Interlaboratory Reproducibility of Standardized Hemagglutination Inhibition Assays.

The hemagglutination inhibition (HI) assay is a prominent and commonly accepted method used to determine quantitative antibody titers for influenza virus. However, the reproducibility and consistency of this assay may be affected by several factors, including its reliance on biological reagents that are difficult to standardize, such as red blood cells. This report assesses HI assay performance across three accredited, global laboratories when using test virus and a human serum panel aliquoted and distributed from a centrally located reagent stock. The panel of human sera comprised samples with expected low, medium, and high HI titers against two influenza viruses: A/H1N1/California/07/2009 and B/Victoria/Brisbane/60/2008. HI analysis followed a consensus test protocol. Overall, the HI assay reproducibility within each laboratory was high for both influenza strains, with a within-assay run and intraday precision of 100%. Interlab reproducibility was assessed by comparing the geometric mean titer (GMT) of each sample at each laboratory to the consensus GMT of the sample. A/H1N1 had 100% interlab reproducibility, and none of the individual laboratory GMT values exceeded a 2-fold difference compared to the consensus GMT in any tested sample. B/Victoria had an overall reproducibility of 83%. The results demonstrate that with standardization of key reagents and the use of a common protocol by trained staff, the biologically based HI assay can provide similar results between geographically dispersed laboratories. IMPORTANCE Licensure of influenza vaccines relies on the hemagglutination inhibition (HI) assay as the primary method to determine quantitative functional antibody titers. The HI assay is also widely used for influenza virus surveillance, characterization, and epidemiology studies. However, the HI assay has a notable lack of reproducibility and consistency. If serology results are required from multiple concurrent studies supporting the development and regulatory approval of a product, the testing capacity of any given testing laboratory may be exceeded and data from more than one testing laboratory included in regulatory filings. Thus, understanding the reproducibility of HI assay results over time and between testing laboratories is necessary to support a robust clinical trial serology data set. Our results demonstrate that with standardization of key reagents and use of a common protocol by experienced and trained staff, the biologically based HI assay can provide similar results between geographically dispersed laboratories.

Pharmacokinetics and biodistribution of RGD-targeted doxorubicin-loaded nanoparticles in tumor-bearing mice.

We report the biodistribution and pharmacokinetics (PK) of a cyclic RGD-doxorubicin-nanoparticle (NP) formulation in tumor-bearing mice. The NP core was composed of inulin multi-methacrylate with a targeting peptide, cyclic RGD, covalently attached to the NPs via PEG-400. Seventy-two percent of the doxorubicin was attached to the NP matrix via an amide bond; 28% of doxorubicin was entrapped as unconjugated drug. The PK of total, unconjugated and metabolized doxorubicin was examined for 5 days following intravenous (i.v.) administration of the NP formulation (250 microg doxorubicin equiv.), revealing a bi-exponential fix with a terminal half-life of 5.99 h. In addition, the biodistribution studies revealed decreasing drug concentrations over time in the heart, lung, kidney and plasma and accumulating drug concentrations in the liver, spleen and tumor. The drug concentration in these latter tissues peaked between 24 and 48 h with the liver, spleen and tumor containing 56, 3.5 and 1.8% of the administered dose at t=48 h, respectively. In contrast to all of the organs studied, the tumors contained high levels of a doxorubicin metabolite.

A Phase I/II Trial of BNC105P with Everolimus in Metastatic Renal Cell Carcinoma.

PURPOSE: BNC105P inhibits tubulin polymerization, and preclinical studies suggest possible synergy with everolimus. In this phase I/II study, efficacy and safety of the combination were explored in patients with metastatic renal cell carcinoma (mRCC). EXPERIMENTAL DESIGN: A phase I study in patients with clear cell mRCC and any prior number of therapies was conducted using a classical 3 + 3 design to evaluate standard doses of everolimus with increasing doses of BNC105P. At the recommended phase II dose (RP2D), patients with clear cell mRCC and one to two prior therapies (including ≥ 1 VEGF-TKI) were randomized to BNC105P with everolimus (arm A) or everolimus alone (arm B). The primary endpoint of the study was 6-month progression-free survival (6MPFS). Secondary endpoints included response rate, PFS, overall survival, and exploratory biomarker analyses. RESULTS: In the phase I study (N = 15), a dose of BNC105P at 16 mg/m(2) with everolimus at 10 mg daily was identified as the RP2D. In the phase II study, 139 patients were randomized, with 69 and 67 evaluable patients in arms A and B, respectively. 6MPFS was similar in the treatment arms (arm A: 33.82% vs. arm B: 30.30%, P = 0.66) and no difference in median PFS was observed (arm A: 4.7 mos vs. arm B: 4.1 mos; P = 0.49). Changes in matrix metalloproteinase-9, stem cell factor, sex hormone-binding globulin, and serum amyloid A protein were associated with clinical outcome with BNC105P. CONCLUSIONS: Although the primary endpoint was not met in an unselected population, correlative studies suggest several biomarkers that warrant further prospective evaluation.

Encapsulation of protamine sulphate compacted DNA in polylactide and polylactide-co-glycolide microparticles.

This paper details the development and characterisation of a DNA-loaded microsphere system for gene delivery purposes. Encapsulation of DNA in microspheres was carried out by precondensation of the DNA with protamine sulphate, followed by encapsulation in a polymeric microsphere using a water-oil-water (w/o/w) solvent evaporation-emulsion process. The polymers used were polylactide and polylactide-co-glycolide. The amount of DNA encapsulated in the microsphere formulation was dependent on the weight ratio of protamine sulphate: DNA, with higher ratios (0.87:1 or 2:1) giving better encapsulation efficiencies (> or =62%). Additionally, compared to non-compacted DNA, the encapsulation of a protamine sulphate:DNA complex increased protection of the DNA against nuclease and decreased shear effects during processing. The release profile of DNA was shown to be dependent on the polymer type and polymer molecular weight. DNA was released relatively quickly from the microspheres and the released DNA primarily existed in a relaxed state. However, in vitro transfection experiments indicated that the DNA was still active upon release from the microspheres.

In vitro study of GDNF release from biodegradable PLGA microspheres.

Glial cell line-derived neurotrophic factor (GDNF) is a protein with potent trophic actions on dopaminergic neurons, which is under investigation as a therapeutic agent for the treatment of neurodegenerative disorders, including Parkinson's disease. The aim of this work was to develop GDNF-loaded microspheres, which could be implanted by stereotaxy in the brain and could offer an alternative strategy in the treatment of Parkinson's disease. A w/o/w extraction-evaporation technique was chosen to prepare protein-loaded microspheres. An in vitro release study of the protein was required to assess the retention of integrity and the performance of the microsphere formulation with regard to sustained release. In order to assess the in vitro release profile of the GDNF-loaded microspheres, a preliminary study was performed to select an appropriate buffer for GDNF stabilization, using experimental designs. GDNF was measured by both enzyme-linked immunosorbant assay (ELISA) and radioactivity using (125)I-GDNF. The GDNF-loaded microsphere release profile was assessed in a low continuous flow system, and showed a sustained release over 56 days of biologically active GDNF at clinically relevant doses.

A phase II clinical trial of the vascular disrupting agent BNC105P as second line chemotherapy for advanced Malignant Pleural Mesothelioma.

BNC105P is a tubulin polymerisation inhibitor that selectively disrupts tumour vasculature and suppresses cancer cell proliferation. This agent has exhibited preclinical and phase I activity in Malignant Pleural Mesothelioma (MPM). This phase II, single arm trial investigated the efficacy and safety of BNC105P as second line therapy in MPM. Participants had progressive MPM after first line pemetrexed/platinum chemotherapy, ECOG PS 0-1, adequate organ function, and measurable disease. BNC105P 16 mg/m(2) was administered intravenously on day 1 and 8 every 21 days until progression or undue toxicity. The primary endpoint was centrally reviewed objective response rate (RR). Tumour response was assessed every two cycles using modified RECIST. 30 patients were enrolled in 10 months, predominantly male (90%), ECOG PS 1 (77%), epithelioid histology (67%), and non-metastatic disease (67%). All patients received at least one dose of study drug, with a median of 2 cycles. No significant haematologic, biochemical, or cardiac adverse events (AEs) were observed. Grade 3 or 4 AEs occurred in 10 patients (33%). There were 2 deaths on study: 1 cardiorespiratory, the other to pneumonia. We observed 1 partial response (3%); 13 patients had stable disease (43%). Median progression free survival was 1.5 months (95% CI 1.4-2.4); median overall survival was 8.2 months (95% CI 3.8-11.9). BNC105P was safe and tolerable. The sole response was insufficient to warrant further research as a single agent.

Clinical, pharmacodynamic, and pharmacokinetic evaluation of BNC105P: a phase I trial of a novel vascular disrupting agent and inhibitor of cancer cell proliferation.

PURPOSE: To determine the recommended phase II dose and evaluate the safety and toxicity profile and pharmacokinetic (PK) and pharmacodynamic (PD) effects of BNC105P, an inhibitor of tubulin polymerization that has vascular disrupting and antiproliferative effects. EXPERIMENTAL DESIGN: BNC105P was administered as a 10-minute infusion on days 1 and 8 of a 21-day cycle in a first-in-human phase I study. A dynamic accelerated dose titration method was used for dose escalation. Plasma concentrations of BNC105P (phosphate prodrug) and BNC105 (active agent) were determined. PD assessments were carried out using dynamic contrast enhanced (DCE)-MRI and analysis of a blood-borne biomarker. RESULTS: Twenty-one subjects with advanced solid tumors were enrolled on 6 dose levels (range: 2.1-18.9 mg/m(2)). The recommended dose level was 16 mg/m(2) and was well tolerated. BNC105P (prodrug) rapidly converted to BNC105 with a half-life of 0.13 hours. Plasma concentrations of BNC105 generally increased in proportion to dose with a half-life of 0.57 hours. Pharmacodymanically active plasma levels were obtained with a dose dependant reduction in the levels of polymerized tubulin (on-target action) being observed in PBMCs. DCE-MRI also indicated blood flow changes in the tumor lesions of a number of subjects. CONCLUSIONS: BNC105P has a favorable toxicity profile at the recommended dose of 16 mg/m(2) and is associated with PD changes consistent with its known mechanism of action. Phase II studies in renal cancer and mesothelioma have commenced.

A novel in vitro delivery system for assessing the biological integrity of protein upon release from PLGA microspheres.

PURPOSE: The development of a novel in vitro system is required to assess the stability and release kinetics of a protein microsphere formulation used for drug delivery to the brain. METHODS: Microspheres containing lysozyme as model protein were prepared using a (w/o/w) emulsion-solvent evaporation process. Both the active and total (active + inactive) encapsulation efficiencies and release profiles were determined. The biologic activity of lysozyme was measured using bacterial cell lysis; total protein content was measured using a 125I-radiolabel. A novel in vitro apparatus was developed to determine kinetics over a sustained time period (>30 days). RESULTS: The microencapsulation technique allowed an entrapment of active lysozyme at 80 +/- 4% and a sustained (>42 days) in vitro release. The kinetics study showed that the novel in vitro system was able to detect the release of low amounts (ng) of protein. To improve the stability of the protein within microspheres and allow the release of biologically active lysozyme, a basic additive ( Mg(OH)2 ) was successfully encapsulated. CONCLUSIONS: This novel in vitro system was appropriate to study protein microsphere release kinetics. In addition, the model is cost-effective and mimes brain physiological conditions more closely than previous models.