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Perfusion studies with labeled chemotherapeutic agents
Perfusion Studies with Labeled Chemotherapeutic Agents R. POISSON, M.D., R. BEAUDRY, M.D., J.R. MOORE, M.D. AND E.J. TABAH, M.D. blcGill University
INTROBUCTION The administration of chemotherapeutic cytotoxic agents to a tumor-bearingarea of the body, isolated from its blood supply, 7 is a very attractive approach to c h e m o t h e r a p y for the following reasons: 1. It permits the employment of toxic compounds in a c o n c e n t r a t i o n much greater than allowed by the usual systemic administration since the escape of the agent into highly sensitive areas (bone marrow, gastrointestinal tract) is minimized. TM This is particularly so when biologically short-lived anticancer agents are used, s, 14 2. |t offers an opportunity to alter the regional physiologic conditions of the perfusate. Such factors as a change in ptt, pCO2, oxygen tension, 2, 13 temperature ~s and flow rate ~ may influence the vascular system and tissues in the perfused circuit. This in turn could enhance the tumorcidal activity of the cytotoxic agents to a degree that could not be achieved by the usual systemic administration. 3. Regional perfusion also offers the opportunity of altering the physiologic conditions in the systemic circulation in order to minimize the effect of leakage and absorption of the drug by health), tissue without interfering with its tumorcida] effect in the perfused area. This may be done by: a. The employment of a systemic antagonist 9 (a specific antidote) to neutralize the amount of chemotherapeutic agent which has escaped from the perfused circuit. From the Department of Surgery, Royal Victoria Hospital, and the Department of Experimental Surgery, McGill University, Montreal, Canada. Supported by a research grant from the National Cancer Institute of Canada, and in part by the Cancer Research Society of Montreal. Submitted for publication March 8, 1963. 56
b. The use metabolism of tivity of the perfused area
of hypothermia z3 to reduce the tissues and minimize the cytotoxic agents outside and, in particular, to protect
the acthe the
bone marrow.
c. Bone marrow aspiration and post-perfusion re-infusion, 2s when a considerable leakage is anticipated. The ultimate effectiveness of regional perfusion is dependent on several factors: (1) the degree of isolation obtained; (2) the absorption of the chemotherapeutic agent in an active form by the tissue cells; (3) the degree of susceptibility of various tissues to the chemotherapeutic agents. 1sit is we]l known that some tissues are more susceptible than others to these agents. 27 More and more cytotoxic drugs are produced by biochemists, and eventually better selective agents are going to be discovered; yet the most effective technique for their administration, and the proper physical and biochemical environments to p o t e n t i a t e their chemotherapeutic effect to an optimum degree, are largely unknown. 19 If one desires to investigate the uptake of v a r i o u s tumors some concept of the normal tissue uptake should be established first. The present c o m p a r a t i v e studies were undertaken on dogs (1) to find out the differential " u p t a k e " in the various normal tissues of the extremities; (2) to study the effect of flow rate on the uptake; (3) to determine whether the uptake could be improved by the continuous administration of the &ug into the arterial line of the perfusion pump oxygenator circuit, as opposed to the intermittent or "all at o n c e " method of drug administration. We also h a d in mind the presumptuous idea of correlating the differential tissue uptake, as measured by the C TM activity in the tissue samples, with the selective tissue toxicity as encountered clinically. The differential tissue uptake of two alkylating agents, namely nitrogen mustard and cyclophosphamide, was studied. JSR
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M A T E R I A L S AND METItODS Methyl his ( 2 - c h l o r o e t h y l C TM) amine hydrochloride (HN 2) and 2-his (chloroethyl) amino-tetrahydro-2-oxide C~4 hydrate (cyclophosphamide) were used in these tracer studies. The dose was 3.745 rag. or roughly 25,000,000 counts per experiment. The formulas for these compounds are illustrated in Figures I and 2. It i s a p p a r e n t thatwith tlN z the C ~4 was labeled in the biologically active portion of the molecule, whereas with cyclophosphamide the C ~4 was not attached to. the alkyl group. Mongrel dogs, weighing between I5 and 20 kg., were anesthetized with intravenous Nembutal, 1 ml. per 5 pounds body weight. 2000 ml. of fresh blood from a donor dog was used to fill the pump o x y g e n a t o r heat exchange circuit. All animals were given heparin as an anticoagulant in a dose of 1 mg. per kg. of body weight. Using the hind limb, a vertical incision was made over the superficial and common femoral v e s s e l s at the groin. A dissection and ligation of as many collaterals as p o s s i b l e was carried out, after which the femoral artery and vein were cannulated. A tight rubber tourniquet was applied, as high as p o s s i b l e , proximal to the site of cannulation, and isolation perfusion started. With this technique leakage from or into the general circulation was not a problem. The radioactive drugs were diluted ~n saline j u s t before being administered into thff arterial
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line of the extracorporeat circuit, either by means of an infusion pump or all at once with a syringe. In all these experiments, the temperature was kept at 104°F. and the delivery of oxygen and CO 2 was maintained at a constant ratio. The only variable was the flow rate, which was kept at 90 cc. per minute for the first group of dogs, 180 cc. per minute for the second and 360 cc. per minute for the third. In the fourth group of animals a continuous infusion of drug into the arterial line of the perfusion circuit was maintained for a period of 30 minutes at 90 cc. per minute, after which the perfusion was allowed to continue for an additional 30 minutes. The duration of the perfusion was 60 minutes in all four groups of experiments. After completion of the perfusion, the perfused blood was drained from the limb and biopsy samples of nerve, muscle, bone marrow, fat, skin and tendon were taken from standard locations in the leg of each animal, which was then sacrificed. To measure the amount of radioactivity in the t i s s u e samples and the plasma we u s e d a liquid scintillation counter. This technique, although more time consuming than other methods, permits a more precise and reliable measurement of C ~4 activity in the t i s s u e s , aThe liquid scintillation technique requires a r e l a t i v e l y c o l o r l e s s sample, so that the light r e s u l t i n g from the interaction of the beta particles with the phosphor in solution may reach the phototube. Therefore, only a small amount of tissue is used (50 rag. of tissue was found satisfactory). The t i s s u e s , placed in a vial, are d i s s o l v e d in 0.5 ml. of ityamine for two days and then placed in an incubator at 80°C. for three to four hours. When the t i s s u e s are well d i s s o l v e d 0.5 ml. of ethanol is added, followed by 19 ml. of PPO, which is a commercial phosphor preparation contained in an organic phase. In t h e scintillation counter each vial is counted three times for 10 minutes. The mean value is taken. P r o v i d e d quenching is not too high we have found, after several pilot studies, that this method of measuring the amount of radioactivity in the t i s s u e s is quite s a t i s f a c t o r y and gives reproducible results. The observed counts have to be corrected on account of (1) q u e n c h i n g by the proteins, (2) the ethanol and Hyamine content of the vials. Correction for quenching is affected by adding to the o b s e r v e d count the difference between it and the known number of counts of an internal standard. The b a c ' k g r o u n d count in the scintillation counter is also determined and subtracted from the reading obtained for each vial.
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RESULTS The r e s u l t s are e x p r e s s e d in terms of counts per minute per 50 rag. of t i s s u e for a standard quantity of administered r a d i o a c t i v e chemotherapeutic agent. To e x p r e s s the distribution or the uptake of t h e s e drugs in terms of p e r c e n t a g e per gram o[ a given t i s s u e would be interesting; however, we have found too many v a r i a b l e s and too many correction factors coming into play. The distribution of r a d i o a c t i v i t y in normal t i s s u e s alter injection o[ C $4 nitrogen mustard and c y c l o p h o s p h a m i d e into the arterial line of the perfusion circuit is summarized in F i g u r e s 3 to 9. T h e s e arbitrary numbers r e p r e s e n t the mean value obtained from a s e r i e s of five experiments at a given flow rate. The t i s s u e uptake, as m e a s u r e d by C~a a c t i v i t y , provides a r e l a t i v e l y c o n s t a n t pattern of the distribotion of t h e s e drugs in the various t i s s u e s . 3,s --%I 1'4 £~ [ . F [
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With a high flow rate (360 ml./min.) there was some edema noticed in the limb at the time b i o p s y s a m p l e s were taken following perfusion. With a flow rate of 180 m]. per minute no edema was encountered. Below 90 ml. per minute the v e n o u s return was very poor and proved difficult to keep c o n s t a n t for a period of one hour with our pump. In all the e x p e r i m e n t s the nerve t i s s u e s h o w e d s i g n i f i c a n t l y higher counts, e s p e c i a l l y when t|N 2 was used. This a g r e e s with the work of Mahaley et al. ~s Muscle t i s s u e came next. With c y c l o p h o s p h a m i d e the counts in the m u s c l e b i o p s i e s tended to be about the s a m e as in the nerve t i s s u e . T i s s u e s showing the l o w e s t C la a c t i v i t y with both drugs were tendon and fat, and esp e c i a l l y the intra-articular fat. T h i s is p o s s i b l y related to the poor blood s u p p l y of t h e s e t i s s u e s . It must be mentioned that, for t e c h n i c a l reas o n s , the bone marrow biopsy sample in all Co'c r,z~t~.d per
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PERF'USION O F C H E M O T t t E R A P E U T I C AGENTS
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DISC [ISSION Before studying the uptake of drugs by tumors some k n o w l e d g e of normal t i s s u e uptake should be obtained. We have shown that chemotherapeutic a g e n t s are taken up differentially" by various t i s s u e s . Some t i s s u e s have a greater affinity than others for t h e s e a g e n t s . 3' is, ~ , ~ 7 In our experiments the uptake of normal tiss u e s , as measured by C ~a a c t i v i t y , p r o v i d e s a r e a s o n a b l y a c c u r a t e and constant pattern of the distribution of nitrogen mustard and c y c l o p h o s phamide in the various t i s s u e s of a d o g ' s leg. T h i s d o e s not appear to be a matter of simple diffusion, nor is it j u s t a phenomenon r e l a t e d to the blood s u p p l y of a given t i s s u e . The alkylating c h e m o t h e r a p e u t i c a g e n t s a p p e a r to have ~n unusual affinity for nerve t i s s u e , 1 5 both ceniral and peripheral, but the blood s u p p l y of nerve t i s s u e is certainly no greater than that of bone marrow or muscle, and y e t a s e l e c t i v e uptake s e e m s to take place. Although our s t u d i e s are not purely q u a n t i t a t i v e , we have found, contrary to other i n v e s t i g a t o r s , that the peripheral nerves s e l e c t i v e l y c o n c e n t r a t e d the alkylating agent better than any other t i s s u e . Clinically this c o r r e l a t e s with the well known s e l e c t i v e neurotoxic e f f e c t s of nitrogen mustard and its d e r i v a t i v e s as m a n i f e s t e d by cerebral edema, peripheral neuritis and the r e l i e f of pain following perfusion. With c y c l o p h o s p h a m i d e the uptake in the bone marrow was surprisingly low; it was found to be c o n s i d e r a b l y higher with I1N 2. Whether this is due to the fact that c y c l o p h o s p h a m i d e has a bone marrow spm'ing e f f e c t or whether it
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is not 100 per cent " a c t i v e " when used regionally, s i n c e it has not p a s s e d through the liver, is difficult to s a y . This finding may a l s o be due to the position of the C 14, which is not in the alkyl portion of the molecule. 1° Variation in the flow rate did n o t affect the uptake of the c y c l o p h o s p h a m i d e to any s i g n i f i c a n t d e g r e e . As far as nitrogen mustard is concerned, a low flow rate p r e d u c e d an uptake two to four times greater than when a high flow rate w a s used. T h i s was a c o n s t a n t finding. With HN 2 the use of tt{e infusion pump i n c r e a s e d the uptake of the drug, but no appreciable change was n o t i c e d in the uptake of r a d i o a c t i v e c y c l o p h o s p h a m i d e . Another outstanding feature of t h e s e comparative s t u d i e s is the fact that the degree of uptake in t i s s u e s of high mitotic rate, like the bone marrow and the skin, is only m o d e r a t e l y e l e v a t e d , w h e r e a s in the nerve and m u s c l e tiss u e s , where there is r e l a t i v e l y little cell multiplication, the uptake is high. A d i s t i n c t i o n should be made b e t w e e n the two terms " b i n d i n g " and " u p t a k e . " It is one
60
POISSON, BEAUDRY, MOORE and TABAH
thing to s p e a k in terms of uptake and distribution of t h e s e chemotherapeutic a g e n t s , as we have done, but this does not n e c e s s a r i l y imply that the drug is chemically bound to the n u c l e i c a c i d s . ']'he biologically' active portion of the drug taking part in the p r o c e s s of a l k y l a t i o n is the one we are interested in, s i n c e it is the part of the drug that is c h e m o t h e r a p e u t i c a l l y effective. Even when the C TM is located in the a c t i v e portion of the c y t o t o x i c agent, it would be incorrect to a s s u m e that the degree of r a d i o a c t i v i t y measured in t i s s u e samples is the binding itself. It measured the uptake of the drug into the various t i s s u e s . Of course, the a c t u a l binding is a p a r t of the uptake and is a direct function of the a l k y l a t i n g action of the compound, The uptake n e c e s s a r i l y i n c l u d e s the percentage of the drug that is adsorbed in the interstitial s p a c e s and on the cell membranes, whether the r a d i o a c t i v i t y is counted in d e s i c c a t e d or in fresh t i s s u e s a m p l e s . It is also quite probable that some of the drug which has a l r e a d y penetrated the c e l l s is adsorbed on mitochondria and c y t o p l a s m i c i n c l u s i o n s , without n e c e s s a r i l y taking an active part in producing any biochemical l e s i o n s of c h e m o t h e r a p e u t i c value. Alkylation must be a s e l e c t i v e and a dynamic p r o c e s s ; the n u c l e i c acids of c e l l s undergoing rapid division are more s u s c e p t i b l e to interaction with the a l k y l a t i n g agents than are the nucleic a c i d s of normal r e s t i n g c e l l s , ttowever, it is n o t u n r e a s o n a b l e to think that there is a r e l a t i o n s h i p between the uptake and the actual binding. In order for a chemotherapeutic agent to be bound to the right target it has first of all to be d e l i v e r e d into the right t i s s u e and then, above all, to be present inside the c e l l s . Here s e l e c t i v e a l k y l a t i o n may or m a y not take place depending on the metabolic n e e d s . A more accurate e s t i m a t i o n of the actual binding would be a determination of the r a d i o a c t i v i t y in the precipitated protein fraction of the t i s s u e sample. In our experiments a l o w flow rate e n h a n c e d the uptake of the chemotherapeutic a g e n t s in the t i s s u e s . T h i s is probably due to the prevention of edema formation and the exposure of the agent to the t i s s u e s for a longer period of time. We have a l s o found with a continuous infusion of HN 2 there was better utilization of the drug, bolus formation was avoided and a c o n s t a n t amount of b i o l o g i c a l l y active drug was maintained in the circuit. SUMMARY P e r f u s i n g dog hind legs with C la labeled nitrogen mustard and C TM l a b e l e d c y c i o p h o s -
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phamide we have found: 1. A r e l a t i v e l y c o n s t a n t pattern in the uptake of t h e s e drugs in the v a r i o u s tissues a n a l y z e d , the uptake being measured as the a c t i v i t y of the C 14 in the various t i s s u e samp i e s . A d i s t i n c t i o n is made here between uptake and binding. 2. The uptake in the peripheral nerves was a l w a y s very high with both drugs. 3. WithltN z the lower the f l o w r a t e the highet the uptake. 4. C o n t i n u o u s infusion of tIN 2 into the arterial line as opposed to intermittent injection has a b e n e f i c i a l effect on the uptake. We would like to express our sincere thanks to Merck, Shazp and Dohme of Canada Limited, Montreal, for their supply of C TM labeled nitrogen mustard and Frank W. Ilorner L i m i t e d , Montreal, for the C ~4 t,abeled cyclophospham ide. REFERENCES i. Ausman, R.K., and Aust, J.B.: Studies in isolated perfusion chemotherapy. 1.Nitrogen mustard. Ann. Surg., 153:527, 1961. 2. Ausman, R.K., Gemmell, S.J., and Aust, J.B.: Effect of oxygen on tile uptake of nitrogen mustard in isolated perfusion. Proc. Am. Assoc. Cancer Research, 3:92, 1960. 3. Boone, I.U., Rogers, B.S., Maxfield, j.R., and Storer, J.B.: Nitrogen mustard and triethylene melamine content in normal and turnout tissues after intra-arteriat and intrave~.ous injection in rats. Cancer Research, 17: 1120, I957. 4. Chen, P.S.: Liquid scintillation counting of C14 a n d H 3 in plasma and serum. Proc. Soc. Exper. Biol. & Med., 98:546, 1958. 5. Clarkson, B. and Lawrence, W., Jr.: Perfvsion anti infusion tecnniques in cancer chemotherapy. M. Clin. North America, 45:689, 1961. 6. Cohn, P.: The distribution of radioactivity in tissues of the rat following the administration of a nitrogen mustard derivative. Brit. J. Cancer, ] 1:258, 1957. 7. Creech, O., Krementz, E.T., and Ryan, R.F.: Chemotherapy of cancer: Regional perfusion utilizing an extracorporeal circuit. Ann. Surg., 148:616, 1958. 8. Creech, O., K r e m e n t z , E.T., Ryan, R.F., Reemtma, K., and Winhlad, J.N.: Experiences with isolation-perfusion techniques in the treatment of cancer. Ann. Surg., 149:627, 1959. 9. lt a t i b o g l u , I., Mihich, E., Moore, G.E., and Nichol, C.A.: Use of sodium thiosulphate as a ~,eutratizing agent during regional administration of nitrogen mustard: An experimental study. Ann. Surg., 994:1001, 1962. 10. Itenderson, I: Discussion of paper by Shingleton and Parker (reference 23). Cancer Chemother. Rep., 10:37, 1960. 11. ltottinger, G.C., Ryan, R.F., Delgado, J.P., and Reemtsma, K.: Physiology of extraeorporeal circulation: Studies of blood flow, oxygen
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consumption and metabolism ir~ the isolated an;' perfused e x t r e m i t y . Surg. Forum, 10:80, 1959. 12. Kit, S., and Griffin, A.C.: Cellular metabolism and cancer: A lleview. Cancer llesearch, 18: 621, 1958. 13. Krementz, E.T., ttarlin, 1t., and Knudson, L.: The enhancement of chemotherapy by increased tissue oxygen tension. Cancer Chemother, ltep., 10:125, 1960. 14. Mahaley, M.S., and Woodhall, B.: An evaluation of plasma levels of alkylating agents during regional chemotherapeutic perfusions. J. Surg. Research, 1:285, 1961. 15. Mahaley, M.S., Huneycutt H., Boone, S., and Woodhall, B.: Localization of methyl bis (2-chloroethyl-1, 2-C TM) amine hydrochloride in nervous tissue after intravenous injection or regional cerebral perfusion in dogs. Cancer Chemother. Rep. 1I:29, I961. 16. Nadkarni, M.V., Burdge, D.C., and Smith, P.K.: Uptake of radioactivity fr~)m Tem-Ct4 and g.lycine-2-Cl'~ by neoplastic and control t~ssues. Proc. Am. Assoc. Cancer Research, 2:330. 1958. 17. Nadkarni, M.V., and Smith, P.K.: The distribution of radioactivity following administration of tri-ethyleneimino-triazine C1~ in tumour bearing and c o n t r o l mice. Cancer R e s e a r c h , 14:559, 1954. 18. l~ochlin, D.B., Thaxter, T.It., Dickerson, A.G., and Shiner, J.: The effect of tissue tempera-
19. 20. 21. 22.
23. 24. 25.
26.
27.
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ture on the binding of alkytating agents in the isolation perfusion treatment of cancer. Surg., Gynec. & Ohst., 1t3:555, 1961. Ross, W.C:J.: Biological Alkylating A g e n t s . Butterworths, London, 1962. Ryan, R.F.: Dosage of chemotherapeutic agents for use with perfusion t e c h n i q u e s . Cancer Chemother. Rep., 10:47, 1960. Sholes, D.M.: P e l v i c perfusion with nitrogen mustard for cancer: A neurological complication- Am. J. Obst. & G y n e c . , 80:481, 1960. Shingleton, W. and Sanders, A.P.: Experimental abdominal perfusion with C TM labeled nitrogen mustard. Cancer Chemother. Rep., I6:557, 1962. Shingteton, W.W., and Parker, R.T.: Abdominal perfusion with hypothermia and hyperthermia. Cancer Chemother. Rep., 10:35, 1960. Sprague, C.: Bone marrow replantation. Cancer Chemother. Rep., 10:109, 1960. S t e h l i u , J.S., Clark, R.L., and White, E.C.: Regional chemotherapy via intra-arterial perfusion for malignant neoplasms. Bull. Soc. lnternat,Chir., I9:543, 1960. Stehlin, J.S., Clark, R.L., White, E.G., Smith, J.L., Griffin, A.C., J e s s e , R.tI., ar..d Healey, J.E.: Regional chemotherapy for cancer. Ann. 5urg., 15]:605, 1960. ~l'oodhall, t3., M a h a l e y , M.S., Boone, S., and Huneycutt, It.: The effect of chemotherapeutic agents upon p e r i p h e r a l nerves. J. Surg. Research, 2:37, 1962.
INTROBUCTION The administration of chemotherapeutic cytotoxic agents to a tumor-bearingarea of the body, isolated from its blood supply, 7 is a very attractive approach to c h e m o t h e r a p y for the following reasons: 1. It permits the employment of toxic compounds in a c o n c e n t r a t i o n much greater than allowed by the usual systemic administration since the escape of the agent into highly sensitive areas (bone marrow, gastrointestinal tract) is minimized. TM This is particularly so when biologically short-lived anticancer agents are used, s, 14 2. |t offers an opportunity to alter the regional physiologic conditions of the perfusate. Such factors as a change in ptt, pCO2, oxygen tension, 2, 13 temperature ~s and flow rate ~ may influence the vascular system and tissues in the perfused circuit. This in turn could enhance the tumorcidal activity of the cytotoxic agents to a degree that could not be achieved by the usual systemic administration. 3. Regional perfusion also offers the opportunity of altering the physiologic conditions in the systemic circulation in order to minimize the effect of leakage and absorption of the drug by health), tissue without interfering with its tumorcida] effect in the perfused area. This may be done by: a. The employment of a systemic antagonist 9 (a specific antidote) to neutralize the amount of chemotherapeutic agent which has escaped from the perfused circuit. From the Department of Surgery, Royal Victoria Hospital, and the Department of Experimental Surgery, McGill University, Montreal, Canada. Supported by a research grant from the National Cancer Institute of Canada, and in part by the Cancer Research Society of Montreal. Submitted for publication March 8, 1963. 56
b. The use metabolism of tivity of the perfused area
of hypothermia z3 to reduce the tissues and minimize the cytotoxic agents outside and, in particular, to protect
the acthe the
bone marrow.
c. Bone marrow aspiration and post-perfusion re-infusion, 2s when a considerable leakage is anticipated. The ultimate effectiveness of regional perfusion is dependent on several factors: (1) the degree of isolation obtained; (2) the absorption of the chemotherapeutic agent in an active form by the tissue cells; (3) the degree of susceptibility of various tissues to the chemotherapeutic agents. 1sit is we]l known that some tissues are more susceptible than others to these agents. 27 More and more cytotoxic drugs are produced by biochemists, and eventually better selective agents are going to be discovered; yet the most effective technique for their administration, and the proper physical and biochemical environments to p o t e n t i a t e their chemotherapeutic effect to an optimum degree, are largely unknown. 19 If one desires to investigate the uptake of v a r i o u s tumors some concept of the normal tissue uptake should be established first. The present c o m p a r a t i v e studies were undertaken on dogs (1) to find out the differential " u p t a k e " in the various normal tissues of the extremities; (2) to study the effect of flow rate on the uptake; (3) to determine whether the uptake could be improved by the continuous administration of the &ug into the arterial line of the perfusion pump oxygenator circuit, as opposed to the intermittent or "all at o n c e " method of drug administration. We also h a d in mind the presumptuous idea of correlating the differential tissue uptake, as measured by the C TM activity in the tissue samples, with the selective tissue toxicity as encountered clinically. The differential tissue uptake of two alkylating agents, namely nitrogen mustard and cyclophosphamide, was studied. JSR
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Vol. 4, No. 2 - F e b r u a r y , 1964 .jC*H CH 3
-
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PERFUSION OF CHEMOTHERAPEUTIC AGENTS
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METHYL-BIS The c a r b o n tag indicated by' t h e
a~,pears asterisks.
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12 C H L O R O E T H Y L ) on either o r bolh
of
AMINE the ethyl groups
Fig. 1
M A T E R I A L S AND METItODS Methyl his ( 2 - c h l o r o e t h y l C TM) amine hydrochloride (HN 2) and 2-his (chloroethyl) amino-tetrahydro-2-oxide C~4 hydrate (cyclophosphamide) were used in these tracer studies. The dose was 3.745 rag. or roughly 25,000,000 counts per experiment. The formulas for these compounds are illustrated in Figures I and 2. It i s a p p a r e n t thatwith tlN z the C ~4 was labeled in the biologically active portion of the molecule, whereas with cyclophosphamide the C ~4 was not attached to. the alkyl group. Mongrel dogs, weighing between I5 and 20 kg., were anesthetized with intravenous Nembutal, 1 ml. per 5 pounds body weight. 2000 ml. of fresh blood from a donor dog was used to fill the pump o x y g e n a t o r heat exchange circuit. All animals were given heparin as an anticoagulant in a dose of 1 mg. per kg. of body weight. Using the hind limb, a vertical incision was made over the superficial and common femoral v e s s e l s at the groin. A dissection and ligation of as many collaterals as p o s s i b l e was carried out, after which the femoral artery and vein were cannulated. A tight rubber tourniquet was applied, as high as p o s s i b l e , proximal to the site of cannulation, and isolation perfusion started. With this technique leakage from or into the general circulation was not a problem. The radioactive drugs were diluted ~n saline j u s t before being administered into thff arterial
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14 H Y D R A T E
57
line of the extracorporeat circuit, either by means of an infusion pump or all at once with a syringe. In all these experiments, the temperature was kept at 104°F. and the delivery of oxygen and CO 2 was maintained at a constant ratio. The only variable was the flow rate, which was kept at 90 cc. per minute for the first group of dogs, 180 cc. per minute for the second and 360 cc. per minute for the third. In the fourth group of animals a continuous infusion of drug into the arterial line of the perfusion circuit was maintained for a period of 30 minutes at 90 cc. per minute, after which the perfusion was allowed to continue for an additional 30 minutes. The duration of the perfusion was 60 minutes in all four groups of experiments. After completion of the perfusion, the perfused blood was drained from the limb and biopsy samples of nerve, muscle, bone marrow, fat, skin and tendon were taken from standard locations in the leg of each animal, which was then sacrificed. To measure the amount of radioactivity in the t i s s u e samples and the plasma we u s e d a liquid scintillation counter. This technique, although more time consuming than other methods, permits a more precise and reliable measurement of C ~4 activity in the t i s s u e s , aThe liquid scintillation technique requires a r e l a t i v e l y c o l o r l e s s sample, so that the light r e s u l t i n g from the interaction of the beta particles with the phosphor in solution may reach the phototube. Therefore, only a small amount of tissue is used (50 rag. of tissue was found satisfactory). The t i s s u e s , placed in a vial, are d i s s o l v e d in 0.5 ml. of ityamine for two days and then placed in an incubator at 80°C. for three to four hours. When the t i s s u e s are well d i s s o l v e d 0.5 ml. of ethanol is added, followed by 19 ml. of PPO, which is a commercial phosphor preparation contained in an organic phase. In t h e scintillation counter each vial is counted three times for 10 minutes. The mean value is taken. P r o v i d e d quenching is not too high we have found, after several pilot studies, that this method of measuring the amount of radioactivity in the t i s s u e s is quite s a t i s f a c t o r y and gives reproducible results. The observed counts have to be corrected on account of (1) q u e n c h i n g by the proteins, (2) the ethanol and Hyamine content of the vials. Correction for quenching is affected by adding to the o b s e r v e d count the difference between it and the known number of counts of an internal standard. The b a c ' k g r o u n d count in the scintillation counter is also determined and subtracted from the reading obtained for each vial.
58
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POISSON El:At DR~I, ,MOORE and TABAI{ - ~ I LI t A L E
Corrected
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Vol. 4, No. 2
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RESULTS The r e s u l t s are e x p r e s s e d in terms of counts per minute per 50 rag. of t i s s u e for a standard quantity of administered r a d i o a c t i v e chemotherapeutic agent. To e x p r e s s the distribution or the uptake of t h e s e drugs in terms of p e r c e n t a g e per gram o[ a given t i s s u e would be interesting; however, we have found too many v a r i a b l e s and too many correction factors coming into play. The distribution of r a d i o a c t i v i t y in normal t i s s u e s alter injection o[ C $4 nitrogen mustard and c y c l o p h o s p h a m i d e into the arterial line of the perfusion circuit is summarized in F i g u r e s 3 to 9. T h e s e arbitrary numbers r e p r e s e n t the mean value obtained from a s e r i e s of five experiments at a given flow rate. The t i s s u e uptake, as m e a s u r e d by C~a a c t i v i t y , provides a r e l a t i v e l y c o n s t a n t pattern of the distribotion of t h e s e drugs in the various t i s s u e s . 3,s --%I 1'4 £~ [ . F [
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With a high flow rate (360 ml./min.) there was some edema noticed in the limb at the time b i o p s y s a m p l e s were taken following perfusion. With a flow rate of 180 m]. per minute no edema was encountered. Below 90 ml. per minute the v e n o u s return was very poor and proved difficult to keep c o n s t a n t for a period of one hour with our pump. In all the e x p e r i m e n t s the nerve t i s s u e s h o w e d s i g n i f i c a n t l y higher counts, e s p e c i a l l y when t|N 2 was used. This a g r e e s with the work of Mahaley et al. ~s Muscle t i s s u e came next. With c y c l o p h o s p h a m i d e the counts in the m u s c l e b i o p s i e s tended to be about the s a m e as in the nerve t i s s u e . T i s s u e s showing the l o w e s t C la a c t i v i t y with both drugs were tendon and fat, and esp e c i a l l y the intra-articular fat. T h i s is p o s s i b l y related to the poor blood s u p p l y of t h e s e t i s s u e s . It must be mentioned that, for t e c h n i c a l reas o n s , the bone marrow biopsy sample in all Co'c r,z~t~.d per
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February,.1964
PERF'USION O F C H E M O T t t E R A P E U T I C AGENTS
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DISC [ISSION Before studying the uptake of drugs by tumors some k n o w l e d g e of normal t i s s u e uptake should be obtained. We have shown that chemotherapeutic a g e n t s are taken up differentially" by various t i s s u e s . Some t i s s u e s have a greater affinity than others for t h e s e a g e n t s . 3' is, ~ , ~ 7 In our experiments the uptake of normal tiss u e s , as measured by C ~a a c t i v i t y , p r o v i d e s a r e a s o n a b l y a c c u r a t e and constant pattern of the distribution of nitrogen mustard and c y c l o p h o s phamide in the various t i s s u e s of a d o g ' s leg. T h i s d o e s not appear to be a matter of simple diffusion, nor is it j u s t a phenomenon r e l a t e d to the blood s u p p l y of a given t i s s u e . The alkylating c h e m o t h e r a p e u t i c a g e n t s a p p e a r to have ~n unusual affinity for nerve t i s s u e , 1 5 both ceniral and peripheral, but the blood s u p p l y of nerve t i s s u e is certainly no greater than that of bone marrow or muscle, and y e t a s e l e c t i v e uptake s e e m s to take place. Although our s t u d i e s are not purely q u a n t i t a t i v e , we have found, contrary to other i n v e s t i g a t o r s , that the peripheral nerves s e l e c t i v e l y c o n c e n t r a t e d the alkylating agent better than any other t i s s u e . Clinically this c o r r e l a t e s with the well known s e l e c t i v e neurotoxic e f f e c t s of nitrogen mustard and its d e r i v a t i v e s as m a n i f e s t e d by cerebral edema, peripheral neuritis and the r e l i e f of pain following perfusion. With c y c l o p h o s p h a m i d e the uptake in the bone marrow was surprisingly low; it was found to be c o n s i d e r a b l y higher with I1N 2. Whether this is due to the fact that c y c l o p h o s p h a m i d e has a bone marrow spm'ing e f f e c t or whether it
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animals w a s taken from the mid-slaaft of the tibia and c o n s e q u e n t l y was more adult and fatty in nature°
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is not 100 per cent " a c t i v e " when used regionally, s i n c e it has not p a s s e d through the liver, is difficult to s a y . This finding may a l s o be due to the position of the C 14, which is not in the alkyl portion of the molecule. 1° Variation in the flow rate did n o t affect the uptake of the c y c l o p h o s p h a m i d e to any s i g n i f i c a n t d e g r e e . As far as nitrogen mustard is concerned, a low flow rate p r e d u c e d an uptake two to four times greater than when a high flow rate w a s used. T h i s was a c o n s t a n t finding. With HN 2 the use of tt{e infusion pump i n c r e a s e d the uptake of the drug, but no appreciable change was n o t i c e d in the uptake of r a d i o a c t i v e c y c l o p h o s p h a m i d e . Another outstanding feature of t h e s e comparative s t u d i e s is the fact that the degree of uptake in t i s s u e s of high mitotic rate, like the bone marrow and the skin, is only m o d e r a t e l y e l e v a t e d , w h e r e a s in the nerve and m u s c l e tiss u e s , where there is r e l a t i v e l y little cell multiplication, the uptake is high. A d i s t i n c t i o n should be made b e t w e e n the two terms " b i n d i n g " and " u p t a k e . " It is one
60
POISSON, BEAUDRY, MOORE and TABAH
thing to s p e a k in terms of uptake and distribution of t h e s e chemotherapeutic a g e n t s , as we have done, but this does not n e c e s s a r i l y imply that the drug is chemically bound to the n u c l e i c a c i d s . ']'he biologically' active portion of the drug taking part in the p r o c e s s of a l k y l a t i o n is the one we are interested in, s i n c e it is the part of the drug that is c h e m o t h e r a p e u t i c a l l y effective. Even when the C TM is located in the a c t i v e portion of the c y t o t o x i c agent, it would be incorrect to a s s u m e that the degree of r a d i o a c t i v i t y measured in t i s s u e samples is the binding itself. It measured the uptake of the drug into the various t i s s u e s . Of course, the a c t u a l binding is a p a r t of the uptake and is a direct function of the a l k y l a t i n g action of the compound, The uptake n e c e s s a r i l y i n c l u d e s the percentage of the drug that is adsorbed in the interstitial s p a c e s and on the cell membranes, whether the r a d i o a c t i v i t y is counted in d e s i c c a t e d or in fresh t i s s u e s a m p l e s . It is also quite probable that some of the drug which has a l r e a d y penetrated the c e l l s is adsorbed on mitochondria and c y t o p l a s m i c i n c l u s i o n s , without n e c e s s a r i l y taking an active part in producing any biochemical l e s i o n s of c h e m o t h e r a p e u t i c value. Alkylation must be a s e l e c t i v e and a dynamic p r o c e s s ; the n u c l e i c acids of c e l l s undergoing rapid division are more s u s c e p t i b l e to interaction with the a l k y l a t i n g agents than are the nucleic a c i d s of normal r e s t i n g c e l l s , ttowever, it is n o t u n r e a s o n a b l e to think that there is a r e l a t i o n s h i p between the uptake and the actual binding. In order for a chemotherapeutic agent to be bound to the right target it has first of all to be d e l i v e r e d into the right t i s s u e and then, above all, to be present inside the c e l l s . Here s e l e c t i v e a l k y l a t i o n may or m a y not take place depending on the metabolic n e e d s . A more accurate e s t i m a t i o n of the actual binding would be a determination of the r a d i o a c t i v i t y in the precipitated protein fraction of the t i s s u e sample. In our experiments a l o w flow rate e n h a n c e d the uptake of the chemotherapeutic a g e n t s in the t i s s u e s . T h i s is probably due to the prevention of edema formation and the exposure of the agent to the t i s s u e s for a longer period of time. We have a l s o found with a continuous infusion of HN 2 there was better utilization of the drug, bolus formation was avoided and a c o n s t a n t amount of b i o l o g i c a l l y active drug was maintained in the circuit. SUMMARY P e r f u s i n g dog hind legs with C la labeled nitrogen mustard and C TM l a b e l e d c y c i o p h o s -
JSR
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February,
1964
phamide we have found: 1. A r e l a t i v e l y c o n s t a n t pattern in the uptake of t h e s e drugs in the v a r i o u s tissues a n a l y z e d , the uptake being measured as the a c t i v i t y of the C 14 in the various t i s s u e samp i e s . A d i s t i n c t i o n is made here between uptake and binding. 2. The uptake in the peripheral nerves was a l w a y s very high with both drugs. 3. WithltN z the lower the f l o w r a t e the highet the uptake. 4. C o n t i n u o u s infusion of tIN 2 into the arterial line as opposed to intermittent injection has a b e n e f i c i a l effect on the uptake. We would like to express our sincere thanks to Merck, Shazp and Dohme of Canada Limited, Montreal, for their supply of C TM labeled nitrogen mustard and Frank W. Ilorner L i m i t e d , Montreal, for the C ~4 t,abeled cyclophospham ide. REFERENCES i. Ausman, R.K., and Aust, J.B.: Studies in isolated perfusion chemotherapy. 1.Nitrogen mustard. Ann. Surg., 153:527, 1961. 2. Ausman, R.K., Gemmell, S.J., and Aust, J.B.: Effect of oxygen on tile uptake of nitrogen mustard in isolated perfusion. Proc. Am. Assoc. Cancer Research, 3:92, 1960. 3. Boone, I.U., Rogers, B.S., Maxfield, j.R., and Storer, J.B.: Nitrogen mustard and triethylene melamine content in normal and turnout tissues after intra-arteriat and intrave~.ous injection in rats. Cancer Research, 17: 1120, I957. 4. Chen, P.S.: Liquid scintillation counting of C14 a n d H 3 in plasma and serum. Proc. Soc. Exper. Biol. & Med., 98:546, 1958. 5. Clarkson, B. and Lawrence, W., Jr.: Perfvsion anti infusion tecnniques in cancer chemotherapy. M. Clin. North America, 45:689, 1961. 6. Cohn, P.: The distribution of radioactivity in tissues of the rat following the administration of a nitrogen mustard derivative. Brit. J. Cancer, ] 1:258, 1957. 7. Creech, O., Krementz, E.T., and Ryan, R.F.: Chemotherapy of cancer: Regional perfusion utilizing an extracorporeal circuit. Ann. Surg., 148:616, 1958. 8. Creech, O., K r e m e n t z , E.T., Ryan, R.F., Reemtma, K., and Winhlad, J.N.: Experiences with isolation-perfusion techniques in the treatment of cancer. Ann. Surg., 149:627, 1959. 9. lt a t i b o g l u , I., Mihich, E., Moore, G.E., and Nichol, C.A.: Use of sodium thiosulphate as a ~,eutratizing agent during regional administration of nitrogen mustard: An experimental study. Ann. Surg., 994:1001, 1962. 10. Itenderson, I: Discussion of paper by Shingleton and Parker (reference 23). Cancer Chemother. Rep., 10:37, 1960. 11. ltottinger, G.C., Ryan, R.F., Delgado, J.P., and Reemtsma, K.: Physiology of extraeorporeal circulation: Studies of blood flow, oxygen
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P E R F U S I O N O F C t t E M O T t l E R A P E U T I C AGENTS
consumption and metabolism ir~ the isolated an;' perfused e x t r e m i t y . Surg. Forum, 10:80, 1959. 12. Kit, S., and Griffin, A.C.: Cellular metabolism and cancer: A lleview. Cancer llesearch, 18: 621, 1958. 13. Krementz, E.T., ttarlin, 1t., and Knudson, L.: The enhancement of chemotherapy by increased tissue oxygen tension. Cancer Chemother, ltep., 10:125, 1960. 14. Mahaley, M.S., and Woodhall, B.: An evaluation of plasma levels of alkylating agents during regional chemotherapeutic perfusions. J. Surg. Research, 1:285, 1961. 15. Mahaley, M.S., Huneycutt H., Boone, S., and Woodhall, B.: Localization of methyl bis (2-chloroethyl-1, 2-C TM) amine hydrochloride in nervous tissue after intravenous injection or regional cerebral perfusion in dogs. Cancer Chemother. Rep. 1I:29, I961. 16. Nadkarni, M.V., Burdge, D.C., and Smith, P.K.: Uptake of radioactivity fr~)m Tem-Ct4 and g.lycine-2-Cl'~ by neoplastic and control t~ssues. Proc. Am. Assoc. Cancer Research, 2:330. 1958. 17. Nadkarni, M.V., and Smith, P.K.: The distribution of radioactivity following administration of tri-ethyleneimino-triazine C1~ in tumour bearing and c o n t r o l mice. Cancer R e s e a r c h , 14:559, 1954. 18. l~ochlin, D.B., Thaxter, T.It., Dickerson, A.G., and Shiner, J.: The effect of tissue tempera-
19. 20. 21. 22.
23. 24. 25.
26.
27.
61
ture on the binding of alkytating agents in the isolation perfusion treatment of cancer. Surg., Gynec. & Ohst., 1t3:555, 1961. Ross, W.C:J.: Biological Alkylating A g e n t s . Butterworths, London, 1962. Ryan, R.F.: Dosage of chemotherapeutic agents for use with perfusion t e c h n i q u e s . Cancer Chemother. Rep., 10:47, 1960. Sholes, D.M.: P e l v i c perfusion with nitrogen mustard for cancer: A neurological complication- Am. J. Obst. & G y n e c . , 80:481, 1960. Shingleton, W. and Sanders, A.P.: Experimental abdominal perfusion with C TM labeled nitrogen mustard. Cancer Chemother. Rep., I6:557, 1962. Shingteton, W.W., and Parker, R.T.: Abdominal perfusion with hypothermia and hyperthermia. Cancer Chemother. Rep., 10:35, 1960. Sprague, C.: Bone marrow replantation. Cancer Chemother. Rep., 10:109, 1960. S t e h l i u , J.S., Clark, R.L., and White, E.C.: Regional chemotherapy via intra-arterial perfusion for malignant neoplasms. Bull. Soc. lnternat,Chir., I9:543, 1960. Stehlin, J.S., Clark, R.L., White, E.G., Smith, J.L., Griffin, A.C., J e s s e , R.tI., ar..d Healey, J.E.: Regional chemotherapy for cancer. Ann. 5urg., 15]:605, 1960. ~l'oodhall, t3., M a h a l e y , M.S., Boone, S., and Huneycutt, It.: The effect of chemotherapeutic agents upon p e r i p h e r a l nerves. J. Surg. Research, 2:37, 1962.