Drug-Based Lead Discovery: The Novel Ablative Antiretroviral Profile of Deferiprone in HIV-1-Infected Cells and in HIV-Infected Treatment-Naive Subjects of a Double-Blind, Placebo-Controlled, Randomized Exploratory Trial.

UNLABELLED: Antiretrovirals suppress HIV-1 production yet spare the sites of HIV-1 production, the HIV-1 DNA-harboring cells that evade immune detection and enable viral resistance on-drug and viral rebound off-drug. Therapeutic ablation of pathogenic cells markedly improves the outcome of many diseases. We extend this strategy to HIV-1 infection. Using drug-based lead discovery, we report the concentration threshold-dependent antiretroviral action of the medicinal chelator deferiprone and validate preclinical findings by a proof-of-concept double-blind trial. In isolate-infected primary cultures, supra-threshold concentrations during deferiprone monotherapy caused decline of HIV-1 RNA and HIV-1 DNA; did not allow viral breakthrough for up to 35 days on-drug, indicating resiliency against viral resistance; and prevented, for at least 87 days off-drug, viral rebound. Displaying a steep dose-effect curve, deferiprone produced infection-independent deficiency of hydroxylated hypusyl-eIF5A. However, unhydroxylated deoxyhypusyl-eIF5A accumulated particularly in HIV-infected cells; they preferentially underwent apoptotic DNA fragmentation. Since the threshold, ascertained at about 150 µM, is achievable in deferiprone-treated patients, we proceeded from cell culture directly to an exploratory trial. HIV-1 RNA was measured after 7 days on-drug and after 28 and 56 days off-drug. Subjects who attained supra-threshold concentrations in serum and completed the protocol of 17 oral doses, experienced a zidovudine-like decline of HIV-1 RNA on-drug that was maintained off-drug without statistically significant rebound for 8 weeks, over 670 times the drug's half-life and thus clearance from circulation. The uniform deferiprone threshold is in agreement with mapping of, and crystallographic 3D-data on, the active site of deoxyhypusyl hydroxylase (DOHH), the eIF5A-hydroxylating enzyme. We propose that deficiency of hypusine-containing eIF5A impedes the translation of mRNAs encoding proline cluster ('polyproline')-containing proteins, exemplified by Gag/p24, and facilitated by the excess of deoxyhypusine-containing eIF5A, releases the innate apoptotic defense of HIV-infected cells from viral blockade, thus depleting the cellular reservoir of HIV-1 DNA that drives breakthrough and rebound. TRIAL REGISTRATION: ClinicalTrial.gov NCT02191657.

Drug-induced reactivation of apoptosis abrogates HIV-1 infection.

HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal antivirals that eliminate viral infection by destroying infected cells. A drug-based drug discovery program, based on these compounds, is warranted to determine the potential of such agents in clinical trials of HIV-infected patients.

Results of a phase II trial of S-1 as first-line treatment of metastatic pancreatic cancer (CESAR-study group).

PURPOSE: S-1, an oral fluoropyrimidine derivative, has previously demonstrated anticancer efficacy in pancreatic cancer (PC), predominantly in Asian populations. This study evaluated the antitumor effect and safety of S-1 in Caucasian patients with metastatic PC. METHODS: Chemotherapy-naïve patients received S-1 orally at 30 mg/m(2) twice daily (BID) for 2 weeks, repeated every 3 weeks. Primary endpoint was ORR. Secondary endpoints included PFS, OS and safety assessment. The trial had a Simon's two-stage design with 22 patients evaluable for efficacy in stage 1 and an additional 18 patients in stage 2, if ≥3/22 patients had a confirmed response at the first stage. RESULTS: Three out of 27 patients showed PR, however, detection of asymptomatic brain metastases in one of them prevented this study from proceeding to stage 2. The median PFS and OS for all patients was 3.5 and 9.1 months, respectively. The median duration of disease control for patients with SD or PR (n = 17) was 4.3 months. S-1 was well tolerated; fatigue was the most frequent grade 3/4 adverse event. CONCLUSIONS: Efficacy data of PFS and OS are at least comparable to gemcitabine, the current standard of care. S-1 is active in Caucasian patients with metastatic PC.

Phase III study of pemetrexed plus carboplatin compared with etoposide plus carboplatin in chemotherapy-naive patients with extensive-stage small-cell lung cancer.

PURPOSE: Following a phase II trial in which pemetrexed-platinum demonstrated similar activity to that of historical etoposide-platinum controls, a phase III study was conducted to compare pemetrexed-carboplatin with etoposide-carboplatin for the treatment of extensive-stage small-cell lung cancer (ES-SCLC). PATIENTS AND METHODS: Chemotherapy-naive patients with ES-SCLC and an Eastern Cooperative Oncology Group performance status of zero to 2 were randomly assigned to receive pemetrexed-carboplatin (pemetrexed 500 mg/m(2) on day 1; carboplatin at area under the serum concentration-time curve [AUC] 5 on day 1) or etoposide-carboplatin (etoposide 100 mg/m(2) on days 1 through 3; carboplatin AUC 5 on day 1) every 3 weeks for up to six cycles. The primary objective of the study was noninferiority of pemetrexed-carboplatin overall survival with a 15% margin. RESULTS: Accrual was terminated with 908 of 1,820 patients enrolled after results of a planned interim analysis. In the final analysis, pemetrexed-carboplatin was inferior to etoposide-carboplatin for overall survival (median, 8.1 v 10.6 months; hazard ratio [HR],1.56; 95% CI, 1.27 to 1.92; log-rank P < .01) and progression-free survival (median, 3.8 v 5.4 months; HR, 1.85; 95% CI, 1.58 to 2.17; log-rank P < .01). Objective response rates were also significantly lower for pemetrexed-carboplatin (31% v 52%; P < .001). Pemetrexed-carboplatin had lower grade 3 to 4 neutropenia, febrile neutropenia, and leukopenia than etoposide-carboplatin; grade 3 to 4 thrombocytopenia was comparable between arms and anemia was higher in the pemetrexed-carboplatin arm. CONCLUSION: Pemetrexed-carboplatin is inferior for the treatment of ES-SCLC. Planned translational research and pharmacogenomic analyses of tumor and blood samples may help explain the study results and provide insight into new treatment strategies.

[Principles and practice of clinical phase I studies]. / Planung und Durchführung klinischer Phase-I-Studien.

There is clear evidence that the epidemiologic importance of malignant diseases will continue to rise in the future. This creates the ethical obligation to further intensify the search for improved systemic treatment options. This review is focussed on principles and practices of phase I trials. The trial methodology outlined here is not only used for first-in-human doses of new chemical entities but also for the investigation of new treatment schedules and combinations. The primary endpoints as well as preclinical requirements are addressed. Although for formal reasons antitumor activity cannot be an endpoint in phase I studies the careful observation and documentation of potential anticancer efficacy is of utmost importance in these early clinical trials. Signals of antitumor activity may serve as important guidance for subsequent clinical development plans. Inclusion criteria, dose escalation schemes, and the importance of the concept of the maximum tolerable dose (MTD) are addressed together with ethical principles of exposure to novel chemical agents.

Correlations of mRNA expression and in vitro chemosensitivity to enzastaurin in freshly explanted human tumor cells.

PURPOSE: Enzastaurin (LY317615) is a novel serine/threonine kinase inhibitor, targeting Protein Kinase C-beta (PKC-beta), and PI3K/AKT pathways to inhibit angiogenesis and tumor cell proliferation. The aims of this study were to determine whether Enzastaurin has direct antitumor activity against freshly explanted tumor cells and to correlate mRNA expression of genes related to the proposed mechanism of action of enzastaurin with in vitro chemosensitivity. EXPERIMENTAL DESIGN: Freshly biopsied tumor cells were studied using soft-agar cell cloning experiments (SACCE) to determine the in vitro chemosensitivity to enzastaurin. An aliquot of the same tumor specimens was shock-frozen and total RNA was isolated for standardized multiplex rt-PCR experiments for gene expression of PKC-beta1, PKC-beta2, IL-8, IL-8RA, IL-8RB, Glycogen Synthase Kinase 3 beta (GSK-3beta) and TGF-beta1. Correlations, threshold optimization, sensitivity, specificity, and efficiency were analyzed using the appropriate statistical methodologies. RESULTS: Seventy-two tumor samples were collected and 63 were fully evaluable. Low levels of mRNA expression of GSK-3beta and high levels of mRNA expression of IL-8 were highly significantly correlated with chemosensitivity to enzastaurin. Optimization analyses demonstrated threshold values of 4,000 copies for IL-8 and three copies for GSK-3beta relative to 10(4) copies of beta-actin. However, no correlation between mRNA expression of PKC-beta1, PKC-beta2, IL-8RA, IL-8RB and chemosensitivity to enzastaurin was observed. Expression of TGF-beta1 mRNA was not detectable in the specimens investigated. CONCLUSIONS: mRNA expression levels of IL-8 and GSK-3beta correlate with antitumor activity of enzastaurin. These results form a rational basis for clinical trials to evaluate the expression of these genes as potential predictors for treatment outcome after enzastaurin chemotherapy.

The pharmacokinetics, toxicities, and biologic effects of FK866, a nicotinamide adenine dinucleotide biosynthesis inhibitor.

BACKGROUND: FK866 is a potent inhibitor or NAD synthesis. This first-in-human study was performed to determine the maximum-tolerated dose, toxicity profile, and pharmacokinetics on a 96-h continuous infusion schedule. MATERIALS AND METHODS: Twenty four patients with advanced solid tumor malignancies refractory to standard therapies were treated with escalating doses of FK866 as a continuous, 96-h infusion given every 28 days. Serial plasma samples were collected to characterize the pharmacokinetics of FK866. Further blood samples were collected for the measurement of plasma VEGF levels. RESULTS: There were 12 women and 12 men with a median age of 61 (range 34-78) and a median KPS of 80%, received a 4-day of infusion of FK866 at dose levels of 0.018 mg/m2/h (n=3), 0.036 mg/m2/h (n=3), 0.072 mg/m2/h (n=3), 0.108 mg/m2/h (n=4), 0.126 mg/m2/h (n=6), and 0.144 mg/m2/h (n=5). Thrombocytopenia was the dose limiting toxicity, observed in two patients at the highest dose level and one patient at the recommended phase II dose of 0.126 mg/m2/h No other hematologic toxicities were noted other than mild lymphopenia and anemia. There was mild fatigue and grade 3 nausea; the latter was controlled with antiemetics and was not a DLT. Css (the mean of the 72 and 96 h plasma concentrations) increased in relation to the dose escalation. The study drug did not significantly affect plasma concentrations of VEGF. There were no objective responses, although four patients had stable disease (on treatment for 3 months or greater). CONCLUSIONS: The recommended phase II dose is 0.126 mg/m2/h given as a continuous 96-h infusion every 28 days. The dose limiting toxicity of FK866 is thrombocytopenia. Pharmacokinetic data suggest an increase in the plasma Css in relation to the escalation of FK866.

In vitro chemosensitivity of freshly explanted tumor cells to pemetrexed is correlated with target gene expression.

AIM OF THE STUDY: mRNA expression of genes involved in the mechanism of action of pemetrexed was correlated with in vitro chemosensitivity of freshly explanted human tumor specimens. EXPERIMENTAL DESIGN: Chemosensitivity to pemetrexed was studied in soft-agar. Multiplex rtPCR experiments for reduced folate carrier (RFC), folate receptor-alpha (FR-alpha), folylpolyglutamate synthetase (FPGS), thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide formyl transferase (GARFT), mrp4, and mrp5 were performed in parallel. Correlations, threshold optimization, sensitivity, specificity, and efficiency were analyzed using the appropriate statistical methodologies. RESULTS: In 61 samples, low levels of TS, GARFT, DHFR, and mrp4 gene expression significantly correlated with chemosensitivity to pemetrexed. Optimization analyses demonstrated threshold values of 144 copies for TS and six copies for mrp4 relative to 10(4) copies of beta-actin. CONCLUSIONS: These results form a rational basis for the design of clinical trials to evaluate the expression of these enzymes as predictors for treatment outcome.

Antitumor activity of enzastaurin (LY317615.HCl) against human cancer cell lines and freshly explanted tumors investigated in in-vitro [corrected] soft-agar cloning experiments.

Enzastaurin (LY317615.HCl) is an antiproliferative agent targeted specifically against PKC-beta. We have investigated the antitumoral effects of Enzastaurin against human cancer cell lines and freshly explanted human tumor tissue. Ten human cancer cell lines (NSCLC, colon, and thyroid) and human tumor specimens from 72 patients were used for in vitro studies in a cloning assay (HTCA). Cell lines and primary tumor cells were exposed to Enzastaurin for either 1 h or 7 days, or for 1 h or 21 days. At clinically achievable concentrations of Enzastaurin, inhibition of cell growth was observed for lung, colorectal, and thyroid cancer cell lines in a concentration dependent manner. Patient specimens exposed 1 h or 21 days to 1,400 nM Enzastaurin demonstrated inhibition rates of 24 and 32%, respectively. Marked inhibitory effects were observed in breast, thyroid, head/neck, non-small cell lung cancer, pancreatic cancer, and melanoma. In addition to its established antiangiogenic effects, Enzastaurin has direct antitumor activity against established human cancer cell lines and primary tumor specimens. This warrants further clinical development in tumors which have been identified to be potentially sensitive to Enzastaurin.

The role of Alimta in the treatment of malignant pleural mesothelioma: an overview of preclinical and clinical trials.

Malignant pleural mesothelioma (MPM) is a rare tumor, but its annual incidence is rapidly increasing. While most patients have locally advanced disease at presentation, to date there is no proven standard chemotherapy for MPM that has shown to increase survival in randomized studies. Therefore, the development of new therapeutic agents is urgent for this disease. Alimta is a novel, multitargeted antifolate with activity as single agent in patients with MPM. Recently, a phase III trial showed that the combination of Alimta with cisplatin resulted in a significantly increased efficacy compared with cisplatin alone. In addition, vitamin supplementation reduced treatment associated toxicities with no apparent affect on activity. This review summarizes preclinical and clinical data to define the future role of Alimta in the treatment of patients with MPM.