INTRODUCTION:

During the last two decades there has been a remarkable increase interest in sustained release of drug delivery systems¹. This has been due to various factors like the prohibitive cost of developing new drug entities, expiration of existing international patents, discovery of new polymeric materials suitable for prolonging the drug release, and improvement in the therapeutic efficacy and safety achieved by these delivery systems ². Novel drug delivery systems are capable of releasing its drug contents at either predetermined time or at specific site. They are able to provide very precise control over drug release for a prolonged period of time eliminating the need for frequent dosing and minimizing side effects, thereby increasing patient compliance and comfort.

Pulsatile drug delivery systems are a type of novel drug delivery systems, which were expected to give bimodal release of drugs. This is required because many of the disease states continuous administration of drugs are undesirable and such pulsed dosing could negate the first pass metabolism, gastric irritation and drug instability³.

This pulsatile dosage form ie., capsules (PULCIN CAP) which consists of water soluble cap and water insoluble body. The drug formulation is sealed within the capsule body by means of hydrogel plug. When the PULSIN CAP is swallowed the water soluble cap dissolves in the gastric juice and exposed hydogel plug begins to swell. At predetermined time after ingestion the swollen plug is ejected out and the encapsulated drug formulation is then released into alimentary tract, where it is dissolved in gastro intestinal fluid and then absorbed into blood stream⁴.

Asthma is a predisposition to chronic inflammation of the lungs in which the airways (bronchi) are reversibly narrowed. Asthma affects a total of 350 million peoples across the world. As the people grow older this will found to rise⁵.

Terbutaline sulphate is an effective broncho dilator and relatively short acting ß₂– adrenergic agonist. It has shorter biological half life of 3-4 hours and it is effective at a low oral dose of 5mg. Terbutaline sulphhate produces many of its pharmacological effects by activation of adenyl cyclase, the enzyme which catalyses the conversion of adenosine triphosphte to cyclic adenosine monophosphte. Terebutaline is incompletely absorbed from the GIT and also subject to extensive first pass metabolism by sulphate conjugation in the liver and possibly by the gut wall. It is used in the treatment of Asthma, chronic bronchitis, emphysema and other bronchopulmonary disorders involving bronchospasm, in doses which do not produce marked cardiac acceleration⁶.

Incorporation of drugs in polymeric matrices is a widely used approach for prolonging the drug release. Matrix devices have many advantages over the other controlled release devices as they cannot undergo sudden dose dumping. Matrix devices gives a higher initial release rate and can be made to release the drug at a nearly constant rate. As the terbutaline sulphate having shorter biological half life and low oral dose, an attempt was made to develop pulsatile drug delivery system containing terbutaline sulphate for controlling the conditions like nocturnal Asthma⁷. Here swellable polymers were used as hydrogel plugs and placed between the doses of drug-lactose mixture within the cross-linked gelatin capsules.

MATERIALS AND METHODS:

Terbutaline Sulphate a gift sample from Astra Zeneca, Bangalore, Sodium carboxy methyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose were obtained from Loba Chemicals Pvt. Ltd., Mumbai. Sodium Alginate from Degussa India Pvt. Ltd., Mumbai. All other chemicals used in this study were of analytical grade.

PREPARATION OF CROSSLINKED HARD GELATIN CAPSULES:

Hard gelatin capsules about 200 in number of size-2 were taken and their body was separated from the cap, then body was placed on a wire mesh. Taken 25ml of 37% v/v of formaldehyde solution in a 100ml beaker and kept in empty desiccator. Added 2.5 gms of potassium permanganate to this. A wire mesh was kept on top of the beaker and placed the bodies of the capsules over on it and immediately closed with a filter paper tied around its mouth⁸.

The bodies of capsules are allowed to react with vapours of formaldehyde for 4 hours. Then they were removed and kept on a filter paper in a hot air oven at 45^oC for 48 hours in order to complete the reaction between formaldehyde and gelatin will take place. Finally they were kept in an open atmosphere for a week to evaporate the residual formaldehyde present in it. Then these capsule bodies were capped with caps (not exposed to formaldehyde) and were stored in a polythene bag⁸.

Evaluation Tests for empty gelatin capsules:

Various physical and chemical tests were carried out simultaneously for both formaldehyde treated and untreated capsules. The identification attributes like colour, odour, lockability, stickyness and shape was checked manually⁹. The size of the capsules ie length, external diameter and thickness was determined by using screw gauge⁹. The average weight of the randomly selected capsules was determined by using single pan electronic balance⁹. The percent moisture content was determined by weighing accurately 1 gm of sample capsules and kept in an oven for drying at 40^oC – 65^oC for 17 hours. Then capsules were cooled in a descicator till room temperature and reweighed. The moisture content was calculated on the basis of loss on drying⁸. The solubility of the empty gelatin capsules was done by stirring the capsule in a beaker containing 100ml of dissolution medium by using magnetic stirrer. The dissolution medium taken was water and buffers (pH 1.2 and 7.2). The time at which capsule dissolves or forms soft fluffy mass was noted⁸.

Chemical Test:

This test is a modification of the limit test for formaldehyde (method A, IP 1996) carried out for absorbed tetatnus, pertusis and diphtheria vaccine.

Standard formaldehyde solution was prepared by diluting a suitable volume of formaldehyde solution with water to get a concentration of 0.002%w/v.

The sample formaldehyde solution is prepared by cutting the body of the capsules into small pieces and transferred to a beaker containing distilled water. This was stirred by using magnetic stirrer for 1hour to solubilize free formaldehyde. Then it was filtered into 50 ml volumetric flask, washed with distilled water and the volume was made up with the washings.

Procedure:

Added 9 ml of water to 1ml of sample of solution. Taken 1ml of this solution and added 4ml of water and 5ml of acetyl acetone reagent, warmed this mixture on water bath for 40^oC then allowed to stand for 40 min. Similarly standard solution was also prepared by using standard formaldehyde solution and compared the intensity of colour developed was compared.

FORMULATION OF SUSTAINED RELEASE PULSIN CAPSULES:

Preparation of physical mixture of drug and polymer:

Accurately weighed 1gm of terbutaline sulphate was geometrically diluted with 19gms of lactose in a mortar and this mixture is stored in polythene bag⁸.

The various formulations by using different polymers as hydrogel plugs are given in table-1.

Filling of capsules:

Hard gelatin capsules of size 2 with formalin treated body and untreated cap were taken for filling. 20 capsules for each formulation were prepared by manual method as follows.

The cap and body of the known weight of capsule was separated individually by hand. The drug-lactose mixture (50mg) equivalent to 2.5mg of drug (terbutaline sulphate) was filled in to the body of capsule; this forms the second dose of the drug. The quantity of the polymer as specified in table-1, for different formulations was weighed and filled above drug mixture, then pressed tightly with a glass plunger; this forms plug2 of the formulation. Similarly first dose of the drug lactose mixture was filled in the capsule body and polymer plug is placed to form plug1 of the capsule. Now the drug-lactose mixture was weighed and placed in soluble cap to form the immediate release (dose) of drug. Finally the cap was locked into the capsule body and stored in tightly packed container for further studies.⁸

Table 1. Composition of different formulations

Sl. No	Formulation Code	Polymer used	Amount of polymer used / plug
1	PC-1	Sodium carboxy methyl cellulose	50 mg
2	PC-2	Hydroxy Propyl Methyl Cellulose (4000cps)	50 mg
3	PC-3	Hydroxy Propyl Methyl Cellulose (1000cps)	50 mg
4	PC-4	Hydroxy Propyl Methyl Cellulose (1000cps)	30 mg

EVALUATION OF DRUG LOADED CAPSULES:

Compatibility studies were conducted using IR spectroscopy and TLC of drug alone, and with polymer. Various physico-chemical properties such as content uniformity, weight variation, disintegration, dissolution and stability studies were determined on prepared capsules.

Individual weights of 20 capsules were determined by using an electronic single pan balance. The percentage moisture loss was determined by keeping the weighed capsules in a desiccator containing anhydrous calcium chloride. After 3 days, the capsules were taken out and re-weighed; the percentage moisture loss was calculated using formula (initial weight-final weight/ initial weight) x 100 ⁹.

Content uniformity:

The contents of the capsule was emptied into 100ml volumetric flask containing 50ml distilled water and shaken well until the drug was completely dissolved. The solution was filtered and the volume was made up to the mark quantitatively with distilled water to get stock solution A.

1.0 ml of p-nitroaniline solution (0.2% w/v in 10% v/v Hcl) was added to 25ml volumetric flask followed by 1.0 ml of sodium nitrite (0.5% w/v in water). The two solutions were mixed well and allowed to stand at room temperature for 10 minutes.

1.0 ml of methanol was next added and mixed well to eliminate the excess of nitrous acid that was formed insitu, followed by the addition of 1ml of stock solution A.

2.0 ml of sodium hydroxide solution (6%w/v in water) was added into the above flask. The orange colour was formed within 2 minutes. Make up the volume to 25ml with distilled water. The absorbance of the resulting solution was measured at 450 nm using UV-Visible spectrophotometer and a concentration was estimated¹⁰.

Disintegration Test:

6 capsules were placed in each of the six tubes of the basket. Perforated plastic discs were then placed on the top of the capsules and the buffer (pH 1.2) was used as dissolution media. Then the disintegration apparatus was operated and test was carried out for all the formulations⁹.

In-Vitro dissolution Studies:

The dissolution studies were carried out by rotating basket method as specified in USP by using Electro lab, tablet dissolution tester. One capsule was taken and placed in the basket, and it was immersed into the dissolution medium (37^oC ± 0.5^oC) carefully by avoiding air bubbles and the basket was rotated at 100rpm.

The dissolution was first carried out in 500 ml of pH 1.2 buffer for 2 hours and in 500 ml of pH 7.2 buffer for the remaining period of 10 hours. At each intervals of 1 hour, 10 ml of the dissolution media was withdrawn and same volume was replaced by dissolution media. The amount of drug dissoluted in withdrawn media can be estimated as earlier¹⁰.

Stability Studies:

The selected formulations were packed in an air tight polythene bag and stored at 60^oC and 45^oC for a period of 3 and 10 weeks respectively. The samples were withdrawn at the end of 5^th ,8^th and 10^th week in case formulations stored at 45^oC and 1^st , 2^nd and 3^rd week in case formulations stored at 60^oC.

All the samples were evaluated for any physical change, drug content, disintegration time and In vitro dissolution test.

RESULTS AND DISCUSSION:

The sustained release pulsatile capsules of terbutaline sulphte were prepared by hand filling method. In order to sustain the release of drug crosslinking of capsules by formalin treatment has been employed to modify the solubility of gelatin capsules. Exposure of capsules to aqueous formaldehyde vapours results in an unpredictable decrease in solubility of capsules (gelatin) owing to cross-linkage of the gelatin molecule initiated by the aldehyde.

All the empty capsules were lockable type, odourless, soft and sticky when touched with wet finger. After formalin treatment, there were no significant changes in the capsules except for the stickiness. The body of the capsules was hard and non sticking even when touched with wet finger.

The physico- chemical evaluation data of empty gelatin capsules presented in table 2 indicates that the thickness of the capsule ranges from 0.14 – 0.19 mm. The capsules with formalin treated showed higher thickness compared to untreated. The individual weights of each capsule are quite uniform and cross-linking did not show any significant change in weight. Average weight of capsules was in the range of 97.22 to 98.42 mg. The length of the cap was 10.85mm where as body was 18.12 and 18.25mm for formalin treated and untreated respectively. The external diameter was found to 6.74 and 6.81 for formalin treated and untreated respectively. The percentage moisture was 12.69 for formalin treated and 15.73 for untreated, this may be due to cross linking that leads to decrease in percentage moisture.

Table 2. Physical characteristics of empty gelatin capsules with and without cross-linking*.

Type of Capsules	Length (mm)		External diameter (mm)		Thickness (mm)		Length of Capsules (mm)		Avg Weight (mg)	Percent moisture
Type of Capsules	Cap	Body	Cap	body	Cap	body	Before Locking	After Locking	Avg Weight (mg)	Percent moisture
Formalin Treated	--	18.12	--	6.74	--	0.19			97.22	12.69
Untreated	10.85	18.25	7.64	6.81	0.18	0.15			98.42	15.73

*Each value is a mean and standard deviation of six determinations.

Table 3. Amount of Physical mixture and drug loading per capsule

Formulation Code	Amount of Physical mixture filled (mg)	Amount of drug per capsule (mg)	Percentage drug content
SRC-1	150.4 ± 1.2	7.46 ± 2.3	99.33 ± 2.5
SRC-2	152.5 ± 1.3	7.51 ± 1.5	100.13 ± 2.6
SRC-3	151.4 ± 2.1	7.49 ± 1.7	99.86 ± 2.4
SRC-4	149.6 ± 1.9	7.44 ± 1.1	99.20 ± 2.1

Table 4. In-Vitro dissolution profile of various formulations*.

Time (hrs)	Cumulative Percentage drug released ±SD
Time (hrs)	PC-1	PC-2	PC-3	PC-4
1	32.57 ± 3.2	34.65 ± 3.0	32.82 ± 2.5	32.17 ± 3.2
2	39.52 ± 1.1	38.25 ± 2.5	35.00 ± 1.6	36.24 ± 1.7
3	43.16 ± 2.2	47.13 ± 2.7	38.42 ± 3.4	43.26 ± 2.0
4	51.34 ± 2.9	51.32 ± 2.1	42.00 ± 3.1	52.35 ± 1.1
5	60.45 ± 3.0	58.76 ± 1.9	48.00 ± 1.1	64.40 ± 1.8
6	67.96 ± 2.1	63.13 ± 2.0	50.00 ± 2.4	66.59 ± 1.3
7	81.50 ± 1.2	74.69 ± 1.6	55.12 ± 1.1	83.71 ± 2.1
8	91.42 ± 1.5	90.30 ± 1.2	61.32 ± 2.2	93.91 ± 2.4
9	98.39 ± 1.1	92.45 ± 3.1	67.19 ± 1.3	96.95 ± 2.1
10	100.00 ± 2.3	98.05 ± 2.3	71.89 ± 1.5	99.61 ± 3.0

*Each value is a mean and standard deviation of six determinations.

Table 5. Stability data of all the formulations

Formulations	Stability tests	Storage Temp 60^oC			Storage Temp 45^oC
Formulations	Stability tests	1week	2week	3week	5week	8week	10week
PC-1	Drug Content	7.51	7.48	7.44	7.49	7.46	7.42
PC-1	Disintegration Time (min)	14.52	13.60	14.22	15.36	15.25	14.34
PC-2	Drug Content	7.36	7.42	7.45	7.40	7.38	7.41
PC-2	Disintegration Time (min)	13.42	15.26	14.24	13.55	14.42	14.29
PC-3	Drug Content	7.35	7.31	7.28	7.37	7.35	7.32
PC-3	Disintegration Time (min)	14.23	15.46	13.44	15.55	13.42	15.29
PC-4	Drug Content	7.48	7.45	7.39	7.49	7.46	7.41
PC-4	Disintegration Time (min)	15.23	13.26	13.48	15.46	14.32	13.39

The solubility studies on both formalin treated and untreated capsule shells was carried out for 24 hrs. The study shows in case of untreated capsules both body and cap were dissolves within 15 minutes, where as in formalin treated capsules only cap dissolves within 15 minutes and the body remained intact.

The qualitative test performed for free formaldehyde showed that the sample solution was not more intensely coloured than the standard solution, inferring that less than 20µg of free formaldehyde is present in 25 capsule bodies. Hence it is considered as a valid container for the drug dispersed in hydrophilic polymer matrix intended for sustained release preparation.

The compatibility studies done by FTIR and TLC studies indicated that there is no chemical interaction between the drug and excipients used.

The amount of physical mixture was found to be 149.6 to 152.5 mg as per the combination drug, diluents and hydrogel plug of polymer. The amount of terbutaline sulphate in each capsule was in between 7.44 to 7.51 mg and it equals to three individual doses separated by hydrogel plug. The theoretical drug loading for each dose is equivalent to 2.5mg. The percentage drug content was from 99.2–100.8, the data is shown in table-3.

The disintegration test showed that the caps of the capsules were disintegrated within 15 minutes; while the body of the capsules was remain unchanged even after 60 minutes.

From the in vitro dissolution studies it was found that all the caps were separated from the body thereby releasing the immediate release dose of 2.5mg within 15 minutes. The polymer plugs swelled in alkaline medium and the plug ejection time was found to be between 4-5hrs in all formulations. About 5-10% of the first loading dose was found to be released even before the plug was ejected. It may be due to the diffusion of the loading dose through the hydrogel plug into the dissolution medium. The dissolution profiles of all the formulations showed a higher initial release rate, which is a characteristic property of the matrix formulations. The drug release followed first order kinetics and their release rate constants were found to be 0.0933, 0.0921, 0.095 and 0.06 hr^-1for PC-I, PC-2,PC-3 and PC-4 respectively (Fig-1, Table-4).

Figure-1. Invitro dissolution profile of various formulations.

The stability studies showed no change in the physical changes and appearance. The data given in table 5 indicates that there is no significant changes in content estimation, disintegration time and dissolution profile.

CONCLUSIONS:

The present work is an attempt to develop sustained release pulsatile capsules of terbutaline sulphate on the lines of novel drug delivery systems. The first step in preparing such type of drug delivery system is to prepare capsules with soluble cap and insoluble body. It was done by exposing the separated body of capsule to formaldehyde vapours, which causes a crosslinkage of the gelatin molecule making it insoluble.

These systems are capable of releasing its contents at predetermined time. The system consists of water soluble cap and insoluble body of the capsule. The drug-lactose mixture was placed in the body of capsule and plugged with a polymeric hydrogel plug in a series. Then the cap is fixed to the body of the capsule. On oral ingestion of the dosage form, the cap dissolves in the gastric medium and immediate relese dose was released. Then hydrogel plug1 swells and the swollen plug is ejected from the body of the capsule releasing the first dose of a drug. Finally the plug2 was swelled and second dose of a drug was released at a predetermined rate.

The results of the excipient compatibility studies revealed that there is no chemical interaction between the drug and excipients and the drug is maintained in its original state.

The next objective of preparing the sustained release pulsatile capsules of terbutaline sulphate was achieved by filling the physical mixture of the drug and the hydrophilic polymers as plugs into the above capsules. The physical mixture was made by mixing the drug with diluent and different polymers.

The formulations were subjected for various physicochemical tests, disintegration and dissolution studies. In vitro dissolution profile revealed that in all the formulations the cap was dissolved within 15 minutes, and then there was a higher initial release of drug followed by a slow and a nearly constant release of drug over a period of 8 to 10 hours depending on the type and amount of polymer used. The drug release followed first order kinetics, indicated that the release rate dependent on the amount of drug remaining in the dosage form.

From the accelerated stability data it was observed that there was no significant changes in the physical properties of the capsules, drug content, disintegration time and the dissolution studies, hence the formulations are quite stable.

Finally to conclude just mixing of the drug with a blend of polymers and filling the mixture into cross linked gelatin capsules prolongs the release of drug. When the two doses of drug were placed between the two polymer plugs within the crosslinked capsules showed sustained release behavior.

REFERENCES:

1. Nichlolas, Lordi G. Sustained release Dosage forms. In theory and practice of industrial pharmacy. Bombay: Varghees publications. 3^rd Edn.(lachman L, Lieberman HL, Kaning JL,eds);1987:430-456.

2. Langer,M.A Joseph R.Robinson. Sustained release drug delivery system. In Remington^,s pharmaceutical sciences. Mack publishing company.17^ththth edn;(Alfonoso R Gennaro Ed);1980:1645-61.

3. Scherer RP. Drug Delivery Systems Brochure on pulsincap 1991.

4. Popli H, Sharma S.N. Trends in oral sustained release formulations. Eastern pharmacist,1989 Aug:99-103.

5. Serafin, “Drugs used in the treatment of Asthama.” In “The Pharmacological basis of therapeutics,” A.G Gilman (ED). 9^th Edn. McGraw Hill, New York,1996,pp662-664.

6. R.S Sathoskar and S.D Bhandarkar, “Pharmacotherapy of Bronchial Asthama.” In “Pharmacology and Pharmacotherapeutics,” R.S Sathoskar (Ed.)13^th Edn. Popular Prakashan,Bombay, 1993,pp.310-315.

7. Ballaard BE. An overview of prolonged action drug dosage form. In Sustained and controlled release drig delivery system,2^ndedn(Robinsonj.r.,ed)Marcel dekker,New York,1986,pp96-135.

8. Ronnic Millender, “capsule shell composition and manufacturing”. In “Multiparticular oral drug delivery”, (Isaac Glebre scllasic ed), New York. Marcel Dekker;357-380.

9. Lachmann L, Libermann, Sustained release dosage forms In The Theory of Industrial Pharmacy. Lachmann (ed.), Bombay: Varghees Publications.3^rd edn;1987:365.

10. Reddy M.N, Sankar.DG, Rao.GD and Sreedhar.K. “Spectrophotometric determination of salbutamol and Terbutaline” Eastern Pharmacist.127-28,(1991).

Received on 09.02.2011 Modified on 26.02.2011

Research J. Pharm. and Tech. 4(9): Sept. 2011; Page 1389-1393

MATERIALS AND METHODS:

Formalin Treated