Aminopterin,

In view of the well-documented inhibition of dihydrofolate reductase by aminopterin (325), methotrexate (326) and related compounds it is generally accepted that this inhibitory effect constitutes the primary metabolic action of folate analogues and results in a block in the conversion of folate and dihydrofolate (DHF) to THF and its derivatives. As a consequence of this block, tissues become deficient in the THF derivatives, and this deficiency has many consequences similar to those resulting from nutritional folate deficiency. The crucial effect, however, is a depression of thymidylate synthesis with a consequent failure in DNA synthesis and arrest of cell division that has lethal results in rapidly proliferating tissues such as intestinal mucosa and bone marrow (B-69MI21604, B-69MI21605). 

In the treatment of human neoplastic diseases methotrexate has largely supplanted aminopterin in chemotherapy, due to the better therapeutic index of the former in experimental animals, although this superiority over (325) has not been conclusively demonstrated in man. 

Aminolevulinic acid dehydratase 3-aminotriazole toxicity to, 1, 139 Aminopterin—see Folic acid, 4-amino-Aminopyrine as antipyretic, 1, 172 biological activity, 5, 295 Aminyl, dimethyl-ESR, 7, 19 Amiphenazole... 

The cell fusion mixture is transferred to a culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium). Unflised myeloma cells are unable to grow as they lack HGPRT. Unflised normal spleen cells can grow but their proliferahon is limited and they eventually die out. The hybridoma cell can proliferate in the HAT medium as the normal spleen cell supplies the enzyme which enables the hybridoma to utilize extracellular hypoxanthine. 

Inhibition of nucleobase synthesis (2). Tetrahydrofolic acid (THF) is required for the synthesis of both purine bases and thymidine. Formation of THF from folic acid involves dihydrofolate reductase (p. 272). The folate analogues aminopterin and methotrexate (ame-thopterin) inhibit enzyme activity as false substrates. As cellular stores of THF are depleted, synthesis of DNA and RNA building blocks ceases. The effect of these antimetabolites can be reversed Ltillmann, Color Atlas of Pharmacology 2000 Thieme All rights reserved. Usage subject to terms and conditions of iicense. 

Successful fusion (2) is a rare event, but the frequency can be improved by adding polyethylene glycol (PEG). To obtain only successfully fused cells, incubation is required for an extended period in a primary culture with HAT medium (3), which contains hypoxan-thine, aminopterin, and thymidine. Amino-pterin, an analogue of dihydrofolic acid, competitively inhibits dihydrofolate reductase and thus inhibits the synthesis of dTMP (see p. 402). As dTMP is essential for DNA synthesis, myeloma cells cannot survive in the presence of aminopterin. Although spleen cells are able to circumvent the inhibitory effect of aminopterin by using hypoxanthine and thymidine, they have a limited lifespan and die. Only hybridomas survive culture in HAT medium, because they possess both the immortality of the myeloma cells and the spleen cells metabolic side pathway. 

A Stock solution Vortex 45.4 mg aminopterine (4-aminopteroyl glutamic acid, sodium salt) in 5 ml ddH20 and dissolve the sofid by dropwise addition of 1M NaOH. Fill up to 100 ml and adjust pH to 7.5. Filter through a 0.2-pm filter and store at -20 °C. 

A novel approach to the production of antimalarial drugs has been described taking advantage of the in vivo transformation of pteridines by resident enzymes <2005AAC3652>. It was shown that simple precursors such as 2,4-diamino-6-hydroxymethylpterin can be converted into aminopterin or methotrexate, depending upon the precursor chosen, and that the dihydrofolate reductase inhibitors so formed were active against Plasmodium falciparum. 

Fusion with the cells compensates for this deficiency. When fused and unfused cells are incubated in the presence of the folic acid antagonist aminopterin, the de novo synthesis of purines and pyrimidines for DNA is blocked. Cells deficient in HGPRT die, whereas hybrid cells are able to bypass aminopterin blockage by metabolism of hypoxanthine and thymidine added to the medium. In the generation of mouse hybridomas, an number of myelomas deficient in HGPRT are available, all originating from MOPC 21, a spontaneous myeloma from the BALB/c mouse strain. 

Rat plasmacytomas are available, derived from the ileocecal lymph nodes of the LOU/C strain. The first rat-rat hybridoma was described by Galfre and coworkers in 1979 using the aminopterin sensitive line R210Y3.Agl.2.3 (Y3), which secretes immunoglobulin light chains. Further nonproducing rat myelomas YB2/0 and IR983F have since been derived. 

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