J Pharm Pharmaceut Sci (www.cspscanada.org) 8(3):430-466, 2005

Canadian Society for Pharmaceutical Sciences

Proceedings of the 8th Annual Symposium

May 30-June 2, 2005

Toronto, Ontario, Canada

Invited Speakers

SESSION 1: Plenary.

Accelerating Drug Discovery & Development: Opportunities & Pitfalls.

Elizabeth B. Vadas, InSciTech Inc., Dorval, Quebec, Canada

The pharmaceutical industry is facing an unprecedented number of simultaneous challenges today. These include weak pipelines, cultural differences and redundancies following mergers, increased regulatory pressures, drug safety issues, potential price controls and limited access to market due to formulary decisions. In an increasingly competitive environment, the industry must become not only more efficient but also more effective both in the areas of discovery and development. Business models, which were successful in the past, may need to be re-evaluated and changed to meet the demands of the current regulatory and business environment. This presentation will focus on the opportunities and pitfalls inherent in new scientific approaches, new technologies and business models. Avoiding the pitfalls and exploiting the opportunities can set the stage for more effective drug development and a streamlined regulatory process resulting in speed to market while keeping patient safety in focus.

Molecular Pharmaceutics in Drug Discovery and Development: A New Era.

Gordon L. Amidon, College of Pharmacy, The University of Michigan, Ann Arbor, Michigan, USA.

During the past ten years, the Pharmaceutical and particularly the Biopharmaceutical Sciences have undergone a molecular revolution. From the solid-state properties of drugs to polymer and nano-systems to biopharmaceutics, the pharmaceutical sciences have progressed from a more less empirical science to molecular design and analysis. The Biopharmaceutical sciences in particular have progressed from empirical fitting of drug absorption profiles based on plasma levels, to mechanistic physiological models for absorption processes, to molecular membrane transporters and drug targeting. With the sequencing of the human genome and the availability of sequence analysis methodologies, expression arrays and proteomics, the role of gene expression in the complex processes of drug absorption, systemic availability, metabolism, elimination, and distribution i.e. ADME as well as drug targeting, is being unraveled at a molecular level. The advances in expression and proteomic technologies in particular allows for the determination of the expression of various transporters and enzymes in cells and tissues and is being used as basis for the development of tissue and cell specific targeting strategies. This modern molecular approach will be illustrated with recent results from our laboratory focused on the identification of transport and cellular activating enzyme for the prodrug valacyclovir. We have previously shown that valacyclovir is transported into epithelial cells via a peptide transporter. Recently we have identified and determined the crystal structure of a new esterase enzyme, Valacyclovirase, an á/b fold serine protease enzyme, as the activating enzyme for two important antiviral prodrugs valacyclovir and valganciclovir. This presentation will provide an overview of the evolution of biopharmaceutics and recent results from our laboratory focused on the transport and activation of nucleoside prodrugs.

Crystal Engineering in Pharmaceutical Sciences: Opportunities for Materials Design by Co-Crystals.

Örn Almarsson, Pharmaceutical Chemistry, TransForm Pharmaceuticals, Inc.

The search for and evaluation of crystal forms for pharmaceutical products is gaining visibility, both within pharmaceutical science and with the public. A number of public discussions have taken place recently about crystal form patents and their role in product life-cycle management and industry competitiveness. The prominent issue of the Ritonavir (Norvir®) polymorph appearance in 1998, after the product was on the market for two years, highlighted the challenges of understanding polymorphism in pharmaceutical systems. New scientific insights, including crystal form prediction efforts, and the advent of novel high-throughput technology developments are fuelling the discussion about best practices in discovery and analysis of crystal forms of pharmaceutical compounds. The lecture will broadly survey the field of crystal engineering, relevance to pharmaceutical science and the emergence of pharmaceutical co-crystals.

SESSION 2: Structure-based Drug Design and Discovery

Impact of structural information in the discovery of specific inhibitors of the Human Papillomavirus E1-E2 protein-protein interaction.

Nathalie Goudreau, Boehringer Ingelheim (Canada) Ltd. Research & Development, Laval, Québec. Canada.

Papillomaviruses are a family of small ds-DNA viruses that induce benign and malignant hyperproliferative lesions of the differentiating epithelium. The HPV genome encodes eight well-characterized proteins, only two of which, the E1 and E2 proteins, are required for replication. Viral DNA replication is initiated by the cooperative binding of E1 and E2 to the HPV origin. Assembly of this E1-E2-origin complex is dependent on the binding of E2 to the origin as well as on a critical protein-protein interaction between E1 and E2. Screening of our corporate compound collection allowed the identification of a series of inhibitors that was capable of antagonizing the interaction between the E1 and E2 proteins. These inhibitors featured an indandione spiro-fused onto a substituted tetrahydrofuran ring. Extensive SAR studies allowed to improve the potency by more than 1000-fold, leading to the first series of low nanomolar inhibitors of the HPV11 E1-E2 protein-protein interaction. Using a combined approach of NMR and computational chemistry, we were also able to determine the absolute stereochemistry of the active species originating from the initial racemic lead. Isothermal titration calorimetry and fluorescence studies showed that these inhibitors act by binding to the N-terminal transactivation domain (TAD) of E2. In addition, X-ray co-crystal data on the E2-TAD complexed with an indandione inhibitor allowed the identification of an appealing binding pocket. These findings were subsequently used to establish a ligand displacement assay, in order to discover novel inhibitors of the E1-E2 interaction that specifically targeted the E2-TAD. A uHTS campaign using this specific displacement assay led to the identification of a new “drug-like” series of inhibitors. The potency of these inhibitors was further improved through subsequent SAR efforts. Finally, NMR and computational chemistry were used to generate a complex model for this novel series of HPV E1-E2 interaction inhibitors.

SMART Drug Design: Novel Small-Molecule: Inhibitors of Oncogenic Protein Kinases.

Tomi K. Sawyer, ARIAD Pharmaceuticals, Cambridge, Massachusetts, USA.

Emerging strategies in cancer therapy are exploiting knowledge of signal transduction mechanisms and functional relationships to cancer cell proliferation, survival, angiogenesis, invasion, and metastasis. Oncogenic protein kinases have become the most dramatic focus of drug discovery strategies to advance novel small-molecule inhibitors. Examples include Bcr-Abl, CDK family, EGFR family, Kit, MAPK family, PDGFR family, Raf, Src family, and VEGFR family. Such oncogenic protein kinases correlate to different signal transduction pathways, and in several instances, there exists a possible advantage for multiple protein kinase inhibition by drug design efforts using both structure- and screening-guided lead compound generation and optimization. Both classic and newly emerging templates have provided a plethora of chemically diverse small-molecule inhibitors. Noteworthy has been drug discovery efforts focused on Src, the first oncogenic protein kinase identified nearly twenty-five years ago, and a promising therapeutic target for cancer metastasis, hematological malignancies and bone diseases. The development of highly potent and uniquely selective Src kinase inhibitors using SMART drug design technology will be described.

Docking to Model Binding Sites.

Brian Shoichet, University of California San Francisco, USA.

Molecular docking is widely used to screen large compound collections for novel lead molecules that complement a receptor of known structure. Docking energy functions are approximate and many degrees of freedom are under-sampled. To understand where algorithms can be improved, we have turned to model systems where predictions can be tested in detail. These are simplified, small buried cavities where the interactions are dominated by one particular energy term. Thus, the L99A cavity in T4 lysozyme is dominated by non-polar complementarity, the L99A/M102Q cavity has a single hydrogen bond acceptor, and the W191G cavity in cytochrome C peroxidase is dominated by a single ionic interaction. Predicted ligands are being tested for binding, geometry, and protein motion using x-ray crystallography. We are using a cycle of theory development and experimental testing in these systems, where mis-predicted ligands and geometries are as informative as correct predictions.

Fragment Based Drug Discovery Using Rational Design.

Harren Jhoti, Founder & Chief Scientific Officer, Astex Technology, Cambridge, United Kingdom

Fragment-based discovery has recently emerged as a new approach for the generation of novel therapeutic agents. The use of high throughput X-ray crystallography, as well as NMR, in fragment-based discovery approaches will be exemplified. Methodology that utilizes high throughput X-ray crystallography and NMR to screen fragment libraries will be described. In particular, libraries of ‘molecular fragments’ have been generated and screened using these approaches, resulting in novel chemical leads being identified for several therapeutic targets. This approach for lead generation has distinct advantages over conventional bioassay-based screening in that very low-affinity fragments with novel structures can be identified. These “hits” can then be rapidly optimized for potency and DMPK properties using iterative cycles of medicinal chemistry and structure-based drug design. The development of novel lead compounds using this approach will be described for targets such as the cyclin-dependant kinases, key proteins involved in cancer.

SESSION 3: Process Development.

Single-Pot Manufacturing Process Using Microwave-Vacuum Drying.

Tarun Makadia, GlaxoSmithKline Canada, Pharmaceutical Development, Mississauga, Ontario, Canada.

Single-pot processors may be used for mixing, granulating and drying pharmaceutical granulations in a single vessel. Single-pot processing technology for pharmaceutical industry is slowly gaining popularity since mid-1980s and now a number of marketed products are manufactured using this technology. This presentation is designed to provide overview of single-pot technology and GlaxoSmithKline Canada’s experience with single-pot manufacturing process technology being used at the Canadian manufacturing facility to manufacture a product for world market. The presentation will include process parameters, such as microwave power, vacuum level, intermittent mixing during drying, and their impact on the drying process.

Emerging Technologies: SCF & Aseptic Isolator Technology in the Production of Microparticulate Suspensions for Parenteral Delivery.

Michael Vachon, Process Development, SkyePharma Inc., Ile des Soeurs, Verdun, Québec, Canada

Parenteral drug product development has historically been typified by the “white-room” manufacture of an autoclave-sterilized, aqueous drug solution. With the advent of new drug therapies, many of these compounds are characterized as having poor solubility or insolubility in water as well as inherent thermal sensitivity, thereby presenting a problem in formulating drugs using traditional approaches. Water-insolubility problems can delay or completely block the development of many new drugs, or prevent the much-needed reformulation of certain currently marketed drugs. In today’s drug development climate, the need for rapid generation of proof-of-concept parenteral formulations for early stage clinical evaluation is critical to project-viability decision making. Exploiting the small-scale preparation capabilities of Insoluble Drug Delivery™ technology permits a wide variety of poorly water-soluble drugs to be developed as micron or sub-micron sized particulate aqueous suspensions for parenteral administration. This is achieved for instance by coupling super critical fluids (SCF) drug particle generation and the aseptic manufacture of product formulations using barrier isolation technology. IDD™ formulations display a capacity for high drug loading, narrow particle size distribution, and almost spherical particle morphology through the use of surface-stabilizing, bio-compatible phospholipids. This approach can lead to innovative therapeutic applications with low volume doses and permits delivery by a number of routes of parenteral administration including intravenous, intramuscular, subcutaneous, and intradermal. This presentation discusses the particle microstructure model of IDD™ drug suspensions, the contained aseptic manufacturing processes leading to rapid generation of in-vivo discovery batches, and regulatory items related to IDD™-SCF products produced in this manner.

Application of Advanced Measurement Techniques To the Fluid Bed Drying of Pharmaceutical Granule.

Todd Pugsley, Chemical Engineering, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.

The batch-wise drying of pharmaceutical granule in a fluidized bed is an important step in the production of certain solid-dosage pharmaceuticals. The most common method for control of this process is to monitor for changes in outlet air or product temperature. A limiting value for either one of these quantities is used to indicate the endpoint of the drying process. Temperature monitoring gives no information about the quality of the fluidization inside the vessel. Control of the fluidized state as drying proceeds is accomplished manually by an operator who observes the behaviour of the bed through a sight-glass. While this method results in an acceptable product quality for the synthetic drugs currently manufactured, the production of high potency or peptide-based drug products may be more sensitive to operation in a non-optimal fluidized state. Our group has been carrying out research for the past several years with the objectives of: (i) improving the fundamental understanding of batch fluidized bed dryers containing pharmaceutical granule; (ii) developing an on-line monitoring and control tool for maintaining the dryer in an optimal fluidized state. With respect to the first objective, we have applied tomographic imaging techniques to better understand the influence of such process variables as particle size distribution, bed loading, and gas velocity on the fluidized bed hydrodynamics. The focus of the second objective has been on the use of a high-frequency pressure transducer to monitor pressure fluctuations inside the bed as drying proceeds. The advanced statistical test that we have applied in the analysis of the pressure fluctuations is able to provide an early warning of a non-optimal fluidized state that would allow an operator or an automatic control system to take action. The current paper will first describe the research infrastructure in my laboratory and pilot plant areas that supports the research projects described above. Much of this infrastructure is unique in Canada. Some of the key results from the research will be presented and the contribution of the research to ongoing and future PAT initiatives within the pharmaceutical industry will be discussed. The paper will close with a discussion of possible future research directions in my laboratory.

Application of Lean Sigma Principles to Increasing the Yield of a Liquid Suspension Product.

Rodney Whale, GlaxoSmithKline, Mississauga, Ontario, Canada.

GSK introduced the Lean Sigma program to improve efficiencies of manufacturing processes that result in improved supply chain performance. On of the key components of Lean Sigma is to start with collecting baseline data on the current process: this was done on a liquid suspension product in 2000. Several areas for improvement were identified: documentation, packaging, testing and yield improvements. The yield improvement project is described, utilizing Lean Sigma principles to support process modifications that resulted in significant financial and efficiency benefits.

SESSION 4: The Discovery-Development Interface.

The Importance Of Materials Sciences In Early Development.

Sophie-Dorothée Clas, Pharmaceutical Research &Development, Merck Frosst Canada & Co., Kirkland, Quebec, Canada.

The appearance of new physical forms of the drug substance with significantly different bioavailability and stability can have a serious effect on the selection of the form for safety studies and formulation development. Different forms of the drug substance can result in different exposures of the dosage form. Changes such as conversion to the amorphous form, interconversion of polymorphs, conversion to hydrates, and salt dissociation, etc. can not only affect the chemical stability of the compound, among other properties, but may also result in very different bioavailabilities. The goal of any dosage form (whether for early safety studies or the final product) is to ensure that there is reproducible, safe exposure of the active pharmaceutical ingredient for clinical efficacy. Using examples, this presentation will seek to show the impact that crystal form changes of the drug substance can have on drug development, selection of the optimum form for development, stability and/or bioavailability of the product.

Integrating the Results of Physiochemical Characterization into Preclinical Development: Biopharmaceutics Models.

Alice Loper, Pharmaceutical Technologies and Clinical Packaging, Adolor Corporation, Exton, Pennsylvania, USA

Preclinical models integrate the results of materials science and in vitro biological system investigations into mechanistic studies of oral absorption and disposition. Equilibrium solubility measurements in carefully selected solvent systems suggest the design of preclinical studies to evaluate potential food effects. Data from preclinical models can justify early clinical intervention with lipophilic formulations, self-emulsifying or micellar drug delivery systems or particle size reduction. Studies of lipophilicity and partition coefficient can be used as a justification for exploration of hepatoportal vein and intestinal lymphatic absorption in conscious rodent models. The results provide a rationale for attempting to influence rate or extent of absorption with formulation. The relevance of the in vitro pH-solubility profile must be evaluated in complex physiological systems. The tendency of the unionized compound either to precipitate at some point in the gastrointestinal (g.i.) tract or to be solubilized by endogenous mixed micelles of bile salts and lipids will determine future development approaches. Solid-state properties may produce only transient differences in rate and extent of dissolution prior to in vitro equilibration, but can profoundly affect drug availability at the site of absorption in the g.i. tract. Selection of appropriate physical forms for development, as well as prospects for modified or controlled release formulations, can be assessed with the aid of models with chronic intestinal ports. In vitro biological systems provide complementary data for mechanistic assessments of carrier-mediated processes or biological barrier properties for further elucidation of the interaction of drug and formulation in preclinical model systems.

Fast-Track Approaches to Phase I Formulation. Amale Hawi, Pharmaceutical Development.

A. Hawi Consulting, Ltd., Ridgefield, Connecticut, USA.

Development of formulation for Phase I studies can be challenging in particular when drug substance supplies are limited and/or when the project is fast-tracked to the clinic. The need to accommodate a wide dosing range adds yet another degree of complexity particularly for poorly water soluble compound intended for oral delivery, the route of choice for drug administration. In that regard, understanding the compound intrinsic physicochemical (e.g. solubility, partition, stability) and biopharmaceutical properties (e.g. metabolism, permeability, PK) and the factors that limit its absorption and bioavailability are the corner stone of rational formulation development. It is possible to adopt a minimalist approach to early formulation development through effective integration of preclinical and pre-formulation data. Such approaches need to balance speed while providing meaningful information as the outcome of the first-time-in-man studies is often the basis for further development decisions. The role of study design and data analysis in the selection of the appropriate formulation will be discussed.

New Paradigms In Early Drug Development - What Role for Human Microdosing?

Steve Matheson, Business Development, Pharmaceutical Profiles, Nottingham, United Kingdom.

The Global Pharmaceutical Industry is amidst a challenging era in its history. Producing new drugs continues to be an expensive and time consuming process. Social and Economic pressures demand new medicines faster and at a cost competitive price. Yet, for all its innovation, the Pharmaceutical Industry is arguably inefficient. In 2002 only 17 New Molecular Entities (NME’s) were registered with the FDA and the cost of drug development has been estimated to be in the region of $900 million. Why do new drugs cost so much?? A high proportion of this figure can be attributed to previous development failures. Additionally, upwards of 30% of new drugs in clinical development fail due to poorly defined human pharmacokinetics. The Drug Development process rapidly requires new paradigms to enhance candidate selection prior to Phase 1 clinical trails. Human Microdosing represents one such model that can easily be incorporated into development plans with excellent results. Microdosing exploits favorable Regulatory environments in Europe and now the USA to enable pharmacokinetic evaluations in man to be undertaken prior to safety and tolerability in Phase 1. This presentation will focus on the use of Microdosing as a tool in Early Development. The definition of such Phase 0 studies will be explored including the Preclinical and analytical requirements to make a successful study. Finally, the presentation will examine a real study example and exemplify the power of this new paradigm in Drug Development.

SESSION 5: PAT/Pharmaceutical Analysis.

Droplet Sizing Determination And Spray Pattern Analysis For Nasal Sprays.

Herman Lam, Analytical Technologies, GlaxoSmithKline, Mississauga, Ontario, Canada.

Time evolution of the droplet size and the spray pattern can be valuable information to enhance the understanding of the spraying process and hence drug delivery of nasal spray products. Advances in the laser diffraction measurement for droplet sizing coupled with precise mechanical control over the actuation of the nasal spray have shed new light into the dynamic of the spray cycle. Some of the challenges such as multiple scattering and vignetting encountered from using instruments designed for particle sizing for droplet sizing have been overcome. In addition, the fast and non-intrusive high-speed imaging with laser illumination will gradually replace the traditional spray pattern analysis of capturing the spray on a thin layer chromatographic plate. The analytical techniques for droplet sizing and spray pattern analysis, verification of the performance of the instruments, factors affecting the droplet size and the spray pattern, and the correlation between the time evolution of the droplet size and the spray pattern will be presented.

Prospects For In-Process Testing By Laser-Induced Breakdown Spectroscopy (LIBS).

Louis St-Onge, National Research Council of Canada, Industrial Materials Institute, Boucherville, Québec, Canada.

Laser-induced breakdown spectroscopy (LIBS) combines, in a single step, laser ablation of the sample and atomic emission spectroscopy from the resulting plasma. This analytical technique is capable of providing quantitative elemental analyses in real time, with little or no preparation of the sample, and without physical contact. LIBS can therefore meet the demands of process analytics, as was demonstrated in various industries. In 2001, a commercial LIBS instrument was introduced to the pharmaceutical sector for the at-line analysis of solid dosage forms. In this presentation, examples will be given of recent research aimed at extending the applicability of such an instrument to other sample types, such as loose powder, liquids, creams, and gels. Although LIBS is indeed directly applicable to samples in such a wide range of physical states, it is also true that the quantitative LIBS response for a given analyte will generally depend on the matrix. In collaboration with the Food & Drug Administration, a study was conducted recently using a well-controlled set of furosemide solid dosage forms in order to better understand the matrix effects that result from changes to the formulation or to manufacturing parameters. The main parameter that was found to affect the LIBS response for the drug was the lactose/avicel ratio. Such an understanding of matrix effects may facilitate at-line product characterization during manufacturing changes.

Discovery, Identification and Verification of Cancer Markers for Endometrial Cancer.

Jingzhong Guo, Terence J. Colgan, Leroi DeSouza, Mary Joe Rodrigues, Alex D. Romaschin, K.W. Michael Siu^; Department of Chemistry and Centre for Research in Mass Spectrometry, York University, Toronto, Ontario, Canada; Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; Biochemistry, Toronto General Hospital, Toronto, Ontario, Canada.

Endometrial carcinoma (EmCa) is a cancer of the uterine epithelium. It is the most common malignancy for women in the U.S. with 40,320 cases projected in 2004. The pathologic diagnosis of EmCa (in fact, almost all human cancers) is currently based upon the microscopic recognition of cellular and tissue phenotypes. Advances in proteomic analysis techniques, especially those that are based on high-resolution mass spectrometry, now permit the recognition of both normal and diseased tissues and cellular phenotypes by means of biomarkers in protein/peptide profiles. We are currently engaging in the proteomics of endometrium and EmCa, and in developing methodologies and technologies in mass spectrometry (MS). At present, over 400 consented tissue and tumor samples have been banked and histologically classified. Eleven potential cancer markers have been recognized and identified; a number of them have been independently verified using immunohistochemistry. Marker discovery is carried out by (1) comparing protein expression profiles after fast separation using solid-phase extraction or selective adsorption on functionalized surfaces (e.g. protein chips of Ciphergen); and (2) comparing relative abundances of tryptic peptides labeled with isotope-coded tags, cICAT and iTRAQ, after nanoLC separation. Methodology No. 1 is relatively easy to implement, but tends to give fewer and is more amenable to smaller proteins; in addition, protein identification requires offline multidimensional chromatography. Methodology No. 2 is technically more demanding, with 2D LC separation (first dimension of SCX followed by second dimension of reverse-phase nanoLC) and protein/peptide labeling, large-scale protein identification and quantification by isotope-dilution MS is achieved, although it is labor-intensive and throughput is medium as protein identification is repeated in every run. The pros and cons of these two technologies will be compared.

Process Applications Of Near-Infrared Spectroscopy Within GlaxoSmithKline.

Dwight Walker, Novel Analytical Technology, GlaxoSmithKline, Research Triangle Park, North Carolina, USA.

Process Analytical Technology (PAT) has recently been moved to the forefront of the pharmaceutical industry. One of the major technologies applied in this effort is near infrared (NIR) spectroscopy. GlaxoSmithKline (GSK) has been using NIR for process measurements in many applications ranging from initial active pharmaceutical ingredient (API) manufacturing through final drug form. When choosing the apply NIR to process there a number of factors that must be considered to ensure a successful deployment of the technology. In this presentation, a number of case studies will be covered that highlight the considerations that one must make to ensure success.

Chemical Imaging and TeraHertz Spectroscopy.

Dan Klevisha, Bruker Optics, Milton, Ontario, Canada.

Not available at time of publication.

SESSION 6: Formulation and Drug Delivery.

Intestinal Lymphatic Transport Of Water-Insoluble Drugs.

Kishor Wasan, Faculty of Pharmaceutical Sciences, the University of British Columbia, Vancouver, British Columbia, Canada.

Over 75% of commercially available drugs are formulated for oral administration. However, one of the major factors limiting the effectiveness of orally administered drugs is poor absorption from the gastrointestinal tract or extensive pre-systemic clearance through hepatic first-pass metabolism. For poorly water-soluble drugs, slow dissolution rate in the primarily aqueous contents of the gastrointestinal tract presents a significant barrier to absorption. One strategy for improving the absorption of these drugs involves administration in a lipid-based delivery system, which presents the drug to the gastrointestinal tract in a solubilized form, thus eliminating poor aqueous solubility, and slows dissolution rate as barriers to absorption. The gastrointestinal lymphatic system is a specific transport pathway through which dietary lipids, fat-soluble vitamins and water-insoluble compounds can gain access to the systemic circulation. Drugs transported by way of the gastrointestinal lymphatic system bypass the liver and avoid potential hepatic first-pass metabolism. Lymphatic delivery of immunomodulatory agents and low therapeutic index drugs used in the treatment of cancer cell metastases and HIV presents an opportunity to maximize therapeutic benefit while minimizing general systemic drug exposure. Furthermore, lymphatic drug transport may promote drug incorporation into the body’s lipid-handling system, thus offering the potential to manipulate drug distribution and residence time within the body. This talk will discuss the potential mechanisms by which drugs can be targeted through the lymphatic system.

Strategies for Fast and Efficient Development of Oral MR Formulations.

Avinash G. Thombre, Pfizer Global R&D, Groton, Connecticut, USA

Seven strategies for the fast and efficient development of oral modified release (MR) formulations are outlined and illustrated with examples/case studies. The strategies include critically assessing the need for MR, using appropriate pre-clinical data and pharmacokinetic simulations to rationally design the dosage form, and, instead of a trial-and-error approach, selecting the most appropriate MR technology to eliminate expensive clinical iterations.

Time and Cost Effective Development of First Time in Human Formulations.

Kwok Chow, Patheon Inc, Mississauga, Ontario, Canada.

A first time in human (FTIH) formulation is defined by the clinical goal, budget, drug substance characteristics (and availability), biopharmaceutics, and available delivery technologies for the new chemical entity. Simple straight forward (oral) formulation techniques such as powder in bottles or simplified capsules may be employed to conserve drug substance, reduce development time and save money in an early drug development program. However, a conventional formulation can be more cost and time effective for a FTIH or proof-of-concept study if the same or similar formulation (e.g. tablet, capsule, liquid, suspension or nasal spray) is used for subsequence clinical studies or eventually as the commercial product. If required, more advanced manufacturing technologies or formulation delivery systems may be considered for unstable, difficult to process (e.g. cohesive) or difficult to deliver (e.g. high dosage, low solubility and/or low permeability) molecules. A multidisciplinary approach with careful examination of the drug substance characteristics and the technologies/formulations of interest will help developing and executing a practical FTIH pharmaceutical development program. A time and cost effective FTIH formulation program is most likely delivered by an experienced and cohesive project team with sound scientific knowledge and project planning/management skills. A versatile pharmaceutical development infrastructure and a flexible yet reliable development quality assurance program are vital for the clinical formulation to reach the clinic on time and budget.

Accelerated Development of Liquids and Semisolids.

Amyn Sayani, Pharmaceutical Development, GlaxoSmithKline Inc., Mississauga, Ontario, Canada.

Product development can be a costly and time-consuming activity, typically taking 6-9 years after a molecule is selected for further development. Formulation and process development scientists have the opportunity to reduce timelines by working smarter and applying various tools at their disposal. The use of a rational design of experiments, for example, in understanding the effect of variables that affect a formulation or process can significantly help the product development scientist to reduce the number of experiments necessary to draw meaningful interpretations of the data. Additionally, the use of first-intent or generic formulations and analytical methods that have been evaluated carefully in indicator studies can further reduce timelines during product development. Different analytical tools for measurement of physical stability of formulations, e.g. rheology and thermal analysis, may also provide information faster than the typical approach of placing product on stability in conventional chambers. This presentation will use case studies of the development of various dosage forms to demonstrate the utility of various tools and approaches to accelerate drug development.

SESSION 7: DMPK Technologies Accelerating Drug Discovery & Development.

Recent Advances in Extrapolating Preclinical Pharmacokinetic Data to Humans.

Christopher A. Evans, Investigator, Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, King of Prussia, Pennsylvania, USA.

Prediction of human pharmacokinetic parameters is often conducted based on in vivo preclinical pharmacokinetic data generated during lead optimization in drug discovery. However, to date, the relative ability to accurately predict human pharmacokinetics from the preclinical species typically used in the pharmaceutical industry has not been presented. This study was conducted to comprehensively survey the available literature on intravenous pharmacokinetic parameters in the rat, dog, monkey, and human, and to compare common methods for extrapolation of intravenous pharmacokinetic parameters, identify the most appropriate species to use in pharmacokinetic lead optimization, and to ascertain whether adequate prospective measures of predictive success are currently available. Based on an exhaustive literature survey, 103 non-peptide xenobiotics were identified with intravenous pharmacokinetic data in rat, dog, monkey, and human; both body weight- and hepatic blood flow-based methods were used for scaling of clearance. The results from this investigation indicate that (1) monkey is the most qualitatively and quantitatively predictive species for human clearance, volume of distribution, and half-life; (2) generation of data in 3 versus 2 preclinical species does not always improve predictivity; and (3) some commonly used prospective measures of predictive success, including correlation coefficient and allometric exponent, do not accurately forecast allometric predictivity. The observations in this investigation have major implications for pharmacokinetic lead optimization and for prediction of human disposition from in vivo preclinical data, and support the continued use of nonhuman primates in preclinical pharmacokinetics.

Application of PK/PD Modeling in Drug Development.

Amarnath Sharma, Clinical Pharmacokinetics &Pharmacodynamics, Pfizer Inc., New London, Connecticut, USA.

PK/PD characterization during early clinical development is critical for the selection of better compounds and their efficient development. A relatively new paradigm in the clinical drug development is to establish proof of mechanism for new compounds by evaluating PK/PD in patients and/or in experimental models in healthy subjects in early Phase 1 studies followed by PK/PD in dose-ranging proof of efficacy study in patients. The major requirements to characterize PK/PD relation of a compound are i) validated biomarkers for therapeutic effects and/or toxicity and ii) understanding of pharmacologic behavior of the drug and pathophysiology of the disease. This talk will discuss the PK/PD modeling of direct and indirect responses and will present the examples to demonstrate the application of PK/PD concepts in clinical drug development.

Metabolism Issues in Drug Development.

Stephen Clarke, Drug Metabolism and Pharmacokinetics, GlaxoSmithKline Pharmaceuticals, Welwyn, United Kingdom.

Over the last ten to twenty years the pharmaceutical industry has recognised the importance of drug metabolism and pharmacokinetics (DMPK) properties on the developability of new chemical entities (NCE). Over that time much effort has been expended to select compounds that are likely to have suitable DMPK properties in man. The targeted properties typically being good oral bioavailability, half-life consistent with once a day dosing and a low propensity to cause drug-drug interactions (both by limiting inhibition of ADME process and involvement of single sensitive elimination processes). Most pharmaceutical companies have a toolbox of in vitro and in vivo techniques and computational or other property to structural relationship that they can employ. Alternatively there are many contract research organisations that offer such services. The industrialisation of these tools has led to unprecedented quantities of data (with a disproportionate increase in understanding), but a clear reduction in adverse DMPK as a cause for attrition. Yet drug metabolism issues continue to occur, some of which may be exacerbated by the intensive lead optimisation screening. This talk will discuss these themes with a particular focus on the issue of qualification of human metabolites in toxicological evaluation of NCEs.

In Vivo Molecular Imaging: Potential To Delivery Better PK/PD Correlation.

Susanta Sarkar, Molecular Imaging Center of Excellence, GlaxoSmithKline, King of Prussia, Pennsylvania, USA.

Not available at time of publication.

SESSION 8: Current Drug Regulatory Issues.

Emerging Regulatory Issues at Health Canada.

Brian C. Foster, Office of Science, Therapeutic Products Directorate, Health Canada, Ottawa, Ontario, Canada.

The regulatory process is under continual flux to meet the strong public demand for timely access to “safe” therapeutic products. With this demand for faster access to new and safer products come new challenges for both the drug industry along with those in the related pharmaceutical sciences and the regulators. This includes a greater workload associated with the need to continually improve and meet emerging issues, demands for new therapies such as drug-covered stents while maintaining critical objectivity. A response to a drug is not consistent among patients in a population and in some cases is not consistent on a day-to-day basis within a patient. This talk will explore some of the safety issues including adverse drug events related to drug-food-natural health product-nutrient/drug interactions, drug resistance, emerging diseases, and what may be faced when “omic” information (metabolomic, pharmacogenomic, proteomic) is required or used in the development of a new drug where the effects of variation in the number and specificity of the receptors and on the other systems are affected by the disease and/or the drug. The role of gender, ethnic differences and age when assessing efficacy and safety of drugs will also be explored.

Regulation of Clinical Trials in Canada.

Jim Gallivan, Clinical Trials & Special Access Programme, Therapeutic Products Directorate, Health Canada, Ottawa, Ontario, Canada.

In September 2001, Health Canada introduced new regulations governing the conduct of clinical trials in Canada. These regulations sought to create an environment more conducive to clinical drug research and development in Canada, enhance the participation and safety of clinical trial subjects, strengthen interactions with clinical trial sponsors and improve accountability. Direct responsibility for the trial extended to the sponsor and a sponsor was redefined to include individual researchers as well as industry. Review times were shortened from 60 day to 30 days, with a 7-day target for many Phase I studies. The new regulations also provided a framework for inspections and adverse event reporting. Guidance for clinical trial sponsors describing the procedures for clinical trial applications was published in June 2003. This presentation will present an overview of the regulations and the review of clinical trial applications.

Regulatory Implications of the International Harmonization of Standards.

Mike Ward, International Programs Division, Office of Science, Health Canada, Ottawa, Ontario, Canada.

Technical guidelines and standards define generally acceptable approaches on how to comply with underlying policies and governing statutes and regulations, including in the Health sector those related to the development, approval and surveillance of pharmaceuticals. With a continuing trend towards the globalization of markets and issues, international harmonization is playing an increasing important role in the regulation of products at the regional and national level. Harmonization activities in the context of drug regulation are centered around the harmonization of drug registration requirements, that is, the standards specifying the type, quantity and sometimes the manner of generating the data necessary to support the safety, quality and efficacy evaluation of a drug. The International Conference on Harmonisation, or ICH, represents the most important and successful harmonization initiatives in the pharmaceutical sector, with the development of over 40 guidelines on drug registration requirements (for both chemical and biotechnologically derived products), including technical guidances, electronic transmission standards, a medical dictionary of regulatory terms and, most recently, a common format for marketing application. As ICH guidelines play a crucial role in the development, registration and marketing of pharmaceuticals internationally, an understanding of ICH is crucial to understanding drug regulation. This presentation will provide an overview of ICH within an international and evolving regulatory environment.

QTc Prolongation and Regulatory Guidance: Safety Pharmacology, Clinical Trials, and the Product Monograph.

Colette Strnad, Therapeutic Products Directorate, Health Canada, Ottawa, Ontario, Canada.

Excessive prolongation of the QTc interval is conducive to the occurrence of torsade de pointes, a polymorphic ventricular tachyarrhythmia that can progress to ventricular fibrillation and sudden cardiac death. Identification of the QTc prolongation liability of new drugs is therefore an important objective of contemporary drug development programmes. The International Conference on Harmonisation (ICH) is preparing a guideline on non-clinical safety pharmacology studies to assess the potential effects of drugs on ventricular repolarization in laboratory animals and in vitro preparations. Another ICH guideline is providing recommendations on the assessment of QTc prolongation liability in clinical trials, including specialized clinical pharmacology studies to characterize the magnitude, dose-dependency, and time course of drug-induced QTc prolongation. The association of a drug with QTc prolongation liability has important regulatory implications for approval and Product Monograph content.

POSTER PRESENTATIONS: Accelerating Drug Discovery & Development.

1 Immobilization Of Whole Cell Penicillin Gacylase On Chitosan.

Daryoush Abedi, N. Tavakoli, H. Korbekandy, M. R. Baghernejad; Department Of Pharmaceutical Biotechnology; Department Of Pharmaceutics, Faculty Of Pharmacy, Isfahan University Of Medical Sciences, Isfahan, Iran.

Purpose: Penicillin acylase (PAs) catalyses the hydrolysis of amide/acyl bond in penicillin molecules and produces the âlactam nucleus, 6-aminopenicillanic acid (6-APA) and the corresponding side chain. Current research is focused on the immobilization of this industrially valuable biocatalyst, either in the form of isolated enzyme, or whole cell enzyme by different techniques. Entrapment of whole cell enzyme is one of the methods of choice. Chitosan has many useful features such as hydrophilicity, biocompatibility and biodegradability, which attract its use as an immobilizing agent. The objective of this study was to evaluate the immobilization of E. coli containing PAs on a chitosan support. by permeabilizing the cell using CTAB and then entrapping on chitosan. Methods: An aqueous suspension of E. coli ATCC 11105 was prepared and diluted by a solution of N-cetyl-N,N,N-trimethyl ammonium bromide (CTAB) to increase permeability of the cells. The resultant microbial suspension was gently stirred for 45 min at room temperature and was added to a solution containing chitosan, acetic acid and glutaraldehyde while stirring for more 4h. The activity of PAs was determined using pH-Stat method. Results: The activity of immobilized enzyme was shown to decrease initially with the incubation time up to 3h and remained almost constant thereafter. The immobilized enzyme activity was highest at pH 7.8. The activity profiles of the free and immobilized PAs showed that the maximum enzyme activities were around pH 8.5 and 8.0 for the immobilized and free enzymes, respectively. Conclusions: Whole cell PAs on chitosan support was successfully done and the immobilized enzyme displayed the same activity even after 20 weeks indicating no leakage of enzyme and very stable immobilization.

2 The Role Of PXR In 2-AAF-Mediated Induction OfMrp2, Bcrp, Oatp2 And Cyp3a11 In Mice.

Alexander Anapolsky, Shirley Teng, Micheline Piquette-Miller; Department Of Pharmaceutical Sciences, University Of Toronto, Ontario, Canada.

Purpose. Activation of the Pregnane X receptor (PXR) mediates the induction of several drug transporters and metabolizing enzymes. In vitro studies indicate alterations in several of these genes after exposure to the hepatocarcinogen, 2acetylaminofluorene (2-AAF). Thus, we hypothesized that PXR may play a role in the in vivo induction of gene expression by 2-AAF. We examined the expression of mrp2, oatp2, cyp3a11, cyp1a2 as well as bcrp. Methods. Wild-type (+/+) and PXR-null (-/-) C57BL/6 mice were injected i.p. for 7 days with 150 and 300 mg/kg of 2-AAF suspended in corn oil. Levels of mRNA isolated from liver were measured by RT-PCR and normalized to b-actin. Results. The hepatic mRNA levels of mrp2 and bcrp were induced (p<0.001) 2- to 3-fold in (+/+) mice in a dose-dependent manner, but not in (-/-) mice, confirming the involvement of PXR in induction of these genes. Similarly, the hepatic mRNA levels of oatp2 were induced (p<0.001) 4fold in (+/+) mice, but not in (-/-) mice. Cyp3a11 mRNA was induced (p<0.001) 3- to 4-fold in (+/+) mice, but not in (-/-) mice. Despite the lack of significance, the hepatic mRNA levels of cyp1a2 were induced 3- to 4-fold in (+/+) mice compared to control. No induction of cyp1a2 was observed in (-/ -) mice. Conclusions. These results suggest that PXR is responsible for the up-regulation of drug efflux transporters and biotransformation enzymes in the liver during pre-neoplastic changes. Moreover, novel findings demonstrate that PXR plays a role in regulation of the drug efflux transporter, bcrp.

3 PXR-Mediated Regulation Of Hepatic Transporter Expression By Tamoxifen.

Shirley Teng, Micheline Piquette-Miller; Department Of Pharmaceutical Sciences, Leslie Dan Faculty Of Pharmacy University Of Toronto, Toronto, Ontario, Canada.

Purpose. The pregnane X receptor (PXR) is a nuclear receptor transcription factor that regulates the expression of many genes involved in drug metabolism and transport. Tamoxifen is a widely-used estrogen receptor antagonist for the treatment of breast cancer. It has been reported that tamoxifen can alter the expression of CYP3A and P-glycoprotein, which are known PXR target genes. Thus our purpose was to determine if tamoxifen can modulate hepatic gene expression via the activation of PXR. Methods. Wild-type (+/+) and PXR-null (-/-) female C57BL/6 mice were treated with 50mg/kg tamoxifen or corn oil vehicle control i.p. for 10 days. Total hepatic RNA was isolated and reverse-transcribed for determination of mRNA levels by RT-PCR. Results. Treatment of mice with tamoxifen resulted in a significant induction of hepatic CYP3A11 and the transporters MRP2, MRP3, BSEP, OATP2, MDR1a, MDR2, BCRP and NTCP mRNA in +/+ but not -/- mice. The expression of PXR itself was also induced in +/+ mice. On the other hand, mRNA levels of MDR1b remained unchanged in both +/+ and -/- mice. Conclusions. Tamoxifen can alter the expression of numerous hepatic transporters via a PXR-dependent mechanism, possibly by acting as an activating ligand. These findings may have clinical implications in terms of drug interactions and the alteration of drug disposition during chemotherapy with tamoxifen.

4 Breast Cancer Resistant Protein (BCRP) Is Involved In The Disposition Of The Hypoglycaemic Drug, Glyburide.

Javad Behravan, Christelle Gideon, Gideon Koren^, Micheline Piquette-Miller; Department Of Pharmaceutical Sciences; Hospital For Sick Children, The University Of Toronto, Toronto, Ontario, Canada.

Purpose: Breast Cancer Resistance Protein (BCRP) is highly expressed in placental syncytiotrophoblasts. It has been shown by several groups that the second generation hypoglycemic drug, glyburide does not significantly cross the placeta of pregnant women. We hypothesized that BCRP functions to protect the fetus by preventing the glyburide transport across placenta. Methods: BCRP, P-glycoprotein (PGP), Multidrug Resistance Protein1 (Mrp1), Mrp2 and Mrp3 transfected cell lines (MCF7/ MX, MCF7/ADR, Hela/MRP1, MDCK/MRP2 and MDCK/MRP3) were seeded on 24-well plates for accumulation studies. The cellular accumulation of [³H]glyburide following a 60-minute incubation was monitored by liquid scintillation counting. Novobiocin (300 mM), verapamil (100 mM) and indomethacin (200 mM) were used as the inhibitors of BCRP, PGP and MRP drug transporters, respectively. Results: Cellular accumulation of [³H]glyburide by BCRP over-expressing cell monolayers was dramatically increased in the presence of novobiocin (15795 ± 829 versus 1441 ± 401 pmol/mL/mg protein, P<0.01). Indomethacin significantly increased the accumulation of [³H]glyburide in MRP3 over-expressing cell line (15394 ± 1142 versus 10724 ± 588 pmol/mL/mg protein, P<0.05), but not in the MRP1 and MRP2 over-expressing cell lines. Conclusion: These studies indicate that glyburide is a substrate of BCRP and MRP3, and that novobiocin and indomethacin can inhibit the BCRP or MRP3-mediated transport of glyburide.

5 In Vivo Release Characterization Of A Novel Implantable Paclitaxel Loaded Lipid Polymer System.

Vessela Vassileva, Christine Allen, Micheline Piquette-Miller, Department Of Pharmaceutical Sciences, University Of Toronto, Toronto, Ontario, Canada.

Purpose: To characterize the in vivo release profile of a novel implantable polymer delivery system (PoLi). Methods: CD-1 female mice were surgically implanted intraperitoneally (IP) with 14C-PTX loaded PoLi formulations composed of high drug to matrix ratio and low drug to matrix ratio. Mice were kept in metabolic cages for 24h periods over the course of 2 weeks. Feces and urine were collected at the end of each 24h time period and 14C-PTX was measured by scintillation counting. At the end of the study period, animals were sacrificed and visually inspected for signs of infection, inflammation and capsid formation; implants and tissues were collected, weighed and solubilized for the determination of 14C-PTX. Results: During the course of the study period, animals appeared healthy. Post-mortem examination did not reveal observable signs of infection, inflammation, local irritation or capsid formation. The high drug:matrix and low drug:matrix PoLi-PTX implants provided a sustained, zero-order release of 11.0 + 2.1 mg/kg/day (5.3 + 0.2 % per day) and 1.0 + 0.3 mg/kg/ day (3.5 + 0.6 % per day) over the 2 week period, respectively. Conclusions: We have demonstrated that the novel PoLi PTX-loaded implant system provides sustained release of PTX. The implant material was found to be non-toxic and biocompatible with minimal or no capsid formation. Since localized therapy reduces systemic drug exposure and increases therapeutic efficacy, our drug delivery system may be of clinical significance in the treatment of solid tumours.

6 In Vitro Characterization Of A Novel Polymer-Lipid Implant System For The Combination Delivery Of Paclitaxel And Carboplatin.

Emmanuel Ho, Christine Allen, Micheline Piquette-Miller; Department Of Pharmaceutical Sciences, University Of Toronto, Toronto, Ontario, Canada.

Purpose: Current chemotherapeutic treatments tend to fail due to the inability to provide therapeutic concentrations at the disease site. Our research groups have developed a novel biomedical implant system for the simultaneous delivery of both paclitaxel (PTX) and carboplatin (CPT). Our main objective was to characterize and evaluate the utility of this implant system. Methods: Implant systems containing PTX only and implants containing both PTX and CPT were incubated for 72 hrs in the presence of SKOV-3 cells. MTT was performed to determine implant biocompatibility and efficacy. Release of ¹⁴C-PTX from implants was determined using scintillation counting while release of CPT from implants was determined by ICP-AES. Results: A zero-order constant release of 0.92 ± 0.03 pg/day PTX over 5 days was delivered from a 10 mg sized PTX- implant with a cumulative dosage release of 8.55 ± 1.04 pg PTX. PTX released from the PTX- implant system was active; effectively inhibiting SKOV-3 cell growth with an IC₅₀ of 211 ng/ml PTX. The 2-in-1 combination implant was also active and displayed an IC₅₀ of 140.2 ng/ml for PTX and an IC₅₀ of 7.56 mg/ ml for CPT. Conclusions: Overall, the novel polymer-lipid implant system was found to be biocompatible and efficacious in providing a continuous release of PTX and CPT. The 2-in-1 implant system displayed to be more effective in reducing the viability of SKOV-3 cells in comparison to the PTX-only implant system. This 2-in-1 implant system may likely offer advantages and clinical utility in the treatment of ovarian cancer.

7 Methoxy Poly (Ethylene Glycol)-Block-Poly ( -Valerolactone) Copolymers For Formulation Of Hydrophobic Drugs.

Helen Lee, Faquan Zeng, Mike Dunne, Christine Allen, Leslie Dan Faculty Of Pharmacy, Department Of Pharmaceutical Sciences; Faculty Of Applied Science And Engineering, Division Of Engineering Science; University Of Toronto, Toronto, Ontario, Canada.

PURPOSE: The intravenous administration of hydrophobic drugs has been a challenge due to their low water solubility and high affinity for plasma protein. Nano-sized polymeric micelles formed from amphiphilic copolymers are capable of encapsulating hydrophobic agents, acting as drug solubilizers and carriers simultaneously. We have prepared a biocompatible, biodegradable amphiphilic diblock copolymer based on methoxy-poly(ethylene glycol) (MePEG) and poly(dvalerolactone) (PVL) for the preparation of micellar drug carriers. METHODS: MePEG-b-PVL copolymers were synthesized using MePEG as the macroinitiator via a metal-free cationic polymerization method. The composition of the copolymers and their molecular weight distributions were determined by ¹H NMR and GPC analyses. The thermal properties of the copolymers were obtained by DSC analysis and the critical micelle concentrations were measured with an established fluorescence-based method. Micelles (+/- paclitaxel) were prepared by the evaporation method and their size distributions were evaluated by dynamic light scattering. RESULTS: Six MePEG-b-PVL copolymers with different hydrophilic and hydrophobic block lengths were synthesized. The copolymers were found to have narrow molecular weight distributions (M_w/ M_n < 1.15). In an aqueous media, the MePEG-b-PVL copolymers formed micelles with effective diameters ranging from 25nm to 100nm depending on the copolymer composition. A hydrophobic anticancer agent, paclitaxel, was encapsulated in the MePEG-b-PVL micelles, increasing the aqueous solubility of this drug by more than 500 fold. CONCLUSION: The amphiphilic MePEG-b-PVL diblock copolymers were synthesized in the absence of a metal-based catalyst. The MePEG-b-PVL copolymer micelles were found to be less than 200 nm in size and capable of solubilizing paclitaxel.

8 Development And Application Of An Orthotopic Rat Liver Transplantation (OLT) Model Using The Three Cuff Technique.

Anjaneya Chimalakonda, Reza Mehvar; School Of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, Texas, USA.

Purpose. The use of methylprednisolone (MP) for the treatment of acute liver rejection is associated with toxicities in non-target tissues. Therefore, selective delivery of MP to the liver may improve its efficacy and alleviate its side effects. Our aim was to develop an OLT model and test the effect of MP and its liver-targeted dextran prodrug (DMP) on acute rejection. Methods. A model of OLT was optimized in our lab using the three-cuff technique for vascular anastomosis. Following OLT in an acute rejection combination (Sprague-Dawley liver donors and Wistar recipients), recipients were administered a single intravenous 10 mg/kg equivalent dose of MP or DMP or with saline (control). Blood samples were collected on days 3, 6, and 9. Plasma concentration of allograft rejection markers, alkaline phosphatase (ALP) and bilirubin were measured. Results. OLT was performed in our lab with an average portal vein clamping time of 17 min and one week survival of 85%. Treatment of rats with either MP or DMP significantly (p<0.05) decreased the plasma concentration of ALP and bilirubin. However, DMP treatment was significantly more effective in reducing plasma concentration of these markers compared to MP. In addition, plasma concentration of lactate dehydrogenase, a marker of cellular integrity of the liver, was significantly attenuated only by DMP treatment. Conclusion. Acute rejection of the liver following OLT was significantly attenuated by DMP compared to the parent drug MP. Therefore, targeted delivery of MP as dextran prodrugs may favorably influence the outcome of liver transplantation.

9 The Effect Of Bcl-Xl Antisense Oligonucleotide And Apoptosis-Inducing Drugs On Human Umbilical Vein Endothelial Cell Survival And Growth.

Jessica Chong, John K. Jackson, Helen M. Burt, UBC Faculty Of Pharmaceutical Sciences, Vancouver, British Columbia, Canada.

PURPOSE Cancerous tissues induce angiogenesis, the formation of new capillary blood vessels, to obtain an adequate supply of oxygen and nutrients. This allows for the growth of the tumor, invasion of local organs, and the escape of cancer cells through the new blood vessels into the circulation and other organs. Capillary growth occurs via growth factor induced proliferation of capillary endothelial cells. It is hypothesized that growth factors may up-regulate the pro-survival bcl-xL protein (a member of the apoptosis controlling bcl-2 family of proteins) in the cells. Therefore, antisense oligonucleotides (ASO’s) targeted against bcl-xL may prevent the up-regulation of the protein so that apoptotic cell death may occur and angiogenesis may be inhibited. The objective of this study was to investigate the effect of varying concentrations of bclxL ASO’s in the presence or absence of apoptosis-inducing drugs on the growth of HUVEC cells in vitro. METHODS HUVEC cells cultured in T-75 flasks in 15 mL of EBM-2 media at 37°C were seeded and cultured undisturbed for two days on 96well plates. Cells were then incubated in serum-free media (SFM) containing the transfection agent Lipofectin with various concentrations of bcl-xL ASO’s for three hours, followed by reapplication of serum-containing media EBM-2. This process was repeated the following day. In some experiments, the apoptosis inducing drugs, paclitaxel (10 nM), camptothecin (20 nM), and doxorubicin (100 nM) were applied to ASO or control-treated cells to study combination effects. On day seven, cell survival was measured using an MTS assay. RESULTS HUVEC cell growth was inhibited by treatment with bcl-xL ASO’s in a concentration dependent manner. In combination experiments, the addition of an apoptosis-inducing drug had additive effects on ASO growth inhibition, demon-strating that the bcl-xL ASO’s had effectively bound to the bcl-xL mRNA to inhibit the production of the essential pro-survival protein. At 50 nM bcl-xL ASO, cell viability was inhibited by 46%. Cell viability was further inhibited by 63%, 66%, and 87%, by the addition of paclitaxel (10 nM), camptothecin (20 nM), and doxorubicin (100 nM), correspondingly. CONCLUSION Bcl-xL ASO’s may be effective agents to inhibit the replication of HUVEC cells and angiogenesis. The combined effect of bcl-xL ASO’s with an apoptosis-inducing drug is additive and effective in inhibiting the survival of HUVEC cells. ACKNOWLEDGEMENT JMC was supported by a scholarship from the Merck Company Foundation.

10 Pravastatin Reverses The Down-Regulating Effect Of Inflammation On Adrenergic Receptors: A Report On Pravastatin-Propranolol-Inflammation Interaction.

John D. Clements, Fakhreddin. Jamali; Faculty Of Pharmacy & Pharmaceutical Sciences, University Of Alberta, Edmonton, Alberta, Canada.

Purpose. Inflammatory conditions reduce electrocardiographic (ECG) response to cardiac â-adrenergic antagonists such as propranolol. We hypothesize that pravastatin, an anti-cholesterol drug, may reverse this, and that it may correct Th1/Th2 immune imbalance associated with the Th1-skewed Pre-Adjuvant Arthritis model (Pre-AA). Methods. PR-interval response to propranolol, a measure of cardiac conduction, was measured in four groups of rats (n=14-16/group): Healthy/ Placebo, Arthritis/Placebo, Healthy/Statin, and Arthritis/Statin. Day 0: 38 mg/kg Mycobacterium butyricum in squalene, or placebo. Day 4: 6 mg/kg of pravastatin twice daily, or placebo. Day 8: final ECG measurement. Results. As expected, response to propranolol was reduced in inflamed rats. Interestingly, however, treatment with pravastatin reversed the down-regulation so that it enabled the inflamed rat to maintain expected propranolol response: Area Under the % Effect Curve (%.min) was 714±214 in Healthy/Placebo, 256±249 in Arthritis/Placebo, 1534±367 in Healthy/Statin, and 1713±393 in Arthritis/Statin. Significantly fewer rats had detectable IFN-ã in Arthritis/Statin versus Arthritis/Placebo. Pravastatin does not appear to correct the elevated plasma propranolol concentrations associated with Pre-AA. Conclusion. Inflammation lowered drug response despite increased plasma propranolol concentration. Pravastatin reversed the effects of inflammation on propranolol’s PR-interval response. Restoration of cardiac response with pravastatin was not associated with a correction of plasma propranolol levels, but coincides with attenuation of the inflammatory cytokine interferon-ã. This indicates that response may be related to the anti-inflammatory effects of pravastatin. Patients in inflammatory status may benefit from statins during cardiovascular treatment. Supported by: CIHR and RX&D Health Research Foundation.

11 Preliminary Report On The Effect Of The AngiotensinReceptor Blocker, Valsartan, On The PharmacodynamicsOf Verapamil In Early Adjuvant Arthritis In The Rat.

Nigel Dagenais, Fakhreddin Jamali; Faculty Of Pharmacy,University Of Alberta, Edmonton, Alberta, Canada.

Purpose: Inflammation can reduce drug response to cardiovascular drugs such as verapamil. Recently, angiotensin II the major effector molecule of the renin-angiotensin-aldosterone system has been shown to act as a pro-inflammatory mediator. Conversely, various clinical and animal studies have shown angiotensin II interruption to have multifarious anti-inflammatory effects. The purpose of this study is to investigate whether the angiotensin receptor blocker, valsartan, can reverse the inflammation-induced diminished PR interval prolongation to verapamil. Methods: Pre-adjuvant arthritis (pre-AA) conditions were achieved by injecting into the rats tail base 10 mg of Mycobacterium butyicum suspended in squalene (day 0). Control rats received 0.2 ml of sterile normal saline injections. Four groups of male Sprague-Dawley rats (230-250 grams, n=4-5/ group) are used for this study: Control/Valsartan treated, Control/Vehicle, Pre-AA/Valsartan and Pre-AA/Vehicle. The experiment is carried out for 12 days. On day 6, treatment is initiated with rats receiving 30 mg/kg p.o. b.i.d. of valsartan suspended in polyethylene glycol 400 or vehicle alone b.i.d. for 6 days. On day 12, after baseline measurement, each rat is dosed with 25 mg/kg p.o. of verapamil solution and ECG measurements are taken at 20,40,60,80,100,120,180 and 240 minutes post-dosing and PR intervals measured. Results: Verapamil prolonged PR-interval by 6-7% in controls at peak. Pre-AA diminished the PR interval prolonging effect of verapamil by 10%, 7%, and 4% at 60, 80 and 180 minutes, respectively (p<0.05). Valsartan treatment of pre-AA rats resulted in reversal of diminishing effect of inflammation so that in response to verapamil, Pre-AA/Valsartan exhibited significantly greater prolongation of RP interval than Pre-AA/Vehicle group (1192 ± 320 vs 168 ± 256 p=0.032) based on area under the effect curve. Conclusion: Valsartan treatment seems to be able to reverse the inflammation-induced diminished response to verapamil due perhaps to an anti-inflammatory action, however, further study is required.

12 Effect Of COX-2 Selective Nonsteroidal Anti-Inflammatory Drugs (NSAIDs) On Kidney Function Of Inflamed Rats.

Sam Harirforoosh, Fakhreddin Jamali; Faculty Of Pharmacy And Pharmaceutical Sciences, University Of Alberta, Edmonton, Alberta, Canada.

Purpose: Inflammation alters glomerular function. Recently, we have shown that rofecoxib, celecoxib, diclofenac, and flurbiprofen, but not meloxicam, reduce urinary electrolyte excretion. Since NSAIDs are mainly used in the treatment of inflammatory conditions, we studied the effect of NSAIDs on electrolyte excretion in inflamed rats. Methods: Placebo, rofecoxib (10 mg/kg), or meloxicam (3 mg/kg) was administered orally as single doses to normal or pre-adjuvant arthritic rats. Urine and blood samples were collected. Nitrite, BUN, serum creatinine, and electrolyte concentrations were measured. Results: Nitrite, BUN, and serum creatinine were increased on day 9 or 13 in the inflamed groups. Sodium and potassium excretion rates were not affected by inflammation. However, treatment with rofecoxib, but not meloxicam, significantly decreased sodium excretion [from 1.62 ± 0.95 to 0.98 ± 0.56 mmol/min (p < 0.011) in normal and from 1.38 ± 0.41 to 0.62 ± 0.41 mmol/min (p < 0.005) in inflamed rats]. Potassium excretion was decreased from 2.55 ± 0.95 to 1.63 ± 0.82 mmol/min in the normal rats that received rofecoxib (p < 0.023). Conclusion: Inflammation alters kidney function demonstrated by an increase in BUN and serum creatinine. However, inflammation does not influence the urinary electrolyte excretion. Rofecoxib decreases sodium and potassium excretion rate. Meloxicam seems to be without any significant effect on kidney function. Since the pattern of kidney effect of the examined NSAIDs in inflamed rats is similar to that of previously reported healthy rats, one may conclude that inflammation does not exacerbate the adverse effect.

13 Gelucire 44/14 Improves Bioavailability Of Neusilin-Loaded Meloxicam By Substantially Increasing Solubility.

Ali Aghazadeh-Habashi, Fakhreddin Jamali; Equitech Corporation And Faculty Of Pharmacy, University Of Alberta, Edmonton, Alberta, Canada.

Purpose: Meloxicam (MEL) is a selective COX-2 inhibitor nonsteroidal anti-inflammatory drug. MEL has a low oral bioavailability (F) due, perhaps, to its poor solubility. F is even lower under acute pain condition when the vagal nervous system is suppressed (F, available brands 0.03-0.04). We studied the physicochemical properties of a Gelucire-Neusilin Mel formulation with F=0.39 in a vagally suppressed rat model. Methods: A solid formulation was prepared by loading MEL on magnesium aluminum silicate (Neusilin) and mixing with Gelucire 44/14. The formulation was tested using differential scanning calorimetry (DSC), dissolution rate and solubility in simulated gastric fluid (pH 1.2). The absolute bioavailability of the formulation (1 mg/kg via a plastic gastric gavage followed by 0.5 mL water) was also confirmed vs i.v. doses (n=4) in vagally suppressed rats (20 mg/kg i.p. propantheline 2 and 1 h before dosing). MEL was assayed using HPLC. Results: The absolute bioavailability of the formulation was confirmed to be 0.39, 10-fold greater than formulations without Gelucire-Neusilin. In 0.5, 1 and 2 h, 32±0.40, 41±2.1 and 56±0.80% of MEL dose were released from the formulation, respectively. Solubility at pH 1.2 for MEL as pure powder, and Neusilinloaded loaded in the absence and presence of Gelucire was 0.04±0.00, 3.9±0.94 and 351±86 mg%, respectively. DSC data indicated an interaction between MEL and Neusilin and/or Gelucire since the endometric peak pertaining to the drug peak disappeared. Conclusion: The poor bioavailability of MEL is due to its low aqueous solubility. Although Neusilin significantly improves solubility of MEL, adding of Gelucire results in drastic improvement in the solubility of this poorly absorbed drug. This is the likely reason behind improved absorption of MEL in the rat.

14 Reduced Hepatic Cyp Enzymes By Pro-Inflammatory Mediators In Early Phase Adjuvant Arthritis: A New Approach To The Use Of An Animal Model Of Inflammation For Pharmacokinetic Studies.

Spencer Ling, Fakhreddin Jamali; Faculty Of Pharmacy And Pharmaceutical Sciences, University Of Alberta, Edmonton, Alberta, Canada.

Purpose. Clearance of the efficiently metabolized drug, verapamil, is reduced in patients with rheumatoid arthritis (RA). Like RA, adjuvant arthritis (AA) in the rat causes increased expression of pro-inflammatory mediators which is associated with suppression of hepatic metabolic processes hence reduces drug clearance. AA however, requires two weeks to develop and subjects animals to significant pain. We confirm that the early phase of adjuvant arthritis (pre-AA), is associated with little or no pain and discomfort as compared with fully developed adjuvant arthritis, and also reduces verapamil clearance as seen in RA. Furthermore, we assess the role of pro-inflammatory mediators in inflammation-induced suppression of hepatic CYP enzymes. Methods. Rats were induced with pre-AA and then monitored for symptoms of arthritis, and levels of the pro-inflammatory mediators, serum nitrite, C-reactive protein (CRP), and tumor necrosis factor alpha (TNFá). On day 6, CYP1A and CYP3A content were determined by Western blot as well as total cytochrome P450 content and verapamil pharmacokinetics were assessed. Results. Verapamil plasma concentrations were significantly elevated in pre-AA rats while signs of pain and arthritis were absent. Serum nitrite, CRP and TNFá levels were also significantly elevated within days of adjuvant injection and increases of pro-inflammatory mediators were significantly associated with reductions in hepatic cytochrome P450, CYP3A and CYP1A content. Conclusion. Pre-AA is marked by reduced verapamil clearance due perhaps to increased pro-inflammatory mediators and down-regulation of hepatic CYP enzymes. Hence, pre-AA is a relatively painless and distress-free model of inflammation suitable for pharmacokinetic studies. This work was presented at the Cytokines and Inflammation conference, San Francisco, January 27-28, 2005.

15 Designing A New Questionnaire To Evaluate Asthma Morbidity.

Naghmeh Foroutan, Minoo Habibi, Jamshid Salamzadeh; School Of Pharmacy, Shaheed Beheshti University Of Medical Sciences, Tehran, Iran; Shaheed Labafinezhad Teaching Hospital, Shaheed Beheshti University Of Medical Sciences, Tehran, Iran

Purpose: This study was designed to construct a new comprehensive questionnaire for determining asthma morbidity over the preceding one year. Methods: Asthmatic patients aged over 5 years, referred for their routine follow-up to an asthma clinic, between October 2003 - September 2004, were enrolled the study. A questionnaire including 9 critical questions were used to determine asthma morbidity. Internal consistency of the new questionnaire was assessed using the Kendall’s rank correlation analysis. 20 covariates consisting of demographic characteristics, history of diseases and medication, socioeconomic status and compliance of asthmatic patients were also chosen and their relationship with asthma morbidity was investigated. Final model building was performed using a multiple ridge regression analysis. Results: 299 patients (male=112, female=187) with median (interquartile range) age of 55 (43-67) years entered the study. Results revealed a rational internal consistency for the new questionnaire (pd”0.05). Covariates including asthma duration (year), treatment step and patients compliance could remain in the final model (r=0.46, p<0.0001). Patients with a longer history of asthma, those in higher steps of asthma treatment and interestingly asthmatics with a higher level of compliance had higher morbidity. Conclusion: Our findings show that the new questionnaire is valid enough to be used to determine morbidity caused by asthma over the preceding one year. It could be applied to evaluate the impact of asthma management initiatives on the community or amongst large groups of asthmatic patients. External validity of this questionnaire needs to be assessed by further studies.

16 Molecular Expression Of Chemokine Receptors InBrain Microvessel And Glial Cell Culture Systems.

Mera Guindy, Patrick T. Ronaldson, Manisha Ramaswamy,Reina Bendayan; Department Of Pharmaceutical Sciences,Leslie Dan Faculty Of Pharmacy, University Of Toronto,Toronto, Ontario, Canada.

Background: Human immunodeficiency virus type 1 (HIV-1) infection of the brain may result in HIV-1 encephalitis (HIVE), a chronic neurodegenerative condition (Kaul et al. 2001). Current evidence suggests that chemokine receptors (i.e., CXCR4, CCR5) may be involved in glial cell injury and neurotoxicity associated with HIVE (Persidsky & Gendelman, 2003). The goal of this project is to investigate gene and protein expression of CXCR4 and CCR5 in brain cellular targets of HIV-1 infection (i.e., astrocytes, microglia) as well as brain microvessel endothelial cells. Methods: Primary cultures of rat and human astrocytes, rat microglia, and human brain endothelial cells (HBEC) as well as correspondent rat brain cell lines were used. Gene and protein expression were determined by RT-PCR and immunoblotting analysis respectively. Results: RT-PCR analysis revealed the presence of CXCR4 and CCR5 mRNA in all cell culture systems studied. Immunoblotting analysis using a CXCR4 polyclonal antibody and a CCR5 monoclonal antibody 3A9, detected bands of appropriate size for the CXCR4 heterodimer (i.e., 59 kDa, 90 kDa) and CCR5 (63 kDa) in primary cultures of rat astrocytes, microglia and MLS-9 cells respectively. Conclusions: These findings suggest that cultured brain microvessel endothelial and glial cells express chemokine receptors known to participate in HIVE pathogenesis. Further work will be undertaken to determine if the activations of these receptors by viral coat proteins such as gp120 could lead to glial cell injury in vitro. Supported by a Merck-Frosst Summer Scholarship, CIHR, and the OHTN.

17 Arsenite Modulates Aryl Hydrocarbon Receptor-Regulated Gene Expression By Increasing ReactiveOxygen Species.

Sara Houlihan, Reem Elbekai, Ayman El-Kadi, Faculty OfPharmacy, University Of Alberta, Edmonton, Alberta,Canada.

Purpose: Recently, we demonstrated the ability of As³⁺ to alter the capacity of AhR ligands to induce the bioactivating phase I and the detoxifying phase II xenobiotic metabolizing enzymes. Since As³⁺ has been shown to exert its toxicity, at least partly, by the generation of reactive oxygen species (ROS), we evaluated the role of metal-induced ROS on the expression of Cyp1a1, QOR, and GST Ya. Methods: Hepa 1c1c7 cells were treated with As³⁺ (5 mM), in the presence or absence of TCDD (1 nM), an AhR ligand. Results: As³⁺ caused perturbations in glutathione redox status, a marker of cellular oxidative stress. Although it inhibited the induction of Cyp1a1 activity by TCDD, Cyp1a1 mRNA levels were potentiated. Pre-treatment with the antioxidant N-acetylcysteine (NAC) did not alter Cyp1a1 mRNA expression but completely abrogated the inhibition of Cyp1a1 activity. In parallel, when cellular GSH was depleted with L-buthionine-[S,R]-sulfoximine (BSO), Cyp1a1 mRNA expression was further potentiated whereas Cyp1a1 activity was further inhibited. On the other hand, As³⁺, alone or in the presence of TCDD, enhanced QOR and GST Ya activities and mRNA levels, an effect that was completely abrogated with NAC pretreatment and markedly potentiated in the presence of BSO. Pretreatment with the DNA transcription suppressor, actinomycin-D, abolished the induction of QOR and GST Ya mRNA levels by the metal, indicating a requirement for de novo mRNA synthesis. Conclusion: Our data clearly show that As³ induce oxidative stress which modulates Cyp1a1 activity by post-transcriptional mechanisms, but induces QOR and GST Ya activities at the transcriptional level. Acknowledgements: This work was supported by NSERC and the Faculty of Pharmacy and Pharmaceutical Sciences at the University of Alberta. S. Houlihan was the recipient of the Merck National Summer Student Research Scholarship.

18 Transcriptional And Post-Transcriptional Regulation Of Cytochrome P450 1a1 (Cyp1a1) By Heavy Metals.

Hesham M. Korashy, Ayman O.S. El-Kadi; Faculty Of Pharmacy & Pharmaceutical Sciences, University Of Alberta, Edmonton, Alberta, Canada.

Purpose: Recently, we have shown that heavy metals, particularly, mercury (Hg²⁺), lead (Pb²⁺), and copper (Cu²⁺), which are ranked highly as hazardous and toxic substances in the environment, increased the constitutive and inducible expression of Cyp1a1, a carcinogen-activating xenobiotic metabolizing enzyme, at the mRNA and protein levels. Heavy metals, however, showed inhibitory effect on the inducible Cyp1a1 activity. Yet, the mechanisms involved remain unknown. The aim of this work is to explore the molecular mechanisms involved in the modulation of Cyp1a1 by heavy metals. Methods: Murine hepatoma Hepa 1c1c7 cells were treated with Hg²⁺, Pb²⁺, or Cu²⁺ in the presence or absence of TCDD, a potent Cyp1a1 inducer. The Cyp1a1 mRNA and protein levels were measured using Northern and Western blot analyses, respectively. Results: (i) Time-dependent effect study showed that, at the constitutive level, all metals significantly induced Cyp1a1 mRNA which was apparent 3 h after treatment and reached the steady-state after 12 h. At the inducible level, Hg²⁺ and Pb²⁺ potentiated, while Cu²⁺ decreased TCDD-induced Cyp1a1 mRNA in a time-dependent manner, (ii) Cyp1a1 mRNA and protein decay assays showed that metals did not significantly alter the mRNA half-life; however, decreased the degradation rate of its protein, and (iii) The increase in Cyp1a1 mRNA by heavy metals was completely blocked by the transcriptional inhibitor, actinomycin D; whereas was further increased by the translational inhibitor, cycloheximide, implying that metals increased de novo RNA synthesis. Conclusions: Heavy metals modulate the expression of Cyp1a1 gene at the transcriptional and post-transcriptional levels. Acknowledgments: This work was supported by NSERC. H.M.K. is the recipient of CIHR / Rx&D Graduate Scholarship Award.

19 Acute Effects Of 17 -Estradiol And DHEA On 5-HT_1A And GABA_A Receptors In The Brain Of Ovariectomized Rats.

Nicolas Morin, Maryvonne Le Saux, Thérèse Di Paolo; Molecular Endocrinology Research Center (CHUL) And Faculty Of Pharmacy, Laval University, Quebec City, Quebec, Canada.

Purpose The present study sought if DHEA, a neurosteroid and precursor of estradiol, has similar activity as estradiol on 5-HT_1A and GABA_A receptors. Methods Two weeks after ovariectomy, female Sprague-Dawley rats were injected sub-cutaneous with vehicle (0.3% gelatin, 0.9% NaCl), 17ß-estradiol (80 µg/kg) or DHEA (3 mg/kg) and killed 15, 30, 45 and 60 min. after. 5-HT_1A receptor stimulation was measured using R(+)-8-OH-DPAT stimulated [³⁵S]-GTPγS binding autoradiography. Specific binding to GABA_A receptors was assessed by autoradiography with [³H]-Flunitrazepam. Results In the brain regions investigated, [³⁵S]-GTPγS specific binding remained unchanged after 17ß-estradiol treatment such as the cortex and dorsal raphe whereas DHEA decreased it in the dorsal raphe. R-(+)-8-OH-DPAT stimulated [³⁵S]-GTPγS specific binding in the cortex and in raphe decreased after estradiol or DHEA treatment. In the brain regions assayed, [³H]-Flunitrazepam specific binding was increased only by DHEA in the hippocampus. Striatal [³H]-Flunitrazepam specific binding decreased following DHEA but not 17ß-estradiol treatment. Conclusion DHEA and 17ß-estradiol decreased 5-HT_1A receptor coupling in the cortex and in the raphe nucleus whereas GABA_A receptors increased in the hippocampus and decreased in the striatum after DHEA. Hence in distinct brain regions, acutely DHEA such as 17ß-estradiol modulated brain 5-HT_1A whereas GABA_A receptors was only affected by DHEA. (Merck SSRP Awardee)

20 Growth Hormone-Releasing Peptides (GHRPs), Ligands Of CD36, Are Involved In The Activation Of NF B Transcription Factor.

Heba Muhey, Louis-Dominic Tremblay, Huy Ong, MarcServant; Faculty Of Pharmacy, University Of Montreal,Montreal, Quebec, Canada.

Purpose: Oxidized LDL (OxLDL) is known to be a major player in the development of atherosclerosis. It binds to several scavenger receptors including SR-A, CD36 and Lox-1, to induce proatherosclerotic genes. It was recently demonstrated that the set of genes up-regulated by a CD36-OxLDL interaction included type I interferon (IFN), suggesting that the IFN Regulatory Factor (IRF) family member and NF-kappa B transcription factors might be activated by OxLDL. In addition, we have recently reported that GHRPs as ligands of CD36 featured significant anti-atherosclerotic properties. Methods: In order to directly address whether IRF-3 was activated by GHRPs we performed Electromobility shift assays, Western blot analysis and Native-PAGE assays on protein extracts derived from PMA-differentiated-monocyte-derived macrophages (U937) treated with growth hormone-releasing hexapeptide, hexarelin. Results: We observed a significant induction of the Interferon-Stimulated Gene (ISG)56, a protein regulated by IRF-3 by GHRPs . However, we were not able to detect any activation of IRF-3 using these classical assays. Another possibility was the acti-vation of the transcription factor NF-kB. We verified the steady state level of the NF-kB’s inhibitor, IkBa, in cells treated with OxLDL and hexarelin. Our results showed the degradation of IkBa following OxLDL treatment. Importantly, GHRPs were shown to activate NF-kB. Conclusion: Altogether, our data suggests that GHRPs upon their binding to CD36 are able to activate the NF-kB transcription factor which is known to play a key role in the atherosclerotic development.

21 Design, Synthesis And Development Of ProdigiosinAnalogues For Breast Cancer Chemotherapy.

Jasmine Regourd, Alison Thompson; Department OfChemistry, Dalhousie University Halifax, Nova Scotia,Canada.

Purpose: Most drugs used against cancer are “anti-proliferative” rather than “anti-cancer”, meaning that they simply cause the death of both cancerous and normal cells. The distribution of anti-cancer drugs around the body is often omnipresent and side-effects are frequently severe. Our aim is to design, synthesis and development prodigiosin analogues for breast cancer chemotherapy. Method: Prodigiosin is a parent member of the 4-methoxypyrrolyldipyrromethene family of natural products isolated from certain Serratia, Streptomyces and Bacillus bacterial strains. Our interest is in the design, synthesis and evaluation of an entirely new class of molecules consisting of the prodigiosin skeleton coupled to a porphyrinogenic unit for the treatment of breast cancer. Prodigiosin is an effective inhibitor of JAK-3 which is expressed in primary tumours such as breast cancer. Breast cancer cells have elevated levels of intracellular copper ions compare to normal cells, and prodigiosin induces double-strand DNA cleavage through copper-mediated oxidative mechanisms. Prodigiosins exhibit some selectivity for breast cancer, and porphyrins are accumulated in tumour tissues. Results: A range of precursors to A- and C-ring porphyrin-appended prodigiosins analogues have been synthesized. Porphyrin appendages was attached via amide, ether and ester linkages. Derivatives with a variety of alkyl spacers are envisaged through changing the chain length between the porphyrin and the prodigiosin. Conclusion: It is hoped that porphyrin-coupled prodigiosin molecules will be selective towards breast cancer cells and result in elevated levels of selective cytotoxicity, a reduction in general toxicity and consequently in side-effects. Keywords: prodigiosin, breast cancer, porphyrins jasmine.regourd@dal.ca

22 Peceol^® Increases The Gastrointestinal Absorption Of Amphotericin B (AmpB) By Increasing Ampb Transport Through The Mesenteric Lymph Duct And Decreasing Cellular Multidrug Resistance 1 (Mdr1) mRNA And P-Glycoprotein Protein (PGP) Expression.

Verica Risovic, Kristina Sachs-Barrable, Michael Boyd, Kishor Wasan; Division Of Pharmaceutics And Biopharmaceutics, Faculty Of Pharmaceutical Sciences, The University Of British Columbia, Vancouver, British Columbia, Canada; Acute Care Animal Unit, Koerner Pavilion, University Of British Columbia, Vancuver, British Columbia, Canada.

Purpose: The purpose of this study was to determine how the incorporation of amphotericin B (AmpB) into a glyceride-rich excipient (Peceol^®) significantly increased AmpB’s gastrointestinal absorption in male Sprague Dawley rats. Methods: Following an overnight fast (12 h) rats were divided into two treatment groups and received a single-dose oral gavage (1 ml total volume) at 0700h of either:, DOC-AmpB (5 mg AmpB/kg; n=6 at each time point) or AmpB incorporated into 100% Peceol^® (Peceol-AmpB; 5 mg AmpB/kg; n=6 at each time point). Mesenteric lymph samples were obtained at 0-4 hr, 4-6 hr and 6-8 hr intervals following the oral gavage and analyzed for drug by HPLC. In a second series of studies, Caco2 cells were seeded at 10,000 cells/cm² and when the cells reached 80% confluency they were treated for one week with 0.25% (v/v) Peceol^® or media alone (control). Following treatment, cells were harvested and mdr1 mRNA and PGP protein expression were determined by RT-PCR and Western Blot analysis respectively. Results: A significantly greater amount of AmpB was transported through the mesenteric lymph duct for all the time intervals following the administration of Peceol-AmpB compared to the administration of DOC-AmpB (7.9±1.5 µg AmpB/ml of lymph for Peceol-AmpB vs. 1.5±0.5* µg AmpB/ml of lymph for DOC-AmpB over 0-8 h interval; n=6 for each treatment group; *p<0.05 vs. Peceol-AmpB). A significantly lower mdr1 mRNA (35-40% decrease compared to non-treated cells; n=6) and PGP protein expression within Caco2 cells were observed following one week of treatment with Peceol 0.25% (v/v) compared to control. Conclusions: Taken together these findings suggest that Peceol increases the gastrointestinal absorption of AmpB by increasing the amount of drug that is transported through the mesenteric lymph duct and by decreasing mdr1 mRNA and PGP protein expression resulting in lower PGP-mediated AmpB efflux. Acknowledgements: Funding was provided from a CIHR Operating Grant (MOP-49432). Portions of this work were presented at the 2004 AAPS Annual Meeting in Baltimore, MA, USA in November 2004.

23 Assessing The Antifungal Activity And Renal And Hepatic Toxicity Of Amphotericin B Lipid Complex (ABLC; Abelcet ) In Combination With Caspofungin In Experimental Systemic Asperigillosis.

Olena Sivak, Karen Bartlett, Verica Risovic, Eugene Choo, Fawziah Marra^, D. Scotty Batty, Kishor Wasan; Division Of Pharmaceutics And Biopharmaceutics, Faculty Of Pharmaceutical Sciences, The University Of British Columbia, Vancouver, British Columbia, Canada.; Acute Care Animal Unit, Koerner Pavilion, University Of British Columbia; Enzon Pharmaceuticals Inc., New Jersey, USA.

Purpose: The purpose of this study was to assess the antifungal activity and renal and hepatic toxicity of Amphotericin B Lipid Complex (ABLC; Abelcet^Ò) following co-administration of Caspofungin to rats infected with Asperigillus fumigatus. Methods: Asperigillus fumigatus inoculum (1.3-2.3 x 10⁷ colony forming units [CFU]) was injected via the femoral vein; 48 h later male albino Sprague-Dawley rats (350-400 g) were administered either a single intravenous (IV) dose of Fungizone^Ò (1 mg AmpB/kg), ABLC (1 or 5 mg AmpB/kg) or an equivalent volume of normal saline (vehicle control) once daily for 4 days. Rats were further randomized into groups to receive 3 mg/kg Caspofungin or physiologic saline IV once daily for 4 days.. To assess antifungal activity Brain, Lung, Heart, Liver, Spleen and Kidney sections were homogenized with normal saline (2 ml) and a 0.1-ml aliquot was spread plated onto a Sabourand dextrose agar plate. The plates were incubated for 48 h at 37⁰ C, at which time the numbers of CFU were determined and corrected for tissue weight. To assess renal and hepatic toxicity, serum creatinine and aspartate aminotransferase levels were determined. Results: Fungizone and ABLC at a dosing regiment of 1 mg/kg i.v. once daily for four consecutive days and Caspofungin at a dosing regiment of 3 mg/ kg i.v. once daily for four consecutive days had similar effectiveness in decreasing the total number of Aspergillus Fumigatus CFUs found in all organs analyzed compared to non-treated controls. A combination of ABLC (1 mg/kg i.v. x 4 days) and Caspofungin (3 mg/kg i.v. x 4 days) significantly decreased the total number of Aspergillus fumigatus CFUs found in all organs analyzed than Caspofungin therapy alone compared to non-treated controls. ABLC at a dosing regiment of 5 mg/kg i.v. once daily for four consecutive days was more effective in decreasing the total number of Aspergillus fumigatus CFUs found in all organs analyzed compared to Fungizone and ABLC at 1 mg/kg and Caspofungin at 3 mg/kg. However, a combination of ABLC (5 mg/kg i.v. x 4 days) and Caspofungin (3 mg/kg i.v. x 4 days) was not more effective than ABLC at 5 mg/kg or the combination of ABLC at 1 mg/kg and Caspofungin 3 mg/kg in reducing the total number of Aspergillus fumigatus CFUs compared to controls . Except for non-treated infected control rats, none of the treatment groups tested displayed a greater than 50% increase in serum creatinine concentrations from baseline. In addition, only Fungizone at a dosing regiment of 1 mg/kg i.v once daily for four consecutive days displayed a greater than 50% increase in AST concentration from baseline. Conclusions: Taken together, these findings suggest that ABLC at 5 mg/kg once daily x 4 days appears to be the best therapeutic choice in this animal model. Acknowledgements: Funding was provided with a grant-in-aid from Enzon Pharmaceuticals Inc. Portions of this work were presented at the 2004 Annual AAPS Meeting in Baltimore, MA, USA in November 2004.

24 Synthesis Of Non-Natural L-Threonine DerivativesAnd Their Incorporation Into The Oxazolone Ring Of Jadomycin.

B. David Jakeman, Spring Farrell, College Of Pharmacy,Dalhousie University, Halifax, Nova Scotia, Canada.

Purpose Many therapies for cancer rely strongly on natural products which have anti-tumor activity. Due to the increasing incidence of drug resistance, and the severity of side effects associated with most of the current chemotherapy drugs, one aspect of our research has been driven towards finding compounds with comparable anti-tumor activities and lower cyto-toxicities. To this end we have investigated the formation of novel jadomycins by rationally altering the growth media. Methods Non-natural amino acids were synthesized and fed to cultures of Streptomyces venezuelae ISP5230. Cultures were extracted and subsequently analyzed by electrospray ionization mass spectrometry (ESI-MS). Results O-methoxy-L-threonine and O-methoxy-methylene-L-threonine were synthesized and fed to S. venezuelae ISP5230. Analysis by ESI-MS of the culture extracts indicated unequivocally that the non-natural amino acids had been incorporated into the jadomycin aglycone. Conclusion Our discovery that nonnatural amino acids are incorporated into the aglycone of jadomycin has opened a new avenue of jadomycin research to understand the scope of the insertion reaction and develop novel jadomycins with improved bioactivity. Keywords Natural products, anticancer and antibacterial activity, structure-activity.

25. Activity Of Actrostaphylos Uva-Ursi On Cytochrome P450 Family -Mediated Metabolism And P-Glycoprotein Function In Human Cell Lines.

C.H. Yu, B.M. Chauhan, J.T. Arnason,^. R.J. Marles, A. Krantis, I. Scott, Brian C. Foster; Centre For Research In Biopharmaceuticals And Biotechnology, University Of Ottawa, Ottawa, Ontario, Canada; Natural Health Products Directorate, Health Canada, Ottawa, Ontario, Canada; Therapeutic Products Directorate, Health Canada, Ottawa, Ontario, Canada.

Purpose: Natural Health Products (NHPs) may affect drug metabolism enzymes and transport proteins potentially affecting the safety and efficacy of the drug or other NHPs. This study was undertaken to characterize the effect of Actrostaphylos uva-ursi cytochrome P450 family -3A4/5/7, 2C19, and CYP19 -mediated metabolism, and P-glycoprotein (Pgp) transport. Methods: Bulk (3) and capsulated (2) A. uvaursi was obtained from commercial outlets. The capsules were batched and herbal samples were ground to a common consistency. Aqueous and methanol extracts (25 mg/ml in DW and 5mg/ml in methanol) were prepared fresh 3A4/5/7, 2C19 and 19-mediated metabolism determined using in vitro bioassays. Pgp transport function was determined with rhodamine uptake test in human monocytes (THP-1) and human Caco-2 cells. All products were analyzed by HPLC for arbutin, gallic acid, myrcitrin, isoquercetin. Results: Our data indicates that both aqueous and methanol extracts of all five A. uva-ursi products show high inhibition, with the exception of the methanolic extracts against 3A4 and 19 which had low to moderate activity. The aqueous extracts of A. uva-ursi show a inhibitory effect at 1 hr and an inductive effect at 18 hrs on uptake for both cell lines. With the exception of gallic acid, similar levels of the examined biomarkers were found in the five products. Conclusions: These herbal products have pharmacological properties including the potential capacity to affect drug safety and efficacy. Further studies are warranted against a wider range of cytochrome P450 isozymes and to determine if these effects are clinically significant.

26 Preparation Of Monodispersed Polymeric Micellar Formulations For Efficient Encapsulation Of Cyclosporine A (CyA) Through A Co-Solvent Evaporation Method.

Sara Elhasi, Rashida Gulamhusein, Hamidreza Montazeri Aliabadi, Afsaneh Lavasanifar; Faculty Of Pharmacy And Pharmaceutical Sciences, University Of Alberta, Edmonton, Alberta, Canada

Purpose: To investigate the effect of micellization procedure on the particle size and capacity of drug loading in micelles of poly(ethylene oxide)-block-poly(caprolactone) (PEO-b-PCL). Methods: PEO-b-PCL block copolymers with PCL average molecular weights of 5000, 13000 and 24000 gmol^-1 were synthesized by ring opening polymerization of å-caprolactone (different feed ratios) using methoxy polyethylene glycol (5000 gmol^-1) and stannous octoate as initiator and catalyst, respectively. Prepared block copolymers were characterized for their average molecular weights and polydispersity by ¹H NMR and gel permeation chromatography (GPC). Micelles of PEO-b-PCL block copolymers were prepared through a co-solvent evaporation method using tetrahydrofuran, acetone and acetonitile as the organic co-solvent phase. Cyclosporine A (CyA), a poorly water soluble peptide, was loaded in PEO-b-PCL micelles with an identical method to the self assembly process. Prepared micelles were characterized for their average diameter and polydispersity of the micellar population as well as drug content by dynamic light scattering and HPLC, respectively. The effect of self assembly conditions such as polymer concentration, type of organic solvent, the organic to aqueous phase ratio and their order of addition on the micellar size, polydispersity, and encapsulation efficiency were determined. Results: The size of self assembled structures was shown to be affected by the polarity of the co-solvent system. Addition of organic to aqueous phase at a low organic to aqueous phase ratio, was shown to be the more effective approach in obtaining PEO-b-PCL micelles with an average diameter of less than 100 nm and minimum polydispersity. Interestingly, the same approach resulted in a higher aqueous solubility (2 mg/mL) and encapsulation efficiency (76%) for CyA in PEO-b-PCL based nanocarriers. Conclusion: Self assembly conditions may be optimized to achieve PEO-b-PCL nanoparticles of appropriate size, narrow polydispersity, and maximum encapsulation efficiency. Acknowledgments: This study was supported in part by Natural Sciences and Engineering Council of Canada (NSERC) (Grant no. G121210926). SE was supported by Libyan Government scholarship. RG was supported by Rx & D HRF/CIHR summer student scholarship. HM was supported by Rx & D HRF/CIHR graduate student research scholarship.

27 The Effect Of Block Copolymer Structure On The Internalization Of Polymeric Micelles By Human Breast Cancer Cells.

Abdullah Mahmud, Afsaneh Lavasanifar; Faculty Of Pharmacy And Pharmaceutical Sciences, University Of Alberta, Edmonton, Alberta, Canada.

Purpose: To assess the effect of hydrophilic/hydrophobic block chain lengths on the internalization of poly(ethylene oxide)block-poly(å-caprolactone) (PEO-b-PCL) micelles by cancer cells. Methods: PEO-b-PCL block copolymers with varied PEO and PCL chain lengths were synthesized, assembled to polymeric micelles and labeled with a hydrophobic fluorescent probe (DiI) by physical encapsulation method. Confinement of the fluorescent probe within the micellar structure was evidenced following DiI transfer to lipid vesicles. The extent of micellar uptake by cancer cells was investigated through their incubation with MCF-7 cells followed by measurement of the fluorescent emission intensity of DiI (ë=550 nm) in the separated lysed cells. The mechanism of micellar uptake was investigated by pretreatment of MCF-7 cells with chlorpromazine (8 ìg/mL) and cytochalasin B (5 ìg/mL). Results: PEO-b-PCL micelles lowered the extent and rate of DiI internalization by cancer cells. For polymeric micelles with 5000 g.mol^-1 of PCL and varied PEO molecular weights of 2000, 5000 and 13000 g.mol^-1, maximum uptake was observed at 5000 g.mol^-1 of PEO. For polymeric micelles with 5000 g.mol^-1 of PEO and varied PCL molecular weight of 5000, 13000 and 24000 g.mol¹, maximum uptake was observed at 13000 g.mol^-1 of PCL. Cytochalasin B and chlorpromazine reduced the cellular uptake of PEO-b-PCL micelles, pointing to the involvement of macropinocytosis and clathrin mediated endocytosis mechanisms in the uptake of polymeric micelles by cancer cells. Conclusion: Chemical tailoring of the core/shell forming blocks may be used to design polymeric micelles with optimal properties for individual drug targeting requirements at a cellular level.

28 Polymeric Micelles For The Solublization And Delivery Of Cyclosporine A: Pharmacokinetics And Biodistribution.

Hamidreza Montazeri Aliabadi, Dion Brocks, AfsanehLavasanifar; Faculty Of Pharmacy And PharmaceuticalSciences, University Of Alberta, Edmonton, Alberta,Canada.

Purpose: To assess the potential of methoxy poly(ethylene oxide)-b- poly(e-caprolactone) (PEO-b-PCL) micelles to modify the pharmacokinetics and tissue distribution of cyclosporine A (CsA). Methods: PEO-b-PCL block copolymers were synthesized by ring opening polymerization of å-caprolactone using methoxy polyethylene glycol (5000 gmol^-1) and stannous octoate as initiator and catalyst, respectively. An optimized method has been developed to achieve PEO-b-PCL micelles of < 100 nm capable of efficient drug encapsulation. Drug-loaded PEO-b-PCL micellar solutions in isotonic medium were prepared and administered intravenously to healthy Sprague-Dawley rats (catheterized under halothane anesthesia into the right jugular vein the day before the experiment). In the pharmacokinetic study, whole blood samples were collected and assayed for CsA, and resultant pharmacokinetic parameters of the polymeric micelle formulation were compared to its commercially available intravenous formulation (Sandimmune^®). For biodistribution study, the same polymeric micelles and Sandimmune^® were administered intravenously and blood, plasma, and tissues were harvested and assayed for CsA. Results: In the pharmacokinetic assessment, a 6.1 fold increase in the area under the blood concentration versus time curve (AUC) was observed for CsA when given as polymeric micellar formulation as compared to Sandimmune^®. The volume of distribution and clearance of CsA as PEO-b-PCL formulation were observed to be 10.0 and 7.6 fold lower, respectively, compared to the commercial formulation. No significant differences in t_1/2 or MRT could be detected. In the biodistribution study, analysis of tissue samples indicated that the mean AUC of CsA in polymeric micelles was lower in liver, spleen and kidney (1.4, 2.3 and 1.4-fold, respectively). Similar to the pharmacokinetic study in these rats the polymeric micellar formulation gave rise to 5.6 and 4.8-fold increases in the AUC of CsA in blood and plasma, respectively. Conclusion: Our results show that PEO-b-PCL micelles can effectively solubilize CsA, at the same time confining CsA to the blood circulation and restricting its access to tissues such as kidney, perhaps limiting the onset of toxicity. Acknowledgements: The authors would like to thank Parvin Mahdipoor, Shahram Ala and Tara Spencer for technical assistance. DRB was funded by Canadian Institutes of Health Research (Grant MOP-67169). HM was supported by Rx & D HRF/CIHR graduate student research scholarship.

29 Pharmacokinetics Of Amiodarone In Combination With Ketoconazole.

Anooshirvan Shayeganpour, Dion Brocks; Faculty Of Pharmacy And Pharmaceutical Sciences, University Of Alberta, Edmonton, Alberta, Canada

Purpose: Amiodarone (AM) is a class III antiarrhythmic drug that undergoes extensive metabolism in liver and intestine by CYP3A, and which is a substrate of P-glycoprotein (P-gp). In this study we investigated the effect of ketoconazole (KTZ), a presumed inhibitor of CYP3A and P-gp, on the plasma pharmacokinetics of AM in the rat. Method: Single doses of AM were administered to four groups of jugular vein-cannulated Sprague-Dawley rats as either iv injection (25 mg/kg, n=12) or oral gavage (50 mg/kg, n=10), with and without oral KTZ. After dosing, serial plasma samples were obtained, followed by analysis using a validated HPLC method for AM and desethylamiodarone (DEA). Noncompartmental pharmacokinetic analysis was performed on the plasma concentration vs. time data. Results: In rats given iv AM, KTZ caused significant increases of 60% in mean AUC_0-inf (CO, 18.5; KTZ, 29.6 mg×h/L), and significant decreases in mean clearance (CO, 1390; KTZ, 889 mL/h/kg) and mean Vdss (CO, 40.9; KTZ 20.2 L/kg). After oral AM, a 206% higher mean AUC_0-inf (CO, 13.6; KTZ, 28.0 mg×h/L) was observed in KTZ treated rats. No significant difference was observed in oral AM mean t_max after KTZ administration. Mean C_max of oral AM significantly increased by 137% in KTZ treated rats (CO, 723; KTZ, 1716 ng/mL). Oral bioavailability of AM increased 27% (CO, 0.37; KTZ, 0.47) after KTZ. KTZ did not significantly influence the t_1/ of AM after iv or oral AM. DEA concentrations were low in all 2 rats. Conclusion: The pharmacokinetics of AM were markedly affected by KTZ. The changes are consistent with a KTZ-associated reduction in efficiency of CYP3A and/or P-gp activity. Funded by CIHR.

30 Effect Of Some Formulation Variables On TheProperties Of Ibuprofen Prolonged Release Microspheres.

Noushin Bolourtchian, Reyhaneh Astaneh; ShaheedBeheshti University Of Medical Sciences, Tehran, Iran;University Of Alberta, Edmonton, Alberta, Canada.

Purpose: The aim of this investigation was to determine the influence of formulation variables on the in vitro drug release and micromeritic properties of the microspheres prepared by solvent evaporation method. Methods: Prolonged release microspheres of ibuprofen with Eudragit RS were prepared using an o/w emulsion solvent evaporation technique. Chloroform and gelatin were used as the polymer solvent and emulsifier, respectively. The effects of three variables including the drug: polymer ratio, gelatin concentration and the emulsion internal phase viscosity on the drug content and particle size distribution of microspheres were examined. The in vitro drug release rate from prepared microspheres and the release kinetics were also studied. Results: The results dem-onstrated that although the drug: polymer ratio did not affect the size distribution significantly, but had a considerable effect on the drug content of microparticles. However, particle size distribution of microspheres was more dependent on the gelatin (emulsifier) concentration and the internal phase viscosity. Based on the results, most prepared microspheres showed a burst effect in the first two hours of drug release followed by a slower release rate. The presence of uncovered drug crystals on the surface of microparticles could explain this burst effect. The kinetics evaluation of release profiles showed that Higuchi equation is the main model fitted for the data. Conclusion: Microspheres with different properties and drug release rates could be obtained by using various formulation variables in the solvent evaporation technique. Keywords: Ibuprofen, Microsphere, Formulation Variables

31 Preparation Of An Injectable Implant For Peptide Delivery: Effect Of Polymer Molecular Weight On Its Release Behaviour.

Reyhaneh Astaneh, Mohamad Erfan, Hamid Mobedi, Hamidreza Moghimi; Shaheed Beheshti University Of Medical Sciences, Tehran, Iran; Iran Polymer And Petrochemical Institute, Tehran, Iran; Dentistry/Pharmacy Center, University Of Alberta, Edmonton, Alberta, Canada.

Purpose: Injectable implants, are novel drug delivery systems that look very promising in protein delivery. These systems are injectable polymeric solutions that solidify after injection, and release their drug in a controlled manner. The main problem related to these systems is usually their burst effect. In this study the effect of polymer molecular weight on release behavior of an injectable implant containing Luprolide Acetate (LA) (a model peptide) was examined. Methods: Poly (lactideco-glycolide) 50:50 (PLGA 50:50) with molecular weights of 12000 and 48000 were used in this investigation. Polymeric solutions containing 3 % w/w LA were prepared and their release behavior was studied using an in-house model diffusion system especially designed to allow study of the release behavior of the system in both liquid and solid phases. Experiments were performed at 37 °C for one month. Results: Results showed that molecular weight affects burst effect significantly. However, the steady- state release rate looked nearly the same, regardless of molecular weight. The amount of drug release at first 24 hours for lower MW polymer, 32% ± 0.485% (X± SD, n=3) was significantly (P<0.05) higher than that of higher MW, 13% ± 0.078% (X ± SD, n=3). This should be due to increased polymer chain interaction and therefore, reduced diffusion coefficient of drug in higher molecular weight polymer. Conclusion: These data show that it is possible to control the burst effect by optimizing MW of the polymer which is used for preparation of injectable implant solution, while keeping the steady-state release rate constant. Key words: Injectable Implant, Burst effect, Peptide and Controlled Drug Deivery Systems

32 Thwarting Bacterial Resistance Through NovelEnzymatic Targets.

Charles Borissow, Joseph Lam, David Jakeman; CollegeOf Pharmacy, Dalhousie University, Halifax, Nova Scotia,Canada; Department Of Microbiology, University OfGuelph, Guelph, Ontario, Canada.

Purpose The number of antibiotic resistant strains of bacteria are on the increase and a novel approach to targeting these organisms is required. RmlA is an enzyme essential for the production of L-rhamnose (a sugar vital for the viability of many bacteria) and is one such target. We aim to synthesize a range of novel sugar nucleotide diphosphonate analogues and test them for inhibition of RmlA. We will use this information, together with modeling studies, to develop a structure-activity relationship allowing the production of more potent inhibitors. Methods A series of bisphosphonate analogues have been prepared from tetraisopropylmethylene bisphosphonate by nucleophilic substitution at the methylene carbon. The deprotected bisphosphonates were coupled to glucose and rhamnose and subsequently thymidine using a number of strategies including: (i) Traditional and microwave enhanced Mitsunobu coupling; (ii) Glycosylation from the corresponding glycosyl iodide; (iii) Nucleophilic substitution at 5’-position of thymidine; (iv) via In situ generation of chlorophosphonates The compounds were tested against recombinant RmlA cloned from Pseudomonas aeruginosa Results The novel chemical synthesis of sugar nucleotide bisphosphonates will be presented together with selected biological activity data. The biological results will be rationalized through structural models based on crystallographic studies. Conclusions The synthesis and biological evaluation of a series of novel sugar nucleotide bisphosphonates gives us greater insight into the structural requirements necessary for inhibition of RmlA. Novel approaches for the development of lipophilic pro-drug forms of the sugar nucleotide bisphosphonates will be presented.

33 Improved Piroxicam Dissolution Through Inclusion Complex With Dimethyl-ß-Cyclodextrin.

Thitima Chuchome, Nattha Kaewnopparat, Sanae Kaewnopparat, Lawan Sripong, Helmut Viernstein; Department Of Pharmaceutical Chemistry, Department Of Pharmaceutical Technology, Faculty Of Pharmaceutical Sciences, Prince Of Songkla University, Hat-Yai, Songkhla, Thailand; Department Of Pharmaceutical Chemistry, Faculty Of Pharmaceutical Sciences, Silpakorn University, Nakorn Prathom, Thailand; Institution Of Pharmaceutical Technology And Biopharmacy, University Of Vienna, Vienna, Austria.

PURPOSE. To enhance the solubility and dissolution rate of piroxicam via complexation with dimethyl-ß-cyclodextrin (DMßCD) and to characterize the physicochemical properties of piroxicam-dimethyl-ß-cyclodextrin systems. METHODS. Inclusion complexes of piroxicam and dimethy-ß-cyclodextrin in 1:1 and 1:2 molar ratio molar ratios were prepared by kneading, coevaporation, and freeze-drying method. The solubility, dissolution, differential scanning calorimetry, X-ray powder diffractometry, FT-IR spectroscopy were studied. RESULTS. The solubility of piroxicam increases linearly with the increasing concentration of DMßCD. This solubility diagram can be classified as type A_L as according to Higuchi-Connors equation which the apparent stability constant of this complex was found to be 183.4 M^-1. From FITR studies, N-H or O-H stretching vibration of piroxicam was not detected in the freeze-dried system which is X-ray amorphous, suggesting that there was an interaction between drug and DMßCD. The kneaded system and coevaporated system also show some diffraction peaks, indicating the absence of an amorphous state that may be attributed to the recrytallization of drug during the preparation process. The dissolution profiles showed that inclusion complexes exhibited higher rates of dissolution than the physical mixtures and pure drug. The freeze-dried systems in 1:1 and 1:2 molar ratio exhibited the fastest dissolution followed by 1:2 and 1:1 inclusion complexes prepared by kneading method and the increases of 28.2-, 28-, 26.6- and 26-fold in drug dissolution within 3 minutes were observed, respectively, compared to pure drug. CONCLUSION. The inclusion complex prepared by freeze-drying technique was achieved complexation which exhibited the fastest dissolution rate.

34 Physicochemical Characterization Of Fast Release Curcuminoids-Polyvinylpyrrolidone K-30 Coprecipitates.

Nattha Kaewnopparat, Sanae Kaewnopparat, Amaravadee Jangwang, Daungkhae Maneenaun, Thitima Chuchome^, Philip Molyneux, Department Of Pharmaceutical Technology; Department Of Pharmaceutical Chemistry, Faculty Of Pharmaceutical Sciences, Prince Of Songkla University, Hat Yai, Thailand.

PURPOSE. To increase curcuminoids solubility and dissolution by solid dispersion technique with polyvinylpyrrolidone K-30 (PVP K-30) as a carrier. The solubility, dissolution and physicochemical properties were evaluated. METHODS. Curcuminoids-PVP K-30 coprecipitates in weight ratios of 1:2, 1:4, 1:5, 1:6 and 1:8 were prepared by solvent method. The solubility, dissolution, differential scanning calorimetry, X-ray powder diffractometry and FT-IR spectroscopy were studied. RESULTS. The solubility of curcuminoids-PVP K-30 coprecipitates was markedly higher than pure curcuminoids and physical mixtures. The O-H stretching vibration of curcuminoids in all coprecipitates disappeared, indicating that the intermolecular hydrogen bonding between curcuminoids and PVP K-30 occurred. The X-ray amorphous was obtained in all coprecipitates. So, PVP K-30 might inhibit the crystal growth of curcuminoids during the evaporation process and the hydrogen bonding between curcuminoids and PVP K-30 would inhibit curcuminoids crystallization. The DSC thermograms of coprecipitates indicated the formation of an amorphous solid solution. All coprecipitates exhibited faster dissolution rate than that of pure curcuminoids and physical mixtures. The 1:6 and 1:8 curcuminoids-PVP K-30 coprecipitates gave the fastest dissolution rate which curcuminoids completely dissolved within 15 minutes. Storage of 1:6 curcuminoids-PVP K-30 coprecipitates at ambient temperature for one year did not significantly change in the dissolution and curcuminoids also still in the amorphous form. CONCLUSIONS. The solubility and dissolution of curcuminoids are markedly enhanced by solid dispersion technique. This may be due to the formation of soluble complex and amorphous solid solution. The dissolution profile and the amorphous form of 1:6 curcuminoids-PVP K30 coprecipitates did not change after one year of storage.

35 Piroxicam Permeation Across Caco-2 Monolayers: Comparison Between Pure Drug And Solid Dispersion.

Suthimaln Ingkatawornwong, Nattha Kaewnopparat, Vimon Tantishaiyakul, Shinji Yamashita, Department Of Pharmaceutical Technology; Department Of Pharmaceutical Chemistry, Faculty Of Pharmaceutical Sciences, Prince Of Songkla University, Hat Yai, Thailand; Faculty Of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka, Japan.

PURPOSE. To evaluate the permeation of pure piroxicam compared to piroxicam solid dispersion across Caco-2 monolayers instead of in vivo study by using side-by-side diffusion chamber which develop for studying the dissolution-permeation relationships from oral solid dosage forms. METHODS. Piroxicam-PVP K-30 solid dispersion in ratio of 1:4 was prepared by solvent method. Dissolution of pure piroxicam and piroxicam-PVP K-30 solid dispersion were determined using the chamber containing 8 ml of transport medium pH 6.5 as the dissolution medium. The permeation studies of piroxicam-PVP K-30 solid dispersion compared to pure piroxicam across Caco-2 monolayers were determined. The permeability coefficients of piroxicam across Caco-2 monolayers using transport medium pH 6.5 or transport medium plus 4.5%w/v of bovine serum albumin were also evaluated. RESULTS. The dissolution studies indicated that the dissolution rate was markedly increased in the solid dispersion compared with pure piroxicam. The percentage of piroxicam dissolved in 15 minutes was about 10 fold higher than pure piroxicam. The dissolution/permeation studies showed that the permeation of piroxicam across Caco-2 monolayers; expressed as percentage of dose; from solid dispersion was higher than pure piroxicam which about 10-fold higher in the first 15 minutes. The permeability coefficients of piroxicam across Caco-2 monolayers using transport medium pH 6.5 or transport medium plus 4.5%w/v of bovine serum albumin were 6.86x10^-5 and 1.93x10^-4 cm/sec respectively. CONCLUSIONS. There was marked difference in the piroxicam permeation across Caco-2 monolayers compared between pure piroxicam and its solid dispersion. It was a good relationship between piroxicam dissolution and permeation across Caco-2 monolayers.

36 Prediction Of Pgp-ATPase Interaction And Rhodamine 123 Efflux Inhibitory Activities Of Propafenone Analogs Using PLS Analysis.

Vimon Tantishaiyakul, Wibul Wongpuwarak, Faculty Of Pharmaceutical Sciences, Prince Of Songkla University, Hat-Yai, Thailand.

Purpose. A method for the prediction of ATPase interaction and rhodamine 123 efflux inhibitory activity of propafenone analogs using multivariate partial least squares projections (PLS) analysis was investigated. Methods. Three dimensional structures of compounds were built using Chem3D Ultra ver. 7 software and molecular calculations were directly performed using the Gaussian 03 W program on the Chem3D window. Geometry optimization was performed initially by molecular mechanics force field and subsequently using the AM1 Hamiltonian of a semiempirical quantum chemical method. The obtained geometries were then optimized on the basis of the ab initio Hartree-Fock method at the 3-21G level. Molecular descriptors were calculated using Dragon and ChemSketch software. Results. Good statistical models were observed for both ATPase interactions and efflux inhibitory activities, resulting in R² values ranging from 0.894 to 0.990 and Q² values ranging from 0.858 to 0.969. The root mean squared error of prediction (RMSEp) values for the external test sets ranged from 0.189 to 0.253. Moriguchi octanol-water partition coefficient (MLOGP) was not selected as a descriptor for ATPase interaction but it was a significant descriptor for efflux inhibitory activity. Conclusion. Since different molecular properties were selected to describe ATPase interaction and rhodamine 123 efflux inhibitory activity, together with the relatively low correlation between log(1/K_a) and log(1/EC₅₀), R² of 0.69, these two properties may not be well associated and other factors besides ATPase interaction are probably involved with the efflux inhibition. Keywords: Propafenone, PLS, ATPase interaction, Rhodamine 123 Efflux, P-glycoprotein The manuscript of this work is accepted for publication in Journal Molecular Structure: THEOCHEM

37 Controlled Release Of Alendronate From Polymeric (PLGA/PEG) Films.

Karen Long, John Jackson, Ke Duan, Rizhi Wang, Helen Burt; Faculty Of Pharmaceutical Sciences, University Of British Columbia, Vancouver British Columbia, Canada.

Bisphosphonates (such as alendronate) increase net bone density by decreasing bone resorption. Bisphosphonate films and coatings on orthopaedic implants are being explored to determine whether local administration of bisphosphonates may enhance bone growth. Purpose: To characterize the encapsulation and drug release characteristics of alendronate in biodegradable films made from poly(lactide-co-glycolide) (PLGA). The specific aims were to develop an HPLC assay for alendronate and to investigate the effect of blending polyethyleneglycol (PEG) in PLGA films on drug release rates. Methods: Polymer films were prepared by suspending finely ground sodium alendronate in a 10% (w/v) polymer solution in dichloromethane containing 0, 10, 20 or 30% PEG 600. 100 ìL aliquots were dispersed onto 1 cm² Teflon^® squares and dried. For drug release studies, films (n=5) were suspended in PBS at 37°C with agitation. Sample aliquots were withdrawn at regular time points. The alendronate amino group was labelled with fluorescamine at pH 10 and quantitated by reverse phase HPLC. Results: The analytical method was validated over a concentration range of 1-500 ìg/mL. Alendronate release from PLGA films was characterized by an initial burst phase (Day 1) followed by slower, more sustained release over 9 days. Films containing PEG released approximately 90% of alendronate within 3 days, whereas films composed of PLGA alone had released only ~45% of the drug over the same period. Conclusion: PLGA films offer a biocompatible, biodegradable matrix suitable for the controlled release of alendronate. Incorporation of PEG into PLGA films increases the alendronate encapsulation efficiency and rate of drug release. Three Keywords Bisphosphonate, controlled release, PLGA

38 Evaluation Of Correlation Between Viscosity, Pump Spray Type And Droplet Size Distribution Of Nasal Sprays Using Sympatec Sprayer^®.

Payam Moslemi, Leila Mohammadyari Fard, Masoomeh Nabavi; R&D Department, Jaber Ebne Hayyan Pharmaceutical Co., Tehran, Iran.

Purpose. Considering factors such as droplet size distribution (DSD) and viscosity in preformulation studies of nasal sprays is really important because of their effect on deposition pattern and residence time of drug in the nasal cavity. In this study the effect of viscosity and type of pump spray on DSD of final formulation has been investigated. Methods. Different concentrations of Avicel RC-591 was dispersed in distilled water to achieve formulations with different viscosities. The prepared formulations were filled into the bottles with different commercially available spray pumps. DSD of nasal sprays was determined by laser diffraction using a Sympatec Sprayer. Data were subjected to statistical analysis of variance (ANOVA). Probability values of <0.05 were considered as statistically significant. Results. The results show that an increase in viscosity causes an increase in droplet mass median diameter. It seems that the samples with the viscosities higher than 15 cps which produce larger droplet sizes and non-uniform spray clouds are not suitable. Also different pump sprays cause different DSD for the same product which is related to the viscosity of preparation (samples with higher viscosity cause greater difference in DSD). Conclusion. The factors such as type of pump spray and viscosity of final formulation which affects the DSD of a nasal spray should be considered as important parameters in development of such platforms.

39 Quantitative Evaluation Of Ethylcellulose And Investigation Of The Drug Release Mechanism From Ethylcellulose Coated Multiparticulate Systems.

Pankaj Rege, Kurt Fegely, Ali R. Rajabi-Siahboomi, Colorcon, Modified Release Technologies, Colorcon, West Point, Pennsylvania, USA.

Purpose: The ability to quantify ethylcellulose content can be a powerful technique during formulation development to assist scale up of process parameters. The purpose of this study was two-fold: (1) Analytically quantify ethylcellulose deposited from an aqueous ethylcellulose dispersion on drug loaded Nu-pariels, and (2) Understand mechanism of drug release from barrier coated systems using drugs of different solubilities. Methods: Sugar spheres were drug layered using a Glatt GPCG3 and then coated with Surelease in the Wurster unit of the GPCG-3. During the coating of the ethylcellulose film, the product bed temperature was maintained at 40-42ºC by controlling the inlet air temperature. For the ethylcellulose content evaluation, coated beads were ground and the ethylcellulose was extracted into tetrahydrofuran. The extracted samples were analyzed by GPC/HPLC technique. Results: There was a strong correlation between the actual and theoretical ethylcellulose content (r² = 0.9999). Release rate of drug decreased with increasing Surelease applied. Interestingly, in case of chlorpheniramine maleate release, Surelease weight gain affected the release profile of drug, by affecting the onset of drug release. However, once drug release was initiated, the release profiles were similar irrespective of the amount of Surelease applied. Conclusions: Adjusting the Surelease film thickness allowed altering the onset of drug release, providing controlled release of the drug. Success in the analytical quantification of ethylcellulose on may be used as a tool to validate process efficiency of an aqueous ethylcellulose dispersion coating system.

40 Development And Characterization Of Ketorolac Tromethamine Loaded Polylactic-Co-Glycolic Acid Microspheres For Parenteral Delivery.

V Sinha, Aman Trehan, University Institute OfPharmaceutical Sciences, Panjab University, Chandigarh, India.

Purpose: To prepare ketorolac tromethamine loaded microspheres of poly lactic-co-glycolic acid (PLGA 85/15) and its blends with polycaprolactone (PCL) Methods: Ketorolac tromethamine was encapsulated in poly lactic-co-glycolic acid (PLGA 85/15) microspheres by o/w emulsion solvent evaporation technique. In order to tailor the release profile of drug for several days’ blends of PLGA 85/15 were prepared with polycaprolactone (PCL) in different ratios. Results: Higher encapsulation efficiency was obtained with microspheres made with pure PLGA 85/15. These formulations were characterized for particle size analysis by Malvern mastersizer, which revealed particle size in range of 12-22 micron for microspheres made with polymer blends and 8 micron in case of pure PLGA 85/15. Surface topography was studied by scanning electron microscopy and differential scanning calorimetric (DSC) studies were also conducted to study any drug polymer interactions. The results revealed that in case of microspheres made with 1:3 (PLGA85/15: PCL) almost the entire drug was released in a week whereas in batches made with pure PLGA85/ 15 and 3:1 (PLGA 85/15: PCL) more than 80% of the drug was released in 60 days as compared to 96% in 60 days in case of 1:1 (PLGA85/15: PCL). In vivo pharmacodynamic studies were also carried out which revealed that KT microspheres were able to sustain release for up to 5 days. Conclusion: From the study it was concluded that with careful selection of different polymers and their combinations we can tailor the release of ketorolac tromethamine in such a way that its levels are maintained for long periods so that this can obviate the need for painful injections given number of times per day.

41 Nanoengineered Liposome-Based Multimodal Contrast Agent For Computed Tomography And Magnetic Resonance Imaging.

Jinzi Zheng, Gregory Perkins, David Jaffray, Christine Allen; Department Of Medical Biophysics, University Of Toronto, Toronto, Ontario, Canada; Department Of Radiation Physics, Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada; Department Of Radiation Oncology, University Of Toronto, Toronto, Ontario, Canada; Department Of Pharmaceutical Sciences, University Of Toronto, Toronto, Ontario, Canada; Department Of Pharmaceutical Sciences, University Of Toronto, Toronto, Ontario, Canada.

Purpose: Recent developments in the field of multimodality imaging have created a need for contrast agents that are effective for use across imaging modalities. In the context of radiation therapy, the development of a multimodal contrast agent that provides prolonged enhancement in computed tomography (CT), magnetic resonance (MR) imaging and cone-beam CT would facilitate tumour localization and delineation for treatment planning, delivery and follow-up. The objective of this study is to develop and evaluate the feasibility of em-ploying nano-sized stealth liposomes to encapsulate conventional iodine and gadolinium based contrast agents for CT and MR imaging applications. Methods: The physico-chemical characteristics of a liposome-encapsulated iohexol and gadoteridol formulation were assessed in terms of agent loading efficiencies, vehicle size and morphology, in vitro stability and release kinetics. The imaging properties of the liposome formulation were assessed based on T₁ and T₂ relaxivity measurements, and in vivo CT and MR imaging in a lupine animal model. Results: The average agent loading levels achieved were 26.5 ± 3.8 mg/mL of iodine and 6.6 ± 1.5 mg/mL of gadolinium. The mean diameter of the liposomes was found to be 74.4 ± 3.3 nm. Evaluation of the in vitro release profile of the encapsulated agents revealed that less than 10% of the total iohexol and gadoteridol were released over a 15-day incubation period in physiological buffer at 37ºC. Significant in vivo contrast enhancement was achieved and maintained in both CT and MR for the liposome formulation over 3.5 hours (81.4 ± 13.05 differential Hounsfield units in CT and 731.9 ± 144.2 differential signal intensity in MR was measured in the aorta 200 minutes following injection). Conclusion: This study demonstrates the feasibility of engineering a stable liposome-based multimodal contrast agent for use in CT and MR. This multimodal contrast agent, with prolonged in vivo residence time and imaging efficacy, has the potential to bring about improvements in the fields of medical imaging and radiation therapy, particularly for image registration based therapy planning and image guidance in therapy delivery. Acknowledgement: A portion of the following work has been presented (oral presentation) during the Medical Imaging Symposium held February 12-17, 2005 in San Diego, CA, USA as part of the annual meeting of SPIE – The International Society of Optical Engineering.

42 Accelerated Drug Excipient Compatibility Study: A Streamlined Approach to Support Selection of Solid Dosage Form Composition.

Ravi Sharma, Marie Di Maso, Thanh Hoang; Merck Frosst Canada and Co., Montreal, Quebec, Canada.

Objective: Identification of solid dosage form composition at the early stages of a drug development program traditionally involves a thorough drug excipient compatibility study to assess the impact of different excipients on the chemical stability of the active pharmaceutical ingredient. The process is labour-intensive, time consuming and often requires large quantities of bulk drug. In addition, meaningful differences in the stability of targeted formulations of a drug may not be observed within a short period even when stored at highly stressed conditions (i.e. 40ºC/75%RH). To streamline drug development, with limited bulk drug availability, an accelerated drug excipient compatibility approach that has been proposed in the literature [1] has been evaluated using Compound A as a model drug candidate. The results from this approach were compared with those obtained with an actual formulated compound A tablet stability study. Method: The excipient compatibility study is based on binary/ternary drug-excipient blends with 10% drug load, 20% added water and stored at 50°C in sealed vials for 3 weeks. The formulation development stability study consisted of a tablet formulation with the similar excipients used in the compatibility study stressed at 25°C/ 60% RH and 40°C/75%RH over 6 months. Analysis was carried out using a stability indicating HPLC method. Results: Chemical degradation patterns observed in the excipients compatibility studies correlated well with the data obtained for the formulation stored at long terms and accelerated stress conditions. Conclusion: In general, this approach addressed several key factors impacting possible drug excipient interactions and allowed for a quick and more focused approach to selection of solid dosage form composition. [1] Serajuddin, A.B.T., et. Al; J. of Pharm. Sci, Vol. 88 (1999), No. 7, pp 696-704

43 Nanoparticles For Pulmonary Drug Delivery.

Leticia Ely, Raimar Loebenberg, Warren Finlay, Wilson Roa, University Of Alberta, Edmonton, Alberta, Canada.

Purpose: Pulmonary inhalation is becoming an attractive route of administration for a large range of drug substances. The deposition of an aerosol in the lungs depends on its Mass Median Aerodynamic Diameter (MMAD). The aim of this study was to optimize the incorporation of nanoparticles into carrier particles with an appropriate MMAD for lung delivery. Methods: Butyl-cyanoacrylate nanoparticles were incorporated into lactose carrier particles using a Büchi 190 mini spray dryer. In brief: 7 g lactose, 125 mg PEG, 125 mg L-leucine were dissolved in 100 mL of water, 2 mL of a 10 mg/mL nanoparticle suspension was added and spray dried between 120 and 140 °C. The size of the nanoparticles was determined using a Malvern Zetasizer HAS 3000. The carrier particles were measured using a Zeiss LSM confocal laser-scanning microscope. The dispersibility of the spray-dried powders was determined using a Mark II Anderson impactor in combination with a high efficiency dry powder inhaler. Results: The MMAD of the carrier particles after optimization was 2.8 to 3.6 microns, which is an adequate size for deep lung deposition. Generally, the size of the nanoparticles was not significantly changed by the spray drying process. However, in some experiments clusters of nanoparticles were observed. Conclusion: The present study shows that nanoparticles incorporated into carrier particles have the potential to be delivered to the lungs if the carrier has an appropriate particle size. This method can be used to apply nanoparticle based drug delivery strategies to the pulmonary route of administration.

44 Comparison Of Solubilty And Dissolution Data To Establish In Vitro/In Vivo Correlations For Glyburide Formulations.

Hai Wei, Raimar Loebenberg, Faculty Of Pharmacy AndPharmaceutical Sciences, University Of Alberta,Edmonton Alberta, Canada.

Purpose: The purpose of this study was to compare solubility and dissolution data as input function into the ACAT model. Previous studies have shown that dissolution profiles acquired in biorelevant dissolution media can be used to establish in vitro/in vivo correlations (IVIVCs) using the advanced compartmental absorption and transit model (ACAT). In the present study we investigated the possibility to use solubility data only as input function into the ACAT model to predict the oral performance of different glyburide formulations. Methods: The solubility of glyburide was determined in different dissolution media at different pH values. The dissolution behavior of two glyburide formulations (3.5 mg and 5 mg) was tested using apparatus 2, USP 28. The dissolution tests were performed at different pH values and using a multi-pH gradient method. The drug permeability was studied using caco-2 cells. The prediction of the fraction dose absorbed for each formulation was performed using GastroPlus^TM. The results of the simulations were compared with clinical data. Results: The solubility of the glyburide was pH-dependent. The drug release profiles showed differences in the different dissolution media. The permeability was determined to be 3.5e4 cm/sec. The different sets of in vitro data (solubility and dissolution) were used together with the permeability data as input-function into the ACAT model. The simulation results for the 3.5 mg formulation successfully predicted the average AUC and C_max of a clinical study if solubility or dissolution data were used. However, for 5 mg formulation only the solubility data were able to predict the oral performance. Conclusion: Using the ACAT model, this study showed that solubility data exhibited a better predictability for the different glyburide formulations then dissolution data. Key words: dissolution, IVIVC, permeability, in vitro/in silico

45 Improving Cyclosporin A Permeability Across Caco2 Cell Monolayers By Using Vitamin B₁₂-Targeted Polymeric Micelles.

Mira Francis, Mariana Cristea, Françoise Winnik; Faculty Of Pharmacy; Department Of Chemistry, University Of Montreal, Montreal, Quebec, Canada; Petru Poni Institute Of Macromolecular Chemistry, Iasi, Romania.

Purpose. To exploit the ability of vitamin B₁₂ (VB₁₂)-modified dextran-g-poly(ethyleneglycol)cetyl ether (DEX-g-PEG-C₁₆) polymeric micelles to enhance the transport of encapsulated poorly-water soluble drugs across Caco-2 intestinal cells following receptor-mediated endocytosis. Methods. VB₁₂-modified DEX-g-PEG-C₁₆ was synthesized by linking VB₁₂ to a DEX-g-PEG-C₁₆ copolymer via a 2,2’(ethylenedioxy)bis(ethylamine) spacer. Cyclosporin A (CsA), a poorly-water soluble immunosuppressant, was selected as model drug. CsA-loaded polymeric micelles were prepared by a dialysis procedure. The level of CsA incorporation was assayed by HPLC. Caco-2 cells were grown in Transwell^® plates until a tight monolayer was formed. Micelles-loaded CsA was added to the apical compartment and aliquots were withdrawn from the basal chamber. CsA transport was studied in the absence and presence of intrinsic factor (IF). Results. The level of VB₁₂ substitution on DEX-g-PEG-C₁₆ copolymer reached 1.68% (w/w). Polymeric micelles showed 22% entrapment efficiency for CsA. Following 24 h of transport, the apical to basal transport of CsA loaded in VB₁₂-modified DEX-g-PEG-C₁₆ polymeric micelles was higher than that in unmodified micelles. In the case of VB₁₂-targeted micelles, the amount of transported CsA increased by 1.8 and 2.3 times in absence and presence of IF, respectively, compared to unmodified micelles. The extent of CsA within Caco-2 cells was higher in the case of VB₁₂-modified micelles. Conclusions. VB₁₂-modified polymeric micelles enhanced the permeability of CsA across intestinal cells, a promising feature for the development of novel targeted polymeric drug carriers for the oral delivery of poorly-water soluble drugs. This work has been presented at the 2004 AAPS annual meeting on November 10 and was financially supported by NSERC and Rx&D.

46 Purification And Characterization Of JadQ And JadS:Enzymes Involved In Jadomycin B Biosynthesis.

Adam Priddle; David Jakeman, Dalhousie UniversityCollege Of Pharmacy, Halifax, Nova Scotia, Canada.

Purpose: To study the nucleotidyltransferase, JadQ, and glycosyltransferase, JadS, which catalyze key reactions in the biosynthesis of jadomycin B. Analysis of these enzymes may facilitate the incorporation of modified carbohydrate residues into the jadomycin aglycone and subsequently enable the determination of structure-activity parameters for jadomycin B analogs. Objectives: Purification of JadQ and JadS enzymes; Assay of catalytic activity. Methods: E. coli were transformed and grown to express JadQ and JadS proteins. These proteins were extracted and purification was attempted by various means including: Ammonium sulfate precipitation, Hydrophobic interaction chromatography, Affinity chromatography using Nickel-NTA spin columns, Anion exchange chromatography, Substrate analog affinity experiment using UDP coated beads to attempt bind and elute JadS. Results: JadQ and JadS were successfully over-expressed in E. coli. The His-tagged JadS was purified in a non-denatured form. JadQ and JadS formed inclusion pellets on cell lysis, precluding further isolation. Conclusions and future directions: JadQ and JadS are difficult proteins to purify and will require further purification. Altering the lysis conditions may improve the protein solubility. Thereafter, size exclusion chromatography could be attempted, but would probably provide only coarse purification. If purification of native protein proves impossible, an attempt could be made to re-nature the enzymes after purification by gradual removal of denaturant. Acknowledgments: Dr. David Jakeman’s Research Group, Merck Foundation for Summer Student Awards.

47 Synthesis Of Sub-Micrometer Lipobeads Using Non-Toxic Solvents.

Sean Buck, Peter Pennefather; Faculty Of Pharmacy,University Of Toronto And Cytophotonics Inc. Toronto,Ontario, Canada.

Purpose. There is considerable interest in developing new forms of liposome and hydrogel microspheres for applications varying from drug delivery to biological sensors. Our group has been exploring ways of combining these two technologies in the form of Lipobeads, a lipid bilayer shell anchored on the surface of a hydrogel core. In the past, Lipobeads have been synthesized using toxic organic solvents and surfactants. Their size distribution has been only roughly controlled by stirring. Shear forces generated by stirring typically generate beads of 10 mm or greater in diameter (see Buck et al., Biomacromol., 2004). For designing an injectable form of Lipobeads there is a need to make smaller beads with a narrower size distribution using minimally toxic reagents. Method. Here we describe how Lipobeads can be produced utilizing mineral and canola oils as the organic phase. We have eliminated surfactant sensitivity by the use of the natural lipid, egg PC and produced smaller sized beads using the extrusion method. Bilayer structure was determined using fluorescently labeled lipids and confocal microscopy the quenching of as described by (Ng et al. Biophys. J., 2004). Results. Extrusion of several different monomers using canola oils or mineral oil this oil phase produced nano sized spheres (800 nm spheres using 200 nm filter pore size) of lower dispersity than those synthesized by normal mechanical processes. Quenching analysis confirmed bilayer nature of Lipobead coating. Conclusion. We have successfully generated sub-micrometer sized Lipobeads suitable for an injectable formulation and with a higher surface to volume ratio.

48 Chlamydia Infection Of Hepatocytes And The Role Of Fatty Acid.

Guqi Wang, Jing Wang, Guangming Zhong, Frank Burczynski; Faculty Of Pharmacy, University Of Manitoba,Winnipeg, Manitoba, Canada; Department OfMicrobiology, University Of Texas, San Antonio, Texas, USA.

INTRODUCTION: Chlamydia is a ubiquitous and obligate intracellular bacterial pathogen that must replicate within a cytoplasmic vacuole of eukaryotic cells and capable of infecting different tissues. We explored the possibility of hepatocyte chlamydia infection and the role of long-chain fatty acids (LCFA) in chlamydial infection. METHODS: The Chlamydia trachomatis serovar LGV2 organisms were used to infect rat hepatoma cells (1548), primary rat hepatocytes, Chang liver cells, and liver fatty acid binding protein (L-FABP) transfected Chang cells. Status of chlamydial infection was monitored by immunofluorescence microscopy. In LCFA uptake experiments, Chang cells were plated on 24 well plates. Following infection (24 hrs), cultures were incubated with BSA-[³H]-palmitate for 210 min at 37^oC. The BSA-[³H]-palmitate uptake was detected by measuring radioactivity in cell lysates made from the chlamydia-infected or mock-infected cultures. L-FABP was detected by Western blot and L-FABP transcript by RT-PCR. RESULTS: Chlamydial antigens were detected in all cell types following infection but only Chang cells were permissive for productive chlamydial infection. RT-PCR and Western blot data showed that only Chang cells had undetectable L-FABP expression. Expressing L-FABP in Chang cells caused earlier cell necrosis after infection. Total LCFA clearance by Chang cells was greater (3.24 ± 0.120 ul/min10⁶cells) in infected cells and than control (2.64 ± 0.0742 ul/min10⁶cells; mean ± SE; n=4; p < 0.01). CONCLUSION: Chlamydia infection resulted in an enhanced uptake of LCFA. An intact well-defined Chlamydia inclusion body was present in hepatocytes lacking L-FABP. Results suggest that LCFA may play a pivotal role during intracellular host infections.

49 Structure-Function Relationships Of Antibiotic And Anticancer Peptides And Proteins By NMR Spectroscopy.

Ray Syvitski, David Jakeman, College Of Pharmacy, Dalhousie University, Halifax, Nova Scotia, Canada.

Purpose To determine the three-dimensional structure of several membrane-associated bioactive peptides and proteins by NMR spectroscopy and correlate the three-dimensional structure to biological function. Systems studied include the ectodomain of p14 protein, one of the smallest transmembrane-fusion proteins known, the antimicrobial peptide pleurocidin from winter flounder and the competence stimulating peptide from Streptococcus mutants responsible for the formation of a biofilm (dental plaque). Methods Peptide samples were prepared in appropriate solvent (dimethyl sulfoxide, dodecylphosphatidyl choline (DPC) micelles in aqueous buffer, or trifluoro ethanol (TFE)) and spectra recorded on a Bruker Avance 500 MHz NMR spectrometer. Two dimensional datasets were analyzed using Sparky software and simulated annealing experiments were run using Xplor software to determine three dimensional structures. Results We have discovered and determined the structure of a small loop in the N-terminal ecto domain of p14. The pleurocidin peptide folds into an amphipathic a-helix in DPC micelles as do the competence stimulating peptides, UA159 and JH1005 in TFE. Conclusion The loop in the P14 ecto domain is proposed to be involved in initiating membrane fusion. The amphipathic pleurocidin a-helical peptide is thought to disrupt membrane integrity whilst the difference in length of the a-helical nature of UA159 and JH1005 is responsible for the altered ability of these peptides to stimulate biofilm formation. Consideration of site-directed mutants of these peptides upon activity will also be discussed. NMR spectroscopy, structure-function, membrane bound peptides and proteins.

50 Comparative Study Of Topical Liposomal And Electroporation Mediated Cutaneous Gene Delivery In Mice.

Narges Shaterian, Ildiko Badea, Marianna Foldvari,College Of Pharmacy And Nutrition, University Of Saskatchewan, Saskatoon, Saskatchewan, Canada.

Purpose: To compare topical liposomal and electroporation delivery methods evaluated by expression of luciferase and IFNg levels in mouse skin in vivo. Methods: Expression of luciferase after different electroporation conditions and formulations containing pMASIA-Luc model plasmid was analyzed in skin homogenates of CD1 mice. The ideal topical electroporation conditions were developed through a series of experiments to determine the optimum voltage, pulse duration, skin collection time post-electroporation and electrode type. Delivery of IFNg gene using pGTmCMV-IFN.GFP bicistronic plasmid coding for IFNg was assessed in mice after topical application with or without electroporation using ELISA. Results: Evaluation of luciferase concentration in the skin after topical treatment with plasmid solution (1-s-dna-t), cationic liposomal formulation (2-fl16-dna-t), cationic complex (3-fldcdna-t) followed by electroporation showed low luciferase expression (all around 50 pg/cm²) whereas after intradermal injection of the above three formulations followed by electroporation increased luciferase expression to about 180, 150 and 120 pg/cm², respectively. Topical treatments without electroporation resulted in higher luciferase expression (all around 240 pg/cm²). Experiments with IFNg-coding plasmid indicated twofold higher IFNg background levels in animals receiving electroporation, indicative of tissue damage, and no increase in expression after treatment. However, after topical DNA solution and topical liposomal formulation treatment (2fl16-dna-t) IFNg expression increased by 125 and 380%, respectively, compared to buffer treated control. Conclusion: These experiments indicate that tissue damage caused by electroporation may be detrimental to protein expression in the skin. Topical treatment with liposomal DNA without electroporation may provide sufficient level of delivery and expression without tissue damage.

51 Validating The Assumptions Of An Ontogeny Model Of Hepatic CYP2E1 And CYP1A2 Enzyme- Mediated Drug Clearance.

Fawzy Elbarbry, Jane Alcorn College Of Pharmacy And Nutrition, Saskatoon, Saskatchewan, Canada.

Purpose: We proposed to evaluate underlying assumptions of a mathematical model that describes the ontogeny of hepatic Cytochrome P450 (CYP) enzyme activity. We tested the hypothesis that changes in V_max account for age-dependent changes in intrinsic clearance, and activity assessments (V_max) with one substrate can be applied to all substrates of the enzyme. Methods: Cyp2e1 and Cyp1a2 ontogenesis were determined in male Sprague-Dawley rat hepatic microsomes at fetal, postnatal and adult ages. Body and liver weights and hepatic microsomal protein content were measured to calculate age-dependent hepatic scaling factors. Enzyme kinetics (V_max and K_M) were characterized for Cyp2e1 and Cyp1a2 using specific probe substrates. As well, enzyme ontogenesis was assessed at the mRNA (real time RT-PCR) and protein (immunoblotting) expression levels. Immunohistochemistry (IHC) was used to study the temporal and zonal kinetics of enzyme expression in developing livers. Results & Conclusion: Age-dependent changes in V_max were significantly different (P£ 0.05) between the different age groups , while K_M values were similar at all stages of postnatal maturation except at neonatal ages £ 5 days. Microsomal protein contents increased with postnatal age with dramatic increases after day 14 of age. The results showed a similar pattern of postnatal change in Cyp2e1 and Cyp1a2 enzyme activity with changes in their respective mRNA and protein expression. IHC showed homogenous distribution of enzyme expression in early ages which seems to focus toward the perivenous region with puberty and adulthood. Further work will determine whether the model may predict in vivo hepatic metabolic clearance. Keywords: Pharmacokinetic models, Cytochrome P450, Immunoblotting, RT-PCR, IHC, Ontogeny.

52 Alterations Due To Acute Inflammation On The Pharmacokinetics And Pharmacodynamics Of Th Calcium Channel Blocker, Nifedipine.

Genevieve Godreau, Kassem Abouchehade, Husam M. Younes. School Of Pharmacy, Memorial University Of Newfoundland, St. John’s, Newfoundland, Canada.

Purpose: Cytokines such as Interferon-gamma (IFN-g) have been reported to interfere with the disposition of various cardiovascular drugs affecting their therapeutic outcomes. This study investigates the possible alterations in the pharmacokinetics and pharmacodynamics of the calcium channel blocker, Nifedipine (NF) under acute inflammation induced by IFN-g injections Methods: Male Sprague-Dawley rats were catheterized under surgery and their jugular veins were used for collecting blood samples while their carotid arteries were used for blood pressure measurements. Teflon coated wires were attached for ECG monitoring. After recovery, rats were divided into two groups (n = 4 per group). The inflammation group was injected subcutaneously with 0.15 ml of murine IFN-g (80,000 U) 12 hours prior to NF oral administration (3mg/Kg). The control group was injected with 0.15 ml saline. Blood samples were collected and analyzed for NF using a validated HPLC method. Blood pressure measurements and ECG intervals were recorded prior to each sample collection. Results: NF reached peak plasma concentrations within 20 min in both groups. The C_max of NF was significantly raised from 543 ± 47 ng/ml in controls to 2045 ± 481 ng/ml in the inflammation group. A significant higher AUC values in the inflammation group compared to control (1341 ± 326 vs 685 ± 251 ng/ml hr) were also recorded. Inflammation group showed 3 folds reduction in their mean arterial pressure compared to the normal group. Conclusion: Inflammation may have altered the metabolic, hemodynamic and baroreflex mechanisms that regulate systemic blood pressure and prevented abnormal fluctuations in vivo. Acknowledgements: G. Godreau was supported by the National Summer Students Research Program funded by the Merck Company Foundation.

53 A Comparative Interim Analysis Of Fatigue And Cognition In Relapsing-Remitting Multiple Sclerosis (RRMS) Patients Receiving Three Different Interferon Medications (Rebif^®, Avonex^®, Or Betaseron^®).

Colin Gramlich, Karen Bergen, Maria Melanson, Michael Namaka; Faculty of Pharmacy, The University of Manitoba, Winnipeg, Manitoba, Canada.

Purpose: A single centre, phase IV, comparative, open-label interim analysis study was conducted in 25 participants diagnosed with relapsing remitting multiple sclerosis (RRMS) that met the pre-established inclusion/exclusion criteria. It was hypothesized that the use of disease modifying interferon drugs Rebif^®, Avonex^® and Betaseron^® will significantly improve the fatigue and/or cognitive status of participants with RRMS. To determine 1) if treatment with Rebif^®, Avonex^® and Betaseron^® results in a statistically significant improvement in fatigue and/or cognition status compared to control values recorded at baseline and 2) if treatment with Rebif^® produces the greatest clinical improvement in fatigue and/or cognition function compared to Avonex^® and Betaseron^®. Methods: The study consists of four individual visits per patient at months 0, 3, 9, 15. The first two visits comprise a baseline of fatigue and cognition whereas the third and fourth visits comprise the fatigue and cognition status of the patients with treatment. Fatigue was measured using the Modified Fatigue Impact Scale whereas cognition was measured using the Brief Repeatable Battery of Neuropsychological Tests in Multiple Sclerosis. Results: The preliminary results obtained from this study suggest that interferon treatment does improve the fatigue and cognitive status of patient with RRMS. Furthermore, the results also suggest that Rebif^® may produce enhanced beneficial effects on fatigue and cognition compared to Avonex^® and Betaseron^®. Conclusion: The presence of disease-induced symptoms such as fatigue and cognition may be a significant factor that contributes to the preferential treatment selection amongst available interferon products.

54 Corporate Social Responsibility And Pharmaceutical Pricing.

Chris Macdonald, Trevor Coffrin, Department Of Philosophy, Saint Mary’s University Halifax, Nova Scotia, Canada.

Purpose Our purpose is to assess whether Canadian pharmaceutical companies have a social responsibility to provide cheap or free pharmaceuticals to those who would otherwise be unable to afford them. Methods We survey the relevant literature on pharmaceutical pricing and Corporate Social Responsibility. We examine ethical arguments based upon affluence, special capacity, special opportunity, the demands of “the rule of rescue,” as well as the special significance of health-related products and services, and find all of these arguments lacking. We also consider counter-arguments rooted in the recognized constraints of competitive domains, obligations of managers to shareholders, and the demands of justice in a mixed-market economy. Our focus is explicitly and solely on corporate decision-making, and whether good corporate citizens ought to feel obligated to take unilateral action. Results We argue that a range of background conditions must be taken into consideration. In a mixed-market economy that respects the value of intellectual property rights, the legal and social context of corporate decision-making are crucial. Decisions about Corporate Responsibility cannot be made in the abstract, but must take their cues from extant social norms and conventions. Conclusion We conclude that Canadian pharmaceutical companies do not have a substantive ethical responsibility to contribute significantly to the provision of cheap or free pharmaceuticals to developing nations. This is not to say that it is not commendable when individual companies do engage in charitable acts; it is merely to say that given current background conditions, such acts cannot be considered ethically mandatory for responsible corporations.

55 Hepatic Iron Removal By Deferiprone And Desferrioxamine In Iron-Loaded Rats.

Jasmina Novakovic , A. Tesoro , J.J. Thiessen, M. Spino, R. Sandhu, J. Connelly, Leslie Dan Faculty Of Pharmacy, University Of Toronto, Toronto, Ontario, Canada; Apopharma, Toronto, Ontario, Canada

Purpose: Deferiprone (L1) and desferrioxamine (DFO) are currently available drugs for treatment of iron overload. Combining L1 and DFO therapy is advantageous in iron-loaded patients. It is not clear if the effect is additive or synergistic. The study purpose was to estimate potential benefits of combined long-term treatment in iron-loaded rats. Methods: Test articles: Deferiprone (Apotex), Desferal (Novartis). Animals: Sprague Dawley rats (N=19), body weight 200-250 grams. Housing and diet: Two per cage, water and regular rodent diet ad libitum. Iron-loading (N=15): Iron-dextran, intra-peritoneally, 100 mg/kg twice weekly for four weeks, followed by equilibration of one week. Chelator treatment: Group #1 (N=4): 60 mg/kg DFO subcutaneously; Group #2 (N=4): 100 mg/kg deferiprone by oral gavage; Group #3 (N=4): DFO and deferiprone; Group #4 (N=3): Control iron-loaded, drug-free; Group #5 (N=4): Control naïve. Test articles administration: Five times weekly (Monday-Friday, 127 days, 89 doses). End point: Euthanization and exsanguination, blood collection, organs and tissues isolation for histology, TEM, and iron determination by ICP-MS. Results: Validity of iron-loading procedure is confirmed by significant elevation of heart, liver, intestine, spleen and skeletal muscle iron in Fe-controls vs Naïve rats, by histological and TEM observations. Conclusion: All three treatments led to decrease in tissue iron. No synergism was detected. For heart and liver iron removal the order was: L1+DFO e” L1 > DFO. Combined L1 and DFO treatment was the most beneficial for the hepatic iron removal.

56 Effect Of HIV-1 Viral Envelope Glycoprotein Gp120 On The Functional Expression Of P-Glycoprotein (P-Gp) In Cultured Glial Cells.

Patrick Ronaldson, Manisha Ramaswamy, ReinaBendayan, Department Of Pharmaceutical Sciences, Leslie Dan Faculty Of Pharmacy, University Of Toronto, Toronto, Ontario, Canada.

Purpose: Exposure of cultured glial cells to HIV-1 envelope glycoprotein gp120 may activate chemokine receptors (i.e., CXCR4, CCR5) and induce pathological events associated with HIV-1 encephalitis (HIVE) (i.e., production/secretion of cytokines and nitric oxide). An obstacle to the pharmacological treatment of HIVE is the functional expression of ATP-binding cassette (ABC) drug transporters (i.e., P-gp) known to export antiretroviral drugs from cellular targets of HIV-1 infection (i.e., astrocytes, microglia) (Lee et al. 2001; Ronaldson et al. 2004). This project investigates the effect of gp120 treatment on Pgp functional expression in primary cultures of rat astrocytes. Methods: Primary cultures of rat astrocytes were incubated in the presence of 1.0 nM gp120 (i.e., 6, 12, 24 h). Cytokine (i.e., TNF-a, IL-1b, IL-6) and inducible nitric oxide synthase (iNOS) levels were measured by semiquantitative RT-PCR analysis. P-gp gene and protein expression were measured by RTPCR and immunoblotting analysis respectively. Transport properties of radiolabelled digoxin, an established P-gp substrate, were investigated at 37°C in primary cultures of astrocytes grown as monolayers. Results: Semiquantitative RTPCR analysis demonstrated increased expression of TNF-á, IL1â, IL-6, and iNOS mRNA in primary cultures of rat astrocytes treated with 1.0 nM gp120, suggesting in vitro glial cell injury. Following gp120 treatment (24 h), P-gp protein expression was decreased (4.7 fold) and digoxin accumulation (1 h) was significantly enhanced (1.8 fold) compared to control untreated cells. Conclusions: Gp120 treatment can modulate the functional expression of P-gp in cultured rat astrocytes suggesting that complex drug-transporter interactions may occur during the pathological response associated with HIVE. Supported by CIHR, Ontario HIV Treatment Network, and an Ontario HIV Treatment Network Studentship.

57 The Effect Of Modulating Valproyl-1-O-Acyl Glucuronide On Valproic Acid-Induced 15-F_2tIsoprostane Levels In Rats.

Vincent Tong, Xiaowei Teng, Thomas K.H. Chang, Frank Abbott, Faculty Of Pharmaceutical Sciences, The University Of British Columbia, Vancouver, British Columbia, Canada.

Purpose. Oxidative stress has been associated with valproic acid (VPA) treatment in rats. This study investigates the effect of inhibiting VPA-1-O-acyl glucuronide (VPA-G) formation on VPA-induced oxidative stress in rats. Methods. Adult male Sprague-Dawley rats (n = 4 - 8/group) were pretreated with or without [(1S)-endo]-(-)-borneol (1 mmol/kg) or salicylamide (2 mmol/kg) at 0.5 hr prior to VPA treatment (500 mg/kg, ip). At 0.5 hr after VPA administration, animals were sacrificed and blood and liver obtained. Liver VPA-G levels were determined by LC/MS. ROS levels in plasma and liver were measured using an EIA method for 15-F_2t-isoprostane (15-F_2t-IsoP). Results. As expected, (-)-borneol and salicylamide reduced the levels of VPA-G by » 90% and 75%, respectively. VPA alone elevated plasma 15-F_2t-IsoP from 35 ± 4 (control) to 93 ± 8 pg/mL. (-)-Borneol and salicylamide pretreatment attenuated this increase of plasma 15-F_2t-IsoP to 60 ± 4 pg/mL and 46 ± 6 pg/mL, respectively. Total liver 15-F_2t-IsoP levels were elevated 2-fold by VPA when compared to control, and this increase was attenuated by (-)-borneol and salicylamide similar to control levels. (-)-Borneol and salicylamide alone did not elevate 15-F_2t-IsoP levels compared to vehicle control. Conclusion. The inhibition of VPA-G in rats is apparently associated with a decrease in VPA-induced formation of 15-F_2t-IsoP.

58 Hemodyanmic Effects Of Diltiazem In Rats Following Single And Repeated Subcutaneous Injection In Vivo.

Pollen Yeung, Angelita Alcos, Jinglan Tang, Ban Tsui, Pharmacokinetics & Metabolism Laboratory, College Of Pharmacy, Dalhousie University, Halifax, Nova Scotia, Canada.

Purpose: To compare the hemodynamic effect of diltiazem following single and repeated subcutaneous injection using an in vivo rat model. Methods: Male SD rats (n = 5 – 6 per group) weighing between 350 - 450 g were used. Each rat received either a single 20 mg/kg dose of diltiazem (Biovail Corp, Mississauga, Ont. Canada) by s.c. injection or 5 mg/kg s.c. bid for 5 doses. Two separate control groups were used (n = 5 – 6 per group) and each rat received saline as described for the treatment groups. Hemodynamic measurements were recorded continuously for each animal before and following treatment for up to 6 h. Results: Diltiazem decreased SBP from 138 +/- 9 to 124 +/- 8 mmHg (10%) following the single dose and from 126 +/-17 to 104 +/- 10 mm Hg (17%) following the 5^th and last dose. The maximum effect occurred in less than 1 h for both dosing regiments. On the other hand, the effects on DBP were greater following the single dose (26% vs 17%). Paradoxically, there was a reduction in HR following the multiple doses (12 %), but a close to 9% increase in HR following the single dose. Conclusion: Hemodynamic effects of diltiazem were both qualitatively and quantitatively different between the single dose and repeated (Supported in part by a grant-in-aid from CIHR/NSHRF/PEF Regional Partnership Program).

59 New Improvements In Ion Mobility Spectrometry(IMS) For Cleaning Validation.

John Carroll, Tri Le, Reno Debono, Smiths Detection,Warren, New Jersey, USA.

Purpose To date, many pharmaceutical companies have investigated and adopted ion mobility spectrometry (IMS) to replace HPLC in cleaning validation. IMS offers the advantages of faster, simpler, lower-cost analyses. This paper reports on progress in three areas that will broaden the range of pharmaceutical applications of IMS. These areas of development are increased dynamic range through split injections, aqueous sample analysis, and detergents analysis. Methods Samples were analyzed by IMS using high performance injection (HPI) as the means of sample introduction. The HPI methods, especially the temperature programming, were optimized with regard to precision, linearity, and sensitivity. Results 1. Increased dynamic range: The API diazepam was detected at concentrations spanning three orders of magnitude with RSDs less than 4% using split ratios up to 100:1 on the HPI. The usual linear range is about one decade. 2. Aqueous sample analysis: HPI methods using programmed temperature ramping have been devised to analyze APIs in aqueous solutions with RSDs less than 4% and a sample throughput of 60 samples per hour. This avoids the problems associated with water for traditional IMS sample introduction. 3. Detergents analysis: A ten-fold improvement in sensitivity, broader linear range, and improved precision was achieved for detergents analysis through the use of an HPI method designed to separate in time the vaporization of different surfactant components. Conclusion Progress in these area will enable IMS to meet an increased variety of cleaning validation needs.

60 Ion Mobility Spectrometry (IMS) Delivers Fast, Successful Method Validation For Cleaning Verification.

Azhar Mahmood, John Carroll, Reno Debono, Smiths Detection, Warren, New Jersey, USA.

Purpose To develop a validated ion mobility spectrometry (IMS) cleaning verification method to determine the pharmaceutical active diphenhydramine HCl (DPH), and to illustrate the advantages of using IMS over HPLC.Methods DPH was analyzed by IMS using high performance injection (HPI) as the means of sample introduction. The IMS method was evaluated with regard to specificity, linearity, precision, accuracy, and stability. The results for these validation parameters were compared to criteria used in an established HPLC method for the determination of DPH. The potential time and cost savings of switching from HPLC to IMS was examined.Results The IMS method met or exceeded all the validation criteria. No interference from solvent, swabs, or excipients was observed. Linearity was demonstrated from 20-150% of the action level with R² = 0.9955. The precision, as measured by RSD, was 1.8% at the action level and 8.3% at the LOQ. Recoveries for swab samples, mock samples, and standards aged 48 hours ranged from 93-105%. The LOD and LOQ were 0.009 mg/mL and 0.022 mg/mL, respectively. Each sample took 12 seconds to analyze. The sample throughput, limited by the autosampler, was 60 samples per hour. Conclusion The IMS method met or exceeded all the validation criteria of the cleaning validation protocol. The time needed to complete the analyses for the validation tests using IMS was approximately 2.5 hours compared to 17.5 hours when using HPLC, a savings of nearly two working days.

61 Use Of A Freezer Mill For Preparation Of Tissue Samples For Analysis Of Paclitaxel By LC-MS/MS.

Hongwen Chen, Richard Liggins, Kaiyue Wang, Pierre Signore, Leanne Embree, Angiotech Pharmaceuticals, Inc., Vancouver, British Columbia, Canada.

Purpose: The present study describes the use of a freezer mill to replace a rotor-stator homogenizer for tissue pulverization in a quantitative LC-MS/MS assay. Methods: Fat pad, ligament, capsule, meniscus and cartilage tissues from rabbit knee joints were used in this study. Samples were pulverized using a liquid nitrogen-cooled freezer mill and extracted with an aqueous acetonitrile solution. Normal phase chromatography and electrospray ionization with tandem mass spectrometry detection were used for quantitation. The quantitation was based on a 10 point calibration curve (1 to 1000 ng/mL). The assay was evaluated for accuracy and precision using control tissues with the addition of paclitaxel at 5, 100 and 1000 ng/ mL (n=5 per concentration). Results: The operation conditions of the freezer mill was optimized. Accuracy and precision of paclitaxel from the five independently prepared tissue samples of each tissue type were evaluated. Values were within or close to the acceptable limits for the lower limit of quantitation (LLOQ) for bioanalytical methods. Paclitaxel was detected at 0.9 to 12 ng/mL in tissue samples from animals exposed to 450 µg paclitaxel locally delivered for 3 days. Conclusions: The freezer mill provided a test article that was easier to filter with better homogeneity, and higher throughput compared to the homogenizer for these tissues. Further evaluation of this method to establish the range is required. Incorporation of an internal standard and improvements to the extraction procedure are being assessed.

62 Synthesis Of 4-Deuteromethyl Nevirapine To Study The Role Of A Quinone Methide Metabolite That May Be Responsible For Nevirapine-Induced Skin Rash.

Baskar Mannargudi, Jack. P. Uetrecht, Faculty OfPharmacy, University Of Toronto, Toronto, Ontario, Canada.

Purpose: Nevirapine, an anti-HIV drug, is associated with a high incidence of skin rash and hepatotoxicity. Recently we developed an animal model of nevirapine-induced skin rash in female Brown Norway rats. Metabolic studies suggested that oxidation of the methyl group to form 12-hydroxynevirapine might be involved in the mechanism. This metabolite has the potential to be further metabolized to a reactive quinone methide. To check this hypothesis we decided to synthesize an analogue, 4-deuteromethyl nevirapine. This 4deuteromethyl nevirapine should have chemical properties similar to nevirapine, but oxidation of the methyl group to the 12-hydroxy metabolite should be slower because of the deuterium isotope effect. Methods: After attempts with other methods, the synthesis of 4-deuteromethyl nevirapine was accomplished by treating nevirapine with potassium tertbutoxide (2 equivalents) in deuterated dimethyl sulfoxide at 140 ⁰C for 48 hours. Other reaction conditions attempted to make this compound will also be discussed. Results: The chemical synthesis of 4-deuteromethyl nevirapine was accomplished successfully in one step from nevirapine. The yield was 97%, and the product was fully characterized by proton and carbon NMR as well as mass spectroscopy. Conclusions: The 4-deuteromethyl nevirapine was synthesized in gram quantities and was subsequently used for metabolism and toxicity studies. This work was supported by a grant from the Canadian Institutes for Health Research.

63 Chromatographic Separation Of Griseofulvin Metabolites From The Parent Molecule Is An Important Factor In The Development Of A Rugged And Selective LC-MS/MS Method For Plasma Griseofulvin.

Ernest Wong, Sladjana Radjevic, Nick Peng, Anita Towers,Nicola Hughes; Biovail Contract Research, Toronto,Ontario, Canada.

Purpose Griseofulvin is an antifungal agent. It is extensively metabolized in vivo to inactive metabolites. HPLC assays are currently available that separate Griseofulvin from its metabolites, however, these methods require excessively long run times. . Our aim was to use the selectivity of LC-MS/MS to develop a fast method suitable for pharmacokinetic application. Methods Griseofulvin, and its internal standard were extracted from human plasma (0.1 mL) using Waters HLB SPE cartridges. Chromatographic separation was carried out using reverse phase conditions. Analytes were detected using an API 300 mass spectrometer equipped with a heated nebulizer ion-source in positive-ion MRM mode. Results An LC-MS/ MS assay for Griseofulvin was validated in human plasma with a run time of 2.5 minutes. However, upon application in a biostudy, an interference peak was observed at the later sampling time-points in every subject but not in calibration standards and Quality Control samples. It was concluded that this interference peak was a metabolite of Griseofulvin, and the method needed to be modified to improve selectivity. This was accomplished by adjusting the proportions of the mobile phase, a 5-fold dilution of the extracted samples and an extension of the chromatographic run time to 4 minutes. These modifications led to the development of a very rugged bioanalytical method, which was successfully used in a pharmacokinetic biostudy. Conclusions The selectivity of mass spectrometry alone is not sufficient for an optimal method for Griseofulvin in human plasma, as chromatographic separation of the analyte from its metabolites is required.

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