Effect of Fluosol-DA® on the response of intracranial 9L tumors to X rays and BCNU

Inr. J. Rodiarion Oncology Biol. Phvs.. Vol. 15, pp. 1187-I Printed in the U.S.A. All rights reserved.

192 Copyright

0360-3016/88 $3.00 + .OO 0 1988 Pergamon Press plc

??Original Contribution

EFFECT

BEVERLY

OF FLUOSOL-DA@ ON THE RESPONSE OF INTRACRANIAL 9L TUMORS TO X RAYS AND BCNU

A. TEICHER,

PH.D.,’

TERENCE

S. HERMAN,

M.D.*

AND CHRISTOPHER

M. ROSE,

M.D.3

‘Division of Cancer Pharmacology, ’ Joint Center for Radiation Therapy, Dana-Farber Cancer Institute, Section of Radiation Oncology, 44 Binney St.. Boston, MA 02 115: and 3Department of Radiation Therapy, St. Joseph Medical Center, Alameda & Buena Vista, Burbank, CA 9 1505 Treatment with a perfluorochemical emulsion combined with breathing a 95% or 100% oxygen atmosphere has been shown to be an effective adjuvant to radiation therapy in several animal tumor systems. Similarly, the addition of treatment with a perfluorochemical emulsion combined with breathing a high oxygen atmosphere has been shown to improve the response of several animal tumor systems to treatment with BCNU. We now report results of the use of the perfluorochemical emulsion, Fluosol-DA, and carbogen breathing with single dose radiation treatment, BCNU and combined drug and radiation treatment in intracranially implanted 9L gliosarcoma. The median enhancement in life span produced by Fluosol-DA and carbogen breathing in addition to radiation was 2 days at 10 Gy and 6 days at 20 Gy compared to radiation treatment alone. In the group receiving 20 Gy with Fluosol-DA and carbogen breathing, 2 of 20 lived 120 days. Treatment with a single intraperitoneal injection of BCNU (10 mg/kg) on day 7 post tumor cell implantation produced an increase in life span of 2 days compared to untreated control animals. The combination of drug treatment with Fluosol-DA and carbogen breathing produced an increase in life span of 26 days, which was significantly different from BCNU treatment with air breathing (p < 0.001). Finally, when BCNU and Fluosol-DA and carbogen were combined with radiation treatment (20 Gy), an increase in life span of nearly 85 days compared to untreated controls was produced, with 47% (9 or 19) surviving 120 days. These results suggest that this combination might be effective in the treatment of malignant brain tumors. Fluosol-DA,

BCNU, X rays, 9L gliosarcoma,

Brain tumors.

these approaches look promising, none has yet come into routine clinical use. In this report, results of the use of Fluosol-DA and carbogen breathing with single dose radiation and BCNU in the 9L gliosarcoma model system are presented. Two of the major difficulties in the therapy of malignant brain tumors are the regional and clonal heterogeneity of human brain tumors within individual patients and the problems of the blood-brain banier.‘i,‘6,28 The heterogeneity of brain tumors is responsible, at least in part, for the development of resistance to chemotherapeutic agents. Intrinsic variation in chemosensitivity has been demonstrated for human glioma cells, and lowdose nitrosourea therapy has been shown to rapidly result in resistant cell populations.‘8 Blood flow and bloodto-tissue transport of small molecules are also important variables determining the efficacy of chemotherapy. There is not much variability in tumor blood flow among

INTRODUCTION

Multimodality therapy for malignant brain tumors, including surgery, radiation therapy, and chemotherapy, has been investigated for over 20 years. Radiation therapy significantly prolongs patient surviva1.14*17,31 BCNU (carmustine) also produces statistically significant survival benefit. Procarbazine, methyl-CCNU, CCNU, and streptozotocin are about as effective as BCNU.*,i7,i8 Patient survival longer than 18 months is specifically related to the addition of chemotherapy to radiation and surgery.*,i7,i8 Several approaches are under investigation to improve the efficacy of radiation therapy of malignant brain tumors.14,31 These approaches include the addition of radiosensitizers, hyperbaric oxygen, unconventional fractionation schemes, heavy particle radiotherapy, interstitial brachytherapy, and hyperthermia. Although some of

This work was supported by NC1 grant 5POl-CA19589, ACS-NY grant CH-340 and a grant from Alpha Therapeutics Corp., Los Angeles, CA 90032. Reprint requests to: Beverly A. Teicher, Ph.D., Dana-Farber Cancer Institute, 44 Binney St., Boston, MA 02 115.

BCNU, 1.3-bis(2-chloroethyl)1-nitrosourea. Accepted for publication 18 May 1988.

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intracerebral tumor models and intracerebral tumor blood flow is generally slightly higher than that to normal brain white matter.2 However, blood flow within individual tumors varies considerably and is frequently lowest within central or necrotic tumor regions. The blood-totissue ratios for the concentration of small molecules varies widely over a range of 1- 100 times the normal brain value.2.3 Treatment with a perfluorochemical emulsion while breathing a 95% or 100% oxygen atmosphere is an effective adjuvant to radiation therapy in several animal tumor systems.‘5,22*25-27 Similarly, the BCNU effect is enhanced by perfluorochemical emulsion treatment and oxygen in several animal tumor systems.7,8,11.23,24 We now report our results with the use of the perfluorochemical emulsion, Fluosol-DA, and carbogen breathing with radiation, BCNU or combined drug and radiation treatment in intracranially implanted 9L gliosarcoma. This study was carried out with a goal of developing a combination treatment which may be effective and have clinical applicability. METHODS

AND

MATERIALS

Materials

Fluosol-DA 20%* was obtained. BCNU (carmustine) was obtained from the Dana-Farber Cancer Institute pharmacy. Carbogen? is 95% oxygen and 5% carbon dioxide. Tzlmov 9L/SF gliosarcoma cells were obtained as a gift from Dr. Dennis Deen (University of California, San Francisco, CA).’ 9L cells were maintained in culture in 01MEMS supplemented with 10% fetal bovine serumg and antibiotics. For experiments, 9L cells (4 X 104) in 10 ~1 of media without serum were implanted intracranially in male Fischer 344 rats** weighing 200-250 gm, on day 0. Briefly, each rat was anesthetized with sodium pentobarbital. A midline scalp incision was made, and a hole was bored through the skull with a 23 gauge needle at a point 2 mm behind the right coronal suture and 2 mm lateral to the midline. The cell suspension was injected into the right frontal lobe at a depth of 3 mm from the dural surface. The needle was removed and the hole filled with dental cement. The scalp was sutured with 4-O nylon.8.‘2 Survival experiments On day 7 after tumor implantation, 1.5 ml (6.0-7.5 ml/kg) of Fluosol-DA was injected into the tail vein of tumor-bearing rats. The animals were then allowed to * Green Cross. Japan, distributed by Alpha Therapeutic Co., Los Angeles. CA. t Medical Technical Gases, Medford. MA. $ Grand Island Biological Co., Grand Island, NY.

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breathe air or were placed in chambers with circulating carbogen. One hr later, the animals were treated with 10 or 20 Gy to the cranium?? (13’Cs gamma rays: dose rate, 0.9 gray/min). The radiation chamber was continuously flushed with air or carbogen. The shielded portion of the animal received less than 2% of the delivered radiation dose. The animals were anesthetized (Nembutal; 60 mg/ kg) during the radiation treatment. BCNU (10 mg/kg) was administered as a single dose intraperitoneally in 0.09% buffered saline (0.2 ml) immediately after Fluosol-DA administration or was prepared in the Fluosol-DA and both agents were given as a single intravenous injection to tumor-bearing animals on day 7 post-tumor cell implantation. In some groups, Fluosol-DA ( 12 ml/kg) was injected into the tail vein immediately prior to the drug. The animals were then allowed to breathe air or were placed in a circulating carbogen atmosphere for 1 hr. Some groups received both drug and radiation. The sequence of treatment was Fluosol-DA, then BCNU and carbogen breathing followed by a single dose of 20 Gy. Survival of animals was monitored daily. Animals that were moribund or unable to reach food or water were killed by CO? inhalation. The data are presented as percent increase in life span of treated compared to untreated control animals. At 120 days post treatment, surviving animals were sacrificed.

Survival was calculated from tumor cell implantation using the Kaplan-Meier method.6 The two-tailed log rank test was used to determine whether differences between survival curves were statistically significant (defined asp I 0.05). RESULTS

Table 1 shows the results obtained when Fluosol-DA and carbogen breathing were added to radiation therapy of intracranially implanted 9L tumors. Treatment with 10 Gy and 20 Gy resulted in increases in median life span of 2 days and 6 days compared to untreated controls, respectively. The addition of carbogen breathing to the X ray treatment resulted in median life spans which were 4 days with 10 Gy and 10 days with 20 Gy longer than those of untreated controls. The combination of Fluosol-DA and carbogen breathing produced increases in median life span of 7 days with 10 Gy and 18 days with 20 Gy compared to untreated control animals. The enhancement in life span produced by Fluosol-DA and carbogen breathing in this tumor system was 5 days at 10 Gy and 12 days at 20 Gy compared to radiation treat$ Sterile Systems Inc., Logan, UT. ** Taconic Farms, Germantown, NY. tt Gamma Cell 40, Atomic Energy of Canada, Ltd.

Fluosol-DA@ on response of tumors to BCNU 0 B. A. TEICHERet al.

Table 1. Survival of Fischer 344 rats implanted intracranially with 9L gliosarcoma and treated with single dose radiation c Fluosol-DA +- carbogen breathing Treatment

Survival,” days

group

Controls 10 Gyd 20 Gy 10 Gy/carbogen e 20 Gy/carbogen 10 Gy/Fhrosol-DA/carbogen 20 Gy/Fluosol-DA/carbogen

f

% increase in life span b

29 31 35

-

(0/2O)C

33 39

14 (O/18) 34 (O/19)

36 47

24 (O/19) 62 (2/20)

7 (O/15) 21 (O/15)

a Median survival in days. b Percent increase in life span compared to untreated control animals. c Number of 120-day survivors over the total number of animals receiving that treatment. d Radiation was delivered as a single dose to the cranium on day 7 after implantation of 4 X lo4 9L tumor cells. e Carbogen breathing was maintained for 1 hr prior to and during radiation treatment. f Fluosol-DA (1.5 ml) was injected into the tail vein immediately prior to the initiation of carbogen breathing.

ment with air breathing (p < 0.01 and 0.001, respectively). Figures 1 and 2 show Kaplan-Meier survival distribution plots for untreated animals and animals treated with 10 or 20 Gy of X rays alone, with carbogen breathing or with FTuosol-DA and carbogen breathing. Treatment with 10 Gy and air or carbogen breathing for 1 hr prior to and during irradiation had very little effect on the survival distribution of these animals (Fig. 1). However, in the treatment group receiving Fluosol-DA in ad-

Time

(days)

Fig. 1. Kaplan-Meier plot of the survival distribution for rats bearing intracranial 9L tumors. The groups shown are: untreated controls (0); 10 Gy (0); 10 Gy with carbogen breathing for 1 hr prior to and during treatment (m); and 10 Gy preceded by Fluosol-DA with carbogen breathing for 1 hr prior to and during treatment (0).

80 m .;

60

G 8

40

20

20

40

60

80

Time

(days)

100

120

140

Fig. 2. Kaplan-Meier plot of the survival distribution for rats bearing intracranial 9L tumors. The groups shown are: untreated controls (0): 20 Gy (0); 20 Gy with carbogen breathing for 1 hr prior to and during treatment (H); and 20 Gy preceded by Fluosol-DA with carbogen breathing for 1 hr prior to and during treatment (0).

dition to carbogen breathing, there was a subpopulation of about 30% of the animals which showed a survival benefit from the treatment combination. Treatment with 20 Gy, either alone or with carbogen breathing, improved the survival ofthe majority ofthe animals but did not increase the number of long-term survivors. Adding Fluosol-DA to treatment with 20 Gy and carbogen breathing improved the median survival and, again, there appeared to be a subpopulation of about 30% of the animals which showed increased long-term survival. and carIn the group receiving 20 Gy with Fluosol-DA bogen breathing, 2 of 20 animals lived 120 days. Treatment with a single intraperitoneal injection of BCNU ( 10 mg/kg) on day 7 post tumor cell implantation produced an increase in median life span of 2 days compared to untreated control animals (Table 2). Adding carbogen breathing or F~UOSO~-DA and air breathing to treatment with BCNU resulted in increases in median life span of 5 and 13 days compared to untreated control animals, respectively. The combination of drug treatment and Fluosol-DA with carbogen breathing produced an increase in median life span of 28 days compared to untreated control animals, and was significantly different from BCNU treatment alone (p < 0.001). The Kaplan-Meier survival distribution for animals in these treatment groups is shown in Figure 3. Although treatment with a single dose of BCNU, BCNU followed by 1 hr of carbogen breathing or BCNU preceded by administration of Fluosol-DA improved the median survival of the treated animals, there was only slight improvement in long-term survival. When the complete treatment combination of Pluosol-DA followed by BCNU and 1 hr of carbogen breathing was given, there was again improvement in median survival, and in a subpopulation

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Table 2. Survival of Fischer 344 rats implanted intracranially with 9L gliosarcoma and treated with BCNU f Fluosol-DA + carbogen breathing Treatment

Survival,” days

group

% increase in life spanb -

BCNUC BCNU/carbogen” BCNU/Fluosol-DA f BCNU/Fluosol-DA/carbogen

32 34 37 45 60

BCNU in Fluosol-DA BCNU in Fluosol-DA/carbogen

49 66

53 (O/9) 106 (3/9)

117

266 (9/19)

Controls

BCNU/X-rays/Fluosol-DA/ carbogen g

(O/19)

6 (O/9)* 15 (O/9) 41 (O/9) 87 (2/9)

a Median survival in days. b Percent increase in life span compared to untreated control animals. ’ BCNU ( 10 mg/kg) was administered as a single dose i.p. on day 7 after implantation of 4 X 1O49L tumor cells. * Number of 120-day survivors over the total number of animals receiving that treatment. e Carbogen breathing was maintained for 1 hr immediately after drug injection. f Fluosol-DA ( 12 ml/kg) was injected into the tail vein immediately prior to injection of the drug. g A single dose of radiation (20 Gy) was administered to the cranium 1 hr after Fluosol-DA and BCNU administration with 1 hr of carbogen breathing prior to and during radiation treatment.

of about 40% of the animals there was improved longterm survival. When BCNU was prepared in Fluosol-DA and thereby the two agents given as a single injection, BCNU in Fluosol-DA with air breathing resulted in an increase

November 1988, Volume 15, Number 5 in median life span of 17 days compared to untreated control animals (Table 2). When carbogen breathing was added, the increase in median life span was 34 days compared to untreated control animals. Although preparing BCNU in Fluosol-DA resulted in increases in median life span which were greater than Fluosol-DA plus the drug intraperitoneally, these differences were not statistically significant. The Kaplan-Meier survival distribution plot of the data from these treatment groups shows that preparing BCNU in Fluosol-DA increased both the median survival and the long-term survival of the animals (Fig. 4). The addition of 1 hr of carbogen breathing time immediately after administration of the BCNU in Fluosol-DA led to a further increase in both median survival and in the number of long-term survivors. Finally, when BCNU and Fluosol-DA with carbogen breathing were combined with radiation treatment (20 Gy), an increase in median life span of nearly 85 days was produced, with 47% (9 of 19) surviving 120 days. The Kaplan-Meier survival distribution for this treatment group indicates a marked improvement in both median survival time and in the number of long-term survivors (Fig. 4). The combination of 20 Gy with BCNU, Fluosol-DA and carbogen breathing was a significant improvement in treatment outcome when compared to either 20 Gy with Fluosol-DA and carbogen breathing or BCNU with Fluosol-DA and carbogen breathing (p < 0.00 1 and 0.00 1, respectively). DISCUSSION Fluosol-DA and carbogen breathing have been shown to enhance the effects of radiation therapy in both single

80

0. 0 20

40

60 Time

a0

100

120

140

(days)

Fig. 3. Kaplan-Meier plot of the survival distribution for rats bearing intracranial 9L tumors. The groups shown are: untreated controls (0); BCNU (10 mg/kg) (0); BCNU ( 10 mg/kg) followed by 1 hr of carbogen breathing (m); BCNU (10 mg/kg) preceded by Fluosol-DA (0); and BCNU ( 10 mg/kg) preceded by Fluosol-DA and followed by 1 hr of carbogen breathing (A).

20

40

60 Time

60

100

120

1 IO

(days)

Fig. 4. Kaplan-Meier plot of the survival distribution for rats bearing intracranial 9L tumors. The groups shown are: untreated controls (0); BCNU (10 mg/kg) (0); BCNU (10 mg/kg) in Fluosol-DA (m); BCNU (10 mg/kg) in Fluosol-DA followed by 1 hr of carbogen breathing (0); and 20 Gy preceded by BCNU (10 mg/kg) and Fluosol-DA with 1 hr of carbogen breathing prior to and during radiation (A).

Fluosol-DA@ on response of tumors to BCNU 0 B. A. TEICHERet al.

dose and fractionated regimens in several different aniAlthough the 9L gliosarmal tumor systems. ‘5*22*25-27 coma implanted intracranially has a very small radiobiologically hypoxic fraction (O-3%).9,‘3.3o,35 a small but significant effect on median survival was seen upon the addition of Fluosol-DA and 1 hr carbogen breathing prior to and during radiation treatment at day 7 post tumor cell implantation. Martin et al.,” using day 15 9L tumors and only 15 min of carbogen breathing prior to irradiation, did not find a significant improvement in median survival of Fluosol-DA and carbogen treated animals compared to animals treated with radiation alone. This difference may be due to a difference in the level of oxygenation in the more mature tumors or to the difference in carbogen breathing time prior to irradiation. The potential effects of Fluosol-DA on the antitumor activity of the nitrosoureas are complex, involving oxygen delivery, drug delivery (pharmacokinetics), alterations in blood flo~,~ and perhaps activation of an immune component.5 The combination of Fluosol-DA and carbogen breathing with BCNU treatment produced a IO-fold increase in tumor growth delay of the FSaII fibrosarcoma.23,24 Kokinai et al. and Kuwomura et al.,‘,’ using Fluosol-43 (20 mg/kg), a similar perfluorochemical emulsion, treated animals bearing the 9L gliosarcoma with a single dose of 13.3 mg/kg of BCNU and 3 hrs of carbogen breathing. They found a significant increase in mean life span (11 1%) in the animals treated with the triple combination compared with untreated control animals and a 55% increase in life span compared with animals treated with BCNU alone. More recently, Martin et al., ‘* using day 15 9L tumors, a Fluosol-DA dose of 10 ml/kg and 10 mg/kg of BCNU i.p. followed by 3 hrs of carbogen breathing, found a smaller but significant increase in median life span in the animals receiving the complete treatment compared to those receiving the drug and air breathing. In the current study, a comparison was made between administering BCNU (10 mg/kg) and Fluosol-DA (6.0-7.5 ml/kg) as separate injections (Fig. 3) and preparing the BCNU in the Fluosol-DA with subsequent administration as a single injection (Fig. 4). The prior preparation of the drug in the Fluosol-DA led to a better treatment outcome with both air breathing

1191

and carbogen breathing. Most probably this improvement represents an improved bioavailability of the drug when prepared in Fluosol-DA. The combination of a nitrosourea and radiation has been examined previously in several tumor systems. In the KHT sarcoma, the addition of CCNU to radiation led to significantly enhanced tumor cell killing. When evaluated by isoeffect plot analysis, the combination data points fell well below the envelope of additivity, indicating a supra-additive interaction between these two agents.‘9-2’ An even greater interaction was observed in the KHT sarcomas when misonidazole was combined with CCNU prior to irradiation in both single dose and fractionated regimens.“-” In the RIF- 1 tumor, in a variety of drug and radiation schedules, BCNU and radiation produced mainly additive results.” A slight survival prolongation was evident in a large clinical trial of glioblastoma multiformi treated with radiation plus BCNU versus radiation alone. 29 Specifically, in the 9L tumor system, subtoxic doses of BCNU were shown to enhance the killing of tumor cells in culture when administered prior to irradiation. 32 The magnitude of this enhancement was critically dependent upon the time interval between administration of BCNU and radiation. The effect in tissue culture was maximal when BCNU preceded the radiation by 16 hr. A 16 hr interval was also shown to be optimal for treating intracerebral 9L tumors with daily doses of BCNU and radiation in a 2 week fractionation schedule.33,34 In the present study, the time interval between drug injection and radiation delivery was 1 hr. In this single dose protocol, this time interval was chosen to optimize the effect of Fluosol-DA and carbogen breathing both on the drug treatment and on the subsequent X ray treatment. Fluosol-DA and carbogen breathing improved the treatment outcome of the individual therapies (X rays or BCNU). In the animals receiving the complete combined modality treatment, nearly 50% of the animals survived 120 days. The data presented here provide some indication that the addition of Fluosol-DA and carbogen or oxygen breathing to treatment of malignant brain tumors in man with a nitrosourea and radiation may lead to improved treatment outcome.

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