Cystatin C Outperforms Creatinine in Predicting Cefepime Clearance in Pediatric Stem Cell Transplant Recipients.

Pediatric hematopoietic stem cell transplant (HSCT) patients are at risk of developing both sepsis and altered kidney function. Cefepime is used for empiric coverage post-HSCT and requires dose adjustment based on kidney function. Since cefepime's antimicrobial efficacy is determined by the time free concentrations exceed bacterial minimum inhibitory concentration (MIC), it is important to assess kidney function accurately to ensure adequate concentrations. Serum creatinine (SCr) is routinely used to estimate glomerular filtration rate (eGFR) but varies with muscle mass, which can be significantly lower in HSCT patients, making SCr an inaccurate kidney function biomarker. Cystatin C (CysC) eGFR is independent of muscle mass, though steroid use increases CysC. Objectives of this study were to describe how eGFR impacts cefepime pharmacokinetic/pharmacodynamic (PK/PD) target attainment in pediatric HSCT patients, to investigate which method of estimating GFR (SCr, CysC, combined) best predicts cefepime clearance, and to explore additional predictors of cefepime clearance. Patients admitted to the pediatric HSCT unit who received ≥2 cefepime doses were prospectively enrolled. We measured total cefepime peak/trough concentrations between the second and fourth cefepime doses and measured SCr and CysC if not already obtained clinically within 24h of cefepime samples. eGFRs were calculated with Chronic Kidney Disease in Children U25 equations. Bayesian estimates of cefepime clearance were determined with a pediatric cefepime PK model and PK software MwPharm++. Simple linear regression was used to compare cefepime clearance normalized to body surface area (BSA) to BSA-normalized SCr-, CysC-, and SCr-/CysC-eGFRs, while multiple linear regression was used to account for additional predictors of cefepime clearance. For target attainment, we assessed the percentage of time free cefepime concentrations exceeded 1x MIC (%fT>1x MIC) and 4x MIC (%fT>4x MIC) using a susceptibility breakpoint of 8 mg/L for Pseudomonas aeruginosa. We enrolled 53 patients (ages 1 to 30 years, median 8.9 years). SCr- and CysC-eGFRs were lower in patients who attained 100% fT>1xMIC compared to those who did not attain this target: 115 versus 156 mL/min/1.73m2 (p = .01) for SCr-eGFR and 73.5 versus 107 mL/min/1.73m2 (p < .001) for CysC-eGFR. SCr-eGFR was weakly positively correlated with cefepime clearance (adjusted [a]r2= 0.14), while CysC-eGFR and SCr-/CysC-eGFR had stronger positive correlations (ar2 = 0.30 CysC, ar2 = 0.28 combo. There was a weak, significant linear association between increasing CysC-eGFR and decreased %fT>1xMIC (ar2 = 0.32) and %fT>4xMIC (ar2 = 0.14). No patients with a CysC-eGFR >120 mL/min/1.73 m2 achieved 100% fT>1xMIC or 50% fT>4x MIC. In multiple regression models, underlying diagnosis of hemoglobinopathy (in all models) and being pretransplant (in SCr and combined models) were associated with increased cefepime clearance, while concomitant use of calcineurin inhibitors was associated with decreased cefepime clearance in all models. Overall, the combo-eGFR model with timing pretransplant, hemoglobinopathy, and use of calcineurin inhibitors had the best performance (ar2 = 0.63). CysC-based eGFRs (CysC alone and combined) predicted cefepime clearance better than SCr-eGFR, even after considering steroid use. Increasing CysC eGFR correlated with decreased probability of PD target attainment, raising concerns for underdosing at high eGFRs. CysC should be included when estimating kidney function to provide adequate dosing of cefepime in pediatric HSCT patients.

Artesunate acts through cytochrome c to inhibit growth of pediatric AML cells.

Artesunate is a derivative of artemisinin, an active compound isolated from Artemisia annua which has been used in Traditional Chinese Medicine and to treat malaria worldwide. Artemisinin derivatives have exhibited anti-cancer activity against both solid tumors and leukemia. The direct target(s) of artesunate are controversial; although, heme-bound proteins in the mitochondria have been implicated. We utilized computational modeling to calculate the predicted binding score of artesunate with heme-bound mitochondrial proteins and identified cytochrome c as potential artesunate target. UV-visible spectroscopy showed changes in the absorbance spectrum, and thus protein structure, when cytochrome c was incubated with artesunate. Artesunate induces apoptosis, disrupts mitochondrial membrane potential, and is antagonized by methazolamide in pediatric AML cells indicating a probable mechanism of action involving cytochrome c. We utilized a multi-disciplinary approach to show that artesunate can interact with and is dependent on cytochrome c release to induce cell death in pediatric AML cell lines.

FDA Approaches in Monitoring Drug Quality, Forces Impacting the Drug Quality, and Recent Alternative Strategies to Assess Quality in the US Drug Supply.

Since the US Food and Drug Administration (FDA) began monitoring the quality of pharmaceutical manufacturing by enforcing current good manufacturing practices roughly 60 years ago, forces related to the global economy have changed, rendering the task of monitoring quality more difficult. Alternative strategies by groups like Valisure, LLC, and the University of Kentucky Drug Quality Study to monitor the quality of the currently circulated US drug supply through end-product testing and screening have resulted in several concerning findings. Given the successful approaches of identifying quality defects in pharmaceuticals by non-regulatory bodies, and considering the changing landscape and pressures on manufacturing, the FDA, large buying groups, and the US Department of Defense should consider these alternative strategies as a means to augment current regulatory activities.

AACC Guidance Document on Laboratory Testing for the Assessment of Preterm Delivery.

Identifying women with preterm labor who will go on to deliver prematurely is crucial to improving outcomes for mother and baby and for saving healthcare resources. Even among those with symptoms, the number of women who deliver preterm is low, and thus the low positive predictive value (PPV) and high negative predictive value (NPV) associated with available biomarkers does not substantially reduce the uncertainty of the clinical diagnosis. While there is some promise in the use of fetal fibronectin (fFN), interleukin 6 (IL-6), or placental alpha microglobulin 1 (PAMG-1) for predicting preterm birth (PTB), their use is unlikely to provide considerable clinical value in populations with a low prevalence. To provide real clinical benefit, a biomarker must demonstrate a high PPV to allow identification of the minority of symptomatic women who will deliver prematurely. As none of the currently available biomarkers exhibit this performance characteristic, we do not recommend their routine clinical use in populations with a pre-test probability of PTB of <5%. Limiting biomarker testing to only high-risk women identified on the basis of cervical length or other characteristics will increase the pre-testprobability in the tested population, thereby improving PPV. PAMG-1 is associated with a higher PPV than fFN and may show clinical utility in populations with a higher pre-test probability, but further work is required to conclusively demonstrate improved outcomes in this patient group.

KEAP1 Is Required for Artesunate Anticancer Activity in Non-Small-Cell Lung Cancer.

Artesunate is the most common treatment for malaria throughout the world. Artesunate has anticancer activity likely through the induction of reactive oxygen species, the same mechanism of action utilized in Plasmodium falciparum infections. Components of the kelch-like ECH-associated protein 1 (KEAP1)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway, which regulates cellular response to oxidative stress, are mutated in approximately 30% of non-small-cell lung cancers (NSCLC); therefore, we tested the hypothesis that KEAP1 is required for artesunate sensitivity in NSCLC. Dose response assays identified A549 cells, which have a G333C-inactivating mutation in KEAP1, as resistant to artesunate, with an IC50 of 23.6 µM, while H1299 and H1563 cells were sensitive to artesunate, with a 10-fold lower IC50. Knockdown of KEAP1 through siRNA caused increased resistance to artesunate in H1299 cells. Alternatively, the pharmacological inhibition of NRF2, which is activated downstream of KEAP1 loss, by ML385 partially restored sensitivity of A549 cells to artesunate, and the combination of artesunate and ML385 was synergistic in both A549 and H1299 cells. These findings demonstrate that KEAP1 is required for the anticancer activity of artesunate and support the further development of NRF2 inhibitors to target patients with mutations in the KEAP1/NRF2 pathway.

Submillisecond Dynamics of Mastoparan X Insertion into Lipid Membranes.

The mechanism of protein insertion into a lipid bilayer is poorly understood because the kinetics of this process is difficult to measure. We developed a new approach to study insertion of the antimicrobial peptide Mastoparan X into zwitterionic lipid vesicles, using a laser-induced temperature-jump to initiate insertion on the microsecond time scale and infrared and fluorescence spectroscopies to follow the kinetics. Infrared probes the desolvation of the peptide backbone and yields biphasic kinetics with relaxation lifetimes of 12 and 117 µs, whereas fluorescence probes the intrinsic tryptophan residue located near the N-terminus and yields a single exponential phase with a lifetime of 440 µs. Arrhenius analysis of the temperature-dependent rates yields an activation energy for insertion of 96 kJ/mol. These results demonstrate the complexity of the insertion process and provide mechanistic insight into the interplay between peptides and the lipid bilayer required for peptide transport across cellular membranes.

Neurofibrillar tangle surrogates: histone H1 binding to patterned phosphotyrosine peptide nanotubes.

Living cells contain a range of densely phosphorylated surfaces, including phospholipid membranes, ribonucleoproteins, and nucleic acid polymers. Hyperphosphorylated surfaces also accumulate in neurodegenerative diseases as neurofibrillar tangles. We have synthesized and structurally characterized a precisely patterned phosphotyrosine surface and establish this assembly as a surrogate of the neuronal tangles by demonstrating its high-affinity binding to histone H1. This association with nucleic acid binding proteins underscores the role such hyperphosphorylated surfaces may play in disease and opens functional exploration into protein-phosphorylated surface interactions in a wide range of other complex assemblies.

Quantum dots encapsulated within phospholipid membranes: phase-dependent structure, photostability, and site-selective functionalization.

Lipid vesicle encapsulation is an efficient approach to transfer quantum dots (QDs) into aqueous solutions, which is important for renewable energy applications and biological imaging. However, little is known about the molecular organization at the interface between a QD and lipid membrane. To address this issue, we investigated the properties of 3.0 nm CdSe QDs encapsulated within phospholipid membranes displaying a range of phase transition temperatures (Tm). Theoretical and experimental results indicate that the QD locally alters membrane structure, and in turn, the physical state (phase) of the membrane controls the optical and chemical properties of the QDs. Using photoluminescence, ICP-MS, optical microscopy, and ligand exchange studies, we found that the Tm of the membrane controls optical and chemical properties of lipid vesicle-embedded QDs. Importantly, QDs encapsulated within gel-phase membranes were ultrastable, providing the most photostable non-core/shell QDs in aqueous solution reported to date. Atomistic molecular dynamics simulations support these observations and indicate that membranes are locally disordered displaying greater disordered organization near the particle-solution interface. Using this asymmetry in membrane organization near the particle, we identify a new approach for site-selective modification of QDs by specifically functionalizing the QD surface facing the outer lipid leaflet to generate gold nanoparticle-QD assemblies programmed by Watson-Crick base-pairing.

Dynamics of the gel to fluid phase transformation in unilamellar DPPC vesicles.

The dynamics of the gel to fluid phase transformation in 100 nm large unilamellar vesicles (LUV) of 1,2-dipalmitoyl(d62)-sn-glycero-3-phosphocholine (d62-DPPC), has been studied by laser-induced temperature-jump initiation coupled with time-resolved infrared spectroscopy and by MD simulations. The infrared transients that characterize the temperature dependent phase transformation are complex, extending from the nanosecond to the millisecond time scales. An initial fast (submicrosecond) component can be modeled by partial melting of the gel domains, initiated at pre-existing defects at the edges of the faceted structure of the gel phase. Molecular dynamics simulations support the model of fast melting from edge defects. The extent of melting during the fast phase is limited by the area expansion on melting, which leads to a surface pressure that raises the effective melting temperature. Subsequent melting is observed to follow highly stretched exponential kinetics, consistent with collective relaxation of the surface pressure through a hierarchy of surface undulations with different relaxation times. The slowest step is water diffusion through the bilayer to allow the vesicle volume to grow along with its expanded surface area. The results demonstrate that the dominant relaxation in the gel to fluid phase transformation in response to a large T-jump perturbation (compared to the transition width) is fast (submicrosecond), which has important practical and fundamental consequences.