《药物化学》课程文献资料（Medicinal Chemistry）GLIVEC（STI571, IMATINIB）,A RATIONALLY DEVELOPED,TARGETED ANTICANCER DRUG，Nat Rev Drug Disc 2002

REVIEWS GLIVEC (STI571,IMATINIB). A RATIONALLY DEVELOPED TARGETED ANTICANCER DRUG Renaud Capdeville,Elisabeth Buchdunger,Juerg Zimmermann and Alex Matter n the early 1980s.it became apparent that the work of pioneers such as Robert Weinbera Mariano Barbacid and many others in identifying cancer-causing genes in humans was opening the door to a new era in anticancer research.Motivated by this,and by dissatisfaction with the limited efficacy and tolerability of available anticancer modaities,adrug discovery programme was initiated with the aim of rationally developing targeted anticancer therapies.Here,we describe how this programme led to the discovery and continuing development of Givec Gleevec in the United States),the first selective tyrosine-kinase inhibitor to be approved for treatment of a cancer. The molecular conse ence of this inter-c ts such a price of high or regre n of t di The discoveryo rucial fo ch. For th mpls,butasdcsaibcdbeioci da) e that thi d by red in cal a :BCR-ABL d from uman leu Theshortene ta for n I stud击i d with th do:10-103m3 specific genetic change URE REVIEWS OLUME I IULY 2002 2002 Nature Publishing Group

© 2002 Nature Publishing Group REVIEWS Until the early 1980s, drug discovery programmes for cancer were focused almost exclusively on DNA synthesis and cell division, and resulted in agents such as antimetabolites, alkylating agents and microtubule destabilizers. These drugs showed efficacy, but at the price of high toxicity due to lack of selectivity. Also, resistance was frequently observed after initial stabiliza￾tion or regression of the disease. The discovery of cancer-causing genes, later called oncogenes, repre￾sented a radical departure — all of a sudden, genes were identified that were uniquely associated with cancerous cells. The molecular epidemiology of these genes was established over many years of studying clinical tumour samples, but as described below, it was clear at the out￾set that chronic myelogenous LEUKAEMIA (CML) — a haematological stem-cell disorder that is characterized by excessive proliferation of cells of the myeloid lineage — represented a particularly interesting case. Target selection: BCR–ABL CML is characterized by a reciprocal translocation between chromosomes 9 and 22 (REF. 1). The shortened version of chromosome 22, which is known as the Philadelphia chromosome, was discovered by Nowell and Hungerford2 , and provided the first evidence of a specific genetic change associated with human cancer. The molecular consequence of this inter-chromosomal exchange is the creation of the BCR–ABL gene, which encodes a protein with elevated tyrosine-kinase activity. The demonstration that Bcr–Abl as the sole oncogenic event could induce leukaemias in mice3–5 has estab￾lished BCR–ABL as the molecular pathogenic event in CML. As the tyrosine-kinase activity of BCR–ABL is crucial for its transforming activity 6 , the enzymatic activity of this deregulated gene could plausibly be defined as an attractive drug target for addressing BCR–ABL-positive leukaemias. For the first time, a drug target was identified that clearly differed in its activity between normal and leukaemic cells. It was conceivable that this enzyme could be approached with classical tools of pharma￾cology, as its activity — the transfer of phosphate from ATP to tyrosine residues of protein substrates — could clearly be described and measured in biochemical as well as cellular assays. Furthermore, cell lines that were derived from human leukaemic cells with the same chromosomal abnormality were available. Such cell lines were instrumental for in vitro and animal studies, which laid the groundwork for the clinical trials. So, the essential tools were assembled to go forward with the aim of identifying potent and selective inhibitors of the ABL tyrosine kinase. GLIVEC (STI571, IMATINIB), A RATIONALLY DEVELOPED, TARGETED ANTICANCER DRUG Renaud Capdeville, Elisabeth Buchdunger, Juerg Zimmermann and Alex Matter In the early 1980s, it became apparent that the work of pioneers such as Robert Weinberg, Mariano Barbacid and many others in identifying cancer-causing genes in humans was opening the door to a new era in anticancer research. Motivated by this, and by dissatisfaction with the limited efficacy and tolerability of available anticancer modalities, a drug discovery programme was initiated with the aim of rationally developing targeted anticancer therapies. Here, we describe how this programme led to the discovery and continuing development of Glivec (Gleevec in the United States), the first selective tyrosine-kinase inhibitor to be approved for the treatment of a cancer. NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | JULY 2002 | 493 Novartis Oncology, Novartis Pharma AG, S-27 2.033, CH-4002 Basel, Switzerland. Correspondence to R.C. e-mail: renaud.capdeville@ pharma.novartis.com doi:10.1038/nrd839 LEUKAEMIA Leukaemia is an uncontrolled proliferation of one type of white blood cell (leukocyte)

REVIEWS 6 N 6 yboth solubility and or thi c),wh ising cand te for dinical development H fo of the to the yt'(p hed theu tubiltasontcrasopad BL by (His)3 nel of P四 kin the c to inb the ompound w that mak roportion of Pha lism y activity of G ec was sh mila inhibitory ac fodrug-likenes T lead com oliferative activityo nd- rop a high potential for diversity,allowing aple chem leukacmia cells from with treat ed o the BCR-ABLin his point d with e'flag-methyl'led to ific for B 093 prepared originally showed poor oral bioavailability mouse model of CMLbased on retroviral p21 494 JULY 2002 VOLUME 1 2002 Nature Publishing Group

© 2002 Nature Publishing Group APOPTOSIS Programmed cell death. SYNGENEIC MODEL An animal model in which the injected tumour cells are derived from the same animal species as the host animal. 494 | JULY 2002 | VOLUME 1 www.nature.com/reviews/drugdisc REVIEWS and low solubility in water. The attachment of a highly polar side chain (an N-methylpiperazine) was found to improve markedly both solubility and oral bioavailability. To avoid the mutagenic potential of aniline moieties, a spacer was introduced between the phenyl ring and the nitrogen atom. The best com￾pound from this series was a methylpiperazine deriva￾tive that was originally named STI571 (imatinib, now known as Glivec or Gleevec), which was selected as the most promising candidate for clinical development9,10 (FIG. 1d). Docking studies11 and X-ray crystallography12 showed that binding of Glivec occurs at the ATP-binding site. Analysis of the crystal structure12 showed that Glivec inhibits the ABL kinase by binding with high specificity to an inactive form of the kinase. The need for the kinase to adopt this unusual conformation, which favours binding, might contribute to the high selectivity of the compound. Unexpectedly, these analy￾ses indicated that the N-methylpiperazine group (added to increase drug solubility) also interacted strongly with ABL by means of hydrogen bonds to the backbone carbonyl group of isoleucine (Ile)360 and histidine (His)361. In an in vitro screen against a panel of protein kinases, the compound was found to inhibit the autophosphorylation of essentially three kinases: BCR–ABL, c-KIT and the platelet-derived growth factor (PDGF) receptor (TABLE 1). More recently, activity against ARG kinase has also been reported13. Pharmacological profile In collaboration with Brian Druker, the selective inhibitory activity of Glivec was shown at the cellular level on the constitutively active p210BCR–ABL tyrosine kinase14. Subsequently, a similar inhibitory activity was also shown on other ABL fusion proteins, such as p185BCR–ABL (REFS 15,16) and TEL (ETV6)–ABL15. The inhibition of autophosphorylation of BCR–ABL was closely related to the antiproliferative activity of Glivec. Incubation with submicromolar concentra￾tions of Glivec selectively induced APOPTOSIS in BCR–ABL-positive cell lines, and induced cell killing in primary leukaemia cells from patients with Philadelphia-chromosome-positive (Ph+) CML and acute lymphoblastic leukaemia14,16–20. In in vivo experiments, once daily intraperitoneal treatment with 2.5–50 mg kg–1 of Glivec, started one week after injecting BCR–ABL-transformed 32D cells into SYNGENEIC mice, caused dose-dependent inhibition of tumour growth14. In nude mice implanted with KU812 cells, oral treatment with 160 mg kg–1 daily in three divided doses for 11 consecutive days was associ￾ated with continuous blockage of p210BCR–ABL tyrosine phosphorylation, and resulted in tumour-free survival of the animals20. The antitumour effect of Glivec was specific for BCR–ABL-expressing cells, as no growth inhibition occurred in mice that were given injections of U937, a BCR–ABL-negative myeloid cell line. Recently, Glivec was shown to be orally active in a mouse model of CML, based on retroviral p210BCR–ABL Medicinal chemistry The starting point for every medicinal-chemistry pro￾ject is a lead compound with a given pharmacological activity. However, the biological activity of a molecule must be complemented by other properties that make the molecule a good drug — it is estimated that a large proportion of molecules fails in late stages of drug development due to drug–drug interactions or poor ADME (absorption, distribution, metabolism and excretion) features. Not detecting these liabilities early in the drug discovery process can be extremely costly and time consuming. On the basis of physical and calculated properties for known drugs, criteria for ‘drug-likeness’ have been established7 . In the case of Glivec, a lead compound was identi￾fied in a screen for inhibitors of protein kinase C (PKC). This compound — a phenylaminopyrimidine derivative — had promising ‘lead-like’ properties8 and a high potential for diversity, allowing simple chem￾istry to be applied to produce compounds with more potent activity or selectivity. Strong PKC inhibition in cells was obtained with derivatives bearing a 3′-pyridyl group at the 3′-position of the pyrimidine (FIG. 1a). During the optimization of this structural class, it was observed that the presence of an amide group on the phenyl ring provided inhibitory activity against tyro￾sine kinases, such as the BCR–ABL kinase (FIG. 1b). At this point, a key observation from analysis of struc￾ture–activity relationships was that a substitution at position 6 of the diaminophenyl ring abolished PKC inhibitory activity completely. Indeed, although the introduction of a simple ‘flag-methyl’ led to loss of activity against PKC, the activity against protein tyro￾sine kinases was retained or even enhanced (FIG. 1c). However, the first series of selective inhibitors that was prepared originally showed poor oral bioavailability N N N N H N N N N N R1 H H 6 O N N N N N R1 H H O H3C N N N N H H N O N N a b c d Figure 1 | Summary of the chemical optimization. The core structure of the lead compound, a phenylamino derivative, is indicated in black. a | The addition of a 3′-pyridyl group (blue) at the 3′-position of the pyrimidine enhanced the cellular activity. b | An amide group (red) attached to the phenyl ring provided activity against tyrosine kinases. c | A ‘flag methyl’ (purple) attached to the diaminophenyl ring abolished the undesirable protein-kinase-C inhibitory activity. d | The final attachment of an N-methyl piperazine moiety (green) markedly increased the solubility and oral bioavailability

REVIEWS ransduction of transplanted bone marrow.Survival of Fundamental phenotypic features in BCR-ABL ty pro link Table 1|Cellular nconstitutieaciationofS7 A长 IC(M) V-AB 0.1-0.3 STAT5 activa 210 月A g of the TEL-ARG 0.5 ttarget of BCR- DGF recepto DGF recepte arker ofBCR-ABL kina d. C-KT FLT3 10 id ha c-FMS and w-ims >10 2100 ≥100 IGF-1epo understood.Recently.BCR-ABL has ent kin lation o Inhibition of MAPK activation ented downre PDGF dependen 0.1- Gliv n of AKT activation 0.1-4 to re 0.26 nof-FOS mRNA expression 0.3-1 >100 dcnan lates for cli▣ al testing o BCR-ABL-transtected 32D.MO-7e.BaF3 cels genes anc BCR-ABL-postive human loukaemia linesl 0.1- BaF3 TEL-PDGF receptor ontrast t U-87 human glioma -15 -343om CML was H526 human SCLC,SCF stimulated 0.8 e that evolve Human GIST882 line <1 om p. Human mast-cel leuka 0.01-0 f bl sis th atients with diagno is approxin 1, RE REVIEWS DRUG DISCOVER OLUME 200 2002 Nature Publishing Group

© 2002 Nature Publishing Group NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | JULY 2002 | 495 REVIEWS Fundamental phenotypic features in BCR–ABL￾positive cells involve resistance to apoptosis, enhanced proliferation and altered adhesion properties. The impact of Glivec on some known downstream signalling molecules of BCR–ABL has been examined. A link between constitutive activation of STAT5 (signal trans￾ducer and activator of transcription 5) and enhanced viability of BCR–ABL-transformed cells has been shown22,23. Glivec had a profound inhibitory effect on STAT5 activation in vitro and in vivo21–23. Furthermore, inhibition of the BCR–ABL kinase activity by Glivec in BCR–ABL-expressing cell lines and fresh leukaemic cells from CML patients induced apoptosis by supp￾ressing the capacity of STAT5 to activate the expression of the anti-apoptotic protein BCL-XL 23. The adaptor molecule CRKL is a prominent target of BCR–ABL, and its tyrosine phosphorylation has been a useful marker of BCR–ABL kinase activity24. As expected, a decrease in tyrosine phosphorylation of CRKL has been observed in Glivec-treated cell lines, and has also served as an indicator of BCR–ABL kinase activity in patients (see below). There is increasing evidence that cell-cycle regula￾tion is disturbed in BCR–ABL-positive cells; however, the underlying molecular mechanisms are poorly understood. Recently, BCR–ABL has been shown to promote cell-cycle progression and activate cyclin￾dependent kinases by interfering with the regulation of the cell-cycle inhibitory protein p27 (REF. 25). Glivec pre￾vented downregulation of p27 levels in BCR–ABL￾expressing cells25,26. The effects of Glivec on cytoskeletal changes and adhesion have been investigated using BCR–ABL￾transfected fibroblasts27. Glivec was shown to restore normal architecture and to increase adhesion in this model of BCR–ABL expression. Clinical development in CML Because of the three known targets of Glivec, many potential cancers can be speculated to be good candi￾dates for clinical testing of this new drug. However, in most cancers, tumorigenesis is complex and involves the disruption of multiple genes and signalling pathways. By contrast, CML can be considered as one of the few examples of a malignancy in which a single signalling￾pathway defect is thought to cause the disease. In addi￾tion, in contrast to most of the solid tumours, for which the measurement of tumour response is complex, phar￾macodynamic response in CML can be measured easily using blood leukocyte count as the end point. For these reasons, CML was selected as the first indication for Phase I clinical testing. Clinically, CML is a chronic disease that evolves through three successive stages, from the chronic phase to the end stage of blast crisis that resembles acute leukaemia (FIG. 2). Overall, the median survival time of patients with newly diagnosed CML is approximately 5–6 years with an interferon-based treatment regimen. The first trial with Glivec was a Phase I study in patients with chronic-phase, and subsequently also with blast￾phase, CML. In this trial, patients were treated with doses transduction of transplanted bone marrow. Survival of animals was significantly prolonged, together with a marked improvement in peripheral-white-blood-cell counts and splenomegaly21. Table 1 | Cellular profile of Glivec Assay IC50 (µM) Inhibition of autophosphorylation v-ABL 0.1–0.3 p210BCR–ABL 0.25 p185BCR–ABL 0.25 TEL–ABL 0.35 TEL–ARG 0.5 PDGF receptor 0.1 TEL–PDGF receptor 0.15 c-KIT 0.1 FLT3 > 10 c-FMS and v-fms > 10 EGF receptor > 100 c-ERBB2 > 100 Insulin receptor > 100 IGF-1 receptor > 100 v-SRC > 10 JAK2 > 100 Inhibition of MAPK activation PDGF dependent 0.1–1 SCF dependent 0.1–1 Inhibition of AKT activation SCF dependent 0.1–1 Inhibition of IP release PDGF induced 0.25 Inhibition of c-FOS mRNA expression PDGF induced 0.3 –1 EGF, FGF or PMA induced > 100 Antiproliferative activity* 32D, MO-7e, BaF3 cells > 10 BCR–ABL-transfected 32D, MO-7e, BaF3 cells < 1 BCR–ABL-positive human leukaemia lines|| 0.1–1 BaF3 TEL-ARG 0.5 BALB/c 3T3 v-SIS (PDGF autocrine) 0.3 BaF3 TEL–PDGF receptor < 1 U-87 human glioma‡ ~1.5 U-343 human glioma‡ ~1.5 MO-7e, SCF stimulated ~0.1 H526 human SCLC, SCF stimulated§ 0.8 Human GIST882 line¶ < 1 Human mast-cell leukaemia line HMC-1# 0.01–0.1 Glivec concentrations that cause 50% inhibition (IC50) are given13–20,47,48,53,54,61,63,66. EGF, epidermal growth factor; FGF, fibroblast growth factor; FLT3, fms-related tyrosine kinase 3; IGF-1, insulin-like growth factor-1; IP, inositol phosphate; MAPK, mitogen-activated protein kinase; PDGF, platelet￾derived growth factor; PMA, phorbol 12-myristate 13-acetate; SCF, stem-cell factor; SCLC, small-cell lung cancer. *Antiproliferative experiments were carried out in 10% fetal calf serum, except for those that were carried out in ‡ 5% human-platelet poor plasma or under § serum-free conditions. ||K562, KU812, MC-3, MBA-1, KBM-5, Z-33, Z-119, Z-181. ¶Expresses the activating KIT mutation K642E (lysine 642 to glutamic acid). # Expresses the activating KIT mutation V560G (valine 560 to glycine)

REVIEWS Chronie phas Advanced pha igure 2|Clinical course of chronic myel nous let anging from 25 to1,000mg per day,and noma multinational studies hav of the ,and tre the rate of b of then respon sociated In addition,eff c chronic- hBCR-ABI diagnos 60 anging from 4001 nost fr ntly reported advers events were mile bsequent studie However,rare but 1000 100 o88 0 80 200 400 600 10d Chronic petients -Upper lmit of nommel range of WBCs nship of Glivec in CML (Phase I studyl.Using the keuko he me the dos atat de 400 40JULY2002 VOLUM正1 ature.com/reviews/drugdis 2002 Nature Publishing Group

© 2002 Nature Publishing Group 496 | JULY 2002 | VOLUME 1 www.nature.com/reviews/drugdisc REVIEWS Subsequently, three large multinational studies have been carried out in 532 patients with late chronic-phase CML in whom previous interferon therapy had failed31, in 235 patients with accelerated-phase CML32, and in 260 patients with myeloid blast crisis33. Treatment was given at a dose of 400 mg in the chronic-phase trial and 600 mg in the two other studies. The results of these three studies indicated that the rate of both haemato￾logical and cytogenetic response increased as the treat￾ment was started earlier in the course of the disease (FIG. 4). Importantly, the achievement of a haematolog￾ical and/or cytogenetic response was associated with improved survival and progression-free survival31–33. In the chronic-phase study, in which patients started treatment within a median of 32 months after their initial diagnosis, the estimated probability of being free of progression at 18 months was 89.2%31. The most frequently reported adverse events were mild nausea, vomiting, oedema and muscle cramps. However, rare but serious adverse events, such as liver ranging from 25 to 1,000 mg per day, and no maximal tolerated dose was identified, despite a trend for a higher frequency of GRADE III–IV ADVERSE EVENTS at doses of 750 mg or higher. On the other hand, a clear dose–response relationship with respect to efficacy was described in patients with chronic-phase CML.At doses of 300 mg or higher, 98% of the patients achieved a complete haematological response, and trough serum levels were above the concentrations required for in vitro activity28,29. Subsequently, a mathematical modelling of the relation￾ship between dose and response, as measured by leuko￾cyte counts after four weeks of therapy, confirmed that doses of 400 mg and higher were optimal in inducing a haematological response30 (FIG. 3). In addition, effective inhibition of the BCR–ABL kinase was documented in patient samples by inhibition of the phosphorylation sta￾tus of the downstream target CRKL27. From this study, doses ranging from 400 mg (for chronic-phase patients) to 600 mg (for advanced-phase CML) were recom￾mended for subsequent studies. GRADE III–IV ADVERSE EVENTS For each adverse event that is associated with a specific treatment, grades are assigned and defined using a scale from 0 to V. Grade III, severe and undesirable adverse event; grade IV, life-threatening or disabling adverse event. 1,000 0 200 400 600 Dose (mg) Model-fitted line WBC counts (× 109 –l 1) 800 1,000 100 10 1 Chronic patients Upper limit of normal range of WBCs Figure 3 | Dose–response relationship of Glivec in CML (Phase I study). Using the leukocyte (white blood cell; WBC) count after 28 days of treatment as a pharmacodynamic marker, the relationship between dose and response was modelled using an Emax model , which makes the assumption that once the maximal effect is achieved (Emax), increasing the dose further does not translate into additional benefit. The data indicate that at doses of 400 mg per day or higher, all the patients are predicted to achieve a reduction of their leukocyte counts within normal range below 10 x 109 l –1 . Adapted with permission from REF. 30 © (2001) American Society of Clinical Oncology. CML, chronic myelogenous leukaemia. Chronic phase Median 4–6 years stabilization Accelerated phase Advanced phases Median duration up to 1 year Blastic phase (blast crisis) Median survival 3–6 months Figure 2 | Clinical course of chronic myelogenous leukaemia

REVIEWS the drug itse thefirst Phase I study). ps虹 小吧 nding of the 52 been descrbed,which are summ A erated phese n=181)Blast crisis n be bc R-ABL but alternative sig 语nh d natient c5 20成0。 rence mplit sI.gene an An BT 15 51( nine ion well as further mutat in t gato these m vary in bserved by ATP.The know ge of the crystal structur ructure vity of mut ABL to Gliv nacti ionioopwhiaht ins the highly sistance to gliv might also be related to phar the ATP-bindingite This been NATURE REVIEWS OLUME 2002 Nature Publishing Group

© 2002 Nature Publishing Group NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | JULY 2002 | 497 REVIEWS observed by Kuriyan and co-workers12 in a complex between mouse c-Abl and a Glivec analogue, and can￾not bind ATP. The knowledge of the crystal structure allows a better understanding of the decreased sensi￾tivity of mutated BCR–ABL to Glivec, and can be a powerful tool in the design of new BCR–ABL inhibitors that maintain inhibitory activity against these mutated kinases. Resistance to Glivec might also be related to phar￾macokinetic factors. Glivec is a substrate of the multi￾drug-resistance-associated P-glycoprotein (PgP). toxicity or fluid-retention syndromes, were also reported. Neuropaenias and thrombopaenias were more common in patients with advanced disease, which indicates that haematological toxicity might be related more to an underlying compromised bone￾marrow reserve than to toxicity of the drug itself through inhibition of c-KIT-driven haematopoiesis. Taken together, these findings have established Glivec as a safe and effective therapy in all stages of CML, and were the basis for marketing approval by the FDA on 10 May 2001 — less than three years after the start of the first Phase I study (FIG. 5). Resistance. In CML blast crisis, even though the rate of haematological responses with Glivec is high, these responses are usually short lived, and most patients will ultimately develop resistance and undergo disease pro￾gression. A prerequisite to optimally develop strategies to prevent or overcome this resistance is to get a good understanding of the potential mechanisms of resis￾tance in these patients. On the basis of preclinical and clinical data that are available at present, several potential mechanisms of resistance have been described, which are summarized in BOX 1. They can be categorized into two main groups: mechanisms whereby BCR–ABL is reactivated and cell proliferation remains dependent on BCR–ABL sig￾nalling, and mechanisms whereby the BCR–ABL protein remains inhibited by Glivec, but alternative signalling pathways become activated. BCR–ABL overexpression and BCR–ABL gene ampli￾fication has been shown in p210BCR–ABL-transformed mouse haematopoietic Ba/F3 cells that are resistant to Glivec34,35, as well as in human BCR–ABL-positive leukaemia lines LAMA84 and AR230 (REFS 35,36). In treated patients, there is now increasing evidence that amplification of the BCR–ABL gene and mutations in the BCR–ABL kinase domain are two common mechanisms of resistance to Glivec. The occurrence of these mechanisms was first reported by Sawyers’ group37. In a study of 11 patients with blast crisis and overt clinical resistance when treated with Glivec, 3 had amplification of the BCR–ABL gene and 6 had a point mutation in the ABL kinase domain, which resulted in a T315I (threonine 315 to isoleucine) amino-acid sub￾stitution. Following this initial report, the T315I muta￾tion as well as further mutations in the ABL kinase domain have been reported by various invest￾igators38–41. Even though these mutations vary in their type and frequency, it is speculated that they might all lead to a reactivation of BCR–ABL-driven signal trans￾duction. To understand the molecular mechanism by which such mutations might cause resistance to Glivec, current studies are using X-ray crystallography to analyse the three-dimensional structure of a complex between the drug and the human c-ABL kinase domain. Glivec binds to an unusual, inactive confor￾mation of ABL with the amino terminus of the activa￾tion loop, which contains the highly conserved DFG (asparagine-phenylalanine-glycine) motif, folded into the ATP-binding site42. This conformation has been Haematological response Major cytogenic response % of patients 100 80 60 40 20 0 Late chronic phase, IFN failure (n = 524) Accelerated phase (n = 181) Blast crisis (n = 229) 95 69 30.6 60 24 16.2 Figure 4 | Haematological and cytogenetic response in CML: Phase II data. In all studies, results are expressed as the percentage of responding patients among the patients for whom the diagnosis of the correct phase of chronic myelogenous leukaemia (CML) was confirmed after a central review of the data. A major cytogenetic response combines both complete (0% Ph+ metaphases) and partial (1–35% Ph+) responses. Haematological response was defined as complete haematological response (CHR) in the chronic￾phase study, and as either a CHR, a marrow response or a return to chronic phase (RTC) in the advanced-phase studies, all to be confirmed after at least four weeks. In the chronic-phase study, CHR was defined as white blood cells <10 x 109 l –1, platelets <450 x 109 l –1, myelocytes and metamyelocytes <5% in blood, no blasts and promyelocytes in blood, basophils <20% and no extramedullary involvement. In advanced-phase studies, CHR was defined as neutrophils = 1.5 x 109 l –1, platelets = 100 x 109 l –1, no blood blasts, marrow blasts <5% and no extramedullary disease. A marrow response was defined by the same criteria as for CHR, but with neutrophils = 1 x 109 l –1 and platelets = 20 x 109 l –1 . An RTC was defined as <15% blasts in marrow and blood, <30% blasts and promyelocytes in marrow and blood, <20% basophils in blood and no extramedullary disease. IFN, interferon; Ph+, Philadelphia chromosome positive

REVIEWS Clinical deve 二 Typical dev ent tin very Typically-8 years .chronic mvelooenous le tha ary to fully assess the the ot these of Glivec,bin rly dias AGP).It has beer increased levels of AGB which would lead to insuffi with low- ose cytarabin This study,kno IS'study).ha nding that AGP p m CMLpa ts failed to of l th te a bet the cl Glivec on the proliferation of e,h The shown that the of G nous ant aluating the wit arabine and other singl This is the first report to identify dysregulation of genes phase or ad 9 JULY2002 VOLUM正1 2002 Nature Publishing Group

© 2002 Nature Publishing Group 498 | JULY 2002 | VOLUME 1 www.nature.com/reviews/drugdisc REVIEWS that are unrelated to BCR–ABL signalling, and further studies will be necessary to fully assess the significance of these findings and their relevance to CML patients. Current and future development in CML The activity of Glivec in patients with newly diag￾nosed CML is being further investigated by a large randomized Phase III study to compare first-line ther￾apy with Glivec against standard interferon in combi￾nation with low-dose cytarabine. This study, known as the ‘IRIS’ study (International Randomized study of Interferon versus STI571), has enrolled 1,106 patients. The results of an interim analysis with a median fol￾low-up of 14 months indicate a better tolerability and a superior efficacy of first-line Glivec compared with interferon and low-dose cytarabine in terms of cyto￾genetic response, haematological response and, more importantly, time to progression to accelerated phase or blast crisis46. Preclinical studies have shown that the combination of Glivec with various anticancer agents might have syn￾ergistic effects. Consequently, several Phase I/II studies are evaluating the feasibility of combining Glivec with interferon, polyethylene glycol (PEG)ylated interferon, cytarabine and other single-agent or combination chemotherapy regimens, in patients with either chronic￾phase or advanced CML. Accordingly, the uptake of Glivec was reduced in Glivec-resistant LAMA84 cells in association with an overexpression of the PgP protein. Sensitivity to Glivec was recovered when cells were treated with the PgP inhibitor verapamil35. At clinically relevant concentra￾tions of Glivec, binding to plasma proteins is approxi￾mately 95%, mostly to albumin and α1-acid glycoprotein (AGP). It has been suggested that a potential mecha￾nism of resistance might relate to this high binding to increased levels of AGP, which would lead to insuffi￾cient availability of free drug for antileukaemic activity 43. However, the clinical significance of this hypothesis is uncertain, in particular in view of the finding that AGP purified from CML patients failed to block the effect of Glivec on the proliferation of leukaemic cells44. Recently, Hofmann et al.45 studied a small group of patients with Ph+ acute lymphoblastic leukaemia who were resistant to Glivec by using DNA-microarray expression profiling. They described an association between the occurrence of resistance to Glivec and upregulation of genes encoding proteins such as Bruton tyrosine kinase and two ATP synthetases (ATP5A1 and ATP5C1), and downregulation of other genes, such as the pro-apoptotic gene BAK1 and the cell-cycle-control gene INK4B45(also known as p15). This is the first report to identify dysregulation of genes June 1998 – First patient with CML treated. 1990 – Lead compound identified in a screen for inhibitors of PKC. 1996 – In vivo activity shown in BCR–ABL-transformed cells in syngeneic mice. June 2000 – Phase III trials initiated. November 2001 – Approved in Europe and Japan for CML. May 2001 – Approved by the FDA for CML. June 1999 – Phase II trials initiated. 1992 – First batch of Glivec synthesized. February 2001 – NDA submitted to FDA for CML. February 2002 – Approved by the FDA for GIST. Discovery Clinical development N N N N H H N O Discovery Clinical development Typically ~8 years Typically ~7 years Typical development timeline Glivec development timeline Figure 5 | Key points in the discovery and development of Glivec. The clinical development was particularly rapid, as can be seen by comparison with the typical drug discovery and development times shown in the inset. An NDA for Glivec was submitted just two years and nine months after treatment of the first patient with CML, and FDA approval was given less than three months after application. CML, chronic myelogenous leukaemia; GIST, gastrointestinal stromal tumour; NDA, new drug application; PKC, protein kinase C

REVIEWS of resistance to alivee in CML Other -KIT-expressingoIn human systemic C of BCR-ABL Mutations in RCR-ARI kin correct binding of Glive Efflux of Glivec (for e mple,by Pep. ciated MDR protein) Protein binding of Glivee (for,tocirculating AGP) BCR-ABL-independent mechanisms(BCR-ABL is inactivated) at n of c-KI Activation of signalling p vs do m of BCR-ABL 8 Activation of leukaemogenic pathways unrelated to BCR-AB ibito arge f 0.1 uM (REE 61).The tor fo m-cell f or(SCF c-KTT,a mer hibition of SC inhibit of MAPK c-Kr with a marked incr e mi in the 5 UM (RE 61的 Development inK-pvGG ted in va us ot hum tumours. it will be imnortant to determine the activ status o crine activatio of thi and acute myeloge. of th c-kIT DO expre utated c ned from the two stru ally sim PDGF-d am to that fou of PDGF cellular e mbryona mic s and inhi tissue.There is increasin his GIST lin ell culture role tum kina d/o asest (REF 57).Two larg g the forr urs by the hu the effect sof tw (400 toohcbeansotnudc URE REVIEWS OLUME 2002 Nature Publishing Group

© 2002 Nature Publishing Group NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | JULY 2002 | 499 REVIEWS Other c-KIT-expressing tumours. In human systemic mastocytosis, most cases show a point mutation in codon 17 of c-KIT, which results in a D816V (aspartic acid 816 to valine) amino-acid substitution in the kinase-2 domain of c-KIT. Interestingly, this mutated c-KIT is resistant to inhibition by Glivec53,58. Expression of c-KIT and SCF has been reported in a retrospective small-cell lung cancer (SCLC) series, indicating that SCLC growth might involve an AUTOCRINE loop. Inhibition of c-KIT activation by transfection of a dominant-negative c-KIT gene results in loss of growth-factor independence59,60. Furthermore, the c-KIT/PDGF-receptor inhibitor AG1296 inhibits growth of SCLC cells in serum-con￾taining medium60. In H526 SCLC cells, pretreatment with Glivec inhibited SCF-mediated c-KIT activation with an IC50 (half-maximal inhibitory concentration) of 0.1 µM (REF. 61). The compound also blocked downstream signal transduction, as evidenced by inhibition of SCF-mediated activation of MAPK and AKT, and potently inhibited SCF-mediated growth in serum-free medium, with a marked increase in apop￾tosis. Glivec also inhibited the growth of SCLC cell lines in a dose-dependent fashion when grown in serum-containing medium; however, the average IC50 was in the range of 5 µM (REFS 61,62). Although c-KIT expression has been documen￾ted in various other human tumours, including acute myelogenous leukaemia, ovarian and testicular cancer, it will be important to determine the activa￾tion status of the receptor and its importance in the pathogenesis (for a review, see REF. 58). Furthermore, it needs to be explored whether pharmacological inhi￾bition of PARACRINE or autocrine activation of this kinase will be successful therapeutically. Exploratory clinical studies are continuing at present in patients with c-KIT-expressing SCLC and acute myeloge￾nous leukaemia. PDGF receptor as a target The third target of Glivec is the PDGF-receptor tyro￾sine kinase. Cellular studies have shown potent inhibi￾tion of the two structurally similar PDGF-α and PDGF-β receptors (PDGFR-α and PDGFR-β), as well as blockade of PDGF-mediated cellular events47,63. PDGF is a connective-tissue-cell mitogen with in vivo functions that include embryonal development, wound healing and control of interstitial-fluid pressure in soft connective tissue. There is increasing evidence that the PDGF ligand–receptor system also has an important role in tumorigenesis64. Paracrine and/or autocrine activation of the PDGFR kinase has been postulated in numerous malignancies, and the pres￾ence of PDGF autocrine loops is most well docu￾mented in gliomas65. Glivec inhibited the in vitro and in vivo growth of cells with autocrine PDGF signalling, including the formation of tumours by the human glioblastoma lines U343 and U87, which had been injected into the brains of nude mice66. The inhibitory effects were mediated predominantly through promo￾tion of growth arrest rather than apoptosis. c-KIT is another target In addition to various oncogenic forms of the BCR–ABL tyrosine kinase, Glivec also inhibits the receptor for stem-cell factor (SCF) — c-KIT, a member of the type III group of receptor kinases. Preclinical studies have established that the drug blocks c-KIT autophosphorylation, as well as SCF-stimulated down￾stream signalling events, such as activation of the mito￾gen-activated protein kinases (MAPKs) ERK1 and ERK2, and AKT (also known as protein kinase B)47,48. Development in c-KIT-positive GISTs. Gastrointestinal stromal tumours (GISTs) represent a rare subset of soft￾tissue sarcomas that involve the gastrointestinal tract and are thought to be derived from the interstitial cells of Cajal. Scientific rationale for the use of Glivec in the treatment of these tumours comes from the landmark work of Hirota et al.49, who first identified somatic gain￾of-function mutations in the c-KIT gene in patients with GIST. Oncogenic c-KIT mutations in GISTs have been localized to the extracellular domain, kinase domains 1 and 2 and predominantly in the juxtamembrane domain of the c-KIT protein50–52.As c-KIT serves as a phenotypic marker of GISTs and has a key role in their pathogenesis, it provides an ideal target for molecular-based therapy. The first evidence that Glivec might inhibit GIST cells that express mutated c-KITwas obtained from studies in a mast-cell leukaemia line expressing a mutated c-KIT similar to that found in GISTs48,53. Furthermore, Glivec rapidly and completely abolished constitutive phospho￾rylation of c-KIT in the human cell line GIST882, which expresses an activating c-KIT mutation in the first part of the cytoplasmic split-tyrosine-kinase domain, and inhib￾ited proliferation in this GIST line54. Similarly, a primary GIST cell culture that expressed a c-KIT exon 11 juxta￾membrane mutation was also inhibited by Glivec54. As reported recently, a pronounced tumour response was first observed in a single patient with progressing GIST55. Following this case report, the high level of effi￾cacy of Glivec in GIST has been shown in two subsequent Phase I (REF. 56) and Phase II studies (REF. 57). Two large Phase III studies are being carried out at present to com￾pare the effectiveness of two doses of Glivec (400 mg or 800 mg daily). On the basis of the Phase II data, the FDA approved the use of Glivec for GISTs on 1 February 2002. AUTOCRINE Describes an agent secreted from a cell that acts on the cell in which it is produced. PARACRINE Describes an agent secreted from a cell that acts on other cells in the local environment. Box 1 | Mechanisms of resistance to Glivec in CML BCR–ABL-dependent mechanisms (cells remain dependent on BCR–ABL signalling) • Amplification of BCR–ABL gene • Mutations in BCR–ABL kinase domain prevent correct binding of Glivec • Efflux of Glivec (for example, by PgP-associated MDR protein) • Protein binding of Glivec (for example, to circulating AGP) BCR–ABL-independent mechanisms (BCR–ABL is inactivated) • Activation of signalling pathways downstream of BCR–ABL • Activation of leukaemogenic pathways unrelated to BCR–ABL AGP,α1-acid glycoprotein; CML, chronic myelogenous leukaemia; MDR, multidrug resistant; PgP, P-glycoprotein

REVIEWS (DES ahighly re ing ch GF in ide (CO AI DGF-B well as prima y DES nd n or PDG of th on ch which pDC bited gro R RB. ment wi th Glv and co ative di and de ed TEL FR fusi gs for th as yet un r gen .0t ting the in this partic .by e with th he cen d CE in the regula add ng too higl nical data available so far in CML,GISTand that ing PR C-KI nd into titi.. Is and st dise cell in this tumour model dono early phases of lopment of a molecul aonyclundtrtodmalig capillary endothelial ells,pericytes and smooth-muscl other malignancies that involve any of these signalling 500 JULY 2002 VOLUME 1 2002 Nature Publishing Group

© 2002 Nature Publishing Group 500 | JULY 2002 | VOLUME 1 www.nature.com/reviews/drugdisc REVIEWS cells79, but might also be influenced indirectly through paracrine action on PDGF-responsive stromal and perivascular cells, which are a principal source of vascu￾lar endothelial growth factor (VEGF)80. PDGF has also been shown to induce the expression of VEGF in endothelial cells, which in turn causes an autocrine VEGF loop81.Anti-angiogenic activity of Glivec has been shown in vitro through inhibition of serum-stimulated capillary sprouting from rat aorta, and in vivo in a subcu￾taneous implant model in which the drug inhibited PDGF- and also VEGF- and basic fibroblast growth factor (bFGF)-stimulated vascularization82. Blockade of PDGFR signalling by Glivec has also been shown to inhibit angio￾genesis and tumour growth in an experimental model of bone metastasis83. Glivec treatment of nude mice injected with PC-3MM human prostate-cancer cells into the tibia inhibited tumour-cell growth and induced apoptosis, both in tumour cells and tumour-associated endothelial cells. The effects were pronounced when mice were treated with the combination of Glivec and taxol. Interestingly, immunohistochemical studies showed that tumour cells growing in the bone (but not those in surrounding musculature) expressed high levels of PDGF-α, PDGF-β, PDGFR-α and PDGFR-β. Tumour￾associated endothelial cells within the bone also expressed PDGFR-α and PDGFR-β. These data indi￾cate that inhibition of the PDGFR in combination with chemotherapy might provide a new approach for the treatment of bone metastasis. Conclusion The discovery and development of Glivec has shown that is possible to produce rationally designed, mole￾cular-targeted drugs for the treatment of a specific cancer. The research programme has also clearly shown that it is possible to define in vitro and animal models with high predictive quality, as the results of the subsequent clinical studies have largely corrobo￾rated the preclinical findings. The predictive quality was achieved in this particular case by using models with identical genetic abnormalities as those found in man. The case of Glivec also shows that compounds that do not only affect one, but two or more targets (which is frequently the case), can be beneficial in allowing several diseases with differing molecular abnormalities to be addressed, without paying too high a price in terms of toxicity. The clinical data available so far in CML, GIST and chronic myeloproliferative disorders that involve rearrangement of the PDGFR gene indicate that the inhibition of BCR–ABL, c-KIT and PDGFRs can be achieved with Glivec in humans, and translated into clinically meaningful patient benefit. Providing clinical ‘proof of concept’, these data validate the initial hypothe￾sis of this programme, and underscore the importance of rationally selecting the target diseases to be consid￾ered in the early phases of development of a molecule such as Glivec. Beyond these reasonably well-understood malig￾nancies, Glivec could have potential in the treatment of other malignancies that involve any of these signalling Autocrine PDGFR activation is also well docu￾mented in tumour cells of dermatofibrosarcoma pro￾tuberans (DFSP), a highly recurrent, infiltrative skin tumour that is characterized by a chromosomal rearrangement involving chromosomes 17 and 22. The resulting fusion-gene product collagen I, α1 polypep￾tide (COL1A1)–PDGF-β triggers the autocrine stimu￾lation of the PDGFR67. COL1A1–PDGFβ-transformed fibroblasts, as well as primary DFSP and giant-cell fibrosarcoma cell cultures, were inhibited by Glivec in vitro and in vivo 67–69. The main mechanism by which Glivec affected DFSP tumour growth was through induction of apoptosis69. Preliminary data indicate that Glivec might also be active in patients with DFSP70. Relatively little is known about the ligand-indepen￾dent activation of PDGFR. However, rearrangement of PDGFRβ has been described in chronic myeloprolifer￾ative diseases. The best known of these is the t(5;12) chromosomal translocation in chronic myelomono￾cytic leukaemia (CMML), in which PDGFRβ, which is located on chromosome 5, is fused to the TEL gene on chromosome 12. Transformation of haematopoietic cells occurs through oligomerization of the TEL– PDGFR-β fusion protein, which causes ligand-indepen￾dent constitutive activation of the PDGFR kinase71. Glivec inhibited the growth of cells expressing TEL–PDGFRβ15, and in transgenic mice that expressed the TEL–PDGFRβ, treatment with Glivec inhibited tumour formation and prolonged survival of the ani￾mals72. A remarkable haematological and complete cytogenetic response has been observed in two patients with chronic myeloproliferative disorders associated with a t(5;12) translocation — one of them with a well-characterized TEL–PDGFR fusion gene and the second with a rearranged PDGFR gene with an as yet unidentified partner gene73. Other explor￾atory clinical trials are being carried out in gliomas and in prostate cancer. Targeting the tumour microenvironment An alternative strategy to influence tumour growth is to interfere with the tumour stroma and microvasculature. Paracrine PDGF signalling in the connective-tissue tumour stroma has been described in various types of solid tumour64. Several lines of evidence indicate a role for PDGF in the regulation of interstitial fluid pressure (IFP)74–76. As most solid tumours have an increased IFP, pharmacological reduction might be a way to increase the uptake of anticancer drugs into tumours77. Recent experiments have shown that Glivec significantly reduced tumour IFP in subcutaneously growing PROb rat-colon carcinomas, and a concomitant increase in trans-capillary transport of a radiolabelled tracer com￾pound into the tumour interstitium was observed78. These effects were mediated by inhibition of the express￾ion of PDGFR on blood vessels and stromal cells, as tumour epithelial cells in this tumour model do not express PDGFRs. The angiogenic activity that has been described for PDGF might not only be explained by its direct effects on capillary endothelial cells, pericytes and smooth-muscle

REVIEWS far feasible.This point with multipl ch in ses such nty of the coml activation of the molecular target in their tumour,as also being investigated o ST1-571 ST-57I 02 47. 21641 -2298 ,ag0a 61221-22E133 d he (P N M 34. 2) ang BOR-AB 1188121 CR- of the M& . LUME 2002 Nature Publishing Grour

© 2002 Nature Publishing Group NATURE REVIEWS | DRUG DISCOVERY VOLUME 1 | JULY 2002 | 501 REVIEWS far as is technically feasible. This point has been cru￾cial in the successful outcome of the CML, GIST and CMML trials. The activity of Glivec in more common cancers with multiple and more complex molecular abnormalities remains to be determined, and is the objective of continuing research in diseases such as SCLC, prostate cancer and gliomas. The potential activity of the combination of Glivec with other sig￾nal-transduction inhibitors or anticancer agents is also being investigated. pathways, or through targeting of the tumour micro￾environment. However, most human cancers are likely to be heterogeneous with regard to molecular abnor￾malities, such as oncogene activation, and involve multiple signalling pathways in addition to either c￾KIT and/or the PDGFR. Consequently, careful atten￾tion will have to be paid in designing clinical trials in these more complex indications as to how patients should be selected on the basis of the expression or activation of the molecular target in their tumour, as 1. Rowley, J. D. A new consistent abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and giemsa staining. Nature 243, 290–293 (1973). 2. Nowell, P. C. & Hungerford, D. A. A minute chromosome in human chronic granulocytic leukemia. Science 132, 1497 (1960). 3. Daley, G. Q., Van Etten, R. A. & Baltimore, D. Induction of chronic myelogenous leukemia in mice by the p210Bcr/Abl gene of the Philadelphia chromosome. Science 247, 824–830 (1990). 4. Kelliher, M. A. et al. Induction of chronic myelogenous leukemia in mice by the v-Abl and Bcr/Abl. Proc. Natl Acad. Sci. USA 87, 6649–6653 (1990). 5. Heisterkamp, N. et al. Acute leukaemia in Bcr/Abl transgenic mice. Nature 344, 251–253 (1990). 6. Lugo, T. G. et al. 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Online links DATABASES The following terms in this article are linked online to: Cancer.gov: http://www.cancer.gov/cancer_information/ acute lymphoblastic leukaemia | acute myelogenous leukaemia | chronic myelogenous leukaemia | ovarian cancer | prostate cancer | small-cell lung cancer | testicular cancer LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ ABL | Abl | AGP | AKT | albumin | ARG kinase | ATP5A1 | ATP5C1| BAK1 | BCL-XL | BCR | Bcr | bFGF | Bruton tyrosine kinase | COL1A1 | CRKL | EGF receptor | c-ERBB2 | ERK1 | ERK2 | FGF | c-FMS | v-fms | IGF-1 receptor | INK4B | insulin receptor | JAK2 | c-KIT | p27 | PDGF-α | PDGF-β | PDGFR-α | PDGFR-β | P-GP | PKC | SCF | v-SRC | STAT5 | TEL | VEGF Medscape DrugInfo: http://promini.medscape.com/drugdb/search.asp cytarabine | Glivec | interferon | taxol | verapamil FURTHER INFORMATION FDA: http://www.fda.gov/default.htm Access to this interactive links box is free online