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A comparative real time study of stability of cream and ointment formulations of clobetasol propionate in drug stores of Bangladesh.

Introduction

Clobetasol propionate, (11p,16P)-21-chloro-9-fluoro-11-hydroxy-16-methyl-17-(1-oxopropoxy)-pregna- 1,4-diene-3,20-dione, is a synthetic corticosteroid normally indicated for topical dermatologic use. Clobetasol, an analog of prednisolone, has a high degree of glucocorticoid activity and a slight degree of mineralocorticoid activity. Clobetasol propionate is a super-high potency corticosteroid indicated for the relief of the inflammatory and pruritic manifestations of corticosteroid-responsive dermatoses. Various formulations of clobetasol propionate are currently used to treat used to treat moderate to severe plaque-type psoriasis due to its antiinflammatory, anti-pruritic, vasoconstrictive and immunomodulating properties (Lowe et al., 2005).

The activity and stability of a drug is superior in a hydrocarbon base in semisolid preparation (Block, 2006).

But, patient acceptability is low due to greasy nature of the base. The difference between creams and ointments is based on their chief components. The former is water-based while the latter is oil-based. The vehicle of the topical steroid influences the strength of the medication. Given the same topical steroid, the following list represents the strengths of the medication, from highest to lowest: Ointment >Creams >Lotions >Solutions. Creams are quickly absorbed by the skin. Ointments on the other hand stay longer on the surface of the skin and release the drug for a prolonged period of time. Since these are not easily absorbed by the skin, the ointments are suitable to use on dry skin. They will keep the skin moist for longer periods thus promoting faster healing. Skin absorbs creams more quickly, i.e. creams promote skin dryness. So they are better used on oily and greasy skin conditions.

The shelf life of topical semisolid medications is mainly determined by several factors like packaging format, presence of preservative, formulation (e.g. ointment or cream), storage condition (temperature, humidity). The in-use stability depends on the type of formulation, type of the packaging materials, frequency of opening of the container, etc. The stability of creams, which are generally oil in water emulsions are much more prone to microbial contamination as the continuous aqueous phase is a much more favorable medium for growth than the fatty continuous phase of an ointment base. Therefore the in-use shelf-life of creams could be shorter than that for ointments even if a preservative is present. For sterile semi-solid medicines, the in-use shelf-life is based on the presentation, area of application and clinical use of the product. NHS pharmacy services of UK recommend for non-sterile ointments packing in tubes and an in-use shelf-life of 6-months, and an in-use shelf-life of 3-months for creams from first opening (Lowe, 2001).

In the present study, the differences in the physical and chemical parameters of Clobetasol propionate topical formulations (cream and ointment) in a simultaneous long-term real time stability study in drug stores of Bangladesh was investigated. According to World Health Organization (WHO) guidelines, the recommended long-term stability testing condition in Bangladesh (climate zone IVA) should be at 300C and 65 % RH (Anon, 2009). But, the climatic condition of Bangladesh for long-term stability of drugs is not ideal throughout the year. It may be pointed out in this regard that the weather conditions of Bangladesh are hot and humid from about March till November, and cool and dry from December till February. Most drug stores in the rural areas as well as a substantial number of drug stores in urban areas do not control temperature and humidity while maintaining their drug products. As a result, studies on the real time stability of a number of drugs need to be monitored for any degradation products to occur, leading to loss of the drug in question as well as variability in concentration and prescribed dosage.

Materials and methods

Chemicals and Reagents

Reference standard of clobetasol propionate (micronised) was purchased from Newchem S.P.A., Italy). Methanol (MeOH) and monobasic sodium phosphate was purchased from Merck KGaA (Darmstadt, Germany), acetonitrile from Lab Scan Analytical Science (Lab-Scan Ltd., Ireland), and ortho-phosphoric acid ([H.sub.3]P[O.sub.4]) (85%) from Riedel-de-Haen (Seezle, Germany). Clobetasol propionate (cream and ointment forms) were purchased and maintained under ambient conditions for the experimental time period (24 months) at Orion Laboratories, Ltd, Dhaka.

Instruments

The quantitative determinations were carried out in HPLC system-Agilent Technologies 1200 series (of Agilent Technologies, Waldbronn, Germany) equipped with an Agilent DAD-G 1315B Diode-Array multibeam UV-visible detector (wavelength 240 nm was used), Agilent GA1322A degasser, Agilent G1316A column Thermostat, and an Agilent G1312A Thermostatted auto-sampler. The column was Luna [C.sub.18] (5[micro]m, 4.6 x 250 mm). The operating software was Agilent Chemstation[TM] for LC and LC-M/S systems. Mobile phases were filtered through membrane filter 47mm PVDF 0.2 [micro]m paper of Pall Corporation. The sample and standard solutions were filtered through disc filter containing 15mm 0.2 [micro]m PVDF paper of Pall Corporation. pH was measured with a pH meter (HANNA, Singapore). The accelerated stability chamber was Tharmostabil[TM] Guiseppe S.N.C., Italy.

Analytical Methods for the Quantification of Active Ingredients

The analysis was run according to the official methods of United States Pharmacopoeia (USP) for clobetasol propionate (Anon., 2007).

Stability Study

The stability testing may be divided into three categories, i.e. Real time stability test, Accelerated stability test, and Microbial stability test.

Testing Frequency

The ICH (International Conference on Harmonization) guidelines (Anon., 2003) prescribed the testing frequency of a stability study of pharmaceutical dosage forms. Real time study for drug substances require a proposed re-test period of at least 12 months, the frequency of testing at the long-term storage condition should be every 3 months over the first year, every 6 months over the second year, and annually thereafter through the proposed re-test period. But, the accelerated storage condition, a minimum of three time points, including the initial and final time points (e.g., 0, 3, and 6 months), from a 6-month study is recommended.

Storage Condition

The temperature and humidity were recorded regularly at the same time in a record book. The seasonal variation of room temperature and humidity was observed. The accelerated stability over six months was done at 400C and 75% relative humidity (Anon, 2003).

Microbial Stability Tests

The microbial tests were done to find out if any microbial contamination occurred over the storage period of 2 years. The microbial examinations were done according to the British and European Pharmacopeias (BP and EP) (Anon., 2005a; Anon., 2005b). Two types of examinations were done: 'tests for microbial contamination of non-sterile products' and 'tests for specific microbes'. It is mentioned in ICH (International Conference on Harmonization) guidelines that certain quantitative chemical attributes (e.g. assay, degradation products, and preservative content) for a drug substance or product can be assumed to follow zero-order kinetics during long-term storage (Anon., 2003). Qualitative attributes and microbiological attribute may also be necessary for evaluation of impact of long-term storage.

Relationship Between Product Stability with Storage Period

We assumed a regression model:

y = [b.sub.o] + [b.sub.1] x

Here, y = dependent variable, shelf life of the product

x = independent variable, observation period (time)

[b.sub.o] = the constant term, intercept

[b.sub.1] = slope

In this regression model, the percentages of active ingredient of the particular formulation after a certain period of time were plotted against time.

Results and discussion

Storage Condition

According to ICH guidelines (Anon., 2003), a drug substance should be evaluated under storage conditions for its thermal stability and, if applicable, its sensitivity to moisture should be ensured. Storage condition should be in long-term studies: 25[degrees]C [+ or -] 2[degrees]C/60% RH [+ or -] 5% RH or 30[degrees]C [+ or -] 2[degrees]C/65% RH [+ or -] 5% RH for a minimum period of 12 months, and for accelerated stability studies: 40[degrees]C [+ or -] 2[degrees]C/75% RH [+ or -] 5% RH for a minimum period of 6 months. But, our stability study was done under normal long-term storage conditions in a room without any air or humidity control system, which is the actual situation in most urban and all rural drug stores of Bangladesh. The mass population of the country obtains medications from this type of drug stores. As a result, such type of stability study is very essential for public health considerations. In the present study, the humidity and temperature were recorded regularly. It was observed that the variation of seasons impacts greatly on the storage condition (temperature and humidity) of the medications. The real time study was also compared with a controlled accelerated stability study for a period of six months.

The variations of temperature were recorded regularly. The summery of the observations is represented in Table 1. The whole year was divided into three categories according to temperature and relative humidity of the air. It was observed that the highest temperature and humidity was observed during the months of June to August.

Chemical and Physical Stability Study

Stability study results of creams and ointments are represented in the Table 2. The experiments were done according to ICH guidelines (Anon., 2003). From the data, it was observed that the changes of potency of both the formulations were within the specified limit (i.e. not less than 90% of the initial value) in both of the real time study for 2 years and the accelerated stability study for 6 months. Both the formulations were stable over 2 years and with their desired physicochemical properties (e.g. physical stability, smoothness, no gel formation or phase separation) and chemical properties (e.g. potency, pH).

The peak chromatogram of clobetasol propionate in HPLC was standard in nature (Fig.1). It is clear that the both the formulations in the experimental conditions were stable enough over two years. The peak of the active component was free from massive degradation (Figs. 2, 3). Some degradation may have occurred but these were within the specified limits. The degraded products were clearly separated from the main analyte; the USP (United States Pharmacopoeia) tailing factor (Anon., 2005c) of the clobetasol propionate was 1.1 (limit 1 to 1.5).

Microbiological Stability Study

The products were non-sterile formulations. Therefore, microbial contamination tests were done according to the guidelines of British and European Pharmacopoeias (Anon., 2005a; Anon., 2005b). The specified limit for these types of products is not more than 100 colony forming units (cfu) per gram. It was observed that the sealed products contain a few (< 10 cfu per gram) or almost no contamination before or after 2 years study periods. And the in-use packs (which were opened once a month) also had less than 10 cfu per gram of cream or ointments.

The tests for specific microbes of BP (Anon., 2005a) were done and there was no Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, or Salmonella typhimurium in the tested samples. So, it may be assumed that the products were free from microbial contamination. One of the causes of the low microbial count may be due to presence of preservatives in the formulations as well as the antimicrobial nature of the active ingredient.

Relationship Between Product Stability with Storage Period

The relationship between product stability and the storage period are represented in the Fig. 4 and Fig. 5. It was observed that the product stability of ointment formulation is not time dependent ([R.sup.2] value was only 0.93). Whereas in creams, time had a significant impact on the stability of the formulation (significance level < 0.001) with a strong linearity ([R.sup.2] value was more than 0.99). So, it may be said that stability of ointment was independent of time and the stability of a cream depended on the storage period, i.e. the active ingredient of clobetasol cream was degraded by 0.21 units (%) for a unit change of storage period (month).

Clobetasol propionate ointment base is composed mainly of propylene glycol, sorbitan sesquioleate, and white petrolatum (70%). Since the formulation contains very little water, hydrolysis did not occur. But, as the cream formulation contains about 80% water, the possibility of hydrolysis and other degradation is higher than ointment.

It was observed that the ointment formulation of clobetasol propionate was more stable and showed very slow time-independent degradation kinetics over a long time. But, the cream formulation showed time-dependent degradation kinetics, even though the extent of degradation was within the specified limits. However, drug stores, particularly in the rural areas of Bangladesh retain drugs in their stores for more than two years and sometimes, even after the expiry period of the drug. In those cases, the seasonal variation of Bangladesh and the lack of optimal storage conditions in drug stores of the country (particularly absence of maintenance of proper temperature and humidity) may be a threat for drug stability especially in water containing (cream) preparations. The local drug stores of the country need to be more cautious to keep the materials to serve patients with a quality drug for optimum activity.

[FIGURE 1 OMITTED]

Mili-anodic output (mAU) versus retention time was plotted here. The retention time of clobetasol propionate was 5.98 minutes.

[FIGURE 2 OMITTED]

Mili-anodic output (mAU) versus rentione time was plotted here. The retention time of clobetasol propionate was 5.6l minutes. There was no visible degradation in the park chromatogram of clobetasol ointment.

[FIGURE 3 OMITTED]

The theoretical concentration of the test solution was approximately 0.04 mg/ ml.

Mili-anodic output (mAU) versus retention time was plotted here. The retention time of clobetasol propionate was 5.74 minutes. The peak chromatogram of clobetasol propionate was seen to be clearly separated from the peaks of degraded products.

[FIGURE 4 OMITTED]

The amount of clobetasol propionate (% of initial value) present in the ointment is plotted against storage period (in month). It is observed that the degradation kinetics of clobetasol propionate in ointment preparation is not linear ([R.sup.2] value = 0.93) and it is not time-dependent.

[FIGURE 5 OMITTED]

The amount of clobetasol propionate (% of initial value) present in the cream formulation is plotted against storage period (in month). It is observed that the degradation kinetics of clobetasol propionate in cream preparation is linear ([R.sup.2] value = 0.99) and it is time-dependent.

References

Anon., 2003. ICH guidelines, ICH Topic Q1C, Stability Testing of New Dosage Forms; and Q1A (R2), Stability testing of new drug substances and products. International Conference on Harmonization, IFPMA, Geneva.

Anon., 2005a. British Pharmacopoeia, Appendix XVIB A357, The Stationery Office, London.

Anon., 2005b. European Pharmacopoeia 5.0, Section 2.6.13, Microbial contamination of non-sterile products, Council of Europe, Strasbourg, France.

Anon., 2005c. Validation of compendial methods. In: United States Pharmacopoeia 28. United States Pharmacopoeial Convention Inc., Rockville, MD,USA. pp: 2748-2751.

Anon., 2007. United States Pharmacopoeia 30--National Formulary 25 (USP 30- NF 25). United States Pharmacopoeial Convention Inc., Rockville, MD,USA. pp: 1787-1788.

Anon., 2009. Stability testing of active pharmaceutical ingredients and finished pharmaceutical products. In: World Health Organization (WHO) Technical Report Series, No. 953.

Block, L.H., 2006. Medicated Topicals, In: Hendrickson R., Remington The science and practice in pharmacy, 21st edn., Lippincott Williams and Wilkins, USA. pp: 871-888.

Lowe, N., Feldman, S.R., Sherer, D., Weiss, J., Shavin, J.S., Lin, Y.L., Foley, V., and Soto, P., 2005. Clobetasol propionate lotion, an efficient and safe alternative to clobetasol propionate emollient cream in subjects with moderate to severe plaque-type psoriasis. Journal of Dermatological Treatment, 16: 158-164.

Lowe, R.A., 2001. Storage, stability and in-use shelf-life guidelines for non-sterile medicines, QA service of Specialist Pharmacy services, National Health Service of UK.

(1) Alok Kumar Paul, (2) Sukalyan Kumar Kundu, (2) Shamima Ahsan, Farhana Jamal, Rasheda Ahmed, Nusratun Nahar, Ishtiaq Ahmad, Rownak Jahan, Mohammed Rahmatullah,

(1) Faculty of Life Sciences, University of Development Alternative,Dhanmondi, Dhaka-1205, Bangladesh. (2) Department of Pharmacy, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh.

Corresponding Author: Dr. Mohammed Rahmatullah, Pro-Vice Chancellor University of Development Alternative House No. 78, Road No. 11A (new) Dhanmondi R/A, Dhaka-1205 Bangladesh Email: rahamatm@hotmail.com Fax: 88-02-8157339
Table 1: Record of temperature and relative humidity
during the real time study.

                                     Seasons

Parameters           Summer       Rainy season    Autumn and winter

Month               March-May      June-August    September-January

Temperature       28 [+ or -] 2   27 [+ or -] 3    22 [+ or -] 2.5
([degrees]C)
(mean [+ or -]
standard
deviation)

Relative          71 [+ or -] 2   75 [+ or -] 3     65 [+ or -] 2
humidity (%
RH)(mean
[+ or -]
standard
deviation)

Table 2: A comparative chemical stability of clobetasol propionate
cream and ointment in both the real time and accelerated condition

Test           Storage               Formulation
parameters     condition

Change of      Real time             Ointment
potency (%)    study                 Cream

[mean          Accelerated           Ointment
[+ or -] (%)   study                 Cream
RSD] n=3
               Real time             Ointment
ph (average    study                 Cream
value) n=3     Accelerated           Ointment
               study                 Cream

Test           Storage period (months)
parameters
               Initial               Three

Change of      100 [+ or -] 0.26     99.91 [+ or -] 1.81
potency (%)    100 [+ or -] 0.29     99.56 [+ or -] 2.94

[mean          100 [+ or -] 0.26     99.68 [+ or -] 0.98
[+ or -] (%)   100 [+ or -] 0.29     99.12 [+ or -] 1.07
RSD] n=3
               7.84                  7.89
ph (average    5.53                  5.61
value) n=3     7.84                  7.9
               5.53                  5.64

Test           Storage period (months)
parameters
               Six                   Nine

Change of      99.08 [+ or -] 0.83   98.68 [+ or -] 1.02
potency (%)    98.58 [+ or -] 0.86   98.04 [+ or -] 0.98

[mean          98.94 [+ or -] 1.45   --
[+ or -] (%)   98.16 [+ or -] 1.12   --
RSD] n=3
               7.95                  7.98
ph (average    5.68                  5.73
value) n=3     7.97                  --
               5.7                   --

Test           Storage period (months)
parameters
               Twelve                Eighteen

Change of      98.03 [+ or -] 1.32   97.92 [+ or -] 0.97
potency (%)    97.74 [+ or -] 2.74   96.18 [+ or -] 1.04

[mean          --                    --
[+ or -] (%)   --                    --
RSD] n=3
               8.05                  8.11
ph (average    5.78                  5.80
value) n=3     --                    --
               --                    --

Test           Storage period (months)
parameters
               Twenty Four

Change of      97.27 [+ or -] 0.13
potency (%)    95.10 [+ or -] 0.43

[mean          --
[+ or -] (%)   --
RSD] n=3
               8.18
ph (average    5.85
value) n=3     --
               --
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Title Annotation:Original Article
Author:Paul, Alok Kumar; Kundu, Sukalyan Kumar; Ahsan, Shamima; Jamal, Farhana; Ahmed, Rasheda; Nahar, Nusr
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:9BANG
Date:May 1, 2010
Words:2969
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