Abstract. The predominant driver of bioanalysis in supporting drug development is the intended use of the data. Ligand-binding assays (LBA) are widely used for the analysis of protein biotherapeutics and target ligands (L) to support pharmacokinetics/pharmacodynamics (PK/PD) and safety assessments. For monoclonal antibody drugs (mAb), in particular, which non-covalently bind to L, multiple forms of mAb and L can exist in vivo, including free mAb, free L, and mono-and/or bivalent complexes of mAb and L. Given the complexity of the dynamic binding equilibrium occurring in the body after dosing and multiple sources of perturbation of the equilibrium during bioanalysis, it is clear that ex vivo quantification of the forms of interest (free, bound, or total mAb and L) may differ from the actual ones in vivo. LBA reagents and assay formats can be designed in principle to measure the total or free forms of mAb and L. However, confirmation of the forms being measured under the specified conditions can be technically challenging. The assay forms and issues must be clearly communicated and understood appropriately by all stakeholders as the program proceeds through the development process. This paper focuses on monoclonal antibody biotherapeutics and their circulatory L that are either secreted as soluble forms or shed from membrane receptors. It presents an investigation into the theoretical and practical considerations for total/free analyte assessment to increase awareness in the scientific community and offer bioanalytical approaches to provide appropriate PK/PD information required at specific phases of drug development.

Botulinum neurotoxin A is a category A bioterrorism agent. Current antitoxin therapies are scarce and produce adverse reactions. XOMA 3AB consists of 3 IgG1 monoclonal antibodies (MAbs), each with a distinct human or humanized variable region, which bind to distinct epitopes on botulinum neurotoxin serotype A. This first-in-human study evaluated the safety and pharmacokinetics (PK) of escalating doses of XOMA 3AB administered intravenously (i.v.) to healthy adults. In this double-blind placebo-controlled dose escalation study, 3 cohorts of 8 healthy subjects received a single intravenous dose of XOMA 3AB or placebo at a 3:1 ratio. Follow-up examinations included physical examinations, hematology and chemistry blood tests, electrocardiograms, and pharmacokinetics. Pharmacokinetic parameters were estimated using noncompartmental methods. There were no infusion discontinuations or hypersensitivity reactions. Two or more subjects experienced headache, hyperglycemia, or anemia; none was dose related. All adverse events (AEs) were mild to moderate except for an episode of exercise-induced elevation of a subject's creatine phosphokinase (CPK) level, unrelated to XOMA 3AB. Concentration-time plots demonstrated a peak in MAb concentrations 1 to 2 h after completion of the infusion, after which the levels declined in a biexponential decay pattern for all analytes. For each MAb, the maximum concentration of drug in serum (Cmax) and the area under the concentration-time curve from 0 to infinity (AUCinf) increased as the dose increased. Clearance of the humanized mouse MAb was more rapid than that of the two fully human MAbs, particularly at the lowest dose. None of the MAbs was immunogenic. At the doses administered, XOMA 3AB was well tolerated. These safety findings support further investigation of XOMA 3AB as a potential agent for botulism treatment and postexposure prophylaxis. (This study has been registered at ClinicalTrials.gov under registration no. NCT01357213.).

Abstract. Continuous improvement in bioanalytical method development is desired in order to ensure the quality of the data and to better support pharmacokinetic (PK) and safety studies of biotherapeutics. One area that has been getting increasing attention recently is in the assessment of "free" and "total" analyte and the impact of the assay format on those assessments. To compliment these considerations, the authors provide a critical review of available literature and prospectively explore methods to mitigate the potential impact of anti-drug antibody on PK assay measurement. This challenge is of particular interest and importance since biotherapeutic drugs often elicit an immune response, and thus may have a direct impact on quantification of the drug for its PK and safety evaluations.

We have examined several analogs of 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in an animal model of osteoporosis (ovariectomized rats) to identify a compound with a greater therapeutic range than 1,25-(OH)(2)D(3) for treatment of this bone disease. Here, we report that one analog, Ro-26-9228, had a bone-protecting effect but did not induce hypercalcemia at a wide concentration range. Analysis of biochemical markers and the bone histomorphometry of analog-treated rats suggested that Ro-26-9228 acted by inhibiting bone resorption and increasing the number of differentiated osteoblasts. To determine the basis for the segregation between hypercalcemia and bone-protecting action, we examined gene expression in tissues that regulate calcium homeostasis. We found that 1,25-(OH)(2)D(3) induced 24-hydroxylase mRNA expression in the duodena of ovariectomized rats, but Ro-26-9228 did not. Furthermore, in the duodena of intact animals, 1,25-(OH)(2)D(3) induced a significant increase in calbindin D 9K and plasma membrane calcium pump 1 mRNAs, but Ro-26-9228 had no effect on these mRNAs. On the other hand, the osteoblast-specific gene products osteocalcin and osteopontin were significantly up-regulated in trabecular bone by both the natural hormone and Ro-26-9228. Further investigation of gene-regulatory events in trabecular bone revealed that both 1,25-(OH)(2)D(3) and Ro-26-9228 up-regulated TGF beta1 and beta2 mRNAs. We concluded that the unique properties of Ro-26-9228 include preferential gene regulation in osteoblasts over duodenum and effective induction of growth factors in bone.

The vitamin D analog, 1alpha-fluoro-16-ene-20-epi-23-ene-26,27-bishomo-25-hydroxyvitamin D(3) (Ro-26-9228) is tissue selective, with a gene regulation preference for bone over duodenum in vivo. In the human osteoblast-like cells, hFOB, the vitamin D receptor (VDR)-mediated transcriptional potencies of Ro-26-9228 and 1,25-dihydroxyvitamin D(3) (1,25D(3)) were similar, but in the intestinal cells, Caco-2, transcriptional potency of Ro-26-9228 was 10-50 times lower. We hypothesized that transcriptional activation of the VDR by Ro-26-9228 in the two cell types is regulated differently, and compared VDR extracted from hFOB or Caco-2 cells for their abilities to interact with a p160 coactivator [glucocorticoid receptor-interacting protein (GRIP)] and with retinoid X receptor (RXR) by pull-down assays. 1,25D(3) had similar potencies to induce interactions of VDR from the two cell types with these partners of transcription. In contrast, Ro-26-9228 induced interaction of osteoblastic VDR with RXR and GRIP but did not induce these interactions with VDR from Caco-2 cells. Further studies revealed that in hFOB cells the unoccupied VDR was cytoplasmic and proteasome sensitive, and that ligand treatment caused a rapid accumulation of the VDR in the chromatin. Both cytoplasmic and chromatin-associated ligand-bound VDR from hFOB cells had the abilities to interact with GRIP. In contrast, in Caco-2 cells, unoccupied VDR was localized in both the cytoplasm (70%) and the chromatin (30%). In Caco-2 cells, the cytoplasmic VDR was proteasome resistant, and neither 1,25D(3) nor Ro-26-9228 induced its binding to GRIP. Only a small fraction of the chromatin-associated VDR was proteasome sensitive, and this fraction was distinguishable by a faster electrophoretic mobility. 1,25D(3) induced an accumulation of the proteasome-sensitive VDR in the chromatin of Caco-2 cells and binding to GRIP. Ro-26-9228 failed to induce accumulation of the proteasome-sensitive VDR in the chromatin or binding to GRIP, but a coincubation of Caco-2 cells with the analog and a proteasome inhibitor restored these abilities. These results suggest that Ro-26-9228 has poor ability to promote the accumulation of a proteasome-sensitive, transcriptionally active VDR isoform in Caco-2 cells, whereas it does not have this limitation in hFOB cells.