Soy Protein Containing Diet Attenuates Murine Drug Exposure and Activity via Hepatic and Intestinal Cytochrome P450 Induction.

Pharmacokinetic variability in drug plasma exposure between different studies within the same species is not unexpected due to a variety of factors (such as differences in formulation, active pharmaceutical ingredient salt form and solid-state, genetic strain, sex, environmental, disease status, bioanalysis methods, circadian rhythms, etc.) although variability from within the same research group typically does not occur to a great degree because these variables are commonly controlled. Surprisingly, a pharmacology proof of concept study with a previously validated tool compound from the literature failed to show expected response in murine glucose-6-phosphate isomerase-induced arthritis model which was tied to compound plasma exposure unexpectedly 10-fold lower than exposure observed from early pharmacokinetic study confirming adequate exposure prior to proof of concept. A systematic series of studies were conducted to investigate causes for exposure difference between pharmacology and pharmacokinetic studies identifying the presence or absence of soy protein in animal chow as the causative variable. Cyp3a11 expression in intestine and liver was determined to increase in a time dependent manner in mice switched to diets containing soybean meal compared with mice on diets without soybean meal. The repeated pharmacology experiments using the soybean meal free diet achieved plasma exposures that were maintained above the EC50 and showed efficacy and proof of concept for the target. This effect was further confirmed with marker CYP3A4 substrates in follow on mouse studies. The role of soy protein containing diets on CYP expression necessitates the inclusion of controlling rodent diet as a variable for preventing possible exposure differences between studies. SIGNIFICANCE STATEMENT: The presence of soybean meal protein in murine diet increased clearance and decreased oral exposure for select cytochrome 3A4 substrates. Related effects were also observed on select liver enzyme expression.

Inhibition of Interleukin-23-Mediated Inflammation with a Novel Small Molecule Inverse Agonist of RORγt.

Blockade of interleukin (IL)-23 or IL-17 with biologics is clinically validated as a treatment of psoriasis. However, the clinical impact of targeting other nodes within the IL-23/IL-17 pathway, especially with small molecules, is less defined. We report on a novel small molecule inverse agonist of retinoid acid-related orphan receptor (ROR) γt and its efficacy in preclinical models of psoriasis and arthritis. 1-(2,4-Dichloro-3-((1,4-dimethyl-6-(trifluoromethyl)-1H-indol-2-yl)methyl)benzoyl)piperidine-4-carboxylic acid (A-9758) was optimized from material identified from a high-throughput screening campaign. A-9758 is selective for RORγt and exhibits robust potency against IL-17A release both in vitro and in vivo. In vivo, we also show that IL-23 is sufficient to drive the accumulation of RORγt+ cells, and inhibition of RORγt significantly attenuates IL-23-driven psoriasiform dermatitis. Therapeutic treatment with A-9758 (i.e., delivered during active disease) was also effective in blocking skin and joint inflammation. Finally, A-9758 exhibited efficacy in an ex vivo human whole blood assay, suggesting small molecule inverse agonists of RORγt could be efficacious in human IL-17-related diseases. SIGNIFICANCE STATEMENT: Using a novel small molecule inverse agonist, and preclinical assays, we show that RORγt is a viable target for the inhibition of RORγt/Th17-driven diseases such as psoriasis. Preclinical models of psoriasis show that inhibition of RORγt blocks both the accumulation and effector function of IL-17-producing T cells.

ABBV-105, a selective and irreversible inhibitor of Bruton's tyrosine kinase, is efficacious in multiple preclinical models of inflammation.

OBJECTIVES: Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase required for intracellular signaling downstream of multiple immunoreceptors. We evaluated ABBV-105, a covalent BTK inhibitor, using in vitro and in vivo assays to determine potency, selectivity, and efficacy to validate the therapeutic potential of ABBV-105 in inflammatory disease. METHODS: ABBV-105 potency and selectivity were evaluated in enzymatic and cellular assays. The impact of ABBV-105 on B cell function in vivo was assessed using mechanistic models of antibody production. Efficacy of ABBV-105 in chronic inflammatory disease was evaluated in animal models of arthritis and lupus. Measurement of BTK occupancy was employed as a target engagement biomarker. RESULTS: ABBV-105 irreversibly inhibits BTK, demonstrating superior kinome selectivity and is potent in B cell receptor, Fc receptor, and TLR-9-dependent cellular assays. Oral administration resulted in rapid clearance in plasma, but maintenance of BTK splenic occupancy. ABBV-105 inhibited antibody responses to thymus-independent and thymus-dependent antigens, paw swelling and bone destruction in rat collagen induced arthritis, and reduced disease in an IFNα-accelerated lupus nephritis model. BTK occupancy in disease models correlated with in vivo efficacy. CONCLUSION: ABBV-105, a selective BTK inhibitor, demonstrates compelling efficacy in pre-clinical mechanistic models of antibody production and in models of rheumatoid arthritis and lupus.

Anti-TNF Thioester Glucocorticoid Antibody-Drug Conjugate Fully Inhibits Inflammation with Minimal Effect on Systemic Corticosterone Levels in a Mouse Arthritis Model.

We describe the discovery of a thioester-containing glucocorticoid receptor modulator (GRM) payload and the corresponding antibody-drug conjugate (ADC). Payload 6 was designed for rapid hepatic inactivation to minimize systemic exposure of nonconjugated GRM. Mouse PK indicated that 6 is cleared 10-fold more rapidly than a first-generation GRM payload, resulting in 10-fold lower exposure and 3-fold decrease in Cmax. The anti-mTNF conjugate ADC5 fully inhibited inflammation in mouse contact hypersensitivity with minimal effects on corticosterone, a biomarker for systemic GRM effects, at doses up to and including 100 mg/kg. Concomitant inhibition of P1NP suggests potential delivery to cells involved in the remodeling of bone, which may be a consequence of TNF-targeting or bystander payload effects. Furthermore, ADC5 fully suppressed inflammation in collagen-induced arthritis mouse model after one 10 mg/kg dose for 21 days. The properties of the anti-hTNF conjugate were suitable for liquid formulation and may enable subcutaneous dosing.

An anti-TNF-glucocorticoid receptor modulator antibody-drug conjugate is efficacious against immune-mediated inflammatory diseases.

Glucocorticoids (GCs) are efficacious drugs used for treating many inflammatory diseases, but the dose and duration of administration are limited because of severe side effects. We therefore sought to identify an approach to selectively target GCs to inflamed tissue. Previous work identified that anti-tumor necrosis factor (TNF) antibodies that bind to transmembrane TNF undergo internalization; therefore, an anti-TNF antibody-drug conjugate (ADC) would be mechanistically similar, where lysosomal catabolism could release a GC receptor modulator (GRM) payload to dampen immune cell activity. Consequently, we have generated an anti-TNF-GRM ADC with the aim of inhibiting pro-inflammatory cytokine production from stimulated human immune cells. In an acute mouse model of contact hypersensitivity, a murine surrogate anti-TNF-GRM ADC inhibited inflammatory responses with minimal effect on systemic GC biomarkers. In addition, in a mouse model of collagen-induced arthritis, single-dose administration of the ADC, delivered at disease onset, was able to completely inhibit arthritis for greater than 30 days, whereas an anti-TNF monoclonal antibody only partially inhibited disease. ADC treatment at the peak of disease was also able to attenuate the arthritic phenotype. Clinical data for a human anti-TNF-GRM ADC (ABBV-3373) from a single ascending dose phase 1 study in healthy volunteers demonstrated antibody-like pharmacokinetic profiles and a lack of impact on serum cortisol concentrations at predicted therapeutic doses. These data suggest that an anti-TNF-GRM ADC may provide improved efficacy beyond anti-TNF alone in immune mediated diseases while minimizing systemic side effects associated with standard GC treatment.

Optimization of Drug-Linker to Enable Long-term Storage of Antibody-Drug Conjugate for Subcutaneous Dosing.

To facilitate subcutaneous dosing, biotherapeutics need to exhibit properties that enable high-concentration formulation and long-term stability in the formulation buffer. For antibody-drug conjugates (ADCs), the introduction of drug-linkers can lead to increased hydrophobicity and higher levels of aggregation, which are both detrimental to the properties required for subcutaneous dosing. Herein we show how the physicochemical properties of ADCs could be controlled through the drug-linker chemistry in combination with prodrug chemistry of the payload, and how optimization of these combinations could afford ADCs with significantly improved solution stability. Key to achieving this optimization is the use of an accelerated stress test performed in a minimal formulation buffer.

Discovery of ABBV-154, an anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody-Drug Conjugate (iADC).

Stable attachment of drug-linkers to the antibody is a critical requirement, and for maleimide conjugation to cysteine, it is achieved by ring hydrolysis of the succinimide ring. During ADC profiling in our in-house property screening funnel, we discovered that the succinimide ring open form is in equilibrium with the ring closed succinimide. Bromoacetamide (BrAc) was identified as the optimal replacement, as it affords stable attachment of the drug-linker to the antibody while completely removing the undesired ring open-closed equilibrium. Additionally, BrAc also offers multiple benefits over maleimide, especially with respect to homogeneity of the ADC structure. In combination with a short, hydrophilic linker and phosphate prodrug on the payload, this afforded a stable ADC (ABBV-154) with the desired properties to enable long-term stability to facilitate subcutaneous self-administration.

Design and Development of Glucocorticoid Receptor Modulators as Immunology Antibody-Drug Conjugate Payloads.

Glucocorticoid receptor modulators (GRM) are the first-line treatment for many immune diseases, but unwanted side effects restrict chronic dosing. However, targeted delivery of a GRM payload via an immunology antibody-drug conjugate (iADC) may deliver significant efficacy at doses that do not lead to unwanted side effects. We initiated our α-TNF-GRM ADC project focusing on identifying the optimal payload and a linker that afforded stable attachment to both the payload and antibody, resulting in the identification of the synthetically accessible maleimide-Gly-Ala-Ala linker. DAR 4 purified ADCs were shown to be more efficacious in a mouse contact hypersensitivity model than the parent α-TNF antibody. Analysis of P1NP and corticosterone biomarkers showed there was a sufficient therapeutic window between efficacy and unwanted effects. In a chronic mouse arthritis model, α-TNF-GRM ADCs were more efficacious than both the parent α-TNF mAb and an isotype control bearing the same GRM payload.

Discovery of ABBV-3373, an Anti-TNF Glucocorticoid Receptor Modulator Immunology Antibody Drug Conjugate.

Using a convergent synthetic route to enable multiple points of diversity, a series of glucocorticoid receptor modulators (GRM) were profiled for potency, selectivity, and drug-like properties in vitro. Despite covering a large range of diversity, profiling the nonconjugated small molecule was suboptimal and they were conjugated to a mouse antitumor necrosis factor (TNF) antibody using the MP-Ala-Ala linker. Screening of the resulting antibody drug conjugates (ADCs) provided a better assessment of efficacy and physical properties, reinforcing the need to conduct structure-activity relationship studies on the complete ADC. DAR4 ADCs were screened in an acute mouse contact hypersensitivity model measuring biomarkers to ensure a sufficient therapeutic window. In a chronic mouse arthritis model, mouse anti-TNF GRM ADCs were efficacious after a single dose of 10 mg/kg i.p. for over 30 days. Data on the unconjugated payloads and mouse surrogate anti-TNF ADCs identified payload 17 which was conjugated to a human anti-TNF antibody and advanced to the clinic as ABBV-3373.

The Bcl-2 family antagonist ABT-737 significantly inhibits multiple animal models of autoimmunity.

The Bcl-2 family of proteins plays a critical role in controlling immune responses by regulating the expansion and contraction of activated lymphocyte clones by apoptosis. ABT-737, which was originally developed for oncology, is a potent inhibitor of Bcl-2, Bcl-x(L), and Bcl-w protein function. There is evidence that Bcl-2-associated dysregulation of lymphocyte apoptosis may contribute to the pathogenesis of autoimmunity and lead to the development of autoimmune diseases. In this study, we report that ABT-737 treatment resulted in potent inhibition of lymphocyte proliferation as measured by in vitro mitogenic or ex vivo Ag-specific stimulation. More importantly, ABT-737 significantly reduced disease severity in tissue-specific and systemic animal models of autoimmunity. Bcl-2 family antagonism by ABT-737 was efficacious in treating animal models of arthritis and lupus. Our results suggest that treatment with a Bcl-2 family antagonist represents a novel and potentially attractive therapeutic approach for the clinical treatment of autoimmunity.

Fit-for-purpose LC-MS/MS quantification of leukotoxin and leukotoxin diol in mouse plasma without sample pre-concentration.

LC/MS quantification of leukotoxin (LTX) and leukotoxin diol (LTXdiol) in plasma has been previously reported, however large sample volumes are required for achieving stated assay Lower Limit of Quantification (LLOQ). Reported here is a fit-for-purpose LC/MS method that reduces plasma volume from 700 to 25 µL and omits pre-concentration steps. These improvements make for a method with increased utility in mouse studies offering limited sample volumes. Additionally, omitting pre-concentration steps streamlines sample processing, which can now be completed in under 10 min. This method can be used to quickly answer if the ratio of LTX to LTXdiol changes with the dose of the therapeutic drug so this could be used as a potential biomarker for correlating PK/PD effects. No extensive assay characterization was performed before application to an exploratory in-life study. Basal levels of LTX and LTXdiol in plasma were quantified by LC-MRM across 10 individual mice, and the average signal-to-noise was 36 for LTX and 3039 for LTXdiol, with CVs of 29.4% and 15.2%, respectively. Addition of LTX and LTXdiol reference standard at 5, 25, and 75 ng/mL into pooled mouse plasma was quantifiable within 30% relative error using a surrogate matrix calibration curve ranging from 0.8 to 200 ng/mL. The average ratio of LTX to LTXdiol across the 10 mice was 0.32, consistent with previous reports. Finally, the method was applied to a mouse PK/PD study to monitor LTX/LTXdiol kinetics after a single oral dose of a soluble epoxide hydrolase inhibitor. The mean plasma ratio of LTX to LTXdiol increased up to 10-fold by 3 h post-dose followed by a decrease to near pre-dose levels by 24 h, consistent with transient inhibition of sEH-mediated conversion of LTX to LTXdiol. The method improvements described here will make subsequent quantification of LTX and LTXdiol in mouse studies significantly easier.

NRF2 activator A-1396076 ameliorates inflammation in autoimmune disease models by inhibiting antigen dependent T cell activation.

Nuclear factor (erythroid-derived 2) like 2 (NRF2) is a nuclear transcription factor activated in response to oxidative stress that induces a gene program that dampens inflammation and can limit cell damage that perpetuates the inflammatory response. We have identified A-1396076, a potent and selective NRF2 activator with demonstrated KEAP1 binding and modulation of cellular NRF2 mediated effects. In vivo administration of A-1396076 inhibits inflammation across several rodent models of autoimmunity when administered at or before the time of antigen challenge while also inducing NRF2 modulated gene transcription in the liver of the animals. It was not effective when administered after the time of antigen challenge or in a T cell independent model of arthritis induced by passive transfer of anti-collagen antibodies. A-1396076 inhibited antigen dependent T cell activation as measured by IFN-γ production in an ex vivo re-stimulation assay and following anti-CD3 challenge of MOG-sensitized mice. A-1396076 reduced costimulatory molecule expression on dendritic cells in the lungs of OVA LPS challenged mice suggesting that the mechanism of T cell inhibition was mediated at least partially by interfering with antigen presentation. These data suggest that NRF2 activation may be an effective strategy to dampen inflammation for treatment of autoimmune disease.

Development of Orally Efficacious Allosteric Inhibitors of TNFα via Fragment-Based Drug Design.

Tumor necrosis factor α (TNFα) is a soluble cytokine that is directly involved in systemic inflammation through the regulation of the intracellular NF-κB and MAPK signaling pathways. The development of biologic drugs that inhibit TNFα has led to improved clinical outcomes for patients with rheumatoid arthritis and other chronic autoimmune diseases; however, TNFα has proven to be difficult to drug with small molecules. Herein, we present a two-phase, fragment-based drug discovery (FBDD) effort in which we first identified isoquinoline fragments that disrupt TNFα ligand-receptor binding through an allosteric desymmetrization mechanism as observed in high-resolution crystal structures. The second phase of discovery focused on the de novo design and optimization of fragments with improved binding efficiency and drug-like properties. The 3-indolinone-based lead presented here displays oral, in vivo efficacy in a mouse glucose-6-phosphate isomerase (GPI)-induced paw swelling model comparable to that seen with a TNFα antibody.