Leonard Liebes

Reactive Oxygen Species Generation and Mitochondrial Dysfunction in the Apoptotic Response to Bortezomib, a Novel Proteasome Inhibitor, in Human H460 Non-small Cell Lung Cancer Cells

Bortezomib, a proteasome inhibitor, shows substantial anti-tumor activity in a variety of tumor cell lines, is in phase I, II, and III clinical trials and has recently been approved for the treatment of patients with multiple myeloma. The sequence of events leading to apoptosis following proteasome inhibition by bortezomib is unclear. Bortezomib effects on components of the mitochondrial apoptotic pathway were examined: generation of reactive oxygen species (ROS), alteration in the mitochondrial membrane potential (Δψm), and release of cytochrome c from mitochondria. With human H460 lung cancer cells, bortezomib exposure at 0.1 μm showed induction of apoptotic cell death starting at 24 h, with increasing effects after 48–72 h of treatment. After 3–6 h, an elevation in ROS generation, an increase in Δψm, and the release of cytochrome c into the cytosol, were observed in a time-dependent manner. Co-incubation with rotenone and antimycin A, inhibitors of mitochondrial electron transport chain complexes I and III, or with cyclosporine A, an inhibitor of mitochondrial permeability transition pore, resulted in inhibition of bortezomib-induced ROS generation, increase in Δψm, and cytochrome c release. Tiron, an antioxidant agent, blocked the bortezomib-induced ROS production, Δψm increase, and cytochrome c release. Tiron treatment also protected against the bortezomib-induced PARP protein cleavage and cell death. Benzyloxycarbonyl-VAD-fluoromethyl ketone, an inhibitor of pan-caspase, did not alter the bortezomib-induced ROS generation and increase in Δψm, although it prevented bortezomib-induced poly(ADP-ribose) polymerase cleavage and apoptotic death. In PC-3 prostate carcinoma cells (with overexpression of Bcl-2), a reduction of bortezomib-induced ROS generation, Δψm increase was correlated with cellular resistance to bortezomib and the attenuation of drug-induced apoptosis. The transient transfection of wild type p53 in p53 null H358 cells caused stimulation of the bortezomib-induced apoptosis but failed to enhance ROS generation and Δψm increase. Thus ROS generation plays a critical role in the initiation of the bortezomib-induced apoptotic cascade by mediation of the disruption of Δψm and the release of cytochrome c from mitochondria.

Intravital observation of Plasmodium berghei sporozoite infection of the liver.

Plasmodium sporozoite invasion of liver cells has been an extremely elusive event to study. In the prevailing model, sporozoites enter the liver by passing through Kupffer cells, but this model was based solely on incidental observations in fixed specimens and on biochemical and physiological data. To obtain direct information on the dynamics of sporozoite infection of the liver, we infected live mice with red or green fluorescent Plasmodium berghei sporozoites and monitored their behavior using intravital microscopy. Digital recordings show that sporozoites entering a liver lobule abruptly adhere to the sinusoidal cell layer, suggesting a high-affinity interaction. They glide along the sinusoid, with or against the bloodstream, to a Kupffer cell, and, by slowly pushing through a constriction, traverse across the space of Disse. Once inside the liver parenchyma, sporozoites move rapidly for many minutes, traversing several hepatocytes, until ultimately settling within a final one. Migration damage to hepatocytes was confirmed in liver sections, revealing clusters of necrotic hepatocytes adjacent to structurally intact, sporozoite-infected hepatocytes, and by elevated serum alanine aminotransferase activity. In summary, malaria sporozoites bind tightly to the sinusoidal cell layer, cross Kupffer cells, and leave behind a trail of dead hepatocytes when migrating to their final destination in the liver.

Phase I Clinical and Pharmacokinetic Study of BMS-247550, a Novel Derivative of Epothilone B, in Solid Tumors

Purpose: The purpose of this study was to determine the maximum tolerated dose, toxicity, and pharmacokinetics of BMS-247550 administered as a 1-h i.v. infusion every 3 weeks. Experimental Design: Patients with advanced solid malignancies were premedicated and treated with escalating doses of BMS-247550. Blood sampling was performed to characterize the pharmacodynamics and pharmacokinetics of BMS-247550. Results: Twenty-five patients were treated at six dose levels ranging from 7.4 to 59.2 mg/m2. At 50 mg/m2, 4 of 9 patients (44.4%) had dose-limiting toxicity (neutropenia, abdominal pain/nausea). At 40 mg/m2 (the recommended Phase II dose), 2 of 12 patients (16.7%) had dose-limiting neutropenia. Overall, the most common nonhematological toxicity was fatigue/generalized weakness (grade 3–4 seen in 9.0% of patients), followed by neurosensory deficits manifested as peripheral neuropathy and by gastrointestinal discomfort. At 40 mg/m2, the incidence of grade 3 fatigue, abdominal pain, diarrhea, and neuropathy was 7.7%. Grade 1–2 neuropathy was observed in all patients enrolled and treated at 40 mg/m2. Two patients with paclitaxel-refractory ovarian cancer, one patient with taxane-naïve breast cancer, and another patient with docetaxel-refractory breast cancer had objective partial responses (lasting 6.0, 5.3, 3.0, and 4.5 months, respectively). The mean pharmacokinetic parameter values during course 1 for clearance, volume of distribution, and apparent terminal elimination half-life at the 40 mg/m2 (recommended Phase II dose) dose level were 21 liters/h/m2, 826 liters/m2, and 35 h (excluding one outlier of 516 h), respectively. Values during course 1 and course 2 were similar. Conclusions: The recommended dose for Phase II evaluation of BMS-247550 is 40 mg/m2, although more long-term observations are needed. BMS-247550 has advantages over taxanes in relation to drug resistance and warrants further study.

Phase I trial of low-dose continuous topotecan infusion in patients with cancer: an active and well-tolerated regimen.

PURPOSE The objective of this trial was to define the maximum-tolerated dose (MTD) of topotecan for a 21-day infusion schedule, repeated every 28 days, in patients with cancer. PATIENTS AND METHODS Cohorts of four patients received continuous ambulatory infusions of topotecan in escalated duration with doses beginning at 0.20 mg/m2/d for 7 days. Forty-four patients with a histologic diagnosis of cancer refractory to standard therapy were treated with infusions of topotecan for a total of 115 cycles and 1,780 patient-days of infusion. The median number of treatment cycles per patient was two (range, one to eight). All patients were heavily pretreated with chemotherapy and/or radiation. RESULTS The dose-limiting toxicity (DLT) was myelo-suppression, with thrombocytopenia greater than neutropenia seen at the dose level of 0.70 mg/m2/d for 21 days. At the MTD of 0.53 mg/m2, ten patients were treated for a total of 20 courses, resulting in one episode of grade 4 thrombocytopenia and leukopenia, one grade 3 thrombocytopenia, and two grade 3 leukopenias. This dose regimen was well tolerated, with minimal nonhematologic toxicity. Local infusion port complications developed in two patients and two had bacteremia, including one patient with repeated local skin infections. Objective responses were observed in this heavily pretreated population for patients with ovarian cancer (two partial responses and one mixed response in six patients), breast cancer (one partial response and one mixed response in two patients), and for one patient each with renal and non-small-cell lung cancer (two partial remissions). CONCLUSION Twenty-one-day topotecan infusion is well tolerated at 0.53 mg/m2, with dose-intensity exceeding other schedules for administration of topotecan. The DLT is hematologic, with thrombocytopenia somewhat exceeding leukopenia. Objective responses were observed in seven patients with breast, ovarian, renal, and non-small-cell lung cancer.

Pharmacokinetics, Safety, and Antiviral Effects of Hypericin, a Derivative of St. John's Wort Plant, in Patients with Chronic Hepatitis C Virus Infection

ABSTRACT Hypericin is a natural derivative of the common St. Johns wort plant, Hypericum perforatum. It has in vitro activity against several viruses, including bovine diarrhea virus, a pestivirus with structural similarities to hepatitis C virus (HCV). We conducted a phase I dose escalation study to determine the safety and antiviral activity of hypericin in patients with chronic HCV infection. The first 12 patients received an 8-week course of 0.05 mg of hypericin per kg of body weight orally once a day; 7 patients received an 8-week course of 0.10 mg/kg orally once a day. At the end of the 8-week period of treatment, no subject had a change of plasma HCV RNA level of more than 1.0 log10. Five of 12 subjects receiving the 0.05-mg/kg/day dosing schedule and 6 of 7 subjects receiving the 0.10-mg/kg/day dosing schedule developed phototoxic reactions. No other serious adverse events associated with hypericin use occurred. The pharmacokinetic data revealed a long elimination half-life (mean values of 36.1 and 33.8 h, respectively, for the doses of 0.05 and 0.1 mg/kg) and mean area under the curve determinations of 1.5 and 3.1 μg/ml × hr, respectively. In sum, hypericin given orally in doses of 0.05 and 0.10 mg/kg/d caused considerable phototoxicity and had no detectable anti-HCV activity in patients with chronic HCV infection.

Topical TLR7 Agonist Imiquimod Can Induce Immune-Mediated Rejection of Skin Metastases in Patients with Breast Cancer

Purpose: Skin metastases of breast cancer remain a therapeutic challenge. Toll-like receptor 7 agonist imiquimod is an immune response modifier and can induce immune-mediated rejection of primary skin malignancies when topically applied. Here we tested the hypothesis that topical imiquimod stimulates local antitumor immunity and induces the regression of breast cancer skin metastases. Experimental Design: A prospective clinical trial was designed to evaluate the local tumor response rate of breast cancer skin metastases treated with topical imiquimod, applied 5 d/wk for 8 weeks. Safety and immunologic correlates were secondary objectives. Results: Ten patients were enrolled and completed the study. Imiquimod treatment was well tolerated, with only grade 1 to 2 transient local and systemic side effects consistent with imiquimods immunomodulatory effects. Two patients achieved a partial response [20%; 95% confidence interval (CI), 3%–56%]. Responders showed histologic tumor regression with evidence of an immune-mediated response, showed by changes in the tumor lymphocytic infiltrate and locally produced cytokines. Conclusion: Topical imiquimod is a beneficial treatment modality for breast cancer metastatic to skin/chest wall and is well tolerated. Importantly, imiquimod can promote a proimmunogenic tumor microenvironment in breast cancer. Preclinical data generated by our group suggest superior results with a combination of imiquimod and ionizing radiation and we are currently testing in patients whether the combination can further improve antitumor immune and clinical responses. Clin Cancer Res; 18(24); 6748–57. ©2012 AACR.

A Phase II Trial of Sorafenib in Metastatic Melanoma with Tissue Correlates

Background Sorafenib monotherapy in patients with metastatic melanoma was explored in this multi-institutional phase II study. In correlative studies the impact of sorafenib on cyclin D1 and Ki67 was assessed. Methodology/Principal Findings Thirty-six patients treatment-naïve advanced melanoma patients received sorafenib 400 mg p.o. twice daily continuously. Tumor BRAFV600E mutational status was determined by routine DNA sequencing and mutation-specific PCR (MSPCR). Immunohistochemistry (IHC) staining for cyclin D1 and Ki67 was performed on available pre- and post treatment tumor samples. The main toxicities included diarrhea, alopecia, rash, mucositis, nausea, hand-foot syndrome, and intestinal perforation. One patient had a RECIST partial response (PR) lasting 175 days. Three patients experienced stable disease (SD) with a mean duration of 37 weeks. Routine BRAFV600E sequencing yielded 27 wild-type (wt) and 6 mutant tumors, whereas MSPCR identified 12 wt and 18 mutant tumors. No correlation was seen between BRAFV600E mutational status and clinical activity. No significant changes in expression of cyclin D1 or Ki67 with sorafenib treatment were demonstrable in the 15 patients with pre-and post-treatment tumor samples. Conclusions/Significance Sorafenib monotherapy has limited activity in advanced melanoma patients. BRAFV600E mutational status of the tumor was not associated with clinical activity and no significant effect of sorafenib on cyclin D1 or Ki67 was seen, suggesting that sorafenib is not an effective BRAF inhibitor or that additional signaling pathways are equally important in the patients who benefit from sorafenib. Trial registration Clinical Trials.gov NCT00119249

Toxicity of Combined Ganciclovir and Zidovudine for Cytomegalovirus Disease Associated with AIDS: An AIDS Clinical Trials Group Study

OBJECTIVE To assess the toxicity, efficacy, and pharmacology of combined zidovudine and ganciclovir therapy in patients with the acquired immunodeficiency syndrome (AIDS) and serious cytomegalovirus (CMV) disease. DESIGN Prospective, phase I multicenter trial (ACTG 004) with patients grouped by previous study drug history. SETTING Three university-based AIDS Clinical Trials Units sponsored by the National Institute of Allergy and Infectious Diseases (NIAID). PATIENTS Forty-one patients with AIDS-related CMV disease. Previous therapy with either zidovudine or ganciclovir was allowed. INTERVENTIONS Patients were treated with zidovudine, 600 to 1200 mg/d; or, if on ganciclovir maintenance, ganciclovir, 5 mg/kg body weight; blood was sampled for pharmacokinetic studies. The other drug was then administered to the patient with blood sampling and, finally, the two drugs in combination were given. Patients were continued on both drug therapies with dose reduction of zidovudine only for grade 3 or 4 toxicity. MEASUREMENTS AND MAIN RESULTS Forty patients were eligible. Hematologic toxicity was frequent, with 9 of the 10 patients requiring dose reductions for grade 3 or 4 toxicity at zidovudine doses of 1200 mg/d. With zidovudine doses of 600 mg/d, 82% experienced such hematologic toxicity. Median survival was 6 months; 10 patients developed intercurrent infection and 19, progressive CMV disease. Pharmacokinetic variables (alpha and beta half-lives, volume of distribution, clearance) were not affected in combination therapy. CONCLUSION The combination of zidovudine and ganciclovir is poorly tolerated in patients with AIDS and serious-CMV disease, with 82% developing severe to life-threatening hematologic toxicity. Such toxicity is not a result of pharmacologic interactions, drug metabolism, or excretion.

Activity and Pharmacodynamics of 21-Day Topotecan Infusion in Patients With Ovarian Cancer Previously Treated With Platinum-Based Chemotherapy

PURPOSE Twenty-one-day topotecan infusion was administered as second-line therapy in patients with previously treated ovarian cancer (based on our prior favorable phase I experience) to determine its activity, time to progression, and pharmacodynamics. PATIENTS AND METHODS Ovarian cancer patients with measurable lesions and one prior platinum-containing regimen were eligible. Topotecan 0.4 mg/m(2)/d 21-day continuous ambulatory intravenous infusion, with appropriate dose modifications for toxicity, was administered every 28 days. Weekly blood levels of topotecan and topoisomerase-1 (topo-1) levels in peripheral-blood mononuclear cells (PBMCs) were determined for pharmacodynamic correlation. RESULTS Twenty-four patients were entered onto the study (six cisplatin-refractory, five relapsing within < 6 months and 13 relapsing > 6 months after platinum-based therapy). A total of 128 cycles of topotecan (median, four cycles per patient; range, one to 12 cycles) were administered. The major toxicity was neutropenia (29% grade 3 in all cycles and 4% grade 4). One episode of grade 4 thrombocytopenia (4%) occurred. Fifty-two percent of the patients had anemia that required transfusions. Eight of 23 patients with measurable disease (35%; 95% confidence interval [CI], 15% to 54%) had partial responses (PRs) lasting longer than 1 month. Two of these patients had minor residual computed tomographic changes but had clinical complete remissions that lasted up to 53 weeks while they were not undergoing further therapy. One patient with nonmeasurable disease had a PR (by CA-125 criteria) that lasted 6 months, for an overall response rate of 38% in nine of 24 patients (95% CI, 18% to 57%). The median time to progression was 26 weeks. Pharmacodynamic analysis demonstrated a statistically significant decrease in free PBMC topo-1 level at weeks 2 and 3 of drug administration. There was a strong statistical correlation between the decrease in free topo-1 levels and increasing area under the curve (AUC) for topotecan. This was confirmed in a pharmacodynamic model. CONCLUSION Twenty-one-day infusion is a well-tolerated method of administering topotecan. Pharmacodynamic studies demonstrate correlations between (1) the week of infusion and the PBMC topo-1 level, (2) the AUC of topotecan and the decrease in topo-1 levels, and (3) the change in topo-1 level and the neutrophil nadir. The objective response rate of 35% to 38% (95% CI, 15% to 57%) in this small multicenter study is at the upper level for topotecan therapy in previously treated ovarian cancer. Prolonged topotecan administration therefore warrants further investigation in larger, randomized studies comparing this 21-day schedule with the once-daily-for-5-days schedule.

Construction of a well-defined multifunctional dendrimer for theranostics.

A dendrimer-based building block for theranostics was designed. The multifunctional dendrimer is polyamide-based and contains nine azide termini, nine amine termini, and fifty-four terminal acid groups. Orthogonal functionalization of the multifunctional dendrimer with a near-infrared (NIR) cyanine dye afforded the final dendrimer that shows fluorescence in the NIR region and no toxicity toward T98G human cells. The synthetic strategy described here might be promising for fabricating the next generation of materials for theranostics.