Friday, September 18, 2015

Drugs in Clinical Pipeline: Spebrutinib

Spebrutinib [N-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide] is a covalent, highly selective, orally active small molecule inhibitor of Bruton’s agammaglobulinemia tyrosine kinase (BTK). Spebrutinib forms a covalent bond with Cys481 in Btk and potently inhibits Btk in biochemical (IC50apparent less than 0.5 nM) and cellular assays (EC50 1-10 nM) including alpha-IgM stimulation of BCR signaling, B cell proliferation and activation. A quantitative pharmacodynamic assay to determine the level of Spebrutinib bonded to Btk in vitro or in vivo was developed and this drug-target engagement by Spebrutinib was shown to correlate directly with inhibition of Btk enzyme activity and substrate phosphorylation [1].

The activity of Spebrutinib is as follows:

Tec family members sharing a homologous Cys (Btk, BMX, Itk, Tec and Txk)

IC50 (BTK enzyme assay) = 5.9 nM
IC50 (BMX enzyme assay) = 0.7 nM
IC50 (ITK enzyme assay) = 36 nM
IC50 (TEC enzyme assay) = 6.2 nM
IC50 (TXK enzyme assay) = 8.9 nM

Biochemical Activity against Src Family Kinases

IC50 (Brk  enzyme assay) = 2430  nM
IC50 (c-Src  enzyme assay) = 1729  nM
IC50 (Csk  enzyme assay) = greater than 10000  nM
IC50 (Fyn  enzyme assay) = 7146  nM
IC50 (Hck  enzyme assay) = 14460  nM
IC50 (Lck  enzyme assay) = 9079  nM
IC50 (Lyn  enzyme assay) = 4401  nM
IC50 (Yes  enzyme assay) = 723 nM

Common Name: Spebrutinib
Synonyms: CC-292; CC292; CC 292; AVL292; AVL-292; AVL 292
IUPAC Name: N-(3-((5-fluoro-2-((4-(2-methoxyethoxy)phenyl)amino)pyrimidin-4-yl)amino)phenyl)acrylamide
CAS Number: 1202757-89-8
Mechanism of Action: Kinase Inhibitor; BTK Inhibitor
Indication: Various Cancers; Anti-inflammatory Agents; Treatment for Rheumatoid Arthritis
Development Stage: Phase II
Company: Avila Therapeutics, Inc.\Celegene

Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. Bruton’s tyrosine kinase (Btk) is a kinase expressed exclusively in B cells and myeloid cells and has a well characterized, vital role in B cells highlighted by the human primary immune deficiency disease, X-linked agammaglobulinemia (XLA), which results from mutation in the Btk gene. Specifically, Btk plays an essential role in the B cell receptor (BCR) signaling pathway. Antigen binding to the BCR results in B cell receptor oligomerization, Syk and Lyn kinase activation, followed by Btk kinase activation. While BCR signaling is essential in the normal development and function of B cells, several pathologies have been attributed to dysregulated BCR activity. These include diseases of autoreactivity, such as that observed in lupus, multiple sclerosis, and rheumatoid arthritis, in which B cells inappropriately break self-tolerance to produce antibodies contributing to autoimmune disease. BCR signaling also contributes to several B cell malignancies, such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma, and subsets of diffuse large B cell leukemia [1].

Biochemical kinase assays may overestimate the potency of Spebrutinib due to high ATP concentrations found in the cellular environment, hence cell activity for several of these closely related kinase family members was assessed. Spebrutinib demonstrated a high degree of selectivity against kinases with a cysteine in a homologous position as Cys481 in Btk (Epidermal growth factor receptor (A431 cell line), Itk (Jurkat cells), Janus kinase 3 (CTLL-2 cell line), EC50 = 1-4 uM). To demonstrate specific inhibition of Btk in cells, Spebrutinib was evaluated in Ramos cells, which express an intact BCR signaling pathway that is activated robustly by addition of anti-IgM. Spebrutinib potently inhibited Btk autophosphorylation on Tyr223 (EC50 = 8 nM), phosphorylation of the Btk substrate, PLCγ2, as well as activation of the downstream kinase extracellular signal-regulated kinase, all previously reported to be sensitive to Btk inhibition. It is noteworthy that while Spebrutinib inhibited autophosphorylation of Btk, it had no effect on the phosphorylation of Btk on Tyr551, a site phosphorylated by Lyn and Syk and required for Btk activation. These data demonstrate Spebrutinib is selective for Btk and does not inhibit the Src-family kinases upstream of Btk in the BCR signaling pathway [1].

Moreover, consistent with its covalent mechanism of action, Spebrutinib provided prolonged inhibition of kinase activity hours after the drug was removed from cells. In contrast to reversible inhibition with the potent Btk inhibitor Dasatinib, for which kinase activity had almost completely returned 6 hours after drug removal, recovery of Btk activity following a 1-hour exposure to Spebrutinib continued to be suppressed ~8 hours in drug-free media. Since Btk exposed to Spebrutinib is irreversibly bound and inhibited, the return of Btk-dependent signaling relies on the appearance of new Btk protein as a result of protein synthesis in a Spebrutinib-free environment [1].

Phase I Trials

A phase 1 trial investigated the safety, dose limiting toxicities (DLT), and clinical activity of Spebrutinib monotherapy in subjects with relapsed or refractory (R/R) CLL or non-Hodgkin's lymphoma.  This interim analysis focused on the safety and clinical activity in subjects with CLL and small cell lymphocytic leukemia (SLL). Eligible subjects with R/R (= 1 prior therapy) CLL/SLL were treated with monotherapy Spebrutinib in a dose-escalation study with doses ranging from 125 mg to 1000 mg QD and BID dose levels of 375 mg and 500 mg.  As a maximum tolerated dose was not established, CLL patients have been enrolled in an early dose expansion cohort of 750 mg QD and preliminary recommended phase 2 dose expansion cohort at 500 mg BID. All subjects received continuous dosing in 28-day cycles until progressive disease or intolerable toxicity. Clinical activity was investigator assessed per the 2008 iwCLL criteria.

Although the sample size is small, subjects treated at 375 mg or 500 mg BID showed continued lymph node size reduction over time from cycle 2 (mean reduction of 42% and 45%, respectively) to cycle 7 (mean reduction of 60% and 71%, respectively). The most common treatment-emergent AEs (= 10% of subjects) were diarrhea (59.7%), fatigue (37.5%), neutropenia (26.4%), thrombocytopenia (26.4%), nausea (26.4%), pyrexia (22.2%), headache (19.4%), cough (19.4%), upper respiratory infection (16.7%), peripheral edema (15.3%), abdominal pain (15.3%), dizziness (13.9%), muscle spasms (13.9%), contusion (13.9%), anemia (12.5%), pneumonia (12.5%), sinusitis (12.5%), and urinary tract infection (11.1%) [2]. It was concluded that Spebrutinib is well tolerated as an oral daily therapy. Single-agent therapy with Spebrutinib is sufficient to achieve high nodal and partial response rates in relapsed/refractory CLL subjects, including those with high-risk genomic features.

Researchers report the in vitro effects of the Spebrutinib as a single agent and in combination with several targeted small molecule inhibitors in Diffuse Large B-Cell Lymphoma (DLBCL), T-Cell Lymphoma (TCL), and Hodgkin Lymphoma (HL). DLBCL cell lines (GC: SUDHL6, SUDHL10, Farage, and OCI-LY19; and ABC: OCI-LY3) were treated with increasing concentrations of Spebrutinib (0.1-20 µM) alone and in combination with AKT inhibitor (AZD5363) and dual PI3K/mTOR inhibitor (BEZ235) for 24-72 hours.

Researchers analyzed cell viability with MTT assay and expression of NFκB, AKT, mTOR, MEK, and PARP by Western blot analysis. Treatment with Spebrutinib resulted in a dose dependent decrease in cell viability in all DLBCL lines. The 50% inhibitory concentration (IC50) for Farage, OCI-LY19, SUDHL6, and SUDHL10 were 2.2 µM, 6.3 µM, 7.2 µM, and 3.3 µM respectively. Furthermore, treatment with 2.5 to 10 µM of Spebrutinib effectively reduced phosphorylation of mTOR, AKT, and MEK, while it increased phosphorylation of NFκB (p65) and cleaved PARP in Farage, OCI-LY19, and SUDHL10. Treatment with Spebrutinib in combination with BEZ235 (PI3K/mTOR inhibitor) showed synergistic cell death in Farage cells (CI value at IC50, 75, and 90: 0.842, 0.731, and 0.661, respectively). Further, Spebrutinib treatment in combination with AZD5363 (AKT inhibitor) also resulted in synergistic cell death in Farage cells (CI value at IC50, 75, 90: 0.772, 0.771, 0.698). Additionally, results demonstrated the cytotoxic effect of Spebrutinib in HL and TCL cell lines. Collectively, these results suggests that Spebrutinib is active as a single agent in TCL and HL and novel/novel combinations with PI3K/mTOR or AKT inhibitors may have potential therapeutic value in the treatment of DLBCL [3].

1. Evans, E. K.; et. al. Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans. J Pharmacol Exp Ther 2013, 346(2), 219-228.
2. Brown J. R.; et. al. 1630 Phase 1 Study Of Single Agent CC-292, a Highly Selective Bruton’s Tyrosine Kinase (BTK) Inhibitor, In Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL). 55th ASH Annual Meeting and Exposition, 2013, Dec 7-10, New Orleans LA.
3. Cerulli, R. A.; et. al. The Bruton’s Tyrosine Kinase Inhibitor CC-292 in Diffuse Large B-Cell Lymphoma (DLBCL), T-Cell Lymphoma (TCL), and Hodgkin Lymphoma (HL): Induction of Cell Death and Examination of Rational Novel/Novel Therapeutic Combinations. Blood 2014, 124(12).
4. Efficacy and Safety Study of CC-292 Versus Placebo as Co-therapy With Methotrexate in Active Rheumatoid Arthritis. NCT01975610 (retrieved 15-09-2015).
5. A Phase IB Study Of The BTKi CC-292 Combined With Lenalidomide In Adults Patients With Relapsed/Refractory B-Cell Lymphoma (CLEAR). NCT01766583 (retrieved 15-09-2015).
6. Novel Combinations of CC-122, CC-223, CC-292, and Rituximab in Diffuse Large B-cell Lymphoma. NCT02031419 (retrieved 15-09-2015).