CN113713086A - Composition containing recombinant III-type humanized collagen and preparation method and application thereof - Google Patents
Composition containing recombinant III-type humanized collagen and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a composition containing recombinant III-type humanized collagen and a preparation method and application thereof. Specifically, the composition comprises the following components in percentage by mass: 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, and can be used for preparing products such as eye drops and the like. The product of the invention can obviously improve the tear secretion of the rat with the xerophthalmia model, prolong the tear film rupture time and effectively relieve the symptoms of the xerophthalmia.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a composition containing recombinant III-type humanized collagen, and a preparation method and application thereof.
Background
Dry eye refers to a disorder of eye secretion caused by various factors and mainly manifested as dry eyes, often accompanied by itching, foreign body sensation, burning sensation in eyes, photophobia, blurred vision, and fluctuation of vision. Common symptoms comprise dry eyes, easy tiredness, eye itching, foreign body sensation, pain and burning sensation, viscous secretion, aversion to wind, photophobia and sensitivity to external stimulation; sometimes, eyes are too dry, basic tears are insufficient, and reflex tears are stimulated to secrete, so that frequent tears are caused; in more severe cases, the eyes become inflamed, engorged, keratinized, and the corneal epithelium is broken and the filaments are adhered, and the damage can cause keratoconjunctival lesion and affect the vision for a long time.
In the modern, young people use electronic products for a long time, the work intensity is high, the young people face computers for a long time, and the people chase after in the dark at night, xerophthalmia is easy to generate, and irreversible damage can be caused to the vision in the long past. There is therefore a need to develop more formulations to alleviate or cure dry eye, giving the patient more treatment options.
Disclosure of Invention
Problems to be solved by the invention
Aiming at the problem that the current human eyes are excessively easy to cause dry eye, the invention provides a composition (such as eye drops) containing the recombinant type III humanized collagen, which is used for preventing and/or treating the dry eye and provides more product choices for patients. The invention also provides a preparation method and application of the composition.
Means for solving the problems
In a first aspect, the present invention provides 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.
Further, the pH adjuster 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, and the protective agent comprises disodium ethylene diamine tetraacetate.
Preferably, the composition 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 composition 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.
Further, in the above composition, the recombinant humanized collagen type iii comprises n repeats of the sequence represented by SEQ ID No.1, n is an integer of 1 or more, wherein when n is an integer of 2 or more, the repeats are directly linked.
Preferably, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32, wherein when n is an integer of 2 or more, the repeated sequences are directly connected.
Preferably, n is 16, and the repeated sequences are directly connected.
In a second aspect, the present invention provides a process for preparing the above composition, 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.
In a third aspect, the present invention provides a product comprising the above composition and optionally pharmaceutically acceptable excipients.
In a fourth aspect, the present invention provides the use of the above composition in the manufacture of a product.
Further, the above-mentioned product is a topical product, preferably an ophthalmic product, more preferably an eye drop.
ADVANTAGEOUS EFFECTS OF INVENTION
Through the implementation of the technical scheme, the invention shows that the composition (such as eye drops) containing the recombinant III type humanized collagen can obviously improve the tear secretion amount of a rat with a dry eye syndrome model, prolong the tear film rupture time and effectively relieve the symptoms of the dry eye syndrome.
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FIG. 1 shows the results of the test of the effect of each formulation and control on the adhesion activity of NIH3T3 cells.
Detailed Description
The following describes embodiments of the present invention, but the present invention is not limited to these embodiments. The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims, and embodiments and examples obtained by appropriately combining the technical means disclosed in the respective embodiments and examples are also included in the technical scope of the present invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In the present specification, a numerical range represented by "a value to B value" or "a value to B value" means a range including the end point value A, B.
The recombinant type III humanized collagen of the present invention is described in patent application No. 201811438582.6 entitled "polypeptide, method for producing the same, and use thereof". The collagen has good hydrophilicity, the amino acid composition of the collagen is 100 percent same as the corresponding part of the amino acid sequence of the natural collagen, the collagen can not generate immunological rejection and anaphylactic reaction when being applied to human bodies, can be widely applied to the industries of biological medicine and cosmetics, and can realize the function of the natural protein in the human bodies.
Reference throughout this specification to "some particular/preferred embodiments," "other particular/preferred embodiments," "some particular/preferred aspects," "other particular/preferred aspects," or the like, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.
In one embodiment, the recombinant type iii humanized collagen of the present invention comprises N repeats of the sequence represented by SEQ ID No.1 (consisting of 30 amino acids, GERGAPGFRGPAGPNGIPGEKGPAGERGAP from segment N to segment C), N being an integer of 1 or more; wherein when n is an integer of 2 or more, the repeated sequences are directly connected.
In a preferred embodiment, the recombinant humanized type iii collagen of the present invention comprises n repeats of the sequence shown in SEQ ID No.1, n being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32; wherein when n is an integer of 2 or more, the repeated sequences are directly connected.
In a more preferred embodiment, the recombinant type III humanized collagen of the present invention comprises 16 repeats of the sequence shown in SEQ ID No.1, with direct linkage between the repeats.
In one embodiment, the composition of the invention comprises the following components in mass percent: 0.05-2.1% of pH regulator, 0.2-1.0% of osmotic pressure regulator, 0.05-0.8% of stabilizer, 0.001-2% of recombinant III type humanized collagen, 0.02-0.1% of optional protective agent and the balance of water; 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, and the protective agent comprises disodium ethylene diamine tetraacetate.
In one embodiment, the composition of the invention comprises the following components in mass percent: 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 III type 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.
In a preferred embodiment, the composition of the invention comprises the following components in mass percent: 0.7-1.1% of anhydrous sodium dihydrogen phosphate, 0.5-1% of anhydrous disodium hydrogen phosphate, 0.2-1.0% of sodium chloride, 1880.3-0.8% of poloxamer, 0.001-2% of recombinant III type humanized collagen and the balance of water.
In a more preferred embodiment, the composition of the invention comprises the following components in mass percent: 1.0% of anhydrous sodium dihydrogen phosphate, 0.8% of anhydrous disodium hydrogen phosphate, 0.34% of sodium chloride, 1880.5% of poloxamer, 0.005% of recombinant type III humanized collagen and the balance of water.
In one embodiment, the composition of the invention comprises the following components in mass percent: 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.
In a preferred embodiment, the composition of the invention comprises the following components in mass percent: 1880.03-0.08% of poloxamer, 0.02-0.2% of hydroxypropyl methylcellulose, 0.02-0.1% of ethylene diamine tetraacetic acid disodium, 0.8-1.0% of sodium chloride, 0.05-0.3% of anhydrous disodium hydrogen phosphate, 0.001-2% of recombinant III type humanized collagen and the balance of water.
In a more preferred embodiment, the composition of the invention comprises the following components in mass percent: 1880.05% of poloxamer, 0.1% of hydroxypropyl methylcellulose, 0.05% of ethylene diamine tetraacetic acid, 0.9% of sodium chloride, 0.09% of anhydrous disodium hydrogen phosphate, 0.005% of recombinant III type humanized collagen and the balance of water.
Unless otherwise indicated, instruments, reagents, materials, laboratory animals and the like used in the present invention are commercially available in a conventional manner.
Example 1: prescription screening and formulation analysis of compositions containing recombinant type III humanized collagen
1. Prescription groping
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 of formulation 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) |
| |
Transparent and free of impurities | Transparent and |
| Prescription | ||
| 10 | Transparent and free of impurities | Transparent and |
| Prescription | ||
| 11 | Transparent and free of impurities | Transparent and |
| 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 of formula 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 of formula 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 that prescription 5/10/11/13 is effective in treating dry eye symptoms and increasing/restoring ocular lacrimal gland secretion.
Sequence listing
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Claims (10)
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.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN117264047A (en) * | 2023-09-21 | 2023-12-22 | 南京巴凯星生物科技有限公司 | Recombinant III type humanized collagen amino acid sequence and preparation method and application thereof |
| CN117264047B (en) * | 2023-09-21 | 2024-05-17 | 南京巴凯星生物科技有限公司 | Recombinant III type humanized collagen amino acid sequence and preparation method and application thereof |
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