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1.
Acta Oncol ; 59(7): 825-832, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32347139

ABSTRACT

Background: Retrospective studies have suggested that chemotherapy-induced leukopenia is associated with improved recurrence-free or overall survival. The SBG 2000-1 trial was designed to verify the favorable prognosis associated with chemotherapy-induced leukopenia in early breast cancer. Patients not experiencing chemotherapy-induced leukopenia were randomized into standard dosed or individually escalated chemotherapy doses based on the grade of leukopenia after a first standard dose.Patients and methods: 1452 women in Sweden and Denmark with operable node-positive or high-risk node-negative breast cancer aged 18-60 years were recruited to participate in this trial. Participants received a first FEC cycle at standard doses (600/60/600 mg/m2). Patients (n = 1052) with nadir leukopenia grade 0-2 after the first cycle were randomized between either 6 standard FEC or 6 tailored FEC courses with doses of epirubicin and cyclophosphamide escalated during courses 2 and 3 and thereafter aimed at achieving grade 3 leukopenia. Patients with nadir leukopenia grade 3-4 after the first course continued treatment with standard FEC. Results of the randomized comparison has been published previously. The present study focuses on chemotherapy-induced leukopenia as a covariable with outcome in randomized and non-randomized patients. The prognostic value of leukopenia after course 3, was studied in a Cox model adjusted for cumulative doses of epirubicin and cyclophosphamide. The association of chemotherapy-induced leukopenia with prognosis was a preplanned secondary endpoint for this trial.Results: The eight-year distant disease-free survival was 73%, 77%, 78% and 83% for patients with leucocyte nadir grade 0, 1, 2 and 3-4, respectively. Higher degree of leukopenia was highly significantly associated to improved distant disease-free survival (HR 0.84, 95% CI 0.74-0.96, p = .008) and overall survival (HR 0.87 (0.76-0.99, p = .032).Conclusion: This prospective study confirms that chemotherapy-induced leukopenia is a covariable with outcome in primary breast cancer, even after adjustment for chemotherapy doses.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Breast Neoplasms/drug therapy , Leukopenia/blood , Leukopenia/chemically induced , Adolescent , Adult , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Breast Neoplasms/surgery , Chemotherapy, Adjuvant , Cyclophosphamide/administration & dosage , Cyclophosphamide/adverse effects , Disease-Free Survival , Epirubicin/administration & dosage , Epirubicin/adverse effects , Female , Fluorouracil/administration & dosage , Fluorouracil/adverse effects , Humans , Leukocyte Count , Middle Aged , Prospective Studies , Survival Rate , Treatment Outcome , Young Adult
2.
J Pharm Biomed Anal ; 185: 113256, 2020 Jun 05.
Article in English | MEDLINE | ID: mdl-32229402

ABSTRACT

Host cell proteins (HCPs) in biotherapeutics can be identified by the use of enzymatic digestion and LC-MS/MS analysis. However, the major challenge is that HCPs are often present at very low levels in relation to the protein drug (low ppm-levels). In this study, the ProteoMiner™ Enrichment Kit (Bio-Rad) was evaluated as a strategy to enable identification of HCPs by LC-MS/MS by enrichment of low-abundant HCPs and a simultaneous depletion of the high-abundant product protein. A recombinant protein produced in Chinese hamster ovary (CHO) cells was spiked with six standard proteins at varying concentrations (10-1000 ppm). The sample was split into two aliquots; one that was prepared with the ProteoMiner™ Enrichment Kit and one control, where the enrichment procedure was omitted. The ProteoMiner™ Enrichment Kit was combined with the ProteoMiner Sequential Elution Large-Capacity Kit (Bio-Rad), eluting the proteins into four fractions. The samples were then digested with trypsin and analyzed with LC-MS/MS. In addition, multiple reaction monitoring (MRM) was applied to obtain an estimate of the protein abundance of HCPs and spiked proteins. The results demonstrated that with the untargeted LC-MS/MS method, 30 HCPs and four of the six spiked standard proteins were identified in the four fractions. The spiked standard proteins were identified down to 30 ppm in the ProteoMiner treated samples, while no HCPs and only the most abundant standard protein (≈1000 ppm) were identified in the non-enriched control sample. MRM assays were developed for 14 out of the 30 identified HCPs. All targeted HCPs and five of the six standard proteins were detected in all fractions as well as in the control sample by MRM. There was an acceptable agreement between estimated concentrations of spiked standard proteins and expected values. An 80-700 fold enrichment of individual HCPs was observed in the fractions. In conclusion, the results clearly demonstrated that the ProteoMiner technology can be used for enriching HCPs in biotherapeutics, enabling their identification by LC-MS/MS.


Subject(s)
Biological Products/analysis , Quality Control , Technology, Pharmaceutical/methods , Animals , Biological Products/chemistry , CHO Cells , Chromatography, High Pressure Liquid/methods , Cricetulus , Feasibility Studies , Recombinant Proteins/analysis , Recombinant Proteins/chemistry , Tandem Mass Spectrometry/methods
3.
Bioanalysis ; 11(15): 1389-1403, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31490106

ABSTRACT

Aim: Accumulation of heparan sulfate (HS) is associated with the neurodegenerative disorder Mucopolysaccharidosis type IIIA (MPS IIIA). Here, we compare HS levels in brain and cerebrospinal fluid (CSF) of MPS IIIA mice after treatment with a chemically modified sulfamidase (CM-rhSulfamidase). Materials & methods: Two LC-MS/MS methods were adapted from literature methodology, one to measure HS metabolites (HSmet), the other to measure digests of HS after heparinase treatment (HSdig). Results: The HSmet and HSdig methods showed similar relative reduction of HS in brain after CM-rhSulfamidase administration to MPS IIIA mice and the reduction was reflected also in CSF. Conclusion: The results of the two methods correlated and therefore the HSdig method can be used in clinical studies to determine HS levels in CSF from patients with MPS IIIA.


Subject(s)
Brain/metabolism , Chromatography, Liquid/methods , Clinical Chemistry Tests/methods , Heparitin Sulfate/cerebrospinal fluid , Mucopolysaccharidosis III/cerebrospinal fluid , Tandem Mass Spectrometry/methods , Animals , Brain/drug effects , Hydrolases/pharmacology , Hydrolases/therapeutic use , Mice , Mucopolysaccharidosis III/drug therapy
4.
Breast Cancer Res Treat ; 168(2): 349-355, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29190004

ABSTRACT

PURPOSE: Although adjuvant polychemotherapy improves outcomes for early breast cancer, the significant variability in terms of pharmacokinetics results in differences in efficacy and both short and long-term toxicities. Retrospective studies support the use of dose tailoring according to the hematologic nadirs. METHODS: The SBG 2004-1 trial was a randomized feasibility phase II study which assessed tailored dose-dense epirubicin and cyclophosphamide (EC) followed by docetaxel (T) (group A), the same regimen with fixed doses (group B) and the TAC regimen (group C). Women aged 18-65 years, ECOG PS 0-1 with at least one positive axillary lymph node were randomized 1:1:1. The primary endpoint of the study was the safety and feasibility of the treatment. Toxicity was graded according to CTC-AE version 3.0. The design and short-term toxicity have been previously published. Here, we report safety and efficacy data after 10 years of follow-up. RESULTS: A total of 124 patients were included in the study. After a median follow-up of 10.3 years, the probability for 10-year survival was 78.5, 75.1, and 63.4% and for relapse free survival 64.1, 71.0, and 59.5% for groups A, B, and C, respectively. There were no cases of clinically diagnosed cardiotoxicity or hematologic malignancies. No patient was lost to follow-up. CONCLUSIONS: In this randomized phase II trial, tailored dose adjuvant chemotherapy was feasible, without an increased risk for long-term adverse events after a median follow-up of 10 years.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Long-Term Care/methods , Neoplasm Recurrence, Local/prevention & control , Adult , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Breast/pathology , Breast/surgery , Breast Neoplasms/pathology , Breast Neoplasms/surgery , Cardiotoxicity/epidemiology , Cardiotoxicity/etiology , Chemotherapy, Adjuvant/methods , Disease-Free Survival , Dose-Response Relationship, Drug , Feasibility Studies , Female , Follow-Up Studies , Hematologic Neoplasms/chemically induced , Hematologic Neoplasms/epidemiology , Humans , Lymphatic Metastasis/pathology , Mastectomy , Middle Aged , Neoplasm Recurrence, Local/epidemiology , Neoplasm Recurrence, Local/pathology , Prospective Studies , Young Adult
5.
Radiother Oncol ; 122(1): 72-78, 2017 01.
Article in English | MEDLINE | ID: mdl-27866849

ABSTRACT

PURPOSE: To determine the inter-observer variation in delineating the coronary arteries as organs at risk (OAR) in breast cancer (BC) radiotherapy (RT) and how this variation affects the estimated coronary artery radiation dose. METHOD: Delineation of the left main and the left anterior descending coronary artery (LMCA and LAD), and the right coronary artery (RCA), by using the heart atlas by Feng et al., was performed by three radiation oncologists in 32 women who had received adjuvant RT for BC. Centres of the arteries were calculated and distances between artery centres were measured and the artery radiation doses were estimated. The intraclass correlation coefficient (ICC) was used to quantify the variability in doses. RESULTS: Along the extent of RCA, the median distance between centres of arteries varied from 2 to 9mm with similar patterns over pairs of oncologists. For the LMCA-LAD the median distance varied from 1 to 4mm. The estimated maximum radiation doses showed an ICC variation from 0.82 to 0.97. CONCLUSION: The coronary arteries can be reliably identified and delineated as OARs in BC RT. The spatial variance is limited and the total variation in radiation dose is almost completely determined by the between patient variation.


Subject(s)
Breast Neoplasms/radiotherapy , Coronary Vessels/diagnostic imaging , Organs at Risk/diagnostic imaging , Radiotherapy Planning, Computer-Assisted/methods , Adult , Aged , Aged, 80 and over , Female , Humans , Middle Aged , Observer Variation , Radiotherapy Dosage
6.
Anticancer Res ; 35(10): 5491-7, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26408714

ABSTRACT

AIM: To elucidate the impact of different forms of radiation toxicities (esophagitis, radiation pneumonitis, mucositis and hoarseness), on the survival of patients treated with curatively intended radiotherapy for non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Data were individually collected retrospectively for all patients diagnosed with NSCLC subjected to curatively intended radiotherapy (≥50 Gy) in Sweden during the time period 1990 to 2000. RESULTS: Esophagitis was the only radiation-induced toxicity with an impact on survival (hazard ratio=0.83, p=0.016). However, in a multivariate model, with clinical- and treatment-related factors taken into consideration, the impact of esophagitis on survival was no longer statistically significant (hazard ratio=0.88, p=0.17). CONCLUSION: The effect on survival seen in univariate analysis may be related to higher radiation dose and to the higher prevalence of chemotherapy in this group. The results do not suggest that the toxicities examined have any detrimental effect on overall survival.


Subject(s)
Adenocarcinoma/mortality , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Squamous Cell/mortality , Esophagitis/mortality , Lung Neoplasms/mortality , Radiation Injuries/mortality , Radiation Pneumonitis/mortality , Radiotherapy/adverse effects , Adenocarcinoma/pathology , Adenocarcinoma/radiotherapy , Adult , Aged , Aged, 80 and over , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/radiotherapy , Carcinoma, Squamous Cell/pathology , Carcinoma, Squamous Cell/radiotherapy , Esophagitis/diagnosis , Esophagitis/etiology , Female , Follow-Up Studies , Humans , Lung Neoplasms/pathology , Lung Neoplasms/radiotherapy , Male , Middle Aged , Neoplasm Staging , Prognosis , Radiation Injuries/diagnosis , Radiation Injuries/etiology , Radiation Pneumonitis/diagnosis , Radiation Pneumonitis/etiology , Retrospective Studies , Survival Rate
7.
Anticancer Res ; 35(5): 2493-501, 2015 May.
Article in English | MEDLINE | ID: mdl-25964522

ABSTRACT

Malignant pleural mesothelioma (MPM) is a rare but aggressive malignancy mainly localized to the pleura. Malignant mesothelioma grows highly invasive into surrounding tissue and has a low tendency to metastasize. The median overall survival (OS) of locally advanced or metastatic disease without treatment is 4-13 months but, during recent years, improvement in survival has been achieved since treatment for patients with mesothelioma has improved with better palliative care, systemic medical treatment, surgery and improved diagnostics methods. The present review aims at describing available data from randomized trials considering systemic medical treatment for this patient category.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Lung Neoplasms/drug therapy , Mesothelioma/drug therapy , Pleural Neoplasms/drug therapy , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Combined Modality Therapy , Disease-Free Survival , Humans , Lung Neoplasms/pathology , Mesothelioma/pathology , Mesothelioma, Malignant , Palliative Care , Pleural Neoplasms/pathology , Randomized Controlled Trials as Topic
8.
Acta Oncol ; 50(3): 329-37, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21299448

ABSTRACT

UNLABELLED: The SBG 2000-1 trial is a randomised study that investigates if dose-tailored adjuvant FEC therapy based on the individual's leukocyte nadir value can improve outcome. The study has included 1535 women with medium and high-risk breast cancer. PATIENTS AND METHODS: After a first standard dosed FEC course (5-fluorouracil 600 mg/m(2), epirubicin 60 mg/mg(2) and cyclophosphamide 600 mg/m(2)), patients who did not reach leukopenia grade III or IV were randomised to standard doses (group standard) or doses tailored to achieve grade III leukopenia (group tailored) at courses 2-7. Patients who achieved leukopenia grade III or more after the first course were not randomised but continued on standard doses (group registered). RESULTS: Both planned and actually delivered number of courses (seven) were the same in all three arms. The relative dose intensity was increased by a factor of 1.31 (E 1.22, C 1.43) for patients in the tailored arm compared to the expected on standard dose. Ninety percent of the patients in the tailored arm achieved leukopenia grade III-IV compared with 29% among patients randomised to standard dosed therapy. Dose tailoring was associated with acceptable acute non-haematological toxicity with more total alopecia, nausea, vomiting and fatigue. CONCLUSION: Dose tailoring according to leukopenia was feasible. It led to an increased dose intensity and was associated with acceptable excess of acute non-haematological toxicity.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Breast Neoplasms/drug therapy , Carcinoma/drug therapy , Leukopenia/chemically induced , Precision Medicine , Adult , Breast Neoplasms/surgery , Carcinoma/surgery , Chemotherapy, Adjuvant/methods , Cyclophosphamide/administration & dosage , Cyclophosphamide/adverse effects , Dose-Response Relationship, Drug , Epirubicin/administration & dosage , Epirubicin/adverse effects , Feasibility Studies , Female , Fluorouracil/administration & dosage , Fluorouracil/adverse effects , Humans , Individuality , Leukopenia/epidemiology , Leukopenia/prevention & control , Mastectomy , Maximum Tolerated Dose , Middle Aged , Precision Medicine/methods , Scandinavian and Nordic Countries , Severity of Illness Index , Treatment Outcome
9.
Acta Oncol ; 50(1): 35-41, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21174610

ABSTRACT

The aim of the study was to evaluate the feasibility of tailored and dose-dense epirubicin and cyclophosphamide followed by docetaxel as adjuvant breast cancer therapy. Material and methods. Patients with node-positive breast cancer received either four cycles of biweekly and tailored EC (epirubicin 38-60-75-90-105-120 mg/m(2), cyclophosphamide 450-600-900-1200 mg/m(2)) followed by four cycles of docetaxel (60-75-85-100 mg/m(2)) (arm A) or the same regimen with fixed doses (E(90)C(600) + 4 → T(75) + 4) (arm B) or docetaxel, doxorubicin and cyclophosphamide (T(75)A(50)C(500)) every three weeks for six cycles (arm C). All patients received G-CSF support and prophylactic ciprofloxacin. Results. One-hundred and twenty-four patients were randomised in the study. In the A, B and C arm, 17% 19% and 3% of the patients had one or more cycles delayed due to side-effects whereas 24%, 5% and 15% experienced a grade 3 infection or febrile neutropenia. After the introduction of an extra week between the EC and T parts in the A and B arms, grade 3 hand-foot-skin reactions were reduced from 5 to 0.2%. Twenty-nine percent (A and B) and 20% (C) of the patients were hospitalised due to side-effects. Discussion. Dose-dense and tailored EC/T can be given with manageable toxicity and is after adjustment presently studied in the phase III Panther trial.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Breast Neoplasms/drug therapy , Adult , Aged , Breast Neoplasms/pathology , Chemotherapy, Adjuvant , Cyclophosphamide/administration & dosage , Cyclophosphamide/adverse effects , Docetaxel , Dose-Response Relationship, Drug , Doxorubicin/administration & dosage , Doxorubicin/adverse effects , Drug Administration Schedule , Drug Combinations , Epirubicin/administration & dosage , Epirubicin/adverse effects , Feasibility Studies , Female , Humans , Middle Aged , Neoplasm Staging , Patient Selection , Taxoids/administration & dosage , Taxoids/adverse effects , Treatment Outcome
10.
J Chromatogr B Analyt Technol Biomed Life Sci ; 878(26): 2427-32, 2010 Sep 15.
Article in English | MEDLINE | ID: mdl-20732836

ABSTRACT

The stearoyl-CoA desaturase (SCD) activity is involved in regulation of metabolism, energy storage, and membrane fluidity. However, only few cellular assays have been developed. We describe a simple and robust method to quantitate SCD activity and its inhibition in primary rat hepatocytes. Hepatocytes assimilate stearic acid, with or without modification by SCD, into its lipid pool. To measure the extent of this conversion primary rat hepatocytes were cultivated 4h or overnight with [1-(14)C]18:0 and extracellular fatty acids were washed out. Total cell lipids were then hydrolyzed and extracted. Recoveries of 18:0 were secured with a modified Folch method by addition of 0.1% Triton X-114 to the samples. The extracted fatty acids were dissolved in 85% ethanol and separated by reverse phase HPLC, which took 10 min including column recovery time. [1-(14)C]18:0 and [1-(14)C]18:1(n9) were detected and quantified by on-line flow scintillation analysis. Incubation of the cells with SCD inhibitors resulted in decreased ratios of 18:1/18:0 in dose-dependent manners. The improvements enabled us to establish a novel robust assay based solely on HPLC analysis of cellular SCD activity, which was developed in 12-well format.


Subject(s)
Chromatography, High Pressure Liquid/methods , Fatty Acids/chemistry , Hepatocytes/enzymology , Stearoyl-CoA Desaturase/metabolism , Animals , Cells, Cultured , Enzyme Inhibitors/pharmacology , Fatty Acids/isolation & purification , Fatty Acids/metabolism , Male , Rats , Rats, Sprague-Dawley , Stearic Acids/chemistry , Stearic Acids/metabolism , Stearoyl-CoA Desaturase/antagonists & inhibitors , Stearoyl-CoA Desaturase/chemistry
11.
J Pharm Biomed Anal ; 51(3): 649-57, 2010 Feb 05.
Article in English | MEDLINE | ID: mdl-19815360

ABSTRACT

The separation, isolation and identification of drug metabolites from complex endogenous matrices like urine, plasma and tissue extracts are challenging tasks. Metabolites are usually first identified by mass spectrometry and tentative structures proposed from product ion spectra. In many cases mass spectrometry cannot be used to determine positional isomers and metabolites have to be fractionated in microgram amounts for analysis by NMR. To overcome the difficulties associated with separation and isolation of drug metabolites from biological matrices, a new two-dimensional liquid chromatography system has been developed. The retention times of 45 acidic, basic and neutral compounds were determined on liquid chromatographic columns with different stationary phases in order to identify two columns with highly different selectivity to be used for two-dimensional liquid chromatography. Drug metabolites of three model compounds were first generated in vitro with liver microsomes and then compared with potential metabolites formed by oxidation with hydrogen peroxide catalyzed by meso-tetra (4-sulphonatophenyl) porphine (porphine). The results showed that the porphine system could be used as a complementary system for the generation of phase I microsomal metabolites with high yield of some metabolites in a less complex matrix. The two-dimensional liquid chromatography system was used to separate and isolate microsomal and porphine generated drug metabolites in off-line and on-line mode. Finally, to verify the utility of the developed system, urine samples were spiked with metabolite standards of model compounds for separation in the two-dimensional system. Excellent separations were obtained with an amide column in the first dimension and a pentafluorophenylpropyl (PFPP) column in the second dimension. The metabolites were successfully separated from each other as well as from the complex biological matrix. The results demonstrate the applicability of the system for fractionation of drug metabolites but it could also be used in many other analytical purposes, especially for basic compounds. Trace levels of metabolites were successfully separated in the on-line mode which failed in the off-line mode.


Subject(s)
Chemistry, Pharmaceutical/trends , Pharmaceutical Preparations/isolation & purification , Pharmaceutical Preparations/metabolism , Animals , Chemistry, Pharmaceutical/methods , Chromatography, Liquid/methods , Chromatography, Liquid/trends , Microsomes, Liver/chemistry , Pharmaceutical Preparations/chemistry , Rats
12.
Acta Oncol ; 47(4): 740-6, 2008.
Article in English | MEDLINE | ID: mdl-18465343

ABSTRACT

PURPOSE AND PATIENTS: During the period from January 1990 to January 1996 a total of 953 patients with lymph node negative primary breast cancer were randomised to oral pamidronate (n=460) 150 mg twice daily for 4 years or no adjuvant pamidronate (n=493) in order to investigate whether oral pamidronate can prevent the occurrence of bone metastases and fractures. The patients received adjuvant chemotherapy, loco-regional radiation therapy, but no endocrine treatment. RESULTS: During the follow-up period the number of patients with pure bone metastases was 35 in the control group and 31 in the pamidronate group. The number of patients with a combination of bone and other distant metastases were 22 in the control group and 20 in the pamidronate group. The hazard rate ratio for recurrence in bone in the pamidronate group compared to the control group was 1.03 (95% confidence interval 0.75-1.40) and p=0.86. No effect was observed on overall survival. In a small subgroup of 27 patients from the study, 12 of whom were treated with pamidronate a significant bone preserving effect was observed on bone mineral density in the lumbar spine, but not in the proximal femur. CONCLUSION: The results from the trial do not support a beneficial effect of oral pamidronate on the occurrence of bone metastases or fractures in patients with primary breast cancer receiving adjuvant chemotherapy.


Subject(s)
Adenocarcinoma/prevention & control , Adenocarcinoma/secondary , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bone Neoplasms/prevention & control , Bone Neoplasms/secondary , Breast Neoplasms/drug therapy , Diphosphonates/therapeutic use , Adenocarcinoma/pathology , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Breast Neoplasms/pathology , Cyclophosphamide/administration & dosage , Diphosphonates/adverse effects , Epirubicin/administration & dosage , Female , Fluorouracil/administration & dosage , Humans , Methotrexate/administration & dosage , Middle Aged , Pamidronate
13.
Toxicology ; 235(1-2): 27-38, 2007 Jun 03.
Article in English | MEDLINE | ID: mdl-17434249

ABSTRACT

Liver microsome and hepatocyte-mediated biotransformation of three oral antileishmanial 2-substituted quinolines were investigated. One quinoline contains an n-propyl group (1) and the other a propenyl chain functionalized at the gamma position either by a nitrile (2) or an alcohol (3). The different isoforms of rat cytochrome P450 responsible for biotransformation of 1 were also investigated. Compounds 2 and 3 mainly reacted with glutathione, preventing further metabolism. Compound 3 however, the reaction being reversible, could be released from glutathione and take alternative reaction pathways. Microsomal incubations of 1 mainly led to hydroxylation of the side chain, involving many cytochromes, predominantly CYP2B1, CYP2A6 and CYP1A1 (at more than 80%). In contrary, minor metabolites hydroxylated on the quinoline ring involved a few cytochromes. The hydroxylated products of 1 were conjugated with glucuronic acid in rat hepatocyte incubations.


Subject(s)
Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme System/metabolism , Hepatocytes/metabolism , Leishmania/drug effects , Mass Spectrometry , Microsomes, Liver/metabolism , Quinolines/metabolism , Trypanocidal Agents/metabolism , Animals , Aryl Hydrocarbon Hydroxylases/metabolism , Biotransformation , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2A6 , Cytochrome P-450 CYP2B1/metabolism , Cytochrome P-450 Enzyme System/genetics , Drug Stability , Glucuronides/metabolism , Glutathione/metabolism , Half-Life , Hepatocytes/enzymology , Humans , Hydroxylation , In Vitro Techniques , Kinetics , Male , Microsomes, Liver/enzymology , Mixed Function Oxygenases/metabolism , Molecular Structure , Quinolines/chemistry , Quinolines/pharmacology , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolism , Trypanocidal Agents/chemistry , Trypanocidal Agents/pharmacology
14.
Eur J Cancer ; 43(5): 877-84, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17306974

ABSTRACT

We compared the efficacy of CEF (cyclophosphamide, epirubicin, and fluorouracil) against CMF (cyclophosphamide, methotrexate, and fluorouracil) in moderate or high risk breast cancer patients. We randomly assigned 1224 patients with completely resected unilateral breast cancer to receive nine cycles of three-weekly intravenous CMF or CEF. Patients were encouraged to take part in a parallel trial comparing oral pamidronate 150 mg twice daily for 4 years versus control (data not shown). Substitution of methotrexate with epirubicin significantly reduced the unadjusted hazard for disease-free survival (DFS) by 16% (hazard ratio 0.84; 95% CI; 0.71-0.99) and for overall survival by 21% (hazard ratio 0.79; 95% CI; 0.66-0.94). The risk of secondary leukaemia and congestive heart failure was similar in the two groups. Overall CEF was superior over CMF in terms of DFS and OS in patients with operable breast cancer without subsequent increase in late toxicities.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Cyclophosphamide/administration & dosage , Cytarabine/administration & dosage , Epirubicin/administration & dosage , Female , Fluorouracil/administration & dosage , Humans , Methotrexate/administration & dosage , Middle Aged , Treatment Outcome
15.
J Clin Oncol ; 24(31): 4956-62, 2006 Nov 01.
Article in English | MEDLINE | ID: mdl-17075113

ABSTRACT

PURPOSE: To compare the efficacy of ovarian ablation versus chemotherapy in early breast cancer patients with hormone receptor-positive disease. PATIENTS AND METHODS: We conducted an open, randomized, multicenter trial including premenopausal breast cancer patients with hormone receptor-positive tumors and either axillary lymph node metastases or tumors with a size of 5 cm or more. Patients were randomly assigned to ovarian ablation by irradiation or to nine courses of chemotherapy with intravenous cyclophosphamide, methotrexate, and fluorouracil (CMF) administered every 3 weeks. RESULTS: Between 1990 and May 1998, 762 patients were randomly assigned, and the present analysis is based on 358 first events. After a median follow-up time of 8.5 years, the unadjusted hazard ratio for disease-free survival in the ovarian ablation group compared with the CMF group was 0.99 (95% CI, 0.81 to 1.22). After a median follow-up time of 10.5 years, overall survival (OS) was similar in the two groups, with a hazard ratio of 1.11 (95% CI, 0.88 to 1.42) for the ovarian ablation group compared with the CMF group. CONCLUSION: In this study, ablation of ovarian function in premenopausal women with hormone receptor-positive breast cancer had a similar effect to CMF on disease-free and OS. No significant interactions were demonstrated between treatment modality and hormone receptor content, age, or any of the well-known prognostic factors.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/radiotherapy , Neoplasms, Hormone-Dependent/drug therapy , Neoplasms, Hormone-Dependent/radiotherapy , Ovary/radiation effects , Premenopause , Adult , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Breast Neoplasms/metabolism , Breast Neoplasms/surgery , Chemotherapy, Adjuvant , Cyclophosphamide/administration & dosage , Disease-Free Survival , Drug Administration Schedule , Female , Fluorouracil/administration & dosage , Follow-Up Studies , Humans , Methotrexate/administration & dosage , Middle Aged , Neoplasms, Hormone-Dependent/metabolism , Neoplasms, Hormone-Dependent/surgery , Odds Ratio , Radiotherapy, Adjuvant , Survival Analysis
16.
Pharmacol Rep ; 58(3): 341-52, 2006.
Article in English | MEDLINE | ID: mdl-16845208

ABSTRACT

The introduction of combinatorial chemistry and robotics for high throughput screening has changed the way drugs are discovered today compared with 10-15 years ago when fewer compounds were tested in animal or organ models. The introduction of new analytical techniques, especially liquid chromatography/mass spectrometry (LC/MS) has made it possible to characterize the chemical properties, permeability, metabolic stability and metabolic fate of a large number of screening hits for further development in a funnel-like manner. The purpose of this contribution is to discuss principles and recent strategies for metabolite identification and to give an introduction to biotransformation studies. Metabolites are experimentally generated with the use of animal and human recombinant expressed enzymes, and different liver and other tissue fractions like microsomes and slices. For separation and identification of structurally diverse metabolites, LC/MS and tandem mass spectrometry (LC/MS/MS) techniques are commonly used. The LC/MS and LC/MS/MS techniques are rapid, sensitive, easy to automate and robust, and therefore, they are the methods of choice for these studies. The outcome of the metabolite identification studies is detection of metabolites that could be pharmacologically active and contribute to the efficacy of a new chemical entity (NCE), and elimination of compounds that form reactive intermediates and/or toxic metabolites that could cause adverse effects of NCE. If such information is available at an early stage during the drug discovery process, the chemical structure of the compound may be modified to reduce the risk of idiosyncratic and/or adverse drug reactions during clinical development.


Subject(s)
Drug Design , Pharmaceutical Preparations/metabolism , Animals , Chromatography, Liquid/methods , Deuterium Exchange Measurement , Granulosa Cell Tumor , Humans , Hydroxylation , Isotopes , Knowledge Bases , Luminescent Measurements , Mass Spectrometry/methods , Microsomes, Liver/metabolism , Software , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry/methods
17.
J Pharm Biomed Anal ; 40(5): 1121-30, 2006 Mar 18.
Article in English | MEDLINE | ID: mdl-16307862

ABSTRACT

An important step in the drug development process is identification of enzymes responsible for metabolism of drug candidates and determination of enzyme kinetic parameters. These data are used to increase understanding of the pharmacokinetics and possible metabolic-based drug interactions of drug candidates. The aim of the present study was to characterize the cytochrome P450 enzymes and enzyme kinetic parameters for metabolism of BVT.2938 [1-(3-{2-[(2-ethoxy-3-pyridinyl)oxy]ethoxy}-2-pyrazinyl)-2(R)-methylpiperazine], a potent and selective 5HT2c-receptor agonist. The enzyme kinetic parameters were determined for formation of three main metabolites of BVT.2938 using human liver microsomes and expressed cytochrome P450 (CYP) isoforms. The major metabolite was formed by hydroxylation of the pyridine ring (CL(int)=27 microl/mgmin), and was catalysed by both CYP2D6*1 and CYP1A1, with K(m) values corresponding to 1.4 and 2.7 microM, respectively. The results from enzyme kinetic studies were confirmed by incubation of BVT.2938 in the presence of the chemical inhibitor of CYP2D6*1, quinidine. Quinidine inhibited the formation of the major metabolite by approximately 90%. Additionally, studies with recombinant expressed CYP isoforms from rat indicated that formation of the major metabolite of BVT.2938 was catalysed by CYP2D2. This result was further confirmed by experiments with liver slices from different rat strains, where the formation of the metabolite correlated with phenotype of CYP2D2 isoform (Sprague-Dawley male, extensive; Dark Agouti male, intermediate; Dark Agouti female, poor metabolizer). The present study showed that the major metabolite of BVT.2938 is formed by hydroxylation of the pyridine ring and catalysed by CYP2D6*1. CYP1A1 is also involved in this reaction and its role in extra-hepatic metabolism of BVT.2938 might be significant.


Subject(s)
Cytochrome P-450 Enzyme System/analysis , Animals , Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Female , Humans , In Vitro Techniques , Isoenzymes/analysis , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Kinetics , Male , Microsomes, Liver/enzymology , Microsomes, Liver/metabolism , Pharmaceutical Preparations/metabolism , Piperazines/analysis , Pyrazines/analysis , Rats , Rats, Sprague-Dawley , Recombinant Proteins/metabolism
18.
J Chromatogr B Analyt Technol Biomed Life Sci ; 828(1-2): 14-20, 2005 Dec 15.
Article in English | MEDLINE | ID: mdl-16198158

ABSTRACT

Metabolic fingerprinting of biofluids like urine is a useful technique for detecting differences between individuals. With this approach, it might be possible to classify samples according to their biological relevance. In Part 1 of this work a method for the comprehensive screening of metabolites was described, using two different liquid chromatography (LC) column set-ups and detection by electrospray ionization mass spectrometry (ESI-MS). Data pretreatment of the resulting data described in is needed to reduce the complexity of the data and to obtain useful metabolic fingerprints. Three different approaches, i.e., reduced dimensionality (RD), MarkerLynx, and MS Resolver, were compared for the extraction of information. The pretreated data were then subjected to multivariate data analysis by partial least squares discriminant analysis (PLS-DA) for classification. By combining two different chromatographic procedures and data analysis, the detection of metabolites was enhanced as well as the finding of metabolic fingerprints that govern classification. Additional potential biomarkers or xenobiotic metabolites were detected in the fraction containing highly polar compounds that are normally discarded when using reversed-phase liquid chromatography.


Subject(s)
Chromatography, Liquid/methods , Data Interpretation, Statistical , Mass Spectrometry/methods , Multivariate Analysis , Spectrometry, Mass, Electrospray Ionization/methods , Urinalysis/methods , Animals , Biomarkers/urine , Male , Rats , Rats, Wistar
19.
Article in English | MEDLINE | ID: mdl-16181815

ABSTRACT

Complex biological samples, such as urine, contain a very large number of endogenous metabolites reflecting the metabolic state of an organism. Metabolite patterns can provide a comprehensive signature of the physiological state of an organism as well as insights into specific biochemical processes. Although the metabolites excreted in urine are commonly highly polar, the samples are generally analyzed using reversed-phase liquid chromatography mass spectrometry (RP-LC/MS). In Part 1 of this work, a method for detecting highly polar metabolites by hydrophilic interaction liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) is described as a complement to RP-LC/ESI-MS. In addition, in an accompanying paper (Part 2), different multivariate approaches to extracting information from the resulting complex data are described to enable metabolic fingerprints to be obtained. The coverage of the method for the screening of as many metabolites as possible is highly improved by analyzing the urine samples using both a C(18) column and a ZIC-HILIC column. The latter was found to be a good alternative when analyzing highly polar compounds, e.g., hydroxyproline and creatinine, to columns typically used for reversed-phase liquid chromatography.


Subject(s)
Biomarkers/urine , Chromatography, Liquid/methods , Spectrometry, Mass, Electrospray Ionization/methods , Urinalysis/methods , Animals , Biomarkers/analysis , Male , Rats , Rats, Wistar
20.
Rapid Commun Mass Spectrom ; 19(12): 1637-43, 2005.
Article in English | MEDLINE | ID: mdl-15915474

ABSTRACT

Most proteins in blood plasma bind ligands. Human serum albumin (HSA) is the main transport protein with a very high capacity for binding of endogenous and exogenous compounds in plasma. Many pharmacokinetic properties of a drug depend on the level of binding to plasma proteins. This work reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Warfarin, iopanoic acid and digitoxin were chosen as site-specific probes that bind to the main sites of HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. The advantages of nanoESI-MS for these studies are the sensitivity, the absence of labeled molecules and the short method development time.


Subject(s)
Pharmaceutical Preparations/metabolism , Serum Albumin/metabolism , Spectrometry, Mass, Electrospray Ionization/methods , Binding Sites , Digitoxin/metabolism , Glyburide/metabolism , Humans , Iopanoic Acid/metabolism , Naproxen/metabolism , Protein Binding , Warfarin/metabolism
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