POTENTIATION OF ANTINEOPLASTIC COMPOUNDS BY ORAL DIMETHYL SULFOXIDE IN

TUMOR-BEARING RATS*

Joel Warren, Miriam R. Sacksteder, Harriet Jarosz, Bruce Wasserman, and Peter E. Andreotti

The Leo Goodwin Institute for Cancer Research Nova University

Fort Lauderdale. Florida 33314

INTRODUCTION

Although dimethyl sulfoxide (DMSO) has been known for over 100 years, it is only in the last decade that its capacity for rapid water substitution has been investi- gated with pharmacological agents in mammals.' A wide variety of analgesics, administered in or with DMSO, were found to be transported across cellular and vascular membranes at an accelerated rate, and often with enhanced effectiveness.2 However, a flood of frequently uncritical studies, combined with observations of toxic damage when large doses were administered to dogs and rabbits,3 led the Food and Drug Administration to suspend clinical research on DMSO temporarily from 1965 to 1967. This action had a salutary effect, and more deliberate and thorough re- search continues to indicate that DMSO may uniquely modify a variety of physio- logical, metabolic, and immunological responses when parenteral or oral adminis- tration results in sustained blood and tissue levels.2

Its weak ability to act as an H+ donor, and thus not to participate in reactions that favor alkylation, led us to investigate the capacity of DMSO to potentiate the antineoplastic effects of cyclophosphamide (CTX), an alkylating derivative of nitrogen mustard.

The rodent tumor selected for these studies (INRL-1871) responds to several antineoplastic drugs, such as chlorambucil, 6-mercaptopurine, 5-fluorouracil, and amethopterin. Tumor development is consistent. A predictable, fatal leukemia follows i.p. inoculation; a solid nonmetastatic lymphosarcoma results from subcutaneous deposition of malignant The antigenicity of NRL-187 1 ap- pears to be quite weak.

We have investigated the therapeutic effect of parenteral and oral CTX in rats that are ingesting DMSO.

MATERIALS AND METHODS

Animals. Fischer 344 rats (obtained from Texas Inbred Mouse Co.) were accli- mated for a few days upon arrival, and were used when approximately 70 g in weight. They were caged in groups of 5 to 10, with even sex distribution, and were maintained on Purina laboratory chow and water ad libitum.

Chemicals. DMSO, reagent grade (Mallinckrodt), dissolved to a 1% or a 2% (w/v) solution in tap water, was used to fill graduated water bottles fitted with sipper

*This work was supported in part by contracts PH43-65-95 and N01-CP-23261 with the National Cancer Institute, National Institutes of Health.

194

Warren et al.: Antineoplastic Compounds 195

tubes, which were placed through the cage top. Fresh solutions were made every 24- 48 hours. Dimethylacetamide (Eastman) was fed at a concentration of 1 % after 2% was found to be toxic. Cyclophosphamide, generously provided by the Mead Johnson Co., was diluted in sterile distilled water for injection or prepared daily in tap water for oral administration.

Tumors. The strain of spontaneous rat lymphatic leukemia used in these studies first appeared in a germ-free Fischer 344 rat.' This line, designated as NRL-1871, resembles the poorly differentiated mononuclear cell leukemias described by Mo- loney and colleagues" in conventional rats. It produces only a weak antigenic reaction, and attempts to demonstrate an immunological cell-mediated response to the tumor have been f r ~ i t l e s s . ~ For the past three years, NRL-1871 has been propa- gated by inoculation of 10% spleen homogenates or washed buffy coat suspensions into barrier-maintained Fischer rats, in which it invariably produced fatal leukemia within 12-18 days when injected i.p. The incubation and survival times were roughly proportional to the number of cells in the inoculum. When injected subcutaneously, a lymphosarcoma appeared in 10-14 days a t the site of injection, and grew rapidly with little metastasis. Metastases are rare, possibly because rats die between the twenty-first and twenty-eighth day of a terminal leukemia, with white blood count (WBC) in excess of 107/mm3. The effectiveness of drug therapy in this tumor system was measured by the parameters of the WBC, average tumor diameter, and mean survival time.

Measurement of Alkylating Activity of Cyclophosphamide Metabolites. We have used the procedure described by Sladek,8 with slight modifications. Solid tissues were minced with scissors after being washed with phosphate-buffered saline (PBS) and homogenized to 10% suspensions in a reciprocating syringe. Heparinized blood and free urine were used without treatment. Samples (0.3 ml) were placed in centrifuge tubes with 2.7 ml Sorensen phosphate buffer (pH 7.4). One ml 5.5% ZnSO, . 7 H,O and 1 ml4.5% Ba(OH), . 8H,O were then added, and after centrifu- gation, 3.0 ml of the supernatant was transferred to screw-capped test tubes that contained 1.0 ml Walpoles acetate buffer (pH 4.0). To the samples was added 0.4 ml 5.0% solution of 4(p-nitrobenzyl) pyridine in acetone; the tubes were capped, placed in a boiling water bath for 20 min, and cooled to room temperature. Working in reduced light, we added 7.0 ml 5:2 ethyl acetate:acetone solution and 1.5 ml 5.0 M NaOH, mixed the samples well, and allowed the phases to separate. Using a Pasteur pipette, we placed aliquots from the organic phase in cuvettes and measured absorption at 540 m l .

RESULTS

Effect of Prolonged Feeding of DMSO on Normal and Leukemic Rats. The reported single toxic dose of DMSO by the oral route in rats that weigh 200 g is be- tween 16 and 24 g/kg.O We used considerably lower concentrations (0.5%-2.0%) for continual feeding. At these levels the animals exhibited moderate polydipsia and polyuria, which gradually disappeared within 4-14 days after treatment was begun. A slight amount of hyperactivity was noticeable for the first few days, after which the behavior of treated rats was indistinguishable from that of water-fed controls. The daily rate of ingestion of 1% and 2% DMSO in five individually caged rats that a t the start of the experiment weighed 65-75 g is shown in FIGURE 1. We have assumed that the average consumption of 2% DMSO was 15 m1/24 hours, or 300 mg/24 hours. (A similar consumption rate for cyclophosphamide fed together with DMSO has been used to estimate mg/kg/24 hours of ingested CTX.)

196 Annals New York Academy of Sciences

ml

2o 18 t 0 Woter 0 I% DMSO H 2% DMSO Averoge of 5 onimols fw each solution.

Initial Averoge Weight = 7 0 I gms.

It can be seen that after an initial acclimatization period, the ingestion rate and weight gain in rats fed these concentrations of the compound were essentially similar to those of animals fed water alone for three weeks. A moderate lymphocytosis fre- quently developed 4-6 days after feeding with DMSO was initiated, but this gradually subsided over the next 10-14 days, after which there was no significant difference in the WBC of treated and control animals for as long as 74 days. No significant changes in the daily RBC, hemoglobin, or hematocrit levels were seen in rats fed 2% DMSO for 40 days. Ingestion of this concentration continuously for over 300 days yielded no gross evidence of toxicity or loss of fertility, and no signs of histopathological damage other than slight centrilobular infiltrations of hepatic fat.

When 1% or 2% DMSO was fed to over 600 Fischer rats prior to the intraperi- toneal inoculation of from 1,OOO to 10,OOO leukemic cells and was continued until the death of the animals, there was no difference between the hematological findings or survival times of these rats and of rats that ingested water alone. The growth rate of solid tumors that resulted from the subcutaneous inoculation of NRL-1871 spleen cells was not modified by ingested 2% DMSO.

Efect of Ingested DMSO on the Toxicity of Cyclophosphamide. Whether administered parenterally or ingested ad lib., the toxicity of CTX is somewhat greater in DMSO-treated rats. The extent of the increase is related to drug concen- tration and the nature of the clinical symptoms that are being evaluated.

Parenteral CTX, in single or repeated doses, induced a more severe leukopenia in healthy rats that were fed 2% DMSO prior to and after the administration of CTX. In the experiment illustrated in FIGURE 2, healthy rats (70 g) were fed 2% DMSO or water (beginning on day 0 ) and asingle i.p. inoculation of either 14,70, or 140 mg/kg was given to these groups of rats on day 5 . As controls, five rats were fed DMSO or

Warren et a/.: Antineoplastic Compounds I97

water only for the duration of the experiment. The WBC of individual rats was de- termined on the days shown, and was averaged for the group of five rats on each par- ticular regimen.

The temporary elevation in WBC caused by oral DMSO alone is evident. For a given dose of CTX, the leukopenia was greater and more prolonged in rats that were ingesting DMSO. Four out of five animals given 140 mg DMSO/kg died; none in the water group succumbed on this dose. The WBC in all 35 surviving animals had returned to normal by 18 days.

When CTX was incorporated in the drinking water, the toxicity threshold for the drug in water- or DMSO-fed rats was not as well defined as in the case of parenteral administration. For example, the weight gain of the DMSO group, measured for a period of 21 days, was markedly reduced (TABLE 1). In rats that weighed 70 g or more, the eventual mortality rates for doses up to 17 mg/kg/24 hours were the same. Chronic leukopenia in rats that are fed CTX tends to be somewhat greater in DMSO-fed rodents for the first three weeks of treatment, after which animals will survive with a WBC of less than 1500 cells/mm3 for 40-50 days, in a state of ex- treme debility. Oral CTX at doses between 8 and 21 mg/kg/24 hours was considerably more toxic in weanling rats (45-60 g) and caused death in 3-7 weeks. The concomitant ingestion of DMSO, however, did not significantly change the sur- vival time or dose-related mortality (TABLE 2). Additional observations of the effect of DMSO on the toxicity of other antineoplastics suggests that the toxicity of low or very high doses of a drug for rats is not significantly altered by ingestion of DMSO.lD There appears, however, to be a midrange in which the mortality that results from a given dose of CTX is increased by 30-50% when the animal is fed DMSO. This does not appear to be true with other antitumor drugs.1°

WBC x 10’

10- I

A/ DMSO 2% . Adlib

6-

4-

-.- Woler only 0 A 0 CTX and Water A 0 CTX and OMS0

2-

I 1 I I I 1 10 12 14 16 18 20 0 2 4 6 8

DAYS

FIGURE 2. Potentiating effect of oral DMSO on cyclophosphamide-induced leukopenia in the rat.

198 Annals New York Academy of Sciences

TABLE 1 EFFECT OF ORAL CYTOXAN AND DMSO ON WATER INTAKE,

WEIGHT GAIN, AND LEUKOTOXICITY IN NORMAL RATS

Mortality W BC Average Average Intake DMSO Total Water Weight Days 22 Days Intake Gain of CTX

mg/kg ml g None none 460 65.6 8,940 10,460 0/10 None 2% 829 60.4 9,890 8,550 0/10

4.3 none 607 67.3 8,500 6,410 0/10 4.3 2% 618 68.5 6,940 3,820 0/10 8.6 none 415 66.4 6,010 4,240 1/10 8.6 2% 48 1 8.7 5,040 550 1/10

10.8 none 370 31.7 3,690 1,725 0/10 10.8 2% 418 16.8 3,280 2,050 1/10 17.2 none 374 26.0 2,320 1,500 2/10 17.2 2% 342 8.0 2,840 1,033 2/10

When it was observed that DMSO potentiated the antitumor effect of oral CTX, it became necessary to investigate the comparative ingestion rates of CTX in water and in DMSO. Groups of 5 rats were fed CTX a t levels of 2 ,4 ,8 , 16 and 32 mg/kg/ 24 hours from calibrated water bottles, and the daily consumption was measured to the nearest 0.5 ml. The rate of drug ingestion was always accelerated in DMSO during the first week of treatment, after which the volume of intake was the same with either diluent (FIGURE 3). Because the consumption patterns were similar for the 2 and 4 mg groups, and for the 8, 16, and 32 mg groups, we have combined these groups in FIGURE 3. I t will be seen that concentrations of CTX greater than 8 mg/ kg124 hours caused a progressive diminution in the daily intake of drug in water or in DMSO. By the tenth day the WBC of these rats was 1,000/mm3, and they ap- peared too debilitated to ingest any kind of liquid nourishment. In the high-dose groups, 9 out of 15 had died by the third week of the experiment. In contrast, ani- mals have been fed CTX in 2% DMSO at 2-4 mg/kg/24 hours for 72 days with normal weight gain and no indication of weakness or alopecia, but their WBC stabi-

TABLE 2 COMPARATIVE SURVIVAL TIMES OF RATS FED CTX IN WATER AND IN 2% DMSO

Days from Start of Drug 16-20 21-25 26-30 31-35 36-40 41-45 46-50 Solvent CTX*

mg/kg/24 hours

8.4

12.6

2 1 3/10 3 3 1 1 8/10 DMSO

H 2 0 1 5 2 1 9/10 DMSO 1 2 1 4 2 10/10

H2O

I 5 2 1 10/10 I 3 5 1 10/10

1 1 3 5 10/10 1 1 4 3 10/10

H2O

H2O

DMSO

DMSO 1

16.8

21.0

*CTX solutions were prepared fresh daily.

Warren et al.: Antineoplastic Compounds 199

mt

2ot I 2 8 4 wK/24 hn.

A Cyclophosphomide 0 Cyclophosphomide

in 2% OMSO in Water

A m q e of Smk/group 9

DAYS

FIGURE 3. Daily consumption by Fischer 344 rats of cyclophosphamide, in water or in 2% DMSO. There was an average of 5 rats in each group.

lized at between 4,000 and 7,000/mm3, or approximately 3,000-4,000 less than the median for normal Fischer rats of this age.

Effect of Oral DMSO on the Therapeutic Eficiency of Parenteral CTX. Four- teen experiments have been performed in which CTX at levels between 15 and 120 mg/kg was injected in single or double doses into leukemic rats that had been receiving DMSO ad l ib. 6 days before treatment and continued to receive it for 50 days. These yielded inconclusive data of the type shown in TABLE 3. The cure rate at

TABLE 3

AS RELATED TO INGESTION OF DIMETHYL SULFOXIDE

S u r v i v a I Rate Average Time

CURATIVE DOSAGE OF CYCLOPHOSPHAMIDE I N LEUKEMIC RATS

Treatment* Cytoxan

(i.P.1 DMSO

w / k g days - - 01 10 23 2% - o/ 10 23 - 68 10/10 5 0

34 9/10 50

- 8.5 4/ 10 30 2% 68 10/10 50 2% 34 9/ 10 50 2% 17 9/10 5 0

~

- 17 5/10 33

2% 8.5 4/10 33

*DMSO was started o n day 0. CTX was given on days 6 and 8.

200 Annals New York Academy of Sciences

TABLE 4 EFFECT OF CTX A N D DMSO IN RAT LEUKEMIA

WHEN TREATMENT WAS DELAYED FOR 12 DAYS AFTER INFECTION

Treatment* Survival DMSO CTX Rate Time Group

mg/kg/24 hours days 18 18

6 none 2.1 Of10 24 3 2% 2.1 1/10 18

4 2% 4.2 0110 23

1 none none 019 2 2% none 0110

7 none 4.2 3/10 20

8 none 8.4 1/10 28 5 2% 8.4 10/10 35

*CTX was administered on the twelfth day after injection and continued for 22 days. The drug and DMSO were combined in drinking water.

the 17 mg level was 50% in the water group and 90% with DMSO. The overall sur- vival rate however, was not markedly enhanced by ingestion of 2% DMSO. This weak potentiation against leukemia (the same has been found for solid tumors) was surprising in view of the manner in which DMSO augmented the blastic effects of daily CTX in normal rats (FIGURE 2.) We suspect, however, that continued daily administration of parenteral CTX for 7-10 doses will result in a greater therapeutic effect and possibly a better gauge of its potentiation by oral DMSO.

Efec t of Oral DMSO on the Efffcacy of Oral CTX. It was found that the NRL- 1871 tumor system would respond to CTX administered in the drinking water of Fischer rats. This observation led us to investigate the efficacy of combining CTX and DMSO for ingestion ad lib. With this regimen, tolerated doses of CTX (2-10 mg/kg/24 hours) were more effective than they were in rats given water alone. The results of a typical experiment are shown in TABLE 4. CTX feeding was delayed until the height of leukemia, on the twelfth day after injection, and was continued for the next 22 days. Of 40 rats that received 2 and 4 mg/kg, 36 died of leukemia. At 8.4

TABLE 5 EFFECT OF CTX AND DMSO ON SURVIVAL OF RATS WITH NOVA LYMPHOSARCOMA*

Treatment Survival Average Survival Time Group DMSO CTX Rate To Tumor To Death

mg/kg/24 hours days 5 none none 0110 17 23.8

10 2% none 0110 18 22.9 4 none 0.21 0110 21.9 31.2 9 2% 0.2 1 2/10 20.3 27.8 3 none I .05 0110 25.8 35.6 8 2% 1.05 319 25.0 35.0 1 none 4.2 9/10 23 (1) 35 (1) 1 none stopped at 30 days 519

4.2 10/10 6 2% I I 4 - -

- - 6 2% stopped at 30 days 9/10

*CTX was given ad lib. in drinking water, from 6 days after the inoculation of 1,OOO tumor cells.

Warren et al.: Antineoplastic Compounds 20 1

mg/kg, 1 out of 10 water-fed rats survived, whereas all of the DMSO group lived for 35 days after treatment was discontinued. The WBC was normal at this time in all eleven survivors.

The efficacy of CTX against solid tumors was also consistently enhanced by the feeding of DMSO. In the regimen shown in TABLE 5, CTX was added to the drinking water on day 6 and continued for 30 days. Of 10 rats on CTX and DMSO, 9 survived without tumors for another 60 days and were negative at autopsy. Fatal tumors rapidly appeared in 4 out of 9 in the “water group” after the drug was discontinued.

In the experiment illustrated in FIGURE 4, oral CTX at three levels was first given on the first appearance of tumors (day 12) in all rats, and was continued for another 9 days when all 60 rats had become free of palpable neoplasms. They recurred within 2 days after the drug was discontinued, but were always delayed in the rats on DMSO, in proportion to the dosage of CTX.

Similar prolonged suppression was seen when leukemia was treated with com- parable drug regimens. Fatal NRL-187 1 leukemia in untreated control rats develops precipitously on days 10 and 12 (FIGURE 5, lines A and B). When CTX, in relatively low doses, was fed continuously from day 6 until the death of all rats, the onset was delayed in proportion to the concentration of CTX; maximum delays always ap- peared in the animals that received CTX in 2% DMSO. We have never obtained complete cures of NRL-187 1 leukemia with CTX at levels below 8 mg/kg/24 hours.

Efect of Oral CTX-DMSO in Experimental Meningeal Leukemia. Enhance- ment of the transport of antitumor drugs across the “blood-brain” barrier has be- come important with the increased incidence of human meningeal leukemia. It has been suggested that this is the result of the prolonged survival time of acute leukemia patients on drug therapy, which results in sequestered leukemia infiltrates in the central nervous system.

The apparent ability of DMSO to facilitate intercellular transport of CTX led us to investigate its ability to potentiate this drug against an experimentally produced

- A

4 ring water J 1 -B - _ _ - - - ---*-.7,-.

5 10 15 20 75

DAYS POST CYTOXAN

FIGURE 4. Effect of DMSO on the suppression and reappearance of rat lymphosarcoma after cessation of cyclophosphamide therapy. Group A were water-fed controls; group B, DMSO-fed controls; and groups C to H were treated with CTX: C, 2.1 mg/kg/24 hours, in water; D, 2.1 mg/kg/24 hours, in 2% DMSO, E, 6.3 mg/kg/24 hours, in water; F, 4.2 mg/kg/ 24 hours, in DMSO, G , 4.2 mg/kg/24 hours, in 2% DMSO; and H, 6.3 mg/kg/24 hours, in 2% DMSO.

202 Annals New York Academy of Sciences

40-

n 0 X I- Z 3 8 30- -I -I W 0

8 J

m w 20- t z

to-

i I I I I I I I

- A 5 C D E F G H

mg/kg/

CTX storied 6 doys ofterinoculotion of io00 leulremio cells I . P

I I 1 1 I 1- 2 4 6 8 10 12 14 16

DAYS POST CYTOXAN

FIGURE 5 . Effect of DMSO on suppression of rat leukemia by subcurative doses of CTX.

meningeal leukemia. Two experiments have been completed, and others are in progress. An initial titration of NRL-1871 cells to test their capacity for meningeal multiplication is shown in the upper portion of TABLE 6. When as few as 10 cells were inoculated i.c. into Fischer rats that weighed 60 g, they caused death from men- ingitis within 18-26 days; larger inocula resulted in a decreased incubation time. On the basis of these findings, we infected 70 rats with 1,OOO NRL cells by the intracere- bra1 route and administered oral CTX in water or in 2% DMSO, from the second to the twenty-fifth day. All 50 treated animals remained a symptom-free. Meningitis appeared within 10 days after the drug was discontinued, however, first in the water- fed groups and subsequently in those that were receiving DMSO. All the rats that

Warren et al.: Antineoplastic Compounds 203

were ingesting water had succumbed on 4.2 mg CTX/kg/24 hours when the DMSO- treated animals were symptom-free. These, however, finally sickened and died 4-5 days later. These results, while encouraging, were obtained with marginal doses of CTX; experiments in progress with higher levels should be more informative.

Lack of Potentiation by Oral Dimethylacetamide ( D M A ) . Because this com- pound has certain similarities to DMSO and shares its solvating properties, we investigated its ability to potentiate CTX under the same conditions as were em- ployed for DMSO. Preliminary experiments confirmed the finding of Weiss and Orzellll that DMA is more toxic when fed to rats.

A DMA concentration of 1 %, fed ad lib., was the maximum tolerated by Fischer rats, and even this amount produced a marked depression in motor activity and a transient leucocytosis similar to that resulting from oral DMSO. DMA (I%), alone or mixed with CTX a t levels of 8, 16, and 24 mg/kg/24 hours, was first fed to rats 6 days after the i.p. inoculation of 1,0oO leukemic cells and was continued until-all the animals succumbed to leukemia. There was no prolongation of survival time in the DMA groups over that of the water-fed controls. DMSO (2%) included in the same experiment prolonged survival time by 4-6 days (25-28%) over that of the water-fed groups.

Pharmacological Studies

We have investigated the basis for the effect of DMSO by measuring the level of CTX metabolites, designated as cytoxyl amines (CTX amines) for convenience, in the tissues, blood, and urine of rats given CTX by parenteral and oral routes in water and in 2% DMSO.

DMSO Does Not Increase C T X Conversion by Liver Tissue (Hepatic Mixed Oxidase Function) in Vitro. Fresh rat liver homogenate was incubated with CTX (2- 5 mg/ml) in glucose-Ringer's solution, which contained 1 or 2% DMSO. There was no evidence of an increased alkylating activity in the presence of I-2% DMSO when samples were assayed over a 5-hour period. On the contrary, the initial 15-60 min

TABLE 6 DELAYED REAPPEARANCE OF MENINGEAL LEUKEMIA

IN RATS FED CTX AND DMSO

Mortality and Average Survival Treated Treatment StODDed* CTX Number of Cells DMSO

in Inoculum mg/kg/24 hours days

23 19

10

16 100

14 1,000 I0,OOO

1 ,ooo - - 10/10 18 1,000 2% - - 10/10 18 1,000 2% 2.1 0110 10/10 36 1,000 2% 4.2 Of10 10/10 41 1,000 2% 6.3 Of10 lo/ 10 41

313 313 313 313

- - - - - - - - - - - -

~

1,000 - 2.1 0110 10/10 35 1 ,OOO 4.2 0110 10/10 37 ~

*CTX was fed from the second through the twenty-fifth day after the intracerebral inocu- lation of leukemic cells.

204 Annals New York Academy of Sciences

30 60 120 180 240 300 360 MINUTES

FIGURE 6. Lack of effect of DMSO on conversion of CTX by liver homogenates in vitro. o = 2 mg CTX in Ringer’s solution; A = 2 mg CTX in Ringer’s solution with 1% DMSO; = 2 mg CTX in Ringer’s solution with 2% DMSO.

conversion rates appeared to be somewhat diminished (FIGURE 6). Three experi- ments of this nature failed to provide any support for the conclusion that DMSO potentiates CTX by increasing its rate of activation by hepatic microsomes.

Conversion of Parenteral CTX in Rats Fed DMSO. We then measured the ap- pearance of alkylating activity in rats fed DMSO. After a single i.p. dose (5 mg). analysis of the pooled tissues of 5 rats sacrificed at 15, 30,60,90 and 120 min yielded the data shown in FIGURE 7. These animals had been drinking water or 2% DMSO for 4 days before the inoculation of the drug. The concentration of CTX amines in the liver was not enhanced by DMSO. The blood levels in DMSO-treated animals, however, were 2-3 times greater than in controls. Peak urine concentrations were essentially the same, but were reached earlier in the DMSO group. FIGURE 8 illustrates the findings in a similar experiment, in which the alkylating activity in NRL-1871 lymphosarcoma was also determined in a group of 5 rats. Again, the blood levels of CTX amines were significantly higher than in water-fed controls. Of greatest interest was the finding of a roughly twofold higher concentration of alky- lating activity in tumor tissue. In two additional experiments of this kind, the tumors of DMSO-fed rats consistently contained more CTX amines.

Conversion of Oral CTX in Rats Fed DMSO. The results of similar investiga- tions of alkylating activity in the blood, urine, and tumors of rats fed DMSO or water substantiated the hypothesis that enhancement of antineoplastic drug action by DMSO results from improved intracellular drug transport. Rats were fed 2% DMSO or water alone for 4-6 days, after which CTX, at calculated levels of 8.4 mg/kg/24 hours, was added to the water bottles. Blood, urine, and tumor levels of

Warren et al.: Antineoplastic C o m p o u n d s 205

CTX amines were measured twice daily in groups of 3-5 rats for 1-2 weeks. Moderate but consistently higher levels of CTX amines were present in the blood, urine (FIGURE 9), and tumors (FIGURE 10) of the DMSO-treated groups. Because of rapid regression of the tumors, it was not possible to continue these assays beyond 3-5 days.

DISCUSSION

The foregoing demonstrates that tumor-bearing, inbred rats that continually in- gest low concentrations of DMSO become physiologically altered, so that the thera- peutic effects of an alkylating agent (cyclophosphamide) are increased. The an- tiblastic action of cyclophosphamide is also enhanced, which results in some lowering of the toxicity threshold of this compound. The admittedly inconclusive data suggest, however, that at selected dosages drug enhancement can be achieved without an increase in toxicity.

This potentiation is associated with elevated concentrations of antineoplastic cytoxyl amines in the circulation and in the tumor. Total conversion of cyclophos- phamide by hepatic tissue does not seem to be increased. This agrees with Sladek’s conclusion that hepatic oxidase activity, the site of formation of cytoxyl amines, operates a t its maximum level in the normal rat.*

0 H,O -Liver

TIME POST CTX INJECTION, MINUTES

FIGURE 7. Cytoxyl amine levels in liver, blood, and urine of rats fed DMSO for 4 days be- fore i.p. injection of 5 mg CTX.

206 Annals New York Academy of Sciences

H20- Blood 0 DMSO- Blood 0 Tumor 0 Tumor A Urine A Urine

Blood 8 Tumor I I

Abs

I 20

I 15

I10

I05

I00

15 30 60 I20 TIME POST CTX INJECTION, MINUTES

Urine I00

90

80

70

60

50

40

30

2 0

10

FIGURE 8. Cytoxyl amine levels in blood, tumor and urine of rats fed DMSO for 4 days be- fore treatment with 5 mg CTX.

Among several questions that remain unanswered are the following: What is the minimum concentration of DMSO that will effectively potentiate cyclophos- phamide? Is total eradication of neoplastic cells enhanced by DMSO? Is an increase in systemic toxicity, and particularly in leukodepression, an inevitable price of DMSO potentiation? Will the enhanced tumor permeability yield higher therapeutic indices without a significant increase in whole body damage? Answers to these should be forthcoming in the near future.

Treatment of human cancer with combinations of oral DMSO and antitumor compounds is both feasible and attractive. Because of potential toxicity problems, it must be- approached, however, not only with caution but also under circumstances in which the maximum amount of information can be obtained on the mode of action of DMSO.

[NOTE ADDED IN PROOF: Since this report was presented, we have extended these studies to 16 antineoplastic compounds which operate through differing mechanisms. Each of the drugs was administered intraperitoneally a t 4 different single or repeated dose levels, beginning 10 days after implantation of a tumor, and at a time when the neoplasms were well advanced. Three compounds, cytosine arabinoside (NSC-63878), vincristine (NSC-67574), and 5-fluorouracil (NSC- 19893)

Warren et al.: Antineoplastic Compounds 207

0 HzO Blood 0 DMSO- Blood

Urine 0 Urine CTX 6 4mg/K 24hrs

I 200-

1100- 1,

I I I I I I I 1 I I I I I I I I 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8

AM PM DAYS

FIGURE 9. Cytoxyl arnine levels in the blood and urine of rats fed CTX in 2% DMSO and in water.

Abs. -

1.50 -

1.40 -

1.30 -

1.20 -

I . 10-

1.00 -

t

0 HzO - Blood 0 DMSO-Blood A Tumor A Tumor

I I I I I 1 Start I 2 3 4 5

DAYS

FIGURE 10. Cytoxyl arnine levels in NRL lymphosarcoma and blood of rats fed CTX (8.4 rng/kg/24 hours), in 2% DMSO and in water.

208 Annals New York Academy of Sciences

were ineffective in rats bearing the NRL-1871 lymphoma under the test conditions. The following drugs significantly prolonged survival times: 6-mercaptopurine (NSC- 755), methotrexate (NSC-740), chlorambucil (NSC-3088). vinblastine (NSC- 49842), procarbazine (NSC-772 13), CCNU (NSC-79037), MCCNU (NSC-95441), BCNU (NSC-409962), daunomycin (NSC-82151), nitrogen mustard (NSC-762), dianhydrogalactitol (NSC-132313). norbornyl (NSC-88 106), and adriamycin (NSC- 123 127).

Although several compounds were more effective in DMSO-fed rats than in water-fed controls a t a specific dosage, there was no T/C ratio in the former which exceeded that of all four control dose levels by 25 or more. Thus, these results re- semble those obtained when cyclophosphamide was administered by the i.p. route. As might be anticipated, when treatment was initiated 2, 4, or 6 days after tumor implantation, the therapeutic indices were higher in both water- and DMSO-treated rats. More time will be required to establish the duration of tumor-free survival in these early-treated animals.

The toxicity of parenterally injected antineoplastics was estimated from the number of rats out of a group of 10 that died before tumors had appeared in the un- treated controls. On this admittedly crude basis, only one drug, chlorambucil, was significantly more toxic in DMSO-treated rats.

In the case of all active compounds, the median tumor diameter decreased faster in animals that were drinking DMSO. This clinical improvement was not associated with a prolongation of survival. These findings will be described in detail in a subse- quent publication .]

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