1. INTRODUCTION:

Oral route of drug delivery is considered the favored and most user friendly means of drug administration having the highest degree of patient compliance, as a result of which much efforts are aimed to identify orally active candidates that would provide reproducible and effective plasma concentrations in vivo^[1,2].

Pulsatile system is amongst one of them and gaining a lot of interest as it increasing patient compliance by means of providing time specific and site specific drug delivery system, thus providing spatial and temporal delivery. Pulsed or Pulsatile drug release is defined as rapid and transient release of a certain amount of drug molecules within a short time period immediately after a predetermined off release timing of medication regimens improve outcome in selected chronic conditions^[^3-5,]. Diseases where in constant drug levels are not preferred, but need a pulse of therapeutic concentration in a periodic manner, act as stimuli for the development of “Pulsatile Drug Delivery Systems”. Pulsatile release dosage forms release drug in pulsatile form.

Lag time is defined as the time between when a dosage form is placed into an aqueous environment and the time at which the active ingredient begins to get released from the dosage form; precisely lag time is an interval of no drug release followed by rapid drug release. Pulsatile drug delivery is useful in chrono pharmacotherapy of diseases following circadian rhythm in their pathophysiology, avoiding the first pass metabolism e.g protein and peptides, drugs for which the tolerance is rapidly established, for targeting specific site in intestine e.g. colon, for time programmed administration of harmone and drugs, for drugs having short half life.

Montelukast sodium is a hygroscopic, optically active, and white to off-white powder. Montelukast sodium is freely soluble in ethanol, methanol, and water and practically insoluble in acetonitrile. Montelukast is a leukotriene receptor agonist (LTRA) used for the maintenance treatment of asthma and to relieve symptoms of seasonal allergies. It is usually administered orally once a day. Montelukast is a CystLT₁antagonist; it blocks the action of leukotriene receptor CysLT₁in the lungs and bronchial tubes by binding to it. This reduces the bronchoconstriction otherwise caused by the leukotriene and results in less inflammation. Because of its method of operation, it is not useful for the treatment of acute asthma attacks. Again because of its very specific mechanism of action it does not interact with other asthma medications such as theophylline. This study attempts to design and evaluate a time controlled chronomodulated drug delivery system of Montelukast sodium with different disintegrant and diluents for the treatment of nocturnal asthma. It was aimed to have a lag time of five hours i.e., the system is taken at bed time and expected to release the drug after a period of five hr when the asthma attacks are more prevalent.

2. MATERIALS AND METHODS:

Montelukast sodium was received as gift sample from Aurobindo Pharma Ltd., Hyderabad. MCC PH 102 was purchased from Brahmar Cellulose., Pharmatose, Ac-di-sol, Sodium starch glycolate, Cross povidone were collected from FMC Biopolymer., Mannitol, HPMC, Xanthan gum, Sodium alginate, Magnesium stereate were collected from S.D.Fine chemicals, and Cellulose acetate phthalate was collected from Ranbaxy fine chemicals. Ltd.

UV-Visible Spectrophotometer– Shimazdu., I.R Moisture Analyzer, Digital Weighing balance – Sartorius., Tapped Density Tester USP, Dissolution test apparatus, dessicator –Electrolab.,10 Station Compression Machine- Cadmach., Hot air oven, Stability Chambers-Thermolabs., Analytical Sieve Shaker, Rapid dryer-Retsh., PH meter- Lab India instruments., Digital melting point apparatus-Avon engg works., Fourier transform infrared spectrophotometer- Bruker, Germany, Vernier Calipers- Digimatic caliper., Mechanical stirrer–Remi motors, Sieves–United engineering.

2.1 Preformulation studies:

The following preformulation study was carried out

2.1.1 Drug Excipient Compatibility study by IR-spectroscopy:

In this study, KBr disc method was employed to conduct IR studies on pure drug and the physical mixture containing the diluent blend containing micro crystalline cellulose: Mannitol, superdisintegrant Crosscaremellose sodium, and the hydrogel forming polymers including HPMC K4M, HPMC E10M, Xanthan gum, Sodium alginate. The sample was powdered and mixed with KBr (1:100). The mixture was then compressed into transparent disc under high pressure using hydraulic pellet press. This disc was placed in FT-IR spectrophotometer using sample holder and IR spectrum was recorded in the range of 400 to 4000 cm-1. Any changes in the chemical composition after combining with the excipients were the investigated with IR spectral analysis.

2.1.2 Preparation of Montelukast sodium granules:

Different formulations containing Montelukast sodium were prepared by varying diluents and superdisintegrants. All the ingredients except magnesium stearate were weighed and passed through 40#. The mixture was taken into a mortar and granules were prepared by hand granulation by tirturation with pestle using water as binder. The obtained granules were dried in a rapid dryer for 15-20 min. Magnesium stearate was passed through 60# and added to dried granules

2.2 Evaluation of Montelukast sodium granules ^[6-8]:

2.2.1 Bulk density ^[6]:

Bulk density was determined by pouring gently 25 gm of sample (Montelukast sodium granules) into 100 ml graduated cylinder. The volume occupied by the sample was recorded. Bulk density was calculated as:

Bulk density = weight of sample in grams / volume occupied by the sample

2.2.2 Tapped density ^[7]:

Tapped density was determined by using Electro lab density tester, which consists of a graduated cylinder mounted on a mechanical tapping device. An accurately weighed sample of granules was carefully transferred to the cylinder (USP II). Typically, the initial volume was noted, and the sample is then tapped (500, 750, or 1250 tapping) until no further reduction in volume is noted or the percentage of difference is not more than 2%. A sufficient number of taps were employed to assure reproducibility for the material. Tapped volume was noted and tapped density is calculated using following formula

Tapped density = wt. of sample in grams / Tapped volume

2.2.3 Carr’sindex and Hausner’s ratio ^[7]:

Both the Carr’s index and the Hausner’s ratio were determined by using bulk density and the tapped density of granules.

Carr’s index (%): Carr’s index was calculated using the formula:

Carr’s index = Tapped density – bulk density / Tapped density *100

Hausner’s ratio: It was calculated using the following formula:

H.R = Tapped density / Bulk density

2.2.4 Angle of Repose ^[7]:

The angle of repose of granules was determined by the fixed funnel method. The accurately weight granules were taken in the funnel. The height of the funnel was adjusted in such a way the tip of the funnel just touched the apex of the powder blend. The granules were allowed to flow through the funnel freely on to the surface. The diameter of the granules cone was measured and angle of repose was calculated using the following equation.

Tan θ = h/r

θ = Tan-¹h/r

2.2.4 Friability: Montelukast sodium granules were subjected to friability in Roches friabilator. 2grams of granules which were retained on sieve#16 were subjected to 100 revolutions in friabilator. Then granules were withdrawn from the friabilator, and received, the retained granules on sieve no 16 were weighed and noted as final weight. The % Friabilty was calculated from the following formulae.

Initial weight – Final weight

% Friability = --------------------------------------------- X 100

Initial weight

2.2.5 In-vitro dissolution studies^[^8]:

In- vitro dissolution studies were performed for all the prepared granules by using USP dissolution apparatus I. The dissolution test was carried for a period of 30 min., at 100 rpm using 900ml of 6.8 P^H phosphate buffer with 0.5% SLS as the dissolution medium maintained at 37±0.5ºC. At appropriate time intervals (5, 10, 15, 20, 30 minutes) 10 ml of the sample was withdrawn and replaced with the same volumes of dissolution medium. The absorbance of the samples was measured at 346nm against blank using UV spectrophotometer to determine the amount of drug release.

2.3 Preparation of cross-linked gelatin capsules:

2.3.1 Formaldehyde treatment ^[9]:

Bodies of hard gelatin capsules (Size 3) were placed on a wire mesh. 25 ml of formaldehyde (15%v/v) was taken into a desiccators and potassium permanganate was added to it until vapors were produced. The wire mesh containing the bodies was then exposed to formaldehyde vapors.

The caps were not exposed leaving them water- soluble. The desiccators were tightly closed. The reaction time was optimized by removing capsule bodies at different time intervals and dried at 50ºC for 30 min to ensure completion of reaction between gelatin and formaldehyde vapors. The bodies were then dried at room temperature to ensure removal of residual formaldehyde. These capsule bodies capped with untreated caps and stored in a polythene bag.

Various physical and chemical tests carried out simultaneously for formaldehyde treated and untreated capsules.

2.4 Formulation of modified pulsincap drug delivery system ^[10-11]:

Formaldehyde treated hard gelatin capsules of size 3 were chosen for formulation. The bodies and caps were separated manually. The granules composed of Mannitol:MCC(1:1) as diluent, Cross caremellose sodium (Ac-di-sol) as disintegrant were filled into capsule body which was pre treated with formaldehyde vapour. Montelukast granules were prepared by using suitable diluent and disintegrants.120 mg granules equivalent to 10 mg Montelukast sodium were accurately weighed and filled in treated capsule bodies by hand filling method. The capsule bodies containing granules were then plugged with different polymers like HPMC K4M, HPMC E 10M, Sodium alginate, Xanthan gum separately at different concentrations (1-30mg,2-40mg, 3-50mg).The resulting bodies were sealed with a cap precoated with the enteric coating polymer CAP. So formulation selected for pulsincap system. Untreated caps were fitted to filled and plugged capsule bodies. The joint of capsule body and cap was sealed with 5% ethanol solution.

2.5 Coating of capsules with cellulose acetate phthalate ^{[9, 12]}:

The filled capsules were completely coated with 5% cellulose acetate phthalate to prevent variable gastric emptying. Coating was repeated until a 10% increase in weight was obtained. %weight gain of the capsules before and after coating was determined.

5%w/v solution of CAP was prepared using acetone: ethanol (8:2) as solvent and 0.5%w/v dibutyl phthalate as plasticizer. Dip coating method was followed to coat the filled capsules. Coating was repeated until an expected weight gain i.e 10% was obtained after drying of coated capsules. This coating resists 0.1 N Hcl and prevents the drug release in stomach.

Upon reaching the intestine at greater PH i.e 6.8, the CAP coating dissolves as a result the capsule cap dissolves and the hydrogel plug gets exposed to dissolution media. The capsule body remains intact due to formaldehyde treatment for 12hrs. The hydrogel plug ejects on swelling and releases the drug from the capsules after a specific lag time.

2.6 Evaluation of modified pulsincap ^[13-16]:

2.6.1 Weight variation:

10 capsules were selected randomly from each batch and weighed individually for weight variation and test was performed according to official method. The average percentage weight variation should be within pharmacopoeial limits of ±10%.

2.6.2 Drug content:

10 capsules of Montelukast were utilized for the assay. The drug was extracted using 6.8 P^H phosphate buffer with 0.5% SLS. The solution was filtered, made up to the volume with 100 ml 6.8 P^HPhosphate buffer with 0.5% SLS, diluted suitably and analyzed spectrophotometrically by measuring the absorbance at 346nm.

2.6.3 Lag time:

Lag time is the total time period after which the plug is ejected out of the capsule body and the drug releases immediately. Lag time was determined visually using buffer PH 1.2 with 0.5% SLS (for 2 hrs) and 6.8 with 0.5% SLS further. For lag time determinations USP II apparatus was used. Capsules were placed in dissolution media using sinkers. The temperature was maintained at 37ºC and paddle speed at 50 rpm. The time at which the polymers plug ejected out was observed visually and noted.

2.6.4 In-vitro release profile ^[16]:

Dissolution studies were carried out by using USP I dissolution test apparatus (Basket) method. 900 ml of dissolution media was and temperature was maintained at 37±0.5ºC and rpm 100. Capsules were placed in a basket so that the capsule should be immersed completely in dissolution media but not float. In order to stimulate the PH changes along the GI tract two dissolution media with PH 1.2, 6.8 with 0.5% SLS were sequentially used. Dissolution studies were performed in PH 1.2 medium (0.1 N HCL) with 0.5% SLS for 2 hrs. After 2 hrs the dissolution medium was replaced with PH 6.8 phosphate buffer with 0.5% SLS. 10 ml of samples were withdrawn for every 30 min time interval (0.5hr, 1hr, 1.5hr, 2hr, 2.5hr, 3hr, 3.5hr, 4hr, 4.5hr, 5hr, 5.5hr, 6hr) and replaced with fresh dissolution media. The withdrawn samples were analyzed at 346nm by UV absorption spectroscopy.

Table 1: Composition of Montelukast sodium capsules with different concentrations of polymer plugs, diluents and disintegrants:

Ingredients	A1	A2	A3	B1	B2	B3	C1	C2	C3	D1	D2	D3
Drug	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4	10.4
Mcc	49.2	49.2	49.2	49.2	49.2	49.2	49.2	49.2	49.2	49.2	49.2	49.2
Mannitol	49.2	49.2	49.2	49.2	49.2	49.2	49.2	49.2	49.2	49.2	49.2	49.2
Ac-di-sol	10	10	10	10	10	10	10	10	10	10	10	10
Mg.stereate	1.2	1.2	1.2	1.2	1.2	1.2	1.2	1.2	1.2	1.2	1.2	1.2
HPMC K4M	170±10	215±15	330±10	-	-	-	-	-	-	-	-	-
HPMC E10M	-	-	-	165±15	215±15	240±15	-	-	-	-	-	-
Sodium alginate	-	-	-	-	-	-	250± 10	275± .10	315± 15	-	-	-
Xanthan gum	-	-	-	-	-	-	-	-	-	150±10	175±10	200± 10

Tablet 2: Evaluation results of modified pulsincap:

Capsules with different polymers	%Drug content	Average weight	Lag time(hrs)
A1	98.25	288.64±3.51	4
A2	96.52	330.56±2.62	3
A3	99.62	440.84±4.55	5
B1	95.65	290.15±2.45	3
B2	97.54	333.15±3.50	3
B3	98.38	358.25±1.95	4
C1	97.95	368.55±4.95	4
C2	98.58	382.75±5.54	4
C3	98.65	395.77±4.44	4
D1	96.56	265.55±5.25	2
D2	98.65	290.62±2.54	3
D3	97.85	315.55±4.55	3

Table 3: In-vitro drug release studies of pulsincap formulations with HPMC K4M hydrogel plugs

Dissolution media	Time (hrs)	% Cumulative drug release
Dissolution media	Time (hrs)	A1	A2	A3
0.1N Hcl +0.5%SLS	1	0	0	0
0.1N Hcl +0.5%SLS	2	0	0	0
6.8 PH phosphate buffer + 0.5%SLS	3	0	0	0
	3.5	67.5±0.5	0	0
	4	98.5±0.5	0	0
	4.5	98.5±0.5	58.5±0.5	0
	5	98.5±0.5	97.5±0.5	0
	5.5	98.5±0.5	97.5±0.5	72±0.5
	6	98.5±0.5	97.5±0.5	98.5±0.5

Table 4: In-vitro drug release studies of pulsincap formulations with HPMC E10M hydrogel plugs

Dissolution media	Time (hrs)	% Cumulative drug release
Dissolution media	Time (hrs)	B1	B2	B3
0.1N Hcl +0.5%SLS	1	0	0	0
0.1N Hcl +0.5%SLS	2	0	0	0
6.8 PH phosphate buffer + 0.5%SLS	3	0	0	0
	3.5	55.8±0.4	0	0
	4	96.5±0.5	0	0
	4.5	96.5±0.5	76.5±0.5	55±0.5
	5	96.5±0.5	97.5±0.5	97±0.5
	5.5	96.5±0.5	97.5±0.5	97±0.5
	6	96.5±0.5	97.5±0.5	97±0.5

3. RESULTS AND DISCUSSION:

Asthma attack follows circadian rhythm and hence chronomodulated pulsatile drug delivery systems are more preferable for effective management of asthma. Initially the granules were formulated with four different diluents (Micro crystalline cellulose (MCC), Mannitol, Lactose, Crospovidone) alone and in combination. The granules were subjected to flow properties and subjected to friability test. All the granules exhibited excellent flow properties. The granules containing the diluent MCC showed adequate mechanical strength and granules formulated with the diluent Mannitol fail to meet friability criteria. The granules containing equivalent to 10mg Montelukast sodium were filled into hard gelatin capsule and subjected to in-vitro dissolution studies. The dissolution rate was found to be dependent on the composition of diluent. The granules containing MCC: Mannitol (1:1) offered high dissolution rate. Further the influence of super disintegrant on dissolution characteristics was investigated and Croscaramellose sodium was found to be effective super disintegrant compared to SSG. So the granules containing MCC: Mannitol as diluent and Croscaramellose sodium as superdisintegrant were selected and subjected to further studies.

Modified pulsincap technology was used to achieve the required lag time. The capsule bodies were subjected to cross linking by exposing to formaldehyde vapor. The diameter of the capsules was observed before and after treatment of formaldehyde vapor. From the observation it was found that there was decrease in dimensions of capsule after formaldehyde treatment. To select hydrogel plug, four different polymers with three different concentrations (30mg, 40mg, 50mg) were employed. The preformulation parameter drug excipient compatibility study was conducted by subjecting the samples to IR spectra studies. The principle peaks corresponding to Montelukast sodium were observed in all mixtures formulated with the selected excipients. Thus these studies indicated the drug and excipient are compatible with each other. The granules along with different hydrogen plugs were filled into formaldehyde treated capsules and the composition was showed in Table 1. The capsules were evaluated for various quality control tests and results are reported in Table 2.

Table 5: In-vitro drug release studies of pulsincap formulations with Sodium alginate hydrogel plugs

Dissolution media	Time (hrs)	% Cumulative drug release
Dissolution media	Time (hrs)	C1	C2	C3
0.1N Hcl +0.5%SLS	1	0	0	0
0.1N Hcl +0.5%SLS	2	0	0	0
6.8 PH phosphate buffer + 0.5%SLS	3	0	0	0
	3.5	0	0	0
	4	0	0	0
	4.5	62±0.5	55.5±0.5	0
	5	96.8±0.5	97±0.5	0
	5.5	96.8±0.5	97±0.5	82±0.5
	6	96.8±0.5	97±0.5	98.5±0.5

All the capsules fulfill the quality control requirements. From the Lag time determination study of the formulations, it was found that the weight of the hydrogel plug increases the lag time. it was also found that satisfactory lag time of minimum5-5.30 hr scan be given by HPMC K4M 50 mg and Sodium alginate 50 mg.

Fig 1: In- vitro drug release profile of pulsincap formulations with HPMC K4M hydrogel plugs:

Table 6: In-vitro drug release studies of pulsincap formulations with Xanthum gum hydrogel plugs

Dissolution media	Time (hrs)	% Cumulative drug release
Dissolution media	Time (hrs)	D1	D2	D3
0.1N Hcl +0.5%SLS	1	0	0	0
0.1N Hcl +0.5%SLS	2	0	0	0
6.8 PH phosphate buffer + 0.5%SLS	3	62.5±0.5	0	0
	3.5	95.5±0.5	68±0.5	0
	4	96±0.5	97±0.5	67.5±0.5
	4.5	96±0.5	97±0.5	98.5±0.5
	5	96±0.5	97±0.5	98.5±0.5
	5.5	96±0.5	97±0.5	98.5±0.5
	6	96±0.5	97±0.5	98.5±0.5

Capsules were subjected to in-vitro dissolution studies in 0.1N Hcl+0.5%SLS for two hours and 6.8 PH phosphate buffer +0.5%SLS for four hours. In-vitro drug release profiles are showed in 3-6. The required lag time was observed from the formulations containing HPMC K4M. It might be due to the alterations in swelling index. The comparative in-vitro drug release profile observed from the pulsatile drug delivery systems formulated with different concentrations of polymers was shown in Figure 1. The lag time was found to be increased with increase in the concentration of polymer

4. CONCLUSION:

The dissolution characteristics of Montelukast sodium are dependent on the composition of granules. The granules containing Mannitol: MCC (1:1) showed high dissolution rate. The lag time was dependent on the polymer present in the hydrogel plug. The polymer HPMC K4M (50mg) exhibited the desired lag time. Thus the change in the composition, the chronomodulated pulsatile drug delivery system of Montelukast sodium can be readily developed

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Received on 04.06.2014 Modified on 05.08.2014

Res. J. Pharm. Dosage Form. and Tech. 6(4):Oct.- Dec.2014; Page 225-229