WO2008070009A2

WO2008070009A2 - Treating solid tumors and monocytic leukemia using topoisomerase inhibitors in liposomes

Info

Publication number: WO2008070009A2
Application number: PCT/US2007/024709
Authority: WO
Inventors: William Zamboni
Original assignee: Alza Corporation
Priority date: 2006-12-01
Filing date: 2007-11-30
Publication date: 2008-06-12
Also published as: WO2008070009A3

Abstract

A method of treating a solid tumor or leukemia is described. More specifically, a method of treating a solid tumor or leukemia with liposome-entrapped topoisomerase inhibitor is provided. In one embodiment, the method involves treating leukemia by evaluating a subject's monocytes and/or neutrophils, and administering at least one topoisomerase inhibitor entrapped in liposomes having an outer surface coating of hydrophilic polymer chains based upon the evaluation. In another embodiment, the method involves treating a solid tumor, by selecting a treatment regimen based on the age and/or ratio of total body weight to ideal body weigh of the patient.

Description

METHOD OF TREATING SOLID TUMORS AND MONOCYTIC LEUKEMIA

TECHNICAL FIELD

[0001] Methods of treating solid tumors and of treating leukemia, specifically monocytic leukemia, are provided. More specifically, a method of treating a solid tumor or leukemia with a liposome-entrapped topoisomerase inhibitor is provided.

BACKGROUND

[0002] Cancer is a global killer of humans with breast cancer and colon cancer among the leaders with many other types killing modest amounts of humans yearly. Two blood cancers are of interest because there is no known cure, i.e. myeloma and leukemia. Each year, nearly 27,000 adults and more than 2,000 children in the United States learn that they have leukemia for which there is no cure only palliative treatment. [0003] Leukemia is a type of cancer that has two characteristics. One is that certain cells in the body become abnormal. Second is that after this development, the human body keeps producing large numbers of these abnormal cells. In most types of leukemia, the abnormal cells are white blood cells. The leukemia cells usually look different from normal blood cells, and they do not function properly. [0004] The major forms of leukemia are divided into four categories.

Myelogenous and lymphocytic leukemia each have acute and chronic forms. The terms myelogenous or lymphocytic denote the cell type involved. Acute leukemia is a rapidly progressing disease that affects mostly cells that are unformed or primitive (not yet fully developed or differentiated). These immature cells cannot carry out their normal functions. Chronic leukemia progresses slowly and permits the growth of greater numbers of more developed cells. In general, these more mature cells can carry out some of their normal functions. Thus, the four major types of leukemia are: acute or chronic myelogenous, and acute or chronic lymphocytic leukemia. The ability to measure specific features of cells has led to further subclassification of the major categories of leukemia. The categories and subsets allow the physician to decide what treatment works best for the cell type and how quickly the disease may develop. [0005] Since this cancer is complex in nature and not susceptible to any specific therapy, there are a number of treatments employed including chemotherapy when drugs are employed, biological with the use of interferon, radiation and bone marrow transplants.

[0006] Major advances in the use of carrier vehicles delivering pharmacologic agents and enzymes to sites of disease have occurred the past ten years (Zamboni WC. Liposomal, nanoparticle, conjugated formulations of anticancer agents. Invited Review. Clin Cancer Res 11(23);8230-8234:2005; ABI 007, Drugs R.D. 5 (3); 155-159:2004; Drummond, D. C₁ 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. C₁ 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). The primary types of carrier-mediated anticancer agents are liposomes and nanoparticles. The theoretical advantages of liposomal and nanoparticle encapsulated and carrier-mediated drugs are increased solubility, prolonged duration of exposure, selective delivery of entrapped drug to the site of action, increase efficacy, decrease toxicity, and potentially overcoming resistance associated with the regular anticancer agent. The development of pegylated liposomes such as Stealth^® liposomal doxorubicin (Doxil^®) was based on the discovery that incorporation of polyethylene glycol (PEG) modified lipids into liposomes yields preparations with 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 AS₁ ed. New York, Marcel Dekker, Inc. 63-87;2005). As more existing anticancer agents go off patent these agents will be most likely be evaluated in some type of liposome or carrier-mediated formulation. In addition, antiangiogenesis agents, antisense oligonucleotides, and enzymes represent rational candidates for liposomal and nanoparticle formulations (Park, J. W., Benz, C. C₁ & Martin, F. J. Future directions of liposome- and immunoliposome-based cancer therapeutics. Semin Oncol 31(6 Suppl 13); 196-205:2004). [0007] Liposomes can alter both the tissue distribution and the rate of clearance of the drug by making the drug take on the pharmacokinetic characteristics of the carrier. The clearance of liposomes and nanoparticlies 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 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 liposome does not prevent uptake by the RES, but simply reduces the rate of uptake (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). The factors associated with the pharmacokinetics and pharmacodynamics 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.

[0008] S-CKD602 is a STEALTH^® liposomal (SL) formulation (phospholipid covalently bound to methoxypolyethylene glycol) of CKD-602, a camptothecin (CPT) analogue. CKD-602 (7-(2-(N-isopropylamino)ethyl)-(20S)-camptothecin) inhibits topoisomerase I₁ thereby preventing DNA replication causing apoptosis. Once in the tumor, the liposomes are localized in the extracellular fluid (ECF) surrounding the tumor cell, but do not enter the cell (Harrington, K. J., Lewanski, C. R., Northcote, A. D., Whittaker, J., Wellbank, H., Vile, R. G., Peters, A. M., & Stewart, J. S. Phase l-ll study of pegylated liposomal cisplatin (SPI-077) in patients with inoperable head and neck cancer. Ann Oncol 12 (4);493-496:2001a; Harrington, K. J., Mohammadtaghi, S., Uster, P. S., Glass, D., Peters, A. M., Vile, R. G., & Stewart, J. S. Effective targeting of solid tumors in patients with locally advanced cancers by radiolabeled pegylated liposomes. Clin Cancer Res 7 (2); 243-254:2001 b). Thus, for the liposomes to deliver the active form of the anticancer agent, such as doxorubicin in the case of Doxil, the drug must be released from the liposome into the tumor ECF and then diffuse into the cell (Zamboni WC. Use of microdialysis in preclinical and clinical development. In: Handbook of Pharmacokinetics and Pharmacodynamics of Anti-Cancer Drugs, 1^st Ed, Figg WD, McLeod H, eds. Humana Press. 2004; 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 B103) in a preclinical tumor model of melanoma. Cancer Chemother Pharmacol 53 (4); 329-336:2004). As a result, the ability of the liposome to carry the anticancer agent to the tumor and release it into the ECF are equally important factors in determining the antitumor effect of liposomal encapsulated anticancer agents. Non-liposomal CKD-602 (NL-CKD602) is approved in South Korea in relapsed ovarian cancer as a first line agent in small cell lung cancer (SCLC). The cytotoxicity of camptothecin analogues is related to the duration of exposure in the tumor.

[0009] The U.S. patent literature has many disclosures of liposomes that are useful for treating tumors, including breast, ovarian and colon tumors. CKD-602 and other topoisomerase inhibitor-entrapped liposomes are disclosed in U.S. Pat. Nos. 6,355,268 and 6, 465,008, which are incorporated by reference herein. It appears from a review of the foregoing that liposomes entrapped with a topoisomerase inhibitor have not been reported for the treatment of human leukemia.

[00010] 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 human leukemia.

[00011] There is also a need for improved methods for treating solid tumors in patients. As mentioned above, liposome-entrapped drugs for treating solid tumors have demonstrated clinical benefits, particularly in providing an accumulation of drug at the tumor site. However, clinical observations have also shown some interpatient variability in the pharmacokinetic (PK) disposition of liposome-entrapped therapeutic agents. There is a need to identify factors associated with the PK variability and to provide effective therapeutic regimens for effective tumor treatment. SUMMARY

[00012] In one aspect, a method of treating leukemia by administering at least one topoisomerase inhibitor entrapped in liposomes having an outer surface coating of hydrophilic polymer chains is provided.

[00013] In another aspect, a method of treating monocytic leukemia comprising administering at least one topoisomerase inhibitor entrapped in liposomes having an outer surface coating of hydrophilic polymer chains is provided.

[00014] In still another aspect, a method of treating leukemia is provided, where a subject's monocytes and/or neutrophils are evaluated, and administering at least one topoisomerase inhibitor entrapped in liposomes having an outer surface coating of hydrophilic polymer chains based upon the evaluation.

[00015] In another aspect, a method of selecting a treatment regimen in a human subject bearing a solid tumor is provided. The method involves determining the age of the patient, determining the ratio of total body weight to ideal body weight of the patient, and selecting a therapeutic treatment regimen based on the age and ratio.

[00016] In another aspect, a method of treating a solid tumor in provided. The method involves determining the age of the patient, determining the ratio of total body weight to ideal body weight of the patient, and selecting a therapeutic treatment regimen based on the age and ratio. In one embodiment, the tumor is an advanced solid tumor or is a refractory solid tumor. In one embodiment, the selected treatment regimen comprises administering a topoisomerase inhibitor entrapped in liposomes having an outer surface coating of hydrophilic polymer chains.

[00017] 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

[00018] Figure 1. The absolute neutrophil count (ANC) profile for all patients on cycle 1 after administration of S-CKD602. ANC values for individual patients are represented by the open circles and are connected by the solid lines. [00019] Figure 2. The monocyte profile for all patients on cycle 1 after administration of S-CKD602. Monocyte values for individual patients are represented by the open circles and are connected by the solid lines.

[00020] Figure 3. The relationship between % decrease in ANC and monocytes at the nadir after administration of S-CKD602. Individual values for % decrease in ANC and monocytes at the nadir are represented by the open circles. The % decrease in

ANC and monocytes within a patient are connected by the solid line. The mean value for % decrease in ANC and monocytes are represented by the solid square and are connected by the bold line.

[00021] Figure 4. The relationship between the ANC and monocytes rate of reduction in at the nadir after administration of S-CKD602. Individual values are represented by the open circles. Values within a patient are connected by the solid line.

The mean values are represented by the solid square and are connected by the bold line.

[00022] Figure 5. The ANC profile for all patients on cycle 1 after administration of

NL-CKD602. ANC values for individual patients are represented by the open circles and are connected by the solid lines.

[00023] Figure 6. The monocyte profile for all patients on cycle 1 after administration of NL-CKD602. Monocyte values for individual patients are represented by the open circles and are connected by the solid lines.

[00024] Figure 7. The relationship between ANC and monocytes % decrease at the nadir after administration of NL-CKD602. Individual values are represented by the open circles. Values within a patient are connected by the solid line. The mean values are represented by the solid square and are connected by the bold line.

[00025] Figure 8. The relationship between the ANC and monocytes rate of reduction in at the nadir after administration of NL-CKD602. Individual values are represented by the open circles. Values within a patient are connected by the solid line.

[00026] Figure 9. The relationship between ANC and monocytes % decrease at the nadir after administration of S-CKD602 in patients < 60 and ≥ 60 years of age. For patients < 60 years of age, individual values are represented by the open circles and values within a patients are connected by the solid line. For patients > 60 years of age, individual values are represented by the open triangles and values within a patients are connected by the dashed line. The mean values for patients < 60 and > 60 years of age are represented by the solid circle and triangle are connected by the bold lines.

[00027] Figure 10. The relationship between the ANC and monocytes rate of reduction at the nadir after administration of S-CKD602 in patients < 60 and > 60 years of age. For patients < 60 years of age, individual values are represented by the open circles and values within a patients are connected by the solid line. For patients > 60 years of age, individual values are represented by the open triangles and values within a patients are connected by the dashed line. The mean values for patients < 60 and >

60 years of age are represented by the solid circle and triangle are connected by the bold lines.

[00028] Figure 11. The relationship between the sum total AUC of S-CKD602 and

% decrease in monocytes at the nadir in all patients. Individual patient values are represented by the open circles.

[00029] Figure 12. The relationship between the AUC of sum total S-CKD602 and

% decrease in monocytes at the nadir in patients > 60 and > 60 years of age. Individual patient values are represented by the open circles.

[00030] Figure 13. There was no relationship between the AUC of sum total S-

CKD602 and % decrease in ANC at the nadir. Individual patient values are represented by the open circles.

[00031] Figure 14. There was no relationship between the AUC of released CKD-

602 and % decrease in ANC at the nadir. Individual patient values are represented by the open circles.

[00032] Figure 15. The relationship between % decrease in monocytes and the

AUC of released CKD-602 in plasma. For patients < 60 and > 60 years of age individual values are represented by the open circles and triangles, respectively. Linear regressions for all the data, patients < 60 years of age, and patients > 60 years of age are represented by the solid (R² = 0.51), (R² = 0.54), and ^'" lines (R² = 0.49), respectively. [00033] Figure 16. The relationship between the rate of reduction in monocytes and the AUC released CKD-602 in plasma. For patients < 60 and > 60 years of age individual values are represented by the open circles and triangles, respectively. Linear regressions for all the data, patients < 60 years of age, and patients > 60 years of age are represented by the solid (R² = 0.58), (R² = 0.68), and ^"" lines (R² = 0.55), respectively.

[00034] Figure 17. The relationship between % decrease in monocytes and the recovery CKD-602 in urine. For patients < 60 and > 60 years of age individual values are represented by the open circles and triangles, respectively. Linear regressions for patients < 60 years of age (R² = 0.82) and patients > 60 years of age (R² = 0.30) are represented by the solid lines.

[00035] Figure 18. The relationship between the rate of reduction in monocytes and the recovery CKD-602 in urine. For patients < 60 and > 60 years of age individual values are represented by the open circles and triangles, respectively. Linear regressions for patients < 60 years of age (R² = 0.91) and patients > 60 years of age (R²

= 0.38) are represented by the solid lines.

[00036] Figure 19 shows the age related effects on the PK disposition of the topoisomerase inhibitor CKD602 entrapped in liposomes sum total in plasma of patients age less than 60 or greater than or equal to 60.

[00037] Figures 20A-20B shows the plasma concentration, in ng.mL, of the topoisomerase inhibitor CKD602 entrapped in liposomes (circles), released from the liposomes (free form, diamonds), and the sum total of both entrapped drug and free drug (squares) in two exemplary patients.

[00038] Figure 21 shows the relationship between liposome-entrapped topoisomerase inhibitor CKD602 and plasma sum total area under the curve (AUC), in ng/mL-h, as a function of dose, in mg/m2, in patients age less than 60 (closed circles) or greater than or equal to 60 (open circles).

[00039] Figure 22 shows the relationship between body composition and topoisomerase inhibitor CKD602 plasma sum PK, by graphing the AUC/Dose as a function of ratio of total body weight to ideal body weight (TBW/IBW) for patients age less than 60 (closed circles) or greater than or equal to 60 (open circles). [00040] Figure 23 is a table presenting the PK parameters for the sum total of topoisomerase inhibitor CKD602 for patients with linear and nonlinear PK.

[00041] Figure 24 illustrates the factors affecting the PK disposition of liposome entrapped anti-tumor agents.

[00042] Figure 25 shows the age, PK, and PD (pharmacodymanic) relationships of liposome entrapped anti-tumor agents.

DETAILED DESCRIPTION

[00043] As indicated above, a method of treating leukemia is provided. Leukemia is a disease or disorder that is well known to those skilled in the art and is characterized by the uncontrolled growth of blood cells. The common types of leukemia are divided into four categories: acute or chronic myelogenous, involving the myeloid elements of the bone marrow (white cells, red cells, megakaryocytes) and acute or chronic lymphocytic, involving the cells of the lymphoid lineage. One particular leukemia is monocytic leukemia. The term "leukemia" 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 leukemia unless otherwise indicated.

[00044] More specifically, a method of treating leukemia with a liposome- entrapped topoisomerase inhibitor is provided. 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 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.

[00045] A method of treating leukemia, such as monocytic leukemia, by evaluating a subject's monocytes and/or neutrophils, and administering at least one topoisomerase inhibitor entrapped in liposomes having an outer surface coating of hydrophilic polymer chains based upon the evaluation is provided. In one embodiment, evaluating the subject's monocytes and/or neutrophils comprises obtaining a count of one or both of the cells types in the blood of the subject, preferably before treatment and/or after treatment with the liposome composition. In another embodiment, the composition of the liposome-entrapped topoisomerase inhibitor and/or the dosage of the liposome- entrapped topoisomerase inhibitor is adjusted based upon the evaluation of a subject's monocytes and/or neutrophils. For example, the dosage of the liposome-entrapped topoisomerase inhibitor is adjusted to alter the monocyte and/or neutrophil count in the blood of a subject. In a preferred embodiment, the dosage of composition of the liposome-entrapped topoisomerase inhibitor is adjusted to decrease the number of monocyte cells in the subject suffering from a leukemia. [00046] In another example, the composition of the liposome-entrapped topoisomerase inhibitor is altered, based on the subject's monocyte and/or neutrophil count, by selecting a different topoisomerase inhibitor, e.g., an inhibitor other than CKD- 602, or altering the lipid composition of the liposome. Alternative 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- methylenedioxycamptothecin or 9-chloro-10,11-methylenedioxycamptothecin, irinotecan, topotecan, (7-(4-methylpiperazinomethylene)-10,11-ethylenedioxy-20(S)- camptothecin, 7-(4-methylpiperazinomethylene)-10,11-methylenedioxy-20(S)- camptothecin. The topoisomerase inhibitor can also be a topoisomerase l/ll inhibitor, such as θ-f^-CdimethylaminoJ-ethyljaminol-S-hydroxy-yH-indeno^.i-clquinolin-y-on e dihydrochloride, azotoxin or 3-methoxy-11 H-pyrido[3',4'-4,5]pyrrolo[3,2-c]quinoline-1 ,4- dione.

[00047] 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 Il compounds, such as doxorubicin. It will be appreciated that a topoisomerase inhibitor Il compound is one that inhibits or reduces the action of topoisomerase Il enzyme. A topoisomerase inhibitor I compound is one that inhibits or reduces the action of topoisomerase I enzyme. A topoisomerase l/ll inhibitor refers to any compound that inhibits or reduces the action of both topoisomerase I enzyme and topoisomerase Il enzyme. [00048] It will be appreciated that the dose and dosing regimen can be varied to optimize the treatment of the leukemia. As noted above, the dose of the topoisomerase inhibitor can be adjusted higher or lower to achieve a desired decrease in monocyte count and/or a desired modified ANC/monocyte ratio. Alternatively, the dosing regimen can be modified to achieve a desired decrease in monocyte count and/or a desired modified ANC/monocyte ratio. 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 for treatment of monocytic leukemia, monocytopenia without inducing neutropenia. Normal cell counts of monocytes and neutrophils are know to those of skill in the art and readily determined from various medical reference books. [00049] It will also be appreciated that the method can additionally include administration of a liposome-entrapped topoisomerase inhibitor in conjunction with a second therapeutic agent, in free or liposome-entrapped form. In one embodiment, a drug, such as granulocyte-macrophage colony-stimulating factor or granulocyte colony- stimulating factor, effective to raise a subject's absolute neutrophil count is administered. It will also be appreciated that leukemia patients may be additionally treated with various chemotherapeutic agents. [00050] In another aspect, a method of treating a solid tumor is provided.

EXAMPLES

[00051] The following example further illustrates the invention described herein and are in no way intended to limit the scope of the invention.

Example 1

METHODS

Patient Population.

[00052] The pharmacokinetic and pharmacodynamic analyses of S-CKD602 were performed as part of a phase I study in patients with refractory solid tumors performed at the University of Pittsburgh Cancer Institute, Pittsburgh, PA. Patients enrolled on this study were 18 years of age or older with a histological or cytological confirmed malignancy for which no curative or effective therapy was available were eligible for this study. Other eligibility criteria included a Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2, adequate bone marrow, hepatic, and renal function as evidenced by the following: absolute neutrophil count (ANC) > 1500/μL, platelets > 100,000/μL, total bilirubin < 1.5 x upper limit of the institutional normal range (ULN), aspartate aminotransferase (AST) < 1.5 x the ULN if liver metastases were not present and < 4 x the ULN if liver metastases were present, and absence of microscopic hematuria. Prior irradiation to brain metastases was allowed if the patient's neurological status was stable 4 weeks after irradiation. Prior treatment with camptothecin analogues except S-CKD602 was permitted. Written informed consent, approved by the Institutional Review board of the University of Pittsburgh Medical Center, was obtained from all patients before they entered the study. The pharmacodynamic analyses of non-liposomal CKD-602 (NL-CKD602) were performed as part of a phase I study in patients with refractory solid tumors and a phase Il study in patients with refractory ovarian cancer performed in South Korea.

Dose and Administration

[00053] In a phase I study S-CKD602 was administered IV over 1 h once every 3 wk at doses from 0.1 to 2.5 mg/m². In the phase I study, NL-CKD602 was administered IV over 0.5 h daily for 5 days every 3 wk at doses from 0.5 to 0.9 mg/m²/d. In the phase Il study, NL-CKD602 was administered IV at 0.5 mg/m²/d every 3 wk.

Blood Counts

[00054] ANC and monocyte counts were obtained at least once per wk on cycle 1 of the S-CKD602 and NL-CKD602 studies. Additional counts were obtained as clinically required. The % decrease in ANC and monocytes at nadir was calculated using the standard formula [(Pre value - nadir) / Pre-value] x 100. The rate of decrease in ANC and monocytes was calculated as % decrease divided by the number of days to nadir.

Pharmacokinetic Sampling and Processing.

[00055] For S-CKD602, serial plasma samples were obtained prior to administration to1 , 2, 3, 4, 6, 17, 24, 48, 72, and 96 h after administration, and on days 8 and 15 of cycle 1. Plasma samples were processed to measure concentrations of encapsulated, released, and sum total (encapsulated + Released) CKD602. Urine was collected in 24 h aliquots from 0 to 96 h after administration of S-CKD602 and processed to measure sum total CKD-602. All forms of CKD-602 total (lactone + hydroxyl acid) were measured by LC-MS/MS. Area under the plasma concentration versus time curves for all forms of CKD-602 were calculated using the log trapezoidal method.

Statistical Analysis.

[00056] Statistical analysis was performed comparing all parameters for paired data using the Wilcoxon signed ranked test. Statistical analysis was performed comparing all parameters for non-paired data using the Two Sample T-test. All analysis was performed using the SPSS version 10.0 (Chicago, IL).

RESULTS

[00057] After administration of S-CKD602 in all patients, the % decrease in ANC and monocytes were 42 ± 30% and 58 ± 34 %, respectively (P = 0.003). For S-CKD602 in all patients, the ratio of % decrease in monocytes to ANC within a patient was 2.1 ± 2.0. For S-CKD602 in patients < 60 years of age the % decrease in ANC and monocytes were 43 ± 31% and 58 ± 26 %, respectively (P = 0.001). For S-CKD602 in patients > 60 years of age the % decrease in ANC and monocytes were 41 ± 31 % and 45 ± 36 %, respectively (P = 0.50). There was no difference in the % decrease in ANC and monocytes after administration of NL-CKD602 (P > 0.05). The linear positive- correlation between % decrease in monocytes and released CKD-602 AUC in patients < 60 years of age (R² = 0.54) and > 60 years of age (R² = 0.49) was similar. The linear positive-correlation between the % decrease in monocytes and the amount of CKD-602 recovered in the urine was stronger in patients < 60 years of age (R² = 0.82) compared with > 60 years of age (R² = 0.30).

Neutropenia and Monocytopenia Associated with S-CKD602

[00058] To evaluate differential effects of S-CKD602 on neutrophils and monocytes, the % decrease and rate of decrease of ANC and monocytes were evaluated in the blood of patients administered S-CKD602. The ANC profile for all patients on cycle 1 after administration of S-CKD602 is presented in Figure 1. The monocyte profile for all patients on cycle 1 after administration of S-CKD602 is presented in Figure 2. The mean ± SD day of ANC and monocyte nadir after administration of S-CKD602 was 15.3 ± 5.0 days and 8.6 ± 3.3 days, respectively (P = 0.008). The parameters describing the neutropenia and monocytopenia administration of S-CKD602 are summarized in Table 1.

Table 1. Summary of ANC and Monocytes Parameters after Administration of S-CKD602 and NL-CKD602

a p = 0.001 " P = 0.0001 c,d p > ^• 0.05 e p = 0.003

Φ = 0.001 ^g p > 0.05 h p = 0.005 ¹P = 0.03

J P > 0.05 ^k P = 0.02 m p > 0.05

[00059] The relationship between ANC and monocytes % decrease at the nadir after administration of S-CKD602 is presented in Figure 3. After administration of S- CKD602, the % decrease in ANC and monocytes were 42 ± 30% and 58 ± 34 %, respectively (P = 0.001). For S-CKD602, the ratio of % decrease in monocytes to ANC within a patient was 2.1 ± 2.0. The relationship between the ANC and monocytes rate of reduction in at the nadir after administration of S-CKD602 is presented in Figure 4. For S-CKD602, the rate of reduction in ANC and monocytes were 3.2 ± 2.4 %/d and 7.4 ± 4.7 %/d, respectively (P < 0.0001). For S-CKD602, the ratio of the rate of reduction of monocytes to ANC within a patient was 3.9 ± 5.3.

Neutropenia and Monocytopenia Associated with NL-CKD602 [00060] To determine if the increased monocytopenia as compared with neutropenia was associated with the liposomal carrier or the encapsulated CKD-602, the % decrease and rate of decrease of ANC and monocytes were evaluated in the blood of patients administered NL-CKD602. The ANC profile for all patients on cycle 1 after administration of NL-CKD602 is presented in Figure 5. The monocyte profile for all patients on cycle 1 after administration of NL-CKD602 is presented in Figure 6. The mean ± SD day of ANC and monocyte nadir after administration of NL-CKD602 was 14.0 ± 3.5 days and 9.2 ± 3.0 days, respectively (P > 0.05). The parameters describing the neutropenia and monocytopenia administration of NL-CKD602 are summarized in Table 1.

[00061] The relationship between ANC and monocytes % decrease at the nadir after administration of NL-CKD602 is presented in Figure 7. After administration of NL- CKD602, the % decrease in ANC and monocytes were 86 ± 11 % and 87 ± 12 %, respectively (P > 0.05). After administration of NL-CKD602, the ratio of % decrease in monocytes to ANC within a patient was 1.0 ± 0.2. The relationship between the ANC and monocytes rate of reduction in at the nadir after administration of NL-CKD602 is presented in Figure 8. After administration of NL-CKD602, the rate of reduction in ANC and monocytes were 6.2 ± 1.8 %/d and 10.3 ± 3.5 %/d, respectively (P > 0.05). After administration of NL-CKD602, the ratio of the rate of reduction of monocytes to ANC within a patient was 1.7 ± 0.7.

Age Related Effects on Neutropenia and Monocytopenia Associated with S-CKD602 [00062] To evaluate age-related effects on the relationship between neutropenia and monocytopenia after administration of S-CKD602, the % decrease and rate of decrease of ANC and monocytes were evaluated in the blood of patients < 60 and > 60 years of age. Categorizing patients as < 60 or > 60 years of age was based on previous studies reporting a reduced clearance of STEALTH liposomal anticancer agents in patients > 60 years of age compared with patients < 60 years of age. The mean ± SD age of patients in groups < 60 and > 60 years of age were 49.2 ± 6.3 years and 70.8 ± 6.0 years, respectively (P = 0.01). The parameters describing the neutropenia and monocytopenia administration of S-CKD602 in patients < 60 and > 60 years of age are summarized in Table 1.

[00063] The relationship between ANC and monocytes % decrease at the nadir after administration of S-CKD602 in patients < 60 and > 60 years of age is presented in Figure 9. After administration of S-CKD602, the % decrease in ANC and monocytes in patients < 60 years of age were 43 ± 31 % and 73 ± 26 %, respectively (P = 0.003). The ratio of % decrease in monocytes to ANC within a patients < 60 years of age was 2.5 ± 2.5. After administration of S-CKD602, the % decrease in ANC and monocytes > 60 years of age were 41 ± 31 % and 45 ± 36 %, respectively (P = 0.5). The ratio of % decrease in monocytes to ANC within a patients > 60 years of age was 1.7 ± 1.3. [00064] The relationship between the ANC and monocytes rate of reduction at the nadir after administration of S-CKD602 in patients < 60 and > 60 years of age is presented in Figure 10. After administration of S-CKD602, the rate of decrease in ANC and monocytes in patients < 60 years of age were 3.3 ± 2.1 %/d and 9.7 ± 4.3 %/d, respectively (P = 0.001). The ratio of the rate of reduction in monocytes to ANC within a patients < 60 years of age was 5.4 ± 7.5. The rate of decrease in ANC and monocytes > 60 years of age were 3.1 ± 2.7 %/d and 5.4 ± 4.1 %/d, respectively (P = 0.005). The ratio of rate of reduction in monocytes to ANC within a patients ≥ 60 years of age was 2.5 ± 1.1.

Pharmacodynamic Relationships of S-CKD602 Exposure and Monocytopenia and Neutropenia

[00065] To evaluate the pharmacodynamic relationship between S-CKD602 and bone marrow suppression, the relationship between S-CKD602 exposure and monocytopenia and neutropenia were evaluated . The relationship between the sum total AUC of S-CKD602 and % decrease in monocytes at the nadir in all patients is presented in Figure 11. This relationship is indicative of an Emax relationship. The relationship between the AUC of sum total S-CKD602 and % decrease in monocytes at the nadir in patients > 60 and > 60 years of age is presented in Figure 12. These relationships are indicative of an Emax relationship. There appears to be a greater reduction in the monocytes at the nadir in patients < 60 years of age compared with patients > 60 years of age. There was no relationship between the AUC of sum total S- CKD602 or released CKD-602 and % decrease in ANC at the nadir (Figures 13 and 14).

Relationship between Monocytes and Pharmacokinetic Disposition of S-CKD602 [00066] To evaluate how monocytes may affect the pharmacokinetics of S-

CKD602, the relationship between the % decreased and rate of reduction of monocytes and release of CKD-602 from S-CKD602 in plasma was compared. The relationship between % decrease and rate of reduction in monocytes and the AUC released CKD- 602 in plasma are presented in Figures 15 and 16, respectively. In all patients, patients < 60 years of age, and in patients > 60 years of age there was a linear relationship between the % decrease in monocytes and rate of reduction in monocytes and the AUC of released CKD-602.

[00067] As the primary clearance pathway of NL-CKD602 and thus released CKD-

602 is renal elimination, the relationship between the % decreased and rate of reduction of monocytes and recovery of CKD-602 in the urine after administration of S-CKD602 were evaluated. The relationship between % decrease and rate of reduction in monocytes and the recovery CKD-602 in urine are presented in Figures 17 and 18, respectively. In all patients, patients < 60 years of age, and in patients > 60 years of age there was a linear relationship between the % decrease in monocytes and rate of reduction in monocytes and the recovery of CKD-602 in urine. There was a stronger relationship between the % decrease and rate of reduction of monocytes in patients < 60 years of age compared with patients > 60 years of age.

DISCUSSION

[00068] Several preclinical and clinical studies were performed evaluating factors associated with the plasma and tumor disposition of pegylated liposomal anticancer agents. It was shown that the liposomal formulation must release active-drug from the liposome into the tumor ECF to achieve antitumor activity. Thus, the factors affecting the delivery of liposomal agents to the tumor and the release of drug into the tumor ECF are both important factors affecting the antitumor activity of these agents. Pegylated liposomal anticancer agents provides pharmacokinetic advantages in plasma, tumor, and tumor ECF compared to non-liposomal form of the drug, which is consistent with the improved antitumor efficacy of liposomal agents (Strychor S, Eiseman JE, Joseph E, Parise RA, Tonda ME, Yu NY, Engber C, Zamboni WC. Plasma, tissue, and tumor disposition of STEALTH liposomal CKD-602 (S-CKD602) and non-liposomal CKD-602, a camptothecin analogue, in mice bearing A375 human melanoma xenografts. Proceedings of AACR 47(3064); 721 :2006; 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); 562:2005). After administration of pegylated liposomal anticancer agents, the peak concentration of drug in tumors occur later and the exposures are more prolonged than in normal tissues. The higher tumor exposure of pegylated liposomal anticancer agents in ovarian xenografts compared with melanoma xenografts is consistent with a higher number of monocytes and dendritic cells in ovarian xenografts (Zamboni WC, Eiseman JE, Strychor S, Rice PM, Joseph E, Potter DM, Shurer J, Walsh DR, Parise RA, Tonda ME, Yu NY, Engber C, Basse PH. Relationship between the plasma and tumor disposition of STEALTH liposomal CKD-602 and macrophages/dendritic cells (MDC) in mice bearing human tumor xenografts. Proceedings of AACR 47(5449); 1280:2006). This is also consistent with ovarian xenografts being more sensitive to liposomal anticancer agents compared with melanoma xenografts.

[00069] To determine if the findings in the preclinical tumor models are consistent with the disposition of liposomal anticancer agents in patients, the relationship between monocytes and the pharmacokinetics of a pegylated liposomal anticancer agent was evaluated as part of a phase I study in patients with refractory solid tumors. The data suggested that monocytes are more sensitive to pegylated liposomal anticancer agents as compared with neutrophils and that the increased sensitivity is related to the liposomal formulation. In addition, the release of drug from the pegylated liposome in the plasma was related to the reduction in monocytes. Thus, the process by which monocytes take up the liposomes results in the release of active drug and toxicity to the monocytes. The results of the preclinical and clinical studies suggested that monocytes and macrophages of the RES play an important role in the distribution of liposomes to the tumor, the release of active-drug into the tumor ECF, and the catabolism of liposomes and the release of drug in the blood.

[00070] Infiltrating mononuclear cells play an important role in many types of cancer (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, lnt J Gynecol Cancer 13;435-443:2003). Malignant ascites is a 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; HeIaI TA, Alia AE, Laban MA, Fahmy RM. lmmunophenotyping of tumor-infiltrating mononuclear cells in ovarian carcinoma. Pathol Oncol Res 10(2);80-4:2004). 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). Monocytes and macrophages have also been used in clinical trials of adoptive immunotherapy. Conversely, monocytes and macrophages may also indirectly foster tumor growth by providing the cellular machinery necessary for progression and metastases, such as proteases, angiogenic factors, and substances enhancing tumor growth and down-regulating the immune response to the tumor. 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. [00071] Little is known about functional changes in the characteristics of monocytes, macrophages, and dendritic cells during aging or in cancer. Animal studies indicate a profound unresponsiveness of macrophages to polysaccharide antigens in aged mice (Plowden J, Renshaw-Hoelscher M, Engelman C, Katz J.Sambhara S. Innate Immunity in aging: Impact on macrophage function. Aging Cell 10;161-167:2004; Chelvarajan RL, Collins SM, Van Willigen JM. The unresponsiveness of aged mice to polysaccharide antigens is a result of a defect in macrophage function. J. Leukcyte Biol 77;503-512:2005; MelicharB, Savary CA, Patenia R, Templin S, Melicharova K, Freedman RS. Phenotype and antitumor activity of ascetic fluid monocytes in patients with ovarian carcinoma. Int. J. Gynecol Cancer 13;435-439:2003). While functions of macrophages are known to be variably impaired in patients with cancer, few studies have systematically measured changes in monocyte numbers and/or functions. Thus, it is important to develop phenotypic measures of monocytes and macrophages and RES activity.

[00072] Melichar and colleagues evaluated the major subtypes, surface phenotype characteristics, and antitumor activity of ascitic fluid monocytes and macrophages in patients with ovarian cancer. Monocytes and macrophages from malignant ascites had phenotypic features similar to these cells in peripheral blood. Treatment of ascitic monocytes and macrophages with IFN-gamma or IL-2 produced significant cytotoxic/cytostatic activity. Treatment of ascitic monocytes and macrophages with IL- 12, paclitaxel, granulocyte-monocyte colony stimulating factor (GM-CSF), or tumor necrosis factor-alpha (TNF-alpha) did not produce cytotoxic/cytostatic activity. Further studies are needed to better define their function of monocytes and macrophages, especially related to the treatment of ovarian cancer using liposomal anticancer agents. [00073] The results of this study suggest that monocytes are more sensitive to S-

CKD602 as compared with neutrophils and that the increased sensitivity is related to the liposomal formulation and not the encapsulated CKD-602. The relationship between changes in monocytes and the pharmacokinetic disposition of S-CKD602 suggests that the monocytes engulf liposomal anticancer agents via their phagocytic function as part of the RES which causes the release of drug from the liposome and cytotoxicity to the monocytes. The sensitivity of monocytes to S-CKD602 suggests that S-CKD602 may be an effective agent in the treatment of monocytic leukemias. There are age related factors associated with the pharmacodynamic interaction between S-CKD602 and monocytes. v

Example 2

[00074] In another study, S-CKD602 was administered intravenously at 0.1-2.5 mg/m2 for 21 days to patients with solid tumors. Concentrations of encapsulated (E), released (R), and sum total (E+R) CKD-602 in plasma and CKD-602 in urine were measured by LC-MS/MS. Area under the plasma concentration versus time curve (AUC) was calculated and normalized by dose (AUC/dose). The ratio of total body weight to ideal body weight (TBW/IBW) was calculated as a measure of body composition.

[00075] The doses of liposome-entrapped drug (S-CKD602) were 0.10, 0.15, 0.20,

0.25, 0.30, 0.40, 0.50, 0.65, 0.85, 1.1 , 1.7, 2.5 and 2.1 mg/m² (MTD). Blood was taken for PK plasma sampling in cycle 1 at 0 to 6 h, 24 h, 48 h, 72 h, 96 h, day 8 and day 15. [00076] Pharmacokinetic (PK) measurements included (i) CKD-602 total (= lactone

+ hydroxy acid) in plasma; (ii) liposome encapsulated, released, and sum total (= encapsulated + released) CKD-602 in plasma; (iii)CKD-602 total in urine, (iv) compartmental PK Analysis in ADAPT II, (iv) Linear and non-linear elimination; (v) Area under the plasma cone, versus time curve (AUC) was normalized by dose. [00077] The patient related factors included (i) Age groups were < 60 and > 60 years old (yo) based on median age; and (ii) body composition based on ratio of total body weight (TBW) to ideal body weight (IBW).

[00078] Statistics: AUC/dose for patients less than (<) and greater than (>) 60 yo were analyzed using non-paired t-test. AUC/dose vs TBW/IBW was analyzed using multiple linear regressions.

[00079] Results are shown in Figure 19, Figures 20A-20B, Figure 21 , Figure 22,

Figure 23, Figure 24, and Figure 25. The mean standard deviation of S-CKD602 sum total AUC/dose in patients less than age 60 (n = 13) and greater than or equal to age 60 (n = 17) years of age (yo) were 4.5 ± 5.0 and 11.2 ± 11.0 (μg/mL^«h)/(mg/m²), respectively (P<0.05). Controlling for age, there was a statistically significant inverse relationship between TBW/IBW and S-CKD602 AUC/dose where low TBW/IBW was associated with high AUC/dose in both age groups (P<0.05). [00080] The cumulative amount of CKD-602 recovered in the urine was 2.2-fold higher in patients less than age 60 compared with greater than or equal to age 60. [00081] These data suggest that patients greater than or equal to age 60 have a reduced clearance of S-CKD602 and reduced release of CKD-602 from S-CKD602 compared with patients less than age 60. In addition, patients less than age 60 and greater than or equal to age 60 with a lean body composition have a reduced tissue distribution and an increased plasma exposure of S-CKD602. Thus, older patients have a reduced capacity to eliminate liposome agents due to age-related impairment of the RES. In addition, body composition alters the disposition of liposomes, in particular liposomes having a surface coating of hydrophilic polymer chains.

[00082] 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 of treating a human having leukemia, the method comprising administering to said human a liposome comprising at least one topoisomerase inhibitor entrapped in said liposome.

2. The method of Claim 1 , wherein the leukemia is monocytic leukemia.

3. The method of Claim 1 , wherein the liposome is composed of at least about 20 mole percent of a vesicle-forming lipid and at least about 1 mole percent of a vesicle- forming lipid derivatized with a hydrophilic polymer, said polymer being distributed on both sides of the liposomes' bilayer membrane.

4. The method of Claim 3, wherein entrapped in the liposome is a topoisomerase inhibitor at a concentration of at least about 0.10 micromole drug per micromole lipid.

5. The method of Claim 1 , wherein the liposome has an inside/outside ion gradient sufficient to retain the topoisomerase inhibitor within the liposome at a specified concentration.

6. The method of Claim 1 , wherein the topoisomerase inhibitor is a topoisomerase I inhibitor selected from the group consisting of camptothecin and camptothecin derivatives.

7. The method of Claim 6, wherein the camptothecin derivative is selected from the group consisting of 9-aminocamptothecin, 7-ethylcamptothecin, 10- hydroxycamptothecin, 9-nitrocamptothecin, 10,11-methylenedioxycamptothecin, 9- amino-10,11-methylenedioxycamptothecin, and 9-chloro-10,11- methylenedioxycamptothecin.

8. The method of Claim 6, wherein the camptothecin derivative is selected from the group consisting of irinotecan, topotecan, (7-(4-methylpiperazinomethylene)-10,11- ethylenedioxy-20(S)-camptothecin, 7-(4-methylpiperazinomethylene)-10,11 - methylenedioxy-20(S)-camptothecin and 7-(2-N-isopropylamino)ethyl)-(20S)- camptothecin.

9. The method of Claim 1 , wherein the topoisomerase inhibitor is a topoisomerase l/ll inhibitor selected from the group consisting of 6-[[2-(dimethylamino)- ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinolin-7-on e dihydrochloride, azotoxin and 3- methoxy-11 H-pyridoβ'^'ΛSlpyrroloβ^-clquinoline-i ,4-dione.

10. The method of Claim 3, wherein the hydrophilic polymer is polyethyleneglycol having a molecular weight between 500-5,000 daltons.

11. The method of Claim 3, wherein the vesicle-forming lipid is selected from the group consisting of hydrogenated soy phosphatidylcholine, distearoylphosphatidylcholine and sphingomyelin.

12. A method of treating leukemia, the method comprising the steps of: evaluating a subject's monocytes and/or neutrophils; and administering at least one topoisomerase inhibitor entrapped in a liposome having an outer surface coating of hydrophilic polymer chains based upon the evaluation.

13. The method of Claim 12, wherein the leukemia is monocytic leukemia.

14. The method of Claim 12, wherein the composition of the liposome is adjusted based upon the evaluation.

15. The method of Claim 12, wherein the dosage of the liposome-entrapped topoisomerase inhibitor is adjusted based upon the evaluation.

16. The method of claim 12, wherein said evaluating comprises obtaining a count of the number of monocytes and/or neutrophils in the subject's blood.

17. A method of treating a solid tumor in a patient, comprising determining the age of the patient, determining the ratio of total body weight to ideal body weight of the patient, and selecting a therapeutic treatment regimen based on the age and ratio.

18. The method of claim 17, wherein the patient has a solid tumor refractory to treatment with a chemotherapeutic agent administered in free form (non-liposome- entrapped).

19. The method of claim 17, wherein said selecting comprises selecting a dose of a therapeutic agent.

20. The method of claim 17, wherein said selecting comprises selecting a dose and/or dosing schedule of a therapeutic agent.

21. The method of any one of claims 17-20 further comprising administering a therapeutic agent entrapped in a liposome.

22. The method of claim 21 , wherein the therapeutic agent is a topoisomerase inhibitor.