INTRODUCTION:

Methylprednisolone is glucocorticoids used as anti-inflammatory or immunosuppressive agents; glucocorticoids can inhibit leukocyte infiltration at the site of inflammation, interfere with mediators of inflammatory response, and suppress humoral immune responses¹. Absorption oral bioavailability 80-99%. Orally 7.5 to 60 mg methylprenisolone daily given as a single dose in the morning or every other day as needed for asthma control²

A prodrug is a pharmacological substance (drug) administered in an inactive (or significantly less active) form. Once administered, the prodrug is metabolized in vivo into an active metabolite, a process termed bioactivation. Prodrugs are usually designed to improve oral bioavailability^3,4, with poor absorption from the gastrointestinal tract usually being the limiting factor, methylprednisolone (MPD) for targeting inflammatory sites, and reducing dosages and administration frequency through increasing drug circulation time.

The objective of the present study is to formulate MPD-PECTIN-ES100 matrix enteric coated tablets⁵ that could provide a controlled delivery of MPD to the colonic region for the treatment of inflammation related to colon⁶.

MATERIALS AND METHODS:

Materials

Methylprenisolone was gift sample from Haustus biotech Pvt. Ltd., Una (H.R) India. Eudragit S100 was gift samples from Amishi Drugs and Chemicals, Ahmadabad, India. All other chemicals used were of analytical grade.

Methods

Preparation of Matrix enteric coated Tablets

Matrix enteric coated tablets using pectin was prepared by dry granulation method. MPD, PECTIN and Eudragit S100, Excipients other than glidant and lubricant were accurately weighed, passed through 60-mesh sieve and mixed in a poly bag for 5-10 minutes. The obtained blend was lubricated with talc and magnesium stearate for another 5 minutes and the resultant mixture was directly compressed into tablets with 8 mm round flat punches using 16-station rotary tabletting machine (CEMACH). The final weight of the tablet was adjusted to 300 mg. The compositions of the methylprenisolone matrix enteric coated tablets are given in Table 1.

Powder characterization

The prepared powder mixtures of different formulations were evaluated for angle of repose, bulk density (apparent and tapped) and compressibility index. Mostly we preferred fixed funnel method was employed to measure the angle of repose (θ) and it was calculated using the following formula:

Tan θ = h/r

In which, θ is angle of repose, h is height of the cone and r is radius of the cone base. Angle of repose less than 30⁰ shows the free flowing of the material. The compressibility index (Carr’s Index) is a measure of the propensity of a powder to be compresse⁷d. It is determined from the bulk and tapped densities and is calculated using the following formulas:

Carr’s Index = [(ρ_{tap -}ρ_b) / ρ_tap] / ×100

In which, ρ_b is bulk density and ρ_tap is tapped density.

Evaluation of Physical Parameters

The obtained formulations were studied for their physical properties like weight variation, hardness and friability. For estimating weight variation, 20 tablets of each formulation were weighed using an Electronic weighing balance (AW 120, Shimadzu Corporation, Japan). The hardness of six tablets was measured using Monsanto tablet hardness tester (Lab Hosp). Friability was determined on ten tablets in a Roche friabilator (Electrolab, Mumbai, India) for 4 min at 25 rpm.

Determination of drug content

For estimation of drug content in our formulation, ten tablets were crushed, and 100 mg of the powder was accurately weighed and transferred to a 100-ml volumetric flask. Initially about 50 ml of mobile phase (organic solvents) was added to the volumetric flask and allowed to stand for 6-8 h with intermittent sonication to ensure complete solubility of the drug. Then the volume was made up to 100 ml with mobile phase, filtered and analyzed for MPD content by the UV-method or HPLC method^8.9.

In Vitro Dissolution Study

The release of MPD from matrix enteric coated tablets was carried out using USP XXIV Type II (paddle method) dissolution apparatus (Electro lab, TDT-08L) at a rotation speed of 50 rpm, and a temperature of 37±0.5°C. In order to simulate the gastrointestinal transit conditions, the tablets were subjected to different dissolution media¹⁰. Initially, the drug release was carried out for 2 hrs in 0.1 N HCl, 2 hrs in buffer pH 5.5 and finally in phosphate buffer pH 7.4 or 6.7 up to 24 hrs¹¹. The samples were filtered, by passing through 0.45 µm membrane filters (Millipore, USA) and analyzed spectrophotometrically at 243 nm.

Drug- Polymer Interaction Studies

To study the possible interaction between MP and polymers, DSC study was carried out on pure dug and optimized formulation and the thermograms were obtained using DSC (Perkin-Elmer, Shelton, U.S).¹² The analyses were performed under nitrogen (nitrogen flow rate 50 ml/min) in order to eliminate oxidative and pyrrolytic effects at a standard heating rate of 15ºC/minute over a temperature range of 50ºC - 350ºC. The infrared spectra of MP and optimized formulation recorded between 400 to 4000 cm^-1on FTIR to detect the drug-excipient interactions¹³. The IR spectra for the test samples were obtained using KBr disk method using an FTIR spectrometer (Perkin Elmer FT-IR, Perkin Elmer Inst. USA). The resultant spectra were compared for any possible changes in the peaks of the FT-IR spectra.

Stability Studies

To estimate the drug and formulation stability, stability studies were done according to ICH and WHO guidelines. Optimized formulation F18 sealed in aluminum packaging coated inside with polyethylene, and various replicates were kept in the humidity chamber maintained at 45 ^oC and 75% RH for six months¹⁴. Samples were collected after three and six months of storage and analyzed for the drug content and in vitro dissolution rate.

In vivo X-ray Imaging studies

X-ray imaging technique was used to monitor tablet in-vivo study throughout the GI system. The inclusion of radio-opaque material into the solid dosage form enables it to be visualized by the use of X-rays. By incorporating barium sulphate into the pharmaceutical dosage forms, it is possible to follow the movement, location and integrity of the dosage form after oral administration by placing the subject under a fluoroscope and taking a series of X-rays at various time points. Three healthy human volunteers, male, with an age limit of 22-30 years and 50-70 kg body weight, were participated in in-vivo studies. They should be non-alcoholics, non-smokers and have not taken any drugs. The purpose of the study was fully explained and volunteers had given their written consent¹⁵. Each subject ingested barium sulphate containing optimized formulation (F8) orally with 200 ml water, after an overnight fast. The tablets were visualized using X-ray. Abdominal radiographs were taken after 30 min, 3, 6, 8 and 24 h in all subjects. The volunteers were served with food; 2 h (breakfast) and 4 h (lunch) after the administration of the tablet¹⁶. The protocol of the X-Ray imaging study of colon specific matrix enteric coated tablets of methylprenisolone.

Phase 1 trials of methylprenisolone matrix enteric coated tablets

Under the guidance of G.N.V. Chandra Sekhar and Dr. Govind (MBBS.MD) with human ethical committee permission (Approval No. 312-03/JIPS/JNG/IHEC/2011). These are the earliest trials in the life of a treatment. They are usually small trials, recruiting anything up to about 22 patients, although often a lot less. The trial may be open to people with ulcerative colitis. When laboratory testing shows that a new treatment might help treat ulcerative colitis, phase 1 trials are done to find out

· The safe dose range

· The side effects

· How the body copes with the drug

· The treatment ulcerative colitis

Patients are recruited very slowly onto phase 1 trials. So although they don't recruit many patients, they can take a long time to complete. The first few patients to take part (called a 'cohort' or group) are given a very small dose of the drug. If all goes well, the next group have a slightly higher dose. The dose is gradually increased with each group. The researchers monitor the effect of the drug, until they find the best dose to give. This is called a dose escalation study. In a phase 1 trial, you may have lots of blood tests, as the researchers look at how the drug is affecting you¹⁷. They also look at how your body copes with, and gets rid of the drug. They record any side effects.

People taking part in phase 1 trials often has advanced ulcerative colitis and usually had all the treatment available to them. They may benefit from the new treatment in the trial, but many won't. Phase 1 trials aim to look at doses and side effects. This work has to be done first, before we can test the potential new treatment to see if it works. Phase 1 trials are important because they are the first step in finding new treatments for the future.

RESULTS AND DISCUSSION:

Powder characterization

The powder mixtures of different formulations were evaluated for angle of repose, bulk density (apparent and tapped), compressibility index and their values were shown in Table 1.The apparent and tapped bulk density values ranged from 0.312 to 0.365 and 0.384 to 0.469 respectively. The results of angle of repose and % Carr’s index ranged from 27.12±1.13 to 32.12±1.84 and 18.75 to 22.17 respectively. The results of angle of repose (<35) and compressibility index (<23) indicates fair to passable flow properties of the powder mixture (16).

Table 1 Composition of different enteric coated polymers to cover the Methylprednisolone matric tablets

Ingredients	Quantity (mg) present in the coat formulation
Ingredients	F1	F2	F3	F4	F5
Cellulose Acetate Phthalate	40	-	-	-	-
HPMC-P	-	40	-	-	-
Pectin	-	-	40	-	-
Eudragit L-100	-	-	-	40	-
PVP	-	-	-	-	40
Microcrystalline cellulose	156	156	156	156	156
Talc	4	4	4	4	4
Magnesium stearate	2	2	2	2	2
Total Weight	200	200	200	200	200

Evaluation of Physical Parameters

The physical properties of MPD-pectin-eudragit-S100 matrix enteric coated tablets are given in Table 2. In weight variation test, the pharmacopoeial limit for the tablets of not more than 5% of the average weight. The average percentage deviation of all tablet formulations was found to be within the above mentioned limit and hence all formulations passed the uniformity of weight as per official requirements (India Pharmacopoeia, 1996). The hardness of the tablets was found to be in the range of 5.0-5.6 kg/cm². Another measure of tablets strength is friability. Conventional compressed tablets that loss less than 1% of their weight are generally considered acceptable. The percentage friability for all formulations was below 1%, indicating that the friability is within the prescribed limits¹⁸. The tablets were found to contain 96.8±1.74 to104.2±0.35% of the labeled amount indicating uniformity of drug content. The physical properties like weight variation, thickness, hardness and friability of all formulations were complied with pharmacopoeial standards, so all the tablets were with acceptable physical characteristics.

Table 2. Composition of 1% Eudragit S100 with different concentration of pectin coats used to cover Methylprednisolone matrix tablets

Ingredients	Quantity (mg) present in the coat formulation
Ingredients	F16	F17	F18	F19	F20
Eudragit S100	20	20	20	20	20
Pectin	10	20	30	50	60
Microcrystalline cellulose	164	154	144	124	114
Talc	4	4	4	4	4
Magnesium stearate	2	2	2	2	2
Total Weight	200	200	200	200	200

Methylprenisolone matrix enteric coated tablets assay

AT WS DT P Avg. Wt

% =----- x ----------x --------- x ----------x------------------ X 100

AS DS WT 100 Label Claim

Where:

AT = Peak Area of Methylprenisolone obtained with test preparation

AS = Peak Area of Methylprenisolone obtained with standard preparation

WS = Weight of working standard taken in mg

WT = Weight of sample taken in mg

DS = Dilution of Standard solution

DT = Dilution of sample solution

P = Percentage purity of working standard

System Suitability Results:

1). Tailing factor Obtained from the standard injection is 1.4

2). Theoretical Plates Obtained from the standard injection is 5323.1

Assay Results:

3291413 10 0. 3 10 10 99.8 103

------------x-----x -------x-----x -------x---------x-------x 100= 99.7%

3296013 10 10 41.2 0.3 100 25

Figure 1 Methylprenisolone matrix enteric coated tablets assay chromatograph

In Vitro Dissolution Study

The cumulative mean percent of MPD released from matrix enteric coated tablets containing different type of enteric coated polymers, Formulations with pectin with eutragit S100 of high viscosity formed swollen gel matrix enteric coated with substantial integrity and the drug release was in a controlled manner which could be due to the better control of water and drug diffusion. In comparison with other enteric coated polymers, the tablet lacks strength and was eroded quickly after swelling , comparison to HPMC-P and EC showed negligible drug release in the initial lag period and followed by controlled release for 24 hrs, which is the normal residence time of solid dosage form in the colon From F1 to F5 was found to vary from .16±1.02 to 19.2 ±2.86 after 5 h of testing in simulated gastric and intestinal fluids and the percent drug release was increased gradually after 5 hrs and it was found to be 101.6±2.14 to 28.62±2.36 in 24 hrs (Figure 2). This indicates that a minimal amount of the drug (<20%) is released in the physiological environment of stomach and small intestine and maximum drug release (>80%) was observed in colonic region. From the above formulations, the optimized formula F8 showed the 6 % drug release in the initial lag period (5 hrs) followed by 101% drug release for 24 hrs in a controlled manner¹⁹.

Thus the formulation F8 was considered better among other formulations to produce colon specific drug delivery of MPD. The drug delivery systems targeted to the colon should not only protect the drug from being released in the physiological environment of stomach and small intestine, but also release the drug in colon.

Figure 3 shows the release profiles of MPD from the matrix enteric coated tablets containing varying amounts of pectin. The drug release kinetics studies revealed high correlation coefficient values for zero order than first order indicating that the drug release from matrix enteric coated tablets followed zero order profile²⁰. The high regression value of Higuchi model ensured that the release of drug from matrix enteric coated tablets followed diffusion mechanism.

Figure 2 Release profile of Methylprenisolone from matrix enteric coated tablets prepared by using different types of enteric coated polymers

Figure 3 Release profile of Methylprenisolone from matrix enteric coated tablets prepared by using different percentages of pectin with 20mgEudragit S100

Drug - Polymer Interaction Studies

DSC studies were performed to understand the nature of the drug in the formulated tablets. Thermograms of the pure drug and optimized formulation (F8) are shown in Figure 4. A sharp endothermic peak corresponding to the melting point of MPD was found at 232.5°C . An endothermic peak corresponding to the melting point of MPD in optimized formulation was observed at 231.3°C. Thermogram of the optimized formulation did not show any significant shift in the endothermic peak, indicating that there was no physical change in drug in the pectin with eutragit S 100 matrices²¹.

Figure 4 DSC thermograms of 1) MPD 2) Optimized formulation F8

However, some additional peaks were observed with physical mixtures, which could be due to the presence of polymers. These results suggest that there is no interaction between the drug and polymers used in the present study.

Stability Studies

In view of the potential utility of the formulation, stability studies were carried out at 45 °C and 75% RH for six months (climatic zone IV condition for accelerated testing) to assess their stability. After storage of three months and six months, the formulation was subjected to a drug assay and in vitro dissolution studies²². The data showed that there was no significant change in formulation in the sense of drug content and dissolution behavior.

Table 3 Characterization of powder mixture

Formulation code	Angle of Repose	Bulk density	Tapped Bulk density	% Carr’s Index
F1	29.12±1.24	0.321	0.402	20.149
F2	31.23±1.32	0.332	0.412	19.417
F3	30.35±1.35	0.312	0.386	19.170
F4	29.56±1.46	0.323	0.398	18.844
F5	27.12±1.13	0.325	0.405	19.753
F6	30.35±1.35	0.365	0.469	22.174
F7	32.12±1.84	0.344	0.436	21.100
F8	30.65±1.35	0.332	0.412	19.417
F9	29.56±1.86	0.315	0.402	21.641
F10	32.12±1.23	0.312	0.384	18.750

Table 4 Stability studies MPD Colon Specific Eudragit-pectin Matrix enteric coated Tablets F18

Time (h)	Cumulative percentage MPD release (Mean ±S.D.)
Time (h)	Before storage	*After 3 months	*After 6 months
0	0.00±0.00	0.00±0.00	0.00±0.00
2	1.15±0.37	1.99±0.33	1.95±0.25
4	2.89±0.73	3.11±0.45	3.91±0.13
5	6.95±0.56	7.25±0.16	6.93±0.14
8	30.11±0.11	41.43±0.36	44.34±0.18
12	39.23±0.67	44.29±0.51	46.15±0.58
18	80.05±0.70	81.10±0.23	81.19±0.58
24	96.37±0.75	95.34±0.59	94.98±0.86
% Assay	99.09±0.56	98.11±0.35	98.80±0.27

* Not Significant at 0.05 LS

In vivo X-ray Imaging studies

X-ray studies were carried out on the F8 formulation tablets, in order to see the matrix enteric coated tablets throughout the GI system. Barium sulphate was used as the marker. The position of the tablets in the body was monitored at different time points. The abdominal radiographs showed that, the tablets remained intact in the stomach in all subjects. The transit time of the tablets throughout the GI system was variable. The position of tablets at different time points is shown in the X-ray images of tablet throughout the GI system (Figure 5). The in vivo results showed that the tablets (F8) reached the colon without disintegrating in the upper region of the GI system in all subjects. From the abdominal radiographs, taken at different time points, the tablets entered the colon, varying between 3-6 h for all volunteers after tablet administration. The X-ray images showed that the tablets slowly disintegrated throughout the colon after reaching it. These results are in agreement with the results of Ashford et al. who observed that the gastric emptying times of 0.6–2.9 h, small intestinal transit times of 1.6–8.5 h and colonic arrival time of 3.1–9.6 h while evaluating pectin as a compression coat for colonic drug delivery, using gamma scintigraphy (figure -5) .

Figure 5.Positions of the MPD Colon Specific Eudragit-PECTIN matrix enteric coated Tablets F8 throughout the GI Tract at Different Time Points show in x-ray image

Results of methylprenisolone matrix enteric coated tablets

Phase 1 trials:-

Methylprenisolone matrix enteric coated tablets are the earliest trials in the life of a new treatment. When laboratory testing shows that a new treatment might help treat ulcerative colitis, phase 1 trials are done to find out the first few patients to take part are given a very small dose 4mg of the drug. If all goes well, the next group have a slightly higher dose 8mg. The dose is gradually increased with each group up to 16mg. The researchers monitor the effect of the drug, they find the best dose to give is 16mg. They record any side effects are Oedema, muscle weakness, hypersensitivity reactions, necrosis, the people taking part in phase 1 trials often have advanced ulcerative colitis and have usually had all the treatment available to them. They may benefit from the new treatment in the trial, Phase 1 trials are important because they are the first step in finding new treatments for the future.

CONCLUSION:

Formulation of methylprenisolone matrix enteric coated type colon drug delivery system with acceptable physical characteristics. Pectin - Eudragit matrix enteric coated tablets are capable of protecting the drug from being released in the upper region of GI system, i.e. stomach and small intestine²³. Based on in vitro drug release studies using dissolution apparatus, F18 formulation showed the significant level of drug release in the colon. The drug release from above formulation followed zero order profile and the mechanism of drug release from matrix enteric coated tablets followed super case II transport. DSC and FTIR spectral studies showed that there is no interaction between the drug and excipients and the accelerated stability studies showed the stability of formulation (figure 6).

Table 5 Patients list of phase 1 trials of methylprenisolone matrix enteric coated tablets

S.NO	Phase 1 trials Patients list	SMALLDOSE	MEDIUM DOSE-	OPTIMUMDOSE	SIDE EFFECTS
1	Patient-1	4	8	16	Giddiness
2	Patient-2	4	8	16	Nausea, vomiting
3	Patient-3	4	8	16	Skin atrophy
4	Patient-4	4	8	16	Necrosis
5	Patient-5	4	8	16	Edema
6	Patient-6	4	8	16	GIT effects
7	Patient-7	4	8	16	Muscle weakness
8	Patient-8	4	8	16	Neckrosis
9	Patient-9	4	8	16	Skin atrophy
10	Patient-10	4	8	16	Hypertension
11	Patient-11	4	8	16	Nausea, vomiting
12	Patient-12	4	8	16	Giddiness
13	Patient-13	4	8	16	Muscle weakness
14	Patient-14	4	8	16	Edema
15	Patient-15	4	8	16	GIT effects
16	Patient-16	4	8	16	Necrosis
17	Patient-17	4	8	16	Skin atrophy
18	Patient-18	4	8	16	Hypertension
19	Patient-19	4	8	16	Edema
20	Patient-20	4	8	16	Giddiness
21	Patient-21	4	8	16	Muscle weakness
22	Patient-22	4	8	16	Nausea, vomiting