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Ann Thorac Surg 2006;82:1033-1037
© 2006 The Society of Thoracic Surgeons
a Department of Cardiothoracic Surgery, St. Antonius Hospital, Nieuwegein, the Netherlands
b Department of Thoracic and Vascular Surgery, University Hospital Antwerp, Antwerp, Belgium
c Laboratory of Experimental Oncology, Catholic University Leuven, Leuven, Belgium
d Laboratory of Cancer Research and Clinical Oncology, University of Antwerp, Antwerp, Belgium
e Department of Pharmacology and Pathophysiology, Utrecht Institute of Pharmaceutical Sciences, Utrecht, the Netherlands
Accepted for publication April 5, 2006.
* Address correspondence to Dr van Putte, St. Antonius Hospital, Department of Cardiothoracic Surgery, Koekoekslaan 1, NL-3430 EM Nieuwegein, the Netherlands (Email: bvanputte{at}yahoo.com).
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Abstract |
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METHODS: Thirty rats had ILuP during 30 minutes using gemcitabine perfusate levels of 1.3, 2.7, 4.0, 5.3, and 6.7 mg/mL. Another 37 rats underwent ILuP with gemcitabine perfusate levels of 6.7 mg/mL during 6 minutes followed by a 5-minute flush and 30 or 60 minutes of reperfusion, while two other groups had ILuP and delayed clamp release for 30 or 60 minutes followed by a 5-minute flush. All effluent and lung samples were stored for later analysis. Results were evaluated using Friedmann two-way analysis and two-way analysis of variance.
RESULTS: At 6 minutes, steady-state of gemcitabine uptake was achieved for all inflow concentrations and a linear relation (r = 0.933, p < 0.0001) between effluent and lung levels was observed. Delayed clamp release resulted in significantly higher lung levels compared with immediate restoration of blood circulation after ILuP (456% at 30 minutes and 828% at 60 minutes).
CONCLUSIONS: Effective gemcitabine lung levels are already achieved after 6 minutes of ILuP with 6.7 mg/mL followed by delayed clamp release during 30 minutes instead of the clinically applied 30 minutes ILuP.
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Introduction |
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Five phase I trials on ILuP have been published that evaluated melphalan and doxorubicin in patients with resectable and irresectable disease [2–6]. The study settings in these trials imply several efficacy-limiting circumstances that have to be solved. First, all studies showed a wide range in final lung levels, probably resulting in subtherapeutic concentrations in a significant number of patients. In a recent paper, we hypothesized that interhuman differences in pulmonary intravascular volume are responsible for variations in initial perfusate levels [7]. Therefore, we proposed a dilution method for assessment of the pulmonary intravascular volume to calculate the drug dose needed [7]. Second, all human studies evaluated single-drug treatment. However, in vitro and in vivo studies from our institution showed significant synergistic actions combining melphalan and gemcitabine [8]. Third, owing to its invasive nature, the efficacy of clinical ILuP is limited by a single and short exposure of 30 minutes only. Longer periods of clamping the pulmonary vasculature may increase the risk on ischemia reperfusion injury clinically known as acute respiratory distress syndrome [9]. Studies evaluating repetitive exposure are under investigation.
In this study, we hypothesized that part of the drug taken up by lung tissue during ILuP is exchanged during the washout period and during restoration of the blood circulation after release of the vascular clamps, resulting in limitation of the exposure time and efficacy. Furthermore, the precise inflow gemcitabine perfusate levels resulting in saturation of lung tissue are not known. Therefore, we investigated gemcitabine uptake into the lung at different inflow perfusate concentrations and studied the effect of delayed clamp release on gemcitabine lung levels.
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Material and Methods |
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Technique of Left Isolated Lung Perfusion
The technique of ILuP was extensively described before [10]. Briefly, anesthesia was induced with isoflurane, nitrous oxide, and oxygen during 3 minutes. Rats were intubated by translaryngeal illumination and connected to the ventilator. Isoflurane was titrated between 0.5% and 1.5% according to muscle relaxation, heart rate, and pupil size. Ventilation was accomplished with a volume-controlled ventilator at a rate of 70 strokes/min and a tidal volume of 10 mL/kg. A left posterolateral thoracotomy was performed, and a rib retractor was placed in the fourth intercostal space. After dissection of the inferior pulmonary ligament, the left lung was held anteriorly. The hilum was dissected free under microscopic view.
Two 16G angiocatheters were placed through the chest wall to facilitate cannulation of the pulmonary vessels. Cannulation of the common trunk vein instead of separate cannulation of both pulmonary veins has been previously described [7]. In short, the pulmonary artery and common trunk vein were proximally clamped with curved microclips simultaneously, before another two clamps were placed distally on both the pulmonary artery and the inferior and superior pulmonary veins. Then, two PE-10 perfusion catheters were introduced into the chest through both angiocatheters. The pulmonary artery and common trunk vein were both cannulated between two clamps without blood loss while the cannulas were secured by a 4-0 silk tie after insertion. After removing the distally placed clamps, the venous cannula lies in the common trunk vein to discard venous effluent coming from both the inferior and superior pulmonary veins. Effluent was collected in a 500 µL K2-EDTA (ethylenediamine tetra-acetic acid) Microtainer (BD, Franklin Lakes, New Jersey). Perfusate was delivered with a flow rate of 0.5 mL/min through the flushed arterial catheter, and buffered starch (Haes Steril 6%; Fresenius Kabi, Schelle, Belgium) was used for all perfusions. Perfusate temperature was controlled at 37°C throughout the whole perfusion period. Rats were placed on a heating pad immediately after induction, and body temperature was kept constantly between 36°C and 37°C.
Gemcitabine and Gemcitabine Processing and Measurement
A high-performance liquid chromatographic method has been used and validated for the determination of gemcitabine in plasma and wet lung tissue. Standard samples of blanc plasma were spiked with gemcitabine (100 ng to 100,000 ng) and extracted in the same way as the other samples and used for a calibration curve [11]. Within-run and between-run precisions were less than 10%, and average accuracies were between 90% and 110%.
Gemcitabine Assay by High-Performance Liquid Chromatographic-Utraviolet
Separation was achieved on a Chrompack Spherisorb ODS-2 reversed phase column (250 x 4.6 mm, 5 µm) (Varian, Palo Alto, CA). The mobile phase used was Pic B7 reagent (Waters Corp, Milford, MA) in 15% methanol (pH = 3.1) with a flow rate of 1.0 mL/min. Gemcitabine is detected by ultraviolet detection at 270 nm.
Experimental Setting
Experiment 1
Thirty rats were randomly divided into five groups (Fig 1). All groups had left ILuP during 30 minutes with a flow rate of 0.5 mL/min using inflow gemcitabine perfusate concentrations of 1.3, 2.7, 4.0, 5.3, and 6.7 mg/mL (groups 1 through 5, n = 6 each). No flush procedure was performed. Effluent samples were taken at different time points during perfusion. Lungs were harvested at the end of 30 minutes of ILuP. All samples were stored at –80°C for later gemcitabine analysis.
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Three groups of rats underwent left ILuP using 6.7 mg/mL gemcitabine under identical conditions as in experiment 1 during 6 minutes followed by a 5-minute flush with buffered starch (group 6, n = 5) and 30 minutes (group 7, n = 5) and 60 minutes (group 8, n = 5) restoration of normal blood circulation. Lungs were harvested immediately after restoration.
In three separate groups, left ILuP using 6.7 mg/mL gemcitabine was followed by delayed clamp release for 30 minutes (group 9, n = 5), 60 minutes (group 10, n = 5), and 60 minutes followed by a 5-minute flush (group 11, n = 6). Lungs were harvested immediately after delayed clamp release (groups 9 and 10) and after flush (group 11).
Finally, one additional group of rats (group 12, n = 6) underwent ILuP during 6 minutes using the same inflow gemcitabine concentration. Lungs were harvested immediately after the end of ILuP.
All lungs were collected and stored at –80°C for later gemcitabine analysis.
Statistics
To assess the distribution of the substance of interest, comparisons were made between all concentrations at the time points of sampling in all treatment groups, using the Friedmann two-way analysis of variance test. Furthermore, two-way ANOVA analysis was applied as the statistical analysis for comparison of the areas under the curve (gemcitabine lung concentrations) in function of time. Statistical significance was accepted at p less than 0.05.
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Results |
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The middle line in Figure 4 shows gemcitabine lung levels after ILuP 6.7 mg/mL for 6 minutes followed by 60 minutes of delayed clamp release and a 5-minute washout. Lung levels are significantly higher for as long as 60 minutes after ILuP compared with immediate washout and clamp release, as shown in the lower line (456% at 30 minutes, p = 0.002, and 828% at 60 minutes, p = 0.03).
Comparison of the areas under the curve in Figure 4 showed also significantly higher lung levels (p < 0.05) during delayed clamp release (middle line, 103,000 µg · g-1 · min-1) compared with restored blood circulation after washout (lower line, 29,300 µg · g-1 · min-1).
No significant differences in wet-to-dry ratios were shown after ILuP during blood flow occlusion and after immediate restoration of circulation (lower versus middle line; Table 1).
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Comment |
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Experiment 1 shows a rapid increase of gemcitabine effluent levels for all concentrations delivered (Fig 2).
In contrast to these findings from gemcitabine perfusate levels implying steady state at 6 minutes, we observed that after 6 minutes only 70% of the gemcitabine tissue levels are achieved compared with 30 minutes of ILuP (Fig 4). Possibly, the interstitial compartment is saturated at 6 minutes while the intracellular compartment is not. Because lung tissue is homogenized before gemcitabine analysis, total drug level is therefore less than the initial interstitial concentration. This phenomenon of lack of saturation at 6 minutes can not be explained by the dilutional effect of lung edema because the wet-to-dry ratio was significantly lower at 6 minutes compared with 30 minutes ILuP (p = 0.024), predicting a higher dilutional effect at 30 minutes.
Although tissue saturation was not achieved at 6 minutes, it is quite interesting to realize that this gemcitabine lung level (2.3 ± 0.34 mg/g) is not significantly different from the concentration measured in a former toxicity study (2.5 ± 1.8 mg/g) after 30 minutes of ILuP 320 mg/kg (5.3 mg/mL inflow concentration) [12]. In a recent paper, we showed significantly longer survival in rats with micrometastatic disease after 30 minutes of ILuP 320 mg/kg compared with untreated control rats [8].
Furthermore, cellular gemcitabine uptake is irreversible, suggesting that only interstitial drug is flushed away during washout. Briefly, gemcitabine requires a one-way protein-mediated transport for efficient cell entry. Once gemcitabine has entered the cell, it is phosphorylated to gemcitabine diphosphate and triphosphate before incorporation into the DNA.
The gemcitabine assay used in this experiment does not detect phosphorylated (intracellular) gemcitabine, explaining the decrease in tissue levels after 6 minutes perfusion followed by delayed clamp release (Fig 4).
In summary, a perfusion time shortened to 6 minutes followed by delayed clamp release of 30 to 60 minutes resulted in effective lung levels, less lung edema, and lower drug costs and drug amount needed per patient.
A linear relation was observed between gemcitabine lung levels and the inflow gemcitabine perfusate levels, suggesting that gemcitabine uptake is only dependent on passive diffusion or that saturation levels of active transport mechanisms are not achieved in the concentration-escalating schedule applied (Fig 3). During perfusion, gemcitabine is exchanged from the intravascular to the interstitial compartment and from there into the intracellular compartment. At the end of ILuP, both the interstitial and intracellular compartments contain gemcitabine and biochemical analysis measures the cumulated concentration of both compartments together after homogenization. Based on these findings, we can only conclude that the two compartments together have not been fully saturated under the (dose-escalating) circumstances applied. Further experiments are necessary to differentiate between interstitial and intracellular drug levels to achieve intracellular saturation while minimizing interstitial exposure. However, Minchin and colleagues [13] did achieve tissue saturation using doxorubicin at 20 nmol/mL, suggesting that both the interstitial and the intracellular compartment had been saturated.
One of the major limitations of ILuP is the short exposure time of the lung to the drug infused. In experiment 2 we confirmed our hypothesis that a major part of the drug taken up during ILuP is quickly exchanged into the circulation during the washout interval and during reperfusion. Delayed clamp release resulted in significantly higher lung levels for as long as 30 and 60 minutes after finishing ILuP compared with immediate restored blood circulation. No significant differences in wet-to-dry ratio were shown between the two modalities (Table 1) excluding a dilutional effect on tissue concentrations. Probably, the main part of the drug is returned from the interstitium into the vascular compartment based on simple diffusion, suggesting that part of the drug did not enter the cells. This assumption is indirectly confirmed by observations with doxorubicin. Minchin and coworkers [14] showed a considerably slower efflux of doxorubicin than uptake after 10 minutes of ILuP, suggesting that intracellular drug binding occurs faster compared with gemcitabine.
Prolonged pulmonary circulatory arrest increases the risk of ischemia-reperfusion injury, clinically manifested as acute respiratory distress syndrome [9]. Based on our findings that effective gemcitabine lung levels are already achieved after 6 minutes of ILuP with 6.7 mg/mL followed by delayed clamp release during 30 to 60 minutes instead of the clinically applied 30 minutes of ILuP, we advocate diminishing perfusion time to 6 minutes followed by delayed clamp release of no longer than 30 minutes to avoid reperfusion injury and acute respiratory distress syndrome.
In conclusion, effective gemcitabine lung levels are already achieved after 6 minutes of ILuP with 6.7 mg/mL followed by delayed clamp release during 30 minutes instead of the clinically applied 30 minutes ILuP. This approach results in less capillary leakage and lower drug costs and drug amount needed per patient. Furthermore, delayed clamp release prevents the washout phenomenon, resulting in higher drug exposure for as long as 60 minutes after ILuP. Therefore, we advocate decreasing perfusion time to 6 minutes followed by delayed clamp release for a period of 30 minutes to obtain higher lung levels and diminish the risk of reperfusion injury.
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Acknowledgments |
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References |
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  B. P. van Putte, M. Grootenboers, W.-J. van Boven, J. M. H. Hendriks, P. E. Y. van Schil, G. Guetens, G. De Boeck, G. Pasterkamp, F. Schramel, and G. Folkerts Pharmacokinetics of Gemcitabine when Delivered by Selective Pulmonary Artery Perfusion for the Treatment of Lung Cancer Drug Metab. Dispos., April 1, 2008; 36(4): 676 - 681. [Abstract] [Full Text] [PDF]
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