NZ511852A

NZ511852A - Injectable anthelmintic formulation

Info

Publication number: NZ511852A
Application number: NZ511852A
Authority: NZ
Inventors: Se-Hoon Kim; Kyu-Boem Han; Byoung-Sun Chang; Hoo-Kyun Choi
Original assignee: Lg Chem Investment Ltd
Priority date: 1998-11-23
Filing date: 1999-11-22
Publication date: 2003-08-29
Also published as: AU1584400A; WO2000031098A1; BR9915559A; CN1354754A; KR20000033304A; CN1166676C; KR100315465B1; MXPA01005123A; AU753532B2

Abstract

An injectable anthelmintic formulation comprising: a) a water-insoluble anthelmintic, b) a surfactant comprising a block copolymer of ethylene oxide (EO) and propylene oxide (PO), c) ethanol and d) a water-soluble solvent. The insoluble anthelmintic is selected from avermectin or ivermectin. It is included in the formulation in a range from 0.5 to 2.0% (w/v).

Description

<div class="application article clearfix" id="description"> WO 00/31098 PCT/KR99/00704 1 INJECTABLE ANTHELMINTIC FORMULATION CROSS REFERENCE TO RELATED APPLICATION This application is based on application No. 98-50135 filed in the Korean Industrial Property Office on November 23, 1998, the content of which is 5 incorporated hereinto by reference. BACKGROUND OF THE INVENTION (a) Field of the Invention The present invention relates to an injectable anthelmintic formulation, and more particularly, to an injectable antheliminitic formulation with increased io water solubility and bioavailibility of an active substance. (b) Description of the Related Art It is well known that ivermectin (or avermectin) is highly effective against both internal and external parasites located in a variety of mammals including cows, pigs, horses, etc. (R.W. Burg, et al., Antimicrobial Agents and 15 Chemotherapy (1979) 361-367; J.C. Chabala, et al., J. Med. Chem. (1980) 23, 1134-1136; J .A. Lasota, et al., Annu. Rev. Entimol. (1991) 36. 91-117). In order to stabilize and solubilize the invermectin (or avermectin), many injectable formulations have been developed and used today. Among them, two representative formulations are described hereinafter. 20 In U.S. Patent No. 4,389,397 to Lo and Williams, ivermectin dissolves in water using a non-ionic surfactant such as polysorbate 80 (Tween 80), and the resulting mixture is stabilized with a cosolvent such as glycerol formal, propylene glycol, glycerine, or polyethylene glycol, and a substrate such as benzyl alcohol, lidocaine, parabens, or choline. According to this patent, ivermectin is able to 25 be stabilized in an aqueous solution. However, no description of the pharmacokinetics and efficiency of the invermectin formulation in animal body are given in this application. Further, in European Patent No. 535,734 A1 to Chen and Williams, a non-aqueous carrier such as hydrogenated castor oil or triacetin is used to 51 2 (followed by page 2 increase the duration of ivermection in animal body up to 42 days. However, since this foumulation is an oil-based solution without water, the viscosity of the solution is almost 500 cps (centipoise; measured by Brookfield viscometer with a spindle #2 at 25 °C, 60 rpm) which is considerably higher than that of the typical 5 water-soluble solution of ivermectin. Therefore, the major drawbacks of European Patent are difficulty of filtration and poor injectability. Other problem probably encountered during manufacturing this formulation includes the high power requirement to mix the solution at high rpm between 400 and 4000. In the case where the non-aqueous solution is used as injectable, 10 stability and solubility problems of ivermectin may be solved but following problems can be encountered: high viscosity; poor injectability; possibility of tissue damage or irritation where the injection is administered; precipitation of active components at the area of injection. Accordingly, there is an increasing demand for better aqueous injectable 15 formulation in which one of the insoluble anthelmintics, ivermectin or avermectin, is not only soluble while maintaining a stable state, but also quickly and efficiently absorbable in the animal body. SUMMARY OF THE INVENTION It is an object of the present invention to provide an injectable 20 anthelmintic formulation with a high level of water solubility and enhanced bioavailability; and/or to provide an injectable anthelmintic formulation with enhanced rate and efficiency absorption characteristics; or to at least provide the public with a useful choice. 25 These and other objects may be achieved by an injectable anthelmintic formulation. The injectable anthelmintic formulation includes a water-insoluble anthelmintic, a surfactant, a cosolvent and a water-soluble solvent. The formulation may further include additives such as an antioxidant and a preservative. The surfactant includes a block copolymer of ethylene oxide and so propylene oxide. 'iponz - 3 JUN 2003 51 2a (followed by page 3 More specifically, the present invention provides an injectable anthelmintic formulation comprising: a water-insoluble anthelmintic; a surfactant comprising a block copolymer of ethylene oxide (EO) and propylene oxide (PO); ethanol; and a water-soluble solvent. BRIEF DESCRIPTION OF THE DRAWINGS iponz -3 JUNM03 WO 00/31098 PCT/KR99/00704 3 The accompanying drawing, which is incorporated in and constitutes a part of the specification, illustrates an example of the invention, and, together with the description, serves to explain the principles of the invention: FIG. 1 is a graph of comparing pharmacokinetics in swine between the 5 formulation of the present invention and the commercially available Ivomec formulation (Merck & Co. INC. of the United States). DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel injectable anthelmintic formulation. The formulation includes a water-insoluble anthelimintic, a io surfactant, a cosolvent and a water-soluble solvent. The formulation may further include additives such as an antioxidant and a preservative. As the insoluble anthelmintic in the formulation of the present invention, it is possible to use ivermectin or avermectin, or derivatives thereof. The formulation includes from 0.5 to 2.0%(w/v) of ivermectin or avermectin, or 15 derivatives thereof. In the formulation of the present invention the surfactant and the cosolvent act as solubilizer. In other words, the surfactant and the cosolvent facilitate the water-insoluble anthelmintic to be dissolved in the water-soluble solvent. 20 The surfactant may be a water-soluble copolymer that is listed in the pharmacopoeia. The water-soluble copolymer has both hydrophilic and hydrophobic properties. The water-soluble copolymer includes a block copolymer of propylene oxide (PO) and ethylene oxide (EO). The block copolymer has (EO)x(PO)y(EO)x as a unit, where the ratio of x to y is between 25 0.75 and 2.0. The copolymer, which average molecular weight is between 9000 Da and 20000 Da, is presolubilized in water before use. The concentration of copolymer in water is between 5 and 25%. If the concentration of the copolymer is more than 25%, the viscosity of the mixed solution is increased and the solubility may be reduced. Whereas, the concentration thereof is less than 30 5%, the stability of the resulting injectable formulation is not guaranteed. The cosolvent may be alcohol, and more preferably, ethanol. The concentration of cosolvent is from 1 to 50 % (w/v) in the formulation of the 4 present invention. If the amount of the cosolvent is more than 50 %(w/v), the animals may feel a pain and stability of the resulting injectable formulation is reduced. Whereas, the amount thereof is less than 1 % (w/v), it is difficult to facilitate the water-insolublej anthelmintic to dissolve in the water-soluble solvent. 5 The water-soluble solvent may be water. In addition, it is possible that the formulation of the present invention may contain a variety of additives such as an antioxidant and a preservative. For example, it is possible to use butylated hydroxytoluene (BHT) as the antioxidant ranging from 0.001 to 0.5 % (w/v), and benzyl alcohol as the 10 preservative agent ranging from 0.5 to 10 %(w/v). For preparing the formulation of the present invention, it is possible to use a stepwise mixing method or a direct mixing method. In the stepwise mixing method, ivermectin or avermectin is immediately dissolved in the solvent with additives. This solution is then added to another 15 solution in which the surfactant is pre-dissolved in water, thereby producing the injectable formulation of the present invention. In the direct mixing method, all components of the formulation are simultaneously mixed together to produce the injectable formulation of the present invention. The stepwise mixing method is preferable because the anthelmintic 20 agent can be completely dissolved in the solvent for a short period of time, while the other direct mixing method needs more than 24 h to solubilize the anthelmintic agent completely. In both methods, the pH of the solution is adjusted to 6.8 to 7.4 while the concentration of components in the injectable formulation is properly adjusted 25 with water for injection. Finally, the prepared solution is sterilized by filtration. The injectable formulation of the present invention prepared according to the above mentioned process remains stable for more than one year at both 4t and room temperature without any phase separation or precipitation. Pharmacokinetics studies of the formulation of the present invention and 30 commercially available Ivomec (Merck & Co., INC. of the United States) using swine show that the absorption rate of the formulation of the present invention to reach the concentration in blood is approximately twice faster than that of iponz - 3 JUN 2003 Ivomec (Merck & Co. INC. of the United States). However, the formulation of the present invention and Ivomec show similar degradation patterns. That is, the invermectin concentrations in blood for both cases are reduced to the same levels within 4 to 5 days and no significant differences between the two solutions are detected thereafter. Since physical and pharmacokinetic properties of the injectable formulation of the present invention may vary according to the composition change of the present invention, the effects of ethanol concentration change in the formulation of the present invention on ivermectin solubility, viscosity of the formulation, and ivermectin pharmacokinetic were tested. It was showed that ivermectin solubility and viscosity of the formulation increase at high ethanol concentration. Furthermore, pharmacokinetic property was also changed depending on the ethanol concentration of the formulation. However, such changes were not so significant when they were compared with Ivomec's properties. In conclusion, it was determined that a retention time of ivermectin in blood for the formulation of the present invention is almost identical to Ivomec, while the absorption of ivermectin in the present invention occurs approximately one day earlier than Ivomec and the absorption efficiency of the present formulation is roughly twice than that of Ivomec. In addition, it was observed that the tested animals experienced no abnormal changes with increased absorption efficiency of ivermectin. In other words, no toxicity or other side effects were observed among the tested animals as a result of the high absorption efficiency of ivermectin. These characteristics strongly indicate that the injectable formulation of the present invention is superior to the prior art, judging from its improved pharmacokinetic data. In order to demonstrate, the efficacy of the present formulation for both endo- and ecto-parasites, efficacy tests were conducted using Ivomec as a control. Various swine including piglets, growing & finishing pigs (swine), and sows were used which wee artificially infected with Ascaris suum and Trichuris suis (endo-parasites), and Sarcoptes scabiei (an ecto-parasite). Test showed that the formulation of the present invention is either identical or superior to iponz -3 JUN 2003 WO 00/31098 PCT/KR99/00704 6 Ivomec. In more detail, the parasite egg positive rate in growing & finishing pigs (swine) was reduced to 0% in 2 weeks after the treatment of Ivomec, while it took just 1 week using the formulation of the present invention. In case of 5 growing & finishing pigs (swine) which were artificially infected, the parasite egg positive rates in one week after anthelmintic treatment started were as follows: 86% for Ascaris suum and 57% for Trichuris suis in no treatment groups; 80% for Ascaris suum and 0.0% for Trichuris suis in Ivomec group; 50% for Ascaris suum and 20% for Trichuris suis in the test group with the formulation of the io present invention. These percentages decreased to 0.0% in two weeks in both treatment groups. The dead parasites were excreted between 1 and 3 days after the treatment with formulation of the present invention, while the killed parasites were observed between 1 and 8 days with Ivomec treatment. This is another good example showing the superior characteristics of the present 15 invention for the removal of endo-parasites. Meanwhile, removal of ecto-parasites in the. piglets was also tested. The rate of pathological change for skin ailment in no treatment group was increased from 0.58 to 1.67 in 1 week and further augmented to 4.20 in 3 weeks after test started. However, in the piglets treated with the formulation of the 20 present invention, the rate of pathological change for skin ailment was sharply decreased from 1.82 to 0.07 in 1 week and maintained at 0.13 after three weeks, which changes are quite similar to those rates of 1.06, 0.13 and 0.07 in case of the Ivomec treatment. As shown in the above tests, the formulation of the present invention is 25 at least equivalent or superior to Ivomec with respect to the pharmacokinetics and the efficacy as anthelmintic agent. Moreover, as described above, the present formulation is stable in aqueous phase for a considerable period of time without any physical changes such as phase separation or precipitation. The present invention will be further described in details by the following 30 examples. However, the invention can be utilized in various ways and is not intended to be confined to the following examples. I. Manufacture of Ivermectin Injectable Solution Using WO 00/31098 PCT/KR99/00704 7 Direct Mixing Method [Example 1] 2 ml of benzyl alcohol (Korean Pharmacopoeia grade) was added to a 300 ml flask which was pre-sterilized for 30 min at 121 °C. Followings were then 5 added to the flask all at once: 0.01 g BHT (butylated hydroxytoluene, Korean Pharmacopoeia grade); 10 ml ethanol (Korean Pharmacopoeia grade); 70 ml water; 1.17 g ivermectin; 10 g Poloxamer 407 (BASF of Germany). Subsequently, a 5cm magnetic bar was introduced to the flask and stirred at 300 to 400 rpm. 10 The resulting mixture became clear with the generation of bubbles therein, and completely clear solution was obtained after approximately 24 h stirring. Next, appropriate amount of water was added to the solution (at an amount to result in a desired final volume of the solution) to complete the mixing process and the solution underwent vacuum suction filtering using 0.2 y of filter 15 paper (Wattman Filter Paper of the U.S.), thereby producing a fully sterilized ivermectin injectable solution of 1%(w/v). A viscosity of the resulting solution was found to be 69 cps (centipoise; Brookfield viscometer spindle #CP-42, 6rpm) at room temperature. [Example 2] 20 Ivermectin injectable solution was prepared by the same procedure in Example 1 except that 1 % (w/v) of ivermectin, 10.0 % (w/v) of poloxamer 407, 20.0 % (w/v) of ethanol, 0.01 % (w/v) of BHT, 2.0 % (w/v) of benzyl alcohol, and balance of water were used. [Example 3] 25 Ivermectin injectable solution was prepared by the same procedure in Example 1 except that 0.5 % (w/v) of ivermectin, 10.0 % (w/v) of poloxamer 407, 15.0 % (w/v) of ethanol, 0.01 % (w/v) of BHT, 2.0 % (w/v) of benzyl alcohol, and balance of water were used. [Example 4] 30 Avermectin injectable solution was prepared by the same procedure in Example 1 except that 1 % (w/v) of avermectin, 10.0 % (w/v) of poloxamer 407, 15.0 % (w/v) of ethanol, 0.01 % (w/v) of BHT, 2.0 % (w/v) of benzyl alcohol, and WO 00/31098 PCT/KR99/00704 8 balance of water were used. [Example 5] Avermectin injectable solution was prepared by the same procedure in Example 1 except that 0.5 % (w/v) of avermectin, 10.0 % (w/v) of poloxamer 407, 5 15.0 % (w/v) of ethanol, 0.01 % (w/v) of BHT, 2.0 % (w/v) of benzyl alcohol, and balance of water were used. II. Manufacture of Ivermectin Injectable Solution Using Stepwise Mixing Method [Example 6] 10 First step: 10 g of Poloxamer 407 (BASF of Germany) was added to a 300 ml flask which was pre-sterilized for 30 min at 121 °C, followed by 70 ml water addition. Next, a 5cm magnetic bar was transferred into the flask and stirred at 300 to 400 rpm. Mixing continued approximately 70 min until the Poloxamer 407 was completely dissolved in water, thereby producing a clear first 15 mixed solution. The first mixed solution was then filtered using a 0.2 y filter and finally the resulting first solution having a viscosity at room temperature of roughly 9.75 cps (centipoise; Brookfield viscometer spindle #CP-42, 6rpm) was obtained. Second step: Approximately 30 min later after the above first step of 20 mixing process, a second mixing process was performed. Adding 2 ml of benzyl alcohol (Korean Pharmacopoeia grade) in a 25 ml fiask which was pre-sterilized for 30 min at 12TC, 0.01g of BHT (Korean Pharmacopoeia grade), 10 ml of ethanol (Korean Pharmacopoeia grade), and 1.17 g of ivermectin were transferred to the flask. Next, a 5 cm magnetic bar was transferred to the flask 25 and stirred at 300 to 400 rpm. Mixing continued approximately 10 min until all the contents in the flask were dissolved, thereby producing a clear second mixed solution. The second mixed solution was then filtered using a 0.2 y filter. Third step: The first mixed solution and the second mixed solution were then transferred to a sterilized container and mixed for approximately 10 min to 30 give a final mixed solution. Next, small amount of water was added to the final solution (at an amount to result in a desired final volume of the solution) to complete the mixing process, thereby producing a fully sterilized ivermectin WO 00/31098 PCT/KR99/00704 9 injectable solution of 1%(w/v). [Example 7] Ivermectin injectable solution was prepared by the same procedure in Example 6 except that 1.0 % (w/v) of ivermectin, 10.0 % (w/v) of poloxamer 407, 5 5.0 % (w/v) of ethanol, 0.01 % (w/v) of BHT, 2.0 % (w/v) of benzyl alcohol, and balance of water were used. [Example 8] Ivermectin injectable solution was prepared by the same procedure in Example 6 except that 0.5 % (w/v) of ivermectin, 20.0 % (w/v) of poloxamer 407, 10 10.0 % (w/v) of ethanol, 0.01 % (w/v) of BHT, 2.0 % (w/v) of benzyl alcohol, and balance of water were used. [Example 9] Avermectin injectable solution was prepared by the same procedure in Example 6 except that 1.0 % (w/v) of avermectin, 10.0 % (w/v) of poloxamer 407, 15 15.0 % (w/v) of ethanol, 0.01 % (w/v) of BHT, 2.0 % (w/v) of benzyl alcohol, and balance of water were used. [Example 10] Avermectin injectable solution was prepared by the same procedure in Example 6 except that 0.5 % (w/v) of avermectin, 10.0 % (w/v) of poloxamer 407, 20 15.0 % (w/v) of ethanol 0.01 % (w/v) of BHT, 2.0 % (w/v) of benzyl alcohol, and balance of water were used. The injectable formulations produced in the above Examples as described underwent various tests as described hereinbelow. [Test 1] Pharmacokinetics Comparison Tests 25 Between Injectable Formulation of the Present Invention and Ivomec (Merck & Co. INC. of the United States) The injectable formulation was manufactured according to the method of Example 6. It consists of 1.0% of ivermectin, 10.0% of Poloxamer 407, 15.0% of ethanol, 2.0% of benzyl alcohol, 0.01% of BHT, and sterilized water making 30 up the remaining portion of the solution. The formulation of the present invention and Ivomec were injected in two separate test groups of growing & finishing pigs (swine) at an amount of 300 jig per kilogram body weight. Next, WO 00/31098 PCT/KR99/00704 10 blood samples were taken at every predetermined time. The blood samples were then centrifuged for 20 min at 3000 rpm to separate the plasma from the blood, after which the plasma was stored at -20°C. In order to analyze a pharmacodynamics of the two tested formulations in pigs, the plasma samples 5 were thawed and assayed using a modified method of Montigny's fluorescence derivatization-HPLC analysis method (J. Pharm. Biomed. Anal. (1990)_8, 507-511). After adding 1 ml acetonitrile to 1 ml of each plasma sample, the resulting mixtures were centrifuged for 10 min at 3000 rpm. The supernatant io was then subjected to solid phase extraction. That is, in solid phase extraction, the samples were treated with in Sep-Pak C18 cartridges (Waters of the U.S.). After the cartridges were fully dried with nitrogen gas, the purified samples were slowly eluted with 5ml chloroform and then dried up using nitrogen gas. Subsequntly, each dried sample was dissolved with 100 fd of 1-15 methyiimidazole: acetonitrile mixture (1:2, v/v) and 150 ]A of trifluroacetic anhydride: acetonitrile mixture (1:2, v/v) was added thereto such that the ivermectin isolated was derivatized for better measurement. 100 i& of this derivatized solution was then analyzed with HPLC (TSO P1000, U.S.A) with a fluorescence detector (TSP F3000, U.S.A.). Peak measurement was done 20 under a 374 nm excitation wavelength and a 475 nm emission wavelength conditions. 0.2% of acetic acid-methyl alcohol-acetonitrile mixture at a volume ratio of 4:32:64 was used for a mobile phase in Nova-Pak C18 (4 y , 3.9 X 150 mm, Waters, U.S.A.), and a mobile phase flow rate was 1.5 ml/min. A concentration of the ivermectin in the samples was determined by 25 comparing HPLC peak area of the plasma samples from the pigs injected by the present formulation and Ivomec, with standard curve prepared by addition of 0.5, 5, and 50 ng of ivermectin (Sigma, U.S.A.) to 1 ml of plasma taken from the pigs not treated with any anthelmintic. The treatment and measurement of the samples were simultaneously performed without any delay and blood samples 30 taken from one pig were analyzed all at once. To reduce variation errors in pre-treatment of samples, standard samples were simultaneously prepared and used every time of analysis. 511852 11 At this time the peak of derivatized ivermectin B1a derivative appeared at about 10 min retention time in HPLC. Also a small peak appeared at about 8 min, which represents ivermectin B1b derivative. The concentration of ivermectin in blood samples was determined by 5 comparing combined areas of two peaks with those of ivermectin peak areas in the standard curve. Everytime the analysis was performed, standard blood sera were prepared by adding different concentrations of ivermectin and treated with the same method as for the test sample treatment. These newly prepared standards were used to determine the concentration of the test samples. 10 The standard curve equation (linear regression equation) between ivermectin concentrations and HPLC peak area was: area = 1.729 x 10"4 x ivermectin concentration + 0.10039; and a linear regression coefficient (R2) was 0.998. Using this linear equation, the ivermectin concentrations in the blood samples were determined and the results of which for both the formulation of the 15 present invention and Ivomec are shown in FIG. 1. As shown in the drawing, the maximum concentration of ivermectin in plasma samples from pigs injected with the formulation manufactured according to Example 6 is twice higher than that of Ivomec. However, 4 or 5 days later after injection, concentrations of ivermectin for both the present formulation and 20 Ivomec are substantially reduced to equal levels. Such a different pharmacokinetics of ivermectin is because of the composition difference between Ivomec and the formulation of the present invention. Compared with the Ivomec, the formulation of the present invention is more quickly absorbed in the blood and shows superior pharmacodynamic 25 property. This suggests that, when identical amounts of ivermection are used, the ability of the formulation of the present invention to kill parasites is significantly better than that of Ivomec. [Test 2] Comparison Tests of Injectability Between Injectable Formulation of the Present Invention and Other Various Commercial Products 30 Containing Ivermectin An injectability comparison test was performed using the injectable formulation of the present invention (I), Ivomec (Merck Co.) (II), Ivomec-F (Merck iponz -3 JUN2003 WO 00/31098 PCT/KR99/00704 12 Co.) (Ill), Baymec (Bayel Co.) (IV), Abamec (Daesung, Korea) (V), and Dectomax (Pfizer) (VI). 3 ml of each above formulation was transferred to a Greenject-5 (5 ml) syringe (Green Cross Pharmaceutical Corp., Korea, 23 gauge, 1") and average 5 injectability values were compared using an injectability measurement device (Test Stand Model-2252, CPU Gauge Model 9500, AIKOH Engineering Co., Ltd., Japan). The values are shown in Table 1 below. As shown in the table, the injectability value of formulation II (Ivomec) was the lowest at 0.53 kg, while the value of formulation IV (Baymec) was the highest at 1.73 kg. The injectability 10 value for the formulation of the present invention, formulation I was approximately in between the highest and lowest values. In conclusion, the force needed during injection of the present formulation is similar to that of typical product in the market. Table 1 Formulation I II III IV V VI Injectability [Kg] 0.96 0.53 0.81 1.73 1.27 1.32 15 [Test 3] Viscosity Measurement Ivomec and the Formulation of the Present Invention Containing Different Concentrations of Ethanol We have studied how physical characteristics such as viscosity and solubility of the formulation of the present invention vary according to the 20 composition change of special component. For viscosity test, a variety of the formulations of the present invention with different concentrations of ethanol (5, 10, 15, and 20%) were prepared according to the method of Example 6. Using a Brookfield Visometer (spindle # CP-42, 6 rpm, 25°C), viscosities of the present formulations and Ivomec were measured. The results are shown 25 in Table 2 below. As shown in the table, the viscosity of the present formulation increases with increase of ethanol level. The time to reach a clear solubilization of all components increases as the concentration of the ethanol decreases. Table 2 Formulation Formulation of Present Invention Type Ivomec (ethanol content %) WO 00/31098 PCT/KR99/00704 13 5% 10% 15% 20% Viscosity (cps*) 36.6 39.9 43.8 55.4 59.3 * cps: centipoise (determined by Brookfield Viscometer spindle #CP-42, 6 rpm, 25 °C) [Test 4] Pharmacokinetics of Ivomec and the Formulation of the Present invention Containing Different Concentrations of Ethanol The formulation of the present invention manufactured according to 5 Example 6 and Ivomec were injected to two separate test groups of growing & finishing pigs (swine) at an amount of 300 tig per kilogram of body weight. Blood samples were taken at predetermined time intervals after the injection and using the method described in [Test 1], ivermectin concentration changes in the blood were measured. The results are shown in Table 3 below. io As shown in the table, average maximum concentrations of ivermectin in the blood are ranged from 1.7 to 2.1-times of that for Ivomec and the time for Ivomec to reach its peak is 36 h, while the formulation of the present invention takes 2 to 5 h depending on the amount of ethanol in the present formulation. Although it is not shown in Table 3, the reduction rate of ivermectin 15 concentration after the peak seems to be correlated with the increase of ethanol level in the formulation. When ethanol content is less than 10%, the reduction of ivermectin concentration in case of the formulation of the present invention is almost identical to that in case of Ivomec in 5 days. Table 3 Formulation Maximum Ivermectin Concentration in Blood* Time to Reach Maximum Concentration (h) Ivomec 1.0 36.0 Present Formulation (5% Ethanol) 2.1 3.5 Present Formulation (10% Ethanol) 2.0 4.0 WO 00/31098 PCT/KR99/00704 14 Present Formulation (15% Ethanol) 1.7 4.3 Present Formulation (20% Ethanol) 1.9 5.3 * Relative maximum blood concentration of ivermectin compared with Ivomec As shown from the above, we can recognize that the results of both [Test 1] and [Test 5] are similar. That is, the absorption rate of the formulation of the present invention is approximately twice higher than that of Ivomec. In 5 addition, the reduction rate of ivermectin concentration in the blood of the formulation of the present invention is slower than that of Ivomec so that the maintnenance of the ivermectin concentration in the blood by the present formulation is nearly identical to that by Ivomec. These results suggest that identical amounts of ivermectin are used, the formulation of the present 10 invention can be more effective to kill parasites (it is proved by [Test 5]). Since the above characteristics were not tested in case where an excessive amount of ethanol (over 20%) is used, it is preferable to use less than 20% ethanol in the present formulation. [Test 5] Efficacy in Tests against Endo and Ecto-Parasites with Ivomec and 15 the Formulation of the Present Invention Containing Different Concentrations of Ethanoi For the efficacy test against endo-parasites, sows and growing & finishing pigs infected with Ascaris suum and Trichuris suis were used, and for the efficacy test against ecto-parasites, piglets infected with Sarcoptes scabiei 20 were used. Each pig was injected with Ivomec or the present formulations (0.3 mg/kilogram of body weight). Feed and water to the test pigs were provided in normal pattern and amount according to the schedule in local pig farms. 40 piglets were artificially infected with Ascaris suum (100 infectious eggs) and Trichuris suis (100 infectious eggs). Infection was made twice in two weeks 25 period. After artificial infection, any excreted egg was inspected every day. The test results are described below. (5-1) Efficacy and safety tests with growing & finishing pigs WO 00/31098 PCT/KR99/00704 15 Test results of artificially infected growing & finishing pigs showed that parasitic eggs positive rates, one week after test started were as follows: 0% for the formulation of the present invention; 0% for a double dosage of the present formulation; 17% for Ivomec; and 33% for the control group of untreated pigs. 5 After 2 weeks, the parasitic egg positive rates for all pigs treated with the formulation of the present invention and Ivomec became 0%, whereas the percentage for the control group of pigs increased to 50%. A parasitic egg reduction was 100% for the pigs treated with either Ivomec or the present formulation, while it was 0% for the untreated control 10 group of pigs. An excretion of dead parasites was 0% for the control group of pigs, while it was between 23.3 and 26.7% for the pigs treated with Ivomec or the formulation of the present invention. Further, in all of treated pigs, no abnormalities in the area of injection (e.g., fever, inflammation) and unusual behavior were observed. 15 Table 4 Formulation* Number of pigs Measurement** Parasitic egg positive rate (%) Parasitic Egg reduction (%) Excretion of dead parasites (%) 0 weeks 1 week 2 weeks 3 weeks Present formulation 30 33 0 0 0 100 8/30 (26.7) Present formulation X 2 30 33 0 0 0 100 7/30 (23.3) Ivomec 30 33 17 0 0 100 7/30 23.3) Control Group 30 33 33 50 50 0 0/30 (0.0) "Injection amount of the present formulation and Ivomec: 0.3 mg/kg body weight Injection amount of X2: 0.6 mg/kg body weight "Inspection of parasitic eggs and severity of diarrhea were performed before treatment and 1, 2, and 3 weeks after treatment; survival rate was observed over the entire period; and excretion of dead parasites was observed everyday until parasites were no longer excreted. WO 00/31098 PCT/KR99/00704 16 (5-2) Efficacy test with artificially infected growing & finishing pigs The excretion of dead parasites was observed between the first and third days among the pigs injected with the formulation for the present invention, whereas among the pigs injected with Ivomec, dead parasites were excreted 5 between 1st and 8th days after treatment. Five weeks after the treatment of anthelmintic agents all the pigs underwent post-mortem examination to check the existence of parasites. A few parasites were observed in the small intestine of the untreated pigs, while no parasite was detected in the pigs treated with the formulation of the present invention. Furthermore, to check the efficacy of the 10 present formulation against Ascaris suum, the pigs at the time of treatment and roughly 1-5 five weeks prior to treatment were artificially infected with approximately 100 larvae. No larvae were observed among the pig tested, suggesting that the formulation of the present invention is also effective to kill this parasite. 15 In addition, no abnormalities in the area of injection (e.g., fever, inflammation) and unusual behavior were observed among the pigs tested. No injection marks or inflammation in the area of injection were detected during post-mortem examination. Table 5 Formulation* No. of pigs Excretion of dead parasites Post-mortem examination results Present formulation 10 1-3 days after injection no parasites Present formulation X 2 10 1-3 days after injection no parasites Ivomec 10 1-8 days after injection no parasites Control group 7 none 100% infection injection amount of the present formulation and Ivomec: 0.3 mg/kg body weight Injection amount of the present formulation X 2: 0.6 mg/kg body weight WO 00/31098 PCT/KR99/00704 17 (5-3) Efficacy test in growing & finishing pigs artificially infected with the endo-parasite, Ascaris suum Three test groups of pigs were artificially infected with Ascaris suum. At 5 the start of the test, Ascaris suum eggs were detected in all of the pigs of every test group. However, after one week, it was shown that the control group of untreated pigs had an egg positive rate of 86%, the group of pigs treated with the formulation of the present invention had the rate of 40-50%, and the group of pigs treated with Ivomec had the rate of 80%. Between 2nd and 5th weeks, the io egg positive rate of control group was between 86 and 100%, whereas in the groups treated with either Ivomec or the formulation of the present invention, a 0% egg positive rate was observed, which means that all the parasites were terminated (see table 6). Table 6 Formulation* No. of pigs Egg positive rate (%) Egg reduction rate (%) 0 week 1 week 2 weeks 3 weeks 4 weeks 5 weeks Present formulation 10 100 40 O 0 0 0 100 Present formulation X 2 10 100 50 0 0 0 0 100 Ivomec 10 100 80 0 0 0 0 100 Control group 7 100 86 100 86 86 86 0 injection amount of the present formulation and Ivomec: 0.3 mg/kg body weight Injection amount of the present formulation X 2: 0.6 mg/kg body weight The feces of pigs tested were examined and it was observed that 100 to 10,560 Ascaris suum eggs per 1g (EPG; egg/gram) of feces were detected at the beginning of the test. Among the pigs treated with either Ivomec or the formulation of the present invention, the number of parasite eggs detected in 20 feces dropped significantly after the first week, with the no eggs being detected after two weeks. However, parasite eggs were continuously observed in control WO 00/31098 PCT/KR99/00704 18 group of pigs. The results of this test are shown in Table 7 below. Table 7 Formulation* No. of pigs Test Item** EPG (Ascaris suumlTrichuris suis) 0 week 1 week 2 weeks 3 weeks 4 weeks 5 weeks Present formulation 10 778+ 1,053 52+69 0+0 0+0 0+0 0+0 Present Inventive formulation X 2 10 2,156+ 3,095 99+120 0+0 0+0 0+0 0+0 Ivomec 10 4,188+ 3,025 168+198 0+0 0+0 0+0 0+0 Control group 10 2,069+ 1,121 971+551 1,058+ 904 483+ 348 248+ 216 410+ 374 * Injection amount of the present formulation and Ivomec: 0.3 mg/kg body weight Injection amount of the present formulation X 2: 0.6 mg/kg body weight **A set amount from 1-2g of feces was collected, and the number of eggs was counted using MacMaster egg counting chamber, and this number was divided by the amount of feces (5-4) Efficacy test in growing & finishing pigs artificially infected 5 with the endo-parasite, Trichuris suis Three test groups of pigs were artificially infected with Trichuris suis. At the start of the test, a egg positive rate was between 29 and 100% for this parasite. After one week, it was determined that the control group of untreated pigs had an egg positive rate of 57%, the group of pigs treated with he 10 formulation of the present invention had an egg positive rate of 20%, and the group of pigs treated with Ivomec had 0% rate. From 2 to 5 weeks after treatment, the egg positive rate of control group was between 0 and 86%, but in the groups of pigs treated with either Ivomec or the formulation of the present invention, no Trichuris suis eggs were detected. Table 8 below outlines the 15 results of this test. Table 8 Formulation* No. Egg positive rate (%) Egg WO 00/31098 PCT/KR99/00704 19 of pigs 0 week 1 week 2 weeks 3 weeks 4 weeks 5 weeks reduction rate (%) Present formulation 10 90 20 0 0 0 0 100 Present formulation X 2 10 100 20 0 0 0 0 100 Ivomec 10 60 0 0 0 0 0 100 Control group 7 29 57 86 29 0 0 0 "Injection amount of the present formulation and Ivomec: 0.3 mg/kg body weight Injection amount of the present formulation X 2: 0.6 mg/kg body weight The feces of pigs tested were also examined. From 0 to 215 Trichuris suis eggs per 1g (EPG; egg/gram) of feces were detected. Up to three weeks, Trichuris suis eggs were detected in the feces of the control group of pigs, while the number of Trichuris suis eggs in feces of the groups treated with the present formulation or Ivomec decreased significantly after one week and no eggs being detected after two weeks. The results are shown in Table 9 below. WO 00/31098 PCT/KR99/00704 20 Table 9 Formulation* No. of pigs Test Item** EPG (Ascaris suumlTrichuris suis) 0 week 1 week 2 weeks 3 weeks 4 weeks 5 weeks Present formulation 10 113+68 34+59 0+0 0+0 0+0 0+0 Present formulation X 2 10 113+25 29+49 0+0 0+0 0+0 0+0 Ivomec 10 72+77 0+0 0+0 0+0 0+0 0+0 Control group 10 25+56 53+77 174+259 98+218 0+0 0+0 * Injection amount of the present formulation and Ivomec: 0.3 mg/kg body weight Injection amount of the present formulation X 2: 0.6 mg/kg body weight **A set amount from 1-2g of feces was collected, and the number of eggs was counted using MacMaster egg counting chamber, and this number was divided by the amount of feces (5-5) Efficacy test against ecto-parasites in piglet Piglets were used to conduct the efficacy test of the present formulation 5 against the ecto-parasite, Sarcoptes scabiei. In the control group of untreated piglets, the rate of skin pathological change at the start of test was 0.58, and increased to 1.67 after one week and further increased to 4.20 after three weeks. In the group of piglets treated with the formulation of the present invention, the rate of skin pathological change was reduced from 1.82 to 0.7 after one week 10 and further decreased to 0.13 after three weeks. In the group of piglets treated with Ivomec, the rate of skin pathological change was reduced from 1.06 to 0.13 after one week and 0.07 after three weeks. Test results are shown in Table 10. WO 00/31098 PCT/KR99/00704 21 Table 10 Formulation* No. of pigs Weight (M+SD)kg** Body weight increase (0-3 weeks) kg Ecto-parasites pathological change 0 week 1 week 3 weeks 0 week 1 week 3 weeks Present formulation 15 7.6+ 1.17 9.5+ 0.89 14.2+ 1.54 6.6 1.82 0.07 0.13 Present formulation X2 15 6.4+ 0.82 8.4+ 0.92 12.7+ 1.30 6.3 1.79 0.31 0.17 Ivomec 15 6.6+ 0.64 8.6+ 0.88 12.6+ 1.95 6.0 1.06 0.13 0.07 Control group 15 5.3+ 0.52 6.9+ 0.73 11.0+ 0.74 5.7 0.58 1.67 4.20 * Injection amount of the present formulation and Ivomec: 0.3 mg/kg body weight Injection amount of the present formulation X2: 0.6 mg/kg body weight "Body weight measurement and inspection of ecto-parasites were conducted at 0,1, 3 weeks M : average weight; SD : standard deviation (5-6) Efficacy test against endo and ecto-parasites in sow Sows were used to conduct the efficacy test of the present formulation 5 against the endo and ecto-parasites. No diarrhea was seen among the sows tested at 1, 2 and 3 weeks. Dead parasites were detected in the feces of the sows injected with the formulation of the present invention after two days and no more parasites were found after 7 days. In the case where the sows were treated with Ivomec, the parasites begun to be excreted after three days and 10 were no longer detected after 8 days. Further, with regard to ecto-parasites, all the parasites were removed in the sows treated with either the present formulation or Ivomec, whereas no significant improvement was detected in the untreated control group of sows. In addition, in all of treated sows, no abnormalities in the area of 15 injection (e.g., fever, inflammation) and unusual behavior were observed. The treated sows were also examined during post-mortem examination. No marks WO 00/31098 PCT/KR99/00704 22 or infection were detected in the injection area. The results of this test are shown in Table 11 below. Table 11 Formulation* No. of sows Parasite egg positive rate (%) Parasite egg reduction rate** (%) Parasite reduction rate** (%) Ectoparasite rate of Destruction ** (%) 0 week 1 week 2 weeks 3 weeks Present formulation 20 50 50 0 0 100 50 100 Present formulation X 2 10 30 5 0 0 100 40 100 Ivomec 10 40 20 10 0 100 40 100 Control group 20 40 40 35 40 0 0 0 * Injection amount of the present formulation and Ivomec: 0.3 mg/kg body weight Injection amount of the present formulation X 2: 0.6 mg/kg body weight ** Parasite excretion rate is the percentage of sows excreting parasites over total sows in each group; parasite egg reduction rate is the percentage of sows without egg excretion over initial egg excreting sows in each group; and ecto-parasite rate(%) of destruction is determined by the skin trouble and sow's status of rubbing the farm partition. 5 The formulations of the present invention as described above are extremely stable in aqueous phase and do not undergo phase separation. Further, as shown in a number of clinical tests, the formulations of the present invention have a better pharmacokinetic property and an equivalent or better efficacy against both endo and ecto-parasites than the prior art. Other 10 physicochemical properties of the formulations of the present invention include their long stability, similar viscosity, and injectability compared to the prior art. Finally, no physical damage or other side effects were observed in the area of injection with the use of the present formulation. While the present invention has been described in detail with reference 15 to the preferred embodiments, those skilled in the art will appreciate that various modifications and substitutions can be made thereto without departing from the WO 00/31098 PCT/KR99/00704 23 spirit and scope of the present invention as set forth in the appended claims. t ♦ </div>

Claims

<div class="application article clearfix printTableText" id="claims"> 'A WHAT WE CLAIM IS: 24

1. An injectable anthelmintic formulation comprising: an water-insoluble anthelmintic; a surfactant comprising a block copolymer of ethylene oxide (EO) and 5 propylene oxide (PO); ethanol ; and a water-soluble solvent.

2. The formulation of claim 1 wherein the insoluble anthelmintic is selected from avermectin and invermectin, and is included in the formulation in a range 10 from 0.5 to 2.0% (w/v).

3. The formulation of claim 1 wherein the surfactant is contained in the formulation in a range from 5 to 25% (w/v).

4. The formulation of claim 3 wherein the ethanol . is contained in the formulation in a range from 1 to 50% (w/v). 15

5. The formulation of claim 1 further comprising additives.

6. The formulation of claim 5 wherein the additives include 0.001 to 0.5%(w/v) of butylated hydroxytoluene and 0.5 to 10%(w/v) of benzyl alcohol.

7. The formulation of claim 1 wherein the block copolymer is a (EO)x(PO)y(EO)x unit, where the ratio of x to y is in a range from 0.75 to 2.0 and 20 the average molecular weight is between 9000 Da and 20000 Da.

8. The formulation of claim 1 wherein the anthelmintic is active against both endo and ecto-parasites.

9. An injectable anthelmintic formulation as defined in claim 1 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawing. iponz - 3 JUN 2003 </div>