Therapy of Spontaneous Mouse Tumors : Failure of Additional Inorganic Compounds

THERAPY OF SPONTANEOUS MOUSE TUMORS : FAILURE OF ADDITIONAL INORGANIC COMPOUNDS

MILLARD C. MARSH (From the State Institute lor the Study of Malignant Dieease, Burton T. Simpson, Director) The experiments to be reported continue the use of a few chemical elements and a large variety of chemical compounds in attempts at therapy, or checking the growth, of spontaneous epithelial mouse tumors. Three papers have previously appeared (1, 2, 3). For the use of organic dyes, the crudes and intermediates of coal tar concerned in the dye industry, and certain inorganic compounds, hitherto discussed, some more or less plausible reason existed. They had either been administered to tumor animals to some apparent therapeutic advantage or were members of or related to groups of chemicals so used. None but negative results attended these attempts at therapy. The compounds t,o which the work has now been extended, and which form the subject of this paper, have no group unity, and it is doubtful if a single intelligent specific reason could be alleged for suspecting them of any activity against neoplasms which would not be equally likely in almosl any soluble substance of known constitution. From this point of view they might seem a group apart. This distinction, such as it is, tends to disappear on further examination. If the experimental work with epithelial tumors on which has been based some presumption in favor of therapeutic value for the various agents hitherto used is reviewed, it will be found that this basis is indeed very slender. It is founded largely on transplanted tumors, and as a guide to therapy of the mammary tumor the results should be discarded, except where negative. It is true that agents which failed of any effect on these growths could hardly be expected to influence spontaneous tumors. Many apparent, positive results, however, were entirely misleading, and much confusion and waste would have been avoided had spontaneous tumors only been used. No doubt the supply of these was scanty. The truth is that the nominal clues which have been followed were obtained with unqualified material, were more apparent than real, and break down when tested against the spontaneous tumor. Most of this so-called chemotherapy is little more than a groping in the dark. In cancer research, however, it is perforce necessary to grope; if not in the light then in the dark, or in whatever twilight exists. Guides to chemotherapy here scarcely exist. Without seeking them further, a number of common or readily available compounds, chiefly inorganic, containing chemical elements not hitherto included in this work, were? assembled, omitting for obvious reasons most gases, radio-elements, and many rare earths. These were tried therapeutically in the Sam(: Ivay as the earlier chemicals, and with the same result, 572 PHERAPY OF SPONTANEOUS MOUSE TUMORS 573

METHODSAND TECHNIC Reference is made to previous papers (2, 3) for details, which are briefly summarized here. The tumors used were all spontaneous growths in albino mice of Strain 3, nearly all of which were non- breeders, never mated. The chemicals were administered daily, intra- venously and subcutaneously when the solvent was water, subcutaneously when alcohol was used as a solvent, and by intratumoral injection for sodium chloride brine. Distilled water was the solvent for all solutes except carbon trichloride, which was dissolved in absolute alcohol. Treatment was usually continued until death. Dosage was an approximate maximum, determined by experiments on over 400 normal mice. Except for two lead salts, intravenous injection was used with every aqueous solution and continued as long as the tail veins permitted; it was then replaced by subcutaneous injection. So far as mere trauma due to the needle is concerned, the tail veins may be pierced daily during the expectancy of life of a tumor mouse. The intravenous injection of a solution, or even of water, causes some local reaction and tends to limit the number of such treatments, In many cases only a few can be given. With almost every agent subcutaneous administration alone was used on one or more animals. The mouse was weighed and the tumor measured initially and each week or oftener thereafter. The tumors were from 3.5 to 13 mm. in diameter, with only 5 above 10 mm. The initial weight varied from 22 to 34 gm., averaging 26.8. Weights of mice are omitted from the table. At death, of 94 tumor mice, 7 showed an increase over the

TABLE1: Chemicals E,mployed

1. Alum, potassic 27. Magnesium tartrate 2. Aluminum sodium chloride 28. Manganese chloride 3. Barium chloride 29. Mercuric chloride 4. Barium hydroxide 30, Molybdenum anhydride 5. Caesium alum 31. Nickel chloride 6. Calcium bromide 32. Osmium tetroxide (osmic acid) 7. Calcium chloride 33. Potassium bichromate 8. Calcium hydroxide 34. Potassium carbonate 9. Carbon trichloride 35. Potassium chloride 10. Chromium chloride 36. Potassium hydroxide 11. Chromium trioxide 37. Potassium iodide 12. Copper (ic) chloride 38. Rhodium chloride 13. Copper sulphate 39. Silver nitrate 14. Erbium nitrate 40. Silver sulphate 15. Hydrogen sulphide 41. Silver tartrate 16. Iodine 42. Sodium bromide 17. Iodine and potassium iodide 43. Sodium carbonate 18. Iridium tetrachloride 44. Sodium chloride 19. Lead acetate 45. Sodium hydrosulphide 20. Lead formate 46. Sodium molybdate 21. Lead nitrate 47. Sodium sulphate 22. Lithium carbonate 48. Sodium sulphide 23. Lithium chloride 49. Sodium polysulphide 24. Lithium hydroxide 50. Sodium tungstate 25. Magnesium chloride 51. Tantalum potassium fluoride 26. Magnesium sulphate 52. Thallium (ous) nitrate 574 MILLARD C. MARSH

TABLEI11: Inl~wen~u~and Subcutaneous Treatment of rYpoataneoua Tumor Mice

Diameter of Tumor Number of (Millimeters) Doses Dura- Sur- %of DO~~W. tion viva1 Total Dosage Begin- Maxi- Serial &lute orml.) Int. Subc. Daye Daye (mg.) ning Death mum

~~ ~ ~ __ ~~ - 1 6.0 5.0 24 25 20 105. 5. 9. 10. I1 0.5 0.5 10 53 03 04 31.5 8. 11. 14. 2 1 .o 1.0 5 50 56 57 55. 8. 18.5 20. 11 10.0 10.0 20 20 21 200. 7.5 10. 3 1.0 0.6 46 19 66 108 32.5 8. 9. I1 1 .o 0.4 21 21 22 8.4 10. 12. 13.5 4 0.125 0.8 74 74 75 37. 0. 10. 11 0.125 0.5 98 98 99 49. 8. 9. 10. 5 sat. 0.3 9 19 28 30 7. 10. 11. (1 Sat. 1.0 25 25 26 10. 17. 0 5.0 5.0 30 25 55 50 275. 10. 17.5 19.5 11 5.0 4.0 39 39 40 156. 8. 15. 17. 7 5.0 2.5 25 14 40 41 97.5 8. 22. I1 1.0 2.0 40 40 41 80. 7. 10.5 8 Set. 0.4 3 30 34 35 9. 21. 11 Sat. 0.4 46 40 47 6. 14. 0 1.0 0.75 37 37 38 27.75 8. 8. 10.5 I1 1.o 0.60 24 25 26 12. 6. 15. 16. 10 1.0 1.0 19 84 103 104 103. 8. 26. I1 1.0 1.0 24 24 25 24. 8. 13. 11 0.1 0.2 13 32 46 47 9. 9. 23. I1 0.1 0.1 37 38 39 3.7 7. Very large 12 0.2 0.2 7 21 28 29 5.6 6. 9.5 10.5 I1 0.1 0.1 25 26 27 2.5 0. 8. 9. 13 0.1 0.1 41 37 79 80 7.8 10. 19. 41 0.1 0.1 50 51 52 5. 7. 22. 14 1.0 1.0 7 34 41 42 41. 6. 18.5 'I 6.0 5.0 14 14 15 70. 7.5 10. 15 0.5' 0.05 91 10 103 104 5. rnl. 8. 16. 17. I1 0.5' 1 .o 50 53 53 60. 'I 8. 24. 16 Sat. 1-2 748 55 56 110. 6.5 14. 17 a 44 40 47 4 7. 13. 14. 11 8 53 55 56 6 9.5 12. 14.5 18 0.25 0.25 20 35 55 56 13.75 10. 23. I1 0.5 0.5 43 43 44 21.5 6. 8.5 9.5 19 1.0 0.5 16 7 23 24 11.5 10. 11.5 11 1.5 1.6 25 20 27 37.6 11. 10. 20 1 .o 1.0 32 32 33 32. 3.5 5. 0.5 11 1.0 1.0 33 33 35 33. 7. 16. 17. 22 Sat. 0.3 21 13 34 35 9.5 8.5 11. 11 sat. 0.1 30 30 31 10. 18. 20. 23 5.0 5.0 75 24 99 100 495. 8.5 25. 26. 11 10.0 10.0 25 25 26 250. 8. 12.5 14. 24 0.5 0.5 9 29 38 39 19. 5.5 7.5 8. 11 1.0 0.5 31 31 33 15.5 4. 12. 25 0.5 1.0 53 29 84 85 84. 8. 26. 11 10.0 10.0 26 26 27 260. 9. 11. 14. 26 1.0 1.0 60 10 71 72 70. 0. 15. I1 10.0 8.0 107 108 109 856. 7. 14. 16. 27 Sat. 0.2 38 5 43 44 9. 12. 13. 11 Sat. 1 .o 34 38 39 7.5 12.

10.5 per cant maximum. Volrrtile. *O.M KI, 041. '1.36 KI, 081. '29.9 KI, 171. * 72. KI, 421. THERAPY OF SPONTANEOUS MOUSE TUMORS 575

TABLEI1 (Continued) Diameter of Tumor Number of (Millimeters) Doses Dura- Bur- % of Dose (mg. tion viva1 Total Dosage Begin- Maxi- Serial Solute or ml). Int. Subc. Days Days (mg.) ning Death mum 28 0.5 0.5 46 3 50 51 24.5 0. 8. 10. 16 0.75 0.525 28 28 30 14.7 9. 13. 1U. 29 0.025 0.05 0 33 39 40 1.95 6.5 10. I1 0.025 0.025 46 47 48 1.5 8. 23. 24. 30 Sat. 2.0 19 19 20 10. 11. 12.5 31 0.1 0.1 23 65 90 8.8 7.5 20, 11 0.1 0.3 20 20 27 7.8 9. 12.5 32 0.05 0.05 7 109 116 120 5.8 4. 11. II 0.05 0.1 8 108 110 130 11.0 5. 35. I1 1.0 1 .o 57 57 58 67. 5. 7. 33 0.25 0.2 19 20 39 40 7.8 7. 11. 12. 11 0.25 0.125 76 77 78 9.5 7. 10. 11. 34 0.5 0.5 45 32 78 79 38.5 6. 27. 29. I1 6.0 2.0 23 23 24 40. 9. 15. 35 0.5 0.5 78 0 80 81 39. 8.5 30. 'I 20.0 15.0 36 36 38 725. 5. 12. 30 0.6 0.6 21 30 52 53 25.5 9.5 24. 11 1.0 1 .o 24 26 30 24. 3. 9. 37 1.0 5.0 34 9 43 44 215. 9.5 21. I1 5.0 15.0 37 37 39 555. 5. 8. 38 0.1 0.1 18 63 81 82 8.1 8.5 10. 11. 11 0.5 0.4 32 33 34 123 5.5 10. 39 0.25 0.25 7 44 53 64 12.75 7.5 12. I1 2.0 1.0 35 35 30 36. 6. 7. 40 Sat. 0.05 48 48 49 7. 18. 19. 41 Sat. 0.3 28 28 29 5. 8. 42 10.0 10.0 24 17 42 43 410. 10. 22. II 25.0 25.0 37 38 39 925. 6.5 11. 43 2.5 2.5 20 13 39 40 97.5 12.5 20. ll 10.0 5.0 24 24 25 120. 8. 12. 44 20.0 10.0 7 86 93 94 930. 9.5 22. 23. '1 Hat. 0.05 33 33 34 11. Seetext 45 0.04 0.04 47 02 110 112 4.36 10. 28. 31. 11 0.26 0.26 61 62 63 16.25 11. 16. 18. 46 2.0 4.0 69 60 109 110 436. 13. 27. 29. 11 2.0 6.0 60 51 62 300. 6. 7.6 47 10.0 20.0 60 81 137 138 2740. 10. 36. 39. 11 20.0 60.0 42 43 44 2520. 6. 17. 18. 48 0.076 0.075 62 80 146 146 10.65 8. 20. 23. I1 0.5 0.6 80 81 82 40. 8. 32. 36. 49 0.16 0.15 60 18 69 70 10.20 9.6 13. 19. I1 1.0 1.0 76 76 77 76. 8.5 12. 13. 60 1.0 2.0 01 6 68 69 134. 8. 24. I1 1.0 2.0 66 68 69 132. 7. 21. 61 Sat. 0.3 23 10 33 34 9.5 12. 14. 11 Bat. 0.4 32 32 33 6. 14. 62 0.126 0.126 53 ' 2 67 69 0.876 8. 10.6 I1 0.25 0.25 17 18 19 4.25 8. 10. 11. initial weight and 87 had lost or failed to gain weight; during treatment 43 of these 87 made a temporary gain. The maximum loss of weight at death was 32 per cent; the average was 13 per cent, being somewhat in excess of that of untreated tumor mice on account of the addition of the effect of treatment to the tumor cachexia. The subcutaneous 576 MILLARD C. MARSH injections often brought about severe reactions-inflammation, adhe- sions, and ulceration-until the animal fell into a miserable condition, dying of the treatment as well as the disease. This is reflected in the loss of weight. Controls, mainly of academic interest, are discussed briefly in previous papers (2, 3). There were no contemporary controls. Judg- ment of results as negative is based on progressive' growth and the entire absence of any consistent regression or cessation of growth. The significant results from the 232 tumor mice used are contained among the 98 tumor mice of Table 11. The latter refers serially to Table I, which lists alphabetically and numbers all the chemicals. Each line of Table I1 represents one tumor mouse. The dose is stated in milligrams of solute for all save saturated solutions, where it is given in milliliters of solution without carrying out the total dosage. The weights and percentage of solute refer to the anhydrous salt. The duration is the period from the first to the last dose, and survival that from the first dose to death. Dose and total dosage refer to the solute and not the volume injected, which may be easily reckoned. The maximum size attained by the tumor is stated separately if greater than the size at death. Sodium Chloride; Intravenous injections of fairly strong sodium chloride solutions (20 per cent) are soon brought to an end by the tissue reaction, and subcutaneous injections do not affect the tumor unless there is actual contact with it. In that case, with a saturated solution, tumor tissue is killed and shriveled to a black residue. The efficient intratumoral dose is a dangerous medication, promptly fatal if by vessel puncture it reaches the blood stream quickly, and the danger rapidly increases with increase of the dose. If it can be kept in the tumor, the latter is rapidly destroyed by repeated doses. Using a daily dose of 0.1 to 0.2 milliliters distributed in the tumor mass, the tumors of 17 mice were treated. Six animals died immediately or within a few hours, and one after six days. The remaining 10 received on the average 24 treatments and survived thirty-five days (12-52). When they died, 3 of the animals had slight remnants of tumor visible, while 7 were clinically free of tumor and appeared cured. They survived one to thirty-six days (average eleven) after treatment ceased. The tumors were well grown, 7 to 18 mm. in diameter, averaging 13 mm. With sodium chloride brine administered in this way, it is prob- able that early tumors, with smaller vessels, could all be removed in safety. While this may be regarded as a cure of a sort, the method of obtaining it is an inferior form of operation, is caustic rather than medicinal, probably painful, and apparently offers no useful contri- bution to human therapy. For these reasons no attempt has yet been made to define more precisely its possibilities, DrsaussIoN There is little to be said. None of the chemicals revealed any sign of therapeutic action with the exception of sodium chloride, and that THERAPY OF SPONTANEOUS MOUSE TUMORS 577 acted only as a caustic. If the spontaneous tumor disappoints, it does not mislead, and there were no equivocal results. With the present list the definite chemical compounds used in this work total about 250, including 66 elements. With the exception of lead, selenium, uranium (4),and iodine, no element was used alone, and the results apply only to compounds. One pictures the metal or base-forming part as the presumptive active agent but is spared this and other interesting ques- tions in the absence of any action. It is obvious that the result, while definite, is but narrowly instructive, since the methods and technic, which could be endlessly varied, might be maladapted. Before desist- ing, it seems desirable for if not incumbent 011 cancer research, despite the unpromising outlook, to accumulate a large body of these negations, based primarily on the use of the simplest chemicals. When the dis- covery is made that it is only combinations of these or others that, begin to be effective, whether by catalysis, complementary or supple- mentary action, one may be sure that the part played by each item of this combination will require a thorough experimental overhauling. One feels virtually certain what the result will be before these experiments are begun; and that perhaps something ought to be said in defense of undertaking them. To let this feeling act as a limitation is to adopt a reactionary attitude, and to ignore the well known fact that much of value in experimental research comes from its by-products, its unforeseen chances, and its accidents. Even aside from this, pes- simism over the present gloomy outlook for a medicinal therapy should have a better foundation in experiment than in theory. SUMMAT~Y Therapy of spontaneous mammary tumors of mice was attempted with fifty chemicals. Most of them were common, familiar inorganic compounds. Common salt brine used as a caustic had marked effective- ness. All the other agents failed. Note: Acknowledgment is made to Dr. Jeanne Shore for skilful assistance in the laboratory work.

R EFERENCE~

1. SIMPSON,BURTON T., AND MARSH, MILLARDC. : Chemotherapeutic expeyiments with coal-tar dyes on spontaneous mouse tumors, J. Caneer Research 10: 50, 1926. 2. MARSH,MILLARD C., AND SIMPSON,&rRTON T.: Chemotherapeutic attempts with coal- tar derivatives on spontaneous mouse tumors, J. Cancer Research 11: 417, 1927. Contains leading references. 3. SIMPSON,BURTON T., AND MARSH,MILLARD C. : Therapy of spontaneous mouse cancer. Failure of tuberculin, karkinolysin, and some inorganic compounds therein, Ann. Surg. 93 : 169, 1931 (Ewing Memorial Number). 4. Annual Reports of the (New York) State Institute for the Study of Malignant Disease 16: 21,1926; 18: 17-18, 1928.