Single-molecule dynamics of lysozyme processing distinguishes linear and cross-linked peptidoglycan substrates.

The dynamic processivity of individual T4 lysozyme molecules was monitored in the presence of either linear or cross-linked peptidoglycan substrates. Single-molecule monitoring was accomplished using a novel electronic technique in which lysozyme molecules were tethered to single-walled carbon nanotube field-effect transistors through pyrene linker molecules. The substrate-driven hinge-bending motions of lysozyme induced dynamic electronic signals in the underlying transistor, allowing long-term monitoring of the same molecule without the limitations of optical quenching or bleaching. For both substrates, lysozyme exhibited processive low turnover rates of 20-50 s(-1) and rapid (200-400 s(-1)) nonproductive motions. The latter nonproductive binding events occupied 43% of the enzyme's time in the presence of the cross-linked peptidoglycan but only 7% with the linear substrate. Furthermore, lysozyme catalyzed the hydrolysis of glycosidic bonds to the end of the linear substrate but appeared to sidestep the peptide cross-links to zigzag through the wild-type substrate.

Phase II study of gemcitabine plus docetaxel in advanced pancreatic cancer: a Hoosier Oncology Group study.

OBJECTIVE: To determine the response rate, duration of response and survival with weekly gemcitabine plus docetaxel in metastatic or unresectable pancreatic cancer. METHODS: Forty patients were enrolled, and 38 patients were evaluable for survival and toxicity. Thirty-seven patients were evaluable for response. Nine patients (24%) had locally advanced disease and 29 (76%) had metastatic disease at the time of enrollment. Median Eastern Cooperative Oncology Group performance status was 1. Patients received gemcitabine 750 mg/m(2) i.v. and docetaxel 35 mg/m(2) i.v. weekly for 3 out of 4 weeks for a maximum of 6 cycles. RESULTS: Patients received a median of 4 cycles (range 1-6) of chemotherapy. An objective response was obtained in 10 patients (27%) with a median duration of 17 weeks. Median survival was 7 months, and 1-year survival was 19.3%. Eight patients experienced at least one form of grade 4 toxicity and 27 patients experienced at least one type of grade 3 toxicity. CONCLUSIONS: The combination of gemcitabine and docetaxel is a well-tolerated regimen with clinical efficacy. The ultimate role of this combination versus single-agent gemcitabine can only be determined by a randomized phase III trial.

Carboxylesterases expressed in human colon tumor tissue and their role in CPT-11 hydrolysis.

PURPOSE: The purpose is to develop new analytical methods to study the expression profile of CPT-11 carboxylesterases and topoisomerase I in colon tumor samples and understand the impact of their expression on CPT-11 metabolism in chemotherapy. EXPERIMENTAL DESIGN: We investigated 24 colon tumors for expression of carboxylesterases CES1A1, CES2, CES3, hBr-3, and topoisomerase I genes by real-time PCR and correlated the gene expression with activity assays. The relative abundance of the carboxylesterase isoenzymes and topoisomerase I genes was determined by real-time PCR. Activity assays performed on colon tumor extracts included CPT-11 hydrolase, 4-methylumbelliferyl acetate hydrolase, and topoisomerase I activity assays. Additionally, nondenaturing activity gel electrophoresis with activity staining showed the distribution of carboxylesterases. RESULTS: We detect the expression of CES1A1, CES2, and CES3 carboxylesterase genes in human colon tumors. We were unable to detect the hBr-3 (also called hCE-3) in human liver, colon, or brain. We find large interindividual variation, >/=150-fold, for both CES1A1 and CES3 genes, 23-fold for CES2, and 66-fold for topoisomerase I. Only CES2 gene expression correlated with the carboxylesterase activity assays (P < 0.01) with CPT-11 and 4-methylumbelliferyl acetate as substrates. Nondenaturing activity gel electrophoresis showed that CES2 was the most predominant activity. Topoisomerase I gene expression significantly correlated with topoisomerase I activity (P < 0.01) in the colon tumors, but interindividual variation was very high. CONCLUSIONS: We conclude that CES2 is the most abundant carboxylesterase in colon tumors that is responsible for CPT-11 hydrolysis. This pilot study reinforces the hypothesis that there is a large interindividual variation in expression of carboxylesterases that may contribute to variation in therapeutic outcome and/or toxicity of CPT-11 therapy for colon cancer.

Phase I trial of carboplatin and paclitaxel with escalating doses of oral topotecan in patients with solid tumors.

Carboplatin, paclitaxel, and topotecan have activity against a variety of cancers. This phase I study was designed to determine the maximum tolerated dose of oral topotecan when given in combination with carboplatin and paclitaxel. Eligibility criteria were as follows: Karnofsky Performance score greater than or equal to 80; and adequate hepatic, renal, and bone marrow function. Patients received paclitaxel 175 mg/m2 intravenously followed by carboplatin AUC 5 iv on day 1 every 3 weeks for up to 6 cycles. Cohorts of 3 to 5 were treated with escalating doses of oral topotecan on days 1 to 5, initially at 0.75 mg/m2 then 1 mg/m2 and 1.25 mg/m2 in subsequent cohorts. Thirteen patients were treated. Three of three patients in cohort 1 had grade IV neutropenia, with one neutropenic fever and one patient requiring a platelet transfusion. In cohort 2, three of five patients had grade III/IV neutropenia including two with neutropenic fever. Four patients required blood transfusions and one required platelet transfusions. In cohort 3, three of five had grade III/IV neutropenia, one of five had grade IV thrombocytopenia, and one patient required blood transfusions. In conclusion, this three-drug regimen resulted in significant cumulative myelosuppression in the doses and schedule tested in this phase I trial. Subsequent combinations of these drugs should focus on alternate doses or schedules.

Homocysteine and methylmalonic acid: markers to predict and avoid toxicity from pemetrexed therapy.

The purpose of this study was to identify predictive factors for severe toxicity caused by antifolate-chemotherapy using pemetrexed (ALIMTA, LY231514), as a model. Data on potential predictive factors for severe toxicity from pemetrexed were collected from 246 patients treated between 1995 and 1999. Multivariate stepwise regression methods were used to identify markers predictive of severe toxicity. Using a multiple logistic regression model allowed us to quantify the relative risk of developing toxicities and to generate a validated clinical hypothesis on ways to improve the safety profile of pemetrexed. Pretreatment total plasma homocysteine (tHcy) levels significantly predict severe thrombocytopenia and neutropenia with or without associated grade 3/4 diarrhea, mucositis, or infection. Pretreatment methylmalonic acid (MMA) levels significantly and independently predict grade 3/4 diarrhea and mucositis; however, these toxicities are still predicted by tHcy alone. Patients with elevated baseline levels of tHcy alone, or of both tHcy and MMA, were found to have a high risk of severe toxicity that led us to postulate that reducing tHcy would result in a reduction of severe toxicity with no harm to efficacy. This study points out for the first time the importance of pretreatment tHcy levels in predicting severe toxicity associated with an antifolate and sets the stage for a prospective clinical intervention to protect patients from pemetrexed-induced severe toxicity and possibly improve the drug's efficacy. Antifolates as a class have been associated with sporadic severe myelosuppression with gastrointestinal toxicity. Although infrequent, a combination of such toxicities can carry a high risk of mortality. This phenomenon had been unpredictable until now. Our work shows that by measuring tHcy, one can identify patients that are at risk of toxicity before treatment. Most importantly, decreasing homocysteine levels via vitamin supplementation leads to a better safety profile of pemetrexed and possibly to an improved efficacy.