INTRODUCTION:

Budesonide is a novel synthetic corticosteroid and drug of the first choice in the treatment of inflammatory bowel disease, especially in the treatment of ulcerative colitis and Crohn’s disease.¹ Budesonide have half-life of 2-4 h with an oral bio availability of 10%. Inflammatory bowel diseases can be treated more effectively by local delivery of drug targeted to the colon.

Colonic drug delivery is also useful for enhanced systemic absorption of drugs because of less hostile environment existing in the colon compared to stomach and small intestine. Budesonide undergoes approximately 85% first pass metabolism.² To overcome this drawback, the present study was undertaken to investigate the colon targeted drug delivery system of budesonide through core in cup tablet.³ Due to the distal location of the colon in the gastrointestinal tract, pulsatile drug delivery should prevent drug release in the stomach and small intestine and produce a gradual onset of drug release upon entry into the colon.⁴Hence in the present study, core in cup tablet of Budesonide was designed with the intention of delivering the drug in the colon region for effective treatment of inflammatory bowel disease. Optimized tablet formulations demonstrated good potential to deliver the drug to the colon by successfully exhibiting a lag time of 5 h during in vitro drug release study.⁵ An in vivo evaluation study conducted to ascertain pharmacokinetic parameters in rabbits revealed that the onset of drug absorption from the Core-in-cup tablets was significantly delayed compared to that from the pure Budesonide suspension.

MATERIALS AND METHODS:

The in vivo study of the optimized formulations were performed as per the guidelines approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of social Justice and Empowerment, Government of India. Prior approval by Institutional animals ethics committee was obtained for conduction of experiments (Ref: IPT /IAEC/1053/PO/Re/S/076/CPCSEA, Dated 27-12-2020). Pure Budesonide suspension and optimized core in cup tablet prepared in the laboratory conditions and chosen on the basis of lag time achieved, in-vitro release studies and stability conditions were chosen as dosage forms for administration.

Preparation of Budesonide core tablets:

After preliminary experiments, the optimized formulation of core tablets was obtained. The core tablets were prepared by wet granulation method. The required quantities of Budesonide, PVPK-30 (as a binder), Moringa olifera gum (as a polymers) and lactose (as a diluent) were weighed and mixed uniformly and prepared a wet mass by addition of binder solution. The wet mass was passed through sieve number #12 and allowed to drying for 30 minutes in a tray dryer for 60⁰C. The dried granules were passed through the sieve number #16 and finally lubricated with talc and magnesium stearate. The obtained dry granules were weighed into individual tablets and finally compressed into the tablet by 16 station rotary tablet compression machine using 9mm flat punches.

Preparation of cup tablet:

The cup formulations were formulated by direct compression technique. In which the required quantities of Eudragit RS100 and microcrystalline cellulose, were weighed and mixed uniformly and finally the powder mixture was compressed by 16 station rotary tablet compression machine by using special punch designed and fabricated, to prepare cup tablets. The newly designed upper 12mm punch has protrusion and lower punch (12mm) remains flat faced.

Preparation of core in cup tablet:

The cups were placed in a 12mm die cavity and core tablet was inserted into the cups and compressed with 12mm flat faced punches⁶.

Enteric coating:

Core in cup tablet were further coated with enteric coating polymer (cellulose acetate phthalate) by spray coating method. 10% cellulose acetate phthalate in 8:2 (v/v) mixture of acetone: ethanol plasticized with dibutyl phthalate (0.75%), was used as a coating solution. Talc (0.1% w/v) was added as antiadherant and the solution was stirred for 15 min. Placed the core in cup tablets into a coating pan, the coating solution was sprayed over the tablets by R&D coater, rotating with a speed of 15 rpm, the pressure of the spray gun was maintained at 0.1 M.Pa and the air temperature was maintained at 35-40⁰C. The tablets were coated to a 5%, 7.5% and 10% w/ w total weight gain.

In vivo evaluation:

Subject selection:

Twelve New Zealand healthy rabbits with a mean age of 10±2 weeks and with a mean body weight of 3±0.2kg were used in this study. Each group consisted of six rabbits (n=6) each and were subjected for overnight fasting, it was taken care that there was no stress on the animals⁷. Rabbits were randomly divided into two groups for different sampling time and each group was housed in one cage. Food and water were available ad libitum at all times during the experiment. The study was conducted in a crossover design with 2 weeks washout periods in between the two experiments. The animal dose of Budesonide was calculated relevant to human dose by using the following formula. The above dosage form was administered through gastric intubation method⁸.

Human dose of Budesonide = 9mg.

Human dose × Animal weight

Animal dose = ----------------------------------------

Human weight

= 9x3/70= 0.385 mg= 0.4mg

Blood sampling:

About 1ml of blood samples were collected from the tracheal lobular vein of the rabbit using and the blood was stored in screw top heparinized plastic tubes, the sampling time for blood was done at 0 mins (Predose), 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 24hr and 48 hr. The plasma was immediately separated by aspiration after centrifugation at 4000rpm for 5 minutes and frozen at -20ºC until analyzed by LC-MS/MS method ⁹.

Determination of Pharmacokinetic Parameters:

Various pharmacokinetic parameters such as peak plasma concentration (Cmax), time at which peak occurred (Tmax), area under the curve (AUC), elimination rate constant (Kel), biological half-life (t½) and mean residence time (MRT) were calculated using the noncompartmental pharmacokinetics data analysis software PK Solutions 2.0™ (Summit Research Services, Montrose, CO, USA). The pharmacokinetic parameters of the tested formulations were statistically analyzed using paired sample’s t-test for normal distributed results of Cmax, Ka, Ke, MRT and AUC0-α value. All tests were performed at 0.001 level of significance¹⁰.

Estimation of Budesonide in plasma (LC-MS/MS method:

Chromatographic conditions:

A summary of the chromatographic and mass spectrometric conditions is as follows^11-12:

HPLC: Shimadzu Nxera X2

Mass: Shimadzu LCMS 8050

Ion source: Turboion spray

Polarity: Positive ion mode.

Detection ions:

Budesonide: 431.00*amu (parent), 323.20* amu (product)

Budesonide–D8: 439.20* amu (parent), 323.25* amu (product)

Column: Waters Acquity, C18, 2.1X100mm, 1.7μm.

Column oven temperature: 40.0°C

Peltier temperature: 15°C

Volume of injection: 20μl

Split ratio: 50:50

Retention times: Budesonide: 2.50 to 3.70 minutes

Budesonide–D8: 2.50 to 3.70

minutes

HPLC Run time: 4.51 minute

Mass Run time: 4 minutes

Preparation of working standard solutions:

1. Preparation of Budesonide stock solution:

Budesonide working standard equivalent to 5mg of Budesonide was weighed and transferred in to a 5ml volumetric flask and dissolved in methanol. The solution was made up to the volume with methanol. The concentration of resulting solution was calculated by considering the purity of Budesonide. The solution was labeled and stored in a cold room at 2-8°C. The stock solution was diluted methanol to get a concentration about 1μg/ml.

2. Preparation of Budesonide –D8 as internal standard stock solution:

Budesonide – D8 working standard equivalent to 5mg of Budesonide – D8 was weighed and transferred into a 5 ml volumetric flask and dissolved in methanol. The solution was made up to the volume with methanol. The concentration of resulting solutions was calculated by considering the purity of Budesonide –D8. The solutions were labeled and stored in a cold store at 2-8°C.

Calibration curve standards:

1. Preparation of stock dilutions of standard Budesonide solution:

Stock dilutions of Budesonide ranging from 1ng/ml to 240ng/ml were prepared with 50% acetonitrile in water using dilutions of main stock solution prepared for calibration curve standards.

2. Spiking of plasma for calibration curve standards:

Concentrations of Budesonide ranging from 50pg/ml to 12000pg/ml with screened K2EDTA human plasma using final dilutions from the Table 5.6 and were labeled as CC1 to CC8. The calibration curve standards were prepared fresh for each validation run.

Sample preparation:

Step 1: Blank, calibration curve standards and the subject samples were withdrawn from the deep freezer and allowed them to thaw. The thawed samples were vortexed to ensure complete mixing of the contents. To 3ml of plasma sample in a ria vial, 30μl of Budesonide –D8 (150ng/ml) was added. To plasma blank and pre-dose (0.0hr), 30ul of 50% acetonitrile in water solution was added. The samples were vortexed to ensure complete mixing of contents.

Step 2: Orochem C18 100mg/1ml cartridge were taken, (new cartridge for each sample) on to a positive pressure processor and the following procedure was followed:

1. Conditioning:

1ml methanol and followed by 1ml of water was added.

2. Loading:

The sample was applied and allowed it to dry for about 1 minute under positive pressure.

3. Rinsing:

The cartridge was rinsed with 1ml of water followed by 1ml of 2% formic acid in water followed by 1ml of 5% methanol in water and was allowed to dry under positive pressure for about I minute.

4. Elution:

The drug was eluted into 1ml of acetonitrile and was allowed to dry under positive pressure for approximately 1minute. The organic layer was evaporated under a stream of nitrogen gas at 50°C. The residue was reconstituted with 0.3ml of mobile phase and vortexed. The samples were transferred in to auto-injector vials and loaded the vials in to auto sampler.

20ul of sample was injected in to LC-MS/MS system. Analyte Concentrations of stock dilutions of standard Budesonide solution with plasma were shown in Table1.

Data processing:

The chromatograms were obtained by using the computer-based lab solution software, version 5.8 supplied by the Schimadzu Corporation. The concentrations of the unknown samples have to be calculated from the equation using regression analysis of spiked plasma calibration standard with 1/x2 as weighting factor.

y = mx + c; Where, y = Ratio of Budesonide peak area and Budesonide –D8 peak area (Analyte area/ISTD area); x = concentration of Budesonide; m = slope of the calibration curve; c = y-axis intercept value. Linear regression analysis equation of stock dilutions of standard Budesonide solution with plasma is y = 8E-0.0005.

RESULTS AND DISCUSSION:

In the present study, pH-dependent polymer (Eudragit RS100) with an overcoat of Cellulose acetate phthalate was suitable for adequately sustained drug release and to protect Budesonide from being released in the upper region of the GI system.

In the present investigation-controlled release formulations of core tablets for per-oral administration were prepared through wet granulation technique by using moringa olifera gum. The granules were characterized with respect to angle of repose, bulk density, tapped density, Carr's index, and Hausner ratio. The angle of repose was less than 30° (27.24 ± 0.08 °), indicating good flow behavior. Similarly, bulk density and tap density were found to be from 0.540 ± 0.008 g/ml and .681±0.008g/ml, depicting good flow properties of the granules. The Carr's index of all formulation batches was in the acceptable range (14.97 ±0.08). The Hausner ratio was found to be (1.17±0.05) less than 1.25 indicates good flowability.

The weight of each tablet was determined to be within the range of 200±5mg in order to maintain the relatively constant volume and surface area. All the formulated preparations were subjected to weight variation, hardness, friability and drug content. All tablets complied I. P. weight variation test requirement. The hardness was found to be in between 4 - 5kg. The tablets satisfied USP friability requirement, as the % friability values are less than 1%. The percent drug content was found to be within 98 - 102% of the labeled amount and hence complied drug content requirement. The core tablet was kept in a core tablet and further core-in-cup tablet was coated with 10% w/w Cellulose acetate phthalate. Core-in-cup tablet with a coating level of 10% w/w showed a lag time of 5hr corresponds to time required to reach colonic region.

During dissolution studies, it was observed that, the enteric coated core in Cup tablets was intact for 2 hrs in pH 1.2, and also in intestinal pH 7.4. With all the formulations, there was absolutely no drug release in pH 1.2 and also in intestinal pH 7.4., thus indicating the efficiency of 10% CAP for enteric coating.

The In vitro drug release studies indicate that the optimized formulation was a promising system targeting Budesonide to the colon. The obtained results showed the capability of the system in delaying drug release for a programmable period of time and the possibility of exploiting such delay to attain colon targeting. The in vivo experiments were conducted as per the protocol and procedure described earlier. The ability of core in cup tablet as a drug delivery system to release drugs in a predetermined time release manner was investigated in rabbits after oral administrations was investigated. Bioanalytical methods employed for the quantitative determination of drugs and their metabolites in biological matrix (plasma, urine, saliva, serum etc) play a significant role in evaluation and interpretation of pharmacokinetic data. For the successful conduct of pharmacokinetic study, the development of selective and sensitive bioanalytical methods plays an important role for the quantitative evaluation of drugs and their metabolites (analytes). The LC-MS/MS methods were highly sensitive and suitable for the detection of drug in plasma even in low concentrations. Calibration curves were constructed from blank sample (plasma sample processed without IS), blank+IS samples and eight point calibration standards for Budesonide in plasma. Plasma concentrations of Budesonide at different times were calculated and are shown in Table 3 and in Fig 2. Pharmacokinetic parameters such as absorption rate constant, elimination rate constant, half life, AUC, and MRT were calculated from the plot of time versus plasma concentration and subjected to statistical analysis and the results were shown in Table 5. The results from the oral administration of Pure Budesonide suspension indicated the maximum plasma concentration (Cmax) 47.89±0.05ng/ml at 2 hr (Tmax) while pulsatile formulations administration exhibited the maximum plasma concentration (Cmax) of 53.80±0.03ng/ml after an initial lag time of 5 hrs. The oral administration of Budesonide resulted in a low and quite variable AUC of 154.1±1.44 ng/ml/hr, whereas the optimized core-in-cup tablets resulted in AUC of 918.2.±3.11ng/ml/hr. The mean residence time of optimized core -in-cup tablets administration (28.9±0.09hrs) was found to be more than oral administration (8±0.03hrs). The bioavailability of optimized formulation was enhanced six times compared with pure drug suspension.

CONCLUSION:

An in vivo evaluation study conducted to ascertain pharmacokinetic parameters in rabbits revealed that the onset of drug absorption from the Core-in-cup tablets was significantly delayed compared to that from the pure Budesonide suspension. In the present study, optimized core-in-cup tablets formulation shown drug release over a period of 5-17 hrs, consistent with requirements for chrono pharmaceutical drug delivery, was achieved. Thus, core-in-cup tablets formulation parameters could be modified to modulate the drug release time in accordance with chronotherapeutic objectives.

Table 1: Composition of optimized Budesonide Core tablets

Ingredients	Core tablet (mg)
Budesonide	9
Povidone	10
Moringa olifera gum	75
Lactose	102
Magnesium stearate	2
Talc	2
Total	200

Table 2: Composition of optimized Budesonide Cup tablets

Ingredients	Cup tablets(mg)
Eudragit RS100	400
MCC	42
Mg. stearate	4
Talc	4
Total	450

Figure 1: Calibration Curve for Estimation of Budesonide in Plasma

Table: 3. Analyte Concentrations of Stock Dilutions of Standard Budesonide Solution with Plasma

Sl. No.	Sample Name	Analyte Concentra tion (ng/ml)	Analyte peak area	IS Peak Area	Area Ratio	Calculated Concentration (ng/ml)	Accuracy (%)
1	RSS	-	502981	412031	1.22	14610.232	-
2	Plasma Blank	-	-	4172	-	N/A	N/A
3	Blank+IS TD	0	-	891458	-	N/A	N/A
4	CC1	50	4068	938751	0.00	50.84	101.72
5	CC2	100	7881	974867	0.01	95.73	95.76
6	CC3	300	26922	1036372	0.03	309.912	103.31
7	CC4	900	81904	1112911	0.07	879.843	97.77
8	CC5	2000	179072	1073112	0.16	1996.281	99.82
9	CC6	4000	350834	1062488	0.33	3951.201	98.79
10	CC7	8000	767292	1153923	0.67	7957.786	99.48
11	CC8	12000	1156678	1115547	1.04	12409.494	103.42

Figure: 2. Chromatograms of reserve stock solution of Standard Budesonide Solution with Plasma

Figure: 3. Chromatograms of Plasma blank

Table: 4. Plasma Concentration of Budesonide following oral administration of core-in-cup tablets and pure drug suspension

Time (h)	Plasma concentration of Budesonide core-in-cup tablets (ng/ml) (Mean ± S.D)	Plasma concentration of pure drug suspension (ng/ml) (Mean ± S.D)
0.0	0	0
0.5	0	9.12±0.03
1.0	0	22.34±0.02
2.0	0	47.89±0.05
4.0	0	28.12±0.04
6.0	11.67±0.02	11.59±0.03
8.0	26.78±0.03	05.45±0.02
10.0	42.89±0.02	01.59±0.03
12.0	53.80±0.03	0.34±0.04
16.0	39.27±0.02	0. 1±0.03
20	29.86±0.03	0.1±0.03
24.0	24.21±0.02	0.1±0.02
32.0	11.9±0.04	0.08±0.02
48	2.54±0.02	0.03±0.02

Figure: 4. Concentration-Time Curve of Budesonide following of core-in-cup tablets and pure drug suspension

(·) Budesonide following oral administration of pure drug suspension

(·) Budesonide following oral administration of core-in-cup tablets

Table: 5. Statistical Treatment of Pharmacokinetic Parameters (Mean±S.D.) of following oral administration of pure drug suspension and core-in-cup tablets of Budesonide

Pharmacokinetic parameter	Pure drug suspension	Core-in-cup tablets	Calculated value of ‘t’
Cmax(ng/ml)	47.89 ±0.05	53.80±0.03	14.16***
MRT (h)	8 ±0.03	28.9 ±0.09	22.14***
Tmax(h)	2.00 ±0.005	12 ±0.08	6. 42***
Kel(h-1)	0.122 ±0.003	0.086 ±0.002	3.43***
Ka(h-1)	0.220 ±0.002	0.191 ±0.003	09.46***
AUC0-∞(ng h/ml)	154.1±1.44	918.2.±3.11	124.26***

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Received on 23.07.2021 Modified on 13.09.2021

Research J. Pharm. and Tech 2022; 15(10):4553-4558.

DOI: 10.52711/0974-360X.2022.00764