INTRODUCTION:

Introduction^[2,3,4]:

The human endogeneous intestinal microflora is a positive health asset that crucially influences the normal structural and functional development of mucosal immune system. In genetically susceptible individuals, some components of flora become hostile and contribute to pathogenesis of various intestinal disorders such as inflammatory bowel diseases (IBD_S) including Ulcerative colitis (UC). The manipulation of the disturbed flora to enhance the beneficial component using probiotic and concomitant treatment of inflammation using Mesalazine a golden standard anti-inflammatory agent represents a promising therapy. About two third of patients with UC have inflammatory involvement in distal left side colon hence, the topical treatment in the form of rectal gels containing the two actives is more effective in inducing faster remission and minimizing relapses^[1]. These gels due to their proximal spread to target site can act as an adjuvant to existing oral modalities of treatment of UC. The uniqueness of our work lies in the nonconventional approach of combination of Mesalazine and fourth generation probiotic cultures of Lactobacilli species for their topical treatment.

MATERIAL AND METHODS:

Materials:

Mesalazine as gift sample generously provided by Wallace Pharmaceutical Pvt. Ltd., Ponda, Goa. Pure Lactobacilli culture of L. plantarum (NCIM 2083) was procured from National Collection of Industrial Microorganisms (NCIM), Pune. Bacteriological media, polymers such as Carbopols, polyethylene glycol and synthetic membrane were procured from reputed local vendors. All the solvents and reagents used were of high purity and quality and the formula with active drug and Excipient in different concentrations is as per table No. 1

Table No. 1Formulation of probiotic gel of L. plantarum containing Mesalazine with Carbopol 940 and PEG 200/400/600.

Sr. no.	Ingredient	Weight in gm
1	Mesalazine	1
2	L. plantarum	0.01
3	Carbopol 940	1
4	PEG 200/400/600	24.20/22.40/21.60
5	Propylene glycol	15.52
6	Triethanolamine	q.s. for pH adjustment
7	Distilled water	q.s. to 100 gm

Method:

Preparation of lyophilized powders of Lactobacilli cultures. The authentic bacterial cultures were harvested on selective nutrient media (MRS broth) using laboratory shaker and employing ambient environmental conditions. The harvested cells were lyophilized using a suitable polysaccharide carrier. The free flowing powders so obtained were characterized for viable cell count by Standard Plate Count (SPC) method. Also their susceptibility/compatibility to the anti-inflammatory drug Mesalazine was assessed by reading zone of inhibition in comparison with standard antibiotic Ampicillin.

Preparation of gels:

The gels were prepared in three stages. The first step involved solubilization of Mesalazine using propylene glycol and polyethylene glycol (PEG 200,400 and 600 seperately). In the second stage, the Carbopol 940 gel bases were prepared individually. The solubilized Mesalazine was then dispersed in the individual Carbopol bases. In third stage lyophilized powders of probiotic cultures were incorporated in to the medicated gels.

Evaluation of formulation^[6,7,8,10]:

Appearance of gels:

All the gel formulations were observed for homogeneity, color, texture, and general appearance.

Determination of pH values of gels:

The pH of specified quantity of each gel was measured using a pH meter. The pH meter was calibrated before each use with standard pH 4, 7 and 10 buffer solutions.

Determination of Spreadability of gels:

Spreadability of gel formulations was done to determine spreading nature of gel formulations. Gel formulations must have good spreading nature to enhance the absorption of gels and also good flowing ability of gel through rectal route. This apparatus was fabricated in our laboratory. The apparatus consisted of two glass slides (7.5 × 2.5 cm), one of which was fixed onto the wooden board and the other was movable, tied to a thread which passed over a pully, carrying a weight.

Rheological study of gels:

Rheological study was done to determine the viscosity of gel formulations. Since, as viscosity increases the drug release from the gels decreases and hence the absorption in colon also decreases.

Drug content:

The quantity of gel equivalent to 10 mg of Mesalazine was dissolved in 100 ml methanol. From this solution, 1 ml samples were withdrawn and diluted to 10 ml with phosphate buffer pH 6.8. The samples were analyzed spectrophotometricaly at a wavelength of 331.0 nm. The concentration of Mesalazine in each sample was determined from a previously prepared standard curve.

Viable bacterial cell count in experimental gel formulations:

Total viable count of bacterial cells was counted to determine the expected number of viable probiotic microorganisms in the prepared probiotic gel and probiotic gels with Mesalazine formulations to deliver the expected number of viable bacterial cells in the colon. For this the SPC method was used to count the viable number of bacterial cells.1 gm gel was dissolved in sterile distilled water and serial dilutions were prepared. From this 0.1ml solution is suspended on MRS broth petriplates and incubated for 24 hr. Also the sterile petriplates of MRS medium were inoculated aseptically with 1ml of diffusion media at the end in-vitro diffusion study. The plates were incubated in glass chamber for 24 hours and the colonies so grown were counted using Quebec colony counter.

In- vitro diffusion study:

These studies were carried out using both healthy sheep rectal membrane and synthetic cellophane membrane mounted in the Keshary- Chien diffusion cell containing 25 ml phosphate buffer (pH 6.8 and pH 7.2) in recipient compartment. Amount of Mesalazine diffused from different gels was estimated at 331nm and 331.5nm respectively for both pH over period of 7 hours.

RESULTS AND DISCUSSION:

Viable bacterial cell count of probiotic cultures of Lactobacillus in gel formulations:

Total viable count of different Lactobacillus culture cells in all the gel formulations given in table No. 2 and was almost same (10⁹-10¹⁰). This range is requisite for delivery in the target site.

Table No2 Data for Viable count of Lactobacillus plantarum in experimental formulations

Sr. no.	Formulation	Viable cell count
1	940 PEG 200	3×10⁹
2	940 PEG 400	1×10⁹
3	940 PEG 600	1×10⁹

Total viable count of Lactobacillus culture cells in each gel formulation was found in expected number, which requires giving expected therapeutic action.

Table No. 3 Average cumulative % drug diffusion of Mesalazine after 7 hr. through cellophane membrane in 6.8 pH phosphate buffer.

Sr. No.	Time in hour	Avg. cumulative % drug diffusion
Sr. No.	Time in hour	M1	M2	M3
1	0.5	19.571±2.54	18.038±1.65	17.693±1.83
2	1	27.526±3.24	24.194±2.45	23.409±2.07
3	2	38.508±1.25	35.705±0.97	32.802±1.69
4	3	46.299±0.98	41.813±2.09	39.246±2.04
5	4	56.879±0.45	52.145±2.41	45.883±3.01
6	5	64.520±0.63	60.176±1.87	49.597±3.11
7	6	76.680±2.45	67.297±1.52	56.497±2.21
8	7	85.665±2.14	72.176±1.63	59.424±2.13

In-vitro drug diffusion study:

These studies were conducted using diffusion media, (phosphate buffer pH 6.8) and two different membranes synthetic(cellophane) and natural (sheep colonic mucosal membrane).Table No. 3 reports the findings for diffusion from gel formulations prepared with carbopol 940 and PEG 200,400 and 600. The same are presented in Graph No. 1

Graph No.1 Graph showing avg. cumulative % drug diffusion of formulation M1, M2 and M3.

Hence by considering all evaluation parameters and in-vitro diffusion study the formulations of carbopol 940 as a base and PEG 200 as cosolvent is selected as best formulation.

Hence carbopol 940 and PEG 200 used for formulations with other Lactobacillus strains.

SUMMARY AND CONCLUSION:

Inflammatory bowel diseases or IBD_Sis a collective term encompassing related but distinct, chronic inflammatory disorders of the gastrointestinal tract such as Crohn’s disease, ulcerative colitis, microscopic colitis and collageneous colitis. The Crohn’s disease and ulcerative colitis are the most common. Presently there is no medication available that can cure ulcerative colitis and the patients typically experience periods of relapse followed by period of remission, which may last for months or even for years. Thus the goals of treatment are to induce early remission, maintain these remissions and minimize side effects of treatment and improve the quality of life. The literature review suggested that the medications used for treating ulcerative colitis are mainly anti-inflammatory agents such as 5-amino salicylic acid (5-ASA). Mesalazine which contains 5-ASA has been considered the golden standard drug for treatment of ulcerative colitis. Similarly many reports are available justifying the use of probiotic cultures for the treatment of IBD_S including ulcerative colitis. These probiotic cultures especially those of species Lactobacillus are thought to beneficially affect the host by correcting the disturbed balance between the residential gut flora and pathogenic bacteria causing infection. Hence, in the present work, a novel combination of the drug Mesalazine and the Lactobacillus species (L. plantarum) was employed in the formulation of colonic gel, which is meant for local and/or systemic action in the left-side colitis treatment. The in vitro evaluation of these probiotic gel formulations showed satisfactory gel characteristics including viscosity, spreadability, drug content uniformity and diffusion of drug Mesalazine in a sustained manner (over a period of 7 hours) through both artificial as well as natural mucous membrane (sheep colonic mucosa). Also, the viable count of lyophilized cultures of Lactobacillus species was requisite when combined with drug Mesalazine.^[1,5,9]

REFERENCE:

1. Gionchetti, P., Ranzi, T., Trallori, G., Valpiani, D., Campieri, M., 1999. A new Mesalazine gel enema in the treatment of left-sided ulcerative colitis: A randomized controlled multicentre trial. Aliment Pharmacol. Ther.13, 381-388.

2. Shanahan, F., 2004. Probiotics in inflammatory bowel disease-therapeutic rationale and role. Advanced Drug Delivery Reviews 56, 809-818.

3. Reid, G., Jass, J., Sebulsky, M. T., McCormick, J. K., 2003. Potential uses of prbiotics in clinical practice. Clinical Microbiology Reviews. 658-672.

4. Ardizzone S., Porro G. B., 1998. A Practical guide to the management of distal ulcerative colitis. Drugs, 55, 519-542(24).

5. Taumola, E., Playne, M., Salminen, S., 2001.Quality assurance criteria for probiotic bacteria. Am J Clin Nutr. 73, 393S-398S.

6. Stadler, M., Viernstein, H., 2003. Optimization of a formulation containing viable lactic acid bacteria. Int. J.Pharm. 256,117-122.

7. Jon A Vanderhoof, Probiotic: future direction, 2001. American journal of clinical neutrition, 73, No. 6, 1152-1155.

8. Huis, H. J., 1998. Selection criteria for application of probiotic microorganisms in man and animal. Indian Drugs .35, 602-603.

9. Gaskins, H. R., Deplancke, B., 2001.Microbial modulation of innate defense: goblet cells and the intestinal mucus layer. Am J Clin Nutr.73, 1131S–41S.

10. Elda, H., 2001. Lactobacillus plantarum 299 v reduces irritable bowel bloating Am. J. Cli. Nutri. 73: 380S-385S

Received on 27.12.2011 Modified on 14.01.2012

Research J. Pharm. and Tech. 5(3): March 2012; Page 387-389