AU2988300A - Liposome composition and method for administration of a radiosensitizer - Google Patents

Description

WO 00/45845 PCT/USOO/03359 LIPOSOME COMPOSITION AND METHOD FOR ADMINISTRATION OF A RADIOSENSITIZER Field of the Invention The present invention relates to a composition and method for administration of a radiosensitizer to enhance radiation treatment as a part of cancer therapy. Backcground of the Invention 10 Radiation treatment has become a conventional part of cancer therapy. One shortcoming to radiotherapy, however, is the destruction to normal, healthy tissue surrounding the tumor which occurs during treatment. Another shortcoming is that after cessation of treatment, recurrence of the tumor can 15 and does occur. Recurrence of the tumor has been partly attributed to the presence of radioresistant hypoxic tumor cells, and the enhancement of radiation dose to damage the hypoxic tumor tissue is often necessary. However, to save normal, healthy tissue, a reduction in the total radiation 20 dose would be desirable. Obviously, these two factors are contradictory. Therefore, the use of certain drugs and chemicals that preferentially sensitize hypoxic tumor cells to radiation, radiosensitizers, are employed. Radiosensitizers are chemical agents that have the 25 capacity to increase the lethal effects of radiation when administered in conjunction with radiation and there are a variety of radiosensitizers that act by more than one mechanism. One class of radiosensitizers that directly alter the macromolecular apparatus determining the radiosensitivity 30 are halogenated pyrimidines. The halogenated pyrimidines include 5-chlorodeoxy-uridine (CudR), 5-bromodeoxyuridine (BudR) and 5-iododeoxy-uridine (IudR). These radiosensitizers are incorporated into the DNA in the tumor cell in place of thymine and render the cell more susceptible to inactivation 35 by radiation. In cancer radiotherapy, the usefulness of inducing an increase in radiosensitivity by selective incorporation of a 1 WO 00/45845 PCTIUSO0/03359 halogenated pyrimidine is limited by several factors. First, the drug must be present for a period long enough to allow the cells to pass through at least one DNA synthesis cycle since halogenated pyrimidines are incorporated only in cells in the 5 S phase. While tumor cells often multiply faster than normal tissues, the fact that certain tumors may have doubling times varying from days to weeks complicates the planning of therapy. Second, the degree of radiosensitization is directly related to the degree of thymidine substitution. Hence, only 10 a prolonged infusion of the drug in free form will maximize its incorporation. Third, it is not only the extent of sensitization that is important, but rather the total number of cells that must be sensitized to obtain any significant effect on the tumor. Fourth, the rapid hepatic degradation is and dehalogentaion must be overcome. One approach to overcoming some of these limitations has been to encapsulate the halogenated pyrimidine in liposomes. However, these attempts have not been entirely satisfactory because the radiosensitizers tend to leak out of the liposomes 20 rapidly and the liposomes have poor stability in serum. Summary of the Invention Accordingly, it is an object of the invention to provide a composition and method for administration of a 25 radiosensitizer for radiotherapy. It is a further object of the invention to provide a method for achieving radiosensitization of a tumor with a single, weekly dose. It is a further object of the invention to provide a 30 composition which achieves a two-fold higher distribution of the radiosensitizer in the tumor tissue than in the normal, healthy tissue. In one aspect, the invention includes a method for administering a radiosensitizer to a tumor. The method 35 includes preparing liposomes comprised of (i) a vesicle forming lipid; (ii) between 1-20 mole percent of a vesicle forming lipid derivatized with a hydrophilic polymer chain, 2 WO 00/45845 PCT/USOO/03359 and (iii) between 1-15 mole percent of a radiosensitizer derivatized with a lipid moiety linked to the radiosensitizer; and administering the liposomes to a tumor bearing patient. 5 In one embodiment, the radiosensitizer is 5-iodo 2 'deoxyuridine or 5-bromo-2 'deoxyuridine. In another embodiment, the lipid moiety is a fatty acid or a saturated fatty acid. In other embodiments, the lipid moiety is selected from lauric acid, myristic acid, palmitic 10 acid, stearic acid, arachidic acid, behenic acid and lignoceric acid. In a preferred embodiment, the radiosensitizer is 5 iodo-2'-deoxyuridine and the lipid moiety is palmitic acid. The radiosensitizer is derivatized with a second lipid 15 moiety, in another embodiment of the invention. For example, the radiosensitizer is 5-iodo-2'-deoxyuridine and the lipid moieties are palmitic acid. The hydrophilic polymer chain included in the lipid composition and derivatized to a lipid moiety, in one 20 embodiment, is polyethyleneglycol. In another aspect, the invention includes a method for preparing a liposome composition including a radiosensitizer, by mixing in a lipid solvent (i) a vesicle-forming lipid; (ii) between 1-20 mole percent of a vesicle-forming lipid 25 derivatized with a hydrophilic polymer chain, and (iii) between 1-15 mole percent of a radiosensitizer derivatized with a lipid moiety linked to the radiosensitizer; and adding an amount of a second solvent selected (i) to achieve a lipid solvent amount greater than 10 weight percent and less than 30 about 50 weight percent and (ii) to obtain a liposome size less than that obtained at another lipid solvent amount, where the lipid solvent and the second solvent are miscible in the resulting hydration mixture. In one embodiment, the lipid solvent is an alcohol, such 35 as methanol, ethanol or butanol. The second solvent, in one embodiment, is water. In another aspect, the invention includes a liposome composition for administration of a radiosensitizer. The composition includes liposomes comprised of (i) a vesicle 3 WO 00/45845 PCTUSOO/03359 forming lipid; (ii) between 1-20 mole percent of a vesicle forming lipid derivatized with a hydrophilic polymer chain, and (iii) between 1-15 mole percent of a radiosensitizer derivatized with a lipid moiety linked to the 5 radiosensitizer. The composition is formed by (a) mixing components (i), (ii) and (iii) in a lipid solvent; and (b) adding a selected amount of a second solvent, said selected amount effective (i) to achieve a lipid solvent amount greater than 10 weight percent and less than about 50 weight io percent and (ii) to obtain a liposome size smaller than that obtained a lipid solvent amount other than said selected amount, said lipid solvent and said second solvent being miscible at the selected amount of second solvent. These and other objects and features of the invention 15 will be more fully appreciated when the following detailed description of the invention is read in conjunction with the accompanying drawings. Brief Description of the Drawings 20 Fig. 1 is a synthetic reaction scheme for synthesis of 3',5'-dipalmitoyl-5-iodo- 2 '-deoxyuridine; Fig. 2 is a plot of liposome size, in nm, as a function of weight percent ethanol during hydration of the liposome lipids; 25 Fig. 3 is a phase diagram for a lipid, solvent, second solvent mixture, showing a preferred operating region when the solvent is ethanol and the second solvent is water; Fig. 4 is a plot showing tumor volume, in mm 3 , for mice bearing firbrosarcoma tumors and treated with radiation alone 30 (#7, closed triangles), with 0.5 ml (#1, closed, inverted triangles) or 1 ml (#3, closed circles) of dpIUdR-entrapped liposomes, and with dpIUdR liposomes at various dosages in combination with radiation (#2, 4, 5, 6); and Figs. 5A-5B are plots showing survival of tumor-bearing 35 mice (Fig. SA) and percentage change in tumor volume (Fig. 5B) following treatment with radiation alone (solid triangles) or with combined therapy of liposome-entrapped dpIUdR and radiation (solid squares). 4 WO 00/45845 PCTIUSOO/03359 Detailed Description of the Invention I. Definitions As used herein, "dpIUdR" refers to 3',5'-dipalmitoyl- 5 iodo-2' -deoxyuridine. 5 As used herein, "IUdR" refers to 5-iodo-2'-deoxyuridine. II. Lipid Derivatized Radiosensitizer As discussed above, radiation sensitizers are compounds that are capable of being incorporated into cellular DNA and 10 subsequently enhancing the damage caused by ionizing radiation when solid tumors are treated with radiation therapy. Two such radiation sensitizers contemplated for use in the present invention are 5-iododeoxyuridine and 5-bromodeoxyuridine. These compounds behave as an analog of thymidine and in the 15 cell undergo phosphorylation and ultimate incorporation into DNA in place of thymidylate. In the present invention the radiosensitizer is derivatized with a lipid moiety for incorporation into the lipid bilayer of a liposome. A wide variety of lipid moieties 20 are suitable for incorporation into a liposome lipid bilayer and include fatty acids, monoacylglycerols, diacylglycerols, fatty alcohols, cholesterol, and phospholipids. These lipids are merely exemplary, and it will be appreciated that any compound which when derivatized to the radiosensitizer renders 25 the radiosensitizer more lipophilic is suitable. In a preferred embodiment, the radiosensitizer is derivatized with a fatty acid, which in preferred embodiments has between 2-24 carbon atoms, more preferably between 10-20 carbon atoms. Suitable fatty acids include saturated fatty acids such as 30 lauric acid (C12) , myristic acid (C14) , palmitic acid (C16), stearic acid (C18), arachidic acid (C20), behenic acid (C22), lignoceric acid (C24) and unsaturated fatty acids, such as oleic acid (C18). It will be appreciated by those of skill in the liposome 35 art that the length of the acyl tail on the lipid moiety is selected according to the degree of compatibility desired with the vesicle-forming lipids forming the liposome lipid bilayer. Lipid moieties having an acyl chain length within one or two carbons of the length of the acyl chain on the vesicle-forming 5 WO 00/45845 PCT/USOO/03359 lipid will result in a more uniform lipid bilayer with the derivatized radiosensitizer more strongly retained. This feature can be used to tailor release of the derivatized radiosensitizer from the bilayer, if desired. 5 The radiosensitizer can be derivatized with one lipid moiety, or in a preferred embodiment of the invention, with two lipid moieties. More lipid moieties may also be possible in some situations. The position at which the radiosensitizer is derivatized is variable, with the proviso that the compound io must retain therapeutic activity after release from the lipid derivative or, in the cases where the lipid derivative is retained, the therapeutic activity is retained when in the derivatized form. In studies performed in support of the invention, 5-iodo 15 2'deoxyuridine, referred to herein as IUdR, was derivatized with a 16-carbon fatty acid, palmitic acid, at two positions on the ribose sugar of IUdR, as shown in Fig. 1. In the reaction scheme shown in the figure and detailed in Example 1, IUdR was reacted with a small excess of palmityl chloride and 20 4-dimethylpyridine catalyst in pyridine or in pyridine/chloroform as the solvent to yield 3',5'-dipalmitoyl 5-iodo-2'-deoxyuridine, referred to herein as dpIUdR. As will be described below, the dpIUdR was incorporated into liposomes and tested in vivo for therapeutic efficacy as a 25 radiosensitizer. III. Method of Preparing the Liposome Composition In one aspect, the invention includes a method of preparing a liposome composition suitable for use in 30 administering a lipid-derivatized radiosensitizer, as exemplified by dpIUdR. One feature of therapeutic liposome compositions is the drug/lipid ratio, where a high drug/lipid ratio is preferred to achieve a therapeutic efficacy with as low of a lipid burden to the patient as possible. Preparation 35 of a lipid-derivatized compound poses formulation issues not encountered with conventional, non-lipid derivatized compounds since the lipid tail on the compound contributes to the lipid bilayer and the total lipid content. As the total lipid content increases, sizing of the liposomes by conventional 6 WO 00/45845 PCT/US00/03359 techniques, such as extrusion through polycarbonate membranes, becomes difficult and limits the drug/lipid ratio which can be obtained. This point is illustrated by the data set forth in Table 5 1. Liposomes were prepared according to the procedure set forth in Example 2, where the lipids hydrogenated soy phosphatidyl choline (HSPC) and methoxy-polyethylenelglycol distearyl-phosphatidyl-ethanolamine (mPEG-DSPE) and the lipid derivatized dpIUdR were dissolved in ethanol in molar ratios 10 of 89/5/6, 87.5/5/7.5 and 85/5/10. The ethanol-lipid solutions were hydrated with an aqueous second solvent to a final ethanol amount of 10.1 weight percent. The liposome suspensions were extruded through polycarbonate disc membranes to achieve a target liposome size of about 100 nm. The 15 liposome particle size in each suspension was then measured using quasi-elastic light scattering and the results are shown in Table 1. As seen in Table 1, liposomes prepared with 6 and 7.5 mole percent dpIUdR could be sized by extrusion through the 20 membranes. However, at 10 mole percent dpIUdR, the target liposome size of around 100 nm could not be achieved, as the liposomes could no longer be readily extruded at the higher lipid content. 25 Table 1 Mole% Conc. dpIUdR Conc. drug/lipid liposome size dpIUdR (mg/ml) phospholipids ratio after (mg/ml) extrusion (nm) 6 2.35 1.50 1.57 90 6 3.48 2.19 1.59 120 7.5 2.06 1.10 1.87 102 10 3.37 1.17 2.88 143 This problem of liposome sizing at higher loading of a lipid-derivatized drug was overcome by preparing the liposomes 30 according to a method now to be described. The method provides a means to achieve a high drug/lipid ratio while retaining the ability to obtain the desired liposome size, 7 WO 00/45845 PCT/USOO/03359 either through use of the method alone or in combination with a second sizing step such as extrusion. In the method, the vesicle-forming lipid and the lipid derivatized compound are mixed in a lipid solvent. As used 5 herein, "lipid solvent" refers to an organic solvent in which the lipid components of the liposome are soluble, at any temperature. Exemplary lipid solvents include alcohols, such as methanol, ethanol, butanol, etc. and low molecular weight polyols, such as glycerol, propyleneglycol and ethyleneglycol. 10 The lipids are added to the solvent in the desired molar ratio and mixed until dissolved, with heating as necessary. An amount of a second solvent is then added to the lipid solvent/lipid mixture to form a hydration mixture. The second solvent, as used herein, refers to a solvent that is miscible 15 with the lipid solvent in some proportion, and must be miscible with the lipid solvent in the resulting hydration mixture. The second solvent is added to the lipid solvent mixture in an amount sufficient to bring the weight percentage of the lipid solvent to a selected point which is greater than 20 about 10 weight percent but less than about 50 weight percent of the lipid solvent, more preferably to a lipid solvent concentration in the range of 15-45 weight percent, most preferably to a lipid solvent concentration in the range of 20-40 weight percent, to obtain liposomes having a desired 25 size, as will now be illustrated. As described in Example 3, the lipids HSPC, mPEG-DSPE and dpIUdR were dissolved in ethanol in an 89:5:6 molar ratio. This lipid stock solution was used to prepare liposome suspensions by hydrating an amount of the lipid solution with 30 a second solvent, an aqueous buffer. Liposome suspensions were prepared in triplicate at final ethanol weight percentages of 8.1, 10.1, 12.2, 14.3, 16.5, 20.8, 25.3, and 44.1. The average size of the liposomes in each sample was measured by quasi-elastic light scattering, and the results are shown in 35 Table 2. 8 WO 00/45845 PCT/US0O/03359 Table 2 Sample f Liposome Size in nm at weight Percent Ethanol No. 8.1 10.1 12.2 114.3 [16.5 20.8 25.3 44.1 1 51200 1410 226 197 84 53700 1000000 12500 2 71 46 78 119 149 27300 11000 10500 3_ _ 405 78 157 1360 84 11400 21400 12500 AVERAGE 17225 511 154 559 106 30800 1344133 11833 In viewing the last row of Table 2, it is apparent that 5 there is a minimum in the liposome particle size at 16.5 weight percent ethanol. This data is shown graphically in Fig. 2 and the trough in liposome size beginning at about 10 weight percent and ending at about 25 weight percent is apparent. A profile such as the one in Fig. 2 of lipid size 10 as a function of ethanol content can be employed to determine the ethanol amount needed to obtain a particular liposome size. For example, the minimum particle size of 106 nm is obtained by hydrating the lipid-ethanol mixture to 16.5 weight percent ethanol. A larger particle size is obtained by 15 hydrating to achieve more or less ethanol, according to the profile. Establishing such a profile, the target amount of lipid solvent to achieve a minimum particle size or a selected particle size is determined for any mixture of lipids and solvents, as will be further illustrated below. 20 Fig. 3 is a phase diagram showing the operating region for formation of the liposomes in accordance with the invention. In the diagram, the shaded region corresponds to formation of liposomes where the lipid solvent amount is between about 10-50 weight percent and the weight percent of 25 the lipids is between 0.1-15. Depending on the lipid mixture and the lipid solvent, the point at which a minimum in liposome particle size occurs within the operating region can be determined. The liposome suspensions in the operating region are visually clear and contain submicron size 30 liposomes. It will be appreciated that the operating region will vary slightly according to the lipid, solvent and drug components. One of skill in the art can readily conduct a study like that set forth in Example 3 and in Table 2 to determine the amount of lipid solvent that yields a minimum in 9 WO 00/45845 PCT/US0O/03359 the liposome size. Liposomes formed by the above-described method can be, depending on the lipid solvent amount in the hydration mixture, at a minimum particle size. Thus, in some cases, it 5 may not be necessary to further size the liposomes via extrusion or other technique. In some cases, it may be desirable to further process the liposomes, for example by extrusion. The method of preparation is particularly useful for incorporation of lipid-derivatized drugs into liposomes at 10 a high drug-to-lipid ratio, where obtaining a pharmaceutically useful particle size of between 90-150 nm is difficult due to an inability to extrude the mixture, as discussed above. Formation of liposomes according to the invention overcomes this limitation, since the liposomes are at or near to the 15 desired particle size upon hydration with the second solvent, and any further size processing is minimized. Thus, a higher lipid-derivatized drug load can be employed while still being able to achieve the desired liposome size. This feature of the invention is illustrated by the study 20 described in Example 4. Liposomes were prepared using the lipids HSPC, mPEG-DSPE and dpIUdR, where the molar amounts of the formulations were 89/5/6, 85/5/10, 80/5/15, 70/5/25 and 55/5/40. The lipids, including the dpIUdR, were dissolved in ethanol and then hydrated with sufficient water to bring the 25 ethanol concentration in each mixture to 16.5 weight percent (20 volume percent) . Each liposome suspension was then extruded as described in the Example and after extrusion, the average particle size of the liposomes in each suspension was measured. The results are shown in Table 3. 30 Table 3 Mole percent Conc. dpIUdR Drug/lipid ratio Liposome size dpIUdR (mg/ml) (nm 6 1.25 1.67 99 10 2.32 3.17 95 15 4.09 4.65 103 25 6.90 8.91 142 40 *composition could not be extruded 10 WO 00/45845 PCTIUSOO/03359 Liposomes containing 6, 10 and 15 mole percent dpIUdR were readily sized to about 100 nm when prepared according to the method of the invention by hydrating the lipid mixture to an amount of lipid solvent that gives a minimum particle size. 5 Importantly, the liposomes are formed at the minimum particle size with a drug-to-lipid ratio of between about 1.5 and 5, more generally between about 2-4. In a preferred embodiment of the invention, liposomes having the desired particle size are prepared using the method to a drug-to-lipid ratio of 10 greater than about 4, as achieved with the liposome composition having 15 mole percent dpIUdR. It will be appreciated that a variety of lipids are suitable for use in the present invention. The studies performed in support of the invention using HSPC and mPEG-DSPE 15 are merely illustrative, and any of the vesicle-forming lipids known to those of skill in the field of liposomes are contemplated. It is also possible to provide a targeted liposome composition containing the entrapped radiosensitizer, by 20 including targeting moieties such as antibodies, to the distal ends of the PEG chains or on the outer surface of the liposomes, as is known in the art. IV. Method of Administering the Composition 25 Liposomes prepared according to the invention and including dpIUdR were administered to animals to determine its effectiveness of the liposome composition as a radiosensitizer. The liposome formulation was composed of HSPC, mPEG-DSPE and dpIUdR in a molar ratio of 89:5:6. The 30 liposome particle diameter was approximately 100 nm, with the dpIUdR prodrug incorporated into the liposome lipid bilayer by insertion of the palmitic acid chains into the bilayer. After delivery of the liposome vehicle to a tumor, hydrolysis of the prodrug releases IUDR into the tumor interstitial space where 35 it can enter the cells and become incorporated into the DNA. The radiosensitizing potential of the dpIUdR liposomes was evaluated in two murine tumor models, the RIF-1 fibrosarcoma model and the human head-and-neck xenograft KB tumor in nude mice. In the fibrosarcoma model, the animals 11 WO 00/45845 PCT/USOO/03359 were inoculated with the tumor and were used in the study after the tumor volume was greater than about 100 mm 3 . The tumor-bearing animals were treated according to one of several regimens set forth in Table 4. 5 Table 4 Regimen Liposome Dose Radiation Dose Days of liposome Dose Adminstration' 1 0.5 ml None -1, +2 2 0.5 ml 5 x 4 Gy -1, +2 3 1 ml None -1, +2 4 1 ml 5 X 4 Gy -1, +2 5 0.5 ml 5 x 4 Gy -3, 0 6 1 ml 5 x 4 Gy -3, 0 7 none 5 x 4 Gy na 8 (saline none None none control) 'indicated as days preceding (e.g., -1) or following (e.g., +2) the first day of radiation treatment, taken as day 0. 10 The results of animals treated with regimens 1-8 of Table 4 are shown in Fig. 4. In the figure, the arrow heads along the x-axis on days 1-5 indicate the days on which a radiation treatment of 4 Gy was given. The data shows that treatment 15 with dpIUdR containing liposomes and radiotherapy (regimens 2 (closed diamonds), 4 (open squares) , 5 (open triangles), 6 (open inverted triangles) delays the growth of the tumor by as much as 30% over the same time period when compared to radiation alone (closed triangles) . The data further shows 20 that dosing one day prior to radiation therapy is sufficient for the dpIUdR to reach the tumor site and become incorporated into the tumor cell DNA, as evidenced by regimen 2 where the 0.5 ml liposome dose was administered just one day prior to the first radiation treatment and then on day 2 of the study. 25 In the KB tumor model study, the animals were dosed concurrent with initiation of radiation therapy. The tumor bearing animals were divided into two test groups, one group receiving radiation therapy alone (9 Gy in 3 fractions) and the other group receiving a dose of the liposome composition 30 on day zero, e.g., the day of radiation therapy. The group 12 WO 00/45845 PCT/USOO/03359 receiving the liposome composition also received a radiation treatment of 9 Gy in 3 fractions. The results of the KB tumor model study are shown in Figs. SA-5B, where Fig. 5A shows the survival of KB-tumor 5 bearing mice, taken as the percentage of tumors not reaching three tumor volume doublings as a function of number of days into the study. The data shows that when using dpIUdR in combination with radiation, the number of mice surviving three weeks after treatment is about 35% greater than the mice that 10 were treated with radiation alone. Fig. 5B shows the percentage change in tumor volume for the test mice, and indicated that dpIUdR in combination with radiation significantly delays tumor growth. At 20 days, the tumor growth of the group receiving radiation alone was more 15 than 200% greater than the tumors of those receiving the combination treatment. The liposomes of the invention containing the radiosensitizer are administered to patients by any of the known procedures utilized for administering liposomes. The 20 liposomes can be administered intravenously, intraperitoneally, intramuscularly, intratumorally, or subcutaneously as a buffered, aqueous solution. Any pharmaceutically acceptable aqueous buffered or other vehicle can be utilized as long as the liposome structure and activity 25 are retained. The dosage for a mammal, including a human, will vary depending on the tumor type, tumor stage, and condition of the patient. Dosage levels for radionucleotides are well established and serve as a guideline for determining the 30 dosage required for the liposome composition of the invention. In a preferred embodiment of the invention, the liposome composition is administered once or twice per week prior or during radiation therapy. In another preferred embodiment of the invention, the liposome composition is effective to 35 achieve a two-fold higher distribution into the tumor tissue than in normal tissue. V. Examples The following examples illustrate the compositions and 13 WO 00/45845 PCT/USOO/03359 methods of the invention and in no way are intended to be limiting. Materials: The following materials were obtained from the 5 indicated source: partially hydrogenated soy phosphatidylcholine (Vernon Walden Inc., Green Village, NJ); cholesterol (Solvay Pharmaceuticals, The Netherlands); ethanol (Quantum Chemical Co. Tuscola, IL) and histidine (Seltzer Chemicals, Carlsbad, CA). mPEG-DSPE was prepared as described 10 in the literature (for example, Zalipsky, S., et al, Bioconjugate Chem., 4:296-299 (1993)). Methods QUELS: quasi-elastic light scattering was performed using a 15 Brookhaven Instruments Model Bl-200SM (Brookhaven Instruments Corporation, Holtsville, NY). Example 1 Synthesis of 3',5'-dipalmitoyl-5-iodo-2'-deoxyuridine 20 Palmityl chloride was added slowly to a solution of deoxyuridine in pyridine or in pyridine/chloroform and 4 dimethylpyridine as a catalyst. The solution was mixed until yellow in color and then left to stand overnight for precipitation. The mixture was dissolved in chloroform and 25 washed with 10% aqueous citric acid solution and saturated sodium bicarbonate solution. The chloroform was removed and methanol was added to yield a white precipitate, identified as 3', 5 '-dipalmitoyl-5-iodo-2'-deoxyuridine (66% yield). The synthetic reaction scheme is shown in Fig. 1. 30 Example 2 Conventional Liposome Preparation The lipids hydrogenated soy phosphatidyl choline (HSPC) and distearoylphosphatidylcholine derivatized with methoxy 35 polyethyleneglycol (DSPE-mPEG) and dpIUdR in molar ratios of 89/5/6, 87.5/5/7.5 and 85/5/10 were each dissolved in ethanol at 70 0 C until complete dissolution was achieved (about 1 hour). The lipid mixtures were hydrated with sufficient 10% sucrose solution at 70 0 C to achieve an ethanol weight 14 WO 00/45845 PCT/USOO/03359 percentage of 10.1 (12.5% v/v percent). The mixtures were stirred for one hour to form liposome suspensions. The liposome suspensions were extruded through polycarbonate membranes to achieve a uniform size of 100 nm. 5 The suspensions were then diafiltered against 10% sucrose solution to reduce the ethanol concentration to below 400 ppm. The suspensions were then ultrafiltered to above the target drug concentration, assayed for drug concentration and diluted to target by adding 10 mM histidine buffer and adjusting the 10 pH to 6.5. The liposome size of each formulation was determined by Quasi-elastic light scattering and the particle size as a function of weight percent dpIUdR is shown in Table 1. 15 Example 3 Liposome Preparation According to Method of the Invention The lipids hydrogenated soy phosphatidyl choline (HSPC) and distearoylphosphatidylcholine derivatized with methoxy polyethyleneglycol (DSPE-mPEG) and dpIUdR in molar ratio of 20 89/5/6 were dissolved in ethanol at 70 0 C until complete dissolution was achieved (about 1 hour). This lipid stock solution, after diluting as necessary to maintain a fixed lipid concentration, was hydrated with a 10% sucrose solution at 70 0 C to vary the ethanol concentration from 8-44 weight 25 percent. Each of the liposome mixtures were stirred for one hour to form liposome suspensions. The liposome particle size of each mixture was determined using quasi-elastic light scattering and the results are shown in Table 2. 30 Example 4 Liposome Preparation at 16.5 Weight Percent Lipid Solvent The lipids hydrogenated soy phosphatidyl choline (HSPC) and distearoylphosphatidylcholine derivatized with methoxy polyethyleneglycol (DSPE-mPEG) and dpIUdR in a molar ratios of 35 89/5/6, 85/5/10, 80/5/15, 70/5/25 and 55/5/40 were dissolved in ethanol at 700C until complete dissolution was achieved (about 1 hour). The lipid mixtures were hydrated with sufficient 10% sucrose solution at 700C to achieve a 16.5 weight percentage ethanol content in the hydration mixture 15 WO 00/45845 PCT/USOO/03359 (20% (v/v) ethanol) . The mixtures were stirred for one hour prior to extruding the mixtures according to the procedure described in Example 2 and measuring the liposome particle sizes by quasi-elastic light scattering. The results are 5 shown in Table 3. Although the invention has been described with respect to particular embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention. 16

Claims (23)

1. A liposome composition for use in administration of a radiosensitizer to a tumor in a subject, comprising 5 liposomes comprised of (i) a vesicle-forming lipid; (ii) between 1-20 mole percent of a vesicle-forming lipid derivatized with a hydrophilic polymer chain, and (iii) between 1-15 mole percent of a radiosensitizer derivatized with a lipid moiety linked to the radiosensitizer. 10

2. The composition according to claim 1, wherein the radiosensitizer is 5-iodo-2'deoxyuridine or 5-bromo 2'deoxyuridine. 15

3. The composition according to claim 1 or claim 2, wherein the lipid moiety is a fatty acid.

4. The composition according to claim 1 or claim 2, wherein the lipid moiety is a saturated fatty acid. 20

5. The composition according to claim 4, wherein the lipid moiety is selected from lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid and lignoceric acid. 25

6. The composition according to claim 1, wherein the radiosensitizer is 5-iodo-2'-deoxyuridine and the lipid moiety is palmitic acid. 30

7. The composition according to claim 1, wherein the radiosensitizer is derivatized with a second lipid moiety.

8. The composition according to claim 7, wherein the radiosensitizer is 5-iodo-2'-deoxyuridine and the lipid 35 moieties are palmitic acid.

9. The composition according to any of the preceding claims, wherein the hydrophilic polymer chain is polyethyleneglycol. 17 WO 00/45845 PCTIUSOO/03359

10. Use of a composition according to any of the preceding claims for the manufacture of a medicament for administration of a radiosensitizer to a subject. 5

11. A method for preparing a liposome composition according to any one of the preceding claims, comprising mixing in a lipid solvent (i) a vesicle-forming lipid; (ii) between 1-20 mole percent of a vesicle-forming lipid derivatized with a hydrophilic polymer chain, and (iii) io between 1-15 mole percent of a radiosensitizer derivatized with a lipid moiety linked to the radiosensitizer; and adding a selected amount of a second solvent, said selected amount effective (i) to achieve a lipid solvent amount greater than 10 weight percent and less than about 50 15 weight percent and (ii) to obtain a liposome size smaller than that obtained a lipid solvent amount other than said selected amount, said lipid solvent and said second solvent being miscible at the selected amount of second solvent. 20

12. The method according to claim 11, wherein the lipid solvent is an alcohol.

13. The method according to claim 12, wherein the lipid solvent is methanol, ethanol or butanol. 25

14. The method according to claim 11, wherein the second solvent is water.

15. A liposome composition for use in administration of 30 a radiosensitizer to a subject, comprising liposomes comprised of (i) a vesicle-forming lipid; (ii) between 1-20 mole percent of a vesicle-forming lipid derivatized with a hydrophilic polymer chain, and (iii) between 1-15 mole percent of a radiosensitizer derivatized 35 with a lipid moiety linked to the radiosensitizer; said liposomes obtainable by (a) mixing components (i), (ii) and (iii) in a lipid solvent, and (b) adding a selected amount of a second solvent, said selected amount effective (i) to achieve a lipid solvent amount greater than 10 weight percent 18 WO 00/45845 PCT/USOO/03359 and less than about 50 weight percent, and (ii) to obtain a liposome size smaller than that obtained a lipid solvent amount other than said selected amount, said lipid solvent and said second solvent being miscible at the selected amount 5 of second solvent.

16. The composition according to claim 15, wherein the radiosensitizer is 5-iodo-2'deoxyuridine or 5-bromo 2 'deoxyuridine. 10

17. The composition according to claim 15 or claim 16, wherein the lipid moiety is a fatty acid.

18. The composition according to claim 15 or claim 16, 15 wherein the lipid moiety is a saturated fatty acid.

19. The composition according to claim 18, wherein the lipid moiety is selected from lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid and 20 lignoceric acid.

20. The composition according to claim 15, wherein the radiosensitizer is 5-iodo-2'-deoxyuridine and the lipid moiety is palmitic acid. 25

21. The composition according to claim 20, wherein the radiosensitizer is derivatized with a second lipid moiety.

22. The composition according to claim 21, wherein the 30 radiosensitizer is 5-iodo-2'-deoxyuridine and the lipid moieties are palmitic acid.

23. The composition according to any of the claims 15 22, wherein the hydrophilic polymer chain is 35 polyethyleneglycol. 19