Target modulation and pharmacokinetics/pharmacodynamics translation of the BTK inhibitor poseltinib for model-informed phase II dose selection

Abstract

The selective Bruton tyrosine kinase (BTK) inhibitor poseltinib has been shown to inhibit the BCR signal transduction pathway and cytokine production in B cells (Park et al. Arthritis Res. Ther. 18, 91, https://doi.org/10.1186/s13075-016-0988-z , 2016). This study describes the translation of nonclinical research studies to a phase I clinical trial in healthy volunteers in which pharmacokinetics (PKs) and pharmacodynamics (PDs) were evaluated for dose determination. The BTK protein kinase inhibitory effects of poseltinib in human peripheral blood mononuclear cells (PBMCs) and in rats with collagen-induced arthritis (CIA) were evaluated. High-dimensional phosphorylation analysis was conducted on human immune cells such as B cells, CD8 + memory cells, CD4 + memory cells, NK cells, neutrophils, and monocytes, to map the impact of poseltinib on BTK/PLC and AKT signaling pathways. PK and PD profiles were evaluated in a first-in-human study in healthy donors, and a PK/PD model was established based on BTK occupancy. Poseltinib bound to the BTK protein and modulated BTK phosphorylation in human PBMCs. High-dimensional phosphorylation analysis of 94 nodes showed that poseltinib had the highest impact on anti-IgM + CD40L stimulated B cells, however, lower impacts on anti-CD3/CD-28 stimulated T cells, IL-2 stimulated CD4 + T cells and NK cells, M-CSF stimulated monocytes, or LPS-induced granulocytes. In anti-IgM + CD40L stimulated B cells, poseltinib inhibited the phosphorylation of BTK, AKT, and PLCγ2. Moreover, poseltinib dose dependently improved arthritis disease severity in CIA rat model. In a clinical phase I trial for healthy volunteers, poseltinib exhibited dose-dependent and persistent BTK occupancy in PBMCs of all poseltinib-administrated patients in the study. More than 80% of BTK occupancy at 40 mg dosing was maintained for up to 48 h after the first dose. A first-in-human healthy volunteer study of poseltinib established target engagement with circulating BTK protein. Desirable PK and PD properties were observed, and a modeling approach was used for rational dose selection for subsequent trials. Poseltinib was confirmed as a potential BTK inhibitor for the treatment of autoimmune diseases.Trial registration: This article includes the results of a clinical intervention on human participants [NCT01765478].

Conflict of interest statement

The authors declare no competing interests.

© 2021. The Author(s).

Figures

Figure 1

The structure of poseltinib. (A) Chemical structure of poseltinib (C26H26N6O3 as free base; molecular weight, 470.52). (B) In silico analysis of binding between poseltinib and BTK (left) and a docking module within the active site of crystallized BTK (right). (C) Biochemical assay of the effects of poseltinib and its biotinylated probe on BTK. The data represent the mean ± S.D (nM).

Figure 2

High-dimensional analysis of BTK-dependent phosphorylation in stimulated immune cells revealed a preferential impact on B cells. (A) Scattergrams of 96 nodes in the absence (orange) or presence (blue) of 30 µM poseltinib in B cells, CD8 + memory T cells, CD4 + memory T cells, NK cells, neutrophils and monocytes 15 min following stimulation. B cells, T cells and neutrophils showed multiple nodes with substantial stimulation, with poseltinib having the largest impact on B cells. On scattergram for PO4 phospho fingerprint analysis, X and Y axis indicates “Log2 ratio stimultated/control” (B) Poseltinib-mediated inhibition of phosphorylation of BTK (Y223), AKT (S473), and PLCγ2 (Y759) in B cells after 2, 5, 15, 60 min treatment. (C) Poseltinib-mediated inhibition of IL-2 induced STAT-5 phosphorylation in CD4 + T cells, CD8 + T cells and NK cells after 2, 5, 15, 60 min treatment.

Figure 3

Effects of HM71035 on B cell antigen-presentation function. (A) Biochemical activity on hBTK, B cell inhibitory efficacy, and BTK occupancy in B cell line or whole blood of HM71035 and poseltinib. (B) B cells that were preactivated in vivo were isolated and incubated with HM71035 for 1 h before being used as antigen-presenting cells to OVA-323–339-specific transgenic T cells. Activation of T cells was inferred from IL-2 production and (C) CD69 upregulation, which were inhibited by HM71035. (D) Expression levels of the costimulatory markers CD80 and CD86 on B cells were inhibited by HM71035 in a dose-dependent manner.

Figure 4

Dose-dependent inhibition of arthritis severity by poseltinib in a rat model of collagen-induced arthritis (CIA). (A) Normal rat PBMC were treated with poseltinib at the indicated concentrations for 1 h, and then free BTK was detected using a biotinylated poseltinib probe, as described in the Materials and Methods. BTK occupancy (%) was calculated from the free BTK percentage, which was compared with that of the untreated control group. The bars represent the ± S.D of independent experiments. *p < 0.5, ** p < 0.1 and *** p < 0.001 vs. the nontreated control group. (B–E) CIA-induced rats were administered the indicated dose of poseltinib for 9 days starting 6 days after injection of type II collagen emulsion. (B) The clinical scores and (C) body weight loss of rats treated with the drug for 9 days were estimated. (D) Histopathological index analysis of both hind legs was conducted on the final day to assess ankle joint damage. (E) Representative images of H&E and Safranin-O staining of the hind legs in CIA model rats taken under a light microscope for histopathological assessment. Bone erosion ( →) was quantified a subchondral and bone destruction and synovitis (*) was scored degree of pannus formation, including synovial inflammation, hypertrophy, interstitial edema, and fibrosis (H&E). Cartilage damage (□) was semiquantitatively scored as an intensity of Safranin-O staining (red). The data are presented as the mean ± SEM. *p < 0.5, ** p < 0.1 and *** p < 0.001, **** p < 0.0001 vs. the CIA control group (one-way ANOVA for C; Kruskal–Wallis test for B,D).

Figure 5

Pharmacologic inhibition of BTK and its correlation with efficacy. (A,B) Whole blood was collected from the CIA-induced rats at the 0, 4, 12, 24 h after final drug administration, and then PBMCs and plasma were isolated. BTK phosphorylation was detected by immunoblotting and normalized to the total BTK level, and BTK occupancy was measured by ELISA using a biotinylated probe as described in the Materials and Methods. The concentration of poseltinib in the plasma was below the LLOQ at 4 h after administration. Full-length blots are presented in Supplementary Fig. 1. (C) Correlations between drug efficacy (clinical arthritis score) and the PD markers BTK phosphorylation percentage (left)/drug efficacy and BTK occupancy percentage (right). Linear regression was performed by using GraphPad Prism 6. The data are presented as the mean ± S.D.

Figure 6

PK/PD relationship of poseltinib in healthy volunteers. (A) Healthy volunteers were administered for 5, 20, 40, or 60 mg/kg poseltinib twice a day (BID). Whole blood was collected 0, 1, 2, 8, 12, 24, and 48 h after drug administration for PK analysis. The data are shown as the mean ± S.D. (B) BTK occupancy (%) by measuring free BTK from human PBMC lysate was measured by ELISA at the indicated times. Each dot indicates an individual subject, and the lines on the dot plots indicate the mean values. Data from 18 of the 24 subjects from the healthy donor study, excluding 6 participants who were administered placebo, are shown.

Figure 7

Simulations based on the PK/PD model constructed from phase I trial data from human volunteers illustrating predicted occupancy as a function of time for 1 to 40 mg QD doses of poseltinib.

Figure 8

Simulated clinical efficacy (ACR20 responders, %) of poseltinib. Virtual populations in a QSP model for RA were fit to observed clinical trial data (dark blue) for therapeutics with different mechanisms of action. The model was qualified by predicting the clinical response to fostamatinib (FOS, light blue), which was not used for model calibration. The error bars represent the 95% CI of the observed data (see the Supplemental Table 1 for trial details). Virtual populations were considered acceptable if they were within the 95% CI of the observed data. With respect to poseltinib response (red), virtual populations were either unbiased (A), biased to exhibit a maximal response to 30 mg (B), biased to exhibit a maximal response to 30 mg with minimal difference from 10 mg (C), or biased to exhibit a maximal response to 30 mg with maximal difference from 10 mg (D). Various doses of poseltinib were simulated and compared against the weighted average placebo response (light gray) in the clinical trials used for model calibration and qualification (Supplemental table 2). ADA adalimumab, TCZ tocilizumab, RTX rituximab, ANA anakinra, TFA tofacitinib, FOS100 100 mg fostamatinib, FOS150 150 mg fostamatinib.