Long-acting slow-release compound preparation for preventing and treating schistosomiasis

Abstract

The invention provides a long-acting slow-release compound preparation for preventing and treating schistosomiasis. The compound preparation comprises two active ingredients, namely niclosamide and praziquantel, the niclosamide accounts for 10-90% of the weight of the compound preparation; praziquantel accounts for 10%-90% of the weight of the compound preparation; the coating further comprises a biodegradable high polymer material and a dispersion medium. Optionally, a wetting agent; the biodegradable high polymer material accounts for 12.5%-14.3%; the dispersion medium accounts for 50.0%-57.1%; and the wetting agent accounts for 0-5.7%. The compound preparation can maintain long-acting slow release after being administrated once, has excellent prevention and control and treatment effects on schistosomiasis, particularly has good prevention and treatment effects on larvae and / or adults of schistosomiasis, blocks a transmission chain of the schistosomiasis, protects human beings and animals from schistosomiasis infection, and has huge economic benefits and social benefits.

Description

Technical Field

[0001] The invention relates to the technical field of antiparasitic drug preparations, and in particular to a compound injection preparation for animals for long-term prevention and treatment of schistosomiasis. Background Art

[0002] Schistosomiasis, as a neglected tropical disease, is a zoonotic parasitic disease that seriously endangers human health. Schistosomiasis is mainly caused by infection with Schistosoma mansoni, Schistosoma japonicum, Schistosoma mekongi, Schistosoma jennerii and related Schistosoma haematobium. The World Health Organization (WHO) proposes that schistosomiasis control should mainly reduce the disease through regular, large-scale population treatment with praziquantel. At present, the only first-line drug used for the prevention and control of schistosomiasis in humans and animals is praziquantel. Although it has a good therapeutic effect, it needs to be used repeatedly due to its rapid metabolism and half-life of only a few hours. In addition, the scale of group medication is very large, which greatly increases the possibility of praziquantel resistance, so the development of other new anti-schistosomiasis drugs has attracted much attention.

[0003] The life cycle of schistosomes is divided into seven developmental stages: adult, egg, miracidium, mother sporocyst, daughter sporocyst, cercariae, and schistosomiasis. Among them, cercariae are the only stage that infects humans and animals, and are also the most vulnerable stage in the life cycle of schistosomes (more than 98% of schistosome cercariae float on the water surface in a static manner). As a snailicide recommended by WHO, niclosamide (Niclosamide, Bayer73, Mitral, Yumosheng, Xuefang-67, etc.) also has a strong killing effect on schistosome cercariae and miracidia. When the concentration of niclosamide film-forming oil is 6.25ng / mL, all cercariae die after 1 minute. However, niclosamide and its ethanolamine salt have strong acute toxicity to many fish and amphibians, and are easy to cause environmental pollution. However, niclosamide has little toxicity to mammals such as humans, cattle, sheep, birds and plants. As early as the early 1970s, niclosamide began to be used clinically to treat tapeworms in humans and animals. Moreover, niclosamide will be affected by the soil environment, pH value, light exposure time, ambient temperature, etc., which will affect its degradation and thus affect its snail-killing effect. In summary, niclosamide is safe for use by humans and livestock, and can avoid environmental pollution.

[0004] In recent years, many studies have used niclosamide to prepare dosage forms to prevent the invasion of schistosoma cercariae. Such as niclosamide skin paint, 1% niclosamide ethanolamine salt spray, niclosamide controlled-release film, niclosamide topical anti-penetration lotion, etc., can all achieve good protection. However, the efficacy of these dosage forms is short-lasting and needs to be reused many times, and the topical dosage forms are mostly applied to the skin and cannot protect against mucosal infections (for example, livestock drinking water containing cercariae). There are still deficiencies in the application of these preparations on site. For this reason, our laboratory has developed a niclosamide in-situ solidification long-acting injection, which has been granted an invention patent (Chinese invention patent 201710813925.1). However, niclosamide is only effective against schistosoma cercariae and has no effect on schistosoma adult worms.

[0005] In summary, although there are existing technologies for treating schistosomiasis, there is a lack of long-term technology for preventing and treating schistosomiasis infection and disease progression, and there is also a lack of technology for long-term treatment of schistosomiasis with a single dose. Therefore, it is urgent to provide a compound sustained-release injection with better long-term efficacy against both adult schistosomes and cercariae. Summary of the invention

[0006] In order to overcome the defects of the above-mentioned prior art in lacking a long-term technology for preventing schistosomiasis infection and disease progression, the first purpose of the present invention is to provide a compound preparation; the second purpose of the present invention is to provide a method for preparing the compound preparation; the third purpose of the present invention is to provide the use of the compound preparation in the preparation of anti-schistosomiasis drugs.

[0007] The purpose of the present invention is achieved by the following technical means:

[0008] In one aspect, the present invention provides a compound preparation, comprising a first active ingredient, niclosamide, and a second active ingredient, praziquantel;

[0009] Wherein, based on 100% by weight of the compound preparation, the niclosamide accounts for 10% to 90% by weight of the compound preparation; the praziquantel accounts for 10% to 90% by weight of the compound preparation.

[0010] In the above-mentioned compound preparation, preferably, the compound preparation further comprises a biodegradable polymer material and a dispersion medium; optionally, the compound preparation further comprises a wetting agent;

[0011] Among them, the biodegradable polymer material accounts for 12.5% ​​to 14.3% of the weight of the compound preparation; the dispersion medium accounts for 50.0% to 57.1% of the weight of the compound preparation; optionally, the wetting agent accounts for 0% to 5.7% of the weight of the compound preparation.

[0012] In the above-mentioned compound preparation, preferably, the biodegradable polymer material is selected from one or more combinations of polylactic acid-glycolic acid copolymer (including ester end-capping, terminal carboxyl group and terminal hydroxyl group), polycaprolactone, polytrimethylene carbonate, L-polylactic acid-polycaprolactone copolymer, L-polylactic acid (including ester end-capping, terminal carboxyl group and terminal hydroxyl group), D-polylactic acid (including ester end-capping, terminal carboxyl group and terminal hydroxyl group), racemic polylactic acid (including ester end-capping, terminal carboxyl group and terminal hydroxyl group), but is not limited thereto.

[0013] In the above-mentioned compound preparation, preferably, the average molecular weight of the polylactic acid-glycolic acid copolymer is 0.8w to 16w.

[0014] In the above-mentioned compound preparation, preferably, the average molecular weight of the polycaprolactone is 0.6w to 12w.

[0015] In the above-mentioned compound preparation, preferably, the average molecular weight of the polytrimethylene carbonate is 1.0w to 14w.

[0016] In the above-mentioned compound preparation, preferably, the average molecular weight of the poly(L-lactic acid) is 0.6w-15w.

[0017] In the above-mentioned compound preparation, preferably, the average molecular weight of the dextrorotatory polylactic acid is 0.8w-15w.

[0018] In the above-mentioned compound preparation, preferably, the average molecular weight of the racemic polylactic acid is 1w to 15w.

[0019] In the above-mentioned compound preparation, preferably, the biodegradable polymer material is composed of a polylactic acid-glycolic acid copolymer with an average molecular weight of 0.8w to 16w and a racemic polylactic acid with an average molecular weight of 1w to 15w; and the mass ratio of the polylactic acid-glycolic acid copolymer to the racemic polylactic acid is in the range of 1:10 to 10:1.

[0020] In the above-mentioned compound preparation, preferably, the biodegradable polymer material is composed of a polylactic acid-glycolic acid copolymer with an average molecular weight of 0.8w to 16w and a polycaprolactone with an average molecular weight of 0.6w to 12w; and the mass ratio of the polylactic acid-glycolic acid copolymer to the polycaprolactone is in the range of 1:10 to 10:1.

[0021] In the above-mentioned compound preparation, preferably, the wetting agent is selected from one or more combinations of Tween 80 and polyethylene glycol 400, but is not limited thereto.

[0022] In the above-mentioned compound preparation, preferably, the dispersion medium is selected from a combination of one or more of triacetin, N-methylpyrrolidone, and benzyl benzoate, but is not limited thereto.

[0023] In the above-mentioned compound preparation, preferably, the wetting agent is selected from Tween 80 and / or polyethylene glycol 400, but is not limited thereto.

[0024] In the above-mentioned compound preparation, preferably, based on 100% by weight of the compound preparation, the compound preparation comprises:

[0025]

[0026] On the other hand, the present invention also provides a method for preparing the above-mentioned compound preparation, which comprises the following steps:

[0027] The biodegradable polymer material is dissolved in a dispersant, and then the first active ingredient niclosamide and the second active ingredient praziquantel are added, and optionally, a wetting agent is added, and the mixture is stirred evenly to obtain a compound preparation.

[0028] In another aspect, the present invention also provides use of the above-mentioned compound preparation or the compound preparation prepared by the above-mentioned method in the preparation of anti-schistosomiasis drugs.

[0029] In the above use, preferably, the schistosomes include adults and / or larvae of schistosomes.

[0030] Beneficial effects of the present invention:

[0031] The compound preparation of the present invention has the effect of long-acting sustained-release anti-schistosomiasis drug. The sustained-release dosage form can maintain the effect of long-term prevention and control of schistosomiasis by administering once, especially has good prevention and treatment effects on larvae and / or adults of schistosomiasis, blocks the transmission chain of schistosomiasis, protects humans and animals from schistosomiasis infection, and has huge economic and social benefits. After injection, it can not only cure cattle and other livestock infected with schistosomiasis, but also provide a relatively long-term effect of preventing reinfection with schistosomiasis, saving the amount of medicine used and manpower and material resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a comparison chart of the in vitro release results of prescriptions 1, 2 and 5 in the examples of the present invention. DETAILED DESCRIPTION

[0033] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is now described in detail below, but it should not be construed as limiting the scope of implementation of the present invention; the raw materials used in the following examples are all commercially available in the art unless otherwise specified; the methods used are all conventional preparation methods in the art unless otherwise specified.

[0034] Embodiments 1 to 11:

[0035] The present embodiment provides a compound preparation for injection into an animal for long-term prevention and treatment of schistosomiasis and a preparation method thereof. The compound preparation consists of a first active ingredient, niclosamide, a second active ingredient, praziquantel, a biodegradable polymer material, a dispersion medium and an optional wetting agent.

[0036] (1) The ingredients and formulas of the biodegradable polymer materials and dispersants of Examples 1 to 5 are shown in Table 1. The preparation method of the compound preparation of the embodiment comprises:

[0037] According to the formula in Table 1, the biodegradable polymer material is dissolved in a dispersant, a wetting agent is further added, and then 0.5 g of niclosamide and 0.5 g of praziquantel are added, and the mixture is stirred well to obtain a compound preparation.

[0038] In Table 1, the average molecular weight of the polylactic acid-glycolic acid copolymer is 1.6w; the average molecular weight of the racemic polylactic acid is 4w; and the average molecular weight of the polycaprolactone is 6w.

[0039] Table 1: Formula table of Examples 1 to 5

[0040]

[0041] * Injectability refers to the ease with which a preparation passes through an injection needle. A “+” indicates that the preparation is easier to inject and a “-” indicates that the preparation is more difficult to inject.

[0042] (2) The components and formulas of the biodegradable polymer materials of Examples 6 to 10 are shown in Table 2. The preparation method of the compound preparation of the example comprises:

[0043] According to the formula in Table 2, the biodegradable polymer material was dissolved in 2.0 g of dispersant N-methylpyrrolidone, 0.2 g of wetting agent Tween 80 was further added, and then 0.5 g of niclosamide and 0.5 g of praziquantel were added, and the mixture was fully stirred to obtain a compound preparation.

[0044] Table 2: Formula table of Examples 6 to 10

[0045]

[0046] Needle-pass test:

[0047] The compound preparations prepared in Examples 1 to 10 above were subjected to an acupuncture test, and the specific method was as follows:

[0048] Add the compound preparation into the syringe to simulate the actual injection process, and apply a certain force to push the solution through the syringe and needle. The greater the pushing force, the worse the needle permeability.

[0049] The experiment found that different biodegradable polymer materials and different dispersants have an effect on the viscosity (permeability) of the compound preparation. With the same dispersant, the permeability of the single-component biodegradable polymer material is relatively poor, while the permeability of the multi-component biodegradable polymer material is relatively good. With the same multi-component biodegradable polymer material, the permeability of the dispersant composed of N-methylpyrrolidone and triacetin is relatively good. The permeability of Examples 1 and 5 is the best.

[0050] In vitro release experiment:

[0051] Compound preparations 1, 2 and 5 were selected for in vitro release study. First, two buffers needed for in vitro release study were prepared, namely 0.5% Tween phosphate buffer (pH 7.4) and 0.5% SDS phosphate buffer (pH 7.4). The preparation steps are as follows:

[0052] Take 1.36 g of potassium dihydrogen phosphate, add 79 mL of 0.1 mol / L sodium hydroxide solution, dilute to 200 mL with water, add 1 g of Tween 80 and mix well to obtain 0.5% Tween phosphate buffer (pH 7.4).

[0053] Take 1.56 g of sodium dihydrogen phosphate dihydrate, add 79 mL of 0.1 mol / L sodium hydroxide solution, dilute to 200 mL with water, add 1 g of SDS and mix well to obtain 0.5% SDS phosphate buffer (pH 7.4).

[0054] After completing the preparation of the buffer solution required for the in vitro release study, the steps for conducting the in vitro release study using the preparation example are as follows:

[0055] For compound preparations 1 to 5, take 0.2 mL of each preparation, add it to a 50 mL EP tube with a stopper, and add 30 mL of 0.5% Tween phosphate buffer (compound preparations 3 to 5 are placed at 37°C for 5 minutes before adding). Oscillate at a speed of 150 r / min in a constant temperature oscillator at 37±1°C, take samples at regular intervals, take the release medium and filter it through a 0.22 μm microporous membrane, take the filtrate, and measure the OD value at 270 nm and 340 nm respectively; calculate the drug concentration in the release medium according to the standard curve, and calculate the cumulative release rate according to formula (1).

[0056]

[0057] In formula (1), R n represents the cumulative release on day n, R n-1 represents the cumulative release on day n-1, C n represents the concentration of release medium on day n. Figure 1 shown.

[0058] According to the results of in vitro release experiments Figure 1 It can be seen that the release time of prescription 1 is the longest, the in vitro release of niclosamide in prescription 1 is more than 7 months, and the in vitro release of prescription 5 is less than 1 month. Therefore, Example 1 was selected for in vivo pharmacokinetic experiments.

[0059] Experimental Example 1: Pharmacokinetic Experiment

[0060] Six male SD rats were subcutaneously injected with the compound preparation obtained in Example 1 at a dose of 300 mg / kg body weight. 1 ml of blood was collected before administration (0h) and 0.08d (2h), 0.33d (8h), 1d (24h), 3d, 7d, 14d, 21d, 30d, 45d, 60d, 90d, 120d, 150d and 180d after administration, respectively, and placed in a heparin-coated test tube, centrifuged at 3500 rpm for 10 minutes, and 0.4 ml of plasma was collected and stored at -80°C for analysis. The UPLC method was used to determine the blood drug concentrations of niclosamide (NCA) and praziquantel (PZQ) in rats. The UPLC chromatographic conditions are as follows:

[0061] Chromatographic column: Waters CORTECS UPLC T3 (2.1 mm×100 mm, 1.6 μm); column temperature: 25°C (room temperature); detection: 216 nm and 340 nm; mobile phase: methanol / 0.1% formic acid water (68 / 32, v / v); flow rate: 0.2 mL / min; injection volume: 3 μL; internal standard: 1 μg / mL (anthracene). The experimental results are shown in Table 3 below.

[0062] Table 3: Pharmacokinetic data of Example 1

[0063]

[0064] It can be seen from the experimental results in Table 3 that the praziquantel and niclosamide of Example 1 are released in rats for at least one month and are still being released in the body.

[0065] Experimental Example 2: In vitro efficacy test on cercariae

[0066] At room temperature of 25°C, the hatched fresh cercariae were placed in 48-well plates, with 20-30 cercariae in each well. The wells contained 0, 0.1, 1, 10, 100, 1000, and 10000 ng / L of homemade pharmaceutical solution, respectively. Each concentration was repeated 3 times, and the cercariae mortality was observed under a microscope at 5, 10, 30, and 60 minutes, respectively. The results are shown in Table 4.

[0067] Table 4 Experiment on killing cercariae of compound prescription

[0068]

[0069] The experimental results in Table 4 show that the compound preparation of the present invention has a stronger effect on killing cercariae than > niclosamide > praziquantel.

[0070] It should be particularly pointed out that the above embodiments are only examples for clear description, and are not limitations on the implementation methods. For professionals in this field, many changes can be derived on the basis of the above description. It is not necessary and impossible to enumerate all the changes here. However, those changes that are obviously derived from them are still within the scope of protection of the present invention.

Claims

1. A compound preparation, characterized in that: The compound preparation comprises a first active ingredient, niclosamide, and a second active ingredient, praziquantel; Wherein, based on 100% by weight of the compound preparation, the niclosamide accounts for 10% to 90% by weight of the compound preparation; the praziquantel accounts for 10% to 90% by weight of the compound preparation.

2. The compound preparation according to claim 1, characterized in that: The compound preparation further comprises a biodegradable polymer material and a dispersion medium; optionally, the compound preparation further comprises a wetting agent; Among them, the biodegradable polymer material accounts for 12.5% ​​to 14.3% of the weight of the compound preparation; the dispersion medium accounts for 50.0% to 57.1% of the weight of the compound preparation; optionally, the wetting agent accounts for 0% to 5.7% of the weight of the compound preparation.

3. The compound preparation according to claim 2, characterized in that: The biodegradable polymer material is selected from one or more combinations of polylactic acid-glycolic acid copolymer (including ester end-capping, terminal carboxyl group and terminal hydroxyl group), polycaprolactone, polytrimethylene carbonate, L-polylactic acid-polycaprolactone copolymer, L-polylactic acid (including ester end-capping, terminal carboxyl group and terminal hydroxyl group), D-polylactic acid (including ester end-capping, terminal carboxyl group and terminal hydroxyl group), racemic polylactic acid (including ester end-capping, terminal carboxyl group and terminal hydroxyl group); Preferably, the average molecular weight of the polylactic acid-glycolic acid copolymer is 0.8w to 16w; Preferably, the average molecular weight of the polycaprolactone is 0.6w to 12w; Preferably, the average molecular weight of the polytrimethylene carbonate is 1.0w to 14w; Preferably, the average molecular weight of the L-polylactic acid is 0.6w to 15w; Preferably, the average molecular weight of the dextrorotatory polylactic acid is 0.8w to 15w; Preferably, the average molecular weight of the racemic polylactic acid is 1w to 15w.

4. The compound preparation according to claim 3, characterized in that: The biodegradable polymer material is composed of a polylactic acid-glycolic acid copolymer with an average molecular weight of 0.8w-16w and a racemic polylactic acid with an average molecular weight of 1w-15w; and the mass ratio of the polylactic acid-glycolic acid copolymer to the racemic polylactic acid is in the range of 1:10-10:

1.

5. The compound preparation according to claim 3, characterized in that: The biodegradable polymer material is composed of a polylactic acid-glycolic acid copolymer with an average molecular weight of 0.8w-16w and a polycaprolactone with an average molecular weight of 0.6w-12w; and the mass ratio of the polylactic acid-glycolic acid copolymer to the polycaprolactone is in the range of 1:10-10:

1.

6. The compound preparation according to claim 2, characterized in that: The dispersion medium is selected from a combination of one or more of triacetin, N-methylpyrrolidone, benzyl benzoate, and pyrrolidone; Preferably, the wetting agent is selected from Tween 80 and / or polyethylene glycol 400.

7. The compound preparation according to claim 2, characterized in that: Taking the weight components of the compound preparation as 100%, the compound preparation comprises:

8. A method for preparing the compound preparation according to any one of claims 1 to 7, comprising the following steps: The biodegradable polymer material is dissolved in a dispersant, and then the first active ingredient niclosamide and the second active ingredient praziquantel are added, and optionally, a wetting agent is added, and the mixture is stirred evenly to obtain a compound preparation.

9. Use of the compound preparation according to any one of claims 1 to 7 or the compound preparation prepared by the method according to claim 8 in the preparation of anti-schistosomiasis drugs.

10. The use according to claim 9, characterized in that: The schistosomes include adults and / or larvae of schistosomes.

Images