Journal of Dermatology and Clinical Research

Transcription

1 Centrl Journl of Dermtology nd Clinicl Reserch Reserch Article Compositions for Prevention/ Prophylctic Tretment of Poison Ivy Dermtitis Mhmoud A. ElSohly -3 *, Wseem Gul,, Mohmed S. Adel-Bkky 4, Susn P. Mnly nd Mohmmd K. Ashfq ElSohly Lortories, Inc., 5-Industril Prk Drive, USA Ntionl Center for Nturl Products Reserch, University of Mississippi, USA 3 Deprtment of Phrmceutics nd Drug Delivery, University of Mississippi, USA 4 Deprtment of Phrmcology, Al-Azhr University, Ciro, Egypt *Corresponding uthor Mhmoud A. ElSohly, ElSohly Lortories, Inc., 5 Industril Prk Drive, xford, MS 38655, United Sttes of Americ, Tel: ; Fx: ; Emil: Sumitted: 7 July 8 Accepted: 4 August 8 Pulished: 5 August 8 Copyright 8 ElSohly et l. PEN ACCESS Keywords Prophylctic; HDC-Phenyllninte; HDC- Hemiglutrte; HDC-4-(4-Aminophenyl)-Butyrte; Urushiol Astrct Allergic contct dermtitis (ACD) to poison ivy nd relted species of the fmily Ancrdice is mjor cuse of occuptionl dermtitis in the United Sttes. Although the condition is not life thretening, it imposes significnt risks to individuls with outdoor occuptions nd those engged in outdoor ctivities. This results in significnt helthcre costs nd loss of productivity. Efforts to prevent poison ivy ACD hve een pursued for yers, nd trils to produce desensitiztion through dministrtion of smll doses of the llergens (urushiols) hve een lrgely unsuccessful nd therefore, ndoned. Topicl locking gents pplied to the skin to protect it from contct with the urushiols re of limited vlue nd require ppliction efore exposure to the llergen. Development of n effective prophylctic tretment to poison ivy ACD is, therefore, of high vlue. This mnuscript reports on the development of wter solule derivtives of the sturted congener on the poison ok urushiol (3-n-heptdecylctechol, HDC) for prophylctic tretment of poison ivy ACD. Three compounds were prepred, nmely the HDC-phenyllninte (), the HDC-hemiglutrte (), nd the HDC-4-(4-minophenyl)-utyrte (3). These derivtives were shown to produce tolernce ginst sensitiztion to poison ivy urushiol in guine pigs (the niml model for llergic contct dermtitis) when dministered prior to sensitiztion of nimls (i.e. nïve guine pigs). Furthermore, upon IM dministrtion of compound 3 to nimls with estlished sensitivity to poison ivy, the product resulted in lmost complete desensitiztion to urushiol. This suggests tht these compounds re good cndidtes for development s prophylctic tretment for poison ivy ACD. ABBREVIATINS ACD: Allergic Contct Dermtitis; ACS: Americn Chemicl Society; EtH: Ethnol; HDC: 3-n-Heptdecylctechol; HDC-APB: HDC-4-(4-minophenyl)-utyrte; HDC-HG: HDC-Hemiglutrte; IM: Intrmusculr INTRDUCTIN Contct dermtitis to poison ivy (Toxicodendron rdicns), poison ok (T. diversiloum), nd poison sumc (T. vernix) ffects -5 million Americns every yer [] nd is the primry cuse of occuptionl dermtitis in the United Sttes []. The prevlence of poison ivy nd poison ok sensitivity in the generl dult popultion rnges from 5% to 7% [3,4]. Pek frequency for sensitiztion occurs etween ges 8-4 [5]. Genetic susceptiility to urushiol sensitivity suggests tht 8% of children who re orn to two urushiol sensitive prents will ecome sensitive [6]. utdoor ctivities s well s outdoor occuptions such s fire fighting, forestry nd griculture re t high risk for incurring significnt medicl expenses nd worker s disility. Ech fire seson, pproximtely one third of forestry workers in Cliforni, regon nd Wshington re disled y poison ok dermtitis [7]. This disorder is very well known to most emergency nd primry cre physicins nd dermtologists [8]. ther gener of the plnt fmily Ancrdicee with dermtogenic constituents include Ancrdium (cshew nuts), Semicrpus (Indi ink tree), Metopium (poison wood), nd Mngifer (mngo). The llergenic components in most of these plnts re 3-n-lk-(en)-yl ctechols with C-5 or C-7 side chins nd different degrees of unsturtion (-3 olefinic onds) [9- ]. Urushiol is typicl of such llergenic components present in poison ivy, poison ok, nd the Asin lcquer tree [3]. It hs ctechol ring sustituted with C5 or C7 hydrocron chin t the 3 or 4 positions, either sturted or hving one, two or three unsturted onds [4]. Both the ctecholic ring nd the liphtic chin re proven to ply importnt roles in llergenicity of urushiols [5-7]. Contct of these ctechols with the skin of susceptile individul s results in sensitiztion to ll urushiols of the plnt fmily Ancrdicee [8]. nce sensitivity is developed, it is difficult, if not impossile, to eliminte. Current tretment for contct dermtitis is primrily symptomtic. For ptients with severe cses, tpering dose of orl corticosteroids such s prednisone my e used. Prednisone is corticosteroid hormone (glucocorticoid) which suppresses the immune system s response to vrious diseses to reduce symptoms such s swelling nd llergic-type rections. However, ville dosepcks of corticosteroids re of little use since they deliver smll doses of corticosteroid for too short period of time nd often result in reound rection [9]. Cite this rticle: ElSohly MA, Gul W, Adel-Bkky MS, Mnly SP, Ashfq MK (8) Compositions for Prevention/Prophylctic Tretment of Poison Ivy Dermtitis. J Dermtolog Clin Res 6(): 5.

2 Centrl There hve een multiple desensitiztion regimens since the 95 s using extrcts of poison ivy/ok; none re relily effective [,]. The techniques consisted of ingestion or prenterl injection of vrious formultions of urushiol. Although some reports descried success, the level of desensitiztion ws vrile nd not durle. In ddition, the regimens produced inflmmtion of mucous memrne, cutneous, nd systemic side effects, hence, this pproch hs een lrgely ndoned. The precutneous sorption of urushiol is similr to tht of other lipophilic sustnces. These molecules preferentilly enter the skin through the intercellulr lipids of the strtum corneum. Any sustnce tht locks the contct of urushiol with the strtum corneum nd prevents its entry would likely offer some protection. Mny commercil products hve een developed nd tested for their effectiveness in preventing urushiol dermtitis, nd these experiments hve een pulished [,-8]. Presently, only few sustnces offer some relistic enefits. Furthermore, the consumer of these products is ssumed to hve prior knowledge tht the person will e exposed to poison ivy so topicl ppliction could e pplied to the skin eforehnd. Such is not the cse with most situtions when poison ivy is encountered. Therefore, the development of prophylctic tretment for poison ivy dermtitis is more relistic nd more effective strtegy, if it could e chieved. Hyposensitiztion y dministrtion of plnt extrcts is not regulrly otined. It requires lrge doses nd months or yers to e produced, nd sensitivity is rpidly regined upon cesstion of such tretment [8, ]. The enefits nd sfety of the use of Rhus extrcts (contining the ctive llergenic ingredient urushiols) for these purposes hve een topics of dispute since they were first dministered in 97. Severl reviews pertining to the clinicl use of Rhus extrcts nd llergens hve een written [,9,3]. The reson for the lck of ctivity of dministered urushiols in the free form might e due to the high rectivity of the ctechol moiety of the urushiols with plsm proteins. nce sored, the urushiols ind irreversily with the proteins nd ecome dectivted. From our erlier studies, we concluded tht it might e necessry for the urushiols to ind to cell memrnes to e effective in the production of tolernce or the prophylctic tretment of poison ivy dermtitis. Thus, conjugte of poison ivy urushiol ound to cell memrnes ws prepred y spiking the urushiol solution into suspension of lood cell memrnes from lyzed nd wshed lood cells nd then re-injected the suspension into donor nimls [8]. We hve shown in tht study tht tolernce ws produced y the dministrtion of 3-n-pentdecylctechol (the sturted congener of poison ivy urushiol) coupled to red lood cell memrnes in guine pigs. The treted group ws tolernt to 3-n-pentdecylctechol for the weeks of the study. Hving succeeded in tht pproch, we theorized tht dministrtion of n urushiol ester might e more effective in tht some of the ester could hydrolyze t the surfce of the lood cells resulting in free urushiol which could ind to the memrne. Tolernce to poison ivy urushiol in the guine pig model ws ccomplished y IV injection of the dicette esters of poison ivy nd ok urushiols in nϊve guine pigs, nd complete desensitiztion or hyposensitiztion ws ccomplished in sensitized nimls y the sme tretment [3]. The efficcy of ElSohly et l. (8) Emil: orl dministrtion of poison ivy nd poison ok urushiols ws compred with the use of the respective esterified derivtives for desensitizing sensitive guine pigs [3]. The esterified derivtives produced greter degree of hyposensitiztion thn ws produced y the free urushiols. However, the effect of the orlly dministered preprtion ws not sustntil. We hve concluded tht for the urushiol esters to e most effective, prentrl dministrtion is necessry. We, therefore, conducted study to evlute the potentil for single-dose regimen to e effective for hyposensitiztion to poison ivy urushiol dermtitis [33]. Hyposensitiztion ws ccomplished in single intrmusculr dose of mg. This puliction dels with the development of group of polr derivtives of urushiol (or its sturted congener) (Figure ) tht would e dministered s slts of these derivtives in n queous solution for successful product for the prevention of poison ivy/poison ok contct dermtitis. Furthermore, this product could e used to provide tolernce to urushiols sensitiztion when dministered to nïve individuls (i.e. prevents sujects from ever eing sensitized to urushiols upon first exposure), or to provide desensitiztion to urushiol (i.e. locks the rection of sensitive individul upon exposure to urushiol) when dministered to lredy sensitized individuls. MATERIALS AND METHDS The novel gents (Figure ) were synthesized, purified, nd chrcterized y spectrl nlysis techniques following the procedures outlined in US Ptent #8,486,998 B [34]. A guine pig contct dermtitis model ws used for in vivo efficcy studies. Chemicls All chemicls nd solvents were of ACS grde (Sigm-Aldrich, Milwukee, WI, USA). Animls Hrtley strins of guine pigs (n=4) were otined from Hrln (Indinpolis, IN 469). The nimls were divided into 5 groups (n=8/group) nd treted s descried elow. These nimls were kept under controlled environment with hour dy nd night cycle nd provided feed nd wter d liitum. The niml study protocol ws pproved y the IACUC of the University of Mississippi (IACUC protocol# 6-7). Study design (See digrm in Figure ) A. Production of tolernce to poison ivy/ok urushiol in nïve guine pigs using wter solule derivtive of 3-n - heptdecylcdetol: The 5 groups of nimls were treted s follows: Group I. Animls in this group were given 3 μl of the compound ELI (HDC-APB) (3) vi the intrmusculr (IM) route, in ech hind leg, contining the equivlent of 3 mg HDC/leg. Two weeks lter, these nimls were sensitized with urushiol ( μl cetone contining. mg of urushiol) on the skin of the dorsl side of the neck region. Two weeks lter, the nimls were chllenged with urushiol (5 μl volume cetone contining 3. μg, or 4.5 μg or 6. μg) on the dominl skin. The vehicle ws 5μL of cetone (see Figure 3). The nimls were J Dermtolog Clin Res 6(): 5 (8) /8

3 Centrl chllenged three times fter sensitiztion. (viz; first chllenge (Test #) t two weeks post sensitiztion, second chllenge (Test #) conducted t four weeks post sensitiztion nd third chllenge (Test #3) ws conducted twelve weeks post sensitiztion). Group II. Animls were given 3 μl of the compound ELI (HDC-phenyllninte) () vi the IM route in ech hind leg, contining the equivlent of 3 mg HDC/leg. This ws followed y sensitiztion nd then chllenging following the sme procedure s for Group I. Group III. Animls were given 3 μl of the compound ELI--83 (HDC-HG) () vi the IM rout in ech hind leg, contining the equivlent of 3 mg HDC/leg. This ws followed y sensitiztion nd then chllenging following the sme procedure s for Group I. Group IV. Animls were given 3 μl of vehicle (5% Ethnol) vi the IM route in ech of the hind legs. This ws followed, two weeks lter, y sensitiztion nd dominl skin test s descried for Group I. Group V. Animls in this group were given PBS (3 μl in ech of the hind legs) vi the IM route. Two weeks lter, the nimls were sensitized nd then chllenged following the sme protocol descried for Group I. B. Desensitiztion to poison ivy/ok urishiol of lredy sensitive guine pigs using wter solule derivtives of 3-n-heptdecylctechol: The control niml groups, Groups IV nd V, vehicle nd phosphte uffer control groups respectively, from the tolernce study (untreted with the derivtives), with estlished sensitivity (see Figures 4 nd 5) were used in this study to determine if IM dministrtion of one of the wter solule derivtives would desensitize lredy sensitive nimls. Animls of Group IV were given 6 μl of vehicle divided into two doses of 3 μl in ech hind leg. Animls of Group V were injected with 6 μl (3 μl in ech hind leg) of ELI (3) solution contining the equivlent to mg/ml of HDC (6 mg/niml dose). After rest period of pproximtely weeks, nimls in oth groups were chllenged y topicl ppliction of 3 doses of urushiol (3. μg, 4.5 μg, nd 6. μg ech dissolved in 5 μl of cetone). At the 4, 48, nd 7 hours post topicl chllenge, skin lesions were oserved nd scored. The severity of erythem nd edem ws oserved nd scored ccording to the Drize scoring system [35] s shown in Tle. The scores were recorded t the 4, 48, nd 7 hrs post urushiol skin ppliction. Sttisticl nlysis Erythem-edem scores for different groups were nlyzed y one-wy ANVA followed y the Tukeys test for multiple comprisons using the Grph Pd prism 5. softwre (L Joll, CA). A P-vlue of less thn.5 ws considered to show significnt difference in the treted nd untreted groups. RESULTS AND DISCUSSIN In previous investigtions [34,36-37], we hve shown tht tolernce, s well s desensitiztion, re ccomplished y the injection of the cette esters of poison ivy urushiol. In H N H N NH C 7 H 35 HDC-Phenyllninte () (ELI--78-) H ElSohly et l. (8) Emil: NH C 7 H 35 HDC-Hemigluturte (HDC-HG) () (ELI--83) C 7 H 35 HDC-4-(4-minophenyl)utyrte (HDC-APB) (3) (ELI--57-3) NH Figure Structures of HDC-Phenyllninte (ELI-7-78-) (), HDC-Hemigluturte (HDC-HG) (ELI--83) (), nd HDC-4-(4- minophenyl) utyrte (HDC-APB) (ELI--57-3) (3). Figure Schemtic study design showing tretments given t vrious times during the experiment. 6. µg urushiol 5 µl Acetone H 4.5µg urushiol 3.µg urushiol Figure 3 Sites on the dominl surfce of skin for ppliction of urushiol chllenge doses dissolved in 5 μl cetone vehicle. continution of this work, nd to develop etter drugs for the prophylctic tretment of contct dermtitis cused y poison ivy, poison ok, nd relted species of the fmily Ancrdicee, we hve prepred wter solule slts of mino cid esters nd dicroxylic cid esters of the sturted congener of poison ok nd tested their ility to convey tolernce nd desensitiztion, J Dermtolog Clin Res 6(): 5 (8) 3/8

4 Erythem+Edem score 4 h Erythem+Edem score 48 h Erythem+Edem score 7 h Centrl Acetone 3ug 4.5ug 6ug A Figure 4 (A-C): Test # -Guine pigs in different groups were injected IM with different tretments: Group I: HDC-APB (ELI--57-3), Group II: HDC-phenyllninte (ELI--78-), Group III: HDC-HG (ELI--83), Group IV: 5% EtH, nd Group V: PBS. Two weeks post tretment; guine pigs were sensitized with. mg urushiol on neck re. Two weeks fter sensitiztion, the guine pigs were chllenged with cetone, or cetone contining 3., 4.5, or 6. µg urushiol t ech of the four dominl sites. Erythem+edem scores for the nimls were tulted. Dt represents group mens ± SEM. Significntly different from Group V (PBS) t the sme chllenge dose Significntly different from group IV (5%EtH) t the sme chllenge dose using the guine pig model of contct dermtitis. Three derivtives of n-heptdecylctechol (HDC) were prepred, nmely the phenyllninte (), the hemiglutrte () esters, nd the 4-(4-minophenyl)-utyrte (3) (Figure ). These compounds were formulted in queous solutions contining 5% ethnol t the equivlent of 6 mg/ml of HDC for ech derivtive. Two studies were crried out using 5 groups of nimls (8 guine pigs/group). Study #: tolernce study A) Production of tolernce to poison ivy/ok urushiol in nïve guine pigs using wter solule derivtives of 3-n-heptdecylctedol: Groups I-III were injected with the three derivtives (one derivtive/group) t totl dose of 6 mg/niml (.3 ml of the derivtive solution per hind leg of the niml for totl dose of.6 ml). Group IV ws injected with the vehicle B C Erythem+Edem score 4 h.5.5 Erythem+Edem score 48 h.5.5 ElSohly et l. (8) Emil: nd group V with phosphte uffer s controls. Two weeks fter dosing, ll nimls were skin sensitized with poison ivy urushiol ( mg urushiol pplied to the shved dorsl side of the neck). Susequently, the nimls were checked for sensitiztion y the ppliction of three doses of urushiol (3, 4.5, nd 6 µg ech in 5 μl of cetone) to the shved dominl skin. A solvent control ws lso used. Three tests for induction of tolernce were crried out s follows: Test #: In the first test, nimls were chllenged two weeks fter sensitiztion. Guine pigs in groups IV nd V (PBS or 5% ethnol tretment, respectively) showed vrying degrees of erythem nd edem with urushiol chllenge doses of 3., 4.5, nd 6. μg. In contrst, guine pigs in group I (ELI--57-3) showed only slight rection t the 6. μg urushiol chllenge. Animls in group II (ELI--78-) nd group III (ELI--83) showed no rection t 3. nd 4.5 μg urushiol chllenges. In Group I, t the 6. μg urushiol chllenge, very slight rection ws oserved. Erythem+Edem score 7 h cetone 3 ug 4.5 ug 6 ug Group V Group IV Group III Group II group I A B C Figure 5 (A-C): Test # - Guine pigs in different groups were injected IM with different tretments: Group I: ELI--57-3, Group II: ELI- -78-, Group III: ELI--83, Group IV: 5% EtH, nd Group V: PSB. Two weeks post tretment; guine pigs were sensitized with. mg urushiol on neck re. Four weeks fter sensitiztion, the guine pigs were chllenged with cetone, or cetone contining 3., 4.5, or 6. µg urushiol t ech of the four dominl sites. Erythem+edem scores for the nimls were tulted. Dt represents group mens ± SEM. significntly different from Group V (PBS) t the sme chllenge dose. significntly different from group IV (5% EtH) t the sme chllenge dose. J Dermtolog Clin Res 6(): 5 (8) 4/8

5 Centrl Tle : Drize scoring system used to score the severity of erythem nd edem. Skin Lesion served Score No Erythem Very Slight Erythem (rely perceptile) Well Defined Erythem Moderte to Severe Erythem 3 Severe Erythem (eet red) Eschr Formtion (deep 4 injury) No Edem Very Slight Edem (rely perceptile) Well Defined Edem (edges of re defined y definite rising) Moderte Edem (re rised pproximtely mm) 3 Severe Edem (rised more thn mm nd extending 4 eyond re of exposure) Mximum Summed Erythem nd Edem Scores = 8 A sttisticlly significnt (P <.5) reduction in skin rection scores ws oserved in the treted niml groups I, II, nd III, scompred with those of the control groups IV nd V (Figure 4A). At 4 hours, the skin rection scored in the vehicle treted group ws lower thn tht of PBS treted t the chllenged score of 3. nd 4.5 μg urushiol. However, there ws no difference in the scores of these two groups t the chllenge dose of 6. μg. As expected, the chllenge (cetone vehicle) showed no rection (Figure 4A). At 48 hours, the skin rection to the urushiol chllenge in groups IV nd V showed the dose response phenomenon: the rection score to the urushiol dose 3. μg eing the lowest, followed y 4.5 μg, nd 6. μg eing the highest. However, in groups I, II, nd III, no or miniml rection ws oserved with 3., 4.5, or 6. μg urushiol chllenge. No erythem or edem ws oserved in the three prophylctic treted groups. In the PBS nd vehicle treted nimls, the skin rection scores were higher with the incresing concentrtion of urushiol chllenge (Figure 4B). The treted groups I, II, nd III did not show ny rection to the urushiol chllenge dose of 3. or 4.5 μg. A slight rection ws oserved t 6. μg urushiol dosge in groups I nd II. The rection scores of the treted groups were significntly different (P <.5) from tht of the control groups IV nd V (Figure 4B). The oservtions t 7 hours remined similr to those oserved t 48 hours. No to miniml skin rection ws oserved in groups I, II, nd III. The control groups showed skin rection scores for groups IV nd V to e comprle ut tended to e slightly higher in the PBS group (Figure 4C). Sttisticlly, the skin rection scores of the treted groups (I, II, nd III) were significntly different (P <.5) from the vehicle or PBS treted groups (IV nd V) t the respective doses of urushiol chllenge. Comprison of lesion scores of the PBS nd vehicle groups t different time points indictes tht mximum erythem nd edem were oserved t 48 hours. At 7 hrs, the lesions tended to suside, s compred to those t 48 hours. In ll groups, no skin rection ws oserved t the site of cetone (without urushiol) ppliction on the skin, indicting tht the rection ws exclusively ginst urushiol. ElSohly et l. (8) Emil: Test #: In the second test, guine pigs were chllenged four weeks fter sensitiztion (two weeks fter the first chllenge). At 4 hours, the skin rection scores of the nimls in groups I, II, nd III, prophylcticlly treted with ELI--57-3, ELI--78-, nd ELI--83 respectively, were significntly lower (P <.5) thn those in group IV (vehicle, 5% EtH) or group V (PBS) t the respective doses of urushiol chllenge (Figure 5A). At 48 hours, very slight rection ws oserved in the treted groups I, II, nd III to 4.5 nd 6. μg doses of urushiol. No rection ws seen t the 3. μg dose of urushiol in these groups. These scores were significntly (P <.5) lower thn those of vehicle (group IV) nd PBS (group V) (Figure 5B). At 7 hours, the rection tended to regress. The skin rection scores in the treted groups I, II, nd III were comprly lower thn those in the control groups IV nd V. However, they re not sttisticlly significnt (Figure 5C). Test #3: The third test ws conducted pproximtely seven weeks fter the second test. At 4 hours, Groups I, II, nd III did not show erythem or edem t the site of urushiol chllenge t ny of the three doses used. Animls in groups IV (vehicle) nd V (PBS) showed dose response to the urushiol chllenge t doses of 3., 4.5, nd 6. μg. The skin rection scores of the treted groups I, II, nd III were significntly lower (P <.5) thn the control groups IV nd V (Figure 6A). At 48 hours, the skin rection scores to the three doses of the urushiol chllenge in Groups I, II, nd III remined significntly lower compred to those of the control groups IV nd V (Figure 6B). At 7 hours, the skin rection ws similr to tht seen t 48 hours (Figure 6C compred with 6B). The skin rections to the three doses of urushiol chllenge remined significntly lower compred to those of the control groups IV nd V (Figure 7). A visile difference ws oserved in erythem t the sites of topicl ppliction of urushiol in groups IV nd V, s compred to groups I, II, nd III. Study #: Desensitiztion study B) Desensitiztion to poison ivy/ok urushiol of lredy sensitive guine pigs using the wter solule prodrug of 3-n-heptdecylctechol HDC-APB (ELI--57-3) The control niml groups IV nd V, (5% EtH nd PBS control groups respectively), from the tolernce study, with estlished sensitivity (see Figure 6, groups IV nd V) were used in this study to determine if IM dministrtion of one of the wter-solule derivtives prepred would desensitize lredy sensitive nimls. Animls of group IV were injected 6 μl (3 μl in ech hind leg) of ELI (HDC-4-(4-minophenyl)-utyrte ester) solution t 57 mg/kg. Animls in group V were given 6 μl of vehicle (5% ethnol in wter) divided in two doses of 3 μl in ech hind leg. After rest period of pproximtely weeks, nimls in oth groups were chllenged y topicl ppliction of 3 doses of urushiol (3 μg, 4.5 μg, nd 6. μg ech dissolved in 5 μl of cetone). At the 4, 48, nd 7 hours post topicl chllenges, skin lesions were oserved nd grded s descried erlier. J Dermtolog Clin Res 6(): 5 (8) 5/8

6 Centrl The photomicrogrph in Figure 8 shows tht t 4 hours, no skin rection ws oserved in the nimls of Group IV treted with ELI In Group V (5% EtH vehicle control), no skin rection ws oserved with 3. μg urushiol chllenge. However, t the 4.5 nd 6. μg urushiol chllenges, skin rections were oserved. At the 48 hours post topicl chllenge with urushiol, the nimls in group IV (treted with ELI--57-3) showed no rection with 3. μg, nd slight rection with the 4.5 nd 6. μg urushiol chllenges. In contrst, the nimls in group V (5% EtH) showed dose response rection to 3., 4.5, nd 6. μg of urushiol chllenge. The skin rection score of the treted group (group IV) were significntly lower thn those of group V (5% EtH treted group V). Figure 9 shows the severity of skin rection score in groups IV nd V. At the 7 hours post topicl chllenge, the ELI treted nimls showed sudued rection t the highest does (6. μg) of urushiol chllenge. At lower doses, no rection ws visile. In contrst, 5% EtH nimls showed rection to ll three doses of urushiol. A pronounced rection to urushiol chllenge ws oserved t the highest dose (6. μg). Comprison of skin rection scores of group IV nimls were significntly (p<.5) lower thn those of the 5% EtH treted group. This indictes tht n IM injection of the test compound (ELI , HDC-APB) effectively desensitized previously rective nimls. This study is composed of two portions, one relted to the induction of tolernce to nïve nimls towrds sensitiztion to poison ivy urushiol nd the other relting to the development of desensitiztion to poison ivy in nimls lredy sensitive to urushiol. In the tolernce study, the skin lesion scores in Groups I, II, nd III in ll the three tests were negligile compred to the control Groups IV nd V (untreted). This indictes tht n intrmusculr injection of ny of the three test compounds protected the nimls ginst sensitiztion to poison ivy dermtitis. All three compounds were eqully effective, s no remrkle difference ws oserved in the skin lesion scores of these three groups. The skin rection of Groups IV nd V in Test # nd Test # were not s severe compred to those oserved in Test #3. It is possile tht the high sensitizing dose of urushiol (.mg) on the neck my hve cused stte of nergy. This condition is oserved in ptients of tuerculosis (TB), who re urdened with huge mount of TB ntigen, ut show no rection to the intrderml TB test (flse negtive). However, rest period of weeks etween sensitiztion nd Test #3 perhps reversed the nergic stte to norml rective stte. Thus, in Test #3, nimls in the Groups IV nd V (untreted) exhiited strong skin rections to urushiol chllenge, while nimls in Group I, II, nd II were protected due to the prophylctic tretment. Therefore, tolernce ws produced, which protected the guine pigs ginst sensitiztion to poison ivy urushiol, y the dministrtion of the test compounds to the nimls prior to ttempted sensitiztion. In order to determine if the test compounds re lso cple of inducing desensitiztion to poison ivy urushiol, groups IV nd V, which re lredy sensitive to urushiol were used in desensitiztion experiment. Group IV ws treted with one of A B ElSohly et l. (8) Emil: C Figure 6 (A-C): Test #3 - Guine pigs in different groups were injected IM with different tretments: Group I: ELI--57-3, Group II: ELI- -78-, Group III: ELI--83, Group IV: 5% EtH, nd Group V: PBS. Two weeks post tretment; guine pigs were sensitized with. mg urushiol on neck re. Eleven weeks fter sensitiztion, the guine pigs were chllenged with cetone, or cetone contining 3., 4.5,or 6. µg urushiol t ech of the four dominl sites. Erythem+edem scores for the nimls were tulted. Dt represents group mens ± SEM. significntly different from Group V (PBS) t the sme chllenge dose. significntly different from group IV (5%EtH) t the sme chllenge dose. the test compounds (ELI--57-3, HDC-APB) while Group V ws dministered the vehicle. Two weeks fter tretment, the nimls were skin tested. Figure 9 shows tht, t 4 hours, no skin lesions were oserved in nimls tht were given ELI (HDC-APB). In the vehicle group, no skin rection ws oserved with 3. μg urushiol. Very slight erythem ws oserved t 4.5 µg nd 6. µg urushiol. At the 48 post hrs topicl chllenge, there ws dose response of skin rection to with 3., 4.5, 6. µg urushiol in the vehicle group, wheres the ELI (HDC-APB) group showed no or very slight erythem. At the 7 hours post urushiol chllenge, in the vehicle group (5% EtH), the skin rections to 3., 4.5, nd 6. µg urushiol ws more pronounced s to compre to the 4 nd 48 hr. However, in the group treted with ELI--57-3, there ws no skin rection in response to the urushiol chllenge t 3. nd 4.5 µg ut slight rection t 6. µg. Significnt reduction in skin rection to the urushiol chllenge ws oserved t 48 nd 7 hrs in the treted (ELI--57-3) group (see Figure 9). This shows tht tretment of lredy sensitive nimls with one of the test compounds (ELI--57-3, HDC-APB) resulted in desensitiztion of the treted nimls to poison ivy urushiol. J Dermtolog Clin Res 6(): 5 (8) 6/8

7 ElSohly et l. (8) Emil: Centrl Figure 7 Skin rections of groups I through V t 4 hours post ppliction of poison ivy urushiol in Test #3.Skin rection of guine pigs t 4 hours post chllenge of test #3. Guine pigs in different groups were treted IM s follows: Group I: ELI -57-3; Group II: ELI -78-; Group III: ELI -83; Group IV: 5% EtH, nd Group V: PBS. Two weeks post IM tretment; guine pigs were sensitized with. mg urushiol on neck re. Eleven weeks fter sensitiztion, the guine pigs were chllenged with cetone or cetone contining 3., 4.5, or 6. µg urushiol t ech of the four dominl sites. Figure 8 Skin rection to poison ivy urushiol chllenge t 7 hours. Previously sensitized guine pigs were treted IM with ELI (HDC-APB) or 5% EtH. 3 4 h 4 h 7 h 3. ug 4.5 ug 6. ug Erythem +Edem score 5% ETH ELI % ETH ELI % ETH ELI Figure 9 Guine pigs were sensitized first with. mg urushiol in neck re. Two weeks fter Test #3, the guine pigs of Group IV (previously treted with EtH) nimls were injected IM with ELI--57-3, while group V guine pigs (previously treted with PBS) were now given IM injection of 5% EtH. Two weeks post tretment, oth groups were chllenged with µl of cetone contining 3., 4.5, or 6. µg urushiol t ech of the four dominl sites. Erythem nd edem score t ech site ws recorded t 4, 48 nd 7 hours post urushiol chllenge. Dt represents men score ± SEM, for group IV (ELI treted) or group V (5 %EtH treted) in the grph. (p<.5) compred to PBS tretment t 48 nd 7 hours t the sme dose of urushiol chllenge. (p<.)compred to PBS tretment t 7 hours t the sme dose of urushiol chllenge. *Acetone ws not dministered to ny of the guine pigs, due to the lck of rectivity seen in the tolernce study. Results from the desensitizing study lso showed visile reduction in erythem nd edem t the site of topicl ppliction of urushiol in the group treted with HDC-APB. This shows tht tretment of lredy sensitive nimls with one of the test compounds (HDC-APB) resulted in desensitiztion of the treted nimls to poison ivy urushiol. These results complement our previous work where guine pigs were desensitized with urushiol cette vi the intrvenous (IV) route nd multiple or single dose of tretments vi the orl route [3,36-37]. However, in this study, HDC-APB ws dministered s single dose vi the intrmusculr (IM) route. CNCLUSIN The tolernce induction portion of this study showed tht n IM dministrtion of ny of the wter solule prodrugs (6 mg equivlent HDC/niml) induced tolernce to the test groups, wherey no (or very slight) skin rection ws oserved in the treted groups s compred to the control (untreted) groups. J Dermtolog Clin Res 6(): 5 (8) 7/8

8 Centrl All compounds were eqully efficcious. Furthermore, tretment of lredy sensitive nimls with one of the test compounds (HDC-APB) resulted in desensitiztion of the treted nimls to poison ivy urushiol. This shows tht the test compounds re cple of producing tolernce when dministered to nïve nimls, nd desensitiztion when dministered to lredy sensitive nimls, ElSohly et l. (8) Emil: mking them good cndidte drugs for prophylctic tretment of poison ivy contct dermtitis. ACKNWLEDGEMENTS This project ws supported y NIH STTR grnt funds from R4-AR nd R4-AR nd prtilly supported y Hpten Sciences, Inc. Cite this rticle ElSohly MA, Gul W, Adel-Bkky MS, Mnly SP, Ashfq MK (8) Compositions for Prevention/Prophylctic Tretment of Poison Ivy Dermtitis. J Dermtolog Clin Res 6(): 5. J Dermtolog Clin Res 6(): 5 (8) 8/8