Telmisartan Nanosuspension for Inhaled Therapy of COVID-19 Lung Disease and Other Respiratory Infection

Abstract

Vaccine hesitancy and the occurrence of elusive variants necessitate further treatment options for coronavirus disease 2019 (COVID-19). Accumulated evidence indicates that clinically used hypertensive drugs, angiotensin receptor blockers (ARBs), may benefit patients by mitigating disease severity and/or viral propagation. However, current clinical formulations administered orally pose systemic safety concerns and likely require a very high dose to achieve the desired therapeutic window in the lung. To address these limitations, we have developed a nanosuspension formulation of an ARB, entirely based on clinically approved materials, for inhaled treatment of COVID-19. We confirmed in vitro that our formulation exhibits physiological stability, inherent drug activity and inhibitory effect against SARV-CoV-2 replication. Our formulation also demonstrates excellent lung pharmacokinetics and acceptable tolerability in rodents and/or non-human primates following direct administration into the lung. Thus, we are currently pursuing clinical development of our formulation for its uses in patients with COVID-19 or other respiratory infections.

Graphical Abstract

INTRODUCTION

The outbreak of the coronavirus disease 2019 (COVID-19) pandemic has spurred global efforts to contain the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for the disease. Although vaccination is the pre-eminent public health strategy, vaccine hesitancy and the occurrence of increasingly more elusive variants have reinforced the need for effective treatment strategies, especially those that prevent the need for hospitalization. Treatment targeted specifically to prevent the acute respiratory distress syndrome (ARDS) caused by COVID-19 offers both short-term and long-term utility in the event of escape variants of SARS-COV-2 and/or other respiratory pandemics. Accumulated evidence suggests that clinically used hypertensive drugs, angiotensin receptor blockers (ARBs), may mitigate deleterious lung pathology, ARDS, in patients with COVID-19 presumably via modulation of the renin-angiotensin system (RAS) perturbed by the disease.^1–5 Specifically, ARBs can shift the pro-inflammatory angiotensin II (ANG II)-dominant pathological state of ARDS towards an anti-inflammatory angiotensin converting enzyme 2 (ACE2)-dominant state.⁶ Further, potential inhibitory roles of ARBs on replication of SAR-CoV-2 have been recently suggested and experimentally validated in vitro.^7–9 Thus, ARBs could exert meaningful therapeutic intervention in COVID-19 lungs in a multi-modal manner.

However, universal use of oral ARB formulations poses safety concerns due to the established systemic adverse effects, particularly for those with normal blood pressure or hypotension.^10–13 Additionally, unlike the original use for systemic pressure-reducing effects, a very high oral dose is likely needed to achieve desirable therapeutic concentrations in the lung tissue to yield meaningful clinical outcomes. Inhalable formulations may elegantly enhance dose flexibility as well as reduce overall dose and/or dosing frequency, therefore minimizing undesired systemic exposure of drug payloads.¹⁴ To this end, we have developed and extensively characterized an inhalable nanosuspension formulation of a clinically used ARB, telmisartan, which can be administered directly into the lung via nebulization. Of note, nebulized nanoformulations, such as drug nanosuspensions, can effectively reach the deep lung when appropriate aerodynamic properties are achieved.^{15, 16}

MATERIALS AND METHODS

Materials

Telmisartan was purchased from Biosynth International, Inc. (San Diego, CA) and stored at 4 °C before use. Free drug telmisartan solution (FD-TEL, Semintra^®) was purchased from Boehringer Ingelheim Vetmedica, Inc. (St. Joseph, MO). Polysorbate 80 (Tween^® 80) was purchased from VWR Life Science (Rouses Point, NY). Water and methanol for HPLC analysis, dithiothreitol, Fura-2-acetoxymethyl and Krebs solution were purchased from Fisher Scientific (Hampton, NH). UltraPureTM DNase/Rnase-Free distilled water, HEPES-buffered saline (HBS), collagenase, papain, and bovine serum albumin (BSA) were purchased from Invitrogen (Carlsbad, CA). Minimum Essential Medium (MEM, 1×), fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were purchased from Thermo Fisher Scientific – GibcoTM (Carlsbad, CA), and 0.25% Trypsin was purchased from Corning (Corning, NY). ANG II was purchased from Sigma Aldrich (St. Louis, MO). Smooth Muscle Cell Medium was purchased from ScienCell (Carlsbad, CA), and SmGM-2 SingleQuots were purchased from Lonza (Basel, Switzerland).

Formulation and physicochemical characterization of inhalable polysorbate 80-coated telmisartan nanosuspensions (INH-TEL)

Twelve-milligram polysorbate 80 was dissolved in 3 mL ultrapure water and then 1, 3, 4 or 5 mg telmisartan was added. The resulted suspension was sonicated with a Branson 2800 ultrasonic bath (Branson Ultrasonic Corp., Danbury, CT) for 15 minutes at room temperature (R.T.) and a spoon of stainless-steel beads (0.5 mm, Next Advance, Troy, NY) was added before loading on to a Tissue Lyser LT (Qiagen, Hilden, Germany) and milling for up to 10 hours with the oscillation speed of 50 s⁻¹. Some batches were subjected to the second pulverization step where the obtained suspension was homogenized with a probe sonicator (Vibra-Cell^™ VCX500, Sonics & Materials, Newtown, CT) for 1 hour (pulse: 1s on and 1s off, amplitude: 40%). Subsequently, the mixture was centrifuged at 900 x g for 10 minutes to remove large aggregates. This first centrifuge step also removes metal pieces released from the beads during the milling process, if any. The supernatant was centrifuged at 17,000 x g for 10 minutes to remove the polysorbate 80 micelles, and the pellet was resuspended in 1 mL ultrapure water. Finally, the suspension was freeze-dried and stored as a white powder (i.e., INH-TEL) at R.T. for future uses.

The hydrodynamic diameter/polydispersity index (PDI) and ζ-potential were measured by dynamic light scattering and laser Doppler anemometry, respectively, using a Nanosizer ZS90 (Malvern Instruments, Southborough, MA). Transmit electron microscopy (TEM, Hitachi H7600, Tokyo, Japan) was performed to determine the morphology of INH-TEL. The drug content in INH-TEL was determined with HPLC (LC-2030C 3D, Shimadzu, Columbia, MD) equipped with Luna^® Omega 5 μm Polar C18 column (Phenomenex^®, Torrance, CA) at a flow rate of 1 mL/min and an injection volume of 25 μL. The fluorescence detector of HPLC was set to the excitation and emission wavelengths at 300 nm and 360 nm, respectively, for the detection of telmisartan. To determine the relative amounts of telmisartan and polysorbate 80 in INH-TEL, the final product was lyophilized and measured for the total dry mass. The amount of polysorbate 80 was then calculated by subtracting the mass of telmisartan, determined by HPLC, from the mass of the lyophilized powder.

Physiological stability of INH-TEL in bronchoalveolar lavage fluid (BALF)

To assess the physiological stability of INH-TEL-NS, BALF was collected from C57BL/6 mice. After euthanizing mice, a small hole was cut in the trachea, an 18 G metal tube adaptor was inserted into the trachea through the hole and then three successive volume of 1 mL of DPBS was instilled and gently aspirated to be pooled. The pooled solution was centrifuged at 1,500 x g for 10 minutes at 4℃ and the and the supernatant was collected and lyophilized for future use. The lyophilized BALF and INH-TEL were rehydrated with DPBS to prepare a mixed solution containing 0.06 mg/mL BALF solid content and 0.6 mg/mL INH-TEL (based on the telmisartan concentration). The INH-TEL in BALF solution was incubated at R.T. and the hydrodynamic diameters and PDI values were measured over time using a Nanosizer ZS90.

Drug release kinetics of INH-TEL

The lyophilized INH-TEL were rehydrated in DPBS to a telmisartan concentration of 5 μg/mL and 1 mL INH-TEL resuspension was loaded into a dialysis bag (MWCO = 3 kDa). The dialysis bag was then immersed in 20 mL DPBS supplemented with 0.05% polysorbate 80 (to generate an artificial sink condition) and placed in a shaking incubator (Incu-Shaker^™, Newport Beach, CA) at 150 rpm and 37 °C. One milliliter solution was taken out for HPLC analysis to determine the telmisartan content and the same volume of fresh DPBS was replenished at each time point.

In vitro drug activity of INH-TEL

Primary pulmonary arterial smooth muscle cells (PASMCs) were isolated from rats as previously described.^{17, 18} Briefly, the heart and lung were excised and transferred to a petri dish containing HBS. The intrapulmonary arteries were isolated, and the tissue was digested for 20 minutes in reduced Ca2+ HBS containing collagenase (type I, 1750 U/mL), papain (9.5 U/mL), BSA (2 mg/mL), and dithiothreitol (1 mM) at 37°C. Individual smooth muscle cells were isolated from the digested tissue via trituration and plated on glass coverslips. Cells were cultured in Smooth Muscle Cell Medium containing SmGM-2 SingleQuots supplemented with 1% P/S for 24 – 48 hours and placed into low-serum media (Smooth Muscle Cell Medium containing 0.5% FBS and 1% P/S) 16 – 24 hours before beginning experiments. Intracellular calcium concentration ([Ca2+]i) was measured as previously described.^{17, 18} Briefly, PASMCs were loaded with 5 μM Fura-2-acetoxymethyl for 1 hour at 37°C before being placed in a temperature-controlled (37 °C) laminar flow chamber in a live cell Ca2+ imaging system (Intracellular Imaging Inc., Cincinnati, OH). Cells were perfused with warmed modified Krebs solution bubbled with 16% O2; 5% CO2 gas at 37 °C. At the beginning of each experiment, cells were perfused for 15 minutes to allow for establishment of a stable baseline. [Ca2+]i was calculated from the F340/F380 ratio using a calibration curve created with known Ca2+ concentrations. Cells were pretreated with DPBS only, FD-TEL or INH-TEL at a final telmisartan concentration of 50 μM for 30 minutes prior to exposure to ANG II (10–7 M).

In vitro anti-viral efficacy of INH-TEL against SARS-CoV-2

The assessment of in vitro anti-viral efficacy was conducted by the University of Pennsylvania High-throughput Screening Core.^{19, 20} Calu-3 cells (ATCC, HTB-55) grown in MEM supplemented with 1% non-essential amino acids, 1% P/S, 2mM L-glutamine, and 10% FBS were plated on a 384 well assay plate (Corning, Corning, NY). On the next day, 1μL of INH-TEL was resuspended in an aqueous buffer and added as an 8-point dose response with three-fold dilutions between test concentrations in triplicate, starting at a final concentration of 33.3 μg/mL. In parallel, cells were treated with FD-TEL at a concentration range for comparison, and the negative control (0.2% DMSO, n = 32) were included on each assay plate. Calu3 cells were pretreated with INH-TEL for 2 hours prior to infection. In BSL3 containment, SARS-CoV-2 (isolate USA WA1/2020) diluted in serum-free growth medium was added to plates to achieve a multiplicity of infection (MOI) = 0.5. Cells were incubated continuously with INH-TEL and SARS-CoV2 for 48 hours. Cells were fixed with 4% formaldehyde for 15 minutes at room temperature, washed 3 times with PBS, blocked with 2% BSA (w/v) in PBS supplemented with 0.1% triton-x-100 (PBST) and incubated with primary antibody against dsRNA (J2) diluted in PBST overnight at 4 °C. Cells were washed 3 times with PBST and incubated with a secondary antibody (anti-mouse Alexa Fluor^® 488) and Hoechst 33342 for 1 hour at room temperature. Cells were washed 3 times with PBST and imaged on an automated microscope (ImageXpress Micro, Molecular Devices, San Jose, CA) at 10X, four sites per well. The total number of cells and the number of infected (dsRNA+) cells were measured using the cell scoring module (MetaXpress 5.3.3, Molecular Devices), and the percentage of infected cells (dsRNA+ cells/cell number) per well was calculated. SARS-CoV-2 infection at each drug concentration was normalized to aggregated DMSO plate control wells and expressed as percentage-of-control (POC; % Infection sample/Avg % Infection DMSO control). A non-linear regression curve fit analysis with Prism 8 (GraphPad Software, La Jolla, CA) of POC Infection and cell viability versus the log10 transformed concentration values were used to determine the IC50/IC90 values for Infection and CC50 values for cell viability. Selectivity index (SI) was calculated as a ratio of drug’s CC50 and IC50 values (SI = CC50/IC50).

Pharmacokinetics study with mice

The lyophilized INH-TEL was reconstituted in ultrapure water to a telmisartan concentration of 0.05 mg/mL and intratracheally administrated into the lungs of C57BL/6 mice at a telmisartan dose of 0.1 mg/kg. Mice were euthanized at predetermined time points post-administration, plasma and lungs were harvested, and drug content was analyzed by HPLC as described earlier.

Pharmacokinetics and tolerability studies with non-human primates (NHPs)

Dosing of NHPs and subsequent sample collection were conducted at BIOQUAL, Inc. (Rockville, MD). The lyophilized INH-TEL was reconstituted in 0.45% saline to a telmisartan concentration of 0.833 mg/mL and intratracheally administrated into the lungs of two cynomolgus macaques (CM1 and CM2) at a dosage of 2.5 mg/animal using a laryngo-tracheal mucosal atomization device (MADgic, Teleflex^®, Morrisville, NC). In parallel, the other macaque (CM3) received daily oral administration of FD-TEL solution at a telmisartan dose of 1 mg/kg. The blood samples and lung tissues were collected at predetermined time points listed in Table S1. Drug contents in the plasma and lungs were measured by HPLC as described earlier. The blood samples were subjected to complete blood count and blood chemistry assays. The livers, kidneys, and lungs were formalin-fixed, paraffin embedded, and sectioned for hematoxylin and eosin (H&E) staining for histopathologic analysis by a board-certified pathologist (KT).

Statistical analysis

Statistical analyses were performed with Student’s t-test and one-way analysis of variance (ANOVA), followed by the Tukey’s multiple comparisons tests, for two-group and multiple (i.e., more than two)-group comparisons, respectively, using Prism (v.8.4.2, GraphPad Software).

RESULT AND DISCUSSION

Physicochemical properties of INH-TEL.

We used a modified top-down method^{21, 22} to formulate an inhalable telmisartan formulation (INH-TEL) composed of TEL drug nanosuspension core stably coated by polysorbate 80, one of the rare polymer-based surfactants approved by the FDA for respiratory use.²³ We conducted physicochemical characterization of INH-TEL freshly prepared by an optimized two-step pulverization method (i.e., 10-hour milling, followed by 1-hour probe sonication) where the hydrodynamic diameters and ζ-potentials were measured to be 322 ± 15 nm (polydispersity index or PDI = 0.24 ± 0.03) and −2.9 ± 0.5 mV, respectively (Figure 1A and Table 1). The hydrodynamic diameters of INH-TEL fall in the size range of 100 – 400 nm reported for polysorbate 80-stabilized nanosuspension formulations prepared with other water-insoluble drugs.^24–27 We found that omitting or shortening either the milling or the probe sonication step resulted in marked increases in the hydrodynamic diameters and the PDI values and/or dramatic reduction in the final yield (Table S2). On the other hand, physicochemical properties and yield were comparable when nanosuspensions were prepared by the optimized pulverization method while varying the telmisartan-to-polysorbate 80 ratios at a fixed polysorbate 80 concentration of 4 mg/ml (Table S3). The ratio between telmisartan and polysorbate 80 in INT-TEL was determined to be roughly 4:1 (Table 1).

Figure 1. Physicochemical properties and in vitro anti-virus activity of INH-TEL.

(A) Hydrodynamic diameter of freshly prepared (black) and lyophilized-rehydrated (red) INH-TEL measured by DLS. (B) Representative transmission electron micrographs of freshly prepared (left) and lyophilized-rehydrated (right) INH-TEL. Scale bar = 100 nm. (C) Colloidal stability of INH-TEL in a physiological lung environment determined by the changes of particle hydrodynamic diameters in mouse BALF at 37 °C over time (n = 3 independent experiments). (D) Cumulative in vitro release of the drug payloads (i.e., telmisartan) from INH-TEL in DPBS supplemented with 0.05% polysorbate 80 over time (n = 3 independent experiments). (E) In vitro drug activity (i.e., inhibition of intracellular calcium spike induced by ANG II) of INH-TEL in comparison to FD-TEL. (F) In vitro inhibitory effect on SARS-CoV-2 replication (red) and cytotoxicity (black) of INH-TEL in Calu-3 cells (n = 3 independent experiments). *p < 0.05 (one-way ANOVA).

Table 1.

Physicochemical properties of freshly prepared and lyophilized-rehydrated INH-TEL.

INH-TEL	Hydrodynamic diameter (nm)	PDI	ζ-potential (mV)	Drug loading density (%)
Fresh	322 ± 15	0.24 ± 0.03	−2.9 ± 0.5	82 ± 3
Lyophilized-rehydrated	351 ± 9	0.25 ± 0.04	−4.0 ± 1.0	‐

We then found that lyoprotectant-free lyophilization and subsequent reconstitution (i.e., rehydration) did not yield particle aggregates and resulted in only moderate changes in hydrodynamic diameters (351 ± 0.9 nm; PDI = 0.25 ± 0.04) and ζ-potentials (−4.0 ± 1.0 mV) (Figure 1A and Table 1). Likewise, transmission electron microscopy revealed that both freshly prepared and lyophilized-rehydrated INH-TEL possessed rod-shaped morphology with similar geometric sizes (Figure 1B). The findings here underscore that INH-TEL could be stored long-term in a powder form prior to reconstitution in an aqueous vehicle solution for inhaled administration. INH-TEL also demonstrated excellent colloidal stability in a physiologically relevant lung environment, mouse BALF, for at least up to 2 hours, as evidenced by negligible changes in hydrodynamic diameters and PDI (Figure 1C). In parallel, we conducted an in vitro drug release study using Dulbecco’s phosphate-buffered saline (DPBS) supplemented with 0.05% polysorbate 80 as an artificial sink condition where nearly 90% of telmisartan was released within the first 5 hours (Figure 1D). The rapid drug release may be beneficial for managing acute pathological conditions that require prompt drug action, such as ARDS triggered by respiratory pathogens.

In vitro pharmacological activities of INH-TEL.

We next tested whether the drug release from INH-TEL preserved its inherent drug activity by assessing the ability to prevent ANG II-mediated elevation of intracellular calcium ion concentration ([Ca2+]i) in lung smooth muscle cells. ANG II binding to its cell surface receptor (i.e., ANG II type 1 receptor) activates the voltage-gated Ca2+ channels to elevate [Ca2+]i, which is effectively inhibited by ARBs.^28–30 We found that the ANG II-mediated transient [Ca2+]i spike was equally and entirely abrogated when cells were treated with dose-matched free drug telmisartan (FD-TEL) or INH-TEL (Figure 1E), suggesting that the drug activity of INH-TEL was fully retained. To test our hypothesis that INH-TEL might provide anti-viral efficacy, we then assessed the ability of INH-TEL to deter SARS-CoV-2 replication in vitro.^{19, 20} We found that the viral replication was inhibited by INH-TEL in a dose-dependent manner (up to the telmisartan concentration of 33.3 μg/mL) in Calu-3 cells without incurring significant cytotoxicity (Figure 1F). Of note, Calu-3 has been confirmed for expression of the cell surface portal for SARS-CoV-2 (i.e., ACE2) and susceptibility to the viral infection accordingly.³¹ Our observation agrees with recent reports demonstrating inhibitory effects of various ARBs, including telmisartan, against SARS-CoV-2 replication in Vero-E6 or Caco-2 cells.^7–9 In relevance to these findings, it has been shown that intracellular calcium is essential for viral assembly and budding of SARS-CoV,³² the virus responsible for outbreak of severe acute respiratory syndrome in 2003, and that ARB reduces viral spread by preventing release of several enveloped viruses from infected cells.^33–35 More recently, the potential role of ARBs in blocking the main protease of SARS-CoV-2 essential for viral replication and transcription has been suggested,⁸ but further investigation is warranted to fully unravel the mechanism(s) of inhibition. Although ARBs were initially speculated to upregulate ACE2 to promote viral infection and disease severity, recent independent studies refuted such a hypothesis.³⁶

We note that while FD-TEL exhibited modest dose-dependent anti-viral effect (Figure S1), telmisartan was markedly more effective when combined with the polysorbate 80 as a nanosuspension (i.e., INH-TEL; Figure 1F). As the anti-viral effect of the two ingredients of INH-TEL appears to be synergistic, direct pulmonary administration is likely the most practical dosing method to dispatch both components simultaneously to the apical lung surface. Importantly, the highest viral load in SARS-CoV-2, as well as numerous other respiratory viral infections, occurs in the apical lung. The exact mechanism of the enhanced in vitro anti-viral effect enabled by the addition of polysorbate 80 is yet to be unraveled. However, we speculate that it may be linked to an anti-viral effect of its hydrolysis product, oleic acid, which has been previously shown to inactivate several enveloped virus.^37–40

Pharmacokinetics of INH-TEL following intratracheal administration into the lungs of mice and cynomolgus macaques

We went on to test our hypothesis that direct administration of INH-TEL into the lung would provide high telmisartan concentrations in the lung. To test this, we intratracheally administered INH-TEL into the lungs of C57BL/6 mice at a telmisartan dose of 0.1 mg/kg and compared the drug content in the lung and the plasma at different time points. Of note, INH-TEL was reconstituted to include < 0.02% w/w polysorbate 80 to meet the FDA cut-off for respiratory use.²³ We found that telmisartan content was about an order of magnitude greater in the lung compared to the plasma at 1- and 12-hour post-administration (Figure 2A). To complement this mouse study, we recently conducted a small pharmacokinetic study using NHPs (i.e., cynomolgus macaques) in which we compared locally administered INH-TEL versus oral FD-TEL. Specifically, two macaques were intratracheally treated with INH-TEL at a telmisartan dose of 2.5 mg per animal (0.81 – 0.87 mg/kg) and lung tissues were harvested at 0.5 or 8-hour post-administration for the assessment of drug content in the lung. As a clinically relevant control, one macaque received daily oral FD-TEL for 7 days at a telmisartan dose of 1 mg/kg and lung tissue was harvested 2 hours after the final dose. The 2-hour time point was selected based on a previous human study demonstrating the median time to maximum plasma concentration of systemically administered telmisartan to be 0.5 – 2 hours.⁴¹ Macaques that received intratracheal INH-TEL, regardless of the time of lung harvest, exhibited an at least 10-fold and up to 40-fold greater drug content in the lung compared to the macaque received 7 daily oral FD-TEL (Figure 2B). In parallel, we monitored plasma pharmacokinetics of these animals. The plasma drug content of the animals that received intratracheal INH-TEL was transiently elevated but quickly reduced to the level on par with or lower than the steady-state plasma drug content observed with the animal under the daily oral FD-TEL regimen (Figure 2C). In agreement with our mouse study (Figure 2A), telmisartan content was markedly and significantly greater in the lung compared to the plasma at both 0.5- and 8-hour post-administration of the intratracheal INH-TEL (Figure 2D). In contrast, the drug content was significantly greater in the plasma than in the lung 2 hours after the final (i.e., 7th) oral FD-TEL administration (Figure 2D).

Figure 2. Pharmacokinetics of INH-TEL following intratracheal administration into the lungs of wild-type C57BL/6 mice and cynomolgus macaques.

(A) Lung and plasma concentrations of telmisartan 1 and 12 hour(s) after a single intratracheal administration of INH-TEL at a telmisartan dose of 0.1 mg/kg into the lungs of wild-type C57BL/6 mice (n = 5 mice per group). (B-D) Two macaques, CM1 (3.08 kg) and CM2 (2.86 kg), received a single intratracheal (IT) administration of INH-TEL at a fixed telmisartan dose of 2.5 mg (0.81 – 0.87 mg/kg, calculated based on the body weights) and one macaque (CM3; 3.08 kg) received daily oral gavage (OG) administration of FD-TEL at a telmisartan dose of 1 mg/kg for 7 days. (B) Telmisartan content in the lung tissues from CM1 and CM2 harvested at 0.5- and 8-hour post-administration of INH-TEL into the lung, respectively, and from CM3 harvested 2 hours after the last (i.e., 7th) daily oral administration of FD-TEL. (C) Plasma pharmacokinetics of telmisartan in CM1 and CM2 after receiving a single intratracheal administration of INH-TEL and CM3 after receiving the 6th daily oral administration of FD-TEL. Plasma pharmacokinetics were monitored until CM1 and CM2 were euthanized to harvest lung tissues and up to 12 hours for CM3. (D) Relative telmisartan content in the lung tissue versus the plasma harvested from CM1 and CM2 at 0.5- and 8-hour post-administration of INH-TEL into the lung and from CM3 at 2-hour post-administration of the last (i.e., 7th) oral FD-TEL dose. *p < 0.05, ***p < 0.005 and ****p < 0.001 (one-way ANOVA).

We note that the NHP study was conducted as a comparison in extremes where we sought to evaluate the tolerability of a very high dose of locally, lung-administered telmisartan in NHPs. Employing a 10-fold safety factor⁴² recommended by the FDA, we chose the intratracheal telmisartan dose at 10 times the efficacious dose predicted from our mouse study (0.1 mg/kg). Based on the reference body and lung weights of different species,⁴³ the intratracheal telmisartan dose of 2.5 mg corresponds to a human equivalent dose of 125 mg. On the other hand, the oral dose was given at a lower level in NHPs in order to allow us to obtain a well-tolerated steady-state dose. Specifically, the 1-mg/kg oral NHP dose converts to the human equivalent of 19.4 mg for a reference human body weight of 60 kg⁴², which is 25% to 50% of the standard oral daily dose of 40 – 80 mg given to patients with hypertension. Clearly, using a ~6.5-fold greater local dose resulted in up to a 40-fold greater drug amount delivered to the lung (Figure 2B). Assuming lung concentrations scale linearly with oral dose, human equivalent oral doses of ~780 mg/day would be required to achieve the same level of lung exposure in these NHPs.

Histopathological analysis of lungs of macaques that received intratracheal INH-TEL

We next assessed tolerability of intratracheally administered INH-TEL in NHPs. Specifically, lung tissues harvested from three macaques at the respective times of pulmonary drug content analysis (Figure 2C) were subjected to paraffin section and hematoxylin & eosin staining. Sections were taken from the peripheral and central cranial, peripheral and central caudal, and peripheral and central midportion of both right and left lungs of each of the three animals. The lung slides were then scored in a blinded manner for edema, composite inflammation, increased bronchus-associated lymphoid tissue (BALT), reactive epithelial changes, alveolar collapse, and interstitial fibrosis by a board-certified pathologist (KT). The evaluation revealed no significant histopathologic differences between INH-TEL intratracheally administered at an extremely highly local dose and oral FD-TEL given at a well-tolerated dose with acceptable tolerability in the lung tissues (Figure 3). We note that mild alveolar collapse observed shortly after the intratracheal administration of INH-TEL was quickly resolved (Figure 3). We also conducted blood biochemistry analysis at the times of lung harvest and respective baselines (i.e., prior to the administration). We found that most of the biochemical readouts were comparable before and after the treatments (Table 2), underscoring that our formulation did not exert significant systemic toxicity. The high drug level of lung exposure achieved in NHPs resulted in circulating plasma levels that are commensurate with well-tolerated systemic telmisartan levels after oral dosing, ensuring systemic safety; of note, human dosing with 120 mg tablets has been reported to yield a Cmax of 1.635 μg/mL.⁴⁴ Notably, the human experience with telmisartan also indicates that increased dosage is not associated with greater blood pressure reductions, but rather with longer duration of effect.⁴⁴ Thus, the transient systemic exposure of telmisartan observed after the intratracheal administration of INH-TEL (Figure 2C) is not expected to yield adverse events. Further, the lung exposure shown to be well tolerated in NHPs is well beyond any level expected to be needed in humans.

Figure 3. Histopathological analysis of lung tissues from macaques after receiving either intratracheal INH-TEL or oral FD-TEL.

Lung tissues were harvested at different time points after a single intratracheal administration of INH-TEL at a fixed telmisartan dose of 2.5 mg (0.81 – 0.87 mg/kg) or after the 7th oral gavage administration of FD-TEL at a telmisartan dose of 1 mg/kg.

Table 2.

Blood biochemistry panel of macaques received either intratracheal INH-TEL or oral FD-TEL.

Macaque ID	CM1		CM2		CM3
Administration	IT		IT		OG
Treatment	INH-TEL (2.5 mg)		INH-TEL (2.5 mg)		FD-TEL (1 mg/kg)
Time	B.L.†	0.5 h§	B.L.†	8 h§	B.L.†	D6 B.L.‡	D7 B.L.‡	2 h§
ALP (U/L)	137	128	135	147	164	150	158	155
ALT (U/L)	64	61	15	51	86	131	177	176
AST (U/L)	44	47	23	235	85	86	163	222
Albumin (g/dL)	3.8	3.5	3.7	3.8	3.5	3.4	3.6	3.5
Total Protein (g/dL)	6.8	6.3	6.7	7	6.9	6.2	6.4	6.2
Globulin (g/dL)	3	2.8	3	3.2	3.4	2.8	2.8	2.7
Total Bilirubin (mg/dL)	0.2	0.2	0.1	0.3	0.1	0.2	0.2	0.2
Bilirubin-Conjugated (mg/dL)	0	0	0	0.1	0	0.1	0.1	0
BUN (mg/dL)	31	27	19	20	26	22	26	27
Creatinine (mg/dL)	0.7	0.6	0.6	0.5	0.7	0.6	0.8	0.8
Cholesterol (mg/dL)	133	127	125	137	125	106	103	104
Glucose (mg/dL)	79	100	87	100	97	77	85	59
Calcium (mg/dL)	9	8.8	9.1	8.7	9.6	8.9	9.7	9.3
Phosphorus (mg/dL)	3.9	3.7	3.7	5.6	5	3.1	3	3.2
Chloride (mmol/L)	104	106	109	113	105	105	103	108
Potassium (mmol/L)	3.3	3.8	3.5	3.7	3	3.8	4.1	3.4
Sodium (mmol/L)	146	146	148	149	146	147	145	148
ALB/GLOB ratio	1.3	1.3	1.2	1.2	1	1.2	1.3	1.3
BUN/Creatinine Ratio	44.3	45	31.7	40	37.1	36.7	32.5	33.8
Bilirubin-Unconjugated (mg/dL)	0.2	0.2	0.1	0.2	0.1	0.1	0.1	0.2
NA/K Ratio	44	38	42	40	49	39	35	44
Hemolysis Index	N	+	N	+++	N	+	N	N
Lipemia Index	N	N	N	N	N	N	N	N

^†Baseline levels prior to the intratracheal administration of INH-TEL or the first oral administration of FD-TEL.

^‡Baseline levels prior to 6th or 7th daily oral dose of FD-TEL.

^§Levels at different times post-administration of intratracheal INH-TEL or of the last (i.e., 7th) oral administration of FD-TEL.

CONCLUSION

In summary, we have developed a surface-stabilized nanosuspension formulation of telmisartan suitable for long-term storage and shipping in a powder form and experimentally confirmed its physiological stability, unperturbed drug activity and inhibitory potential against SARS-CoV-2 infection. Further, our formulation demonstrates excellent lung pharmacokinetics and acceptable local and systemic tolerability as revealed by our NHP studies. To this end, we are currently continuing our effort toward the clinical development of our formulation to be nebulized for treating patients with COVID-19 or ARDS associated with other respiratory infections.