Preparation method for triamcinolone acetonide acetate ultradeformable nanometer lipid vesicle spraying agent and application to target hypertrophic scars

A technology of triamcinolone acetonide acetate and nano-lipids, which is applied in aerosol delivery, medical preparations containing active ingredients, and pharmaceutical formulas. There are no reports on the preparation of vesicle sprays, etc., and the therapeutic effect is remarkable and the effect is long-lasting.

Inactive Publication Date: 2014-12-03

SHANGHAI SEVENTH PEOPLES HOSPITAL

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[0006] The preparation of triamcinolone acetonide acetate flexible nano-lipid vesicles has been reported abroad, but the preparation of triamcinolone acetonide acetate...

Abstract

The invention discloses a preparation method for a triamcinolone acetonide acetate ultradeformable nanometer lipid vesicle spraying agent and application to target hypertrophic scars. The triamcinolone acetonide acetate ultradeformable nanometer lipid vesicle spraying agent is composed of the following compositions in percent by weight: 0.05%- 0.5% of triamcinolone acetonide acetate, 0.5%- 10% of phosphatide, and 0.2%- 50% of a softener. The prepared triamcinolone acetonide acetate ultradeformable nanometer lipid vesicle spraying agent has the average particle size of 50-500 nm. By optimizing the above ratio, the medicine can be relatively highly concentrated at a scar skin nidus area, and the composition ratio comprises, in percent by weight, 0.1% of triamcinolone acetonide, 4% of phosphatide, and 0.77% of sodium cholate. The prepared triamcinolone acetonide acetate ultradeformable nanometer lipid vesicle spraying agent solves many problems caused by multi-point injection in scar and triamcinolone acetonide external application, and is substantial in the hypertrophic-scar targeting effect.

Application Domain

Organic active ingredientsAerosol delivery +1

Technology Topic

DrugChemistry +6

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Examples

Experimental program(4)

Example Embodiment

[0024] Example 1 Screening of phospholipid ratio of triamcinolone acetonide acetate flexible nanolipid vesicle spray

[0025] Prescription group distribution ratio (w/w/%) and dosage:

[0026]

[0027] The distribution ratio of the two prescriptions (w/w/%) and dosage:

[0028]

[0029]

[0030] The three-group distribution ratio (w/w/%) and dosage of prescription:

[0031]

[0032] Preparation

[0033] Quantitatively weigh the phospholipids and triamcinolone acetonide in the above recipes into an eggplant-shaped bottle, add 30ml of a mixed solvent of chloroform-methanol (1∶1, v/v), in a constant temperature water bath at 37°C, and evaporate the organic solvent under reduced pressure with a water pump , A uniform lipid-like film is formed on the inner wall of the bottle, and nitrogen is injected. Adding the aqueous solution of sodium cholate with the prescribed content and rotating the membrane for 2 hours to obtain a suspension of flexible nanolipid vesicles. Under the condition of ice-water bath, probe-type intermittent ultrasound for 15 minutes, and filtration with 0.22 μm microporous membrane to prepare uniform flexible nano lipid vesicles. The flexible nano-lipid vesicles are mixed with corresponding distilled water to prepare triamcinolone acetonide acetate flexible nano-lipid vesicle spray. Steam sterilization, filtration, and aseptic dispensing into spray bottles.

[0034] Morphological observation

[0035] Take a small amount of triamcinolone acetonide acetate flexible nano-lipid vesicle spray prepared from various prescriptions, drop it on the copper mesh with supporting film, and after a little drying, use 3% phosphotungstic acid for negative staining. The filter paper absorbs the excess liquid on the edge of the copper mesh. Slightly dry, observe through transmission electron microscope. figure 1 Electron micrograph of triamcinolone acetonide acetate flexible nano-lipid vesicle spray (×20000), figure 1 It can be seen that the triamcinolone acetonide acetate flexible nano-lipid vesicle spray prepared by each prescription is in the shape of a sphere or an ellipse, and the lipid vesicles prepared in the first, second, and third prescriptions have a particle size of 100-200 nm.

[0036] Encapsulation rate comparison

[0037] Take 1.5ml of the triamcinolone acetonide acetate flexible nano-lipid vesicle spray prepared by the above recipes in a centrifuge tube, 8×10 5 Centrifuge at r/min for 2h at 4°C. Take the supernatant and determine the amount of free drug C1 by HPLC. In addition, 1.5ml of the triamcinolone acetonide acetate flexible nano-lipid vesicle spray prepared by the above prescriptions was added to an appropriate amount of 20% TritonX-100, and the total amount of the drug C was determined by HPLC 2 , Encapsulation rate = (C 2 -C 1 )/C 2 ×100%.

[0038] Assay

[0039] Chromatographic conditions: Diamonsil C column 18 Column (4.6mm×250mm, 5μm), mobile phase is methanol-water (70:30), column temperature 25°C, flow rate 1.0ml/min, detection wavelength 240nm, injection volume 20μl.

[0040] Preparation of the standard curve: accurately weigh 25mg of the reference substance, place it in a 50ml volumetric flask, add 35ml of methanol to dissolve, dilute to the mark with water, and shake it up as a stock solution. Dilute with mobile phase, diffusion cell receiving solution, and scar blank skin tissue homogenate to prepare corresponding standard curves. Take the average peak area A as the ordinate and the concentration of the reference substance C as the abscissa to perform linear regression. The standard curve equation for determining the encapsulation rate of lipid vesicles is A=44826C+3246.6 (n=5, r=0.9999), linear range It is 0.1~50μg/ml.

[0041] The encapsulation rate experiment measured that the encapsulation rate of prescription one triamcinolone acetonide acetate flexible nanolipid vesicles was (67.04±3.77%), and the encapsulation rates of prescription two and prescription three were (44.68±3.24)% and (68.31), respectively. ±4.57)%.

[0042] As the weight percentage of phospholipids in the prescription increases, the encapsulation efficiency of flexible nanolipid vesicles increases, but when the weight percentage of phospholipids is higher than 4%, the encapsulation efficiency does not increase significantly. In summary, the phospholipid prescription of the flexible nanolipid vesicles is 4%.

Example Embodiment

[0043] Example 2 Screening of sodium cholate ratio of triamcinolone acetonide acetate flexible nanolipid vesicle spray

[0044] Prescription group distribution ratio (w/w/%) and dosage:

[0045]

[0046] The distribution ratio of the two prescription groups (w/w/%) and dosage:

[0047]

[0048] Distribution ratio of the three prescriptions (w/w/%) and dosage:

[0049]

[0050]

[0051] Preparation

[0052] Quantitatively weigh the phospholipids and triamcinolone acetonide in the above recipes into an eggplant-shaped flask, add 30 ml of a mixed solvent of chloroform-methanol (1∶1, v/v), in a constant temperature water bath at 37°C, and evaporate the organic solvent under reduced pressure by a water pump , A uniform lipid-like film is formed on the inner wall of the bottle, and nitrogen is injected. Adding the aqueous solution of sodium cholate with the prescribed content and rotating the membrane for 2 hours to obtain a suspension of flexible nanolipid vesicles. Under the condition of ice-water bath, probe-type intermittent ultrasound for 15 minutes and filtration with 0.22 μm microporous membrane to prepare uniform flexible nanolipid vesicles. The flexible nano-lipid vesicles are mixed with corresponding distilled water to prepare triamcinolone acetonide acetate flexible nano-lipid vesicle spray. Steam sterilization, filtration, and aseptic dispensing into spray bottles.

[0053] Morphological observation

[0054] Take a small amount of triamcinolone acetonide acetate flexible nano-lipid vesicle spray prepared from various prescriptions, drop it on a copper mesh with a supporting membrane, and after a little drying, use 3% phosphotungstic acid for negative staining. Slightly dry, observe through transmission electron microscope. The results showed that the triamcinolone acetonide acetate flexible nano-lipid vesicle spray prepared by each prescription was in the shape of a sphere or an ellipse, and the lipid vesicles prepared by prescriptions one, two and three had particle sizes of 100-200nm.

[0055] Deformability investigation

[0056] Under the action of external pressure, the performance of the flexible nano-lipid vesicle spray of triamcinolone acetonide acetate prepared from various prescriptions was observed through deformation through a microporous membrane with a pore size of 0.06μm. First record the time required for 5ml of distilled water to pass through the microporous membrane, and then record the time required for 5ml of triamcinolone acetonide acetate flexible nanolipid vesicle spray to pass through the same membrane to calculate the relative permeation rate P=Vtf/Vwater×100%. The relative permeation rate of triamcinolone acetonide acetate flexible nanolipid vesicle spray prepared from various prescriptions under certain pressure was investigated. The results of the deformability of each prescription are shown in Table 1. It can be seen that the deformability of the spray prepared increases with the increase of the sodium cholate content, but the relative rate of prescription three does not increase significantly compared with prescription one.

[0057] Table 1 Deformability test results of sprays prepared from various prescriptions (n=4, )

[0058]

[0059] *P<0.05, compared with prescription two

[0060] Encapsulation rate comparison

[0061] Take 1.5ml of the triamcinolone acetonide acetate flexible nano-lipid vesicle spray prepared by the above prescriptions in a centrifuge tube, 8×10 5 Centrifuge at r/min for 2h at 4°C. Take the supernatant and determine the amount of free drug C by HPLC 1. In addition, 1.5ml of the triamcinolone acetonide acetate flexible nano-lipid vesicle spray prepared by the above formulas was added to an appropriate amount of 20% TritonX-100, and the total amount of the drug C was determined by HPLC 2 , Encapsulation rate = (C 2 -C 1 )/C 2 ×100%.

[0062] The encapsulation rate experiment measured that the encapsulation rate of prescription one triamcinolone acetate flexible nanolipid vesicles was (67.04±3.77%). The encapsulation rates of prescription two and prescription three were (71.15±2.04)% and (51.81), respectively. ±1.52)%.

[0063] As the weight percentage of sodium cholate in the prescription increases, the deformability of the flexible nanolipid vesicles increases, but the encapsulation efficiency decreases. In summary, the sodium cholate formula of flexible nanolipid vesicles is 0.77%.

[0064] Combining the results of Examples 1 and 2, when the formulation ratio is 4% phospholipid and 0.77% sodium cholate, the spray particle size, encapsulation efficiency and deformability are all better.

Example Embodiment

[0065] Example 3 Drug release experiment on isolated scar skin

[0066] Skin preparation

[0067] Selected patients with hypertrophic scars with a disease course of more than 1 year, relatively smooth local scars, no ulceration, an area of 3cm×3cm or more, requiring surgical resection, and informed consent before surgery. After the scar skin is removed by surgery, the subcutaneous fat is removed, rinsed with saline, and stored at -20°C for later use.

[0068] Penetration experiment

[0069] The modified Franz diffusion cell is used to fix the skin between the supply tank and the receiving tank, the stratum corneum faces the supply tank, and the effective penetration area is 0.952cm 2. The supply pool was put into prescription one to prepare 0.1% triamcinolone acetonide acetate flexible nanolipid vesicle spray, 0.1% triamcinolone acetonide acetate nanolipid vesicles, 0.1% triamcinolone acetonide solution, and commercially available triamcinolone acetonide milk Each ointment is 0.5g, and 4.2ml of 20% ethanol saline is added to the receiving pool. The temperature of the water bath is (37.0±0.5)℃, the magnetic stirring speed is 300r/min, all the receiving solution is drawn at 0, 2, 4, 6, 9, and 12 hours, and the same volume of fresh receiving solution is added at the same time. The acceptance is determined by HPLC The content of triamcinolone acetonide in the liquid, and the cumulative permeation Q (μg/cm 2 ) And transdermal flow rate J (μg cm -2 ·H -1 ).

[0070] Drug retention in the skin

[0071] After the penetration test for 12 hours, wipe the skin of the diffusion site with a cotton ball dipped in methanol to remove the excess drug. Precisely weigh the skin, cut into pieces, add 5ml of 20% ethanol and normal saline, homogenize in a homogenizer for 5min, 1×10 4 Centrifuge for 10 min at r/min. Take 200μl of homogenate, place it in a 10ml centrifuge tube, add 200μl of 70% methanol, mix well, add 2ml of ethyl acetate-n-hexane (4:1), vortex for 2min, let stand for 10min, centrifuge at 3000r/min for 10min, and draw The upper organic phase is placed in a 10ml cone-bottomed test tube. Then add 2.0 ml of ethyl acetate-n-hexane (4:1) to the above-mentioned test tube with a stopper, and perform a second extraction. Combine the two extracts in a water bath at 40°C and evaporate to dryness under nitrogen flow, reconstitute with 200μl of mobile phase, 1×10 4 Centrifuge at r/min for 10 min, filter, and determine the content of triamcinolone acetonide in the supernatant by HPLC.

[0072] The standard curve equation of the receiver solution measured by HPLC is A=44713C-71.065 (n=5, r=0.999), and the linear range is 0.1-10μg/ml; the standard curve equation for the determination of scar skin homogenate is A=38763C+29712(n = 6, r = 0.9987), the linear range is 1-100 μg/ml.

[0073] The transdermal characteristics of different triamcinolone acetonide preparations in vitro scar skin, see figure 2. See Table 2 for the transdermal flow of different triamcinolone acetonide preparations in isolated scar skin and the amount of drug retained in the skin.

[0074] figure 2 As shown in Table 2, the 12-hour cumulative permeation of triamcinolone acetonide acetate flexible nanolipid vesicle spray is (7.84±0.99) μg/cm 2 , Which are 2.20 and 4.43 times that of ordinary nanolipid vesicles and commercial creams respectively (P<0.05).

[0075] Table 2 The transdermal flux and the amount of drug retained in the skin of the isolated scar skin of triamcinolone acetonide preparation (n=4, )

[0076]

[0077] *P<0.05, compared with 0.1% triamcinolone acetonide cream

PUM

Property	Measurement	Unit
The average particle size	50.0 ~ 500.0	nm
The average particle size	100.0 ~ 200.0	nm
Particle size	100.0 ~ 200.0	nm

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Preparation method for triamcinolone acetonide acetate ultradeformable nanometer lipid vesicle spraying agent and application to target hypertrophic scars

AI-Extracted Technical Summary

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Abstract

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