Medicarpin and millepurpan, two flavonoids isolated from Medicago sativa, induce apoptosis and overcome multidrug resistance in leukemia P388 cells.

BACKGROUND: High consumption of flavonoids has been associated with a decrease risk of cancer. Alfalfa (Medicago sativa) leaves have been widely used in traditional medicine and is currently used as a dietary supplement because of their high nutrient content. We previously reported the cytotoxic activity of alfalfa leaf extracts against several sensitive and multidrug resistant tumor cell lines. HYPOTHESIS/PURPOSE: We aimed to determine whether medicarpin and millepurpan, two isoflavonoids isolated from alfalfa leaves, may have pro-apoptotic effects against drug-sensitive (P388) and multidrug resistant P388 leukemia cells (P388/DOX). STUDY DESIGN/METHODS: Cells were incubated with medicarpin or millepurpan for the appropriate time. Cell viability was assessed by the MTT assay. DNA fragmentation was analyzed by agarose gel electrophoresis. Cell cycle analysis was realized by flow cytometry technics. Caspases 3 and 9 activities were measured using Promega caspACE assay kits. Proteins and genes expression were visualized respectively by western-blot using specific antibodies and RT-PCR assay. RESULTS: P-glycoprotein-expressing P388/DOX cells did not show resistance to medicarpin (IC50 ≈ 90 µM for P388 and P388/DOX cells) and millepurpan (IC50 = 54 µM and 69 µM for P388 and P388/DOX cells, respectively). Treatment with medicarpin or millepurpan triggered apoptosis in sensitive as well as multidrug resistant P388 cells. These effects were mediated through the mitochondrial pathway by modifying the balance pro/anti-apoptotic proteins. While 3 µM doxorubicin alone could not induce cell death in P388/DOX cells, concomitant treatment with doxorubicin and subtoxic concentration of medicarpin or millepurpan restored the pro-apoptotic cascade. Each compound increased sensitivity of P388/DOX cells to doxorubicin whereas they had no effect in sensitive P388 cells. Vinblastine cytotoxicity was also enhanced in P388/DOX cells (IC50 = 210 nM to 23 and 25 nM with medicarpin and millepurpan, respectively). This improved sensitivity was mediated by an increased uptake of doxorubicin in P388/DOX cells expressing P-gp. P-gp expression was not altered by exposure to medicarpin and millepurpan. CONCLUSION: These data indicate that medicarpin and millepurpan possess pro-apoptotic properties and potentiate the cytotoxicity of chemotherapy drugs in multidrug resistant P388 leukemia cells by modulating P-gp-mediated efflux of drugs. These flavonoids may be used as chemopreventive agents or as sensitizer to enhance cytotoxicity of chemotherapy drugs in multidrug resistant cancer cells.

Effect of doxorubicin/pluronic SP1049C on tumorigenicity, aggressiveness, DNA methylation and stem cell markers in murine leukemia.

PURPOSE: Pluronic block copolymers are potent sensitizers of multidrug resistant cancers. SP1049C, a Pluronic-based micellar formulation of doxorubicin (Dox) has completed Phase II clinical trial and demonstrated safety and efficacy in patients with advanced adenocarcinoma of the esophagus and gastroesophageal junction. This study elucidates the ability of SP1049C to deplete cancer stem cells (CSC) and decrease tumorigenicity of cancer cells in vivo. EXPERIMENTAL DESIGN: P388 murine leukemia ascitic tumor was grown in BDF1 mice. The animals were treated with: (a) saline, (b) Pluronics alone, (c) Dox or (d) SP1049C. The ascitic cancer cells were isolated at different passages and examined for 1) in vitro colony formation potential, 2) in vivo tumorigenicity and aggressiveness, 3) development of drug resistance and Wnt signaling activation 4) global DNA methylation profiles, and 5) expression of CSC markers. RESULTS: SP1049C treatment reduced tumor aggressiveness, in vivo tumor formation frequency and in vitro clonogenic potential of the ascitic cells compared to drug, saline and polymer controls. SP1049C also prevented overexpression of BCRP and activation of Wnt-ß-catenin signaling observed with Dox alone. Moreover, SP1049C significantly altered the DNA methylation profiles of the cells. Finally, SP1049C decreased CD133(+) P388 cells populations, which displayed CSC-like properties and were more tumorigenic compared to CD133(-) cells. CONCLUSIONS: SP1049C therapy effectively suppresses the tumorigenicity and aggressiveness of P388 cells in a mouse model. This may be due to enhanced activity of SP1049C against CSC and/or altered epigenetic regulation restricting appearance of malignant cancer cell phenotype.

The anticancer effect of cytotoxin 1 from Naja atra Cantor venom is mediated by a lysosomal cell death pathway involving lysosomal membrane permeabilization and cathepsin B release.

The cytotoxin family of cobra venom proteins, also called cardiotoxins, can activate both necrotic and apoptotic cell death pathways in cancer cells. Cytotoxin 1 (CTX1)from Naja atra Cantor venom is a 60 amino acid, 6698 Da protein with as yet untested anticancer efficacy and cell selectivity. We tested the toxicity of CTX1 on a number of cancer cell lines (MCF-7, P388, K562, and H22) and on one normal human cell line (16HBE). The rank order of cytotoxicity was MCF-7 > P388 ≈ K562 >H22 ≈ 16HBE, indicating that the effect of CTX1 on certain cancer cell types was relatively selective.Treatment with CTX1 greatly prolonged the survival of P388 ascites tumors bearing KM mice compared to cyclophosphamide treatment. Cell viability, apoptosis, and lysosomal permeability assays all demonstrated that CTX1 induced dose- and time-dependent cell death, with most cells exhibiting the morphological and biochemical features of late apoptosis and necrosis. Mitochondrial membrane potential was lost in CTX1-treated P388 cells. In addition, CTX1 induced an increase in both lysosomal membrane permeability and cathepsin B protease activity. These analyses reveal that CTX1 possesses significant and selective anticancer activity, likely by inducing programmed cell death through mitochondrial and/or lysosomal pathways.

Crystal structure of SsfS6, the putative C-glycosyltransferase involved in SF2575 biosynthesis.

The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C-glycosyltransferase (C-GT) that forms the C-C glycosidic bond between the sugar and the SF2575 tetracycline-like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT-B fold wherein the TDP and docked putative aglycon are consistent with the overall C-glycosylation reaction. As one of only a few existing structures for C-glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C-glycosylation.

Extracellular ATP induces CD44 shedding from macrophage-like P388D1 cells via the P2X7 receptor.

The P2X7 receptor (P2X7R) is a nucleotide receptor expressed predominantly on hemopoietic, bone, and epithelial cells. The P2X7R can be activated by extracellular ATP and induces the influx of calcium, releases cytokines, and participates in cell proliferation and apoptosis. CD44 is an adhesion molecule. The effects of CD44 include cell-cell and cell-matrix adhesion interactions, lymphocyte activation, and cell migration. Many studies have shown that P2X7R and CD44 play important roles in hematological malignancies, but no study exists regarding the relationship between P2X7R and CD44. In the present study, we characterized P388D1 cells for the surface expression of CD44 and analyzed ATP-induced shedding. The data showed that P388D1 cells express CD44. Incubation of P388D1 cells with ATP induced a rapid loss of CD44 from the P388D1 cell surface. In addition, using a receptor inhibitor and P2X7R short hairpin RNA, we showed that the loss of CD44 is mediated via the P2X7R. Finally, we demonstrated that activation of P2X7R by ATP induces CD44 shedding.

Overexpression of apoptosis-associated speck-like protein in P388D1 murine lymphoma cells affects metastatic properties.

Apoptosis-associated speck-like protein (ASC) is a bipartite adaptor molecule that participates in inflammation and apoptosis. ASC silencing has been observed in a significant proportion of human cancers. Here, we examined the role of ASC overexpression in the metastasis of the P388D1 murine lymphoma cell line to the liver, lung, spleen and kidney. First, we determined that the P388D1 cells express ASC. Then, ASC overexpression in P388D1 was achieved by transfecting pEGFP-ASC-C2 into the P388D1 cells. Furthermore, after the ASC-overexpressing P388D1 cells were injected into DBA/2 mice through the vena caudalis, their metastasis to the lung and the liver was significantly reduced in the pEGFP-ASC-C2-transfected group. These data indicate that ASC overexpression affects the in vivo metastatic properties of P388D1 cells.

Interaction potential of the endothelin-A receptor antagonist atrasentan with drug transporters and drug-metabolising enzymes assessed in vitro.

PURPOSE: Atrasentan is a highly potent and selective endothelin receptor A (ET(A)) antagonist under development for the treatment of prostate cancer. Only little data exist on its interaction with drug-metabolising enzymes and drug transporters possibly influencing its safety and effectiveness. Our study evaluated whether atrasentan can induce the expression of relevant human drug transporters and cytochrome P450 isozymes (CYPs), whether it retains its efficiency in multidrug resistant cell lines, and whether it inhibits P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). METHODS: Induction of transporters and enzymes was quantified at the mRNA level by real-time RT-PCR in LS180 cells and for P-gp also at the protein level by Western blot. P-gp inhibition was evaluated by calcein assay in P388/dx and L-MDR1 cells and BCRP inhibition in MDCKII-BCRP cells by pheophorbide A efflux. Substrate characteristics were evaluated by growth inhibition assays in MDCKII cells overexpressing particular ABC-transporters. RESULTS: Atrasentan profoundly induced several CYPs and drug transporters (e.g. 12-fold induction of CYP3A4 at 50 µM). It was a moderate P-gp inhibitor (IC(50) in P388/dx cells = 15.1 ± 1.6 µM) and a weak BCRP inhibitor (IC(50) in MDCKII-BCRP cells = 59.8 ± 11 µM). BCRP or P-gp overexpressing cells were slightly more resistant towards antiproliferative effects of atrasentan. CONCLUSIONS: Our data provide a comprehensive analysis of the induction profile of atrasentan and its interaction with P-gp and BCRP. The profound induction effects stress the need for thorough assessment of its interaction potential in vivo.

On the use of fluorescence probes for detecting reactive oxygen and nitrogen species associated with photodynamic therapy.

Fluorescent probes are frequently employed for the detection of different reactive oxygen and nitrogen species formed during the irradiation of photosensitized cells and tissues. Investigators often interpret the results in terms of information provided with the different probes without examining specificity or determinants of fluorogenic reactions. We examine five fluorescent probes in a cell-free system: reduced 2',7'-dichlorofluorescein, dihydroethidine, dihydrorhodamine, 3'-(p aminophenyl) fluorescein (APF), and 4',5'-diaminofluorescein. Of these, only APF demonstrates a high degree of specificity for a single reactive species. There is a substantial influence of peroxidase activity on all fluorogenic interactions. The fluorescence of the photosensitizing agent also must be taken into account in evaluating results.

Enhancement of doxorubicin concentration in the M5076 ovarian sarcoma cells by cucurbitacin E co-treatment.

Cucurbitacin E increases the doxorubicin (DOX) level in M5076 ovarian sarcoma via suppressed DOX efflux in vitro. An increase in DOX induced antitumor activity by cucurbitacin E in vivo has been reported previously. This paper attempts to clarify the mechanism of cucurbitacin E induced increments in the antitumor activity of DOX. MK-571, a multidrug resistance associated protein (MRP) inhibitor, significantly suppressed DOX efflux from M5076 ovarian sarcoma cells. The combination of cucurbitacin E with MK-571 also inhibited DOX efflux, whereas the efficacy was the same in each treatment. Namely, the inhibition of DOX efflux by cucurbitacin E was expected to be related to MRP. In contrast, it appeared that the effect of cucurbitacin E on DOX permeability did not relate to P-gp. The cucurbitacin E co-treatment significantly increased DOX concentration in the tumor within a short time after DOX administration, whereas the same treatment decreased the DOX concentration in normal tissues. The different effects of cucurbitacin E between tumor and normal tissues was speculated to be related to differences in DOX transport system on cell membrane. In DOX therapy, cucurbitacin E co-treatment was expected to increase DOX induced antitumor activity without an increase in adverse reactions due to DOX.

Heparin inhibits P388D1 cells adherence and metastasis to peripheral lymph nodes in vitro and in vivo.

Heparin is known to attenuate tumor metastasis mainly by inhibiting the interaction between L-selectin and its ligand. However, the mechanism of heparin on lymphoma is unclear. This report demonstrates that both L-selectin/h IgG chimeric protein and heparin treatment can significantly inhibit the adhesion of P388D1 cells onto lymphatic sinusoids and marginal sinusoids in vitro, that heparin can attenuate P388D1 cell homing to lymph nodes in vivo at 12 hours, and that heparin significantly reduced P388D1 cells metastasis to lymph nodes 18 days after injection. These results indicate that heparin may act as a ligand for L-selectin on the P388D1 macrophage-like lymphoma cell line to attenuate tumor growth and metastasis.

Functional evaluation of multidrug resistance transporter activity in surgical samples of solid tumors.

Determination of multidrug resistance (MDR) activity of tumor cells could provide important information for the personalized therapy of cancer patients. The functional calcein assay (MultiDrug Quant Assay, Solvo Biotechnology, Budaörs, Hungary) has been proven to be clinically valuable in hematological malignancies by determining the transporter activity of MDR protein 1 (MDR1, ATP-binding cassette protein [ABC] B1, P-glycoprotein-170) and MDR-related protein 1 (MRP1, ABCC1). In this study, we evaluated if the same functional test was adaptable for the analysis of MDR activity in solid tumors. For this purpose, tissue specimens of human colorectal cancer samples were subjected to limited enzymatic digestion by collagenase to provide a single-cell suspension; dead cells were excluded by 7-aminoactinomycin D staining, and epithelial cancer cells were detected by Cy5-conjugated anti-BerEP4 monoclonal antibody. The transporter functions of MDR1 and MRP1 in viable epithelial cells were assessed by flow cytometry detecting the intracellular accumulation of calcein dye after exposing cells to various MDR inhibitors. Collagenase disintegration preserved the MDR activity and the antigenicity of tumor cells. Thus using the extended calcein assay provided sufficient viable and functionally active tumor cells from surgical biopsies to determine the functional MDR activity. In conclusion, the newly described modified calcein assay may be applicable for evaluating the MDR phenotype in solid tissue specimens from colorectal forceps biopsy to surgical samples.

Synthesis of ferulic ester dimers, functionalisation and biological evaluation as potential antiatherogenic and antiplasmodial agents.

Oxidative dimerization of ferulic acid methyl ester afforded dihydrobenzofuran derivative and new linear compound identified by X-ray crystallography. The gallate derivatized dihydrobenzofuran analogue was obtained and all compounds were evaluated for potential antiatherogenic, antiplasmodial (best IC(50)=0.8 microM) and cytotoxic activities.

Group V phospholipase A2-derived lysophosphatidylcholine mediates cyclooxygenase-2 induction in lipopolysaccharide-stimulated macrophages.

Activation of macrophages and macrophage cell lines by bacterial LPS elicits a delayed phase of PG biosynthesis that appears to be entirely mediated by cyclooxygenase-2 (COX-2). In previous work, we found that a catalytically active group V secreted phospholipase A(2) (sPLA(2)-V) was required for COX-2 induction, but the nature of the sPLA(2)-V metabolite involved was not defined. In this study, we identify lysophosphatidylcholine (lysoPC) as the sPLA(2)-V downstream mediator involved in COX-2 induction by LPS-stimulated macrophages. Inhibition of sPLA(2)-V by RNA interference or by the cell-permeable compound scalaradial blocked LPS-induced COX-2 expression, and this inhibition was overcome by incubating the cells with a nonhydrolyzable lysoPC analog, but not by arachidonic acid or oleic acid. Moreover, inhibition of sPLA(2)-V by scalaradial also prevented the activation of the transcription factor c-Rel, and such an inhibition was also selectively overcome by the lysoPC analog. Collectively, these results support a model whereby sPLA(2)-V hydrolysis of phospholipids upon LPS stimulation results in lysoPC generation, which in turn regulates COX-2 expression by a mechanism involving the transcriptional activity of c-Rel.

Mouse CD40-transfected cell lines cannot exhibit the binding and RANTES-stimulating activity of exogenous heat shock protein 70.

Here we demonstrate the inducible mouse Hsp72 binds markedly to lymphoid neoplastic macrophage-like P388D1 cells. To examine whether mouse CD40 can play a role in signaling exogenously administered HSP70 in a fashion similar to that of human CD40, we established mouse CD40-transfectants of both human 293 cells and murine-pro-B cell line Ba/F3. A small portion of mouse CD40 expressed on 293-derived transfectants was the mature form with a signal-transducible C-terminal domain, whereas a majority of expressed antigen showed the molecular size smaller than we expect. Flow cytometry showed that mouse Hsp72, but neither its deletion variants nor the related Escherichia coli DnaK, bound to the 293-derived transfectants regardless of CD40 expression. CD40 molecules expressed on the transfectants showed the binding of soluble form of CD40L but this binding was not inhibited by excess amount of HSP70. CD40L, but not any HSP70 recombinant proteins, stimulated the production of chemokine RANTES in the transfectants. Furthermore, no RANTES production was induced by HSP70-RCMLA complex in the transfectants, although it binds to 293-derived cells in a CD40-independent manner. No interaction between mouse CD40 and HSP70 recombinant proteins was detected by using the Ba/F3-derived transfectants that express the mature form of mouse CD40. The present results imply that mouse CD40 expressed on the transfectants differs from its human homolog in the binding of exogenously administered HSP70.

Isoquino[4,5-bc]acridines: design, synthesis and evaluation of DNA binding, anti-tumor and DNA photo-damaging ability.

Several novel isoquino[4,5-bc]acridine derivatives have been designed and synthesized. Their DNA-binding, anti-tumor and DNA-photo-damaging properties were investigated. A4 exhibited the highest anti-tumor activities against both A 549 (human lung cancer cell) and P388 (murine leukemia cells). All these compounds were found to be more cytotoxic against P388 than against A549. Under 365-nm light irradiation, A3 damaged plasmid DNA pBR322 at <2 microM and cleaved DNA from form I to 100% form II by 50 microM. The mechanism studies revealed that A3 damaged DNA by electron transfer mechanism and singlet oxygen species.

Binding with serum components favorably affects cellular uptake of 111In-oligonucleotide in a leukemia cell line.

PURPOSE: The influence of serum components on the intracellular uptake of an 111In-oligonucleotide (ODN) against mdr1 mRNA was investigated in the murine leukemia cell line, P388/S, and its mdr1-overexpressed P388/R. METHODS: 111In-ODNs naked and vectorized with lipids were analyzed for binding with serum components using high-performance liquid chromatography (HPLC). 111In-ODN was incubated in albumin and transferrin solutions. 111In-DTPA and 111In-mononucleotide were incubated in serum. Degradation of naked 111In-ODN was detected in phosphate buffered saline (PBS) and serum containing endonuclease S1. Cellular uptakes of naked and vectorized 111In-ODN in the above cells were examined with and without fetal calf serum (FCS). RESULTS: Time-dependent binding of naked and vectorized 111In- ODN with serum components was observed throughout 24 hours. Transchelation of 111In to transferrin was not detected. HPLC profiles of 111In-DTPA and 111In-mononucleotide did not change in serum. Degradation of 111In-ODN by S1 was less remarkable in serum than in PBS. Specific accumulation of vectorized 111In-ODN in P388/R cells was achieved in culture with and without 10% FCS. CONCLUSIONS: This study verified the intense binding of ODN with serum components, leading to no inhibition on ODN intracellular specific uptake. Binding with serum components protects 111In-ODN from degradation by endonuclease and thus may facilitate ODN transmembrane delivery.

Cycloimide bacteriochlorin p derivatives: photodynamic properties and cellular and tissue distribution.

Reactive oxygen species generated by photosensitizers are efficacious remedy for tumor eradication. Eleven cycloimide derivatives of bacteriochlorin p (CIBCs) with different N-substituents at the fused imide ring and various substituents replacing the 3-acetyl group were evaluated as photosensitizers with special emphasis on structure-activity relationships. The studied CIBCs absorb light within a tissue transparency window (780-830 nm) and possess high photostability at prolonged light irradiation. The most active derivatives are 300-fold more phototoxic toward HeLa and A549 cells than the clinically used photosensitizer Photogem due to the substituents that improve intracellular accumulation (distribution ratio of 8-13) and provide efficient photoinduced singlet oxygen generation (quantum yields of 0.54-0.57). The substituents predefine selective CIBC targeting to lipid droplets, Golgi apparatus, and lysosomes or provide mixed lipid droplets and Golgi apparatus localization in cancer cells. Lipid droplets and Golgi apparatus are critically sensitive to photoinduced damage. The average lethal dose of CIBC-generated singlet oxygen per volume unit of cell was estimated to be 0.22 mM. Confocal fluorescence analysis of tissue sections of tumor-bearing mice revealed the features of tissue distribution of selected CIBCs and, in particular, their ability to accumulate in tumor nodules and surrounding connective tissues. Considering the short-range action of singlet oxygen, these properties of CIBCs are prerequisite to efficient antitumor photodynamic therapy.

Transcriptional regulation of CD1D1 by Ets family transcription factors.

CD1 molecules are MHC class I-like glycoproteins specialized in presenting lipid/glycolipid Ags to T cells. The distinct cell-type specific expression of CD1D1 plays an important role in the development and function of NKT cells, a unique subset of immunoregulatory T cells. However, the mechanisms regulating CD1D1 expression are largely unknown. In this study, we have characterized the upstream region of the CD1D1 gene and identified a minimal promoter region within 200 bp from the translational start site of CD1D1 that exhibits cell-type specific promoter activity. Analysis of this region revealed an Ets binding site critical for CD1D1 promoter activity. Gel shift assays and chromatin immunoprecipitation experiments showed that Elf-1 and PU.1 bind to the CD1D1 promoter. Furthermore, we found that gene disruption of Elf-1 resulted in decreased CD1D1 expression on B cells but not other cell types, whereas conditional activation of PU.1 negatively regulated CD1D1 expression in PU.1-deficient myeloid cells. These findings are the first to demonstrate that Ets proteins are involved in the transcriptional regulation of CD1D1 and that they may function uniquely in different cell types.

Cellular recognition of paclitaxel-loaded polymeric nanoparticles composed of poly(gamma-benzyl L-glutamate) and poly(ethylene glycol) diblock copolymer endcapped with galactose moiety.

Poly(gamma-benzyl L-glutamate) (PBLG)/poly(ethylene glycol) (PEG) diblock copolymer endcapped with galactose moiety (abbreviated as GEG) was synthesized and characterized for study of liver-specific targeting. From dynamic light scattering measurement, particle sizes of copolymeric nanoparticles were decreased with an increase of PEG in the copolymer. The morphology of GEG-3 nanoparticles observed by transmission electron micrograph was observed as almost spherical shapes and ranged about 50-300 nm. From the structural characterization using 1H nuclear magnetic resonance, both characteristic peaks of PBLG and PEG were visible in CDCl3 but the characteristic peaks of PBLG were invisible in D2O, indicating that GEG block copolymers are found to the core-shell type nanoparticles in water with PBLG innercore and PEG outershell, exposing that galactose moiety of GEG block copolymers are outerwards oriented on the nanoparticle surfaces. By galactose-specific aggregation test of particles using beta-galactose specific lectin, and flow cytometry measurement, specific interaction between asialoglycoprotein receptors (ASGPR) of HepG2, human hepatoma cell line, and galactose moieties of the GEG nanoparticles was confirmed. From cell cytotoxicity test, HepG2 cells with ASGPR are more sensitive to paclitaxel (TX)-loaded nanoparticles than free TX whereas, P388 cells, murine leukemia cell line, and SK-Hep 01, human hepatoma cell line, without ASGPR is less sensitive to TX-loaded nanoparticles than free TX, suggesting that specific interaction between HepG2 cells and galactose moiety of the nanoparticles occurred.

Preclinical safety, pharmacokinetics and antitumor efficacy profile of liposome-entrapped SN-38 formulation.

BACKGROUND: SN-38, 7-ethyl-10-hydroxycamptothecin, is a biologically active metabolite of irinotecan. Its poor solubility restricted its development as an anticancer agent. We have developed an easy-to-use liposome-entrapped SN-38 (LE-SN38) and evaluated its toxicology, pharmacokinetics and antitumor efficacy profile. MATERIALS AND METHODS: Toxicity and pharmacokinetics studies were conducted in CD2F1 mice and beagle dogs. Therapeutic efficacy studies were performed in murine leukemia (P388 and P388/ADR) and in a human pancreatic (Capan-1) tumor models. RESULTS: Multiple dose administration (i.v. x 5) of LE-SN38 indicated a maximum tolerated dose (MTD) of 5.0 and 7.5 mg/kg/day for male and female mice, respectively. The MTD of LE-SN38 in dogs was 1.2 mg/kg. The elimination half-life (t1/2) of SN-38 in mouse plasma was 6.38 h with volume of distribution (VdSS) 2.55 L/kg. In dogs, t1/2 and VdSS were 1.38-6.42 h and 1.69-5.01 L/kg; respectively. P388 tumor-bearing mice dosed with LE-SN38 at 5.5 mg/kg (i.v. x 5) showed 100% survival. LE-SN38 at 4 or 8 mg/kg (i. v. x 5) inhibited 65% and 98% tumor growth, respectively, in a human pancreatic tumor model. CONCLUSION: LE-SN38 showed a favorable pharmacokinetics profile and can be administered safely at therapeutically effective doses.