An analysis of 1-(2-chloroethyl)-1-nitrosourea activity at the cellular level

Abstract

The effect of five different 1-(2-chloroethyl)-1-nitrosoureas on the growth of cultured P388 cells has been analyzed in terms of physical, chemical, and kinetic parameters that are related to the mechanism of action of this class of cancer chemotherapeutic agent. This study correlates structure with activity at the cellular level by using a dose function that is related to the amount of active species, the (2-chloroethyl)diazonium ion, that is formed during the period of exposure of cells to drug rather than to the initial drug dose. 1-(2-Chloroethyl)-1-nitrosourea analogues that rapidly enter the P388 cells are shown to have the same activity relative to the amount of active species formed. When analyzed in this way, activity is not influenced by the structure of the N-3 substituent, lipophilicity, or carbamoylating activity. The agents 1-(2-chloroethyl)-1-nitrosourea (CNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) all produce a 50% cell growth inhibition at 6 to 7 microM active species formed per cell volume. Chlorozotocin required a twofold higher effective dose to produce the same toxic effect. This decreased activity is attributed to the slow uptake of the water-soluble chlorozotocin into P388 and L1210 cells relative to the rate of chlorozotocin conversion to active species in medium. The yields to 2-chloroethanol from CNU, BCNU, and chlorozotocin were shown to be the same, indicating that these agents generate the same yield of alkylating intermediate at 37 degrees C and pH 7.4.

Intracellular activation of cytotoxic agents: kinetic models for methylnitrosoureas and N-methyl-N'-nitro-N-nitrosoguanidine in cell culture

“…The cell culture cytotoxicity of the nitrosoureas MNU and streptozotocin is shown to be equal when referenced to the amount of methylation product formed within the cell during the assay incubation period. A similar observation was made with (chloroethyl)nitrosourea derivatives by using the same P388 cell growth rate assay (22) or 9L cell colony forming efficiency assay (21).…”

“…When the cytotoxic activity is related to the amount of intracellular methylation [P4], the ED50 values for MNU and streptozotocin are between 140 and 180 µ . According to the same assay, the EDjJPJ values for several (chloroethyl)nitrosoureas are between 6 and 7 µ (22).…”

“…The cytotoxicity experiments have been described in detail previously (22). Briefly, two different types of experiments were performed.…”

“…Equation 4 can be simplified further if the activation rates in medium and the cell are equal, kí2 = kM, and the fractional cell volume is small, to give eq 5. The latter equation is applicable to alkylnitrosoureas (21,22).…”

“…The properties of these reactions are important in the development of a kinetic model that represents the amount of active species formed in the cell. Previous studies developed a model for (chloroethyl) nitrosoureas (21,22), which is extended in this report to an agent, MNNG, that undergoes selective intracellular activation.…”