CN107137345A - A kind of liposome gel formulation and its preparation and use for suppressing scar proliferation - Google Patents

A kind of liposome gel formulation and its preparation and use for suppressing scar proliferation Download PDF

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CN107137345A
CN107137345A CN201710377078.9A CN201710377078A CN107137345A CN 107137345 A CN107137345 A CN 107137345A CN 201710377078 A CN201710377078 A CN 201710377078A CN 107137345 A CN107137345 A CN 107137345A
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liposome
imiquimod
phospholipid
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preparation
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时军
张慧迪
吴艳婷
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Guangdong Pharmaceutical University
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
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    • 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
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    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes

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Abstract

The present invention provide it is a kind of be used to suppressing the imiquimod liposome gel formulation of scar proliferation, the liposome gel formulation includes liposome, gel rubber material and glycerine, and described liposomal encapsulated have imiquimod bulk drug.Present invention simultaneously provides the preparation and use of the liposome gel formulation.The imiquimod liposome gel formulation of the present invention shows that, with obvious sustained release and Transdermal absorption facilitation effect, compared with imiquimod cream, Percutaneous permeability and skin accumulation are significantly improved through tablets in vitro；Compared with other anti-scar proliferation pharmaceutical preparations or therapeutic scheme of report, recurrence rate is low, and toxic side effect incidence is low, and can be applied to the position of any scar proliferation of body.The lipidosome gel of the present invention has more advantage as Drug Percutaneous Absorption carrier.

Description

Liposome gel preparation for inhibiting scar hyperplasia and preparation and application thereof
Technical Field
The invention relates to a liposome gel preparation, in particular to an imiquimod liposome gel preparation, and also provides a preparation method and application of the preparation, belonging to the field of medical pharmacy.
Background
Hypertrophic Scars (HS) are a progressive, pathological and difficult-to-reverse skin disease, which often occurs during the repair of deep wounds (e.g., surgery, burns and scalds) and is caused by excessive dermal fibrosis due to massive microangiogenesis, fibroblast proliferation, and excessive collagen deposition. In the treatment scheme of hypertrophic scars, surgical excision and drug treatment means are commonly used in clinic at present. The surgical treatment usually requires skin grafting and radiation or laser irradiation auxiliary treatment, and the average recurrence rate reaches more than 50%. The therapeutic drugs mainly comprise steroid hormones (local injection or smearing), antimetabolites (local injection or oral administration)) and silicon preparations (smearing or spraying), wherein the hormone hormones have quick response, but have local side effects and systemic reactions such as skin atrophy, local telangiectasia, pigmentation and the like after long-term application; antimetabolites are generally anticancer preparations, have prominent toxic and side effects, and influence the metabolism and activity of normal skin tissue cells; the silicon preparation has no side effect and good patient compliance, but is effective only on the parts (such as limbs) where pressure is maintained.
Imiquimod is an imidazole quinoline drug, is the only interferon-induced immune response regulator in artificially synthesized small molecular compounds, and has the functions of resisting virus and tumor and regulating immunity. Imiquimod has unique effect, does not have direct antiviral activity, does not cause direct and nonspecific cytolytic destruction, but stimulates various immune cells to generate a series of cytokines and induces various cytokines by reducing the competitive inhibition effect of Toll-like receptors TLR7 and TLR8, thereby enhancing the immunoregulation effect. Imiquimod is mainly used for treating keloid, condyloma acuminatum, genital wart and the like in clinic. Due to the poor solubility of imiquimod, the available dosage forms are limited, and the products sold on the market at present are only creams and creams.
The liposome has the characteristics of reduced drug toxicity, targeted positioning, small particle size, good compatibility with biological membranes and the like, and the drug is prepared into the liposome, so that the stability and retention of the drug in organs are increased, and the curative effect of the drug is more remarkable. The gel is a semisolid preparation containing dissolved, suspended or emulsion drug, and can be tightly combined with action site for a long timeThe adhesive is broken, has better biological adhesive breaking property, and is simple to prepare and comfortable to use. The gel is thick liquid or semisolid preparation prepared by the medicine and auxiliary materials capable of forming gel and is in a uniform, suspension or emulsion type. It has the advantages of good air permeability, high bioavailability, good stability, less adverse reaction, and convenient application. Liposome gel mixes liposome and gel matrix, thereby increasing the stability of liposome, improving the applicability of liposome as external preparation, and providing slow release and controlled release of encapsulated drug. The liposome is used as common nanometer administration carrier, and has unique advantages of promoting drug penetration capability of scar heterogeneous epidermis and enhancing drug dermal retention property, and the aqueous administration environment satisfies the requirement of wound healing on O2The requirements of (a). In the research, the imiquimod is prepared into the liposome gel, so that the solubility of the imiquimod is increased, the retention amount of the imiquimod in a dermis layer is increased, and the effect of inhibiting scars by the imiquimod is better exerted. There is no current literature report on imiquimod liposome gel formulations.
Disclosure of Invention
One of the purposes of the invention is to provide a liposome gel preparation for inhibiting scar hyperplasia, wherein the liposome gel preparation comprises liposome, gel material and glycerol, the liposome is encapsulated with imiquimod bulk drug, and the liposome comprises phospholipid, cholesterol and vitamin E.
In a preferred embodiment, the phospholipid is composed of one or more phospholipids selected from the group consisting of egg yolk lecithin, cephalin, inositol phospholipid, serine phospholipid, nucleotide phospholipid, soybean lecithin, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylglycerol, hydrogenated soybean lecithin, and hexadecylphosphatidylcholines. Preferably, the phospholipid is composed of one or a mixture of egg yolk lecithin and hydrogenated soybean lecithin. Experiments show that the phase transition temperature of various phospholipid materials is different, and the preparation temperature is higher than the phase transition temperature. The experiment preferably uses one or a mixture of egg yolk lecithin and hydrogenated soybean lecithin.
In a preferred embodiment, the gel material is selected from one or more of poloxamer 188, poloxamer 407, carbomer 934 and carbomer 980.
In a preferred embodiment, the liposome microparticles have a particle size of 100 to 1000 nm.
In a preferred embodiment, the weight part ratio of cholesterol to phospholipid is 1-3: 2-6, the weight part ratio of imiquimod bulk drug to liposome (drug-lipid ratio) is 1: 10-60, and the amount of vitamin E accounts for 2-7% of the total mass fraction.
In another aspect, the invention provides a preparation method of an imiquimod liposome gel preparation for inhibiting hypertrophic scars.
The inventor firstly determines the transdermal absorption performance of imiquimod, discusses the relationship between the oil-water distribution coefficient of imiquimod and the transdermal absorption rate of imiquimod, screens out a reasonable prescription process and a reasonable feeding ratio by taking the entrapment rate, the particle size and the form as examination indexes, preferably selects imiquimod liposome with higher entrapment rate, and finally prepares the imiquimod liposome into liposome gel.
The inventor carries out liposome preparation methods in the early stage from injection methods, film dispersion methods, pH gradient methods, ultrasonic injection methods-pH gradient methods, film dispersion methods-pH gradient methods and the like, and finds that the film dispersion method-pH gradient methods are most suitable for industrial production, the prepared liposome solution has proper particle size (100-1000 nm) and high entrapment rate (> 80%).
The method provided by the invention comprises the following steps:
(a) accurately weighing a certain amount of imiquimod bulk drug, phospholipid material, cholesterol and vitamin E, adding trichloromethane, and stirring to dissolve the components to obtain a clear and transparent solution;
(b) placing the clear transparent solution obtained in the step (a) into a container, carrying out reduced pressure rotary evaporation to remove the solvent, so that phospholipid forms a layer of uniform film on the wall of the container, adding phosphate buffer solution, hydrating overnight to obtain milky suspension, adjusting the pH value with alkaline solution to form pH gradient difference, incubating, and carrying out ultrasonic treatment under ice bath to obtain imiquimod liposome;
(c) filtering the liposome obtained in the step (b) (filtering by a 0.8 mu m microporous filter membrane, filtering the filtrate by a 0.45 mu m microporous filter membrane, and repeating for 3 times) to obtain a liposome solution;
(d) adding a polymer gel material into the liposome solution, adding glycerol after complete swelling, and uniformly grinding to obtain the liposome.
In a preferred embodiment, in the above-mentioned process step (b), the temperature is 50 ℃ and the rotational speed is 30 r.min-1And carrying out reduced pressure rotary evaporation for 15-45 minutes, standing for 2 hours to fully swell and hydrate the cholesterol, wherein the pH value of the phosphate buffer solution is 6.0-7.0, the alkaline solution is 1mol/L NaOH, and the ultrasonic time under ice bath is 15-45 minutes to obtain the imiquimod liposome. More preferably, in the above-mentioned process step (b), the temperature is 50 ℃ and the rotational speed is 30 r.min-1And carrying out reduced pressure rotary evaporation for 30 minutes, standing for 2 hours to fully swell and hydrate the cholesterol, and carrying out ultrasonic treatment for 30 minutes in ice bath to obtain the imiquimod liposome.
In a preferred embodiment, in the method, the weight part ratio of cholesterol to phospholipid is 1-3: 2-6, the weight part ratio of imiquimod bulk drug to liposome (drug-to-lipid ratio) is 1: 10-60, the amount of vitamin E accounts for 2-7% of the total mass fraction, the incubation temperature is 30-70 ℃, the incubation time is 20-100 minutes, the pH gradient difference is 1-5, and the mass ratio of gel material to glycerol is 1-50: 1-5; preferably, the mass ratio of cholesterol to phospholipid is 1: 3-5, the medicine-lipid ratio is 1: 20-40, the using amount of vitamin E is 4-6%, the incubation temperature is 40-60 ℃, the incubation time is 50-90 minutes, the pH gradient difference is 1-3, the mass ratio of the gel material to glycerol is 1-20: 1-2; most preferably, the mass ratio of cholesterol to phospholipid is 1:5, the drug-to-lipid ratio is 1:20, the dosage of vitamin E is 6%, the incubation temperature is 50 ℃, the incubation time is 70 minutes, the pH gradient difference is 3, and the mass ratio of the gel material to the glycerol is 3: 1.
In a further aspect, the invention provides the use of a liposome gel formulation of the invention in the preparation of a medicament for inhibiting hypertrophic scarring.
The liposome gel preparation is directly applied to scar hyperplasia parts in clinical application, in particular to deep wound parts without or with inflammation after operation and burn and scald. The imiquimod transdermal gel preparation is prepared by encapsulating medicines by using phospholipid and other materials with good human physiological compatibility, can effectively improve the solubility of imiquimod, increase the retention amount of the imiquimod in a dermis layer, better play the role of the imiquimod in inhibiting scars, has low recurrence rate and lower toxic and side effect incidence rate compared with other reported anti-scar hyperplasia medicinal preparations or treatment schemes, and can be applied to any scar hyperplasia part of a body.
In addition, the preparation method of the liposome gel preparation for inhibiting hypertrophic scars, provided by the invention, has the following advantages: 1) the preparation has no toxic solvent residue, and does not contain auxiliary materials which can cause skin irritation or other adverse reactions; 2) compared with the anti-scar hyperplasia drug sold on the market, the anti-scar hyperplasia drug has the advantages of small toxic and side effects, good patient compliance, ideal transdermal speed and effective regulation of skin immune cell factors; 3) the preparation technology can be industrially implemented, and the prepared liposome gel has proper viscosity and good stability and meets the requirements of pharmacopoeia.
Drawings
FIG. 1 is a graph of transdermal rate constants and lg in one embodiment of the present inventionPThe schematic diagram of the fitted curve is shown,Pthe transdermal property of the imiquimod is closely related to the oil-water distribution coefficient of the imiquimod under the condition of a solvent medium with the pH value of 4.5-7.4, and the penetration of the imiquimod can be predicted by measuring the oil-water distribution coefficient of the drugSkin properties.
Fig. 2 is a transmission electron microscope image of liposome particles in the process of forming the liposome gel preparation in one embodiment of the present invention, and it can be seen from the image that the particles are uniform in size, the outer edges are circular, and the shape is regular.
FIG. 3 is a distribution diagram of liposome particle size during formation of a liposome gel formulation according to an embodiment of the present invention, showing that the mean particle size is about 335 nm, which is in accordance with a normal distribution.
FIG. 4 is a graph of the transdermal permeation kinetics of a liposomal gel formulation with a conventional imiquimod gel in one embodiment of the present invention, where the liposomal gel formulation of the present invention is represented; □, it can be seen that the liposome gel formulation of the present invention has a significantly better drug release profile than the conventional imiquimod gel.
Detailed Description
The liposome gel preparation provided by the invention comprises liposome, gel material, glycerol and the like, wherein the liposome is encapsulated with the following examples to facilitate better understanding of the invention, but the invention is not limited by the following examples. The experimental procedures in the following examples are conventional unless otherwise specified.
The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. Unless otherwise indicated, percentages and parts are by weight.
Example 1: research on imiquimod apparent oil-water distribution coefficient and correlation between imiquimod apparent oil-water distribution coefficient and transdermal absorption rate constant
Placing n-octanol into conical flask with stopper, saturating with buffer salt with pH of 4.5, 5.0, 5.8, 6.5, 7.4, shaking at 33 + -0.5 deg.C for 24 hr, 3500 r.min-1Centrifuging for 20min, and separating n-octanol from the buffer solution. Accurately weighing imiquimod 1.5mg, dissolving n-octanol pre-saturated with buffer solutions of different pH values, and standing at 25 ml, in a volumetric flask, performing constant volume to obtain a series of concentrations of 60 mug-mL-1. Taking the buffer solution saturated by n-octanol and the imiquimod n-octanol solution, putting the buffer solution and the imiquimod n-octanol solution into a conical flask according to the volume ratio of 1:1, and sealing the conical flask. Placing a part of the solution in the conical flask in a constant temperature shaking table at 33 + -0.5 deg.C, shaking for 72h, swirling another part of the conical flask for 40 min, placing in a constant temperature water bath at 33 + -0.5 deg.C, standing for 72h, taking out, and sealing at 3500 r.min-1Centrifuging for 20min, taking the buffer salt layer, and fixing the volume. The concentration of the buffer salt phase was measured by HPLC to calculate the oil-water distribution coefficient, and the results are shown in Table 1.
P= (C-Cw)/Cw equation 1
Wherein,Pis the apparent oil-water distribution coefficient of imiquimod; c is the initial drug concentration in the saturated n-octanol of the buffer solution, and Cw is the drug concentration in the buffer solution after equilibrium.
Placing rat skin between the two half tanks with stratum corneum facing upwards, clamping, and preventing liquid leakage, and effective permeation area of 2.25 cm2The volume of the receiving pool is 15mL, the temperature is 33 +/-0.5 ℃, and the stirring speed is 350 r.min-1Samples were taken at 1, 2, 4, 6, 8, 12, 24h, 1mL each time, while making up 1mL, with a receiving solution of buffer salt at pH = 6.5. Calculating to obtain Qn, drawing the Qn-t equation, and obtaining the result shown in the table 2.
Equation 2
Wherein, Qn: accumulating the amount of the permeated drug in unit area at the nth hour; cn: the concentration sampled at the nth hour; v: receiving a volume of liquid; a: the effective diffusion area of the diffusion cell.
TABLE 1 determination of the oil-water partition coefficient of imiquimod in n-octanol-buffer solutions of different pH values
PApparent oil-water partition coefficient for imiquimod
The experimental result shows that the imiquimod is in the range of pH 5.8-7.3, and 1 < lgPLess than 3, showing that the medicine has good lipophilicity and poor water solubility, and lg measured by the shaking bed method and vortex methodPThe difference is not obvious, and lg is shown when the pH value of the solvent medium is between 4.5 and 7.4PIncreasing with increasing pH.
TABLE 2 measurement of the percutaneous absorption Rate constant, slope is the percutaneous Rate constant (C:)J）
From table 2, it can be seen that the transdermal absorption rate constant of imiquimod is maximal around pH 5.8.
The correlation between the oil-water distribution coefficient of imiquimod and the transdermal rate constant is researched, the result is shown in fig. 3, the inventor finds that the transdermal performance of imiquimod is closely related to the oil-water distribution coefficient of imiquimod in a solvent medium with the pH value of 4.5-7.4 through experimental research, and the transdermal performance of the drug can be predicted by measuring the oil-water distribution coefficient of the drug.
Example 2: prescription screening of imiquimod liposomes
Taking liposome encapsulation efficiency as an investigation index, selecting L9(34) The liposome prescription is optimized by orthogonal design of factor level, the preparation process is that the incubation temperature is 40 ℃, the incubation time is 50 min, the pH gradient difference is 3, the phospholipid/cholesterol mass ratio (A), the drug-lipid ratio (B) and the VE dosage (%) (C) are considered, the imiquimod liposome is prepared by adopting a film dispersion-pH gradient method, and orthogonal test is carried out to determine the optimal feeding ratio of each component, and the formula factor level is shown in Table 3.
Table 3: level of prescription factor
Accurately weighing a certain amount of imiquimod bulk drug, phospholipid material, cholesterol and vitamin E, adding a proper amount of trichloromethane, and stirring to dissolve the components to obtain a clear and transparent solution; placing the solution in 500ml eggplant-shaped bottle at 50 deg.C and 30r min-1And carrying out rotary evaporation under reduced pressure to form a layer of uniform film on the wall of the container by phospholipid, adding a phosphate buffer solution with pH of 6.5, rotating for 30 minutes, hydrating overnight to obtain milky suspension, adjusting the pH value with 1mol/L NaOH solution to form a pH gradient difference, incubating, carrying out ultrasonic treatment under ice bath to obtain imiquimod liposome, filtering the obtained liposome with a 0.8 mu m microporous filter membrane, filtering the filtrate with a 0.45 mu m microporous filter membrane, and repeating for 3 times to obtain a liposome solution. The results of the orthogonal tests are shown in Table 4 and the results of the analysis of variance are shown in Table 5.
Table 4: orthogonality test and results
Table 5: analysis of variance results
From the experimental results in tables 4 and 5, when imiquimod is used to prepare liposome, factors A and B in the formula have significant influence on the liposome, and it can be known that B > A > C and the optimal combination is A3B1C3, namely, the phospholipid-cholesterol ratio is 5:1, the drug-lipid ratio is 20:1 and the VE dosage is 6%.
Example 3: preference of preparation Process
The ratio of phospholipid to cholesterol is 5:1, and the medicine-to-lipid ratio is20:1, wherein the dosage of VE is 6 percent, and L is selected9(34) The formula process is screened by orthogonal design of factor level, the process factors mainly comprise incubation temperature (A), incubation time (B) and pH gradient difference (C), and the factor level is shown in Table 6.
Table 6: level of orthogonality of the preparation process
Table 7: analysis of variance results
Table 8: quadrature test and results of the preparation Process
The experimental results in tables 7 and 8 show that the factors A and B have significant influence on the preparation process, the A > B > C according to the deviation square sum sequence, the optimal combination is A2B2C3, namely the temperature is 50 ℃, the incubation time is 70min, and the pH gradient difference is 3.
The best process is verified: 5 replicate validation experiments were performed according to the preferred formulation to prepare 5 batches of liposomes. Accurately weighing 50 mg of cholesterol, 200 mg of phospholipid (S100), 6 mg of VE and 10 mg of imiquimod, adding 20mL of trichloromethane, and stirring to dissolve the components to obtain a clear and transparent solution; placing the solution in a 500ml eggplant-shaped bottle, rotating at the rotation speed of 30 r.min < -1 > at the temperature of 50 ℃, evaporating the solvent under reduced pressure in a rotating manner to form a layer of uniform film on the wall of the container, adding a phosphate buffer solution with the pH value of 6.5, rotating for 30 minutes, placing for 2 hours to fully swell and hydrate cholesterol, adjusting the pH value with a 1mol/L NaOH solution to form a pH gradient difference of 3, incubating at the temperature of 50 ℃ for 70 minutes, performing ultrasonic treatment for 30 minutes in an ice bath to obtain imiquimod liposome, filtering the obtained liposome with a 0.8 mu m microporous filter membrane, filtering the filtrate with a 0.45 mu m microporous filter membrane, and repeating for 3 times to obtain a liposome solution. The obtained imiquimod liposome solution is milky semitransparent, uniform in color, slightly opalescent and good in liquidity. The results show that the particle size of 5 batches of prepared liposome is in normal distribution, the average particle size is 355 nm, the entrapment rate (87.86 +/-0.0153)%, and the RSD is 0.96%.
Example 4: examination of the leakage Rate of Imquimod liposomes
Under the optimal preparation process, the prepared imiquimod liposome is respectively placed in a refrigerator at 4 ℃ and stored at room temperature of 25 ℃, the content of the liposome is measured by sampling at 0 d, 30 d and 60 d respectively, and the leakage rate P in the storage process is calculated according to the following formula. Each set of experiments was repeated three times and averaged.Wherein, W1、W2The encapsulation amount during preparation and the encapsulation amount after a certain storage time are respectively. The results are shown in Table 9.
Table 9: imiquimod liposome leakage rate
Example 5: content determination and stability preliminary investigation of imiquimod liposome gel preparation
Preparation of imiquimod liposome gel and common gel preparation: weighing 9400.3 g of carbomer and 0.002 g of ethylparaben, adding into the prepared liposome solution, swelling completely, adding 0.1 g of glycerol, and grinding uniformly to obtain the imiquimod liposome gel preparation. Weighing 10 mg of imiquimod, adding a buffer solution with pH 6.5 to dissolve, and fixing the volume to 10 mL; adding carbomer 0.3 g and ethylparaben 0.002 g, swelling completely, adding glycerol 0.1 g, and grinding to obtain imiquimod common gel.
Content determination of imiquimod liposome gel preparation: accurately weighing 0.3 g each of imiquimod liposome gel preparation and common gel preparation, placing in a 25 mL volumetric flask, adding 10mL methanol solution, dissolving, performing ultrasonic treatment for 5 min, and diluting with distilled water to desired volume. Filtering, precisely sucking the filtrate, and performing content determination on a high performance liquid chromatograph. The determination result shows that the content of the imiquimod liposome gel preparation (3.03 +/-0.25) mg.g-1(n = 3), average sample recovery 96.54% (n = 9, RSD value 4.91%). The stability RSD value is 3.85%, and the reproducibility RSD value is 2.16%.
Preliminary investigation of stability of imiquimod liposome gel formulation:
the characteristics are as follows: the gel is light white, uniform, fine and translucent, has no sticky block, can keep colloidal at normal temperature, is not dried or liquefied, and has good spreading performance.
Viscosity: the gel has moderate viscosity.
pH value: 3 g of the product is taken and diluted by 10mL of distilled water, and the mixture is uniformly stirred, and the measured pH value is between 6.0 and 7.0.
And (3) centrifugal test: taking 3 batches of samples, each 5 g, 4500 r.min-1Centrifuging for 30 min, and homogenizing without coagulation and stratification.
One or more verification test results show that the preparation process conditions of the imiquimod liposome gel preparation are stable and reliable.
Example 6 in vitro transdermal permeation kinetics examination of Imquimod Liposol gel formulations
In vitro skin preparation comprises cutting neck of male Kunming mouse (18-22 g), removing abdominal skin, scraping abdominal hair, removing excessive fat and connective tissue, cleaning with normal saline, drying with filter paper, spreading skin, and storing in refrigerator at-20 deg.C for one week.
Transdermal absorption experiment: recovering mouse skin to room temperature, washing with physiological saline, blotting surface liquid with filter paper, and placing in Franz diffusion cell (effective drug release area is 1.766 cm)2Receiving reservoir volume 15 mL) and the stratum corneum toward the supply chamber. In the diffusion cell, the receiving chamber was filled with a phosphate buffer solution containing 30% ethanol at a pH of 6.5 to remove air bubbles and bring the receiving medium into close contact with the skin. The temperature of the receiving medium is (33 +/-0.5) DEG C, and the magnetic stirring speed is 350r min-1. 0.3 g of imiquimod liposome gel preparation and imiquimod gel are respectively added into a supply room for each group, 1mL of receiving solution (equivalent isothermal receiving solution is supplemented at the same time) is precisely absorbed from a receiving room sampling tube after 1, 2, 4, 6, 8, 10, 12 and 24 hours after the test, and 20 muL of filtrate is taken for sample injection and measurement after the receiving solution is filtered by a 0.22μm microporous filter membrane. Substituting the peak area into a standard curve to calculate the concentration of the drug, and calculating the cumulative permeation amount per unit area (Q) at each time point according to the formula (2)n, µg·cm-2) Drawing Qn-t equation, run in parallel 6 times, and take the average. The results are shown in FIG. 4.
(2)
Wherein Q isnAccumulating the amount of the permeated drug in unit area at the nth hour; cnThe concentration sampled at the nth hour; and the effective diffusion area of the A diffusion cell.
Example 7: imiquimod liposome transdermal gel preparation
The formula is as follows: 50 mg of cholesterol, 200 mg of egg yolk lecithin (S100), 16 mg of vitamin E, 10 mg of imiquimod, 10mL of phosphate buffer (pH =6.5), 9400.3 g of carbomer, 0.002 g of ethylparaben and 0.1 g of glycerol.
The preparation method comprises the following steps: accurately weighing 50 mg of cholesterol, 200 mg of yolk lecithin (S100), 16 mg of vitamin E and 10 mg of imiquimod, adding 20mL of trichloromethane, and stirring to dissolve the components to obtain a clear and transparent solution; placing the solution in a 500ml eggplant-shaped bottle, rotating at the rotation speed of 30 r.min < -1 > at the temperature of 50 ℃, evaporating the solvent under reduced pressure in a rotating manner to form a layer of uniform film on the wall of the container, adding phosphate buffer solution with the pH value of 6.5, rotating for 30 minutes, hydrating overnight to obtain milky white suspension, adjusting the pH value to 10 with 1mol/L NaOH solution, incubating for 30 minutes at 40 ℃, performing ultrasonic treatment under ice bath to obtain imiquimod liposome, filtering the obtained liposome with a 0.8 mu m microporous filter membrane, filtering the filtrate with a 0.45 mu m microporous filter membrane, and repeating for 3 times to obtain liposome solution. Weighing 9400.3 g carbomer and 0.002 g ethylparaben, adding into the prepared liposome solution, swelling completely, adding 0.1 g glycerol, grinding, packaging, spreading on a backing material, and sterilizing to obtain imiquimod liposome gel preparation.
Example 8: imiquimod liposome transdermal gel preparation
The formula is as follows: 50 mg of cholesterol, 200 mg of hydrogenated soybean lecithin (S100), 16g of vitamin E, 10 mg of imiquimod, 10mL of phosphate buffer (pH =6.5), 9400.3 g of carbomer, 0.002 g of ethylparaben and 0.1 g of glycerol.
The preparation method comprises the following steps: accurately weighing 50 mg of cholesterol, 200 mg of hydrogenated soybean lecithin (S100), 16g of vitamin E and 10 mg of imiquimod, adding 20mL of trichloromethane, and stirring to dissolve the components to obtain a clear and transparent solution; placing the solution in 500ml eggplant-shaped bottle at 50 deg.C and 30r min-1Evaporating solvent under reduced pressure to form a uniform film on the wall of the container, adding phosphate buffer solution with pH of 6, rotating for 30 min, hydrating overnight to obtain milky suspension, and adjusting pH with 1mol/L NaOH solution to desired value9, incubating at 50 ℃ for 45 minutes, performing ultrasonic treatment in an ice bath to obtain imiquimod liposome to obtain the imiquimod liposome, filtering the obtained liposome through a 0.8 mu m microporous filter membrane, filtering the filtrate through a 0.45 mu m microporous filter membrane, and repeating the steps for 3 times to obtain a liposome solution. Weighing 9400.3 g carbomer and 0.002 g ethylparaben, adding into the prepared liposome solution, swelling completely, adding 0.1 g glycerol, grinding, packaging, spreading on a backing material, and sterilizing to obtain imiquimod liposome gel preparation.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Claims (10)

1. A liposomal gel formulation for inhibiting scar hyperplasia, wherein the liposomal gel formulation comprises liposomes, a gel material, and glycerol, the liposomes encapsulating an imiquimod drug substance, and the liposomes comprising a phospholipid, cholesterol, and vitamin E.
2. The liposomal gel formulation of claim 1 wherein the phospholipid is comprised of one or more phospholipids selected from the group consisting of egg yolk lecithin, cephalin, inositol phospholipid, serine phospholipid, nucleotide phospholipid, soy lecithin, distearoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylglycerol, hydrogenated soy lecithin, and hexadecylphosphatidylcholine.
3. The liposomal gel formulation according to claim 1 or 2, wherein the gel material is selected from one or more of poloxamer 188, poloxamer 407, carbomer 934 and carbomer 980.
4. The liposomal gel formulation of claim 1 or 2 wherein the particle size of the liposomal microparticles is 100 to 1000 nm.
5. The liposome gel preparation of claim 1 or 2, wherein the weight part ratio of cholesterol to phospholipid is 1-3: 2-6, the weight part ratio of imiquimod bulk drug to liposome (drug-lipid ratio) is 1: 10-60, and the amount of vitamin E accounts for 2-7% of the total mass fraction.
6. A method of preparing the liposomal gel formulation of claim 1, the method comprising:
(a) providing an imiquimod bulk drug, a phospholipid material, cholesterol and vitamin E, adding trichloromethane, and stirring to dissolve all the components to obtain a clear and transparent solution;
(b) placing the clear transparent solution obtained in the step (a) into a container, carrying out reduced pressure rotary evaporation to remove the solvent, so that phospholipid forms a layer of uniform film on the wall of the container, adding phosphate buffer solution, hydrating overnight to obtain milky suspension, adjusting the pH value with alkaline solution to form pH gradient difference, incubating, and carrying out ultrasonic treatment under ice bath to obtain imiquimod liposome;
(c) filtering the liposome obtained in the step (b) to obtain a liposome solution;
(d) adding a polymer gel material into the liposome solution, adding glycerol after complete swelling, and uniformly grinding to obtain the liposome.
7. The method for preparing the liposome gel formulation of claim 6, wherein the rotation time in step (b) is 15-45 minutes, the pH of the phosphate buffer solution is 6.0-7.0, the alkaline solution is 1mol/L NaOH, and the ultrasound time in ice bath is 15-45 minutes.
8. The preparation method of the liposome gel preparation as claimed in claim 6 or 7, wherein the weight portion ratio of cholesterol to phospholipid is 1-3: 2-6, the weight portion ratio of imiquimod raw material drug to liposome (drug-lipid ratio) is 1: 10-60, and the amount of vitamin E accounts for 2-7% of the total weight.
9. The method for preparing the liposome gel preparation according to claim 6 or 7, wherein the difference of the pH gradient is 1 to 5, the incubation temperature is 30 to 70 ℃, and the incubation time is 20 to 100 minutes.
10. Use of the liposomal gel formulation of claim 1 in the preparation of a medicament for inhibiting hypertrophic scarring.
CN201710377078.9A 2017-05-25 2017-05-25 A kind of liposome gel formulation and its preparation and use for suppressing scar proliferation Pending CN107137345A (en)

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