In-Vivo Investigation of the Irritation potential of Lamivudine and Stavudine liposomal gels in rabbits
R Pai, D Kusum, D Kshama, S Fattepur
histological assessment, in vivo skin irritation, lamivudine, liposomalgels, stavudine
R Pai, D Kusum, D Kshama, S Fattepur. In-Vivo Investigation of the Irritation potential of Lamivudine and Stavudine liposomal gels in rabbits. The Internet Journal of Pharmacology. 2008 Volume 6 Number 2.
Effective antiretroviral therapy is required on a long-term basis to maintain viral suppression and reduce disease progression in HIV infections. An attempt has been made to implement the principles of novel drug delivery to antiretrovirals in order to overcome the drawbacks of existing therapy. Liposomal gels of lamivudine and stavudine respectively were developed for the transdermal drug delivery. The irritation potential of drug loaded and blank formulations was investigated on shaved rabbit skin, in vivo, and compared to that of 5% sodium lauryl sulphate (SLS) solution (a known irritant) as the positive control and untreated portion of the skin as a negative control. Skin irritation was assessed by visual observations and histological evaluations. We found that the drug loaded and blank liposomal gels caused no significant epidermal irritation and erythema. Thus we conclude that these liposomal gels may be used safely as transdermal drug delivery vehicles for the selected antiretrovirals.
Source of Support
Department of Science and Technology, New Delhi, India for financial support for the project
Human Immunodeficiency Virus (HIV) is a retrovirus, which causes irreversible destruction of the immune system, leading to the occurrence of AIDS. Effective antiretroviral therapy is required on a long-term basis to maintain viral suppression and reduce disease progression. Currently available anti-HIV drugs bear some significant drawbacks such as relatively short half-life, low bioavailability, poor permeability and undesirable side effects. Efforts have been made to design drug delivery systems for anti-HIV agents to obtain an effective controlled delivery of the drug. An attempt has been made to deliver the antiretrovirals via transdermal route by implementing the principles of novel drug delivery. Transdermal delivery offers a number of advantages such as the avoidance of the first pass effect that usually follows oral delivery . A noninvasive zero – order delivery via transdermal route is desirable for controlled delivery of antiretrovirals. Also vesiculation of the chosen antiretrovirals viz., lamivudine and stavudine will alleviate the toxicity of these molecules. Gel formulations containing liposomal dispersions are being developed for transdermal delivery of drugs . A range of drugs can be solubilised in the gels , with the possibility of delivering them into and through the skin.
The skin is comprised of different layers, each with distinct properties and functions. The main role of the outer layer, the epidermis, is to act as a barrier, preventing the ingress of harmful chemicals and microbes into the body, while restricting the loss of water and other ions from the body to the environment. Cells in the uppermost layer, the stratum corneum (SC), are cornified and embedded in a lipid matrix . It is this impervious layer that forms the barrier to drug penetration into the skin. It is this layer, therefore, that will be the first point of contact with any topically applied product. Most of the substances interact with skin constituents and might cause reversible or irreversible damage to the skin cells, which must be investigated.
Skin irritation is defined as a non-immunological local inflammatory reaction which is usually reversible, and is characterized by erythema and edema, following a single or repeated application of a chemical to the same cutaneous site . All substances can cause irritation to all individuals, and the factors that determine whether a substance will produce irritation in a certain individual include the age, sex, anatomical site, and other factors particular to the individual, as well as the concentration, dose, vehicle, and chemical properties of the irritant, the manner and area of application, and duration of contact. The local inflammatory reaction following perturbation of the epidermal barrier (either by exposure to a physical or chemical irritant) can appear within minutes to hours after the insult, and is believed to be initiated by the release of primary cytokines from keratinocytes . There is a range of non-invasive techniques to assess skin irritation or damage, the simplest being visual irritation scoring , whose main disadvantage is the occurrence of subjective readings. More objective methods such as the measurement of transepidermal water loss (TEWL) and skin blood flow (SBF) using an evaporimeter and laser Doppler velocimetry (LDV), respectively, are required and are widely used in the evaluation of skin irritation .
Histological assessment on skin biopsies taken at the end of studies, although invasive, helps to visualize the different layers of the skin and provide more information on skin reactions when evaluating any damaging effects of topically applied preparations . In the mid 1940s, published a method for assessing the skin irritation caused by topically applied substances in rabbits, which involves the occluded application of test chemicals to rabbit skin for 24 hour duration . Such a method, however, is not considered always reliable for predicting the true irritation potential of topical preparations in man, as demonstrated by Phillips et al. The method used in our study has taken into account recommendations from the literature cited above, but has been optimized for the testing of a semisolid preparation such as the liposomalgel, and does not involve occlusion of the test site.
So far no published data is available on the skin irritation potential of antiretrovirals. The skin irritation data for liposomal formulations of lamivudine and stavudine will explore the possibilities of further development of these drugs into transdermal drug delivery systems. In this study we report on investigations into the irritation effects of liposomalgels of selected antiretrovirals in rabbits, following repeated applications. The liposomal gel consisted of liposomes made up of soyalecithin and cholesterol. These liposomes were loaded with selected antiretrovirals i.e. either lamivudine or stavudine. The drug-loaded liposomes were incorporated into aqueous base of Hydroxy Propyl Methyl Cellulose (HPMC)  to obtain the liposomal gels. Poly Ethylene Glycol PEG-400 was used as a permeation enhancer. The liposomal gels of lamivudine and stavudine and their corresponding blank formulations (total four formulations) were tested in rabbits against a positive control (standard irritant – 5% SLS aqueous solution)  and a negative control. The site of liposomal gel application was visually examined through out the test period and at the end of the study the animals were sacrificed and the skin samples were subjected for histopathological evaluation.
Materials And Methods
Hydrogenated soya phosphatidylcholine (PC) and cholesterol (CHOL), were obtained from Sigma Chemical Company (St. Louis, MO). Lamivudine and stavudine was kindly donated by from CIPLA India Ltd and Karnataka Antibiotics Pvt. Ltd., Karnataka, India, methanol, chloroform, hydroxy propyl methyl cellulose, polyethylene glycol – 400, sodium lauryl sulphate were of analytical grade, and obtained from commercial sources. Millipore Millique water was used throughout the experiments. Healthy male rabbits were obtained from an authorized breeder and the investigational protocol for animal experimental procedures adhered to the “Principles of Laboratory Animal Care” All animal procedures were conducted in accordance with the institutional ethical committee guidelines. The animals were caged in a room with standardized environmental conditions (20 ± 2˚C, 35 – 45% RH) and a constant day/night cycle. They were fed dehydrated pellets and received water ad libitum throughout the study.
Preparation of Liposomal gels
Liposomes of lamivudine and stavudine were prepared by employing thin film hydration technique . The lipid phase was prepared by dissolving accurately weighed quantities of the PC and CHOL, in the chloroform-methanol mixture (2:1 v/v), in a 250 ml round bottom flask. The solvent mixture was removed from the lipid phase by rotary evaporation at 45 ± 2°C in a Rotary Vacuum Evaporator, to obtain a thin film of lipids on the wall of the flask. Subsequently, the flask was kept overnight to ensure the complete removal of residual solvent. The drug solution was prepared by dissolving the drug in water. The dry lipid film was then hydrated with this drug solution at a temperature of 40 ± 2°C. The dispersion was left undisturbed at room temperature for 2-3 hours to allow complete swelling of the lipid film and hence to obtain vesicular suspension. The best liposomal formulation was selected for incorporation into the aqueous base of HPMC. Hydroxy Propyl Methyl Cellulose (optimized concentration 6% w/v) was hydrated with water on a magnetic stirrer (500 rpm) at room temperature for 4 hrs to get a smooth dispersion. The stirring was halted and dispersion was made to stand overnight at room temperature, to achieve complete hydration of the polymer and to remove entrapped air bubbles (Gabriela et al 2005). If any lumps of partially wetted HPMC were present at this stage the dispersion was discarded and fresh batch was prepared. The liposomal formulation of antiretrovirals was then suspended into hydrogel base by slow speed agitation. Poly Ethylene Glycol – 400 (5% w/v) was used as the permeation enhancer. The optimized formulation of liposomal gel was selected for further investigation (Table1 and Table 2). The blank formulations were prepared in a similar manner but excluding the drug. The positive control was prepared by dissolving 5% w/v of SLS powder in distilled water. Liposomal gels with the following compositions were prepared:
Skin Irritation Studies
Male rabbits weighing 2 to 2.5 Kg, 6 to 8 months old were chosen for the study. These animals were maintained in separate cages till the commencement of the study. Four areas were chosen (top right, bottom right, top left and bottom left) on the dorsal part of each rabbit  for the treatment with the drug loaded formulation, blank formulation, positive control (SLS) and the negative control (no treatment) respectively. Twenty-four hours prior to the first application, the animal’s backs were shaven to result in an approximate area of 3cm2 on the four portions mentioned above, with clippers. Their skin was then checked for cuts, and they were allowed to rest overnight. The rabbits were treated topically with their respective formulations once daily on day 1 of the 7-day study. Amount of the gel applied was corresponding to 10.5 mg of lamivudine dose per 1.5 kg body weight of the rabbit and for stavudine it was 2.8mg. The amount of blank formulation applied was same as the corresponding drug loaded formulation. 5% SLS solution was applied as positive control. All treatment sites were then covered with sterile gauze and secured with surgical tape to prevent grooming and removal of the formulation from the skin. Every day visual assessments were made. Further, prior to the fresh application, the gauze was taken off and the treated area was gently wiped with water-soaked gauze to remove any residual vehicle from the skin surface .
The gels and the positive controls were applied to rabbits for 7 days taking all the precautions mentioned earlier; visual measurements were taken on all the days; erythema was quantified every day, skin biopsies were taken at the end of the study and cross-sections of the skin were examined to investigate histological changes, if any, caused by the different preparations (Table3).
Visual assessment of skin irritation
The site of liposomal gel application was visually examined for cutaneous irritation/reaction and a score for erythema (redness) was given as follows: 0, no reaction; 1, weak spotty or diffuse erythema; 2, weak but well perceptible erythema covering the total exposure area; 3, moderate erythema; 4, severe erythema with edema; 5, very severe erythema with epidermal defects (vesicles, erosions, etc.). Such quantification of erythema and edema was similar to that used by Frosch et al. Similar scoring was followed for allergic reactions and ulceration.
At the end of day 7, the animals were sacrificed and approximately 3 cm2 skin biopsies from all treated and untreated sites were taken and preserved in 10% formalin solution for at least 48 h. Following dehydration through a graded series of alcohols and embedding in paraffin wax, sections were cut of from each sample, parallel to the direction of hair growth, stained with haematoxylin and eosin (H&E) and examined with a light microscope . Each cross-section sample was photographed at the midpoint and halfway to the edge on either side of the midpoint, and scored to give an average for each biopsy sample. Parameters such as thickening of the epidermis, thickening of the SC (hyperkeratosis), parakeratosuis, acanthosis were observed. The dermis layer was observed for sweat glands, sebaceous glands, hair follicles, edema, and hypertrophy of muscle fibers, inflammation, fibrosis, congestion of blood vessels. Their intensity scored (see Table 4). Mean histological score was then calculated for each treatment as follows:
where A; B; C; etc. are the scored histological parameters, e.g. hyperkeratosis, a; b; c etc. are the frequency of occurrence of a particular score for a histological parameter in one rabbit and N is the number of rabbits. Score of 10 was regarded as absence of undue reactions to rabbit skin, and a score of >21 was regarded as unacceptable damage .
In the above study the visual assessment of application sites on the rabbits exhibited following results. Ertyhma, edema, allergy or ulceration were not observed both with liposomal gel formulations of lamivudine and stavudine in comparison with the negative control. Scoring was 0, for erythema, edema, allergic reactions and ulceration. As expected the area of the application of SLS resulted in scoring of 4 each for erythema and ulceration, and a score of 2 and 3 for allergy and edema respectively Table – 4. The histopathological evaluation of untreated skin indicated thin stratum corneum, distinct boundary between epidermis and dermis, and hair follicle base. As compared to the positive control no histopathological changes were observed in case of blank formulation. This is to be expected, as the components used in formulation i.e, soyalecithin, cholesterol hydroxypropyl methylcellulose and PEG-400 do not exhibit any skin irritation in the concentrations used, in topical application. The histopathological evaluation of rabbit skin treated with lamivudine liposomal gel and stavudine liposomal gel exhibited thin stratum corneum, distinct boundary between epidermis and dermis, and hair follicle base. Where as the skin samples treated with standard irritant exhibited considerable damage to the epidermis, with hyperkeratosis and intercellular edema. The microscopic sections of drug loaded and blank formulations, did not show any hyperkeraotosis, parakeratosuis and acanthosis in the epidermal layer. The dermal layer was characterized by numerous sweat glands, sebaceous glands and hair follicles. Edema of the dermis was not seen. Hypertrophy of muscle fibers, inflammation, fibrosis and congestion of blood vessels were totally absent for the skin samples treated with the formulations (score 0) Where as all of the above pathological changes were apparently observed with the skin sample of positive control exhibiting an average score of 15 (Table- 5).
Transdermal drug delivery is the most commonly used route of administration to achieve controlled delivery of a number of drugs. But evaluating the skin irritation potential and skin deposition of the developed formulation are important criteria. Skin irritation is a complex mechanism involving epidermal and dermal cells interacting with one other and with blood cells, under the influence of cytokines generated mainly by the epidermal keratinocytes that have been activated following the application of an irritant, or a physical perturbation to the epidermal barrier . This usually leads to cutaneous vasodilatation and is manifested by erythema and local swelling. Irritation may be caused by binding of the chemical moiety to keratin in the SC, via ionic and/or hydrophobic interactions, and by concomitant protein denaturation resulting in swelling of the SC  or it may be due to lipid removal from the SC which exposes more binding sites on the keratin . Removal of lipids leads to irreversible loss in SC structure; hence, reversible damage to the skin by different substances is thought to be due to its interaction with the horny layer proteins . Increased skin permeation is required in order to achieve suitable drug concentrations in or through the skin, is not achieved without side effects, and seems to come hand in hand with increased skin irritation, as demonstrated by Kanikkannan and Singh . There have been a few irritancy studies in animal using Tween 20, the major constituent of a number of gels, found erythema, edema, and skin thickening when Tween 20 was applied daily to rabbit skin for 10 days; however, Tavss et al. reported a severe irritation within 1 day with a 10% SLS solution in human volunteers. In vitro tests using human skin showed a 0.2% increase in swelling of the SC after 1 hour incubation with 2% w/v Tween 20, compared to a 32% change with SLS . In the present study we did not come across any skin irritation reaction by the antiretrovirals, as lamivudine and stavudine were entrapped with in liposomal formulation and these liposomes are suspended in the aqueous gel base.
The authors wish to thank Prof. B.G Shivananda, Principal Al-Ameen College of Pharmacy and the Management, Al-Ameen College of Pharmacy Bangalore, India for their continuous support. We are also thankful to Cipla India Ltd and Karnataka Antibiotics Pvt. Ltd, Karnataka, India for generously providing the gift samples of Lamivudine. The authors are thankful to Mr. Anil Dandu, Benson Mathai, Amarnath K, Research Scholars, Department of Pharmacology, Al-Ameen College of Pharmacy, Bangalore, for their support and help throughout the study. Our thanks are also due to