Development of a New Method to Evaluate the Biodistribution of Antibodies Using Non-Radioactive Metal Labeling and Inductively Coupled Plasma Mass Spectrometry.

PURPOSE: The use of radiolabeled compounds is associated with a number of limitations. Therefore, a new method for the radioisotope-free evaluation of antibody distribution using metal labeling and inductively coupled plasma-mass spectrometry (ICP-MS) was developed herein. METHODS: Indium-labeled monoclonal antibodies were administrated intravenously to tumor-bearing mice and cynomolgus monkeys, and antibody concentrations in plasma and tissues were measured by ICP-MS. The results were compared with those obtained using a ligand binding assay (LBA) and radioisotope-labeled antibody administration. Indium-, terbium-, holmium-, and yttrium-labeled cetuximab were co-administered to one C57BL/6 J mouse for simultaneous PK and tissue distribution evaluations. RESULTS: The administration of a radioactive or non-radioactive indium-labeled anti-human interleukin-6 receptor (hIL-6R) antibody to tumor-bearing hIL-6R transgenic mice resulted in similar plasma antibody concentration-time profiles by ICP-MS, a ligand binding assay (LBA), and gamma-ray detector. Liver, kidney, brain, spleen, and tumor concentrations of antibodies measured by ICP-MS were similar to those after the administration of radiolabeled anti-hIL-6R antibodies. Following the administration of indium-labeled cetuximab to cynomolgus monkeys, plasma antibody concentrations measured by ICP-MS were similar to those measured by LBA, and antibody concentrations in organs were evaluable by ICP-MS. The PK of all metals were similar to antibody PK evaluated by LBA, and concentrations in each tissue were equivalent among metals. CONCLUSIONS: The assessment of antibody distribution using ICP-MS is a novel alternative to the traditional radiolabeled approach. It facilitates the assessment of antibody distribution in the early stages of drug discovery and accelerates the assessment of target engagement.

In vivo-in vitro correlation of antitumor activity of heat shock protein 90 (HSP90) inhibitors with a pharmacokinetics/pharmacodynamics analysis using NCI-N87 xenograft mice.

The in vitro antitumor activity (e.g. IC50) of anticancer drugs is important for selecting candidate compounds for in vivo drug efficacy study in the early stage of drug discovery. In this study, we investigated the relationship between in vitro IC50 and in vivo EC50 using six heat shock protein 90 (HSP90) inhibitors.IC50 of each compound was calculated from in vitro cell proliferation assays using the NCI-N87 cancer cell line. Each compound was administered to NCI-N87 xenograft mice, and EC50 and the maximum tumour-killing rate constant were calculated from pharmacokinetics/pharmacodynamics analyses using plasma concentrations and tumour volumes.IC50 obtained in vitro was poorly correlated with EC50 obtained in vivo, while a good correlation (r = 0.856) was observed between them when corrected with the unbound fraction ratio.The results of this study using of HSP90 inhibitors as model compounds suggest importance of the consideration of an unbound fraction to evaluate the relationship between IC50 and EC50. These results will contribute to improvement in the prediction accuracy of in vivo drug efficacy from in vitro activity and the efficiency of drug discovery research.

Exploitation of Elevated Extracellular ATP to Specifically Direct Antibody to Tumor Microenvironment.

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.

Retinal toxicity induced by small-molecule Hsp90 inhibitors in beagle dogs.

Heat shock protein 90 (Hsp90) is a constitutively expressed molecular chaperone and plays an important role in the folding of client proteins with key regulatory roles in growth, survival, differentiation and metastasis. Because inhibition of Hsp90 degrades multiple oncogenic client proteins, it is considered to be an attractive anticancer therapy, and clinical trials of several Hsp90 inhibitors have been carried out. In the present study, two structurally distinct Hsp90 inhibitors, CH5164840 and CH5449302, were orally administered to beagle dogs to evaluate systemic toxicity. CH5164840 induced symptoms that suggest visual disorder, and ophthalmological observation and electroretinography (ERG) revealed loss of pupillary light reflex and abnormal waveforms, respectively. Histopathological examination showed changes in the photoreceptor cell layer and the outer nuclear layer of retina. On the other hand, while there were no clinical symptoms related to visual disorder, animals treated with CH5449302 showed similar abnormalities of ERG responses and histopathological changes in the photoreceptor cell layer and the outer nuclear layer of retina. The visual symptoms and abnormalities of ERG responses were noted at an earlier stage or lower dose than other toxicities in both compounds. Considering that two structurally distinct Hsp90 inhibitors induced a retinal toxicity in dogs after repeated administration, and that visual disorders were also reported in some clinical trials of Hsp90 inhibitors, it would seem highly likely that Hsp90 inhibition induces retinal toxicity. Also, our study indicated that a detailed ocular examination to evaluate the safety of Hsp90 inhibitors would be useful in both preclinical and clinical studies.

Assessing the impact of HER2 status on the antitumor activity of an HSP90 inhibitor in human tumor xenograft mice using pharmacokinetic-pharmacodynamic modeling.

The purpose of this study is to assess the impact of human epidermal growth factor receptor 2 (HER2) status on the antitumor activity of CH5164840, an orally available heat shock protein 90 (HSP90) inhibitor, using pharmacokinetic-pharmacodynamic modeling. Athymic mice, each implanted with one of eight human tumor xenografts, were treated with CH5164840 once daily at doses of 3.13 to 50 mg/kg. Plasma concentrations of CH5164840 were described by a one-compartment model with first-order absorption rate. Time profiles of tumor growth inhibition in the eight xenograft models were well captured by an indirect response model with a maximum tumor-killing rate constant (Emax model). Threshold plasma concentrations for tumor stasis, which are determined by multiple pharmacodynamic parameters, Emax, EC50 and tumor growth rate constant, were significantly lower in HER2-positive tumors (1.96-3.85 µM) than in HER2-negative tumors (4.48-23.4 µM). The results suggest that CH5164840 was more efficacious in HER2-positive tumors than in HER2-negative tumors in terms of the lower effective concentration of the drug in preclinical animal models.