"FORMULATION AND EVALUATION OF MORONIC ACID LOADED TRANSDERMAL PATCHES"

Objective : To prepare Transdermal patches of Moronic acid along with various polymers for controlled release action. Methods : Suitable method such as Solvent Casting Technique of Film Casting Technique are used for the preparation of Transdermal patch. Results : The prepared Transdermal patches were transparent, smooth, uniform and flexible. The method adopted for the preparation of the system was found satisfactory. Conclusion : Various formulations were developed by using hydrophilic and hydrophobic polymers like HPMC E5 and EC respectively in single and combinations by solvent evaporation technique with the incorporation of penetration enhancer such as dimethylsulfoxide and dibutyl phthalate as plasticizer. Formulation F7 containing an equal ratio of HPMC E5: EC (5:5) showed maximum and sustained release of 86.814±0.262 within 24 h. Kinetic models were used to confirm the release mechanism of the formulations. Moronic acid release from the patches F1 to F7 followed non Fickian diffusion rate controlled mechanism.


INTRODUCTION
Conventional systems of medication that require multi-dose therapy are having many problems. The controlled drug delivery is a newer approach is to deliver the drug in to systemic circulation at a predetermined rate. Our system should duplicate continuous intravenous infusion, which not only bypasses hepatic 'first pass' elimination but also maintains a constant, prolonged and therapeutically effective drug level in the body [1]. Transdermal drug delivery system constitutes one of the most important routes for new drug delivery system. Transdermal delivery of drugs offers several advantages over conventional delivery methods. Transdermal delivery, that traditionally uses a patch containing drug substance pressed onto the skin, is non-invasive, convenient and painless, and can avoid gastrointestinal toxicity (e. g. peptic ulcer disease) and the hepatic first-pass metabolism [2].
Bevirimat, a derivative of the related Triterpenoids betulinic acid, is under development as an anti-HIV drug; however, moronic acid has shown better antiviral profiles in vitro than bevirimat. A particular moronic acid derivative showed potent anti-HIV activity with EC50 values of 0.0085 μM against NL4-3, 0.021 μM against PI-R (a multiple protease inhibitor resistant strain), and 0.13 μM against FHR-2 (an HIV strain resistant to (bevirimat). This derivative has become a new lead for clinical trials and is also active against the herpes simplex virus [4].

Method
Suitable method such as Solvent Casting Technique of Film Casting Technique are used for preparation of Transdermal patch.

Determination of solubility [5, 6]
An excess amount of drug was taken and dissolved in a measured volume of distilled water in a volumetric flask to get a saturated solution. The solution was kept for 24 h at room temperature for the attainment of equilibrium. These solutions were kept for sonication and then the supernatant was filtered using a 0.45-micron whatmann filter paper to separate the undissolved drug particles and diluted suitably and the concentration of Moronic acid in the filtrate was determined spectrophotometrically by measuring at 300 nm.

Determination of partition coefficient [7]
The partition coefficient of the drug was determined by taking equal volumes of 1-octanol and aqueous solution in a separating funnel. In case of water-soluble drugs, a drug solution was prepared in distilled water, and in case of water-insoluble drugs, a drug solution of was prepared in 1-octanol. Standard solution of the drug was prepared in this phosphate buffer pH 7.4 solution. Octanol (10 ml) was added to an equal volume of this standard drug solution in a separating funnel and was kept for 24 h at 37±0.5 °C with intermittent shaking. Finally, the buffer solution was separated, clarified by centrifugation and assayed for drug content.

FT-IR [8]
In the preparation of film formulation, drug and polymer may interact as they are in close contact with each other, which could lead to the instability of drug, Preformulation studies regarding the drug-polymer interaction are therefore very critical in selecting appropriate polymers. FT-IR spectroscopy was employed to ascertain the compatibility between Moronic acid and the selected polymers. The pure drug and drug with excipients were scanned separately.

Procurement of standard drug
Moronic acid was procured from Tokyo Chemical Industry Co. Ltd.

DSC of moronic acid
Purity profile of the drug was determined by using Differential Scanning Calorimetry (DSC). The latter can be assessed by the melting behavior observed in the recorded thermogram. The main application of DSC to purity relies on the notion that impurities reduce the melting temperature of the drug. The melting temperature is a strong indication of drug purity for carrying out DSC of the model drug, 2 mg of sample was placed in an aluminum pan. The pan was crimped using punching press. The sample pan was placed in pan holder of the DSC machine. The sample was run at a ramp rate of 10 °C/min from 25 °C to 300 °C with a flow rate of 60 ml/min for nitrogen [1].

Calibration curve of moronic acid
The standard calibration curve was constructed to obtain a regression line equation to be used for finding out the concentration of drug in samples. Two calibration curves of the drug were plotted; one by RP-HPLC method and one by UV spectrophotometer. Calibration curve by RP-HPLC method was used for assay of drug in gel matrix for entrapment efficiency studies. The other one was plotted by UV spectrophotometer using Ethanolic phosphate buffer (pH 7.4) for carrying out in vitro drug release studies.

Physical appearance
All the prepared patches were visually inspected for color, clarity, flexibility and smoothness.

Thickness uniformity
The aim of the present study was to check the uniformity of thickness of the formulated films. The thickness of the film was measured at 3 different points using a digital caliper and an average thickness of three reading was calculated.

Weight uniformity [9]
For each formulation, three randomly selected patches were used. For the weight variation test, 3 films from each batch were weighed individually and the average weight was calculated.

Folding endurance [10]
The folding endurance was measured manually for the prepared films. A strip of film (5 x 5 cm) was cut and repeatedly folded at the same place till it broke. The number of times the film could be folded at the same place without breaking/cracking gave the value of folding endurance.

Percentage moisture absorption [11]
The films were weighed accurately and placed in the desiccators containing 100 ml of a saturated solution of potassium chloride, which maintains 80-90% RH. After 3 d, the films were taken out and weighed. The study was performed at room temperature. The percentage moisture absorption was calculated using the formula: Percentage moisture absorption = Final Weight-Initial Weight/Initial Weight X 100

Percentage moisture loss
The films were weighed accurately and kept in a desiccators containing anhydrous calcium chloride. After 3 d, the films were taken out and weighed. The moisture loss was calculated using the formula: Percentage moisture loss = Final Weight-Initial Weight/Initial Weight X 100

Water vapors transmission rate [12]
Glass vials of 5 ml capacity were washed thoroughly and dried to a constant weight in an oven. About 1 gm of fused calcium chloride was taken in the vials and the polymer films of 1.44 cm 2 were fixed over the brim with the help of an adhesive tape. Then the vials were weighed and stored in a humidity chamber of 80-90 % RH condition for a period of 24 h. The vials were removed and weighed at the time interval of 24 h for three consecutive days to note down the weight gain.
Water vapour transmission rate = Final Weight-Initial Weight/Time X Area X 100

Tensile strength [13]
Tensile strength of the film was determined with Universal strength testing machine (Hounsfield, Slinfold, Horsham, U. K.). The sensitivity of the machine was 1 gram. It consisted of two load cell grips. The lower one was fixed and the upper one was movable. The test film of size (4 × 1 cm2) was fixed between these cell grips and force was gradually applied till the film broke. The tensile strength of the film was taken directly from the dial reading in kg. Tensile strength is expressed as follows; Tensile strength = Tensile load at Break/Cross-Sectional Area             Weighing three films from the same batch and calculating the average weight. The dry films were digitally weighed. The films were uniformly 0.363-0.417 g in weight, with a minimal standard variation.

CONCLUSION
UV spectroscopy was used to analyse Moronic acid. Moroninic acid (pH 7.4) showed maximum absorption at 215 nm. R2 was 0.992, suggesting a linear relationship between drug concentration and absorbance. In this case, R2 = 0.998, which indicates a linear connection between drug concentration and absorbance. It means the patches will be less fragile when applied to the skin and fold well. A little moisture keeps the patch firm and prevents dry, brittle regions. For example, HPMC E5 patches released more than EC patches, perhaps owing to HPMC patches' high water vapour permeability and EC's hydrophobicity. For better and longer release, the monolithic system was updated using HPMC E5 and EC. Within 24 h, Formulation F7 with HPMC E5:EC (5:5) released 86.8140.262. Less than a week later, the kinetic simulations verified the release F1-F7 have non-Fickian moronic acid release. Evaporated solventbased moronic acid transdermal patch Studies on healthy animals can evaluate pharmacokinetics. In vitro/in vivo correlation may achieve batch-to-batch stability.

ACKNOWLEDGMENT
It's our privilege to express profound sense of gratitude and cordial thanks to our respected chairman Mr. Anil Chopra, Vice Chairperson Ms. Sangeeta Chopra and Pro-Chairman Mr. Prince Chopra, St. Soldier Educational Society, Jalandhar for providing the necessary facilities to complete this research work.

AUTHORS CONTRIBUTIONS
All the authors have contributed equally.

CONFLICTS OF INTERESTS
There are no conflicts of interest.