Plasma and cerebrospinal fluid pharmacokinetics of vodobatinib, a neuroprotective c-Abl tyrosine kinase inhibitor for the treatment of Parkinson's disease.

BACKGROUND: Preclinical evidence suggests that c-Abl is critical in the pathogenesis of Parkinson's Disease (PD). Vodobatinib (K0706) is a potent, specific Abl kinase inhibitor currently being developed for the treatment of PD. In previously reported studies, nilotinib, a multikinase c-Abl inhibitor, did not show clinical activity as evidenced by no improvement of symptoms or the rate of decline after one to six months of treatment at the maximum permissible dose, presumably because of insufficient CNS penetration. Here we report clinical PK and safety data for vodobatinib. OBJECTIVES: To determine safety, plasma PK, and CSF penetration of vodobatinib in healthy volunteers and PD subjects following oral administration, and compare CSF levels to in vitro concentrations required for c-Abl inhibition relative to data reported for nilotinib. METHODS: Inhibition of c-Abl kinase activity and c-Abl binding affinity were first assessed in vitro. Healthy human volunteers and PD patients received various oral doses of vodobatinib once-daily for seven and fourteen days respectively, to assess safety, and plasma and CSF PK. RESULTS: In in vitro assays, vodobatinib was more potent (kinase IC50 = 0.9 nM) than nilotinib (kinase IC50 = 15-45 nM). Administration of vodobatinib 48, 192 and 384 mg to healthy subjects for 7 days yielded mean Cmax, CSF values of 1.8, 11.6, and 12.2 nM respectively, with the two highest doses exceeding the IC50 over the entire dosing interval. Cavg, CSF values were 6-8 times greater than the IC50. Comparable CSF levels were observed in PD patients. All doses were well tolerated in both cohorts. CONCLUSION: Based on achieved CSF concentrations, the potential for c-Abl inhibition in the brain is substantially higher with vodobatinib than with nilotinib. The CSF PK profile of vodobatinib is suitable for determining if c-Abl inhibition will be neuroprotective in PD patients.

Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues.

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

Real-time observation of protein aggregates in pharmaceutical formulations using liquid cell electron microscopy.

Understanding the properties of protein-based therapeutics is a common goal of biologists and physicians. Technical barriers in the direct observation of small proteins or therapeutic agents can limit our knowledge of how they function in solution and in the body. Electron microscopy (EM) imaging performed in a liquid environment permits us to peer into the active world of cells and molecules at the nanoscale. Here, we employ liquid cell EM to directly visualize a protein-based therapeutic in its native conformation and aggregate state in a time-resolved manner. In combination with quantitative analyses, information from this work contributes new molecular insights toward understanding the behaviours of immunotherapies in a solution state that mimics the human body.

Development and Validation of Electrochemiluminescence Assays to Measure Free and Total sSLAMF7 in Human Serum in the Absence and Presence of Elotuzumab.

Elotuzumab is a first in class humanized IgG1 monoclonal antibody for the treatment of multiple myeloma (MM). Elotuzumab targets the glycoprotein signaling lymphocyte activation molecule family 7 (SLAMF7, also described as CS1 or CRACC) which is expressed on the surface of myeloma cells and a subset of immune cells, including natural killer cells. A soluble version of SLAMF7 (sSLAMF7) has also been reported in MM patients but has not been evaluated as a potential biomarker following therapeutic intervention. In order to measure serum levels of sSLAMF7, two immunoassays were developed to monitor changes in circulating sSLAMF7 before and after elotuzumab treatment. Free (drug-unbound) and total (drug-bound and unbound) electrochemiluminescence (ECL) ELISA assays were developed and validated following a fit for purpose (FFP) methodology. Both assays met analytical acceptance criteria for precision, drug interference, dilution linearity, spike recovery, parallelism, and stability. Both exhibited the range and sensitivity necessary to measure clinical samples with an LLOQ of 51.2 pg/mL and ULOQs of 160 (free) and 800 ng/mL (total). Previously described assays were unable to detect sSLAMF7 in healthy individuals. However, due to the increased sensitivity of these new assays, low but measurable sSLAMF7 levels were detected in all normal healthy sera evaluated and were significantly elevated in MM patients. Cohort statistics revealed a significant increase of circulating sSLAMF7 in MM patients versus normal controls and both significant decreases in free and increases in total levels of protein post-elotuzumab treatment.

Anti-PEG antibody bioanalysis: a clinical case study with PEG-IFN-λ-1a and PEG-IFN-α2a in naive patients.

BACKGROUND: Extensive use of polyethylene glycol (PEG) in consumer products necessitates the assessment of anti-PEG antibodies (APAb). METHODS: In clinical trials comparing PEG-IFN-λ to PEG-IFN-α, conventional bridge and direct assays were assessed. RESULTS & CONCLUSION: The bridge assay detected IgM and IgG APAb reactive with common PEG sizes and derivatives at sufficient sensitivity, 15-500 ng/ml. Of subjects evaluated, 6% of PEG-IFN-λ and 9% of PEG-IFN-α subjects had persistent APAb while 60% of PEG-IFN-λ and 33% of PEG-IFN-α subjects had persistent anti-interferon antibodies (AIAb). Pre-existing APAb and AIAb prevalence was comparable (approximately 10% of subjects). APAb were earlier onset, less frequent, less persistent and lower titer than AIAb. No associated hypersensitivity events were reported.

Development and Fit-for-Purpose Validation of a Soluble Human Programmed Death-1 Protein Assay.

Programmed death-1 (PD-1) protein is a co-inhibitory receptor which negatively regulates immune cell activation and permits tumors to evade normal immune defense. Anti-PD-1 antibodies have been shown to restore immune cell activation and effector function-an exciting breakthrough in cancer immunotherapy. Recent reports have documented a soluble form of PD-1 (sPD-1) in the circulation of normal and disease state individuals. A clinical assay to quantify sPD-1 would contribute to the understanding of sPD-1-function and facilitate the development of anti-PD-1 drugs. Here, we report the development and validation of a sPD-1 protein assay. The assay validation followed the framework for full validation of a biotherapeutic pharmacokinetic assay. A purified recombinant human PD-1 protein was characterized extensively and was identified as the assay reference material which mimics the endogenous analyte in structure and function. The lower limit of quantitation (LLOQ) was determined to be 100 pg/mL, with a dynamic range spanning three logs to 10,000 pg/mL. The intra- and inter-assay imprecision were ≤15%, and the assay bias (percent deviation) was ≤10%. Potential matrix effects were investigated in sera from both normal healthy volunteers and selected cancer patients. Bulk-prepared frozen standards and pre-coated Streptavidin plates were used in the assay to ensure consistency in assay performance over time. This assay appears to specifically measure total sPD-1 protein since the human anti-PD-1 antibody, nivolumab, and the endogenous ligands of PD-1 protein, PDL-1 and PDL-2, do not interfere with the assay.

An integrated multiplatform bioanalytical strategy for antibody-drug conjugates: a novel case study.

BACKGROUND: The bioanalytical strategy for antibody-drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker. METHODS: The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC. RESULTS & CONCLUSION: Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure-response relationship.

Optimization of Rolling-Circle Amplified Protein Microarrays for Multiplexed Protein Profiling.

Protein microarray-based approaches are increasingly being used in research and clinical applications to either profile the expression of proteins or screen molecular interactions. The development of high-throughput, sensitive, convenient, and cost-effective formats for detecting proteins is a necessity for the effective advancement of understanding disease processes. In this paper, we describe the generation of highly multiplexed, antibody-based, specific, and sensitive protein microarrays coupled with rolling-circle signal amplification (RCA) technology. A total of 150 cytokines were simultaneously detected in an RCA sandwich immunoassay format. Greater than half of these proteins have detection sensitivities in the pg/ml range. The validation of antibody microarray with human serum indicated that RCA-based protein microarrays are a powerful tool for high-throughput analysis of protein expression and molecular diagnostics.

Conjugated critical reagent characterization for ligand-binding assays: using MALDI-TOF-MS as an orthogonal tool to assess assay performance.

Large-molecule biotherapeutics are forming an increasingly large percentage of emerging pharmaceutical pipelines. These molecules present specific challenges to the bioanalysts charged with measuring in vivo concentrations of the biotherapeutic. The challenges are typically met using ligand-binding assays in support of pharmacokinetic, pharmacodynamic and immunogenicity assays. Ligand-binding assays employ complex biological molecules that specifically recognize the biotherapeutic for quantitation. Generally, a minimum of one of these critical reagents must be chemically modified to generate a signal that is measured in the assay. Once chemically modified it is necessary to characterize the reagent prior to use in an assay. The concentration, purity and molar incorporation ratio of chemical modification are key characteristics. This article presents mass spectral techniques for determining the molar incorporation ratio. Case studies are provided to demonstrate the time and cost savings that can be realized with timely and detailed characterization of critical reagents for ligand-binding assays.

Measuring biotherapeutics with endogenous counterparts and pre-existing antibodies: an interferon case study.

BACKGROUND: This article presents case study data demonstrating the importance of having a thorough understanding of matrix-interference in support of global clinical trials. METHODS/RESULTS: A ligand-binding assay used in the measurement of interferon lambda in human serum was transferred from the reference laboratory to a US-based comparator laboratory and then to a China-based comparator laboratory. The method was successfully validated at each laboratory, however, during cross-validation, there were notable differences, including 30-60% difference in incurred study sample results. The differences were attributed to matrix factors included in the serum pool used to prepare standards and quality controls. Newly procured serum (n = 75 individuals) was tested for assay interference. 12% contained either pre-existing antibodies (auto-antibodies) or were identified as pharmacokinetic assay outliers. CONCLUSION: Prescreening of serum to exclude reactive individuals resulted in successful cross-validation and the establishment of a high integrity pharmacokinetic assay in support of global clinical trials.

Targeting an acid labile aspartyl-prolyl amide bond as a viable alternative to trypsin digestion to generate a surrogate peptide for LC-MS/MS analysis.

BACKGROUND: FGF21-AdPKE is a fusion protein and functionally inactivated in vivo by cleavage around the C-terminus. It is important to quantify the intact active protein in serum. RESULTS & DISCUSSION: Taking advantage of a uniquely acid-labile aspartyl-prolyl amide bond, we developed an acid hydrolysis procedure based on heating FGF21-AdPKE in dilute formic acid to generate a surrogate peptide encompassing the last 17 amino acids at the C-terminus. The monkey serum samples were extracted with an immunocapture procedure with an antibody specific for AdPKE. The calibration range was 200-50000 ng/ml. The assay accuracy and precision were between 92.8-99.8% and 3.9-14.5%, respectively. The method was applied to analyze incurred serum samples from a cynomolgus monkey toxicokinetic study involving administration of FGF21-AdPKE. CONCLUSION: A method of combining immunocapture and acid hydrolysis to quantify a therapeutic protein in biological fluids was developed.

Roux-en-Y gastric bypass corrects hyperinsulinemia implications for the remission of type 2 diabetes.

CONTEXT: Roux-en-Y gastric bypass (RYGB) has been shown to induce rapid and durable reversal of type 2 diabetes. OBJECTIVE: The aim of the study was to investigate a possible mechanism for the remission of type 2 diabetes after RYGB. DESIGN: A cross-sectional, nonrandomized, controlled study was conducted. Surgery patients were studied before RYGB and 1 wk and 3 months after surgery. SETTING: This study was conducted at East Carolina University. SUBJECTS: Subjects were recruited into three groups: 1) lean controls with no surgery [body mass index (BMI) < 25 kg/m²; n = 9], 2) severely obese type 2 diabetic patients (BMI > 35 kg/m²; n = 9), and 3) severely obese nondiabetic patients (BMI > 35 kg/m²; n = 9). INTERVENTION: Intervention was RYGB. RESULTS: One week after RYGB, diabetes was resolved despite continued insulin resistance (insulin sensitivity index was approximately 50% of lean controls) and reduced insulin secretion during an iv glucose tolerance test (acute insulin response to glucose was approximately 50% of lean controls). Fasting insulin decreased and was no different from lean control despite continued elevated glucose in the type 2 diabetic patients compared with lean. CONCLUSIONS: After RYGB, fasting insulin decreases to levels like those of lean control subjects and diabetes is reversed (fasting blood glucose < 125 mg/dl). This leads us to propose that 1) exclusion of food from the foregut corrects hyperinsulinemia and 2) fasting insulin is dissociated from the influence of fasting glucose, insulin resistance, and BMI. The mechanisms for reversal of diabetes in the face of reduced insulin remain a paradox.