Ebola virus antibody decay-stimulation in a high proportion of survivors.

Neutralizing antibody function provides a foundation for the efficacy of vaccines and therapies1-3. Here, using a robust in vitro Ebola virus (EBOV) pseudo-particle infection assay and a well-defined set of solid-phase assays, we describe a wide spectrum of antibody responses in a cohort of healthy survivors of the Sierra Leone EBOV outbreak of 2013-2016. Pseudo-particle virus-neutralizing antibodies correlated with total anti-EBOV reactivity and neutralizing antibodies against live EBOV. Variant EBOV glycoproteins (1995 and 2014 strains) were similarly neutralized. During longitudinal follow-up, antibody responses fluctuated in a 'decay-stimulation-decay' pattern that suggests de novo restimulation by EBOV antigens after recovery. A pharmacodynamic model of antibody reactivity identified a decay half-life of 77-100 days and a doubling time of 46-86 days in a high proportion of survivors. The highest antibody reactivity was observed around 200 days after an individual had recovered. The model suggests that EBOV antibody reactivity declines over 0.5-2 years after recovery. In a high proportion of healthy survivors, antibody responses undergo rapid restimulation. Vigilant follow-up of survivors and possible elective de novo antigenic stimulation by vaccine immunization should be considered in order to prevent EBOV viral recrudescence in recovering individuals and thereby to mitigate the potential risk of reseeding an outbreak.

Disruption of memory B-cell trafficking by belimumab in patients with systemic lupus erythematosus.

OBJECTIVES: Autoreactive memory B cells contribute to chronic and progressive courses in autoimmune diseases like systemic lupus erythematosus (SLE). The efficacy of belimumab (BEL), the first approved biologic treatment for SLE and lupus nephritis (LN), is generally attributed to depletion of activated naïve B cells and inhibition of B cell activation. BEL's effect on memory B cells (MBCs) is currently unexplained. We performed an in-depth cellular and transcriptomic analysis of BEL's impact on the blood MBC compartment in patients with SLE. METHODS: A retrospective meta-analysis was conducted, pooling flow cytometry data from four randomized trials involving 1245 patients with SLE treated with intravenous BEL or placebo. Then, extensive MBC phenotyping was performed using high-sensitivity flow cytometry in patients with mild/moderate SLE and severe SLE/LN treated with subcutaneous BEL. Finally, transcriptomic characterization of surging MBCs was performed by single-cell RNA sequencing. RESULTS: In BEL-treated patients, a significant increase in circulating MBCs, in a broad range of MBC subsets, was established at week 2, gradually returning to baseline by week 52. The increase was most prominent in patients with higher SLE disease activity, serologically active patients, and patients aged ≤18 years. MBCs had a non-proliferating phenotype with a prominent decrease in activation status and downregulation of numerous migration genes. CONCLUSION: Upon BEL initiation, an increase of MBCs was firmly established. In the small cohort investigated, circulating MBCs were de-activated, non-proliferative, and demonstrated characteristics of disrupted lymphocyte trafficking, expanding on our understanding of the therapeutic mechanism of B cell-activating factor inhibition by BEL. TRIAL REGISTRATION: ClinicalTrials.gov NCT00071487, NCT00410384, NCT01632241, NCT01649765, NCT03312907, NCT03747159.

Longitudinal modeling of efficacy response in patients with lupus nephritis receiving belimumab.

Belimumab was approved for active lupus nephritis (LN) in adults in the European Union and patients ≥ 5 years of age in the USA based on a Phase 3, double-blind, placebo-controlled, 104-week study. The study evaluated the efficacy of belimumab plus background standard therapy in adults with active LN using an intravenous (IV) dose of 10 mg/kg. A longitudinal analysis of Primary Efficacy Renal Response (PERR) and Complete Renal Response (CRR) was performed to assess whether patients with high proteinuria at the start of belimumab treatment would benefit from a higher dose. Responder probability was modeled as a logistic regression with probability a function of time and treatment (belimumab or placebo). Dropout risk at each visit was incorporated into a joint model of efficacy response; only efficacy data prior to dropout events (belimumab discontinuation, treatment failure, or withdrawal) were included. Average belimumab concentration over the first 4 and 12 weeks and baseline proteinuria were considered as continuous covariates. In general, renal response (PERR and CRR) over time was higher in patients receiving belimumab than in those receiving placebo. Baseline proteinuria was considered the most relevant predictor of renal response, with reduced efficacy in patients with increased proteinuria for both belimumab or placebo treatment. For belimumab-treated patients, belimumab exposure was not found to be an important predictor of renal response. In conclusion, the 10 mg/kg IV dose was considered appropriate in all patients and there was no evidence to suggest a higher response would be achieved by increasing the dose.

Assessment of the safety, pharmacokinetics and pharmacodynamics of GSK3335065, an inhibitor of kynurenine monooxygenase, in a randomised placebo-controlled first-in-human study in healthy volunteers.

GSK3335065 is an inhibitor of kynurenine monooxygenase (KMO) being developed for the treatment of acute pancreatitis. Healthy male volunteers were administered ascending doses of GSK3335065 or matched placebo as a single intravenous bolus injection to assess safety, tolerability, pharmacokinetics and pharmacodynamics. GSK3335065 displayed an apparent volume of distribution between 20.6 L and 44.6 L, a clearance between 0.462 L/h and 0.805 L/hr and a terminal half-life between 31.3 and 34.5 hr. In the single subject who received 1.3 mg GSK3335065, changes in tryptophan pathway metabolites were observed consistent with the changes seen in preclinical species suggesting that KMO enzyme activity was partially inhibited. However, a broad complex ventricular tachycardia was observed in this subject, which was judged to be a Serious Adverse Event (SAE) and resulted in early termination of the study. While development of GSK3335065 was subsequently discontinued, significant confounding factors hinder a clear interpretation that the tachycardia was directly related to administration of the compound.

Population model-based analysis of the memory B-cell response following belimumab therapy in the treatment of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by B-cell hyperactivity and breach of tolerance. Autoreactive memory B cells, which have a decreased activation threshold and the ability to survive in absence of antigen, are believed to contribute to chronicity in autoimmune diseases like SLE. Belimumab, the first approved biological treatment of active SLE and lupus nephritis, reduces B cells dependent on B-lymphocyte stimulator protein (BLyS) for survival, whereas memory B cells are spared; several studies reported circulating memory B-cell concentrations increase following BLyS neutralization. This analysis investigated the effect of dose, demographics, and disease status on memory B-cell response after starting belimumab treatment. Population pharmacodynamic models were fitted to a pooled dataset from seven belimumab SLE trials. The optimal model was selected using maximum likelihood methods and was then refit to the data using Bayesian analysis and used to simulate memory B-cell response by belimumab dose and covariate subgroups. At the belimumab approved doses (10 mg/kg intravenously every 4 weeks, 200 mg subcutaneously every week), circulatory memory B cells increase in the first 4-8 weeks after belimumab initiation, typically returning to baseline levels over 76 weeks. The model analysis suggested belimumab stimulates memory B-cell transition from lymphoid and/or inflamed tissues into the circulation, rather than inhibiting trafficking in the reverse direction. Baseline BLyS and anti-double-stranded deoxyribonucleic acid antibody concentrations were statistically identifiable covariates of memory B-cell response, although their impact on predicting size and response duration was small.

The effect of 24-week belimumab treatment withdrawal followed by treatment restart in patients with SLE: an open-label, non-randomised 52-week study.

BACKGROUND: Treatment goals for patients with systemic lupus erythematosus (SLE) include minimising disease activity and reducing the risk of flares. Although belimumab is effective at reducing disease activity and risk of severe flares, it was previously unknown what the clinical effects were upon treatment discontinuation. The objective of this study was to assess the impact of temporary withdrawal of intravenous (IV) belimumab in patients with SLE. METHODS: This multicentre, open-label, non-randomised, 52-week study (GSK Study BEL116027; NCT02119156) recruited patients with SLE and stable low disease activity, of whom those on belimumab 10 mg/kg IV plus standard therapy either discontinued belimumab for 24 weeks and then restarted belimumab 10 mg/kg IV every 4 weeks (q4w) for 28 weeks (treatment holiday [TH] group), or continued on belimumab 10 mg/kg IV plus standard therapy q4w for 52 weeks (treatment continuation [TC] group). The primary endpoint was median time to first Safety of Estrogens in Lupus Erythematosus National Assessment-SLE Disease Activity Index (SELENA-SLEDAI) Flare Index flare. Secondary and other endpoints included rate of any flare, time to severe flare, time to renal flare and rebound (SELENA-SLEDAI score exceeding parent study baseline). Data on rebound phenomenon in patients with any disease level of SLE who had permanently withdrawn from further belimumab treatment (long-term discontinuation group [LTD]) were also assessed. Safety was assessed. RESULTS: The primary endpoint was not evaluable in the TH (n = 12) and TC (n = 29) groups as fewer than half of patients flared. Unadjusted flare rates per patient-year were 1.0 during treatment discontinuation and 0.3 during treatment restart (0.6 overall) in the TH group and 0.6 in the TC group; there were no severe or renal flares. No TH patients rebounded; 2 (6.9%) TC patients rebounded; 2 (5.1%) patients in the LTD group rebounded. There were no new safety signals. CONCLUSIONS: Twenty-four-week belimumab discontinuation did not appear to increase the risk of flares or rebound in patients with low SLE disease activity; flare rates were low in both groups. Further studies may help to fully determine the effect of belimumab discontinuation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02119156 . Registered on April 21, 2014.

Pharmacokinetics of Belimumab in Children With Systemic Lupus Erythematosus.

The phase 2 placebo-controlled, double-blind PLUTO trial characterized the pharmacokinetics of belimumab plus standard systemic lupus erythematosus (SLE) therapy in patients with childhood-onset SLE (cSLE) and demonstrated similar efficacy and safety to that in adult SLE. Patients with active cSLE aged 5-17 years were randomized to intravenous belimumab 10 mg/kg every 4 weeks (n = 53). A linear 2-compartment population pharmacokinetics (popPK) model with first-order elimination was developed, and an exploratory exposure-response analysis assessed the impact of between-patient exposure variability on clinical response (SLE Responder Index 4 [SRI4]) in week 52, and occurrence of serious adverse events during the study. The popPK model estimated clearance of 158 mL/day, steady-state volume of distribution of 3.5 L, terminal half-life of 16.3 days, and distribution half-life of 0.8 days in the overall population. Fat-free mass (FFM) better characterized the pharmacokinetics than total body weight and was more consistent with allometric scaling theory; belimumab pharmacokinetics were largely determined by FFM. Age, sex, disease activity, and concomitant medication had no impact on pediatric belimumab exposure after accounting for body size. Individual and median steady-state pediatric pharmacokinetic profiles were similar to known adult profiles and pediatric exposure estimates for belimumab 10 mg/kg intravenously were consistent with adult exposures. Exposures were similar between SRI4 responders and nonresponders, and patients who did or did not experience a serious adverse event. There was no clinically relevant correlation between exposure and efficacy or safety, confirming belimumab 10 mg/kg intravenous dose every 4 weeks as appropriate for pediatric patients with cSLE.

Control of translation initiation: a model-based analysis from limited experimental data.

We have built a detailed kinetic model of translation initiation in yeast and have used a novel approach to determine the flux controlling steps based on limited experimental data. An efficient parameter estimation method was adapted in order to fit the most uncertain parameters (rate constants) to in vivo measurements in yeast. However, it was found that there were many other sets of plausible parameter values that also gave a good fit of the model to the data. We therefore used random sampling of this uncertain parameter space to generate a large number of diverse fitted parameter sets. A compact characterization of these parameter sets was provided by considering flux control. In particular, we suggest that the rate of translation initiation is most strongly influenced by one of two reactions: either the guanine nucleotide exchange reaction involving initiation factors eIF2 and eIF2B or the assembly of the multifactor complex from its constituent protein/tRNA containing complexes. It is hoped that the approach presented in this paper will add to our understanding of translation initiation pathway and can be used to identify key system-level properties of other biochemical processes.

Population Pharmacokinetic Modelling of Ceftazidime and Avibactam in the Plasma and Epithelial Lining Fluid of Healthy Volunteers.

OBJECTIVES: Our objective was to develop population pharmacokinetic (PK) models for ceftazidime and avibactam in the plasma and epithelial lining fluid (ELF) of healthy volunteers and to compare ELF concentrations to plasma PK/pharmacodynamic (PD) targets. METHODS: Plasma and ELF population PK models were developed for ceftazidime and avibactam concentration data from 42 subjects (NCT01395420). Two- and three-compartment plasma PK models were fitted to ceftazidime and avibactam plasma PK data, and different plasma-ELF linked models were evaluated. Using best-fitting models, plasma and ELF concentration-time profiles were simulated for 1000 subjects. ELF concentration-time profiles for ceftazidime/avibactam 2000-500 mg every 8 h were compared with plasma PK/PD targets for ceftazidime (50% of time above [fT >] 8 mg/l) and avibactam (50% fT > 1 mg/l). RESULTS: Three-compartment PK models best fitted the plasma concentration data for ceftazidime and avibactam. ELF data for both drugs were best described by a direct response (instantaneous equilibrium) model. Ceftazidime plasma-ELF relationships were best described by a saturable Michaelis-Menten model. For avibactam, departure from plasma-ELF relationship linearity was more modest than for ceftazidime. ELF:plasma penetration ratios of both ceftazidime (52%) and avibactam (42%) at plasma concentrations relevant for efficacy (~ 8 mg/l for ceftazidime and ~ 1 mg/l for avibactam) were greater than previously calculated using non-compartmental area under the curve (AUC) methods, which average across the entire concentration range. Ceftazidime and avibactam ELF exposures exceeded their respective plasma PK/PD time-above-threshold targets by the dosing interval mid-point in most subjects. CONCLUSIONS: This compartmental modelling analysis suggests ELF exposures of both ceftazidime and avibactam exceed levels required for efficacy in plasma.

Modelling of tumour growth and cytotoxic effect of docetaxel in xenografts.

One of the major sources of information on physiological and pathophysiological effects in pre-clinical oncology studies is the xenografted tumour animal model. However, measurement of tumour volume over time potentially masks a range of biological changes that the xenograft is undergoing. In this paper a mechanistic model of tumour growth in xenografts is presented that can be used to investigate the mode of drug action with respect to phenotypic changes. The model encapsulates key histological biomarkers and spatial constraints. The unknown model parameters are first shown to be uniquely identifiable from the proposed experimental studies, and then estimated from the resulting data using the anti-cancer agent docetaxel.

The dynamics of water exchange in gadolinium DOTA complexes studied by transition path sampling and potential of mean force methods.

The mechanism of water exchange at the Gd centre of the two isomers of [Gd(iii)DOTA](-) (gadolinate(1-), [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetato(4-)-N1,N4,N7,N10,O1,O4,O7,O10]) has been explored using transition path sampling and potential of mean force methods to sample those regions of phase space inaccessible to standard molecular dynamics simulation. We find that there are definite differences in the details of the solvent rearrangement accompanying the exchange of the capping water molecule for the two isomers. We conclude that these solvent effects, rather than any differences in the binding energy of the capping water, are central in determining the exchange rate. We find that the potential of mean force studies yield absolute and relative rates of water exchange for the two isomers that are in good agreement with experiment.

Exploring reaction pathways with transition path and umbrella sampling: application to methyl maltoside.

The transition path sampling (TPS) method is a powerful approach to study chemical reactions or transitional properties on complex potential energy landscapes. One of the main advantages of the method over potential of mean force methods is that reaction rates can be directly accessed without knowledge of the exact reaction coordinate. We have investigated the complementary nature of these two differing approaches, comparing transition path sampling with the weighted histogram analysis method to study a conformational change in a small model system. In this case study, the transition paths for a transition between two rotational conformers of a model disaccharide molecule, methyl beta-D-maltoside, were compared with a free energy surface constrained by the two commonly used glycosidic (phi,psi) torsional angles. The TPS method revealed a reaction channel that was not apparent from the potential of mean force method, and the suitability of phi and psi as reaction coordinates to describe the isomerization in vacuo was confirmed by examination of the transition path ensemble. Using both transition state theory and transition path sampling methods, the transition rate was estimated. We have estimated a characteristic time between transitions of approximately 160 ns for this rare isomerization event between the two conformations of the carbohydrate. We conclude that transition path sampling can extract subtle information about the dynamics not apparent from the potential of mean force method. However, in calculating the reaction rate, the transition path sampling method required 27.5 times the computational effort than was needed by the potential of mean force method.