ANALOGS OF PTEROYLGLUTAMATE (fOLATE) 583 



concentration in the liver remaining approximately constant for at least 3 

 weeks after a single intravenous dose. It is noteworthy that some tissues, 

 such as the lung and spleen, do not pick up much of the analog, and that 

 the kidney loses the analog relatively more rapidly than the liver. Werk- 

 heiser (1960) likewise found that the liver retains aminopterin for long pe- 

 riods, while the intestine does not. In mice given a lethal dose of aminop- 

 terin 1 hr after a protective injection of folate, the liver folate reductase 

 activity is depressed 96% after 24 hr and remains at this level for 7 days, 

 following which there is a slow recovery (Werkheiser, 1962). The intestinal 

 enzyme is similarly inhibited but recovers faster. The loss of aminopterin 

 from the intestine is characterized by a half-life of 60 hr, but the liver shows 

 two components with half-lives of 60 hr and 90 days, respectively. It was 

 suggested that rapidly proliferating cells are dependent on folate reductase 

 activity and that the disappearance of the inhibition is faster in such tissue 

 because of the more rapid turnover of cells; in other words, the 60 hr com- 

 ponent would arise from proliferating tissue while the 90 day component 

 would relate to nonproliferating tissue. If this is the case, the binding of 

 aminopterin to the enzyme in vivo must be essentially irreversible. These 

 results all point to a very high degree of specificity in the binding and inhi- 

 bition, and confirm the major site of attack as being on folate reduction. 

 Further evidence comes from the reduced urinary folinate levels in rats on 

 25 //g/day of aminopterin (Nichol and Welch, 1950). Less direct evidence 

 is provided by Nichol (1954) and Broquist et al. (1953), who showed that 

 resistant streptococci or leukemic cells have a much greater ability to pro- 

 duce folinate from folate than do normal cells. It is possible that this in- 

 creased activity allows enough active folinate to be formed to enable the 

 cells to grow and multiply in the presence of the analogs, but it is probable 

 that this is not the only mechanism of resistance to these agents. 



The nature of the binding of these folate analogs to the reductase has 

 not been fully elucidated, but Zakrzewski (1963) has determined the ther- 

 modynamic characteristics for the dissociation of the EI complexes (see ac- 

 companying tabulation). The inhibitions by the substituted pteridines are 

 competitive. The K^ values for aminopterin were taken from Werkheiser 



