EXHIBIT 1001
`
`

`

`(12) United States Patent
`Derrieu
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 8,501,799 B2
`Aug. 6, 2013
`
`U3008501799B2
`
`(54) PHARMACEUTICAL COMPOSITION
`CONTAINING AN N-PHENYLPYRAZOLE
`DERIVATIVE, AND USE THEREOF FOR
`PREPARING A TOPICAL VETERINARY FOR
`FLEA CONTROL
`
`(75)
`
`Inventor: Guy Dern'en, Cagnes sur Mer (FR)
`
`(73) Assignee: Virbac, Carros (FR)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl.No.:
`
`13/132,996
`
`(22) PCT Filed:
`
`Dec. 16, 2008
`
`(86) PCTNo.:
`
`PCT/FR2008/001756
`
`§371(C)(1),
`(2), (4) Date:
`
`Aug. 17, 2011
`
`(87) PCT Pub. No.: W02010/070210
`
`PCT Pub. Date: Jun. 24, 2010
`
`(65)
`
`Prior Publication Data
`
`US 2012/0029043 A1
`
`Feb. 2, 2012
`
`(51)
`
`Int. Cl.
`A01N 47/02
`A 01N 25/02
`A 0IP 7/04
`A61K 31/415
`A61K 4 7/34
`A 61K 9/00
`A 6IP 33/14
`(52) us. Cl.
`USPC ......................... 514/407; 548/356.1; 424/405
`
`(2006.01)
`(2006.01 )
`(2006.01 )
`(2006.01)
`(2006.01)
`(2006.01 )
`(2006.01 )
`
`(58) Field of Classification Search
`None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`5/2002 Etchegaray ................... 514/407
`6,395,765 B1 *
`9/2004 Etchegaray et a1.
`.......... 514/407
`6,797,724 B2 *
`FOREIGN PATENT DOCUMENTS
`W0 97/ 12521
`4/1997
`
`W0
`
`OTHER PUBLICATIONS
`
`Definition of Prevent, Princeton University “About WordNet.”
`WordNet. Princeton University. 2010. <http://wordnet.princeton.
`edu> accessed Sep. 7, 2012*
`International Search Report and Written Opinion for International
`Applicaton No. PCT/FR2008/001756, mailed Sep. 30, 2009.
`Meria] Animal Health Limited; “Summary of product and character-
`istics. Frontline Spot on Cat”; Internet Article; [online]; [Retrieved on
`Aug. 26, 2008]; Retrieved from the Internet <URL: http://www.vmd.
`gov.uk/espcsite/Documents/ 138 1 28.doc; XP007905533; 7 pages.
`
`* cited by examiner
`
`Primary Examiner — Sreeni Padmanabhan
`Assistant Examiner — Irina Neagu
`(74) Attorney, Agent, or Firm — Alston & Bird LLP
`
`(57)
`
`ABSTRACT
`
`The invention relates to a liquid pharmaceutical composition
`that contains N-phenylpyrazole derivative as an active prin-
`ciple, benzyl alcohol, and an appropriately selected organic
`solvent, and to the use of such a composition for preparing a
`topically applied antiparasitic veterinary drug for preventing
`and/or treating flea infestation in pets, in particular, in dogs
`and cats.
`
`15 Claims, No Drawings
`
`

`

`US 8,501,799 B2
`
`1
`PHARMACEUTICAL CONIPOSITION
`CONTAINING AN N-PHENYLPYRAZOLE
`DERIVATIVE, AND USE THEREOF FOR
`PREPARING A TOPICAL VETERINARY FOR
`FLEA CONTROL
`
`FIELD OF THE INVENTION
`
`The present invention relates to a liquid pharmaceutical
`composition containing, as active principle, an N—phe-
`nylpyrazole derivative, benzyl alcohol and a suitably selected
`organic solvent, and also to the use of such a composition for
`the preparation of an antiparasitic veterinary medicament for
`topical use for preventing and/or treating flea infestations on
`pets, in particular cats and dogs.
`
`BACKGROUND OF THE INVENTION
`
`Pets are oflen infested with one or more parasites that feed
`on blood, such as cat or dog fleas, ticks, or mange.
`Fleas are Wingless insects, with a laterally compressed
`body and highly developed legs, adapted for jumping. They
`are ectoparasites, which suck the blood of mammals or birds.
`The approximately 2000 listed species belong to the order of
`Siphonaptera. Two species offlea are commonly encountered
`in Europe; they are the cat flea (Ctenocephalidesfelis) and the
`dog flea (Ctenocephalides canis) which live in the animals’
`fur. The cat flea, which is the most common, is capable of
`reproducing both on cats and dogs. It can also attack man and
`other pets, but cats are the main host responsible for infesta-
`tion when cats and dogs live in the same environment.
`Fleas have a complex life cycle with four distinct stages:
`egg, larva, nymph and adult. They mate within the first 8 to 48
`hours following acquisition by the host, after their first blood
`meal. The females thus begin to lay 24 to 48 hours after this
`first blood meal. The adult flea generally lays on the animal.
`The eggs laid on the animal do not remain thereon, but fall to
`the floor. Under optimum conditions, the female may lay
`more than 25 eggs per day. She will lay several hundred in her
`lifetime. After a few days, a white, hairy, worm-like larva
`about 1.5 mm long is born. The larva feeds on organic debris,
`larval remains and dry blood excreted by the adults. The larval
`state lasts for l to 3 weeks, ifthe conditions are favorable (18°
`to 27° C. and 70% relative humidity). The larva then spins a
`cocoon and transforms into a nymph. Normally, the nymph
`develops in 1 to 2 weeks, but passage to the adult state may
`take up to 1 year, if the conditions are unfavorable. The adult
`flea (small and black) emerges from the cocoon when it
`detects vibrations, heat and a higher concentration of carbon
`dioxide, which takes place during the passage of a cat, a dog
`.
`.
`. or a human! It then jumps onto the victim, feeds imme-
`diately on its blood and rapidly becomes engorged, taking a
`lighter reddish—brown color. The adult flea lives for 6 to 12
`months. Without food, it can survive for up to 2 months.
`Flea bites cause itching, in animals and in man. Flea saliva
`(secreted at each bite) may also, depending on the individual,
`lead to immediate or delayed allergic reactions. These reac-
`tions are reflected by various skin lesions and itching. Two
`types of flea-related dermatosis are distinguished, namely
`pulicosis and flea-bite allergic dermatitis. Whereas, in both
`cases, the dermatosis results from a more or less pronounced
`infestation with fleas, it is only in the second case that an
`allergic phenomenon is associated. Flea-bite allergic derma-
`titis (FBAD) is the most frequent cause ofpruritus in dogs. In
`France, in adult dogs, it thus represents close to half the
`pruriginous dermatoses. Close to 80% of dogs presenting
`FBAD also have atopic dermatitis. Reciprocally, tWo out of
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`three atopic dogs have FBAD. It is thus likely that atopic dogs
`are predisposed to developing a flea-bite allergy and that
`infestation with fleas is a triggering factor for atopic derma-
`titis. This justifies the need for very rigorous antiflea control
`in the case of atopic dogs or dogs belonging to at-risk breeds.
`Furthermore, FBAD is probably the main cause of reappear-
`ance of pruritus in the case of desensitized atopic dogs.
`Fleas ofthe genus Ctenocephalides are moreover interme-
`diate hosts ofDipylidium caninum, which is a parasitic worm
`ofthe small intestine of cats and dogs. The carnivore becomes
`infested by swallowing the parasitized fleas. This infestation
`may lead to anal pruritus, engorgement of the anal sacs, and
`also to dermatitis of the perineal region. This is why it is
`occasionally recommended to regularly worm animals in
`addition to combating fleas.
`Similarly, ticks (Rhipicephalus sp., Ixodes sp., Dermacen—
`tor sp.,Amblyomma sp., etc.) may also cause the animal stress
`and be harmful to its health. They may also be harmful to man.
`However, the most serious problem concerning ticks is that
`they are a vector of pathogens that affect animals as much as
`man. Among the major diseases that need to be avoided,
`mention may be made of Borellioses (Lyme’s disease caused
`by Borellia burgdorferi), Babesioses (piroplasmoses caused
`by Babesia sp.) and Rickettsioses. Ticks may also release
`toxins with paralyzing and inflammatory, and occasionally
`fatal, properties.
`Mange (Demodex sp., Sarcoptes sp., Otodectes sp., etc.) is
`particularly difficult to combat since there are very few effi-
`cient active materials. It requires frequent treatments.
`Infestation with these various parasites, and most particu—
`larly with fleas, thus represents a major health problem for the
`infested animals and imposes the need for suitable treatments.
`The treatment should in particular not only have immediate
`eflicacy (fast-acting) but also prolonged eflicacy over time
`(remanence) so as to avoid, on the one hand, repeated treat-
`ments, and, on the other hand, any risk of infestation and/or
`reinfestation for a prolonged period. The flea, in particular,
`must be eliminated before it reproduces and begins to lay.
`Many insecticidal substances that are more or less active
`and more or less expensive exist. Resistance phenomena
`appear associated with their use, and this is especially the case
`during the use of carbamates, organophosphorus compounds
`and pyrethroids.
`Moreover, patent applications EP 0 295 117 and EP 0 352
`944 describe a large family ofN-phenyl—pyrazoles with a very
`broad spectrum of activity, including antiparasitic activity.
`Although effective, N-phenylpyrazole derivatives, and in
`particular 5—amino-1-[2,6-dichloro-4~(trifluoro-methyl)phe—
`nyl]-4-(t1ifluoromethylsulflnyl)-lH-pyrazole-S-carbonitrile
`(fipronil), are occasionally difficult to formulate since they do
`not always have sufficient solubility in the excipients conven-
`tionally used for the preparation of ready-to-use liquid anti-
`parasitic compositions.
`Specifically, products that are active against blood- sucking
`parasites, and in particular against fleas, may especially be in
`the form of liquid compositions (pipettes) or solutions for
`applying to the skin, also known as “Spot-On solutions”, to be
`applied very easily, in a single topical application directly to
`the animal’s skin, generally between the shoulder blades.
`However, in this type of composition, fipronil is often
`difficult to formulate and may lead to crystallization. In order
`to overcome this problem, it has already been proposed, espe-
`cially in patent application EP 0 881 881, to formulate N-phe-
`nylpyrazole derivatives in solvent medium in the presence of
`a crystallization inhibitor and a C1-C4 alcohol. The product
`Frontline® Spot—On Chat et Chien, sold in France by the
`company Merial SAS, is based on this technology.
`
`

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`US 8,501,799 B2
`
`Although such compositions are suitable for preventing the
`crystallization problems of these particular active principles,
`they are, however, not entirely satisfactory as regards the
`duration ofprotection they give the animal. In the case of the
`product Frontline® Spot-On Chat et Chien especially, the
`duration of protection against new flea infestations stated by
`the manufacturer is limited to 4 weeks in the case of cats and
`to 2 months in the case of dogs. However, antiparasitic effi—
`cacy tests conducted according to the current standards do not
`make it possible to reproduce the prolonged eflicacy results,
`and the product does not therefore always have entirely sat-
`isfactory remanence.
`
`SUMMARY OF THE INVENTION
`
`It is thus in order to overcome all the problems encountered
`with the antiparasitic products currently available on the mar-
`ket and to provide a product that can effectively prevent and
`treat flea infestations on pets, both cats and dogs, that the
`Applicant has developed the product that is the subject of the
`invention. The Applicant particularly set itself the aim of
`providing a product for preventing and treating flea infesta—
`tions on pets that is easy to formulate and easy to apply, while
`at the same time being fast-acting and more remanent than the
`products currently available on the market.
`Another obj ect of the invention is to provide such compo-
`sitions that are easy to use irrespective of the animal species,
`the size of the animal or the nature of its coat.
`
`Another object ofthe invention is to provide efficient com-
`positions that do not necessitate wetting of the entire animal.
`These objects are achieved by the antiparasitic pharmaceu—
`tical composition that forms the subject ofthe present inven-
`tion.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Unexpectedly, the antiparasitic pharmaceutical composi—
`tion ofthe invention provides eflective and prolonged activity
`in the treatment and protection ofpets in the form of a ready-
`to-use solution that is easy to use.
`.
`Thus, one subject of the present invention is a liquid phar-
`maceutical composition, characterized in that it contains:
`as active principle, 5-amino-1-[2,6-dichloro—4—(trifluo-
`romethyl)phenyl] -4-(trifluoromethyl sulfinyl)- 1 H-pyra-
`zole—3—carbonitrile (fipronil),
`at least 5% (weight/volume) of benzyl alcohol, and
`at least 50% (weight/volume) of an organic solvent chosen
`from propylene glycol monomethyl ether, dipropylene
`glycol n-butyl ether, ethylene glycol monomethyl ether,
`ethylene glycol monoethyl ether, diethylene glycol
`monoethyl ether and propylene glycol, and mixtures
`thereof,
`it being understood that said composition is free of C1—C4
`alcohol.
`
`According to the invention, said pharmaceutical composi-
`tion is intended in particular to be applied to cats or to dogs.
`In the pharmaceutical composition in accordance with the
`invention, fipronil preferably represents from 1% to 20%
`(weight/volume) and even more preferentially from 5% to
`15% (weight/volume). It should be understood, however, that
`these amounts are given as a guide and that they may be
`adjusted according to the formulation requirements, in par-
`ticular with regard to the effective doses as a function of the
`animal to be treated and its weight.
`According to one preferred embodiment of the invention,
`benzyl alcohol represents from 5% to 40% (weight/volume)
`and even more preferentially from 25% to 35% (weight/
`volume).
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`The organic solvent(s) mentioned above are used in the
`amounts necessary to adjust the pharmaceutical composition
`to the required final volume. The weight/volume percentage
`of the organic solvent or of the mixture of organic solvents
`depends on the nature (and thus on the density) of the solvent
`or solvent mixture used.
`Among said organic solvents, diethylene glycol monoethyl
`ether (DGME) is most particularly preferred.
`The pharmaceutical composition used in accordance with
`the invention may also contain one or more excipients that
`may be chosen, for example, from surfactants, thickeners,
`dyes, fragrances and antioxidants, among which examples
`that may be mentioned, in a non-limiting manner, include
`butylhydroxyanisole, butylhydroxy—toluene, propyl gallate,
`ascorbyl palmitate and rosemary extracts, and mixtures
`thereof.
`
`When it is (they are) present, the antioxidant(s) preferably
`represent fi'om 0.005% to 2% approximately (weight/vol-
`ume) and even more preferentially from 0.01% to 0.1%
`approximately (weight/volume).
`Besides fipronil, the pharmaceutical composition may also
`contain one or more additional antiparasitic active principles.
`Additional antiparasitic active principles that may especially
`be mentioned include acaricides such as amitraz or cymiaz-
`ole, flea and tick growth inhibitors, also known as “IGR” for
`“Insect Growth Regulators”, such as pyriproxyfen and ethox-
`azole, endoparasiticides such as avermectins and derivatives
`thereof, for instance ivermectin, abamectin, doramectin and
`moxydectin, milbemycins, and also compounds that are
`active against pet ectoparasites and sand flies.
`Such combinations of active agents may be useful for
`broadening the spectrum of action of the composition in
`accordance with the present invention.
`The pharmaceutical composition in accordance with the
`invention may be readily prepared by simple dilution of
`fipronil and optionally of the additional antiparasitic active
`principle(s) inbenzyl alcohol and the organic solvent(s) used.
`After its preparation, the pharmaceutical composition is
`preferably conditioned in single-dose pipettes.
`Another subject of the present patent application is the use
`of a liquid pharmaceutical composition as described previ-
`ously for the preparation of an antiparasitic veterinary medi-
`cament for topical application for preventing (protecting)
`and/or treating flea infestations on pets, in particular on cats
`or dogs.
`According to this use, said medicament is intended to be
`applied by direct application to the animal’s skin, on the
`shoulder blades or along a dorsal line starting from the base of
`the tail and going up to the neck.
`The amount of medicament to be administered may range
`from 0.3 to 1.5 ml approximately, preferably from 0.5 ml
`approximately for cats and from 0.3 to 6.0 ml approximately
`for dogs, as a function of the weight of the animal under
`consideration and the dosage.
`The volume to be applied according to the invention should
`preferably correspond to a unit dose of fipronil ranging from
`0.3 to 60 mg per kg of body weight and even more preferen-
`tially from 5 to 15 mg per kg of body weight.
`Thus, according to one preferred embodiment ofthe inven-
`tion, said medicament is intended to administer a unit dose of
`flpronil ranging from 0.3 to 60 mg per kg ofbody weight and
`even more preferentially from 5 to 15 mg per kg of body
`weight.
`Besides the preceding provisions, the invention also com—
`prises other provisions that will emerge from the description
`that follows, which refers to an example of preparation of a
`pharmaceutical composition in accordance with the inven-
`
`

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`5
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`US 8,501,799 B2
`
`tion, and also to examples demonstrating the efficacy of said
`composition in the treatment of fleas on cats and dogs.
`these
`It should be clearly understood, however,
`that
`examples are given purely as illustrations ofthe subject of the
`invention, of which they do not in any way constitute a limi-
`tation.
`
`Example 1
`
`Antiflea Composition
`
`The following antiflea composition was prepared, by
`simple dissolution of fipronil in the mixture of the other
`constituents of the composition:
`
`fipronil
`benzyl alcohol
`hutylhydroxyanisole
`butylhydroxytoluene
`diethylene glycol monoethyl ether
`
`10.0 g
`30.0 g
`0.02 g
`0.01 g
`qs 100 ml
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`After preparation, this composition may be conditioned
`directly in single—dose pipettes.
`
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`Example 2
`
`Study of the Immediate and Prolonged Efficacy of a
`Composition in Accordance with the Invention
`Against Fleas on Dogs
`
`In this example, a study was performed to determine and
`compare the immediate and prolonged efficacy of two
`fipronil-based topical compositions:
`a composition A in accordance with the invention as
`described above in Example 1;
`the product sold under the name Frontline® Spot-on dog
`by the company Mérial.
`1) Materials and Methods
`a) Type of Study
`This is a randomized blind controlled eflicacy study, per-
`formed in parallel on 3 groups of eight dogs.
`b) Animals Used and Maintenance Conditions
`The dogs used in this study were male or female adult
`domestic dogs, more than 4 months old, ofmixed breeds, but
`mainly of short-hair European breed, weighing between 2 and
`20 kg. Before the start of the study, all the dogs were checked
`to ensure that they were in good health, that they were not
`infested with fleas and that the females were not pregnant. All
`the dogs were wormed and acclimatized to the living condi—
`tions for at least 7 days before the start of the study.
`During the acclimatization period and throughout the study
`period, the dogs were kept in an air-conditioned room, each
`dog being confined in an individual enclosure of dimensions
`1.9 mx2.97 m without litter and without possible contact
`between the various dogs engaged in the study. The identifi-
`cation number, the group number and the type ofcomposition
`administered were noted on the outside of each enclosure.
`The temperature of the room was maintained at about 20°
`C.=4° C. The dogs were subjected to an alternation of 12
`hours of light and 12 hours of darkness.
`The animals were fed once a day with commercial dog
`kibbles sold under the trade name Eukanuba® by the com-
`pany lams, a division of Foodcorp., according to the manu-
`facturer’ s recommendations, and they were given free access
`to fresh drinking water.
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`c) Compositions Tested
`Composition A in accordance with the invention was com-
`pared with the product Frontline® Spot-on dog containing
`10% (g/100 m1) offipronil and a mixture of excipients. It was
`used as supplied by the manufacturer.
`d) Treatments
`Group 1: Treatment with compositionA in a proportion of
`0.67 ml per dog for dogs weighing between 2 and 10 kg, and
`in a proportion of 1.34 ml per dog for dogs weighing between
`more than 10 kg and 20 kg,
`Group 2: Treatment with the product Frontline® Spot-on
`dog in a proportion of 0.67 ml per dog for dogs weighing
`between 2 and 10 kg, and in a proportion of 1.34 ml per dog
`for dogs weighing between more than 10 kg and 20 kg,
`Group 3: Negative control: no treatment.
`The treatment was applied topically, between the dogs’
`shoulder blades, in a single application at the start ofthe study
`(D=0).
`e) Flea Infestations/Measurement ofthe Efficacy ofthe Treat-
`ments
`
`6 days before the start of the study (D=—6), all the dogs
`were infested with about 100 laboratory fleas, of the strain
`Ctenocephalis felis, of male or female sex. The fleas were
`then counted 4 days before the start of the treatment (D=-4).
`To do this, all the fleas present on an animal are harvested by
`combing the dog and then counted after combing. The num-
`ber of fleas is thus determined. Afier counting, the fleas are
`destroyed and a new batch of about 100 fleas is placed on the
`animal the day prior to administration of the treatment (D=—
`1).
`The number of fleas still alive 2 days after the administra-
`tion of the test composition (D=2) was then counted.
`The dogs were again infested with a known amount offleas
`(about 100) 7 days (D=7), 14 days (D=14), 21 days (D=21),
`28 days (D=28), 35 days (D=35), 42 days (D=42), 49 days
`(D=49), 56 days (D=56), 63 days (D=63), 70 days (D=70), 77
`days (D=77), 84 days (D=84) and 91 days (D=91) after
`administration of the treatment.
`
`Counting of the fleas that were still alive was then per-
`formed 48 hours after each of these new infestations (D=9;
`D=16; D=23; D=30', D=37; D=44; D=51; D=58; D=65;
`D=72; D=79; D=86 and D=93).
`On each counting, the efficacy of the treatment was calcu-
`lated according to the following equation:
`% eflicacy=100x(NPVC—NTVT)/NPVC
`in which:
`
`NPVC is the geometric average of the number of live fleas
`counted on the dogs of group 3 (control);
`NPVT is the geometric average of the number of live fleas
`counted on the dogs of a group that has received a
`treatment (group 1 or 2).
`A treatment is said to be effective ifthe percentage efficacy
`is greater than or equal to 95%.
`2) Results
`The average results obtained are given in Table I below:
`
`TABLE I
`
`GROUP 1
`GROUP 2
` Days (Composition A) (Frontline ® Spot—on dog)
`
`
`
`D = 2
`99.7
`100.0
`D = 9
`100.0
`100.0
`D = 16
`99.8
`100.0
`D = 23
`100.0
`100.0
`D - 30
`100.0
`100.0
`D - 37
`100.0
`100.0
`D - 44
`99.9
`100.0
`
`

`

`US 8,501,799 B2
`
`7
`TABLE I-continued
`
`GROUP 1
`GROUP 2
`
`Days
`(Composition A)
`(Frontline ® Spot-on dog)
`D - 51
`100.0
`100.0
`D - 58
`100.0
`99.6
`D - 65
`99.9
`99.3
`D - 72
`99.8
`98.2
`D - 79
`98.9
`96.4
`D - 86
`99.2
`85.5
`
`97.1D - 93 75.4
`
`
`These results show that:
`
`composition A in accordance with the present invention
`remains effective (determination 48 hours after the
`infestation) for 13 weeks (D=93) against flea infesta-
`tions on dogs;
`the product Frontline® Spot-on dog remains effective (de-
`termination 48 hours after infestation) only for 1 1 weeks
`(D=79) against flea infestations on dogs.
`The efficacy and the better remanence ofthe compositionA
`in accordance with the present invention are thus clearly
`demonstrated.
`
`Example 3
`
`Study of the Efficacy of a Composition in
`Accordance with the Invention Against Fleas on
`Dogs
`
`In this example, a study was performed to determine and
`compare the efficacy of two fipronil—based topical composi-
`tions:
`a composition A in accordance with the invention and as
`described above in Example 1;
`the product sold under the name Frontline® Spot—on dog
`by the company Mérial.
`1) Materials and Methods
`a) Type of Study
`This is a randomized blind controlled efficacy study per-
`formed in parallel on 3 groups of six dogs.
`b) Animals Used and Maintenance Conditions
`The dogs used in this study were male or female mongrels
`belonging to the species Canisfirmiliaris, more than 6 months
`old, weighing between 6 kg and 25 kg. Before the start of the
`study, all the dogs were checked to ensure that they were in
`good health and that they were not infested with fleas. All the
`dogs were wormed and acclimatized to the living conditions
`for at least 7 days before the start of the study.
`The dogs were also checked to ensure that they had not
`received any topical flea treatment in the 12 weeks preceding
`the start of the study.
`the
`During the acclimatization period and throughout
`study, the dogs were kept in an air-conditioned room, each
`dog being confined in an individual enclosure of dimensions
`1.9 mx2.97 in without litter and with no possible contact
`between the various dogs engaged in the study. The identifi-
`cation number, the group number and the type ofcomposition
`administered were noted on the outside of each enclosure.
`The temperature of the room was maintained at about 20°
`C.:4° C. The dogs were subjected to an alternation of 12
`hours of light and 12 hours of darkness.
`The animals were fed once a day with commercial dog
`kibbles sold under the trade name Ultradog Superwoofby the
`company Nola, a division of Foodcorp., according to the
`manufacturer’ 5 recommendations, and were given free access
`to fresh drinking water.
`
`8
`
`c) Compositions Tested
`CompositionA in accordance with the invention was com-
`pared with the product Frontline® Spot-on dog containing
`10% (g/l 00 ml) of fipronil and a mixture of excipients. It was
`used as supplied by the manufacturer.
`d) Treatments
`Group 1: Treatment with compositionA in a proportion of
`0.067 ml per kg of body weight,
`Group 2: Treatment with the product Frontline® Spot-on
`dog in a proportion of 0.067 ml per kg of body weight,
`Group 3: Negative control: no treatment.
`The treatment was applied topically, between the dogs’
`shoulder blades, in a single application at the start ofthe study
`(D=0).
`e) Flea Infestations/Measurement ofthe Efficacy ofthe Treat-
`ments
`
`6 days before the start of the study (D=—6), all the dogs
`were infested with about 100 laboratory fleas, of the strain
`Ctenocephalis felis, of male or female sex. The fleas were
`then counted 5 days before the start of the treatment (D=— 5).
`To do this, all the fleas present on an animal are harvested by
`combing the dogs and then counted after combing. The num—
`ber of fleas is thus determined. After counting, the fleas are
`destroyed and a new batch of about 100 fleas is placed on the
`animal the day prior to administration of the treatment (D=—
`2).
`The number of fleas still alive 1 day after administration of
`the composition (D=l) was then counted.
`The dogs were again infested with a known amount offleas
`(about 100) 7 days (D=7), 14 days (D=14), 21 days (D=21),
`35 days (D=35), 42 days (D=42), 49 days (D=49) and 56 days
`(D=56) after administration of the treatment.
`The fleas that were still alive were then counted 24 hours
`afier each of these new infestations (D=8; D=15; D=22;
`D=36; D=43; D=50 and D=57).
`At each counting, the efficacy of the treatment was calcu-
`lated according to the following equation:
`
`% efficacy=1 00x (NPVC—NPvD/NPVC
`in which:
`NPVC is the geometric average of the number of live fleas
`counted on the dogs of group 3 (control);
`NPVT is the geometric average of the number of live fleas
`counted on the dogs of a group that has received a
`treatment (group 1 or 2).
`A treatment is said to be effective ifthe percentage efficacy
`is greater than or equal to 95%.
`2) Results
`The average results obtained are given in Table 11 below:
`
`TABLE 11
`
`GROUP 1
`GROUP 2
`Days
`(Composition A)
`(Frontline ® Spot-on dog)
`
`D = 1
`92.2
`84.4
`D - 8
`99.5
`99.8
`D - 15
`100.0
`99.8
`D - 22
`99.6
`100.0
`D = 29
`100.0
`98.7
`D = 36
`100.0
`96.9
`D - 43
`99.2
`96.8
`D :- 50
`97.0
`93.7
`D - 57
`89.4
`74.8
`
`These results show that:
`
`composition A acts more quickly than the product Front-
`line® Spot-on dog (comparison ofthe % efiicacy values
`at D=1);
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`4o
`
`45
`
`50
`
`55
`
`60
`
`65
`
`

`

`9
`
`10
`
`US 8,501,799 B2
`
`composition A in accordance with the present invention
`remains effective (determination 24 hours after infesta-
`tion) for 7 weeks (D=50) against flea infestations on
`dogs;
`the product Frontline® Spot-on dog remains effective (de-
`termination 24 hours after the infestation) only for 6
`weeks (D=43) against flea infestations on dogs.
`The speed of action and the better remanence of composi-
`tion A in accordance with the present invention are thus
`clearly demonstrated.
`
`Example 4
`
`Study of the Efficacy of a Composition in
`Accordance with the Invention Against Fleas on Cats
`
`In this example, a study was performed to determine and
`compare the efficacy of two fipronil-based topical composi-
`tions:
`
`a composition A in accordance with the invention and as
`described in Example 1;
`the product sold under the name Frontline® Spot—on cat by
`the company Mérial.
`1) Materials and Methods
`a) Type of study
`This is a randomized, blind controlled efficacy study per-
`formed in parallel on 3 groups of six cats.
`This study was performed according to the Good Labora-
`tory Practices for the evaluation ofveterinary products GENV/
`MC/CHEM/(98)l7; decree of Jan. 28, 2005, published in the
`Oflicial Gazette of Feb. 20, 2005) and according to the guide-
`line recommendations of the Committee for Veterinary
`Medicinal Products (CVMP): “Guidelinesfiir the testing and
`evaluation ofthe eficacy ofantiparasitic substancesfor the
`treatment andprevention oftick andflea infestations in dogs
`and cats”, EMEA/CVMP/OOS/ZOOO—Rev. 2).
`b) Animals Used and Maintenance Conditions
`The cats used in this study were male or female adult
`European domestic cats, from 7 months to 2 years old, weigh-
`ing on average 6720.] kg. All the cats were acclimatized to
`the living conditions for at least 9 days before the start of the
`study.
`During the acclimatization period and throughout the study
`period, the cats were kept in an air—conditioned room, each cat
`being confined in individual cages. The identification num—
`ber, the group number and the type of composition adminis-
`tered were noted on each cage. The temperature of the room
`was maintained at about 23 ° C.:2° C. with a relative humidity
`of 60: 1 0%.
`
`The animals were fed once a day (except for Sundays) with
`commercial cat kibbles, sold by the company Harlan-Teklad
`under the manufacturer’s recommendations, and they were
`given free access to fresh drinking water.
`c) Compositions Tested
`Composition A in accordance with the invention was com-
`pared with the product Frontline® Spot—on cat containing
`10% (g/ 100 ml) offipronil and a mixture of excipients. It was
`used as supplied by the manufacturer.
`d) Treatments
`Group 1 : Treatment with composition A in a proportion of
`0.5 ml per cat, i.e. 50 mg of fipronil per animal,
`Group 2: Treatment with the product Frontline® Spot-on
`cat in a proportion of 0.5 ml per cat, i.e. 50 mg of flpronil per
`animal,
`Group 3: Negative control: no treatment.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`The treatment was applied topically, between the shoulder
`blades of the cats, in a single application at the start of the
`study (D=0).
`e) Flea Infestations/Measurement ofthe Efficacy ofthe Treat-
`ments
`
`Nine days before the start of the study (D=—9), all the cats
`were infested with about 50 laboratory fleas, of the strain
`Ctenocephalis felis, of male or female sex. The fleas were
`then counted before the start of the treatment so as to check
`
`the capacity of each of the cats to host fleas. To perform
`counting, all the fleas present on an animal are harvested by
`combing the cat, and are then counted after combing. The
`number of fleas is thus determined. After counting, the fleas
`are destroyed and a new batch of about 50 fleas is placed on
`the animal the day prior to administration of the treatment
`(D=—1).
`The first infestation took place the day before the treatment
`(D=—1).
`The reinfestations then took place on D=7, D=l4, D=21,
`D=28, D=35, D=42 and D=49.
`The fleas were counted 48 hours after administration ofthe
`treatment and then 48 hours after each of the reinfestations
`
`(D 9, D 16, D 23, D 30, D 37, D 44 and D—51).
`To do this, all the fleas present on an animal are harvested
`by combing the cat, and are then counted after combing. The
`number of live fleas is thus determined. Afler each counting,
`50 new live fleas are placed on the cat.
`At each counting, the efficacy of the treatment was calcu-
`lated according to the following equation:
`
`% eflicacy=100x(NPVC—~NPVT)/NPVC
`in which:
`
`NPVC is the geometric average of the number of live fleas
`counted on the cats of group 3 (control);
`NPVT is the geometric average of the number of live fleas
`counted on the cats of a group that has received a treat-
`ment (groups 1 or 2).
`A treatmen