Cyanamide promotes the antitumor effect of cyclophosphamide more than its toxic effect

Abstract

Cyclophosphamide (cyclophosphan, CP) was synthesized in the mid-20th century, but it is still one of widely used antitumor drugs in clinical practice. CP itself is inactive, but, in the body, it is hydroxylated by liver microsomal monoamine oxidases to 4-hydroxycyclophosphamide; after opening the rings, this metabolite is converted into tautomeric aldophosphamide, which nonenzymatically dissociates, forming active alkylating compounds: phosphoramide mustard (PM) and acrolein. A significant proportion of acrolein interacts with glutathione in the cytoplasm and is thereby inactivated, whereas the less reactive PM penetrates into the nucleus and alkylates DNA and other macromolecules [1, 2]. The antitumor CP activity is primarily associated with PM, whereas acrolein is assumed to be responsible for some toxic effects of CP, in particular, in the case of lung [3] and urinary bladder [4] damage. Apart from acrolein, some other CP metabolites display insignificant antitumor activity; these are chloracetaldehyde (it is formed after enzymatic splitting off the CP “war-heads,” chlorethyl radicals) and carboxycyclophosphamide, a product of aldophosphamide oxidation by another enzyme, aldehyde dedydrogenase (ADG), before it degrades into PM and acrolein [2, 5, 6]. Hence, to improve the antitumor and reduce the toxic effects of CP, the activating and inactivating reactions of CP metabolism could be stimulated or inhibited, respectively. Since ADG is the main factor of CP inactivation, we attempted to improve the CP effect by using ADG inhibitors. The results obtained showed that cyanamide, a specific ADG inhibitor, administered at nontoxic doses along with CP, improved the CP therapeutic effect [6]. However, these results had no clinical impications, because ADG inhibitors were supposed to damage stem cells to a greater extent than tumor cells by producing degradation products of 4-hydroxy- and aldophosphamide, namely, PM and acrolein [5, 6]. We used a number of experimental models to study the effect of cyanamide on the antitumor and toxic activities of CP, and the data obtained showed that the above suggestion was unjustified. Male A/Sn and CBA mice carrying intramuscular transplants of the HA-1 hepatocellular tumor [7] and RLS lymphosarcoma [8], respectively, were used in our experiments. Apart from a solid node in the area of transplantation, the HA-1 tumor formed numerous metastases in the liver, which caused animal death. In mice treated with CP alone, the growth of primary nodes and that of intrahepatic metastases was somewhat inhibited, but the lifespan of the animals remained almost unchanged (increased by 17%) (Table 1). Cyanamide itself had no effect on these parameters, but, in combination with the same dose of CP, cyanamide inhibited the growth of both primary and metastatic tumors significantly, and the lifespan of mice with tumors was at least 80% increased. In mice with RLS lymphosarcoma, the effect of CP at a dose of 100 mg/kg body weight on the tumor growth was insignificant, whereas when administered at a still tolerable dose of 300 mg/kg body weight, CP inhibited tumor growth significantly and increased the animal lifespan. In combination with cyanamide (70 mg/kg body weight), CP at a dose of 100 mg/kg body weight caused the same therapeutic effect as CP alone used at a dose of 300 mg/kg body weight (Table 2). This dose ratio was used to compare the general toxic effects of CP alone and CP in combination with cyanamide on the CBA male mice weighing 29‐30 g. Before the experiment, aliquots of blood were obtained from the tale tip to determine the number of leukocytes. Eight animals were injected intravenously with CP at a dose of 300 mg/kg body weight, and nine animals were injected with CP at a dose of 100 mg/kg body weight 15 min after intraperitoneous administration of cyanamide. Blood was also sampled daily from the 2nd to the 7th day after the substances were administered. A com