Transdermal delivery of insulin from poloxamer gel: ex vivo and in vivo skin permeation studies in rat using iontophoresis and chemical enhancers

Journal of Controlled Release 89 (2003) 127–140 www.elsevier.com / locate / jconrel

Transdermal delivery of insulin from poloxamer gel: ex vivo and in vivo skin permeation studies in rat using iontophoresis and chemical enhancers Omathanu Pillai, Ramesh Panchagnula* Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research ( NIPER), Sector-67, S. A.S. Nagar, 160062 Punjab, India Received 25 October 2002; accepted 31 January 2003

Abstract Gels are considered to be the most suitable delivery vehicle for iontophoresis, as they can be easily amalgamated with the iontophoretic delivery system and can also match the contours of the skin. Insulin was used as a model peptide for large peptides in the molecular weight range of 3–7 kDa. A gel formulation of insulin was formulated using poloxamer 407 and was evaluated by ex vivo and in vivo skin permeation studies in rat with chemical enhancer and / or iontophoresis. The poloxamer gel was physically and chemically stable during the storage period. In ex vivo studies, both linoleic acid and menthone in combination with iontophoresis showed a synergistic enhancement of insulin permeation. The plasma insulin concentration (PIC) was highest with linoleic acid pre-treatment, in agreement with ex vivo permeation studies, but the reduction in plasma glucose levels (PGL) was comparable to iontophoresis. Menthone pre-treatment resulted in rapid attainment of peak PIC, but the reduction in PGL was less than other treatment groups. There was no direct relation between PIC and PGL and is attributed to the fact that the action of insulin in mediated by a cascade of cellular mechanisms, before a reduction in PGL is observed. However, iontophoresis either alone or in combination with linoleic acid produced a reduction in PGL to the extent of 36–40%. A combination of chemical enhancers and iontophoresis caused greater skin irritation than when either of them was used alone.  2003 Elsevier Science B.V. All rights reserved. Keywords: Poloxamer 407 gel; Combination strategy; Menthone; Linoleic acid; Plasma glucose levels; Plasma insulin concentration; PK–PD relationship

1. Introduction Peptide and protein drugs have emerged as an important class of therapeutic agents due to the *Corresponding author. Tel.: 191-172-2146-8286; fax: 191172-214-692. E-mail address: [email protected] (R. Panchagnula).

significant advancements made in recombinant DNA technology. At present, the parenteral route remains to be the only option to systemically deliver peptide / protein drugs, which in turn is associated with poor patient compliance, because of the discomforts of frequent injections and the risk of infection and inflammation at the site of injection [1]. The main bottleneck lies in achieving significant blood levels

0168-3659 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0168-3659(03)00094-4

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through non-parenteral routes, as they suffer from poor membrane permeability and proteolytic degradation at most of the biomembranes [2]. The transdermal route has a proven commercial success due to good patient compliance for a number of drugs having short biological half-lives [3]. Further, skin lacks or has much less proteolytic activity [4]. These two factors coupled together offer an attractive opportunity for delivery of peptide / protein drugs. The large molecular size and charged nature of these drugs necessitates the use of enhancement strategies to overcome the barrier posed by stratum corneum (SC) and several such strategies are under active investigation [5]. Iontophoresis, that uses a small electric current to drive charged molecules across the skin holds good promise for delivery of peptide / protein drugs [6]. One of the technical issues that remain to be resolved is the development of suitable formulations that can serve as a link between the drug and the device [7]. Most of the studies in the literature have been carried out with solutions, but to hold promise for further clinical application, gel systems would be the most suitable formulation. The gel systems can match the contours of the skin and at the same time may also be easily amalgamated with the iontophoretic delivery system. Poloxamer 407 is a polyoxypropylene–polyoxyethylene non-ionic surface-active block co-polymer composed of approximately 70% of ethylene oxide and 30% of propylene oxide with a molecular weight of 115 000 da [8]. One of the characteristics of poloxamer solutions (20–30%) is their reversible thermal gelation property (i.e., solution at low temperature and gel at room temperature), which offer many advantages, such as; convenience in handling and ease of application [9]. In addition, this property can be exploited for refillable unit dose iontophoretic drug delivery systems. The widespread application of poloxamer gel in topical delivery systems is due to the reversible sol–gel property that allows cool solution to flow onto the skin and permit good contact with skin on formation of a non-occlusive gel at the body temperature. The gel can also be easily removed from the skin on washing with cold water. Further, due to its ability to form hydrogel, it shows good electrical conductivity, and absorbs sweat gland secretions, which is irritating under long-term occlusion [10].

From the standpoint of peptide / protein formulation, the negative thermo rheological behaviour facilitates accurate preparation of a homogeneous peptide formulation that can be stored under refrigerated conditions [11]. In our preliminary studies we found that poloxamer 407 forms a gel at pH 3.6 (the optimized conditions for iontophoretic transport of insulin) with acceptable viscosity and release characteristics. It would be difficult, if not impossible, to achieve sufficient permeation of large peptides like insulin with iontophoresis per se, signifying the use of combination strategies involving chemical enhancers and iontophoresis [5]. A number of chemicals are known to enhance the transdermal permeation of drugs, which are classified based on their structure and mechanism of action in skin [12]. Terpenes and fatty acids are naturally occurring compounds that have been demonstrated to enhance the permeation of a number of drug molecules [13,14]. Earlier in our laboratory, we found that menthone and linoleic acid showed good penetration enhancement of insulin solution across rat skin (unpublished data). The combined use of chemical enhancers and iontophoresis will aid in moderating the iontophoretic delivery as well as increasing the transport efficiency of large peptides like insulin. The optimized physicochemical and electronic parameters were used in this investigation for studying the ex vivo and in vivo permeation of insulin from gel both in the presence and absence of chemical enhancers along with iontophoresis. Insulin, which is a polypeptide, was used as a model peptide for large peptides in the molecular weight range of 3–7 kDa. It is used in the treatment diabetes mellitus and has immense therapeutic and commercial importance [15].

2. Materials and methods

2.1. Chemicals Human insulin (28.04 IU / mg) was a gratis sample from Eli Lilly (USA). [ 125 I]Insulin (80–100 mCi / g) was provided by the Board of Radiation and Isotope Technology (BRIT, India). Poloxamer 407 was from

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BASF (Germany). Ethanol was procured from Merck (Germany); linoleic acid (Sigma, USA) and menthone (Fluka, Germany) were used as 5% (v / v) solution in ethanol. Streptozotocin was purchased from Calbiochem (USA). Commercial glucose estimation kit (Accurex, India) and human specific radioimmunoassay (RIA) kit (Linco Research, USA) were used in the study for estimation of blood glucose levels and insulin levels, respectively. The RIA kit was specific for human insulin and had less than 0.1% cross-reactivity for rat insulin. All other chemicals and reagents used were of analytical grade. Ultra pure water prepared using reverse osmosis by passing water through Elgastat (ELGA, UK) was used in all experiments and had a resistivity of 18 MV or greater.

2.2. Gel preparation and evaluation The poloxamer gel was prepared by ‘cold process’ as reported in the literature [16]. Insulin (75 IU / ml) was dissolved in citrate phosphate buffer (pH 3.6), which contained 0.05 M sodium chloride, urea (2 mg / ml as a deaggregating agent) and sodium azide (0.002%, w / v, to prevent any microbial growth). To this solution, poloxamer (25%, w / v) was slowly added with stirring at 4 8C and left in the refrigerator overnight. Rheograms were recorded using a Rehocal DVIII1 programmable rheometer (Brookfield Engineering, USA) using the smallest spindle (RV 007) and the measurements were made periodically on gel stored in refrigerator up to 45 days. The chemical stability of insulin in gel was studied for 60 days by a HPLC method developed in our laboratory. Briefly, insulin was separated on an RP C-18 ˚ 100 mm; Deltapak, column (23150 mm, 300 A; Waters, USA) using acetonitrile and sodium sulfate (pH 2.3) as the mobile phase (25:75). The response (absorbance at 214 nm) was linear in the concentration range of 1–100 mg / ml.

2.3. Ex vivo permeation studies All experiments were conducted according to the protocol approved by the Institutional Animal Ethics Committee (IAEC). A female Sprague–Dawley rat

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(200–250 g) was sacrificed by excessive ether anesthesia and the hair was removed from the dorsal portion using an animal hair clipper (Aesuclap, Germany). After harvesting the full thickness skin, the fat adhering on the dermis side was removed using a scalpel and isopropyl alcohol. Finally, the skin was washed in tap water and stored at 220 8C in aluminium foil packing. The skin was used within a week. Phosphate-buffered saline (0.1 M; pH 7.4) was sonicated for 30 min and was placed in the receptor compartment of unjacketed Franz diffusion cells (area 0.79 cm 2 ), followed by equilibration for overnight at 3760.2 8C with a stirring speed of 900 rpm in a heating-stirring module (Perme Gear, USA). The thawed skin piece was mounted in the Franz diffusion cell with the SC side facing the donor compartment and was equilibrated for 1 h. The gel formulation (500 ml) was applied using a positive pressure pipette to the donor compartment and current (0.5 mA / cm 2 ) was applied either continuously for 6 h or in periodic fashion through platinum electrodes (2 cm30.5 mm diameter) using a six-channel power supply unit (Ultra pure Scientifics, Mumbai, India). During periodic iontophoresis, the current was applied using an on / off ratio of 1:1 for 12 h (in six cycles). The anode was placed in the donor compartment and cathode was placed in the receptor compartment. When chemical enhancers were used, 500 ml of ethanolic solution of either menthone (5%, v / v) or linoleic acid (5%, v / v) were applied to the donor compartment and pre-treated for 2 h. The chemical enhancers were removed and the skin was washed with water and blotted using tissue paper. Then, the poloxamer gel was placed in the donor compartment followed by passive permeation or current application. The samples were periodically withdrawn from the receptor compartment, up to 48 h, and counted on an automatic gamma scintillation counter (1470, Wallac, Finland). The area exposed to the gel formulation was washed with water, dried, cut and weighed. The cut piece was then digested using tissue solubiliser (NCS–II, Amersham, UK) in a shaker water bath (Julabo, Germany) at 37 8C and 100 rpm for overnight. From the skin homogenate, 200 ml were used for radioactive counting. Based on the ratio of ‘hot’ to ‘cold’ insulin, the amount of insulin in the samples was calculated.

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2.4. In vivo studies Female Sprague–Dawley rats (250–300 g) were housed in the central animal facility of NIPER, and diabetes was induced by intraperitoneal injection of streptozotocin (65 mg / kg body weight). The plasma glucose levels (PGL) were estimated after 48 h to ensure that diabetes has been induced and the PGL was allowed to stabilize for 3 weeks. Only rats whose PGL.250 mg / dl were considered as diabetic and used in the study. The animals were fasted for 12 h before the experiment and water was supplied ad libitum. To the control groups, a subcutaneous (s.c) injection of insulin (1 IU / kg) was administered. In the case of transdermal permeation studies, the rats were fixed supinely and an animal hair clipper was used to remove the abdominal hair and the skin was wiped with dilute isopropyl alcohol. The teflon rings (1.532 cm i.d.) were fixed to the rat’s body using polyacrylate glue and the distance between the two rings was 10 mm. The animals were made repose for 2 h to allow the glucose levels to stabilize, which had risen due to fixation. The gel formulation (2 ml) was applied using a positive pressure pipette to one of the teflon compartments, while to the other compartment, a blank gel was applied. The platinum electrodes were kept in place using tight fit caps on the teflon ring to make contact with the gel and the current was applied using a constant power supply. The anode was placed in the drug compartment, while the cathode was placed in the other compartment. In the passive treatment group, no current was applied. In another set of experiments, the skin was pre-treated for 2 h with either 5% (v / v) of linoleic acid or menthone in ethanol. The enhancer was then removed using a tissue paper and the skin was washed with water. To the cleaned skin surface, gel was applied and either passive or iontophoretic permeation was carried out. The blood samples (0.3 ml) were withdrawn from the retro-orbital plexus periodically up to 12 h and the plasma was separated by centrifugation. One aliquot of the plasma was used for estimation of PGL and the other aliquot was used for estimation of insulin.

2.5. Histological examination Normal Sprague–Dawley rats were subjected to

one of the following treatments; iontophoresis (0.5 mA / cm 2 for 6 h), chemical enhancer pretreatment (5%, v / v, menthone, linoleic acid) alone or followed by iontophoresis using blank poloxamer gel. The treated skin area was excised and stored in 50% formalin, followed by dehydration through a graded series of alcohols. Then, the skin was treated with antemedia and embedded in paraffin. A skin section of 5-mm thickness was cut from each sample and stained with hematoxylin–eosin for microscopic examination. The treated skin sections were observed under a light microscope and a scoring system was used to assess the changes in skin [17].

2.6. Data treatment The cumulative amount of insulin permeated was plotted against time and the flux was calculated from the straight-line portion of the curve [18]. The intercept of the straight line on the x-axis gave the lag time in hours. For the calculation of skin affinity values, the method described by Panchagnula and Patel [19] was followed. The drug concentration in the skin (mg / mg) was divided by the drug in the receptor compartment at the end of the experiment (in mg / mg; taking the density of receptor solution as 1 g / ml). The enhancement ratio was calculated by dividing the flux in presence of chemical enhancer and / or iontophoresis with the flux in absence of enhancers or iontophoresis. All experiments were done in triplicate and the values are expressed as mean6S.E.M. unless specified. The passive permeation experiments served as control in ex vivo studies. From the plasma concentration–time curve of insulin, the pharmacokinetic parameters of Cmax and T max were calculated, and the area under the curve (AUC) was calculated using the trapezoidal method. The initial glucose level before treatment was taken as 100% and the plasma glucose levels at other time points were represented as a percent glucose levels from the initial. Using the percent plasma glucose reduction versus time curves, the minimum PGL of initial (Cmin% ) and the time (T min ) to achieve the same was noted. The percent decrement in plasma glucose from 0 to 12 h was calculated using a method reported by Tozaki et al. [20]. All data were subjected to either one-way ANOVA or t-test at a significance level of P,0.05.

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3. Results and discussion

3.1. Physical and chemical stability of gel formulation The poloxamer gel exhibited sufficient viscosity (6000–7000 cps) and there was no appreciable change in viscosity, when analysed up to 45 days. For topical formulations, the yield value must be low to permit easy handling and facilitate spreading on the skin. At the same time the yield value is expected to be high enough to hold the gel on the skin. In this regard, poloxamer gels have been reported to exhibit pseudoplastic behavior with an anti-clockwise hysteresis loop [9]. When stored in the refrigerator, the insulin content in poloxamer gel was $98% up to 30 days and reduced to 89% in 60 days. It has been earlier reported in the literature that poloxamer 407, in addition to serving as a good delivery vehicle, may also prevent denaturation of proteins and retain their biological activity [21,22]. However, further studies are required to investigate the mechanism by which poloxamer stabilizes proteins.

3.2. Ex vivo permeation studies Fig. 1 shows the permeation profile of insulin from poloxamer gel, when subjected to current application (continuous and periodic) and chemical

Fig. 2. Flux enhancement ratio of insulin from poloxamer 407 gel after chemical enhancer pre-treatment and or iontophoresis. Ion, iontophoresis; Me, menthone pre-treatment; Mel, menthone pretreatment followed by iontophoresis; LOA, linoleic acid pretreatment; LOAI, linoleic acid pre-treatment followed by iontophoresis. Each value represents mean6S.E.M. (n53).

enhancer pre-treatment followed by either passive or iontophoretic facilitated permeation. The permeation of insulin was highest with the combination of linoleic acid and iontophoresis. The flux enhancement (Fig. 2) was slightly higher with periodic iontophoresis compared to continuous current application (though not statistically significant; P.0.05) due to the alternate polarisation and depolarisation of skin in the former [23]. The lag time and skin affinity was reduced significantly (P,0.05) with

Fig. 1. Transdermal permeation of insulin from poloxamer 407 gel. P, passive; Ion, iontophoresis; Me, menthone pre-treatment; Mel, menthone pre-treatment followed by iontophoresis; LOA, linoleic acid pre-treatment; LOAI, linoleic acid pre-treatment followed by iontophoresis. Each data point represents mean6S.E.M. (n13).

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Table 1 Skin permeation parameters of insulin from poloxamer-407 gel using chemical enhancers and / or iontophoresis Treatment

Lag time (h)

Flux (mg / cm 2 / h)

Cumulative amount permeated (mg)

Skin affinity

Passive Iontophoresis a Periodic iontophoresis b Menthone c Menthone1TI d Linoleic acid c Linoleic acid1TI d

8.40 (1.94) 0.26 (0.09) 4.64 (2.74) 1.20 (0.02) 1.51 (0.72) 0.65 (0.49) 1.63 (0.20)

1.46 (0.18) 2.77 (0.04) 3.77 (0.48) 5.57 (0.13) 20.97 (0.63)* 8.08 (0.20) 31.79 (0.67)*

51.27 (2.63) 97.43 (3.63) 107.62 (4.15) 210.78 (2.64) 322.00 (5.55) 244.38 (30.21) 411.44 (21.80)*

18.85 (3.53) 31.47 (9.23) 31.94 (15.00) 10.57 (0.38)* 3.34 (1.38)* 5.28 (2.37)* 2.72 (0.83)*

Pre-treatment with enhancers was followed by iontophoresis; *Statistically significant (P,0.05). All the values are mean (n53) with S.E.M. in parentheses. a 0.5 mA / cm 2 for 6 h. b Current (0.5 mA / cm 2 ) was applied periodically using 1:1 on / off ratio for 12 h. c Concentration was 5% (v / v) in ethanol; pre-treated for 2 h. d TI is transdermal iontophoresis.

both chemical enhancer and / or iontophoresis compared to passive permeation. As is evident from Table 1, the flux and total amount of insulin permeated was highest with linoleic acid in combination with iontophoresis. Synergistic enhancement in flux was observed, when iontophoresis was combined with either linoleic acid or menthone (Fig. 2). All the skin permeation parameters observed with gel formulation were comparable to those obtained from insulin solution in our earlier studies (data not shown). In a hydrogel, the three-dimensional network provides sufficient rigidity, while the highly hydrated microscale environment facilitates mass transfer [24]. Although, a number of ionic polymers are known to exist as hydrogels, they retard the release of large peptides like insulin by binding to the ionic side chains of the polymer backbone [25]. Ideally, the gel matrix should not retard the release of large molecules like peptides / proteins, since it has to further diffuse through the highly impermeable stratum corneum. Poloxamer 407 is a non-ionic block copolymer, which is intermediate between hydrophilic and hydrophobic polymers [26]. It forms a themoreversible hydrogel [27] created by the aqueous solution of polyoxy(ethylene oxide)-b-poly(propylene oxide)-poly(ethylene oxide). This gel formation is a result of micellar entanglement and packing with an outer aqueous environment (hydrated PEO chains) and inner hydrophobic core

(PPO chains); making it suitable for the delivery of both hydrophilic and hydrophobic drugs. It is important to note that macroviscosity of the gel may appear to offer resistance to diffusion of drug, but the drug release is mainly governed by the microviscosity of the aqueous channels [28]. Erosion of the gel is the main controlling mechanism in poloxamers and is dependent on the water uptake into the gel as well as the boundary layer created by the static or stirring conditions [28]. Hence, the molecular size of the drug may have relatively little influence on the release rate, when there is a high uptake of water into the gel. When electric current is applied, the chemo-mechanical properties of the gel may be altered due to the associated electroosmotic flow, which substantially changes the structure and the composition of the gel [29]. This ultimately has a pronounced effect on drug release from the gel. Further, at pH 3.6, the electroosmotic flow in the skin is predominantly from the cathode to the anode (receptor to donor compartment), resulting in increased aqueous channels in the gel, which aids insulin release. In the absence of iontophoresis, slow uptake of water by the gel may alter the microviscosity, which results in release of insulin from the gel. Earlier, Banga and Chien [10] have studied the release and skin permeation of three model peptides (insulin, calcitonin and vasopressin) of varying molecular size using hydrogel matrices, where the diffusion coefficient from

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polyacrylamide gel decreased as a function of molecular size. The authors noted that it is necessary to control the swelling of hydrogels to avoid complicated swelling behavior, unlike the poloxamer gel used in our study. According to the lipid–protein partition theory [30], the chemical enhancers act at either the lipid bilayer and / or the protein domain; the solvents aid in the partitioning of the enhancers in the skin and therefore the choice of solvent can have an influence on the action of penetration enhancers. In our earlier studies, ethanol was found to show a significant synergistic enhancement in combination with iontophoresis among a variety of solvents [31]. From FT-IR studies, it was observed that ethanol extracted skin lipids and as a result reduced the diffusive resistance for iontophoretic transport of insulin. In case of terpenes, oxygen-containing terpenes have been reported to be effective enhancers for hydrophilic drugs compared to hydrocarbon terpenes [32]. We found that menthone showed maximum enhancement of insulin solution among a group of oxygencontaining terpenes [33]. There was no synergism in enhancement of insulin permeation when ethanol was used as a solvent for menthone (ethanol showed an enhancement ratio of 14 in comparison to 15 for the menthone–ethanol combination). Although both ethanol and menthone act at the same site in the skin, they differ in their mechanism of action. Ethanol produces its effects by slowly extracting the lipids from the skin, while menthone more readily alters the lipid bilayer [34]. As a consequence, ethanol’s effect on the skin is attenuated in the presence of menthone, and this was confirmed by FT-IR spectroscopy, where the percent decrease in the area of lipid peak was less for the menthone–ethanol combination in comparison to ethanol alone [33]. Terpenes form pools in the skin and increase the conductivity of skin due to the creation of additional ion-permeable pathways [35] and this leads to synergistic enhancement in combination with iontophoresis. Unsaturated cis fatty acids form ‘kinks’ in the skin and cause phase separation of skin lipids through which water and polar molecules can permeate [36,37]. This phase separation creates a favourable atmosphere for the iontophoretic transport of drugs; thereby resulting in synergistic enhancement. We earlier investigated a homologous series of unsatu-

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rated cis fatty acids and found that, in the presence of iontophoresis, the permeability of insulin was highest with linoleic acid (two double bonds). Beyond linoleic acid, the increase in number of double bond, leads to increased disruption of lipid bilayer [38], which leads to no further increase in the iontophoretic permeation of insulin through the highly perturbed and tortuous skin.

3.3. In vivo permeation studies The plasma insulin concentrations (PIC) and PGL are shown in Figs. 3 and 4 of various treatment groups. Table 2 shows the pharmacokinetic (PK) and pharmacodynamic (PD) parameters on subcutaneous and transdermal administration of insulin. As shown in Fig. 3, there was corresponding increase and decrease in PIC and PGL, respectively, with s.c administration of insulin. The PGL was reduced to 28% of its initial value and returned to baseline value by 8 h. The passive transdermal permeation resulted in low PIC and the PGL remained in the baseline and there was only 9% decrement in PGL over a period of 12 h (Table 2). On the other hand, iontophoresis resulted in higher peak PIC than passive permeation (Fig. 4) and the PGL reduced to 64% of its initial value in 2 h; which further continued to decrease even after stopping the current. This is attributed to the formation of insulin depot in skin [39], as was evident from the skin affinity values of insulin in ex vivo permeation studies (Table 1). Menthone pretreatment either alone or in combination with iontophoresis resulted in rapid attainment of insulin levels, but the levels were lower than iontophoresis. The extent of insulin permeated was relatively less in menthone treatment group compared to other treatment groups. Presumably, the faster attainment of peak PIC also led to a rapid clearance from the systemic circulation. With respect to PGL, there was a time lag before a reduction could be seen; however, the combination of menthone and iontophoresis did not provide higher levels of PIC or greater reduction in PGL, when compared to iontophoresis alone. There was no significant difference (P.0.05) in PK and PD parameters between menthone pretreatment with and without iontophoresis. Linoleic acid pre-treatment in combination with iontophoresis resulted in higher PIC among all the treatment

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Fig. 3. Plasma glucose levels and plasma insulin concentration in control groups. (a) Subcutaneous administration; (b) passive permeation from poloxamer 407 gel; (c) iontophoretic permeation of insulin from poloxamer 407 gel. Each data point represents mean6S.E.M. (n53).

groups, but the reduction in PGL was similar to iontophoresis (Table 2). All the treatment groups (except menthone pre-treatment and passive permeation) showed comparable extent of insulin permeation and higher PIC compared to s.c administration. However, the glucose reduction in all the transder-

mal treatment groups was less than that seen with s.c control group. As most of the endogenous peptides are eliminated rapidly from the systemic circulation, determination of plasma concentration is difficult and hence pharmacological activity is empirically used as a substitute for drug’s effect. In the case of insulin, a number of reports have shown that the AUC of the effect curve is not directly proportional to the AUC of the plasma concentration–time profile [40]. Moreover, the PK–PD relationship in endogenous peptides is complicated by the receptor mediated uptake, dose dependency and intracellular metabolism, multiple as well as differential effects on different tissues in the body [41–43]. Insulin tends to aggregate during transport in the skin, as the pH varies from 4 to 7 from epidermis to dermis resulting in entrapment inside the skin; which is slowly cleared by the microcirculation at dermo-epidermal junction [39,44,45]. Hence, it is not surprising that the PIC continue to increase, while PGL decrease even after stopping the current. Several investigators have reported that there is no direct relation between magnitude of glucose reduction and blood insulin levels on iontophoretic delivery of insulin [39,46]. It is important to note that earlier in vivo studies have used either high insulin concentration, alteration of skin barrier either by physical removal or by use of depilatory application in varying diabetic animal models with different current profiles using solution [39,46–52]. We achieved a significant reduction in PGL with milder chemical enhancers (menthone and linoleic acid) either alone or in combination with iontophoresis using a gel formulation. The lack of a direct correlation between ex vivo and in vivo results may be attributed to the difference in skin hydration levels [50] and the faster recovery of skin to the action of chemical enhancers in in vivo compared to ex vivo experiments. Moreover, the higher amount of insulin found in ex vivo permeation studies using radiochemical methods is an overestimation, as it includes both the degraded and intact insulin; whereas, RIA (used in in vivo) detects only immunologically active insulin species [46]. Nevertheless, the study demonstrates that either chemical enhancer or iontophoresis alone and in combination is useful to improve the rate and or

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Fig. 4. Plasma glucose levels and plasma insulin concentration of after pre-treatment with chemical enhancers alone or in combination with iontophoresis. (a) Menthone pre-treatment; (b) menthone pre-treatment followed by iontophoresis; (c) linoleic acid pre-treatment; (d) linoleic acid pre-treatment followed by iontophoresis. Each data point represents mean6S.E.M. (n53).

Table 2 Pharmacokinetic and pharmacodynamic parameters of insulin after in vivo administration in diabetic rats Treatment

Cmax a insulin (mU / ml)

Cmin b (% PGL of initial)

a T max of insulin (h)

b T min of PGL (h)

AUC ( 0 – 12 ) c (mU / ml).h

%D( 0 – 12

Subcutaneous Passive Iontophoresis e Menthone f Menthone1TI g Linoleic acid f Linoleic acid1TI g

110.42 (5.07) 51.00 (10.14) 141.00 (9.00)* 86.00(18.00) 115.00(28.00) 208.00(75.00)* 220.00 (25.00)*

28.00 84.12 64.22 71.95 72.08 60.00 66.17

4.00 (2.00) 10.00 (2.00) 8.67 (1.76) 1.67 (0.33)* 1.15 (1.53)* 6.00 (1.15) 4.30 (2.03)

2.50 (1.50) 10.00 (2.02) 8.70 (3.33) 6.67 (2.91) 10.67 (1.33) 6.33 (3.18) 6.01 (1.15)

856.99 (70.82) 353.83 (82.00) 941.32 (54.13) 529.35 (28.86) 522.16 (28.05)** 851.00 (104.00) 1188.00 (233.00)

55.96 (11.02)* 9.92 (12.50) 28.81 (11.21) 16.54 (10.78) 11.95 (2.14) 13.19 (2.20) 28.66 (8.40)

(14.22)* (12.01) (20.00) (11.12) (4.45)** (14.23) (13.89)**

d h)

*Statistically significant (P,0.05); **statistically not significant (P.0.05). PGL, plasma glucose level; TI, transdermal iontophoresis. All the values are mean (n53) with S.E.M. in parentheses. a The parameters were directly read from insulin concentration versus time plot. b The parameters were directly read from plasma glucose level (% of initial) versus time plot. c This indicates the area under the curve of plasma insulin versus time plot and was calculated using trapezoidal method. d %D5(12AUC / 123100) 100. The AUC used in the formula was from the plasma glucose reduction curve. e A current of 0.5 mA / cm 2 was applied for 6 h. f Pre-treatment was carried out for 2 h. g Pre-treatment was followed by iontophoresis (0.5 mA / cm 2 for 6 h).

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extent of insulin permeation, but at the same time a higher PIC may result in saturation of cellular uptake mechanisms leading to no direct relation between insulin permeation and PGL reduction.

3.4. Histological examination The morphological changes in skin after subjecting the skin to chemical and or current treatment are shown in Fig. 5. In the case of blank gel treatment, clearly defined SC could be seen; with well-woven structures and the inflammatory cell infiltration observed may be due to the response elicited by poloxamer. On application of current, the cell structures loosened with increased cell infiltration in dermis and in some skin samples, degeneration of appendages were also observed (Table 3). With menthone pre-treatment, there was destruction of SC and sub-epidermal edema; while in the presence of iontophoresis, there was further destruction of SC layer (Fig. 5c,d). Pre-treatment with linoleic acid resulted in swollen sub-epidermis and dermis was more fluid with loosened structures, which further amplified on application of current (Fig. 5e,f). Overall, the total irritation score was highest with the combination of linoleic acid and iontophoresis (Table 3), which also showed maximum enhancement in both ex vivo and in vivo permeation studies. Although, the irritation scores are subjective, they serve as useful tool to compare qualitatively the effects of various treatments on skin [17]. Earlier studies have shown that enhancers, which cause significant enhancement, also produced high skin irritation [53,54]. When a combination of chemical enhancers and iontophoresis is used, the latter has a more predominant role in causing synergistic enhancement and skin irritation [31,33,55]. However, the skin irritation depends on the concentration of the enhancer and the duration of application. Jiang et al. [56] have shown using TEWL and ultrastructural studies that skin regained its barrier property within 48 h after application of oleic acid and iontophoresis. The co-application of chemical enhancer and iontophoresis may be beneficial, where the latter can be used to modulate the permeation of former and reduce the application time for chemical enhancers. Another potential strategy to reduce skin irritation is to combine two chemical enhancers, which act by

different mechanisms. This, when coupled to iontophoresis can result in use of reduced concentration of penetration enhancers, that shows maximum enhancement with less skin irritation. For example, in the case of insulin, menthone gave rapid insulin levels, while linoleic acid pre-treatment resulted in a high extent of insulin permeation; therefore the combination of these two enhancers at low concentrations may be advantageous not only from a pharmacokinetic viewpoint but also in terms of reducing the skin irritation potential. The rat skin is more permeable than human skin [57] and may provide an overestimation of skin penetration of drugs and enhancers (leading to increased skin irritation). Therefore, the relevance of the findings from this study need to be further confirmed using human skin.

4. Conclusions Gels are clinically acceptable delivery systems for iontophoresis in terms of stability, ease of handling and refilling of iontophoretic patches. Poloxamer 407 gel was found suitable for transdermal delivery of insulin. The combination of chemical enhancers and iontophoresis resulted in synergistic enhancement of insulin permeation and also showed synergism in causing skin irritation. There was no direct correlation between the ex vivo and in vivo permeation results. The PK–PD correlation is complicated by a number of factors and there is a need to develop suitable PK–PD models to explain the permeation of endogenous peptides through skin. Future clinical application would depend on how far the dose efficiency is improved without compromising the skin barrier integrity using combination strategies.

Acknowledgements The Department of Science and Technology (DST), India, supported this work through a research grant. The authors appreciate the support of Board of Radiation and Isotope Technology (BRIT), Mumbai, India, for supplying the radioiodinated insulin for the study. Eli Lilly, USA, supplied human insulin and

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Fig. 5. Light microscopic photographs (3250) after chemical enhancer pre-treatment and / or iontophoresis. (a) Blank poloxamer gel treatment; (b) iontophoresis for 6 h (0.5 mA / cm 2 ); (c) menthone pre-treatment for 2 h; (d) menthone pre-treatment followed by iontophoresis; (e) linoleic acid pre-treatment; (f) linoleic acid pre-treatment followed by iontophoresis.

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Table 3 Skin irritation scoring on skin treated with chemical enhancers and / or iontophoresis Parameters

Epidermis liquefaction Sub-epidermal edema Dermis Collagen fiber swelling Inflammatory cell infiltration Skin appendages degeneration Total irritation score

Treatment Control a

0.5 mA / cm 2 b

Menthone c

Menthone 1TI d

Linoleic acid c

Linoleic acid1TI d

0–1 0

0–1 2

0 2

1–2 3

1–2 2

1–2 2–3

2 2 0 4–5

2 2 1 5–6

2–3 0–1 0 5–6

3 1–2 1–2 9–12

1–2 2–3 0 6–8

3–4 1–2 1–2 8–13

TI, transdermal iontophoresis. Grade: 0, no change; 1, very slight; 2, slight; 3, moderate; 4, marked change. a Control was skin treated with blank poloxamer gel. b Current was applied for 6 h after application of blank polaxmer gel. c Pre-treatment with 5% (v / v) of penetration enhancer in ethanol for 2 h. d Pre-treatment with enhancers was followed by iontophoresis for 6 h using blank poloxamer gel.

poloxamer 407 was a gift sample from BASF, Germany.

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