This invention relates to combinations, especially synergistic
combinations of an avermectin or a milbemycin which is selectamin with a bis-aryl
compounds which is fipronil and their use in combating parasite, especially helminth,
The avermectins are a group of broad spectrum antiparasitic
agents referred to previously as the C-076 compounds. They are produced by fermenting
a strain of the micro-organism Streptomyces avermitilis under aerobic conditions
in an aqueous nutrient medium containing inorganic salts and assimilable sources
of carbon and nitrogen. The isolation and the chemical structure of the eight individual
components which make up the C-076 complex is described in detail in
British Patent Specification 1573955
The C-076 complex comprises eight distinct but closely
related compounds described as C-076 A1a, A1b, A2a, A2b, B1a, B1b, B2a and B2b.
The "a" series of compounds refers to the natural avermectins wherein the 25-substituent
is (S)-sec-butyl and the "b" series to those wherein the 25-substituent is isopropyl.
The designations "A" and "B" refer to avermectins wherein the 5-substituent is methoxy
or hydroxy, respectively, and the numeral "1" refers to avermectins wherein a double
bond is present at the 22-23 position, and numeral "2" to avermectins wherein there
is a single bond between C-22 and C-23 and wherein there is a 23-hydroxy substituent.
European Patent Applications 0214731
there are described preparations of compounds related to the naturally-occurring
avermectins but having a group at the 25-position other than the isopropyl or (S)-sec-butyl
groups found in the original avermectin compounds disclosed in
British Patent Specification 1573955
. Such compounds may be prepared by fermentation of particular strains
of Streptomyces avermitilis in the presence of organic acids or derivatives
thereof. Production of such avermectins is described in
Journal of Antibiotics (1991), 44, No. 3, pp 357-365
The milbemycins form another group of related macrolides
which are distinguished from the avermectins in lacking a sugar residue attached
at the C-13 position. Examples of such compounds are described in
UK patent 1390336
European patent publications 170006
. In addition to these fermentation products a large number of publications
describe compounds derived semisynthetically from them, many of which possess useful
antiparasitic activities. Some of this chemistry is reviewed in
Macrolide Antibiotics, Omura S., Ed., Academic press, New York (1984
) and by
Davies, H.G., Green, R.H. in Natural product Reports (1986), 3, 87-121
Chem. Soc. Rev., 1991, 20, 271-339
. It is known that the avermectins and milbemycins and their derivatives
are active as antiparasitic agents.
Other avermectin and milbemycin derivatives which may be
mentioned are disclosed in the following patents, applications, and their equivalents:
European - 214 731
410 615 623 137
The terms avermectins and milbemycins used herein includes
both naturally occurring compounds and synthetic derivatives thereof, especially
those mentioned in the art cited herein.
Bis-aryl compounds having insecticidal and acaricidal properties
are also known. These compounds comprise directly joined aromatic rings which may
include at least one nitrogen atom. Examples of this class of compounds include
N-phenyl pyrazole derivatives having arthropodicidal, plant nematodicidal anthelmintic
and anti-protozoal properties.
describes compositions containing a 1-phenyl pyrazole derivative and a
macrocyclic lactone endectoparasiticide.
Cutler, S. L. in the Journal of Small Animal Practice, (1998 Feb) 39 (2) 86-7
., "Ectopic Psoroptes cuniculi infestation in a pet rabbit.", discusses
treatment with ivermectin injection followed by fipronil application.
It has now been found that certain avermectins and milbemycins
display unexpected synergy with members of the above-mentioned bis-aryl compounds
with respect to their antiparasitic activity, allowing more effective control of
parasites, especially those affecting livestock and companion animals. Examples
of such parasites incude heartworm and various ectoparasites in companion animals.
This is particularly unexpected as impairment of biological performance would be
anticipated for such a combination, given the reported mode of action of avermectin
and milbemycin compounds as agonists at the invertebrate glutamate gated chloride
channel- receptor complex and the antagonist activity of the bis-aryl compounds
at this site.
According to one aspect of the invention, there is provided
a composition comprising a combination of a bis-aryl compound and an avermectin
or milbemycin or derivative thereof, and if necessary a suitable pharmaceutical
or veterinary carrier, wherein the bis-aryl compound is fipronil and the avermectin
or milbemycin is selamectin. The invention also includes the use of such a composition
in medicine, for instance for treating or preventing parasitic infestations in humans
or animals, including infestation by heartworm and other parasites in companion
animals such as dogs and cats.
Another aspect of the invention is the administration to
an animal of a bis-aryl as defined above compound and an avermectin or milbemycin
as defined above either in combination in the same composition, or via separate
treatments, either substantially simultaneously or at spaced intervals, for treating
or preventing a parasitic infestation. Different modes of administration can be
envisaged by the skilled person where the bis-aryl compound is administered separately
from the avermectin or milbemycin compound.
Another aspect to the invention is the use of a composition
comprising an avermectin or milbemycin as defined above and a bis-aryl compound
as defined above in the manufacture of an antiparasitic medicament.
Another aspect of the invention is a pharmaceutical pack
comprising a bis-aryl compound as defined above and an avermectin or milbemycin
as defined above.
For use in mammals, including humans, the compounds, either
alone or in the combinations mentioned above, can be administered alone, but will
generally be administered in admixture with a pharmaceutically or veterinarily acceptable
diluent or carrier selected with regard to the intended route of administration
and standard pharmaceutical practice. For example, they can be administered orally,
including sublingually, in the form of tablets containing such excipients as starch
or lactose, or in capsules or ovules either alone or in admixture with excipients,
or in the form of elixirs, solutions or suspensions containing flavouring or colouring
agents. The compounds could be incorporated into capsules or tablets for targetting
the colon or duodenum via delayed dissolution of said capsules or tablets for a
particular time following oral administration. Dissolution could be controlled by
susceptibility of the formulation to bacteria found in the duodenum or colon, so
that no substantial dissolution takes places before reaching the target area of
the gastrointestinal tract. The compounds can be injected parenterally, for example,
intravenously, intramuscularly or subcutaneously. For parenteral administration,
they are best used in the form of a sterile aqueous solution or suspension which
may contain other substances, for example, enough salt or glucose to make the solution
isotonic with blood. They can be administered topically, in the form of sterile
creams, gels, suspensions, lotions, ointments, dusting powders, sprays, drug-incorporated
dressings or via a skin patch. For example they can be incorporated into a cream
consisting of an aqueous or oily emulsion of polyethylene glycols or liquid paraffin,
or they can be incorporated into an ointment consisting of a white wax soft paraffin
base, or as hydrogel with cellulose or polyacrylate derivatives or other viscosity
modifiers, or as a dry powder or liquid spray or aerosol with butane/propane, HFA
or CFC propellants, or as a drug-incorporated dressing either as a tulle dressing,
with white soft paraffin or polyethylene glycols impregnated gauze dressings or
with hydrogel, hydrocolloid, alginate or film dressings. The compounds could also
be administered intraocularly as an eye drop with appropriate buffers, viscosity
modifiers (e.g. cellulose derivatives), preservatives (e.g. benzalkonium chloride
(BZK)) and agents to adjust tenicity (e.g. sodium chloride). Such formulation techniques
are well-known in the art. In some instances the formulations may advantageously
also contain an antibiotic. All such formulations may also contain appropriate stabilisers
For veterinary use, compounds can be administered as a
suitably acceptable formulation in accordance with normal veterinary practice and
the veterinary surgeon will determine the dosing regimen and route of administration
which will be most appropriate for a particular animal.
For topical application dip, spray, powder, dust, pour-on,
spot-on, emulsifiable concentrate, jetting fluid, shampoos, collar, tag or harness
may be used. Such formulations are prepared in a conventional manner in accordance
with standard veterinary and pharmaceutical practice. Thus capsules, boluses or
tablets may be prepared by mixing the active ingredient with a suitable finely divided
diluent or carrier, additionally containing a disintegrating agent and/or binder
such as starch, lactose, talc, or magnesium stearate. A drench formulation may be
prepared by dispersing the active ingredients in an aqueous solution together with
dispersing or wetting agents and injectable formulations may be prepared in the
form of a sterile solution or emulsion. Pour-on or spot-on formulations may be prepared
by dissolving the active ingredients in an acceptable liquid carrier vehicle, such
as butyl digol, liquid paraffin or non-volatile ester with or without addition of
a volatile component such as isopropanol.
Alternatively, pour-on, spot-on or spray formulations can
be prepared by encapsulation to leave a residue of active agent on the surface of
the animal. These formulations will vary with regard to the weight of active compound
depending on the species of host animal to be treated, the severity and type of
infection and type and body weight of the host. The combinations may be administered
continuously, particularly for prophylaxis by known methods. Generally for oral,
parenteral and pour-on administration a dose of from about 0.001 to 10mg per kg
of animal body weight given as a single dose or in divided doses for a period of
from 1 to 5 days will be satisfactory but of course there can be instances where
higher or lower dosage ranges are indicated and such are within the scope of this
As an alternative the combinations may be administered
with the animal feedstuff and for this purpose a concentrated feed additive or premix
may be prepared for mixing with the normal animal feed.
For use as an insecticide and for treating agricultural
pests the compounds are applied as sprays, dusts, pour-on formulations, emulsions
and the like in accordance with standard agricultural practice.
For human use the combinations are administered as a pharmaceutically
acceptable formulation in accordance with normal medical practice.
The combinations of compounds of the invention may be formulated
as described above as a mixture, alternatively the avermectin or milbemycin compound
and the bis-aryl compound may be administered as separate doses and such treatment
is still within the scope of the invention.
The compositions, treatments, etc, of the invention may
be combined with other agents, treatments, etc. useful against certain other diseases
or in the reduction or suppression of other symptoms. Examples of such agents (which
are provided by way of illustration and should not be construed as limiting) include
other antiparasitics, eg lufenuron, imidacloprid, organophosphates, pyrethroids;
antihistamines, eg chlorpheniramine, trimeprazine, diphenhydramine, doxylamine;
antifungals, eg fluconazole, ketoconazole, itraconazole, griseofulvin, amphotericin
B; antibacterials, eg enroflaxacin, marbofloxacin, ampicillin, amoxycillin; anti-inflammatories
eg prednisolone, betamethasone, dexamethasone, carprofen, ketoprofen; dietary supplements,
eg gamma-linoleic acid; and emollients. Therefore, the invention further provides
a product containing a compound of the invention and one or more selected compounds
from the above list as a combined preparation for simultaneous, separate or sequential
use in the treatment of conditions mediated by parasites, and the related methods
of treatment, etc.
The avermectin or milbemycin is selamectin described in
International Patent Application PCT/EP94/00095
The bis-aryl compound is 5-amino-3-cyano-1-(2,6-dichloro-4-trifluoromethylphenyl)-4-trifluoromethylsulphinylpyrazole),
a compound known as fipronil described in
When used as pesticidal agent in a host animal the compositions
of the invention may be applied to the human or animal host patient at a typical
amount of 0.001-10 mgs/kg of body weight of avermectin or milbemycin compound. The
weight ratio of avermectin or milbemycin compound to the aryl pyrazole will generally
be in the range of 1 to 100, preferably 1 to 10. The synergistic effects of certain
combinations allow a reduced dosage of both compounds to be employed to obtain a
given antiparasitic effect, reducing risks of unwanted side effects, toxicity and
development of resistance to the compounds concerned and longer duration of action.
The combination therapy of the invention is effective in
treating a variety of conditions caused by endoparasites including, in particular,
helminthiasis which is most frequently caused by a group of parasitic worms described
as nematodes and which can cause severe economic losses in swine, sheep, horses
and cattle as well as affecting domestic animals and poultry. The most common genera
of nematodes infecting the animals referred to above are Haemonchus,
Trichostrongylus, Ostertagia, Teladorsagia, Nematodirus,
Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia,
Strongylus, Trichonema and Dictyocaulus. The combinations are
also effective against other nematodes which affect various species of animals including,
for example:- Dirofilaria in dogs and various parasites which can infest
livestock, companion animals such as cats and dogs and also humans including gastrointestinal
parasites such as Ancylostoma, Uncinaria, Necator,
Ascaris, Strongyloides, Trichinella, Capillaria,
Toxocara, Toxascaris, Trichuris, Enterobius and parasites
which are found in the blood or other tissues and organs such as filarial worms
and the extra intestinal stages of Strongyloides, Toxocara and
Trichinella. They are especially useful in treating heartworm in companion
The combinations are also of value in treating ectoparasite
infections including particular arthropod ectoparasites of host humans, animals
and birds such as ticks, mites, lice, fleas, blowfly, biting insects and migrating
dipterous larvae which can affect cattle and horses. The combinations are also insecticides
active against household pests such as the cockroach, clothes moth, carpet beetle
and the housefly as well as being useful against arthropod pests of stored grain
and of agricultural plants such as spider mites, aphids, caterpillars and against
migratory orthopterans such as locusts.
The efficacy of compositions according to an embodiment
of the invention against heartworm (Dirofilaria immitis) is demonstrated
by the following Example.
Infective L3 larvae of Dirofilaria immitis were
recovered from previously infected Aedes egypti and cultured in vitro
to the L4 stage by techniques described by
D. Abraham et al (J. Parasit. 73(2) 1987, pp 377-383
Testing of compounds took place when >95% of the larvae
had moulted to the L4 stage. The assay system consisted of a 96 well microtitre
plate in which 79 mcl of assay media, 1 mcl test compound and 20 mcl assay media
containing 15 to 20 L4 D. immitis were dispensed. Each test compound was dissolved
in dimethylsulphoxide (DMSO) and dilutions were made using this solvent. The test
microtitre plates were kept at 37 degrees centigrade under an atmosphere comprising
5% CO, in air for 72 h.
The effects of each compound at each concentration and
combination was assessed by comparing the motility of L4 larvae in control and treated
wells. Microscopic observations were made at 2, 4, 24, 48 and 72 hours on each well.
Observations were scored according to the levels of larval motility, ranging from
0 (all dead) to 5 (all normal motility).
The above procedure was carried out using solutions containing
the compounds shown in Table 1 below at the stated concentrations. Compound (1)
is 5-oximino 22,23-dihydro-25-cyclohexylavermectin B1 monosaccharide (selamectin),
disclosed as Example 5 in
Table 1 In vitro L4 Heartworm (D.immitis)
Observations (hours post treatment)
Key - Observation Score:
0 = No motility, larvae dead
1= Motility in at least 1 larva, all others dead
2 =≥80% of larvae dead, others sluggish
3 = < 80% of larvae dead, others sluggish
4 = All larvae (or most) motile but sluggish
5 = Normal in appearance and motility.
It can be seen from the results given in Table 1 that,
whereas compound (1) alone had only a modest effect in causing mortality or paralysis
and fipronil alone had no observable effect at all at concentrations of 10 mcg/ml,
the combination of these compounds was very effective.
All publications mentioned above are herein incorporated
by reference in their entirety.
Reference to treatment herein includes preventative, palliative
and curative treatments.