Adduct-specific monoclonal antibodies for the measurement of cisplatin-induced DNA lesions in individual cell nuclei

The anticancer drug cisplatin executes its cytotoxic activity via formation of intra- and interstrand crosslinks in DNA. The relative contribution of structurally defined cisplatin adducts to induce apoptosis and the cellular processing of these lesions is still poorly understood mostly due to the lack of sensitive analytical tools for in vivo studies. Here we describe a new method to establish and characterize monoclonal antibodies (Mab) for structurally defined DNA adducts. The two major reaction products of cisplatin, the guanine–guanine (Pt-[GG]) and adenine–guanine (Pt-[AG]) intrastrand crosslinks are recognized by Mab R-C18 and R-B3, respectively. Both antibodies were employed in an immuno-cytological assay allowing the quantification of drug-induced lesions in individual cell nuclei at clinically relevant doses. Analyzing various tissues of cisplatin-treated C57Bl/6 mice the accumulation of Pt-(GG) was highest in kidney tubular cells compared with 30, 50 and 90% lower levels in kidney stroma, liver and peripheral blood cells, respectively. Adduct kinetics revealed that wild type mouse cells remove up to 80% of the crosslinks in contrast to their complete persistence in nucleotide excision repair-deficient (XPC(−/−)) mice. The aptitude of the immunoassay for human molecular dosimetry studies was demonstrated by measuring adduct levels in tumor biopsies from patients treated with cisplatin

Науково-теоретична конференція «Гармонізація науки і вищої освіти в інформаційному суспільстві»

У Києві 30−31 березня 2011 року в Національному авіаційному університеті відбулася науково-теоретична конференція «Гармонізація науки і вищої освіти в інформаційному суспільстві»

Extensive Metabolism and Hepatic Accumulation of Gemcitabine After Multiple Oral and Intravenous Administration in Mice

ABSTRACT: In a clinical study with oral gemcitabine (2,2-difluorodeoxycytidine, dFdC), we found that gemcitabine was hepatotoxic and extensively metabolized to 2,2-difluorodeoxyuridine (dFdU) after continuous oral dosing. The main metabolite dFdU had a long terminal half-life after oral administration. Our hypothesis was that dFdU and/or phosphorylated metabolites of gemcitabine accumulated in the liver after multiple oral dosing. In this study, mice were treated with oral or i.v. dFdC at a single dose (1qd؋1d) or at multiple doses once daily for 7 days (1qd؋7d) or seven times daily (7qd؋1d). Blood, liver, kidneys, and lungs were collected at several time points. Urine samples were collected after i.v. dFdC, and peripheral blood mononuclear cells were collected 7qd؋1d dosing of dFdC. The nucleosides dFdC and dFdU as well as the nucleotides gemcitabine monophosphate (dFdC-MP), diphosphate, and triphosphate (dFdC-TP) and dFdU monophosphate, diphosphate (dFdU-DP), and triphosphate (dFdU-TP) were simultaneously quantified by high-performance liquid chromatography with ultraviolet and radioisotope detection. We demonstrate that phosphorylated metabolites of both dFdC and dFdU are formed in mice, primarily consisting of dFdC-MP, dFdC-TP, and dFdU-TP. Multiple dosing of dFdC leads to substantial hepatic and renal accumulation of dFdC-TP and dFdU-TP, which have a more pronounced liver accumulation after oral than after i.v. dosing. The presence of dFdC-MP, dFdC-TP, and dFdU-TP in plasma and urine suggests efflux of these potentially toxic metabolites. Our results show that dFdU, dFdC-TP, and dFdU-TP accumulate in the liver after multiple dosing of dFdC in mice and might be associated with hepatotoxicity of oral dFdC in patients. Gemcitabine (2Ј,2Ј-difluorodeoxycytidine, dFdC), a pyrimidine nucleoside anticancer drug, is used in the treatment of patients with a variety of solid tumors Alternatively, dFdC is deaminated to 2Ј,2Ј-difluorodeoxyuridine (dFdU) by cytidine deaminase (CDA), which is highly expressed in human liver and mice kidney In a clinical study, dFdC was orally administered in continuous dosing regimens at low dose levels in patients with advanced solid tumors . The exposure to dFdC was low because of extensive first-pass metabolism to dFdU. Additionally, we found that the triphosphate form of dFdU (dFdU-TP) was formed at high exposure levels in peripheral blood mononuclear cells (PBMCs). One patient treated with 8 mg of oral dFdC once daily for 14 days of a 21-day cycle developed lethal hepatic toxicity during the second cycle. Pathological examination revealed severe drug-induced liver necrosis. Pharmacokinetic analysis demonstrated that dFdU has a long terminal half-life (t 1/2 ) (ϳ89 h) and appeared to accumulate in the liver of patients. Based on these findings, we hypothesized that continuous daily oral dosing of dFdC results in liver accumulation of dFdU and/or phosphorylated metabolites in patients, possibly associated with the hepatotoxicity of dFdC. We recently found that dFdU is Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.021048. ABBREVIATIONS: dFdC, 2Ј,2Ј-difluorodeoxycytidine (gemcitabine); dCK, deoxycytidine kinase; dFdC-MP, gemcitabine monophosphate; dFdC-TP, gemcitabine triphosphate; dFdC-DP, gemcitabine diphosphate; dFdU, 2Ј,2Ј-difluorodeoxyuridine; CDA, cytidine deaminase; dFdU-TP, dFdU triphosphate; PBMC, peripheral blood mononuclear cell; hCNT1, human concentrative nucleoside transporter type 1; PK, pharmacokinetics; 1qdϫ1d, single dose on day 1; 1qdϫ7d, once daily dosing for 7 days; 7qdϫ1d, seven times daily dosing for 1 day; dFdU-MP, dFdU monophosphate; dFdU-DP, dFdU diphosphate; THU, tetrahydrouridine; AP, alkaline phosphatase; HPLC, high-performance liquid chromatography; AUC, area under the curve

Left and right ventricular longitudinal strain-volume/area relationships in elite athletes.

We propose a novel ultrasound approach with the primary aim of establishing the temporal relationship of structure and function in athletes of varying sporting demographics. 92 male athletes were studied [Group IA, (low static-low dynamic) (n = 20); Group IC, (low static-high dynamic) (n = 25); Group IIIA, (high static-low dynamic) (n = 21); Group IIIC, (high static-high dynamic) (n = 26)]. Conventional echocardiography of both the left ventricles (LV) and right ventricles (RV) was undertaken. An assessment of simultaneous longitudinal strain and LV volume/RV area was provided. Data was presented as derived strain for % end diastolic volume/area. Athletes in group IC and IIIC had larger LV end diastolic volumes compared to athletes in groups IA and IIIA (50 ± 6 and 54 ± 8 ml/(m(2))(1.5) versus 42 ± 7 and 43 ± 2 ml/(m(2))(1.5) respectively). Group IIIC also had significantly larger mean wall thickness (MWT) compared to all groups. Athletes from group IIIC required greater longitudinal strain for any given % volume which correlated to MWT (r = 0.4, p < 0.0001). Findings were similar in the RV with the exception that group IIIC athletes required lower strain for any given % area. There are physiological differences between athletes with the largest LV and RV in athletes from group IIIC. These athletes also have greater resting longitudinal contribution to volume change in the LV which, in part, is related to an increased wall thickness. A lower longitudinal contribution to area change in the RV is also apparent in these athletes

Correlation between the dose of cisplatin (exposure: 2 h; 37°C) and the level of specific intrastrand crosslinks in the nuclear DNA of cultivated human Jurkat T cells

<p><b>Copyright information:</b></p><p>Taken from "Adduct-specific monoclonal antibodies for the measurement of cisplatin-induced DNA lesions in individual cell nuclei"</p><p>Nucleic Acids Research 2006;34(6):e47-e47.</p><p>Published online 29 Mar 2006</p><p>PMCID:PMC1420801.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Adduct levels were measured after immunostaining with anti-(Pt-DNA) Mab R-C18 (Pt-[GG] lesions (), or R-B3 specific for Pt-(AG) () and quantitative ICA image analysis (Materials and Methods). Antibody-derived signals from individual nuclei were normalized for the actual DNA content of each cell. Values are given as AFU and represent means ± SE of 200 analyzed cells per dose. Correlation: (A) = 0.96; (B) = 0.91

Radio-chromatographic analysis of the relative amounts of Pt-(GG) and Pt-(AG) intrastrand crosslinks in DNA preparations Pt 1–3 without antibody selection () or after the immuno-trapping procedure with Mab R-B3 (), Mab R-C7 () or Mab R-C18 ()

<p><b>Copyright information:</b></p><p>Taken from "Adduct-specific monoclonal antibodies for the measurement of cisplatin-induced DNA lesions in individual cell nuclei"</p><p>Nucleic Acids Research 2006;34(6):e47-e47.</p><p>Published online 29 Mar 2006</p><p>PMCID:PMC1420801.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The -axes represent the P-derived radioactive signals (c.p.m.), the -axes give the elution time (min)

Multiparameter Flow Cytometry Assay for Quantification of Immune Cell Subsets, PD‐1 Expression Levels and PD‐1 Receptor Occupancy by Nivolumab and Pembrolizumab

We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD-1, and PD-1 receptor occupancy. Cell subsets include CD4+ and CD8+ -T-cells, B-cells, natural killer cells, classical-, intermediate- and non-classical monocytes, and myeloid- and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within- and between subject variations. The lower limit of quantification was 5.0% of PD-1+ cells. Samples were stable for at least 153 days of storage at -80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD-1+ immune cells, and number of PD-1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8+ -T-cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle. © 2019 International Society for Advancement of Cytometry