INTRODUCTION:

Inflammation and ulcers (sores) in the digestive system are symptoms of the inflammatory bowel disease (IBD) known as ulcerative colitis. The innermost lining of your large intestine, often known as the colon and rectum, is impacted by ulcerative colitis. In the majority of people, symptoms typically appear gradually rather than abruptly. Draining and perhaps life-threatening consequences are possible with ulcerative colitis. Although there is no known cure, there are a number of innovative treatments that can significantly lessen the disease's signs and symptoms and result in a long-lasting remission¹. Typically, either drug therapy or surgeries are used as treatments. The treatment of ulcerative colitis may be successful with a variety of drug classes. Some individuals may not respond adequately to some drugs. The initial line of treatment for ulcerative colitis is frequently anti-inflammatory drugs, which are suitable for the majority of patients with this condition.

The enteric coated drug-delivery technology (ECDDT), which offers complete enteric protection without the need for a separate enteric coating, is described in recent technical research. ECDDT can provide quicker development timelines and decreased programme risk by doing away with the preparation and application stages required for enteric coating². It has been demonstrated that this cutting-edge technology makes it possible to develop products that require enteric protection and/or targeted release to the upper gastrointestinal tract by enabling the oral delivery of sensitive molecules and acting as a valuable tool for product development.

Making mini-tablets that meet the USP dissolution Q values of Mesalamine is the most difficult aspect of the study to prevent dose dumping. A factorial design was used to develop mini-tablets based on Mesalamine. To investigate the effect of independent factors on dependent variables, a two-factor, three-level factorial design was used with Stat-Ease Design Expert v12. According to the experimental design, nine formulations were generated. Excipient levels in factorial designs were chosen in accordance with the Handbook of Pharmaceutical Excipients³. Mini-tablets were compressed and evaluated for precompression and post compression parameters before being encapsulated in enteric-coated capsules. In Vitro drug release, Release Kinetics, Stability and in -vivo study for the determination of pharmacokinetic parameters were conducted on a mini-tablet in an enteric-coated capsule.

MATERIALS AND METHODS:

Mesalamine and Diazepam were obtained as gift sample from Remedium Laboratories Pvt. Ltd, Hyderabad. Enteric Coated Capsule was obtained from XPRS Nutra, West Jordan. Microcrystalline Cellulose, Crospovidone, HPMC K4M, Magnesium stearate and Talc was purchased from Signet Excipients Pvt.Ltd, Bangalore. Gradient grade methanol, ammonium acetate, Propionic anhydride, formic acid (GR Grade) and methyl t-butyl ether were purchased from Merck (Worli, Mumbai, India). All other chemicals used in the study were of analytical grade.

Assessment of enteric coated capsules^4-7

Enteric coated capsules were assessed for maintaining the integrity in acidic pH. 6 units with 250mg of Mesalamine drug in each capsule were subjected to dissolution for 2 Hours in 0.1N Hydrochloric acid according to USP Dissolution method. Specification of NMT 1% release of Mesalamine after 2 Hours according to Mesalamine USP Monograph. Dissolution Conditions (Volume: 750mL, Medium: 0.1N Hydrochloric Acid, Temperature: 37°, RPM: 100, Time: 2 Hours, Intervals (Hours) After 2 Hours: Apparatus: USP Type II)

Compatibility study of drug and excipients using FT-IR:

Mesalamine and Excipients compatibility were evaluated using Perkin Elmer. Potassium Bromide pellet method is employed for the current study.

Formulation of Mini-tablets with factorial design approach:

Mesalamine based Mini Tablets were prepared by using factorial design. A 2-factor three-level factorial design was employed using Stat-Ease Design Expert v12 to study the effect of independent variables on dependent variables. A total of 9 formulations were prepared according to the experimental design. The amount of HPMC K4M (X1-Low Level:10% and High Level 20%) and Crospovidone (X2-Low Level 2 and High Level 5) were selected as independent variables. The time dissolution response of drug in 1hr (Q1), 2hr (Q2) and 6 hrs (Q3) was selected as dependent variable. Excipient levels in factorial design were selected according to handbook of pharmaceutical excipients 6^th Edition³.

Formulation development:

All the ingredients were weighed according to the quantities specified in Table 1 and passed through #60mesh separately. Then the ingredients were mixed in geometrical order and compressed into tablets of mg by using 8-station rotary mini press tablet machine using 5mm punch. Compressed tablets were filled into Enteric Coated capsule of size#0. Each capsule contains 5 mini-tablets. The dose of each capsule is 250mg.

Evaluation of Mini-tablets:

Pre-compression:

Bulk density, Tapped density, Angle of repose, Compressibility Index and Hausners ratio were evaluated according to USP General chapter <1174> to assess the flow property of the blend before compression.

Post-compression:

Compressed tablets were evaluated for post compression parameters according to the procedure mentioned in the following USP general chapters Weight Variation and Drug Content <905>, Hardness, Thickness and Friability <1217>.

In-vitro drug release study:

In-Vitro dissolution was performed according to the procedure mentioned in Mesalamine USP Monograph. Mini-Tablets encapsulated enteric coated capsules were assessed for maintaining the integrity in 0.1N HCL acidic pH for 2 hours while maintaining dissolution conditions (Apparatus 2, 750mL, 100 RPM; 37°± 5°C; followed by (7.2 pH Phosphate Buffer; 960mL, 100RPM; 37°± 5°C; 8 Hours) at sampling intervals of 1, 2, 4, 6, 8 Hours). Samples obtained were determined by UV-visible spectrophotometer at 214 nm.

Table 1: Mesalamine Mini-tablets formulations

S. No	Ingredient (mg)	MF1	MF2	MF3	MF4	MF5	MF6	MF7	MF8	MF9
1	Mesalamine	50	50	50	50	50	50	50	50	50
2	HPMC K4M	17	8.5	8.5	17	12.75	12.75	17	8.5	12.75
3	Crospovidone	2.97	2.97	4.25	1.7	2.97	1.7	4.25	1.7	4.25
4	Microcrystalline Cellulose 112	13.76	22.26	20.98	15.03	18.01	19.28	12.48	23.53	16.73
5	Magnesium Stearate	0.43	0.43	0.43	0.43	0.43	0.43	0.43	0.43	0.43
6	Talc	0.85	0.85	0.85	0.85	0.85	0.85	0.85	0.85	0.85
Total Tablet Weight (mg)		85	85	85	85	85	85	85	85	85

Stability studies:

Stability studies were performed according to the ICH guidelines Q1A (R2) for optimized formulation. The stability study is an indicative method for determination of durability of quality and quantity of therapeutic agents with the passes of time under the influence of various atmospheric conditions such as temperature, humidity, light and to establish a re-test period for the drug substance or a shelf life for the drug product and recommended storage conditions at 40°C ± 2°C/75% RH ± 5% RH.

Differential scanning colorimetry (DSC):

Further stability studies were confirmed by Differential Scanning Calorimetry. Thermal analysis of Optimized Formulation before and after stability was performed using thermal analyzer (Mettler Toledo-DSC 3) Temperature axis and cell constant were calibrated by utilizing indium (In). This technique allows a rapid assessment of possible interaction by disclosing transition in exhibition, dissipation of endothermic or exothermic peaks, and transition in the pertinent enthalpy standards in thermal curves of drug-excipients combinations.

Release kinetics:⁸

The drug release kinetics study was conducted on optimized formulation related to various kinetic models, namely, zero-order kinetics, first-order kinetics, Higuchi and Korsmeyer-Peppas models, and then the regression analysis (R²) and diffusion coefficient (n) were determined.

Animal ethics:

All animal experiments approved and performed in Jeeva Life Sciences accordance with the guidelines of Institutional Animal Ethics Committee (CPCSEA Registration No: CPCSEA/IAEC/JLS/19/02/23/008).

Animal husbandry and maintenance:

Healthy adult male New Zealand rabbits weighing (3–3.5 kg) of 6-month-old, were obtained from Animal house. Each cage (815 mm × 500 mm × 340 mm) holds one rabbit. These rabbits are housed in a specific room. The air filtration rate is 10-20 air changes per hour; the temperature is 20°-26°C. The humidity is 40-70%, and the fluorescent light is dark for 12 h (08:00-20:00) and then 12 h every day. These rabbits were housed in an environment where they could eat and drink freely and adapted to the environment for at least 2 weeks before experiment. Only healthy animals were assigned for these studies according to OECD Guidelines 404. Approval to carry out these studies was obtained from the Institutional Animal Ethics Committee and an experiment was performed in compliance with the Principles of Laboratory Animal Care (NIH Publication 85-23, revised 1985).

In vivo study of Mesalamine:

LC-MS/MS conditions:

The LC system was manufactured by (Agilent Technologies, model series 1200). Mass spectrometric detection was performed on an API 3200 quadrupole instrument (ABI-SCIEX). Data were processed on Analyst 1.5.1 software package (SCIEX). Turbo ion spray negative mode with Unit Resolution, MRM was used for the detection.

Mass spectrometer conditions:

Mass parameters were optimized as source temperature 650 °C, nebulizer gas 20 psi, heater gas 30 psi, curtain gas 30 psi, CAD gas 4 psi, IS voltage-2000 volts, source flow rate 600 µL/min without split, entrance potential 10 V, Collision cell exit potential (CXP) 12 V, Declustering potential (DP) 50V, collision energy(CE) 35 V for mesalamine and diazepam respectively.

Chromatographic conditions:

Chromatography was performed on INERTSIL ODS-C18 (150 x 4.6 mm, 5µm) column. The mobile phase used as10mM ammonium acetate: methanol, 85:15 v/v at the flow rate of 0.4 mL/min. Injection volume was 10µL, column temperature was 40°Cwith an isocratic elution mode. [24, 25]

Calibration and quality control standards:

Calibration curve of mesalamine was prepared within the concentration range of 2.00-1500.00 ng/mL (2, 4, 10, 75, 150, 300, 600, 900,1200 and 1500 ng/mL) for mesalamine >0.998). The calibration curve consisted of one replicate of 10 non-zero standards.

Sample preparation:

Liquid-liquid extraction was used for extraction of drug and IS. For this purpose, 10 μL internal standard solution (15 ng/mL of Diazepam) was added to10µL of plasma sample (respective concentration) into vial. To this, 2.5 µL of derivatisation solution (10%propionic anhydride in methanol) was added and vortexed briefly. After that,10 µL of 0.5% formic acid was added into each tube and vortexed briefly again. Then, 3 mL of methyl t-butyl ether was added and vortexed for 10 minutes. Samples were then centrifuged for 5 minutes at 4000 rpm at 20°C. Supernatant from each sample was transferred into vial and evaporated to dryness. This was followed by reconstitution with 800µL of reconstitution solution (10 mM ammonium acetate: methanol, 85:15 v/v).and vortex briefly. From this, 2.5 μL of sample was injected into the LC–MS/MS system through the autosampler.

Application of the method:

The LC–MS/MS procedure was developed to determine budesonide in rabbit plasma 0–48 h. After an initial period of acclimatisation for two weeks to laboratory conditions, the rabbits were randomly divided into 2 groups of 3 subjects each. All the rabbits were fasted for twelve hours with impromptu access to water before the experiment. Dose of drug was administered according to Animal Equivalent Dose Calculations.

Maximum dose per day- 9mg/Day/60kg- 80mg/kg

AED = 0.80/0.324 = 246.91 mg

Group 1: Administered with pure drug

Group 2: Administered with formulated Mini-tablet in enteric coated capsule.

These rabbits were administered with pure drug, dissolving in normal saline. To dose Budesonide Mini-tablet in enteric coated capsule, a soft plastic dosing tube may be used. insert the filled capsule into the dosing tube so that the short end of the capsule protrudes slightly from the tip of the tube. Grasp the head firmly with one hand about the maxilla. Insert the dosing tube containing the capsule behind the incisors. Then slide the dosing tube straight into the back of mouth. Now eject the capsule by pushing the plunger then remove the dosing tube and close the rabbit’s mouth. Stroke the neck gently to facilitate swallowing. Animal had access to food 4 h after dose administration. Concerning 0.5 ml of blood sample was withdrawn from marginal ear vein into heparinized Eppendorf tubes at time intervals of 0 (pre-dose), 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 20, 24, 30, 36 and 48 hours post administration.

RESULTS AND DISCUSSION:

Assessment of enteric coated capsules:

Samples were collected at respective intervals and analysed in UV. There are no traces of drug found at the end of 2 hours. Results indicates that capsules are having integrity and resistance in acidic pH. Capsules were found to be an ideal candidate for enteric drug delivery system.

Compatibility study of drug and excipients using FTIR:

The FT-IR spectrophotometer was used to identify the possibilities of any interaction between the formulation components. As showed in Figure 1, there was no substantial differentiation in the FT-IR spectra of the drug and when compared to the spectra of the Formulation mixture.

IR spectrum of Mesalamine shows a broad peak at 3102.64 cm^-1 may be due to O-H stretching, 3437.24 cm^-1 N-H₂ stretching, 1619.73 cm^-1 may be due to carboxylic acid COO-stretching, 1650.77 cm^-1 may be due to C=C stretching, From IR Spectra interpretation of drug was done with individual excipients and formulation mixture and observed that there is no appreciable change in the positions of the characteristic bands. Since there is no change in the nature and position of the bands in the formulation, it can be concluded that the drug maintains its identity without going any chemical interaction with the excipients used.

Evaluation of Mini-tablets:

Precompression studies:

Results were found within the acceptable limits according to the pharmacopeial limits and showed good flow properties for all the formulations.

Post compression parameters:

Results obtained in Table 3; were found within the acceptable limits for friability and weight variation. Thickness was found in the range of 1.82-2.28mm; Hardness was found in the range of 1.9-2.8 kg/cm² and Drug content was fund in the range of 97.58-100.21 %.

Statistical analysis by design expert software:

The response Q1h, Q2h and Q6h also followed interactive (2FI) model and Design-Expert® was shown in Figure 2.

Response 1: Dissolution @ 1 Hour (%):

Dissolution@1 Hour (%) = +36.38722 + -1.45800 * HPMC K4M + 8.20000 * Crospovidone + -0.286000 * HPMC K4M * Crospovidone

The codes and actual equation of Dissolution for 1 Hour (%) suggests that factor A (% HPMC K4M), and factor B (% Crospovidone) have positive dominant effect. This is further interpreted from ANOVA results and equation that increase in Crospovidone concentration in proportion to HPMC K4M will result in significant increase in release rate to achieve the desired Q points.

Response 2: Dissolution @ 2 Hour (%):

Dissolution @ 2 Hour (%) = 46.03333 + -1.41000 * HPMC K4M + 10.27778 * Crospovidone + -0.386667 * HPMC K4M * Crospovidone

The codes and actual equation of Dissolution for 2 Hour (%) suggests that factor A (% HPMC K4M). AB has negative dominant effect and factor B (% Crospovidone) and have positive dominant effect. This is further interpreted from ANOVA results and equation that increase in Crospovidone concentration in proportion to HPMC K4M will result in significant increase in release rate to achieve the desired Q points.

Response 3: Dissolution @ 6 Hour (%):

Dissolution @ 6 Hour (%) = 85.06056 + -0.807000 * HPMC K4M + 7.83667 * Crospovidone + -0.324667 * HPMC K4M * Crospovidone

The codes and actual equation of Dissolution for 6 Hour (%) suggests that factor A (% HPMC K4M), AB has negative dominant effect and factor B (% Crospovidone) have positive dominant effect. This is further interpreted from ANOVA results and equation that increase in Crospovidone concentration in proportion to HPMC K4M will result in significant increase in release rate to achieve the desired Q points.

Upon Interpretation of data, it was observed that rapid release rates were higher in formulations prepared with higher concentrations of Crospovidone in proportion to the HPMC K4M (MF2, MF3, MF8 and MF9). Retardation of release rates were observed with formulations prepared with higher concentrations of HPMC K4M in proportion to the Crospovidone (MF1, MF4, MF5, MF6 and MF7). A multi-criteria decision optimization of approaches was employed for formulation optimization with desired responses (Table 2). Optimization was performed with constraints of Q1h in the range of NMT-35%, Q2h in the range of 35-60% and Q6h in the range of NLT 80%, to get optimized formula solutions with higher desirability function.

Figure 2: Response surface plot for dissolution and optimization

Table 2: Mesalamine Mini-tablets in enteric coated capsules compositions with their observed responses

		Factor 1	Factor 2	Response 1	Response 2	Response 3
Std	Run	A:HPMC K4M	B:Crospovidone	Dissolution @ 1 Hour	Dissolution @ 2 Hours	Dissolution @ 6 Hours
		%	%	%	%	%
6	1	20	3.5	15.12	25.2	73.72
4	2	10	3.5	43.77	55.06	95.2
7	3	10	5	45.8	62.75	98.96
3	4	20	2	11.96	22.69	71.59
5	5	15	3.5	28.88	39.56	82.07
2	6	15	2	23.24	36.34	79.46
9	7	20	5	18.95	30.43	75.68
1	8	10	2	30.23	43.41	85.13
8	9	15	5	35.87	49.56	88.24

Table 3: Point Prediction and Confirmation

Solution 6	Optimized Formulation	Predicted Mean	Predicted Median	SD	95% PI low	95% PI high
Dissolution @ 1 Hour	30.68	31.6345	31.6345	2.80457	14.9981	48.2709
Dissolution @ 2 Hour	44.09	43.0073	43.0073	1.93581	30.3998	55.6149
Dissolution @ 6 hours	86.53	85.0822	85.0822	1.32622	76.4448	93.7196

HPLC chromatograms of pharmacokinetic study:

The chromatograms and retention times of Blank plasma, Mesalamine, Internal Standard (Diazepam), plasma spiked with Mesalamine and Diazepam are shown in Figure 4. From the retention times of all the plasma samples, it has been observed that blank plasma has no interference from endogenous substance at the retention times of IS and analyte. The retention time minutes for IS and minutes for Mesalamine showing good resolution between IS and analyte. The retention time for the plasma samples collected after 2 hours from the subject administered via oral administration are found to be similar indication no interference between the analyte and plasma.

Standard linearity curve of Mesalamine hydrochloride:

The calibration curve results observed over the concentration range of 2 to 1500 ng/ml were satisfactory. The regression equation was found to be y = 9432x + 8184 with a regression coefficient of 0.9999. The linearity of results was depicted in Figure 5. The present study also estimates the significant PK parameters of the Mesalamine pure drug and Mesalamine Mini-tablet in enteric coated capsule (Optimized formulation). The Mean plasma drug concentration of both formulations at various time points is illustrated in Figure 6.

Time vs. plasma concentration plots of the pure drug and optimized formulation was observed. Plotted Area under the curve indicates the extent of drug absorption inside the body. In comparison with the present results, higher value of T_max (10 Hours) and T1/2 (9 Hours) was observed for the Optimized formulation with C_max value of (35.24 ± 0.45 ng/mL), AUC _(0-48)(411.58 ± 1.45 hr.ng/mL) and Ke (0.077 ± 0.010 hr^-1) it may be due to delayed release formulation. Pharmacokinetic analysis of Mesalamine pure drug plasma concentration-time data provided the following pharmacokinetic parameters like C_max value ranging (53.65 ± 0.85 ng/mL), T_max value (5.0 Hours), AUC _(0-48) value (440.46 ± 2.16 h.ng/mL), Half Life (4.5 Hours) and other pharmacokinetic parameters are depicted in Table 4.

Figure 5: Linearity of Mesalamine

Figure 6: Mean plasma concentration time profile in rabbits (Optimized formulation)

Table 4: Pharmacokinetic parameters (Optimized formulation)

Parameters	Mesalamine	Mesalamine Mini-tablet
C_max (ng/mL)	53.65 ± 0.85	35.24 ± 0.45
T_max (Hours)	5.0	10
AUC _(0-48)(h.ng/mL)	440.46 ± 2.16	411.58 ± 1.45
T_1/2(Hours)	4.5 ± 0.25	9.0 ± 1.0
Ke (hr^-1)	0.154 ± 0.018	0.077 ± 0.010

CONCLUSION:

The physicochemical properties of the produced mini-tablets were satisfactory. The Mini-tablets were developed with the use of 3² full factorial design and optimisation methodologies. Based on statistical information gathered via a factorial design approach, Design-Expert® software given output that Mesalamine Mini-tablet in enteric coated capsule with 11.87 5 HPMC K4M and 2.41 % Crospovidone as optimized formula. The drug content, in vitro dissolution, and DSC of the improved formulation were all within acceptable ranges, according to short-term accelerated stability studies. Thus, it can be stated that the formulation is stable. ECDDT demonstrates to be a potential method for delivering medications that are sensitive to acid without enacting the enteric coating techniques included in commercialised formulations. It is evident from the data obtained, demonstrates the variability in pharmacokinetic parameters like T_max, C_max, T1/2 (Hours), AUC_(0-48) and Ke. Henceforth, the newly developed Mini-tablet in enteric coated capsule could be utilized clinically for the treatment of ulcerative colitis, alternatively with cost-effectiveness.

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Received on 13.05.2024 Modified on 18.06.2024

Research J. Pharm. and Tech 2024; 17(8):4035-4042.

DOI: 10.52711/0974-360X.2024.00626