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 (Mw/ Mn
< 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-HT1A And GABAA 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-HT1A and GABAA
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-HT1A receptor stimulation was measured using R(+)-8-OH-DPAT
stimulated [35S]-GTPγS binding autoradiography. Specific
binding to GABAA 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-HT1A receptor coupling in the cortex and
in the raphe nucleus whereas GABAA 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-HT1A
whereas GABAA 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; AbelcetÒ)
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 FungizoneÒ (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 t1/2
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 AUC0-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 AUC0-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 tmax after KTZ
administration. Mean Cmax 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 t1/ 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 AL
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/Ka)
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 GastroPlusTM. 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 Cmax
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 37oC. 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 Vmax account for age-dependent
changes in intrinsic clearance, and activity assessments (Vmax) 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 (Vmax and KM)
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 Vmax
were significantly different (P£ 0.05) between the different age groups ,
while KM 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 Cmax 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-F2tIsoprostane
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-F2t-isoprostane (15-F2t-IsoP). Results. As
expected, (-)-borneol and salicylamide reduced the levels of VPA-G by » 90% and
75%, respectively. VPA alone elevated plasma 15-F2t-IsoP from 35 ± 4
(control) to 93 ± 8 pg/mL. (-)-Borneol and
salicylamide pretreatment attenuated this increase of plasma 15-F2t-IsoP
to 60 ± 4 pg/mL and 46 ± 6 pg/mL, respectively. Total liver 15-F2t-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-F2t-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-F2t-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 5th 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.
Published by the Canadian Society for Pharmaceutical Sciences.
Copyright © 1998 by the Canadian Society for Pharmaceutical Sciences.
CSPS Home | JPPS Home | Search | Subscribe to JPPS