Biomarkers of erythropoiesis response to intravenous iron in a crossover pilot study in unexplained anemia of the elderly.

Anemia is common in older adults, but often unexplained. Previously, we conducted a randomized, controlled trial of intravenous (IV) iron sucrose to study its impact on the 6-minute walk test and hemoglobin in older adults with unexplained anemia and ferritin levels of 20-200 ng/mL. In this report, we present for the first time the response of hemoglobin, as well as the dynamic response of biomarkers of erythropoiesis and iron indices, in a pooled analysis of the initially IV iron-treated group of 9 subjects and the subsequently IV iron treated 10 subjects from the delayed treatment group. We hypothesized that there would be a reproducible hemoglobin response from IV iron, and that iron indices and erythropoietic markers would reflect appropriate iron loading and reduced erythropoietic stress. To investigate the biochemical response of anemia to IV iron, we studied the dynamics of soluble transferrin receptor (STfR), hepcidin, erythropoietin (EPO), and iron indices over 12 weeks after treatment. In total, all 19 treated subjects were evaluable: 9 from initial treatment and 10 after cross-over. Hemoglobin rose from 11.0 to 11.7 g/dL, 12 weeks after initiating IV iron treatment of 1000 mg divided weekly over 5 weeks. We found early changes of iron loading after 1-2 IV iron dose: serum iron increased by 184 mcg/dL from a baseline of 66 mcg/dL, ferritin by 184 ng/mL from 68 ng/mL, and hepcidin by 7.49 ng/mL from 19.2 ng/mL, while STfR and serum EPO declined by 0.55 mg/L and 3.5 mU/mL from 19.2 ng/mL and 14 mU/mL, respectively. The erythroid response and evidence of enhanced iron trafficking are consistent with the hypothesis that IV iron overcomes iron deficient or iron-restricted erythropoiesis. These data provide new insight that iron-restricted erythropoiesis is a potential and targetable mechanism for patients diagnosed with unexplained anemia of the elderly and offers support for larger prospective trials of IV iron among anemic older adults of low to normal ferritin.

First-in-Human Study of Bamlanivimab in a Randomized Trial of Hospitalized Patients With COVID-19.

Therapeutics for patients hospitalized with coronavirus disease 2019 (COVID-19) are urgently needed during the pandemic. Bamlanivimab is a potent neutralizing monoclonal antibody that blocks severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) attachment and entry into human cells, which could potentially lead to therapeutic benefit. J2W-MC-PYAA was a randomized, double-blind, sponsor unblinded, placebo-controlled, single ascending dose first-in-human trial (NCT04411628) in hospitalized patients with COVID-19. A total of 24 patients received either placebo or a single dose of bamlanivimab (700 mg, 2,800 mg, or 7,000 mg). The primary objective was assessment of safety and tolerability, including adverse events and serious adverse events, with secondary objectives of pharmacokinetic (PK) and pharmacodynamic analyses. Treatment-emergent adverse event (TEAE) rates were identical in the placebo and pooled bamlanivimab groups (66.7%). There were no apparent dose-related increases in the number or severity of TEAEs. There were no serious adverse events or deaths during the study, and no discontinuations due to adverse events. PKs of bamlanivimab is linear and exposure increased proportionally with dose following single i.v. administration. The half-life was ~ 17 days. These results demonstrate the favorable safety profile of bamlanivimab, and provided the initial critical evaluation of safety, tolerability, and PKs in support of the development of bamlanivimab in several ongoing clinical trials.

Clinical development and evaluation of a VEGF-D assay in plasma from patients with metastatic colorectal cancer in the RAISE study.

BACKGROUND: Vascular endothelial growth factor (VEGF)-D was identified as a potential predictive biomarker for ramucirumab efficacy in second-line metastatic colorectal cancer using a research use only (RUO) assay. We describe results with a new assay for detecting VEGF-D in human plasma. METHODS: In RAISE (Clinical Trial Registration: NCT01183780), 1072 patients were randomized 1:1 to ramucirumab or placebo plus FOLFIRI. All patients were then randomized 1:2 to marker exploratory (ME) and marker confirmatory (MC) groups, and those with plasma samples were analyzed accordingly. A new assay validated for investigational use only (IUO) was used to measure VEGF-D levels in plasma, which were analyzed for correlation with overall and progression-free survival (OS/PFS). IUO assay data were compared with historical RUO assay data. RESULTS: ME subset analyses determined the optimal cutpoint of 5.4 ng/mL for defining high/low VEGF-D subgroups. In the combined ME/MC placebo arms, OS/PFS were numerically greater for patients with low vs high VEGF-D (OS: 12.8 vs 11.1 months; PFS: 5.6 vs 4.2 months). In patients with high VEGF-D, ramucirumab vs placebo demonstrated a numerically greater improvement in OS and PFS. Differential efficacy by VEGF-D level was statistically significant for PFS, but not OS. CONCLUSION: In patients with high VEGF-D, ramucirumab demonstrated a greater improvement in OS and PFS vs placebo; however, baseline VEGF-D level was not predictive of ramucirumab OS benefit using VEGF-D assay for IUO. The RAISE intent-to-treat results remain valid.

Analytical Characterization of an Enzyme-Linked Immunosorbent Assay for the Measurement of Transforming Growth Factor ß1 in Human Plasma.

BACKGROUND: The transforming growth factor ß (TGF-ß)-signaling pathway has emerged as a promising therapeutic target for many disease states including hepatocellular carcinoma (HCC). Because of the pleiotropic effects of this pathway, patient selection and monitoring may be important. TGF-ß1 is the most prevalent isoform, and an assay to measure plasma levels of TGF-ß1 would provide a rational biomarker to assist with patient selection. Therefore, the objective of this study was to analytically validate a colorimetric ELISA for the quantification of TGF-ß1 in human plasma. METHODS: A colorimetric sandwich ELISA for TGF-ß1 was analytically validated per Clinical and Laboratory Standards Institute protocols by assessment of precision, linearity, interfering substances, and stability. A reference range for plasma TGF-ß1 was established for apparently healthy individuals and potential applicability was demonstrated in HCC patients. RESULTS: Precision was assessed for samples ranging from 633 to 10822 pg/mL, with total variance ranging from 28.4% to 7.2%. The assay was linear across the entire measuring range, and no interference of common blood components or similar molecules was observed. For apparently healthy individuals, the average TGF-ß1 level was 1985 ± 1488 pg/mL compared to 4243 ± 2003 pg/mL for HCC patients. Additionally, the TGF-ß1 level in plasma samples was demonstrated to be stable across all conditions tested, including multiple freeze-thaw cycles. CONCLUSIONS: The ELISA described in this report is suitable for the quantification of TGF-ß1 in human plasma and for investigational use in an approved clinical study.

TGF-beta signalling-related markers in cancer patients with bone metastasis.

We measured transforming growth factor (TGF)-beta-dependent biomarkers in plasma and in peripheral blood mononuclear cells (PBMCs) to identify suitable pharmacodynamic markers for future clinical trials with TGF-beta inhibitors. Forty-nine patients with bone metastasis were enrolled in the study, including patients with breast (n=23) and prostate cancer (n=15). Plasma TGF-beta1 levels were elevated in more than half of the cancer patients (geometric mean 2.63 ng ml(-1)) and positively correlated with increased platelet factor 4 (PF4) levels, parathyroid-related protein (PTHrP), von Willebrand Factor (vWF) and interleukin (IL)-10. PBMC were stimulated ex vivo to determine the individual biological variability of an ex vivo assay measuring pSMAD expression. This assay performed sufficiently well to allow its future use in a clinical trial of a TGF-beta inhibitor.

Optimization of analytical and pre-analytical variables associated with an ex vivo cytokine secretion assay.

PURPOSE: Measurements of cytokine release in whole blood after ex vivo stimulation are useful in drug development. The components contributing to variation within such assays have not been clearly defined. Therefore, we characterized the sources of variability within an ex vivo stimulation assay for TNF-alpha release. METHOD: Fresh whole blood or mononuclear cells from a cell preparation tube were added to silanized, screw-top tubes with a final concentration of 1 microg/mL lipopolysaccharide (LPS). Each tube was purged with 95% air/5%CO2 and incubated 4 or 6 h at 37 degrees C in a metabolic water bath. Plasma TNF-alpha was next measured in supernatants by immunoassay. Total method variability was assessed in 10 normal donors each drawn in the morning and afternoon over 3 days. Four additional samples were pre-treated with dexamethasone to investigate inhibition of TNF-alpha release. RESULTS: Our analysis indicated precise temperature control, the timing and duration of stimulation, and the surface properties of the stimulation vessel most significantly influenced assay performance. A comparison of multiple anticoagulants indicated that careful consideration should be taken in selecting the optimal anticoagulant. The estimated total assay CV for all anticoagulants tested was less than 33.81%. The analytical variability (stimulation and measurement) was less than 25.88% CV. The one exception was mononuclear cells collected in sodium heparin. The total variability estimate incorporated day-to-day, diurnal, inter-donor, tube-to-tube and immunoassay variability. Using our optimized conditions, TNF-alpha release was inhibited by dexamethasone with a mean IC50 of 33.3 +/- 4.6 nM. CONCLUSIONS: We have described an optimal set of conditions for collection, storage and processing of an ex vivo cytokine stimulation assay. These conditions were selected for operational feasibility, minimal imprecision and elimination of potential confounding factors. The end result is a more robust method that can be applied to clinical drug development.