Buthionine sulfoximine pretreatment potentiates the effect of isolated lung perfusion with doxorubicin

ORIGINAL

ARTICLES: GENERAL THORACIC

Buthionine Sulfoximine Pretreatment Potentiates the Effect of Isolated Lung Perfusion With Doxorubicin Jeffrey L. Port, MD, Steven N. Hochwald, MD, Hong-Yue Wang, MD, and Michael E. Burt, MD, PhD The Thoracic Oncology/Surgical Metabolism Laboratory, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York New York

Background. Although surgical resection remains the mainstay of treatment for metastatic pulmonary sarcoma, 5-year survival approaches only 25%. Chemotherapy has been limited by tumor resistance and systemic toxicity. We assessed the efficacy of r-buthionine-SR-sulfoximine, an inhibitor of glutathione synthesis, as a sensitizer for isolated lung perfusion. Methods. In experiment 1, sarcoma-bearing rats (n = 20) received either buthionine sulfoximine via intraperitoneal injection or Hespan. After the last injection, tumor glutathione levels were measured. In experiment 2, rats (n = 60) were injected with sarcoma intravenously. On day 6, animals were pretreated with either buthionine sulfoximine or Hespan intraperitoneally. On day 7, rats underwent isolated lung perfusion (Hespan or doxorubicin) or intravenous therapy (Hespan or doxorubicin). On day 14, tumor nodules were counted.

Results. Buthionine sulfoximine effectively depleted tumor glutathione. Animals treated with intravenous therapy had no response to therapy, whereas those animals treated with doxorubicin isolated lung perfusion alone had a limited response. Buthionine-sulfoximine pretreatment in combination with doxorubicin isolated lung perfusion led to a 13-fold reduction in tumor nodules and 5 complete responses. Conclusions. Buthionine-sulfoximine pretreatment in combination with doxorubicin isolated lung perfusion is superior to intravenous doxorubicin and doxorubicin isolated lung perfusion alone for the treatment of metastatic pulmonary sarcoma.

he m a n a g e m e n t of metastatic p u l m o n a r y soft tissue sarcoma is a significant clinical problem. To date, s t a n d a r d systemic c h e m o t h e r a p y holds little promise for long-term survival [1]. Surgical resection r e m a i n s the s t a n d a r d of care for metastatic p u l m o n a r y sarcoma, yet 5-year survival after complete resection a p p r o a c h e s only 25% [1-4]. Treatment failures are usually local and stem from micrometastatic disease present at the time of resection [4], the high prevalence of sarcoma chemoresistance [5], a n d the toxicity of doxorubicin, which often precludes effective systemic c h e m o t h e r a p y [6]. A t t e m p t s have been m a d e to increase the efficacy of antineoplastic t h e r a p y with locoregional delivery systems using highdose c h e m o t h e r a p y [7-9]. This t h e r a p y produces increased local tissue levels with minimal systemic exposure. O t h e r a p p r o a c h e s have focused on selective, preferential t u m o r sensitization to chemotherapy, to reduce t u m o r resistance. Glutathione m a y play a significant role in t u m o r resistance to c h e m o t h e r a p y and radiation [10, 11]. Buthionine sulfoximine (BSO) is a potent inhibitor of glutathione

synthesis [10-14]. In this study, we have c o m b i n e d isolated doxorubicin lung perfusion with BSO for the treatm e n t of p u l m o n a r y metastases. We hypothesize that the delivery of high-dose local c h e m o t h e r a p y in conjunction with glutathione depletion m a y potentiate t u m o r cytotoxicity while reducing u n w a n t e d systemic side effects.

T

Presented at the Thirty-first Annual Meeting of The Society of Thoracic Surgeons, Palm Springs, CA, Jan 30-Feb l, 1995. Address reprint requests to Dr Burt, Department of SurgeD,, Memorial Sloan-KetteringCancer Center, 1275 York Ave, New York, NY 10021. © 1995 by The Society of Thoracic Surgeons

(Ann Thorac Surg 1995;60:239-44)

Material and Methods

Animal Care Animals (male Fischer F344 rats; Charles River, Kingston, NY) were treated in accordance with the A n i m a l Welfare Act and the NIH " G u i d e for the Care a n d Use of Laboratory A n i m a l s " (NIH publication 85-23, revised 1985). Experiments were a p p r o v e d by the Institutional Animal Care and Use C o m m i t t e e of Memorial SloanKettering Cancer Center. All rats were allowed access to standard laboratory rat chow (Purina Rat Chow; Ralston Purina, St. Louis, MO) and water ad libitum. Housing was t e m p e r a t u r e controlled and p r o v i d e d a 12-hour light/dark cycle.

Preparation of Tumor Cells The t u m o r is a m e t h y l c h o l a n t h r e n e - i n d u c e d sarcoma that has been serially p a s s e d subcutaneously in the flank of F344 rats a n d is well characterized. The t u m o r cells 0003-4975/95/$9.50 0003-4975(95)00362-0

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Ann Thorac Surg 1995;60:239-44

were harvested fresh from a t u m o r - b e a r i n g animal, and a single cell suspension p r e p a r e d on the day of injection. The t u m o r is characterized by r a p i d growth a n d local invasion after subcutaneous implantation. P u l m o n a r y metastases are i n d u c e d in a reproducible fashion after intravenous injection.

i n d u c e d sarcoma cells via the right external jugular vein. On day 6 after inoculation, rats received p r e t r e a t m e n t consisting of an intraperitoneal (IP) injection (2 PaL/injection) of H e s p a n or BSO (2 mmol/kg) every 8 hours for three doses. Two hours after the last IP dose animals were treated with isolated lung perfusion (ILP) (doxorubicin or Hespan) or intravenous injection (doxorubicin or Hespan). Pretreatment a n d treatment for all groups were as follows: group I (n = 6) received H e s p a n IP a n d H e s p a n intravenously; group II (n = 6) received BSO IP a n d doxorubicin intravenously (25 gg in 0.5 mL); group III (n = 12) received H e s p a n IP and H e s p a n ILP; group IV (n - 12) received BSO IP a n d H e s p a n ILP; group V (n = 12) received H e s p a n IP and doxorubicin ILP; a n d group VI (n - 12) received BSO IP and doxorubicin ILP. Those groups that u n d e r w e n t ILP were per(used either with doxorubicin (10 ~g/mL) for 5 m i n u t e s followed b y a 2.5-minute H e s p a n w a s h o u t or with H e s p a n ILP alone for 7.5 m i n u t e s at a rate of 0.5 m L / m i n (Syringe I n f u s e p u m p 22; H a r v a r d Apparatus). Those animals that u n d e r w e n t intravenous t h e r a p y were injected t h r o u g h their right jugular vein with either doxorubicin (25/~g in 0.5 mL) or 0.5 mL of Hespan. On day 14 after inoculation, all groups were e u t h a n i z e d a n d their left lungs were stained with India ink for identification of p u l m o n a r y metastases [16].

Isolated Lung Perfusion Isolated left lung perfusion was p e r f o r m e d by the m e t h o d previously established in this laboratory [7]. Animals were anesthetized with pentobarbital (50 mg/kg) intraperitoneally. Under direct visualization the animals were i n t u b a t e d with a 16-gauge intravenous catheter placed over a guidewire [15] and then placed on a volume ventilator (Rodent Ventilator m o d e l 683; Harvard Apparatus, South Natick, MA). Ventilation was m a i n t a i n e d at a tidal volume of 10 mL/kg, with 100% 02 s u p p l e m e n t e d with 0.5% halothane at a rate of 75 strokes/min. The left side of the chest was s h a v e d and p r e p a r e d with a p o v i d o n e - i o d i n e 10% solution, a n d then entered through a fourth intercostal incision. The left p u l m o n a r y artery and vein were visualized u n d e r an operative microscope (OpMi-1,16x; Carl Zeiss, Wotan, Germany). A PE-10 catheter (Becton Dickinson & Co, Parsippany, NJ) was inserted into the p u l m o n a r y artery for infusion. A p u l m o n a r y v e n o t o m y was p e r f o r m e d and the effluent suction collected by a catheter placed in proximity to the venotomy. At the completion of the perfusions both the v e n o t o m y and arteriotomy were r e p a i r e d with 9-0 nylon suture (Ethicon, Somerville, NJ) a n d the p u h n o n a r y circulation was restored. Through a separate puncture wound, a 16-gauge catheter connected to a 3-mL syringe (Becton Dickinson & Co) was introduced into the left chest cavity to facilitate lung reexpansion. The thoracotomy incision was closed in three layers. W h e n animals recovered, their chest tubes and endotracheal tubes were removed.

Experiment 1: BSO Dose Response Twenty male F344 rats (250 to 300 g) were r a n d o m i z e d into five groups (n - 4/group) to d e t e r m i n e the appropriate dose of L-buthionine-SR-sulfoximine (Schweizerhall Inc, Plainfield, NJ) to be used for therapeutic studies. All animals were injected subcutaneously in the right flank with 1 × 107 viable m e t h y l c h o l a n t h r e n e - i n d u c e d sarcoma cells. W h e n the tumors reached approximately 5% t u m o r b u r d e n by weight, a dose response was performed to d e t e r m i n e the dose of BSO that maximally decreased tissue glutathione levels. Animals u n d e r w e n t intraperitoneal injections (2 mL/injection) every 8 hours for 24 hours. Animals were r a n d o m i z e d into the following five t r e a t m e n t groups: control (Hespan), group I (0.5 m m o l / k g of BSO), group II (2.0 m m o l / k g of BSO), group III (4.0 m m o l / k g of BSO), and group IV (6.0 m m o l / k g of BSO). Two hours after the last injection, animals were e u t h a n i z e d and tissues were harvested.

Experiment 2: Efficacy Sixty male F344 rats (200 to 250 g) were r a n d o m i z e d into six groups. On day zero, all groups u n d e r w e n t an intravenous injection of 5 × 10 ~"viable methylcholanthrene-

Glutathione Processing and Analysis The tumors were excised, weighed, a n d i m m e d i a t e l y h o m o g e n i z e d (Brinkmann Instruments, Westbury, NY) in 5% 5-sulfosalicylic acid (Fisher Scientific). A laparotomy was p e r f o r m e d and the portal vein was c a n n u l a t e d with a 22-gauge a n g i o g r a p h y catheter. The liver was per(used with 10 mL of ice-cold saline solution to wash out red blood cells. A s e g m e n t of the right lobe then was excised, weighed, and h o m o g e n i z e d in 5% 5-sulfosalicylic acid. Samples were spun in a microfuge centrifuge (Fisher Scientific), and the s u p e r n a t a n t was derivatized according to a modification of the m e t h o d of N e w t o n and associates [17]. One h u n d r e d twenty microliters of 2 m m o l / L diethyle n e t r i a m i n e pentaacetic acid (Sigma) was a d d e d to 360 ~zL of sample. One h u n d r e d microliters of 2 mol/L Tris-HC1 (pH 9) then was a d d e d , followed i m m e d i a t e l y by 5 ~tL of 0.1 mol/L m o n o b r o m o b i m a n e (Calbiochem, LaJolla, CA). The reaction was allowed to p r o c e e d in the dark for 20 minutes and then was s t o p p e d with 15 ~tL of glacial acetic acid. Samples were quantitated by highperformance liquid c h r o m a t o g r a p h y (Waters Associates, Milford, MA) using fluorescence detection. The system consisted of a p u m p series 501, gradient controller 680, data m o d u l e 746, a n d fluorescence detector 420-AC. One h u n d r e d micro|iters of the s a m p l e was injected into a 5-~tm C18 (4.6 × 250 mm) Ultrasphere ODS (Beckman) column using a dual solvent system. Solvent A consisted of 9% methanol a n d 0.25% acetic acid (pH 3.9), and solvent B consisted of 90% methanol and 0.25% glacial acetic acid (pH 3.9). One h u n d r e d percent solvent A was run for 12 minutes at I mL/min., followed by 94% solvent A and 6% solvent B for 12 minutes. Then 100% solvent B was run for 25 minutes followed by reequilibration with solvent A. Curves were c o m p a r e d with external stan-

Ann Thorac Surg 1995;60:239-44

PORT ET AL ISOLATED LUNG PERFUSION "vVITHBSO PRETREATMENT

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Fig 1. Liver glutathione levels after treatment with buthionine su(t ~ oximine (BSO) (n = 4 rats/group; dose: group I, 0.5 mmol/kg; group II, 2.0 mmol/kg; group IIL 4.0 mmol/kg; group IV, 6.0 mmol/kg). (~p < 0.05 versus CTL; ~'p < 0.05 versus group 1; CTL control group.)

Fig 2. Tumor glutathione levels after treatment with buthionine sulfoximine (BSO) (n = 4 rats/group; dose: group L 0.5 mmol/kg; group IL 2.0 mmol/kg; group III, 4.0 mmol/kg; group IV, 6.0 mmol/kg). ('~p < 0.05 versus CTL; bp < 0.05 versus group I; CTL = control group.)

d a r d s of g l u t a t h i o n e (Sigma) d e r i v i t i z e d in a similar fashion.

ther in g r o u p s III a n d IV in c o m p a r i s o n w i t h g r o u p s I a n d II (p ~ 0.05).

Chemicals

E x p e r i m e n t 2: Efficacy T h e r e was no i m m e d i a t e p e r i o p e r a t i v e m o r t a l i t y in any of the groups. Two a n i m a l s in g r o u p III d i e d on day 13 after i n o c u l a t i o n with massive, bilateral, p u l m o n a r y tum o r r e p l a c e m e n t . T r e a t m e n t r e s p o n s e is d e p i c t e d in Table 1. T h e r e was no difference in t r e a t m e n t - r e l a t e d mortality a m o n g the groups. All g r o u p s h a d m a s s i v e t u m o r r e p l a c e m e n t of their u n t r e a t e d right l u n g s (Fig 3). T h e r e was no r e s p o n s e to t r e a t m e n t n o t e d in t h e left lungs of those a n i m a l s that r e c e i v e d either H e s p a n or d o x o r u b i c i n i n t r a v e n o u s t h e r a p y ( g r o u p s I a n d II) or H e s p a n ILP (groups III and IV). T h o s e a n i m a l s that r e c e i v e d d o x o r u b i c i n ILP (groups V a n d VI) h a d a decrease in the n u m b e r of t u m o r n o d u l e s in their t r e a t e d left l u n g s in c o m p a r i s o n w i t h g r o u p s I to W (p ~ 0.01). T h e r e was a substantial difference in r e s p o n s e b e t w e e n g r o u p s V a n d VI as well. T h e r e w e r e 223 ± 97 n o d u l e s in the t r e a t e d left l u n g s of a n i m a l s that r e c e i v e d H e s p a n p r e t r e a t m e n t with d o x o r u b i c i n ILP (group V) a n d 16 +_ 22 n o d u l e s in the t r e a t e d left l u n g s of a n i m a l s that r e c e i v e d p r e t r e a t m e n t w i t h BSO a n d d o x o r u b i c i n ILP (group VI;

All c h e m i c a l s w e r e of high purity ( h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y grade). Statistical A n a l y s i s All data are p r e s e n t e d as m e a n ~- s t a n d a r d deviation. A n a l y s i s was p e r f o r m e d by o n e - w a y analysis of v a r i a n c e or u n p a i r e d t test w h e r e a p p r o p r i a t e . Significance was d e f i n e d as p less t h a n or e q u a l to 0.05.

Results

E x p e r i m e n t 1: D o s e R e s p o n s e The b o d y w e i g h t s a n d p e r c e n t t u m o r b u r d e n by w e i g h t w e r e similar b e t w e e n groups. Body w e i g h t s of the control g r o u p w e r e 280 m 13 g, and for g r o u p s I t h r o u g h IV w e r e 277 _+ 21, 274 _+ 17, 277 + 14, a n d 279 _+ 18 g, r e s p e c t i v e l y (p = not significant). The p e r c e n t t u m o r b u r d e n in all g r o u p s r a n g e d f r o m 2% to 4% (p = not significant). B u t h i o n i n e - s u l f o x i m i n e t r e a t m e n t d e c r e a s e d liver a n d t u m o r g l u t a t h i o n e levels in a d o s e - d e p e n d e n t m a n n e r (Figs 1, 2). Liver g l u t a t h i o n e level for the control g r o u p was 20.60 _+ 0.60 txmol/g, a n d for g r o u p s I t h r o u g h IV the levels w e r e 8.60 + 4.24, 2.31 ÷ 0.23, 1.62 + 0.54, a n d 1.90 -- 1.35 /~mol/g, respectively. Liver g l u t a t h i o n e levels in rats that r e c e i v e d BSO ( g r o u p s I t h r o u g h IV) w e r e significantly d e c r e a s e d in c o m p a r i s o n with the control g r o u p (p ~ 0.05). In addition, liver g l u t a t h i o n e levels w e r e f u r t h e r d e c r e a s e d in g r o u p s lI t h r o u g h IV w h e n c o m p a r e d w i t h g r o u p I (p _< 0.05). T u m o r g l u t a t h i o n e level in the control g r o u p was 2.26 + 0.07 # m o l / g , a n d the levels in g r o u p s I t h r o u g h IV w e r e 0.39 _~ 0.12, 0.28 _+ 0.09, 0.21 + 0.10, a n d 0.16 _+ 0.03 /~mol/g, respectively. T u m o r g l u t a t h i o n e levels in g r o u p s l t h r o u g h IV w e r e d e c r e a s e d in c o m p a r i s o n w i t h the control g r o u p (p < 0.05). In addition, t u m o r g l u t a t h i o n e levels w e r e d e c r e a s e d fur-

Table 1. Response to Therapy Group I (n -

ll (n lII (n IV (n V (n VI (n -

6) 6) 10) 12) 12) 12)

Pretreatment

Treatment

Left Lung Nodules

Hespan IP BSO IP Hespan IP BSO IP Hespan IP BSO IP

Hespan IV DOX IV Hespan [LP Hespan ILP DOX ILP DOX ILP

>500 >500 >500 >500 223 _+ 97~ 16 _+ 22a'b

"~p 0.05versus groups [, [I, llI, IV. b p < 0.05 versus group V. DOX = doxorubicin; BSO buthionine sulfoximine; CR - complete response; ILP isolated lung perfusion; IP intraperitoneaI; IV - intravenous.

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PORT ET AL iSOLATED L U N G PERFUSION WI~IH BSO PRETREATMENT

A

C

B

D

Fig 3. A single, representative, posterior view of the lun~;s of the four treatment groups that underwent isolated lung perfusion (ILP): (A) Hespan intraperitoneally/Hespan ILP (group liD, (B) BSO intraperitoneally/Hespan ILP (group/VL (C) Hespan intraperitoneally/ doxorubicin ILP (group V), (i9) BSO intraperitoneally/doxorubicin ILP (group VI).

p -~ 0.01). In addition, there was a complete response to therapy in 5 of the animals in group VI with no complete responses in group V.

Comment Soft tissue sarcomas metastasize almost exclusively to the lung, and the lung is often the sole site of recurrence after treatment [18]. Although surgery remains the mainstay of treatment for metastatic pulmonary sarcoma, 5-year survival after complete resection only approaches 25% [1]. Residual microscopic pulmonary disease is thought to be responsible for local recurrence after complete resection [4]. Combination chemotherapy has been implemented to treat metastatic pulmonary, sarcoma I1]. Doxorubicinbased regimens have proved to be the most efficacious [6]. However, doxorubicin-associated cardiac toxicity [6] and the frequent development of drug-resistant tumor cell populations after repeated courses of doxorubicin have limited the systemic use of this drug [19]. Attempts have been made to potentiate the cytotoxic effects of chemotherapy while minimizing systemic toxicity. Ideally, a strategy is needed that would produce preferential sensitization of tumor cells while providing simultaneous protection of normal tissue. To address the clinical problem of systemic toxicity of chemotherapy, we have developed a technique of ILP with doxorubicin in the rat that results in minimal

A n n Thorac S u r g 1995;60:239-44

systemic drug toxicity, operative morbidity, and mortality [7]. With this model, we are capable of raising pulmonary levels of doxorubicin to 20 times that of comparable systemic doses with a sevenfold reduction in heart concentrations. Although this technique holds the promise of delivering high-dose local chemotherapy without adverse systemic exposure, there is still a real concern regarding local toxicity. Studies examining isolated doxorubicin lung perfusion in dogs noted significant lung damage at low drug concentrations [8]. The ability to increase doxorubicin tumor cytotoxicity at lower concentrations might further reduce the toxicity to surrounding normal tissue. The acquired resistance of cancer cells to antitumor drug therapy is frequently a major cause of treatment failure in patients [19]. Chemotherapy is administered routinely at near maximum tolerated doses. It is usually not possible to increase the dose of an agent by even twofold [5]. Therefore, the degree of drug resistance necessary for a tumor to become refractory to conventional therapy is minimal. The mechanism by which tumor cells develop resistance to antineoplastic therapy has been the focus of considerable research efforts. The ability to overcome this resistance offers the potential for increased chemotherapeutic efficacy at lower tissue levels and with less systemic toxicity. A n u m b e r of mechanisms have been proposed to explain tumor drug resistance. There is increasing evidence to support a pivotal role for glutathione in the resistance of tumors to chemotherapy and radiation [10-14]. Glutathione is the major component of the intracellular nonprotein sulfhydryl p o o l and acts as a detoxifying agent by scavenging free radicals. Glutathione has been shown to be important in the detoxification of chemotherapeutic agents such as doxorubicin that produce activated oxygen species such as hydrogen peroxide, superoxide, and hydroxyl groups [20, 21]. The recent discovery of chemoresistant h u m a n tumor cells that contain higher levels of glutathione and glutathione synthetic activity in comparison with chemosensitive tumor cells as well as normal cells suggests that glutathione may be an important factor capable of providing tumor resistance to conventional cancer treatment [22]. Pharmacologic manipulation of tissue glutathione levels may allow augmentation of antineoplastic therapy. Buthionine sulfoximine is a potent inhibitor of ~,-glutamylcysteine synthetase, an enzyme catalyzing the ratelimiting step in glutathione synthesis [9-13J. Buthionine sulfoximine has been shown to increase the effectiveness of numerous chemotherapeutic agents in vitro and in vivo [10-14]. It was found that BSO pretreatment of mice bearing drug-resistant leukemia cells before chemotherapy led to an increased survival [11[. A relationship between ovarian cancer drug resistance and glutathione activity has also been established [14]. This resistance has been shown to be reversed by BSO depletion of tumor cell glutathione levels. It is therefore possible that the clinical use of BSO in combination with chemotherapy might provide a means of overcoming drug resistance in

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PORT ET AL 1SOLATEDLUNG PERFUSIONW1THBSO PRETREATMENT

tumors. However, the administration of BSO might sensitize normal tissue to systemic c h e m o t h e r a p y and increase u n w a n t e d toxicities. Ideally, a system that delivered locoregional c h e m o t h e r a p y without systemic leak could capitalize on BSO p r e t r e a t m e n t and tu m or sensitization without increasing systemic toxicity. This study examined the effect of the addition of BSO p r e t r e a t m e n t in conjunction with doxorubicin ILP for the treatment of metastatic p u l m o n a r y sarcoma. The first e x p e r i m e n t was p e r f o r m e d to d e t e r m i n e the appropriate dose of BSO p r e t r e a t m e n t in our animal model. Other studies previously have e x a m i n e d the pharmacokinetics of BSO treatm e n t and have d e m o n s t r a t e d a substantial reduction in tissue glutathione level lasting several days after a similar BSO dosing r e g i m e n [11, 13]. We anticipated that if glutathione did play a significant role in methylcholant h r e n e - i n d u c e d sarcoma drug resistance, that short-term glutathione depletion would be a d e q u a t e to demonstrate increased efficacy with ILP. Pretreatment of animals with 2 m m o l / k g (× 3 doses) of BSO over 24 hours depleted liver and t u m o r glutathione levels maximally. This dose then was used to d e te r m in e if BSO p r e t r e a t m e n t would increase the efficacy of doxorubicin ILP. There were no differences in mortality a m o n g the groups. This indicates that BSO p r e t r e a t m e n t can be administered safely in conjunction with ILP. In addition, this c o m b i n e d t r e a t m e n t modality was highly effective for the t reat m en t of metastatic p u l m o n a r y sarcoma. There was a 13-fold reduction in tu m o r nodules in 7 animals and a complete response in 5 animals that were pretreated with BSO followed by doxorubicin ILP in comparison with all other therapies. The group pretreated with H e s p a n followed by doxorubicin ILP had only a limited response to therapy with no complete responses. No other groups had a response to therapy. These experiments indicate that BSO p r e t r e a t m e n t potentiates the efficacy of doxorubicin delivered by ILP. In summary, 2 m m o l / k g of BSO IP given in three doses over 24 hours is effective in depleting tu m o r glutathione levels in our model. Using this dose of BSO, we have d e m o n s t r a t e d that ILP with doxorubicin c o m b i n e d with BSO p r e t r e a t m e n t is superior therapeutically to doxorubicin ILP alone or to intravenous doxorubicin with BSO pretreatment. Isolated lung perfusion with BSO pretreatm e n t offers the potential for a selective increase in t u m o r sensitization and cytoxicity with minimal systemic exposure, In addition, these benefits may be achieved at lower doses of chemotherapy, which potentially would reduce local toxicity to normal tissue. In conclusion, the addition of BSO p r e t r e a t m e n t m a y prove useful for future clinical studies examining the efficacy of ILP for the t r e at m en t of metastatic p u l m o n a r y disease.

2. Jablons D, Steinberg SM, Roth JA, et al. Metastectomy for soft tissue sarcoma. J Thorac Cardiovasc Surg 1989;97:695705. 3. Casson AG, Putnam JB, Natarajan G, et al. Five-year survival after pulmonary metastectomy for adult soft tissue sarcoma. Cancer 1992;69:662-8. 4. Huth JF, Holmes EC, Vernon SE, et al. Pulmonary resection for metastatic sarcoma. Am J Surg 1980;140:9-14. 5. Samuels BL, Murray JL, Cohen MB, et al. Increased glutathione peroxidase activity in a human sarcoma cell line with inherent doxorubicin resistance. Cancer Res 1991;51:521-7. 6. Casper ES, Gaynor JJ, Hajdu SI, et al. A prospective randomized trial of adjuvant chemotherapy with bolus versus continuous infusion of doxorubicin in patients with high grade extremity soft tissue sarcoma and an analysis of prognostic factors. Cancer 1991;68:1221-9. 7. Weksler B, Ng B, Lenert JT, Butt M. Isolated single-lung perfusion with doxorubicin is pharmacokinetically superior to intravenous injection. Ann Thorac Surg 1993;56:209-14. 8. Baciewicz FA, Arredondo M, Chaudhuri B, et al. Pharmacokinetics and toxicity of isolated perfusion of lung with doxorubicin. J Surg Res 1991;50:124-8. 9. Johnston MR, Christensen CW, Minchin RF, et al. Isolated total lung perfusion as a means to deliver organ-specific chemotherapy: long term studies in animals. Surgery 1985; 98:35-44. 10. Mitchell JB, Cook JA, DeGraff W, Glastein E, Russo A. Glutathione modulation in cancer treatment: will it work? Int J Radiat Oncol Biol Phys 1989;16:1289-95. 11. Meister A. Glutathione deficiency produced by inhibition of its synthesis, and its reversal: applications in research and therapy. Pharmacol Ther 1991;51:155-94. 12. Russo A, DeGraff W, Friedman N, Mitchell J. Selective modulation of glutathione levels in human normal versus tumor cells and subsequent differential response to chemotherapy drugs. Cancer Res 1986;46:2845-8. 13. Terradez P, Asensi M, De La Vega C, et al. Depletion of glutathione in vivo by buthionine sulfoximine: modulation by the rate of cellular proliferation and inhibition of cancer growth, Biochem ] 1993;292:477-83. 14. Ozols RF, Louie KG, Plowman J, et al. Enhanced melphalan cytotoxicity in human ovarian cancer in vitro and in tumor bearing nude mice by buthionine sulfoximine depletion of glutathione. Biochem Pharmacol 1987;36:147-53. 15. Weksler B, Ng B, Lenert JT, Burt M. A simplified method for endotracheal intubation in the rat. J Appl Physiol 1994;76: 1823-5. 16. Wexler H. Accurate identification of experimental pulmonary metastasis. J Natl Cancer Inst 1966;36:641-5. 17. Newton GL, Dorian R, Fahey RC. Analysis of biological thiols: derivitization with monobromobimane and separation by reverse-phase high-performance liquid chromatography. Anal Biochem 1981;114:383-7. 18. Potter DA, Glenn J, Kinsella T, et al. Patterns of recurrence in patients with high-grade soft-tissue sarcomas. J Clin Oncol 1985;3:353- 66. 19. Dusre L, Mimnaugh G, Myers CE, Sinha BK. Potentiation of doxorubicin cytotoxicity by buthionine sulfoximine in multidrug-resistant human breast tumor cells. Cancer Res 1989; 49:511-5. 20. Meijer C, Mulder NH, Bosscha HT, Zijlstra JG, de Vries EG. Role of free radicals in an Adriamycin resistant human small cell lung cancer cell line. Cancer Res 1987;47:4613-7. 21. Meijer C, Mulder NH, de Vries EG. The role of detoxifying systems in resistance of tumor cells to cisplatin and Adriamycin. Cancer Treat Rev 1990;17:389-407. 22. Godwin A, Meister A, O'Dwyer P, et al. High resistance to cisplatin in human ovarian cancer cell lines is associated with marked increase of glutathione synthesis. Proc Natl Acad Sci USA 1992;89:3070-4.

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PORTET AL ISOLATED LUNG PERFUSION WITH BSO PRETREATMENT

1995;60:239-44

DISCUSSION DR THOMAS M, EGAN (Chapel Hill, NC): What do you think the cardiac toxicity would be in vivo, and have you looked at that in any surviving rats that you have perfused? DR PORT: There is evidence that glutathione may provide cardioprotection from agents such as doxorubicin. Therefore, there has been considerable hesitancy in using BSO, an inhibitor of glutathione, with systemic doxorubicin therapy. We have demonstrated in our model of isolated lung perfusion that there is very little systemic leak when perfusions are performed with doxorubicin. We have shown that the cardiac outputs of rats perfused with doxorubicin are preserved in comparison with intravenously treated animals. Doxorubicin isolated lung perfusion with BSO pretreatment is therefore an ideal treatment model. DR FRANK A. BACIEWICZ (Detroit, M1): 1 enjoyed your paper. You have done previous studies at your institution with doxorubicin alone and showed almost no tumor in the lung. In this study did you select less than a maximum dose of doxorubicin so that tumor is present and a response can be seen with the BSO? If you used higher doses of doxorubicin, were there better results in concert with the BSO? Third, did you do a contralateral pneumonectomy to look at the toxicity in this model?

DR PORT: Our previous studies used much higher doses of doxorubicin for perfusions. In those studies we achieved a complete response to therapy. To assess the efficacy of BSO pretreatment, we needed to lower the dose of doxorubicin to the point where only a partial response was evident. In response to assessing local toxicity, we have not yet performed those experiments. DR LARRY R. KAISER (Philadelphia, PA): I enjoyed your paper. You have done a lot with this model. You have treated with the BSO I believe at day 6 after injection of your tumor cells. I know the methylcholanthrene-induced sarcoma is quite an aggressive tumor. Do you have gross nodules that can be seen at day 7 when you did your isolated perfusion, or is there only microscopic disease at that point? DR PORT: Gross disease is first evident by day 10. DR KAISER: In how many days would the injection of tumor cells that you used here kill these animals if untreated? DR PORT: Animals injected with this number of cells would succumb to disease by 21 days.

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