Anti-Amnesic Activity of Vitex negundo in Scopolamine Induced

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Pharmacology & Pharmacy, 2010, 1, 1-38 Published Online July 2010 in SciRes (http://www.SciRP.org/journal/pp/)

TABLE OF CONTENTS

Volume 1

Number 1

July 2010

Anti-Amnesic Activity of Vitex negundo in Scopolamine Induced Amnesia in Rats A. Kanwal, J. Mehla, M. Kuncha, V. G. M. Naidu, Y. K. Gupta, R. Sistla…………………………………………………………1

Development and Evaluation of a New Interpenetrating Network Bead of Sodium Carboxymethyl Xanthan and Sodium Alginate for Ibuprofen Release R. Ray, S. Maity, S. Mandal, T. K. Chatterjee, B. Sa…………………………………………………………………………………9

Preparation and Evaluation of Rapidly Disintegrating Fast Release Tablet of Diazepam-Hydroxypropyl-β-Cyclodextrin Inclusion Complex T. K. Giri, B. Sa…………………………………………………………………………………………………………………18

The Porcine Pulmonary Surfactant Protein A (pSP-A) Immunogenicity Evaluation in the Murine Model S. de Cássia Dias, F. L. dos Santos, D. Sakauchi, D. Iourtov, I. Raw, F. S. Kubrusly……………………………………………27

New Design of Biopharmaceuticals through the Use of Microalgae Addressed to Global Geopolitical and Economic Changes. Are You Ready for New Development in Biopharma? A. B. Avagyan……………………………………………………………………………………………………………………33

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Pharmacology & Pharmacy, 2010, 1, 1-8 doi:10.4236/pp.2010.11001 Published Online July 2010 (http://www.SciRP.org/journal/pp)

Anti-Amnesic Activity of Vitex negundo in Scopolamine Induced Amnesia in Rats Abhinav Kanwall,2, Jogender Mehla3, Madhusudana Kunchal, Vegi Ganga Modi Naidul, Yogendra Kumar Gupta3, Ramakrishna Sistla1* 1

Division of Pharmacology, Indian Institute of Chemical Technology (IICT), Hyderabad, India; 2National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad, India; 3Department of Pharmacology, All India Institute of Medical Sciences (AIIMS), New Delhi, India. Email: [email protected] Received June 8th, 2010; accepted July 12th, 2010.

ABSTRACT In the present study we investigated the anti-amnesic activity of Vitex negundo in scopolamine induced amnesia in rats. Wistar rats (180-200 g) were trained on active avoidance task. Each animal received session of 15 trials with inter trial duration of 15 s for 5 days. Scopolamine (3 mg/kg, i.p) was administered at different time periods on the basis of stages of memory i.e acquisition, consolidation and retention in different groups (n = 6). Effect of Vitex negundo extract was evaluated and compared to a standard drug, Donepezil. Significant (p < 0.05) increase in the avoidance response on the 5th session has been observed as compared to 1st session in control group. Scopolamine treatment significantly (p < 0.05) reduced the avoidance response compared to control. Extract treated groups shown significant (p < 0.05) increase in number of avoidance responses as compared to scopolamine treated groups. Increased oxidative stress in brain after scopolamine treatment, as observed by increase in MDA & decrease in GSH & SOD, was lowered in the groups treated with extracts. AChE activity was also improved after V. negundo treatment. Results of the study have shown that V. negundo treated groups decrease the phenomenon of amnesia by increasing learning of memory through antioxidant effect and decreasing AChE activity. Keywords: Vitex negundo, Amnesia, Acetylcholinestrase, Scopolamine, Learning and Memory, Oxidative Stress

1. Introduction The Memory is the most important function of the brain. Memory is the process by which organisms are able to record their experiences and use this information to adapt their responses to the environment. Hence it is vital for survival [1]. Central cholinergic system is considered as the most important neurotransmitter involved in regulation of cognitive functions [2]. Impaired cognitive functions are the major features of Alzheimer disease (AD) [3]. Presence of acetylcholine within the neocortex is sufficient to ameliorate learning deficits and restore memory [4]. The prevalence of AD increases with the age (65 yrs) from 2% to 30-45% in those over 85 yrs [5]. AD and stroke together rank as the third most common causes of death [6]. The incidence of AD for those aged 65yrs and older was 3.24 per 1000 individuals in a year [7]. One study in India showed that, the median survival time determined to be 3.3 yrs for patients with dementia and 2.7 yrs for patients with AD [8]. Scopolamine, a Copyright © 2010 SciRes.

nonselective muscarinic cholinergic antagonist, is a wellknown centrally acting cholinergic probe, which causes impairment in learning [9]. In addition, scopolamine also causes increase in cognitive impairment in healthy elderly subjects compared to young adults [10]. The treatment with AChE inhibitors and muscarinic receptors agonists which increases cholinergic neurotransmission causes an improvement in cognitive deficits in AD [11]. Besides reducing cholinergic activity, oxidative stress plays an important role and is one of the major causes for memory loss in AD [12,13] Extensive research is going on different plants all around the world as plant extracts have a relatively higher therapeutic window, lesser side effects and are economical. Plant extracts may also provide a source of new compound as many synthetic drugs have been originated from herbal sources. Vitex negundo, a deciduous shrub belonging to family Verbenaceae that comprises 75 genera and nearly 2500 species, chiefly occurs in Pakistan, India and Srilanka. Though almost all PP

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Anti-Amnesic Activity of Vitex negundo in Scopolamine Induced Amnesia in Rats

parts of the plant are used, the extract from leaves and the roots is the most important in the field of phytomedicine and is sold as drugs. The leaf extract is used in Ayurvedic and Unani system of medicine [14]. Water extract of mature fresh leaves exhibited anti-inflammatory, analgesic and antihistamine properties [15]. Literature survey of V. negundo revealed the presence of volatile oil, triterpenes, diterpenes, sesquiterpenes, lignan, flavonoids, flavones glycosides, iridoid glycosides, and stilbene derivative [14]. Lignans, one class of natural compounds present in V. negundo, showed anti-cholinesterase activity in in-vitro [14]. However no studies were conducted to explore the effect of V. negundo extract against memory impairment in in-vivo. In the process of learning and memory, three important stages have been suggested viz., acquisition, consolidation and recall of the learned task [16]. The Scopolamine hydrobromide is an anticholinergic drug, which produces amnesia by reducing the levels of acetylcholine, which is considered to be an important neurotransmitter for the learning and memory. Therefore, the present study was aimed to investigate the anti-amnesic effect of V. negundo aqueous extract on scopolamine administered at different stages of active avoidance learning in rats.

2. Materials and Methods 2.1 Materials Aqueous extract of the plant Vitex negundo was obtained from Amruta herbals Pvt Limited, Indore (M.P), India, (Batch no. AHVN/556.) along with the copy of certificate of analysis. Scopolamine hydrobromide, Thiobarbituric acid (TBA), Glutathione, DTNB, Acetylthiocholine all were purchased from Sigma-Aldrich (Bangalore, India). SOD kit was purchased from Fluka. Other chemical and reagents are of analytical grade.

2.2 Animals Male Wistar rats weighing between 180-200 g were obtained from National Institute of Nutrition, Hyderabad. The animals were housed in an animal facility of Indian Institute of Chemical Technology (IICT). The animal house maintained at 20 ± 2°C and 50-60% relative humidity. A 12-hour dark/light cycle was maintained throughout the study. Air changes were maintained with 5µ HEPA filter. Rats had free access to food (pellet diet supplied from M/s Petcare India Ltd., Bangalore) and water ad libitum. This study protocol was approved by the Institutional Animal Ethics Committee of Indian Institute of Chemical Technology, Hyderabad.

2.3 Behavioral Test 2.3.1 Two-Way Active Avoidance with Negative (Punishment) Reinforcement The animals were trained on Active Avoidance Task in Copyright © 2010 SciRes.

an automatic reflex conditioner with two-way shuttle box (Ugo Basile, Italy). The rats were treated orally with the standard drug through an intragastric feeding tube. Similarly the plant extract were administered for 14 days. For this purpose each rat is placed in a compartment separated from the other one by a guillotine door in the shuttle box. Exploration period of 2 min is given initially. There after, the trial start. In each trial the animal is subjected to a light for 30 s followed by a sound stimulus for 10s. Immediately after the sound stimulus, the rat receives a single low intensity foot shock (0.5 mA; 3 s) from 10th day to 14th through the floor grid if it does not transfer to the other shock free compartment. Infrared sensors monitor the transfer time from one compartment to another, which is recorded as avoid (after the stimulus of either light alone or both light and sound) and escape (after the foot shock) response. Each animal received a daily session of 15 trials with an inter-trial duration of 15 s for 5 days i.e., a maximum of 75 trials. The rats were evaluated on the basis of their performance in the last session i.e., in the 5th session for their decrease in amnesic activity and increased learning and memory. The criterion for improved cognitive activity was taken as significant increase in the avoidance response on 5th session (retention) compared to 1st session.

2.4 Scopolamine Induced Loss of Memory in Rat Acquisition: scopolamine was administered 5 min prior to 1st Trial on 1st session. Consolidation: scopolamine was administered 5 min after the 15th (i.e., last) trial on 1st session (Training session). Retention: scopolamine was administered 5 min prior to the 1st trial on the last session i.e., 5th session (Training session). Dementia effect of scopolamine was evaluated on the basis of significant decrease in number of avoidance response in the treated groups as compared to that of control group in the last session i.e., 5th session.

2.5 Treatment Schedule The animals were divided into eight different groups (n = 6). Scopolamine (3 mg/kg, i.p) was administered at different time periods in the three groups (GR-2, GR-3, and GR-4) as follows: Group I (GR-1)–Saline (control). Group II (GR-2)–scopolamine was administered 5 min prior to 1st Trial on 1st session (Training session). Group III (GR-3)–scopolamine was administered 5 min after the 15th (i.e., last) trial on 1st session (Training session). Group IV (GR-4)–scopolamine was administered 5 min prior to the 1st trial on the last session i.e., 5th session. Group V (GR-5)–Standard drug, Donepezil (5 mg/kg) PP


is given to GR-2 rats prior to 1 hour of 1st trial. Group VI (GR-6)–Similar to GR-2 but rats were pre-treated with plant extract for 14 days. Group VII (GR-7)–Similar to GR-3 but rats were pre-treated with plant extract for 14 days. Group VIII (GR-8)–Similar to GR-4 but rats were pretreated with plant extract for 14 days.

2.6 Biochemical Estimation of Markers of Oxidative Stress On day 14th following the behavioral testing, animals were sacrificed and the brain tissues were quickly removed, cleaned with ice-cold saline and stored at –80°C for biochemical estimation. 2.6.1 Preparation of Brain Homogenate Brain-tissue samples were thawed and homogenized with 10 times (w/v) ice-cold 0.1 M phosphate buffer (pH 7.4). Aliquots of homogenates from the rat brains were separated and used to measure protein, lipid peroxidation and glutathione. The remaining homogenates were centrifuged at 10,000 rpm for 15 min and the supernatant was then used for enzyme assay. Superoxide dismutase was determined within 24 h. 2.6.2 Estimation of Malondialdehyde (MDA) Aliquotes of 0.5 ml distilled water and 1.0 ml 10% TCA were added to a volume of 0.5 ml brain tissue homogenate, mixed well and centrifuged at 3000 rpm for 10 min. To 0.2 ml supernatant, 0.1 ml thiobarbituric acid (TBA) (0.375%) was added. The total solution was placed in a water bath at 80ºC for 40 min and then cooled to room temperature. The absorbance of the clear supernatant was measured at 532 nm in spectrophotometer [17]. 2.6.3 Estimation of Superoxide Dismutase (SOD) The SOD activity of the brain tissue was analyzed by using the SOD Assay kit (Fluka). For the assay, 200 µl of working solution, 20 µl of dilution buffer and 20 µl of enzyme working solution was added. Incubate the plate at 37°C for 20 min. Absorbance was read at 450 nm using a microplate reader. 2.6.4 Measurement of Glutathione Pipette out 100 µl of the brain supernatant and add 50 µl of O-ophthaldehyde (100 µl/ml). Incubate at room temperature for 15 min. The flouroscent complex formed was read at an excitation wavelength of 350 nm and emission wavelength of 420 nm [18]. 2.6.5 Estimation of Cholinergic Status in the Rat Brain The cholinergic marker, acetylcholinesterase was estimated in the whole brain according to the method of [19]. Briefly, the brains of the rats were removed over ice and the brain was separated using fine forceps. The tissue was then homogenized in 100 mM phosphate buffer. 0.1 Copyright © 2010 SciRes.

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ml of this homogenate was incubated for 5 min with 2.7 ml of phosphate buffer and 0.1 ml of DTNB. Then, 0.1 ml of freshly prepared acetylthiocholine iodide, pH 8 was added and the absorbance was read at 412 nm for 3 min at 30, 60, 90, 120, 150 and 180 sec.

2.7 Statistical Analysis All data were expressed as mean ± SD. The significance of difference among the values of control, scopolamine treated, standard drug and extract treated groups for each session was determined by ANNOVA (one-way) followed by Dunnett’s test. The difference between values on 1st session and 5th session of the same group was analyzed by student’s t-test.

3. Results 3.1 Selection of the Dose One single dose (300 mg/kg) of the herbal extract has been selected after the initial pilot study. This pilot study was done by taking limited number of Wistar rats. In the pilot study three different doses (100, 300 & 900 mg/kg) were taken. Based on initial data (data not shown) from active avoidance test 300 mg/kg was selected for the main study. It was also seen that animals with higher dose (900 mg/kg) tolerated the shock and remained at one place, which is not acceptable for the avoidance test. However, with lower dose (100 mg/kg) there was no significant difference in the number of avoidances between different groups of animals.

3.2 Automatic Reflex Conditioner There was a significant (p < 0.05) increase in avoidance response on 5th session (6.4 ± 1.67) as compared to 1st session (3.0 ± 1.00) in the control group (Table 1). All groups except GR-3 have shown significant (p < 0.05) increase in avoidance response compared to their first session data. Significant (p < 0.05) reduction of avoidance response was observed in scopolamine treated group (GR-2) compared to control group (GR-1). However standard drug (donepezil) treatment and extract feeding (GR-5 and GR-6) significantly (p < 0.05) increased the avoidance response in their first session compared to their corresponding scopolamine treated group (GR-2). This reflects the effectiveness of donepezil as well as aqueous extract during scopolamine induced memory loss. However donepezil group (GR-5) showed improved response compared to extract treated group (GR-6) (5.8 ± 0.83 vs 4.8 ± 0.44) at the end of 5th session. While extract treatment showed significant (p < 0.05) improvement of avoidance response at the end of 5th session in GR-7, no improvement was observed in GR-8. PP


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Table 1. The number of avoidance responses in control (GR-1), scopolamine (GR-2, 3, 4) and drug treated (GR-6, 7, 8) groups (Mean ± SD, n = 6) S.NO.

GROUPS

DAY1

DAY2

DAY3

DAY4

DAY5

1

GR-1

03 ± 1.00

3.6 ± 1.14

4.2 ± 0.83

05 ± 0.70

6.4 ± 1.67**

2

GR-2

2.4 ± 0.55

03 ± 1.00

3.4 ± 1.14

3.8 ± 0.83

4.2 ± 0.83**,†

3

GR-3

3.2 ± 1.09

2.6 ± 0.54

3.4 ± 0.54

3.4 ± 0.89

3.2 ± 0.44

4

GR-4

3.4 ± 0.54

3.6 ± 0.89

4.2 ± 0.83

4.6 ± 0.54

4.8 ± 0.44*

5

GR-5

03 ± 0.70

04 ± 100

4.4 ± 0.54

5.4 ± 0.89

5.8 ± 0.83**, Ψ

6

GR-6

3.4 ± 0.54

3.8 ± 0.83

4.2 ± 0.83

4.8 ± 0.83

4.8 ± 0.44**,†, Ψ

7

GR-7

3.6 ± 0.54

3.6 ± 0.54

3.8 ± 0.44

4.4 ± 1.52

4.6 ± 0.54*, Ψ

8

GR-8

3.6 ± 0.89

3.8 ± 0.44

4.4 ± 0.54

4.8 ± 0.44

5.4 ± 0.54**

*

p < 0.05 vs. Day 1; **p < 0.01 vs. Day 1; †p < 0.05 vs. Standard drug (GR-6); Ψp < 0.05 vs. corresponding scopolamine treated group

3.3 Markers of Oxidative Stress in Rat Brain 3.3.1 Malondialdehyde (MDA) levels Scopolamine treatment (GR-2, GR-3 and GR-4) significantly (p < 0.05) increased the brain MDA level compared to control (GR-1) group (Figure 1). However only GR-3 showed significant (p < 0.05) change compared to GR-1. Standard drug (GR-5) and aqueous extract of V. negundo (GR-6, GR-7 and GR-8) treatment significantly (p < 0.05) decreased brain MDA level compared to their corresponding scopolamine treated groups (GR-2, GR-3 and GR-4). 3.3.2 Glutathione (Gsh) Levels Brain GSH level was decreased significantly (p < 0.05) in scopolamine treated groups (GR-2, GR-3 and GR-4) compared to control (GR-1) (Figure 2). However standard drug (GR-5) and aqueous extract of V. negundo (GR-6, GR-7 and GR-8) treatment significantly (p < 0.05) increased brain GSH level compared to their corresponding scopolamine treated groups (GR-2, GR-3 and GR-4). 3.3.3 SOD Activity SOD Activity has been expressed in % inhibition rate. Scopolamine treatment decreased brain SOD activity significantly (p < 0.05) in GR-2 and GR-3 groups but not in GR-4 (Figure 3). No improvement of SOD activity was observed in V. negundo extract treated groups (GR-6, GR-7 and GR-8) compared to their corresponding scopolamine treated groups (GR-2, GR-3 and GR-4). However standard drug (GR-5) increased the SOD activity significantly (p < 0.05) compared to the corresponding scopolamine treated group (GR-2). 3.3.4 AChE Activity Acetylcholinestrase activity was estimated by the Vmax Copyright © 2010 SciRes.

values as shown in Figure 4. The scopolamine treated groups have more Vmax values as compared to control group. Significant (p < 0.05) increased of AChE activity

†

†

††

††

*p < 0.05 vs. control group †p < 0.05 vs. corresponding scopolamine treated group ††p < 0.01 vs. corresponding scopolamine treated group

Figure 1. Effect of aqueous extracts of Vitex negundo (300 mg/kg body wt.) on MDA levels in brains on Session 5 on different groups (Mean ± SD, n = 6)

†

††

††

††


Figure 2. Effect of aqueous extracts of Vitex negundo (300 mg/kg body wt.) on Glutathione levels in brains on Session 5 on different groups (Mean ± SD, n = 6)

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Anti-Amnesic Activity of Vitex negundo in Scopolamine Induced Amnesia in Rats ††


Figure 3. Effect of aqueous extracts of Vitex negundo (300 mg/kg body wt.) on SOD levels in brains on Session 5 on different groups (Mean ± SD, n = 6)

†† ††

††


Figure 4. Effect of aqueous extracts of Vitex negundo (300 mg/kg body wt.) on AChE levels in brains on Session 5 on different groups (Mean ± SD, n = 6)

was observed in scopolamine treated groups (GR-2 and GR-3, not in GR-4) compared to control (GR-1) (Figure 4). However standard drug (GR-5) and aqueous extract of V. negundo (GR-6 and GR-7, not in GR-8) treatment significantly (p < 0.01) decreased brain AChE activity compared to their corresponding scopolamine treated groups (GR-2, GR-3 and GR-4).

4. Discussion V. negundo possesses many medicinal properties. Leaves of V. negundo have been investigated for its anti-inflammatory activity [15,20]. Telang et al. first noticed non-steroidal anti-inflammatory (NSAID) activity of V. negundo. Similarly, fresh leaves of V. negundo have been suggested to possess anti-inflammatory and pain suppressing activities. Antinociceptive activity study of ethanolic leaf extract of V. negundo showed that it possesses both central and peripheral analgesic activity [21]. V. negundo has been also used in adjuvant therapy to standard anti-inflammatory drugs [22]. Literature survey of V. negundo also revealed the presence of lignans derivative, which is responsible for anti-cholinesterase acCopyright © 2010 SciRes.

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tivity in in-vitro [14]. LD50 dose of V. negundo leaf extract is 7.58 g/kg which practically falls in the non- toxic dose range [23]. The administration of the antimuscarinic agent scopolamine produces transient memory deficit. Also, scopolamine has been shown to impair memory retention when given to rat shortly before training in an avoidance task. The ability of a range of different cholinergic agonist drugs to reverse the amnesic affects of scopolamine is now well documented in animals and human volunteers [24]. The scopolamine amnesia test is widely used as primary screening test for so called anti-Alzheimer drugs [24]. Here, scopolamine is given at different time of training sessions and trials. Such protocol is adapted to distinguish between the three different stages of memory i.e. acquisition, consolidation and retention. In the preliminary screening of the present study showed that the improvement in learning and memory tasks in the shuttle-box was only observed at a dose of 300 mg/kg body wt. Therefore, the aqueous extract with 300 mg/kg was evaluated in more details. The avoidance responses shown by the animals were due to their ability to learn the task, which reflects the cognitive function. The task was investigated by using the scopolamine-induced dementia with the aqueous herbal extract of V. negundo. The animals were treated with scopolamine at different time intervals of trial in the sessions according to the different stages of the memory. Scopolamine administered 5 min prior to 1st trial on 1st session was for the acquisition whereas scopolamine administered 5 min after the 15th (i.e., last) trial on 1st session was for the consolidation stage of the memory. Similarly for the requisition (recall) the scopolamine was administered 5 min prior to the 1st trial on the last session i.e., 5th session. To evaluate the effect of the herbal aqueous extract of V. negundo, scopolamine was administered in the similar pattern in the pretreated herbal extract animals. The efficacy and potency of the extract was compared with the vehicle control and standard drug (Donepezil) group. From the behavioral test i.e. two way shuttle box active avoidance test, it is clearly seen that there was a general decrease in the performance in the active avoidance in the scopolamine treated groups. The memory loss effect of scopolamine is more prominent compared to the control group. The aqueous herbal extract of V. negundo improved the memory loss effect of scopolamine in all three events like acquisition, consolidation and retention. As scopolamine-induced memory loss was more prominent in acquisition period, we administered standard drug, donepezil with this group. In comparison with Donepezil, the extract treated group had almost equal avoidance responses which indicates therapeutic efficacy of V. negundo against memory loss. The present study therefore demonstrates the probable PP

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mechanism by which V. negundo enhanced the anti-amnesic activity by increasing the performance of learning and memory. It had been suggested that the varying degrees of behavioral impairments are associated with aging and age associated neurodegenerative diseases. Oxidative stress due to free radicals generation is responsible for producing the neuronal changes mediating these behavioral deficits [25]. Oxidative stress in brain generates oxygen radicals like superoxide anion, hydroxyl radical, and hydrogen peroxide, which act on polyunsaturated fatty acids in brain, thereby propagating the lipid peroxidation [26]. The major antioxidant and oxidative freeradical scavenging enzymes like glutathione, SOD and catalase plays an important role to reduce oxidation stress in brain. In the present study rats after scopolamine treatment showed a significant increase in the brain levels of malondialdehyde, which is the measure of lipid peroxidation and free radical generation. At the same time there was a significant reduction in levels of glutathione, a tripeptide found in all cells, which reacts with free radicals to protect cells from superoxide radical, hydroxyl radical and singlet oxygen [27]. Pre-treatment of V. negundo reduced the MDA levels and increased GSH content in brain after scopolamine treatment. Scopolamine reduced the SOD activity in brain. SOD is the only enzyme that uses the superoxide anions as the substrate and produces hydrogen peroxide as a metabolite. Super oxide anion is more toxic than H2O2 and has to be removed. Pretreatment with V. negundo significantly prevented the reduction of SOD activity in brain during scopolamine treatment. Our results also suggest that the aqueous extract of V. negundo reduced oxidative stress by reducing lipid peroxidation and increasing the endogenous antioxidant enzymes in brain. Other important activity has been shown by the extract is that it has acetylcholinetrase (AChE) inhibiting activity. This activity tends to allow the more retention of acetylcholine in the brain, which is important for the cognitive functions, learning and memory. In conclusion, the present study demonstrates that aqueous V. negundo extract has potential therapeutic effects on improving the anti-amnesic activity in rats through inhibiting lipid peroxidation, augmenting endogenous antioxidant enzymes and decreasing acetylcholinestrase (AChE) activity in brain. Further study is warranted to find its potential use in humans.

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5. Aknowledgements

[13] M. A. Lovell, W. D. Ehmann, S. M. Butler and W. R. Markesberg, “Elevated Thiobarbituric Acid Reactive Substances and Antioxidant Enzyme Activity in the Brain in Alzheimer’s Disease,” Neurology, Vol. 45, No. 8, 1995, pp. 1594-1608.

Authors are very thankful to the Project Director, National Institute of Pharmaceutical Education and Research, Hyderabad and Director Indian Institute of Chemical Technology, Hyderabad for supporting this work. We are also thankful to Amruta Herbals Pvt Limited, Indore (M.P), India for providing the plant material.

[15] M. G. Dharmasiri, J. R. Jayakodym, G. Galhenam, S. S. Liyanagem and W. D. Ratnasooriyam, “Anti Inflammatory and Analgesic Activities of Mature Fresh Leaves of Vitex negundo,” Jounal of Ethnopharmacology, Vol. 87,


[14] U. H. Azhar and M. Abdul, “Enzymes Inhibiting Lignans from Vitex negundo,” Chemical and Pharmaceutical Bulletin, Vol. 52, No. 11, 2004, pp. 1269-1272.

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Anti-Amnesic Activity of Vitex negundo in Scopolamine Induced Amnesia in Rats No. 2-3, 2003, pp. 199-206. [16] A. C. Guyton and J. E. Hall, “Textbook of Medical Physiology,” Harcourt Asia Pte Ltd, Singapore, 1999. [17] H. Ohkawa, N. Ohishi and K. Yagi, “Assay for Lipid Peroxides in Animal Tissues by Thiobarbituric Acid Reaction,” Analytical Biochemistry, Vol. 95, No. 2, 1979, pp. 351- 358. [18] P. J. Hissin and R. Hilf, “A Fluorometric Method for Determination of Oxidized and Reduced Glutathione in Tissues,” Analytical Biochemistry, Vol. 74, No. 1, 1976, pp. 214-226. [19] G. L. Ellman, D. K. Courtney, V. Andres and R. M. Featherstone, “A New and Rapid Colorimetric Determination of Acetylcholinesterase Activity,” Biochemical Pharmacology, Vol.7, No. 2, 1961, pp. 88-95. [20] R. S. Telang, S. Chatterjee and C. Varshneya, “Studies on Analgesic and Anti-Inflammatory Activities of Vitex negundo Linn,” Indian Journal of Pharmacology, Vol. 31, No. 5, 1999, pp. 363-366. [21] R. K. Gupta and V. R. Tandon, “Antinociceptive Activity of Vitex-Negundo Linn Leaf Extract,” Indian Journal of Pharmacology, Vol. 49, No. 2, 2005, pp. 163-170.


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[22] R. K. Gupta and V. R. Tandon, “Vitex negundo Linn (VN) Leaf Extract as an Adjuvant Therapy to Standard AntiInflammatory Drugs,” The Indian Journal of Medical Research, Vol. 124, No. 4, 2006, pp. 447-450. [23] M. N. Ghosh, “Fundamentals of Experimental Pharmacology,” Scientific Book Agency, Calcutta, 1984. [24] M. H. V. Kumar and Y. K. Gupta, “Antioxidant Property of Celastrus Paniculatus Willd: A Possible Mechanism in Enhancing Cognition,” Phytomedicine, Vol. 9, No. 4, 2002, pp. 302-311. [25] C. I. Cantuti, B. Shukitt-Hale and J. A. Joseph, “Neurobehavioural Aspects of Antioxidants in Aging,” International Journal of Developmental Neuroscience, Vol. 18, No. 4-5, 2000, pp. 367-381. [26] T. Coyle and P. Puttfarcven, “Oxidative Stress, Glutamate and Neurodegenerative Disorder,” Science, Vol. 262, No. 5134, 1993, pp. 89-695. [27] J. B. Schulz, J. Linderau and J. Dichgans, “Glutathione, Oxidative Stress and Neurodegeneration,” European Journal of Biochemistry, Vol. 267, No. 16, 2000, pp. 49044911.

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Graphical Abstract


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Development and Evaluation of a New Interpenetrating Network Bead of Sodium Carboxymethyl Xanthan and Sodium Alginate for Ibuprofen Release Rajat Ray, Siddhartha Maity, Sanchita Mandal, Tapan K. Chatterjee, Biswanath Sa The Division of Pharmaceutics, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India. Email: [email protected] Received June 6th, 2010; accepted July 8th, 2010.

ABSTRACT Interpenetrating network (IPN) beads of sodium carboxymethyl xanthan (SCMX) and sodium alginate (SAL) were prepared by ionotropic gelation process using AlCl3 as a cross-linking agent. The effect of different formulation variables like total polymer concentration, gelation time, concentration of cross-linking agent, and drug load on the extent of release of ibuprofen (IBP), a non steroidal anti-inflammatory drug, was examined. The formation of IPN structure was examined using Fourier Transform Infra-red (FTIR) analysis and the compatibility of the drug in the bead was evaluated through FTIR, X-ray diffraction (XRD) and Differential Scanning Calorimetry (DSC) analyses. While increase in the concentration of total polymer, gelation time, and drug load decreased the drug release in both acidic (pH-1.2) and phosphate buffer (PB) solution (pH-6.8), increase in the concentration of cross-linking agent tended to increase the drug release. However, from all the formulations, the drug release in acidic medium was considerably slow and a maximum 14% of the loaded drug was released in 2 h. Complete drug release was achieved in PB solution within 210 to 330 min depending upon the formulation variables. The release of the drug followed non-Fickian transport process in acidic medium and case-II transport mechanism in PB solution and these release behaviour correlated well with the kinetics of dynamic swelling of IPN beads. The study indicated that the IPN beads of SCMX and SAL could be a suitable dosage form to minimize the drug release in acidic solution and to control the drug release in PB solution depending upon the need. Keywords: IPN Bead, Ibuprofen, Drug Release, Kinetics, Swelling

1. Introduction Among the most abundant natural polymers, polysaccharides are widely used in pharmaceutical dosage forms as excipients like suspending agents, emulsifying agents, tablet binders, gelling agents. With the advent of macromolecular chemistry, the use of polysaccharides has been extended towards new applications in pharmaceutical, biomedical, and agricultural fields. Although naturally available polysaccharides exhibit certain limitations in terms of their reactivity and processibility, these can be overcome by modification of the polysaccharides through either physical or chemical cross-linking, grafting with other materials and developing hydrogels or interpenetrating network (IPN) structures. Since the homopolymers alone can not meet divergent Copyright © 2010 SciRes.

demand in terms of properties and performances, development of IPN appears to be a better approach [1] and one of the easiest ways for modification of the properties of polysaccharides. IPN consists of two polymers, each in network form, which can be cross-linked in the presence of each other to give a three dimensional network structure [2] and hence, combine the properties of two cross-linked polymers in a network form [3]. IPNs are thus emerging as a rapidly developing branch of polymer blended technology and are finding applications in artificial implants, dialysis, membranes, drug delivery systems [4], and in agricultural field [5]. Sodium alginate (SAL), a hydrophilic biopolymer obtained from brown sea weeds, is a polysaccharide composed of varying proportions of D-mannuronic acid (M) and L-guluronic acid (G) residues which are arranged in PP

10

Development and Evaluation of a New Interpenetrating Network bead of Sodium Carboxymethyl Xanthan and Sodium Alginate for Ibuprofen Release

MM or GG blocks interspersed with MG blocks [6]. Its unique property of forming water insoluble calcium alginate gel through ionotropic gelation with Ca2+ ions in a simple and mild condition has made possible to encapsulate both macromolecular agents [7] and low molecular weight therapeutic agents [8,9] in calcium alginate beads. However in physiological environment, calcium alginate beads tend to have poor mechanical stability [10]. To overcome this limitation, IPN beads of SAL with gelatin or egg albumin [2], polyvinyl alcohol-graftedpolyacrylamide [11], N,O-Carboxymethyl chitosan [12] for controlled drug delivery, and with gelatin [5] for controlled release of pesticides have been developed. Although xanthan gum, a polysaccharide obtained from Xanthomonas campestris, can not form gel beads, its Na-salt of carboxymethyl derivative is able to form gel beads through ionotropic gelation with Al3+ ions [13]. Sodium carboxymethyl xanthan (SCMX) beads have been found capable of encapsulating albumin [14] and diltiazem hydrochloride [15]. However hitherto there are no reports on IPN beads of SCMX with SAL for drug release study. The objective of the present work was to develop a new IPN bead composed of SCMX and SAL and to evaluate the beads for encapsulation and release behavior of ibuprofen (IBP).

2. Experimental 2.1 Materials Ibuprofen (Indian Pharmacopoeia) and xanthan gum were obtained as gift samples from respectively M/S Albert David Limited and M/S Deys Medical Stores (Mfg). Pvt. Limited, Kolkata, India. Sodium alginate (Mol. wt. 240kDa), AlCl3·2H2O (SD Fine Chem Pvt. Ltd, Mumbai, India), Monochloro acetic acid (Loba Chemie Pvt. Ltd, Mumbai, India) and all other analytical grade reagents were obtained commercially and used as received.

2.2 Preparation of Sodium Carboxymethyl Xanthan (SCMX) Xanthan gum was derivatised to SCMX having O-carboxymethyl substitution of 0.8 following the method reported previously [13]. In brief, required amount of xanthan gum was dispersed in ice cold solution of 45% w/v sodium hydroxide. The dispersion was kept at 5-8°C with continuous stirring for 1h. Monochloroacetic acid solution (75% w/v) was added with stirring in the reaction mixture and the temperature was raised slowly to 15-18°C. After 30 min, the temperature was raised to 75°C and maintained for additional 30 min. The reaction mixture was, then cooled to room temperature, cut into small pieces and dried at 50°C. The dried product was Copyright © 2010 SciRes.

milled, washed with 80% v/v methanol and again dried.

2.3 Preparation of Interpenetrating Network (IPN) Bead Required amount of ibuprofen (IBP) was homogenously dispersed in an aqueous solution of SCMX and SAL. The resulting dispersion was extruded through 21 G flat-tip hypodermic needle into AlCl3 solution. Gelation of the beads was carried out for different periods of time. The beads were, then collected by filtration, washed with deionized water, dried at 45°C in a hot air oven to constant weight and kept in a dessicator until used. The beads were prepared using the following variables: 1) Keeping the drug load constant at 50% w/w of total polymer and the concentration of AlCl3 constant at 2% w/v, the total polymer concentration was varied from 2-4% w/v (SCMX to SAL weight ratio 1:1) and the gelation time was varied from 0.5 to 2 hour. 2) Keeping the drug load constant at 50% w/w of total polymer, the gelation time at 0.5 hour and total polymer concentration 3% w/v, concentration of AlCl3 was varied from 2-8% w/v. 3) Keeping the total polymer concentration fixed at 3% w/v, gelation time at 0.5 hour, and AlCl3 concentration at 2% w/v, drug load was varied from 20-60% w/w of total polymer. The composition of beads is shown in Table 1. Each formulation was prepared in duplicate. Table 1. Composition and drug entrapment efficiency (DEE) of sodium carboxymethyl xanthan (SCMX) and sodium alginate (SAL) IPN beads SCMX%: SAL%

Concentration Drug load Gelation of AlCl3 (% w/w of time (hr) (% w/v ) total polymer)

DEE (Mean ± SD, n = 4)

1:1

50

0.5

2

93.46 ± 2.18

1.5:1.5

50

0.5

2

97.22 ± 2.45

2:2

50

0.5

2

99.50 ± 2.86

1:1

50

2

2

91.15 ± 1.92

1.5:1.5

50

2

2

94.25 ± 3.37

2:2

50

2

2

95.31 ± 2.72

1.5:1.5

50

0.5

4

97.25 ± 1.38

1.5:1.5

50

0.5

8

97.65 ± 3.84

1.5:1.5

20

0.5

2

98.86 ± 1.26

1.5:1.5

40

0.5

2

99.16 ± 1.74

1.5:1.5

60

0.5

2

96.96 ± 2.52

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2.4 Fourier Transform Infrared (FTIR) Analysis

content) × 100.

FTIR spectra of SCMX, SAL and drug free IPN bead were recorded in a FTIR spectrophotometer (Perkin- Elmer, model Spectrum RX-1, UK). Each sample was mixed with KBr and converted into disc at 100 kg pressure using a hydraulic press. The spectra were recorded within 4000-400 cm-1 wave numbers. Similarly, the FTIR spectra of IBP and drug loaded IPN beads were recorded.

2.9 In-Vitro Drug Release Study

2.5 Powder X-Ray Diffraction (XRD) Analysis Qualitative XRD studies were performed using an X-ray diffractometer (Bruker D8 advanced powder diffractometer, USA). Pure IBP and powdered beads were scanned from 5° to 55° diffraction angle (2θ) range under the following conditions: Source, Ni-filtered Cu-Kα (λ = 1.54) radiation; voltage, 40 kV; Current, 40 mA; scan speed, 16°/min

2.6 Differential Scanning Calorimetry (DSC) Study

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In-vitro drug release study was carried out in acidic solution 0.1 (N) HCl (pH 1.2) and in USP PB solution (pH 6.8) using USP-II dissolution rate test apparatus (model TDP-06P Electro Lab, Mumbai, India). 20 mg beads were placed in 500 ml acidic solution or 500 ml PB solution (37 ± 1°C) and rotated with paddle at 75 rpm. Aliquot was withdrawn at different times and replenished immediately with the same volume of fresh solution. Undiluted or suitably diluted withdrawn samples were analyzed spectrophotometrically at 220 nm for acidic solution and 222 nm for PB solution. The amount of drug released in acidic solution and PB solution were calculated from the calibration curves drawn respectively, in 0.1 (N) HCl and PB solution (pH 6.8). Each release study was conducted four times.

2.10 Swelling Study

DSC thermograms of IBP and powdered beads were obtained in the following way: A weighed amount (about 6 mg) of sample was kept in a hermetically sealed aluminium pan and heated at a scan speed of 10°C /min over a temperature range of 35°C310°C in a Differential Scanning Calorimeter (PerkinElmer, model Pyris Diamond TG/DTA, UK ) which was calibrated against indium. A nitrogen purge (20 ml/min) was used throughout the runs.

Dried drug-free IPN beads (50 mg) were immersed in 25ml acidic solution (pH 1.2) at 37°C. The beads were removed at different times by filtration and blotted carefully to remove excess surface water. The swollen beads were weighed. The swelling ratio of the beads were determined using the following formula: Swelling ratio = (weight of swollen beads-weight of dry beads)/weight of dry beads Swelling ratio of the beads in PB solution (pH 6.8) was determined in a similar way.

2.7 Photomicrograph

2.11 Statistical Analysis

Photomicrograph of IPN beads were taken at 4X magnification with an optical microscope (Leica DM 2500P) fitted with a camera (Cannon Power Shot S-80, Japan).

Each formulation was prepared in duplicate, and each analysis was duplicated. Effect of formulation variables on drug release was tested for significance level by using analysis of variance (ANOVA: single factor and two factor) with the aid of Microsoft® Excel 2003. Difference was considered significant when p < 0.05.

2.8 Drug Entrapment Efficiency IPN beads (20 mg) were accurately weighed in an electronic balance (Precisa XB 600 MC, Precisa Instrument Ltd; Switzerland), immersed in 250 ml USP phosphate buffer (PB) solution (pH 6.8), and shaken for 2h on a mechanical shaker. The beads were crushed and further shaken for 1h. The solution was filtered and an aliquot following suitable dilution was analyzed at 222 nm in a UV-Visible spectrophotometer (model Cary-50 Bio-spectrophotometer, VARIAN, Australia)) and the content of the beads was determined using a calibration curve constructed using PB solution of pH 6.8. The reliability of the above analytical method was judged by conducting recovery analysis at three levels of spiked drug solution in the presence or absence of the polymers for three consecutive days. The recovery averaged 98.45 ± 2.68%. DEE was determined using the following relation: DEE (%) = (Determined drug content/Theoretical drug Copyright © 2010 SciRes.

3. Results & Discussion 3.1 Formation of IPN IPN beads composed of SCMX and SAL were prepared by inotropic gelation process using AlCl3 as a common cross-linking agent for both the polymers. Formulation of IPN structure was verified by FTIR analysis (Figure 1). FTIR spectrum of SCMX showed the presence of bands corresponding to asymmetric and symmetric carboxylate anions at respectively 1605 cm-1 and 1419 cm-1, a broad band at 3419 cm-1 corresponding to stretching vibration of hydroxyl group, a peak at 1327 cm-1 corresponding to C = O stretching of carboxymethyl group. These results are similar to the findings reported earlier [14]. The spectrum of SAL showed the bands characteristics of PP

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carageenan beads [17]. Moreover, neither the concentration of cross-linking agent (AlCl3) nor the gelation time had any appreciable effect on morphology of IPN beads. The shape of the beads did not change even when the drug load was varied from 20 to 60% w/w of total polymer.

3.3 Compatibility of Drug in IPN Bead Figure 1. FTIR spectra of (a) SCMX, (b) SAL, (c) drug free IPN bead

asymmetric and symmetric carboxylate anions at respectively 1612 cm-1 and 1420 cm-1, a broad peak corresponding to the stretching of hydroxyl group at 3468 cm-1. Similar spectrum of SAL has been reported elsewhere [16]. The FTIR spectrum of drug-free IPN beads showed peaks at 1639 cm-1 and 1425 cm-1 respectively for asymmetric and symmetric carboxylate anions and a peak at 3405 cm-1 for hydroxyl group. Moreover, the peak at 1327 cm-1 assigned for carboxymethyl group was retained. Comparison of the spectra, however, demonstrated shift of the peaks of carboxylate anions to higher wave numbers. The shift of carboxylate bands confirms the formation of complex between the two polymers and Al3+ ions through physical cross-linking. These results suggest the formation of IPN structure wherein both the polymers are present in cross-linked condition.

Compatibility of IBP in IPN beads was studied using FTIR, XRD and DSC analyses. The characteristics bands corresponding to C=O stretching and –OH stretching of IBP appeared in FTIR spectrum respectively at 1720 cm-1 and 2956 cm-1. The above two bands were also detected at the same positions in the spectrum of drug-loaded IPN beads (Figure 3). XRD analysis showed reflection to the interplanner distances of 14.41, 7.24, 5.32, 5.01, 4.72, 4.65, 4.39, 3.98 and 3.63 Å respectively at 6.13, 12.21, 16.64, 17.68, 18.78, 19.06, 20.20, 22.30 and 24.52˚ 2θ. Drug–loaded IPN beads also exhibited the same reflections at the same 2θ degrees (Figure 4). The result indicates that the crystallinity of the drug in IPN beads was retained and no amorphization of the drug took place. Comparison of DSC thermograms revealed that the

3.2 Morphology of IPN Bead The composition of IBP-loaded IPN beads has been shown in Table1. The beads were prepared with a SCMX to SAL weight ratio of 1:1 but in different total polymer concentration (1%:1%, 1.5%:1.5%, 2%:2%) and gelling in AlCl3 solution (2-8% w/v) for different periods of time (0.5 to 2 h). Although the shapes of the wet beads were spherical, the shapes distorted after drying. The surface of dried beads was rough and folded (Figure 2) and was due to shrinkage of the beads during the drying process. Similar shape distortion has been reported for chitosan/

Figure 2. Photo micrographs of ibuprofen-loaded IPN beads, prepared under different conditions. (a) SCMX: SAL 1%:1%, 2% w/v AlCl3, 0.5 h, (b) SCMX: SAL 1.5%: 1.5%, 4% w/v AlCl3, 2 h, (c) SCMX: SAL 2%:2%, 8% w/v AlCl3, 0.5 h Copyright © 2010 SciRes.

Figure 3. FTIR spectra of (a) Ibuprofen (b) Ibuprofen-loaded IPN bead

Figure 4. X-ray diffractograms of (a) Ibuprofen (b) Ibuprofen-loaded IPN bead PP


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melting endothermic peak of IBP at 79°C also appeared in the DSC curve of drug loaded IPN bead (Figure 5). These studies indicated that apparently no interactions between the drug and the polymers took place during the formation of IPN beads.

3.4 DEE of IPN bead DEE of IPN beads tended to increase as the total polymer concentrations was increased from 2-4% w/w keeping SCMX to SAL weight ratio constant at 1:1 (Table 1). Two way analysis of variance (ANOVA-2) revealed significant difference (F2,3 > Ftabular at 0.05 level) in DEE in IPN beads prepared with increasing polymer concentrations although no significant difference was noted within the batches of each formulation. Same observations were noted with IPN beads which were prepared by gelling in 0.5% AlCl3 solution for two different gelation times. Increase in DEE with increase in total polymer concentrations is related to the higher rigidity of the matrices of IPN beads. Higher encapsulation efficiency of cefadroxyl has been reported for IPN beads prepared using SAL and gelatin or egg albumin [2]. Concentrations of cross-linking agent did not produce any appreciable change in DEE of IPN beads prepared with a total polymer concentration of 3% w/v keeping SCMX to SAL weight ratio constant at 1:1. The results of one way analysis of variance (ANOVA-1) revealed no significant difference in DEE (F2,3 < Ftabular at 0.95 level) of IPN beads prepared with various concentrations of AlCl3 . Similar independence of DEE on the extent of cross-linking has been reported for ketorolac loaded IPN beads composed of sodium carboxymethyl cellulose and gelatin [18]. The time of gelation, however, had an impact on DEE which tended to decrease as gelation time was increased (Table 1). The results are in agreement with the reports of other workers [2]. Although the solubility of IBP in aqueous medium is very less, prolonged exposure in the gelation medium may cause greater leaching of the drug from IPN beads resulting in decreased DEE. IPN beads having 20 to 60% w/w of IBP were prepared using 3% w/w total polymer concentration and gelling for 0.5 h in 2% w/v AlCl3 solution. DEE was found to vary within 96.96 to 99.16% (Table 1). No significant effect of drug loading on DEE was observed. Similar non-dependence of DEE on % of drug loading has been reported for IPN beads composed of sodium carboxymethyl cellulose and gelatin.

3.5 In-Vitro Drug Release 3.5.1 Effect of Polymer Concentration Release of IBP from IPN beads, prepared using increased polymer concentrations (SCMX: SAL weight ratio 1:1) and gelling for 0.5 h in 2% AlCl3 solution, have been represented in Figure 6. Drug release in acidic medium Copyright © 2010 SciRes.

Figure 5. DSC thermograms of (a) Ibuprofen (b) Ibuprofen-loaded IPN bead

Figure 6. Release profiles of Ibuprofen in acidic solution (open symbols) and phosphate buffer solution (closed symbols) from IPN beads prepared using different concentration of SCMX and SAL and gelling in 2% w/v AlCl3 solution for 0.5 h. Key: SCMX: SAL = () 1%:1%, (∆) 1.5%:1.5%, () 2%:2%. Maximum SEM = 1.24(n = 4)

was slow and 8.82 to 14.09% of the loaded drug was released in 2 h. In PB solution (pH 6.8), complete drug release was achieved in 210 min to 300 min depending upon the total polymer concentration in the beads. Increase in total polymer concentration from 2 to 4% w/w decrease the drug release in both the dissolution media. The derived properties obtained from drug release profiles indicated that the area under the curves (AUCs), determined using trapezoidal rule, in acidic dissolution medium decreased as the polymer concentration in IPN beads increased. Similarly, the time required for 50% (t50%) and 80% (t80%) drug release in PB solution increased and AUCs decreased with increase in polymer concentration in the beads. Similar trend in drug release was observed from IPN beads which were prepared by gelling for 2 h (Table 2). Drug release from hydrophilic polymeric beads depends upon the type of matrix used as well as its rigidity [11]. Increase in total polymer concentration results in a more entangled or more compact gel system with a greater cross-linking density in the PP


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Table 2. Derived properties of drug release in different dissolution media from IPN beads prepared using different polymer concentrations and gelling for 2 h in 2% w/v AlCl3 solution SCMX%: SAL%

In acidic solution

In phosphate buffer solution

AUC (% mg · min/ml) (Mean ± SD, n = 4)

t50% (min) (Mean ± SD, n = 4)

t80% (min) (Mean ± SD, n = 4)

AUC (% mg·min/ml) (Mean ± SD, n = 4)

1:1

885.6 ± 56.31

94.8 ± 5.20

143.5 ± 15.21

14336.1 ± 25.63

1.5:1.5

729.2 ± 35.50

108.5 ± 8.75

174.1 ± 10.11

12553.9 ± 27.75

2:2

574.5 ± 46.21

132.4 ± 6.31

209.6 ± 8.65

10564.5 ± 14.02

matrix [12]. As a result, the rigidity of gel matrix increases and free volume of the matrix decreases [19]. This hinders easy transport of drug molecules through the matrix and reduces drug release from the matrix. 3.5.2 Effect of Swelling of IPN Bead The release of a drug from a polymeric matrix is controlled by the swelling behaviour of the polymer. To study the effect of swelling of IPN beads on drug release, swelling ratio of beads was measured in terms of water uptake at selected time intervals and the results have been represented in Figure 7. While the swelling ratio of the IPN beads was very low in acidic solution, the same property increased considerably in PB solution (pH 6.8). The main functional group present in both the polymers that undergoes cross-linking with Al3+ ions is –COOH group. In acidic solution, –COOH group remains protonated and exerts insignificant electrostatic repulsive force. As a result, the beads swell to very less extent. At higher pH value of PB solution, –COOH group undergoes ionization which exerts electrostatic repulsion between the ionized groups, and results in higher swelling. Moreover, upon ionization, the counter ion concentration inside the polymeric network increases, and an osmotic pressure difference exists between the internal and external solutions of the beads. The increased osmotic pressure is balanced by the swelling of the beads [20]. The higher the swelling of the polymers, the higher is the drug release from the IPN beads. Thus the slower release of IBP in acidic solution and faster release in PB solution are related to the swelling behaviour of IPN beads in the respective dissolution media. It was further observed that increase in total polymer concentration from 2 to 4% w/v decreased the swelling of IPN beads in both the media. At low polymer concentration, the polymeric network is loose with a greater hydrodynamic free volume which allows more of the liquid to be absorbed and produces higher swelling. This, in turn, facilitates transport of the drug molecule through the matrix and causes higher drug release [21]. On the other hand, at higher polymeric concentration, opposite phenomenon takes place resulting in slower release of drug. Copyright © 2010 SciRes.

3.5.3 Effect of Concentration of AlCl3 The effect of the concentration of the cross-linking agent (AlCl3) on the release profiles of the drug was studied with IPN beads prepared using 3% w/v total polymer concentration and gelling for 0.5 h in 2-8% AlCl3 solution. Figure 8 showed that as the concentration of AlCl3

Figure 7. Swelling ratios of IPN beads, in acidic solution (open symbols) and phosphate buffer solution (closed symbols), prepared using different concentration of SCMX and SAL and gelling in 2% w/v AlCl3 solution for 0.5 h. Key: SCMX: SAL = () 1%:1%, (∆) 1.5%:1.5%, () 2%:2%

Figure 8. Release profiles of ibuprofen in acidic solution (open symbols) and phosphate buffer solution (closed symbols) from IPN beads prepared using SCMX: SAL = 1.5%: 1.5% and gelling for 0.5 h in different concentration of AlCl3 solution. Key: () 2% w/v, (∆) 4% w/v, () 8% w/v. Maximum SEM = 0.81 (n = 4) PP


was increased during the preparation of beads, the release of drug increased in both the dissolution media. Statistical analysis in terms of ANOVA-1 also confirmed this phenomenon as the AUCs in acidic medium increased and t50%, t80% decreased and AUCs increased in PB solution significantly. This unusual release behaviour could be explained in the following way. When the IPN beads were prepared with higher concentration of AlCl3, a thick outer gel layer might have been formed along the periphery of the beads. The thicker outer gel layer provided higher diffusional resistance to further influx of Al3+ ions resulting in the formation of inhomogeneous gel beads and less densely cross-linked matrix in the core of the beads. During dissolution study, once the outer thick gel layer swelled, quick drug release occurred from the beads. At lower concentration of AlCl3, Al3+ ions diffuse more uniformly into the beads and form homogenous gel beads resulting in slow drug release. 3.5.4 Effect of Gelation Time The derived properties obtained from drug release profiles (Table 3) indicated that increase in gelation time decreased the drug release appreciably. The higher the gelation time, the greater is the cross-linking density and rigidity of the matrix which resulted in a fall in drug release. 3.5.5 Effect of Drug Load The effect of drug load on the release dynamics of IBP was studied using IPN beads prepared using 3% w/v total polymer concentration (SCMX:SAL in a weight ratio 1:1) and gelling for 0.5 h in 2% w/v AlCl3 solution, and the results are shown in Figure 9. Increase in drug load from 20 to 60% w/w of total polymer decreased the drug release in both the dissolution media. Generally, higher drug load provides higher concentration gradient between the drug in the dosage form and the external dissolution medium and results in faster drug release. The release of a drug is governed not only by drug diffusion

15

through the polymeric network but also by the relaxational process of the polymer on solvent penetration. Low drug load in IPN beads forms larger pore fraction resulting in higher swelling and consequently faster drug release. On the other hand, at higher drug load, larger crystalline domain of drug is formed in the beads. This causes reduction as well as shrinkage of pores of the matrix and results in fall in drug release. Decrease in drug release with increase in drug load from various IPN beads have been reported [5,18,22-23]. 3.5.6 Release Kinetics Drug release from a swellable matrix primarily depends on the degree of gelation, hydration, chain relaxation, and erosion of polymer. To understand the mode of drug transport through the IPN beads, the release data were fitted to the classical power law expression [24] Mt/Mα = Ktn where Mt and Mα are, respectively, the amount of drug released at time t and at infinite time, K represents a constant incorporating structural and geometrical characteristics of the dosage forms, n denotes the diffusion exponent indicative of the mechanism of drug release. Values of n ranging from 0.45 to 0.5 indicate Fickian or diffusion controlled release, values of n ranging from 0.5 to 0.89 indicate non-Fickian or anomalous release, and values of n ranging from 0.89 to 1.0 indicate Case-II transport mechanism. By applying least squares method to release data, the values of n were estimated and have been shown in Table 4 along with the correlation co-efficient (r2). The results indicate that drug release in acidic medium followed non-Fickian mechanism and in PB solution drug release occured following case-II transport mechanism. When the swelling data of drug-free IPN beads were fitted to the above power law expression, it was found that swelling in acidic medium took place following the non-Fickian mechanism and that in PB solution followed Case-II transport mechanism (Table 4).

Table 3. Effect of gelation time on derived properties of drug release in different dissolution media from IPN beads prepared using different polymer concentration and gelling in 2% w/v AlCl3 solution for different periods of time In acidic solution


Gelation time 0.5 h Gelation time 2 h SCMX%: SAL%

Gelation time 0.5 h

AUC AUC t50% (min) (% mg · min/ml) (% mg · min/ml) (Mean ± SD, (Mean ± SD, (Mean ± SD, n = 4) n = 4) n = 4)

Gelation time 2 h

AUC t80% (min) (% mg · min/ml) (Mean ± SD, (Mean ± SD, n = 4) n = 4)

t50% (min) (Mean ± SD, n = 4)

t80% (min) (Mean ± SD, n = 4)

AUC (% mg · min/ml) (Mean ± SD, n = 4)

1%:1%

970.1 ± 22.63

885.4 ± 56.31

77.5 ± 7.86

111.1 ± 11.53 13151.5 ± 31.46

94.8 ± 5.20

143.5 ± 15.21 14336.1 ± 25.63

1.5%:1.5%

702.2 ± 27.15

729.2 ± 35.50

94.3 ± 10.45

148.1 ± 9.45

11117.7 ± 20.46

108.5 ± 8.75

174.1 ± 10.11 12553.9 ± 27.75

2%:2%

565.5 ± 13.36

574.5 ± 46.21

119.7 ± 12.61 176.4 ± 14.63

9230.4 ± 26.81

132.4 ± 6.31

209.6 ± 8.65


10564.5 ± 14.02

PP


16

Table 4. Kinetic data (n) and correlation coefficient (r2) of (A) drug release and (B) swelling of IPN beads prepared by gelling in 2% w/v AlCl3 solution for 0.5 h In acidic solution


SCMX:SAL

A

n

r2

n

r2

1%:1%

0.73

0.997

1.26

0.989

1.5%: 1.5%

0.69

0.996

1.28

0.994

2%:2%

0.87

0.993

1.30

0.989

1%:1%

0.69

0.877

1.40

0.996

1.5%:1.5%

0.64

0.935

1.33

0.982

2%:2%

0.80

0.907

1.23

0.923

due to poor swelling of the beads. Complete drug release was achieved in PB solution (pH 6.8) at different periods of time depending on the formulation variables and the release followed case II transport process due to swelling and erosion of the beads. The results of the study indicate that high drug-loaded IPN beads can be prepared using SCMX and SAL by ionotropic gelation process and could be used to minimize the release of IBP in acidic medium and to modulate the drug release in PB solution (pH 6.8).

REFERENCES [1]

M. Changez, K. Burugapalli, V. Koul and V. Chowdary, “The Effect of Composition of Poly (Acrylic Acid)-Gelatin Hydrogel on Gentamycin Sulphate Release in Vitro,” Biomaterials, Vol. 24, No. 4, 2003, pp. 527-536.

[2]

A. R. Kulkarni, K. M. Soppimath, T. M. Aminabhavi and W. E. Rudzinski, “In-Vitro Release Kinetics of Cefadroxyl Loaded Sodium Alginate Interpenetrating Network Beads,” European Journal of Pharmaceutics and Biopharmaceutics, Vol. 51, No. 2, 2001, pp. 127-133.

[3]

T. T. Hsieh, K. H. Hsieh, G. P. Simon and C. Tiu, “Interpenetrating Polymer Networks of 2-Hydroxylethyl Methacrylate Terminated Polyurethanes and Urethanes,” Polymer, Vol. 40, No. 11, 1999, pp. 3153-3163.

[4]

A. K. Bajpai, J. Bajpai and S. Shukla, “Water Sorption through Semi-Interpenetrating Polymer Network with Hydrophilic and Hydrophobic Chains,” Reactive and Functional Polymers, Vol. 50, No. 1, 2002, pp. 9-21.

[5] Figure 9. Effect of drug load on release of ibuprofen in acidic solution (open symbols) and phosphate buffer solution (closed symbols) from IPN beads prepared using SCMX:SAL = 1.5%:1.5% and gelling for 0.5 h in 2% AlCl3 solution. Key: drug load, () 20% w/v, (∆) 40% w/v, () 60%w/v of total polymer. Maximum SEM = 1.27 (n = 4)

A. Roy, A. K. Bajpai and J. Bajpai, “Designing Swellable Beads of Alginate and Gelatin for Controlled Release of Pesticide (Cypermethrin),” Journal of Macromolecular Science, Part A, Vol. 46, No. 9, 2009, pp. 847-859.

[6]

4. Conclusions

P. Aslani and R. A. Kennedy, “Studies on Diffusion in Alginate Gels 1. Effect of Cross-Linking with Calcium or Zinc Ions on Diffusion of Acetaminophen,” Journal of Controlled Release, Vol. 42, No. 1, 1996, pp. 75-82.

[7]

T. L. Bowersock, H. HogenEsch, M. Suckow, P. Guimond, S.Martin, D. Borie, S.Torregrosa, H. Park and K.Park, “Oral Vaccination of Animals with Antigens Encapsulated in Alginate Microspheres,” Vaccine, Vol. 17, No. 13-14, 1999, pp. 1804-1811.

[8]

M. L. Gonzalez-Rodriguez, M. A. Holgado, C. Sanchez-Lafuente, A. M. Rabasco and A. Finni, “Alginate/ Chitosan Particulate Systems for Sodium Diclofenac Release,” International Journal of Pharmaceutics, Vol. 232, No. 1-2, 2002, pp. 225-234.

[9]

A. Halder, S. Maiti and B. Sa, “Entrapment Efficiency and Release Characteristics of Polyethyleneimine-Treated or Untreated Calcium Alginate Beads Loaded with Propranolol,” International Journal of Pharmaceutics, Vol. 302, No. 1-2, 2005, pp. 84-94.

B

SCMX-SAL interpenetrating network beads were prepared by inotropic gelation method using Al3+ ions as cross-linking agent for both the polymers. Formation of IPN structure was verified by FTIR analysis and the absence of drug-polymer interaction in IPN beads was confirmed by FTIR, XRD, and DSC analysises. DEE of IPN beads were found to be reasonably high (91.15 to 99.50%) and was not affected by formulation variables except the gelation time, the increase of which tended to decrease DEE. While the release of IBP decreased in both acidic (pH 1.2) and PB solution (pH 6.8) with increase in total polymer concentration, gelation time, and drug load, the drug release increased in both the media with increase in the concentration of AlCl3. However, all the formulations showed considerably low release in acidic medium and the release followed non-Fickian transport mechanism Copyright © 2010 SciRes.

[10] N. P. Desai, A. Sojomihardjo, Z. Yao, N. Ron and P. Soon-Shiong, “Interpenetrating Polymer Networks of Alginate and Polyethylene Glycol for Encapsulation of Is-

PP

Development and Evaluation of a New Interpenetrating Network bead of Sodium Carboxymethyl Xanthan and Sodium Alginate for Ibuprofen Release lets of Langerhans,” Journal of Microencapsulation, Vol. 17, No. 6, 2000, pp. 677-690. [11] S. G. Kumber and T. M. Aminabhavi, “Preparation and Characterisation of Interpenetrating Network Beads of Poly (Vinyl Alcohol)-Grafted-Poly (Acrylamide) with Sodium Alginate and their Controlled Release Characteristics of Cypermethrin Pesticides,” Journal of Applied Polymer Science, Vol. 84, No. 3, 2002, pp. 552-560. [12] Y. H. Liu, H.-F. Lian, C.-K. Chung, M.-C. Chen and H. -W. Sung, “Physically Cross-Linked Alginate/N,O-Carboxymethyl Chitosan Hydrogels with Calcium for Oral Delivery of Protein Drugs,” Biomaterials, Vol. 26, No. 14, 2005, pp. 2105-2213. [13] B. Sa and M. Setty, “Novel Gel Microbeads Based on Natural Polysaccharides,” Indian Patent, No. 224992, 31 October 2008. [14] S. Maiti, S. Roy, B. Mondal, S. Sarkar and B. Sa, “Carboxymethyl Xanthan Microparticles as a Carrier for Protein Delivery,” Journal of Microencapsulation, Vol. 24, No. 8, 2007, pp. 743-756. [15] S. Ray, S. Maiti and B. Sa, “Preliminary Investigation on the Development of Diltiazem Resin Complex Loaded Carboxymethyl Xanthan Beads,” AAPS PharmSciTech, Vol. 9, No. 1, 2008, pp. 295-301. [16] C. M. Setty, S. S. Sahoo and B. Sa, “Alginate-Coated Alginate-Polyethyleneimine Beads for Prolonged Release of Furosemide in Simulated Intestinal Fluid,” Drug Development and Industrial Pharmacy, Vol. 31, No. 4-5, 2005, pp. 435-446. [17] P. Piyakulawat, N. Praphairaksit, N. Chantarasiri and N. Muangsin, “Preparation and Evaluation of Chitosan/CarRageenan Beads for Controlled Release of Sodium Diclofenac,” AAPS PharmSciTech, Vol. 8, No. 4, 2007, pp. 1-10. [18] A. P. Rokhade, S. A. Agnihotri, S. A. Patil, N. N. Mal-


17

likarjuna, P. V. Kulkarni and T. M. Aminabhavi, “SemiInterpenetrating Polymer Network Microspheres of Gelatin and Sodium Carboxymethyl Cellulose for Controlled Release of Ketorolac Tromethamine,” Carbohydrate Polymers, Vol. 65, No. 3, 2006, pp. 243-252. [19] S. A. Agnihotri and T. M. Aminabhavi, “Development of Novel Interpenetrating Network Gellan Gum-Poly (Vinyl Alcohol) Hydrogel Microspheres for the Controlled Release of Carvedilol,” Drug Development and Industrial Pharmacy, Vol. 31, No. 6, 2005, pp. 491-503. [20] K. S. Soppimath, A. R. Kulkarni and T. M. Aminabhavi, “Chemically Modified Polyacrylamide-G-Guar Gum Based Crosslinked Anionic Microgels as PH Sensitive Drug Delivery Systems: Preparation and Characterization,” Journal of Controlled Release, Vol. 75, No. 3, 2001, pp. 331-345. [21] R. V. Kulkarni and B. Sa, “Novel PH-Sensitive Interpenetrating Network Hydrogel Beads of Carboxymethyl Cellulose-(Polyacryl Amide-Grafted-Alginate) for Controlled Release of Ibuprofen: Preparation and Characterization,” Current Drug Delivery, Vol. 5, No. 4, 2008, pp. 256-264. [22] S. Benita, A. Barkai and Y. U. Pathak, “Effect of Drug Loading Extent on the in Vitro Release Kinetic Behaviour of Nifedipine from Polyacrylate Microspheres,” Journal of Controlled Release, Vol. 12, No. 3, 1990, pp. 213-222. [23] K. S. Soppimath, A. R. Kulkarni and T. M. Aminabhavi, “Controlled Release of Antihypertensive Drug from the Interpenetrating Network Poly (Vinyl Alcohol)-Guar Gum Hydrogel Microspheres,” Journal of Biomaterials Science Polymer Edition, Vol. 11, No. 1, 2000, pp. 27-43. [24] P. L. Ritger and N. A. Peppas, “A Simple Equation for Description of Solute Release. II Fickian and Anomalous Release from Swellable Devices,” Journal of Controlled Release, Vol. 5, No. 1, 1987, pp. 37-42.

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Preparation and Evaluation of Rapidly Disintegrating Fast Release Tablet of Diazepam-Hydroxypropyl-β-Cyclodextrin Inclusion Complex ——Rapidly Disintegrating Fast Release Tablet Tapan Kumar Giri, Biswanath Sa* Centre for Advanced Research in Pharmaceutical Sciences, Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India. Email: [email protected] Received June 1st, 2010; accepted July 8th, 2010.

ABSTRACT This study was undertaken to develop tablets of diazepam-hydroxypropyl-β-cyclodextrin inclusion complex that disintegrate within 3 minutes and release 85% of drug within 30 minutes to provide rapid action of the drug through oro-mucosal route. Formation of inclusion complex was verified using X-ray diffraction and differential scanning calorimetric studies. Enhanced of aqueous solubility, as evident from phase solubility study, and dissolution of the drug were related with the formation of inclusion complex. Among the various formulations, tablet containing inclusion complex of drug/hydroxypropyl-β-cyclodextrin in a molar ratio of 1:2, and a combination of microcrystalline cellulose/lactose in a ratio of 4:1 disintegrated in 13 seconds and released 85% drug within 9 minutes. Addition of 10% w/w polyvinyl pyrrolidone in the tablet formulation further enhanced the drug release. Accelerated stability study indicated that mean dissolution time of the drug from the tablet did not change significantly within 6 months. Keywords: X-Ray Diffraction, Phase Solubity, Dissolution Efficiency, Mean Dissolution Time, Stability

1. Introduction Though conventional oral and parenteral routes are used widely to achieve systemic action of drugs, various mucosae are being explored as possible alternative routes for drug delivery. Since the invention of nitroglycerin sublingual tablets, the oral mucosal route is drawing attention of both academia and industries as a substitute drug delivery approach. Several constraints like difficulty in swallowing experienced by many paediatrics and geriatrics [1], and in chewing by edentulous [2]; nausea and vomiting experienced with certain drugs when released in stomach [3]; degradation and metabolism of susceptible drugs in gastrointestinal tract [4]; tissue necrosis and irritation from repeated administration of parenterals [5], high expenses due to sterile manufacturing [6] are avoided through oromucosal delivery of drugs. In certain diseases like epilepsy, rapid onset of drug action is necCopyright © 2010 SciRes.

essary to suppress convulsion and terminate seizures. Thus early termination of seizures by initiating therapy as soon as possible, preferably at home, has been emphasized as a key to minimize morbidity of these seizures [7-9]. Benzodiazepines are used for the acute management of severe seizures and have a rapid onset of action once delivered into the central nervous system and are safe. [10] Diazepam, a benzodiazepine, is included in the “WHO Essential Drug list” for the treatment of convulsion and epileptic seizure [11-14]. Although intravenous therapy is the most rapid way to suppress epileptic convulsion, it may produce toxic manifestation due to excessive drug concentration [15,16], requires great care and caution to avoid thrombophlebitis and irritation [17] and may not be feasible where adequate medical facilities are not available in the immediate vicinity. While absorption of diazepam from intramuscular route is poor and erratic PP

Preparation and Evaluation of Rapidly Disintegrating Fast Release Tablet of Diazepam-Hydroxypropyl-β-Cyclodextrin Inclusion Complex

[18], the time to reach peak plasma concentration following oral administration is 1-2 hours [19] and is accompanied with acid hydrolysis and extensive liver metabolism [10]. If diazepam is formulated in a rapidly disintegrating fast dissolving tablet dosage form, the high vascularity and rich blood supply of oral mucosa [20] may provide rapid absorption and faster onset of action [21] and could enable a patient for self medication even without the aid of water in a situation where onset of convulsion is apprehended. Two principle criteria appear to be important for developing rapidly disintegrating fast dissolving tablets: 1) disintegration time preferably < 3 minutes [22] and 2) rapid drug dissolution: time required for 85% dissolution (t85%) less than 30 minutes [23]. Valuable research reports for formulation of rapidly disintegrating tablets are available [24]; also, various technologies for improving dissolution property of poorly water soluble drugs have been documented to enhance bioavailability following oral absorption [25]. Among the various strategies, formulation of solid dispersions with hydrophilic carriers especially polyethylene glycols (PEGS) have been successfully used for enhancing dissolution of poorly water soluble drugs [26-29]. However, development of dosage forms like tablet and capsule using the solid dispersion encounters problems in pulverization/sifting of the solid dispersion which are usually soft and tacky and exhibits poor flow properties. In recent years, inclusion complexes of poorly water soluble drugs with cyclodextrins especially hydroxypropyl-β-cyclodextrins (HPβCD) have become popular to enhance the solubility and bioavailability of drugs. Loftsson [30] reported that the solubility of diazepam and various poorly water soluble drugs improved in solution with the natural cyclodextrins and their derivatives. Subsequent studies also shows that the incorporation of hydrophilic polymer such as sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose and polyvinyl pyrrolidone increase the solubilizing effect of the cyclodextrins and reduce the amount of cyclodextrin required. The objective of this study was to develop a rapidly disintegrating fast dissolving tablet of diazepam that can disintegrate in less than 3 minutes and release/dissolve 85% of the drug within 30 minutes in the oral cavity. The initial part of this work involved preparation of diazepam-HPβCD inclusion complex by kneading method and characterization of the complex using X-ray diffraction(XRD) and differential scanning calorimetric (DSC) studies. The subsequent phase involved the preparation of tablets of diazepam-HPβCD complex by direct compression method to meet the specified time limits of disintegration and drug dissolution. Finally the optimized tablets were subjected to accelerated stability study. Copyright © 2010 SciRes.

19

2. Experimental 2.1 Materials Diazepam (East India Pharmaceutical Works Ltd., Kolkata, India), Saccharin-Na, Crosscarmellose sodium (AcDisol), Microcrystalline Cellulose (Avicel, PH-102) [Dey’s Medical Stores (Mfg.) Ltd., Kolkata, India], Hydroxypropyl beta cyclodextrin (HPβCD) [Dr Reddy’s Lab, Hyderabad, India] were obtained as gift samples. Mannitol, lactose monohydrate and sorbitol (Merc, India), PVPK30 (Qualigens, Mumbai, India), magnesium stearate and all other ingredients were obtained commercially and used as received.

2.2 Preparation of Solid Complexes Solid inclusion complexes of diazepam-HPβCD were prepared in 1:1 and 1:2 ratio by kneading method with and without the addition of polyvinyl pyrrolidone (PVP). PVP was added at a concentration of 10% (w/w) of the solid complex. Mixture was triturated for one hour in a mortar with a small volume of water to obtain a homogeneous paste. During the process, the water content of the paste was empirically adjusted to maintain the consistency of the paste. The paste was dried at 45°C for 48 hours, pulverized and passed through sieve # 100.

2.3 Thermal Analysis Differential thermal analysis of diazepam, HPβCD, diazepam/HPβCD physical mixture, and inclusion complex were carried out using Perkin-Elmer instrument (Pyris Diamond TG/DTA, Singapore) equipped with a liquid nitrogen subambient accessory. About 4 mg samples were kept in aluminum pan, hermetically sealed and scanned at a rate of 5°/min between 30-210°C under nitrogen atmosphere.

2.4 X-Ray Diffraction Study X-ray powder diffraction patterns of diazepam, HPβCD, diazepam/HPβCD physical mixture, and inclusion complex were conducted with a X-ray powder diffractometer (Rigaku-MiniFlax, Tokyo, Japan.) using a copper Kα target with a nickel filter at 30 kV voltage, 15 mA current and at scanning speed of 1°/min over a 2θ range of 5°-60°.

2.5 Phase Solubility Study Phase solubility studies were carried out by adding excess amounts of drug to 10 ml of USP phosphate buffer solution (pH 5.8) containing various concentrations of HPβCD (3-15 mM) in stoppered conical flasks. The flasks were shaken at 50 revolutions per minute in a shaking incubator (Model KMC 8480 SL, Vision Scientific Company, Ltd., Seoul, South Korea) at 37 ± 0.5°C PP

20


until equilibrium (about 90hours) was reached. The resulting mixtures were filtered and aliquots, following suitable dilutions, were analyzed using a spectrophotometer (Genesyis, 10 UV, Thermo Electron Corporation, Wisconsin, USA.) at 230 nm against blanks prepared in the same concentration of HPβCD in USP phosphate buffer solution (pH 5.8). Phase solubility studies were conducted with and without the addition of PVP. The PVP was added at a concentration of 0.5% w/v to the solution containing HPβCD. The solubility experiments were conducted in triplicate.

2.6 Preparation of Placebo Tablet by Direct Compression Method Microcrystalline cellulose (MCC), lactose or mannitol or sorbitol and crosscarmellose sodium were mixed without drug for 10 minutes. The resulting mixture was lubricated with magnesium stearate and mixed for 5 minutes. The final powder mixture was then compressed into tablet using concave punches (approx 9.5 mm diameter) in a 10 station Minipress tablet machine (RIMEK, Karnavati Engineering Ltd, Gujarat, India).

2.7 Preparation of Tablet with Drug by Direct Compression Method MCC, lactose or mannitol or sorbitol and crosscarmellose sodium were mixed with drug (as such or solid inclusion complexes) for 10 minutes. The resulting mixture was lubricated with magnesium stearate and mixed for 5 minutes. The final powder mixture was then compressed into tablet using concave punches (approx 9.5 mm diameter) in a 10 station Minipress tablet machine (RIMEK, Karnavati Engineering Ltd, Gujarat, India).

adding 10ml of fresh medium. The amount of drug released was determined from the calibration curve. The reliability of the above analytical method was judged by conducting recovery analysis in the presence or absence of the excipients. Low, middle, and high concentrations of drug solution were spiked and recovery was found to vary from 99.05 to 100.83%.

2.10 Stability Study The stability of the drug in the optimized tablet was assessed by keeping the tablets in a sealed glass bottle and subsequently placing the bottle in a Stability Test Chamber (Humidity Cabinet, Testing instruments manufacturing company, Kolkata) at 40°C and 75% RH for different periods of time. The tablets were analyzed immediately (0 month), and after 1, 3 and 6 months for appearance, hardness, friability, drug content, disintegration time and dissolution profiles of the drug.

3. Results and Discussion Diazepam-HPβCD inclusion complexes were prepared by kneading method. The resulting complex was analyzed by XRD, DSC, and phase solubility studies. Thermal behavior of diazepam, HPβCD, diazepam/ HPβCD physical mixture, and diazepam/HPβCD inclusion complex are shown in Figure 1. The DSC thermogram of diazepam shows one sharp endothermic peak at 133°C which corresponds to the melting point of the drug. The DSC trace of HPβCD did not show any endothermic peak. The thermal event of the physical mixture demonstrated the appearance of a sharp peak having diminished intensity at 133°C which was assigned to the melting point of the drug. The DSC thermogram of the inclusion

2.8 Measurement of Disintegration Time Disintegration times were measured using a modified disintegration test method. [31] To determine disintegration time, 10ml of USP phosphate buffer solution (pH 5.8) was taken in a petridish (10 cm diameter) and a tablet was carefully placed in the centre and agitated mildly. Time for the tablet to completely disintegrate into fine particles was noted using a stop watch.

2.9 Measurement of in Vitro Drug Release Dissolution profiles of the diazepam tablets were determined using USP II dissolution rate test apparatus (model TDP-06P, Electrolab, Mumbai, India).The dissolution medium was 500ml of USP phosphate buffer solution (pH 5.8) maintained at 37 ± 0.5°C and stirring speed was 50 rpm. At appropriate time intervals, 10ml samples were withdrawn, suitably diluted, and analyzed spectrophotometrically for diazepam content at 230 nm. The initial volume of dissolution medium was maintained by Copyright © 2010 SciRes.

Figure 1. Differential scanning calorimetric thermograms of a) diazepam, b) HPβCD, c) physical mixture, d) inclusion complex PP


complex revealed that although the intensity of the melting endotherm of the drug at 133°C decreased to a great extent, it was not abolished completely. The decrease in endothermic peak of the drug was due to entrapment of the crystalline drug within the cavity of HPβCD. This indicates the formation of an inclusion complex of diazepam with HPβCD. During the preparation of solid dispersion using melting method [32] solvent/co-evaporation method [33], the crystalline drugs are completely converted into amorphous form and consequently, no thermal event is evident in the DSC scan. On the other hand the endothermic melting peak of a crystalline drug may not be abolished, although may be diminished to a great extent, when a drug forms solid complex with HPβCD by kneading method [32]. The XRD patterns of drug, HPβCD, physical mixture, and inclusion complex are shown in Figure 2. The diffractogram of the drug exhibited a series of intense peaks due to its crystalline structure. The XRD pattern of HPβCD was an amorphous halo. The diffractogram of the physical mixture appeared to represent the superimposition of each components spectrum although the drug crystallinity reduced considerably. A no. of sharp peaks, although of reduced intensity, were still present in the diffractogram of the solid complex .Close examination of the diffractogram revealed that many peaks of the drug disappeared(2θ of 26.12°, 26.69°, 27.56°, 28.28°) and many new peaks emerged (2θ of 25.67° and 26.99°). Similar to the DSC results, the XRD analysis does not show diffraction pattern of drugs when the solid dispersions are prepared by melting method [32] or solvent/ co-evaporation method [33]. However, the inclusion complexes prepared by kneading process still show peaks on the diffractogram [32]. These results indicate the formation of inclusion complex. Phase solubility diagram (Figure 3) demonstrated a linear increase in the aqueous solubility of diazepam with the concentration of HPβCD. The improved solubility of various poorly soluble drugs through complexation with HPβCD is well documented in scientific journals [34,35]. The complexation of diazepam with HPβCD was type AL [36] and as the slope of the straight line of concentration of diazepam verses concentration of HPβCD plots was < 1, the complexation took place in 1:1 molar ratio. The apparent stability constant (KC) was calculated from the slope of the linear plot of the phase solubility diagram following the equation KC = Slope/So (1-Slope), where So is the solubility of the drug in the absence of HPβCD. The value of KC (Table 1) indicated that the complex was quite stable. Tablets formulated using MCC and low-substituted hydroxypropyl cellulose has been reported to disintegrate and dissolve rapidly in the saliva of humans [37]. However, such tablets provide a gritty mouth feel due to the Copyright © 2010 SciRes.

21

Figure 2. X-ray diffraction pattern of a) diazepam, b) HPβCD, c) physical mixture, d) inclusion complex

Figure 3. Phase solubility diagrams of diazepam-hydroxypropyl β-cyclodextrin complex in the presence (■) and absence (◆) of PVP Table 1. Effect of PVP on stability constant (KC) and solubilizing efficiency of diazepam-HPβCD complexes† KC (M-1)

Solubilizing Efficiency.‫٭‬

D-HPβCD

499.087

3.880

D-HPβCD-PVP

515.094

4.271

Sample

†HPβCD indicates hydroxypropyl β-cyclodextrin; PVP, polyvinyl pyrrolidone; and D, diazepam. ‫٭‬Ratio of drug solubility in USP buffer solution (pH 5.8) (15 mM) of cyclodextrin (with or without PVP) to drug solubility in buffer.

presence of insoluble crystalline cellulose [38]. To formulate tablets that can disintegrate rapidly and reduce gritty mouth feel characteristics, several water soluble diluents like sorbitol, manitol and lactose were selected, and 9 placebo tablets containing of MCC and PP


22

either sorbitol, manitol or lactose in the ratios of 1:1, 2:1, and 4:1 were prepared by direct compression method. The compositions of the placebo tablets are shown in Table 2. The blended powder of each formulation exhibited good flowability as evident from the measurement of angle of repose (measured by conventional method) that varied from about 31.3° to 34° (Table 3). Angle of repose below 40° is an indication of good flowability of powder/granules [39]. The compression force during tabletting was adjusted in such a way that the hardness (measured using Monsanto type hardness tester) of the tablets, each weighing 300 mg, was 2 Kg-F. The friability of the tablets (determined using Friabilator, Veego instrument, Mumbai, India) was found between 0.03 to 0.29% that was below 1% indicating sufficient mechanical integrity and strength of the placebo tablets. The disintegration time of tablets (P1) which were prepared using MCC and sorbitol in 1:1 ratio was 42.85 seconds. The tablets P2 and P3 which consisted of MCC and sorbitol in a ratio of 2:1 and 4:1 respectively disintegrated in 29.03 and 27.34 seconds. The result indicates that increase in MCC/sorbitol ratio decreased the disin-

tegration time of the tablets and this decrease was found significant at 95% confidence limit (p < 0.05). MCC is considered as one of the most versatile excipients in tablet manufacturing. In addition to its performance as diluents and dry binder, it is also regarded as an excellent disintegrant for tablets prepared by direct compression method. This property is related to its wicking action due to which water penetrates into the tablet and the developed hydrostatic pressure causes break down of the tablet. The rate and extent of water penetration is related to the porosity (determined using laboratory pycnometer) it provides in the tablets. The greater the amount of MCC, the greater will be the porosity in the tablet matrix. Table 3 shows that as the ratio of MCC/sorbitol increased, the porosity of the tablets increased significantly (p < 0.05) that provided faster penetration of water. Determination of wetting time (determined by the method described by Bi et al. [40]) demonstrated that the wetting time of the tablets decreased significantly (p < 0.05) with increase in the amount of MCC (Table 3) indicating faster penetration of water into the tablets. Similar observations were noted for the tablets prepared using MCC/manitol (tablets P4, P5, P6) and MCC/lactose (tablets P7, P8, P9).

Table 2. Composition of placebo tablets (without drug) prepared by direct compression method Ingredients(mg/tablet)

P1

P2

P3

P4

P5

P6

P7

P8

P9

144.25

193

230.8

144.25

193

230.8

144.25

193

230.8

Lactose

-

-

-

-

-

-

144.25

96.5

57.7

Mannitol

-

-

-

144.25

96.5

57.7

-

-

-

Sorbitol

144.25

96.5

57.7

-

-

-

-

-

-

7.5

7.5

7.5

7.5

7.5

7.5

7.5

7.5

7.5

Mg-St.

1

1

1

1

1

1

1

1

1

Saccharin-Na

3

3

3

3

3

3

3

3

3

300

300

300

300

300

300

300

300

300

MCC

Crosscarmellose-Na

Total

Table 3. Physical characteristics of placebo tablets (without drug) prepared by direct compression method P1

P2

P3

P4

P5

P6

P7

P8

P9

Angle of Repose

34.03 (0.33)

33.56 (0.39)

31.47 (1.4)

33.99 (0.29)

32.66 (0.69)

32.49 (1.13)

35.38 (0.8)

32.37 (1.02)

31.29 (0.96)

Porosity

9.99 (0.13)

15.21 (0.21)

22.39 (0.21)

17.26 (0.14)

21.28 (0.16)

26.17 (0.28)

19.31 (0.19)

24.34 (0.42)

31.87 (0.7)

Disintegration time, seconds

42.85 (1.89)

29.03 (0.67)

27.34 (0.62)

43.29 (0.77)

18.76 (0.25)

15.3 (0.9)

26.49 (1.36)

17.25 (0.17)

13.23 (0.46)

Wetting time, seconds

119.92 (1.63)

92.69 (2.1)

84.78 (1.85)

82.56 (1.86)

52.69 (2.81)

42.53 (1.7)

80.67 (1.78)

50.3 (1.07)

35.43 (1.62)

Figures in parentheses indicate ± SD; n = 6 for disintegration time and ± SD, n = 3 for Angle of Repose, porosity and wetting time


PP


It was also noted that the type of soluble diluents influenced the porosity, wetability and disintegration time of the tablets considerably. The tablets prepared with MCC/lactose combination exhibited higher % of porosity, and shorter wetting and disintegration times followed by the tablets prepared with MCC/manitol and MCC/sorbitol. Change of soluble excipient from sorbitol to mannitol to lactose significantly increased (p < 0.05) the porosity and decreased the wetting time and disintegration time of the tablets. These observations were found in each of the ratios of MCC/soluble diluents. The solubility data [41] indicate that aqueous solubility of the above used excipients increased in the following order: lactose > mannitol > sorbitol. Higher solubility of lactose was responsible for its rapid solution. As lactose dissolves quickly it creates pores rapidly encouraging penetration of water into the tablets and this led to quick disintegration of the tablets. Decrease in solubility of the excipients delayed both the wetting and disintegration of tablets. The subsequent study involved the preparation of drug-loaded tablets that will disintegrate rapidly and provide rapid dissolution of the drug contained therein. Considering the results of the above study, the formula of tablet P9 which consisted of MCC/lactose in a ratio of 4:1 and disintegrated in the shortest period of time (13.2 seconds) was selected for incorporation of diazepam or diazepam-HPβCD inclusion complex. The composition of the tablets is represented in Table 4 and the physical characteristics of the blended powder and the tablets are shown in Table 5. Statistical analysis in the form of Students t-test revealed that the angles of repose of the blended powder containing either diazepam or inclusion complex of diazepam-HPβCD in 1:1 and 1:2 molar ratios did not Table 4. Composition of tablets prepared by direct compression method using either diazepam or inclusion complex Ingredients (mg/tablet)

Q1

Q2

Q3

Q4

Q5

MCC

226.8

207.136

204.768

187.472

183.1392

Lactose

56.7

51.784

51.192

46.868

45.7848

HPβCD

-

24.58

24.58

49.16

49.16

PVP

-

-

2.96

-

5.416

Diazepam

5

5

5

5

5

7.5

7.5

7.5

7.5

7.5

Mg-St.

1

1

1

1

1

Saccharin-Na

3

3

3

3

3

300

300

300

300

300

Crosscarmellose-Na

Total


23

Table 5. Physical characteristics of tablets containing either diazepam or its inclusion complex Q1

Q2

Q3

Q4

Q5

Angle of Repose

31.45 (0.84)

31.64 (1.26)

34.15 (0.8)

32.59 (0.88)

35.44 (0.77)

Porosity

31.13 (0.42)

33.47 (0.11)

30.14 (0.11)

31.14 (0.08)

27.82 (0.04)

Disintegration time, seconds

13.12 (1.32)

12.84 (0.97)

14.09 (0.42)

13.3 (1.06)

19.06 (0.92)

Wetting time, seconds

36.22 (1.12)

34.24 (1.06)

40.42 (0.73)

38.57 (1.05)

56.52 (2.13)

t85%,minutes

125.43 (1.25)

10.24 (0.17)

8.86 (0.13)

8.98 (0.007)

7.48 (0.30)

DE10minutes

21.10 (0.59)

54.68 (1.23)

59.44 (0.35)

57.66 (0.92)

62.56 (0.16)

Figures in parentheses indicate ± SD; n = 6 for disintegration time and ± SD, n = 3 for Angle of Repose, porosity, wetting time, t85% and DE10minutes.

change significantly (p < 0.05) from that of the blended powder used to prepare placebo tablet P9. Similarly, no significant changes were noted (p < 0.05) in porosity, disintegration time and wetting time of the tablets due to incorporation of either the drug or its inclusion complexes. The release profiles of the drug from various tablets are shown in Figure 4. While the time required for 85% (t85%) of diazepam to be released from tablet Q1 was 125.4 minutes, the same from the tablet (Q2) containing inclusion complex of diazepam in 1:1 molar ratio drastically reduced to 10.24 minutes. For the tablets Q4 containing diazepam/ HPβCD in a molar ratio of 1:2, the t85% further reduced to 8.98 minutes. In addition to one point comparison using t85%, the entire drug release profiles were compared using dissolution efficiency (DE) to ascertain the differences in

Figure 4. Dissolution profiles of diazepam from various tablet formulations PP


24

the release of the drug from various tablets. The DE is defined as the area under the dissolution curve upto a certain time, t, expressed as a percentage of the area of the rectangle described by 100% dissolution in the same time [42]. t    y.dt    100% D.E   0  y100 .t     

where y is the drug percent dissolved in time t. DE can have a range of values depending on the time interval chosen. However, while comparing a set of data, a constant time interval should be selected. In the present study, DE10minutes (dissolution efficiency upto 10 minutes) were calculated from the dissolution profile of each tablet and used for comparison. It was found that in comparison to DE10minutes obtained from tablet containing diazepam, a 2.59 and 2.73 fold increase in DE10minutes were obtained from the tablets prepared using diazepam/ HPβCD inclusion complex in molar ratios of 1:1 and 1:2 respectively. These increase in DE10minutes were found to be statistically significant (p < 0.05). Faster release of diazepam from the tablets prepared using its inclusion complexes was related to the enhanced solubility of the drug because of the formation of complex with HPβCD. In another two batches of tablets namely Q3 and Q5 containing respectively 1:1 and 1:2 diazepam-HPβCD complex, PVP, a hydrophilic polymer was added at a concentration of 10% w/w of the solid complexes (Table 4) to investigate its effect on the various physical characteristics which are shown in Table 5. Incorporation of PVP in the formula increased the angle of repose of the blended powders and decreased the porosity of the tablets significantly (P < 0.05). Moreover, PVP increased both the disintegration time and wetting time of the tablets significantly (p < 0.05). The larger the amount of

PVP, the higher the disintegration time and the wetting time. PVP which acts as a binder densified the powder resulting in decreased flowability of the powder blend and reduced the porosity of the tablets. Reduced porosity, in turn, protracted the wetting and the disintegration of the tablets. Addition of PVP in the tablet formulations, however, further reduced t85% and increased DE indicating that drug dissolution took place faster than that from the tablets without containing PVP. Addition of PVP in the complex also increased the solubility linearly having a slope < 1 as evident from phase solubility diagram (Figure 3). At each point of determination, the solubility of the drug was higher than that produced by the complex in absence of PVP. The stability constant was also found to be higher (Table 1). The potentiated solubility of diazepam in complex form in the presence of PVP was evaluated by determining the solubilization efficiency, which is defined as ratio of solubility of drug in USP phosphate buffer solution (pH 5.8) of 15 mM HPβCD (with or without PVP) and the solubility of the drug in buffer solution. Table 1 show that while HPβCD without PVP produced 3.88 fold increases, the same in presence of PVP produced 4.27 fold increases in the solubility of diazepam. It, therefore, appears that presence of hydrophilic polymer like PVP enhances the solubilizing efficiency of HPβCD. Accelerated stability test on the tablets (Q5) was conducted at 40°C and 75% RH in accelerated stability test chamber (Humidity Cabinet, Testing instruments manufacturing company, Kolkata) and the physical characteristics of the tablets observed after different periods of time are shown in Table 6. The physical characteristics such as appearance, friability and drug content did not change and were confined within the specified limits upto 6 months of time. The disintegration time and t85% of the tablets upto one month storage did not change significantly when compared to those of the fresh tablets. However, marginal

Table 6. Various characteristics of Diazepam tablets stored at 40°C and 70% RH Storage Appearance

Friability (%)

Content (mg)

DT (seconds)

t85% (minutes)

MDT (minutes)

White,round-flat tablet

< 1%

95-105%