Formulation and In-Vitro Evaluation of Ethyl Cellulose Microspheres by Novel Quasiemulsification Method for Colonic Delivery of Metronidazole

Prasanta Kumar Choudhury¹*, Padala Narasimha Murthy¹, Suvakanta Dash², Rana Mazumder³ and Dhruba Sankar Goswami⁴

¹Department of Pharmaceutical Technology, Royal College of Pharmacy and Health Sciences, Andhapasara Road, Berhampur-760002, Ganjam, Orissa, India.

²Girjananda Choudhury Institute of Pharmaceutical Sciences, Guwahati, Assam, India

³Dept. of Pharmaceutics, Calcutta Institute of Pharmaceutical Technology and Allied Health Sciences, Howrah-711316, West Bengal, India.

⁴Dept. of Pharmaceutics, S.D. College of Pharmacy, Barnala, Punjab, India

ABSTRACT:

The present study was aimed to develop and evaluate different polymeric microspheres for colon-specific delivery of Metronidazole for better treatment of colonic diseases. Different approaches for colon-specific drug delivery have been studied over the last decade, including pro-drugs, polymeric coating using pH sensitive or bacterial degradable polymers and matrices. In this research work we present colon-specific ethyl cellulose microspheres to deliver active molecules to the colonic region, which combine pH-dependent and controlled release properties. Microspheres were prepared by modified Novel Quasiemulsification solvent-diffusion method to study the effect of ethyl cellulose on drug release with different proportions of metronidazole and ethyl cellulose. Prepared microspheres of ethyl cellulose were evaluated for size, morphology, sphericity study, percentage yield, loose surface crystal study, drug content and entrapment efficiency. In vitro drug release study was conducted by buffer change method to mimic Gastro Intestinal environment. The investigations revealed that microspheres prepared with metronidazole: ethyl cellulose ratio (1:2) show only 19.394 ±0.67% drug release in first 5 hours and 46.72 ±0.69% in 12 hours, which prove the potentiality of ethyl cellulose for colonic delivery of drugs.

KEYWORDS: Colon drug delivery; Controlled-release; pH-dependent release, Novel Quasiemulsification Solvent-diffusion technique.

INTRODUCTION:

Site specific drug delivery to colon has a number of important implications in the field of pharmacotherapy. These include the topical treatment of colonic disorders, such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). Moreover, a new perspective for the oral delivery of peptides, proteins and other labile drugs into the lower gastrointestinal tract (GIT) has been developed since it has been demonstrated that the colon has a less adverse enzymatic activity than the other regions of GIT. A colonic delivery system could be of additional value when a delay in systemic absorption is therapeutically desirable, as occurs, for example, in disorders that are affected by circadian rhythms (asthma or arthritis). Therefore, the specific colonic delivery of drugs would allow either the reduction of the total administered dose to the patient, decreasing their possible adverse effects, or

the improvement of the oral bioavailability of some molecules. Over the last few years, different approaches have been reported in order to achieve specific colonic drug delivery which includes coating with pH-sensitive polymers, coating with biodegradable polymers, fabrication of pro-drugs, timed-release systems and embedding in biodegradable matrices and hydrogels¹.

Drugs, which are used for the treatment of diseases associated with the colon, require the passage of the formulation in an intact form through the stomach and small intestine and the release of the whole amount of drug into the colon. The conventional dosage form normally dissolves in the stomach or small intestine and gets absorbed from these regions of the GIT; thus, very less amount of the drug reaches the colon. To obtain maximum therapeutic efficacy, it becomes necessary to deliver the agent to the target site in the optimal amount for a right period of time, thereby causing little toxicity and minimal side effects. Attempts have been made to achieve colon specific drug delivery using polysaccharides, pH sensitive polymers; Wakerly et al. (1997) have investigated pectin/ethyl cellulose film coated tablets where as Rama Prasad et al. (1998) have proposed matrix tablets of guar gum for colon-specific drug delivery.

Colonic diseases are important causes of death by protozoal infections in the developing world and even in advanced countries. Hence, in the present study metronidazole (MNZ) was selected as a model drug which has extremely broad spectrum of protozoal and antimicrobial activity. It is clinically effective in colonic diseases, both locally and systemically². Based on the observations, an attempt has been made to develop ethyl cellulose microspheres for metronidazole by modified Novel Quasiemulsification solvent-diffusion method to deliver active molecule to the colonic region, which combines pH-dependent and controlled drug release properties.

MATERIALS AND METHODS:

M/s Diamond Drugs Pvt Ltd, Howrah, WB, India, generously supplied metronidazole as a gift sample. Ethylcellulose-LR (EC-LR), Span 80 and Tween 80 were procured from S.D. fine-chem. Ltd., Mumbai, India. Light liquid paraffin was obtained from Loba Chemie Ltd, Mumbai. All other solvents and reagents were of analytical grade procured from local suppliers.

PREPARATION OF ETHYL CELLULOSE MICROSPHERES:

Ethyl cellulose (EC) microspheres were prepared by Novel Quasiemulsification solvent-diffusion method³. A solution of EC in ethanol (2%, 4% and 8%) containing drug (1g) was added to liquid paraffin containing emulgent (Span 80), while stirring, at a speed of 1500 rpm. The emulsion was stirred for 5 to 6 hours at 25°C to 30°C. Subsequently, a suitable amount of petroleum ether was added to the dispersion. The microspheres suspended in liquid paraffin were filtered and collected. The resultant microspheres were washed with water followed by petroleum ether to remove traces of liquid paraffin. The microspheres were dried at ambient temperature and desiccated under vacuum. The batch was coded as ECQ. Many batches were prepared using these methods, but only those batches and conditions that led to the batches having approximately the same mean geometric diameter are given here.

IN VITRO CHARACTERIZATION OF MICROSPHERES:

Drug-Polymer Interaction Study by FTIR Analysis:

To eliminate the possibility of polymers interfering with the analysis of drug, Infra-red spectrum was taken by using the FTIR model Shimdzu-840-os, Japan by scanning the sample in potassium bromide (KBr) discs. Before taking the spectrum of the sample, a blank spectrum of air background was taken. The sample of pure drug, pure polymer and the formulations containing both the drug and polymer were scanned

Determination of the Yield of the Microspheres:

The yield was calculated using the equation:

Yield of microspheres (%) =

Weight of the microspheres (mg) X 100------ Equation 1 [Drug (mg) + Polymer (mg)]

Particle Size Analysis:

Particle size distribution of the microspheres was determined by optical microscopy using calibrated ocular eyepiece¹¹. Fifty microspheres were observed and the geometric mean diameter was calculated using the equation:

X_g= 10 X [(n_i X log X_i) / N] ----------------------Equation 2

Where Xgis geometric mean diameter, n_iis no of particles in the range, X_iis the midpoint of range, and N is total no of particles analyzed.

Determination of Shape and Sphericity:

Morphological appearance and surface characteristics of the microspheres were studied by dispersing the microspheres in liquid paraffin and observing under 100X magnification in an optical microscope¹¹.

The particle shape was measured by computing circularity factor (S). The tracings obtained from optical microscopy were used to calculate area (A) and perimeter (P), which are used to calculate the circularity factor (S) by using the equation¹²:

S = P² / (12.56 X A) --------------------------------Equation 3

Determination of drug content:¹³

The amount of drug present in the Ethyl cellulose microspheres prepared by quasiemulsion method was determined by a method reported by Thanoo et al (1992). A weighed quantity of the microspheres was extracted with methanol for 24 hr, and drug concentration in supernatant was determined spectrophotometrically at 313.5 nm (UV 1700 Shimadzu, Japan).

Drug Entrapment efficiency: (DEE %)

Entrapment efficiency of the microspheres was calculated using the formula

DEE % = Practical Drug Loading X 100 ----Equation 4

Theoretical Drug Loading

Loose surface crystal study:¹⁴

Loose surface crystal study was performed to observe the excess drug present on the surface of microspheres. From each batch 100mg of microspheres were shaken in 100 ml of phosphate buffer, pH 7.4 for 5 minutes and then filtered through Whattman filter paper 41. The amount of drug in the filtrate was determined spectrophotometrically at 319 nm and calculated as percent of total drug content. This estimates the surface entrapment of the drug by the microspheres.

In Vitro Drug Release from Microspheres:

In vitro drug release studies were carried out using USP dissolution rate test apparatus (basket apparatus, 100 rpm, 37 ± 0.1°C) by buffer change technique¹⁵. Microspheres bearing MNZ were suspended in simulated gastric fluid (SGF), pH 1.2 (500 ml), for 1 hr. The dissolution media was then replaced with mixture of simulated gastric fluid and simulated intestinal fluid (SIF), pH 4.5 (500 ml) for next two hours, then for next two hours simulated intestinal fluid (SIF), pH 6.8 (500 ml) and the release study was continued further in simulated intestinal fluid (500 ml) pH 7.4.

Samples were withdrawn periodically and compensated with an equal amount of fresh dissolution media. The samples were analyzed for drug content by measuring absorbance at 319.5 nm using UV spectrophotometer (UV 1700, Shimadzu, Japan).

Drug Release data model fitting:

The suitability of several equations that are reported in the literature to identify the mechanisms for the release of drug was tested with respect to the release data up to the first 50% drug release. The data were evaluated according to the following equations:

Zero order model¹⁶

Mt = M₀+ K₀t-----------------------------------------Equation 5

Higuchi model¹⁷

Mt = M₀ +K_H t ^0.5-------------------------------------Equation 6

Korsmeyer-Peppas model¹⁸

Mt = M₀ + K_Ktⁿ---------------------------------------Equation 7

Where Mt is the amount of drug dissolved in time t. M₀ is the initial amount of the drug. K₀ is the Zero order release constant, K_H is the Higuchi rate constant, K_K is a release constant and n is the release exponent that characterizes the mechanism of drug release.

RESULTS AND DISCUSSION:

Microspheres of ethyl cellulose loaded with Metronidazole (MNZ) were successfully prepared by the Novel Quasiemulsification solvent-diffusion method using light liquid paraffin in the external phase. The effect of drug polymer ratios was analyzed in order to optimize the formulation. It was observed that by changing drug: polymer ratio the shape, size as well as the entrapment efficiency of formulations considerably influenced. The yield of microencapsulation process was increased with increase in ethyl cellulose concentration in the formulations. The microspheres were discrete and fairly spherical in shape while the surface roughness was slightly increased with the incorporation of the drug. Excellent microspheres were produced when the process was carried out with drug: ethyl cellulose ratio 1:2 while the shape of the microspheres was distorted and some of them fused with each other when ethyl cellulose ratio was decreased. The drug particles appeared on the surface of the microspheres when they were prepared with drug: polymer ratio 2:1.

Table 1 formulation composition of ethyl cellulose microspheres

Batch code.	Amount of drug (mg)	Amount of polymer(mg)	Drug: polymer ratio	Quantity of ethanol(ml)	Quantity of liquid paraffin (ml)
ECQ1	1000	500	2:1	25	50
ECQ2	1000	1000	1:1	25	50
ECQ3	1000	2000	1:2	25	50
ECQ 4	1000	3000	1:3	25	50
ECQ 5	1000	4000	1:4	25	50
ECQ 6	1000	5000	1:5	25	50

Table 2 Evaluation parameters of ethyl cellulose microspheres

Batch code	Drug : polymer ratio	Yield (%)	Particle size (µm)	Circularity factor (S)	Loose surface crystal study (surface entrapment)	Entrapment efficiency (%)
ECQ1	2:1	72.33 ± 3.74	21.76 ± 3.33	1.06 ± 0.030	23.044 ± 3.19	79.02±4.88
ECQ2	1:1	95.11± 2.48	25.84 ± 1.43	1.05 ± 0.005	19.118 ± 3.78	86.96±2.49
ECQ 3	1:2	99.29 ± 4.71	28.36 ± 2.00	1.06 ±0.025	16.714 ± 4.22	98.796±4.68
ECQ 4	1:3	97.00 ± 3.27	30.2 ± 2.30	1.13 ± 0.018	32.347 ± 4.10	79.02±6.05
ECQ 5	1:4	81.00 ± 2.41	32.3 ± 2.43	1.10 ± 0.011	24.236 ± 4.08	73.35±5.34
ECQ 6	1:5	78.08 ± 3.24	34.8 ± 2.45	1.08 ± 0.026	29.442 ± 4.01	98.47±4.89

Values are expressed as Mean average ± SD (n=3)

TABLE-3: Dissolution kinetics and the model fittings (R and K values) for all the formulations.

Formulation Code	t50 ( hr )	Zero order		Higuchi square root		Korsmeyer-Peppas
Formulation Code	t50 ( hr )	K₀	R²	K_H	R²	K_K	R²	n value
ECQ1	9.72	12.973	0.9874	29.820	0.9792	5.255	0.9840	0.9139
ECQ2	12.5	9.348	0.9917	23.909	0.8892	22.6416	0.9854	0.9959
ECQ3	14.17	8.925	0.9828	21.251	0.9185	14.161	0.9693	0.7905
ECQ4	14.19	11.633	0.9941	29.890	0.8910	5.558	0.9964	1.0966
ECQ5	14.83	9.126	0.9908	30.862	0.9668	5.340	0.9912	1.1071
ECQ6	15.64	9.213	0.9834	30.991	0.8547	5.445	0.9920	1.3136

K₀–Zero Order rate constant, K_H - Rate constant Higuchi Model, K_K - Rate constant Peppas Model, R2 – Correlation coefficient.

Particle size of the microspheres was determined using optical microscopic method. Mean particle size was found to be 21.76 ± 3.33µm in case of microspheres having drug: ethyl cellulose ratio 2:1 while it was significantly increased to 34.8±2.45µm with drug: ethyl cellulose ratio (1:5) (Table 1). The size of the microspheres is controlled by the size of the dispersed droplets of Ethyl cellulose in liquid paraffin. When the concentration of the ethyl cellulose in the formulation was increased, there was increment in the size of dispersed droplets that resulted in the formation of microspheres having bigger particle size. With increase in the polymer ratio in the formulations the mean particle size of all the formulations increased as shown in Table 1. All the microsphere formulations have the circularity factor nearest to “1” which proves that they are almost spherical in shape (Table 1).

The microspheres showed better entrapment efficiency with increase in polymer ratio. Highest entrapment efficiency was observed for the formulation ECQ3 99.29±4.71 (Table 1). As the drug: polymer ratio was increased from 2:1 to 1:2 the surface entrapment of the drug on the microsphere surfaces was decreased which is suitable for the colonic delivery of the drugs and the surface entrapment of drug shows a less amount of drug lose due to the process variables but with further increase in polymer ratio the surface entrapment was increased. It may be due to the dispersion of the drug in polymer layer more evenly rather than entrapped into to the polymer layer.

The microspheres were subjected to in-vitro drug release rate studies in SGF (pH 1.2) for 1 hour and in mixture of SGF and SIF (pH 4.5) for the next 2 hours in order to investigate the capability of the formulation to withstand the physiological environment of the stomach and small intestine. The MNZ percent released from the microspheres of drug: ethyl cellulose ratio 1:2 after 12 h studies is 46.72 %. The amount of MNZ released during first 5 h studies was found to be 29.993 ± 1.22 %, 16.342 ± 1.13 %, 19.790 ± 0.48 %, 14.495 ± 0.77 %, 19.394 ± 0.67 % and 10.003 ± 0.88 % for ECQ1, ECQ2, ECQ3, ECQ4, ECQ5, and ECQ6 respectively (Figure. 6) which attests the ability of ethyl cellulose to remain intact in the physiological environment of stomach and small intestine. The little amount of the drug, which is released during 5 h release rate studies, is due to the presence of un-entrapped drug on the surface of the microspheres. The release of the drug was much faster during the 6-12 hour study period. It is due to the fact that during the initial period (0-5 h) the strength of the barrier was too high to be broken and during 6-12 hour period the network was somewhat loosened which facilitated the release of drug (Figure 6.). Basing on all the evaluation parameters studied, like %yield, particle size, sphericity, surface entrapment, entrapment efficiency and in vitro drug release the formulation ECQ3 was found to be the ideal formulation.

CONCLUSIONS:

The efficacy of the ethyl cellulose was evaluated for colon targeted drug delivery by fabricating it into microspheres. The microspheres of ethyl cellulose prepared by modified quasiemulsion method were capable of providing protection to the drug in the hostile environment of upper gastrointestinal tract and released the drug at the target site. The in vitro drug release studies of ethyl cellulose microspheres revealed that very less amount of the drug was released in the physiological environment of stomach and small intestine. Hence these data attests the potentiality of ethyl cellulose for colon-specific delivery of the drugs.

ACKNOWLEDGEMENTS:

We express our sincere thanks to Royal College of Pharmacy and Health Sciences, Berhampur, Orissa for their support in providing the facilities to complete the work. We also extend our thanks to M/s Diamond drugs Pvt Ltd, Howrah, W.B. for providing us gift samples of metronidazole.

REFERENCES:

1. Rubinstein A, Approaches and opportunities in colon-specific drug delivery. Crit Rev Ther Drug Carrier Syst. 1995; 12(2-3): 101-49.

2. Chourasia MK and. Jain SK. Design and Development of Multiparticulate System for Targeted Drug Delivery to Colon, Drug Delivery. 2004; (11):201–7

3. Ranga Rao KV, Devi KP. Swelling controlled release systems: recent developments and application. Int J Pharm. 1988; 48:1-16.

4. Chourasia MK, Jain SK. Pharmaceutical appraoaches to colon targeted drug delivery systems. J Pharm Pharmaceut Sci (www.ualberta.ca/~csps) 2003; 6(1): 33-66

5. Ashford M, Fell JT, Attwood D, Sharma H and Woodhead P.J. An in vivo investigation into the suitability of pH-dependent polymers for colonic targeting. Int J Pharm, 1993; 95: 193-9

6. Mishra B, et al. Development of chitosan-alginate microcapsules for colon specific delivery of metronidazole. Indian Drugs, 2003; 40 (12): 695-700.

7. Chourasia MK and Jain SK. Potential of Guar Gum Microspheres for Target Specific Drug Release to Colon. Journal of Drug Targeting. August 2004; 12 (7): 435–442

8. Gohel MC, Parikh RK, Amin AF, Surati AK. Preparation and Formulation Optimization of Sugar Cross linked gelatin Microspheres of Diclofenac Sodium. Indian J. Pharm Sci., 2005; 67(5): 575-81

9. Chowdary KPR, Rao YS. Preparation and evaluation of mucoadhesive microspheres of Indomethacin. Indian J. Pharm Sci. 2003; 65(1): 49-52

10. Kawashima et al. Preparation and in vitro characterization of Eudragit RL100 microsphere containing 5 –fluorouracil. Indian J. Pharm Sci. 2003; 60(2): 107-9

11. Chourasia MK, Jain SK. Design and Development of Multiparticulate System for Targeted Drug Delivery to Colon. Drug Delivery. 2004; 11: 201–7

12. Shabaraya AR, Narayanacharyulu R. Design and evaluation of chitosan microspheres of metoprolol tartarate for sustained release. Indian J. Pharm Sci. 2003; 65(3): 250-2

13. Bhumkar DR, Maheswari M, Patil VB and Pokharkar VB. Studies on effect of variabilities by response surface methodology for Naproxen microspheres. Indian drugs. 2003; 40 (8): 455-461

14. Mishra B, Jayanta B, Sankar C. Development of chitosan-alginate microcapsules for colon specific delivery of Metronidazole. Indian drugs. 2003; 40(12): 695-700

15. Abu-Izza K, Garcia LC and Robert D. Preparation and evaluation of zidovudine loaded sustained release microspheres: optimization of multiple response variables. J Pharm. Science. 1996; 85(6): 572-4

16. Danbrow M, Samuelov Y, Zero order drug delivery from double –layered porous films: release rate profiles from ethyl cellulose, hydroxypropyl cellulose and polyethylene glycol mixtures, Journal of Pharmacy and pharmacology. 1980; 32: 463-470.

17. Higuchi T, Mechanism of sustained-action medication: theoretical analysis of rate of release of solid drugs dispersed in solid matrices. Journal of pharmaceutical sciences. 1963; 52:1145-1149

18. Korsmeyer RW, Gurny R, Doelker EM, Buri P, Peppas NA. Mechanism of solute release from porous hydrophilic polymers. International Journal of Pharmacy. 1983; 15: 25-35.

Received on 29.05.2010

Accepted on 12.06.2010

Research Journal of Pharmaceutical Dosage Forms and Technology. 2(4): July-August 2010, 206-214