Formulation and Characterization of Two Nystatin Lozenge Formulations as a New Pharmaceutical Dosage form in The Syrian Market

 

Suhail H. Alghanem*, Dimah N. Ibraheem, Basem M. Mansour, Aktham N. Shraiba

Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy,

Al-Andalus University, Tartous, Syria.

*Corresponding Author E-mail: sg4@au.edu.sy

 

ABSTRACT:

The aim of this study was to prepare Nystatin lozenges (suitable for many age categories, easy to administrate and preferable in many medical cases) using a variety of excipients, then evaluate these formulations In vitro to select the best one for later use to treat Candida Albicans ( the cause of oral candidiasis). Two formulations were prepared, in F1 (only Arabic gum was used) while in F2 (Arabic gum with propylene glycol were used). A comparison was performed to evaluate the two formulations. The results of our comparison indicated that the F2 formula was the best (disintegrating time: 21.37±0.88 min, Td = 40.59 min is the time required to release 63.2% of the Nystatin content). Nystatin is released from the F2 formulation according to zero order kinetics (linearity between the amount of drug released with time).

 

KEYWORDS: Nystatin, Lozenges, In vitro, release, Arabic gum.

 

 


INTRODUCTION: 

Oral candidiasis is a common fungal infection that affects the oral mucosa, this lesion is caused by the Candida. Albicansyeast, which is a component of the oral microflora (about 30-50% of people carry this organism, and its rates increase with the age of the patient)1–3. In fact, it is isolated from the oral cavity of healthy people, and it is controlled with the rest of the components of the microflora by the hostimmunity, but any change in the microflora environment will give this pathogen "Candida Albicans" an opportunity to invade any site4. The risk factors for the host that increase the chance of infection of the oral mucosa are: Impaired function of the salivary glands, wearing a dental prosthesis, oral mucosal disorder, taking medications (long-term use of broad-spectrum antibiotics - corticosteroids - antidepressants - antitumors – immunosuppressants), age (common in newborns and the elderly), endocrine changes (diabetes mellitus - pregnancy - kidney failure – hyperthyroidism), nutritional factors (high carbohydrate diet - iron

 

 

deficiency anemia), some disease states such as cancer - infection with the human immunodeficiency virus (HIV). The elimination of predisposing factors is an important strategy in the treatment of oral candidiasis1,5–8.

 

At the present time, there are many local or systemic drugs for treating oral candidiasis. Some of the systemic antifungal drugs are: triaconazole - fluconazole - and itraconazole6, which are suitable for patients who do not respond or can't tolerate local treatment, as well as patients who have a high risk of developing systemic infections. However, numerous drug interactions and decreased susceptibility of species other than Candida Albicans towards azoles limit the administration of systemic antifungal agents6,9.Local antifungal medications such as nystatin, amphotericin B, miconazole and clotrimazole are recommended as a first-line treatment for uncomplicated cases of oral candidiasis6,10–12.

 

In the year 1950 Nystatin "figure (1)" was discovered by Hazen and Brown. The melting point of pure Nystatin was found to be between 158-160°C13. It is considered the first choice for the oral candidiasistreatment11, also oral Nystatin is  the first drug administered orally as an antifungal prophylaxis14. The increased use of Nystatin is associated with the increasing number of candidiasis cases in patients with cancer, AIDS and other diseases. Nystatin has efficacy for the treatment of most infections caused by Candida species. The effectiveness of Nystatin comes from its ability to interfere with ergosterol (a sterol found in the plasma membranes of fungal cells)12, and form pores that allow K+ ions to pass through, resulting in the death of fungal cells15.

 

 

Figure 1. General structure of nystatin 16

 

Nystatin is not absorbed through the skin or mucous membranes, and its absorption through the gastrointestinal tract is very little, so there are no drug-drug interactions and systemic adverse effects. The use is safe for children, including newborns and infants17–19. Nystatin remains controversial in determining its class (according to the Biopharmaceutical Classification System "BCS") due to limited information on solubility and permeability data20.

 

The treatment dose for adults and children is 100,000 units 4 times a day after food, usually for 7 days (continued for 48 hours after lesions have resolved) 19,21.Nenoff, et al. determined in their research the minimum inhibition concentrations (MICs) for Nystatin in a range (0.625 to 1.25 µg/ml) for Candida albicans22.

 

Nystatin is available in the Syrian market in several pharmaceutical dosage forms, including oral drops, suspensions, and film-coated tablets. It is also present in the form of creams and is included in many formulations of ointments, creams, vaginal ovules, and suppositories. Hence the importance of our study is to suggest a formulation of Nystatin lozenges for many reasons: the formulation of Nystatin as lozenges is absent in the Syrian pharmaceutical market, it is suitable for many age groups, its sweet taste encourages children to administrate it and many physicians including dentists prefer to prescribe Nystatin as lozenges because of the slow release of its contents23, which achieves the therapeutic goal of Nystatin.

 

lozenges are solid pharmaceutical forms divided into multiple doses, placed in the oral cavity and by sucking for a local effect in the oral cavity and throat, it usually contains one or more active substances in a sweetened and flavored base, the goal is to dissolve or slowly disintegrate when sucked into the mouth. Lozenges are used for patients who have difficulty swallowing other solid oral forms of the drug, in adults and children who do not prefer to take the drug in its liquid pharmaceutical forms due to the unpleasant taste of the drug substance, as well as, the case of drug substances that must be released slowly with the administration of a constant amount of the drug inside the oral cavity or to cover the throat membranes with the drug solution. The lozenges are usually well tolerated24–31.

 

MATERIALS AND METHODS:

The raw and standard Nystatin was obtained by Unipharma Company for Pharmaceutical Industries - Syria, sucrose, propylene glycol (Scharlab S.L, Gato Perez, 33-P.I. Mas d'en Cisa 08181 Sentmenat Spain), Arabic gum (Titan, Biotech LTD, Bhiwadi-301019, Rajasthan, India), sodium lauryl sulfate and sodium hydroxide. Many devices also was used: silicon mold (was designed by us and made by a small specialized laboratory in Damascus, Syria), Erweka for hardness model (D-63150 Heusen stamm / Germany, Typ: TBH  225 Serial No: 126074. 132e), Erwika for disintegrating model (D-63150 Heusen stamm/ Germany, Typ: ZT 221, Serial No: 128932.0afc) and Erwika for dissolution to study drug release model (D-63150 Heusen stamm/ Germany, Typ: DT 620, Serial No: 126039.1266).

 

MOLD DESIGN:

A silicon mold was designed by our research group to prepare 50 lozenges with a weight of 1g each, depending on the density of sucrose and the components of the formula. The dimensions of the holes in the mold were (12.36 mm in diameter, 5 mm in height, the volume was 0.6 cm3). After that we printed it relying on a small specialized laboratory in Damascus, Syria. Figure (2) shows the mold used to prepare the lozenges.

 

Figure 2. The silicon mold used to prepare the lozenges

 

Formulation and Preparation of Nystatin Lozenges:

Two formulations were listed to prepare Nystatin lozenges, F1 (that contained Arabic gum only), F2 (that contained Arabic gum and propylene glycol) beside to others components in these two formulations necessary to prepare lozenges29. Formulation F1 was hard lozenges while F2 was soft one. Table (1) shows the components of this two formulations.

 

Table 1: Formulations of Prepared Nystatin Lozenges

Components

Formulation F1

Formulation F2

Sucrose

80 g

60 g

Nystatin

10 g

10 g

Propylene glycol

-----

20 g

Arabic gum solution (30%)

10 g

-----

Arabic gum

-----

10 g

Final weight

100 g

100 g

 

Preparation of the formulation F1: first of all, the solution of Arabic gum at a concentration of 30% was prepared, then the other ingredients were weighted, grinded and mixed very well. After that a paste with a suitable consistency was obtained and used to prepare lozenges by silicon mold. Finally, these lozenges were dried in a dry and dark place. Formulation F1 was hard lozenges29.

 

Preparation of the formulation F2: first the ingredients were weighted, then sucrose was grinded, then propylene glycol was added (this step was done using a water path at 78°C temperature) after the sucrose dissolved, we added Arabic gum, Nystatin respectively with good stirring and mixing until getting suitable consistency paste which will be used to prepare lozenges by silicon mold. Finally, these lozenges were dried in a dry and dark place. Formulation F2 was soft lozenges29.

 

Physiochemical Characterizes of Nystatin Lozenges:

A number of tests were carried out on the prepared Nystatin lozenges to compare between the two prepared formulations, these tests included: hardness, weight variation, drug content uniformity, disintegration and drug release23,29,32,33.

 

Nystatin Lozenges Assay:

A spectrophotometric assay of nystatin was applied11,13,16,34. We prepared a standard of series of Nystatin with concentrations (20µg/ml – 15µg/ml – 12.5µg/ml – 10µg/ml – 7.5µg/ml) where the absorbance of these solutions was measured using a device Spekol (1200/1, Analytik jena, Germany) at a wavelength of 293nm and plot the resulting graph and get the equation of this straight graph, which was (y = 0.0467x - 0.0771, and the value of the coefficient of determination R2 = 0.9998 ).

 

In vitro Drug Release Study:

Drug release study of Nystatin from lozenges was applied according to FDA requirements listed in the table number (2). Erwika for dissolution test was used for this test. The medium temperature was 37°C± 2.

 

 

Table 2. FDA Requirements for Nystatin Release Study from its Lozenges

USP Apparatus

Paddle II

Speed or rotating paddle

75 rpm

Medium of release

Purified Water with 0.1% SLS

Volume of medium release (ml)

900

Recommended Sampling Times (minutes)

15, 30, 45, 60 and 90.

 

The curves of Nystatin release were drawn for both formulations F1 and F2 (12 lozenges were tested and the average amount of Nystatin released was calculated and represented graphically as a function of time). The  kinetic of  release module was determined  depending on the value of coefficient of determination R2, four modules were examined: zero order, first order, Higuchi and Weibull. Table (2) shows the equations of four modulus used to study the kinetic release of Nystatin and parameters for each module35–38.

 

Table 2. Equations and parameters of modules used to study kinetic release of Nystatin from prepared lozenges formulations F1&F2

Module

Equation

Module Parametrs

Zero order

Q = k0t

k0

First order

Ln(100 – Q) =

Ln Q0 – k1t

k1

Higuchi module

Q = kHt1/2

kH

Weibull module

Log[-Ln (1 - m)] = b * Log t – Log a

Td, b

Where: a = (Td)b

 

RESULTS AND DISCUSSION:

WEIGHT VARIATION:

Table (3) summarizes the results of physiochemical tests we obtained. With regard to the weight uniformity test for both formulations F1 and F2, the average weight was (0.958g ±0.024) for F1 formulation, while it was (0.982 g ±0.033) for F2 formulation. The results of this test meet the British Pharmacopoeia requirements.

 

Nystatin Content Uniformity:

The determination of the Nystatin content was carried out on both formulations F1 and F2. The test was done according to the British Pharmacopoeia requirements. The average content of Nystatin was (96.83mg±2.69) for formulation F1 and (98.43mg±2.69) for formulation F2 (Table 2). The results of this test meet the British Pharmacopoeia requirements.

 

Hardness Test:

We applied this test on Formulation F1 only because the formulation F2 was soft (propylene glycol existed as a plasticizer), the results showed (Table 3) that the average hardness value for 20 samples was (20.409 KP ±0.807).

 

Diegrating Test:

All six samples from each formulations were disintegrated. The results in Table 2 showed that the average time of disintegrating was (12.52min ±0.43) for formulation F1 and (21.37min±0.88) for the formulation F2.

 

Table 3. physiochemical parameters of Nystatin formulations (F1&F2)

 

F1

F2

Weight variation (g)

0.958 ± 0.024

0.982 ± 0.033

Nystatin content (mg)

96.83 ± 2.69

98.43 ± 2.45

Hardness (KP)

20.409 ± 0.807

-----

Disintegrating time (min)

12.52 ± 0.43

21.37 ± 0.88

 

In vitro Drug Release Test:

The figure (3) indicates the In vitro drug release profiles of Nystatin from its lozenges formulations F1 and F2.

 

Figure 3. In vitro Drug Release profiles of Nystatinfrom its lozenges formulations F1&F2

 

The kinetic release order of Nystatin from its lozenges formulations F1 and F2wasdetermined. The results in table (4) indicates that Nystatin released from F1 by first order kinetic (R2 = 0.9140, there was a linearity  correlation between napierian logarithm of resident content of Nystatin "LnQ" and time "t"), while Nytatin released from F2 by zero order kinetic (R2 = 0.9612, there was a linearity correlation between released content "Q" and time "t").

 

Table 4. Coefficient of determination R2 Values of Nystatin release kinetic modules from its formulations F1&F2

Kinetic module

F1

F2

Zero order

0.5648

0.9612

First order

0.9140

0.8869

Higuchi

0.8481

0.9313

Weibull

0.9665

0.9723

 

According to the results showed in table (4), the Weibull module could be used suitable to explain the release of Nystatin from its formulations F1 and F2 (R2 value was 0.9665, 0.9723 in order). Parameters of this module were used to compare between Nystatin release from F1 and F2 formulations, the results showed in table (5). F2 release curve was S-shape (sigmoid) "b value ˃ 1" with upward curvature followed by a turning point as it appeared in figure (3), while it was parabolic with formulation F1 (b value ˂ 1) with a higher initial slope and after that consistent with the exponential35,39. The time needed to release 63.2% of Nystatin from its lozenges formulations F1 and F2 was: 9.71 min and 40.59 min in order. The role of propylene glycol in the formulation F2 was binder, stabilizer, humectants and antimicrobial and preservative agent (10% percentage in formulation F2)29,40. The amount of Arabic gum in the formulation F2 was 10mg in each lozenge (10% in percentage), while it was 3mg  in the formulation F1 (3% in percentage), so the good binding and strength in the formulation F2 (Arabic gum and propylene glycol), all that lead to length the time of disintegrating more than the lozenges in formulation F1 and slow the release rate of Nytatin from this formulation.

 

Table 5. Weibull module parameters of Nystatin form its lozenges formulations F1&F2

Wiebull module Parameters

F1

F2

b (shape parameter)

0.6807

1.6774

a

4.698

498.884

Td (min)

9.71

40.59

Td: Time needed to release 63.2% of Nystatin form lozenges

 

CONCLUSION:

General view on  the characteristics of Nystatin and its applications assures that Nystatin lozenges – which is  absent in the Syrian medical market– may be a good candidate to treat more cases in various age categories (like children). In  this research, we succeeded in preparing and characterizing  Nystatin lozenges by preparing two formulations F1 (only Arabic gum was used) and F2 (Arabic gum with propylene glycol were used). The results of the study showed that the F2 formula was better in terms of the applied physicochemical tests and the slow release of Nystatin, where propylene glycol plays an important role in this formulation in combination with Arabic gum. This is an important step in introducing this formulation as a pharmaceutical dosage form in the Syrian market, there is a need to follow up on further In vitro tests on this lozenges, and In Vivo tests must be conducted to evaluate its therapeutic efficacy.

 

ACKNOWLEDGMENTS:

The authors would like to thank Universal Pharmaceutical Industries "Unipharma", Damascus, Syria for providing the raw and standard Nystatin that was used in the research, and they also would like to thank the Faculty of Pharmacy at Al-Andalus University for Medical Sciences, Tartous, Syria for their support in the completion of this research.

 

 

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Received on 21.07.2022            Modified on 17.09.2022

Accepted on 21.10.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(4):1639-1643.

DOI: 10.52711/0974-360X.2023.00268