RESUMO
RATIONALE: Physicians' adherence to prescribing evidence-based inpatient and outpatient therapies for chronic obstructive pulmonary disease (COPD) is low, and there is a paucity of information about the utility of admission order sets for patients with COPD exacerbations. OBJECTIVES: To determine if implementation of a locally designed, evidence-based, multidisciplinary computer physician order entry set in the electronic health record improves the quality of physician pharmacologic prescribing for patients hospitalized for COPD exacerbations. METHODS: This study was performed before and after implementation of a computerized order set for patients hospitalized for COPD exacerbations. The primary outcome was the rate of zero prescribing errors by physicians for inpatient and discharge drugs for COPD over a 1-year period before implementation and for 6 months after implementation. Errors were defined as no therapy or inappropriate therapy in the following categories: antibiotic, systemic corticosteroid, short-acting bronchodilator, long-acting bronchodilator, and inhaled corticosteroid. Secondary outcomes included mean physician pharmaceutical prescribing error rate; types of errors; hospital lengths of stay; and unscheduled physician visits, emergency department visits, rehospitalizations, and deaths within 30 days from discharge. MEASUREMENTS AND MAIN RESULTS: There were 194 COPD exacerbation admissions during the 1-year preimplementation period and 81 admissions during the 6-month postimplementation period. Compared with the preimplementation period, the percentage of patients receiving all recommended pharmacologic therapies for the 6 months after implementation increased from 18.6% to 54.3% (P < 0.001). The mean number of errors decreased from 1.76 to 0.65 (P < 0.001). Antibiotic and systemic corticosteroid errors decreased from 39% to 16% (P < 0.001) and from 58% to 28% (P < 0.001), respectively. Fewer patients were discharged without a short-acting bronchodilator (13.9% vs. 2.5%; P = 0.005), a long-acting bronchodilator (16.5% vs. 7.4%; P = 0.047), or inhaled corticosteroid (18% vs. 9.9%; P = 0.089). Improvements were sustained over the 6-month postimplementation period. Hospital length of stay decreased from 4 (±3) days preimplementation to 2.9 (±1.9) days postimplementation (P = 0.002). There were no significant differences in 30-day clinical outcomes, including the rates of unscheduled physician or emergency department visits, rehospitalizations, or deaths. CONCLUSIONS: Computerized multidisciplinary admission order set implementation for patients hospitalized for a COPD exacerbation improved physicians' adherence to evidence-based pharmacologic treatment, and they were associated with reductions in length of hospital stay.
Assuntos
Fidelidade a Diretrizes/estatística & dados numéricos , Prescrição Inadequada/prevenção & controle , Sistemas de Registro de Ordens Médicas , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Corticosteroides/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Broncodilatadores/uso terapêutico , Progressão da Doença , Feminino , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Minnesota , Melhoria de QualidadeRESUMO
Cbl is one of the major tyrosine-phosphorylated proteins in Bcr-Abl-expressing cells. A direct association between the SH2 domain of Bcr-Abl and tyrosine-phosphorylated Cbl has been demonstrated. The purpose of this study was to determine if and how unphosphorylated Cbl and Bcr-Abl may associate. Interactions between Cbl and Bcr-Abl were investigated in yeast two- and three-hybrid systems, gel overlay assays, and immunoprecipitates from mammalian cells expressing wild-type and the Y177F mutant of Bcr-Abl. No direct interaction between Bcr-Abl and unphosphorylated Cbl was observed. Bcr-Abl did, however, associate with Grb2, an adaptor protein that binds tyrosine 177 of Bcr-Abl. Additionally, Grb2 interacted with Cbl. In a yeast three-hybrid assay, Grb2 mediated an interaction between Cbl and Bcr-Abl that was dependent on a functional Grb2 binding site. This interaction was confirmed in vitro using purified proteins. In cells expressing Bcr-Abl with a mutation in the Grb2 binding site, binding of Cbl to Bcr-Abl was significantly reduced, but Cbl tyrosine phosphorylation was maintained. Imatinib treatment of these cells further reduced but did not abrogate Cbl binding, reflecting residual kinase activity. Multiple phosphotyrosine-dependent and -independent interactions stabilize the interaction between Cbl and Abl. Grb2 or another, yet unidentified, protein may mediate an initial interaction between Cbl and Bcr-Abl that is independent of Cbl tyrosine phosphorylation. Following this initial interaction, Cbl can then become tyrosine phosphorylated and interact with the SH2 domain of Bcr-Abl, further stabilizing the complex.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Fusão bcr-abl/metabolismo , Fosfotirosina/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ubiquitina-Proteína Ligases , Animais , Benzamidas , Proteína Adaptadora GRB2 , Humanos , Mesilato de Imatinib , Camundongos , Fosforilação , Piperazinas/farmacologia , Proteínas/química , Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-cbl , Pirimidinas/farmacologia , Técnicas do Sistema de Duplo-Híbrido , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies.
Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Apoptose/efeitos dos fármacos , Benzamidas/farmacocinética , Benzamidas/farmacologia , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Piperazinas/farmacocinética , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
The BCR-ABL tyrosine kinase is the defining feature of chronic myeloid leukemia (CML) and its kinase activity is required for induction of this disease. Current thinking holds that BCR-ABL forms a multi-protein complex that incorporates several substrates and adaptor proteins and is stabilized by multiple direct and indirect interactions. Signaling output from this highly redundant network leads to cellular transformation. Proteins known to be associated with BCR-ABL in this complex include: GRB2, c-CBL, p62(DOK), and CRKL. These proteins in turn, link BCR-ABL to various signaling pathways indicated in cellular transformation. In this study we show that a triple mutant of BCR-ABL with mutations of the direct binding sites for GRB2, CBL, p62(DOK) and CRKL, is defective for transformation of primary hematopoietic cells in vitro and in a murine CML model, while it retains the capacity to induce IL-3 independence in 32D cells. Compared to BCR-ABL, the triple mutant's ability to activate the MAP kinase and PI3-kinase pathways is severely compromised, while STAT5 phosphorylation is maintained, suggesting that the former are crucial for the transformation of primary cells, but dispensable for transformation of factor dependent cell lines. Our data suggest that inhibition of BCR-ABL-induced leukemia by disrupting protein interactions could be possible, but would require blocking of multiple sites.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia/genética , Leucemia/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sítios de Ligação , Transformação Celular Neoplásica , Feminino , Proteína Adaptadora GRB2/metabolismo , Interleucina-3/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/genética , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de SinaisRESUMO
BMS-354825 (dasatinib) and AMN107 (nilotinib) are potent alternate Abl inhibitors with activity against many imatinib mesylate-resistant BCR-ABL kinase domain (KD) mutants, except T315I. We used N-ethyl-N-nitrosourea (ENU)-exposed Ba/F3-p210(BCR-ABL) cells to compare incidence and types of KD mutants emerging in the presence of imatinib mesylate, dasatinib, and nilotinib, alone and in dual combinations. Although ENU is expected to induce mutations in multiple proteins, resistant clones were almost exclusively BCR-ABL KD mutant at relevant concentrations of nilotinib and dasatinib, consistent with a central role of KD mutations for resistance to these drugs. Twenty different mutations were identified with imatinib mesylate, 10 with nilotinib (including only 1 novel mutation, E292V) and 9 with dasatinib. At intermediate drug levels the spectrum narrowed to F317V and T315I for dasatinib and Y253H, E255V, and T315I for nilotinib. Thus, cross-resistance is limited to T315I, which is also the only mutant isolated at drug concentrations equivalent to maximal achievable plasma trough levels. With drug combinations maximal suppression of resistant clone outgrowth was achieved at lower concentrations compared with single agents, suggesting that such combinations may be equipotent to higher-dose single agents. However, sequencing uniformly revealed T315I, consistent with the need for a T315I inhibitor, to completely block resistance.