Ann Thorac Surg 1996;62:848-852
© 1996 The Society of Thoracic Surgeons
Original Articles: General Thoracic
Isolated Lung Perfusion With FUDR in the Rat: Pharmacokinetics and Survival
Jeffrey L. Port, MD,
Bruce Ng, MS,
Jennifer L. Ellis, MD,
Sumihiko Nawata, MD,
Jeffrey T. Lenert, MD,
Michael E. Burt, MDPhD
Thoracic Oncology/Surgical Metabolism Laboratory, Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York
Accepted for publication April 28, 1996.
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Abstract
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Background. Although surgical resection remains the mainstay of treatment for metastatic pulmonary colorectal cancer, 5-year survival approaches only 30% to 40%. We have developed a model of isolated left lung perfusion (ILP) with FUDR (2`-deoxy-5-fluorouridine) for the treatment of pulmonary colorectal metastases. FUDR ILP toxicity and pharmacokinetics were evaluated and compared with continuous intravenous infusion in the rat.
Methods. Toxicity was first evaluated in F344 rats (n = 17) after left ILP (20-minute perfusion at 0.5 mL/min) with 21 mg/mL (n = 11), 28 mg/mL (n = 2), 35 mg/mL (n = 2), and 70 mg/mL (n = 2) of FUDR. Animals were followed up and weights recorded for 14 days postoperatively before a right pneumonectomy was performed to evaluate the effect of FUDR perfusion on left lung function. In the second study, 32 rats (n = 8/group) underwent: systemic FUDR (intravenous), or ILP with 7, 14, and 21 mg/mL respectively (ILP 7, ILP 14, and ILP 21 groups). Left lungs and serum were analyzed for FUDR and 5-fluorouracil by high-performance liquid chromatography.
Results. Rats perfused with doses of FUDR greater than 21 mg/mL died perioperatively. All animals perfused at 21 mg/mL survived until day 14, and 8/11 survived a right pneumonectomy. Rats that survived ILP resumed normal weight gain and grooming habits within 1 week. Pharmacokinetic evaluation demonstrated that ILP at 21 mg/mL maximally elevated total lung FUDR and 5-fluorouracil levels (508.5 ± 96.4 µg/g lung) in comparison with the ILP 14, ILP 7, and intravenous groups (299.1 ± 44.8, 116.0 ± 21.1, and 7.5 ± 4.1 µg/g lung, respectively) (p< 0.05). Serum FUDR levels were 10.5 ± 6.8, 1.3 ± 0.5, 2.31 ± 1.1, and 1.2 ± 0.4 µg/g lung (p = not significant) for intravenous, ILP 7, ILP 14, and ILP 21 groups, respectively.
Conclusions. Isolated left lung perfusion with FUDR is well tolerated to a maximum dose of 21 mg/mL and results in significantly higher FUDR and 5-fluorouracil lung levels with low serum levels compared with intravenous treatment. These higher pulmonary levels may offer advantages in the treatment of pulmonary colorectal metastases.
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Introduction
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It is estimated that there will be approximately 140,000 new cases of colorectal adenocarcinoma in 1995 with more than 55,000 related deaths [1]. In approximately 2% to 4% of these patients, isolated pulmonary metastases will develop [2]. Systemic chemotherapy, which is often limited by host toxicity, holds little promise for long-term survival [3]. Although pulmonary resection has been the standard of therapy for isolated pulmonary disease, 5-year survival approaches only 30% [4–7].
A model of isolated left lung perfusion (ILP) in the rat has been established in our laboratory for the treatment of metastatic pulmonary sarcoma [8]. Isolated left lung perfusion with doxorubicin has been effective in the treatment of metastatic pulmonary sarcoma while minimizing systemic toxicity [9, 10]. These results have been encouraging and have led to further investigation of ILP with FUDR (2`-deoxy-5-fluorouridine) for the treatment of pulmonary colorectal metastases.
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Material and Methods
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Animals (male F344 rats; Charles Rivers, Kingston, NY) were treated in accordance with the Animal Welfare Act and the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (NIH publication 85-23, revised 1985). Experiments were approved by the Institutional Animal Care and Use Committee, Memorial Sloan-Kettering 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 temperature controlled and provided a 12-hour light/dark cycle.
Isolated Lung Perfusion
Isolated left lung perfusion was performed by the method previously established in this laboratory [8]. Briefly, animals were anesthetized with pentobarbital (50 mg/kg) intraperitoneally. Under direct visualization, the animals were intubated with a 16-gauge intravenous (IV) catheter placed over a guidewire and then placed on a volume ventilator (Rodent Ventilator model 683; Harvard Apparatus, South Natick, MA) [11]. Ventilation was maintained at a tidal volume of 10 mL/kg, with 100% O2 supplemented with 0.5% halothane at a rate of 75 strokes/min. The left chest was shaved and prepared with a povidone-iodine 10% solution, and then entered through a fourth intercostal incision. The left pulmonary artery and vein were visualized under an operative microscope (OpMi-1, 16x; Carl Zeiss, Wotan, Germany). A PE-10 catheter (Becton Dickinson & Co, Parsippany, NJ) was inserted into the pulmonary artery for FUDR infusion. A pulmonary venotomy was performed for effluent retrieval. Those animals evaluated in toxicity and survival experiments had a catheter placed in proximity to the venotomy for effluent collection. At completion of the perfusion, both the venotomy and arteriotomy were repaired with 9-0 nylon suture (Ethicon, Somerville, NJ) and the pulmonary 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. When animals recovered, their chest tubes and endotracheal tubes were removed. In animals that underwent pharmacokinetic study, a PE-90 catheter (Becton Dickenson & Co) was inserted into the pulmonary vein for effluent collection. After these acute experiments, animals were sacrificed by intraperitoneal injection of pentobarbital (150 mg/kg).
Experiment 1
Seventeen male F344 rats (200 to 250 g) were randomized into four groups for toxicity trials with FUDR (Floxuridine; Roche Laboratories, Nutley, NJ) via ILP. Toxicity was evaluated after ILP with 21 mg/mL (n = 11), 28 mg/mL (n = 2), 35 mg/mL (n = 2), and 70 mg/mL (n = 2) of FUDR in a buffered hespan solution (BHE). Each group underwent left lung ILP with FUDR for 20 minutes at a rate of 0.5 mL/min (Syringe Infusepump 22; Harvard Apparatus) followed by a 10-minute BHE flush at the same rate. Rats that survived were followed up and their weights were recorded. On day 14, animals underwent a right pneumonectomy to evaluate the effect of FUDR perfusion on left lung function. Subsequently, all animals were sacrificed and histologic evaluation of perfused left lungs was performed.
Experiment 2
Thirty-two male F344 rats (200 to 250 g) were randomized into four treatment groups (n = 8/group): IV, ILP 7, ILP 14, and ILP 21. The IV group received systemic IV FUDR (1 mg•kg-1•day-1) for 7 days via an osmotic minipump infusion (Alzet minipump model 2ML1; Alza Corp, Palo Alto, CA). This dose corresponds to a human dosing schedule of 0.17 mg•kg-1•day-1 [12]. The osmotic minipumps were loaded with FUDR, heparin (500 units), and BHE and then connected to PE-50 (Becton Dickenson & Co) catheters the evening before placement. The pumps were primed by incubation overnight in a 37°C BHE bath. Before pump placement, the animals were anesthetized with intraperitoneal pentobarbital (50 mg/kg), and 500 units of penicillin were injected subcutaneously. The pumps were placed in the subcutaneous space of the anterior abdominal wall. The pumps' catheter tips were tunneled subcutaneously into a right neck incision and inserted into the right external jugular vein. The incisions were closed and animals were observed for 7 days before sacrifice. On day 7, the animals were sacrificed by pentobarbital overdose. Serum was drawn via abdominal aortic puncture. The pumps were removed and checked for proper drug delivery. The left lungs were excised and weighed, and samples were stored at -70°C for later FUDR and 5-fluorouracil (5-FU) analysis.
Isolated left lung perfusion groups underwent ILP for 20 minutes with 7, 14, or 21 mg/mL of FUDR infused at 0.5 mL/min, followed by a BHE flush for 10 minutes at 0.5 mL/min. The ILP groups had a PE-90 catheter inserted into the pulmonary vein for collection of pulmonary effluent samples. The pulmonary vein effluent samples were collected at 2-minute intervals throughout the 20-minute FUDR infusion, and then every 5 minutes during the flush. Serum samples were drawn by direct intracardiac puncture at the completion of the perfusions. Animals were then sacrificed, and the left lungs were excised, weighed, and frozen at -70°C for later FUDR and 5-FU analysis.
Sample Processing and Measurement
Pulmonary effluent and serum samples were collected and centrifuged, and the supernatant was stored at -70°C for subsequent high-performance liquid chromatographic analysis [13]. Lung tissue samples were homogenized with a Brinkmann homogenizer (Brinkmann Instruments, Westbury, NY) in saline solution to a sample ratio of 1:5. Bromouridine (Sigma Inc, St. Louis, MO) was used as an internal standard (1 mg/mL). Twenty-five microliters of standard, 50 µL of perchloric acid, and 50 µL of potassium hydroxide were added to 200 µL of tissue homogenate or pulmonary effluent supernatant. Samples were vortexed and centrifuged to precipitate proteins. Samples were analyzed by high-performance liquid chromatography. A Perkin Elmer pump series 410 LCI, an autosampler ISS 200, and an LC75 spectrophotometric detector (Perkin Elmer Corp, Plainview, NJ) were used for analysis. Fifty microliters of sample was injected into a C18, reverse-phase, Extrasil column (Phenomenex, Torrance, CA). The mobile phase consisted of 25 mmol/L potassium phosphate and 2% acetonitrile at a flow rate of 1 mL/min. Curves were compared with standard curves prepared from plasma and lung.
Statistics
Statistical analysis was performed by one-way analysis of variance. Data are expressed as mean ± standard error of the mean and significance is defined as p less than 0.05.
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Results
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Toxicity and Survival Experiment
Animals perfused with doses greater than 21 mg/mL of FUDR died perioperatively (Table 1
). Those rats perfused at 70 mg/mL of FUDR died before the completion of the perfusion. Rats perfused with 35 and 28 mg/mL survived several hours after ILP before dying. All animals perfused with 21 mg/mL survived the operation. After ILP with 21 mg/mL, animals lost weight for 2 days and then recovered, gaining weight until the completion of the study on day 14 (Fig 1). Eight of 11 animals (73%) perfused at 21 mg/mL survived a right contralateral pneumonectomy on day 14. Histologic evaluation revealed normal left lung architecture after perfusion with 21 mg/mL of FUDR (Fig 2).
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Fig 2. . Histologic evaluation of a left lung perfused with 21 mg/mL of FUDR. Note the normal lung architecture.
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Pharmacokinetics
Animals undergoing placement of the osmotic pumps for IV perfusion of FUDR (IV group) survived for 7 days and received the total 7-day dose of FUDR as confirmed by aspiration of pump contents at the completion of the study.
Levels of FUDR in the pulmonary venous effluent in ILP groups reached a plateau after 2 to 4 minutes (Fig 3) and were constant throughout the 20 minutes of FUDR perfusion. After the 20 minutes of FUDR perfusion, a BHE washout was performed for 10 minutes. At the completion of this washout, no FUDR was detected in the venous effluent. The FUDR extraction ratio for all perfusion groups was not significantly different and was equal to less than 1% of all drug delivered.
Serum FUDR in all treatment groups was analyzed (Fig 4). A small amount of systemic leak was noted in all ILP groups, which was not significantly different than IV serum levels.
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Fig 4. . Serum FUDR levels after intravenous (iv) FUDR or FUDR isolated left lung perfusion (ILP) therapy. (ILP 7 = isolated lung perfusion with 7 mg/mL of FUDR; ILP 14 = isolated lung perfusion with 14 mg/mL of FUDR; ILP 21 = isolated lung perfusion with 21 mg/mL of FUDR; n.s. = not significant.)
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Total lung FUDR and 5-FU levels were measured for all treatment groups (Fig 5). Total lung FUDR and 5-FU levels were significantly elevated in groups ILP 14 and ILP 21 (299.1 ± 44.8 and 508.5 ± 96.4 µg/g lung) in comparison with the ILP 7 and IV treatment groups (116.3 ± 21.1 and 7.5 ± 4.1 µg/g lung) (p < 0.05). Total lung levels were also significantly elevated in ILP 21 animals in comparison with ILP 14 animals (p < 0.05).
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Fig 5. . Total FUDR and 5-fluorouracil (5-FU) lung levels after intravenous (iv) FUDR or FUDR isolated left lung perfusion (ILP) therapy. (ILP 7 = isolated lung perfusion with 7 mg/mL of FUDR; ILP 14 = isolated lung perfusion with 14 mg/mL of FUDR; ILP 21 = isolated lung perfusion with 21 mg/mL of FUDR.)
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Comment
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The treatment of metastatic pulmonary colorectal cancer poses a significant clinical problem [2]. Although operation remains the gold standard of therapy, 5-year survival approaches only 30% to 40% [4–7]. Surgical treatment failures appear to stem from the presence of micrometastatic disease present at the time of surgical resection. Systemic chemotherapy has offered little survival benefit for patients with metastatic disease [14]. Overall response rates of 20% with median survival of 8 to 10 months have been reported. The disappointing results with chemotherapy are related to the inability to deliver adequate cytotoxic doses without incurring extensive host toxicity [15, 16]. The fluorinated pyrimidines, FUDR and 5-FU, are among the most active agents in the treatment of colorectal adenocarcinoma. The use of these drugs, however, can result in extensive myelosuppression and gastrointestinal distress [3].
Isolated lung and liver perfusion systems have been developed to deliver high doses of local chemotherapy while minimizing systemic toxicity [8, 17]. Isolated single-lung perfusion in an animal model has been developed in our laboratory. To date, ILP with doxorubicin has been efficacious in the treatment of metastatic pulmonary sarcoma with minimal systemic toxicity [9, 10]. These results have been encouraging and have motivated us to investigate the use of ILP with FUDR for the treatment of metastatic pulmonary colorectal cancer.
Acute toxicity and survival experiments were initially undertaken to determine the maximum dose of FUDR ILP compatible with animal survival. These experiments established 21 mg/mL of FUDR delivered via ILP to be the maximum dose compatible with survival. All animals perfused at this dose survived ILP and 8/11 animals subsequently survived a contralateral right pneumonectomy on day 14. Histologic evaluation also confirmed that this concentration of FUDR delivered via ILP leads to minimal acute toxicity as evidenced by a lack of significant histologic changes. Isolated left lung perfusion with FUDR at 21 mg/mL at a rate of 0.5 mL/min for 20 minutes will deliver a total dose of 210 mg. This dose is equivalent to 840 mg/kg for a 250-g animal. The reported median lethal dose for rats after an IV bolus injection of FUDR is 670 mg/kg. These first experiments demonstrate that high-dose FUDR ILP can be performed with animal survival at lethal systemic doses. Furthermore, these doses were not toxic to the local environment acutely.
The pharmacokinetic profile of FUDR via ILP was compared with IV FUDR therapy. Isolated left lung perfusion with FUDR at 21 mg/mL maximally elevated total lung FUDR and 5-FU levels in comparison with all other groups. The amount of systemic leak evidenced in our study with ILP was minimal in all ILP groups. These serum levels compared favorably with IV FUDR-treated groups.
It has been previously established in an ex-vivo lung perfusion model that the lung is an inefficient extractor of FUDR. Further, the lung is unable to catabolize FUDR beyond 5-FU [18]. The amount of FUDR extracted by the lung in our study with ILP was less than 1% of total FUDR delivered. This is similar to previously reported values. The relatively inefficient uptake of FUDR by the lung combined with the lung's minimal catabolic clearance may have important clinical implications for the treatment of metastatic pulmonary colorectal cancer.
In conclusion, FUDR ILP is well tolerated to a maximum concentration of 21 mg/mL for 20 minutes at a rate of 0.5 mL/min. At this concentration, total FUDR and 5-FU lung levels are significantly elevated with low serum levels in comparison with IV FUDR therapy. The higher pulmonary levels achieved in this model, combined with the favorable pharmacokinetics as a result of the lung's inefficient FUDR extraction as well as the lung's inability to detoxify FUDR, may offer advantages in the treatment of pulmonary colorectal metastases.
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Footnotes
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Address reprint requests to Dr Burt, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021.
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Abstract
Introduction
