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Nat Struct Biol 9:117–120 Zimmermann J, Buchdunger E, Mett H, Meyer T, Lydon NB, Taxler P (1996) Phenylamino-Pyrimidine (PAP)-derivatives: a new class of potent and highly selective PDGF-receptor autophosphorylation inhibitors. Bioorg Med Chem Lett 6:1221–1226 Zimmermann J, Buchdunger E, Mett H, M eyer T, Lydon NB (1997) Potent and selective inhibitors of the Abl kinase Phenylamino- pyrimidine (PAP) derivatives. Bioorg Med Chem Lett 7:187–192 102 Chapter 5 · Signal Transduction Inhibitors in Chronic Myeloid Leukemia Contents 6.1 Medical Treatment of CML 103 6.2 Imatinib Mesylate 103 6.2.1 Clinical Efficacy 103 6.2.2 Side Effects of Imatinib 106 6.2.3 Dosage of Imatinib 107 6.2.4 Imatinib in Combination 108 6.2.5 Imatinib Resistance 109 6.2.6 Prediction of Response 109 6.3 Novel Bcr-Abl Inhibitors in Clinical Trials 109 6.3.1 Nilotinib (AMN107) 109 6.3.2 Dasatinib (BMS 354825) 111 References 111 Abstract. Leukemias have t raditionally served as model systems for research on neoplasia because of the easy availability of cell mater ial from blood and marrow for diagnosis, monitoring, and studies on pathophysio- logy. Beyond these more technical aspects, chronic myeloid leukemia (CML) became the first neoplasia in which the elucidation of the genotype led to a rationally designed therapy of the phenotype. Target ing of the pathogenetically relevant Bcr-Abl tyrosine kinase with the inhibitor imatinib has induced remissions with almost complete disappearance of any signs and sym- ptoms of CML. This therapeutic success has triggered an intensive search for suitable targets in other cancers and has led to the development of numerous inhibitors of potential targets now being studied in preclinical and clinical trials worldwide. Imatinib mesylate has been the first selective inhibitor of Bcr-Abl employed in patients. Its routine use has been considered a revolution in the treatment of CML. 6.1 Medical Treatment of CML The first drug reported to be active in CML was arsenic in 1865. Currently, arsenic has been reintroduced into CML management as second-line treatment in combination w ith imatinib. Therapy remained palliative during most of the last century and included splenic irra- diation, various cytostatic agents, of which busulfan was standard for almost three decades, and intensive combination therapy. The intention of the treatment became curative with the introduction of stem cell transplantation in the 1970s (Goldman and Melo 2003). At the same time, interferon a (IFN) in combination with hydroxyurea or low-dose cytarabine (ara-C) offered the prospect of prolonging survival, particularly in low-risk patients and in patients who achieve a cytogenetic remission (Bonifazi et al. 2001; Hehlmann et al. 1994, 2003). 6.2 Imatinib Mesylate 6.2.1 Clinical Efficacy In an effort to identify compounds which could selec- tively inhibit the aberrantly enhanced tyrosine kinase Bcr-Abl, imatinib mesylate, a 2-phenylaminopyrimidine derivative, was identified (see Chap. 1, entitled Chronic Myeloid Leukemia – A Brief History). Imatinib compet- Treatment with Tyrosine Kinase Inhibitors Andreas Hochhaus itively inhibits the ATP binding site of Bcr-Abl tyrosine kinase and, by inhibiting tyrosine phosphorylation blocks the Bcr-Abl signal transduction cascade. It is highly selective for inhibiting Bcr-Abl, ABL, PDGF-R al- pha and beta, ARG, and c-kit (Buchdunger et al. 2000) without inhibiting the proliferation of BCR-ABL-negative cells (Druker et al. 1996). Imatinib is well absorbed from the gut. Peak plasma levels are reached after 2–4 h and bioavailability is 98%. A single oral dose of 400 mg/day produces a steady state plasma concentration which exceeds the minimal required concentration for inhibiting cellular phosphorylation and causes lysis of BCR-ABL-positive cell lines in vitro. The mean elimination half-time of imatinib is 13– 16 h. Excretion is primarily via the feces (Druker et al. 2001b). Imatinib is 95% protein bound, predominantly to albumin and a 1 glycoprotein. Metabolism is mainly through the action of the cytochrome P450 (CYP) iso- form CYP3A4. The main metabolite (N-demethylated piperazine derivative) has similar in vitro potency to the parent compound. In a phase I study 83 IFN refractory patients in chronic phase (CP) were treated with imatinib (Druker et al. 2001 b). The median duration of IFN pretreatment was 8.5 months (1 week to 8.5 years) and the median duration of imatinib therapy was 310 days (17–607 days). Complete hematologic response (CHR) was noted in 53 of 54 patients treated with more than 300 mg of imatinib (criteria: leukocytes <10´ 10 9 /l and platelets <450´ 10 9 /l for at least 4 weeks). Hematologic responses were attained generally within the first 4 weeks of imatinib therapy and were durable in 51 of 53 patients with a median follow-up of 265 days (17–468 days). A major cytogenetic remission (MCR, Ph+ metaphases <35%) was noted in 17 (31%), being complete in 7 patients (13%). The median time to best cytogenetic response was 148 days (48–331 days). The side effects (i.e., nausea, diarrhea, myalgias, and periorbital edema) were relatively frequent (25– 43%) but mostly mild (WHO grades I and II). In some patients abnormal liver function tests were noted. An initial drop of hemoglobin of 1–2 g/d l, which was dose related, occurred frequently. Leukopenia and thrombocytopenia (WHO grade III) occurred in 14% and 16%, respectively, and was not dose limiting. The highest dose of imatinib administered was 1000 mg daily and the maximum tolerable dose was not defined. In a second phase I study 58 patients with myeloid (n=38) or lymphoid blast crisis (BC) or Ph-positive acute lymphoblastic leukemia (ALL, n=20) were treated with 300–1000 mg of imatinib daily (Druker et al. 2001a). The median age was 48 years (range, 24 to 76). Additional chromosomal abnormalities were noted in 58% and 65%, respectively. Twenty-one patients with myeloid BC (55%) achieved a hematologic response, which was complete in four p atients (11%). In 12 patients (32%) less than 5% blasts were noted in the bone marrow. In patients with lymphoid BC, hematologic response rate was 70%, which was complete in 20%. In 11 patients (55%) less than 5% blasts were noted in the bone marrow. Seven of 58 patients (12%) attained MCR, which was complete in five patients (3 and 2 patients, respectively from each group). Response rates were not closely related to the administered doses. Of the 21 patients with myeloid BC who had attained a hematologic response, nine patients relapsed after a median of 84 days (42–194 days). All but one of the patients with lymphoid BC relapsed after a median of 58 days. The side-effect profiles were comparable to the aforementioned study in CP CML. Overall, 16 patients died due to disease progression. Phosphorylation of CRK-oncogene-like protein (CRKL), a major substrate of Bcr-Abl kinase, was markedly reduced in leukemic cells, demonstrating the effect of imatinib on its target. Phase II trials were conducted in BC (n=260), accelerated phase (AP) (n =235), and CP after IFN resistance or intolerance (n =532). According to the original pub- lications, in patients with BC hematolog ic response rate was 52% (complete in 8%), MCR occurred in 16%, with 7% of the responses being complete (Sawyers et al. 2002). Time to progression and median survival were significantly shorter in pretreated patients. In patients with AP imatinib induced sustained hematologic responses lasting at least 4 weeks in 69% (complete in 34%) (Talpaz et al. 2002). MCR rate was 24%. Estimated 12-month overall survival was 74%. In IFN refractory or intolerant patients in CP CML imatinib induced CHR in 95%, MCR in 60%, with 41% of the responses being complete (Kantarjian et al. 2002). The median time to onset of CHR was 0.7 months, of MCR 2.9 months. Updated results are prov ided in Table 6.1 (Silver et al. 2004). Phase-II data were confirmed by a large ex- panded access program with more than 7,000 patients (Hensley et al. 2003). A subsequent phase III randomized controlled trial (IRIS – International Randomized Study of Interferon and STI571) in 1,106 patients with newly diagnosed CML in CP recruited between June 2000 and January 2001, has shown the superiority of imatinib, 400 mg/ 104 Chapter 6 · Treatment with Tyrosine Kinase Inhibitors day, over the combination of IFN and cytarabine in all relevant endpoints. At 18 months the hematologic and cytogenetic response rates in the imatinib arm were 97% and 87%, respectively, which is much hi gher than comparable figures for the IFN/cytarabine arm (69% and 22%, respectively); the toxicity with imatinib was lower. Time to progression to blast phase, duration of progression-free survival irrespective of the prognostic-factor score at the time of study entry, and the per- ceived occurrence of adverse events were advantageous for primary imatinib therapy (O’Brien et al. 2003). Due to the large numbers of crossovers from IFN to imatinib, a long-term comparis on of both therapies is impossible. A 60-month update of the imatinib group showed complete hematologic remissions in 98%, partial cytogenetic remissions in 92%, and complete cytogenetic remissions in 87% of cases (Druker et al. 2006) (Figs. 6.1, 6.2). Annual rate of relapse was 3.3, 7.5 and 4.8% in the first three years and decreased to 1.5 and 0.9% in the fourth and fifth year after start of treatment. The time to complete hematologic remission was much shorter with imatinib (about 90% after 3 months) than with IFN. Similar to the effects observed with IFN, the achievement of complete cytogenetic remissions was fol- lowed in most patients by a continuous decline of BCR- ABL transcript levels which continues up to 42 months. Major parameters with favorable prognostic impact were any cytogenetic response after 6 months (Fig. 6.3) and major cytogenetic response (Fig. 6.4) after 12 months of therapy (Druker et al. 2003; Hughes et al. 2003). Quality-of-life analysis has demonstrated advantages of imatinib compared with IFN + cytoarabine as first- line treatment of CP CML. In addition, patients who cross over to imatinib from IFN-based therapies experience a significant improvement in quality of life (Hahn et al. 2003). There are a number of questions that have been answered definitively by the IRIS study. The study has shown that in terms of hematologic and cytogenetic responses, progression-free sur vival, and side effects, tol- erability, and quality of life, imatinib is superior to IFN plus low-dose c ytarabine. However, two important related questions have not definitely been answered by this a 6.2 · Imatinib Mesylate 105 Table 6.1. Rates of hematologic and cytogenetic responses with imatinib (Phase II-studies, update 2004, Kantarjian et al. 2002; Sawyers et al. 2002; Silver et al. 2004; Talpaz et al. 2002) Recruited Complete hematologic response (%) Major cytogenetic response (£ 35% Ph-positive metaphases) (%) Complete cytogenetic response (%) Chronic phase 532 96 66 55 Accelerated phase 235 40 28 20 Myeloid blast crisis 260 8.7 16 7.4 Fig. 6.1. Estimated response to first-line imatinib. CHR, complete hematologic response; MCR, major cytogenetic response (Ph+ £ 35%); CCR, complete hematologic response (Druker et al. 2006; O’Brien et al. 2003) Fig. 6.2. Estimated CCR to first-line Imatinib by Sokal Group (Guilhot 2004; O’Brien et al. 2003) study, namely (1) is imatinib superior to IFN plus cytarabine in terms of long-term survival? and (2) what will be the outcome of imatinib-treated patients in the longer term? By extrapolation from survival data of IFN-treated CML patients who achieved complete cytogenetic remissions, a 10-year survival rate of at least 51% was estimated for imatinib-treated patients (Hasford et al. 2005). As- suming the relat ionship between CCR and survival with IFN holds good for imatinib, the much higher CCR rates with imatinib therapy will result in an estimated 6.23 life- years gained compared w ith treatment with IFN plus low-dose cytarabine (Anstrom et al. 2004). It can be concluded that imatinib is superior to IFN with regard to response rate, progression-free survival and adverse effects. Comparison with historic data indi- cates a clear sur vival advantage of imatinib compared to IFN in patients with early CP CML (Kantarjian et al. 2003). Another critical issue of course is whether imatinib can cure CML. Current in vitro and in vivo data suggest that dormant or “quiescent” nondiv iding BCR-ABL- positive stem cells are not responsive to imatinib and may produce relapse after withdrawal of imatinib (Dru- ker et al. 2006; Graham et al. 2002). 6.2.2 Side Effects of Imatinib The majorit y of imatinib-treated patients experience adverse events at some time. Most events are of mild to moderate grade. The most frequently reported drug-related adverse events are nausea, vomiting, edema, and muscle cramps. Edema is most frequently periorbital or in lower limbs and is manageable with diuretics. The frequency of severe edema in phase I–III trials was 2–5%. Some of the adverse events observed are at- tributable to local or general fluid retention including pleural effusion, ascites, pulmonary edema, and rapid weight gain with or without superficial edema. The incidence of edema was dose and age related; it was about 20% higher for patients who received imatinib 600 mg/ day vs. 400 mg/day and for patients > 65 years of age (Table 6.2). Myelosuppression is common in CML patients treated with imat inib (more common in patients with advanced disease and in the init ial phase of therapy). In the phase III randomized trial of newly diag nosed patients in the CP treated with imatinib at 400 mg/day, grade 3 neutropenia (ANC <1 ´ 10 9 /l) was experienced by 11% of patients, grade 4 neutropenia (ANC <0.5´ 10 9 /l) occur red in 2% of patients, grade 3 thrombocytopenia (platelets < 50´ 10 9 /l) occurred in 6.9% of patients, and grade 4 thrombocytopenia (platelets <10´ 10 9 /l) occurred in less than 1% of patients (O’Br ien et al. 2003). Both in vitro and in vivo data indicate that inhibition of normal hematopoiesis during imatinib treatment is minimal; it is seen primarily as neutropenia and is large- ly restricted to high doses (Deininger et al. 2003). The much lower rate of infectious complications obser ved as compared to that expected in patients with a similar level of myelosuppression induced by conventional che- motherapy may be related to the lack of mucous mem- brane damage in patients on imatinib. Most patients, even patients with advanced phase CML, experience recovery of normal blood counts during continuous therapy with imatinib. Interventions with hematopoietic growth factors are under investigation. 106 Chapter 6 · Treatment with Tyrosine Kinase Inhibitors Fig. 6.3. Prognostic value of any cytogenetic response (CyR) at 6 months (Guilhot 2004; O’Brien et al. 2003) Fig. 6.4. Progression-free survival on first-line imatinib by molecular response (MR) at 12 months (Guilhot 2004; O’Brien et al. 2003) 6.2.3 Dosage of Imatinib The recommended dosage of imatinib is 400 mg/day for patients in CP CML and 600 mg/day for patients with AP or BC CML. Consecutive cohorts of patients treated with 400 mg and 600 mg imatinib demonstrated the advantage of 600 mg/day in advanced disease. Retrospec- tive analysis of prognostic factors showed that the 400 mg and 600 mg cohorts were well matched. Dose increases from 400 mg to 600 mg/day in patients with CP CML or from 600 mg to 800 mg/day in patients with advanced disease may be considered for patients with progressive disease, if a satisfactory hematologic response is not achieved after > 3 months of treatment, if cytogenetic remission is not achieved after 6–12 months of treatment or if a previously achieved hematologic or cytogenetic remission is lost (in the ab- sence of severe nonleukemia-related neutropenia or thrombocytopenia). The increase of imatinib dosage has been previously shown to improve response in patients with accelerated disease. Accelerated disease was found to have higher response rates with 600 mg imatinib than with 400 mg. Kantarjian et al. (2004) have reported in a historical comparison that higher cytogenetic and molecular remission rates can be achieved in shorter time intervals with an imatinib dosage of 800 mg daily as compared to 400 mg in CP CML. The disadvantage of the higher imatinib dose is a higher rate of adverse effects, in particular myelosuppression and fluid retention. It is unknown a 6.2 · Imatinib Mesylate 107 Table 6.2. Management of side effects from imatinib (Garcia-Manero et al. 2003) Side effect Management Nausea and/or emesis Avoid taking imatinib on an empty stomach Antiemetics (e.g., ondansetron at a dose of 8 mg orally or prochlorperazine at a dose of 10 mg orally 30 min prior to intake of imatinib) Adequate fluid intake Diarrhea Loperamide at a dose of 2 mg orally after each loose bowel movement (up to 16 mg daily) or diphenoxylate atropine at a dose of 20 mg orally daily in 3–4 divided doses Skin rashes Avoid sun exposure Topical steroids (e.g., 0.1% triamcinolone cream topically as needed) Systemic steroids (e.g., prednisone at a dose of 20 mg orally daily for 3–5 days) Muscle cramps Electrolyte substitution Tonic water (quinine) Mg 2+ replacements Bone aches Cox-2 inhibitors (e.g., celecoxib at a dose of 200 mg orally daily or rofecoxib at a dose of 25 mg orally daily) Liver function abnormalities Hold imatinib Resume within 1–2 weeks Consider decreasing the dose (no less than 300 mg orally daily) Myelosuppression Anemia Erythropoietin as needed Neutropenia G-CSF as needed Thrombocytopenia Hold if platelets £ 40 ´ 10 9 /L High-dose folic acid Interleukin-11 as needed Resume at lower dose level (no less than 300 mg orally daily) whether the effect of high-dose imatinib is sustained and provides a survival benefit. The dosage of imatinib should be adjusted or treat ment interrupted if severe neutropenia or thrombocytopenia occurs. Dose increase is suggested in case of suboptimal response, which is defined as: 1. Failure to achieve a complete hematologic response after 3 months, 2. failure to achieve any cytogenetic response after 6 months, or 3. failure to achieve a major cytogenetic response after 12 months of imatinib therapy (Druker et al. 2003; Hughes et al. 2003). High-dose imatinib therapy was tested in CML patients after IFN failure and in newly diagnosed patients. Cyto- genetic and molecular responses were achieved faster with 800 mg imatinib/day (Cortes et al. 2003; Kantarjian et al. 2004). The TIDEL multicenter study in Australia examined the effect of 600 mg/day among newly diagnosed patients with CP CML. Dose escalation to 800 mg/day was allowed if patients did not achieve a CHR at 3 months, an MCR at 6 months, a CCR at 9 months, or became PCR negative at 12 months. Additionally, patients who did not achieve hematologic or cytogenetic responses following dose escalation to imatinib 800 mg/day were allowed to add intermittent ara-C to their regimen. When compared with historical data from the IRIS study, a significantly higher proportion of patients in the TIDEL study achieved an MCR and CCR (Hughes et al. 2004). 6.2.4 Imatinib in Combination Combinations of imatinib with other drugs have been extensively analyzed invitro and have shown that a number of drugs are synergistic with imatinib in vitro. Of particular interest were the combinations of imatinib with IFN or low-dose ara-C. The feasibility of the combinations of imatinib with IFN (Pegasys, Peg-Intron) and low-dose cytarabine has been shown in phase I and II studies (Baccarani et al. 2004; Hochhaus et al. 2002). On the basis of these studies, randomized trials were designed by national study groups in Germany, France, UK, and USA to compare imatinib monotherapy at 400 mg with imatinib in various combinations (IFN, cytarabine) and dosages (600 mg, 800 mg). The first of these studies, the German CML Study IV, started re- cr uitment in July 2002 and compared imatinib 400 mg/d with imatinib+IFN, imatinib+cytarabine and imatinib after IFN failure in newly diagnosed patients with CP CML. By June 2006, 810 patients were randomized. According to the Hasford score, 35% of patients were low risk, 54% intermediate risk, and 11% high risk. Rate of progression was rare, within the first year 13/335 patients (6 low, 3 intermediate, 4 high risk; 4%) progressed to BC, 4 of them revealed clonal evolution (complex aberrant karyotype, n=3; +8, n=1), two other BCR-ABL mutations. Within the second year 3/232 patients progressed to BC. During the first year of treatment imatinib therapy was stopped due to side effects or resistance in 6% of patients in the imatinib 400 mg arm, in 2% of patients in the imatinib+IFN arm, and in 2% of patients in the imatinib+cytarabine arm. IFN was stopped in 21% and cytarabine in 18% of patients. The interim analysis of a prospective randomized trial with imatinib and imatinib in combination for newly diagnosed pat ients with CML has proven the feasibility of imatinib combinations in addition to high response and low progression rates (Berger et al. 2004). In September 2003, the French study was started which compares imatinib monotherapy at 400 mg vs. imatinib at 600 mg vs. imatinib plus IFN (Pegasys) vs. imatinib plus low-dose cytarabine. After an observation period, it is planned to reduce the study to two arms. The UK study compares imatinib monotherapy at 400 mg vs. imatinib at 800 mg vs. imatinib plus IFN (Pe- gasys). The USA study is focusing on the comparison of 400 mg and 800 mg imatinib therapy only. The emergence of resistance to imatinib monotherapy has led to a search for downstream targets of the Bcr-Abl kinase that may mediate the altered growth properties of BCR-ABL-transformed cells. Identification of signaling pathways downstream of ABL tyrosine kinase may increase our understanding of the pathogen- esis of CML and suggest strategies to improve clinical treatment of the disease. Farnesyl transferase inhibitors enhance the antiproliferative effects of imatinib against BCR-ABL-expressing cells, including imatinib-resistant cells. Cells resistant to imatinib because of amplification of BCR-ABL remain sensitive to tipifarnib and lonafarnib and co- treatment of these cells with imatinib plus farnesyl transferase inhibitors leads to enhanced antiproliferative or proapoptotic effects even in cells that are resistant to imatinib based on the expression of a Bcr-Abl kinase domain mutation (T315I) that is completely in- 108 Chapter 6 · Treatment with Tyrosine Kinase Inhibitors sensitive to imatinib. Although this mutant is sensitive to lonafarnib, the addition of lonafar nib to imatinib yielded no increase in antiproliferative effects. These results raise crit ically important issues of how and when to molecularly targeted agents should be combined for optimal results. Although the discussion that follows will focus on CML and Bcr-Abl signal transduction, this paradigm could be applied to any agent that targets signaling pathways (Peters et al. 2001). Early clinical studies using a combination of imatinib and farnesyl transferase inhibitors in advanced phase CML patients demonstrated feasibility but showed only moderate activity, probably due to clonal evolution with novel molecular or cytogenetic aberra- tions in addition to BCR-ABL not responding to farnesyl transferase inhibitors (Cortes et al. 2003). It is shown that the PI3-kinase/Akt pathway is a critical contributor to survival/proliferation of BCR-ABL- transformed cells. The serine/threonine protein kinase mTOR (mammalian target of rapamycin) is a downstream component of the PI3-Kinase/Akt pathway, and plays an important role in controlling cell g rowth and proliferation. The mTOR pathway is constitutively activated by Bcr-Abl in CML cells. Two of its known sub- strates, ribosomal protein S6 and 4E-BP1, are constitutively phosphorylated in a Bcr-Abl-dependent manner in BCR-ABL-expressing cell lines and CML cell lines. These data suggest that Bcr-Abl may regulate transla- tion of critical targets in CML cells via mTOR. The effect of rapamycin in three different imatinib- resistant Bcr-Abl mutant cell lines (Ba/F-BCR-ABL T315I, G250E, M351T) has been described. Rapamycin alone inhibited proliferation to a degree that would be predicted if mTOR was a critical downstream effector of Bcr-Abl, while the combination of low-dose rapamycin with imatinib markedly enhanced this growth inhibitory effect. The synergy between rapamycin and imatinib, occur- ring at doses well below typical serum levels obtained during monotherapy with each of these agents repre- sents a st rong argument in favor of investigating the clinical activ ity of the combinat ion (Ly Chi et al. 2003). 6.2.5 Imatinib Resistance Several questions remain open, notably those concern- ing the development of imatinib resistance, which is rare in early CP, but increases in frequency along the course of the disease (Hochhaus and La Rosée 2004). Essentially two mechanisms underlie the development of imat inib resistance: 1. Mutations of the ATP-binding site of the Bcr-Abl tyrosine kinase, and 2. clonal evolution with aberrant karyotypes ultimately leading to BC. Pharmacological mechanisms including activation of multidrug resistance proteins may cause variations in the individual intracellular imatinib concentrations, which may contribute to the development of resistance. etailed sequence analysis of the Bcr-Abl k inase domain has been performed to elucidate which mutations are responsible for the development of imatinib resistance. More than 30 different mutations have been rec- ognized which are detailed elsewhere (Hochhaus and La Rosée 2004; Shah et al. 2004). The prognostically most- serious mutations concern the so-called P-loop domain of the tyrosine kinase. P-loop mutations have been asso- ciated with an especially poor prognosis (Branford et al. 2003), but cessation of imatinib therapy and alternative therapy with other drugs seem to be able to improve prognosis. Novel methods are available to screen for small clones of mutated leukemic cells, e.g., denaturing high-performance liquid chromatography (D-HPLC). The impac t of the results of such assays needs to be explored in prospective clinical trials (Soverini et al. 2005). 6.2.6 Prediction of Response Attempts have been made to develop prognostic models to predict the outcome of CML patients on imatinib therapy. In CP patients after IFN failure, a low neutro- phil count and poor cytogenetic response at 3 months were identified as independent factors by investigators of the Hammersmith Hospital in London (Marin et al. 2003), but data are conflicting and were not confirmed by others (Lahaye et al. 2005). 6.3 Novel Bcr-Abl Inhibitors in Clinical Trials 6.3.1 Nilotinib (AMN107) Nilotinib (AMN107) is a novel aminopyrimidine, available as an oral formulation. It is a competitive inhibitor of the protein tyrosine k inase activity of Bcr-Abl and a 6.3 · Novel Bcr-Abl Inhibitors in Clinical Trials 109 prevents the activation of Bcr-Abl-dependent mitogenic and antiapoptotic pathways (e.g., PI-3 kinase and STAT5), resulting in death of the Bcr-Abl-induced phenotype. In cellular autophosphorylation assays and mediated cell proliferation, nilotinib is a highly potent inhibitor of the Bcr-Abl tyrosine kinase. An important feature of nilotinib is its ability to inhibit most imatinib-resistant mutant forms of BCR-ABL. Based on this activity, nilotinib may provide therapeutic benefit in patients w ith CML who have developed resistance to imatinib therapy due to mutations within the Bcr-Abl kinase. The effects of nilotinib on Bcr-Abl autophosphorylation have been evaluated in K562 and KU-812F human leukemia cell lines, which naturally express Bcr-Abl, as well as with p190 or p210-BCR-ABL transfected murine hematopoietic 32D and p210-BCR-ABL transfected Ba/F3 cells. In addition, the compound has been evaluated for effects on autophosphorylation in a panel of Ba/ F3 cells, expressing different mutant forms of the Bcr- Abl kinase. Nilotinib potently inhibits the Bcr-Abl kinase in cell lines derived from human leukemic CML cells and from transfected murine hematopoietic cells, with IC 50 values in the range of 20 nM to 60 nM. Nilotinib also potently inhibited most of the imatinib-resistant mutant forms of BCR-ABL. Thus, M237I, M244V, L248V, G250A (E, V), Q252H, E255D (K), E275K, E276G, E281K, K285N, E292K, F311V, F317L (C, V), D325N, S348L, M351T, E355 (G), A380S, L387F, M388L, F486S are inhibited with IC 50 values in the 1–200 nM range, Y253H, E255R (V), and F359C (V), are inhibited with IC 50 values between 200 nM and 800 nM, leaving only the T315I mutant un- affected by nilotinib at concentrations < 8000 nM. The selectivity of nilotinib as a protein kinase inhibitor has been demonstrated by its lack of appreciable activity (IC 50 value for inhibition of cell proliferation >3000 nM) against a panel of Ba/F3 cells transfected to express a variety of different kinases (Weisberg et al. 2005). Excellent efficacy in models of myeloproliferative disease was observed. In an acute model in which NOD-SCID mice were injected with murine 32D cells harboring the firefly luciferase gene and transfected to be dependent upon p210 Bcr-Abl, nilotinib (100 mg/kg QD) markedly reduced tumor burden, as assessed by noninvasive imaging. Furthermore, nilotinib (75 mg/ kg p.o., QD) prolonged the survival and reduced tumor burden, as assessed by spleen weights, of mice having either p2 10 or mutant (E255V, M351T) Bcr-Abl myeloproliferative disease. Nilotinib was also evaluated in a disease model using primary hematopoietic cells, in which mice were transplanted with bone-marrow cells transfected to express Bcr-Abl. Treated animals showed reduced morbidity and had spleen weights within the normal range. Similar, although slightly reduced efficacy was observed in mice receiving bone-marrow transplants after infection with either E255V or M351T Bcr-Abl (Weisberg et al. 2005). A Phase I/II study of nilotinib is currently ongoing in adult patients with imatinib-resistant CML in CP, AP, or BC relapsed/refractory Ph+ acute lymphoblastic leukemia, and other hematological malignancies. The phase I portion of this study has completed its enroll- ment and the phase-II portion is currently ongoing. During the phase I part of this study, 119 patients were initially treated in dose cohorts from 50 mg to 1200 mg on a once daily schedule with intrapatient dose escalation, and subsequently a twice daily dosing schedule with 400 mg and 600 mg cohorts. For the phase II part of the study, the 400 mg BID dose was selected on the basis of safety and acceptability; this improved serum exposure over once daily dosing in the phase I patients. In the Phase I study nilotinib was orally administered after a light breakfast and a 2-h fast in sequent ial cohorts of patients at escalating once daily doses of 50, 100, 200, 400, 600, 800, and 1200 mg. Since a limited increase in serum exposure to nilot inib occurred at higher dose levels, the protocol was amended to add a twice daily dosing schedule of 400 mg (800 mg/day) and 600 mg (1200 mg/day). Nilotinib doses, up to 1200 mg orally once per day or 400 mg orally twice 110 Chapter 6 · Treatment with Tyrosine Kinase Inhibitors Fig. 6.5. Chemical structure of imatinib, nilotinib, and dasatinib (Shah et al. 2004; Weisberg et al. 2005) [...]... 18 60 70 23 23 2 881 28 47 62 31 32 3 867 28 37 48 46 31 4 485 15 35 40 51 28 5 2 14 7 19 18 71 41 6 57 2 16 22 73 32 7 4 – – – – – 5-year probability of leukemia-free survival (LFS), survival, transplant-related mortality (TRM), and relapse incidence (RI) a 7 .4 · Prognostic Indicators for Transplant Outcome permits correction for patients with a high busulfan level to prevent potential toxicity) This... time 7 months) Response 8/17 15 /44 14. 8 months CP1 15% > CP1 12% Day 100 = 0% 3 deaths Day 100 = 0% Day 100 = 11% 1 yr 28.5% 2/15 NRM 30 months 36 months 42 months 12 months 30 months Median Followup 20 months 41 % at 18 months 5/17 Relapse at 2 years CP1 22% > CP1 64% 85% at 5 years 4 deaths from relapse 49 % at 5 years 1 relapse 3 BCR-ABL pos (median 8 months) DFS 24/ 44 6/17 CP1 70% > CP1 56% 85% at... of interferon and low-dose ara-C versus STI571 (imatinib) in patients with newly-diagnosed chronic phase chronic myeloid leukemia N Engl J Med 348 :9 94 10 04 Peters DG, Hoover RR, Gerlach MJ et al (2001) Activity of the farnesyl protein transferase inhibitor SCH66336 against BCR/ABL-induced murine leukemia and primary cells from patients with chronic myeloid leukemia Blood 97: 140 4– 141 2 Sawyers CL, Hochhaus... dasatinib has been shown to be a more potent inhibitor of Bcr-Abl (260-fold), c-kit (eightfold), PDGFRb (60-fold), and SRC (>1000-fold) than imatinib Dasatinib inhibits the Bcr-Abl kinase with an in vitro IC50 of 3 nM In cellular assays, dasatinib killed or inhibited the proliferation of all Bcr-Abl-dependent leukemic cell lines tested In vitro results suggest that dasa- 111 tinib is effective in reducing... Russo D, Pane F, Saglio G (20 04) GIMEMA Working Party on Chronic Myeloid Leukemia Imatinib and pegylated human re- 112 Chapter 6 · Treatment with Tyrosine Kinase Inhibitors combinant interferon-a2b in early CP chronic myeloid leukemia Blood 1 04: 4 245 42 51 Berger U, Engelich G, Reiter A, Hochhaus A, Hehlmann R and the German CML Study Group (20 04) Imatinib and beyond – the new CML-Study IV A randomised controlled... CML 186 24 (Crawley et al 2005) (Ruiz-Arguelles et al 2005) Bu/Cy/Fd Various Protocol 24 CP 118 CP1 26 CP2 30 AP 12BC Disease Response 41 yrs 18/ 24 molecular remissions 5/ 24 Cytogenetic 50 yrs 113 Sib 20 other related 52 VUD 24 Sib 86.9% Overall Age Donor 15 months Median Followup 35 months 8% Day 100 = 3.8% 2 yr = 18.8% NRM OS 20/ 24 at 27 months 92% at 27 months 33% at 3 yrs CP 43 % at 3 yrs 54% at 3... The interim efficacy analysis of the phase-I/II study revealed complete hematologic response in 92% of CP, 51% of AP, and 6% of BC patients Complete cytogenetic response was achieved in 35%, 14% , and 6% of patients, respectively (Kantarjian et al 2006) 6.3.2 Dasatinib (BMS 3 548 25) Dasatinib (BMS-3 548 25) is a potent, orally active inhibitor of the Bcr-Abl, c-KIT, and SRC family kinases It belongs to... Ottmann OG (2006) Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL N Engl J Med 3 54: 2 542 –2551 Lahaye T, Riehm B, Berger U, Paschka P, Müller MC, Kreil S, Merx K, Schwindel U, Schoch C, Hehlmann R, Hochhaus A (2005) Response and resistance in 300 patients with BCR-ABL-positive leukemias treated with imatinib in a single center: a 4. 5-year follow-up Cancer 103:1659–1669 Ly Chi,... 79:276–282 Ruiz-Arguelles GJ, Gomez-Almaguer D, Morales-Toquero A, GutierrezAguirre CH, Vela-Ojeda J, Garcia-Ruiz-Esparza MA, Manzano C, Karduss A, Sumoza A, de-Souza C, Miranda E, Giralt S (2005) The early referral for reduced-intensity stem cell transplantation in patients with Ph1 (+) chronic myelogenous leukemia in chronic phase in the imatinib era: results of the Latin American Cooperative Onco- a References... 10 18/32 patients 31/ 34 A/W (median follow-up of 100 months) Meloni et al 1990 (updated 2000) 21 Unmanipulated PBSC 11/17 patients 5-year survival of 56% Hoyle et al 19 94 20 Unmanipulated PBSC 13/20 patients 5-year survival of 75 ± 42 % Pigneux et al 1999 120 Unmanipulated PBSC/ MSC Not available 5-year survival of 58 ± 6% McGlave et al 19 94 316 Unmanipulated PBSC 70/207 patients 5-year survival of 71% . 23 2 881 28 47 62 31 32 3 867 28 37 48 46 31 4 485 15 35 40 51 28 5 2 147 19187 141 6 57 2 16 22 73 32 7 4 –––– 5-year probability of leukemia-free survival (LFS), survival, transplant-related mortality. constitutively activated by Bcr-Abl in CML cells. Two of its known sub- strates, ribosomal protein S6 and 4E-BP1, are constitutively phosphorylated in a Bcr-Abl-dependent manner in BCR-ABL-expressing. Lan- cet 359 :48 7 49 1 von Bubnoff N, Veach DR, Miller WT, Li W, Sanger J, Peschel C, Born- mann WG, Clarkson B, Duyster J (2003) Inhibition of wild-type and mutant Bcr-Abl by pyrido-pyrimidine-type