Jonathan W. Nyce

Dehydroepiandrosterone and Structural Analogs: A New Class of Cancer Chemopreventive Agents

Publisher Summary Over the past several years, several therapeutic effects of dehydroepiandrosterone (DHEA) in laboratory mice and rats have been demonstrated. These include cancer preventive, possible antiautoimmune, and antiatherogenic effects as well as antiobesity and antidiabetic activities. However, certain simplicity in the mechanism of the action of the DHEA class of steroids is now beginning to emerge. The well-documented capacity of DHEA to inhibit mammalian glucose-6-phosphate dehydrogenase (G6PDH)—the rate limiting enzyme in the pentose phosphate pathway, a major source of cytosolic NADPH—now appears central to the mechanism of the cancer preventive action of this steroid. Several laboratories have undertaken determinations of DHEAS plasma levels in women with breast cancer and in matched controls. The predominance of evidence in these case-control studies suggests that the women with breast cancer have subnormal DHEAS plasma concentrations. However, as in all case-control studies, it is uncertain whether the presence of the measured abnormality proceeds or is a consequence of the disease.

Clinical Potential of Respirable Antisense Oligonucleotides (RASONs) in Asthma

The human genome project, as well as advances in our understanding of asthma susceptibility, are yielding novel candidate targets for disease intervention. The normalization of up-regulated gene expression may treat or improve the disease outcome. However, only some of these gene product targets may be ‘tractable’, i.e. amenable to blockade by small, orally active, organic molecules. The remainder have been termed ‘non-tractable’.For over a decade, antisense oligonucleotides (ASONs) have been used as tools to evaluate the importance of specific gene products in vitro. In recent years evidence has accumulated indicating their potential as a viable new therapeutic approach in their own right, being able to block ‘non-tractable’ targets as well as ‘tractable’ targets.Distribution, cell-specific uptake, and effectiveness of aerosolized phosphorothioate ASONs are currently being evaluated in animal models. The results demonstrate broad distribution throughout the lung, and uptake by all of the cell types examined to date. Functionality has been demonstrated against diverse targets, including nuclear transcription factors, tyrosine kinases, G-protein coupled receptors, cytokine receptors, growth factors, and chemokines.EPI-2010, a respirable ASON (RASON) against the adenosine A1 receptor, is the first test case for this new class of respiratory therapeutics. The rationale for EPI-2010 is that overactivity of the adenosine-signaling pathway in asthmatic lungs contributes to airway inflammation and hyperresponsiveness. EPI-2010 binds to the initiation codon of the adenosine A1 receptor mRNA, and thereby blocks translation and targets the message for degradation by RNase. EPI-2010 is apparently metabolized locally by endogenous nucleases confining its activity to the airways. Phase I clinical trials have shown EPI-2010 to be well-tolerated, with indications of efficacy.In conclusion, one important application of RASONs is in addressing up-regulated disease targets, only some of which are ‘tractable’ by small molecules. It is hoped that this will yield new therapeutic options to the benefit of patients with asthma and allergic disorders.

Discovery and development of respirable antisense therapeutics for asthma.

Respirable antisense oligonucleotides (RASONs) represent a novel class of respiratory therapeutic molecules with the potential to specifically address the challenges posed by the successes of the Human Genome Program, namely, the need to rapidly identify the critical pulmonary disease-relevant drugable targets from the vast pool of 30,000-40,000 human genes and to discover and develop drugs that specifically attack these targets. We have shown that EPI-2010, a RASON targeting the adenosine A1 receptor, a G-protein coupled receptor that has been implicated in the regulation of three major determinants of asthma, can be delivered directly to the target disease tissue as an aerosol formulation. In vivo efficacy, absorption, distribution, metabolism, and excretion (ADME), and safety studies of inhaled EPI-2010 employing animal models of human asthma suggest that the RASON approach enables the specific delivery of efficacious, safe, and long-acting doses of phosphorothioate oligonucleotides to the respiratory tract. Moreover, these data indicate that RASONs truly have the potential to address the respiratory drug discovery bottleneck of the postgenomic era, that is, the ability to rapidly validate disease targets and develop pulmonary disease therapeutics for these validated targets.

Gene silencing in mammalian cells by direct incorporation of electroporated 5-methyl-2′-deoxycytidine 5′-triphosphate

DNA methylation is an important process contributing to transcriptional regulation in animal and plant cells. We observed that electroporation of Chinese hamster V-79 cells in the presence of millimolar concentrations of 5-methyl-2′-deoxycytidine 5′-triphosphate (5mdCTP) led to high-level direct incorporation of this nucleotide into DNA polymer. Under optimum conditions, approximately 2×108 molecules of 5mdCTP were calculated to have been incorporated into each unit genome (6 pg of DNA). Since a diploid mammalian genome contains approximately 1.2–1.5×109 cytosines, this level of 5mdCTP incorporation corresponds to substitution of up to 16.6% of total cytosines by 5-methylcytosine, or about 100–150 new methylated cytosines per average gene. The pattern of genomic methylation produced under these conditions differed from that produced physiologically in that 5mdCTP was substituted into DNA at random cytosines, superimposing a novel methylation pattern upon that derived from the normal enzyme-driven postreplicational process. This novel pattern of methylation showed no preference for CpG dinucleotides, but was nevertheless found capable of silencing HPRT gene expression and producing a condition of resistance to 6-thioguanine. The epigenetic nature of this gene silencing event was shown by the very high level of reexpression of HPRT mRNA following exposure of HPRT− cells to the demethylating agent 5-azadeoxycytidine.

RASONs: a novel antisense oligonucleotide therapeutic approach for asthma

Inhalation based approaches enable the local delivery of antisense oligonucleotides (ASONs) to the respiratory tract and thus facilitate the ability of ASONs to target and modulate the activity of discordantly expressed respiratory disease genes. Studies involving EPI-2010, a respirable antisense oligonucleotide (RASON), targeting the adenosine A1 receptor, a G-protein-coupled-receptor (GPCR) that plays an important role in the aetiology of asthma, demonstrate that ASON therapeutics can be delivered directly to the lung as an aerosol. EPI-2010 has been shown to inhibit adenosine A1 receptor expression and significantly improve allergen-induced airway obstruction and bronchial hyper-responsiveness in animal models of human asthma. Absorption, tissue distribution, metabolism and excretion (ADME) and safety studies of aerosolised EPI-2010 suggest that phosphorothioate RASONs can be delivered to target respiratory tissues in low, safe, efficacious and long-acting doses. This supports the concept that RASONs offer the potential to address a variety of respiratory targets including those for which approaches employing systemic distribution and systemic bioavailability of the therapeutic agent may be undesirable. In addition, our studies with EPI-2010 indicate that the RASON approach may represent a technology that is uniquely positioned to address the challenges of the post-genome era in respiratory drug discovery, since it enables simultaneous in vivo target validation and antisense therapeutic discovery in an accelerated timeframe.

Respirable Antisense Oligonucleotide (RASON) Therapy for Allergic Asthma

A new technology for treating respiratory disease, respirable antisense oligonucleotides (RASONs), has recently been developed by our group. RASONs are short, single-stranded nucleic acids, generally modified to reduce degradation. They differ from traditional drugs, which usually antagonise preformed proteins already functioning in a disease process. Instead, RASONs can attenuate the expression of disease-associated genes by targeting the messenger RNA (mRNA). Delivered directly to the target tissue, the lung, they avoid the problems of ineffective delivery encountered by other routes of administration. When an adenosine A1 antisense oligonucleotide was delivered to the lungs of allergic rabbits with up-regulated A1 adenosine receptors, desensitisation to the bronchoconstrictor effects of adenosine, histamine and a common aeroallergen (dust mite) occurred. The effect on A1 receptors persisted on average for nearly 7 days. RASON (the phosphorothioate antisense oligonucleotide EPI-2010) administered in low dosage was evenly distributed throughout the lung (with no detectable systemic active metabolites), and was excreted primarily in urine. These results demonstrate that RASONs can be efficiently and effectively delivered to the peripheral lung. They potently and selectively attenuate the expression of disease-associated genes, an approach to therapy which is now being extended to other potentially important mediators of bronchial asthma.

Induction of peroxisomal enzymes in rat liver by dehydroepiandrosterone sulfate

Male F-344 rats, when treated with either 150 mg/kg or 300 mg/kg body weight of DHEAS for 14 days, produced a dose-dependent increase in liver weight and peroxisomal beta-oxidation activity, characteristic of peroxisomal proliferation. Contrary to previous observations in vitro, we also found a significant increase in catalase activity in rat liver with the higher dose of the steroid. Furthermore, the in vivo induction of peroxisomal beta-oxidation by DHEAS observed in our study was significantly less than reported in vitro, and also unlike previously reported in vitro results, was approximately equivalent to DHEA administered in vivo.

Autoradiographic evidence that intrastriatal administration of adenosine A1 receptor antisense oligodeoxynucleotide decreases adenosine A1 receptors in the rat striatum and cortex

In this study, the effect of a phosphorothioated A(1) adenosine receptor antisense oligodeoxynucleotide on A(1) receptor density and mRNA in the striatum and cortex of rats was determined. Receptor autoradiography and in situ hybridization revealed a reduction in striatal and cortical A(1) receptor density and cortical A(1) receptor mRNA, respectively, in antisense-treated brains but not in those treated with a mismatch oligonucleotide. There was no change in A(2) receptor binding. These data imply that the corticostriatal pathway synthesizes A(1) receptors and transports them to its terminals.

Hypotensive effects of [d-Tyr27,36,d-Thr32]neuropeptide Y(27–36)

An analogue of the 10 C-terminal amino acids of neuropeptide Y (NPY) containing three D-isomeric substitutions (27-36-D) has been synthesized and its cardiovascular activity studied in Sprague-Dawley (SD) and spontaneously hypertensive (SHR) rats. Intravenous administration of 1000 nmol/kg 27-36-D decreases MAP in SHR (-59.9 +/- 5.0 mmHg) and SD rats (-44.4 +/- 4.7 mmHg). The hypotension produced by 1000 nmol/kg 27-36-D diminished by 71.2% following pretreatment with the histamine receptor antagonist diphenhydramine, although histamine depletion with compound 48/80 does not significantly alter this hypotension. These data suggest that NPY (27-36)-D produces a profound and sustained hypotension in two strains of rat which is partially attributable to activity at histamine receptors.

Intravenous NPY (27–36)-d decreases cardiac output in conscious Sprague-Dawley rats

Intravenous (IV) administration of NPY (27-36)-D, a substituted carboxyterminal fragment of neuropeptide Y (NPY), decreases mean arterial pressure (MAP) in normo-and hypertensive rats by a mechanism partially involving histamine receptors. The purpose of this study is to further characterize the cardiovascular effects of NPY (27-36)-D. NPY (27-36)-D dose-dependently decreased MAP, cardiac output, and stroke volume without significantly altering peripheral resistance. Myocardial contractility diminished by 151.2 +/- 31.8, 529.6 +/- 182.5, and 495.4 +/- 66.7 mmHg/s2 in rats treated with 300, 500, and 750 nmol/kg NPY (27-36)-D, respectively. Therefore, NPY (27-36)-D modifies MAP, in part, by a reversible negative inotropic effect on the heart.