The effect of irradiation on the biodistribution of radiolabeled pegylated liposomes

Int. J. Radiation Oncology Biol. Phys., Vol. 50, No. 3, pp. 809 – 820, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/01/$–see front matter

PII S0360-3016(01)01508-5

BIOLOGY CONTRIBUTION

THE EFFECT OF IRRADIATION ON THE BIODISTRIBUTION OF RADIOLABELED PEGYLATED LIPOSOMES KEVIN J. HARRINGTON, M.R.C.P., F.R.C.R.,*† GAIL ROWLINSON-BUSZA, PH.D.,* KONSTANTINOS N. SYRIGOS, M.D., PH.D.,* PAUL S. USTER, PH.D.,‡ RICHARD G. VILE, PH.D.,† A. MICHAEL PETERS, F.R.C.P., F.R.C.R.,§ AND J. SIMON W. STEWART, F.R.C.P., F.R.C.R.㛳 *ICRF Oncology Unit and §Department of Imaging, Imperial College of Science, Technology and Medicine, Hammersmith Hospital, London, United Kingdom; †Molecular Medicine Program, Mayo Clinic, Rochester, MN; ‡SEQUUS Pharmaceuticals Inc., Menlo Park, CA; 㛳Department of Radiotherapy, Charing Cross Hospital, London, United Kingdom Purpose: The effect of total-body irradiation (TBI) on the biodistribution and pharmacokinetics of 111In-DTPAlabeled pegylated liposomes (IDLPL) was evaluated in tumor-bearing nude mice as part of an ongoing effort to develop liposome-targeted radiosensitizers. Methods and Materials: Mice received TBI (2 Gy or 5 Gy) according to two protocols: (1) to test the effect of radiation delivered 30 min before liposome injection on the time course of IDLPL biodistribution to tumor and normal tissues over 96 h; (2) to test the effect of radiation at times ranging from 72 h to 1 h before liposome injection on tumor and normal tissue uptake of IDLPL at 24 h. Tumor and tissue/organ levels of liposome uptake were measured by dissection and quantitation in a gamma counter. Results: For most tissues (tumor, liver, kidney, lung, skin, heart, and central nervous system), irradiation did not alter IDLPL biodistribution. Splenic uptake appeared to be increased by TBI, but further analysis revealed that this effect was due to reduced splenic weight in irradiated mice. IDLPL uptake was increased in the small intestine, stomach, musculoskeletal system, female reproductive tract, and adrenal glands in irradiated mice. Conclusion: These findings suggest that concomitant administration of liposomal radiosensitizers during radical radiotherapy is likely to be safe. However, caution should be exercised in situations in which significant volumes of small intestine or hemopoietic tissue will be irradiated. © 2001 Elsevier Science Inc. Biodistribution, Radiotherapy, Pegylated liposome, Radiosensitizer.

INTRODUCTION

viously been shown to alter the uptake of macromolecules, such as monoclonal antibodies, into tumors (8 –10) and may influence the biodistribution and pharmacokinetics of pegylated liposomes, either favorably (by increasing tumor deposition and/or decreasing normal tissue deposition) or unfavorably (by decreasing tumor deposition and/or increasing normal tissue deposition). Thus, detailed information on these potential interactions will be a critical part of the development of pegylated liposome encapsulated radiosensitizing agents. We have recently demonstrated that localized single fraction tumor irradiation at doses between 5 Gy and 20 Gy has no effect on tumor deposition of radiolabeled pegylated liposomes in a human tumor xenograft model (11). Here, we report the effect of low-dose (2 Gy and 5 Gy) single fraction total-body irradiation (TBI) on the pharmacokinetics and biodistribution of 111In-DTPA-labeled pegylated liposomes (IDLPL) in xenograft tumors and a wide range of normal tissues in nude mice.

Liposomes are phospholipid bilayer membrane-bound vesicles that can encapsulate a wide variety of substances either within their lipid membranes or their central aqueous cores (1, 2). Polyethylene glycol-derivatised (pegylated) liposomes escape rapid clearance by the reticuloendothelial system (RES) (3, 4) and, consequently, have a prolonged circulation half-life and the ability to accumulate in and deliver therapeutic agents to tumors in a number of preclinical and clinical settings (reviewed in Ref. 5). A number of novel therapeutic approaches using pegylated liposomal agents are under investigation, including strategies for targeted delivery of radiosensitizers to tumors during concomitant chemoradiotherapy (CCRT) (6, 7). This approach offers the twin attractions of increasing drug deposition in the local tumor and reducing local and systemic drug delivery to dose-limiting normal tissues. However, radiotherapy (RT) has preReprint requests to: Dr. K. J. Harrington, Molecular Medicine Program, Guggenheim 1836, Mayo Clinic, 200 1st Street SW, Rochester, MN 55902. E-mail: [email protected]

Accepted for publication 29 January 2001.

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METHODS AND MATERIALS Nude mice and tumor model Female nude mice of mixed genetic background were used in these experiments. The animals were bred under specific pathogen-free conditions at the Imperial Cancer Research Fund Animal Breeding Unit, South Mimms, Herts, UK. Thereafter, the animals were housed in sterile filter-top cages on sterile bedding, and maintained on an irradiated diet and autoclaved, acidified water (pH 2.8). The human tumor KB cell line was derived from a male patient with a poorly differentiated squamous cell cancer of the floor of mouth and tongue and established in cell culture in 1954 (12). KB xenograft tumors were established as previously described (13). Briefly, 5 ⫻ 106 KB tumor cells in 100 ␮L of culture medium without fetal calf serum were injected s.c. into the right flank of nude mice. All mice were aged between 8 and 12 weeks and weighed approximately 20 –25 g at the time of the experiments. Mice were maintained in accordance with the Medical Research Council guidelines (Responsibility in the Use of Animals for Medical Research, 1993). Tumors were allowed to grow for 14 –20 days. Because previous data have shown that tumor size acts as a significant influence on tumor uptake of pegylated liposomes in this model (14), the tumors used in the various study groups were of comparable sizes. Liposome labeling with 111In-oxine Diethylenetriaminepentaacetic acid (DTPA, Janssen Chimica, Geel, Belgium) entrapped in a proprietary pegylated liposome matrix was provided by SEQUUS™ Pharmaceuticals, Inc. (Menlo Park, CA). STEALTH liposomes are a registered trademark and have been described previously (13). The lipid composition of the liposomes was as follows: [hydrogenated soybean phosphatidylcholine (56.2%), cholesterol (38.3%), and N-(carbamoyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3phospho-ethanolamine sodium salt (MPEG-DSPE) (5.3%) (values expressed in % molar ratio, stated to one decimal place such that the total does not equal 100%)]. Their mean diameter was approximately 96 nm. This liposome matrix is essentially identical to that which is in clinical use as pegylated liposomal doxorubicin (Caelyx™Doxil™, ALZA Corporation, Palo Alto, CA), the agent that has entered Phase I/II clinical CCRT trials (6, 7). Liposomes were radiolabeled as described previously (13). Briefly, 5 mL of DTPA-containing pegylated liposomes were incubated for 1 h with 0.5 mL of 111In oxine (18.5 MBq) (Amersham International plc, Amersham, United Kingdom). During this period, a solution of 1.5 g of ethylenediaminetetraacetic acid (EDTA) (BDH Ltd, Poole, UK) in 10 mL of sterile water (Fresenius Health Care Group, Basingstoke, UK) was prepared and added to 250 mL of 5% dextrose. At the end of the 1-h incubation period, 0.25 mL of the resulting solution was added to the pegylated liposome solution to chelate any residual unencapsulated 111In-oxine and promote its prompt excretion following i.v. administration.

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Before administration, the efficiency of encapsulation of 111 In was measured by running a 10-␮L sample on a 20-mL Sephadex G-50 (Pharmacia, Uppsala, Sweden) column, as described previously (13). Liposome administration proceeded if the encapsulation efficiency exceeded 90%. Total-body irradiation In view of the technical difficulties inherent in attempting to irradiate individual mouse organs/tissues, it was decided to use TBI to ensure delivery of uniform irradiation doses. Animals were irradiated using a 111 TBq 137Cs source (CIS bio international, Gif-sur-Yvette, France). Before commencing animal experiments, dosimetric calibration of the irradiator was performed by exposing lithium fluoride thermoluminescent dosimeters (TLDs) (Nuclear Enterprises, Reading, United Kingdom) to doses of 2 Gy, 5 Gy, 10 Gy, 15 Gy, and 20 Gy and reading them in a Toledo 654 TLD reader (D. A. Pitman, Weybridge, United Kingdom). All TLD chips used in these studies had previously been calibrated at doses between 2 Gy and 20 Gy using a 6-MV linear accelerator (Varian, United Kingdom) in the Department of Clinical Oncology, Hammersmith Hospitals NHS Trust (data not shown). Thereafter, groups of 5 unanesthetized KB tumor-bearing mice were positioned in a shallow plastic box with breathing holes cut in the lid, and this was positioned in the irradiator with its center on the central axis of the 137Cs source. The mice received single-fraction TBI at doses of either 2 Gy or 5 Gy at a dose rate of 1.85 Gy/min. Control animals were treated in an identical fashion to irradiated animals, apart from the radiation exposure itself. TBI was delivered according to two protocols. In the first study, TBI was delivered 30 min before an i.v. injection of IDLPL, and groups of 5 animals were dissected 1, 24, 48, 72, and 96 h postinjection. Henceforth, this study will be referred to as the “time-course study.” In the second study, groups of 5 mice received TBI at 72, 48, 24, 6, and 1 h before i.v. injection of IDLPL, and all the animals were dissected at 24 h postinjection. This time point was selected because it has previously been shown to be the time of maximal liposome deposition in most normal tissues and KB xenograft tumors (13). Henceforth, this study will be referred to as the “interval study.” Biodistribution and pharmacokinetics of IDLPL after TBI Animals received 100 ␮L of IDLPL containing 0.37 MBq of radioactivity as an i.v. bolus injection via a lateral tail vein according to the protocols detailed above. At the time of dissection, animals were anesthetized with isoflurane (Abbott Laboratories Ltd, Queenborough, Kent, United Kingdom) in an anesthetic jar and killed by direct cardiac puncture and aspiration of the maximal blood volume (up to 1.2 mL), followed by cervical dislocation to ensure humane death. Maximal exsanguination was performed in an attempt to minimize the contribution of radiolabeled liposomes in the intravascular compartment to the measured levels of liposome uptake for the individual tissues. Tissue samples, except for blood, urine, and brain, were rinsed with

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Table 1. Biodistribution of IDLPL in unirradiated control mice in time-course study of effect of total-body irradiation; data expressed in % injected dose per gram (mean ⫾ SD)

Blood Urine Tumor Liver Spleen Lung Kidney Bladder Esophagus Stomach Ileum Colon Gallbladder Pancreas Uterus Ovary Skin Heart Adrenal Bone Muscle Brain Spinal cord

1h

4h

24 h

48 h

72 h

96 h

31.6 ⫾ 3.4 44.8 ⫾ 28.5 0.92 ⫾ 0.24 6.1 ⫾ 2.0 7.9 ⫾ 1.9 7.2 ⫾ 2.6 6.1 ⫾ 1.4 1.2 ⫾ 0.2 2.5 ⫾ 0.5 0.84 ⫾ 0.14 1.2 ⫾ 0.2 1.0 ⫾ 0.3 1.2 ⫾ 0.6 1.4 ⫾ 0.4 1.6 ⫾ 0.3 1.4 ⫾ 0.4 1.1 ⫾ 0.4 4.0 ⫾ 1.6 4.4 ⫾ 1.3 1.7 ⫾ 0.3 0.41 ⫾ 0.16 0.62 ⫾ 0.19 1.8 ⫾ 0.7

19.6 ⫾ 2.3 8.6 ⫾ 4.2 1.5 ⫾ 0.4 8.6 ⫾ 2.4 8.4 ⫾ 1.0 5.6 ⫾ 2.3 5.1 ⫾ 2.7 1.2 ⫾ 0.3 1.9 ⫾ 0.5 0.76 ⫾ 0.21 1.2 ⫾ 0.2 1.6 ⫾ 0.4 0.75 ⫾ 0.23 1.0 ⫾ 0.3 2.5 ⫾ 0.7 1.3 ⫾ 0.5 1.6 ⫾ 0.5 2.2 ⫾ 0.8 4.0 ⫾ 0.9 0.76 ⫾ 0.09 0.32 ⫾ 0.10 0.48 ⫾ 0.11 1.5 ⫾ 0.8

7.4 ⫾ 2.1 5.2 ⫾ 3.2 4.9 ⫾ 2.4 19.8 ⫾ 3.4 17.5 ⫾ 4.0 1.9 ⫾ 0.5 6.3 ⫾ 1.8 0.92 ⫾ 0.24 4.0 ⫾ 1.1 1.3 ⫾ 0.3 2.2 ⫾ 0.4 1.7 ⫾ 0.4 0.51 ⫾ 0.18 0.87 ⫾ 0.26 4.9 ⫾ 2.2 3.2 ⫾ 0.5 4.9 ⫾ 1.4 1.4 ⫾ 0.6 3.4 ⫾ 1.3 1.0 ⫾ 0.5 0.31 ⫾ 0.10 0.18 ⫾ 0.06 0.38 ⫾ 0.30

1.8 ⫾ 0.9 2.3 ⫾ 1.0 4.3 ⫾ 1.8 18.1 ⫾ 3.7 15.8 ⫾ 5.4 1.4 ⫾ 0.3 5.6 ⫾ 1.4 0.72 ⫾ 0.17 1.2 ⫾ 0.5 1.1 ⫾ 0.3 1.2 ⫾ 0.4 1.9 ⫾ 0.6 0.61 ⫾ 0.22 0.40 ⫾ 0.21 6.3 ⫾ 1.9 1.9 ⫾ 0.6 5.3 ⫾ 2.1 0.93 ⫾ 0.31 1.2 ⫾ 0.3 0.65 ⫾ 0.25 0.28 ⫾ 0.16 0.04 ⫾ 0.02 0.13 ⫾ 0.09

0.32 ⫾ 0.12 8.6 ⫾ 3.9 3.0 ⫾ 1.2 17.0 ⫾ 2.6 13.6 ⫾ 3.9 0.81 ⫾ 0.26 4.9 ⫾ 1.6 0.56 ⫾ 0.12 1.3 ⫾ 0.6 0.63 ⫾ 0.24 1.1 ⫾ 0.3 2.1 ⫾ 0.5 0.47 ⫾ 0.20 0.48 ⫾ 0.28 4.3 ⫾ 2.4 1.8 ⫾ 0.4 4.9 ⫾ 1.7 0.79 ⫾ 0.18 1.2 ⫾ 0.2 0.67 ⫾ 0.31 0.24 ⫾ 0.13 0.01 ⫾ 0.01 0.04 ⫾ 0.01

0.05 ⫾ 0.01 4.5 ⫾ 2.6 1.8 ⫾ 0.4 13.5 ⫾ 4.3 13.5 ⫾ 2.8 0.57 ⫾ 0.23 4.4 ⫾ 1.0 0.73 ⫾ 0.28 1.1 ⫾ 0.4 0.43 ⫾ 0.19 1.0 ⫾ 0.4 1.6 ⫾ 0.4 0.38 ⫾ 0.30 0.33 ⫾ 0.16 5.1 ⫾ 3.0 2.4 ⫾ 0.8 4.0 ⫾ 1.1 0.52 ⫾ 0.23 0.94 ⫾ 0.13 0.72 ⫾ 0.20 0.18 ⫾ 0.10 0.01 ⫾ 0.00 0.03 ⫾ 0.02

PBS containing heparin (1000 U/L), blotted dry, and weighed in preweighed scintillation tubes (Sterilin, Stone, United Kingdom). The levels of contained radioactivity were determined in a Canberra Packard Minaxi 5550 gamma counter (Pangbourne, Berks, United Kingdom). Standards of the injected material were made in triplicate and used to correct for physical decay of the 111In.

in the different study groups (Table 4) (p ⬎ 0.1 for all comparisons). The circulation half-life (t1/2␤) of IDLPL in each group was determined by means of the P.Fit program (Biosoft, Cambridge, United Kingdom), which fitted the blood data for the first 96 h to the biexponential decay equation: y⫽Pe⫺␣x⫹Qe⫺␤x

Statistical analysis The levels of uptake of IDLPL in the various tissues at each time point for the 2 Gy and 5 Gy groups were compared with unirradiated control animals by the Student’s t test. In addition, the Student’s t test was used to compare tumor and splenic weights in the various groups; p values ⬍ 0.05 were considered significant. RESULTS Time-course study The data on the biodistribution of IDLPL over a period of 96 h for the unirradiated control animals are presented in Table 1. The corresponding data for the groups of animals which received TBI at doses of 2 Gy and 5 Gy administered 30 min before injection of IDLPL are shown in Tables 2 and 3, respectively. TBI at doses of either 2 Gy or 5 Gy had no significant effect on tumor localization of IDLPL, as compared with the unirradiated controls. Because tumor size can affect liposome uptake, the weights of the tumors in the unirradiated control, 2 Gy, and 5 Gy TBI groups were compared. There were no statistically significant differences in tumor weights

TBI had no demonstrable effect on the blood clearance of IDLPL in this study with t1/2␣ and t1␤ values of 0.20, 0.22, and 0.23 h and 12.3, 12.3, and 12.7 h for unirradiated controls, 2 Gy TBI and 5 Gy TBI, respectively (r2 ⬎ 0.999 for each fit of the data). As can be seen in Tables 2 and 3, at each TBI dose there were 6 time points for comparison with the data for unirradiated control animals. This represents a very large number of statistical comparisons. Few of the comparisons were significantly different from control data with most tissues showing no significant effect of irradiation at either of these clinically relevant doses. There was no evidence of increased liposome uptake in kidney, lung, liver, colon, pancreas, heart, spinal cord, or skin (p ⬎ 0.1 at each dose and time point). In some tissues (brain and gallbladder), the levels of liposome uptake at a single time point were significantly higher in irradiated animals. The lack of a consistent pattern or evidence of dose-dependence suggests that such observations are unlikely to be clinically relevant. Similarly, in the esophagus, the occurrence of a significant difference after both 2 Gy and 5 Gy at the 48-h time point is of uncertain relevance in the absence of other altered

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Table 2. Biodistribution of IDLPL in time-course study of the effect of 2 Gy total-body irradiation; data expressed in % ID/g (mean ⫾ SD)

Blood Urine Tumor Liver Spleen Lung Kidney Bladder Esophagus Stomach Ileum Colon Gallbladder Pancreas Uterus Ovary Skin Heart Adrenal Bone Muscle Brain Spinal cord

1h

4h

24 h

48 h

72 h

96 h

35.0 ⫾ 2.8 65.1 ⫾ 35.0 1.0 ⫾ 0.2 6.4 ⫾ 1.5 7.1 ⫾ 1.0 6.2 ⫾ 1.4 6.1 ⫾ 1.3 1.2 ⫾ 0.3 2.5 ⫾ 0.6 0.71 ⫾ 0.21 1.1 ⫾ 0.2 1.2 ⫾ 0.2 1.5 ⫾ 0.5 1.0 ⫾ 0.2 1.4 ⫾ 0.4 1.5 ⫾ 0.2 1.0 ⫾ 0.3 3.4 ⫾ 0.5 4.0 ⫾ 0.8 1.7 ⫾ 0.5 0.43 ⫾ 0.14 0.73 ⫾ 0.24 2.26 ⫾ 0.44

21.2 ⫾ 2.4 3.3 ⫾ 1.0 1.3 ⫾ 0.3 10.0 ⫾ 2.0 9.9 ⫾ 1.6 5.9 ⫾ 1.5 4.9 ⫾ 0.8 0.89 ⫾ 0.28 1.9 ⫾ 0.7 0.72 ⫾ 0.14 1.6 ⫾ 0.2 1.1 ⫾ 0.7 0.66 ⫾ 0.46 1.1 ⫾ 0.4 1.8 ⫾ 0.6 1.8 ⫾ 0.3 1.7 ⫾ 0.2 2.4 ⫾ 0.8 3.5 ⫾ 1.2 1.5 ⫾ 0.2‡ 0.52 ⫾ 0.12* 0.54 ⫾ 0.18 1.4 ⫾ 1.0

7.5 ⫾ 2.3 5.1 ⫾ 2.7 5.0 ⫾ 1.2 22.7 ⫾ 4.7 30.6 ⫾ 4.9† 2.4 ⫾ 0.3 7.0 ⫾ 1.6 1.1 ⫾ 0.4 4.4 ⫾ 2.4 1.8 ⫾ 0.6 2.8 ⫾ 0.6 1.6 ⫾ 0.3 0.95 ⫾ 0.76 1.4 ⫾ 0.9 7.9 ⫾ 4.2 2.4 ⫾ 1.6 3.8 ⫾ 1.8 1.5 ⫾ 0.1 3.3 ⫾ 1.1 1.9 ⫾ 0.4* 0.64 ⫾ 0.42 0.27 ⫾ 0.14 0.32 ⫾ 0.16

0.90 ⫾ 0.39 8.9 ⫾ 3.6 3.8 ⫾ 1.0 20.9 ⫾ 3.9 23.1 ⫾ 5.4 1.1 ⫾ 0.4 4.9 ⫾ 0.5 3.3 ⫾ 4.1 3.2 ⫾ 1.4† 1.6 ⫾ 0.2* 2.5 ⫾ 0.6 1.9 ⫾ 0.8 0.65 ⫾ 0.52 0.88 ⫾ 0.76 9.5 ⫾ 4.2 2.6 ⫾ 1.1 6.1 ⫾ 2.7 0.72 ⫾ 0.22 2.5 ⫾ 0.4‡ 1.2 ⫾ 0.5 0.52 ⫾ 0.32 0.04 ⫾ 0.01 0.11 ⫾ 0.08

0.11 ⫾ 0.08 5.8 ⫾ 3.7 3.5 ⫾ 1.2 23.4 ⫾ 9.4 23.4 ⫾ 12.1 0.92 ⫾ 0.63 4.8 ⫾ 1.3 1.0 ⫾ 0.5 1.5 ⫾ 0.6 1.3 ⫾ 0.6 2.5 ⫾ 1.5 1.5 ⫾ 0.4 1.3 ⫾ 0.8* 0.88 ⫾ 0.31 4.7 ⫾ 2.8 2.5 ⫾ 1.5 4.4 ⫾ 2.2 0.93 ⫾ 0.16 3.3 ⫾ 2.0* 0.94 ⫾ 0.26 0.37 ⫾ 0.16 0.04 ⫾ 0.06 0.05 ⫾ 0.05

0.04 ⫾ 0.01 2.5 ⫾ 0.5 1.8 ⫾ 0.4 11.6 ⫾ 3.8 11.6 ⫾ 2.3 0.59 ⫾ 0.32 4.0 ⫾ 0.9 1.5 ⫾ 0.6 1.9 ⫾ 1.2 0.76 ⫾ 0.14† 1.1 ⫾ 0.2 0.89 ⫾ 0.12*§ 0.9 ⫾ 1.0 0.50 ⫾ 0.14 7.3 ⫾ 4.4 3.1 ⫾ 1.0 3.5 ⫾ 0.7 0.48 ⫾ 0.07 2.2 ⫾ 1.7 0.90 ⫾ 0.21 0.62 ⫾ 0.65 0.01 ⫾ 0.00 0.02 ⫾ 0.02

* p ⬍ 0.05 in comparison with unirradiated controls. p ⬍ 0.01 in comparison with unirradiated controls. ‡ p ⬍ 0.001 in comparison with unirradiated controls. § Indicates value in irradiated group significantly lower than in unirradiated controls. †

values. This finding may be partly explained by a low control uptake value at this time point. For some tissues, there was an increase in deposition of IDLPL after TBI (Fig. 1). The most striking example of this phenomenon was the marked effect of TBI on the splenic uptake of IDLPL (Fig. 1A). Splenic uptake was significantly higher than unirradiated controls at 24 h for the 2-Gy group (30.6 ⫾ 4.9 vs. 17.5 ⫾ 4.0% ID/g, p ⬍ 0.01). For the 5-Gy group, splenic uptake was significantly higher than unirradiated controls at 4 h (12.3 ⫾ 2.8 vs. 8.4 ⫾ 1.0% ID/g, p ⬍ 0.02), 24 h (34.1 ⫾ 5.3 vs. 17.5 ⫾ 4.0% ID/g, p ⬍ 0.001), 48 h (41.8 ⫾ 13.2 vs. 15.8 ⫾ 5.4% ID/g, p ⬍ 0.01), and 72 h (33.5 ⫾ 10.6 vs. 13.6 ⫾ 3.9% ID/g, p ⬍ 0.01). At dissection, the spleens of the mice that had received TBI appeared to be smaller than those of the unirradiated control group. Therefore, the splenic weights in the control, 2-Gy and 5-Gy TBI groups were compared, and the data for splenic uptake were recalculated in terms of whole organ uptake values. There was a significant reduction in the splenic weight in irradiated animals as compared to unirradiated controls, with the effect most marked at the 5-Gy dose level (Fig. 2A). Splenic weight fell rapidly, with significant reduction after 4 h at the 5-Gy dose, reaching a nadir at 24 – 48 h before gradual recovery. The rapidity of onset and reversibility of this phenomenon supports the hypothesis that radiation-induced cell death of splenic lymphocytes was responsible. There was no significant increase in the whole organ uptake values between the irradiated and unirradiated

groups, confirming that the measured increase in splenic uptake was an apparent rather than a true effect (p ⬎ 0.1 for all comparisons) (Fig. 2B). There was evidence of increased localization of IDLPL in the small intestine of irradiated mice (Fig. 1C). For the 5-Gy group, liposome uptake in the small intestine was significantly increased at 4 h, 24 h, 48 h, and 72 h. Similarly, uptake in the stomach was significantly increased at the 48 –96 h time points. As regards the musculoskeletal system, there was a small increase in uptake of IDLPL at early time points (before 24 h) in bone and muscle tissue after both 2 Gy and 5 Gy TBI. The levels of uptake in the adrenal gland were significantly higher than controls for both 2 Gy and 5 Gy TBI groups at the 48 h and 72 h time points (Fig. 1D). The results for the tissues of the female genital tract showed evidence of increased localization of IDLPL in the uterus and ovarian tissue at 24, 48, and 72 h after 5 Gy TBI (Fig. 1E). However, it should be noted that there was considerable variability of these data, which may have been due, at least in part, to variations in the state of oestrus of the mice in different groups. However, the state of oestrus was not examined, and no data were available on this variable. Interval study For these studies, the data from the 24 h time point of the TBI time-course study were used as controls. The corresponding data for the groups of animals that received TBI at doses of 2 Gy and 5 Gy administered at various time points

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Table 3. Biodistribution of IDLPL in time-course study of the effect of 5 Gy total-body irradiation; data expressed in % injected dose per gram (mean ⫾ SD)

Blood Urine Tumor Liver Spleen Lung Kidney Bladder Esophagus Stomach Ileum Colon Gallbladder Pancreas Uterus Ovary Skin Heart Adrenal Bone Muscle Brain Spinal cord

1h

4h

24 h

48 h

72 h

96 h

28.0 ⫾ 3.6 83.7 ⫾ 38.5 0.81 ⫾ 0.47 5.1 ⫾ 1.2 6.2 ⫾ 1.0 6.6 ⫾ 1.8 5.1 ⫾ 1.3 1.0 ⫾ 0.4 2.1 ⫾ 0.8 0.90 ⫾ 0.19 1.1 ⫾ 0.2 0.72 ⫾ 0.20 0.49 ⫾ 0.30 1.1 ⫾ 0.2 1.2 ⫾ 0.2 1.8 ⫾ 0.6 1.3 ⫾ 0.3 3.6 ⫾ 0.7 3.9 ⫾ 1.6 1.8 ⫾ 0.6 0.72 ⫾ 0.20* 0.77 ⫾ 0.22 1.5 ⫾ 0.7

19.8 ⫾ 2.5 7.7 ⫾ 7.4 1.9 ⫾ 0.5 9.5 ⫾ 1.7 12.3 ⫾ 2.8* 6.2 ⫾ 1.8 4.5 ⫾ 1.2 0.93 ⫾ 0.20 2.1 ⫾ 0.7 0.72 ⫾ 0.19 1.9 ⫾ 0.2‡ 1.4 ⫾ 0.9 0.91 ⫾ 0.53 1.1 ⫾ 0.5 2.3 ⫾ 0.5 1.7 ⫾ 0.6 1.4 ⫾ 0.5 2.3 ⫾ 0.9 4.8 ⫾ 2.1 1.7 ⫾ 0.2‡ 0.57 ⫾ 0.16† 0.52 ⫾ 0.18 1.1 ⫾ 0.3

6.9 ⫾ 0.8 5.9 ⫾ 1.6 4.8 ⫾ 1.1 22.8 ⫾ 5.1 34.1 ⫾ 5.3‡ 2.9 ⫾ 1.3 6.9 ⫾ 1.3 0.97 ⫾ 0.17 3.4 ⫾ 0.8 1.6 ⫾ 0.4 3.2 ⫾ 0.4† 1.8 ⫾ 0.5 0.46 ⫾ 0.19 1.4 ⫾ 0.9 8.6 ⫾ 2.4* 3.0 ⫾ 1.3 6.0 ⫾ 1.3 1.7 ⫾ 0.3 3.1 ⫾ 0.6 1.7 ⫾ 0.3 0.56 ⫾ 0.15* 0.33 ⫾ 0.09† 0.17 ⫾ 0.03

1.6 ⫾ 0.9 9.2 ⫾ 2.9 5.3 ⫾ 1.9 23.7 ⫾ 5.2 41.8 ⫾ 13.2† 1.1 ⫾ 0.2 6.5 ⫾ 1.6 1.3 ⫾ 0.5* 2.5 ⫾ 0.5‡ 1.7 ⫾ 0.3* 3.3 ⫾ 1.3* 2.1 ⫾ 0.5 0.48 ⫾ 0.39 0.51 ⫾ 0.17 10.7 ⫾ 1.8* 4.2 ⫾ 1.2† 6.2 ⫾ 1.5 1.3 ⫾ 0.7 3.2 ⫾ 0.6‡ 1.7 ⫾ 0.5† 0.55 ⫾ 0.32 0.06 ⫾ 0.05 0.18 ⫾ 0.13

0.07 ⫾ 0.04 5.6 ⫾ 2.7 3.5 ⫾ 1.1 15.4 ⫾ 2.7 33.5 ⫾ 10.6† 0.58 ⫾ 0.14 5.1 ⫾ 0.9 0.77 ⫾ 0.15* 1.6 ⫾ 0.6 1.1 ⫾ 0.2* 1.8 ⫾ 0.2† 1.4 ⫾ 0.1†§ 0.60 ⫾ 0.41 0.39 ⫾ 0.15 6.2 ⫾ 3.1 3.0 ⫾ 0.7† 5.6 ⫾ 0.7 0.67 ⫾ 0.16 1.7 ⫾ 0.2† 1.1 ⫾ 0.3 0.34 ⫾ 0.19 0.01 ⫾ 0.00 0.01 ⫾ 0.03

0.04 ⫾ 0.03 5.7 ⫾ 5.1 2.1 ⫾ 0.8 11.6 ⫾ 1.8 13.9 ⫾ 2.8 0.38 ⫾ 0.04 3.7 ⫾ 0.8 0.61 ⫾ 0.11 0.82 ⫾ 0.25 0.62 ⫾ 0.17 1.1 ⫾ 0.6 0.64 ⫾ 0.19†§ 0.28 ⫾ 0.21 0.21 ⫾ 0.08 4.6 ⫾ 5.1 2.4 ⫾ 1.1 3.8 ⫾ 1.5 0.41 ⫾ 0.11 0.64 ⫾ 0.34 0.70 ⫾ 0.30 0.23 ⫾ 0.03 0.01 ⫾ 0.00 0.04 ⫾ 0.07

* p ⬍ 0.05 in comparison with controls. p ⬍ 0.01 in comparison with controls. ‡ p ⬍ 0.001 in comparison with controls. § Indicates value in irradiated group significantly lower than in untreated controls. †

between 72 h and 1 h before injection of IDLPL are shown in Tables 5 and 6, respectively. As with the time-course study, there was no significant difference in the levels of tumor localization of IDLPL between unirradiated controls and animals that had received TBI. Again, account was taken of the possible effect of tumor weight on these data. There were no statistically significant differences (p ⬎ 0.1 for all comparisons) in the mean tumor weights for the unirradiated controls and the 2 Gy and 5 Gy TBI groups (Table 7). There was no demonstrable effect of 2 Gy and 5 Gy TBI on the blood levels of 111In-DTPA-labeled pegylated liposomes at 24 h postinjection. The only significant difference was seen at the 2 Gy dose delivered 24 h before liposome injection (3.6 ⫾ 2.2 vs. 7.4 ⫾ 2.1% ID/g, p ⬍ 0.05). As can be seen in Tables 5 and 6, there were few differences between unirradiated control and irradiated animals for most normal tissues. Specifically, there was no evidence of altered uptake in lung, liver, esophagus, gall-

bladder, pancreas, heart, spinal cord, brain, or adrenal (p ⬎ 0.1 at each dose and time point). In some tissues (stomach, ileum, colon, skin, bladder, uterus, and ovary), the levels of liposome uptake at one or two time points were significantly higher or lower than controls in irradiated animals. In such cases, the lack of a consistent pattern or evidence of dose dependence suggests that such observations are unlikely to be clinically relevant. In contrast, the levels of renal uptake were significantly reduced after TBI doses of 2 Gy and 5 Gy delivered 48 and 72 h before liposome injection (p ⬍ 0.05 for each comparison). It is possible that such data may reflect acute radiation-induced alterations of renal blood flow. In the musculoskeletal system there was evidence of alteration of the biodistribution of IDLPL in those mice irradiated at 72, 48, and 24 h before administration, although this effect only appeared to be consistent for the 5 Gy TBI dose level. Once again, TBI appeared to increase IDLPL uptake in the spleen. Although there seemed to be a trend toward

Table 4. Tumor weights in time-course study of biodistribution of 111In-DTPA-labeled pegylated liposomes after total-body irradiation; data expressed in grams (mean ⫾ SD) Group

1h

4h

24 h

48 h

72 h

96 h

Control 2 Gy 5 Gy

0.72 ⫾ 0.31 0.63 ⫾ 0.40 0.57 ⫾ 0.22

0.56 ⫾ 0.28 0.49 ⫾ 0.31 0.64 ⫾ 0.26

0.52 ⫾ 0.19 0.69 ⫾ 0.28 0.62 ⫾ 0.20

0.64 ⫾ 0.16 0.73 ⫾ 0.21 0.53 ⫾ 0.27

0.49 ⫾ 0.26 0.47 ⫾ 0.17 0.70 ⫾ 0.23

0.58 ⫾ 0.25 0.62 ⫾ 0.28 0.46 ⫾ 0.24

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Fig. 1. Effect of total-body irradiation (TBI) on time course of 111In-DTPA-labeled pegylated liposomes (IDLPL) biodistribution in selected normal tissues: (A) spleen; (b) stomach; (C) small intestine; (D) adrenal gland; (E) ovary. Data are presented as mean % ID/g (⫾ SD).

increased uptake after 2 Gy TBI, no statistically significant differences were seen (p ⬎ 0.1 for all comparisons). However, at the 5-Gy dose level there was a striking increase in the level of uptake in the spleen relative to unirradiated controls when the irradiation was delivered at 6 h (36.2 ⫾ 7.2 vs. 17.5 ⫾ 4.0% ID/g, p ⬍ 0.002) or 24 h (55.0 ⫾ 26.2 vs. 17.5 ⫾ 4.0% ID/g, p ⬍ 0.02) before the injection of the IDLPL. This effect was absent at the 48 and 72 h time points (p ⬎ 0.1). In line with the data presented for the time-course study above, the effect of splenic weight on these data were assessed. The splenic weights were significantly reduced after 2 Gy TBI at 1 h and 6 h and after 5 Gy TBI at 1, 6, and 24 h before IDLPL injection (Fig. 3A). There was no significant difference in whole organ splenic liposome lev-

els in the irradiated and unirradiated control animals, except at the 5 Gy dose for animals irradiated 1 h before liposome injection, in which case the splenic uptake in the irradiated animals was lower than the control group (1.8 ⫾ 0.5 vs. 2.9 ⫾ 0.8% ID, p ⬍ 0.05) (Fig. 3B). DISCUSSION In recent years, CCRT approaches have been shown to yield clinical benefit in patients with head and neck (15, 16), lung (17–19), esophageal (20), anal (21), and cervical cancers (22–24). In addition, there is interest in the use of CCRT in patients with bladder (25, 26) and pancreatic cancers (27, 28), sarcomas (29), and gliomas (30, 31).

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Fig. 2. Effect of total-body irradiation (TBI) at doses of 2 Gy and 5 Gy on (A) splenic weight; (B) radiolabeled liposome uptake during the time-course study.

However, in a number of these studies, the reported clinical benefits have been achieved at the expense of increased acute and late radiation toxicity, presumably as a consequence of sensitization of normal tissues to the effects of radiation. Therefore, attempts to target delivery of radiation sensitizers preferentially to tumor tissues by using pegylated liposomes represents a possible means of circumventing this problem. The cancers listed above are likely to represent the most appropriate clinical systems in which to test the efficacy and safety of concomitant pegylated liposomal agents and RT in the first instance. As such, the findings reported here have considerable importance for the future development of this approach.

As in previous studies using doses between 5 Gy and 20 Gy (11), there was no alteration of tumor uptake of liposomes at these clinically relevant radiation doses of 2 Gy and 5 Gy. Such data contrast with studies investigating the effect of tumor irradiation on uptake of MAB (8 –10). However, it must be borne in mind that these results are derived from a single animal model, and the effects of irradiation on tumor microvascular permeability and interstitial fluid pressure may be heterogeneous and tumor dependent. In comparison with uptake data from other studies, the levels of tumor uptake documented with this animal model were relatively low (up to 6% ID/g compared to up to 15–20% ID/g for conventional [32–34] and up to 8 –10%

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Table 5. Biodistribution of IDLPL in interval study of the effect of 2 Gy total-body irradiation; data expressed in % injected dose per gram (mean ⫾ SD)

Urine Blood Tumor Liver Spleen Lung Kidney Bladder Esophagus Stomach Ileum Colon Gallbladder Pancreas Uterus Ovary Skin Heart Adrenal Bone Muscle Brain Spinal cord

⫺72 h

⫺48 h

⫺24 h

⫺6 h

⫺1 h

4.7 ⫾ 1.7 5.7 ⫾ 1.2 4.1 ⫾ 1.0 19.5 ⫾ 2.5 16.0 ⫾ 1.4 2.3 ⫾ 1.0 4.0 ⫾ 0.5* 1.1 ⫾ 0.2 2.3 ⫾ 1.2 1.4 ⫾ 0.3 1.9 ⫾ 0.2 1.6 ⫾ 0.4 1.0 ⫾ 0.6 0.81 ⫾ 0.49 5.1 ⫾ 2.3 3.5 ⫾ 1.5 7.7 ⫾ 1.6** 0.96 ⫾ 0.14 2.9 ⫾ 0.4 1.5 ⫾ 0.3 0.75 ⫾ 0.33* 0.11 ⫾ 0.04 0.26 ⫾ 0.14

5.3 ⫾ 1.6 5.7 ⫾ 0.3 4.2 ⫾ 2.2 17.3 ⫾ 2.9 18.1 ⫾ 8.2 1.3 ⫾ 0.3 4.0 ⫾ 0.5* 1.2 ⫾ 0.8 2.4 ⫾ 0.7 1.4 ⫾ 0.1 1.6 ⫾ 0.3 1.5 ⫾ 0.8 0.8 ⫾ 1.0 0.43 ⫾ 0.20 4.4 ⫾ 2.9 1.9 ⫾ 0.5 7.2 ⫾ 1.9 0.97 ⫾ 0.19 1.9 ⫾ 0.3 1.5 ⫾ 0.4 0.61 ⫾ 0.26 0.08 ⫾ 0.02 0.18 ⫾ 0.12

6.4 ⫾ 4.9 3.6 ⫾ 2.2* 4.0 ⫾ 4.3 16.1 ⫾ 5.9 18.5 ⫾ 7.3 2.2 ⫾ 0.8 4.8 ⫾ 0.6 1.8 ⫾ 1.4 2.9 ⫾ 1.3 1.9 ⫾ 0.3† 4.0 ⫾ 1.2† 2.5 ⫾ 0.6 1.2 ⫾ 0.9 0.62 ⫾ 0.30 13.1 ⫾ 5.6* 3.6 ⫾ 1.9 6.2 ⫾ 1.6 1.1 ⫾ 0.5 4.2 ⫾ 1.8 2.4 ⫾ 0.8* 0.41 ⫾ 0.24 0.12 ⫾ 0.06 0.54 ⫾ 0.41

7.9 ⫾ 3.4 5.3 ⫾ 1.0 4.7 ⫾ 0.8 18.3 ⫾ 3.0 23.3 ⫾ 2.3 2.2 ⫾ 0.3 5.5 ⫾ 0.8 1.3 ⫾ 0.3 2.4 ⫾ 0.8 1.7 ⫾ 0.2 3.0 ⫾ 0.5 2.3 ⫾ 0.4 1.0 ⫾ 0.3 1.9 ⫾ 0.3 6.5 ⫾ 2.9 3.7 ⫾ 0.5 5.8 ⫾ 0.3 1.1 ⫾ 0.3 2.3 ⫾ 0.9 1.5 ⫾ 0.2 0.40 ⫾ 0.15 0.15 ⫾ 0.06 0.48 ⫾ 0.20

9.5 ⫾ 5.6 5.6 ⫾ 2.1 4.7 ⫾ 1.4 20.0 ⫾ 1.2 23.5 ⫾ 7.3 2.6 ⫾ 0.7 5.1 ⫾ 1.7 0.92 ⫾ 0.09 2.3 ⫾ 1.4 1.2 ⫾ 0.3 3.2 ⫾ 1.3 1.7 ⫾ 0.6 0.75 ⫾ 0.31 1.7 ⫾ 1.2 6.1 ⫾ 2.7 2.2 ⫾ 0.9 6.4 ⫾ 1.8 1.1 ⫾ 0.4 2.5 ⫾ 0.9 1.3 ⫾ 0.5 0.38 ⫾ 0.14 0.18 ⫾ 0.08 0.38 ⫾ 0.10

* p ⬍ 0.05 in comparison with unirradiated controls—24-h time point from Table 1. p ⬍ 0.01 in comparison with unirradiated controls—24-h time point from Table 1.

†

ID/g for other sterically stabilized (including pegylated) [35–39] liposomes). Previous histologic studies using this animal model have demonstrated that tumor necrosis develops in tumors greater than 0.75 g and that this phenomenon is associated with a significant reduction in liposome uptake (14). Therefore, further studies in different tumor types and in tumors with different patterns of vascularization may provide further useful information on the effect of RT on tumor uptake of liposomes. Significantly, there was no measurable effect of RT in a wide range of normal tissues, including the lung, liver, esophagus, colon, pancreas, gallbladder, heart, central nervous system, and skin. These data are relevant because these tissues can be important radiation dose-limiting structures in certain clinical scenarios. For example, in patients with intrathoracic tumors, such as bronchogenic or esophageal cancers, the dose-limiting late normal tissue structures for radical RT are the adjacent ipsilateral and contralateral lung, esophagus, and spinal cord. Therefore, the fact that RT does not increase pegylated liposome deposition in any of these tissues provides reassurance that increased normal tissue toxicity arising from targeted radiosensitisation would not be likely. In the context of head and neck cancer, in which Phase I/II studies of pegylated liposomal radiosensitizers have already been reported (6, 7), cutaneous and mucosal radiation toxicity are dose-limiting. Although this animal model did not permit accurate quantitation of the effect of TBI on IDLPL uptake in the mucosae of the upper aerodigestive tract, the lack of any effect on cutaneous and esophageal liposome localization support the premise that CCRT

will be feasible in SCCHN. In addition, the lack of an effect of irradiation on the deposition of IDLPL in the brain suggests that use of liposome-targeted agents would not cause an excess risk of brain necrosis during CCRT for head and neck cancer (e.g., nasopharyngeal cancer in which the temporal lobes receive a significant radiation dose) or gliomas. For anal, bladder, cervical, and prostatic cancers, the dose-limiting late normal tissue structures for radical RT are the bowel and bladder. The data showing no change in localization of IDLPL in colon and bladder are reassuring but the finding of increased liposome uptake in the small intestine is of concern. Similarly, for primary tumors in the upper gastrointestinal tract (pancreatic or biliary tree cancer), increased liposome deposition in the small intestine and stomach would pose the threat of increased acute and late bowel toxicity. The significance of the effects of RT on liposome uptake into muscle and bone are uncertain. Although these tissues can manifest both acute (myositis, myelosuppression) and late (pathologic fractures, marrow failure) radiation toxicities, these are rarely significant. In this regard, the lack of any evidence of increased toxicity to the musculoskeletal system during studies of liposomal doxorubicin with RT in patients with lung and head and neck cancers and sarcomas (7, 40) offers reassurance that this issue is unlikely to be of clinical significance. The mechanisms underlying the observed increases in liposome uptake in certain tissues are not clear. The most dramatic data were observed for the spleen and, initially, it was thought that this effect was due to increased splenic vascular permeability secondary to RT. However, as de-

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Table 6. Biodistribution of IDLPL in interval study of the effect of 5 Gy total-body irradiation; data expressed in % injected dose per gram (mean ⫾ SD)

Urine Blood Tumor Liver Spleen Lung Kidney Bladder Esophagus Stomach Ileum Colon Gallbladder Pancreas Uterus Ovary Skin Heart Adrenal Bone Muscle Brain Spinal cord

⫺72 h

⫺48 h

⫺24 h

⫺6 h

⫺1h

4.7 ⫾ 3.5 7.1 ⫾ 1.2 4.9 ⫾ 1.9 16.9 ⫾ 4.9 19.7 ⫾ 9.4 1.6 ⫾ 0.4 3.8 ⫾ 1.9* 1.0 ⫾ 0.5 3.0 ⫾ 2.8 1.7 ⫾ 0.8 1.5 ⫾ 0.6 2.3 ⫾ 1.1 1.3 ⫾ 1.2 1.3 ⫾ 0.7 5.4 ⫾ 4.2 2.3 ⫾ 0.8 6.5 ⫾ 1.7 1.0 ⫾ 0.5 3.5 ⫾ 1.0 2.6 ⫾ 0.8† 1.0 ⫾ 0.5* 0.11 ⫾ 0.07 0.24 ⫾ 0.11

5.5 ⫾ 0.9 5.4 ⫾ 1.7 4.8 ⫾ 1.4 19.1 ⫾ 3.9 20.8 ⫾ 6.5 1.3 ⫾ 0.3 4.4 ⫾ 0.8* 1.6 ⫾ 0.7 3.0 ⫾ 0.9 1.6 ⫾ 0.2 1.9 ⫾ 0.4 2.2 ⫾ 0.3 1.1 ⫾ 1.8 0.77 ⫾ 0.48 6.5 ⫾ 1.0 2.9 ⫾ 0.5 4.8 ⫾ 1.1 1.0 ⫾ 0.1 2.4 ⫾ 0.3 2.1 ⫾ 0.5† 0.82 ⫾ 0.45* 0.07 ⫾ 0.01 0.17 ⫾ 0.05

9.5 ⫾ 5.9 5.3 ⫾ 2.8 5.8 ⫾ 2.8 16.2 ⫾ 4.8 55.0 ⫾ 26.2* 3.7 ⫾ 1.9 4.6 ⫾ 0.7 0.90 ⫾ 0.23 2.1 ⫾ 1.2 1.1 ⫾ 0.5 2.3 ⫾ 0.7 1.0 ⫾ 0.2†‡ 0.67 ⫾ 0.75 0.79 ⫾ 0.21 3.1 ⫾ 2.5 2.7 ⫾ 1.5 4.4 ⫾ 1.8 1.1 ⫾ 0.3 2.7 ⫾ 1.3 1.4 ⫾ 0.4 0.40 ⫾ 0.16 0.18 ⫾ 0.07 0.56 ⫾ 0.34

8.4 ⫾ 4.1 6.4 ⫾ 1.1 5.9 ⫾ 2.2 16.6 ⫾ 2.5 36.2 ⫾ 7.2† 2.3 ⫾ 0.4 4.4 ⫾ 0.7 0.74 ⫾ 0.35*‡ 2.5 ⫾ 0.6 1.2 ⫾ 0.3 2.0 ⫾ 0.3 2.2 ⫾ 0.7 0.70 ⫾ 0.40 1.2 ⫾ 0.5 4.8 ⫾ 1.1 2.3 ⫾ 0.6 4.5 ⫾ 1.3 1.2 ⫾ 0.4 2.7 ⫾ 0.5 1.4 ⫾ 0.2 0.47 ⫾ 0.10 0.17 ⫾ 0.06 0.48 ⫾ 0.18

6.9 ⫾ 2.8 8.7 ⫾ 1.6 5.2 ⫾ 2.0 19.1 ⫾ 1.8 27.2 ⫾ 8.2 2.8 ⫾ 0.3 4.7 ⫾ 1.1 0.79 ⫾ 0.21*‡ 2.3 ⫾ 0.8 1.0 ⫾ 0.4 2.7 ⫾ 0.4 2.8 ⫾ 0.7 1.0 ⫾ 1.3 0.55 ⫾ 0.30 5.8 ⫾ 1.7 2.4 ⫾ 0.3*† 4.2 ⫾ 1.1 1.4 ⫾ 0.2 3.7 ⫾ 0.6 1.7 ⫾ 0.5 0.30 ⫾ 0.06 0.24 ⫾ 0.08 0.47 ⫾ 0.23

* p ⬍ 0.05 in comparison with unirradiated controls—24 h time point from Table 1. p ⬍ 0.01 in comparison with unirradiated controls—24 h time point from Table 1. ‡ Indicates value in irradiated group significantly lower than in untreated controls. †

tailed above, this apparent increase in uptake, expressed in terms of % ID/g, disappeared when account was taken of the significant reductions in splenic weight after RT. Therefore, perhaps the most likely explanation of these data are that irradiation resulted in massive depletion of splenic lymphocytes, which are exquisitely sensitive to RT, without significantly affecting vascular permeability. The increased tissue localization of IDLPL in the small intestine, stomach, musculoskeletal system, female reproductive tract (especially the uterus), and adrenal gland cannot be explained so easily. There exists the possibility that some of the documented increase may have been caused by increased intravascular, as opposed to extravascular, accumulation of liposomes, perhaps mediated by the hyperemia induced by a direct effect of RT on blood vessels or by an indirect effect of RT on the autonomic nervous system. However, this explanation would not account for the changes seen at 48 h or later, at which time the blood liposome levels had decreased such that they were equal to or less than those in most tissues. In addition, the exsanguination of the mice at the time of

sacrifice was designed to minimize this possible effect of blood-borne liposomes on the measured tissue levels of liposome uptake. Another possible common link between some of these tissues (gastrointestinal tract, bone marrow, and female reproductive tract), which might offer an explanation for these findings, is that they contain rapidly proliferating cell populations which are sensitive to RT. In the case of the small intestine, cell death in the epithelial cell population may have caused increased vascular permeability, or, alternatively, inflammatory changes may have been responsible for the increased liposome deposition in this tissue. No histologic studies were performed to confirm the presence of cell death in this population or, indeed, to look for evidence of inflammatory change. Another possibility is that radiation-induced lymphocyte deletion in the small intestine may have reduced the weight of this tissue relative to the control group. This hypothesis is not easy to test because the entire small intestine was not dissected and counted in the gamma counter but, rather, a single loop of intestine was taken. The reason for the increased levels of

Table 7. Tumor weights in interval study of biodistribution of 111In-DPTA-labeled pegylated liposomes after total-body irradiation; data expressed in grams (mean ⫾ SD) Group 2 Gy 5 Gy

⫺72 h

⫺48 h

⫺24 h

⫺6 h

⫺1 h

0.42 ⫾ 0.18 0.38 ⫾ 0.18

0.63 ⫾ 0.22 0.45 ⫾ 0.14

0.59 ⫾ 0.19 0.60 ⫾ 0.25

0.47 ⫾ 0.26 0.39 ⫾ 0.17

0.67 ⫾ 0.31 0.53 ⫾ 0.23

Note: The mean tumor weight (⫾ SD) of the control group was 0.52 ⫾ 0.19 g.

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Fig. 3. Effect of total-body irradiation (TBI) at doses of 2 Gy and 5 Gy on (A) splenic weight; (B) radiolabeled liposome uptake during the interval study. Note: The 24-h time point from the time-course study was used as the control group.

liposome deposition in the musculoskeletal system are not readily apparent. Whereas radiation-induced cell death of hematopoietic cells in the bone marrow of the femur may have been responsible for the increased levels seen in the bones, this does not explain the increased levels seen in the quadriceps femoris muscle. As discussed above, the female mice used in these studies may have had rapidly dividing cells in the female reproductive tract, although no specific data were available on their state of estrus. The lack of a clear pattern of a dose dependence in this effect (a more dramatic effect at 2 Gy than 5 Gy in the interval study) may

point to the fact that variations between the treatment groups may have played a part in this finding. As regards the effect of TBI on increasing liposome uptake into the adrenal glands, this was only seen at 48 and 72 h in the time-course study. This may indicate that the effect was a result of increased adrenal blood flow in response to the stress of TBI. However, this possibility does not adequately explain the absence of a similar effect for the animals irradiated 72 and 48 h before liposome injection in the interval study. Although these studies have documented an increase in liposome uptake in certain tissues after RT, they do not

Irradiation and biodistribution of pegylated liposomes

necessarily predict an increase in normal tissue toxicity in combination with RT. Such an occurrence would need there to be increased release of the liposomal contents under the influence of tissue factors and macrophages. The studies reported here do not provide data on this important issue that should be the basis for future biodistribution and therapeutic studies. It is also important to recognize the fact that the experiments performed here do not simulate standard RT practice that involves fractionated treatment for 5 days per week over a period of up to 7 weeks. Therefore, it is possible that during such fractionated courses of RT the vascular volume and permeability in normal tissues may be altered such that liposome extravasation may either increase or decrease. Indeed, such changes may underlie

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the somewhat increased cutaneous and mucosal toxicity reported in clinical studies combining RT and pegylated liposomal doxorubicin. Unfortunately, the nature of this murine model precluded delivery of fractionated RT over a prolonged period. However, this issue is clearly of great importance and should form the basis of further preclinical and clinical study. Therefore, in summary, the findings of these studies suggest that CCRT approaches using pegylated liposomes are likely to be safe. However, the documented increases in liposome uptake in the gastrointestinal and musculoskeletal systems mean that considerable caution should be exercised in the development of such treatment strategies where there will be significant volumes of small intestine, stomach, or bone marrow within the irradiated area.

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