Intolerant Chronic Myelogenous Leukemia Research Articles

The European Medicines Agency Review of Bosutinib for the Treatment of Adult Patients With Chronic Myelogenous Leukemia: Summary of the Scientific Assessment of the Committee for Medicinal Products for Human Use

On March 27, 2013, a conditional marketing authorization valid throughout the European Union was issued for bosutinib (Bosulif) for the treatment of adult patients with chronic-phase, accelerated-phase, and blast-phase Philadelphia chromosome positive (Ph⁺) chronic myelogenous leukemia (CML) previously treated with one tyrosine kinase inhibitor or more and for whom imatinib, nilotinib, and dasatinib are not considered appropriate treatment options. Bosutinib is a kinase inhibitor that targets the BCR-ABL kinase. The recommended dose is 500 mg of bosutinib once daily. The main evidence of efficacy for bosutinib was based on a CML subgroup analysis of study 3160A4-200, a phase I/II study of bosutinib in Ph⁺ leukemia in imatinib-resistant or intolerant CML. The subgroup was defined based on the presence of a BCR-ABL kinase domain mutation that would be expected to confer resistance to dasatinib (F317, E255) or nilotinib (E255, Y253, F359) and expected to have sensitivity to bosutinib or based on the presence of medical conditions or prior toxicities that may predispose the patient to unacceptable risk in the setting of nilotinib or dasatinib therapy. A conditional marketing authorization was granted because of the limited evidence of efficacy and safety currently supporting this last-line indication.

Efficacy and safety of nilotinib in Japanese patients with imatinib-resistant or -intolerant Ph+ CML or relapsed/refractory Ph+ ALL: a 36-month analysis of a phase I and II study

Although the tyrosine kinase inhibitor (TKI) imatinib is often used as first-line therapy for newly diagnosed chronic myelogenous leukemia (CML), some patients fail to respond, or become intolerant to imatinib. Nilotinib is a potent and selective second-generation TKI, with confirmed efficacy and tolerability in patients with imatinib-resistant or -intolerant CML. A phase I/II study was conducted in Japanese patients with imatinib-resistant or -intolerant CML or relapsed/refractory Ph+ acute lymphoblastic leukemia. Thirty-four patients were treated with nilotinib for up to 36 months. Major cytogenetic response was achieved in 15/16 patients (93.8%) with chronic-phase CML within a median of approximately 3 months. Major molecular response was achieved in 13/16 patients (81.3%). These responses were sustained at the time of the most recent evaluation in 13 patients and 11 patients, respectively. Hematologic and cytogenetic responses were also observed in patients with advanced CML. The BCR-ABL mutation associated with the most resistance to available TKIs, T315I, was observed in three patients. Common adverse events included rash, nasopharyngitis, leukopenia, neutropenia, thrombocytopenia, nausea, headache and vomiting. Most adverse events resolved following nilotinib dose interruptions/reductions. These results support the favorable long-term efficacy and tolerability of nilotinib in Japanese patients with imatinib-resistant or -intolerant chronic-phase chronic myeloid leukemia.

Nilotinib: A Novel, Selective Tyrosine Kinase Inhibitor

The development of tyrosine kinase inhibitors (TKIs) for the treatment of chronic myelogenous leukemia (CML) was based on the discovery that CML stem and progenitor cells overexpress the abnormal fusion protein kinase BCR-ABL. The prototype TKI, imatinib, selectively inhibits BCR-ABL, as well as several other kinases, including stem cell factor receptor (KIT), discoidin domain receptor (DDR), platelet-derived growth factor receptor (PDGFR), and colony-stimulating factor receptor-1 (CSF-1R). Although the management of CML improved dramatically with the introduction of imatinib, not all patients benefit from treatment because of resistance or intolerance. Consequently, research efforts have focused on developing more potent TKIs with the ability to circumvent imatinib resistance. Nilotinib, a second-generation oral TKI, was rationally designed based on the crystal structure of imatinib to be highly active against a wide range of imatinib-resistant BCR-ABL mutants and is approved for the treatment of newly diagnosed or imatinib-resistant or -intolerant CML, and has shown superiority over imatinib in first-line treatment for newly diagnosed CML. Furthermore, the activity of nilotinib against KIT and PDGFRα has led to its evaluation in advanced gastrointestinal stromal tumors (GIST). The purpose of this review is to describe the development of nilotinib, providing a structural explanation for the differential activity of nilotinib and imatinib in GIST. Activity of nilotinib against KIT and PDGFR and emerging evidence of differences in cellular uptake between nilotinib and imatinib are discussed.

P-glycoprotein and Breast Cancer Resistance Protein Influence Brain Distribution of Dasatinib

The novel tyrosine kinase inhibitor dasatinib (Sprycel; BMS-354825) is approved for use in imatinib (Gleevec; STI 571)-resistant or -intolerant chronic myelogenous leukemia and may be useful for other tumors in the central nervous system (CNS). The objective of this study was to investigate the role of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in modulating the CNS penetration of dasatinib. Results from the in vitro studies indicate that cellular delivery of dasatinib is significantly limited by active efflux due to both P-gp and BCRP. Permeability studies indicated greater permeability in the basolateral-to-apical direction than in the apical-to-basolateral direction due to active efflux by P-gp or BCRP. Selective inhibitors of P-gp and BCRP, such as (R)-4-((1aR,6R,10bS)-1,2-difluoro-1,1a,6,10b-tetrahydrodibenzo-(a,e)cyclopropa(c) cycloheptan-6-yl)-alpha-((5-quinoloyloxy)methyl)-1-piperazineethanol, trihydrochloride (zosuquidar; LY335979) and 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12alpha-octahydropyrazino1',2': 1,6pryrido3,4-bindol-3-yl)-propionic acid tert-butyl ester (Ko143), were able to restore the intracellular accumulation and abolish the directionality in net flux of dasatinib. In vivo brain distribution studies showed that the CNS distribution of dasatinib is limited, with the brain-to-plasma concentration ratios less than 0.12 in wild-type mice, which increased approximately 8-fold in Mdr1a/b(-/-) Bcrp1(-/-) mice. Dasatinib brain distribution was significantly increased in Mdr1a/b(-/-) mice and when wild-type mice were pretreated with LY335979. Simultaneous inhibition of P-gp and BCRP by elacridar [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] (GF120918) resulted in a 5-fold increase in brain concentration. These in vitro and in vivo studies demonstrate that dasatinib is a substrate for the important efflux transporters p-glycoprotein and BCRP. These transport systems play a significant role in limiting the CNS delivery of dasatinib and may have direct implications in the treatment of primary and metastatic brain tumors.

Dasatinib Is An Effective Inhibitor of Proliferation and Inducer of Apoptosis in the KASUMI Cell Line Bearing the T(8;21)(q22;q22) and the N822K KIT Mutation

Introduction. Within the group of core binding factor (CBF) AML, the presence of the t(8;21)(q22;q22) confers a favorable prognosis based on high complete remission rates and high survival probabilities. However within this subgroup the presence of KIT mutations and in some studies specifically mutations at codon 816 in exon 17 have been associated with inferior event free survival, relapse free survival, cumulative incidence of relapse and overall survival. Dasatinib a dual SRC/ABL kinase inhibitor is an active agent already approved for the treatment of imatinib resistant or intolerant chronic myelogenous leukemia which has shown in vitro activity against KIT exon 17 mutations including the D816 imatinib resistant mutation. The aim of the present study was the investigation of the activity of dasatinib on cell proliferation and apoptosis of leukemic cell lines with or without KIT mutations.Materials and methods. The leukemic cell lines ME-1, NB4 and KASUMI were cultured in RPMI. Following RNA extraction RT-PCR was performed for the amplification of the extracellular (exon 8,9), transmembrane/juxtamembrane (exon 10,11) and tyrosine kinase 2 domains (exon 17,18) of c-Kit.Following sequencing only the KASUMI cell line derived from a t(8;21)(q22;q22) AML was found to bear the N822K KIT mutation at exon 17, also described in patients'samples. The KASUMI, the K562 cell line bearing the t(9;22) used as a positive control and the NB4 cell line without KIT mutations used as a negative control, were subsequently cultured under the presence of dasatinib at the concentrations of 1nM, 10nM, 100nM, 500 nM. Cell proliferation, was determined at 24, 48, 72 h using the Cell Proliferation Elisa, BrDU protocol and apoptosis was determined by the method of annexin using flow cytometry at the same time points.Results The BrDU value of K562 cells at 48h without the drug was 1.046 significantly higher compared to those of cells cultured under the presence of Dasatinib at 1nM, 10nM, 100nM, 500 nM (0.6485, 0,5647, 0,4770, 0.4755 respectively) (p<0.001). Similarly the BrDU value of K562 cells without the drug at 72h was 1.320 significantly higher to those under the presence of the drug at 10, 100, 500 nM (0.8137, 0.7292, 0.6637 respectively) (p<0.001). The level of apoptosis was significantly induced by the drug at all concentrations at 24h(p<0.001) and at the concentrations of 10nM, 100nM, 500 nM at 48h (p<0.001) but not at 72h.Ôhere was no effect of the drug on the proliferation and apoptosis of the NB4 cell line. In the KASUMI cells there was a significant reduction of the BrDU values by the presence of dasatinib at the concentrations of 10nM, 100nM, 500nM at 48h (0.9517 vs 0.6462, 0.5653, 0.3467, p=0.038, 0.011, 0.002 respectively). The same was true at the concentrations of 100nM and 500nM at 72h (0.9538 vs 0.2412, 0.1907, p=0.002, 0.004 respectively). Dasatinib significantly increased the level of apoptosis of the KASUMI cells at 24h at 1nM, 10nM, 100nM (2.45 vs 1.41, 1.71, 2.18, p<0.001, <0.001, 0.026 respectively) At 48h dasatinib significantly increased the level of apoptosis at the concentrations of 1nM, 10nM, 100nM (0.84vs 1.03, 1.49, 2.81, p=0.02, p<0.001, p<0.001 respectively). At 72h there was a significant induction of apoptosis by the drug at all concentrations (0.16 vs 1.11, 1.94, 2.93, 1.88 p<0.001)Conclusion. Dasatinib is an effective suppressor of proliferation and inducer of apoptosis of the KASUMI cell line with the t(8;21)(q22;q22) and the N822K KIT mutation. These encouraging results need to be confirmed on patients' cells with the view to integrate the drug in conventional chemotherapy regimens in future clinical trials.

Efficacy and safety of dasatinib in imatinib-resistant or -intolerant patients with chronic myeloid leukemia in blast phase

Dasatinib is an inhibitor of BCR-ABL and SRC-family kinases for patients with imatinib-resistant or -intolerant chronic myelogenous leukemia (CML). In this international phase II trial, dasatinib was administered orally (70 mg twice daily) to patients with myeloid blast phase (MBP, n=109) or lymphoid blast phase (LBP, n=48) CML. After a minimum follow-up of 12 months (range 0.03-20.7 months), major hematologic responses were induced in 34% (MBP-CML) and 35% (LBP-CML) of patients. Major cytogenetic responses were attained in 33% (MBP-CML) and 52% (LBP-CML) of patients and complete cytogenetic responses were attained in 26 and 46%, respectively. Median progression-free survival was 6.7 (MBP-CML) and 3.0 (LBP-CML) months. Median overall survival was 11.8 (MBP-CML) and 5.3 (LBP-CML) months. Overall, dasatinib had acceptable tolerability. Fluid retention events were more frequent in the MBP-CML than the LBP-CML cohort: pleural effusion occurred in 36 and 13% (all grades) and 15 and 6% (grades 3/4), respectively. Other non-hematologic side effects were primarily grade 1/2; grade 3/4 events were recorded in <or=6% of patients, except febrile neutropenia (15%). Cytopenias were noted in the majority of patients, and were manageable with dose interruptions/reductions. Dasatinib is associated with a promising rate of response in this high-risk population.

Dasatinib dosing strategies in Philadelphia chromosome-positive leukemia

Imatinib resistance has emerged as a significant clinical issue in the treatment of chronic myelogenous leukemia (CML) and Philadelphia chromosome positive acute lymphocytic leukemia (Ph+ ALL). Imatinib intolerance is also a concern. Dasatinib is approved for the treatment of patients with imatinib-resistant or -intolerant CML or Ph+ ALL and has helped address these concerns. Here, we review the clinical profile of dasatinib and discuss dosing strategies to manage these adverse events. References were obtained through literature searches on PubMed as well as from the Proceedings of Annual Meetings of the American Society of Clinical Oncology, the American Society of Hematology, and European Hematology Association. Initial studies of dasatinib showed that 70 mg twice-daily resulted in impressive hematologic and cytogenetic responses in patients with all phases of CML and Ph+ ALL. Aside from hematologic events, the most common drug-related toxicities (mostly grade 1-2) in dasatinib clinical trials were fluid retention (including pleural effusion), diarrhea, skin rash, headache, hemorrhage, fatigue, nausea and dyspnea. A Phase III dose optimization study showed that the safety profile of dasatinib is improved and efficacy maintained when administered on a 100 mg once-daily dosing schedule compared with the 70 mg twice-daily schedule in patients with chronic phase (CP) CML. The safety profile of dasatinib is manageable. A 100 mg once-daily dose is now approved for use in patients with CP CML. A dose of 70 mg twice daily remains the recommended dosage for use in patients with advanced phase CML or Ph+ ALL.

Nilotinib in patients with imatinib-resistant or -intolerant chronic myelogenous leukemia in chronic phase (CML-CP): Updated phase II results

7010 Background: Nilotinib, a potent and highly selective BCR-ABL inhibitor, has been approved in both the US and Europe for the treatment of patients (pts) with CML-CP and accelerated phase CML (CML-AP) who are resistant or intolerant to prior therapy, including imatinib. Methods: This phase 2, open-label study was designed to evaluate the efficacy and safety of nilotinib in pts with Philadelphia chromosome-positive (Ph+) imatinib-resistant or -intolerant CML-CP. Nilotinib was administered at 400 mg twice daily (BID), with the possibility to dose escalate to 600 mg BID for inadequate responses. The primary endpoint was the rate of major cytogenetic response (MCyR). Results: Included in this analysis were 321 pts (71% imatinib-resistant; 29% imatinib-intolerant). 72% of pts received prior imatinib doses > 600 mg. Of the 206 pts who did not have complete hematological remission (CHR) at baseline, 158 (77%) achieved CHR during nilotinib therapy. Overall, MCyR was observed in 184 pts (57%); 41% had complete CyR. MCyR was observed in 125 (55%) of the 227 imatinib-resistant pts and in 59 (63%) of the 94 imatinib-intolerant pts. The median time to CHR and MCyR was 1.0 and 2.8 months, respectively. The majority of pts (84%) maintained MCyR for at least 18 months. At 18 months, the estimated overall survival rate was 91%. Treatment with nilotinib is ongoing in more than half of pts enrolled. The most frequent grade 3/4 laboratory abnormalities were thrombocytopenia (28%), neutropenia (30%), and asymptomatic serum lipase elevation (15%). Grade 3/4 non-hematologic AEs were rare and included rash, headache, and diarrhea. A low incidence of QT prolongation (QTcF > 500 msec, < 1%) was observed overall. The safety profile of nilotinib has not changed with increased follow up. There was minimal cross-intolerance between nilotinib and imatinib in pts intolerant to prior imatinib treatment. Conclusions: The present study confirms that nilotinib induces significant and durable responses in CML- CP pts with imatinib-resistance or -intolerance. Nilotinib is well tolerated, with minimal occurrence of grade 3/4 AEs and minimal cross-intolerance to imatinib, and is a valuable new treatment option for this pt population. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Bristol-Myers Squibb, Novartis Bristol-Myers Squibb, Novartis Bristol-Myers Squibb, Innovive, Merck, Novartis, Wyeth

Analysis of Glucose Profiles in Imatinib-Resistant or Intolerant Chronic Myelogenous Leukemia (CML) Patients (pts) Treated with Nilotinib: Lack of Correlation between Glucose Levels and Nilotinib Efficacy.

Background: Nilotinib is an oral selective BCR-ABL inhibitor, more potent than imatinib (IC50 <30 nM) and active against 32/33 BCR-ABL imatinib-resistant mutants. Recent phase I/IIa study results demonstrated that nilotinib is well tolerated and has significant efficacy in imatinib-resistant or intolerant Ph+CML pts. Although nilotinib and imatinib have similar chemical structure and mechanism of action, differences in safety profiles, including effects on glucose metabolism, have been observed. In the pivotal phase II nilotinib study, the incidence of Grade 3/4 hyperglycemia was 10.7% in CP and 4.4% in AP pts. No pts required dose adjustment and/or interruption/discontinuation due to hyperglycemia. A recent publication involving small number of pts (n=13) in the phase I trial, suggested that blood glucose (BG) levels could be used as an indirect predictor for response to nilotinib (1).Methods: Based on this observation, a post-hoc analysis of BG levels in the pivotal phase II study investigating safety and efficacy of nilotinib in imatinib-resistant or -intolerant CML pts was conducted.Results: This analysis included 447 pts [51% male; median age 58 (21–85) yrs]. BG levels were collected once weekly for the first 2 mos and then once every 2 wks for the remainder of the study period. Only 20% were fasting samples. The median treatment durations were 341 (CP) and 190 days (AP). Although some pts has increase in non-fasting BG levels during nilotinib treatment, mean BG for the entire cohort (n=477) were within normal fasting range (mean 82.5±61.0 mg/dL). At baseline, 2 (0.6%) CP and 1 (0.8%) AP pt had Grade 3/4 hyperglycemic events (BG >250 mg/dL). During the nilotinib treatment period, 12.5% of CP and 6.7% of AP pts had Grades 3/4 hyperglycemic events. No episodes of diabetes-related serious adverse events occurred (ketoacidosis, hyperosmolar coma or diabetes-related hospitalization) and no pt discontinued nilotinib due to hyperglycemia. CP pts who presented with elevated BG had higher body mass index (BMI >30), p=0.0442. There was no significant increase in mean body weight during the treatment period in either CP or AP pts. There was no correlation between high BG levels (Grade 3/4) and response to nilotinib treatment in either the CP or AP cohorts.Conclusion: There were no clinically significant changes in mean BG levels of pts receiving nilotinib. The highest BG levels occurred in pts with increased BMI. Although an increase in the incidence of Grade 3/4 hyperglycemia was observed, no hyperglycemia complications occurred. Importantly, no correlation was found between BG levels and nilotinib response. Based on the results from this large phase II study, BG levels cannot be used to predict hematological or cytogenetic responses to nilotinib. Breccia M, et al. Leuk Res. 2007 Mar 21; [Epub ahead of print]CP (n=320)AP (n=127)BL %Tx %PBL %Tx %PDiabetes mellitus10--6.3--Insulin2.23.20.0252.42.4nsOral anti-diabetic5.69.30.00024.75.5nsBG (mg/dL)73.2 ±57.082.6 ±61.4

A phase II study of nilotinib administered to patients with imatinib resistant or intolerant chronic myelogenous leukemia (CML) in chronic phase (CP), accelerated phase (AP) or blast crisis (BC) who also failed dasatinib

7038 Background: Nilotinib and dasatinib are two new second generation tyrosine kinase inhibitors used in the treatment of imatinib-resistant/intolerant CML. There is limited data regarding the efficacy and safety of these therapies when given sequentially. Methods: This phase II open-label study was designed to evaluate the safety and efficacy of nilotinib at a dose of 400mg BID in CML-CP, -AP, and -BC pts who previously received and either failed or were intolerant to imatinib and dasatinib. Results: A total of 42 pts are reported - CP (16), AP (9), and BC (17 total; 13 myeloid, 4 lymphoid). Overall 10 (24%) pts had extramedullary disease at baseline. For all pts, median time from first diagnosis was 16.8 (0.6–265) months. The median duration of nilotinib exposure was 81 days with median dose intensity of 800mg/day. A total of 13 (31%) pts with dasatinib failure remain on treatment, 29 (69%) discontinued (6 for AEs, 16 for disease progression). CP: Of the 16 pts, 5 (31%) had a MCyR (3 complete, 2 partial). Complete hematologic response (CHR) was reported in 5/13 (39%) pts without CHR at baseline. Disease progression only occurred in 2 pts. AP: 2/9 (22%) pts had a return to chronic phase (RTC), 6 pts were not evaluable and there was 1 death. Disease progression occurred in 5 pts. BC: 3/17 (18%) achieved CHR, 1 (6%) had RTC, and 4 (24%) pts had disease progression. For all pts, the most common Grade 3/4 AEs reported were thrombocytopenia (26%), neutropenia (24%), and anemia (7%) pts. Conclusions: Nilotinib has significant clinical activity in CML-CP, AP, and BC patients who have failed imatinib and dasatinib. Nilotinib is safe and well tolerated, consistent with its kinase selectivity profile. No significant financial relationships to disclose.

Nilotinib is associated with minimal cross intolerance to imatinib in patients with imatinib-intolerant chronic myelogenous leukemia (CML) in chronic phase (CP)

7039 Background: Nilotinib is a highly selective Bcr-Abl tyrosine kinase inhibitor that is 30-fold more potent than imatinib and is an important therapeutic option for patients (pts) who have either imatinib-resistant or -intolerant CML disease. Results of a subset of pts with Ph+ CML-CP who received nilotinib for imatinib-intolerance are reported Methods: Pts were part of a phase II open-label study evaluating the safety and efficacy of nilotinib in imatinib-resistant or -intolerant CML-CP. Imatinib intolerance was defined as no MCyR and discontinuation of imatinib due to Grade 3/4 AEs or persistent (&gt; 1 mo) or recurrent Grade 2 AE (recurred &gt; 3 times) despite optimal supportive care. The proportion of pts achieving MCyR was the primary endpoint, and safety and toxicity were secondary endpoints. Planned starting dose was nilotinib 400mg BID. Results: Of the 316 pts with CML-CP enrolled, 95 (30%) pts had imatinib-intolerance for either non-hematologic and/or hematolgic AEs. Some pts had more then one AE satisfying the criteria for intolerance. The frequency of intolerant symptoms for imatinib and nilotinib is shown in the table below. Only 2/80 (3%) pts with non-hematologic imatinib-intolerance experienced a recurrence of similar symptoms during nilotinib therapy. 33 patients entered the study due to hematologic intolerance (neutropenia, thrombocytopenia) and only 7/33 (21%) developed similar problems. In 86 pts with intolerance and at least 6 mos of follow up, 55% of these patients achieved MCyR, similar to that previously reported for imatinib-resistant patients. Conclusions: These are the first reported results evaluating cross intolerance symptoms to TKIs. The results demonstrate that nilotinib is effective in imatinib-intolerant CML-CP and has a very low rate of cross- intolerance, further supporting the excellent tolerability of nilotinib No significant financial relationships to disclose. [Table: see text]

A Phase II Study of Nilotinib, a Novel Tyrosine Kinase Inhibitor Administered to Patients with Imatinib Resistant or Intolerant Chronic Myelogenous Leukemia (CML) in Chronic Phase (CP), Accelerated Phase (AP) or Blast Crisis (BC) Who Have Also Failed Dasatinib Therapy.

Nilotinib is a potent, highly selective, aminopyrimidine inhibitor which in vitro is 30-fold more potent than imatinib and active against 32/33 imatinib resistant cell lines with Bcr-Abl mutations. This open-label study was designed to evaluate the efficacy and safety as defined by hematologic and cytogenetic response rates (HR/CyR) of nilotinib administered at a daily dose of 400 mg bid to patients who previously received and either failed or were intolerant to imatinib and dasatinib. A total of 50 patients were enrolled and included CP (n=26), AP (n=10), and BC (n=14) patients. Of the 14 BC patients 10 were myeloid and 4 were lymphoid. Overall 8 (16%) patients had extramedullary disease at baseline. The median age was 57 (range 19–78) years and the median time from first diagnosis of CP, AP, or BC to treatment was 65 (range 8–213) months. The median duration of nilotinib exposure was 226 (range 3–379) days. A total of 28 (56%) patients remain on treatment, 22 (44%) discontinued (6 for adverse events, 11 for disease progression, 3 withdrew consent, and there were 2 deaths related to cerebral hemorrhages). Complete (HR) was reported in 9 of the 20 (45%) CP patients who did not have a CHR at baseline. Of the 26 CP patients, 8 (31%) had a major CyR (2 complete and 6 partial), 2 (8%) patients had minimal responses, and 9 (35%) patients had no response. Disease progression occurred in one CP patient and 6 patients were not evaluable. Confirmed hematologic responses were observed in 2 of 10 (20%) AP patients who had a return to chronic phase (RTC), 7 patients were not evaluable and there was one patient death. Of the 10 AP patients 1 (10%) each had CyR (partial, minor, minimal and no response). Of the 14 BC patients 1 had a CHR (7%), 2 (14%) had a return to chronic phase (RTC), 6 (43%) were not assessable for response, and 5 (36%) had progressive disease. Overall the most frequent Grade 3 or 4 adverse events reported were thrombocytopenia in 12 (24%), neutropenia in 11 (22%), and anemia in 5 (10%) patient. In summary, nilotinib has significant clinical activity and an acceptable safety and tolerability in CML-CP, AP, and BC patients who were resistant or intolerant to imatinib and have also failed dasatinib therapy.

Clinical pharmacokinetics (PK) of AMN107, a novel inhibitor of Bcr-Abl, in healthy subjects and patients with imatinib resistant or intolerant chronic myelogenous leukemia (CML) or relapsed/refractory Ph+ acute lymphocytic leukemia (Ph+ALL)

3095 Background: AMN107, a selective and potent inhibitor of Bcr-Abl tyrosine kinase, has demonstrated activity against imatinib resistant forms of Bcr-Abl in patients with CML or Ph+ALL in a phase I study. This study characterizes clinical PK of AMN107 for the safe and effective use of the drug. Methods: Serum samples were collected in 119 patients with CML or Ph+ALL treated with once (QD) or twice daily (BID) oral AMN107 (doses of 50 to 1200 mg/day). PK parameters were derived from serum concentration-time data by non-compartmental methods. In separate multi-period crossover studies 92 healthy subjects were treated with single doses of AMN107 to assess the food effect on PK, and to assess the potential for drug-drug interactions with medications which inhibit (ketoconazole) or are metabolized by CYP3A4 (midazolam). Results: Steady-state AMN107 PK in patients were stable over time, and showed a dose-dependency with less than dose-proportional increases in systemic exposure at dose levels higher than 400 mg QD. Daily exposure at 400 mg BID, which maintains serum AMN107 levels at least 30 times higher than the IC50 of cellular phosphorylation of Bcr-Abl, was greater than at 800 mg QD. The effective half-life was 15 hours. Bioavailability of AMN107 administered with a high-fat meal was increased by 82% compared to fasting. CYP3A4 inhibition by concomitantly administered ketoconazole produced 3-fold increases in systemic exposure to AMN107, whereas AMN107 appeared a weak inhibitor of CYP3A4 with 30% increase in systemic exposure of midazolam given concomitantly. Conclusions: Systemic AMN107 levels at an oral AMN107 dose of 400 mg BID in patients with CML or Ph+ALL were well above the IC50 of Bcr-Abl. The food/drug interaction results provide further guidance for the safe use of AMN107 in the clinical setting. [Table: see text]

Phase II Study of Low-Dose Decitabine in Patients With Chronic Myelogenous Leukemia Resistant to Imatinib Mesylate

To determine the activity of decitabine, a DNA methylation inhibitor, in imatinib-refractory or intolerant chronic myelogenous leukemia. Thirty-five patients were enrolled in this phase II study (12 in chronic phase, 17 in accelerated phase, and six in blastic phase). Decitabine was administered at 15 mg/m2 intravenously over 1 hour daily, 5 days a week for 2 weeks. DNA methylation was measured using a LINE1 bisulfite/pyrosequencing assay. Complete hematologic responses were seen in 12 patients (34%) and partial hematologic responses in seven patients (20%), for an overall hematologic response rate of 54% (83% in chronic phase, 41% in accelerated phase, and 34% in blastic phase). Major cytogenetic responses were observed in six patients (17%), and minor cytogenetic responses were seen in 10 patients (29%) for an overall cytogenetic response rate of 46%. Median response duration was 3.5 months (range, 2 to 13+ months). Myelosuppression was the major adverse effect, with neutropenic fever in 28 (23%) of 124 courses of therapy. LINE1 methylation decreased from 71.3% +/- 1.4% (mean +/- standard error of the mean) to 60.7% +/- 1.4% after 1 week, 50.9% +/- 2.4% after 2 weeks, and returned to 66.5% +/- 2.7% at recovery of counts (median, 46 days). LINE1 methylation at the end of week 1 did not correlate with subsequent responses. However, at day 12, the absolute decrease in methylation was 14.5% +/- 3.0% versus 26.8% +/- 2.7% in responders versus nonresponders (P = .007). Decitabine induces hypomethylation and has clinical activity in imatinib refractory chronic myelogenous leukemia. We hypothesize that the inverse correlation between hypomethylation 2 weeks after therapy and response is due to a cell death mechanism of response, whereby resistant cells can withstand more hypomethylation.

Hypomethylation Dynamics Following Decitabine Therapy in Chronic Myelogenous Leukemia.

Decitabine (5-aza-2′-deoxycytidine, DAC) is a cytidine analogue that inhibits DNA methylation by trapping DNA methyltransferases. DAC is cytotoxic at high doses, hypomethylating at low doses, and has clinical activity in myeloid malignancies that appears to be optimal at low doses. In a phase II study, we have been evaluating the activity of single agent DAC (at 15 mg/m2 IV over one hour daily, 5 days a week for two weeks) in Imatinib refractory or intolerant chronic myelogenous leukemia (CML). Thirty five patients were enrolled (12 in chronic phase [CP], 17 in accelerated phase [AP] and 6 in blastic phase [BP]). Complete hematologic responses were seen in 12 patients (34%), partial hematologic responses in 7 patients (20%) and hematologic improvement in 4 patients (11%), for an overall hematologic response rate of 65% (83% in CP, 59% in AP and 50% in BP). Major cytogenetic responses were observed in 7 patients (20%), and minor cytogenetic responses were seen in 9 patients (26%) for an overall cytogenetic response rate of 46%. Median response duration was 3 months, ranging from 2 to 13+ months, and responses are ongoing in 4 patients. The only common grade 3 or 4 toxicities observed were related to myelosuppression. Neutropenic fever developed in 18 patients (51%), in 27/115 (23%) courses of therapy. There were two deaths on study, both related to thrombocytopenia and hemorrhage. We used a bisulfite/pyrosequencing assay to measure DNA methylation across LINE repetitive elements in peripheral blood mononuclear cells as a surrogate marker for global methylation in CML. LINE methylation had a mean +/− standard error of the mean of 71.2+/−1.2% prior to therapy (range 56.5–85.8). Methylation decreased to 60.1+/−1.44% after 1 week, 49.7+/−2.9 after 2 weeks, 50.1+/−2.7% after 3 weeks and returned to 67.2+/−2.1% at recovery of counts (median, 46 days). The dynamics of hypomethylation following DAC (decrease of about 10% at week 1, 10–20% at weeks 2 and 3 and recovery by week 6) were similar after cycle 2 and cycles >2, with no evidence of cumulative hypomethylation or development of resistance to the hypomethylating effect of DAC. LINE methylation at the end of week 1 did not correlate with subsequent responses. However, the degree of LINE hypomethylation at the end of therapy (day 12) was paradoxically higher in patients who did not subsequently respond to therapy. Thus, the absolute decrease in methylation was 17.7% vs. 28.6% in responders vs. non-responders (p=0.009) and the relative decrease in methylation was 25.0% vs. 39.5% responders vs. non-responders (p=0.016). These results are consistent with a cell death mechanism of response, whereby the most hypomethylated cells die rapidly in patients sensitive to therapy, while cells resistant to DAC can withstand higher degrees of hypomethylation without cell death. Consistent with this concept, in patients who responded and subsequently lost their response while on therapy, the degree of hypomethylation achieved in the cycle immediately prior to relapse was similar or higher than that achieved in prior cycles, indicating that DAC resistance in this population is not pharmacologic, but likely biologic resistance to hypomethylation-induced cell death. We conclude that DAC induces hypomethylation in-vivo and has significant clinical activity in Imatinib refractory CML. The inverse correlation between hypomethylation at day 12 and response is consistent with a cell death mechanism of action of DAC in CML.

The SRC/ABL Inhibitor BMS-354825 Overcomes Resistance to Imatinib Mesylate in Chronic Myelogenous Leukemia Cells through Multiple Mechanisms.

BCR-ABL is an oncogenic tyrosine kinase expressed in chronic myelogenous leukemia (CML) cells and is the main target of the tyrosine kinase inhibitor imatinib mesylate. Imatinib-based CML therapy induces hematological and cytogenetic remission in early phase CML patients whereas more advanced patients frequently develop resistance to imatinib by multiple mechanisms, including mutations in the BCR-ABL kinase domain and over-expression of tyrosine kinases that are not inhibited by imatinib. These observations suggest that dual inhibition of src and abl kinases may circumvent imatinib resistance and provide more effective therapy for CML. BMS-354825 is a novel tyrosine kinase inhibitor that inhibits both abl and src kinases at low nM concentrations and is currently being clinically evaluated in imatinib resistant or intolerant CML patients. Our earlier studies demonstrated that increased expression of the src-related kinase Lyn in BCR-ABL expressing K562 cells was associated with imatinib resistance in this cell model and some CML patients. To determine whether inhibition of SRC/ABL kinases differentially affects imatinib sensitive K562 (BCR-ABL +, Lyn −) and resistant K562R (BCR-ABL +, Lyn +) cells were treated with imatinib or BMS-354825 before analysis of cell growth, survival and signaling. BMS-354825 induced apoptosis in both K562 and K562R cells which correlated with inhibition of both Lyn activation and BCR-ABL signaling (CrkL). BMS-354825 effectively reduced both K562 and K562R tumor growth in nude mice whereas imatinib had minimal effects on K562R tumors. Clinical specimens from imatinib resistant CML patients (with and without BCR-ABL kinase mutations) were treated with imatinib or BMS-354825 and analyzed for changes in Lyn and Hck activation. While imatinib had minimal inhibitory effects on Lyn/Hck activation, BMS-354825 completely suppressed Lyn/Hck phosphorylation which correlated with its greater anti-tumor activity in CML samples. BCR-ABL tyrosine phosphorylation was not inhibited by imatinib in Cos cells co-expressing BCR-ABL and Lyn kinase and loss of imatinib sensitivity was totally dependent on Lyn kinase activity. BMS-354825 reduced both Lyn and BCR-ABL activation in co-expressing cells, suggesting that Lyn-mediated phosphorylation plays a direct role in imatinib resistance. We conclude that dual inhibition of SRC/ABL kinases in CML cells by BMS-354825 overcomes resistance to imatinib in vitro and in vivo and induces anti-tumor effects in CML patient specimens resistant to imatinib through expression of imatinib-inactivating BCR-ABL kinase mutations as well as other resistance mechanisms.