Showing papers on "Doxorubicin published in 2001"

Reduced Cardiotoxicity and Preserved Antitumor Efficacy of Liposome-Encapsulated Doxorubicin and Cyclophosphamide Compared With Conventional Doxorubicin and Cyclophosphamide in a Randomized, Multicenter Trial of Metastatic Breast Cancer

Abstract: Purpose To determine whether Myocet (liposome-encapsulated doxorubicin; The Liposome Company, Elan Corporation, Princeton, NJ) in combination with cyclophosphamide significantly reduces doxorubicin cardiotoxicity while providing comparable antitumor efficacy in first-line treatment of metastatic breast cancer (MBC). Patients and methods Two hundred ninety-seven patients with MBC and no prior chemotherapy for metastatic disease were randomized to receive either 60 mg/m(2) of Myocet (M) or conventional doxorubicin (A), in combination with 600 mg/m(2) of cyclophosphamide (C), every 3 weeks until disease progression or unacceptable toxicity. Cardiotoxicity was defined by reductions in left-ventricular ejection fraction, assessed by serial multigated radionuclide angiography scans, or congestive heart failure (CHF). Antitumor efficacy was assessed by objective tumor response rates (World Health Organization criteria), time to progression, and survival. Results Six percent of MC patients versus 21% (including five cases of CHF) of AC patients developed cardiotoxicity (P =.0002). Median cumulative doxorubicin dose at onset was more than 2,220 mg/m(2) for MC versus 480 mg/m(2) for AC (P =.0001, hazard ratio, 5.04). MC patients also experienced less grade 4 neutropenia. Antitumor efficacy of MC versus AC was comparable: objective response rates, 43% versus 43%; median time to progression, 5.1% versus 5.5 months; median time to treatment failure, 4.6 versus 4.4 months; and median survival, 19 versus 16 months. Conclusion Myocet improves the therapeutic index of doxorubicin by significantly reducing cardiotoxicity and grade 4 neutropenia and provides comparable antitumor efficacy, when used in combination with cyclophosphamide as first-line therapy for MBC.

Pegylated liposomal doxorubicin: metamorphosis of an old drug into a new form of chemotherapy.

Abstract: Pegylated liposomal doxorubicin (Doxil, Caelyx) is a formulation of doxorubicin in poly(ethylene glycol)-coated (stealth) liposomes with a prolonged circulation time and unique toxicity profile. We review the preclinical and clinical pharmacology as well as recent clinical data obtained in specific cancer types. Doxil liposomes retain the drug payload during circulation and accumulate preferentially in tissues with increased microvascular permeability, as often is the case of tumors. Doxil toxicity profile is drastically different from that of doxorubicin, and is characterized by dominant and dose-limiting mucocutaneous toxicities, mild myelosupression, minimal alopecia, and no apparent cardiac toxicity. Although the single maximum tolerated dose (MTD) of Doxil is actually lower than that of conventionally administered doxorubicin, the cumulative MTD dose of Doxil may be substantially greater than that of free doxorubicin. Doxil is probably one of the most active agents in AIDS-related Kaposi's sarcoma an...

Cyclophosphamide, doxorubicin, and paclitaxel enhance the antitumor immune response of granulocyte/macrophage-colony stimulating factor-secreting whole-cell vaccines in HER-2/neu tolerized mice.

Abstract: Tumor-specific immune tolerance limits the effectiveness of cancer vaccines. In addition, tumor vaccines alone have a limited potential for the treatment of measurable tumor burdens. This highlights the importance of identifying more potent cancer vaccine strategies for clinical testing. We tested immune-modulating doses of chemotherapy in combination with a granulocyte/macrophage-colony stimulating factor (GM-CSF)-secreting, HER-2/neu (neu)-expressing whole-cell vaccine as a means to treat existing mammary tumors in antigen-specific tolerized neu transgenic mice. Earlier studies have shown that neu transgenic mice exhibit immune tolerance to the neu-expressing tumors similar to what is observed in patients with cancer. We found that cyclophosphamide, paclitaxel, and doxorubicin, when given in a defined sequence with a GM-CSF-secreting, neu-expressing whole-cell vaccine, enhanced the vaccine's potential to delay tumor growth in neu transgenic mice. In addition, we showed that these drugs mediate their effects by enhancing the efficacy of the vaccine rather than via a direct cytolytic effect on cancer cells. Furthermore, paclitaxel and cyclophosphamide appear to amplify the T helper 1 neu-specific T-cell response. These findings suggest that the combined treatment with immune-modulating doses of chemotherapy and the GM-CSF-secreting neu vaccine can overcome immune tolerance and induce an antigen-specific antitumor immune response. These data provide the immunological rationale for testing immune-modulating doses of chemotherapy in combination with tumor vaccines in patients with cancer.

In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576.

Abstract: The overexpression of P-glycoprotein (P-gp) on the surface of tumor cells causes multidrug resistance (MDR). This protein acts as an energy-dependent drug efflux pump reducing the intracellular concentration of structurally unrelated drugs. Modulators of P-gp function can restore the sensitivity of MDR cells to such drugs. XR9576 is a novel anthranilic acid derivative developed as a potent and specific inhibitor of P-gp, and in this study we evaluate the in vitro and in vivo modulatory activity of this compound. The in vitro activity of XR9576 was evaluated using a panel of human (H69/LX4, 2780AD) and murine (EMT6 AR1.0, MC26) MDR cell lines. XR9576 potentiated the cytotoxicity of several drugs including doxorubicin, paclitaxel, etoposide, and vincristine; complete reversal of resistance was achieved in the presence of 25-80 nM XR9576. Direct comparative studies with other modulators indicated that XR9576 was one of the most potent modulators described to date. Accumulation and efflux studies with the P-gp substrates, [3H]daunorubicin and rhodamine 123, demonstrated that XR9576 inhibited P-gp-mediated drug efflux. The inhibition of P-gp function was reversible, but the effects persisted for >22 h after removal of the modulator from the incubation medium. This is in contrast to P-gp substrates such as cyclosporin A and verapamil, which lose their activity within 60 min, suggesting that XR9576 is not transported by P-gp. Also, XR9576 was a potent inhibitor of photoaffinity labeling of P-gp by [3H]azidopine implying a direct interaction with the protein. In mice bearing the intrinsically resistant MC26 colon tumors, coadministration of XR9576 potentiated the antitumor activity of doxorubicin without a significant increase in toxicity; maximum potentiation was observed at 2.5-4.0 mg/kg dosed either i.v. or p.o. In addition, coadministration of XR9576 (6-12 mg/kg p.o.) fully restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice. Importantly all of the efficacious combination schedules appeared to be well tolerated. Furthermore, i.v. coadministration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel. These results demonstrate that XR9576 is an extremely potent, selective, and effective modulator with a long duration of action. It exhibits potent i.v. and p.o. activity without apparently enhancing the plasma pharmacokinetics of paclitaxel or the toxicity of coadministered drugs. Hence, XR9576 holds great promise for the treatment of P-gp-mediated MDR cancers.

Cancer gene therapy using a survivin mutant adenovirus

Abstract: We have constructed a replication-deficient adenovirus encoding a nonphosphorylatable Thr(34)-->Ala mutant of the apoptosis inhibitor survivin (pAd-T34A) to target tumor cell viability in vitro and in vivo. Infection with pAd-T34A caused spontaneous apoptosis in cell lines of breast, cervical, prostate, lung, and colorectal cancer. In contrast, pAd-T34A did not affect cell viability of proliferating normal human cells, including fibroblasts, endothelium, or smooth muscle cells. Infection of tumor cells with pAd-T34A resulted in cytochrome c release from mitochondria, cleavage of approximately 46-kDa upstream caspase-9, processing of caspase-3 to the active subunits of approximately 17 and 19 kDa, and increased caspase-3 catalytic activity. When compared with chemotherapeutic regimens, pAd-T34A was as effective as taxol and considerably more effective than adriamycin in induction of tumor cell apoptosis and enhanced taxol-induced cell death. In three xenograft breast cancer models in immunodeficient mice, pAd-T34A suppressed de novo tumor formation, inhibited by approximately 40% the growth of established tumors, and reduced intraperitoneal tumor dissemination. Tumors injected with pAd-T34A exhibited loss of proliferating cells and massive apoptosis by in situ internucleosomal DNA fragmentation. These data suggest that adenoviral targeting of the survivin pathway may provide a novel approach for selective cancer gene therapy.

Cumulative and Irreversible Cardiac Mitochondrial Dysfunction Induced by Doxorubicin

Abstract: Interference with mitochondrial calcium regulation is proposed to be a primary causative event in the mechanism of doxorubicin-induced cardiotoxicity. We previously reported disruption of mitochondrial calcium homeostasis after chronic doxorubicin administration (Solen et al. Toxicol. Appl. Pharmacol, 129: 214-222, 1994). The present study was designed to characterize the dose-dependent and cumulative interference with mitochondrial calcium regulation and to assess the reversibility of this functional lesion. Sprague Dawley rats were treated with 2 mg/kg/week doxorubicin s.c. for 4-8 weeks. With succinate as substrate, cardiac mitochondria isolated from rats after 4 weeks of treatment with doxorubicin expressed a lower calcium loading capacity compared with control. This suppression of calcium loading capacity increased with successive doses to 8 weeks of treatment (P < 0.05) and persisted for 5 weeks after the last doxorubicin injection, and was corroborated by dose-dependent and irreversible histopathological changes. Preincubation of mitochondria with tamoxifen, DTT, or monobromobimane did not reverse the diminished calcium loading capacity caused by doxorubicin. In contrast, incubation with cyclosporin A abolished any discernible difference in mitochondrial calcium loading capacity between doxorubicin-treated and saline-treated rats. The decrease in cardiac mitochondrial calcium loading capacity was not attributable to bioenergetic changes in the electron transport chain, because the mitochondrial coupling efficiency was not altered by doxorubicin treatment. However, the ADP/ATP translocase content was significantly lower in mitochondria from rats that received 8 weeks of doxorubicin treatment. These data indicate that doxorubicin treatment in vivo causes a dose-dependent and irreversible decrease in mitochondrial calcium loading capacity. Suppression of adenine nucleotide translocase content may be a key factor altering the calcium-dependent regulation of the mitochondrial permeability transition pore, which may account for the cumulative and irreversible loss of myocardial function in patients receiving doxorubicin chemotherapy.

Randomized phase II trial of pegylated liposomal doxorubicin (DOXIL/CAELYX) versus doxorubicin in the treatment of advanced or metastatic soft tissue sarcoma : a study by the EORTC Soft Tissue and Bone Sarcoma Group

Abstract: CAELYX/DOXIL, pegylated liposomal doxorubicin, has shown antitumour activity and reduced toxicity compared with standard doxorubicin in other tumour types. In this prospective randomised trial, 94 eligible patients with advanced soft-tissue sarcoma (STS) were treated, 50 with CAELYX (50 mg/m(2) by a 1 h intravenous (i.v.) infusion every 4 weeks) and 44 with doxorubicin (75 mg/m(2) by an i.v. bolus every 3 weeks). Histological subtypes were evenly matched, 33% were leiomyosarcoma (CAELYX: 18; doxorubicin: 13). Primary disease sites were well matched. CAELYX was significantly less myelosuppressive, only 3 (6%) patients had grade 3 and 4 neutropenia, versus 33 (77%) on doxorubicin; febrile neutropenia occurred in 7 (16%) patients given doxorubicin, but only 1 (2%) given CAELYX. 37 (86%) patients on doxorubicin had grade 2-3 alopecia, but only 3 (6%) on CAELYX, and the major toxicity with CAELYX was to the skin. Palmar-plantar erythrodysesthesia with CAELYX was grade 1: 4 (8%) patients, grade 2: 11 (22%) patients, grade 3: 9 (18%) patients and grade 4: 1 (2%) patient. Other non-haematological grade 3 and 4 toxicities were rare. Confirmed responses were observed with both agents: CAELYX: complete response (CR) 1 (uterine), partial response (PR) 4 (response rate (RR) 10%); and doxorubicin: CR 1, PR 3 (RR of 9%); with the best response being stable disease (NC) in 16 and 18 patients, respectively. The reason for the low response rate is unknown, but it may be due partly to a high proportion of gastrointestinal stromal tumours. In conclusion, CAELYX has equivalent activity to doxorubicin in STS with an improved toxicity profile and should be considered for further investigation in combination with other agents such as ifosfamide.

Inhibition of NF-κB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin

Abstract: The transcription factor NF-kappaB has anti-apoptotic properties and may confer chemoresistance to cancer cells. Here, we describe human pancreatic carcinoma cell lines that differ in the responsiveness to the topoisomerase-2 inhibitors VP16 (20 microM) and doxorubicin (0.3 microM): Highly sensitive T3M4 [corrected] and PT45-P1 cells, and Capan-1 and A818-4 cells that were almost resistant to both anti cancer drugs. VP16, but not doxorubicin, transiently induced NF-kappaB activity in all cell lines, whereas basal NF-kappaB binding was nearly undetectable in T3M4 [corrected] and PT45-P1 cells, but rather high in Capan-1 and A818-4 cells, as demonstrated by gel-shift and luciferase assays. Treatment with various NF-kappaB inhibitors (Gliotoxin, MG132 and Sulfasalazine), or transfection with the IkappaBalpha super-repressor, strongly enhanced the apoptotic effects of VP16 or doxorubicin on resistant Capan-1 and 818-4 cells. Our results indicate that under certain conditions the resistance of pancreatic carcinoma cells to chemotherapy is due to their constitutive NF-kappaB activity rather than the transient induction of NF-kappaB by some anti-cancer drugs. Blockade of basal NF-kappaB activity by well established drugs efficiently reduces chemoresistance of pancreatic cancer cells and offers the potential for improved therapeutic strategies.

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

Abstract: 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

Modulation of Hsp90 function by ansamycins sensitizes breast cancer cells to chemotherapy-induced apoptosis in an RB- and schedule-dependent manner. See: E. A. Sausville, Combining cytotoxics and 17-allylamino, 17-demethoxygeldanamycin: sequence and tumor biology matters, Clin. Cancer Res., 7: 2155-2158, 2001.

Abstract: 17-allyl-aminogeldanamycin (17-AAG) is an ansamycin antibiotic that binds to a highly conserved pocket in the Hsp90 chaperone protein and inhibits its function. Hsp90 is required for the refolding of proteins during cellular stress and the conformational maturation of certain signaling proteins. 17-AAG has antitumor activity in cell culture and animal xenograft models and is currently in clinical trial. It causes an RB-dependent G 1 arrest, differentiation, and apoptosis. RB-negative cells arrest in mitosis and undergo apoptosis. Hsp90 plays an important role in the cellular response to environmental stress. Therefore, we tested whether the regulation of Hsp90 function by 17-AAG could sensitize cells to cytotoxic agents. 17-AAG sensitized tumor cells to Taxol and doxorubicin. Taxanes cause growth arrest in mitosis and apoptosis. The addition of 17-AAG to cells after exposure to Taxol significantly increased both the activation of caspases 9 and 3 and apoptosis. In cells with intact RB, exposure to 17-AAG before Taxol resulted in G 1 arrest and abrogated apoptosis. Schedule dependence was not seen in cells with mutated RB, because both agents blocked cells in mitosis. Schedule- or RB-dependence was also not observed when cells were treated with 17-AAG and doxorubicin, a DNA-intercalating agent that acts on different phases of the cell cycle. These findings suggest that inhibition of Hsp90 function by 17-AAG enhances the apoptotic effects of cytotoxic agents. The sequence of drug administration and the RB status significantly influence efficacy.

Enhanced delivery of doxorubicin into the brain via a peptide-vector-mediated strategy: saturation kinetics and specificity.

Abstract: Doxorubicin delivery to the brain is often restricted because of the poor transport of this therapeutic molecule through the blood-brain barrier (BBB). To overcome this problem, we have recently developed a technology, Pep:trans, based on short natural-derived peptides that are able to cross efficiently the BBB without compromising its integrity. In this study, we have used the in situ mouse brain perfusion method to evaluate the brain uptake of free and vectorized doxorubicin. Doxorubicin was coupled covalently to small peptide vectors: L-SynB1 (18 amino acids), L-SynB3 (10 amino acids), and its enantio form D-SynB3. We first confirmed the very low brain uptake of free radiolabeled doxorubicin, which is most likely due to the efflux activity of the P-glycoprotein at the level of the BBB. Vectorization with either L-SynB1, L-SynB3, or D-SynB3 significantly increased the brain uptake of doxorubicin (about 30-fold). We also investigated the mechanism of transport of vectorized doxorubicin. We show that vectorized doxorubicin uses a saturable transport mechanism to cross the BBB. The effect of poly(L-lysine) and protamine, endocytosis inhibitors, on the transport across the brain was also investigated. Both inhibitors reduced the brain uptake of vectorized doxorubicin in a dose-dependent manner. These studies indicate that the transport of vectorized doxorubicin appears to occur via an adsorptive-mediated endocytosis.

D-24851, a novel synthetic microtubule inhibitor, exerts curative antitumoral activity in vivo, shows efficacy toward multidrug-resistant tumor cells, and lacks neurotoxicity.

Abstract: N-(pyridin-4-yl)-[1-(4-chlorbenzyl)-indol-3-yl]-glyoxyl-amid (D-24851) is a novel synthetic compound that was identified in a cell-based screening assay to discover cytotoxic drugs. D-24851 destabilizes microtubules and blocks cell cycle transition specifically at G2-M phase. The binding site of D-24851 does not overlap with the tubulin binding sites of known microtubule-destabilizing agents like vincristine or colchicine. In vitro, D-24851 has potent cytotoxic activity toward a panel of established human tumor cell lines including SKOV3 ovarian cancer, U87 glioblastoma, and ASPC-1 pancreatic cancer cells. In vivo, oral D-24851 treatment induced complete tumor regressions (cures) in rats bearing Yoshida AH13 sarcomas. Of importance is that the administration of curative doses of D-24851 to the animals revealed no systemic toxicity in terms of body weight loss and neurotoxicity in contrast to the administration of paclitaxel or vincristine. Interestingly, multidrug-resistant cell lines generated by vincristine-driven selection or transfection with the Mr 170,000 P-glycoprotein encoding cDNA were rendered resistant toward paclitaxel, vincristine, or doxorubicin but not towards D-24851 when compared with the parental cells. Because of its synthetic nature, its oral applicability, its potent in vitro and in vivo antitumoral activity, its efficacy against multidrug-resistant tumors, and the lack of neurotoxicity, D-24851 may have significant potential for the treatment of various malignancies.

Inhibition of cyclooxygenase-2 aggravates doxorubicin-mediated cardiac injury in vivo

Abstract: The clinical use of doxorubicin, an anthracycline chemotherapeutic agent, is limited by cardiotoxicity, particularly when combined with herceptin, an antibody that blocks the HER2 receptor. Doxorubicin induces cyclooxygenase–2 (COX-2) activity in rat neonatal cardiomyocytes. This expression of COX-2 limits doxorubicin-induced cardiac cell injury, raising the possibility that the administration of a prostaglandin may protect the heart during the in vivo administration of doxorubicin. Doxorubicin (15 mg/kg) administered to adult male Sprague Dawley rats induced COX-2 expression and activity in cardiac tissue. Prostacyclin generation measured as the excretion of 2,3-dinor-6-keto-PGF1α also increased, and this was blocked by a COX-2 inhibitor, SC236. In contrast, administration of a COX-1 inhibitor SC560 at a dose that reduced serum thromboxane B2 by more than 80% did not prevent the doxorubicin-induced increase in prostacyclin generation. Doxorubicin increased cardiac injury, detected as a rise in plasma cardiac troponin T, serum lactate dehydrogenase, and cardiomyocyte apoptosis; this was aggravated by coadministration of SC236 but not SC560. The degree of injury in animals treated with a combination of doxorubicin and SC236 was attenuated by prior administration of the prostacyclin analogue iloprost. These data raise the possibility of protecting the heart during the administration of doxorubicin by prior administration of prostacyclin.

Anthracyclines: selected new developments.

Abstract: Anthracycline antibiotics play an important role in cancer chemotherapy. The need for an improvement of their therapeutic index has stimulated an ongoing search for anthracycline analogues with improved properties. Analogue development was originally limited by a lack of information on the cellular drug target, nevertheless almost 20 years ago the mechanism of action of doxorubicin and daunorubicin was revealed and DNA topoisomerase II was recognised to be their main cellular target. Several anthracyclines interfere with topoisomerase II functions by stabilizing a reaction intermediate in which DNA strands are cut and covalently linked to tyrosine residues of the enzyme. Investigations on the sequence specificity of doxorubicin in vitro and in nuclear chromatin of living cell have led to a molecular model of drug receptor on the topoisomerase II-DNA complex. Anthracyclines are likely placed at the interface between the DNA cleavage site and the active site of the enzyme, forming a DNA-drug-enzyme ternary complex. Moreover, a quite detailed structure-function relationship has been established for anthracyclines. First, drug intercalation is necessary but not sufficient for topoisomerase II poisoning; second, the removal of the 4-methoxy and 3'-amino substituents greatly increases the drug activity and third, the 3' substituent of the sugar moiety markedly influences the sequence selectivity of anthracycline-stimulated DNA cleavage. These relationships have been exploited during the last decade by several groups, including ours, in the search for new anthracycline drugs with lower side effects and higher activity against resistant cancer cells. This review will focus on areas of the anthracycline field including synthesis of new analogues, new strategies of synthesis and recent developments in the area of drug delivery.

Inhibition of Insulin-like Growth Factor I Receptor Increases the Antitumor Activity of Doxorubicin and Vincristine against Ewing’s Sarcoma Cells

Abstract: Innovative treatment modalities are needed for Ewing's sarcoma (ES), a neoplasm with a disappointingly low survival rate despite the use of aggressive multimodal therapeutic approaches. We and others (D. Yee et al., J. Clin. Investig., 86: 1806-1814, 1990; K. Scotlandi et al., Cancer Res., 56: 4570-4574, 1996) have previously shown the existence and the pathogenetic relevance of an autocrine loop, mediated by the insulin-like growth factor-I receptor (IGF-IR), which is crucial for survival and proliferation of ES cells in vitro. Moreover, we reported that the IGF-IR-blocking monoclonal antibody (MAb), alphaIR3, as well as suramin, a drug that can interfere with growth factor by binding to the receptors, inhibited both the tumorigenic and the metastatic ability of ES cells in athymic mice. In this study, we analyzed whether agents that can block the IGF-IR-mediated loop are of value in association with conventional cytotoxic drugs for the design of more effective therapeutic regimens. Both alphaIR3 MAb and suramin treatment significantly increased the antitumor in vitro effects of doxorubicin and vincristine, two drugs with a leader action on ES. These findings were obtained by both simultaneous and sequential treatments. Analysis of the proliferation rate and of apoptosis revealed that alphaIR3 MAb and suramin significantly enhanced the G(1)-phase rate induced by doxorubicin, without substantially affecting doxorubicin-G(2)-M-blockage of cell cycle, and significantly increased the induction of apoptosis, which confirmed that the specific blockage of IGF-IR deprives ES cells of an important tool for the prevention of drug-induced apoptosis. Moreover, combination treatments of doxorubicin plus alphaIR3 MAb significantly increase the doxorubicin-induced impairment of the ability of ES cells to form colonies in soft agar. In conclusion, we showed that, in ES, the blockage of IGF-IR by a neutralizing MAb or by suramin may greatly potentiate the antitumor activity of conventional chemotherapeutic drugs.

Doxorubicin-peptide conjugates overcome multidrug resistance

Abstract: A well-known mechanism leading to the emergence of multidrug-resistant tumor cells is the overexpression of P-glycoprotein (P-gp), which is capable of lowering intracellular drug concentrations. To overcome this problem, we tested the capability of two peptide vectors that are able to cross cellular membranes to deliver doxorubicin in P-gp-expressing cells. The antitumor effect of peptide-conjugated doxorubicin was tested in human erythroleukemic (K562/ ADR) resistant cells. The conjugate showed potent dose-dependent inhibition of cell growth against K562/ADR cells as compared with doxorubicin alone. Doxorubicin exhibited IC50 concentrations of 65 microM in the resistant cells, whereas vectorized doxorubicin was more effective with IC50 concentrations of 3 microM. After treatment of the resistant cells with verapamil, the intracellular levels of doxorubicin were markedly increased and consequent cytotoxicity was improved. In contrast, treatment of resistant cells with verapamil did not cause any further enhancement in the cell uptake nor in the cytotoxic effect of the conjugated doxorubicin, indicating that the conjugate bypasses the P-gp. Finally, we show by the in situ brain perfusion method in P-gp-deficient and competent mice that vectorized doxorubicin bypasses the P-gp present at the luminal site of the blood-brain barrier. These results indicate that vectorization of doxorubicin with peptide vectors is effective in overcoming multidrug resistance.

Reversal of multidrug resistance by the P-glycoprotein modulator, LY335979, from the bench to the clinic.

Abstract: Multidrug resistance may be conferred by P-glycoprotein (Pgp, ABCB1) or the multidrug resistance associated protein (MRP). These membrane proteins are members of the ATP binding cassette transporter superfamily and are responsible for the removal from the cell of several anticancer agents including doxorubicin. Modulators can inhibit these transporters. LY335979 is among the most potent modulators of Pgp with a Ki of 59 nM. LY335979 is selective for Pgp, and does not modulate MRP-mediated resistance by MRP1 (ABCC1) and MRP2 (ABCC2). LY335979 significantly enhanced the survival of mice implanted with Pgp-expressing murine leukemia (P388/ADR) when administered in combination with either daunorubicin, doxorubicin or etoposide. Coadministration of LY335979 with paclitaxel compared to paclitaxel alone significantly reduced the tumor mass of the Pgp-expressing UCLA-P3.003VLB lung carcinoma in a xenograph model and delayed the development of tumors in mice implanted with the parental drug-sensitive UCLA-P3 tumor. LY335979 was without significant effect on the pharmacokinetics of these anticancer agents. This may be due impart to its poor inhibition of four major cytochrome P450 isozymes important in metabolizing doxorubicin and other oncolytics. The selectivity and potency of this modulator allows the clinical evaluation of the role of Pgp in multidrug resistance. LY335979 is currently in clinical trials.

Sphingosine generation, cytochrome c release, and activation of caspase-7 in doxorubicin-induced apoptosis of MCF7 breast adenocarcinoma cells.

Abstract: Treatment of human breast carcinoma MCF7 cells with doxorubicin, one of the most active antineoplastic agents used in clinical oncology, induces apoptosis and leads to increases in sphingosine levels. The transient generation of this sphingolipid mediator preceded cytochrome c release from the mitochondria and activation of the executioner caspase-7 in MCF7 cells which do not express caspase-3. Bcl-x(L) overexpression did not affect sphingosine generation whereas it reduced apoptosis triggered by doxorubicin and completely blocked apoptosis triggered by sphingosine. Exogenous sphingosine-induced apoptosis was also accompanied by cytochrome c release and activation of caspase-7 in a Bcl-x(L)-sensitive manner. Furthermore, neither doxorubicin nor sphingosine treatment affected expression of Fas ligand or induced activation of the apical caspase-8, indicating a Fas/Fas ligand-independent mechanism. Our results suggest that a further metabolite of ceramide, sphingosine, may also be involved in mitochondria-mediated apoptotic signaling induced by doxorubicin in human breast cancer cells.

Connexin 43 (cx43) enhances chemotherapy-induced apoptosis in human glioblastoma cells.

Abstract: Stable re-expression of connexin 43 (cx43) in human glioblastoma suppresses transformation and tumorigenicity. The present study was designed to examine the role of cx43 in chemotherapy-induced apoptosis. Expression of cx43 in human glioblastoma cells significantly increased sensitivity to several common chemotherapeutic agents, including etoposide, paclitaxel (Taxol) and doxorubicin, compared with control-transfected cells. The increased sensitivity to chemotherapeutic agents resulted from apoptosis as evidenced by Hoechst dye staining, TUNEL assay and annexin V assay. These cx43-mediated effects were coupled with decreased expression of the specific apoptosis inhibitor bcl-2. Over-expression of bcl-2 in cx43-transfected cells partially confers the resistance to apoptosis induced by etoposide, suggesting that the cx43-mediated apoptosis to chemotherapeutic agents is regulated in part through the down-regulation of bcl-2 expression. Furthermore, the cx43-mediated apoptosis in response to chemotherapeutic drugs may not be linked to increased gap junctional communication in cx43-transfected cells. Our results demonstrate a new role of cx43 in the mediation of apoptosis during chemotherapy. © 2001 Wiley-Liss, Inc.

Novel Peptide Conjugates for Tumor-Specific Chemotherapy§

Abstract: One of the major problems in cancer chemotherapy are the severe side effects that limit the dose of the anticancer drugs because of their unselectivity for tumor versus normal cells. In the present work, we show that coupling of anthracyclines to peptides is a promising approach to obtain selectivity. The peptide−drug conjugate was designed to bind to specific receptors expressed on the tumor cells with subsequent internalization of the ligand−receptor complex. Neuropeptide Y (NPY), a 36-amino acid peptide of the pancreatic polypeptide family, was chosen as model peptide because NPY receptors are overexpressed in a number of neuroblastoma tumors and the thereof derived cell lines. Daunorubicin and doxorubicin, two widely used antineoplastic agents in tumor therapy, were covalently linked to NPY via two spacers that differ in stability: an acid-sensitive hydrazone bond at the 13-keto position of daunorubicin and a stable amide bond at the 3‘-amino position of daunorubicin and doxorubicin. Receptor binding...

Sequence-dependent Enhancement of Cytotoxicity Produced by Ecteinascidin 743 (ET-743) with Doxorubicin or Paclitaxel in Soft Tissue Sarcoma Cells

Abstract: Ecteinascidin 743 (ET-743) is a potent antitumor agent from the Caribbean tunicate Ecteinascidin turbinata and is presently in clinical trials for human cancers. To better understand how ET-743 might be used clinically, the present study used SRB assays to examine the cytotoxicity resulting from combining ET-743 with three other antineoplastic agents: doxorubicin (DXR), trimetrexate, and paclitaxel in different administration schedules in two soft tissue sarcoma cell lines, HT-1080 and HS-18, in vitro. Concurrent exposure of ET-743 with DXR resulted in synergistic interactions in both cell lines. Addition of ET-743 for 24 h before DXR was the most effective cytotoxic regimen against both cell lines. Morphological study by fluorescence microscopy revealed that combination treatment of both cells with ET-743 and DXR induced apoptosis. Exposure to paclitaxel before ET-743 was also an effective regimen. These results encourage studies of the combination of ET-743 and DXR in the treatment of soft tissue sarcoma, because each of these agents have activity in this disease.

The synthesis of a prodrug of doxorubicin designed to provide reduced systemic toxicity and greater target efficacy.

Abstract: Doxorubicin (Dox) can provide some stabilization in prostate cancer; however, its use is limited because of systemic toxicities, primarily cardiotoxicity and immunosuppression. The administration of a prodrug of doxorubicin, designed to permit selective activation by the tumor, would reduce general systemic exposure to the active drug and would thereby increase the therapeutic index. Prostate specific antigen (PSA) is a serine protease with chymotrypsin-like activity that is a member of the kallikrein gene family. PSA's putative physiological role is the liquefaction of semen by virtue of its ability to cleave the seminal fluid proteins semenogelins I and II. Serum PSA levels have been found to correlate well with the number of malignant prostate cells. The use of a prodrug which is cleaved by the enzyme PSA in the prostate should in principle produce high localized concentrations of the cytotoxic agent at the tumor site while limiting systemic exposure to the active drug. Cleavage maps following PSA treatment of human semenogelin were constructed. Systematic modification of the amino acid residues flanking the primary cleavage site led to the synthesis of a series of short peptides which were efficiently hydrolyzed by PSA. Subsequent coupling of selected peptides to doxorubicin provided a series of doxorubicin-peptide conjugates which were evaluated in vitro and in vivo as targeted prodrugs for PSA-secreting tumor cells. From these studies we selected Glutaryl-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-Dox, 27, as the peptide-doxorubicin conjugate with the best profile of physical and biological properties. Compound 27 has a greater than 20-fold selectivity against human prostate PSA-secreting LNCaP cells relative to the non-PSA-secreting DuPRO cell line. In nude mouse xenograft studies, 27 reduced PSA levels by 95% and tumor weight by 87% at a dose below its MTD. Both doxorubicin and Leu-Dox (13) were ineffective in reducing circulating PSA and tumor burden at their maximum tolerated doses. On the basis of these results, we selected 27 for further study to assess its ability to inhibit human prostate cancer cell growth and tumorigenesis.

In vitro induction of apoptosis of neoplastic cells in low-grade non-Hodgkin's lymphomas using combinations of established cytotoxic drugs with bendamustine.

Abstract: BACKGROUND AND OBJECTIVES. Regulation of apoptotic cell death is being increasingly recognized as a mechanisms by which cytostatic agents mediate tumor cell death. Preliminary clinical studies with bendamustine, an alkylating agent with a purine nucleus, provide strong evidence that this drug is a highly effective cytostatic in low grade lymphomas. Therefore, we investigated the in vitro activity of bendamustine in combination with other established cytotoxic drugs. DESIGN AND METHODS. 2 lines (DOHH-2, WSU-NHL) and mononuclear cells (MNC) from patients with leukemic low-grade B-non-Hodgkin's lymphoma (NHL) (n=10), T-NHL (n=7) and chronic lymphocytic leukemia (CLL) (n=12). Apoptosis (7-AAD), depolarization of mitochondrial membrane potential (MMP, JC-1), caspase-3-activity (FIENA) and cell proliferation (XTT/WST-1) were determined. Several incubation times and drug dosages (for IC(30/50/75/90)) were studied. Synergistic, additive or antagonistic effects were calculated by a median plot effect and the combination index (CI) method. RESULTS. In general, combinations of bendamustine with mitoxantrone or doxorubicin resulted in antagonistic effects in the tested cell lines and the MNC from the patients. CI-calculation failed in these cases since there was not a sufficient dose response. On the other hand, the combination of bendamustine with 2-CdA showed synergistic in vitro activity on the tested cell lines, neoplastic lymphocytes from patients with peripheral T-cell lymphomas and partially on MNC from patients with CLL and B-NHL. The antagonism of the combination of bendamustine and anthracyclines appeared to be due to inhibition of depolarization of mitochondrial-membrane potential and caspase-3-activity during apoptosis of the studied cell lines. INTERPRETATION AND CONCLUSIONS. In conclusion, our results suggest that schedules using combinations of bendamustine and anthracyclines should not be recommended for the treatment of low-grade NHL, whereas bendamustine combined with 2-CdA could be considered for the development of future treatment strategies.