CN115487139B - Puerarin gellan gum ionic in-situ gel eye drops and preparation method thereof - Google Patents
Puerarin gellan gum ionic in-situ gel eye drops and preparation method thereof Download PDFInfo
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- CN115487139B CN115487139B CN202211118746.3A CN202211118746A CN115487139B CN 115487139 B CN115487139 B CN 115487139B CN 202211118746 A CN202211118746 A CN 202211118746A CN 115487139 B CN115487139 B CN 115487139B
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- puerarin
- gellan gum
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- situ gel
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0048—Eye, e.g. artificial tears
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
Abstract
The invention discloses puerarin gellan gum ionic in-situ gel eye drops and a preparation method thereof, wherein the eye drops take water as a solvent and 100mL of the eye drops comprise the following raw material components: puerarin 0.3-1.5g; 0.4-0.55g of gellan gum; 4-10g of solubilizer; 0.05-0.25g of pH regulator; osmotic pressure regulator 2-4.5g; 0.1-0.3g of antioxidant; 0-0.2g of bacteriostat; the solubilizer is polyvinylpyrrolidone. The puerarin gellan gum ionic in-situ gel eye drops have the advantages of good stability, small irritation, good bioavailability, high intraocular drug concentration and long duration.
Description
Technical Field
The invention relates to the technical field of eye drops, in particular to puerarin gellan gum ionic in-situ gel eye drops and a preparation method thereof.
Background
The physiological anatomy of the eye is complex and there are many physiological barriers such as tears, cornea, conjunctiva, blood-aqueous humor and blood-retina. Clinically, the eye diseases are mainly treated by oral administration, local administration and injection administration, the oral administration or injection administration needs to penetrate through blood-eye barriers, the administration dosage is large, and toxic and side effects are easy to generate when the effective treatment concentration is achieved, so that the local eye administration becomes a necessary trend. Ophthalmic topical administration forms include eye drops, ointments, gels, implants, and vitreous injections. Gel or ointment has the defects of inconvenient administration, inaccurate dosage, easy eye pasting and the like, and has poor patient acceptance; both implants and intravitreal injections are invasive treatments, and patients have low acceptance, and certain chronic diseases (such as age-related macular degeneration) require repeated injections, which have many risks such as endophthalmitis, vitreous hemorrhage, and retinal detachment. Therefore, compared with other administration dosage forms and modes, the eye drops have the advantages of simple and convenient use, good patient compliance, high safety and the like, and are the most commonly used treatment means in clinic.
The traditional eye drops are generally in the form of aqueous solution of medicines, can quickly run off from the ocular surface due to tear flushing and blink reflex after entering eyes, and have low bioavailability because of the existence of various physiological barriers such as cornea and the like, and the medicines are not easy to enter ocular tissues. There is an urgent need to develop new formulations for the eye to resist tear dilution while facilitating administration, and to have a certain permeability to the cornea and sclera. Research shows that the novel eye drug delivery system such as nano particles, micelles, liposome, in-situ gel and the like has good application prospect in the aspects of improving bioavailability and reducing adverse reaction. Wherein the in situ gel is more advantageous in terms of drug delivery that is more water soluble and has a larger dose.
The in-situ gel is in a liquid state before administration, and when the external environment condition changes, the phase change from solution to gel occurs, and the in-situ gel can be divided into temperature, pH and ion sensitivity according to different response conditions. The dosage form can well control the dosage of the drug, has long residence time in eyes and has been widely used for the eye delivery of drugs. The temperature-sensitive in-situ gel is prepared by changing the polymer from solution to gel at the eye surface temperature (34 ℃) in response to temperature change, and more polymer, such as poloxamer and cellulose derivatives, is used. The pH sensitive in-situ gel is a preparation which generates a gelling reaction when the pH of the environment is changed, and chitosan and carbomer are common polymers, but the carbomer solution is highly acidic, so the pH sensitive in-situ gel is usually combined with a tackifier such as methyl cellulose to reduce the use concentration and the eye irritation. Ion-sensitive in situ gels are polymers that undergo a solution-to-gel phase change when contacted with cations in tear fluid, and commonly used polymers are gellan gum and sodium alginate. The ionic in-situ gel has the optimal development prospect by taking Gellan Gum (Gellan Gum) as a matrix. The low concentration gellan gum forms anionic polysaccharide in aqueous solution, and when the ionic strength of the environment increases, if the gellan gum contacts tear fluid, the solution agent is changed into gel agent, cations in the tear fluid increase, and the gel forming proportion also increases. As an in-situ gel matrix for eyes, the gellan gum has the outstanding characteristics of (1) low dosage: generally less than 0.6%, and has small irritation to eyes; (2) ease of administration, rapid gel formation: the low concentration gellan gum aqueous solution is a low viscosity solution, shows very good fluidity, and after the gellan gum is spotted into eyes to touch a trace amount of tear, cations in the tear induce the gellan gum to form chain bridges from random coils and turn into double helices, and then the double helices are aggregated to form a bonding area to form gel. (3) comfort enhancement, even distribution: the artificial tear has shearing and diluting characteristics, namely, the viscosity is reduced by stirring or frequent blinking of eyelid, and the viscosity is increased to be gelatinous after the artificial tear is static, so that the artificial tear is beneficial to ocular administration and distribution on the surface of the eyeball.
The medicinal gellan gum is a microbial edible gum originally developed by CP Kelco company in U.S. in the age of 20 th century, is a linear anionic heteropolysaccharide, and is formed by connecting glucose, glucuronic acid and rhamnose into quaternary repeating units in the molar ratio of 2:1:1 to form a polymer main chain, wherein the relative molecular mass is about 0.5 multiplied by 10 6 The structural formula is shown as the formula (1), and the compound has been used for researching various medicines.
Puerarin is an isoflavone compound derived from root of Pueraria lobata Ohwi of Leguminosae, and is slightly soluble in water, and has molecular formula of C 21 H 20 O 9 The relative molecular weight is 416.38, and the structural formula is shown in the formula (2). Pharmacological studies show that puerarin has the functions of resisting oxidation, resisting inflammation, resisting sugar, dilating coronary vessels, improving microcirculation, protecting optic nerve cells and the like, and can be used for treating eye diseases such as glaucoma, retinal vascular obstruction, diabetic retinopathy and the like. Puerarin can inhibit beta receptor of ciliary epithelial cells, reduce aqueous humor production, reduce intraocular pressure, promote blood circulation, protect optic nerve, and be used for treating retinal artery and vein embolism and ocular fundus ischemic diseasesTreatment of disease. After the glaucoma is treated by the systemic injection administration mode, the blood flow conditions of the ocular artery, the short ciliary artery and the central retinal artery are observed by using the Doppler blood flow imaging technology, and the results show that the blood flow speed of each artery is obviously increased, the vascular resistance is obviously reduced, and the ischemic optic nerve damage caused by the glaucoma and the embolism of retinal blood vessels are relieved. The retina of a diabetic retinopathy patient can highly express inflammatory factors such as interleukin 1, tumor necrosis factor alpha and the like, so that inflammatory reaction is caused, retina is damaged, meanwhile, the level of malondialdehyde is increased, the activity of superoxide dismutase is reduced, and the oxidation stress reaction is shown.
At present, puerarin preparation comprises injection and eye drops, and because puerarin is slightly dissolved in water, high-concentration propylene glycol is added into the injection to assist dissolution, and adverse reaction is easily caused by long-term intravenous injection; the common solution is easy to run off after ocular administration, is difficult to permeate into eyes, and has low bioavailability. Therefore, there is a need to design new drug delivery systems to improve drug bioavailability for application in the treatment of ocular disorders. In the study of puerarin dissolved in a solution of polyamide-amine polymer to obtain a mixed solution of the puerarin and the polyamide-amine polymer, the pharmacokinetics of the anterior chamber of rabbit eyes shows that compared with the puerarin solution, the AUC of the mixed solution (0-∞) The improvement is 2 times. The blank contact lens is synthesized by taking hydroxyethyl methacrylate, methyl acrylate and polyvinylpyrrolidone as matrixes, and then soaked in 0.802 mg.mL -1 The puerarin phosphate buffer solution is prepared into a drug-loaded preparation, and the result shows that the pre-cornea residence time of the contact lens is 6.04 times of that of the puerarin solution, but the bioavailability is not obviously different from that of the 1% puerarin solution, which also shows that the contact lens can reach the same intraocular drug concentration at a lower dosageDegree.
In the aspect of in-situ gel preparation, 21% of poloxamer 407 and 5% of poloxamer 188 are used as matrixes to prepare puerarin temperature-sensitive in-situ gel, and compared with puerarin solution, the eye residence time of the preparation is prolonged by 7.50 times; also, the pH-sensitive in-situ gel prepared by taking 0.1% carbomer 940 and 0.4% hydroxypropyl methylcellulose as matrixes is studied, and the results of a pharmacokinetics test show that the AUC of the gel group (0-24h) Is 2.17 times of the solution group, and remarkably improves the bioavailability. In addition, the patent uses poloxamer 407 and poloxamer 188 to prepare puerarin temperature-sensitive in-situ gel, compared with intravenous injection puerarin injection, the in-situ gel preparation obviously enhances the retinal blood flow intensity after eye administration, and simultaneously the medicine concentration in retina and choroid is also obviously improved; there are also patents for preparing pH-sensitive in-situ gel of puerarin by using carbomer as matrix, and compared with puerarin solution, the in-situ gel preparation not only has stronger ocular hypotensive effect, but also remarkably improves microcirculation, reduces plasma viscosity and ensures ocular blood flow supply.
The ion-sensitive in-situ gel requires a lower concentration of polymer than the temperature-sensitive in-situ gel; in comparison to pH sensitive in situ gels, ion sensitive in situ gels can adjust the prescribed pH to the eye optimum pH, so minimal ocular irritation is achieved. For example, the in situ gel of bendazac lysine ion prepared by using gellan gum as matrix, and the pharmacokinetic study shows that compared with the commercial eye drops, the in situ gel of bendazac lysine ion max And AUC (0-t) The two times of the two times are respectively improved by 2.38 and 2.20; also, research shows that the brinzolamide ionic in-situ gel prepared by taking gellan gum as a matrix has no obvious stimulus to rabbit eyes, and compared with brinzolamide solution, the ionic in-situ gel preparation remarkably enhances the strength and duration of intraocular pressure reduction; and the only in-situ gel eye drops approved by the FDA in the United states at present, namely timolol maleate ionic in-situ gel, is a preparation taking gellan gum as a matrix.
However, no literature or patent report on puerarin ionic in-situ gel exists at present.
Disclosure of Invention
Aiming at the problems of poor ocular bioavailability, frequent use and poor patient compliance of puerarin eye drops in the prior art, the invention provides puerarin gellan gum ionic in-situ gel eye drops with good bioavailability, high intraocular drug concentration and long duration and a preparation method thereof.
The technical scheme of the invention is as follows:
the puerarin gellan gum ionic in-situ gel eye drops takes water as a solvent and 100mL of the eye drops comprises the following raw material components:
the solubilizer is polyvinylpyrrolidone.
The ophthalmic preparation applied to the ocular surface has limited residence time, and the release speed of the ophthalmic preparation is not too slow, so that the prepared ophthalmic preparation has the dual characteristics of quick release and continuous release to produce better technical effects.
In the puerarin gellan gum ionic in-situ gel eye drop, the concentration of gellan gum is 0.4-0.55g/mL, the concentration of the solubilizer is specifically 4-10g/mL of polyvinylpyrrolidone, the concentration of puerarin is 0.3-1.5g/mL, and the gellan gum, the solubilizer and the puerarin with specific concentrations act together, so that the puerarin gellan gum ionic in-situ gel eye drop has the dual characteristics of quick release and sustained release, the medicine is released relatively quickly, the medicine is absorbed into eyes through corneas and sclera to play a role, the higher concentration in eyes can be maintained for a long time, and the long-time intraocular effect is ensured.
Preferably, the pH regulator is tromethamine.
Regarding the pH regulator, it is generally reported that a buffer salt system, such as the conventional puerarin common solution drop eye solution is a buffer solution system containing sodium salt, but the invention adopts gellan gum to prepare in-situ gel for eyes, and ionic in-situ gel can be converted into gel from liquid when encountering sodium, potassium, magnesium, calcium and other ions, so that the invention adopts tromethamine and the like as the pH regulator to avoid.
The pH of normal tears is about 7.4, and the normal tears have a certain buffer capacity, and after eye drops enter eyes, the normal tears are diluted by tears, and the buffer function of the tears can neutralize ophthalmic solution, so obvious uncomfortable feeling can be relieved. But when the eye drops are too sour, proteins of the eye mucosa can be coagulated; too alkaline can harden or dilate the epithelial cells of the ocular mucosa. Thus, an eye drop with too high or too low a pH can be irritating to the eye. Irritation caused by improper pH can increase tear secretion, leading to rapid loss of drug and even damage to the cornea. The pH of the eye drops can generally be in the range of pH 5.5 to 7.8. The puerarin solution is an acidic solution, but stability tests show that impurities are increased when the pH of the preparation is regulated to be below 6.0.
Preferably, the pH value of the puerarin gellan gum ionic in-situ gel eye drops is 6.2-7.4; osmotic pressure was 260-320mOsmol/kg.
The proper pH value and osmotic pressure can reduce the irritation of eye drops to eyes, thereby reducing the secretion of tears and leading gellan gum to stay on the surface of eyes for a long time. The gellan gum of 0.4-0.55g/mL has the advantages of quick effect and capability of preventing the concentration from being reduced due to drug loss caused by tear flushing.
Preferably, the osmotic pressure regulator is at least one of mannitol, glycerol and 1, 2-propylene glycol; the antioxidant is at least one of vitamin C and sodium metabisulfite; the bacteriostatic agent is at least one of benzalkonium chloride, benzalkonium bromide and parahydroxybenzoate.
Further preferably, the osmotic pressure regulator is mannitol; the antioxidant is vitamin C.
The bacteriostatic agent, which can also be called a preservative, is used for preventing bacteria from breeding after the eye drops are packaged and deteriorating the eye drops. Because some patients may be sensitive to the preservative and cause eye discomfort, the preservative component can be removed, and the package with single daily dose can be changed, and the eye mask can be used as soon as possible after unsealing.
A preferred technical scheme is as follows:
the puerarin gellan gum ionic in-situ gel eye drops takes water as a solvent and 100mL of the eye drops comprises the following raw material components:
the solubilizer is polyvinylpyrrolidone; the pH regulator is tromethamine; the osmotic pressure regulator is mannitol; the antioxidant is vitamin C.
The invention also provides a preparation method of the puerarin gellan gum ionic in-situ gel eye drops, which comprises the following steps:
(1) Adding part of water into the gellan gum with the formula amount, and stirring for swelling;
(2) Dissolving the solubilizer and puerarin with partial water, and stirring uniformly;
(3) Dissolving other auxiliary materials with the rest part of water, and adding the solution in the step (2); or other auxiliary materials are directly added into the solution in the step (2) for dissolution and are stirred uniformly;
(4) Adding the solution obtained in the step (3) into the gellan gum swelled in the step (1), or adding the gellan gum swelled in the step (1) into the solution obtained in the step (3), supplementing the balance of water, and stirring until the solution is clear and transparent, thus obtaining the puerarin gellan gum ionic in-situ gel eye drops.
Preferably, the water added in the step (1) is 50-60% of the total water; the temperature is 90+/-5 ℃; the stirring swelling time is 15-20 min.
Preferably, in the step (4), after stirring until the solution is clear and transparent, the solution is kept at a temperature of more than 40 ℃ and filtered for sterilization.
The filter sterilization can be performed by using a filter membrane with a pore size of 0.1-0.22 μm.
Because the gellan gum can dissolve quickly when meeting water with higher temperature, the gellan gum is firstly prepared into a solution with high concentration at higher temperature during preparation, and meanwhile, the influence of the high temperature on the medicine is relatively small because the main medicine puerarin is not contained; and then dissolving and mixing the main medicine and other components, and adding the main medicine and the other components into the gellan gum solution. Finally, the filtration and sterilization are required to be carried out at a temperature above 40 ℃, because the viscosity of the eye drop solution is relatively much smaller than that of the eye drop solution at a temperature of about 25 ℃ (the viscosity is smaller as the temperature of the gellan gum solution is higher, for example, the viscosity is about 8cP at 35 ℃ and about 120cP at 25 ℃ at 10 rpm), the viscosity is below 4cP at 40 ℃, and the filtration can be carried out smoothly by the microporous filter membrane.
Compared with the prior art, the invention has the beneficial effects that:
compared with the puerarin common solution eye drops in the prior art, the puerarin gellan gum ion type in-situ gel eye drops can keep the concentration of the medicine in aqueous humor at a higher level for a longer time, greatly improve the highest medicine concentration and accumulated medicine quantity, increase the bioavailability, reduce the use frequency of the medicine and improve the compliance of patients.
Drawings
Fig. 1 is a graph of the results of the measurement of the effect of different ionic strengths on the viscosity of formulation 2 in example 1 (34 ℃,10rpm, n=4).
Fig. 2 is a graph of the results of the measurement of the effect of different ionic strengths on the viscosity of formulation 3 in example 1 (34 ℃,10rpm, n=4).
Fig. 3 is a graph of the results of the measurement of the effect of different ionic strengths on the viscosity of formulation 4 in example 1 (34 ℃,10rpm, n=4).
Fig. 4 is a graph of the results of the measurement of the effect of different ionic strengths on the viscosity of formulation 5 in example 1 (34 ℃,10rpm, n=4).
Fig. 5 is a graph of the results of the measurement of the effect of different ionic strengths on the viscosity of formulation 6 in example 1 (34 ℃,10rpm, n=4).
FIG. 6 is a graph showing the results of the viscosity measurement of the ratio of the shear rate to the artificial tear volume of the formulation 3 in example 1 at 40:21 (34 ℃ C., n=4).
Figure 7 is a graph of cumulative permeability versus time for the different puerarin formulations prepared in example 2 in rabbit ex vivo sclera (n=3,)。
FIG. 8 is a graph showing the in vitro cumulative release rate versus time of various puerarin formulations prepared in example 3 (n=4,)。
figure 9 is a graph of cumulative permeability versus time for the different puerarin formulations prepared in example 3 in rabbit ex vivo sclera (n=4,)。
fig. 10 is a graph of in vitro cumulative permeability versus time of puerarin in situ gel and solution eye drops obtained in example 5 in rabbit ex vivo cornea (n=4, )。
FIG. 11 is a graph showing the concentration of puerarin in situ gel and eye drop solution prepared in example 6 in aqueous humor of rabbit eye versus time (n=8,note that: * P<0.01,*p<0.05, in situ gel vs solution).
Detailed Description
The main auxiliary materials and the reasonable range thereof in the prescription are mainly searched under the condition of 1% puerarin, and the problems of low solubility in puerarin water and release and permeation rate are solved firstly.
Puerarin is slightly soluble in water, and the saturated solubility of Puerarin in a dissolution medium shown in table 1 is not high, so that a solubilizer is required to be added to meet the requirement of preparation concentration.
TABLE 1 solubility of Puerarin in various media (34 ℃ C.)
Taking puerarin with concentration of 1% as an example, 5% hydroxypropyl-beta-cyclodextrin or polyvinylpyrrolidone K30 (PVP-K30) can dissolve 1% puerarin, i.e. if the preparation reaches the expected content, the preparation can be wrapped by adding high polymer materials hydroxypropyl-beta-cyclodextrin and PVP-K30 to increase the solubility. It has been reported that adding an amount of tween 80 to the formulation also increases the solubility. By looking at the limits of ophthalmic excipients provided by the FDA mesh and the nootropic mesh in the united states, it was learned that the use limit of hydroxypropyl-gamma-cyclodextrin in ophthalmic formulations was 1.5% (hydroxypropyl-beta-cyclodextrin can be referenced to this limit), PVP-K30 was 15% and tween was 4%. In view of the fact that the long-term administration of the eye part can cause irritation to the eye part and influence the safety and effectiveness of the medicine, PVP-K30 or Tween 80 is adopted as a solubilizer for further test.
Tween 80 is a surfactant which can not only solubilize but also promote the permeation of drugs in theory, but in practice, in puerarin, in vitro scleral permeation experiments of rabbits show that Tween 80 does not play a role in promoting permeation, so that the Tween 80 is not added. See example 2.
Table 1 shows that the saturation solubility of puerarin at 34 ℃ is slightly higher than 1% at 5% PVP-K30, and the puerarin solution prepared at the concentration of puerarin can be 6% aiming at PVP-K30 concentration of 1% in the process of storage, and the puerarin solution is refrigerated at 4 ℃ for one year without drug precipitation because the preparation needs to be stored for a long time. For puerarin with concentration lower than 1% or higher than 1%, the dosage of PVP-K30 can be reduced or increased appropriately.
Ophthalmic formulations for topical applications generally have a limited residence time, so it is considered that the formulation is not released too slowly and has the dual characteristics of rapid and sustained penetration into the sclera and cornea to produce better technical results. That is, the invention can release the medicine quickly, and can be absorbed into eyes through cornea and sclera, and can maintain high concentration in eyes for a long time, thus ensuring long-time intraocular effect.
The gellan gum with concentration of 0.4-0.55% is effective and can prevent drug loss and concentration drop caused by tear flushing. By matching with proper pH value and osmotic pressure, the stimulus to eyes is reduced, so that secretion of tears can be reduced, and gellan gum can stay on the surface of eyes for a long time.
Although gellan gum can increase the residence time of the formulation on the ocular surface, since human blinks frequently, tears are continuously secreted and the loss of the formulation is accelerated, the drug should be released from the formulation as soon as possible and penetrate the sclera, cornea and into aqueous humor relatively quickly. The in vitro release rate and the in vitro scleral penetration rate of the preparation are taken as investigation indexes, the in vitro release and penetration conditions of the puerarin by the gellan gum and the PVP-K30 with different concentrations are investigated, and the study shows that the in vitro release of the puerarin is reduced with the increase of the PVP-K30 and the gellan gum, but the accumulation release amount of 10 hours tends to be consistent in the initial period (0.5-4 hours). The expression of the PVP-K30 on the penetration of rabbit isolated sclera is not completely consistent with the in vitro release, and the PVP-K30 content is increased (10 percent) and the gellan gum is added, so that the penetration of puerarin is greatly affected. That is to say, the concentration changes of PVP-K30 and gellan gum can all cause certain influence on the in vitro scleral penetration of the puerarin rabbits, the PVP-K30 and the gellan gum are controlled within a proper range, the PVP-K30 and the gellan gum are within 10 percent, the gellan gum can be released quickly, and the puerarin has quick and sufficient penetration capacity. Example 3 can be seen in particular.
Stability research shows that puerarin is sensitive to temperature and can be influenced by illumination. Generally, the stability of the preparation can be maintained for at least two years, so that the problems of continuous reduction of pH, yellowing and deepening of color and luster and the like caused by oxidation due to various reasons in the production process of puerarin preparations are solved. The stability is examined by taking the appearance, pH, osmotic pressure, content and related substances of the preparation as indexes, and the limit is referred to puerarin injection standard in the two parts of Chinese pharmacopoeia 2020 (impurity peaks exist in a chromatogram of a sample solution, the peak areas of the impurity I and the impurity II are not more than 1.5 times (1.5%) of the main peak area of a control solution, the peak areas of other single impurities are not more than 0.5 times (0.5%) of the main peak area of the control solution, and the sum of the peak areas of all impurities is not more than 3 times (3.0%) of the main peak area of the control solution). In the early stability test, no antioxidant is added into the preparation, and test results show that under the illumination condition of 4500+/-500 lx, the content of puerarin in the preparation is slightly reduced, the content of other single impurities is increased, and the impurity I, II and other indexes are not greatly changed; at the high temperature of 60 ℃, the content of the preparation is obviously reduced, related substances are obviously increased, other single impurities exceed the limit of 0.5 percent, the pH is obviously reduced, and the preparation solution is gradually deepened from light brown; under the acceleration test condition of the temperature of 40+/-2 ℃ and the humidity of 25+/-5%, other single impurities exceed the limit (0.5%) in 2 months, the content and the pH of the preparation are obviously reduced when the other single impurities reach 1.0% in 3 months, but the total impurities I and II do not exceed the limit, and the total impurities also exceed 3.0% in 6 months; placing for 6 months at 25+ -2deg.C and relative humidity of 40% + -5%, wherein the content of the preparation is obviously reduced, the content of related substances is increased, and other single impurities are overrun; the samples of the same batch are placed in a refrigerator at the temperature of 4 ℃ for 3 months, and all indexes are not changed obviously. In the above test process, the temperature was found to have a large influence on the stability of the puerarin gellan gum ionic in-situ gel, and the pH of the preparation was also lowered with the increase of impurities, presumably producing some acidic impurities during storage. The antioxidant such as sodium metabisulfite or vitamin C is added to inhibit the oxidation of the medicine, so that the influence of the pH and the antioxidant on the stability of the preparation is examined through experiments. The test result shows that the vitamin C has better antioxidation effect, for 1% puerarin in-situ gel preparation, 0.2% vitamin C can obviously inhibit the generation of impurities, after accelerating for 3 months, the content of the preparation, related substances, pH and the like slightly change, but compared with a prescription without antioxidant, the stability is obviously enhanced, and all indexes are within limits. It was also found that when the pH was below 6.0, the formulation produced more impurities, so the pH of the final formulation was adjusted to between 6.2-7.4. In the intermediate condition test (30 ℃), each index of the preparation is in a qualified range in 6 months, and other single impurities are out of limit in acceleration of 6 months at 40 ℃. See example 9.
In conclusion, the puerarin gellan gum ion type in-situ gel preparation is sensitive to temperature, and the puerarin gellan gum ion type in-situ gel preparation can be influenced by illumination, so that the puerarin gellan gum ion type in-situ gel preparation is stored in a shady and cool place for a long time.
Example 1
Different formulations were formulated as in table 2 below and different amounts of gellan gum and polyvinylpyrrolidone K30 (PVP-K30) were screened.
TABLE 2 gelling ability of different adjuvant ratios
Note that: +: gelatinizing, but shaking and easy dispersion; ++: rapidly gelling and shaking and is not easy to be dispersed.
The preparation method comprises the following steps: adding gellan gum into purified water at about 90 ℃ to prepare gellan gum solution with the weight percentage content of 0.8%, and stirring and swelling completely. And (3) dissolving PVP-K30 and puerarin (the final concentration is 1%) with a proper amount of water according to the numerical values in the table, then adding a proper amount of the 0.8% gellan gum solution, stirring uniformly, regulating the isoosmotic property by mannitol, regulating the pH value to about 7.0 by tromethamine, supplementing the volume to 20mL by deionized water, and mixing uniformly to obtain the product.
Gel ability measurement: 2mL of artificial tear (34 ℃ C., aqueous sodium chloride solution 6.7g.L) -1 Sodium bicarbonate aqueous solution 2.0 g.L -1 0.8 g.L of calcium chloride dihydrate aqueous solution -1 ) Placed in a stoppered tube, incubated in a water bath at 34℃and 100. Mu.L of in situ gel was added to the tube, and gel formation and dissolution were observed and recorded.
In addition, the change of viscosity of different prescriptions along with tear ratio (ionic strength) and rotating speed (shear rate) can be measured by using a Brookfield DV-2T viscometer. Since the eyes can continuously secrete tears after administration, in-situ gel and artificial tears with different proportions are uniformly mixed, the mixture is added into a sample cup, after balancing for 5min, the conditions that the temperature is 34 ℃ and the rotation speed is 10rpm and the viscosity changes with time are measured, and each sample is measured for 4 times. Taking prescriptions 2-6 as examples, the viscosity of the gel changes along with the ionic strength, as shown in figures 1-5, before artificial tears are not added, the viscosity of the gel in situ is about 8cP, and after artificial tears with different proportions are added, the viscosity can greatly change along with the change of the ionic strength. This is a process that increases or decreases with increasing tear fluid volume, which characterizes ionic in situ gels. The formulation has the highest overall viscosity at a volume ratio of 40:21 with artificial tear. The effect of shear rate on prescription viscosity is also very high, taking prescription 3 as an example, the viscosity change of the prescription and artificial tear is shown in fig. 6 when the volume ratio of the prescription to the artificial tear is 40:21, the higher the shear rate is, the smaller the viscosity display is, which proves that the gellan gum can show the characteristic of shear dilution in the artificial tear, namely, the viscosity is reduced due to stirring or frequent blinking of eyelid, the viscosity is increased again to be gelatinous when the gellan gum is stationary, and the administration to eyes and the distribution on the surface of eyeball are facilitated.
When the concentration of the gellan gum is 0.30% or below, the gelation ability is weaker, when the concentration of the gellan gum is 0.60% or above, the gellan gum is sticky at room temperature, and because the gellan gum needs to be mixed with PVP-K30, PVP-K30 is a high molecular polymer and has certain viscosity, and the optimal concentration range of the gellan gum is 0.40% -0.50% on the basis that the viscosity of the prescription is not too high when comprehensively considered. The results of the influence of the ionic strength in the tears on the viscosity of the prescriptions of the puerarin gellan gum preparation are shown in figures 1 to 5, and as can be seen from the figures, the viscosity of each prescription is in a state of increasing firstly and then decreasing as the proportion of the artificial tears increases, and the viscosity is the largest when the proportion of the gellan gum preparation to the artificial tears is 40:21; the gellan gum has a concentration of 0.45% and 0.50% and a higher anti-tear dilution capacity than that of 0.40%.
The experimental results of the proportions of the gellan gum and PVP-K30 can be used as the basis for selecting the weight percentage content of the gellan gum, the gellan gum can be preferably 0.4-0.5%, and the PVP-K30 can be preferably 6-8%.
Example 2
Tween 80 is a surfactant which not only solubilizes but also has been shown to promote drug penetration. Formulations were prepared as shown in table 3 below, and the in vitro sclera penetration of rabbits at different levels of tween 80 was compared.
TABLE 3 prescriptions containing Tween 80 at different concentrations
The preparation method comprises the following steps: adding proper amount of purified water at about 90 ℃ into gellan gum to prepare gellan gum solution with the weight percentage content of 0.8%, and stirring and swelling completely; and dissolving PVP-K30 and puerarin (with a final concentration of 1%) in a prescribed amount with a proper amount of purified water, stirring uniformly, adding a proper amount of gellan gum solution, stirring uniformly, adding mannitol to adjust the isotonicity, adjusting the pH with tromethamine, and adding purified water to 20mL. Stirring uniformly to obtain the final product.
Rabbit ex vivo sclera penetration study: taking healthy New Zealand white rabbits without eye diseases, killing eyeballs, removing superfluous tissues on the surfaces, separating sclera, and washing with normal saline for later use. The experiment used a home-made, arcuate port Franz permeation cell, including a receiving cell and a feeding cell. The water bath circulation temperature of the pipeline outside the receiving tank is set to be 34 ℃, and bubbles in the pipeline outside the receiving tank are exhausted. A magnetic stirrer and about 3mL of ringer's solution as a release medium are added into the receiving tank, the sclera is stretched at the tank mouth of the infiltration tank, the outer surface of the sclera faces the supply tank, and the spring clip is fixed to exhaust bubbles in the receiving tank. Puerarin preparation and artificial tear are mixed uniformly in a ratio of 40:7, a pipette sucks 94 μl, and the mixture is added into a supply tank, 100 μl is sampled from a receiving tank at 1, 2, 4, 6, 8 and 10h respectively, and then isothermal and equal volume of blank release medium is added. The samples were subjected to HPLC determination, the chromatographic conditions were as follows: diamond C 18 Columns (4.6 mm. Times.150 mm,5 μm); mobile phase: acetonitrile-1% glacial acetic acid (15:85); detection wavelength: 249nm; flow rate: 1.0 mL/min -1 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature: 30 ℃; sample injection amount: 20. Mu.L. Cumulative permeability (%) is plotted against time t.
The results are shown in FIG. 7. The results showed that there was no significant difference in 1% spitting and 4% tween in vitro penetration. Increasing the amounts of PVP-K30 and gellan gum tended to decrease the permeability, and adding Tween on this basis did not promote permeation, whereas the Tween-containing formulations were less permeable. Tween 80 does not play a role in promoting permeation, and in view of the fact that the auxiliary materials of the ophthalmic preparation are not excessive, PVP-K30 is only used as a solubilizer, and the preparation does not contain Tween 80.
Example 3
Formulations were prepared as shown in Table 4 below, and the formulations containing PVP-K30, gellan gum, and hydroxypropyl-beta-cyclodextrin at different concentrations were compared for in vitro release and penetration on the sclera of rabbits.
TABLE 4 formulations of PVP-K30, gellan gum, hydroxypropyl-beta-cyclodextrin at different concentrations
The preparation method comprises the following steps: adding gellan gum into purified water at about 90deg.C, stirring and swelling completely; and (3) dissolving PVP-K30 and puerarin (the final concentration is 1%) with purified water, uniformly stirring, adding appropriate amount of gellan gum solution, uniformly stirring, adjusting the isoosmotic property with mannitol, adjusting the pH with tromethamine, and adding purified water to 20mL. Stirring uniformly to obtain the final product.
In vitro release experiments: and respectively precisely transferring 0.5mL of puerarin preparation, placing into a dialysis bag with a molecular retention of 14KD, fastening the mouth of the dialysis bag, and placing into a penicillin bottle. 20mL of artificial tear was added as a diffusion medium, and the vial was placed in a constant temperature shaker at 34℃and oscillation frequency of 100rpm. 1mL is sampled at regular intervals (0.5, 1, 2, 4, 6, 8 and 10 hours), and isothermal equal volume artificial lacrimal eyes are timely supplemented. And after the sample is centrifuged, measuring the content of the medicine by adopting a high performance liquid phase method.
Rabbit ex vivo sclera penetration study: the procedure is as described in the previous examples.
The results are shown in FIG. 8. The results show that with increasing amounts of PVP-K30 and gellan gum, the in vitro release of puerarin decreased in the initial period (0.5-4 h), but the cumulative release amount over 10h tended to be consistent. When the concentration of gellan gum is increased to 0.6% and PVP-K30 is increased to 13% (formulation 8), the cumulative release (total released) for 10 hours is significantly reduced (P < 0.01). At 8% hydroxypropyl-beta-cyclodextrin and 30% PVP-K (formula 9), the release amount in the initial period was relatively low, but the cumulative release amount for 10 hours was not significantly low (P > 0.05).
The permeation result on the rabbit isolated sclera is not completely consistent with the in vitro release, the PVP-K30 content is increased (10%), the permeation of puerarin by 0.45% of gellan gum (formula 7) has a great influence, and the cumulative permeation amount of 10 hours is 71% of that of formula 3 (PVP-K30 6% and gellan gum 0.45%). The PVP-K30 content increased to 13% and gellan gum increased to 0.6% (formulation 8) was more pronounced, with a 10h cumulative permeation of only 42% of formulation 3. Whereas 0.6% gellan gum plus 8% hydroxypropyl-beta-cyclodextrin (formulation 9) greatly reduced the scleral penetration of puerarin, with a cumulative penetration of only 17% of formulation 3 for 10 hours. The results are shown in FIG. 9. That is, hydroxypropyl-beta-cyclodextrin is not suitable as a solubilizer for puerarin, has a strong coating property, and has poor penetration on sclera. The concentration changes of PVP-K30 and gellan gum all have a certain influence on the in vitro scleral penetration of puerarin rabbits, wherein the influence of PVP-K30 is stronger, for example, as can be seen in FIG. 9, prescriptions 2 and 3 are 6% PVP-K30, prescriptions 4 and 5 are 8% PVP-K30, and the penetration of puerarin solutions without gellan gum is similar to that of 6% or 8% PVP-K30 respectively. So that both should be controlled within a suitable range (PVP-K30 is preferably within 10% and gellan gum is preferably within 0.55%) to provide a faster release and a rapid and adequate penetration.
Example 4
The puerarin gellan gum ionic in-situ gel eye drops have the following formula:
the preparation method comprises the following steps: adding gellan gum into purified water at about 90deg.C, stirring and swelling completely; and dissolving PVP-K30 with proper amount of purified water, adding puerarin, glycerol, trometamol and parahydroxybenzoate, stirring for dissolving, adding into the solution of the gellan gum, adding purified water to 100mL, filtering with a 0.22 μm microporous filter membrane, and packaging to obtain puerarin with a weight percentage content of 0.3% and a final weight percentage concentration of gellan gum of 0.4%. The pH was 6.2 and the osmotic pressure was 261mOsmol/kg.
Example 5
The puerarin gellan gum ionic in-situ gel eye drops have the following formula:
the preparation method comprises the following steps: adding the gellan gum with the prescription amount into 60mL of purified water at about 90 ℃ and stirring and swelling completely; puerarin, PVP-K30, mannitol, trometamol and benzalkonium chloride are put into a small bottle, and about 30mL of purified water is taken and stirred for dissolution. Adding gellan gum solution into the solution, stirring, washing with water, adding constant volume, filtering with microporous membrane of 0.22 μm at 40deg.C, and packaging. The puerarin weight percentage content is 1.0%, and the final weight percentage concentration of the gellan gum is 0.45%. The pH was 7.2 and the osmotic pressure was 300mOsmol/kg.
Rabbit ex vivo corneal penetration study: glutathione-ringer's solution is used as osmotic medium to protect isolated cornea from excessive hydration. Other experimental procedures were as described for the ex vivo sclera of rabbits.
The results are shown in figure 10, which shows that the cumulative permeabilities of cornea of self-made gellan gum group and solution group (containing 6% PVP-K30) at 6h are 37% and 39%, respectively, and there is no significant difference (P > 0.05) between the two, and the permeabilities meet the zero order kinetic characteristics. It can be seen in connection with example 3 that the permeability of the sclera is superior to that of the cornea because the cornea is histologically divided into 5 layers from outside to inside, whereas the sclera has only 3 layers, and the cornea structure is relatively complex and the cell arrangement structure is compact; further hydration of the cornea during the test also increases the thickness of the cornea, making drug transmembrane difficult and less permeable than the sclera.
Because the in-vitro permeation experiment is carried out in the vertical supply tank, the solution preparation cannot be lost, so that the in-situ gel can prolong the retention time of the ocular surface and improve the concentration of the medicine in the eye. Further pharmacokinetic studies in the eye are therefore necessary, see example 6.
Example 6
The puerarin gellan gum ionic in-situ gel eye drops have the following formula:
The preparation method comprises the following steps: adding the gellan gum with the prescription amount into 50mL of purified water at about 90 ℃ and stirring and swelling completely; putting puerarin and PVP-K30 into a small beaker, adding 30mL of purified water at about 50 ℃ for stirring and dissolving, adding trometamol and mannitol for stirring uniformly, adding the solution into the gellan gum solution, and adding the solution into the small beaker after washing for 2-3 times with about 10mL of water. Adding purified water to 100mL, filtering with 0.22 μm microporous membrane at 50-60deg.C, and packaging. The puerarin percentage content is 1%, and the final mass percentage concentration of the gellan gum is 0.45%. The pH was 6.9 and the osmotic pressure was 286mOsmol/kg.
Rabbit aqueous humor pharmacokinetic experiments: 16 healthy New Zealand rabbits of 2.5-3.0kg are randomly divided into two groups, one group is puerarin gellan gum in-situ gel group, the other group is puerarin solution group, and 8 are each. New Zealand rabbits were taken and 25% uratam (1 g.kg) was intravenously injected at the auricular margin -1 ) Anesthesia, exposing the skin around the eyes, fixing the head, placing on a constant temperature pad (37 ℃). Firstly, mydriasis (compound topiramate eye drops), an eyelid opening device is used for opening an eyelid, a 25G injection guide needle is inserted into a liquid outlet end of a microdialysis probe from a bevel end hole of the guide needle until the liquid outlet end is penetrated out from the other end of a needle head, the guide needle is removed, the position of the microdialysis probe is adjusted, a dialysis membrane window is completely immersed in the anterior chamber, and a threading hole is closed by a medical adhesive. Then the microdialysis pump was used to push a pH7.4 ringer's buffer salt solution (flow rate 1.0. Mu.L.min) -1 ) And (3) filling the microdialysis probe, and balancing for 1h to fully recover the state of the eye. After the eyeball state was recovered, the tears on the eyeball surface were gently wiped dry with a medical cotton swab, 40 μl of the preparation (puerarin about 0.4 mg) was instilled into one eye, and the other eye was left blank. After administration 1Microdialysis samples were collected every 30min within 0 h.
Liquid phase conditions: chromatographic column: agilent ZORBAX SB-C18 (3.0X105 mm,3.5 μm); mobile phase: 0.1% formic acid water (a): acetonitrile (B), gradient elution: 0.01 to 0.10min,85 percent of A;0.10 to 1.20min,85 to 75 percent of A;1.20 to 2.80 minutes, 75 to 5%A;2.80 to 4.50 minutes, 5 percent of A;4.50 to 4.51min,5 to 85 percent of A;4.51 to 5.80 minutes, 85 percent of A; flow rate: 0.6mL min -1 The method comprises the steps of carrying out a first treatment on the surface of the Sample injection amount: 5.0. Mu.L; column temperature: 35 ℃.
Mass spectrometry conditions: the genistein is used as an internal standard, an anion electrospray ion source under a multiple reaction monitoring mode is adopted, and the parameters are set as follows: ion spray voltage: -4500V; ion source temperature: 600 ℃; collision air pressure: 8psi; air curtain air pressure: 20psi; atomization gas pressure: 55psi; auxiliary air pressure: 60psi; the monitoring ion pairs of puerarin are 415.1 to 267.1 respectively; injection voltage: -10V; collision voltage: -45V; collision cell ejection voltage: -10V; monitoring ion pairs of internal standard: 269.2→133.1; injection voltage: -7V; collision voltage: -42V; collision cell ejection voltage: -10V.
And (3) the measured drug concentration is used for calculating the real drug concentration of the aqueous humor in a substitution way according to the probe recovery rate measured in advance, then the pharmacokinetic parameters are calculated by processing with DAS2.0 pharmacokinetic software, and the two groups of data are subjected to comparative analysis. Results of the concentration-time curves of the drugs in aqueous humor are shown in fig. 11, and table 5 (n=8). The results showed that the concentration of puerarin solution in aqueous humor of eye drops was always at a relatively low level, the area under the concentration-time curve (AUC 0-t ) The maximum concentration (Cmax) was 0.77mg/L at 3.13 mg/L.min, whereas the in situ gel prepared in example 6 of the present invention maintained aqueous humor drug concentration at a higher drug concentration level for a longer period of time, with an AUC of 13.78 mg/L.min and a Cmax of 2.90mg/L. The AUC of the in situ gel was 4.40 times that of the commercial eye drops and Cmax was 3.77 times that of the commercial eye drops, with a very significant difference (p<0.01). On the pharmacokinetic profile, the concentration of drug in aqueous humor at each time point was not different between the two formulations at the first and last time points (p>0.05 With significant differences at the second and last penultimate time points (p<0.05 At other time points, both formulations were extremely significantly different(p<0.01). Research shows that the puerarin gellan gum ionic in-situ gel obviously improves the drug concentration in aqueous humor, has better ophthalmic drug delivery capability, obviously improves intraocular bioavailability, and shows good clinical application prospect.
Table 5. Pharmacokinetic parameters of puerarin gellan gum in situ gel and solution eye drops in rabbit eyes comparison (n=8)
Note that: * In situ gel VS solution P <0.05; * In situ gel VS solution P <0.01.
Example 7
The puerarin gellan gum ionic in-situ gel eye drops have the following formula:
the preparation method comprises the following steps: adding the prescription amount of gellan gum into about 60mL of purified water, heating to 90-95 ℃, and stirring until the mixture is clear; and adding proper amount of water into puerarin, PVP-K30, 1, 2-propylene glycol, tromethamine and sodium metabisulfite, stirring uniformly, and adding into the gellan gum solution. Then the purified water 10mL is added into the mixed solution by twice swashing the liquid medicine bottle. Adding purified water to 100mL, maintaining the temperature at 50-60deg.C, filtering with 0.22 μm microporous membrane, and packaging to obtain final concentration of puerarin 1.2% and gellan gum 0.50%. The pH was 6.7 and the osmotic pressure was 291mOsmol/kg.
Example 8
The puerarin gellan gum ionic in-situ gel eye drops have the following formula:
the preparation method comprises the following steps: adding the gellan gum with the prescription amount into about 60mL of purified water, heating to about 90 ℃, stirring and shaking uniformly; and adding other adjuvants and puerarin into 20mL purified water at about 50deg.C, stirring to dissolve, and adding into the gellan gum solution. And adding a small amount of purified water into the mixed solution after twice washing the liquid medicine bottle. Adding purified water to 100mL, and passing through 0.22 μm microporous filter membrane at 40deg.C to obtain puerarin 1.5%, and gellan gum 0.55%. The pH was 7.4 and the osmotic pressure was 320mOsmol/kg.
Rabbit eye irritation test: taking 4 healthy New Zealand rabbits with qualified quarantine, and each half of male and female rabbits. The control was administered to the left eye and the right eye of each rabbit using autologous left and right side controls. The animal was allowed to tilt his head to the right, the left eye was allowed to tilt upward, the lower eyelid of one eye was gently lifted, and 1 drop (about 50. Mu.L) of the test sample (puerarin ophthalmic gellan gel prepared in example 8) was directly dropped into conjunctival sac, and the eyelid was gently closed for 10s. The right eye was added dropwise with a 0.9% aqueous nacl solution as a control in the same manner. The administration was continued for 14 days. Eye irritation response was examined and scored with ophthalmoscopes prior to the first dose per day and 1, 2, 4, 24, 48 and 72 hours after the last dose. The ocular irritation test showed no irritation.
Example 9
The puerarin gellan gum ionic in-situ gel eye drops have the following formula:
the preparation method comprises the following steps: adding gellan gum into 500-600mL of purified water at about 90deg.C, stirring and swelling completely; dissolving puerarin and PVP-K30 in 350mL purified water, adding mannitol, tromethamine and benzalkonium chloride, and stirring. Adding appropriate amount of high concentration gellan gum solution into the solution, adding the rest purified water, stirring, filtering with microporous membrane of 0.22 μm at 40deg.C, and packaging. The puerarin weight percentage content is 1.0%, and the final weight percentage concentration of the gellan gum is 0.45%. The pH was 7.0 and the osmotic pressure was 294mOsmol/kg.
Stability test: the preparation is packaged into light blue high-density polyester eye drops bottles to be sealed (5 mL each) and then used as a sample for preliminary influence factor tests and acceleration tests,
1. influence factor test
(1) High temperature test: the samples were placed in an incubator at 40℃and 60℃for 15 days, respectively. And sampling at 0, 5, 10 and 15 days to examine each index of the preparation.
(2) Strong light irradiation test: the samples were placed in an illumination incubator and left for 15 days under the conditions of an illuminance of 4500 lx.+ -. 500lx, and samples were taken on days 5, 10, and 15, and each index of the samples was examined.
2. Acceleration test: the samples were placed in a constant temperature incubator at 40.+ -. 2 ℃ and a relative humidity of 25.+ -. 5% for 6 months, and were sampled at 1, 2, 3, and 6 months, respectively, and each index of the samples was examined.
Test results:
influence factor test:
(1) High temperature test: the results are shown in Table 6.
The results show that after the preparation is placed at a high temperature of 60 ℃ for 15 days, the content of the preparation is obviously reduced, the contents of impurities I, II and total impurities are all within the limit range, but the content of other single impurities exceeds the limit, the pH is obviously reduced, the osmotic pressure slightly changes, but the temperature has obvious influence on the stability of the preparation within the human eye bearing range, and the preparation is kept in a shady place while avoiding a high-temperature environment.
(2) Strong illumination test: the results are shown in Table 7.
The result shows that when the preparation is placed for 15 days under the condition of 4500+/-500 lx, the pH, the content and related substances of the preparation are changed, the content of single impurities is obviously increased although the preparation is not out of limit, and if the preparation is exposed to strong light for a long time, the single impurities can be out of limit, so the preparation should be preserved in a dark place.
Acceleration and results of intermediate test conditions:
the results are shown in Table 8. The results show that compared with the initial preparation, after the preparation is accelerated for 3 months at 40 ℃, the osmotic pressure of the preparation is not obviously changed, the pH and the content of the preparation are slightly reduced, the impurity content is increased, and after the preparation is accelerated for 6 months, other single impurities exceed the limit of 0.5 percent. Compared with 40 ℃, after accelerating for 3 months at 30 ℃, the pH, content reduction and impurity increase degree of the preparation are smaller than those of the preparation under accelerating for 40 ℃, the osmotic pressure has no obvious change, and after accelerating for 6 months, each index has little change, and the preparation is stable. The appearance of the preparation is gradually deepened from light brown to darker brown, and the oxidative discoloration of vitamin C can be related. Although the impurity content is increased and the pH is reduced, the method is still within the limit.
TABLE 6 stability of Puerarin gellan gum in situ gel at high temperature of 60℃
TABLE 7 stability of Puerarin gellan gum in situ gel under light conditions
TABLE 8 results of in situ gel acceleration test and intermediate test of Puerarin gellan gum
The foregoing embodiments have described the technical solutions and advantages of the present invention in detail, and it should be understood that the foregoing embodiments are merely illustrative of the present invention and are not intended to limit the invention, and any modifications, additions, substitutions and the like that fall within the principles of the present invention should be included in the scope of the invention.
Claims (9)
1. The puerarin gellan gum ionic in-situ gel eye drops is characterized by taking water as a solvent and 100 mL by weight, and comprises the following raw material components:
puerarin 0.3-1.5 g;
gellan gum 0.4-0.55 g;
4-10 parts of solubilizer g;
pH regulator 0.05-0.25 and g;
osmotic pressure regulator 2-4.5 g;
antioxidant 0.1-0.3 g;
bacteriostat 0-0.2. 0.2 g;
the solubilizer is polyvinylpyrrolidone K30; the pH regulator is tromethamine; the pH value of the puerarin gellan gum ionic in-situ gel eye drops is 6.2-7.4.
2. The puerarin gellan gum ionic in situ gel eye drop of claim 1, wherein the osmotic pressure is 260-320 mOsmol/kg.
3. The puerarin gellan gum ionic in situ gel eye drop of claim 1, wherein the osmotic pressure regulator is at least one of mannitol, glycerol and 1, 2-propanediol; the antioxidant is at least one of vitamin C and sodium metabisulfite; the bacteriostatic agent is at least one of benzalkonium chloride, benzalkonium bromide and parahydroxybenzoate.
4. The puerarin gellan gum ionic in situ gel eye drop of claim 1, wherein the osmotic pressure regulator is mannitol; the antioxidant is vitamin C.
5. The puerarin gellan gum ionic in situ gel eye drop according to claim 1, wherein the puerarin gellan gum ionic in situ gel eye drop comprises the following raw material components by taking water as a solvent and 100 mL:
puerarin 0.3-1.5 g;
gellan gum 0.4-0.55 g;
4-10 parts of solubilizer g;
pH regulator 0.05-0.25 and g;
osmotic pressure regulator 2-4.5 g;
Antioxidant 0.1-0.3 g;
bacteriostat 0-0.2. 0.2 g;
the solubilizer is polyvinylpyrrolidone K30; the pH regulator is tromethamine; the osmotic pressure regulator is mannitol; the antioxidant is vitamin C.
6. A method for preparing the puerarin gellan gum ionic in-situ gel eye drops according to any one of claims 1-5, comprising the following steps:
(1) Adding part of water into the gellan gum with the formula amount, and stirring for swelling;
(2) Dissolving the solubilizer and puerarin with partial water, and stirring uniformly;
(3) Dissolving other auxiliary materials with the rest part of water, and adding the solution in the step (2); or other auxiliary materials are directly added into the solution in the step (2) for dissolution and are stirred uniformly;
(4) Adding the solution obtained in the step (3) into the gellan gum swelled in the step (1), or adding the gellan gum swelled in the step (1) into the solution obtained in the step (3), supplementing the balance of water, and stirring until the solution is clear and transparent, thus obtaining the puerarin gellan gum ionic in-situ gel eye drops.
7. The method for preparing puerarin gellan gum ionic in-situ gel eye drops according to claim 6, wherein the water added in the step (1) is 50-60% of the total water; the temperature is 90+/-5 ℃; the stirring swelling time is 15-20 min.
8. The method for preparing puerarin gellan gum ionic in-situ gel eye drops according to claim 6, wherein in the step (4), the solution is stirred until the solution is clear and transparent, and the solution is kept at more than 40 ℃ for filtration and sterilization.
9. The method for preparing puerarin gellan gum ionic in-situ gel eye drops according to claim 8, wherein filtering sterilization is performed by adopting a filter membrane with a pore size of 0.1-0.22 μm.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101564374A (en) * | 2008-04-25 | 2009-10-28 | 北京和润创新医药科技发展有限公司 | Medicinal in situ forming eye gel |
| CN101579305A (en) * | 2008-05-14 | 2009-11-18 | 北京和润创新医药科技发展有限公司 | Ready-to-use puerarin ophthalmic gel |
| CN104688672A (en) * | 2014-12-29 | 2015-06-10 | 浙江莎普爱思药业股份有限公司 | Application of puerarin gel eye drop in preparation of drugs for treating ischemic ocular fundus diseases |
| CN109966245A (en) * | 2019-04-03 | 2019-07-05 | 浙江省医学科学院 | A kind of brimonidine tartrate gellan gum type situ-gel eye drops and preparation method |
-
2022
- 2022-09-13 CN CN202211118746.3A patent/CN115487139B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101564374A (en) * | 2008-04-25 | 2009-10-28 | 北京和润创新医药科技发展有限公司 | Medicinal in situ forming eye gel |
| CN101579305A (en) * | 2008-05-14 | 2009-11-18 | 北京和润创新医药科技发展有限公司 | Ready-to-use puerarin ophthalmic gel |
| CN104688672A (en) * | 2014-12-29 | 2015-06-10 | 浙江莎普爱思药业股份有限公司 | Application of puerarin gel eye drop in preparation of drugs for treating ischemic ocular fundus diseases |
| CN109966245A (en) * | 2019-04-03 | 2019-07-05 | 浙江省医学科学院 | A kind of brimonidine tartrate gellan gum type situ-gel eye drops and preparation method |
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| Title |
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| H.Qi,et al.Development of a poloxamer analogs/carbopol-based in situ gelling and mucoadhesive ophthalmic delivery system for puerarin.International Journal of Pharmaceutics.2007,第337卷178–187. * |
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