ABSTRACT
In two crossover studies with 12 (6 males/6 females) healthy young volunteers each, we compared the bioavailability of Neoimmun capsules with the microemulsion Neoral and the influence of a fat-rich breakfast on the bioavailability of Neoimmun. Each volunteer received a single dose of 200 mg cyclosporine A in each period. Blood samples were taken up to 24 h and analysed for cyclosporine A by high-performance liquid chromatography (HPLC) and photometric detection. The pharmacokinetic parameters were determined by non-compartmental analysis. The treatments were tested for bioequivalence and significant differences. The bioavailability of Neoimmun was significantly lower compared to Neoral, albeit Neoimmun met the bioequivalence criterion (90% confidence interval of AUC 0.80-0.94) or missed the criterion only marginally (90% confidence interval of c (max) 0.75-0.91). The bioavailability of Neoimmun as determined by area under the blood concentration-time curve (AUC) increased by nearly 20% after a fat-rich breakfast. However, mean peak concentrations after food were only higher in male subjects, whereas mean peak concentrations in female subjects were lower compared to fasting administration. In conclusion, our data show that Neoimmun exhibits a lower bioavailability than the microemulsion Neoral and that food has a significant but variable and sex-dependent impact on the bioavailability of Neoimmun capsules.
Subject(s)
Cyclosporine/pharmacokinetics , Dietary Fats , Immunosuppressive Agents/pharmacokinetics , Adult , Area Under Curve , Biological Availability , Capsules , Chromatography, High Pressure Liquid , Cross-Over Studies , Cyclosporine/administration & dosage , Cyclosporine/blood , Emulsions , Fasting , Female , Food-Drug Interactions , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/blood , Male , Reference Values , Sex Factors , Therapeutic EquivalencyABSTRACT
A multicentric phase 2 study was conducted to determine the efficiency and the tolerance of imatinib mesylate in children with chronic myelogenous leukemia (CML) in advanced phase of the disease, in relapse after stem cell transplantation, or in case of failure to an interferon alpha-based regimen. In all, 30 children from eight European countries were enrolled. In 18 children assessable for hematologic response, imatinib mesylate induced complete hematologic response in eight (80%) of the 10 patients included in chronic phase and in six (75%) of eight enrolled in advanced phase of the disease with acceptable toxicity. In 27 patients assessable for cytogenetic response, imatinib mesylate induced disappearance of Philadelphia chromosome-positive bone marrow cells in 12 (60%) of 20 children included in chronic phase and in two (29%) of seven included in advanced phase. A reduction of the bcr-abl/abl ratio to less than 10(-4) was achieved in 11 (50%) of the children included in chronic phase. Estimated 12-month overall survival rate was 95% (95% CI, 87-100%) for the patients included in chronic phase and 75% (95%CI, 45-100%) for those enrolled in advanced phase. Imatinib mesylate is well tolerated and molecular remission can be achieved in children with CML.
Subject(s)
Antineoplastic Agents/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/therapeutic use , Pyrimidines/therapeutic use , Stem Cell Transplantation , Adolescent , Benzamides , Child , Child, Preschool , Chronic Disease , Drug Administration Schedule , Europe , Female , Follow-Up Studies , Humans , Imatinib Mesylate , Infant , Male , Recurrence , Remission Induction , Survival Rate , Treatment OutcomeABSTRACT
An isocratic online-enrichment HPLC-assay was developed allowing for the simple and fast separation and quantitation of STI-571 and its main metabolite N-desmethyl-STI (N-DesM-STI) in plasma, urine, cerebrospinal fluid (CSF), culture media and cell preparations in various concentrations using UV-detection at 260 nm. The analytical procedure consists of an online concentration of STI-571 and N-DesM-STI in the HPLC system followed by the elution on a ZirChrom-PBD analytical column. Time of analysis is 40 min including the enrichment time of 5 min. The detection limit is 10 ng/ml in plasma, CSF, culture medium (RPMI) and 25 ng/ml in urine for both STI-571 and N-DesM-STI. The intra-day precision, as expressed by the coefficient of variation (CV), in plasma samples ranges between 1.74 and 8.60% for STI-571 and 1.45 and 8.87% for N-DesM-STI. The corresponding values for urine measurements are 2.17-7.54% (STI-571) and 1.31-9.51% (N-DesM-STI). The inter-day precision analyzed over a 7-month time period was 8.31% (STI-571) or 6.88% (N-DesM-STI) and 16.45% (STI-571) or 14.83% (N-DesM-STI) for a concentration of 1000 ng/ml in plasma and 750 ng/ml in urine, respectively. Moreover, we demonstrate that with an alternative, but more time and labor consuming sample preparation and the implementation of electrochemical detection, a detection limit < 10 ng/ml can be achieved. The method described was used to perform pharmacokinetic measurements of STI-571 and N-desmethyl-STI in patient samples and for kinetic measurements of intracellular STI-571 and N-DesM-STI following in vitro incubation.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Chromatography, High Pressure Liquid/methods , Enzyme Inhibitors/pharmacokinetics , Piperazines/pharmacokinetics , Pyrimidines/pharmacokinetics , Antineoplastic Agents/blood , Antineoplastic Agents/cerebrospinal fluid , Antineoplastic Agents/urine , Benzamides , Culture Media , Electrochemistry , Enzyme Inhibitors/blood , Enzyme Inhibitors/cerebrospinal fluid , Enzyme Inhibitors/urine , HL-60 Cells , Humans , Imatinib Mesylate , Piperazines/blood , Piperazines/cerebrospinal fluid , Piperazines/urine , Pyrimidines/blood , Pyrimidines/cerebrospinal fluid , Pyrimidines/urine , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, UltravioletSubject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/therapeutic use , Pyrimidines/therapeutic use , Aged , Benzamides , Drug Administration Schedule , Female , Humans , Imatinib Mesylate , Male , Middle Aged , Neoplasm, Residual/diagnosis , Remission Induction/methodsABSTRACT
We sought to determine dynamics of BCR-ABL mRNA expression levels in 139 patients with chronic myelogenous leukemia (CML) in early chronic phase, randomized to receive imatinib (n=69) or interferon (IFN)/Ara-C (n=70). The response was sequentially monitored by cytogenetics from bone marrow metaphases (n=803) and qualitative and quantitative RT-PCR from peripheral blood samples (n=1117). Complete cytogenetic response (CCR) was achieved in 60 (imatinib, 87%) vs 10 patients (IFN/Ara-C, 14%) after a median observation time of 24 months. Within the first year after CCR, best median ratio BCR-ABL/ABL was 0.087%, (imatinib, n=48) vs 0.27% (IFN/Ara-C, n=9, P=0.025). BCR-ABL was undetectable in 25 cases by real-time PCR, but in only four patients by nested PCR. Median best response in patients with relapse after CCR was 0.24% (n=3) as compared to 0.029% in patients with continuous remission (n=52, P=0.029). We conclude that (i) treatment with imatinib in newly diagnosed CML patients is associated with a rapid decrease of BCR-ABL transcript levels; (ii) nested PCR may reveal residual BCR-ABL transcripts in samples that are negative by real-time PCR; (iii) BCR-ABL transcript levels parallel cytogenetic response, and (iv) imatinib is superior to IFN/Ara-C in terms of the speed and degree of molecular responses, but residual disease is rarely eliminated.
Subject(s)
Antineoplastic Agents/administration & dosage , Fusion Proteins, bcr-abl/genetics , Interferon-alpha/administration & dosage , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Piperazines/administration & dosage , Pyrimidines/administration & dosage , Adult , Aged , Antimetabolites, Antineoplastic/administration & dosage , Benzamides , Cross-Over Studies , Cytarabine/administration & dosage , Cytogenetics , Female , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/epidemiology , Male , Middle Aged , Prognosis , Prospective Studies , RNA, Messenger/metabolism , Recurrence , Risk Factors , Treatment OutcomeABSTRACT
A significant proportion of chronic myeloid leukemia (CML) patients achieve a major cytogenetic remission (MCR) to imatinib therapy after failing interferon (IFN) alpha-based protocols. We sought to determine levels of residual disease in patients with MCR using various molecular methods and to establish a relation between residual BCR-ABL transcript levels and rate of relapse in complete cytogenetic remission (CCR). Response was measured by conventional cytogenetic analysis, hypermetaphase and interphase fluorescence in situ hybridization (HM-FISH, IP-FISH) of bone marrow (BM) cells, qualitative nested and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for BCR-ABL transcripts. We investigated 323 peripheral blood (PB) and BM samples from 48 CML patients who achieved a complete (Ph+ 0%; n=41) or partial (Ph+ 1-34%; n=7) cytogenetic remission after 3-20 months of imatinib therapy. Prior to imatinib, 35 patients were in chronic phase (CP), eight in accelerated phase (AP), four in myeloid and one in lymphoid blast crisis. HM-FISH results correlated with ratios BCR-ABL/ABL in PB and BM. In patients with CCR, residual disease was detectable by HM-FISH (31%), IP-FISH (18%), and RT-PCR (100%). During follow-up, BCR-ABL became undetectable in two patients (one CP, one AP) by both nested and quantitative RT-PCR. CCR is ongoing in 30 evaluable patients, 11 patients have relapsed. At the time of best response, median ratios BCR-ABL/ABL were 2.1% (range 0.82-7.8) in patients with subsequent relapse and 0.075% (range 0-3.9) in patients with ongoing remission (P=0.0011). All 16 CP patients, who achieved ratios BCR-ABL/ABL <0.1% as best molecular response are in continuous remission, while 6/13 patients (46%) with ratios >/=0.1% have relapsed (P=0.0036). We conclude that: (i) in patients with CCR to imatinib, HM-FISH and RT-PCR usually reveal residual BCR-ABL+ cells; (ii) RT-PCR results derived from PB and BM are comparable in CP CML; and (iii) low levels of residual disease with ratios BCR-ABL/ABL &<0.1% are associated with continuous remission.
Subject(s)
Antineoplastic Agents/therapeutic use , Interferon-alpha/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Neoplasm, Residual/diagnosis , Piperazines/therapeutic use , Pyrimidines/therapeutic use , Adult , Aged , Benzamides , Bone Marrow/metabolism , Bone Marrow/pathology , Drug Resistance, Neoplasm , Female , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Humans , Imatinib Mesylate , In Situ Hybridization, Fluorescence , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Middle Aged , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Prognosis , Protein-Tyrosine Kinases/antagonists & inhibitors , RNA, Messenger/analysis , RNA, Neoplasm/genetics , Remission Induction , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Survival RateABSTRACT
Patients with refractory or relapsed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) rarely have prolonged responses to salvage therapy, including imatinib, resulting in a short opportunity for potentially curative stem cell transplantation. To identify minimal residual disease (MRD) parameters predictive of imminent relapse, we quantitated Bcr-Abl expression by real-time PCR in peripheral blood (PB) and bone marrow (BM) of 24 Ph+ALL patients after achieving a complete response and MRD minimum. The ratio of Bcr-Abl and glyceraldehyde-3-phosphate dehydrogenase copies, magnitude of increase and velocity of increase were evaluated regarding subsequent time intervals to relapse, death or censoring. High Bcr-Abl levels >/=5 x 10(-4) in PB (n=23) and >/=10(-4) in BM (n=18) were significantly associated with short time periods to relapse. Bcr-Abl increases >2 logarithmic units (log) in PB, but not in BM preceded short-term relapse. The velocity of Bcr-Abl increases predicted response duration in PB (cutoff: 1.25 log/30 days) and BM (0.6). Bcr-Abl level and velocity of increase in BM as well as magnitude of increase in PB correlated with remaining periods of survival and predicted relapse within 2 months in nine of 10, 10 of 11 and four of four patients, respectively. Thus, these MRD parameters may guide timing and intensity of therapeutic modifications.
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasm, Residual/diagnosis , Piperazines/therapeutic use , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Pyrimidines/therapeutic use , RNA, Messenger/analysis , Benzamides , Bone Marrow/metabolism , Bone Marrow/pathology , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Humans , Imatinib Mesylate , Neoplasm Recurrence, Local/diagnosis , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/metabolism , Neoplasm, Residual/drug therapy , Neoplasm, Residual/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , RNA, Neoplasm/genetics , Remission Induction , Reverse Transcriptase Polymerase Chain Reaction , Salvage Therapy , Survival Rate , Treatment OutcomeABSTRACT
Imatinib has marked antileukemic activity in advanced Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL), but secondary resistance develops rapidly, reflecting the limitations of single-agent therapy. Experimental data suggest that interferon-alpha (IFN-alpha) enhances the antileukemic activity of imatinib. We therefore examined combined imatinib and low-dose IFN-alpha in six patients with Ph+ALL who were ineligible for stem cell transplantation. All patients had received imatinib for 0.5-4.8 months prior to IFN-alpha, for relapsed (n=3) or refractory (n=1) Ph+ALL or as an alternative to chemotherapy following severe treatment-related toxicity (n=2). Five patients were in hematologic remission (CR) with minimal residual disease (MRD+), one patient was refractory to imatinib. Four of the five MRD+ patients are alive in CR after a median treatment duration of 20 (11-21) months. Two of these patients are in continuous CR 21 months after imatinib was initiated, while the other two patients experienced an isolated meningeal relapse that was successfully treated with additional intrathecal chemotherapy. Sustained molecular remissions were achieved in three patients and are ongoing 13 and 10.5 months after central nervous system (CNS) relapse and 6 months after starting concurrent IFN-alpha and imatinib, respectively. Marrow relapse occurred in one of the five MRD+ patients. Combination treatment was associated with a complete marrow response of 5 months duration in the imatinib-refractory patient. Imatinib combined with low-dose IFN-alpha may achieve prolonged hematologic and molecular remissions in a subset of patients with advanced Ph+ALL, who are not candidates for allogeneic SCT. CNS prophylaxis is necessary and may enhance the antileukemic activity of imatinib and IFN-alpha.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Interferon-alpha/administration & dosage , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Aged , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Benzamides , Female , Humans , Imatinib Mesylate , Interferon-alpha/adverse effects , Male , Middle Aged , Piperazines/administration & dosage , Piperazines/adverse effects , Pyrimidines/administration & dosage , Pyrimidines/adverse effects , Time Factors , Treatment OutcomeABSTRACT
In BCR-ABL-positive cells, the transcription factor STAT-5 is constitutively activated by tyrosine phosphorylation. STAT-5 activation results in upregulation of bcl-X(L) and increased resistance to induction of apoptosis. Here, we investigated the effects of imatinib mesylate and cytosine arabinoside (Ara-C) on STAT-5 tyrosine-phosphorylation, cellular proliferation and induction of apoptosis in cell lines and primary hematopoietic cells. Imatinib mesylate treatment strongly suppressed STAT-5 tyrosine-phosphorylation in K562 and primary CML blasts. In contrast to JAK-2 and PI-3-kinase inhibition, exposure of K562 cells to imatinib mesylate resulted in obvious suppression of proliferation. Reduced cell growth was due to specific induction of caspase activation followed by apoptotic cell death. In addition, we investigated the effects of Ara-C on STAT-5 tyrosine-phosphorylation. Exposure to Ara-C resulted in significant downregulation of STAT-5 tyrosine-phosphorylation and inhibition of DNA binding. Treatment of K562 cells with Ara-C in combination with imatinib mesylate revealed synergistic effects at the level of STAT-5 tyrosine-phosphorylation and DNA binding, Hck tyrosine-phosphorylation, cell growth and induction of apoptosis. Overall, in this report we demonstrate that STAT-5 tyrosine-phosphorylation is a specific target of imatinib mesylate and Ara-C. Our results suggest that, in combination therapy, inhibition of STAT-5 tyrosine-phosphorylation may be responsible for synergistic or additive effects on BCR-ABL-positive cells.
Subject(s)
Antineoplastic Agents/pharmacology , Cytarabine/pharmacology , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Milk Proteins , Piperazines/pharmacology , Protein-Tyrosine Kinases/metabolism , Pyrimidines/pharmacology , Trans-Activators/metabolism , Tyrosine/metabolism , Apoptosis/drug effects , Benzamides , Blotting, Western , Caspases/metabolism , Cell Division/drug effects , DNA/metabolism , Down-Regulation , Drug Synergism , Fusion Proteins, bcr-abl , Humans , Imatinib Mesylate , In Situ Nick-End Labeling , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Phosphorylation , Poly(ADP-ribose) Polymerases/metabolism , Precipitin Tests , STAT5 Transcription Factor , Signal Transduction/drug effects , U937 CellsABSTRACT
We report the response to the ABL kinase inhibitor imatinib mesylate (STI571) in a patient with chronic myeloid leukemia (CML) who relapsed twice after dose-reduced allogeneic stem cell transplantation (alloSCT) for B lymphoid blast crisis (BC) and failed to develop an antileukemic response despite grade 3 graft-versus-host disease (GvHD). Complete hematologic, cytogenetic and molecular responses were achieved within 9 weeks of therapy and are maintained after 27 months. Extensive chronic skin GvHD necessitating immunosuppressive therapy developed after 14 months. This case illustrates the ability of imatinib to induce sustained hematologic and molecular remissions in some patients relapsing with advanced stage CML after alloSCT.
Subject(s)
Antineoplastic Agents/administration & dosage , Blast Crisis/drug therapy , Hematopoietic Stem Cell Transplantation , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/administration & dosage , Pyrimidines/administration & dosage , Adult , Benzamides , Humans , Imatinib Mesylate , Male , Recurrence , Remission Induction , Transplantation Chimera , Transplantation, HomologousABSTRACT
Chronic myelogenous leukemia (CML) is characterized by aberrant trafficking of malignant hematopoietic progenitor cells in the peripheral blood. Expression of the cell adhesion molecule CD62L was reported to be significantly lower in CML patients than in normal controls. We studied whether the transcription of CD62L in CML cells is dependent on the activity of the BCR-ABL tyrosine kinase. Following addition of the Abelson (ABL) tyrosine kinase inhibitor imatinib (formerly STI571) to two BCR-ABL-positive cell lines (BV173, SD-1), we observed a dose-dependent increase in CD62L RNA levels of up to 45-fold by a quantitative, real-time polymerase chain reaction and an increase in the amount of cell surface-bound CD62L of up to 18-fold by quantitative flow cytometry, respectively. These data are validated by an increased CD62L expression in the bone marrow of patients (n=6) with advanced CML who received imatinib. Restoration of defective cell adhesion mediated via the CD62L pathway may be one mechanism of action of imatinib in BCR-ABL-positive leukemias.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/analysis , Enzyme Inhibitors/pharmacology , Fusion Proteins, bcr-abl/analysis , Gene Expression Regulation, Leukemic/drug effects , L-Selectin/biosynthesis , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Neoplasm Proteins/biosynthesis , Piperazines/pharmacology , Pyrimidines/pharmacology , Benzamides , Dose-Response Relationship, Drug , Humans , Imatinib Mesylate , L-Selectin/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Neoplasm Proteins/genetics , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Tumor Cells, Cultured/drug effectsABSTRACT
Imatinib has pronounced antileukemic activity in Ph+ALL, although responses are usually short. To determine whether imatinib may facilitate allogeneic SCT in relapsed or refractory Ph+ALL, we evaluated 46 consecutive, not previously transplanted patients who were enrolled in phase II studies of imatinib. Of 30 patients eligible for SCT, 22 (73%) were actually transplanted. Ten patients were in complete hematologic remission (CHR) (n = 5) or had a complete marrow response (CMR) (n = 5) at the time of SCT, 12 patients had again relapsed or were refractory. After SCT, 18 patients were in complete remission, one patient was refractory, three patients died prior to response assessment. Seven patients (32%) are in ongoing complete remission with a median follow-up of 9.4 (range 1.7-23.8) months. Seven patients (32%) relapsed a median of 5.2 months after SCT. Transplant-related mortality (TRM) was 36%. Probability of disease-free survival (DFS) is 25.5 +/- 9.8% overall and 51.4 +/- 17.7% when SCT was performed in CHR or CMR, compared with 8.3 +/- 8% for SCT during overt leukemia (P = 0.06). In conclusion, imatinib is a well-tolerated salvage therapy prior to allogeneic SCT in patients with Ph+ALL, but requires that SCT be performed within a few weeks of starting treatment to avoid resistance. Disease status at time of transplantation is an important determinant of DFS and TRM.
Subject(s)
Antineoplastic Agents/administration & dosage , Hematopoietic Stem Cell Transplantation/mortality , Piperazines/administration & dosage , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Pyrimidines/administration & dosage , Adolescent , Adult , Antineoplastic Agents/toxicity , Benzamides , Bone Marrow Transplantation/adverse effects , Bone Marrow Transplantation/methods , Bone Marrow Transplantation/mortality , Disease-Free Survival , Female , Graft Survival , Hematopoietic Stem Cell Transplantation/adverse effects , Hematopoietic Stem Cell Transplantation/methods , Humans , Imatinib Mesylate , Male , Middle Aged , Piperazines/toxicity , Pyrimidines/toxicity , Remission Induction/methods , Salvage Therapy , Survival Analysis , Therapeutic Equivalency , Transplantation, Homologous/adverse effects , Transplantation, Homologous/methods , Transplantation, Homologous/mortality , Treatment OutcomeABSTRACT
Selective inhibition of the BCR-ABL tyrosine kinase by imatinib (STI571, Glivec/Gleevec) is a promising new therapeutic strategy in patients with chronic myelogenous leukemia (CML). Despite significant hematologic and cytogenetic responses, resistance occurs, particularly in patients with advanced disease. We sought to determine the underlying mechanisms. Sixty-six patients with CML in myeloid blast crisis (n = 33), lymphoid blast crisis (n = 2), accelerated phase (n = 16), chronic phase (n = 13), and BCR-ABL-positive acute lymphoblastic leukemia (n = 2) resistant to imatinib were investigated. Median duration of imatinib therapy was 148 days (range 6-882). Patients were evaluated for genomic amplification of BCR-ABL, overexpression of BCR-ABL transcripts, clonal karyotypic evolution, and mutations of the imatinib binding site in the BCR-ABL tyrosine kinase domain. Results were as follows: (1) Median levels of BCR-ABL transcripts, were not significantly changed at the time of resistance but 7/55 patients showed a >10-fold increase in BCR-ABL levels; (2) genomic amplification of BCR-ABL was found in 2/32 patients evaluated by fluorescence in situ hybridization; (3) additional chromosomal aberrations were observed in 19/36 patients; (4) point mutations of the ABL tyrosine kinase domain resulting in reactivation of the BCR-ABL tyrosine kinase were detected in 23/66 patients. In conclusion, although the heterogeneous development of imatinib resistance is challenging, the fact that BCR-ABL is active in many resistant patients suggests that the chimeric oncoprotein remains a good therapeutic target. However, patients with clonal evolution are more likely to have BCR-ABL-independent mechanisms of resistance. The observations warrant trials combining imatinib with other agents.
Subject(s)
Antineoplastic Agents/therapeutic use , Chromosome Aberrations/drug effects , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/therapeutic use , Fusion Proteins, bcr-abl/genetics , Genes, abl/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Piperazines/therapeutic use , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/therapeutic use , Benzamides , DNA Mutational Analysis , DNA Primers/chemistry , DNA, Neoplasm/metabolism , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Mutation , Neoplasm Recurrence, Local/genetics , Polymerase Chain Reaction , Protein-Tyrosine Kinases/genetics , Treatment OutcomeABSTRACT
The degree of tumor load reduction as measured by cytogenetic response is an important prognostic factor for chronic myelogenous leukemia (CML) patients on therapy. We sought to determine whether BCR-ABL transcript levels can predict chromosomal response. Residual disease was evaluated in 120 CML patients in chronic phase (CP) treated with the selective tyrosine kinase inhibitor imatinib after resistance or intolerance to interferon alpha (IFN). Median time of therapy was 401 days (range 111-704). BCR-ABL and total ABL transcripts were measured in 486 peripheral blood (PB) specimens with a real time RT-PCR approach using fluorescent-labeled hybridization probes (LightCycler technology) and results were expressed as the ratio BCR-ABL/ABL. Cytogenetic response was determined in 3-monthly intervals: From 101 evaluable patients, 42 achieved a complete (CR, 0% Philadelphia chromosome (Ph)- positive metaphases), 18 a partial (PR, 1-34% Ph+), 13 a minor (MR, 35-94% Ph+), and 26 no response (NR, >94% Ph+). All PB samples were RT-PCR positive. The proportion of Ph+ metaphases and simultaneous BCR-ABL/ABL ratios correlated with r = 0.74, P < 0.0001. In order to investigate whether early molecular analysis may predict cytogenetic response, quantitative RT-PCR data obtained after 1 and 2 months of therapy were compared with cytogenetic response at 6 months. BCR-ABL/ABL ratios after 1 month were not predictive, but results after 2 months correlated with the consecutive cytogenetic response (P = 0.0008). The probability for a major cytogenetic response was significantly higher in patients with a BCR-ABL/ABL ratio <20% after 2 months of imatinib therapy. We conclude that: (1) quantitative determination of residual disease with real time RT-PCR is a reliable and sensitive method to monitor CML patients on imatinib therapy; (2) BCR-ABL/ABL ratios correlate well with cytogenetic response; (3) in IFN-pretreated patients all complete responders to imatinib have evidence of residual disease with the limited follow-up available; and (4) cytogenetic response at 6 months of therapy in CP patients is predictable with real time RT-PCR at 2 months.
Subject(s)
Antineoplastic Agents/therapeutic use , Fusion Proteins, bcr-abl/genetics , Interferon-alpha/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Piperazines/therapeutic use , Pyrimidines/therapeutic use , RNA, Messenger/analysis , Adult , Aged , Aged, 80 and over , Benzamides , Bone Marrow/metabolism , Bone Marrow/pathology , Drug Resistance, Neoplasm , Female , Fusion Proteins, bcr-abl/metabolism , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Male , Middle Aged , Neoplasm, Residual , Prognosis , Protein-Tyrosine Kinases/antagonists & inhibitors , RNA, Neoplasm/genetics , Recurrence , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Survival RateABSTRACT
We examined safety and efficacy of STI-571 in 24 bcr/abl-positive patients with CML post PBSCT. At start of STI-571 therapy, nine patients presented in blast crisis (BC) or in accelerated phase (AP), and 15 in chronic phase (CP). Patients were evaluated for hematologic, cytogenetic and molecular response, survival and toxicity. In general, STI-571 was well tolerated in this heavily pretreated group of patients with a non-hematologic and hematologic toxicity profile similar to that observed in a previous phase I trial at comparable doses. Five of nine patients with CML in transformation (AP, BC) were evaluable for hematologic response. Two of five patients had transient reductions in WBC and blasts, and three patients achieved a sustained hematologic response (>4 weeks). Cytogenetic analysis in these patients revealed numerical and/or structural responses. In CML chronic phase, STI-571 induced complete hematologic responses in all patients and major cytogenetic responses in 61% of patients with a complete cytogenetic response rate of 46%. This report indicates that STI-571 is a safe and effective drug in heavily pretreated patients. No apparent additional side-effects were noted in this patient cohort. The high rate of complete hematologic and complete cytogenetic responses in CP patients is remarkable, as intensive treatment approaches plus IFN-alpha failed to be efficient in achieving long-term stabilization of CML in this patient cohort.
Subject(s)
Antineoplastic Agents/administration & dosage , Fusion Proteins, bcr-abl/antagonists & inhibitors , Hematopoietic Stem Cell Transplantation , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/therapy , Piperazines/administration & dosage , Pyrimidines/administration & dosage , Adult , Antineoplastic Agents/adverse effects , Benzamides , Blood Cell Count , Combined Modality Therapy , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/adverse effects , Female , Humans , Imatinib Mesylate , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Male , Middle Aged , Piperazines/adverse effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/adverse effects , Transplantation, Autologous , Treatment OutcomeABSTRACT
We describe the clinical activity of the ABL kinase inhibitor STI571 in a patient with accelerated phase of chronic myeloid leukemia (CML) relapsing after a second allogeneic BMT and with minimal levels of donor chimerism. STI571 resulted in rapid elimination of leukemic cells with ensuing prolonged severe leukopenia and neutropenia complicated by neutropenic fever and colitis. Subsequent hematopoietic recovery was driven by donor derived cells and was associated with grade 3 graft-versus-host disease (GVHD). STI571 induced sustained hematological and cytogenetic remission combined with controllable GvHD, therapeutic goals not achieved by two preceding allogeneic transplants and repeated donor lymphocyte transfusions (DLT).
Subject(s)
Antineoplastic Agents/therapeutic use , Bone Marrow Transplantation , Hematopoietic Stem Cell Transplantation , Leukemia, Myeloid, Accelerated Phase/drug therapy , Neoplasm Recurrence, Local/drug therapy , Piperazines/therapeutic use , Pyrimidines/therapeutic use , Salvage Therapy , Antineoplastic Agents/adverse effects , Antineoplastic Agents, Alkylating/therapeutic use , Benzamides , Bone Marrow Transplantation/adverse effects , Colitis/chemically induced , Combined Modality Therapy , Enzyme Inhibitors/adverse effects , Enzyme Inhibitors/therapeutic use , Female , Graft Survival , Graft vs Host Disease/etiology , Graft vs Leukemia Effect , Humans , Hydroxyurea/therapeutic use , Imatinib Mesylate , Immunosuppression Therapy , Interferon-alpha/therapeutic use , Leukemia, Myeloid, Chronic-Phase/drug therapy , Leukemia, Myeloid, Chronic-Phase/therapy , Lymphocyte Transfusion , Middle Aged , Neoplasm, Residual , Neutropenia/chemically induced , Piperazines/adverse effects , Pyrimidines/adverse effects , Remission Induction , Transplantation Conditioning , Transplantation, HomologousABSTRACT
A reversed-phase ion-pairing high-performance liquid chromatography method for the detection and separation of SAM486A in human plasma and urine is described. Precipitation of proteins was used for plasma sample preparation. Enrichment of SAM486A on a 5 micro C18 column using 0.05 M NaH(2)PO(4) and 0.005 M pentan-sulfonic acid (pH 3.0) as eluent was followed by isocratic elution onto a 5 micro C18 analytical column using 0.01 M NaH(2)PO(4) and 0.005 M pentan-sulfonic acid (pH 3.0) as eluent. Analysis time was 23.0 +/- 0.1 min. The separation parameters were: capacacity factor = 6.21; plates/m = 15,002; peak tailing = 2.076. The method is linear between 5 ng/ml (detection limit) and 1000 ng/ml.
Subject(s)
Adenosylmethionine Decarboxylase/antagonists & inhibitors , Amidines/analysis , Antineoplastic Agents/analysis , Enzyme Inhibitors/analysis , Indans/analysis , Chromatography, High Pressure Liquid/instrumentation , Chromatography, High Pressure Liquid/methods , Humans , Reproducibility of ResultsABSTRACT
To elucidate factors responsible for altered proliferation of preneoplastic hepatocytes in rat hepatocarcinogenesis in vivo, EGF-stimulated DNA synthesis of normal and nodular hepatocytes in primary culture was studied. In addition, the influence of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated to clarify whether this potent tumor promoter differentially affects normal and nodular hepatocyte cultures. Unexpectedly it was found that in nodular hepatocytes spontaneous and EGF-stimulated DNA synthesis was enhanced with increasing cell density while DNA synthesis was inhibited in dense cultures of normal hepatocytes. Mitogenic responses were detected both by [3H]thymidine incorporation into DNA and by 5-bromo-2'-deoxyuridine labeling indices. TCDD (1 nM) acted as a mitoinhibitor both in normal and in nodular hepatocytes. The results suggest marked differences in growth behavior of nodular versus normal hepatocyte cultures probably due to paracrine stimulation by growth factors and altered cell-cell interaction.
Subject(s)
Liver/cytology , Liver/pathology , Precancerous Conditions/pathology , Animals , Bromodeoxyuridine/metabolism , Cell Count , Cells, Cultured , DNA/biosynthesis , Epidermal Growth Factor/pharmacology , Liver/metabolism , Male , Mitogens/pharmacology , Polychlorinated Dibenzodioxins/toxicity , Precancerous Conditions/chemically induced , Rats , Rats, Wistar , Thymidine/metabolismABSTRACT
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a widespread environmental contaminant and the most potent agonist of the aryl hydrocarbon receptor (AhR). Persistent activation of the AhR has been shown to be responsible for most TCDD-mediated toxic responses, including liver tumour promotion. However, the mechanisms responsible for these complex toxic reactions are still unknown. TCDD (1 nM) has previously been shown to reduce DNA synthesis of primary hepatocyte cultures and cell contact inhibition of confluent WB-F344 cells. The latter model was used to study early effects of TCDD on protein tyrosine kinase c-Src in confluent WB-F344 cells. It was found that TCDD decreased cytosolic c-Src (protein and tyrosine kinase activity) after 20-60 min, and increased c-Src in the membrane fraction. Membrane translocation of c-Src occurred in the presence of 100 microM cycloheximide and was observed after treatment with 1 nM TCDD or 50 nM 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin. Under these conditions epidermal growth factor (EGF) receptor tyrosine phosphorylation was also studied. As expected, its phosphorylation was low in confluent cells but was significantly enhanced by TCDD treatment. Pretreatment of WB-F344 cells for 1 h with 1 microM geldanamycin, which disrupts cytosolic heat shock protein Hsp90 complexes with AhR and Src, abolished TCDD-mediated Src translocation and TCDD-mediated reduction of cell contact inhibition. The WB-F344 cell model appears to be very useful to study TCDD effects on protein tyrosine kinases and of signaling pathways responsible for modulation of the cell cycle by TCDD.
Subject(s)
Cell Membrane/drug effects , Liver/drug effects , Polychlorinated Dibenzodioxins/toxicity , Proto-Oncogene Proteins pp60(c-src)/metabolism , Animals , Benzoquinones , Cell Membrane/metabolism , Cells, Cultured , Cycloheximide/pharmacology , Cytochrome P-450 CYP1A1/metabolism , DNA/biosynthesis , Enzyme Inhibitors/pharmacology , ErbB Receptors/drug effects , ErbB Receptors/metabolism , Immunoblotting , Lactams, Macrocyclic , Liver/metabolism , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/drug effects , Quinones/pharmacology , Rats , Rats, Inbred F344 , Time Factors , Tyrosine/metabolismABSTRACT
Functions and regulation of selected human UDP-glucuronosyltransferases (UGT1A1, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B15) are summarized. Evidence for at least two PAH-inducible UGTs (UGT1A6 and UGT1A9) is presented, which, however, are also constitutively expressed in a tissue- and cell-specific manner. These isoforms have recently been characterized to conjugate planar and bulky phenols, respectively. Using a selective RT-PCR method, UGT1A6 expression was detected in a variety of tissues (liver, kidney, lung, intestine, and pharyngeal mucosa). PAH-inducible UGTs may cooperate in the metabolism of phenolic metabolites of benzo(a)pyrene. Studies with stably expressed isoforms suggest that UGT1A9 is responsible for the formation of benzo(a)pyrene-3.6-diphenol diglucuronide, the major biliary metabolite of benzo(a)pyrene.