Adriamycin and Daunorubicin: a Comparison of Antitumor Activities and Tissue Uptake in Mice Following 1

Home , Daunorubicin

[CANCER RESEARCH 33, 1837-1844, August 1973] Adriamycin and Daunorubicin: A Comparison of Antitumor Activities and Tissue Uptake in Mice following 1

Herbert S. Schwartz and Gerald B. Grindey

Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203

SUMMARY at C- 14 of AM. This difference in structure confers some differences in biotransformation and distribution (1â€”5,14, The effects of adriamycin (AM) and daunorubicin (DM) 15, 21, 22, 31), although the mechanisms of action of the have been compared in DBA/2 mice bearing an ascitic 2 drugs appear to be similar (8, 12, 20). More strikingly, murine lymphocytic leukemia (P-288). Over a range of the agents have marked differences in therapeutic ef doses (either a single dose, or I dose daily for 5 days), ficacy in experimental animal tumor systems (7, 19, 23, 28) the i.p. administration of AM increased the average sur and in preliminary clinical trials (30). vival times of leukemic mice from about 12 days (controls) Differences in efficacy have not been explained hereto to 25 days or more, and 10 to 20% of the animals were fore on a biological, biochemical, or pharmacodynamic long-term survivors. In contrast, DM increased survival basis (15). In fact, when comparisons of the drugs have time only about 40% or less, with no long-term survivors. been made to correlate antitumor activity with mech Mice given whole-body irradiation (250 R) or cyclophos anisms, the results have indicated that these agents phamide (150 mg/kg) prior to tumor implantation showed should be equipotent or that DM should be the more ef markedly decreased differential responses to these drugs. fective antitumor drug. In contrast, antitumor studies When AM was administered daily for 5 days, concurrently have generally indicated a broader spectrum of activity with methotrexate to mice bearing Ll2lO or P-288, the in and a more favorable therapeutic index for AM. creased average survival times due to AM alone were re In this study, we used transplantable murine lympho duced by the drug combination in both tumors. cytic leukemia P-288 (18), which shows a remarkable re Tissue concentrations of the agents were determined by sponse to AM but only minimal growth inhibition after fluorometric analysis. Uptake of AM by P-288 in normal treatment with DM. The pharmacological bases for the and irradiated hosts was generally less than that of DM different responses to AM and DM have been investigated. during the early times after administration, although such differences diminished by 3 to 4 hr and after high MATERIALS AND METhODS doses. There were no marked differences in uptake of AM and DM by liver and thymus; in contrast, DM was Mice. Female DBA/2J mice (17 to 21 g) were obtained found in spleen at concentrations about twice those of from the breeding colony of this Institute or were pur AM. These concentrations were reduced when mice were chased from The Jackson Laboratory, Bar Harbor, given irradiation and P-288 cells prior to administration Maine. The P-288 lymphocytic neoplasm originally de of the drugs. The results indicate that DM may be more scribed by Potter (18) was obtained for this Department toxic to the spleen and, therefore, more immunosuppres from Dr. C. J. Kensler (Arthur D. Little, Inc., Cam sive than AM. This difference may account for the greater @ efficacy of AM in various transplanted tumors. DN: R H AM:RzOH H INTRODUCTION

2 and DM are similar anthracycline aminoglycosi dic antibiotics. As shown in Chart I, the compounds differ only by the addition of a hydroxyl group on the side chain

1 This work was supported by USPHS Core Program Grant CA 13038 from the National Cancer Institute and by an allocation from American Cancer Society Institutional Grant IN-54-L-l7 to Roswell . H CI Park Memorial Institute.

2 The abbreviations used are: AM, adriamycin; DM, daunorubicin (daunomycin, rubimycin). NH, Received October 17, 1972; accepted April 18, 1973. Chart I. Structures of DM and AM

AUGUST 1973 1837

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1973 American Association for Cancer Research. Herbert S. Schwartz and Gerald B. Grindey bridge, Mass.). Leukemia Ll2lO used in this laboratorycells (8 determinations for each drug). In normal rat tis haswereReagents. been described previously (16).sues, the recoveries of DM and AM respectively, andand AM (Farmitalia Co., Milan, Italy; Lot 23)liver, 80 and 78%; spleen, 7 1 and 69%; and thymus, 92 were:methotrexateDM (Lots 142 and 148), cyclophosphamide, and94%. When corrected for internal quench, recoveries ofand were kindly provided by the Drug Researchliver, 94%; spleen, 85%; and thymus, 99%. Recoveries mouse(Bethesda,Development Branch of the National Cancer InstituteAM and DM from P-288 cells and from normal Md). DM was contaminated with traces oftissues for the current studies were within the ranges de AMquench.and (demonstrable chromatographically), and both AMscribed above, but were not corrected for tumorandDM were contaminated with their respective aglyconesChromatography. Mice bearing ascitic P-288 afterofother fluorescent materials. The total contaminationcells were treated with AM or CM (10 mg/kg) 1 day werefluorescenteach was estimated at about 2 to 5%, assumingtumor implantation (108 cells/inoculum). Cells AgNO3:freshlyequivalence. Solutions of AM and DM werewashed as described above and extracted with 1-butanol.keptprepared routinely, protected from light, andisoamyl alcohol or with 0.3 N HCI in 50% plates,wascold or frozen until used. Reagent grade AgNO3The extracts were spotted (50 @zl)on Silica Gel G theburg,purchased from J. T. Baker Chemical Co. (Phillips activated 14 to 18 hr at 120Â°,and chromatographed in N. J.) and isoamyl alcohol was from Fisher Scien solvent systems described by Bachur (2), i.e., CCI4: meth tific Co. (Pittsburgh, Pa.). Precoated Silica Gel G (1000anol: H2O (80: 20: 3) and CCI4: methanol:glacial ace asfrom@tm) thin-layer chromatography plates were purchasedtic acid (8 : 20 :4). Standards included tissue blanks, bylnstruments.Analtech, Inc. (Newark, Del.).well as AM and DM and their aglycones (prepared mm).General Irradiation was administered by aacid hydrolysis in 0.1 N HC1 at 75Â°for 30 by20 Electric Model 250 Maxitron set at 250 kVp andStandard deviation and t tests were as described (26).ter.ma with a 0.25-mm copper and 1.0-mm aluminum fil Snedecor aVictoreenThe dose was calculated in roentgens in air with thesame Model 570 condenser R meter placed in ventilatedRESULTS50-miunit used to irradiate the mice i.e., 12 (2rpm). polypropylene tubes on an aluminum turntable anMaxitron,The axis of the turntable was set 50 cm from theThe P-288 tumor is lethal in all DBA/2 mice after and all tubes were equidistant from the axisi.p. inoculum of 10 or more cells. For evaluation of ther andcells,Assay from each other.apeutic efficacy, mice were inoculated with 106 12leased of AM and DM, Both AM and DM are re which resulted in an average survival time of about dailymayfrom their binding to DNA by Ag@ (20, 24) anddays. Chart 2 shows the effects of treatment with 5 tomethodbe extracted quantitatively into isoamyl alcohol. Ai.p. doses of AM or DM. Over the range from 0.25 eachin based on this technique was recently described2.0 mg/kg, AM prolonged the average survival at detail (24). Briefly, tumor cells from 3 or more micedose level, compared to that of 0.9% NaCl solution were washed in H 20 and 0.9% NaCI solution, as de treated controls (p < 0.01). At the higher doses, pro scribedgfor by Kessel et al. (1 1), and centrifuged at 300 x 5Or cellweight30 sec in tared centrifuge tubes. The packed was determined and the cells were suspended in an equal volume of 0. 1% sodium lauryl sulfate. An ali 401- quot (0. 1 ml; 10 or 50 mg, wet weight) was then added to a 33% (w/v) solution of AgNO3 and shaken for 10 mm at 301- 40 in thedark.For a determination of drug uptake in normal tissues, 3ormice were killed by cervical dislocation, and organs from 201- more mice were removed and dropped into ethanol at The frozen tissues were weighed and homogenized - -I- - I0 (1ogin I .0 ml (pooled spleen and pooled thymus) or 2.0 ml liver) H20 with 0.2 ml 33% (w/v) AgNO3 and shaken for 10 mm at 4Â°as before. Two ml of isoamyl alcohol â€˜/ ,â€˜@â€˜. â€˜ â€˜ â€˜ were added to each sample, and tubes were again shaken SALINE 0.125 025 0.5 1.0 2.0 4.0 at 4Â°for 10 mm in the dark. Fluorescence in the organic CONTROLS phase was measured with a Farrand ratio fluorometer mq I kg Cx 5) (primary filters 3387 and 5562; secondary filters 3384 and Chart 2. Average survival of DBA/2 mice bearing P-288 ascites 9780). Tissue blanks and standard concentrations of AM tumor and treated with AM or DM administered i.p. once daily for 5 days. Tumors were implanted (10' cells) on Day 0, and treatment corn and DM (0.2 to 10 @tg/ml) were extracted through the . .. . menced on Day I. Each point represents 10 to 15 treated mice and 30 procedure for each determination. In the previous study controls. Vertical bars S.D.; numbers in parentheses, number of 50-day (24), average (Â±S.D.) recoveries of DM and AM added survivors over the total number treated per group. Long-term survi to L 1210 ascites cells were 89.9 Â±6. 1 and 86.6 Â±5. 1%, vors included in the calculation of average survival time were those that respectively, for the range 0.2 to 10 @gdrug per 50 mgo survived 50 days. Saline, 0.9% NaCI solution.

1838 CANCER RESEARCH VOL. 33

Comparison ofAM and DM longation was as much as 140% more than that of con (2/5) @ trols; furthermore, 20% of these animals survived 50 C,, 50 Am days or longer. In contrast, DM produced no long-term >- a4 Dmf survivors, and survival times were increased by only 40% 40 (p < 0.05) at just 1 dosage level, I.0 mg/kg. -I (1/5) In experiments shown in Chart 3, drugs or 0.9% NaC1 4 > 30 solution were administered once 24 hr after transplanta > tion of the tumor. Average survival time was doubled by AM at dosages of from 3.3 to 10 mg/kg, whereas no U) 20 single dosage of DM had a comparable effect. More Li over, 4 of 15 @micereceiving the higher dosages of AM 0 4 were 50-day survivors. None of those treated with DM 10 were long-term survivors. Li Previous studies (16, 17) have shown that the contribu 0 â€˜-â€œ tion of an immunological response to the therapeutic SALINE . 0.62 1.25 2.5 5.0 10 20 CONTROLS effects of drugs cannot be discounted because the mg/kg (xl) tumor seems compatible on the basis of its viability upon Chart 3. Average survival of DBA/2 mice bearing P-288 ascites tu transplantation of a small number of cells. For elimination mor and treated with AM or DM administered as a single i.p. dose on of the possible contribution of transplantation immunity Day I. Tumors were implanted (10' cells) on Day 0. Symbols are as de in our studies, the drugs were reevaluated in immunosup scribed in the legend of Chart 2. There were 5 treated mice per group, pressed mice that were prepared by pretreatment with and 10 mice were given 0.9% NaCI solution (saline). ionizing irradiation or cyclophosphamide (6, 16, 17). Animals were given whole-body irradiation (250 R) 24 hr 50 NOT IRRADIATED IRRADIATED prior to tumor transplantation (106 cells), and 24 hr there after they received a single dose of AM, DM, or 0.9% NaCl solution i.p. The results of these studies (Chart 4) in 40 Am@ dicated that irradiation alone shortened the average -J control survival time from I I.8 days (30 mice: unirra 30 Dmf diated 0.9% NaC1 solution-treated controls) to 10.6 > days (30 mice: 250 R, 0.9% NaCI solution); this difference was significant at p < 0.01, indicating the participation U) 20 Li of a weak immune response in this transplanted tumor 0 @ system. 4 I0 Unirradiated control mice (Chart 4) were placed in Li the irradiation apparatus and were constrained for the same period of time as were the irradiated animals. AM SALINE 1.25 2.5 5.0 SALINE 1.25 2.5 5.0 (2.5 and 5.0 mg/kg) doubled the average survival time, CONTROLS CONTROLS with 4 of 20 mice surviving 50 days. DM again produced mgI kg( x I) only a small increase in average survival time (28% at 2.5 Chart 4. Average survival of DBA/2 mice bearing P-288 ascites mg/kg) in unirradiated mice, with no 50-day survivors. tumor and treated with a single i.p. dose of AM or DM. Animals, ir The effect of irradiation in these studies was to reduce radiated (250 R) or unirradiated (as described in â€œMaterialsand survival time of AM-treated mice without a comparable Methodsâ€•) on the day prior to transplantation (Day 0), were treated with reduction in mice treated with DM (Chart 4). This resulted a single i.p. dose of AM or DM (10/group) or with 0.9% NaCI solution in the complete elimination of any differences in survival (saline) (30/group). Symbols are as described in the legend of Chart 2. times between the 2 agents (p > 0. 1) although both AM and DM increased survival time by a small but significant treatment with AM, survival was reduced from a range of (p < 0.01) amount above that of the irradiated tumor con 27.5 to 36.2 days (5 of 30 mice were long-term survivors) to trols given only 0.9% NaCI solution. a range of 12.7 to 15.9 days, by pretreatment with cyclo Results similar to those obtained with irradiation were phosphamide. After treatment with DM, survival was re also obtained when cyclophosphamide was used to sup duced from a range of 15.2 to 16.0 days (no long-term sur press the immune response (6). Mice (10/group) were vivors) to a range of 9.2 to 12.7 days by cyclophosphamide. given a single dose of cyciophosphamide (150 mg/kg, The data illustrate again the marked reduction in tumorici i.p.) 24 hr prior to receiving P-288 (106 cells). AM and dal activity of AM in immunosuppressed mice. As indicated DM, in doses ranging from 1.25 to 5.0 mg/kg, were then below, this change cannot be related to toxicity, although administered 24 hr after the inoculum. Like irradiation, this may be a factor after DM is administered in combina cyciophosphamide reduced the survival time of control as tion with cyclophosamide or irradiation. well as drug-treated mice. Average survival time of con For determination of whether the reduction of survival trol groups was reduced from 13.7 Â± 1.55 to 10.1 Â±3.21 times after irradiation or cyclophosphamide treatment, days by cyclophosphamide alone (p < 0.05). Following particularly in AM-treated mice, was due to increased tox

AUGUST 1973 1839

icity, the combinations were administered to mice that U) had received no tumor implants. For these comparisons, -J S Am â€”I the same schedules were maintained; 48 hr after they Li Dm. were given irradiation (250 R) or cyclophosphamide C,) a (150 mg/kg), mice received a single high dose of AM or Li . DM in the therapeutic range (5 mg/kg). All 16 mice that C-) received AM in combination with irradiation or cyclophos 4 0 phamide survived more than 50 days without evident toxicity, whereas those 16 receiving DM in the combina tion survived only I I to 20 days. 0 Methotrexate, which is also an immunosuppressive j@I , 20 agent, was administered daily for 5 days concurrently with AM to DBA/2 mice bearing P-288 or L12l0. L12l0 HOURS was selected because it is less immunizing than P-288 in Chart 7. Uptake of AM and DM (I mg/kg; i.p.) by P-288 ascites DBA/2 mice, and results indicated (Chart 5) that AM is cells at various times after administration. DBA/2 mice bearing 7- to 9- less effective against L12l0 than against P-288 (Chart 6). day-old P-288 were given drugs i.p. in a volume adjusted to 10 mI/kg. Methotrexate itself has little effect on either tumor in Points, cells pooledfrom 3 to 5 mice. DBA/2 mice under the conditions selected for these cx periments. At optimal schedules (27), methotrexate 30 alone is effective against both L12l0 and P-288 and MTX,rnrj/kgx5 doily might enhance the antitumor activities of AM and DM. However, daily treatment is highly immunosuppressive and, as used here, is less selective for tumoricidal activity. lao This is indicated in our experiments in which methotrex I ate diminished the therapeutic effects of AM in mice with either P-288 or Ll210. These results, as well as those with irradiation and cy @ I0 clophosphamide, are consistent with the possibility that immune defenses of the host may contribute to the effec tiveness of AM in transplantable tumors and that agents

@ â€”@-@ Â£ that diminish this response also decrease the effective 0 0.5 I 2 4 ness of AM. Adriomycin,mg/kg a 5 doys The tissue concentrations of AM and DM were esti Chart 5. Average (Ave.) survival of DBA/2 mice bearing Ll210 mated after various treatment regimens. Chart 7 shows ascites and treated concurrently with AM and methotrexate (MTX) the uptake by P-288 after administration of single low administered once daily for 5 days. Tumors were implanted on Day 0 doses of AM and DM (I mg/kg). There is little difference and treatment commenced on Day I . Points, average survival of 5 between the 2 agents in these tumors except that DM is treatedand 10 controlmice. initially taken up more rapidly. Chart 8 shows the uptake by P-288 at I hr after i.p. doses of AM and DM ranging MTX,mq/kgx5doily from 0.5 to 10 mg/kg. For determination of whether prior irradiation of the host would affect drug uptake by P-288, experiments were

0 carried out using the same schedule shown in Chart 4, .8 but the tumor inoculum was increased to 108 cells to

0 permit uptake measurements when the mice were treated 24 hr later. The results shown in Table I indicate a rapid

Cl) uptake of DM 1 hr after a small dose (I mg/kg, i.p.) was administered to unirradiated mice. Under similar conditions, the concentration of AM in these cells is con siderably lower, and it is further reduced by prior irra diation of the host animals. This effect of irradiation can also be seen at 3 hr. By 4 hr, however, the concentration of DM in the tumor cells of irradiated mice decreases to that of AM (15 to 18 zg/g packed cells; data not shown). Adriomycin, mg/kg a 5 doys Chart 6. Average (Aye.) survival of DBA/2 mice bearing P-288 as Similar experiments (Table 1) were carried out with cites and treated concurrently with AM and methotrexate (MTX) ad animals receiving single high doses (10 mg/kg, i.p.). During ministered once daily for 5 days. Conditions are the same as described the 1st 3 hr after drug administration, the concentrations in Chart 5. Long-term survivors (not shown) were included in the calcu of DM also were higher than those of AM in P-288, lation of average survival time as surviving 50 days. but the effects of irradiation were considerably less pro

I840 CANCER RESEARCH VOL. 33

the loss of efficacy of the drug after low doses of irradia 400 tion. To determine whether a change in biotransformation of the drugs was affected by prior irradiation, we pre pared extracts (0.3 N HC1 in 50% 1-butanol) from tu mor cells taken at 1 and 3 hr after administration of the @ 300 single high dose (10 mg/kg). The extracts were chroma -J tographed on Silica Gel G as described in â€œMaterials and Li C) Methods. Under these conditions, there was no evidence a of hydrolysis in similarly prepared extracts from aqueous Li solutions of AM and DM. In the 1-hr series, there was a @ 200 IAm marked reduction in the fluorescent material in fast mi LDm. grating zones of both AM and DM in extracts from the irradiated hosts. The RF of these zones (0.7 to 0.9) in a, both the acid and neutral solvent systems suggests that these may be aglycones (2, 3) and that their formation was a, diminished by irradiation. By 3 hr, however, there were @, 100 no marked differences between P-288 extracts from ir radiated and unirradiated mice, and the inhibitory ef fect of radiation noted at I hr was no longer apparent for either DM or AM. No further attempt has been made to recover the products or estimate the amounts formed for 00@ 2 4 6 8 10 the present investigations. In another series of experiments, untreated mice (3 to 4/ Chart 8. Uptake of varying doses of AM and DM by P-288 ascites group) bearing 7 to 10-day-old tumors were given either cells in DBA/2 mice at I hr after i.p. administration. The conditions are as described in the legend of Chart 7. AM or DM (2 mg/kg) and were killed 18 to 20 or 42 to 44 hr later. Tumor cells were collected, washed, and cx tracted with Ag@: isoamyl alcohol. The extracts and Table I washes were chromatographed on silica gel. Results Concentrations of A M and DM in P.288 in irradiated and showed that only I component, unchanged AM, was cx unirradiated DBA /2 mice tracted from cells by the Ag@: isoamyl alcohol method. AM and DM were administered i.p. to mice (3 or 4 per group) I or 3 Two components were extracted from cells of DM hr before sacrifice. Animals were irradiated and inoculated with P-288 treated mice: the original DM and a derivative presumed (108 cells) at 48 and 24 hr. respectively, before the drugs were adminis tered. to be the reduced component, daunorubicinol (2, 3). Re extraction of the cells with HCI :ethanol and chroma in tography then showed fluorescent material at RF's (@g/g)IrradiationConditionsConcentrations P.228 comparable to those of the aglycone and the reduced aglycone of DM but showed only traces of fluorescense (250DM+ R)Dose (mg/kg)Time (hr)AM comparable to the RF of AM aglycone. No fluorescent materials were found in the cell-free ascitic fluid or 42 washes. The observed differences in metabolism are con 63+â€”I II 1II 34 sistent with those reported by Meriwether and Bachur 409 (14), and do not account for the differences in therapeutic 486+â€”10 101 1244 256 efficacy of the 2 drugs. In other studies of drug uptake, AM and DM (5 32 36+â€”I I3 314 42 mg/kg) were administered to normal mice; and liver, spleen, and thymus were extracted with Ag@: isoamyl al 543 cohol. As shown in Chart 9, uptake by liver was initially â€”10 103 3347 469 510 rapid and then declined. Thymus took up less drug, and in neither tissue was there a marked difference between nounced than in the previous experiments with lower do the concentrations of AM and DM. In the spleen, how ses. ever, DM was taken up more rapidly and was concen The results suggest that low doses of AM have no trated to a level at least twice that of AM. In a recent re greater therapeutic efficacy than DM in both irradiated port, Yesair et al. (3 1) also showed that mouse spleen and unirradiated animals. The uptake at higher doses, concentrates DM (and its metabolites) more than it does in contrast, does not explain the therapeutic effectiveness AM and that this differential may be retained for at least of AM in unirradiated mice and, moreover, the relatively 24 hr. high concentrations of AM found in tumors of mice ir The effect of prior irradiation and tumor implantation radiated 48 hr prior to treatment do not seem to explain on drug uptake by spleen were estimated 4 hr after treat

AUGUST 1973 I841

ment with either AM or DM. The results in Table 2 tions are consistent with the suggestion that the thera show that both irradiation and P-288 implantation reduce peutic value of AM may be enhanced by the immunity of the uptake of AM and DM. In addition, the combina the host toward tumor antigens. tion results in a further decrease in splenic uptake. In another series of experiments, uptake of AM and DM (or their metabolites) was similar in the P-288 tumor and in liver and thymus of tumor-free mice, indicating DISCUSSION that both agents had equivalent access to the tumor as well as to these tissues. In the spleen, however, there In this study, we have demonstrated a marked thera was a greater uptake of DM, confirming the eather obser peutic advantage of AM, compared to DM, in the treat vation by Yesair et al. (31). ment of ascitic murine leukemia P-288. The striking dif In mice subjected to prior irradiation at low, immuno ference in the effects of AM and DM is apparently re suppressive doses (16, 17), the uptake of both AM and lated more to the host response to the tumor than to a DM was reduced in the spleen. Even so, the concentra differential tumor susceptibility. This was shown in ani tions of DM were still higher than those of AM. Di mals whose immune defenses were impaired by cy minished uptake in these animals is probably due to se clophosphamide and by whole-body irradiation prior to lective sensitivity of a portion of the splenic cell popula tumor implantation and drug treatment. Under such tion and its subsequent depletion after irradiation. The conditions, the effects of AM and DM were indistinguish uptake of AM and DM by P-288 was also reduced by able. In a further comparison of the effects of AM on 2 prior irradiation of the host. This effect, however, was tumors, P-288 and Ll2lO, AM had markedly less thera seen primarily at early intervals and after low doses of peutic value in the less-immunizing Ll210. Moreover, the drugs. For these reasons it does not seem to offer a when the mice were treated concurrently with the im suitable explanation for the diminished therapeutic ef munosuppressive drug methotrexate, the efficacy was ficacy of AM in P-288. The delayed uptake by these again reduced in both P-288 and L1210. These observa cells may be related instead to decreased metabolic rates and altered distributions that have been reported after Amo such low doses of irradiation (9, 13). Dm. The biotransformation of DM and daunorubicinol to their respective aglycones requires the microsomal

4- 20 THYMUS LIVER enzyme system which is inhibited for long periods of time -C by relatively low doses of ionizing radiation and cyclo a, 16 4) phosphamide (8, 9). It seems reasonable to expect that the same enzyme complex also hydrolyzes AM to its re 4- 12 4) spective aglycone, but the rate of hydrolysis appears to be much less for AM than for DM or its reduced form, a, daunorubicinol (3, 4). There are several implications that a, are apparent from these observations. The reduced rate of aglycone formation, especially for DM after irradia tion, may be related to the increased lethality of DM in @ 0 /2 I 2 4 0 /2 2 4 0I... 1/2 I 2 4 irradiated (and cyclophosphamide-treated) hosts, sug HOURS gesting to us that detoxification is diminished by de I.@ creased rates of aglycone formation. These results also Chart 9. Uptake of AM or DM (5 mg/kg, i.v.) by tissues of DBA/2 suggest the possibility that diminished rates of hydrolysis mice at varying times after administration. Points, tissue pooled from 3 mice. may alter cardiac toxicity, at least the acute form that re portedly occurs in the hamster (15). Whether this effect would bear on the more serious delayed cardiac toxicity in Table2 patients is not known. Concentrations of A M and DM in spleen of DBA /2 mice The relatively greater uptake of DM in spleen is con AM and DM (5 mg/kg, iv.) were given to mice (3 to 4 per group) 4 hr sistent with the possibility that it may be the more immuno before sacrifice. Animals were irradiated and inoculated with P-288 at 48 suppressive drug (7, 10). Similarly, a recent report by and 24 hr, respectively, before the drugs were administered. Razek et al. (19) showed that DM was considerably more in toxic to the normal hematopoietic stem cells from the fern (.ig/g)IrradiationP-288(250ConditionsConcentrations spleen oral marrow of AKR mice than was AM. In another re cent study by Casazza Ct al. (7), tumors were induced with Moloney murine sarcoma virus and challenged with AM or cells)AMDMâ€”â€”11.519.2+â€”5.08.8â€”+7.913.3++<1.03.3R)(l0 DM. The authors noted that cell-mediated immunity may be affected differently by these agents but were unable to exclude the possibility of a more potent or more prolonged action of AM against the tumor cells. There is, at the present time, no substantial evidence that

I842 CANCER RESEARCH VOL. 33

AM is more effective than DM as a direct tumoricidal ical Toxicity of Adriamycin: A New Antineoplastic Antibiotic. Toxi agent. Using HeLa cells, Kim and Kim (12) demonstrated col.Appl.Pharmacol.,21:287â€”301,1972. that DM is considerably more effective than AM in reduc 6. Bonmassar, E., Bonmassar, A., Vadlamudi, S., and Goldin, A. Anti ing cell viability. In Ll210, Wang et al. (29) showed that genic changes of Ll210 Leukemia in Mice Treated with 5-(3,3 Di both antibiotics inhibited precursor incorporation into DNA, methyl-l-triazeno)imidazole-4-carboxamide. Cancer Res., 32: 1446â€” 1450, 1972. RNA, and protein without significant differences but, in 7. Casazza, A. M., Di Marco, A., and Di Cuonzo, G. Interference of vitro, DM was the more effective inhibitor. Similar results Daunomycin and Adriamycin on the Growth and Regression of were obtained with leukemic cells from patients. L12l0 Murine Sarcoma Virus (Moloney) Tumors in Mice. Cancer Res., 31: cells, made resistant to one or the other drug, were cross 1971â€”1976,1971. resistant, and polymerase isolated from this tumor was 8. Donelli, M. G., Franchi, G., and Rosso, R. The Effect of Cytotoxic equally sensitive to AM and DM. The recent studies by Agents on Drug Metabolism. European J. Cancer. 6: 125-126, 1970. Meriwether and Bachur ( I4) also showed that precursor 9. Du Bois, K. H. Inhibition by Radiation of the Development of Drug incorporation into DNA and RNA of L1210 cells was Detoxification Enzymes. Radiation Res., 30: 342â€”350,1967. slightly more sensitive to DM than to AM and, in addition, 10. Isetta, A. M., Intini, C., and Soldati, M. On the lmmunodepressive Action of Adriamycin. Experientia, 27: 202â€”204,1971. that the uptake by the cells was substantially greater for 11. Kessel, D., Botterill, V., and Wodinsky, I. Uptake and Retention of DM. Daunomycin by Mouse Leukemic Cells as Factors in Drug Response. Such studies with L12l0 suggest that, if there is a Cancer Res., 28: 938-941, 1968. difference in tumoricidal activity, DM should be the more 12. Kim, S. H., and Kim, J. H. Lethal Effect of Adriamycin on the Divi effective drug against this leukemia. Other studies, how sion Cycle of HeLa Cells. Cancer Res., 32: 323â€”325,1972. ever, demonstrate that the reverse is actually the case and 13. Lindop, P. J. Tissue Effects of Radiation in Relation to Radiotherapy. that, under various optimizing schedules, AM is the su Current Topics Radiation Res., 6: 293â€”324,1970. perior therapeutic agent (23). In the L1210 system, there 14. Meriwether, W. D., and Bachur, N. R. Inhibition of DNA and RNA is also a small contribution of the host immunological de Metabolism by Daunorubicin and Adriamycin in L1210 Mouse Leu fenses to the therapeutic effects of various drugs (16, 17), kemia. Cancer Res., 32: 1137â€”1142,1972. and it seems likely that this provides the small advantage 15. Mhatre, R. M., Herman, E. H., Waravdekar, V. S., and Lee, I. P. for AM in therapeutic efficacy. Distribution and Metabolism of Daunomycin, Adriamycin, and N- Acetyldaunomycin in the Syrian Golden Hamster. Biochem. Med., In summary, the therapeutic advantage of AM, as com 6:445-453, 1972. pared to DM, may be the consequence of a lesser depres 16. Mihich, E. Modification of Tumor Regression by Immunologic sion of host-immune mechanisms. This is consistent with Means. Cancer Res., 29: 2345â€”2350,1969. presently available evidence that: (a) both are equipotent 17. Mihich, E., and Kitano, M. Differences in the Immunogenicity of at the level of tumor target cells, both in animals and in Leukemia Ll2lO Sublines in DBA/2 Mice. Cancer Res., 31: 1999â€” culture; (b) uptake in tumor cells is similar or favors DM, 2003,1971. both in animals and in culture; (c) in lymphoid organs, 18. Potter, M. Variations in Resistance Patterns in Different Neoplasms. AM is less toxic to marrow stem cells and is concentrated Ann. N. Y. Acad. Sci., 76: 630-642, 1958. less in spleen; and (d) the therapeutic response to the 2 19. Razek, A., Valeriote, F., and Vietti, T. Survival of Hematopoietic and Leukemic Colony-forming cells in Vivofollowing the Administra drugs is similar after immunosuppression. tion of Daunorubicin or Adriamycin. Cancer Res., 32: 1496-1500, 1972. 20. Rusconi, A. Different Binding Sites for Actinomycin and Daunomycin. AC KNOWLEDGMENTS Biochim. Biophys. Acta, 123: 627-629, 1966. 21. Rusconi, A., and Calendi, E. Action of Daunomycin on Nucleic Acid We are grateful to Edwin Kelly, Mikolaj Dyl, and Alice Atwood for Metabolism in HeLa Cells. Biochem. Biophys. Acta, 119: 413-415, 1966. their skilled assistance, and to Dr. J. F. Holland and Dr. E. Mihich for their valued suggestions. 22. Rusconi, A., Di Fronzo, G., and Di Marco, A. Distribution of Tn tiated Daunomycin in Normal Rats. Cancer Chemotherapy Rept., 52 (Part1):331â€”335,1968. REFERENCES 23. Sandberg, J. S., Howsen, F. L., DiManco, A., and Goldin, A. Com panison of the Antileukemic Effect in Mice of Adniamycin (NSC I. Alberts, D. S., Bachur, N. R., and Holtzman, J. L. The Pharmaco 123127) with Daunomycin (NSC-82151). Cancer Chemotherapy kinetics of Daunomycin in Man. Clin. Pharmacol. Therap., 12: 96â€” Rept., 54: (Part I): 1â€”7,1970. 104, 1971. 24. Schwartz, H. S. A Fluormetnic Assay for Daunomycin and Adniamy 2. Bachur, N. R. Daunorubicinol: A Major Metabolite of Daunorubicin. cm in Animal Tissues. Biochem. Med., in press. J. Pharmacol. Exptl. Therap., 177: 573â€”578,1971. 25. Schwartz, R. S. Are Immunosuppressive Anticancer Drugs Self 3. Bachur, N. R., and Hoffman, D. H. Daunorubicin Metabolism: Es defeating? Cancer Res., 28: 1452â€”1454,1968. timation of Daunorubicin Reductase. Brit. J. Pharmacol., 43: 828â€” 833, 1971. 26. Snedecor, G. W. In: Statistical Methods, Ed. 5, pp. 42-52. Ames, 4. Bachur, N. R., Moore, A. L., Bernstein, J. G., and Liu, A. Tissue Iowa: Iowa State College Press, 1956. Distribution and Disposition of Daunomycin (NSC 82151) in Mice: 27. Straus, M. J., Mantel, N., and Goldin, A. Effects of Priming Dose Fluorometric and Isotopic Method. Cancer Chemotherapy Rept., Schedules in Methotrexate Treatment of Mouse Leukemia L1210. 54 (Part I): 89-94, 1970. Cancer Res.,31:1429â€”1433,1971. 5. Bertozzoli, C., Chieli, T., Ferni, G., Ricevuti, G., and Solcia, E. Clin 28. Venditti, J. M., Abbott, B. J., DiMarco, A., and Goldin, A. Effective

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1973 American Association for Cancer Research. Adriamycin and Daunorubicin: A Comparison of Antitumor Activities and Tissue Uptake in Mice following Immunosuppression

Herbert S. Schwartz and Gerald B. Grindey

Cancer Res 1973;33:1837-1844.

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