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Biomedical subjects

John E Murphy

Publications and source records attributed to John E Murphy.

15 recordsLinked to original sources
PubMed2007

Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion.

Production of coagulation factor VIII (FVIII) by recombinant cell lines is limited by its failure to reach or maintain the native conformation in the endoplasmic reticulum. This results in significant cytoplasmic degradation and/or aggregation of the misfolded product. The molecular chaperone Hsp70 was overexpressed in an attempt to increase the recombinant FVIII (rFVIII) secretion. The characteristics of increased Hsp70 expression were investigated by comparing a clone of BHK-21 cells expressing rFVIII (rBHK-21(host)) to a chaperone clone derived by transfection of the host clone with human Hsp70 (rBHK-21(Hsp70)) in small-scale batch cell cultures. To aid this investigation a number of fluorescence based cellular apoptosis assays were developed and optimized. These assays demonstrated sub-populations of rBHK-21(host) cells that were apoptotic in nature and were identified prior to the loss in plasma membrane integrity. Dual staining for intracellular rFVIII and caspase-3 activation showed a reduction in intracellular rFVIII in rBHK-21(host) cells that correlated with a significant increase in active caspase-3, suggesting that apoptosis was a factor limiting rFVIII secretion. In sharp contrast there was more intracellular rFVIII and less active caspase-3 in rBHK-21(Hsp70) cell cultures. Moreover when grown in batch culture, rBHK-21(Hsp70) cells released rFVIII of higher specific activity (active FVIII protein/total FVIII protein), suggesting improved product quality. Thus, increased expression of HSP70 led to an increased yield of a secreted recombinant protein by inhibition of apoptosis and promoting proper conformational maturation of rFVIII in sub-optimal bioreactor conditions.

Animals↗
PubMed2006

Predictability of vancomycin trough concentrations using seven approaches for estimating pharmacokinetic parameters.

PURPOSE: Seven methods for estimating vancomycin pharmacokinetic parameters were studied to determine which method best predicted measured concentrations for patients at a community teaching hospital. METHODS: Data from adult patients who were given vancomycin and had at least one steady-state trough concentration measured were retrospectively reviewed. Data analyzed included laboratory test values, concomitant medications, weight, height, sex, age, laboratory cultures, medical procedures performed, vancomycin dose and interval, measured vancomycin concentrations, and time of measurement. Relevant data were used in seven predictor methods that estimate volume of distribution, vancomycin clearance, and elimination rate constant to determine which yielded the best predictions of actual measured concentrations in the patient population. RESULTS: Data from 189 patients were included in the analyses. The coefficients of determination for the methods ranged from 0.114 to 0.234. Bias ranged from -5.90 to 0.69 mg/L, and precision ranged from 6.05 to 8.08. The Matzke method had the best combination of the least bias and best precision. Predictions were within 2.5 and 5 mg/L of measured concentrations 18.0-43.9% and 43.4-66.1% of the time, respectively. The percentage of predictions within 25% and 50% of measured concentrations ranged from 7.9% to 31.2% and from 18.0% to 48.1%, respectively. Ten (5.3%) patients had trough concentrations exceeding 20 mg/L, and 11 (5.8%) had trough concentrations of < or = 3 mg/L. CONCLUSION: The seven methods studied for estimating vancomycin pharmacokinetic parameters varied widely in predicting vancomycin trough concentrations compared with measured serum concentrations and were not sufficiently reliable to replace therapeutic monitoring of vancomycin serum concentrations.

Anti-Bacterial Agents↗
PubMed2006

Use of centrally developed pharmacoeconomic assessments for local formulary decisions.

PURPOSE: The distribution, content, timeliness, use, and influence of pharmacoeconomic assessments (PEAs) of drugs in New Zealand public hospitals were examined. METHODS: In April 2005, a questionnaire-based, cross-sectional survey was sent to chief pharmacists at all 29 New Zealand hospitals employing a pharmacist. The questionnaire asked pharmacists about the use and influence of PEAs in their hospitals' formulary decision-making process. Answers were given using a scale of 1 to 6, with 1 being the most positive response. RESULTS: Of the 29 surveys mailed, 24 (83%) were completed. Data on 12 PEAs were analyzed. Assessments were seen and summaries read in most hospitals (median, 77% and 65%, respectively). Full documents were read in fewer hospitals (35%). In general, the PEAs were considered moderately easy to understand, provided a concise summary, and contained adequate detail of the methodology. Of the 24 respondent hospitals, 21 had assessment processes for new medicines; hence, a total of 252 hospital evaluations of Pharmaceutical Management Agency (PHARMAC)-assessed drugs were possible. A total of 132 possible evaluations (52%) were undertaken. More evaluations (106 [42%]) took place before PHARMAC's PEAs were distributed and fewer (26 [10%]) after distribution. Where used, the PEAs appeared to have a modest effect on hospital decisions. CONCLUSION: The provision of 12 PEAS by PHARMAC to hospitals in New Zealand had only a modest influence on their formulary decision-making process, mostly due to the lack of timeliness of the PEAs. The timely delivery of centrally developed PEAs may be essential to generating a greater effect on the formulary decisions at a wider level.

Decision Making↗
PubMed2006

Performance of drug-drug interaction software for personal digital assistants.

BACKGROUND: Personal digital assistants (PDAs) allow healthcare professionals to check for potential drug-drug interactions (DDIs) at the point of care, reducing the need to consult traditional references. However, PDAs can only be as effective as the software programs they use. OBJECTIVE: To examine the ability of DDI software programs manufactured for Palm OS-compatible PDAs in detecting clinically important DDIs. METHODS: Eight PDA software programs were assessed for sensitivity, specificity, and positive and negative predictive values for 16 well-documented DDIs contained within 6 simulated patient profiles. RESULTS: Sensitivity of the software programs ranged from 0.81 to 1.0, specificity ranged from 0.52 to 1.0, positive predictive values ranged from 0.62 to 1.0, and negative predictive values ranged from 0.88 to 1.0. Five programs scored perfect sensitivity scores: DrugIx, ePocrates Rx, ePocrates Rx Pro, Lexi-Interact, and the Tarascon pocket Pharmacopoeia. Of these, the ePocrates programs scored the highest in specificity (0.9), while Lexi-Interact and the Tarascon pocket Pharmacopoeia scored considerably lower (0.52). MosbyIx was the only program to score a 1.0 in specificity; however, its sensitivity was just 0.81. CONCLUSIONS: ePocrates Rx and ePocrates Rx Pro scored greater than or equal to 90% in regard to both sensitivity and specificity, making them the most reliable in detecting the clinically relevant interactions studied without the distraction of detecting those of no clinical significance. In addition, ePocrates Rx is updated regularly and is easily accessible on the Internet at no cost.

Computers, Handheld↗
PubMed2006

Expression of human coagulation factor VIII in a human hybrid cell line, HKB11.

Loss of coagulation factor VIII (FVIII) function results in a bleeding disorder, hemophilia A, which requires FVIII replacement therapy. Owing to its large size and complexity, the expression level of recombinant FVIII is two to three orders of magnitude lower than other recombinant proteins produced in mammalian cell lines. To understand cellular factors limiting FVIII expression, we studied the expression of FVIII in a human cell line, HKB11 (a hybrid cell line of HEK293 and a human B cell line). In comparison with other cell lines, such as HEK293 and BHK-21, HKB11 showed increased FVIII expression levels. With unamplified, pooled stable cells, FVIII expression in HKB11 cells was 8- to 30-fold higher than the other cell lines tested. In this study, HKB11 clones expressing varying levels of FVIII were analyzed and FVIII secreted from these clones had similar specific activity. Characterization of these clones by immunofluorescence staining, Western blotting analysis, and flow cytometry showed that high-producing cells not only secreted more active FVIII but also accumulated more FVIII protein intracellularly. FVIII expression appears to be controlled by the rates of transcription, translation, and secretion, but transcription and translation may play more important roles than secretion in determining expression level in HKB11 cells. FACS analysis of live cells showed that the high-producing clones also had more FVIII on the HKB11 cell surface than low-producing cells, thus opening the possibility of using FACS to select high-producing cell lines. Expression levels of the chaperone protein Hsp70 and antiapoptotic proteins such as Bcl-2 and Bcl-xL were similar among HKB11 clones with different FVIII productivity. In conclusion, HKB11 is an efficient host cell line for expression of FVIII and possibly other recombinant proteins. Systematic approaches, such as gene expression profiling by DNA microarray, will be necessary to understand the global changes in the cells producing recombinant proteins.

Biotechnology↗
PubMed2006

Evaluation of the performance of drug-drug interaction screening software in community and hospital pharmacies.

BACKGROUND: Computerized drug-drug interaction (DDI) screening is widely used to identify potentially harmful drug combinations in the inpatient and outpatient setting. OBJECTIVE: To evaluate the performance of drug-drug interaction (DDI) screening software in identifying select clinically significant DDIs in pharmacy computer systems in community and hospital pharmacies. METHODS: Ten community pharmacies and 10 hospital pharmacies in the Tucson metropolitan area were invited to participate in the study in 2004. To test the performance of each of the systems used by the pharmacies, 25 medications were used to create 6 mock patient profiles containing 37 drug-drug pairs, 16 of which are clinically meaningful DDIs that pose a potential risk to patient safety. Each profile was entered into the computer pharmacy system, and the system response in terms of the presence or absence of a DDI alert was recorded for each drug pair. The percentage of correct responses and the sensitivity, specificity, positive predictive value, and negative predictive value of each system to correctly classify each drug pair as a DDI or not was calculated. Summary statistics of these measures were calculated separately for community and hospital pharmacies. RESULTS: Eight community pharmacies and 5 hospital pharmacies in the Tucson metropolitan area agreed to participate in the study. The median sensitivity and median specificity for community pharmacies was 0.88 (range, 0.81-0.94) and 0.91 (range, 0.67-1.00), respectively. For hospital pharmacies, the median sensitivity and median specificity was 0.38 (range, 0.15-0.94) and 0.95 (range, 0.81-0.95), respectively. CONCLUSION: Based on this convenience sample of 8 community pharmacies and 5 hospital pharmacies in 1 metropolitan area, the performance of community pharmacy computer systems in screening DDIs appears to have improved over the last several years compared with research published previously in 2001. However, significant variation remains in the performance of hospital pharmacy computer systems, even among systems manufactured by the same vendor. Future research should focus on improving the performance of these systems in accurately and precisely identifying DDIs with a high probability of resulting in true potential adverse effects on clinical outcomes and creating a low .noise. ratio associated with false-positive alerts.

Clinical Pharmacy Information Systems↗
PubMed2005

Prediction of gentamicin peak and trough concentrations from six extended-interval dosing protocols for neonates.

PURPOSE: The ability of six extended-interval gentamicin dosing protocols to achieve desired peak and trough concentrations in neonates was evaluated using simulated calculations based on pooled patient data. METHODS: The demographic and pharmacokinetic data of 293 neonates age one week or less from three previously published studies were pooled and applied in each of six published protocols. The data collected, including weight, dosage, dosing interval, and measured serum or plasma gentamicin concentrations, were used to determine gentamicin clearance, elimination-rate constant, and distribution volume. Peak and trough gentamicin concentrations were calculated and compared with several therapeutic ranges that aim to achieve high peak concentrations for maximum efficacy and low trough concentrations for minimum toxicity. RESULTS: All protocols resulted in more than 95% of peak concentrations exceeding 5 mg/L. Peaks exceeded 10 mg/L in 26-42% of the simulations. Most of the protocols resulted in high trough concentrations (0.5-1.5 mg/L). Trough concentrations were below 1 mg/L in 51-83% of the simulations. CONCLUSION: Simulated calculations based on pooled patient data found that six gentamicin dosing protocols for neonates would likely yield acceptable peak concentrations and a trough concentration of < or = 1 mg/L for at least 50% of patients. It may be sufficient to monitor only the trough concentrations when applying these protocols.

Anti-Bacterial Agents↗
PubMed2003

Treatment of experimental asthma by long-term gene therapy directed against IL-4 and IL-13.

The clinical manifestations of allergic asthma are believed to result from a dysregulated, T helper 2 lymphocyte (Th2)-biased response to antigen. Although asthma symptoms can be controlled acutely, there is a need for a therapy that will address the underlying immune dysfunction and provide continuous control of chronic airway inflammation. The Th2-type cytokines, IL-13 and IL-4, have been demonstrated to play a crucial role in asthma pathogenesis and their selective neutralization results in the alleviation of asthmatic symptoms in mouse models. The activity of both of these cytokines can be inhibited by a mutant IL-4 protein, IL-4 receptor antagonist (IL-4RA), and thus, continual IL-4RA therapy might be beneficial in treatment of chronic asthma. To explore the potential utility of long-term gene therapy for the treatment of asthma we used a recombinant adeno-associated virus (AAV) vector to deliver and provide sustained expression of IL-4RA in vivo. We show that AAV-mediated delivery of IL-4RA to the airways of mice reduces airway hyperresponsiveness (AHR) and airway eosinophilia triggered by either IL-13 or IL-4. Furthermore, AAV-delivered IL-4RA, expressed either systemically or in the airways of mice following allergen sensitization, significantly inhibited development of airway eosinophilia and mucus production and reduced the levels of asthma-associated Th2 cytokines and AHR in the experimental mouse model of allergic asthma. Thus, gene therapy can be a potential therapeutic option to treat and control chronic airway inflammation and asthmatic symptoms.

Animals↗
PubMedDate not supplied

Workload and availability of technology in metropolitan community pharmacies.

OBJECTIVE: To assess workload characteristics and the presence of prescription processing technologies within metropolitan community pharmacies in the United States. DESIGN: Cross-sectional postal survey. SETTING: 18 metropolitan statistical areas (MSAs) in the United States. PARTICIPANTS: 3000 managers located in community pharmacies processing at least 500 third-party claims per month for four major pharmacy benefits managers. INTERVENTIONS: 34-item survey instrument designed to collect data about the pharmacy including demographics, workload issues, handling of drug-drug interactions (DDIs), and pharmacists' attitudes toward computerized DDI alerts. MAIN OUTCOME MEASURES: Workload (hours of operation, prescription volume, staffing hours, prescription processing intensity) and prescription processing technologies (telecommunication systems, automated counting/filling or verification devices, number of computer terminals, and computer software vendors). RESULTS: Overall, 736 usable surveys were returned (response rate, 25.3%). On average, respondents reported a volume of approximately 1340 prescriptions per week processed at a rate of almost 17 prescriptions per hour. Independent pharmacies processed approximately 3 prescriptions per hour more than chain pharmacies even though a statistically equal or slightly lower proportion of those pharmacies had automated technologies. The presence of technology was generally high for all pharmacies, particularly countertop tablet/capsule-counting devices and telefacsimile machines. The most common software vendors differed considerably between chain and independent pharmacies. CONCLUSION: The number of prescriptions processed per hour and number of technologies available increased with the total weekly volume of prescriptions processed in this national survey. A majority of pharmacies had at least one type of automated prescription processing technology and an automated telecommunication system for accepting new or refill prescriptions. Independent pharmacies processed more prescriptions per hour than did chains but did so with fewer categories of technologies.

Automation↗
PubMedDate not supplied

Community pharmacy managers' perception of computerized drug-drug interaction alerts.

OBJECTIVE: To examine community pharmacists' attitudes toward computerized drug-drug interaction (DDI) alerts and identify factors associated with more favorable perceptions of these alerts. DESIGN: Cross-sectional postal survey. SETTING: 18 metropolitan statistical areas (MSAs) in the United States. PARTICIPANTS: 3000 community pharmacy managers. INTERVENTIONS: 34-item survey instrument designed to collect data about the pharmacy including demographics, workload issues, handling of DDIs, and pharmacists' attitudes toward computerized DDI alerts. MAIN OUTCOME MEASURE: Responses to items concerning community pharmacy managers' attitudes toward DDI alerts and factors associated with more favorable attitudes toward these alerts. RESULTS: A total of 736 usable surveys were returned (25.3% response rate). Pharmacy managers generally disagreed that DDI alerts were a waste of time (mean +/- SD, 2.1 +/- 1.1 on a scale of 1, strongly disagree, to 6, strongly agree). However, they were not completely confident that their computer systems provided them with meaningful DDI alerts (mean +/- SD, 4.5 +/- 1.2). They were confident in their ability to identify DDIs (mean +/- SD, 4.9 +/- 0.9) and discuss DDIs with physicians (mean +/- SD, 5.2 +/- 0.7). Pharmacy software that provided detailed DDI information as well as the ability to customize DDI alerts were associated with more favorable perceptions of DDI alerts. CONCLUSION: Despite being presented with a large proportion of clinically unimportant alerts, community pharmacy managers did not believe DDI alerts were meaningless or a waste of time. Incorporation of features that streamline DDI alerts may improve their effectiveness in community pharmacy practice.

Attitude to Computers↗