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Ann Thorac Surg 1998;65:1420-1425
© 1998 The Society of Thoracic Surgeons
a Department of Thoracic and Cardiovascular Surgery, University of Berne, Berne, Switzerland
b Memorial Sloan Kettering Cancer Center, New York, New York, USA
Accepted for publication December 8, 1997.
Address reprint requests to Dr Furrer, Thoracic and Vascular Surgery, Kantonsspital, CH-7000 Chur, Switzerland
e-mail: (markus.furrer{at}ksc.chur.ch)
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Abstract |
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Methods. Isolated lung perfusion with and without doxorubicin was performed on white pigs during 15 minutes either by a single-pass system (n = 7) or by a recirculating-blood perfusion system (n = 7). Three animals with endovenous drug application served as controls. Leakage was assessed using isotopic tracers. Perfusion-induced lung tissue injury was determined by postperfusion chest radiographs, by angiotensin-converting enzyme-to-protein ratio in the plasma and in the bronchioalveolar lavage fluid, and by wet-to-dry weight ratio and histologic examination of lung biopsy specimens at 20 and 50 minutes. Doxorubicin concentration in lung tissue and plasma was compared between the three study groups.
Results. All isolated lung perfusion studies were successfully performed without significant systemic leakage (<0.6%). Wet-to-dry weight ratio was significantly lower after single-pass as compared with recirculating-blood perfusion and endovenous drug application at both time points (5.0 ± 1.1 and 5.3 ± 0.8 for single-pass versus 6.6 ± 1.1 and 6.9 ± 0.5 for recirculating-blood versus 6.6 ± 0.2 and 5.9 ± 0.7 for the control group, respectively; p < 0.05). Angiotensin-converting enzyme-to-protein plasma ratio in the single-pass group was significantly lower only at 20 minutes (6.3 ± 2.4 versus 9.3 ± 1.0 versus 9.7 ± 1.9, respectively; p < 0.05) but not at 50 minutes. Angiotensin-converting enzyme-to-protein ratio in bronchoalveolar lavage fluid, histology of lung biopsy specimens, and chest radiographs did not differ significantly between the three groups. Doxorubicin lung tissue concentration was not significantly different after single-pass (17.5 µg/g) and recirculating-blood perfusion (21.9 µg/g), but was significantly higher than after endovenous drug application (3.0 µg/g; p < 0.01).
Conclusions. Both isolated lung perfusion techniques resulted in a sixfold to sevenfold higher doxorubicin lung tissue concentration than after endovenous application. Isolated lung perfusion-induced lung injury was similar for both techniques, but recirculating-blood perfusion appeared to result in more acute lung injury and was technically more demanding than single-pass perfusion.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentFootnotesAcknowledgmentsReferences |
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Isolated perfusion techniques were developed to deliver an increased amount of drug to the target tissue while reducing the systemic side effects of chemotherapy [8–13]. Two different techniques for isolated lung perfusion (ILP) have been described: the single pass system (SP), which discards the venous effluent after passage through the perfused tissue, and the recirculating-blood circuit (RB), which collects the venous effluent in a reservoir to be reinfused after oxygenation [9–11, 14–16]. Although SP perfusion facilitates drug kinetic studies by ensuring a constant drug concentration in the perfusate, the use of oxygenated blood in an RB system might be more physiologic and more effective because hypoxic tumor areas are avoided. Moreover, longer perfusion periods with smaller amounts of perfusate and drugs might be applied with the RB system [14, 17].
To determine benefits and drawbacks of isolated SP and RB lung perfusion using doxorubicin as the cytostatic agent, we evaluated the technical practicability in the pig and assessed doxorubicin concentration and tissue injury by chest radiography, repetitive withdrawal of blood samples, lung biopsy specimens, and bronchoalveolar lavage fluid (BALF) at various time intervals during and after the surgical procedure.
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Material and methods |
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Among the total of 17 animals, 7 had SP perfusion (3 without and 4 with 50 mg/m2 doxorubicin [Adriblastin; Pharmacia, Milano, Italy]), 7 had RB perfusion (4 without and 3 with 50 mg/m2 doxorubicin), and 3 animals served as controls and had endovenous doxorubicin application (50 mg/m2) (IV group).
The animals were premedicated with ketamine hydrochloride, 10 mg/kg intramuscularly, and standard endotracheal intubation (Portex blue line 7.0 to 7.5) was performed. Anesthesia was maintained by O2 and NO2-halothane administration. Perioperative monitoring included on-line measurements of arterial blood pressure obtained by open cannulation (6F catheter) of the common carotid artery. For infusions and blood sample collections, a 6F venous catheter was introduced through the left external jugular vein. All animals underwent a standard anterolateral left thoracotomy through the fifth intercostal space.
Anesthesia, monitoring, and surgical procedures were identical for animals receiving doxorubicin by intravenous administration (500 ml of 6% buffered hetastarch via caval catheter during 15 min).
Technique of isolated lung perfusion
The left pulmonary artery and the left superior and inferior pulmonary veins were dissected and encircled with tapes. After intravenous administration of heparin (2 mg/kg), a 16F metal-tipped, right-angled cannula was introduced in the left pulmonary artery through a pursestring suture after proximal occlusion of the vessel. With an angulated Satinsky clamp placed across the left atrium, phlebotomies were performed separately to the superior and inferior pulmonary veins. Pulmonary artery pressure was measured on-line during perfusion by a 4F catheter introduced into the distal pulmonary artery. The left lung was ventilated during perfusion. At the end of the perfusion the perfusate was discarded and the lung was washed with 0.5 L of buffered hetastarch solution during 5 minutes to remove unbound doxorubicin before blood flow was reestablished to the left lung. The pulmonary artery cannula was removed, and the artery and vein were repaired with 7-0 monofilament sutures. Protamine (corresponding to the dose of heparin) was administered after restoration of blood flow.
Recirculating-blood perfusion
The perfusion circuit included a roller pump (Cobe Perfusions System; Lakewood, CO), an oxygenator-reservoir, and a heat exchanger (D 701 Masterflo 34 Infant Hollow Fibre Oxygenator; Dideco, Mirandola, Italy). The venous effluent was drained into the cardiotomy reservoir (Polystan Kardiotomiereservoir F 30; Polystan A/S, Vaerlose, Denmark). The priming volume consisted of 200 mL of autologous blood and 200 mL of 6% buffered hetastarch solution. The perfusion was performed at normothermia (37°C) for 15 minutes at a flow rate of 70 to 120 mL/min, while the on-line measurement of the pulmonary artery pressure did not exceed 20 mm Hg. In 3 animals doxorubicin (50 mg/m2) was added to the perfusate.
Single-pass system
Isolated lung perfusion was carried out by continuous pulmonary arterial infusion of 6% buffered hetastarch with a flow rate of 100 mL/min for 15 minutes while the venous effluent was discarded. In 4 animals doxorubicin (50 mg/m2) was added to the hetastarch infusion.
Leakage detection during isolated lung perfusion
Perfusate leakage ratio during perfusion was checked using a radionuclide technique. Twenty microcuries (0.74 Mbq) of iodine-131–labeled human serum albumin was injected intravenously to establish a baseline count level. After a period of 5 minutes for the establishment of equilibrium, 200 µCi (7.4 Mbq) of iodine-131–labeled human serum albumin was added to the perfusate. Blood samples from the systemic circulation were obtained at 0, 5, 10, 15, 20, and 30 minutes after the inition of lung perfusion.
The leakage factor was calculated from the following formula:
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Assessment of lung tissue injury
For determination of protein and angiotensin-converting enzyme (ACE) levels, blood samples were collected at the beginning of perfusion (0 minutes), at the end of washout (20 minutes), and after the release of the pulmonary circulation (50 minutes). In addition, the same parameters were measured simultaneously in BALF harvested from the left upper lobe by injection of 10 mL of NaCl 0.9% into the left upper main bronchus, followed by aspiration under bronchoscopic control. The ratio of ACE to protein was calculated by comparing enzyme levels to avoid a bias emerging from dilution effects of blood and BALF samples during perfusion.
Lung biopsy specimens were also harvested at 0, 20, and 50 minutes. For this purpose left upper lobe wedge resections were performed and used for determination of wet-to-dry weight ratio and histologic examination. Wet-to-dry weight ratio was calculated after drying the pulmonary tissue for 3 days at 80°C. Histologic examination was performed in a blinded way by the same pathologist (H.J.A.). The findings from each specimen were described individually, and tissue injury was graded using the following score system: 0 = no visible damage, 1 = minimal damage, 2 = obvious damage, and 3 = severe pathologic alterations.
All animals underwent postperfusion chest radiography at the end of the procedure to evaluate radiologic findings of the perfused lung.
Pharmacokinetics
For determination of the doxorubicin level, perfusate samples were collected at 5-minute intervals during perfusion and washout. Blood samples from the systemic circulation were collected at the end of perfusion and at 2 minutes after reestablishment of pulmonary circulation. For drug tissue analyses, biopsy specimens were harvested from the left lower lobe at the end of ILP.
High-performance liquid chromatography analysis was performed with a modification of the procedure reported by Baciewicz and associates [10] with a model 501 pump (Waters Associates, Milford, MA), a model 712 autosampler (Waters), an RP-18 (5 µm) LiChrospher 100 precolumn (Merck, Dietikon, Switzerland), a Nucleosil 7 µm C6H5 column (ET 250/8/4; Macherey Nagel, Oensingen, Switzerland), and a model Fluor LC304 fluorescence detector (Linear Instruments, Reno, NV). Excitation and emission wavelengths were set to 482 and 550 nm, respectively. The mobile phase was prepared from a mixture of 16 mmol/L ammonium formiate and acetonitrile (70:30) to which 2 mL/L dimethylamine was added, followed by adjusting the pH to 4 with formic acid. The flow rate was 1.2 mL/min. Aqueous standard solutions of doxorubicin and daunorubicin (internal standard) were used (100 µg/mL each). Quantitation was based on multilevel internal calibration using peak areas.
Doxorubicin measurement in plasma and perfusate
Calibrator (five levels in the concentration range 0.1 to 5 µg/mL) and control samples (1.0 µg/mL) were prepared in blank swine plasma. Extraction was effected with C 18 Isolute cartridges (International Sorbent Technology, Hengoed Mid Glamorgan, UK), which were conditioned by rinsing three times with 1 mL methanol followed by three times with 1 mL water. One-milliliter sample aliquots (plasma or perfusate, diluted with swine plasma if necessary) were mixed with 20 µL of internal standard solution and applied to the columns. The columns were rinsed with 1 mL of water. Elution occurred first with 50 µL dimethyl sulfoxide and then with 0.5 mL of methanol containing 10% formic acid. The eluate was condensed (40°C under air) to a volume of about 250 µL. For analysis, aliquots of 100 µL were injected. Extraction recovery and interday reproducibility (n = 10) and intraday imprecision (n = 6) at the 1 µg/mL drug level were determined to be 84.5%, 9.55%, and 9.27%, respectively.
Doxorubicin measurement in tissue homogenate
Calibrators (five levels in the range of 0.4 to 8 µg/mL homogenate) and controls (2 µg/mL) were prepared from blank swine lung tissue. Two mL of 0.2 mmol/L potassium dihydrogenphosphate buffer (pH 3.8) and 0.5 g of tissue were homogenized at 15,000 rpm with a Polytron PT 3000 homogenizer (Kinematica, Littau, Switzerland). Two hundred fifty microliters of homogenate and 15 µL of internal standard solution were mixed and incubated for 15 minutes at 37°C. Two hundred fifty microliters of an equivolume mixture of methanol and formic acid was added and the samples were incubated for 15 minutes at 37°C and centrifuged at 15,000 rpm for 3 minutes. The pellet was resuspended with water and the methanol-formic acid mixture (100 µL each) was vortex mixed and centrifuged. The two supernatant fractions were combined, spiked with 25 µL of dimethyl sulfoxide; condensed (45°C under air) to a volume of 250 µL, and again centrifuged at 15,000 rpm. For analysis, aliquots of 150 µL of the clear supernatant were injected. Extraction recovery, interday reproducibility (n = 6), and intraday imprecision (n = 8) at the 2 µg/mL drug level were determined to be 76.3%, 5.05%, and 3.89%, respectively.
Statistical analysis
Indicators of lung tissue injury (wet-to-dry weight ratio, ACE-to-protein ratio in plasma and BALF, and pathologic ranking of histologic findings) were compared using the Kruskal-Wallis test for three-group comparisons; for comparison of drug concentration, the unpaired Student’s t test was applied. Statistical significance was accepted at a p level less than 0.05.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentFootnotesAcknowledgmentsReferences |
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Lung tissue injury
Chest radiography
All animals had weak infiltrations of the perfused lung, suggesting pulmonary edema, without differences between SP and RB. Nonperfused animals of the IV group also showed discrete bilateral infiltrations.
Histology
Hemorrhagic lung edema, dystelectasis, and slight bronchitic infiltrations were observed in all specimens. The histologic assessment did not reveal differences between ILP with and without doxorubicin. The mean pathologic grading of the biopsy specimens harvested at the end of ILP and after reestablishment of pulmonary circulation was 1.3 versus 1.7 for SP and 1.2 and 1.5 for RB, respectively (Table 1). This difference was not significant, the histologic alterations being comparable with the findings in control animals (1.0 and 2.3, respectively).
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Angiotension-converting enzyme-to-protein ratio
Animals submitted to SP demonstrated significantly lower plasma ratio compared with RB and IV at 20 minutes (p < 0.05), but at 50 minutes the difference did not reach the level of statistical significance (see Table 1). The ACE-to-protein ratio in the BALF samples did not demonstrate significant differences between the three groups.
Pharmacokinetics
Doxorubicin perfusate and tissue concentration
The doxorubicin level of the perfusate and of the effluent during RB and SP perfusion as well as lung tissue levels (at the end of ILP) are illustrated in Figure 1. Mean drug tissue concentration was comparable between RB ILP and SP ILP (21.9 µg/g [26.2 µg/g and 17.5 µg/g] versus 17.5 ± 4.8 µg/g). In contrast, the mean drug tissue level was 3.0 ± 0.8 µg/g after termination of intravenous doxorubicin infusion.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentFootnotesAcknowledgmentsReferences |
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The cannulation technique was identical for both procedures. In all 14 animals complete isolation (leakage <0.5%) of the perfusion circuit was achieved, and all procedures could be successfully performed. Using isotopic tracers we would have been able to identify even minimal leakage, as shown by an increase in the plasma activity of the animals during perfusion. In previous studies [9–13, 15, 19], plasma drug concentration was measured for identification of leakage. This method, however, may not be appropriate because minimal leakage might occur despite undetectable plasma drug levels. In 1 animal of the RB group a back-bleeding into the perfusion circuit did dilute the perfusate, but correction of the perfusate’s concentration corresponding to the increased volume was not performed. This animal, however, was excluded from the comparison of drug tissue level.
The major advantage of a recirculating perfusion system is the use of oxygenated blood representing the most physiologic perfusate. Recirculating-blood perfusion, however, requires a technically more demanding system, including the entire set-up of the extracorporal circulation technique. Besides the type of perfusate, the inflow pressure during perfusion is another important factor related to the occurrence of lung injury. One significant difference regarding the application of the two ILP systems is linked to the pulmonary artery pressure measured during perfusion. In the pump-free SP system, the in-flow pressure was dependent on the hydrostatic pressure of the infusion system and was found to be less than 10 mm Hg in our experimental model. In contrast, the perfusion pressure in the RB system correlated with the pump rate of the perfusion circuit. In accordance to other protocols of RB ILP [10], the perfusion pressure was strictly limited to 20 mm Hg in our setting, thus respecting the physiologic pulmonary artery pressure in pigs. To achieve the same flow rate of 100 mL/min in both ILP groups, perfusion pressure in RB animals was approximately 10 mm Hg higher than in SP animals.
Evaluation of perfusion-induced lung injury was restricted to determination of acute tissue reaction. Our preliminary studies have demonstrated severe inflammatory damage occurring after vascular cannulation in pigs, irrespective of the administration of cytostatic drugs or the perfusion technique applied. Baciewicz and associates [10] showed that biopsy specimens harvested 2 hours and 24 hours after perfusion showed the same histologic findings as those taken at the end of the perfusion, suggesting that the latter adequately reflects perfusion-induced histologic changes. Chest radiography at the end of the procedure, wet-to-dry weight ratio, and histology of biopsy specimens as well as ACE-to-protein ratio in plasma and BALF are indicative of pulmonary edema and lung injury [20–22]. Our results demonstrate that these indicators of acute lung injury were independent of the administration of doxorubicin, but the significantly higher wet-to-dry weight ratio after RB as compared with SP perfusion suggests a higher degree of acute lung injury induced by RB. Severity of pulmonary edema was also more pronounced during RB as judged by the clinical impression during bronchoscopy. The lower plasma ACE-to-protein ratio after SP perfusion as compared with RB perfusion may also reflect a less important inflammatory response in the SP groups [20–22]. The correlation of wet-to-dry weight ratio and plasma ACE-to-protein ratio indicates that the latter might be used as an indicator of acute lung tissue damage. In this study no definite conclusions can be drawn from BALF ACE levels or BALF ACE-to-protein ratio, considering the important dispersion of individual values found in this limited number of animals.
No distinct difference between the two ILP groups was evident in the pathologic grading of lung histology. Moderate hemorrhagic pulmonary edema and interstitial infiltration were the major findings in almost all specimens. This is in accordance with other reports [10], but because minor hemorrhagic edema also has been demonstrated in nonperfused animals, the biopsy technique (clamping of pulmonary tissue for wedge resection) might also have contributed to this histopathologic feature.
These findings of some lesser degree of lung tissue injury using a gravity perfusion technique might be explained by the increased vascular resistance provoked by the RB circuit using an oxygenator and heat exchanger as well as a roller pump. Further, the activation of complement, leukocyte, and platelets, as seen in all cardiopulmonary perfusion techniques, might have induced pulmonary injury.
Mean doxorubicin tissue concentration at the end of perfusion was comparable between RB and SP ILP (21.9 versus 17.5 µg/g). Both ILP systems resulted in a sixfold to sevenfold higher tissue concentration of doxorubicin as compared with the intravenous application of the same drug dose. Baciewicz and associates [10] observed in dogs a mean tissue concentration of 20.6 µg/g at an initial perfusate concentration of 7.6 µg/mL after 45 minutes of ILP. In our setting we obtained the same high tissue concentration with only 15 minutes of ILP and only a slight decrease of doxorubicin concentration measured in the effluent perfusate during SP perfusion. This finding suggests that doxorubicin uptake in the lung might also be limited in pigs at a plateau of 4 to 5 µg/mL in the perfusate as shown in dogs [10]. The low extraction rate of doxorubicin and the short perfusion time might explain why no difference in pharmacokinetics was demonstrated between RB and SP perfusions. In contrast to the modest extraction rate observed in canine and porcine isolated perfusion settings, Weksler and associates [12] demonstrated that doxorubicin lung concentration increased linearly with rising perfusate concentration in rats. They observed a 40-fold higher tissue concentration as compared with pigs and dogs [10]. These species-related differences in doxorubicin tissue concentration underline the need for pharmacokinetic studies of ILP in large-animal models before clinical application.
The estimated peak blood level in patients receiving 50 to 90 mg/m2 of doxorubicin is about 1.0 µg/ml [23]. In our experimental setting, we measured blood levels of 0.7 to 0.8 µg/mL after endovenous application of 50 mg/m2 of doxorubicin. Considering the fact that in an animal model complete tumor response was achieved by a fivefold increase of lung tissue level [18], the sixfold to sevenfold increase in tissue concentration of doxorubicin obtained by ILP in large animals appears to be promising for clinical trials.
In conclusion, our results indicate that both SP and RB ILP were highly effective regarding lung tissue concentration as compared with intravenous administration of the same drug dose. In this setting, SP and RB perfusion showed similar results regarding pharmacokinetics, if ILP was performed with the same drug dose, flow rate, and perfusion time. Recirculating-blood perfusion resulted in more acute lung injury than SP. Our results suggest that further studies should focus on ILP using the SP modality; this technique is easier to perform and may have the potential of being applied through endovascular systems, which would obviate invasive surgical interventions.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsCommentFootnotesAcknowledgmentsReferences |
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We acknowledge the assistance of Max Lanz and Giula Vucic (technical support), Regula Theurillat (pharmacologic studies), and Dr Gert Prinzen and Helga Bockhoff (methodologic support).
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Footnotes |
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