Chemotherapy Investigations in Cancer with Reference to the Influence of Certain Organic Dibasic Acids, Diamino Compounds and Nitro Compounds on T Umors in Mice 1)

Chemotherapy Investigations in Cancer With Reference to the Influence of Certain Organic Dibasic Acids, Diamino Compounds and Nitro Compounds on T umors in Mice 1). L. \Voodhouse, Ph.I)., _XI.Sc. (From the Cancer Research Laboratory, The Medical School, Ui2iversitv of Birmi,gham, Birmingham, England) (Received for publication December 16, 1946)

Although knowlcdge of many aspects of cancer, I. Growths produccd (m mice by rcpcated appli- including thc chemistry of the ccll, is rapidly ex- cations of benzpyrcne in acetone to the skin. The panding, it is still impossiblc to hazard any but very tests wcrc comlnenced xvhcn the warts had been tentative hypotheses for guidance in selecting com- visible for several weeks and had an appearance sug- pounds for testing possible therapeutic value against gesting cpithelionla. Such tumors rarely regrcss even tumors. In thc past, substances havc becn investi- in the mixed strain of mice employed for inducing gated, in the majority of cases, on an empirical basis. the growths. A reasonable approach would be to observe thc pro- lI. Transplants from a spindle-celled sarcolna perties of members of various c/asses of compounds. originally induced with dibenzanthraccnc in this la- It might be possible in this way to eliminate many boratory and previously described (8). An inbred categories and obtain information indicating what but unnamed strain of mouse was used, in the leg types are likely to form a basis for yielding effective muscle of which this tumor grows regularly. The material. tumors are suitable for tests about 7 days after ino- Therefore, though aware that in thc abscnce of culation. 'l'hc transplants utilized were those after sound guiding principles such experiments can be some lnt0 or more passages. explorative only in nature, we have carried out tests III. Transl)lants of "carcinoma 63" which had on various groups of compounds from this point of becn through about 20 passages in this laboratory view. Some of these which may bc of interest to when these tests were commenced. other workers are briefly dcscrit~ed in this contri- B. lnjcctions.--In the experiments described bution which deals with thc action of some substi- here injcetions were chiefly subcutalaeous or intra- tuted succinic acids, a number of amino COlnplexes peritoneal, at a distance froln the tumor, twicc or and certain nitro componnds on thrcc types of three tilnes each week. Intravenous injections have mouse tumor. been given where the reaction suggcsted that the li- Boyland (2), by fccding ecrtain dibasic acids, ob- mited nmnber of injections possible by this route tained cvidcncc of distinct inhibitory cffccts on might be effective. There is evidence that some sub- mousc tmnors, onc of the most active being malonic stances, quite ineffective by other routes, arc inhibi- acid. Thc scrics of substituted suecinic acids were tory or even curative against certain mouse tulnors made available by Mr. Garfield 'l'homas (biochemist when injected intravenously (11). The various or- to Queen Elizabeth IIospital, Birmingham). "Ille ganic acids were easily soluble as such, or a sohfl)le amino compounds were supplied chiefly 1)y Mcssrs. sodium salt could easily hc made, and they proved May and Bakcr and in some instances are among quite without toxic effects when injected into con- the substances investigated by Boyland (3) who, trol animals in relatively large alnounts. \Vhcn the however, cmph)ycd different ,~.umors and technics. more poisonous sul)stanccs, e.g. the nitrated series, They all possess trypanocidal properties. Complcxes wcrc tested, the lethal dose was determined and with several of the amino compounds with lmeleic doses of one-fifth to one-tenth lethal were given in acids were also tested, for some of which I am in- a volume of 0.2 ml. for the subcutaneous injections debted to Prof. M. Staeey, Chemistry Department, and 0.10 ml. for the intravenous ones. \Vhen the University of Birmiugham. substance was not soluble enough to afford a suit- TECI IN ICAL METI IODS able aqueous solution a colloidal suspension stabi- lized bv a small amoullt of gum or gelatine, was A. Tumors and animals uscd.--The tumors used employed. for these experiments were: 398

The correlation of nmncrical designations with general toxic propertics. 'l'hus the tumor-ilfllibiting chemical compounds is as follows: action of carcinogenic hydrocarbons given subcu- taneously, appears to bc largely incidental to the gcn- 654 4,4'diaminostilbene eral disturbance of the metabolism of the animal 736 4,4'-diamidinodiphcnylcthcr 688 4,4'-diaminodiphcnylether (11). In order to control inhibition tests adequate- 875 4,4'-diamidinodiphcnylurca diacctatc ly, therefore, two control series arc rcquircd, one in 800 4,4'-diamidino-1,5-dephcnoxypentanc which the weight and health of noncancerous mice dihydrochloridc 971 4,4'-diaminoazobcnzcnc reccMng similar injections are observed, and another 952 1)isulphanilamide of tulnor-bcaring mice which arc allowed to continue 967 4,4'-diaminodiphenylthiourea without treatment. C. Control tests.--It has bccn shown that inauy An estimate of the effects of treatment of the experiments dcsigncd to demonstrate the inhibitory skin cpithcliomas was made by comparing the size

TABLE I: T~,:sTs \VITII DERIVATIVES OF DIBASIC ACIDS ON .~|ICl,; BEARING TRANSPI.ANTED '])IBENZANTIIRACENE' SARCO,~L~,S. (136TII-lqSTn GllAFTS.) 7TH-9TII DAY AI.'TER INOCUI_ATION. SUBCUTANEOUS INJECTIONS OF 2 MGM.

Final av. Initial Final wt. of av. wt. av. wt. No. of No. of ItllllOIs, of anim.ds, of aninmls, Group Treatlnel,t anilnals injections gin. gm. gin. Result 1 Succinic acid ~ 10 3.7 30 32 No inhibition 2 Methyl succinic acid 10 11 2.8 ~ 33 35 Some inhibition 3 Phcnyl succinic acid 8 8 3.85 29 32 No inhibition 4 Controls to 10 Gps. 1, ;.,~ 3, 10 -- 3.6~ 32 35 5 Cyclohcxyl succinic acid 8 8 2.7 z~8.S 33 Sol]lC inhibition 6 Asymdimcthyl succinic acid 10 9 3.63 32 35 No inhibition 7 Cyclohcxvl-1 -acctvl-1 -carboxvl 2.72 35 38 Some inhibition succinic acid 10 10 8 Controls to 4, 5, 6 l0 ~ 3.8 31 33 -- 9 1-I lvdroxvl- 1-carboxvl- cvclo- 3.8 30.6 30 No inhibition hexane 9 10 10 Malonic acid 6 10 3 0 30 32 Some inhibition 11 Glutaric acid 6 10 3.9 34 36 No inhibition 12 Controls to 9, 10, l l 10 10 3.8 34 37 TABI,r, II: TESTS wrrH CO~IPI.F,XES ON Mine wrrn TRANSPI,ANTED DIm.:NZ:~,x'rm~ACF.XE SARCONIA (180"rn-lq0T~I) TRANSI'I,ANTS). SUBCUTANEOUS IXJEeTIOS,'S 0.20 ML. = 0.~ MGM.

Av. Av. initial final Av. wts. wt. ot wt. of of No. of No. of animals. ,'/nimals tmnors GrouF T,'entmcnt animals injections gin. gin. gin. Ilesult 1 654 6 8 32 34 3.7 No inhibition 2 736 6 8 32 32 3.8 No inhibition 3 Ribonuclcic acid complcx with 736 7 7 30.6 32 2.4 Definite inhibition 4 688 5 8 31 33.5 3. ~ No inhibition 5 Ribonuclcic acid complex with 688 6 7 30.6 32 2.6 Definite inhibition 6 967 6 9 28.4 29. ~ 2.1 Definite inhibition 7 875 6 8 8 Desoxyribonuclcic acid complex 30.6 32 3.5 No inhibition with 875 6 8 25.6 30.~ 3.6 No inhibition 9 Desoxwmcleic acid complex witl{ 688 7 7 3 ~ 36.5 2.7 Definite inhibition 10 800 6 7 33 34 3.8 No inhibition 11 971 6 8 28 29.5 3.4 No inhibition 12 952 6 7 ~0 33 3.7:5 No inhibition 13 Prontosil R 5 8 25.3 27 3.5 No inhibition Average tumor weight in 4 tumm' v,nlrol gl'OllpS --- 3.75 glil. power of chcmicals on thc growths of animal tumors of the tumors at thc base bcforc and after the injec- have bccn invalidated by disregard for thc concomi- tions. In the experiments with "Carcinoma 63" tile tant effect on the growth rate of the host and that vohnncs of the nodules wcrc computed, whilc for apparent inhibition mu:st bc rcvicwed in relation to thc sarcomas, since it was difficult to get a satisfac-

tory value for the initial size, the final weight of No. 688 ...... 9.at the tumors after removing normal tissue was com- Dcsoxyribonuclcic acid complex with 688 ...... 8.8 Ribonuclcic acid complex with 688 ...... 6.3 pared in the injected and control series. No. 971 ...... 9.0 RESULTS Controls ...... 10.2 The series of nitro compounds gave the follow- Data for the tests with the dibasic acids and mg results: amino compounds on the sarcoma are givcn in Nitrovanillin (and vanillin) 2,4-dinitrophenol, Table I and II. Full details arc not given for the nitroguanidinc, and fluorenc wcrc inert when test- other experiments since most of the substances pro- cd either on the dibenzanthraccnc tumors or on car- duccd only slight or no inhibition. cinoma 63. Somc inhil)itory action was demonstrat- \Vhcn tested by intravcnous injections (2 mgm. ed against either tmnor by 2-ni,':_rofluorcne. in 0.10 ml.) on the transplanted dibenzanthracene sarcoma (1 ~0th to 160th grafts), commencing 7 days DISCUSSION after implantation and administered in 5 to 6 injcctions in a period of 3 weeks, the following acid com- The majority of the substances tested showed no pounds gave no cvidence of inhibitory action: inhibitory action on thc tumors by either method of injcction, and none caused entire regression of any Succinic acid, phcnyl succinic acid, asymctric dimcthyl succinic acid, 1-hydroxyl, 1-cart)oxyl cyclo hcxane, glutaric tumor. The distinct inhibitory action of malonic acid. acid, which contrasts wi~]~ the inactivity of succinic acid and glutaric acid, confirms the findings of Boy- The following were somcwhat inhibitory as indi- land (2) who observed 3 to 4 times the inhibition cated by the average final wcights of the tumors by malonic compared with glutaric t)v feeding mice compared with those of control animals. The health bearing grafted sarcomas or Sl)ontancous carinomas. of the animals was not impaired by the injections. The cffcc,t does not appear to 1)e due to any action _\Icthyl succinic acid (Tumor wt. --- 2.41 gin.) oll the general condition of the 1nice, which main- Cyclolmxyl succinic acid (tumor wt. ~ 2.jrl gin.') rained or increased thcir weight during the experi- Cyclohcxyl,l-acctyl,l-carboxylic (tumor wt. = 2.74 gin.) mCllt. .Xlalonic acid ...... (tumor wt. -- 3.0 gm.) Controls ...... (tumor wL-- 3.4 gin.) It is of interest ttiat 3 other colnpounds in this group also had inhibitory action at 1cast as effective Methyl succinic acid, cyclo-hexyl succinic acid and as malonic acid, including the dibasic acids contain- cyclo hexyl,l-acetyl,l-carboxylic acid, also inhibited ing the cyclohcxyl structure. the growth of the benzpyrcne-induccd papillomas. ~l'his is not retained when the structure is altered The ratios of average final size to initial size of the so that only one carboxyl group remains. Thc general tumors in groups of 5 to 8 animals .injected sub- properties were similar for each type of tumor with cutaneously for 5 weeks (10 to 12 injections of 2 the exception that methyl succinic acid (lid not ap- mgm.) were: pear to be effective against carcinoma 63. Crabtrce. Methyl succinic ...... 14 (5) who found that malonic, acid did not delay the Cyclohcxyl succinic ...... 13 production of skin tumors in mice painted with a Cyclohcxyl:l-acetyl-1 -carboxyl ...... 1Jr solu~ion of 3,4-bcnpyrcnc, states that it does not Controls ...... 19 combine with SH groups, having been used in bio- The two latter compounds were also inhibitory chemical studies as an inhibitor of succinic dchv- towards carcinoma 63 but methyl succinic acid was drogcnase, towards which it functions as a compet- inert as were the other compounds of this series itive substratc with: cffcctiveness depending on the tested. ratio of its concentration to that of succinic acid. In the experiments with this series of compounds It might be of interest to test whether this series of injected sut)cutancously into animals bearing carci- acids might act in a somewhat analogous way ,to- noma 63, the following compounds proved to be wards the enzyme. slightly inhibitory, the others being quite inactive. On examining the rcsults with the amino com- The ratios for the average final size of the tumors pounds there are 2 points of interest. Firstly, our sar- compared with the average initial size is given for coma was somewhat less responsive to the action of the several groups of active substances. the chemicals than the carcinoma and, also proba- Compour.d Ratio bly, than ,the tumor utilized by Boyland (3). Thus No. 736 ...... 9.0 with his technic those compounds which gave an Ribonucleic acid complex with 736 ...... 6.8 inhibition of 8 to 20 per cent had little effect on our

sarcoma, while the more effective compound 967 Methyl succinic acid, cyclohexyl succinic acid, (4'4'-diaminodiphcnylthiourca) produced some inhi- cyclohexyl acetyl-l-carboxylic acid, malonic acid, 4'4'- bition on this strain. Secondly, the 2 complcxes diaminodiphenylthiourea, 4,4'-diaminoazobenzene, with ribonucleic acid gavc evidence of dcfinite in- 4,4'-diaminodiphenylether complex with ribonucleic hibitory power. The desoxyribo complex with 688 acid, 4'4'-diaminodiphenylethcr complex with desoxy- was also activc in a lesser degree. In view of the small ribonuclcie acid, and 2-nitrofluorene. number of compounds tested it vdould be un- wise to speculate on what part thc ribonucleic acid ACKNOWLEDGMENT may play in such action. It may be pointcd out, however, that Parsons (9,10) has shown that the nu- This work was carried out under the aegis of the Bir- mingham Branch Of the British Cancer Campaign and cleotides adenylic acid and guanylic acid produce the author is grateful to the Director, Professor I laswell changes in the blood and tissues of normal and tu- Wilson, for facilities and advice. mor-bearing micc and an inhibitory action on mouse tumors. Bertolotto (1) asserted that a purified "nu- REFERENCES clcoprotein B" isolatcd from the samc type of tumor 1. BFRTOLOTTO, U. L'azione di nucleoproteidi sullo (Ehrlich adenocarcinoma) caused inhibi)tion of tu- sviluppo dcl cancro del topo. Tumori, 6:601-619. mor growth. 1932. Among thc nitro dcrivatives the activity of nitro- 2. BOYLAND, E. Experiments on tile Chemotherapy of fluorenc was sufficient to classify it as somcwhat Cancer. 4. Further Experiments with Aldehydes and Their Derivatives. Biocheln. ]., 34:1196-1201. inhibitory towards thc sarcoma. Nitro compounds, 1940. particularly dinitrophenol and dinitrocresol, have 3. BOYLAND, E. Experiments on the Chemotherapy the property of accclcrating metabolism. The ef- of Cancer. 6. The Effects of Aromatic Bases. Bio- fect of dinitrophenol on white rats by fceding and chem., l.,. 40:55-58. 1946. injection was tcsted by Emge, \Vulff and Tainter 4. BISCnOFF, F., and LONC, M. L. The Influence of Inducing Drastic Changcs in Body Metabolism (6), who f~ no action on the growth ratc of im- upon the Growth of Sarcoma 180. Am. J. Can- plantcd tumor but an increase in thcir vascularity cer, 31:67-71. 1937. and certain ccllular changes. Bischoff and Long (4) 5. CRXWrREE,H. G. Influence of Unsaturatcd Dibasic also found no significant change in thc tumors of Acids on the Induction of Skin Tumors by Chcmi- micc (Sarcoma 180) after fccding or injection of cal Carcinogens. Cancer Rescarch, 5:346-351.. 1945. dinitrophenol. Krcybcrg (7) observcd carlier pro- 6. EMGE, L. A., \Vutrr, L. M. R., and TAmTER, M. duction of skin tumors and a greatcr number of tu- L. Effects of Dinitrophenol on an Experimental mors in micc painted with coal tar in the scrics re- Sarcoma of the White Rat. Proc. Soc. Exper. ceiving 0.10 to 2 mgm. dinitrocresol daily in food. Biol. & Med., 31:152-154. 1933. 7. KREYrERr L. Influence of Dinitrokresol on the SUMMARY Development of Tar Tumors in Mice. Am. J. Cancer, 36:51-55. 1939. The cffects of subcutaneous and intravenous in- 8. McDoNALD, S., JR., and \VooDHOUSE, D. L. The jections of a scrics of substitutcd succinic acids have Histology of Transplants from a Strain of 1:2:5:6 Dibenzanthracene Mouse Sarcoma. J. Path. & been obscrved on the growth of a dibenzanthracenc- Bact., 47:615-623. 1938. , induced, transplanted mousc sarcoma, and of sub- 9. PARSONS, L. Doxo'rrIx'. Tissue Changes in Experi- cutaneous injections on benzpyrene-induced pap- mental Mice Treated with Pentose Nucleotides. illomas and on carcinoma 63. J. Path. & Bact., 57:9-20. 1945. 10. I'ARSONS, L. D., and VV'OLFF, B. Nucleotidcs and The effects of subcutancous injcctions of a series Experimental Sarcoma in Mice. (Report from Can- of amino compounds, and a number of nucleic acid cer Research Department of the London Hospital.) complexes with thcsc, havc been observcd on micc 22nd Annual Report, British Empire Cancer Cam- with carcinoma 63 and dibcnzanthracene-'induccd paign. 1945, pp. 44-45. 11. STm,,rER, S. Effect of a Carcinogenic Hydrocarbon sarcomas, and the effects of subcutaneous injections on Manifest Malignant Tumors in Mice. Eradica- of some nitro organic compounds on thesc tumors tion of Transplanted Leukemia in Mice and At- have also bccn studied. Thc cxperimcnts were con- tempts at Inhibition of Other Manifest Malignant trollcd by "wcight control" animals and "tumor Tumors in Mice by Treatment with 9:10-Dimethyl- controls". None of these substances caused com- 1:2-benzanthracenc. (With a Summary in Danish) Translated from Danish by ~ Hans Anderson. plete regression of any ot~ the tumors but a dcfinitc (Acta. path. et microbiol. Scandinav., Supplement inhibitory effect of the following was observed: XLVII )

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1947 American Association for Cancer Research. Chemotherapy Investigations in Cancer. With Reference to the Influence of Certain Organic Dibasic Acids, Diamino Compounds and Nitro Compounds on Tumors in Mice

D. L. Woodhouse

Cancer Res 1947;7:398-401.

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