Michael R. Van Scott

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.

Cardiac and Vasular Changes in Mice After Exposure to Ultrafine Particulate Matter

Increased ambient air particulate matter (PM) concentrations are associated with risk for myocardial infarction, stroke, and arrhythmia, and ultrafine PM (UFPM) might be particularly toxic to the cardiovascular system. Recent epidemiological studies are beginning to offer mechanistic insights, yet the rodent model remains a valuable tool to explore potential mechanisms. This article reviews a series of studies from our laboratory demonstrating the promise of mouse models to link health effects to biological mechanisms. Specifically, data from 6- to 10-wk-old male ICR mice exposed to intratracheal instillation of 100 μ g of UFPM collected from the Chapel Hill, NC airshed are described. Studies of ischemia/reperfusion, vascular function, and hemostasis are described. In summary, UFPM exposure doubles the size of myocardial infarction attendant to an episode of ischemia and reperfusion while increasing postischemic oxidant stress. UFPM alters endothelial-dependent and -independent regulation of systemic vascular tone; increases platelet number, plasma fibrinogen, and soluble P-selectin levels; and reduces bleeding time, implying enhanced thrombogenic potential. Taking these findings together, this model of acute UFPM exposure in the mouse indicates that UFPM induces a prothrombotic state and decreases vasomotor responsiveness, thereby offering insight into how UFPM could contribute to vascular events associated with thrombosis and ischemia and increasing the extent of infarction.

Sense and antisense: therapeutic potential of oligonucleotides and interference RNA in asthma and allergic disorders.

Advances in our understanding of asthma pathogenesis and delineation of the human genome project are yielding novel candidate targets for therapeutic intervention. In parallel with target identification, the past decade has produced novel approaches to normalizing expression genes that are upregulated in disease processes. Single-stranded antisense oligonucleotides and double-stranded short-interfering RNA molecules, which specifically mark target transcripts for degradation, are being investigated for their ability to modulate disease processes. In both cases, the targets are RNA transcripts, and not protein; therefore, all types of molecular gene products can be inhibited, including historically undrugable species such as transcription factors and phosphatases. Various RNA interference strategies have been successfully tested in vitro and in animal models of disease, and data is beginning to accumulate from human clinical trials.EPI-2010, a 21-mer phosphorothioate against the adenosine A1 receptor promoter region, has completed preclinical pharmacology testing and its initial clinical trials. The rationale for EPI-2010 is that overactivity of the adenosine signaling pathway in asthmatic lungs contributes to airway inflammation and hyperresponsiveness. Phase I/IIa clinical trials have shown EPI-2010 to be safe and well-tolerated, with modest indications of efficacy in patients with mild asthma.

A circadian clock in murine bone marrow-derived mast cells modulates IgE-dependent activation in vitro

Circadian rhythm is expressed in most organisms, and many functions and parameters in the immune system are associated with time-of-day. However, it is largely unknown if local circadian clocks in immune cells directly control physiological outcomes. We hypothesized that a circadian clock in murine bone marrow derived mast cells (BMMCs) modulates IgE-dependent activation in vitro. Mature BMMCs, grown from bone marrow of C57BL/6 mice, were synchronized with serum rich media (50% horse serum). Total RNA was harvested from BMMCs at 4 h intervals for up to 72 h following synchronization and expression of circadian genes (mPer1, mPer2, Bmal1, Rev-erbα, and Dbp) was measured by quantitative PCR. Serum shock synchronized expression of circadian genes (mPer2, Bmal1, Rev-erbα, and Dbp) in BMMCs. Synchronized BMMCs stimulated via the high affinity IgE receptor (FcεRI) at different time intervals display circadian rhythms in IL-13 and IL-6 mRNA expression. The expression of fcer1a gene and FcεRIα protein displayed a circadian pattern following serum shock, with mean periods of 18.9 and 28.6 h, respectively. These results demonstrate that synchronized BMMCs provide an in vitro model to study circadian mechanism(s) associated with allergic disease and that circadian oscillation of cytokine production following IgE-dependent activation is at least in part due to circadian oscillation of FcεRIα.

Systemic administration of an Fcγ–Fcε-fusion protein in house dust mite sensitive nonhuman primates

Crosslinking Fc(epsilon)RI and FcgammaRIIB receptors inhibits mast cell and basophil activation, decreasing mediator release. In this study, a fusion protein incorporating human Fcgamma and Fc(epsilon) domains, hGE2, was shown to inhibit degranulation of human mast cells and basophils, and to exhibit efficacy in a nonhuman primate model of allergic asthma. hGE2 increased the provocative concentration of dust mite aeroallergen that induced an early phase asthmatic response. The treatment effect lasted up to 4 weeks and was associated with reduction in the number of circulating basophils and decreased expression of Fc(epsilon)RI on repopulating basophils. Repeat hGE2 dosing induced production of serum antibodies against human Fcgamma and Fc(epsilon) domains and acute anaphylaxis-like reactions. Immune serum induced histamine release from human IgE or hGE2-treated cord blood-derived mast cells and basophils in vitro. These results indicate that repeat administration with hGE2 induced an antibody response to the human molecule that resulted in activation rather than inhibition of allergic responses.

Biomarkers of Disease and Treatment in Murine and Cynomolgus Models of Chronic Asthma

Background Biomarkers facilitate early detection of disease and measurement of therapeutic efficacy, both at clinical and experimental levels. Recent advances in analytics and disease models allow comprehensive screening for biomarkers in complex diseases, such as asthma, that was previously not feasible. Objective Using murine and nonhuman primate (NHP) models of asthma, identify biomarkers associated with early and chronic stages of asthma and responses to steroid treatment. Methods The total protein content from thymic stromal lymphopoietin transgenic (TSLP Tg) mouse BAL fluid was ascertained by shotgun proteomics analysis. A subset of these potential markers was further analyzed in BAL fluid, BAL cell mRNA, and lung tissue mRNA during the stages of asthma and following corticosteroid treatment. Validation was conducted in murine and NHP models of allergic asthma. Results Over 40 proteins were increased in the BAL fluid of TSLP Tg mice that were also detected by qRT-PCR in lung tissue and BAL cells, as well as in OVA-sensitive mice and house dust mite-sensitive NHP. Previously undescribed as asthma biomarkers, KLK1, Reg3γ, ITLN2, and LTF were modulated in asthmatic mice, and Clca3, Chi3l4 (YM2), and Ear11 were the first lung biomarkers to increase during disease and the last biomarkers to decline in response to therapy. In contrast, GP-39, LCN2, sICAM-1, YM1, Epx, Mmp12, and Klk1 were good indicators of early therapeutic intervention. In NHP, AMCase, sICAM-1, CLCA1, and GP-39 were reduced upon treatment with corticosteroids. Conclusions and clinical relevance These results significantly advance our understanding of the biomarkers present in various tissue compartments in animal models of asthma, including those induced early during asthma and modulated with therapeutic intervention, and show that BAL cells (or their surrogate, induced sputum cells) are a viable choice for biomarker examination.

Identification and Characterization of GAL-021 as a Novel Breathing Control Modulator.

Background: The authors describe the preclinical pharmacological properties of GAL-021, a novel peripheral chemoreceptor modulator. Methods: The ventilatory effects of GAL-021 were characterized using tracheal pneumotachometry (n = 4 to 6), plethysmography (n = 5 to 6), arterial blood gas analyses (n = 6 to 11), and nasal capnography (n = 3 to 4) in naive animals and those subjected to morphine-induced respiratory depression. Morphine analgesia in rats was evaluated by tail-flick test (n = 6). Carotid body involvement in GAL-021 ventilatory effects was assessed by comparing responses in intact and carotid sinus nerve–transected rats. Hemodynamic effects of GAL-021 were evaluated in urethane-anesthetized rats (n = 7). The pharmacological profile of GAL-021 in vitro was investigated using radioligand binding, enzyme inhibition, and cellular electrophysiology assays. Results: GAL-021 given intravenously stimulated ventilation and/or attenuated opiate-induced respiratory depression in rats, mice, and nonhuman primates, without decreasing morphine analgesia in rats. GAL-021 did not alter mean arterial pressure but produced a modest increase in heart rate. Ventilatory stimulation in rats was attenuated by carotid sinus nerve transection. GAL-021 inhibited KCa1.1 in GH3 cells, and the evoked ventilatory stimulation was attenuated in Slo1 −/− mice lacking the pore-forming &agr;-subunit of the KCa1.1 channel. Conclusions: GAL-021 behaved as a breathing control modulator in rodents and nonhuman primates and diminished opioid-induced respiratory depression without compromising opioid analgesia. It acted predominantly at the carotid body, in part by inhibiting KCa1.1 channels. Its preclinical profile qualified the compound to enter clinical trials to assess effects on breathing control disorders such as drug (opioid)-induced respiratory depression and sleep apnea.

Pulmonary allergic reactions impair systemic vascular relaxation in ragweed sensitive mice.

Asthma is often associated with cardiovascular complications, and recent observations in animal models indicate that induction of pulmonary allergic inflammation increases susceptibility of the myocardium to ischemia and reperfusion injury. In this study, we used a murine model of allergen sensitization in which aspiration of allergen induces pulmonary and systemic inflammation, to test the hypothesis that pulmonary exposure to allergen alters vascular relaxation responses. BALB/C mice were sensitized by intraperitoneal injection of ragweed and challenged by intratracheal instillation of allergen. Airway hyperreactivity and pulmonary inflammation were confirmed, and endothelium-dependent and -independent reactivity of thoracic aorta rings were evaluated. Ragweed sensitization and challenge induced airway hyperreactivity to methacholine and pulmonary inflammation, but did not affect constrictor responses of the aortic rings to phenylephrine and K+ depolarization. In contrast, maximal relaxation of aortic rings to acetylcholine and sodium nitroprusside decreased from 87.6±3.9% and 97.7±1.2% to 32±4% and 51±6%, respectively (p<0.05). The sensitivity to acetylcholine was likewise reduced (EC₅₀=0.26±0.05 μM vs. 1.09±0.16 μM, p<0.001). The results demonstrate that induction of allergic pulmonary inflammation in mice depresses endothelium-dependent and -independent vascular relaxation, which can contribute to cardiovascular complications associated with allergic inflammation.

Hyperosmolarity-Induced Dilation and Epithelial Bioelectric Responses of Guinea Pig Trachea in Vitro: Role of Kinase Signaling

Exercise-induced airway obstruction is thought to involve evaporative water loss and hyperosmolarity of the airway surface liquid. Hyperosmolar challenge of the epithelium of isolated, perfused guinea pig trachea rapidly alters transepithelial potential difference (Vt), and it elicits smooth muscle relaxation mediated by epithelium-derived relaxing factor (EpDRF). In many cell types, protein kinases mediate responses to hyperosmolarity and regulatory volume increase. In this study, inhibitors were used to investigate the involvement of kinases and phosphatases in bioelectric responses of epithelium to hyperosmolarity and their possible relationship to EpDRF-mediated relaxation. After contraction of the perfused trachea with extraluminal methacholine, d-mannitol applied intraluminally (≤80 mosM) increased Vt and elicited dilation of the smooth muscle with a similar concentration-dependence; higher concentrations decreased Vt. In tracheas exposed to 30 mosM d-mannitol (∼EC50), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580) and SKF 86002 [6-(4-fluorophenyl)-2,3-dihydro-5-(4-pyridyl)imidazo[2,1-b]thiazole] (p38 inhibitors) potentiated the dilation, whereas SP 600125 [anthra[1,9-cd]pyrazol-6(2H)-one-1,9-pyrazoloanthrone] and dicumarol [c-Jun NH2-terminal kinase (JNK) inhibitors], chelerythrine [nonselective protein kinase C (PKC) inhibitor], and NaAsO2 (mitogen-activated protein kinase stress inducer) and Na3VO4 (protein tyrosine phosphatase inhibitor) inhibited the hyperpolarization. Large increases in the phosphorylation of p38 and JNK occurred at concentrations higher than those needed to elicit functional responses. The phosphatidylinositol 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002) and Na3VO4 did not affect the Vt responses, but they inhibited methacholine-induced constriction; SP 600125 and dicumarol potentiated, and chelerythrine inhibited, methacholine-induced epithelial hyperpolarization. These results suggest that JNK, PKC, and phosphatase(s) are involved in hyperosmolarity-induced hyperpolarization of the tracheal epithelium but that p38 is involved in EpDRF-mediated relaxation.

Nocturnal thoracoabdominal asynchrony in house dust mite-sensitive nonhuman primates

Nocturnal bronchoconstriction is a common symptom of asthma in humans, but is poorly documented in animal models. Thoracoabdominal asynchrony (TAA) is a noninvasive clinical indication of airway obstruction. In this study, respiratory inductive plethysmography (RIP) was used to document nocturnal TAA in house dust mite (HDM)-sensitive Cynomolgus macaques. Dynamic compliance (Cdyn) and lung resistance (RL) measured in anesthetized animals at rest and following exposure to HDM allergen, methacholine, and albuterol were highly correlated with three RIP parameters associated with TAA, ie, phase angle of the rib cage and abdomen waveforms (PhAng), baseline effort phase relation (eBPRL) and effort phase relation (ePhRL). Twenty-one allergic subjects were challenged with HDM early in the morning, and eBPRL and ePhRL were monitored for 20 hours after provocation. Fifteen of the allergic subjects exhibited gradual increases in eBPRL and ePhRL between midnight and 6 am, with peak activity at 4 am. However, as in humans, this nocturnal response was highly variable both between subjects and within subjects over time. The results document that TAA in this nonhuman primate model of asthma is highly correlated with Cdyn and RL, and demonstrate that animals exhibiting acute responses to allergen exposure during the day also exhibit nocturnal TAA.