Antibodies against the erythroferrone N-terminal domain prevent hepcidin suppression and ameliorate murine thalassemia.

Erythroferrone (ERFE) is produced by erythroblasts in response to erythropoietin (EPO) and acts in the liver to prevent hepcidin stimulation by BMP6. Hepcidin suppression allows for the mobilization of iron to the bone marrow for the production of red blood cells. Aberrantly high circulating ERFE in conditions of stress erythropoiesis, such as in patients with ß-thalassemia, promotes the tissue iron accumulation that substantially contributes to morbidity in these patients. Here we developed antibodies against ERFE to prevent hepcidin suppression and to correct the iron loading phenotype in a mouse model of ß-thalassemia [Hbb(th3/+) mice] and used these antibodies as tools to further characterize ERFE's mechanism of action. We show that ERFE binds to BMP6 with nanomolar affinity and binds BMP2 and BMP4 with somewhat weaker affinities. We found that BMP6 binds the N-terminal domain of ERFE, and a polypeptide derived from the N terminus of ERFE was sufficient to cause hepcidin suppression in Huh7 hepatoma cells and in wild-type mice. Anti-ERFE antibodies targeting the N-terminal domain prevented hepcidin suppression in ERFE-treated Huh7 cells and in EPO-treated mice. Finally, we observed a decrease in splenomegaly and serum and liver iron in anti-ERFE-treated Hbb(th3/+) mice, accompanied by an increase in red blood cells and hemoglobin and a decrease in reticulocyte counts. In summary, we show that ERFE binds BMP6 directly and with high affinity, and that antibodies targeting the N-terminal domain of ERFE that prevent ERFE-BMP6 interactions constitute a potential therapeutic tool for iron loading anemias.

AAV-mediated expression of galactose-1-phosphate uridyltransferase corrects defects of galactose metabolism in classic galactosemia patient fibroblasts.

Classic galactosemia (CG) is a rare disorder of autosomal recessive inheritance. It is caused predominantly by point mutations as well as deletions in the gene encoding the enzyme galactose-1-phosphate uridyltransferase (GALT). The majority of the more than 350 mutations identified in the GALT gene cause a significant reduction in GALT enzyme activity resulting in the toxic buildup of galactose metabolites that in turn is associated with cellular stress and injury. Consequently, developing a therapeutic strategy that reverses both the oxidative and ER stress in CG cells may be helpful in combating this disease. Recombinant adeno-associated virus (AAV)-mediated gene therapy to restore GALT activity offers the potential to address the unmet medical needs of galactosemia patients. Here, utilizing fibroblasts derived from CG patients we demonstrated that AAV-mediated augmentation of GALT protein and activity resulted in the prevention of ER and oxidative stress. We also demonstrate that these CG patient fibroblasts exhibit reduced CD109 and TGFßRII protein levels and that these effectors of cellular homeostasis could be restored following AAV-mediated expression of GALT. Finally, we show initial in vivo proof-of-concept restoration of galactose metabolism in a GALT knockout mouse model following treatment with AAV-GALT.

Marstacimab, a tissue factor pathway inhibitor neutralizing antibody, improves coagulation parameters of ex vivo dosed haemophilic blood and plasmas.

INTRODUCTION: Tissue factor pathway inhibitor (TFPI) is an endogenous inhibitor of the extrinsic pathway that negatively regulates thrombin production during coagulation. Under haemophilic conditions, where the intrinsic coagulation pathway is impaired, inhibition of TFPI may improve clotting. AIM: We investigated the ex vivo effects of a human TFPI neutralizing antibody, marstacimab (previously PF-06741086), in coagulation assays including rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay, performed in haemophilic whole blood and plasmas. We compared the effects of marstacimab to the effects of recombinant coagulation factors and investigated the reproducibility of marstacimab in restoring haemostasis by comparing its effect in whole blood collected from the same study participants on differing days. METHODS: Citrated whole blood and plasmas obtained from haemophilia participants were supplemented ex vivo with vehicle, marstacimab, recombinant FVIII (rFVIII) or recombinant factor IX (rFIX) and analysed in ROTEM, TGA and the dPT assay using low tissue factor concentrations to trigger coagulation. RESULTS: Marstacimab induced pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in angle. Similarly, participant plasmas supplemented with marstacimab exhibited improvements in TGA parameters, including reduced lag times, increased peak thrombin concentrations and reductions in dPT clotting time. Concentrations of marstacimab tested showed activity comparable to addition of rFVIII or rFIX and were reproducible. CONCLUSIONS: These studies show the ex vivo potency of marstacimab in restoring haemostasis in whole blood and plasmas from haemophilia participants and comparability to ex vivo reconstitution with recombination coagulation factors.

Noncovalent stabilization of the factor VIII A2 domain enhances efficacy in hemophilia A mouse vascular injury models.

An important negative regulator of factor VIIIa (FVIIIa) cofactor activity is A2 subunit dissociation. FVIII molecules with stabilized activity have been generated by elimination of charged residues at the A1-A2 and A2-A3 interfaces. These molecules exhibited reduced decay rates as part of the enzymatic factor Xa generation complex and retained their activities under thermal and chemical denaturing conditions. We describe here the potency and efficacy of 1 such stability variant, D519V/E665V, derived from B domain-deleted FVIII (BDD-FVIII). The major effect of A2 stabilization was on cofactor activity. D519V/E665V potency was increased twofold by the 2-stage chromogenic assay relative to BDD-FVIII. D519V/E665V demonstrated enhanced thrombin generation responses (fivefold by peak thrombin) relative to BDD-FVIII. In vivo consequences of enhanced cofactor activity of D519V/E665V included >fourfold increased maximal platelet-fibrin deposition after laser injury and twofold increased protection from bleeding in acute and prolonged vascular injury model in hemophilia A mice. These results demonstrate that noncovalent stabilization of the FVIII A2 subunit can prolong its cofactor activity, leading to differential enhancement in clot formation over protection from blood loss in hemophilia. The FVIII molecule described here is the first molecule with clear efficacy enhancement resulting from noncovalent stabilization of the A2 domain.

Field Study and Correlative Studies of Factor IX Variant FIX-R338L in Participants Treated with Fidanacogene Elaparvovec.

BACKGROUND: Fidanacogene elaparvovec, an adeno-associated virus-based gene therapy vector expressing the high-activity factor IX (FIX) variant FIX-R338L, is in development for hemophilia B. One-stage clotting (OS) assays and chromogenic substrate (CS) assays are commonly used to measure FIX-R338L variant activity. Data from ongoing trials suggest FIX activity varies between different OS and CS assays. MATERIAL AND METHODS: To better understand FIX-R338L activity in clinical samples, an international multisite field study was conducted across a central laboratory and 18 local laboratories, using standard protocols, reagents, and instrumentation, with individual participant samples from a phase 1/2a study of fidanacogene elaparvovec. RESULTS: Unlike the wild-type FIX control, FIX-R338L activity was higher with the OS silica-based assay versus OS ellagic acid-based and CS assays. Variation in FIX activity was greater at the lowest activity levels. Activated FIX (FIXa) in plasma could result in higher OS assay activity or increased thrombin generation, which could overestimate FIX activity. However, FIXa was not detected in the participant samples, indicating that it was not contributing to the OS assay differences. Since individuals on gene therapy may receive exogenous replacement FIX products, replacement products were spiked into patient plasma samples to target a therapeutic concentration. Exogenous FIX was additive to endogenous FIX-R338L, with no interference from FIX-R338L. CONCLUSION: These results demonstrate FIX-R338L activity can be measured with OS and CS assays in clinical laboratories and provide insight into assay variability when measuring FIX with endogenously produced FIX-R338L. The findings may help establish best practices for measuring FIX-R338L activity (Clinicaltrials.gov identifier: NCT02484092).

Rational design of a fully active, long-acting PEGylated factor VIII for hemophilia A treatment.

A long-acting factor VIII (FVIII) as a replacement therapy for hemophilia A would significantly improve treatment options for patients with hemophilia A. To develop a FVIII with an extended circulating half-life, but without a reduction in activity, we have engineered 23 FVIII variants with introduced surface-exposed cysteines to which a polyethylene glycol (PEG) polymer was specifically conjugated. Screening of variant expression level, PEGylation yield, and functional assay identified several conjugates retaining full in vitro coagulation activity and von Willebrand factor (VWF) binding.PEGylated FVIII variants exhibited improved pharmacokinetics in hemophilic mice and rabbits. In addition, pharmacokinetic studies in VWF knockout mice indicated that larger molecular weight PEG may substitute for VWF in protecting PEGylated FVIII from clearance in vivo. In bleeding models of hemophilic mice, PEGylated FVIII not only exhibited prolonged efficacy that is consistent with the improved pharmacokinetics but also showed efficacy in stopping acute bleeds comparable with that of unmodified rFVIII. In summary site-specifically PEGylated FVIII has the potential to be a long-acting prophylactic treatment while being fully efficacious for on-demand treatment for patients with hemophilia A.

Hemostatic efficacy of marstacimab alone or in combination with bypassing agents in hemophilia plasmas and a mouse bleeding model.

Background: Patients with hemophilia have deficiencies in intrinsic coagulation factors and can develop inhibitors that limit the effectiveness of replacement coagulation factors. Marstacimab, a human monoclonal antibody, binds and inhibits the human tissue factor pathway inhibitor. Marstacimab is currently under development as a potential prophylactic treatment to prevent bleeding episodes in patients with hemophilia A and B. Objective: To assess the effects of marstacimab alone or in combination with the bypassing agent recombinant factor FVIIa (rFVIIa) or activated prothrombin complex concentrate (aPCC) on thrombin generation and bleeding. Methods: Marstacimab and/or rFVIIa or aPCC were added to hemophilic A or B plasma or nonhemophilic plasma in vitro. Hemostatic activity was measured using the thrombin generation assay. In vivo effects were assessed using a mouse acute bleeding model. Male hemophilia A mice were dosed with marstacimab plus aPCC before tail clip; blood loss was quantified by measuring hemoglobin. Results: Marstacimab plus rFVIIa or aPCC slightly increased peak thrombin levels compared with either agent alone. This increase was within the reported range for nonhemophilic plasma and did not exceed levels observed in nonhemophilic plasma treated with marstacimab alone. Hemophilia A mice that received 200 U/kg aPCC had significantly reduced bleeding (62%) compared with vehicle-treated mice (p < 0.05), and marstacimab plus aPCC reduced bleeding by 83.3% compared with vehicle (p= 0.0009). Conclusions: Marstacimab alone or with bypassing agents increased hemostasis in hemophilia plasma without generating excessive thrombin. The hemostatic activity of marstacimab plus aPCC was confirmed in hemophilia A mice.

Community pharmacy and pharmacist staff call center: assessment of medication safety and effectiveness.

OBJECTIVE: To determine proof of concept for use of a network of pharmacists to evaluate the safety of medications. DESIGN: Pilot, comparative, prospective evaluation. SETTING: Community pharmacies and a pharmacist-staffed call center in Arizona during January through August 2006. PATIENTS: Patients filling prescriptions for ipratropium or tiotropium bromide at 1 of 55 Arizona pharmacies were encouraged to call a pharmacist-staffed call center. A total of 67 patients contacted the center and 41 participated. INTERVENTION: A network of community pharmacies and a call center were used to collect data on patients receiving one of two medications for the treatment of chronic obstructive pulmonary disease. Pharmacists in the community pharmacies recruited patients who presented with a prescription or requested a refill for one of the medications. The call center was used to collect patient data. Patients provided data on medication use, completed the chronic respiratory questionnaire (CRQ), and were encouraged to call the center to report health problems. After 30 days, patients were called to determine whether they experienced any adverse events while taking their medication and the CRQ was readministered. MAIN OUTCOME MEASURE: Knowledge gained on the feasibility of the model using pharmacists to assess drug safety. RESULTS: A total of 67 (6.7%) of a possible 995 patients contacted the call center about participating in the study. Approximately one-half (n = 28) of the 55 pharmacies had one or more patients contact the center about the study. A total of 41 patients met inclusion/exclusion criteria and were enrolled. Six (15%) patients reported an adverse effect, including one serious adverse event (acute glaucoma). CONCLUSION: This study provides limited evidence that community pharmacies and a pharmacist-staffed call center can be used to assess medication safety; however, a number of issues need to be examined to determine whether the approaches can be sufficiently effective.

Assessment of galactose-1-phosphate uridyltransferase activity in cells and tissues.

Galactosemias are a family of autosomal recessive genetic disorders resulting from impaired enzymes of the Leloir pathway of galactose metabolism including galactokinase, galactose uridyltransferase, and UDP-galactose 4-epimerase that are critical for conversion of galactose into glucose-6-phosphate. To better understand pathophysiological mechanisms involved in galactosemia and develop novel therapies to address the unmet need in patients, it is important to develop reliable assays to measure the activity of the Leloir pathway enzymes. Here we describe in-depth methods for indirectly measuring galacose-1-phosphate uridyltransferase activity in cell culture and animal tissues.

Factor IX assay discrepancies in the setting of liver gene therapy using a hyperfunctional variant factor IX-Padua.

BACKGROUND: Limited information exists regarding the factor IX (FIX) coagulant activity (FIX:C) measured by different assays following FIX-Padua gene therapy. OBJECTIVE: Assess for the first time FIX:C in five commonly used coagulation assays in plasma samples from hemophilia B subjects receiving FIX-Padua gene transfer. METHODS: FIX:C was compared between central (n = 1) and local laboratories (n = 5) in the study, and across four commonly used FIX:C one-stage assays and one FIX:C chromogenic assay. For comparison, samples of pooled congenital FIX-deficient plasma spiked with purified recombinant human FIX (rHFIX)-Padua protein or rHFIX (nonacog alfa) to obtain FIX:C concentrations from ~20% to ~40% were tested. RESULTS: FIX:C results at local laboratories strongly correlated with central laboratory results. However, absolute values at the central laboratory were consistently lower than those at local laboratories. Across five different FIX:C assays, a consistent pattern of FIX:C was observed for subjects receiving fidanacogene elaparvovec-expressed gene transfer. Use of Actin FSL activated partial thromboplastin time (APTT) reagent in the central laboratory resulted in lower FIX:C values compared with other APTT reagents tested. The chromogenic assay determined lower FIX:C than any of the one-stage assays. The rHFIX-Padua protein-spiked samples showed similar results. In contrast, FIX:C results for rHFIX-nonacog alfa measured within 25% of expected for all one-stage assays and below 25% in the chromogenic assay. CONCLUSIONS: Assay-based differences in FIX:C were observed for fidanacogene elaparvovec transgene product and rHFIX-Padua protein, suggesting the variable FIX:C determined with different assay reagents is inherent to the FIX-Padua protein and is not specific to gene therapy-derived FIX-Padua.

Prescribers' knowledge of and sources of information for potential drug-drug interactions: a postal survey of US prescribers.

BACKGROUND: Given the high prevalence of medication use in the US, the risk of drug-drug interactions (DDIs) and potential for patient harm is of concern. Despite the rise in technologies to identify potential DDIs, the ability of physicians and other prescribers to recognize potential DDIs is essential to reduce their occurrence. The objectives of this study were to assess prescribers' ability to recognize potential clinically significant DDIs and to examine the sources of information they use to identify potential DDIs and prescribers' opinions on the usefulness of various DDI information sources. METHODS: A postal questionnaire was developed to assess prescriber knowledge of medications that may interact and prescribers' usual sources of DDI information. Recipients were asked to classify 14 drug pairs as 'contraindicated', 'may be used together but with monitoring' or 'no interaction'. A response option of 'not sure' was also provided. The questionnaires were sent to a national sample of 12 500 prescribers based on past history of prescribing drugs associated with known potential for DDI, who were identified using data from a pharmacy benefit manager covering over 50 million individuals. RESULTS: Usable questionnaires were obtained from 950 prescribers. The percentage of prescribers who correctly classified specific drug pairs ranged from 18.2% for warfarin and cimetidine to 81.2% for paracetamol (acetaminophen) with codeine and amoxicillin, with 42.7% of all combinations classified correctly. The number of drug pairs correctly classified by the prescribers ranged from 0 to 13. For half of the drug pairs over one-third of the respondents answered 'not sure'; among those drug pairs, two were contraindicated. When asked what source was used to learn more about a potential DDI, a quarter of the prescribers reported using personal digital assistants and another quarter used printed material. The majority of the prescribers (68.4%) reported that they were usually informed by pharmacists about their patients' potential exposure to DDIs. Compared with the prescribers who used other sources, those who used computerized DDI alerts as their usual source of DDI information consistently gave a lower rating score to the five statements that assessed the usefulness of the information. CONCLUSION: This study suggests that prescribers' knowledge of potential clinically significant DDIs is generally poor. These findings are supported by other research and emphasize the need to develop systems that alert prescribers about potential interactions that are clinically relevant. Physicians most commonly reported learning about potential DDIs from pharmacists, suggesting further work is needed to improve the drug-prescribing process to identify potential safety issues earlier in the medication use process.

Cell surface staining of recombinant factor VIII is reduced in apoptosis resistant BHK-21 cells.

We have recently demonstrated an increase in recombinant factor VIII (rFVIII) secretion from BHK-21 cells (rBHK-21(host)) following an over-expression of the chaperone protein heat shock protein 70 (Hsp70) (rBHK-21(Hsp70)) due to an inhibition of apoptotic cell death and an increased cellular expression of rFVIII [Ishaque, A., Thrift, J., Murphy, J.E., Konstantin, K., 2007. Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion. Biotechnol. Bioeng. Biotech. Bioeng. 97, 144-155]. In the present study we investigated the difference in adherence of rFVIII to the cell membrane surface by comparing changes in cell viability and extent of phosphatidylersine (PS) exposure in apoptosis between rBHK-21(host), rBHK-21(Hsp70), and parental BHK-21 cells devoid of rFVIII expression (BHK-21(native)) during batch cell culture experiments. The Zenon technique was used to double stain for cell surface and intracellular rFVIII using flow cytometric Guava PCA analysis. By this quantitative analysis intracellular rFVIII was shown to decrease in rBHK-21(host) cells as the cell viability declined while the rFVIII cell surface staining increased. Conversely, rBHK-21(Hsp70) cell cultures displayed higher cell viability and intracellular rFVIII with less cell surface rFVIII staining. Time dependent increases of rFVIII adherence to the surface of rBHK-21(host) cells and its reduction on the surface of rBHK-21(Hsp70) cells was also confirmed by fluorescence microscopy. Furthermore, greater rFVIII cell surface staining correlated with an increase in detectable PS exposure on the surface of BHK-21(native) batch cell cultures. However, PS exposure could not be identified to the same extent on rBHK-21(host) cells despite a similar decline in cell viability between rBHK-21(host) and BHK-21(native) batch cultures. Any exposed PS on rBHK-21(host) cells was most likely masked by secreted rFVIII, mimicking the effect on activated platelets where the externalization of PS also occurs, and serves as a ligand for FVIII activation in the blood coagulation cascade. Taken together we have identified that rFVIII sequestration on the membrane surface is another potential limitation to rFVIII productivity and one which can also be alleviated by reduction of apoptosis in a clone expressing human HSP70.

Impact of medication packaging on adherence and treatment outcomes in older ambulatory patients.

OBJECTIVE: To evaluate medication adherence and treatment outcomes in elderly outpatients using daily-dose blister packaging (Pill Calendar) compared with medications packaged in bottles of loose tablets. DESIGN: Randomized controlled trial. SETTING: Ambulatory care clinics at Ohio State University Medical Center, Columbus; University of Arizona Health Science Center, Tucson; and Riverside Methodist Hospital Family Medicine Clinic, Columbus, Ohio, from July 1, 2002, to December 31, 2004. PATIENTS: 85 individuals 65 years of age or older being treated with lisinopril for hypertension. INTERVENTION: Patients were randomly assigned to receive lisinopril in either daily-dose blister packaging (Pill Calendar) or traditional bottles of loose tablets. MAIN OUTCOME MEASURES: Adherence was assessed by prescription refill regularity and medication possession ratio (MPR). Treatment outcome and use of medical services were assessed by medical record review of blood pressure and morbidity associated with poorly controlled hypertension. RESULTS: Patients receiving lisinopril in the daily-dose blister packaging (Pill Calendar) refilled their prescriptions on time more often (P = 0.01), had higher MPRs (P = 0.04), and had lower diastolic blood pressure (P = 0.01) than patients who had their medications packaged in traditional bottles of loose tablets. CONCLUSION: Providing medications in a package that identifies the day each dose is intended to be taken and provides information on proper self-administration can improve treatment regimen adherence and treatment outcomes in elderly patients.

Translational Pharmacokinetic/Pharmacodynamic Characterization and Target-Mediated Drug Disposition Modeling of an Anti-Tissue Factor Pathway Inhibitor Antibody, PF-06741086.

Tissue factor pathway inhibitor (TFPI) exhibits multiple isoforms, which are known to present in multiple locations such as plasma, endothelium, and platelets. TFPI is an endogenous negative modulator of the coagulation pathway, and therefore, neutralization of TFPI function can potentially increase coagulation activity. A human monoclonal antibody, PF-06741086, which interacts with all isoforms of TFPI is currently being tested in clinic for treating hemophilia patients with and without inhibitors. To support clinical development of PF-06741086, pharmacokinetics (PK) and pharmacodynamics of PF-06741086 were characterized in monkeys. In addition, a mechanistic model approach was used to estimate PK parameters in monkeys and simulate PK profiles in human. The results show that PF-06741086 exhibited target-mediated drug disposition and had specific effects on various hemostatic markers including diluted prothrombin time, thrombin generation, and thrombin-antithrombin complex in monkeys after administration. The model-predicted and observed human exposures were compared retrospectively, and the result indicates that the exposure prediction was reasonable within less than 2-fold deviation. This study demonstrated in vivo efficacy of PF-06741086 in monkeys and the utility of a rational mechanistic approach to describe PK for a monoclonal antibody with complex target binding.

Student use of flipped classroom videos in a therapeutics course.

PURPOSE: To evaluate the extent of student use of flipped classroom videos. METHODS: This was a cross-sectional study conducted in a college of pharmacy therapeutics course in the Unites States. In one section of the course (four sessions) all content was provided in the form of lecture videos that students had to watch prior to class. Class time was spent discussing patient cases. For half of the sessions, there was an electronic quiz due prior to class. The outcome measure was video view time in minutes. Adequate video view time was defined as viewing ≥75% of total video duration. Video view time was compared with or without quizzes using the Wilcoxon signed-rank test. RESULTS: There were 100 students in the class and all were included in the study. Overall, 74 students had adequate video view time prior to session 1, which decreased to 53 students for session 2, 53 students for session 3, and 36 students for session 4. Median video view time was greater when a quiz was required [80 minutes (IQR: 38-114) versus 69 minutes (IQR: 3-105), p < 0.001]. The mean score on the exam was 84 ± 8 points (out of 100). There was a significant association between video view time (per 50% increment) and score on the exam (coefficient 2.52; 95% CI: 0.79-4.26; p = 0.005; model R2 = 7.8%). CONCLUSION: Student preparation prior to the flipped classroom is low and decreases with time. Preparation is higher when there is a quiz required.

Pharmacy Educators' Knowledge of Medication Safety and Their Perception Toward Its Integration into the Doctor of Pharmacy Curriculum in Saudi Arabia.

Objective. To assess pharmacy educators' knowledge of medication safety and their perception toward its integration into the PharmD curriculum in Saudi Arabia. Methods. A survey was administered to pharmacy educators at a college of pharmacy and its affiliate hospital. Knowledge, training, and perception toward integrating medication safety into the PharmD curriculum were evaluated. Results. More than 50% of respondents indicated that medication safety should be covered within selected courses, and 65% indicated that such courses should be mandatory. Pharmacy practice educators had significantly higher levels of knowledge about medication safety than their nonpractice counterparts. Perceptions toward medication safety integration into the curriculum varied significantly by general discipline, academic degree, years of experience, and gender. Conclusion. Pharmacy educators in Saudi Arabia understand the importance of medication safety and its integration into the curriculum. Further studies are needed to guide curricular change to achieve this integration.

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.

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.

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.