Journal of the Arnerican Mosquito Control Association' ll(l)i29-34, 1995Copyright @ 1995 by the American Mosquito Control Association, Inc.

INSECT REPELLENT FORMULATIONS OFN.N-DIETFfYL-M-TOLUAMIDE (DEET) IN A

LIPOSPHERE SYSTEM: EFFICACY AND SKIN UPTAKE

ABRAHAM J, DOMB,' ALEXANDER MARLINSKY,MANOJ MANIAR, AND LIMOR TEOMIM

Department of Pharmaceutical Chemistry, School of Pharmacy-Faculty of Medicine,The Hebrew [)niversity of Jerusalem, Jerusalem 91120 Israel

ABSTRACT. Novel formulations for deet in liposphere microdispersion in the form of lotion wereprepared from natural solid triglycerides and phospholipids dispersed in buffer solution. The formulations"ont ini.tg 6.5, lO, and 20% Get were efective as a repellent against the common aggressive bitingmosquitois, Aedes'aegypti and Anopheles stephensi, for up to 6 h. The acute dermal absorption of thel0% ioaded formulation was conducted in rabbits using 'aCJabeled deet. 'aClabeled deet, l0% in alcoholsolution or in liposphere microdispersion was applied to the intact rabbit skin undera porous nonirritatingcover for 7 dayi. Plasma levels ofradioactivity were determined for 24 h, and daily for a total of 7 days.

The taC-deet blood levels following intravenous bolus administration were also measured. The bioavail-

ability of deet from I 0% ethanol solution was 45%, whereas the bioavailability of deet from lipospheres*"r i6%. a 3-fold reduction in the amount of deet absorbed. Examination of the rabbits during theexperiment and after necropsy showed no evidence of toxicity or irritation. The l0% deet-liposphereformulation was stable at room temperature for at least I year.

INTRODUCTION

Insects such as mosquitoes, flies, and fleas area significant factor in the spread ofserious dis-eases including malaria and encephalitis. Theyare a general nuisance to humans and animals.Efforts to repel these insect pests, other thanphysical means, are generally effective for lessthan 3 h. A common means for repelling insectsconsists of applying the compound N,N-diethyl-m-toluamide (deet) to the skin. The commer-cially available liquid deet formulations containbetween l5 and l00o/o deet and they are not rec-ommended for use on children. The toxicity ofdeet has been extensively reported and relatedto its high skin absorption after topical admin-istration (Reuveni and Yagupsky 1982, Snod-grass et al. 1982, Robbins and Cherniak 1986,Mody 1989, Mody et al. 1989, Lipscomb et al.1992, Clem et al. 1993). Previous studies havedemonstrated that as much as 50% of a topicaldose of deet was systematically absorbed (Snod-gmss et al. 1982, Robbins and Cherniak 1986,Mody 1989, Lipscomb et al. 1992). Severalmethods have been reported for increasing theduration of deet effectiveness (Gupta and Rut-ledge 1989, Raden 1989, Schreck and Kline1989). These methods used polymer solutionsthat form a film on skin, complexes of deet witharomatic proton donor carriers, or acrylate-based

t Corresponding author, afrfiated with the DavidBloom Center for Pharmacy, The Hebrew Universityof Jerusalem.

2 Present address: Houghten Pharmaceuticals, SanDiego, CA 92121.

microcapsules dispersed in oil in water emulsion.However, none showed a significant improve-ment over alcoholic solutions ofdeet (Gupta andRutledge 1989, Schreck and Kline 1989).

A novel encapsulation system, referred to asliposphere, was developed for the delivery ofbioactive compounds by parenteral injection orfor topical application (Amselem et al. 1992a,1992b; Domb 1993a, 1993b; Masters and BerdeI 994). The system is an aqueous microdispersionof all-natural, solid, water-insoluble sphericalmicroparticles of 0.2-100 pm in diameter. Theliposphere microparticles are made of hydro-phobic solid triglycerides having a layer ofphos-pholipids embedded on the surface of the par-ticle. The solid core contains the bioactivecompound dissolved or dispersed in the solid fatmatrix. The system has been effective in con-trolled delivery of antibiotics and anti-inflam-matory agents for 3-5 days after a single injection(Domb 1993a, 1993b). Lipospheres have alsobeen used for the delivery of vaccines (Amselemei al. 1992a, 1992b) and local anesthetics (Mas-ters and Berde 1994).

In an effort to develop a new topical formu-lation for deet that possesses reduced skin ab-sorption as well as an increase in the duration ofrepellency, we have encapsulated deet into li-pospheres and studied its skin absorption dy-namics and duration ofaction. We hypothesizedthat encapsulation of deet will reduce its contactsurface area with skin and reduce its evaporationrate from the skin surface, resulting in reduceddermal uptake and extended repellent activity.We report the preparation ofa deet-encapsulatedliposphere system, its repellent activity, and the

29

０ Jounxer or rxe AMTRTcAN Moseurro Corvrnol Assocr,moN Vor. ll, No. I

systemic absorption of topically administereddeet-liposphere formulations.

MATERIAIS AND METHODS

Materials: Deet was a gift from Morflex Inc.(Greensboro, NC) and contained 95olo N,N-di-ethyl-m-toluamide and 5olo related isomers. Ra-diolabeled deet (carbonyl C-14) 12.92 mCi,/mmolwith a radiochemical purity of > 98.60lo was pur-chased from New England Nuclear (Boston, MA).Hydrogenated vegetable oil (Sterotex@, meltingpoint 65'C) was purchased from Capital CityProducts (Columbus, OH). I€cithin (Centrolex-D@) was purchased from Central Soya (FortWayne, IN). Dibasic sodium phosphate andmonobasic potassium phosphate were purchasedfrom EM Science (Cherry Hill, NJ). Propylpar-aben and methylparaben were obtained fromNapp Chemicals Inc. (Lodi, NJ). All water usedin this study was deionized. Absolute ethanolwaspurchasedfrom Quantum (Newark, NJ). NewZnaland white rabbits, 16 males weighing 4 kg,were purchased from Myrtle's Rabbitry (JacksonStation, TN).

Preparation of liposphere formulations.' For-mulations containing l09o deet were prepared bythe addition of deet (10 g) and propylparaben(0.05 g) to 12.5 g of melted hydrogenated vege-table oil, followed by the addition of 72.5 mlphosphate buffer (pH 7.4, O.O5 M) containingmethylparaben (0.10 g) and lecithin (5 g). Themixture was homogenized for 2 min using a Sil-verson L4 homogenizer (Silverson Machines,Waterside, England) to form a homogeneous dis-persion and then cooled to room temperature toform a smooth, easy-to-apply lotion. Formula-tions containing 0 and 6.59o deet were preparedsimilarly; the formulation containing 20o/o deetwas prepared using 20% hydrogenated vegetableoil.

Preparation of radiolabeled doses.' The intra-venous doses contained 160 pCt/8O mg of deet.The deet-liposphere formulation contained 20pCi per gram of l09o formulation (20 ttCt/lDOmg deet). The radiolabeled deet liposphere for-mulation was prepared from deet (20 pCt/100mg), hydrogenated vegetable oil, phosphate bufler, and phospholipid as described above. Thealcoholic solution was prepared by the dilutionof radiolabeled deet with ethanol to form a l0o/osolution containing 2O pCi/lOO mg deet.

Characterization of lipospheres: Particle sizeof the liposphere formulations was determinedusing a Coulter LSl00 particle size analyzer. TheSEM analysis was conducted on a Philips 70scanning electron microscope. The content ofthephospholipid on the surface oflipospheres wasdetermined by the trinitrobenzenesulfonic acid(TNBS) method using liposphere formulations

containing phosphatidylethanolamine (PE) (New1990). Deet content in lipospheres was deter-mined by gas chromatogaphy (GC) conductedon a Hewlett Packard 5890 GC with a HP 33964integrator and a flame ionization detector.An HP Ultra2 (25 m x 0.2 mm x 0.ll mm),methyl phenyl silicone 506 column was used. Thefollowing conditions were used: detector and in-jector temperatures were 260'C. column tem-perature was 150"C, and helium gas at 40 psi.Viscosity measurements of formulations wereconducted using an RTV Brookfield viscometer(Brookfield I-abs., Stoughton, MA) using a Hal-ipath spindle # B.

S t orage stability : T en prcent deet-liposphereformulations (10 g) packed in l0-ml glass con-tainers were stored at 4'C and 25'C in 600lo hu-midity cabinets and the particle size, deet con-tent, viscosity, and appearance were monitoredfor 12 months. Samples were analyzed in trip-licate at 0, l, 3, 6, and, 12 months of storage.

Eftcacy stu.dies: The residual efficacy of li-posphere formulations was evaluated on 3 vol-unteers who had given informed consent. Theformulations were applied to the skin and ex-posed to mosquitoes. The time of 100% repel-lency (zero biting) was the index for determiningthe effectiveness of a formulation.

The formulations were applied on 4locationson the arm of volunteers at a concentration of2.5 mg/cm2 on a total area of 12 cm2 skin surface.Mosquitoes were placed in a screen-bottomed(18 mesh netting, l0-cm2 exposure arca) l-oz.cylindrical cup, made of clear polymethacrylate,containing l5 5-I5-day-old female mosquitoesdisplaying host-seeking behavior with access tothe skin through the netting. The forearm wasplaced on the mosquito netting for l0 min every30 min and the number of biting mosquitoes(evident by a blood meal) was recorded. Prior toany efficacy experiment the mosquitoes weretested on untreated skin to confirm their host-seeking behavior. Two mosquito species weretested: Aedes aegypti (Linn.) and, Anopheles ste-phensi Liston, both aggressive biters.

Skin absorption studies: The pharmacokineticprofile of deet in rabbits was determined afterintravenous administration (Group IV) and top-ical administration of l0o/o deet in ethanol (GroupA) and l0o/o deet in lipospheres (Group B). Six-teen rabbits were used in the study; 8 rabbitsreceived intravenously 2O p.Cr/lo mglkg of ra-diolabeled deet in alcohol (Group IV). After awashout period of 3 days, 8 of the 16 rabbitsreceived a l-g/kg topical dose of l07o deet inethanol (Group A) and 8 rabbits received a l-glkg topical dose of l09o deet in lipospheres (GroupB). A summary of the experimental groups isshown in Table l.

Preparation of the rabbits for the study: TllLe

M A R C H 19 9 5 DeE-r REpEus.rr rN l LrpospnsRE Sysrprrr

Table l. Experimental groups used in the rabbit skin absorption study.

Experimental group

Intravenous (IV) Ａ

AdministrationNo. of rabbitsFormulationDose administered

(formulation)Dose administered (deet)Dose administered (pCi)

IV ―b lo od lev els

8

10 0//o in alcoh o l

O .l m 1/kg10 m g/kg20 μC i

top ical- lip o sp h ere

8

100//o in lip o sp here

l g/kg10 0 m g/kg20 μC i

to p ical――alco h ol

8

109/o in alco h o l

l y 雑10 0 m g/kg20 μC i

animals were housed in metabolic cages for the7 days of the study and allowed free access tofood and water. Prior to application ofthe topicaldoses, the back and neck of the rabbits wereshaved. On the day of the study, the adminis-tration area was outlined with a 0.25-in.-highstrip of polystyrene tape (secured to the animalwith paper tape) in order to form a barrier forthe dose.

Administration of radiolabeled doses: Intra-venous doses containing one-tenth ofthe deet ofthe topical dose, but the same amount of radio-activity, were administered via the marginal earvein over 15 sec and flushed with 0.906 NaCl.Topical administration was accomplished by ap-plication of the material to a 64-cm2 square areaof rabbit skin, which approximates l0o/o of thebody surface area in a 4-kg rabbit. Rabbits re-ceived deet-liposphere formulation dose of Igkg over a 8 x 8-cm2 skin area. A nonocclusivecloth was then secured to the top ofthe barrierwall in order to collect evaporated deet, and thebody of the rabbit wrapped to prevent removalof the patch. An Elizabethan collar was placedon the rabbit to inhibit animal access to the ad-ministration area. The residual dose was re-moved from the skin by washing and quantitatedafter 24 h.

Collection of samples: A heparin lock wasplaced in the ear of the rabbit for collection ofthe first several blood samples after administra-tion ofthe intravenous and topical doses. Beforedrawing the samples, the lock was cleared ofhep-ainized saline. Samples of 1.0 ml of blood weredrawn at 0, 5, 10, 15, 30, 45,60, and 90 min,and at 2, 3, 4, 6, 6.5, 7, 7.5, 8, 9, and l0 h forthe intravenous doses. A sample was drawn im-mediately before administration of the topicaldose for validation of the washout period. Aftertopical administration, blood samples were drawnat 0, 5, 15, 30, 45,60,90, 120,150, 190, and210 min, andat4,5,6,8, 12, 16, and 24 h, anddaily for a total of7 days.

Urine was collected 24h after intravenous ad-ministration and after l, 2, and,3 days in each

of the rabbits administered a topical dose. Therabbits were sacrificed by isoflurane overdose af-ter 7 days. The application sites of the skin werecollected and frozen at -70'C until analysis.

Determination of radioactivity in specimens:The specimens (blood, urine, skin, patch, andwashings from dose removal) were homogenizecand a known aliquot treated with tissue solubi-lizer (Soluable@, New England Nuclear) and de-colorized with 30% hydrogen peroxide (Sigma,St. Louis, MO) according to the manufacturer'sdirections. The samples were then suspended inscintillation cocktail and the radioactivity quan-titated by liquid scintillation counting. Scintil-lation counting was performed according to stan-dard operating procedures on a BeckmanLS6000 I C liquid scintillation counter (BeckmanInstruments, Colombia, MD) equipped with in-ternal quench and eftciency adjustments. Eachsample was analyzed in triplicate and the meantaken as the true value.

Data analysis.' The recovery of the radioac-tivity administered was calculated based uponthe radioactive content ofthe organs tested, skin,patch removed, and urinary excretion. The ab-solute bioavailabi-lity was computed from the areaunder the curve (AUC) of a plot of radioactivecontent in the blood stream versus time, usingpiecewise trapezoidal integration. The meanAUCs of the group of rabbits receiving the top-ical applications were compared to the AUCs ofthe group of animals receiving the intravenousadministration (each was corrected for dose) us-ing the following formula:

9o absolute bioavailability: {(AUC [topical/(AUc UVD] x 100.

RESULTS AND DISCUSSION

Preparation and characterization of dea- load-ed liposp heres : The liposphere microdispersionscontaining deet incorporated in solid triglycerideparticles were prepared by the melt method usingcommon natural ingredients in one step without

M A R C H 19 9 5 DSET REppu.evr rN I LrpospnsRE Svsrpu

Timeafter

appli-

Table 3. Repellency effectiveness ofdeet-liposphere formulations.'

70000

官 60000

言 500000. 4000 0

呂 300001~ 20000

― Er EthanoL Top cal

― EthanoL IV

Time (hours)

Fig. 3. Blood concentrations @PM/ml) after in-travenous and topical administration of l0% deet inethanol solution. Ten percent deet solution (l ml, 20pCt/ml) administered intravenously (IV) or topically(surface area 64 cm2) to rabbits. Results are average of8 rabbits.

also based on the assumption that the rate ofelimination is not dose dependent and that elim-ination after bolus intravenous administration isthe same as elimination after topical adminis-tration.

Table 4 summarizes the total urinary contentof taC after intravenous and topical administra-tion of deet formulations. About 74o/o of tlrleIV-administered dose was collected in the urine, and39 and l9Vo of the topically administered doseswere collected for the alcoholic and liposphereformulations, respectively. Optimal collection ofurine was difficult in the metabolic cages used inthis experiment as evident from the large vari-ation in urine collection (74 + 23).Assumingthat the error in urine collection is similar in allexperiments, the difference in radioactivity con-tained in the urine after topical administrationofthe liposphere dosage form is about 50% thatofthe alcoholic dose, which corresponds to theblood bioavailability calculations. The totalamount of deet recovered from skin (washing ofresidual dose and extraction from skin) was sim-ilar for both formulations, indicating that bothformulations were similarly exposed to the skin

Number biting Aedes aegyptiand, A nopheles stephensi

０２０６

】

ｎ

　ｎ

Ｃａ

・的

．皿

U n ―

trcat, C o n‐

ed 2 tr012

o/o deet informulation Refer-

ence3

15 2 (5

30 5 (4

60 3 (5

90 3 (5

120 5 (5

150 5 (3

180 3 (5

2 10 4 (4

24 0 5 (3

2 70 3 (4

30 0 4 (6

380 5 (6

0 (0 ) 0 (0) 0 (0)0 (0) 0 (0 ) 0 (0)0 (0) 0 (0 ) 0 (0)0 (0) 0 (0 ) 0 (0 )0 (0) 0 (0 ) 1 (1)0 (0) 0 (0 ) 0 (1)0 (0) 0 (0) 1 (2)0 (0 ) 0 (0) 1 (1)0 (2) 0 (0) 2 (1)0 (2) 0 (0)1 (2) 0 (0)

0 (0)

4 (0 )5 Q )5 (3)5 (3)5 (5)5 (5)4 (4)5 (3)3 (4)4 (3)4 (5)5 (5)

0 (0)0 (0 )0 (0 )0 (0 )0 (0 )0 0 )

トロ凹０

(1)(1)(2)

2 (2)3 (2)

' Cups with mosquitoes were placed for l0 min every 30min on the skin of volunteers treated with formulation (2.5mglcm2), and the number ofbites were recorded. The resultsare average of 3 independent tests (cup placements), fie resultsin parentheses arc of An. stephensi.

2 Untreated test was without any treatment, Control was adeet-free liposphere formulation.

3 Reference formulation was 1096 by weight ofdeet in eth-anol.

deet l0o/o in lipospheres (Group B). Figure 4 rep-resents the blood concentration following topicaladministration data from Fig. 3 so that they maybe compared directly on an expanded concen-tration axis. The AUC after topical dosing was24,494 DPM/ml for Group A (l0o/o alcohol so-lution) and 8,444 DPM/ml for group B (lipo-sphere formulation). Therefore, the absolute bio-availability ofdeet from a l0% ethanol solutionwas 45olo, whereas the bioavailability from li-pospheres was only 160/o, a 3-fold reduction inthe amount ofdeet absorbed. This result is basedon the assumptions that the absorption of deetfrom lipospheres and ethanol vehicles is a com-parison of absorption only. The conclusion is

Table 4. Recovery of taC-deet in urine and skin of rabbits after intravenous (IV) and topicaladministration of alcoholic or liposphere formulation.t

Percent recovery + SD

Dose type Urine sttn 2 T o talIV ‐―alco h o l

T o p ical―alco h o l

T op ical- lip o sp h crc

74 ± 2 3

39 ± 5 (53)19 ± 8 (2 6)

74 ± 2 3３

９

一　＋一＋一

２

２

± 3 (6 5)± 8 (38)

I Results are % of administered dose, the numbers in parentheses are the corrected values for 74% recovery after intravenousadministration.

' Combined radioactivity from washings and skin tissue content.

Jourxer or rHr Arr,cnrcex Moseurro Corvrnol Assocr,r.nox

一Ｅヽこ」０）

Ｏｏｏ一０

こ↓トロロ０

A E ] ] . m

. U f f i

1 6

Time (hours)

Fig. 4. Blood concentrations (DPM,/ml) after top-ical administration of I 0% deet in ethanol or liposphereformulation. Formulations l0% containing deet (l ml,20 pCilml) administered topically (surface area 64 crn2)to rabbits. Results are average of 8 rabbits.

and thus the results are comparable. The non-recovered deet after topical administration isprobably due to evaporation of deet from thenonocclusive patches to the environment. Thelower deet recovery of the liposphere formula-tion can be interpreted as more deet is releasedto the environment resulting in more repellentactivity.

Conclusions: Encapsulation ofdeet in a novelstable liposphere microdispersion system result-ed in improved efficacy and reduced dermal ab-sorption. Deet-containing lipospheres (10%) wereeffective against mosquitoes for at least 3.5 h.The deet absorption through skin from these for-mulations was a third of that from alcoholic so-lution for the same concentration. The improvedefficacy and reduced dermal absorption of deet,in addition to its composition of only 1006 deetcontent encapsulated in common natural ingre-dients and the simple method of preparation,make this formulation attractive for consumeruse.

ACKNOWLEDGMENTS

This study was supported by LittlePoint Cor-poration, Cambridge, MA. We thank JosephKnowles, Marie Rock, and Denise Hannibal fortheir contribution.

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Clem, J. R., D. F. Havemann and M. A. Raebel. 1993.Insect repellent (N,N-diethyl-m-toluamide) cardio-vascular toxicity in an adult. Ann. Pharmacother.27:289-293.

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Gupta, R. K. and L. C. Rutledge. 1989. Iaboratoryevaluation of controlled release repellent formula-tions on human volunteers under three climatic reg-imens. J. Am. Mosq. Control Assoc. 5:52-55.

Lipscomb, J. W., J. E. Kramer and J. B. Leikin. 1992.Seizure following briefexposure to the ins€ct repel-lent N,N-diethyl-m-toluamide. Ann. Emerg. Med.2l:315-317.

Masters, D. and C. Berde. 1994. Drug delivery toperipheral nerves, pp. 443-455. fn: A. J. Domb (ed.).Polymer site-specific pharmacotherapy. Wiley,Chichester.

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New, R. R. C. 1990. Liposomes-a practical ap-proach. Oxford Univ. Press, Oxford.

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Schreck, C. E. and D. L. Kline. 1989. Personal pro-tection aforded by controlled release topical repel-lents and permethrin-treated clothing against naturalpopulations of Aedes taeniorhynchus. J. Am. Mosq.Control Assoc. 5:77-80.

Snodgrass, H. L., D. C. Nelson and M. H. Weeks.1982. Dermal penetration and potential for placen-tal transfer of the insect repellent N,N-diethyl-m-toluamide. Am. Ind. Hyg. Assoc. J.43:747-753.