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Since the first patch was commenced in 1981, for clinical benefits, industry interest and regulative precedence it predicted a strong market for transdermal therapeutic systems. This route is particularly suitable for drugs used in long-term therapy like hypertension and diabetes. Captopril and Lisinopril, an orally effective angiotensin-I converting enzyme inhibitors, routinely used to cure hypertension and congestive heart failure. Captopril has a short elimination half life and its plasma half life in man ranges from 1.6-1.9 h [6-8]. Captopril and Lisinopril being antihypertensive agents needs long time administration. Results of post market observation on Captopril and Lisinopril had shown that number of the patients had to withdraw therapy because of the unwanted effects [14-15]. In present market these drugs are available only in the form of oral tablets and have slow and incomplete absorption; moreover food may affect oral absorption of both drugs by up to 25-40% [9-12]. Major problem related to oral therapy includes irregular bio-distribution throughout the body, an absence of drug targeting specificity, the requirement of a large dose to get high blood concentration and unwanted effects due to such high doses . These drugs have stability problems due to the oxidation reaction, which converts Captopril and Lisinopril into Captopril disulphide and Lisinopril disulphide respectively. A recent study had shown that the oxidation rate of Captopril and Lisinopril in dermal homogenates is considerably less than that in intestinal homogenates [15-16]. For hypertensive patients, medication become an essential part of life and noncompliance of therapy may result in chronic complication. Worldwide frequency estimate for hypertension may be as much as 1 billion people, and concerning 7.1 million deaths per year may be attribute to hypertension. To overcome the problems encountered with oral delivery like imperfect absorption, less oral bioavailability, and for the effective treatment of chronic hypertension, alternative transdermal route of administration suggested valuable. Being medication essential part of life, the success of the treatment depends on patient compliance too. Seeing as transdermal dosage forms can reduce the fluctuations of plasma drug concentration and increase patient compliance, the development of the transdermal therapeutic systems for these drugs has become research interest of late [17,18].
Captopril and Lisinopril were scanned in the UV wavelength region of 200-400 nm for maximum absorption (Î» max). The Î»maxes were found to be at 227 nm and 217 nm for Captopril and Lisinopril respectively that were same as reported value. Linear relationships were observed between the concentration and absorbance values (Table 4.1.1 & Table 4.2.1) in the range of 5 to 30 µg/ml (Slope = 0.0138, R2 = 0.9945) for Captopril and same 5 to 30 µg/ml (slope = 0.0358, R2 = 0.9966) for Lisinopril. (Fig 4.1.1 & Fig 4.2.1)
Though it was hypothesized that skin is permeable to the lipophilic moieties of low molecular weight, in reality the extent of transdermal permeation is a composite parameter influenced by many factors. In addition to molecular weight, partition co-efficient, solubility, the pka value that determines the extent of ionization, is of prime importance. As Doh et al suggested, drug candidates for transdermal dosage form should have molecular weight around 200~500 Da . Captopril and Lisinopril having molecular weights 217.29 and 405.5 Da respectively, well fits into this range. Various physicochemical parameters of Captopril and Lisinopril were investigated and the results listed in Table 4.1.2 & 4.2.2. Both drug candidates showed good aqueous solubility. The experimentally determined partition coefficients (octanol/phosphate buffer) were found to be 0.335 ± 0.0090 and 0.675 ± 0.0124 for Captopril & Lisinopril respectively, which indicated that these drugs have less affinity towards the lipid compared to phosphate buffer, which is not favorable for transdermal permeation. Although, the lipophilicity of a drug moiety is an intrinsic character therefore cannot be modified.
At pH 7.4 mammalian skins are negatively charged and ionic forms have low affinity to skin. Captopril and Lisinopril, being an acidic drug with the pKa value of 3.481 and 3.85 respectively, were largely ionized which decreased its affinity toward the skin . However ionization make favorable for iontophoresis. In the process of iontophoresis, the ionized moieties are actively forced, using low intensity current through natural pore pathways of skin at the same time the unionized fraction could pass the unbroken horny layers by passive diffusion resulting in enhanced permeation . As the permeation rate completely depends on the energy so controlling the supply of the current can control the delivery rates . The diffusion cell was modified, to simulate the physiological condition, where both the electrodes were placed on the same side of skin and receiving chamber filled with phosphate buffer, reflected the body.
Proper selection of electrode is very vital in iontophoresis as in this method the electronic current gets converted to ionic current at the electrode-solution interface. The electrodes like stainless-steel, platinum or carbon graphite do not contribute in the electrochemistry thus the inert electrochemistry forces the water in the reservoir to become fuel for the electrochemistry. Because the oxidation take place at the anode and reduction at the cathode, hydrogen and hydroxyl ions are generated; this results to drop in pH at the anode and rise at the cathode. That change in the pH may lead to irritation or burns at the site. In addition, there may also chance for degradation of drug at the electrode. Problem does not occur with silver/silver chloride reversible electrodes, which take parts in electrochemistry and chosen for this study .
The main factor that affects the in-vitro transdermal permeation is the barrier properties of the skin. The present study carried out using porcine ear skin, obtained from local slaughterhouse. As the barrier properties of the skin varies with age of the skin, dermal thickness, hair follicle depth, factors controlled by experimentally. Same ear skin was used for iontophoresis as well as passive permeation studies to control the variability of results by skin. Porcine skin has similar thickness of stratum corneum as human skin. Also the hair follicle density of pig and human skin (about 11 hair follicles/cm2) is similar which is higher in other mammalians. Using a number of compounds, it has been shown that the pig skin has found the closed permeability characteristics to that of human skin. In-vitro studies with human skin have been also shown to correlate fine with in-vivo studies in pigs. In one more study the skin permeability of nicorandil was find out across excised skin samples from hairless mouse, guinea pig, pig, hairless rat, dog and human. Among six species, the permeability was highest in hairless mouse while that in pig and human skin was found in good agreement. It was observed that pig and human skin had similar barrier thickness, surface lipid and morphological features .
Among the various factors that affect skin permeability, the concentration of the actives in the delivery system is the most crucial. To evaluate this effect of concentration and effect of current densities the experiments were designed at three different drug concentrations and current densities (Table 4.1.3 &Table 4.2.3).
Fig 4.1.8 & Fig 4.1.9 shows passive and iontophoresis permeation profile of Captopril at different donor concentrations. The passive profiles are linear at all concentration levels indicating the permeation kinetics was more or less zero order. The rate of permeation increased with increasing donor drug load. This is expected, as increase in the donor drug concentration enhances the concentration gradient, that is the driving force of mass transport . In contrast iontophoretic profiles are less linear showing the involvement of some other factors along with concentration. In iontophoresis though ionic repulsion is the dominant process, there is also a convective transport of the materials toward the direction of flow of current, moreover the permeability of skin changes. The total flux of a solute during iontophoresis is the sum of fluxes due to electro repulsion, convective flow, and passive diffusion . Similar results were found to be in case of Lisinopril. (Fig 4.2.8 & Fig 4.2.9).
The total ¬‚ux of a solute during iontophoresis is the sum of ¬‚uxes due to electro-repulsion, convective flow, and passive diffusion . At pH 7.4, Lisinopril (pKa 3.85) and Captopril (pKa= 3.48) acquired negative charges and decided to delivered from cathodal chamber. Since the isoelectric point of the skin varies between 3 and 4, at physiological pH, the volume ¬‚ow was directed toward the cathode. Hence at pH 7.4, only passive and electro-repulsive ¬‚uxes were expected to contribute to the permeation. There are also possibilities to oppose the permeation from the cathodal compartment by electro-osmotic ¬‚ow . In our study, iontophoretic profile indicates the initial permeation was high however the permeation rate declined in the later hours. This was unexpected as the voltage gradually dropped with time and therefore the magnitude of electro-osmotic opposition was expected to be lesser in the later part of the study. The contrary of result suggested the contribution of a factor that negatively influenced the permeation as time passed. This is also possible that as the current flows, the cathodal electrode (Ag/AgCl) obtained a steady ¬‚ow of electrons, which lead to the release of negatively charged chloride ions. As time passed, the concentration of this newly liberated chloride ions were probably to increase in the cathodal portion. A chloride ion, being much smaller than the drug ion, was a powerful competitor, which diminished the transport efficiency of the drugs, since the drug candidates were negatively charged and chloride ions act as competitor .
Though steady state flux is considered to be the most therapeutically relevant parameter, permeability and diffusion parameters are also very important for comparison purpose . It is evident that as the concentration increase in donor the permeability coefficients decrease. Result for the permeability and diffusion coefficients of Captopril & Lisinopril in different systems in our study for passive diffusion and iontophoresis are provided in Table 4.1.12 & 4.2.12, which found to obey above hypothesis.
Table 4.1.13 & Table 4.2.13 depict the enhancement in iontophoretic flux compared to the passive flux of same donor concentration. Enhancements were highest at the lowest drug load and lowest at the highest drug load for both the drugs. To analyze the net benefit of electrical energy, the active fluxes of drug at various donor drug loads was compared with the corresponding passive value. (Table 4.1.13 & Table 4.2.13) The iontophoretic contribution was found to be slightly more at higher donor concentrations.
Pikal el al suggested that permeability of skin also changes under influences of current . To observe the effect of current densities on the transport of Captopril and Lisinopril through pig ear skin, experiment was carried out at three different current densities and result was found that iontophoretic drug transport increased with the increasing current densities (Fig 4.1.10 & 4.2.10).
The relationship between current density and flux of drugs may be described by Faraday's law which is represent by following equation 
Ji = ti It / Zi F
where Ji, Zi are the flux and charge of drugs at particular pH, It is the applied current density, F is the Faraday's constant, ti is a proportionality constant. Since in the experimental conditions ti and Zi i.e., charge of the drug was kept constant, then by above equation flux is directly proportional to the current density. Literature survey suggested that disordering of intracellular lipid of stratum corneum by increasing of current density may cause increase in drug transport . Some researchers suggested that possibility may be also that the electro-osmotic volume flow increases with an increase an current densities  which leads to increase in the flux of the drug.
To assess the combine effect of chemical enhancers and iontophoresis on transdermal delivery of selected drugs, permeation was carried using permeation enhancers with iontophoresis. Dimethyl sulfoxide (DMSO), menthol, oleic acid, peppermint oil, poly ethylene glycol, and sodium lauryl sulphate were used as permeation enhancers. These chemical substances temporarily disrupting the barrier properties of skin and known as accelerants or sorption promoters can enhance drug permeation. DMSO is one of the earliest and most commonly used as penetration enhancers, it changes the intercellular keratin conformation, from Î± helical to ß sheet [147-148] and enhances the permeation. The possible mechanism of action of oleic acid suggested as it increases the fluidity of the lipid structure by penetrating into it, that is again supported by its polar end and bent structure and peppermint oil probably operates due to opening of tight junctions [149-150]. Mechanisms of action for the permeation enhancement by propylene glycol is suggested by keratin solvation within stratum corneum by competition for the hydrogen bond binding sites with water and the intercalation in the polar head groups of the lipid bilayers . Anionic surfactant like sodium lauryl sulphate open up the protein controlled polar pathway by uncoiling and extending the alpha-keratin helices and swelling the stratum corneum . Menthol reported for penetration enhancing effect by changing of dense barrier structure of the stratum cornea . Cumulative amount permeation of drugs with and without enhancers provided in Fig 4.1.24-25 & Fig 4.2.24-25. Diffusion and permeability coefficients increase in the order of Pure drug < PEG < SLS < Oleic acid < Menthol < Peppermint oil < DMSO. The diffusion and permeability coefficients of both drugs with DMSO were found highest in our study, moreover with the iontophoresis synergistic effect was found (Table 4.1.29 and Table 4.2.29). Results shows that overall permeability using enhancers considerably higher that of passive values. Benefits by enhancers and enhancement ratio are provided in Table 4.1.30 and Table 4.2.30.
Comparative figure of steady state fluxes with different enhancers and iontophoresis are shown in Fig 4.1.26 & 4.2.26. Steady state fluxes increase in the following order Pure drug < PEG < SLS < Oleic acid < Menthol< Peppermint oil < DMSO<Iontophoresis < (Ionto+PEG) < (Ionto+SLS) < (Ionto+Oleic acid) < (Ionto+Menthol)< (Ionto+Peppermint oil) < (ionto+DMSO). Steady state flux was found highest (14.878 ± 0.707mmol/cm2/hr) with combine effect of DMSO and iontophoresis and benefit by this combine effect was 11.533 mmol/cm2/hr for Captopril which was also followed by Lisinopril. Iontophoresis considerably increase permeation with all permeation enhancers.
Most of the studies related to transdermal iontophoresis are focused on aqueous solutions, due to the complex nature of the drug delivery. Gels can be easily integrate with the iontophoretic delivery system and match the contours of the skin so it considered to be the most appropriate delivery vehicles for iontophoresis. Several other advantages of gels over liquids include easy fabrication into the device, aptness with the electrode design, deformability into skin contours, superior occlusion, and better stability. Furthermore, the high proportion of water used in gel formulations can in turn provide a helpful electro-conductive base for clinical use. A hydrogel formulation might also be helpful in minimizing skin hydration during the period of medication and might reduce the convective flow which often accompanies iontophoretic delivery. One more advantage of a hydrogel preparation would be the ease of application [138, 154, 155].
After passive and iontophoretic in-vitro permeation of Captopril and Lisinopril from solutions, we found that iontophoresis could significantly enhance the permeation rate as compared to its passive diffusion. However, skin permeation of a drug from dosage form is much more complex than that from a solution, because in drug stay in more intimate contact with the excipients in dosage form, that affect its release profile. Since the driving force of passive diffusion is the concentration gradient of the free drug between the skin surface and plasma so enough free drug concentration must be available on the surface of the skin ensure a high concentration gradient, hence it is essential to ensure that the drug is not irreversibly bound to the dosage form components. For the formulations of gels hydroxyl propyl methyl cellulose were used. HPMC being a neutral polymer did not show any interaction with the drug. Drug content of prepared gel were determined and found to be uniform. The measured viscosity of the gel was about 1.4 Pa/S for Captopril and 1.3 Pa/S for Lisinopril (Brookfield viscometer, 12 rpm), suitable for transdermal application.
The FT-IR studies were carried out to check compatibility of drug and exipients. Spectra of pure drug Captopril, and Captopril gel formulation recorded individually and no interaction observed. FT-IR spectra of Captopril revealed the presence of characteristic peak at 1384.8 Cm-1 because of C-N stretching due to the presence of tertiary amine group. Peak at 1747.4 Cm-1 because of C = O stretching and vibration due to the presence of carboxylic group. Peak at 2565 Cm-1because of S-H vibration because of the presence of thiol group. Peak at 1454 cm-1 because of C-H bending. Spectra also shows presence of weak peaks at 2877 cm-1 that could be attribute to asymmetric C-H stretching vibration of captopril anions. After comparing the IR spectra of gel formulation, it was observed that the IR spectra lack any sign of probable interactions. (Figure 4.1.35-36)
The FTIR studies were performed for the pure drug Lisinopril, and Lisinopril gel formulation individually and the spectra showed no interaction. FT-IR spectra of Lisinopril exposed the presence of characteristic peak of O-H stretching around 3333 cm-1, N-H stretching around 3557.85cm-1 , sp3 C-H stretching at 2957cm-1, C=O stretching around 1612 cm-1 and C-O stretching around 1045 cm-1 , and the spectra of Gel formulation revealed the presence of characteristic peaks of N-H stretching, O-H stretching, sp3C-H stretching, C=O stretching and C-O stretching. Thus it was confirmed that the IR spectra lack any sign of probable interactions. (Figure 4.2.35-36)
The short term stability studies of gel formulations were performed at different temperature and humidity according to ICH guidelines and various performance parameters i.e., physical appearance, pH, viscosity, drug content and drug releases were evaluated. Results indicate that there were no significant change occurred during storage at different temperature and humidity. (Table 4.1.37 & 4.2.37)
Since one of the purposes of our study was to evaluate plasma concentration profile of both drugs in rabbits, it was required to have some basic idea about the permeability of drugs with respect to the rabbit skin also. So many studies revealed that the barrier properties of pig skin are close to the human skin  whereas the permeability of rabbit skin is much higher among all laboratories animals . This is confirmed in our study by comparative permeability data of pigskin and rabbit skin (Fig 4.1.32-33 and Fig 4.2.32-3). Furthermore the comparison of passive and iontophoretic permeation of both drugs from rabbit and pig skin shown in Fig 4.1.29-31 & Fig 4.2.29-31. As we assumed that higher follicular density and water content would favor iontophoresis in rabbit skin, it was found true as after the study have performed. Permeation enhancement by iontophoresis was found to be higher in case of rabbit skin than pig skin.
Steady state fluxes, permeability coefficients, and enhancement ratios were determined and provided in Table 4.1.36 and Table 4.2.36. Enhancement by iontophoresis 6.265 folds for Captopril and 4.615 folds for Lisinopril were found in our study using rabbit skin it as slightly higher than that of pigskin. Plasma concentration profile of Captopril and Lisinopril using passive diffusion and iontophoresis can be seen in Figures 4.1.34 & 4.2.34. It was observed that the Captopril and Lisinopril concentration after iontophoresis increase much rapidly than that of passive diffusion. Very less amount of Captopril and Lisinopril determined in plasma after passive diffusion but as expected it was considerably higher when iontophoresis was used in the study.
In our study the maximum plasma concentration were achieved 909 ± 28 ng /ml and 45.076 ± 1.924 ng /ml for Captopril & Lisinopril respectively at the end of 8th hour. Results indicate that the target permeation rates for both drugs could be achieved by iontophoresis with increasing the area of application in an appreciable range.
The required minimum administered area is calculated by formula  Areq= Jtarget / JSS , Where Areq represents required administered area, Jtarget Target flux and JSS steady state flux obtained in our study. The non compartmental analysis of the pharmacokinetic data indicate that to meet the demand of maintenance therapy for 60 kg individual 1488 Î¼g/h of Captopril and 26.7 g/h of Lisinopril must be supplied to the systemic circulation[158-159]. The in-vitro iontophoretic fluxes of drug formulations through rabbit skin were found to be 251.471 g/hr.cm2 and 6.402 g/hr.cm2 in our studies. So the required minimum area for Captopril 5.917 cm2 and for Lisinopril 4.17 cm2 can achieve the target. As the patch in the market usually have wider area (10 cm2 and above), it can be expected to achieve the target. Overall results looked quite promising for transdermal delivery of Captopril and Lisinopril.