Methotrexate, assay

The methotrexate assay [130] is described in some detail, as it can serve as an example of the chemiluminescence immunoassay method in general [131],... 

We saw in Chapter 3 that bisubstrate reactions can conform to a number of different reaction mechanisms. We saw further that the apparent value of a substrate Km (KT) can vary with the degree of saturation of the other substrate of the reaction, in different ways depending on the mechanistic details. Hence the determination of balanced conditions for screening of an enzyme that catalyzes a bisubstrate reaction will require a prior knowledge of reaction mechanism. This places a necessary, but often overlooked, burden on the scientist to determine the reaction mechanism of the enzyme before finalizing assay conditions for HTS purposes. The importance of this mechanistic information cannot be overstated. We have already seen, in the examples of methotrexate inhibition of dihydrofolate, mycophenolic acid inhibiton of IMP dehydrogenase, and epristeride inhibition of steroid 5a-reductase (Chapter 3), how the [5]/A p ratio can influence one s ability to identify uncompetitive inhibitors of bisubstrate reactions. We have also seen that our ability to discover uncompetitive inhibitors of such reactions must be balanced with our ability to discover competitive inhibitors as well. 

Recently, Choy et al. also reported that LDHs are an efficient drug reservoir for folate derivatives [187]. Folic acid derivatives, folinic acid and methotrexate (MTX), have been successfully hybridized with Mg/Al LDHs by ion-exchange reactions. Cellular uptake tests with the MTX-LDH hybrids were carried out in the fibroblast (human tendon) and osteosarcoma (SaOS-2) cell lines by in vitro assay. They found that the LDH not only plays a role as a biocompatible delivery matrix for drugs but also facilitates a significant increase in the delivery efficiency. 

Drug/Lab test interactions Methotrexate, pyrimethamine, and most antibiotics invalidate folic acid and vitamin Bi2diagnostic microbiological blood assays. 

W.M. Spees, T.P.F. Gade, G.L. Yang, W.P. Tong, W.G. Bornmann, R. Gorlick, J.A. Koutcher, An F-19 magnetic resonance-based in vivo assay of solid tumor methotrexate resistance Proof of principle, Clin. Cancer Res. 11 (2005) 1454-1461. 

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

Albertioni E, Rask C, Eksborg S, Poulsen JH, Petters-son B, Beck O, et al. Evaluation of clinical assays for measuring high-dose methotrexate in plasma. Clin Chem 1996 42 39-44. 

M.-L. Chen, HPLC assay and pharmacokinetics of methotrexate, Ph.D. Thesis, University of Illinois, 1982. 

Adjuvant chemotherapy with cyclophosphamide, methotrexate, and fiuoro-uracil (CMF) in early breast cancer prolongs relapse-free survival and overall survival in premenopausal patients, but has only a slight effect in postmenopausal patients (B9). Because chemotherapy causes ovarian suppression (D2) and breast cancer is responsive to endocrine therapy, it is important to know if, and to what extent, the effect of chemotherapy is mediated by ovarian suppression. The efficacy of ERP/PRP assays in predicting the response of breast cancer to chemotherapy has been studied by many investigators in their search for a marker that would prepict the tumor response. 

Caimes, DA. Evems, W.E. High-performance hquid chromatographic assay of methotrexate, 7-hydrox-ymethotrexate, 4-deoiQ -4-amino-N10-methylpteroic acid and sulfamethoxazole in serum, urine and cerebrospined fluid. J.Chromatogr., 1982, 231, 103—110... 

FBS, split twice per week by incubation for 5 min with an enzyme-free cell dissociation buffer, and reseeded at a density of 1-2 x 10 cells/cm. Before an assay, the cells are seeded in methotrexate at 2 x 10 cells/well in flat-bottomed 96-well tissue culture plates, allowed to attach for 6h, washed with 250-pL/well PBS, and incubated overnight in methotrexate containing endotoxin-free BSA (0.1%, w/v). 

Tayab Z R, Balthasar J P (2004). Development and validation of enzyme-linked immunosorbent assays for quantification of anti-methotrexate IgG and Fab in mouse and rat plasma. J. Immunoassay Immunochem. 25 335-344. 

DHFR) inhibitor classes (e.g., methotrexate and methylprednisolone), they induced the AMLl/ETO abrogation signature at low nanomolar concentration. Hit confirmation was done by cell-based assays, where the compounds induced myeloid differentiation, dramatically reduced cell viability, and ultimately induced apoptosis. 

Fig. 2. ADPR-transferase activity in permeabilized cells. Conditions for growing cells and adding drugs was as described for Fig. 1 except that [ H]-thymidine was not present. At the times indicated cells were recovered, permeabilized and assayed for transferase activity [5]. The cell growth media contained either 100 juAf methotrexate alone ( ) or 100 yM methotrexate and 25 nM hypo-xanthine (O)...

---