As with any inhaled drug, and especially for a novel drug class such as oligonucleotides (ONs), pre-clinical and clinical safety assessment is challenging. With this in mind, a panel of internationally recognized lung experts from academic clinical laboratories, as well as industry-employed clinicians, with experience in drug development and pulmonary toxicology was convened by the Oligonucleotide Safety Working Group's (OSWG) Inhalation Subcommittee. The panel was asked whether the toxicities observed in pre-clinical animal studies would be anticipated in humans, to identify the strengths and weaknesses of current methods for detecting such effects in clinical trials and to make recommendations for human safety risk management of inhaled ONs.
There is a diversity of structures, chemistries, and mechanisms of action for ON therapeutics, but most of the members of this drug class can be categorized on the basis of whether or not they target mRNA or proteins. Antisense oligonucleotides (ASO), short-interfering RNA (siRNA), antagomirs, microRNA mimetics, and DNAzymes are part of the RNA-targeting group, while immunostimulatory sequences (ISS), aptamers, and decoys are members of the protein-targeting group. The toxicology and pharmacokinetics of ONs following systemic administration (e.g., intravenous and subcutaneous routes of administration) used in a variety of indications have been well characterized, and a large body of information is available through the regulatory databases and the broad scientific literature (Levin et al., 1998
; LEVIN, 1999
; Levin et al., 2001
; Sazani et al., 2010
). For example, the mechanisms of toxicity for RNA-targeting ONs can be subdivided into hybridization-independent and hybridization-dependent effects. Hybridization-independent toxicities are due to interactions between the ON drug and proteins, which are unrelated to Watson and Crick base pairing to RNA. Hybridization-dependent toxicities arise because the ON hybridizes to cellular RNA using the normal base-pairing principles which can lead to side effects associated with inhibition of the intended target (referred to as exaggerated pharmacology) or inhibition of unintended RNA targets (referred to as off-target effects). The majority of toxicities observed for ASOs and siRNAs tested to date fall into the hybridization-independent category and are believed to mainly result from the ON's chemistry or the composition of the delivery system (Levin et al., 2001
). For example, the most common modification used in ON compounds, phosphorothioate (PS) linkages (and other related backbone alterations) typically strengthen the polyanionic character of the molecule and render it more reactive. This, in addition to the greater tissue persistence, translates into more pronounced non-specific effects, such that systemic administration of PS ASOs result in various forms of toxicity largely unrelated to the mechanism of action (i.e., hybridization-independent). The most frequent and well studied hybridization-independent effects associated with PS ON administration are immune related, and the tendency to stimulate pro-inflammatory reactions, principally occurring in tissues containing the highest concentration of ONs (KRIEG, 2000
; Levin et al., 2001
Some pharmaceutical companies and investigators have explored alternative dosing routes. Currently the only 2 approved ON products are for local administration to the eye (CROOKE, 1998
; Ng and Adamis, 2005
). As for local delivery to the lungs, there is a relatively small amount of information on efficacy, deposition, and tolerability by this route. Furthermore, the Division of Pulmonary, Allergy, and Rheumatology Products at the US Food and Drug Administration (FDA) has reviewed only six ON drug candidates with representatives of the ASO, siRNA, and ISS types through investigational new drug (IND) applications or pre-IND meetings (DIA Conference, 2010
), thus limiting the regulatory database of information available to this division.
Based on published information (Templin et al., 2000
; Ali et al., 2001
; Guimond et al., 2008
) in rodent and primate species and the personal experiences of the Inhalation Subcommittee members, a list has been assembled of key findings that have been observed in non-clinical animal toxicity studies of inhaled ONs, which are generally limited to the respiratory tract and lung, the tissue of major accumulation.
With the understanding that the type and/or severity of findings may differ among ON subclasses (e.g. different backbone chemistries, duration of administration, and target species, certain types of changes in the lungs have been commonly observed). A summary of these key findings, which are typically dose related and reversible upon termination of treatment and which primarily occur at high toxicological doses is listed below.
- Alveolar macrophage “accumulation” (i.e., reflecting increased numbers and prominence upon light microscopy);
- Interstitial macrophages and mononuclear cell infiltration and accumulation in the lung parenchyma, more than the upper airway tissues and trans-bronchial lymph nodes;
- Occasional observations of hemorrhage, possibly secondary to tissue inflammation; and
- Fibroplasia and metaplasia in the lung or associated tissues (e.g., trachea, lymph nodes), usually with relatively pronounced inflammation.
One of the primary challenges in advancing these molecules into clinical trials is the observation of such findings and whether the findings represent safety concerns for humans. In addition, although toxicity has not been reported in normal subjects to date, there may be increased susceptibility in patients with diseased lungs, for example, due to impaired epithelial barrier function. However, there have been no reports of increased lung inflammation in patients following inhalation of ONs (Ball et al., 2003
; Gauvreau et al., 2006
; DeVincenzo et al., 2008
; Gauvreau et al., 2008
; DeVincenzo et al., 2010
). However, the inhaled doses have been low and the clinical trials have been of relatively short duration (i.e., less than 1 month); thus, the effects of prolonged exposure to ONs to human lungs remain undetermined. However, an important concern is that the techniques for monitoring lung toxicity may be insensitive to detect early clinical changes similar to those seen in animals. Although similar histopathological changes have been observed in other target organs (e.g., liver and kidney) when ONs are delivered via other routes of administration (e.g., parenteral), these other tissues can be monitored with increasingly sensitive biomarkers able to detect even earlier functional perturbation, whereas the technologies to monitor subtle pathologic changes in the lungs are less advanced.
This position paper addresses these issues and summarizes the panel discussion and outlines the consensus points and recommendations from the experts and the members of the Inhalation Subcommittee.
The Inhalation Oligonucleotide Subcommittee
Following the April 2007 Drug Information Association (DIA) meeting on oligonucleotide therapeutics in Bethesda, Maryland, the Oligonucleotide Safety Working Group (OSWG) was set up with representatives from both industry and regulatory authorities. Several subcommittees have been formed to deal with genotoxicity, off-target effects, immunostimulation, exaggerated pharmacology, safety pharmacology, reproductive toxicity, and carcinogenicity. The Inhalation Oligonucleotide Subcommittee, formed in 2009, has been discussing the main toxicology issues/challenges relating to the non-clinical development of inhaled oligonucleotides.