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.

Combination of cassette-dosing and microsampling for reduced animal usage for antibody pharmacokinetics in cynomolgus monkeys, wild-type mice, and human FcRn transgenic mice.

PURPOSE: The aim of this study was to develop a useful antibody PK evaluation tool using a combination of cassette-dosing and microsampling in mice and monkeys in order to reduce the number of animals used. METHODS: Cetuximab, denosumab, infliximab, and a mixture of the three antibodies, i.e., cassette-dosing, were administered intravenously to cynomolgus monkeys, C57BL/6J mice, and homozygous human neonatal Fc-receptor transgenic (Tg32) mice. Mouse blood was collected from one animal continuously via the jugular vein at nine points. RESULTS: In cynomolgus monkeys, infliximab showed faster elimination in the cassette-dosing group than in the single-dose group. Anti-drug antibody production was observed, but the PK parameters of the clearance and distribution volume were similar in both groups. In C57BL/6J and Tg32 mice, each of the plasma concentrations-time profiles after cassette-dosing were similar to those after single dosing. PK evaluation using a combination of cassette-dosing and microsampling in mice may reduce the number of mice used by approximately 90% compared with the conventional method. CONCLUSIONS: The combination of antibody cassette-dosing and microsampling is a promising PK evaluation method as a high-throughput and reliable with reduced numbers of mice and cynomolgus monkeys.

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.

Predicting Method for the Human Plasma Concentration-Time Profile of a Monoclonal Antibody from the Half-life of Non-human Primates.

Efficiency (speed and cost) and animal welfare are important factors in the development of new drugs. A novel method (the half-life method) was developed to predict the human plasma concentration-time profile of a monoclonal antibody (mAb) after intravenous (i.v.) administration using less data compared to the conventional approach; moreover, predicted results were comparable to conventional method. This new method use human geometric means of pharmacokinetics (PK) parameters and the non-human primates (NHP) half-life of each mAb. PK data on mAbs in humans and NHPs were collected from literature focusing on linear elimination, and the two-compartment model was used for analysis. The following features were revealed in humans: 1) the coefficient of variation in the distribution volume of the central compartment and at steady state of mAbs was small (22.6 and 23.8%, respectively) and 2) half-life at the elimination phase (t1/2ß) was the main contributor to plasma clearance. Moreover, distribution volume showed no significant correlation between humans and NHPs, and human t1/2ß showed a good correlation with allometrically scaled t1/2ß of NHP. Based on the features revealed in this study, we propose a new method for predicting the human plasma concentration-time profile of mAbs after i.v. dosing. When tested, this half-life method showed reasonable human prediction compared with a conventional empirical approach. The half-life method only requires t1/2ß to predict human PK, and is therefore able to improve animal welfare and potentially accelerate the drug development process.

Simplified Method to Determine the Efflux Ratio on P-Glycoprotein Substrates Using Three-Compartment Model Analysis for Caco-2 Cell Assay Data.

PURPOSE: Multiple time-point sampling is required in transcellular transport studies to accurately calculate the appropriate efflux ratio (ER). Our study sought to develop a simplified method to determine the ER in Caco-2 cells. METHODS: The equation for the ER was derived from a three-compartment model of apical to basal and basal to apical transport. Transcellular transport studies were conducted with 10 non-P-glycoprotein (P-gp) and 6 P-gp substrates in Caco-2 cells, and the ER was calculated using this equation. RESULTS: The equation for the ER used the concentration ratio in the receiver compartment at the same time-point; therefore, the ER can theoretically be calculated using only a single point. The ER of all non-P-gp substrates tested was close to 1 at all sampling times. The ERs of cyclosporine A calculated from the concentration ratio at 30, 60, 90, and 120 min incubation were 2.93, 6.43, 7.12, and 9.57, respectively, and the ER at 120 min was almost identical to the theoretical value (9.62) calculated using three-compartment model analysis. The other 5 P-gp substrates showed a similar tendency. Single-point sampling can be used to accurately calculate ER at 120 min. CONCLUSIONS: Single-point sampling is a promising approach for calculating appropriate ERs in the drug discovery stage.

Three-Compartment Model Analysis with Minimal Sampling Points in the Caco-2 Permeability Assay.

The purpose of this study was to establish a modified method of three-compartment model analysis that minimized the sampling frequency. A Caco-2 permeability assay was performed on ten structurally diverse compounds with passive diffusion. A three-compartment model was analyzed by a conventional method and a method with fewer sampling points, called the simplified method, using concentration-time profiles in the donor, intracellular, and receiver compartments. The concentration-time profiles in all compartments were well described by the conventional method. The calculated unbound fraction of intracellular (fu2) and apparent permeability coefficient (Papp) were 0.0107-1.22 and 0.886-146 × 10-6 cm/s, respectively. The simplified method also described the concentration profiles in the compartments of all ten compounds except one, ibuprofen. The difference in values calculated by the simplified method compared to the conventional method was between -7 and 7% for fu2 and between -6 and 42% for Papp. These results suggested that the parameter values from the simplified method were comparable with those from the conventional method. The simplified method may be a promising approach to improve the throughput of three-compartment model analyses of Caco-2 permeability assays in the early stages of drug discovery.

Pharmacodynamic Actions of a Long-Acting PTH Analog (LA-PTH) in Thyroparathyroidectomized (TPTX) Rats and Normal Monkeys.

Hypoparathyroidism is a disease of chronic hypocalcemia and hyperphosphatemia due to a deficiency of parathyroid hormone (PTH). PTH and analogs of the hormone are of interest as potential therapies. Accordingly, we examined the pharmacological properties of a long-acting PTH analog, [Ala(1,3,12,18,22) , Gln(10) ,Arg(11) ,Trp(14) ,Lys(26) ]-PTH(1-14)/PTHrP(15-36) (LA-PTH) in thyroparathyroidectomized (TPTX) rats, a model of HP, as well as in normal monkeys. In TPTX rats, a single intravenous administration of LA-PTH at a dose of 0.9 nmol/kg increased serum calcium (sCa) and decreased serum phosphate (sPi) to near-normal levels for longer than 48 hours, whereas PTH(1-34) and PTH(1-84), each injected at a dose 80-fold higher than that used for LA-PTH, increased sCa and decreased sPi only modestly and transiently (<6 hours). LA-PTH also exhibited enhanced and prolonged efficacy versus PTH(1-34) and PTH(1-84) for elevating sCa when administered subcutaneously (s.c.) into monkeys. Daily s.c. administration of LA-PTH (1.8 nmol/kg) into TPTX rats for 28 days elevated sCa to near normal levels without causing hypercalciuria or increasing bone resorption markers, a desirable goal in the treatment of hypoparathyroidism. The results are supportive of further study of long-acting PTH analogs as potential therapies for patients with hypoparathyroidism. © 2016 American Society for Bone and Mineral Research.

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.

Possible protective role of pregnenolone-16 alpha-carbonitrile in lithocholic acid-induced hepatotoxicity through enhanced hepatic lipogenesis.

Lithocholic acid (LCA) feeding causes both liver parenchymal and cholestatic damages in experimental animals. Although pregnenolone-16 alpha-carbonitrile (PCN)-mediated protection against LCA-induced hepatocyte injury may be explained by induction of drug metabolizing enzymes, the protection from the delayed cholestasis remains incompletely understood. Thus, the PCN-mediated protective mechanism has been studied from the point of modification of lipid metabolism. At an early stage of LCA feeding, an imbalance of biliary bile acid and phospholipid excretion was observed. Co-treatment with PCN reversed the increase in serum alanine aminotransferase (ALT) as well as alkaline phosphatase (ALP) activities and hepatic hydrophobic bile acid levels. LCA feeding decreased hepatic mRNA levels of several fatty acid- and phospholipid-related genes before elevation of serum ALT and ALP activities. On the other hand, PCN co-treatment reversed the decrease in the mRNA levels and hepatic levels of phospholipids, triglycerides and free fatty acids. PCN co-treatment also reversed the decrease in biliary phospholipid output in LCA-fed mice. Treatment with PCN alone increased hepatic phospholipid, triglyceride and free fatty acid concentrations. Hepatic fatty acid and phosphatidylcholine synthetic activities increased in mice treated with PCN alone or PCN and LCA, compared to control mice, whereas these activities decreased in LCA-fed mice. These results suggest the possibility that PCN-mediated stimulation of lipogenesis contributes to the protection from lithocholic acid-induced hepatotoxicity.