Novel dual Src/Abl inhibitors for hematologic and solid malignancies
Importance of the field: c-Src and Bcr-Abl are two non-receptor or cytoplasmic tyrosine kinases (TKs) that play important roles in the development of solid and hematological malignancies. Indeed, Src is overexpressed or hyperacti- vated in a variety of solid tumors, while Bcr-Abl is the causative agent of chronic myeloid leukemia (CML), where Src is also involved. The two enzymes share significant sequence homology and remarkable structural resemblance. Areas covered in this review: ATP-competitive compounds originally devel- oped as Src inhibitors, showed to be also potent Abl inhibitors. Dasatinib, the first dual Src/Abl inhibitor approved by the US FDA in 2006 for the treat- ment of imatinib-resistant CML, is currently being tested in several clinical tri- als for the treatment of different solid tumors. SKI-606 and AZD0530 are two other important dual Src/Abl inhibitors extensively tested in animal models and in clinical trials, but not entered into therapy yet.
What the reader will gain: In this review we will report the latest results regarding dasatinib, SKI-606 and AZD0530, but also the knowledge on new compounds that have appeared in the literature in the last few years, includ- ing AP24163, AP24534, XL228, DC2036. We will focus on the most recent clinical trials or on preclinical studies that are in progress on these small- molecule TK inhibitors that represent a targeted therapy with high potential against cancer.
Take home message: Molecularly targeted therapies, including the inhibition of specific TKs hyperactivated or overexpressed in many human cancers, could be less toxic than the classical non-specific cytotoxic chemotherapeutic agents; they could offer important therapeutic effects, especially if used in association with other agents such as monoclonal antibodies.
Keywords: Brc-Abl, dual inhibitors, hematologic malignancies, solid tumors, Src, tyrosine kinases
1. Introduction
Src is a non-receptor or cytoplasmic tyrosine kinase (TK), belonging to a family with nine currently identified members. Src affects cell proliferation, differentiation, migration, invasion, apoptosis and angiogenesis, by interaction with a diverse array of molecules, including growth factor receptors, cell–cell adhesion receptors, integ- rins and steroid hormone receptors. In normal cells it remains in an inactive state, being only transiently activated during the multiple cellular events in which it is involved (Figure 1). By contrast, Src is overexpressed and/or hyperactivated in a large variety of cancer cells and is probably a strong promoting factor for the development of metastatic cancer phenotypes [1,2]. Moreover, Src plays an important role in osteoclast activation and bone resorption, which are often aberrantly activated in Abl inhibitors. Based on the similarity between Abl and Src and on the critical role of both enzymes in the pathogenesis of CML, small molecules acting as dual Src/Bcr-Abl inhibi- tors could possess enhanced activity against CML compared with selective Bcr-Abl inhibitors, i.e., imatinib and niloti- nib [7]. Moreover, dual Src/Bcr-Abl inhibitors have demon- strated antiproliferative activity against a number of solid tumor cell lines, probably due to their action on Src and its downstream pathways, thus paving the way to preclinical and clinical assays for the treatment of different malignancies. The majority of dual Src/Abl inhibitors were discovered through a rational design approach, usually by modification of Src inhibitors, helped by molecular modeling techniques and by an understanding of the X-ray structure of both enzymes.
In this review we will report the latest results regarding dasatinib, SKI-606, AZD0530, AP24534, XL228 and DC2036, already in clinical trials; but also the knowledge on new compounds that have appeared in literature in the last few years, including AP24163, benzotriazines and pyrazolo-pyrimidines, which so far have been tested only in animal models.
The Bcr-Abl inhibitor imatinib represents (since 2001) an important drug for CML treatment, but relapse after an initial response has been observed in some patients, especially in the advanced phases of the disease. Multiple mechanisms of resis- tance have been identified, even if the dominant one seems to be represented by amino acid point mutations in the kinase domain of the enzyme [6]. For this reason, the search for new targeted inhibitors by both pharmaceutical companies and universities is very active.
Abl shares significant sequence homology and remarkable structural resemblance in its active state with Src family mem- bers. Indeed, several ATP-competitive compounds originally developed as Src inhibitors have also proven to be potent the setting of bone metastases. Given the role of Src in all these functions, its inhibition would be predicted to have a broad therapeutic benefit in patients with Src-dependent cancers [3]. Bcr-Abl, encoded by the aberrant gene Bcr-Abl on the chro- mosome Philadelphia, is another cytoplasmic TK and repre- sents the causative agent of chronic myeloid leukemia (CML) (see [4] for a recent update). Bcr-Abl, differently from its cellular counterpart, c-Abl, is constitutively activated and promotes multiple transduction cascades, leading to growth, proliferation and survival of hematopoietic cells; it also plays a role in defective DNA repair, alteration of cellular adhesion and inhibition of apoptosis [5]. Since it has been demonstrated that the prototypic non-receptor TK c-Abl, normally present in cells, is not a vital enzyme, selective inhi- bition of all Abl activity is a potential treatment modality for CML.
2. Dasatinib
Dasatinib, Sprycel® (BMS-354825) (Figure 2, compound 1) is a 2-(aminopyrimidinyl)thiazole-5-carboxamide derived from the optimization of 2-acylamino-5-carboxamidothiazoles pre- viously identified as Lck inhibitors, in which the 2-acyl func- tionality has been substituted with a variety of heterocycles, such as the pyridine or the pyrimidine rings; the latter, in combination with solubilizing basic side chain, led to the identification of some very interesting compounds, including dasatinib [8].
2.1 Dasatinib and hematologic malignancies
2.1.1 Dasatinib and CML
Dasatinib (Bristol-Myers Squibb) is the first and only dual Src/Bcr-Abl inhibitor approved by the US FDA for the treatment of CML patients resistant or intolerant to imatinib, in any disease phase (i.e., chronic, accelerated and terminal blast crisis), or Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients.
Dasatinib is chemically unrelated to imatinib and is approximately 325-fold more potent than the latter at inhib- iting Bcr-Abl in vitro [9]; moreover, it shows inhibitory activity against Src family kinase members greater (IC50 0.5 nm/l) than against Abl (IC50 1 nm/l). It also inhibits c-Kit, PDGFR, EphA2 and other kinases [6]. In CML progenitor cells, it inhi- bits both Bcr-Abl-dependent and Bcr-Abl-independent Src activity and also other downstream signaling pathways includ- ing MAPK, Akt, and STAT5 [10]. After several cellular and preclinical studies, the effects of dasatinib in patients in different stages of CML and Ph+ ALL have been explored in the START (Src/Abl Tyrosine Kinase Inhibition Activity Research Trials) program, articulated in several phases, which led to the approval of the drug by FDA in 2006; the study is still ongoing for further results [11]. The approved dose regimen of dasatinib was 70 mg twice daily.
The agent is well tolerated and has shown clinical activity in patients. However, recently the approved dose for dasatinib in patients with CML-chronic phase (CP) was updated to 100 mg once daily; this causes fewer severe adverse events, including thrombocytopenia and pleural effusion, compared with those caused by the 70-mg twice-daily regimen [12,13]. By contrast, the dose remains 70 mg twice daily for patients with advanced CML or Ph+ ALL, because, due to the short half-life of the drug, the response durability has not been confirmed [14].
At the moment about 45 clinical trials for the use of dasa- tinib alone or in combination with other agents in CML patients are planned, ongoing or just completed, with the aim to obtain more clinical information, including the rate of patient responses at different doses, efficacy in patients with Bcr-Abl point mutations, progression-free survival, safety after treatment, effects of dose reductions, interruptions and discontinuations, and the adverse event rate.
It has been demonstrated that dasatinib can cross the blood–brain barrier, showing therapeutic response in a mouse model of CML and interestingly also in CML patients with CNS involvement.Indeed, results from two clinical trials (ClinicalTrials.gov NCT00108719 and NCT00110097) indicated that dasatinib possesses promising therapeutic potential in managing intra- cranial leukemic disease and substantial clinical activity in patients who experience CNS relapse during imatinib therapy [15].
However, dasatinib brain concentrations were from 12- to 31-fold lower than those in plasma; the therapeutic benefit was attributed to the high activity of the compound against Abl and Src, but could represent a limitation for the dasatinib therapy of other less sensitive CNS tumors [15]. Very recently, Chen and colleagues demonstrated that P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) play an important role in limiting the CNS penetration of dasatinib. In detail, they observed that brain penetration of dasatinib is increased in specific knockout mice (lacking the expression of P-gp and BCRP) or in wild-type (WT) mice pretreated with P-gp and BCRP inhibitors [16].
Results from an international Phase II trial with dasatinib (70 mg twice daily) in patients with imatinib-resistant or- intolerant CML in blast phase indicated that the compound is associated with a promising rate of response in this high-risk population, with an acceptable tolerability [17].
Results from a Phase III study (NCT00123487) that com- pared the efficacy and safety of dasatinib 140 mg once daily versus 70 mg twice daily in patients with CML in accelerated phase resistant or intolerant to imatinib have been reported, and indicated that dasatinib 140 mg once daily has similar efficacy to dasatinib 70 mg twice daily, but with an improved safety profile [18].
After 2 years of follow-up in a Phase II study (START-R) (NCT00103844), dasatinib demonstrated durable responses and improved response and progression-free survival rates relative to high-dose imatinib [19].Other trials (NCT00123474, NCT00101660, and NCT00103844) focused on the analysis of responses to dasa- tinib treatment in patients with pre-existing Bcr-Abl muta- tions: high response rates were achieved with different mutations, except the gatekeeper T315I mutant, including highly imatinib-resistant mutations in the P-loop region [20]. Regarding Abl mutants, the spectrum of mutations arising with dasatinib in in vitro mutagenesis studies and also in vivo is narrow, represented by F317V and T315I mutations [21]. Martinelli and co-investigators reported that unfortunately Ph+ patients who already harbor imatinib-resistant Bcr-Abl kinase domain mutations tend to develop additional muta- tions associated with resistance to dasatinib and to nilotinib (an Abl inhibitor approved for CML therapy). They found that 83% of cases of relapse after an initial response are asso- ciated with emergence of newly acquired mutations. However, the spectra of mutants conferring resistance to dasa- tinib or nilotinib are small and non-overlapping, except for T315I [22].
2.1.2 Dasatinib and lymphoma
Dasatinib has been also tested on lymphoma cells especially on diffuse large B-cell lymphoma (DLBCL), the most DLBCL (NCT00918463), non-Hodgkin’s lymphoma (NCT00550615), or other lymphoma tumor types.
2.2 Dasatinib and solid tumors
The central role of c-Src in many signaling transduction path- ways combined with the frequent overexpression of c-Src in human epithelial cancer makes this TK an attractive target for tumor therapy. On the basis of recent preclinical studies, dasatinib was shown to be a promising agent for clinical trials in different malignancies, including breast, prostate, lung, pancreatic, and head and neck, CNS (including glioblas- toma), ovarian cancers, mesothelioma, sarcomas (including chondrosarcoma), neuroblastoma, and unspecified adult and childhood solid tumors. Initial pharmacokinetic data show that levels required to inhibit Src activity are clinically achievable and that the drug is well tolerated [3].
2.2.1 Dasatinib and breast cancer
Dasatinib selectively inhibits growth of breast cancer cells, including those of the basal-type/triple-negative breast cancer, categorized as aggressive and lacking effective treatment (i.e., hormonal manipulation or monoclonal antibodies). Different studies provide the rationale for the clinical development of dasatinib in the treatment of this breast cancer type [25]. More- over, it has been recently reported that dasatinib synergizes with doxorubicin, a commonly used chemotherapeutic, to block growth, migration, and invasion of different breast cancer cells, including the highly metastatic, triple-negative MDA-MB-231 cell line [26].
Phase I and II clinical trials are planning or ongoing to vali- date the role of dasatinib in various breast cancer subtypes, either as a single agent or in combination with other drugs, including paclitaxel (taxol) (NCT00820170), fulvestrant + MK-0646 (an estrogen antagonist + a monoclonal antibody for IGF-1R) (NCT00903006), letrozole (NCT00696072) or exemestane (NCT00767520) (two aromatase inhibitors), ixabepilone (an epothilone B analogue) (NCT00924352), capecitabine (a pro- drug of 5-fluorouracil) (NCT00452673), and bevacizumab (a monoclonal antibody for VEGF-A) (NCT00792545). Other specific trials aimed at evaluating the activity of dasatinib, alone (NCT00410813) or in combination with zolendronic acid (a bisphosphonate) (NCT00566618) in the treatment of breast cancer bone metastasis are also in progress.
2.2.2 Dasatinib and prostate cancer
Src plays also an important role in signaling processes involved in prostate tumor progression that lead to the transi- tion to androgen-independent cell growth and to bone metas- tasis. Inhibition of Src, which is a vital enzyme in the regulation of bone remodeling, could offer an important strat- egy for managing bone metastasis in this disease (for recent reviews, see [27,28]).
Gallick and colleagues demonstrated that dasatinib inhibited growth and lymph node metastasis of prostate cancer in an orthotopic mouse model [29]. Moreover, Corey and colleagues reported that the drug inhibited the growth of bone metastases of prostate cancer in SCID (severe com- bined immunodeficiency) mice injected with C4 — 2B CaP (prostatic carcinoma) cells and decreased osteolysis in animals [30].
Recent results from a Phase II clinical trial in patients with advanced castration-resistant prostate cancer (CRPC) support a favorable effect of dasatinib on bone metastasis. Indeed, chemotherapy-naive men with CRPC and increasing prostate-specific antigen were treated with dasatinib 100 or 70 mg twice daily. Lack of disease progression was achieved in 43% patients at week 12 and in 19% patients at week 24; moreover, reduction of urinary N-telopeptide, a marker of bone resorption that predicts for skeletal-related events, was observed in 80% of patients with bone metastases [31].
2.2.3 Dasatinib and colon cancer
Src expression has been shown to be increased in approxi- mately 80% of colorectal cancer specimens, lymph nodes and liver compared with normal colon epithelium; moreover, Src activation may mediate resistance to chemotherapy in this malignancy. Independently from the stage of the disease, increased Src activity levels have been also associated with poor prognosis.
Recognition of Src in colorectal cancer has led to several tri- als of Src inhibitors alone or in combination with other agents including
gemcitabine, cetuximab and oxaliplatin. In general, Phase I studies have reported the unique toxicities of pleural effusion or, more rarely, pericardial effusion and atypical pulmonary infiltrates [32].
In oxaliplatin-resistant colon cancer cell lines, Src activity is constitutively increased. In a mouse model of colorectal liver metastases, treatment with oxaliplatin also results in chronic Src activation. The combination of dasatinib and oxaliplatin results in significantly smaller tumors compared with single- agent treatment, corresponding with reduced proliferation and angiogenesis. These results suggest that Src inhibitors com- bined with oxaliplatin may have efficacy in metastatic colon cancer and may provide the first indication of a molecular phenotype that might be susceptible to such combinations [33].
2.2.4 Dasatinib and gastrointestinal stromal tumors (GISTs)
Dasatinib is also being tested in clinical trials as first-line treatment (NCT00568750) of GISTs that are characterized by activating mutations of c-Kit, a transmembrane TK [34]. In this tumor, dasatinib activity is probably due to inhibition of c-Kit rather than Src or Abl.
2.2.5 Dasatinib and pancreatic cancer
Src is overexpressed in 70% of pancreatic adenocarcinomas. Molecular or pharmacological inhibition of Src expression and activity decreases tumor progression and metastasis of human pancreatic adenocarcinoma cells in an orthotopic nude mouse model. In detail, in animals bearing WT tumors treated with dasatinib, tumor size was decreased and incidence of metastases was significantly reduced, compared with con- trols. These results demonstrate that Src activation contributes to pancreatic tumor progression in this model, offering Src as a candidate for targeted therapy [35].
Dasatinib in a dose-dependent manner also inhibits EphA2, a receptor TK that is overexpressed in many solid malignancies, including pancreatic cancer. In vivo treatment with dasatinib decreased EphA2 phosphorylation in BxPC-3 xenografts, suggesting that dasatinib might have activity in pancreatic cancer due to EphA2 inhibition, besides its effects on Src [36].
2.2.6 Dasatinib and liver cancer
Some recent studies showed that liver cancer cell lines from patients exhibited increased expression of Src and that the proliferation levels of four human liver cancer cell lines were sensitive to dasatinib. These data indicate that Src antagonism with specific inhibitors may be an attractive treatment paradigm for patients with hepatocellular carcinoma [37]. Two clinical trials with dasatinib are ongoing in patients with liver cancer (NCT00459108 and NCT00608361).
2.2.7 Dasatinib and lung cancer
Biological studies showed that Src inhibition by dasatinib causes antiproliferative effects on non-small cell lung cancer (NSCLC) cells in vitro, since the drug induces cell cycle arrest and apoptosis and decreases cell invasion [38]. Furthermore, dasatinib inhibited tumor growth in HCC827 GR (gefiti- nib-resistant NSCLC) cell xenografts to a significantly greater extent than did treatment with gefitinib alone, indicating a rationale for clinical use of the compound in gefitinib- resistant NSCLC, especially with Met amplification [39]. Other studies support further clinical investigation aimed to evaluate the efficacy of dasatinib in combination with cisplatin [40] or with STAT3 inhibitors [41] in the treatment of NSCLC.
There are now 10 ongoing clinical trials (one just termi- nated, NCT00564876) or planned for patients with NSCLC; some are in combination with other agents, including erloti- nib and bevacizumab. Results from a Phase I/II study of dasa- tinib in combination with erlotinib in advanced NSCLC cancer patients have been very recently reported by Haura and colleagues: the co-administration of the two drugs is tol- erable, with adverse effects consistent with the two agents; dis- ease control and inhibition of plasma angiogenesis markers were also observed [42].
Dasatinib has also been tested in a Phase II clinical trial in patients with chemosensitive relapsed small cell lung cancer, but unfortunately no objective response was recorded among the 43 eligible and treated patients. Common reasons for removal of patients from protocol treatment were progres- sive disease (65%) and adverse events, and the study was terminated [43].
2.2.8 Dasatinib and CNS tumors: glioblastoma Glioblastoma is the most common and aggressive form of pri- mary brain tumor. Deregulated signaling via Src has been shown to underlie glioma-related proliferation, angiogenesis, migration and survival [44]. Dasatinib at low nanomolar con- centrations decreased levels of phospho-Src, Akt, and ribo- somal protein S6 in human glioblastoma cell lines and also reduced the invasive potential of the cells.
On the other hand, blocking Src alone is unlikely to trans- late into a significant clinical benefit; thus, strategies for combining Src inhibitors with potential synergistic therapeu- tic modalities are in study. Dasatinib in combination with temozolomide more effectively increased the therapeutic effi- cacy of temozolomide than when dasatinib was combined with carboplatin or irinotecan [45].
Golub and colleagues, using a bead-based method that they developed for detecting phosphorylation of both WT and mutant TKs, confirmed that
Src is frequently phosphorylated in glioblastoma cell lines and is also activated in primary glioblastoma patient samples. They also showed that dasatinib inhibits glioblastoma cell viability and migration in vitro and tumor growth in vivo; using engineered TKs alleles, the inves- tigators confirmed that Src is indeed the relevant target of dasatinib, which inhibits many TKs, in glioblastoma cells [46]. Dasatinib is currently in Phase I/II trials in this type of very aggressive tumor, alone (NCT00423735) or in combina- tion with other agents, including temozolomide, lomustine,bevacizumab and erlotinib.
2.2.9 Dasatinib and melanoma
Dasatinib blocks growth, migration and invasion of human melanoma cells [47] indicating that Src and downstream sig- naling proteins are implicated as having key roles in this can- cer [48,49]. A recent clinical study reported that two metastatic melanoma patients with the L576P Kit mutation had marked reduction (> 50%) or elimination of tumor F18-fluorodeoxy- glucose (FDG)-avidity by positron emission tomography (PET) imaging (a powerful tool to investigate the suppression of tumor metabolic activity) after dasatinib treatment. These data support the selective inhibitory effect of dasatinib against cells harboring the most common Kit mutation in melanoma. Naturally, in this case dasatinib effects are at least in part due to Kit inhibition [50].
Two Phase II clinical trials with dasatinib in patients with advanced melanomas are in progress (NCT00700882, NCT00436605) together with other Phase I/II trials in combination with different drugs, including dacarbazine and bevacizumab.
2.2.10 Dasatinib and neuroblastoma
Neuroblastoma is a neuroendocrine tumor, arising from neural crest elements of the sympathetic nervous system. It represents the most common extracranial solid cancer in childhood, characterized by a poor prognosis. A recent preclinical study reported that even if dasatinib inhibited neuroblastoma cell growth in vitro, in orthotopic mouse models the antitumor effect of dasatinib was only partial. This observation highlighted the importance of testing candi- date drugs in animal environments mimicking the human tumor [51].
With regard to solid tumors in general, results from a Phase I dose-escalation and pharmacokinetic study of dasati- nib in metastatic tumors refractory to standard therapies or for which no effective standard therapy exists have been very recently reported. The obtained data indicated rapid absorp- tion, dose proportionality and lack of drug accumulation. Although no objective tumor responses were seen, durable stable disease was observed in 16% of patients. The drug is well tolerated, with a safety profile similar to that previously reported in leukemia patients, although with far less hemato- logic toxicity. Doses of 120 mg of dasatinib twice daily in 28-day cycles either for 5 consecutive days followed by 2 non-treatment days every week or as continuous, twice-daily 70 mg, have been administered [52].
Preclinical data have indicated that dasatinib is metabolized primarily through cytochrome P450 3A4 (CYP3A4) and may cause QT prolongation. Johnson and colleagues performed a Phase I pharmacokinetic and drug-interaction study of dasati- nib in patients with advanced solid tumors to assess the safety of a once-daily dasatinib regimen, alone or in combination with the potent CYP3A4 inhibitor ketoconazole. Hemato- logic toxicities of dasatinib alone were markedly less than those observed in patients with leukemia, whereas non- hematologic toxicities were similar, as previously reported by Demetri [52]; the dose-limiting toxic effect for dasatinib was pleural effusion. Co-administration of ketoconazole led to a marked increase in dasatinib exposure, which was correlated with an increase in corrected QT (QTc) values of approxi- mately 6 ms. Even if no adverse cardiac events were observed, co-administration of dasatinib with CYP3A4 inhibitors or agents that prolong the QTc interval should be avoided if possible [53].
3. Bosutinib
Using a high-throughput yeast-based assay, Wyeth Pharma- ceuticals researchers identified a family of 3-quinolinecarboni- triles as interesting Src inhibitors. Subsequent SAR studies indicated that an anilino group at C4, a carbonitrile group at C3 and an alkoxy groups at C6 of the quinoline were all crucial for optimal activity. The introduction at C7 of a 3-(4-methylpiperazin-1-yl)propoxy group led to the most interesting compound of this series, bosutinib (SKI-606) (Figure 2, compound 2) [54].
3.1 Bosutinib and CML
Bosutinib is an orally active dual Src/Abl inhibitor [55]. It has shown 200-fold higher potency than imatinib for Bcr-Abl and is active against a number of Bcr-Abl mutants but not on T315I [11]. Recently, the Superti-Furga group demonstrated that in primary CML cells, SKI-606 targets not only Src and Abl kinases but also > 45 novel tyrosine and serine/ threonine kinases, including TEC family kinases, apoptosis- linked STE20 kinases and CAMK2 G, implicated in myeloid leukemia cell proliferation [56]. In vivo experiments confirmed SKI-606 activity in CML imatinib-resistant models where resistance was not caused by mutations, as well as in cells carrying the Y253F, E255K, and D276G mutations [57].
Bosutinib is being evaluated in some Phase III trials in patients with CML-CP who had failed imatinib. Results from a Phase II trial are at the moment encouraging. Indeed, of 84 imatinib-resistant patients, 40% achieved a major cyto- genetic response (MCyR) (which includes complete and partial cytogenetic responses); among these, 29% showed a complete cytogenetic response (CCyR) after only 6 months of follow-up. Of the 22 patients intolerant to imatinib, 59% achieved MCyR, including 50% with CCyR [58].
3.2 Bosutinib and solid tumors
Bosutinib is also being studied for the treatment of solid tumors; it is orally effective in nude mouse xenograft models, including colorectal [59] and breast tumors, as well as in several in vivo models of metastasis [60].
3.2.1 Bosutinib and breast cancer
Mice implanted with breast cancer MDA-MB-231 basal cells and receiving bosutinib (150 mg/kg) developed tumors of significantly smaller volume (45 — 54%) compared with control animals. Analysis of lungs, liver, and spleen showed a significant decrease of tumor metastasis in animals receiving the drug versus controls, at a dose that was well tolerated. Bio- logical analysis of primary tumors showed that these effects were due to the ability of bosutinib to block tumor cell prolif- eration, angiogenesis, growth factor expression, and inhibition of Src-mediated signaling pathways in vivo [61]. Subsequent studies demonstrated that the compound inhibits protein net- works involved in controlling breast cancer cell motility and invasion, rather than proliferation [62]. Bosutinib is currently in Phase I/II clinical development for the treatment of breast cancer, alone (NCT00319254) or in combination with other agents, including exemestane (NCT00793546), capecitabine (NCT00959946) and letrozole (NCT00880009).
3.2.2 Bosutinib and pancreatic cancer
Recently, bosutinib activity on a panel of human pancreatic tumor xenografts has been evaluated. Surgically resected human pancreatic tumors were implanted into female nude mice and randomized to bosutinib versus control. Of 15 patient tumors, three were found to be sensitive to bosuti- nib. In sensitive tumors, bosutinib resulted in increased apo- ptosis and inhibition of phosphorylation of key signaling molecules downstream Src. Even if the percentage of sensitive tumors is quite low, these results may aid the clinical develop- ment of bosutinib and other Src inhibitors in pancreatic cancer [63]. A Phase I/II clinical trial of SKI-606 in association with gentamicin for the treatment of pancreatic cancer is starting (NCT01025570).
3.2.3 Bosutinib and other tumors
The compound is also tested against different solid tumors for which no effective therapy is available. Bosutinib, like dasatinib, appears to synergize with specific chemotherapy agents in melanoma cell lines, but no in vivo study has yet been reported.
4. AZD0530
4- Anilinoquinazolines represent a well-known class of protein kinase inhibitors, including EGFR, VEGFR, p38MAPK, and cyclin-dependent kinase 2. Varying the substitution pattern, AstraZeneca researchers identified AZD0530 (saracatinib) (Figure 2, compound 3) as a potent, orally available, dual Src/ Abl inhibitor. It displays excellent pharmacokinetic parameters in animals, with good aqueous solubility and moderate binding to plasma proteins. Moreover, it inhibits the tumor growth in a c-Src-transfected 3T3-fibroblast xenograft model in vivo and led to a significant increase in animal survival in an orthotopic model of human pancreatic cancer [64].
Ottmann reported that AZD0530 specifically inhibits the growth of CML cells and Ph+ ALL cell lines, but not the Ph- ALL; probably the antiproliferative effect of AZD0530 results from the inhibition of both Src and Bcr-Abl kinases, with consequent downregulation of survival signaling path- ways (STAT5, Erk, PI3IK/Akt) in Ph+ cells resistant or sensitive to imatinib [65]. Actually, very recently, AstraZeneca researchers, who discovered the compound, pointed out that AZD0530’s activity on Abl is not currently considered suffi- cient to provide a strong rationale for its clinical development in Bcr-Abl-driven leukemias [66].
The compound inhibited tumor growth in different xenograft models, even if a complete overlap with its antiproliferative effects tested in vitro has been not observed and further investigation on AZD0530 in other tumor mod- els is warranted to define its biochemical and anticancer activ- ity. Obtained data suggest that the compound may provide clinical benefit by preventing or delaying tumor progression through inhibition of tumor cell migration and invasion, rather than by reduction of primary tumor growth [67].
The compound is currently being tested in a number of Phase II clinical trials for the treatment of different solid tumors, usually advanced and metastatic, including breast, pros- tate, ovarian, endometrial, skin (melanoma), pancreatic, gastric, colorectal, head and neck, NSCLC and osteosarcoma, alone or in combination with conventional chemotherapeutic agents.
4.1 AZD0530 and breast cancer
AZD0530 inhibits Src activity in different breast cancer cell lines [3] and interestingly suppressed the motile and invasive nature of endocrine-resistant breast cancer cells.Treatment of the latter with AZD0530 in combination with tamoxifen resulted in a reduction of Src and FAK activity together with a complete abrogation of their in vitro invasive behavior [67].
AZD0530 and anastrozole, an antiestrogen used to treat estrogen receptor (ER)-alpha-positive breast cancer, alone or in combination were tested in vitro and in vivo on suitable xenografts. AZD0530 monotherapy initially retarded xenograft growth, but drug resistance rapidly emerged, while the com- bined regimen anastrozole/AZD0530 reduced drug resistance and showed greater antitumor efficacy, providing the rationale for clinical investigation of anastrozole-AZD0530 therapy for postmenopausal ER-positive breast cancer [68].
The compound is being tested in Phase I/II clinical trials in patients with metastatic or advanced breast cancer (NCT00559507), also in combination with zoledronic acid for the treatment of bone metastasis (NCT00558272) or in with AZD2171 (cediranib), a potent VEGFR inhibitor in patients with advanced solid tumor, including breast cancer (NCT00475956).
4.2 AZD0530 and prostate cancer Chang and colleagues studied the involvement of Src and Abl in prostate cancer cells and showed that while both Src and Abl are expressed in all prostate cancer cell lines, Src but not Abl is hyperactivated. AZD0530 inhibited Src activation in a rapid and dose-dependent manner in cells and reduced orthotopic DU145 xenograft growth by 45% in mice [69].
AZD0530 completely inhibited metastasis in mice bearing orthotopic tumors from castration-independent prostate tumor cell line clones and also retarded osteolytic lesions in a mouse model of bone metastatic prostate cancer [27].
Moreover, the compound inhibits tumor metastasis in SCID mice implanted with GRP (gastrin-releasing peptide)-autocrine prostate cancer cells, which represent a new neuropeptide- autocrine model of androgen-insensitive prostate cancer with aggressive behaviour and enhanced mobility [70].
A Phase II trial of AZD0530 in patients with advanced CRPC showed that the treatment was generally well tole- rated, but possessed little clinical efficacy as monotherapy. Strong preclinical evidence warrants further investigation of AZD0530 in earlier-stage prostate cancer or as combination therapy [71].
4.3 AZD0530 and pancreatic cancer
Very recent articles reported that AZD0530 treatment signif- icantly inhibits tumor growth in a subset of human pancreatic tumor xenografts, downregulating Src and its downstream sig- naling proteins, including FAK, paxillin, and STAT-3 [72], and that the compound (25 mg/kg/day for 5 days in mice) reduced Src activity in both hypoxic and non-hypoxic BxPC3 (a pancreatic carcinoma cell line) tumor regions [73].
A recent Phase I clinical trial of AZD0530 in healthy men demonstrated that inhibition of Src by this compound reduces osteoclastic bone resorption in humans [74]. Another Phase I trial in patients with cancer showed that AZD0530 inhibited phosphorylation of Src substrates and significantly reduced levels of markers of osteoclastic bone resorption in a dose-dependent manner [75].
5. ARIAD compounds
5.1 Purine derivatives
Ariad Pharmaceuticals researchers synthesized several trisubsti- tuted purine-based compounds that were found to be potent dual Src/Abl ATP-binding site inhibitors [76]. Very recently, guided by mutagenesis studies and molecular modeling techni- ques, they designed a series of novel N9-arenethenyl purines, characterized by a trans vinyl linkage at N9 that projects hydro- phobic substituents into the selectivity pocket near the ATP binding site of the enzymes. Extensive SAR studies led to the discovery of potent and orally bioavailable inhibitors, some of which, including AP24163 (Figure 2, compound 4), possessed efficacy in mice bearing subcutaneous xenografts of Src Y527F expressing NIH 3T3 cells. The compounds elicited dose- dependent tumor shrinkage with complete tumor regression observed at the highest dose (100 mg/kg) [77]. Interestingly, AP24163 in enzymatic assay also inhibited the highly resistant T315I mutant and suppressed in vitro resistance [78].
5.2 Imidazo-pyridazines
AP24534 (Figure 2, compound 5) is a low-nanomolar Src/Bcr- Abl inhibitor, potently active also on the T315I and other mutants: for this reason it has been called a pan-Bcr-Abl inhibitor by Ariad researchers. The compound has been designed on the basis of X-ray crystallographic studies on the Abl kinase domain; specifically, the imidazo[1,2-b]pyridazine core occupies the adenine pocket of the enzyme and the ethynyl linkage allows the accommodation of the T315I side chain.
AP24534 suppressed Bcr-Abl(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Enzymatic, cellular and preclinical data supported clinical evaluation of AP24534 for treatment of CML [79]. The compound is currently in a Phase I trial (NCT00660920) to determine the safety and doses of oral administration in patients with refractory or advanced CML and other hematologic malignancies.
6. XL228
XL-228 (structure currently proprietary), synthesized by Exelixis Pharmaceuticals, is a multi- kinase inhibitor that targets Src family kinases, IGF-1R, Aurora A, and Bcr-Abl (including the T315I mutant) in the low nanomolar range [80]. In xenograft models, XL228 demonstrated more activity than imatinib and dasatinib, providing the rationale for its clinical development in patients with drug-resistant CML. Preliminary data from an ongoing Phase I trial of XL228 were presented in a poster session at the 13th Congress of the European Hematology Association in Copenhagen, Denmark, on 13 June 2008. This compound has been administered at doses in the range of 0.45 — 7.2 mg/kg. All subjects, also bearing the T315I mutation, have demonstrated stable or decreasing white blood cell counts, with manageable adverse effects. XL228 is being currently tested in two Phase I clinical trials, one for treatment of CML or Ph+ ALL (NCT00464113), and the other for advanced malignancies, including lymphoma (NCT00526838).
7. Benzotriazines
TargeGen researchers reported a series of 3-aminobenzo[1,2,4] triazine derivatives as potent c-Src/Abl dual inhibitors, includ- ing TG100435, (Figure 2, compound 6). Derivatives showed activity in a murine CT-26 carcinoma cell assay and in a pulmonary metastases model. Compound 6 was evaluated in an A549 human NSCLC xenograft model, along with tarceva as positive control. Notably, at 25 mg/kg i.p. it showed potency comparable to tarceva at 80 mg/kg p.o.; and at the higher dose of 40 mg/kg, it showed 86% reduction in tumor burden compared with the vehicle control [81].
The corresponding pyrrolidine N-oxide, TG100855 (Figure 2, compound 7), is the predominant metabolite of 6 in human, dog and rat and is from two- to ninefold more potent than the parent compound, so increasing the overall TK inhibition in animal models after oral administration of 6 [82].
8. DCC-2036
DCC-2036 (structure currently proprietary), synthesized by Deciphera Pharmaceuticals, is an Abl inhibitor that also tar- gets Src family kinases such as Lyn and Hck. It showed anti- proliferative and apoptotic activity on Ba/F3 cells expressing WT Bcr-Abl as well as T315I Bcr-Abl, with IC50 values\ < 10 nM, without significant inhibition of parental Ba/F3 cells. When dosed at 100 mg/kg/day, it significantly pro- longed the survival of mice with CML-like myeloproliferative disease induced by retroviral expression of WT Bcr-Abl and T315I in bone marrow, showing good bioavailability and a safety profile [83]. A Phase I study started in 2009 to evaluate the safety and tolerability of once-daily continuous oral dosing of this com- pound in patients with treatment-resistant or -intolerant CML or Ph+ ALL, including patients with the T315I gatekeeper amino acid mutation. 9. Pyrazolo[3,4-d]pyrimidines 4-amino substituted Recently, a new library of pyrazolo[3,4-d]pyrimidines was synthesized and found to block Abl and Src phosphorylation in the nanomolar range in enzymatic assays. This new class of inhibitors induces apoptosis and possesses antiproliferative activity in different solid tumor cell lines such as epidermoid carcinoma A431 cells, the breast cancer 8701-BC cells, osteosarcoma SaOS-2 cells and prostate cancer PC3 cells [84-87]. Some compounds have been also tested in in vivo assays: SI83 (Figure 2, compound 8) by inhibiting Src phosphoryla- tion, decreased in vivo osteosarcoma tumor mass in a mouse model and showed selectivity for osteosarcoma, since it had far less effect in primary human osteoblasts. These results show that human osteosarcoma had Src-dependent proliferation and that modulation of Src activity may be a therapeutic target [88]. Two other derivatives of the same series, namely SI34 and SI35 (Figure 2, compounds 9 and 10) have been evaluated against ARO cells, a human anaplas- tic thyroid cancer cell line. These compounds demonstrated an ability to reduce proliferation, as well as increasing cell death, in this cell line. To improve the biopharmaceutical properties of the compounds, characterized by low water sol- ubility, a liposome formulation was prepared. When entrapped in unilamellar liposomes, SI34 and SI35 exerted their cytotoxic effects even at lower doses and after shorter incubation time either in ARO or other thyroid cancer cell lines. Moreover, the growth of tumor xenografts induced in SCID mice was inhibited by i.v. administration of 25 -- 50 mg/kg of the drug liposomal formulation [89]. Some other C-6 unsubstituted pyrazolo-pyrimidines synthesized by the same group have been assayed in a medullo- blastoma cell line. This tumor is the most common malignant brain tumor in children, still lacking an effective treatment. Compounds S29 and SI163 (Figure 2, compounds 11 and 12) demonstrated antiproliferative activity by inhibit- ing Src phosphorylation, as well as inducing apoptosis. Interestingly, in a mouse model the administration of 100 mg/kg p.o. of S29 was able to significantly reduce tumor growth of medulloblastoma cell xenograft. After 40 days of treatment, a ~ 25% reduction of the tumor burden was observed in the treated group compared with controls [90]. 10. Expert opinion Work in the field of TK inhibitors is extremely difficult; now that encouraging results have been obtained, researchers are beginning to understand not only what has already been achieved, but also what remains to be done. The journey towards a fuller understanding of the complex biological sys- tems involved is likely to be long. Therapeutic progress in CML is a success for molecular medicine and imatinib repre- sents the most exciting example of a TK inhibitor which, through targeting Bcr-Abl, the etiologic agent of CML, might effectively cure this disease. Patients suffering from CML nowadays are expected to live longer than patients diagnosed 10 years ago. However, disease eradication and further advances in CML treatment are still the highest priority. After the successes achieved in CML, scientists understood that various levels of resistance can develop in patients over the course of their treatment; consequently, research must focus on overcoming this. Sometimes, efforts to predict potential emerging resistance can be more profitable than aiming to discover a new, improved drug. Moreover, as reported by the most eminent clinicians in this field despite the activity of all the above-mentioned agents, curing CML will ultimately depend on the development of agents capable of targeting Bcr-Abl CML stem cells; efforts aimed at achieving this goal are ongoing [91]. Molecularly tar- geted therapies, including the inhibition of specific TKs hyperactivated or overexpressed in many human cancers, could be less toxic than the classical nonspecific cytotoxic chemotherapeutic agents that do not distinguish malignant from the non-malignant cells. With the recognition that drugs focused on hematological cancer could be also used against solid malignancies, work has been carried out in this direction, with some positive results already being obtained. Unfortunately, in solid tumors abnormalities of a single TK are rare (e.g., Kit in GIST) and a specific inhibitor targeting only one kinase (or one family of kinases) would not be sufficiently effective to fight the disease. Moreover, even if Src is widely implicated in cancer progres- sion, Src activity alone is unlikely to cause oncogenic transformation. Preclinical and clinical studies suggested that monotherapy with single-targeted agents such as monoclonal antibodies specific for a single growth factor may produce only subopti- mal results, since multiple signaling pathways are activated in solid tumors [92]. Dual inhibitors (Src/Abl inhibitors) and even more multitargeted inhibitors, such as dasatinib or bosu- tinib (that inhibit a high number of kinases), especially if used in association with other agents such as monoclonal antibod- ies, could offer important therapeutic effects. As an example, recent evidence indicates that antiangiogenic agents (bevacizu- mab) can normalize the complicated and disorganized tumor vasculature to enhance drug delivery. Indeed, the dual Src/Abl inhibitors reported here are being tested in different preclini- cal or clinical studies in combination with classic chemother- apeutic agents, since Src inhibition would increase the therapeutic effects of cytotoxic drugs. The rationale for target- ing multiple pathways is also justified by the resistance that a drug-targeted enzyme could develop to the drug itself through multiple mechanisms, including amino acid mutations, constitutive activation of alternative signaling pathways, and inactivation of phosphatases to amplify the kinase activity [93]. In our opinion, in the near future it will be possible to test different ‘cocktails’ of drugs, at least for the most aggressive malignancies; so a potent dual Src/Abl inhibitor could be added to the conventional combined therapies, e.g., cisplatin + antracicline + 5-fluorouracil, used nowadays for the treatment of breast cancer. If this approach is explored, caution should be observed regarding the potential toxicity of the combined regimens, since, as previously reported in this article, the metabolism of some TK inhibitors could interfere with or be modified by other drugs such as the cytochrome P450 inhibitors. Comparisons of clinical trial results should also be under- taken with caution due to the differences in protocols and baseline patient demographics. New clinical trials are ongoing to better define the possible long-term benefit of dual Src/Abl inhibitors. Recently synthesized compounds, for which preclinical studies are just starting, could offer further opportunities to develop other drugs that are active in hema- tological and solid malignancies. Data remain scarce, but investigators are confident that knowledge will be rapidly obtained. The discovery of new dual Src/Abl inhibitors or in general multitargeted kinase inhibitors for the treatment of different cancers is of particular interest, mainly because the simplified view of ‘one drug, one target’ is clearly not always true [94]. Systems biology, the analysis of the relationship among the elements in the cell in response to genetic and environmental perturbations, aims ultimately to develop predictive models of human diseases and could result in an important tool to dis- cover new drugs [95]. The advantages of using a cell systems biology approach include the possibility to test a large number of potential drug targets simultaneously and to scan multiple disease models and mechanisms, leading to the improvement of hit quality (smaller libraries, pre-qualified for drug-like qualities) and reducing the time and expense of target valida- tion prior to screening [96]. Moreover, with this approach it may be also possible to predict drug side effects [97]. An interesting example in the field of Bcr-Abl inhibitors was recently reported by Sylvester and colleagues, who developed a bead-based activity screen for small-molecule inhibitors of signal transduction in chronic myelogenous leukemia cells [98]. The use of systems biology together with systems pharma- cology that studies drug action in the context of the whole genome [99] could facilitate the discovery of new drugs,RK 24466 including new dual- or multitargeted kinase inhibitors.