes, and then is that 30～50r/min slowly stirs at speed of agitator, begins cooling, stops when being cooled to 31 ℃ stirring.Make lisozima sterile milk paste formulation product.
(7) aseptic packaging, check, warehouse-in.
Embodiment 4, the aseptic emulsifiable paste Cavia porcellus of lisozima Buehler experiment
Get the aseptic emulsifiable paste of lisozima of embodiment 1 as test sample.Described drug concentration percentage ratio is mass percent.
Get 40 of healthy male guinea pigs, be divided into immediately 20 of 10 of excipient matched groups, 2,10 of 4-dinitrochlorobenzene positive controls and the aseptic emulsifiable paste test sample of lisozima groups by body weight, minute box is raised.24h is with the about 3 * 3cm of Cavia porcellus left of spine rib abdominal part area before the medicine
2Scrape hair, positive controls takes by weighing 1%2,4-dinitrochlorobenzene 0.2g, and excipient matched group and test sample matched group take by weighing respectively each 0.2g of the aseptic emulsifiable paste of lisozima, is applied to depilation district, Cavia porcellus left side, softly with cellophane and 2.5 * 2.5cm
2Two layers of gauze cover, again with fixture with drug blockage, fixing.Use the medicine of warm water cleaning application area after 6 hours.Sensitization 1 time is repeated with the same manner in the 7th day and the 14th day, for 3 times.Depilation district, 14 days right sides uses first warm water cleaning administration place skin after this sensitization, then positive controls take by weighing 0.1%2,4-dinitrochlorobenzene 0.2g, excipient matched group and test sample matched group take by weighing respectively each 0.2g of the aseptic emulsifiable paste of lisozima, be coated with and depilation district, Cavia porcellus right side, softly with cellophane and 2.5 * 2.5cm
2Two layers of gauze cover, again with fixture with drug blockage, fixing.6 hours with warm water cleaning smear zone medicine.Excite remove medicine after 24h, 48h examine skin erythema, edema and other abnormal responses, press table 1 pair erythema and edema the scoring, calculate irritated incidence rate.Judge the anaphylaxis occurrence degree according to table 2.
The standards of grading of table 1 skin allergy degree
| Dermoreaction intensity | Score value |
| Erythema | |
| Without erythema | 0 |
| Slight visible erythema | 1 |
| Moderate erythema | 2 |
| Serious erythema | 3 |
| Swollen property erythema | 4 |
| Edema | |
| Without edema | 0 |
| Mild edema | 1 |
| Intermediate edema | 2 |
| Serious edema | 3 |
| Maximum total mark | 7 |
Reaction meansigma methods=(erythema forms total points+edema and forms total points)/total number of animals.
Table 2 skin hypersensitivity evaluation criterion test sample group and excipient contrast
| Anaphylaxis incidence rate (%) | Classification | Anaphylaxis intensity |
| 0-8 | I | Hyposensitiveness |
| 9-18 | II | Slight sensitization |
| 29-64 | III | Moderate sensitization |
| 65-80 | IV | Strong sensitization |
| 81-100 | V | Extremely strong sensitization |
The number of animals of anaphylaxis incidence rate=occur skin erythema or edema/animal subject number.
Duration of test, observe Cavia porcellus every day, and the aspects such as external feature, the mental status, behavioral activity and the breathing of Cavia porcellus show no obvious abnormalities.For the first time before the sensitization, last sensitization and weigh when exciting and calculate every group of each body weight
Each group compares with vehicle group, as a result no significant difference.The results are shown in Table 3
The aseptic emulsifiable paste of table 3 lysozyme is on the impact of Cavia porcellus body weight gain
| Group | First sensitization | Last sensitization | Excite |
| The excipient matched group | 341.3±5.0 | 375.2±5.0 | 408.8±5.4 |
| Positive controls | 343.9±12.0 | 377.5±11.9 | 410.9±12.0 |
| The test sample group | 345.4±18.6 | 379.0±18.4 | 412.3±18.5 |
Annotate: matched group n=10, test sample group n=20
Test sample group and excipient matched group Cavia porcellus lasing region skin erythema and edema all do not occur at each time point, and the reaction meansigma methods is 0, and the anaphylaxis incidence rate is 0, and sensitivity response is negative.Positive controls Cavia porcellus lasing region skin erythema and edema all occur removing medicine 24h, 48h, and the reaction meansigma methods is respectively 1.7 and 1.8, and the anaphylaxis incidence rate is 100%, is extremely strong sensitization.The results are shown in Table 4
The initiatively skin allergy scoring of table 4 Cavia porcellus
Under this experiment condition, the aseptic emulsifiable paste of lisozima to Cavia porcellus without obviously initiatively sensitization of skin effect.
Embodiment 5, the test of the aseptic emulsifiable paste rabbit of lisozima skin irritation
Get the aseptic emulsifiable paste of lisozima of embodiment 3 and do this experiment as test sample.Experimental result such as Fig. 1-shown in Figure 8:
Wherein, Fig. 1, subcutaneous group, epidermis, Skin appendages be complete, has no hyperemia, degeneration, necrosis and inflammatory reaction (head/intact skin medication side); Fig. 2, epidermis is complete, without hyperemia and inflammatory cell infiltration (head/intact skin control sides); Fig. 3, subcutaneous group, epidermis, Skin appendages be complete, has no hyperemia, degeneration, necrosis and inflammation (tail/intact skin medication side); Fig. 4, epidermis is complete, without hyperemia and inflammatory cell infiltration (tail/intact skin control sides); Fig. 5, epidermis injury has inflammatory cell infiltration and exudate (left front/damaged skin medication side); Fig. 6, epidermis injury has inflammatory cell infiltration and exudate (left front/the damaged skin control sides); Fig. 7, epidermis injury has inflammatory cell infiltration and exudate (right front/damaged skin medication side); Fig. 8, epidermis injury has inflammatory cell infiltration and exudate (right front/the damaged skin control sides).
Get 8 of the rabbit of healthy adult, ♀ ♂ half and half.Normal skin is evaded hair in front 24 hours of administration with the electricity consumption of back part of animal spinal column diamond wool, and the depilation area is 3 * 3cm
2The damaged skin depilating method prepares with normal skin.The depilation district is with 75% ethanol partly sterilised, with local " # " shape cut of aseptic syringe needle depilation, take oozing of blood as degree.Adopt consubstantiality left and right sides self-contrast method.Use first 75% ethanol partly sterilised during the administration of normal skin group, take by weighing each 0.5g of test sample and excipient, be applied to respectively rabbit right, left side depilation district (administration area 3 * 3cm
2), then cover with double gauze and one deck cellophane, sealed, fixed with fixture again.Use the medicine of warm water cleaning application area after 4 hours.Each administration should be continuously in the administration of same position, and the damaged skin group gives medicine with method.Administration every day 1 time, successive administration 7 days.The last administration removes after the tested material 1,24,48,72h observes and the irritant reaction situations such as position erythema and edema smeared in record, and recovery situation and the recovery time of above-mentioned variation.The air tap inserting method is put to death 1/2 animal after observing end, and then ♀ ♂ half and half fixes institute's bark fetching skin tissue with 4% neutral formalin solution, carries out histopathological examination after the HE dyeing.The residue animal continues to observe 14 days, again carries out in case of necessity histopathological examination.According to observed and recorded erythema and edema etc. before table 5 couple removal medicine 1h and the rechallenge, and to erythema and edema scoring.Calculated to put each observing time and respectively organized integral mean value, then calculated and observe every animal integrated value in the time limit, carried out the stimulus intensity evaluation by table 6.
Table 5 skin irritation reaction standards of grading
| Irritant reaction | Score value |
| Erythema | |
| Without erythema | 0 |
| Slight erythema (reluctantly as seen) | 1 |
| Moderate erythema (obviously as seen) | 2 |
| The severe erythema | 3 |
| The aubergine erythema forms to slight eschar | 4 |
| Edema | |
| Without edema | 0 |
| Mild edema (reluctantly as seen) | 1 |
| Intermediate edema (obviously protuberance) | 2 |
| Severe edema (cutaneous protuberance 1mm, profile is clear) | 3 |
| Serious edema (cutaneous protuberance 1mm above and have expansion) | 4 |
| High total mark | 8 |
Table 6 skin irritation intensity evaluation
| Score value | Estimate |
| 0-0.49 | Nonirritant |
| 0.5-2.99 | Slight zest |
| 3.0-5.99 | The moderate zest |
| 6.0-8.0 | Strong and stimulating |
Duration of test, the rabbit general status is good, before each administration, medicine is removed behind the 1h and the last administration remove behind the medicine 1,24,48 and 72h and convalescent period observe and observe the medication part after finishing and have no the symptoms such as obvious erythema, edema, pigmentation, petechia, pachylosis or epidermatic atrophy, the scoring score value is 0.
Pathologic finding as seen, intact skin group medication (seeing accompanying drawing 1, accompanying drawing 3) side undertissue, epidermis, adnexal structure are complete, clear, have no congested degeneration, necrosis and inflammatory reaction, relatively have no obvious difference with the excipient control sides; Control sides (seeing accompanying drawing 2, accompanying drawing 4) epidermis is complete, without hyperemia and inflammatory cell infiltration etc.; Damaged skin group medication side (seeing accompanying drawing 5, accompanying drawing 7) is drawn materials and is organized all visible epidermis injury, inflammatory reaction and exudate, relatively has no notable difference with the excipient control sides.Due to the mechanical damage, infer with medicine irrelevant in the time of may preparing with damaged skin.The visible epidermis injury of damaged skin control sides (seeing accompanying drawing 6, accompanying drawing 8), inflammatory cell infiltration and exudate.
Under this experimental condition, the aseptic emulsifiable paste of lisozima to the rabbit skin histology without the obvious stimulation effect.
Claims (2)
1. lisozima external use sterile emulsifiable paste for the treatment of skin ulcer, raw materials quality part comprises following component:
A: 50～80 parts of lisozimas, enzymatic activity 6200～20000 units/mg;
B: 50～80 parts of span, 30～60 parts of poloxamers, 30～50 parts of tweens, 20～40 parts of sorbitol, 10～20 parts of sodium dihydrogen phosphate, 1～10 part of ethyl hydroxybenzoate;
C: 220～260 parts of liquid paraffin, 30～50 parts of glyceryl monostearates, 10～30 parts of hexadecanol, 10～30 parts of octadecanol, 70～100 parts of white vaseline;
D: 449～500 parts of sterilized water;
Make as follows:
(1) in 100,000 grades of clean areas, take by weighing B by proportioning and organize each component, add an amount of sterilized water dissolving, as water;
(2) in 100,000 grades of clean areas, take by weighing C by proportioning and organize each component, melting in 65～70 ℃ of water-baths is as oil phase;
(3) in 100,000 grades of clean areas, take by weighing A component lisozima by proportioning, add the sterilized water dissolving of surplus, after 0.22 μ m membrane filtration degerming, gained lisozima feed liquid is delivered in hundred grades of sterilized tanks of sterile workshop, room temperature is for subsequent use;
(4) oil phase that the water that step (1) is made and step (2) make was sterilized 30 minutes under 121～125 ℃ of conditions respectively, was delivered to respectively in hundred grades of sterilized tanks of sterile workshop and deposited, and is for subsequent use 65～70 ℃ of insulations;
(5) under hundred grades of aseptic conditions, the lisozima feed liquid that step (3) is made and step (4) insulation water for subsequent use mixes, and stirs, and it is for subsequent use to be warming up to 65～70 ℃ of insulations, gets the lisozima aqueous phase liquid;
(6) under hundred grades of aseptic conditions, under stirring step (4) insulation oil phase for subsequent use is joined in the lisozima aqueous phase liquid of step (5), under 65～70 ℃ of conditions of temperature, 1000～1750r/min low velocity shear is 8～10 minutes first, and then 2500～2880r/min sheared 4～5 minutes at a high speed; Stop to shear, open and stir, rotating speed is 60～90r/min, continues to stir 5～10 minutes, and then is that 30～50r/min slowly stirs at speed of agitator, begins cooling, stops when being cooled to 30～35 ℃ stirring, and makes lisozima sterile milk paste formulation product.
2. the lisozima external use sterile emulsifiable paste for the treatment of skin ulcer as claimed in claim 1 is characterized in that raw materials quality part comprises following component:
A: 50～60 parts of lisozimas, enzymatic activity 12000～16000 units/mg;
B: 50～60 parts of Arlacel-40s, 30～50 parts of F68s, 30～40 parts of tween 80s, 20～30 parts of sorbitol, 10～15 parts of sodium dihydrogen phosphate, 1～5 part of ethyl hydroxybenzoate;
C: 220～240 parts of liquid paraffin, 30～40 parts of glyceryl monostearates, 10～20 parts of hexadecanol, 10～20 parts of octadecanol, 70～80 parts of white vaseline;
D: 450～470 parts of sterilized water.
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| CN104758310B (en) * | 2015-04-01 | 2018-10-12 | 山东司邦得制药有限公司 | Compound montmorillonite sucrose ointment and its preparation method and application |
| CN104771750A (en) * | 2015-04-01 | 2015-07-15 | 山东司邦得制药有限公司 | Compound montmorillonite lysozyme powder as well as preparation method and application thereof |
| CN105963689A (en) * | 2016-05-11 | 2016-09-28 | 山东司邦得制药有限公司 | Compound hydrochloric acid lysozyme ointment and preparation method and application thereof |
| CN105963761B (en) * | 2016-05-11 | 2019-03-22 | 山东司邦得制药有限公司 | A kind of lisozima adhesive bandage and its preparation method and application |
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