The pharmacokinetic profile of brensocatib and its effect on pharmacodynamic biomarkers including NE, PR3, and CatG in various rodent species.

Brensocatib is a novel, oral, selective, reversible inhibitor of dipeptidyl peptidase 1 (DPP1), which activates several neutrophil serine proteases (NSPs), including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG) in the bone marrow during the early stage of neutrophil maturation. These NSPs are associated with pathogen destruction and inflammatory mediation; their dysregulated activation can result in excess secretion of active NSPs causing damaging inflammation and contributing to neutrophil-mediated inflammatory and autoimmune diseases. Pharmacological inhibition of DPP1 in the bone marrow could therefore represent an attractive strategy for these neutrophil-driven diseases. A completed Phase 2 trial in non-cystic fibrosis bronchiectasis patients (ClinicalTrials.gov number NCT03218917; EudraCT number: 2017-002533-32) indeed demonstrated that administration of brensocatib attenuated the damaging effects of chronic inflammation by inhibiting the downstream activation of NSPs. To support a range of preclinical programs and further understand how rodent species and strains may affect brensocatib's pharmacokinetic (PK) profile and its pharmacodynamic (PD) effects on NE, PR3, and CatG, an extensive naïve dosing study with brensocatib at different dosing levels, frequencies, and durations was undertaken. Dose-dependent PK exposure responses (AUC and Cmax) were observed regardless of the rodent species and strain. Overall, mice showed greater reduction in NSP activities compared to rats. Both mice and rats dosed once daily (QD) had equivalent NSP activity reduction compared to BID (twice a day) dosing when the QD dose was 1.5-times the BID daily dose. For both mouse strains, CatG activity was reduced the most, followed by NE, then PR3; whereas, for both rat strains, PR3 activity was reduced the most, followed by CatG, and then NE. Maximum reduction in NSP activities was observed after ∼7 days and recoveries were nearly symmetrical. These results may facilitate future in vivo brensocatib study dosing considerations, such as the timing of prophylactic or therapeutic administration, choice of species, dosage and dosing frequency.

Brensocatib, an oral, reversible inhibitor of dipeptidyl peptidase 1, mitigates interferon-α-accelerated lupus nephritis in mice.

Neutrophils have been implicated in initiating and perpetuating systemic lupus erythematosus and the resultant kidney damage in lupus nephritis (LN) patients, in part through an excessive release of neutrophil serine proteases (NSPs). NSP zymogens are activated by dipeptidyl peptidase 1 (DPP1) during neutrophil maturation and released by mature neutrophils in response to inflammatory stimuli. Thus, a potential strategy to attenuate disease progression in LN would be to inhibit DPP1. We tested whether brensocatib, a highly selective and reversible DPP1 inhibitor, could mitigate LN progression in an interferon-alpha (IFNα)-accelerated NZB/W F1 mouse model. To confirm brensocatib's pharmacodynamic effect on NSPs in this mouse strain, repeated dose studies were conducted for 7 and 14 days in naïve NZB/W F1 mice via oral gavage twice a day. Brensocatib at 2 and 20 mg/kg/day achieved a significant reduction in bone marrow NSP activities after 7 days of daily administration. To initiate LN disease progression, the mice were injected with an IFNα-expressing adenovirus. After 2 weeks, three brensocatib doses (or vehicle) were administered for 6 more weeks. Throughout the 8-week study, brensocatib treatment (20 mg/kg/day) significantly reduced the occurrence of severe proteinuria compared to the vehicle control. Brensocatib treatment also entailed a significant reduction in the urine albumin-to-creatinine ratio, indicating decreased kidney damage, as well as a significant reduction in blood urea nitrogen level, suggesting improved renal function. Based on kidney histopathology analysis, brensocatib treatment significantly lowered both the renal tubular protein score and the nephropathy score compared to the vehicle group. A trend towards reduced glomerulonephritis score with brensocatib treatment was also observed. Lastly, brensocatib significantly reduced LN mouse kidney infiltration by various inflammatory cells. In conclusion, these results suggest that brensocatib alters disease progression in LN mice and warrant further evaluation of DPP1 inhibition in LN.

Dipeptidyl peptidase-1 inhibition with brensocatib reduces the activity of all major neutrophil serine proteases in patients with bronchiectasis: results from the WILLOW trial.

BACKGROUND: Brensocatib is an oral, selective, reversible inhibitor of dipeptidyl peptidase-1 (DPP-1), responsible for activating neutrophil serine proteases (NSPs) including neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CatG). In chronic inflammatory lung diseases such as non-cystic fibrosis bronchiectasis (NCFBE), neutrophils accumulate in the airways resulting in excess active NSPs that cause damaging inflammation and lung destruction. METHODS: The 24-week WILLOW trial (NCT03218917) was a randomized, double-blind, placebo-controlled, parallel-group trial in patients with NCFBE conducted at 116 sites across 14 countries. In this trial, treatment with brensocatib was associated with improvements in clinical outcomes including time to first exacerbation, reduction in exacerbation frequency and a reduction in NE activity in sputum. An exploratory analysis of NE activity in white blood cell (WBC) extracts and NE, PR3 and CatG activity in sputum was conducted to further characterize brensocatib's effect and identify potential correlated effects. RESULTS: NE, PR3 and CatG activities were reduced in sputum and NE activity was reduced in WBC extracts in a dose-dependent manner after four weeks of brensocatib treatment, with a return to baseline four weeks after the end of treatment. Brensocatib produced the greatest reduction in the sputum activity of CatG, followed by NE and then PR3. Positive correlations among the sputum NSPs were observed both at baseline and in response to treatment, with the strongest correlation among the sputum NSPs for NE and CatG. CONCLUSIONS: These results suggest a broad anti-inflammatory effect of brensocatib underlying its clinical efficacy observed in NCFBE patients. TRIAL REGISTRATION: The study was approved by the corresponding ethical review boards of all participating centers. The trial was approved by the Food and Drug Administration and registered at clinicaltrials.gov (NCT03218917) on July 17, 2017 and approved by the European Medicines Agency and registered at the European Union Clinical trials Register (EudraCT No. 2017-002533-32). An independent, external data and safety monitoring committee (comprising physicians with pulmonary expertise, a statistician experienced in the evaluation of clinical safety, and experts in periodontal disease and dermatology) reviewed all adverse events.

An optimized method of extracting and quantifying active Neutrophil serine proteases from human whole blood cells.

PURPOSE: Neutrophil serine proteases (NSPs) are implicated in numerous inflammatory diseases. Thus, a robust methodology to monitor and quantify NSPs is important to study disease progression and evaluate the effect of pharmacological interventions. A comparison of the various methods used to extract NSPs from neutrophil granulocytes has not been published, providing the impetus to conduct this method optimization and comparison study. METHODS: Two NSP recovery methodologies were evaluated on samples from five human donors: zymosan stimulation and cell pellet extraction. For the zymosan stimulation method, 1 mL donor blood was added to zymosan and samples were incubated at 37°C for 30 min while shaking. Samples were then centrifuged, and the plasma was collected for quantitation of NSP activity. For the cell pellet extraction procedure, 2 mL whole blood samples were centrifuged into white blood cell pellets following red blood cell lysis. To each pellet, three sequential lysis steps were performed using either 0.05% Nonidet P-40 Substitute (NP40) or 0.02% Triton X-100 lysis buffers under agitation followed by centrifugation. NSP activities were quantified using an exogenous peptide substrate specific to each of the three NSPs being analyzed: neutrophil elastase, cathepsin G, and proteinase 3. RESULTS AND DISCUSSION: The zymosan stimulation method resulted in lower recovery of active NSPs and was unable to stimulate significant release of active cathepsin G. In contrast, the NP40 pellet extraction method showed consistent inter-donor NSP release with greater recoveries of active NSPs than the Triton method or the zymosan stimulation method. Overall, the pellet extraction procedure provided 13.3-fold greater recovery of active neutrophil elastase, 283-fold greater recovery of active cathepsin G, and 2.9-fold greater recovery of active proteinase 3 than the zymosan method. CONCLUSION: The NP40 cell pellet extraction method resulted in greater extraction of active NSPs compared to the other methods investigated here, which may allow for a more accurate and complete biomarker profile when evaluating human clinical samples.

Development and Preclinical Evaluation of New Inhaled Lipoglycopeptides for the Treatment of Persistent Pulmonary Methicillin-Resistant Staphylococcus aureus Infections.

Chronic pulmonary methicillin-resistant Staphylococcus aureus (MRSA) disease in cystic fibrosis (CF) has a high probability of recurrence following treatment with standard-of-care antibiotics and represents an area of unmet need associated with reduced life expectancy. We developed a lipoglycopeptide therapy customized for pulmonary delivery that not only demonstrates potent activity against planktonic MRSA, but also against protected colonies of MRSA in biofilms and within cells, the latter of which have been linked to clinical antibiotic failure. A library of next-generation potent lipoglycopeptides was synthesized with an emphasis on attaining superior pharmacokinetics (PK) and pharmacodynamics to similar compounds of their class. Our strategy focused on hydrophobic modification of vancomycin, where ester and amide functionality were included with carbonyl configuration and alkyl length as key variables. Candidates representative of each carbonyl attachment chemistry demonstrated potent activity in vitro, with several compounds being 30 to 60 times more potent than vancomycin. Selected compounds were advanced into in vivo nose-only inhalation PK evaluations in rats, where RV94, a potent lipoglycopeptide that utilizes an inverted amide linker to attach a 10-carbon chain to vancomycin, demonstrated the most favorable lung residence time after inhalation. Further in vitro evaluation of RV94 showed superior activity to vancomycin against an expanded panel of Gram-positive organisms, cellular accumulation and efficacy against intracellular MRSA, and MRSA biofilm killing. Moreover, in vivo efficacy of inhaled nebulized RV94 in a 48 h acute model of pulmonary MRSA (USA300) infection in neutropenic rats demonstrated statistically significant antibacterial activity that was superior to inhaled vancomycin.

Prostanoid receptor subtypes involved in treprostinil-mediated vasodilation of rat pulmonary arteries and in treprostinil-mediated inhibition of collagen gene expression of human lung fibroblasts.

Treprostinil (TRE) is a potent pulmonary vasodilator with effects on other pathological aspects of pulmonary arterial hypertension. In this study, the prostanoid receptors involved in TRE-induced relaxation of isolated rat pulmonary arteries and TRE-induced inhibition of increased gene expression in collagen synthesis and contractility of human lung fibroblasts were determined. TRE (0.01-100 µM) relaxed prostaglandin F2α-precontracted rat pulmonary arteries which was attenuated by denudation of the vascular endothelium. TRE-induced relaxation was predominantly blocked by the IP receptor antagonist RO3244194 (1 µM), with slightly greater inhibition in endothelium-denuded tissue. At higher TRE concentrations (> 1 µM), the DP1 receptor antagonist BW A868C (1 µM) also inhibited relaxation reaching significance above 10 µM. In contrast, the EP3 receptor antagonist L798106 (1 µM) accentuated TRE-induced relaxation of pulmonary arteries with intact endothelium. In human lung fibroblasts, the EP2 receptor antagonist PF-04418948 (1 µM) blocked transforming growth factor ß1 (TGF-ß1)-increased expression of collagen synthesis (COL1A1 and COL1A2) and fibroblast contractility (ACTG2) genes in presence of TRE (0.1 µM). In conclusion, the IP receptor located on rat pulmonary vascular smooth muscle and endothelium is the primary receptor mediating vasorelaxation, while the DP1 receptor present on the rat endothelium is involved only at higher TRE concentrations. In human lung fibroblasts, the EP2 receptor is the dominant receptor subtype involved in suppression of increased collagen synthesis and fibroblast contractility gene expression induced by TGF-ß1 in the presence of TRE.

Therapeutic administration of inhaled INS1009, a treprostinil prodrug formulation, inhibits bleomycin-induced pulmonary fibrosis in rats.

Idiopathic pulmonary fibrosis is a progressive and lethal disease and while there are now two approved drugs (Esbriet® and Ofev®) additional effective treatments are still needed. Recently, prostacyclin analogs such as iloprost and treprostinil (TRE) have been shown to exert some protection against bleomycin-induced pulmonary fibrosis in mice when administered in a prophylactic regimen. In this study, we evaluated the effect of the inhaled treprostinil prodrug hexadecyl-treprostinil (C16TR) formulated in a lipid nanoparticle (INS1009) administered therapeutically in a fibrotic rat model. Male Fischer 344 rats challenged with intra-tracheal saline instillation were then treated with daily inhaled phosphate buffered saline (PBS) while rats challenged with bleomycin sulfate (3.5-4.0 mg/kg) instillation were treated with either daily inhaled PBS, daily inhaled INS1009 (10, 30, or 100 µg/kg), or twice-daily orally with the anti-fibrotic compound pirfenidone (100 mg/kg). Dosing started on day 10 post-bleomycin challenge and continued until day 27 after bleomycin. Lungs were harvested 24 h after the last dose of treatment for evaluation of lung hydroxyproline content and pulmonary histology. Lung hydroxyproline content increased from 421 µg/lung lobe in saline challenged and PBS treated animals to 673 µg/lung lobe in bleomycin challenged and PBS treated rats. Treatment of bleomycin challenged rats with 10, 30, or 100 µg/kg INS1009 dose-dependently reduced lung hydroxyproline content to 563, 501, and 451 µg/lung lobe, respectively, and pirfenidone decreased hydroxyproline content to 522 µg/lung lobe. Histologically, both INS1009 (100 µg/kg) and pirfenidone (100 mg/kg) reduced the severity of subepithelial fibrosis. Single dose pharmacokinetic (PK) studies of inhaled INS1009 in bleomycin challenged rats showed dose-dependent increases in lung C16TR concentration and plasma TRE on day 10 post-bleomycin challenge. Multiple dose PK studies of inhaled INS1009 showed dose-dependent increases only in lung C16TR concentration on day 27 post-bleomycin challenge. We also investigated the effects of TRE on the cytokine transforming growth factor-ß1 (TGF-ß1)-stimulated collagen gene and protein expressions in cultured human lung fibroblasts, assessed by real-time PCR and Sirius Red staining, respectively. In human fibroblasts, TRE (0.001-10 µM) inhibited TGF-ß1 (20 ng/mL)-induced expression of collagen mRNA and protein in a concentration-dependent manner. These results demonstrated that inhaled INS1009, administered in a therapeutic dosing paradigm, dose-dependently (10-100 µg/kg) inhibited bleomycin-induced pulmonary fibrosis in rats. This effect may involve direct actions of TRE in suppressing collagen expression in lung fibroblasts.

Discovery of a Novel Piperidine-Based Inhibitor of Cholesteryl Ester Transfer Protein (CETP) That Retains Activity in Hypertriglyceridemic Plasma.

Herein we describe the discovery and characterization of a novel, piperidine-based inhibitor of cholesteryl ester transfer protein (CETP) with a core structure distinct from other reported CETP inhibitors. A versatile synthesis starting from 4-methoxypyridine enabled an efficient exploration of the SAR, giving a lead molecule with potent CETP inhibition in human plasma. The subsequent optimization focused on improvement of pharmacokinetics and mitigation of off-target liabilities, such as CYP inhibition, whose improvement correlated with increased lipophilic efficiency. The effort led to the identification of an achiral, carboxylic acid-bearing compound 16 (TAP311) with excellent pharmacokinetics in rats and robust efficacy in hamsters. Compared to anacetrapib, the compound showed substantially reduced lipophilicity, had only modest distribution into adipose tissue, and retained potency in hypertriglyceridemic plasma in vitro and in vivo. Furthermore, in contrast to torcetrapib, the compound did not increase aldosterone secretion in human adrenocortical carcinoma cells nor in chronically cannulated rats. On the basis of its preclinical efficacy and safety profile, the compound was advanced into clinical trials.

Discovery and Characterization of Allosteric WNK Kinase Inhibitors.

Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.

Small-molecule WNK inhibition regulates cardiovascular and renal function.

The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.

Discovery of N-[5-(6-Chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide, a Cortisol-Sparing CYP11B2 Inhibitor that Lowers Aldosterone in Human Subjects.

Human clinical studies conducted with LCI699 established aldosterone synthase (CYP11B2) inhibition as a promising novel mechanism to lower arterial blood pressure. However, LCI699's low CYP11B1/CYP11B2 selectivity resulted in blunting of adrenocorticotropic hormone-stimulated cortisol secretion. This property of LCI699 prompted its development in Cushing's disease, but limited more extensive clinical studies in hypertensive populations, and provided an impetus for the search for cortisol-sparing CYP11B2 inhibitors. This paper summarizes the discovery, pharmacokinetics, and pharmacodynamic data in preclinical species and human subjects of the selective CYP11B2 inhibitor 8.

Structure-Activity Relationships, Pharmacokinetics, and in Vivo Activity of CYP11B2 and CYP11B1 Inhibitors.

CYP11B2, the aldosterone synthase, and CYP11B1, the cortisol synthase, are two highly homologous enzymes implicated in a range of cardiovascular and metabolic diseases. We have previously reported the discovery of LCI699, a dual CYP11B2 and CYP11B1 inhibitor that has provided clinical validation for the lowering of plasma aldosterone as a viable approach to modulate blood pressure in humans, as well normalization of urinary cortisol in Cushing's disease patients. We now report novel series of aldosterone synthase inhibitors with single-digit nanomolar cellular potency and excellent physicochemical properties. Structure-activity relationships and optimization of their oral bioavailability are presented. An illustration of the impact of the age of preclinical models on pharmacokinetic properties is also highlighted. Similar biochemical potency was generally observed against CYP11B2 and CYP11B1, although emerging structure-selectivity relationships were noted leading to more CYP11B1-selective analogs.

Aldosterone synthase inhibition: cardiorenal protection in animal disease models and translation of hormonal effects to human subjects.

BACKGROUND: Aldosterone synthase inhibition provides the potential to attenuate both the mineralocorticoid receptor-dependent and independent actions of aldosterone. In vitro studies with recombinant human enzymes showed LCI699 to be a potent, reversible, competitive inhibitor of aldosterone synthase (K i = 1.4 ± 0.2 nmol/L in humans) with relative selectivity over 11ß-hydroxylase. METHODS: Hormonal effects of orally administered LCI699 were examined in rat and monkey in vivo models of adrenocorticotropic hormone (ACTH) and angiotensin-II-stimulated aldosterone release, and were compared with the mineralocorticoid receptor antagonist eplerenone in a randomized, placebo-controlled study conducted in 99 healthy human subjects. The effects of LCI699 and eplerenone on cardiac and renal sequelae of aldosterone excess were investigated in a double-transgenic rat (dTG rat) model overexpressing human renin and angiotensinogen. RESULTS: Rat and monkey in vivo models of stimulated aldosterone release predicted human dose- and exposure-response relationships, but overestimated the selectivity of LCI699 in humans. In the dTG rat model, LCI699 dose-dependently blocked increases in aldosterone, prevented development of cardiac and renal functional abnormalities independent of blood pressure changes, and prolonged survival. Eplerenone prolonged survival to a similar extent, but was less effective in preventing cardiac and renal damage. In healthy human subjects, LCI699 0.5 mg selectively reduced plasma and 24 h urinary aldosterone by 49 ± 3% and 39 ± 6% respectively (Day 1, mean ± SEM; P < 0.001 vs placebo), which was associated with natriuresis and an increase in plasma renin activity. Doses of LCI699 greater than 1 mg inhibited basal and ACTH-stimulated cortisol. Eplerenone 100 mg increased plasma and 24 h urinary aldosterone while stimulating natriuresis and increasing renin activity. In contrast to eplerenone, LCI699 increased the aldosterone precursor 11-deoxycorticosterone and urinary potassium excretion. CONCLUSIONS: These results provide new insights into the cardiac and renal effects of inhibiting aldosterone synthase in experimental models and translation of the hormonal effects to humans. Selective inhibition of aldosterone synthase appears to be a promising approach to treat diseases associated with aldosterone excess.

Discovery and in Vivo Evaluation of Potent Dual CYP11B2 (Aldosterone Synthase) and CYP11B1 Inhibitors.

Aldosterone is a key signaling component of the renin-angiotensin-aldosterone system and as such has been shown to contribute to cardiovascular pathology such as hypertension and heart failure. Aldosterone synthase (CYP11B2) is responsible for the final three steps of aldosterone synthesis and thus is a viable therapeutic target. A series of imidazole derived inhibitors, including clinical candidate 7n, have been identified through design and structure-activity relationship studies both in vitro and in vivo. Compound 7n was also found to be a potent inhibitor of 11ß-hydroxylase (CYP11B1), which is responsible for cortisol production. Inhibition of CYP11B1 is being evaluated in the clinic for potential treatment of hypercortisol diseases such as Cushing's syndrome.

Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors.

Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11beta-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC(50) values of 1.6+/-0.1 and 9.9+/-0.9 nM (mean+/-SEM, n=3-6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K(i) values of 0.8+/-0.04 and 2.2+/-0.2 nM (n=3-4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.

Optimization of halopemide for phospholipase D2 inhibition.

Halopemide, which was identified by HTS to inhibit phospholipase D2 (PLD2), provided the basis for an exploratory effort to identify potent inhibitors of PLD2 for use as inflammatory mediators. Parallel synthesis and purification were utilized to rapidly identify orally available amide analogs derived from indole 2-carboxylic acids with superior potency versus PLD2.