Thymidylate synthase expression in patients with colorectal carcinoma using a polyclonal thymidylate synthase antibody in comparison to the TS 106 monoclonal antibody.

Colorectal cancer is one of the most common human cancers, for which 5-fluorouracil (5FU) is usually part of the treatment. Thymidylate synthase (TS), the target enzyme for 5FU, can be predictive for the outcome of 5FU-based therapy. TS levels in tumor samples can be determined with radiochemical enzyme assays, RT-PCR, and immunohistochemical staining. We validated TS immunohistochemistry with a polyclonal rabbit anti-human TS antibody using the avidin-biotin method. This antibody can be used on paraffin-embedded, formalin-fixed material using an antigen retrieval method with citrate buffer and microwave treatment. The antibody shows a granular cytosolic staining pattern. The reproducibility in cross-sections from colorectal tumors from 50 patients was 90% and the interobserver variability was acceptable with a kappa of 0.45. On Western blotting it detects purified TS at 36 kD, while in 5FU-treated cells the ternary complex between FdUMP, TS, and 5, 10-methylene-tetrahydrofolate is clearly visible at 38 kD, with no other interfering bands. In a separate set of tumors, immunostaining was compared with enzyme levels; Western blots correlated with enzyme levels. Because both this polyclonal antibody and the monoclonal antibody TS-106 are being used for large-scale studies, we also determined whether they could be used interchangeably. No differences were observed. This polyclonal antibody is specific and gives reproducible results. A study on a larger scale is ongoing to determine the role of TS as a predictive parameter in patients with colorectal cancer treated either with postoperative adjuvant 5FU/levamisole or with surgery only.

Immunoreactive dUMP and TTP pools as an index of thymidylate synthase inhibition; effect of tomudex (ZD1694) and a nonpolyglutamated quinazoline antifolate (CB30900) in L1210 mouse leukaemia cells.

The inhibition of thymidylate synthase (TS) as a drug development target has received much attention in recent years, and several compounds have reached clinical evaluation. During drug development, the effectiveness of target inhibition can be assessed by determination of the perturbations of deoxythymidine 5-triphosphate (TTP) and deoxyuridine 5'-monophosphate (dUMP) pools in drug-treated cells. Rapid, sensitive, and reproducible radioimmunoassays for TTP pools and immunoreactive dUMP pools have been developed to meet our requirement for the rapid assessment of TS inhibition by quinazoline antifolates. The assays can be carried out on 1-2 million cells, and require minimal sample preparation. The limit of detection for TTP is 1 pmole/10(6) cells and for immunoreactive dUMP ("dUMP"), 3.0 pmole/10(6) cells, both assays being performed on the same cell extract. TTP and "dUMP" pools have been measured in mouse L1210 leukaemia cells treated with the quinazoline antifolates ZD1694 (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl )-N-methylamine]-2-thenoyl)-L-glutamic acid) and CB30900 (N-[N-[4-[N-[(3,4-dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)methyl ]-N-prop-2- ynylamino]-2-fluorobenzoyl]-L-gamma-glutamyl]-D-glutamic acid). Unlike ZD1694, CB30900 is a TS inhibitor that does not rely on polyglutamation for activity. In L1210 cells, both compounds caused a rapid inhibition of TTP pools in a dose- and time-related manner. Greater than 90% TS inhibition was achieved following a 4-hr exposure to each compound at equitoxic doses (up to 100 times the IC50 determine by a 48-hr growth inhibition assay). For both compounds, this was accompanied by a 5-10-fold increase in "dUMP" pools. For ZD1694, neither the TTP pool or "dUMP" levels were normalised when cells were resuspended in a drug-free medium for 4 hr and, at the higher doses studied, TS was still inhibited after a 16-hr period in the absence of drug. This is consistent with the formation and intracellular retention of potent polyglutamated forms of ZD1694. In contrast, TS activity as determined by repletion of the TTP pools and normalisation of "dUMP" levels were demonstrated for CB30900. However, at a high dose (50 microM, equivalent to 250 times the IC50), retention of TS inhibition was observed following 4 hr, but not 16 hr in the absence of drug. The radioimmunoassays described will prove useful to further define the extent and time-course of TS inhibition by novel antifolate compounds, and will also provide valuable in vitro and in vivo pharmacodynamic information on established antimetabolites when used alone or in combination with other drugs and modulators.

The pharmacokinetics of subcutaneous bolus cytosine arabinoside in an arachis oil plus aluminium distearate suspension.

An attempt was made to create a delayed release preparation of cytosine arabinoside (araC) which could be administered subcutaneously, and would produce plasma levels similar to steady state infusion concentrations. A thixotropic suspension of araC in arachis oil and aluminium distearate was formulated. This preparation was similar to that previously used with bleomycin oil suspension and procaine penicillin. Two hundred mg/ml of araC in arachis oil containing varying amounts of aluminium distearate were administered firstly to New Zealand White rabbits and then to patients with acute myelogenous leukaemia. This preparation was well tolerated by both rabbits and patients but did not delay the release of araC from the subcutaneous tissues.

Techniques for the measurement of methotrexate in biological samples.

Techniques available for the measurement of methotrexate (MTX) in biological fluids are reviewed. The importance of measuring MTX in plasma following high-dose MTX treatment is now well recognized and renewed interest in the metabolism and pharmacokinetics of the drug has prompted the development of new techniques of analysis. Results obtained with our radioimmunoassay are presented and compared with results obtained by other methods.