In a 4-week rat study, a nucleotide prodrug antiviral agent containing a synthetic non-natural amino acid (C-331) induced a dose-related vacuolation in the brain's white matter tracts without concurrent neuronal loss, gliosis, or neurological deficits. Routine safety pharmacology studies showed no effects. Penetration of the drug through the blood-brain barrier was negligible. Rat-specific metabolites were not evident. A follow-up 13-week rat study with a 4-week interim assessment was performed using oral doses of 0, 100, 300, or 1000 mg/kg/day for 4 or 13 weeks. Intermittent, transient clonic convulsions (<1 min) occurred in 1 male rat at 100 mg/kg/day (3 observations) and 5 animals at 1000 mg/kg/day during the dosing phase but were not observed during recovery; their relationship to brain vacuolation remains uncertain. No test article-related neurobehavioral or open-field changes, or ophthalmic abnormalities were noted. In all treated groups, widespread vacuolation predominantly affecting white matter was observed in the cerebellum, thalamus, and striatum. The severity of vacuolation increased over time and persisted after the 4-week recovery period. Based on ultrastructural evaluation, the vacuolation resulted from myelin sheath splitting (intramyelinic edema) without axonal degeneration. The vacuolation was considered to be an adverse effect based on the severity and extent of the changes and the potential for causing oligodendrocyte dysfunction. An NOAEL in the rat was not identified. Because of multiple unknowns, including the lack of consistent neurobehavioral signs, species specificity, low exposure multiples, and the potential human significance, further development of C-331 was terminated. The study describes the unusual presentation of species-specific, drug-induced splitting of myelin sheaths/intramyelinic edema without clear correlative neurofunctional effects.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PAs) and progressive increase in pulmonary vascular resistance leading to right ventricular failure. Although several drugs are approved for the treatment of PAH, mortality rates remain high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. METHODS: The expression of the RGD (arginylglycylaspartic acid)–binding integrin α5β1 was assessed in PAs, PA smooth muscle cells, and PA endothelial cells from patients with PAH and controls using NanoString, immunoblotting, and Mesoscale Discovery assays. RNA sequencing was conducted to identify gene networks regulated by α5β1 inhibition in PAH PA smooth muscle cells. The therapeutic efficacy of α5β1 inhibition was evaluated using a novel small molecule inhibitor and selective neutralizing antibodies in Sugen/hypoxia and monocrotaline rat models, with validation by an external contract research organization. Comparisons were made against standard-of-care therapies (ie, macitentan, tadalafil) and sotatercept and efficacy was assessed using echocardiographic, hemodynamic, and histological assessments. Ex vivo studies using human precision-cut lung slices were performed to further assess the effects of α5β1 inhibition on pulmonary vascular remodeling. RESULTS: We found that the arginine-glycine-aspartate RGD-binding integrin α5β1 is upregulated in PA endothelial cells and PA smooth muscle cells from patients with PAH and remodeled PAs from animal models. Blockade of the integrin α5β1 or depletion of the α5 subunit downregulated FOXM1 (forkhead box protein M1)–regulated gene networks, resulting in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. We demonstrated that α5β1 integrin blockade safely attenuates pulmonary vascular remodeling and improves hemodynamics and right ventricular function and matched or exceeded the efficacy of standard of care and sotatercept in multiple preclinical models. Ex vivo studies further validated its potential in reversing advanced remodeling in human precision-cut lung slices. CONCLUSIONS: These findings establish α5β1 integrin as a pivotal driver of PAH pathology and we propose its inhibition as a novel, safe, and effective therapeutic strategy for PAH.

Inhibition of integrin αvβ6 is a promising approach to the treatment of fibrotic disease such as idiopathic pulmonary fibrosis. Screening a small library combining head groups that stabilize the bent-closed conformation of integrin αIIbβ3 with αv integrin binding motifs resulted in the identification of hit compounds that bind the bent-closed conformation of αvβ6. Crystal structures of these compounds bound to αvβ6 and related integrins revealed opportunities to increase potency and selectivity, and these efforts were accelerated using accurate free energy perturbation (FEP+) calculations. Optimization of PK parameters including permeability, bioavailability, clearance, and half-life resulted in the discovery of development candidate MORF-627, a highly selective inhibitor of αvβ6 that stabilizes the bent-closed conformation and has good oral PK. Unfortunately, the compound showed toxicity in a 28-day NHP safety study, precluding further development. Nevertheless, MORF-627 is a useful tool compound for studying the biology of integrin αvβ6.

Introduction: Pulmonary arterial hypertension (PAH) is marked by pathological remodeling of distal arteries, driven by hyperplasia of pulmonary arterial smooth muscle cells (PASMCs), dysfunction of pulmonary arterial endothelial cells (PAECs), and increased deposition of the extracellular matrix (ECM). Hypothesis: Considering the crucial role of ECM in vascular restructuring, we investigated the potential of therapeutically targeting a5b1, a fibronectin-binding integrin involved in cell proliferation and angiogenesis, for PAH treatment. Aim: To evaluate the therapeutic potential of targeting a5b1 for the treatment of PAH. Methods: We have developed potent, orally bioavailable small molecule inhibitors (SMis) and monoclonal antibodies (mAbs) targeting a5b1 to evaluate its therapeutic potential. The effect of a5b1 inhibition was assessed in cultured PASMCs, human precision-cut lung slices (PCLS), and a rat Sugen/hypoxia PAH model. Results: PAH patients exhibited increased expression of fibronectin and a5b1 in distal pulmonary arteries. Selective inhibition of a5b1 in cultured PASMCs modulated multiple pathways involved in cell cycle regulation at both transcriptional and post-transcriptional levels, thereby blocking cellular proliferation. PCLS treated with a5b1 inhibitors showed decreased expression of pathways involved in ECM deposition and reduced levels of smooth muscle cell markers. In the rat Sugen/hypoxia PAH model, a5b1 inhibition with either an SMi or mAb significantly improved cardiac and vascular function by reducing pulmonary arterial wall thickness, right ventricular hypertrophy, and fibrosis. Cardiac improvement is further evidenced by a reduction in circulating NT-proBNP and NT-proANP levels. Conclusion: These findings reveal a previously underappreciated role for a5b1 in PAH pathogenesis and support the potential of a5b1 inhibition as a disease-modifying therapeutic strategy for managing PAH.

Mast cells play a central role in the pathophysiology of allergic inflammation. Mast cell activation, migration, proliferation and survival are dependent on KIT (CD117) signaling. THB001 is a potent and selective inhibitor of wild type KIT being studied clinically as a therapy for mast cell driven diseases. The current study correlated target engagement, mast cell depletion and efficacy in an in vivo model of anaphylaxis. Potency and selectivity were assessed in kinase-dependent Ba/F3 cell proliferation assays. Plasma pharmacokinetics, the pharmacodynamic effect on mast cells in the ear, and inhibition of Evans blue dye extravasation in response to allergen sensitization and challenge were assessed following 28 days of once daily oral administration of 2.5, 10 and 50 mg/kg/day THB001 in a rat model of passive cutaneous anaphylaxis (PCA). THB001 potently inhibited KIT in cells (IC50 0.02 μM) and had 48- and >100-fold selectivity against CSF-1R and PDGFR alpha/beta, respectively. In the PCA model, THB001 trough plasma concentrations exceeded the protein binding adjusted Ba/F3 IC50 for KIT by approximately 2x, 5x and 20x at 2.5, 10 and 50 mg/kg/day, respectively. THB001 resulted in a dose-dependent depletion of dermal mast cells correlating to efficacy at all doses tested, achieving 93% inhibition at 50 mg/kg/day (p<0.0001). Efficacy at the highest THB001 dose was similar to the antihistamine positive control desloratadine and approached the level of response in non-sensitized animals. THB001, a potent and selective inhibitor of wild type KIT, demonstrated efficacy in a rat PCA model at clinically relevant exposures.

Pulmonary arterial hypertension (PAH) is characterized by the narrowing of pulmonary arteries (PAs), causing right ventricular (RV) failure and premature death. Pulmonary artery smooth muscle cells (PASMCs) from PAH patients exhibit increased proliferation and resistance to apoptosis in response to extracellular matrix remodeling. Integrins are known to be involved in all these processes. Our hypothesis is that integrin signaling promotes PASMCs proliferation and apoptosis resistance, leading to PA remodeling, RV maladaptive hypertrophy and fibrosis, resulting in RV failure in PAH. Using NanoString, we identified members of the fibronectin-binding integrins (FnBIs) family as the most abundantly expressed in PAH-PASMCs. We confirmed the changes in FnBIs expression levels by Western blot (WB) in distal PAs, PASMCs and decompensated RVs from PAH patients compared to controls. Pharmacological inhibition of FnBIs decreased PAH-PASMCs proliferation (WB PLK1; Ki67), resistance to apoptosis (WB Survivin; Annexin V) and was associated with a decreased activation of FnBIs downstream signaling pathways FAK and ILK. In adult rat cardiomyocytes and human RV fibroblasts (RVFbs), FnBIs inhibition decreased phenylephrine-induced hypertrophy (f-actin labelling) and TGFβ1-induced RVFbs activation (WB αSMA, COL1). In both monocrotaline and sugen/hypoxia rats, pharmacological inhibition of FnBIs alone or in combination with macitentan and tadalafil improved RV function (mPAP, CO, TAPSE) and vascular remodeling (EVG). In the PA banding rat model, inhibition of FnBIs attenuated RV failure (CO, TAPSE, RVEDP). In conclusion, FnBIs signaling promotes maladaptive remodeling of the pulmonary vasculature and RV in PAH.

Supplementary Data from Targeting DNA Repair in Chronic Lymphocytic Leukemia Cells with a Novel Acyclic Nucleotide Analogue, GS-9219

Abstract Background Integrin α4β7 regulates the recruitment of T cells to intestinal mucosa through its interaction with mucosal addressin cell adhesion molecule (MAdCAM)-1. Disruption of this interaction has been clinically validated for the treatment of inflammatory bowel diseases (IBD) by the anti-α4β7 antibody vedolizumab. The current study was aimed at elucidating the preclinical efficacy of MT-103, a potent and selective small molecule α4β7 inhibitor in the clinically relevant CD4+CD45RBhi T cell transfer (TCT) colitis mouse model. Methods Mice were administered either MT-103 via subcutaneously implanted minipumps across 3 dose cohorts (3, 10, or 30 mg/ml), or an antibody specific for IL12p40 (25 mg/kg) or α4β7 (30 mg/kg), through intraperitoneal injections for 7 weeks. Colitis development was evaluated via readouts including body weights, gross colon weight by length ratios, colonic tissue gene expression, and histopathological scores. Results MT-103 significantly inhibited the development of colitis in mice at all 3 doses equivalently, as evident from significantly improved gross colon weight/length ratios in comparison to vehicle controls which presented with moderate-severe colitis (Figure 1A). MT-103 treatment also led to a significant protection from weight loss that occurred in vehicle-treated mice. Importantly, mice treated with MT-103 exhibited remarkably improved histopathology scores, including inflammation, hyperplasia, and gland loss compared to vehicle controls (Figure 1B). Furthermore, broad gene transcriptional analysis showed that MT-103 broadly suppressed pro-inflammatory pathways and processes within colonic tissues. Specifically, MT-103-induced downregulation of Ifng, Il17a, and Il1b, besides reduced Cd3 as a surrogate metric of T cell trafficking inhibition to the colon, and conversely enabled upregulation of anti-inflammatory cytokine Il10. The colitis-protective effects of MT-103 were equivalent, if not superior, to the saturating doses of α4β7 mAb or anti-IL12p40 mAb when compared to their respective vehicle controls. Conclusion Treatment with MT-103 protects from colitis by restoring colonic tissue homeostasis as measured by body fitness and an array of immunological and histopathological assessments. These proof-of-concept data demonstrate an α4β7-specific small molecule, MT-103, can occlude pathogenic T cells from initiating disease in a chronic IBD model, equivalent to an α4β7 blocking antibody.

Administration of a novel and selective small molecule integrin alpha v beta 6 inhibitor, MORF-627, to young cynomolgus monkeys for 28 days resulted in the rapid induction of epithelial proliferative changes in the urinary bladder of 2 animals, in the absence of test agent genotoxicity. Microscopic findings included suburothelial infiltration by irregular nests and/or trabeculae of epithelial cells, variable cytologic atypia, and high mitotic rate, without invasion into the tunica muscularis. Morphologic features and patterns of tumor growth were consistent with a diagnosis of early-stage invasive urothelial carcinoma. Ki67 immunohistochemistry demonstrated diffusely increased epithelial proliferation in the urinary bladder of several monkeys, including those with tumors, and alpha v beta 6 was expressed in some epithelial tissues, including urinary bladder, in monkeys and humans. Spontaneous urothelial carcinomas are extremely unusual in young healthy monkeys, suggesting a direct link of the finding to the test agent. Inhibition of integrin alpha v beta 6 is intended to locally and selectively block transforming growth factor beta (TGF-beta) signaling, which is implicated in epithelial proliferative disorders. Subsequent in vitro studies using a panel of integrin alpha v beta 6 inhibitors in human bladder epithelial cells replicated the increased urothelial proliferation observed in monkeys and was reversed through exogenous application of TGF-beta. Moreover, analysis of in vivo models of liver and lung fibrosis revealed evidence of epithelial hyperplasia and cell cycle dysregulation in mice treated with integrin alpha v beta 6 or TGF-beta receptor I inhibitors. The cumulative evidence suggests a direct link between integrin alpha v beta 6 inhibition and decreased TGF-beta signaling in the local bladder environment, with implications for epithelial proliferation and carcinogenesis.

Abstract Objective Vedolizumab has been shown to reduce the antibody response to an oral cholera, but not intramuscular hepatitis B vaccine in healthy subjects (Wyant, Gut 2015). The generation of an immune response to an oral antigen (Ag) begins at inductive sites such as Peyer’s patches (PP) followed by cell migration to effector sites in the gut. The integrin α4β7 is known to play a major role in cell trafficking to gut-associated lymphoid tissues. This study assessed whether MT-102, a potent and selective small molecule α4β7 inhibitor has similar effects on immunizations to those reported in the clinic for vedolizumab. Methods An in vivo immunization model system was established to determine the effects of MT-102, dosed via osmotic minipump, on the development of a gut-specific immune response. Mice were orally immunized with cholera toxin (CTX), and anti-CTX antibodies were quantified in fecal and plasma samples. The impact of MT-102 on lymphocytes within the PP of immunized mice was determined by flow cytometry. Results MT-102 delayed the production of anti-CTX IgA and IgG antibodies as measured in fecal samples without perturbing total gut IgA levels. Furthermore, MT-102 also inhibited the levels of Ag-specific antibodies in the plasma following repeated CTX oral immunizations. Blocking α4β7 induced a decrease in the frequency of B cells in the PP of treated mice compared to the control group. Conclusion α4β7-specific inhibitor, MT-102, effectively diminished the mucosal antibody response to an orally delivered Ag in mice. These results are consistent with what has been reported clinically in vedolizumab-treated subjects and thus establishes that potent small molecule inhibitors of α4β7 can replicate the pharmacology of an antibody.

Introduction: MORF-057 is a potent and selective α4β7 integrin inhibitor being developed as an oral treatment for patients with inflammatory bowel disease. In a previously reported Phase 1 study in healthy subjects (NCT04580745) MORF-057 was well tolerated and demonstrated favorable pharmacokinetic (PK) and pharmacodynamic (PD) properties at doses up to 100 mg twice daily (BID). This study evaluated second generation formulation and the multidose assessment to 200 mg BID. Methods: An immediate release formulation in capsules at dosage strengths of 25 and 100 mg was used for this study. This was a two-part healthy volunteer study where Part 1 investigated the safety and PK of a single administration of 25 mg (fasted) and 100 mg (fasted, fed) MORF-057. Part 2 investigated the safety, PK and PD of a single dose of 200 mg MORF-057 followed, after washout, by 14 days of repeat dosing at 200 mg BID. Blood samples to assess PK (Part 1 and 2) and receptor occupancy (RO; Part 2 only) of α4β7 and α4β1 integrins were obtained prior to the first dose and 12 hours post-dose. Changes in lymphocyte subsets (LS) and expression of C-C Motif Chemokine Receptor 9 (CCR9) mRNA in blood were also measured in Part 2 of the study. (Figure) Results: A total of 20 subjects were enrolled in the study (n=8 in Part 1; n=12 in Part 2). Three non-serious adverse events (AEs) were reported. No AEs were deemed related to MORF-057, and no safety signals were identified. Approximately dose proportional exposures of MORF-057 was observed following single administration at doses from 25 to 200 mg. MORF-057 was rapidly absorbed with a Tmax ranging from 2-4 hours. The high fat meal delayed the absorption resulting in a slight decrease in AUC (23%) and Cmax (49%) and an increase in Ctrough (46%). Saturating α4β7 RO (99%) was achieved at 12 hours following a single 200 mg dose and was sustained over multiple doses at 200 mg BID. α4β1 RO was below the limit of quantitation. The B cells and T cell subsets, and expression of CCR9 mRNA in blood, were elevated at Day 19 during BID dosing. (Table) Conclusion: Single and multiple dose MORF-057 was well tolerated in this study. Compared with data from a previous study, biomarker responses are consistent between 100 and 200 mg BID suggesting saturation of biomarker effect. MORF-057 demonstrated a favorable PK and PD profile supporting further clinical development.Figure 1.: α4β7 receptor occupancy (RO) Following Multiple Oral Administration of 200 mg BID MORF-057 Data presented as boxplots with central line representing the median value; α4β7 RO was measured at trough (12 h post dose) after a single administration (Day1_12h) and during repeat dose administration at 200 mg BID (Day6_12h, Day12_12h, Day19_12h). Table 1. - Summary of Plasma MORF-057 PK Parameters Following Single Dose Administration AUC, Cmax, and C12, values are presented as geometric mean (geometric CV%). Tmax is presented as median (min, max) PharmacokineticParameter (Unit) Part 1 Period 1100 mg MORF-057 IRCapsules (Fasted)(N=8) Part 1 Period 2100 mg MORF-057 IRCapsules (Fed)(N=7) Part 1 Period 325 mg MORF-057 IRCapsules (Fasted)(N=7) Part 2200 mg MORF-057 IRCapsules (Fasted)(N=9) AUC0-inf (h*ng/mL) 2050 (51.2) 1660 (41.1) 604 (36.5) 3770 (30.3) Cmax (ng/mL) 545 (45.9) 299 (48.7) 139 (38.6) 970 (46.6) C12 (ng/mL) 19.0 (81.9) 35.1 (98.6) 5.98 (61.8) 40.0 (44.1) Tmax (h) 2.01 (1.02, 4.02) 4.00 (1.50, 4.00) 1.50 (1.00, 2.50) 2.50 (1.52, 4.02) NA = Not Applicable.

Background Ovarian cancer (OC) is the most lethal gynecologic malignancy. Despite the initial high response rate to chemotherapy, most patients relapse. Immunotherapy offers potential for long-term remission but single checkpoint inhibition benefits less than 15% of patients. Growing evidence suggests that immune-checkpoint blockade (ICB) is enhanced when combined with therapies that target tumor tolerance. Transforming growth factor beta (TGF-β) is associated with resistance to immunotherapy and tumor tolerance. Integrin αvβ8 controls cell-type-specific activation of TGF-β and αvβ8 antagonism promotes anti-tumor immunity leading to tumor regression in ICB refractory tumors.1,2 We explored the impact of αvβ8 inhibition to restore ICB response in a murine ovarian carcinoma model and performed blood cytokine profiling to search for pharmacodynamic markers of response to treatment. Methods Bioinformatic analysis on bulk and single-cell levels of public OC datasets was performed to evaluate ITGB8 and TGF-β-related gene signatures. qPCR was used for the detection of TGF-β and ITGB8 expression in ID8, a murine ovarian carcinoma model (ID8-Luc-mCh-Puro.TD1). ID8 tumors unresponsive to PD-1/L1 inhibition were established to mimic advanced-stage disease. The efficacy of αvβ8 mAbs in combination with PD-L1 blockade was evaluated. Mice were evaluated for survival for 65 days. Blood samples were harvested before treatment and on days 1,8 and 14. Plasma was analyzed via Luminex for a total of 30 ­­­cytokines. Transcriptomic analyses of MC38 and EMT6 mouse tumors were performed by bulk RNA seq. Results In high-grade serous OC, ITGB8 expression is associated with shorter overall survival and increased gene signatures of TGF-β1/3 pathways. The ID8 murine ovarian carcinoma model is unresponsive to PD-1/L1 inhibition and expresses Itgb8 together with TGF-β 1/3. The combination of αvβ8 and PD-L1 mAbs led to complete tumor regression in 9/10 mice relative to 0/10 in the PD-L1 group, resulting in superior survival (P=0.0001) (Fig 1 A, B). The combination therapy led to upregulation of blood granzyme B (P=0.05), IL-27(P=0.03), and IL-1a (P= 0.008) at 14-day post-treatment and transient upregulation of CCL3 (P<0.0001) and CCL7 (P= 0.0005) at 8-day post-treatment. Cross-analysis of tumor tissues from the colon and breast syngeneic models after treatment showed similar transcript upregulation for cytokines. Conclusions Inhibition of αvβ8 renders advanced ID8 tumors sensitive to immune checkpoint therapy, leading to tumor eradication and superior survival. These data provide evidence that a treatment modality targeting αvβ8-mediated TGF-β 1/3 pathways may enhance patients' sensitivity to checkpoint therapies. Plasma blood-based biomarkers serve as a non-invasive method for response assessment to αvβ8-based therapy. References Reszka-Blanco NJ, Yadav V, Krumpoch M, Cappellucci L, Cui D, Dowling JE, et al. Inhibition of integrin avß8 enhances immune checkpoint induced anti-tumor immunity by acting across immunologic synapse in syngeneic models of breast cancer. AACR; Cancer Res 2021;81(13_Suppl): Abstract nr 1559 Dodagatta-Marri E, Ma H-Y, Liang B, Li J, Meyer DS, Chen S-Y, et al. Integrin avß8 on T cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy. Cell Report. 2021; 36(1): 109309