WO2008079240A1 - Use of liposome-entrapped agents for treating tumors - Google Patents

Use of liposome-entrapped agents for treating tumors Download PDF

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WO2008079240A1
WO2008079240A1 PCT/US2007/025963 US2007025963W WO2008079240A1 WO 2008079240 A1 WO2008079240 A1 WO 2008079240A1 US 2007025963 W US2007025963 W US 2007025963W WO 2008079240 A1 WO2008079240 A1 WO 2008079240A1
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cells
tumor
res
modulating
liposome
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PCT/US2007/025963
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French (fr)
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William Zamboni
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Alza Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • a method of treating tumors is provided. More specifically, a method of treating tumors by increasing the exposure of a tumor to a liposome-entrapped agent comprising the step of modulating the composition of cells in a subject's reticuloendothelial system (RES) is described. In addition, a method of treating tumors by increasing the release of drug from a liposome in a tumor comprising the step of modulating the composition of cells in a subject's RES is described. Further, a method of treating tumors by increasing the sensitivity of a tumor to treatment with a liposome- entrapped agent comprising the step of modulating the composition of cells in a subject's RES is described.
  • RES reticuloendothelial system
  • a method of treating tumors by individualizing treatment of a tumor comprising the steps of (1) evaluating the composition of RES cells in the tumor, and (2) determining a treatment regimen based on said evaluating, wherein said treatment regimen comprises administering at least one liposome-entrapped agent is described.
  • variable antitumor responses within a single patient may be associated with inherent differences in tumor vascularity, capillary permeability, and/or tumor interstitial pressure that result in variable delivery of anticancer agents to different tumor sites (Jain, RK. Delivery of molecular medicine to solid tumors. Science, 271(5252);1079-1080:1996b; Zamboni, W. C, Houghton, P. J., Hulstein, J. L., Kirstein, M., Walsh, J., Cheshire, P. J., Hanna, S. K., Danks, M. K., & Stewart, C. F.
  • Liposomal and nanoparticle are carrier formulations of anticancer agents and were developed to increase the tumor delivery; however, exact factors associated with the increased tumor delivery of nanoparticles and liposomes are unclear (Zamboni WC. Liposomal, nanoparticle, conjugated formulations of anticancer agents. Invited Review. Clin Cancer Res 11(23);8230-4:2005; Drummond, D.
  • Liposomal pharmacokinetics Classical, sterically-stabilized, cationic liposomes and immunoliposomes. In Liposomes: Rational Design, Janoff AS 5 ed. New York, Marcel Dekker, Inc. 63-87;2005).
  • the disposition of the STEALTH-encapsulated drug is dictated by characteristics of the liposome, such as, size, surface charge, membrane lipid packing, steric stabilization, dose, and route of administration, all of which alters the plasma pharmacokinetic profile and tissue distribution of the drug .
  • liposomes extravasate through leaky capillary beds of tumors and lodge into the interstitial spaces among tumor cells, where they release the encapsulated drug (Maeda, H., Wu, J., Sawa, T., Matsumura, Y., & Hori, K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release 65 (1-2); 271-284:2000).
  • the active form of the anticancer agent must be released from the liposome into the tumor extracellular fluid (ECF) and then penetrate into the cell.
  • ECF tumor extracellular fluid
  • RES reticuloendothelial system
  • the RES uptake of conventional or non-pegylated liposomes results in their rapid removal from the blood and accumulation in tissues involved in the RES, such as the liver and spleen. Uptake by the RES usually results in irreversible sequestering of the encapsulated drug in the RES, where it can be degraded. In addition, the uptake of the liposomes by the RES may result in acute impairment of the RES and toxicity.
  • S-CKD602 is a STEALTH liposomal formulation (phospholipid covalently bound to methoxypolyethylene glycol) of CKD-602, a camptothecin analogue (Zamboni, W. C, Gervais, A. C, Egorin, M. J., Schellens, J. H., Zuhowski, E. G., Pluim, D., Joseph, E., Hamburger, D. R., Working, P. K., Colbern, G., Tonda, M. E., Potter, D. M., & Eiseman, J. L.
  • CKD-602 Plasma and tumor disposition of STEALTH Liposomal CKD-602 (S-CKD602) and non- liposomal CKD-602, a camptothecin analogue, in mice bearing A375 human melanoma xenograft. Proceedings of AACR-NCI-EORTC 2005, #B 173). CKD-602 inhibits topoisomerase I, thereby preventing DNA replication and causing apoptosis.
  • Non- liposomal CKD-602 has demonstrated clinical activity and has been approved in South Korea in relapsed ovarian cancer and first line small cell lung cancer at a dose of 0.5 mg/m2 IV per day for five days, repeated every 3 weeks (Lee JH, Lee JM, Lim KH, Kim JK, Ahn SK, Bang YJ, Hong CL Preclinical and phase I clinical studies with CKD-602, a novel camptothecin derivative. Ann N Y Acad Sci 922;324-5:2000).
  • camptothecin analogues The cytotoxicity of camptothecin analogues is related to the duration of exposure in the tumor (Zamboni WC, Gajjar AJ, Houghton PJ, Mandrell TD, Einhaus SL, Danks MK, Rogers WP, Heideman RL, Stewart CF.
  • a topotecan 4-hour intravenous infusion achieves cytotoxic exposure throughout the neuroaxis in the nonhuman primate model: implications for the treatment of children with metastatic medulloblastoma.
  • a method of treating tumors in a subject is described, preferably a human tumor.
  • RES reticuloendothelial system
  • a method of treating tumors by increasing the release of drug from a liposome in a tumor comprising the step of modulating the composition of cells in a subject's RES is described.
  • a method of treating tumors by increasing the sensitivity of a tumor to treatment with a liposome-entrapped agent comprising the step of modulating the composition of cells in a subject's RES is described.
  • a method of treating tumors by individualizing treatment of a tumor comprising the steps of (1) evaluating the composition of RES cells in the tumor, and (2) determining a treatment regimen based on said evaluating, wherein said treatment regimen comprises administering at least one liposome-entrapped agent is described.
  • FIG. 1 Concentration versus time profiles of CKD-602 in plasma, tumor homogenate, and tumor ECF after administration of S-CKD602 in mice bearing A375 human melanoma xenografts.
  • Plasma profiles consist of sum total, encapsulated, and released CKD-602.
  • Plasma and tumor sum total concentration versus time point represents the mean of three mice.
  • the individual CKD-602 concentration in the tumor ECF is represented by the open diamonds.
  • the mean concentration in the tumor ECF at each collection interval is represented by the solid diamonds and is connected by the dashed line.
  • FIG. 1 Concentration versus time profiles of CKD-602 in plasma, tumor homogenate, and tumor ECF after administration of S-CKD602 in mice bearing SKOV-3 human ovarian xenografts.
  • Plasma profiles consist of sum total, encapsulated, and released CKD-602.
  • Plasma and tumor sum total concentration versus time point represents the mean of three mice.
  • the individual CKD-602 concentration in the tumor ECF is represented by the open diamonds.
  • the mean concentration in the tumor ECF at each collection interval is represented by the solid diamonds and is connected by the dashed line.
  • FIG. 3 Immunohistochemical staining using FITC-co ⁇ jugated cdl Ib antibody and polyethylene glycol-conjugated cdl Ic antibodies as measurements of cells of the RES in SKOV-3 human ovarian (top figure) and A375 human melanoma xenografts (bottom figure) obtained at 75 h after administration of S-CKD602 .
  • the cdl Ib antibody is primarily expressed on macrophages, DC, natural killer cells, and granulocytes. Detection of these cells using the immuno-fluorescence staining antibody cdl Ib emits the color green.
  • the cdl Ic antibody is expressed on DC and lymph node T- cells after activation and emits a red color upon staining of these cells.
  • APC antigen presenting cells
  • a conjugate of cdl Ib + cdl Ic was used. This conjugate emits a yellow color upon staining.
  • Figure 4A and 4B Time profiles of cdl Ib and cdl Ic percent staining in
  • the individual % staining of cdl Ib is represented by the open circles.
  • the mean % staining of cdl Ic at each sample time point is represented by the closed circles and is connected by the solid line.
  • the individual % staining of cdl Ic is represented by the open triangles.
  • the mean % staining of cdl Ic at each sample time point is represented by the closed triangle and is connected by the dashed line.
  • FIG. 5A and 5B Time profiles of cdl Ib and cdl Ic percent staining in spleen obtained from mice bearing A375 human melanoma ( Figure 3A) or SKOV-3 human ovarian xenografts ( Figure 3B) from 0 to 75 h.
  • the individual % staining of cdl Ib is represented by the open circles.
  • the mean % staining of cdl Ic at each sample time point is represented by the closed circles and is connected by the solid line.
  • the individual % staining of cdl Ic is represented by the open triangles.
  • the mean % staining of cdl Ic at each sample time point is represented by the closed triangle and is connected by the dashed line.
  • FIG. 6A and 6B Time profiles of cdl Ib and cdl Ic percent staining of liver obtained from mice bearing A375 human melanoma ( Figure 3A) or SKOV-3 human ovarian xenografts ( Figure 3B) from 0 to 75 h.
  • the individual % staining of cdl Ib is represented by the open circles.
  • the mean % staining of cdl Ic at each sample time point is represented by the closed circles and is connected by the solid line.
  • the individual % staining of cdl Ic is represented by the open triangles.
  • the mean % staining of cdl Ic at each sample time point is represented by the closed triangle and is connected by the dashed line.
  • Tumors are well known to those skilled in the art and are characterized by an abnormal growth of tissue resulting from uncontrolled, progressive multiplication of cells and serving no physiological function; also referred to as a neoplasm.
  • Commonly known types of tumors include, but are not limited to liver, brain, spleen, ovarian, kidney, breast, lung, colon and prostrate.
  • Tumors can be solid or one that forms in the plasma, such as multiple myeloma, primary or secondary, and are sometime drug resistant.
  • the term "tumors" as it is used in this specification is meant to be construed broadly and is not intended to be limited to any particular type of tumor unless otherwise indicated.
  • a method for increasing the exposure of a tumor to a liposome-entrapped agent comprising modulating the composition of cells in the reticuloendothelial system (RES).
  • the modulating comprises modulating the composition of cells in the RES by administering a compound that alters the composition of cells in the RES.
  • Cells in the reticuloendothelial system include, but are not limited to macrophages, dendritic cells, and monocytes.
  • the modulating further comprises modulating the composition of cells in the RES to achieve an increase in the number of RES cells present in a solid tumor, preferably the modulating achieves an increase in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
  • the modulating further comprises modulating the composition of cells by allowing a physiologic change in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
  • a method of increasing the release of drug from a liposome in a tumor comprising modulating the composition of cells in the reticuloendothelial system.
  • the modulating comprises modulating the composition of cells in the RES by administering a compound that alters the composition of cells in the RES.
  • Cells in the reticuloendothelial system include, but are not limited to macrophages, dendritic cells, and monocytes.
  • the modulating further comprises modulating the composition of cells in the RES to achieve an increase in the number of RES cells present in a solid tumor, preferably the modulating achieves an increase in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
  • the modulating further comprises modulating the composition of cells by allowing a physiologic change in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
  • a method for increasing sensitivity of a tumor to treatment with a liposome-entrapped agent comprising modulating the composition of cells in the reticuloendothelial system.
  • the modulating comprises modulating the composition of cells in the RES by administering a compound that alters the composition of cells in the RES.
  • Cells in the reticuloendothelial system include, but are not limited to macrophages, dendritic cells, and monocytes.
  • the modulating further comprises modulating the composition of cells in the RES to achieve an increase in the number of RES cells present in a solid tumor, preferably the modulating achieves an increase in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
  • the modulating further comprises modulating the composition of cells by allowing a physiologic change in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
  • a method for individualizing treatment of a tumor comprising evaluating the composition of RES cells in the tumor, and determining a treatment regimen based on said evaluating, wherein said treatment regimen comprises administering at least one liposome-entrapped agent.
  • a liposome-entrapped agent is a liposome-entrapped topoisomerase inhibitor.
  • the evaluating comprises determining the number of RES cells, preferably the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
  • topoisomerase inhibitors include, but are not limited to, topoisomerase I inhibitors such as camptothecin and camptothecin derivatives.
  • the camptothecin derivative can be 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, 10,11-methlyenedioxycamptothecin, 9- amino-10,11 -methylenedioxy camptothecin or 9-chloro-10,l 1- methylenedioxycamptothecin, irinotecan, topotecan, (7-(4-methylpiperazinomethylene)- 10,1 l-ethylenedioxy-20(S)-camptothecin, 7-(4-methylpiperazinomethylene)-10,l 1- methylenedioxy-20(S)-camptothecin.
  • a preferred topoisomerase I inhibitor is CKD-602.
  • the topoisomerase inhibitor can also be a topoisomerase I/II inhibitor, such as 6-[[2- (dimethylamino)-ethyl]amino]-3-hydroxy-7H-indeno[2, 1 -c]quinolin-7-on e dihydrochloride, azotoxin or 3-methoxy-l lH-pyrido[3',4'-4,5]pyrrolo[3,2-c]quinoline- 1 ,4-dione.
  • the liposome-entrapped topoisomerase inhibitor excludes liposome-entrapped doxorubicin. In another embodiment, the liposome- entrapped topoisomerase inhibitor excludes liposome-entrapped topoisomerase inhibitor II compounds, such as doxorubicin. It will be appreciated that a topoisomerase inhibitor II compound is one that inhibits or reduces the action of topoisomerase II enzyme. A topoisomerase inhibitor I compound is one that inhibits or reduces the action of topoisomerase I enzyme. A topoisomerase I/II inhibitor refers to any compound that inhibits or reduces the action of both topoisomerase I enzyme and topoisomerase II enzyme.
  • cytotoxic agents are particularly useful as the entrapped agent in liposomes targeted for neoplastic disease indications.
  • the drug may be an anthracycline antibiotic selected from doxorubicin, daunorubicin, epirubicin and idarubicin and analogs thereof.
  • the cytotoxic drug can be a nucleoside analog selected from gemcitabine, capecitabine, and ribavirin.
  • the cytotoxic agent may also be a platinum compound selected from cisplatin, carboplatin, ormaplatin, and oxaliplatin.
  • the cytotoxic agent may be a topoisomerase 1 inhibitor selected from the group consisting of topotecan, irinotecan, SN-38, 9-aminocamptothecin and 9-nitrocamptothecin.
  • the cytotoxic agent may be a vinca alkaloid selected from the group consisting of vincristine, vinblastine, vinleurosine, vinrodisine, vinorelbine and vindesine. It will be appreciated that cytotoxic agents are well known to those of skill in the art and readily determined from various medical reference books. [00027] It will be appreciated that the dose and dosing regimen can be varied to optimize the treatment of the tumor.
  • the dose of the anticancer agent can be adjusted higher or lower to achieve a desired modulation in the composition of cells in the RES.
  • the dosing regimen can be modified to achieve a desired increase in the number of macrophage cells, dendritic eels, and/or monocyte cells in the tumor.
  • the dosing regimen can comprise an escalating dose for a particular period of time, followed by a constant or decreasing dose for a second period of time.
  • the dosing regimen can be designed to achieve, in one embodiment a physiologic change in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor. Normal cell counts of macrophages, dendritic cells, and monocytes are know to those of skill in the art and readily determined from various medical reference books.
  • the method can additionally include administration of a liposome-entrapped agent in conjunction with a second therapeutic agent, in free or liposome-entrapped form.
  • the second agent can be any therapeutic agent mentioned herein, including other drug compounds (in one example chemotherapeutics) as well as biological agents, such as peptides, antibodies, and the like.
  • the second agent can be administered simultaneously with or sequential to administration of the targeted-liposomes, by the same or a different route of administration.
  • the objectives of this study were to evaluate the relationship between plasma and tumor disposition of S-CKD602 and the cells of the RES in mice bearing human melanoma and ovarian tumor xenografts.
  • the microdialysis methodology was used to evaluate the tumor ECF disposition of released CKD-602 after administration of S-CKD602. Materials and Methods Mice
  • mice were handled in accordance with the Guide to the Care and Use of Laboratory Animals (National Research Council, 1996), and studies were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh Medical Center.
  • Mice female CB-17 SCID, 4-6 weeks of age, and specific pathogen free), were obtained from the NCI Animal Production Program (Fredrick, MD), and were allowed to acclimate to the animal facilities at the University of Pittsburgh for 1 week prior to initiation of study.
  • mice were housed in microisolator cages and allowed Teklad LM-484 autoclavable rodent chow (Harlan Tekla Diets, Madison, WI) or ISDPRO RMH3000 irradiated rodent chow (PMI Nutrition International, Inc., Brentwood, MO) and were ad libitum. Body weights and tumor size were measured twice weekly and clinical observations were made twice daily.
  • A375 human melanoma xenografts and SKO V-3 human ovarian xenografts were obtained from the DCTD Tumor Repository (Fredrick, MD) and were mouse antigen production test-negative.
  • A375 or SKOV-3 tumors were passed in CB- 17 SCID mice, as approximately 25 -mg fragments implanted subcutaneously on the right flank by aseptic techniques. Tumor volumes were calculated from the formula: length x (width) 2 /2, where length is the largest diameter and width is the smallest diameter perpendicular to the length.
  • Pharmacokinetic and microdialysis studies were performed when the tumors were approximately 1000 to 1500 mm 3 (1 to 1.5 g) in size.
  • the formulation of S-CKD602 used in this study is the same as used in the phase I study of S-CKD602 (15).
  • the liposome of S-CKD602 contains hydrogenated soy phosphatidylcholine and cholesterol. Methoxypolyethylene glycol is covalently bound to phosphatidylethanolamine and a component of the lipid bilayer.
  • the mean particle size of the liposomes is approximately 110 nm.
  • Liposomal encapsulation of CKD-602 exceeds 90%.
  • the drug-to-lipid ratio is approximately 14 mg of CKD-602 per milligram of lipid.
  • the concentration of CKD-602 in the dosing solution is 1 mg/ml.
  • S-CKD602 was administered at 1 mg/kg IV push xl via a tail vein in mice bearing A375 human melanoma xenograft or SKO V-3 human ovarian xenograft (20). This dose is one-half the maximum tolerated dose (MTD) in mice (20).
  • the doses of S-CKD602 refer to actual doses of CKD-602.
  • mice bearing A375 human melanoma and SKOV-3 human ovarian xenografts Due to limited sample volume, the pharmacokinetic and microdialysis studies of S-CKD602 were performed in separate groups of mice bearing A375 tumors.
  • the blood samples were centrifuged at 12,000 x g for 4 min.
  • the plasma was processed to measure encapsulated, released, and sum total (encapsulated + released) CKD602. Tumors samples were obtained for measurement of sum total CKD602.
  • mice 3 per time point
  • heparinized blood samples approximately 0.8 to 1 mL
  • the HPLC system consisted of a Finnigan Specta Systems AS3000 autosampler and P4000 quarternary pump (Thermo Finnigan, Waltham, MA) with a Phenomenex Synergi Hydro-RP 8OA (4 um, 100 x 2 mm) analytical column (Phenomenex, Torrance, CA).
  • the isocratic mobile phase consisted of 0.1% formic acid in methanol: water (35:85, v/v) and was pumped at 0.2 ml/min. Injection volume was 10 ⁇ l and the run time was 10 min.
  • Standard curves for CKD were constructed by plotting the analyte to LS. ratio versus the known concentration of the analyte in each standard. Standard curves were fit by linear regression with 1/y 2 weighting and back calculation of CKD concentrations.
  • the cdl Ib antibody is primarily expressed on macrophages, DC, natural killer cells, and granulocytes. Detection of these cells using the immunofluorescence staining antibody cdl Ib emits the color green. The cdl Ic antibody is expressed on DC and lymph node T-cells after activation and emits a red color upon staining of these cells.
  • APC antigen presenting cells
  • the F4/80 monoclonal antibody reacts with an approximately 125 kDa transmembrane protein on mouse macrophages.
  • the F4/80 antigen is expressed by subpopulations of mature macrophages and is by some regarded as the best marker for this population of cells.
  • other cell types such as Langerhans cells and liver Kupffer cells have been reported to express this antigen.
  • some cytokines down regulate the expression of F4/80 resulting in lack of F4/80 antigen on a subpopulation of macrophages.
  • Cells stained with the F4/80 antibody emit a green color. Staining results were calculated as the percentage of total area with positive staining (% staining) in each sample. In addition, the mean +/- SD of percent staining from 0 to 72h was calculated.
  • S-CKD602 in mice bearing A375 human melanoma and SKO V-3 human ovarian xenografts The concentration versus time profile of sum total, encapsulated, and released CKD-602 mice bearing A375 human melanoma and SKOV-3 human ovarian xenografts are presented in Figure 1.
  • the pharmacokinetic parameters are summarized in Table 1. Since the extrapolated area associated with the estimated AUC from 0 to infinity was greater than 10% the AUC are reported as AUC from 0 to the last measurable time point. After administration of S-CKD602, the disposition of sum total, encapsulated, and released CKD-602 were similar in mice bearing SKOV-3 and A375 xenografts.
  • mice bearing SKOV-3 and A375 there was a prolonged exposure of sum total, encapsulated, and released CKD-602.
  • concentration versus time profiles of released CKD-602 in plasma was consistent with the profiles of sum total and encapsulated drug suggesting that there is relatively constant release of CKD-602 in the plasma.
  • the percent encapsulation of CKD-602 in mice bearing SKOV-3 and A375 was 73% and 84%, respectively.
  • the overall tumor disposition of CKD-602 was measured, as measured by sum total CKD-602 in tumor homogenates, and disposition of CKD-602 in tumor ECF, as estimated by using microdialysis methodology, after administration of S- CKD602 in mice bearing A375 human melanoma and SKO V-3 human ovarian xenografts.
  • concentration versus time profile of sum total CKD-602 in tumor and CKD-602 in tumor ECF in mice bearing A375 human melanoma and SKO V-3 human ovarian xenografts are presented in Figure 1.
  • the pharmacokinetic parameters are summarized in Table 1.
  • the concentration versus time profiles of sum total CKD-602 in tumor were similar in the SKO V-3 and A375 xenografts. In both mice models, there was a prolonged exposure of sum total CKD-60.
  • the sum total CKD-602 AUC in tumor was higher in SKOV-3 (15,698 ng/mL «h) compared with A375 (14,141 ng/mL*h) xenografts.
  • the ratio of tumor sum total AUC to plasma sum total AUC was 1.6-fold higher in mice bearing human SKOV-3 xenografts compared with A375.
  • the active drug must be released from the liposome into the tumor ECF, thus microdialysis was used to evaluate the disposition of released CKD-602 in tumor ECF.
  • the concentration versus time profile of CKD-602 in tumor ECF in mice bearing A375 human melanoma and SKOV-3 human ovarian xenografts are presented in Figure 1. In both tumor models, the concentration versus time profile of CKD-602 in tumor ECF was consistent with the profile of sum total CKD-602 in tumor.
  • the difference in the CKD- 602 measured in samples obtained from the tumor homogenate and tumor ECF may be due to the slow release of CKD-602 from the liposome and the subsequent binding of CKD-602 to proteins within the tumor matrix because the tumor ECF samples were obtained using microdialysis methodology which can only recovery released non-protein bound drug due to the molecular weight cut off of the probe ( ⁇ 20 kD).
  • the concentration of CKD-602 in tumor ECF varied approximately 10-fold at individual time points during each of the collection intervals.
  • the % staining of cdl Ib and cdl Ic in control tumors and in tumors at 27 h, 51 h, and 75 h after administration of S-CKD602 in A375 and SKOV-3 xenografts is presented in Figures 4A and 4B, respectively.
  • the cbl Ib and cdl Ic staining were relatively constant compared with the SKOV-3 xenografts.
  • the cbl Ib staining was variable from 0 to 75 h and the cdl Ic staining decreased 4-fold from 0 to 45 h and then increased 2-fold at 75 h.
  • mice bearing SKO V-3 The mean ⁇ SD % staining of cdl Ib and cdl Ic in spleen obtained from mice bearing SKO V-3 were 4.2 ⁇ 2.2 % and 1.3 ⁇ 0.7 %, respectively.
  • Malignant ascites is frequently associated with advanced ovarian cancer (Loercher AE, Nash MA, Kavanagh JJ, Platsoucas CD, Freedman RS.
  • monocytes and macrophages are the primary cells of the RES, which has also been called the mononuclear phagocytic system. Macrophages appear to be important in epithelial ovarian cancer as they are frequently the dominant population of leukocytes in the peritoneal fluid of patients with malignant ascites. Monocytes circulate in peripheral blood and can be induced by a variety of stimuli to adhere to the vascular endothelium and migrate into tissues where they differentiate into specialized cells, macrophages or dendritic cells.
  • the tumor associated macrophages appear to participate in the immunologic antitumor defense mechanism through direct cytotoxic and cytostatic activities or indirect activities through the release of cytokines, stimulating the adaptive immune response by antigen presentation, or producing factors with anti-angiogenic activity (e.g., angiostatin).
  • monocytes and macrophages and the RES may represent key targets for a variety of therapeutic interventions and may be prognostic factors in ovarian cancer.
  • the number of macrophages and dendritic cells in tumors as measured by cdl Ib, cdl Ic, and F4/80 staining are highly variable and change over time.
  • the low expression of F4/80 in the tumors relative to cdl Ic may indicate that the cells stained with cdl Ic are either DCs or immature macrophages. It is unclear if the changes in cells of the RES are due to physiological movement and function of these cells or related to direct or indirect cytotoxicity associated with S-CKD602 and/or released CKD-602 in the tumor.
  • monocytes are more sensitive to S-CKD602 as compared with neutrophils in patients and that the increased sensitivity is related to the liposomal formulation and not the encapsulated CKD-602.
  • the relationship between the reduction of monocytes in blood and the pharmacokinetic disposition of S-CKD602 in plasma of patients suggests that the monocytes engulf liposomal anticancer agents which cause the release of drug from the liposome and toxicity to the monocytes.
  • the changes in RES cells in the tumor and tissue in this study may be related to S-CKD602.
  • RES Refractory ovarian cancer
  • liposomal anticancer agents such as STEALTH liposomal doxorubicin (Doxil) and S- CKD602.
  • Doxil liposomal doxorubicin
  • S- CKD602. liposomal anticancer agents
  • the modulation of RES activity may be a tumor selective approach to increase the exposure of liposomal agents in tumors and the release of the active drug from the liposome in ovarian tumors.
  • This methodology may have a direct effect on the treatment of ovarian cancer using DOXIL, S-CKD602, and other liposomal anticancer agents, build on the current advantages of liposomal and nanoparticle agents, act as a catalyst to develop RES induction as a novel target for treatment of ovarian cancer, and guide the development of future liposomal and nanoparticle agents (Ozols RF, Schwartz PE, Eifel PJ. Ovarian cancer, fallopian tube carcinoma, and peritoneal carcinoma.

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Abstract

A method of treating tumors is provided. More specifically, a method of treating tumors by increasing the exposure of a tumor to a liposome-entrapped agent comprising the step of modulating the composition of cells in a subject's reticuloendothelial system (RES) is described. In addition, a method of treating tumors by increasing the release of drug from a liposome in a tumor comprising the step of modulating the composition of cells in a subject's RES is described. Further, a method of treating tumors by increasing the sensitivity of a tumor to treatment with a liposome-entrapped agent comprising the step of modulating the composition of cells in a subject's RES is described. Still further, a method of treating tumors by individualizing treatment of a tumor comprising the steps of (1) evaluating the composition of RES cells in the tumor, and (2) determining a treatment regimen based on said evaluating, wherein said treatment regimen comprises administering at least one liposome-entrapped agent is described.

Description

USE OF LIPOSOME-ENTRAPPED AGENTS FOR TREATING TUMORS
TECHNICAL FIELD
[0001] A method of treating tumors is provided. More specifically, a method of treating tumors by increasing the exposure of a tumor to a liposome-entrapped agent comprising the step of modulating the composition of cells in a subject's reticuloendothelial system (RES) is described. In addition, a method of treating tumors by increasing the release of drug from a liposome in a tumor comprising the step of modulating the composition of cells in a subject's RES is described. Further, a method of treating tumors by increasing the sensitivity of a tumor to treatment with a liposome- entrapped agent comprising the step of modulating the composition of cells in a subject's RES is described. Still further, a method of treating tumors by individualizing treatment of a tumor comprising the steps of (1) evaluating the composition of RES cells in the tumor, and (2) determining a treatment regimen based on said evaluating, wherein said treatment regimen comprises administering at least one liposome-entrapped agent is described.
BACKGROUND
[0002] It is currently unclear why within a patient with solid tumors there can be a reduction in the size of some tumors while other tumors can progress during or after treatment, even though the genetic composition of the tumors is similar (Balch, C. M., Reintgen, D. S., Kirkwood, J. M., & et al. Cutaneous Melanoma. In Cancer: Principles and Practice in Oncology, 5th Ed, Devita VT, Hellman S, and Rosenberg SA, eds. Lippincott-Raven. 1947; 2006). Such variable antitumor responses within a single patient may be associated with inherent differences in tumor vascularity, capillary permeability, and/or tumor interstitial pressure that result in variable delivery of anticancer agents to different tumor sites (Jain, RK. Delivery of molecular medicine to solid tumors. Science, 271(5252);1079-1080:1996b; Zamboni, W. C, Houghton, P. J., Hulstein, J. L., Kirstein, M., Walsh, J., Cheshire, P. J., Hanna, S. K., Danks, M. K., & Stewart, C. F. Relationship between tumor extracellular fluid exposure to topotecan and tumor response in human neuroblastoma xenograft and cell lines. Cancer Chemother Pharmacol 43 (4); 269-276: 1999a). However, studies evaluating the intra-tumoral concentration of anticancer agents and factors affecting tumor exposure in preclinical models and patients are rare ( Blochl-Daum B, Muller M, Meisinger V, Eichler HG., Fassolt A, & Pehamberger H. Measurement of extracellular fluid carboplatin kinetics in melanoma metastases with microdialysis. Br J Cancer 73(7); 920-924:1996; Muller, M., Mader, R. M., Steiner, B., Steger, G. G., Jansen, B., Gnant, M., Helbich, T., Jakesz, R., Eichler, H. G., & Blochl-Daum, B. 5-fluorouracil kinetics in the interstitial tumor space: clinical response in breast cancer patients. Cancer Res 57 (13); 2598-2601: 1997). Liposomal and nanoparticle are carrier formulations of anticancer agents and were developed to increase the tumor delivery; however, exact factors associated with the increased tumor delivery of nanoparticles and liposomes are unclear (Zamboni WC. Liposomal, nanoparticle, conjugated formulations of anticancer agents. Invited Review. Clin Cancer Res 11(23);8230-4:2005; Drummond, D. C, Meyer, O., Hong, K., Kirpotin, D. B., & Papahadjopoulos, D. Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors, Pharmacol Rev 51(4);691-743: 1999; Papahadjopoulos, D., Allen, T. M., Gabizon, A., Mayhew, E., Matthay, K., Huang, S. K., Lee, K. D., Woodle, M. C, Lasic, D. D., Redemann, C. Sterically stabilized liposomes: improvements in pharmacokinetics and antitumor therapeutic efficacy. Proc Natl Acad Sci U.S.A 88 (24);11460- 11464:1991; D'Emanuele, A. & Attwood, D.Dendrimer-drug interactions. Adv Drug Deliv Rev 57(15) 2147-2162:2005; ABI 007, Drugs R.D. 5 (3); 155-159:2004). Thus, there is impending need to develop and implement techniques and methodologies to evaluate the disposition and exposure of, anticancer agents, especially carrier formulations, within the tumor matrix.
[0003] The theoretical advantages of liposomal-encapsulated drugs are increased solubility, prolonged duration of exposure, selective delivery of entrapped drug to the site of action, improved therapeutic index, and potentially overcoming resistance associated with the regular anticancer agent. The development of STEALTH liposomes was based on the discovery that incorporation of polyethylene glycol (PEG)-lipids into liposomes yields preparations with prolonged circulation in the blood and superior tumor delivery compared to conventional liposomes composed of natural phospholipids ( Allen, T. M. & Martin, F. J. Advantages of liposomal delivery systems for anthracyclines. Semin Oncol, 31 (6 Suppl 13); 5-15:2004; Allen, T. M. & Stuart, D. D. Liposomal pharmacokinetics: Classical, sterically-stabilized, cationic liposomes and immunoliposomes. In Liposomes: Rational Design, Janoff AS5 ed. New York, Marcel Dekker, Inc. 63-87;2005). The disposition of the STEALTH-encapsulated drug is dictated by characteristics of the liposome, such as, size, surface charge, membrane lipid packing, steric stabilization, dose, and route of administration, all of which alters the plasma pharmacokinetic profile and tissue distribution of the drug . In theory liposomes extravasate through leaky capillary beds of tumors and lodge into the interstitial spaces among tumor cells, where they release the encapsulated drug (Maeda, H., Wu, J., Sawa, T., Matsumura, Y., & Hori, K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. J Control Release 65 (1-2); 271-284:2000). For anticancer agents encapsulated in liposomes to be an effective treatment in patients with solid tumors, the active form of the anticancer agent must be released from the liposome into the tumor extracellular fluid (ECF) and then penetrate into the cell.
[0004] The clearance of nanoparticles and liposomes has been proposed to occur by uptake of these agents by the monocytes and macrophages of the reticuloendothelial system (RES). The RES uptake of conventional or non-pegylated liposomes results in their rapid removal from the blood and accumulation in tissues involved in the RES, such as the liver and spleen. Uptake by the RES usually results in irreversible sequestering of the encapsulated drug in the RES, where it can be degraded. In addition, the uptake of the liposomes by the RES may result in acute impairment of the RES and toxicity. The presence of the PEG coating on the outside of the STEALTH liposome does not prevent uptake by the RES, but simply reduces the rate of uptake. However, the factors associated with the clearance of conventional and pegylated liposomes and mechanisms by which steric stabilization of liposomes decreases the rate of uptake by the RES are unclear and have not been extensively evaluated. In addition, the relationship between RES in tumors and the tumor disposition of liposomal anticancer agents has not been evaluated.
[0005] S-CKD602 is a STEALTH liposomal formulation (phospholipid covalently bound to methoxypolyethylene glycol) of CKD-602, a camptothecin analogue (Zamboni, W. C, Gervais, A. C, Egorin, M. J., Schellens, J. H., Zuhowski, E. G., Pluim, D., Joseph, E., Hamburger, D. R., Working, P. K., Colbern, G., Tonda, M. E., Potter, D. M., & Eiseman, J. L. Systemic and tumor disposition of platinum after administration of cisplatin or STEALTH liposomal-cisplatin formulations (SPI-077 and SPI-077 B 103) in a preclinical tumor model of melanoma. Cancer Chemother Pharmacol 53 (4); 329- 336:2004; Zamboni WC, Friedland DM, Ramalingam S, Edwards RP, Stoller RG, Belani CP, Strychor S, Ou YC, Tonda ME, Ramanathan RK. Final results of a phase I and pharmacokinetic study of STEALTH liposomal CKD-602 (S-CKD602) in patients with advanced solid tumors. Proceedings of ASCO 24(2013);82s:2006; Zamboni WC, Strychor S, Joseph E, Walsh DR, Parise RA, Tonda ME, Yu NY, Engber C, Eiseman JE. Plasma and tumor disposition of STEALTH Liposomal CKD-602 (S-CKD602) and non- liposomal CKD-602, a camptothecin analogue, in mice bearing A375 human melanoma xenograft. Proceedings of AACR-NCI-EORTC 2005, #B 173). CKD-602 inhibits topoisomerase I, thereby preventing DNA replication and causing apoptosis. Non- liposomal CKD-602 has demonstrated clinical activity and has been approved in South Korea in relapsed ovarian cancer and first line small cell lung cancer at a dose of 0.5 mg/m2 IV per day for five days, repeated every 3 weeks (Lee JH, Lee JM, Lim KH, Kim JK, Ahn SK, Bang YJ, Hong CL Preclinical and phase I clinical studies with CKD-602, a novel camptothecin derivative. Ann N Y Acad Sci 922;324-5:2000). The cytotoxicity of camptothecin analogues is related to the duration of exposure in the tumor (Zamboni WC, Gajjar AJ, Houghton PJ, Mandrell TD, Einhaus SL, Danks MK, Rogers WP, Heideman RL, Stewart CF. A topotecan 4-hour intravenous infusion achieves cytotoxic exposure throughout the neuroaxis in the nonhuman primate model: implications for the treatment of children with metastatic medulloblastoma. Clinical Cancer Research 4; 2537-2544:1998; Furman WL, Stewart CF, Poquette CA, Pratt CB, Santana VM, Zamboni WC, Bowman LC, Ma MK, Hoffer FA, Meyer WH, Pappo AS, Walter AW, Houghton PJ. Direct translation of a protracted irinotecan schedule from xenograft model to phase I trial in children. J Clin Oncol 17;1815-1824:1999). In animal models, a 3-10 fold increase in therapeutic index with STEALTH formulation versus non-liposomal CKD-602 was observed (Yu NY, Conway CA, Pena RLS. Improvement in therapeutic index by STEALTH CKD-602 vs free CKD-602 and topotecan in human tumor xenografts. Proceedings of AACR 46(2396); 562:20050). [0006] Consequently, there is a need for an anti-cancer drug for humans that mitigates the above mentioned disadvantages of current drug therapy and effectiveness against tumors in a subject, preferably a human tumor.
SUMMARY
[0007] In one embodiment, a method of treating tumors in a subject is described, preferably a human tumor.
[0008] In another embodiment, a method of treating tumors by increasing the exposure of a tumor to a liposome-entrapped agent comprising the step of modulating the composition of cells in a subject's reticuloendothelial system (RES) is described.
[0009] In another embodiment, a method of treating tumors by increasing the release of drug from a liposome in a tumor comprising the step of modulating the composition of cells in a subject's RES is described.
[00010] In another embodiment, a method of treating tumors by increasing the sensitivity of a tumor to treatment with a liposome-entrapped agent comprising the step of modulating the composition of cells in a subject's RES is described.
[00011] In yet another embodiment, a method of treating tumors by individualizing treatment of a tumor comprising the steps of (1) evaluating the composition of RES cells in the tumor, and (2) determining a treatment regimen based on said evaluating, wherein said treatment regimen comprises administering at least one liposome-entrapped agent is described.
[00012] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following descriptions. BRIEF DESCRIPTION OF THE DRAWINGS
[00013] Figure 1. Concentration versus time profiles of CKD-602 in plasma, tumor homogenate, and tumor ECF after administration of S-CKD602 in mice bearing A375 human melanoma xenografts. Plasma profiles consist of sum total, encapsulated, and released CKD-602. Plasma and tumor sum total concentration versus time point represents the mean of three mice. Microdialysis samples (n = 3 to 4 mice per interval) were obtained from 0-2 h, 24 to 27 h, 48 to 51 h, and 72 to 75 h after administration of S-CKD602. The individual CKD-602 concentration in the tumor ECF is represented by the open diamonds. The mean concentration in the tumor ECF at each collection interval is represented by the solid diamonds and is connected by the dashed line.
[00014] Figure 2. Concentration versus time profiles of CKD-602 in plasma, tumor homogenate, and tumor ECF after administration of S-CKD602 in mice bearing SKOV-3 human ovarian xenografts. Plasma profiles consist of sum total, encapsulated, and released CKD-602. Plasma and tumor sum total concentration versus time point represents the mean of three mice. Microdialysis samples (n = 3 to 4 mice per interval) were obtained from 0-2 h, 24 to 27 h, 48 to 51 h, and 72 to 75 h after administration of S- CKD602. The individual CKD-602 concentration in the tumor ECF is represented by the open diamonds. The mean concentration in the tumor ECF at each collection interval is represented by the solid diamonds and is connected by the dashed line.
[00015] Figure 3. Immunohistochemical staining using FITC-coηjugated cdl Ib antibody and polyethylene glycol-conjugated cdl Ic antibodies as measurements of cells of the RES in SKOV-3 human ovarian (top figure) and A375 human melanoma xenografts (bottom figure) obtained at 75 h after administration of S-CKD602 . The cdl Ib antibody is primarily expressed on macrophages, DC, natural killer cells, and granulocytes. Detection of these cells using the immuno-fluorescence staining antibody cdl Ib emits the color green. The cdl Ic antibody is expressed on DC and lymph node T- cells after activation and emits a red color upon staining of these cells. To detect antigen presenting cells (APC), a conjugate of cdl Ib + cdl Ic was used. This conjugate emits a yellow color upon staining. [00016] Figure 4A and 4B. Time profiles of cdl Ib and cdl Ic percent staining in
A375 human melanoma (Figure 3A) and SKOV-3 human ovarian xenografts (Figure 3B) from 0 to 75 h. The individual % staining of cdl Ib is represented by the open circles. The mean % staining of cdl Ic at each sample time point is represented by the closed circles and is connected by the solid line. The individual % staining of cdl Ic is represented by the open triangles. The mean % staining of cdl Ic at each sample time point is represented by the closed triangle and is connected by the dashed line.
[00017] Figure 5A and 5B. Time profiles of cdl Ib and cdl Ic percent staining in spleen obtained from mice bearing A375 human melanoma (Figure 3A) or SKOV-3 human ovarian xenografts (Figure 3B) from 0 to 75 h. The individual % staining of cdl Ib is represented by the open circles. The mean % staining of cdl Ic at each sample time point is represented by the closed circles and is connected by the solid line. The individual % staining of cdl Ic is represented by the open triangles. The mean % staining of cdl Ic at each sample time point is represented by the closed triangle and is connected by the dashed line.
[00018] Figure 6A and 6B. Time profiles of cdl Ib and cdl Ic percent staining of liver obtained from mice bearing A375 human melanoma (Figure 3A) or SKOV-3 human ovarian xenografts (Figure 3B) from 0 to 75 h. The individual % staining of cdl Ib is represented by the open circles. The mean % staining of cdl Ic at each sample time point is represented by the closed circles and is connected by the solid line. The individual % staining of cdl Ic is represented by the open triangles. The mean % staining of cdl Ic at each sample time point is represented by the closed triangle and is connected by the dashed line.
DETAILED DESCRIPTION
[00019] As indicated above, a method of treating tumors in a subject is provided, preferably a human subject. Tumors are well known to those skilled in the art and are characterized by an abnormal growth of tissue resulting from uncontrolled, progressive multiplication of cells and serving no physiological function; also referred to as a neoplasm. Commonly known types of tumors include, but are not limited to liver, brain, spleen, ovarian, kidney, breast, lung, colon and prostrate. Tumors can be solid or one that forms in the plasma, such as multiple myeloma, primary or secondary, and are sometime drug resistant. The term "tumors" as it is used in this specification is meant to be construed broadly and is not intended to be limited to any particular type of tumor unless otherwise indicated.
[00020] In one embodiment, a method for increasing the exposure of a tumor to a liposome-entrapped agent is provided, comprising modulating the composition of cells in the reticuloendothelial system (RES). The modulating comprises modulating the composition of cells in the RES by administering a compound that alters the composition of cells in the RES. Cells in the reticuloendothelial system include, but are not limited to macrophages, dendritic cells, and monocytes. The modulating further comprises modulating the composition of cells in the RES to achieve an increase in the number of RES cells present in a solid tumor, preferably the modulating achieves an increase in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor. The modulating further comprises modulating the composition of cells by allowing a physiologic change in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
[00021] In another embodiment, a method of increasing the release of drug from a liposome in a tumor is provided, comprising modulating the composition of cells in the reticuloendothelial system. The modulating comprises modulating the composition of cells in the RES by administering a compound that alters the composition of cells in the RES. Cells in the reticuloendothelial system include, but are not limited to macrophages, dendritic cells, and monocytes. The modulating further comprises modulating the composition of cells in the RES to achieve an increase in the number of RES cells present in a solid tumor, preferably the modulating achieves an increase in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor. The modulating further comprises modulating the composition of cells by allowing a physiologic change in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor. [00022] In another embodiment, a method for increasing sensitivity of a tumor to treatment with a liposome-entrapped agent is provided, comprising modulating the composition of cells in the reticuloendothelial system. The modulating comprises modulating the composition of cells in the RES by administering a compound that alters the composition of cells in the RES. Cells in the reticuloendothelial system include, but are not limited to macrophages, dendritic cells, and monocytes. The modulating further comprises modulating the composition of cells in the RES to achieve an increase in the number of RES cells present in a solid tumor, preferably the modulating achieves an increase in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor. The modulating further comprises modulating the composition of cells by allowing a physiologic change in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
[00023] In yet another embodiment, a method for individualizing treatment of a tumor is disclosed, comprising evaluating the composition of RES cells in the tumor, and determining a treatment regimen based on said evaluating, wherein said treatment regimen comprises administering at least one liposome-entrapped agent. One example of a liposome-entrapped agent is a liposome-entrapped topoisomerase inhibitor. The evaluating comprises determining the number of RES cells, preferably the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
[00024] Exemplary liposome-entrapped topoisomerase inhibitors are described in
U.S. Patent Nos. 6,355,268 and 6,465,008, which are incorporated herein by reference. Specifically, but not exclusively, incorporated herein by reference is the description of a method for preparing liposomes containing a topoisomerase inhibitor, and the materials used in preparation of liposomes. Preparation of liposomes and selection of materials for preparing liposomes, is well known in the art, as exemplified in U.S. Patent Nos. 6,355,268 and 6,465,008. Topoisomerase inhibitors include, but are not limited to, topoisomerase I inhibitors such as camptothecin and camptothecin derivatives. For example, the camptothecin derivative can be 9-aminocamptothecin, 7-ethylcamptothecin, 10-hydroxycamptothecin, 9-nitrocamptothecin, 10,11-methlyenedioxycamptothecin, 9- amino-10,11 -methylenedioxy camptothecin or 9-chloro-10,l 1- methylenedioxycamptothecin, irinotecan, topotecan, (7-(4-methylpiperazinomethylene)- 10,1 l-ethylenedioxy-20(S)-camptothecin, 7-(4-methylpiperazinomethylene)-10,l 1- methylenedioxy-20(S)-camptothecin. A preferred topoisomerase I inhibitor is CKD-602. The topoisomerase inhibitor can also be a topoisomerase I/II inhibitor, such as 6-[[2- (dimethylamino)-ethyl]amino]-3-hydroxy-7H-indeno[2, 1 -c]quinolin-7-on e dihydrochloride, azotoxin or 3-methoxy-l lH-pyrido[3',4'-4,5]pyrrolo[3,2-c]quinoline- 1 ,4-dione.
[00025] In one embodiment, the liposome-entrapped topoisomerase inhibitor excludes liposome-entrapped doxorubicin. In another embodiment, the liposome- entrapped topoisomerase inhibitor excludes liposome-entrapped topoisomerase inhibitor II compounds, such as doxorubicin. It will be appreciated that a topoisomerase inhibitor II compound is one that inhibits or reduces the action of topoisomerase II enzyme. A topoisomerase inhibitor I compound is one that inhibits or reduces the action of topoisomerase I enzyme. A topoisomerase I/II inhibitor refers to any compound that inhibits or reduces the action of both topoisomerase I enzyme and topoisomerase II enzyme.
[00026] Other liposome-entrapped agents are contemplated and provided. In one embodiment, cytotoxic agents are particularly useful as the entrapped agent in liposomes targeted for neoplastic disease indications. The drug may be an anthracycline antibiotic selected from doxorubicin, daunorubicin, epirubicin and idarubicin and analogs thereof. The cytotoxic drug can be a nucleoside analog selected from gemcitabine, capecitabine, and ribavirin. The cytotoxic agent may also be a platinum compound selected from cisplatin, carboplatin, ormaplatin, and oxaliplatin. The cytotoxic agent may be a topoisomerase 1 inhibitor selected from the group consisting of topotecan, irinotecan, SN-38, 9-aminocamptothecin and 9-nitrocamptothecin. The cytotoxic agent may be a vinca alkaloid selected from the group consisting of vincristine, vinblastine, vinleurosine, vinrodisine, vinorelbine and vindesine. It will be appreciated that cytotoxic agents are well known to those of skill in the art and readily determined from various medical reference books. [00027] It will be appreciated that the dose and dosing regimen can be varied to optimize the treatment of the tumor. As noted above, the dose of the anticancer agent can be adjusted higher or lower to achieve a desired modulation in the composition of cells in the RES. Alternatively, the dosing regimen can be modified to achieve a desired increase in the number of macrophage cells, dendritic eels, and/or monocyte cells in the tumor. For example, the dosing regimen can comprise an escalating dose for a particular period of time, followed by a constant or decreasing dose for a second period of time. The dosing regimen can be designed to achieve, in one embodiment a physiologic change in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor. Normal cell counts of macrophages, dendritic cells, and monocytes are know to those of skill in the art and readily determined from various medical reference books.
[00028] It will also be appreciated that the method can additionally include administration of a liposome-entrapped agent in conjunction with a second therapeutic agent, in free or liposome-entrapped form. The second agent can be any therapeutic agent mentioned herein, including other drug compounds (in one example chemotherapeutics) as well as biological agents, such as peptides, antibodies, and the like. The second agent can be administered simultaneously with or sequential to administration of the targeted-liposomes, by the same or a different route of administration.
EXAMPLES
[00029] The following example further illustrates the invention described herein and are in no way intended to limit the scope of the invention.
Example 1
[00030] The objectives of this study were to evaluate the relationship between plasma and tumor disposition of S-CKD602 and the cells of the RES in mice bearing human melanoma and ovarian tumor xenografts. The microdialysis methodology was used to evaluate the tumor ECF disposition of released CKD-602 after administration of S-CKD602. Materials and Methods Mice
[00031] All mice were handled in accordance with the Guide to the Care and Use of Laboratory Animals (National Research Council, 1996), and studies were approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh Medical Center. Mice (female CB-17 SCID, 4-6 weeks of age, and specific pathogen free), were obtained from the NCI Animal Production Program (Fredrick, MD), and were allowed to acclimate to the animal facilities at the University of Pittsburgh for 1 week prior to initiation of study. Mice were housed in microisolator cages and allowed Teklad LM-484 autoclavable rodent chow (Harlan Tekla Diets, Madison, WI) or ISDPRO RMH3000 irradiated rodent chow (PMI Nutrition International, Inc., Brentwood, MO) and were ad libitum. Body weights and tumor size were measured twice weekly and clinical observations were made twice daily.
Tumor Lines
[00032] A375 human melanoma xenografts and SKO V-3 human ovarian xenografts were obtained from the DCTD Tumor Repository (Fredrick, MD) and were mouse antigen production test-negative. A375 or SKOV-3 tumors were passed in CB- 17 SCID mice, as approximately 25 -mg fragments implanted subcutaneously on the right flank by aseptic techniques. Tumor volumes were calculated from the formula: length x (width)2/2, where length is the largest diameter and width is the smallest diameter perpendicular to the length. Pharmacokinetic and microdialysis studies were performed when the tumors were approximately 1000 to 1500 mm3 (1 to 1.5 g) in size.
Formulation and Administration
[00033] The formulation of S-CKD602 used in this study is the same as used in the phase I study of S-CKD602 (15). The liposome of S-CKD602 contains hydrogenated soy phosphatidylcholine and cholesterol. Methoxypolyethylene glycol is covalently bound to phosphatidylethanolamine and a component of the lipid bilayer. The mean particle size of the liposomes is approximately 110 nm. Liposomal encapsulation of CKD-602 exceeds 90%. The drug-to-lipid ratio is approximately 14 mg of CKD-602 per milligram of lipid. The concentration of CKD-602 in the dosing solution is 1 mg/ml. S-CKD602 was administered at 1 mg/kg IV push xl via a tail vein in mice bearing A375 human melanoma xenograft or SKO V-3 human ovarian xenograft (20). This dose is one-half the maximum tolerated dose (MTD) in mice (20). The doses of S-CKD602 refer to actual doses of CKD-602.
Sample Processing for Plasma and Tissue Pharmacokinetic Studies
[00034] Pharmacokinetic and microdialysis studies were performed in mice bearing A375 human melanoma and SKOV-3 human ovarian xenografts. Due to limited sample volume, the pharmacokinetic and microdialysis studies of S-CKD602 were performed in separate groups of mice bearing A375 tumors. For the pharmacokinetic studies, mice (n = 3 per time point) were euthanized with carbon dioxide, and heparinized blood samples (approximately 0.8 to 1 mL) were collected by cardiac puncture prior to treatment, and at 5 min, and 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 17, 24, 48, and 72 h after administration for S-CKD602. The blood samples were centrifuged at 12,000 x g for 4 min. The plasma was processed to measure encapsulated, released, and sum total (encapsulated + released) CKD602. Tumors samples were obtained for measurement of sum total CKD602.
[00035] The collection of released CKD602 from S-CKD602 in tumor ECF was obtained using microdialysis methodology (Zamboni WC. Use of microdialysis in preclinical and clinical development. In: Handbook of Pharmacokinetics and Pharmacodynamics of Anti-Cancer Drugs, 1st Ed, Figg WD, McLeod H, eds. Humana Press. 2004). In mice bearing A375 human melanoma xenografts, microdialysis samples (n = 3 to 4 mice per interval) were then obtained every 20 min from 0 to 2 h and every 30 min at 24 to 27 h, 48 to 51 h, and 72 to 75 h. At the end of each microdialysis procedure, plasma and tumor samples were obtained and processed as described above. [00036] The pharmacokinetic and microdialysis studies were performed in the same mice bearing SKOV-3 human ovarian xenografts. In addition, the number of sample time points from 5 min to 6 h were reduced based on the results from mice bearing A375 xenografts. For the pharmacokinetic studies, mice (n = 3 per time point) were euthanized with carbon dioxide, and heparinized blood samples (approximately 0.8 to 1 mL) were collected by cardiac puncture prior to treatment, and at 0.25 h and 4 h after administration for S-CKD602. Microdialysis samples (n = 3 to 4 mice) were obtained every 20 min. from 0 to 2 h, 24 to 28 h, 48 to 52 h and 72 to 76 h. At the end of each microdialysis procedure, plasma and tumor samples were obtained and processed as described above.
Analytical Studies.
[00037] An LC/MS assay was used to measure the camptothecin total (sum of lactone and hydroxy acid) forms of encapsulated, released, and sum total CKD-602 in plasma, sum total CKD-602 in tumor and CKD-602 in tumor ECF after administration of S-CKD602. (Zamboni WC, Hamburger DR, Jung LL, Jin R, Joseph E, Strychor S, Sun SL, Egorin MJ, Eiseman JL. Relationship between systemic exposure of 9-nitrocamptothecin and its 9-aminocamptothecin metabolite and tumor response in human colon tumor xenografts. Clin Cancer Res l l(13):4867-74, 2005).
[00038] The HPLC system consisted of a Finnigan Specta Systems AS3000 autosampler and P4000 quarternary pump (Thermo Finnigan, Waltham, MA) with a Phenomenex Synergi Hydro-RP 8OA (4 um, 100 x 2 mm) analytical column (Phenomenex, Torrance, CA). The isocratic mobile phase consisted of 0.1% formic acid in methanol: water (35:85, v/v) and was pumped at 0.2 ml/min. Injection volume was 10 μl and the run time was 10 min. Column eluant was analyzed with a ThermoFinnigan aQa mass spectrometer (Thermo Quest, San Jose, CA) operating in electrospray positive mode electron ionization monitoring CKD and deuterated CKD (D7-CKD) at 434.1 mlz and 441.2 m/z, respectively. The insert probe temperature was set at 4000C with 3 kV ion spray voltage and 20 V orifice voltage. Nitrogen gas flow was fixed at 75 p.s.i. at the tank head unit. The system was operated with ThermoFinnigan Xcaliber software. The CKD to LS. ratio was calculated for standards by dividing the analyte peak area by that of the LS. Standard curves for CKD were constructed by plotting the analyte to LS. ratio versus the known concentration of the analyte in each standard. Standard curves were fit by linear regression with 1/y2 weighting and back calculation of CKD concentrations.
Pharmacokinetic Analysis
[00039] The areas under the sum total, encapsulated, and released plasma concentration versus time curves (AUC) of CKD-602 from 0 to infinity and 0 to the last measurable concentration versus time point were calculated using the log trapezoidal method (Rowland M and Tozer T (eds). Clinical pharmacokinetics: concepts and applications. Lea and Febiger, Philadelphia 1999). The percent encapsulation of CKD- 602 in plasma was calculated using the following equation: [(sum total AUC - released AUC) / the sum total AUC] x 100.
Staining of RES Cells in Tumor and Tissues
[00040] Immunohistochemical staining of tumor, liver, spleen, kidney and brain were performed in mice bearing A375 and SKOV-3 xenografts. The collection of these sample tissues were obtained prior to administration of S-CKD602 and at the end of the microdialysis collection interval (i.e. 27 h, 51 h, and 75 h after S-CKD602 administration). The tissue samples were immediately fixed in Tissue-Tek O. CT. Compound, an embedding medium for frozen tissue, and placed in liquid hexane at - 8O0 C.
[00041] The cryosections were stained with FITC-conjugated cdl Ib antibody and polyethylene glycol-conjugated cdl Ic antibodies as measurements of cells of the RES (Tumor-localization by adoptively transferred, interleukin-2-activated NK cells leads to destruction of well-established lung metastases. Int J Cancer. 2003 JuI l;105(4):512-9; Basse PH, Whiteside TL, Herberman RB. Cancer immunotherapy with interleukin-2- activated natural killer cells.Mol Biotechnol. 2002 Jun;21(2): 161-70; Melichar B, Savary CA, Patenia R, Templin S, Melicharova K, Freedman RS. Phenotype and antitumor activity of ascitic fluid monocytes in patients with ovarian carcinoma. Int J Gynecol Cancer 13;435-443:2003). The cdl Ib antibody is primarily expressed on macrophages, DC, natural killer cells, and granulocytes. Detection of these cells using the immunofluorescence staining antibody cdl Ib emits the color green. The cdl Ic antibody is expressed on DC and lymph node T-cells after activation and emits a red color upon staining of these cells. To detect antigen presenting cells (APC), a conjugate of cdl Ib + cdl Ic was used. This conjugate emits a yellow color upon staining. The F4/80 monoclonal antibody reacts with an approximately 125 kDa transmembrane protein on mouse macrophages. The F4/80 antigen is expressed by subpopulations of mature macrophages and is by some regarded as the best marker for this population of cells. However, other cell types such as Langerhans cells and liver Kupffer cells have been reported to express this antigen. It has been shown that some cytokines down regulate the expression of F4/80 resulting in lack of F4/80 antigen on a subpopulation of macrophages. Cells stained with the F4/80 antibody emit a green color. Staining results were calculated as the percentage of total area with positive staining (% staining) in each sample. In addition, the mean +/- SD of percent staining from 0 to 72h was calculated.
Statistics
[00042] Statistical analysis was performed on all paired data using the Wilcoxon signed ranked test. Statistical analysis was performed on all non-paired data using the Two Sample T-test. All analysis was performed using the SPSS version 10.0 (Chicago, IL).
Results
Plasma Disposition
[00043] The plasma disposition of CKD-602 was compared after administration of
S-CKD602 in mice bearing A375 human melanoma and SKO V-3 human ovarian xenografts. The concentration versus time profile of sum total, encapsulated, and released CKD-602 mice bearing A375 human melanoma and SKOV-3 human ovarian xenografts are presented in Figure 1. The pharmacokinetic parameters are summarized in Table 1. Since the extrapolated area associated with the estimated AUC from 0 to infinity was greater than 10% the AUC are reported as AUC from 0 to the last measurable time point. After administration of S-CKD602, the disposition of sum total, encapsulated, and released CKD-602 were similar in mice bearing SKOV-3 and A375 xenografts. In both mice models, there was a prolonged exposure of sum total, encapsulated, and released CKD-602. In addition, the concentration versus time profiles of released CKD-602 in plasma was consistent with the profiles of sum total and encapsulated drug suggesting that there is relatively constant release of CKD-602 in the plasma. The percent encapsulation of CKD-602 in mice bearing SKOV-3 and A375 was 73% and 84%, respectively.
Table 1. Plasma, Tumor, and Tumor ECF Pharmacokinetic Parameters for S-CKD602 in Mice Bearing A375 Human Melanoma and SKOV-3 Human Ovarian Xenografts
Parameters Units S-CKD602 in S-CKD602 in
A375 SKOV-3
Plasma
Sum Total AUC (0-72h) ng/mL-h 201,222 144,379 Encapsulated AUC (0- ng/mL-h 164,857 96,692
72h)
Released AUC (0-72h) ng/mL-h 33,186 40,065
Tumor
Sum Total AUC (0-72h) ng/mL-h 14,141 15,698 ECF AUC (0-74h) ng/mL-h 187 440
Time>lng/mL in Tumor h >72 72
ECF Tumor Disposition
[00044] The overall tumor disposition of CKD-602 was measured, as measured by sum total CKD-602 in tumor homogenates, and disposition of CKD-602 in tumor ECF, as estimated by using microdialysis methodology, after administration of S- CKD602 in mice bearing A375 human melanoma and SKO V-3 human ovarian xenografts. The concentration versus time profile of sum total CKD-602 in tumor and CKD-602 in tumor ECF in mice bearing A375 human melanoma and SKO V-3 human ovarian xenografts are presented in Figure 1. The pharmacokinetic parameters are summarized in Table 1. The concentration versus time profiles of sum total CKD-602 in tumor were similar in the SKO V-3 and A375 xenografts. In both mice models, there was a prolonged exposure of sum total CKD-60. The sum total CKD-602 AUC in tumor was higher in SKOV-3 (15,698 ng/mL«h) compared with A375 (14,141 ng/mL*h) xenografts. Moreover, the ratio of tumor sum total AUC to plasma sum total AUC was 1.6-fold higher in mice bearing human SKOV-3 xenografts compared with A375. These results suggest that there is an increased delivery of S-CKD602 in SKOV-3 xenografts compared with A375.
[00045] For liposomal formulations of anticancer agents to achieve antitumor effects, the active drug must be released from the liposome into the tumor ECF, thus microdialysis was used to evaluate the disposition of released CKD-602 in tumor ECF. The concentration versus time profile of CKD-602 in tumor ECF in mice bearing A375 human melanoma and SKOV-3 human ovarian xenografts are presented in Figure 1. In both tumor models, the concentration versus time profile of CKD-602 in tumor ECF was consistent with the profile of sum total CKD-602 in tumor. The difference in the CKD- 602 measured in samples obtained from the tumor homogenate and tumor ECF may be due to the slow release of CKD-602 from the liposome and the subsequent binding of CKD-602 to proteins within the tumor matrix because the tumor ECF samples were obtained using microdialysis methodology which can only recovery released non-protein bound drug due to the molecular weight cut off of the probe (< 20 kD). In both tumor models, the concentration of CKD-602 in tumor ECF varied approximately 10-fold at individual time points during each of the collection intervals. These results suggest that CKD-602 has a relatively constant but highly variable release from the STEALTH liposome.
[00046] The tumor ECF AUC in mice bearing A375 and SKO V-3 xenografts were
0.19 and 0.44 μg/mL«h, respectively. The ratio of tumor ECF AUC to tumor sum total AUC was 2.2-fold higher in mice bearing human SKOV-3 xenografts compared with A375. These results suggest that there is an increased release of CKD-602 in SKOV-3 xenografts compared with A375.
Staining of RES Cells in Tumors and Tissue
[00047] To evaluate the relationship between the tumor and tissue disposition of S-
CKD602 and the RES in mice bearing SKOV-3 and A375 xenografts, immunohistochemical staining of tumor, liver, spleen, kidney and brain was performed using cdl Ib, cdl Ic, and F4/80 antibodies as measurements of cells of the RES. Representative immunohistochemical staining results using cdl Ib and cdl Ic antibodies at 75 h after administration of S-CKD602 in A375 and SKOV-3 xenografts are presented in Figure 3. The % staining of cdl Ib and cdl Ic in control tumors and in tumors at 27 h, 51 h, and 75 h after administration of S-CKD602 in A375 and SKOV-3 xenografts is presented in Figures 4A and 4B, respectively. In A375 xenografts, the cbl Ib and cdl Ic staining were relatively constant compared with the SKOV-3 xenografts. In SKOV-3 xenografts, the cbl Ib staining was variable from 0 to 75 h and the cdl Ic staining decreased 4-fold from 0 to 45 h and then increased 2-fold at 75 h. As depicted in figures 3 and 4 and the mean staining of cdl Ic from 0 to 75 h was higher in SKOV-3 compared with A375. Mean ± SD % staining of cdl Ib in A375 and SKOV-3 tumors were 4.1 ± 1.7 % and 4.7 ± 2.2 %, respectively (P > 0.05). Mean ± SD % staining of cdl Ic in A375 and SKOV-3 tumors were 1.3 ± 0.7 % and 4.4 ± 2.6 %, respectively (P < 0.0001). Consistent with the cdl Ic staining, the staining of F4/80 was X and Y in A375 and SKOV-3 xenografts, respectively.
[00048] The % staining of cdl Ib and cdl Ic in control spleens and in spleens at 27 h, 51 h, and 75 h after administration of S-CKD602 in mice bearing A375 and SKOV-3 xenografts are presented in Figures 5A and 5B, respectively. In both studies, the staining of cdl Ic was more variable than cdl Ib. The mean ± SD % staining of cdl Ib and cdl Ic in spleen obtained from mice bearing A375 were 4.2 ± 1.7 % and 1.3 ± 0.7 %, respectively. The mean ± SD % staining of cdl Ib and cdl Ic in spleen obtained from mice bearing SKO V-3 were 4.2 ± 2.2 % and 1.3 ± 0.7 %, respectively. The cdl Ib and cdl Ic staining in A375 compared with SKOV-3 bearing mice were not statistically different (P > 0.05).
[00049] The % staining of cdl Ib and cdl Ic in control liver and in liver at 27 h, 51 h, and 75 h after administration of S-CKD602 in mice bearing A375 and SKOV-3 xenografts are presented in Figures 6A and 6B, respectively. In both studies, the staining of cdl Ib and cdl Ic in liver was more variable in mice bearing SKOV-3 xenografts compared with A375 xenografts. The mean ± SD % staining of cdl Ib and cdl Ic in liver obtained from mice bearing A375 were 0.25 ± 0.07 % and 0.33 ± 0.09 %, respectively. The mean ± SD % staining of cdl Ib and cdl Ic in liver obtained from mice bearing SKOV-3 were 0.32 ± 0.05 % and 0.45 ± 0.07 %, respectively.
[00050] Overall, the staining of cdl Ib, cdl Ic, F4/80 in the spleen was greater than in SKOV-3 or A375 xenografts (P < 0.05). In addition, the staining of cdl Ib, cdl Ic, and F4/80 in spleen and tumor were significantly greater than in the liver in mice bearing both tumor models (P < 0.05V
Discussion [00051] This is the first report evaluating the relationship between the tumor and tissue disposition of a liposomal agent and the presence of the RES. After administration of S-CKD602, the plasma sum total, encapsulated, and released CKD-602 were similar in SKOV-3 and A375 xenografts. There was a higher relative exposure of sum total CKD- 602 in tumor and increased exposure of released CDK-602 in tumor ECF of mice bearing SKOV-3 compared with A375 xenografts. In addition, the percent staining of cdl Ic in SKOV-3 and A375 xenografts were 4.4 +/- 2.6% and 1.3 +/- 0.7%, respectively (P < 0.0001). The increased tumor delivery and release of CKD-602 from S-CKD602 in SKOV-3 human ovarian xenografts compared with A375 human melanoma xenografts was consistent with increased cdl Ic staining in SKOV-3 suggesting that variability in the RES may affect the tumor disposition of liposomal anticancer agents. In addition, these results are consistent with previous studies reporting that ovarian tumor xenografts were 3 -fold more sensitive to SKOV-3 compared with melanoma xenografts. Thus, the presence of the RES in tumors may be a factor affecting the tumor sensitivity of S- CKD602 and other liposomal and nanoparticle anticancer agents in ovarian cancer and other solid tumors.
[00052] Infiltrating mononuclear cells play an important role in many types of cancer. Malignant ascites is frequently associated with advanced ovarian cancer (Loercher AE, Nash MA, Kavanagh JJ, Platsoucas CD, Freedman RS. Identification of IL- 10-producing HLA-DR-negative monocyte subset in malignant ascites of patients with ovarian carcinoma that inhibits cytokine protein expression and proliferation of autologous T cells. J Immunol 163(11);6251-60: 1999; Zavadova E, Loercher A, Verstovsek S, Verschraegan CF, Micksche M, Freedman RS. The role of macrophages in antitumor defense of patients with ovarian cancer. Hematol Oncol Clin North Am 13(1);135-44:1999; Zamboni WC, Maruca LJ, Strychor S, Zamboni BA, Friedland DM, Ramalingam S, Edwards RP, Stoller RG, Belani CP, Ramanathan RK. Pharmacodynamic study of Stealth liposomal CKD-602 (S-CKD602) and monocytes in patients with refractory solid tumors. Submitted to ASCO'07). Ascites is characterized by variable numbers of exfoliated tumor cells and activated mesothelial cells, as well as by many mononuclear leukocytes, monocytes and macrophages, and lymphocytes. The monocytes and macrophages are the primary cells of the RES, which has also been called the mononuclear phagocytic system. Macrophages appear to be important in epithelial ovarian cancer as they are frequently the dominant population of leukocytes in the peritoneal fluid of patients with malignant ascites. Monocytes circulate in peripheral blood and can be induced by a variety of stimuli to adhere to the vascular endothelium and migrate into tissues where they differentiate into specialized cells, macrophages or dendritic cells. The tumor associated macrophages appear to participate in the immunologic antitumor defense mechanism through direct cytotoxic and cytostatic activities or indirect activities through the release of cytokines, stimulating the adaptive immune response by antigen presentation, or producing factors with anti-angiogenic activity (e.g., angiostatin). Thus, monocytes and macrophages and the RES may represent key targets for a variety of therapeutic interventions and may be prognostic factors in ovarian cancer.
[00053] The number of macrophages and dendritic cells in tumors as measured by cdl Ib, cdl Ic, and F4/80 staining are highly variable and change over time. The low expression of F4/80 in the tumors relative to cdl Ic may indicate that the cells stained with cdl Ic are either DCs or immature macrophages. It is unclear if the changes in cells of the RES are due to physiological movement and function of these cells or related to direct or indirect cytotoxicity associated with S-CKD602 and/or released CKD-602 in the tumor. It has been found that monocytes are more sensitive to S-CKD602 as compared with neutrophils in patients and that the increased sensitivity is related to the liposomal formulation and not the encapsulated CKD-602. In addition, the relationship between the reduction of monocytes in blood and the pharmacokinetic disposition of S-CKD602 in plasma of patients suggests that the monocytes engulf liposomal anticancer agents which cause the release of drug from the liposome and toxicity to the monocytes. Thus, the changes in RES cells in the tumor and tissue in this study may be related to S-CKD602.
[00054] In other studies, the highest tissue exposure of CKD-602 after administration of S-CKD602 were in the spleen > liver > tumor > kidney. The exposures in the spleen and liver are consistent with these tissues being the primary sites of the RES. However, the staining of RES cells in this study was significantly higher in the spleen and tumor compared with the liver (P = 0.001). The difference between the exposure of CKD-602 and the RES staining in the liver may be related to the disposition of released and liposomal encapsulated CKD-602 in the plasma of mice. It has been found that approximately 20% of CKD-602 is released from S-CKD602 in plasma of mice and that the highest tissue exposures of non-liposomal CKD-602 occur in the liver and kidney, which are the primary organs for the elimination of camptothecins (Sparraboom A and Zamboni WC. Topoisomerase I Inhibitors. In Chabner BA and Longo DL, editors. Cancer Chemotherapy and Biotherapy: Principles and Practice, Fourth Edition, Lippincott Williams & Wilkins, 2005). Thus, some of the exposure in the liver after administration of S-CKD602 is related to released CKD-602. The relatively low staining of the RES cells in the liver compared with spleen and tumor is unclear. The distribution of S-CKD602 to the brain may be associated with the presence of RES cells.
[00055] There is an urgent need to identify new targets and improve treatment for patients with both newly diagnosed and refractory ovarian cancer and other solid tumors. Induction of the RES is a novel tumor-selective targeting strategy to increase response and decrease toxicity in the treatment of ovarian cancer using liposomal anticancer agents such as STEALTH liposomal doxorubicin (Doxil) and S- CKD602. Of note, there are approximately 50 liposomal and nanoparticle anticancer agents currently in development for which RES induction may prove to be advantageous. The modulation of RES activity may be a tumor selective approach to increase the exposure of liposomal agents in tumors and the release of the active drug from the liposome in ovarian tumors. This methodology may have a direct effect on the treatment of ovarian cancer using DOXIL, S-CKD602, and other liposomal anticancer agents, build on the current advantages of liposomal and nanoparticle agents, act as a catalyst to develop RES induction as a novel target for treatment of ovarian cancer, and guide the development of future liposomal and nanoparticle agents (Ozols RF, Schwartz PE, Eifel PJ. Ovarian cancer, fallopian tube carcinoma, and peritoneal carcinoma. In Cancer: Principles and practice of oncology, 5th Ed, DeVita VT, Hellman S, and Rosenberg SA, eds. Lippincott-Raven. 1502-1534; 1997; Grever MR, & Chabner BA. Cancer drug development. In Cancer: Principles and practice in oncology, 5th Ed, Devita VT, Hellman S, and Rosenberg SA, eds. Lippincott-Raven. 385; 2006; Bookman MA. Standard treatment in advanced ovarian cancer in 2005: the state of the art. Int J Gynecol Cancer 15(3);212-20:2005; Aichele P, Zinke J, Gorde L, Schwendener RA, Kaufmann SHE, Seiler P. Macrophages of the splenic marginal zone are essential for trapping of blood-borne particulate antigen but dispensable for induction of specific T cell responses. J Immunol 171(3);1148-1155:2003; Markman M, Gordon AN, McGuire WP, Muggia FM. Liposomal anthracycline treatment for ovarian cancer. Semin Oncol 31;91 -105:2004). This methodology may also be used to individualize DOXIL and S- CKD602 treatment in patients with ovarian cancer based on tumor specific RES activity. [00056] 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.

Claims

What is claimed is:
1. A method for increasing the exposure of a tumor to a liposome-entrapped agent, comprising: modulating the composition of cells in the reticuloendothelial system (RES).
2. A method of increasing the release of drug from a liposome in a tumor, comprising modulating the composition of cells in the reticuloendothelial system.
3. A method for increasing sensitivity of a tumor to treatment with a liposome- entrapped agent, comprising modulating the composition of cells in the reticuloendothelial system.
4. The method of claim 1, 2, or 3, wherein said modulating comprises modulating the composition of cells in the RES by administering a compound that alters the composition of cells in the RES.
5. The method of claim 4, wherein the cells are selected from the group consisting of macrophages, dendritic cells, and monocytes.
6. The method of claim 1, 2, or 3, wherein said modulating comprises modulating the composition of cells in the RES to achieve an increase in the number of RES cells present in a solid tumor.
7. The method of claim 6, wherein the modulating achieves an increase in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
8. The method of claim 1, 2, or 3, wherein said modulating comprises modulating the composition of cells by allowing a physiologic change in the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
9. A method for individualizing treatment of a tumor, comprising evaluating the composition of RES cells in the tumor, and determining a treatment regimen based on said evaluating, wherein said treatment regimen comprises administering at least one liposome-entrapped agent.
10. The method of claim 9, wherein the at least one liposome-entrapped agent is a topoisomerase inhibitor.
11. The method of claim 10, wherein the topoisomerase inhibitor is a camptothecin analog.
12. The method of claim 9, wherein said evaluating comprises determining the number of macrophage cells, dendritic cells, and/or monocyte cells in the tumor.
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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2000023052A1 (en) * 1998-09-16 2000-04-27 Alza Corporation Liposome-entrapped topoisomerase inhibitors
WO2003043584A2 (en) * 2001-11-20 2003-05-30 University Of Kentucky Research Foundation Engineered liposomal particles containing core-loaded pro-drugs for the controlled release of camptothecins

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WO2000023052A1 (en) * 1998-09-16 2000-04-27 Alza Corporation Liposome-entrapped topoisomerase inhibitors
WO2003043584A2 (en) * 2001-11-20 2003-05-30 University Of Kentucky Research Foundation Engineered liposomal particles containing core-loaded pro-drugs for the controlled release of camptothecins

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STRYCHOR SANDRA; EISEMAN JULIE L; JOSEPH ERIN; TONDA MARGARET E; YING NU Y; ENGBERS CHARLES; ZAMBONI WILLIAM C: "Plasma, tissue and tumor disposition of STEALTH (R), liposomal CKD-602 (S-CKD602) and non-liposomal CKD-602, a camptothccin analogue, in mice bearing A375 human melanoma xenografts", PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH ANNUAL MEETING, vol. 47, April 2006 (2006-04-01), pages 721, XP001538110 *
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