Anti-inflammatory Disease Therapies

Developing Therapies for Inflammatory Disease: Animal Models of Respiratory Diseases (COPD and Asthma)

Sarah Bolton, Department of Pathology, Safety Assessment UK, AstraZeneca R&D, Leicestershire, UK

Animal models are frequently used to test candidate drugs and to give confidence that a test compound will work in human disease. However, many of these animal models are too reductive and will model, at best, maybe only one aspect of a human condition, suggesting there is a clear disconnect between the disease and the experimental animal paradigm. The differences between human systems and rodents are significant and include differences in basic anatomy, cell types, turnover rates, and pathological consequences of a particular insult. It is also very difficult to model the complex contributions of both genetic and environmental factors. Respiratory diseases such as chronic obstructive pulmonary disease (COPD) and asthma are also subject to animal modeling using various inhaled challenges such as ovalbumin or lipopolysaccharide, but these are particularly focused on leucocyte infiltration. More clinically relevant challenges such as tobacco smoke or house dust mite are also used, but even these animals may not mimic key structural changes seen in the human tissue. Recently, several publications have highlighted the potential of using multifactorial models, or even genetically predisposed rodents such as the spontaneously hypertensive rat, to provide more relevant endpoints. However, to get more information from these experiments, what is being modeled and how the target relates to human disease need to be better understood. These animal models can have additional utility in toxicology studies. Drugs are not given to healthy humans, and it is becoming apparent that safety studies performed on naive animals may not always be appropriate. Investigation into the effects of some candidate drugs in a diseased lung setting using the animal models should be considered for inclusion in the safety assessment studies.

Developing Therapies for Inflammatory Diseases: Inhaled Delivery—Part 1

Dave Hassall, GlaxoSmithKline, Stevenage, Hertfordshire, UK

Therapies using the inhaled route have been in existence for many centuries. Modern-day medicines for the treatment of airway diseases such as asthma and COPD have advanced considerably, providing both symptomatic relief of bronchoconstriction and anti-inflammatory therapies through a variety of mechanisms that include β₂ agonists, muscarinic receptor antagonists, and steroids. Yet there remains considerable unmet medical need for many patients who are unresponsive to current medications. This has prompted a shift in emphasis away from these more traditional approaches, and current efforts are now focused on the resolution of chronic inflammation within the lung. For novel anti-inflammatory inhaled therapies, there is a greater focus on the required molecular properties early on in the discovery phase as well as the pharmacology of human cell systems and human lung tissues. Generating pharmacokinetics, toxicokinetics, and pharmacokinetics/pharmacodynamics in preclinical species to aid clinical dose prediction by the inhaled route is also important. Depending on the final molecular characteristics, the drug can be administered either via nebulization or by dry-powder devices. Advances in dry-powder technology suggest future potential targeted delivery to central or peripheral locations in the patient lung. This, coupled with a better understanding of inflammatory disease endpoints, should provide greater confidence in delivering the right dose to the right location and the use of a broader assessment of clinical efficacy to demonstrate a clear clinical benefit. It is only when all of these challenges are overcome that we will be able to affect the underlying disease directly and thereby decrease overall morbidity and mortality.

Developing Therapies for Inflammatory Diseases: Inhaled Delivery—Part 2

Deon Hildebrand, GlaxoSmithKline, Ware, Hertfordshire, UK

The development of inhaled anti-inflammatory drugs poses several challenges, including innate immunity, pathology, safety margins, and biomarkers. The lung’s innate immune system can respond promptly to inhaled drugs as it does to environmental pollutants and pathogens. Consequently, inhaled anti-inflammatory drugs must exert their therapeutic effects locally or enter the systemic circulation without eliciting a significant innate immune response. Deposited drugs may induce pathology in the respiratory tract of animals. The adversity of the pathology is often obscure because the structural and functional impact on the respiratory tract is largely unknown. As a result, adversity is usually a subjective judgment. The judgment can nonetheless jeopardize active clinical trials or precipitate the demise of otherwise promising candidates, particularly in the lung, because very large safety margins are imposed by some regulators when drug-induced pathology in the lung is deemed to be adverse. As a result, attrition rates rise with substantial financial implications, particularly in late-phase preclinical development. It is noteworthy that the safety margins are in part based on an assumption that only a small proportion of delivered drugs reaches the animal’s lungs, whereas in humans, the whole dose reaches the lung. The proportion is assumed to be similar for all drug candidates. The assumptions can be challenged, for example, by measuring the deposition fractions of each drug candidate explicitly, but it is not an easy task. Before embarking on this measurement, one should keep in mind that the deposition fraction is calculated from the estimated achieved dose, and it is therefore imperative to first accurately estimate that dose level. The safety margins are also in part imposed because drug-induced pathology in the lung is not possible to monitor in humans because of a lack of biomarkers. An urgent need exists for good biomarkers, including imaging modalities that can monitor the onset, progression, and reversibility of pathologies in the lungs of humans and animals. There is also a need to better understand the mechanisms underlying drug-induced pathologies in the respiratory tract. For dry-powder formulations, particle-mediated mechanisms may be involved in addition to more traditional parent and/or metabolite-based mechanisms. However, the identification of particle-mediated effects is a challenge, not least because concurrent control groups do not always control for the particle load that is delivered to the lungs of treated animals. In conclusion, the challenges faced by inhaled anti-inflammatory drug candidates are diverse and substantial. The challenges are not unique to anti-inflammatories but are encountered with many different inhaled drug classes. Novel innovative approaches to preclinical development are required to confront and overcome these challenges.

Therapeutic Targets in Multiple Sclerosis: Lesson from Human Pathology and Animal Models

Wolfgang Brück, Professor of Neuropathology, Head, Department of Neuropathology, University Medical Center Göttingen, Georg-August University Gottingen, Germany

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that leads to focal destruction of myelin, acute axonal damage/loss of axons, and reactive astrogliosis. The irreversible axonal loss is thought to be the major correlate of chronic disability in MS. So far, the current approved immunomodulatory or immunosuppressive treatments for MS are mainly targeting different components of the peripheral immune system. Novel therapeutics that enter the central nervous system (CNS) and affect the CNS-resident inflammation, inhibit myelin and axonal damage, or even induce repair of MS lesions are not currently available.

The present lecture aims at defining and characterizing new therapeutic targets in MS with respect to structural features and immunopathologic heterogeneity of MS lesions, pathological characteristics of relapsing-remitting versus progressive MS, and observations in animal models of MS. Promising new therapeutic targets in MS may include (1) oligodendrocyte precursor cells as a reservoir of myelinating cells inducing remyelination; (2) CNS-resident inflammatory cells such as astrocytes or microglia that may play an important role in lesion development and progression; (3) cortical MS lesions, which are abundant in progressive MS; and (4) the immunopathologic heterogeneity of early MS lesions that requires individual therapeutic approaches independent of the clinical phenotype of the patients.

Retinoid X Receptor Signaling in Inflammation and Remyelination

Jeffrey K. Huang, PhD, Department of Veterinary Medicine, MS Society Cambridge Centre for Myelin Repair, University of Cambridge, UK

In MS, the natural ability for resident oligodendrocyte precursor cells (OPCs) to differentiate and regenerate lost myelin sheaths, a process called remyelination, becomes progressively impaired. To understand how genes and signaling pathways regulate CNS remyelination, we performed focal experimental demyelination in rats followed by laser capture microdissection of lesions and microarray/bioinformatic analysis and generated a comprehensive gene expression profile of CNS remyelination. The molecular signature of remyelination, based on differential gene expressions, indicates that communication between the inflammatory environment and adult OPCs in the injured brain is crucial for successful repair. We found that the retinoic X receptor, RXR-gamma, was highly expressed during CNS remyelination. Reverse transcription polymerase chain reaction and in situ hybridization revealed its expression by oligodendrocyte lineage cells and macrophages in lesions. Administration of the retinoid X receptor agonist, 9 cis-retinoic acid, promoted oligodendrocyte differentiation in cultured oligodendrocytes and resulted in a significant increase in myelination in oligodendrocyte-neuron co-cultures and in experimentally demyelinated rats. Our data suggest that the enhancement of retinoid X receptor signaling promotes CNS remyelination and might be a useful therapeutic strategy to treat demyelinating diseases.

Immune Regulatory Neural Stem Cells and CNS Diseases

Stefano Pluchino, MD, PhD, Department of Clinical Neurosciences, Cambridge Centre for Brain Repair and Cambridge Stem Cell Initiative, University of Cambridge, UK

Compelling evidence exists that somatic stem cell–based therapies protect the CNS from chronic inflammation-driven degeneration, such as that occurring in experimental autoimmune encephalomyelitis, MS, and cerebral ischemic/hemorrhagic stroke. However, while it was first assumed that stem cells may act through direct replacement of lost/damaged cells, it has now become clear that they are able to protect the damaged nervous system through a number of “bystander” mechanisms other than cell replacement. In immune-mediated experimental demyelination and stroke—both in rodents and nonhuman primates—we and others have shown that transplanted neural stem/precursor cells (NPCs) possess a constitutive and inducible ability to mediate efficient bystander myelin repair and axonal rescue. Yet a comprehensive understanding of the multiple mechanisms by which NPCs exert their therapeutic impact is lacking. We envisage that the remarkable therapeutic plasticity of NPCs results from their capacity to engage highly sophisticated programs of horizontal cell-to-cell communication at the level of the (micro)environment, and we attribute a key role to the transfer of secreted membrane vesicles from (donor) NPCs to (recipient) neighboring cells. We are starting to define whether this form of communication is biologically relevant for NPCs and look forward to establishing whether it is associated with cell-to-cell trafficking of noncoding RNAs and indeed on elucidating its molecular signature and therapeutic significance for MS. We believe that the true innovation of this approach lies in its unique peculiarity to look into an innate cellular mechanism with the visionary focus of translating the knowledge of basal stem cell functions into innovative high-impact clinical therapeutics for MS.

Preclinical Safety Challenges of Developing Oligonucleotide-based Drugs

Tom McKevitt and Joel Parry, Safety Assessment, GSK R&D, Ware, UK

Although the number of oligonucleotide-based drugs in clinical development for anti-inflammatory indications is currently relatively small, the class-related toxicities associated with these drugs are seen regardless of the target and may be discussed in general terms. These toxicities include (1) plasma protein binding (e.g., inhibition of Factor H in the primate, resulting in complement activation), (2) general immunostimulatory effects (believed to be mediated through interaction with receptors of the innate immune system), (3) accumulation-driven degenerative changes in tissues such as the kidney, and (4) mechanism-related exaggerated pharmacology and potential for off-target messenger RNA interactions. The accumulation of oligonucleotide-based drug-related material in tissue macrophages (e.g., Kupffer cells in the liver) is also frequently observed, manifest as the presence of basophilic granules and hypertrophy.

Relative to systemically administered oligonucleotide-based drugs, the current safety knowledge base for topical delivery to the lungs is limited. However, a number of the class-related effects highlighted are relevant for the inhaled route. Indeed, the Inhaled Oligonucleotide Subcommittee of the Oligo Safety Working Group, Industry/Regulatory discussion forum, has highlighted lung toxicities observed in preclinical safety studies to be generally reminiscent of the immunostimulatory and macrophage effects. Key findings observed in short-term inhalation toxicology studies have included foamy macrophage accumulation (containing basophilic granules) and mononuclear cell infiltration, mainly in the interstitium but also in the proximal lymphoid tissues and upper airways. At higher doses, fibroplasia and metaplasia, most likely secondary to more pronounced inflammation, have also been reported. These effects tend to occur at lower dose levels in rodents than in nonhuman primates.

Clinical development of systemically administered and inhaled single-stranded oligonucleotide-based drugs (e.g., RNAse H-dependent antisense) has focused on simple solutions, so-called gymnotic delivery. However, the dose levels required to achieve efficacy are relatively high. GSK’s experience for inhaled double-stranded small interfering RNAs (siRNAs) suggests that novel delivery agents will be required to achieve meaningful target gene knockdown in the lungs with this class of oligonucleotide-based drugs, although this view is not universal. Similarly, many of the siRNAs in clinical development for indications requiring systemic administration are using delivery agents. The use of delivery agents, such as liposomes or nanoparticles, adds another layer of complexity to development and brings its own safety challenges. Based on experience with systemically administered formulated oligonucleotide-based drugs, dose-limiting toxicities are likely to be primarily associated with the delivery agent rather than the oligonucleotide-based drugs’ payload. Thus, the challenge is to identify a delivery agent that is both efficacious and well tolerated, to ensure a therapeutic index more favorable than that seen with gymnotic delivery.

There are a number of opportunities to characterize key class toxicities early in the discovery phase to allow early deselection of lead oligonucleotide-based drugs with less favorable safety profiles for systemic and inhaled administration. An overview of early safety screens typically employed to characterize systemically administered oligonucleotide-based drugs, along with an outline of GSK’s R&D strategy to achieve this for inhaled oligonucleotide-based drugs, will be given.

Stem Cells as Anti-inflammatory Therapies: Nonclinical Safety Strategies

Michaela Sharpe, Drug Safety Research and Development, Pfizer Ltd, Sandwich, UK

Stem cell therapies have the potential to establish a new clinical paradigm for the treatment of inflammatory diseases, and pressures are building to accelerate their development. But with new classes of therapy come new challenges for establishing safety. Although there are three classes of stem cell lines currently being used in the development of stem cell therapies, embryonic stem cell lines, induced pluripotent stem cell lines, and adult stem cell (ASC) lines, it is ASC therapies and in particular mesenchymal stem cell (MSC) therapies that are predominantly being developed and trialed as therapies for inflammatory conditions. MSCs were originally considered for therapy based on their multilineage differentiation capacity, but more recently, the focus has shifted to exploring the ability of MSCs to exert their biological function through tropic mechanisms, and the majority of clinical studies on the utility of stem cell therapies for the treatment of inflammatory conditions have used the immunomodulatory characteristics of MSCs.

The safety profile of a stem cell therapy depends on many factors, including the type of cell therapy, the differentiation status and proliferation capacity of the cells, the route and/or site of administration, the long-term survival of the product and/or engraftment, the need for repeated administration, the disease to be treated, and the age of the population. Potential risks include tumor formation, unwanted immune responses, and the risk of biodistribution and ectopic engraftment. Overall, stem cell therapies represent a great hope for many diseases, but a thorough evaluation of the potential risks is essential before wider clinical application.