By inquiry, the pH range of the ophthalmic preparation is 6-8, and the optimal pH is 7.04; the osmotic pressure range is about 300 isotonic, the formula and the proportion are inspected and the two index detection results are screened, the qualified samples of the two indexes correspond to the formula example 5, the samples are filtered by a 0.22 mu m filter membrane and then are subpackaged, and the observation of the character visible foreign matters is carried out.

Formulation example 5 was expanded to examine the effect of different amounts of hydroxypropyl methylcellulose and poloxamer 188 on the formulation as follows:

through the experiments, in the study of the prescription example 5 series, the use amount of poloxamer 188 exceeds 0.08, and when the use amount reaches 0.09, the eye drops can generate precipitates in about 7 days. When poloxamer 188 reaches 0.1, the eye drops can directly generate precipitates, and the eye drops are turbid. Since the recombinant type III humanized collagen used in the present invention has extremely high water solubility, precipitation occurs, which should be caused by protein denaturation.

In the cases offormulation 10/11/13, it was found that no precipitation occurred when poloxamer 188 was used at 0.5 without using hydroxypropylmethyl cellulose, poloxamer 188, or disodium edetate system. In the search for these formulations, precipitation occurred only when poloxamer 188 reached around 1. Therefore, the preliminary judgment can be made that the addition of the disodium ethylene diamine tetraacetate and the hydroxypropyl methylcellulose can reduce the use of the poloxamer 188, so that the effect of stabilizing the solution can be achieved under the low concentration. Incidentally, when the concentration of poloxamer 188 reached 0.09, the protein was affected.

2. Prescription screening and formulation analysis

As inquired, the pH range of the ophthalmic preparation is 6-8, and the optimal pH is 7.04; the osmotic pressure range is about 300 isotonic, so the formulation analysis is preferably carried out according to the prescription example 5/10/11/13.

Preparation process of formula example 5: according to the formula, poloxamer 188, hydroxypropyl methylcellulose, ethylene diamine tetraacetic acid disodium, sodium chloride and anhydrous disodium hydrogen phosphate are weighed and dissolved in the formula amount of water for injection; sterilizing at 115 deg.C for 30min, proportionally adding recombinant type III humanized collagen filtered by 0.22 μm filter membrane, mixing, measuring pH and osmotic pressure, and packaging.

Preparation process of formula example 10/11/13: dissolving anhydrous sodium dihydrogen phosphate, anhydrous disodium hydrogen phosphate, sodium chloride and poloxamer 188 in the formula amount of water for injection; sterilizing at 115 deg.C for 30min, proportionally adding recombinant type III humanized collagen filtered by 0.22 μm filter membrane, mixing, measuring pH and osmotic pressure, and packaging.

2.1 clarity examination

Taking 1mL of the product, and detecting the clarity and color of the eye drops according to the first method of 0902 of the four general rules of the Chinese pharmacopoeia 2015 edition and the first method of 0901 of the four general rules of the Chinese pharmacopoeia 2015 edition.

Sample (I)	Clarity of the solution	Colour(s)
			Prescription 5	Transparent and free of impurities	Transparent andcolorless
Prescription
	10	Transparent and free of impurities	Transparent andcolorless
Prescription
	11	Transparent and free of impurities	Transparent andcolorless
Prescription
	13	Transparent and free of impurities	Transparent and colorless

From the above experimental results, it can be seen that the product has good clarity, is colorless and transparent, and meets the standards of pharmacopoeia for eye drops.

2.2 measurement of Nitrogen content

20mL of a control example (0.005g of recombinant III type humanized collagen dissolved in 100g of water) and 5/10/11/13 of a prescription example are precisely weighed, placed in a digestion tube of a dry full-automatic nitrogen determinator, then 0.3g of anhydrous sodium sulfate and 5 drops of 30 v/v% copper sulfate solution are added, 2.0mL of sulfuric acid with the concentration higher than 98 v/v% is added along the bottle wall dropwise, the digestion tube is added into the digester and kept for 5min at 150 ℃, 5min at 350 ℃ and 60-80 min at 400 ℃ until the solution becomes clear green, then digestion is continued for 10min, and the digestion tube is taken out, cooled and the nitrogen content is directly determined by the full-automatic nitrogen determinator.

Sample (I)	Determination of content
		Comparative example	100.0％
Prescription
	5	99.2％
Prescription
	10	99.5％
Prescription
	11	98.9％
Prescription
	13	98.8％

Through nitrogen content measurement, it can be seen that, in the prepared prescription composition, the nitrogen content measurement result is greater than 98%, which proves that the protein has good stability in the prescription and the content measurement is qualified.

2.3 protein Activity assays

(1) Comparative examples, prescription examples and prescription example controls were prepared according to the following table

(2) 100 μ L of each protein solution and a blank PBS solution control were added to a 96-well plate and allowed to stand at room temperature for 60 min.

(3) Adding 10 into each hole⁵In the form of a cultureThe NIH3T3 cells in good state were incubated at 37 ℃ for 60 min.

(4) Each well was washed 4 times with PBS.

(5) The absorbance of OD492nm was detected with LDH detection kit (Roche, 04744926001). According to the value of the blank control, the adherence rate of the cells can be calculated. The calculation formula is as follows: cell adherence rate ═ 100%/(positive well-blank well). The anchorage rate of the cells can reflect the activity of the collagen. The higher the activity of the protein, the better the external environment can be provided for the cells in a short time, and the cells are attached to the wall.

As shown in FIG. 1, it is understood that the cell adhesion activity offormula 5/10/11/13 is similar to that of the control, while no cell adhesion activity was detected in the control 5 '/10'/11 '/13', and the effect offormula 5/10/11/13 on the cell adhesion activity was small.

Through the study of the above examples, the formulation and preparation process of the recombinant type III humanized collagen-containing composition were determined as follows:

example 2: pharmacodynamic study of composition containing recombinant type III humanized collagen

1. Pharmacodynamic experimental method

(1) Female rats of 6-8 weeks, 25 in total, were selected and divided into 5 groups. One of the saline (containing no recombinant type III humanized collagen) control groups and the administered group 4 (formula 5/10/11/13, respectively).

(2) The rats of the control group and the administration group were injected with scopolamine hydrobromide of 0.5mL and 3mg/mL subcutaneously for 4 times a day, respectively at 8, 12, 16 and 20 points, continuously for 14 days, and a rat xerophthalmia model was established. Meanwhile, the rats in the control group and the administration group were dropped with physiological saline or the composition of the prescription example 5 times a day for 28 days at 8, 11, 14, 17 and 20 points, respectively.

(3) Tear secretion experiments and tear film rupture time experiments are respectively carried out on the rats of each group at 0d, 7d, 14d, 21d and 28d before model building and after drug administration, and data are recorded. The tear secretion test and tear film rupture time test methods are as follows:

tear secretion test: the amount of the lacrimal fluid secreted from the eyes of the rats was measured by performing a lacrimal fluid secretion test using phenol red cotton thread at the same time period of 0d, 7d, 14d, 21d, and 28d before the molding and after the administration. The eye surface of the rat was not treated with anesthetic (the secretory function of the major lacrimal gland was tested), and the phenol red cotton thread was held by forceps, placed in the conjunctival sac of the lower eyelid of the rat at 1/3, left for 30 seconds and removed. The length of the cotton wet was measured and recorded.

Tear film break up time test: 1 μ L of 10g/L sodium fluorescein was added dropwise to the conjunctival sac and the eyelids were closed and observed under a slit-lamp microscope under cobalt blue light. After 3 times of brief eyes, the time is counted by starting with the last 1 time of brief eyes, and the time until the cornea generates the 1 st black spot is the tear film rupture time.

2. Results of the experiment

Tear secretion test results are given in the following table:

tear film break up time(s) test results are given in the following table:

as can be seen from the results of pharmacodynamic experiments, after the administration of the formulation example 5/10/11/13, the secretion of the tears of the rats is obviously recovered, and the secretion of the tears before the model building can be basically approached within about 28 days. Meanwhile, the tear film rupture time is obviously prolonged compared with that of a control group, and is close to the data before model building. The above results demonstrate thatprescription 5/10/11/13 is effective in treating dry eye symptoms and increasing/restoring ocular lacrimal gland secretion.

Sequence listing

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<120> a composition containing recombinant type III humanized collagen and preparation method and application thereof

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<170> SIPOSequenceListing 1.0

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Gly Glu Arg Gly Ala Pro Gly Phe Arg Gly Pro Ala Gly Pro Asn Gly

1 5 10 15

Ile Pro Gly Glu Lys Gly Pro Ala Gly Glu Arg Gly Ala Pro

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Claims

1. A composition comprising the following components in mass percent: 0.05-2.1 percent of pH regulator, 0.2-1.0 percent of osmotic pressure regulator, 0.05-0.8 percent of stabilizer, 0.001-2 percent of recombinant III type humanized collagen, 0.02-0.1 percent of optional protective agent and the balance of water.

2. The composition of claim 1,

the pH regulator comprises at least one of anhydrous disodium hydrogen phosphate and anhydrous sodium dihydrogen phosphate;

the osmotic pressure regulator comprises sodium chloride;

the stabilizer comprises at least one of poloxamer 188 and hydroxypropyl methylcellulose;

the protective agent comprises disodium ethylenediaminetetraacetate.

3. The composition according to claim 1 or 2,

the composite material comprises the following components in percentage by mass: 1.2-2.1% of pH regulator, 0.2-1.0% of osmotic pressure regulator, 0.3-0.8% of stabilizer, 0.001-2% of recombinant type III humanized collagen and the balance of water, wherein the pH regulator is anhydrous disodium hydrogen phosphate and anhydrous sodium dihydrogen phosphate, the osmotic pressure regulator is sodium chloride, and the stabilizer is poloxamer 188;

or

The composite material comprises the following components in percentage by mass: 0.05-0.3% of pH regulator, 0.8-1.0% of osmotic pressure regulator, 0.05-0.28% of stabilizer, 0.02-0.1% of protective agent, 0.001-2% of recombinant III type humanized collagen and the balance of water, wherein the pH regulator is anhydrous disodium hydrogen phosphate, the osmotic pressure regulator is sodium chloride, the stabilizer is poloxamer 188 and hydroxypropyl methylcellulose, and the protective agent is disodium ethylene diamine tetraacetate.

4. The composition according to any one of claims 1 to 3, wherein the recombinant humanized collagen type III comprises n repeats of the sequence shown in SEQ ID No.1, n being an integer of 1 or more, wherein when n is an integer of 2 or more, the repeats are directly linked.

5. The composition of claim 4, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32, and wherein when n is an integer greater than or equal to 2, the repeating sequences are directly linked.

6. The composition of claim 4 or 5, wherein n is 16 and the repeats are directly linked.

7. A process for the preparation of a composition according to any one of claims 1 to 6, comprising the steps of:

s1: dissolving the formula amount of pH regulator, osmotic pressure regulator, stabilizer and optional protective agent in the formula amount of water, and sterilizing;

s2: adding the recombinant III type humanized collagen filtered by the filter membrane according to the proportion, and uniformly mixing to obtain the collagen.

8. A product comprising a composition according to any one of claims 1 to 6 and optionally pharmaceutically acceptable excipients.

9. Use of a composition according to any one of claims 1 to 6 in the manufacture of a product.

10. Use according to claim 9, wherein the product is a topical product, preferably an ophthalmic product, more preferably an eye drop.