Available online at www.sciencedirect.com
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`"3' ScienceDirect
`
`Veterinary Parasitology 150 (2007) 196-202
`
`"°‘°'i"""
`parasitology
`
`._..__.._:.__
`www.elsevier.com/locate/vetpar
`
`Topical formulations of metaflumizone plus arnitraz to
`treat flea and tick infestations on dogs
`
`S. Sabnis *, J. Zupan, M. Gliddon
`Fort Dodge Animal Health, P.0. Box 5366, Princeton, NJ 08543-5366, USA
`
`Abstract
`
`A topical spot-on solution was developed for treating pets that contained of active ingredients metaflumizone and amitraz
`and intended for use as an ectoparasiticide. The formulation vehicle system was designed by balancing the following three
`attributes of various solvents: evaporation/drying, surface spreading, and percutaneous absorption. The solvents were selected
`by evaluating the solubilization capacity of individual solvents with respect to the above active ingredients. The evaporation
`rates of various solvent systems were then determined. The visual observations of the treatment sites was also performed a day
`after treating the dogs to understand the cosmetic effect of various solvent systems. The lead formulations dried off within a
`day after application with no noticeable residue at the treatment site, while others produced appreciable powdery residue or a
`large wet and oily spot. The stability of the lead formulations was also evaluated over 2 years to demonstrate a 2-year shelf life
`of this product.
`© 2007 Elsevier B.V. All rights reserved.
`
`Keywords: Metafiumizone: Amitraz; Ectoparasiticide; ProMeris®; ProMeris Duo”; Fleas; Ticks
`
`1. Introduction
`
`In the past the ability to develop topical dosage
`forms, often creams and ointments, has been described
`as art. With the availability of multifunctional
`adjuvants and sophisticated machines, the technology
`has advanced significantly in recent years. Topical
`formulations present unique and challenging demands
`in that (1) they should have both chemical and physical
`stability for at least 2 years, (2) have components that
`are nonirritating, non-sensitizing and non-allergenic,
`(3) are cosmetically acceptable and preferably cosme-
`tically elegant, and (4) are efficacious because of their
`ability to deliver therapeutic levels of drug over a
`
`"‘ Corresponding author. Tel.: +1 732 631 5902:
`fax: +1 732 631 5934.
`E-mail address: sabniss@pt.fdah.com (S. Sabnis).
`
`0304-4017/3 — see front matter © 2007 Elsevier B.V. All rights reserved.
`doi: l 0.] 01 6/j.vetpar.2007.08.044
`
`defined period of time. The realization that changes in
`vehicles/adjuvants can significantly change efficacy
`has permanently altered the way pharmaceutical
`scientists view topical formulations. The fomiulator
`is required to design dosage forms that target follicles
`or other specific skin regions, select vehicles that
`optimize drug—skin interactions or vehicles that release
`drugs for sustained periods of time (Osborne and
`Amann, 1990).
`Since percutaneous absorption is common to both
`topical and transdennal products,
`there has been a
`tendency to view this event as being of comparable
`significance in both delivery modalities. For transder—
`mal products, an optimal drug flux across the skin
`without appreciable drug build—up in the skin is ideal,
`while for topical products an optimal drug build-up in
`the skin with little or no drug flux through the skin is
`most desirable. Topical formulations usually contain
`several excipients
`such as permeation enhancers,
`
`Exhibit 1016A
`
`Merial V. Virbac
`
`IPR2014-01279
`
`

`
`S. Sabnis et al./ Veterinary Parasitology 150 (2007) 196-202
`
`197
`
`that partition into the skin in
`spreading agents, etc.
`accordance with their physicochemical properties.
`Certain excipients change the integrity of the stratum
`comeum (Comwell and Barry, 1993). When this occurs,
`the ease with which the active ingredients diffuse
`through this tissue is affected. Some components
`evaporate during the course of delivery, systematically
`increasing the drug concentration in the remaining
`vehicle/solvent. This means that
`localization within
`
`the
`skin depends upon the unique properties of
`formulations. Depending upon the specific formulation
`used, a high degree of drug accumulation in the skin
`may be achieved even at very low flux values (Shah
`et al., 1992).
`This report addresses the challenges involved in the
`development of an efficacious topical Spot-on formula-
`tion containing a novel active ingredient, metaflumi-
`zone,
`for use as an ectoparasiticide for domestic
`animals. Spot-on formulations are typically small
`volume liquids that are applied in one or two spots
`on the back of the animal at a location that cannot
`
`be licked off by the animal’s tongue (Klink et al., 1998).
`Metafiumizone (IUPAC hydrazinecarboxamide, 2-[2-
`(4—cyanophenyl)- l -[3—(trifl uoromethyl)phenyl]ethyli-
`dene]-N—[4-(trifluoromethoxy)phenyl]) is an excellent
`neuronal
`sodium channel antagonist. A neuronal
`sodium channel antagonist
`functions by preventing
`the ability of a neuron cell to transfer sodium ions across
`the cell membrane. A neuron cell thus affected is not
`
`capable of propagating a neuronal impulse, resulting in
`paralysis, and ultimately mortality in the target pest
`(Heaney et al., 2004). Metaflumizone is highly
`efficacious against
`fleas (e.g. Ctenocephalides sp.)
`and can also be used in combination with an acaricide,
`such as amitraz,
`for
`treatment against
`ticks (e.g.,
`Rhipicephalus sanguineus, Dermacemor variabilis,
`Amblyomma americanum,
`Ixodes scapularis, etc.)
`(Heaney et al., 2004; Rugg et al., 2007).
`The objective of the present study was to develop a
`spot-on solution for a metatlumizone plus amitraz
`combination for dogs at a dose of 20 mg/kg of
`each active ingredient
`(ProMeris®/ProMeris Duo®
`for Dogs, Fort Dodge Animal Health, Overland Park,
`KS). The formulation vehicle system was designed by
`balancing three attributes: (l) evaporation/drying, (2)
`surface spreading, and (3) percutaneous absorption.
`Since spot-on formulations are applied in low dose
`volumes, good solubility of the active ingredients in
`each of the solvents of the formulation was imperative.
`Therefore,
`all potential vehicles were tested for
`solubilization capacity followed by drying and spread-
`ing characteristics.
`
`2. Materials and methods
`
`2.1. Saturation solubility study
`
`To evaluate the solubility of each active ingredient in
`each of the potential solvents, several binary mixtures of
`one active ingredient and one vehicle were prepared in
`20 ml scintillation vials. This was done by measuring
`approximately 4 g of the active ingredient and adding
`6 g of the following solvents to the vials: 2-(2-
`butoxyethoxy)ethyl acetate, 2—butoxyethyl acetate, 2-
`phenoxyethanol, benzyl
`alcohol, benzyl benzoate,
`cineole
`(eucalyptol), diethylene glycol monoethyl
`ether, dimethyl sulfoxide, dipropylene glycol methyl
`ether, fatty alcohol ethoxylate surfactant,
`'y-hexalac-
`tone, heptyl acetate,
`isopropyl myristate,
`isopropyli—
`denc glycerol, N,N-diethyl—m—toluamide, oleic acid,
`polyethylene glycol 400, propylene glycol diacetate,
`and propylene glycol methyl ether acetate. The sample
`mixtures were then shaken on a Cole Palmer orbital
`shaker (Series 51300) at 200 rpm for 48 h at 22-25 °c.
`The system was designed to ensure that excess solid
`remained at all
`times in the samples to provide a
`saturated solution. The supernatant
`(approximately
`lml) was filtered using a 0.8 p.m nylon filter disc
`and assayed for active ingredient concentration by
`methods described elsewhere (unpublished method).
`
`2.2. Effect of co-solvent concentrations on
`evaporation rate
`
`The experimental formulations were set to contain
`15% (w/v) of each of the active ingredients. The ideal
`formulation is a balance between the properties of
`solubilization, depot in the skin, evaporation/drying and
`spreading on the surface of the skin. Since the actives
`were required to depot in the skin rather than penetrate
`through the skin,
`the level of the skin partitioning
`component was minimized to 20% (w/v) of
`the
`formulation total. The levels of
`the solubilizing
`component, the surface spreading component and the
`evaporation/drying component were optimized by
`performing a design of experiments exercise (Anderson
`and Whitcomb, 1998). The level of the solubilizing but
`nonevaporating and nonspreading component was
`varied between 10 and 25% (w/v). The spreading
`component was varied between 0 and 30% (w/v) and the
`volatile component was varied between 10 and 25% (w/
`v). Sixteen mixtures were prepared in 20 ml scintilla-
`tion vials wherein the concentrations of these compo-
`nents were varied such that the extreme limits as well as
`
`the central regions of the three components could be
`
`

`
`198
`
`S. Salmi: et al./ Veterinary Parasitology 150 (2007) 196-202
`Table I
`
`Saturation solubility results of metaflumizone and amitraz in various
`
`solvents” '
`Solvent
`Metaflumizone Amitraz
`solubility
`solubility
`
` (%, w/w) (%, w/w)
`
`
`23.2
`9.68
`2-(2-Butoxyethoxy)ethyl acetate
`26.6
`11.20
`2-Butoxyethyl acetate
`14. 1
`1 .08
`2-Phenoxyethanol
`20.7
`1.57
`Benzyl alcohol
`24.4
`1.58
`Benzyl benzoate
`24.7
`0.47
`Cineole (Eucalyptol)
`11.1
`8.57
`Diethylene glycol monoethyl ether
`14.8
`37.13
`Dimethyl sulfoxide
`21.4
`10.85
`Dipropylene glycol methyl ether
`13.3
`5.28
`Fatty alcohol ethoxylate surfactant
`31.1
`24.18
`'y—I-lexalactone
`28.5
`11.38
`Heptyl acetate
`16.2
`0.44
`Isopropyl myristate
`8.29
`1.43
`lsopropylidene glycerol
`33.9
`31 .22
`N,N—Diethy1—m—to1uamide
`7.53
`0.73
`Oleic acid
`,
`5.10
`1.54
`Polyethylene glycol 400
`15.4
`12.64
`Propylene glycol diacetate
`
`
`18.26Propylene glycol methyl ether acetate 25.4
`
`“ The values represent means of two individual observations for
`each solvent and active ingredient.
`
`3.2. Co-solvent concentration effect on
`~ evaporation rate
`
`All the mixtures were prepared such that vehicles
`composed of 70 parts of the mixture and the two actives
`contributed to 15 parts each. The different mixtures
`used in this experiment and the resultant evaporation
`rates are presented in Table 2. A response surface plot
`was constructed of
`the evaporation rate versus
`concentration of each of the vehicle component
`(Fig. 1).
`As seen from Fig. 1, changes in the surface spreading
`component had considerable effect on the overall
`evaporation rate of the mixture, while altering the
`relative concentrations of the solubilizing component
`and the evaporating component did not have as much
`impact on the overall
`rates of evaporation. The
`component ratio in the formulation for the commercial
`product was chosen as follows:
`a plateau region,
`depicted by a dotted ellipse in Fig. 1. The area within the
`ellipse defines where (1)
`the evaporation rate was
`sufficiently high, and (2) a moderate change in the
`mixture’s composition did not dramatically alter the
`resultant mixture’s evaporation rate. Various solvent
`ratios within this region were attempted and cosmetic
`evaluations were performed. The results are described
`in the next section.
`
`studied. The evaporation rates were determined by
`studying the change in weight due to evaporation of the
`solutions over a 24-h period at 25 °C. A surface contour
`plot of the evaporation rate versus concentrations of
`various mixture components was thus obtained.
`
`2.3. Post-application cosmetic evaluation of the
`formulations
`’
`
`Since the formulations were designed for domestic
`pets, it was essential to have (1) quick drying of the
`dose (within 1 day) after application, and (2) no drug
`residue at the site of application. However, satisfying
`these conditions became a complex issue as the rapidly
`evaporating formulations tended to leave noticeable drug
`residue on the hair coat and the solvent systems that did
`not show any drug precipitate tended to remain wet for an
`extended period of time. The experiments were focused
`on striking a balance between the rates of evaporation,
`spreading and permeation of the mixture by sequentially
`changing the concentrations of the components respon-
`sible for these attributes. Three beagle or mix-breed dogs
`were selected per treatment group and housed indoors in
`individual pens. The experimental spot-on fonnulations
`were applied on the back of the neck as one spot at a rate
`of 1.34 ml/10 kg body weight. Visual observations of the
`application sites were made 4, 24, and 48 h post-
`application. The final fonnulation was optimized so that
`no drug precipitate or wet spot was observed at the
`application site at 24 h.
`
`2.4. Stability evaluation of the optimized product
`
`The stability of the optimized product was studied to
`determine its shelf life. The formulation was packaged
`in amber glass vials with Tefion® faced rubber stoppers
`and stored at the 25 °C and 60% relative humidity (RH)
`for up to 24 months. The samples were periodically
`tested for
`the active ingredient concentrations by
`established assay methods (Fort Dodge Animal Health
`company documents).
`
`3. Results
`
`3.]. Saturation solubility study
`
`A number of solvents were evaluated with respect to
`the solubilization properties (Table 1). The solubility of
`the active ingredients varied significantly amongst
`various solvents. The solvents that dissolved high levels
`of both the actives were deemed suitable for further
`
`evaluation for formulation development.
`
`

`
`S. Sabnis er al./ Veterinary Parasitology 150 (2007) 196-202
`
`199
`
`Table 2
`
`Design matrix for optimization of vehicle system and resultant evaporation rates
`
`Absorption
`Solubilizing component
`Surface spreading
`Evaporating
`Evaporation rate
`
`enhancer (%, w/w)
`(%, w/w)
`component (%, w/w)
`component (%, w/w)
`(mg/h cmz)
`20
`10
`30
`10
`1.139
`20
`10
`25
`15
`1.050
`20
`10
`20
`20
`0.994
`20
`10
`15
`25
`0.960
`20
`15
`25
`10
`1.118
`20
`15
`20
`15
`0.963
`20
`15
`15
`20
`0.899
`20
`15
`I0
`25
`1.139
`20
`20
`20
`10
`0.990
`20
`20
`15
`15
`0.716
`20
`20
`10
`20
`0.602
`20
`20
`5
`25
`0.343
`20
`25
`15
`10
`0.758
`20
`25
`10
`15
`0.668
`20
`25
`5
`20
`0.577
`
`25 0 2520 0.546
`
`
`
`
`3.3. Past-application cosmetic evaluation of the
`formulations
`
`The concentrations of (1) solubilizing component,
`(2) evaporating/drying component, and (3) surface
`spreading component were sequentially changed within
`the boundaries identified by the dotted circle in
`evaporation rate experiment (Fig. I) discussed above.
`The cosmetic appearance 24 h after application on the
`animal’s back was then evaluated. Photos of some
`
`representative formulations are presented in Figs. 2—7.
`Fig.2 illustrates the effect ofincluding high levels of the
`solubilizing component. A distinct oily patch was
`visible even after 24 h post-application. In Fig. 3, a
`white powdery residue of the active ingredients was
`
`observed on the hair shafts at the site of application as a
`result of high levels of evaporating components. In
`Fig. 4, the effect of addition of high level of surface
`spreading component is illustrated. Here the product
`spread over a larger area on the back of the animal and
`was still wet after a 24 h. Some precipitate was also
`observed on the hair shafts. All the above conditions
`
`would render the product unacceptable by the con-
`sumer. Upon optimization of the levels of different
`components in the formulation,
`the change in the
`resultant cosmetic effect is evident in Figs. 5-7. These
`photos show the effect on dogs with different colored
`haircoat. The product appears to have essentially dried
`within 24 h with no visible product
`residue or
`precipitate.
`
`DESIGN-EX PERT Plot
`2 axis = Evaporation rutc
`X] axis = Solubilizing component 10 ~ 25% u/v
`X2 axis
`Ewaporating component 10 — 25% \\I\'
`.\'3 axis = Surface spreading component (I — 30% w/\’
`
`138'
`I07.
`
`Evaporation rate
`(nig.lir'‘.cm':)
`
`086
`0.65
`
`
`
`Selected combination range,
`where each individual
`component does not have a
`significant impact on the
`evaporation rate
`
`X1. 210%
`Soluhilnxnp: tnu: ,...n.~m
`
`x3_o7/2.-
`I-Zuipmnimg cum [HVIICIH
`
`sum: spreading
`component
`
`Fig. 1. The surface contour plot of evaporation rates of various solvent systems.
`
`

`
`200
`
`S. Sabnix et al./ Veterinary Parasitology J50 (2007) 196-202
`
`
`
`Fig.2. The cosmetic effect ofinclusion of high level of the solubiliz-
`ing component.
`
`Fig. 5. The cosmetic evaluation of the optimized formulation——-
`example 1.
`
` Fig. 6. The cosmetic evaluation of the optimized formulation—
`
`Fig. 3. The cosmetic effect of inclusion of high level of the evaporat-
`ing component.
`
`example 2.
`
`
`
`Fig. 4. The cosmetic effect of inclusion of high level of the surface
`spreading component.
`
`Fig. 7. The cosmetic evaluation of the optimized formulation-
`example 3.
`
`

`
`S. Sabnis et aL/ Veterinary Parasitology J50 (2007) 196-202
`
`'
`
`20]
`
`% W/v
`16.5
`16.1
`15.6
`15.2
`14.8
`14.3
`13.9
`
`13.4 13.0
`
`After topical application in dogs and cats, the drug
`can be transported into the haircoat, the skin itself and
`the circulation. Kramer and Mencke (2001) have
`identified the skin surface as the effective carrier of
`
`the active ingredient after dermal application and the
`one producing the ectoparasiticidal effect of imidaclo—
`prid. Furthermore,
`topical ectoparasiticidal products
`have been shown to distribute all over the body of dogs
`and cats within as little as 12h after application.
`Similar results were obtained for this formulation as
`
`well, wherein after application of the product between
`the shoulder blades of the dogs,
`the product was
`detectable at
`the base of the tail within a day and
`was detectable at significant levels even after 28 days
`post—application (DeLay et al., 2007). The other
`likely channel of distribution for the active ingredients
`is the skin. Although the transdermal uptake of the
`actives into the bloodstream was found to be negligible
`for this formulation of metaflumizone plus amitraz
`(DeLay et al., 2007),
`the possibility of distribution
`and depot of the actives in the stratum comeum
`and deeper in the epidermis cannot be eliminated.
`Low angle X—ray diffraction studies have revealed the
`lipid—protein compartmentalization in the stratum
`comeum. The intercellular spaces in the epidermal
`region are composed of lipid bilayers (Friberg et al.,
`1990). The lipid composition in dog has been reported
`to be primarily nonpolar in nature, specifically sterol
`esters (42%), wax diesters (32%), and free sterols (9%),
`while polar lipids are about 7% (Sharaf et al., 1977). As
`a result, the transport of a lipophilic moiety parallel to
`the lipid bilayers is
`fast,
`the same magnitude as
`would be seen in a liquid with minimal resistance
`(Friberg et al., 1990). This phenomenon can be further
`exploited by utilizing organic solvents
`that are
`miscible with lipids of the skin. The vehicle system
`for this metaflumizone plus amitraz formulation was
`selected to facilitate
`the distribution of active
`
`ingredients across the skin. The distribution and
`depot of the active ingredients in the stratum comeum
`and epidermal
`layers of the skin has two distinct
`advantages:
`(1) prolongation of efficacy and (2)
`minimal loss of efficacy due to bathing or because of
`rain. The selection of a balanced vehicle system
`further prevented loss of actives by minimizing their
`precipitation on the hair coat. As a result most of the
`active ingredients were partitioned into the skin layers
`producing maximum therapeutic effect. The photo-
`graphs of
`the application sites
`treated with the
`optimized fonnulation (Figs. 5-7) showed negligible
`drug residue. It was further demonstrated that
`the
`vehicles selected for this formulation also provided
`
`0.0 2.0 4.0
`
`6.0 8.0
`
`10.0 12.0 14.0 l6.0 18.0 20.0 21024.0
`Months
`
`Fig. 8. Stability of metaflumizone and amitraz spot-on formulation
`packaged in amber glass vials and stored at 25 "C and 60% RH.
`
`3.4. Stability evaluation of the optimized
`formulation
`
`Since the typical shelf life of a topical parasiticide
`product is 2 years (Rathbone et al., 2000), the shelf life
`stability of the optimized formulation was studied for 2
`years. The results (Fig. 8) show that at least 95% of each
`active ingredient remains at the end of 2 years based on
`the lower 95% confidence intervals obtained from the
`
`regression analysis (P < 0.25). Therefore, a 2-year shelf
`life is appropriate for this product.
`
`4. Discussion
`
`The criteria for selecting an appropriate formulation
`for a topical flea treatment for dogs and cats are good
`solubility of the active ingredients, good adhesion to the
`skin, good spreading properties, good local, and
`systemic tolerance, stability, and compliance within
`regulatory standards (Kramer and Mencke, 2001). The
`saturation solubility experiments with a variety of
`solvents allowed the forrnulators to choose the solvents
`
`that dissolved both the actives at high levels. At 30% w/
`v (i.e., 15% of each of the active ingredients), the drug
`loading in the formulation was quite high but this
`allowed the product to be applied at low volumes on the
`animals. This makes the formulation more appealing
`and cosmetically elegant. Our experiments involving
`higher concentrations of active ingredients did not yield
`better results. These observations corroborate well with
`
`studies involving other actives. For example, Kramer
`and Mencke (2001) have reported that a 10% solution of
`imidacloprid was more effective than a 20% or a 30%
`solution applied at
`the same dose level. The 10%
`solution spread more effectively over the skin than the
`concentrated solutions thus providing better insecticidal
`activity.
`
`

`
`202
`
`S. Sabnis er al./ Veterinary Parasitology I50 (2007) I96-202
`
`the product, which is
`adequate stability for
`essential aspect of an efficacious product.
`
`an
`
`References
`
`Anderson, M.J., Whitcomb. P.J., 1998. Find the most favorable
`formulations. Chem. Eng. Prog. 94, 63—67.
`Cornwell, P.A., Bany, B.W., 1993. The routes of penetration of ions
`and 5-fiuorouracil across human skin and the mechanisms of
`action of terpene skin permeation enhancers. Int. J. Pharm. 94,
`189-194.
`DeLay, R.L., Lacoste, E., Mazzasalma, T., Blond-Riou, F., 2007.
`Pharmacokinetics of metaflumizone and amitraz in the plasma
`and hair of dogs following topical application. Vet. Parasitol.
`150, 251-257.
`Friberg, S., Kayali, l., Margosiak, M., Osborne, D., Ward, A., 1990.
`Stratum comeum structure and transport properties. In: Osborne,
`D.W., Amann, AH. (Eds.), Topical Drug Delivery Formulations.
`Marcel Dekker, lnc., New York, pp. 29—45.
`Heaney, K., Dunney, S., Rugg, D., 2004. Use of neuronal sodium
`channel antagonists for the control of ectoparasites in home-
`othennic animals. European Patent Application EPl4l320l A2.
`
`Klink, P.R., Ferguson, T.H., Magruder, J.A., 1998. Formulation of
`veterinary dosage forms. In: llardee, G.E., Baggot, J.D. (Eds),
`Development and formulation of veterinary dosage forms. Marcel
`Dekker, Inc., New York, pp.
`l45—229.
`Kramer, F., Mencke, N. (Eds). 2001. Flea Biology and Control.
`Springer, Berlin, pp. 99-106.
`Osborne, D.W., Amann, A.l-l. (Eds), l990. Topical Drug Delivery
`Formulations. Marcel Dekker, lnc., New York, p. iii.
`Rathbone, M.J., Shen, 1., Fawcett, J.P., Ogle, C.R., Burggraaf, S.,
`Bunt, C.R., 2000. Stability testing of veterinary drug products.
`In: Rathbone. M.J., Gurny, R. (Eds.), Controlled Release Veter-
`inary Drug Delivery. Elsevier Science B.V., The Netherlands,
`pp. 333-352.
`Rugg, D., Hair, J.A., Everett, R.E., Cunningham, J.R., Carter, L., 2007.
`Confirmation of the efficacy of a novel formulation of metallu-
`mizone plus amitraz for the treatment and control of fleas and ticks
`on dogs. Vet. Parasitol. 150. 209-218.
`Shah, V.P., Behl, C.R., Flynn, G.L., Higuchi, W., Schaefer, l.H., 1992.
`Principles and criteria in the development and optimization of
`topical therapeutic products. Int. J. Pharm. 82, 2l—28.
`Sharaf, D., Clark, S., Downing, D., I977. Sldn surface lipids of the
`dog. Lipids 12, 786-790.