Enhancement of helminth treatment

Abstract

The present invention provides a method of treating a liver fluke infection in a mammal in need thereof, the method comprising administering an effective amount of difeniratel in combination with an effective amount of closurone.

Description

Technical Background

[0001] Liver flukes are helminthic parasites that cause huge economic losses in livestock farming, primarily through infection with Fasciola hepatica or its tropical counterpart, Fasciola gigantica. Since all known drugs active against Fasciola hepatica are also active against Fasciola gigantica, the two liver flukes will be collectively referred to as Fasciola spp. hereinafter.

[0002] Fasciola infections primarily affect farmed ruminants; however, they are not selective for definitive hosts and successfully infect many mammals, including humans. In farmed ruminants, fascioliasis causes billions of dollars in economic losses worldwide each year. McVeigh et al., Trends in Parasitology, March 2018, Vol. 34, No. 3, 184-196.

[0003] Liver flukes have a complex life cycle. In their definitive hosts, Fasciola can be divided into three stages, the early immature stage, the immature stage, and the mature stage (often called the adult stage). In sheep, the early immature form occurs in flukes 1-3 weeks old, the immature form occurs in flukes 4-7 weeks old, and the mature form occurs in flukes 8 weeks old and older. In cattle, the early immature form occurs in flukes 1-5 weeks old, the immature form occurs in flukes 6-9 weeks old, and the mature form occurs in flukes 10 weeks old and older.

[0004] Some anthelmintics are only effective against adult worms and require more frequent applications, while many current anthelmintics have lost their chemopreventive advantage against fascioliasis due to the development of resistance.

[0005] In Australia, resistance of Fasciola hepatica to several anthelmintics has been demonstrated in the field and in the laboratory ((1990) Drug resistance in Fasciola hepatica. See Boray JC, Martin PJ, Roush RT (eds.) Resistance of parasites to antiparasitic drugs. MSD AGVET, Rahway, NJ, pp 51–60 and D. Boray JC, De Bono Drug resistance in Fasciola hepatica. Outteridge PM, Richards RB (eds.) Australian advances in veterinary science (1989). Australian Veterinary Association, pp 166–169). Long-term regular use of salicylates in sheep in endemic areas of New South Wales has been shown to select for resistant strains of Fasciola hepatica. These resistant strains have been shown to maintain their resistance in cattle and over several generations of sheep. Ten of 17 isolates (58.8%) from different geographical areas developed resistance to the recommended dose of iosamide, with significant secondary resistance to closantel. Resistance to the drugs was mainly observed in immature liver flukes, but rarely in adults. Better treatments for liver fluke infections are needed. Summary of the invention

[0006] The present invention provides a method for treating liver fluke infection in a mammal in need of treatment, the method comprising administering an effective amount of difenitide in combination with an effective amount of clorsulon. The synergistic combination of difenitide and clorsulon will have advantages in efficacy against sensitive and resistant Fasciola.

[0007] Diamphenethide, also known by its chemical name N-[4-[2-[2-(4-acetylaminophenoxy)ethoxy]ethoxy]phenyl]acetamide. The preparation of diamphenethide is well known in the art. For example, US3896235. Diamphenethide has been used against liver flukes and is rapidly metabolized by deacetylation to a compound called DAMD. Diamphenethide has been found to be highly effective against early immature flukes up to 6 weeks of age, but its activity gradually decreases as the flukes mature. Fairweather et al., The Veterinary Journal 1999, 158, 81-112. Diamphenethide is marketed under the trade name Marketed for the treatment of liver fluke infections in sheep, it was withdrawn from the market in the early 1980s. The recommended oral dose for sheep is 80-120 mg / kg.

[0008] Clorsulon, also known by its chemical name 4-amino-6-(1,2,2-trichlorovinyl)benzene-1,3-disulfonamide. The preparation of clorsulon is well known in the art. For example, US4064239. Clorsulon is believed to be effective against mature flukes and is more effective against cattle than sheep. The combination of clorsulon and ivermectin is known as Ivomec. or sold under other trade names for the treatment of mature liver fluke in cattle.

[0009] The term "liver fluke infection" refers to infection with Fasciola hepatica or Fasciolagigantica. Unless otherwise indicated, the term includes infection with flukes at any stage of maturity or a combination of stages.

[0010] The term "mammal" refers to a warm-blooded vertebrate having hair or fur and females secreting milk to feed their young, including humans. Specific mammals are ruminants, such as cattle, goats, sheep, bison, African buffalo, water buffalo, antelope, deer, moose, elk and giraffe. The term mammal also includes so-called pseudo-ruminants, such as llamas and alpacas. The term mammal also includes horses and donkeys. More specifically, mammals are understood to be goats, sheep, bison, African buffalo, water buffalo and cattle. More specifically, mammals are sheep and cattle.

[0011] Mammals in need of the present treatment can be readily identified by a skilled clinician. For example, symptoms may include fever, malaise, abdominal pain, eosinophilia, hepatomegaly, and abnormal liver tests. Infection may also be detected by antibody tests, including ELISA tests, particularly of blood and milk. Infection may also be diagnosed by examination of fecal specimens, including ELISA tests. In addition, modeling based on season, rainfall, temperature, and other local conditions may be used to predict when liver fluke infection is more likely to occur.

[0012] As used herein, the terms "in combination with..." as well as "combination" and "systemic combination" refer to the administration of difenidide before, during or after the administration of clorsulon. That is, in the combination herein, difenidide and clorsulon are administered to a mammal in combination sequentially or simultaneously. Typically, sequential administration refers to the administration of the other drug within three days of the administration of difenidide or clorsulon. More typically, difenidide and clorsulon are administered within one day relative to each other, and even more typically within the same day. Sequential administration may also refer to the administration of another drug within 1 hour, or even minutes, of the administration of difenidide or clorsulon. For convenience, difenidide and clorsulon are administered simultaneously.

[0013] An effective amount of difenidide in combination with an effective amount of clorsulon is administered orally or parenterally. In some embodiments, an effective amount of difenidide in combination with an effective amount of clorsulon is administered orally to a mammal. In some embodiments, an effective amount of difenidide in combination with an effective amount of clorsulon is administered parenterally to a mammal.

[0014] Oral administration may include adjuvants conventionally used in the field of formulations, and thus may be processed in a known manner to provide, for example, solutions, emulsions, soluble powders, powder mixtures, granules or microcapsules in polymeric materials. These formulations, preparations or compositions containing difenitide and clorsulon alone or together may optionally include solid or liquid adjuvants and are prepared in a manner known in the art, for example, by mixing and / or grinding the active ingredients with adjuvants (e.g., solvents, solid carriers, etc.). Oral administration may also be accomplished by drenching, gavage, tablets, capsules or feed.

[0015] The terms "an effective amount of difenidide" and "an effective amount of clorsulon" refer to the amount of difenidide and clorsulon required to eliminate, substantially eliminate, slow or halt the development of a liver fluke infection when the combination herein is administered.

[0016] In certain embodiments, the effective amount of difenidone in the combination herein is 20-120 mg / kg (e.g., 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 65 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 85 mg / kg, 90 mg / kg, 95 mg / kg, 100 mg / kg, 105 mg / kg, 110 mg / kg, 115 In some embodiments, the effective amount of clorsulon in the combination herein is 1-10 mg / kg (e.g., 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, 5 mg / kg, 5.5 mg / kg, 6 mg / kg, 6.5 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 8.5 mg / kg, 9 mg / kg, 9.5 mg / kg or 10 mg / kg). In certain embodiments, the effective amount of difenidide in the combination herein is 20-80 mg / kg (e.g., 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, or 80 mg / kg), and the effective amount of clorsulon in the combination herein is 1-10 mg / kg (e.g., 1.5 mg / kg, 3.5 mg / kg, 5 mg / kg, 7 mg / kg, or 10 mg / kg). In certain embodiments, the effective amount of difenidide in the combination herein is 30-80 mg / kg, 40-80 mg / kg, 50-80 mg / kg, 60-80 mg / kg, or 70-80 mg / kg. In certain embodiments, the effective amount of clorsulon in the combination herein is 1.5-10 mg / kg, 3-10 mg / kg, 5-10 mg / kg, or 7-10 mg / kg. In certain embodiments, the effective amount of difenidide in the combination herein is 30-60 mg / kg, 40-60 mg / kg or 40-50 mg / kg. In certain embodiments, the effective amount of clorsulon in the combination herein is 3-10 mg / kg, 5-10 mg / kg, 6-10 mg / kg or 7-10 mg / kg.

[0017] In certain embodiments, in the treatment of sheep, the effective amount of difenidide in the combination herein is 20-80 mg / kg, and the effective amount of clorsulon in the combination herein is 3.5-10 mg / kg. In certain embodiments, in the treatment of sheep, the effective amount of difenidide in the combination herein is 20-60 mg / kg, and the effective amount of clorsulon in the combination herein is 5-10 mg / kg. In certain embodiments, in the treatment of sheep, the effective amount of difenidide in the combination herein is 35 mg / kg, and the effective amount of clorsulon in the combination herein is 5 mg / kg.

[0018] In certain embodiments, in the treatment of cattle, the effective amount of difenidide in the combination herein is 40-120 mg / kg, and the effective amount of clorsulon in the combination herein is 3.5-10 mg / kg. In certain embodiments, in the treatment of cattle, the effective amount of difenidide in the combination herein is 40-80 mg / kg, and the effective amount of clorsulon in the combination herein is 5-10 mg / kg. In certain embodiments, in the treatment of cattle, the effective amount of difenidide in the combination herein is 70 mg / kg, and the effective amount of clorsulon in the combination herein is 10 mg / kg.

[0019] The present invention provides a method for treating liver fluke infection in a mammal in need of treatment, the method comprising administering an effective amount of difenidine in combination with an effective amount of clorsulon. In other embodiments, the mammal is a ruminant. In other embodiments, the mammal is selected from sheep and cattle. In other embodiments, the mammal is a sheep. In other embodiments, the mammal is a cattle. In other embodiments, the effective amount of difenidine in the combination is 20-80 mg / kg (e.g., 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg or 80 mg / kg). In other embodiments, the effective dose of clorsulon in the combination is 1-10 mg / kg (e.g., 1.5 mg / kg, 3.5 mg / kg, 5 mg / kg, 7 mg / kg or 10 mg / kg). In certain embodiments, the effective amount of difenidide is 30-80 mg / kg, 40-80 mg / kg, 50-80 mg / kg, 60-80 mg / kg or 70-80 mg / kg. In certain embodiments, the effective amount of clorsulon is 1.5-10 mg / kg, 3-10 mg / kg, 5-10 mg / kg or 7-10 mg / kg.

[0020] The present invention also relates to the use of a combination of difennilide and clorsulon in the preparation of a medicament for treating liver fluke infection. In other embodiments, the difennilide used in the combination is 20-80 mg / kg (e.g., 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg or 80 mg / kg). In other embodiments, the clorsulon used in the combination is 1-10 mg / kg (e.g., 1.5 mg / kg, 3.5 mg / kg, 5 mg / kg, 7 mg / kg or 10 mg / kg). In certain embodiments, the effective amount of difennilide is 30-80 mg / kg, 40-80 mg / kg, 50-80 mg / kg, 60-80 mg / kg or 70-80 mg / kg. In certain embodiments, the effective amount of clorsulon is 1.5-10 mg / kg, 3-10 mg / kg, 5-10 mg / kg or 7-10 mg / kg.

[0021] Various aspects of the disclosure are set forth in the following numbered clauses:

[0022] Clause 1. A method of treating a liver fluke infection in a mammal in need of such treatment, the method comprising administering an effective amount of difenitide in combination with an effective amount of clorsulon.

[0023] Item 2. The method of Item 1, wherein the mammal is selected from sheep and cattle.

[0024] Clause 3. The method of Clause 1, wherein the mammal is a sheep.

[0025] Clause 4. The method of Clause 1, wherein the mammal is a cow.

[0026] Clause 5. The method of any one of Clauses 1-4, wherein the effective amount of difenidide is 20-80 mg / kg, 30-80 mg / kg, 40-80 mg / kg, 50-80 mg / kg, 60-80 mg / kg or 70-80 mg / kg.

[0027] Clause 6. The method of any one of Clauses 1-5, wherein the effective amount of difenidide is 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg or 80 mg / kg.

[0028] Clause 7. The method of any one of Clauses 1-6, wherein the effective amount of clorsulon is 1-10 mg / kg, 1.5-10 mg / kg, 3-10 mg / kg, 5-10 mg / kg, or 7-10 mg / kg.

[0029] Clause 8. The method of any one of Clauses 1-7, wherein the effective amount of clorsulon is 1.5 mg / kg, 3.5 mg / kg, 5 mg / kg, 7 mg / kg or 10 mg / kg.

[0030] Clause 9. The method of any one of Clauses 1 to 8, wherein the liver fluke is Fasciola hepatica.

[0031] Clause 10. The method of any one of Clauses 1-9, wherein the liver fluke is Fasciolagigantica.

[0032] Clause 11. The method of any one of Clauses 1-10, wherein the liver fluke infection is an early immature stage liver fluke infection.

[0033] Clause 12. The method of any one of Clauses 1-10, wherein the liver fluke infection is an immature stage liver fluke infection.

[0034] Clause 13. The method of any one of Clauses 1-10, wherein the liver fluke infection is a mature stage liver fluke infection.

[0035] Item 14. Use of a combination of an effective amount of difenitide and an effective amount of clorsulon for treating liver fluke infection in a mammal in need of such treatment.

[0036] Item 15. The use according to Item 14, wherein the mammal is selected from sheep and cattle.

[0037] Clause 16. The use according to Clause 14, wherein the mammal is a sheep.

[0038] Clause 17. The use according to Clause 14, wherein the mammal is a cow.

[0039] Clause 18. The use according to any one of Clauses 14 to 17, wherein the effective amount of difenidide is 20-80 mg / kg, 30-80 mg / kg, 40-80 mg / kg, 50-80 mg / kg, 60-80 mg / kg or 70-80 mg / kg.

[0040] Clause 19. The use according to any one of Clauses 14 to 18, wherein the effective amount of difenidide is 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg or 80 mg / kg.

[0041] Clause 20. The use according to any one of Clauses 14 to 19, wherein the effective amount of clorsulon is 1-10 mg / kg, 1.5-10 mg / kg, 3-10 mg / kg, 5-10 mg / kg or 7-10 mg / kg.

[0042] Clause 21. The use according to any one of Clauses 14 to 20, wherein the effective amount of clorsulon is 1.5 mg / kg, 3.5 mg / kg, 5 mg / kg, 7 mg / kg or 10 mg / kg.

[0043] Clause 22. The use according to any one of Clauses 14 to 21, wherein the liver fluke is Fasciola hepatica.

[0044] Clause 23. The use according to any one of Clauses 14 to 21, wherein the liver fluke is Fasciola gigantica.

[0045] Clause 24. The use according to any one of Clauses 14 to 23, wherein the liver fluke infection is an early immature stage liver fluke infection.

[0046] Clause 25. The use according to any one of Clauses 14 to 23, wherein the liver fluke infection is an immature stage liver fluke infection.

[0047] Clause 26. The use according to any one of Clauses 14 to 23, wherein the liver fluke infection is a mature stage liver fluke infection.

[0048] Clause 27. Use of a combination of an effective amount of difenitide and an effective amount of clorsulon in the preparation of a medicament for treating a liver fluke infection in a mammal in need of such treatment.

[0049] Clause 28. The use according to Clause 27, wherein the mammal is selected from sheep and cattle.

[0050] Clause 29. The use according to Clause 27, wherein the mammal is a sheep.

[0051] Clause 30. The use according to Clause 27, wherein the mammal is a cow.

[0052] Clause 31. The use according to any one of Clauses 27-30, wherein the effective amount of difenidide is 20-80 mg / kg, 30-80 mg / kg, 40-80 mg / kg, 50-80 mg / kg, 60-80 mg / kg or 70-80 mg / kg.

[0053] Clause 32. The use according to any one of Clauses 27 to 31, wherein the effective amount of difenidide is 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg or 80 mg / kg.

[0054] Clause 33. The use according to any one of Clauses 27 to 32, wherein the effective amount of clorsulon is 1-10 mg / kg, 1.5-10 mg / kg, 3-10 mg / kg, 5-10 mg / kg or 7-10 mg / kg.

[0055] Clause 34. The use according to any one of Clauses 27 to 33, wherein the effective amount of clorsulon is 1.5 mg / kg, 3.5 mg / kg, 5 mg / kg, 7 mg / kg or 10 mg / kg.

[0056] Clause 35. The use according to any one of Clauses 27 to 34, wherein the liver fluke is Fasciola hepatica.

[0057] Clause 36. The use according to any one of Clauses 27 to 34, wherein the liver fluke is Fasciola gigantica.

[0058] Clause 37. The use according to any one of Clauses 27 to 36, wherein the liver fluke infection is an early immature stage liver fluke infection.

[0059] Clause 38. The use according to any one of Clauses 27 to 36, wherein the liver fluke infection is an immature stage liver fluke infection.

[0060] Clause 39. The use according to any one of Clauses 27 to 36, wherein the liver fluke infection is a mature stage liver fluke infection.

[0061] Item 40. A synergistic combination of difenitide and clorsulon for use in treating a liver fluke infection in a mammal in need of such treatment.

[0062] Clause 41. The combination of Clause 40, wherein the mammal is selected from the group consisting of sheep and cattle.

[0063] Clause 42. The combination of Clause 40, wherein said mammal is a sheep.

[0064] Clause 43. The combination of Clause 40, wherein said mammal is a bovine.

[0065] Clause 44. The combination of any one of Clauses 40-43 comprising 20-80 mg / kg, 30-80 mg / kg, 40-80 mg / kg, 50-80 mg / kg, 60-80 mg / kg or 70-80 mg / kg of difenidide.

[0066] Clause 45. The combination of any one of Clauses 40 to 44 comprising 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg or 80 mg / kg of difenidide.

[0067] Clause 46. The combination of any one of Clauses 40 to 45 comprising 1-10 mg / kg, 1.5-10 mg / kg, 3-10 mg / kg, 5-10 mg / kg or 7-10 mg / kg clorsulon.

[0068] Clause 47. The combination of any one of Clauses 40 to 46 comprising 1.5 mg / kg, 3.5 mg / kg, 5 mg / kg, 7 mg / kg or 10 mg / kg clorsulon.

[0069] Clause 48. The combination of any one of Clauses 40-47, wherein the liver fluke is Fasciola hepatica.

[0070] Clause 49. The combination of any one of Clauses 40-47, wherein the liver fluke is Fasciola gigantica.

[0071] Clause 50. The combination of any one of Clauses 40-49, wherein the liver fluke infection is an early immature stage liver fluke infection.

[0072] Clause 51. The combination of any one of Clauses 40-49, wherein the liver fluke infection is an immature stage liver fluke infection.

[0073] Clause 52. The combination of any one of Clauses 40-49, wherein the liver fluke infection is a mature stage liver fluke infection.

[0074] Clause 53. The method of any one of clauses 1 to 13, the use of any one of clauses 14 to 39, or the combination of any one of clauses 40 to 52, wherein difenitide and clorsulon are administered sequentially.

[0075] Clause 54. The method of any one of clauses 1 to 13, the use of any one of clauses 14 to 39, or the combination of any one of clauses 40 to 52, wherein difenitide and clorsulon are administered simultaneously.

[0076] Clause 55. The method of any one of Clauses 1-4, wherein the effective amount of difenidide is 30-60 mg / kg, 40-60 mg / kg or 40-50 mg / kg.

[0077] Clause 56. The method of Clause 55, wherein the effective amount of difenidide is 30 mg / kg, 40 mg / kg, 50 mg / kg or 60 mg / kg.

[0078] Clause 57. The method of Clause 55 or Clause 56, wherein the effective amount of clorsulon is 3-10 mg / kg, 5-10 mg / kg, 6-10 mg / kg or 7-10 mg / kg.

[0079] Clause 58. The method of any one of Clauses 55-57, wherein the effective amount of clorsulon is 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg.

[0080] Clause 59. The method according to Clause 3, wherein the effective amount of difenidide is 20-60 mg / kg, and the effective amount of clorsulon is 5-10 mg / kg.

[0081] Clause 60. The method of Clause 4, wherein the effective amount of difenidide is 40-80 mg / kg and the effective amount of clorsulon is 5-10 mg / kg.

[0082] Clause 61. The method of any one of Clauses 55-60, wherein the liver fluke is Fasciola hepatica.

[0083] Clause 62. The method of any one of Clauses 55-60, wherein the liver fluke is Fasciola gigantica.

[0084] Clause 63. The method of any one of Clauses 55-60, wherein the liver fluke infection is an early immature stage liver fluke infection.

[0085] Clause 64. The method of any one of Clauses 55-60, wherein the liver fluke infection is an immature stage liver fluke infection.

[0086] Clause 65. The method of any one of Clauses 55-60, wherein the liver fluke infection is a mature stage liver fluke infection.

[0087] Clause 66. The use according to any one of Clauses 14 to 17, wherein the effective amount of difenidide is 30-60 mg / kg, 40-60 mg / kg or 40-50 mg / kg.

[0088] Clause 67. The use according to Clause 66, wherein the effective amount of difenidide is 30 mg / kg, 40 mg / kg, 50 mg / kg or 60 mg / kg.

[0089] Clause 68. The use according to Clause 66 or Clause 67, wherein the effective amount of clorsulon is 3-10 mg / kg, 5-10 mg / kg, 6-10 mg / kg or 7-10 mg / kg.

[0090] Clause 69. The use according to any one of Clauses 66-68, wherein the effective amount of clorsulon is 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg.

[0091] Clause 70. The use according to Clause 16, wherein the effective amount of difenidide is 20-60 mg / kg and the effective amount of clorsulon is 5-10 mg / kg.

[0092] Clause 71. The use according to Clause 17, wherein the effective amount of difenidide is 40-80 mg / kg and the effective amount of clorsulon is 5-10 mg / kg.

[0093] Clause 72. The use according to any one of Clauses 66 to 71, wherein the liver fluke is Fasciola hepatica.

[0094] Clause 73. The use according to any one of Clauses 66 to 71, wherein the liver fluke is Fasciola gigantica.

[0095] Clause 74. The use of any one of Clauses 66-71, wherein the liver fluke infection is an early immature stage liver fluke infection.

[0096] Clause 75. The use of any one of Clauses 66-71, wherein the liver fluke infection is an immature stage liver fluke infection.

[0097] Clause 76. The use of any one of Clauses 66-71, wherein the liver fluke infection is a mature stage liver fluke infection.

[0098] Clause 77. The use according to any one of Clauses 27 to 30, wherein the effective amount of difenidide is 30-60 mg / kg, 40-60 mg / kg or 40-50 mg / kg.

[0099] Clause 78. The use according to Clause 77, wherein the effective amount of difenidide is 30 mg / kg, 40 mg / kg, 50 mg / kg or 60 mg / kg.

[0100] Clause 79. The use according to Clause 77 or Clause 78, wherein the effective amount of clorsulon is 3-10 mg / kg, 5-10 mg / kg, 6-10 mg / kg or 7-10 mg / kg.

[0101] Clause 80. The use according to any one of Clauses 77-79, wherein the effective amount of clorsulon is 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg.

[0102] Clause 81. The use according to Clause 29, wherein the effective amount of difenidide is 20-60 mg / kg and the effective amount of clorsulon is 5-10 mg / kg.

[0103] Clause 82. The use according to Clause 30, wherein the effective amount of difenidide is 40-80 mg / kg and the effective amount of clorsulon is 5-10 mg / kg.

[0104] Clause 83. The use according to any one of Clauses 77 to 82, wherein the liver fluke is Fasciola hepatica.

[0105] Clause 84. The use according to any one of Clauses 77 to 82, wherein the liver fluke is Fasciola gigantica.

[0106] Clause 85. The use of any one of Clauses 77-82, wherein the liver fluke infection is an early immature stage liver fluke infection.

[0107] Clause 86. The use of any one of Clauses 77-82, wherein the liver fluke infection is an immature stage liver fluke infection.

[0108] Clause 87. The use of any one of Clauses 77-82, wherein the liver fluke infection is a mature stage liver fluke infection.

[0109] Clause 88. The combination of any one of Clauses 40-43, wherein the effective amount of difenidide is 30-60 mg / kg, 40-60 mg / kg or 40-50 mg / kg.

[0110] Clause 89. The combination of Clause 88, wherein the effective amount of difenidide is 30 mg / kg, 40 mg / kg, 50 mg / kg or 60 mg / kg.

[0111] Clause 90. The combination of Clause 88 or Clause 89, wherein the effective amount of clorsulon is 3-10 mg / kg, 5-10 mg / kg, 6-10 mg / kg or 7-10 mg / kg.

[0112] Clause 91. The combination of any one of clauses 88-90, wherein the effective amount of clorsulon is 3 mg / kg, 4, mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg.

[0113] Clause 92. The combination of Clause 42, wherein the effective amount of difenidide is 20-60 mg / kg (eg, 35 mg / kg) and the effective amount of clorsulon is 5-10 mg / kg (eg, 5 mg / kg).

[0114] Clause 93. The combination of Clause 43, wherein the effective amount of difenidide is 40-80 mg / kg (eg, 70 mg / kg) and the effective amount of clorsulon is 5-10 mg / kg (eg, 10 mg / kg).

[0115] Clause 94. The combination of any one of Clauses 88-93, wherein the liver fluke is Fasciola hepatica.

[0116] Clause 95. The combination of any one of Clauses 88-93, wherein the liver fluke is Fasciola gigantica.

[0117] Clause 96. The combination of any one of Clauses 88-93, wherein the liver fluke infection is an early immature stage liver fluke infection.

[0118] Clause 97. The combination of any one of Clauses 88-93, wherein the liver fluke infection is an immature stage liver fluke infection.

[0119] Clause 98. The combination of any one of Clauses 88-93, wherein the liver fluke infection is a mature stage liver fluke infection.

[0120] Clause 99. The method of any one of clauses 55-60, the use of any one of clauses 66-71 or 77-82 or the combination of any one of clauses 88-93, wherein difenidide and clorsulon are administered sequentially.

[0121] Clause 100. The method of any one of clauses 55 to 60, the use of any one of clauses 14 to 39, or the combination of any one of clauses 88 to 93, wherein difenitide and clorsulon are administered simultaneously.

[0122] Clause 101. The method of clause 1, the use of clause 14 or clause 27, or the combination of clause 40, wherein the effective amount of difenidide is 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 60 mg / kg or more, 70 mg / kg or more, 80 mg / kg or more, 90 mg / kg or more, 100 mg / kg or more, 110 mg / kg or more, or 120 mg / kg or more.

[0123] Clause 102. The method of clause 1, the use of clause 14 or clause 27, or the combination of clause 40, wherein the effective amount of difenidide is 120 mg / kg or less, 110 mg / kg or less, 90 mg / kg or less, 80 mg / kg or less, 70 mg / kg or less, 60 mg / kg or less, 50 mg / kg or less, 40 mg / kg or less, 30 mg / kg or less, or 20 mg / kg or less.

[0124] Clause 103. The method of clause 1, 101 or 102, the use of clause 14, 27, 101 or 102, or the combination of clause 40, 101 or 102, wherein the effective amount of clorsulon is 1 mg / kg or more, 1.5 mg / kg or more, 2 mg / kg or more, 2.5 mg / kg or more, 3 mg / kg or more, 3.5 mg / kg or more, 4 mg / kg or more, 4.5 mg / kg or more, 5 mg / kg or more, 5.5 mg / kg or more, 6 mg / kg or more, 6.5 mg / kg or more, 7 mg / kg or more, 7.5 mg / kg or more, 8 mg / kg or more, 8.5 mg / kg or more, 9 mg / kg or more, 9.5 mg / kg or more, or 10 mg / kg or more.

[0125] Clause 104. The method of clause 1, 101 or 102, the use of clause 14, 27, 101 or 102, or the combination of clause 40, 101 or 102, wherein the effective amount of clorsulon is 10 mg / kg or less, 9.5 mg / kg or less, 9 mg / kg or less, 8.5 mg / kg or less, 8 mg / kg or less, 7.5 mg / kg or less, 7 mg / kg or less, 6.5 mg / kg or less, 6 mg / kg or less, 5.5 mg / kg or less, 5 mg / kg or less, 4.5 mg / kg or less, 4 mg / kg or less, 3.5 mg / kg or less, 3 mg / kg or less, 2.5 mg / kg or less, 2 mg / kg or less, 1.5 mg / kg or less, or 1 mg / kg or less.

[0126] Clause 105. The method of Clause 1, the use of Clause 14 or Clause 27, or the combination of Clause 40, wherein the effective amount of difenidide is 20-80 mg / kg and the effective amount of clorsulon is 3.5-10 mg / kg.

[0127] Clause 106. The method of any one of clauses 101-105, the use of any one of clauses 101-105, or the combination of any one of clauses 101-105, wherein the mammal is a sheep.

[0128] Clause 107. The method of any one of clauses 101-105, the use of any one of clauses 101-105, or the combination of any one of clauses 101-105, wherein the mammal is a cow.

[0129] Clause 108. The method, use or combination of Clause 106, wherein the effective amount of difenidide is 35 mg / kg and the effective amount of clorsulon is 5 mg / kg.

[0130] Clause 109. The method, use or combination of Clause 107, wherein the effective amount of difenidide is 70 mg / kg and the effective amount of clorsulon is 10 mg / kg.

[0131] Clause 110. The method of any one of clauses 101-109, the use of any one of clauses 101-109, or the combination of any one of clauses 101-109, wherein the liver fluke is Fasciola hepatica.

[0132] Clause 111. The method of any one of clauses 101-109, the use of any one of clauses 101-109, or the combination of any one of clauses 101-109, wherein the liver fluke is Fasciolagigantica.

[0133] Clause 112. The method of any one of clauses 101-111, the use of any one of clauses 101-111, or the combination of any one of clauses 101-111, wherein the liver fluke infection is an early immature stage liver fluke infection.

[0134] Clause 113. The method of any one of clauses 101-111, the use of any one of clauses 101-111, or the combination of any one of clauses 101-111, wherein the liver fluke infection is an immature stage liver fluke infection.

[0135] Clause 114. The method of any one of clauses 101-111, the use of any one of clauses 101-111, or the combination of any one of clauses 101-111, wherein the liver fluke infection is a mature stage liver fluke infection.

[0136] Clause 115. The method, use or combination of any of the preceding clauses, wherein the method, use or combination is 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more effective in treating liver fluke infection.

[0137] Clause 116. The method, use or combination as described in any of the preceding clauses, wherein the therapeutic efficacy of said method, use or combination is 85-99%.

[0138] Clause 117. The method, use or combination as described in any of the preceding clauses, wherein the therapeutic effect of said method, use or combination is 85-96%.

[0139] Clause 118. The method, use or combination of any one of clauses 115 to 117, wherein the assessment of therapeutic efficacy is based on the number of liver flukes expelled from the liver of the mammal compared to an untreated control.

[0140] Clause 119. The method, use or combination of clause 118, wherein the liver having the gall bladder and common bile duct is evaluated 40-80 days after treatment (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 days after treatment).

[0141] The following examples demonstrate the improved therapeutic efficacy of the combination of difenidone and clorsulon in sheep and cattle. It should be understood that these examples are intended to be illustrative and not limiting.

[0142] In the following examples, all animals were confirmed free of Fasciola hepatica infection and were assessed as clinically healthy by a veterinarian prior to inclusion in the study. The dosage was administered orally using a disposable syringe of appropriate size.

[0143] Trematode counts and body weights were transformed to logarithms (only after adding a constant of 1 to the trematode counts), and the distribution of the transformed data was examined to determine whether it met the assumption of normality. Analysis of variance (ANOVA) was used to analyze differences between treatment groups, as the log-transformed trematode counts and log-transformed body weights as well as the (untransformed) trematode measurements met the assumption of normality.

[0144] The arithmetic mean values ​​of trematode counts, trematode measurements, and body weights were summarized (A 平均值 ), geometric mean (G 平均值 ), median and range. A constant of 1 was used to calculate the geometric mean of the trematode counts.

[0145] The percentage efficacy of each treatment group was calculated as follows:

[0146] Efficacy % = 100 × (FC C -FC T ) / FC C

[0147] Among them, FC C is the arithmetic mean of the trematode counts in the control group, FC T is the arithmetic mean of the fluke counts in the treatment groups. This formula can also be implemented using the geometric mean of the fluke counts; these are calculated using log-transformed data, adding 1 as a constant to each fluke count and then subtracting it after transformation.

[0148] A synergistic effect can be concluded in the following cases:

[0149] (1-Efficacy D+C )<(1–Efficacy D )×(1–EfficacyC )

[0150] Where D = difenidol, C = clorsulon, and the efficacy is measured as an absolute number (not %).

[0151] Note that expected synergy is defined as:

[0152] 1-Efficacy (no synergy expected) = (1-Efficacy D )×(1–Efficacy C ).

[0153] Example 1

[0154] A representative difenidide and clorsulon formulation is provided in Example 1. A suspension of difenidide is prepared as follows: Place propylene glycol in a glass beaker. Add the two parabens and stir until dissolved. Add polysorbate 80, antifoam emulsion, and water and stir until homogeneous. Very slowly add Avicel RC591 while stirring. Then use Ultra Homogenize for a few minutes, slowly add difenac while stirring, and use Ultra again. Homogenize for at least 10 minutes to obtain a suspension with the following composition:

[0155] 1. Difenidol 15% (w / v)

[0156] 2. Propylene glycol 5% (w / v)

[0157] 3. Methyl paraben 0.11% (w / v)

[0158] 4. Propylparaben 0.04% (w / v)

[0159] 5. Polysorbate 80 0.5% (w / v)

[0160] 6. Avicel RC 591 1.2% (w / v)

[0161] 7. Antifoaming emulsion 0.6% (w / v)

[0162] 8. Add demineralized water to 100.

[0163] A suspension of clorsulon was prepared as follows: Heat water, add the two parabens, and stir until dissolved. Cool to room temperature, add SDS and antifoam emulsion, and stir until homogeneous. Very slowly add Avicel RC591 while stirring. Then use Ultra- Homogenize for a few minutes

[0164] Slowly add clorsulon while stirring and use Ultra Homogenize again for at least 10 minutes to obtain a suspension with the following composition:

[0165] 1. Clorsulon 3.5% (w / v)

[0166] 2. Methyl paraben 0.11% (w / v)

[0167] 3. Propylparaben 0.04% (w / v)

[0168] 4. SDS (sodium dodecyl sulfate) 0.1% (w / v)

[0169] 5. Avicel RC 591 1.2% (w / v)

[0170] 6. Antifoaming emulsion 0.5% (w / v)

[0171] 7. Add demineralized water to 100.

[0172] Example 2

[0173] Concurrent treatment with difenitide and clorsulon

[0174] Evaluation of synergistic effects against immature stages of Fasciola hepatica infection in sheep

[0175] This study was designed to evaluate the efficacy of difenidone (40 mg / kg) combined with clorsulon (3.5 mg / kg), difenidone alone (40 mg / kg), and clorsulon alone (3.5 mg / kg) against Fasciola hepatica in sheep at day 42. This was a controlled, blinded, randomized study using 3-4 month old Merino lambs.

[0176] On day 0, sheep were experimentally infected with 200 metacercariae of Fasciola hepatica "Sunny Corner" (laboratory strain reference number Fashep-66). The number of metacercariae required for infection was prepared in a solution of water and 4% carboxymethylcellulose. Water was administered orally after each infectious dose. Syringes were then checked for residual metacercariae after infection and the dose given to sheep was determined to be 93.5% to 100% (average 98.3%) of the target infectious dose.

[0177] Sheep were randomly divided into treatment groups according to body weight on day 41 and dosed as shown in Table 2.1.

[0178] Table 2.1

[0179] Group treat Dosage (mg / kg) n 1 No treatment - 5 2 Diphenhydramine 40 5 3 Closulon 3.5 5 4 Difenitide and clorsulon 40 and 3.5 5

[0180] Body weight was measured one day before treatment, and the volume was determined and rounded to the nearest 0.1 mL. On day 42, before feeding, individual animals were treated only once according to the treatment groups in Table 2.1. Animals were returned to their pens after treatment and fed within 1 hour. Untreated control animals were fed at the same time.

[0181] The liver with gall bladder and intact common bile duct was collected from each animal and processed for F. hepatica counts on day 91. The efficacy was evaluated based on the number of F. hepatica removed from the liver.

[0182] An ANOVA model was used to compare fluke counts between treatment groups. The model was applied to the logarithmic transformation of the counts. Treatment group had one fixed effect (treatment group).

[0183] All control sheep in Group 1 were positive for Fasciola hepatica infection with a geometric mean of 87.02. The percentage efficacy of the treated sheep was compared with the percentage efficacy of the sheep in the untreated control group as shown in Table 2.2 below.

[0184] Table 2.2

[0185] Group treat <![CDATA[Fluke count 1 > <![CDATA[Therapeutic efficacy % 2 > <![CDATA[Fluke count 3 > <![CDATA[Therapeutic efficacy % 4 > 1 No treatment control 90.2 – 87.0 – 2 Diphenhydramine 57.6 36.1 50.9 41.5 3 Closulon 81.8 9.3 79.7 8.4 4 Difenitide and clorsulon 18.2 79.8 11.3 87.0

[0186] 1 Arithmetic mean; 2 The arithmetic mean is used; 3 Geometric mean; 4 Using geometric mean

[0187] As shown in Table 2.3, pairwise comparisons showed that the fluke counts in the concurrently treated groups were significantly reduced compared to the untreated group and the single agent treated groups.

[0188] Table 2.3

[0189]

[0190] The expected and observed efficacy % of concurrent treatment against Fasciola hepatica is shown in Table 2.4.

[0191] Table 2.4

[0192]

[0193] The observed efficacy of concurrent treatment with difenidone and clorsulon was higher than the expected additive effect, demonstrating their synergistic effect.

[0194] Example 3

[0195] Difenitide and clorsulon are administered simultaneously

[0196] Evaluation of synergistic effects against early immature stages of Fasciola hepatica infection in sheep

[0197] This study evaluated the efficacy of difenidone (20 mg / kg) combined with clorsulon (10 mg / kg), difenidone alone (20 mg / kg), and clorsulon alone (10 mg / kg) against Fasciola hepatica in sheep at day 14. This was a controlled, blinded, randomized study using sheep.

[0198] Sheep were experimentally infected with 200 metacercariae of Fasciola hepatica "Sunny Corner" (laboratory strain reference number Fashep-66) on day 0. The number of metacercariae required for infection was prepared in water and 2% carboxymethylcellulose solution. Water was administered orally after each infection dose.

[0199] Sheep were randomized into treatment groups based on body weight on day 13 and dosed as shown in Table 3.1. Animals were treated with a single dose on day 14 as shown in Table 3.1.

[0200] Table 3.1

[0201]

[0202]

[0203] Body weight was measured one day before treatment, and the volume was determined and rounded to 0.1 mL. On day 14, before feeding, individual animals were treated only once according to the treatment groups in Table 3.1. After treatment, the animals were returned to their pens and fed. At the same time, untreated control animals were fed.

[0204] The liver with gall bladder and intact common bile duct was collected from each animal and processed for F. hepatica counts on day 91. The efficacy was evaluated based on the number of F. hepatica removed from the liver.

[0205] An ANOVA model was used to compare fluke counts between treatment groups. The model was applied to the logarithmic transformation of the counts. Treatment group had a fixed effect.

[0206] All control sheep in Group 1 were positive for Fasciola hepatica infection with a geometric mean of 137.3. The percentage efficacy of the treated sheep was compared with the percentage efficacy of the sheep in the untreated control group as shown in Table 3.2 below.

[0207] Table 3.2

[0208]

[0209] 1 Arithmetic mean; 2 The arithmetic mean is used;3 Geometric mean; 4 Using geometric mean

[0210] As shown in Table 3.3, in terms of fluke counts, pairwise comparisons indicated that fluke counts were significantly lower in the concurrently treated groups compared with the untreated group and the single-agent treated groups, whereas there were no significant differences between the single active treatments.

[0211] Table 3.3

[0212]

[0213] The expected and observed efficacy % of concurrent treatment against Fasciola hepatica is shown in Table 3.4.

[0214] Table 3.4

[0215]

[0216] The observed efficacy of concurrent treatment with difenidone and clorsulon was higher than the expected additive effect, demonstrating their synergistic effect.

[0217] Example 4

[0218] Difenitide and clorsulon are administered simultaneously

[0219] Evaluation of synergistic effects against immature stages of Fasciola hepatica infection in cattle

[0220] This study was designed to evaluate the efficacy of difenidone (60 mg / kg) combined with clorsulon (5 mg / kg), difenidone alone (60 mg / kg), and clorsulon alone (5 mg / kg) against Fasciola hepatica in cattle at day 49. This was a controlled, blinded, randomized study.

[0221] On day 0, cattle were experimentally infected with a single isolate of approximately 382 Fasciola hepatica metacercariae from the Sunny Corner (Fashep-66) isolate. The required number of metacercariae was prepared in 2% carboxymethylcellulose. Syringes were checked for residual metacercariae after infection. Infectious material was administered orally, with water following each infectious dose. Cattle were randomized into treatment groups based on body weight on day 48 and dosed as shown in Table 4.1.

[0222] Table 4.1

[0223] Group treat Dosage (mg / kg) n 1 No treatment - 5 2 Diphenhydramine 60 5 3 Closulon 5 5 4 Difenitide and clorsulon 60 and 5 5

[0224] Body weight was measured one day before treatment. All treatments were measured by volume and rounded to the nearest 0.1 mL. On day 49, before feeding, individual animals were treated only once according to the treatment groups in Table 4.1. Animals were returned to their pens after treatment and fed within 1 hour. Untreated control animals were fed at the same time.

[0225] The liver with gall bladder and intact common bile duct was collected from each animal and processed for F. hepatica counts on day 91 or 92. The efficacy was evaluated based on the number of F. hepatica removed from the liver.

[0226] The percent efficacy of the treated cattle was compared with the percent efficacy of cattle in the untreated control group as shown in Table 4.2 below.

[0227] Table 4.2

[0228] Group treat <![CDATA[Fluke count 1 > <![CDATA[Therapeutic effect % 2 > <![CDATA[Fluke count 3 > <![CDATA[Therapeutic efficacy % 4 > 1 No treatment control 84.0 – 83.5 – 2 Diphenhydramine 73.8 12.1 61.8 26.0 3 Closulon 72.6 13.6 70.7 15.3 4 Difenitide and clorsulon 5.2 93.8 4.5 94.6

[0229] 1 Arithmetic mean; 2 The arithmetic mean is used; 3 Geometric mean; 4 Using geometric mean

[0230] As shown in Table 4.3, with respect to fluke counts, pairwise comparisons indicated that fluke counts were significantly lower in the concurrently treated groups compared to the untreated group and the single agent treated groups, and the single active treatments were not significantly different from each other.

[0231] Table 4.3

[0232]

[0233] The expected and observed % efficacy of concurrent treatment against Fasciola hepatica is shown in Table 4.4.

[0234] Table 4.4

[0235]

[0236] The observed efficacy of concurrent treatment with difenidone and clorsulon was higher than the expected additive effect, demonstrating their synergistic effect.

[0237] Example 5

[0238] Difenitide and clorsulon are administered simultaneously

[0239] Evaluation of synergistic effects against early immature stages of Fasciola hepatica infection in cattle

[0240] This study evaluated the efficacy of difenidone (40 mg / kg) combined with clorsulon (7 mg / kg), difenidone alone (40 mg / kg), and clorsulon alone (7 mg / kg) against Fasciola hepatica in cattle at day 21. This was a controlled, blinded, randomized study.

[0241] On day 0, cattle were experimentally infected with a single isolate of approximately 350 Fasciola hepatica metacercariae from the Sunny Corner (Fashep-66) isolate. The required number of metacercariae was prepared in 2% carboxymethylcellulose. Syringes were checked for residual metacercariae after infection. Infectious material was administered orally, with water following each infectious dose. Cattle were randomized into treatment groups based on body weight on day 20 and dosed as shown in Table 5.1.

[0242] Table 5.1

[0243] Group treat Dosage (mg / kg) n 1 No treatment - 5 2 Diphenhydramine 40 5 3 Closulon 7 5 4 Difenitide and clorsulon 40 and 7 5

[0244] Body weight was measured one day before treatment. All treatments were measured by volume and rounded to the nearest 0.1 mL. On day 21, before feeding, individual animals were treated only once according to the treatment groups in Table 4.1. Animals were returned to their pens after treatment and fed within 1 hour. Untreated control animals were fed at the same time.

[0245] The liver with gall bladder and intact common bile duct was collected from each animal and processed for F. hepatica counts on day 91 or 92. The efficacy was evaluated based on the number of F. hepatica removed from the liver.

[0246] The percent efficacy of the treated cattle was compared with the percent efficacy of cattle in the untreated control group as shown in Table 5.2 below.

[0247] Table 5.2

[0248] Group treat <![CDATA[Fluke count 1 > <![CDATA[Therapeutic efficacy % 2 > <![CDATA[Fluke count 3 > <![CDATA[Therapeutic efficacy % 4 > 1 No treatment control 78.2 – 73.2 – 2 Diphenhydramine 54.2 30.7 53.1 27.5 3 Closulon 45.2 42.2 42.4 42.1 4 Difenitide and clorsulon 3.8 95.1 2.5 96.6

[0249] 1 Arithmetic mean; 2 The arithmetic mean is used; 3 Geometric mean; 4 Using geometric mean

[0250] As shown in Table 5.3, with respect to fluke counts, pairwise comparisons indicated that fluke counts were significantly lower in the concurrently treated groups compared to the untreated group and the single agent treated groups, and the single active treatments were not significantly different from each other.

[0251] Table 5.3

[0252]

[0253]

[0254] The expected and observed % efficacy of concurrent treatment against Fasciola hepatica is shown in Table 5.4.

[0255] Table 5.4

[0256]

[0257] The observed efficacy of concurrent treatment with difenidone and clorsulon was higher than the expected additive effect, demonstrating their synergistic effect.

Claims

1. A method of treating a liver fluke infection in a mammal in need of such treatment, the method comprising administering an effective amount of difenitide in combination with an effective amount of clorsulon.

2. The method of claim 1, wherein: The mammal is selected from sheep and cattle.

3. The method of claim 1, wherein: The mammal is a sheep.

4. The method of claim 1, wherein: The mammal is a cow.

5. The method according to any one of claims 1 to 4, wherein: The effective amount of difenidide is 20-80 mg / kg.

6. The method according to any one of claims 1 to 5, wherein: The effective dose of difenidone is 20mg / kg, 30mg / kg, 40mg / kg, 50mg / kg, 60mg / kg, 70 mg / kg or 80mg / kg.

7. The method according to any one of claims 1 to 6, wherein: The effective amount of clorsulon is 3-10 mg / kg.

8. The method according to any one of claims 1 to 7, wherein: The effective amount of clorsulon is 3.5 mg / kg, 5 mg / kg, 7 mg / kg or 10 mg / kg.

9. The method according to any one of claims 1 to 8, wherein: The liver fluke is Fasciola hepatica.

10. The method according to any one of claims 1 to 8, wherein: The liver fluke is Fasciolagigantica.

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