Frederick S. Philips

THE TOXIC EFFECTS OF 6‐MERCAPTOPURINE AND RELATED COMPOUNDS

The present review of the toxic effects of 6-mercaptopurine (6-MP) in experimental mammals has two primary aims. The first and more practical purpose concerns risks to be anticipated during therapeutic use. The second aim concerns the specific cytocidal effects of 6-MP as a possible contribution to the elucidation of its mechanism of action. As such, the study is a continuation of work of this laboratory by which i t has been possible to differentiate the cytotoxic effects of folic acid antagonists such as aminopterinl and certain diaminopyrimidines2s2a from those of the nitrogen mustards and mustardlike compounds, such as HN2 and TEM; and from those of 2,6-diaminopuIn the present instance, tissue changes induced by 6-MP are compared with those caused by 2,6-diaminopurine (2,6-DAP) and by other purine analogs. Certainly if the action of 6-MP is to be explained in terms of a specific metabolite antagonism, then such an exposition must draw sustenance not only from differences in toxic properties between 6-MP and, for example, 2,6-DAP1 but must also reconcile similarities in the effects of these compounds. Toxicity studies. FIGURE 1 provides a list of the purines employed and presents graphically their relative potency in terms of toxicity in Swiss male mice. In most instances, two schedules of dosage have been used, i.e., single and five successive daily injections. The agents are listed in an order largely based on their relative potency by chronic administration. The bars topped by arrows refer to the fact that the highest dose employed, the quantity indicated by the height of the serrated column, is less than LDso. Data are taken from previous reports45 , 6. and from unpublished observations of the author^.^ The graph illustrates that there is more than a hundredfold difference in potency between the two extreme members of the series. Moreover, 6-MP is neither the most nor least toxic of its close structural analogs. The extent by which the toxicity of each compound is enhanced by cumulative action can be appreciated by comparing the LDE0 of single doses with that of multiple doses. It may be seen that most of the compounds, including 6-MP, exhibit relatively minor cumulative activity, i.e., the LDso in mgm./kg./day for five daily doses, when expressed as a fraction of the single median lethal dose, ranges between one fourth in the case of adenine to more than one half in the case of 6-chloro-

THE TOXICITY OF ACTINOMYCIN D

The high toxicity of the actinomycins in mammals was first described after the isolation of the A complex from Streptomyces antibioticus by Waksman and Woodruff,’ whose early work directed attention to what appeared a t first a selective atrophy of the mammalian spleet1.2,~ These investigators also showed that doses not greatly exceeding the LDSo killed, within less than 24 hours. Somewhat more delayed fatalities were caused by lower single doses or by repeated daily injections of small fractions of the acute LDbo. Anorexia, diarrhea, disturbances of locomotion, and weakness were features of chronic poisoning. Hackmann’s later studies of the C complex showed that the spleen was not the only susceptible lymphoid tissue: thymus and lymph nodes were equally or, possibly, more sensitive sites of damage.4t6 He also reported mitotic inhibition in the crypts of Lieberkuhn, as well as degeneration and desquamation of the intestinal epithelium. Untoward clinical effects such as stomatitis, nausea, and diarrhea were considered by him to be related manifestations of gastrointestinal damage. Our present interest in these compounds stemmed primarily from the description given by Hackmann and by Schmidt-Kastner of the biosynthetic actinomycin F1 (KSJ . 6 J Since F1 had antitumor activity but was significantly less toxic than earlier actinomycins, it seemed worthwhile to learn whether or not it might be distinguished by other qualitatively different pharmacological effects. For a more meaningful comparison, we decided to become familiar first with the toxic properties of actinomycin D8t9 in different laboratory animal species. This is the subject of the present communication, which extends the earlier observations of other

Effect of 4-Amino-Pteroylglutamic Acid (Aminopterin) on Early Pregnancy.

Summary 4-amino-pteroylglutamic acid was found to be lethal to the early embryo of mice and rats. Fetal death was observed with doses which temporarily caused depletion of the bone marrow but no other significant signs of intoxication in mothers.

Binding of [G-3H]-7,12-dimethylbenz(α)anthracene to DNA of normal and of rapidly dividing hepatic cells of rats

Abstract The administration of tritium-labeled 7,12-dimethylbenz(α)anthracene (DMBA) to rats at 24 h after partial hepatectomy resulted in its binding to DNA in small intestine and regenerating liver. The binding in regenerating liver, which began 3 h after i.v. administration of DMBA, reached a maximum at 6 h and declined at 24 h; the uptake in intestinal DNA was similar but there was no decline for 48 h. The DMBA-induced inhibition of DNA synthesis paralleled uptake; both were dose dependent. More than twice as much DMBA was bound in vitro to DNA that had been isolated from livers of immature rats or from those undergoing regeneration than to DNA from livers of young adults. Such a difference was also observed in vivo. The possibility that the binding of DMBA is causally related to inhibition of DNA synthesis and carcinogenesis is discussed. Two forms of binding were apparent. In one the attractive forces were weak. In the other the binding resisted extensive dialysis, solvent extraction and denaturation. That the tightly bound form involved unchanged hydrocarbon was shown by direct gas-chromatographic analysis of the DNA-DMBA complex. The amount of DMBA tightly bound after i.v. injection of biologically active doses of 12.5–25 mg/kg corresponded to about 10·103–20·103 molecules per regenerating rat liver cell genome. This kind of interaction with DNA, which may be causally related to the mechanism of DMBA-induced oncogenesis, does not appear to require prior metabolic activation of the agent.

Effects of 4-amino-pteroylglutamic acid in dogs with special reference to megaloblastosis.

Summary The administration of 4-amino-PGA to dogs produced a sprue-like syndrome with diarrhea, peripheral leucopenia, depletion of bone marrow with increased numbers of hyper segmented polymorphonuclears and giant metamyelocytes, abnormal nuclear disintegration of normoblasts, change of nuclear pattern in erythroid elements and megalo-blasts. Doses given were lethal and animals succumbed with anorexia, weight loss, dehydration, and hemorrhagic diarrhea following severe ileitis and ulcerative colitis. It is suggested that the syndrome results from antagonism of folic acid.

Effects of N-Methylformamide and Related Compounds in Mouse Sarcoma 180.

Summary and Conclusions 1. Formamide and its more potent N-methyl derivative have been described as transient inhibitors of the growth of mouse sarcoma 180. This effect is not a property of formamides in general since other compounds containing the formamide moiety have failed to affect the growth of the tumor. 2. N-methylformamide has been shown to exert its effects when therapy is started either 24 or 96 hours after implantation of the tumor or when the agent is given in a single, large dose. Further, the compound has been found equally effective whether given by the oral or intraperitoneal route. 3. Histologic changes of a non-specific nature have been described in tumors from animals treated with N-methylformamide. 4. In view of the structural resemblance between formamide and urethane, the actions of both agents have been compared. Urethane, like formamide, inhibits the growth of S-180; however, treatment with the former substance causes toxic manifestations not encountered in mice given formamide. 5. Several conceivable mechanisms have been proposed to account for the inhibitory effects of the formamides. Further studies of these suggested modes of action may provide useful information concerning biochemical mechanisms involved in the growth of tumors. 6. In view of the hepatotoxicity of formamide and its N-methyl derivative it is not expected that these agents will prove therapeutically useful.

Actions of 2,6-diaminopurine in mice, rats, and dogs.

Summary The administration of large doses of 2,6-diaminopurine in mice and rats can result in death within a few hours. In dogs, large doses caused protracted vomiting, hemorrhagic diarrhea, dehydration and death within 48 hours. Doses permitting survival of rats and dogs for 24 hours or longer produced depletion of bone marrow and damage to epithelium of colon and ileum. The course of intoxication and changes in bone marrow permit a differentiation between the actions of 2,6-diammopurine and a folic acid-antagonist like 4-aminopteroylglutamic acid.

Susceptibility of Intestinal Crypt Epithelium to Hydroxyurea and Other Antitumor Agents

The outstanding limitation in the effective use of most antitumor agents is selective damage in normal cell renewal systems in vivo. These systems contain undifferentiated or partially differentiated precursors of various mature cells such as leukocytes, erythrocytes, villar epithelium of intestine, sperm, and non-germinal components of epidermis. Normally the systems operate to maintain a steady balance between the birth of differentiated descendants and their continuous loss from the organism (6). The susceptibility of two of the cell renewal populations, that is, hematopoietic tissues and digestive tract epithelium, commonly determines the upper limit of doses of antitumor drugs. It is likely that lesions in the digestive tract are the major determinant of maximal dosage since these give rise to distressing symptoms such as oropharyngitis, nausea, and abdominal cramps and cause alarming signs of intoxication such as diarrhea and ulceration of pharyngeal and intestinal mucosa.

Hydroxyurea: inhibition of DNA-synthesis and selective susceptibility of S-phase cells in vivo.

The cytotoxic effects of hydroxyurea and of related hydroxamic acid derivatives in vivo are briefly described. They occur selectively in tissues with high rates of cell renewal and they are of brief duration. Tissue concentration of hydroxyurea diminish rapidly as the result of renal excretion and metabolism; there is a close temporal relation between the physiological disposition of the agents and the cytotoxic changes. Hydroxyurea induces an immediate inhibition of DNA synthesis in proliferating tisdsues such as thymus, small intestine, and regenerating liver. Autoradiographic studies of mouse duodenum using tritiated thymidine have shown that the lethal susceptibility to hydroxyurea is restricted to cells in the S-phase of the mitotic cycles. Cells in G 1 , G 2 , and M are not damaged by the agent.