Development, Optimization and Characterization of Buccal Pullulan Based Disulfiram Film for Alcohol Dependence

A variety of biological, psychological, and social symptoms are indicative of alcohol dependence. It is a maladaptive pattern of alcohol consumption that results in either distress or impairment that is clinically significant, or both. Alcohol-dependent people often organize their days around obtaining, consuming, and feeling the effects of alcohol. Alcohol dependence may be indicated by a history of neglecting other pleasures and obligations at work, home, or school, as well as by a loss of control over the quantity or pattern of use. Another indicator of alcohol dependence is use that continues despite knowledge of the negative effects of alcohol consumption. If one or more of the a forementioned are present, it is a sign that alcohol dependence may be suspected. . Problems at work, interpersonal issues brought on by alcohol consumption, and the emergence of a physical or mental illness frequently linked to alcohol consumption all point to the possibility of heavy alcohol consumption and should raise suspicions of alcohol dependence. Acamprosate, naltrexone, and disulfiram are a few of the drugs that can be used to treat alcoholism over the long term.

Acamprosate:

Acamprosate is an anti-craving drug used to treat alcoholism over the long term. It functions as a functional glutamatergic NMDA antagonist, according to the theory. According to meta-analyses, it works better than a placebo at sustaining abstinence and averting relapse. For mild to moderate hepatic dysfunction, it is comparatively safe. It comes in 333 mg tablet form. Acamprosate's typical daily dosage is 1332 mg (body weight < 50 kg; dosing schedule: Tab. Acamprosate (333 mg) 1-1-2, one tablet each in the morning and afternoon, and two tablets at bedtime) to 1998 mg (body weight > 50 kg; dosing schedule: Tab. Acamprosate (333 mg) 2-2-2, two tablets thrice a day).

Disulfiram:

Disulfiram is a deterrent drug used to treat alcoholism over the long term. It is an irreversible inhibitor that inhibits aldehyde dehydrogenase, which leads to the disulfiram ethanol reaction (DER), which is the accumulation of acetaldehyde when alcohol is consumed.
Disulfiram should never be started without the patient's written informed consent. Patients taking disulfiram should also be advised to stay away from anything that contains alcohol, such as alcoholic drinks, lotions for shaving, foods that contain vinegar, and drugs like metronidazole. Disulfiram is typically taken at a dose of 250 mg daily. At least 24 hours should pass between the last alcohol dosage and the first disulfiram dosage. The dose should ideally be given under supervision. If 250 mg per day does not produce DER in a patient, the dosage can be raised to 500 mg per day, and then to 750 mg per day. However, before increasing the dosage, one should make sure compliance is maintained. Before starting disulfiram, liver function tests (particularly SGOT and SGPT levels) should be performed at baseline. For the first two months of treatment, these tests should be performed every two weeks. It can then be carried out once every three months.

Naltrexone:

Another anti-craving medication for the long-term treatment of alcoholism is naltrexone. It is believed to work by blocking opioid receptors, which stops alcohol's euphoric and rewarding effects from being mediated by opiate receptors. Nartrexone taken orally has been demonstrated to decrease relapses to heavy drinking. 50 mg of oral Naltrexone is administered daily. Before beginning naltrexone, baseline liver function tests are advised. To check for the development of hepatic side effects of the medication, liver function tests should be performed on a regular basis (monthly for the first three months, and then once every three months after that). Furthermore, acamprosate and naltrexone can be used together. Additionally, disulfiram can be used with acamprosate and naltrexone.^[1]

Treatment duration:

Regarding the length of time that drugs used in the long-term phase of managing alcoholism should be taken, there is little agreement. It is recommended that these drugs be taken continuously for nine to twelve months. If both the patient and the clinician think it is appropriate, these can be continued for even longer. The length of treatment can be determined by a number of factors, including the patient's confidence in their ability to live without the assistance of medications, their risk of relapsing, and their level of rehabilitation.

Oral film:

Originally developed in the late 1970s to help elderly and pediatric patients who had trouble swallowing tablets and capsules, oral film technology is currently popular in the pharmaceutical industry because it is less fragile than other oral dosage forms. dosage precision, quick release, and simplicity of use. One of the most popular methods of administering drugs is orally since it is more practical, economical, and simple to use, all of which increase patient compliance. Disulfiram is one of the FDA-approved medications for treating alcoholism, so this is the study's primary goal. Disulfiram taken orally may increase the risk of side effects both during the long-term management phase and when ethanol is consumed. However, it should be taken under the supervision of a qualified professional. Innovative drug delivery methods, such as buccal film, speed up absorption through the buccal mucosa and deliver the medication straight to the blood. Compared to other dosage forms, our disulfiram buccal film will increase patient compliance and have fewer side effects because we made it with a lower dose. ^[2,3]

MATERIALS AND METHODS :

Table .1 Materials used

Table .2 Equipment’s used

Scope of the work:

• Disulfiram is one of the FDA-approved medications that can be used to treat alcohol dependence
• Disulfiram tablets taken orally may raise your risk of adverse effects both during the long-term management phase and also when consuming ethanol.however, it should be taken under the supervision of a qualified professional.
• Disulfiram is an BCS CLASS 2 drug with low solubility and high permeability in order to improve the solubility it will be made into solid dispersion in the buccal film formulation
• So, the buccal film formulation may better serve the purpose of delivery of the medicine for alcohol dependence in the buccal region and may improve the bioavailability of dosage form as buccal film compared to conventional dosage forms like tablets.

Objectives of the work :

• To determine the characteristics of the drug and excipients through preformulation studies.
• To prepare solid dispersion with disulfiram.
• To evaluate the solid dispersion formulation.
• To prepare the buccal film with disulfiram solid dispersion formulation.
• To evaluate the buccal film .

Plan of work :

Pre formulation studies

• Standard curve for disulfiram
• Solubility studies
• FT IR studies

Formulation of Solid dispersion

• Solvent evaporation method

Evaluation of solid dispersion

• Solubility
• Drug content
• Percentage drug release

Formulation of fast dissolving buccal film

• Optimization of variables
• Preparation of mouth dissolving film

Evaluation of fast dissolving film

• Weight of the film
• Thickness of film
• Ph
• Folding endurance
• Content uniformity
• In vitro disintegrating time
• Ex vivo permeation study

Stability study

Experimental procedure:

Experimental protocol:

Preformulation study:

Description: The description of drug was observed using the visual examination by the appearance of colour ,odour and existing forms. ^[4]

Disulfiram standard curve:

Preparation of standard stock solution :

 In a 100 ml volumetric flask, 10 mg of the medication was weighed and dissolved. Methanol was added to the volume to reach the desired level of 100 ug/ml. To achieve additional dilution, this solution was utilized as a standard stock solution. ^[13]

Preparation of standard plot for disulfiram:

Disulfiram standard plot preparation: Using methanol, dilutions of 2,4,6,8,10, and 12 ug/ml solution were made from the stock solution. and absorbance was measured by using uv spectrophotomer at 216 nm.

Studies on solubility:

Solubility studies:

Saturated solubility study

The drug's saturation solubility was measured using a variety of solvents, such as organic solvents, distilled water, and phosphate buffer (pH 7.4). 50 mL of distilled water or a buffer with the necessary pH was added to a 100 mL volumetric flask. More medication was added to each volumetric flask and covered with aluminum foil. These volumetric flasks were connected in an orbitally shaking water bath.The temperature was maintained at approximately 37±0.5°C for the duration of the investigation by applying 50 rpm of shaking for 48 hours. The final samples were then filtered using syringe filters with pore sizes of 0.22 µm. Following suitable dilutions with the same solvent, the filtrates were gathered, and a UV-visible spectrophotometer (UV-1800, Shimadzu Corporation, Japan) was used to measure the drug's absorbance at the solvent's pre-scanned λmax.The drug's standard curve in each pertinent solvent was then used to calculate the concentration from the absorbance.  ^[6]

 FTIR Studies:

Through FTIR spectrum analysis, the potential interactions between the drug and excipients (disulfiram, pullulan, and fast dissolving film mixture) are further examined. The ftir spectrum of both pure drugs and drug-excipient combinations was obtained using a scanning range of 450–4000 cm-1, with different techniques applied to different samples^{.  [7]}                   

Formulation of solid dispersion :

A precisely weighed amount of disulfiram and polyethylene glycol 1500 were placed in a china dish, dissolved in an adequate amount of ethanol, and heated to between 40 and 50°C. The sample was then placed in a desiccator and evaluation was done after 24 hours. ^[8]

Evaluation of solid dispersion :

Solubility :

To assess solubility, a surplus of solid dispersion was added to the solvent (water, phosphate buffer, pH 6.8) at room temperature, and the mixture was shaken occasionally for 48 hours. To analyze the supernatant, a Shimadzu UV 2450 double beam spectrophotometer was used. If necessary, dilution of the sample will be done.(either 100 or 1000-fold).

Drug content:

10 mg of drug solid dispersion was dissolved in 10 ml of ethanol, found as above-mentioned disulfiram content. following filtration, the filtrate's disulfiram content was assessed using UV spectroscopy at 216 nm. If necessary, ethanol was used to achieve the proper dilution.

Weight taken=weight equivalent ×Average weightlabel claim

Assay=Sample absorbancespecific absorbance×1000×Sample dilutionWeight taken×Average weight

Percent drug release:

Usp apparatus 1 (basket) was used to measure the percentage of drug release. A solid dispersion containing 50 mg of disulfiram was added to each vessel 900 ml of phosphate buffer (pH 6.8). after each sample was placed in a basket (wrapped in muslin cloth) at 37°C and spinning at 50 rpm. After a certain amount of time, 10 milliliters of the sample are taken out and subjected to UV spectroscopy with an appropriate dilution to determine the percentage of drug release.

Amount of drug release=Concentration ×900×101000

Percentage drug release=Amountlabel Claim×100

Optimization of mouth dissolving film:

By using Central composite design

In a study there are 2 independent variables and 3 responses. Central composite design constructed using Pullulan (X1),ssg (x2) and response like weight variation (y1),drug release (y2),invitro disintegration time (y3).

Table 3. Central composite design

X1= pullulan (mg)

X2= sodium starch glycolate (mg)

As per CCD the developed formulation were investigated for Response variables and Weight variation (Y1), DrugRelease( Y2) and Invitro disintegration Time(Y3) on the selected formulation variables. Amount of Pullulan (X1), ssg (X2). All the generated polynomial coefficient followed the second order polynomial model to explore the possibilities of significant interactions between the studied response as shown in the Eqn..

Y= β₀+ β₁A+ β₂B+ β₁₂AB+ β₁₁A²+ β₂₂B²

Design matrix consist of 13 runs was constructed Where,

• β0 – Intercept
• β₁, β₂, β₁₂, β₁₁, β₂₂ – regression coefficient
• A,B – the coded levels of the independent variable
• AB – linear interaction term
• A², B² – Quadratic terms ^{[9,10,11,12,13]}

Preparation of mouth dissolving film:

• Based on the optimization formulation was proceeded.
• Prepared solid dispersion of disulfiram was weighed on the electronic balance.
• Buccal film forming solution was prepared by dissolving pullulan,sodium starch glycolate,menthol,citricacid,propyleneglycol in distilled water in a beaker.
• Temperature was adjusted in magnetic stirrer to 50? .
• Weighed amount of solid dispersion were added to the buccal film forming solution.
• Preparation was allowed to run for 50-60 min in a magnetic stirrer.
• Preparation was allowed to cool and casted on petri plate .
• Preparation was placed in hot air oven and heated at 40 ?.and evaluation was done. ^[8,14,15]

FORMULATION OF OPTIMIZED BATCH F7

Evaluation of oral dissolving films:

Weight of films:

Using an analytical balance, the average weight of the oral fast-dissolving films was calculated. ^[16]

Thickness of films:

A micrometer screw gauge was used to measure the film's thickness,and an average of the three readings was determined . ^[17]

pH value:

By dissolving the film in 10 milliliters of distilled water, the pH value was ascertained.and a measurement of the obtained solution's pH was done. ^[18]

Folding endurance:

To investigate the film's elasticity during handling and storage, folding endurance is crucial.One film was folded repeatedly in the same spot until it broke to test the films' folding endurance. This is thought to demonstrate strong film qualities. A 2 x 2 cm piece of film was cut uniformly, then folded repeatedly in the same spot until it brokes. ^[19]

Content uniformity:

Uniformity of drug content

The UV-vis spectrophotometer was used to estimate the drug content for each oral film solution batch .Distilled water was used for dilution of 1ml of the preparation in a 100 ml volumetric flask and again 1 ml is diluted with 10 ml water  . At 216 nm, its absorbance was measured and the reading was noted.

Weight taken=weight equivalent ×Average weightlabel claim

Assay=Sample absorbancespecific absorbance×1000×Sample dilutionWeight taken×Average weight

IN VITRO DISSOLUTIONSTUDY :

Using a usp basket apparatus (type I) and 200 ml of phosphate buffer (pH 6.8) at 50 rpm and 37 ± 0.5 ?C, this study was carried out in triplicate. Periodically, 5 ml aliquots were taken out and replaced with an equal volume of the same fresh medium. A UV vis spectrophotometer was used to measure the amount of drug released at 216 nm after samples were filtered through a 0.45, millipore filter. A standard calibration curve of disulfiram in phosphate buffer (pH 6.8) Q5 min (% drug release in 5 min in phosphate buffer pH 6.8) was used to determine the cumulative amount of drug release against time.

Amount of drug release=Concentration ×900×101000

Percentage drug release=Amountlabel Claim×100

Ex vivo permeation study:

The methodology used in this study was identical to that outlined in the in vitro drug release study. In this study, goat buccal tissue—obtained from the nearby slaughterhouse as soon as the goat was killed—was used in place of the dialysis membrane. After that, it was cautiously transported to the lab while being maintained in a pH 6.8 phosphate buffer solution. Using a sharp knife, the buccal membrane was safely separated from the underlying membrane. The donor and receptor compartments were separated by the membrane. The donor compartment was filled with a 4 cm² area of the film that contained 0.798 mg of disulfiram in which each film occupies 1.77 ml of oral film solution ( 1ml contains 0.451 mg of drug content). The Franz diffusion cell's temperature was kept at 37 ± 1 °C. The receptor medium was saline phosphate (pH 6.8). For eight hours, the sampling was carried out at prearranged intervals, and the amount of drug that permeated was measured using a UV spectrophotometer set to 216 nm. ^[22,23,24]

jss=QA×T

Stability study:

As a long-term fixed time stability study in aluminum sachets at 40+_2? c /75+_5% RH storage conditions, the stability studies of the optimized otf formulation showed no discernible difference in the desired parameters, including appearance, surface ph, drug content, and in vitro disintegration time over a period of three months . ^[25]

RESULTS AND DISCUSSION :

Optimization and results.

TABLE 5. OPTIMIZATION RESULTS

Fig  1. Weight variation contour plot

Fig 2.   Weight variation 3d plot

Weight variation.

The highest weight was recorded in run 7, and no variables had an impact on weight.For response Y1, a quadratic model was proposed, and the p-value was determined to be significant.

ANOVA for Quadratic model

Response 1: weight variation

Table 6. Weight variation anova

In comparison to the pure error, the Lack of Fit is not significant, according to the Lack of Fit F-value of 0.77. A large Lack of Fit F-value has a 56.87% probability of being caused by noise. We want the model to fit, so a non-significant lack of fit is good.

Predicted R2 of 0.8454 is in reasonable agreement with the adjusted R2 of 0.9262 the difference is less than 0.2.

Perturbation:

A Perturbation plot shows how sensitive the response to each factor while keeping other factors constant. Each line (A&B) represents how the response changes when only the factor is varied while other remains fixed.

Interpretation of Perturbation Graph:

Factor A1( Pullulan) starts slowly and increases upto a peak and then decreases slowly it means the Response R1 increases with A upto a point and then Decreases when A continues to increase. So there is an optimum value of A around the middle region.

Factor B has the smaller slope and the mild curve it means factor B has lesser effect on the response compare to A.  Since A’s curve is steeper than B. A influences R1 ( Weight variation) more effectively.

Drug release:

Fig 3. drug release % contour plot

Fig 4. drug release % 3d surface

Drug release %

Because the concentration of sodium starch glycolate was higher in run 7,8 than in the others, the release was deemed to be good.
For response y2, a quadratic model was proposed. Additionally, the p-value was determined to be significant (p < 0.05).

ANOVA for Quadratic model

Response 2: Drug Release

Table 7.Drug Release Anova

Model terms are considered significant when the P-value is less than 0.0500. B and A2 are important model terms in this instance. The model terms are not significant if the values are higher than 0.1000. Model reduction could make your model better if it contains a lot of unnecessary terms (apart from those needed to support hierarchy).

In comparison to the pure error, the Lack of Fit is not significant, according to the Lack of Fit F-value of 0.66. A large Lack of Fit F-value has a 61.86% probability of being caused by noise. We want the model to fit, so a non-significant lack of fit is good.

In vitro disintegrating time

Fig 5. in vitro disintegration time contour plot

Fig 6. in vitro disintegration time 3d surface

In vitro disintegration time:

In runs 6 and 7, As the concentration of ssg has increased, the in vitro disintegration time has decreased.

For response y3, a quadratic model was proposed, and the p value (0.0020) was determined to be significant.

ANOVA for Quadratic model

Response 3: in vitro disintegration time

Table 8.in vitro disintegration time anova

Preformulation study:

DESCRIPTION :

The drug's pure form was described as a white, crystalline powder.

STANDARD CURVE OF DISULFIRAM :

The disulfiram calibration curve was plotted against the absorbance of various disulfiram concentrations in phosphate buffer (pH 7.4) at 216 nm.For the calibration of disulfiram in methanol, the linear correlation coefficient was found.disulfiram complies with Beer's law when its concentration falls between 2 and 12 mcg/ml.

Table 9. standard curve of disulfiram

Fig7. standard curve of disulfiram

Solubility studies: At room temperature, the drug's solubility was examined using water, organic solvents, and buffers. (Studies of saturated solubility).

Solubility studies

Table 10. solubility studies

FT-IR studies:

FTIR spectra of Disulfiram

Fig 11. FTIR  spectra of disulfiram

Table 11. FTIR spectra table of disulfiram

Fig 12. FTIR spectra of pullulan

Table 12. FTIR spectra table of pullulan

Fig 13. FTIR  spectra of polyethylene glycol 1500

Table 13. FTIR spectra table of polyethylene glycol 1500

Fig 14.FTIR  spectra of citric acid

Table 14. FTIR  spectra table of citric acid

Table 15. FTIR spectra table of propylene glycol

Fig 15. FTIR  spectra of propylene glycol

Fig 16. FTIR  spectra of mannitol

Table 16. FTIR spectra table of mannitol

Fig 16. FTIR  spectra of premixture

Table 16. FTIR spectra table of premixture

Evaluation of solid dispersion:

Solubility:

Table 18: Solubility of solid dispersion

n=3,Mean±s.d

Drug content :

Table 19, Drug content of solid dispersion

n=3,Mean±s.d

Percent drug release:

Table 20: Percent drug release of solid dispersion

n=3,Mean±s.d

Fig 18. Percent drug release at 60 min (%)

Evaluation of Mouth dissolving film:

Weight variation:

The average weight of the OTF batches was found to be between 40 and 53 mg..

Uniformity of thickness:

All batches' mean thickness values was found to be between 0.45 and 0.67 mm.

Folding endurance:

Pullulan-based otf with polyethylene glycol 1500 as plasticizer was found to between 72-100

Surface pH:

The otf batches' surface pH was found to be 6.2-6.8.

Uniformity of content :

It was discovered that each batch contained nearly the same amount of medication in 4cm² area of the film that contained 0.798 mg of disulfiram in which each film occupies 1.77 ml of oral film solution ( 1ml contains 0.451 mg of drug content).

In vitro dissolution study:

In phosphate buffer, all batches were examined for dissolution and nearly had a range of 81.6-91.83% drug release within  5 minutes

In vitro disintegration study:

An indication of the drug's desired onset of action for an oral film formulation is provided by an in vitro disintegration study. disulfiram-loaded pullulan-based oral film was disintegrated in vitro at a predetermined time interval in phosphate buffer (pH 6.8). As the concentration of sodium starch glycolate increased, the disintegration time of otf decreased . The mean time for complete disintegration was found to be 18 seconds for the F7 formulation for the total disappearance of the film into solution .

Ex vivo permeation study:

This study was conducted using the buccal mucosa of a freshly excised goat.The formulation F7 was chosen for the study of ex vivo permeation. The flux average was found to be 30.46 mcg/cm²/hr over 8 hrs.

Stability study :

The outcome of expedited stability studies conducted in compliance with ICH guidelines. For formulation F7, stability studies were conducted.For three months, the formulation was exposed to an accelerated temperature of 40 ? Weight variation, surface ph, and percentage drug release are calculated monthly to test the stability of the optimized formulation.

Formulation results:

Table 21, Formulation results

n=3,Mean±s.d

Stability study

Table 22, Stability study

n=3,Mean±s.d

Fig 19. Disintegration time ( sec) for different formulations

Fig 20.In vitro Drug release % for different  formulations

Ex vivo study:

Fig 21. Goat head

Fig 22. Goat buccal tissue

Fig 23. Working of Franz diffusion cell apparatus