INTRODUCTION:

Colon-related disorders are now far more common than they were in the recent past. 5-Aminosalicylic Acid (5-ASA) is preferred for the treatment of a number of colonic illnesses, including IBD (inflammatory bowel disease), CD (Crohn's disease), and ulcerative colitis.¹

If none of these conditions are addressed early on, they might all develop into colorectal cancer. In order to prevent early absorption and drug loss, several attempts have been undertaken over the past ten years to create an effective formulation that releases the 5-ASA exactly in the colon without releasing it elsewhere in the gastrointestinal tract (GIT).^2,3

The Prier literature predicted a variety of methods, including the use of pH- or dependent on time polymers in matrix tablets, or colonic microflora degradable. Due to this molecule's extreme bio variability, only few of these studies were able to establish a link between in vitro-in vivo release. Because pH is a very fluctuating physiological parameter in IBD patients, using a strictly pH-dependent method for colon targeting has limitations.⁴

Through the inhibition of cyclooxygenase, reduction of prostaglandin synthesis, and suppression of interleukin-1 (IL-1) and tumour necrosis factor-a (TNF-a) synthesis, 5-ASA functions as an anti-inflammatory drug. If medications had a proper release pattern and were directed at the site of action, some chronic severe illnesses may be treatable more successfully. One of the most promising methods that might increase the 5-ASA's efficacy with fewer adverse effects is targeted distribution utilising a multiparticulate system.⁵

The therapeutic use of 5-ASA is nonetheless constrained by significant biovariability and high dosage. Therefore, it is urgent to design an oral solid dose form that is tailored specifically for the colon. In this context, pellets have benefits including predictable stomach transit time, little local discomfort, increased bioavailability, decreased dosage dumping, and ease of coating, making them suited for controlled drug administration.⁶

METHODS AND MATERIALS:

Materials:

5-aminosalicylic acid was procured from Chemi S.P.A, Italy. BASF Chemicals (Mumbai, India) provided Stearic acid (Kolliwax S Fine), Hydrogenated Castor oil (Kolliwax HCO), Glyceyl Monosterate (Kolliwax GMS). Sugar Spheres was purchased from Colorcon Pvt Ltd,Goa. Ethylcellulose (Ethocel 7 Cps) and Hypromellose E6 Supplied from Dow Chemicals,United States. Other excipients used to prepare coating dispersion were of standard pharmaceutical grade and all chemical reagents of analytical grade.

FORMULATION AND DEVLOPMENT:

Procedure for coating:

Seal coating:

Preparation of seal coating solution:

The sugar spheres were given a 3% seal coating using a 2% weight per volume solution of HPMC E6 in isopropyl alcohol (IPA): Dichloromethane (DCM) (50:50), which was agitated for 30 minutes to create a transparent solution.

Procedure for seal coating of Sugar spheres:

The needed number of sugar spheres were dispensed, weighed, and preheated for 10 minutes before the seal coating solution was applied to the core sugar pellets. Pellets were then dried for 15 minutes at 35-45°C.

Drug layering:

Preparation of Drug layering Dispersion:

Weighing was done on each ingredient. The necessary amount of Isopropyl Alcohol and Dichloromethane (80:20) was taken, kept under stirring, and then Ethyl cellulose, Hypromellose E6, and Mesalamine were added to the solution's vortex under the homogenizer and homogenised for 20 min. at 1500–3000rpm. The remainder of the glyceryl monosteate, stearic acid, and hydrogenated castor oil are next added in a systematic manner. Afterward, talc was added, mixed for 40 minutes, and the dispersion was passed through 100mesh. Drug stacking dispersion has a 15% weight-to-weight solid strength.

Procedure for Drug layering of seal coated sugar spheres:

Consider sugar spheres with a seal coating that have been loaded into a fluidized bed processing pan. The medicine was loaded there by dispersing it on the sugar spheres. The table No. 1 contained a parameter for medication layering.

Controlled Release coating of drug layered pellets:

Preparation of Controlled Release coating solution:

Finally, ATBC is dispersed in the solution and agitated for an additional 10 minutes to complete the formation of the solution. Ethyl cellulose 7cps and Hypromellose are distributed in IPA water combination in the ratio 90:10 parts. Pellets should gain 3% w/w in weight on average.

Procedure for Controlled Release coating:

Consider loading controlled release coating solution into drug-layered sugar spheres that were loaded into a fluid bed processing pan. The table No. 1 contained a parameter for controlled release coating.

Table 1: Parameter for Fluid Bed Processor

Process Parameters	Seal Coating	Drug Layering	Controlled Release
Inlet Temperature (◦C)	35-45	50-65	55-60
Product Temperature (◦C)	30-35	38-50	35-50
Exhaust Temperature (◦C)	28-30	30-50	30-48
Air Flow (CFM)	10—15	10—30	10—20
Atomization Air pressure (bar)	1.0 -3.0	1.0-3.0	1.0-3.0
Spray Rate (g/min)	3-5	5—15	5—8

Table 2: Composition

S. No	Formula	MEZ -1		MEZ -2		MEZ -3		MEZ -4		MEZ -5
	Ingredients	mg/cap	%w/w	mg/cap	%w/w	mg/cap	%w/w	mg/cap	%w/w	mg/cap	%w/w
	Seal Coating
1	Sugar Spheres (40-45)	50.00	14.28	50.00	14.28	55.00	15.71	45.00	12.85	47.00	13.43
2	Hypromellose E6	1.35	0.39	1.35	0.35	1.35	0.39	1.35	0.39	1.35	0.38
3	Talc	0.15	0.04	0.15	0.04	0.15	0.04	0.15	0.04	0.15	0.04
4	Dichloromethane	14.25	-	14.25	-	14.25	-	14.25	-	14.25	-
5	Isopropyl Alcohol	14.25	-	14.25	-	14.25	-	14.25	-	14.25	-
	Drug Layering
6	Mesalamine	250.00	71.43	250.00	71.43	250.00	71.43	250.00	71.43	250.00	71.43
7	Stearic acid	7.50	2.14	5.00	1.43	0.00	0.00	5.00	1.43	2.50	0.71
8	Hydrogenated Castor oil	5.00	1.43	2.50	0.71	5.00	1.43	5.00	1.43	2.50	0.71
9	Glyceyl Monosterate	5.00	1.43	2.50	0.71	5.00	1.43	5.00	1.43	2.50	0.71
10	Ethylcellulose 7 cps	18.75	5.36	23.00	6.57	18.44	5.27	18.23	5.21	18.75	5.36
11	Hypromellose E6	0.00	0.00	0.00	0.00	0.00	0.00	5.00	1.43	10.00	2.85
12	Talc	2.00	0.57	5.00	1.43	5.00	1.43	5.00	1.43	5.00	1.42
13	Isopropyl Alcohol	1485.00	-	1447.00	-	1447.13	-	1320.34	-	1320.34	-
14	Dichloromethane	165.00	-	160.79	-	160.79	-	330.09	-	330.09	-
	Film coating
15	Ethyl cellulose 7 Cps	6.2	1.75	6.47	1.85	6.03	1.72	6.16	1.76	6.16	1.76
16	Hypromellose E6	3.02	0.89	3.02	0.86	3.02	0.86	3.08	0.88	3.06	0.87
17	Acetyl tributyl citrate	1.03	0.29	1.01	0.29	1.01	0.29	1.03	0.29	1.03	0.29
18	Isopropyl alcohol	175.60	-	171.86	-	171.86	-	175.60	-	175.60	-
19	Water	19.51	-	19.10	-	19.10	-	19.51	-	19.51	-
	Coated Weight	350.00	100.00	350.00	100.00	350.00	100.00	350.00	100.00	350.00	100.00

RESULT AND DISCUSSION:

Pre-formulation study:

Characterization of physicochemical properties of drug:

Organoleptic properties:

Physical appearances: Pink colored needle shaped crystal powder.

Odour: Odourless.

Melting point by Capillary tube Melting point instrument:

Mesalamine API Melting Point was found to be 283°C.

Solubility analysis:

Water and mesalamine are very slightly soluble in each other. However, the pH solubility tests were performed at 37°C throughout a range of pH, and the findings are presented in table no. 3.

Table 3: pH Dependent Solubility at 37°C (mg /250 mL)

Different Media	Solubility (mg/250mL)
0.1 N HCL	1176.3
pH 2.0 HCL buffer	505.5
pH 3.0 Citrate buffer	376.0
pH 4.5 Acetate buffer	357.0
pH 4.5 Phosphate buffer	339.7
pH 6.0 Phosphate buffer	472.7
pH 6.8 Phosphate buffer	1011.3

According to the preceding table, mesalamine is more soluble in 0.1 N HCL, less soluble in the pH range of 2 to 6, and more soluble starting at pH 6.8.

Micrometric Properties:

Bulk density:

The following formulas were used for the calculation of prepared mesalamine pellets' bulk density (BD) and tapped density (TD) for all of the formulations.^7,9

Bulk density= Weight of the powder (g)/ Volume of the powder (ml).

Tapped density= Mass/Tapped Volume

Compressibility Index (Carr’s index):

The capacity of a powder to flow can be assessed by contrasting its bulk density (BD), tapped density (TD), and packing down rate. Mesalamine pellets were made, and the compressibility index (Carr's index) was determined using the formula below

Carr’s index (%) = [(TBD – BD) × 100] / TBD

The specification states that a Carr's index value "between" 5 and 15 indicates outstanding flow and a value between 12 and 16 indicates good flow. Values "between" 18 and 21 denote merely passable, while values "between" 23 and 25 denote subpar. Very poor is defined as "between" 33 and 38, and extremely poor as "greater than" 40. ^9,10

Hausner’s ratio:

The Hausner’s ratios of prepared mesalamine pellets were determined by following formula

Hausner’s ratio=TD/BD

Specifications state that values below 1.25 denote good flow (=20% of Carr's index), whereas values above 1.25 denote bad flow (=33% of Carr's index). Glidant must be added between 1.25 and 1.5 to improve flow.¹¹

Table 4 Micrometric Properties of Mesalamine API

Sr. No.	Properties	Result
1	Bulk Density	0.226g/ml
2	Tap Density	0.529 g/ml
3	Compressibility Index	57.27%
4	Hausners Ratio	2.34

Particle Size Distribution of API by Electromagnetic Sieve Shaker:

Table 5: Particle Size Distribution of Mesalamine API

Mesh size	Micron	Empty weight	Sample weight	Retains	% Retains	%Cumulative
18#	1000µm	428	450	22	43.14	43.14
20#	841µm	426	430	4	7.84	50.98
25#	707µm	428	435	7	13.72	64.7
30#	595µm	418	428	9	17.65	82.35
40#	400µm	398	403	5	9.8	92.15
60#	250µm	382	386	4	78.84	99.99
80#	177µm	370	370	0	0	99.99
100#	149µm	368	368	0	0	99.99
Pan	NAP	374	374	0	0	99.99

Loss on Drying: 0.15% w/w

Analysis of drug:

Calibration Curve Method:

λ max were found

7.5 Phosphate Buffer = 230 nm

Standard curve of Mesalamine

Table 6: Standard Calibration Data of Drug A in 7.5 Phosphate Buffer

Concentration (ppm)	Absorbance
0	0
1	0.06
3	0.197
5	0.329
7	0.469
10	0.671
12	0.816

Fig No 1.: Standard curve of Model API in pH 7.5 Phosphate buffer

The resultant calibration curve obtain from above data is depicted in above figure and the equation of the line was y = 0.067x with an R2 value of 0.9996 indicating that the method was linear over the concentration range 0 – 12 μg/ml.

Drug–Excipients compatibility studies:

Taken in a ratio of 1:1 in transparent USP type I glass vials, the active component and excipients intended for this product were exposed to 40ºC/75% RH (closed samples) for four weeks.

Sample Preparation:

Add drug and excipient mix with spatula and fill it in vial close with rubber lid.

Sample collection and analysis:

Samples were taken at the conclusion of the fourth week from a closed environment at 40°C and 75% relative humidity, and they were then examined for impurities and associated chemicals. During the processing of the sample, the entire contents of the vials must be consumed.

Table 7: Compatibility Study of Mesalamine and Excipients

Sr. No	Composition	Physical Appearance
Sr. No	Composition	Initial Description	Final Description
1.	Mesalamine	Slight Pink colour	No colour Change
2.	Mesalamine + Ethylcellulose 7 Cps	Very slight pink colour	No colour Change
3.	^Mesalamine+ ^{Hypromellose E6 Cps}	Very Slight Pink Colour	No colour Change
4.	^Mesalamine+ ^{Stearic acid (Kolliwax} S Fine)	Very slight pink colour	No colour Change
5.	Mesalamine + Hydrogenated Castor oil (Kolliwax HCO)	Very slight pink colour	No colour Change
6.	Mesalamine + Glyceryl Monosterate (Kolliwax GMS)	Very slight pink colour	No colour Change
7.	Mesalamine + Sugar spheres	Very slight pink colour	No colour Change
8.	Mesalamine + Acetyl tributyl citrate	Very slight pink colour	No colour Change
9.	Mesalamine + Talc (Luzenac)	Very slight pink colour	No colour Change
10.	^{Mesalamine + Ethylcellulose 7 Cps}+ ^{Hypromellose E6 Cps}+ ^{Stearic acid}(Kolliwax S Fine) + + Hydrogenated Castor oil (Kolliwax HCO) +Sugar spheres+ Acetyl tributyl citrate+Talc(Luzenac)	Very slight pink colour	No colour Change
11.	^{Ethylcellulose 7 Cps}+ ^{Hypromellose E6 Cps}+ ^{Stearic acid (Kolliwax S}Fine) +Hydrogenated Castor oil (Kolliwax HCO) +Sugar spheres+ Acetyl citrate+ Talc (Luzenac)	White Colour Powder	No colour Change

Drug –Excipient study by Differential Scanning Calorimetry:

The melting point of mesalamine, 283⁰C, was clearly visible in the DSC analysis. The thermal behaviour of the physical mixture of mesalamine and other excipients (283⁰C) was the same as that of the individual component. Additionally, DSC data demonstrated that the thermogram was superimposed in the physical mixture of mesalamine and excipients. When mesalamine and excipients were physically combined, there was no discernible change in the melting endotherm.⁸

As a result, it was determined from the DSC analysis that Mesalamine and the other excipients employed in the formulation did not interact. The Figure included the DSC thermogram.

Fig 2: Differential Scanning Calorimetry of Mesalamine API

Fig 3: Differential Scanning calorimetry of Physical mixture of Mesalamine and Excipients

Characterization of Mesalamine pellets:

Appearance of Pellets: The shape and colour of the pellets are spherical, with some having a somewhat reddish appearance.

Particle Size Distribution:

Table 8 Particle Size Distribution of Different Formulations

Batch No:		MEZ 1	MEZ 2	MEZ 3	MEZ 4	MEZ 5
MESH SIZE	MICRONS	% RETAINS
18#	1000µm	41	72	56	77	65
20#	850µm	34	21	27	16	26
25#	707µm	13	4	14	3	6
30#	595µm	5	2	5	3	3
40#	400µm	4	0	0	0	0
Pan	NAP	0	0	0	0	0

Micrometric properties of pellets

Formulations	Bulk density (g/ml)	Angle of Repose (◦)	Friability (%)	Coating Process Efficiency (%)
MEZ 1	0.76	22.3	0.34	80%
MEZ 2	0.71	25.5	0.58	90%
MEZ 3	0.73	18.5	0.22	85%
MEZ 4	0.78	21.7	0.14	88%
MEZ 5	0.76	30.5	0.55	89%

Drug content determination By Assay:

Chemicals Required: Mesalamine, potassium dihydrogen phosphate, sodium hydroxide, sodium acetate trihydrate, tetra butyl ammonium hydrogen sulphate, acetonitrile and miliq water.

Preparation of 1N Sodium Hydroxide: 4.0g of sodium hydroxide pellets should be weighed, dissolved in 100 ml of milli-Q water, and thoroughly stirred.

Preparation of pH 6.6 Phosphate Buffer: In 900ml of milli-Q water, weigh and dissolve 1.9g of sodium acetate trihydrate and 4.6g of tetrabutyl ammonium hydrogen sulphate. After that, adjust the pH to 6.6 with 1N sodium hydroxide and reduce the volume to 1000ml of milli-Q water. Take a sonicator to degas the solution after filtering it through a 0.45m Nylon membrane filter.

Preparation of Mobile Phase:

Mix pH 6.6 buffer and Acetonitrile in the ratio of 800:200(v/v) and degas.

Diluent Preparation (pH 7.5 Buffer):

Measure and dissolve 6.8g of potassium dihydrogen orthophosphate in 900ml of mill Q water. Then, raise the pH to 7.5 by adding 1N sodium hydroxide and bring the volume to 1000ml.

Blank Preparation:

Use diluent as Blank

Chromatographic condition:

Column: Waters symmetry C18 (4.6 x 150)mm, 3.5µm

Elution Mode: Isocratic

Flow Rate: 1.5ml/minute 73

Injection Volume: 10µL

Column oven Temperature: 30◦C

Sample Temperature: 8^◦C

Wavelength: 240nm

Run time: 6 minutes

Retention time: about 3 minutes

Standard Preparation:

Precisely weigh 50mg of mesalamine functioning standard, transfer to a 100ml volumetric flask, add around 70ml of diluent, sonicate to disperse the substance, and bring the quantity up to the desired level with the diluent. Put 5ml of the resulting solution in a 25 ml volumetric flask, fill the remaining space to the specified level with diluent, and shake the flask vigorously (100ppm).

Sample preparation: Crush the small pieces into fine powder by weighing 5 capsules and estimating the median weight of the full content. To obtain the covering material, the powder must also be in its finest form possible. Put the tablet powder, which is equal to 200mg of mesalamine, into a volumetric flask and weigh it. Add around 70ml of diluent, shake it mechanically for 30 minutes, then sonicate it with intermittent shaking in the same volumetric flask for 30 minutes. Once the solution has cooled to room temperature, add more diluent to get the volume up to the desired level. Place a 0.45m PVDF material syringe filter over this solution to filter it. Transfer 5ml of the filtration into a volumetric flask with a volume of 100m and add diluent (100ppm) to fill the remaining space. Well blended mixture into the HPLC apparatus.

Procedure: Separately inject equal volume (about 10µL) of the blank, standard preparation and sample preparation into the chromatograph, record the chromatograms and measure the response of major peak.¹²

% Assay of Mesalamine = 𝐴𝑡/ 𝐴𝑆 𝑋 𝑊/𝑠 100 𝑋 5/ 25 𝑋 100 /𝑊𝑡 𝑥 100/ 5 𝑋 𝐴𝑊 /𝐿𝐶 𝑋 P

Table 9: Assay of Different Formulations

Formulations	Assay (%)
MEZ 1	82.4
MEZ 2	94.3
MEZ 3	95.3
MEZ 4	100.5
MEZ 5	101.3

In vitro release studies of Mesalamine pellets:

Medium: 0.05M pH 7.5 Phosphate buffer prepared by dissolving 6.8g of monobasic potassium phosphate and 1g of sodium hydroxide in water to make 1000ml of Solution and modify with 10N Sodium hydroxide to a pH of 7.50±0.

Procedure: Determine the amount of Mesalamine dissolved from UV absorbance at the wavelength of most absorbance at approximately 330nm on filtered quantities of the answer below take a look at certainly diluted with medium, if Necessary, in evaluation with a today's solution having an acknowledged interest of USP mesalamine RS withinside the identical medium.¹³

Table 10: In vitro release studies of Mesalamine pellets

Media	pH 7.5 Phosphate Buffer
Time (Hrs)	%Cumulative Drug Release
Time (Hrs)	MEZ 1	MEZ 2	MEZ 3	MEZ 4	MEZ 5
0	0	0	0	0	0
1	20	27	33	23	57
2	35	40	50	41	82
4	49	55	69	73	103
6	70	74	81	86	103
8	79	81	90	99	104
12	80	88	96	100	106

Fig 4: In vitro Drug Release Batch F1 to F5

CONCLUSION:

A study on "Formulation and Characterization of coclon targeted Mesalamine pellets" is described in the current article.

Mesalamine, an amino salicylate, is prescribed to treat inflammatory bowel disorders such ulcerative colitis and crohn's disease. By reducing the formation of prostaglandins (PG) in the colon and by blocking the enzyme cyclooxygenase, mesalamine reduces inflammation. Inflammatory bowel disease treatment of choice is amino salicylate.

In this formulation, various concentrations of polymers and release retardants, including stearic acid, ethylcellulose, hypromellose, and hydrogenated castor oil, are employed to manage the release over an extended period of time. Drug stacking into sugar spheres is prevented from dumping doses by employing polymer in the matrix. Drug coated pellets with an additional extended-release coating were added to manage the initial drug release and prevent product degradation.

All of the excipients applied in the formulation of the pellets were compatible with the medication Mesalamine, as shown by preformulation tests such as characterization of pharmaceutical active excipients, Differential Scanning calorimetry investigations, and accelerated stability experiments.

Mesalamine pellets were created using five different strategies. The formulation and characterisation of MEZs 1 to 5 are carried out.

The pellets' shapes are satisfactory; some of them are rounded or oval, while the remaining few are fashioned like dumbbells.

The release retardant has a maximum drug level of 101.7% in the pellets, which were created using a different polymer concentration.

The formulation MEZ-IV was the optimum formulation, according to the experiments on drug release conducted in vitro. The 12-hour in vitro drug release is consistent. (1hour. 23%, 2nd hr. 41%, 4hr. 73, 6hr. 86, 8hr. 99%, and 12hr. 100%)

ACKNOWLEDGEMENT:

We express our gratitude to the teachers and Principal of Loknete Dr. J. D. Pawar College of Pharmacy, Manur, Tal. Kalwan, for their valuable guidance and support.

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Received on 22.05.2023 Modified on 27.11.2023

Asian J. Pharm. Res. 2024; 14(2):107-113.

DOI: 10.52711/2231-5691.2024.00018