Formulation and Evaluation of Microspheres as an effective Colon Targeting Drug Delivery System

Alka Singh¹*, Shalu Verma², Tarun Parashar², Kiran Dobhal², Gauree Kukreti¹

¹School of Pharmaceutical Sciences and Technology, Sardar Bhagwan Singh University,

Dehradun, Uttarakhand - 248161, India.

²Uttaranchal Institute of Pharmaceutical Sciences, Uttaranchal University,

Dehradun, Uttarakhand - 248007, India.

*Corresponding Author E-mail: alkasingh1790@gmail.com

ABSTRACT:

The present work comprised of the preparation of coated HPMC capsules of meloxicam microspheres in order to target the drug release in colon resulting in increased absorption and subsequent bioavailability. The meloxicam microspheres were developed in nineteen different batches using HPMC, combination of EC with HPMC, Eudragits (S100, RS100 and E100) in different drug and polymer ratios by solvent evaporation method. The formulation of different batches were examined by various studies i.e. percentage yield, surface morphology (SEM), particle size analysis, entrapment efficiency, drug compatibility with polymers using FTIR and in-vitro drug release determinations. The complex of meloxicam with β-cyclodextrin increased the solubility of the drug accompanying its in-vitro release. The microspheres filled HPMC capsules were coated with eudragit S100 which proved to be an effective method for drug targeting to the colon. A 2² factorial design showed that a very significant increase in release of the drug using polymers i.e. HPMC, E.C with HPMC, Eudragit S100, Eudragit RS100, Eudragit E100 could be obtained by the exclusive manipulation of two variables i.e. surfactant and polymer concentrations. The drug loaded microspheres showed percentage drug entrapment as 35.09-62.50% in A₁-A₄, 76.29-87.78% in B₁-B₄, 81.36-92.70% in S₁-S₄, 65.47-69.4% in RS₁-RS₄ and 68.91-78.08% in E₁-E₃. The pH 7.4 phosphate buffer and simulated colonic fluid were used for the in-vitro release tests. The best drug release profiles were seen with formulations A₃, B₂, RS₁, S₁ and E₂ (containing different ratios of drug: polymer) coated with 15% (w/v) Eudragit S-100 solution.

KEYWORDS: Meloxicam, Microspheres, Colon, Target delivery.

INTRODUCTION:

By preventing drug release and absorption in the stomach and small intestine of target drug delivery, as well as by preventing the bioactive agent from degrading in either of the dissolution sites, the colon specific drug delivery system (CDDS) is able to protect the drug being delivered to the colon.¹ Oral absorption of drugs poses a serious limitation in the gastrointestinal tract (GIT), whereas CDDS is necessary to be protected from the hostile environment of the upper GIT.²

Colon disorders(ulcerative colitis, Chron's disease, carcinomas, and various infections) are cured by oral drug administration because it achieve high local concentration and reduced adverse drug reactions or unneeded systemic absorption.^3,4,5

Due to the colon's abundance of lymphoid tissue and the quick local generation of antibodies triggered by the uptake of antigens by colonic mucosal mast cells, vaccine administration is made more effective.^6,7Peptide and protein drugs are suitable for colon absorption due to reduction of digestive enzyme diversity.^8,9

MATERIALS AND METHODS:

Meloxicam (M), Ehyl cellulose (EC, Fine Chem. Labs. Mumbai), HPMC K4M (Signet chemical corporation), Eudragit S 100(Evonik Pharma), Eudragit RS 100(Purchased by local supplier), Eudragit E 100 (Purchased by local supplier), Acetone (Rankem), Methanol (Rankem), Iso propyl alcohol (IPA, Rankem), Talc (Rankem), HPMC capsules (Gift sample by Pfizer), Iso propyl alcohol (Rankem), Benzyl alcohol (CDH), Dichloromethane (Rankem), Tween – 80(Rankem), β-cyclodextrin (BC, Hi Media Laboratories Pvt. Ltd).

Table 1: Quantitative formulation design of different batches

Sr. No

Batch

Ingredients

RS1

RS2

RS3

RS4

M and BC

1:1

Lyophilized M and BC

1:2

HPMC

2gm

3gm

EC and HPMC

1.5:0.5

Eudragit RS 100

200mg

400mg

Tween 80

0.2ml

0.4ml

0.2ml

0.4ml

0.2ml

0.4ml

0.2ml

0.4ml

0.2ml

0.4ml

0.2ml

0.4ml

Acetone

10ml

Dichloro methane

20ml

Ethanol

20ml

10ml

10.

Benzyl alcohol

0.4ml

11.

Petroleum ether

50ml

Determination of λmax of the drug: Prepared stock solution of 100μg/ml solution of meloxicam. Different dilutions were scanned between 200-400nm using Systronics 2205 double beam UV/visible spectrophotometer.^10,11

Preparation of standard calibration curve:

Prepared stock solution of 10μg/ml solution of meloxicam in pH 7.4 phosphate buffer. Absorbance of different dilution were recorded at 364nm and obtain standard calibration curve.^12,13The same procedure was repeated to obtain standard calibration curve of Meloxicam in Simulated Colonic Fluid.

Fourier Transformation Infra-red Spectroscopy (FT-IR)¹³:

FTIR spectrophotometer was used to conduct the drug-polymer compatibility investigation. Lyophilized meloxicam and cyclodextrin-meloxicam complex were prepared and Multiple regression analysis for 2² factorial designs.

Multiple regression analysis for 2² factorial design¹⁴:

In a 2^k factorial design, each factor was assigned with two levels i.e. low (-1) and high (+1). The responses obtained from 2^k factorial design studies were subjected to multiple regression analysis. The polynomial equations were determined using the form:

Y = b₀ + b₁X₁ + b₂X₂

Where Y = the dependent variables, eg: % entrapment efficiency (Y1), release at pH 7.4 phosphate buffer (Y2), release at simulated colonic fluid (Y3). The simplified model was then used to create three dimensional response surface and contour plots to investigate the effect of independent variables. The factorial design was subjected to different formulations using Design expert 8.0 (DX8 Trail).

Preparation of microspheres:

A) Microspheres of HPMC:

The drug complex of BC and polymer (1:1) was dissolved in 40ml of solvent (1:1 ratio of dichloromethane and ethanol). Add this mixture in to light liquid paraffin having tween 80 as surfactant and rotate at 1500rpm in propeller. This procedure were followed similar for all batches.¹⁵

B) Microspheres of EC and HPMC:

Mix EC and HPMC in the ratio of 1.5:0.5. BC complex was dissolved in 40ml of at the above solvent.

C) Microspheres of Eudragit RS100:

The drug complex of BC and Eudragit RS 100 polymer (1:1) were dissolved in 20ml of solvent (Acetone and ethanol, 1:1) using ultra sonicator for 1 hour.

D) Microspheres of Eudragit S100:

The drug complex of BC and eudragit S 100 polymer was dissolved in 20ml solvent (Isopropyl alcohol and acetone, 1:1) using ultra sonicator for 2 hour.

E) Microspheres of Eudragit E100:

The drug complex of beta-cyclodextrin and eudragit E100 polymer was dissolved in 20ml of solvent (acetone and ethanol, 1:1) using ultra sonicator for 1.5 hour.

Coating of capsules:

HPMC capsules were coated using eudragit S100 i.e. PEG 4000 and eudragit S100 in purified water and isopropyl alcohol respectively. A polymer solution of 15% w/v Eudragit S100 in IPA and 20% w/v aqueous solution of PEG 4000 were formulated for use in final coating solution. The final coating solution contained 74.4ml of eudragit S100 (15% w/v) and 9.2ml of 20% (w/v) of PEG 4000 added with 16.4ml of IPA to produce a total volume of 100ml. The capsules were incorporated in tablet coating machine for coating. The coating material was sprayed over the capsule shell with a flow rate of 0.5ml/sec.¹⁵

Percentage Yield and Entrapment Analysis:

U.V. Spectrophotometer was used for the drug entrapment studies. A dilution of microspheres of was compared to standard absorbance to determine the absorbance of the filtered content.^16,17,18

Drug encapsulation efficiency (%)= Practical drug Absorbance/ Theoritical drug Absorbance x 100

Particle size analysis and Surface morphology:

For each batch of microspheres, the average diameter and particle size dispersion were calculated. SEM, Model Quanta FEI 200F, was used to analyses the surface morphology and particle size of meloxicam microspheres through A photomicroscope (QUASMO, Quality Scientific and Ambala).

Coating Durability Test:

To determine the coating's durability in the various pH ranges of the gastrointestinal tract, the disintegration research was conducted for two hours in a pH 1.2 hydrochloric acid buffer, followed by four hours in a pH 6.8 phosphate buffer, and finally two hours in a pH 7.4 phosphate buffer.^19,20,21

In-vitro drug release:

The United States Pharmacopoeia (USP) XXIV dissolution testing equipment II was used to assess the release kinetics of Meloxicam microspheres (Basket method) by using 900 ml of pH 1.2 hydrochloric acid buffer, simulated stomach fluid, pH 6.8 phosphate buffer, simulated intestinal fluid, pH 7.4 phosphate buffer, and simulated colonic fluid at 370.5°C and 100 rpm.²²

RESULT AND DISCUSSION:

Beer's Lambert's Law was found to be obeyed by the medication at concentrations between 2 and 10 g/ml, according to a graph that produced a straight line in the end at pH phosphate buffer.

The obtained values were Y= 0.065x, r²₌0.980 in 7.4 phosphate buffer and Y= 0.086x, r²₌0.972 in simulated colonic fluid. Fig 1 (A)peaks were at 1618 cm^-1 (C=O streching), 3286 cm^-1 (secondary –NH or –OH), prominent bends such as 836 and 569 cm^-1 (-CH aromatic ring bending and hetero aromatic) and 1344, 1120 cm^-1 (S=O streching) respectively. The presence of all characteristic peaks of Meloxicam showed in combinations with the β cyclodextrin complex in Fig 1(B) and with HPMC K4M in Fig1 (C). It shows The given study showed that no chemical interaction and changes took place.

F

G

E

Fig. 1: FT-IR spectra of Meloxicam and excipients A) Meloxicam B) meloxicam- β cyclodextrin complex C) HPMC K4M with Meloxicam

D) Ethyl cellulose with Meloxicam E) FTIR spectra of Eudragit S 100 with Meloxicam F) Eudragit RS 100 with Meloxicam G) Eudragit E 100 with Meloxicam

Fig. 2: SURFACE MORPHOLOGY A: Scanning Electron Microscopy of formulation A_3,B: Scanning Electron Microscopy of formulation B_2, C: Scanning Electron Microscopy of formulation S_1,D: Scanning Electron Microscopy of formulation S_1,E: Scanning Electron Microscopy of formulation E₂

The maximum and minimum % yield was found 81.06% in batch A4 and 44.2% in batch B4 respectively. Conclusively the % yield increased with polymer and surfactant concentration used in formulation.

Drug entrapment efficiency:

Table 2: Percentage drug entrapment of batch S₁-S₄ and RS₁-RS₄

Formulation	S₁	S₂	S₃	S₄	RS₁	RS₂	RS₃	RS₄
Absorbance	0.524	0.531	0.584	0.597	0.421	0.426	0.443	0.447
Entrapment (%)	81.36	82.45	90.68	92.70	65.47	66.20	68.78	69.40

Table 3: Percentage drug entrapment of batch E₁- E₃

Formulation	E₁	E₂	E₃
Absorbance	0.490	0.506	0.443
Entrapment (%)	76.07	78.57	68.78

Entrapment efficiency also increased with increase in polymer concentration. The high entrapment efficiency of meloxicam could be attributed to its poor aqueous solubility enhanced by complexation.^23,24

Stability studies:Dissolution release profile of formulation A₃, B₂, RS₁, S₁, E₂ on stability

Table 4: Comparative release kinetic data of formulation A₃, B₂, RS₁, S₁, E₂ on stability at different time interval (90 Days).

S. No.	TIME (HRS)	% DRUG RELEASE			TIME (HRS)	% DRUG RELEASE	TIME (MIN)	% DRUG RELEASE
S. No.	TIME (HRS)	A₃	B₂	RS₁	TIME (HRS)	S₁	TIME (MIN)	E₂
1.	1	28.27	22.60	23.01	1	20.95	5	18.91
2.	2	34.98	39.98	28.54	2	29.74	10	35.16
3.	3	37.12	43.54	32.712	3	32.21	15	46.09
4.	4	38.98	54.86	35.19	4	37.89	20	57.81
5.	5	44.61	62.13	41.52	5	45.95	25	71.91
6.	6	49.98	62.87	42.45	7	59.93	30	85.85
7.	7	55.17	67.59	46.58	9	56.94	35	97.89
8.	8	61.56	68.94	54.04	11	70.02
9.	9	68.42	71.04	58.95	13	71.18
10.	10	72.78	72.25	69.25	15	72.89
11.	11	81.26	72.98	70.82	17	73.08
12.	12	87.39	74.67	80.93	20	73.36
13.					24	75.89

Fig. 3: Comparative release profile of formulation A_3,B₂,RS₁,S₁,E₂ on stability

The different formulation were analyzed via particle size, percentage yield, entrapment efficiency, surface morphology and in-vitro dissolution. The optimized formulations using 2² factorial designs were consequently filled in HPMC capsules and coated with 15% (w/v) solution of eudragit S100. The in-vitro dissolution studies were carried out on dissolution apparatus (Electro lab, TDT 06L, 6 basket) in pH 7.4 phosphate buffer and in simulated colonic media respectively. It led to the maximum release of the drug with A₃, B₂, RS₁ and S₁ batches in simulated colonic fluid and consequently interpreted that such formulations may produce effective targeting to the colon with sustained release parameters while E₂ batch got immediately released (99.04% in simulated colonic fluid). Conclusively such formulation (E₂) may produce effective targeting to colonic tissues for better, quick and significant release.The optimized formulations A₃, B₂, RS₁, S₁, E₂ were subjected to stability studies. The stability study indicated that the prepared formulations were stable and retained their pharmaceutical properties at 45^°±2^°C and 75±5% relative humidity over a period of 90 days.

CONCLUSION:

Nineteen batches of meloxicam-targeted microspheres for the colon were created in the current study employing a variety of polymers, including HPMC, EC, Eudragit S100, Eudragit RS100, and Eudragit E100 in varying ratios. λ_max was determined by spectrophotometric scan of the drug which was found to be 364 nm. The FTIR analysis concluded no interactions occurred between the drug with its various excipients by unaltering the characteristic peaks of the drug. The surface morphological studies carried out with scanning electron microscopy (SEM) confirmed smooth and spherical surfaces of the microspheres. The microspheres of eudragit E100 was exemplified to posses hollow cavities. Drug entrapment efficiency lied in the range of 35.04-62.50% (for A1-A4), 76.29- 87.78% (for B1-B4), (81.36-92.70% (for S1-S4), 65.47-69.4% (for RS1-RS4), 68.91-78.08% (for E1-E3). The optimized batches obtained from factorial analysis were A₃, B₂, RS₁, S₁ and E₂ and their optimization exemplified good desirability enabling the best batches to be optimized. The durability test for coated capsules with eudragit S100 was performed at pH 1.2 for 2 hrs followed by pH 6.8 for 2 hrs. The test confirmed no drug release could occur from the coated HPMC capsules in these media. The in-vitro best releases of the drug from optimized batches at pH 7.4 were 87.36, 74.54, 81.23, 76.87 and 98.98% for A₃, B₂, RS₁, S₁ and E₂ respectively at different time intervals.

The stability studies performed for optimized batches as per ICH guidelines under officially prescribed conditions showed that these formulations were stable and thus complied with dose uniformity criteria.

The current investigation suggested that employing various polymers enclosed in HPMC capsule shells coated with eudragit S100, microspheres may be delivered for both immediate and sustained release of meloxicam in the colon and such formulation design can be applied to drugs of various categories which may pave the way to future researchers aiming at optimized pharmacological effectiveness with reduced toxic parameters.

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Received on 13.12.2021 Modified on 16.05.2022

Research J. Pharm. and Tech 2023; 16(4):1919-1924.

DOI: 10.52711/0974-360X.2023.00315