Liia D. Vainchtein

Carcinogen and Anticancer Drug Transport by Mrp2 in Vivo: Studies Using Mrp2 (Abcc2) Knockout Mice

The ATP-binding-cassette (ABC) transporter multidrug resistance protein (MRP) 2 (ABCC2) forms a natural barrier and efflux system for various (conjugates of) drugs, other xenotoxins, and endogenous compounds. To obtain insight in the pharmacological and physiological functions of Mrp2, we generated Mrp2 knockout mice, which were viable and fertile but suffered from mild hyperbilirubinemia due to impaired excretion of bilirubin monoglucuronides into bile. The mice also had an 80-fold decreased biliary glutathione excretion and a 63% reduced bile flow. Levels of Mrp3 (Abcc3) in liver and Mrp4 (Abcc4) in kidney of Mrp2-/- mice were approximately 2-fold increased. After oral administration of the food-derived carcinogens [14C]PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine) and [14C]IQ (2-amino-3-methylimidazo[4,5-f]quinoline) plasma values were 1.9- and 1.7-fold higher in Mrp2-/- mice versus wild-type mice, respectively, demonstrating the role of Mrp2 in restricting exposure to these compounds. At a high dose of 50 mg/kg of the drug [3H]methotrexate, the plasma area under the curve for i.v. administration was 1.8-fold higher in Mrp2-/- mice (1345 ± 207 versus 734 ± 81 min ·μg/ml). No clear plasma concentration difference arose at low dose (1 mg/kg). Subsequently, Mdr1a/b/Mrp2 knockout mice were generated. Their biliary excretion of doxorubicin after i.v. administration (5 mg/kg) was 54-fold decreased (0.32 ± 0.13 versus 17.30 ± 6.59 nmol/g liver in wild type), and a role for both Mdr1a/b and Mrp2 in this process was revealed. Our results demonstrate that the Mrp2-/- mouse provides a valuable tool for studies of the impact of Mrp2 on behavior of drugs and other toxins, especially when combined with other ABC transporter knockout mice.

Enhanced resolution triple‐quadrupole mass spectrometry for ultra‐sensitive and quantitative analysis of the investigational anticancer agent EO9 (apaziquone) and its metabolite EO5a in human and dog plasma to support (pre)‐clinical studies of EOquin® given intravesically

A highly sensitive and selective liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to quantify the experimental anticancer agent EO9 and its metabolite EO5a in biological matrices. A 200-microL aliquot of human/dog plasma was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and extracted with 1.25 mL of ethyl acetate. Dried extracts were reconstituted in 0.1 M ammonium acetate/methanol (7:3, v/v) and 20-microL volumes were injected onto the LC system. Separation was achieved on a 150 x 2.1 mm C18 column using an alkaline eluent (1 mM ammonium hydroxide/methanol (gradient system)). The detection was performed by a Finnigan TSQ Quantum Ultra equipped with an electrospray ionization source operated in positive mode and enhanced mass resolution capability. It demonstrated improved sensitivity with a factor 10-20 for EO9 and EO5a over a 3-decades dynamic range, with acceptable accuracy and precision, when compared with the previously described assay for EO9 and EO5a, developed by our group, using an API 2000. The assay quantifies a range from 0.5 to 500 ng/mL for EO9 and EO5a using 200-microL human plasma and dog samples. The described mass resolution method was successfully applied for the evaluation of the pharmacokinetic profile of EO9 and its metabolite EO5a in human and dog plasma.

Ultrasensitive Bioanalytical Assays for Cytotoxic Drugs: Focus on Locally Administered Anti-Cancer Agents

Local administration routes have been investigated to reduce the systemic toxicity and to increase the local ef- ficacy of cytotoxic drugs. Some examples of local administration strategies are cutaneous, intraperitoneal, intrathecal and intravesical chemotherapy. When administered locally, high local drug concentrations can be achieved with increased lo- cal efficacy and, conditionally that only small amounts of drug are absorbed into the bloodstream, low systemic toxicity. Our main purpose is to make an inventory and to comment on the availability of ultrasensitive bioanalytical assays that could determine traces of the drugs that may have passed into the bloodstream, e.g. after local application, and which may lead to the systemic toxicity. We conclude that in the last years, multiple ultrasensitive assays have been designed capable to quantitatively determine very low levels of cytotoxic agents e.g. systemically reached after local administration. Most methods are based on the hyphenated liquid chromatography with tandem mass spectrometric detection.

1577PGERMLINE TYMS GENOTYPE IS HIGHLY PREDICTIVE IN PATIENTS WITH ADVANCED COLORECTAL AND GASTROESOPHAGEAL CANCER INDEPENDENT OF FLUOROPYRIMIDINE PHARMACOLOGY: RESULTS FROM TWO PROSPECTIVE TRANSLATIONAL STUDIES

ABSTRACT Aim: Germline gene polymorphisms may impact clinical outcome in patients with gastrointestinal malignancies, but there are no data on the interaction between chemotherapy pharmacokinetics (PK) and gene polymorphisms, to assess their independent predictive value. Methods: Two studies prospectively assessed 44 gene polymorphisms in 16 genes (TYMS, MTHFR, GSTP1, -M1, -T1, DPYD, XRCC1, XRCC3, XPD, ERCC1, RECQ1, RAD54L, ABCB1, ABCC2, ABCG2, UGT2B7) by Sanger sequencing in 64 patients with advanced colorectal cancer (CRC) receiving capecitabine/oxaliplatin chemotherapy, and 76 patients with advanced gastroesophageal (GE) cancer receiving epirubicin/cisplatin/capecitabine (ECC) chemotherapy, respectively. Plasma concentrations of all anticancer drugs were sampled for up to 24 hours, analyzed using validated LC-MS/MS (capecitabine), HPLC (epirubicin) and flameless atomic absorption spectrometry (platinum), and results submitted to population PK analysis using non-linear mixed effects modeling. We calculated the association between gene polymorphisms, anticancer drug exposure, radiological tumor response, progression-free survival (PFS), overall survival (OS) and chemotherapy-related toxicity using appropriate statistical tests. Results: Patients with a low clearance of the active metabolite 5FU had an increased risk of neutropenia in both groups (p A and A2846T were associated with diarrhea (p Conclusions: Carriers of the TYMS high-expression genotype have a markedly inferior outcome when receiving capecitabine-based chemotherapy for advanced colorectal or gastroesophageal cancer. Therapeutic targeting of this molecularly-defined subgroup of patients should be explored to improve their prognosis. Disclosure: All authors have declared no conflicts of interest.

Simultaneous Quantitative Analysis of EO9 (Apaziquone) and its Conversion Products EO5a and EO9-Cl in Human and Dog Urine by High-Performance Liquid Chromatography Coupled with Electrospray Tandem Mass Spectrometry

A sensitive and specific LC-MS/MS assay for the quantitative determination of anticancer agent EO9 and its conversion products EO5a and EO9-Cl in human and dog urine is presented. A 20-μL-urine aliquot was spiked with a mixture of deuterated internal standards EO9-d3 and EO5a-d4 and diluted with 180 �L 0.1 M ammonium acetate - methanol (7:3, v/v). Next, 25 μL-volumes were injected onto the HPLC system. Separation was achieved on a 150 � 2.1 mm C18 column using an alkaline eluent (1 mM ammonium hydroxide - methanol (gradient system)). Detection was executed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range from 0.1 �g/mL to 50 � g/mL for EO9, from 0.2 � g/mL to 50 � g/mL for EO5a and 0.1 � g/mL to 4 � g/mL for EO9-Cl using 20 μL of stabilized urine samples. Validation results demonstrate that EO9, EO5a and EO9-Cl concentrations can be accurately and precisely quantified in human and dog urine. This assay is used now to support pre-clinical and clinical pharma- cologic studies with intravesically administered EO9.