Preclinical evaluation of ELP-004 in mice

Abstract

This study provides a detailed understanding of the preclinical pharmacokinetics and metabolism of ELP-004, an osteoclast inhibitor in development for the treatment of bone erosion. Current treatments for arthritis, including biological disease-modifying antirheumatic drugs, are not well-tolerated in a substantial subset of arthritis patients and are expensive; therefore, new treatments are needed. Pharmacokinetic parameters of ELP-004 were tested with intravenous, oral, and subcutaneous administration and found to be rapidly absorbed and distributed. We found that ELP-004 was non-mutagenic, did not induce chromosome aberrations, non-cardiotoxic, and had minimal off-target effects. Using in vitro hepatic systems, we found that ELP-004 is primarily metabolized by CYP1A2 and CYP2B6 and predicted metabolic pathways were identified. Finally, we show that ELP-004 inhibits osteoclast differentiation without suppressing overall T-cell function. These preclinical data will inform future development of an oral compound as well as in vivo efficacy studies in mice.

Keywords: CYP450; metabolism; mice; osteoclast; pharmacokinetics; preclinical.

FIGURE 1

(A) Structure of ELP‐004. (B) Plasma concentration–time profiles and elimination half‐life slopes following a single i.v. dose of 2 mg/kg ELP‐004 (n = 3 per timepoint). See Table S6 for individual data values. (C) BSA binding assay.

FIGURE 2

(A) Reconstructed ion chromatograms displaying the unchanged drug and its metabolites from LC–MS/MS analysis following 1‐h incubation of 10 μM of ELP‐004 in NADPH, NADH, UDPGA, DTT, PAPS fortified liver S9 from human (HL, left), and mouse (ML, right). (B) In vitro metabolic pathways of ELP‐004 in human and mouse.

FIGURE 3

(A) Monocytes were isolated from the bone marrow of male DBA/1 mice (n = 3). Cells were stimulated with RANKL and mCSF and treated with escalating doses of ELP‐004 ex vivo. Cells were stained for the TRAP expression (top) or plated on matrix‐coated plates to assess bone resorption activity (bottom). Area of resorption pits was quantified (right). Groups were analyzed using repeated measures ANOVA with the Geisser–Greenhouse correction; multiple comparisons were performed using Tukey's post hoc test. (B) Single cell suspensions of splenocytes were prepared from male DBA/1 mice (n = 5). T cells were stimulated ex vivo with anti‐CD3 and anti‐CD28 and treated with increasing doses of ELP‐004. Secreted cytokines were assessed using a multi‐analyte ELISA (MSD plate). Cytokine concentrations were using compared using repeated measures one‐way ANOVA with a Dunnett post hoc test for multiple comparisons. Data are presented as mean ± SD. *p < .05; **p < .01; ****p < .0001.