Paolo M. Renzi

Antisense Therapy against CCR3 and the Common Beta Chain Attenuates Allergen-induced Eosinophilic Responses

RATIONALE The drug product TPI ASM8 contains two modified phosphorothioate antisense oligonucleotides designed to inhibit allergic inflammation by down-regulating human CCR3 and the common beta chain (beta(c)) of IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors. OBJECTIVES This study examined the effects of inhaled TPI ASM8 on sputum cellular influx, CCR3 and beta(c) mRNA and protein levels, and the airway physiologic response after inhaled allergen. METHODS Seventeen subjects with mild atopic asthma were randomized in a crossover study to inhale 1,500 microg TPI ASM8 or placebo by nebulizer, once daily for 4 days. On Day 3, subjects underwent allergen inhalation challenge. Sputum samples were collected before and after allergen. CCR3 and beta(c) protein levels were measured by flow cytometry, mRNA was measured using real-time quantitative polymerase chain reaction, and the FEV1 was measured over 7 hours after challenge. MEASUREMENTS AND MAIN RESULTS Compared with placebo, TPI ASM8 inhibited sputum eosinophil influx by 46% (P = 0.02) and blunted the increase in total cells (63%) after allergen challenge. TPI ASM8 significantly reduced the early asthmatic response (P = 0.04) with a trend for the late asthmatic response (P = 0.08). The allergen-induced (Day 2 to Day 3) levels of beta(c) mRNA and CCR3 mRNA in sputum-derived cells were inhibited by TPI ASM8 (P = 0.039 and P = 0.054, respectively), with no significant effects on the cell surface protein expression of CCR3 and beta(c) (P > 0.05). No serious adverse events were reported. CONCLUSIONS TPI ASM8 attenuates the allergen-induced increase in target gene mRNA and airway responses in subjects with mild asthma. Clinical trial registered with www.clinicaltrials.gov (NCT 00264966).

The CCR3 Receptor Is Involved in Eosinophil Differentiation and Is Up-Regulated by Th2 Cytokines in CD34+ Progenitor Cells

The involvement of chemokines in eosinophil recruitment during inflammation and allergic reactions is well established. However, a functional role for chemokines in eosinophil differentiation has not been investigated. Using in situ RT-PCR, immunostaining, and flow cytometric analysis, we report that human CD34+ cord blood progenitor cells contain CCR3 mRNA and protein. Activation of CD34+ progenitor cells under conditions that promote Th2 type differentiation up-regulated surface expression of the CCR3. In contrast, activation with IL-12 and IFN-γ resulted in a significant decrease in the expression of CCR3. Eotaxin induced Ca2+ mobilization in CD34+ progenitor cells, which could explain the in vitro and in vivo chemotactic responsiveness to eotaxin. We also found that eotaxin induced the differentiation of eosinophils from cord blood CD34+ progenitor cells. The largest number of mature eosinophils was found in cultures containing eotaxin and IL-5. The addition of neutralizing anti-IL-3, anti-IL-5, and anti-GM-CSF Abs to culture medium demonstrated that the differentiation of eosinophils in the presence of eotaxin was IL-3-, IL-5-, and GM-CSF-independent. These results could explain how CD34+ progenitor cells accumulate and persist in the airways and peripheral blood of patients with asthma and highlight an alternative mechanism by which blood and tissue eosinophilia might occur in the absence of IL-5.

Dose–response effects of TPI ASM8 in asthmatics after allergen

To cite this article: Gauvreau GM, Pageau R, Séguin R, Carballo D, Gauthier J, D’Anjou H, Campbell H, Watson R, Mistry M, Parry‐Billings M, Killian K, Renzi PM. Dose–response effects of TPI ASM8 in asthmatics after allergen. Allergy 2011; 66: 1242–1248.

Multitargeted Approach Using Antisense Oligonucleotides for the Treatment of Asthma

Abstract:  Asthma is characterized by inflammation and hyperresponsiveness related to the accumulation of inflammatory cells, particularly eosinophils, within the airways. We tested the hypothesis that a multitargeted approach is better than a single‐targeted approach in a rat model of asthma. We simultaneously delivered oligonucleotides (ODNs) targeting the chemokine receptor CCR3 and the common beta chain subunit of the receptors for IL‐3, IL‐5, and GM‐CSF at the time of ovalbumin challenge in sensitized Brown Norway rats. Fewer eosinophils were detected in bronchoalveolar lavage (BAL) of rats treated with both ODNs as compared to each ODN alone. Moreover, airway responsiveness to LTD4 was significantly decreased at lower doses in the 2 ODN‐treated groups compared to a single ODN. As ODN therapy has raised concerns of toxicity we therefore examined ODNs prepared with modified DNA bases, specifically 2′amino, 2′deoxyadenosine (DAP) in place of adenosine. In vivo, administration of individual DAP‐ODN was efficacious in inhibiting airway hyperresponsiveness, whereas delivery of 2 DAP‐ODNs (targeting CCR3 and common beta chain) reduced the influx not only of eosinophils but also lymphocytes and macrophages in the lungs of rats as compared to the unmodified ODNs. Blocking multiple inflammatory pathways simultaneously is more effective in preventing eosinophilia and airway hyperresponsiveness than inhibiting either pathway alone. The challenges associated with the development of a product containing two oligonucleotides in humans are discussed.

Comparison of inflammatory cell profile and Th2 cytokine expression in the ethmoid sinuses, maxillary sinuses, and turbinates of atopic subjects with chronic sinusitis

Chronic sinusitis is a common disease characterized by persistent inflammation of the sinus mucosa. This study was undertaken to investigate immunopathologic findings in biopsy specimens from the ethmoid sinuses, maxillary sinuses, and inferior nasal turbinates of 14 allergic subjects with chronic sinusitis. The composition of the inflammatory infiltrate in the three tissue sites was examined by immunocytochemistry with anti-CD3 (total T cells), anti-CD4 (helper T cells), anti-CD8 (suppressor T cells), anti-MBP (eosinophils), antitryptase (mast cells), and antichymase (mast cells) antibodies. These revealed a significant increase in the T-cell helper/suppressor ratio and eosinophils in the ethmoid sinus mucosa compared with those in the maxillary sinus mucosa and the inferior turbinate. Eosinophil numbers were also higher in the maxillary sinus than in the inferior turbinate. Mast cells were present in significantly higher numbers in the ethmoid sinus and inferior turbinate biopsy sections than in the maxillary sinus. With antisense, radiolabeled riboprobes, we used in situ hybridization to examine the expression of interleukin-4 and interleukin-5 transcripts. The density of cells expressing interleukin-4 transcripts was significantly higher in the inferior turbinate biopsy sections than in those from the ethmoid and maxillary sinuses. In addition, the number of interleukin-4 mRNA—positive cells was higher in the ethmoid than in the maxillary sinus mucosa. The density of interleukin-5 mRNA—positive cells was significantly higher in the ethmoid and maxillary sinuses than in the inferior turbinate. The results of this study indicate (1) a more intense inflammatory response in the ethmoid sinus than in the maxillary sinus and inferior turbinate in allergic chronic sinusitis and (2) different inflammatory responses in the upper airways that are dependent on the anatomic site. These findings have potential implications in the design of new therapeutic interventions for allergic chronic sinusitis. (Otolaryngol Head Neck Surg 1998;118:804–9.)

Advantageous toxicity profile of inhaled antisense oligonucleotides following chronic dosing in non-human primates.

TPI ASM8 and TPI 1100 are two products containing modified phosphorothioate antisense oligonucleotides (AONs), which are undergoing development for the treatment of asthma and chronic obstructive pulmonary disease (COPD), respectively. TPI ASM8 is comprised of two AONs, one targeting the human chemokine receptor 3 (CCR3) and the other targeting the common beta-chain of the IL-3/IL-5/GM-CSF receptors. TPI 1100 is also a dual-AON compound targeting the phosphodiesterase (PDE) 4 and 7 isotypes. For both products, the AONs are present in a 1:1 ratio by weight. Both products will be administered by inhalation to patients, and TPI ASM8 is currently undergoing Phase 2 clinical trials. As part of the safety assessment of both products, the toxicity and disposition (i.e., pharmacokinetics of the AON components in plasma and tissues) were investigated in 14-day inhalation studies in monkeys at doses ranging from 0.05 to 2.5mg/kg/day. Results indicated that both products were safe and well tolerated at all dose levels. Reversible treatment-related alterations were only observed at the high dose levels tested and were limited to changes in the respiratory tract which were characterized primarily by the presence of alveolar macrophages in the absence of a generalized inflammatory response. Plasma pharmacokinetic profiles showed very low plasma concentrations, and no plasma accumulation was observed after repeated doses. While significant amounts of the AONs of both TPI ASM8 and TPI 1100 were measured in trachea and lung, only limited amounts of the AONs could be measured in kidney and liver, which, in combination with the low plasma level data, is indicative of very low systemic exposure. Taken together, these results demonstrate that these two new AON-based products are safe and that delivery via the inhaled route achieves localized deposition in the pulmonary tract with very limited systemic exposure and reduced toxicity compared to other routes of AON administration.

Characterization of Antisense Oligonucleotides Comprising 2′-Deoxy-2′-Fluoro-β-d-Arabinonucleic Acid (FANA)

Abstract:  Antisense oligonucleotides (AON) are being developed for a wide array of therapeutic applications. Significant improvements in their serum stability, target affinity, and safety profile have been achieved with the development of chemically modified oligonucleotides. Here, we compared 2′‐deoxy‐2′‐fluoro‐β‐D‐arabinonucleic acid (FANA)‐containing AONs with phosphorothioate oligodeoxynucleotides (PS‐DNA), 2′‐O‐methyl‐RNA/DNA chimeras and short interfering RNAs (siRNA) with respect to their target knockdown efficacy, duration of action and resistance to nuclease degradation. Results show that two different configurations of FANA/DNA chimeras (altimers and gapmers) were found to have potent antisense activity. Specific target inhibition was observed with both FANA configurations with an estimated EC50 value comparable to that of an siRNA but 20‐to 100‐fold lower than the other commonly used AONs. Moreover, the FANA/DNA chimeras showed increased serum stability that was correlated with sustained antisense activity for up to 4 days. Taken together, these results indicate that chimeric FANA/DNA AONs are promising new tools for therapeutic gene silencing when increased potency and duration of action are required.

Report of an Expert Panel to Review the Socio-Economic Models and Related Components Supporting the Development of Canada-Wide Standards (CWS) for Particulate Matter (PM) and Ozone To the Royal Society of Canada

Canada-Wide Standards (CWS) for particulate matter (PM) and ozone may be the most ambitious environmental standards ever proposed in Canada. They have attracted considerable attention and debate. This report addresses the validity of the socioeconomic modeling aspects of the Canada-Wide Standards development process. Socioeconomic considerations are addressed in one of eight principles underlying the development and attainment of CWS, according to a CWS subagreement signed by the Canadian Council of Ministers of the Environment (CCME). Principle 3.1.7 states that measures to attain agreed-upon Canada-Wide Environmental Standards will be determined in a sustainable development context, recognizing environmental and socio-economic considerations. This work has been previously published by the Royal Society of Canada, Ottawa (Ontario), in June 2001, appearing as “Review of Socio-Economic Models and Related Components Supporting the Development of Canadian Standards for Particulate Matter and Ozone,” ISBN 0-920064-73-6. A CCME Framework for Socio-economic Analyses in Setting Environmental Standards (CCME, 1998) describes procedures and information requirements for socioeconomic assessments of potential or proposed environmental standards. This framework states that while it may not be possible or necessary to carry out all of the analytical steps because of time, data, or resource constraints, a partial assessment can produce information that is useful for policy deliberations. The framework also notes that socioeconomic findings are not intended to be prescriptive concerning decisions about environmental standards because other input factors such as toxicity, epidemiology, ecological consequences and geographical distribution of effects, and other equity considerations are also necessary and important to an informed choice with respect to standard setting. Socioeconomic considerations are also specified under Government of Canada regulatory policy requiring federal regulatory authorities to demonstrate that the benefits of regulatory requirements are greater than their costs. Regulatory authorities must “ensure that the benefits outweigh the costs to Canadians, their governments and businesses. In particular, when managing risks on behalf of Canadians, regulatory authorities must ensure that the limited resources available to government are used where they do the most good” (Government of Canada, 1999). This implies not only that benefits should be greater than costs, but also that benefits minus costs, or net benefits are to be maximized, which means an attempt should be made to make standards efficient. The objective of the expert panel process was to provide an independent, expert review and critique of the socioeconomic analyses (SEA)—in this case a cost-benefit analysis (CBA)—conducted in developing the Canada-Wide Standards on PM and ozone. Through a review of the models and associated data and assumptions used in the analyses, the panel was asked to produce a report to address the following questions: a. What are the strengths, merits, limitations, gaps, and the degree of uncertainties of the proposed approaches, models, and their inputs and outputs? b. By what means could the models and analytical approaches be improved, so as to minimize uncertainties and maximize the relevance, reliability, and utility of outputs? c. What other approaches and/or tools could be used to conduct these analyses? The benefits and costs associated with Canada-Wide Standards for PM and ozone are highly uncertain and controversial. Uncertainties are associated with each step in the analysis of benefits and costs—including the link between emission reductions and ambient air quality, the extent to which human health and the environment are affected by changes in ambient levels of pollutants, the economic values (as measures of preferences) associated with improvements in environmental and human health, accuracy of the emissions inventory and projections of what this inventory and other factors will be in a future baseline, and the scope and magnitude of economic costs associated with emission reductions, both to industry and to society. With uncertainties so pervasive, analysts are required to make many choices and assumptions in estimating costs and benefits. For example, while it is clear that the epidemiological association between PM and excess mortality is consistent and robust, there are many remaining gaps in current understanding of the relative toxicity of PM components and gaseous copollutants and the magnitude of potential life-shortening effects. These uncertainties introduce possible biases into the estimation of PM-related health benefits. The panel draws the following major conclusions from its review of the CBA undertaken for the development of CWS for PM and ozone and from the academic and policy literature relevant to this topic. In drawing these conclusions, the panel views the use of a structured approach to the examination of costs and benefits as a positive development in Canadian regulatory policy analysis. 1. The CWS socioeconomic analysis (SEA) was in fact limited to a cost-benefit analysis (CBA). Because the CWS implementation of the SEA process was judged by the panel to be limited to a CBA, it was reviewed as such. CBA is just one of many available decision support tools. The requirements of CBA in the CWS process depend on whether the purpose is to select an ambient air quality standard or to guide and evaluate the implementation process. The extent to which the results of the CBA can inform the CWS decision process is limited for various reasons including the following: • Provinces are required to establish implementation plans to ensure that CWS will be met. Therefore, when doing a prospective CBA, the measures to be implemented (i.e., revised emission control regulations) are necessarily undetermined, as are the compliance levels for the revised control regulations that will be achieved in various provinces. • The distributional impacts of both costs and benefits have not been assessed. The panel acknowledges that the CWS Development Committee for PM and Ozone describes the cost estimation as “preliminary and, in some instances, a cursory analysis used to provide a macro level order of magnitude perspective on the costs associated with the various optional levels for PM and ozone CWSs” and notes that caution should be exercised in their interpretation (Canada-Wide Standards Development Committee for PM and Ozone, 1999). Deficiencies in Canadian data and modeling capabilities and limited time and resources restricted the scope of the analysis that could be undertaken for the CWS process. 2. Credible CBA should be conducted to support the development of Canada-wide standards. The panel recommends that CBA be used to inform decision makers about the projected costs and anticipated benefits of CWS. CBA needs to be designed to distinguish between the costs and benefits of meeting alternative PM and ozone standards within the limits of current science. There are potential overlaps in the estimation of costs and benefits for PM and ozone because emission control strategies will impact both PM and ozone levels and it is not clear which components of the air pollution mix are responsible for the various health effects. These uncertainties in the CBA need to be clearly communicated. At its best, CBA provides the decision maker with a systematic identification, estimation, and measure of uncertainty of monetary values for the relevant costs and benefits of interest to decision makers and stakeholders. To be fully informative, the CBA results provided to stakeholders and decision makers need to adequately analyze and explain the major sources of uncertainty in the inputs of the CBA model projections and their likely effect on model outputs. 3. In view of the importance of the proposed regulatory decision, the CBA performed for the CWS for PM and ozone is deficient in relation to the state-of-the-art for CBA. If the CWS CBA was intended to provide an adequate basis for balancing costs and benefits and for influencing where the CWS should be set, this CBA was not up to the task. If the objective was strictly to confirm that costs were not exorbitant for CWS that were deemed to be both technically feasible and associated with some substantial benefits, then this CBA provided contributions toward those judgments. When judged against the elements of process and structure of CBA required for credibility as indicated in conclusions 4 and 5 that follows, the panel finds that the CWS CBA does not satisfy these requirements and does not meet a reasonable level of quality for a CBA to support a decision of this import. While the CWS CBA has some value as a scoping analysis and provides a limited degree of guidance for decision-makers, it requires substantial improvement to meet the criteria for credibility. 4. The process for using CBA in CWS needs improvement. A Discussion Paper on Particulate Matter (PM) and Ozone Canada-Wide Standard Scenarios for Consultation prepared by the Canada-Wide Standards Development Committee for PM and Ozone (May 1999) states that “in selecting PM and ozone CWS level scenarios for stakeholder considerations, an attempt was made to balance the anticipated benefits of improved air quality with the technological feasibility and costs of achieving those improvements.” But it is apparent that the standard CBA procedure of comparing incremental benefits for tighter standards with incremental costs was not done for the CWS CBA process. Timelines for the analysis were exceedingly short, and the CBA effort appeared to be underfunded, resulting in short-cuts that substantially reduced the credibility of the analysis. Reporting and communicating the CBA results was also ineffective, particularly in terms of conveying a clear understanding of what was done and why it was done as it was. 5. The panel

Depletion of CD8+ T cells enhances pulmonary inflammation but not airway responsiveness after antigen challenge in rats

BACKGROUND CD8+ (OX-8+) T cells may suppress airway inflammation and airway responsiveness after allergen challenge. OBJECTIVE We studied the effects of depletion of OX-8+ T cells on allergen-induced lung eosinophilia and airway responsiveness in the Sprague-Dawley rat. METHODS Sprague-Dawley rats were sensitized to ovalbumin and challenged by aerosol 14 days later. Test animals received either low-dose (2 mg, n = 9) or high-dose (3 mg, n = 7) OX-8 monoclonal antibody (mAb), whereas controls (n = 8) received BALB/c ascites fluid. A fourth group of animals (n = 10) was not sensitized to ovalbumin and also received ascites fluid. Twenty-four hours after ovalbumin challenge, responsiveness to methacholine was measured, and lung inflammation was assessed in the large airways and small airways and parenchyma. RESULTS Circulating and airway CD8+ T cells were decreased by OX-8 mAb administration with greatest changes in animals treated with high-dose OX-8 mAb compared with controls (blood: 1.0% +/- 3.6% vs 18.7% +/- 3.9%, p < 0.05); (large airways: 2.5% +/- 1.2% vs 13.8% +/- 1.2%, p < 0.05). Ovalbumin challenge resulted in increases in macrophages and neutrophils in the small airways and parenchyma of sensitized compared with unsensitized rats (p < 0.05). High-dose OX-8 mAb further increased total leukocytes, attributable to increases in neutrophils and eosinophils, retrieved from the large airways and small airways and parenchyma compared with other groups (p < 0.05). Airway responsiveness to methacholine was not significantly different between control and ovalbumin-challenged animals and was not augmented by OX-8 pretreatment. CONCLUSION CD8+ T cells modulate the extent of allergen-induced airway inflammation. However, the enhancement of inflammation was not sufficient to affect airway responsiveness.

Effect of nedocromil sodium on allergen-induced airway responses and changes in the quantity of airway smooth muscle in rats

BACKGROUND Allergen exposures induce growth of airway smooth muscle in the Brown Norway rat. OBJECTIVE The purpose of the study was to examine the role of mediators associated with the early and late responses in the induction of airway smooth muscle growth. METHODS Nedocromil sodium was administered to block early and late responses in ovalbumin-sensitized and ovalbumin-challenged rats undergoing single or multiple challenges (5 times at 5-day intervals) with ovalbumin. Airway smooth muscle was quantitated by morphometry on lungs removed 2 days after the final challenge. RESULTS Nedocromil sodium administered before ovalbumin challenge blocked both the early and late responses. When administered 2 hours after ovalbumin challenge, it also blocked the late response. Rats undergoing challenge with aerosolized ovalbumin five times at 5-day intervals were also treated with nedocromil before (n = 10) or 2 hours after (n = 10) each ovalbumin inhalation, respectively. The quantity of airway smooth muscle standardized for size was greater after ovalbumin challenge (0.069 +/- 0.005) compared with saline controls (0.033 +/- 0.003, p < 0.005). Nedocromil significantly reduced the airway smooth muscle (0.036 +/- 0.003, p < 0.005) when administered before ovalbumin. However, the airway smooth muscle in rats that received nedocromil 2 hours after ovalbumin challenge (0.046 +/- 0.003), although lower than in ovalbumin-challenged rats (p < 0.01), was still significantly higher than in saline-treated rats (p < 0.05). CONCLUSIONS Allergen-induced early responses, late responses, and airway inflammation are antagonized by nedocromil. The mediators of both the early and late responses contribute to allergen-induced airway smooth muscle growth, a process that can be prevented by administration of nedocromil.