Formulation And Evaluation of Dissolving Oral Film Containing Glutathione as Skin Lightening Agent

       
           
       
Fig. 1:- Structure of Glutathione

Pentonic acid, 2-amino-5-[(R)-(caroxymethylamino)-3-mercapto-1-oxopropan-2ylamino]- oxo, (S); N-(N-L-?-Glutamyl-l-cysteinyl) glycine.

Glutathione, a small, water-soluble thiol-tripeptide with low-molecular weight, is made from three amino acids: Glutamate, Cysteine, and Glycine. Glutathione is commonly found in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSH). Its biological function serves as a potent antioxidant in human body. Melanin, a skin pigment, consists of blackish-brown eumelanin and reddish-yellow pheomelanin. Higher pheomelanin proportion will make skin brighter. Literature search on search engines revealed scarce results the fact that glutathione use as a skin-whitening agent is still rare and is yet to be researched extensively on human skin. The limitations of oral clinical trials are evident since the ability of GSH as an intact molecule decreases as it passes through the gastrointestinal tract. To pass the intestinal tract, glutathione has to be broken down into three amino acid components prior to absorption. Several animal studies showed that intact glutathione molecules may be absorbed through the small intestine, thus increasing plasma glutathione level. This would not be achieved if amino acid component is given in equivalent amount.

Inhibition of tyrosinase (the key enzyme of melanogenesis):

•Direct inhibition: Thiol group binding with the copper-containing active site of the enzyme

•Indirect inactivation: Exerted via the antioxidant effect of glutathione that leads to

•quenching of free radicals and peroxides

•Switching production of eumelanin to phaeomelanin

•Modulation of the depigmenting properties of other antimelanogenic principles

Table No.1

       
           
       
Fig. No. 2 :- Oral Dissolving Film

The saliva containing the dissolved or dispersed medicament is then swallowed and the drug is absorbed in the normal way. Some drugs are absorbed from the mouth, pharynx, and esophagus as the saliva passes down into the stomach & it may produce rapid onset of action. In such cases, the bioavailability of the drugs is significantly greater than those observed from conventional tablets dosage form. The urge for the development of a mouth dissolving film is patients who find swallowing standard tablets and capsules troublesome for one purpose or another can benefit from fast dissolving dosage forms.

Special Features of Oral Dissolving Films

?Film should be thin and elegant.

?It should be available in various size and shapes.

?It should be Unobstructed.

?It should adhere to the oral cavity easily.

?It should processes fast disintegration without water.

?It should release rapidly.

Advantages of Oral Dissolving Film

?It is convenient form of dosing.

?It does not require water.

?There is no risk of chocking.

?With dosage form, we can mask the taste easily.

?It enhances stability.

?Improved patient compliance.

?The drug enters the systemic circulation with reduced hepatic first pass effect.

?It is site specific and has a local action.

?Availability of large surface area that leads to rapid disintegration and dissolution within oral cavity.

?Serves dose accuracy in comparison to liquid dosage form.

Disadvantages of Oral Dissolving Film

?The disadvantage of ODF is that high dose cannot be incorporated into the strip. The dose should be between 1-30 mg.

?There remain a number of technical limitations with use of film strips; the thickness while casting the film.

?The other technical challenge with these dosage forms is achieving dose uniformity.

?Packaging of films requires special equipment’s and it is difficult to pack.

?The drug should have pleasant taste.

Plan of Work

Hyperpigmentation is caused by the eposure to ultraviolet radiation, resulting in the formation of reactive oxygen and nitrogen species between cells. Oral antioxidants will reduce melanogenesis by suppressing those free radicals. Glutathione may affect skin pigmentation by inhibiting tyrosinase activity during melanogenesis, either directly or indirectly. Direct inactivation is done by binding to the active site of enzymes containing copper ion, while indirect inactivation eliminates free radicals and peroxides in ant oxidative manners. During melanogenesis, glutathione converts eumelanin to pheomelanin and modulates depigmentation of melanocytotoxic agents.

Selection of Drug and Excipient:

Drug candidate L-Glutathione obtained from Aseschem. Pvt. Ltd. Jodhpur, Rajasthan. Stevia was obtained from Dipa Enterprises, Aurangabad. Hydroxypropyl methyl cellulose, citric acid, sodium lauryl sulphate was obtained from Shreeyash Institute of Pharmaceutical Education and Research. Chemicals were the entire analytical grade. Glutathione, a small, water-soluble thiol-tripeptide with low-molecular weight, is made from three amino acids: Glutamate, Cysteine, and Glycine. Glutathione is capable of preventing damage to important cellular components caused by reactive oxygen species such as free radicals, peroxides, lipid peroxides, and heavy metals. HPMC, short for hydroxypropyl methylcellulose, is a semisynthetic, inert, viscoelastic polymer used as eye drops, as well as an excipient and controlled-delivery component in oral medicaments, found in a variety of commercial products. Citric acid is an organic compound with the chemical formula HOC(CH?CO?H)?. It is a colorless weak organic acid. It occurs naturally in citrus fruits. Citric acid stimulation was able to significantly increase salivary flow rate, pH value, sAA activities, sAA specific activity and sAA amount (including glycosylated and non-glycosylated sAA amount) in healthy children (P<0>

Sample: 500mg of glutathione. Blank: 50ml of metaphosphoric acid Mode: Direct Titration

Titrant: 0.1N Iodine VS End point detection: Visual

Analysis: Dissolve the sample in 50ml of metaphosphoric acid and titrate with titrant. Calculate the percentage of glutathione () in portion of glutathione taken.

Result = [{VU - Vb} * N * F * 100] / W Where

VU = Titrant volume of sample (ml) Vb = Titrant volume of blank (ml) N = Titrant Normality

F = Equivalence Factor

W = Weight of Sample (mg)

Acceptance Criteria: 98.0% - 101% on the dried basis.

METHOD:

Solvent Casting Method

       
           
       
Fig. 6:- Casting of Solvent in drug Solution

Colour, flavour, and taste are the required organoleptic attributes for a fast dissolving formulation. The formulation should have acceptable organoleptic palatable characteristics because it will dissolve in the mouth. Hue helps patients accept a Formulation, and when oral films are given to youngsters, they should have appealing Colour. Therefore, the colour of the formulation should be consistent and appealing. Visual inspection can be used to assess colour. The smell is another organoleptic characteristic. The flavour added to the recipe should give it a pleasing aroma. By using a flavouring agent, the smell of the polymer, medication, and any other Excipient should be concealed. Taste is another crucial element that needs to be considered. A unique person is used to assess the taste.

Table No. 3

Surface pH Test

Thickness

Micrometer screw gauges or calibrated digital Vernier Calipers are used to measure film Thickness.

The recommended range for film thickness is 5-200 mm. It is important to determine uniformity in the thickness of the film since this is directly Related to the accuracy of the dose distribution in the film. The thickness should be asses sed at five distinct points (four corners and one in the centre).

Folding Endurance

Folding endurance gives the brittleness of a film. The method followed to determine endurance value is that the film specimen (2 × 2 cm2) are repeatedly folded at the same place until it breaks or a visible crack is observed. The number of times the film is folded without breaking or without any visible crack is the calculated folding endurance value.

Drug Content Uniformity

Any standard assay method specified for the specific API in any of the standard Pharmacopoeia can be used to determine this. By measuring the API content in each individual strip, content consistency is Assessed. 85 to 115 percent is the maximum level of content uniformity.

In Vitro Disintegration Test

When an oral film comes in contact with saliva or water, it begins to disintegrate at that point. The time of disintegration should be in the range of 5 to 30 seconds for a film that dissolves quickly. Disintegration time can be investigated using a USP (United States Pharmacopoeia) disintegration device. Another approach involves dipping the film in 25 ml of water in a beaker to visually assess the disintegration time. When the film begins to crack or dissolve, the beaker should be gently shaken and the moment noted.

Dryness/Tack

In all there have been eight stages identified for film drying and these are set-to-touch, dust-free, tack-free (surface dry), dry-to touch, dry-hard, dry-through (dry-to-handle), dry-to-recoat, and dry print-free. Tack is the tenacity with which the strip adheres to an accessory (a piece of paper) that has been pressed into contact with strip. Instruments are also available for this study.

Tensile Strength

Tensile strength is the maximum stress applied to a point at which the strip specimen breaks. It is calculated by the applied load at rupture divided by the cross-sectional area of strip as given in the equation below:

Tensile strength = Load at failure × 100/Strip thickness × Strip width

Percent Elongation

When stress is applied on a film (2 × 2 cm2) sample it gets stretched, this is referred to strain. Strain is basically the deformation of strip before it gets broken due to stress. It is measured by using hounsfield universal testing machine. Generally elongation of strip increases as the plasticizer content increases. It is calculated by the formula:

% Elongation = Increase in length of strip × 100/Initial length of strip

Tear Resistance

Tear resistance is the resistance which a film offers when some load or force is applied on the film specimen. The load mainly applied is of very low rate 51 mm/min. The unit of tear resistance is Newton or pounds-force. In other words it is the maximum force required to tear the specimen.

Young's Modulus

Young's modulus or elastic modulus is the measure of stiffness of strip. It is represented as the ratio of applied stress over strain in the region of elastic deformation as follows: Young's modulus = Slope × 100/Strip thickness × Cross head speed

Hard and brittle strips demonstrate a high tensile strength and Young's modulus with small elongation.

In Vitro Dissolution Studies

Dissolution is defined as the amount of drug substance that goes into the solution per unit time under standardized conditions of liquid/solid interface, temperature, and solvent concentration. The standard basket or paddle apparatus described in any of the pharmacopoeia can be used for dissolution testing. The selection of dissolution medium will essentially depend as per the sink conditions and highest dose of API. The temperature of dissolution medium should be maintained at 37 ± 0.5°C and rpm at 50.

When the paddle apparatus is employed, it has a disadvantage that oral films have a tendency to float over the dissolution medium.

Contact Angle

Contact angle measurement predicts the wetting behavior, disintegration time, and dissolution of oral film. These measurements are performed with help of goniometer (AB Lorentzen and Wettre, Germany) and the measurements should be done at room temperature. The water used to determine contact angle should be double distilled water.A drop of double distilled water is placed on the surface of dry film. Images of water droplet are recorded within 10 s of deposition by means of digital camera. Digital pictures can be analyzed by image 1.28v software (NIH, USA) for angle determination.

Transparency

To determine transparency of oral film, a simple ultraviolet (UV) spectrophotometer can be used. The film specimen is placed on the internal side of spectrophotometer cell. The transparency of films is calculated as follows:

Transparency = (log T600)/b = ?€c

Where T600 is the transmittance at 600 nm and b is the film thickness (mm) and c is concentration.

Scanning Electron Microscopy

To study the surface morphology of film between different excipients and drug scanning, electron microscopy can be used. The film sample should be placed in sample holder and at ×1000 magnification; various photomicrographs can be taken using tungsten filament as an electron source.

Permeation Studies

Even though permeability of oral mucosa is 4-1000 times greater than that of skin, permeation studies should be carried out. To study the permeability, modified Franz diffusion cell can be used along with porcine buccal mucosa. The Franz diffusion cell consists of a donor and a receptor compartment. In between the two compartments, mucosa is mounted and the size of the mucosa should be of the same size as that of the head of receptor compartment. The receptor compartment is filled with buffer and maintained at 37 ± 0.2°C and to maintain thermodynamics a magnetic bead stirring at a speed of 50 rpm is used. A film specimen moistened with a few drops of simulated saliva should be kept in contact with mucosal surface. The donor compartment should consist of 1 ml simulated saliva fluid of pH 6.8. At particular interval, samples are withdrawn and replaced by same amount of fresh medium. By suitable analytical method, percentage of drug permeated can be determined.

Percentage Moisture Loss

To determine percentage moisture loss films of area 2 × 2 cm2 are cut and weighed accurately on an electronic balance. After weighing, the films were kept in desiccators containing fused anhydrous calcium chloride. The films should be kept for 72 h in the desiccator. After 72 h, they are taken out and again weighed and the percentage moisture loss of films was measured by using the formula:

Percent moisture loss = (Initial weight ? Final weight)/Initial weight × 100

The percentage moisture loss studies are done to determine physical stability and integrity of the film. Determination of percentage yield of buccal patches Percentage yield of buccal patches can be calculated by the following formula:

% yield = Mass of the buccal patches obtained/Total weight of drug and polymer × 100

Stability Study

Stability study should be carried out according to the International Conference on Harmonization (ICH) guidelines. The prepared formulation was wrapped in a special way. Firstly, it was wrapped in a butter paper then above it an aluminum foil was wrapped and the packing should be placed in an aluminum pouch and make it heat sealed. The storage conditions at which formulations are kept should be 30°C/60% relative humidity (RH) and 40°C/75% RH. After 3 months, the films were evaluated for drug content, disintegration time, and physical appearance observation.

Storage And Packaging Of ODF:

Oral dissolving films can be packed using single pouches, blister card with multiple units, multiple-unit dispenser, and continuous roll dispenser. There are certain patented packaging systems for fast dissolving films such as Rapidcard by Labtec and Core-peel by Amcor flexible. The rapid card is of same size as a credit card and holds three films on each side. Every dose can be taken out individually.