CANCER CHEMOTHERAPEUTIC PROPERTIES AND TOXECOLOGEC EFFECTS OF CiS- PLATINUM”) BIAMMINO DICHLORIDE Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY RECHARD J. KOCIBA 1970 ‘ This is to certifg that the thesis entitled CANCER CHElv’TOT‘iERAPEUTI-C PROPERTIES AKD TOXICOLOGIC EF‘ ECTS OF CIS-PLATINUM(II) DIAMKINO" DICHLQRIDE presented by Richard J. Kociba 9' has been accepted towards fulfillment T of 'the requirements for Ph.D. Pathology degree in j/MJ M Major professor (7 Date May 1‘3, 1970 0-169 t‘ ‘ ¢ . » ‘9‘ A a WW Ms” . -x . I 1 ~ ,, Fl 2‘ . ,. i A it _~ .: . he” 1 “ 5 ps. I 3,4 i“ l M" ’ I ’ *‘ i 1" .' k m L I‘; . NIH” \ SEC) 0 c" .7097 c “ ' ,1: l) (—44,: mil: 2 1333 W. 33:: ' ‘ , tita- I 0‘3301132908 M’fl'fi‘ ' {~- W075 ”A 4"?" Ij'fi'lf‘, ABSTRACT CANCER CHEMOTHERAPEUTIC PROPERTIES AND TOXICOLOGIC EFFECTS OF CIS-PLATINUM(II) DIAMMINO DICHLORIDE. by Richard J. Kociba The cancer chemotherapeutic pr0perties of the compound Cis- PlatinumH>n=m 449 cu mouooaeaH muo>H>uom A.wx\.wav wouamaeaH muam .02 .oz .au one usuooa oaaoo AH>unm om hon ouow mHHoo A Serial ToxicitygStudy of Cis-Pt.(II) in Male Rats l. Hematqlgggc and Serum Chemistgy Alterations. Following a single IP injection of 12.2 mg./kg. Cis—Pt.(II) a marked leukOpenia occurred. Figure 14 illustrates this decrease in total leukocytes which was most severe at 3 days postinjection. This was followed by a regenerative increase in which the counts returned to the normal range by Day 5 through Day 7. By Day 9 the circulating leukocyte counts were elevated above the normal range. Determinations made on Day 20 and Day 28 showed a gradual return of the total leukocyte count to the normal range. Exami- nation of the neutrophil and lymphocyte counts also revealed maximal depression at 3 days after injection, followed by a regenerative increase and finally a return to the normal range. The number of circulating platelets was also depressed most severely on Day 3 postinjection. A maximal depression of reticulocytes occurred by Day 4 postinjection (Figure 15). This was followed by a regenerative increase, in which the reticulocyte counts returned to the normal range. Determination of circulating erythrocytes, packed cell volumes and hemo- globin values revealed no marked alterations following injection of Cis-Pt.(II). Examination of blood urea nitrogen (BUN) values (Figure 16) revealed a sharp increase by Day 3—Day 4, followed by a return to the normal range. 36 mu 9"..- ------------------- an ill fifl % 5" .............. .' MORTALITY 4g 30 20 . L; 4 [1.] LU '0 LOG. MG./KG. ClS-PT.(II) Figure 13. Determination of LD50 following the fitting of the data to a straight line by the method of least squares. LD50 estimated at 12.2 mg./kg. Cis-Pt.(II) for the male rat. LDgo and LDlo can also be estimated at 26.5 mg./kg. and 5.5 mg./kg., respectively. 37 200‘ PLATELETS % 100W" 0 , :p—pq' pug..— LYMPHOCYTES NEUTROPHILS 2001 - . TOTAL . ' [Emms 100 00000000000000.0000...coo 00 on ....... :0 I oooooooooooooooo coco-:0... 0 .4 0 . v . v v J I— l 2 3 4 5 5 7 3 9 20 28 DAYS Figure 14. Serial alterations observed in blood leuko- cytes and platelets following a single IP injection of 12.2 mg./kg. Cis—Pt.(II) on Day 0. All values converted to relative percentage of the normal values (100% i_l std. dev.) estab- lished for the control rats used in this study. All plotted points signify mean :1 std. dev. 38 momma vowel % ovvfivvvfiv~:l—U-fll—I— j 2 r EM. I % : 0 - - - - . f rT - :I—v—Oh— i 200-] «Ulcuocms 10 ........................................ "‘L‘” % . 0- . . f . .fi‘l-w—Il I'- 123456799 20'73 Figure 15. Serial alterations in blood reticulocytes, erythrocytes, packed cell volumes and hemoglobin values follow- ing a single IP injection of 12.2 mg./kg. Cis-Pt.(II) on Day 0. All values converted to relative percentage of normal values (100% 1L1 std. dev.) established for the control rats used in this study. All plotted points signify mean i. 1 std. dev. 39 V v v v ‘ T V '1 v fiH~ 1233557ééfi'2'0”2'8 Figure 16. Serial alterations in blood urea nitrogen, glucose, albumin and total protein values following a single IP injection of 12.2 mg./kg. Cis-Pt.(II) on Day 0. All values converted to relative percentage of normal valubs (100% i;l std. dev.) established for the control rate used in this study. All plotted points signify mean :_1 std. dev. 40 Serum glucose levels were elevated 2-4 days postinjection, followed by a return to normal (Figure 16). Serum albumin levels were inconsistently elevated following injection of Cis-Pt.(II), whereas total protein levels were depressed from Day 1- Day 4 postinjection. Later determinations showed both albumin and total protein values near the normal range of values (Figure 16). Total bilirubin values and uric acid values were in the lower portion of the normal range of values (Figure 17). Calcium and inorganic phos- phorus values remained in the normal ranges. Figure 18 shows changes in 3 serum enzymes and cholesterol values following injection of 12.2 mg./kg. Cis-Pt.(II). Cholesterol values fluc- tuated only to a small degree, and in most cases stayed within normal limits. Lactic dehydrogenase values, alkaline phosphatase values and glu- tamic oxaloacetate transaminase values were irregularly decreased follow- ing intoxication with Cis-Pt.(II), and in no case were any values increased above normal levels (Figure 18). 2. Bone Marrow. Injection of 12.2 mg./kg. Cis-Pt.(II) caused a general shift to the right, with a relative decrease of the immature pro- normoblasts and myeloblasts in the bone marrow. By Day 2, swelling and dissolution of hematOpoietic cells, vacuolation of cytOplasm, fragmenta- tion of cytOplasm and arrested mitotic activity were noted in bone marrow elements. Maximal depletion of the bone marrow occurred on Day 3, with the general hypocellularity of the marrow accompanied by engorgement of dilated sinusoids with erythrocytes. 41 mom 1 m... % 0 w v - w— - v a r - :l—I-Vh-v—n 20 mm Bllnwlu 100R oooooo o oooooooooooooooo o ooooooooo o ooooooooo l ooooooooooo o oooooooooo o o o o of v ' fi f— v ' JH* 1231567§9fi20 28 DAYS Figure 17. Serial alterations in total-bilirubin, uric acid, calcium and inorganic phosphorus following the IP injec- tion of 12.2 mg./kg. Cis-Pt.(II) on Day 0. All values con- verted to relative percentage of the normal values (100% i 1 std. dev.) established for the control rats used in this study. All plotted points signify mean :_1 std. dev. 42 GLUTAMIC WUATE lO TRANSAMINASE 99 ALKNJNE ..__, . _ii._- -. _ MSMTASE l socooco10:200..oooop‘oofiepof'ooeooooooopooooeoccasioooooooooOM % . . r + fi fi—fiHW lmT'c oo‘o—o-ooooooooeooooooooooo coco. 7:70:00 ' 3:... I “WEMSE - ———— _u——3___._!! _ {ILL -- i O i; *4. “a v .—+ w —,‘I—I—-fl—I—- 20 CHOLESTEROL - .—- _- ..-.--_____.____-_.___ 0/0 10 cos oooooooooooooo .___..-‘;.- ----- '- ooooo 0.00:0 oooooo ; 0:: 0 Figure 18. Serial alterations in serum enzymes-and cho- lesterol values following a single IP injection of 12.2 mg./kg. Cis-Pt.(II) on Day 0. All values converted to relative percen- tage of normal values (100% :_l~std. dev.) established for the control rate used in this study. All plotted points signify mean i l std. dev. 43 Rats surviving the LDSO dose began to show regenerative activity throughout the depleted bone marrow by 4-5 days after injection. There was a marked shift to the left as the new stem cells appeared in the marrow. From Day 9 through Day 20 the bone marrow continued to show evidence of increased regenerative activity. 3. Thymus and Lymphoid Tissue. The involution of lymphoid tissue in the body was most easily followed in the thymus (Figure 19). Moderate atrOphy was noted by Day 2, with further involution through Day 4. As the thymus underwent this involution, the cortical portion shrank some- what more rapidly than the medulla, and during the period of cortical contraction the lymphocytes appeared more numerous in the medulla than in the cortex, thus tending to reverse the normal histologic pattern. Maximal involution of lymphoid elements caused the thymus to be small and fibrous on Day 3-Day 4. Rats surviving the LD50 showed thymuses which were increased in size, and histologically exhibited hyperplastic changes. As a result of this lymphoid hyperplasia the cortex reassumed its normal lymphocytic appearance, while the medulla became more conspicu-l ous for its reticular cells and epithelial elements. 4. ‘Spleen. A progressive reduction in the size of the spleen (Figure 20) resulted from the disappearance of lymphocytes with atrOphy of the Malpighian corpuscles. The ultimate stage in the regression of the spleen was noted at Day 3—Day 4 after_injection, at which time the disappearance of lymphocytes from the Malpighian corpuscles and the disap- pearance of myeloid and erythroid precursors from the red pulp gave the organ a somewhat fibrous appearance. The spleens of rats surviving the LD50 dosage showed excessive lym- phoid regeneration, with the enlargement of the Malpighian corpuscles. 44 Histologic appearance of thymus on Day 4 Figure 19. following IP injection of 12.2 mg./kg. Cis-Pt.(II) on Day 0. Note extreme involution of the cortical area (C) due to lymphoid depletion. H & E stain. x 125.1 45 Figure 20. Histologic appearance of spleen on Day 4 following IP injection of 12.2 mg./kg. Cis-Pt.(II) on Day 0. Note depletion of lymphocytes from Malpighian corpuscle. H & E stain. x_250. 46 In the red pulp, clusters of primitive hemat0poietic cells reappeared, with megakaryocytes, granulocytes and erythrocytes found around these areas. These regenerative changes were most pronounced from Day 9 through Day 20. 5. Intestinal Tract. Clinical evidence of diarrhea and alterations of the intestinal tract were consistently indicative of intestinal injury following the.LD50 dosage of Cis-Pt.(II). The lesions were most severe in the small intestine. By Day 1 after injection the villi of the intes- tinal mucosa were edematous and the lining cells were swollen and distended with clear vacuoles (Figure 21). At Day 3-Day 4 when the lesions were most severe, the crypts of Lieberkuhn appeared as cyst-like spaces, lined by flat, elongated cells. The mucosal surface was irregularly denuded, or showed patchy areas of hypOplasia (Figure 22). In later stages a slight fibrous reaction sometimes led to deposits of connective tissue in the villi which tended to become shorter, broader and stubby. The maximal epithelial alterations and sloughing coincided in time with the development of fluid distention and mucous diarrhea. The intestinal epithelium of rats surviving the LDSO dosage showed patchy areas of epithelial hyperplasia, with the crypts lined by hyper- chromatic cells with increased mitotic activity. 6. Urinary System. Kidneys of rats killed on Day 2 after injection had minimal evidence of tubular vacuolation and dilatation. Irregulari- ties in staining characteristics were also noted in the tubular epithelium, suggesting early tubular necrosis. By Day 3 there was further tubular necrosis, with hyaline casts present in the tubular lumina. Extensive loss of tubular elements was noted by Day 4, especially in the deeper aspects of the renal cortex (Figure 23). 47' L_,iiiiii,,, Figure 21. Histologic appearance of intestinal epi- thelium on Day 1 following IP injection of 12.2 mg./kg. Cis-Pt.(II) on Day 0. Note vacuolation of cytoplasm of some cells and the dilatation of lymphatic channels. Mitotic activity has.been inhibited in the crypts of Lieberkuhn. H 5 E stain. x 125. 48 Figure 22. Histologicxappearance of intestinal epi- thelium on Day 4 following 1? injection of 12.2 mg./kg. Cis—Pt.(II) on Day 0. Note hypoplasia of epithelium and cyst—like appearance of crypts as a result 'of inhibition of normal cell replication. H 6 E stain. x 63. 49 Rats surviving the LD50 dosage showed variable degrees of tubular epithelial regeneration. The tubular alterations, which were most severe at Day 4, closely paralleled the elevation in the BUN values at this time. 7. Respiratory System. No consistent alterations were observed in the upper or lower respiratory tract following the LDSO dosage of CIS‘PC.(II). 8. CirculatorySystem. Some evidence of congestion was noted in various organs, such as the thymus, following administration of the LD50 dosage. No histologic evidence of cardiac injury was observed. 9. Male Repgoductive System. Some sections of the testes possibly suggested a temporary arrestment of normal spermatogenesis, but to fully evaluate any injury to the testes, additional studies are indicated. Other factors, such as marked weight loss, have been shown to diminish pituitary activity (Mulinos and Pomerantz, 1940) and must be considered. 10. Central Nervous System. The brain showed no histologic abnor- malities following the LD50 dosage of Cis-Pt.(II). Work by Jean Allen (1970) has indicated this compound does not cross the blood-brain barrier to any great extent. ll. Liver. No consistent changes were observed in the liver which could have been attributed to treatment with Cis-Pt.(II). Variable degrees of glycogen depletion were observed. 12. Musculoskeletal System. No abnormalities were observed in sec- tions of skeletal muscle, cartilage or osseous tissue which were examined. 50 Figure 23. Histologic appearance of renal tubules on Day 4 following IP injection of 12.2 mg./kg. Cis-Pt.(II) on Day 0. Note the extensive tubular sloughing and presence of hyaline casts in lumina of tubules. H & E stain. x 125. lllll lllllll . .lJI-Il. Ill ..ll’ \I‘l Ell 51 13. Endocrine System. No consistent histologic changes were noted in thyroid, parathyroid, pancreas or adrenal glands. The pituitary gland was not studied. DISCUSSION The initial experiments utilized the Dunning Ascitic Leukemia to evaluate the cancer chemotherapeutic prOperties of-Cis—Pt.(II). This transplantable neoplasm, first described by Dunning and Curtis (1957), has proven very useful both as a screening test for potential anticancer agents (Jones et aZ., 1958) as well as for quantitative pharmacological studies of cancer chemotherapeutic agents. The remarkable uniformity of neOplastic development, survival time and extent of organ infiltra— tion all serve as critical parameters for evaluating the effects of candidate compounds on this neOplasm. The CCNSC has chosen the DAL as one of a select few transplantable neOplasms for use in cancer chemotherapy studies. The CCNSC has pub- lished recommended protocols (CCNSC, 1962) to be followed during the transplantation of this neOplasm, as well as during the evaluation of any cancer chemotherapeutic effects which a candidate compound may exhibit against the develOpment of this neOplasm. Any candidate compound which extends the survival time of the DAL bearing rats by a minimum of 25% following treatment from Day 1 through Day 5 postimplantation is con- sidered significant by the CCNSC protocols. Any candidate compound giv- ing these minimal results is then considered to warrant additional study in regard to its cancer chemotherapeutic properties. The results of this study, in which the compound Cis-Pt.(II) was administered in the form of a single IP injection on Day 1 gave results which far surpassed the criteria established by the CCNSC. 52 II‘ llllll'll 1 .. I III‘ Illll.|||‘ l | {I 1 ll] ‘ t lilll Ill.- ll III. III \\ 53 Thus a single injection of Cis-Pt.(ll) on Day 1 gave results compar- able to repeated treatments from Day 1 through Day 5 with various alky- lating agents, such as the nitrogen mustards or cyclophosphamide which have been very effective against Dunning Leukemia (Table 5). AmethOpterin (an antifolate), 6-mercaptopurine ribonucleoside (purine analog) and mitomycin C have been somewhat effective against Dunning Leukemia. It has been reported as being refractory to treatment with 6—mercaptopurine (purine analog), urethane (ethylcarbamate), 5-fluorouracil (pyrimidine analog), actinomycin D, colchicine or hydrocortisone (Skipper and Schmidt, 1962). The challenge dose of 5 x 106 DAL cells implanted IP into all Day 30 survivors failed to reestablish the neOplastic process in any of the rats previously treated with Cis-Pt.(II) subsequent to implantation of DAL. This indicated these rats had been exposed to DAL, had successfully overcome the neOplasia, and had a protective immunity to the challenge dose of DAL cells. In contrast, all rats which had not been initially implanted with DAL cells and then treated with Cis-Pt.(II) died approxi- mately 2 weeks following the challenge dose of DAL cells. Postmortem examination disclosed the presence of terminal DAL in all these rats. Gross and microscOpic examinations of the treated rats which were refrac- tory to DAL cell reimplantation were conducted on Day 30' (60 days from initial DAL implantation) and failed to disclose any evidence of neo- plastic cells. The successful delayed treatment of DAL with a single injection of Cis—Pt.(II) on Day 4 or Day 7 offered additional evidence supporting the potential cancer chemotherapeutic prOperties of this compound. Comparable data in reference to the activity of other cancer chemotherapy agents used in the delayed treatment of DAL are scant. However, Skipper and 54 Table 5. Therapeutic indices of various compounds against Dunning Leu- kemia and Walker tumor systems 4 Tumor Walkerf» Walker Dunning Dunning ‘ Dunning Form Subcut. Subcut. Subcut. Subcut. Subcut. Days Treated 1-5 7—11 1-5 7-11 1—5 Route IP IP IP IP IP Days Assessed 18—21 23-28 28 1-30 28 Tumor Tumor Life Criteria Weight weight Survival Span Survival Compound Nitrogen mustard 2 l 0 2 3 Nitromin 9 2 3 7 7 Chlorambucil lO 9 2 8 2 dl-Phenylalanine mustard 14 22 3 6 19 l-Phenylalanine mustard 14 4O 3 7 18 Mannitol mustard 2 1 0 3 2 Benzimidazole mustard 7 6 2 8 8 Chloroquine mustard 0 0 O 0 O Triethylene— melamine 7 5 2 l Triethylenephos- phoramide 8 5 2 7 3 Cyclophosphamide 19 28 4 26 9 Busulfan O O O 0 O Amethopterin O O 2 0 - 6-Mercapt0purine 1 4 O 0 - 6-Mercapt0purine ribonucleoside Azoserine Urethane 5-Fluorouracil Hydrocortisone Actinomycin D Mitomycin C Colchicine ..- OOOOOOOH ... OO‘OOOOON ObOOOOOb ODOOOOOO l “'T *Zero (0) indicates those agents which failed to inhibit tumor growth to 40% of controls (or increase life Span by 40%) at the LDl dosage. In comparing therapeutic indices it is essential that considers ion be given to differences in protocols employed. The reader is referred to the ori— ginal source of these data (Skipper and Schmidt, 1962) for additional information. 55 Schmidt (1962) have compiled quantitative data indicating the delayed treatments (Day 7 through Day 11) with the various alkylating agents were most effective against the subcutaneous form of Dunning Leukemia (Table 5). Mitomycin C was somewhat effective, while 6-mercaptopurine, urethane, 5—fluorouracil, actinomycin D, colchicine and hydrocortisone were ineffective. Postmortem examination of the DAL implanted rats which were treated during the more advanced stages of development of the neOplasm (Day 4 or Day 7 treatment) revealed the presence of adhesions between adjacent abdominal viscera. This indicated the DAL had undergone a regression following treatment with Cis-Pt.(II). The Walker 256 Carcinosarcoma has also been recommenced as a trans- plantable neOplasm to be used in cancer chemotherapy studies (CCNSC, 1962). This neoplasm has a high rate of cell division in that the cell pOpulation doubles every 1.7 days (Bertalanffy and Lau, 1962). The fine structure of this neOplasm has been well described (Fisher and Fisher, 1961). These factors, plus its high rate of reproducibility (100%), relative lack of spontaneous regression (2%) and its ability to metasta- size to all parts of the body make the Walker tumor ideal for cancer chemotherapy studies. The recommended CCNSC protocol is based on implantation of the neo- plastic cells on Day 0 followed by treatment on a daily basis from Day 3 through Day 6. All rats are killed on Day 7, and any candidate com- pound which inhibits tumor develoPment to 60% or less when compared to nontreated tumor rats is considered significant, and thus warrants addi- tional study. In this study a single 1? injection of Cis-Pt.(II) on Day 3 caused a marked inhibition of the neOplasm, so that by Day 7 tumor development had been inhibited to less than 20% of the nontreated tumors. l III III 56 Skipper and Schmidt (1962) listed the chemotherapeutic agents which were effective in inhibiting the early develOpment of the subcutaneous Walker tumor. Included in this group were the nitrogen mustards, 6- mercaptopurine, 6-mercaptopurine ribonucleoside and mitomycin C. Inef- fectual compounds included amethOpterin, urethane, 5-fluorouracil, hydro- cortisone, actinomycin D and colchicine (Table 5). Delayed treatment of rats bearing the Walker tumor on Day 7 caused a marked regression of the tumor at the initial site of implantation. In addition, it increased the survival time of the rats and reduced metastasis to other areas of the body. Comparison of the other cancer chemotherapeutic agents used in delayed treatment (Day 7 through Day 11) revealed that the same compounds which were previously listed as possesSing activity against the early stages of the Walker tumor were also effective in the later stages of development. Compounds found ineffective in the early treatment of the Walker tumor were also ineffective against the later stages. The LD50 of the compound Cis-Pt.(II) was calculated to be 12.2 mg./kg. for the male rat. The LDgo and LD10 could also be calculated to be 26.5 mg./kg. and 5.5 mg./kg., respectively. The CCNSC (1964) has stated the LDlO, as read from plotted dosage-mortality data, can be employed as a reproducible maximum tolerated dose (MTD). This MTD has been considered just as important as the tumor-response data when calcu- lating the maximum effectiveness or therapeutic index (TI) of a drug. When increase in life span_of the tumor bearing host has been employed as the end point in drug evaluation, the TI has been defined as follows: L2l9_(Nontumor bearing animals) ILS40 (Dose giving 40% increaSe in life span) )1“ ll Ill 57 In reference to this study where the DAL was used as the test tumor the minimal TI could thus be calculated as follows: TI - 5.5 mgplkg. - 2.75 2.0 mg./kg. Since therapeutic dosages lower than 2 mg./kg. Cis-Pt.(II) were not util- ized in this study the actual TI may be considerably greater than the value which was calculated. Skipper and Schmidt (1962) have compared the T1 of various agents used in the treatment of DAL and Walker tumor and have listed various alkylating agents as having therapeutic indices ranging from a high of 40 down to 0 (Table 5). The antitumor prOperties and toxicologic effects of Cis-Pt.(II) are strikingly similar to those of alkylating agents and x-irradiation. The term "radiomimetic" was introduced by Dustin (1947) to describe certain chemicals which induced cytological effects similar to those observed after x-irradiation. Elson (1955) has listed a series of prOperties which such compounds may have in common with x-irradiation. One of the best known radiomimetic chemicals is nitrogen mustard. The observed pathologic effects of Cis-Pt.(II) suggested its bio- logic activity was not limited to any one tissue of the body, but resulted in alterations in those tissues having rapid turnover times. Examination of data compiled by Leblond and Walker (1956) supplemented by data pub- lished by Bertalanffy and Lau (1962) and also Schalm (1965) has permitted the listing of the turnover times of the cells comprising the more actively dividing tissues of the rat: Blood granulocyte - 0.04 days Blood lymphocyte - 0.3 days Intestinal epithelium - 0.6-1.6 days 58 Bone marrow myeloid series - 2.5 days Blood reticulocytes - 1.8 days Bone marrow erythroid series - 2.5 days Thymic lymphocyte - 2.5 days Gastric epithelium - 1.8-6.5 days Seminiferous epithelium - 16-27 days Epidermis - 20-35 days Blood erythrocyte - 60-80 days The turnover time for the ne0plastic cells of the Walker tumor has been estimated to be 1.7 days (Bertalanffy and Lau, 1962). As the resultant histologic and hematologic alterations following a toxic dose of Cis-Pt.(II) included panleukocytopenia, reticulocytepenia, atrOphy of intestinal epithelium, atrOphy of the thymus and bone marrow repression, it was apparent that the degrees of tissue susceptibility were directly prOportional to the rate of regeneration of the cells com- prising that tissue. As a result those tissues having turnover times comparable to that of neOplastic cells showed the most pronounced alterations. The observed histologic alterations following intoxication with Cis-Pt.(II) were similar to those reportedly caused by the nitrogen and sulfur mustards (Graef et aZ., 1948) or by x-irradiation (Upton, 1963). Hematologic alterations were somewhat similar to those reported for other commonly used cancer chemotherapeutic agents such as the nitrogen and sulfur mustards, urethane (Dustin, 1947), 6-mercaptopurine (Clarke et aZ., 1953) and mitomycin C (Philips, Schwartz and Sternberg, 1960). The number of circulating lymphocytes and neutrOphils (short turnover times) was rapidly depressed to low levels by Day 3 after injection of Cis-Pt.(II). This was followed by a rapid regenerative increase which 59 created a temporary leukocytosis which eventually returned to the normal range. The number of circulating platelets was not depressed as much as the leukocytes following intoxication with Cis-Pt.(II). Megakaryocytes have been known to be relatively more resistant to the toxic effects of radio- mimetic agents (Kindred, 1947). While the production of reticulocytes was temporarily inhibited, the number of circulating erythrocytes was not altered. This could be explained by the longer life Span of the rat erythrocyte (60-80 days) and the lack of any evidence indicating hemolysis of erythrocytes following Cis-Pt.(II) intoxication. If the compound Cis-Pt.(II) were capable of hemolyzing erythrocytes it would be eXpected to cause an increase in the bilirubin level; however, this was not the case. Packed cell volumes and hemoglobin levels were unchanged except for a slight elevation which occurred at the time coincident with dehydration and enteritis (Day 4). Total protein levels in the blood include an extremely complex mix- ture of simple and conjugated proteins. Clinically, serum proteins are divided into 2 large classes, albumins and globulins. The albumin frac- tion is synthesized mainly in the liver, whereas the globulin fraction contains some constituent proteins which have been synthesized in lymphoid cells (gamma globulins). The observed decrease in total protein following intoxication with Cis-Pt.(II) may have been a reflection of the lymphoid depression which occurred at this time. Albumin levels were essentially unchanged, and the slight elevations may have been due to the dehydration and enteritis. I .I Illll. l 4 ill" (I ‘I ‘I III I ll ll II. I! l .411 A l llll‘ll’ l' | Ill IIIllI Illinll'l lilil 60 Uric acid levels in the blood normally reflect the exogenously derived sources of purines and endogenously formed purines from nucleoprotein metabolism. The serum levels of uric acid following intoxication with Cis-Pt.(II) remained in the lower part of the normal range established for the control rats. Intoxication with Cis—Pt.(II) apparently had no effect on serum levels of calcium or inorganic phosphorus, as all values remained in the normal range. The circulating level of cholesterol is normally the result of intes- tinal absorption and hepatic synthesis balanced by degradation and hepatic utilization of cholesterol as the precursor of bile acids and other sterols. In this study blood levels of cholesterol were not altered by Cis-Pt.(II). Lactic dehydrogenase (LDH) is normally the enzyme of the Embden- Meyerhof glycolytic pathway which reversibly catalyzes the oxidation of lactate to pyruvate. The enzyme is particularly abundant in hepatic, cardiac and muscular tissues and has been shown to be elevated following myocardial infarction, hepatitis and muscular trauma. As the activity of LDH in the erythrocytes'is 100 times that of serum, any hemolysis will alter the LDH value. Alkaline phosphatase is an enzyme that is most active between pH 8 and pH 10 and can utilize a variety of phosphomonoesters as substrates. Alkaline phosphatase is formed in bone by osteoblasts, and is also present in liver, kidney and intestine. Elevated serum levels accompany bone conditions in which there is excessive osteoblastic or chondroblastic activity. In hepatic dysfunction, the interference with biliary excretion of the enzyme produces an elevation of the serum level. 61 Glutamic oxaloacetate transaminase (GOT) normally catalyzes the trans— fer of the amino group from the amino acid (glutamic acid) to oxaloacetate and thus plays an important role in the metabolism of amino acids. High levels of GOT are normally present in cardiac muscle, liver, skeletal muscle, kidney and erythrocytes. Increased serum levels of GOT have been noted following cellular injury to any of these tissues. In this study, serum levels of LDH, alkaline phosphatase and GOT were higher in the control rats than in treated rats. This suggested the absence of any extensive injury to those tissues containing high enzymic levels, such as hepatic or myocardial tissues, and may indicate that Cis-Pt.(II) has a general inhibitory effect on enzymes. Glucose levels in the blood are normally controlled by hormonal factors. Epinephrine increases blood glucose levels by promoting glyco- genolysis. Adrenocortical hormones also increase blood glucose levels by promoting gluconeogenesis in the liver. The resultant hyperglycemia noted following intoxication with Cis-Pt.(II) may have been the result of stress-induced hormonal—mediated interactions which increased the blood glucose levels. Some degree of hyperglycemia may also have been associated with the dehydration and enteritis which occurred following the intoxication. The blood urea nitrogen (BUN) level is quantitatively the most important nonprotein nitrogenous constituent. It is the chief end product of protein metabolism and normally is excreted entirely by the kidneys. Hence its blood concentration is directly related to the protein content of the diet and the renal excretory capacity.- The elevated BUN levels occurred at a time coincident with the development of tubular necrosis (Day 3—Day 4) and probably reflected the tubular injury associated with the excretion of Cis—Pt.(II). The work of Allen (1970) has indicated 62 Cis—Pt.(II) is excreted via the urinary route. Eventually the BUN values returned to the normal range as the renal tubules underwent regeneration (Day 20-Day 28). In simple terms, toxicity as a consequence of the use of antitumor agents is a reflection of the fact that most of the therapeutically use- ful compounds are cytotoxic agents that affect normal as well as neOplastic cell replication, and in general these agents have a low therapeutic index. The compound Cis-Pt.(II) apparently will not be different in this respect from other cytotoxic agents.‘ As far as is known, neOplastic cells synthesize DNA and divide in essentially the same way as their normal counterparts. In fact, cell cycle times are not too dissimilar, although the prOportion of cells in active proliferation and nonproliferation would be different. Toxicity and recovery from toxicity would then be dependent upon the distribution of normal and neOplastic cells which are in these pools (of proliferat- ing or nonproliferating cells). The tissues of the body most frequently affected by cytotoxic agents are the bone marrow, gastrointestinal tract and lymphoid organs. The cellular constituents of these tissues all have high rates of renewal so that agents exerting their cytotoxic effects on DNA synthesis or on mitosis will encounter relatively large numbers of susceptible cells. As a result panhemocytopenia and intestinal denuda- tion are observed clinically after the use of cytotoxic agents. In this respeCt Cis-Pt.(II) apparently possesses the same cytotoxicity as the other antitumor agents. Fortunately, the high rate of renewal of the cells most susceptible to cytotoxic agents means that once the offending agent is removed the regenerative process is quite rapid. Here again, my work indicated this to be the case following the use of Cis-Pt.(II), as rats surviving the LD50 showed regeneration in the affected tissues. 63 Thus the biologic action of Cis—Pt.(II) is apparently more similar than dissimilar to the other cytotoxic agents which have been used as oncostatic agents. Much effort has been expended in the elucidation of the specific action of the cytotoxic agents that are employed as cancer chemotherapeutic agents. Brookes and Lawley (1964) have studied the mechanism of action of the alkylating agents, and have shown that sulfur mustard exerts a nucleOphilic attack on the N-7 of guanine residues in DNA which labilized the glycosidic bond and subsequently caused the release of guanine from the DNA. They also showed that sulfur mustard could react with 2 guanine residues to produce a cross-linking, which they postulated to be the primary cytotoxic action. In a subsequent report Lawley and Brookes (1967) noted similar cross—linking was obtained following the addition of-triethylenemelamine. Ruddon and Johnson (1968) have reported that DNA treated with nitrogen mustard had a decreased template activity for RNA polymerase and DNA polymerase. In Spite of the considerable data compiled thus far, the view that the primary chemotherapeutic effect of alkylating agents results from their attack on DNA is not universally accepted (Heidelberger, 1969). Wheeler and Alexander (1964) found no correlation between the extent of alkylation of the DNA and chemotherapeu- tic effect. Apparently not all guanine residues in DNA are equally reactive to sulfur mustard. Until the nature of this specificity is better understood, the mechanism of action of the alkylating agents will remain somewhat obscure (Heidelberger, 1969). A metabolite can be defined as some naturally occurring compound produced during metabolism, and an antimetabolite can be defined as a compound related structurally to the metabolite which prevents its utili- zation by competing with it for an enzyme. 64 The antifolates were one of the first antimetabolites to be utilized in cancer chemotherapy. Folic acid is normally reduced to tetrahydro- folate by the enzyme dihydrate reductase, which is powerfully inhibited by antifolates such as amethOpterin (Methotrexate). Tetrahydrofolate reacts with formaldehyde (or other compounds at the same oxidation level) to give isomeric formyl or methylene tetrahydrofolates, which are the‘ coenzymes of all one—carbon metabolism. Consequently, these coenzymes are required for 2 steps of purine synthesis and for thymidylate synthetase. The extensive efforts by Baker and Meyer (1969) have made it possible to achieve a remarkable degree of specificity in the inhibition of dihy- drate reductase enzyme isolated from L1210 tumor cells, but not affecting the enzyme isolated from mouse liver, spleen or intestine. The general group of purine antimetabolites includes a large number of analogs of the naturally occurring purine bases (adenine and guanine). Various aspects relating to these compounds have been reviewed by Heidelberger (1967). Zimmerman and Greenberg (1965) have shown 8-azoguanine will inhibit protein synthesis as a result of being incorporated into RNA to give some sort of fraudulent messenger RNA or transfer RNA. Another purine antimetabolite, 6-mercaptopurine, has been shown to competitively inactivate a pyrophosphorylase enzyme which normally catalyzes the con- version of hypoxanthine and guanine to their correSponding nucleotides (Brockman, 1965). Brockman and Chumley (1965) have also reported that 6-mercaptopurine inhibited the formation of phosphoribosylamine which is the first step in purine biosynthesis. This inhibition was of the negative feedback type which is also exhibited by the naturally occurring purines. The pyrimidine antimetabolites include analogs of the pyrimidine bases that normally occur in the nucleic acids (uracil, thymine and cyto- sine). The only pyrimidine antimetabolite which is chemotherapeutically 65 active is S-fluorouracil. With all other pyrimidine analogs, the nucleo- side (S-iodo-Z-deoxyuridine or IUdR) has been shown to be incorporated into nucleic acids (Prusoff, Bakhle and McCrea, 1963). Additional studies have also shown the inhibition of various enzymes necessary for nucleic acid synthesis. In addition to the alkylating agents and antimetabolites, a large number of additional chemotherapeutic agents have been subjected to studies to determine the Specific cytotoxic action. Mitomycin C is an antibiotic which has the potential to cross-link complementary Strands of the double helix DNA (Iyer and Szybalski, 1963). Actinomycin D has been shown to block the synthesis of RNA (Sartorelli, 1964). Administration of L- asparaginase apparently depletes the serum of the amino acid asparagine, thereby causing selective starvation of tumors for which L-asparagine is an essential nutrient. Thus L-asparaginase chemotherapy utilizes a some- what different biologic approach than the majority of oncostatic agents which have as their basic mechanism of action some cytotoxic prOperty. The preceding discussion has served as a prelude to the final point to be discussed; namely the question as to how the compound Cis-Pt.(II) exerts its oncostatic effects. Data presented in this study have sug- gested a strong parallelism between the oncostatic preperties (and toxi- cologic effects) of Cis-Pt.(II) and those compounds, such as the alkylating agents, which can be referred to as cytotoxic agents. The oncostatic properties of these cytotoxic agents are based on the indiscriminate ability to interfere with those processes necessary for cellular replication. The hypothesis that CiS-Pt.(II) exerts its oncostatic effect by virtue of its general cytoinhibitory properties has been amply demonstrated by the work of Howle and Gale (1970), in which they Showed the inhibition 66 of incorporation of isotopic precursors into DNA, RNA and proteins. A further definition of the basic mechanism.of action has been the subject of several studies which are Still in progress. SUMMARY AND CONCLUSIONS A 3—phase study of the oncostatic prOperties and toxicologic effects of the compound Cis-Pt.(II) was conducted. Phase 1 utilized the Dunning Ascitic Leukemia as a test tumor to evaluate the oncostatic prOperties of Cis-Pt.(II) and yielded data which showed that Day 1 treatment of DAL—bearing rats with 2 mg./kg. or 4 mg./kg. Cis-Pt.(II) extended the survival time to at least 60 days. This was in contrast to the expected 10-14 day survival time of untreated DAL-bearing rats. Delayed treatment of DAL—bearing rats on Day 4 or Day 7 caused a regression of the neo— plastic process and also extended the survival time. Phase 2 utilized the intramuscular Walker Carcinosarcoma 256 in a further evaluation of the oncostatic prOperties of Cis-Pt.(II). A single IP injection of 2 mg./kg. or 4 mg./kg. Cis-Pt.(II) on Day 3 after tumor implantation caused a marked inhibition of the neOplastic process. This same therapeutic regimen administered on Day 7 after tumor implantation also inhibited the neOplastic process at the site of initial implantation and also decreased the rate of metastasis and increased the survival time of the tumor-bearing rats. Phase 3 was an attempt to define the toxicologic parameters of Cis-Pt.(II), and the LDSO was determined to be 12.2 mg./kg. in the male rat. The LD90 and LDlO were calculated to be 26.5 mg./kg. and 5.5 mg./kg., respectively. A minimal therapeutic index was calculated to be 2.75 (based on limited data available from trials which utilized the DAL). 67 68 The histologic alterations subsequent to the IP injection of 12.2 mg./kg. Cis—Pt.(II) were most pronounced in those tissues having cellular constituents which have short turnover times. Thymic atrophy (due to lymphocytic depletion), Splenic depletion of lymphoid elements, intestinal epithelial denudation and bone marrow repression were most severe at 2-4 days after intoxication. Renal tubular necrosis and sloughing also occurred at this time, possibly as a result of tubular damage associated with the renal excretion of Cis-Pt.(II). Rats surviving the intoxication showed regeneration of the cellular constituents in those tissues which were affected. PanleukocytOpenia, reticulocytopenia and blood platelet depression were also most severe at about Day 3 after injection of 12.2 mg./kg. Cis—Pt.(II). This was followed by a regenerative leukocytosis and reticu- locytosis in rats surviving the intoxication. Erythrocyte counts, PCV and hemoglobin values were not depressed by the toxic dosage of Cis-Pt.(II), and serum bilirubin levels were not elevated. Serum albumin levels were essentially unchanged, whereas the depres- sion of total serum protein levels occurred at a time coincident with maximal lymphoid depletion. Serum levels of uric acid, calcium, inorganic phosphorus, lactic dehydrogenase, alkaline phOSphatase and glutamic oxaloacetate transaminase were not elevated subsequent to the injection of 12.2 mg./kg. Cis-Pt.(II). A Slight hyperglycemia occurred at a time coincident with the occur- rence of enteritis and dehydration. Levels of BUN were elevated at a time (Day 3-4) coincident with the observation of tubular necrosis in the kidney. 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Rev., Exptl. Path., 2, (1963): 199-240. Wheeler, C., and Alexander, J.: Studies with mustards. 5. In vivo fixa- tion of Cl4 labeled alkylating agents by bilaterally grown sensi- tive and resistant tumors. Cancer Res., 24, (1964): 1331-1337. Zimmerman, E., and Greenberg, 8.: Inhibition of protein synthesis by 8-azoguanine. Mol. Pharmacol., 1, (1965): 113-125. VITA Richard J. Kociba was born in Harbor Beach, Michigan, on April 8, 1939. He graduated from Our Lady of Lake Huron High School, Harbor Beach, Michigan. In 1959, after service in the United States Army, he enrolled as a student in Port Huron Junior College, Port Huron, Michigan. The following year he transferred to Michigan State University, where the Bachelor of Science degree was awarded with honors in 1964. In March, 1966, the Doctor of Veterinary Medicine degree was awarded with honors. From March, 1966, to March, 1967, the author was employed in a private veterinary practice and diagnostic laboratory at Milford, Indiana. In April, 1967, he joined the Faculty of the College of Veterinary Medicine at Michigan State University. In March, 1969, the author was awarded the Master of Science degree in Pathology. A Postdoctoral Fellowship was awarded by the National Institutes of Health for the completion of the Doctor of PhilOSOphy degree in Pathology. 73 .‘sfl {If -' HICHIGRN STRTE UNIV. LIBRRRIES 1|HIHNIHHIIWIHWIWIMIIIWIMWIHIIIWHI 31293102915539