7-hydroxy-methotrexate

Methotrexate acts by inhibition of dihydrofolate reductase, the enzyme requisite for the reduction of dihydrofolic acid (3) to 5,6,7,8-tetrahydrofolic acid (4). In turn, (4) is a precursor to a series of enzyme cofactors (5-7) essential for the transfer of one carbon unit necessary for the biosynthesis of purines and pyrimidines and hence, ultimately, DNA. As an inhibitor of dihydrofolate reductase, methotrexate kills cells during the S phase of the cell cycle, when the cells are in the log phase of growth. Unfortunately, this cytotoxicity is non-selective, and rapidly proliferating normal cells, e.g., gastrointestinal epithelium cells and bone marrow, are dramatically affected as well. In addition, recent use of high dose methotrexate therapy with leucovorin rescue has led to additional clinical problems arising from a dose-related nephrotoxic metabolite, 7-hydroxy methotrexate (8). Finally, the very polar nature of methotrexate renders it virtually impenetrable to the blood-brain barrier, which can necessitate direct intrathecal injection in order to achieve therapeutic doses for the treatment of CNS tumours. 

Slordal L, Sager G, Jaeger R, Aarbakke J. Interactions with the protein binding of 7-hydroxy-methotrexate in human serum in vitro. Biochem Pharmacol 1988 37(4) 607-11. 

Antimetabolites - Two reports on the metabolism of methotrexate (MTX) to 7-hydroxy-methotrexate (7-OH MTX) have appeared. The major metabolite of MTX in rabbits was found to be 7-OH MTX. The clearance of 7-OH MTX from plasma was slower than that of MTX itself. In a study of the uptake and metabolism of MTX in a MTX-sensitive human acute lymphoblastic leukemia cell line, MOLT4, it was shown that 7-OH MTX and MTX are converted to poly glutamates and that 7-OH MTX interferes with MTX accumulation and metabolism. Phase I clinical trials of 10-... 

Fig. 3 Venn-diagram for selected compounds interacting with the key MDR-related ABC transporters. MDR-substrate anticancer agents. Abbreviations VCR vincristine, VP-16 etoposide, STER steroids, TAM tamoxiphen, TKI-INHIB tyrosin kinase inhibitors e.g. STI-571, DOX doxorubicine or adriamycin, DNR daunorubicin, EPIR epirubicin, MX mitoxantrone, TOPOT topotecan, iridotecan, BISANT bisanthrone, COLCH colchicin, ACT-D actinomycin D, MYTOM mytomycin, TX methotrexate, CPHAM cyclophosphamide, CHLB chlorambucil, CARM carmustine, LCV leucovorin, HUR hydroxy urea, CISPL cisplatin, TAXOL paclitaxel. (Reproduced from [4])...

Methotrexate is readily filtered by the kidneys, and renal clearance is influence by both tubular secretion [139, 140, 141, 142] and tubular reabsorption [142]. Intravenous administration of methotrexate 140-350 mg/kg [<6h infusion] results in 70-94% of the dose appearing in the urine over 24 h [143]. In contrast, when methotrexate is administered as a 24-h continuous infusion, 60% of the dose is excreted in the mine during the 24-h infusion [144]. Approximately 10% of the dose is excreted in the urine as 7-hydroxymethotrexate [143,144]. The 7-hydroxy metabohte is important since it may contribute to the renal toxicity of methotrexate [148] and this moiety becomes a significant metabolite when methotrexate doses are 50 mg/kg or greater [145]. Following oral administration of methotrexate a lesser fraction of the dose is recovered in the urine than following intravenous administration [141]. This may reflect the dose-dependent incomplete absorption of methotrexate [141,146,147]. Methotrexate is highly bound to plasma proteins. 

The 96-well plate SPE procedures have become very popular. In Ch. 11.4, applications of SPE in 96-well plate format were described for LOR and DCL [29, 64-65]. Many other examples are available in literature. The use of SPE in a 384-well plate format was reported for the determination of methotrexate and its 7-hydroxy metaboUte in human plasma and urine [106]. The work-up time for the 384-well plate is similar as for the 96-well plate. 

G. Rule, M. Chappie, J.D. Henion, A 384-well SPE forLC-MS dS determination of methotrexate and its 7-hydroxy metabolite in human urine and plasma. Anal. Chem., 73 (2001)439. 

Rule, G. Henion, J. A 384-Well Solid-Phase Extraction for LC/MS/MS Determination of Methotrexate and its 7-Hydroxy Metabolite in Human Urine and Plasma, Ann/. Chem. 73,439 43 (2001). 

NATA, N-L-acetyltryptophanamide BSND, benzenesulfonamide BSFN, benzenesulfonate Sta, statine BZD, benzamidine CFM, coformycin MTX, methotrexate TMP, trimethoprim PT, pterin NPT, N5-deazapterin THP, tetrahydropyrrole Bz, benzene ADA, adenosine deaminase HCAIl, human carbonic anhydrase II TS, thymidyiate synifaase DHFR, dihydrofolate reductase MBP, mannose bindnig protein HEI, hydroxyethylene inhibitor HPR, (6R)-6-hydroxy-l,6 dihydropurine riboside (R),R configuration (S),S configuration NA, not available or not applicable. 

Gel prepared from N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) crosshnked with sodium tripolyphosphate and conjugated with the apo-transferrin These gels showed triggered release of methotrexate disodium (MTX) at low tumor pH. [24]... 

The remaining 59 patients, with relatively low serum bile acid concentrations (SGLC = 2.2 0.3,GC = 2.2 0.3, 3a-hydroxy bile acids = 10.3 0.9), included patients with mild liver disease, intestinal dysfunctions such as Crohn s disease, coeliac disease and gastroenteritis, acute lymphatic leukemia treated with methotrexate, contaminated small bowel syndrome and three patients with benign recurrent intrahepatic cholestasis (BRIC). 

A solution of modified chitosan, iV-[(2-hydroxy-3-trimethylammonium)propyl]chito-san chloride was combined with sodium tripolyphosphate in order to prepare the microgels and methotrexate disodium (MTX), a cyctotoxic drug utilized in the treatment of cancer was loaded into the gel. Further treatment allowed the hydrogel to undergo a lowering of their pH within the cell from 7.4 to 5.0 which enhances the speed at which the MTX would be released into the lower pH environment of the tumor cells (124). 

Figure lO.Obtusifolin hit docked in the active site of DHFR. The two hydroxy groups match the amino groups in the pteridine ring of methotrexate... 

The patients with pityriasis rubra pilaris had not responded to topical retinoic acid, oral vitamin A (750 000 I.U. for 6 weeks), topical keratolytics, topical cr-hydroxy acids, or systemic methotrexate. 

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