R E V I E W
`
`doi: 10.1111/sji.12069
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Inhibitors of BTK and ITK: State of the New Drugs for
`Cancer, Autoimmunity and Inflammatory Diseases
`
`L. Vargas*, A. Hamasy*†, B. F. Nore*‡ & C. I. E. Smith*
`
`Abstract
`
`*Department of Laboratory Medicine, Clinical
`Research Center, Karolinska Institutet,
`Karolinska University Hospital, Huddinge,
`Sweden; †Department of Clinical Biochemistry,
`College of Pharmacy, Hawler Medical University,
`Erbil, Kurdistan Region, Iraq (none of the work
`was carried out in Hawler Medical University,
`but the author receives salary from this
`university); and ‡Department of Biochemistry,
`School of Medicine, Faculty of Medical Sciences,
`University of Sulaimani, Sulaimani, Kurdistan
`Region, Iraq
`
`Received 18 April 2012; Accepted in revised
`form 5 May 2013
`
`Correspondence to: C. I. Edvard Smith, Karolinska
`Institutet, Clinical Research Center,
`level 5
`Novum, SE-141 86 Huddinge, Sweden. E-mail:
`edvard.smith@ki.se
`
`BTK and ITK are cytoplasmic tyrosine kinases of crucial importance for B and
`T cell development, with loss-of-function mutations causing X-linked agam-
`maglobulinemia and susceptibility to severe, frequently lethal, Epstein–Barr
`virus infection, respectively. Over the last few years, considerable efforts have
`been made in order to develop small-molecule inhibitors for these kinases to treat
`lymphocyte malignancies, autoimmunity or allergy/hypersensitivity. The ratio-
`nale is that even if complete lack of BTK or ITK during development causes
`severe immunodeficiency, inactivation after birth may result in a less severe
`phenotype. Moreover, therapy can be transient or only partially block the activity
`of BTK or ITK. Furthermore, a drug-induced B cell deficiency is treatable by
`gamma globulin substitution therapy. The newly developed BTK inhibitor
`PCI-32765, recently renamed Ibrutinib, has already entered several clinical trials
`for various forms of non-Hodgkin lymphoma as well as for multiple myeloma.
`Experimental animal studies have demonstrated highly promising treatment
`effects also in autoimmunity.
`ITK inhibitors are still under
`the early
`developmental phase, but it can be expected that such drugs will also become
`very useful. In this study, we present BTK and ITK with their signalling
`pathways and review the development of the corresponding inhibitors.
`
`Introduction
`
`BTK and ITK are TEC family kinases (TFKs) and loss-of-function
`mutations cause human disease
`
`Before reviewing the newly developed inhibitors of BTK
`and ITK, we provide a background to these tyrosine
`kinases. In the first section, we discuss their identification
`and the effect of inactivating mutations. In the following
`section, we describe the intracellular signalling pathway of
`BTK and ITK and summarize what is known about their
`regulation. This is followed by the description of the
`inhibitors.
`TFKs, consisting of BTK, BMX (ETK), ITK, TEC and
`TXK (RLK),
`form the second largest family of non-
`receptor kinases in humans, the largest being the SRC
`family. The TFK ancestor emerged already prior to the
`evolution of metazoans and shows evidence of differential
`evolutionary wiring [1–3]. BTK and ITK contain an
`N-terminal Pleckstrin homology (PH) domain, followed
`by a Tec homology (TH), Src homology (SH)-3, -2 and -1
`(catalytic) domains [4–6]. As depicted in Fig. 1, the TH
`domain consists of an N-terminal Zn2+-binding BTK
`motif, and one or two proline-rich motifs [4, 7–9].
`
`All the mammalian TFKs were identified in the 1990s.
`ITK [10, 11] and BTK [12, 13] were each cloned
`independently by two groups. This family of kinases soon
`received wide interest, owing to the fact
`that BTK
`mutations cause an X-linked form of B-lymphocyte
`deficiency (X-linked agammaglobulinemia, XLA) in man
`[13–16]. Today, more than 1000 patients with known
`mutations exist in the BTKbase registry [17]. In mice,
`mutations
`cause
`the phenotypically milder X-linked
`immunodeficiency (Xid) [18, 19]. In mice, it seems as if
`the TEC kinase has a unique compensatory role, because,
`while the TEC kinase is also expressed in human B
`lymphocytes, in mice, the double knockout of BTK and
`TEC causes an XLA-like phenotype. In contrast, TEC
`single-knockout mice do not have an overt phenotype [20].
`ITK deficiency in humans is much less common and was
`not reported until 16 years after the identification of
`mutations in BTK. Thus, ITK is one of the several genes in
`which loss-of-function mutations cause susceptibility to
`severe, often fatal, Epstein–Barr virus infections [21].
`BTK is constitutively expressed in myeloid and lym-
`phoid cells but absent in T cells and in mature plasma cells
`[22]. It is found during all stages of the B cell lineage up
`until the plasma-cell stage, where it is absent in the most
`
`130
`
`Ó 2013 John Wiley & Sons Ltd
`
`

`

`L. Vargas et al.
`
`BTK and ITK Inhibitors 131
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Figure 1 Schematic representation of BTK and ITK showing membrane binding and regulatory tyrosine phosphorylation sites. BTK and ITK have
`similar domain organization, with the difference that BTK has two proline-rich repeats (PRR) in the Tec homology domain (TH). The highly conserved
`BTK motif binds a Zn2+ ion, which stabilizes the PH domain. The PH domain binds phosphatidylinoisitol-3,4,5-trisphosphate (PIP3), which is generated
`by PI3K (Fig. 2). In the PH domain of BTK, there is a 27 amino acid insertion not found in ITK (marked in blue). The SH3 domain binds to proline-rich
`regions, while the SH2 domain interacts with phosphorylated tyrosine residues forming reversible signalling complexes. The depicted trans-
`phosphorylated tyrosine residue in the catalytic domain has an activating function, whereas the role of the autophosphorylated tyrosine in the SH3 domain
`is less defined.
`
`mature form. Mutations cause a differentiation block at the
`stage of pre-B cells, with mature B cells being very few and
`non-responsive to foreign antigens, even if rare patients can
`have close to normal numbers [14, 23–25].
`for
`crucial
`ITK is
`less widely expressed and is
`T-lymphocyte development, as initially shown by knock-
`ing out the gene in mice [26]. More in-depth studies
`revealed that both ITK-deficient mice [5, 6, 27] and the
`few patients with ITK mutations analysed so far show
`almost complete absence of invariant natural killer T cells.
`While several different genetic defects show susceptibility
`to severe EBV infection, it was recently reported that ITK
`deficiency is clinically distinct
`from both signalling
`lymphocyte activation molecule (SLAM)-associated protein
`(SAP) and X-linked inhibitor of apoptosis protein (XIAP)
`deficiency [28]. ITK may also be a crucial host factor
`needed for the development of an HIV infection [29].
`Further studies have shown that ITK-deficient mice have
`drastically reduced lung inflammation, eosinophil infiltra-
`tion and mucous production in response to ovalbumin-
`induced induction of allergic asthma [30]. Therefore,
`inhibition of T cell activation has been one of the strategies
`for developing immunosuppressive agents to treat autoim-
`mune disorders and inflammation [31]. Suppression of host
`immune functions by blocking T cell activation is also a
`successful modality for preventing organ transplant rejec-
`tion [32].
`In contrast to BTK, ITK is not constitutively expressed.
`Thus, the corresponding transcript was initially identified
`from an IL-2-dependent mouse T cell line [11]. This means
`that even if BTK and ITK frequently are considered to be
`analogs,
`selectively
`expressed in T-
`and B cells,
`respectively, their differential expression with regard to
`
`the need for inducibility shows that this is not entirely
`true. This difference is also likely to influence the
`treatment effect of inhibitors.
`
`Signalling pathways of BTK and ITK and target diseases
`
`TFKs play central, but diverse, modulatory roles in various
`cellular processes. They participate in signal transduction
`in response to virtually all types of extracellular stimuli
`that are transmitted by growth factor receptors, cytokine
`receptors, G-protein-coupled receptors, antigen receptors
`and integrins [33, 34]. As illustrated in Fig. 2 (Step 1),
`following BCR, TCR stimulation, SRC family kinases are
`activated, leading to the phosphorylation of immunore-
`ceptor tyrosine activation motifs (ITAMs) of the CD79
`(Ig-a, Ig-b) and CD3 complex chains. Similarly, PI3K is
`activated to catalyse the conversion of membrane-associated
`PIP2 to PIP3 leading to BTK/ITK recruitment to the
`plasma membrane through the interaction of
`its PH
`domain with PIP3 [35]. Concomitantly, the phosphoryla-
`tion of Ig-a and Ig-b ITAMs leads to the recruitment of
`SYK/ZAP70 kinase via SYK SH2 domains. Following
`activation, BTK/ITK ignites multiple downstream signals
`generating pleiotropic effects (Fig. 2, step 2): (1) PLCc
`activation, generation of
`second messengers,
`such as
`inositol [1,4,5]-triphosphate (IP3), diacylglycerol (DAG)
`and calcium, (2) Cell proliferation, differentiation, apop-
`tosis and survival [16, 36]. However, the molecular basis of
`many of these pathways is not fully understood, and many
`interacting molecules remain to be isolated. Using affinity
`purification combined with tandem mass spectrometry, we
`have recently characterized the interaction of BTK with an
`ankyrin-repeat domain protein [37] and the protein 14-3-3,
`
`Ó 2013 John Wiley & Sons Ltd
`
`

`

`132 BTK and ITK Inhibitors
`
`L. Vargas et al.
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Step 1
`
`TCR
`
`Step 2
`
`BCR
`
`~
`
`PIP2 ---+ PIP3
`
`I
`
`PLCy2/l
`
`IP3
`
`\
`i
`*
`
`~
`
`(lg-a) {lg-fi)
`
`.-8~
`.-8
`
`l
`
`r---- CypA
`
`PKC
`Pin- I -------,
`
`/
`
`DAG
`½
`
`PKC - (Ca2+)
`
`Figure 2 BTK and ITK activation. B cell receptor (BCR) and BTK signalling components are depicted to the left and T cell receptor (TCR) to the right
`upon engagement of BCR and TCR, SRC family kinases including LYN and LCK are activated leading to phosphorylation of immunoreceptor tyrosine
`activation motifs (ITAMs) (Step 1). Activated PI3K converts PtdIns-4,5-bisphosphate (PIP2) into PIP3, which tethers BTK/ITK to the membrane
`(Fig. 1), where they phosphorylate regulatory tyrosine residues in PLCc2 and PLC, c1 respectively (Step 2). The increasing PLCc activity results in the
`production of the secondary messengers DAG and IP3 inducing activation of the transcription factors NF-jB (active mainly in B cells) and NF-AT (active
`mainly in T cells). Endogenous BTK/ITK inhibitors (PKC, Pin-1/CypA) regulate their activity affecting cellular responses such as cell survival, apoptosis,
`adhesion, migration and proliferation.
`
`which regulate nucleo-cytoplasmic shuttling [38] and
`attenuate signalling [39].
`To date, almost only B cell-derived tumours have been
`treated with the newly developed inhibitor for BTK,
`Ibrutinib (see below). The rationale is that tumours,
`similar to non-transformed B cells, may be dependent on
`BCR signalling for their survival. However, tumours with
`activating mutations downstream of BTK are likely to be
`resistant, and this also seems to be the case [40]. Although
`animal models of autoimmunity show very promising
`outcomes from Ibrutinib treatment, the drug has not yet
`been used in clinical studies in man. Allergies and other
`forms of hypersensitivity and inflammatory diseases with a
`strong B cell component could also become targets. Target
`diseases for ITK inhibitors are less defined and will await
`studies in relevant animal models. As always, potential side
`effects have to be balanced against the benefit of treatment.
`
`BTK inhibitors
`
`LFM-A13
`
`(alpha-
`The leflunomide metabolite analog (LFM-A13)
`cyano-beta-hydroxy-beta-methyl-N-(2,5-ibromophenyl)-pro-
`pen-amide) (Table 2) is one of the first rationally designed
`antileukemic agents targeting BTK [41, 42]. This small-
`molecule inhibitor binds non-covalently to the catalytic site
`
`of BTK in a reversible manner (half-maximal inhibitory
`concentration (IC50) = 17.2 lM for human BTK in vitro
`and IC50 = 2.5 lM for recombinant BTK). It does not
`affect the enzymatic activity of other protein tyrosine
`kinases, including EGFR, HCK, IRK JAK1, JAK3 and
`IRK at concentrations of 278 lM [41]. However, even if the
`molecule has been described as a highly specific inhibitor of
`BTK, it can also efficiently affect the activity of other
`kinases such as the erythropoietin receptor, JAK2 and
`downstream molecules [43].
`During the last decade, a plethora of in vitro and in vivo
`studies have suggested that LFM-A13 could act as a dual-
`function anticancer drug with apoptosis-promoting and
`antithrombotic properties [44–46]. In addition, LFM-A13
`also exhibits antiproliferative activity against Her2/Neu-
`overexpressing breast cancer cells [47]. Furthermore, LFM-
`A13 has been reported to prevent acute fatal graft-versus-
`host disease in a murine model of allogeneic bone marrow
`In addition, LFM-A13 has been
`transplantation [48].
`broadly used in vitro to inhibit BTK downstream signalling
`pathways (Fig. 2) and further to elucidate the role of SFKs
`[49–51]. On neutrophils, for example, it has been shown
`that LFM-A13 also negatively affects the translocation of
`Rac-2, RhoA, ADP ribosylation factor-1, TEC, BMX and
`BTK induced by fMet-Leu-Phe [52]. Moreover, LFM-A13
`could block the endogenous phosphorylation of Myd88
`adapter-like (Mal) on tyrosine in cells
`treated with
`
`Scandinavian Journal of Immunology, 2013, 78, 130–139
`
`

`

`L. Vargas et al.
`
`BTK and ITK Inhibitors 133
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`
`macrophage-activating lipopeptide-2 or LPS [53] and
`LFM-A13 inhibited Heme oxygenase (HO-1) induction
`by the classical TLR4 ligand LPS in cell cultures [54].
`Other reports show that LFM-A13 by inhibiting TFKs,
`rescues the suppression of TCR-induced CD25 expression
`in Jurkat cells [55]. In primary myeloma-bearing immu-
`nodeficient mice, LFM-A13 inhibited osteoclast activity,
`prevented myeloma-induced bone resorption and moder-
`ately suppressed myeloma growth [56]. Administration of
`LFM-A13 is not toxic to mice, rats or dogs at daily dose
`levels as high as 100 mg/kg [57]. However, as mentioned,
`rather high doses are needed for a pharmacological effect,
`and we are not aware of any ongoing, or planned, clinical
`studies.
`
`Dasatinib
`
`Dasatinib, BMS-354825 or Sprycel [N-(2-chloro-6-meth-
`ylphenyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-meth-
`ylpyrimidin-4-ylamino)thiazole-5-carboxamide] (Table 2) is
`an orally available, dual ABL/SRC tyrosine kinase inhibitor
`(TKI), which was developed to treat patients with chronic
`myelogenous leukaemia (CML), who had failed, or were
`intolerant to, therapy with Imatinib BCR-ABL1 and SFK
`TKI [58, 59]. Other diseases in which bone metastases are
`frequent (e.g. breast or prostate tumours) could also benefit
`from the addition of Dasatinib to standard-of-care treat-
`ments [60, 61].
`Native targets of Dasatinib in CML cells have been
`identified using a chemical proteomics approach [62–64].
`Besides ABL and SRC kinases, BTK and TEC, but not
`ITK, were recognized as major binders
`inhibited by
`nanomolar concentrations. In addition, the gatekeeper
`residue as the critical determinant of Dasatinib suscepti-
`bility has been detected with the help of structure-based
`mutagenesis experiments. Mutation of Thr-474 in BTK to
`Ile and Thr-442 in TEC to Ile conferred resistance to
`Dasatinib, whereas mutation of the corresponding residue
`in ITK (Phe-435) to Thr sensitized the otherwise insen-
`sitive ITK [64]. Other studies have shown that Dasatinib
`induces apoptosis in primary chronic lymphocytic leukae-
`mia (CLL) cells blocking LYN kinase activity [65, 66].
`Moreover, Dasatinib decreased levels of the activated,
`phosphorylated forms of AKT, ERK1/2 and p38 and
`reduced the expression of the antiapoptotic proteins MCL-1
`and BCL-XL [67]. Thus, it seems that Dasatinib as a single
`agent has activity in relapsed and refractory CLL [66].
`In line with the role of TFKs in lymphoid and myeloid
`cells, Dasatinib inhibited the secretion of several immu-
`nomodulators [68, 69]. The observed inhibition of TFKs
`predicts immunosuppressive (side) effects of this drug and
`may offer therapeutic opportunities for inflammatory and
`immunological disorders [64, 70, 71]. However, further
`experiments are required to describe the exact mechanism
`of the above-mentioned hypothesis.
`
`Ó 2013 John Wiley & Sons Ltd
`
`In summary, this compound has been approved by the
`FDA for the treatment of patients with CML in all phases
`or Ph+-ALL, who were resistant, or intolerant, to therapy,
`with Imatinib. In Europe, it has been approved for therapy
`of patients with CML who are resistant, or intolerant, to
`Imatinib [59]. However, the drug has not been used to
`clinically interfere with TFKs.
`
`Ibrutinib (PCI-32765)
`
`(1-{(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-
`Ibrutinib,
`pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl}prop-2-en-1-
`one) (Table 2), is a selective and irreversible small-molecule
`BTK inhibitor that inhibits BCR signalling in human B
`cells. It was originally named PCI-32765 and re-named
`Ibrutinib by the World Health Organization (WHO) and
`the United States Adopted Name (USAN) Council. Orally
`administered Ibrutinib has demonstrated to be particularly
`active in different B cell malignancies including CLL,
`mantle
`cell
`lymphoma
`(MCL), diffuse
`large B cell
`lymphoma (DLBCL) and multiple myeloma (MM) [72–75].
`Ibrutinib inactivates BTK through covalent binding to
`the active site (Cys-481) in the ATP-binding domain of
`BTK with IC50 of 0.5 nmol/L [76]. Several TFKs with
`homology to BTK, including BMX and ITK, have similar
`cysteine residues that might also be irreversibly inhibited
`by Ibrutinib. Other kinases that can also be sensitive to
`Ibrutinib at nanomolar concentrations include BLK, TEC,
`EGFR, ERBB2, HER2, HER4 and JAK3 [72, 76, 77].
`
`Ibrutinib as a potential drug for B cell malignancies
`
`The use of Ibrutinib in preclinical and clinical trials
`appears to be a promising new strategy for treatment of B
`cell malignancies (Table 1). The in vivo effect of ibrutinib
`has been demonstrated in patients with CLL. Recent
`reports have shown that
`Ibrutinib inhibits CLL cell
`survival and proliferation as well as induces CLL apoptosis
`[77, 78]. In addition, treatment of CD40- or BCR-
`activated CLL cells with Ibrutinib results in inhibition of
`BTK tyrosine phosphorylation and also effectively abro-
`gates downstream survival pathways activated by this
`kinase, including ERK1/2, PI3K and NF-jB [74, 77].
`Ibrutinib also acts by modulating the interaction
`between CLL cells and their microenvironment. For
`example,
`it inhibits activation-induced proliferation of
`CLL cells and effectively blocks survival signals, which are
`provided externally to CLL cells from the microenviron-
`ment (CD40L, BAFF, IL-6, IL-4 and TNFa, fibronectin)
`engagement and stromal cell contact, as well as migration
`in response
`to tissue-homing chemokines
`(CXCL12,
`CXCL13) [79]. Moreover, the secretion of BCR-dependent
`cytokines such as CCL3 and CCL4 is effectively decreased
`both in vitro and in patients with CLL treated with
`Ibrutinib [78].
`
`

`

`134 BTK and ITK Inhibitors
`
`L. Vargas et al.
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Table 1 Clinical trials of Ibrutinib in B cell malignancies.
`
`Disease
`
`Study description
`
`Drugs and doses
`
`Study phase
`
`CLL and
`SLL
`
`CLL, SLL
`
`Ibrutinib (420 mg)
`
`II
`
`ROR CLL,SLL
`with 17p deletion
`CLL, SLL in patients
`older than 65 or
`have 17p deletion
`
`Ibrutinib (420 mg)
`
`Ibrutinib (420 mg)
`
`CLL, SLL
`
`ROR
`CLL, SLL
`ROR
`CLL,SLL
`
`Ibrutinib (420 mg)
`VS chlorambucil
`Ibrutinib (420 mg)
`VS Ofatumumab
`Ibrutinib (420 mg) +
`Rituximab+
`Bendamustine
`Ibrutinib
`
`II
`
`II
`
`III
`
`III
`
`III
`
`II
`
`Estimated
`patients/age
`
`30
`≥18 years
`
`111
`≥18 years
`86
`≥65 years
`and ≥ 18 year
`for 17p deletion
`272
`≥65 years
`350
`≥18 years
`580
`≥18 years
`
`Objective
`
`Study duration
`
`Clinical trials.gov
`identifier
`
`Impact on
`leukemia cell
`trafficking
`and death
`ORR,PFS, OS
`
`2012–2015
`
`NCT01752426
`
`2013–2016
`
`NCT01744691
`
`ORR, OS, PFS
`
`2011-2015
`
`NCT01500733
`
`ORR, PFS
`
`2013–2016
`
`NCT01722487
`
`PFS, OS, ORR
`
`2012–2015
`
`NCT01578707
`
`PFS,OS, ORR
`
`2012–2018
`
`NCT01611090
`
`PFS, ORR,OS
`
`2012–2014
`
`NCT01589302
`
`NCT01325701
`
`CLL, SLL,
`B-PLL
`DLBCL
`
`FL
`
`ROR CLL,SLL,
`B-PLL
`ROR
`DLBCL
`Refractory FL
`
`MCL
`
`MCL
`
`ROR
`MCL
`ROR
`MCL
`MCL
`
`B cell
`neoplasm
`MM
`
`WM
`
`Recurrent mature B
`cell neoplasm
`Relapsed or relapsed
`and refractory MM
`WM
`
`Ibrutinib (560 mg)
`
`Ibrutinib
`(560 mg)
`Ibrutinib (560 mg)
`
`Ibruinib (560 mg)
`VS Temsirolimus
`Ibrutinib (560 mg)
`VS Temsirolimus
`Ibrutinib (560 mg) +
`Rituximab+
`Bendamustine
`Ibrutinib (420,
`560 mg)
`Ibrutinib (420,
`560, 840 mg)
`Ibrutinib
`
`II
`
`II
`
`II
`
`III
`
`III
`
`III
`
`I
`
`II
`
`II
`
`75
`≥18 years
`60
`≥18 years
`110
`≥18 years
`110
`≥18 years
`280
`≥18
`280
`≥18 years
`520
`≥65 years
`
`24
`≥20 years
`164
`≥18 years
`33
`≥18 years
`
`Efficacy and safety
`
`2011–2014
`
`ORR,OS, FPS
`
`2013–2016
`
`NCT01779791
`
`ORR, PFS, OS
`
`2012–2015
`
`NCT01599949
`
`PFS, OS
`
`2012
`
`PFS,ORR, OS
`
`2012–2017
`
`NC2012-
`000601-74
`NCT01646021
`
`PFS,OS, ORR
`
`2013–2019
`
`NCT01776840
`
`Safety and
`pharmacokinetic
`Efficacy and safety
`
`2012–2014
`
`NCT01704963
`
`2012–2016
`
`NCT01478581
`
`ORR, safety
`
`2012–2014
`
`NCT01614821
`
`Follicular lymphoma (FL), overall response rate (ORR), progression-free survival (PFS), overall survival (OS), chronic lymphocytic leukemia (CLL), small
`lymphocytic lymphoma (SLL), multiple myeloma (MM), mantle cell lymphoma (MCL), diffused large B cell lymphoma (DLBCL), B cell prolymphocytic
`leukemia (B-PLL), relapsed or refractory (ROR), Waldenstr€oms macroglobulinemia (WM).
`
`The initial phase I study of Ibrutinib enrolled patients
`with B cell lymphomas including CLL demonstrated that a
`dose of 420 mg is as efficient as 840 mg. In fact, the
`occupancy and inhibition of BTK were similar in both
`doses, so 420 mg was selected for further studies to
`minimize adverse effects [77]. Administration of Ibrutinib
`(420 mg/day)
`in patients with CLL induces a rapid
`shrinkage of enlarged lymph nodes and symptomatic
`improvement within the first few weeks of treatment [73].
`The expected pattern of initial rapid nodal response with
`sometimes marked lymphocytosis was also observed. This
`increase in lymphocyte count was transient and could be
`typically resolved after the first few months of therapy [72,
`In the 2012 American Society of Hematology
`77].
`meeting, it was reported that Ibrutinib induces an overall
`
`response rate (ORR) of 68% in previously untreated CLL
`patients, aged 65 or older, and an ORR of 71% in
`previously treated patients [72].
`It has been recently reported that BTK is highly
`expressed in malignant plasma cells from patients with
`MM [80, 81]. This is in contrast to the most mature
`normal plasma cells, where BTK is not expressed [22]. In
`MM models, Ibrutinib reduced osteoclast formation and
`bone resorption and also inhibited BTK-mediated osteo-
`clastogenesis induced by M-CSF and RANKL [80]. The
`chemokine and cytokine secretion from bone marrow
`stromal cells (BMSCs) and osteoclasts was significantly
`decreased by Ibrutinib, and it blocks SDF-1-induced
`adhesion and migration [81]. Furthermore, it also inhibited
`MM cell growth triggered by IL-6 or coculture with
`
`Scandinavian Journal of Immunology, 2013, 78, 130–139
`
`

`

`L. Vargas et al.
`
`BTK and ITK Inhibitors 135
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Table 2 Chemical structure of BTK and ITK inhibitors.
`
`Structure
`
`BTK inhibitors
`
`BMSCs in vitro. In addition, Ibrutinib inhibits MM cell–
`induced osteolysis of implanted human bone chips in SCID
`mice [80].
`Ibrutinib also shows promising and encouraging results
`as a single-agent drug in MCL. Thus, Ibrutinib produced
`ORR of 66% in phase II study for patients receiving
`(560 mg/day) [82]. Co-administration of Ibrutinib with
`the proteasome inhibitor Bortezomib resulted in a syner-
`gistic effect and induced MCL cell-death [74].
`
`Ibrutinib as a therapeutic agent for autoimmune diseases
`
`Ibrutinib shows promising activity against experimental
`autoimmune diseases, particularly in rheumatoid arthritis
`(RA).
`In mice with collagen-induced arthritis
`(CIA),
`administration of 12.5 mg/kg effectively reduced arthritic
`symptoms after few days of treatment [72, 83]. These
`studies show that in CIA and collagen antibody-induced
`arthritis, Ibrutinib markedly reduced inflammation, bone
`resorption and cartilage destruction. Moreover, the infil-
`tration of
`inflammatory cells, cytokine and chemokine
`levels in synovial fluid are decreased [83, 84]. In the MRL-
`Fas (lpr) lupus model, treatment with Ibrutinib inhibits
`autoantibody production and renal impairment [76].
`
`Compound name
`
`Ibrutinib
`(PCI-32765)
`MW 440.5
`
`Dasatinib
`MW 488
`
`LFM-A13
`MW 360
`
`AVL-292
`MW 423.17
`
`Compound 3 (Sanofi)
`MW 297.38
`
`Compound 7 (Sanofi)
`MW 375.44
`
`Pharmacokinetics
`
`Ibrutinib is administered orally and rapidly absorbed.
`Between 1 and 2 hours after drug administration, it reaches
`the mean plasma concentration. It has a short half-life of 2-
`3 hours, but BTK inhibition will remain for 24 hours.
`Generally, the drug is well tolerated, with mild adverse
`effects like cough, fatigue, diarrhoea and infectious com-
`plications, which could be managed easily (Table S1) [85].
`
`ITK inhibitors
`
`..
`
`AVL-292
`
`AVL-292 is a new orally available potent compound that
`selectively inhibits BTK (Table 2). It also binds to Cys-481
`at the kinase domain, sustaining BTK inhibition for
`24 hours. Human clinical trials with AVL-292 have dem-
`onstrated that the drug is safe and like other BCR inhibitors,
`the drug caused lymphocytosis within few weeks of the
`treatment [73, 84]. AVL-292 also showed a promising effect
`in animal models with rheumatoid arthritis. Currently,
`according to ClinicalTrials.Gov, there are two studies
`evaluating AVL-292, one recruiting for non-Hodgkin
`lymphoma, CLL and Waldenstr€om’s macroglobulinemia
`(WM), and a second, active, but not recruiting, studying the
`safety and pharmacokinetic of this drug [84].
`
`BMS-509744
`MW 623.83
`
`?'.,
`
`-(_('yl/"'.)\,
`
`¥ (cid:173)
`
`0
`,,l
`""
`
`ITK inhibitors
`
`The crystal structure of the ITK kinase domain was
`elucidated for the phosphorylated and non-phosphorylated
`
`kinase domain bound to staurosporine, a potent broad-
`spectrum kinase inhibitor [86]. These structures are highly
`useful
`for the design of selective ITK inhibitors and
`provide insight into the influence of inhibitor binding and
`
`Ó 2013 John Wiley & Sons Ltd
`
`

`

`136 BTK and ITK Inhibitors
`
`L. Vargas et al.
`
`. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`ITK [86].
`conformation of
`phosphorylation on the
`Together, Met-398, Phe-403, Ala-407, Met-410 and
`Met-411 (from the a-C-helix), Phe-374 (from the Gly-
`rich loop), and Val-424, Leu-433 and Lys-391 define an
`extensive hydrophobic pocket lying adjacent to the ITK
`active site [86]. The size and shape of this pocket are
`different in SFKs as well as in Ser/Thr kinases such as
`CDK2. Although residues Met-398, Phe-403 and Met-410
`are conserved in ITK and BTK, Phe-435 is unique to ITK
`[86]. This residue is therefore identified as a gatekeeper (or
`blocker) of this extensive hydrophobic pocket. An inhibitor
`of the other TEC family members (BTK, TXK/RLK, TEC
`and BMX) may exploit the smaller threonine residue at
`this position, either via a hydrogen bonding interaction or
`by accessing the hydrophobic pocket
`[86]. A broad-
`spectrum inhibitor of all TFKs could be achieved using
`small lipophilic groups positioned close to residue Phe-435
`role in non-cytotoxic
`[86] and may have a general
`immunosuppression.
`A high-throughput screen campaign of a compound
`collection by Bristol-Myers Squibb and Boehringer Ingel-
`heim (Table 2) led to the identification of potent ITK
`inhibitors (Table S2) from a distinct scaffold series of
`2-aminothiazoles [87, 88] and benzimidazoles [89–91],
`respectively. Further development of benzimidazoles have
`demonstrated selective inhibition of ITK, improved the
`cellular and functional potency as well as the drug-like
`properties, with the 10n and 10o compounds as excellent
`agents for proof-of-concept studies [92].
`Novel ITK inhibitors based on (4- or 5-aryl)pyrazolyl-
`indole scaffolds were also found to be selective for ITK over
`other kinases
`[93]. Molecular modelling predictions
`showed that pyrazolyl-indoles are inhibitors of
`ITK.
`Docking models of various pyrazolyl-indole derivatives
`with ITK indicated a common binding mode, which
`involved three hydrogen bonds in the hinge region of ITK
`between: (i) N–H of the pyrazole ring with the carbonyl of
`Glu-436 of ITK, (ii) N of pyrazole and N–H of Met-438 of
`ITK and (iii) N–H of the indole ring with the carbonyl of
`Met-438 of ITK [93]. It was also apparent from docking,
`that among the three series examined, 5-benzylpyrazolyl-
`indoles (compound 34) show the best binding to ITK, and
`were followed in the decreasing order by 4-phenylpyraz-
`olyl-indoles (compound 13) and 5-phenylpyrazolyl-indoles
`(compound 24) (Table S2). On the other hand, compound
`44, which was developed by chemical optimization of an
`initial high-throughput screening hit,
`inhibited ITK’s
`activity with an IC50 in the nanomolar
`range [94].
`Compound 44 substantially reduced pro-inflammatory
`immune responses in vitro and in vivo after systemic
`administration in two acute
`contact hypersensitivity
`models [94].
`In another study, a series of ITK inhibitors, active in
`nanomolar concentrations, were synthesized based on
`indolylindazole libraries. The potential of this series of
`
`compounds was confirmed through in vivo tests in an
`anti-CD3-IL2 mouse model. The intravenous administra-
`tion of highly potent ITK inhibitor 11o (Table S2) resulted
`in dose-dependent, efficient suppression of IL-2 at 10 mg/
`kg [95].
`The discovery of a new series of potent and selective
`novel ITK inhibitors based on 3-aminopyride-2-ones has
`also been reported [96]. These inhibitors were identified
`by structure-based design,
`starting from a fragment
`generated de novo, the 3-aminopyrid-2-one motif. Among
`various derivatives, the compound 7v (Table S2) illustrates
`the fact that fragment-like de novo starting point can
`rapidly evolve into biologically inhibitors of ITK, with
`good potency and selectivity profile. Other derivatives 7w,
`7x and 7y also show potent inhibition of ITK (Table S2)
`[96].
`Another selective inhibitor, 7-benzyl-1-(3-(piperidin-1-
`yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]qui-
`noxalin-6(5H)-one
`(CTA056), was developed through
`screening of 9600 compounds,
`followed by molecular
`modelling, and extensive structure–activity relationship
`studies [97]. CTA056 (Table S2) exhibits the highest
`inhibitory effects towards ITK,
`followed by BTK and
`BMX. Among the 41 cancer cell lines analysed, CTA056
`selectively targets acute lymphoblastic T cell leukaemia and
`cutaneous T cell lymphoma [97].
`Recently, novel and selective thienopyrazole inhibitors
`of ITK were generated by combining structure-based
`design and medicinal chemistry at Sanofi (Table 2) (Table
`S2). The most potent compounds crystallized with the
`target kinase ITK and also with SYK [98]. The mul