Many known compounds can enhance drug absorption through the skin, but their modes of action are still not fully understood.
Delivery of drugs through the skin has many attractions, including increased patient acceptability, avoidance of gastrointestinal disturbances, and first pass metabolism of the drug.1, 2 In fact, this route is not commonly used because of the skin'S inherent barrier properties.3 It is generally difficult to get even a potent drug to pass through the skin at a sufficient rate to deliver a therapeutic dose. Permeation enhancers must be developed to improve the permeability of drugs across the skin.4
For the last two decades, researchers have attempted to identify specific chemicals or combinations of chemicals that might act to enhance permeation.5 Much of this work has been carried out by pharmaceutical companies, which regard the results of their work as proprietary. Consequently, a great deal of cited work is found in patents and pharmaceutical literature.6 And, although many permeation enhancers are now known, their modes of action are still not fully understood.7
Recently, researchers have investigated the use of terpenes such as menthol, d-limonene, citral, and cineole as permeation enhancers.8 Terpene compounds are derived from plant essential oils and combine good permeation enhancing abilities with low skin irritancy and low systemic toxicity. Terpenes have been extensively researched as permeation enhancers for various drug candidates.9
These compounds increase drug permeation by disrupting the highly ordered structure of intercellular lipids and improving the partitioning of solutes in the stratum corneum. Zhao and Singh reported the mechanism of in vitro permeation enhancement of tamoxifen using terpenes. These researchers combined propylene glycol with three separate compounds-eugenol, limonene, and menthone-and studied the effects of each combination on the permeation of tamoxifen through porcine skin. Limonene produced the highest permeation enhancement of the three terpenes studied.10
Sapra and colleagues investigated the role of volatile oil pretreatment on permeation of ion-paired diclofenac sodium from the transdermal films. They used volatile oils containing terpenes, including cardamom oil (cineole), lemon oil (limonene), clove oil (eugenol), turpentine oil (longifolene), and eucalyptus oil (aromandrene). Cardamom oil produced the maximum in vitro permeation of ion-paired diclofenac sodium.11
Verapamil hydrochloride is a calcium channel blocker used extensively in clinical medicine to treat angina pectoris and hypertension. It is 90% absorbed in the gastrointestinal tract, and, although the oral dose is high (40-80 mg), bioavailability is only 10% to 20% of the absorbed drug, indicating extensive first-pass metabolism in the liver. An additional concern is its short plasma half-life of 4 � 1.5 h.12
Many researchers have worked on the skin permeation of verapamil hydrochloride using different penetration enhancers across skin models.13, 14 To the best of our knowledge, however, there is no report on the skin permeation of verapamil hydrochloride using cineole, d-limonene, and citral as penetration enhancers across three skin models: rat, mouse, and human cadaver skin.
Materials and Methods
Verapamil hydrochloride IP was obtained from Torrent Pharmaceuticals (Ahmedabad, India); cineole, d-limonene, and citral were purchased from Sigma-Aldrich (St. Louis). Swiss albino mice and rats were purchased from the National Institute of Nutrition (Hyderabad, India). Human cadaver skin from the chest region was obtained from 35-and 36-year-old males at Civil Hospital (Raichur, India). Double distilled water was used throughout the study; all other chemicals used were of analytical grade.
Figure 1. Permeation of Verapamil Hydrochloride
An excess amount of the drug was taken and dissolved in a measured amount of distilled water in a glass vial to obtain a saturated solution. The system was kept at rest for 24 hours at 37oC to assist in the attainment of equilibrium. The supernatant was filtered and assayed spectrophotometrically.
Ten mL of octonol were added to an equal volume of aqueous solution of the drug of known concentration in a separating funnel and kept for 24 hours at 37oC with intermittent shaking. Finally, the aqueous layer was separated, clarified by centrifugation at 2,000 rpm, and assayed. The apparent partition coefficient was calculated with and without terpenes using the following equation:
Preparation of Mouse and Rat Skin
Permission from the institutional ethical committee was obtained to use mouse, rat, and human skin for this project. Swiss albino mice and rats eight to nine weeks old were used for the study. The animals were sacrificed by cervical dislocation just before use. Each animal'S abdominal skin section was carefully cut, lifted, and separated from the adhering fatty tissues and visceral materials. The excised skin was thoroughly washed three times with double distilled water, and a 2.63 cm2 surface area of skin was mounted on the donor compartment of the diffusion cell, with the stratum corneum facing the donor compartment.15
To prepare the human cadaver skin, we used the same method we reported in our previous paper.16 Skin samples were removed from the chest region within 48 hours after death. The skin was cut carefully, lifted, and separated from fatty tissues; visceral materials and the skin section were washed with double distilled water to remove extractives. To separate the epidermal layer from the remaining skin, we immersed each skin section in water heated to 60oC for 30 seconds. The epidermis was teased from the dermis using forceps. A separated epidermal layer of 2.63 cm2 surface area was used for the permeation study. The remaining skin samples were stored in aluminum foil at -20oC until the next experiment, which was conducted within two hours. It has been reported that prolonged freezing of the skin has no effect on permeability of the drug through skin.17 The skin was left at room temperature for two hours prior to experimentation.
Vertically assembled Keshary-Chien diffusion cells with a downstream volume of 20 mL were used for the study. The receptor compartment was filled with 20 mL of double distilled water, the magnetic stirrer was set at 100 rpm, and the whole assembly was maintained at 37�0.5oC. The mouse/rat/ human cadaver skin (2.63 cm2) was fixed to the donor compartment with the help of an adhesive. Skin pretreatment involved applying terpenes (10%, 15%, and 20% weight/volume of donor solution) to the stratum corneum surface, and 1 mL of the drug solution in distilled water (2 mg/mL) was poured into the donor compartment. The amount of drug permeated was determined by withdrawing 5 mL samples at specific time intervals of 24 hours. The volume withdrawn was replaced with an equal volume of fresh distilled water, and samples were analyzed in an ultraviolet spectrophotometer at 229 nanometers.
Figure 2. Influence of Terpenes
The selected terpenes were tested for skin irritation studies in rats. A modification of the method followed by Rajesh and Pandit was used.18 The abdominal hair of seven rats was removed 48 hours before the study. An aqueous solution of formalin (0.8%) was applied as a standard irritant, and terpenes (20% weight/volume of donor drug solution) were applied to the nude skin. After 24 hours, the animals were checked for any signs of erythema and edema. A skin score was noted using visual observations.
Data presented are the averages of three readings plotted as percent drug permeated per unit surface area of the skin against time. The slope of the linear portion of the plot was calculated as the steady-state flux (JSS). In experiments where steady state could not be achieved, the average permeation rate was calculated by dividing the amount of drug permeated by the duration of the experiment.16 The enhancement factor (EF) was calculated using the following equation:19
Results and Discussion
The solubility of any drug in a given vehicle determines the active concentration at which the drug could be presented onto the skin surface. Hence, a good solubility in a chosen vehicle ensures the movement of a given drug through delivery systems. Because water is the most acceptable vehicle, we determined the solubility of the drug in distilled water to be 55.5 mg/mL.
The physicochemical characteristics that facilitate percutaneous absorption of drugs include high lipid solubility, as indicated by a high octanol/water partition coefficient (PC) value. A lipid/water PC value of one or greater is generally required for good permeation, because the drug must be dissolved in the lipids of the skin before it enters blood circulation. The PC values obtained are presented in Table 1 (p. 40). The PC value of the drug was found to be 0.41 without terpenes, indicating poor permeability. But the PC values increased in the presence of terpenes: 0.75, 0.92, and 0.70 for cineole; 0.62, 0.86, and 0.58 for dlimonene; and 0.58, 0.79, and 0.53 for citral at 10%, 15%, and 20% concentration levels, respectively-all higher values than those that were obtained with the drug alone (Table 1, p. 40). We observed increasing PC values when terpene concentration was increased from 10% to 15%; at 20% concentration, however, values decreased.
Chemical permeation enhancers can increase skin permeability using various mechanisms, including increasing solubility, increasing partitioning into the stratum corneum, fluidizing the crystalline structure of the stratum corneum, and causing dissolution of stratum corneum lipids. The enhancement in absorption of lipophilic drugs is apparently due to the partial leaching of epidermal lipids by chemical enhancers, resulting in improvement of the transdermal/ transfollicular penetration. The composition of intercellular lipids undergoes a solid-liquid phase transition at 40oC; it is possible that some penetration enhancers act to disrupt the structure of intercellular lipids and lower the phase transition temperature, thereby increasing the permeability of the skin to more polar drugs.20, 21
In our investigation, the calculated flux for drug permeation in the absence of terpenes was 1.5968 X 10-2, 1.1368 X 10-2, and 1.0080 X 10-2 mg/cm2/hour across mouse, rat, and human cadaver skins, respectively, at the end of 24 hours (see Table 1 and Figure 1, pages 40 and 39, respectively). But the presence of terpenes enhanced the permeation rate. We observed an increase in flux, from 7.7353 X 10-2 to 10.4285 X 10-2 mg/cm2/hour, when the concentration of cineole was changed from 10% to 15% (P<0.05);>-2 mg/cm2/hour across mouse skin. Similar trends were seen for d-limonene and citral. Among the three terpenes used, cineole (15%) showed the most significant permeation rate in human skin (flux of 10.9885 X 10-2 mg/cm2/hour; P<0.05)>-2 mg/cm2/hour). The order of permeation enhancement for terpenes was cineole>d-limonene>citral (P<0.05)>10%>20% (see Figures 2, 3, and 4, pages 41, 42, and 53, respectively).
The literature on transdermal delivery is replete with data obtained from skin permeation experiments carried out with various animal skin models. Data obtained from different skin models fluctuate, however, and actual estimation is possible only if these models can be graded according to their resistance to the entry of drug molecules. Permeation of drugs across the skins of common laboratory animals such as rats, mice, and rabbits has been found to be significantly higher than human skin; this may be due to differences in lipid composition and organization in the stratum corneum.22
Figure 3. Permeation of Verapamil Hydrochloride Across Mouse Skin
Figure 4. Permeation Enhancement of Verapamil Hydrochloride
In our study, we have used three different skin models-mouse, rat, and human cadaver-in order to investigate their resistance to the entry of drugs into the body. Compared to mouse and rat skins, human skin offered high resistance to the entry of the drug, in the following order: human cadaver skin>rat skin>mouse skin. The flux values for mouse, rat, and human skin were 1.5968 X 10-2, 1.1368 X 10-2, and 1.0080 X 10-2mg/cm2/hour, respectively, without terpenes. In the presence of terpenes, human skin offered low resistance (see Table 1, p. 40); among the selected terpenes, cineole at 15% concentration showed the highest drug permeation rate and lowest resistance (flux of 10.9885 X 10-2 mg/cm2/hour). In general, permeation enhancement caused by all three terpenes was greater with human skin than with mouse and rat skins. Greater drug flux in the presence of terpenes may be due to greater partitioning into the stratum corneum. Terpenes are lipophilic in nature, and this lipophilic characteristic may be involved in the extraction of intercellular lipids.23 Hence, an increase in flux can be attributed to lipid extraction. The relatively lower flux at higher concentrations (20%) of terpenes may be due to the ability of excess lipids to retain the drug in the stratum corneum.
The results of the skin irritation study are shown in Table 2 (p. 41). Formalin (standard) showed average scores of 2.57 and 3.42 for erythema and edema, respectively, indicating moderate to severe erythema and edema. Edema was almost absent in the case of terpenes, but a slight erythema-an average score of 1.0-was observed with citral. Cineole and d-limonene showed no erythema, with average scores of 0.71 and 0.86, respectively. The U.S. National Institute for Occupational Safety and Health interprets scores of 0 to 0.9 as non-irritant and safe for intact human skin contact, whereas scores ranging from 1 to 1.9 indicate a mild irritant requiring protective measures.24 Thus, we concluded that the selected terpenes were less irritating and safer at certain concentrations, although these conclusions should be further confirmed by a larger sample size.
Conclusion
Based upon the above study it can be said that among the selected terpenes, cineole at 15% concentration (weight/ volume of donor solution) was effective in enhancing the permeation rate of vera-pamil hydrochloride to 10.9885 X 10-2 mg/cm2/hour; hence, the required permeation rate could be achieved with the aid of cineole by varying the area of application of a transdermal patch within an appreciable range. �
Kulkarni is at the BLDEA'S College of Pharmacy in Bijapur, India; Dr. Hiremath and Sagar are at the V.L. Department of Pharmacy in Raichur, India. Reach them by contacting Kulkarni at
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