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.

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.

Characterisation of cough evoked by inhaled treprostinil and treprostinil palmitil.

Cough is induced by inhaled prostacyclin analogues including treprostinil (TRE), and, at higher doses, treprostinil palmitil (TP), a prodrug of TRE. In this report, we have investigated mechanisms involved in TRE- and TP-induced cough, using a dry powder formulation of TP (TPIP) to supplement previous data obtained with an aqueous suspension formulation of TP (TPIS). Experiments in guinea pigs and rats investigated the prostanoid receptor subtype producing cough and whether it involved activation of sensory nerves in the airways and vasculature. Experiments involved treatment with prostanoid, tachykinin and bradykinin receptor antagonists, a cyclooxygenase inhibitor and TRE administration to the isolated larynx or intravenously. In guinea pigs, cough with inhaled TRE (1.23 µg·kg-1) was not observed with an equivalent dose of TPIP and required higher inhaled doses (12.8 and 35.8 µg·kg-1) to induce cough. TRE cough was blocked with IP and tachykinin NK1 receptor antagonists but not with EP1, EP2, EP3, DP1 or bradykinin B2 antagonists or a cyclooxygenase inhibitor. TRE administered to the isolated larynx or intravenously in rats produced no apnoea or swallowing, whereas citric acid, capsaicin and hypertonic saline had significant effects. The mechanisms inducing cough with inhaled TRE likely involves the activation of prostanoid IP receptors on jugular C-fibres in the tracheobronchial airways. Cough induced by inhaled dry powder and nebulised formulations of TP occurs at higher inhaled doses than TRE, presumably due to the slow, sustained release of TRE from the prodrug resulting in lower concentrations of TRE at the airway sensory nerves.

An overview of the biology of a long-acting inhaled treprostinil prodrug.

Treprostinil (TRE) is a prostanoid analog pulmonary vasodilator drug marketed with subcutaneous, intravenous (i.v.), oral, and inhaled routes of administration for the treatment of pulmonary arterial hypertension (PAH). Due to its short half-life, TRE requires either continuous infusion or multiple dosing, which exacerbates its side effects. Therefore, a long-acting prostanoid analog that maintains the positive attributes of TRE but has fewer TRE-related side effects could be of clinical benefit. In this report, we describe the discovery, preclinical development, and biology of the TRE ester prodrug, treprostinil palmitil (TP), which is formulated in a lipid nanoparticle (LNP) for administration as a nebulized inhaled suspension (TPIS). In screening assays focused on the conversion of prodrug to TRE, TP (16 carbon alkyl chain) had the slowest rate of conversion compared with short-alkyl chain TRE prodrugs (i.e., 2-8 carbon alkyl chain). Furthermore, TP is a pure prodrug and possesses no inherent binding to G-protein coupled receptors including prostanoid receptors. Pharmacokinetic studies in rats and dogs demonstrated that TPIS maintained relatively high concentrations of TP in the lungs yet had a low maximum plasma concentrations (Cmax) of both TP and, more importantly, the active product, TRE. Efficacy studies in rats and dogs demonstrated inhibition of pulmonary vasoconstriction induced by exposure to hypoxic air or i.v.-infused U46619 (thromboxane mimetic) over 24 h with TPIS. Cough was not observed with TPIS at an equivalent dose at which TRE caused cough in guinea pigs and dogs, and there was no evidence of desensitization to the inhibition of pulmonary vasoconstriction in rats with repeat inhaled dosing. TPIS was also more efficacious than i.v.-infused TRE in a sugen/hypoxia rat model of PAH to inhibit pulmonary vascular remodeling, an effect likely driven by local activities of TRE within the lungs. TPIS also demonstrated antifibrotic and anti-inflammatory activity in the lungs in rodent models of pulmonary fibrosis and asthma. In a phase 1 study in healthy human participants, TPIS (referred to as INS1009) had a lower plasma TRE Cmax and fewer respiratory-related side effects at equimolar doses compared with inhaled TRE. We have now formulated TP as an aerosol powder for delivery by a dry powder inhaler (referred to as treprostinil palmitil inhalation powder-TPIP), and as an aerosol solution in a fluorohydrocarbon solvent for delivery by a metered dose inhaler. These options may reduce drug administration time and involve less device maintenance compared with delivery by nebulization.

Inhaled Treprostinil-Prodrug Lipid Nanoparticle Formulations Provide Long-Acting Pulmonary Vasodilation.

Treprostinil (TRE), a prostanoid analogue approved in the USA for the treatment of pulmonary arterial hypertension, requires continuous infusion or multiple dosing sessions per day for inhaled and oral routes of administration due to its short half-life. The inhaled drug is known to induce adverse systemic and local effects including headache, nausea, cough, and throat irritation which may be due at least in part to transiently high drug concentrations in the lungs and plasma immediately following administration [1]. To ameliorate these side effects and reduce dosing frequency we designed an inhaled slow-release TRE formulation. TRE was chemically modified to be an alkyl prodrug (TPD) which was then packaged into a lipid nanoparticle (LNP) carrier. Preclinical screening in a rat model of hypoxia-induced pulmonary vasoconstriction led to selection of a 16-carbon alkyl ester derivative of TRE. The TPD-LNP demonstrated approximately 10-fold lower TRE plasma Cmax compared to inhaled TRE solution while maintaining an extended vasodilatory effect. The favorable PK profile is attributed to gradual dissociation of TPD from the LNP and subsequent conversion to TRE. Together, this sustained presentation of TRE to the lungs and plasma is consistent with a once- or twice-daily dosing schedule in the absence of high Cmax-associated adverse events which could provide patients with an improved treprostinil therapy.

Inhaled hexadecyl-treprostinil provides pulmonary vasodilator activity at significantly lower plasma concentrations than infused treprostinil.

INS1009 is a long acting pulmonary vasodilator prodrug of treprostinil (TRE) that is formulated in a lipid nanoparticle for inhaled delivery by nebulization. This study examined the ability of INS1009 to inhibit vasoconstriction in the pulmonary vasculature of rats and dogs and the extent to which local activity within the lung contributes to its activity. Rats received a single dose of INS1009 by nose-only inhalation or were given a continuous intravenous (i.v.) infusion of TRE, followed by an i.v. challenge of the thromboxane mimetic pulmonary vasoconstrictor U46619 and the increase in pulmonary arterial pressure (PAP) was measured. In beagle dogs, INS1009 was given by inhalation via face mask and TRE was given by continuous i.v. infusion; vasoconstriction was then induced by inhaled hypoxia with reduction of FIO2 to 0.10. Changes in the dog's right ventricular pulse pressure (RVPP) were measured using implanted telemetry probes. Blood samples were collected in rats and dogs immediately after the challenge to measure the plasma TRE concentration. Exposure of rats to inhaled INS1009 (0.5, 3.0 and 20.9 µg/kg) inhibited the U46619-induced increase in PAP at all doses up to 6 h with statistically significant inhibition up to 24 h with the pooled dose-response data. The concentration of TRE in the plasma at which PAP was reduced by 50% was approximately 60-fold lower for INS1009 (EC50 = 0.08 ng/mL) as compared to i.v. TRE (EC50 = 4.9 ng/mL). In dogs, INS1009 (2.7-80.9 µg/kg) inhibited the hypoxia-induced increase in RVPP at all doses up to 6 h with activity once again observed with the pooled dose-response of 10 µg/kg and higher at 24 h. The concentration of TRE in the plasma at which RVPP was reduced by 50% was approximately 550-fold lower for INS1009 (EC50 = 0.0075 ng/mL) as compared to i.v. TRE (EC50 = 4.1 ng/mL). These studies, in two species and by two different pulmonary vasoconstrictor challenges, demonstrate that inhaled INS1009 not only has long-acting vasodilatory effects but also that the local activity within the lung contributes to this response. Therefore, INS1009 may offer the opportunity to effect pulmonary vasodilation for long periods but with substantially lower systemic exposure than infused TRE.

Preclinical Pharmacology and Pharmacokinetics of Inhaled Hexadecyl-Treprostinil (C16TR), a Pulmonary Vasodilator Prodrug.

This article describes the preclinical pharmacology and pharmacokinetics (PK) of hexadecyl-treprostinil (C16TR), a prodrug of treprostinil (TRE), formulated in a lipid nanoparticle (LNP) for inhalation as a pulmonary vasodilator. C16TR showed no activity (>10 µM) in receptor binding and enzyme inhibition assays, including binding to prostaglandin E2 receptor 2, prostaglandin D2 receptor 1, prostaglandin I2 receptor, and prostaglandin E2 receptor 4; TRE potently bound to each of these prostanoid receptors. C16TR had no effect (up to 200 nM) on platelet aggregation induced by ADP in rat blood. In hypoxia-challenged rats, inhaled C16TR-LNP produced dose-dependent (0.06-6 µg/kg), sustained pulmonary vasodilation over 3 hours; inhaled TRE (6 µg/kg) was active at earlier times but lost its effect by 3 hours. Single- and multiple-dose PK studies of inhaled C16TR-LNP in rats showed proportionate dose-dependent increases in TRE Cmax and area under the curve (AUC) for both plasma and lung; similar results were observed for dog plasma levels in single-dose PK studies. In both species, inhaled C16TR-LNP yielded prolonged plasma TRE levels and a lower plasma TRE Cmax compared with inhaled TRE. Inhaled C16TR-LNP was well tolerated in rats and dogs; TRE-related side effects included cough, respiratory tract irritation, and emesis and were seen only after high inhaled doses of C16TR-LNP in dogs. In guinea pigs, inhaled TRE (30 µg/ml) consistently produced cough, but C16TR-LNP (30 µg/ml) elicited no effect. These results demonstrate that C16TR-LNP provides long-acting pulmonary vasodilation, is well tolerated in animal studies, and may necessitate less frequent dosing than inhaled TRE with possibly fewer side effects.