Utility of Cellular Measurements of Non-Specific Endocytosis to Assess the Target-Independent Clearance of Monoclonal Antibodies.

Past studies have demonstrated higher clearance for monoclonal antibodies possessing increased rates of non-specific endocytosis. However, this metric is oftentimes evaluated indirectly using biophysical techniques or cell surface binding studies that may not provide insight into the specific rates of cellular turnover. Furthermore, few examples evaluating non-specific endocytosis have been reported for a therapeutic antibody that reached clinical assessment. In the current report, we evaluated a therapeutic human immunoglobulin G2 monoclonal antibody targeted against the interleukin-4 receptor alpha chain (IL-4Rα) that exhibited elevated target independent clearance in previous Phase 1 and 2 studies. We confirmed high non-specific clearance of the anti-IL-4Rα antibody as compared to a reference antibody during pharmacokinetic assessments in wild type mice where target-mediated disposition was absent. We then developed a cell-based method capable of measuring cellular protein endocytosis and demonstrated the anti-IL-4Rα antibody exhibited marked non-specific uptake relative to the reference compound. Antibody homology modeling identified the anti-IL-4Rα antibody possessed positive charge patches whose removal via targeted mutations substantially reduced its non-specific endocytosis. We then expanded the scope of the study by evaluating panels of both preclinical and clinically relevant monoclonal antibodies and demonstrate those with the highest rates of non-specific uptake in vitro exhibit elevated target independent clearance, low subcutaneous bioavailability, or both. Our results support the observation that high non-specific endocytosis is a negative attribute in monoclonal antibody development and demonstrate the utility of a generic cell-based screen as a quantitative tool to measure non-specific endocytosis of protein therapeutics at the single-cell level.

Evidence supporting the use of therapeutic drug monitoring of ganciclovir in transplantation.

PURPOSE OF REVIEW: This review describes current knowledge of ganciclovir (GCV) and valganciclovir (ValGCV) pharmacokinetic/pharmacodynamic characteristics, highlighting the likely contribution from host genetic factors to interpatient variability. The evidence and challenges surrounding optimization of drug dosing through therapeutic drug monitoring (TDM) are examined, with recommendations made. RECENT FINDINGS: Pharmacokinetic studies of current dosing guidelines have shown high interindividual and intraindividual variability of GCV concentrations. This is sometimes associated with a slow decline in cytomegalovirus (CMV) viral load in some transplant recipients. A high incidence of GCV-associated myelosuppression has limited the use of this drug in the transplant setting. Patient groups identified to benefit from GCV TDM include pediatric patients, cystic fibrosis with lung transplantation, obese with kidney transplantation, and patients with fluctuating renal function or on hemodialysis. The emergence of refractory resistant CMV, particularly in immune compromised patients, highlights the importance of appropriate dosing of these antivirals. Host genetic factors need to be considered where recently, two host genes were shown to account for interpatient variation during ganciclovir therapy. Therapeutic Drug Monitoring has been shown to improve target antiviral-level attainment. The use of TDM may guide concentration-based dose adjustment, potentially improving virological and clinical outcomes. However, evidence supporting the use of TDM in clinical practice remains limited and further study is needed in the transplant cohort. SUMMARY: Further studies examining novel biomarkers are needed to guide target concentrations in prophylaxis and treatment. The use of TDM in transplant recipients is likely to improve the clinical efficacy of current antivirals and optimize outcomes in transplant recipients.

Novel modelling approaches to predict the role of antivirals in reducing influenza transmission.

To aid understanding of the effect of antiviral treatment on population-level influenza transmission, we used a novel pharmacokinetic-viral kinetic transmission model to test the correlation between nasal viral load and infectiousness, and to evaluate the impact that timing of treatment with the antivirals oseltamivir or baloxavir has on influenza transmission. The model was run under three candidate profiles whereby infectiousness was assumed to be proportional to viral titer on a natural-scale, log-scale, or dose-response model. Viral kinetic profiles in the presence and absence of antiviral treatment were compared for each individual (N = 1000 simulated individuals); subsequently, viral transmission mitigation was calculated. The predicted transmission mitigation was greater with earlier administration of antiviral treatment, and with baloxavir versus oseltamivir. When treatment was initiated 12-24 hours post symptom onset, the predicted transmission mitigation was 39.9-56.4% for baloxavir and 26.6-38.3% for oseltamivir depending on the infectiousness profile. When treatment was initiated 36-48 hours post symptom onset, the predicted transmission mitigation decreased to 0.8-28.3% for baloxavir and 0.8-19.9% for oseltamivir. Model estimates were compared with clinical data from the BLOCKSTONE post-exposure prophylaxis study, which indicated the log-scale model for infectiousness best fit the observed data and that baloxavir affords greater reductions in secondary case rates compared with neuraminidase inhibitors. These findings suggest a role for baloxavir and oseltamivir in reducing influenza transmission when treatment is initiated within 48 hours of symptom onset in the index patient.

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H.

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.

Detection of Ganciclovir-Resistant Cytomegalovirus in a Prospective Cohort of Kidney Transplant Recipients Receiving Subtherapeutic Valganciclovir Prophylaxis.

Cytomegalovirus infection during antiviral prophylaxis occurs in transplant recipients despite individualized regimens based on renal function. Fifty kidney transplant recipients were assessed between 2016 and 2019 for valganciclovir dosing, ganciclovir exposure, cytomegalovirus infection, and genotypic resistance markers during the first year posttransplant. Ganciclovir plasma concentrations were measured using mass spectrometry. Population pharmacokinetics was used to determine individual ganciclovir exposure and to evaluate the ability of manufacturer dosing guidelines to meet therapeutic target daily area under the curve (AUC24) of 40 to 50 µg·h/mL. Full-length UL54 and UL97 were assessed using high-throughput sequencing in cytomegalovirus DNA-positive patient specimens. Valganciclovir doses administered to recipients with creatinine clearance of <40 mL/min were higher than specified by guidelines, and they were lower for recipients with creatinine clearance of ≥40 mL/min. The mean ganciclovir AUC24 was 33 ± 13 µg·h/mL, and 82% of subjects did not attain the therapeutic target. Pharmacokinetic simulations showed that the guidelines similarly could not attain the therapeutic target in 79% of individuals. Cytomegalovirus breakthrough occurred in 6% (3/50) of recipients, while 12% (6/50) developed late-onset infection. The mean AUC24s of recipients with (n = 3) and without (n = 47) infection were not significantly different (P = 0.528). However, one recipient with an AUC24 of 20 µg·h/mL acquired two UL97 ganciclovir resistance mutations. Current prophylaxis guidelines resulted in subtherapeutic ganciclovir exposure in several study recipients, including the emergence of resistance genotypes. IMPORTANCE This study examined the pharmacokinetics and viral genomic data from a prospective cohort of kidney transplant recipients undergoing valganciclovir prophylaxis for cytomegalovirus (CMV) prevention. We showed for the first time using high-throughput sequencing the detection of ganciclovir resistance mutations in breakthrough CMV infection during subtherapeutic plasma ganciclovir as indicated by the pharmacokinetic parameter daily area under the curve (AUC24). In addition, we found that current valganciclovir dosing guidelines for CMV prophylaxis are predicted to attain therapeutic targets in only 21% of recipients, which is consistent with previous pharmacokinetic studies. The novel findings of resistance mutations during subtherapeutic ganciclovir exposure presented here can inform future studies investigating the dynamics of drug selection pressure and the emergence of resistance mutations in vivo.

An overview of systematic reviews on the pharmacological randomized controlled trials for reducing intracranial pressure after traumatic brain injury.

BACKGROUND: There is a need for an overview of systematic reviews (SRs) examining randomized clinical trials (RCTs) of pharmacological interventions in the treatment of intracranial pressure (ICP) post-TBI. OBJECTIVES: To summarize pharmacological effectiveness in decreasing ICP in SRs with RCTs and evaluate study quality. METHODS: Comprehensive literature searches were conducted in MEDLINE, PubMed, EMBASE, PsycINFO, and Cochrane Library databases for English SRs through October 2020. Inclusion criteria were SRs with RCTs that examined pharmacological interventions to treat ICP in patients post-TBI. Data extracted were participant characteristics, pharmacological interventions, and ICP outcomes. Study quality was assessed with AMSTAR-2. RESULTS: Eleven SRs between 2003 and 2020 were included. AMSTAR-2 ratings revealed 3/11 SRs of high quality. Pharmacological interventions included hyperosmolars, neuroprotectives, anesthetics, sedatives, and analgesics. Study samples ranged from 7 to 1282 patients. Hyperosmolar agents and sedatives were beneficial in lowering elevated ICP. High bolus dose opioids had a more deleterious effect on ICP. Neuroprotective agents did not show any effects in ICP management. CONCLUSIONS: RCT sample sizes and findings in the SRs varied. A lack of detailed data syntheses was noted. AMSTAR-2 analysis revealed moderate to high quality in most SRs. Future SRs may focus on streamlined reporting of dosing and clearer clinical recommendations. PROSPERO-Registration: CRD42015017355.

Development of an immunoassay for aglycosylated murine IgG1 in mouse serum via generation of a specific tool antibody.

Aim: To develop a method for the quantitation of effector functionless mouse surrogate IgG1 drug molecules in mouse matrices. Materials & methods: A panel of antibodies that bound specifically to N297G mutation-containing mouse IgG molecules was generated in rats. The panel was screened to identify an antibody that could be used as both the capture and detection reagent in an electrochemiluminescent immunoassay. Results & conclusion: The quantitative assay developed with the N297G-specific antibody passed acceptance criteria across multiple IgG1 fragment crystallizable (Fc)-containing protein formats and provides accurate quantitation of the total levels of mouse surrogate protein Fc present in in vivo mouse serum samples. These results are useful in understanding drug integrity and the development of precise pharmacokinetic/pharmacodynamic relationships.

Exercise interventions for older adults with Alzheimer's disease: a systematic review and meta-analysis protocol.

BACKGROUND: The growing societal and economic impact of Alzheimer's disease (AD) is further compounded by the present lack of disease-modifying interventions. Non-pharmacological intervention approaches, such as exercise, have the potential to be powerful approaches to improve or mitigate the symptoms of AD without added side effects or financial burden associated with drug therapies. Various forms and regiments of exercise (i.e., strength, aerobic, multicomponent) have been reported in the literature; however, conflicting evidence obscures clear interpretation of the value and impact of exercise as an intervention for older adults with AD. The primary objective of this review will be to evaluate the effects of exercise interventions for older adults with AD. In addition, this review will evaluate the evidence quality and synthesize the exercise training prescriptions for proper clinical practice guidelines and recommendations. METHODS: This systematic review and meta-analysis will be carried out by an interdisciplinary collective representing clinical and research stakeholders with diverse expertise related to neurodegenerative diseases and rehabilitation medicine. Literature sources will include the following: Embase, PsychINFO, OVID Medline, and Ovid MEDLINE(R) and Epub Ahead of Print, In-Process & Other Non-Indexed Citations and Daily. Inclusion criteria are participants with late onset AD and structured exercise interventions with prescribed duration, frequency, and intensity. The primary outcome of this study will center on improved or sustained cognitive functioning. Secondary outcomes will include institutionalization-related outcomes, ability in activities of daily living, mood and emotional well-being, quality of life, morbidity, and mortality. Analysis procedures to include measurement of bias, data synthesis, sensitivity analysis, and assessment of heterogeneity are described in this protocol. DISCUSSION: This review is anticipated to yield clinically meaningful insight on the specific value of exercise for older adults with AD. Improved understanding of diverse exercise intervention approaches and their specific impact on various health- and function-related outcomes is expected to guide clinicians to more frequently and accurately prescribe meaningful interventions for those affected by AD. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42020175016 .

Patient-Derived Cytomegaloviruses with Different Ganciclovir Sensitivities from UL97 Mutation Retain Their Replication Efficiency and Some Kinase Activity In Vitro.

Mutations in the cytomegalovirus UL97 kinase gene contribute to antiviral resistance. Mutations A594S and G598D from two clinical isolates were analyzed, and bacterial artificial chromosome (BAC)-engineered A594S recombinant cytomegalovirus exhibited a ganciclovir-resistant phenotype on plaque reduction. Viral replication was comparable to that of the wild type. Cell-based kinase activity and autophosphorylation of ectopically expressed proteins showed that mutants retained some kinase activity. This study showed that patient-derived cytomegalovirus with different ganciclovir sensitivities retained replication efficiency and exhibited some kinase activity in vitro.

Peer-Reviewed Validation of a Comprehensive Framework for Disaster Evaluation Typologies.

INTRODUCTION: The Comprehensive Framework for Disaster Evaluation Typologies, developed in 2017 (CFDET 2017), aims to unify and facilitate agreement regarding the identification, structure, and relationships between various evaluation typologies found in the disaster setting. A peer-reviewed validation process sought input from international experts in the fields of disaster medicine, disaster/emergency management, humanitarian/development, and evaluation. This paper discusses the validation process, its results, and outcomes.Research Problem:Previous frameworks, identified in the literature, lack validation and consistent terminology. To gain credibility and utility, this unique framework needed to be validated by international experts in the disaster setting. METHODS: A mixed methods approach was designed to validate the framework. An initial iterative process informed an online survey which used a combination of a five-point Likert scale and open-ended questions. Pre-determined consensus thresholds, informed by a targeted literature review, provided the validation criteria. RESULTS: A sample of 33 experts from 11 countries responded to the validation process. Quantitative measures largely supported the elements and relationships of the framework, and strongly supported its value and usefulness for supporting, promoting, and undertaking evaluations, as well as its usefulness for teaching evaluation in the disaster setting. Qualitative input suggested opportunities to strengthen and enhance the framework. There were limited responses to better understand the barriers and enablers of undertaking disaster evaluations. A potential for self-selection bias of respondents may be a limitation of this study. The attainment of high consensus thresholds, however, provides confidence in the validity of the results. CONCLUSION: For the first time, a framework of this nature has undergone a rigorous validation process by experts in three related disciplines at an international level. The modified framework, CFDET 2018, provides a unifying framework within which existing evaluation typologies can be structured. It gives evaluators confidence to choose an appropriate strategy for their particular evaluation in the disaster setting and facilitates consistency in reporting across the different phases of a disaster to better understand the process, outcomes, and impacts of the efficacy and efficiency of interventions. Future research could create a series of toolkits to support improved disaster evaluation processes and to evaluate the utility of the framework in the real-world setting.

Systematic review of ganciclovir pharmacodynamics during the prevention of cytomegalovirus infection in adult solid organ transplant recipients.

Human cytomegalovirus (CMV) represents the most common infection among recipients of solid organ transplants (SOTs). Previous meta-analysis showed 0.8% of SOT recipients developed CMV disease whilst receiving valganciclovir (ValGCV) prophylaxis. However, the clinical utility of monitoring ganciclovir (GCV) blood concentrations is unclear. We systematically reviewed the association between GCV concentrations during prophylaxis and the incidence of CMV. MEDLINE and EMBASE databases were searched for studies between 1946 and 2018, where GCV pharmacokinetics and incidence of CMV viraemia or disease in SOT were available. Research designs included randomised trials, comparative, prospective cohort, retrospective, or case report studies. Only human adult studies were included, with English language restriction. The 11 studies that met the eligibility criteria included 610 participants receiving GCV or ValGCV prophylaxis. Quality assessment showed 2/4 randomised trials, 4/6 cohort studies, and 1/1 case report were of high quality. Despite dose adjustments for renal impairment, mean GCV exposures for patients were heterogeneous and ranged between 28 and 53.7 µg·h/mL across three randomised trials. The incidence of CMV infection and disease ranged from 0% to 50% and 0% to 3.1%, respectively, with follow up between 3 to 9 months. One study showed statistical power in determining relationship, where GCV exposure at 40 to 50 µg·h/mL in high-risk SOT recipients was associated with a reduced risk of viraemia. Clinical monitoring for GCV exposure can be applied to high-risk SOT recipients during ValGCV prophylaxis; however, further studies are needed to determine the utility of monitoring in all SOT recipients.

Periodontal pathogens in the placenta and membranes in term and preterm birth.

INTRODUCTION: Preterm birth is a common cause of adverse neonatal and childhood outcomes. It is commonly associated with infection of the maternal-fetal interface. The relationship between periodontitis and preterm labour is controversial. METHODS: Control placental tissues from uncomplicated term births were compared with those from spontaneous preterm births for incidence of common periodontal bacteria. A chi-square analysis was used to compare the populations, with significance determined at p=<0.05. RESULTS: The study group comprised 29 control women who had an uncomplicated term birth, 25 delivered by caesarean section and 4 vaginal deliveries, and 36 women with a spontaneous preterm labour and subsequent delivery at less than 34 weeks gestation. There were significant (p=<0.05) differences between the preterm and term groups maternal age with 28.7 compared to 32.0 years old respectively. There was no significant (p=>0.05) differences between the groups fetal risk factors or co-morbidities, except the preterm group had a significantly higher (p=<0.05) rate of premature rupture of membrane (PROM). There were significantly (p=<0.01) more Fusobacterium spp. in the placentas from term births than preterm births. DISCUSSION: This study found that the common periodontal pathogen, Fusobacterium spp., is not detected more in placentas from preterm birth and may potentially be lower, possibly resulting from bacterial ecological factors in term placentas.

Layer-by-layer assembly of brushes of vertically-standing enzymatic nanotubes.

Brushes of vertically-standing enzyme-containing nanotubes are prepared onto planar surfaces by a combination of hard-templating and layer-by-layer assembly. The nanotubes have a core-shell morphology made of two compartments, one for mechanical rigidity, the other containing ß-lactamase for bioactivity. We demonstrate inclusion of the enzymatic component either in the core or in the shell part of the nanotubes. Kinetic studies reveal that both types of systems are bioactive but that the activity is significantly better preserved over long time periods when ß-lactamase is incorporated in the core of the nanotubes.

Uptake of Long Protein-Polyelectrolyte Nanotubes by Dendritic Cells.

Anisotropic nanostructures, such as nanotubes, incorporating bioactive molecules present interesting features for application as drug delivery carriers. Here, we present the synthesis of layer-by-layer (LbL) nanotubes including protein (ovalbumin) layers and go from simple to more complex synergetic combinations of synthetic and natural polyelectrolytes, leading to structures with tunable properties. The rigidity in organic and aqueous media, the stability in buffer solution and the uptake of different LbL tubes by dendritic cells (DCs) are analyzed to contrast size and chemistry. The most rigid studied systems appear as the best candidates to be internalized by cells, regardless of the chemistry of their outermost layers. The successful transport of long protein-loaded robust rigid nanotubes to the cytoplasm of DCs paves the way for their use as new cargo for the delivery of large amounts of antigen to such cells.

The effects of Gua sha on symptoms and inflammatory biomarkers associated with chronic low back pain: A randomized active-controlled crossover pilot study in elderly.

OBJECTIVE: To address the challenges for trialing with elderly and the lacking of valid sham/placebo control, a randomized crossover pilot study is designed and its feasibility on elderly subjects is evaluated. DESIGN: A pilot randomized crossover study was conducted with hydrocollator-based hot pack therapy as active control. Pain intensity, physical disability, depression, general health status, and salivary biomarkers were assessed as outcome measures. RESULTS: Despite there was no significant difference observed between any outcome measures attained by the two interventions, several important differences were noted during the one-week follow-up period. The magnitudes of pain reduction (21-25% versus 16-18%) and disability improvement (45-52% versus 39-42%) were greater in the Gua sha-treated group than the hot pack group. Both treatments were shown to improve flexion, extension and bending movements of the lower back, whereas areas of improvement varied between the two interventions. Decreasing trends were observed in both tumor necrosis factor-alpha (TNF-α) and heme-oxygenase-1 (HO-1) levels following Gua sha. However, rebounds of the biomarkers were observed one week following hot pack. Furthermore, in response to Gua sha, the decrease of TNF-α was strongly correlated with the improvement of physical disability, whereas the physical disability was correlated with the VAS pain intensity. CONCLUSION: It demonstrated a feasible clinical trial protocol for evaluating the effectiveness of Gua sha and other therapeutic modalities. Gua sha may exhibit a more long-lasting anti-inflammatory effect relative to hot pack for pain relief and improved mobility in elderly patients with chronic low back pain.

Disaster Metrics: A Comprehensive Framework for Disaster Evaluation Typologies.

Introduction The frequency of disasters is increasing around the world with more people being at risk. There is a moral imperative to improve the way in which disaster evaluations are undertaken and reported with the aim of reducing preventable mortality and morbidity in future events. Disasters are complex events and undertaking disaster evaluations is a specialized area of study at an international level. Hypothesis/Problem While some frameworks have been developed to support consistent disaster research and evaluation, they lack validation, consistent terminology, and standards for reporting across the different phases of a disaster. There is yet to be an agreed, comprehensive framework to structure disaster evaluation typologies. The aim of this paper is to outline an evolving comprehensive framework for disaster evaluation typologies. It is anticipated that this new framework will facilitate an agreement on identifying, structuring, and relating the various evaluations found in the disaster setting with a view to better understand the process, outcomes, and impacts of the effectiveness and efficiency of interventions. METHODS: Research was undertaken in two phases: (1) a scoping literature review (peer-reviewed and "grey literature") was undertaken to identify current evaluation frameworks and typologies used in the disaster setting; and (2) a structure was developed that included the range of typologies identified in Phase One and suggests possible relationships in the disaster setting. RESULTS: No core, unifying framework to structure disaster evaluation and research was identified in the literature. The authors propose a "Comprehensive Framework for Disaster Evaluation Typologies" that identifies, structures, and suggests relationships for the various typologies detected. CONCLUSION: The proposed Comprehensive Framework for Disaster Evaluation Typologies outlines the different typologies of disaster evaluations that were identified in this study and brings them together into a single framework. This unique, unifying framework has relevance at an international level and is expected to benefit the disaster, humanitarian, and development sectors. The next step is to undertake a validation process that will include international leaders with experience in evaluation, in general, and disasters specifically. This work promotes an environment for constructive dialogue on evaluations in the disaster setting to strengthen the evidence base for interventions across the disaster spectrum. It remains a work in progress. Wong DF , Spencer C , Boyd L , Burkle FM Jr. , Archer F . Disaster metrics: a comprehensive framework for disaster evaluation typologies. Prehosp Disaster Med. 2017;32(5):501-514.