1. INTRODUCTION:

Chronotherapeutics discusses about the treatment method where the in vivo drug release is programmed to match the rhythms of disease such that to obtain the required therapeutic outcomes and abate side effects^[1,2]. 1-4 Circadian rhythms are self-sustaining, endogenous oscillations that occur with a periodicity of about 24 hours and are synchronized according to internal biologic clocks related to the sleep-wake cycle. The best-known circadian rhythms include body temperature, hormone secretion, metabolism, sleep or wake cycle^[3,4]. Chronotherapeutic delivery system is required to be formulated to suit drug release conditions in various diseases like cardiovascular diseases. Asthma is disease of lung airways (bronchi) characterized by hyper active responsiveness to a variety of stimuli and in this condition the airways constricts and becomes inflamed with excess mucus lining the passage.

The stimuli which may trigger the asthmatic condition includes exposure to allergens, cold air, moist air, emotional stress etc^[5,6]. Nocturnal asthma is condition where an exacerbation of the asthma is observed during the night times, such that worsening the functioning of the lung with increased airway response^[7,8,9]. Usually, asthma attacks are more predominant in early mornings so it is annoying for a patient to take medicine at late night, hence in this condition there is a demand for pulsatile drug delivery system which release the drug at required time at proper site with efficient therapeutic action^[10,11]. Pulsatile drug delivery system offers various advantages like it provides extended activity, reduced side effects and dosage frequency, reduction in dose size, improved patient compliance^[12]. These systems have a peculiar mechanism of delivering the drug rapidly completely after a “lag time,” i.e., a period of “no drug release.”^[13].

2. MATERIAL AND METHOD:

Doxofylline was gifted by Delta Finochem Pvt. Ltd, Nashik. Microcrystalline cellulose, Sodium Hydroxide, Potassium dihydrogen phosphate, Disodium hydrogen phosphate, Isopropyl alcohol was gifted by Loba chemie Pvt. Ltd., Mumbai. Talc, Hydrochloric Acid was obtained by Merck chemie Pvt. Ltd., Mumbai. Eudragit (S-100) was obtained by Research lab fine chem. Industries, Mumbai.

2.1 Methodology:

2.1.1 Formulation of Doxofylline Core Tablet^[14]:

The composition of trial batches B1 to B3 containing super disintegrant (3 to 6%) is shown in table. The core tablets are formulated by using 400mg of drug molecule, sodium starch Glycolate as super disintegrate, microcrystalline cellulose as diluent, talc and magnesium stearate as Glidant and Lubricant respectively. All ingredients used in formulation including Doxofylline, SSG and MCC were passed through a #40 sieve and dry blended for 15 minutes. Pre-lubricated this dry mixed blend with pre-sifted talc through #40 sieve and mixed for 5 minutes. Lubricated this blend with pre sifted magnesium stearate through #60 sieve and mixed for 3 minutes. Final lubricated blend was directly compressed using 9.5 mm round; concave punch by using rotary tablet compression machine .Total weight of core tablet was fixed as 450mg. The tablets are prepared by using 9.5 mm flat punch. Then the prepared core tablets are subjected to coating by using various compositions of polymers.

Table No 1: Composition of Core tablets

Ingredients	Quantity per tablet[mg]
Ingredients	B1	B2	B3
Doxofylline	400	400	400
Sodium Starch Glycolate	9	18	27
Microcrystalline cellulose	36.5	27.5	18.5
Magnesium Stearate	2.25	2.25	2.25
Talc	2.25	2.25	2.25
Total Weight (mg)	450	450	450

2.1.2 Preparation of coated tablets:

Prepared core tablets were coated using different concentrations of Eudragit® S 100 polymer solutions (i.e. 4%, 5% and 6%) in Ethanol and Isopropyl alcohol in 1:3 proportion. Dibutyl phthalate was used as plasticizer. Dip coating technique was used for the coating of tablet and tablets were dried at 50°C for 10 h. before further evaluation.

2.1.3 Formulation of Coated tablets (F1 to F9) ^[15,12]:

The composition of core tablet batches B1 to B3 containing super disintegrant (3 to 6%) is shown in table. The core tablets are formulated by using 400mg of drug molecule, sodium starch Glycolate as super disintegrant, microcrystalline cellulose as diluent, talc and magnesium stearate as Glidant and Lubricant respectively .Total weight of core tablet was fixed as 450mg. The tablets are prepared by using 9.5mm flat punch. Formulated core tablets were coated using different concentrations of Eudragit® S 100 polymer solutions (i.e. 4%, 5% and 6%) in Ethanol and Isopropyl alcohol in 1:3 proportion. Dibutyl phthalate (10%w/v) was used as plasticizer.. Dip coating technique was used for the coating of tablet and tablets were dried at 50°C for 10 h. before further evaluation The concentration of coating polymer for F1, F4 and F7 was 4% w/v in Ethanol and isopropyl alcohol in 1:3 proportion. The concentration of coating polymer for F2, F5 and F8 was 5%. The concentration of coating polymer for F3, F6 and F9 was 6%.

Table No. 2: Composition of Coated Tablets. (F1 to F9)

Ingredients	Quantity per tablet [mg]
Ingredients	F1	F2	F3	F4	F5	F6	F7	F8	F9
Doxofylline IP	400	400	400	400	400	400	400	400	400
Sodium Starch Glycolate	9	9	9	18	18	18	27	27	27
Dibasic calcium phosphate	36.5	36.5	36.5	27.5	27.5	27.5	18.5	18.5	18.5
Magnisium stearate	2.25	2.25	2.25	2.25	2.25	2.25	2.25	2.25	2.25
Talc	2.25	2.25	2.25	2.25	2.25	2.25	2.25	2.25	2.25
Total weight	450	450	450	450	450	450	450	450	450
Eudragit S100 (coating polymer) in %	4	5	6	4	5	6	4	5	6

3. EVALUATION PARAMETERS:

3.1 Pre compression evaluation parameters of core Tablet:

Powder was evaluated for their physical parameters such as Bulk Density, Tapped density, Compressibility index, Hausner’s ratio and Angle of repose.

a) Angle of repose (θ):^[16]

The frictional forces in loose powder mixture can be measurement by the angle of repose. This is the maximum angle possible between the surface of a pile of powder or powder and the horizontal plane.

tan θ = h/r

θ = tan-1(h/r)

Where, the θ is the angle of repose, h = height, r = radius

The powder was allowed to flow through the funnel fixed to a stand at definite height. The angle of repose was then calculated by measuring the height and radius of the heap of powder formed.

b) Bulk Density:^[17]

The ratio of mass to volume is known as Bulk Density (BD) of material. The volume of packing recorded as,

Weight of sample

Bulk Density = ------------------------------------

Volume occupied by sample

c) Tapped Density:^[17]

The tapped density (TD) was determined by tapped density apparatus i.e. (Lab. Tech Instrument)

Weight of sample

Tapped Density (g/ml) = ------------------------------------

Tapped Volume of sample

d) Compressibility index and Hausner’s ratio:^[17]

The Compressibility Index and Hausner’s Ratio are measures of the propensity of a powder to be compressed. As such, they are measures of the relative importance of interparticulate interactions. In a free-flowing powder, such interactions are generally less significant, and the bulk and tapped densities will be closer in value. The Compressibility index and Hausner’s ratio is calculated by given equation:

Tapped Density - Bulk Density

Compressibility index (%) = ------------------------------------ X 100

Tapped Density

Hausner’s ratio = ------------------------------------

Bulk Density

3.2 Post compression evaluation parameters of Core Tablet:

The prepared tablets were evaluated for hardness, friability, uniformity of weight, drug content and in vitro Dissolution study.

a) Hardness:^[18]

The hardness of the tablets was measured by using Monsanto hardness tester. The hardness was measured in terms of kg/cm².

b) Friability:^[18]

The Friability of the tablet was determined using Roche friabilator. It is expressed in percentage. Ten tablets were initially weighed (W_initial) and transferred into the friabilator. The friabilator was operated at 25 rpm for 4 mins. The tablets were weighed again (W_final). The % friability was then calculated by

W (Initial) –W (final)

F = ----------------------------------------X 100

W (initial)

c) Weight Variation:^[17]

Twenty tablets were weighed individually. Average weight was calculated from the total weight of all tablets. The individual weights were compared with the average weight. The percentage difference in the weight variation should be within the acceptable limits. The percent deviation was calculated using the following formula:

Individual weight - Average Weight

% Deviation = ----------------------------------- x 100

Average weight

d) Thickness:^[16]

The thickness of tablets was measured by using Vernier caliper along with the standard deviation.

e) Disintegration time:^[17]

The test was carried out on 6 tablets using tablet disintegration tester and 6.8pH phosphate buffer at 37°C ± 2°C was used as a disintegration media and the time in seconds taken for complete disintegration of the tablet with no palatable mass remaining in the apparatus was measured.

f) Drug Content:^[17]

The drug content can be determined by triturating 20 tablets and powder equivalent to 100mg was added in 100ml of 6.8 pH Phosphate buffer solution. Followed by shaking for 30min. and solution was filtered. The filtered solution was diluted 6.8 pH phosphate buffer and the absorbance of resultant solution was measured spectrophotometrically at 274nm.

g) In vitro Dissolution study:^[18]

The following conditions were maintained during the in vitro dissolution study of Doxofylline tablets. The apparatus used for dissolution parameter is (Type II Paddle method). The dissolution medium is (Phosphate buffer pH 6.8) whose volume is (900ml). Temperature sets to (37º±0.5ºC) with speed of rotation at (50 rpm).

Dissolution medium 5 ml was withdrawn at 2 min interval. The volume withdrawn was replaced by volume of fresh dissolution medium to maintain sink condition. Each sample was filtered and diluted up to 10 ml. The filtered sample was analyzed at spectrophotometrically at 274nm.The release study was performed in triplicate and cumulative percentage of drug release was calculated

3.3 Post compression evaluation parameters of Coated Tablet:

The prepared tablets were evaluated for hardness, uniformity of weight, and drug content. Same procedure was followed as given in Section .3.2

In vitro Dissolution study:^[18]

In vitro dissolution study was performed using USP Type II dissolution apparatus (paddle type) at speed of 50rpm 0.1 N HCl of pH 1.2 was first used for 2 hr, which was then replaced with phosphate buffer pH 6.8 and kept up to 10 hr. Aliquot of dissolution medium (5 ml) were withdrawn at specific time intervals of 1 hr and filtered each with Whatman filter paper. Equalamount of fresh dissolution medium was replaced immediately after each withdrawal. The amount of drug present in each sample was determined by UV-spectrophotometer at 274 nm.

4. RESULT AND DISCUSSION:

4.1 Result of Pre compression parameters of core Tablet:

Angle of response (θ):^[16]

The results obtained for angle of response of all the formulation are B1 (30±1.033), B2 (29±1.063), B3 (32±1.021). The values were found to be in the range of 29.106º to 32.102º respectively. The data showed good flow property of mixture.

Bulk Density and Tapped density:^[17]

The results obtained for angle of repose of all the formulations are B1 (0.79±0.02), B2 (0.77±0.13), B3 (0.67±0.02). The bulk density for all the formulations range from 0.67±0.02 and 0.79±0.02gm/cm³ respectively and tapped dnsity for all the formulations are B1 (0.96±0.11), B2 (0.96±0.11), B3 (0.87±0.09) range from 0.87±0.09 to 0.96±0.11. The values obtained lies within the acceptable range and not large differences found between bulk density and tapped bulk density. This result helps in calculating the % compressibility of the powder.

Hausner’s ratio:^[17]

The results obtained for all the formulations are B1 (1.21±0.04), B2 (1.19±0.033), B3 (1.25±0.032). The values were found to be in the range of 1.19 to 1.25. All the formulations showed the hausner‘s ratio within the range.

Carr’s Index:^[17]

This percentage compressibility of powder was determined by Carr‘s compressibility index. The results obtained for all the formulations are B1 (17.7±0.011), B2 (17.3±0.03), B3 (18.11±0.012). The percentage compressibility for all the nine formulations lies within the range of 17.3 to 18.11. This showed acceptable compressibility.

4.2 Evaluation of post compression parameters of core tablet

Weight variatin test:^[17]

The percentage weight variation for all the formulations are B1 (451±2.48), B2 (453±1.23), B3 (448±3.65). All the tablet passed weight variation test as the percentage weight variation was within the pharmacopoeia limits of 5%. It was found to be form 448±3.65mg to 453±1.25mg. The weight of all the tablet was found to be uniform (Table No.4)

Thickness:^[16]

The thickness of tablet was measured by using Vernier caliper by picking the tablet randomly. The mean values are are B1 (5.67±0.3), B2 (5.69±0.34), B3 (5.68±0.32). The values are almost uniform in all formulations. Thickness was found in range from 5.62 mm to 5.68 mm respectively.

Hardness test:^[18]

The results of hardness are B1 (4.2±0.5), B2 (4.4±0.45), B3 (4.1±0.6). Hardness test was performed by Monsanto tester. Hardness was maintained to be within 4.2kg/cm² to 4.6kg/cm².

Friability test:^[18]

The study results are B1 (0.12±0.021), B2 (0.19±0.036), B3 (0.21±0.082) was found in the range from 0.19% to 0.42%.

Disintegration test:^[18]

The study results are B1 (30±2.98), B2 (24±2.75), B3 (18±2.45) was found in the range 18 sec-31 sec. From the result of in vitro disintegration time of core tablet it was found that as the percentage of super disintegrants increased (2 to 6%), the disintegration time decreased. Minimum disintegration time (18±2.94 sec) was observed with formulation B3 containing 6% sodium starch glycolate.

Drug content uniformity:

The content uniformity was performed for all the nine formulations and results are B1 (97.98±2.31), B2 (99.23±4.25), B3 (99.11±3.22).Three trials for each formulation were analysed spectro photometrically. The drug content of the tablet was found ―between (98.43% to 101.4%) of Doxofylline. The results indicated that all formulations the drug content was uniform.

In-vitro Dissolution studies:

The in-vitro release of Doxofylline core tablet formulation B1 to B3 from the in-vitro dissolution data show in the (Table No.3 and fig, no. 1) All the three formulations were release more than 90% drug in 10 min which is acceptable for core tablet.

Table No. 3: In vitro drug release profile of core Tablet.

Time in (min)	% cumulative drug release
Time in (min)	B1	B2	B3
0	0	0	0
2	14.17	20.12	17.24
4	36.12	46.11	49.34
6	60.19	78.13	81.32
8	80.44	88.12	90.18
10	92.12	97.19	98.99

Fig. No. 1: %CDR Cumulative Percentage Drug Release of Core tablet

4.3 Evaluation of post compression parameters of coated tablet:

The prepared coated matrix tablets were evaluated for various parameters like Uniformity of weight, Hardness, Friability, in vitro drug release, Thickness and Drug content.

Weight variation test:^[17]

All the tablet passed weight variation test as the percentage weight variation was within the pharmacopoeial limits. This indicates the weight of all the was found to be uniform. (Table No.4)

Thickness:^[16]

The mean values are shown in (Table No. 4) the values are almost uniform in all formulations.

Hardness test:^[18]

The results of hardness are given in (Table No.4). Hardness was maintained to be within 6.4 kg/cm² to 7.8 kg/cm² which is acceptable for coated tablets.

Drug content uniformity:^[17]

The drug content of the tablet was found ―between (97.21% to 101.4%) of Doxofylline. The results indicated that all formulations the drug content was uniform.(Table No. 4)

Table No.4: Results of post compression parameters of coated tablet.

Formulation/Parameters	Average weight (mg)	Hardness (kg/cm2)	Friability (%)	Thickness (mm)	Drug content (%)
F1	556± 2.48	6.4±0.5	0.32±0.011	6.45±0.06	99.47±2.61
F2	569±3.21	7.5±0.7	0.36±0.036	6.65±0.05	98.94±3.25
F3	635±2.65	6.7±0.6	0.41±0.082	7.00±0.13	97.12±3.22
F4	577±3.20	7.0±0.5	0.33±0.152	6.54±0.02	99.22±3.23
F5	600±2.64	6.7±0.8	0.43±0.095	7.23±0.24	101.41±2.64
F6	598±2.86	7.8±0.5	0.39±0.091	7.70±0.02	99.13±3.22
F7	582±2.59	6.5±0.6	0.44±0.032	6.64±0.04	97.47±4.39
F8	600±1.48	6.9±0.4	0.34±0.076	7.46±0.067	98.72±4.21
F9	599±2.99	7.6±0.7	0.42±0.045	7.95±0.051	97.23±4.75

(Mean ± S.D., n=3)

Table No. 5: In vitro drug release profile of coated Tablet.

Time (hrs)	% Cumulative drug release
Time (hrs)	F1	F2	F3	F4	F5	F6	F7	F8	F9
1.	0	0	0	0	0	0	0	0	0
2.	0	0	0	0	0	0	0	0	0
3.	0	0	0	0	0	0	0	0	0
4.	0	0	0	0	0	0	0	0	0
5.	11.33	0	0	10.76	7.11	0	6.16	0	0
6.	32.31	19.27	11.42	41.61	39.23	0	34.48	11.56	0
7.	80.11	46.33	50.64	81.34	64.66	40.23	58.53	34.51	20.23
8.	97.44	75.34	80.47	98.55	78.18	55.51	94.48	79.56	66.41
9.		94.52	96.44		92.12	98.66		95.44	97.39

Figure No. 2: % Cumulative Percentage Drug Release of Coated tablet

5. CONCLUSION:

The formulation consisted of a core tablet containing a drug Doxofylline and outer layer of enteric coated polymer Eudragit S100. From all the batches, it was concluded that Formulation F6 was the ideal formulation with lag time of 6 hrs followed by burst release of drug and also meeting all specifications of pre-compression and post compression parameters. Thus the dosage forms can be taken at bedtime so that the content will be released in the morning hours i.e. at the time of symptoms. The present study provides a new alternative which is cheaper and biocompatible formulation of Doxofylline in the treatment of nocturnal asthma. Hence, the present once a day formulation of Doxofylline may improve the patient compliance with cost reduction.

6. REFERENCE:

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3. Moore R.Y. Entertainment pathways and the functional organization of the circadian system. Brain Res.1996;111: 103-119.

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7. Hetzel MR, Clark TJ. Comparison of normal and asthmatic circadian rhythms in peak expiratory flow rate. Thorax. 1980; 35: 1002-1007.

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10. Philip E Silkoff. and Richard J Martin. Pathophysiology of nocturnal asthma. Annals Of Allergy, Asthma and Immunology. 1998;81: 378-387.

11. Anamika Singh et al. Pulsatile Drug Delivery System: an Approach of Medication according to Circadian Rhythm. Journal Of Applied Pharmaceutical Science. 2012; 02 (03): 166-176.

12. G. Sree Lakshmi, Gowthamy Priya, kumar Sravan et al. Formulation and evaluation of chrono modulated pulsatile drug delivery system of doxofylline. Int. J. of Pharmacy and Analytical Research. 2017; Vol-6(3): 526-531.

13. Arora Shweta, Ali. J, Ahuja Alka, Baboota Sanjula and Qureshi J. Pulsatile Drug Delivery Systems: An Approach for Pulsatile Drug Delivery Systems: An Approach for Controlled Drug Delivery. Indian Journal of Pharmaceutical Sciences. 2006;68(3): 295-300

14. Patil Ajit, Payghan Santosh, disouza John. Formulation and evaluation of Enteric Coated tablets of Azithromycin dehydrate. International Journal of Chem Tech Research. 2011; 3(3): 1479-1484.

15. Mehta Rohit, Chawla Anuj, Sharma Pooja, Pawar Pravin. Formulation and evaluation of Eudragit S-100 coated naproxen matrix tablets for colon – targeted drug delivery system. J. Adv. Pharm. Tech. Res. 2019;4(1): 31-41.

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17. Indian Pharmacopoeia, published by controller of publication and Ministry of Health, Volume II, 2007, 2010; 459-463.

18. United States Pharmacopoeia, The National Formulary, 20th Edition, United State Pharmacopoeial Convention INC, Washington, 2002; 711, 2011.

Received on 04.12.2019 Modified on 24.03.2020

Research J. Pharm. and Tech. 2020; 13(12):6170-6175.

DOI: 10.5958/0974-360X.2020.01076.8