Interact CardioVasc Thorac Surg 2009;8:635-638. doi:10.1510/icvts.2008.194720
© 2009 European Association of Cardio-Thoracic Surgery
Institutional report - Experimental |
Drug uptake in a rodent sarcoma model after intravenous injection or isolated lung perfusion of free/liposomal doxorubicin ,
Cai Chenga,d,
Amina Haoualab,
Thorsten Kruegera,
Francois Mithieuxa,
Jean Y. Perentesa,
Solange Petersc,
Laurent A. Decosterdb and
Hans-Beat Risa,*
a Division of Thoracic and Vascular Surgery, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
b Division of Clinical Pharmacology and Toxicology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
c Division of Oncology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
d Department of Cardiothoracic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Received 18 September 2008;
received in revised form 24 February 2009;
accepted 24 February 2009
 Presented at the 16th European Conference on General Thoracic Surgery, Bologna, Italy, June 8–11, 2008. 
Financial support was provided by a grant Nr 310000-118222/1 from the Swiss National Science Foundation and from the Foundation Naef. Doxorubicin (Adriblastin®) was provided by Pharmacia and Upjohn, Dübendorf, Switzerland, LiporubicinTM was provided by CT Sciences SA, Lausanne, Switzerland.
*Corresponding author. Service de Chirurgie Thoracique et Vasculaire, Centre Hospitalier Universitaire Vaudois, Rue du Bugnon 46, 1011 Lausanne, Switzerland. Tel.: +41 21 3142408; fax: +41 21 3142358.
E-mail address: hans-beat.ris{at}chuv.ch (H.-B. Ris).
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Abstract
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The distribution of free and liposomal doxorubicin (LiporubicinTM) administered by intravenous injection (IV) or isolated lung perfusion (ILP) was compared in normal and tumor tissues of sarcoma bearing rodent lungs. A single sarcomatous tumor was generated in the left lung of 35 Fischer rats, followed 10 days later by left-sided ILP (n=20) or IV drug administration (n=12), using 100 µg and 400 µg free or liposomal doxorubicin, respectively. The tumor and lung tissue drug concentration was measured by HPLC. Free doxorubicin administered by ILP resulted in a three-fold (100 µg) and 10-fold (400 µg) increase of the drug concentration in the tumor and normal lung tissue compared to IV administration. In contrast, ILP with LiporubicinTM resulted in a similar drug uptake in the tumor and lung tissue compared to IV injection. For both drug formulations and dosages, ILP resulted in a higher tumor to lung tissue drug ratio but also in a higher spatial heterogeneity of drug distribution within the lung compared to IV administration. ILP resulted in a higher tumor to lung tissue drug ratio and in a more heterogeneous drug distribution within the lung compared to IV drug administration.
Key Words: Isolated lung perfusion; Intravenous drug application; Sarcoma; Chemotherapy; Doxorubicin; Liposomal encapsulation
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1. Introduction
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Isolated lung perfusion (ILP) has been recognized as an attractive treatment concept against lung metastases since it specifically delivers a cytostatic agent to the target organ while sparing the systemic circulation. Doxorubicin-based ILP was shown to cause a high drug uptake in the lungs with minimal leakage in the systemic circulation and toxicity [1–5]. However, we previously observed that the penetration of doxorubicin or LiporubicinTM – a pegylated liposomal encapsulated formulation of doxorubicin – administered by ILP was significantly lower in sarcomas grown in rodent lungs compared to normal lung parenchyma [6]. We have also recently shown that doxorubicin-based ILP can lead to a heterogeneous drug distribution within the perfused lung and a high variability in drug distribution between animals [7, 8]. This could, in part, explain the inconsistent results obtained in patients undergoing ILP for lung metastases, irrespective of the cytostatic agent and drug dose used.
Here, we hypothesized that the concentration, formulation and administration of doxorubicin affects its distribution in normal and tumor tissues and could help optimize the tumor to lung tissue drug ratio. Using our previously described sarcoma model, we compared the tumor/normal parenchyma drug uptake following IV or ILP administration of doxorubicin and LiporubicinTM. For each condition, we determined the drug uptake in tumors and lung parenchyma by HPLC, the tumor/lung ratio and the drug distribution variability in different lung compartments.
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2. Material and methods
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Male Fischer rats (Charles River, France) weighing 250–300 g were used. They were treated in accordance with the Animal Welfare Act, the National Institute of Health ‘Guidelines for the Care and Use of Laboratory Animals’ and according to the Local Ethical Committee of the University of Lausanne.
Thirty-five Fischer rats underwent tumor implantation in their left lung using a syngeneic methylcholanthrene-induced sarcoma (MCA) cell line [9] followed 10 days later by isolated lung perfusion (ILP, n=20) or intravenous administration (IV, n=12) of free or liposomal encapsulated doxorubicin (LiporubicinTM) as previously described [6, 9]. ILP and IV drug administration were performed for both drug formulations at equimolar drug doses of 100 µg and 400 µg, respectively. Sixty minutes following ILP and 70 min following IV injection, the animals were sacrificed and the lungs were harvested. Drug concentrations in the tumor and the normal surrounding lung were assessed by high performance liquid chromatography (HPLC) as previously described [6, 7, 10, 11]. In addition, the concentrations of doxorubicin in tissues were separately determined in the upper, middle and lower part of each perfused lung to assess the coefficient of variation (CV%) as previously described [6]. Three untreated animals served as controls for histological and immunohistochemical analysis.
Doxorubicin concentrations in tumors and normal lung parenchyma were compared for both doxorubicin formulations using a Student's t-test for unrelated samples. Variabilities in doxorubicin lung tissue levels were expressed as the coefficients of variation of tissue concentration in the three parts of perfused lungs for both doxorubicin formulations and drug doses and were compared using the Student's t-test. A bidirectional hypothesis was applied and significance accepted at P<0.05.
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3. Results
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3.1. Controls
Histological assessment of untreated tumors revealed a well-circumscribed sarcomatous tumor in each lung, mainly formed by large undifferentiated cells. Immunostaining for von Willebrand factor revealed an extensive vascular network composed of small vessels and capillaries and present throughout the tumors as previously described [6]. Spontaneous necrosis was observed in <2% of the tumor volume for each case analyzed. Tumor growth within the pleural cavity was not identified in any of the animals.
3.2. Drug concentrations in tumor and lung tissue (Tables 1 and 2)
Free doxorubicin administered by ILP resulted in a three-(100 µg) to 10-fold (400 µmg) increase of drug concentration in the tumor (P=0.048) and in the lung (P=0.0002) compared to IV drug administration. However, doxorubicin concentrations in tumors were always lower than in lung tissue in all animals, for both drug doses and modes of drug administration assessed.
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Table 1 Tissue concentration, ratio of tumor to normal tissue drug concentration and coefficient of variation (CV%) of free and liposomal encapsulated doxorubicin after isolated lung perfusion (mean values±S.D.)
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Table 2 Tissue concentration, ratio of tumor to normal tissue drug concentration and coefficient of variation (CV%) of free and liposomal encapsulated doxorubicin after IV drug application (mean values±S.D.)
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In contrast, LiporubicinTM resulted for each drug dose in similar drug concentrations in the tumor and normal lung tissues following ILP or IV administration. Interestingly, the increase of LiporubicinTM drug dose did not affect tumor or normal tissue drug concentration following ILP but significantly increased these parameters following IV application. LiporubicinTM concentrations in tumors were always lower than in the normal lung tissue, for both drug doses and both modes of drug administration assessed.
3.3. Ratio of tumor to lung tissue drug concentration (Fig. 1)
To determine how the doxorubicin administration, formulation or dose affected its penetration in tumors, we determined the tumor to normal tissue drug ratio for each of the previous experiments. Free doxorubicin: increasing the drug dose during ILP significantly improved the tumor to lung tissue drug ratio (P=0.003). This holds not true for IV application of free doxorubicin. Comparing IV application with ILP, the ratio was significantly higher for ILP at a drug dose of 400 µg (P=0.02) but not at a dose of 100 µg.
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Fig. 1. Ratio of tumor to lung tissue drug concentration in sarcoma bearing lungs for ILP and IV drug administration of 100 µg and 400 µg doxorubicin and equimolar-dosed LiporubicinTM.
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LiporubicinTM: ILP revealed for both drug doses a higher tumor to lung tissue drug ratio than IV application although the differences were not significant. After ILP, both drug doses resulted in a similar ratio; after IV administration, the ratio was higher with the higher drug dose but not statistically significant (P=0.06).
3.4. Spatial drug distribution after ILP and IV drug administration
ILP led to a wide inter-animal variability and a heterogeneous spatial drug distribution within the perfused lung for both doxorubicin formulations at both drug doses as demonstrated by the large coefficients of variation (CV%) (Table 1). Both, inter-animal variability and spatial drug distribution was less pronounced after IV drug application, for both drug formulations (Table 2).
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4. Discussion
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Since advanced sarcoma often presents with lung metastases without extra thoracic tumor manifestation, doxorubicin-based ILP has been assessed under clinical and experimental conditions as an alternative method for the delivery of high-dose chemotherapy while minimizing systemic toxicity. Although effective drug delivery to the perfused lung was obtained, ILP has failed to demonstrate effective tumor control in clinical trials [1–3]. We have previously shown that ILP with doxorubicin results in an uneven distribution of drug within the perfused lung [6–8] and that little doxorubicin penetrates tumors in a rodent model of sarcoma metastasis to the lung [6, 7]. Fluorescence microscopic assessment revealed that doxorubicin fails to cross the endothelial barrier of vessels within the tumors during ILP in this model [7].
Here, we compared the drug uptake in tumors and normal lung parenchyma, the tumor to normal lung tissue drug ratio and the pattern of drug distribution in the lung after ILP and IV drug administration of doxorubicin and equimolar dosed LiporubicinTM in a rat model of sarcoma metastasis to the lungs. Histological assessment and immunostaining for von Willebrand's factor showed that untreated sarcomatous tumors were well-circumscribed, had a rich vascular network consisting of fine branching capillaries witnessing the presence of a well developed tumor vascularization and did not present spontaneous necrosis.
Doxorubicin administered by ILP resulted in a three-(100 µg) to 10-fold (400 µg) increase in drug concentration in the lung and tumor tissues compared to IV administration. However, both modes of doxorubicin administration resulted in a consistently lower drug uptake in tumors than in normal lung tissue, for both drug doses applied. The highest tumor to lung tissue drug ratio was obtained with 400 µg of doxorubicin administered by ILP, however, the spatial distribution of doxorubicin in the lung parenchyma after ILP was highly variable within animals and between animals as previously reported in rodent [7] and porcine [8] models.
We then conducted the same experiments using a pegylated liposomal encapsulated form of doxorubicin (LiporubicinTM). Liposomal delivery systems for anthracyclines have shown several advantages for intravenous application compared with the administration of the free drug. They have been shown to improve drug delivery to tumors while decreasing toxicity to normal tissues [12–14]. The tumor-targeting mechanism relies on the size of liposomes, which render them difficult to extravasate through the capillaries of normal tissues outside of the reticulo-endothelial system.
In our study, we found that LiporubicinTM-based ILP resulted for both drug doses in a similar tumor and lung tissue drug uptake compared to IV application which may be explained by the specific pharmacokinetic profile of liposomal doxorubicin [12–14]. However, ILP with LiporubicinTM revealed a consistently lower drug uptake in tumors and lung tissue compared to equimolar-dosed doxorubicin. This could be due to the microvascular properties of the sarcoma model used in this study. Indeed, previous work in various tumor types has shown that the convective forces involved in drug distribution can be limited by the high intratumoral interstitial fluid pressure. In this situation, the major driving force becomes diffusion which is mostly affected by the drug molar mass. Here, while the size of LiporubicinTM may increase its specificity for the tumor vasculature compared to doxorubicin, it may also limit its distribution because of the increase in size and molar mass. Finally, another element that could limit the distribution of LiporubicinTM administered by ILP is the absence of enzymes that have the capacity to de-pegylate LiporubicinTM and liberate doxorubicin in the lung perfusion solute.
Conversely, LiporubicinTM-based ILP revealed for both drug doses a higher tumor to lung tissue drug ratio than IV application although the differences were not significant due to the great inter-animal variability after ILP. In addition, the tumor to lung tissue drug ratio was not significantly different between doxorubicin and LiporubicinTM-based ILP. These findings suggest that LiporubicinTM may have advantages for drug escalating ILP schedules since an increase of the drug dose may allow an enhanced tumor drug uptake without exposing the lung parenchyma to excessive doxorubicin concentrations and to a risk of doxorubicin-induced lung toxicity.
Finally, similarly to doxorubicin, LiporubicinTM administered by ILP was associated with a higher heterogeneity of doxorubicin distribution and inter-animal variability compared to IV application implicating that this phenomenon is related to the mode of drug delivery and cannot be alleviated by liposomal encapsulation of doxorubicin.
In conclusion, ILP administration of both doxorubicin formulations resulted in a trend for better tumor to lung tissue drug ratio than IV administration with a significant increase in this ratio for free doxorubicin at a dose of 400 µg. Although LiporubicinTM administered by ILP did not cause a higher tumor to lung tissue drug ratio compared to IV, its lung concentrations were significantly lower than that of doxorubicin. The latter could thus bear an advantage for investigational ILP protocols with drug-escalating schedules.
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Abstract
1. Introduction
