Role of volatile oil pretreatment and skin cholesterol on...
Transcript of Role of volatile oil pretreatment and skin cholesterol on...
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
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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
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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
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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
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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
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-g 40 0 ';!. 30
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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.
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