Assessment of Inhaled Treprostinil Palmitil, Inhaled and Intravenous Treprostinil, and Oral Selexipag in a Sugen/Hypoxia Rat Model of Pulmonary Arterial Hypertension.

Treprostinil palmitil (TP), a long-acting inhaled pulmonary vasodilator prodrug of treprostinil (TRE), has beneficial effects in a Sugen5416/hypoxia (Su/Hx) rat model of pulmonary arterial hypertension (PAH) that compare favorably to the oral phosphodiesterase 5 inhibitor (PDE5) sildenafil. In this study in male Sprague-Dawley rats, a dry powder formulation of TP (TPIP) was compared with inhaled and intravenous TRE and oral selexipag to evaluate inhibition of hemodynamic and pathologic changes in the lungs and heart induced by Su/Hx challenge. Su (20 mg/kg) was injected subcutaneously followed by 3 weeks of Hx (10% O2/balance N2) and then initiation of test article administration over 5 weeks with room air breathing. Hemodynamics and histopathology were measured at the end of the study. Su/Hx challenge approximately doubled the mean pulmonary arterial blood pressure (mPAP) and the Fulton index, decreased cardiac output (CO), doubled the wall thickness and muscularization of the small (10-50 µm) and medium (51-100 µm) sized pulmonary arteries, and increased the percentage of obliterated pulmonary blood vessels. Even though inhaled TRE (65 µg/kg, 4× daily), intravenous TRE (810 ng/kg/min), and oral selexipag (30 mg/kg, twice daily) provided some beneficial effects against the Su/Hx challenge, the overall benefit was generally greater with TPIP at high dose (117 µg/kg, once daily). These results demonstrate that TPIP compares favorably to inhaled and intravenous TRE and oral selexipag with respect to inhibition of the pathophysiological changes induced by Su/Hx challenge in rats. SIGNIFICANCE STATEMENT: Treprostinil palmitil (TP) is a long-acting pulmonary vasodilator prodrug of treprostinil (TRE) formulated for inhaled administration by dry powder [treprostinil palmitil inhalation powder (TPIP)]. Comparison of the activity of TPIP, inhaled and intravenous TRE, and oral selexipag in a Sugen5416/hypoxia (Su/Hx) rat model of pulmonary arterial hypertension demonstrated that each of these drugs exert protection against the hemodynamic and histopathological changes induced by the Su/Hx challenge, with the greatest effect on these changes produced by TPIP.

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.

Treprostinil palmitil, an inhaled long-acting pulmonary vasodilator, does not show tachyphylaxis with daily dosing in rats.

BACKGROUND: Treprostinil palmitil (TP) is an inhaled long-acting pulmonary vasodilator prodrug of treprostinil (TRE) that has been formulated for delivery as a suspension (treprostinil palmitil inhalation suspension; TPIS) and as a dry powder (treprostinil palmitil inhalation powder; TPIP). In humans, tachyphylaxis is frequently observed with continuous intravenous (IV) or subcutaneous (SC) infusion of TRE and requires dosage escalation to maintain activity. The aim of the present study was to determine whether tachyphylaxis occurs with repeat daily administration of inhaled TPIS. METHODS: Experiments were performed in male Sprague-Dawley rats prepared with a telemetry probe implanted into the right ventricle to measure the change in right ventricular pulse pressure (ΔRVPP) induced by exposure to a 10% oxygen gas mixture. TPIS (6 mL) at concentrations of 0.25, 0.5, and 1 mM was given by nose-only inhalation using an Aeroneb Pro nebulizer, either as a single administration or daily for 16 or 32 consecutive days. In studies involving consecutive daily administrations of TPIS, the delivered TP dosage was 140.3 µg/kg at 1 mM and ranged from 40.2 to 72.2 µg/kg at 0.5 mM. A separate cohort of telemetered rats received continuous IV infusion of TRE via an Alzet mini-pump at a dosage rate of 250 ng/kg/min for 16 days. Blood and lung tissue samples were obtained, and the concentration of TRE in the plasma and TRE and TP in the lungs were measured approximately 1 h after TPIS administration. RESULTS: Dose-response studies with TPIS administered as a single administration inhibited the hypoxia-induced increase in RVPP in both a concentration-dependent (0.25, 0.5, and 1 mM) and time-dependent (1-24 h) manner. TPIS, given QD or BID at inhaled doses ranging from 40.2 to 140.3 µg/kg for 16 or 32 consecutive days, produced statistically significant (P < .05) inhibition of the increase of RVPP due to hypoxia over the full duration of the dosing periods. By contrast, the inhibition of the hypoxia-induced increase in RVPP observed with IV TRE infusion (250 ng/kg/min) disappeared after 16 days of infusion. The plasma concentrations of TRE were significantly higher after IV TRE (range, 2.85-13.35 ng/mL) compared to inhaled TPIS (range, 0.22-0.73 ng/mL) CONCLUSIONS: There was no evidence of tachyphylaxis with repeat daily dosing of TPIS for a period of up to 32 days. The absence of tachyphylaxis with TPIS is likely related to its local vasodilatory effects within the lungs, combined with an absence of sustained high plasma concentrations of TRE.

Development and Characterization of Treprostinil Palmitil Inhalation Aerosol for the Investigational Treatment of Pulmonary Arterial Hypertension.

Treprostinil palmitil (TP) is a prodrug of treprostinil (TRE), a pulmonary vasodilator that has been previously formulated for inhaled administration via a nebulizer. TP demonstrates a sustained presence in the lungs with reduced systemic exposure and prolonged inhibition of hypoxia-induced pulmonary vasoconstriction in vivo. Here, we report on re-formulation efforts to develop a more convenient solution-based metered-dose inhaler (MDI) formulation of TP, a treprostinil palmitil inhalation aerosol (TPIA) that matches the pharmacokinetic (PK) and efficacy profile of a nebulized TP formulation, treprostinil palmitil inhalation suspension (TPIS). MDI canisters were manufactured using a two-stage filling method. Aerosol performance, formulation solubility, and chemical stability assays were utilized for in vitro evaluation. For in vivo studies, TPIA formulations were delivered to rodents using an inhalation tower modified for MDI delivery. Using an iterative process involving evaluation of formulation performance in vitro (TP and excipient solubility, chemical stability, physical stability, and aerosol properties) and confirmatory testing in vivo (rat PK and efficacy, guinea pig cough), a promising formulation was identified. The optimized formulation, TPIA-W, demonstrates uniform in vitro drug delivery, a PK profile suitable for a once-daily administration, efficacy lasting at least 12 h in a hypoxic challenge model, and a significantly higher cough threshold than the parent drug treprostinil.

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.

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.

Preclinical Investigation of a Lipoglycopeptide Dry Powder Inhalation Therapy for the Treatment of Pulmonary MRSA Infection.

The increased prevalence of pulmonary methicillin-resistant Staphylococcus aureus (MRSA) infection in patients living with cystic fibrosis (CF) is concerning due to a correlation with reduced life expectancy and lack of available treatment options. RV94 is a next generation lipoglycopeptide designed for pulmonary delivery that preclinically demonstrated high potency against MRSA in planktonic and protected colonies and improved pulmonary clearance relative to same class molecules. Here, RV94 was formulated into a dry powder for inhalation (DPI) to investigate the localized treatment of pulmonary MRSA presented in a potentially more convenient dosage form. RV94 DPI was generated using a spray-drying process with 12.5 wt% trileucine and demonstrated aerosol characteristics (2.0 µm MMAD and 73% FPF) predictive of efficient pulmonary deposition. In vivo PK from a single dose of RV94 DPI delivered by inhalation to rats yielded lung levels (127 µg/g) much greater than the MRSA minimum inhibitory concentration (0.063 µg/mL), low systemic levels (0.1 µg/mL), and a lung t1/2 equal to 3.5 days. In a rat acute pulmonary MRSA model, a single dose of RV94 DPI delivered by inhalation either up to seven days prior to or 24 h after infection resulted in a statistically significant reduction in lung MRSA titer.

Treprostinil palmitil inhibits the hemodynamic and histopathological changes in the pulmonary vasculature and heart in an animal model of pulmonary arterial hypertension.

Treprostinil palmitil (TP) is a long-acting inhaled pulmonary vasodilator prodrug of treprostinil (TRE). In this study, TP was delivered by inhalation (treprostinil palmitil inhalation suspension, TPIS) in a rat Sugen 5416 (Su)/hypoxia (Hx) model of pulmonary arterial hypertension (PAH) to evaluate its effects on hemodynamics, pulmonary vascular remodeling, and cardiac performance and histopathology. Male Sprague-Dawley rats received Su (20 mg/kg, s.c), three weeks of Hx (10% O2) and 5 or 10 weeks of normoxia (Nx). TPIS was given during the 5-10 week Nx period after the Su/Hx challenge. Su/Hx increased the mean pulmonary arterial blood pressure (mPAP) and right heart size (Fulton index), reduced cardiac output (CO), stroke volume (SV) and heart rate (HR), and increased the thickness and muscularization of the pulmonary arteries along with obliteration of small pulmonary vessels. In both the 8- and 13-week experiments, TPIS at inhaled doses ranging from 39.6 to 134.1 µg/kg, QD, dose-dependently improved pulmonary vascular hemodynamics, reduced the increase in right heart size, enhanced cardiac performance, and attenuated most of the histological changes induced by the Su/Hx challenge. The PDE5 inhibitor sildenafil, administered at an oral dose of 50 mg/kg, BID for 10 weeks, was not as effective as TPIS. These results in Su/Hx challenged rats demonstrate that inhaled TPIS may have superior effects to oral sildenafil. We speculate that the improvement of the pathobiology in this PAH model induced by TPIS involves effects on pulmonary vascular remodeling due to the local effects of TRE in the lungs.