December 1, 2005 (Vol. 25, No. 21)
Utilization of Oligonucleotides to Block Expression in Inflammatory Pathways
Treating complex, multifactorial diseases is one of the top challenges in clinical practice today. Like many other chronic diseases that burden the healthcare system, multiple signaling pathways are responsible for the variety of clinical symptoms of respiratory disease.
Currently, there are two pharmacologic approaches to treating respiratory disease. The first involves drugs that block the expression of a specific target in a single pathway. The second approach is nonspecific and has a broad effect on multiple pathways. Single pathway approaches do not benefit from potential synergistic effects derived by multiple pathway intervention. While the non-specific approach can produce synergistic effects, it also can lead to unwanted side effects because of unrelated targets.
This tutorial discusses a unique pharmacologic approach to treating respiratory disease pioneered by Topigen Pharmaceuticals (Montreal). Topigen’s approach addresses the issue of pathway redundancy by blocking the expression of specific genes in multiple pathways.
The company’s first-in-class drug, ASM8, is designed to inhibit two distinct but overlapping pathways that are important in asthma and allergic inflammation. The drug is composed of therapeutic oligonucleotides that are administered topically, directly into the lungs by inhalation. The expectation is to improve efficacy and safety compared to current therapies by blocking genes directly at the desired site of action. This approach bypasses the safety concerns associated with systemic oligonucleotides.
Multi-Pathway Target Knockdown for Allergic Inflammation
The clinical features of allergic airway disease are chronic inflammation with airflow obstruction, airway hyper reactivity, and remodeling. The two pathways involved are the recruitment of immune and inflammatory cells and the activation and persistence of those cells. These immune and cellular responses are mediated through a number of cytokines and chemokines.
Most of the allergic cellular recruitment is mediated through the cytokine IL-5 and several chemokines that work through CCR3, the CC chemokine receptor. CCR3 is involved in eosinophil recruitment, proliferation, and progenitor differentiation at the site of respiratory inflammation. Blocking CCR3 has multiple effects because several chemokines that require the receptor are increased in the airways of allergy and asthma patients.
The cytokines IL-3, IL-5, and GM-CSF are key regulators in inflammation and hematopoiesis. Together, they activate eosinophils, mast cells, and macrophages, typical cells found in inflammation. They cause eosinophils to release pro-inflammatory cytoplasmic granules, lipid mediators, and other cytokines. All of these may contribute to tissue damage, and airway hyper reactivity and remodeling seen in asthmatic airways. Silencing the expression of all three cytokine receptors by targeting their common beta subunit (c) is more effective than previous cytokine approaches, which focused on neutralizing only IL-5. (Figure 1)
ASM8: Therapeutic Oligonucleotides
ASM8 blocks the expression of the receptors for cytokines IL-3, IL-5, and GM-CSF and CCR3 with a single drug product. The drug is composed of 1:1 mixture of two modified phophorothioate oligonucleotides. One 19-mer oligonucleotide blocks the mRNA for the c -subunit common to all 3 cytokines. The second 21-mer blocks the mRNA for CCR3 (Figure 2).
The synergistic effects of knocking down two major pathways with ASM8 has been demonstrated in in vitro studies using human cell lines and in cells obtained from monkeys and allergic humans. In vivo studies in animal models of asthma (brown Norway rats) demonstrated significant reduction in airway hyper reactivity and eosinophil recruitment after allergen challenge. Each oligonucleotide demonstrated efficacy alone, but the combination produced the best results, at lower doses in both models.
Corticosteroids are first-line therapy for patients with asthma. Steroids have systemic distribution and run the risk of side effects, especially with long-term use and high dosages. ASM8 has demonstrated <1% systemic distribution in rats, monkeys, and humans. In comparison to the corticosteroid, budesonide, the rat equivalent of ASM8 has shown better efficacy on eosinophilic inflammation, even when the steroid was administered at exponential higher doses. (Figure 3). The half-life of the oligonucleotides in the lungs and the length of efficacy of ASM8 make it a once-per-day product, which should increase patients' compliance with therapy.
ASM8 is delivered topically via a nebulizer and can also be delivered by inhalers. In validation studies, the nebulizer produced the correct particle size for good delivery into the lung tissue. Additionally, the stability of oligonucleotides in powder or in aqueous solutions makes them well-suited for the therapy of chronic respiratory diseases.
Phase I results demonstrated that ASM8 is well-tolerated in humans with no serious adverse events, no effects on respiratory symptoms, laboratory values, or pulmonary function. The plasma levels of ASM8 in the subjects indicated low systemic bioavailability, decreasing the risk of toxicity. Further, ASM8 was found in the sputum at 6 and 12 hours after administration, which confirms previous publications of persistence and metabolism of oligonucleotides within the lungs.
Future Development of Therapeutic Oligonucleotides
Topigen announced a new generation of oligonucleotide chemistry called FANA (Fluoro Arabino Nucleic Acid). The backbone of FANA, arabinonucleic acid, is a stable stereoisomer of RNA. This structure allows strong binding while increasing RNAase H activity and thus mRNA degradation. The result is a more efficacious, potent, and durable drug effect compared to older therapeutic oligonucleotides.
Topigen has a FANA-based drug in preclinical development called PD3 for the treatment of Chronic Obstructive Pulmonary Disease (COPD). Similar to asthma, COPD is a complex disease involving multiple pathways. PD3 blocks the expression of isotypes of a family of cAMP-dependent phosphodiesterases involved in inflammation and tissue damage in COPD.
A multi-pathway approach to treating complex diseases makes practical use of the knowledge gained since therapeutic oligonucleotides were introduced 20 years ago. Blocking specific genes in multiple pathways with oligonucleotides may produce synergistic effects and help to improve drug safety and efficacy when compared to older therapeutic approaches. Combined with topical delivery, newer generation therapeutic oligonucleotides have the potential to be potent and durable drugs with improved safety margins.