Troy Harasym

Ratiometric dosing of anticancer drug combinations: Controlling drug ratios after systemic administration regulates therapeutic activity in tumor-bearing mice

Anticancer drug combinations can act synergistically or antagonistically against tumor cells in vitro depending on the ratios of the individual agents comprising the combination. The importance of drug ratios in vivo, however, has heretofore not been investigated, and combination chemotherapy treatment regimens continue to be developed based on the maximum tolerated dose of the individual agents. We systematically examined three different drug combinations representing a range of anticancer drug classes with distinct molecular mechanisms (irinotecan/floxuridine, cytarabine/daunorubicin, and cisplatin/daunorubicin) for drug ratio–dependent synergy. In each case, synergistic interactions were observed in vitro at certain drug/drug molar ratio ranges (1:1, 5:1, and 10:1, respectively), whereas other ratios were additive or antagonistic. We were able to maintain fixed drug ratios in plasma of mice for 24 hours after i.v. injection for all three combinations by controlling and overcoming the inherent dissimilar pharmacokinetics of individual drugs through encapsulation in liposomal carrier systems. The liposomes not only maintained drug ratios in the plasma after injection, but also delivered the formulated drug ratio directly to tumor tissue. In vivo maintenance of drug ratios shown to be synergistic in vitro provided increased efficacy in preclinical tumor models, whereas attenuated antitumor activity was observed when antagonistic drug ratios were maintained. Fixing synergistic drug ratios in pharmaceutical carriers provides an avenue by which anticancer drug combinations can be optimized prospectively for maximum therapeutic activity during preclinical development and differs from current practice in which dosing regimens are developed empirically in late-stage clinical trials based on tolerability. [Mol Cancer Ther 2006;5(7):1854–63]

In vivo maintenance of synergistic cytarabine:daunorubicin ratios greatly enhances therapeutic efficacy

We demonstrate here that cytarabine and daunorubicin, a standard drug combination used in the treatment of leukaemia, exhibits drug ratio-dependent synergistic antitumor activity in vitro and in vivo. A cytarabine:daunorubicin molar ratio of 5:1 displayed the greatest degree of synergy and minimum antagonism in a panel of 15 tumor cell lines in vitro. Co-encapsulating cytarabine and daunorubicin inside liposomes maintained the synergistic drug ratio in plasma for 24h post-injection. Liposome-encapsulated cytarabine:daunorubicin combinations exhibited drug ratio-dependent in vivo efficacy with the 5:1 molar drug ratio (designated CPX-351) having the greatest therapeutic index, despite using sub-MTD daunorubicin doses. CPX-351 exhibited superior therapeutic activity compared to free-drug cocktails, with high proportions of long-term survivors, consistent with in vivo synergy. The therapeutic advantage of CPX-351 was associated with prolonged maintenance of synergistic drug ratios in bone marrow. These results indicate that in vitro informatics on cytarabine:daunorubicin cytotoxicity can be translated in vivo to optimize the efficacy of anticancer drug combinations by controlling the exposure of drug ratios with drug delivery vehicles.

In Vitro and in Vivo Characterization of Doxorubicin and Vincristine Coencapsulated within Liposomes through Use of Transition Metal Ion Complexation and pH Gradient Loading

Purpose: There is an opportunity to augment the therapeutic potential of drug combinations through use of drug delivery technology. This report summarizes data obtained using a novel liposomal formulation with coencapsulated doxorubicin and vincristine. The rationale for selecting these drugs is due in part to the fact that liposomal formulations of doxorubicin and vincristine are being separately evaluated as components of drug combinations. Experimental Design: Doxorubicin and vincristine were coencapsulated into liposomes using two distinct methods of drug loading. A manganese-based drug loading procedure, which relies on drug complexation with a transition metal, was used to encapsulate doxorubicin. Subsequently the ionophore A23187 was added to induce formation of a pH gradient, which promoted vincristine encapsulation. Results: Plasma elimination studies in mice indicated that the drug:drug ratio before injection [4:1 doxorubicin:vincristine (wt:wt ratio)] changed to 20:1 at the 24-h time point, indicative of more rapid release of vincristine from the liposomes than doxorubicin. Efficacy studies completed in MDA MB-435/LCC6 tumor-bearing mice suggested that at the maximum tolerated dose, the coencapsulated formulation was therapeutically no better than liposomal vincristine. This result was explained in part by in vitro cytotoxicity studies evaluating doxorubicin and vincristine combinations analyzed using the Chou and Talalay median effect principle. These data clearly indicated that simultaneous addition of vincristine and doxorubicin resulted in pronounced antagonism. Conclusion: These results emphasize that in vitro drug combination screens can be used to predict whether a coformulated drug combination will act in an antagonistic or synergistic manner.

Modulating the Therapeutic Activity of Nanoparticle Delivered Paclitaxel by Manipulating the Hydrophobicity of Prodrug Conjugates

A series of paclitaxel prodrugs designed for formulation in lipophilic nanoparticles are described. The hydrophobicity of paclitaxel was increased by conjugating a succession of increasingly hydrophobic lipid anchors to the drug using succinate or diglycolate cross-linkers. The prodrugs were formulated in well defined block copolymer-stabilized nanoparticles. These nanoparticles were shown to have an elimination half-life of approximately 24 h in vivo. The rate at which the prodrug was released from the nanoparticles could be controlled by adjusting the hydrophobicity of the lipid anchor, resulting in release half-lives ranging from 1 to 24 h. The diglycolate and succinate cross-linked prodrugs were 1-2 orders of magnitude less potent than paclitaxel in vitro. Nanoparticle formulations of the succinate prodrugs showed no evidence of efficacy in HT29 human colorectal tumor xenograph models. Efficacy of diglycolate prodrug nanoparticles increased as the anchor hydrophobicity increased. Long circulating diglycolate prodrug nanoparticles provided significantly enhanced therapeutic activity over commercially formulated paclitaxel at the maximum tolerated dose.

Drug ratio–dependent antitumor activity of irinotecan and cisplatin combinations in vitro and in vivo

Irinotecan and cisplatin are two established anticancer drugs, which together constitute an effective combination for treating small-cell lung cancer. We investigated whether the efficacy of this combination could be improved by controlling drug ratios following in vivo administration. Irinotecan and cisplatin combinations were evaluated systematically for drug ratio–dependent synergy in vitro using a panel of 20 tumor cell lines. In vitro screening informatics on drug ratio–dependent cytotoxicity identified a consistently antagonistic region between irinotecan/cisplatin molar ratios of 1:2 to 4:1, which was bordered by two synergistic regions. Liposomal co-formulations of these two agents were developed that exhibited plasma drug half-lives of ∼6 hours and maintained a fixed drug ratio for more than 24 hours. Drug ratio–dependent antitumor activity was shown in vivo for these liposome formulations, and irinotecan/cisplatin ratios between 5:1 and 10:1 were identified as therapeutically optimal. The relationship between irinotecan/cisplatin ratio and in vivo efficacy was consistent with in vitro drug ratio dependency results. Superior antitumor activity was observed for the liposome-encapsulated 7:1 molar ratio of irinotecan/cisplatin (designated CPX-571) compared with the free-drug cocktail in all models tested. Further efficacy studies in a range of human tumor xenografts, including an irinotecan-resistant model, showed that both liposomal agents contributed to the overall efficacy in a manner consistent with in vivo synergy. These results show the ability of drug delivery technology to enhance the therapeutic activity of irinotecan/cisplatin combination treatment by maintaining synergistic ratios in vivo. CPX-571, a fixed-ratio formulation of irinotecan and cisplatin, is a promising candidate for clinical development. [Mol Cancer Ther 2009;8(8):2266–75]

Increased preclinical efficacy of irinotecan and floxuridine coencapsulated inside liposomes is associated with tumor delivery of synergistic drug ratios.

Whether anticancer drug combinations act synergistically or antagonistically often depends on the ratio of the agents being combined. We show here that combinations of irinotecan and floxuridine exhibit drug ratio-dependent cytotoxicity in a broad panel of tumor cell lines in vitro where a 1:1 molar ratio consistently provided synergy and avoided antagonism. In vivo delivery of irinotecan and floxuridine coencapsulated inside liposomes at the synergistic 1:1 molar ratio (referred to as CPX-1) lead to greatly enhanced efficacy compared to the two drugs administered as a saline-based cocktail in a number of human xenograft and murine tumor models. When compared to liposomal irinotecan or liposomal floxuridine, the therapeutic activity of CPX-1 in vivo was not only superior to the individual liposomal agents, but the extent of tumor growth inhibition was greater than that predicted for combining the activities of the individual agents. In contrast, liposome delivery of irinotecan:floxuridine ratios shown to be antagonistic in vitro provided antitumor activity that was actually less than that achieved with liposomal irinotecan alone, indicative of in vivo antagonism. Synergistic antitumor activity observed for CPX-1 was associated with maintenance of the 1:1 irinotecan:floxuridine molar ratio in plasma and tumor tissue over 16-24 h. In contrast, injection of the drugs combined in saline resulted in irinotecan:floxuridine ratios that changed 10-fold within 1 h in plasma and sevenfold within 4 h in tumor tissue. These results indicate that substantial improvements in the efficacy of drug combinations may be achieved by maintaining in vitro-identified synergistic drug ratios after systemic administration using drug delivery vehicles.

Preclinical pharmacology, toxicology and efficacy of sphingomyelin/cholesterol liposomal vincristine for therapeutic treatment of cancer

Purpose: To establish the pharmacodynamic relationships between drug biodistribution and drug toxicity/efficacy, a comprehensive preclinical evaluation of sphingomyelin/cholesterol (SM/chol) liposomal vincristine and unencapsulated vincristine in mice was undertaken. Methods: Pharmaceutically acceptable formulations of unencapsulated vincristine and liposomal vincristine at drug/lipid ratios of 0.05 or 0.10 (wt/wt) were evaluated for toxicity, antitumor activity and pharmacokinetics following intravenous administration. Results: Mice given liposomal vincristine at 2 mg/kg vincristine had concentrations of vincristine in blood and plasma at least two orders of magnitude greater then those achieved after an identical dose of unencapsulated drug. One day after administration of the liposomal vincristine, there were at least tenfold greater drug quantities, relative to unencapsulated vincristine, in the axillary lymph nodes, heart, inguinal lymph nodes, kidney, liver, skin, small intestines and spleen. Increased plasma and tissue exposure to vincristine as a result of encapsulation in SM/chol liposomes was not associated with increased drug toxicities. Treatment of the murine P388 ascitic tumor with a single intravenous dose of unencapsulated drug at 2, 3 and 4 mg/kg, initiated 1 day after tumor cell inoculation, resulted in a 33 to 38% increase in lifespan. In contrast, long-term survival rates of 50% or more were achieved in all groups treated with the SM/chol liposomal vincristine formulations at doses of 2, 3 and 4 mg/kg. At the 4 mg/kg dose, eight of ten and nine of ten animals survived past day 60 when treated with SM/chol liposomal vincristine prepared at the 0.05 and 0.1 drug/lipid ratios, respectively. Conclusions: Overall, increased and prolonged plasma concentrations of vincristine achieved by liposomal encapsulation were correlated with dramatically increased antitumor activity in comparison with the unencapsulated drug, but no correlations could be established between pharmacokinetic parameters and toxicity.

Intratumor distribution of doxorubicin following i.v. administration of drug encapsulated in egg phosphatidylcholine/cholesterol liposomes

Abstract Purpose: A pharmacological evaluation of an egg phosphatidylcholine/cholesterol (55:45 mole ratio, EPC/Chol) liposome doxorubicin formulation was carried out. The objective was to define liposomal lipid and drug distribution within sites of tumor growth following intravenous (i.v.) administration to female BDF1 mice bearing either Lewis lung carcinoma, B16/BL6 melanoma, or L1210 ascitic tumors. Methods: Mice were injected i.v. with EPC/Chol liposomal doxorubicin, and plasma and tumor levels of lipid and drug were determined 1, 4 and 24 h later with radiolabeled lipid and fluorimetry or fluorescence microscopy, respectively. In addition, single-cell suspensions of the Lewis lung and B16/BL6 tumors were prepared and the presence of macrophages was determined with an FITC-labeled rat antimouse CD11b (MAC-1) antibody. Results: For mice bearing the Lewis lung solid tumors, there was a time-dependent accumulation of liposomal lipid, with a plateau of approximately 500 μg lipid/g tumor at 48 h. In contrast, the apparent plateau (μg doxorubicin/g tumor) for doxorubicin was achieved at 1 h and remained constant over a 72-h time course. In comparison with free drug administered at the maximum tolerated dose (MTD, 20 mg/kg) doxorubicin levels in tumors were two- to threefold greater when the drug was administered in liposomal form. The increase in drug delivery was comparable for both solid tumors. With animals bearing the L1210 ascitic tumor, drug exposure was as much as ten times greater (in comparison with free drug) when doxorubicin was administered in liposomes. An evaluation of single-cell suspensions prepared from the two solid tumors suggested that more than 98% of the tumor-associated drug and liposomal lipid was not tumor cell-associated. Histological studies with the Lewis lung carcinoma, however, revealed that a proportion of the drug did colocalize with tumor-associated macrophages. Analysis of cells obtained from mice bearing ascitic tumors showed that more than 80% of the cell-associated drug could be removed by procedures designed to remove adherent cells. Conclusion: The results summarized here suggest drug concentrations within a solid tumor, such as the Lewis lung carcinoma, are constant over time when the drug is given in a “leaky” EPC/Chol formulation. The results also suggest that liposomal lipid within sites of tumor growth is primarily localized within the interstitial spaces or tumor-associated macrophages.

Leukemia-selective uptake and cytotoxicity of CPX-351, a synergistic fixed-ratio cytarabine:daunorubicin formulation, in bone marrow xenografts

The objective of this study was to examine the pharmacodynamic basis for the potent preclinical and clinical anti-leukemic activity of CPX-351, a nano-scale liposome formulation of cytarabine and daunorubicin co-encapsulated at a synergistic 5:1 molar ratio. A bone marrow-engrafting CCRF-CEM leukemia model in Rag2-M mice was utilized to correlate the therapeutic and myelosuppressive properties of CPX-351 with bone marrow delivery and drug uptake in leukemia cells relative to normal bone marrow cell populations. When administered to mice bearing CCRF-CEM human leukemia xenografts, CPX-351 ablated bone marrow (BM) leukemic cells to below detectable levels for multiple weeks, whereas the free-drug cocktail only transiently suppressed leukemia growth. In contrast to the activity against leukemia cells, CPX-351 and free-drug cocktail induced similar myelosuppression in non-tumor-bearing BM. In leukemia-laden BM, drug concentrations were markedly elevated for CPX-351 over free-drug cocktail and the first dose of CPX-351, but not free-drug cocktail, potentiated BM drug accumulation for subsequent doses. Confocal fluorescence microscopy revealed that CPX-351 liposomes are taken up by CCRF-CEM cells and subsequently release drugs intracellularly. The improved in vivo efficacy of CPX-351 appears related to increased and prolonged exposure of synergistic cytarabine:daunorubicin ratios in BM, and the selective killing of leukemia may arise from direct liposome-leukemia cell interactions. These features may also have broader applicability in the treatment of other haematological malignancies.

Lipid-based formulations of antisense oligonucleotides for systemic delivery applications.

Publisher Summary Increasing the specificity of therapeutic drugs and improving their delivery to sites of disease are primary goals of todays pharmaceutical industry. One of the most exciting advances in recent years has been the development of antisense technologies, which are capable of modulating protein expression with exquisite specificity. Unfortunately, this class of drugs is particularly sensitive to nuclease degradation, is eliminated rapidly from the circulation after intravenous administration, and is severely limited in its ability to penetrate through cellular membranes unaided. Attempts to address these problems through medicinal chemistry have produced several key advances. However, chemical alterations to improve one property of the molecule often affect other properties, potentially in a negative manner. The past decade has seen extensive use of liposomes and lipid-based delivery systems to improve the pharmacological properties of a variety of drugs. The principal benefits afforded therapeutic agents by liposomal encapsulation are enhanced plasma-circulation lifetimes, increased delivery to sites of disease, and changes in tissue distribution, which can result in reduced toxic side effects. Liposomal preparations of doxorubicin (DOXIL) and daunorubicin (DaunoXome) have been approved for the treatment of HIV-associated Kaposis sarcoma, whereas lipid-based formulations of amphotericin B (AmBisome, ABELCET, AMPHOTEC) are successful clinical products employed in the treatment of fungal infections. It is not surprising, therefore, that considerable interest has been on developing lipid-based delivery systems to overcome the problems associated with the systemic administration of DNA- and RNA-based therapeutics. The intent of this chapter is to introduce the reader to the various lipid-based formulations applied to polynucleic acid drugs, with emphasis on the generation and characterization of delivery vehicles for intravenous applications. The majority of examples apply to antisense oligodeoxynucleotides (ASODN) as they represent the most widely available class of DNA-based drugs.