Christopher A. Slapak

An Increase in the Expression of Ribonucleotide Reductase Large Subunit 1 Is Associated with Gemcitabine Resistance in Non-Small Cell Lung Cancer Cell Lines

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.

Phase II study of enzastaurin, a protein kinase C beta inhibitor, in patients with relapsed or refractory diffuse large B-cell lymphoma.

PURPOSE Protein kinase C beta (PKCbeta) was identified by gene-expression profiling, preclinical evaluation, and independent immunohistochemical analysis as a rational therapeutic target in diffuse large B-cell lymphoma (DLBCL). We conducted a multicenter phase II study of a potent inhibitor of PKCbeta, enzastaurin, in patients with relapsed or refractory DLBCL. PATIENTS AND METHODS Enzastaurin was taken orally once daily until disease progression or unacceptable toxicity occurred. Study end points included freedom from progression (FFP) for > or= two cycles (one cycle = 28 days), objective response, and toxicity. RESULTS Fifty-five patients (median age, 68 years) were enrolled. Patients had received a median number of two prior therapies (range, one to five); six patients relapsed after high-dose therapy and autologous stem-cell transplantation. Only one grade 4 toxicity (hypomagnesemia) occurred. Grade 3 toxicities included fatigue (n = 2), edema (n = 1), headache (n = 1), motor neuropathy (n = 1), and thrombocytopenia (n = 1). No grade 3 or 4 neutropenia occurred. No deaths or discontinuations due to toxicity were reported. Fifteen patients completed less than one cycle of therapy. Twelve of 55 patients (22%; 95% CI, 13% to 46%) experienced FFP for two cycles, and eight patients remained free from progression for four cycles (15%; 95% CI, 6% to 27%). Four patients (7%; 95% CI, 2% to 18%), including three complete responders and one patient with stable disease, continue to experience FFP 20+ to 50+ months after study entry. CONCLUSION Treatment with enzastaurin was well-tolerated and associated with prolonged FFP in a small subset of patients with relapsed or refractory DLBCL. Further studies of enzastaurin in DLBCL are warranted.

A Phase I Trial of a Potent P-Glycoprotein Inhibitor, Zosuquidar.3HCl Trihydrochloride (LY335979), Administered Orally in Combination with Doxorubicin in Patients with Advanced Malignancies

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/m2 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/m2 of zosuquidar and 75 mg/m2 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.

Tumor Survivin Is Downregulated by the Antisense Oligonucleotide LY2181308: A Proof-of-Concept, First-in-Human Dose Study

Purpose: Enhanced tumor cell survival through expression of inhibitors of apoptosis (IAP) is a hallmark of cancer. Survivin, an IAP absent from most normal tissues, is overexpressed in many malignancies and associated with a poorer prognosis. We report the first-in-human dose study of LY2181308, a second-generation antisense oligonucleotide (ASO) directed against survivin mRNA. Patients and Methods: A dose-escalation study evaluating the safety, pharmacokinetics, and pharmacodynamics of LY2181308 administered intravenously for 3 hours as a loading dose on 3 consecutive days and followed by weekly maintenance doses. Patients were eligible after signing informed consent, had exhausted approved anticancer therapies and agreed to undergo pre- and posttreatment tumor biopsies to evaluate reduction of survivin protein and gene expression. Results: A total of 40 patients were treated with LY2181308 at doses of 100 to 1,000 mg. Twenty-six patients were evaluated at the recommended phase 2 dose of 750 mg, at which level serial tumor sampling and [11C]LY2183108 PET (positron emission tomography) imaging demonstrated that ASO accumulated within tumor tissue, reduced survivin gene and protein expression by 20% and restored apoptotic signaling in tumor cells in vivo. Pharmacokinetics were consistent with preclinical modeling, exhibiting rapid tissue distribution, and terminal half-life of 31 days. Conclusions: The tumor-specific, molecularly targeted effects demonstrated by this ASO in man underpin confirmatory studies evaluating its therapeutic efficacy in cancer.

Phase I Study of Docetaxel in Combination with the P-Glycoprotein Inhibitor, Zosuquidar, in Resistant Malignancies

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.

Modulation by LY335979 of P-glycoprotein function in multidrug-resistant cell lines and human natural killer cells.

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.

Oral Administration of Gemcitabine in Patients with Refractory Tumors : A Clinical and Pharmacologic Study

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 (∼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.

Results of a Phase 1 Study of AME-133v (LY2469298), an Fc-Engineered Humanized Monoclonal Anti-CD20 Antibody, in FcγRIIIa-Genotyped Patients with Previously Treated Follicular Lymphoma

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/m2 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. Clin Cancer Res; 18(5); 1395–403. ©2012 AACR.

Phase I Study of Oral Gemcitabine Prodrug (LY2334737) Alone and in Combination with Erlotinib in Patients with Advanced Solid Tumors

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. Clin Cancer Res; 17(18); 6071–82. ©2011 AACR.

Role of P-glycoprotein in dolastatin 10 resistance

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.