In-vivo study of Budesonide mini-tablets in enteric coated capsules for the treatment of ulcerative colitis

Donepudi Pradyumna*, Lankalapalli Srinivas, Nallamothu Krishna

GITAM School of Pharmacy, GITAM (Deemed to be) University, Visakhapatnam, India.

*Corresponding Author E-mail: pradyu1986@gmail.com

 

ABSTRACT:

Budesonide, a corticosteroid, was used as a model drug to study the effects of independent variables like HPMC K4M as a release retarding polymer at 2-6% and Crospovidone as a super disintegrant at 2-5% on in vitro drug release to meet desired Q points of dissolution. Current study used a 32 factorial design (Stat-Ease Design Expert v12) to formulate a mini-tablet in an enteric-coated capsule to treat ulcerative colitis utilising ECDDT and mini-tablets. An in vivo study was conducted in New Zealand rabbits to determine the pharmacokinetic parameters for the optimized formulation (BF4-O). 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. Pharmacokinetic analysis of Budesonide pre drug plasma concentration–time data provided the following pharmacokinetic parameters like Cmax value ranging (1.42 ± 2.12 ng/mL), Tmax value (4.0 Hours), AUC value (12.77 ± 0.15 h.ng/mL), Half Life (3.5 Hours) and other pharmacokinetic parameters are depicted. Study demonstrates the variability in pharmacokinetic parameters like Tmax, Cmax, T1/2 (Hours), AUC 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.

 

KEYWORDS: Budesonide, Mini-Tablets, Pharmacokinetic, In-Vivo, BF4-O, ECDDT.

 

 


INTRODUCTION: 

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 decrease programme risk by doing away with the preparation and application stages required for enteric coating. 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 development1. Budesonide is used for the prevention of asthma symptoms2-3.

 

 

 

Making mini-tablets that meet the USP dissolution Q values of Budesonide4 is the most difficult aspect of the study to prevent dose dumping. A factorial design was used to develop mini-tablets based on Budesonide. 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 6th Edition; Handbook of Pharmaceutical Excipients 5. Mini-tablets were compressed and evaluated for precompression and post compression parameters before being encapsulated in enteric-coated capsules.

 

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, BF4-O was thought to be an optimized formulation. The drug content, in vitro dissolution, and DSC of the improved formulation (BF4-O) 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 commercialized formulations. As a result, the goal of this study was to evaluate the pharmacokinetics of an optimized Budesonide formulation (BF4-O) of reported in literature6.

 

MATERIALS AND METHODS:

The Budesonide and Betamethasone standards were obtained as a gift sample from Remedium Laboratories Private Limited. Gradient grade methanol, acetonitrile (ACN), and formic acid (GR Grade) were purchased from Merck (Worli, Mumbai, India). All other chemicals used in the study were of analytical grade.

 

Experimental Design

Budesonide 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) and Crospovidone (X2) were selected as independent variables. Excipient levels in factorial design were selected according to handbook of pharmaceutical excipients 6th Edition5. The time dissolution response of drug in 1hr (Q1), 2hr (Q2) and 6 hrs (Q3) was selected as dependent variable6. The optimized formulation which will fit into the model is (BF4), based on the response surface plots reported (Table 1).

 

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 studies7 according to OECD Guidelines 4047. Approval to carry out these studies was obtained from the Institutional Animal Ethics Committee and an experiment was performed in compliance with the Principles8 of Laboratory Animal Care (NIH Publication 85-23, revised 1985).

 

In vivo Study of Budesonide

LC-MS/MS conditions

The liquid chromatograph was an Alliance 2695 system consisting of a quaternary pump, solvent degasser, auto sampler and column heater (Waters Corporation, Milford, USA). Chromatographic separation was achieved on a Waters XBridge MS C18 column (3.5 mm, 2.1 mm x 50mm) in combination with a protecting guard column (3.5 mm, 2.1 mm x 5mm) of the same type (Waters Corporation, Milford, USA). The mobile phase was water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The following gradient program, time (% A – % B), was applied: 0min (90–10), 5min (10–90), 8.5 min (10–90), 9.5 min (90–10), 13.0 min (90–10). The flow rate was 0.3 mL/min and the column temperature was maintained at 35 ℃.9,10

 

Mass spectrometer conditions

Detection was carried out using a Quattro Premier XE triple quadrupole mass spectrometer equipped with an ESCI Multi-Mode Ionization Source (Waters Corporation). The mass spectrometer operated in positive electrospray ionization mode (ESI+) with the following conditions: capillary voltage 3.25kV; source and desolvation temperature 145 8C and 500 8C, respectively; desolvation and cone gas (nitrogen) flow 1000L/h and 100 L/h, respectively. Acquisition was performed in Selected Reaction Monitoring mode, monitoring two specific transitions for each compound, with a dwell time of 100ms. Data analysis9-10 was performed using MassLynx 4.1 Software (Waters Corporation).


 

Table 1: Budesonide mini-tablets formulations

S.No

Ingredient (mg)

BF1

BF2

BF3

BF4

BF5

BF6

BF7

BF8

BF9

1

Budesonide

3

3

3

3

3

3

3

3

3

2

HPMC K4M

0.8

1.6

2.4

0.8

1.6

2.4

1.6

0.8

2.4

3

Crospovidone

0.8

2

1.2

2

1.2

2

0.8

1.2

0.8

4

Microcrystalline Cellulose 112

34.8

32.8

32.8

33.6

33.6

32

34

34.4

33.2

5

Magnesium Stearate

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

6

Talc

0.4

0.4

0.4

0.4

0.4

0.4

0.4

0.4

0.4

Total tablet weight (mg)

40

40

40

40

40

40

40

40

40


Sample preparation

The internal standard solution was prepared diluting BET in water: acetonitrile (1:1) at a concentration of 50ng/mL. Add 20mL of BET internal standard solution to 0.5 mL of plasma. Activate the Strata-X cartridge (1 mL, 30mg) (Phenomenex, Torrance, USA) with 1mL of methanol and wash with 1mL of water. Load the sample on the cartridge, at a flow speed of 2mL/min. Wash the cartridge with 3mL of a solution of 5% methanol in water. Dry the cartridge under low vacuum conditions for 30 s. Elute the analytes with 1.5 mL of methanol at a flow speed of 2mL/min, and completely remove the remaining solvent through vacuum. Evaporate the eluate to dryness under nitrogen stream and heating at 35 8C. Dissolve the sample in 100mL of a mixture of water: acetonitrile (1:1) containing 1% formic acid and vortex for 30 s. Transfer the reconstituted extract to autosampler vial for LC-MS/MS analysis9-10.

 

Preparation of calibration standards and Quality Control Samples

The linearity of the method was assessed with five-points matrix-matched calibration curves, obtained by spiking pooled blank samples of plasma with standard solutions of BUD 0, 0.25, 0.5, 1, 5 and 10 ng/mL, and freshly prepared during different days. Peak area ratios between each analyte and BET (internal standard) were plotted against their concentration ratios, and a linear regression least square regression model was applied. The quality control samples were tested by analyzing blank samples fortified at three concentrations (0.5, 1 and 5ng/mL) for both analytes9-11.

 

Application of the method

The LC-MS/MS procedure was developed to determine Budesonide in rabbit plasma 0–24 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- 0.15mg/kg

AED = 0.15/0.324 = 0.46 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), 1, 2, 3, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 11, 12, 13, 14, 16, 20 and 24 hours post administration12.

 

RESULTS AND DISCUSSION:

HPLC Chromatograms of Pharmacokinetic Study

The chromatograms and retention times of Blank plasma, Budesonide, Internal Standard (Betamethasone), plasma spiked with Budesonide and Betamethasone are shown in Figure 1-4 and Table 2. 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 Internal standard (IS) and analyte. The retention time minutes for IS and minutes for Budesonide 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.

 

 

Figure 1: Chromatogram of Blank plasma

 

Figure 2: Chromatogram of Budesonide

 

Figure 3: Chromatogram of Betamethasone

 

Figure 4: Chromatogram of plasma spiked with Analyte and Internal Standard

 

                Table 2: Details of Chromatograms

S. No

Name of the sample

Retention time (mins)

1

Blank Plasma

0.0

2

Analyte (Budesonide)

8.943

3

Betamethasone (Internal standard)

6.379

4

Budesonide + Betamethasone

8.972 + 6.358

 

Standard Linearity Curve of Budesonide Hydrochloride

The calibration curve results observed over the concentration range of 0.25-10 ng/ml were satisfactory. The regression equation was found to be y = 32353x – 91.05 (Figure 5) with a regression coefficient of 1.000. The linearity of results was depicted in Table 3.

 

Table 3: Linearity for Budesonide

S. No

Conc. (ng/ml)

Peak Area

1

0.25

8954

2

0.5

16212

3

1

31240

4

5

161222

5

10

323732

 

 

 

Figure 5: Linearity of Budesonide

 

Table 3: QC Samples for Budesonide

Amount Added (ng/mL)

Amount Found

0.5

1

5

0.51

1.1

4.91

0.48

0.91

5.12

0.53

0.99

4.89

0.55

0.82

5.14

0.5

1.01

4.95

0.49

1.11

5.08

Mean

0.51

0.99

5.015

Standard Deviation

0.03

0.11

0.11

Table 4: Experimental mean plasma concentration values of pure drug

First group of rabbits administered with pure drug (Budesonide)

Conc. (ng/ml)

Time in Hours

Subject

1

2

3

Mean

Standard

Deviation

0

0

0

0

0

0

1

0.65

0.75

0.7

1

0.05

2

1.06

0.98

1.04

2

0.04

3

1.29

1.32

1.36

3

0.04

4

1.47

1.39

1.4

4

0.04

4.5

1.35

1.38

1.41

4.5

0.03

5

1.21

1.3

1.26

5

0.05

5.5

1.12

1.28

1.16

5.5

0.08

6

1.09

1.08

1.15

6

0.04

6.5

1.03

1.06

1.02

6.5

0.02

7

0.98

1.03

0.99

7

0.03

7.5

0.96

0.94

0.98

7.5

0.02

8

0.85

0.89

0.91

8

0.03

8.5

0.79

0.84

0.78

8.5

0.03

9

0.68

0.76

0.65

9

0.06

10

0.51

0.64

0.58

10

0.07

11

0.49

0.54

0.5

11

0.03

12

0.44

0.5

0.45

12

0.03

13

0.33

0.37

0.32

13

0.03

14

0.31

0.27

0.29

14

0.02

16

0.17

0.21

0.19

16

0.02

20

0.09

0.07

0.08

20

0.01

24

0.03

0.06

0.08

24

0.03

 

 

Table 5: Experimental mean plasma concentration values of

optimized formulation BF4-O

Second group of rabbits administered with Budesonide mini-tablet in enteric coated capsules (Optimized formulation               BF4-O)

Conc. (ng/ml)

Time in

hours

Subject

1

2

3

Mean

Standard

deviation

0

0

0

0

0

0

1

0.00

0.00

0.00

0.00

0.00

2

0.01

0.01

0.01

0.01

0.01

3

0.10

0.01

0.10

0.10

0.01

4

0.32

0.01

0.32

0.32

0.01

4.5

0.41

0.01

0.41

0.41

0.01

5

0.48

0.01

0.48

0.48

0.01

5.5

0.58

0.01

0.58

0.58

0.01

6

0.71

0.01

0.71

0.71

0.01

6.5

0.74

0.01

0.74

0.74

0.01

7

0.64

0.02

0.64

0.64

0.02

7.5

0.58

0.02

0.58

0.58

0.02

8

0.53

0.02

0.53

0.53

0.02

8.5

0.51

0.03

0.51

0.51

0.03

9

0.45

0.02

0.45

0.45

0.02

10

0.39

0.02

0.39

0.39

0.02

11

0.35

0.02

0.35

0.35

0.02

12

0.27

0.03

0.27

0.27

0.03

13

0.23

0.02

0.23

0.23

0.02

14

0.20

0.02

0.20

0.20

0.02

16

0.16

0.03

0.16

0.16

0.03

20

0.10

0.02

0.10

0.10

0.02

24

0.06

0.03

0.06

0.06

0.03

 

 

The present study also estimates the significant PK parameters of the Budesonide Pure Drug and Budesonide Mini-Tablet in Enteric Coated Capsule (Optimized Formulation BF4-O). 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 Tmax (6.5 Hours) and T1/2 (5.5 Hours) was observed for the Optimized formulation BF4-O with Cmax value of (0.74 ± 1.18 ng/mL), AUC(0-24) (12.77 ± 0.15 hr.ng/mL) and Ke (0.126 ± 0.012 hr-1) it may be due to delayed release formulation. The pharmacokinetic analysis of Budesonide pre drug plasma concentration–time data provided the following pharmacokinetic parameters like Cmax value ranging (1.42 ± 2.12 ng/mL), Tmax value (4.0 Hours), AUC value (5.79 ± 0.18 h.ng/mL), Half Life (3.5 Hours) and other pharmacokinetic parameters are depicted in Table 6.

 

Table 6: Pharmacokinetic parameters

Parameters

Budesonide pure drug

Budesonide mini-tablet in enteric coated capsules (Optimized formulation BF4-O)

Cmax (ng/mL)

1.42 ± 2.12

0.74 ± 1.18

Tmax (Hours)

4.0

6.5

AUC(0-24) (h.ng/mL)

12.77 ± 0.15

5.79 ± 0.18

T1/2 (Hours)

3.5 ± 0.5

5.5 ± 0.5

Ke (hr-1)

0.198 ± 0.016

0.126 ± 0.012

 

 

 

Figure 6: Mean plasma concentration time profile after single dose administration in rabbits.

 

 

CONCLUSION:

It is evident from the data obtained, demonstrates the variability in pharmacokinetic parameters like Tmax, Cmax, T1/2 (Hours), AUC 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 27.01.2024            Modified on 24.02.2024

Accepted on 30.03.2024           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(4):1758-1762.

DOI: 10.52711/0974-360X.2024.00279