Drugs in Clinical Pipeline: GDC-0834 | Treatment of Rheumatoid Arthritis | Antiinflammatory Agent | BTK Inhibitor

GDC-0834 [(R)-N-(3-(6-((4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenyl)amino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide] is a potent, selective small molecule inhibitor of Bruton's tyrosine kinase (BTK). Its development followed SAR studies on CGI-1746, another potent and selective BTK inhbitor. GDC-0834 maintained the potency and selectivity of CGI-1746, but with much improved PK in preclinical animal models.  GDC-0834 inhibits BTK in vitro with an IC₅₀ of 0.006 uM and has an EC₅₀ of 0.060 uM in the cell based CD86 assay. In human whole blood, GDC-0834 demonstrated potent inhibition of both anti-IgE stimulated CD63 expression (basophils) and anti-IgD stimulated CD69 expression (B-cells) with EC₅₀’s of 0.35 and 0.38 uM, respectively [1].

Appreciating its potential for the treatment of rheumatoid arthritis, a single dose IND was filed and GDC-0834 was taken in to a single dose phase I trial in healthy volunteers to quickly evaluate the human pharmacokinetics. In human, GDC-0834 was found to be highly labile at the exo-cyclic amide bond that links the tetrahydrobenzothiophene moiety to the central aniline ring, resulting in insufficient parent drug exposure.

GDC-0834: 2D and 3D Structure

The activity of GDC-0834 is as follows:

IC₅₀ (BTK enzyme assay) = 0.006 uM

EC₅₀ (Cell Based CD86 assay) = 0.06 uM

Common Name: GDC-0834
Synonyms:  GDC-0834; GDC-0834; GDC 0834
IUPAC Name: (R)-N-(3-(6-((4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenyl)amino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide
SMILES:O=C(C1=CC(CCCC2)=C2S1)NC3=CC=CC(C(N=C4NC5=CC=C([C@H]6N(C)CCN(C)C6=O)C=C5)=CN(C)C4=O)=C3C
CAS Number: 1133432-46-8
Mechanism of Action: Kinase Inhibitor; BTK Inhibitor
Indication: Anti-Inflammatory Agent; Treatment of Rheumatoid Arthritis
Development Stage: Pre-Clinical
Company: Gilead Pharmaceutical/Genentech

1H NMR (Estimated) for GDC-0834

References:
1. Young, W. B.; et. al. Potent and selective Bruton's tyrosine kinase inhibitors: discovery of GDC-0834. Bioorg Med Chem Lett 2015, 25(6), 1333-1337 (synthesis and activity).
2. Blomgren, P. A.; et. al. Substituted amides, methods of making, use thereof for the treatment of diseases such as cancer. WO2009039397A2 (synthesis and activity)

Drugs in Clinical Pipeline: ABT-494

ABT-494 [(3S,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine-1-carboxamide] is a second generation Jak kinase inhibitor with high selectivity for Jak1 (IC₅₀ = 0.043 uM) thereby minimizing the potential for side effects related to Jak2 (IC₅₀ = 0.2 uM) and Jak3 (IC₅₀ = 2.3 uM) inhibition. ABT-494 is an ATP competitive inhibitor, and is most potent against Jak1 with concentration at 50% inhibition (IC₅₀) of about 0.045 µM when tested at 0.1 mM ATP and less than 0.003 µM at 0.001 mM ATP [1].

Moreover, ABT-494 displays good selectivity in a panel of more than 60 protein kinases that also includes Jak3. Of the kinases in the panel, 14 kinases have an IC₅₀ below 10 µM, but only 2 non-Jak kinases have IC₅₀'s below 1 µM (Rock1 at 0.55 µM and Rock2 at 0.43 µM).

ABT-494 was engineered for increased selectivity for Jak1 using structural predictions that indicated the potential for differential binding interactions outside the ATP-binding active site of Jak1 but not Jak2. The efficacy and selectivity of ABT-494 were tested in a battery of relevant cellular and in vivo pharmacology assays including bone marrow colony formation, adjuvant induced arthritis (AIA), erythropoietin induced reticulocyte deployment and NK/NKT cell suppression [2]. 

ABT-494 demonstrates approximately 74 fold selectivity for Jak1 over Jak2 in cellular assays dependent on specific, relevant cytokines. ABT-494 is a potent inhibitor of inflammation and bone loss in rat AIA and, compared to Tofacitinib, spares relevant essential physiological processes such as erythropoietin signaling and peripheral NK cell counts at similarly efficacious doses in rats. When dosed orally for 14 days in healthy human subjects ABT-494 did not decrease reticulocyte or NK cell counts at predicted efficacious doses consistent with its pharmacodynamic properties in rats.

The activity of ABT-494 is as follows:

IC₅₀ (JAK1 enzyme assay) = 0.043 uM

IC₅₀ (JAK2 enzyme assay) = 0.2 uM

IC₅₀ (JAK3 enzyme assay) = 2.3 uM
IC₅₀ (TYK2 enzyme assay) = 4.7 uM
IC₅₀ (ROCK1 enzyme assay) = 0.55 uM

IC₅₀ (ROCK2 enzyme assay) = 0.43 uM

Common Name: ABT-494

Synonyms: ABT-494; ABT494; ABT 494

IUPAC Name: (3S,4R)-3-ethyl-4-(3H-imidazo[1,2-a]pyrrolo[2,3-e]pyrazin-8-yl)-N-(2,2,2-trifluoroethyl)pyrrolidine-1-carboxamide

CAS Number: 

Mechanism of Action: Kinase Inhibitor; Janus Kinase 1 Inhibitor

Indication: Anti-inflammatory Agents; Rheumatoid Arthritis Drug; Autoimmune Disease Treatment

Development Stage: Phase II

Company: AbbVie

References:
1. Voss, J. W.; et. al. Jak1 selective inhibitor and uses thereof. WO2015061665A1
2. Voss, J.; et. al. THU0127 Pharmacodynamics of A Novel JAK1 Selective Inhibitor in Rat Arthritis and Anemia Models and in Healthy Human Subjects. Ann Rheum Dis 2014, 73, 222.

Drugs in Clinical Pipeline: Fostamatinib

Fostamatinib [[6-({5-Fluoro-2-[(3,4,5-trimethoxyphenyl)amino]pyrimidin-4-yl}amino)-2,2-dimethyl-3-oxo-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazin-4-yl]methyl dihydrogen phosphate] is an oral prodrug that is rapidly converted to a molecule named Tamatinib (or R406), which is potent and relatively selective inhibitor of Spleen tyrosine kinase (Syk). 

Tamatinib is an ATP-competitive inhibitor of biochemical Syk activity (K_i = 30 nM). The IC₅₀ for this compound is 41 nM, and it inhibits 78% of Syk activity at a concentration of 0.3 uM. Tamatinib also inhibits the isolated enzymes Lyn (IC₅₀ = 63 nM) and Lck (IC₅₀ = 37 nM). Tamatinib have potent anti-inflammatory activity, suggesting a role for Syk inhibition in the treatment of rheumatoid arthritis [1].

Despite similar IC₅₀ values on isolated kinases, Tamatinib shows selectivity in cell-based assays. In mast cells activated by FcεRI-crosslinking, the compound is 20-fold more potent for inhibition of linker for activation of T cells (LAT) tyrosine residue Y191 phosphorylation (a Syk kinase substrate; EC₅₀ approximately 0.08 uM) compared with phosphorylation of Syk itself at the Y352 residue (a Lyn substrate; EC₅₀ greater than 2 uM). Moreover, Tamatinib was identified as a potent inhibitor of FcεRI -dependent mast cell activation (EC₅₀ = 43 nM) in primary human mast cells [1].

Rigel Pharmaceuticals is credited with discovering Tamatinib and Fostamatinib. AstraZeneca announced an exclusive worldwide license agreement with Rigel Pharmaceuticals in February 2010 for the global development and commercialization of Fostamatinib. On June 4, 2013, Astra Zeneca announced they were giving up future development on the compound, and terminated their license with Rigel after early results from a Phase IIb study for Rheumatoid Arthritis.

On Sept 8, 2015 Rigel Pharmaceuticals, Inc. discosed that U.S. Food and Drug Administration (US-FDA) has granted Orphan Drug designation to Fostamatinib, Rigel's oral spleen tyrosine kinase inhibitor which is currently in Phase 3 clinical studies in patients with chronic immune thrombocytopenic purpura or ITP. Rigel's Phase 3 program for Fostamatinib in ITP, called FIT, has surpassed the half-way point in enrollment and Rigel expects the program to read out results in mid-2016.

About ITP

Immune Thrombocytopenic Purpura (ITP) is an autoimmune disease where the immune system attacks and destroys platelets in the blood. This results in abnormally low platelet counts. 

There are two forms of ITP:

a: acute thrombocytopenic purpura, which is most commonly seen in young children;

b: chronic thrombocytopenic purpura, which requires continual follow up care with a hematologist.


50,000-60,000 people suffer from chronic ITP-the majority are women. Antibodies, usually of the IgG type, mediate platelet destruction in ITP. Fostamatinib has a novel mechanism of action, blocking IgG receptor signaling in both macrophages and B cells via SYK kinase.

The activity of Tamatinib is as follows:

IC₅₀ (SYK enzyme assay) = 41 nM; K_i = 30 nM

IC₅₀ (LYN enzyme assay) = 63 nM

IC₅₀ (LCK enzyme assay) = 37 nM

Common Name: Fostamatinib

Synonyms: R935788; R 935788; R-935788; R788; R 788, R-788

IUPAC Name: [6-({5-Fluoro-2-[(3,4,5-trimethoxyphenyl)amino]pyrimidin-4-yl}amino)-2,2-dimethyl-3-oxo-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazin-4-yl]methyl dihydrogen phosphate

CAS Number: 901119-35-5; 1025687-58-4 (disodium salt)

SMILES: 

Mechanism of Action: Kinase Inhibitor; SYK Inhibitor

Indication: Various Cancers; Anti-inflammatory Agents; Treatment for Rheumatoid Arthritis (withdrawn); Treatment for Immune Thrombocytopenic Purpura

Development Stage: Phase III

Company: Rigel Pharmaceuticals

Common Name: Tamatinib

Synonyms: R406; R-406; R 406

IUPAC Name: [6-(5-fluoro-2-(3,4,5-trimethoxyphenylamino)pyrimidin-4-ylamino)-2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one

CAS Number: 841290-80-0

SMILES: 

Mechanism of Action: Kinase Inhibitor; SYK Inhibitor

Indication: Various Cancers; Anti-inflammatory Agents; Treatment for Rheumatoid Arthritis (withdrawn); Treatment for Immune Thrombocytopenic Purpura

Development Stage: Phase III

Company: Rigel Pharmaceuticals

References:
1. Braselmann, S.; et. al. R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation. J Pharmacol Exp Ther 2006, 319(3), 998-1008.

Drugs in Clinical Pipeline: RN486

RN486 [6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one] is a potent, selective, reversible inhibitor of Bruton's tyrosine kinase (BTK). In the enzymatic assay, the compound potently inhibited Btk kinase activity with an IC₅₀ of 4.0 nM. RN486 binds the enzyme in a competitive manner as demonstrated in a time-resolved FRET-based competitive binding assay with an IC₅₀ of 0.3 nM. RN486 was shown to be highly selective when tested against a panel of 369 kinases in the Kinomescan. In the assay, the compound exhibited a strong and competitive binding to Btk with a K_d of 0.31 nM and a high degree of selectivity over almost all other kinases, including Syk and Janus kinase (JAK, K_d = 5.1 uM). The enzyme that was most potently inhibited next to Btk was Ste20-like kinase (SLK, K_d = 0.043 uM), for which the compound showed a 139-fold selectivity [1].

The activity of RN486 is as follows:

IC₅₀ (BTK enzyme assay) = 4.0 nM; K_d = 0.31 nM

K_d (JAK binding assay) = 5.1 uM

K_d (SLK binding assay) = 0.043 uM

Common Name: RN486

Synonyms: RN486; RN 486; RN-486

IUPAC Name: 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one

CAS Number: 1242156-23-5

SMILES: OCc1c(cccc1n1ccc2c(c1=O)c(F)cc(c2)C1CC1)c1cc(Nc2ccc(cn2)N2CCN(CC2)C)c(=O)n( c1)C

Mechanism of Action: Kinase Inhibitor; BTK Inhibitor

Indication: Various Cancers; Anti-inflammatory Agents; Treatment for Rheumatoid Arthritis

Development Stage: Pre-Clinical

Company: Hoffmann-la-Roche

The production and effector function of antibodies are regulated by distinct immunoreceptors on B cells and innate immune cells. The receptors, termed B cell antigen receptor (BCR) and activating Fc receptor (FcR), belong to a family of Ig-like immunoreceptors containing an intracellular immunoreceptor tyrosine-based activation motif (ITAM). ITAMs act to integrate diverse antigen- or Fc-specific signals into a common pathway regulated by nonreceptor tyrosine kinases including Lyn, spleen tyrosine kinase (Syk), and Bruton's tyrosine kinase (Btk) from the sarcoma kinase, Syk, and Tec kinase families. These kinases relay the signals sequentially from ITAMs to phospholipase Cγ2 (PLCγ2) and thus play a critical and nonredundant role in the signal transduction of BCR and FcR. Consequently, loss-of-function mutation in the Btk gene results in severe B cell immunodeficiency and impaired FcR function in both patients with X-linked agammaglobulinemia and mutant mice with X-linked immunodeficiency.

Together, Btk and Syk regulate the signal transduction of ITAM-containing receptors or adaptors that are critical for autoantibody production, effector function, and osteoclast differentiation. Therefore, pharmacological inhibition of these enzymes may affect multiple steps in the pathogenesis of Rheumatoid arthritis (RA) and represent a useful approach for the treatment of the disease. Rheumatoid arthritis (RA) is an autoimmune joint disease characterized by chronic synovial inflammation and progressive joint destruction. The disease is often associated with the appearance of autoantibodies in both blood and inflamed joints. Several of these autoantibodies have emerged as potential arthritogenic factors. For example, anti-glucose-6 phosphate isomerase and anti-type II collagen antibodies, both of which are highly arthritogenic in mice, can be detected in patients with RA. In addition, anticitrullinated protein autoantibodies, the most prevalent in RA, can bind citrullinated fibrinogen in RA joints to form immune complexes, which stimulate macrophages to produce inflammatory cytokines such as TNFα. Lastly, clinical efficacy of B cell-depleting agents in RA strongly implicates autoantibodies as culprits in the pathogenesis of the disease.

When tested in the rat and mouse, RN486 exhibited an excellent pharmacokinetic profile. In the rat, it reached the maximal concentration of 2.5 µM at 4.5 h when dosed orally at 20 mg/kg and showed a half-life of 9.8 h in the blood when administered intravenously. In the mouse, the compound reached the maximal concentration of 6.0 µM at 3 h and a trough concentration of 1.0 µM at 24 h when dosed orally at 30 mg/kg [1].

Important facts about RN486:

a: RN486 Blocks both BCR and FcR Signaling.

b: RN486 Displays a Selective B Cell Inhibitory Profile in BioMAP Systems.

c: RN486 Displays Efficacy on Immune Arthritis Induced by both Active and Passive Immunization in Mice.

d: RN486 Inhibits Inflammation and Bone Erosions in Adjuvant-Induced Arthritis Either Alone or in Combination with Methotrexate.

e: RN486 Reduces Blood Inflammatory Markers in AIA.

Combination therapy with low-dose methotrexate is an important treatment option for patients with RA. Researchers therefore determined the potential of RN486 for combined therapy with methotrexate. To identify a suboptimal or low dose of methotrexate for combination study, researchers tested methotrexate alone in the AIA model at doses ranging from 0.025 to 0.25 mg/kg. Methotrexate displayed a dose-dependent inhibitory effect on both paw inflammation and splenomegaly in AIA rats, attenuating paw swelling by ~50 and 100%, respectively, at 0.075 and greater or equal 0.15 mg/kg. Researchers then tested RN486 and methotrexate at their respective suboptimal doses, 10 and 0.075 mg/kg, alone or in combination in the AIA model to assess the combined effect. As in the monotherapy studies, both RN486 and methotrexate attenuated paw swelling by approximately 50% when tested alone at the suboptimal doses. When combined, the two compounds completely eradicated paw swelling, splenomegaly, and histopathogical changes of inflammation and bone erosions [1].

References:

1. Xu, D.; et. al. RN486, a selective Bruton's tyrosine kinase inhibitor, abrogates immune hypersensitivity responses and arthritis in rodents. J Pharmacol Exp Ther 2012, 341(1), 90-103.

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 (IC_50apparent 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

SMILES: 

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].

References:
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). 55^th 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. ClinicalTrials.gov Efficacy and Safety Study of CC-292 Versus Placebo as Co-therapy With Methotrexate in Active Rheumatoid Arthritis. NCT01975610 (retrieved 15-09-2015).

5. ClinicalTrials.gov 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. ClinicalTrials.gov Novel Combinations of CC-122, CC-223, CC-292, and Rituximab in Diffuse Large B-cell Lymphoma. NCT02031419 (retrieved 15-09-2015).

Drugs in Clinical Pipeline: Filgotinib

Filgotinib [N-[5-[4-[(1,1-Dioxido-4-thiomorpholinyl)methyl]phenyl][1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide] is the first orally-available, selective inhibitor of Janus Kinase 1 (JAK1). Filgotinib, was shown to selectively inhibit JAK1-dependent signaling in cellular and whole blood assays (WBAs) and showed remarkable efficacy in collagen-induced arthritis (CIA) disease models for RA in both mouse and rat.

A detailed structure-activity relationship in triazolopyridine series led to the identification of Filgotinib as one of the lead compounds of the series. Characterization of Filgotinib at the biochemical level indicated a selective inhibition of JAK1 and JAK2 over JAK3 and TYK2 with a rank order potency of JAK1 ~ JAK2 greater than TYK2 and JAK3. IC₅₀ values determined in tyrosine kinase inhibition assays correlated with K_d values determined in ligand displacement assays. It displayed a JAK1/JAK2 inhibitor profile in biochemical assays, but subsequent studies in cellular and whole blood assays revealed a selectivity of ~30-fold for JAK1- over JAK2-dependent signaling. Filgotinib dose-dependently inhibited Th1 and Th2 differentiation and to a lesser extent the differentiation of Th17 cells in vitro. Filgotinib was well exposed in rodents upon oral dosing, and exposure levels correlated with repression of Mx2 expression in leukocytes. Oral dosing of Filgotinib in a therapeutic set-up in a collagen-induced arthritis model in rodents resulted in a significant dose-dependent reduction of the disease progression. Paw swelling, bone and cartilage degradation, and levels of inflammatory cytokines were reduced by Filgotinib treatment. Efficacy of Filgotinib in the collagen-induced arthritis models was comparable to the results obtained with Etanercept [1,2].

Filgotinib is being developed by the Belgian biotech company Galápagos NV, which believes that the JAK1 selective inhibitor Filgotinib is a promising novel therapeutic with potential for oral treatment of Rheumatoid Arthritis, Crohn’s Disease and possibly other immune-inflammatory diseases. Galápagos plans to take Filgotinib into Phase 3 in rheumatoid arthritis by early 2016. Filgotinib has shown best-in-class efficacy and safety in the DARWIN Phase 2B studies in rheumatoid arthritis. In September 2015, AbbVie who was collaborating with Galápagos for Filgotinib decided to pull back, putting their efforts behind in-house JAK1 inhibitor, ABT-494. Filgotinib is now fully owned by Galapagos.


The activity of Filgotinib is as follows:

IC₅₀ (JAK1 enzyme assay) = 10 ± 0.8 nM; K_d = 11 nM

IC₅₀ (JAK2 enzyme assay) = 28 ± 5.4 nM; K_d = 32 nM

IC₅₀ (JAK3 enzyme assay) = 810 ± 180 nM; K_d = 300 nM

IC₅₀ (TYK2 enzyme assay) = 116 ± 39 nM; K_d = ND

Common Name: Filgotinib

Synonyms:  Filgotinib; GLPG0634; GLPG-0634

IUPAC Name: N-[5-[4-[(1,1-Dioxido-4-thiomorpholinyl)methyl]phenyl][1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide

CAS Number: 1206161-97-8

Mechanism of Action: Kinase Inhibitor; JAK1 Inhibitor; Janus Kinase 1 Inhibitor

Indication: Immune-inflammatory Diseases; Treatment of Rheumatoid Arthritis; Treatment of Crohn’s Disease

Development Stage: Phase II

Company: Galápagos NV

The JAKs receive continued interest as therapeutic targets for autoimmune, inflammatory, and oncological diseases. JAKs play critical roles in the development and biology of the hematopoietic system, as evidenced by mouse and human genetics. JAK1 is critical for the signal transduction of many type I and type II inflammatory cytokine receptors.

Four human JAKs have been described: JAK1, JAK2, JAK3, and TYK2. JAKs bind to the intracellular moieties of type I and type II receptors, and JAK homo- or heterodimers become activated upon ligand binding. The JAKs phosphorylate each other followed by phosphorylation of tyrosine residues on the intracellular domains of the receptors. These phosphorylated residues serve as docking sites for STAT transcription factors. JAK phosphorylation of the STAT proteins results in their nuclear translocation and provides transcriptional output for the cytokine ligands. JAKs play important roles in the functioning of the immune system. Mouse and human genetics studies linked deficiencies of JAK1 and JAK3 to severe combined immune deficiency and TYK2 to increased susceptibility to infections. AK2 serves signal transduction for inflammatory cytokines such as IFN-γ, IL-12, IL-23, and GM-CSF. Hence, JAKs have been targeted for their therapeutic potential in immune-inflammatory disorders. In fact, small-molecule JAK inhibitors proved efficacious in a range of animal disease models and have already shown promise in the clinic for organ transplant rejection, rheumatoid arthritis (RA), psoriasis, dry eye disease, myelofibrosis, inflammatory bowel disease, and asthma. Recent findings suggest that JAK1 dominates JAK1/JAK3/γc signaling, suggesting that JAK1 inhibition might be largely responsible for the in vivo efficacy of JAK inhibitors in immune-inflammatory diseases. These results indicate that a selective JAK1 inhibitor could provide an increased therapeutic window allowing for higher dosing and efficacy while avoiding dose-limited pharmacology as observed for the pan-JAK inhibitors [1].

Pre-Clinical Characterization

Characterization of Filgotinib at the biochemical level indicated a selective inhibition of JAK1 and JAK2 over JAK3 and TYK2, whereas cellular and human whole blood assays (WBAs) revealed selectivity for JAK1- over JAK2-dependent signaling in a cellular environment. Filgotinib efficiently blocks cytokine-induced signaling cascades involving JAK1 in several cell lines as well as in human primary cells. Moreover, Th1, Th2, and Th17 differentiation driven by cytokine cocktails, including JAK1-dependent cytokines such as IL-2, IL-4, and IL-6, is also inhibited by Filgotinib. These in vitro findings translate to pharmacodynamic readouts in rodents showing that JAK1 signaling is blocked in vivo as measured by a reduction of Mx2 mRNA levels. Using Tofacitinib and Baricitinib in the same assays for a comparison with Filgotinib further highlighted that Filgotinib selectively inhibits JAK1-dependent signaling in a cellular environment and is more JAK1 selective than the other two drugs.

The pharmacokinetics of Filgotinib was determined in rats and mice. Following i.v. administration, Filgotinib displayed a low to moderate plasma clearance, depending on the species tested. In mice, the total clearance represented 58% of the liver blood flow, and in rats it represented 41%. Steady-state volume of distribution ranged from ~1.7 l/kg in rats to 6 l/kg in mice, implying a significant species difference in volume of distribution. Half-life observed after oral administration was 1.7 h in mice and 3.9 h in rats. Following oral administration, the absolute bioavailability was moderate in rats (45%) and high in mice (~100%).

Furthermore, Filgotinib dose-dependently reduces inflammation, cartilage, and bone degradation in the collagen-induced arthritis (CIA) model in rats and mice [1]. In rats, doses of 1, 3, and 10 mg/kg Filgotinib reduced the clinical score to the same extent as Etanercept at endpoint. Different from the Filgotinib doses tested, the high dose of Etanercept normalized the clinical score already from the start of dosing.

Galapagos has shown that Filgotinib is actually 3 times more selective than AbbVie’s ABT-494.

References:

1. Van Rompaey, L.; et. al. Preclinical characterization of GLPG0634, a selective inhibitor of JAK1, for the treatment of inflammatory diseases. J Immunol 2013, 191(7), 3568-3577.
2. C. J.; et. al. Triazolopyridines as selective JAK1 inhibitors: from hit identification to GLPG0634. J Med Chem 2014, 57(22), 9323-9342.

Drugs in Clinical Pipeline: CG100649

CG100649 [4-(3-(3-fluorophenyl)-5,5-dimethyl-4-oxo-4,5-dihydrofuran-2-yl)benzenesulfonamide] demonstrates a dual mechanism of action on cyclooxygenase-2 (COX2) and carbonic anhydrase (CA) that may result in favorable treatment effects and few adverse gastrointestinal and cardiovascular events [1].

Cyclooxygenase-2 (COX-2) inhibitors have become a common analgesic treatment option for patients with arthritis. However, long-term treatment has been associated with increased cardiovascular risk. With the past withdrawals and rejections of approval for COX-2 inhibitors the treatment options are now very limited. This translates for example to about 10 million osteoarthritis patients in the US who cannot receive COX-2 inhibitors because of concomitant hypertension. And this exemplifies the unmet medical need to develop and offer safe treatment options for this particular patient population. Preclinical data show a dual mechanism of action, which consists of the inhibition of the two enzymes COX-2 and carbonic anhydrase-I/-II (CA-I/II) and through which the cardiovascular risk of COX-2 inhibition might be attenuated [4].

Common Name: CG100649

Synonyms:  CG100649; CG-100649; CG 100649; Polmacoxib

IUPAC Name: (4-(3-(3-fluorophenyl)-5,5-dimethyl-4-oxo-4,5-dihydrofuran-2-yl)benzenesulfonamide

CAS Number: 301692-76-2

Mechanism of Action: Cyclooxygenase-2 Inhibitor; COX2 Inhibitor; Carbonic Anhydrase Inhibitor; CA Inhibitor; Analgesics; Antirheumatic Agents; Enzyme Inhibitors

Indication: Anti-inflammatory

Development Stage: Phase III

Company: Crystal Genomics, Inc. (South Korea)

In mouse models CG100649 inhibits premalignant and malignant colorectal lesions partly through inhibiting tumor cell proliferation. These pre-clinical findings suggest a need for further exploration of CG100649 for CRC prevention and treatment. The long-term safety profile of CG100649, particularly regarding its effect on cardiovascular risk, is yet to be determined [2].

Nonsteroidal anti-inflammatory drugs (NSAIDs) elevate cardiovascular risk by disrupting cyclooxygenase-2 (COX-2)-dependent biosynthesis of prostacyclin (PGI(2)). CG100649 is a novel NSAID proposed to inhibit both COX-2 and carbonic anhydrase (CA)-I/-II. Researchers compared its impact on prostanoid biosynthesis with that of celecoxib, an NSAID purposefully designed to selectively inhibit COX-2. In a controlled, double-blind randomized trial, single oral doses of 2 or 8 mg CG100649, 200 mg celecoxib, or placebo were well tolerated by healthy volunteers (n = 23). Both CG100649 and celecoxib had the effect of depressing urinary excretion of 2,3-dinor-6-keto-PGF(1a) (PGI-M); the effect of CG100649 was dose-dependent and more sustained (up to 240 h after the dose) than that of celecoxib. Neither CG100649 nor celecoxib significantly inhibited COX-1-dependent prostanoid formation. Carbonic Anhydrase (CA) inhibition was not detected after administration of CG100649, despite its partitioning asymmetrically into erythrocytes. CG100649 and celecoxib are both relatively selective inhibitors of COX-2, but they differ in duration of action. Whether they have similar impact on cardiovascular events remains to be determined [3].

Phase I Study

Methodology

In a randomized, double-blind, placebo-controlled, multiple ascending oral dose study that was performed on 8 male and 8 female subjects per dose cohort. Each subject was randomly selected to receive either a single loading dose followed by 6 days of once-daily placebo (n = 4; 2 male and 2 female subjects) or CG100649 (n = 12; 6 male and 6 female subjects). Each subject was administered 1 of 3 sequential dose levels (8-mg loading dose + 2 mg/d, 10-mg loading dose + 4 mg/d, or 12-mg loading dose + 8 mg/d). Blood samples for pharmacokinetic analysis were obtained =480 hours after the last dose. Blood samples for measuring serum thromboxane B2 (TXB2) and ex vivo lipopolysaccharide-stimulated prostaglandin E2 (PGE2) (markers of cyclooxygenase-1 and cyclooxygenase-2 activity, respectively) and urine samples for measuring prostanoid metabolites were collected =21 days after the last dose [1].

Results

During steady state, the median Tmax in blood and plasma after the last dose ranged from 3 to 10 hours and 3.5 to 7.3 hours, respectively. Mean terminal t½ values in blood and plasma ranged from 121 to 203 hours and 100 to 167 hours, respectively. Whole blood concentrations were 50 to 70 times higher than plasma concentrations in all 3 dose cohorts in both male and female subjects. Compared with baseline, serum TXB2 diminished by 68% to 91% at 8 hours after the administration of the last dose in all 3 cohorts (P less than 0.001). Ex vivo lipopolysaccharide-stimulated PGE2 was maximally inhibited (89%-96%; P less than 0.001) by all 3 dose levels on day 7. Urinary prostacyclin metabolite was inhibited by 64% (P less than 0.001) on day 7 (12-24 hours) but only by the highest CG100649 dose. There were no clinically significant drug-related changes in blood pressure between treatment groups. The most frequently encountered adverse events were aphthous stomatitis and dyspepsia.

Conclusion

CG100649 was well tolerated and demonstrated a whole blood concentration that is ~50 to 70 times higher than in plasma in these healthy subjects. CG100649 suppressed TXB2 and PGE2 at all 3 doses, and only the highest dose suppressed the urinary excretion of the urinary prostacyclin metabolite.

References:

1. Kim, M. J.; et. al. Pharmacokinetic, pharmacodynamic, and safety/tolerability profiles of CG100649, a novel COX-2 inhibitor: results of a phase i, randomized, multiple-dose study in healthy Korean men and women. Clin Ther 2015, 37(1), 197-210.

2. Kim, S. H.; et. al. CG100649, a novel COX-2 inhibitor, inhibits colorectal adenoma and carcinoma growth in mouse models. Invest New Drugs 2014, 32(6), 1105-1112.

3. Skarke, C.; et. al. Comparative impact on prostanoid biosynthesis of celecoxib and the novel nonsteroidal anti-inflammatory drug CG100649. Clin Pharmacol Ther 2012, 91(6), 986-993.

4. ClinicalTrials.gov Effects of Dual Cyclooxygenase-2 and Carbonic Anhydrase Inhibition. NCT00780325 (retrieved on 26-04-2015)

5. ClinicalTrials.gov Phase II Study of CG100649 for Primary Osteoarthritis in Male Subjects. NCT00530452 (retrieved on 26-04-2015)

6. ClinicalTrials.gov Phase III Study of CG100649 in Osteoarthritis Patients. NCT01765296 (retrieved on 26-04-2015)