Human Recombinant Alkaline Phosphatase (Ilofotase Alfa) Protects Against Kidney Ischemia-Reperfusion Injury in Mice and Rats Through Adenosine Receptors.

Adenosine triphosphate (ATP) released from injured or dying cells is a potent pro-inflammatory "danger" signal. Alkaline phosphatase (AP), an endogenous enzyme that de-phosphorylates extracellular ATP, likely plays an anti-inflammatory role in immune responses. We hypothesized that ilofotase alfa, a human recombinant AP, protects kidneys from ischemia-reperfusion injury (IRI), a model of acute kidney injury (AKI), by metabolizing extracellular ATP to adenosine, which is known to activate adenosine receptors. Ilofotase alfa (iv) with or without ZM241,385 (sc), a selective adenosine A2A receptor (A2AR) antagonist, was administered 1 h before bilateral IRI in WT, A2AR KO (Adora2a-/- ) or CD73-/- mice. In additional studies recombinant alkaline phosphatase was given after IRI. In an AKI-on-chronic kidney disease (CKD) ischemic rat model, ilofotase alfa was given after the three instances of IRI and rats were followed for 56 days. Ilofotase alfa in a dose dependent manner decreased IRI in WT mice, an effect prevented by ZM241,385 and partially prevented in Adora2a-/- mice. Enzymatically inactive ilofotase alfa was not protective. Ilofotase alfa rescued CD73-/- mice, which lack a 5'-ectonucleotidase that dephosphorylates AMP to adenosine; ZM241,385 inhibited that protection. In both rats and mice ilofotase alfa ameliorated IRI when administered after injury, thus providing relevance for therapeutic dosing of ilofotase alfa following established AKI. In an AKI-on-CKD ischemic rat model, ilofotase alfa given after the third instance of IRI reduced injury. These results suggest that ilofotase alfa promotes production of adenosine from liberated ATP in injured kidney tissue, thereby amplifying endogenous mechanisms that can reverse tissue injury, in part through A2AR-and non-A2AR-dependent signaling pathways.

Discovery of Clinical Candidate 1-{[(2S,3S,4S)-3-Ethyl-4-fluoro-5-oxopyrrolidin-2-yl]methoxy}-7-methoxyisoquinoline-6-carboxamide (PF-06650833), a Potent, Selective Inhibitor of Interleukin-1 Receptor Associated Kinase 4 (IRAK4), by Fragment-Based Drug Design.

Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound 40). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.

Efficacy and Pharmacology of the NLRP3 Inflammasome Inhibitor CP-456,773 (CRID3) in Murine Models of Dermal and Pulmonary Inflammation.

A critical component of innate immune response to infection and tissue damage is the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome, and this pathway and its activation products have been implicated in the pathophysiology of a variety of diseases. NLRP3 inflammasome activation leads to the cleavage of pro-IL-1ß and pro-IL-18, as well as the subsequent release of biologically active IL-1ß, IL-18, and other soluble mediators of inflammation. In this study, we further define the pharmacology of the previously reported NLRP3 inflammasome-selective, IL-1ß processing inhibitor CP-456,773 (also known as MCC950), and we demonstrate its efficacy in two in vivo models of inflammation. Specifically, we show that in human and mouse innate immune cells CP-456,773 is an inhibitor of the cellular release of IL-1ß, IL-1α, and IL-18, that CP-456,773 prevents inflammasome activation induced by disease-relevant soluble and crystalline NLRP3 stimuli, and that CP-456,773 inhibits R848- and imiquimod-induced IL-1ß release. In mice, CP-456,773 demonstrates potent inhibition of the release of proinflammatory cytokines following acute i.p. challenge with LPS plus ATP in a manner that is proportional to the free/unbound concentrations of the drug, thereby establishing an in vivo pharmacokinetic/pharmacodynamic model for CP-456,773. Furthermore, CP-456,773 reduces ear swelling in an imiquimod cream-induced mouse model of skin inflammation, and it reduces airway inflammation in mice following acute challenge with house dust mite extract. These data implicate the NLRP3 inflammasome in the pathogenesis of dermal and airway inflammation, and they highlight the utility of CP-456,773 for interrogating the contribution of the NLRP3 inflammasome and its outputs in preclinical models of inflammation and disease.

A mechanistic pharmacodynamic model of IRAK-4 drug inhibition in the Toll-like receptor pathway.

The TLR pathway has been implicated in the pathogenesis of numerous diseases. IRAK-4 is integral to this pathway, making it a viable target for therapeutic intervention. This paper describes the application of a mechanistic pharmacodynamic model to assess the impact of IRAK-4 inhibition on the TLR-4 pathway. The model uses a minimal number of rate equations, molecular species, and parameters to characterize TLR signal transduction biology, including ligand-receptor interaction, protein complex formation, protein phosphorylation, negative regulation, and cytokine production. The model successfully reproduces the dynamic responses of TNFα to LPS stimulation, the tolerance to sequential LPS bolus dosing, the burst following a LPS bolus or infusion, and the modulation of pathway biomarkers following administration of an IRAK-4 inhibitor. Drug dosing schemes are evaluated for simulated disease states. The results emphasize the significance of LPS kinetics on response dynamics and the utility of a mechanistic model to help translate drug efficacy.

Development and application of a biomarker assay for determining the pharmacodynamic activity of an antagonist candidate biotherapeutic antibody to IL21R in whole blood.

BACKGROUND: In preparation for potential clinical development of Ab-01, an antagonistic antibody directed against the IL21R, studies were undertaken to address translational medicine needs that fall into four categories: 1) development of a pharmacodynamic biomarker assay suitable for use in the clinic, 2) demonstration that Ab-01 has the desired biological activity in vitro and in vivo in cynomolgus monkeys, the preferred safety study species, 3) pre-clinical in vivo proof-of-concept that the assay can be used to detect Ab-01 pharmacodynamic (PD) activity in treated subjects, and 4) comprehensive assessment of the agonistic potential of Ab-01 when cross-linked. This report and a recently published companion report address the first three of these needs. The fourth has been addressed in a separate study. METHODS: Genes that change RNA expression upon ex vivo rhIL21 stimulation of whole blood were identified in human and cynomolgus monkey. The inhibitory effects of exogenously added Ab-01 were measured ex vivo in human and monkey, and the in vivo inhibitory effects of Ab-01 treatment were measured in monkey. RESULTS: Stimulation of whole human blood for 2 hours with rhIL21 induced robust increases in RNA expression of 6 genes. This response was blocked by Ab-01, indicating that the assay is suitable for measuring Ab-01 activity in blood. rhIL21 induced expression of a similar set of genes in cynomolgus monkey blood. This response was blocked with Ab-01, thus demonstrating that Ab-01 has the desired activity in the species, and that safety studies done in cynomolgus monkeys are relevant. Proof -of-concept for using this assay system to detect PD activity in vivo was generated by measuring the response in monkey blood to ex vivo rhIL21 stimulation before and 5 minutes following in vivo Ab-01 administration. CONCLUSIONS: A robust PD biomarker assay suitable for clinical use has been developed in human whole blood. The successful adaptation of the assay to cynomolgus monkeys has enabled the demonstration of Ab-01 activity both in vitro and in vivo in monkey, thus validating the use of this species in safety studies and establishing proof-of-concept for using this PD assay system to aid in dose selection in clinical studies.

Correlation of pharmacodynamic activity, pharmacokinetics, and anti-product antibody responses to anti-IL-21R antibody therapeutics following IV administration to cynomolgus monkeys.

BACKGROUND: Anti-IL-21R antibodies are potential therapeutics for the treatment of autoimmune diseases. This study evaluated correlations between the pharmacodynamic (PD) activity, pharmacokinetics, and anti-product antibody responses of human anti-IL-21R antibodies Ab-01 and Ab-02 following IV administration to cynomolgus monkeys. METHODS: The PD assay was based on the ability of recombinant human IL-21 (rhuIL-21) to induce expression of the IL-2RA gene in cynomolgus monkey whole blood samples ex vivo. Monkeys screened for responsiveness to rhuIL-21 stimulation using the PD assay, were given a single 10 mg/kg IV dosage of Ab-01, Ab-02, or a control antibody (3/group), and blood samples were evaluated for PD activity (inhibition of IL-2RA expression) for up to 148 days. Anti-IL-21R antibody concentrations and anti-product antibody responses were measured in serum using immunoassays and flow cytometry. RESULTS: Following IV administration of Ab-01 and Ab-02 to cynomolgus monkeys, PD activity was observed as early as 5 minutes (first time point sampled). This PD activity had good correlation with the serum concentrations and anti-product antibody responses throughout the study. The mean terminal half-life (t1/2) was approximately 10.6 and 2.3 days for Ab-01 and Ab-02, respectively. PD activity was lost at approximately 5-13 weeks for Ab-01 and at approximately 2 weeks for Ab-02, when serum concentrations were relatively low. The estimated minimum concentrations needed to maintain PD activity were approximately 4-6 nM for Ab-01 and approximately 2.5 nM for Ab-02, and were consistent with the respective KD values for binding to human IL-21R. For Ab-01, there was noticeable inter-animal variability in t1/2 values (approximately 6-14 days) and the resulting PD profiles, which correlated with the onset of anti-product antibody formation. While all three Ab-01-dosed animals were positive for anti-Ab-01 antibodies, only one monkey (with the shortest t1/2 and the earliest loss of PD activity) had evidence of neutralizing anti-Ab-01 antibodies. All three Ab-02-dosed monkeys developed neutralizing anti-Ab-02 antibodies. CONCLUSIONS: For anti-IL-21R antibodies Ab-01 and Ab-02, there was good correlation between PD activity and PK profiles following IV administration to cynomolgus monkeys. Compared with Ab-01, Ab-02 was eliminated markedly faster from the circulation, which correlated with a shorter duration of PD activity.

Interleukin-13 neutralization by two distinct receptor blocking mechanisms reduces immunoglobulin E responses and lung inflammation in cynomolgus monkeys.

Interleukin (IL)-13 is a key cytokine driving allergic and asthmatic responses and contributes to airway inflammation in cynomolgus monkeys after segmental challenge with Ascaris suum antigen. IL-13 bioactivity is mediated by a heterodimeric receptor (IL-13Ralpha1/IL-4Ralpha) and can be inhibited in vitro by targeting IL-13 interaction with either chain. However, in cytokine systems, in vitro neutralization activity may not always predict inhibitory function in vivo. To address the efficacy of two different IL-13 neutralization mechanisms in a primate model of atopic disease, two humanized monoclonal antibodies to IL-13 were generated, with highly homologous properties, differing in epitope recognition. Ab01 blocks IL-13 interaction with IL-4Ralpha, and Ab02 blocks IL-13 interaction with IL-13Ralpha1. In a cynomolgus monkey model of IgE responses to A. suum antigen, both Ab01 and Ab02 effectively reduced serum titers of Ascaris-specific IgE and diminished ex vivo Ascaris-triggered basophil histamine release, assayed 8 weeks after a single administration of antibody. The two antibodies also produced comparable reductions in pulmonary inflammation after lung segmental challenge with Ascaris antigen. Increased serum levels of IL-13, lacking demonstrable biological activity, were seen postchallenge in animals given either anti-IL-13 antibody but not in control animals given human IgG of irrelevant specificity. These findings demonstrate a potent effect of IL-13 neutralization on IgE-mediated atopic responses in a primate system and show that IL-13 can be efficiently neutralized by targeting either the IL-4Ralpha-binding epitope or the IL-13Ralpha1-binding epitope.

IL-13 blockade reduces lung inflammation after Ascaris suum challenge in cynomolgus monkeys.

BACKGROUND: Airway inflammation is a hallmark feature of asthma and a driver of airway hyperresponsiveness. IL-13 is a key inducer of airway inflammation in rodent models of respiratory disease, but a role for IL-13 has not been demonstrated in primates. OBJECTIVE: We sought to test the efficacy of a neutralizing antibody to human IL-13 in a cynomolgus monkey model of lung inflammation. METHODS: Using cynomolgus monkeys (Macaca fascicularis) that are sensitized to Ascaris suum through natural exposure, we developed a reproducible model of acute airway inflammation after segmental A suum antigen challenge. This model was used to test the in vivo efficacy of mAb13.2, a mouse mAb directed against human IL-13, and IMA-638, the humanized counterpart of mAb13.2. Bronchoalveolar lavage (BAL) cells and BAL fluid were collected before and after antigen challenge and assayed for cellular content by means of differential count. RESULTS: Total BAL cell count, eosinophil number, and neutrophil number were all reduced in animals treated with mAb13.2 or IMA-638 compared with values in control animals that were untreated, given saline, or treated with human IgG of irrelevant specificity. In addition, levels of eotaxin and RANTES in BAL fluid were reduced in anti-IL-13-treated animals compared with levels seen in control animals. CONCLUSION: These findings support a role for IL-13 in maintaining lung inflammation in response to allergen challenge in nonhuman primates. CLINICAL IMPLICATIONS: IL-13 neutralization with a specific antibody could be a useful therapeutic strategy for asthma.