EXHIBIT 1016
`
`

`

`
`
`ELSEVIE
`
`Available online at www.5ciencedirect.com
`0‘ 4”
`"3' ScienceDirect
`
`Veterinary Parasitology 150 (2007) 196—202
`
`
`
`“term"
`parasitology
`
`——
`www.clsevier.com/1ocate/vetpar
`
`Topical formulations of metaflumizone plus amitraz to
`treat flea and tick infestations on dogs
`
`S. Sabnis *, J. Zupan, M. Gliddon
`Fort Dodge Animal Health, E0. 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: Metaflumizone; 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).
`
`O304-4017/$ — see front matter © 2007 Elsevier B.V. All rights reserved.
`doi:10.1016/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 formulator
`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 transdermal 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,
`
`

`

`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).
`Metaflumizone (IUPAC hydrazinecarboxamide, 2—[2—
`(4-cyanophenyl)—1-[3—(trifluoromethyl)phenyl]ethyli-
`dene]—N—[4-(t1ifluoromethoxy)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, Dermacentor 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 metaflumizone 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: (1) 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.]. 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—
`dene 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 pm nylon filter disc
`and assayed for active ingredient concentration by
`methods described elsewhere (unpublished method).
`
`2.2. Efi’ect 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% (WW) and the
`volatile component was varied between 10 and 25% (w/
`v). Sixteen mixtures were prepared in 20 m1 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. Sabnis et al./ Veterinary Parasitology 150 (2007) 196—202
`
`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 formulations
`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 formulation 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 Teflon® 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
`
`3.1. 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.
`
`Table 1
`
`Saturation solubility results of metaflumizone and amitraz in various
`solventsa
`
`Solvent
`
`Metaflumizone Amitraz
`solubility
`solubility
`(%, w/w)
`(%, w/w)
`
`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-Hexalactone
`Heptyl acetate
`Isopropyl myristate
`Isopropylidene glycerol
`N,N-Diethyl-m-toluamide
`Oleic acid
`Polyethylene glycol 400
`Propylene glycol diacetate
`Propylene glycol methyl ether acetate
`
`9.68
`11.20
`1.08
`1.57
`1.58
`0.47
`8.57
`37.13
`10.85
`5.28
`24.18
`11.38
`0.44
`1.43
`31.22
`0.73
`1.54
`12.64
`18.26
`
`23.2
`26.6
`14.1
`20.7
`24.4
`24.7
`l 1.1
`14.8
`21.4
`13.3
`31.1
`28.5
`16.2
`8.29
`33.9
`7.53
`5.10
`15.4
`25.4
`
`a The values represent means of two individual observations for
`each solvent and active ingredient.
`
`3.2. Co-solvent concentration effect an
`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.
`
`

`

`S. Sabnis et 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
`10
`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. Post-application cosmetic evaluation of the
`formulations
`
`The concentrations of (l) 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 of including high levels of the
`solubilizing component. A distinct oily patch was
`visible even after 24h 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-EXPERT Plot
`Z axis = Evaporation rate
`X1 axis = Solubilizing component 10 .. 25% w/v
`X2 axis = Evaporating component 10 - 25% wlv
`X3 axis = Surface spreading component 0 - 30% w/v
`
`
`
`
`1.28
`
`1.07
`
`0'86
`0.65
`0.44
`
`-r
`.2
`Evaporation rate
`(mg.hr .cm )
`
`
`
`Selected combination range,
`where each individual
`
`component does not have a
`significant impact on the
`evaporation rate
`
`
`X3,0%
`x1,40%
`[imprinting component
`Solubilizing corn poncnl
`
`X 2, 40%
`$urfacu spreading
`component
`
`Fig. l. The surface contour plot of evaporation rates of various solvent systems.
`
`

`

`200
`
`S. Sabm'x et al./ Veterinary Parasitology 150 (2007) 196—202
`
`
`
`Fig.2. The cosmetic effect of inclusion of high level of the solubiliz-
`ing component.
`
`Fig. 5. The cosmetic evaluation of the optimized formulation——
`example 1.
`
`
`
`”53>
`
`
`Fig. 3. The cosmetic effect of inclusion of high level of the evaporat—
`ing component.
`
`Fig. 6. The cosmetic evaluation of the optimized formulation—
`example 2.
`
`K
`
`Fig. 4. The cosmetic effect of inclusion of high level of the suxface-
`spreading component.
`
`Fig. 7. The cosmetic evaluation of the optimized formulation——
`example 3.
`
`

`

`S. Sabm's et al./ Veterinary Parasitology 150 (2007) 196—202
`
`201
`
`Lower confidence interval fiat
`owm
`
`"/o w/v
`16.5
`16.1
`15.6
`
`15.2
`14.8
`14.3
`13.9
`
`13.4 13.0
`
`0.0 2.0 4.0
`
`6.0 8.0 10.0 12.0 14.0 16018.0 20.0 21026.0
`Months
`
`Fig. 8. Stability of metafiumizone 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 formulators 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.
`
`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 formulation (Figs. 5—7) showed negligible
`drug residue. It was further demonstrated that
`the
`vehicles selected for this formulation also provided
`
`

`

`202
`
`S. Sabnis et al./ Veterinary Parasitology 150 (2007) 196—202
`
`the product, which is an
`adequate stability for
`essential aspect of an efficacious product.
`
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`
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`formulations. Chem. Eng. Prog. 94, 63—67.
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