Indian Journal of Experimental Biology Vol. 38. September 2000. pp. 895-900

Role of volatile oil pretreatment and skin cholesterol on permeation of

ion-paired diclofenac sodium

B Sapra, S Gupta. & A. K Tiwary *

Department of Pharmaceutical Sciences and Drug �cscarch. Punjabi University. Patiala. India- 147002

Receil'ed 27 Jll/r 1999: accepted 13 March 2000

This study was designed to investigate the inlluence of volatile oil pretreated skin on ill \'itro permeation from films containing ionized and dodecylamine ion-paired diclofenac sodium (OS). The involvement of skin cholesterol was investi­gated to determine its possible role in enhancing the permeation of ion-paired OS. Cardamom oil produced the maximum (10 x) ill vitro permeation enhancement for ion-paired OS. The carrageenan induced rat paw oedema reduction (up to 12 hI') by cardamom oil was comparable to that of diclofenac injection (s c) .. Leaching of cholesterol from excised skin in addition

to increased partition coeflicient following volatile oil skin pretreatment appears to be responsible for ill vitro permeation enhancement of OS. Whereas. a mild barrier perturbation crfect due to altered cholesterol levels following pretreatment with volatile oils appears to increase the permeation of ion-paired OS across viable skin. thereby producing signiticant reduction of calTageenan induced paw oedema ..

Transdermal drug delivery offers many biomedical advantages over conventional routes of drug admini­stration. However, at the pH of skin normally experi­enced under physiological conditions, most of the drugs are in their ionized state. The partitioning of a drug through the skin under such a condition, is a function of its intrinsic charge.

The resistance to transdermal flux can be reduced by many methods. Out of the several methods, syn­thesis of lipophilic analogues and the use of permea­tion enhancers are being widely investigated. lon­pairing is a method of increasing the lipophilicity of an ionized permeane. Our earlier investigations have shown that ion-pairing of diclofenac sodium (OS) with dodecylamine increases its partition coefficient between isopropyl myristate and phosphate buffer (PH 6.0). Although, the ill vitro flux from films con­taining ion-paired diclofenac was five times greater as compared to that from films containing ionized OS2, it was much less than that reported from pseudolatex dispersions'. Hence, it was felt necessary to increase the permeation of ion-paired OS.

Terpenes in their pure form are increasingly being investigated for their percutaneous permeation en­hancement effect-l-6. The permeation enhancement following topical application of pure terpenes is re­ported to be due to an increase in the stratum corneum lipid fluidity and perturbation of the integrity or the

* COITespondent author

epidermis6. However, the influence of terpenes on the microconstituents of skin that are involved in main­taining its barrier integrity has not been reported.

This investigation aims at studying the influence of different volatile oils on the permeation of ionized and ion-paired OS from transdermal films. In order to investigate the mechanism of permeation enhance­ment by volatile oils, the role of cholesterol, a marker of epidermal integrity was studied.

Materials and Methods

Oiclofenac sodium (Ciba-Geigy, India), Eudragil L- lOO (Rohm Pharma, Germany), HPMC (Shinetsu, Japan), carrageenan (Spectrochem, India) and dode­cylamine (Lancaster, UK) were used as received. Clove oil was purchased from SO Fine Chemicals, India. Other oils (expressed) were procured from do­mestic manufacturers. Cholesterol was measured by using cholesterol test kit (Span Diagnostics Ltd., Su­rat, fndia). All other chemicals were of AR grade.

Preparation offt/lIIs-Transdermal films with total polymer content of 12% w/w were prepared by mix­ing solutions of eudragit L-IOO and HPMC (9: 1). Eu­dragit selution was prepared in alcohol (1 part). Films containing ionized OS were prepared by dissolving OS in HPMC solution prepared in a mixture of phos­phate buffer (pH 6.0) and alcohol (6: 1). For preparing films containing ion-paired OS, the drug was dis­solved in phosphate buffer (pH 6.0) and rnixed with dodecylamine�. This mixture was then incorporated

896 INDIAN J EXP BIOL, SEPTEMBER 2000

into HPMC solution prepared as above. Polyethylene glycol 400 (15 % w/w of tota l polymer) was added as plasticizer. The films were cast on mercury andl dried at 45 °C for 4 hr. Amount of mercury was standard­ized or maintaining film thickness. Using the formu la, Cp xVd xKe and Cp of 1.9 ).lg/ml7

, Vd of 0.17 UKg . 8 and Ke of 1.482/hr , the required release rate was cal-culated to be 478 ).lg/hr, corresponding to L 1.48 mg/day. Hence, 22.944 mg of drug was loaded into films (3.63 cm2

) to suffice for 48 hr study period. Permeation studies (in vitro)-In vitro permeation

of OS was investigated through dorsal skin of wistar rats excised 24 hr after removing the hair wi th me­chanical clippers. Permeation was carried out in verti­cal Keshary-Chien diffusion cell containing phosphate buffer (pH 7.4) at 37° ± L °C in the receptor com­partment. The stirring speed was 300 rpm. Aqueous formaldehyde (0.2% v/v) and polyethylene glycol 400 (5 % v/v) were added for preservation and mainte­nance of sink condition, respectively. Pretreatment of skin was done by applying 1 ml of volatile oil to the stratum corneum surface immediate ly before applica­tion of the film formulation. OS permeated into the receptor compartment was analyzed at different time intervals at 276 nm9 using Beckrrian spectrophotome­ter (OU 640B).

Anti-inflammatory response in rat paw oe­dema-The anti-inflammatory studies were performed using a plethysmometer to measure catTageenan in­duced paw oedema following the method of Winter et a/. 1° Five wistar albino rats were used in each group. Hairs from the dorsal side (3 cm2)were removed with c lippers. The transdermal film formulation was applied after 12 hr and occluded with an adhesive tape. After 30 min of application of film, 0.1 ml (1 % w/v) solution of carrageenan was administered by intraplanter injec­tion in one hind paw. The other hind paw served as control. The volume of both hind paws was measured at regular intervals up to 24 hr.

The treatments given to different groups were: group I, no drug (control); group II, inj ection ( 15 mg/kg, sc); group III, marketed gel formulation (con­taining 26.615 mg of diclofenac di ethyl ammonium= 22.944 mg of OS) ; group IV , film containing OS at pH 2.0 (unionized); group V, film containing dode­cylamine-OS ion-pair; group VI, f ilm contai ni ng ion­ized OS and sk in pretreated with cardamom oi l; group VII, film containing ion-paired OS and skin pretreated with cardamom oii. Skin was pretreated by applying 1 ml of volatile oil on the shaved portion immediately before application of film fo rmulation.

Effect of skin pretreatment with volatile oils on cholesterol leaching (in vitro)-Whole skin was clamped on the diffusion cell. The receptor fluid was stirred for 4 hr after which it was replaced with fresh phosphate buffer (pH 7.4). Volatile oil (1 ml) was applied on the stratum corneum side and stirring con­tinued for 48 hr. At the end of this period the entire fluid was evaporated under vacuum and analyzed for cholesterol content by using cholesterol kit.

Effect of skin treatment with volatile oils on cho­lesterol in viable skin -Two patches (2 cm2

), one on either side of the spinal cord of wistar rats were pre­pared by shaving and left open for 12 hr. One patch was treated with volatile oil (1 ml) and the area oc­cluded with adhesive tape. The other patch served as control. The animal was sacrificed after 48 hr and both the portions of skin excised. After drying the skin to constant weight at 50°C, homogenizing in chloroform-methanol (1: 1) followed by filtration and concentration (under vacuum), the cholesterol content was determined. All experiments were catTied out in triplicate.

Results

The permeation of ion-paired OS across volatile oil treated skin (Fig. I) is significantly greater (t-test, P < 0.05) than that of ionized OS (Fig. 2). Among the various oils tested, cardamom oil produced the maxi­mum permeation enhancement for ion-paired OS. The

0 +I E

12

10

t: 8 Qj

E Qj u r:: ~ 6 r:: Qj

r:: 0 ~ 4 Qj

E Qj

a. 2

0 Cardamom Lemon Clove T urpcntine Eucalyptus

Dlonized OS • ion-paired OS

Fig. 1- Influen ce of volatile oil pretreatment on permeation en­hancement ratio of ion-paired DS wi th respect to ionized DS and ion-paired DS across non pretreated skin.

SAPRA ET AL. ROLE OF VOLATILE OIL PRETREATMENT AND SKIN CHOLESTEROL ON PERMEATION. 897

permeation profile of ion-paired OS across cardamom oil pretreated skin is depicted in Fig. 3. Increased fl ux of ion-paired OS following skin pretreatment with

8

7

0 6 :;::1

I! -c CD 5 E CD u c ca 4 .J:. c CD c 0 3 :;::1 ca CD

E CD 2 a.

0 Cardamom Clove Lemon Eucalyptus Twpentine

Olonized OS •Ion-paired OS

Fig. 2-Innuence of volati le oil pretreatment on permeation en­hancement ratio of ionized OS with respect to ionized and ion­paired OS across non pretreated skin.

volati le oils is accompanied with an increase in parti­tion coefficient (Fig. 4) and increase in the amount of cholesterol leached from excised skin (Fig. 5). In ad­dition, cholesterol content in viable skin is found to slightly increase after topical application of any vola­tile oil (Fig. 5). This indicates a mild epidermal per­turbation effect. Pharmacodynamic studies indicate that a combination of ion-pairing and cardamom oi l skin pretreatment produces a sustained effect that is better than that obtained by single application of mar­keted gel formulation containing diclofenac dieth­ylammonium (Fig. 6).

Discussion OS exhibits a pKa of 4.0 11

• At pH values greater than 4 .0, it will exist in ionized form. Primary fatty amines being cationic at higher pH values, form ' ion­pairs' with OS to yield a lipophilic molecule. Our earlier investigations showed that among various amines dodecylamine is most effective in enhancing both partition coefficient (isopropyl myristate I phos­phate buffer) and in vitro permeation of OS across rat skin. Film formulation containing ion-paired OS (at pH 6.0) exhibited five times greater in vitro flux than that from films containing ionized OS2

.

In order to further increase the in vitro permeation

'E

20 40 60

Time ( t, hrs)

() 5000 0"

"' 4500 Cl

4000 .3 a1 3500 iii Q) 3000 E Q) 2500 c.. ....

2000 c: :l 0

1500 E < Q) 1000 > ~ 3

500 E :l 0 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1.2 1.6 1.8

Time (Log hr)

Fig. 3-Transformed permeation profile of ion-paired OS from transdermal film across cardamom oil pretreated skin (the initial experi­mentally obtained points that were not linear in cumulative amount permeated Ys time plot were left out and the other points were re­gressed and then a cumulative amount of drug permeated Ys log ti me plot was constructed); Inset: Non-transformed profil e.

898 INDIAN J EXP BlOL, SEPTEMBER 2000

of OS, pretreatment of sk in with diffe rent volat ile oi ls was carried out. The enhancement ratio for various volatile oils followed the order, cardamom> lemon= clove > turpentine >eucalyptus (t-test, P < 0.05). The maximum permeation of DS fro m films containing ion-pairs obtained using cardamom o il pretreated skin is approximately 10 times greater as compared to that from films containing ioni zed DS (Fig. 1). These re­su lts demonstrate the influence of a combinat ion of ion-pairing and volatile o il ski n pretreatment in in­creasing the permeat ion of DS.

It is interesting to note that the permeation of ion­ized DS is increased after sk in pretreatment as com­pared to that across non pretreated skin with carda­mom o il producing the max imum enhancement (Fig. 2). However, the permeation enhancement for ionized DS by the respective o il s was significantl y less than that for ion-paired DS (t-test , P < 0.05). T he order of permeation enhancement for ionized DS was, carda­mom > clove > lemon > eucalyptus > turpentine (t­test, P < 0.05). It is noteworthy, that the permeati on of ionized DS across oi l pretreated skin was significantly greater as compared to that of ion-pa ired DS across non pretreated skin . These results indicate that vola­tile oi ls are capable of enhanci ng the permeation of ionized form of OS and their presence is essential for enha ncing the perc utaneous permeation of hi ghly lipoph ilic ion-paired DS molec ul es.

... Ql .. 4 Ql

E ~ IV a.. c 3 .2 ~ Q)

E 2 Q.l a..

Control Cud&mOm Lemon Clove Turpentine Eucalyptu•

0 Flux (mcg/sq em/log hr) El Diffusivity (sq em/log hr)

Ill Part. Coeff. m Lag time (hr)

Fig. 4-lnfluence of volati le o il pretreatment o n in vitro percuta­neous permeation parameters of ion-paired DS (Note: actual val­ues arc, Flux x 1000; Diffusiv ity xi o-4

; Part ition coefficient xO. I ).

T he change in order of enhancement ratio for ion­pai red and ionized DS can be explained on the basi s of the active constituents of the respecti ve volati le oils. Li monene, the maj or constituent of le mon oi l, has bee n reported to enhance the in vitro transport of lipophilic molec ule, oes tradio l and decrease the transport of hydrophilic molecul e, 5-fluorouracil 12

. .

The present in vesti ga tion also revea ls a highe r en­hance me nt ratio for ion -paired as compared to that for ioni zed DS by le mon oi l. Clove oil contains eugenol, a hydroxyl group containing terpene , that can be more effective in e nhancing the permeation of hydrophi lic drugs by formation of hydrogen bonds. Hence, clove and le mon o il rank second and third in enhancing the permeation of ionized DS and ion­paired DS, respecti ve ly . Similarly, eucalyptus was more effec ti ve than turpentine fo r ionized DS, while the reverse is true fo r ion-pai red DS. T his finding is in agreement with the earlier report that aroman­d rene (the consti tue nt of e ucalyptus oi l) is more ef­fec tive than lon gifo lene (the major constituent of turpentine oi l) in enhanci ng the in vitro transport of 5-fluorouracil 13

. Also, e uca lyptus oi l is reported to be more effective than peppermint o il for 5-flu orouraci 114

• Separation and purificat ion of volatile o il s fo r obtai ning the active constituents in pure form is a cost ly process. T he results of thi s study suggest

16

14

12

10

8

6

4

2

0 Cardamom Lemon Clove Turpentine Eucalyptus

0 Flux (mcg/sq em/log hr) • Chi (leached) wrt control liil Chi (viable skin) % control

Fi g. 5-lnfluence of volatil e oi l skin pretreatment on flu x of ion ­paired DS and cholesterol (Ch i) leached from excised skin and in viable skin (Note: actual va lues are. Flux xI 000; Chi (leached) x i O wrt contro l; Ch i x !O (v iable skin) % control ).

SAPRA ET AL. ROLE OF VOLATILE OIL PRETREATMENT AND SK IN CHOLESTEROL 0 PERMEATION. 899

80

70

60

ns 50 E Q)

-g 40 0 ';!. 30

20

10

0 i -;

Group Group Group Group Group Group GRoup I II III IV V VI VII

0% Oedema at 12 hr • % Oedema at 24 hr

Fig. 6-Reduetion in carrageenan induced oedema after various treatments (re fer ex peri mental section for details).

that it would be possible to increase the permeation of a drug by s imply utili zing a polar or non-polar solvent extract of the oil yielding part of the plant. The choice of extrac ting solvent will depend upon the lipophilic I hydrophilic character of the drug.

Figure 3 (inset) depicts the 'burst release' pattern obtained for permeation of ion-paired DS from films across cardamom oil pretreated skin. Calculation of lag time by extrapolation to X-axis is not possible in such graphs. Hence, an attempt was made to trans­form the d:Jta of Fig. 3 (inset) by using the approach used by Shah et al.t 5 The initial points that were not linear in cumulative amount (CA) Vs time plot were left out. All the other experimentally obtained points were treated through a regression analysi s and a CA Vs log time pl ot was constructed (Fig. 3) that resul ted in a higher R2 value. For comparison purpose, the permeation profi les of DS reported by Vyas et a/ .3 and Huang et a!. t6 were also transformed using this

method. The reported flux of 118 and 74.15 ~tg/cm2/hr upon transformation become 3504.692 and 2976.885 ~g/cm2/log hr. respectively. The highest flux obtained in the present investigation from ion-paired films across cardamom oil pretreated skin is 3775.392 ~g/cm2/log hr. This is 1.07 and 1.26 times greater

3 '6 than that reported by Vyas et al . and Huang et a!." , respec tively.

A comparison of permeation parameters following sk in pretreatment with various volatile oi ls shows that an increase in pmtition coefficient may play a role in increasing the flux (Fig. 4). Diffusivity does not seem to be important because no particular trend is evident. Interestingly, lag time is found to increase following sk in pretreatment as compared to control because of slow redistribution leading to 'conditioning' of the membrane during early stages of diffusion process 13

•

In addition to partition coefficient, vo latile oil treat­ment also increases the a:nount of cholesterol leached from exc ised skin (Fig. 5 ). An alteration of enthalpy and entropy of lipid transitions 17 and perturbation of stratum corneum lipid fluidit/ 8 following skin treat­ment with terpenes have been reported to increase skin pe!·meability and enhance the permeation of drugs. Moreover, increased epidermal cholesterol synthesis is known to accompany altered batTier function and transepidermal water loss following perturbation 19

. Therefore, changes in transepidermal water loss 17

.18 appear to arise from altered cholesterol

levels in skin. Fig. 5 shows increase in the cholesterol content in viable skin after pretreatment with o il s, indicating a mild epidermal perturbation effect. This suggests that pretreatment with volati le oil s increases the permeation of ion-paired DS both by increas ing its partition coefficient and by mild epidermal perturba­tion effect due to their influence on epidermal cho­lesterol. Hence , continuous use of volatile oils at the same site needs to be critically considered. In addi­ti on, thei r effect on other epidermal constituents can ­not be ruled out and needs further investigation.

Fig. 6 shows that oedema :tt 12[11 hour foll ows the order, I = IV > V = III = VI > VII = II (t-test, P < 0.05). Hence, the permeation of ion-paired DS across cardamom oil pretreated skin (gr.Vll) is not signifi­cantly different from that following subc utaneous in­jection (gr. II). Cardamom oil pretreatment also en­hanced the permeation of ion ized DS (gr. VI) and the oedema reduction is comparable to that produced by marketed gel formu latiBn containing an equivalent dose of diclofe nac diethyl ammonium (gr. III). There­fore at the end of 12 hr, single application of the mar­keted gel is significantly less effective than applica­tion of ion-paired DS with cardamom oil pretreat­ment. However, oedema at the end of 24 'hr followed the order, lii = I= IV = V =VI> VII> II (t-test, P < 0.05). No significant difference in oedema between the control group and the group that received an ap­plication of marketed gel at the end of 24 'hr is be­cause the drug is rapidly absorbed and metabolized

900 INDIAN J EXP BIOL, SEPTEMBER 2000

fol low ing application of ge l formulation and hence, does not produce any reduction in oedema after 10 ' hr. Group VII exhibits lower oedema with respect to other groups. Thi s indicates that a combination of ion­pairing and skin pretreatment with cardamom oil en­hances systemic deli very of DS. However, the oe­dema of group VII is s ignificantly higher than that obtained following subcutaneous injec tion . Thi s can be attributed to the susta ined re lease of DS from ion­paired fi lms.

A flux of 118 JJ.g/cm2/h r (transformed flux 3504.692 ~tg/cm2/log hr) has been reported to g ive a Cmax of 1.92 JJ.g/ml and is effect ive in vivo3

. The pre­sent study reveals a hi gher flux in vitro (3775 .92 ~tg/cm2/log hr) from films containing ion-paired DS ac ross cardamom oi l treated skin that is pharmacody­namically effecti ve. Hence, a combination of ion­pairing and cardamom oi l skin pretreatment may pro­vide a new formulation strategy for percutaneous permeation enhancement of DS.

References

Barker N & Hadgraft J, Faci litated percutaneous absorption : A model system. lnt J Pha rm , 8 ( 1981) 193.

2 Rana V, Rai P, Tiwary A K & Gupta S, Enhanced in vit ro percutaneous permeation of dic lofen:.c sodium wi th primary amine and pyrrolidone ion pairs, Indian Drugs, 36 (1998) 2 1.

3 Vyas S P, Gogoi P J & Jain S K. Development and charac­te rization of pseudolatex based transdermal drug delivery system of diclofenac, Drug Dev lnd Pharm, 17 ( 199 1) I 04 1.

4 Santus G C & Baker R W, Transdermal enhancers patent lit­erature, J Contr Rei, 25 ( 1993) I.

5 Kadir R & Barry B W, a-bisabolol, a possible safe penetra­tion enhancer for dermal and transdermal therapeutics. lnt J Pharm, 70 ( 1991 ) 87.

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8 Willi s J V, Kendall M J, Flinn R M, Thunhi ll D P & Welling P G. The pharmacok inetics of diclofenac sodium following intravenous and oral admini stration , Eur J C/in Pharmacal, 16 (1979) 405.

9 Chri st ianah M, Adeyeye C M & Li P K, in Analytical profiles of drug substances, Vol 9, by H. G. Brittain (Academic Press Inc., London) 1978.

10 Winter C A, Ri sley E A & Nuss G W, Carrageenan induced edema in hind paw of the rat as an assay for infl ammatory drugs. Proc Soc Exp Bioi Med, 162 (1963) 544.

II Tomida H, Shinohara M, Ku wada N & Kiryu S, in British Pharmacewicaf Codex, by W. Lund (The Pharmaceuti cal Press, London) 1994, 836.

12 Moghimi H R, Willi ams A C & Barry B W, A lammelar ma­trix for stratum corneum intercellu lar lipids . V. Effects of ter­pene penetration enhancers on the structure and thermal be­haviour of the matrix , tnt J Pharm , 146 ( 1997) 41.

13 Cornwell P A & Barry B W, Sesquiterpene components of vo lat il e oi ls as skin penetration enhancers for hydrophili c permeant 5-lluorouraci l. J Pharm Pharmacal, 46 ( 1994) 26 1.

14 Abdullah D, Ping Q N & Liu G J, The enhancing effect of es­sential oi ls on the penetration of 5-lluorouraci l th rough rat skin , Yao-Hsu eh-Hsueh- Pao, 3 11 ( 1996) 2 14.

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17 Cornwell P A, Barry B W, Bouwstra J A & Gooris G S. Modes of action of terpene penetration enhancers in human ski n: dif­ferential scanning calorimetry, small angle x-ray di ffraction and enhancer uptake studies, tnt J Pharm. 127 ( 1996) 9.

18 Gao S & Singh J, Mechanism of transdermal transport of 5-tluouracil by terpenes: carvone, I, 8 cineole and thymol, tnt J Pharm , l54(1997)67.

19 Ghadially R, Brown B E, Han ley K. Reed J T, Feingold K R & Elias P M, Decreased epidermal lipid synthesis accounts for altered barrier funct ion in aged mice, J In vest Dermatol, I 06 ( 1996) I 064.