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.

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.

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.

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.

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.

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.

Influence of kidney disease on drug disposition: An assessment of industry studies submitted to the FDA for new chemical entities 1999-2010.

In 1998, the United States Food and Drug Administration (FDA) released the first guidance for industry regarding pharmacokinetic (PK) studies in renally impaired patients. This study aimed to determine if the FDA renal PK guidance influenced the frequency and rigor of renal studies conducted for new chemical entities (NCEs). FDA-approved package inserts (APIs) and clinical pharmacology review documents were analyzed for 194 NCEs approved from 1999 to 2010. Renal studies were conducted in 71.6% of NCEs approved from 1999 to 2010, a significant increase over the 56.3% conducted from 1996 to 1997 (P = .0242). Renal studies were more likely to be completed in highly renally excreted drugs (fe ≥ 30%) compared with drugs with low renal excretion, fe < 30% (89.6% vs 65.8%, P = .0015). PK studies to assess the impact of dialysis were conducted for 31.7% of NCEs that had a renal study: a greater proportion of high fe NCEs were studied (44.2% vs 26.0%, P = .0335). No significant change in frequency or rigor of PK studies was detected over time. The majority of NCEs (76.3%) with a renal study provided specific dosing recommendations in the API. The adoption of the 1998 FDA guidance has resulted in improved availability of PK and drug-dosing recommendations, particularly for high fe drugs.

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.

The need for PGY2-trained clinical pharmacy specialists.

The American College of Clinical Pharmacy and other stakeholder organizations seek to advance clinical pharmacist practitioners, educators, and researchers. Unfortunately, there remains an inadequate supply of residency-trained clinical specialists to meet the needs of our health care system, and nonspecialists often are called on to fill open specialist positions. The impact of clinical pharmacy specialists on pharmacotherapy outcomes in both acute care and primary care settings demonstrates the value of these specialists. This commentary articulates the need for postgraduate year two (PGY2)-trained clinical specialists within the health care system by discussing various clinical and policy rationales, interprofessional support, economic justifications, and their impact on quality of care and drug safety. The integrated practice model that has grown out of the American Society of Health-System Pharmacists Pharmacy Practice Model Initiative (PPMI) could threaten the growth and development of future clinical specialists. Therefore, the ways in which PGY2-trained clinical pharmacist specialists are deployed in the PPMI require further consideration. PGY2 residencies provide education and training opportunities that cannot be achieved in traditional professional degree programs or postgraduate year one residencies. These specialists are needed to provide direct patient care to complex patient populations and to educate and train pharmacy students and postgraduate residents. Limitations to training and hiring PGY2-trained clinical pharmacy specialists include site capacity limitations and lack of funding. A gap analysis is needed to define the extent of the mismatch between the demand for specialists by health care systems and educational institutions versus the capacity to train clinical pharmacists at the specialty level.

Global substrate profiling of proteases in human neutrophil extracellular traps reveals consensus motif predominantly contributed by elastase.

Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a "proteolytic signature" from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.

Recommendations for generating, evaluating, and implementing drug-drug interaction evidence.

In October 2009, a 2-day, multistakeholder, national conference was held in Rockville, Maryland, to discuss and propose methods to improve the drug-drug interaction (DDI) evidence base and its evaluation and integration into clinical decision support (CDS) systems. The conference featured participants representing consumers, health care providers, those responsible for relevant policies and guidelines, and developers and vendors of DDI compendia, databases, and CDS systems. One desired outcome of the conference was to prepare recommendations on critical issues surrounding DDI evidence. A set of recommendations was developed to improve the generation, evaluation, and translation of DDI evidence into CDS systems based on presentations by experts and the supporting literature. These recommendations were reviewed initially by conference moderators, speakers, and Scientific Steering and Planning Committee members, and subsequently by all attendees. The following recommendations were developed to increase patient safety by improving the relevance and assessment of DDI evidence: conduct well-designed studies to determine the incidence, outcomes, and patient-level risk factors for DDIs; use a systematic and transparent process for evaluating the DDI evidence in order to estimate the severity and risks of DDIs; and improve the integration of DDI evidence into electronic CDS. Opportunities exist to improve the DDI evidence base, develop and promote a systematic approach for evaluating the evidence, and integrate this evidence into meaningful CDS.

Potentially harmful drug-drug interactions in the elderly: a review.

BACKGROUND: Elderly patients are vulnerable to drug interactions because of age-related physiologic changes, an increased risk for disease associated with aging, and the consequent increase in medication use. OBJECTIVE: The purpose of this narrative review was to describe findings from rigorously designed observational cohort and case-control studies that have assessed specific drug interactions in elderly patients. METHODS: The PubMed and International Pharmaceutical Abstracts databases were searched for studies published in English over the past 10 years (December 2000-December 2010) using relevant Medical Subject Headings terms (aged; aged, 80 and over; and drug interactions) and search terms (drug interaction and elderly). Search strategies were saved and repeated through September 2011 to ensure that the most recent relevant published articles were identified. Additional articles were found using a search of review articles and reference lists of the identified studies. Studies were included if they were observational cohort or case-control studies that reported specific adverse drug interactions, included patients aged ≥65 years, and evaluated clinically meaningful end points. Studies were excluded if they used less rigorous observational designs, assessed pharmacokinetic/pharmacodynamic properties, evaluated drug-nutrient or drug-disease interactions or interactions of drug combinations used for therapeutic benefit (eg, dual antiplatelet therapy), or had inconclusive evidence. RESULTS: Seventeen studies met the inclusion criteria. Sixteen studies reported an elevated risk for hospitalization in older adults associated with adverse drug interactions. The drug interactions included: angiotensin-converting enzyme (ACE) inhibitors and potassium-sparing diuretics, ACE inhibitors or angiotensin receptor blockers and sulfamethoxazole/trimethoprim, benzodiazepines or zolpidem and interacting medications, calcium channel blockers and macrolide antibiotics, digoxin and macrolide antibiotics, lithium and loop diuretics or ACE inhibitors, phenytoin and sulfamethoxazole/trimethoprim, sulfonylureas and antimicrobial agents, theophylline and ciprofloxacin, and warfarin and antimicrobial agents or nonsteroidal anti-inflammatory drugs. One study reported the risk for breast cancer-related death as a function of paroxetine exposure among women treated with tamoxifen. CONCLUSIONS: Several population-based studies have reported significant harm associated drug interactions in elderly patients. Increased awareness and interventions aimed at reducing exposure and minimizing the risks associated with potentially harmful drug combinations are needed.

Student evaluations of the portfolio process.

OBJECTIVE: To evaluate pharmacy students' perceived benefits of the portfolio process and to gather suggestions for improving the process. METHODS: A questionnaire was designed and administered to 250 first-, second-, and third-year pharmacy students at the University of Arizona College of Pharmacy. RESULTS: Although the objectives of the portfolio process were for students to understand the expected outcomes, understand the impact of extracurricular activities on attaining competencies, identify what should be learned, identify their strengths and weaknesses, and modify their approach to learning, overall students perceived the portfolio process as having less than moderate benefit. First-year students wanted more examples of portfolios while second- and third-year students suggested that more time with their advisor would be beneficial. CONCLUSIONS: The portfolio process will continue to be refined and efforts made to improve students' perceptions of the process as it is intended to develop the self-assessments skills they will need to improve their knowledge and professional skills throughout their pharmacy careers.

The science of safety curriculum in US colleges and schools of pharmacy.

OBJECTIVE: To describe the integration of science of safety (SoS) topics in doctor of pharmacy (PharmD) curricula of US colleges and schools of pharmacy. METHODS: A questionnaire that contained items pertaining to what and how SoS topics are taught in PharmD curricula was e-mailed to representatives at 107 US colleges and schools of pharmacy. RESULTS: The majority of the colleges and schools responding indicated that they had integrated SoS topics into their curriculum, however, some gaps (eg, teaching students about communicating risk, Food and Drug Administration [FDA] Sentinel Initiative, utilizing patient databases) were identified that need to be addressed. CONCLUSIONS: The FDA and the American Association of Colleges of Pharmacy (AACP) should continue to collaborate to develop resources needed to ensure that topics proposed by the FDA in their SoS framework are taught at all colleges and schools of pharmacy.