PHARMBIT, XXII (2), July - Dec, 2010

Page 1



Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Chief Editor : Dr. R. N. Gupta Associate Editor : Mr. Manik Ghosh

Editorial Advisory Board Prof. B. K. Gupta Chairman, Gluconate Ltd., Kolkata Dr. Somlak Kongmuang Silpakorn University, Thailand Dr. Roop K. Khar Jamia Hamdard, New Delhi Dr. Sunil K. Gupta, Impex Pharmaceuticals Hayward, USA

Contents l Drug Use in the Elderly : Attention Needed

85

Santanu K Tripathi

l Formulation and Evaluation of Biodegradable

Dental Implants Containing Antibacterial Agents for Periodontitis

l Formulation and Optimization of Mucoadhesive

Nano Drug Delivery System of Acyclovir

94

U.V. Bhosale, V. Kusum Devi

l Quantification of Vinorelbine in Bulk and Injection Dosage

Form by Spectrophotometric Method

Pawan K. Saini, Raman M. Singh, C. L. Jain, Satish C. Mathur, Gyanendra N. Singh

Dr. Malay Chatterjee, Jadavpur University, Kolkata

l Reporting of Adverse Drug Reactions by Consumers :

Dr. P. Ramkumar School of Pharmacy, Malaysia

Hanumantha Rao Potharaju

Dr. U. K. Mazumdar Jadavpur University, Kolkata

Rationale and Potential

l Niosomes : A Potential Drug Delivery System

D. Nagasamy Venkatesh, Anindita De, Vinay Valecha

Dr. Susmita Chanda Roche, San Fransisco, USA

l Comparative Pilot Bioavailability Study of Carbamazepine

Dr. T. R. Krishanan FDA, Canada

A. Ghosh, Neha Sharma, N. R. Biswas

Dr. Suresh K. Saravdekar Health Dept., Maharashtra Dr. Sampad Bhattacharya Sun Pharma Ltd., Baroda Dr. Maya Prakash Singh Wyeth Research, Newyork Mr. Anjani Kumar Cipla Ltd., Mumbai Dr. Shivaji Singh Navtech LLC, Atlanta, USA Dr. P. R. Vavia, ICT, Matunga, Mumbai Dr. G. N. Singh, Director, CIPL, Ghaziabad Dr. P. H. Rao ASCI, Hyderabad Prof. B. G. Shivananda, Principal, Al-ameen College of Pharmacy, Bangalore

88

Krishnananda Kamath, Shripathy D., A. R. Shabharaya

in Nepalese Healthy Volunteers

l Degradation Kinetic and Preformulation

Stability Studies of Curcumin

105

110 115

132

139

Vivek R. Yadava, Sarasija Suresha, Seema Yadavb, Veda Murthy Joshia

l Anthelmintic Activity of the Leaves of Barleria cuspidata 144

Manoj Kumar Parida, Biswajeet Panda, Samikshya Negi, B. Behera, Anasuya Sahoo

l Formulation and Evaluation of Mouth Dissolving

Tablet of Loratidine

147

P. J. Narain, Nimisha, Sanjay Srivastava l Production of Jackwine – A Wine from Ripe Jackfruit

154

l Instructions to Authors

158

Amit K. Tiwari, Ambarish S. Vidyarthi

Editorial Board Members Mr. M. P. Chopra

Mr. Abhimanyu Dev

Miss Anuva Jas

Mr. Alok Kumar

Miss Debarati Sengupta

Miss Veena Vijan


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

EXECUTIVE COMMITTEE OF PHARMACEUTICAL SOCIETY Dept. of Pharmaceutical Sciences, B.I.T., Mesra, Ranchi Patron President Prof. In-charge Co-Prof. In-charge Treasurer Joint Treasurer Chief Editor-Pharmbit Associate Editor Cultural Incharge Co-Cultural Incharge Vice President Secretary Jt. Secretary

: : : : : : : : : : : : :

Dr. Ajay Chakrabarty, Vice Chancellor Dr. B. N. Sinha, HOD, Pharmaceutical Sciences Dr. S. Samanta Dr. (Mrs.) S. M. Verma Dr. S. Jha Dr. (Mrs.) A. Pandey Dr. R. N. Gupta Sri Manik Ghosh Prof. (Mrs.) S.G. Panpalia Mrs. Trishna Bal Varsha Toshniwal Sougata Mishra, Ankita Sharma Saurav Ganguli , Mayuri

ADVISORY COMMITTEE Dr. D. Sasmal Prof. C. M. Prasad Dr. P. R. P. Verma Prof. (Mrs.) S. G. Panpalia Dr. (Ms.) S. Ganguly Dr. (Mrs.) P. M. Mazumdar Dr. J. Venkatesh Shri A. K. Patnaik Sri S. P. Pattanayak Sri B. Sarkar Mrs. Nibha Mishra Sri Dinesh M. Biyani Dr. Animesh Ghosh

Dr. (Mrs.) M. Mukherjee Dr. Jayaram Kumar Sri A Basu Sri Abhimanyu Dev Dr. Sandeep K. Singh Dr. (Mrs.) Neelima Sharma

CLASS REPRESENTATIVES B. S. Nagaraj Niharika Singh Hare Ram Jayshree Dongre Swati Bansal

Anita Pansari Navin Keshwani Sandhya Kumari Reena Manish Kr. Mishra

Rituparna Hore G. Durga Prasad Monika Srivastava Vikash Kumar Yamini Gaur

REVIEWERS PANEL Dr. B. Mishra, Varanasi Dr. Biswajeet Mukherjee, Kolkata Dr. Javed Ali, Delhi Dr. S. K. Kaushal, Kolkata Dr. P. Suresh, Visakapatnam Dr. T. K. Ravi, Coimbatore Dr. S. C. Mondal, Kolkata Dr. P. N. Murthy, Behrampur Dr. S. K. Sharma, Hisar Dr. D. N. Misra, Hisar

Dr. G. Vidyasagar, Bhuj Dr. (Mrs.) Sarsija Suresh, Bangalore Dr. T. V. Narayana, Bangalore Dr. M. P. Rana, Kolkata Dr. Suresh K. Saravdekar, Mumbai Dr. L. K. Ghosh, Kolkata Dr. B. B. Barik, Bhubneshwar Dr. P. K. Manna, Annamalainagar Dr. G. Parthasarthi, Mysore Dr. S. Madhav, Dehradun


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

From the Editor’s Desk It is a matter of great pleasure for us to bring out the XXII, volume of PHARMBIT, biannual scientific journal in time. The journal has been accepted for indexing in Biotechnology & Bio engineering, Applied Microbiology, Neuroscience and Toxicology abstract under “Natural Science database” (www.csa.com) from Jan - June 2008 issue besides indexing in “Chemical Abstract” from Jan - June 2007 issue. In this issue 11 papers are being published covering papers on development of formulation, standardization, pharmacy practice, and pharmacovigilance .More potent & newer drugs in novel drug delivery system are being made available by research Pharmacists. The Pharmacists with qualifications- Ph.D., Pharm.D, M.Pharm, B.Pharm., D.Pharm are available for Pharmacy services. A Pharmacist has the knowledge of drug molecule, research, manufacture, testing, drug action (pharmacology), storage, use, dispensing, distribution, regulatory affairs etc. Now the time has come to provide Pharmacy services (Practice – Patient Counseling) in society by all category of Pharmacists irrespective of their engagement as Research , Manufacturing, Quality Control, teaching ,Drugs Control, Hospital and community Pharmacist. Their educational background is complete in all respect as Pharmacy Professional. Certainly they have to keep themselves abreast with current development and to upgrade their professional skill. So they should read on continuous basis the pharmacy journals i.e. Pharmbit, Pharmatimes, Pharmabiz, Pharma Review, Indian Pharmacist, Indian Journal of Pharmaceutical Sciences, IJPER , IDMA Bulletin ,Indian Drugs, JIHPA etc. As an editor of this journal, I am thankful to all Authors, Reviewers, Editorial Board Members, Advertisers, well wishers, Faculty members and Head, department of Pharmaceutical Sciences and Vice-Chancellor, BIT, Mesra for their support and encouragement in bringing out this edition of PHARMBIT. Lastly we invite suggestions from readers and well wishers to further improve the PHARMBIT. Dr R. N. Gupta


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Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Drug Use in the Elderly : Attention Needed Santanu K Tripathi School of Tropical Medicine, Kolkata

The phenomenal advances in health technologies and drug development in the last half a century have witnessed a steady improvement in the life expectancy, and consequently, there is ballooning in the size of the elderly population throughout the world. India is no exception. According to the estimate of the National Commission on Population, Government of India, the elderly population, aged 60+ years, in India shows a trend of increase from 7 per cent in 2001 to 8.3 per cent in 20111. It is a common knowledge that old people tend to suffer from multiple and more diseases than their younger counterparts. Physiological changes associated with ageing may masquerade as illness. Pharmacokinetics and pharmacodynamics may be altered by normal ageing or disease, heightening the risk of adverse drug reactions (ADR). Super-specialty medicare services being commonplace these days, the elderly people tend to consult different specialists and are frequently treated by several doctors simultaneously. They are thus more likely to be prescribed medications by their doctors and to take multiple agents. Polypharmacy including ‘lateral prescribing’ for multiple co-morbidities in the elderly is a common practice. Secondly, physicians, in general, do not have appropriate training in geriatric pharmacology and often fail to appreciate the need for the same. Further, older citizens are in general rarely enrolled in clinical trials and the prescribing doctor often does not appreciate that evidences available from trials conducted in younger patients may not apply in the elderly. Thus inappropriate drug prescribing in the elderly is quite common2,3 and they are liable to show tendency to imperfect adherence to prescribed treatment, ADRs and drug interactions4,5. A number of drugs are known to be associated with a much higher risk of ADRs in the elderly. Studies show around 16–18 percent higher risk of their prescription per patient year for ambulatory, community-based older patients6. These have caused lot of concerns and the criteria for defining inappropriate medications for the elderly – Beer’s Criteria – have been developed7,8. Wilcox et al9 applied Beer’s criteria to 6,171 non-institutionalized elderly from the 1987 National Medical Expenditure Survey in the US and nearly one-fourth of all elderly people living in the community were found to have been prescribed an inappropriate medication. More recently, Golden et al.10 found that nearly 40% of 2,193 homebound elderly individuals were prescribed an inappropriate drug according to the Beers criteria. Drug utilization review (DUR) programmes have offered safeguards against inappropriate use prescription drugs in the elderly11. DUR programs are not substitutes for careful consideration by physicians and pharmacists. Instead, they are a mechanism for sending alerts about potentially inappropriate medications7. A concurrent DUR program is effective in reducing inappropriate prescribing in the elderly. Mandated by state pharmacy law in the US, DUR applies explicit criteria to patient prescription and medical history information12. The utilization review focuses on safety, appropriateness, effectiveness 85


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

and cost-containment to determine potentially inappropriate prescribing such as drug interactions, contraindications, overdoses, underutilization and overutilization13. In a resource-poor country like India, it is likely that the elderly age group receives relatively scant attention insofar as medical care for chronic diseases they suffer, is concerned. The cost of medical and pharmaceutical care has been escalating enormously, steadily increasing the out-of-pocket (OOP) spending on medicines thereby making it ill-affordable to all patients in general, and it is possible that elderly population in India, are worst hit by this. The obvious economic insecurity the average elderly Indians are confronted with, probably also denies them their rightful access to medical and pharmaceutical care. They tend to take recourse to over-the-counter medications more frequently. Further, in India with multiple alternative health care systems like Ayurveda, Homeopathy, Unani etc prevailing, the seniors may find solution to their health problems in these apparently more economic health care systems. A careful scrutiny of contemporary literature fails to reveal published reports on medication used by the elderly Indians. To the best of our knowledge no data are available regarding the potential problems caused to elderly patients who receive treatment by multiple specialist doctors and consuming medicines as prescribed by each of them. Neither any reports on the prevalence of inappropriate prescribing in the elderly Indians are available. In India, it thus appears unlikely for any scope of review, i.e., DUR programmes, of these multiple prescriptions that converge on an elderly person who is the unwitting recipient for all those medicines. No specific studies addressing OOP expenditure on medicines by the elderly Indians have probably been conducted in recent times. In view of the fact that the elderly population in India is constantly swelling, and we are committed to ensure an optimum quality of life to our senior citizens, it is high time that we pay our attention to this yet neglected area of medication used by the elderly. Special strategies need to be formulated and implemented at the earliest. Health professionals of all categories should join hands to help our older citizens keep away the medication related problems. Efforts to optimize medication use in this population through a partnership between patients, pharmacists and physicians is needed. Clinical pharmacists and medical pharmacologists can assume a ‘bridging’ role in India, in supplementing pharmaceutical care to the elderly subjects exposed to multiple ‘lateral prescribing polypharmacy’. Special DUR programs may link information on geriatric prescribing with the opportunity to change prescribing as appropriate. Such programs can optimize medication management in the elderly by identifying potential problems and empowering health care providers to improve prescribing in this special population. References 1.

National Commission on Population - Technical Group on Population Projections, Registrar General of India (RGI) 1996 : http://populationcommission.nic.in/facts1.htm (Accessed on 11.12.2010)

2.

Gottlieb Scott. Inappropriate drug prescribing in elderly people is common. BMJ. 2004; 329-367.

3.

GAO-US. Prescription drugs and elderly: many still receive potentially harmful drugs despite recent improvements. Report to the Honourable Ron Wyden, House of Representatives. GAO/ HEHS. 1995; 95-152.

4.

Keith Beard. “Adverse Reactions as a Cause of Hospital Admission in the Aged”, Drugs & Aging, Vol. 2, No. 4 ( July/ August. 1992), pp.356-67.

86


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

5.

Nananda Col, James E. Fanale, Penelope Kronholm. “ The Role of Medication Noncompliance and Adverse Drug Reactions in Hospitalizations of the Elderly”, Archives of Internal Medicine, Vol. 150, No. 4 ( Apr. 1990), pp. 84145.

6.

van der Hooft CS, Jong GW, Dieleman JP, et al; Inappropriate drug prescribing in older adults: the updated 2002 Beers criteria--a population-based cohort study. Br J Clin Pharmacol. 2005 Aug;60(2):137-44. [abstract]

7.

Aparasu RR, Mort JR. Inappropriate prescribing for the elderly : Beers’ criteria-based review. Ann Pharmacother 2000; 34: 338-346.

8.

Beers MH. Explicit criteria for determining potentially inappropriate medication use by the elderly. Arch Intern Med. 1997;157:1531-1536.

9.

Wilcox SM, Himmelstein DU, Woolhandler S (1994), Inappropriate drug prescribing for the community-dwelling elderly. JAMA 272(4):292-296.

10. Golden AG, Preston RA, Barnett SD et al. (1999), Inappropriate medication prescribing in homebound older adults. J Am Geriatr Soc 47(8):948-953. 11. Pierre Jean G, Morisan J, Potvin L, Chabot I, Verreault R, Milot A. Effect of drug utilization reviews on the quality of in-hospital prescribing: a quasi-experimental study. BMC Health Services Research 2006, 6:33-43. 12. Monane M, Matthias DM, Nagle BA, Kelly MA (1998a), Improving prescribing patterns for the elderly through an online drug utilization review intervention: a system linking the physician, pharmacist, and computer. JAMA 280(14):1249-1252. 13. Monane M, Nagle B, Kelly MA (1998b), Pharmacotherapy: strategies to control drug costs in managed care. Geriatrics 53(9):51-64.

q

q

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Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Formulation and Evaluation of Biodegradable Dental Implants Containing Antibacterial Agents for Periodontitis Krishnananda Kamath*, Shripathy D., A. R. Shabharaya Srinivas College of Pharmaceutical Sciences, Mangalore, Karnataka, India

ABSTRACT This study was designed to develop and evaluate biodegradable dental implants containing combination of ciprofloxacin (complexed with b-cyclodextrin) and tinidazole for the treatment of periodontitis. The implants were prepared by using biodegradable polymer, poly(e-caprolactone), then evaluated for their physicochemical properties including weight variation, thickness, estimation of drug content, stability and in vitro release studies. The in vitro drug release profiles from implants were investigated using vial and flow through methods. Mean weight and thickness data showed that the different films were uniform. In vitro drug release data indicated that the implant showed an initial burst release followed by sustained release of the drugs. The study suggests that implant containing ciprofloxacin and tinidazole is a potential drug delivery device for the local treatment of periodontitis and shall have good physicochemical properties. Key Words : Periodontits, bCD-Ciprofloxacin complex, Tinidazole, Biodegradable implant. INTRODUCTION Periodontal diseases are infections affecting a significant proportion of people in all populations. The presence of wide diversity of periodontal pathogens such as Porphyromonas gingivalis, Prevotella intermedia and Actinobacillus actinomycetemcomitans are responsible for periodontal destruction. Therefore, an objective of periodontal treatment is to suppress or eliminate sub gingival periodontal pathogens. This is generally achieved through sub gingival debridement, resulting in the reduction of the total bacterial load. However, some patients may experience continued periodontal attachment loss, and this may be due to some periodontal pathogens that are inaccessible during mechanical periodontal therapy1-3. On the other hand, it is conceivable that local or systemic administration of effective antimicrobial agents may enhance the outcome of mechanical therapy. Systemic antimicrobials as such adjuncts to mechanical therapy have had a positive effect on clinical as well as microbiological parameters. But the impact of this approach is reduced by the fact that the antibiotic is normally difficult to maintain in therapeutic concentrations at the site over the course of the treatment period. Moreover, systemic antibiotic therapy carries with it the risk of the host developing resistance4. Due to these negative effects, the use of local drug delivery devices containing antibiotics which can maintain therapeutic concentrations at the site of infection is an approach that may be explored. This could enhance the therapy of periodontal diseases while also reducing side effects5. The presence of wide diversity of periodontal pathogens may be necessitated to use more than one antibiotic, either serially or in combination. The combination therapy using systemic metronidazole along with ciprofloxacin has been already tried. These combinations of drugs provide an excellent elimination of many organisms in adult and juvenile periodontitis that had been treated unsuccessfully alone with tetracycline6. Therefore in this study combinations of drugs are used i.e. bCD-Ciprofloxacin complex and tinidazole used. Pharmacological agents applied locally for the treatment of periodontitis are targeted to several areas 88


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

such as bacteria in periodontal pockets, soft tissue walls of the pocket, and the exposed root cementum which is having a depth beyond 5 mm. Experimental evidence suggests that many forms of local delivery like dentifrices, mouth rinses, gels and irrigation solutions are not able to deliver medications to all these locations. For example, agents in mouth rinses and those used for irrigation do not predictably reach beyond 5 mm into the periodontal pocket7. While systemic antibiotics have a very limited use in treating typical periodontal disease, there has been much interest in local antibiotic delivery. If an antibiotic can be delivered directly to the pocket, without the patient having to take systemic doses, there are far fewer side effects, and fewer chances of resistant bacteria and concentration of the antibiotic at the diseased site is much greater8-10. The most recent local antibiotic therapy introduced consists of small spheres of minocycline. The spheres, which look like a fine powder, are contained in a small blunt plastic needle, and are injected into the pocket. The spheres are bio-adhesive and stick to the pocket wall where they slowly release minocycline over a 14-21 day period. Because the spheres are also biodegradable so it’s removal is not required11. It has been shown that there is significant reduction in pocket depths, as well as reduction in disease activity. PerioChip is a small, orange brown, rectangular chip (rounded at one end) for insertion into periodontal pocket. It contains a substance, which is effective in killing bacteria in the gum (2.5 mg Chlorhexidine gluconate). After scaling, PerioChip is inserted into the pocket and the active ingredient takes effect in killing the bacteria. The chip itself dissolves naturally; hence there is no need for an additional visit to the dentistry to remove it. It is recommended to attend a follow-up visit after three months to check whether any further treatment is required11-12. Therefore, an attempt has been made in this study to develop biodegradable implant containing bCD-ciprofloxacin complex and Tinidazole for the treatment of periodontitis. Ciprofloxacin is a second generation fluroquinolone derivative, exhibiting activity against a wide range of Gram-negative and Gram-positive facultative bacteria as well as periodontal pathogens. Ciprofloxacin, a common drug is used effectively against Actinobacillus actinomycetemcomitans and to determine the effects of ciprofloxacin in chronic gingivitis three doses of ciprofloxacin were administered to establish adequate gingival crevicular fluid and serum concentrations of the agent. According to these results, ciprofloxacin is an alternative drug in subjects with periodontitis13-15. Tinidazole is a close analog of metronidazole and has the antimicrobial spectrum similar to it. Previous studies have shown that it will reach sufficient concentration in serum and, GCF and Gingival tissues to inhibit putative periodontopathic bacteria16. Periodontal pockets have an average depth of 6-8 mm which allows easy placement of drug delivery device. Biocompatible polymers like polylactic acid, Poly (e-caprolactone) and its copolymers have potential usefulness in development of biodegradable intra-pocket drug delivery devices. The drug release from drug delivery system is controlled by a combination of several physical parameters. These include permeation of water into matrix, leaching (extraction or Diffusion) of the drug from the matrix17. This study involves the formulation and evaluation of sustained release implants containing Beta cyclodextrin-Ciprofloxacin complex and Tinidazole. 89


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

MATERIAL AND METHODS Ciprofloxacin and Tinidazole were gift samples supplied by Cadila Healthcare Ltd., Goa. Poly (e-caprolactone) and Polyvinyl pyrrolidone were purchased from Polysciences Inc., USA; Betacyclodextrin (bCD) was purchased from Nippo Shokuhin kaako Co. Ltd., Japan. All the other chemicals were of analytical grade. Preparation of Implants Poly (e-caprolactone) both mol. Wt and PVP (K- 30) were weighed according to the formula given in table-1 and dissolved in dichloromethane. To this bCD-ciprofloxacin complex and tinidazole were added and sonicated. The resultant suspension was poured in to glass mould 5 x 3 cm lined with aluminium foil and allowed to dry for 24 hrs. at room temperature. The resulting film was cut into 0.5 x0.5 cm strips containing approximately 1 mg each of Ciprofloxacin and tinidazole15. Estimation of Drug content Five implants were weighed and taken separately in 100 ml volumetric flask, dissolved in glacial acetic acid, made up the volume to 100.0 ml and filtered absorbance were measured specrtometrically and concentration of ciprofloxacin and Tinidazole were calculated from the calibration curve18. In vitro release studies Vial Method Five implants were taken in a vial containing 5 ml of PBS (pH 7.4). Samples of 1.0 ml were withdrawn periodically at specific interval of time by replacing equivalent fresh buffer. The samples were analyzed spectrophotometrically after suitable dilution with PBS. Concentration of ciprofloxacin and tinidazole were calculated from the calibration curve14. Flow through method A column type flow through assembly was designed in our laboratory which maintains perfect sink conditions facilitating better in vitro evaluation. An intravenous infusion set was attached to the bottle containing phosphate buffer saline (pH 7.4). The flow rate was adjusted to 5 ml/hr. using a flow regulator. Five ml of phosphate buffer saline was always maintained in the donor cell (containing implant) throughout the experiment. The implant was supported in a small nylon mesh (#40). The flow of the buffer was from the bottle through the implant containing cell and so the receiver. The samples of (5ml/hr.) were collected and were analyzed. Concentration of ciprofloxacin and tinidazole were calculated from the calibration curve. An in vitro release drug release plot of time versus cumulative percentage Table-1 : Formula for the preparation of Dental implants drug release was constructed from Quantity Ingredients the data obtained. (in mg) RESULTS Physicochemical characterization of implants The prepared implants were smooth and non-brittle. They had a thickness of 0.54Âą0.03 mm an average weight of 12.5Âą0.5 mg.

Ciprofloxacin b-CD complex (equivalent to 60 mg of Ciprofloxacin) Tinidazole

245 60

Poly (e-caprolactone) (Mol. wt. 35,000)

600

Poly (e-caprolactone) (Mol. wt. 42,500)

600

Poly vinyl pyrollidone (K-30)

90

60


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Table-3 : Data of in vitro drug release from biodegradable implants (Flow through Method)

Table-2 : Data of in vitro drug release from biodegradable implants (Vial Method) Time (Hours)

Cum. % release of Ciprofloxacin

Cum. % release of Tinidazole

0

0.00

0.00

1

9.31

34.87

2

13.53

39.05

3

15.83

43.32

4

17.35

47.10

5

19.38

6

Time (Hours)

Cum. % release of Cum. % release of Ciprofloxacin Tinidazole

0

0.00

0.00

1

3.18

14.62

2

4.93

25.13

3

6.26

32.14

4

7.47

38.52

5

9.20

44.76

6

10.69

49.58

50.76

7

12.36

54.09

20.02

55.56

24

18.61

66.54

7

26.47

64.88

48

27.30

74.88

72

39.08

82.80

8

32.77

65.68

96

47.89

85.21

120

51.39

86.85

24

42.53

78.95

144

54.83

87.11

48

49.22

83.98

168

62.22

87.11

72

54.64

84.15

192

65.90

-

96

58.06

84.28

216

67.33

-

240

68.56

-

120

59.35

88.64

264

69.65

-

144

65.23

88.79

288

72.76

-

168

69.78

89.98

192

72.91

88.91

360

75.20

86.38

528

75.26

87.78

696

78.58

86.01

864

82.92

85.30

1032

84.81

85.36

1200

89.80

86.97

1368

90.19

85.53

91

336

74.44

-

432

76.12

-

528

77.87

-

624

79.87

-

720

81.80

-

816

84.56

-

912

86.00

-

1008

87.41

-

1104

88.13

-

1200

88.89

-

1296

90.33

-

1392

90.75

-

1488

91.15

-


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Figure 1 : Showing normal and tooth with periodontal pocket.

Figure 2 : In Vitro drug released from implants in phosphate buffered saline (pH 7.4) (Vial Method).

In vitro release studies The vial method how ever have few inherent disadvantages such as saturation of the release medium and hence prevalence of non-sink condition. This method therefore fails to mimic in vivo conditions and makes the in vitro-in vivo correlation unrealistic. With the flow through method the release pattern of ciproflixacin and tinidazole were comparable to that of the vial method but, was clearly defined. Figure 2 and 3. Ciprofloxacin and tinidazole were released in triphasic manner from implants characterized by an initial burst effect followed by a slow release and secondary burst effect. The burst effect corresponds to the release of the drug located on or near the surface of implants or release of poorly dispersed drug. The slow release of the drug may be due to medium being diffused in to the polymer matrix where by degradation occurs and the drug diffuses out of implants. The secondary burst effect occurs when the matrix becomes more water soluble which results in erosion and collapse of the matrix. Tinidazole Figure 3 : In Vitro drug released from implants in phosphate buffered saline release was faster and comparatively (pH 7.4) (Flow through Method). higher concentration than ciprofloxacin. Stability studies Stability studies were conducted for acceleration and at room temperature. These studies indicated that degradation rate of drugs increases as the temperature increases. When degradation rates of these two drugs are compared the degradation rate of tinidazole was higher when compared to bCDciprofloxacin complex. DISCUSSION In an attempt to formulate appropriate dental drug delivery system for the treatment of periodontits, it was possible to design biodegradable implant which could maintain drug release in a sustained pattern over a prolonged period of time. The bCD inclusion complex may help to improve the stability of antibiotics and improve the release characteristics as the free drug has to dissociate from the complex and diffuse through the polymeric matrix, besides the bCD inclusion complex is more water soluble and insoluble in dichloromethane, which is used as a solvent for casting the implants resulting in a drug 92


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

suspension rather than a solution. The in vitro release results indicated that biodegradable delivery devices could be suitable for the sustained delivery of anti-infective agents. The release of drugs was function of both the release kinetics and rate of degradation of device. Release was controlled by a combination of several physical parameters. These include permeation of matrix by water, Leaching (extraction or diffusion) of drug from the matrix and erosion of the matrix material. ACKNOWLEDGEMENTS The authors are thankful to M/s. Cadila Healthcare Ltd, Goa for supplying ciprofloxacin and tinidazole as the gift samples. REFERENCES

1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

G Tihomir, N Kamen. Rational antibiotic therapy in the complex treatment of patients with generalized Periodontitis. J. of IMAB. 2009; 2:10-13 AD Haffajee, SS Socransky, JC Gunsolley. Systemic anti-infective periodontal therapy. Ann. J. Periodontol. 2003; 8:115-81. S Jorgens. Efficient Antimicrobial treatment in Periodontal Maintenance Care. J. Am. Dent. Assoc. 2000; 131:12931304. J Slots, TE Rams. Antibiotics in periodontal therapy; advantages and disadvantages. J. Clin. Periodontol. 1990; 17:479-492. M Minabe. Intra-pocket antibiotic therapy using reabsorbable and non-resorbable slow-release devices containing tetracycline. Periodontal Clin. Investig. 2000;22:14-21 TE Rams, D Feik, J Slots. Ciprofloxacin/metronidazole treatment of recurrent periodonttits. J. Dent. Res. 1992; 71:319-327. KS Aithal, N. Udupa. Dental implants of ciprofloxacin and norfloxacin. The Antiseptic, 1995; 92:4-8. TE Rams, J Slots. Local delivery of antimicrobial agents in the periodontal pocket. J. Periodontal, 1996; 10:139–59. GA Mohammed. Formulation of chitosan-based ciprofloxacin and diclofenac film for periodontitis therapy. Trop. J. of Pharma. Res. 2009; 8:33-41 H Yeom. Clinical and microbiological effects of minocycline loaded microcapsules in adult peridontitis, J. Periodontal, 1997; 68;1102-1109. SP Peiman. Local Delivery of Antibiotics; Soleymani DDS, Beverly Hills Periodontics and Implant Dentistry Center. htm. 2008 Mecall. www.drmecall.com. Mecall Periodontics and Dental Implant Center, 2009 BRR Verma, N Udupa. Biodegradable dental implants of doxycycline hydrochloride. The Antiseptic, 1996; 93:126129. R Nagaraju, M Jacob, N Udupa, BRR Verma. Biodegrable dental implants of ciprofloxacin BCD complexes in the treatment of priodontitis, Ind. J. Exp. Biology 1999;37;305-307. R Nagaraju, N Udupa, S Arshad, BRR Verma, Controlled release biodegradable implants of doxycycline and its clinical efficacy. The Antiseptic, 2001; 98:165-169. VS Mastiholimath Formulation and evaluation of ornidazole dental implants for periodontitis. Indian J. Pharm. Sci., 2006;68:68-71 PJ Hanes, Local anti-infective therapy: pharmacological agents. A systematic review. Ann. Periodontol. 2003; 8:7998. P Trivedi P. Simultaneous spectrometric analysis of norfloxacin and tinidazole from tablets. Indian drugs, 1997; 34:190-193.

q

q

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Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Formulation and Optimization of Mucoadhesive Nano Drug Delivery System of Acyclovir U.V. Bhosale*, V. Kusum Devi Al-Ameen College of Pharmacy, Bangalore, India

Abstract Acyclovir is an antiviral drug, used for treatment of herpes simplex virus infections with an oral bioavailability of only 10 to 20 % (limiting absorption in GIT to duodenum and jejunum), half life about 3h, soluble only at acidic pH (pKa 2.27). Mucoadhesive polymeric nanodrug delivery systems of acyclovir have been designed and optimized using 23 full factorial design. Poly (lactic-co-glycolic acid) (PLGA) (50:50) was used as polymer along with polycarbophil (Noveon AA-1) as mucoadhesive polymer and Pluronic F68 as stabilizer. From the preliminary trials, the constraints for independent variables X1 (amount of Poly (lactic-co-glycolic acid), X2 (amount of Pluronic F68) and X3 (amount of polycarbophil) have been fixed. The dependent variables that were selected for study were, particle size (Y1), % drug entrapment (Y2) and %drug release in 12h (Y3). The derived polynomial equations were verified by check point formulation. The application of factorial design gave a statistically systematic approach for the formulation and optimization of nanoparticles with desired particle size, % drug release and high entrapment efficiency. Drug:polymer ratio and concentration of stabilizer were found to influence the particle size and entrapment efficiency of acyclovir loaded Poly (lactic-co-glycolic acid) nanoparticles. The release was found to follow fickian as well as non-fickian diffusion mechanism with zero order drug release for all batches. In vitro intestinal mucoadhesion of nanoparticles increased with increasing concentration of polycarbophil.These preliminary results indicate that acyclovir loaded mucoadhesive poly (lactic-co-glycolic acid) nanoparticles could be effective in sustaining drug release for a prolonged period. Key words : Acyclovir, PLGA, nanoparticles, 23 factorial design, mucoadhesive. Herpes simplex virus (HSV) is a member of family of herpes viridae, a DNA virus. There are two types of Herpes Simplex Viruses (HSV). viz HSV type 1 and type 2. HSV type 1 is the herpes virus that is usually responsible for cold sores of the mouth, the so called, fever blisters. HSV type 2 is the one that most commonly causes genital herpes1. The infection causes painful sores on the genitals in both men and women. In addition, Herpes sores provide a way for HIV to get past the body’s immune defenses and make it easier to get HIV infection. A recent study found that people with HSV had three times the risk of becoming infected with HIV as compared to people without HSV2. Currently the treatments available for herpes simplex are conventional tablets and topical gel for application on outbreaks. The drugs that are commonly used for herpes simplex are acyclovir, valaclovir and famciclovir. Acyclovir, the first agent to be licensed for the treatment of herpes simplex virus infections, is the most widely used drug for infections such as cutaneous herpes, genital herpes, chicken pox, varicella zoster infections. Acyclovir is currently marketed as capsules (200 mg), tablets (200mg, 400mg and 800 mg) and topical ointment1. Oral acyclovir is mostly used as 200 mg tablets, five times a day. In addition, long term administration of acyclovir (6 month or longer) is required in immunocompromised patient with relapsing herpes simplex infection2. The presently available conventional therapy is associated with a number of drawbacks such as highly variable absorption and low bioavailability (10–20%) after oral administration3. Furthermore, with increase in dose, there is decrease in bioavailability. Moreover, 94


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

because the mean plasma half life of the drug is 2.5 h, five times a day administration is required. In order to make oral therapy of acyclovir more patient compliant there is a need of using different approaches like matrix tablets, nanoparticles4 and polymeric films5. The main problem with the therapeutic effectiveness of acyclovir is its absorption which is highly variable and dose dependent thus reducing the bioavailability to 10–20%6. Acyclovir is soluble in acidic pH and is predominantly absorbed from upper gastro intestinal tract (GIT)7. There are indications of its active absorption from the duodenum and jejunum regions of GIT8. The inherent shortcomings of conventional drug delivery and the potential of nanoparticles as drug delivery systems have offered tremendous scope for researchers in this field and is fast moving from concept to reality. Nanoparticles may be used for oral administration of gut-labile drugs or those with low aqueous solubility9. These colloidal carriers have the ability to cross the mucosal barrier as such. In addition they have the potential for enhancing drug bioavailability via particle uptake mechanisms. It was therefore decided to prepare nanoparticles of acyclovir so as to optimize its delivery and overcome its inherent drawbacks. The concept of mucosal adhesives, or mucoadhesives, was introduced into the controlled drug delivery arena in the early 1980s10. Mucoadhesives are synthetic or natural polymers which interact with the mucus layer, covering the mucosal epithelial surface and mucin molecules, constituting a major part of the mucus. They localize the formulation at a particular region of the body thereby improving bioavailability of drugs with low bioavailability. The increased contact time and localization of the drug due to applying nanoparticles of acyclovir, which are made mucoadhesive, enhances its delivery. Possible added advantage of this approach would be increase in bioavailability as well as reduction in frequency of administration. For the present investigation, mucoadhesive polymeric nanodrug delivery systems of acyclovir have been designed and optimized using 23 full factorial design. Poly (lactic-co-glycolic acid) (PLGA) (50:50) was used as polymer along with Polycarbophil (Noveon AA-1) used as mucoadhesive polymer and Pluronic F68 was used as stabilizer. MATERIALS AND METHODS Acyclovir was a gift sample from Ajanta Pharmaceutical Limited, Mumbai India ; poly (D,L lactide-coglycolide) (PLGA 50:50 and PLGA 85:15) were obtained as gift samples from Indena Ltd., Rome, Italy; pluronic F68 and polycarbophil (Noveon AA-1) were procured from StridesArco Lab, Bangalore,India, as gift; acetone, cellophane membrane, were purchased from S.D. Fine Chem. Ltd., Mumbai,India. All other reagents and chemicals used in this study were of analytical grade. Mucoadhesive poly (lactic-co-glycolic acid) (PLGA) nanoparticles Mucoadhesive PLGA nanoparticles were prepared by the solvent deposition method. Acyclovir was dissolved in neutral water at 35-40°C containing a hydrophilic surfactant at various concentrations. A mucoadhesive polymer, polycarbophil was dispersed in this aqueous phase. Organic phase was prepared by solubilizing PLGA in acetone at various concentrations. The organic phase was poured into the aqueous solution drop wise, under stirring (RPM 5000) for 2 h, thus forming a milky colloidal suspension. The organic solvent was then evaporated by using a Rota evaporator. The resultant dispersion was dried using a freeze drying method11. 95


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Experimental design The formulations were fabricated according to a 23 full factorial design, allowing the simultaneous evaluation of three formulation variables and their interaction. The experimental designs with corresponding formulations are outlined in Table-1.The dependent variables that were selected for study were - particle size (Y1), % drug entrapment (Y2) and % drug release in 12 h(Y3). Determination of particle size The particle size and size distribution of the acyclovir loaded mucoahesive PLGA (50:50) nanoparticles were characterized by laser light scattering using Particle size Analyzer (Malvern Mastersizer Hydro-2000 SM, UK). The obscuration level was set between 7 to 11 %, distilled water was used as medium. Determination of Encapsulation Efficiency The free drug was estimated by taking said quantity of formulation in dialysis bag (cellophane membrane, molecular weight cut off 10000-12000 Da, Hi-Media,Mumbai India) which was tied and placed into 100 ml water (pH=7) kept at 37±5° C on magnetic stirrer. At predetermined time intervals, 5 ml of the samples were withdrawn by means of a syringe. The volume withdrawn at each interval was replaced with same quantity of fresh water (pH=7) maintained at 37±5ºC. The samples were analyzed for free drug by measuring the absorbance at 252 nm using UV/Vis spectrophotometer (ShimadzuUV-1700) after suitable dilution. Above described process of withdrawing sample and analysis was continued till a constant absorbance was obtained12. Encapsulated drug was estimated by taking residue formulation remaining behind in dialysis membrane after estimation of free drug content, as described above. Formulation in dialysis bag was added to acetone (10 ml) to dissolve PLGA and filtered. Residue remaining on filter paper was dissolved in 100 ml of water (pH=7) kept at 37±5º C and after removing supernatant, sample was analyzed for drug content by measuring the absorbance at 252 nm using UV/Vis spectrophotometer (Shimadzu UV-1700) after suitable dilution. The percentage of drug entrapped and the percentage of free drug were calculated by following Eqns13. Amount of free drug present in 100mg of formulation % free drug = ------------------------------------------------------------------- × 100 Total amount of drug present in 100mg of formulation

…(1)

Amount of encapsulated drug present in 100mg of formulation % Drug entrapment = ------------------------------------------------------------------------------×100 ...(2) Total amount of drug present in 100mg of formulation Statistical analysis14 The results from factorial design were evaluated using Sigma plot software (Systat Software Inc., Version 3.0, Richmond, CAsoftware). Step-wise backward linear regression analysis was used to develop polynomial equations for dependent variables particle size (Y1) % drug entrapment (Y2) and %drug release in 12 h(Y3) Y=B0+B1X1+B2X2+B3X3+B11X12+B22X22+B33X32+B12X1X2+B13X1X3+B23X2X3+B123 X1X2X3 ...1 Where Y is dependent variable, B0 arithmetic mean response of eight batches, and B1,B2, B3 are estimated coefficient for factor X1,X2 and X3 respectively. The main effects (X1, X2 and X3) represent 96


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

average result of changing one factor at a time from its low and high value. The interaction terms (X1X2), (X1X3), (X2X3) shows how the response changes when three factors are simultaneously changed. The polynomial term (X1X2X3) is included to investigate non-linearity. The validity of the developed polynomial equations was verified by preparing check point formulation (C). Drug release study A quantity of selected factorial formulations equivalent to 25 mg of the drug was taken in the dialysis bag (cellophane membrane, molecular weight cut off 10000-12000 Da, Hi-Media, India).The dialysis bag was then suspended in a flask containing 100 ml of 0.1 N HCl on a magnetic stirrer at 37±0.5º C at 100 rpm. Required quantity (5 ml) of the medium was withdrawn at specific time periods (1, 2, 3, 4, 6, 8, 10, 12, 24, 32 h) and same volume of dissolution medium was replaced in the flask to maintain a constant volume. The withdrawn samples were filtered and then 5 ml filtrate was made up to volume with 100 ml of 0.1 N HCl. The samples were analyzed for drug release by measuring the absorbance at 252nm using UV/Vis spectrophotometer15. Invitro evaluation of intestinal mucoadhesion of nanoparticles The Institutional Animals Ethical Committee (IAEC) of Al-Ameen College of Pharmacy, Bangalore, approved the protocol for the study. Male Sprague Dawley rats weighing 200-250 g were fasted overnight before the experiments, but allowed free access to water. A part of intestine (duodenum and jejunum) was excised under anesthesia and perfused with physiological saline to remove the contents of stomach. The cleaned portion was used immediately after preparation. A 50 mg quantity of mucoadhesive nanoparticles sample that were suspended in phosphate buffer (pH 6.8 )was filled into the cleaned intestine, ligated and then incubated in physiological saline at 37° for 30 min. The liquid content of separated portion of intestine was then removed by injecting the air and the same was perfused with phosphate (pH 6.8) for 2 h, at a flow rate of 1 ml per min. The intestine was cut to open and nanoparticles that remained in it were recovered with phosphate buffer (pH 6.8). The final volume of washing solution was mixed with 10 ml of acetone solution and kept for 2 h for complete digestion of nanoparticles. After filtration through 0.45 mm filter paper, absorbance was determined spectrophotometrically at 252 nm (acyclovir) and gastric mucoadhesion was determined as the % of nanoparticles remaining in intestine after perfusion16. Drug-Polymer Interaction Studies Differential scanning calorimetry (DSC) is one of the most powerful analytical techniques, which offers the possibility of detecting chemical interaction. Acyclovir ( pure drug), PLGA, and physical mixtures of drug and polymer at different ratios (1:1, 1:1.5, 1:2, 1:2.5) were kept at 40 ±2°C/75±5% RH Samples at 0.1, 2, 3 and 6 months were withdrawn and sent for DSC Analysis. Also drug-polymer interaction study for selected formulation of coated and uncoated nanoparticles were evaluated by DSC (PerkinElmer DSC 7, USA). Thermograms of acyclovir, polymer (PLGA), and mucoadhesive nanoparticles, were obtained with 5°C/min of heating rate between the temperature range of 5°C to 280°C. SEM Photomicrographs The morphology of coated and uncoated nanoparticles was examined by scanning electron microscopy (SEM, JSM-5310LV scanning microscope Tokyo, Japan). The nanoparticles were mounted on metal stubs using double-sided tape and coated with a 150 Å layer of gold under vacuum. Stubs were visualized under scanning electron microscope. 97


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

RESULTS AND DISCUSSION Out of all mucoadhesive formulations, developed according to the factorial design and the above described method, MF2, MF5, MF6 and MF8 were found to be free flowing i.e. non sticky but formulations, MF1, MF3, MF4 and MF7 were found to be sticky. All formulation were white and powdery in appearance. The particle size affects the biopharmaceutical, physicochemical and drug release properties of the Figure 1 : Comparison of particle size of mucoadhesive factorial formulations nanopaticles. A graphical representation and check point formulation of the particle size of mucoadhesive nanoparticles obtained is given in Fig.1 Particle size is an important parameter because it has a direct relevance to the stability of the formulation. Larger particles tend to aggregate to a greater extent compared to smaller particles, thereby resulting in sedimentation16. The amount of stabilizer used also had an effect on the properties of nanoparticles. If the concentration of stabilizer is too low, aggregation of the polymer will take Figure 2 : Comparison of % drug entrapment and % drug release in 12 h of place, whereas, if too much stabilizer factorial and check point formulations is used, drug incorporation could be reduced as a result of the interaction between the drug and stabilizer[1]. The effect of the concentration of the polymers tested is negative or positive. A positive effect would imply that increasing the concentration causes the emulsion to have larger droplets, hence leading to larger particles. A negative effect means that increasing the concentration causes the emulsion to be more stable, hence leading to smaller particles16. From Fig 1 and Fig 2 and Table 1, it is revealed that as drug: polymer (acyclovir: PLGA or acyclovir: polycarbophil) ratios increased from 1:0.875 to 1:1.25 (for PLGA) and 1:0.6 to 1:0.9 (for polycarbophil), particle size and drug entrapment efficiency increased significantly. It also revealed that concentration of stabilizer has significant effect on particle size but it has insignificant or negligible effect on drug entrapment efficiency of nanoparticles. This can be explained by observing particle size and % drug entrapment of mucoadhesive factorial formulations MF1, MF2 and MF7, MF8 where drug :polymer (PLGA) ratio increased from 1:0.875 to 1:1.25,with constant concentration of stabilizer (pluronic F68) i.e.0.3% for MF1, MF2 and 0.2% for MF7, MF8 drug entrapment efficiency increased from 89.9 % to 93.7 % and 80.09 % to 86.26 %, 98


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Table 1 : Experimental Design and Parameters for 23 Full Factorial Design Batches Batch Code

Variable level in Coded Form

Particle size (nm)

% Drug entrapment

Drug Release in 12 hrs

Average % Intestinal retention

X1*

X2#

X3§

MF1

1

1

1

1580

93.7

54.04

49.3

MF2

-1

0

1

1210

89.9

59.52

62.1

MF3

1

-1

1

1630

94.1

53.32

45.7

MF4

-1

1

1

1420

88.12

57.35

55.6

MF5

1

1

-1

870

84.12

65.33

56.2

MF6

-1

-1

-1

740

80.16

71.14

67.3

MF7

1

-1

-1

914

86.26

63.72

52.5

MF8

-1

-0.5

-1

810

80.09

67.02

59.7

-0.5

-

-0.5

1107

80.59

64.43

----

C

* For PLGA (50:50) (X1) transformed levels in polymer weight are:–1= 175mg; +1=250mg; -0.5= 193.75mg # For surfactant (Pluronic F68) (X2) transformed levels in % are: –1=0.20%; +1= 0.30%; –0.5=22.5% § For Mucoadhesive polymer (Polycarbophill) (X3) transformed levels in% are:-1=0.10% +1=0.15%; -0.5=11.25%.

respectively, also particle size increased from 1210 to 1580 nm and 810 to 914 nm, respectively. In the same way it can be explained with respective to mucoadhesive factorial formulations MF1,MF5 and MF4, MF8, where drug: polymer ratio increased from 1:0.6 to1:0.9, with constant concentration of stabilizer (Pluronic F68) i.e.0.3% for MF, MF5 and 0.2% for MF4, MF8, drug entrapment efficiency increased from 84.12 % to 93.7 % and 80.09 % to 88.12 %, respectively, also particle size increased from 870 to1580 nm and 810 to 1420 nm, respectively. But it has observed for mucoadhesive factorial formulations MF1, MF3 and MF6, MF8, where stabilizer concentration increased from 0.2% to 0.3%, with constant Drug:Polymer ratio i.e. 1:1.25 for MF1, MF3 and 1:0.875 for MF6, MF8. Particle size decreased from 163 to 1580nm and 810 to740 nm, respectively, but at the same time, there is insignificant or negligible change in drug entrapment efficiency as it changed from 94.1% to 93.7% and 80.09 % to 80.16 %, respectively. Thus it can be concluded that polymer and surfactant concentration has significant effect on particle size. However there is insignificant or negligible effect of surfactant concentration on drug entrapment efficiency. Drug release from nanoparticles and subsequent biodegradation are important for developing successful formulations. The release rate of nanoparticles depends upon i) desorption of the surfacebound/adsorbed drug; ii) diffusion through the nanopaticle matrix; iii) diffusion (in case of nanocapsules) through the polymer wall; iv)nanoparticle matrix erosion: and v) a combined erosion/diffusion process. Thus, diffusion and biodegradation govern the process of drug release17. It is generally anticipated for a bulk eroding polymer such as 50:50 PLGA to give an initial burst release followed by a controlled release, in contrast to the release pattern observed in other controlled release systems for example sustain release tablets, pellets and beads. In cases where there is an initial 99


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

burst effect, the high initial release may be attributed to the presence of crystals of free and weakly bound drug on the surface of the particulate carriers18. The mechanism of drug release from nanoparticles is determined by different physical–chemical phenomena. The exponent n has been proposed as indicative of the release mechanism. In this context, n = 0.43 indicates Fickian release, n = 0.85 indicates a purely relaxation (Case II) and > 0.85 indicates super case II controlled delivery. Intermediate values 0.43 < n < 0.85 indicate an anomalous behavior (non-Fickian kinetics) corresponding to coupled diffusion/polymer relaxation19. The average percentage release was fitted into different release models: zero order, first order and Higuchi’s square root plot. The models giving a correlation coefficient close to unity were taken as the order of release In vitro drug release data of all factorial formulations was subjected to goodness of fit test by linear regression analysis according to zero order and first order kinetic equations, Higuchi’s and Korsmeyer-Peppas models to ascertain the mechanism of drug release. From various parameters determined for drug release from nanoparticles based on Peppas Model, Higuchi Model and Diffusion profile, it is evident that values of ‘r2’ for Higuchi plots of all mucoadhesive factorial formulations are close to unity i.e. linear (drug release by diffusion). Diffusion exponent values ‘n’ of Peppas equation for MF1, MF2 and MF3 are 0.6446, 0.5074 and 0.6435 respectively shows non-fickian diffusion and for MF4, MF5, MF6, MF7 and MF8 are 0.4286, 0.3858, 0.3225, 0.3942 and 0.3311 respectively shows fickian diffusion. Table 2 shows, almost zero order drug release for all factorial formulations as correlation coefficient of zero order close unity than first order. It can be concluded that the different drug release rates may be attributed to different sizes of the nanoparticles. It is expected as the particle size of PLGA nanoparticle is smaller, their surface area will be more and the drug release is faster16. From the data of experimental design and parameters (Table1) for mucoadhesive factorial formulations F1 to F8, polynomial equations for three dependent variables (particle size % drug entrapment and % drug release in 12 h) have been derived using Sigma plot software (Systat Software Inc., Version 3.0, Richmond, CAsoftware.) The equation derived for particle size is: Y1= 1136.7+11.75 X1–36.75 X2 +303.25 X3 +13.25 X1 X2-8.25X2X3+53.25X1X3

...2

The equation derived for % drug entrapment is: Y2= 87.0563+2.4887 X1–0.0862 X2 +4.3987X3 -0.5487X1X2+0.4313X2X3-0.0438X1X3

...3

The equation derived for % drug release in 12 hrs is Y3= 63.07-2.2175 X1+1.1725 X2 -5.2750X3 -0.45X1X2-0.2925X2X3-0.0075X1X3

...4

In Eqn. 2, negative sign for coefficient of X2 indicates that the particle size of nanoparticles increases when concentration of Pluronic F 68 is decreased and positive sign for coefficient of X 1and X3 indicate positive effect of polymer concentrations (PLGA and Polycarbophill) on particle size. In Eqn. 3 positive sign for coefficient of X1 and X3 indicates that the % drug entrapment increases when concentration of PLGA and polycarbophill increases and negative sign for coefficient of X2 indicates that % drug entrapment of nanoparticles increases when concentration of Pluronic F 68 decreases. Also value of coefficient for X2 (-0.0862) shows insignificant or negligible effect of independent variable on dependent variable. 100


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Table 2 : Various Pharmacokinetic Parameters Determined for Drug Release Predicted values Formulation

Particle Size nm

%Drug entrapment

% Drug Release in 12 h

C

1012

83.61

66.03

Observed values Particle Size nm

%Drug entrapment

% Drug Release in 12 h

1107

80.59

64.43

In Eqn. 4 negative sign for coefficient of X1 and X3 indicates that the % Drug release in 12 hrs increases when concentration of PLGA and polycrbophill is decreased and positive sign for coefficient of X2 indicate positive effect of pluronic F68 concentration on % drug release in 12h. Validity of the above equations was verified by designing check point formulation (C). The particle size, % drug entrapment and % drug release in 12 h from the equations derived and those observed from experimental results are summarized in Table 2. The closeness of predicted and observed values for particle size and % drug entrapment indicates validity of derived equations for dependent variables. Graphical presentation of the data can help to show the relationship between response and independent variables. Graphs gave information similar to that of the mathematical equations obtained from statistical analysis. The response surface graphs of particle size and % drug entrapment are presented in Fig. 3 and 4. The response surface plots and contour plots illustrated that as concentration of polymers (PLGA and Polycarbophill) increases, the value of dependent variable i.e. particle size increase and concentration of stabilizer (Pluronic F 68) increases the value of dependent variable i.e. Figure 3 : Response surface plots of effects of factorial variables on % particle size decreases. Drug entrapment. (X1- amount of Poly (lactic-co-glycolic acid), X2- amount of Pluronic F68, X3- amount of polycarbophil.)

Similarly the response surface plots and contour plots for % drug entrapment shows positive effects of independent variable i.e. polymer concentrations (PLGA and Polycarbophill) and negative effect of other independent variable i.e. concentration of stabilizer (Pluronic F 68).

But in contrast to this illustration, the response surface plot and contour plot for % drug release in 12 h shows negative effect of independent variable i.e. polymer concentrations (PLGA and Figure 4 : Response surface plots of effects of factorial variables on Polycarbophill) and positive effect of independent Particle size. (X1- amount of Poly (lactic-co-glycolic acid), X2- amount of Pluronic variable i.e. concentration of stabilizer (Pluronic F68) on % drug release in 12 h. F68, X3- amount of polycarbophil.) 101


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Mucoadhesion involves different kind of interaction forces between mucoadhesive materials and mucous surface, such as electrostatic attraction, hydrogen bonding, Van der Waals forces and mechanical interpenetration and entanglement[18]. Spectrophotometric method (λmax 252 nm) was used to measure in vitro mucoadhesive Figure 5 : Response surface plots of effects of factorial variables on % capacity of developed formulations shows Drug release in 12 h. the % intestinal retention of mucoadhesive (X1- amount of Poly (lactic-co-glycolic acid), X2- amount of Pluronic nanoparticles in the rat intestinal mucosa. F68, X3- amount of polycarbophil.) Table 3: Parameters of Check Point Formulation Formulation

Correlation Coefficient Zero Order

Correlation Coefficient First Order

Kinetic/Diffusion Exponent ‘n’

Correlation Coefficient, (Higuchi model)

MF1

0.989

0.0091

0.6446

0.9897

MF2

0.983

0.004

0.5074

0.9832

MF3

0.975

0.015

0.6435

0.9759

MF4

0.973

0.011

0.4286

0.973

MF5

0.961

0.002

0.3858

0.9618

MF6

0.936

0.004

0.3225

0.936

MF7

0.956

0.006

0.3942

0.956

MF8

0.937

0.001

0.3311

0.937

a

b

c

d

e

Figure 6 : DSC thermograms. (a-Drug, b-Coated Formulation, c-Uncoated Formulation, d-Polymer, e-Drug: Polymer Physical Mixture)

The adhesion properties of nanoparticles increased with increasing concentration of mucoadhesive polymer (Polycarbophil); among various concentrations of polycabophil, better mucoadhesion was observed for MF2 and MF6 formulations as (67.3 %) and (62.1%), respectively (Table 1) DSC gives information regarding the physical properties like crystalline or amorphous nature of the samples. The DSC thermogram of acyclovir (Fig 6a ) shows an exothermic peak at 267.03 corresponding to its melting temperature , which was not detected in the thermograms for Acyclovir loaded coated and uncoated nanoparticles of PLGA 50:50 (Fig 6b and 6c). It has been shown by a couple of authors that when the drug 102


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

does not show its exothermic peak in the formed nanoparticles, it is said to be in the amorphous state19. Hence it could be concluded that in both the prepared PLGA nanoaprticles (coated and uncoated), the drug was present A B in the amorphous phase and may have Figure 7 : SEM of PLGA Nanoparticles. (A- PLGA (Uncoated) , B-PLGA (coated) been homogeneously dispersed in the Scale bar =50 µm, Magnification:10,000× . PLGA matrix. PLGA nanoparticles were prepared by the solvent deposition method,and were characterized as ‘mucoadhesive’ by coating with mucoadhesive polymer, polyacrylic acid (Polycarbophill). The application of factorial design gave a statistically systematic approach for the formulation of nanoparticles with desired particle size, high entrapment efficiency and sustained drug release. Figure 8 : Average % Drug release of Mucoadhesive Factorial Formulations; Drug:polymer ratio and concentration of MF1(--), MF2 (-n-), MF3(- -), MF4(-×-), MF5(-|×-), MF6 (-l-), MF7(-|--), MF8(---) surfactant were found to influence the particle size and % drug release of acyclovir loaded PLGA mucoadhesive nanoparticles. In vitro drug release study of all formulations (MF1 to MF8) showed 57.71% to78.31% drug release in 32 h. The release was found to follow fickian as well as non Fickian diffusion mechanism with almost zero order drug release for all batches. In vitro intestinal mucoadhesion of nanoparticles showed that the adhesion properties of nanoparticles increased with increasing concentration of mucoadhesive polymer (Polycarbophil). These preliminary results indicate that acyclovir loaded mucoadhesive PLGA nanoparticles could be effective in sustaining drug release for a prolonged period. Further studies are needed to confirm its performance in vivo. REFERENCES

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Tran T, Druce JD, Catton MC, Kelly H, Birch CJ. Changing epidemiology of genital herpes simplex virus infection in Melbourne, Australia, between 1980 and 2003. Sex Transm Infect 2004;80:277-79. Emmert DH. Treatment of common cutaneous Herpes Simplex Virus Infections. Am Fam Physician 2000;61:1697704. Wagstaff AG, Faulds D, Goa KL. Aciclovir: a reappraisal of its antiviral activity, pharmacokinetic properties and therapeutic efficacy. Drugs 1994;47:153–205. Ruhnese M, Sandstorm F, Andersson B. Treatment of recurrent genital herpes simplex infection with acyclovir. J Antimicrob Chemother 1985;16:621–28. Fuertes I, Miranda A, Millan M, Caraballo I. Estimation of the percolation thersholds in acyclovir hydrophilic matrix tablets. Eur J Pharm Biopharm 2006;64:336–42. Jalon De EG, Blanco-Prieto MJ, Ygartua P, Santoyo S. Increased efficacy of acyclovir-loaded microparticles against herpes simplex virus type 1 in cell culture. Eur J Pharm Biopharm 2003;56:183–87. Rossi S, Sandri G, Ferrari F, Bonferoni MC, Caramella C. Buccal delivery of acyclovir from films based on chitosan and polyacrylic acid. Pharm Dev Technol 2003; 8:199–208.

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O’Brien JJ, Campoli-Richards DM. Acyclovir: an updated review of its antiviral activity, pharmacokinetic properties and therapeutic efficacy. Drugs 1989;37:233–309.

9. Allemann E, Leroux JC, Gurny R. Polymeric nano- and microparticles for the oral delivery of peptides and peptidomimetics. Adv Drug Deliv Rev 1998;34:171Y189. 10. Smart, JD, KellawayIW, Worthington HEC. J Pharm Pharmacol 1984;36: 295. 11. Kamath KR, Park K. In Encyclopedia of Pharmaceutical Technology, Ed.; Marcel Dekker, New York ;1988,10, 133-64. 12. Fessi, H, Puisieux F, Devissaguet JP, Ammoury N, Benita S. Nanocapsules deposition by interfacial polymer deposition following solvent deplacement. Int J Pharm1989;55:R1–R4. 13.

Freitas MN ,. Marchetti JM. Nimesulide PLA microspheres as a potential sustained release system for the treatment of inflammatory diseases. Int J Pharma 2005;13: 201-11

14. Gohel M, Patel M, Amin A, Agarwal A, Dave R, Bariya N. Formulation design and optimization of mouth dissolve tablets of nimesulide using vacuum drying technique. AAPS Pharm Sci Tech 2004; 5(3):36 15. Suman Ramteke, Umamaheshwari RB, Jain N.K. Clarithromycin Based Oral Sustained Release Nanoparticulate System. Indian J Pharm Sciences.July-August 2006; 479-84 16. Ashish K, Mehta P, Yadav KS, Krutika KS. Nimodipine Loaded PLGA Nanoparticles: Formulation Optimization Using Factorial Design, Characterization and In Vitro Evaluation. Current Drug Delivery 2007; 4:185-93 17. Nixon JR. Release characterization of microcapsules. In F. Lim (Ed.), Biomedical Applications of Microcapsulation. Boca Raton, FL: CRC Press.,1983.. 18. Costa P, Lobo JM. Modeling and comparison of dissolution profiles. E J Pharm Sciences 2001;13:123–33 19. Ritger, PL, Peppas, NA. A simple equation for description of solute release : Fickian and non-Fickian release from nonswellable devices in the form of slabs, spheres, cylinders or discs. J Control Release1992;5:23–36.

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Quantification of Vinorelbine in Bulk and Injection Dosage Form by Spectrophotometric Method Pawan K. Saini1, Raman M. Singh1*, C. L. Jain2, Satish C. Mathur1, Gyanendra N. Singh1 1 Indian Pharmacopoeia Commission, Govt. of India, Sector-23, Rajnagar, Ghaziabad, India 2 Department of Chemistry, M.M.H. College, Ghaziabad, India

ABSTRACT A simple, fast, accurate, precise and economical spectrophotometric method has been developed and validated for the quantitation of the novel anticancer agent vinorelbine in bulk and injection dosage form. Identification was carried out using a UV wavelength at 270 nm in methanol. The method was validated with respect to its specificity, linearity range, accuracy, precision and recovery. The calibration curve was found linear between 1 to 50 μg/ml. The limit of detection and limit of quantification were found 0.0702 and 0.2127 μg/ml, respectively. Percentage recoveries were obtained in the range of 98.42 % and 99.69 %. Regression analysis showed good correlation in the concentration range of 1-50 μg/ml. The proposed method is accurate, precise, specific, reproducible, and fast for quantitation of the vinorelbine in bulk and injection dosage form. Keywords : Vinorelbine tartrate, Spectrophotometer, Method development and validation. INTRODUCTION Vinorelbine is chemically known as 3’,4’-didehydro-4’-de-oxy-c’-norvincalcukoblastine1. The structure of vinorelbine tartrate is given in Fig.12. Vinorelbine is a vinca alkaloid derivative which is marketed under the brand name Navelbine in many countries. It has activity reported against non small cell lung and advanced breast cancer3-7. This semisynthetic compound differs from other vinca alkaloids in structural modifications on the catharantine ring instead of the vidoline ring8. Vinorelbine, like other vinca alkaloids, blocks cell mitosis by interfering with microtubule assembly and by inducing depolymerization of the microtubules9. It have relatively low neurotoxicity as compared with other vinca alkaloids, due to its low affinity for axonal microtubules10. It has been widely employed in combination with cisplatin with or without 5-fluorouracil for the treatment of lung cancer and head/neck carcinomas11, 12. Vinorelbine tartrate and its injection is official only in Indian Pharmacopoeia and is assayed by HPLC method2. Literature survey reveals that few analytical methods were reported for the determination of vinorelbine by high performance liquid chromatography13, (HPLC) with electrospray tandem mass spectrometry 14 and HPLC/MS/MS 15. Though these methods are sensitive, they require expensive instruments and skilled personnel. Despite the availability of sophisticated and sensitive instruments, for routine quantitative analysis, a simple and cost effective analytical method is always preferred. In the present investigation efforts has been made to develop a fast, accurate and precise method for the analysis of vinorelbine in bulk Figure 1 : Chemical Structure of Vinorelbine tartrate and injection dosage form. MATERIALS AND METHODS Chemicals and Reagents Vinorelbine tartrate reference standard and Vinelbine (Vinorelbine 50 mg / 5 ml) injection were 105


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provided by Dabur India Ltd., Sahibabad, (UP). Methanol AR grade was purchased from E. Merck (India) Ltd., Mumbai. HPLC grade water was prepared using Millipore Purification System (Millipore, Molsheim, France, Model Elix-10). Instrumentation A single beam UV-Visible Spectrophotometer (Beckman DU 640B, U.S.A.) was used for analysis and acquisition of data. The instrument have an automatic wavelength accuracy of 0.1 nm and matched quartz cells of 10 mm path length. A Mettler balance (Toledo-AG204, Mettler, Switzerland) was used for weighing of samples. CAUTION – Vinorelbine tartrate is potentially cytotoxic. Great care has been taken in handling the powder and preparing solutions. Preparation of standard solutions Approximately 10 mg of vinorelbine tartrate reference standard was accurately weighed and transferred to a 100 ml volumetric flask. The volume was filled to the mark with methanol, to obtain a concentration of 100 µg/ml. The solution was further diluted to obtain a concentration in the range 1-50 µg/ml. Preparation of sample solution Approximately 10 mg of vinorelbine tartrate bulk was accurately weighed and transferred to a 100 ml volumetric flask and diluted to volume with methanol to obtain a concentration of 100 µg/ml. Further 3.0 ml of this solution was diluted to 10 ml with methanol to obtain a concentration of 30 µg/ml. For analysis of injection, an accurate volume 1.0 ml of Vinorelbine injection (50 mg /5 ml) was taken and transferred to a 100 ml volumetric flask and diluted to volume with methanol to obtain a concentration of 100 µg/ml. The resultant solution was filtered through 0.45 µm nylon filter. Further 3.0 ml of the filtrate was diluted to 10 ml with methanol to obtain a concentration of 30 µg/ml. METHOD VALIDATION Specificity The specificity of an analytical method may be defined as the ability to detect the analyte in the presence of the analyte by-products, or other inactive components, such as dosage form excipients or impurities. This study is performed to demonstrate, non interference from degradation products that are formed during acid stress, base stress and oxidative stress, on the test sample. The method will be stability indicating, if the degradation products do not interfere with the analyte. Accuracy and Precision Precision of the assay was determined by repeatability (intraday) and intermediate precision (inter-day) for 3 consecutive days. Three different concentrations of vinorelbine were analyzed in six independent series in the same day (intra-day precision) and 3 consecutive days (inter-day precision). Every sample was determined in triplicate. The accuracy of the method, which is defined as the nearness of the true value and found value, was evaluated by the following equation: %accuracy = observed concentration / nominal concentration × 100 Linearity The calibration curve was constructed with concentrations (simultaneously prepared) ranging from 106


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1 to 50 µg/ml in methanol. Calibration curves were constructed by plotting the ratio of the mean absorbance versus the concentration. The linearity was assessed by linear regression analysis, which was calculated by the least square method. Limit of Detection and Limit of Quantification Limit of detection and limit of quantitation was calculated by the method based on the standard deviation (SD) of the response and the slope (S) of the calibration curve at levels approximating the LOD and LOQ, LOD = 3.3 (SD/S) and LOQ = 10 (SD/S). Recovery The pre-analyzed samples were spiked with extra 80, 100 and 120 % of the standard vinorelbine and analyzed by the proposed method. The experiment was conducted in triplicate. This was done to check the recovery of the drug at different levels in the formulations. Assay procedure Drug contents were calculated by comparison with the appropriate standard solution of the drug. No interferences due to excipients was observed in the spectra or chromatograms produced. RESULT AND DISCUSSION Analytical method development Different medium were investigated to develop a suitable UV-Visible spectrophotometric method for the analysis of vinorelbine. For selection of medium the criteria employed was sensitivity of the method, ease of sample preparation, solubility of the drug, cost of solvents and applicability of method to various purposes. An optimum absorbance was found at 270 nm of 30 µg/ml solution of vinorelbine in methanol (fig. 2).

Figure 2 : UV- Spectra for identification of vinorelbine

Validation of the proposed method The method was validated with respect to the following parameters given below as per ICH guidelines15:

Figure 3 : Linearity graph of vinorelbine (1–50 µg/ml)

Figure 4 : Linearity curve of vinorelbine

Calibration Curve The linearity of the calibration curves for vinorelbine was calculated and constructed by least square regression method as illustrated previously. The correlation coefficient (r2) for the standard calibration curves for vinorelbine was 0.9997. This indicates linearity of the absorbance in the range of 1–50 µg/ml (Fig 3 & 4). Accuracy and precision The data of accuracy and precision during the intra and inter-day run were found within the acceptance criteria (i.e. %RSD is <2). Inter and intra-day accuracy (expressed as %RSD) ranged from 0.55 to 1.93 for vinorelbine. 107


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Specificity and selectivity In order to confirm the specificity of the method for vinorelbine in the presence of excipients, three solutions of the injection were analysed. As discussed above, the specificity of an UV method is the ability to detect the analytes in the presence of other ingredients. Absorbance was performed under the experimental conditions stated previously. The excipients present in the sample did not exhibit any absorbance, and therefore no interferences were detected. Recovery The proposed method when used for extraction and subsequent estimation of vinorelbine in injection dosage form after adding 80, 100 and 120 % of additional drug afforded recovery of 98-102% as listed in Table 1. Table-1 : Result of analysis of bulk, injection and recovery studies Drug

Found %

Vinorelbine bulk

Amount Added (mg)

99.2

Vinorelbine Injection (10 mg/ ml)

Amount Recovered (mg)

-

98.0

-

% Recovery -

8

7.9

98.75

10

9.8

98.00

12

11.8

98.33

Validation of proposed method The UV spectrophotometeric method was validated using following parameters given in table 2. Table-2 : Validation parameters of vinorelbine Parameters

Methanol

Range (µg/ml)

1- 50

Regression equation

y = 0.0195x - 0.0014

Correlation Coefficient (r2)

0.9997

L. O. D. (µg/ml)

0.0702

L. O. Q (µg/ml)

0.2127

CONCLUSION The proposed method of analysis is simple, fast and can be used for routine analysis of vinorelbine in bulk and pharmaceutical formulations. The sample recovery in the formulation were in good agreement with their label claim and thus suggested non-interference of excipients in estimation and at the same time use of organic solvent for extraction of vinorelbine from formulation was avoided. The recommended procedure is well suited for the assay and evaluation of drugs in pharmaceutical preparations to assure high standards of quality control. ACKNOWLEDGEMENTS The authors are thankful to Dabur Pharma Ltd., Ghaziabad for providing standard and sample of vinorelbine. 108


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REFERENCES

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The Merck Index. 13th ed. NJ. Merck Research Laboratories Publishers. 2001. Page no. 1780.

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Anonymous, Indian Pharmacopoeia 2007, fifth edition, Vol. II, The Indian Pharmacopoeia Commission, Ghaziabad. 2007; Page no: 1241-1244.

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Armand, J. P.; Marty, M. Navelbine: a new step in cancer therapy. Semin Oncol 1989, 16(4), 41-45.

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Budman, D. R. New vinca alkaloids and related compounds. Semin Oncol 1992, 19, 639-645.

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Cvitkovic, E.; Izzo, J. The current and future place of vinorelbine in cancer therapy. Drugs. 1992, 44(4), 36-45.

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Sorensen, J. B.; Vinorelbine. A review of its antitumor activity in lung cancer. Drugs. 1992, 44(4), 60-65.

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Spielmann, M.; Dorval, T.; Turpin, F. et. al. Phase II trial of vinorelbine/ doxorubicine as first-line therapy of advanced breast cancer. Journal of Clinical Oncology, 1994, 12, 1764-1770.

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Potier, P. The synthesis of navelbine, prototype of a new series of vinblastine derivatives. Semin Oncol 1989, 16(4), 2-4.

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Zhou, X. J.; Rahmani R. Preclinical and clinical pharmacology of vinca alkaloids. Drugs. 1992, 44(4), 1-16.

10. Binet, S.; Chaineau, E.; Fellous A. Immunofluorescence study of navelbine, vincristine and vinblastine on mitotic and axonal microtubules. International Journal of Cancer. 1990, 46, 262-266. 11. Kohno, E.; Murase, S.; Nishikata, M.; Okamura, N.; Matzno, S.; Kuwahara, T.; Matsuyama, T. Methods of preventing vinorelbine-induced phlebitis: an experimental study in rabbits Int. J. Med. Sci. 2008, 5, 218-223. 12. Estevez, M. D.; Vieytes, M. R.; Louzao, M. C.; Alfonso, A.; Vilarino, N.; Botana, L. M. The antineoplastic drug vinorelbine activates non-immunological histamine release from rat mast cells. Inflamm. Res. 1997, 46, 119- 124. 13. Puozzo, C.; Ung, H.L.; Zorza, G. A high performance liquid chromatography method for vinorelbine and 4-O-deacetyl vinorelbine: A decade of routine analysis in human blood. J. Pharm. Biomed. Anal. 2007, 44, 144-149. 14. Damen, C. W. N.; Rosing, H.; Tibben, M. M.; Maanen, M. J.; Lagas, J. S.; Schinkel, A. H. et al. A sensitive assay for the quantitative analysis of vinorelbine in mouse and human EDTA plasma by high performance liquid chromatography coupled with electrospray tandem mass spectrometry. Journal of Chromatography B, 2008, 868, 102- 109. 15. de Graeve, J.; van Heugen, J. C.; Zorza, G.; Fahy, J.; Puozzo, C. Metabolism pathway of vinorelbine ( Navelbine) in human: Characterisation of the metabolites by HPLC-MS/MS. J. Pharm. Biomed. Anal. 2008, 47, 47-58. 16. Validation of Analytical Procedures, Methodology, ICH harmonized tripartite guidelines, 1996, 1.

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Reporting of Adverse Drug Reactions by Consumers : Rationale and Potential Hanumantha Rao Potharaju Administrative Staff College of India, Hyderabad. India

ABSTRACT To assess the feasibility of involving consumers in reporting and monitoring adverse drugs reactions (ADR) in India, a household survey was carried out in three states of India, namely Andhra Pradesh, Maharashtra and Uttar Pradesh using a pre-tested questionnaire. Responses were obtained from a total of 566 households in three states together. Also elicited were the opinions of other stakeholders such as (a) hospitals and nursing homes, (b) private medical practitioners, (c) chemists and (d) pharmacovigilance centres with regard to involving household in the reporting of ADRs. A huge 93 per cent of the households are willing to report ADRs. Other stakeholders are also in favour of involving consumers in the reporting of ADRs and believe that it is a good idea.It is concluded that in cases where an ADR is likely to occur at, an ADR form may be given to the consumer at the time of prescribing by doctors or of dispensing by chemists. A prepaid system of providing an inland letter/envelop is a feasible option for encouraging consumers to report the ADRs experienced by them. INTRODUCTION National pharmacovigilance programmes monitor Adverse Drug Reactions (ADRs) and help to improve the safety of medicines prescribed. Under-reporting is a major concern in national pharmacovigilance programmes, especially those dependent on spontaneous reporting1,2. Patients consume medicines at home. Advertising promotes the tendency to self-medicate, which might lead to inappropriate use of medicines and ADRs. In principle, patients/consumers have a vested interest in reporting ADRs. Hence, consumers/patients can be an important source of such reporting3. Patients in the United States were the first to get an opportunity to report ADRs directly to the Food and Drug Administration (FDA) in the 1960s. In April 2003, Dutch patients began to report possible ADRs to LAREB, a foundation separate from the country’s national drug regulatory authority. Denmark allowed patients or relatives to report ADRs from June 2003. In Italy, patients have been able to download a special form to report ADRs to the AIFA (Italian Drug Regulatory Agency) since 2004. A consumer organization, Test-Achats/Test-Aankoop (TA), was established in Belgium in 2006 to accept reports from patients and transfer them to the Federal Agency for Medicines and Health Products (FAMHP). Medicines and Health Related Products Regulatory Agency (MHRA) in the UK made substantial efforts in February 2008 to raise awareness so as to increase the number of reports from patients. The website of Swedish Medical Products Agency (MPA) added an interactive section to enable patients and consumers to report ADRs in June 2008. Norwegian Medicines Agency started accepting electronic reports directly from patients since March 20103. The consumer-focused reporting service in Australia-Adverse Medicines Events Line (AMEL), a telephone hotline was begun in October 20034. KILEN, run by a consumer group in Sweden, has been receiving reports from patients since 1978 and also provides feedback to those submitting reports5. OBJECTIVE India launched the National Pharmacovigilance Programme (NPVP) in April 2004 based on spontaneous reporting. The Government of India was keen to explore different options for improving 110


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the reporting and monitoring of ADRs under NPVP6. The objective of the present study was to assess the feasibility of involving patients/consumers in reporting and monitoring of ADRs in India. Besides consumers (households), a number of other stakeholders were also considered for assessing the options for improving the reporting and monitoring of ADRs under NPVP. METHODS Three states in India, namely Andhra Pradesh, Maharashtra and Uttar Pradesh were selected for the study. In each state, three districts were selected at random. A total of 566 households were interviewed between February and June 2006. About 42% of the households are from urban areas, and 58% are from rural areas. About 73% of the respondents are male. About 21% per cent had studied up to 12 the class and about 29% studied beyond. About 22% are daily wage earners, 24% are in service and 15% are in business. The monthly income of about 30% is below Rs. 2000, and about 56% are in the income group of Rs 2,000-10,000. RESULTS Household Perspective 他 of households had at least one member taking medicines during the past three-month period from the date of survey. Awareness about NPVP among the households in India is less than 2%. About 50% of the respondents rated the problem of ADRs as very important and 45 per cent as important on a five point Likert type scale. The vast majority (93%) mentioned that they are willing to report ADRs. About 84% of the households wanted the ADR forms to be available at hospitals/nursing homes and 6% preferred chemists. While 7% mentioned that they require help in filling up the forms, 19% stated that they could do it on their own (the non-response rate was very high for this question.) About 57% of the respondents preferred hospitals for sending in the filled-up forms, while 22% preferred doctors and 4% preferred chemists. The most preferred mode for sending in the filled-up form is by post (45%), while about 38% preferred delivering it by hand. About 43% suggested the need for creating mass awareness through advertisement in different media and about 21% expressed the need for educating the patient. About 10% mentioned that the ADR forms should be in the local language. Opinion of Other Stakeholders Majority of the other stakeholders contacted in this study are also in favour of involving patients/ consumers in the reporting of ADRs. About 66% out of 148 hospitals and nursing homes have opined that it is a good idea to give the ADR form to the patients in OPD or to the inpatients at the time of discharge for select drugs and ask them to send in the filled-up form in case they experience ADRs. About three fourths of the 172 Private Medical Practitioners (PMPs) contacted for the study are in favour of giving ADR forms to patients coming to them (for a select list of drugs) and asking them to send in the filled-up ADR forms to them or to a designated centre. About 54% out of 189 chemists, felt that it is good idea to give an ADR form to the patients at the time of purchase of selective drugs. Even the pharmacovigilance centres participated in the study were favourably inclined towards the involvement of consumers/patients in ADR reporting. Eleven out of the sixteen peripheral centres and two out of the three regional centres, who responded, supported involvement of consumers in ADR reporting. All the stakeholders emphasized the need for generating awareness using mass media and other appropriate means, among common citizens as well as different stakeholders, before the consumers can be involved in the reporting of ADRs. 111


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DISCUSSION ADR reports have several advantages. They have value as a quantitative indicator of quality and safety7. Patient reports contain data on personal and social consequences8. Medawar9 found individual patient reports much richer in their descriptions of behavioural phenomena and feelings compared to the Yellow Card reports submitted by professionals in the UK. He concluded that though individually such reports may be deficient or exaggerated and sometimes wrong, collectively they reflect good common sense. O’Brien10 found the information on ADRS to be analytical. Jarernsiripornkul11, observed that patient perceptions of potential ADRs provides useful information but GPs do not report all the symptoms told to them by patients. Hence, he recommended that they should be an integral part of any pain management strategy12. A study of users of two popular nonsteroidal anti-inflammatory drugs (NSAIDs) in Australia13 revealed reactions that often evade detection during pre-marketing clinical trials. Reviewing published literature, Blenkinsopp14 observed that reports by patients identified possible new ADRs that had not previously been reported by health professionals. Campbell and Howie15 reported that the patients, who were prescribed a black triangle drug (a new drug put under intensive surveillance by MHRA in UK) increased from 10 per 1000 to 23 per 1000 in two months. A study of 650 adults with self-reported ADRs to statins in the US revealed that it is mostly patients who initiate the discussion with their physicians and concluded that targeting patients is likely to boost the yield of ADR reporting systems16. Disproportionality analysis of two data sets of data on ADRs, namely (a) only form health care providers and (b) from health care providers as well as consumers, by Glaxo Smith Kline in the US, found that in 52.2% of events, the signal was identified earlier when consumer reports were included in the data17. A Dutch study18 mentions that patients reported new suspected reactions to paroxetine (an antidepressant) on an average, 273 days before doctors reported the same reaction. Partnering with in-patients is a promising strategy to prevent adverse drug events. In a prospective study involving 107 inpatients of a teaching hospital in a Boston, 29% of the nurses indicated that at least one medication error was prevented when a patient or family member identified a problem19. However, Lampela20 found a great disparity between the adverse effects identified by the physician and those reported by a group of elderly patients in Finland. It may be because the elderly people tend to neglect adverse drug effects and may consider them to be an unavoidable part of normal ageing. The 2004 European law regarding medicinal products (726/2004/EC Article 22) states that “Patients shall be encouraged to communicate any adverse reaction to health-care professionals.� Aspden21 presented various scenarios (a) clinicians provide appropriate instructions and encouragement to patients for reporting adverse side effects, (b) reporting systems with multiple options capture reports of medication errors from patients and families, and (c) resources to address complications from prescriptions are available around the clock. A Working Group on Patient in the UK proposed methods such as use of posters in outlets that stock patient Yellow Cards, use of advertising, feedback for people and promotion through the media, for reporting of ADRs. It is also necessary that sufficient resources are made available for this purpose22. CONCLUSIONS All over the world there is an increasing trend of involving consumers in the process of health care. Consumer reporting has several advantages like qualitative details; increase in ADRs reported, newer 112


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ADRs being reported, early detection of ADRs and also as a strategy to prevent medication errors. Moreover allowing patients to report demonstrates a necessary attitudinal change towards showing greater respect to those experiencing illness and taking medicines6. None of the countries with patient reporting systems has reported poor quality of patient reports to be an issue23. Recent reports from developing countries also are in favour of consumer reporting of ADRs. Fernandopulle24 has suggested that an independent consumer reporting system complements the present health professional-based system in Sri Lanka. Gunawardena25 used pharmacovigilance based on consumer feedback in Sri Lanka. Ahmed26 emphasized the need for involving consumers in the existing pharmacovigilance programme in Malaysia and Palaian27 suggested a novel approach. The present study suggests a favourable picture on the involvement of consumers in reporting ADRs. Though India is experiencing an IT revolution, many respondents preferred to use the hard copy of the ADR form for filling-up and sending in. This is not surprising as the level of penetration of computers and the Internet at the household level is very low. Hardly 3% of the sample households contacted in the study have access to computer and 2% to the Internet. Hence, a prepaid postage system, inland letter/envelop, may be developed to encourage consumers to report the ADRs directly to NPVP or to the nearby hospital or chemist from which they regularly obtain health care services. Prepaid postage forms are being used for ADR reporting by consumers in Canada, UK, USA and Australia. There should be a wide-spread media campaign on the importance and means of reporting ADRs by consumers, supported by patient education. van Hunsel28 reported that media attention affects drug use and ADR reporting by patients in Netherlands. However, there is a need for piloting before consumer reporting can be made an integral part of the National Pharmacovigilance Programme in India. LAREB in Netherlands piloted it for one year between April 2003 and March 2004 before it decided to continue the reporting station for patients29. It will be useful to understand: (a) What motivates a person to take the trouble to report an ADR? (b) How to create an enabling environment to motivate them? (c) How to make the ADR reporting forms easily accessible to people? (d) What kind of support systems are to be put in place for effective involvement of consumers in ADR reporting and monitoring? ACKNOWLEDGEMENTS The findings presented in this paper are a part of the study on ‘Post-marketing Surveillance and Monitoring Averse Drug Reactions” prepared for the Ministry of Health and Family Welfare, Government of India, under the Capacity Building Project completed by ASCI in 2008 led by Dr. P. H. Rao. The author Mr. A.N. Raju, Mr. Coyalkar Mohandas and R. Krishna Murthy for collection of data. REFERENCES

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Institute of Medicine. Adverse Drug Event Reporting: The Roles of Consumers and Health-Care Professionals, Workshop Summary. Washington, DC: The National Academies Press 2007. pp. 26-33. Consumer reporting of adverse drug reactions. WHO Drug Information 2000; 14(4): 211-215. Herxheimer A, Crombag R. Direct Patient Reporting of Adverse Drug Reactions A Fifteen-Country Survey & Literature review. Paper Series Reference 01-2010/05 Health Action International (Europe) Amsterdam. Netherlands, May 2010. Graham JD. Magic number. The Australian Health Consumer. 2003–4; 2: 32–4. Health Action International. Patients’ reporting of adverse reactions. Outcomes of a seminar organised by Health Action International Europe. Netherlands, 26 May 2005.

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6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

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Options for Post Marketing Surveillance and Monitoring Adverse Drug Reactions. Administrative Staff College of India, Hyderabad, India. 2008. Agoritsas T, Bovier PA, Perneger TV. Patient reports of undesirable events during hospitalization. J Gen Intern Med 2005; 20(10): 922–928. Medawar C, Herxheimer A, Bell A Jofre S. Paroxetine, Panorama and user reporting of ADRs: consumer intelligence matters in clinical practice and post-marketing drug surveillance. Int J Risk Saf Med 2002; 15: 161–169. Medawar C, Herxheimer A. A comparison of ADR reports from professionals and users, relating to risk of dependence and suicidal behaviour with paroxetine. Int J Risk Saf Med 2003; 16: 5–19. O’Brien MC, Yearwood JL. Decisions Surrounding Adverse Drug Reaction Prescribing: Insights from Consumers and Implications for Decision Support. Journal of Research and Practice in Information Technology 2005; 37(1): 57-71. Jarernsiripornkul N, Krska J, Capps PAG Richards RME, Lee A. Patient reporting of potential adverse drug reactions: a methodological study. Br J Clin Pharmacol 2002; 53: 318-325. Jarernsiripornkul N, Krska J, Richards RME, Capps PAG. Patient reporting of adverse drug reactions: useful information for pain management, Eur J Pain 2003; 7(3): 219–224. Mitchell AS, Henry DA, Hennrikus D, O’Connell DL. Adverse drug reactions: can consumers provide early warning? Pharmacoepidemiol Drug Saf 1994; 3: 257–264. Blenkinsopp A, Wilkie P, Wang M, Routledge PA. Patient reporting of suspected adverse drug reactions: Br J Clin Pharmacol 2007; 63(2): 148 – 156. Campbell JRM, Howie JGR. Involving the patient in reporting adverse drug reactions. J R Coll Gen Pract 1988; 38: 370-371. Golomb BA, McGraw JJ, Evans MA, Dimsdale JE. Physician response to patient reports of adverse drug effects: implications for patient-targeted adverse effect surveillance. Drug Saf 2007; 30(8): 669-75. Hammond IW, Rich DS, Gibbs TG. Effect of consumer reporting on signal detection: using disproportionality analysis. Expert Opin. Drug Saf 2007; 6(6): 705-12. Egberts TCG, Smulders M, De Koning FHP, Meyboom RHB, Leufkens HGM. Can adverse drug reactions be detected earlier, BMJ 1996; 313: 530-1. Weingart SN, Toth M, Eneman J, Aronson MD, Sands DZ, Ship AN, Lessons from a patient partnership intervention to prevent adverse drug events. Int J Qual Health Care 2004; 16(6): 499–507. Lampela P, Hartikainen S, Sulkava R, Huupponen R. Adverse drug effects in elderly people -- a disparity between clinical examination and adverse effects self-reported by the patient. Eur J Clin Pharmacol 2007; 63(5): 509-15. Aspden P, Wolcott J, Bootman JL, Cronenwett LR. Editors. Part II. Moving toward a patient-centered integrated medication-use system in Preventing medication errors: quality chasm series. Committee on identifying and preventing medication errors, 2007, pp: 143-148. Medicines and Healthcare Products Regulatory Agency. Patient Reporting of Suspected Adverse Drug Reactions Working Group. Committee on Safety of Medicine, Post Licensing Division. UK, 2005. Blenkinsopp, A., Wilkie, P., Wang, M., Patient reporting of suspected adverse drug reactions: A review of published literature and International experience. Br J Clin Pharmacol, 2007, 63:2 148–156. Fernandopulle RB, Weerasuriya K. What can consumer adverse drug reaction reporting add to existing health professional-based systems? Focus on the developing world. Drug Saf 2003; 26(4): 219-25. Gunawardena, S., Ranganathan, S.S., Fernandopulle R. Pharmacovigilance through consumer feedback (reporting) in the mass treatment of lymphatic filariasis using diethylcarbamazine and albendazole in two districts of Sri Lanka. Trop Med Int Health 2008; 13(9): 1153–1158. Ahmed AM, Izham IM, Subish P. Importance of consumer pharmacovigilance system in developing countries: a case of Malaysia. JCDR 2010; 4:2929-2935. Palaian, S. Alshakka, M. Izham, M. (2010). Developing a consumer reporting program in Malaysia: a novel initiative to improve pharmacovigilance. Pharm World Sci 32:2–6. DOI 10.1007/s11096-009-9342-8. van Hunsel F, Passier A, van Grootheest K. Comparing patients’ and healthcare professionals’ ADR reports after media attention: Br J Clin Pharmacol 2009; 67(5): 558–564. van Grootheest AC, Passier JL, van Puijenbroek EP. Direct reporting of side effects by the patient: favourable experience in the first year. Ned Tijdschr Geneeskd 2005; 149: 529-33.

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Niosomes : A Potential Drug Delivery System D. Nagasamy Venkatesh1*, Anindita De1, Vinay Valecha2 1 JSS College of Pharmacy, Ooty 2 B. S. Anangpuria Institute of Pharmacy, Alampur, Faridabad

ABSTRACT The vesicles formed as a result of admixture of a non-ionic surfactant with cholesterol followed by subsequent hydration are termed as niosomes. The method of preparation of niosomes is similar to liposome technology. However, niosomes exhibit more chemical stability over liposomes (a phospholipid vesicle), as non-ionic surfactants are found to be more stable over phospholipids. Commonly used nonionic surfactants in the formulation of niosomes are polyglyceryl alkyl ether, crown ether, polyoxyethylene alkyl ether, ester lined surfactants, spans and tweens. The formation of niosomes is governed by various process variables which include nature of surfactants, nature of drug and presence of charge inducers. The present review highlights the theoretical concept in niosomal formulation, fabrication techniques of niosomes, and their characterization with therapeutic applications. INTRODUCTION In the period of novel drug delivery system (NDDS), a considerable importance has been devoted on the spatial placement of drugs in chronic conditions. Novel drug delivery system appears to be a promising drug delivery of anticancer and anti-infective agents. They are able to target the drug at the site and provide better drug utilization and therapeutic efficacy. Among the different approaches used for delivering these drugs including liposomes, microspheres, nanospheres, microcapsules and niosomes have received a considerable attention. Niosomes (or) non-ionic surfactants vesicles are microscopic lamellar structures formed as a result of hydrating mixture of cholesterol and non-ionic surfactants1-2 (figure 1). These are formed by self-assembly of non-ionic surfactants in an aqueous media as a spherical, unilamellar system process is rarely spontaneous and requires energy through physical agitation, extrusion or heat3-6. Niosomes and liposomes are equiactive in delivering drug potential and increase the efficacy of drug as compared to free drug. However, niosomes are preferred over liposomes due their high chemical stability and economy7. Encapsulation of drugs into niosomes was similar like liposomes. The former Figure 1 : Niosomes – ‘O’ is osmotically active and increases the stability of entrapped drug. They repres e n ts th e h y d r o p hi l i c head group and ‘–’ hydrophobic tail offer special properties such as handling and storage and do not require any special conditions. Niosomes can entrap both lipophilic and hydrophilic drug moieties and exhibit low toxicity due to their non-ionic property. They improve oral bioavailability of poorly absorbable drugs and enhance the skin permeation of drugs. Due to their flexibility in their structural characterization such as composition, fluidity and size, it can be designed according to therapeutic needs. It improves the performance of drug by enhancing the better availability, and controlled delivery at particular area. Salient features of niosomes over liposomes 1. Niosomes are capable of entrapping drugs similar to liposomes. 2. Their osmotically active nature renders better stability of the entrapped drugs. 115


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Table-1 : Characterization of niosomes S. No.

Parameters

Instruments used

Vesicle size determination and size distribution

a. Malvern master sizer b. Optical microscopy c. Laser diffraction particle size analyzer d. Coulter submicron size analyzer e. Anderson cascade impactor f. Photon correlation spectroscopy

2.

Shape and morphological characterization

a. Polarized light microscopy b. Transmission electron microscopy c. Scanning electron microscopy d. Phase contrast microscopy e. Freez fracture microscopy f. Single angle X-ray diffraction.

3.

Zeta potential and surface charge

a. High performance capillary electrophoresis b. Malvern zeta sizer

4.

Thermal behavior and shape transition

a. Differential scanning calorimetry b. Differential thermal analysis

5.

Lamellarity

a. Optical microscopy b. Transmission electron microscopy

6.

Rheology

a. Ostwald U tube method b. Low shear rheo analyzer

1.

3. The infrastructure of the niosomes consists of both hydrophilic and hydrophobic moieties held together and as a result it can accommodate wide range solubility drugs. 4. Niosomes offer flexibility in their structural characteristics in terms of composition, fluidity, size and it can be fabricated according to desired target area. 5. They offer better or improved oral bioavailability of poorly absorbed drugs and also enhance the penetration of drugs through the skin. 6. They provide controlled delivery of drug at the desired site. 7. Niosomes alters the in vivo behavior of drug by allowing them to attach at the hydrophilic group and also by incorporating hydrophilic moieties in their bilayer. 8. They do not require any special conditions towards the handling and storage conditions. 9. The surfactants used in the formulation of niosomes are biodegradable, biocompatible, nonimmunogenic, stable, lower in cost and as well as less storage problem. 10. The niosomal delivery improves the therapeutic performance of the drug owing to delayed clearance from the circulation, safe guarding the drug from an undesirable biological environment and offering desirable effects to target site. 11. They can be conveniently administered by oral, parental and topical routes. METHODS OF PREPARATION OF NIOSOMES Niosomes exhibit significant difference in their properties depending on the method of preparation and composition of its bilayer. The method of preparation is similar to that of liposomal technology involving the dissolution of cholesterol, non-ionic surfactants and drug in a suitable organic solvent 116


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followed by drying. The thin film was then subjected to hydration with an aqueous phase to obtain niosomal suspension at a critical temperature. Generally the hydration temperature employed for the niosomes should be more than the phase temperature of non-ionic surfactants used. The various techniques involved in the preparation of niosomes are as follows : a) Hand shaking/lipid film formation Surfactant/lipid mixture is dissolved in an organic solvent usually in chloroform or diethyl ether. The organic solvent is further removed by using a rotary flask evaporator under a reduced pressure leads to the formation of surfactant/lipid film. The dried surfactant film was further hydrated with an aqueous solution or buffer solution of drug at 50-60°C (usually above the phase transition temperature of surfactants) for a specified period of time with constant shaking8-11. b) Ether injection method This method employs injection of organic solvents containing surfactants and lipids into an aqueous solution of drug or material to be encapsulated using a syringe type infusion pump at a temperature of 55-65°C under a reduced pressure. The vaporization of ether results in the formation of vesicles. However, the diameter of the vesicles varies with the conditions employed12. Niosomes prepared using this method had shown to possess higher entrapment efficiency over hand shaken and sonicated vesicles. The vesicles formed by this method were approximately 3 fold smaller in their diameter as compared to hand shaken method and displayed a greater entrapment efficiency13-14. c) Reverse phase evaporation This method involves the removal of an organic solvent from oil in water (o/w) emulsion under evaporation. Initially an emulsion is formed as a result of admixture of two phases by bath sonication. The emulsion is dried in a rotary evaporator under reduced pressure leaving the dispersion of niosomes in an aqueous phase. In certain cases emulsion is further hydrated or homogenized to yield niosomes15-16. This method was beneficial in encapsulating large hydrophilic macromolecules. d) Sonication method An aqueous phase was added to the surfactant lipid mixture in an organic solvent using a probe or bath sonication maintained at 60° C for 3 min17-18. This method is useful in particle size reduction of multilamellar vesicles to unilamellar vesicle formed by hand shaking and ether injection methods. However, an increase in sonication time results in simultaneous reduction in vesicle diameter. e) Microfluidisation This method is used to prepare smaller MLV, employing a micro fluidizer to pump the fluid at 10000 psi through micro channels with two streams of fluid to collide at their right angles with an efficient transfer of energy. This process produces vesicles of uniform dimension, smaller size with reproducibility19. f) Transmembrane pH gradient method Cholesterol and surfactant are dissolved in an organic solvent (chloroform). The organic solvent is evaporated under reduced pressure leaving a thin film in a round bottom flask. The film was then hydrated at pH 4 with 300 mM citric acid using vortex mixing. The multilamellar vesicles (MLV’s) formed are frozen and thawed for 3 times followed by sonication. To this suspension, an aqueous solution of drug was added and vortexed. Further the pH of the suspension is raised from 7.0 to 7.2 followed by heating at 60° C for 10 min leads to the formation of niosomes20-21. 117


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g) Bubbling technique It is a unique method and consumes lesser time in the method of preparation of liposomes and niosomes without the usage of organic solvents. The homogenized mixture of cholesterol and surfactant are dispersed in to an aqueous phase maintained at 70°C, followed by mixing micellar solutions for 15 sec with high shear homogenizer followed by bubbling of nitrogen gas22-23. Factors governing vesicle size, entrapment efficiency and release characteristics Niosomes are similar to liposomes assume spherical shape and their structures can be determined by using electron microscopy, photon correlation microscopy, fluorescence microscopy and dynamic light scattering technique24-26, zeta sizer27, confocal laser scanning microscopy28 and poly dispersity study29. Diameter of the vesicles dependent upon the length of alkyl chain surfactant with longer alkyl chain produces larger size vesicles. An increase in the concentration of dicetyl phosphate alters the mean diameter of vesicles slightly. Presence of dicetyl phosphate in the formulation might be responsible for niosomes with diameter greater than 100 nm. Entrapment efficiency Entrapment efficiency is determined after the separation of the unentrapped drug from the entrapped drug by dialysis30, sucrose gradient centrifugation31, gel filtration32, size exclusion chromatography33 and ficoll floatation technique34 and the drug remained entrapped in niosomes is determined using disruption of vesicles using 0.1% triton or 50% n-propanol or 2.5% sodium lauryl sulfate and analyzing the resultant solution by a suitable analytical method35. The percentage drug entrapped is calculated using the formula,

Amount of drug entrapped % Entrapment efficiency = --------------------------------------- Ă— 100 Total amount of drug

Addition of PEG during the process of rehydration step leads to an increase in the encapsulation efficiency of drug. It has been reported by the researchers that the presence of a charge in the membrane structure can increase water uptake, increase in thickness and size with higher entrapment efficiency of lidocaine in niosome36. Positively charged stearylamine has been reported to increase encapsulation efficiency of anionic drug all-trans-retinoic acid37. Similarly, entrapment efficiency of adjuvant-DNA was reported to be more in negatively charged niosomes38. Comparing to Span 40, Span 60 exhibited more entrapment efficiency of primaquine in niosomes39. In addition, sorbitans (Span 80, 40) incorporated niosomes found to exhibit prolonged activity with highest entrapment efficacy. In certain cases the method of preparation of niosomes such as reverse evaporation method can also alters the drug entrapment efficiency over ether injection and film hydration methods. It was observed that the entrapment efficiency increased with an increase in cholesterol content due to increase in vesicle size and width of lipid bilayer. Niosomes prepared with Span 60 showed a maximum entrapment efficacy of methotrexate. In vitro drug release from niosomes The in vitro release study of drug from niosomes are monitored by various techniques including Franz diffusion cell, membrane diffusion technique, sigma dialysis membrane, cellophane dialyzing membrane etc., The niosomal suspension is placed in a 200 ml of buffer solution with constant shaking at 37° C. At a predetermined time interval, the buffer is analyzed for drug content by an appropriate analytical techniques40. In vitro permeation studies have been carried out using Wister rat skin, Hamster 118


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flank skin, new born pig skin etc. The niosomal suspension of primaquine exhibited slower release rate across the dialysis membrane over the free drug. Release kinetics In general the drug release from the niosomal suspension follows first order kinetics41. Moreover, the release of drug from niosomes exhibited a biphasic pattern of release. Inclusion of dicetyl phosphate into niosomes reduced the rate and extent of drug release and the effect being a concentration dependent. A linear regression analysis of release data indicates that the entrapped drug was released by diffusion controlled mechanism. Incorporation of niosomes into a structured gel vehicle showed a slower initial phase compared with niosomes alone probably due to the diffusion restriction imposed by polymeric network of the gel42-43. Niosomal formulation of acyclovir found to follow Higuchi’s diffusion. FACTORS AFFECTING NIOSOMAL FORMULATION Nature of surfactant The formation of niosomes is guided by the hydrophile-lipophile value of non-ionic surfactant. HLB value of surfactant influences the entrapment efficiency; a HLB value of 14-17 is not desirable for niosomes, but a value of 8.6 has higher entrapment efficiency, but HLB value from 8.6 to 1.7 decreases the entrapment efficiency44. The chain length and hydrophilic part of the non-ionic surfactant affect the entrapment efficiency and size of the niosomes. Generally, an increase in the chain length decreases the drug release. An increase in polysorbate 80 concentration decreased the size of the niosomes45. The entrapment efficiency of vesicles obtained from stearyl (C18) surfactants, such as Brij 72, glyceryl monostearate, Span 60 and Tween 61 were significantly higher than lauryl (C12) chain surfactants46-47. Brij 30, tetra glyceryl mono laurate and stearyl surfactant i.e Brij 92 were able to prolong the release of insulin in SGF and SIF48. Tween 40 exhibited a maximum drug release and maximum drug entrapment of ketoconazole49-50. The Tween series surfactants bearing long alkyl chains with hydrophilic moiety in combination with cholesterol at a ratio of 1:1 have exhibited a highest efficiency of water soluble drug. A combination of span20:tween60:tween 80 at a ratio of 1:1:1 exhibited an highest entrapment efficiency of water soluble drug. Span 60 entrapment of primaquine phosphate was significantly more51. Increase in the concentration of Tween decrease drug release with prolonged action of ketoconazole in topical application. Surfactant in plasmid DNA encapsulated niosomal formulation serve as a penetration enhancer by raising the fluidity and reducing the barrier property of stratum corneum52-53. Span 60 provided a higher ketorolac flux across the skin than those prepared with Tween 2054. Span 60 exhibited highest protection against proteolytic enzymes and good stability in presence of sodium deoxycholate55. Encapsulation of estradiol in niosomes with Spans exhibited higher values in terms of permeation in excised rat skin in vitro56. Cholesterol The incorporation of cholesterol into bilayer composition in niosomal suspension induces membrane stabilizing effect and decreases leakiness on the membrane. Increase in the cholesterol content in the vesicles did not affect the transdermal delivery57. Incorporation of cholesterol into bilayer increases the entrapment efficiency of 5,6-Carboxy florescein and reduced permeability by a factor of 1058. Conversely, a decrease in cholesterol content decreases the drug loading. Cholesterol incorporation delayed the drug release in vitro59-60. 119


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Structure of surfactants The geometry of vesicle to be formed is determined by the structure of surfactant which is directly related to the critical packing parameters61. This can be expressed using the following equation, CPP (Critical Packing Parameters) = V/lc × a0 Where, V = volume of hydrophobic group, lc = length of hydrophobic group, a0 = area of hydrophilic head group. Based on the value of CPP, the type of micellar structure formed can be ascertained. CPP < 0.5 leads to the formation of spherical micelles If 0.5 < CPP <1 formation of a bilayer micelles and CPP >1 formation of inverted miscelles. Composition of membrane A stable niosomal formulation can be prepared by addition of different addictives with surfactants and drugs. Niosomes with a different morphology, permeability characters and their stability properties can be modified by manipulating membrane characteristics by addictives62. A typical example of polyhedral niosomes formed from C16G2, retained its shape after addition of low amount of solulan C24 (Cholesteryl poly-24-oxy ethylene ether) that prevents aggregation due to stearic hinderance. The mean size of the niosomes is also influenced by its membrane composition, polyhedral niosomes formed as a result of C16G2:Solulan C24 in the ratio of (91:9 having vesicles higher in size (8.0 ± 0.03 mm) than spherical niosomes formed by C16G2: Cholesterol: Solulan C24 in the ratio of (49:49:2) resulted in smaller size niosomes (6.6 ± 0.2 mm). Incorporation of cholesterol molecule in the niosomal formulation provides rigidity to membrane and reduces the leakage of drug from niosomes63. Nature of drug The physicochemical property of the drug alters the charge and rigidity of bilayer. Drug interacts with surfactants resulting in a development of a charge that undergoes mutual repulsion between surfactant bilayers results in increase in vesicle structure. The aggregation of vesicles is prevented due to the presence of charge in bilayer64. Temperature of hydration Hydration temperature influences shape and size of vesicles. Ideally, it should be above the gel to liquid phase transition temperature of system. Temperature alters the change in niosomal system by affecting the assembly of surfactants into vesicles and shape transformation. Polyhedral vesicles formed by C16G2: solulan C24 (91:9) at 25° C, which on heating undergone spherical vesicle at 48° C, but on cooling produced a cluster of smaller vesicle before changing to polyhedral structures at 35°C. Conversely, vesicle formed by C16G2: cholesterol: solulan C24 in the ratio of (49:49:2) showed no change in shape transformation on heating or cooling. The volume of hydration medium and the time of hydration are the critical facts in determining the shape and size. Improper selection of parameters resulting in the formation of fragile niosomes or drug leakage problems65. Osmotic effect Addition of a hypertonic salt solution to niosomal formulation causes a reduction in the vesicle diameter with concomitant water efflux may be due to pumping out of vesicles. While in case of hypotonic 120


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salt solution a slow release of drug with slight swelling probably due to inhibition of eluting fluid from vesicle and followed by faster release of drug which may be due to mechanical loosening of vesicles under the osmotic stress66. Modified Niosomes Aspasomes Addition of ascorbyl palmitate in combination with cholesterol and a negative charge induce dicetyl phosphate forms aspasomes. Generally, a film hydration method was used for the preparation of aspasomes followed by sonication. Typically cholesterol at a concentration of 45% shows a maximum retardation in drug release. The cholesterol content in the aspasomes has lesser effect on the vesicle size as well as entrapment efficiency and release rate of azidothymidine. Aspasome have an inherent antioxidant property and significantly prevents the disorder caused by reactive oxygen molecules. The transdermal permeation of aspasomes is significantly higher than aqueous solution of drug67. Discomes They are disc shaped structures formed on mixing of solulans (cholesteryl polyoxyethylene) in vesicular dispersion. Discomes are formulated by hand shaking or sonication method, where a mixture of diglyceryl ether, cholesterol and diacetyl phosphate (69:29:2) and followed by incubation with solulan C24 at 74°C. Discomes entrap hydrophilic solute, an example of 5(6)-carboxyfluorescein entrapment in discomes was 3.603 ¹ 2.916% with a release rate of 56% after 24 hours at room temperature68. Therapeutic applications of niosomes Methotrexate Vesicles with larger in size increased the terminal half-life of methotrexate due to increase in the circulation through fenestrated vessels. An increase in mean retention time of methotrexate indicated a sustained drug release69. Niosomes prepared with span 60 showed maximum entrapment efficacy and better pharmacokinetics in mice70. Niosomes containing methotrexate exhibited prolonged blood levels with larger liver uptake and increase brain penetration. The metabolic profile of drug was altered by the niosomal formulation71. The elimination of methotrexate in plasma of mice bearing S 180 tumor was slower in niosomal suspension. A noticeable increase in area under methotrexate concentration time curve and mean residence time of methotrexate in niosomal formulation with a decrease in apparent volume of drug distribution72. A maximum concentration of drug was achieved in lymph by niosomal formulation (55.6%) through intraperitoneal administration, whereas it was only a 6.2% of the dose by intravenous route in a rat model. The niosomal formulations of methotrexate exhibited longer circulation time73. Adriamycin Niosomal adriamycin delayed tumor growth in mice as compared to free drug and offered less chance of cardiac damage74. Cytarabine The antitumor activity of cytarabine in mice was enhanced after encapsulation into niosomes75. Doxorubicin Doxorubicin niosomes exhibited prolonged half-life, prolonged circulation time with altered metabolic and maintenance of antitumor activity76. The vesicle formulation exhibited a superior in vivo safety profile 121


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as compared to free drug and reduced tumor size, but overall less active than free drug77. Niosomal doxorubicin exhibited a slight reduction in IC50 against resistant ovarian cancer cell over free drug78. Palmitoyl muramic acid vesicles containing doxorubicin avoided the liver uptake in mice leading to reduction in liver toxicity79. Clearance of doxorubicin from niosomes was 10 times higher with 6 fold increase in AUC compared to solution form with modest targeting was achieved in solid tumor bearing mice80. Niosomal formulation of doxorubicin delivery is more as compared to free drug in the plasma compartment by intraperitoneal route81. Doxorubicin niosomes exhibited prolonged half-life, prolonged circulation time with altered metabolism and better maintenance of antitumor activity. Bleomycin Niosomal bleomycin was cleared from plasma much slowly than free drug. A significant increase in plasma concentration was achieved than free drug with enhanced tumor bleomycin level82. A niosomal bleomycin exhibited improved anticancer activity due to better delivery of bleomycin to tumor site83. Vincristine The toxicity of vincristine sulphate was reduced after incorporated into niosomes and antitumor activity improved which may be due to better delivery of drug at tumor site84. Daunorubicin Niosomal formulation of daunorubicin clearly exhibited its superiority in terms of its prolonged release85. Niosomal formulation appears to have better impact on initial tumor growth kinetics with increase in volume doubling time against free drug. Niosomes encapsulated daunorubicin decreased the rate of proliferation of sarcomas. This could be due to the slow release of drug and increased duration of circulation time. It can also be postulated that niosomal daunorubicin delivered preferentially to the tumor site by enhancing the cell killing and decrease in toxicity in non–cancerous tissues. Niosomal formulation showed a limited cardiac edema and no cell necrosis as compared to free drug in mice due to decreased accumulation of drug in the heart86. Also in another study, the niosomal formulations were able to destroy the Dalton’s ascetic lymphoma cells in 3 days as compared to free drug in 6 days. An enhanced mean survival time was achieved by the niosomal formulation in mice. Withaferin A Niosomal withaferin A showed a significant increase in the life span as compared to pure drug by increasing the survival rate. A 10mg/kg plain withaferin A showed 42.105 increases in life span, whereas niosome entrapped withaferin showed 57.815 increases in life span. An increase of 1.37 times fold observed with niosomal formulation. Similarly, at a higher dose of 20mg/kg showed an increase in life span up to 89.47 with 2.23 times fold increase in life span. The niosomal entrapped withaferin A delivered preferentially to the tumor site by enhancing cell kill and decreasing the toxicity in non-cancerous cells. This reason may also be supported by the fact that the niosomal formulations offered slow release of drug with longer duration of circulation87. Levonorgestrel Niosomal levonorgestrel exhibited better progestational activity as indicated by endometrial assay and better inhibition with formation of corpora lutea. Acetazolamide Niosomal formulations of acetazolamide doubled the Cmax that obtained upon instillation of 122


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suspension containing same amount of drug. The niosomal formulation showed a longer duration of action with double in AUC89. Niosomal acetazolamide were found to be effective and showed a prolonged decrease in IOP90. A positively charged niosomes showed good corneal permeability and pharmacodynamics were however found to be in appropriate in terms of the corneal toxicity. bioadhesive coated formulation showed lesser toxicity with lowering of intra ocular pressure showed a comparable physiological effect extended up to 6 hours91. Acyclovir The niosomal formulation of acyclovir exhibited significant retardation of drug release compared with free drug. The in vivo study revealed that niosomal dispersion significantly improved the oral bioavailability of drug in rabbits by 2 fold. The niosomal formulation of acyclovir significantly altered the pharmacokinetic properties in rabbits in terms of increase in Tmax, t½, MRT and AUC92. Ketoconazole Niosomal formulation of ketoconazole offered maximum antifungal activity over plain drug and marketed preparation in terms of prolonged action of 48 hours and 72 hours respectively93. On the other hands the niosomes exhibited a potential to reduce the therapeutic dose of ketaconazole in rabbits94. Sodium stibogluconate Niosomal formulation of sodium stibogluconate exhibited an equiactive and increased drug efficiency by an order of magnitude compared with free drug95. The niosomal formulation of sodium stibogluconate was more effective than its free form, presumably because of maintenance of high level in the infected RES96. Minoxidil Niosomes enhanced transdermal bioavailability of minoxidil in hairless mouse skin at very low concentration; thereby it could be used as a carrier for topical delivery of minoxidil in skin disease such as hair loss97. Colchicine Niosomes exhibited a higher encapsulation efficiency of colchicines with an expected reduction in side effects98. Ciprofloxacin and Norfloxacin Niosomal ciprofloxacin and norfloxacin increased the intestinal absorption as compared to plain inclusion complexes99. Flurbiprofen Niosomal formulation demonstrated a significant improvement in bioavailability and antiinflammatory activity over the ointment form of drug100. Sumatriptan succinate Sumatriptan succinate encapsulated niosomes across nasal mucosal using ex vivo animal model exhibited better absorption as compared to plain drug101. Pentoxifylline Niosomal pentoxyfylline showed significant increase by 8 times in locomotor activity of reserpine treated mice and adenosine treated rats. The niosomal formulation of drug produces the same effect 123


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as plain drug at lower dose with a benefit of better distribution of drug in brain by offering a sustained release with decreased metabolism of drug102. Vasoactive intestinal peptide A radiolabelled (I125) vasoactive intestinal peptide (VIP) loaded with glucose bearing mice upon i.v administration to mice exhibited higher VIP brain uptake as compared to free drug103. Bovine serum albumin It was observed that niosomal formulation elicited a significantly higher Ig G titre upon topical application as compared with topically applied alum absorbed bovine serum albumin104. Vasopressin The stability of the peptide (vasopressin) increased significantly upon incorporation into niosomes105. Luteinizing hormone releasing hormone (LHRH) Niosomes were able to protect LHRH being cleared immediately from the injection site in male wister rats106. Monoclonal antibodies Monoclonal antibody conjugated niosomes exhibited targeting of niosomes to specific cell receptors with a high selectivity and specificity107. Niosomes are known to enhance both the humoral and cell mediated immunity to variety of antigens. Such adjuvant activity has been attributed to the depot action of niosome suspension at the site of action, which induces higher Ig G2a titre compared to the aqueous suspension108-110. Viral antigens such as avian rheovirus (Novasomes) licensed for veterinary use111. DNA delivery (immunization) The immune stimulating activity of DNA encoding hepatitis B surface antigen administered by topical application of niosomes in Balb/C mice exhibited comparable results in terms of serum antibody titer when given though intramuscularly15. Topical niosomes elicited a comparable serum antibody titre and endogenous cytokine level as compared to intramuscular recombinant HBsAG and topical liposomes. This study signifies the potential of niosomes as DNA vaccine carriers for effective topical immunization due to enhanced permeation of plasmid across the skin due to high thermodynamic activity gradient of bioactive stratum corneum interface113. Insulin The niosomes composed of brij 92 are able to stabilize insulin against enzymatic degradation114. Tetanus toxoid Topically given tetanus toxoid in niosomal formulation could elicit immune response (anti TT IgG) was equivalent to intramuscularly alum-absorbed TT based immunization. But the immune response from the niosomes elicited weaker115. Ciclopirox olamine Deposition of niosomal ciclopirox olamine into rat skin and its gel was significantly higher than plain drug116. Ketoprofen There was a significant difference observed between free and encapsulated ketoprofen in reducing the carrageenan induced paw edema in albino rats due to accumulation of niosomes at the inflamed tissues117. 124


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Nimesulide The results showed that incase of niosomal preparations there was a significant increase in the anti-inflammatory activity which was lasted for longer period of time as compared to plain drug and marketed formulation52. Cyclopentolate HCl Niosomal cyclopentolate significantly improved ocular bioavailability by modifying the permeability characteristics of conjuctival and scleral membranes51. Harmine Histopathological studies revealed that the toxicity of drug was reduced upon niosomal administration. Harmine in vesicular forms found to be more effective in destroying the intracellular parasites as well as non-hepatotoxic and non-nephrotoxic55. Bacopasaponin C Bacopasaponin C in vesicular systems was found to be very active without any side effects as indicated by normal liver and kidney functions65. Enoxacin Enoxacin encapsulated niosomes permeated at greater rate in mouse skin than liposomal formulation88. Gliclazide In case of gliclazide niosomes, the reduction in blood glucose levels was slow and revealed a maximum reduction with in 4-5 hours and this reduction in blood glucose levels was sustained over longer periods of time up to 12 hours. A significant hypoglycaemic effect (25%) was maintained for a period of 2 to 12 hours over the oral administration of free drug exhibited 2-3 hours123. Rifampicin The in vivo study revealed that a maximum lymph concentration (46.2%) was achieved through the niosomal formulation. Niosomal formulation of rifampicin provides an effective compartmentalization of the drug in the lymph system. A 145 times increase in drug accumulation capacity was also observed in the lungs of albino rats compared to that of free drug93. Diclofenac sodium Anti-inflammatory activity in carrageenan induced paw edema in rat was higher with niosomal preparation administered intraperitoneally and transdermally. Greater retention of diclofenac sodium in the arthritic joint might reduced the potential adverse systemic effects of the drug because of local administration into the diseased area118. Niosomal formulation reduced the GI toxicity and sustained the effect of drug for longer period of time up to 61 hours119. Ketorolac Comparison of plasma level of ketorolac, niosomal preparations exhibited better action with prolonged half-life over free drug120. Indomethacin Inhibition of paw edema volume was 10 times higher than free indomethacin121. Niosomal indomethacin exhibited a greater inhibiting effect on platelet aggregation and ATP release than free drug122. 125


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Salbutamol A niosomal salbutamol exhibited maximum tissue distribution at lungs (75%) in rabbits. Niosomal salbutamol sulphate showed higher tissue drug distribution in lungs (75.62%) and improved or enhanced pharmacokinetic parameters in terms of t½, AUC over conventional dosage forms and pure drug. Dithranol Niosomal preparations found to exhibit improved permeation properties112. Zidovudine A higher concentration of zidovudine in the liver and spleen attained by administration of niosomal formulation with a reducing in the toxicity. The MRT and AUC values reflects the sustained release effects of niosomal formulation showed a significant slower clearance rate with maximum bioavailability. An increase in the half-life was also exhibited by the niosomal formulations30. Iopromide The organ specificity of iopromide encapsulated in niosomes was studied in rats, that it can be targeted to kidney for imaging studies45. Clotrimazole Niosomal clotrimazole exhibited a better antifungal activity and better tolerability at the tissue level in the rat124. Plumbagin The niosomal plumpagin and plumpagin ester showed a better antifertility activity over the plain drug119. Additionally, the niosomal formulation decreased the toxicity of plumpagin with an increase in antifertility activity. CONCLUSION Niosomes are more stable over liposomes and they alter the pharmacokinetic profile of various drugs such as anticancer and antiviral agents that are incorporated to them. Also niosomal delivery of peptides, antigens, NSAID’s have demonstrated a greater potential of targeted delivery. Further the drug delivery of niosomes can be enhanced by applying newer concepts like proniosomes, discomes and aspasome. However, to explore the potential utilization of niosomal delivery system as a promising drug delivery, vigorous research activities are further desirable. REFERENCES

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Comparative Pilot Bioavailability Study of Carbamazepine in Nepalese Healthy Volunteers 1

A. Ghosh1, Neha Sharma2, N. R. Biswas2 Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India 2 B.P. Koirala Institute of Health Sciences, Dharan, Nepal

ABSTRACT Carbamazepine (CBZ) is the primary drug for the treatment of partial and tonic-clonic seizers and also for trigeminal and glossopharyngeal neuralgias..It is designed to assess the comparative bioavailability of different brands of carbamazepine 200 mg extended release tablets in Nepalese Healthy Volunteers (n=6). A total six male volunteers (nonsmokers) ranging age between 18 and 45 years were randomly selected for this study. ZEN RETARD 200 (containing carbamazepine 200 mg ER), Intas Pharmaceuticals, India, was taken as reference preparation (A) and BAZETOL-200XR (containing carbamazepine 200 mg ER), Asian Pharmaceuticals Pvt. Ltd., Nepal, was taken as test preparation (B). This study was a single dose, randomized, two treatment and two-way cross over study, with a wash out period of 14 days. CBZ plasma levels were determined by validated RP-HPLC (Knauer, Germany) method. Cmax, tmax, AUC0-t, AUC0-∞, t1/2, and Kel were calculated by using zero moment non-compartmental pharmacokinetics for the single dose bioavailability study. A statistical analysis using ANOVA and 90% confidence interval (CI) tests was conducted for this study. On the basis of comparison of the AUC0-∞ for carbamazepine after one 200 mg ER dose administration, the relative bioavailability of the Test preparation (B) was 104.96% of that of the Reference preparation (A). 90% confidence interval for Cmax, AUC0-t, and AUC0-∞ values of test preparation were 0.9510 – 1.0123, 0.8259 – 1.2016, and 0.8135 – 1.2256 respectively. On the basis of the pharmacokinetic parameters studied, it can be concluded that the test preparation is bioequivalent with the reference preparation. Keywords : Carbamazepine, Extended Release (ER), Pharmacokinetic Analysis, Bioequivalence. INTRODUCTION Carbamazepine (CAS 298-46-4) is a white to off-white powder, practically insoluble in water and soluble in alcohol and in acetone. The chemical name is 5H dibenz[b,f]azepine-5-carboxamide, with a molecular weight of 236.271, 2. Carbamazepine limits the repetitive firing of action potentials evoked by a sustained depolarization of mouse spinal cord or cortical neurons maintained in vitro. This appears to be mediated by a slowing of the rate of recovery of voltage-activated Na+ channels from inactivation. These effects of carbamazepine are evident at concentrations in the range of therapeutic drug levels in CSF in humans. The effects of carbamazepine are selective at these concentrations, in that there are no effects on spontaneous activity or on responses to iontophoretically applied GABA or glutamate. The carbamazepine metabolite, 10, 11-epoxycarbamazepine, also limits sustained repetitive firing at therapeutically relevant concentrations, suggesting that this metabolite may contribute to the antiseizure efficacy of carbamazepine. The pharmacokinetics of carbamazepine is complex. They are influenced by its limited aqueous solubility and by the ability of many antiseizure drugs, including carbamazepine itself, to increase their conversion to active metabolites by hepatic oxidative enzymes. Carbamazepine is absorbed slowly and erratically after oral administration. Peak concentrations in plasma usually are observed 4 to 8 hours after oral ingestion, but may be delayed by as much as 24 hours, especially following the administration of a large dose. The drug distributes rapidly into all tissues. Approximately 132


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75% of carbamazepine binds to plasma proteins and concentrations in the CSF appear to correspond to the concentration of free drug in plasma3. An HPLC – UV method has described for measurement of carbamazepine in human plasma with UV detection. But the method suffered from lack of clinical application. Hence, the objective of this work is to develop a precise, sensitive and feasible HPLC method with direct PDA-detection in human plasma. In the present study the bioequivalence study of two carbamazepine extended release formulation (Test & Reference) has been carried out by comparing equivalence with respect to the rate and extent of absorption, while the area under concentration time curve (AUC) generally serves as the characteristic of the extent of absorption 4, 5. No single parameter reliably measures the rate of absorption; for instance, the maximal drug concentration (Cmax) has been widely used, but it depends more on the fraction absorbed than the rate of absorbed. The time of maximal concentration (tmax) depends on both absorption and elimination rates6. Materials and methods A total six male volunteers, (nonsmokers) ranging in an age between 18 and 45 years (mean ± SD = 36.00 ± 7.35 years) and a mean weight of 65.67 ± 10.88 kg were randomly selected for this study on the basis of a healthy medical history and a physical examination. The protocol of this study was approved by Institutional Review Board, of BPKIHS, Dharan, Nepal. Before admission to the study each subject was informed of the nature and the risks of the study and a written informed consent was obtained from the volunteers. The study was a single dose, randomized, two treatment and two-way cross over study, with a wash out period of 14 days between the two dosing sessions. In each dosing session, volunteers received either of the test or the reference preparation. Product information Reference Preparation (A) : One Tablet Zen Retard 200 (containing Carbamazepine 200 mg ER) Mfg. by : Intas Pharmaceuticals Selaqul, Dehradun – 248197, India Batch No. : DK 2891 Mfg. Date : September, 2009, Exp. Date : August, 2012 Test Preparation (B) : Tablet BAZETOL-200XR (containing Carbamazepine 200 mg ER) Mfg. by : Asian Pharmaceuticals Pvt. Ltd. Solar Division, Padasari, Vdc-9, Siddharth Nagar, R Upandehi, Nepal Batch No.: BXTF 7015 Mfg. Date: May, 2010, Exp. Date : April, 2012 Each CBZ ER formulation was administered at 08.00 hr following an overnight fasting of 10 hrs. Food was withheld for 4 hrs after the administration of the two CBZ ER formulations. Venous blood samples (2 ml) were taken from the forearm vein at dosing 0, 1, 2, 3, 4, 6, 8, 8, 12, 24, 36, 48, 72, and 144 hrs after dosing. The plasma was immediately separated by centrifugation at 5000 rpm for 15 min and stored at -4ºC. Before assaying, the plasma was allowed to reach room temperature. 0.5 ml of plasma was taken in a 15 ml stopper test tube. 20 µl of Internal Standard (Lamotrigine) was added and mixed for 30 second by vortex mixer. To this 6.00 ml of chloroform was added after 133


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converting the matrix into alkaline pH with 20 µl of 5M sodium hydroxide solution and hand mix for 15 min followed by the centrifuge for 10 min at 5000 rpm. Then 5.00 ml of organic layer was separated in a separate test tube and evaporated to dryness in nitrogen atmosphere at 40-45ºC.The residue was reconstituted with 200 µl of mobile phase and was injected in HPLC system. Peak areas of carbamazepine and internal standard were recorded. CBZ plasma levels were determined by the validated reverse phase high performance liquid chromatographic method. The HPLC system was Knauer, Germany, and it consisted of a solvent delivery pump (Smartline pump 1000), a 20 µl rheodyne injector and a Smartline PDA detector (2800). Integration was done using Clarity-chrome software. The analysis was carried out at room temperature using an analytical column, Eurospher 100-5-C18 (250X4.6) mm, 5µ particle size, with a C18 guard column. The mobile phase consisted of 10 mM phosphate buffer, pH was adjusted to 3.7 with dilute ortho phosphoric acid and acetonitrile (HPLC grade) in the ratio of 65:35 (v/v) and elute at a flow rate of 1 ml/min. The sample was injected through the rheodyne injector system fitted with 20 µl fixed loop. The eluent was monitoring at 215 nm. The method was validated for linearity range, accuracy, and precision and system suitability parameter as per the standard guidelines 7, 8. Plasma concentration-time profile of carbamazepine was determined using the zero moment noncompartrmental pharmacokinetics method. Both the maximum plasma concentration (Cmax) and time to peak plasma concentration (tmax) were obtained directly from the analytical data. The elimination half-life (t1/2) was calculated from the slope of the terminal log linear phase, using the formula 0.693/Kel; where Kel is the apparent elimination rate constant. AUC0-t (where t is the time at which the last quantifiable concentration was observed) was calculated using the trapezoidal rule. AUC0-∞ was calculated according to the following formula: AUC0-∞ = AUC0-t + Clast / Kel, where Clast is the last quantifiable plasma level. Result For each subject, descriptive statistics were used to summarize the estimated pharmacokinetic parameters. For the purpose of bioequivalence study AUC0-t, AUC0-∞ and Cmax values were considered primary variables. Their log-transformed data were analyzed by an analysis of variance (ANOVA), including treatment, period and subject as variables. The bioequivalence analysis was made according to guidance of Committee for Proprietary Medicinal Products9. Test formulation was considered bioequivalent to reference formulation if 90% confidence interval (CI) for the ratio between each parameter fell within the predetermined equivalence range of 80–125%10.

Figure 1 : HPLC Chromatogram of blank plasma.

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Figure 2 : HPLC chromatogram of carbamazepine (2 µg/ml) and IS (2 µg/ml) spiked in human plasma

The described analytical method used for the measurement of carbamazepine was shown to be accurate and sensitive. Fig 1 shows that there were no interferences observed in the chromatogram of plasma samples. The peaks of carbamazepine and internal standard were well resolved (Fig.2). The retention times (RT) of carbamazepine and internal standard were about at 15.5 and 4.5 min respectively. The lower limit of quantification (LLOQ) for carbamazepine in plasma was 0.1 µg/ml. The relation between concentration and peak area ratio (carbamazepine: internal standard) was found to be linear within the range of 0.1 µg/ml to 4 µg/ml (r2=0.995). The linearity curve is shown in fig 3. Quality control points at low, medium, and high levels (0.5, 1.5 and 3 µg/ml) were used to determine stability, absolute recovery, within-day and between-day precision and accuracy. Within-day and between-day precision and accuracy data are summarized in Table 1.

Area Ratio (analyte peak are : IS peak area)

2.5

2

1.5

1 y = 0.5662x + 0.0048 2

0.5

R = 0.995

0 0

1

2

3

4

5

Concentration (mcg/ml)

Figure 3 : Linearity curve in the concentration range of 0.1 – 4 µg/ml spiked in human plasma

Table-1: Within-day and between –day precision and accuracy of HPLC method Nominal (µg/ml)

GM (µg/ml)

SD (±)

DEV (%)

WRP (%)

BRP (%)

N

0.5 0.4962 0.0156 0.7444 2.9933 0.1626 18 1.5 1.4995 0.0671 0.3407 4.5567 0.3215 18 3.0 3.0330 0.3032 -1.1031 9.9614 0.1391 18 GM=grand mean; SD=Standard deviation; DEV=percent deviation from nominal value; WRP=within-run precision; BRP=between-run precision; N=number of total replicate observation.

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Table-2 : Demographic data of six healthy human volunteers Vol. No.

Sex

Age

Height (cm)

Weight (kg.)

1

M

44

157.48

55

2

M

30

157.48

64

3

M

29

162.56

63

4

M

29

165.1

57

5

M

42

160.02

85

6

M

42

177.8

70

Mean

36.00

163.41

65.67

S.D.

7.35

7.65

10.88

Mean plasma concentration versus time curves of carbamazepine after single dose administration of reference and test products to healthy subjects are shown in fig. 4. Table 2 summarizes the demographic and mean health parameters of all the volunteers. 3

Plasma Concentration (µg/ml)

2.5

2

Reference

1.5

Test

1

0.5

Administration of the reference preparation as single dose in the fasting state produced the maximum plasma concentration of 2.907 + 0.084 µg/ml (Cmax) at the time 12.667 + 5.888 hour (tmax) whereas the test preparation as a single dose in the fasting state produced the maximum plasma concentration 2.853 + 0.095 µg/ml (Cmax) at the time 14.667 + 7.448 hour (tmax).

The area under plasma concentration time curve (AUC0-t) for reference and test Figure 4 : Mean (±SD) plasma concentration (n=6) versus time curves of preparation were 174.568 + 32.186 µg carbamazepine after single dose administration of reference and test products. hr/ml and 172.606 + 34.018 µg hr/ml, respectively. 0

0

20

40

60

80

100

120

140

Time (hr)

When administered as a single dose, in the fasting state, the reference preparation produced the area under plasma concentration time curve upto infinity (AUC0-∞) 202.002 ­+ 49.383 µg.hr/ml., whereas administration of the test preparation produced area under plasma concentration time curve upto infinity (AUC0-∞) 212.010 + 64.3935 µg.hr/ml. Administration of the Reference preparation produced the plasma elimination half-life (t1/2) 45.562 + 11.346 hr whereas administration of the Test preparation produced the plasma elimination half-life (t1/2) 50.866 + 16.752 hr Administration of the reference preparation produced the plasma elimination constant (K­el) 0.0160 + 0.0036 hr-1, whereas administration of the test preparation produced the plasma elimination constant (Kel) 0.0148 + 0.0045 hr-1. 136


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Table-3 : Pharmacokinetic Parameters in 06 volunteers with the Test and Reference Preparation

Cmax (µg/ml.)

Reference Preparation (A) (Mean ± SD, n=6) 2.907 ± 0.084

Test Preparation (B) (Mean ± SD, n=6) 2.853 ± 0.095

tmax (hr)

12.667 ± 5.888

14.6667 ± 7.4476

AUC0-t (µg. hr/ml.)

174.568 ± 32.186

172.606 ± 34.0187

AUC0-∞ (µg. hr/ml.)

202.002 ± 49.383

212.010 ± 64.3935

kel (hr-1)

0.0160 ± 0.0036

0.0148 ± 0.0045

t1/2 (hr)

45.562 ± 11.346

50.8657± 16.7519

Relative Bioavailability (%)

100%

104.96%

Pharmacokinetic Parameters

Table-4 : 90% confidence Interval with the Test and Reference Preparation Cmax

AUC 0-t

AUC 0-∞

Untransformed Data

0.9510 – 1.0123

Ln transformed Data

0.9536 – 1.0115

Untransformed Data

0.8259 – 1.2016

Ln transformed Data

0.9581 – 1.037

Untransformed Data

0.8135 – 1.2256

Ln transformed Data

0.8575 – 1.2456

On the basis of comparison of the AUC0-∞ for carbamazepine after 200 mg ER single dose administration, the relative bioavailability of the test preparation of tablet BAZETOL – 200XR was 104.96% of that of the reference preparation, tablet ZEN RETARD 200. The mean pharmacokinetic parameters of both reference and test preparation are summarized in table 3. 90% confidence interval for Cmax, AUC0-t, and AUC0-∞ values of test preparation were 0.9510 – 1.0123, 0.8259 – 1.2016, and 0.8135 – 1.2256, respectively and are summarized in table 4.

The ANOVA at P<0.05, was applied with the variables of treatment, period and subjects for Cmax, AUC0-t, and AUC0-∞ and their log transformed data. The frequency distribution value (F Value) for Cmax & logCmax, AUC0-t & logAUC0-t and AUC0-∞ & logAUC0-∞ was ranged between 0.9001 to 1.2567, 0.6273 to 1.0735 and 0.5764 to 0.9240 respectively which was less than the critical F value at P<0.05 (3.48). So there was no significant difference of the test preparation as that of reference preparation. Discussions The single dose bioequivalence study of tablet BAZETOL – 200XR was conducted in 6 Nepalese adult, male, healthy, human volunteers with one preparation of carbamazepine 200 mg. Values of Cmax, tmax and AUCo-t, were comparable for the reference and the test preparation in the fasting state. Carbamazepine was detected in plasma from 1.0 hour to 144 hours in the reference preparation as well as in the test preparation. Peak plasma levels of carbamazepine with both the preparations were achieved between 8 and 24 hrs The mean peak plasma levels of BAZETOL – 200XR with reference preparation, tablet ZEN RETARD 200 (containing CARBAMAZEPINE 200 mg ER) on the study day ranged between 2.760 – 2.990 µg /ml, while the test preparation of tablet BAZETOL – 200XR ranged between 2.740 – 2.990 µg/ml. On the basis of comparison of the AUC0-∞ for carbamazepine 200 mg after single dose administration, the relative bioavailability of the test preparation of tablet BAZETOL 137


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– 200XR was 104.96 % of that of the reference preparation; tablet ZEN RETARD 200, which is under the acceptable limit for the bioequivalence study (80 – 120%). As shown in table 4, 90% confidence interval values for Cmax, Ln Cmax, AUCo-t, Ln AUC0-t and AUC0-∞, Ln AUC0-∞ of test preparation were within the accepted limit of bioequivalence study (0.8 – 1.25) as compared to reference preparation. Conclusion On the basis of the pharmacokinetic parameters studied, it can be concluded that the test preparation of tablet BAZETOL – 200XR mfg. by Asian Pharmaceuticals Pvt. Ltd. Solar Division, Padasari, Vdc-9, Siddharth Nagar, Rupandehi, Nepal, is bioequivalent with the reference preparation, one tablet ZEN RETARD 200 mfg. by Intas Pharmaceuticals Selaqul, Dehradun - 248197, India. As the study has been done in 6 healthy volunteers, there will be a scope to carry out the study with the higher numbers of volunteers. Acknowledgement The authors are thankful to the Department of Clinical Pharmacology and Therapeutics, B.P.Koirala Institute of Health Sciences, Dharan, Nepal for providing the necessary instrumental facilities to carry out this study. The corresponding author is thankful to Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India for sanctioning the Extra Ordinary Leave to act as an Assiatant Professor in the Department of Clinical Pharmacology and Therapeutics, B.P. Koirala Institute of Health Sciences, Dharan, Nepal under Govt. of India assistance programme. References 1.

Laurence L.Bruton, John S.Lazo, Keith L. Parker. Goodman and Gilman’s the pharmacological basis of therapeutics, eleventh edition.

2.

Physicians Desk Reference.64th edition. Montvale, NJ:PDR Network;2009

3.

Fauci, Braunwald, Kaspar, Hauser et al. Principles of Internal Medicine. 17th ed. McGraw-Hill Companies, Inc. 2010.

4.

Hauschke D, Steinijans V, Diletti E. A distribution free procedure for the statistical analysis of bioequivalence studies. Int J Clin Pharmacol Ther Toxicol. 1990; 30: 37–43.

5.

Schulz HU & Steinijans VW. Striving for standards in bioequivalence assessment: a review. Int J Clin Pharmacol Ther Toxicol. 1992;30:51-56.

6.

Farolfi M, Power JD, Rescigno A. On the determination of bioequivalence. Pharmacol Res. 1999;39:1-4.

7.

U.S. Food and Drug Administration. Guidance for Industry: Bioavailability and Bioequivalence Studies for orally Administered Drug Products- General Considerations. Rockville, MD: Center for Drug Evaluation and Research. 2000.

8.

Shah VP, Midha KK, Sing S. Analytical method validation: bioavailability, bioequivalence and pharmacokinetic studies. Eur J Drug Metab Pharmacokin.1992; 16:249-255.

9.

Committee for Proprietary Medicinal Products (CPMP).Note for Guidance: Investigation of Bioavailability and Bioequivalence. London: Working Party on the Efficacy of the Medicinal Products. 1991.

10. European Agency for the Evaluation of Medicinal Products. Note for guidance on the investigation of bioavailability and bioequivalence CPMP/EWP/QWP/1401/98. 2002.

q

q 138

q


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Degradation Kinetic and Preformulation Stability Studies of Curcumin Vivek R. Yadava, Sarasija Suresha*, Seema Yadavb, Veda Murthy Joshia a Al-Ameen College of Pharmacy, Hosur Road, Bangalore - 560027, India b Department of Pharmacology, Baroda Medical College, M.S. University, Baroda, India

ABSTRACT Curcumin, a naturally occurring poorly-water soluble, highly lipophilic molecule has wide range of pharmacological activities. However, its limited aqueous solubility and degradation at alkaline pH restricts its bioavailability. The purpose of this study to find out degradation kinetics of curcumin under various pH conditions and the stability of curcumin in physiological matrices were investigated. When curcumin was incubated in 0.1 M phosphate buffer and serum-free medium, pH 7.2 at 37째C, about 90% decomposed within 30 min. A series of pH conditions ranging from 1.2 to 10 were tested and the result showed that decomposition was pH-dependent and occurred faster at neutral-basic conditions. It is more stable in medium containing 1 % tartaric acid, ascorbic acid and citric acid improve the stability at pH 7.4; less than 15 % of curcumin decomposed within 1 h and after incubation for 8 h, about 68 % of curcumin is still remained. INTRODUCTION Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3, 5-dione], a low molecular weight polyphenol derived from the rhizomes of turmeric (Curcuma longa Linn.), is a yellow pigment, widely used as a coloring agent and spice in many foods which has been used in Ayurvedic and Chinese medicine for centuries. Turmeric contains bioactive substances the curcuminoids, such as curcumin, demethoxycurcumin, bisdemethoxycurcumin and dimethoxycurcumin1. Interests in this dietary polyphenol has grown in recent years due to its vast array of beneficial pharmacological effects including antioxidant, anti-inflammatory, anticarcinogenic 2-4, hypocholesterolemic 5, antibacterial6, wound healing, antispasmodic, anticoagulant, antitumor7 and hepatoprotective8 activities. It also reported the protective effect of curcumin on 2,4,6-trinitrobenzene sulfonic acid induced colitis in mice, curcumin reduced significantly the degree of both neutrophil infiltration and lipid peroxidation as well serine protease activity9. It is also a potent free radical scavenger, having superoxide anions, singlet oxygen, hydroxyl radicals scavenging and lipid peroxidation inhibitory activities10. Despite the promising biological effects of curcumin, its poor oral bioavailability in both rodents and humans, as reported by several workers11, has restricted its use. It is well known that many drugs show bioavailability problems due to their low water solubility, slow dissolution rate and instability in the gastrointestinal tract. Poor oral absorption12 due to its extremely low aqueous solubility or extensive pre-systemic metabolism may be responsible for the unfavorable pharmacokinetics of this molecule. In rodents, curcumin undergoes avid metabolism by conjugation and reduction and its absorption after oral dosing is characterized by poor systemic bioavailability13-14. Some studies on the curcumin are based on the ionic structure where the keto-enol equilibrium (Figure 1) is present or when it is fully in keto form15 with the resulting properties depending on the 139


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

latter. The reaction of curcumin was carried in 0.1 M phosphate buffer, pH 7.2 at 37°C for 30 min. UV-visible spectra absorbance of curcumin and the reaction mixtures were monitored at 425 nm. From this, we found that curcumin alone in phosphate buffer undergoes loss of the absorbance at 425 nm, indicating that it is unstable at physiological condition in vitro. The present work shows the degradation kinetic studies carried out in aqueous media under a given set of pH conditions imposed, in order to produce the best working conditions to calculate the thermodynamic constants of the curcumin species in water and the stability of curcumin in physiological matrices were investigated. MATERIAL AND METHODS Materials Curcumin was obtained as a gift sample from Natural Remedies Pvt Ltd., Bangalore, India. Methanol, tartaric acid, ascorbic acid and citric acid were purchased from Thomas Baker, Mumbai, India. All other chemicals used were of analytical grade. The quantitative determination of curcumin was performed by UV-visible spectrophotometer (Shimadzu UV-1700) with quartz cells having a 1 cm optical path and was monitored for UV absorption at 425 nm. Preliminary stability studies of curcumin In order to validate the instability of curcumin in aqueous medium and in various pH conditions such as pH 1.2, 6.8 and 7.4, a simple spectroscopic method was adopted. Curcumin is water insoluble, in order to dissolve the curcumin in aqueous medium, co-solvent methanol was used. Lower concentration (4µ/ml or 10.8 µ M) of curcumin was chosen as it shows precipitation in higher concentration in aqueous medium. The effect of acids like citric acid, ascorbic acid and tartaric acid on the stability of curcumin also was determined. Spectrophotometric kinetic study Stock solution was dissolved in 24 ml of freshly prepared buffer solution in 25 ml of volumetric flask. Immediately the absorbance was read at 425 nm against blank and was considered as 100 %. Further, the absorbance measured at regular intervals of 5 min and relative degradation was measured as cumulative percentage curcumin remaining. The graph of cumulative percentage remained verses time was plotted. The above procedure was repeated for different buffers and distilled water. The effect of acids like ascorbic acid, tartaric acid and citric acid were studied by dissolving 1 % w/v of the salts in the respective buffers. Curcumin degradation studies conducted in distilled water and different pH conditions such as pH 1.2, 6.8 and 7.4. Further, the effect of 1 % w/v citric acid, ascorbic acid and tartaric acid on the stability of curcumin in pH 6.8 and 7.4 were also studied. A graph of logarithmic concentration verses time was plotted. The slope of obtained from the graph, was multiplied with 2.303 to get the apparent rate of degradation of curcumin, k, the constant in the respective medium. Further, the t½ was calculated from the below formula. t ½ = 0.693/k where k is apparent rate of degradation. Further the k values were incorporated into the student t test in order to obtain the significant variation. 140


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RESULTS AND DISCUSSION Curcumin behavior in acid and basic media In basic media the solutions produced by curcumin have a red color while in acid media they have a yellow color. The stability of the curcumin in aqueous media depends on the pH of the system, this being a significant factor in its degradation kinetics. Before doing the assessment of the thermodynamic constants of the species in aqueous media, it was decided to study its kinetic behavior within the pH range imposed by the presence of the sodium hydroxide at the concentrations used. O

O

O

OH

HO

O

OMe

MeO

OMe

MeO

H

OH

HO

basic condition

acidic condition

Cum % Curcumin remaining

Figure 1 : Curcumin in acidic and basic conditions 0.6

D. Water pH 1.2 pH 6.8 pH 7.4

100

DW pH 1.2 pH 6.8 pH 7.4

0.5 0.4 0.3

log c

80 60

0.2 0.1

40

0 -0.1 0

20 0 0

20

40

60

Time in min

80

100

20

40

60

-0.2

80

100

120

Time in min

0.6

pH 7.4 pH 7.4+ AA pH 7.4 + TA pH 7.4 + CA

120 100

pH 7.4 pH 7.4+ AA pH 7.4+ta pH 7.4+ca

0.5 0.4 0.3 log c

80 60 40

0.2 0.1

20

0 -0.1 0

0 0

50

100

20

40

-0.2

Tim e in m in

60

80

100

120

Time in min

Figure 2a : Comparative degradation profile of curcumin in pH 7.4 in presence of acids pH 6.8 pH 6.8+ AA pH 6.8 + TA pH 6.8+ CA

110 90 70 50 30

0.4 0.3 0.2 0.1 0

10 -10 0

pH 6.8 pH 6.8+ aa pH 6.8+ta pH 6.8+ca

0.6 0.5

log ( C )

Cum % Curcumin remained

Cum % curcumin Remained

Figure 2 : Comparative degradation profile of curcumin in different buffers

20

40

60

80

0

100

20

40

60 Time in min

Time in min

Figure 2b : Comparative degradation profile of curcumin in pH6.8 in presence of acids

141

80

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Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Kinetic study The curcumin dissolves in NaOH on a basic pH of the system, hence favoring the presence of the most basic species or the one wholly deprotonated; in order to allow formation of quinonoid systems (Fig.1) thus giving a characteristic red coloration to the system. The absorption spectrum evolves as a function of time. Fig. 2 shows the time evolution for the curcumin species under different experimental conditions. In all the conditions curcumin was found be unstable. The graph of logarithm of concentration verses time was plotted from slope of the above graph, apparent rate of degradation (k) was computed by multiplying the slope with 2.303. Another graph of cumulative percentage of curcumin remained verses time was plotted which indicated the relative % of curcumin present at specific interval of time. Further the k values were subjected for student T test in order to find out the significant change in the stability of curcumin in presence of the acids, results are shown in Table 1. Table-1 : Preliminary stability data of curcumin indicating slope, k value and t half Sample

Slope

k- Value

t half ( min)

P value

Level of significance

DW

0.001

0.002303

300.9119

pH 1.2

0.001

0.002303

300.9119

pH 6.8

0.0023

0.005297

130.8312

pH 7.4

0.0066

0.015212

45.59271

pH 7.4+aa

0.0009

0.002073

334.3465

<0.01

Significant

pH 7.4+ta

0.0008

0.001842

376.1398

<0.01

Significant

pH 7.4+ca

0.0023

0.005297

130.8312

<0.01

Significant

pH 6.8+aa

0.0025

0.005758

120.3647

<0.01

Significant

pH 6.8+ta

0.0019

0.004376

158.3747

>0.05

Not Significant

pH 6.8+ca

0.0023

0.005297

130.8312

>0.05

Not Significant

Amongst all the pH conditions, curcumin was found to be more stable in acidic pH of 1.2 where about 75% of curcumin was remained after 105 min (k value, 0.002303). In case of distilled water, pH 6.8 and 7.4, 69.3% (k value, 0.002303), 51.0 % (k value, 0.005297) and 23.61 % (k value, 0.0152) was remained after 105 min indicating that curcumin may undergo hydrolytic reaction and stability decreases with the increasing pH. The effect of 1 % w/v acids such as ascorbic acid, tartaric acid and citric acid on the stability of curcumin in the higher pH conditions of 6.8 and 7.4 were conducted. An improvement in the stability of curcumin in basic conditions was observed. In presence of 1 % w/v citric acid, in pH 7.4, 60.8 % of drug was present after 105 min. Similarly the presence of 1 % w/v ascorbic acid and tartaric acid, about 77.2 % and 78.4 % of curcumin was remained, indicating the stability increase in stability of 3 folds. In case of pH 6.8 buffer, it was observed that curcumin was more stable than in pH 7.4 buffer. The effect of citric acid, tartaric acid and ascorbic acid on the stability of curcumin was found to improve the stability of curcumin to an extent ranging from 68 % from 51 % at the end of 8 hrs. CONCLUSION From the above study it can be concluded that stability of curcumin can be enhanced by the addition of acids such as citric acid, tartaric acid and ascorbic acid. The maximum improvement in stability of 142


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curcumin was observed by the tartaric acid and ascorbic acid in pH 7.4 buffer, which was also supported by the student T test. ACKNOWLEDGEMENTS The authors would like to thank Prof. B.G. Shivananda, Principal, Al-Ameen College of Pharmacy for his kind support and encouragement. This study was supported by SRF grant to Vivek Yadav from Indian Council of Medical Research, India REFERENCE

1. 2. 3. 4.

5. 6. 7. 8. 9.

10. 11. 12. 13. 14.

15.

Ruby AJ, Kuttan G, Babu KD, Rajasekharan KN and Kuttan R. Antitumour and antioxidant activity of natural curcuminoids. Cancer Lett 1995; 94: 79–83. Gescher AJ, Sharma RA and Steward WP. Cancer chemoprevention by dietary constituents: a tale of failure and promise. Lancet Oncol 2001; 2: 371–379. Sharma RA, Gescher AJ and Steward WP. Curcumin: The story so far. Eur J Cancer 2005; 41: 1955–1968. Sharma RA, McLelland HR, Hill KA, Ireson CR, Euden SA, Manson MM, Pirmohamed M, Marnett LJ, Gescher AJ and Steward WP. Pharmacodynamic and pharmacokinetic study of oral curcuma extract in patients with colorectal cancer. Clin. Cancer Res 2001; 7: 1894–1900. Rao DS, Sekhara NC, Satyanarayana MN and Srinivasan M. Effect of curcumin on serum and liver cholesterol levels in the rat. J Nutrition 1970; 100: 1307–1315. Ramaprasad C and Sirsi M. Indian medicinal plants: Curcuma longa; in vitro antibacterial activity of curcumin and the essential oil. J Sci Ind Res 1956; 15C: 239–241. Ammon HPT and Wahl MA. Pharmacology of Curcuma longa. Planta Med 1991; 57: 1–7. Subramanian L and Selvam R. Prevention of CCI4-induced hepatotoxicity by aqueous extract of turmeric. Nutra Res 1999; 19: 429–441. Ukil A, Maity S, Karmakar S, Datta N, Vedasiromoni JR and Das PK. Curcumin, the major component of food flavor turmeric, reduces mucosal injury in trinitrobenzene sulphonic acid-induced colitis. Br J Pharmacol 2005; 139 (2): 209–218. Tonnesen HH and Greenhill JV. Studies on curcumin and curcuminoids. XXII. Curcumin as a reducing agent and as a radical scavenger. Int J Pharm 1992; 87: 79–87. Wahlstrom B and Blennow G. A study on the fate of curcumin in the rat. Acta Pharmacol Toxicol 1978; 43: 86–92. Pan MH, Huang TM and Lin JK. Biotransformation of curcumin through reduction and glucuronidation in mice. Drug Metab Dispos 1999; 27: 486–494. Holder GM, Plummer JL and Ryan AJ. The metabolism and excretion of curcumin (1, 7-bis-(4-hydroxy-3methoxyphenyl)-1, 6-heptadiene-3, 5-dione) in the rat. Xenobiotica 1978; 8: 761–768. Ireson CR, Orr S, Jones DL, Verschoyle R, Lim CK, Luo JL, Howells L, Plummer SM, Jukes R, Williams M, Steward WP and Gescher A. Characterization of metabolites of the chemopreventive agent curcumin in humans and rat hepatocytes and in rat plasma and evaluation of their ability to inhibit cyclooxygenase-2 expression. Cancer Res 2001; 61: 1058–1064. Wang YJ, Pan MH, Chang AL, Hsieh CY, Lin JK. Stability of curcumin in buffer solutions and characterization of its degradation products. J Pharmaceut Biomed Anal 1997; 15:1867-1876.

q

q

143

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Anthelmintic Activity of the Leaves of Barleria Cuspidata *Manoj Kumar Parida1, Biswajeet Panda1, Samikshya Negi2, B. Behera2, Anasuya Sahoo3 1 GLA Institute of Pharmaceutical Research, Mathura, U.P 2 Gayatri College of Pharmacy, Sambalpur, ORISSA 3 Sir Madanlal Institute of Pharmacy, Etawah, U.P.

ABSTRACT Leaves of Barleria cuspidata were dried, powdered and extracted with Petroleum ether, chloroform, ethanol & water in Soxhlet extractor. Anthelmintic activity of these various extracts was evaluated on Indian adult earthworms Pherentima posthuma. Results showed that the chloroform and Petroleum ether extracts of the leaves of Barleria cuspidata show significant level of activities. Key words : Barleria cuspidate, anthelmintic activity, Pheretima posthuma INTRODUCTION Barleria cuspidata commonly known as kantaphul in India (Acanthaceae) is a low spiny shrub of about 1 meter high. This is distributed mainly in dry districts of Circars Decan & Carnatic. It is also found in the some parts of country like dry areas of Mayurbhanj of Orissa. The stem branches are cylindrical and leaves are entire, opposite, long with slender spines. Flowers are axillary, occur solitary in leaf axils or in terminal spikes have a funnel form with 2 petals (the upper one is divided into 4 & lower one is whole). Commonly known as lesser yellow nail dye/spiny barleria. In Oriya it is known as Kantaphul1,2. Table : Profile of Anthelmintic activity of leaves of Barleria cuspidata Group

Albendazole

Petroleum ether extract

Chloroform extract

Ethanol extract

Aqueous extract

Concentration

Time taken in minutes

of extract in %

Paralysis

Death

1.0

30.66±0.88

42.00±3.60

2.5

24.00±1.52

37.00±3.60

5.0

17.33±1.52

28.66±1.85

1.0

77±1.15

88±1.0

2.5

42.33±1.45

53.66±0.87

5.0

26±0.57

37.33±1.20*

1.0

63±1.73

73.66±1.20

2.5

33±1.73

44.33±2.84

5.0

21.66±0.87

37±1.15*

1.0

62±1.15

71.66±0.87

2.5

52±1.15

62.33±0.87

5.0

36±0.57

49±1.15

1.0

75.33±1.45

85±1.73

2.5

66.66±1.20

78±1.52

5.0

51.33±0.87

62±1.15

Results are expressed as Mean from five observations; Control worms were alive up to 24hrs of observation.

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According to ethno pharmacology, Barleria species are used in inflammation, stomach disorder, rheumatism, ottitis fever, toothache, whooping cough, pneumonia, acne, hepatotoxicity, worm infection and diabetes etc. From the pharmacological and ethno medical investigation on uses of Barleria species shows that Barleria cuspidata may have some therapeutic uses. The literature revealed that no such work on antihelmintic activity was reported. So objective of the present research was to prove traditional anthelmintic use of the plant Barleria cuspidata. Materials and Methods Plant materials: The leaves of Barleria cuspidata were collected in the month of September, 2007 from the forest region of Mayurbhanj, Orissa and get authenticated by Botanical survey of India (BSI), Kolkata, West Bengal.(Voucher specimen number: CNH/I-I/ (209)/ 2007/ Tech.II/.) Drugs and Chemicals All the chemicals and solvents were purchased from CDH, New Delhi. Chemicals and solvents were of reagent grade and solvents were purified before use by standard procedures. Albendazole (BANDY,Mankind Pharma Ltd., New Delhi) was used as standard drug throughout the experiment. Chemicals: Petroleum ether, chloroform and ethanol & saline water. Preparation of extracts Dried and coarsely powdered leaves (500 g) of Barleria cuspidata were subjected to extraction in Soxhlet extractor using solvents Petroleum ether, Chloroform and Ethanol successively. The extracts of leaves of Barleria cuspidata were concentrated by vacuum distillation and then dried in open air to yield 3.6%w/w, 3.8%w/w and 6.112%w/w extracts respectively3. Animals Indian adult earthworms (Pheretima posthuma) collected from moist soil and washed with normal saline to remove all faecal matter were used for the anthelmintic study. The earthworms of 3-5 cm in length and 0.1-0.2 cm in width were used for all the experimental protocol due to their anatomical and physiological resemblance with the intestinal roundworm parasites of human beings4,5. Anthelmintic activity All the extracts of Barleria cuspidata were prepared in Tween80 (1%) to obtain 1, 2.5 and 5% concentrations. Solutions of similar concentrations of the reference standard drug Albendazole were also prepared in distilled water. All the extracts & drugs were freshly prepared before starting the experiment. Two ml of each of the above concentration of various extracts and standard drug Albendazole were diluted to 10 ml separately with normal saline. Six groups of six earthworm each were released into petridish contains 10 ml of desired concentrations (1, 2.5 and5%) of extract and standard drug. The petridishes were divided into six groups. Group I consists of normal saline, Group II consists of standard drug Albendazole and Group III to VI consists of four extracts. Each group consists of 1, 2.5 and 5% concentration and to each concentration equal size of adult earthworm of six numbered were released into petridishes. Times were recorded at the time of releasing the earthworms to each concentration. Observations were made for the time taken to paralysis and death of individual worms. Paralysis was said to occur when the worms did not revive even in normal saline. Death was concluded when the worms lost their mobility followed with fading away of their body colors.6,7. 145


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Results and Discussion The data revealed that the chloroform & petroleum ether extract both were showed significant anthelmintic activity at 5% concentrations. Results were compared with standard drug Albendazole at same concentration. It can be concluded that active constituents responsible for antihelmintic activity are present in chloroform & petroleum ether extracts of leaves of Barleria cuspidata. This indicates that the antihelmintic principles are non polar compounds. ACKNOWLEDGEMENT Authors are thankful to the Head, Botanical survey of India (BSI), Kolkata, West Bengal for their help in the authentication of the plant. REFERENCES

1.

Fisher CFC, Flora of Madras Presidency, Law publishers Pvt. Ltd. vol-II, Part-VI, 740

2.

Hooker JD, Flora of British India, vol-IV, 484

3.

Harborne JB, Phytochemical methods. A guide to Modern Techniques of Analysis, Chapman and Hall Publishers, London; 1973.

4.

Thorn GW, Adams RD, Braunwald E, Isselbacher KJ, Petersdrof RG, Harrison’s Principles of Internal Medicine. Mcgraw Hill Co., New York, 1977. P. 1088-1089

5.

Vigar Z. Atlas of Medical Parasitology. 2nd ed. Singapore, P.G. Publishing House; 1984. p. 216-217.

6.

KP Manjunath, H Shivakumar, T Prakash, KS Patil, A Veeranagouda, BHM Jayakumarswamy, JS Venkatesh, NR Rao. Anthelmintic activity of roots of Swertia chirata. Indian J. Nat. Prod. 2006; 22: 8- 10.

7.

VD Tambe, SA Nirmal, RS Jadhav, PB Ghogare, RD Bhalke, AS Girme, RS Bhambar. Anthelmintic activity of Wedelia trilobata leaves. Indian J. Nat. Prod. 2006; 22: 27-29.

q

q

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Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Formulation and Evaluation of Mouth Dissolving Tablet of Loratidine P. J. Narain, Nimisha, Sanjay Srivastava* Mahatma Gandhi Institute of Pharmacy, Lucknow

ABSTARCT In the recent past several novel technologies have emerged with improved performance,improved patient compliance and reduced adverse effects. Loratidine is a long acting tricyclic antihistaminic drug used in the treatment of allergy. In the present study, an attempt has been made to formulate, optimize and evaluate mouth dissolving tablets of Loratidine by direct compression using two superdisintegrants like Ac-Di-Sol and Poly- plasdone in different ratios with microcrystalline cellulose along with directly compressible mannitol to enhance the mouth feel. The prepared batches of the tablets were evaluated for hardness, friability, drug content uniformity, wetting time, water absorption ratio and in vitro dispersion time. The formulation exhibited good disintegration properties with total disintegration time in the range of 26 to 36s. Key words: Mouth dissolving tablets, Loratidine, Ac-di-sol, Polyplasdone and Primojel, direct compression. INTRODUCTION In the recent past several novel technologies have emerged to deliver drugs to patients in more efficient manner. One such technique is formulation of mouth dissolving tablets (MDTs). MDTs are the novel types of tablets that dissolve, disintegrate or disperse in saliva within few seconds. Its demand has been growing over other oral dosage forms among paediatric, geriatric, dysphagic, psychotic and non-cooperative patients and travelers1, 2. It is estimated that 25% of the population find it difficult to swallow tablets and capsules and therefore do not take their medication as prescribed by the doctors resulting in ineffective therapy. The basic approach used in development of MDTs is the use of superdisintegrants, which provide instantaneous disintegration of tablet after putting on tongue, thereby releasing the drug in saliva3-5. The bioavailability of some drugs may be increased due to absorption of drugs in oral cavity and also due to pregastric absorption of saliva containing dispersed drug that pass down into the stomach6.Moreever, the amount of drug subjected to first pass metabolism is reduced in this MDTs. The basic approach used in the formulation of a rapidly disintegrating tablet by all technologies is to maximize the porous structure of the tablet matrix and to incorporate superdisintegrant like cross linked carboxymethyl cellulose, sodium starch glycolate, poly vinyl pyrrolidine etc. in optimum concentration. So, as to achieve rapid disintergration and instantaneous dissolution for the tablet along with good taste masking properties and excellent mechanical strength7-9. The various technologies used to prepare mouth dissolving tablets includes freeze drying, tablet moulding, spray drying and sublimation10,11. Direct compression is the easiest way to manufacture tablets using conventional equipments and resulting in low cost12. Loratidine, an antihistaminic drug is used for acute coryza, allergic rhinitis and chronic idiopathic urticaria. Loratidine is rapidly absorbed after oral administration but bioavailability is low after conventional administration, due to extensive hepatic metabolism13,14. In the present study, rapidly 147


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

disintegrating tablet of loratidine for pediatric and elderly patients was designed with the aim to develop a dosage form that was easy to administer, provided rapid release of drug and also enhanced bioavailability of the drug. Pregastric absorption through mouth, pharynx and esophagus, could enhance the bioavailability by suitable formulation approaches and also provide local action, as the drug releases in saliva and passes down in to the stomach15,16. MATERIALS AND METHODS Materials Loratidine was a gift sample from Shree Ji Pharma Ltd.Ahemedabad, Gujrat. Mannitol, Microcrystalline cellulose, Ac-Di-Sol, Polyplasdone were procured from Merck Ltd., Mumbai and Central drug house, (New Delhi). Preparation of Tablets MDTs of Loratidine were prepared by direct compression as per the formulae given in the Table 1. Ac-Di-Sol and Polyplasdone were used as superdisintegrants17,18. The tablets were formulated employing direct compression method using 10 mm flat faced punches. The drug, diluent, superdisintegrant, sweetener and flavor were passed through sieve number 4019,20. All the above ingredients were properly mixed with 1% of magnesium stearate and 1% talc then passed through mesh number 80, mixed and blended with the initial mixture. The resulting powder blend were compressed into flat tablets21,22. Evaluation of Powder Blend Bulk Density (Db)23 Apparent bulk density (gm/ml) was measured by pouring the pre-sieved weighed powder (passed through standard sieve 20) into a graduated and measuring its volume. Db = M/ V0 M = mass of powder, V0 is the Bulk volume of the powder. Table-1 : Formulation composition of Mouth Dissolving Tablets of Loratidine Batches (mg/tab) Ingredients

A1

A2

A3

A4

A5

A6

A7

A8

Loratidine

10

10

10

10

10

10

10

10

Ac-Di-Sol

2

-

2

4

8

4

8

-

Polyplasdone

-

2

2

4

4

8

-

8

Mannitol

40

40

40

40

40

40

40

40

136

136

134

130

126

126

130

130

Sod Saccharine

2

2

2

2

2

2

2

2

Mint flavour

4

4

4

4

4

4

4

4

Aerosil

4

4

4

4

4

4

4

4

Mag. Stearate

2

2

2

2

2

2

2

2

MCC- Avicel102

Tapped Density (Dt)24 It is determined by placing a graduated cylinder containing a known mass of powder blend on mechanical tapping apparatus, which was operated for a fixed number of taps (100) until the powder bed volume has reached a minimum. Using the weight of drug in cylinder and this minimum volume, the tapped density may be computed. Dt = M/ Vt M mass of powder,Vt tapped volume of the powder. 148


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Angle of repose (q)25 The frictional forces in a loose powder can be measured by the angle of repose; q. It is defined as the maximum angle possible between the surface of a pile of powder and the horizontal plane. A glass funnel was secured with its tip at a given height (H) above the piece of graph paper placed on a horizontal surface. The powder mixture was allowed to flow through the funnel. tan q = h / r , q= tan -1 (h / r) Where, q is the angle of repose, h is the height in cms. Carr’s Index (I) It indicates powder flow properties. It is expressed in percentage and is given by I=

Dt –Db ———— X 100 Dt tapped density of the powder, Db bulk density of powder. Dt

Evaluation of tablets26-29 Tablets were evaluated for hardness, friability, weight variation, thickness, disintegration time in vivo and in vitro, wetting time and dissolution. Hardness The hardness of the tablet was determined using a Monsanto hardness tester. It is expressed in kg/cm2. Friability (F) The friability of the tablet was determined using Roche Friabilator. 20 tablets were initially weighed (Winitial) and transferred into the friabilator.The friabilator was operated at 25rpm for 4 mins. The % friability was then calculated by Winitial – W final F = ­­­­­———————— X 100 Winitial Weight Variation 20 tablets were selected randomly from the lot and average weight was determined using an electronic balance. Tablets were weighed individually and compared with average weight. Disintegration Time (IN -VITRO) One tablet was placed in each of the six tubes of the disintegration test apparatus and disk was added to each tube. The time taken for complete disintegration of the tablet with no palpable mass remaining in the apparatus was measured in seconds. Disintegration Time in (IN- VIVO) The disintegration time in oral cavity of human volunteers was measured by placing the tablets on the tongue until no lumps remain. Wetting Time and Water absorption ratio A piece of tissue paper folded twice was placed in a small Petri dish( internal Diameter of 5 cm) containing 6.5 ml of water. A tablet was placed on the paper, and the time required for complete wetting of the tablet was measured. The wetted tablet was then weighed. 149


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Water absorption ratio ‘R’ was calculated using the equation R= 100 X (Wb-Wa) /Wa, where Wa is weight of tablet before water absorption and Wb is weight of tablet after water absorption. Drug Content 10 tablets were weighed and triturated. The tablet triturate equivalent to 10mg of the drug was weighed accurately, dissolved in 0.1M hydrochloric acid and liquid was filtrated (whatmann No.1 filter paper). The loratidine content in the filtrate was determined by measuring the absorbance spectrophotometrically at 275.5 nm against the reagent blank. In vitro Dissolution Studies The In vitro dissolution study was carried out in USP dissolution test apparatus Type 2 (Electrolab, Modle-TDT 06 N) employing a paddle stirrer at 50 rpm using a dissolution medium of 900 mL of 0.1 M Hydrochloric acid at 37 ± 0.50C. 5 ml sample were withdrawn at specific interval of time and analyzed for drug content by measuring the absorbance spectrophotometrically at 275.5 nm. The volume withdrawn at each time interval was replaced with fresh quantity of the dissolution medium. Cumulative percentage of loratidine released was calculated and plotted against time. Table-2 : Evaluation of Powder Blends of Loratidine and Excipients Formulation

Bulk Density*(gm/ml)

Tapped Density*(gm/ml)

Angle of Repose*(0)

Carr’s Index

A1

0.58 ± 0.067

0.66 ± 0.075

27.06 ± 1.67

0.82

A2

0.57 ± 0.097

0.66 ± 0.084

27.09 ± 1.34

0.86

A3

0.56 ± 0.057

0.65 ± 0.046

26.57 ± 1.56

0.86

A4

0.58 ± 0.057

0.66 ± 0.076

24.62 ± 0.89

0.87

A5

0.6 ± 0.034

0.69 ± 0.027

24.42 ± 1.64

0.86

A6

0.61 ± 0.046

0.69 ± 0.064

24.17 ± 1.75

0.88

A7

0.67 ± 0.047

0.67 ± 0.047

24.51 ± 1.42

0.85

A8

0.58 ± 0.023

0.66 ± 0.087

24.49 ± 1.63

0.87

* The data are expressed as mean ± SD (n=3)

Table-3 : Evaluation of Mouth Dissolving Loratidine Tablets Formulation

Weight Variation*(%)

Hardness* (kg/cm2)

Friability* (%)

InVitroDT (sec)

InVivoDT (Sec)

A1

5.7 ± 0.51

3.2 ± 0.39

0.5 ± 0.14

52-58

60-66

A2

6.0 ± 0.22

3.2 ± 0.67

0.7 ± 0.12

65-70

62-68

A3

5.5 ± 0.34

3.0 ± 0.46

0.6 ± 0.25

58-60

60-65

A4

5.0 ± 0.28

3.0 ± 0.69

0.6 ± 0.15

3-5

3-4

A5

5.4 ± 0.30

3.0 ± 0.59

0.6 ± 0.17

3-5

3-4

A6

5.0 ± 0.54

3.0 ± 0.34

0.6 ± 0.13

5-7

4-5

A7

5.2 ± 0.28

3.0 ± 0.76

0.6 ± 0.21

32-35

25-27

A8

5.0 ± 0.39

3.0 ± 0.94

0.6 ± 0.16

38-45

30-34

* The data are expressed as mean ± SD (n=3)

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Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Table-4 : Dissolution profile for pure drug Loratidine, prepared MDT batch A4 and marketed conventional tablet formulation Time

Cumulative Percentage Drug Release*

(Minutes)

Pure Drug

Batch A4

Marketed Tablet

5

2.0 ± 0.12

87.50 ± 2.27

0.0

10

2.0 ± 0.38

89.05 ± 2.18

2.30 ± 0.42

15

8.0 ± 0.25

91.13 ± 2.76

4.2 ± 0.58

20

15.0 ± 0.15

93.48 ± 3.61

13.6 ± 0.35

25

20.0 ± 0.27

95.76 ± 4.21

20.4 ± 0.14

30

26.0 ± 0.35

98.63 ± 2.47

39.5 ± 0.34

* The data are expressed as mean + SD (n=3)

Figure 1 : .In vitro release for pure drug Loratidine, prepared MDT batch A4 and marketed conventional tablet formulation.

RESULTS AND DISCUSSION Preformulation studies were carried out by using FTIR, spectrophotometry and thin layer chromatography, all showed that loratidine molecules was compatible with excipeints. The result obtained by evaluating the powder blend of drug and excipient is shown in Table 2. Bulk density of various formulation ranges between 0.57-0.61 gm/ml. angle of repose was found in the range of 240-270, and Carr’s index was in the range of 0.82-0.87. From these values, it was evident that powder blends were free flowing and had good compressibility. The results for evaluation of different batches of loratidine mouth dissolving tablets prepared by direct compression method are shown in Table 3. Hardness of all formulation was in range of 3.0-3.2 Kg/cm2. The friability values of none of the 151


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

formulations exceeded 0.7%. It can bear indicates that the tablets were mechanically stable and could handle the rigors of transportation. Weight variation was found within specification of the USP limits. In vitro and in vivo disintegration time for different batches of mouth dissolving tablets was 3 to70 seconds and 3 to 68 seconds respectively. The formulation containing Ac-Di-Sol and Poly-plasdone alone at low concentration (2mg per tablet) showed higher values of 52 to 70 seconds. The formulations containing 4mg of Ac-Di-Sol and Polyplasdone in combination per tablets showed 3-5 seconds. The tablets prepared without combination of Ac-Di-Sol and Poly-plasdone showed higher time. Batch A4 containing 4mg per tablet of Ac-Di-Sol and Poly-plasdone showed minimum 3 to 5 seconds in vitro and in in vivo disintegration time. Hence A4 batch was used for further studies. The rapid disintegration may be due to the rapid uptake of water from medium, swelling and brust effect. Wetting time of batch A4 was found to be 2 seconds. The dissolution profile from pure drug, batch A4 and marketed formulation is shown in Table 4. It was observed that in first 10 minutes, only 2.0% drug was released from pure drug and marketed conventional tablet formulation while it was 89.0% in case of MDT. At the end of 30 minutes, 98.63% drug was released from the batch A4 as compared to the pure drug and conventional tablet formulation in which only 26.0% and 39.5% drug was released respectively (Figure 1). Thus the release rate of loratidine was significantly enhanced by formulating MDTs by using superdisintegrants. Conclusion From the study, it can be concluded that Fast Dissolving Tablets of Loratidine with sufficient crushing strength and disintegration time can be formulated by direct compression by using a combination of Ac-Di-Sol and Polyplasdone as superdisintegrants. It can be conclusively inferred that the mouth dissolving tablet of loratidine would be quite effective in providing quick onset of action without need for water for swallowing or administration. Acknowledgement Free gift samples of Loratidine (Shree Ji Pharma Ltd. Ahemedabad is greatfully acknowledged. REFERENCES 1.

Akihiko L., Masayasu S., Development of oral dosage form for elderly patients: Use of agar as base of rapidly disintegrating tablet.Chem Pharm Bull.1996;44:2132- 2136.

2.

Agarwal G. P., Jain N. K., Study on some fast release products of ibuprofen. Indian Drugs.1998; 26: 226.

3.

Avari J.G., Bhalekar M.Cation exchange resins for taste masking and rapid dissolution of sparfloxacin. Indian Drugs.2004;41(1):19-21.

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Bi Y., Sunada H., Danjo K., Otsuka A, Preparation and evaluation of a compressed tablet rapidly disintegrating in the oral cavity. Chem Pharm Bull.1996; 44: 2121-2127.

5.

Bi Y., Sunada H., Danjo K., Yonezawa Y., Evaluation of rapidly disintegrating tablets prepared by a direct compression method. Drug Dev. Ind. Pharm. 1999;25:571-581.

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Caramella C. Colombo P, La maan A., The role in the disintegration process’, Int J Pharm Technol Prod Manuf. 1984;2: 1-5.

7.

Duru C.A comparative study of the disintegrating efficiency of polysaccharides in a directly compressible tablet formulation.Pharm. Technology Int. 1992; 4(5):15-20.

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8.

Fassihi A.R.Characteristic of hydrogel as disintegrant in solid dose technology’ J Pharm. Pharmacology.1989; 54:59-62

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Halakatti P.K., Manvi F.V. Rapidly disintegrating domperidone tablets. Indian Drugs. 2006;42(7) : 594-597.

10. Chang R.,Guo X., Burnside B.A., Fast- dissolving tablets.Pharm. Technol. Eur.2000; 12(6):52-58. 11. Devi V K, Pai R S., Orodispersible fluconazole tablets-Preparation and evaluation’, Indian Drugs.2006;43(7):548-552. 12. Kuchekar B.S., Mahajan H S, Mouth dissolving tablets: A novel drug delivery system. Pharma Times.2003;35:1-7. 13. Chaudhari P.D., More D.M., Formulation and evaluation of fastdissolving tablets of famotidine. Indian Drugs. 2005;42(10):641-649. 14. Costa P., Sousa J.M., Review : Modeling and comparison of dissolution profiles’, Eur. J. Pharm. 2001;123-133. 15. Feinstein W., Bartilucci A.J., Comparative study of selected disintegrating agents.J Pharm Sciences. 1996;55:332334. 16. Gohel M.C., Bhatt N, Formulation and evaluation of orodispersible taste masked tablets of famotidine. Pharma. Bio. World.2005; 75. 17. Gohel M., Bariya A., Formulation and evaluation of fast dissolving tablets of nimesulide. Pharm. Sci. Tech. 2004; 26; 36. 18. Joshifusa S., Kunio I., Studies of rapidly disintegrating tablets in the oral cavity co-ground mixtures of mannitol with crospovidone.Chem. Pharm. Bull. 2002; 50: 193. 19. Kornblum S, Stoopak S, A new tablet disintegrating agent: Crosslinked polyvinyl pyrollidone J Pharm Sci. 1973;62:4349. 20. Jean P.R., Sam C., Formulation and production of rapidly disintegrating tablets by lyophilisation using hydrochlorothiazide as a model drug. Int. J. Pharm. 1997;152: 215. 21. Kaushik, D., Kuchekar B.S., Mouth dissolving tablets : A review. Indian

Drugs. 2004;41:187-200.

22. Kuchekar B S , Badhan A, Mahajan H S, Mouth dissolving tablets of salbutamol sulphate : A novel drug delivery system. Indian Drugs. 2005; 41(10):25-28. 23. Mahajan H S, Badhan A C, Mouth dissolving tablets ofsumatriptan succinate. Indian J Pharm Sci.2004; 66:238239. 24. Pandey S., Shenoy V, Optimizing fast dissolving dosage form of diclofenac sodium by rapidly disintegrating agents. Indian J Pharm Sci. 2003; 40:197-200. 25. Nayak S M, Gopal K P Design and optimization of fast dissolving tablets for promethazine theoclate. Indian Drugs. 2003; 41(9): 42-47. 26. Patel D M, Jogani P D, Studies in formulation of orodispersible tablets of rofecoxib. Indian J Pharm Sci. 2004;66: 621-624. 27. RajyaguruT H, Induwade N H Novel approach Fast dissolving tablets.Indian Drugs.2002;39:401- 405. 28. Shangraw R, Shah M, Mitrevej A A, new era of tablet disintegrants. Pharm. Technol.1982;4:49-57. 29. Shirwaikar A., Dutta M, Once dailyast dissolving tablets of granistron hydrochloride formulation and in vitro evaluation’, Indian Drugs, 2006, 43(7),576-581.

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Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Production of Jackwine – A Wine from Ripe Jackfruit Amit K. Tiwari, Ambarish S. Vidyarthi Birla Institute of Technology, Mesra, Ranchi, India

Abstract Jackfruit (Artocarpus heterophyllus) is a horticultural crop of almost all parts of India and widely used for culinary purposes. The fruit contains carbohydrates, latex, vitamins and minerals. Sugar content in the fruit varies with the age and parts of fruit. Attempts were made to produce wine (Jackwine) from ripe jackfruit. The maximum alcohol content in the jackwine was 10% (V/V); with a sugar utilization of 14% (TSS). These early results promise the use of this fruit for commercial wine production beside its utilization in vegetable, pickles & other food products. The development of suitable technology based on the process will help the farmer to increase their livelihood and thus, strengthening the economy of rural India. Introduction Jackfruit, a very popular and cheap food in the eastern and southern India, is hardly regarded as a commercial fruit. Jackfruit is widely grown and is one of the largest produce amongst the edible fruits in the central region of the country and has a high local demand (1). It is also grown extensively in the west coast, Assam, West Bengal, Orissa, Bihar and Jharkhand (2). Approximately 40% of the total produce of jackfruit is spoiled in various post harvest stages, in some areas, the jackfruit is fed to cattle. The fruits develop during spring and summer. The fruits become ready to harvest in June or early July. Generally Jackfruit is most acceptable in the very tender stage for cooking as curried vegetable. The fruits ripen when the maximum temperature reaches during the end of summer season. Jackfruits turn brown and deteriorate quickly after ripening. The ripe fruits can be kept for a maximum of 3- 6 weeks at 50 - 550F (11-130C) and relative humidity 85 –95% (3). The fully developed jackfruit may be 20- 90 cm long, 15-50 cm wide and weight ranges from 3 to 40 kg (60 kg under extreme case). All parts contain sticky white latex. The exterior shell (rind) of ripe fruit is greenish yellow color. The interior consists of large bulbs, yellow flesh etc. (4). Ripe fruit has high nutritive value and contains minerals, vitamins, Proteins and carbohydrates. The fruit contains 25-40% pulp of its total weight (5, 6). It is good source of vitamin A, B along with minerals. Recent reports (7) suggest that the bulbs of ripe Jackfruit could be utilized for making jackfruit beverages using fruit Juice. The fruit pulp can be used for the production of Jam. The addition of synthetic flavouring agent such as ethyl and n-butyl esters of 4-hydroxybutric acids at 100 and 120ppm, respectively greatly improves the flavour of the Jackfruit products. The ripe Jackfruit contains good amount of sugar, which may be explored for the commercial production of Vinegar and Wines. Development of food products like fermented beverages especially fruit wine and vinegar from ripe jackfruit using food processing and biotechnological techniques will not only reduce the losses of fruit material but also make the fruit’s product availability beyond the season and generate the money and also more employment opportunities in rural areas. 154


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Materials and Methods Yeast culture preparation Saccharomyces cerevisiae var. ellipsoideus was used for the preparation of wine. Yeast was taken from the Department of Pharmaceutical Sciences, B.I.T., Mesra, Ranchi. Yeast (5 g/L) were cultured at 250C in a presterilized MPYD medium (malt extract 0.3%, peptone 0.5%, yeast extract 0.3%, dextrose 2.0%) for 24 hrs, and 2% (v/v) inoculum was used to inoculate 0.5 L of medium in a 1000 ml conical flask and covered by sterilized cotton stopper. Fermentation process was carried out at 250C for about 2 days as suggested by Reddy & Reddy (8). This yeast culture was used to inoculate the jackfruit pulp to study the wine production. The yeast stack culture was maintained on MPYD agar (malt extract 0.3%, peptone 0.5%, yeast extract 0.3%, dextrose 2.0% and Agar 1.5%) slants at refrigerated temp. Wine preparation The inoculum was prepared by adding the a loopfull slant culture into 25 ml sterile MPYD liquid medium in 100 ml conical flask and incubated in orbital shaker at 300C for 48 hrs. Then this inoculum was mixed with 100 ml jackfruit pulp in a 500ml conical flask. Batch fermentation of the must was done in conical flask by incubating at 25¹10C temperature for 25 days. The fermented samples were analyzed for residual sugar and ethanol content. The concentration of sugar content was determined by Lane & Eynon method (9) measures of Total soluble solids were taken with the help of BM Hand Refractometer. Total acidity was estimated by titration with N/10 NaoH as citric acid (9). Ethanol content in the wine was determined by taking refractive index with the help of table type Refractometer. Yeast fermentation was also studied with different sugar contents using a synthetic medium similar to the jackfruit pulp composition for production of wine. Synthetic medium include sucrose 280 g/L and citric acid as natural acidity 2 g/L. The medium was sterilized at 15 psi for 15 min. Results and Discussion Wine prepared from the pulp obtained from fully ripe fruit. At the time harvesting the TSS of the fruit pulp was 29 °Brix. The fermentation of pulp was done by using yeast culture (Saccharomyces cerevisiae) that converts the simple sugars into ethanol. The result of change in the sugar concentration during the period of fermentation is shown in Table-2. The selection of fruit was based on the maturity level and Total Soluble Solids (TSS) of ripe jackfruit; the results are presented in Table-1. Maturity level on the basis of harvesting time and TSS are shown in Figure 1(a). Changes in sugar concentration was determined on every 5th day, two extra readings were taken to confirm results (Table-2). The decrease amount of sugar was noticed in due course of fermentation using S. cerevisiae suggested the increased amount of ethanol. Total ethanol content was found 10.5% (v/v) after 25th days of fermentation. The appearance of the wine was also acceptable by group of peoples as shown in Figure 1(b). These preliminary results demonstrate the utilization of Jackfruit for commercial wine production beside its conventional uses in vegetable, pickles & other food products. The development of suitable technology based on the process will help the farmer to increase their livelihood and thus, strengthening the economy of rural India. 155


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

(a) (b) Figure 1 : Variation in TSS with the age of the jackfruit (a) and the production of Jackwine (b) Table-1: Quality parameters of ripe Jackfruit S. No.

Fruit harvesting age (in days)

Wt. of fruit in Kg

TSS °Brix

Skin colour of fruit

1

60

3.2

18

Green

2

65

3.5

19.8

Green

3

70

4.0

22.6

Yellowish green

4

75

4.3

27

Yellowish brown

5

80

4.8

29

Yellow

Table-2 : Changes in sugar content during fermentation period

S. No.

Days of Estimation

Initial Sugar (%)

Sugar utilization during fermentation (%)

1

Control

28

0

Alcohol content (%) -

2

5 day

26

2

-

3

10th day

21

7

-

4

15th day

17

11

-

5

20th day

14

14

10.0

6

25th day

14

14

10.5

7

30 day

14

14

10.5

th

th

Conclusions On the basis of ethanol produced, appearance and the acceptability of the wine by group of peoples it can be concluded that the certain maturity level and ripeness of jackfruit (2930 ÂşBrix), are essential for the production of jackwine. The jackwine fermented with yeast Saccharomyces cerevisiae var. ellipsoideus has good flavour and taste, and thus acceptable to a group of people. 156


Pharmbit Vol. XXII, No. 2, Jul - Dec, 2010

Acknowledgement The authors thankfully acknowledge the financial support from Ministry of Food Processing Industries, Government of Indai New Delhi and infrastructure support from Department of Biotechnology, BIT, Mesra, Ranchi. References

1.

JJ Ochse, MJ Soule Jr., MJ Dijikman, and C Welburg. Tropical and Subtropical Agriculture. Macmillan Co.; 1981 pp-652-5.

2.

Jharkhand Opportunity (www.jharkhandyellowpages.net/opportunity.php) (Last Accessed on Dec., 2008)

3.

KK Singh. Farm Information Bull. No.71. Directorate of Extension, Ministry of Agriculture, New Delhi; 1972

4.

Anonymous. Fruits for the Future: Jackfruit. International Centre for Underutilized Crops. Factsheet No.6; 2003.

(www.icuc-iwmi.org/files/News/Resources/Factsheets/jackfruit) (Last Accessed on Dec., 2008) 5.

Anonymous. Extension Bulletin No.22, Indian Institute of Horticultural Research, Bangalore; 1979

6.

JF Morton. Fruits of warm climates. Miami, FL, USA; 1987. http://www.hort.produce.edu/newcrop/morton/jackfruit_ars.html (Last Accessed on Dec., 2008)

7. Anonymous. www.smallindustryindia.com/ publications/pmryprof/food/ch14.pd (Last Accessed on Dec., 2008) 8. LVA Reddy and OVS Reddy. Production and characterization of wine from mango fruits (Mangifera indica Linn.). World J Microbial Biotechnology, 2005, 21, 1345-50. 9.

S Ranganna. Analysis of Fruit and Vegetable Products. 2nd edition, 2003, pp 12-16.

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Instructions to Authors PHARMBIT is an official scientific journal and biannual publication of Pharmaceutical Society of Department of Pharmaceutical Sciences, Birla Institute of Technology, Ranchi.The journal is devoted to publish review and research articles in pharmacy and the related disciplines of pharmaceutical education. Manuscripts will be subjected to peer review process to determine their suitability for publication provided they fulfill the requirements of the journal. After reviewer’s comments the revised manuscript should be submitted by e-mail. Submission of a manuscript to PHARMBIT for publication implies that the same has not been either published or under consideration for publication in another journal. The author should confirm during submission of manuscript. Preparation Of Manuscripts : Resaerch Papers Manuscripts must be prepared in accordance with “Uniform requirements for Manuscripts submitted to Biomedical Journal” developed by International Committee of Medical Journal Editors (October 2001). Manuscripts should be concisely written and conform to the following general requirements: Manuscripts should be typewritten in double- space in A4 sized sheets, only on one side, with a 1.0 inch margin on both sides. Not including illustrations, Research Papers, should not exceed 8-10 pages, Review Articles, 12-15 pages and Short Communications, 4-5 pages. Pages should be numbered consecutively, starting with the title page and the matter arranged in the following order: Title page, Abstract, Introduction, Materials and Methods, Results, Discussion or Results and Discussion, Acknowledgements, References and Illustrations (Tables and Figures including chemistry schemes along with titles and legends). Each table or figure should be on separate sheets. All other section titles should be in capital letters while subtitles in each section shall be in bold face lower case followed by a colon. TITLE PAGE - Title page should contain title of the paper in bold face, title case (font size 14), names of the authors in normal face, upper case (font size 12) followed by the address(es) in normal face lower case. The author to whom all correspondence be addressed should be denoted by an asterisk mark. ABSTRACT - Start on a new page after the title page and should be typed in single-space to distinguish it from the Introduction. Abstracts should briefly reflect all aspects of the study, as most databases list mainly abstracts. Short Communications as well as Review Articles should have an Abstract. INTRODUCTION - Start immediately after the Abstract, as the next paragraph, but should be typed in double-space. The Introduction should lead the reader to the importance of the study; tie-up published literature with the aims of the study and clearly states the rationale behind the investigation. MATERIALS AND METHODS - Start as a continuation to introduction on the same page. All important materials used along with their source shall be mentioned. RESULTS - All findings presented in tabular or graphical form shall be described in this section. The data should be statistically analyzed and the level of significance stated. Results section shall start after materials and methods section on the same page. DISCUSSION - This section should follow results, deal with the interpretation of results, convey how they help increase current understanding of the problem and should be logical. Results and discussion of results can also be combined under one section, Results and Discussion. 158


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