Preclinical Efficacy and Anti-Inflammatory Mechanisms of Action of the Bruton Tyrosine Kinase Inhibitor Rilzabrutinib for Immune-Mediated Disease

Abstract

Bruton tyrosine kinase (BTK) is expressed in B cells and innate immune cells, acting as an essential signaling element in multiple immune cell pathways. Selective BTK inhibition has the potential to target multiple immune-mediated disease pathways. Rilzabrutinib is an oral, reversible, covalent BTK inhibitor designed for immune-mediated diseases. We examined the pharmacodynamic profile of rilzabrutinib and its preclinical mechanisms of action. In addition to potent and selective BTK enzyme and cellular activity, rilzabrutinib inhibited activation and inflammatory activities of B cells and innate cells such as macrophages, basophils, mast cells, and neutrophils, without cell death (in human and rodent assay systems). Rilzabrutinib demonstrated dose-dependent improvement of clinical scores and joint pathology in a rat model of collagen-induced arthritis and demonstrated reductions in autoantibody-mediated FcγR signaling in vitro and in vivo, with blockade of rat Arthus reaction, kidney protection in mouse Ab-induced nephritis, and reduction in platelet loss in mouse immune thrombocytopenia. Additionally, rilzabrutinib inhibited IgE-mediated, FcεR-dependent immune mechanisms in human basophils and mast cell-dependent mouse models. In canines with naturally occurring pemphigus, rilzabrutinib treatment resulted in rapid clinical improvement demonstrated by anti-inflammatory effects visible within 2 wk and all animals proceeding to complete or substantial disease control. Rilzabrutinib is characterized by reversible covalent BTK binding, long BTK residence time with low systemic exposure, and multiple mechanistic and biological effects on immune cells. Rilzabrutinib's unique characteristics and promising efficacy and safety profile support clinical development of rilzabrutinib for a broad array of immune-mediated diseases.

Graphical abstract

FIGURE 1.

Biochemical characterization, selectivity, and potency of rilzabrutinib. (A) Rilzabrutinib has both noncovalent and covalent binding regions, as shown at left, which enables binding with high potency and long residence time to BTK and limits binding to off-target kinases, as shown at right. (B) Kinase selectivity was determined at a rilzabrutinib 1 μM concentration using enzymatic inhibition in a panel of 251 kinases. Data were acquired in duplicate and assessed in two independent experiments. The dendrogram illustrates kinase selectivity with >90% inhibition by rilzabrutinib for six kinases (BTK, RLK, TEC, BMX, BLK, ERBB4) shown in red circles. (C) In vitro kinase selectivity for BTK by rilzabrutinib was further supported by inhibition (IC₅₀) of five kinases by rilzabrutinib with potency <10 nM in an ATP competitive enzyme inhibition in vitro assay (left table). Shown at right are the percent target occupancy values of rilzabrutinib toward the same five kinases at both 1 and 24 h, as evaluated using a biochemical residence time assay. Data were acquired in duplicate at each timepoint and assessed in two independent experiments.

FIGURE 2.

Reversible, covalent binding and durable BTK inhibition with rilzabrutinib in cells. (A) BTK occupancy, potency, and durability was evaluated in human Ramos B cells treated with rilzabrutinib or DMSO, then with a competitive BTK-selective biotinylated probe to measure BTK occupancy by competitive binding. Rilzabrutinib treatment in an eight-point concentration curve (starting at 1 μM; final DMSO concentration 0.1%) resulted in potent BTK occupancy of 8 ± 2 nM (n = 4). In a washout experiment, rilzabrutinib 1 μM treatment was given for 1 h and washed out, and cells were further incubated to measure durability of biotin-labeled BTK-selective probe for 4 or 18 h. BTK occupancy was durable at 72% at 18 h postwashout (n = 2). (B) BTK occupancy in human whole-blood PBMCs treated with rilzabrutinib 2 μM for 1 h; PBMCs were isolated, washed three times, and further incubated for 4 and 18 h postwashout. Cells were labeled with a bodipy-labeled, BTK-selective probe for 1 h, processed, and measured through fluorescent scanning of SDS-PAGE–separated protein blots. BTK occupancy showed strong durability in PBMCs, with 79% BTK occupancy at 18 h postwashout (n = 3).

FIGURE 3.

Platelet aggregation and function in healthy volunteers and ITP patients treated with rilzabrutinib. Plasma from human healthy volunteers [HVs; n = 5 (A)] or ITP patients treated with rilzabrutinib 1 μM [n = 7 (B)] or with ibrutinib 1 μM in HVs [n = 5 (C)] were studied to evaluate their impact on platelet aggregation. Plotted is the percent of maximum platelet aggregation of compound-treated samples normalized to that of untreated samples for each of the indicated platelet agonists and compared using a two-tailed t test versus DMSO control. Only the ibrutinib-treated 2.5 μg/ml collagen group (in C) was statistically significant at *p < 0.05.

FIGURE 4.

Cellular characterization of rilzabrutinib by evaluating inhibition of B cell activation and in vitro IgG and IgM Ab production. (A) Inhibition of B cell activation as measured by anti-IgM–induced CD69 expression on CD20⁺ B cells from human whole blood treated with increasing concentrations of rilzabrutinib, as measured by flow cytometry (n = 4). (B) Inhibition of IgG and IgM production. Rilzabrutinib-treated enriched human B cells were stimulated with CpG (n = 4), TNP-LPS (n = 4–6) or anti-CD40 + IL-21 (n = 2–7) for 7 d. Measurement of IgG and IgM showed that both T-dependent and T-independent Ab production were inhibited by rilzabrutinib. Curves are representative experiments. Table provides IC₅₀ profiles of IgG and IgM production in rilzabrutinib-treated B cells.

FIGURE 5.

Ab-mediated Fc-receptor pathway effects in innate immune cells (A), passive Arthus reaction measured by reduction in diameter (B), and OD of intradermal dye extravasation (C) following Ab challenge. (A) Inhibition of IgG/FcγR–stimulated human monocytes as measured by TNF-α production following treatment with increasing doses of rilzabrutinib (n = 6). Rilzabrutinib treatment resulted in potent inhibition of FcγR-mediated monocyte stimulation with an IC₅₀ of 56 ± 45 nM. (B and C) Sprague Dawley rats were treated for 3 d with oral vehicle, prednisolone (10 mg/kg once daily, positive control), or rilzabrutinib (10, 20, or 40 mg/kg once daily) followed by IgG-mediated Arthus induction (n = 5–8 per group). The reaction was measured 4 h after Ab administration by the diameter of EBD extravasation (B) and dye density was recorded at an OD (C) measurement at 610-nm wavelength.

FIGURE 6.

Effect of rilzabrutinib in a sheep anti-rat GBM Ab-induced lupus nephritis model of female 129X1/SvJ mice. A mouse model of GBM Ab-induced lupus nephritis was treated with vehicle, dexamethasone (1 mg/kg; positive control), or rilzabrutinib (once daily or twice-a-day dosages) on days −1 through 10 (through day 9 for positive control) (n = 10 per group). Representative photomicrograph images for each of the groups are shown in Supplemental Fig. 1. (A) Efficacy evaluations were based on glomerulus, crescents, protein casts scores, and interstitial inflammation compared with vehicle controls (n = 10 per group; n = 9 rilzabrutinib 20 mg/kg once daily). *p < 0.05 ANOVA (Dunnett post hoc) versus vehicle once daily; ^†p < 0.05 Student t test versus vehicle once daily; ^‡p < 0.05 Student t test versus vehicle twice a day. (B) Kidney function was measured by serum BUN levels (n = 7–10 per group). *p < 0.05 by ANOVA (Dunnett post hoc) versus vehicle once daily or Student t test versus vehicle twice a day. (C) Incidence of severe proteinuria (>300 mg/dl) was assessed across all treatment groups (n = 10 per group). Urine protein levels to evaluate severe proteinuria were measured over time, with vehicle control mice showing generally expected increased proteinuria scores over the course of the study. At study termination, 7 out of 10 once daily vehicle mice and 5 out of 10 twice-a-day vehicle mice had >300 mg/dl urine protein. Mice treated with rilzabrutinib (once daily or twice a day) or dexamethasone demonstrated a lower incidence of severe proteinuria, but did not differ significantly in absolute urine protein score from their respective vehicle controls over time.

FIGURE 7.

Evaluation of platelet loss in mouse anti-CD41–induced ITP model. BALB/c mice (n = 8 per group) were treated with vehicle, rilzabrutinib (10, 20, or 40 mg/d once daily), or IVIG 1000 mg/kg i.v. prior to ITP model induction with anti-CD41 (given i.p.). Blood was collected 6 h post–Ab challenge, and platelets were evaluated versus vehicle control. Unmanipulated naive mice were included as a reference control. *p < 0.05, **p < 0.001 compared with vehicle control.

FIGURE 8.

IgE/FcεR–stimulated basophils and a mouse PCA-reaction model. (A) Inhibition of IgE/FcεR–stimulated basophils. Rilzabrutinib-treated human whole blood was stimulated with IgE, then assessed for CD63 activation on CD123⁺/HLA-DR basophils (n = 9). Rilzabrutinib inhibited FcεR activation in basophils with an IC₅₀ of 490 ± 130 nM. (B) PCA-reaction model was induced in BALB/c mice who were treated for 3 d with oral vehicle, prednisolone (10 mg/kg once daily, positive control), cyproheptadine (25 mg/kg once daily i.p., positive control), or rilzabrutinib (20 or 40 mg/kg once daily) followed by IgE-mediated Arthus induction (n = 5 per group). The reaction was measured 4 h after Ab administration by the diameter of EBD extravasation (B) and dye density was recorded at an OD measurement at 610-nm wavelength (C).

FIGURE 9.

Effects of rilzabrutinib on BTK occupancy, PK, and inflammation in CIA in rats. (A) BTK occupancy in female Lewis rat splenocytes was measured following oral administration of 40 mg/kg rilzabrutinib (mean, n = 3 per timepoint per group) and normalized to vehicle control. Rilzabrutinib PK levels were assessed, and spleen percent BTK occupancy was measured by binding of an irreversible fluorescent probe to unbound BTK in splenic cell samples. (B) Female Lewis rats with established CIA were treated orally with vehicle, dexamethasone (positive control), or rilzabrutinib (10, 20, or 40 mg/kg once daily or 20 mg/kg twice a day) on study days 10–20 (arthritis days 1–11; n = 12 per group). On study day 11, efficacy evaluations were performed on ankle diameter. Sustained BTK occupancies ≥79% achieved with twice-a-day dosing resulted in complete reversal of experimental arthritis scores as shown by ankle diameter. All treatment groups were significantly reduced p < 0.05 compared with vehicle control (orange diamond). (C) Mean individual ankle histopathology scores for CIA rats treated orally with vehicle daily (n = 4), dexamethasone 0.075 mg/kg daily (n = 8), or rilzabrutinib 10, 20, or 40 mg/kg once daily or 20 mg/kg twice a day (n = 12 per group) on study days 10–20. *p < 0.05 compared with vehicle daily control. (D) Mean individual ankle histopathology scores for CIA rats treated orally with vehicle twice a day (n = 4) or rilzabrutinib 20 mg/kg twice a day (n = 8) on study days 13–20. ^†p < 0.05 compared with vehicle twice-a-day control. (E) Reversal of experimental arthritis disease was achieved with delayed dosing initiated at study day 13 in established rat CIA model. Error bars represent SD; n = 4 normal controls; and n = 8 per treatment group. ^†p < 0.05 compared with vehicle for rilzabrutinib twice-a-day control. (F) Microcomputed tomography and histopathology imaging showed that joint damage was prevented by treatment with rilzabrutinib.

FIGURE 10.

Rilzabrutinib monotherapy (without CSs) was effective for treatment of canines with naturally occurring PF. As an open-label crossover study, four dogs were treated with rilzabrutinib 15 mg/kg oral once daily (or 30 mg/kg divided twice a day per clinical response and BTK occupancy). (A) A representative 13-y-old, yellow Labrador retriever with an initial cPDAI score of 69.5 began with a 500 mg/d dosage of rilzabrutinib and was escalated to 500 mg every 12 h at week 4. Her cPDAI score was 14.5 at week 8 (80% improvement) and 12 at week 20. Significant decreases in lesions and regrowth of nearly all her coat was achieved by week 20. (B) cPDAI scores over time showed 77–100% improvement with rilzabrutinib dosing as monotherapy (n = 4 dogs). (C) BTK occupancy was measured similar to the above in vivo studies; measurements ranged from 50 to 93% at the 4-h (peak) and 24-h (trough) timepoints. Although within the target range, occupancy did not correlate with partial versus full clinical response.