Stability studies of Hollow Microspheres at Refrigerated, Normal and Accelerated Conditions

 

Surbhi Rohilla*, Dinesh Chandra Bhatt

Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology,

Hisar, Haryana - 125001, India.

 

ABSTRACT:

In the present investigation an attempt was made to focus on the stability aspects of itraconazole hollow microsphere at refrigerated condition, room temperature and at accelerated condition. In stability studies, more emphasis given on the effect of different temperature conditions on appearance, % drug content, % buoyancy and % drug release of formulation over a period of 6 months. Apart from above studies, SEM and FTIR analysis was done to determine any change in morphology or chemical structure. The results showed non-significant changes in pharmaceutical properties. From result findings, it can be concluded that the itraconazole hollow microspheres are stable formulation to sustain the drug in upper GIT for prolonged period of time.

 

 

INTRODUCTION:

Stability studies ensure the quality, safety, and efficacy of products throughout its shelf-life. These studies are preconditioned for approval and acceptance of any pharmaceutical product. For such studies, there are specific planned guidelines to be followed and are regulated by authorities such as ICH, WHO, and or other agencies.^1,2 The stability is the ability of pharmaceutical formulation containing drug products to remain within its physicochemical, pharmaceutical, therapeutic, and toxicological specifications in a prescribed manner.^3,4

 

The stability testing of drug formulation is a complex process, it provides the facts concerning the influence of different environmental conditions overtime on the quality of drug products.⁵

 

Itraconazole is a broad-spectrum antifungal drug that belongs to biopharmaceutical classification II, which means it is having solubility limitations.

 

It suffers from poor bioavailability because of having a narrow absorption window in upper GIT which indicated that the adequate dissolution and absorption of the drug requires an acidic environment. Thus the present work is aimed to formulate hollow microsphere of itraconazole for prolonged gastric retention.^6-8

 

The stability analysis of pharmaceutical products is essential in many aspects including patient safety, legal requirements to fulfill the quality and purity of drug products, and to avoid economical consequences due to the marketing of undesirable products. An attempt was made in the present investigation to focus on the stability aspects of the hollow microsphere system. The purpose of the present investigation is to assess the stability profile of hollow microspheres of ethylcellulose and Eudragit RS100 at three different temperatures i.e. the normal room temperature, freezing temperature, and accelerated temperature condition for 6 months. 

                                                                                                                                       

MATERIALS AND METHODS:

Materials:

Itraconazole and Eudragit Rs100 were generously gifted by Zydus Cadila Healthcare Limited, Ahmedabad, India, and Evonik Degussa India Pvt. Ltd, Mumbai, India respectively. Ethyl Cellulose was procured from Thomas baker Pvt. Ltd., New Delhi. All other reagents and solvents were of analytical grade.

 

Hollow microspheres preparation:

Hollow microspheres containing itraconazole were prepared by the emulsion solvent diffusion evaporation method. Briefly, drug (Itraconazole) and polymer (ethylcellulose and Eudragit RS100) having ratio 6:1 were dissolved simultaneously in (1:1) mixture of ethanol and dichloromethane. The slurry was added slowly to 1.0%w/v solution of  Polyvinyl alcohol-containing Tween-80 (0.2% w/v), forming emulsion (o/w) and stirred continuously on three-blade propeller type mechanical stirrer maintained at 40°C temperature for 2h at a speed of 350rpm. The hollow microspheres were formed by the generation of the internal gaseous phase due to evaporation of solvent which was then diffused out and replaced by water. The hollow microspheres were filtered, washed with distilled water, dried at room temperature, and subsequently stored in a desiccator.^9,10

 

STABILITY STUDIES:

The stability testing of itraconazole loaded hollow microspheres were done according to the International Conference on Harmonization (ICH) guidelines. The formulation was packed in tightly closed glass vials and was kept at three different temperature conditions (refrigerated condition (5±3°C), room temperature (25±2°C) and under accelerated condition (40±2°C/75±5% RH) for six months in a stability chamber. The samples (n=3) were examined critically for physical appearance, drug content, buoyancy, and drug release studies at an interval of 0, 1, 2, 3, and 6 months.^11,12

 

FTIR analysis:

FT-IR analysis was done to discover any chemical change in formulation under a lapse of 6 months. For this, the samples were crushed into a fine powder with KBr and placed it over the sample holder for analysis. The spectrum scan was done over a frequency range of 4000 to 400 cm⁻¹ by using a Fourier transform infrared spectrophotometer (IR Affinity, Shimadzu, Japan).

 

Appearance:

The physical characteristics of the samples were carefully observed for any visual changes in color and clumping/aggregation behaviour. Morphological transformation, if any during the study was checked using a scanning electron microscope.

 

Drug Content:

For drug content determination, the accurately weighed amount of microspheres were crushed and dissolved in 25ml methanol, passed it through a syringe filter of 0.45μm pore size. Diluted it appropriately with methanol and analyzed UV spectrophotometrically (n=3) at 262 nm.¹³ The % buoyancy was calculated by using equation 1:

 

                       Weight of drug-loaded in microspheres

% Drug content = –––––––––––––––––––––––––––––––× 100     eq 1

Weight of microspheres

 

Buoyancy:

The in-vitro Buoyancy studies were done by placing a weighed amount of microspheres in simulated gastric fluid (pH 1.2) in USP dissolution apparatus II maintained at 37℃ at a stirring speed of 50rpm for 10 h.  After 10 h both the buoyant and settled segment was collected separately, dried, and then weighed.¹⁴ The % buoyancy was calculated by using equation 2.

 

                                Weight of buoyant microspheres

% Buoyancy = –––––––––––––––––––––––––––––––––––×100    eq 2

                                  Total weight of microspheres

 

Figure 1 FTIR spectra at different temperature conditions

 

In-vitro Drug Release:

The in-vitro drug release was carried out using USP dissolution apparatus II. The predetermined amount of drug-containing microspheres were filled into the dialysis membrane and immersed in a dissolution medium consist of 900ml of simulated gastric fluid (pH 1.2) maintained at 37±0.5⁰C temperature with a paddle rotation speed of 100rpm. The samples were withdrawn at predetermined time intervals up to 10 h, filtered through a 0.45µm membrane filters, and analyzed UV spectrophotometrically (n=3) at 258nm.¹³

 

RESULTS AND DISCUSSION:

A formulation is said to be stable if its physical and chemical integrity remains intact over a period of time. Ideally, drug product must fulfil the criteria of physical-chemical and microbiological characterization throughout the intended shelf-life.

 

On the interpretation of FTIR spectra, it is clear that all characteristic peaks of the drug are intact. The spectrum after 6 months is as analyzed initially before stability studies (Figure 1). Hence, no major shift or addition of new peaks indicated that no significant chemical interaction between drug and polymers.

 

The results of the visual examination showed white discrete particles with no significant variation over 6 months under different storage conditions. The surface morphology, SEM images indicated the retention of the spherical shape of microspheres, there was no sign of morphological transformation or development of any kind of cracks or rupturing of the surface of stored formulations. While an insignificant aggregation of particles is seen at refrigerated conditions (5±3°C/ 65% RH±5%) (Figure 2). Hence, the formulation complies with stability conditions.

 

Figure 2 SEM images of microspheres: (a) Refrigerated temperature (b) Room temperature (c) Accelerated temperate condition.

 

After 6 months storage period, non-significant loss of drug was found in the samples which may be due to a slight loss of integrity of the system. ^[15] The drug content was found to be maximum at room temperature (Table 1).

 

The % buoyancy was not changed much (<5%) for the stored formulation. Initially, the % buoyancy at the start of the study was 61.1±0.70% which changes to 60.70±1.02, 60.40±1.04, and 59.10±1.29% for increasing order of temperature selected during the study (n=3). Thus, the sample stored at refrigeration temperature shows maximum buoyancy which might be due to aggregation of microspheres stored in refrigerated conditions. The size of the particle has an inverse relationship with density. Hence, a slight increase in buoyancy might be due to an increase in the aggregation of particles.¹⁶

 

Table 1. Stability study evaluation of various parameters (mean±SD)

Parameters	0 month	1 month	2 month	3 month	6 month
Refrigerator conditions (5±3℃)
Visual Appearance	White discrete free flowing particles	No remarkable change	No remarkable change	No remarkable change	No remarkable change
Drug Content	14.32±0.39	14.31±0.12	14.27±0.28	14.26±0.25	14.25±0.25
Buoyancy	61.1±0.70	61.05±0.60	60.98±0.52	60.88±1.25	60.70±0.8
In vitro drug release	78.66±0.96	78.63±1.05	78.54±0.71	78.48±0.88	78.33±1.15
Room temperature (25±2℃)
Visual Appearance	White discrete free flowing particles	No remarkable change	No remarkable change	No remarkable change	No remarkable change
Drug Content	14.32±0.39	14.30±0.20	14.30±0.55	14.27±0.44	14.20±0.63
Buoyancy	61.1±0.70	61.00±0.87	60.92±1.38	60.71±1.29	60.40±1.14
In vitro drug release	78.66±.96	78.50±0.89	78.20±1.46	78.05±1.22	77.32±1.58
Accelerated conditions (40±2 °C/75±5% RH)
Visual Appearance	White discrete free flowing particles	No remarkable change	No remarkable change	No remarkable change	No remarkable change
Drug Content	14.32±0.39	14.30±0.62	14.24±0.15	14.26±0.28	14.06±0.76
Buoyancy	61.1±0.70	61.04±0.80	59.96±1.05	59.59±1.45	59.10±1.29
In-vitro drug release	78.66±0.96	78.44± 0.97	78.26±1.25	77.67±1.45	76.20±1.64

(n=3)

 

No considerable changes are found in the percent drug release from microsphere formulation at different storage conditions. Initially, the hollow microspheres showed 78.66±96% drug release in 10 h while at the end of 6 months the formulation showed 78.33±1.50, 77.32±1.98, and 76.20±1.64%. The release is slightly decreased for formulation kept at accelerated conditions as compared to initial day release which might be due to the formation of rigid surface in the microspheres due to evaporation of residual amount of solvent. The histogram plotted between % drug content, % buoyancy, and % drug release for formulation stored at different temperature conditions shown in figure 3.

 

Figure 3 Histogram showing comparative analysis of % drug content, %buoyancy and %drug release at different temperature conditions (mean±SD (n=3)

 

The results of the stability studies of the itraconazole hollow microsphere illustrated that the microspheres were found to be stable at all temperature conditions but in spite of that, it is most stable at room temperature.

 

CONCLUSION:

The stability of the product is thus, justified by observing the results of the physical and chemical stability of the dosage form. The results of stability studies are comparable to results on the initial day (0 month). Very insignificant (<5%) change was observed in % drug content, % buoyancy, and % drug release. Thus, it may be concluded that hollow microspheres of itraconazole are the suitable delivery system for prolonged drug release at the absorption site and having considerable stability when stored at different temperatures.

 

ACKNOWLEDGEMENT:

The authors are grateful to the Department of Science and Technology, New Delhi, for providing financial assistance under the "INSPIRE" fellowship [IF 150924]. The authors are thankful to SAIF, AIIMS, New Delhi for Scanning Electron Microscopy analysis.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 29.12.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(10):5351-5354.