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ã 2000 Macmil an Publishers Ltd All rights reserved 0950 ±9232/00 $15.00 From oncogene to drug: development of small molecule tyrosine kinase inhibitors as anti-tumor and anti-angiogenic agents 1P®zer Global R&D, Groton, Connecticut, CT 06340, USA The con¯uence of two distinct but related activities in the accomplished to date to understand the molecular past 10 years has dramatically accelerated e€orts pharmacology of small molecule inhibitors of receptor towards the discovery and development of novel drugs tyrosine kinases (Sedlacek, 2000; Fry, 2000; Bridges, to treat cancer. The ®rst is a rapidly emerging 1999; Levitzki, 1999; Lawrence and Niu, 1998). With- understanding that a number of distinct tyrosine kinases out summarizing each of these important reviews, they play roles in diverse but fundamentally important aspects provide an appropriate context for understanding the of tumor progression (growth, survival, metastasis and obstacles and triumphs that have led, very recently, to angiogenesis). The second is the discovery that small the ®rst reproducible, objective clinical responses in molecule compounds have the capacity to potently and cancer patients treated with tyrosine kinase inhibitors.
selectively inhibit the biochemical function of tyrosine The catalytic function of protein tyrosine kinases kinases by competing for ATP binding at the enzyme involves the simple transfer of the gamma phosphate of catalytic site. These observations have been conjoined in ATP to hydroxyl group of a tyrosine residue of major e€orts to bring forward into clinical development proteins (or peptides) encompassing a diversity of novel cancer drugs with the potential to provide both primary sequences and tertiary structures (Songyang clinical ecacy and improved tolerability. The focus of and Cantley, 1998). Each of the substrates in the this review is on the development of small molecule phosphotransfer reaction, the tyrosine hydroxy group tyrosine kinase inhibitors, and does not extend to other and ATP, represent reasonable pharmacological start- approaches that could be applied to disrupt the same ing points for the design of substrate analogs and pathways in clinical tumors (receptor and/or ligand- competitive inhibitors of tyrosine kinases. A diverse set competitive antibodies, intrabodies, antisense ribonucleo- of pharmacophores, including natural products (laven- tides, ribozymes, phosphatase inhibitors or SH2/SH3- dustins and erbstatins) and synthetic tyrosine mimetics, directed agents). Selected tyrosine kinase inhibitors, have all been characterized on the basis of their ability known or believed to be in development in cancer to competitively inhibit tyrosine kinase function treatment trials, are summarized as are some of the (Levitzki, 1999). These compounds tended to have key issues that must be addressed if these compounds are poor potency (particularly in cells), to yield relatively to be developed into clinically useful cancer chemother- ¯at structure-activity relationships, and to be some- apeutic agents. Oncogene (2000) 19, 6574 ± 6583.
what non-speci®c in their kinase inhibition (Fry, 2000).
Attacking this reaction from the other side, by Keywords: tyrosine kinase inhibitors; anti-tumor; anti- identifying compounds that mimic ATP, was originally thought to be even less tractable. As reviewed by Lawrence and Niu (1998), the theoretical obstacles were immense. First, the primary sequence of the ATP- Origin of species ± brief overview of substrate-based binding pocket of all kinases is highly conserved, and therefore selectivity, if not speci®city, represents a signi®cant technical challenge. Secondly, the intracel- Among all non-traditional (non-DNA-directed) cancer lular concentration of ATP can exceed 5 mM, targets for which pharmacological intervention is particularly in tumor cells, while the Km for ATP in feasible, there are none that have generated as much most kinase active sites is in the micromolar range, widespread interest, and have invoked as much thus ensuring full-time saturation by ATP. ATP- resource investment in both the public and private competitive inhibitors would need to exhibit at least sectors in the past 7 years, as have the tyrosine kinases.
nanomolar inhibitory kinetic constants to e€ectively Several excellent recent reviews have described the compete in this circumstance (Lawrence and Niu, functions of various tyrosine kinases in the key 1998). Finally, there are multiple non-kinase ATP- pathways that drive tumor progression, from ®rst dependent enzymes important to normal physiology, genetic insult to disseminated disease (Hanahan and and so an indiscriminant ATP mimetic would likely Weinberg, 2000; Hunter, 2000; Gibbs, 2000). Key have toxicities that were pharmacologically and among these are the receptor tyrosine kinases which initiate signal transduction in tumor cells or endothelial This theoretical logjam was broken in convincing cells following the binding of the growth factors EGF, fashion when the tyrosine kinase inhibitory activities of PDGF and VEGF. There are also several excellent anilinoquinazolines were ®rst described in 1994 by reviews that provide detailed overviews of the work three separate groups (Fry et al., 1994; Ward et al., 1994; Osherov and Levitzki, 1994). For example, the work of Fry et al. (1994) at Warner Lambert revealed that 4-anilinoquinazolines were potent (nM) inhibitors Tyrosine kinase inhibitors in cancer treatment trials of the EGFR tyrosine kinase with good cell activity relapsed on drug between 45 and 81 days. Of 19 and profound biochemical selectivity relative to other responding patients, 10 experienced Grade 3 ± 4 neu- kinases within the tyrosine kinase family. Further tropenia. This response rate, and the incidence of elaboration of structure-activity relationships rich in Grade 3 ± 4 toxicity, compares very favorably to the new possibilities resulted in ATP-competitive inhibitors standard of care cytotoxic chemotherapies for CML.
of the EGFR tyrosine kinase with Ki values in the As such, more de®nitive trials assessing the ecacy and single digit picomolar range. It is interesting to note safety of STI 571 are ongoing in CML.
that the Michaelis-Menten equation could not be used It is interesting to speculate as to the biochemical to derive the Ki values of these molecules. So avid was basis for both the ecacy and the toleration pro®le of the binding of compound to the ATP site, the STI 571. Two other tyrosine kinases potently inhibited conventional approximation that total and free enzyme by STI 571, c-kit and PDGFR, are both believed to play concentrations were equivalent did not apply under important roles in maintaining bone marrow stroma ± these conditions. These accomplishments, which may progenitor cell interactions (Ashman, 1999; Sungaran et be among the most important in pharmacology for the al., 2000). Thus, inhibition of c-kit and PDGFR could last 10 years, were largely achieved by empirical also account for some of the compelling clinical activity screening and iterative medicinal chemistry. Even more of STI 571 in CML, as well as for its toxicity pro®le new chemotypes may emerge as structure-based design (neutropenia). Treatment of a c-kit expressing a human becomes more commonly applied to the identi®cation myeloid leukemia cell line, M-07e, with STI 571 before of both active site- and allosteric site-directed inhibitors stimulation with kit ligand inhibited c-kit autopho- for an ever-widening slate of tyrosine kinase targets.
sphorylation, activation of mitogen-activated protein While these early lead molecules had biopharmaceu- (MAP) kinase, and activation of Akt, with an IC50 of tical properties which were by-and-large incompatible 100 nM (Heinrich et al., 2000). STI 571 was even more with oral bioavailability and good duration of exposure potent in a human mast cell leukemia cell line (HMC-1) in vivo, the results spurred on a number of groups, expressing an activated mutant form of c-kit. Similar which have since identi®ed and developed tyrosine results have also recently been reported in non- kinase inhibitors with signi®cant potential to treat hematopoietic tumor cells (Wang et al., 2000). The ecacy and safety hypotheses for inhibition of c-abl in CML may perhaps only be addressed with a more selective abl tyrosine kinase inhibitor. Given the Selected development candidates ± updates apparent therapeutic bene®t of STI 571, this may be largely an academic question, but one with important implications as one tries to rationalize the desired selectivity pro®les of tyrosine kinase inhibitors most STI 571 (CGP57148B) Among all of the candidates likely to generate both ecacy and safety in humans.
currently in clinical development, perhaps none has provided as much `proof of concept' for the clinical SU101 (le¯unomide; HWA 486) Le¯unomide was ecacy and tolerability of small molecule tyrosine originally described and developed as an inhibitor of kinase inhibitors as has STI 571. Originally disclosed by dihydroorotate dehydrogenase, a key enzyme in the de Novartis as a multitrophic tyrosine kinase inhibitor, novo synthesis of pyrimidines, for use as an immuno- STI 571 was described by Druker et al. (1996); and suppressive or anti-arthritic agent (Bartlett and Druker and Lydon (2000) as having potent activity vs Schleyerbach, 1985; Kuo et al., 1996). Le¯unomide the translocation product bcr-abl, the transforming has shown signi®cant activity as a treatment for tyrosine kinase found in virtually all CML cells rheumatoid arthritis (Smolen and Emery, 2000; Cohen expressing the Philadelphia chromosome (Kurzrock et et al., 2000b), and was launched by Aventis as Arava2 al., 1988; Kelliher et al., 1990). The inhibition of v-abl, in the US and elsewhere beginning in 1998. Extending bcr-abl and PDGFR autophosphorylation by the 2- the work of others (Mattar et al., 1993; Xu et al., 1995), Shawver and co-workers reported that micro- lar concentrations was found to translate to both in molar concentrations of le¯unomide inhibited the vivo anti-tumor activity, and to the inhibition of autophosphorylation of the tyrosine kinase receptors clonogenicity of blasts from CML patients (le Coutre for PDGF and VEGF (Shawver et al., 1997). The et al., 1999; Druker et al., 1996). The results of a compound was also e€ective at blocking mitogenesis clinical trial in which STI 571 was administered to stimulated by both PDGF and EGF, but exogenous CML and ALL patients expressing bcr-abl in their uridine could not reverse the e€ect of le¯unomide on leukemic blasts were most recently summarized in May PDGF mitogenesis, suggesting that inhibition of the 2000 (Talpaz et al., 2000). STI 571 was used to treat 33 receptor tyrosine kinase, and not inhibition of acute leukemia patients, which included 21 myeloid pyrimidine pools, was a key pharmacological activity.
blast crisis CML patients and 12 bcr-abl-positive ALL The inhibition of EGF-induced mitogenesis by le¯u- or lymphoid blast crisis CML patients. Clinical nomide was reversed in part by uridine (Shawver et al., responses, as de®ned by a decrease in the percentage 1997), despite the fact that le¯unomide and close-in of patients achieving reduction in bone marrow blasts analogs also have inhibitory activity vs the EGFR to 15% of pre-treatment levels, were observed in 55% tyrosine kinase (Ghosh et al., 1999).
of myeloid blast crisis patients, with complete responses Le¯unomide/SU101 is clearly a tyrosine kinase in 22% of these patients. The response rates in patients inhibitor with multiple biochemical e€ects, and readily with bcr-abl positive ALL and lymphoid blast crisis of generates a predominant active metabolite (SU0020 or CML were higher (82% with 55% complete responses), A771726; Figure 1) that has a complex inhibitory but all of the patients with lymphoid leukemias pro®le of its own (Hamilton et al., 1999). SU 101 was, Tyrosine kinase inhibitors in cancer treatment trials Figure 1 Structures of selected tyrosine kinase inhibitors in clinical development for cancer nonetheless, progressed into clinical trials by SUGEN clinical ®ndings with AstraZeneca's ZD1839 (Iressa2) (now part of Pharmacia). A Phase I study in cancer have been equally compelling. The pharmacological patients revealed that SU 101 was well-tolerated as a characteristics of Iressa2 were ®rst described in 1996 24 h continuous i.v. infusion at doses up to 443 mg/m2/ (Wakeling et al., 1996; Woodburn et al., 1997) as a wk. At this dose, the plasma concentration of the potent and selective inhibitor of the EGFR tyrosine active metabolite was maintained at levels sucient to kinase. This quinazoline-based compound (Figure 1) is block both PDGFR and EGFR signaling, as well as an ATP-competitive inhibitor of the EGFR tyrosine pyrimidine biosynthesis (Eckhardt et al., 1999). Toxi- kinase (IC50 25 nM) with 50-fold selectivity relative to cities were relatively minor (Grade 1 ± 2 nausea, closely homologous erbB family members (IC50 for vomiting and fever in approximately 20% of all erbB2 1 ± 3 mM) and even greater selectivity for more courses given). Surprisingly, hematopoietic toxicities divergent tyrosine kinases. It demonstrates good and hemolysis, which had been noted in the preclinical cellular potency (80 nM IC50 for inhibition of EGF- experience with SU 101, were not seen in this Phase I dependent mitogenesis) and robust, dose-dependent population. One partial response was seen in 26 anti-tumor ecacy in a variety of human tumor patients receiving an average of two courses each; the xenografts (Woodburn et al., 1997). These results have responding patient received 13 courses (52 infusions) to been most recently extended to show that Iressa2 has treat an anaplastic astrocytoma, and had a notable in vivo ecacy in a diverse human tumor xenograft (450%) reduction in one measurable lesion (Eckhardt models both with (Ciardello et al., 2000) and without et al., 1999). SU 101 has been reported to be in (Sirotnak et al., 2000) highly activated EGFR signaling advanced trials for multiple solid tumor types, but pathways. Of equal interest are the observations that recent disclosures (Garber, 2000) indicate that Phase Iressa2 combines with standard cytotoxic agents III trials in at least one tumor type (glioblastoma) have (platinums, taxanes, topoisomerase I inhibitors, etc.) been abandoned. The status of other trials (ongoing to produce additive or supra-additive anti-tumor Phase II trials for ovarian and NSCLC; planned Phase ecacy in vivo without exacerbation of the toxicity of III trials for prostate, colon and NSCLC) is uncertain the co-administered cytotoxics. The ®ndings that tumor EGFR density does not predict ecacy when the compound is used in conjunction with cytotoxic agents have signi®cantly impacted the development strategy employed by AstraZeneca as Iressa2 moves towards Iressa2 (ZD1839) While STI 571 has provided no- Multiple Phase I trials with Iressa2 have been table clinical proof-of-concept for the clinical ecacy summarized, and the results revealed reasonable and safety of tyrosine kinase inhibitors, the early pharmacokinetics, good toleration and the ®rst signs Tyrosine kinase inhibitors in cancer treatment trials of clinical ecacy when used as a single agent in (Pollack et al., 1999; Goss et al., 2000; Allen et al., patients with advanced disease (Ferry et al., 2000; 2000). Given the overall safety and toleration pro®le of Baselga et al., 2000; Kelly et al., 2000). Following oral Iressa2, AstraZeneca has committed to an aggressive administration of a single dose (50 mg), maximum development strategy, which includes two large Phase plasma drug concentrations (mean 45 ng/ml) occurred III studies to assess the use of Iressa2 in combination 1 ± 5 h post-dose. The mean terminal t1/2 was 34 h.
with cis- or carbo-platinum plus a taxane or Inter-subject variability in exposure was signi®cant gemcitabine in ®rst-line therapy for NSCLC (trials 14 following single and multiple administration (up to and 17), as well as a Phase II trial (trial 16) to con®rm sevenfold at each dose level), but exposure increased the single agent activity of Iressa2 in patients with proportionally with dose, with no apparent change in advanced NSCLC (Kelly et al., 2000). It is important terminal t1/2 across the dose range tested (Kelly et al., to note that these trials do not call for a prospective 2000). In a larger dose-escalation trial, Ferry and selection for patients with tumors with some pre- collaborators administered Iressa2 at doses of 50 ± de®ned level of EGFR over-expression. All epithelial 700 mg once daily, given orally for 14 days followed by tumors express some EGFR, and in the disease target 14 days of observation (Ferry et al., 2000). In total, 64 here, NSCLC, tumors often present with a high patients with advanced disease, who had each proportion of EGFR over-expression (up to 80 ± 90% progressed while on prior chemotherapy, completed in advanced disease). The strategy is also consistent 145 cycles. Cmax and AUC0-24h were proportional with pre-clinical data suggesting that ecacy in drug across the entire dose range (mean values 113 ± combinations may not be determined in large part by 2255 ng/ml and 1.8 ± 38.5 mg.h/ml, respectively). As the level of EGFR over-expression in tumors (Sirotnak in single dose studies, Iressa2 showed a long terminal et al., 2000). Results are expected from these pivotal elimination half-life (mean of 46 h). Iressa2 was very trials in a late-2001 or early-2002 timeframe.
well-tolerated in this study; the most common adverse events were diarrhea and acne-like skin rash (Grade 1 ± OSI-774 (CP-358,774) CP-358,774 is also a potent 2). Acne-like skin rashes have emerged as a common, and selective quinazoline-based inhibitor of the EGFR mechanism-based adverse event for EGFR inhibitors, function (Figure 1). This compound is a reversible, but the speci®c toxicological e€ect in the skin is not yet ATP-competitive inhibitor (IC50 of 2 nM) of the EGFR well understood. Grade 3 ± 4 adverse events were tyrosine kinase, with greater than 500-fold selectivity shown to be rare with Iressa2 treatment, and were against other tyrosine kinases, such as the closely generally ascribed to disease progression. The dose- related erbB2 kinase, as well as v-src, c-abl and the limiting toxicity, de®ned at the 700 mg dose level, was insulin and IGF-1 receptors, (Moyer et al., 1997). CP- Grade 3 diarrhea (Ferry et al., 2000).
358,774 inhibits the autophosphorylation of the EGF A compelling level of ecacy was also revealed in receptor in a variety of EGFR over-expressing tumor these early trials (Ferry et al., 2000). Anti-tumor cells (IC50=20 nM), and produces cell cycle arrest and responses were most evident among the 16 NSCLC apoptosis in multiple cell types (Moyer et al., 1997; patients treated with Iressa2 ± two had an objective Barbacci et al., 1997; Iwata et al., 1997). In vivo, CP- partial response, two patients had signi®cant regression 358,774 e€ectively inhibits EGFR-speci®c tyrosine of disease and two patients had stable disease. Similar phosphorylation in human tumor xenografts (ED50 of pharmacokinetic and safety pro®les were noted in a 10 mg/kg p.o. when given as a single dose) with separate study (Baselga et al., 2000), one that also signi®cant duration of action; daily dosing produces revealed the potential for ecacy from Iressa2 in substantial growth inhibition and regressions in human patients with advanced prostatic and head-neck tumor xenografts (Pollack et al., 1999). Moreover, the cancers. These early results added importantly to the dose-response for tumor growth inhibition shows good proof-of-concept that selective tyrosine kinase inhibi- agreement with the dose-response for inhibition of tors could have signi®cant single agent ecacy, as EGFR-phosphotyrosine in tumors from treated ani- measured by objective tumor regressions, in patients mals. As with Iressa2, CP-358,774 was found to with advanced disease. The clinical observations have generate additive anti-tumor activity when used in therefore recapitulated the pre-clinical data showing combination with cis-platinum and other cytotoxic that Iressa2 increased apoptosis and regressions in agents, without exacerbating the toxicities of the other human tumor xenograft models (Ciardello et al., 2000).
chemotherapeutants (Pollack et al., 1999).
The Iressa2 data indicate that the ecacy of these Clinical studies with CP-358,774 have revealed that agents can be measured using more classically de®ned the agent is well-tolerated at oral doses that achieve clinical endpoints. There will undoubtedly be signi®- plasma concentrations projected to be required for cant value in the use of pharmacodynamic and anti-tumor ecacy in humans (400 ± 500 ng/ml). In one surrogate endpoints to guide dose-intensi®cation or to study, escalating doses were administered orally once pre-select patients for whom other tyrosine kinase every week (Karp et al., 1999). Eighteen patients with inhibitors might represent the most promising treat- advanced solid tumors were treated at ®ve doses (100 ± ment option. Pharmacodynamic endpoints have not 1000 mg) for a maximum period of 24 weeks.
played a major role in the early development of EGFR Toxicities were observed only at doses higher than tyrosine kinase inhibitors, despite the fact that several 200 mg/week, and included mild fatigue, Grade 2 reasonable options exist, including both invasive maculopapular (acneiform) rash, Grade 2 nausea, and techniques (direct measurement of tumor-derived or Grade 2 diarrhea. Like Iressa2, CP-358,774 exhibited normal tissue-derived EGFR phosphotyrosine, phos- intra- and inter-subject variability in exposure, but phorylation of down-stream signaling molecules; dose-proportional increases in exposure were observed apoptosis markers) and non-invasive techniques such throughout the 100 ± 1000 mg weekly dose range.
as PET imaging of metabolically modulated tumors During the ®rst 24 h following a single dose, the Cavg Tyrosine kinase inhibitors in cancer treatment trials (0.9 ± 4.8 mg/ml for 100 ± 1000 mg doses, respectively) has taken over complete responsibility for the devel- was some two- to 10-fold above the projected opment of CP-358,774, which is now formally referred ecacious plasma concentration. No maximally toler- ated dose or dose-limiting toxicity was discerned in this study. In a second Phase I study (Siu et al., 1999), CI-1033 (PD183805) As described above, the selec- patients were given CP-358,774 tablets in a variety of tive and reversible inhibitors of the EGFR tyrosine dose schedules, culminating in daily dosing at the kinase appear to o€er the promise of therapeutic maximally tolerated dose. The target Cavg of 400 ± ecacy coupled to reasonable tolerability. It is 500 ng/ml was achievable at doses at and above important to note, however, that the therapeutic index 100 mg/day on a well-tolerated schedule (Cavg values of neither Iressa2 nor CP-358,774 has yet to be fully following continuous daily dosing at the 50, 100 and elaborated, and that there may be signi®cant proximity 200 mg/day levels were 432, 973 and 2120 ng/ml, between the maximally tolerated doses and the respectively). Dose-limiting diarrhea was encountered ecacious doses for both agents. Moreover, the at the 200 mg/day level. An intermediate dose of ecacy of neither agent has yet to be established in a 150 mg/day was subsequently de®ned as the maximally blinded, placebo controlled study. As such, there tolerated dose (two of three patients had Grade 1 continues to be an opportunity to discover and develop distinctly di€erent EGFR tyrosine kinase inhibitors Siu and co-workers also made e€orts to understand with even greater potential for ecacy and a broader the `characteristic' Grade 1 ± 2 acneiform rash seen in spectrum of activity. CI-1033 is one such distinctly patients treated with CP-358,774, which was limited to di€erent development candidate. As recently reviewed regions of the upper body where adolescent acne is by David Fry of the former Warner Lambert usually manifest (face, back and scalp). Histopathology organization, signaling through the erbB family of of skin biopsies showed subepidermal neutrophilic tyrosine kinase receptors often involves complex cross- in®ltration and epidermal hyperproliferation (Siu et talk among the members of that receptor family (Fry, al., 1999). While the precise cytopathic basis for the 2000). The four family members (EGFR or erbB; acneiform rash has not yet been determined, the erbB2, erbB3 and erbB4) are known to intensify their consistent clinical observations with three di€erent kinase-dependent transforming signals via the forma- agents targeting EGFR function (CP-358,774, Iressa2 tion of heterodimers with each other (Tzahar et al., and Imclone's C-225 antibody) suggest that this is a 1996). There is, therefore, a compelling rationale to mechanism-based ®nding (Siu et al., 1999; Ferry et al., consider the potential utility of nonspeci®c but selective 2000; Cohen et al., 2000b). Skin changes are consis- inhibitors that e€ectively block the function of the erbB tently noted in preclinical studies with rodents exposed family but do not inhibit more structurally diverse to CP-358,774 for extended dosing periods, and these toxicological results are analogous to the skin changes There is also a strong rationale to consider seen in the waved-2 mouse, which has a mutated and irreversible tyrosine kinase inhibitors. The reversible marginally functional EGFR tyrosine kinase (Luetteke inhibitors have apparently generated clinical ecacy with dosing regimens designed to maintain plasma Early ecacy readouts from ongoing Phase II concentrations at fairly high levels for extended periods clinical trials with CP-358,774 have been compelling.
of time. The optimal dosing paradigm for an The agent appears to have a broad potential to treat a irreversible inhibitor would be less likely to require variety of human solid tumors, including NSCLC, prolonged exposure. Moreover, the `absolute ®nality' breast, ovarian and squamous head and neck tumors (Fry, 2000) of the irreversible inhibitors could con- (Bonomi et al., 2000; Allen et al., 2000; Siu et al., 2000; ceivably provide signi®cant advantages in terms of Hammond et al., 2000). For example, in 34 NSCLC antitumor ecacy. To be balanced, a multi-tropic and patients who had failed prior chemotherapy, daily oral irreversible inhibitor would also have the potential to doses of 150 mg CP-358,774 were well-tolerated, with a generate a toxicity pro®le that was di€erent and, maculopapular (acneiform) rash being the most perhaps, without advantages relative to the more common adverse event reported. In 56 total patients selective, reversible inhibitors. Preclinical data suggest evaluable for tumor response, there have been six that irreversible EGFR tyrosine kinase inhibitors can partial responses in the lung and/or liver at 8 weeks generate signi®cant ecacy with good toleration and several patients with stable disease (Bonomi et al., (Vincent et al., 1999), but the ultimate utility of these 2000). In 71 patients with refractory squamous agents can only be determined in clinical trials.
carcinomas of the head and neck, CP-358,774 was Homology modeling of ATP binding to the pocket again found to cause a reversible acneiform rash and of EGFR suggested that the thiol of cys773 would be a Grade 1 ± 2 diarrhea. Of 78 patients evaluable for key potential site for attack by a rationally designed response, there have been at least eight con®rmed irreversible ATP-mimetic. One compound containing partial responses and 23 patients with stable disease an acrylamide functionality at the six position of the 4- (Siu et al., 2000). These preliminary results indicate anilinoquinazoline nucleus (Figure 1) was found to that CP-358,774 is generally well-tolerated and demon- have a profoundly rapid onset and long-lasting strates evidence of single agent anti-tumor activity in inhibition of both EGFR and erbB2 in tumor cells, patients with recurrent head and neck cancer, as well and to be selective relative to non-erbB tyrosine kinases (Fry et al., 1998). When compared to very closely Due to signi®cant interests in both CP-358,774 and related reversible analogs (in which the acrylamide CI-1033, P®zer was directed to divest one of these two double bond was reduced), the 6-substituted irrever- agents as a condition of their acquisition of Warner sible analogs were more potent in vitro and had Lambert in 2000. As such, Oncogene Science (OSIP) signi®cantly greater ecacy in vivo. Further improve- Tyrosine kinase inhibitors in cancer treatment trials ments (addition of substitutions which also improved of FGFR, which occurred at SU 5416 concentrations water-solubility) led to the elaboration of PD 183805/ some 100-fold higher, was found in kinetic experiments CI-1033 (Figure 1). Like its predecessors, this com- to be `mixed' competitive and non-competitive (Mendel pound has excellent (low nM) potency against erbB2 et al., 2000). It has been speculated that the latter result and EGFR in both enzyme- and cell-based assays is due to speci®c biopharmaceutical properties of the (Sherwood et al., 1999). Consistent with a predicted compound, which is both lipophilic and potentially advantage relative to reversible inhibitors, CI-1033 reactive in nature. Consistent with this concept are potently inhibits human tumor xenografts when dosed preliminary observations that the inhibition of VEGF- as infrequently as once per week, and a single dose dependent endothelial cell proliferation by SU 5416 has eliminated the level of EGFR phosphorylation in both a rapid onset and a pseudo-irreversible behavior tumors for longer than 72 h (Vincent et al., 1999).
which may be due to high intracellular levels of Like CP-358,774, CI-1033 combines well in drug compound (Mendel et al., 2000). Inhibition of combinations with cytotoxic agents. Given 24 h after endothelial cell proliferation translated to anti-tumor gemcitabine, CI-1033 produced a signi®cant increase in ecacy in a number of human xenograft and rodent the apoptotic fraction in tumors over treatment with tumor models (Fong et al., 1999). In these studies, no either drug alone (Nelson and Fry, 2000). CI-1033 also data were generated to relate drug exposure (said to be e€ectively decreased the clonogenicity of human tumor very short-lived in rodents), or biochemical inhibition cells taken from patients (Medina et al., 2000), with of VEGFR or PDGFR, to anti-tumor ecacy.
notable responses seen in breast (67%), NSCLC (60%) Interestingly, the ecacy of SU 5416 was found to be and ovarian cancer specimens. CI-1033 Phase I clinical greater in slower-growing vs faster growing solid tumor trials have recently been initiated, but data on xenografts, which led Fong et al. (1999) to speculate pharmacokinetics or safety have not yet been disclosed.
that SU 5416 might bind preferentially to resting vs activated tyrosine kinases on endothelial cells. This Small molecule tyrosine kinase inhibitors targeting would be at odds with other data suggesting that quinazolines bind more avidly to activated kinases (Levitzki and Bohmer, 1998) but, if true, may bode There are multiple tyrosine kinase receptors which well for human ecacy in a majority of clinical appear to have key roles in the generation of new tumor blood vessels and, as such, represent reasonable Phase I studies were carried out in 69 advanced targets for cancer chemotherapy (for excellent recent disease patients, with SU 5416 dosed i.v. twice weekly.
reviews, see Cherrington et al., 2000; Randal, 2000; Patients were treated at 13 dose levels between 4.4 ± Thompson et al., 1999; Hamby and Showalter, 1999).
190 mg/m2/day; at the highest dose, a dose limiting Included among the key tyrosine kinase targets that toxicity (projectile vomiting) was observed (Rosen et have generated the most interest in the scienti®c and al., 1999). Induction of metabolism was noted in all patent (Connell, 2000) literature are PDGFR, VEGFR, patients, either due to the parent drug, a metabolite or FGFR and tie-2. The key development candidates dexamethasone premedication, and the elimination targeting PDGFR, STI 571 and SU101, were described half-life was found to be 55 min (Cropp et al., 1999).
above, though neither compound is likely to reveal the Early signs of ecacy were also apparent, with clinical utility of PDGFR-directed inhibition of objective responses seen in three patients (Kaposi's angiogenesis due to their multiple mechanisms of sarcoma, metastatic basal cell and colorectal cancer); action. Agents that selectively target FGFR and tie-2 seven patients remained on study for more than 6 are not known to be in development, though several months, while two remained on study for greater than drugs targeting VEGFR have inhibitory activity vs 18 months (Rosen et al., 1999; Mendel et al., 2000).
FGFR. As such, the focus of the remainder of this Given these results, SU 5416 has been advanced into overview will be on the clinical candidates targeting multiple Phase II and III at an initial recommended VEGFR. Two high anity receptors for VEGF have dose of 145 mg/m2, which is sucient to produce been identi®ed and characterized on human endothelial systemic exposure comparable to what was required to cells, ¯t-1 and KDR. KDR appears to be expressed yield e€ective tumor growth inhibition in animals primarily on activated endothelial cells and is thought (Cropp et al., 1999). This dose is also within 30% of to be more of a key driver of mitogenic responses the human maximally tolerated dose (190 mg/m2). The commonly found in neovascularizing tumors, while ¯t- ongoing development plan includes large studies in 1 is expressed on multiple other cell types (Plate et al., NSCLC and colorectal cancer to assess the ecacy of 1994; Wedge et al., 2000a). For the purposes of this SU 5416 both as a single agent and in combination review, the terms KDR and VEGFR will be used with standard chemotherapies (Mendel et al., 2000).
interchangeably, unless otherwise speci®ed.
A related agent in development, SU 6668 (Figure 1), combines a less selective inhibitory pro®le (inhibition SU 5416 and SU 6668 The former SUGEN organiza- of FGFR in addition to PDGFR and VEGFR) with a tion (now part of Pharmacia) has clearly set the early more favorable biopharmaceutical pro®le (Laird et al., pace in the race to identify and develop inhibitors of 2000). SU 6668 has a signi®cantly lower Ki for PDGFR the VEGFR tyrosine kinase. E€orts towards this end relative to VEGFR or FGFR (8 nM vs 2.1 and 1.2 mM, have initially focused on the indolin-2-one pharmaco- respectively), a result which appeared consistent with phore (Figure 1). Among the earliest compounds of homology models of the respective active sites, but this class was SU 5416, which was found to be a potent inconsistent with the cellular e€ects of SU 6668 inhibitor of the kinase activities of both VEGFR and (VEGFR-stimulated mitogenesis of endothelial cells PDGFR. Inhibition of these two tyrosine kinases was much more potently inhibited relative to either found to be competitive with ATP, but the inhibition PDGFR or FGFR) (Laird et al., 2000). Like Tyrosine kinase inhibitors in cancer treatment trials SU 5416, SU 6668 was found to be potent and halted and marked regressions could again be induced ecacious in a variety of tumor models. Unlike in these tumors upon re-treatment. While the pre- SU 5416, which was dosed i.p. in a DMSO-based clinical data for both compounds appear to be very vehicle, ecacy was achievable with SU 6668 when promising, Phase I results for neither ZD4190 nor dosed orally each day in a cremaphore-based vehicle.
In Phase I studies, SU 6668 was administered orally once daily to 16 patients with advanced malignancies, PTK 787 Novartis is reported to be developing PTK at dose levels between 100 ± 1600 mg/m2/day (Rosen et 787, which has an anilinophthalazine pharmacophore al., 2000). Nine of 16 patients remained on study for (Bold et al., 2000) related to but distinct from the up to 28 weeks while the remaining seven patients had quinazolines described above (Figure 1). The com- progressive disease. Dose limiting toxicities were not pound is a potent inhibitor of both major human observed, and dose escalation was said to be ongoing.
VEGFR (IC50 values of 37 and 77 nM for KDR and Two patients at 1600 mg/m2 developed liver function ¯t-1, respectively) and, like STI 571, it provides potent abnormalities, but both had potentially confounding (sub-micromolar) inhibition of PDGFR and c-kit but liver disease. Other possible drug related toxicities does not inhibit v-abl, EGFR or FGFR (Wood et al., included nausea, headache, fatigue and changes in 2000). PTK 787 inhibits VEGF-induced KDR auto- bowel movements. Pharmacokinetic data suggested phosphorylation and mitogenesis, and promotes en- that SU 6668 had a moderate-high clearance (78 l/ dothelial cell apoptosis, at a similar concentration day/m2) and a somewhat improved elimination half-life (Wood et al., 2000). The compound also has good of 2.5 h relative to SU 5416 (Rosen et al., 2000). Phase biopharmaceutical properties (plasma concentrations II studies in multiple tumor types have apparently been 41 mM 8 h after administration of a 50 mg/kg oral dose to mice), and impressive antiangiogenic (ED50512.5 mg/kg/day for inhibition of angiogenesis ZD4190 and ZD6474 ZD4190 is a quinazoline-based in a s.c. growth factor implant model) and anti-tumor VEGFR inhibitor (Figure 1) said to have entered activity (signi®cant growth inhibition in six di€erent Phase I in early 2000. ZD6474 is thought to be from human tumor xenograft models at daily oral doses of the same structural class, but AstraZeneca has not yet 25 ± 75 mg/kg) (Wood et al., 2000). A key issue in the disclosed the speci®c structure. ZD4190 inhibits both ®eld of anti-angiogenesis research has long been the KDR and ¯t-1 (IC50 values of 29 and 708 nM, fear that inhibition of tumor angiogenesis would also respectively), and much less potent at inhibiting FGFR impair normal angiogenesis, such as that in wound (approximately 200-fold relative to KDR). The healing. Given that most solid tumors are managed compound is also 30-fold more potent at inhibiting using multi-modality treatments that include surgery, VEGF-mediated endothelial cell growth relative to this has been a theoretical limitation to inhibitors of FGF-stimulated cell growth (IC50 values of 50 and angiogenesis. Interestingly, PTK 787 appears to have 1530 nM, respectively) (Wedge et al., 2000a). In vivo, much less ecacy as an inhibitor of physiological the compound was found to inhibit capillary invasion angiogenesis of wound healing than as an e€ective of cartilage (increased epiphyseal growth plate area), blocker of tumor angiogenesis. Daily dosing of rats up and to inhibit the growth of four human tumor to 50 mg/kg day did not impair the healing or decrease xenografts in a dose-dependent manner with daily oral the tensile strength of full-thickness incisional wounds administration (Wedge et al., 2000a). Direct measure- (Wood et al., 2000). Data on the antitumor activity of ments of tumor vascular endothelial permeability, PTK 787 were recently extended to a renal tumor using contrast medium-enhanced MRI indicated that implant model, which was used to show that the acute ZD4190 treatment produced measurable changes compound could also inhibit both primary tumor in vascular permeability at doses which yielded anti- growth and the emergence of tumor metastasis to the tumor activity during chronic administration (Wedge et lung. Using a non-invasive (color Doppler imaging) al., 1999). ZD6474, the second putative development surrogate endpoint, a commensurate decrease in renal candidate, is unique among small molecule angiogen- artery blood ¯ow could also be observed after chronic esis inhibitors, in that it is found to induce signi®cant treatment (Drevs et al., 2000). Thus, PTK 787 appears regressions in PC-3 tumors of varying size, with to show signi®cant preclinical ecacy, and to produce greatest e€ects being produced in the largest tumors potent anti-tumor e€ects under well-tolerated dosing (Wedge et al., 2000b). An intermittent ZD6474 regimens. The preclinical toxicological pro®le and the treatment schedule, involving withdrawal of compound human pharmacokinetics of this compound have not for 4 weeks, revealed that tumor re-growth could be Table 1 Selected small molecule tyrosine kinase inhibitors in clinical development for cancer Phase II/III in multiple tumors (discontinued?) Tyrosine kinase inhibitors in cancer treatment trials directed. This may perhaps be a non-issue for VEGFR and PDGFR inhibitors, which target transiently, It is clear that the development of newer agents like the focally activated receptors on normal cells. However, tyrosine kinase inhibitors will require new concepts and it is clear that there may be di€erent approaches in clinical paradigms that are distinctly di€erent from dealing with this issue during the development of those used to develop the well-known cytotoxic agents EGFR inhibitors. AstraZeneca has apparently not commonly used in cancer chemotherapy. Some recent incorporated prospective measurements of EGFR commentaries have done an outstanding job at framing over-expression in tumors from patients with NSCLC these development issues (Sausville, 2000; Workman, as an inclusion criteria in their Phase III trials with 2000; Hudes, 1999; Eisenhauer, 1998).
Iressa2. As mentioned previously, one can surmise that Paramount among these is the need for non- the rationale for this strategy was based on both the conventional endpoints in clinical trial design, and for high proportion of NSCLC tumors that over-express the identi®cation, validation and implementation of EGFR, and by preclinical data showing that over- surrogate endpoints which may help direct dose- expression is not predictive of a drug combination anti- modulation during therapy. From the examples provided above, it is clear that for several agents P®zer and Oncogene Science have executed at least (Iressa, CP-358,774; SU 5416), the presumed ecacious one Phase II study with CP-358,774 in head and neck dose in humans is very close to the maximally tolerated cancer patients where EGFR expression levels were dose. None of these agents has yet been in a clinical evaluated as an entry criterion (Siu et al., 2000). The trial designed to probe a broader aspect of ecacious basis of this strategy could be said to reside in the dose range. Given the somewhat poor performance of Genentech development experience with Herceptin2, in preclinical ecacy models in predicting e€ective plasma which all patients entered into clinical trial, and concentrations in humans, organizations developing subsequently all patients receiving the approved these new agents often resort to targeting some commercial product, are ®rst pre-screened to detect multiple of the plasma concentration required to the level of over-expression of erbB2 in their breast generate ecacy in animal models, without ®rst gaining tumors. It is interesting to note that retrospective an understanding as to whether clinical ecacy is dose- analyses attempting to relate the level of erbB2 responsive, or that most patients are not being dosed at expression to clinical response in patients treated with a level well-along on the plateau of the dose-response Herceptin2 (Dowsett et al., 2000) have been incon- curve. Non-invasive approaches (Doppler and contrast sistent and unconvincing. It is somewhat troubling to agent imaging for VEGFR inhibitors) and invasive see that so straight-forward an assay (immunohisto- approaches (tumor and tissue sampling pre- and post- chemistry) applied on a post hoc basis to understand an treatment for EGFR inhibitors) are being developed to agent with so singular a mechanism of action aid in the assessment of minimally and maximally (Herceptin2) has led to so little insight.
e€ective doses during the ®rst days and weeks of One can perhaps begin to guage the obstacles that clinical trial. The development of these surrogate may lie ahead for the development of surrogate endpoints is occurring on a parallel path with the endpoints, which encompasses the application of novel agents themselves, probably too late to help de®ne the technologies applied in a prospective way to drugs with dose-response, the minimally- and maximally-e€ective complex mechanisms of action and pharmacological dose, or the most ecient development paradigm.
e€ects. This is the next frontier for the development of A second key issue is one of the biochemical these new therapies for cancer. As such, the next 5 ± 6 selectivity of tyrosine kinase inhibitors, and the impact years are likely to be as challenging, and as that it may have on both the ecacy and the safety of exhilarating, as have been the past 5 ± 6 years.
the clinical candidate. The current experience with both non-selective tyrosine kinase inhibitors (STI 571 and SU 5416) and selective compounds (Iressa2 and CP- 358,774) suggest that ecacy can be generated with ATP, adenosine triphosphate; EGF/EGFR, epidermal either class of inhibitor, with modest, comparable safety growth factor/EGF receptor; PDGF/PDGFR, platelet- margins. There could be an opportunity to assess the derived growth factor/PDGF receptor; CML, chronic relative merits of EGFR-selective vs. pan erbB inhibitors when comparing the results of the trials with Iressa2, CP-358,774 and CI-1033, but the irreversibility of the latter candidate is likely to confound the I would like to acknowledge the outstanding contributions comparisons of relative therapeutic index. A related of each member of the P®zer Global R&D division and the issue is one of pre-screening patients for the over- P®zer Oncology team for their insights, support, commit- expression of the target at which the tyrosine kinase is Allen LF, Cerna C, Gomez L, Yochmowitz M, Medina ML Bartlett RR and Schleyerbach R. (1985). Intl. J. Immuno- and Weitman S. (2000). Proc. NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy, 384.
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