INTRDUCTION:

Oral drug administration still remains the route of choice for the majority of clinical applications. Some drugs have ideal characteristics for good absorption to occur throughout the gastrointestinal tract, whereas others present difficulties.¹ Rifabutin (RFB) is a potent semi-synthetic spiropiperidly-rifamycin derived from rifamycin-S having high permeability and low solubility.² It possesses a broad spectrum of antibacterial activity and is particularly active against mycobacterium tuberculosis, including rifampicin-resistant strains, and atypical mycobacteria. The antimicrobial action of the RFB results from the inhibition of bacterial DNA-dependent RNA polymerase.³

RFB is clinically used as a standard component of a combination regimen for tuberculosis treatment in HIV infected patients where rifampin therapy is contraindicated.^4-6In order to improve the therapeutics efficacy of RFB, many ways have been explored, like inhalable microparticles^7,8, liposomal drug delivery^9-10.

Number of ways has been explored to use hydrophilic polymers in the preparation of microparticles as Kellaway et al prepared microspheres using HPMC and other polymers for intra-nasal delivery of drugs.¹¹ Martinac et al prepared microspheres of gemfibrozil using chitosan and HPMC to modify its release rate.¹² Santinho et al manufactured casein microparticles by using HPMC as thickener for targeting of the drugs..¹³

Reverchon prepared HPMC based composite microparticles of ampicillian trihydrate using supercritical fluid technology to control release rate of the drug.¹⁴

Aim of the study was to prepare RFB loaded ethyl cellulose/ HPMC microspheres and to evaluate the effect of various processing parameters on the physical characteristics of the microspheres by determining its release kinetics, X-ray diffraction pattern, change in enthalpy and stability in accelerated conditions of temperature.

MATERIALS AND METHODS:

Materials:

Rifabutin (RFB) was supplied by Lupin Ltd (Aurangabad, India). Hydroxypropyl methylcellulose (HPCM E-15 LV) was obtained as gift sample from Colorcon (Goa, India). Ethyl Cellulose (EC) and polyvinyl alcohol (PVA, mol. Wt. 30000-70000) were received as a gift sample from Alembic Ltd. (Vadodara, India). Methanol, hydrochloric acid (HCL) and methylene chloride were procured from Qualigens (Mumbai, India).

Preparation of Microspheres:

Microspheres were prepared by emulsion solvent diffusion method. Briefly, a fixed amount (1 g) of the polymers (EC/HPMC in 1:0, 0.75:0.25) and RFB (1 g) were dissolved in 12ml of 9:3 (v/v) methylene chloride/ methanol mixture. This organic phase was added at room temperature, under constant mechanical stirring (2000 rpm) (Remi Stirrer, Mumbai), to 100 ml of 0.75 % w/v aqueous solution of PVA. Stirring was continued for 30 min. Solid microspheres were collected after filtration, washed with deionized water and dried at room temperature.

Drug Content:

A weighed quantity (10mg) of the samples was dispersed in 10 ml aqueous solution of HCL (0.01 N). It was sonicated for 10 min and centrifuged at 2000 rpm for 10 min.

Figure 1: In-vitro release profile of rifabutin from pure drug and microspheres prepared with EC and EC/HPMC.

Figure 2: SEM photomicrographs of (A) pure rifabutin and microspheres prepared with (B) EC and (C) EC/HPMC.

The supernatant was diluted with suitable quantity of 0.01N HCL and analyzed by UV-Visible Spectrophotometer (Shimadzu UV-1700, Japan) at 281nm. To increase the reliability of the results, it was performed in triplicate.

Dissolution Testing:

Dissolution studies were performed using a LABINDIA Disso 2000 (Mumbai, India) USP dissolution test apparatus type II (Paddle) at a rotating speed of 100 rpm. Samples of pure drug and RFB loaded microspheres (equivalent to 300 mg of rifabutin) were introduced in the dissolution media (0.01 N HCL). The volume and temperature of the dissolution media were 900 ml and 37 ± 0.2 ⁰C, respectively. After set time intervals the samples were collected, filtered through whatman filter papers (No. 1) and concentration of the dissolved drug was determined by an analytically validated method (r² = 0.9995) of UV- Visible Spectrophotometer (Shimadzu UV-1700, Japan) at 281 nm. To increase the reliability of the results, it was performed in triplicate.

Scanning Electron Microscopy (SEM):

Morphological characteristics of the drug and samples were analyzed using JSM-6400 scanning electron microscope (JEOL, Tokyo, Japan). Samples were fixed on aluminum stubs with conductive double sided adhesive tape and coated with gold by sputter coater at 50mA for 50s.

Figure 3: X-RD diffraction pattern of (A) pure rifabutin and microspheres prepared with (B) EC and (C) EC/HPMC.

X-Ray Powder Diffractometry:

The X-ray powder diffraction pattern of the drug and microspheres was collected using copper radiation (40 kV, 30 mA), on Philips Analytical X-RD (Model: PW 3710, Holland), in the range 5 to 60⁰ at a scanning rate of 0.02⁰/min of 2θ.

Differential Scanning Calorimetry (DSC):

Samples were heated in a hermetically sealed aluminum pans and heat runs from 30to 350 ⁰C at a heating rate of 10 ⁰C/ min, using a temperature modulated DSC (TA Instruments, USA, Model: SDT 2960). Nitrogen was employed as blanket gas.

Stability studies:

As per ICH guidelines, microspheres were passed through accelerated stability studies. Weighed quantity of the samples (each 10mg, n=3) were kept for stability studies at 40 ± 2 ⁰c and 75 ± 5% RH for a period of 3 months in environmental test chamber (HMG INDIA, Mumbai). The samples were kept in glass vials sealed with rubber plugs. After 30 and 60, 90 days, the samples were taken out and analyzed for drug content.

Moisture Uptake Study:

A weighted quantity (10 mg) of the microspheres were placed in crucible at accelerated condition of temperature and humidity, 40 ± 2 ⁰C and 75 ± 5% respectively. The gain in weight of drug and samples were determined.

RESULTS AND DISCUSSION:

Preparation of Microspheres:

Study was designed to investigate the influence of some process parameters on the physical characteristics of the microspheres (like morphology, drug content, kinetics of release). Microspheres of rifabutin were prepared using EC and HPMC by emulsion solvent diffusion. EC and HPMC were selected as polymers on the basis of low cost, low toxicity, high stability and compatibility with the drug. Microspheres were prepared at 1000, 1500 and 2000 rpm, in which 2000 rpm was found to be optimum. With increased rate of stirring, size of the microspheres was reduced (as observed under optical microscope). Organic phase (12 ml) containing drug was added to the surfactant solution in 0, 1 and 2min. At faster rate of addition (in 0 min) organic phase did not get sufficient time to diffuse in aqueous phase and results in larger size of the particles; however at slower rate of addition (in 2 min) it got sufficient time to diffuse uniformly in aqueous phase results in smaller size of the microspheres.

Drug Content:

Results of the drug content and entrapment efficiency are given in Table 1. Entrapment efficiency of the drug in polymeric microspheres prepared with EC was found to be optimum (87.64 %); however after addition of HPMC, entrapment efficiency of the drug was decreased (76.42 %). This decrease in the entrapment efficiency of the drug after addition of HPMC may be due to the diffusion of the drug in to the aqueous phase. Increased amount of HPMC (a hydrophilic polymer) may cause decreased interfacial tension between the drug and the aqueous phase, motivates its apparent solubility in aqueous phase resulting in loss of drug.

Dissolution Testing:

Dissolution studies predicted 88% of the drug release at the end of 12 hrs from microspheres prepared using EC, whereas the microspheres prepared using EC /HPMC released more than 90% of the drug at the end of 8 hrs. These results are analogous to that reported by Guyot et al.¹⁴RFB release from EC/ HPMC microspheres exhibit initial slight burst effect and the drug release was improved after 1 hr. HPMC, a cellulose ether is a hydrophilic polymer mostly used as a surfactant in the preparation of nano and microparticles.^11,12In contrast, ethyl cellulose, an ethyl ether of cellulose, is a long chain of β-anhydroglucose units joined together by acetyl linkages. Ethyl cellulose coated dosage forms can be controlled by diffusion through the film and demonstrate poor dissolution. Use of hydrophilic polymer (HPMC) with EC can create pores in its wall causing a continuous release of the drug.

SEM, XRD and DSC Studies:

Figure 2 dictates the results for SEM photomicrographs of the drug and microspheres. Rifabutin is identified with irregular shape, rough surface and large particle size as observed in Figure 2 A. In contrast, microspheres prepared with EC are spherical with smooth surface (Figure 2 B); however microspheres prepared with EC/HPMC are small, spherical with rough surface (Figure 2 C). Rough surface of the EC/HPMC microspheres may be due to the addition of HPMC in preparation.

Figure 4: DSC thermograms of (A) pure rifabutin and microspheres prepared with (B) EC and (C) EC/HPMC.

Figure 3 showed X-ray diffraction patterns of the drug and drug loaded microspheres. Pure RFB did not showed any peak; indicating its amorphous nature. Similarly the microspheres prepared with EC and EC/HPMC also did not showed any peak confirming its amorphous nature.

Thermograms of the drug and drug loaded microspheres are shown in Figure 4. In case of pure drug an endothermic peak is observed at 122.45 ⁰C, corresponding to the melting point of rifabutin. In contrast, no endothermic peak corresponding to the fusion of rifabutin was observed in thermograms of the drug loaded microspheres (EC and EC/HPMC). This disappearance of the endothermic peak may be due to the entrapment of the drug in the polymers.

Moisture uptake and Stability Studies:

Use of HPMC polymers or the preparation method may result in the products with hygroscopic nature. Moisture uptake study was performed to check the hygroscopic nature of the samples. No change in the weight of the samples was observed, denoting its non-hygroscopic nature.

The results of the accelerated stability studies indicated that EC and EC/HPMC microspheres did not show physical changes during the study period and the drug content was found to be more than 98 %. The drug content for the EC and EC/HPMC microspheres were found to be (n=3; mean ± S.D.), at 0 time, 100.00 ± 00.00 %, after 30 days: 99.56 ± 0.07 and 99.49 ± 0.06, after 60 days: 99.38 ± 0.03 and 99.26 ± 0.05, after 90 days: 98.87 ± 0.06 and 98.73 ± 0.09 respectively, indicating that the samples are stable at accelerated conditions.

CONCLUSION:

In the present study, it has been concluded that due to the addition of HPMC in the preparation of microspheres entrapment efficiency reduced, however rate of stirring and rate of addition of organic phase affect the particle size of the microspheres. As compared to the pure drug, microspheres have sustained the drug release. Microspheres are stable at accelerated conditions of temperature and humidity.

ACKNOWLEDGEMENTS:

We are thankful to Lupin Ltd, Aurangabad for providing gift sample of RFB and Alembic, Vadodara for providing excipients and Shivaji University, Kolhapur for getting facilities to perform XRD and DSC.

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Received on 23.11.2008 Modified on 15.04.2009

Research J. Pharm. and Tech.2(2): April.-June.2009,;Page 371-374

Batch	Ratio	Theoretical Drug content (%)	Drug Content (%)^*	Entrapment Efficiency (%)
RFB/EC	1:1	50	43.82 ± 1.27	87.64
RFB/EC/HPMC	1:0.75:0.25	50	38.21 ± 1.08	76.42