Flurala drops as well as preparation method and application thereof

Abstract

The invention discloses a fluralan drop as well as a preparation method and application thereof, and belongs to the field of pharmaceutical preparations. The fluralan drop is prepared from the following components in parts by weight: 5 to 15 parts of fluralan; 2-6 parts of hydrogenated soybean lecithin; 0.2 to 0.7 part of cholesterol; 1 to 4 parts of matrine C12-C16 fatty acid ionic liquid; 15 to 45 parts of short chain alcohol; and 30-70 parts of water. The sophocarpidine fatty acid ionic liquid of fatty acid with a specific carbon chain length is introduced into the ethosome, so that the surface charge of the ethosome is increased, and the stability of the ethosome and the encapsulation efficiency of the flurala sodium are improved. Moreover, due to the introduction of the matrine fatty acid ionic liquid, the transdermal permeation rate of the preparation and the medicine accumulation transdermal permeation amount are also enhanced, the medicine utilization rate is improved, the insecticidal activity of the preparation is remarkably prolonged, and the treatment effect on animal parasitic infection and the administration compliance are further improved.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to flurellana drops and a preparation method and use thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Flurellan, CAS No.: 864731-61-3, chemical name is carbamoylbenzamidephenylisoxazoline, is an isoxazoline drug, which inhibits the activity of glutamate-β-aminobutyric acid and γ-aminobutyric acid gated chloride ion channels in the nervous system of arthropods, leading to depolarization and overexcitement, paralysis and death of parasites. Its insecticidal range includes mites, ticks, siphonaptera, lice, diptera and hemiptera.

[0004] Currently, Merck has launched a topical formulation of flurellana for pet deworming, which can maintain a 12-week therapeutic effect on cat ctenocephala and tick infections after a single administration. The inventors have found that the preparation still has the problem of slow drug transdermal speed and drug transdermal permeability to be improved, and hope to further extend the insecticidal effect. Summary of the invention

[0005] Based on the problems existing in the prior art, the purpose of the present invention is to provide a flurellana drop with high transdermal permeability and longer-term insecticidal activity, so as to improve the therapeutic effect and drug administration compliance of animal parasitic infections.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, a flurellan drops is provided, which is composed of the following components in parts by weight:

[0008] Flurella 5-15 parts;

[0009] Hydrogenated soy lecithin (HSPC) 2-6 parts;

[0010] Cholesterol 0.2~0.7 parts;

[0011] Matrine C12~C16 fatty acid ionic liquid 1~4 parts;

[0012] 15-45 parts of short chain alcohol;

[0013] 30~70 parts of water.

[0014] In one or some embodiments of the present invention, the flurana drops are composed of the following components in parts by weight:

[0015] Flurella 7-10 parts;

[0016] Hydrogenated soy lecithin (HSPC) 2.5~4 parts;

[0017] Cholesterol 0.3~0.6 parts;

[0018] Matrine C12~C16 fatty acid ionic liquid 1.5~3.5 parts;

[0019] 20-35 parts of short chain alcohol;

[0020] 50~60 parts of water.

[0021] In one or some embodiments of the present invention, the matrine C12~C16 fatty acid ionic liquid includes matrine lauric acid ionic liquid ([Mat]C12), matrine myristic acid ionic liquid ([Mat]C14), and matrine palmitic acid ionic liquid ([Mat]C16). The preparation method thereof does not belong to the protection content of the present invention. The preparation method can refer to Chinese patent CN201811467058.1. In the present invention, the matrine C12~C16 fatty acid ionic liquid is prepared by the following method: under an argon atmosphere, 0.01 mol of C12~C16 fatty acid is dissolved in a reactor with 30~40 mL of methanol, 0.01 mol of matrine is dissolved in 50 mL of methanol, and the mixture is added dropwise to the reactor in which the C12~C16 fatty acid is dissolved, and the mixture is heated to 50~80° C., and the ionization modification reaction is carried out for 20~24 hours; after the reaction is completed, the solution is concentrated to about 1 / 8~1 / 10 of the volume of the reaction solution under vacuum conditions, frozen and crystallized, and the crystal product of the matrine C12~C16 fatty acid ionic liquid is obtained by filtering, washing and separation, and the pure matrine C12~C16 ionic liquid is obtained by drying in a vacuum drying oven for 48 hours.

[0022] In one or some embodiments of the present invention, the short-chain alcohol, for example, can be selected from one or more combinations of ethanol, isopropanol, and propylene glycol, preferably a combination of ethanol and a small amount of isopropanol or propylene glycol, to increase the viscosity of the alcohol body and improve the stability of the preparation. In a more specific embodiment of the present invention, the short-chain alcohol is composed of ethanol and propylene glycol, and the mass ratio thereof is 4 to 8:1, preferably 5 to 7:1, for example 6:1.

[0023] In the second aspect of the present invention, a method for preparing the fluranabol drops described in the first aspect is provided, which can be prepared by injection method, ultrasonic method, thin film dispersion method and the like.

[0024] In one or some embodiments of the present invention, the flurana drops are prepared by injection method, which comprises the following steps:

[0025] (1) The prescribed amount of hydrogenated soybean lecithin (HSPC), cholesterol, flurella, and matrine C12-C16 fatty acid ionic liquid were heated at 30-60 °C. o C, soluble in short-chain alcohols;

[0026] (2) Inject the above alcohol solution into a preheated o C in water, and stir evenly to obtain flurellan drops.

[0027] In one or some embodiments of the present invention, in step (1), a prescribed amount of hydrogenated soybean lecithin (HSPC), cholesterol, flurellana, matrine C12-C16 fatty acid ionic liquid is added at 40-50 °C. o C (e.g. 45 o C) soluble in short-chain alcohols.

[0028] In one or some embodiments of the present invention, in step (2), the alcohol solution is injected into a liquid preheated to 40-50 o C (e.g. 45 o C) in water.

[0029] In one or some embodiments of the present invention, in step (2), the alcohol solution is injected into a liquid preheated to 30-60 o C in water, stir at 500-1000 r / min with a magnetic stirring bar during the injection process, and continue stirring for 20-60 min after the injection to obtain flurellan drops.

[0030] The rotation speed of the magnetic stirring bar is preferably 600-800 r / min.

[0031] The stirring time after injection is preferably continued for 20 to 40 minutes, for example 30 minutes.

[0032] In the third aspect of the present invention, there is provided the use of the flurana drops described in the first aspect or the flurana drops prepared by the preparation method described in the second aspect in the preparation of drugs for preventing and treating parasitic infections on the surface and / or in the body of animals, preventing canine heartworm disease and allergic dermatitis caused by fleas.

[0033] In one or some embodiments of the present invention, the animals include but are not limited to cats, dogs, and the like.

[0034] In one or some embodiments of the present invention, the external parasites are fleas, ticks, scabies, ear mites, demodex mites, fleas, lice, etc.; the internal parasites are roundworms, hookworms, whipworms, tapeworms, etc.

[0035] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects:

[0036] The present invention introduces matrine fatty acid ionic liquid of a fatty acid of a specific carbon chain length into the ethosome to increase the surface charge of the ethosome, thereby improving the stability of the ethosome and the encapsulation rate of flurellana. In addition, the introduction of the matrine fatty acid ionic liquid also enhances the transdermal penetration rate and the cumulative transdermal penetration amount of the preparation, improves the utilization rate of the drug, and significantly prolongs the insecticidal activity of the preparation, further improving the therapeutic effect of animal parasitic infection and the compliance of administration. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0038] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0039] The invention provides a flurellane ethosome drops, which use hydrogenated soybean lecithin (HSPC), cholesterol, polyols and water as basic materials of the ethosome, and introduce matrine fatty acid ionic liquid.

[0040] The inventors found that the preparation of flurellane ethosomes using only hydrogenated soy lecithin (HSPC), cholesterol, polyols, and water as basic materials cannot meet the preparation requirements of high stability, high drug loading, and suitable transdermal performance. The inventors tried to introduce different surfactants to further improve the performance of the ethosomes. The inventors introduced ionic surfactants (dioctadecyl dimethyl ammonium chloride), nonionic surfactants (Tween 80), and matrine fatty acid ionic liquids.

[0041] In further research, the inventors found that conventional ionic surfactants (dioctadecyl dimethyl ammonium chloride) and non-ionic surfactants (Tween 80) cannot achieve the above-mentioned improvement in the performance of the alcohol body. The introduced matrine fatty acid ionic liquid can significantly improve the drug encapsulation rate, particle size, particle size distribution and transdermal performance of the alcohol body. In particular, the inventors found that the matrine fatty acid ionic liquid prepared from fatty acids with too low carbon chain numbers (C6~C10) cannot achieve the above-mentioned performance improvement. The introduction of the fatty acid matrine ionic liquid with a carbon chain number of C12~C16 can achieve a higher encapsulation of flurellana and make the alcohol body have a particle size more suitable for transdermal absorption, and the positive charge introduced by the ionic liquid can improve the stability and transdermal performance of the alcohol body, so that the preparation has a faster transdermal absorption rate and a higher cumulative transdermal permeation of the drug, improve the drug utilization rate, and significantly prolong the insecticidal activity of the preparation, further improving the therapeutic effect and drug administration compliance of animal parasitic infections.

[0042] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0043] Reagents and sources

[0044] Matrine: purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0045] The preparation of matrine hexanoate ionic liquid ([Mat]C6), matrine caprylate ionic liquid ([Mat]C8), matrine capric acid ionic liquid ([Mat]C10), matrine laurate ionic liquid ([Mat]C12), matrine myristic acid ionic liquid ([Mat]C14), and matrine palmitic acid ionic liquid ([Mat]C16) can refer to Chinese patent CN201811467058.1.

[0046] Specifically, matrine hexanoate ionic liquid ([Mat]C6) is prepared by the following method:

[0047] Under an argon atmosphere, 0.01 mol of hexanoic acid is dissolved in 35-40 mL of methanol in a reactor, and 0.01 mol of matrine is dissolved in 50 mL of methanol, and the mixture is added dropwise to the reactor in which the hexanoic acid is dissolved, and the mixture is heated to 65-80°C, and the ionization modification reaction is carried out for 20-24 hours; after the reaction is completed, the solution is concentrated to about 1 / 8-1 / 10 of the volume of the reaction solution under vacuum conditions, and the solution is frozen and crystallized, and the crystal product of matrine hexanoic acid ionic liquid is obtained by filtration and washing, and the pure matrine hexanoic acid ionic liquid is obtained by drying in a vacuum drying oven for 48 hours;

[0048] The remaining steps and conditions of the matrine C8-C16 fatty acid ionic liquid are the same as those of the preparation method of the matrine hexanoic acid ionic liquid. The matrine C8-C16 ionic liquid is in liquid state at room temperature.

[0049] Flurellana: purchased from Chongqing Pharmaceutical Preparation Engineering Technology Research Center.

[0050] Dioctadecyl dimethyl ammonium chloride (TA100): purchased from VARISOFT, Germany.

[0051] Hydrogenated soybean lecithin (HSPC): purchased from Avituo (Shanghai) Pharmaceutical Technology Co., Ltd.

[0052] Cholesterol and Tween 80 were purchased from Shanghai Aladdin Reagent Co., Ltd.

[0053] BRAVECTO®, purchased from Merck.

[0054] The remaining reagents are commercially available.

[0055] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0056] Examples 1-6 Preparation of Flurellane Ethosome Drops

[0057] (1) According to Table 1, weigh the prescribed amount of hydrogenated soybean lecithin (HSPC), cholesterol, flurellana, and ionic liquid and heat them at 45 o C is soluble in alcohol;

[0058] (2) Use a syringe to slowly and evenly inject the above alcohol solution into a o C in distilled water and stirred at 800 r / min with a magnetic stirring bar. After injection, stirring was continued for 30 min to obtain flurellan alcohol body drops.

[0059] Comparative Examples 1-7 Preparation of Flurellane Ethosome Drops

[0060] (1) According to Table 1, weigh the prescribed amount of hydrogenated soybean lecithin (HSPC), cholesterol, flurella, ionic liquid or surfactant (Tween 80 or TA-100) and stir at 45 o C is soluble in alcohol;

[0061] (2) Use a syringe to slowly and evenly inject the above alcohol solution into a o C in distilled water and stirred at 800 r / min with a magnetic stirring bar. After injection, stirring was continued for 30 min to obtain flurellan alcohol body drops.

[0062] Table 1. Flurellane ethosome formulation (by weight)

[0063]

[0064] Test Example 1. Analysis of the physicochemical properties of flurellane ethosomes

[0065] (1) Determination of particle size and zeta potential of flurellane ethosomes

[0066] According to the method of the embodiment, three portions of each flurellane alcohol were prepared in parallel as test samples. Each test sample was appropriately diluted, filtered through a 0.22 μm membrane, and the particle size and zeta potential of the sample were measured using a Malvern particle size analyzer. The results are shown in Table 2.

[0067] (2) Determination of encapsulation efficiency of flurellane ethosomes

[0068] According to the method of the embodiment, a suitable amount of flurellane ethosome was placed in a 100 kD ultrafiltration tube. o The mixture was centrifuged at 8000 r / min for 1 h at 400 °C, and an appropriate amount of the centrifugal filtrate and the corresponding flurellanine alcohol body were taken as samples. The flurellanine content was detected by HPLC, and the encapsulation efficiency was calculated according to the following formula. The results are shown in Table 2.

[0069] Encapsulation efficiency (%) = (1-W l / W c )×100%

[0070] Among them, W l is the mass of unencapsulated flurellane, W c is the total mass of flurella in the ethanosomes.

[0071] Table 2. Particle size, particle size distribution and encapsulation efficiency of flurellane ethosomes (n=3)

[0072]

[0073] Table 2 shows that the flurellana alcohol body obtained in each embodiment (E1~E6) of the above-mentioned addition of matrine-C12~C16 fatty acid ionic liquid has a particle size between 60~100 nm, a relatively uniform particle size distribution, a zeta potential of about 20 mV, and an encapsulation rate of more than 80%. The particle size of the flurellana alcohol body prepared in Comparative Example 1 (D1) without adding any ionic liquid or surfactant is much higher than that of the embodiment, about 230 nm, and the particle size distribution is now wider, the zeta potential is closer to neutral, and the flurellana encapsulation rate is too low, only 34.2±3.6%. Comparative Examples 2~4 (D2~D4) using matrine fatty acid ionic liquids with lower carbon chain number fatty acids have larger particle sizes and lower encapsulation rates than the embodiments, only about 50%. The flurellane alcohol body prepared in Comparative Example 5 (D5) with the addition of nonionic surfactant Tween 80 has a large particle size (223.1±26.3 nm), a near-neutral ζ potential, and a low encapsulation efficiency (32.2±4.4%), indicating that the addition of nonionic surfactant Tween 80 is not conducive to the stability and formation of alcohol bodies, and is not conducive to the encapsulation of drugs. The flurellane alcohol body prepared in Comparative Example 6 (D6) with the addition of cationic surfactants also has a large particle size (195.6±15.8 nm) and a low drug encapsulation efficiency (46.9±3.1%). In Comparative Example 7, a relatively high amount of [Mat]C14 (5%) was added, and it was found that excessive use of [Mat]C14 instead led to an increase in the alcohol body particle size and a decrease in the drug encapsulation efficiency.

[0074] (3) Study on the stability of flurellane ethanolsomes

[0075] According to the example method, each fluorouracil alcohol body is placed in 4 o C or 25 o C, the sample status was observed by naked eyes 4h, 7 days and 30 days after preparation, and the particle size and zeta potential were measured according to the particle size and zeta potential measurement method of the flurellane ethanolate in this test example (1) at 30 days. The results are shown in Tables 3 and 4.

[0076] Table 3. Apparent stability results of flurellane ethanolsomes

[0077] Note: “-” represents a uniform milky white liquid without appearance change, stratification or precipitation; “+” represents appearance change, stratification or precipitation.

[0078] Table 4. 30-day 25 results of flurellane ethosomes o C stability test results (n=3)

[0079]

[0080] Combining Table 3 and Table 4, it can be seen that the flurellane alcohols prepared in Examples 1 to 6 were relatively stable in the 30-day stability test, and only the flurellane alcohol of Example 6 was stable in the 25-day stability test. o C. Flurellane alcohols in Comparative Examples 1 to 7 all showed instability during storage. In particular, alcohols in Comparative Examples 1 and 5 showed precipitation only 4 hours after preparation.

[0081] Test Example 2. In vitro skin permeation test of flurellana ethosome drops

[0082] Preparation of ex vivo skin: 6-month-old rabbits were euthanized, the back hair was shaved, the back skin was peeled off and the subcutaneous fat was removed, and the skin was washed with physiological saline and stored in it for future use.

[0083] In vitro transdermal test: The above ex vivo skin was cut into discs with a diameter of about 2.8 cm, with the exposed part of the epidermis facing upwards, and fixed between the cup and the receiving pool of the TPY-2 transdermal diffusion instrument. 0.8 mL of the drops prepared in the examples and comparative examples and the control drug (BRAVECTO®, specification: 0.89 ml) were placed in the cup. 6.5 mL of 30% ethanol saline was added to the receiving pool as the receiving solution. The skin epidermis was in contact with the receiving solution, and the magnetic stirring was set at 200 r / min. The constant temperature water bath was 32 o C. Take 0.5 mL of the receiving solution as the test sample at 1, 2, 4, 8, 12, and 24 h after the start of the transdermal test, and immediately add an equal amount of fresh 30% ethanol saline. The sample to be tested was filtered through a 0.22 μm filter membrane and the content of flurellana was determined using HPLC. The test results are shown in Table 5.

[0084] Table 5. Cumulative transdermal penetration percentage of flurellana in each preparation (%) (n=3)

[0085]

[0086] As can be seen from Table 5, the flurellanose ethosomes prepared in the examples have a higher cumulative transdermal percentage (greater than 80%) than the comparative examples and the control drug BRAVECTO®, and the flurellanose ethosomes prepared in the examples approach the maximum transdermal permeation in a shorter time (between 8 and 12 hours). The rapid and sufficient absorption of the drug in the flurellanose ethosomes of the present invention can achieve rapid onset of efficacy and maintain the efficacy for a longer period of time.

[0087] Test Example 3 Anti-insect Effect of Flurellana Ethosome Drops

[0088] Experimental animals: Dogs that were confirmed to be tick-free by clinical examination and laboratory diagnosis and had not received deworming treatment in the past 6 months; regardless of breed and gender, and aged 8 weeks or above.

[0089] Preparations: Flurellan ethosome drops E2 prepared in Example 2, Flurellan ethosome drops D1 prepared in Comparative Example 1, Flurellan ethosome drops D5 prepared in Comparative Example 5, Flurellan ethosome drops D6 prepared in Comparative Example 6, and control preparation BRAVECTO® (specification: 250 mg Flurellan, 0.4 mL).

[0090] Test method:

[0091] The test animals were divided into 6 groups, 6 in each group, and the above preparations and control preparations were administered respectively. The administration method was as follows: the hair at the shoulder blade of the test dogs was parted, and the drug was dripped onto the skin along the spine. After the drug was completely absorbed, the drug was administered once in total. After the administration, the test dogs were not bathed until the end of the test.

[0092] The dosages were as follows: based on the content of flurellana, 25 mg of flurellana was administered per kilogram of experimental animals.

[0093] Each of the 6 groups of test animals was infested with 50 ticks on the 2nd, 12th, 28th, 56th, and 84th days, and the total number of ticks on the bodies of the test animals was counted 2 days after the infestation to evaluate the anti-tick effect of the flurellana drops. The results are shown in Table 6.

[0094] Table 6. Anti-tick effect of flurellana drops

[0095]

[0096] As can be seen from the table, Flurellan Ethosome Drops E2 has a longer-lasting antiparasitic effect than D1, D5, D6 and BRAVECTO®.

[0097] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A flurana drop, characterized in that: The flurana drops are composed of the following components in parts by weight: Flurella 5-15 parts; 2-6 parts of hydrogenated soy lecithin; Cholesterol 0.2~0.7 parts; Matrine C12~C16 fatty acid ionic liquid 1~4 parts; 15-45 parts of short chain alcohol; 30~70 parts of water.

2. The flurana drops as claimed in claim 1, characterized in that: The flurana drops are composed of the following components in parts by weight: Flurella 7-10 parts; 2.5-4 parts of hydrogenated soy lecithin; Cholesterol 0.3~0.6 parts; Matrine C12~C16 fatty acid ionic liquid 1.5~3.5 parts; 20-35 parts of short chain alcohol; 50~60 parts of water.

3. The flurana drops as claimed in claim 1, characterized in that: The matrine C12-C16 fatty acid ionic liquid is matrine lauric acid ionic liquid, matrine myristic acid ionic liquid or matrine palmitic acid ionic liquid.

4. The flurana drops as claimed in claim 1, characterized in that: The short-chain alcohol is one or a combination of ethanol, isopropanol and propylene glycol.

5. The flurana drops as claimed in claim 4, characterized in that: The short-chain alcohol is a combination of ethanol and isopropanol or propylene glycol.

6. A method for preparing the flurana drops according to any one of claims 1 to 5, characterized in that: The preparation method adopts an injection method, an ultrasonic method or a thin film dispersion method.

7. The method for preparing the flurana drops as claimed in claim 6, characterized in that: The method of preparing flurellana drops by injection comprises the following steps: (1) Add the prescribed amount of hydrogenated soybean lecithin, cholesterol, flurella, and matrine C12-C16 fatty acid ionic liquid at 30-60 °C. o C, soluble in short-chain alcohols; (2) Inject the alcohol solution in step (1) into a preheated o C of water, and stirring to obtain flurellana drops.

8. The method for preparing the flurana drops as claimed in claim 7, characterized in that: In step (1), the prescribed amount of hydrogenated soybean lecithin, cholesterol, flurella, and matrine C12-C16 fatty acid ionic liquid are added at 40-50 °C. o C and soluble in short-chain alcohols.

9. The method for preparing the flurana drops as claimed in claim 7, characterized in that: In step (2), the alcohol solution is injected into a liquid preheated to 40-50 o C in water; In step (2), the alcohol solution is injected into a preheated o C in water, stir at 500-1000 r / min with a magnetic stirring bar during the injection process, and continue stirring for 20-60 min after the injection to obtain flurellan drops.

10. Use of the flurana drops according to any one of claims 1 to 4, or the flurana drops prepared by the preparation method according to claim 6, or the flurana drops prepared by the preparation method according to any one of claims 7 to 9 in the preparation of drugs for preventing and treating parasitic infections on the surface and / or in the body of animals, preventing canine heartworm disease and allergic dermatitis caused by fleas.

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