Pharmacokinetics of CPX-351; a nano-scale liposomal fixed molar ratio formulation of cytarabine:daunorubicin, in patients with advanced leukemia.

Forty-eight patients received CPX-351 (liposome-encapsulated cytarabine:daunorubicin at a 5:1 molar ratio) every other day for 3 doses at 10 dose levels. Pharmacokinetic parameters were dose-independent and exhibited low inter-patient variability. CPX-351 showed a negligible distribution phase and prolonged mono-exponential first-order plasma elimination (t(1/2)∼24 h). The plasma ratio of 5:1 was maintained at all dose levels. Nearly all of the detectable cytarabine and daunorubicin in circulation following CPX-351 administration was in the form of liposome encapsulated drug. Dose-dependent hematopoietic effects had early onset with cytopenias at 12 units/m(2), and a gradual increase in frequency and severity, until single induction complete response was achieved at 43 units/m(2). Non-hematologic effects had onset by 24 units/m(2) with shallow dose-response until maximum frequency and severity were observed at the 101-134 units/m(2) dose levels. Single induction response occurred over a 2.3-fold range of doses indicating that CPX-351 may be useful at high doses for patients suitable for intensive chemotherapy and at reduced doses for patients at increased risk of treatment-related mortality. The unique pharmacologic features of CPX-351 contribute to its promising antileukemic efficacy.

Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol).

The presence of poly(ethylene glycol) (PEG) at the surface of a liposomal carrier has been clearly shown to extend the circulation lifetime of the vehicle. To this point, the extended circulation lifetime that the polymer affords has been attributed to the reduction or prevention of protein adsorption. However, there is little evidence that the presence of PEG at the surface of a vehicle actually reduces total serum protein binding. In this review we examine all aspects of PEG in order to gain a better understanding of how the polymer fulfills its biological role. The physical and chemical properties of the polymer are explored and compared to properties of other hydrophilic polymers. An evidence based assessment of several in vitro protein binding studies as well as in vivo pharmacokinetics studies involving PEG is included. The ability of PEG to prevent the self-aggregation of liposomes is considered as a possible means by which it extends circulation longevity. Also, a "dysopsonization" phenomenon where PEG actually promotes binding of certain proteins that then mask the vehicle is discussed.

Establishment and comprehensive analysis of a new human transformed follicular lymphoma B cell line, Tat-1.

A spontaneously EBV transformed follicular lymphoma (FL) cell line, Tat-1, was established from the lymph node biopsy specimen of a patient with B cell FL, grade 1 in transformation to high grade disease. Tat-1 cells expressed lymphoid markers and developed tumor masses in immunodeficient mice. Bcl-2, Bcl-X(L), Bax and p53 protein expression was revealed by Western blotting. Flow cytometric analysis confirmed P-gp expression. Cytogenetically, the Tat-1 cell line showed identical chromosomal alterations to that of the initial biopsy specimen, among which the most notable were the t(14;18) typical of FL and additional abnormalities involving chromosomes 1, 8 and 13. Multicolor FISH analysis delineated all abnormalities, including a t(1p;8q), a der(8)(8q24::14q32::18q21) and a der(13)(13q32::8q24::14q32::18q21). Further FISH investigations using a locus-specific probe cocktail containing c-myc, IgH and bcl-2 revealed fusion of these three loci on the derivatives 8 and 13, in addition to the derivative 14 IgH/bcl-2 fusion and an extra copy of c-myc on derivative chromosome 1. These results demonstrate an additional example of the deregulation of bcl-2 and c-myc expression through recombination with a single IgH enhancer region. The unusual molecular features of the Tat-1 cell line render it a unique tool for studies focused on cytogenetic alterations, expression of multidrug resistance phenotype and expression of anti-apoptotic proteins in FL.

A comparison of liposomal formulations of doxorubicin with drug administered in free form: changing toxicity profiles.

The anthracycline antibiotic doxorubicin has wide activity against a number of human neoplasms and is used extensively both as a single agent and in combination regimens. In addition to the use of free, unencapsulated doxorubicin, there are two US Food and Drug Administration approved liposomal formulations of doxorubicin currently available, with several additional liposomal formulations being researched either in the laboratory or in clinical trials. The two approved liposomal formulations of doxorubicin have significantly different lipid compositions and loading techniques, which lead to both unique pharmacokinetic and toxicity profiles, distinct from those of the unencapsulated form. This article discusses the toxicities associated with the free form of doxorubicin, as well as those associated with the two most common liposomal formulations, namely Doxil and Myocet. One of the key toxicity issues linked to the use of free doxorubicin is that of both an acute and a chronic form of cardiomyopathy. This is circumvented by the use of liposomal formulations, as these systems tend to sequester the drug away from organs such as the heart, with greater accumulation in liver, spleen and tumours. However, as will be discussed, the liposomal formulations of doxorubicin are not without their own related toxicities, and, in the case of Doxil, may be associated with the unique toxicity of palmar-plantar erythrodysaesthesia. Overall, the use of liposomal doxorubicin allows for a greater lifetime cumulative dose of doxorubicin to be administered, however acute maximal tolerated doses differ significantly, with that of Myocet being essentially equivalent to free doxorubicin, while higher doses of Doxil may be safely administered. This review highlights the differences in both toxicity and pharmacokinetic properties between free doxorubicin and the different liposomal formulations, as have been determined in pre-clinical and clinical testing against a number of different human neoplasms. The need for further testing of the liposomal formulations prior to the replacement of free doxorubicin with liposomal doxorubicin in any established combination therapy regimens, as well as in combination with the newer therapeutics such as monoclonal antibodies is also discussed.

Visualization of bioavailable liposomal doxorubicin using a non-perturbing confocal imaging technique.

Commonly employed tissue processing techniques can significantly alter tissue drug distribution patterns for liposomal encapsulated drugs by virtue of drug leakage via loss of membrane integrity. We report here a method that has been developed to determine the fluorescence of bioavailable doxorubicin (DOX) in tissues after administration of liposomal DOX formulations. A non-perturbing confocal fluorescence microscopy (CFM) technique with image processing analysis was used with unprocessed fresh tissues. This method takes advantage of the fact that considerable quenching occurs when DOX is within liposomes, leading to the selective visualization of the fluorescence due to DOX released from liposomes. We demonstrate that fresh tissue confocal imaging can be applied to provide detailed drug distribution information with improved accuracy and is a superior method for analyzing tissue distribution of liposome entrapped fluorescent agents.

Liposomal and nonliposomal drug pharmacokinetics after administration of liposome-encapsulated vincristine and their contribution to drug tissue distribution properties.

We have determined the pharmacokinetics of liposomal vincristine, in a Lewis lung carcinoma solid tumor model in mice, with the aim of differentiating the contribution of liposomal and nonliposomal (released from liposomes) drug pools to the overall pharmacokinetic profile. Two types of liposomal formulations were used: one composed of 1,2 distearoyl-sn-glycero-3-phosphocholine/cholesterol (Chol) (55/45; mol/mol) and the other composed of sphingomyelin/cholesterol (SM/Chol; 55/45; mol/mol). Vincristine elimination from the circulation after injection of conventional, aqueous formulated vincristine (C-VINC) was characterized by a short half-life (1.36 h), low plasma area under the plasma concentration-time curve (AUC) (0.59 microg x h/ml), and large volume of distribution (145 ml). Total drug elimination from the circulation after liposomal vincristine injection using SM/Chol liposomes was characterized by a prolonged half-life (6.6 h), increased plasma AUC (213 microg x h/ml) and small volume of distribution (2.0 ml). Our results indicate that > or =98% of the total vincristine measured in the plasma of mice administered with liposomal vincristine was encapsulated within the liposomes. The systemic exposure to free drug after administration of liposomal formulations was significantly lower than that observed after the injection of C-VINC. Plasma concentrations of free drug remained between 0.025 and 0.05 microg/ml over 4 h of postinjection for both liposomal formulations. In contrast, concentrations between 0.1 and 0.35 microg/ml were observed following C-VINC administration. Free plasma drug concentrations did not correlate with vincristine tissue distribution properties following administration of liposomal vincristine formulations. Rather, accumulation of vincristine in tissues appeared to be influenced primarily by the drug retention properties of the liposome. While the reduced systemic exposure to free vincristine correlates with reduced toxicity, additional information (such as liposome drug release properties) may be necessary to correlate pharmacokinetic behavior with antitumor activity.

Intermembrane transfer of polyethylene glycol-modified phosphatidylethanolamine as a means to reveal surface-associated binding ligands on liposomes.

In order to explore the use of exchangeable poly(ethylene glycol) (PEG)-modified diacylphosphatidylethanolamines (PE) to temporarily shield binding ligands attached to the surface of liposomes, a model reaction based on inhibition and subsequent recovery of biotinylated liposome binding to streptavidin immobilized on superparamagnetic iron oxide particles (SA magnetic particles) was developed. PEG-lipid incorporation into biotinylated liposomes decreased liposome binding to SA magnetic particles in a non-linear fashion, where as little as 0.1 mol% PEG-PE resulted in a 20% decrease in binding. Using an assay based on inhibition of binding, PEG(2000)-PE transfer from donor liposomes to biotinylated acceptor liposomes could be measured. The influence of temperature and acyl chain composition on the transfer of PEG-diacyl PEs from donor liposomes to acceptor liposomes, consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol and N-((6-biotinoyl)amino)hexanoyl)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (54.9:45:0.1 mole ratio), was measured. Donor liposomes were prepared using 1,2-distearoyl-sn-glycero-3-phosphocholine (50 mol%), cholesterol (45 mol%) and 5 mol% of either PEG-derivatized 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG(2000)), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG(2000)), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG(2000)). Transfer of DSPE-PEG(2000) to the donor liposomes was not detected under the conditions employed. In contrast, DMPE-PEG(2000) was transferred efficiently even at 4 degrees C. Using an acceptor to donor liposome ratio of 1:4, the time required for DMPE-PEG(2000) to become evenly distributed between the two liposome populations (T(EQ)) at 4 degrees C and 37 degrees C was approx. 2 and <0.5 h, respectively. An increase in acyl chain length from C14:0 to C16:0 of the PEG-lipid resulted in a significant reduction in the rate of transfer as measured by this assay. The transfer of PEG-lipid out of biotinylated liposomes was also studied in mice following intravenous administration. The relative rates of transfer for the various PEG-lipids were found to be comparable under in vivo and in vitro conditions. These results suggest that it is possible to design targeted liposomes with the targeting ligand protected while in the circulation through the use of PEG-lipids that are selected on the basis of exchange characteristics which result in exposure of the shielded ligand following localization within a target tissue.

Selective protein interactions with phosphatidylserine containing liposomes alter the steric stabilization properties of poly(ethylene glycol).

Incorporation of 5 mol% poly(ethylene glycol)-conjugated lipids (PEG-lipids) has been shown to extend the circulation longevity of neutral liposomes due to steric repulsion of PEG at the membrane surface. The effects of PEG-lipids on protein interactions with biologically reactive membranes were examined using phosphatidylserine (PS) containing liposomes as the model. Incorporating 15 mol% 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG 2000 into PS liposomes resulted in circulation lifetimes comparable to that obtained with neutral liposomes containing 5 mol% DSPE-PEG 2000. These results suggested that 15 mol% DSPE-PEG 2000 may be effective in protecting PS liposomes from the high affinity, PS-mediated binding of plasma proteins. This was determined by monitoring the effects of PEG-lipids on calcium-mediated blood coagulation protein interactions with PS liposomes. Prothrombin binding and procoagulant activity of PS liposomes could be inhibited >80% when 15 mol% DSPE-PEG 2000 was used. These results are consistent with PS on membrane surfaces forming transient nucleation sites for protein binding that may result in lateral exclusion of PEG-lipids incorporated at <10 mol%. These nucleation sites may be inaccessible when PEG-lipids are present at elevated levels where they adopt a highly compressed brush conformation. This suggests that liposomes with reactive groups and PEG-lipids may be appropriately designed to impart selectivity to protein interactions with membrane surfaces.

Modulation of P-glycoprotein (PGP) mediated multidrug resistance (MDR) using chemosensitizers: recent advances in the design of selective MDR modulators.

Over the past two decades, a number of chemical entities have been investigated in the continuing quest to reverse P-glycoprotein (PGP) mediated multidrug resistance (MDR) in cancer. The complexity of interactions between these agents and the proteins responsible for MDR in conjunction with the challenges associated with developing SAR/QSAR relationships for MDR modulators has hampered our ability to develop agents that modulate MDR with enhanced specificity of target, increased efficacy, and minimized toxicity when coadministered with anticancer drugs. With an increased understanding of the molecular interaction, target-mediated SAR and combinatorial chemistry approaches, newer more selective inhibitors have been recently reported. These agents have shown remarkable promise in preclinical trials; although their ultimate clinical therapeutic utility remains to be established. The emphasis of this review is placed on the current understanding of modulator-drug transport protein interactions and to review the advances in the structure-based design, synthetic efforts and the cellular pharmacology of MDR modulating activity of a number of known PGP inhibitors.

Molecular and pharmacological strategies to overcome multidrug resistance.

Multidrug resistance is a major obstacle to the effective treatment of cancer. Despite vast improvements in our understanding of the mechanisms of drug resistance, relatively few significant advances have been made towards effectively circumventing it in a clinical setting. The ability to modulate multidrug resistance has been complicated by the fact that many human tumors simultaneously exhibit multiple resistance mechanisms. In order to effectively overcome multidrug resistance it will be necessary to design new strategies that combine multiple modulating agents and approaches. This review provides an overview of the major causes of multidrug resistance and summarizes many of the current approaches being taken to overcome it. We also describe how liposomal drug delivery systems can be utilized to aid in achieving these goals.

Assessment of the involvement of CYP3A in the vitro metabolism of a new modulator of MDR in cancer chemotherapy, OC144-193, by human liver microsomes.

The novel substituted imidazole compound, OC144-093 exhibits potent biological activity in vitro and in vivo for reversal of P-glycoprotein (PgP) based resistance to cancer chemotherapy. Its mechanism of action relies upon its inhibitory interaction with the mdr1 gene product, a known mediator of multidrug resistance (MDR). Overlapping substrate specificities and tissue distribution of cytochrome P450 3A (CYP3A) and PgP indicate the potential for drug-drug interactions when modulator and anticancer agent are co-administered. We have examined the metabolism of OC144-093 in vitro using human liver microsomes to determine if CYP3A is involved. Our results show that OC144-093 is converted to one major metabolite (M1) in human liver microsomes which was identified by LCMS to be the O-deethylated derivative. Km and Vmax for O-deethylation were determined as 3.96+/-0.67 microM and 32.08+/-9.73 pmol/mg protein/min, respectively (n=3). Correlation studies conducted in a panel of human livers phenotyped for specific P450 enzyme activity showed a significant relationship between M1 formation and the activity of CYP2C9, CYP2B6, CYP2E1 and CYP3A4. Treatment of microsomes with carbon monoxide gas inhibited M1 formation and diethyldithiocarbamate and ketoconazole (>3 microM), non-specific CYP inhibitors, gave IC50 values of 124.4+/-21.6 microM and 25.3+/-3.2 microM respectively for the inhibition of O-deethylation, also implicating the involvement of CYP enzymes. Specific CYP inhibitors of CYP3A4 were essentially non-inhibitory to M1 formation. We can conclude therefore that OC144-093 is not extensively metabolised in human liver microsomes although conversion to its O-deethylated derivative does occur. Our data indicates that this conversion is not mediated by CYP3A4.

The role for liposomal drug delivery in molecular and pharmacological strategies to overcome multidrug resistance.

When P-glycoprotein (PGP) was first identified as a direct mediator of multidrug resistance (MDR) a great deal of excitement was generated as scientists and clinicians anticipated the ability to successfully treat previously refractory cancers by blocking this drug efflux pump. More than twenty years later there is still minimal evidence that inhibiting PGP will have widespread impact on the chemosensitivity of human tumors. Yet, we know that PGP is over-expressed in many cancers, is associated with poor prognosis in certain tumor types and, if functional, will certainly reduce the accumulation of many common anticancer drugs inside tumor cells exhibiting elevated PGP levels. Similar situations have arisen more recently for other potential mediators of chemosensitivity such as the apoptosis antagonist protein Bcl-2. Bcl-2 has been linked to drug resistance and poor patient prognosis in numerous studies. There has been a great deal of interest in blocking expression or function of this protein to increase the susceptibility of tumor cells to apoptotic stimuli such as chemotherapy. However, preclinical and clinical evidence supporting this approach as a unilateral means of significantly enhancing the response of tumors to chemotherapy is limited. In view of these examples, it would appear likely that similar caveats will be experienced in the future as new molecular targets are identified for potential MDR reversal. Given the ever increasing evidence of genetic diversity in cancer development and progression, it should not be surprising that the development of MDR is also complex and heterogeneous. Consequently, it should also not be surprising that solutions to this problem are unlikely to arise from interventions aimed at any single resistance mechanism. These concepts suggest that new approaches to addressing the various molecular and pharmacological features associated with MDR will be necessary in order to make significant in-roads into improving the clinical activity of current and future anticancer agents. This review summarizes many of the current directions being taken to overcome MDR and how liposomal drug delivery systems may play an important role in achieving this aim.

Effects of Bcl-2 modulation with G3139 antisense oligonucleotide on human breast cancer cells are independent of inherent Bcl-2 protein expression.

We have investigated the effects of transient Bcl-2 down-regulation induced by the Bcl-2 antisense oligodeoxynucleotide (ODN) G3139 (Genta Incorporated) in high Bcl-2 protein expressing, estrogen receptor (ER) positive MCF-7 and low Bcl-2 expressing, ER negative MDA435/LCC6 human breast cancer cells. Treatment with Bcl-2 antisense ODN in vitro caused > 80% reduction of Bcl-2 protein levels in a sequence specific manner for both cell lines. Maximum mRNA reduction was achieved within 24 h of the first antisense ODN exposure whereas full protein down-regulation required antisense exposure over 48 h. This Bcl-2 reduction was associated with 80-95% loss of viable cells compared to untreated cells. Similar cytotoxic effects were observed in both cell lines despite a nine-fold intrinsic difference in Bcl-2 protein expression suggesting that the relative degree of down-regulation of Bcl-2 is more important than the absolute reduction. Cell death associated with G3139 exposure exhibited properties indicative of apoptosis such as mitochondrial membrane depolarization and caspase activation. Combined treatment with G3139 and cytotoxic agents resulted in additive cytotoxicity in both cell lines. However, under most conditions studied, the direct cytotoxic activity of G3139 antisense was not synergistic with the cytotoxic agents. These results suggest that while Bcl-2 clearly constitutes an attractive therapeutic target due to its role in regulating apoptosis in breast cancer cells, additional mechanisms are important in the control of apoptosis arising from exposure to anticancer agents in vitro.

Interaction of polyphemusin I and structural analogs with bacterial membranes, lipopolysaccharide, and lipid monolayers.

Three structural variants (PV5, PV7, and PV8) of the horseshoe crab cationic antimicrobial peptide polyphemusin I were designed with improved amphipathic profiles. Circular dichroism spectroscopy analysis indicated that in phosphate buffer polyphemusin I, PV7, and PV8 displayed the spectrum of a type II beta-turn-rich structure, but, like polyphemusin I, all three variants adopted a typical beta-sheet structure in an anionic lipid environment. Both polyphemusin I and variants were potent broad spectrum antimicrobials that were clearly bactericidal at their minimal inhibitory concentrations. The variants were moderately less active in vitro but more effective in animal models. Moreover, these variants exhibited delayed bacterial killing, whereas polyphemusin I killed Escherichia coli UB1005 within 5 min at 2.5 microg/mL. All the peptides showed similar abilities to bind to bacterial lipopolysaccharide (LPS) and permeabilize bacterial outer membranes. Consistent with this was the observation that all peptides significantly inhibited cytokine production by LPS-stimulated macrophages and penetrated polyanionic LPS monolayers to similar extents. None of the peptides had affinity for neutral lipids as evident from both tryptophan fluorescence spectroscopy and Langmuir monolayer analysis. As compared to polyphemusin I, all variants showed reduced ability to interact with anionic lipids, and the hemolytic activity of the variants was decreased by 2-4-fold. In contrast, polyphemusin I efficiently depolarized the cytoplasmic membrane of E. coli, as assessed using a membrane potential sensitive fluorescent dye 3,3-dipropylthiacarbocyanine (diSC(3)5) assay, but the variants showed a substantially delayed and decreased depolarizing ability. The coincident assessment of cell viability indicated that depolarization of the bacterial cytoplasmic membrane potential by polyphemusin I occurred prior to lethal damage to cells. Our data suggest that increase of amphipathicity of beta-sheet polyphemusin I generally resulted in variants with decreased activity for membranes. Interestingly, all variants showed an improved ability to protect mice both against infection by Pseudomonas aeruginosa and from endotoxaemia.

Multidrug resistance (MDR) in cancer. Mechanisms, reversal using modulators of MDR and the role of MDR modulators in influencing the pharmacokinetics of anticancer drugs.

In recent years, there has been an increased understanding of P-glycoprotein (P-GP)-mediated pharmacokinetic interactions. In addition, its role in modifying the bioavailability of orally administered drugs via induction or inhibition has been also been demonstrated in various studies. This overview presents a background on some of the commonly documented mechanisms of multidrug resistance (MDR), reversal using modulators of MDR, followed by a discussion on the functional aspects of P-GP in the context of the pharmacokinetic interactions when multiple agents are coadministered. While adverse pharmacokinetic interactions have been documented with first and second generation MDR modulators, certain newer agents of the third generation class of compounds have been less susceptible in eliciting pharmacokinetic interactions. Although the review focuses on P-GP and the pharmacology of MDR reversal using MDR modulators, relevance of these drug transport proteins in the context of pharmacokinetic implications (drug absorption, distribution, clearance, and interactions) will also be discussed.

Quantitative fluorescence cytometric analysis of Bcl-2 levels in tumor cells exhibiting a wide range of inherent Bcl-2 protein expression: correlation with Western blot analysis.

BACKGROUND: A protocol to measure a wide range of Bcl-2 protein expression using quantitative fluorescence cytometry (QFCM) in different cell types was developed for use with flow cytometry. Bcl-2 measurements obtained by flow cytometry were correlated with Western blot Bcl-2 measurements to confirm specificity of the Bcl-2-FITC staining. This protocol was applied to measure absolute levels of Bcl-2 protein in different tumor cell lines including Bcl-2-transfected breast carcinoma cell lines and in peripheral blood lymphocytes (PBL). METHODS: HL-60, K562, DOHH2, Jurkat, MDA435/LCC6, MCF7 cell lines, and PBL derived from normal donors were fixed, permeabilized, stained with anti-Bcl-2-FITC antibody and evaluated by QFCM. In parallel, the same cells were evaluated for Bcl-2 protein expression by Western blot analysis. Mitochondrial localization of anti-Bcl-2-FITC antibody inside cells was confirmed using fluorescence imaging microscopy. RESULTS: Bcl-2 expression in different cell types could be accurately quantified based on antibody-binding capacity (ABC) ranging from 12.6 x 10(3) antibody-binding sites in HL-60 cells to 1.64 x 10(6) antibody-binding sites in a Bcl-2-transfected MDA435/LCC6 clone. The data from flow cytometry analysis correlated well with Western analysis (R(2) = 0.78). Bcl-2-FITC staining colocalized with dyes specific for mitochondria. CONCLUSIONS: The Bcl-2 staining protocol described here was shown to be specific, sensitive, and it was able to provide higher resolution as well as more reproducible quantitation of Bcl-2 protein content in cells when compared with Western blot methods. Quantitation of Bcl-2 content in cells by QFCM may be useful for monitoring Bcl-2 expression in cells undergoing various treatments in vitro and in vivo.

In vitro characterization of the anticancer activity of membrane-active cationic peptides. I. Peptide-mediated cytotoxicity and peptide-enhanced cytotoxic activity of doxorubicin against wild-type and p-glycoprotein over-expressing tumor cell lines.

Cationic amphipathic peptides, such as the defensins and cecropins, induce cell death in prokaryotic and eukaryotic cells by increasing membrane permeability. Increased permeability may lead to cell lysis or, alternatively, may produce subtle changes in the membrane's barrier function that promote cell death. The in vitro cytotoxic and lytic activity of short mammalian-derived extended-helical cationic peptides and insect-derived alpha-helical peptides was measured in this study with the objective of establishing the anticancer potential of these agents. Two specific aims were addressed: (i) to assess the activity of peptides against non-malignant cells (sheep erythrocytes and human umbilical vein endothelial cells) versus tumor cells; and (ii) to characterize the cytotoxic activity using multidrug-resistant tumor cell lines in the presence and absence of the anthracycline doxorubicin. Cell lysis assays demonstrated that the lytic activity of the peptides tested was 2->50 times more cytotoxic to tumor cells than to non-malignant cells. Further, the cytotoxic activity of these peptides was equivalent when tested against sensitive and multidrug-resistant cell lines. In addition to their inherent cytotoxic activity, these membrane-active peptides can also augment the in vitro cytotoxic activity of doxorubicin against multidrug-resistant tumor cells.

Molecular and pharmacokinetic properties associated with the therapeutics of bcl-2 antisense oligonucleotide G3139 combined with free and liposomal doxorubicin.

Bcl-2 is a key apoptosis-regulating protein that has been implicated in mechanisms of chemoresistance for a variety of malignancies by blocking programmed cell death. This study investigated the activity of the Bcl-2 antisense oligodeoxynucleotide (AS ODN) G3139 combined with free doxorubicin (F-DOX) or sterically stabilized liposomal doxorubicin (SL-DOX) to determine the role that drug pharmacodistribution properties may have on antitumor activity using a Bcl-2-expressing human breast solid tumor xenograft model. Administration of G3139 was able to delay the growth of MDA435/LCC6 cells compared with control ODN-treated animals; however, in all of the cases, tumors reestablished after AS ODN treatment. Western blot analyses of Bcl-2 levels of solid tumors showed a sequence-specific down-regulation of the Bcl-2 protein after four daily doses of G3139, which correlated with histological evidence of tumor cell death. Interestingly, the expression of Bcl-2 returned to pretreatment levels during the course of subsequent ODN administration, which suggested the development of resistance to continued Bcl-2 ODN treatment. The antitumor activity of ODN given in conjunction with either F-DOX or SL-DOX was also examined. The combination of G3139 and F-DOX was able to suppress the growth of MDA435/LCC6 cells beyond that obtained with either of the treatments given alone, indicative of synergistic action. Examination of the pharmacokinetics of F-DOX with systemic G3139 administration revealed that elevated tumor drug DOX levels were obtained compared with DOX treatment in the absence of G3139. This effect was sequence-specific and plasma DOX levels were unaffected by G3139 treatment, which indicated possible positive ODN-drug interactions at the tumor site. Combining G3139 with SL-DOX further increased the degree of antitumor activity. The improved efficacy of this combination was attributed to increased tumor drug levels that arise from the ability of SL-DOX to passively accumulate in solid tumors. These results suggest that additional benefits of Bcl-2 antisense ODN may be obtained when it is combined with liposomal formulations of anticancer drugs such as DOX.

Discovery and characterization of OC144-093, a novel inhibitor of P-glycoprotein-mediated multidrug resistance.

OC144-093 is a novel substituted diarylimidazole (Mr 495) generated using the OntoBLOCK system, a solid-phase combinatorial chemistry technology, in combination with high-throughput cell-based screening. OC144-093 reversed multidrug resistance (MDR) to doxorubicin, paclitaxel, and vinblastine in human lymphoma, breast, ovarian, uterine, and colorectal carcinoma cell lines expressing P-glycoprotein (P-gp) with an average EC50 of 0.032 microM. Inhibition of MDR by OC144-093 was reversible, but the effect persisted for at least 12 h after removal of compound from the culture medium. OC144-093 had no effect on the response to cytotoxic agents by cells in vitro lacking P-gp expression or expressing a multidrug resistance-associated protein (MRP-1). OC144-093 was not cytotoxic by itself against 15 normal, nontransformed, or tumor cell lines, regardless of P-gp status, with an average cytostatic IC50 of >60 microM. OC144-093 blocked the binding of [3H]azidopine to P-gp and inhibited P-gp ATPase activity. The compound was >50% p.o. bioavailable in rodents and dogs and did not alter the plasma pharmacokinetics of i.v.-administered paclitaxel. OC144-093 increased the life span of doxorubicin-treated mice engrafted with MDR P388 leukemia cells by >100% and significantly enhanced the in vivo antitumor activity of paclitaxel in MDR human breast and colon carcinoma xenograft models, without a significant increase in doxorubicin or paclitaxel toxicity. The results demonstrate that OC144-093 is an orally active, potent, and nontoxic inhibitor of P-gp-mediated multidrug resistance that exhibits all of the desired properties for treatment of P-gp-mediated MDR, as well as for prevention of MDR prior to selection and/or induction of refractory disease.