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Stephan Herbertz,1 J Scott Sawyer,2 Anja J Stauber,2 Ivelina Gueorguieva,3 Kyla E Driscoll,4 Shawn T Estrem,2 Ann L Cleverly,3 Durisala Desaiah,2 Susan C Guba,2 Karim A Benhadji,2 Christopher A Slapak,2 Michael M Lahn21Lilly Deutschland GmbH, Bad Homburg, Germany; 2Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN, USA; 3Lilly Research Laboratories, Eli Lilly and Company, Windlesham, Surrey, UK; 4Lilly Research Laboratories, Eli Lilly and Company, New York, NY, USA Abstract: Transforming growth factor-beta (TGF-β) signaling regulates a wide range of biological processes. TGF-β plays an important role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. There are several pharmacological approaches to block TGF-β signaling, such as monoclonal antibodies, vaccines, antisense oligonucleotides, and small molecule inhibitors. Galunisertib (LY2157299 monohydrate) is an oral small molecule inhibitor of the TGF-β receptor I kinase that specifically downregulates the phosphorylation of SMAD2, abrogating activation of the canonical pathway. Furthermore, galunisertib has antitumor activity in tumor-bearing animal models such as breast, colon, lung cancers, and hepatocellular carcinoma. Continuous long-term exposure to galunisertib caused cardiac toxicities in animals requiring adoption of a pharmacokinetic/pharmacodynamic-based dosing strategy to allow further development. The use of such a pharmacokinetic/pharmacodynamic model defined a therapeutic window with an appropriate safety profile that enabled the clinical investigation of galunisertib. These efforts resulted in an intermittent dosing regimen (14 days on/14 days off, on a 28-day cycle) of galunisertib for all ongoing trials. Galunisertib is being investigated either as monotherapy or in combination with standard antitumor regimens (including nivolumab) in patients with cancer with high unmet medical needs such as glioblastoma, pancreatic cancer, and hepatocellular carcinoma. The present review summarizes the past and current experiences with different pharmacological treatments that enabled galunisertib to be investigated in patients. Keywords: TGF-β, TGF-βRI kinase inhibitor, ALK5, galunisertib, LY2157299, cancer, clinical trials

Purpose: This phase I study was carried out to determine the phase II recommended dose of tasisulam sodium (hereafter, tasisulam), a novel anticancer agent with a unique mechanism of action.Methods: Tasisulam was administered intravenously, every 21 days, in patients with refractory solid tumors using a three-plus-three dose-escalation schema.Results: Fifty-three patients were enrolled; the first 34 were treated with a flat dose of tasisulam of up to 2,400 mg, the dose level at which all three patients had dose-limiting toxicity (DLT). Controlling for C(max) proved important to reduce the risk of toxicity; therefore, we initially focused on identifying which parameters explained C(max) (end-of-infusion concentration) variability. Pharmacokinetic analysis indicated that C(max) negatively correlates with lean body weight (LBW). Thus, the dosing regimen was revised using a LBW-based algorithm targeting a specific C(max). A loading/chronic dose paradigm was then implemented as pharmacokinetic results revealed a long terminal half-life of tasisulam, likely because of its high-affinity albumin binding. C(max)-based dose escalation was stopped at the 420-μg/mL cohort, in which one of the 16 patients had DLT (transient hepatic transaminase elevation); grade 3/4 hematologic toxicity was noted in later cycles in three patients. Although response was not a primary objective, 33% of heavily pretreated patients with post-dose radiological assessments had stable disease.Conclusion: Implementation of a novel targeted C(max)-based dosing regimen allowed for the recommendation of a phase II tasisulam dose (loading dose of 420 μg/mL targeted C(max) with all subsequent doses administered at 65% of chronic dose given every 21 days) despite pharmacological challenges posed by high albumin binding. [ABSTRACT FROM AUTHOR]

Background: Hepcidin plays a central role in iron homeostasis and erythropoiesis. Neutralizing hepcidin with a monoclonal antibody (mAb) may prevent ferroportin internalization, restore iron efflux from cells, and allow transferrin-mediated iron transport to the bone marrow. This multicenter, phase 1 study evaluated the safety, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of a fully humanized mAb (LY2787106) with high affinity for hepcidin in cancer patients with anemia., Methods: Thirty-three patients with hepcidin levels ≥5 ng/mL received LY2787106 either every 3 weeks (19 patients, dose range 0.3-10 mg/kg) (part A) or weekly (14 patients, dose 10 mg/kg) (part B). LY2787106 PK/PD markers of iron and hematology biology were measured., Results: LY2787106 clearance (32 mL/h) and volume of distribution (7.7 L) were independent of dose and time, leading to a dose-proportional increase in concentration with dose. Consistent dose-dependent increases in serum iron, and transferrin saturation were seen at the 3 and 10 mg/kg dose levels, typically peaking within 24 h after LY2787106 administration and returning to baseline by day 8., Conclusions: Our findings indicate that LY2787106 was well tolerated in cancer patients with anemia and that targeting the hepcidin-ferroportin pathway by neutralizing hepcidin resulted in transient iron mobilization, thus supporting the role of hepcidin in iron regulation., Trial Registration: ClinicalTrial.gov, NCT01340976.

This phase 2 study assessed the safety and efficacy of ocaratuzumab, a humanized anti-CD20 monoclonal antibody. Fifty patients with previously treated follicular lymphoma (FL) and a low-affinity genotype of FcγRIIIa received ocaratuzumab 375 mg/m(2) weekly for 4 weeks. Grade 3/4/5 adverse events (AEs) were reported in 11/1/1 patients, respectively. Serious AEs were reported by 11/50 patients, and three discontinued due to AEs. One patient died from aspiration pneumonia due to possibly drug-related nausea and vomiting. Investigator-assessed response rate was 30% (15/50), including four complete responses (CR), three CR unconfirmed (CRu) and eight partial responses (PR). Investigator-assessed median Progression-free survivial (PFS) was 38.3 weeks. Ocaratuzumab's pharmacokinetic profile was similar to that reported for rituximab. Lymphocyte subset analysis showed significant, selective reduction of B-cells during and after ocaratuzumab treatment. Ocaratuzumab at this dose and schedule is active and well tolerated in patients with previously treated FL with low affinity FcγRIIIa genotypes. ClinTrials registry number: NCT00354926.

Purpose: LY2334737 is an oral gemcitabine prodrug. This Phase I study assessed the safety and tolerability of LY2334737 in Japanese patients with solid tumors and evaluated pharmacokinetics (PK), pharmacodynamics, and antitumor activity., Methods: Patients with advanced/metastatic solid tumors received escalating doses of LY2334737 once daily for 14 days, followed by a 7-day drug-free period. Cycles were repeated until discontinuation criteria were met., Results: Of 13 patients treated, 3 received 20 mg/day, 6 received 30 mg/day, 4 received 40 mg/day. On the 40 mg dose, 3 patients experienced dose-limiting toxicities (DLTs): hepatic toxicities (e.g., Grade [G]3/4 transaminase and G1-3 bilirubin elevation) and G4 thrombocytopenia; all 3 showed features of disseminated intravascular coagulation. One additional DLT occurred on the 30 mg dose (G3 transaminase elevation). Exploratory pharmacogenetic analyses identified a genetic variation in the CES2 gene potentially associated with these DLTs. PK data showed no clear relationship between the AUC of gemcitabine and its incorporation into leukocyte DNA; 2 of the 3 DLT patients had high incorporation. Two patients (30 mg/day) achieved stable disease with progression-free survival lasting 135 and 155 days., Conclusions: LY2334737 was tolerated by Japanese patients up to 30 mg/day. The toxicities observed at the 40 mg dose may require the development of alternative dosing schedules.

Purpose: AME-133v is a humanized monoclonal antibody engineered to have increased affinity to CD20 and mediate antibody-dependent cell-mediated cytotoxicity (ADCC) better than rituximab. Safety, pharmacokinetics, and efficacy were assessed in a phase 1/2 trial in patients with previously treated follicular lymphoma (FL)., Patients and Methods: AME-133v was characterized in vitro by ADCC and cell binding assays. A phase 1 study was conducted in which 23 previously treated patients with FL were assigned sequentially to one of five dose-escalation cohorts of AME-133v at 2, 7.5, 30, 100, or 375 mg/m(2) weekly × 4 doses., Results: AME-133v showed a 13- to 20-fold greater binding affinity for CD20 and was 5- to 7-fold more potent than rituximab in ADCC assays. Cell binding assays showed AME-133v and rituximab competed for an overlapping epitope on the CD20 antigen, and AME-133v inhibited binding of biotinylated rituximab to CD20 in a concentration-dependent manner. AME-133v was well tolerated by patients and common related adverse events included chills and fatigue. One patient experienced a dose-limiting toxicity of neutropenia. AME-133v showed nonlinear pharmocokinetics with properties similar to rituximab. Selective reduction of B cells during and after AME-133v treatment was shown by flow cytometry of peripheral blood. A partial or complete response was observed in 5 of 23 (22%) patients and the median progression-free survival was 25.4 weeks., Conclusions: AME-133v was safe and well tolerated at the doses tested. AME-133v showed encouraging results as an anti-CD20 therapy in heavily pretreated FL patients with the less favorable FcγRIIIa F-carrier genotype.

Purpose: LY2334737 is an orally available prodrug of gemcitabine. The objective of this study was to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) of daily administration of LY2334737 with or without erlotinib., Experimental Design: Patients with advanced or metastatic cancer were treated with escalating doses of LY2334737 monotherapy or in combination with continuous daily administration of 100 mg erlotinib. LY2334737 was given once daily for 14 days of a 21-day cycle. The study was extended with a bioequivalence trial to investigate a novel LY2334737 drug formulation., Results: A total of 65 patients were treated in this study. The MTD was 40 mg LY2334737. Fatigue was the most frequent DLT for LY2334737 monotherapy (4 patients) followed by elevated transaminase levels (2 patients), both observed at the 40- to 50-mg dose levels. Among the 10 patients in the combination arm, 2 had DLTs at the 40-mg dose level. These were fatigue and elevated liver enzyme levels. The most common adverse events were fatigue (n = 38), nausea (n = 27), vomiting (n = 24), diarrhea (n = 23), anorexia (n = 20), pyrexia (n = 18), and elevated transaminase levels (n = 14). The pharmacokinetics showed dose proportional increase in LY2334737 and gemcitabine exposure. The metabolite 2',2'-difluorodeoxyuridine accumulated with an accumulation index of 4.3 (coefficient of variation: 20%). In one patient, complete response in prostate-specific antigen was observed for 4 cycles, and stable disease was achieved in 22 patients overall. Pharmacokinetic analysis showed that the 2 investigated LY2334737 drug formulations were bioequivalent., Conclusions: LY2334737 displays linear pharmacokinetics and the MTD is 40 mg with or without daily administration of 100 mg erlotinib. Signs of antitumor activity warrant further development., (©2011 AACR)

Purpose: To determine the toxicity, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of oral gemcitabine (2',2'-difluorodeoxycytidine; dFdC) in patients with cancer., Experimental Design: Patients with advanced or metastatic cancer refractory to standard therapy were eligible. Gemcitabine was administered p.o. starting at 1 mg once daily using dose escalation with three patients per dose level. Patients received one of two dosing schemes: (a) once daily dosing for 14 days of a 21-day cycle or (b) every other day dosing for 21 days of a 28-day cycle. Pharmacokinetics were assessed by measuring concentrations of dFdC and 2',2'-difluorodeoxyuridine (dFdU) in plasma and gemcitabine triphosphate in peripheral blood mononuclear cells, and pharmacodynamics by measuring the effect on T-cell proliferation., Results: Thirty patients entered the study. Oral gemcitabine was generally well-tolerated. The maximum tolerated dose was not reached. Mainly moderate gastrointestinal toxicities occurred except for one patient who died after experiencing grade 4 hepatic failure during cycle two. One patient with a leiomyosarcoma had stable disease during 2 years and 7 months. Systemic exposure to dFdC was low with an estimated bioavailability of 10%. dFdC was highly converted to dFdU, probably via first pass metabolism and dFdU had a long terminal half-life ( approximately 89 h). Concentrations of dFdCTP in peripheral blood mononuclear cells were low, but high levels of gemcitabine triphosphate, the phosphorylated metabolite of dFdU, were detected., Conclusions: Systemic exposure to oral gemcitabine was low due to extensive first-pass metabolism to dFdU. Moderate toxicity combined with hints of activity warrant further investigation of the concept of prolonged exposure to gemcitabine.

Purpose: To determine the maximum tolerated dose, dose-limiting toxicity, and pharmacokinetics of docetaxel infused over 1 hour when given in combination with oral zosuquidar to patients with resistant solid tumors., Experimental Design: In cycle 1, patients received docetaxel alone. In subsequent cycles, zosuquidar was administered with docetaxel, which was escalated from 75 to 100 mg/m2. Zosuquidar was escalated from 100 to 300 mg/m2 every 8 hours on days 1 to 3 for a total of 7 doses, or from 400 to 500 mg every 12 hours for 2 doses administered 2 hours before docetaxel. The pharmacokinetics of docetaxel with and without zosuquidar administration were obtained., Results: Thirty-six of 41 patients completed at least one cycle of docetaxel and zosuquidar. The maximum tolerated dose was docetaxel 100 mg/m2 and zosuquidar 500 mg every 12 hours for 2 doses. The most common toxicity was neutropenia. In 35 patients, zosuquidar produced minimal increases in the docetaxel peak plasma concentrations and area under the curve. Dosing over 3 days with zosuquidar (7 doses) did not show benefit over the 1-day dosing. Of the 36 patients, one patient had a partial response, and 14 patients had disease stabilization., Conclusions: Docetaxel at 75 or 100 mg/m2 and zosuquidar 500 mg 2 hours before docetaxel and 12 hours later is well tolerated. Zosuquidar minimally alters the pharmacokinetics of docetaxel, allowing full dose docetaxel to be given with this P-glycoprotein modulator. A Phase II study with this combination in advanced breast carcinoma is underway.

The mechanisms of resistance to the antimetabolite gemcitabine in non-small cell lung cancer have not been extensively evaluated. In this study, we report the generation of two gemcitabine-selected non-small cell lung cancer cell lines, H358-G200 and H460-G400. Expression profiling results indicated that there was evidence for changes in the expression of 134 genes in H358-G200 cells compared with its parental line, whereas H460-G400 cells exhibited 233 genes that appeared to be under- or overexpressed compared with H460 cells. However, only the increased expression of ribonucleotide reductase subunit 1 (RRM1), which appeared in both resistant cell lines, met predefined analysis criteria for genes to investigate further. Quantitative PCR analysis demonstrated H358-G200 cells had a greater than 125-fold increase in RRM1 RNA expression. Western blot analysis confirmed high levels of RRM1 protein in this line compared with the gemcitabine-sensitive parent. No significant change in the expression of RRM2 was observed in either cell line, although both gemcitabine-resistant cell lines had an approximate 3-fold increase in p53R2 protein. A partial revertant of H358-G200 cells had reduced levels of RRM1 protein (compared with G200 cells), without observed changes in RRM2 or p53R2. In vitro analyses of ribonucleotide reductase activity demonstrated that despite high levels of RRM1 protein, ribonucleotide reductase activity was not increased in H358-G200 cells when compared with parental cells. The cDNA encoding RRM1 from H358-G200 cells was cloned and sequenced but did not reveal the presence of any mutations. The results from this study indicate that the level of RRM1 may affect gemcitabine response. Furthermore, RRM1 may serve as a biomarker for gemcitabine response.

Purpose: Our intention was to (a) to investigate the safety and tolerability of a potent P-glycoprotein modulator, zosuquidar trihydrochloride (LY335979), when administered i.v. alone or in combination with doxorubicin, (b) to determine the pharmacokinetics of zosuquidar and correlate exposure to inhibition of P-glycoprotein function in a surrogate assay, and (c) to compare the pharmacokinetics of doxorubicin in the presence and absence of zosuquidar., Patients and Methods: Patients with advanced malignancies who provided written informed consent received zosuquidar and doxorubicin administered separately during the first cycle of therapy and then concurrently in subsequent cycles. Zosuquidar was given i.v. over 48 h in a cohort-dose escalation manner until the occurrence of dose-limiting toxicity or protocol specified maximum exposure. Doxorubicin doses of 45, 60, 75 mg/m(2) were administered during the course of the trial., Results: Dose escalation proceeded through 9 cohorts with a total of 40 patients. The maximal doses administered were 640 mg/m(2) of zosuquidar and 75 mg/m(2) of doxorubicin. No dose-limiting toxicity of zosuquidar was observed. Pharmacokinetic analysis revealed that, in the presence of zosuquidar at doses that exceeded 500 mg, there was a modest decrease in clearance (17-22%) and modest increase in area under the curve (15-25%) of doxorubicin. This change was associated with an enhanced leukopenia and thrombocytopenia but was without demonstrable clinical significance. The higher doses of zosuquidar were associated with maximal P-glycoprotein inhibition in natural killer cells., Conclusion: Zosuquidar can be safely coadministered with doxorubicin using a 48 h i.v. dosing schedule.

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.

Purpose: The purpose of this study was to investigate the safety and tolerability of Zosuquidar.3HCl, a potent inhibitor of P-glycoprotein (Pgp), when administered p.o. alone and in combination with doxorubicin and to determine whether Zosuquidar.3HCl affects doxorubicin pharmacokinetics and inhibits Pgp function in peripheral blood natural killer lymphocytes., Experimental Design: Patients with advanced nonhematological malignancies were eligible for this Phase I trial. Zosuquidar.3HCl and doxorubicin were administered separately during the first cycle of therapy and then administered concurrently. Zosuquidar.3HCl was administered over 4 days, with doses escalated until the occurrence of dose-limiting toxicity. Subsequently, doxorubicin doses were increased from 45 to 75 mg/m(2). Zosuquidar.3HCl, doxorubicin, and doxorubicinol pharmacokinetics were analyzed, and dual fluorescence cytometry was used to determine the effects of Zosuquidar.3HCl on Pgp function in natural killer cells., Results: A total of 38 patients were treated at nine dose levels. Neurotoxicity was dose-limiting for oral Zosuquidar.3HCl, characterized by cerebellar dysfunction, hallucinations, and palinopsia. The maximum-tolerated dose for oral Zosuquidar.3HCl administered every 12 h for 4 days is 300 mg/m(2). Zosuquidar.3HCl did not affect doxorubicin myelosuppression or pharmacokinetics, and Zosuquidar.3HCl pharmacokinetics were similar in the absence and presence of doxorubicin. Higher plasma concentrations of Zosuquidar.3HCl were associated with greater Pgp inhibition in natural killer cells., Conclusion: Zosuquidar.3HCl can be coadministered with doxorubicin using a 4-day oral dosing schedule, with little effect on doxorubicin toxicity or pharmacokinetics. Further refinement in Zosuquidar.3HCl dosing and scheduling should be explored to optimize Pgp inhibition while minimizing cerebellar toxicity.

Human multidrug resistance protein 1 (MRP1) confers resistance to the Vinca alkaloids, the anthracyclines, and the epipodophyllotoxins. It is also capable of binding to and transporting the glutathione S-conjugate leukotriene C4 (LTC4) in isolated membrane vesicles. To explore species differences that exist between MRP orthologs, we cloned and characterized the mRNA encoding a canine ortholog of human MRP1-designated canine MRP1 (canMRP1). The canMRP1 mRNA encodes a protein of identical length as MRP1. Sequence alignment revealed that canMRP1 was 92% identical to MRP1 and 88% identical to murine mrp1. Five polymorphisms were identified in the canMRP1 cDNA coding sequence, including one resulting in an amino acid change from alanine to serine at aa149 (canMRP1-A and B alleles, respectively). canMRP1 was expressed and functionally characterized in HeLa and A2780 cells. Both alleles conferred an increased resistance to vincristine and etoposide and transported LTC4. The compound LY402913, a modulating agent developed against human MRP1, was able to sensitize canMRP1-expressing cells to vincristine. The modulation of canMRP1 by LY402913 was additionally confirmed by the calcein-AM accumulation assay. LY402913 inhibited the efflux of calcein in canMRP1-expressing cells. Thus, canMRP1 is similar to MRP1 in conferring resistance to vincristine and etoposide, transporting calcein-a.m., and being inhibited by LY402913. However, despite the high degree of sequence identity and functional similarity to MRP1, canMRP1 transgene failed to confer resistance to doxorubicin either in HeLa or A2780 cells. Knowledge of species differences between canine and human proteins will aid in the design of appropriate pharmacokinetic and toxicokinetic studies for the preclinical evaluation of MRP1 modulators.

U-937 human leukemia cells were selected for resistance to doxorubicin in the presence or absence of a specific drug modulator that inhibits the activity of P-glycoprotein (Pgp), encoded by the multidrug-resistance gene (MDR1). Parental cells expressed low basal levels of the multidrug-resistance-associated gene (MRP1) and major vault protein (MVP) mRNAs and no MDR1 mRNA. Two doxorubicin-resistant cell lines were selected. Both drug-resistant cell lines upregulated the MVP mRNA level 1.5-fold within 1 cell passage. The MVP mRNA level continued to increase over time as the doxorubicin selection pressure was increased. MVP protein levels generally paralleled the mRNA levels. The 2 high molecular weight vault protein mRNAs were always expressed at constitutive levels. Fully formed vault particles consisting of the MVP, the 2 high molecular weight proteins and the vault RNA assembled and accumulated to increased levels in drug-selected cells. MVP induction is therefore the rate-limiting step for vault particle formation in U-937 cells. By passage 25 and thereafter, the selected cells were resistant to doxorubicin, etoposide, mitoxantrone and 5-fluorouracil by a pathway that was independent of MDR1, MRP1, MRP2 and breast cancer resistance protein. In summary, U-937 doxorubicin-selected cells are programmed to rapidly upregulate MVP mRNA levels, to accumulate vault particles and to become multidrug resistant., (Copyright 2002 Wiley-Liss, Inc.)

Resistance to chemotherapy by some human tumors may be due to overexpression of membrane-associated transport proteins. The best characterized of these is the multidrug resistance (MDR) transporter, P-glycoprotein (Pgp). The aim of this study was to measure the inhibitory effects of a potent new MDR modulator, (2R)-anti-5-(3-[4-(10,11-difluoromethanodibenzo-suber-5-yl) piperazin-1-yl]-2-hydroxypropoxy)quinoline trihydrochloride (LY335979), in the drug-resistant cell line HL60/VCR and in normal, human CD56(+) lymphocytes. We used flow cytometric methods to detect the accumulation of rhodamine 123 and daunorubicin, fluorescent MDR substrates, in these cells. Our results indicate that LY335979 was 500-1500 times more potent than cyclosporin A or verapamil in restoring Pgp substrate accumulation in the MDR cell line HL60/VCR. Moreover, LY335979 could effectively block Pgp function on isolated CD56(+) lymphocytes (IC(50) = 1.2 nM) or CD56(+) lymphocytes in whole blood (IC(50) = 174 nM). We conclude that LY335979 is among the most potent Pgp inhibitors described and that it maintains significant potency in whole-human blood. These latter findings are important for establishing the dosing regimens of LY335979 for future clinical studies.

Acquired multidrug resistance to anti-cancer agents has been associated with overexpression of the P-glycoprotein and other members of the ATP-binding cassette superfamily. The present studies demonstrate that SCC-25 cells selected for resistance to the alkylating agent cisplatin (CDDP) overexpress the anti-apoptotic Bcl-xL protein. In contrast to parental cells, the SCC-25/CDDP-resistant variant failed to exhibit activation of caspase-3, cleavage of protein kinase C delta, and other characteristics of apoptosis in response to CDDP. Similar results were obtained when SCC-25/CDDP cells were exposed to the structurally and functionally unrelated antimetabolite 1-beta-D-arabinofuranosyl-cytosine (ara-C). Other cells selected for resistance to doxorubicin or vincristine also exhibited overexpression of Bcl-xL and failed to respond to CDDP and ara-C with activation of caspase-3. The results further demonstrate that multidrug-resistant cells exhibit a block in the release of mitochondrial cytochrome c into the cytosol and that this effect is dependent on overexpression of Bcl-xL. The demonstration that lysates from the resistant cells respond to the addition of cytochrome c with activation of caspase-3 confirms that the block in apoptosis is because of inhibition of mitochondrial cytochrome c release. These findings demonstrate that cells respond to diverse classes of anti-cancer drugs with overexpression of Bcl-xL and that this response represents another mechanism of acquired multidrug resistance.

The U-A10 cell line, a doxorubicin-selected variant of human U-937 myeloid leukemia cells, exhibits a redistribution of anthracyclines into a expanded vesicular compartment. The acidic nature of this compartment was confirmed by vital staining with a pH sensitive dye, LysoSensor yellow/blue DND-160. Identification of the vesicular compartment was performed by immunofluorescence analysis. Staining for the LAMP-1 and LAMP-2 antigens showed that the vesicles are enlarged lysosomes that are eccentrically placed near the nucleus of U-A10 cells. By contrast, the expression of the multidrug resistance-associated protein and the P-glycoprotein was observed predominately on the plasma membrane of the drug-resistant cells. The accumulation of daunorubicin into cellular compartments was quantified using radiolabeled drug. Exposing cells to 3[H]-daunorubicin and then isolating intact nuclei showed that nuclei from U-A10 cells accumulated twofold to threefold less anthracycline than nuclei from U-937 cells. However, when nuclei were isolated first and then exposed to 3[H]-daunorubicin, little difference in net nuclear drug accumulation was detected. Cytoplasts prepared from U-A10 and U-937 cells were exposed to 3[H]-daunorubicin to measure cytoplasmic drug accumulation. At external daunorubicin concentrations of 100 ng/mL or higher, cytoplasts from U-A10 cells accumulated significantly more daunorubicin than cytoplasts from U-937 cells. Moreover, studies with the lysosomotropic agent chloroquine showed that U-A10 cells accumulated twofold more chloroquine and showed twofold enhanced sensitivity to this agent as compared with parental U-937 cells. Fluorescence microscopy showed that chloroquine affects vesicular anthracycline sequestration in U-A10 cells with an associated increase in daunorubicin nuclear fluorescence. Although chloroquine did not alter anthracycline cytotoxicity in parental cells, it restored daunorubicin and doxorubicin sensitivity to U-A10 cells. Taken together, these studies demonstrate that U-A10 cells exhibit a redistribution of the lysosomal compartment. The trapping of drug into an expanded acidic vesicular compartment results in decreased nuclear drug accumulation and decreased cytotoxicity. Lysosomotropic agents, such as chloroquine, warrant further study as modulators of this acquired drug-resistance phenotype.

The genome structure and expression of mdr genes were examined in multidrug-resistant sublines of two different murine (DBA/2J) Friend erythroleukemia cell lines, PC4 and C7D, derived by stepwise exposure to increasing concentrations of adriamycin beginning with 5 ng/ml. The PC4 cell lines selected in higher drug concentrations (80-1280 ng/ml) demonstrated amplification of all three mdr genes with preferential amplification of mdr3. Overexpression of the mdr2 and mdr3 genes accompanied their genomic amplification; however, expression of mdr1 was not seen despite amplification. In the C7D cell lines selected with higher drug concentrations (40-160 ng/ ml), amplification and overexpression of mdr1 and mdr2 without mdr3 was observed. Increased expression of mdr1 occurred prior to gene amplification. The distribution of mdr-specific genes in micrococcal nuclease-generated chromatin fractions differing in transcriptionally active sequences and proteins was different between the parent and drug-resistant sublines. An enrichment (two- to threefold) of mdr3 genes in the H1-depleted mononucleosome fraction enriched for actively transcribed genes (e.g., globin) was detected by Southern analysis of chromatin fractions in PC4-80 cells (selected in 80 ng/ml of adriamycin and overexpressing mdr3), compared to the parental cells. mdr3 enrichment was also detected using a new PCR-based method, which examined mdr3 genes and repetitive sequences. Of note, the H1-depleted chromatin fraction from PC4-20 showed enrichment of the mdr3 gene, although mdr3 expression was not detected in the cell line. These studies showed a different pattern of gene amplification and overexpression in genetically related erythroleukemia cell lines selected for resistance to the same chemotherapeutic agent. A change in chromatin organization of mdr genes preceded overexpression and amplification of the mdr3 gene.

The multidrug resistance protein (MRP) is a membrane protein that mediates altered transport of cytotoxic drugs. Although MRP overexpression has been described in doxorubicin-selected human tumor cell lines, the murine PC-V10 and PC-V40 cell lines are members of the only reported series of vincristine-selected cell lines that overexpress mrp. Western blotting, using an antiserum developed against human MRP, demonstrated high-level expression of murine MRP primarily in the plasma membranes in each of the vincristine-selected cell lines. Only PC-V160, selected for high level resistance, demonstrated concomitant overexpression of the P-glycoprotein. As compared with parental cells, each of the drug-selected cell lines demonstrated an energy-dependent, decreased net accumulation of vincristine without any changes in the initial rates of vincristine influx. However, there was an enhanced rate of vincristine loss, 2.3-fold from the PC-V40 cell line and 3.9-fold from the PC-V160 cell line. Selective plasma membrane permeabilization with digitonin equalized vincristine accumulation among the parental, the PC-V40, and the PC-V160 cell lines. No intracellular pH differences were detected among the cell lines. Despite high-level MRP expression, daunorubicin accumulation and the rate of daunorubicin loss in the PC-V40 cells were the same as that observed in parental PC4 cells. Fluorescence microscopy demonstrated no difference in the pattern of subcellular daunorubicin accumulation between parental and PC-V40 cells. These studies demonstrate that murine MRP, overexpressed and found predominantly in the plasma membrane of vincristine-selected PC-V40 cells, is associated with an energy-dependent increased efflux of vincristine, but not with efflux or altered distribution of daunorubicin.

Expression of murine P-glycoprotein (P-gp), encoded by mdrl or mdri3, confers a multidrug resistance phenotype. Higher expression of c-fos and c-jun has also been demonstrated in multidrug resistant human and murine cells. We detected increased expression of c-fos early in the derivation of two series of murine erythroleukemia sublines selected for resistance to vincristine or adriamycin which eventually overexpress mdr3. We speculate that early expression of c-fos prepares cells for overexpression of other genes, such as mdr3, that contribute to the multidrug resistance phenotype.

Acquired resistance to diverse chemotherapeutic agents has been associated with overexpression of the P-glycoprotein. We have selected human U-937 cells for clones resistant to the cytotoxic agents doxorubicin (U-A20) and vincristine (U-V20). The results demonstrate that P-glycoprotein-positive U-A20 and U-V20 cells exhibit resistance to inducers of internucleosomal DNA fragmentation. Although parental U-937 cells responded to ionizing radiation with the DNA laddering characteristic of physiological cell death, the drug-resistant lines were insensitive to this effect. The U-A20 and U-V20 clones were also resistant to endonucleolytic DNA cleavage associated with exposure to tumor necrosis factor or ceramide. Previous work has demonstrated that physiological cell death is inhibited by overexpression of the Bcl-2 protein. However, analysis of Bcl-2 revealed similar levels in the parental and drug-resistant cells. In contrast, we show that U-A20 and U-V20 cells overexpress the Bcl-2-related protein, Bcl-xL. Moreover, studies with a U-937 cell line transfected with a Bcl-XL expression vector confirm resistance to ionizing radiation-induced DNA fragmentation and cell killing. These findings suggest that, unlike Bcl-2, Bcl-XL may be constitutively overexpressed as a result of selection for cytotoxic drug resistance and that Bcl-XL participates in an acquired form of multimodality resistance to chemotherapeutic agents and radiation.

Multidrug-resistant sublines of the murine erythroleukemia cell line PC4 were sequentially selected in increasing vincristine concentrations (5-160 ng/ml). The low- and intermediate-level resistant cell lines, selected in < or = 40 ng/ml of vincristine, demonstrated resistance to Vinca alkaloids and to an epipodophyllotoxin but little or none to an anthracycline. The expression of murine mdr genes, as analyzed by Northern blotting, revealed a baseline expression of murine mdr2 in parental cells that was unchanged in the drug-resistant cell lines. Overexpression of mdr3 was observed only in the highest-level resistant cell line, PC-V160, whereas mdr1 mRNA was not detected in any of the cell lines. The polymerase chain reaction, using mdr3-specific primers, excluded the possibility that low levels of P-glycoprotein expression contributed to the resistance phenotype in the low and intermediate-level resistant cell lines. Northern blot analysis using a human complementary DNA probe for the multidrug resistance-associated protein (MRP) demonstrated overexpression of murine mrp in each of the vincristine-selected sublines. Genomic amplification of the mrp gene was coincident with mrp overexpression. The expression of mrp was also examined in two series of previously characterized doxorubicin-selected cell lines derived from parental PC4 and C7D murine erythroleukemia cells. In contrast to the vincristine-selected cell lines, overexpression of mrp was not detected. These studies demonstrate that, in murine erythroleukemia cells selected for vincristine resistance, overexpression of murine mrp occurred prior to that for murine mdr. In contrast to human MRP, selection for vincristine, but not doxorubicin resistance, resulted in the overexpression of murine mrp.

Dolastatin 10, a cytotoxic pentapeptide isolated from the mollusk Dolabella auricularia, exhibits potent antitumor activity. The present studies demonstrated that sublines of murine PC4 and human U-937 leukemia cells expressing a multidrug resistance (MDR) phenotype are cross-resistant to this agent. We also demonstrated that such resistance was reversed by verapamil. While these findings suggested the involvement of the P-glycoprotein (P-gp) in dolastatin 10 resistance, we performed similar studies in a CHO cell line transfected with the human mdr1 cDNA. Expression of P-gp in the transfected cells was associated with resistance to dolastatin 10 by a verapamil-sensitive mechanism. The demonstration that photoaffinity labeling of P-gp was decreased in the presence of dolastatin 10 further supports the interaction of this cytotoxic peptide with P-gp. Taken together, these findings suggest that resistance to dolastatin 10 is conferred, at least in part, by P-gp and that this cytotoxic peptide is a novel member of the MDR phenotype.

Previous studies have demonstrated that human HL-60 myeloid leukemia cells differentiate in response to phorbol esters. This event is associated with induction of the c-jun early response gene and appearance of a monocytic phenotype. The present studies have examined the effects of vincristine-selected, multidrug resistance on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 cell differentiation. The results demonstrate that multidrug-resistant HL-60 cells, designated HL-60/vinc, fail to respond to TPA with an increase in c-jun transcripts or other phenotypic characteristics of monocytic differentiation. By contrast, treatment of HL-60/vinc cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases, induces c-jun transcription, growth arrest, and expression of the c-fms gene. Studies were also performed with an HL-60/vinc revertant (HL-60/vinc/R) line that has regained partial sensitivity to vincristine. The finding that HL-60/vinc/R cells respond to TPA with induction of a monocytic phenotype, but not c-jun expression, suggests that c-jun induction is not obligatory for monocytic differentiation. Other studies further demonstrate that the jun-B and fra-1 genes are induced by TPA in both HL-60/vinc and HL-60/vinc/R cells, whereas c-fos expression is attenuated in the HL-60/vinc line. Since TPA activates protein kinase C (PKC), we examined translocation of PKC from the cytosol to the membrane fraction. Although HL-60 and HL-60/vinc/R cells demonstrated translocation of PKC activity, this subcellular redistribution was undetectable in HL-60/vinc cells. Activity of the mitogen-activated protein kinase family with associated phosphorylation of c-Jun Y-peptide was markedly diminished in TPA-treated HL-60/vinc cells, but not in response to okadaic acid. Taken together, these findings suggest that vincristine resistance confers insensitivity to TPA-induced differentiation and can include defects in PKC-mediated signaling events and induction of jun/fos early response gene expression.

Twenty-nine patients aged 62-82 years with acute myeloid leukemia (AML) were treated with a 21-day course of continuous infusion cytarabine, oral hydroxyurea, and 1,25-dihydroxyvitamin D3 (calcitriol). Ten patients had an antecedent myelodysplastic syndrome. Calcitriol was continued as the only postremission therapy. Thirteen patients (45%) obtained a complete remission, and 10 patients (34%) had a partial response for an overall 79% response rate. There were three early deaths. The median remission duration was 9.8 months. Overall median survival was 12 months for all patients and 14 months for responding patients. All responding patients had marked bone marrow hypoplasia. Twenty patients received part or all of their chemotherapy as outpatients. This regimen has acceptable toxicity and can result in prolonged remissions in elderly, high-risk patients with AML. The favorable results may be related to the synergistic effect of hydroxyurea, cytarabine, and calcitriol.

Twenty previously untreated patients with advanced colorectal adenocarcinoma were entered on a Phase II trial of 3-day continuous infusion cisplatin (25 mg/m2/day) and 5-fluorouracil (800 mg/M2/day) with oral calcium leucovorin (30 mg/dose) every 6 hours. There were four partial responses (20%) and two complete responses (10%) for a total response rate of 30% (95% confidence limits +/- 20%). Patients received a median of 4.5 cycles of therapy (range 2-9 cycles). Three patients experienced neutropenia; one had a life-threatening infection. One developed neuropathy at 375 mg/M2 cumulative dose. Four patients developed mucositis. Treatment was stopped for one patient with stable disease after 5 cycles because of anorexia and nausea and vomiting; treatment was stopped for four patients because of excessive fatigue. The median duration of responses was 4 months (range 3-6 months). Although this regimen is active, the response rate, cumulative nature of the toxicity, and the requirement for hospitalization led us to conclude that this regimen does not warrant Phase III testing but might be a basis for further Phase II therapeutic trials.

Anthracycline accumulation was evaluated by flow cytometry or radiolabeled drug assays in cells and cytoplasts (enucleated cells) prepared from parental and multidrug-resistant human K562 leukemia cells. Treatment with energy inhibitors, such as dinitrophenol (DNP) or sodium azide/deoxyglucose, led to a marked decrease in daunorubicin accumulation in parental cells and cytoplasts. Another ionophore, monensin, also caused a significant decrease in daunorubicin accumulation; however, ATPase inhibitors ouabain, vanadate, and N-ethylamaleimide had little or no effect. The lysosomatropic agents chloroquine and methylamine caused a moderate decrease in anthracycline accumulation. Fluorescence microscopy showed that the DNP-sensitive daunorubicin uptake occurred in a nonnuclear subcellular compartment. Studies using increasing daunorubicin concentrations demonstrated fluorescence quenching that occurred in the nonnuclear, DNP-sensitive compartment. The effect of inhibitors on the accumulation of rhodamine 123 and acridine orange strongly implicated lysosomes as the principal compartment of this inhibitable daunorubicin accumulation. Cytoplasts from P-glycoprotein containing multidrug-resistant K562 cells demonstrated a verapamil-reversible, decreased daunorubicin accumulation that was observed in resistant whole cells. Verapamil pretreatment of cytoplasts from resistant cells revealed the subcellular DNP-sensitive uptake present in parental cytoplasts. These studies demonstrate that cytoplasts are an effective means to study drug transport in mammalian cells without nuclear drug binding. Parental K562 cells and cytoplasts exhibit an energy-dependent accumulation of daunorubicin into cytoplasmic organelles that is also present in resistant cells and cytoplasts when P-glycoprotein mediated efflux is inhibited.

First-step Adriamycin (doxorubicin)-resistant mutants of the murine erythroleukemia cell line PC4 were cloned from Adriamycin-containing (10 ng/ml) methylcellulose at a frequency of 3 x 10(-4). They demonstrated 1.6- to 2.4-fold stable resistance to Adriamycin. Most were cross-resistant to etoposide, but not to vincristine, and were without enhanced expression of mdr genes, which code for P-glycoproteins. Two different murine erythroleukemia cell lines, PC4 and C7D, were passaged in suspension culture into stepwise increasing amounts of Adriamycin. No high-level resistant mutants were isolated de novo; cells initially displayed low-level resistance to Adriamycin and etoposide. Two stepwise doublings of the drug concentration were needed before PC4 cells acquired vincristine resistance, but there was no detectable overexpression of mdr or a change in anthracycline uptake. In a subsequent doubling of Adriamycin concentration, the cells showed a further increase in resistance to all three drugs and now a decreased anthracycline accumulation. However, there was still no detectable increase in mdr expression as judged by Northern analysis of poly(A)+ enriched RNA and Western blot analysis of membrane proteins. Only after a fourth doubling of Adriamycin concentration did the cells demonstrate enhanced expression of mdr and P-glycoprotein. Equivalent mutants of C7D were selected, but generally at lower Adriamycin concentrations. Verapamil partially lowered resistance, but failed to restore parental susceptibility in any mutant; it caused an increased uptake in those mutants showing decreased anthracycline accumulation, including those that did not overexpress mdr. This study demonstrated different resistance phenotypes among mutants appearing spontaneously under stepwise drug selection; mutants with vincristine resistance and decreased anthracycline uptake preceded those associated with over-expression of P-glycoprotein.

Interferons (IFN) have clinical efficacy in certain hematologic malignancies. Combining IFN with conventional cytotoxic agents has been proposed as a means of improving therapy for diseases such as chronic myelogenous leukemia (CML). In this study, we examined the effect of recombinant interferons alone and in combination with Ara-C on normal and leukemic human hematopoietic progenitor cells (CFU-GM) in vitro. Mononuclear cells from normal bone marrow, peripheral blood of patients with CML, or the acute nonlymphocytic leukemia cell line HL-60 were incubated with alpha-, beta-, or gamma-IFN (0-1,000 units/ml) followed by the addition of Ara-C. The survival of normal CFU-GM was significantly increased if cells were treated with IFN 1 h before 3 h of Ara-C exposure. Similar IFN pretreatment of CML and HL-60 progenitors failed to protect leukemic CFU-GM from Ara-C-induced toxicity. This selective protection of normal CFU-GM may have clinical application.

Twenty-nine evaluable patients with colorectal adenocarcinoma were treated in a phase I-II trial of combination chemotherapy with a 72-h continuous infusion of cisplatin (CDDP) and 5-fluorouracil (5-FU) with an infusion of VP-16 given at 24 and 48 h after the start of therapy. There were five (17 +/- 14%) partial responses lasting 2-6 months (median, 3). Three of these responses occurred among the 10 previously untreated patients. The toxicity of this regimen was pronounced. Four of eight patients with severe neutropenia required hospitalization for infections, two of which were life-threatening; one of six patients with severe thrombocytopenia had a life-threatening hemorrhagic complication; and four patients experienced severe, dose-limiting fatigue. These complications occurred principally with CDDP and VP-16 at doses above 27.5 mg/m2/day and 110 mg/m2/dose, respectively. Mucositis occurred in six patients and limited the dose of 5-FU to 1,300 mg/m2/day. Although the response rate appeared to be high in previously untreated patients, the minimal palliative benefit of treatment and the brief duration of the responses do not compensate for the toxicity observed.