Synthesis of methotrexate-antibody conjugates by regiospecific coupling and assessment of drug and antitumor activities

Abstract

In order to increase the retention of drug activity, regiospecific coupling has been used to synthesize conjugates of methotrexate (MTX, 1) with normal rabbit IgG (NRG) and a mouse anti-human renal cancer monoclonal IgG (Dal K-20). MTX gamma-methyl ester (4) was produced either by selective esterification of MTX or by coupling of 4-amino-4-deoxy-N10-methylpteroic acid (2) with suitable glutamic acid derivatives. The MTX gamma-methyl ester (4) was then converted to the corresponding hydrazide 6. An amide-linked conjugate was formed when the MTX gamma-hydrazide (6) was converted to reactive acylating species 7 by using tert-butyl nitrite or trifluoroacetaldehyde, which were reacted with nucleophilic centers, presumably epsilon-amino groups, in native IgG. A hydrazone-linked conjugate was formed when MTX gamma-hydrazide (6) was reacted directly with IgG that had first been oxidized with periodate to form polyaldehyde IgG. The regiospecifically synthesized conjugates were somewhat more effective inhibitors in vitro of dihydrofolate reductase and of colony formation by human renal cancer (Caki-1) cells than were control nonregiospecific conjugates.

Selective coupling of methotrexate to peptide hormone carriers through a gamma-carboxamide linkage of its glutamic acid moiety: benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate activation in salt coupling.

“…This compound is a potent inhibitor of the enzyme dihydrofolate reductase, which is responsible for recycling 7,8-dihydrofolate to its reduced, physiologically active 6(R)-tetrahydro form (5). Although not all types of tumors are affected by MTX, this cytotoxic agent has been used successfully in chemotherapy for more than 4 decades.…”

Tissue localization of methotrexate-monoclonal-IgM immunoconjugates: Anti-SSEA-1 and MOPC 104E in mouse teratocarcinomas and normal tissues

“…MTX has been coupled to antibody amino groups and carbohydrates either directly or through various intermediate carriers (reviewed [11]; for more recent discussion, see [1,17,18,25,31,32,36,37]). Release of free drug from immunoconjugates has been a persistent problem.…”

“…Originally, conjugation to IgG antibodies gave enhanced specificity, but resulted in lowered drug efficacy and antibody binding [5,7,10,11,15,19,20,24,29]. More recently, several groups have made improved methotrexate conjugates having an efficacy greater than the equimolar drug and little loss of antibody binding capacity [17,18,23,30,31,32,36].…”

Soluble Polymers as Targetable Drug Carriers

“…It was shown that enhanced antibody affinity leads to increased endocytosis of the immunoconjugate and potentiation of cytotoxicity. KRALOvEC et al (1989) used a regiospecific coupling method to bind MTX to a mouse anti-human renal cancer monoclonal IgG . Two conjugates were synthesized.…”