Elzbieta Izbicka

G-quadruplexes as targets for drug design

G-quadruplexes are a family of secondary DNA structures formed in the presence of monovalent cations that consist of four-stranded structures in which Hoogsteen base-pairing stabilizes G-tetrad structures. These structures are proposed to exist in vivo, although direct confirmatory evidence is lacking. Guanine-rich regions of DNA capable of forming G-quadruplex structures are found in a variety of chromosomal regions, including telomeres and promoter regions of DNA. In this review, we describe the design of three separate groups of G-quadruplex-interactive compounds and their interaction with G-quadruplex DNA. Using the first group of compounds (anthraquinones), we describe experiments that provide the proof of concept that a G-quadruplex is required for inhibition of telomerase. Using the second group of compounds (perylenes), we describe the structure of a G-quadruplex-ligand complex and its effect on the dynamics of formation and enzymatic unwinding of the quadruplex. For the third group of compounds (porphyrins), we describe the experiments that relate the biological effects to their interactions with G-quadruplexes.

A Phase II, Pharmacokinetic, and Biological Correlative Study of Oblimersen Sodium and Docetaxel in Patients with Hormone-Refractory Prostate Cancer

Purpose: To determine the antitumor activity and safety of oblimersen sodium, a phosphorothioate antisense oligonucleotide directed to the bcl-2 mRNA, with docetaxel in patients with hormone-refractory prostate cancer (HRPC) and to determine if relevant pharmacokinetic and pharmacodynamic variables of oblimersen or docetaxel influence response to this therapy. Experimental Design: Patients with HRPC were treated with oblimersen sodium by continuous i.v. infusion on days 1 to 8 with docetaxel given i.v. over 1 hour on day 6 every 3 weeks. Plasma samples were analyzed to characterize the pharmacokinetic variables of both oblimersen and docetaxel, and paired collections of peripheral blood mononuclear cells were collected to determine Bcl-2 protein expression pretreatment and post-treatment. Results: Twenty-eight patients received 173 courses of oblimersen (7 mg/kg/d continuous i.v. infusion on days 1-8) and docetaxel (75 mg/m2 i.v. on day 6). Prostate-specific antigen responses were observed in 14 of 27 (52%) patients, whereas 4 of 12 (33%) patients with bidimensionally measurable disease had objective responses. The mean oblimersen steady-state concentration (Css) was a significant determinant of antitumor activity; mean Css values were higher in responders compared with nonresponders (6.24 ± 1.68 versus 4.27 ± 1.22; P = 0.008). The median survival of all patients was 19.8 months. Bcl-2 protein expression decreased a median of 49.9% in peripheral blood mononuclear cells post-treatment, but the individual incremental change did not correlate with either oblimersen Css or response. Conclusions: Oblimersen combined with docetaxel is an active combination in HRPC patients demonstrating both an encouraging response rate and an overall median survival. The absence of severe toxicities at this recommended dose, evidence of Bcl-2 protein inhibition, and encouraging antitumor activity in HPRC patients warrant further clinical evaluation of this combination, including studies to optimize oblimersen Css.

In vitro antitumor activity of the novel marine agent, ecteinascidin-743 (ET-743, NSC-648766) against human tumors explanted from patients.

BACKGROUND Ecteinascidin-743 (ET-743), a member of the ecteinascidin family selected for clinical development, is a tetrahydroisoquinolone alkaloid isolated from the marine ascidian, Ecteinascidia turbinata. This novel compound is a minor groove binding, guanine-specific alkylating agent which also interacts with the microtubule network and blocks cell cycle progression at late S/G2. MATERIALS AND METHODS A soft agar cloning assay was used to determine the in vitro effects of ET-743 against primary human tumor specimens taken directly from patients. A total of 93 evaluable specimens were exposed to ET-743 for one-hour (n = 25) and/or 14-day continuous exposure (n = 92) at concentrations ranging from 0.1 nM to 1 microM. In vitro responses were defined as an inhibition > or = 50% of human tumor colony forming units at a given concentration. RESULTS One-hour exposure to ET-743 at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM and 1 microM induced in vitro responses in 0% (0/17), 6% (1/17), 16% (4/25), 13% (1/8), and 25% (2/8) of specimens, respectively. Continuous exposure to ET-743 at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM and 1 microM, inhibited 0% (0/16), 13% (2/16), 49% (44/90), 62% (47/76), and 77% (58/75) of tumor specimens, respectively. Tumor-specific responses and concentration-dependent relationships were observed with a continuous exposure to ET-743. At 100 nM, the compound inhibited 79% (11/14) breast, 69% (9/13) non-small-cell lung, 58% (7/12) ovary, and 88% (7/8) melanoma specimens. At 1 microM, ET-743 inhibited 100% (14/14) breast specimens, 85% (11/13) non-small-cell lung, 67% (8/12) ovary and 86% (6/7) melanoma specimens. Activity of ET-743 at and above 10 nM was also observed against sarcoma and kidney tumors. At 10 nM concentration and continuous exposure ET-743 demonstrated incomplete cross-resistance with paclitaxel, alkylating agents, doxorubicin and cisplatin. CONCLUSIONS Our data from the cloning assay indicate that the duration of exposure to ET-743 is an important factor in human tumors. Therefore, long-term exposure to ET-743 may be preferred in future clinical trials. The activity of ET-743 in breast, non-small-cell lung, and ovarian cancers as well as in melanoma may deserve further clinical evaluations. The potential of ET-743 in sarcoma and renal tumors might also be considered. In addition, our data indicate that a plasma concentration of 100 nM of ET-743 must be considered as a target during the clinical development of the compound; also the concept of continuous/protracted exposure in clinical trials with ET-743 has to be taken into account.

The effects of cytokines and growth factors on osteoblastic cells

In this short review, some regulatory mechanisms that are involved in the control of normal bone formation are proposed, based on several in vivo and in vitro models our group has utilized recently to study osteoblast differentiation and mineralized bone matrix formation. Of course, these proposals must be assessed in the light of the limitations of the models, which probably represent a simplification of the complex and different ways in which normal mammalian bone is formed at different sites. Nevertheless, it is likely that the same general types of control mechanisms are active in each of the different types of bone formation. In adult humans, bone formation predominantly occurs by remodeling, the process by which bone which has recently been resorbed by osteoclasts is replaced by teams of osteoblasts. Other types of bone formation such as endochondral bone formation and appositional bone formation are also important, particularly during growth and adolescence. The end results of each of these processes are the same, namely a complex mineralized proteinaceous bone matrix. These processes are modulated by systemic hormonal influences, which are particularly important with respect to pituitary hormones and sex steroids during growth and adolescence, and by local cellular microenvironmental differences. The former will not be discussed here. Rather, we will concentrate on the local events and factors which are likely involved in the bone formation process occurring during normal bone remodeling.

Systemic Administration of Acidic Fibroblast Growth Factor (FGF-1) Prevents Bone Loss and Increases New Bone Formation in Ovariectomized Rats

There are no universally accepted agents that will substantially increase bone mass in osteoporotic patients. A number of peptides important in normal bone formation, such as members of the transforming growth factor‐β superfamily, are not satisfactory for this purpose either because their beneficial effects are predominantly local or there is systemic toxicity associated with their administration. We have examined the effects of exogenous fibroblast growth factor‐1 and ‐2 (FGF‐1 and FGF‐2) on bone in vivo, since FGFs have been shown recently to be essential for normal skeletal development. FGF‐1 was injected daily (0.2 mg/kg intravenously) for 28 days into the tail vein of adult female rats immediately following and 6 months after sham operation or ovariectomy (OVX). In rats treated immediately post‐OVX, OVX produced more than a 30% decrease in tibial bone density, which was prevented by FGF‐1 and estrogen. However, FGF‐1 also had an anabolic effect. In sham‐operated rats, FGF‐1 increased bone density to 2‐fold, whereas estrogen had no effect. In rats 6 months post‐OVX, severe bone loss and disruption of trabecular microarchitecture occurred similar to that seen in patients with severe osteoporosis. In these rats, administration of FGF‐1 induced extensive new woven bone formation with new trabecular‐like structures filling much of the marrow spaces, and bone density in the tibial metaphysis increased 3‐fold. FGF‐1 and FGF‐2 were also administered subcutaneously over the calvaria of mice in doses of 2–2000 μg/day for 3 days and shown to produce substantial increases in bone formation when examined morphologically. Thus, we conclude that both local and systemic FGF‐1 increases new bone formation and bone density, and systemic FGF‐1 also appears to restore bone microarchitecture and prevent bone loss associated with estrogen‐withdrawal.

Herbimycin A, a pp60c-src tyrosine kinase inhibitor, inhibits osteoclastic bone resorption in vitro and hypercalcemia in vivo.

Since absence of expression of the c-src gene product in mice indicates that the pp60c-src tyrosine kinase is required and essential for osteoclastic bone resorption, we tested the effects of the antibiotic herbimycin A, which is an inhibitor of pp60c-src on osteoclastic bone resorption in vitro and on hypercalcemia in vivo. We examined the effects of herbimycin A on the formation of bone resorbing osteoclasts in mouse long-term marrow cultures, on isolated rodent osteoclasts and on bone resorption in organ cultures of fetal rat long bones stimulated by parathyroid hormone. We found that herbimycin A in concentrations of 1-100 ng/ml inhibited bone resorption in each of these systems. We determined the effects of herbimycin A (100 ng/ml) on src tyrosine kinase activity in mouse marrow cultures and found that it was decreased. Herbimycin A also decreased elevated blood calcium levels that were induced either by repeated subcutaneous injections of recombinant human interleukin-1 alpha or by a human tumor. There was no evidence for toxicity in any of these culture systems or in mice treated with herbimycin A. A different tyrosine kinase inhibitor that does not inhibit pp60c-src was used as a control and caused none of these effects. These data suggest that pp60c-src tyrosine kinase inhibitors may be useful pharmacologic inhibitors of osteoclastic bone resorption and hypercalcemia.

A Phase I Pharmacokinetic and Biological Correlative Study of Oblimersen Sodium (Genasense, G3139), an Antisense Oligonucleotide to the Bcl-2 mRNA, and of Docetaxel in Patients with Hormone-Refractory Prostate Cancer

Purpose: To assess the feasibility of administering oblimersen sodium, a phosphorothioate antisense oligonucleotide directed to the Bcl-2 mRNA, with docetaxel to patients with hormone-refractory prostate cancer; to characterize the pertinent pharmacokinetic parameters, Bcl-2 protein inhibition in peripheral blood mononuclear cell(s) (PBMC) and tumor; and to seek preliminary evidence of antitumor activity. Experimental Design: Patients were treated with increasing doses of oblimersen sodium administered by continuous i.v. infusion on days 1 to 6 and docetaxel administered i.v. over 1 h on day 6 every 3 weeks. Plasma was sampled to characterize the pharmacokinetic parameters of both oblimersen and docetaxel, and Bcl-2 protein expression was measured from paired collections of PBMCs pretreatment and post-treatment. Results: Twenty patients received 124 courses of the oblimersen and docetaxel combination at doses ranging from 5 to 7 mg/kg/day oblimersen and 60 to 100 mg/m2 docetaxel. The rate of severe fatigue accompanied by severe neutropenia was unacceptably high at doses exceeding 7 mg/kg/day oblimersen and 75 mg/m2 docetaxel. Nausea, vomiting, and fever were common, but rarely severe. Oblimersen mean steady-state concentrations were 3.44 ± 1.31 and 5.32 ± 2.34 at the 5- and 7-mg/kg dose levels, respectively. Prostate-specific antigen responses were observed in 7 of 12 taxane-naïve patients, but in taxane-refractory patients no responses were observed. Preliminary evaluation of Bcl-2 expression in diagnostic tumor specimens was not predictive of response to this therapy. Conclusions: The recommended Phase II doses for oblimersen and docetaxel on this schedule are 7 mg/kg/day continuous i.v. infusion days 1 to 6, and 75 mg/m2 i.v. day 6, respectively, once every 3 weeks. The absence of severe toxicities at this recommended dose, evidence of Bcl-2 protein inhibition in PBMC and tumor tissue, and encouraging antitumor activity in HPRC patients warrant further clinical evaluation of this combination.

Serum Protein Expression Profiling for Cancer Detection: Validation of a SELDI-Based Approach for Prostate Cancer

Multiple studies have reported that analysis of serum and other bodily fluids using surface enhanced laser desorption/ionization time of flight mass spectroscopy (SELDI-TOF-MS) can identify a “fingerprint” or “signature” of spectral peaks that can separate patients with a specific disease from normal control patients. Ultimately, classification by SELDI-TOF-MS relies on spectral differences in position and amplitude of resolved peaks. Since the reproducibility of quantitation, resolution and mass accuracy of the SELDI-TOF-MS, or any high throughput mass spectrometric technique, has never been determined this method has come under some skepticism as to its clinical usefulness. This manuscript describes a detailed design of a three-phase study to validate the clinical usefulness of SELDI-TOF-MS in the identification of patients with prostatic adenocarcinoma (PCA). At the end of this validation study, the usefulness of the general SELDI-TOF-MS approach to identifying patients with PCA will be demonstrated and how it compares with PCA diagnosis by measuring prostate specific antigen.

Antiproliferative effects of recombinant human bone morphogenetic protein-2 on human tumor colony-forming units.

Bone morphogenetic protein-2 (BMP-2) is a differentiation factor for normal osteoblasts. BMP-2 is structurally related to transforming growth factor-β which inhibits cell proliferation and enhances apoptosis. A recent study has shown the presence of BMP-2 receptors on several cancer cell lines. In this study, we attempted to determine if recombinant human BMP-2 (rhBMP-2) can modulate the proliferation of human tumor colony-forming units taken from 113 patients. Tumor cells were cultured in soft agar and continuously exposed to three concentrations of rhBMP-2 (10,100 and 1000 ng/ml) for 14 days in the capillary cloning system. There were 65 evaluable specimens, including 17 breast cancers, 15 ovarian cancers, 14 non-small cell lung cancers and five prostate cancers. Importantly, rhBMP-2 did not stimulate the tumor cell proliferation. A significant inhibition (50% or less survival of tumor colony-forming units) was seen in 16 of 65 specimens (24.6%) at 1000 ng/ml, including five of 14 non-small cell lung cancers, five of 17 breast tumors and two of 15 ovarian tumors. A concentration-response relationship was observed (p< 0.001 by Mantel-extension test). The results of this study encourage further evaluation of the antiproliferative effects of rhBMP-2 against human cancers.

Preclinical and clinical strategies for development of telomerase and telomere inhibitors

BACKGROUND Telomerase is an important enzyme whose activity has been convincingly demonstrated in humans recently. It is required for maintenance of ends of chromosomes (telomeres) during cell division. Since its presence has been selectively demonstrated in dividing cells including tumor cells, it has generated considerable excitement as a potential anti-cancer strategy. DESIGN In this article, we review the current relevant biology of the enzyme, the challenges encountered in the preclinical phase of target development and the current efforts that focus on telomeres and telomerase as therapeutic targets. We also speculate on the potential toxicities and mechanisms of resistance that may be encountered during use of such therapies.