In/ J Radrar~m Ondo~r Bwl Phyv, Vol. Pnnted ,n the 1J.S.A. All nghts reserved.
19, PP. 945-95
I
0360.3016/90 $3.00 + 00 Copyright %I I990 Pergamon Press plc
??Original Contribution
INFLUENCE OF SCHEDULING, DOSE, AND VOLUME OF ADMINISTRATION OF A PERFLUOROCHEMICAL EMULSION ON TUMOR RESPONSE TO RADIATION THERAPY BEVERLY
A. TEICHER, ‘Dana-Farber
PH.D.,‘,’ Cancer
TERENCE
Institute,
S. HERMAN,
M.D.1-2
and ‘Joint Center for Radiation
AND
Therapy,
STEVEN
Boston,
M.
JONES,
B.S.’
MA 02 115
Studies were carried out with a new. concentrated perfluorochemical emulsion (PFCE) of the perfluorochemical F44E (48% V/V). When given at 4, 1.6, or 1 g/kg in undiluted injection volumes iv 1 hr prior to a range of single doses of radiation with inspired carhogen dose modifying factors (DMF’s) based on tumor growth delay (TGD) in the Lewis lung tumor of 2.5, 1.7, and 1.5, respectively, were produced. When the PFC dose was administered in a volume of 0.2 ml, the dose modifying factors produced by 4 g/kg (0.1 ml undiluted) did not change significantly (2.6), but the dose modifying factors produced by 1.6 g/kg (0.04 ml undiluted) and by 1.0 g/kg (0.025 ml undiluted) increased significantly to 2.0 and 1.8 (p < 0.05), respectively. Using the tumor excision assay at 24 hr post treatment in the FSaIIC fibrosarcoma, administration of 6, 4, or 2 g/kg in 0.2 ml injections plus carbogen breathing 1 hr prior to and during treatment resulted in dose modifying factors of 1.5. 1.6, and 1.3, respectively. In a fractionated radiation protocol in the Lewis lung tumor using four daily fractions, a dose of 4 g/kg of PFC on days 1 and 3 proved superior to a dose of 2 g/kg daily (dose modifying factors 2.4 vs. 1.9, p < 0.05). When a fractionated radiation regimen of 3 Gy daily X 5 and carbogen was used, PFC doses of 0.5, 1, 2, and 4 g/kg administered undiluted produced increasing tumor growth delays with increasing dose of PFCE and increasing frequency of administration. In addition, dilutions to 0.2 ml proved significantly more effective. In a 2-week fractionated radiation protocol using 2. 3, or 4 Gy daily X 5 weekly, PFCE given in 0.2 ml volume plus carhogen breathing daily at 4, 1.6, or 1 g/kg produced dose modifying factors of 2.0, 1.9, and 1.6, respectively. Finally, when used in a day 1, 3, and 5 radiation regimen for 3 weeks at 2, 3, or 4 Gy/fraction, 4 g/kg of PFCE given in a volume of 0.2 ml plus carbogen breathing produced a superior dose modifying factor (1.6) as compared with 1.6 or 1 .O g/kg (dose modifying factors 1.4 and 1.3, respectively). These results indicate that PFCE plus carbogen breathing effectively enhances the antitumor effects of both single dose and fractionated radiation. In addition, a significant effect of volume of administration was demonstrated which suggests that some of the efficacy of this perfluorochemical emulsion plus carbogen may be dependent on changes in circulatory dynamics which result in increases in tumor perfusion. Perfluorochemical
emulsion, F44E, Oxygen carrier, Radiosensitizer.
INTRODUCTION
In both normal and tumor tissues. oxygen tension is dependent on supply through the vascular system and removal by metabolism in the tissues. In contrast to normal tissue, the uncontrolled proliferation of malignant cells leads to external pressure on capillaries which in turn causes blood vessels to collapse so that red blood cell passage is restricted (3,4, 10, 1 1, 12,33,40). These constricted vessels contribute to zones of necrosis and areas of hypoxia within tumors. The diameters of tumor vessels are not fixed, however, and irz vitro observations of growing tumors have revealed rapidly opening and closing vessels (7, 41). The therapeutic significance of acute or partial hypoxia as well as chronic hypoxia have recently been discussed ( 1, 2 1).
By radiobiologic methods (20. 31) and by direct pOZ measurements (39,4 1,42) hypoxia has been shown to be present in most animal solid tumor models. Gatenby el al. (5) have measured the oxygen distribution in human squamous cell carcinoma metastases and correlated the oxygen tension in these masses with the response of the tumors to radiation therapy. Twelve of 3 1 tumors in this study had greater than 26% of their volume containing a pOZ less than 8 mm Hg (5). Vaupel et a/. (42) found in human breast cancer xenografts growing in nude rats that with increasing tumor mass, the 02 consumption rate per unit weight significantly decreased, which reflected progressive limitation of the mostly nutritive blood supply. Tissue oxygenation in microareas of human breast cancers xenotransplanted subcutaneously into nude rats was mostly inadequate (5, 6, 42). As a consequence, tissue
Reprint requests to: Beverly A. Teicher, Dana-Farber Cancer Institute. 44 Binney St., Boston, MA 02 115. This work was supported by a grant from the E. I. Du Pont
de Nemours & Co., Chemicals and Pigments Deepwater, N.J. Accepted for publication 26 April 1990. 945
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hypoxia and anoxia were common findings even in very early growth stages. The potential of perfluorochemical emulsions and carbogen breathing to improve the oxygenation of hypoxic regions in the RIF-1 tumor has been investigated using oxygen microelectrodes by Song et ul. (30) and Hasegawa et al. (8). These measurements demonstrated that intratumor ~02 significantly increased when Fluosol-DA (20%) was injected into the animals and the animals breathed carbogen (95% 02/1 5% CO,). Hiraga et ~11. (9) found that cerebral blood flow increased approximately 2-fold following complete blood-PFC exchange and 1.5fold by the partial exchange. A similar 1.5fold increase in flow was also measured in intraparenchymal tumors following partial exchange. Thus. it appears that perfluorochemical emulsions are capable of improving tumor oxygenation both by serving as a carrier for oxygen and by increasing tumor blood flow. The perfluorochemical emulsion Fluosol-DA in combination with breathing a 100% or 95% oxygen atmosphere has been shown to enhance the response of several solid rodent tumors to single dose and fractionated radiation treatment (8, 9, 18. 26. 30. 35, 37, 38, 43). Based on these promising preclinical results, several clinical trials were initiated (14, 27). The fundamental hypothesis is that there should be a gain in the therapeutic ratio if oxygen can be transported effectively to the hypoxic cells in the tumor (2. 7) since most normal tissues are already radiobiologically fully oxygenated. Biocompatible perfluorochemical emulsions with perfluorochemical concentrations up to five times higher than the perfluorochemical content of Fluosol-DA (20%) have recently become available. These new emulsions allow circulating perfluorochemical levels to be varied over a wide range, making it possible to optimize perfluorochemical levels for use with various therapeutic combinations without causing fluid overload of animals. In this report, we have examined several dosage levels and schedules of the concentrated perfluorochemical emulsion* with carbogen breathing and with single dose and fractionated radiation therapy in the Lewis lung carcinoma. Tumor cell survival after single dose radiation therapy was also assessed in the FSalIC fibrosarcoma after administration of several different doses of Therox and carbogen breathing.
METHODS
AND
MATERIALS
Muterials The perfluorochemical (PFC) emulsion containing 48% (v/v) [83% (w/v)] F44E and egg yolk lecithin as the emulsifier in an isotonic buffer was used in these experiments. The particle size of the emulsion is 0.25 pm. The half-life
* Therox, E. I. Du Pont de Nemours Pigments
Dept., Deepwater,
N.J.
& Co., Chemicals
and
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1990. Volume
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X-Ray
4
Dose,
Gray
Fig. I. Growth delay of the Lewis lung carcinoma produced by single dose radiation treatment with various levels of perfluorochemical and carbogen breathing for I hr prior to and during X-ray delivery. The treatment groups were, indicating PFC dose (volume of injection): 4 g/kg (0.2 ml) (0); 4 g/kg (0.1 ml) (m): I .6 g/kg (0.2 ml) (0): 1.6 g/kg (0.04 ml) (0); I g/kg (0.2 ml) (a): I g/kg (0.025 ml) (A); and no PFC ( ‘). Points, mean growth delay, bars, SE.
of this emulsion in circulation is about 2.5 hr and the dwell time of this PFC in tissues is about 7 days (23, 24, 34).
The Lewis lung tumor (29, 3 1, 32) was carried in male C57BL/6J mice.+ For tumor growth delay experiments, 2 X lOh tumor cells prepared from a brei of several stock tumors were implanted i.m. into the gastrocnemius muscles of 8- to lo-week-old males. When the tumors were approximately 100 mm3 in volume (about 1 week after tumor cell implantation), various volumes and schedules of the undiluted or diluted F44E emulsion were administered by tail vein injection. Dilution of the F44E emulsion was made with phosphate buffered saline. The animals were then placed in circulating carbogen (95% O?/ 5% CO?) chambers or were allowed to breathe air. One hour later the animals, while breathing carbogen or air, were treated with a dose of ‘j7Cs y-ray$ to the tumorbearing limb (dose rate. 0.88 Gy/min). Radiation was delivered as a single dose or in multiple fractions. The shielded portion of the animal received less than 2% of the delivered dose. No anesthesia was used during the
* The Jackson Laboratories. Bar Harbor, ME. * Gamma Cell 40. Atomic Energy of Canada, Ltd.
Tumor
response
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to RT 0 B. A. TEICHERa al
radiation treatment. Tumor size was followed by thrice weekly measurements. The experimental end point was the number of days post tumor cell implantation for the tumors to reach a volume of 500 mm3 (28). Untreated tumors reach 500 mm3 in approximately 14 days.
The FSaII fibrosarcoma (22) adapted for growth in culture (FSaIIC) (38) was carried in male C3H/FeJ mice.+ For the experiments. 2 X lo6 tumor cells prepared from a brei of several stock tumors were implanted i.m. into the legs of 8- to lo-week-old male C3H/HeJ mice. When the tumors were approximately 100 mm3 in volume (about 1 week after tumor cell implantation), various doses of the F44E emulsion diluted to 0.2 ml were injected via the tail vein. The animals were then allowed to breathe air or were placed in a circulating atmosphere of 95% O,/ 5% CO1 for 1 hr; then while breathing carbogen or air the animals were treated with a single dose of 13’Cs y-rays of 5, 10, or 15 Gy. No anesthetic was used during radiation treatment. Mice were sacrificed 24 hr after treatment to allow for full expression of cytotoxicity and repair of potentially lethal damage and then immersed in 95% ethanol. The tumors were excised under sterile conditions and sin-
X-Ray
3.75 Dose,
1
5.0 Gray(x4)
gle cell suspensions were prepared for the colony forming assay (36). One week later the plates were stained with crystal violet and colonies of more than 50 cells were counted. The untreated tumor cell suspensions had a plating efficiency of 8- 12%. The results are expressed as the surviving fraction f SE of cells from treated groups compared to untreated controls.
0.1
c
:
lk cn
.-c > ._ >
s
I
I
2.5
Fig. 3. Growth delay of the Lewis lung carcinoma produced by fractionated radiation treatment (daily for 4 days with 2.5. 3.75 or 5.0 Gy/fraction) and either 4 g/kg PFC in 0.2 ml on treatment days 1 and 3, (w); 2 g/kg PFC in 0.2 ml treatment days l-4 (0) or no PFC (0) with carbogen breathing for 1 hr prior to and during X-ray delivery. Points, mean growth delay: bars. SE.
1 .o
E ._
J.
I-
0.01
0
Data analysis
5 X-Ray
10 Dose,
15 Gray
Fig. 2. Survival of FSaIIC cells from FSaIIC tumors treated with various doses of radiation and various doses of pertluorochemical in a volume of 0.2 ml and carbogen breathing for 1 hr prior to and during x-ray delivery. The treatment groups are: 6 g/kg PFC (Cl); 4 g/kg PFC (0); 2 g/kg PFC (m): and no PFC (0). Points. mean of three independent determinations: bars, SE.
5 For the Apple 11 and microcomputer.
Data from the tumor growth delay experiments were analyzed using a computer program written in BASIC.” The program first derives the best-fit curve for each individual set of tumor volume data and then calculates the median, mean, and standard error for each experimental group. The day on which the tumor reached 500 mm3 and the median, mean, and standard error are then derived. A second program provides statistical comparisons between any number of groups using Student’s t test and deriving degrees of freedom and p values. Each experimental group had seven mice. and each experiment was repeated at least once: therefore, the minimum number of tumors examined at each point was 14 (38).
1. J. Radiation Oncology 0 Biology 0 Physics
948
Dose modifying suming zero growth factor relating the same growth delay group (37, 38).
October
RESULTS The PFC emulsion administered at any of the doses examined in these experiments had no effect on the tumor growth or growth delay produced by radiation therapy when the animals breathed air. Single dose radiation was examined with three doses of PFC administered iv either as the undiluted emulsion or adjusted to a volume of 0.3 ml. Carbogen breathing was maintained for 1 hr prior to and during radiation delivery (Fig. 1). The greatest enhancement in tumor growth delay was observed at a PFC dose of 4 g/kg. When this dose was administered in 0.3 ml the DMF was 2.6 f 0.2 and when administered in 0.1 ml (undiluted), the DMF was 2.5 & 0.2. At the lower PFC doses of 1.6 g/kg and 1 g/kg, the volume of administration had a significant effect (p 4 0.05). When administered in 0.2 ml, the DMF’s were 2.0 & 0.1 and I .8 + 0.1 for PFC doses of 1.6 and 1 g/kg. respectively: for the undiluted emulsion the DMF’s for the same doses of PFC were 1.7 + 0. I (0.04 ml) and 1.5 f 0.1 (0.025 ml), respectively. Tumor cell survival was examined in the FSaIIC fibrosarcoma following single dose radiation with various doses of PFC administered in a volume of 0.2 ml with carbogen breathing prior to and during radiation delivery (Fig. 2). As seen above, a maximal degree of dose modification was obtained with a PFC dose of 4 g/kg (DMF = 1.6 ? 0.1). Although at the higher dose of 6 g/kg the DMF
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was essentially the same (1.5 + 0. 1), at the lower dose of 2 g/kg the enhancement of tumor cell killing was significantly decreased (DMF = 1.3 * 0.1, p < 0.05). Several fractionated radiation schemes with various schedules of PFC were studied. In Figure 3, 4-day fractionated x-ray protocols (2.5. 3.75, or 5.0 Gy/fraction) were carried out with PFC administered either on days 1 and 3 at a dose of 4 g/kg or daily at a dose of 2 g/kg, so that the total dose of PFC was the same on both schedules. Carbogen breathing was daily for 1 hr prior to and during X ray delivery in both groups. The alternate day PFC schedule at 4 g/kg proved better (DMF = 2.4 k 0.1 based on slopes of tumor growth daily curves) than daily PFC administration at 2 g/kg (DMF = 1.9 & 0.1). The effect of a wider PFC dosage range with a 3 Gy X 5 daily radiation therapy protocol and carbogen breathing daily is shown in Figure 4. The PFC administered on treatment day 1 only either as the undiluted emulsion or in a volume of 0.2 ml had no effect on the tumor growth delay produced by the radiation and carbogen breathing. Administration of the PFC on treatment days 1. 3. and 5 significantly enhanced tumor growth delay at PFC doses of 2 and 4 g/kg. The greatest enhancement in tumor growth delay was obtained with the PFC administered daily, and this enhancement was significantly better (p < 0.01) than radiation and carbogen breathing over the entire PFC dosage range tested from 0.5 to 4 g/ kg. Volume of administration became a significant variable with daily PFC treatment and injecting the PFC in a volume of 0.3 ml provided a greater tumor growth delay than injecting the undiluted emulsion. Two multiple-week fractionated radiation treatment regimens were also tested. In Figure 5. radiation was de-
factors (DMF’s) were calculated asdelay with 0 Gy. then calculating the control dose to the dose causing the (8 days) achieved in the experimental
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1990. Volume
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Fig. 4. Growth delay of the Lewis lung carcinoma produced by fractionated radiation treatment (daily for 5 days with 3 Gy/fraction) and various doses of PFC delivered in a constant volume of 0.2 ml (solid symbols) or in a variable volume of the undiluted emulsion (open symbols) with carbogen breathing for I hr prior to and during X-ray delivery. PFC was administered i.v. on three different schedules: treatment Day 1 only (0. 0); treatment Days 1. 3. and 5 (A. A): and treatment Days l-5 (H. 0). Points, mean growth delay; bars, SE.
949
Tumor response to RT 0 B. A. TEICHER n al
livered at 2, 3, or 4 Gy daily (5 days per week) for 2 weeks with a PFC volume of 0.2 ml and carbogen breathing administered for 1 hr prior to and during each radiation fraction. The dose modifying factors obtained were 2.0 + 0.2, 1.9 & 0.1, and 1.6 + 0.1 with doses of 4, 1.6, and 1 g/kg, respectively, of PFC based on the slopes of the tumor growth delay curves. The tumor growth delay produced by an alternate radiation fractionated regimen with radiation administered three times per week for 3 weeks. PFC administered in a volume of 0.2 ml, and carbogen breathing for 1 hr prior to and during each fraction is shown in Figure 6. The dose modifying factors obtained with this treatment scheme were 1.6 & 0.1, 1.4 * 0.1. and 1.3 + 0.1 with PFC doses of 4, 1.6, and 1 g/kg, respectively. On this three times per week radiation schedule, 4 g/kg of PFC was significantly more effective than 1.6 g/kg or
: E
4
5 0
1 g/k.
X-Ray
Over the past 5 years, studies from this laboratory (35, 37, 38) and others (8, 13, 15-17, 19, 25, 30) have established that administration of a biologically compatible perfluorochemical emulsion with carbogen or oxygen breathing can enhance the oxygenation and radiosensi-
28
24
,
I
I
2
3
4
Dose,
I
I
3
4
Dose,
i
Gray(x9)
Fig. 6. Growth delay of the Lewis lung carcinoma produced by fractionated radiation treatment (3 times per week (MWF) for
DISCUSSION
X-Ray
I
2
Gray(xl0)
Fig. 5. Growth delay of the Lewis lung carcinoma produced by fractionated radiation treatment (daily for 5 days (M + F) for 2 weeks with 2. 3. or 4 Gy/fraction) and various levels of PFC in a volume of 0.2 ml with carbogen breathing for 1 hr prior to and during X-ray delivery. PFC was administered i.v. with each
treatment at a dose of 4 g/kg (B): 1.6 g/kg (0); 1 g/kg (0) or no PFC (0). Points, mean growth delay: bars, SE.
3 weeks with 2. 3, or 4 Gy/fraction)
and various levels of PFC
in a constant volume of 0.2 ml with carbogen breathing for I hr prior to and with each treatment at a dose of 4 g/kg (0); 1.6 g/kg (m); 1 g/kg (0): or no PFC (0). Points. mean growth delay:
bars. SE.
tization of rodent solid tumors and can increase the antineoplastic effects of radiation in rodent solid tumors. The current studies were designed to define the lowest effective doses of the more concentrated PFCE. and to evaluate its efficacy when used in extended fractionation protocols. We have previously observed (34) that PFC doses of 6 g/kg and higher can result in decreased enhancement of radiation-induced tumor growth delay in murine tumors. This finding was confirmed in the present studies. The PFC dosage range between 1.6 and 4 g/kg of the emulsion appeared to be optimal with single dose radiation and is similar to the 1.6-2.4 g/kg dosage range which is optimal for Fluosol-DA, 20% in the same setting (37). As with Fluosol-DA, the PFCE emulsion plus carbogen breathing was dose modifying with a range of radiation doses in the FSaIIC fibrosarcoma. In the FSaIIC tumor cell survival assay, 4 g/kg with the emulsion plus carbogen breathing produced a greater increase in tumor cell killing (DMF 1.6) than 2.4 g/kg with Fluosol-DA (DMF 1.4; 38). however, 2 g/kg with the emulsion (DMF 1.3) was considerably less effective than 2.4 g/kg with Fluosol-DA. The higher dose requirement for the emulsion may be partially due to the shorter circulating half-life of this emulsion (2-2.5 hr) compared to that of Fluosol-DA (12 hr) since radiation treatment followed administration of PFCE’s by 1 hr. The emulsion was also most effective when administered at 4 g/kg with fractionated radiation, but the difference between the DMF produced by 4 g/kg as opposed to lower doses diminished with increasing length of the radiation protocols. Thus, over 4 days, 4 g/kg every other
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was superior to 2 g/kg daily (DMF 2.4 versus 1.9), but when used with daily X 5 radiation weekly for 2 weeks, 1.6 g/kg proved as good as 4 g/kg (DMF 1.9 & 0.1 vs. 2.0 & 0.2, respectively). Since the PFCE’s are cleared from circulation by the reticuloendothelial system, repeated dosing may saturate the removal mechanism, thus increasing the circulating half life of the emulsion. Frequent dosing over prolonged periods. like the 4-6 weeks typical of therapeutic radiation courses in human patients therefore, may lead to a decrease in the optimal, fractionated emulsion dose. In all cases tested, the enhancement in tumor growth delay achieved was greater if the PFC was administered in a volume of 0.2 ml than as the undiluted 48% emulsion. The effect of volume of administration became statistically significant at the lower PFC doses (Fig. 1). Since the ability of the PFC to carry oxygen should be relatively unrelated to the volume of administration. the effect of volume may reflect alterations in circulatory dynamics which, in turn, cause perfusion changes in the tumors being treated. Thus. the fluid bolus and osmotic load produced by 4 g/kg in 0.2 ml given rapidly iv into the approximately 3 ml circulatory volume of the mouse may be nearly optimal for increasing tumor perfusion. whereas 6 g/kg undiluted (0.15 ml) may represent too great an osmotic load and 4 g/kg in 0.1 ml too small a fluid bolus. Furthermore, because the circulatory change produced by the administered PFCE’s appear to affect therapeutic efficacy importantly when used with radiation and oxygen breathing, this factor day
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may well represent a less than optimum variable in the on-going human trials. Fluosol-DA, given in a dose of 10 ml/kg to an average 70 kg adult with an approximate circulatory volume of 5 liters over 90 min, is delivered at the rate of 0.0016 ml/ ml of blood volume/mitt. In contrast, in the mouse, 2.4 g/kg of Fluosol-DA in 0.3 ml (which we have found to be optimal for this PFCE (37) given over about 20 set into the approximately 3 ml blood volume of the mouse represents a rate of infusion of 0.3 ml/ml of blood volume/ min, or approximately 18%times more rapid rate of administration than in the human patients. We have begun studies which specifically address the importance of infusion rate. and early results appear to confirm the importance of this parameter with both Fluosol-DA and Therox. The most effective clinical use of PFC emulsions/carbogen breathing may well depend on the characteristics of the emulsion including PFC concentration, half-life in circulation. and frequency of administration as well as infusion rate. It is likely that in human beings there is an optimal level of the oxygen carrying PFCE for radiation therapy and that ideally the dose and infusion rate of PFCE used clinically should be adjusted in specific patients to produce an optimal hematocrit/fluorocrit as well as an optimal increase in tumor perfusion and ultimately oxygenation. Use of invasive PO: probes or magnetic resonance spectroscopy to measure tumor oxygenation may be required to optimize PFCE administration.
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29. Shipley, W. V.: Stanley, J. A.; Steel, G. G. Tumor size dependence in the radiation response of the Lewis lung carcinoma. Cancer Res. 35:2488-2493: 1975. 30. Song, C. W.; Lee. I.: Hasegawa. T.: Rhee, J. G.; Lewitt, S. H. Increase in p02 and radiosensitivity of tumors by Fluosol-DA (20%) and carbogen. Cancer Res. 47:442-446; 1987. 31. Stanley, J. A.: Shilpey. W. V.; Steel, G. G. Influence of tumor size on hypoxic fraction and therapeutic sensitivity of Lewis lung tumor. Br. J. Cancer. 36:105-l 13; 1977. 32. Steel. G. G.; Nill, R. P.: Peckham, M. J. Combined radiotherapy-chemotherapy of Lewis lung carcinoma. Int. J. Radiat. Oncol. Biol. Phys. 4:49-52: 1978. 33. Tannock. I. F.: Steel, G. G. Quantitative technique for the study of the anatomy and function of small blood vessels in tumors. J. Natl. Cancer Inst. 42:77 l-782: 1969. 34. Teicher, 9. A.: Herman, T. S.: Jones, S. M. Optimization of perfluorochemical levels with radiation therapy. Cancer Res. 49:2693-2697: 1989. 35. Teicher. 9. A.: Holden. S. A.: Jacobs, J. L. Approaches to defining the mechanism of Fluosol-DA 20% with carbogen enhancement of melphalan antitumor activity. Cancer Res. 47:5 13-528: 1987. 36. Teicher, 9. A.: Rose. C. M. Effects of dose and scheduling on growth delay of the Lewis lung carcinoma produced by the perfluorochemical emulsion, Fluosol-DA. Int. J. Radiat. Oncol. Biol. Phys. I?: 13 1I - 13 13: 1986. 37. Teicher. 9. A.: Rose, C. M. Oxygen-carrying chemical emulsion as an adjuvant to radiation mice. Cancer Res. 44:4285-4288; 1984.
perfluorotherapy in
emulsions 38. Teicher. 9. A.: Rose, C. M. Perfluorochemical can increase tumor radiosensitivity. Science (Wash.. DC) 2’3:934-936: 1984. in tumor tissue of 39. Vaupel. P.; Thews. G. PO? distribution DS-carcinosarcoma. Oncology 30:475-484: 1974. 40. Vaupel, P. Hypoxia 13:399-408: 1977.
in neoplastic
tissue.
Microvasc.
Res.
41. Vaupel, P.: Fortmeyer, H. P.; Runkel. S.: Kallinowski, F. Blood flow, oxygen consumption and tissue oxygenation of human breast cancer xenografts in nude rats. Cancer Res. 47:3496-3503; 1987. 42. Vaupel, P.: Frinak. S.: Bicher. H. I. Heterogeneous oxygen partial pressure and pH distribution in C3H mouse mammary adenocarcinoma. Cancer Res. 4 I :2008-20 13: 198 1. 43. Zhang, W. L.; Pence, D.: Patten, M.: Levitt. S. H.: Song, C. W. Enhancement of tumor response to radiation by FIuosol-DA. Int. J. Radiat. Oncol. Biol. Phys. 10: 172- 175; 1984.
19, PP. 945-95
I
0360.3016/90 $3.00 + 00 Copyright %I I990 Pergamon Press plc
??Original Contribution
INFLUENCE OF SCHEDULING, DOSE, AND VOLUME OF ADMINISTRATION OF A PERFLUOROCHEMICAL EMULSION ON TUMOR RESPONSE TO RADIATION THERAPY BEVERLY
A. TEICHER, ‘Dana-Farber
PH.D.,‘,’ Cancer
TERENCE
Institute,
S. HERMAN,
M.D.1-2
and ‘Joint Center for Radiation
AND
Therapy,
STEVEN
Boston,
M.
JONES,
B.S.’
MA 02 115
Studies were carried out with a new. concentrated perfluorochemical emulsion (PFCE) of the perfluorochemical F44E (48% V/V). When given at 4, 1.6, or 1 g/kg in undiluted injection volumes iv 1 hr prior to a range of single doses of radiation with inspired carhogen dose modifying factors (DMF’s) based on tumor growth delay (TGD) in the Lewis lung tumor of 2.5, 1.7, and 1.5, respectively, were produced. When the PFC dose was administered in a volume of 0.2 ml, the dose modifying factors produced by 4 g/kg (0.1 ml undiluted) did not change significantly (2.6), but the dose modifying factors produced by 1.6 g/kg (0.04 ml undiluted) and by 1.0 g/kg (0.025 ml undiluted) increased significantly to 2.0 and 1.8 (p < 0.05), respectively. Using the tumor excision assay at 24 hr post treatment in the FSaIIC fibrosarcoma, administration of 6, 4, or 2 g/kg in 0.2 ml injections plus carbogen breathing 1 hr prior to and during treatment resulted in dose modifying factors of 1.5. 1.6, and 1.3, respectively. In a fractionated radiation protocol in the Lewis lung tumor using four daily fractions, a dose of 4 g/kg of PFC on days 1 and 3 proved superior to a dose of 2 g/kg daily (dose modifying factors 2.4 vs. 1.9, p < 0.05). When a fractionated radiation regimen of 3 Gy daily X 5 and carbogen was used, PFC doses of 0.5, 1, 2, and 4 g/kg administered undiluted produced increasing tumor growth delays with increasing dose of PFCE and increasing frequency of administration. In addition, dilutions to 0.2 ml proved significantly more effective. In a 2-week fractionated radiation protocol using 2. 3, or 4 Gy daily X 5 weekly, PFCE given in 0.2 ml volume plus carhogen breathing daily at 4, 1.6, or 1 g/kg produced dose modifying factors of 2.0, 1.9, and 1.6, respectively. Finally, when used in a day 1, 3, and 5 radiation regimen for 3 weeks at 2, 3, or 4 Gy/fraction, 4 g/kg of PFCE given in a volume of 0.2 ml plus carbogen breathing produced a superior dose modifying factor (1.6) as compared with 1.6 or 1 .O g/kg (dose modifying factors 1.4 and 1.3, respectively). These results indicate that PFCE plus carbogen breathing effectively enhances the antitumor effects of both single dose and fractionated radiation. In addition, a significant effect of volume of administration was demonstrated which suggests that some of the efficacy of this perfluorochemical emulsion plus carbogen may be dependent on changes in circulatory dynamics which result in increases in tumor perfusion. Perfluorochemical
emulsion, F44E, Oxygen carrier, Radiosensitizer.
INTRODUCTION
In both normal and tumor tissues. oxygen tension is dependent on supply through the vascular system and removal by metabolism in the tissues. In contrast to normal tissue, the uncontrolled proliferation of malignant cells leads to external pressure on capillaries which in turn causes blood vessels to collapse so that red blood cell passage is restricted (3,4, 10, 1 1, 12,33,40). These constricted vessels contribute to zones of necrosis and areas of hypoxia within tumors. The diameters of tumor vessels are not fixed, however, and irz vitro observations of growing tumors have revealed rapidly opening and closing vessels (7, 41). The therapeutic significance of acute or partial hypoxia as well as chronic hypoxia have recently been discussed ( 1, 2 1).
By radiobiologic methods (20. 31) and by direct pOZ measurements (39,4 1,42) hypoxia has been shown to be present in most animal solid tumor models. Gatenby el al. (5) have measured the oxygen distribution in human squamous cell carcinoma metastases and correlated the oxygen tension in these masses with the response of the tumors to radiation therapy. Twelve of 3 1 tumors in this study had greater than 26% of their volume containing a pOZ less than 8 mm Hg (5). Vaupel et a/. (42) found in human breast cancer xenografts growing in nude rats that with increasing tumor mass, the 02 consumption rate per unit weight significantly decreased, which reflected progressive limitation of the mostly nutritive blood supply. Tissue oxygenation in microareas of human breast cancers xenotransplanted subcutaneously into nude rats was mostly inadequate (5, 6, 42). As a consequence, tissue
Reprint requests to: Beverly A. Teicher, Dana-Farber Cancer Institute. 44 Binney St., Boston, MA 02 115. This work was supported by a grant from the E. I. Du Pont
de Nemours & Co., Chemicals and Pigments Deepwater, N.J. Accepted for publication 26 April 1990. 945
Department,
946
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Oncology
0 Biology 0 Physics
hypoxia and anoxia were common findings even in very early growth stages. The potential of perfluorochemical emulsions and carbogen breathing to improve the oxygenation of hypoxic regions in the RIF-1 tumor has been investigated using oxygen microelectrodes by Song et ul. (30) and Hasegawa et al. (8). These measurements demonstrated that intratumor ~02 significantly increased when Fluosol-DA (20%) was injected into the animals and the animals breathed carbogen (95% 02/1 5% CO,). Hiraga et ~11. (9) found that cerebral blood flow increased approximately 2-fold following complete blood-PFC exchange and 1.5fold by the partial exchange. A similar 1.5fold increase in flow was also measured in intraparenchymal tumors following partial exchange. Thus. it appears that perfluorochemical emulsions are capable of improving tumor oxygenation both by serving as a carrier for oxygen and by increasing tumor blood flow. The perfluorochemical emulsion Fluosol-DA in combination with breathing a 100% or 95% oxygen atmosphere has been shown to enhance the response of several solid rodent tumors to single dose and fractionated radiation treatment (8, 9, 18. 26. 30. 35, 37, 38, 43). Based on these promising preclinical results, several clinical trials were initiated (14, 27). The fundamental hypothesis is that there should be a gain in the therapeutic ratio if oxygen can be transported effectively to the hypoxic cells in the tumor (2. 7) since most normal tissues are already radiobiologically fully oxygenated. Biocompatible perfluorochemical emulsions with perfluorochemical concentrations up to five times higher than the perfluorochemical content of Fluosol-DA (20%) have recently become available. These new emulsions allow circulating perfluorochemical levels to be varied over a wide range, making it possible to optimize perfluorochemical levels for use with various therapeutic combinations without causing fluid overload of animals. In this report, we have examined several dosage levels and schedules of the concentrated perfluorochemical emulsion* with carbogen breathing and with single dose and fractionated radiation therapy in the Lewis lung carcinoma. Tumor cell survival after single dose radiation therapy was also assessed in the FSalIC fibrosarcoma after administration of several different doses of Therox and carbogen breathing.
METHODS
AND
MATERIALS
Muterials The perfluorochemical (PFC) emulsion containing 48% (v/v) [83% (w/v)] F44E and egg yolk lecithin as the emulsifier in an isotonic buffer was used in these experiments. The particle size of the emulsion is 0.25 pm. The half-life
* Therox, E. I. Du Pont de Nemours Pigments
Dept., Deepwater,
N.J.
& Co., Chemicals
and
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1990. Volume
19. Number
X-Ray
4
Dose,
Gray
Fig. I. Growth delay of the Lewis lung carcinoma produced by single dose radiation treatment with various levels of perfluorochemical and carbogen breathing for I hr prior to and during X-ray delivery. The treatment groups were, indicating PFC dose (volume of injection): 4 g/kg (0.2 ml) (0); 4 g/kg (0.1 ml) (m): I .6 g/kg (0.2 ml) (0): 1.6 g/kg (0.04 ml) (0); I g/kg (0.2 ml) (a): I g/kg (0.025 ml) (A); and no PFC ( ‘). Points, mean growth delay, bars, SE.
of this emulsion in circulation is about 2.5 hr and the dwell time of this PFC in tissues is about 7 days (23, 24, 34).
The Lewis lung tumor (29, 3 1, 32) was carried in male C57BL/6J mice.+ For tumor growth delay experiments, 2 X lOh tumor cells prepared from a brei of several stock tumors were implanted i.m. into the gastrocnemius muscles of 8- to lo-week-old males. When the tumors were approximately 100 mm3 in volume (about 1 week after tumor cell implantation), various volumes and schedules of the undiluted or diluted F44E emulsion were administered by tail vein injection. Dilution of the F44E emulsion was made with phosphate buffered saline. The animals were then placed in circulating carbogen (95% O?/ 5% CO?) chambers or were allowed to breathe air. One hour later the animals, while breathing carbogen or air, were treated with a dose of ‘j7Cs y-ray$ to the tumorbearing limb (dose rate. 0.88 Gy/min). Radiation was delivered as a single dose or in multiple fractions. The shielded portion of the animal received less than 2% of the delivered dose. No anesthesia was used during the
* The Jackson Laboratories. Bar Harbor, ME. * Gamma Cell 40. Atomic Energy of Canada, Ltd.
Tumor
response
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to RT 0 B. A. TEICHERa al
radiation treatment. Tumor size was followed by thrice weekly measurements. The experimental end point was the number of days post tumor cell implantation for the tumors to reach a volume of 500 mm3 (28). Untreated tumors reach 500 mm3 in approximately 14 days.
The FSaII fibrosarcoma (22) adapted for growth in culture (FSaIIC) (38) was carried in male C3H/FeJ mice.+ For the experiments. 2 X lo6 tumor cells prepared from a brei of several stock tumors were implanted i.m. into the legs of 8- to lo-week-old male C3H/HeJ mice. When the tumors were approximately 100 mm3 in volume (about 1 week after tumor cell implantation), various doses of the F44E emulsion diluted to 0.2 ml were injected via the tail vein. The animals were then allowed to breathe air or were placed in a circulating atmosphere of 95% O,/ 5% CO1 for 1 hr; then while breathing carbogen or air the animals were treated with a single dose of 13’Cs y-rays of 5, 10, or 15 Gy. No anesthetic was used during radiation treatment. Mice were sacrificed 24 hr after treatment to allow for full expression of cytotoxicity and repair of potentially lethal damage and then immersed in 95% ethanol. The tumors were excised under sterile conditions and sin-
X-Ray
3.75 Dose,
1
5.0 Gray(x4)
gle cell suspensions were prepared for the colony forming assay (36). One week later the plates were stained with crystal violet and colonies of more than 50 cells were counted. The untreated tumor cell suspensions had a plating efficiency of 8- 12%. The results are expressed as the surviving fraction f SE of cells from treated groups compared to untreated controls.
0.1
c
:
lk cn
.-c > ._ >
s
I
I
2.5
Fig. 3. Growth delay of the Lewis lung carcinoma produced by fractionated radiation treatment (daily for 4 days with 2.5. 3.75 or 5.0 Gy/fraction) and either 4 g/kg PFC in 0.2 ml on treatment days 1 and 3, (w); 2 g/kg PFC in 0.2 ml treatment days l-4 (0) or no PFC (0) with carbogen breathing for 1 hr prior to and during X-ray delivery. Points, mean growth delay: bars. SE.
1 .o
E ._
J.
I-
0.01
0
Data analysis
5 X-Ray
10 Dose,
15 Gray
Fig. 2. Survival of FSaIIC cells from FSaIIC tumors treated with various doses of radiation and various doses of pertluorochemical in a volume of 0.2 ml and carbogen breathing for 1 hr prior to and during x-ray delivery. The treatment groups are: 6 g/kg PFC (Cl); 4 g/kg PFC (0); 2 g/kg PFC (m): and no PFC (0). Points. mean of three independent determinations: bars, SE.
5 For the Apple 11 and microcomputer.
Data from the tumor growth delay experiments were analyzed using a computer program written in BASIC.” The program first derives the best-fit curve for each individual set of tumor volume data and then calculates the median, mean, and standard error for each experimental group. The day on which the tumor reached 500 mm3 and the median, mean, and standard error are then derived. A second program provides statistical comparisons between any number of groups using Student’s t test and deriving degrees of freedom and p values. Each experimental group had seven mice. and each experiment was repeated at least once: therefore, the minimum number of tumors examined at each point was 14 (38).
1. J. Radiation Oncology 0 Biology 0 Physics
948
Dose modifying suming zero growth factor relating the same growth delay group (37, 38).
October
RESULTS The PFC emulsion administered at any of the doses examined in these experiments had no effect on the tumor growth or growth delay produced by radiation therapy when the animals breathed air. Single dose radiation was examined with three doses of PFC administered iv either as the undiluted emulsion or adjusted to a volume of 0.3 ml. Carbogen breathing was maintained for 1 hr prior to and during radiation delivery (Fig. 1). The greatest enhancement in tumor growth delay was observed at a PFC dose of 4 g/kg. When this dose was administered in 0.3 ml the DMF was 2.6 f 0.2 and when administered in 0.1 ml (undiluted), the DMF was 2.5 & 0.2. At the lower PFC doses of 1.6 g/kg and 1 g/kg, the volume of administration had a significant effect (p 4 0.05). When administered in 0.2 ml, the DMF’s were 2.0 & 0.1 and I .8 + 0.1 for PFC doses of 1.6 and 1 g/kg. respectively: for the undiluted emulsion the DMF’s for the same doses of PFC were 1.7 + 0. I (0.04 ml) and 1.5 f 0.1 (0.025 ml), respectively. Tumor cell survival was examined in the FSaIIC fibrosarcoma following single dose radiation with various doses of PFC administered in a volume of 0.2 ml with carbogen breathing prior to and during radiation delivery (Fig. 2). As seen above, a maximal degree of dose modification was obtained with a PFC dose of 4 g/kg (DMF = 1.6 ? 0.1). Although at the higher dose of 6 g/kg the DMF
I
0.5
19. Number
4
was essentially the same (1.5 + 0. 1), at the lower dose of 2 g/kg the enhancement of tumor cell killing was significantly decreased (DMF = 1.3 * 0.1, p < 0.05). Several fractionated radiation schemes with various schedules of PFC were studied. In Figure 3, 4-day fractionated x-ray protocols (2.5. 3.75, or 5.0 Gy/fraction) were carried out with PFC administered either on days 1 and 3 at a dose of 4 g/kg or daily at a dose of 2 g/kg, so that the total dose of PFC was the same on both schedules. Carbogen breathing was daily for 1 hr prior to and during X ray delivery in both groups. The alternate day PFC schedule at 4 g/kg proved better (DMF = 2.4 k 0.1 based on slopes of tumor growth daily curves) than daily PFC administration at 2 g/kg (DMF = 1.9 & 0.1). The effect of a wider PFC dosage range with a 3 Gy X 5 daily radiation therapy protocol and carbogen breathing daily is shown in Figure 4. The PFC administered on treatment day 1 only either as the undiluted emulsion or in a volume of 0.2 ml had no effect on the tumor growth delay produced by the radiation and carbogen breathing. Administration of the PFC on treatment days 1. 3. and 5 significantly enhanced tumor growth delay at PFC doses of 2 and 4 g/kg. The greatest enhancement in tumor growth delay was obtained with the PFC administered daily, and this enhancement was significantly better (p < 0.01) than radiation and carbogen breathing over the entire PFC dosage range tested from 0.5 to 4 g/ kg. Volume of administration became a significant variable with daily PFC treatment and injecting the PFC in a volume of 0.3 ml provided a greater tumor growth delay than injecting the undiluted emulsion. Two multiple-week fractionated radiation treatment regimens were also tested. In Figure 5. radiation was de-
factors (DMF’s) were calculated asdelay with 0 Gy. then calculating the control dose to the dose causing the (8 days) achieved in the experimental
Ol
1990. Volume
I
1
I
1
2
4
PFC
Dose,g/kg
Fig. 4. Growth delay of the Lewis lung carcinoma produced by fractionated radiation treatment (daily for 5 days with 3 Gy/fraction) and various doses of PFC delivered in a constant volume of 0.2 ml (solid symbols) or in a variable volume of the undiluted emulsion (open symbols) with carbogen breathing for I hr prior to and during X-ray delivery. PFC was administered i.v. on three different schedules: treatment Day 1 only (0. 0); treatment Days 1. 3. and 5 (A. A): and treatment Days l-5 (H. 0). Points, mean growth delay; bars, SE.
949
Tumor response to RT 0 B. A. TEICHER n al
livered at 2, 3, or 4 Gy daily (5 days per week) for 2 weeks with a PFC volume of 0.2 ml and carbogen breathing administered for 1 hr prior to and during each radiation fraction. The dose modifying factors obtained were 2.0 + 0.2, 1.9 & 0.1, and 1.6 + 0.1 with doses of 4, 1.6, and 1 g/kg, respectively, of PFC based on the slopes of the tumor growth delay curves. The tumor growth delay produced by an alternate radiation fractionated regimen with radiation administered three times per week for 3 weeks. PFC administered in a volume of 0.2 ml, and carbogen breathing for 1 hr prior to and during each fraction is shown in Figure 6. The dose modifying factors obtained with this treatment scheme were 1.6 & 0.1, 1.4 * 0.1. and 1.3 + 0.1 with PFC doses of 4, 1.6, and 1 g/kg, respectively. On this three times per week radiation schedule, 4 g/kg of PFC was significantly more effective than 1.6 g/kg or
: E
4
5 0
1 g/k.
X-Ray
Over the past 5 years, studies from this laboratory (35, 37, 38) and others (8, 13, 15-17, 19, 25, 30) have established that administration of a biologically compatible perfluorochemical emulsion with carbogen or oxygen breathing can enhance the oxygenation and radiosensi-
28
24
,
I
I
2
3
4
Dose,
I
I
3
4
Dose,
i
Gray(x9)
Fig. 6. Growth delay of the Lewis lung carcinoma produced by fractionated radiation treatment (3 times per week (MWF) for
DISCUSSION
X-Ray
I
2
Gray(xl0)
Fig. 5. Growth delay of the Lewis lung carcinoma produced by fractionated radiation treatment (daily for 5 days (M + F) for 2 weeks with 2. 3. or 4 Gy/fraction) and various levels of PFC in a volume of 0.2 ml with carbogen breathing for 1 hr prior to and during X-ray delivery. PFC was administered i.v. with each
treatment at a dose of 4 g/kg (B): 1.6 g/kg (0); 1 g/kg (0) or no PFC (0). Points, mean growth delay: bars, SE.
3 weeks with 2. 3, or 4 Gy/fraction)
and various levels of PFC
in a constant volume of 0.2 ml with carbogen breathing for I hr prior to and with each treatment at a dose of 4 g/kg (0); 1.6 g/kg (m); 1 g/kg (0): or no PFC (0). Points. mean growth delay:
bars. SE.
tization of rodent solid tumors and can increase the antineoplastic effects of radiation in rodent solid tumors. The current studies were designed to define the lowest effective doses of the more concentrated PFCE. and to evaluate its efficacy when used in extended fractionation protocols. We have previously observed (34) that PFC doses of 6 g/kg and higher can result in decreased enhancement of radiation-induced tumor growth delay in murine tumors. This finding was confirmed in the present studies. The PFC dosage range between 1.6 and 4 g/kg of the emulsion appeared to be optimal with single dose radiation and is similar to the 1.6-2.4 g/kg dosage range which is optimal for Fluosol-DA, 20% in the same setting (37). As with Fluosol-DA, the PFCE emulsion plus carbogen breathing was dose modifying with a range of radiation doses in the FSaIIC fibrosarcoma. In the FSaIIC tumor cell survival assay, 4 g/kg with the emulsion plus carbogen breathing produced a greater increase in tumor cell killing (DMF 1.6) than 2.4 g/kg with Fluosol-DA (DMF 1.4; 38). however, 2 g/kg with the emulsion (DMF 1.3) was considerably less effective than 2.4 g/kg with Fluosol-DA. The higher dose requirement for the emulsion may be partially due to the shorter circulating half-life of this emulsion (2-2.5 hr) compared to that of Fluosol-DA (12 hr) since radiation treatment followed administration of PFCE’s by 1 hr. The emulsion was also most effective when administered at 4 g/kg with fractionated radiation, but the difference between the DMF produced by 4 g/kg as opposed to lower doses diminished with increasing length of the radiation protocols. Thus, over 4 days, 4 g/kg every other
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was superior to 2 g/kg daily (DMF 2.4 versus 1.9), but when used with daily X 5 radiation weekly for 2 weeks, 1.6 g/kg proved as good as 4 g/kg (DMF 1.9 & 0.1 vs. 2.0 & 0.2, respectively). Since the PFCE’s are cleared from circulation by the reticuloendothelial system, repeated dosing may saturate the removal mechanism, thus increasing the circulating half life of the emulsion. Frequent dosing over prolonged periods. like the 4-6 weeks typical of therapeutic radiation courses in human patients therefore, may lead to a decrease in the optimal, fractionated emulsion dose. In all cases tested, the enhancement in tumor growth delay achieved was greater if the PFC was administered in a volume of 0.2 ml than as the undiluted 48% emulsion. The effect of volume of administration became statistically significant at the lower PFC doses (Fig. 1). Since the ability of the PFC to carry oxygen should be relatively unrelated to the volume of administration. the effect of volume may reflect alterations in circulatory dynamics which, in turn, cause perfusion changes in the tumors being treated. Thus. the fluid bolus and osmotic load produced by 4 g/kg in 0.2 ml given rapidly iv into the approximately 3 ml circulatory volume of the mouse may be nearly optimal for increasing tumor perfusion. whereas 6 g/kg undiluted (0.15 ml) may represent too great an osmotic load and 4 g/kg in 0.1 ml too small a fluid bolus. Furthermore, because the circulatory change produced by the administered PFCE’s appear to affect therapeutic efficacy importantly when used with radiation and oxygen breathing, this factor day
October
1990. Volume 19. Number 4
may well represent a less than optimum variable in the on-going human trials. Fluosol-DA, given in a dose of 10 ml/kg to an average 70 kg adult with an approximate circulatory volume of 5 liters over 90 min, is delivered at the rate of 0.0016 ml/ ml of blood volume/mitt. In contrast, in the mouse, 2.4 g/kg of Fluosol-DA in 0.3 ml (which we have found to be optimal for this PFCE (37) given over about 20 set into the approximately 3 ml blood volume of the mouse represents a rate of infusion of 0.3 ml/ml of blood volume/ min, or approximately 18%times more rapid rate of administration than in the human patients. We have begun studies which specifically address the importance of infusion rate. and early results appear to confirm the importance of this parameter with both Fluosol-DA and Therox. The most effective clinical use of PFC emulsions/carbogen breathing may well depend on the characteristics of the emulsion including PFC concentration, half-life in circulation. and frequency of administration as well as infusion rate. It is likely that in human beings there is an optimal level of the oxygen carrying PFCE for radiation therapy and that ideally the dose and infusion rate of PFCE used clinically should be adjusted in specific patients to produce an optimal hematocrit/fluorocrit as well as an optimal increase in tumor perfusion and ultimately oxygenation. Use of invasive PO: probes or magnetic resonance spectroscopy to measure tumor oxygenation may be required to optimize PFCE administration.
REFERENCES I. Chaplin, D. J.: Durand, R. E.; Olive. P. L. Acute hypoxia in tumors: implications for modifiers of radiation effects. Int. J. Radiat. Oncol. Biol. Phys. 12:1279-1282: 1986. 2. CROS Conference on Chemical Modification-Radiation and Cytotoxic Drugs. Int. J. Radiat. Oncol. Biol. Phys. 8:323808: 1982. 3. Eddy, H. A. Development of the vascular system in the hamster malignant neurilemma. Microvasc. Res. 6:63-82; 1973. 4. Endrich, B.: Antaglietta, M.: Reinhold. H. S.: Gross. J. F. Hemodynamic characteristics in microcirculatory blood channels during early tumor growth. Cancer Res. 39: 1723: 1979. 5. Gatenby. R. A.: Kesler. H. B.; Rosenblum. J. S.: Coia. L. R.: Moldofsky, P. J.; Hartz, W. H.; Broer, G. J. Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. Int. J. Radiat. Oncol. Biol. Phys. 14:831-838; 1988. 6. Groebe. K.; Vaupel, P. Evaluation of oxygen diffusion distances in human breast cancer xenografts using tumor-specific in vivo data: role of various mechanisms in the development of tumor hypoxia. Int. J. Radiat. Oncol. Biol. Phys. 15:69 l-697: 1988. 7. Gullino. P.; Grantham, F.: Coutney. A. Utilization of oxygen by transplanted tumors in iairo. Cancer Res. 27: lO20- 1030: 1967. 8. Hasegawa, T.: Rhee, J. G.: Lewitt, S. H.; Song. C. W. Increase in tumor pOZ by perfluorochemicals and carbogen. Int. J. Radiat. Oncol. Biol. Phys. 13:569-574; 1987.
9. Hiraga, S.; Klubes, P.: Owens, E. S.; Cysack, R. L.: Blasberg, R. G. Increases in brain tumor and cerebral blood flow by blood-perfluorochemical emulsion (Fluosol-DA) exchange. Cancer Res. 47:3296-3302; 1987. 10. Hirst. D. G. Oxygen delivery to tumors. Int. J. Radiat. Oncol. Biol. Phys. 12:1271-1277; 1986. 1 1. Jirtle, R.; Clifton, K. H. The effect of tumor size and host anemia on tumor cell survival after irradiation. Int. J. Radiat. Oncol. Biol. Phys. 4:395-400; 1978. 12. Kjartasson, 1.; Appelgren. L.; Peterson. H. J.: Rosengren. B.: Rudenstan, C. M.; Lewis, D. H. Capillary blood flow, exchange and flow distribution in transplatable rat tumors. In: 7th Europ. Conf. Microcirculation, Aberdeen, Part II. Bibl. Amat. No. 12. Basel: Karger, 1973: 5 19-526. 13. Lee, I.: Lewitt. S. H.: Song. C. W. Effects of Fluosol-DA 20% and carbogen on the radioresponse of SCK tumors and skin of A/J mice. Radiat. Res. I 13:173-182; 1987. 14. Lustig, R.: McIntosh-Lowe. N. L.: Rose. C.; Haas. J.; Krasnow. S.; Spaulding, M.; Prosnitz. L. Phase I-11 study of Fluosol-DA and 100% oxygen breathing as an adjuvant to radiation in the treatment ofadvanced squamous cell tumors of the head and neck (Abstract 193). Proc. 29th Annual ASTRO Meeting, Boston. MA. October. 1987. 15. Martin, D. F.; Porter, E. A.; Fischer, J. J.: Rockwell, S. Effect of a perfluorochemical emulsion on the radiation response of BA 1112 rhabdomyosarcomas. Radiat. Res. 112: 45-53: 1987. 16. Martin, D. F.; Porter. E. A.: Rockwell, S.: Fischer, J. J. Enhancement of tumor radiation response by the combi-
Tumor response to RT 0 B. A
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
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