Research Abstracts

The Journal of Informed Pharmacotherapy 2004;15:400.

Faculty of Pharmaceutical Sciences, University of British Columbia
Alan C. Hayman Summer Student Research Competition 2003

Partial Proceedings from the Faculty of Pharmaceutical Sciences Summer Student Research Program Poster Competition

Abstracts

Heat Treated Fungizone® (HFZ) Maintains Amphotericin B’s (AmB) Antifungal Activity while Decreasing its Renal Cytotoxicity: Role of Fungal Phospholipases
Erin Chew, Stephen D. Lee, Nancy S. Chung and Kishor M. Wasan. Faculty of Pharmaceutical Sciences, University of British Columbia

ESTABLISHING A LIMITED SAMPLING STRATEGY FOR CYCLOSPORINE (NEORAL®) IN PEDIATRIC RENAL TRANSPLANT PATIENTS
Amanda Lai, David S. Lirenman, James Carter, Morrison Hurley, Colin White, Dawn K. Strong, Mary H. H. Ensom. University of British Columbia and Children’s and Women’s Health Centre of British Columbia, Vancouver, BC

NEUSTATE-DEPENDENT GABAERGIC INHIBITION OF SCIATIC NERVE-EVOKED RESPONSES OF DORSAL SPINOCEREBELLAR TRACTRONS
A. Chan, N. Taepavarapruk, S. A. McErlane, S. Chow, and P. J. Soja

EFFECT OF FUNGIZONE® AND ABELCET® ON CETP-MEDIATED TRANSFER OF CHOLESTERYL ESTERS FROM LDL TO HDL
Benny Lau, and Kishor M. Wasan. Faculty of Pharmaceutical Sciences, The University of British Columbia.

USING PROTEOMIC DATABASES FOR THE ANALYSIS OF GENE EXPRESSION IN HUMAN BRONCHIAL EPITHELIUM CELLS IN THE PRESENCE AND ABSENCE OF VIRAL INFECTION
Adeline Chau_, Jeffrey Lau_, Sidney Katz_, Robert Harris_. Faculty of Pharmaceutical Sciences, University of British Columbia and Department of Biological Sciences, Faculty of Science, University of British Columbia, Vancouver, BC.

Health Beliefs as Predictors of Heart Failure Medication Adherence
Kiran Saini1, IvanaDojcinovic1, Phuong Hoang2, Andrew Ignaszewski2, Adrian Levy2, Wolfgang Linden2, and Stephen Shalansky1&2 1Faculty of Pharmaceutical Sciences, The University of British Columbia and 2 St. Paul’s Hospital, Vancouver, BC.

Consequences of the Interaction between a Lipophilic Endotoxin Antagonist, E5564, with Plasma Lipoproteins
Eugene K. Choo1, Daniel P. Rossignol2, Nancy S. Chung1, Kishor M. Wasan1. 1Faculty of Pharmaceutical Sciences, the University of British Columbia. 2Eisai Medical Research, Glenpointe, NJ, USA.

INVESTIGATING POTENTIAL GLUCOSE LOWERING PROPERTIES OF ORGANIC VANADIUM COMPOUNDS AND TRANSITION ELEMENT-MALTOLATO COMPOUNDS
Jeremy Tse1, Violet Yuen1, Chris Orvig2, and John H. McNeill1. 1Faculty of Pharmaceutical Sciences, The University of British Columbia and 2Department of Chemistry, The University of British Columbia.

The encapsulation and release of antisense oligonucleotides from an injectable, controlled release, polymeric paste
Jessica M. Chong, John K. Jackson, and Helen M. Burt.

Formulation Development of FDC-2-4, a Novel Phytostanyl Analogue, into a Non-toxic Aqueous Solution for in vivo Cholesterol Uptake Studies Using Rats
Shabnam Iqbal 1, Simon Wallis 2, Eugene Choo 1, Verica Risovic 1, Kishor M. Wasan 1. 1 Faculty of Pharmaceutical Sciences, The University of British Columbia and, 2 Forbes Medi-Tech, Inc.

Lysophosphatidylcholine mediated recruitment of cardiac luminal LPL requires generation of lysophosphatidic acid
Patsy Yip and Brian Rodrigues. Faculty of Pharmaceutical Sciences, The University of British Columbia

Pharmacological Investigation of Kinase Pathways Activated by Phenylephrine and A-61603 in the Aorta and Tail Arteries of Rats
Perminder Bains1, Irem Mueed1, and Kathleen MacLeod1, Faculty of Pharmaceutical Sciences, The University of British Columbia

THE INDUCTION OF HEMOLYSIS BY MePEG-PCL DIBLOCK POLYMERS: AN INDICATION OF MEMBRANE PERMEABILITY MODIFICATION
Mike Borslein, Jason Zastre, John Jackson, and Helen Burt. Faculty of Pharmaceutical Sciences, The University of British Columbia.

PICTOGRAPHIC INSTRUCTIONS FOR MEDICATIONS: DO OTHER CULTURES INTERPRET THEM ACCURATELY?
Zahra Sadikali 1, LCol Régis Vaillancourt 2, John B. Collins 3, Rosemin Kassam 4. 1 Research Assistant, Faculty of Pharmaceutical Sciences, The University of British Columbia. 2 Directorate of Medical Policy, Pharmacy Policy and Standards, Canadian Forces. 3 Adjunct Professor, Department of Educational Studies, The University of British Columbia. 4 Assistant Professor; Director-Structured Practice Education Programs, Faculty of Pharmaceutical Sciences, The University of British Columbia.

Release of Paclitaxel from poly (lactic-co-glycolic acid) (PLGA) microspheres: Effect of diblock copolymers on drug release
Tawny Hung and Helen Burt. Faculty of Pharmaceutical Sciences, The University of British Columbia

Effect of Warfarin on Haloperidol Lipoprotein and Protein Association within Normolipidemic and Hyperlipidemic Human Plasma
Tiffany Ho 1, Ric M. Procyshyn 1,2 and Kishor M. Wasan 1. 1 Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada 2 Riverview Hospital, Port Coquitlam, British Columbia, Canada.

The Effect Of Complementary And Alternative Medicine Usage Patterns On Warfarin-Related Adverse Events
Esther A. Abd-Elmessih, B.Sc. 1,2; Shelly A.V. To 1,2; Stephen J. Shalansky, Pharm.D. 1,2 ; and Larry Lynd, B.S.P., Ph.D. Candidate 2. 1 Faculty of Pharmaceutical Sciences, The University of British Columbia 2 St. Paul’s Hospital, Vancouver, British Columbia


Heat Treated Fungizone® (HFZ) Maintains Amphotericin B’s (AmB) Antifungal Activity while Decreasing its Renal Cytotoxicity: Role of Fungal Phospholipases
Erin Chew, Stephen D. Lee, Nancy S. Chung and Kishor M. Wasan. Faculty of Pharmaceutical Sciences, University of British Columbia

Introduction: Fungizone® (FZ), an amphotericin B deoxycholate formulation is effective in treating fungal infections but its use is limited by dose-dependent renal toxicity. Previous studies in our lab have shown that heating FZ at 70˚C for 20 minutes substantially reduces AmB-induced renal toxicity without altering the drug’s antifungal activity. However, to date, the reasons for this observation remain unknown.

Rationale: Recent studies have shown that AmB lipid complex (ABLC) was ineffective in killing mutants of C. albicans, a fungal cell line that lacks the ability to secrete fungal phospholipases. However, upon addition of fungal phospholipases to the media of these cells, ABLC’s ability to kill these cells was restored. This was explained by the selective release of AmB at the site of fungal infection through the action of fungal phospholipases.

Purpose: Based on the studies with ABLC, the purpose of this investigation was to determine if the addition of fungal phospholipases to pig kidney cells would restore HFZ’s cytotoxicity to those observed when these cells were treated with FZ.

Methods: LLC-PK1 cells, a pig kidney cell line, were grown in T75 flasks and seeded in 96 well plates at a density of 40,000 cells/cm2. FZ and HFZ treatment solutions containing a concentration of 10, 20, and 50 _g AmB/ml were prepared. HFZ was prepared by heating the drug in a 70˚C water bath for 20 minutes. A concentration of 0.43 units/ml of Fungal Phospholipases A2 was added to all treatment groups and incubated for 1 h at 37˚C before the cells were further incubated for an additional 18 h with different concentrations of FZ and/or HFZ. Following this incubation, an MTS assay was performed to determine the mitochondrial respiration as a function of cell proliferation, thereby indicating the viability of cells post treatment.

Results: HFZ was significantly less toxic than FZ to renal cells. However, the addition of fungal phospholipases to the cell culture increased HFZ cytotoxicity to the levels seen for FZ. FZ treatment resulted in 80% toxicity in LLC-PK1 cells versus control whereas HFZ showed a dose-dependent increase in cytotoxicity from 20% to 50% over AmB concentrations from 10µg/ml to 50µg/ml versus control. In the presence of fungal phospholipases, HFZ renal cytotoxicity was similar to FZ renal cytotoxicity.

Conclusions: The addition of fungal phospholipases to pig kidney cells restores HFZ cytotoxicity to those observed when these cells were treated with FZ.

Acknowledgements: Funding provided with a grant from the Canadian Institutes of Health Research.

ESTABLISHING A LIMITED SAMPLING STRATEGY FOR CYCLOSPORINE (NEORAL®) IN PEDIATRIC RENAL TRANSPLANT PATIENTS
Amanda Lai, David S. Lirenman, James Carter, Morrison Hurley, Colin White, Dawn K. Strong, Mary H. H. Ensom. University of British Columbia and Children’s and Women’s Health Centre of British Columbia, Vancouver, BC

Purpose: In pediatric renal transplant patients, 1) to define the optimal limited sampling strategy (LSS) for cyclosporine (CSA) (Neoral®) monitoring and to test its predictive performance, and 2) to characterize the pharmacokinetic parameters of Neoral®.

Methods: In Phase I, 18 pediatric renal transplant patients were entered into the study, following written informed consent. Upon administration of a steady-state morning Neoral® dose, blood samples were collected at 0, 0.5, 1, 2, 4, 6, and 8 hours post-dose. The whole blood samples were analyzed for CSA by fluorescence polarization immunoassay using a specific monoclonal antibody kit. Pharmacokinetic parameters were determined via non-compartmental analysis using WinNonlin® software. LSSs were determined by multiple regression analysis with forward stepwise elimination using Statistica® software. In Phase II, 16 patients underwent regular clinical monitoring of Neoral® and had blood collected and analyzed according to the above procedures. These data were used to test the predictive performance [measured by the coefficient of determination (r2), bias, and precision] of LSSs developed in Phase I.

Results: Data from Phase I patients were used to derive the following LSSs; the predictive performance of these equations, as tested in Phase II patients, is depicted below:

For Phase I patients (Phase II patients), mean±SD AUCo-τ was 4407.73±1675.41 (5387.56±1963.20) μg*hr/L, Cmax was 1059.94±381.59 (1162.38±414.54) μg/L, Cmin was 161.61±76.11 (196.81±103.88) μg/L, Tmax was 1.39±0.50 (1.69±0.50) hours, and half-life was 4.08±1.10 (5.91±2.27) hours.

Conclusions: The optimal and most clinically feasible LSS for pediatric renal transplant recipients is AUC = 8.38C0 + 3.48C2 + 134.53 and requires 2 blood samples. This is based collectively on patient wait time, number of blood samples required, coefficient of determination, bias, and precision. For the two LSSs requiring only one blood sample, using C2 did not yield a significant advantage over the traditional use of C0 (i.e., trough monitoring) in our pediatric patients. Study findings will be incorporated directly into clinical practice.

NEUSTATE-DEPENDENT GABAERGIC INHIBITION OF SCIATIC NERVE-EVOKED RESPONSES OF DORSAL SPINOCEREBELLAR TRACTRONS
A. Chan, N. Taepavarapruk, S. A. McErlane, S. Chow, and P. J. Soja

Purpose: Monosynaptic responses of dorsal spinocerebellar tract (DSCT) neurons to sciatic nerve stimuli are reduced during active sleep (Soja et al., Soc. Neuroscience Abstracts, Bethesda, 27: 402.12., 2001). Active sleep is comprised of tonic, non-rapid-eye-movement non-(REM) and phasic REM episodes. The present study was performed to determine if active sleep-related suppression of sciatic responses occurs specifically during REM and/or NREM periods of active sleep, and if so, if GABA plays a role in this phenomenon.

Methods: Pre-recorded behavioral state parameters (electro-encephalogram-EEG, electro-occulogram-EOG and electromyogram-EMG) and short-latency (i.e., mono-synaptic, 2-5 ms) sciatic nerve-evoked spike activity of identified DSCT neurons were streamed into a computer program (Spike 2, CED, Inc.). REMs from EOG waveform activity recorded during naturally occurring active sleep were demarcated as envelope gates by a computer algorithm. Graphs depicting the magnitude of sciatic nerve-evoked responses were analyzed around (i.e., before, during and after) REM events of active sleep and compared with response magnitude during the state of wakefulness.

Results: When compared to the state of wakefulness, the magnitude of the monosynaptic sciatic nerve-evoked response obtained from 18 DSCT neurons decreased by 23.7% before and 21.1% after REM events of active sleep (P<0.05, ANOVA). During REM events of active sleep, the monosynaptic sciatic nerve-evoked response magnitude decreased further by 18.7% (P<0.05). Additional data were also analyzed from three other recording experiments where the GABAA receptor antagonist bicuculline was administered juxtacellularly to recorded DSCT neurons by microiontophoresis. When compared to the state of wakefulness, the magnitude of the sciatic response for these DSCT neurons, prior to bicuculline administration, decreased by ~40 % before or during REM events and ~ 30.4% after REM events of active sleep (P<0.05). In the presence of bicuculline, the median response magnitude around REM events of active sleep did not differ when compared with response magnitude obtained during the state of wakefulness.

Conclusions: These data indicate that during active sleep, monosynaptic transmission between primary afferents and DSCT neurons is tonically inhibited during non-REM periods and further suppressed during REM events characteristic of this state. GABA mediates this state-specific inhibition of evoked sensory transmission through the DSCT. The site of this modulation is likely to occur at the central arbors of primary afferent terminals per se and/or the postsynaptic membrane of DSCT neurons.

Acknowledgement: Supported by NIH NS32306, NS41921 (PJS) and CIHR (AC)

EFFECT OF FUNGIZONE® AND ABELCET® ON CETP-MEDIATED TRANSFER OF CHOLESTERYL ESTERS FROM LDL TO HDL
Benny Lau, and Kishor M. Wasan. Faculty of Pharmaceutical Sciences, The University of British Columbia.

Introduction: Fungizone®, the micellular formulation of amphotericin B, is mostly distributed in the lipoprotein-deficient fraction of the plasma, and when administered in this formulation, amphotericin B is associated with significant renal toxicity. In contrast, ABELCET®, the amphotericin B lipid complex formulation, is mostly found bound to HDL (high density lipoprotein) and is associated with much reduced toxicity. In previous studies, it was found that ABELCET® inhibits the transfer of cholesteryl esters and amphotericin B from HDL to LDL (low density lipoprotein), while Fungizone® had no effect.

Purpose: The purpose of this study was to determine whether the transfer of cholesteryl ester by CETP (cholesteryl ester transfer protein) from LDL to HDL is affected by Fungizone® or ABELCET®.

Methods:

Preparing lipoprotein fractions and LTP source

To obtain HDL, human plasma was separated into different lipoprotein fractions by density gradient ultracentrifugation, of which only the HDL fraction was collected. To obtain 3H-LDL, human plasma was incubated overnight with 3H-cholesteryl oleate before separation by density gradient ultracentrifugation, after which the LDL fraction was collected. The lipoprotein fractions were dialyzed in normal saline overnight prior to the assay. The source of CETP was delipidated plasma prepared immediately prior to the assay by the addition of barium chloride and phosphotungstic reagents to human plasma.

CETP transfer assay

Each drug was incubated with the CETP source at 37°C for 90 minutes. For positive controls, TP2 (monoclonal antibody directed against LTP-1) was added instead of a drug. After incubation, HDL and 3H-LDL were added; The amount of each fraction added was equivalent to 10_g of cholesterol. This mixture was incubated again at 37°C for 90 minutes. Manganese and phosphate reagents were then added to precipitate out all 3H-LDL. The supernatant, consisted of only HDL, was counted for radioactivity to determine the amount of cholesteryl ester transferred from LDL.

Results: For Fungizone®, the transfer of cholesteryl ester was decreased from 19% in the control to 7.5% in the positive control (TP2). In the treatment groups, the transfer ranged from 18.4 to 30.4%, none of which were significantly different from the control. For ABELCET®, transfer was decreased from 25.2% in the control to 10.0% in the positive control. In the treatment groups, the transfer ranged from 25.7 to 30.5%, again none of which were significantly different from the control.

Conclusions: Fungizone® does not affect CETP-mediated transfer of cholesteryl ester in either direction between HDL and LDL. In contrast, ABELCET® inhibits transfer from HDL to LDL based on previous findings, but has no effect on transfer from LDL to HDL. This uni-directional inhibition may contribute to the high distribution of ABELCET® in HDL and very low presence in LDL.

Acknowledgements: Funding for this project was provided by the Heart and Stroke Foundation and CIHR.

USING PROTEOMIC DATABASES FOR THE ANALYSIS OF GENE EXPRESSION IN HUMAN BRONCHIAL EPITHELIUM CELLS IN THE PRESENCE AND ABSENCE OF VIRAL INFECTION
Adeline Chau_, Jeffrey Lau_, Sidney Katz_, Robert Harris_. Faculty of Pharmaceutical Sciences, University of British Columbia and Department of Biological Sciences, Faculty of Science, University of British Columbia, Vancouver, BC.

PURPOSE: The objectives of this study are to determine the function of genes expressed during viral infection in Human Bronchial Epithelium (HBE) cells and to establish a list of genes to be used as potential targets for the study of the effects of natural health products such as Echinacea on the inflammatory response.

METHODS: In previous studies, HBE cells were infected with rhinovirus, Platelet Activating Factor (PAF) or both resulting in inflammation. Based on the Affymetrix proteomic system, gene expression in cells infected by the virus was found to be increased in 108 genes and decreased in 16 genes. Gene expression was observed to be increased in 9 genes and decreased in 4 genes in cells infected with PAF only. In this current study, using bioinformatic databases such as Ensembl, Source and OMIM, gene names, chromosomal location, subcellular location and functions were determined. Only genes from the virus vs. control data and from the PAF vs. control data were analyzed.

RESULTS: The function of genes whose expression was either up- or down-regulated during inflammation included those involved in cell adhesion, cell growth, cell signaling, immune response, inflammatory response, kinase function, proteolysis, receptor function, RNA binding, transcription, transferase, and transport function. Based on this initial functions list, more information was sought using proteomic databases for genes involved in receptor function, inflammatory response, immune response, cytokine function, interleukin function, transport, antimicrobial function, cAMP function, and protein kinase. Using this information, the genes chosen to be tested with herbal products were TRIM15, HPGD, C1orf38, VNN2, IL-8, STAT1, CXCL10, LGALS3BP, TNFSF10, TNFRSF6, CCR1, OASL, HLA-B, HLA-G, HLA-DPA1, PLAUR, C3, BTN3A3, and GPR37.

CONCLUSION: The list of HBE cell genes to be tested in the future with natural health products includes 19 genes primarily involved in receptor-, interleukin-, and cytokine function. Future studies will determine if Echinacea and other natural health products reverse the effects of viral infection and inflammation on HBE gene expression.

ACKNOWLEDGMENTS: Funding for this project was provided by the Canadian Institutes of Health Research.

Health Beliefs as Predictors of Heart Failure Medication Adherence
Kiran Saini1, IvanaDojcinovic1, Phuong Hoang2, Andrew Ignaszewski2, Adrian Levy2, Wolfgang Linden2, and Stephen Shalansky1&2 1Faculty of Pharmaceutical Sciences, The University of British Columbia and 2 St. Paul’s Hospital, Vancouver, BC.

BACKGROUND: Programs aimed at improving medication adherence have generally had limited success, at least in part because patients most likely to benefit have not been targeted. The objective of this study was to identify patient characteristics and beliefs associated with non-adherence with medications prescribed for treatment of heart failure.

METHODS: Consenting patients attending the Heart Function Clinic at St. Paul’s Hospital were administered a structured survey including three previously validated health belief questionnaires: the Health Belief Model (including perceived susceptibility, severity, benefits, barriers and physician perception), the Multidimensional Health Locus of Control (including internal, powerful others, and chance scores), and the Beliefs about Medications Questionnaire. The survey also included questions concerning patient demographics, living situation, cultural background, employment status, non-prescription medication use and perceived stress. Clinic records were reviewed to identify duration of heart failure diagnosis, NYHA heart failure class, ejection fraction, and heart failure etiology. Patients were classified as non-adherent if they used <80% of their prescribed heart failure medications over the past year, based on prescription refill records in PharmaNet, the British Columbia centralized prescription database.

RESULTS: Two hundred and ninety-one patients completed the survey. Twenty (6.9%) patients were categorized as non-adherent. Univariate analysis revealed statistically significant differences in use of compliance aids, number of CHF medications taken, daily dosing regimen, and furosemide use between adherent and non-adherent patients. Of the health beliefs surveys employed, only the necessity score from the Beliefs about Medications Questionnaire was significantly different between the groups (20.4± 4.4 for adherent patients versus 17.6± 3.4 for non-adherent patients, P<0.001). Multivariate regression analysis identified the necessity score (OR 0.85 per unit increase, 95% Cl 0.75 – 0.97, P=0.02), frequency of medication taking (OR 0.26 per unit increase, 95% Cl 0.11 – 0.61, P=0.002) and furosemide use (OR 0.25, 95% Cl 0.10 – 0.69, P=0.005) as the only significant independent predictors of non-adherence. A necessity score of ≤12 appeared to be a threshold that discriminated between adherent and non-adherent patients: 50% (3 of 6) of the patients who scored ≤12 were non-adherent, while only 1.1% (3 of 271) of the adherent population had a necessity score ≤12

CONCLUSION: Heart failure patients attending the Heart Function Clinic at St. Paul’s Hospital had very high adherence with CHF medications, making it difficult to identify predictors of non-adherence. In this cohort, the necessity score from the Beliefs about Medications Questionnaire was an independent predictor of non-adherence, and a score of ≤ 12 appears to be a useful threshold for identifying potentially non-adherent patients. However, studies involving a larger number of non-adherent patients are required to further clarify the relationship between health beliefs and medication adherence.

Consequences of the Interaction between a Lipophilic Endotoxin Antagonist, E5564, with Plasma Lipoproteins
Eugene K. Choo1, Daniel P. Rossignol2, Nancy S. Chung1, Kishor M. Wasan1. 1Faculty of Pharmaceutical Sciences, the University of British Columbia. 2Eisai Medical Research, Glenpointe, NJ, USA.

Objective: To assess the pharmacological effect of E5564 when associated with high-density lipoproteins (HDL), low-density lipoproteins (LDL), triglyceride-rich lipoproteins (TRL which consists of very low density lipoproteins and chylomicrons) and plasma proteins (primarily albumin and alpha-1-glycoprotein) within the lipoprotein-deficient fraction (LPDP).

Methods: E5564 Formulation: E5564 (Lot No. L0900006-1), synthesized by Eisai Research Institute, (Andover, MA) was provided as a lyophilized powder. Lipopolysaccharide (LPS) formulation: LPS (Sigma) was dissolved in normal saline and 2mL aliquots of 100ng/mL were stored at -80ºC prior to use. Plasma lipoprotein separation & preparation: The HDL, LDL, TRL and LPDP fractions were separated by density step-gradient ultra-centrifugation. The cholesterol/triglyceride/protein content of each fraction was determined by enzymatic colorimetric assays (ThermoDNA and BioRad respectively). TNF-alpha assay with human whole blood: Fresh human blood was collected into a syringe containing heparin (1:100 v/v). Heparinized blood (400_L) including 10ng/mL LPS and the treatment (10 nm of E5564 mixed with different concentrations of HDL, LDL, TRL and LPDP) were co-incubated with gentle shaking at 37ºC and 5% CO2 atmosphere. After 3 hours, the plates were centrifuged at 1,000xg for 10 minutes at 4ºC. Plasma was then collected and stored at -80ºC until analysis. The plasma samples were analyzed for TNF-_ by an enzyme-linked immunosorbent assay (eBioscience).

Results: A dose response curve was constructed by treating LPS-exposed human whole blood to increasing concentrations of E5564 (0-0.2µM). Plasma TNF- _ concentrations were found to be reduced by greater than 50% compared to control at a E5564 dose of 6.25nM and greater than 95% compared to control at doses higher than 25nM of E5564. In a second set of studies, E5564 (10nM) was co-incubated with physiological concentrations (observed in septic patients) of each lipoprotein/lipoprotein deficient fraction (HDL/LDL/TRL/LPDP) overnight in a water bath set at 37ºC. When incubated with either LDL or TRL, E5564 maintained its activity, reducing plasma TNF- _ concentrations by 50-75% compared to controls. However, when E5564 was pre-incubated with HDL or LPDP fractions, the inhibitory effect of E5564 on TNF-_ release was completely eliminated.

Conclusions: E5564 is a potent inhibitor of LPS-induced TNF- _ release from red blood cells. However, the antagonistic effect of E5564 on TNF- _ release was significantly diminished when it is co-incubated with HDL and plasma proteins from the LPDP fraction, yet it retains its activity when co-incubated with LDL or TRL. Taken together, this data suggests that the mechanism for loss of E5564’s activity may be because of irreversible binding to proteins found in HDL and LPDP fractions.

Acknowledgements: Science Council of BC, Canadian Institute of Health Research and Health Research Foundation Scholarship & Eisai Research Institute.

INVESTIGATING POTENTIAL GLUCOSE LOWERING PROPERTIES OF ORGANIC VANADIUM COMPOUNDS AND TRANSITION ELEMENT-MALTOLATO COMPOUNDS
Jeremy Tse1, Violet Yuen1, Chris Orvig2, and John H. McNeill1. 1Faculty of Pharmaceutical Sciences, The University of British Columbia and 2Department of Chemistry, The University of British Columbia.

Purpose: The objective of this study was to compare the potential glucose lowering properties of bis(curcumin)oxovanadium(IV) (BCOV) with that of bis(ethylmaltolato)oxovanadium(IV) (BEOV). We also tested the hypothesis that various metal ions complexed to the maltolato ligand would produce the same glucose-lowering response at the ED50 dose for bis(maltolato)oxovanadium(IV) (BMOV). A third objective of this study was to determine whether the use of 1% carboxymethylcellulose (CMC) or 3% gum arabic (GA) as suspending agents had any effect on the response of the drugs used.

Methods: Male Wistar rats (190-210g) obtained from Charles River and the U.B.C. Animal Care Centre (ACC) were made diabetic by a single tail vein injection of streptozotocin (STZ) of 60mg/kg. Compounds to be tested were administrated by oral gavage as a suspension. In a cumulative dose response study comparing BCOV with BEOV, rats were initially dosed at 0.4mmol/kg in either 1% CMC or 3% GA and were dosed every 24 hours for 1 week. Changes in dose were made based on blood glucose readings taken prior to dosing. Animals in a chronic study comparing BCOV with BEOV were initially dosed at 0.2mmol/kg/day in 1% CMC for 2 weeks, after which doses were adjusted every 24 hours for an additional 3 weeks. In both the cumulative dose response study and the chronic study, blood samples were taken immediately prior to each dosing. Animals in a study comparing BMOV to tris(thiomaltolato)oxovanadium(III) (TTOV), bis(isomaltolato)oxovanadium(IV) (BIOV), tris(maltolato)chromium(III), cis-bis(maltolato)dioxomolybdenum(VI), bis(maltolato)zinc(II), bis(maltolato)cobalt(II), and bis(maltolato)copper(II) were given a single dose of 0.6mmol/kg of their respective compounds as a suspension in 1% CMC and were monitored for 3 days with blood collections immediately prior to drug administration and at 4, 8, 12, 24, 48, and 72 hours following administration. Plasma samples were analyzed for glucose levels with a Beckman 2 Glucose analyzer. Results were analyzed with GLM ANOVA and one-way ANOVA tests followed by Newman-Keuls test. Values were considered significant if p < 0.05.

Results: In the cumulative dose response study, a comparison of basal and final glucose values found that BEOV in either GA (n=6) or CMC (n=6) significantly lowered plasma glucose (given in mmol/L ± standard error mean) from 26.6±1.3 to 11.0±1.8 and from 26.2±1.1 to 7.5±1.3 respectively. Five and six animals from the BEOV in GA and CMC groups responded (blood glucose <10mmol/L) respectively. Plasma glucose was not significantly lowered in BCOV in GA (n=6), BCOV in CMC (n=6), and diabetic control (n=6) groups with one and three animals responding in the BCOV in GA and CMC groups respectively. There was no significant difference between respective GA and CMC groups. In the chronic study, it was found that 2 weeks of chronic low dose treatment did not significantly lower glucose levels in BEOV treated (n=9), BCOV treated (n=9), and diabetic controls (n=5). Following incremental dosage, plasma glucose levels were significantly lowered in all six animals of the BEOV treated group from 29.3±0.9 to 9.8±1.3. Only one animal in the BCOV group responded. In a comparison of BMOV with the various maltolato compounds, plasma glucose was significantly lowered in at least four consecutive time points in only the BMOV (n=6) and molybdenum (n=6) treated groups. However, only BMOV significantly lowered plasma glucose levels enough for euglycemic effect, with a peak response at 12 hours (26.0±1.1 to 6.3±0.7), where all 6 animals responded. Diabetic controls (n=8), TTOV (n=5), BIOV (n=4), copper (n=8), cobalt (n=8), zinc (n=7), and chromium (n=7) treated groups did not significantly lower plasma glucose levels.

Conclusions: The use of 1% CMC and 3% GA as a suspending agent did not have a significant effect on the biological efficacy of suspensions. BCOV was not an effective glucose lowering compound under these conditions. Our results do not support our hypothesis that varying the metal ion complexed to the maltolato ligand would produce the same glucose-lowering response as BMOV at its ED50 dose. In conclusion, BEOV and BMOV were able to significantly lower glucose levels, suggesting that vanadium is superior amongst transition elements for glucose lowering.

Acknowledgements: Funding for this project was provided by the Merck Company Foundation National Summer Student Research Program 2003 and a grant from CIHR.

The encapsulation and release of antisense oligonucleotides from an injectable, controlled release, polymeric paste
Jessica M. Chong, John K. Jackson, and Helen M. Burt.

Purpose: The daily intravenous administration of clusterin antisense oligonucleotide (ASO-C) has been shown to reduce tumour size in a mouse model of prostate cancer. The purpose of this project was to investigate the in vitro drug release properties of a polymeric paste formulation of ASO-C. Site-directed polymeric drug delivery systems based on an intratumoral injection of a controlled release formulation of the ASO-C are being developed. This would allow for the sustained release of the drug without the need for daily injections.

Methods: ASO-C was combined with a cationic microparticulate (Chitosan) and suspended in a polymeric paste composed of a triblock (TB) copolymer of poly(L-lactide-co-caprolactone)-block-PEG-block poly(L-lactide-co-caprolactone) blended with methoxypolyethylene glycol (MePEG). Two types of ASO-C were used; 2'-O-(2-methoxy)ethyl (MOE) ASO-C and phosphothioated ASO-C. Test tubes contained various ratios of ASO-C to Chitosan suspended in a paste; with 4% w/w Chitosan and 0.4%, 1%, 2%, or 3% w/w ASO-C. A set of ASO-C control samples was made without the inclusion of the Chitosan in the paste. Formulations were incubated in 10 mM Phosphate Buffer Saline Solution (pH 7.4) at 37ºC and samples were taken for daily ASO-C quantitation by ion-exchange HPLC methods. After one month of sampling, one paste fraction from each set, as well as the control samples, were broken down by vortexing and sonicating in 50 mM Phosphate Buffer Solution (pH 10). The supernatant was centrifuged and analyzed to obtain the residual amounts of ASO-C that was in the paste. The three other paste fractions from each set were incubated in 0.4 ug/mL lysozyme in 10 mM Phosphate Buffer Saline Solution (pH 7.4) at 25ºC. The lysozyme was added to determine if the chitosan would be broken down to release any remaining ASO-C.

Results: The percent release of ASO-C was proportional to the ratio of ASO-C to Chitosan in the paste formulation. The ASO-C was released from the Chitosan-containing paste formulations with an initial lag phase of four days, followed by a burst phase of seven days, followed by a slow, sustained release. In contrast, the control samples had an initial burst phase of three days, followed by minimal ASO-C release lasting eleven days. Residual analysis confirmed the presence of unreleased ASO-C.

Conclusion: The injectable polymer paste containing Chitosan/ASO-C complexes offers a potentially suitable formulation for the controlled release of this antisense oligonucleotide. The dose of ASO-C released may be controlled by varying the ratio of ASO-C to microparticulate suspended in the paste.

Acknowledgement: This work was supported by a grant from ARC Pharmaceuticals, Vancouver, B.C.

Formulation Development of FDC-2-4, a Novel Phytostanyl Analogue, into a Non-toxic Aqueous Solution for in vivo Cholesterol Uptake Studies Using Rats
Shabnam Iqbal 1, Simon Wallis 2, Eugene Choo 1, Verica Risovic 1, Kishor M. Wasan 1. 1 Faculty of Pharmaceutical Sciences, The University of British Columbia and, 2 Forbes Medi-Tech, Inc.

Purpose: The purpose of this project was to formulate the water insoluble FDC-2-4 into a 4mg/ml to 8mg/ml aqueous solution using a surfactant. An additional objective was to characterize the formulation and perform in vivo cholesterol uptake studies using rats.

Methods: FDC-2-4 and the surfactant being tested were dissolved using a suitable organic solvent; heat was applied if necessary. The organic solvent was removed by nitrogen evaporation and water was added to the test tubes. The mixtures were visually inspected for cloudiness or particles. The formulation developed was characterized using light microscopy, particle size measurement, zeta potential measurement, and transmission electron microscopy. The formulation was administered to rats to measure cholesterol uptake in the presence of FDC-2-4.

Results: After testing a number of surfactants and organic carrier solvents, a successful formulation was developed using Tween 80. Solutions containing 4mg and 8mg of FDC-2-4 were formulated, with 120mg and 240mg of Tween 80, respectively (per ml of formulation). Microscopy at 40x and 100x magnification revealed no difference in the amount or type of particulate matter between the selected FDC-2-4 formulation and control sample. Zeta potential measurement of the 4mg/ml FDC-2-4 formulation was -5.9mV, -9.8mV, and -4.7mV, with an average zeta potential of -6.8mV +/- 2.7mV. The mean particle size measurement of the 4mg/ml solution was 4.3nm. Administration of the 4mg/ml formulation to rats resulted in a 45.6% inhibition of cholesterol uptake in comparison to the controls.

Conclusions: FDC-2-4 can be solubilized into an aqueous solution using Tween 80. Administration of the developed FDC-2-4 4mg/ml formulation to rats showed a 45.6% inhibition of cholesterol uptake compared to controls.

Acknowledgements: Funding for this project was provided by the Canadian Institutes of Health Research and Forbes Medi-Tech, Inc.

Lysophosphatidylcholine mediated recruitment of cardiac luminal LPL requires generation of lysophosphatidic acid
Patsy Yip and Brian Rodrigues. Faculty of Pharmaceutical Sciences, The University of British Columbia

Purpose: Coronary lipoprotein lipase (LPL) actively metabolizes the triglyceride (TG) core of lipoproteins to fatty acid (FA) for myocardial energy production. As endothelial cells (EC) cannot synthesize LPL, it is synthesized and activated in myocytes and then translocated across the interstitial space onto heparan sulfate proteoglycan (HSPG) binding sites on the luminal surface of EC. The mechanism of LPL transfer from the myocyte to the EC is poorly understood. Recently, the lysophosphatidylcholine (lysoPC) generated following TG breakdown has been shown to augment cardiac luminal LPL. The objective of this study was to examine the mechanism by which lysoPC increases cardiac LPL on the luminal surface.

Methods: Retrograde Langendorff perfusions were performed on control hearts from two treatment groups: 1) LysoPC (1 nM, n=8) and 2) Cycloheximide (CHX, 2 mg/kg, i.p.) with subsequent in vitro treatment by lysoPC (1 nM, n=8). In a separate experiment, cardiac myocytes were incubated with either lysoPC (1 nM, n=5) or lysophospatidic acid (LPA) (1 nM, n=5).

Results: The luminal LPL of hearts perfused with lysoPC increased three fold as compared to control and those treated with CHX prior to lysoPC perfusion. Cardiac myocytes incubated with lysoPC did not demonstrate any change in myocyte LPL but when incubated with LPA significantly enhanced basal and heparin releasable LPL activity.

Conclusions: Since prior treatment with CHX, an agent that blocks myocyte LPL synthesis, prevented the ability of lysoPC to increase luminal LPL, our data suggests that lysoPC mobilizes LPL from the cardiomyocytes. Interestingly, lysoPC mediated increases in luminal LPL was not attributed to changes in the mycoyte LPL suggesting that lysoPC probably undergoes biochemical degradation to form LPA in the EC before exerting its LPL augmenting property. In summary, our data suggest that lysoPC dependent increases in LPA augments myocyte and subsequently luminal LPL. Since exaggerated LPL levels, with ensuing free fatty acid (FFA) supply and utilization, has been implicated in the initiation and progression of heart disease during diabetes, an understanding of mechanisms involved in LPL translocation will help in reducing diabetic heart disease.

Pharmacological Investigation of Kinase Pathways Activated by Phenylephrine and A-61603 in the Aorta and Tail Arteries of Rats
Perminder Bains1, Irem Mueed1, and Kathleen MacLeod1, Faculty of Pharmaceutical Sciences, The University of British Columbia

Purpose: The objective of this study was to determine the relative contributions of Rho kinase (RhoK) and Protein kinase C (PKC) calcium sensitization pathways on smooth muscle contraction in response to _1 adrenoreceptor stimulation in rat aorta and tail arteries. The effect different agonists and receptor subtypes have in relation to this contraction were also examined.

Methods: Concentration dose-response curves to the nonselective _1 adrenoreceptor agonist Phenylephrine (PE 10-9 to 10-5M) and the selective _1A adrenoreceptor agonist A-61603 (10-9 to 10-5) were determined in the presence and absence of the RhoK blocker Y-27632 (10-6M), or the PKC blocker Ro-318220 (3x10-6M) in isolated rat aorta and tail arteries. Contractile responses were expressed as a percentage of the initial maximal response to the agonist. Data was examined by non-linear regression analysis to determining pD2 (-log EC50) and the maximum response (Rmax). Results were evaluated for statistical significance using two-way ANOVA followed by Neuman-Keul’s Multiple Comparison test.

Results: The higher sensitivity of the tail towards A-61603 compared to PE is shown by their respective pD2 values (A-61603 8.1±.1, n=12 and PE 6.1±.1, n=9). Rmax values expressed as %maximum KCl show that the maximum responses are similar (A-61603 Rmax=229.1±36.6 n=6, and PE Rmax=199.1±21.5 n=6). The aorta was more sensitive to PE than A-61603 (PE pD2=7.5±.1 n=11, and A-61603 pD2=6.1±.1 n=12), while the maximal responses were not statistically different (PE Rmax=138.5±11.0 n=6, and A-61603 Rmax=121.2±5.0, n=6).

In aorta, both the RhoK inhibitor Y-27632 and the PKC inhibitor, Ro-318220 antagonized responses to PE and A61603. Both Y-27632 and Ro-318220 shifted the PE dose-response to the right, without affecting the maximum response to this agonist. The change in the PE pD2 value (ΔpD2) produced by Y-27632 (0.7± 0.1) was not significantly different from that produced by Ro-318220 (0.4±0.1). The maximum response to PE in the presence of Y-27632 was 91.1±7.1%, which was not significantly different than that in the presence of Ro-318220 (99.0 ± 5.1%). In contrast, the two antagonists reduced the A61603 Rmax, while having little or no effect on the pD2 value. Y-27632 reduced the A61603 Rmax to 71.8±8.0%, while Ro-318220 reduced it to 82.8 ±13.5% of the response in the absence of the inhibitor. These values were not significantly different.

In the tail artery, Ro-318220 had no significant inhibitory effect on the response to PE (ΔpD2, 0.03 ± 0.2, Rmax, 90.5 ±11.6%). However, Y-27632 shifted the PE dose-response to the right (ΔpD2, 0.8± 0.1) without affecting the Rmax (94.5 ± 2.5%). Y-27632 also shifted the A61603 dose-response curve to the right (ΔpD2, 0.4± 0.1) without significantly affecting the maximum response (90.6±7.3%). However, the change in PE pD2 value was significantly greater than the change in A61603 pD2 value in the presence of Y-27632. Ro-318220 also shifted the dose-response curve of tail artery to A61603 (ΔpD2, 0.4± 0.1) and in addition, significantly reduced the maximum response to 72.1± 13.4% of the response in the absence of A61603.

Conclusions: The increased sensitivity of the tail artery compared with the aorta to the _1A selective agonist A-61603 relative to the non-selective agonist PE is consistent with the evidence that the aorta has mainly _1D receptors, while the tail contains mainly _1A receptors. The results suggest that both the RhoK and the PKC pathways contribute to contractile responses of aorta to both PE and A61603. In contrast, in the tail artery, the RhoK pathway appears to be activated in response to both PE and to A61603, but the extent of the contribution is greater in the case of PE. On the other hand, PKC does not appear to contribute to the response of tail artery to PE, whereas it does appear to play a role in the response to A61603. These data suggest _1D receptors in aorta and _1A receptors in tail artery are both coupled to the signaling pathways leading to activation of PKC and RhoK. The reason that PKC does not appear to contribute to the PE response in tail artery is not clear. However, there could be differences in the interaction of PE and A61603 with the receptor in this artery, leading to differences in subsequent activation of signal transduction pathways.

Acknowledgements: Funding for this project was provided by the Canadian Institute of Health Research, and an operating grand provided by the Heart and Stroke Foundation of BC & Yukon.

THE INDUCTION OF HEMOLYSIS BY MePEG-PCL DIBLOCK POLYMERS: AN INDICATION OF MEMBRANE PERMEABILITY MODIFICATION
Mike Borslein, Jason Zastre, John Jackson, and Helen Burt. Faculty of Pharmaceutical Sciences, The University of British Columbia.

Introduction: Amphipathic diblock copolymers composed of methoxy poly(ethylene glycol)-block-polycaprolactone have been shown to inhibit p-glycoprotein (p-gp) and allow the accumulation of drugs susceptible to p-gp efflux. It is proposed that this inhibitory effect is related to the penetration of the diblocks into cell membranes and subsequent membrane permeability effects; which can alter the activity of p-gp or enhance trans bilayer permeation. The objective of this study was to investigate erythrocyte membrane permeabilization of diblock copolymers measuring the release of hemoglobin.

Methods: Five diblock copolymers, with different methoxy-poly(ethylene glycol) (MePEG) and polycaprolactone (PCL) block lengths, were examined. There was a minimum of three trials (n=3) for each diblock copolymer, each with three replicates of each sample. Fresh human red blood cells (RBCs) were collected and centrifuged; a small fraction was diluted with PBS to a final concentration of 1.0x10-7 cells/mL as determined by a hemacytometer. Serial dilutions of diblock copolymer were made which was added to a RBC suspension and incubated at 370C with gentle mixing for five seconds every ten minutes. Samples were removed at 1, 2, and 4 hour time points. Then centrifuged and assayed for hemoglobin release using UV-Vis spectroscopy (absorbance at 540 nm).

Results: For all five diblock copolymers the induction of hemolysis was both time and concentration dependent. The diblock copolymers with a high ratio of hydrophilic (PEG) to hydrophobic (PCL) caused higher degrees of hemolysis. For 1% copolymer solutions after four hours, percent lysis increases from MePEG12-PCL4 (15%) to the highest MePEG44-PCL4 (40%). MePEG17-PCL10 had very poor solubility at this concentration and so it cannot be compared.

Conclusions: Red blood cells provide a good model for membrane permeation studies as both the extent and rate of hemolysis may be measured. Diblock copolymers composed of MePEG-b-PCL induce hemolysis in RBCs indicative of cell membrane permeation.

Acknowledgements: Funding for this project was provided by a grant awarded to Dr. Helen Burt from Canadian Institute of Health Research.

PICTOGRAPHIC INSTRUCTIONS FOR MEDICATIONS: DO OTHER CULTURES INTERPRET THEM ACCURATELY?
Zahra Sadikali 1, LCol Régis Vaillancourt 2, John B. Collins 3, Rosemin Kassam 4. 1 Research Assistant, Faculty of Pharmaceutical Sciences, The University of British Columbia. 2 Directorate of Medical Policy, Pharmacy Policy and Standards, Canadian Forces. 3 Adjunct Professor, Department of Educational Studies, The University of British Columbia. 4 Assistant Professor; Director-Structured Practice Education Programs, Faculty of Pharmaceutical Sciences, The University of British Columbia.

Background: Dispensing medication is a major service provided by Canadian Forces humanitarian relief missions around the world, often in developing countries. This study tested a set of sixteen pre-developed pictograms to determine whether they accurately communicated the written directions found on medication labels to ethnic respondents who neither speak nor read English, French or Spanish.

Purpose: (1) To determine whether ethnically diverse individuals could understand the pictogram meanings without additional aids such as verbal instructions or explanations, and (2) to identify appropriate modifications to the pictograms to reduce interpretation errors.

Method: Both qualitative and quantitative methods evaluated the pictograms’ interpretability among three ethnic groups: Cantonese, Somali and Punjabi. Standard ANOVAs tested for differences due to ethnicity and other demographics.

Results: Only four of the 16 initial pictograms tested were interpreted correctly by 80% of participants. Relaxing the criterion from 80% to 50% included eight more. Modifications to problem icon elements further improved interpretation accuracy levels by 22% for a ‘best-of-three’ tally of 67.15%. Quantity errors were twice as common as timing, administration route or auxiliary instruction errors.

Conclusions: Participants identified particular pictographic symbols they found confusing or ambiguous. Basic education and time since immigration predicted interpretation accuracy better than ethnicity or any other demographic characteristic.

Acknowledgements: Funding for this project was provided by the Canadian Forces. Recruitment of participants was made possible through the assistance of both Mosaic Settlement Services and the Somali Services Society of British Columbia.

Release of Paclitaxel from poly (lactic-co-glycolic acid) (PLGA) microspheres: Effect of diblock copolymers on drug release
Tawny Hung and Helen Burt. Faculty of Pharmaceutical Sciences, The University of British Columbia

Introduction: Paclitaxel (PTX) may be incorporated at high efficiencies into PLGA microspheres. These microspheres may be employed as injectable, controlled release dosage forms of PTX for use in vivo. However, PTX is released very slowly from these microspheres (less than 5% of total drug released in 1 month). It is proposed that amphipathic diblock copolymers based on polycaprolactone (PCL) or polylactic acid (PLA) and methoxypolyethylene glycol (MePEG) might be incorporated into PLGA microspheres to increase the release rate of PTX.

Methods: Three diblock copolymers were manufactured using MePEG (mol wt 750) with either 2, 5, or 10 units of caprolactone polymerized onto one end (termed PCL2, 5, 10 respectively). One diblock copolymer was manufactured using 20 units of PLA on MePEG (mol wt 2000). Five % PTX-loaded microspheres were manufactured using an oil in water solvent evaporation method from PLGA blended with either zero, 10, 20, 30, or 40% of PCL2, 5, 10 or PLA20 (17 batches in total). Diblock copolymer incorporation into the microspheres was assayed by the gel permeation chromatography (GPC), while paclitaxel encapsulation was determined by reverse-phase HPLC methods. Diblock copolymer miscibility with PLGA was tested by the differential scanning calorimetry (DSC). Paclitaxel release was measured by removing phosphate buffer solution (PBS) with albumin from the incubation test tubes at appropriate times. Drug release was analyzed by HPLC. Residues obtained at the end of the release study were assayed using the GPC to determine the relative amounts of copolymer, PLGA and paclitaxel remaining.

Results: All microspheres manufactured with diblock copolymers gave high yields (80%-90%), except for sets with 40% loaded copolymers (yields at 40%-55%). The GPC analysis showed that PCL2 was poorly encapsulated whereas PCL5, PCL10 and PLA20 were encapsulated at high efficiencies. Paclitaxel incorporation efficiencies, as determined by HPLC, were high (80%-90%), and DSC analysis of glass transition temperatures (Tg) showed that all the diblock copolymers were miscible with PLGA. The paclitaxel release study showed slow drug release for 100% PLGA microspheres (less than 3% in 10 days). However, when loaded with diblock copolymers, the microspheres released paclitaxel more quickly. For the PCL5, PCL10 and PLA20 microspheres, 30% diblock loaded formulations gave a sharper increase in drug release rate than 10% and 20% microspheres. Residues from the release study showed that most of the PCL5, 10 and PLA20 diblocks had dissolved from the PLGA microspheres at 30% loadings. However, significant amounts of diblock copolymer remained in the microspheres at 10% and 20% loadings for all the diblock sets.

Conclusion: Diblock copolymers with higher hydrophobicities (PCL5, 10 and PLA20) were effectively loaded into PLGA microspheres and were found to be miscible. These diblocks may be incorporated into paclitaxel loaded PLGA microspheres to increase the release rate of the drug in a concentration dependent manner.

Effect of Warfarin on Haloperidol Lipoprotein and Protein Association within Normolipidemic and Hyperlipidemic Human Plasma
Tiffany Ho 1, Ric M. Procyshyn 1,2 and Kishor M. Wasan 1. 1 Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada 2 Riverview Hospital, Port Coquitlam, British Columbia, Canada.

Purpose: The objective of this study was to determine the protein and lipoprotein association of Haloperidol in the presence of different concentrations of warfarin in normolipidemic and hyperlipidemic human plasma.

Rationale: Haloperidol is highly bound to plasma proteins albumin and _-1 glycoprotein. It was hypothesized that the co-administration of warfarin, another drug that is extensively bound to albumin and _-1 glycoprotein, may displace Haloperidol from these proteins altering the drug’s plasma distribution.

Methods: Warfarin sodium was pre-incubated in normolipidemic and hyperlipidemic human plasma at different concentrations (1, 5, 10 µg/mL) for 24 hours at 37˚C (n=6). Following the pre-incubation with warfarin, [3H]Haloperidol mixed with unlabeled Haloperidol (total concentration = 18 ng/mL) was incubated in this plasma for 1 hour at 37˚C. Treated plasma samples were separated into four fractions: triglyceride-rich lipoproteins (TRL), low-density lipoproteins (LDL), high-density lipoproteins (HDL), and lipoprotein-deficient plasma (LPDP) by density gradient ultracentrifugation. Each lipoprotein and lipoprotein-deficient fraction was assayed for [3H]Haloperidol by radioactivity. In addition, 100µL of the LPDP fraction from each treated hyperlipidemic plasma sample was pipetted into an ultrafiltration column to distinguish free Haloperidol from protein-bound Haloperidol. The filtrate and filter from the devices as well as 100µL of each LPDP sample were assayed for [3H]Haloperidol by radioactivity to determine the amount of free compared to protein-bound Haloperidol.

Results: In normolipidemic plasma, increasing concentrations of warfarin did not significantly alter the lipoprotein distribution of haloperidol. In hyperlipidemic plasma, increasing concentrations of warfarin correlated with an 11.5% decrease in the amount of haloperidol recovered in the LPDP fraction and a concurrent 4.8% and 5.2% increase in the amount of haloperidol recovered in TRL and LDL fractions, respectively. As warfarin concentration increased, the percentage of protein-bound Haloperidol decreased from 65.8% ± 2.2% (0µg/mL) to 57.0% ± 3.2% (10µg/mL).

Conclusion: In normolipidemic plasma, increasing concentrations of warfarin did not significantly affect the lipoprotein distribution of haloperidol.

In hyperlipidemic plasma, increasing concentrations of warfarin correlated with a significant decrease in amount of haloperidol recovered in the LPDP fraction and concurrent increases in the amount of Haloperidol recovered in the TRL and LDL fractions. Increasing warfarin concentrations significantly decreased the percentage of protein- bound Haloperidol in the LPDP fraction

Acknowledgements: Funding for this project was provided by CIHR and Riverview Hospital

The Effect Of Complementary And Alternative Medicine Usage Patterns On Warfarin-Related Adverse Events
Esther A. Abd-Elmessih, B.Sc. 1,2; Shelly A.V. To 1,2; Stephen J. Shalansky, Pharm.D. 1,2 ; and Larry Lynd, B.S.P., Ph.D. Candidate 2. 1 Faculty of Pharmaceutical Sciences, The University of British Columbia 2 St. Paul’s Hospital, Vancouver, British Columbia

PURPOSE: The purpose of this study was to identify the usage patterns of complementary and alternative medicines (CAMs) amongst a cohort of patients taking warfarin. The risk of warfarin-related adverse outcomes (INR ≥ 4 or bleed) for CAM-users (consistent and fluctuating use) compared to non-CAM-users was calculated. In addition, consumption patterns and risk associated with use of over-the-counter (OTC) medications, vitamins and supplements were identified.

METHODS: Patients taking warfarin were identified through a review of pharmacy, cardiac rehabilitation clinic, anticoagulation clinic and atrial fibrillation clinic records. Those patients consenting to participate kept weekly diaries for a 16-week period, recording exposure to factors potentially increasing the risk of warfarin-related adverse events (INR ≥ 4 or bleed) including: acute illnesses, alcohol consumption, OTC drug use, vitamin K-containing food consumption and CAM use. Patients were also queried as to their daily doses of warfarin, bleeding episodes, and hospitalizations. British Columbia’s provincial prescription database (Pharmanet) was accessed, providing information on prescription medication use during the diary period. Patient INR values were obtained from medical laboratories when such data were available.

RESULTS: The study was completed by 118 patients, totaling 1827 diary weeks, 54 (46%) of whom reported to have used at least one CAM. The most commonly reported interacting CAMs were vitamin E (27%), garlic (9.3%), devil’s claw (3.4%) and parsley (2.5%). There was no statistically significant difference in the proportion of patients experiencing bleeds between non-users 39/73 (53%), consistent users 14/26 (54%) and fluctuating users 8/19 (42%) of CAMs reported to increase the risk of bleeding (P=0.661). Of the 101 patients for whom INR results were available, the time in therapeutic range (TTR) for non-users, consistent users and fluctuating users of CAMs that potentially elevate INRs were 60%, 65% and 56% respectively (P=0.681), and of CAMs that potentially decrease INRs were 61%, 73% and 39% respectively (P=0.208). Although the risk of bleeding was not affected by non-use, consistent use or fluctuating use of OTCs (P=0.251), fluctuating users of acetaminophen were more likely to have elevated INRs (28%) than non-users (8.5%) and consistent users (0%), (P=0.017).

CONCLUSIONS: The use of potentially interacting CAMs was common amongst warfarin patients, and patients often recorded fluctuating usage patterns. The proportion of patients reporting bleeds or having INRs outside therapeutic range was not different between non-users, consistent users and fluctuating users of interacting CAM products. However, fluctuating users of acetaminophen were more likely to experience elevated INRs compared to non-users and consistent users.

ACKNOWLEDGMENTS: Erin Neall, B.Sc. (Pharm) 1,2, Melissa Lo 1,2, B.Sc.(Pharm) 1,2, Biljana Radulovic, B.Sc.(Pharm) 1,2, Curtis R. Sedlak, B.Sc.(Pharm) 1,2, Ivana Dojcinovic 1,2, Kiran Saini1,2. 1 Faculty of Pharmaceutical Sciences, The University of British Columbia, 2 St. Paul’s Hospital, Vancouver, British Columbia 


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