Milenko Cicmil

Mineralocorticoid receptor antagonists attenuate pulmonary inflammation and bleomycin-evoked fibrosis in rodent models.

Accumulating evidence indicates protective actions of mineralocorticoid antagonists (MR antagonists) on cardiovascular pathology, which includes blunting vascular inflammation and myocardial fibrosis. We examined the anti-inflammatory and anti-fibrotic potential of MR antagonists in rodent respiratory models. In an ovalbumin allergic and challenged Brown Norway rat model, the total cell count in nasal lavage was 29,348 ± 5451, which was blocked by spironolactone (0.3-60 mg/kg, p.o.) and eplerenone (0.3-30 mg/kg, p.o.). We also found that MR antagonists attenuated pulmonary inflammation in the Brown Norway rat. A series of experiments were conducted to determine the actions of MR blockade in acute/chronic lung injury models. (1) Ex vivo lung slice rat experiments found that eplerenone (0.01 and 10 µM) and spironolactone (10 µM) diminished lung hydroxyproline concentrations by 55 ± 5, 122 ± 9, and 83 ± 8%. (2) In in vivo studies, MR antagonists attenuated the increases in bronchioalveolar lavage (BAL) neutrophils and macrophages caused by lung bleomycin exposure. In separate studies, bleomycin (4.0 U/kg, i.t.) increased lung levels of hydroxyproline by approximately 155%, which was blocked by spironolactone (10-60 mg/kg, p.o.). In a rat Lipopolysaccharide (LPS) model, spironolactone inhibited acute increases in BAL cytokines with moderate effects on neutrophils. Finally, we found that chronic LPS exposure significantly increased end expiratory lung and decreased lung elastance in the mouse. These functional effects of chronic LPS were improved by MR antagonists. Our results demonstrate that MR antagonists have significant pharmacological actions in the respiratory system.

The preclinical efficacy, selectivity and pharmacologic profile of MK-5932, an insulin-sparing selective glucocorticoid receptor modulator.

Glucocorticoids are used widely in the treatment of inflammatory diseases, but use is accompanied by a significant burden of adverse effects. It has been hypothesized that gene- and cell-specific regulation of the glucocorticoid receptor by small molecule ligands could be translated into a therapeutic with an improved risk-benefit profile. MK-5932 is a highly selective glucocorticoid receptor modulator that is anti-inflammatory in vivo with an improved profile on glucose metabolism: Bungard et al. (2011). Bioorg. Med. Chem. 19, 7374-7386. Here we describe the full biological profile of MK-5932. Cytokine production following lipopolysaccharide (LPS) challenge was blocked by MK-5932 in both rat and human whole blood. Oral administration reduced inflammatory cytokine levels in the serum of rats challenged with LPS. MK-5932 was anti-inflammatory in a rat contact dermatitis model, but was differentiated from 6-methylprednisolone by a lack of elevation of fasting insulin or glucose levels after 7 days of dosing, even at high exposure levels. In fact, animals in the vehicle group were consistently hyperglycemic at the end of the study, and MK-5932 normalized glucose levels in a dose-dependent manner. MK-5932 was also anti-inflammatory in the rat collagen-induced arthritis and adjuvant-induced arthritis models. In healthy dogs, oral administration of MK-5932 exerted acute pharmacodynamic effects with potency comparable to prednisone, but with important differences on neutrophil counts, again suggestive of a dissociated profile. Important gaps in our understanding of mechanism of action remain, but MK-5932 will be a useful tool in dissecting the mechanisms of glucose dysregulation by therapeutic glucocortiocids.

Etanercept ameliorates inflammation and pain in a novel mono-arthritic multi-flare model of streptococcal cell wall induced arthritis

BackgroundThe impact of anti-TNF, corticosteroid and analgesic therapy on inflammation and pain was evaluated in a novel mono-arthritic multi-flare rat Streptococcal Cell Wall (SCW) model using Etanercept, Dexamethasone and Buprenorphine.MethodsMultiple flares of arthritis were induced with an intra-articular injection of SCW in the hind ankle on day 1, followed by intravenous challenges on days 21 and 42. Inflammation and pain were monitored in the hind paws. Cytokine profiling, cell phenotyping, bioluminescence imaging and histopathological evaluation were also performed.ResultsLocal injection of SCW caused a rapid onset of inflammation and pain in the injected ankle which resolved within 4 days (Flare 1). Intravenous injection 20 days after sensitization resulted in an increase in ankle diameter and pain, which partially resolved in 8 days (Flare 2). The subsequent intra-venous injection in the same animals 14 days after resulted in a more chronic disease with inflammation and pain persisting over a period of 10 days (Flare 3). In Flare 2, therapeutic administration of Dexamethasone inhibited paw swelling (95%; P<0.001) and pain (55%; P<0.05). Therapeutic administration of Buprenorphine inhibited pain (80%; P<0.001) without affecting paw swelling (0%). Prophylactic administration of Etanercept in Flare 2 inhibited paw swelling (≥60%; P<0.001) and pain by ≥30%. Expression of IL-1β, IL-6, MCP-1 and CINC was reduced by >50% (P<0.001). Treatment with Etanercept in Flare 3 inhibited paw swelling by 60% (P<0.001) and pain by 25%. Prior treatment with Etanercept in Flare 2 followed by re-administration in Flare 3 led to a complete loss in the efficacy of Etanercept. Systemic exposure of Etanercept corroborated with lack of efficacy. Dexamethasone inhibited inflammation and pain in both Flares 2 and 3 (P<0.001).ConclusionsWe established a novel multi-flare SCW arthritis model enabling drug intervention in different stages of disease. We show for the first time the evaluation of inflammation and pain simultaneously in this model. Etanercept and Dexamethasone inhibited inflammation, pain and proinflammatory cytokines in this model. Taken together, this model facilitates the assessment of anti-rheumatic agents targeting inflammation and pain in the multiple flare paradigm and offers a powerful tool for drug discovery.

Anti-inflammatory actions of Chemoattractant Receptor-homologous molecule expressed on Th2 by the antagonist MK-7246 in a novel rat model of Alternaria alternata elicited pulmonary inflammation

Alternaria alternata is a fungal allergen linked to the development of severe asthma in humans. In view of the clinical relationship between A. alternata and asthma, we sought to investigate the allergic activity of this antigen after direct application to the lungs of Brown Norway rats. Here we demonstrate that a single intratracheal instillation of A. alternata induces dose and time dependent eosinophil influx, edema and Type 2 helper cell cytokine production in the lungs of BN rats. We established the temporal profile of eosinophilic infiltration and cytokine production, such as Interleukin-5 and Interleukin-13, following A. alternata challenge. These responses were comparable to Ovalbumin induced models of asthma and resulted in peak inflammatory responses 48h following a single challenge, eliminating the need for multiple sensitizations and challenges. The initial perivascular and peribronchiolar inflammation preceded alveolar inflammation, progressing to a more sub-acute inflammatory response with notable epithelial cell hypertrophy. To limit the effects of an A. alternata inflammatory response, MK-7246 was utilized as it is an antagonist for Chemoattractant Receptor-homologous molecule expressed in Th2 cells. In a dose-dependent manner, MK-7246 decreased eosinophil influx and Th2 cytokine production following the A. alternata challenge. Furthermore, therapeutic administration of corticosteroids resulted in a dose-dependent decrease in eosinophil influx and Th2 cytokine production. Reproducible asthma-related outcomes and amenability to pharmacological intervention by mechanisms relevant to asthma demonstrate that an A. alternata induced pulmonary inflammation in BN rats is a valuable preclinical pharmacodynamic in vivo model for evaluating the pharmacological inhibitors of allergic pulmonary inflammation.

Pharmacological Evaluation of Selective α2c-Adrenergic Agonists in Experimental Animal Models of Nasal Congestion

Nasal congestion is one of the most troublesome symptoms of many upper airways diseases. We characterized the effect of selective α2c-adrenergic agonists in animal models of nasal congestion. In porcine mucosa tissue, compound A and compound B contracted nasal veins with only modest effects on arteries. In in vivo experiments, we examined the nasal decongestant dose-response characteristics, pharmacokinetic/pharmacodynamic relationship, duration of action, potential development of tolerance, and topical efficacy of α2c-adrenergic agonists. Acoustic rhinometry was used to determine nasal cavity dimensions following intranasal compound 48/80 (1%, 75 µl). In feline experiments, compound 48/80 decreased nasal cavity volume and minimum cross-sectional areas by 77% and 40%, respectively. Oral administration of compound A (0.1–3.0 mg/kg), compound B (0.3–5.0 mg/kg), and d-pseudoephedrine (0.3 and 1.0 mg/kg) produced dose-dependent decongestion. Unlike d-pseudoephedrine, compounds A and B did not alter systolic blood pressure. The plasma exposure of compound A to produce a robust decongestion (EC80) was 500 nM, which related well to the duration of action of approximately 4.0 hours. No tolerance to the decongestant effect of compound A (1.0 mg/kg p.o.) was observed. To study the topical efficacies of compounds A and B, the drugs were given topically 30 minutes after compound 48/80 (a therapeutic paradigm) where both agents reversed nasal congestion. Finally, nasal-decongestive activity was confirmed in the dog. We demonstrate that α2c-adrenergic agonists behave as nasal decongestants without cardiovascular actions in animal models of upper airway congestion.

Abstract A23: Development of a novel preclinical model to delineate the role of tumor microenvironment on mechanism of action and efficacy of PD-1 checkpoint blockade

Background: The cellular environment in which the tumor exists, also known as tumor microenvironment, includes surrounding blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix. This microenvironment can contribute to the growth and metastases of tumors, potentially influencing the efficacy of therapeutic agents. Thus, understanding the contribution of the microenvironment on tumorigenesis may enable us to investigate biology and kinetics between tumor and surrounding environment and improve target selection for standalone or combination therapies. Methods: To interrogate the role of the microenvironment we developed a tumor model in which the tumors can grow inside an air-pouch created in the dorsal part of the mouse. The air-pouch serves as the local microenvironment, which can be modulated by pro or anti-inflammatory stimuli to study the impact of local cells and cytokines on tumor growth as well as the efficacy of therapeutic agents. Results: Using luciferized mouse colon carcinoma (mc38-LUC2) syngeneic cells, we demonstrate that the kinetics of tumor growth within the air-pouch is similar to standard subcutaneously induced tumor models and was quantified via bioluminescence imaging. Additionally, further characterization of the tumor microenvironment was performed using MR and CT imaging, cytokine expression, and cell phenotyping. Furthermore, we also evaluated checkpoint inhibitor agent targeting the PD-1pathway to evaluate its effect on tumor growth and impact on cellular and cytokine microenvironment. Conclusions: Taken together, our novel model can facilitate innovative assessment of the mechanism of action of immunomodulatory agents across multiple oncological malignancies and evaluation of next generation therapeutics. Citation Format: Kalyan Chakravarthy, Raquel Sevilla, Michael Caniga, Weisheng Zhang, Anwar Murtaza, Lily Moy, Milenko Cicmil. Development of a novel preclinical model to delineate the role of tumor microenvironment on mechanism of action and efficacy of PD-1 checkpoint blockade. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A23.

CHAPTER 12:Animal Models: Practical Use and Considerations

Historically, the use of animal models in drug discovery has been integral to the development of novel therapies. Unfortunately, the translatability of animal model data into the clinical arena continues to be subject to seemingly everlasting debate. While there is an increasing focus to improve translatability of a drug discovery employing in-vitro biologically relevant assays with human tissue, many areas of the complex biology can only be adequately explored in an integrated system represented by animal models. For example, in the areas of inflammation and immunology antigen recognition, presentation and subsequent immune response precipitate a disease phenotype involves multiple cell types that orchestrate a time dependent pathology. Furthermore the exploration of the interplay of a drugs pharmacokinetic (PK) profile with the required pharmacodynamic (PD) response is critical for clinical dose regimen guidance and can only be explored in in vivo animal settings. Thus, research scientist continues to require in vivo models to enhance the probability of successfully advancing novel compounds from the bench to human patients. Central to the PKPD modeling is a deep understanding of the utility and limitations of animal models. In this chapter we make a distinction between pathway biology PD models and disease mechanism models. We provide high level guidance on the use of animals in the context of in vitro pharmacology and PK, focusing on preventing the over-interpretation of the data generated in these preclinical in vivo systems. We discuss practical aspects of experimental design and offer useful considerations for the medicinal chemist and other biomedical scientists on how to effectively build a platform of evidence, which incorporates animal data, to support progression of drug targets. Ethics, relevant species choice, group size, statistics, PD end point and PKPD relationship are also addressed in this chapter.

AB0130 Novel Mono-Arthritic Multi-Flare Model of Streptococcal Cell Wall Induced Arthritis: Pharmacological Evaluation with Etanercept

Background To assess the efficacy of novel anti-rheumatic agents on inflammation and pain we developed a novel mono-arthritic multi-flare rat Streptococcal Cell Wall (SCW) model which captures the remitting and chronic phases of arthritis similar to Rheumatoid Arthritis (RA). We have evaluated the impact of anti-TNF and steroid therapy using Etanercept and Dexamethasone to further understand the translatability of this model to human disease. Objectives To assess the effects of Etanercept and Dexamethasone in multiple flares of a novel model of SCW induced arthritis in rats. Methods SCW arthritis was induced in 6-8 week old female Lewis rats with an intra-articular injection in the hind ankle joint on day 1 followed by two intravenous challenges on days 21 and 42 of SCW extract PG-PS 100p. The contralateral paw was used as a negative control. Local inflammation and pain were monitored through the course of the study in the hind paws by measuring paw swelling and withdrawal threshold respectively. In addition, cytokine profiling, cell phenotyping, bioluminescence imaging and histopathological evaluation were also performed in the local joint. Then we assessed the efficacy of Etanercept and Dexamethasone in this model. Results Local injection of SCW caused a rapid onset of inflammation and pain in the injected ankle joint which resolved in 4 days (Flare 1). Systemic intravenous injection 20 days after sensitization resulted in a profound increase in ankle diameter and pain, which resolved in 8 days (Flare 2). A subsequent systemic challenge in the same animals on day 42 resulted in a chronic disease phenotype with inflammation and pain that continued to persist over the period of 10 days (Flare 3). Prophylactic administration of Etanercept in Flare 2 significantly inhibited paw swelling by 60% (p<0.001) and partially inhibited pain by 30% respectively. Production of proinflammatory cytokines IL-1β, IL-6, MCP-1 and CINC was reduced by >70% (p<0.001). Histopathological analysis corroborated with our efficacy data. Furthermore, prophylactic treatment with Etanercept in Flare 3 inhibited paw swelling by 60% (p<0.001) and partially inhibited pain by 25%. Interestingly, prior treatment with Etanercept in Flare 2 followed by a wash out period of 14 days and re-administration in Flare 3 led to a complete loss in efficacy, which could be due to potential immunogenicity. Serum exposure levels of Etanercept corroborated well with the lack of efficacy. Positive control Dexamethasone significantly inhibited inflammation and pain in both Flares 2 and 3 (p<0.001). Conclusions This is the first report to demonstrate a novel multi-flare extended paradigm of the SCW model. This model exhibits certain aspects of the human disease such as reactivating remission and exacerbation flares enabling investigation of RA pathogenesis and drug intervention in different stages of disease progression. We also report that the model can be used to evaluate clinically relevant parameters of inflammation and pain simultaneously. Our data shows that Etanercept and Dexamethasone inhibit inflammation, pain and relevant cytokines in this model. Taken together our novel model can facilitate innovative assessment of anti-rheumatic agents in multiple flares and offers a powerful tool for drug discovery. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2161

Alternaria alternata induced inflammatory lung responses: a novel in vivo PK/PD model

Rationale Asthma is a heterogeneous disorder characterized by several physiologic and immunologic phenotypes. Common environmental allergens such as pollen, house dust mite and mold induce airway inflammation and exacerbate asthmatic symptoms. Traditional rodent models of asthma use multiple sensitizations and challenges with allergens such as OVA and HDM to induce asthma like responses. Alternaria alternata is a fungal allergen linked to the development of severe asthma [1]. This allergen is capable of eliciting robust immune responses in the lungs [2]. In the current study we evaluated a single intratracheal (i.t.) instillation of Alternaria to model immune responses in Brown Norway rats.

Brown Norway ovalbumin model: temporal profile of cytokines

Background The ovalbumin (OVA) sensitized and challenged Brown Norway (BN) rat model is a practical PD model often used to determine the impact of drug treatment on late phase lung inflammation. However, the sole measurement of bronchoalveolar lavage fluid (BALF) inflammatory cells does not always correlate into human efficacy for respiratory diseases such as asthma. Added value to this rodent model may be derived by a deeper understanding of the relationship between disease-related cytokine secretion and inflammation.