Indian Journal of Pharmaceutical Education & Research

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email: [email protected] | Website: www.ijperonline.com. Dr. Srinivasa Murthy [email protected]. Dr. Kulkarni P.K. pkkulk@lycos.com. Dr. Mallikarjuna Rao ...
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Indian Journal of Pharmaceutical Education & Research

Vol. 43(2), Apr-Jun, 2009 Abstracted and Indexed in Science Citation Index Expanded (Scisearch) and Journal Citation reports

A P T I

PAST EDITORS •





Dr. Nagavi B.G. Mysore 1997 - 2006 Dr. Rao M.N.A. Manipal 1995-1996 Dr. Gundu Rao P. Manipal 1985-1995









Dr. Kasture A.V. Nagpur 1981 - 1984 Dr. Saoji A.N. Nagpur 1980 - 1980 Dr. Lakhotiya C. L. Nagpur 1979 - 1980





Dr. Chopde C.T. Nagpur 1978 - 1978 Dr. Gundu Rao P. Manipal 1975 - 1978 Dr. Mithal B. M. Pilani 1967 – 1974

EDITOR–IN–CHIEF

Dr. Sanjay Pai P.N. [email protected]

ASSOCIATE EDITORS Dr. Srinivasa Murthy [email protected]

Dr. Mallikarjuna Rao C. [email protected]

Dr. Kulkarni P.K. [email protected]

Dr. Mueen Ahmed K. K. [email protected]

EDITORIAL OFFICE INDIAN JOURNAL OF PHARMACEUTICAL EDUCATION AND RESEARCH The Official Publication of Association of Pharmaceutical Teachers of India H.Q.: Al-Ameen College of Pharmacy, Opp. Lalbagh Main Gate, Hosur Road, Bangalore 560 027, INDIA Mobile : 91-9448207428 | 91-9242898028 | 91-9845655732 | 91-9880423041 | 91-9448445612 Fax: 080-22225834; 080-22297368 email: [email protected] | Website: www.ijperonline.com

Indian Journal of Pharmaceutical Education & Research

ijper

Vol. 43(2), Apr-Jun, 2009 Abstracted and Indexed in Science Citation Index Expanded (Scisearch) and Journal Citation reports

EDITORIAL ADVISORY BOARD Dr. Miglani B.D., New Delhi. Dr. Murthy R.S.R., Vadodara. Dr. Nagavi B.G., Dubai. Dr. Pulok K Mukherjee, Kolkata. Dr. Rao M.N.A., Hyderabad. Dr. Ravi T.K., Coimbatore. Prof. Shivananda B.G., Bangalore. Dr. Shivakumar H.G., Mysore Dr. Subrahmanyam C.V.S, Hyderabad. Dr. Suresh B., Ooty. Dr. Tipnis H.P., Mumbai. Dr. Udupa N., Manipal. Dr. Vyas S.P., Sagar.

Dr. Betgeri G.V., USA. Dr. Mrs.Claire Anderson, UK. Mr. Frank May, USA. Dr. Gaud R.S., Mumbai. Dr. Goyal R.K., Ahmedabad. Dr. Harkishan Singh, Chandigarh. Dr. Hukkeri V.I., Bangalore. Dr. Jagdeesh G., USA. Dr. Katare O.P., Chandigarh. Dr. Khar R.K., New Delhi. Dr. Madan A. K., Rohtak. Dr. Madhusudhan Rao Y., Warangal. Dr. Manavalan R., Annamalai Nagar. Publication Committee • Pharmaceutics

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Dr. Paradkar A.R., Dr. Sarasija Suresh, Dr. Vavia P.R. Dr. Gopal Krishna Rao, Dr. Raghurama Rao A. • Pharmaceutical Chemistry and Analysis Dr. Valliappan K. Dr. Krishna D.R., Dr. Kshama Devi, • Pharmacology Dr. Sreenivasan B.P. Dr. Ganapaty S., Dr. Salma Khanam, • Pharmacognosy Dr. Swati S.Patil Dr. Nagappa A.N., Dr. Rajendran S.D, • Pharmacy Practice Dr. Shobha Rani R.H. Dr. Raman Dang, Dr. Unnikrishnan M.K., • Pharmaceutical Education Dr. Bhise S.B. Dr. Burande M.D., Dr. Gayathri Devi S., • Pharmaceutical Marketing Dr. Kusum Devi V. Note: The Editor does not claim any responsibility, liability for statements made and opinions expressed by authors. INDIAN JOURNAL OF PHARMACEUTICAL EDUCATION AND RESEARCH The Official Publication of Association of Pharmaceutical Teachers of India H.Q.: Al-Ameen College of Pharmacy Opp. Lalbagh Main Gate, Hosur Main Road, Bangalore - 560027 INDIA Fax: 080-22225834; 080-22297368; email: [email protected] | Website : www.ijperonlline.com

Indian Journal of Pharmaceutical Education & Research

ijper Vol. 43(2), Apr-Jun, 2009

CONTENTS

Invited Article • D. Pharm or Pharm D: A Professional chirality? Professor S. K. Kulkarni ..............................................................................................................................114-116 Articles • Formulation and Modulation of Drug Release from an Intra Vaginal Ring Vidya Iyer and S.S.Poddar….…...…………………………………………………………………………….........117-124 • Impact of Community Pharmacist Provided Patient Education in Asthma Patients on Treatment Outcomes- A Study Anjan Kumar D.S., Ramesh Adepu, G. Parthasarathi and P.A. Mahesh……………………………………...125-133 • Modified Diffusion Apparatus for Evaluation of Transdermal Drug Delivery System M. C. Gohel, R. K. Parikh, R. R. Delvadia, S. A. Nagori, K. G. Sarvaiya, B. H. Patel and V. P. Patel......134-139 • Parallel Combinatorial Synthesis and In Vitro Evaluation of Ester and Amide Prodrugs of Flurbiprofen, Ibuprofen and Ketoprofen. A. Lohade, P. Jain and K. R. Iyer.................................................................................................................140-149 • Taste Masking and Formulation of Ofloxacin Rapid Disintegrating Tablets and Oral Suspension Shishu, Varun Rishi Kapoor, Kamalpreet……………………………………………………….........................150-155 • Stress Stability Studies and the Estimation of Lamotrigine in Pharmaceutical Formulation by Validated RPHPLC Method Prabhat K. Shrivastava and Sushant K. Shrivastava ………………………………………………………........156-161 • Effect of an Antacid on the Oral Pharmacokinetics of Rosiglitazone in Healthy Human Volunteers Suresh Kumar J.N., Prameela D. and Mullangi R…………………………………………………………….....162-165 • Pharmacological Investigation of Protective Effects of Nigella sativa oil in Experimental Diabetic Neuropathy in rats. Mohammad Abid Ansari, Shibli Jameel Ahmad, Razia Khanum, Mohammad Akhtar................................166-176 • Preparation and In Vitro Evaluation of Gastric Floating Microcapsules of Metformin HCl Bipul Nath, L.K.Nath, B.Mazumdar, N.K.Sharma, M.K.Sarkar………………………………………………...177-186 • Interfacial Tension Using A Simple Laboratory Technique Hadkar U.B., and Ravindra R.P………………………………………………………………………………........187-191 • Journal Club as an Effective Teaching Learning Process in Pharmacy Education Jiny Varghese K, Sonal Sekhar M, Asha Jose, Revikumar KG………………………………………………....192-198 • Isolated Cock Ileum: A Tool For Pharmacology Experiments Surendra H. Bodakhe, J.S.Dangi, Alpana Ram, K. P. Namdeo and Kiran S. Bodakhe...............................199-202 • Need of Quality Education for Changing Pharma World Bhupinder Singh and O. P. Katare...............................................................................................................203-215

Indian J.Pharm. Educ. Res. 43(2), Apr-Jun, 2009

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ijper Invited Article D. Pharm or Pharm D: A Professional chirality? Professor S. K. Kulkarni Panjab University, Chandigarh [email protected]

Perspective and Preparedness Douglas Hepler and Linda Strand of the University of Minnesota, USA described the concept of Pharmaceutical care (PC) in 1990 (1). They defined the term PC as the responsible provision of drug therapy for the purpose of achieving definite outcomes that improve the patients quality of life. Further, PC implied that the practitioner (Pharmacist) takes responsibility for patient’s drug related needs and holds him/herself accountable for meeting these needs. In other words pharmacist assumes the responsibility for providing a service of real value and for which he would be paid for. This role is much bigger and responsible than simply dispensing a drug. The definition of pharmaceutical care essentially addressed two issues, namely the patient (as the focus) and the responsibility of the pharmacist for providing real service (practice and the value of it). In order to meet this responsibility, the profession of pharmacy in the USA debated on the issue of “dispensing” to “dispensing and patient care”. The patient care required the pharmacist to know and also have expertise on drug related morbidity and mortality, selection of drug therapy, its optimization and cost effectiveness. It took almost 10 years for the American Association of the Colleges of Pharmacy (AACP) to effectively advocate and mandate the concept and to bring in the changes in the profession’s mission and direction. These included changes in regulations, reimbursement, pharmaceutical education, practice, patient education and expectations, relationships with physicians, responsibility of professional associations, physical structures of pharmacies and attitudes of the pharmacist. The AACP has not only re-defined its goals and mission to advance the quality of pharmacy education and the leadership role of the pharmacist but also

mandated doctor of Pharmacy (Pharm D) degree (a six year program) as the first professional degree. This new professional degree has now completely replaced the traditional B.S. degree in Pharmacy in the United States of America. WHO initiative Almost a decade ago (1997-98) on the occasion of the 50th anniversary, the World Health Organization (WHO) took the initiative to prepare a global health policy framework so that all health professionals are appropriately and adequately prepared to face the health problems of the 21st century. In this endeavor WHO addressed pharmacy and pharmaceutical sciences as a focal point for a global health policy discussion. The WHO constituted a consultative group (called Vancouver consultative group, represented by 22 countries) to prepare a mission paper for the profession and practice of pharmacy and to implement WHA 47.12 resolution. As a member of the team I visited many countries and studied the healthcare requirements particularly the pharmaceutical requirement depending upon the social, economic and cultural backgrounds. The Vancouver consultative group defined the quality of an ideal front-line pharmacist as a “Seven-Star pharmacist” (2). The Seven-Star pharmacist is expected to be a care giver, decision maker for drug therapy, communicator and a community leader. In order to assume these roles he/she has to co-learn and work closely with the other members of the healthcare team (physicians, nurses and health workers). An Indian thought process At the 48th session of the IPC at Chennai (1996), I delivered a plenary lecture entitled “Pharmaceutical Education in tune with times” arguing for the profession to redefine the goals of pharmaceutical education to meet social and economic needs of our country in the context of the global changing scenario (3). Subsequently, at the First World Congress on

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Pharmacy Education at New Orleans, USA I made presentation on the “Status and future challenges of Pharmaceutical Education in India”(4). My presidential address of the 53rd IPC in 2001 at New Delhi called for “Harmonization of Pharmaceutical Education: Preparing a future pharmacist” (5). Essentially, warning the profession to get prepared for the global changes that are going to take place in pharmacy profession and education. Unpreparedness or business as usual approach Unlike in the developed countries of the West, we in India often fall prey to the buzz words without assessing our own requirements, man-power assessment studies, job opportunities, rural vs. urban, and economic disparities. Unfortunately, pharmacy and the role of pharmacist in healthcare requirement did not figure in the National Health Policy draft of Government of India 2001 or 2006. It was not even at the periphery. In the last one decade the technical education particularly, the pharmaceutical education, has expanded goallessly and in unmindful way. There is a great paucity of pharmaceutically qualified teachers and administrators to impact quality education. Moreover, there are no man-power assessment data available. The admissions to undergraduate courses (B. Pharm) have fallen down drastically during 2008-09 session, (nearly 40 % seats are lying vacant across the country and the situation is worst for D. Pharm course where admissions are in single digits in many colleges). The slowing down of economy is expected to affect the job opportunities for the post-graduates (M. Pharm) in the coming years. D. Pharm or Pharm D: a professional chirality? As per the provisions of the Pharmacy Act 1948, D. Pharm (2 years course) is the minimum qualification required to be a registered pharmacist to practice the profession. According to the Pharmacy Council of India (PCI) and All India Council for Technical Education (AICTE) websites there are nearly 425 diploma colleges and 25,000 students are expected to come out every year. Since there are a very few admissions this year across the country, it appears that there is a saturation in the professional job requirement and unemployment of diploma holders. It is a very serious situation and the profession, more so the Pharmacy Council of India should seriously address the issue. Both PCI and AICTE should assess the man-power

requirement before granting permission to open new colleges. Suddenly this year (2008-09), the PCI has given permission to start Pharm D course and some private colleges/management have started this professional course. It is not clear whether the PCI has done any man-power requirement study about the requirement of Pharm Ds in 2014 when the first batch of Pharm D graduates will come out. Will these new breed of professionals go for hospital jobs, in government or private sector, rural or urban sector and will they be equal partners in healthcare delivery team guiding the physicians in selection of drugs? Only time will tell about the future role of Pharm Ds. It is also argued that some of these Pharm Ds will seek greener pastures in developed countries of the West, say in USA or Canada. But in the recently concluded 60th IPC at New Delhi in one of the seminars a speaker from USA emphatically said that the PCI course content of Pharm D is not equivalent to that of the of USA. He also expressed his doubts whether new breed of Indian Pharm Ds will be accepted or considered equivalent to American graduates. In the context of the starting of the Pharm D course on one hand, and less and less number of admissions to D. Pharm course on other, it is time for the profession to debate on the future of D. Pharm course as the minimum qualification for the pharmacist. The undergraduate degree (B. Pharm) and postgraduate degrees (M. Pharm) will also come under professional scrutiny. Unplanned babies have difficult childhood and adult life, particularly when conceived by economically compromised parents. Although we argue that India is going to be the economic superpower in 30 years but we also know that we live in “two Indias”, one ITurban India and the other is rural India, where minimum or basic requirements of primary education, health and food are still day to day issues for millions. At the crossroads again As a concerned pharmaceutical educationist, D. Pharm or/vs Pharm D appear like chiral issues. One an eutomer (desired isomer) and other distomer (an undesired or ballastic isomer). Time will tell us whether both the courses would be needed and/or one would become antagonistic to the development of the other. The recent press release that the competitive GATE examination will not be there for pharmacy graduates

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from 2010 onwards and pharmaceutical education will be out of the AICTE umbrella are equally causes of concern. All these issues need wider professional debate. The profession of pharmacy in India is at the crossroads once again. “Change”, “yes we can”, was the slogan with which Barack Obama won the US presidential election-2008 but the issue before the pharmaceutical profession in India is not just the “change from D Pharm to Pharm D” but change in quality pharmaceutical education to meet the challenges and the needs of the nation in the 21st century. References: 1. Hepler CD, Strand LM. Opportunities and responsibilities in pharmaceutical care. Am J Hosp Pharm 1990; 47: 533-43.

2.

3.

4.

5.

Kulkarni SK. Seven –Star Pharmacist- A global perspective. Pharma Times 1998; 29:11-12. Kulkarni SK. Pharmaceutical Education in tune with times. 48th Indian Pharmaceutical Congress, Chennai 1996. Kulkarni SK. Present Status and Future Challenges of Pharmaceutical Education in India, presented at the First World Congress of Pharmacy Education, New Orleans, USA. Eastern Pharmacist 1998; August: 21-26. Kulkarni SK. Harmonization of Pharmaceutical Education: Preparing a future pharmacist Presidential address 53rd Indian Pharmaceutical Congress, New Delhi, 2001. Pharma Times 2002; 34:13-19.

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Formulation and Modulation of Drug Release from an Intra Vaginal Ring Vidya Iyer* and S.S.Poddar. Department of Pharmaceutics, KMK College of Pharmacy, Cuffee Parade, Mumbai 400005. Email: [email protected] ABSTRACT: Intra Vaginal Rings (IVR), primarily developed for contraception, provide a means of delivering pharmacologically active ingredients for local or systemic availability in a controlled manner. Use of a locally acting spermicidal agent like Nonoxynol-9 (N-9) is the preferred method of achieving reversible non-hormonal contraception. Invariably in most of the spermicidal preparations, N-9 is the active moiety. A non-ionic vaginal contraceptive, N-9 has been available in various dosage forms like foams, jellies and sponges for more than 30 years. However, the use of N-9 has been associated with irritation and ulceration and this makes the vagina highly prone to sexually transmitted diseases. Polyvinylpyrrolidone (PVP) when added to N-9 decreases its irritation potential. No work has been reported on IVR containing N-9 and PVP combination. Hence development of an user-friendly, long acting controlled release spermicidal IVR would be the need of the time. In the present work, N-9 and PVP loaded IVR with reduced irritation potential was formulated. The release profiles of the IVR were found to follow zero order kinetics. Based on the gradient of release, the formulation was optimized for a 20 day therapy to achieve spermicidal concentrations of N-9 in the vagina. It was found in this study that PVP acts as a favourable release modulating aid for N-9 release from the IVR. Keywords: Intravaginal ring, Contraception, Nonoxynol-9, Polyvinylpyrrolidone, Spermicidal, Drug release. INTRODUCTION Although contraception is the need of the day for controlling the population explosion in various developing countries, the choice of contraceptive therapy, especially the female-used available till date is not to the peoples’ full contentment. This is because each of the available female-controlled contraceptive measures suffers some or the other drawback. One of the preferred methods of reversible contraception is the non-hormonal, locally acting vaginal spermicide, lacking systemic side-effects. The widely used vaginal spermicide is Nonoxynol-9, a non-ionic surfactant, which is invariably the active in most of the commercially available products.1 Some of the available OTC products of N-9 are Advantage 24®, Conceptrol® cream, Delfon® foam, Semicid® suppository, Today® pessary, Gynoll® jelly. The major setback of most of these spermicidal preparations is

Indian Journal of Pharmaceutical Education & Research Received on 27/3/2008; Modified on 31/7/2008 Accepted on 14/10/2008© APTI All rights reserved

their short duration of action and poor retention, that questions the contraceptive efficacy of the product and hence puts forth an obligation on the female to use the product quite prior to each time she has an intercourse in order to practise safe sex. The inconvenience caused by this dire requirement may reduce user compliance and this encourages discontinuation of the use of a particular product. Since, spermicidals are one amongst the most commonly used female contraceptives, development of a long acting spermicidal contraceptive dosage form would be beneficial to sexually active females and their partners. N-9 containing contraceptive intravaginal ring is a novel delivery form, which is designed to provide prolonged controlled release of the therapeutic in the vaginal milieu for 20 days, once the ring is placed inside the vagina. The outstanding advantage of this product is that the ring could be inserted several hours prior to sexual activity with no compromise on its contraceptive efficacy. This makes the ring user friendly. Other merits are that the ring is devoid of self

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or partner-consciousness as well as any messiness or leaks, which is very obvious with conventional preparations like creams or gels. Since, longer term protection requires multiple dosing which goes against the user compliance, most of the conventional short term use vaginal contraceptives meant for single use provides an extremely high and unnecessary dose in the range 50 – 108 mg of N-9, which is quite harmful to the vaginal epithelium. It is a well accepted fact that N-9 is unfavourable to the vaginal ecosystem at higher concentration and this detrimental effect increases with rise in the frequency of administration of N-9.2-8 The disruption caused by N9 to the vaginal mucosa compromises the host defence system, leaving the vagina susceptible to several infections including the dreadful HIV.2,3,9 Inspite of these alarming drawbacks of the N-9 products, it is being used by many female subjects because of the lack of better alternatives. Hence it is the need of the time to develop a formulation of N-9 that delivers therapeutic amount of N-9 to vagina and at the same time doesn’t compromise upon the vaginal integrity. N-9 as an individual entity is a potent spermicidal agent. Its potency can be attributed to the fact that N-9 at therapeutic concentration inside the vagina favourably partitions in to the sperm cell membrane compared to the vaginal mucosal cells.10 However, it’s very cytotoxic in nature, as it doesn’t spare the vaginal host epithelium and flora.11 This makes N-9 irritant to the vagina beyond a particular concentration. The therapeutic concentration of N-9 used in the vagina is above the Critical Micelle Concentration (CMC) of N9. Hence, in vivo N-9 exists as micelles rather than its monomeric form. It has been reported that aggregates of certain amphiphilic compounds exert toxic effects on various tissues.12-15 It has also been shown that due to its micelle formation, N-9 can initiate irritation and ulceration of mucosal tissues both in vitro and in vivo, compromising their integrity.16,17 This even leaves the mucosal tissues susceptible to sexually transmitted pathogens. Polyvinylpyrrolidone (PVP), which is one of the most commonly used polymers in pharmaceuticals because of its safety for human use and its hydrophilic properties,18 can serve as an excellent carrier for N-9 due to its membrane seeking and coating properties.19,20

It is reported to increase the CMC of N-9, providing a reduction in its irritation potential, while maintaining the required spermicidal activity. The self-association properties and the consequent irritation potentials of N9 are altered with the combination of N-9 and PVP.21 Eventually, Digenis et al in one of their studies has reported the novel idea of combining N-9 with PVP, which raises the CMC of N-9, thus making it comparatively less irritant at therapeutic concentrations used in vivo.21 With this scenario, the development of a user friendly long acting contraceptive intra vaginal ring (IVR) containing N-9 and PVP is reported in this study. Most of the prospective problems allied with the use of products containing N-9 as contraceptive can be overcome by developing a dosage form that is dictated by a delivery regime, which would provide a therapeutic concentration of N-9 in the vagina for its spermicidal action. This was achieved in the present formulation by optimizing the release profile in vitro. The objective of the present work was (i) to estimate the effect of varied loading of N-9 on its release from the IVR. (ii) to determine the effect of PVP on the release of N-9 from IVR. (iii) to optimize the release of N-9 from IVR, so as to achieve spermicidal concentration in the vagina. (iv) to investigate in to the formulation of a long acting (20-day therapy) local nonirritating contraceptive IVR. MATERIALS AND METHODS Materials: N-9 was supplied as a gift sample by Bliss Chemicals and Pharmaceuticals India Ltd; Silicone elastomer (MDX4-4210) was gifted courtesy Dow Corning. PVP K-30 was purchased from ISP Tech Inc, US. Ring making mold was designed inhouse. Methodology: Designing of mold: The mold for IVR fabricated from stainless steel grade 316 was made of two parts with an inlet for injection. When the upper part of the mold was held on the lower part, a ring cavity was left inside that had the dimension exactly that of the proposed IVR. The mold was so designed as to allow proper flow and curing of the elastomeric blend inside the ring cavity. The standard machining and polishing of the mold faces ensured a microscopic passage just enough to cause escape of air & not the elastomeric blend. The loading of the

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elastomeric blend inside the mold directed its flow confined to the cavity formed inside and not its extension outside. Complete loading of the mold left no unfilled spaces within the cavity, which was ensured by perception of a distinct resistance offered by the elastomeric blend on the piston of the syringe, when attempted to be filled beyond its capacity (2.85 cm3). Loading of Mold: A 5 cm3 glass syringe was used to fill the elastomeric blend into the mold. The inlet of the mold was fabricated so that the outlet nozzle of the syringe could fit in precisely. Ring: N-9 and PVP were mixed in the w/w ratio of 10:1 with a blender. This blend was then incorporated in the silicone elastomer and mixed thoroughly. The drugpolymer blend was then filled inside the ring making mold of the following dimension: 50 mm o.d and 5 mm cross-sectional diameter.22 The formulation filled mold was left undisturbed for curing for 72 hrs. The postcured IVR formulation was then removed from the mold. IVR with different loading doses of N-9 (ranging from 1-5 %) were prepared. The optimized concentration of N-9 and PVP in the IVR was decided by following the factorial design methodology. Evaluation: Drug Release:22 The contraceptive IVR were individually placed in stoppered 100 ml conical flasks containing 10 ml of Vaginal Simulated Fluid (VSF)23 having pH 4.2. The contents of the flasks were gently shaken at a constant rate (100 rpm) for 20 days in an orbital shaker at 37º C. The samples were taken and the release medium was renewed fully every 24 hrs. An aliquot of 1ml was then analysed by UV spectrometry at a wavelength of 224 nm. Release studies were performed in triplicate. The information obtained from the optimized batch was then treated for zero order, first order and Higuchi type release kinetics. Assessment of spermicidal activity of IVR: Fresh human semen was obtained from healthy human male volunteers. Criteria considered for the suitable semen sample were (i) Average sperm concentration: 75 to 90 x 106 /ml. (ii) Normal morphology of more than 60 % and (iii) grade A motility of more than 40 % with (v) more than 70 % sperm viability.24 A daily

aliquot of 0.2 ml from the drug release study was pipetted in to a test tube, followed by addition of 50 µl of human semen. The tube was vortexed for 10 sec and then left undisturbed for 2 min. An aliquot of 10 µl of this was placed on a glass slide and covered.25 This was mounted under 450X (eyepiece adjusted at 10X and the objective at 45X) magnification on an optical microscope and motility of the sperm, if any was observed. While performing the test, a sperm was considered dead, if it lacked any motion, since motionless sperm cannot travel the required distance to fertilize eggs.26 Assessment of irritation potential through hemolytic studies:21 The RBC hemolysis assay has been reported in the screening of topical preparations for their local irritation potential.27 Locally acting vaginal spermicides can be classified as topical agents and also irritation potential of N-9 has previously been reported using the RBC hemolysis assay. The irritation potentials of IVR containing only N-9 and the IVR containing N-9 with PVP were estimated using an RBC hemolysis assay. Blood samples were obtained from healthy human volunteers and the RBCs were isolated by centrifugation, washed and resuspended in VSF. Positive control was prepared by incubating RBC suspension in presence of N-9 with mild shaking to ensure complete hemolysis. Similarly negative control was prepared by incubating 0.5 ml of RBC suspension with 3 ml of 10 % PVP solution. The IVR without (B-I) and with PVP (B-II) were subjected to this test in order to compare the irritation potentials. The aliquots from the dissolution study (experimental samples) were incubated at 37 ºC for 30 min with mild shaking. The experimental samples prepared by mixing thoroughly 0.5 ml RBC suspension with 3 ml aliquot from the dissolution study were centrifuged at 1500 rpm for 3 min and then placed in ice bath to quench the hemolytic reaction. The absorbance of the supernatant of each sample was measured at 576 nm. The percentage of hemolysis (%H) was determined as –

Abs: Absorbance of test sample Abscontrol: Absorbance of negative control.

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Abs100: Absorbance of positive control. RESULTS: Ring: The drug loaded elastomeric blend was compatible with mold and the cured IVR could be removed from the mold easily without sticking to the mold. The elastomer did not shrink on curing and the rings were free from defects like presence of air spaces, uneven texture, uneven surface, presence of extensions. Thus, N-9 loaded IVR could be cast successfully with the desired dimension. Drug Release: i) Drug release from IVR with varied loading doses of N-9: Release profiles describing the daily release of N-9 from matrix-type silicone IVR with different loading doses of N-9 (1, 3 and 5 %) are presented in the adjacent graph (Fig. 1). ii) Drug release from the optimized batch: Release profiles showing the daily release of N-9 from matrix-type silicone IVR (optimized batch) is given in the adjacent graph (Fig. 2). The first-day burst from the IVR represented the release of N-9 initially available at the surface of IVR. The IVR having 3 % N-9 and 10 % PVP released ~2.7 mg of N-9 on the first day before levelling off to ~1.6 mg per day over 2nd to 20th day. Post first day, the gradient release profile of all the batches were similar; with a daily controlled release of ~1.6 mg (amount required in the vagina for contraception). 28,29 Hence the drug release was considered as optimized to achieve spermicidal concentrations of N-9 in vagina, with the credit of zero order release. iii) Effect of PVP on the release of N-9 from IVR: Comparative release profiles describing the daily release of N-9 from matrix-type silicone IVR with and without PVP are presented in the adjacent graph (Fig. 3). It is evident from this graph that the formulation that included PVP bestowed a relatively steady and uniform release profile for a prolonged period of time covering the duration of therapy. Assessment of spermicidal activity of IVR: From the release profile, it was found that the amount of N-9 released daily from the optimally formulated IVR was reasonably above the threshold concentration of N-9 in the vagina needed for the spermicidal activity. In addition, the aliquots of daily release of N-9 from the IVR were found to be spermicidal.

Assessment of irritation potential through hemolytic studies: Inclusion of PVP in the formula resulted in a marked reduction in the RBC hemolysis (Table 1). Thus, PVP may aid in designing a contraceptive IVR with possibility of reduced vaginal irritation. DISCUSSION It has been previously reported that N-9 being a liquid drug, doesn’t necessarily obey Higuchi’s equation for diffusion-controlled release of a homogenously dispersed solid substance from a non-biodegradable, non-swellable polymer network.28 Instead, N-9 continuously re-distributes itself in the matrix during the course of its release. This is to the advantage of achieving zero order release profile inspite of a matrix formulation. This is evident from Fig.2. Hence a long acting IVR releasing steady spermicidal concentration in the vagina could be designed. However, N-9, a leading spermicidal agent has been unfortunately associated with vaginal irritation because of its very nature as a surfactant. However, the irritation potential of N-9 has been well reported to be a function of its frequency of use and dosage.2-8 Consequently, a formulation that delivers N-9 in the vagina exactly to the need and not in excess at any given point of time would be appreciated. This objective was achieved by modulation of N-9 release from a controlled delivery device like IVR, so as to achieve the minimum concentration of N-9 in the vagina that will be spermicidal. The different batches of IVR formulated exhibited an almost similar gradient of release over the duration of therapy irrespective of the loading dose (Fig. 1). The initial burst was the characteristic of all the IVR studied, which could be attributed to the surface-release from the dosage form. However this initial burst declined over the subsequent days to give a steady release. Although the initial burst is found to be a function of the initial loading, the subsequent release is not much affected by the initial loading. Hence, a formulation with a burst just enough to compensate for the lag in the build up of sustained spermicidal concentration over the course of therapy was considered optimized. It was estimated that atleast 1.6 mg of N-9 is required in the vagina to achieve contraception, based on the in vitro data indicating that

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Table No. 1 : Assessment of irritation potential of the IVR with and without PVP Time (day) Percent Hemolysis (%) B-I B-II 1 81.5 50.08 2 43.65 17.85 3 48.24 17.01 4 42.54 17.29 5 48.02 18.09 6 46.18 17.26 7 40.26 18.32 8 47.93 17.66 9 42.06 18.39 10 45.33 17.93

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Fig. 1 : Drug release profile of batches with differing loading dose

Fig. 2 : Drug release profile of the optimized batch

Drug released (mg)

5 4 3 2 1 0 0

2

4

6

8

10

12 14

16 18

20 22

Time (day) N-9 with 10 % PVP

N-9

Fig. 3 : Drug release profile of IVR containing N-9 alone and that with PVP

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200 µgm/ml is the effective spermicidal concentration and a conservative estimate of 8 ml for the volume of vaginal fluid.28,29 Although 1.6 mg is estimated to be the minimum amount of N-9 required in the vagina for contraception, it is quite possible that much less might actually be required in vivo. Estimates of vaginal secretions per day range from 1.5 to 8 ml; at the lower end of this range the IVR would only need to release ~ 0.3 µgm/ml. However, several important clinical issues like fluid dynamism involved in the vaginal milieu, vaginal metabolism are still to be addressed. The IVR investigated in this study provided a concentration of N-9 close to 1.6 mg per day lasting for up to atleast 20 days (Fig. 2). Zero order release was observed inspite of a matrix formulation which is to the advantage of achieving steady release of N-9 in the vagina even after a prolonged period of time. It was found in this study that PVP in addition to serving as a CMC-raising agent for N-9 as reported previously, also acted as a favourable release modulating aid for N-9 release from IVR (Fig. 3). While PVP alone has no inherent sperm toxicity, the formation of N-9/PVP complexes seemed to produce a synergistic response which would cause a more rapid damage to the sperm than any of the two materials alone.30 PVP could possibly form complex with N-9 and thus prevent aggregation of N-9 molecules amongst themselves. Ben-David and Gavendo have shown that PVP at 4.6% w/v concentrations protect red blood cells from osmotic fragility and mechanical injury.20 The membrane-seeking properties of PVP suggest that in addition to raising the CMC of N-9, the PVP polymer, via its cell-membrane coating properties, also provides vaginal and cervical surface coverage coating with N-9 over extended periods of time. It is reported that this effect is brought about by a "coating" or external interaction of PVP with cell membranes. It is evident that inclusion of PVP in the formula resulted in a marked reduction in the RBC hemolysis (Table 1). Eventually, PVP might aid in designing a contraceptive IVR with possibility of reduced vaginal irritation. Existence of N-9 in its monomeric form might aid in its controlled release from the silicone matrix. Hence, PVP might be incorporated in the formulation of IVR to possibly achieve a steady and controlled release of N-9

in the vagina over the duration of the therapy, showing improved efficacy and reduced irritation. Briefly, the benefits of incorporating PVP in the formulation include: • PVP raises CMC of N-9, resulting in reduced irritation due to lesser micelle formation. • PVP has coating and surface/membrane seeking properties, due to which it gives protection against epithelial erosion. • PVP possibly modulates release of N-9, assisting in giving a better constant release of N-9. CONCLUSION: • The release profiles of the IVR were found to follow drug release kinetic of zero order. Based on the gradient of release, the formulation was optimized for a 20 day therapy to achieve spermicidal concentrations of N-9 in the vagina. • N-9 and PVP loaded IVR with reduced irritation potential was formulated. • It was found in this study that PVP, in addition to serving as a CMC-raising agent for N-9 as reported previously, also acted as a favourable release modulating aid for N-9 release from IVR. The release of minute and therapeutically efficacious doses of N-9 in a more precise and controlled manner through the use of a specially designed IVR could defeat the problems of possible poor vaginal retention and occurrence of vaginal epithelium damage. ACKNOWLEDGEMENTS: The authors are thankful to Bliss Chemicals and Pharmaceuticals India Ltd for supplying gift sample of N-9 and to Dow Corning for providing gift sample of Silicone elastomer (MDX4-4210). REFERENCES: 1. OTC Panel, Federal Register, 1980;45:8201482049. 2. Roddy RE, Zekeny L, Ryan KA, Tamouf U, Weir SS, Wong EL. A controlled trial of nonoxynol-9 film to reduce male to female transmission of sexually transmitted diseases. N Engl J Med. 1998;339:504-510.

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Roddy RE, Zekeng L, Ryan KA, Tamoufe U, Tweedy KG. Effect of N-9 gel in urogenital gonorrhea and chlamydial infection: a randomized controlled trial. JAMA 2002;287:1117-1122. Van Damme L, Chandeying V, Ramjee G, et al. Safety of multiple daily applications of COL1492, a nonoxynol-9 vaginal gel, among female sex workers. COL-1492 Phase II Study Group. AIDS 2000;14:85–88. Van L, Niruthisard S, Atisook R. Safety evaluation of nonoxynol-9 gel in women at low risk of HIV infection. AIDS 1998;12(4):433437. Roddy RR, Cordero M, Cordero C, Fortney JA. A dosing study of nonoxynol-9 and genital irritation. Int J STD AIDS 1993;4:165-170. DHHS, FDA, 2003. Raymond EG, Chen PL, Luoto J. Contraceptive effectiveness and safety of five nonoxynol-9 spermcides: A randomized trial. Obstet Gynecol. 2004;103:430-439. Hira SK, Feldblum PJ, Kamanga J, Mukelabai G, Weir SS, Thomas JC. Condom and nonoxynol-9 use and the incidence of HIV infection in serodiscordant couples in Zambia. Int J STD AIDS 1997;8:243-250. Karen Y, Yie W. Spermicidal activity-structure relationship of nonoxynol oligomers: physicochemical basis. Int J Pharm.1995;125:8190. Helenius A, Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta 1975;415(1):29–79. Barwicz J, Christian S, Gruba I. Effects of the aggregation state of amphotericin B on its toxicity to mice. Antimicrob Agents Chemother. 1992;36(10):2310-2315. Yamashita K, Janout V, Bernard E, Armstrong D, Regen S. Micelle/monomer control over the membrane-disrupting properties of an amphiphilic antibiotic. J Am Chem Soc. 1995;117:6249-6253. Levin RJ, Parker AJ. Spermicidal action on the vagina: effects of a new, non-ionic surfactant spermicide (RS37367) on rat vagina electrogenic

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ion transfer and permeability in vitro. Med Sci Res. 1987; 15:1045-1046. Tomecjek L, Reid G, Cuprerus PL et al. Correlation between hydrophobicity and resistance to nonoxynol-9 and vancomycin for urogenital isolates of lactobacilli. FEMS Microbiol Lett 1992;73:101-104. Stafford M, Ward H, Flanagan A, et al. Safety study of nonoxynol-9 as a vaginal microbicide: evidence of adverse effects. J Acquir Immune Defic Syndr Hum Retrovirol. 1998;17(4):327331. Phillips D, Taylor C, Zacharopoulos V, Maguire R. Nonoxynol-9 causes rapid exfoliation of sheets of rectal epithelium. Contraception 2000;62(3):149-154. Robinson B, Sullivan F, Borzelleca J, Schwartz S. PVP: A critical review of the kinetics and toxicology of Polyvinylpyrrolidone. Chelsea, MI: Lewis Publishers, 1990. Digenis GA, Rokem SS, Eckert D, Blecher L. Studies on the association of 14C-povidone-131Iiodine complex with red blood cells and bacterial membranes. In: Digenis GA, Ansell J, editors. Proceedings of the International Symposium on Povidone. Lexington: University of Kentucky,1983:302-311. Ben-David A, Gavendo S. The protective effect of polyvinylpyrrolidone and hydroxyethyl starch on noncryogenic injury to red blood cells. Cryobiology 1972;9:192-197. Philip T, Gustavo F, Paul M, George A. Coprecipitation of nonoxynol-9 with polyvinylpyrrolidone to decrease vaginal irritation potential while maintaining spermicidal potency. AAPS Pharm Sci Tech. 2003;4(3),Article 30. Young A, Mukul S, Brij B. Development of vaginal rings for sustained release of nonhormonal contraceptive and anti-HIV agents. Contraception 2007;76:132-138. Derek H, David F. A vaginal fluid stimulant. Contraception 1999;59:91-95. Daniel P, Saradindu B, Debasis J, Rajkumar M, Debidas G. In Vitro determination of the contraceptive spermicidal activity of a composite extract of Achyranthes aspera and Stephania

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hernandifolia on human semen. Contraception 2006;284-288. Chi-Hyun L, Robert B, Yie W. Comparative in vitro spermicidal activity of chelating agents and synergistic effect with nonoxynol-9 on human sperm functionality. J Pharm Sci. 1996;85(1):9195. Cramer D, Goldman M, Schiff I. J. The relationship of tubal infertility to barrier method and oral contraceptive use. Am. Med.Assoc. 1987;257:2446-2450. Pape W, Hoppe U. In vitro methods for the assessment of primary local effects of topically

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applied preparations. Skin Parmacol. 1991;4(3):205-212. Karl M, David W, Julie R, Chris A. In vitro release of nonoxynol-9 from silicone matrix intravaginal rings. JCR 2003;9:355-364. Lee C, Martha A, Yie W. Characterization of invitro spermicidal activity of chelating agent against human sperm. J Pharm Sci. 1996;85(6):649-654. Walter B, Hawi A, Zavos P, Digenis G. Solubilization and in vitro spermicidal assessment of nonoxynol-9 and selected fractions using rabbit spermatozoa. Pharm. Res. 1991;8:403-408.

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APTI

ijper

Impact of Community Pharmacist Provided Patient Education in Asthma Patients on Treatment Outcomes- A Study Anjan Kumar D.S.1, Ramesh Adepu1*, G. Parthasarathi1 and P.A. Mahesh2 Department of Pharmacy Practice, JSS College of Pharmacy, SS Nagara, Mysore1 Department of Pulmonology, JSS Hospital, Ramanuja Road, Mysore2 Corresponding Author : [email protected]

ABSTRACT Patient education is considered as an essential component in improving knowledge, attitude and practice of the patients towards disease management and improved medication adherence behavior and treatment outcomes. Role of pharmacist as a patient educator in chronic disease is well recognized in western world. Objective: To study the influence of pharmacist provided patient education on asthma patients’ treatment outcomes. Methodology: This was a prospective randomized study. Asthma patients who met the study criteria were randomized into test and control groups. Patients’ knowledge, attitude and practices toward asthma and medication usage were analyzed. Inhaler technique and medication adherence behavior of all the enrolled patients were assessed. The treatment outcome was assessed by using FEV1 in spirometer. Patients in the test group received education about the disease, medications and precautions to take to minimize the triggering factors, where as the patients in control group received education only regarding inhaler usage technique at the baseline. Results: One hundred six asthma patients completed the study. Significant improvement (p 200 ml after 4 puffs of Salbutamol a β2 agonist according to ATS guidelines. (FEV1 = Forced Expiratory Volume in one second) Knowledge, Attitude and Practice (KAP) assessment Validated KAP questionnaire was administered to assess the patients’ perceptions about the disease, medications and their attitudes towards the disease management. The questionnaire was administered to

the test and control group patients at base line and the last follow up. Inhaler check lists Prepared metered dose inhaler and rotahaler checklists were used to assess the inhaler technique of patients. Each checklist consists of ten steps. Each step was recorded as correct or incorrect based on the demonstration of the technique by the patient. Inhaler technique was assessed on each follow-up. Medication adherence assessment Adherence to treatment was assessed in both the test and control groups by dairy keeping method, which is one of the indirect subjective methods of adherence measurement. 6 The medication Adherence of the patients was calculated using the formula: Total no of actual doses the patient has consumed since last appointment %Adherence = _____________________________________x 100 Total no of calculated doses to be consumed since last appointment

Pulmonary function tests Lung function of the study subjects was assessed by Spirometer, which was done at baseline and during all the four follow-ups for both the groups. From the spirometry FEV1 values were used to assess the treatment outcomes. Patient Education Structured patient education was developed for the study purpose included both verbal and printed information about the disease. Education was provided to all the test group patients, at baseline and at every follow-up, and control group patients received the education only at the end of the study. Structured patient education included about the disease, medications, lifestyle modifications required for the better management of the disease and inhaler usage techniques through counseling aids and prepared patient information leaflets. The control group patients received only one time basic education about inhaler usage technique. Stastical Analysis Student ‘t’ test was conducted to assess the statistical significance of the results and ANOVA to analyse the influence of age, sex, education and socioeconomic background.

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RESULTS A total number of 106 asthma patients met the inclusion criteria. These patients were randomized in to test (52) and control (54) groups respectively. Ninety eight patients (49 patients test and 49 patients control) completed the study and eight patients (5 patients in control and 3 patients in test) were dropouts due to non compliance. Demography Out of 98 patients, there were 36 (73%) males in control group and 34 (69%) males in test group. There is no statistical significant difference in the demographic parameters between cases and controls regarding age, gender, education, smoking history, duration of asthma and the baseline spirometry. (Table.1) Analysis of Knowledge, Attitude and Practice results. The analysis of KAP results was assessed by the percentage of patients answering each item correctly at baseline and at final follow-up (before and after the education). A higher percentage improvement of knowledge, attitude and practices of patients in test group was observed compared to the control group. The results are shown in Table 2. Assessment of Inhaler/Rotahaler usage techniques in control and test group patients Assessment of individual steps of MDI usage. In the use of MDI, step 5 (Exhale as much as possible), step 6 (Place the mouth piece in mouth and close lips around it or hold the inhaler 2 to 3 cm from the mouth) step 7 (begin to breath in and then actuate the inhaler once during inspiration), step 8 (continue slow and deep inspiration through mouth) and step 9 (Hold the breath for ten seconds and then breath out) are considered to be critical steps in terms of achieving better therapeutic outcomes. Control group: All the patients performed step 1(removing the cap) and step 3 (holding the MDI upright) correctly in all the follow-ups (100%) at baseline and in subsequent follow-ups. A gradual improvement in the performance of step 4 (Tilt the head back) and step 5 (Exhale as much as possible) was observed. However, no significant improvement in the performance in step 2 (shake the inhaler) and in steps 6, 8, 9 10 was observed. The results are presented in Fig1.

Test group: All the patients of test group who were on MDI performed step 1 (removing the cap) and step 3 (holding the MDI upright) correctly (100%) at baseline. A significant improvement in the performance of steps 2,4,5,6,7,8,9,10 by patients was observed from the first follow up to final follow up. The improvement in MDI performance technique in the test group at each follow up is shown in Fig-2. Inhaler usage technique scores of control and test group patients at baseline and in 1st follow –up, was not very much significant (p>0.05). However from the second follow up the inhaler usage performance of the test group patients was significantly(p< 0.01) improved from the 2nd follow up to 4th follow up. The scores are given in the Table.3. Assessment of individual steps of Rotahaler usage. Steps 5 (Exhale as much as possible), 6 (place the mouth piece in mouth and close lips around it, 7 (Breath in deeply) and 8 (Hold breath for ten seconds and then breath out) are considered to be the critical steps in the rotahaler usage. Control group: All the patients in control group, who were on rotahaler performed step1 (Hold the inhaler upright level) and step2 (take the rotacap and press it firmly such that top end of rota cap is in level with the top of the hole) correctly (100%) at the baseline and in subsequent follow-ups. The rotahaler usage performance in steps 4,5,6,7,8,9 and 10 by the patient was found to be significant. The performance of the control group patients in rotahaler usage technique at each follow –up is shown in Fig-3. Test group: All the patients of test group who were on Rotahaler performed step1 (Hold the inhaler upright level) and step2 (take the rotacap and press it firmly such that top end of rotacap is in level with the top of the hole) correctly (100%) at baseline and in all the subsequent follow-ups. A considerable improvement increase in percentage of patients performing steps 3, 4, 5, 6, 7, 8, 9, 10 on the final follow-up was observed. The improvement in DPI technique in the test group on each follow-up day is shown in Fig-4. At baseline no significant (p>0.05) differences between the rotahaler usage technique scores of test and control groups was observed, however from the 2nd follow-up to 4th follow-up, the scores of test group patients was significantly (p 0.05

36 (73%) 13 (27%)

34 (69%) 15 (31%)

15 (36.61%) 15 (30.61%) 19 (38.77%)

16 (32.65%) 13 (26.53%) 20 (40.81%)

P > 0.05

19 (38.77%) 20 (40.81%) 10 (20.41%)

20 (40.81%) 18 (36.73%) 11 (22.44%)

P > 0.05

8.05 ± 6.07 21 ± 4.1

9.2 ± 6.7 20.5 ± 4.7

P > 0.05 P > 0.05

P > 0.05

Table 2: Analysis of Knowledge, Attitude and Practice results at baseline and final follow-up % of patients % of answered % patients answered correctly (%) Improvement correctly (%) KAP at Final (n=49) KAP at Baseline QUESTIONS follow-up (n=49) (n=49) Control Test Control Test Control Test Do you know, which part of the body is affected in 30.61 34.61 40.81 97.95 10.2 63.34 asthma? What happens to a person during an asthma attack? 14.28 12.24 30.61 61.22 16.33 48.98 Can you name the Signs and Symptoms of 30.61 20.44 44.89 57.14 14.28 36.70 Asthma? Can you name the causes of asthma? 14.28 16.32 32.65 51.02 18.37 34.70 Do you think asthma is a contagious disease? 10.20 12.24 22.44 53.06 12.24 40.82 Do you think asthma is a curable disease? 12.24 14.28 36.73 53.06 24.49 38.78 Do you think knowing about your disease 10.20 8.16 30.61 51.02 20.41 42.86 condition is important to you? Are you taking any medications for your disease? 73.46 77.55 85.71 91.83 12.25 14.28 What type of medications are you receiving for 40.81 44.89 77.55 81.63 36.74 36.74 your Asthma? Do you think taking asthma medications regularly 20.40 18.36 42.85 61.22 22.45 42.86 is important to you? Do you receive any advice regarding the proper 20.40 24.48 51.02 100 30.62 75.82 usage of medications from any one? Do you have any worries about side effects of your 20.40 18.36 51.02 81.63 30.62 63.00 medications? Which form of treatment do you feel comfortable 24.48 22.44 53.06 77.55 28.58 55.11 with? If you are using a metered dose Inhaler, do you shake the canister just before taking puff? 20.40 18.36 44.89 87.75 24.49 69.39

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If you are using a Metered dose Inhaler / Rotahaler, do you wash your mouth after inhalation? If you are using Inhalers, how do you rate the ease of using Metered dose inhaler? Do you purchase all the medicines on prescription at once? Do you take your medication regularly as advised by your physician? Do you skip your medications?

16.32

18.36

53.06

83.67

36.74

65.31

20.40

20.44

51.02

85.71

30.62

65.27

20.40

22.44

44.89

71.42

30.62

48.98

22.44

22.91

51.02

71.42

28.58

48.51

20.40

22.91

44.89

77.55

24.49

54.64

Table3: Inhaler usage techniques scores of control and test group patients from baseline to 4th follow-up. Inhaler checklist Follow ups Total No of steps did correctly p Value (Mean ± SD) Control Vs Test Metered dose Control (n=49) Test (n=49) Base line 4.11 ± 0.99 4.25 ± 1.03 0.44 (NS) Inhaler st l follow up 5.94 ± 1.34 6.42 ± 1.19 0.06 (NS) 2nd follow up 6.00 ± 1.11 7.62 ± 1.11 < 0.001 (S) 3rd follow up 6.11 ± 0.96 8.61 ± 0.73 < 0.001 (S) 4th follow up 6.25 ± 0.85 9.14 ± 0.60 < 0.001 (S) S –Significant (p0.05) by‘t’ test. Table4: Rotahaler usage techniques scores of control and test group from baseline to 4th follow-up. Inhaler checklist Follow ups Total No of steps did correctly p Value (Mean ± SD) Control Vs Test Rotahaler Control (n=9) Test(n=9) Base line 4.72 ± 1.02 4.87 ± 1.15 0.17 (NS) lst follow up 6.62 ± 1.57 7.62 ±1.91 0.05) differences between the medication adherence scores of test and control groups was observed. However from the 3rd follow-up to 4th follow-up significant (p0.05) differences between the FEV1 values of both test and control groups was observed. However from the 2nd follow-up to 4th follow-up significant improvement in FEV1 values was observed in test group compared to the control group. The results are shown on Figure 5.

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DISCUSSION The results of KAP suggest that patients at the baseline possess poor knowledge, attitude and practice on disease and its management. This may be due to inadequate information about the disease and medication usage techniques, patients’ poor interest to know about their disease management. In the present study, the poor awareness of the patients at baseline towards the disease and its management can be attributed to inadequate information provided by the health care professional. At the final follow – up, the percentage improvement of KAP was increased in test group patients compared to the control group patients. This may be because of pharmacist provided patient education to the test group patients. The results suggest that, education provided by health care professional has a positive influence on patient’s disease management attitude. Although education was not provided to the control group patients, improved scoring of KAP in final follow up was observed. This may be due to peer group exchange of the knowledge. Patient education with in the context of the controlled evaluations is capable of improving knowledge, and beneficially altering behavior. Hilton et al. studied the impact of education in 339 patients in a systematically allocated open study. The interventions were maximum education program, a limited education program and no intervention. Improvement in the knowledge was shown in the maximum intervention group.1 These observations were found to be similar to observations made by P.P.Guptha and K.B.Guptha study.2 The results suggest that patients treated by highly qualified health care professionals were found to have more knowledge about the disease, triggering factors, and medication usage technique. Where as poor awareness was observed in the patients who received the treatment from local general practitioners. The results suggest that increased awareness about the disease and management depends upon the education provided by the health care professional. The results of the present study suggest that at base line the critical steps as per the standard check list, such as tilt the head back (step 4), exhale as much as possible (step 5), coordination between actuation and inhalation (step 7), continue inspiration slow and deep (step 8) holding the breath for 10 seconds (step 9) which has greater influence on the therapeutic outcomes were not

performed by many patients at the base line of both control and test groups. The patients who were on rotahaler were also not able to perform certain critical steps such as tilt the head back (step 4), exhale as much as possible (step 5), breath in deeply (step 7) and wait for one minute before taking the second dose (step 9). These steps are considered as crucial steps in increasing the efficacy of drug in asthma patients. As the education was provided to the patients of both groups regarding the inhaler usage techniques at the baseline and followed up regularly at each follow-up for the test group patients, the inhalation usage technique was improved in test group patients. These results was comparable to the results observed by Iman A Basheti et al,6 and also suggest that community pharmacist provided education improved the MDI / Rotahaler usage technique. In an another study Windsor et al studied 267 patients in a randomized open study of the effectiveness of a health education intervention consisting of a 30 minute one to one session, and use of self help guide to asthma control, a 60 minute support group session and two brief telephone reinforcement calls. Over a 12 month period the intervention group had improved inhaler use and adherence, medication adherence, and a total adherence rating.3 These studies suggest that education has a positive influence on inhaler usage technique. The same was demonstrated in the inhaler usage technique scores of the test group. Dairy keeping method was used in the present study to assess the adherence behavior of the enrolled patients using the percentage adherence formula. A significant improvement was observed in the last two follow ups in test group patients compared to control group. This improvement can be attributed to regular monitoring of the patients by the pharmacist. These results were comparable to the findings of Ines Krass et al.11 the results of the study reveal that community pharmacists trained in medication review and providing continuous education improved medication adherence in patients with type 2 diabetes in the intervention group than the control group. These findings suggest that pharmacist medication reviewing and providing continuous education to the patients helps in medication adherence and improves out comes. In the present study, improved adherence in the test group compared to control group

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may be due to patient education imparted by the community pharmacist. Measurement of Therapeutic Outcomes Therapeutic outcomes are considered as goal of any therapy. Awareness of the disease and its management, adherence to the medication shows a positive influence on the therapeutic outcomes. The Forced Expiratory Volume in one second has a clinical relevance and acts as a primary indicator in assessing the airway function. The FEV1 values of test group patients have shown significant improvement compared to the control groups. This is likely due to improved adherence to medication and because of structured patient education, indicating education has a positive influence on therapeutic outcomes. In a major study conducted by Tullio in 1987 demonstrated the impact of pharmacists’ patient education on compliance with asthma therapy though a controlled patient –blinded study.13 Nineteen mild to moderate asthmatic patients who were on “Inhalernaïve” were enrolled in to the study. Patients were randomly assigned to a study or control group. Study group patients were only counselled on inhaler use and its importance in asthma management. In addition, the researchers demonstrated the 11 steps necessary for proper inhaler usage technique and then observed as the patient repeated the process. Control group patients received instructions about the name of the new inhaler, its purpose and the prescribed dosage. No additional information about the use of the inhaler was given. All patients received a patient package insert (PPI) detailing the method of administration for the inhaler. The authors assessed compliance at the next clinic visit through baseline forced expiratory volume at one second (FEV1) and forced vital capacity (FVC) testing, using a Spirometer and by observing patients using their inhalers and counting the number of properly completed steps from the 11-step check list. Based on the study, the authors concluded that the education and demonstration of inhaler usage technique provided by the clinical pharmacist resulted in better patient understanding, increased correct performance of inhaler use, and improved bronchodilation as measured by PFTs. These findings are closer to our study which demonstrated the positive influence of consistent education on knowledge, practices and improved inhaler usage and health out comes.

In a Canadian based study “The Better respiratory education and Asthma Treatment in Hinton and Edson (BREATHE) was conducted to study the influence of structured education by community pharmacists on the treatment outcomes in asthma patients visiting respiratory therapist clinics.14 In the study, eligible patients were randomized in to control and intervention groups. The intervention group patients received education and written asthma action plan from the pharmacist and followed on 2nd week, 4th week and later at monthly interval for next six months. The control group patients did not received any education and written action plan. At the baseline the respiratory therapist assessed the FEV 1 for both intervention and test group patients using spirometer. Despite of education and written action plan, the intervention group patients had shown minimal improvement in the FEV1 scores. This is attributed for a poor follow up by the community pharmacist. This study shows that, limited education may not show significant influence on the treatment outcomes. In another study conducted by Gibson et al, demonstrated that, patients who received self management education plan, written action plan had shown improvement in the health outcomes such as improved lung function, decreased emergency hospital visits, decreased lost working days.15 Further they have also identified that, less intensive education programs such as providing only patient information leaflets does not show considerable influence on the treatment outcomes. In the present study, the observations with respect to inhaler usage technique, medication adherence behavior, and improvement in the lung function tests, indicate that the scores are improved only after 2nd and 3rd follow up. The improvement in the scores can be attributable to the regular education to the test group of patients. CONCLUSION The study concludes that pharmacist provided structured patient education found to have significant influence on improvement in the knowledge, attitude and practices of asthma patients towards the management of the disease. Patients in the test group had shown a significant improvement in their inhaler, rotahaler usage technique, adherence behavior and also

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in the treatment outcomes compared to the control group patients. ACKNOWLEDGEMENTS Sincere thanks to JSS Mahavidyapeetha and Principal, J.S.S. College of Pharmacy, for providing the facilities to conduct the study. Hearty thanks to Delhi Pharmaceutical Trust (DPT) for providing the financial assistance to complete the study. REFERENCES: 1. Hilton. S Sibbald., Anderson H.R and Freeling P. Controlled evaluation of the effects of patient education on asthma morbidity in general practice. Lancet. 1986; I : 26 -29. 2. Prem Prakash Guptha and K.B. Guptha. Awareness about the disease in asthma patient receiving treatment from physicians at different places. The Indian Journal of Chest Diseases and Allied Sciences 2001; 58(2): 1-6. 3. Windsor, R.A., Bailey, W.C., Richards, J.M. et al. Evaluation of the efficacy and cost effectiveness of health education methods to increase medication adherence among the adults with asthma. Am J Public health. 1990; 80: 1519 -1521. 4. Andrew G Weinstein. Should patients with persistent severe asthma be monitored for medication adherence. The Indian journal of chest diseases 2005; 94: 251-257. 5. L.Y.Yong, W.A. Kradjan, Asthma. In: M.A. Kodakimble, B. Joseph Gugliemo, B.K. Aldridge, Robin Correlli, Timothy H Self. Asthma. Applied therapeutics the clinical Use of Drugs. Eighth edition. Published by Lippincott Williams & Wilkins 2005: 23-1-23-27. 6. Iman A Basheti et al. Counseling about Turbuhaler technique: Needs assessment and effective strategies for community pharmacists. Respiratory care 2005; 50(5): 617-623.

7.

Leslie Fish, Collen Lum Lung. Adherence to asthma therapy. Ann Allergy Asthma Immunol 2001; 86:24-30. 8. Bruce G Bender. Overcoming barrier to nonadherence in asthma therapy. J Allergy Clin Immunol 2002; 109:s554-559. 9. John A Gans. Improving Medication Adhernce: Pharmacists should take the Lead. Journal of the American Pharmacists 2003; 43(6): 665. 10. Phillippe Sudre, Stephane Jacquemet, Christophe Uldry, Thomas V Perneger. Objectives, methods and content of patient education programmes for adults with asthma: Systemic review of studies published between 1979 and 1998. Thorax 1999; 54: 681-687. 11. Ines. K, Susan J. Taylor, Carlene Smith, Carol L. Armour. Impact on Medication Use and Adherence of Australian Pharmacists: Diabetes Care Services. J AmPharm Assoc 2005; 45: 33-40. 12. Bheekie A, J.A. Syce, E.G. Weinberg. Peak expiratory flow rate and symptom self- monitoring of asthma initiated from community pharmacies. Journal of Clinical Pharmacy and Therapeutics 2001; 26: 287-296. 13. Tullio PL and Corson ME. Effect of pharmacist counseling on ambulatory patients use of aerosolized brochodilators. Am J Hosp Pharm. 1987; 44: 1802 -1804. 14. Theresa L Charrois, Stephen C Newman, Ambikaipakan Senthilselvan and Ross T Psuyuki. Improving asthma control in the rural setting. The BREATHE study. Canadian Pharmacy Journal. 2006; 139 (4): 44 -50. 15. Gibson PG, Coughlan J, Abramson M, et al. the effects of self management education and regular review in adults with asthma. (Cochrane Review, Latest version, 26th February 1998).

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Modified Diffusion Apparatus for Evaluation of Transdermal Drug Delivery System M. C. Gohel*, R. K. Parikh, R. R. Delvadia, S. A. Nagori, K. G. Sarvaiya, B. H. Patel and V. P. Patel Department of Pharmaceutics and Pharmaceutical Technology, L. M. College of Pharmacy, Ahmedabad 380 009, India. * Corresponding Author: [email protected] ABSTRACT The objective of present study was to develop a relevant in vitro diffusion method to evaluate transdermal drug delivery system of ondansetron hydrochloride. A flow through cell was designed and evaluated. In this method, a glass diffusion cell was connected to a glass beaker (500 ml capacity) containing 300 ml of diffusion medium (phosphate buffer, pH 7.4). The diffusion media was circulated at a flow rate of 80 ml/m. The in vitro drug diffusion in the modified diffusion apparatus was compared with that obtained in the Franz diffusion cell. The drug diffusion curves were dissimilar (f2=43). Experiments with different flow rates, different diffusion media and relevant diffusion barrier may be used to achieve the objective of in vivo in vitro correlation (IVIVC). Key words - Ondansetron hydrochloride, Transdermal patch, Diffusion, Flow through cell. INTRODUCTION Transdermal drug delivery system (TDDS) has attracted attention of scientists in the recent past. Clonidine, nicotine and glyceryl nitrate transdermal products are commercially available.1-2 Transdermal drug delivery systems, in comparison to conventional pharmaceutical dosage forms, offer advantages such as improved systemic bioavailability of active pharmaceutical ingredients, fewer administration frequency, longer duration of action, reduced side effects, steady drug delivery profile, non-invasive means of drug delivery, easy to apply and remove in the event of toxicity. It excludes the variables that affect drug absorption from the gastrointestinal tract such as pH, enzymatic activity and drug-food interactions.3 Transdermal drug delivery system allows delivery of drug into the systemic circulation via permeation through skin layers at a controlled rate. Factors affecting the penetration of the drug molecule into the skin include physicochemical nature of the drug molecule, time of application, site and condition of the skin, type of the formulation and vehicle effect on the Indian Journal of Pharmaceutical Education & Research Received on 12/5/2008 ; Modified on 12/8/2008 Accepted on 18/11/2008 © APTI All rights reserved

properties of stratum corneum. The commercially available products are referred to as drug in adhesive system, matrix system, peripheral adhesive system and reservoir system. In the matrix system, the drug is embedded in polymer layer and a nearly constant drug delivery rate is obtained.4-8 The main components of transdermal patch are backing layer, matrix formers/drug reserviour, rate controlling membranes, pressure sensitive adhesives and release liners. Ideal drug candidate for TDDS must be non-ionic, molecular weight less than 500 daltons, have adequate solubility in oil and water (log P = 1-3), melting point less than 200o and dose less than 50 mg/day. In the present study ondansetron hydrochloride was selected as a model drug for preparation of TDDS. Daily oral dose of ondansetron hydrochloride is 16 mg. Ondansetron hydrochloride is highly lipophilic molecule (log P = 2.14). It exhibit low oral bioavailability (60%) due to hepatic first pass metabolism and its elimination half life is around 4 h.9 In vitro diffusion study is an important test for evaluating TDDS. The currently used in vitro diffusion methods do not mimic the in vivo conditions present

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below the skin.10-12 Hence, modified diffusion apparatus was developed and evaluated to overcome the problem of Franz diffusion cell in terms of automation, sampling and mimicking the flow of blood beneath the skin. MATERIALS AND METHODS Materials Ondansetron hydrochloride I.P. was received as a gift sample from Unimed Pharmaceuticals (India). Eudragit RL 100 was received as a gift sample from Roehm Pharma (India). Citric acid I.P., propylene glycol (PG), polyethylene glycol 400 I.P. (PEG 400) and glycerin I.P. were purchased from S. D. Fine Chem. Pvt. Ltd. (India). Alcohol I.P. was purchased from Baroda Chemical Industries Ltd. (India). Preparation of transdermal patch Eudragit RL 100 (200 mg) was dissolved in 5 ml of ethanol. Citric acid (20 mg), propylene glycol (0.01 ml), polyethylene glycol 400 (0.02 ml), glycerin (0.01 ml) and ondansetron (30 mg) were added to the alcoholic polymeric solution. The mixture was agitated on a magnetic stirrer (Remi equipments, India) at 50 rpm until clear drug solution was obtained. The drug solution was poured in the aluminum mould having an area of 16 cm2. The solution was stored at room temperature for six hours to facilitate evaporation of ethanol. The prepared patch was cut from the centre in round shape having 5.3 cm2 area. The drug loaded patch was sticked to circular polyethylene backing membrane with an area of 9.0 cm2. Natural rubber adhesive was applied on the periphery of polyethylene membrane. Finally, silicone release liner was applied. Modified diffusion apparatus Schematic representation of Franz diffusion cell and modified diffusion apparatus is shown in Fig.1 (a) and (b) respectively. Modified diffusion apparatus is a continuous flow through cell. The cell has receptor compartment with one inlet at the bottom and one outlet at the top. A mini pump, kept in a beaker, was connected to the cell using a plastic tube. The outlet was connected to the glass beaker. The beaker was kept in a water bath maintained at 32±0.5o. A flow regulating valve was kept between the pump and inlet of diffusion cell to regulate the flow of the medium (phosphate buffer, pH 7.4, 80 ml/m). In vitro diffusion study A cellulose acetate membrane (0.22 µ) was supported on O ring of the diffusion cell. The drug loaded patch

was kept on the membrane in such a way that the backing layer was facing towards the donor compartment. The glass beaker was filled with 300 ml phosphate buffer having pH 7.4 and temperature equal to 32±0.5°. Samples (10 ml) were withdrawn at regular interval from the glass beaker for analysis. Ten milliliter fresh phosphate buffer was added immediately after sampling to maintain the volume equal to 300 ml. The absorbance of the samples was measured of 248 nm using a UV-visible spectrophotometer after suitable dilution.13 The diffusion study was also carried out using Franz diffusion cell (80 ml, phosphate buffer pH 7.4, 32±0.5°, 80 RPM). The similarity factor f2 was calculated according to the equation 1 considering the drug diffusion data of modified diffusion apparatus and Franz diffusion cell as reference and test respectively. n f2 = 50 * log {[1 + (n)-1 ∑ (Rt-Tt)2 ] -0.5 *100}--(1) n=1 In vitro drug diffusion data of modified diffusion apparatus were analyzed by different kinetic models in order to evaluate the release mechanism of ondansetron hydrochloride from the patch. A FORTRAN software, developed in-house, was used. The least value of sum of square of residuals (SSR) and Fisher’s ratio (F) were used to select the most appropriate kinetic model. Short term diffusion stability study Formulated ondansetron hydrochloride transdermal patch was stored for 2 months in tightly closed conditions (double polyethylene bags) at ambient conditions (40±2o and 75±5% RH). At the end of second month, the patch was subjected to in vitro diffusion study using modified diffusion study. The procedure employed for the study was identical to that described above. RESULT AND DISCUSSION Ondansetron hydrochloride transdermal patch Cellulose acetate membrane was selected due to uniform thickness and biorelevancy with human skin.14 Polyethylene glycol 400 was selected mainly as a penetration enhancer,15-17 while glycerin and PG were selected as solubility enhancer to avoid the recrystallization of the ondansetron hydrochloride in the patch.18 Citric acid was added as a stabilizer and pore forming agent.19 Modified diffusion apparatus -In vitro drug diffusion from transdermal dosage forms has been extensively

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Table No. 1 - Comparison between Franz diffusion cell and modified diffusion apparatus. Parameters Franz diffusion cell Modified diffusion apparatus Type Static-diffusion cell. Dynamic- close continuous flow through cell. Magnetic stirrer

It is required for uniform mixing of drug in the receptor compartment.

Mimic blood flow Problem of bubble entrapment For poorly soluble drug

No Observed during sampling, creating a barrier for diffusion. Addition of solubility enhancer is compulsory, which may increase the flux.

It is not required due to the presence of reciprocating pump which continuously replaces the reservoir compartment fluid. Yes Not observed as sampling is done from the beaker. One can increase the volume of the receptor compartment by just changing the beaker and adding more medium to avoid the super saturation.

Fig. 1. Diffusion cells for evaluation of transdermal products ; Franz diffusion cell (a), Modified diffusion apparatus (b)

Fig. 2. Comparative drug diffusion profile in Franz diffusion cell and modified diffusion apparatus.

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investigated using the modified Franz cell diffusion system with a synthetic membrane and to some extent using the enhancer cell.20-26 Depending on the solubility of the drug substance, the receptor medium may contain alcohol and/or surfactant. Deaeration is critical to avoid bubble formation at the interface with the membrane. A synthetic membrane often serves as an inert support membrane. Depending on the characteristics of the drug product, it may be possible to conduct the in vitro diffusion test without a synthetic support membrane.27 Because of the value and importance of in vitro drug diffusion study, it is highly desirable to determine the diffusion rate of TDDS. It is expected that given the variety of formulations, sites of applications and release rates no single test procedure would be suitable for the development, biopharmaceutical characterization and quality control of all TDDS. Addicks et al. described a flow-through finite-dose diffusion cell for use in transdermal drug delivery research.28 In the present study a modified diffusion apparatus is proposed for evaluation of TDDS. The apparatus is close type continuous flow through cell used for the evaluation of tablets by Pernarowaski and Searl with certain modifications.29 The major modifications are: (a) O-ring is provided for mounting the membrane (b) receptor compartment was added and (c) an outlet was kept just beneath O ring. Receptor compartment contained 300 ml of phosphate buffer (pH 7.4) as a representative of the actual volume of distribution (Vd). The flow rate was kept at 80 ml/m to provide the sink condition and mimic the blood flow beneath the skin.30 Phosphate buffer of pH 7.4 appears to be more appropriate diffusion medium as compared to the use of 40% PEG 400 as it can act as a penetration enhancer. The exposed area of membrane in Franz cell and modified diffusion apparatus was kept identical, i.e. 5.3 cm2. For investigational purposes, the exposed area may be changed by just changing the diameter of the O ring. For the drugs having poor aqueous solubility, the volume of diffusion media may be increased by replacing the 500 ml beaker by a larger beaker to avoid addition of solubilizer in the diffusion media. Table 1 shows comparison between the Franz diffusion cell and the modified diffusion apparatus. The modified diffusion apparatus allows easy sampling and it eliminates bubble entrapment problem as seen in the Franz diffusion cell. The modified diffusion apparatus

may show good in vivo in vitro correlation since an attempt was been made to mimic the in vivo blood flow condition. In vitro diffusion study The drug diffusion pattern of the formulated batch in Franz diffusion cell and modified diffusion apparatus is shown in Fig. 2. The excipients did not show absorbance at 248 nm. The UV spectrum remained unchanged during in vitro diffusion study, indicating stability of ondansetron hydrochloride during the analytical procedure. The modified diffusion apparatus showed slower drug diffusion as compared to Franz diffusion cell. The probable reason for it could be higher upward force due to vortex formation in Franz diffusion cell. Less than 3.5% of the drug was diffused at the end of 120 m from both the cells. Incomplete drug release was seen at the end of 1440 m. The amount of drug diffused in Franz cell and modified diffusion apparatus was 86% and 75% respectively. The similarity factor f2 between the drug diffusion data of Franz cell and modified diffusion apparatus was 43 indicating dissimilarity.31-32 The in vitro diffusion study data of modified diffusion apparatus were analyzed for establishing kinetics of drug diffusion. Model fitting was done using an inhouse program developed by the authors. Zero-order, first-order, Higuchi, Hixson-Crowell, KorsmeyerPeppas and Weibull models were tested. The best fit was shown by zero order model with least sum of square of residuals (SSR = 27.7) and Fisher’s ratio (F = 2.5). Stability study Short term in vitro diffusion stability study of the formulated ondansetron hydrochloride patch was carried out for 2 months at 40±2o with 75% RH. Unpaired t-test with equal variance indicated statistical insignificant difference in the in vitro drug diffusion at 5% with tcalculated (0.013)< tcritical-two tail (2.06). CONCLUSION In the present study, a modified diffusion apparatus is proposed wherein an effort was made to mimic blood flow. The modified diffusion apparatus showed dissimilar diffusion at 10% level. The membrane facing the receptor compartment is surrounded by the fresh recirculating diffusion medium. The modified diffusion apparatus also allows easy sampling. The drug diffusion study from the modified diffusion apparatus followed

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zero order kinetics. The modified diffusion apparatus may be adopted as a quality control test or for establishment of IVIVC. Further study may be carried out for poorly soluble drug by changing total volume of diffusion media for checking effect of saturation on diffusion of drug. Similarly, liquid circulation rate can also be changed. ACKNOWLEDGEMENT We are thankful to Unimed Pharmaceutical Ltd. and Roehm Pharma for kindly providing the gift samples of ondansetron hydrochloride and Eudragit RL 100 respectively. REFERENCES 1. Jain S, Chourasia M, Sabitha M et al. Development and characterization of transdermal drug delivery systems diltiazem hydrochloride. Drug Delivery 2003;10:169-77. 2. Lipp R, Muller-Fahrow. A use of X-ray crystallography for the characterization of single crystals grown in steroid containing transdermal drug delivery systems. Eur J Pharm Biopharm 1999;47:1333-8. 3. Prausnitz M, Mitragotri S, Langer R. Current status and future potential of transdermal drug delivery. Nature Reviews 2004;3:115-24. 4. Fígen O, Ílbeyí A. Development of a membrane controlled transdermal therapeutic system containing isosorbide dinitrate. Int J Pharm 1999;180:177-83. 5. Mi-Kyeong K, Hong Z, Chi-Ho L, Dae-Duk K. Formulation of a reservoir-type testosterone transdermal delivery system. Int J Pharm 2001;219:51-9. 6. Kandavilli S, Nair V, Panchagnula R. Polymers in transdermal drug delivery system. Pharm Tech 2002;26:62-80. 7. Wolff H. Optimal process design for the manufacture of transdermal drug delivery systems. Pharmaceutical Science and Technology Today 2000;3:173-81. 8. Naik A, Kalia Y, Guy R. Transdermal drug delivery:overcoming the skin’s barrier function. Pharmaceutical Science and Technology Today 2000;3:318-26. 9. Simpson K, Hicks F. Clinical pharmacokinetics of ondansetron-A review. Eur J Clinical Pharmacol 1996;48:774-81.

10. Franz T. Percutaneous absorption on the relevance of in vitro data. J Invest Dermatol 1975;64:190-5. 11. Bosman I, Avegaart S, Lawant A, Ensing K, DeZeeuw R. Evaluation of a modified diffusion cell for in vitro transdermal permeation: Effects of injection height, volume and temperature. J Pharm Biomed Ana 1998;17:493-9. 12. Addicks W, Flynn G, Weiner N. Validation of a flow-through diffusion cell for use in transdermal research. Pharma Res 1987;4:337-41. 13. Llácer J, Gallardo V, Parera A, Ruiz M. Formation of ondansetron polymorphs. Int J Pharm 1999;177:221-9. 14. Dias M, Farinha A, Faustino E, Hadgraft J, Pais J, Toscano C. Topical delivery of caffiene from some commercial formulations. Int J Pharm 1999;182:41-7. 15. Santoyo S, Arellano A, Ygartua P, Martin C. Penetration enhancers effect in vitro percutaneous absorption of piroxicam through rat skin. Int J Pharm 1995;117:219-24. 16. Williams A, Barry B. Skin absorption enhancers. Crit Rev Ther Drug Carr Sys 1992;9:305-53. 17. Ritschel W, Sprockel O. Sorption promoters for topically applied substances. Drugs of Today 1988;24:613-27. 18. Wade A, Weller P. Handbook of Pharmaceutical Excipients. Second edition. London: The Pharmaceutical Press; 1994. 19. Ruff M, Kalidindi S, Sutton J, inventors; Burroughs Wellcome, assignee. Pharmaceutical composition containing bupropion hydrochloride and a stabilizer. US Patent 5358970. 1994 Oct 25. 20. Bonferoni M, Rossi S, Ferrari F, Caramella C. A modified Franz diffusion cell for simultaneous assessment of drug release and washability of mucoadhesive gels. Pharm Dev Technol 1999;4:45-53. 21. Djordjevic J, Michniak B, Uhrich K. Amphiphilic star-like macromolecules as novel carriers for topical delivery of nonsteroidal anti-inflammatory drugs. AAPS PharmSciTech 2003;5:article 26 DOI:10.1208/ps050426. 22. Winkler A, Müller-Goymann C. Comparative permeation studies for δ-aminolevulinic acid and its n-butyl ester through stratum corneum and

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23.

24.

25.

26.

27.

artificial skin constructs. Eur J Pharm Biopharma 2002;53:281-7. Mary L, William E, Sheri L et al. Percutaneous penetration of Aldara™ cream, 5% during the topical treatment of genital and perianal warts. Primary care updae for OB/GYNS 1998;5;151. Gintautas K, Lars L, Karl H, Jürgen A, Hans D. Enhancement of percutaneous penetration of aniline and o-toluidine in vitro using skin barrier creams. Toxicol In vitro 2008;22:812-8. Müller-Goymann C, Alberg U. Modified water containing hydrophilic ointment with suspended hydrocortisone-21-acetate – the influence of the microstructure of the cream on the in vitro drug release and in vitro percutaneous penetration. Eur J Pharm Biopharm 1999;47:139-43. Fares H, Zatz J. Measurement of drug release from topical gels using two types of apparatus. Pharm Tech 1995;19:52-8. Addicks W, Flynn G Weiner N. Cells for use in transdermal research. Pharm Res 1987;4:337-41.

28. Shah V, Elkins J. In vitro release from corticosteroid ointments. J Pharm Sci. 1995;84:1139-40. 29. Pernarowaski W, Searl M. Continuous flow apparatus for the determination of the dissolution characteristics of tablets and capsules. J Pharm Sci 1968;57:1419-21. 30. Chimoskey J. Skin blood flow by 133Xe disappearance validated by venous occlusion plethysmography. J Applied Physiology 1972;32:432-5. 31. Shah V, Tsong Y, Sathe P, Lin J. In vitro dissolution profile comparison-statistics and analysis of the similarity factor f2. Pharm Res 1998;15:889-96. 32. Shah V, Elkins J, Williams R. Evaluation of the test system used for in vitro release of drugs from topical dermatological drug products. Pharm Dev Technol 1999;4:377-85

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Parallel Combinatorial Synthesis and In Vitro Evaluation of Ester and Amide Prodrugs of Flurbiprofen, Ibuprofen and Ketoprofen. A. A. Lohade, P. Jain and K. R. Iyer* Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Kalina, Mumbai 400 098, India. Author for correspondence:[email protected] ABSTRACT The application of the parallel combinatorial synthesis approach for preparation of ester and amide prodrugs of flurbiprofen, ibuprofen and ketoprofen was evaluated. The three non-steroidal anti-inflammatory drugs were each esterified or amidated with five different alcohols or amines, respectively, using dicylcohexylcarbodiimide. A total of fifteen ester and fifteen amide prodrugs were thus synthesized and characterized by melting point, UV and IR spectroscopy and HPLC retention times. The hydrolysis kinetics of the prodrugs in 50% human plasma was followed by HPLC. It was observed that the ester prodrugs were hydrolysed by human plasma with half lives ranging from 0.34 – 35.07 h. In contrast, the amide prodrugs, expectedly were refractory to plasma hydrolysis. The data obtained reflect the utility of parallel combinatorial synthesis for the generation of simple prodrugs. Key words: NSAIDs, parallel combinatorial synthesis, ester prodrugs, amide prodrugs, hydrolysis kinetics. INTRODUCTION The modern drug discovery approach involves the generation and/or evaluation of thousands of compounds to discover a single lead molecule that may progress to a therapeutically effective drug molecule. One of the approaches for the generation of large number of testable compounds is combinatorial chemistry. In this regard, parallel synthesis and splitmix combinatorial synthesis methods have been used to generate millions of compounds for high throughput screening.1 The prodrug approach has been widely used to overcome drug problems related to bad taste, poor aqueous or lipid solubility, inadequate chemical and enzymatic stability, incomplete absorption across a variety of biological membranes, distribution and premature/rapid/slow metabolism to inactive species.2-4 Additionally, the prodrug approach has also been applied in many therapeutic areas for optimization of peptide drugs, anticancer agents, local anesthetics, and non-steroidal anti-inflammatory drugs (NSAIDs).2-4 The NSAIDs share certain therapeutic actions and side effects. Gastric pain, mucosal erosion/ ulceration, blood loss and gastrointestinal (GI) toxicity are the major Indian Journal of Pharmaceutical Education & Research Received on 4/4/2008; Modified on 21/7/2008 Accepted on 1/8/2008 © APTI All rights reserved

drawback of NSAIDs.5 Various prodrug approaches have been used to overcome these drawbacks. One of the approaches is the synthesis of amide derivatives of diclofenac, tolfenamic acid, ibuprofen and indomethacin with the well-known antioxidant cystamine.6 Glycine amides of ketoprofen and several other well-known NSAIDs have also been synthesized and shown to be less irritating to GI mucosa.7 Glucosamine conjugates of meloxicam and flurbiprofen have also been evaluated.8,9 Most of the reported prodrugs have shown comparable antiinflammatory and analgesic activity as that of the parent drug with fewer GI side effects.5-10 The present investigation was carried out to synthesize various amide and ester prodrugs of three NSAIDs viz. ibuprofen, flurbiprofen and ketoprofen, using the parallel combinatorial synthesis approach and further, their potential in vitro reconversion to active drug in 50% human plasma (in 0.01 M phosphate buffer, pH 7.4) was evaluated. The main aim was to evaluate the applicability of the combinatorial synthetic approach to the synthesis of simple prodrugs. MATERIALS AND METHODS Ibuprofen, flurbiprofen and ketoprofen were gifts from Bajaj Life Sciences Pvt Ltd., Mumbai, India, FDC Pvt

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Ltd., Mumbai, India, and Themis Laboratories, Mumbai, India, respectively. Butylamine, propylamine, N, N-dicyclohexylcarbodiimide (DCC), and dimethylaminopyridine (DMAP) were purchased from S. D. Fine Chem. Mumbai, India. All other chemicals used were of analytical grade. All melting points were determined in open capillary tubes and are uncorrected. Combinatorial parallel synthesis of prodrugs: The overall approach for obtaining different prodrugs was to first synthesize the esters or amides in parallel fashion followed by individual work up of the reaction mixtures. The typical procedure for the synthesis of different prodrugs is described below with flurbiprofen as an example (Fig. 1). In five different round bottom flasks a solution of flurbiprofen (1.657 g, 6.7 mmol), DCC (1.537 g, 7.4 mmol), one of the five different alcohols (methanol 275 µl, ethanol 437 µl, n-propanol 557 µl, n-butanol 681 µl or benzyl alcohol 771 µl, all equivalent to 7.4 mmol) and DMAP (0.085 g, 0.692 µmol) in dichloromethane (30 ml) were taken. The flasks were shaken on a water bath at room temperature, until esterification was complete. The reaction was monitored by silica thin layer chromatography (TLC). In case of amide prodrugs of flurbiprofen (Fig. 2) a solution of flurbiprofen (1.657 g, 6.7 mmol) and DCC was allowed to react for 15 min and then one of the five different amines (propylamine 820 µl, isopropylamine 961 µl, butylamine 1.007 ml, benzylamine 1.093 ml or aniline 850 µl, all equivalent to 7.4 mmol) was added and the amidation was carried out similar to the esterification reaction. The flasks were shaken on a water bath at room temperature, until amidation was complete. The reaction was monitored by TLC. After completion of reaction, the precipitated N, Ndicyclohexylurea (DCU) was filtered off and the filtrate was washed successively with water (3 x 30 ml), 5% acetic acid solution (3 x 30 ml), 1% sodium bicarbonate (3 x 30 ml) and water (3 x 30 ml). The organic layer was separated and dried over anhydrous sodium sulphate. The solvent was evaporated and the crude product was purified by column chromatography using hexane: chloroform as mobile phase. The products were then weighed and qualitatively analyzed by UV–visible spectrophotometry (Shimadzu UV-160A), Fourier transformer infrared spectroscopy (Jasco FT/IR- 5300),

and HPLC (Jasco – Borwin, PU-980 pump with UV975 detector). A similar protocol was followed for the synthesis of ester and amide prodrugs of ibuprofen (1.4 g, 6.7 mmol) and ketoprofen (1.726 g, 6.7 mmol). The flurbiprofen ester prodrugs synthesized were designated as flurbiprofen methyl ester (E-Flu-m), flurbiprofen ethyl ester (E-Flu-e), flurbiprofen n-propyl ester (E-Flup), flurbiprofen n-butyl ester (E-Flu-bu), and flurbiprofen benzyl ester (E-Flu-be). Likewise the esters of ibuprofen and ketoprofen were also synthesized and designated as E-Ibu-m, E-Ibu-e, E-Ibup, E-Ibu-bu, E-Ibu-be and E-Ket-m, E-Ket-e, E-Ket-p, E-Ket-bu, E-Ket-be, respectively. Similarly amide prodrugs of flurbiprofen, ibuprofen, and ketoprofen were synthesized using parallel combinatorial technique and abbreviated as propyl flurbiprofenamide (A-Flu-p), isopropyl flurbiprofenamide (A-Flu-ipa), butyl flurbiprofenamide (A-Flu-bu), aniline flurbiprofenamide (A-Flu-ani) and benzyl flurbiprofenamide (A-Flu-be); A-Ibu-p, A-Ibu-ipa, AIbu-bu, A-Ibu-ani, A-Ibu-be and A-Ket-p, A-Ket-ipa, A-Ket-bu, A-Ket-ani and A-Ket-be, respectively. Characterization of prodrugs: For UV spectrophotometric characterization, solutions of the ester and amide prodrugs of flurbiprofen (5 µg/ml), ibuprofen (250 µg/ml) and ketoprofen (10 µg/ml) were prepared in methanol and water (3:1), 0.1 N NaOH and in ethanol, respectively, and scanned in the range of 200 – 400 nm.. FT-IR spectra of various ester and amide prodrugs were obtained using the KBr disc technique. For HPLC characterization, the mobile phases used were acetonitrile: water (50:50 v/v, pH adjusted to 3 using phosphoric acid) and methanol: water (80:20 v/v) for ester and amide prodrugs, respectively. A C-18 Nucleosil (4.6 mm x 15 cm) column was used at a flow rate of 1.2 ml/min. The wavelength used for detection was 254 nm. An aliquot of 20 µl of flurbiprofen and its prodrugs (10 µg/ml), ibuprofen and its prodrugs (1 mg/ml), and ketoprofen and its prodrugs (10 µg/ml) was injected for characterization of the retention times. Hydrolysis kinetics in human plasma: Hydrolysis kinetics of the ester and amide prodrugs was carried out in 50% human plasma (diluted with 0.01 M phosphate buffer, pH 7.4) at 370. The hydrolysis

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reaction was initiated by addition of the esters and amides to a final concentration of 0.2 - 1.4 mM (flurbiprofen and ketoprofen prodrugs were incubated at a final concentration of 0.2 mM, while ibuprofen prodrugs were incubated at a higher concentration of 1.4 mM to allow for HPLC analysis) and the reaction followed by sampling the incubated mixture at periodic intervals of 0, 15, 30, 60, 120, 240, and 480 min. (For amide prodrugs sampling was continued for 24 h). Incubations were terminated by addition of twice the volume of 2% zinc sulphate (in 50% aqueous methanol), centrifuged at 7500xg for 15 min and supernatant was analyzed by HPLC. The mobile phase was acetonitrile: water (50:50) and phosphoric acid was added to adjust the pH~3. The C-18 Waters (3.9 x 300 mm) column was used at a flow rate of 1.2 ml/min. Ketoprofen was used as the internal standard for ibuprofen and flurbiprofen prodrugs, for ketoprofen ester prodrugs flurbiprofen was used as internal standard while for ketoprofen amide prodrugs, celecoxib was used as an internal standard. Hydrolysis kinetics in aqueous buffer (pH 7.4): Control incubations for hydrolysis kinetics of prodrugs of flurbiprofen in human plasma were carried out in 0.01 M phosphate buffer pH 7.4, in a water bath shaker at 37o. The incubation was identical to that described above except for the absence of plasma in these incubations. HPLC conditions were similar to that described above RESULTS The yields of flurbiprofen, ibuprofen and ketoprofen ester prodrugs were found to be in the range of 38.854.7%, 45.55-77.45% and 54.43-67.97%, respectively. The yield of flurbiprofen, ketoprofen and ibuprofen amide prodrugs was found to be in the range of 55.1569.12%, 48.57-64.31% and 48.75-52.63%, respectively. The nature, melting point and percent yield of each prodrug is reported in Table No. 1. The UV-λmax, UV molar absorptivity and IR characteristics (C=O stretching vibration) are reported in Table No. 2. The composition of mobile phase, retention time (min) and t1/2 (in 50% plasma) are as shown in Table No. 3. First order kinetics was observed for the hydrolysis of ester prodrugs in 50% human plasma at 37º (0.01 M phosphate buffer, pH 7.4). The ratio of area of prodrug to internal standard was estimated at time zero and at regular interval after incubation in human plasma. The

log % prodrug remaining was plotted against time. The pseudo first order rate constant (Kobs) was obtained from the slope of linear plots of the log % ratio against time as follows: Slope = - Kobs/2.303, and therefore Kobs = - (2.303 x slope). The corresponding half life (t1/2) was obtained as: t1/2 = 0.693 / Kobs The log % prodrug remaining plotted against time for all ester prodrugs of flurbiprofen, ketoprofen and ibuprofen are given in Figs: 3-5. The t1/2 for hydrolysis of flurbiprofen, ibuprofen and ketoprofen ester prodrugs in 50% human plasma prepared in 0.01 M phosphate buffer pH 7.4 was found to be in the increasing order as follows: E-Flu-bu (9.73 h), E-Flu-p (13.23 h), E-Flu-e (15.19 h), E-Flu-m (20.3 h) and E-Flu-be (35.07 h). E-Ibu-bu (7.59 h), E-Ibu-p (8.63 h), E-Ibu-e (9.64 h), EIbu-m (13.74 h) and E-Ibu-be (20.46 h). E-Ket-be (0.34 h), E-Ket-bu (0.83 h), E-Ket-p (1.69 h), E-Ket-m (2.23 h) and E-Ket-e (3.98 h). On the other hand, none of the amide prodrugs were hydrolyzed to parent drug after incubation in plasma. DISCUSSION Gastric upset caused by NSAIDs is mainly due to the acidic functional group associated with these compounds. To overcome this drawback, a prodrug approach has been used to temporarily mask the acidic functional group of the NSAIDs.11 The idea is to reduce gastric exposure to free acid. After absorption of prodrug, it is expected that the prodrug must be metabolized to release the parent drug in circulation or at the site of action. This can be achieved by breaking the bond between parent drug and promoiety either by enzymatic or by chemical reaction.12 Esters as well as amides can potentially be hydrolyzed in plasma, serum or in liver microsomes and further these can be very easily synthesized if drug molecule contains carboxylic acid functional group.13-15 DCC a type of carbodiimide has been widely used in peptide synthesis for coupling of amino acids. DCC activates the carboxylic acid moiety toward nucleophilic substitution reaction.16-18 Previous studies have reported effectiveness of DCC in esterification as well as amidation reaction for synthesis of ester prodrugs of ibuprofen and indomethacin, etc. 12,19,20 It is also reported that the use of DMAP with DCC in the esterification reaction improves the yield of final product in comparison to use of DCC alone.21 In the

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Table 1 - Nature, melting point and % yield of ester and amide prodrugs of flurbiprofen, ibuprofen and ketoprofen. Amide prodrugs Prodrugs

Nature

A-Flu-p

Ester prodrugs % Yield

Prodrugs

Nature

Semisolid

Melting point (°) -

% Yield

Solid

Melting point (°) 46-48

68.7

E-Flu-m

A-Flu-ipa

Solid

109-110

64.26

E-Flu-e

Oil

-

47.13

A-Flu-bu

Solid

77-79

55.73

E-Flu-p

Oil

-

46.37

A-Flu-ani

Solid

142-144

69.12

E-Flu-bu

Oil

-

38.8

A-Flu-be

Solid

147-148

55.15

E-Flu-be

Oil

-

54.7

A-Ibu-p

Semisolid

-

50.42

E-Ibu-m

Oil

-

66.98

A-Ibu-ipa

Solid

78-80

48.33

E-Ibu-e

Oil

-

77.45

A-Iba-bu

Semisolid

-

47.77

E-Ibu-p

Oil

-

66.55

A-Ibu-ani

Solid

147-149

52.63

E-Iba-bu

Oil

-

45.55

A-Ibu-be

Solid

145-146

48.75

E-Ibu-be

Oil

-

74.67

A-Keto-p

Semisolid

-

61.02

E-Keto-m

Oil

-

54.43

A-Keto-ipa

Semisolid

-

52.12

E-Keto-e

Oil

-

56.43

A-Keto-bu

Semisolid

-

51.66

E-Keto-p

Oil

-

57.75

A-Keto-ani

Semisolid

-

64.31

E-Keto-bu

Oil

-

57.99

A-Keto-be

Semisolid

-

48.57

E-Keto-be

Oil

-

67.97

48.54

Table 2 - UV and IR characterization of ester and amide prodrugs of flurbiprofen, ibuprofen and ketoprofen. Amide prodrugs Ester prodrugs Prodrugs

λ max (nm)

ε (L/cm.mole)

A-Flu-p

245.2

A-Flu-ipa

λ max (nm)

Prodrugs

26800

C=O str. vibration (cm-1) 1645.43

E-Flu-m

245.4

32786

C=O str. vibration (cm-1) 1738.02

245.0

25000

1655.07

E-Flu-e

245.6

34356

1734.16

A-Flu-bu

245.8

21500

1645.43

E-Flu-p

246.2

26864

1734.16

A-Flu-ani

245.4

33000

1660.86

E-Flu-bu

246.4

21500

1734.16

A-Flu-be

245.0

31500

1643.50

E-Flu-be

246.0

31626

1736.09

A-Ibu-p

263.8

275

1647.36

E-Ibu-m

263.5

140

1739.95

A-Ibu-ipa

263.3

235

1643.50

E-Ibu-e

263.7

254

1736.09

A-Iba-bu

264.0

220

1645.43

E-Ibu-p

263.5

302

1736.09

A-Ibu-ani

242.4

450

1658.93

E-Iba-bu

263.7

199

1736.09

A-Ibu-be

263.8

438

1641.57

E-Ibu-be

263.2

443

1738.02

A-Keto-p

254.6

37875

1651.22

E-Keto-m

253.2

27869

1738.02

ε (L/cm.mole)

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A-Keto-ipa

254.8

36025

1649.26

E-Keto-e

253.2

29017

1732.23

A-Keto-bu

255.6

28780

1651.22

E-Keto-p

253.4

48789

1734.16

A-Keto-ani

251.8

30075

1658.93

E-Keto-bu

253.6

28707

1734.16

A-Keto-be

254.2

28715

1657.93

E-Keto-be

253.8

30662

1736.09

Table No. 3 - HPLC characterization of ester and amide prodrugs of flurbiprofen, ibuprofen and ketoprofen. Ester Prodrugs Amide Prodrugs Prodrugs

Retention time (min)

6.025

t1/2 in 50% plasma (h) -

E-Flu-m

10.197

t1/2 in 50% plasma (h) 20.3

A-Flu-ipa

6.007

-

E-Flu-e

13.322

15.19

A-Flu-bu

7.413

-

E-Flu-p

18.072

13.23

A-Flu-ani

7.853

-

E-Flu-bu

25.203

9.73

A-Flu-be

7.062

-

E-Flu-be

23.695

35.07

A-Ibu-p

7.39

-

E-Ibu-m

13.389

13.74

A-Ibu-ipa

8.56

-

E-Ibu-e

18.133

9.64

A-Iba-bu

9.02

-

E-Ibu-p

25.878

8.63

A-Ibu-ani

9.31

-

E-Iba-bu

37.342

7.59

A-Ibu-be

8.78

-

E-Ibu-be

34.860

20.46

A-Keto-p

4.512

-

E-Keto-m

6.923

2.23

A-Keto-ipa

4.447

-

E-Keto-e

8.707

3.98

A-Keto-bu

5.150

-

E-Keto-p

11.462

1.69

A-Keto-ani

5.453

-

E-Keto-bu

15.530

0.83

A-Keto-be

5.095

-

E-Keto-be

15.007

0.34

Prodrugs

Retention time (min)

A-Flu-p

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Fig. 1 - General synthetic scheme for combinatorial synthesis of ester prodrugs of NSAIDs. CH3 DCC COOH + RNH 2 DCM, RT

H3C CH3

CH3 CONHR

H3 C CH3

IBUPROFEN

O

CH3 COOH

O + RNH2

CH3 CONHR

DCC DCM, RT

KETOPROFEN

F

F CH3 CONHR

CH3 DCC COOH + RNH 2 DCM, RT FLURBIPROFEN

R = -C3H7, -Isopropyl ,-C4H9, -CH2C6H5, -C6H5

Fig. 2 - General synthetic scheme for combinatorial synthesis of amide prodrugs of NSAIDs. E-FLU-M

100 Log % Prodrug Remaining

E-FLU-E E-FLU-P E-FLU-BU E-FLU-BE

10 0

120

240

360

480

Time (min)

Fig. 3 - Hydrolysis kinetics of flurbiprofen ester prodrugs in 50% human plasma. Fig 3. The five ester prodrugs of flurbiprofen were incubated in 50% human plasma at a final concentration of 0.2 mM. The hydrolysis was followed by sampling at periodic intervals, after which the incubations were analyzed by RP-HPLC to determine the % prodrug remaining.

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E-IBI-M

100 Log % Prodrug Remaining

E-IBU-E E-IBU-P E-IBU-BU E-IBU-BE

10 0

120

240

360

480

Time (min)

Fig. 4 - Hydrolysis kinetics of ibuprofen ester prodrugs in 50% human plasms. Fig. 4: The five ester prodrugs of ibuprofen were incubated in 50% human plasma at a final concentration of 1.4 mM. The hydrolysis was followed by sampling at periodic intervals, after which the incubations were analyzed by RP-HPLC to determine the % prodrug remaining. E-KET-M E-KET-E

Log % Prodrug Remaining

100

E-KET-P E-KET-BU E-KET-BE

10 0

120

240

360

480

Time (min)

Fig. 5- Hydrolysis kinetics of ketoprofen ester prodrugs in 50% human plasma Fig. 5 : The five ester prodrugs of ketoprofen were incubated in 50% human plasma at a final concentration of 0.2 mM. The hydrolysis was followed by sampling at periodic intervals, after which the incubations were analyzed by RP-HPLC to determine the % prodrug remaining. present investigation, DCC was used for synthesis of ester and amide prodrugs of flurbiprofen, ibuprofen and ketoprofen by combinatorial parallel synthesis technique. Since the purpose of present investigation was only to evaluate the utility of combinatorial chemistry for synthesis of prodrugs and not optimization of the DCC reaction, the reaction conditions for the amidation and esterification of drugs with DCC were not optimized with respect to quantity of DCC, quantity of alcohols/amines, temperature, and time. Thus it is possible that further studies may give higher or improved yields of the ester and amide prodrugs.

UV characterization of all prodrugs showed similar spectral behavior as that of the parent drug, except benzyl ester prodrugs which exhibited a shoulder at about 270-285 nm. However the λmax of benzyl ester prodrug was similar to parent drug. The molar absorptivity values (ε) of all the prodrugs were found to be similar to the parent drug. IR spectra for ester and amide prodrugs showed ester and amide characteristic band, respectively and also showed the absence of the acid functionality band. HPLC characterization revealed the difference in retention times of prodrugs compared to the parent drug. The results of hydrolysis of ester prodrugs in phosphate buffer (pH 7.4) (Control experiment) indicate that these

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prodrugs were not significantly hydrolyzed to parent molecules in the absence of plasma. Previous reports have indicated that hydrolysis of ester prodrugs of ibuprofen in phosphate buffer (pH 7.4), showed half lives (t1/2) of 222.9 h, 98.7 h, 72.4 h and 42.9 h for the Ibu-methyl, Ibu-ethyl, Ibu-propyl and Ibu-butyl esters, respectively.22,23 In another study, the t1/2 of ketoprofen hydroxyethyl ester, ketoprofen hydroxypropyl ester, ketoprofen hydroxybutyl ester, naproxen hydroxyethyl ester, naproxen hydroxypropyl ester and naproxen hydroxybutyl ester were 15, 47, 38, 25, 52, and 65 days, respectively, in phosphate buffer (pH 7.4).24 The present work also indicates that both the ester and amide prodrugs are fairly stable in phosphate buffer (pH 7.4). Bansal et al have reported that t1/2 for the hydrolysis of ibuprofen ester prodrugs in plasma was in increasing order as follows: Ibu-butyl (4.9 h), Ibu-propyl (7.8 h), Ibu-ethyl (10.5 h) and Ibu-methyl ester (11.25 h).22,23 This suggested that the longer alkyl chain of prodrug showed a faster rate of hydrolysis. In another similar study, Rautio et al reported that t1/2 for hydrolysis of ketoprofen hydroxyalkyl ester and naproxen hydroxyalkyl ester prodrugs in 80% human serum was in increasing order24 i.e. ketoprofen hydroxybutyl ester (0.58 h), ketoprofen hydroxypropyl ester (1.15 h) and ketoprofen hydroxyethyl ester (2.06 h), and also naproxen hydroxybutyl ester (1.01 h), naproxen hydroxypropyl ester (2.45 h) and naproxen hydroxyethyl ester (3.73 h). The t1/2 values for ibuprofen prodrugs in our study are slightly higher than reported, most probably because we have used 50% human plasma while in the reported studies hydrolysis was carried out in 100% plasma. However, a similar rank order of hydrolysis half-lives was observed in the present study as in previous reports. The data from this hydrolysis study also suggests that increase in the alkyl chain group of ester results in faster hydrolysis rate and decreases the t1/2 value. However, the benzyl ester of ketoprofen was found to have shorter t1/2 than other esters of ketoprofen. On the other hand benzyl ester of ibuprofen and flurbiprofen was found to have longer t1/2 than other esters of ibuprofen and flurbiprofen, respectively. The behavior of the ketoprofen ester prodrugs in plasma is similar to that reported for benzoic acid esters. The t1/2 values for the hydrolysis of benzoic acid esters in 80% human plasma was in

increasing order as follows: benzyl (19 min), propyl (46 min), methyl (108 min) and ethyl (210 min) ester.5 A similar pattern was observed in ketoprofen ester series of prodrugs. The difference in hydrolysis pattern of ketoprofen ester prodrugs with flurbiprofen and ibuprofen ester prodrug is not known at present and further investigation is required to understand the underlying mechanism for this anomalous behavior. In vitro hydrolysis of the amide prodrugs was carried out using enzymatic hydrolysis in 50% human plasma. The hydrolysis of benzyl ester prodrugs of ketoprofen in human plasma was done at the same time as a positive control, to ascertain that the human plasma used for hydrolysis of amide prodrugs was enzymatically competent. The results revealed that the amide prodrugs of flurbiprofen, ibuprofen and ketoprofen were not hydrolyzed to parent drug in 50% human plasma (no decrease in prodrug peak height and absence of the corresponding parent drug peak in hydrolysis chromatograms). This indicates that the amide prodrugs are stable in 50% human plasma. This may be due to inherent stability of amide bond of the prodrugs as compared to the ester prodrugs. In this regard, procaine and procainamide are ester and amide analogues of p-amino benzoic acid, respectively. Procaine when incubated in plasma is hydrolyzed to pamino benzoic acid within one minute25, while procaineamide shows no hydrolysis after incubation in plasma for nineteen hours.26 The results obtained for the hydrolysis reaction of amide and ester prodrugs of NSAIDs in the present investigation are in agreement with generally accepted fact that esters are much easier to hydrolyze biologically than amides unless the amides are activated as for examples, in the case of N-glycyl derivative midodrin.3 Ester-type of topical anesthetics have been reported to undergo hydrolysis in the presence of esterase present in plasma and thus tend to have shorter half-lives. Amide types of topical anesthetics are mainly hydrolyzed by liver esterase and generally possess longer half-life as compared to ester types of topical anesthetics.27 Docarpamine is an amide-type dopamine prodrug. Conversion rates from docarpamine to dopamine in various rat tissue homogenates were in the order of liver > small intestine > blood. Free dopamine, is mainly produced in the liver.28 It is possible that in our case, the hydrolysis of amide prodrug may occur in

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the liver. However preliminary studies (data not shown) have suggested that amide prodrugs of NSAIDs are stable in liver homogenates. Interestingly, studies have also indicated that in some cases cleavage of the amide bond and release of drug may not be necessary for activity since the amidated analogs have potential antiinflammatory activity, i.e., amide derivative of NSAIDs may not be technically prodrugs. 19,29 CONCLUSION Overall the synthesis of ester and amide prodrugs, and the in vivo bioconversion potential was investigated by incubation of these prodrugs in 50% human plasma. Our results of ester prodrugs are in good agreement with other reported results for ester prodrugs of NSAIDs. In addition in case of amide prodrugs, no hydrolysis in human plasma was observed as expected. This study demonstrates the utility of parallel combinatorial synthesis approach for synthesis of simple ester or amide prodrugs. ACKNOWLEDGEMENTS The authors wish to thank Dr. M. L. Kubal, Wockhardt Ltd., Aurangabad for providing the HPLC column. This research work was supported by AICTE (File No. 8019/RDII/R&D/PHA (213) 2000-01) and DST-“FIST Program”-[SR/FST/LS1-163/2003]. REFERENCES 1. Bennett WD, Christensen JW, Hamaker LK, Peterson ML, Rhodes MR, Sanii H, Fruka A. Handbook of Combinatorial and Solid Phase Organic Chemistry. Kentucky: Advanced Chemtech Inc; 1998. 2. Bundgaard H, ed. Design of Prodrugs. Amsterdam: Elsevier; 1985. 3. Larsen CS, Ostergaard J. Design and Application of Prodrugs. In: Krogsgaard-Larsen P, Liljefors T, Madsen U, ed. Textbook of Drug Design and Discovery, 3rd ed. New York: Taylor and Francis Inc; 2002. 410-58. 4. Silverman RB. The Organic Chemistry of Drug Design and Drug Action. 2nd ed. New Delhi: Academic Press; 2004. 5. Roberts LJ, Morrow JD. Analgesic-antipyretic and anti-inflammatory agents and drugs employed in the treatment of gout. In: Hardman JG, Limbard LE, Gilman AG, ed. Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th ed. New York: McGraw-Hill; 2001. 687-732.

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Shanbhag VR, Crider AM, Gokhale R, Harpalani A, Dick RM. Ester and amide prodrugs of ibuprofen and naproxen: synthesis, antiinflammatory activity, and gastrointestinal toxicity. J Pharm Sci 1992; 81: 149-54. Zovko M, Zorc B, Jadrijevic M, Takac M, Metelko B, Novak P. The Novel Ketoprofenamides: Synthesis and Spectroscopic Characterization. Croatia Chemica Acta 2003; 76 (4): 335-41. Dhaneshwar SS, Ghodeswar BC, Bhojani MR. Synthesis and biological evaluation of glucosamine conjugate prodrug of flurbiprofen. Indian Drugs 2003; 40 (3): 156-9. Hunjra AS, Dharmendra K, Gairola N, Bhojani M, Dhaneshwar SS. Synthesis and biological evaluation of mutual prodrug of mefanamic acid with glucosamine. Indian J Pharm Edu Res 2006; 40(6): 40-4. Friis GJ, Bundgaard H. Design and application of prodrugs. In: Krogsgaard-Larsen P, Liljefors T, Madsen U, ed. Textbook of Drug Design and Discovery, 2rd ed. Amsterdam: Harwood Academic Publishers; 1996. 351-85. Smith FT, Clark CR. Drug latentiation and prodrugs. In: Delgado JN, Remers WA, ed. Wilson and Gisvold’s Textbook of Organic Medicinal Chemistry and Pharmaceutical Chemistry. 10th ed. Philadelphia: Lippincott-Raven Publishers; 1998. 123-38. Nielsen M, Bundgaard, H. Evaluation of glycolamide esters and various esters of aspirin as true aspirin prodrugs. J Med Chem 1989; 32: 72734. Morrison RT, Boyd RT. Organic Chemistry, 6th ed. New Delhi: Prentice Hall of India Pvt. Ltd.; 1996. Furniss BS, Hannaford AJ, Smith PWG, Tatchell A. Textbook of Practical Organic Chemistry, 5th ed. Singapore: Longman Scientific and Technical; 1989. Finar IL. Organic Chemistry, The Fundamental Principles, 6th ed. New Delhi: ELBS-Pearson Eductional Ltd; 1973. Hodson D, Holt G, Wall DK. Diazoketones from the interaction of diazoalkanes with carboxylic acid-dicyclohexylcarbodiimide mixture. J Chem Soc 1970; 971-73.

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prodrug of ketoprofen and naproxen. J Pharm Sci 1998; 87(12): 1622-28. 25. Borde BB, Lief PA, Poet R. The fate of procaine in man following its intravenous administration and methods for estimation of procaine and diethyl amino ethanol. J Pharmacol Exp Ther 1948; 94: 359-66. 26. Mark LC, Kayden HJ, Steele JM, Cooper JR, Berlin I, Rovenstine EA Brodie B. Physiological disposition and cardiac effects of procaine amide. J Pharmacol Exp Ther 1951; 102: 5-13. 27. Nelson D, Garland WA, Breck GD, Trager WF. Quantification of lidocaine and several metabolites utilizing chemical ionization mass spectroscopy and isotope labelling. J Pharm Sci 1977; 66 (8): 1180-89. 28. Yoshikawa M, Nishiyama S, Takaiti O. Metabolism of dopamine prodrug, docarpamine. Hypertens Res 1995; 1: 211-13. 29. Kalgutkar AS, Crews BC, Rowlinson SW, Marnett AB, Kozak KR, Remmel RP, Marnett LJ. Biochemically based design of cyclooxygenase (COX-2) inhibitors. Facile conversion of nonsteroidal antiinflammatory drugs to potent and highly selective COX-2 inhibitors. Proc Natl Acad Sci 2000; 97: 925-30. ***********

17. Doleschall G. Lampert, K. On the mechanism of carboxyl condensations by carbodimiides. Tetrahedron Lett 1963; 18: 1195-99. 18. Carey, FA, Sundburg RJ. Advanced Organic Chemistry – Part B. 4th ed. New York: Kluwer Academic / Plenum Publishers; 2001. 19. Kalgutkar AS, Marnett AB, Crews BC, Remmel R, Marnett LJ. Ester and amide derivatives of the nonsteroidal anti inflammatory drug indomethacin, as selective cyclooxygenase–2 inhibitors. J Med Chem 2000; 43: 2860-70. 20. Hassen A, Alexanina V. Direct room temperature esterification of carboxylic acids. Tetrahedron Lett 1978; 46: 4475-76. 21. Kurzer F, Douraghi-Zadeh K. Advance of chemistry of carbodimiides. Chem Rev 1967; 67: 107-40. 22. Bansal AK, Dubey R, Khar RK. Quantitation of activity of alkyl ester prodrugs of ibuprofen. Drug Develop Ind Pharmacy 1994; 20(12): 2025-34. 23. Bansal AK, Dubey R, Khar RK, Sharma AK. Alkyl ester prodrug for improved topical delivery of ibuprofen. Ind J Exp Bio 2001; 39: 280-83. 24. Rautio J, Taipale H, Gynther J, Vepsalainen J. In vitro evaluation of acyloxyalkyl ester as dermal

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APTI

ijper

Taste Masking and Formulation of Ofloxacin Rapid Disintegrating Tablets and Oral Suspension Shishu*, Varun Rishi Kapoor and Kamalpreet University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, INDIA *Corresponding author: [email protected] ABSTRACT Ofloxacin, widely used antibacterial drug was bitter taste-masked and formulated into two patient compliant dosage forms, rapidly disintegrating tablets and liquid oral suspension. Taste masking was achieved using a pH sensitive polymer Eudragit E-100 (aminoalkylmethacrylate copolymers). Extrusion followed by crushing technique was used to prepare taste-masked granules. Taste masked granules were directly compressed into tablets using microcrystalline cellulose (MCC) as directly compressible filler and sodium starch glycolate (SSG) as a super disintegrant. The tablets were optimized for tensile strength and fast disintegration characteristics by varying the amounts of MCC and SSG. Finally the optimized dosage form was subjected to various evaluation tests like pharmacopoeial tests, in vitro drug release, disintegration time in oral cavity, wetting time and panel testing for taste assessment. A liquid oral suspension was also prepared using taste masked granules and was evaluated for taste by panel testing besides other parameters like drug content, viscosity, pH and resuspendability. Panel testing data collected from 20 healthy human volunteers indicate successful formulation of oral fast disintegration tablets with a good taste and rapid disintegration in oral cavity and excellent palatability of taste masked liquid oral suspension. Keywords: Rapidly disintegrating tablets; taste masked granules; Eudragit E-100; compression method. INTRODUCTION With the advancement in technology and experience, pharmaceuticals are prepared and administered to patients in more compliant and efficient manner. Rapid disintegrating tablets are the new improved dosage forms developed especially for the young and the elderly patients who find inability to swallow tablets and capsules due to under developed muscular, nervous system and dysphagia. These solid dosage forms dissolve or disintegrate rapidly in oral cavity, resulting in solution or suspension that can be swallowed without the need of water. When this type of tablet is placed into the mouth, the saliva serves to rapidly dissolve the tablet usually in about 30 seconds1,2. The critical formulation problem is the masking of bitter taste associated with most of the drugs and this can be overcome by taste masking techniques such as polymer coating, complex formation, granulation, microencapsulation and use of ion exchange resins3.

Indian Journal of Pharmaceutical Education & Research Received on 21/8/2008 ; Modified on 3/12/2008 Accepted on 21/12/2008 © APTI All rights reserved

Also bitterness inhibition can be achieved by the use of selective inhibitor like PA-LG, a combination of phosphatidic acid and β-lactoglobulin that inhibits only bitter taste sensation without affecting other taste modalities like sweetness, sourness and saltiness4. Ofloxacin is widely used antibacterial drug recommended in the treatment of chronic bronchitis, respiratory/ENT infections, nonspecific urethritis, gonorrhoea, atypical pneumonia, leprosy, cervicitis. One of the major drawbacks of this drug is its bitter taste which may give rise to patient noncompliance when formulated as conventional dosage forms. In the present study taste masking of bitter drug ofloxacin was achieved by preparing taste masked granules using a pH-sensitive polymer, Eudragit E1005. These granules were then formulated into rapidly disintegrating tablets using the technique of superdisintegrant addition. Also a suspension dosage form was prepared using taste- masked granules of ofloxacin. MATERIALS AND METHODS Materials - Ofloxacin and Eudragit E-100 were

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obtained as gift sample from Sai Bliss Drug and Pharmaceuticals, Karnal, India and Röhm Pharma, Germany respectively. Microcrystalline cellulose was received as a gift from Panacea Biotech, Lalru, India and Sodium starch glycolate from Kemwell Pvt. Ltd., Bangalore, India. All other chemicals used were of analytical grade. Preparation of taste-masked granules Ofloxacin was thoroughly mixed with varying amounts of powdered Eudragit E-100. Then 10% ethanol was added to this mixture in a glass beaker and a gel was prepared. The prepared gel was manually pressed out using a syringe. After extrusion of the gel, ethanol was removed by evaporation overnight at room temperature. Subsequently the solidified gel was crushed into granules using a pestle and mortar6. The drug: polymer ratio which produced taste masked granules was used for further studies. Formulation of rapidly disintegrating tablets (RDTs) The prepared Eudragit E-100 granules of ofloxacin were compressed into (RDTs) by using microcrystalline cellulose (MCC) as directly compressible binder and sodium starch glycolate (SSG) as the superdisintegrant7. Flavoring was done with menthol to give the tablets more palatable feel. Magnesium stearate was added as the lubricant while mannitol was added as the diluent. Similarly control tablets were prepared using pure drug instead of taste masked granules. Different ratios of the super-disintegrant, SSG and the binder, MCC were investigated for the formulation of ofloxacin RDTs and the ratio that gave the minimum disintegrating time along with acceptable hardness was chosen for the formulation of the final batch of tablets (Table 1). Evaluation of RDTs The force required to break the tablet (tensile strength) was measured using Monsanto hardness tester. Friability was measured with the Roche friabilator. Initial weight of tablet was noted and then was rotated in the friabilator for 4 minutes at 30 rpm. The loss in weight (%) was calculated. To measure wetting time and water absorption ratio a piece of paper tissue folded twice was placed in a petriplate containing 6 ml of water. A pre-weighed tablet was placed on the paper and the time for complete wetting was measured. The wetted tablet was

then weighed and water absorption ratio, R, was determined according to the following formula, R=100(Wa –Wb)/Wb where, Wa and Wb are the weight after and before water absorption, respectively. In vitro dissolution studies were carried out using USP XXIII Dissolution apparatus II (paddle type). A tablet was placed in a basket and rotated at 50 rpm. The release profile was studied both in phosphate buffer at pH 6.8 and hydrochloric acid buffer pH 1.2. For the measurement of in vitro disintegration time a modified dissolution apparatus (paddle type) was used. 900 ml of water maintained at 37°C and stirred with a paddle at 100 rpm was used as the disintegration fluid. A tablet was placed in the sinker and disintegration time was determined at the point at which the tablet disintegrated completely and passed through the screen of the sinker. Disintegration time in oral cavity was also determined. The time required for complete disintegration in the oral cavity of six healthy volunteers was noted who randomly took one tablet without biting and without drinking water8. The taste evaluation was done using spectrophotometric method and by panel testing9,10. For panel testing, 20 healthy human volunteers, of either sex, in the age group of 20-30 years were selected out of 31 volunteers based on the 6-n-propylthiouracil (Prop) taste sensitivity test11. Taste threshold for all the volunteers were determined by making a range of dilutions of Prop. The non-tasters and super-tasters were rejected. Then the selected panel of 20 volunteers was requested to taste the taste-masked RDTs by keeping in the mouth till they disintegrated and rank it on a scale of perception ranging from 0-5. For comparison, RDTs of pure ofloxacin were also subjected to taste evaluation by the panel and the results were compared. Taste masked suspension A suspension dosage form was prepared using the tastemasked ofloxacin granules. Granules were suspended in sodium citrate buffer pH 6.8 with the help of Tween 80. Preservatives were dissolved in sorbitol and the suspension was flavored with strawberry flavor. The prepared suspension was evaluated for taste by panel testing besides other parameters like drug content, viscosity, pH and resuspendability12.

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Table No. 1: Effect of sodium starch glycolate (SSG) and microcrystalline cellulose (MCC) ratio on the disintegration time of ofloxacin RDTs Tablet strength Formulation code SSG: MCC Disintegration time (s) (n=6) (hardness in kg/cm2) OF-1 1:0.5 0.5 7.00 ± 0.94 OF-2 1:1.0 1.0 15.00 ± 1.36 1.5 OF-3 1:2.0 52.00 ± 1.11 OF-4 1:3.0 2.5 58.00 ± 1.31 OF-5 1:4.0 3.0 63.00 ± 0.97 3.5 OF-6 1:5.0 84.00 ± 1.54 4.0 OF-7 1:6.0 86.00 ± 2.01 4.5 OF-8 1:8.0 98.00± 1.73 5.5 OF-9 1:10 119.00 ± 0.63 The highlighted ratio is selected as optimum ratio of SSG and MCC for preparation of RDTs

Table No. 2: Physical properties of ofloxacin RDTs Parameter RDT Weight (mg), n=20 250.00± 1.58 Diameter (cm), n=6 0.95 ± 0.03 Thickness (cm), n=6 0.41 ± 0.1 Hardness (kg/cm2), n=6 2.50 ± 0.21 Friability, n=6 0.20 % Wetting time (s), n=3 45.00 ±0.71 Water absorption ratio ,n=3 15.34 ± 0.84 In vitro disintegration time (s), n=6 58.26± 1.46 Disintegration time in oral cavity (s), n=3 27.16± 1.37 Drug content (mg), n=3 49.26± 0.16

Pure drug tablet 250.00± 1.28 0.95 ± 0.03 0.42 ± 0.1 3.00± 0.20 0.38% 59.00±1.00 29.20±0.88 72.00±1.00 29.16± 1.39 49.16± 0.18

Table No. 3: Data for evaluation of taste of ofloxacin preparations by the panel No. of volunteers rating the formulation as* Formulation 0 1 2 3 4 Tablet of plain drug 1 15 4 Taste masked granules 20 RDT of ofloxacin 17 3 * 0=good, 1=tasteless, 2=slightly bitter, 3=bitter, 4=very bitter, 5=awful

5

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Fig 1: Comparison of in vitro release profiles of pure drug tablet and RDT of ofloxacin in phosphate buffer at pH 6.8 (n = 6)

% Cummulative Release

120 100 80 60 40 20 0 0

1

2 3 Time (min)

4

5

% Release of Pure Drug Tablet % Release of RDT

Fig. 2: Comparison of in vitro release profiles of pure drug tablet and RDT of ofloxacin in HCl buffer at pH 1.2 (n = 6) RESULTS AND DISCUSSION Evaluation of the RDTs Taste masked granules were prepared by granulation technique involving mixing of bitter drug and powdered Eudragit E-100 and then preparation of gel using 10% ethanol followed by extrusion and drying of the gel. This taste masking technique has advantage that it is

simple and does not employ harmful organic solvents as used in microencapsulation or some coating techniques. The minimum concentration among a range of dilutions of a substance at which the volunteer just starts feeling the bitter taste is known as ‘threshold concentration’. The threshold bitterness concentration of ofloxacin as determined by sensory evaluation using a panel of 20

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healthy human volunteers was found to be 100 µg/ml. Spectrophotometric method of analysis for taste of showed that 8.39±0.003 µg/ml of drug (approximately 1/12th the threshold bitterness concentration) was released from taste masked ofloxacin granules in phosphate buffer pH 6.8 after 30 s of contact time. Also, sensory evaluation for taste by the panel confirmed the tasteless characteristics of the granules as none of the subjects felt bitter taste even after keeping in mouth for 20-30 s. Hence, this technique of tastemasking using a pH-sensitive polymer was found to be effective in masking the bitter taste of the drug. The taste masked granules were compressed into rapidly disintegrating tablets. Different ratios of the super-disintegrant, SSG and the directly compressible binder, MCC were also investigated for the formulation of RDTs and the ratio that gave the minimum disintegrating time along with acceptable hardness was chosen for the formulation of the final batch of tablets. Ratios of SSG: MCC below ratio 1:1 gave very less disintegration times (less than 19 s) but the tablet hardness was very low. All the ratios above 1:3 (i.e., Formulations OF-5 to OF-9) gave high disintegration times and therefore, were rejected. Formulation OF-4 gave acceptable hardness as well as rapid disintegration. Therefore, it was selected for the preparation of final batch of RDTs (Table 1). Various physical parameters of this batch of tablets are summarized in Table 2. Also a linear correlation between the disintegration time and wetting time of the RDTs was observed suggesting that wetting is an important step for the disintegration process In phosphate buffer pH 6.8, a cumulative percent drug release of about 2.4% was obtained in 10 min from RDTs of taste-masked ofloxacin (Fig. 1). This amount is much less than the threshold bitterness concentration of ofloxacin (100µg/ml) suggesting that sufficient tastemasking has been achieved and the bitter taste of the drug will not be perceived while the tablet is in the mouth after oral intake. Further, by comparing the release profile of control and RDTs of taste-masked granules at pH 6.8, it was seen that about 99.7% of drug was released from the plain drug tablet in just 2 min while only about 1% was released from the RDTs of taste-masked granules (Fig. 1). Therefore, it can be implied that there exists a

marked difference in the dissolution profiles of pure drug and taste-masked tablets at pH 6.8. At pH 1.2 more than 98 % of the drug was released within 2 min from the RDTs of taste-masked ofloxacin (Fig. 2) indicating that the drug will be released in the acidic pH of stomach. The release profile of the drug from the RDTs of taste masked granules and the RDTs of plain drug was comparable and super imposable at pH 1.2, indicating that the drug will be released at approximately the same rate and to the same extent in the stomach, thus showing that taste-masking the drug with the pH-sensitive polymer does not affect the release of the drug in the stomach and its bioavailability. The hardness and friability of the RDTs (Table 2) were found to be within the limits specified (hardness=3-10 kg/cm and friability up to 2%) for fast disintegrating tablets. The wetting time was found to be 45±0.71 s and the water absorption ratio was 15.34 ± 0.84 (Table 2).The disintegration time of the RDTs in the external medium (58 s) and in the mouth of the volunteers (27 s) was found to be within the limits reported for fast disintegrating tablets (Table 2), thus showing that fast disintegration has been successfully achieved. The results for the taste evaluation of the RDTs by a panel of 20 healthy human volunteers are shown in Table 3. All the volunteers reported the taste-masked granules as tasteless on the perception scale and rated the formulated RDTs as good. This suggests that the bitter taste of ofloxacin in the granules continued to be masked on being compressed into tablets. Also all the volunteers experienced a good mouth feel of the ofloxacin RDTs with a minimum of grit indicating good palatability of the tablets. Evaluation of the Suspension Drug content of the formulated suspension was 98.10 ± 1.79% and the pH was 6.8. The suspension was found to be easily resuspendable and remained so for sufficient period to allow the pouring of a dose. The suspensions prepared using plain drug and the tastemasked granules were subjected to taste evaluation by the same panel of 20 selected volunteers. All 20 volunteers in the panel rated the formulated suspension of ofloxacin as good [0] on the perception scale of 0-5. This suggests that the bitter taste of ofloxacin granules

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excipients. 3rd ed., London: The Pharmaceutical continued to be masked on being suspended in an oral Press; 2000. p. 401-406. dosage form. 6. Ishikawa T, Watanabe Y, Utoguchi N. Matsumoto CONCLUSION M. Preparation and evaluation of tablets rapidly To conclude, efficient taste masking was achieved for disintegrating in saliva containing bitter-tastethe bitter drug ofloxacin using the technique of masked granules by the compression method. granulation. Patient compliant dosage forms i.e. oral Chem Pharm Bull. 1999;47(10):1451-1454. liquid suspension and rapidly disintegrating tablets that 7. Gergely G, Gergely T, Gergely I. Pharmaceutical had good taste were successfully formulated. These Preparation in the form of an effervescent and/or studies suggest that these patient friendly taste masked disintegrating tablet or an instant granule and dosage forms may be useful for children and elderly process of producing it. PCT Int Appl patients. WO9313760 July 22, 1993. ACKNOWLEDGEMENT 8. Bi W, Sunada H, Yonezawa Y, Danzo K, Otsuka Free gifts of Ofloxacin (Sai Bliss Drug A, Iida K. Preparation and evaluation of a Pharmaceuticals, Karnal, India), Eudragit E-100 compressed tablet rapidly disintegrating in the oral (Röhm Pharma, Germany), Microcrystalline cellulose cavity. Chem Pharm Bull. 1996;44(11):2121-2127. (Panacea Biotech, Lalru, India) and Sodium starch 9. Shirai Y, Yamamoto K, Kojima K, Fujioka H, glycolate (Kemwell Pvt. Ltd.) was gratefully Makita H, Nakamura Y. A novel fine granule acknowledged. system for masking bitter taste. Biol Pharm REFERENCES Bull.1993;16(6):172-177. Erratum in: Biol Pharm 1. Fu Y, Yang S, Jeong SH, Kimura S, Park K. Bull 1993 Jun;16(6):619. Orally fast disintegrating tablets: developments, 10. Sjöqvist R, Graffner C, Ekman I, Sinclair W, technologies, taste-masking and clinical studies. Woods JP. In vivo validation of the release rate Crit Rev Ther Drug Carrier Syst 2004;21(6): 433and palatability of remoxipride-modified release 476. suspension. Pharm Res. 1993;10(7):1020-1026. 2. Shishu, Bhatti A. Fast disintegrating tablets of 11. Bartoshuk LM. Bitter taste of saccharin related to diazepam. Indian Drugs 2006;43(8):643- 648. the genetic ability to taste the bitter substance 6-n3. Douroumis D. Practical approaches of taste propylthiouracil. Science 1979;205(4409):934masking technologies in oral solid forms. Expert 935. Opin Drug Deliv. 2007;4(4):417-426. 12. Chandibhamar V, Yadav MR, Murthy RS. Studies 4. Katsuragi Y, Sugiura Y, Lee C, Otsuji K, Kurihara on the development of taste-masked suspension of K. Selective inhibition of bitter taste of various chloroquine. Boll Chim Farm. 2004;143(10):377drugs by lipoprotein. Pharm Res. 1995;12(5):658382. 662. 5. Chang RK, Shukla AJ. Polymethacrylates. In: A. H. Kibbe editor. Handbook of pharmaceutical ***********

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Stress Stability Studies and the Estimation of Lamotrigine in Pharmaceutical Formulation by Validated RP- HPLC Method Prabhat K. Shrivastava and Sushant K. Shrivastavai Department of Pharmaceutics, Institute of Technology, Banaras Hindu University, Varanasi (U.P.) -221 005. 1 For Correspondence: [email protected]

ABSTRACT The aim of the present work to develop validated RP-HPLC method which determines stress stability and concentration of lamotrigine in pharmaceutical preparation as per ICH guidelines. Separation was performed using Cecil liquid chromatography CE-4100-dual piston pump equipped with 20 µl loop and variable W/L UV/Vis detector (λ= 306 nm) and phenomenex, (250 X 4.6 mm) Luna 5µ C 18 (2) 100 A Column. Mobile phase consisted of acetonitrile: methanol: 10mM potassium dihydrogen phosphate buffer (pH -7), (20:20:60 v/v) was used. All the system suitability parameter was found within the range. The method was extensively validated for specificity, linearity, accuracy, precision, recovery, limit of quantitation and detection. The lamotrigine was found to be highly labile to alkaline hydrolysis compared to acid. Lamotrigine degradation was associated with rise in a major degradation product at retention time of 5.4 + 0.05 min. The percent assay of drug was found to be in the range 99.25 –102.75 in pharmaceutical dosage form. Key-words- Reverse Phase High Performance Liquid Chromatography (RP-HPLC); ICH Guidelines; Lamotrigine; Validation; Stress Stability. INTRODUCTION Lamotrigine, 6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5diamine is an antiepileptic drug of the phenyltriazine class, is chemically unrelated to existing antiepileptic drugs.1 Lamotrigine is thought to exert its anticonvulsant effect by stabilizing presynaptic neuronal membranes.2 A literature survey revealed some high-performance liquid chromatographic (HPLC) methods reported for lamotrigine determination from human biological fluids and formulation.3-9 However no HPLC method was found for lamotrigine determination in bulk drug and formulations as a stability indicating assay method. Forced degradation study is performed to check the shelf life of formulated product as well as bulk drug when exposed to different environmental condition such as temperature, humidity, light, etc. according to International Conference on Harmonization (ICH)

guidelines.10-11 To study all types of stability parameters a suitable method of analysis which is stability indicatory and provides an idea on how drug substance, or product degrades, degenerates and behaves under changing conditions is very essential. 1214 This paper describes the development and validation of a stability-indicating method for the assay of lamotrigine in bulk drug as well as tablet. MATERIALS AND METHODS Instrumentation tools: Quantitative HPLC analysis was performed on a highperformance liquid chromatography which consisted of a Cecil- CE-4100-Adept series-Dual piston pump with Cecil-CE-4201- Variable W/L UV/Vis Detector, and manual sampler of microliter syringes (Hamilton 702 NR) –25 µl. The HPLC system was equipped with data acquisition and processing software Cecil-CE-4900power stream chromatography system manager.

Indian Journal of Pharmaceutical Education & Research Received on 20/3/2008; Modified on 3/12/2008 Accepted on 11/2/2009 © APTI All rights reserved

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Materials: Lamotrigine reference standard was obtained as gift sample from Lupin Research Park, Pune. Acetonitrile and methanol (HPLC grade) were obtained from Merck. Potassium dihydrogen orthophosphate, triethylamine, sodium hydroxide, hydrochloric acid and hydrogen peroxide were purchased from Qualigen India Ltd. Water of HPLC grade was obtained by glass distillation of normal distilled water and passed through a 0.22-mm membrane filter. Local commercial tablet formulation (Lamepil – 50, Innova, IPCA laboratories Ltd., Mumbai, India) was used for analysis. Chromatographic conditions: HPLC analytical measurement and separation were performed using phenomenex, (250 x4.6 mm) Luna 5µ C-18 (2) 100 A stainless steel column. The mobile phase consisted of acetonitrile: methanol: 10mM potassium dihydrogen phosphate buffer pH -7 (20:20:60, v/v). The pH -7 of buffer was adjusted with triethylamine. The mobile phase was filtered through a nylon membrane (0.45 µm) and sonicated for 10 min. The chromatography was performed at room temperature using flow rate of 1.5 ml/min. The run time was set to 10 min. Eluents were monitored on UV/Vis detector at a wavelength of 306 nm. The volume of each injection was 20 µl. Preparation of standard solution: Accurately 20 mg of Lamotrigine was weighed and transferred to 100 ml volumetric flask, dissolved into 5 ml of methanol and volume was made up with 0.1 N HCl (200 µg /ml). 10 ml of stock solution was diluted to 100 ml with 0.1 N HCl to get working standard (20 µg/ml). Using working aliquots of standard, solution of 2, 4, 6, 8, 10 and 12 µg/ml were prepared and subjected for analysis. Peak area under curve were observed and plotted against respective concentration and linearity was observed in the range of 2-12 µg/ml. Preparation of solutions for assay validation: System suitability tests were carried out on freshly prepared standard solution in replicates and calculated from standard chromatogram. System precision was evaluated by performing five replicates injections of lamotrigine standard solution. Linearity response for lamotrigine was checked by preparing standard solutions at six different concentration levels ranging from 2-12 µg/ml. The

response ratio (response factor) was determined by dividing the peak area with the respective concentration. The intra- day precision of method was determined with six replicates, of three different concentrations (4, 8, and 12 µg/ml). These studies were also repeated on different days to determine inter-day precision. To determine accuracy, recovery studies were performed with six replicates in the concentration range 4-8 µg/ml with the previously analyzed sample of concentration 4 µg/ml. The limit of quantitation and limit of detection was determined by decreasing the concentration of drug and observed by visual method. 15 Stress studies: Forced degradation studies were performed to provide an indication of the stability-indicating properties and specificity of the method. A degradation sample was prepared by taking 20 mg lamotrigine in three different 100 ml volumetric flask dissolved in 5 ml methanol by shaking and sonicating the flask. In two flasks, volume was adjusted to 100 ml with 1N HCl for acidic degradation and 1N NaOH for basic degradation respectively. Two flasks were refluxed for about six hours. To the third flask, lamotrigine standard, previously stored in oven for 3 day at 1250 C was taken and volume was adjusted to 100 ml with diluent for thermal degradation. The samples were withdrawn at different time interval, allowed to cool to room temperature and treated as follows. The different degradation samples were neutralized (pH 7), acid degradation sample with dilute sodium hydroxide and base degradation sample with hydrochloric acid. The thermal degradation sample was used as such. Samples were further diluted with 0.1N HCl to final concentration in the calibration range. The solutions were filtered through 0.45µm syringe filter during injection and were analyzed for degradation pattern in aforementioned mobile phase (Fig. 1, 2 and 3). The peak area of lamotrigine was plotted on standard calibration curve. Data were acquired, stored, and analyzed with the software ‘‘Cecil-CE-4900-Power stream chromatography system manager”. Preparation of sample solution: Twenty tablets (Lamepil –50, dose- 50 mg) were weighed. The average weight was determined and

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ground to a fine powder. Tablet powder equivalent to 20 mg of lamotrigine were taken and dissolved in 5 ml of methanol and the volume then made up to 100 ml with 0.1N HCl. The resulted solution was sonicated for 10 minute and then filtered through Whatman filter paper No 42. RESULTS AND DISCUSSION A suitability test was applied to representative chromatograms to check various parameters such as 10% Plate Efficiency, 50% Plate Efficiency, peak asymmetry and RSD of peak area. The results obtained were within acceptable limits (Table 1). Thus, the system meets suitability criteria. The six solutions containing lamotrigine at concentrations ranging from 2 to 12 µg/ml were analyzed and found to be linear. The linearity, regression equation, Y= 20.11786 (±0.15) X – 0.0351 (±0.01) and correlation coefficient r2 = 0.9998 was found in the linearity range of 2-12 µg/ml. The linearity response ratio for all the concentaration was found to be equal. The data obtained from precision experiments are given in Table 2 for intra-and inter-day precision studies. The %RSD values for intra-day and inter-day study were < 2.0%, confirming that the method was sufficiently precise. Percentage recovery was calculated from differences between the peak areas obtained for

No. 1. 2. 3. 4.

fortified and unfortified solutions for the accuracy of method. As shown from the data in Table 3, the recovery was found to be in the range of 99 to 101 %. The limit of detection and quantization were found to be 100ng and 200ng respectively. Selectivity of the developed method was assessed by stress studies involving acid, base and thermal revealed that the drug was found to be highly labile to alkaline hydrolysis and degraded to maximum extent of 98.2 %. The reaction in 1M NaOH at 80°C was so fast; the degradation of drug was started after 30 min. Drug degradation was associated with rise in a major degradation product at RT 5.4 + 0.05 accompanied with a drastic reduction in the number of theoretical plates and increased tailing. Complete degradation of the drug was observed in 5 h. The rate of hydrolysis in acid (2M HCl) was very slow as compared to that of alkali. No major degradation product was observed after exposure of drug substance in 100°C in oven for three days but amount of drug decrease very slightly (Table 4). Except alkaline conditions, the drug content was found in the range 95–105% for all stress conditions indicating the stability and specificity of the analytical method. The percentage of lamotrigine in tablet dosage form was found to be 99.25- 102.75 % with RSD less then 2.

Table No. 1 System Suitability Parameters Parameter Value Acceptance Limits 10% Plate Efficiency >6500 More than 6000 50% Plate Efficiency >9500 More than 9000 Peak asymmetry 0.74 Less than 2 RSD of peak area 0.205 Less than 2%

Table No. 2 Precision data evaluated through intra-day and inter-day studies for lamotrigine Inter-day measured Actual concentration (µg/ml) Intra-day measured Concentration* Concentration* (µg/ml)±S.D.; (µg/ml)±S.D.; R.S.D.% R.S.D.% 3.92±0.042; 1.209 4.008±0.057; 1.333 4 7.93±0.053; 0.825 7.99±0.091; 1.108 8 11.95±0.081; 0.650 12.012±0.065; 0.650 12 * Mean of six replicates

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Table No. 3 Recovery studies data for lamotrigine Actual concentration (µg/ml) Calculated concentration (µg/ml)±S.D.; R.S.D.% 3.96±0.046; 1.411 4 6 6.08±0.038; 0.957 8 7.89±0.072; 0.777

*Recovery (%)

99.0 101.0 99.50

* Mean of six replicates Table No. 4 Results of lamotrigine exposed to different degradative pathways Conditions Time Recovery± S.E (%) RT of Degradation Product (min) 1N HCl, reflux 1N NaOH, reflux Thermal (100° C)

5 hr 5 hr 3 days

96.5± 4.0 1.80± 0.8 98.46± 2.5

7.4, 9.4 5.4, 6.39 ---

1

29

Signal [mV]

14 2

-0 -15 -30 00:00

02:00

04:00

06:00

08:00

10:00

Time[mm:ss]

Figure 1 HPLC Chromatogram of Lamotrigine and degradation product after 90 minute degradation in 1N NaOH.

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20 2

Signal [mV]

8

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-4 -16 -28 00:00

02:00

04:00

06:00

08:00

10:00

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Figure 2: HPLC Chromatogram of Lamotrigine and degradation product after 150 minute degradation in 1N NaOH.

22

Signal [mV]

12

2

1

2

-9

-1 9 0 0 :0 0

0 2 :0 0

0 4 :0 0 0 6 :0 0 T im e [m m :s s ]

0 8 :0 0

1 0 :0 0

Figure 3 HPLC Chromatogram of Lamotrigine and degradation product after 240 minute degradation in 1N NaOH. CONCLUSION HPLC method was developed for quantitative determination of lamotrigine in bulk, and tablet dosage form. The method can be used for the routine analysis of lamotrigine in bulk as well as tablet dosage form. The study shows that lamotrigine is a labile molecule in alkali. It is stable to acidic hydrolysis and thermal condition. A stability-indicating method was developed, which separates all the degradation products formed under variety of conditions. The method proved to be simple, accurate, precise, specific and selective. Hence

it is recommended for analysis of the drug and degradation products in stability samples. ACKNOWLEDGEMENTS The authors wish to thanks, Lupin Research Park, Pune for supplying lamotrigine as a gift sample for research work. The financial assistance received from UGC in the form of fellowship is gratefully acknowledged by the author. REFERENCES 1. http://en.wikipedia.org/wiki/Lamotrigine. 2. http://us.gsk.com/products/assets/us_lamictal.pdf

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3.

4.

5.

6.

7.

8.

Emami J, Ghassami N, Ahmadi F. Development and Validation of a New HPLC Method for Determination of Lamotrigine and Related Compounds in Tablet Formulations. J Pharm Biomed Anal 2006; 40 (4): 999-1005. Papadoyannis IN, Zotou AC, Samanidou VF. Solid-phase Extraction study and RP-HPLC analysis of Lamotrigine in Human Biological Fluids and in Antiepileptic Tablet Formulations. J Liq Chromatogr 1995; 18(13): 2593-2609. Ching-Ling C, Chen-His C, Oliver Yoa-Pu H. Determination of Lamotrigine in Small Volumes of Plasma by High-Performance Liquid Chromatography. J Liq Chromatogr B 2005; 817 (2): 199-206. Angelis-Stoforidis P, Morgan DJ, O'Brien TJ, Vajda FJ. Determination of Lamotrigine in Human Plasma by High-Performance Liquid Chromatography. J Chromatogr B Biomed Sci Appl 1999; 727(1-2): 113-8. Yamashita S, Furuno K, Kawasaki H, Gomita Y, Yoshinaga H, Yamatogi Y, Ohtahara S. Simple and rapid analysis of Lamotrigine, A Novel Antiepileptic, In Human Serum by HighPerformance Liquid Chromatography using a Solid-phase Extraction Technique. J Chromatogr B Biomed Sci Appl 1995; 670(2): 354-7. Croci D, Salmaggi A, De Grazia U, Bernardi G. New High-Performance Liquid Chromatographic

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12.

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14.

15.

Method for Plasma/Serum analysis of Lamotrigine. Ther Drug Monit 2001; 23(6): 665-8. Hart AP, Mazarr-Proo S, Blackwell W, Dasgupta A. A rapid cost-effective High-Performance Liquid Chromatographic (HPLC) Assay of Serum Lamotrigine after Liquid-Liquid Extraction and using HPLC conditions routinely used for analysis of Barbiturates. Ther Drug Monit 1997; 19(4): 431435. ICH, Q1A Stability Testing of New Drug Substances and Products, International Conference on Harmonization, Geneva, October 1993. Rawlins EA. Bentley’s Textbook of Pharmaceuticals. 8th Edition, London: Published By Baillier Tindall; 2002, 140. Acharya MM. Pharmaceuticals- Stability Testing and Studies – An Over View. The Eastern Pharmacist 1999; 31-36. Alton’s ME, Pugh J. Kinetics and Product Stability, The Science of Dosage Form Design. London: Churchill Livingstone; 2002, 109. Cartensen, J.T. Modus Operandi for Stability Programme, Drug Stability and Practices, 2nd Edition, Vol-II, Newyork: Marcel Dekker; 1995, 487. ICH, Q2BValidation of Analytical Procedure: Methodology, International Conference on Harmonization, Geneva, March 1996.

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Effect of an Antacid on the Oral Pharmacokinetics of Rosiglitazone in Healthy Human Volunteers Suresh Kumar J.N.1,2, Prameela D.2 and Mullangi R.*3 1

Center for Biotechnology, JNTU, Kukatpally, Hyderabad, 500072 India Deccan School of Pharmacy, Kanchanbagh, Hyderabad, 500 058 India *3Jubilant Biosys, Yeshwanthpur, Bangalore, 560 022 India *Corresponding author: [email protected]

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Abstract The aim of this study was to study the effect of an antacid on the pharmacokinetics of RGZ in healthy human volunteers using a randomized, placebo controlled, 2-way crossover. Each subject received orally either 8 mg of RGZ with a placebo or co-administration with 400 mg of an antacid. Plasma concentrations of RGZ were estimated using a validated LC-MS/MS method. There was no statistically significant difference observed in the pharmacokinetic parameters viz., AUC(0-t), AUC(0-∞), Cmax, Tmax, Kel and t½ of RGZ following co-administration with an antacid. Key words: Rosiglitazone; Antacid; Drug-drug interaction; Humans; Oral pharmacokinetics INTRODUCTION Rosiglitazone (RGZ, Avandia®), chemically (±)-5-[[4[2-(methyl-2-(pyridinylamino) ethoxy]phenyl] methyl] methyl] -2,4-thiazolidinedione is a potent blood glucose lowering agent that reduces insulin resistance in patients with type 2 diabetes1,2. RGZ represents a novel class of molecules known as thiazolidinediones. Thiazolidinediones are active oral hypoglycemic agents by their strong binding to peroxisome proliferator activated receptor gamma (PPARγ)3-6. The agonistic activity at the PPARγ receptor leads to glucose lowering property exhibited by this class of molecules. Being an important member of the novel class of molecules, RGZ has been extensively researched over the last decade. The pharmacokinetic disposition including metabolic profiling has been completely delineated in both animals and human subjects7,8. In humans, [14C] RGZ has revealed a complete absorption of the orally administered drug with absolute bioavailability almost reaching 100%. While urine represented a major route for elimination of the radioactive dose (about 65% of the administered dose), the fecal excretion represented almost 22% of the administered radioactive dose8. Indian Journal of Pharmaceutical Education & Research Received on 20/9/2008; Modified on 14/11/2008 Accepted on 16/1/2009 © APTI All rights reserved

The major Phase I metabolites identified included the N-desmethylrosiglitazone and hydroxy-rosiglitazone, which further undergo extensive conjugation8. Diabetic therapy involves long medication of drugs. During therapy people often take antacids for getting relief from acidity in GI tract. Literature review suggested that antacids are known to cause drug-drug interactions with drugs belong to different therapeutic categories 9-13. In order to evaluate the possible drugdrug interaction between RGZ and an antacid the present study was undertaken to evaluate the effect of an antacid on the pharmacokinetics of the RGZ in healthy human volunteers. MATERIALS Rosiglitazone maleate, 4 mg tablets (Windia, Glaxo Simth Kline, Mumbai, India) and Gelusil MPS tablets (Pfizer Limited, Mumbai, India) were purchased from local pharmacy. Gelusil MPS Tablet which comprises - activated dimethicone: 50 mg, magnesium hydroxide: 250 mg, dried aluminum hydroxide gel 250 mg and magnesium aluminum silicate hydrate: 50 mg. Imepramine with a purity of >99% was obtained from Mars Therapeutics, Hyderabad, India. HPLC grade acetonitrile and methanol were procured from Ranbaxy Fine Chemicals Ltd., New Delhi, India. Dimethylsulphoxide (DMSO) was purchased from

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Sigma-Aldrich, Milwaukee, WI, USA. AR grade formic acid and potassium dihydrogen ortho phosphate were purchased from Qualigens, Mumbai, India. All aqueous solutions including the buffer for the HPLC mobile phase was prepared with Milli Q (Millipore, USA) grade water. Control K2.EDTA human plasma of healthy volunteers was obtained from Cauvery Diagnostics and Blood Bank, Secunderabad, India. METHODS Study design The study was conducted with a double-blind, randomized and 2-way crossover design. The treatments administered were 8 mg RGZ (2 x 4 mg tablets) with placebo and 8 mg RGZ (2 x 4 mg tablets) with Gelusil MPS tablet. Subjects Sixteen healthy male volunteers aged 22-27 years; weighing 54-61 kg and height of 155.8-160.2 cm participated in the study. All the subjects underwent physical examinations and laboratory tests on the blood and urine. All subjects were non-smokers, nonalcoholic, had taken no medication for at least 14 days prior to the study and were admitted to the study after signing the informed consent. This study was approved by institutional ethics committee. Protocol The subjects were randomly divided into two groups and the study was conducted with a double blind 2-way crossover design with a wash-out period of 10 days. Following overnight fasting (~12 h), volunteers received either of the treatments mentioned above. Volunteers were not permitted to take food or drink post drug administration. Blood samples (~3 mL) were drawn at pre-dose and 0.5, 1.0, 1.5, 2, 4, 8, 10 and 24 h postdosing. Following blood collection at each time point, plasma was harvested by centrifuging the blood using Biofuge (Hereaus, Germany) at 12,800 rpm for 5 min and stored at -80±10 °C until analysis. Plasma sample analysis Plasma samples were analyzed for RGZ using a validated LC-MS/MS. Briefly, to an aliquot of 200 µL plasma sample 30 µL of internal standard solution (imepramine, 500 ng/mL) was added, vortexed for 30 sec and extracted with 500 µL of acetonitrile by vortex mixing for 5 min on Vibramax 100 (Heidolph, Germany). The contents were centrifuged at 6000 rpm for 5 min using Biofuge (Heraeus instruments,

Germany) at 10±1°C. Around 200 µL clear supernatant was transferred into sample vial and 10 µL was injected onto the analytical column for analysis. Resolution of analyte and internal standard was achieved on an Amazon ODS-3 C18 (50 x 4.6, 5 µm) maintained at 40°C using acetonitrile:0.2% formic acid buffer (75:25, v/v) at a flow rate of 0.5 mL/min. Tandem mass spectrometry (MS/MS) was performed with a triple quadrupole MDS Sciex (Foster City, CA, USA) API-2000 mass spectrometer equipped with a Turboionspray (ESI) source and the drug was monitored and quantified on analyst 1.4.1 software. Detection of the ions was performed in the multiple reaction monitoring (MRM) mode, monitoring the transition of the m/z 358.0 precursor ion to the m/z 135.3 product ion for rosiglitazone and m/z 281.1 precursor ion to the m/z 86.2 product ion for internal standard. Quadrupoles Q1 and Q3 were set on unit resolution. This method has a reproducible linearity over a range of 1-500 ng/mL. Pharmacokinetic data analysis Plasma concentration-time data of rosiglitazone was analyzed by non-compartmental method using WinNonlin Version 5.1 (Pharsight Corporation, Mountain View, CA) to derive various pharmacokinetic parameters viz., AUC(0-t), AUC(0-∞), Cmax, Tmax, Kel and t½. Statistical analysis The pharmacokinetic parameters obtained from the two treatments were compared using Student’s paired t-test. A value of p