key: cord-0729094-lkd55twg
authors: Sahakijpijarn, Sawittree; Moon, Chaeho; Warnken, Zachary N.; Maier, Esther Y.; DeVore, Jennie E.; Christensen, Dale J.; Koleng, John J.; Williams, Robert O.
title: In Vivo Pharmacokinetic Study of Remdesivir Dry Powder for Inhalation in Hamsters
date: 2020-12-23
journal: bioRxiv
DOI: 10.1101/2020.12.22.424071
sha: ab91dfa3edddd2f9acddfb154217b7d5729bb10a
doc_id: 729094
cord_uid: lkd55twg

Remdesivir dry powder for inhalation was previously developed using thin film freezing (TFF). A single-dose 24-hour pharmacokinetic study in hamsters, a small animal model for SARS-CoV-2, demonstrated that pulmonary delivery of TFF remdesivir can achieve plasma remdesivir and GS-441524 levels higher than the reported EC50s of both remdesivir and GS-441524 (in human epithelial cells) over 20 hours. The half-life of GS-4412524 following dry powder insufflation was about 7 hours, suggesting the dosing regimen would be twice daily administration. Although the remdesivir-Captisol® (80/20 w/w) formulation showed faster and greater absorption of remdesivir and GS-4412524 in the lung, remdesivir-leucine (80/20 w/w) exhibited a greater Cmax with shorter Tmax and lower AUC of GS-441524, indicating lower total drug exposure is required to achieve a high effective concentration against SAR-CoV-2. In conclusion, remdesivir dry powder for inhalation would be a promising alternative dosage form for the treatment of COVID-19 disease.

The coronavirus disease 2019 worldwide pandemic that is caused by Severe Acute 23

Respiratory Syndrome-CoV (SARS-CoV-2) has strained global health care systems. Although most 19 patients experienced only mild respiratory symptoms, the infection can develop into acute respiratory 25 distress syndrome (ARDS), pneumonia and even multi-organ dysfunction which can be lethal [1] . As of 26

December 2020, it has resulted in more than 77 million infected cases and 1.7 million deaths across the 27 world [2] . Several therapeutic agents have been investigated for the treatment of COVID-19 such as 28 remdesivir, favipiravir, lopinavir/ritonavir, darunavir/cobicistat, mesilate/nafamostat, chloroquine/ 29 hydroxychloroquine, camostat, tocilizumab, eculizumab, colchicine, baricitinib, aviptadil [3] and 30 (Charles River, 049LVG) 35-42-days old and weighing between 80 and 130 g (average weight of 102 g) 92 were housed in a 12-hour light/dark cycle with access to food and water ad libitum and were subjected 93 to one week of acclimation to the housing environment. Seventy hamsters were separated into two equal 94 groups (REM-CAP and REM-LEU). 95 TFF powder formulation was passed through a No. 200 sieve (75 μm aperture) to break down large 96 aggregates into fine particles. Precisely weighed quantities of sieved TFF powder were administered to 97 hamsters intratracheally using a dry powder insufflator (DP-4 model, Penn-Century Inc., Philadelphia, PA, 98 USA) connected to an air pump (AP-1 model, Penn-Century Inc., Philadelphia, PA, USA). The dose of 99 remdesivir was targeted to be 10 mg/kg. Each hamster was briefly anesthetized with isoflurane (4% 100 induction, 2% maintenance) and placed on its back on an intubation stand. Its upper incisors were used 101 to secure the hamster using silk at a 45° angle, with continuous delivery of anesthesia through a nose 102 cone. A laryngoscope was used to visualize the trachea, and the blunt metal end of the insufflator device 103 was inserted into the trachea. The sieved TFF powder was actuated into the lung using 3 puffs of the 104 connected pump (200 μL of air per puff). The mass of powder delivered was measured by weighing the 105 device chamber before and after dose actuations. 106

Following powder administration, five hamsters from each group were harvested at each time point 107 (15 mins, 30 mins, 1, 2, 4, 8, 24 hours) . Blood was drawn via cardiac puncture and immediately transferred 108 into a heparinized microtainer (BD, 365985, Lithium Heparin/PST™ Gel,). The blood sample was 109 centrifuged at 10,000 rpm for 1.5 minutes, and the plasma was separated and frozen on dry ice. The 110 hamster was carefully perfused with PBS, the lung was filled with 1 mL of PBS to collect bronchoalveolar 111 lavage (BAL) fluid, and the lung was removed, weighed and frozen. Plasma samples, BAL and lungs were 112 kept frozen and stored at −80 °C until analysis. 113

For plasma samples, remdesivir and its metabolites were extracted according to the following 115 protocol [19] . Briefly, 100 μL of plasma was combined with 100 μL of methanol containing 100 ng/mL of 116 the heavy labeled internal standards for remdesivir and GS-441524. The samples were mixed using a 117 vortex mixer and then centrifuged at 12,000 rpm for 15 minutes. The supernatant was collected and 118 placed in a 96-well plate for LC/MS/MS analysis. 119

For lung tissue samples, remdesivir and its metabolites were extracted according to the following 120 protocol [19] . Briefly, lung tissue samples were added into a 2 mL tube with 3.5 g of 2.3 mm zirconia/silica 121 beads (BioSpec Producs, Bartlesville, OK, USA), and homogenized at 4800 rpm for 20 seconds. After 122 homogenization, 1000 μL of methanol containing 100 ng/mL internal standards for remdesivir and GS-123 441524 was added to the tube. The tube was then vortexed and centrifuged at 12,000 rpm for 15 minutes. 124

The supernatant was placed in a 96-well plate for LC/MS/MS analysis. Calibration standards were 125 prepared for plasma and lung tissue in the same protocol. Remdesivir and GS-441524 standard solutions 126 were spiked into the blank plasma and lung tissue to obtain matrix matched calibrations. The calibration 127 range for plasma and lung tissue was 0.1-1000 ng/ml, and 50-10,000 ng/mL, respectively. The calibration 128 range was selected to bracket sample levels measured. 129

Remdesivir and GS-441524 were separated on an Agilent Poroshell column (2.1 × 50 mm, 2.7 μm) 130 (Agilent, Santa Clara, CA, USA) using a gradient of 0 to 90.25% of acetonitrile with 0.025% trifluoroacetic 131 acid in 5 min at a flow rate of 0.35 mL/min, and a column temperature of 40 °C. In total, 10 μL of each 132 sample was injected for analysis with an Agilent 6470 triple quadrupole LC/MS/MS system (Agilent, Santa 133 Clara, CA, USA). 134

Remdesivir and metabolite GS-441524 plasma and lung concentrations over time data were 136 analyzed by non-compartmental analysis using PKSolver to obtain pharmacokinetic parameters in the 137 hamster model after inhalation of the formulations [32] . Due to the sparse sampling requirements with 138 this animal model and to obtain lung concentrations over time a naïve pooled-data approach was used in 139 which the noncompartmental analysis was fit to the data as if the average of the measured concentrations 140 from the five animals at each time point were taken from a single subject. This was based on the methods 141 previously reported for estimating population kinetics from very small sample sizes [33, 34] . 142

The concentrations of remdesivir and GS-441524 were determined in plasma and lung tissue from 144 healthy hamsters treated with a single 10 mg/kg remdesivir dry powder insufflation of remdesivir/Captisol 145 (REM-CAP) or remdesivir/leucine (REM-LEU) formulation. The resulting lung tissue concentration-versus-146 time curves are shown in Figure 1 , while the corresponding pharmacokinetic parameters are summarized 147

in Table 1 . The Cmax of remdesivir for REM-CAP was 8-fold higher than that of REM-LEU (75.41 ng/mg VS 148 8.71 ng/mg, respectively), while Tmax of remdesivir for REM-CAP was lower compared to REM-LEU (30 149 mins VS 24 hour, respectively). Additionally, REM-CAP exhibited higher AUC0-24 of remdesivir in lungs than The systemic in vivo pharmacokinetics of drug absorption from the lungs was also investigated in 164 hamsters. Figure 2 shows the comparison of mean remdesivir and GS-441524 plasma concentration-time 165

profile from each formulation. The pharmacokinetic parameters following a single dose dry powder 166 insufflation calculated using a non-compartment model are presented in Table 2 

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