Indian Journal of Biochemistry & BiophysicsVol. 41, December 2004, pp. 273-280

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Minireview

Role of protein tyrosine kinase inhibitors in cancer therapeutics

S B Bhise*, Abhijit D Nalawade and Hitesh Wadhawa

Govt. College of Pharmacy, Karad, Maharashtra 415 124'IIndia

Received 19 February 2004; revised 28 September 2004

Protein tyrosine kinases (PTKs) are critical in regulating cell growth and differentiation and are deeply involved inseveral cancers. PTK-inhibitors are mainly ATP-site directed and are finding use in the treatment of several cancers, andmore than 30 such agents are now in phase I-III clinical trials. The ~review focuses mainly on the development ofPTK inhibitors in clinical trials, with special emphasis on imatinib mesylate, a rationally designed, potent oral anticanceragent and selective inhibitor for Abl tyrosine kinase, including Bcr-Abl, C-kit and platelet-derived growth factor-receptortyrosine kinases, which has been implicated in several malignancies, including chronic myeloid leukemia and gastro-intestinal stromal tumour.

Keywords: Protein tyrosine kinase, tyrosine kinase inhibitors, imatinib mesylate, chronic myeloid leukemia, acute myeloidleukemia, anticancer agent, gastrointestinal stromal tumor, epidermal growth factor receptor, vascularendothelial growth factor.

IntroductionRemarkable advances in recent years in the

molecular biology of cancer have resulted indeveloping more rational and effective strategies fordiagnosis and treatment of cancer. One such approachhas been the use of protein kinases (PTKs) in cancertherapeutics. A PTK inhibitor, imatinib mesylate(originally called CGP-57-148 B), promises to be animportant advance in the treatment of patients withchronic myeloid leukemia (CML) and gastrointestinalstromal tumor (GIST)l. PTKs play an important rolein regulating cell growth and division and are ofteninvolved in tumor formatiorr'". Role of PTKinhibitors in cancer therapeutics is an active area ofresearch today. Several PTK inhibitors have been

*Corresponqing authorPh: (02164) 273830 (R).(02164) 271196 (0)

Fax: (02164)271196E-mail: satishbhise@rediffmail.comAbbreviations: CML, chronic myeloid leukemia; AML, acutemyeloid leukemia; EGF, epidermal growth factor; EGFR,epidermal growth factor receptor; VEGF-R, vascular endothelialgrowth factor receptor; VEGF, vascular endothelial growth factor;PTK, protein tyrosine kinase; RTK, receptor tyrosine kinase;GIST, gastro-intestinal stromal tumor; RPTK, receptor PTK; Ph-chromosome, Philadelphia chromosome; Abl, Abelson tyrosinekinase; Bcr, breakpoint cluster region; KDR, vascular endothelialgrowth factor receptor 2; PDGF, platelet-derived growth factor;PDGF-R, platelet-derived growth factor receptor; SFK, Src familyPTK; Csk, C-terminal Src family; C-kit, stem cell factor receptor

, developed, which are now in pre-clinical and clinicalstudies (Table 1). The present review focuses mainlyon the development of PTK inhibitors in clinicaltrials, with special emphasis on imatinib mesylate.

Tyrosine kinases are attractive anticancer targetsApproximately 2000 kinases are known and more

than 90 PTKs have been found in human genome.PTKs can transfer a phosphate group from a donormolecule (usually ATP) to an amino residue of aprotein. They are divided into two main classes,receptor- and non-receptor PTKs. Receptor PTKspossess an extracellular ligand binding domain and anintracellular catalytic domain with intrinsic tyrosinekinase activity and upon ligand binding, the receptortyrosine kinase (RTK) triggers receptor dimerizationand autophosphorylation. The autophosphorylationcreates phosphotyrosine docking sites for proteinswhich transduce signals within the cell". Non-receptor PTKs, which includes Abl, Fes/Fer,Syk/Zap70, Jak, Tee, Fak, Ack, Src and Cskparticipate in a variety of signaling processes,including mitogenesis, cell survival and. immuneresponse".

In multicellular animals, Src-family of non-receptorPTKs, including v-Src/c-Src, Yes, c-Fgr, Lyn, Hck,Fyn, Blk and Trk are distinguished from other groupof PTKs by the absence of extracellular ortransmembrane domains. The Src-farnily of PTKs

274 INDIAN J. BIOCHEM. BIOPHYS., VOL. 41, DECEMBER 2004

Inhibitor

Imatinib

OSI-774 (Tarceva TM)

SU5416ZD 1839 (Iressa TM)

LY-333531FlavopiridolCCI779EIRB 0796SUl1248CEP-701Angiozyme R

PKC412Bryostatin- IISIS 3521 (antisense)CI1040RADOOIE7070Cyc202UCN-OlGEM231 (antisense)SCIO-469BAY 43-9006CEP-1347PTK7871ZK222584PKI 166AGOI3726KRN633CHIR 200131CI-1033 (irreversible)CP-547632EKB-569GW-2016SU6668ZD 6474PKC412

Company

Table I-Protein kinase inhibitors in clinical trials53

Target kinase(s)

Novartis

Roche/Genentech/OSISUGEN/PharmaciaAstraZenecaEli LillyAventisWyeth-AyerstBoehringer IngelheimSUGENlPharmaciaCephalonRibozymeNovartisBristol Myers SquibbISISPfizerNovartisEisaiCyclacelNCIHybridonSciosBayerCephalonNovartis/Schering AGNovartisAgouronKirinChironPfizerlW arner -LambertPfizerWyeth- AyerstGlaxoSmithKlineSUGEN/PharmaciaAstraZenecaNovartis

Abl (c-kit, PDGF-R)

EGF-RKDREGF-RPKC-13CdksmTORp38

PDGF-RTrkVEGF-Rl (Fit-I)PKC and VEGFPKCPKC-aMEKmTORCdksCdksPKC and Chk 1PKAp38

c-rafJunKDREGF-R and HER-2VEGF-R2, PDGF-RVEGF-R1I2,KitVEGF-R1I2, FGF-R, PDGF-REGF-R and HER-2VEGF-R2, FGF-REGF-R and HER-2EGF-R and HER-2KDR (PDGF,R, FGF-R)KDR and EGF-RFlt-3

Status

Phase III; FDA approval 5101for CML and 2/02 for GISTs

Phase IIIPhase IIIPhase IIIPhase IIIIII for retinopathyPhase IIPhase IIPhase IIPhase IIPhase IIPhase IIPhase IIII A for retinopathyPhase I-IIPhase IIIIPhase IPhase IPhase IPhase I.Phase IPhase IPhase IPhase IPhase IPhase 1*Phase IPhase IPhase IPhase IPhase IPhase IPhase IPhase IPhase IPhase IPhase I for AML

Cdk, cyclin-dependent kinase; Chk l , checkpoint kinase 1; Cyc, cyclin epidermal growth factor; FGF-R, fibroblast growth factorreceptor; KDR, kinase domain receptor (also called VEGF-R2); MEK, mitogen-activated protein kinase; PKA, proteinkinaseA;PKC, protein kinase C. *PTK7871ZK222584 currently in phase IIII triaI3?

serves as a central molecular switch that mediates thecommunication between an upstream extracellularsignal and downstream intracellular events such ascell growth and differentiation, cell cycle progressionand cell adhesion and movement'i''!'. They containfunctional domains such as SH4 (Src homology 4), aunique' domain, SH3, SH2, SH1 and a C-terminalregulatory region, which are responsible for theirinteraction with particular receptors and proteintargets. The activity of several Src-family kinaseshave been found to be upregulated by gain of functionmutations or over-expressions in several humanleukemias, breast cancer, Hodgkins disease, Burkitt's

lymphoma and melanoma. Therefore, the Src-familyof PTKs is considered as attractive target for the drugdesign due to their involvement in several cancers '".

C-terminal 'Src kinase (Csk), a cytoplasmic PTK,consistmg of an SH3, an SH2 and a kinase domainspecifically phosphorylates Src-family kinases on aC-terminal Tyr and plays a pivotal role in various cellsignalling. These domains have structuralresemblance with other PTKs, relevant to their activesite cleft, ATP and peptide-binding lobes 13.14 (Fig. 1).

PTKs play a crucial role in signal transductionpathway, known to regulate normal cellular functions.They are considered as attractive targets for the drug

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BHISE et al.: PROTEIN TYROSINE KINASE INHIBITORS IN CANCER THERAPEUTICS275

Fig. I--Structures of Csk catalytic domain and the substrate-docking site [(A: Ribbon structure of Csk with identified residuesin the substrate-docking site shown in a ball-and-stick model.Several loop structures relevant to peptide substrate binding andcatalysis are indicated by arrows; and (B) Surface structure of Csk(yellow) and the substrate-docking site (colored by electrostaticpotential). The active site cleft is indicated 13]

design due to their ATP-binding property15,16. PTKinhibitors are mainly selected as prime targets invarious oncogenic complications.

oral administration and are commonly referred to asEGFR tyrosine kinase inhibitors (EGFR-TKIs). Theycompetitively inhibit binding of ATP to the tyrosinekinase domain of the receptor, thereby inhibiting'EGFR autophosphorylation. EGF and EGFR havedemonstrated therapeutic benefit in several humantumors, including breast, lung, colorectal, ovarian,prostate, head and neck19-21.

EGFR tyrosine kinase inhibitors ZD1839 andOSI-774

ZD1839 [4_(3_chloro-4-fluroanilino)-7 -methoxy-6-(3-morpholinopropoxy) quinazoline], a low molecularmass (447 kD) quinazoline derivative specificallyinhibits Erb Bland Erb B2 tyrosine kinase. It hasdemonstrated clinical antitumor activity in severaltumors, including those derived from hormone-resistant prostrate cancer, ovarian cancer, ductalcarcinoma of the breast, colon, vulval, small cell andnon-small-cell lung cancers22.

24.

OSI-774 ([6,7 _bis_(2_methoxy-ethoxy)-quinazolineA_yl]_[3_ethylphenyl]-amine), a low-molecular quina-zoline derivative has a very similar pharmacologicaland physiological profile to ZD1839. It has shownactivity in ovarian carcinoma, non-small cell lungcancer and head and neck cancers25,26.

PTK inhibitors of vascular endothelial growthfactor (VEGF)

VEGFs -C and -D are the ligands for VEGFreceptors, VEGFR-2 (Flk-lIKDR) and VEGFR-3(Flt-t). Signalling via VEGFR-3 is important for themaintenance and function of kidney glomeruli,ovarian corpus luteum angiogenesis, motor neuronesof spinal cord and central nervous system, as well ashematopoietic stem cells27-29. VEGF stimulatesendothelial cells of blood vessels to proliferate andgrow. This mechanism is called angiogenesis. As theuncontrolled growth of cancer cell is legendary, andrequires a lot of blood flow to the cell area, if theblood vessel growth is re~trained then in theory,cancer growth also can be restrained.

Inhibitors of VEGF and EGF have found use inangiogenesis and playa pivotal role in several tumors,including thyroid carcinoma30,31. Th~refore, theVEGFR families of tyrosine kinases are considered tobe attractive targets for rational drug design

32,33

VEGF-tyrosine kinase inhibitors PTK787 andSU5416

PTK787 or ZK222584, a novel oral angiogenesistyrosine kinase inhibitor was initially designed to

PTK inhibitors of epidermal growth factorreceptor (EGFR)

The EGF receptors are small proteins consistingof an amino-terminal extracellular domain, a singletransmembrane anchoring region and acarboxylterminal intracellular domain. Thesereceptors are found on the surface of many types ofcancer cells. EGFR binds exclusively to the growthfactor, triggering production of tyrosine kinase, whichin turn causes cell to grow and divide17,18.EGFRs area family of four receptors [Erb B1 (also termed EGFRand HER 1), Erb B2 (HER 2), Erb B3 (HER 3) andErb B4 (HER 4)]. Inhibitors of EGFR are active after

276 INDIAN J. BIOCHEM. BIOPHYS., VOL. 41, DECEMBER 2004

inhibit VEGF signal transduction by binding directlyto the ATP-binding sites of VEGFRs34. It readilypenetrates cells and inhibits VEGF-induced (VEGF-R2) autophosphorylation and mitogenesis andpromotes endothelial cell apoptosis. Consistent withits anti-angiogenic activity, it reduces vascularity andblood flow in tumor tissues and does not have a directeffect on tumor cells. It inhibits the growth of thyroidcancer. It is now proceeding into large clinical studiesand is looking more promising, both in terms ofefficacy as well as tolerability35.38.

SU5416 compound (Z-3-[(2,4-dimethylpyrrol-5-yl)methylidenyIJ-2-indolinone) is both lipophilic andpotentiatjy reactive in nature and is metabolized bythe cytochrome P450 enzymes. It was found to be apotent inhibitor of the VEGF-R2, C-kit and Flk-2, andplays a role in tumor angiogenesis. It has shown anti-angiogenic activity in various cancers, including renalcell carcinoma, melanoma and multiple myeloma39-42.

Imatinib, an Abl-specific tyrosine kinase inhibitorChronic myeloid leukemia (CML) is characterized

by the presence of an abnormally short chromosome22 (Ph chromosome), resulting from a reciprocaltranslocation involving the long arms of chromosome9 and 22

43,44.The disease progresses through three

phases: chronic, accelerated and blast crisis. Chronicphase (CP) lasts for about 4-5 years. Initially, thepatients usually have minor symptoms and the canceris usually detected by routine blood tests. Acharacteristic elevation of white blood count andexpression of Ph chromosome are evident. Thetreatment includes interferon therapy alone or withother drugs or bone marrow transplant. Acceleratedphase (AP) is characterized by the increasing numberof blasts in the peripheral blood and the medianduration is usually of 6-9 months. Blast crisis phase(BC) is the most severe form of CML with anincreasing number of blasts in the blood and bonemarrow. Its duration is usually of 3-6 months":",

Imatinib mesylate, chemically designated as 4-[(4-methyl-l-piperazinyl) methyIJ-N-[ 4-methyl-3-[[ 4-(3-pyridinyl)-2-pyrimidinyIJaminophenyIJ benzamidemethanesulfonate48 (Fig. 2) has recently beenapproved as a first-line therapy for patients withchronic, accelerated or blast phase. It acts as ainhibitor of the Bcr-Abl tyrosine kinase in CML andalso inhibits cell-surface protein tyrosine kinase, theplatelet-derived growth factor receptor (PDGF-R) andstern cell factor receptor (C_kit)49.51.The inhibition oftyrosine kinase results in the reduction and

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proliferation of the leukemic cells. US FDA approvedimatinib mesylate (also known as STI-571), apromising new oral treatment for patients with CML(May 2001) and gastro-intestinal stromal tumor[(GIST) Feb 2002J52,53.It is considered as a milestonein cancer treatment, because it is generally welltolerated and causes fewer side effects54,55.

Mechanism of Imatinib

The Bcr-Abl tyrosine phosphokinase enzyme isconstitutively an active kinase which functions bybinding ATP and transferring phosphate from ATP totyrosine residues on various substrates, therebytransmitting intracellular signals independent ofligand binding to growth factor receptors (such asinterleukin-3). Imatinib mesylate occupies the kinasepocket of Bcr-Abl protein, blocking access to ATP,and thus prevents phosphorylation of downstreameffector molecules56,15(Fig. 3).

Clinical effectiveness

Gastro-intestinal stromal tumors (GISTs) are themost common malignant form of sarcoma that arise inthe gastrointestinal tract. In a phase I study, involving40 patients (of whom 36 had GISTs), response was

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BHISE et al.: PROTEIN TYROSINE KINASE INHIBITORS IN CANCER THERAPEUTICS

monitored for 36 patients .. They received imatinibmesylate orally with food, 400 mg once daily (n = 8),300 mg twice daily (n = 8), 400 mg twice daily (n =16) and 500 mg twice daily (n = 8). Patients weremonitored for 3 months after initiation of treatment.An objective response was observed in 25 patients(69.4%), while 19 patients (52.7%) confirmed partialresponse. Unconfirmed partial response was observedin 6 patients (16.6%), while stable disease wasobserved . in 7 patients (19.4%) and diseaseprogression was observed in 4 patients (11.1 %)57,58.

In CML, 150 patients with chronic phasePh-positive, in whom prior IFN-a therapy had failed,were selected for the study, Patients received 400 mgimatinib mesylate orally after breakfast. Dose wasincreased from 400 mg to 600 mg, in the absence ofhematologic response. Patients exhibited thefollowing effects: 44% complete cytogenic response,22% partial cytogenic response and 8% minorcytogenic response at 12 months. The estimatedprogression free survival (PFS) was 89.2% at 12months and 80.2% at 18 months.".

DosingAlthough the recommended dosage of imatinib is

400 mg once daily for patients in chronic phase ofCML and 600 mg once daily for patients inaccelerated or blast crisis phase, but the dose may beincreased (from 400 mg to 600 mg, or 600 mg to 800mg), in the absence of significant toxicity, in thepresence of disease progression, the failure to achieveresponse, or the loss of a previously achievedresponse. If the toxicity is resolved within two weeks,treatment should be resumed at the original dose of400 mg/day and resolved after more than two weeks,or recurred after resuming therapy, the drug should bewithheld, until the event has resolved by usingguidelines for hematologic and nonhematologictoxicity. Treatment should be continued as long as thepatient continues to benefir'"'".Mechanism of relapse

Several mechanisms of clinical resistance havebeen proposed. Some mechanisms involve alterationin Bcr-Abl itself, while others are Bcr-Ablindependent, due to altered drug uptake. Mutationsdiscovered within the PTK domains of Bcr-Abl geneitself has been characterized as a mechanism ofresistance. At the time of disease relapse, mutations ofamino acids (315 and 253, 255 and 351) wereidentified in 60% of patients with kinase domainmutations62-65. To date, 18 different candidates of

277

gleevec resistance mutations (GRMs) have beenidentified in the cells from patients, who becamerefractory to imatinib treatment at 14 different sites,all between Abl codons 244 and 39666-68.Bcr-Ablindependent mechanisms, including imatinibsequestration via its binding to u-I-acid glycoprotein,an acute phase serum protein may be a potentialcausative factor of resistance. There is still much to belearnt about imatinib resistance in long-term relapsedpatients.

SafetyThe most common frequent side effects include

mild to moderate nausea, diarrhoea, myalgias andperipheral edema and less being skin rashes andperipheral edema?". Myelosuppression occurredfrequently and incidence was more common at higherdoses (600 mg/day) in blast crisis and acceleratedphase than in the chronic phase (400 mg/day) inCML 48,70.Hepatotoxicity was. reported with severeelevation of transaminases or bilirubin, however, mostabnormalities were managed with dosage reduction orinterruption (median duration of episode 51 weeks),but may be fatal, though rarely, when co-administeredwith acetaminophen. Cytopenias, includingneutropenia, thrombocytopenia and anemia were

. 1 . d . 11 di 6171consistent y expenence III a stu res ' .

ContraindicationsImatinib is metabolised in the liver by the

cytochrome P450 system. It is contraindicated inpatients with hypersensitivity to the drug. Patientsshould be cautioned to avoid or restrict the use ofover-the-counter and prescription medicationscontaining acetaminophen or any other ingredient inthe product formulation, such as colloidal silicondioxide, crospovidone, magnesium stearate,microcrystalline cellulose, gelatin, iron oxide, red(EI72); iron oxide, yellow (EI72) and titaniumdioxide (EI71)61,70,

ConclusionRole of various PTKs in signal transduction

pathways has become clearly evident in a diversearray of cellular processes, The development of PTKinhibitors blocking signalling pathways is considereda promising approach for drug development. Theclinical efficacy of imatinib has clearly demonstratedthe potential of kinase inhibitors in CML and GIST.The studies of imatinib mesylate provide proof-of-principle' for using aberrant kinases as therapeutictarget in several cancers.

278 INDIAN J. BIOCHEM. BIOPHYS., VOL. 41, DECEMBER 2004

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