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

High accumulation of nivolumab in human breast milk: A case report

Nivolumab is an immunotherapeutic monoclonal antibody (mAb) that is used for the treatment of several types of cancer. The evidence on its use during lactation is lacking. Here, we report on a 39-year-old woman with metastasized melanoma who was treated with 480 mg nivolumab every four weeks during lactation. Breast milk samples were collected over the course of 34 days, including two cycles of nivolumab. The highest measured concentration of nivolumab during the first cycle was 503 ng/mL at day 13. The cumulative relative infant dose (RID) over the first cycle (28 days) was 9.8 %. The highest overall measured nivolumab concentration was 519 ng/mL at day 33, five days after administration of the second nivolumab cycle. Nivolumab seems to accumulate in breast milk over two consecutive cycles, hence the RIDs of consecutive cycles are expected to be higher. To draw further conclusions regarding safety of breastfeeding during nivolumab therapy, more information about the oral bioavailability of nivolumab in newborns, the nivolumab steady-state concentrations in breast milk and its pharmacodynamic effects are needed

Is Higher Docetaxel Clearance in Prostate Cancer Patients Explained by Higher CYP3A? An In Vivo Phenotyping Study with Midazolam

Patients with prostate cancer (PCa) have a lower docetaxel exposure for both intravenous (1.8-fold) and oral administration (2.4-fold) than patients with other solid cancers, which could influence efficacy and toxicity. An altered metabolism by cytochrome P450 3A (CYP3A) due to castration status might explain the observed difference in docetaxel pharmacokinetics. In this in vivo phenotyping, pharmacokinetic study, CYP3A activity defined by midazolam clearance (CL) was compared between patients with PCa and male patients with other solid tumors. All patients with solid tumors who did not use CYP3A-modulating drugs were eligible for participation. Patients received 2 mg midazolam orally and 1 mg midazolam intravenously on 2 consecutive days. Plasma concentrations were measured with a validated liquid chromatography–tandem mass spectrometry method. Genotyping was performed for CYP3A4 and CYP3A5. Nine patients were included in each group. Oral midazolam CL was 1.26-fold higher in patients with PCa compared to patients with other solid tumors (geometric mean [coefficient of variation], 94.1 [33.5%] L/h vs 74.4 [39.1%] L/h, respectively; P =.08). Intravenous midazolam CL did not significantly differ between the 2 groups (P =.93). Moreover, the metabolic ratio of midazolam to 1′-hydroxy midazolam did not differ between the 2 groups for both oral administration (P =.67) and intravenous administration (P =.26). CYP3A4 and CYP3A5 genotypes did not influence midazolam pharmacokinetics. The observed difference in docetaxel pharmacokinetics between both patient groups therefore appears to be explained neither by a difference in midazolam CL nor by a difference in metabolic conversion rate of midazolam

A Phase I dose-escalation study of two cycles carboplatin-olaparib followed by olaparib monotherapy in patients with advanced cancer

Preclinical studies have shown synergistic effects when combining PARP1/2 inhibitors and platinum drugs in BRCA1/2 mutated cancer cell models. After a formulation change of olaparib from capsules to tablets, we initiated a dose finding study of olaparib tablets bidaily (BID) continuously with carboplatin to prepare comparative studies in this patient group. Patients were included in a 3 + 3 dose-escalation schedule: olaparib 25 mg BID and carboplatin area under the curve (AUC) 3 mg*min/mL d1/d22, olaparib 25 mg BID and carboplatin AUC 4 mg*min/mL d1/d22, followed by increasing dose-levels of olaparib from 50 mg BID, 75 mg BID, to 100 mg BID with carboplatin at AUC 4 mg*min/mL d1/d22. After two cycles, patients continued olaparib 300 mg BID as monotherapy. Primary objective was to assess the maximum tolerable dose (MTD). Twenty-four patients with a confirmed diagnosis of advanced cancer were included. Most common adverse events were nausea (46%), fatigue (33%) and platelet count decrease (33%). Dose-level 3 (olaparib 75 mg BID and carboplatin AUC 4 mg*min/mL; n = 6) was defined as MTD. Fourteen out of 24 patients (56%) had a partial response as best response (RECIST 1.1). Systemic exposure of the olaparib tablet formulation appeared comparable to the previous capsule formulation with olaparib tablet AUC0-12 of 16.3 μg/mL*h at MTD. Polymers of ADP-ribose levels in peripheral blood mononuclear cells were reduced by 98.7% ± 0.14% at Day 8 compared to Day 1 for dose-level 3. Olaparib tablets 75 mg BID and carboplatin AUC 4 mg*min/mL for two cycles preceding olaparib monotherapy 300 mg is a feasible and tolerable treatment schedule for patients with advanced cancer

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