Organophosphates (OPs), widely used as insecticides, exhibit severe acute toxicity and pose a significant threat to public health. Current standard therapy, which mainly relies on atropine and oxime reactivators, alleviates symptoms but fails to effectively eliminate circulating toxin or excess acetylcholine (ACh), thus representing a non-causal intervention. This study proposes a novel, efficient synergistic dual-enzyme strategy for the treatment of acute OP poisoning in a Sprague-Dawley rat model. Intravenous co-administration of organophosphorus hydrolase (OPH) from Pseudomonas diminuta, site-specifically PEGylated at its N-terminus, together with recombinant human butyrylcholinesterase (BuChE), effectively mitigated cholinergic crisis and accelerated systemic clearance of residual OPs. The hydrolysis rate of ethyl paraoxon by PEG-OPH is 90.9-fold higher than its binding rate to BuChE. This kinetic advantage enables rapid degradation of free OPs in the circulation during the early intoxication phase, thereby dominating initial toxin clearance and preventing excessive BuChE consumption. Concurrently, the administered BuChE hydrolyzes accumulated ACh due to acetylcholinesterase (AChE) inhibition, helping restore the balance of cholinergic neurotransmission. Compared with BuChE monotherapy, the synergistic regimen reduced the required BuChE dose by up to 83%, while achieving full restoration of respiratory parameters to baseline levels within 30 min post-treatment. Moreover, 24-h open-field testing revealed significantly enhanced locomotor activity in the combination group, as evidenced by a markedly greater total distance traveled compared with either PEG-OPH or BuChE monotherapy. Critically, this dual-enzyme strategy conferred complete protection against lethality, yielding a 100% 7-d survival rate. This work pioneers a therapeutic strategy using exogenous enzymes to simultaneously target both the primary toxin and its pathological downstream metabolite. Our results define a highly efficient, precise, cause-targeted approach with significant translational potential for the management of acute organophosphate poisoning.

The von Hippel-Lindau tumor suppressor (VHL) has been extensively used to develop degraders targeting numerous proteins of interest. However, studies on the rational design of VHL-proteolysis-targeting chimeras (PROTACs) remain scarce. This study aimed to develop strategies to investigate VHL-recruiting PROTACs connecting with varying attachment sites on VHL ligands, which could be utilized for KRAS(G12C) degraders development and expanded to additional targets. We developed a molecular dynamics (MD)-based strategy to explore the stability of ternary complexes induced by KRAS(G12C) PROTACs with four distinct attachment sites of VH032. We found a potent degrader namely YN14-H, linked to hydroxyl group on VH032 benzene ring, exhibited the most superior ability of inducing ternary complexes, reflected by the lowest dissociation constant (K-d) for ternary complex induction and the highest AlphaScreen (AS)-based interaction. YN14-H inhibited cell growth with low nanomolar half maximal inhibitory concentration (IC50) and half maximal degradation concentration (DC50) values as well as >98 % of maximum degradation (D-max) in NCI-H358 and MIA PaCa-2 cells harboring KRAS(G12C)-mutation. Mechanistically, YN14-H significantly induced apoptosis and inhibited the migratory capacity. Notably, YN14-H demonstrated favorable pharmacokinetic properties and excellent antitumor activity in vivo. Furthermore, bromodomain-containing protein 7 (BRD7) and Bruton tyrosine kinase (BTK) degraders attached to distinct sites on VH032 further verified the rationality and universality of our MD-based strategies. Our findings demonstrated that YN14-H could serve as a promising candidate for the treatment of tumors with KRAS(G12C)-mutation and present a strategy for the rational design of VHL-recruiting PROTACs that target additional proteins at distinct attachment sites. (c) 2025 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

Background: Chronic insomnia is associated with elevated cardiovascular disease risk, and current therapeutic options for this condition remain inadequate. Melatonin (MT) combined with cannabidiol (CBD) may exert synergistic effects on improving sleep; the underlying pharmacological drug–drug interactions (DDI) and interspecies differences in their combined actions remain unknown. Purpose: This study aimed to evaluate the pharmacokinetic characteristics of combined drug formulations by utilizing DDI-based approaches so as to underpin the efficacy and safety of the formulation. Methods: Overexpressing hPEPT1 in MDCK cells, multiple species liver microsomes, equilibrium dialysis, and a static DDI model were employed to assess CBD’s effects on MT’s cellular uptake, inhibitory effect, enzymatic phenotype, protein binding, and human AUC changes. Results: CBD significantly increased MT exposure in dogs but caused dose-dependent biphasic changes in rats. MT negligibly affected CBD PK. In vitro, CBD inhibited MT metabolism with species differences: potent competitive inhibition in dogs (IC50 = 3.42 ± 1.30 μM), weaker inhibition in rats/humans (IC50 = 13.54 ± 1.15/16.47 ± 4.23 μM). CBD also demonstrated mechanism-based inhibition (KI = 25.63 μM, Kinact = 0.063 min−1) against human CYP1A2-mediated MT metabolism. Acidic conditions revealed that CBD inhibited PEPT1-mediated MT uptake. CBD exhibits high and MT moderate protein binding. Static model predictions aligned with in vivo dog/rat data project a worst-case human MT AUC increase up to 12-fold. Conclusions: This study identifies the critical role of PEPT1 in MT absorption and elucidates the dual mechanisms of CBD; namely, absorption inhibition and metabolic delay in regulating MT pharmacokinetics, which exhibits interspecies differences.

To mitigate risks in central nervous system (CNS) drug development, we established a high-throughput in vitro blood-brain barrier (BBB) model using LLC-PK1-MOCK and LLC-PK1-MDR1 cells in a Transwell system, aiming to replicate in vivo brain distribution and elucidate permeability mechanisms. Model integrity was assessed via transepithelial electrical resistance (TEER) and efflux functionality using control drugs (atenolol, digoxin). Bidirectional transport studies of 41 compounds quantified permeability (Papp), efflux ratios (ER), and recoveries, while in vivo brain distribution parameters (Kp,uu,brain) were derived from literature and rat studies. The model demonstrated critical BBB features: tight junction integrity (TEER > 70 Ω·cm2), P-gp efflux activity (digoxin ER = 5.10 ~ 17.12), and discrimination of passive diffusion (63.41% of drugs) from transporter-mediated mechanisms (19.5% P-gp substrates). A training set of 20 randomly selected drugs revealed a robust correlation between MDR1-derived Papp(A-B) and Kp,uu,brain (R = 0.8886), with the remaining 21 compounds validating predictive accuracy (≤2-fold error). Four alkaloids exhibiting low recovery (<80%) due to lysosomal trapping were corrected using Bafilomycin A1, aligning their permeability with in vivo outcomes. These results position the LLC-PK1-MOCK/MDR1 model as a reliable surrogate tool for early CNS drug screening, enabling rapid prioritization of candidates based on BBB penetration potential. Its integration into preclinical workflows promises to accelerate the development of therapeutics for neurological disorders.

A small hexameric coiled-coil fusion complex formed by the interaction between α-helical motif of heptad repeat 2 (HR2core) and HR1 core site (HR1core) in human betacoronavirus spike protein plays a crucial role in facilitating membrane fusion. Formation of the six-helix bundle involves the evolutionary conservation of key residues in the HR1core region. Therefore, to recapitulate the native conformation of the HR2core peptide α-helix and the HR1core site conserved residues, we employed hydrocarbon double-stapling. The resulting all-hydrocarbon stapled peptide M2PA shows highly potent and broad-spectrum antiviral activity against SARS-CoV-2 and its emerging variants, as well as other betacoronaviruses tested. Also, M2PA is effective against authentic SARS-CoV-2 infection in vivo, and it possesses outstanding pharmacokinetic properties. This represents the first successful minimization of a pan-coronavirus inhibitor to an HR2core-based α-helical peptide, and as such, M2PA peptide stands as a promising candidate for drug development to combat coronavirus pandemics.

HD561, which was designed to enhance nerve growth, was re-engineered into HD56, a carboxylic acid ester prodrug. The goal of this study was to compare the druggability, species differences, and the correlation between in vitro and in vivo transformation of HD56 to HD561 from a pharmacokinetic (PK) perspective, offering a scientific basis for HD56's clinical research. The bidirectional transmembrane transport of HD56 and HD561 was investigated using Caco-2 cells and LLC-PK1 cells overexpressing MDR1 monolayer cells. Recombinant enzymes and chemical inhibition methods were employed to identify the reaction phenotyping. The conversion of HD56 to HD561 was compared in hepatic and intestinal microsomes, as well as plasma, across different species, including humans, rats, monkeys, and mice with humanized liver. PK studies were conducted in rats, monkeys, and mice with different humanized liver proportions (Hu-URG, Hu-URG-Low, and Hu-URG-High). Finally, an in vivo-in vitro correlation was established between the conversion rate of HD56 to HD561. Results showed that HD56 had better permeability than HD561. HD56 could be hydrolyzed by carboxylesterase 1 to HD561 and be metabolized by cytochrome P450 isoenzymes, while HD561 underwent further metabolism via CYP2C9. Significant species differences existed, and a good in vivo-in vitro correlation was only achieved in humanized mice (r = 0.98). Both in vitro and in vivo PK characteristics of HD56 were remarkably superior to those of HD561, suggesting that HD56 held promise for development. Humanized liver mice serve as a powerful model to address the issue of species differences in ester prodrugs. SIGNIFICANCE STATEMENT: Prodrug HD56 showed superior pharmacokinetic properties compared with the active compound HD561, guiding similar prodrug research. The use of chimeric mice with human hepatocytes, for the first time, to study carboxylesterase (CES) prodrug HD56 provides a model that closely mimics human metabolism. Findings deepen understanding of HD56's behavior and offer a predictive tool for CES prodrugs' metabolic fate, streamlining drug development and improving preclinical accuracy.

HD561, which swas designed to enhance nerve growth, was re-engineered into HD56, a carboxylic acid ester prodrug. The goal of this study was to compare the druggability, species differences, and the correlation between in vitro and in vivo transformation of HD56 to HD561 from a pharmacokinetic perspective, offering a scientific basis for HD56’s clinical research. The bidirectional transmembrane transport of HD56 and HD561 was investigated using Caco-2 cells and LLC-PK1 cells overexpressing MDR1 monolayer cells. Recombinant enzymes and chemical inhibition method were employed to identify the reaction phenotyping. The conversion of HD56 to HD561 was compared in hepatic and intestinal microsomes, as well as plasma, across different species including humans, rats, monkeys, and mice with humanized liver. Pharmacokinetic studies were conducted in rats, monkeys and mice with different humanized liver proportions (Hu-URG, Hu-URG-Low, and Hu-URG-High). Finally, an in vivo-in vitro correlation (IVIVC) was established between the conversion rate of HD56 to HD561. Results showed that HD56 had better permeability than HD561. HD56 could be hydrolyzed by CES1 to HD561 and be metabolized by CYP isoenzymes, while HD561 underwent further metabolism via CYP2C9. Significant species differences existed and a good IVIVC only achieved in humanized mice (r=0.98). Both in vitro and in vivo pharmacokinetic characteristics of HD56 were remarkably superior to those of HD561, suggesting that HD56 held promise for development. Humanized liver mice serve as a powerful model to address the issue of species differences in ester prodrugs.

Objective: The aim was to evaluate the pharmacokinetics and underlying mechanisms of peramivir, a clinically approved antiviral agent for severe influenza, subsequent to airway inhalation in rats, thereby surmounting the constraints associated with the sole currently available intravenous formulation. Methods: Pharmacokinetic and tissue distribution investigations of peramivir were carried out in rats following both intravenous and inhaled administration. In vitro cell models were verified to investigate peramivir’s transmembrane transport and cellular uptake across diverse cell systems. Results: In vivo, peramivir exhibited restricted permeability, predominantly localizing within the alveolar epithelial lining fluid and lung tissue after inhalation, accompanied by minimal systemic dissemination. In vitro, it manifested low permeability across cell models, with no participation of efflux transporters. Despite the low rate of A549 uptake, the underlying uptake transport mechanism was still revealed. Peramivir was verified as an OCTN2 substrate. A robust correlation was observed between the in vitro and in vivo findings. Conclusions: A preclinical pharmacokinetic platform applicable to inhaled medications was established. Inhalation of peramivir augments exposure at the target site while diminishing systemic exposure, presenting potential therapeutic benefits in terms of efficacy and safety and suggesting it as a favorable alternative administration pathway.

Objective: To establish and validate an LC-MS/MS method for the simultaneous determination of curcumin (CUR) as well as its glucuronide conjugate (COG) and sulfate conjugate (COS) in rat plasma. The method was employed to evaluate and compare the pharmacokinetic behaviors of curcumin following oral and intratracheal administration in rats. Methods: Rat plasma samples were separated by chromatography on a C18 column after protein precipitation with acetonitrile. Gradient elution with a mobile phase of 0.5 mM ammonium acetate in acetonitrile was utilized. Mass spectrometry detection incorporated an electrospray ionization (ESI) source, multiple reaction monitoring (MRM), and dual-mode (positive and negative) scanning for quantitative analysis. A total of 12 SD rats were randomly divided into two groups and were orally (20 mg/kg) or intratracheally (10 mg/kg) administrated curcumin, respectively. CUR, COG, and COS concentrations in plasma were measured to assess pharmacokinetic disparities. Results: The method demonstrated linearity within the ranges of 2–400 ng/mL for CUR and COS and 5–1000 ng/mL for COG. Intratracheal administration significantly elevated CUR plasma concentrations compared to oral administration. The exposure of COG was higher than COS following oral administration. Conversely, intratracheal administration resulted in markedly higher COS exposure, with no significant difference in COG exposure after dose normalization between oral and inhalation routes. Conclusions: The established LC-MS/MS method provides a reliable tool for the simultaneous measurement of CUR, COG, and COS in rat plasma, facilitating preclinical pharmacokinetic investigations. The study reveals distinct pharmacokinetic profiles for CUR following oral versus intratracheal administration, suggesting that inhalation may offer superior therapeutic efficacy.

Different from most antiretroviral drugs that act as passive defenders to inhibit HIV-1 replication inside the host cell, virus inactivators can attack and inactivate HIV-1 virions without relying on their replication cycle. Herein, we describe the discovery of a hydrocarbon double-stapled helix peptide, termed D26. D26 is based on the HIV-1 gp41 protein lentiviral lytic peptide-3 motif (LLP3) sequence, which can efficiently inhibit HIV-1 infection and inactivate cell-free HIV-1 virions. It was noted that D26 was highly resistant to proteolytic degradation and exhibited a remarkably extended in vivo elimination half-life. Additionally, relative to its linear, nonstapled version, D26 exhibited much higher exposure in sanctuary sites for HIV-1. Amazingly, this lead compound also demonstrated detectable oral absorption. Thus, it can be concluded that D26 is a promising candidate for further development as a long-acting, orally applicable HIV-1 inactivator for the treatment of HIV-1 infection.

Tetrandrine (TET) has been traditionally used in China as a medication to treat silicosis and has recently demonstrated anti-SARS-CoV-2 potential in vitro. By recognizing the disparity between in vitro findings and in vivo performance, we aimed to estimate the free lung concentration of TET using a physiologically based pharmacokinetic (PBPK) model to link in vitro activity with in vivo efficacy. Comparative pharmacokinetic studies of TET were performed in rats and dogs to elucidate the pharmacokinetic mechanisms as well as discern interspecies variations. These insights facilitated the creation of an animal-specific PBPK model, which was subsequently translated to a human model following thorough validation. Following validation of the pharmacokinetic profile from a literature report on single oral dosing of TET in humans, the plasma and lung concentrations were predicted after TET administration at approved dosage levels. Finally, the antiviral efficacy of TET in humans was assessed from the free drug concentration in the lungs. Both in vivo and in vitro experiments thus confirmed that the systemic clearance of TET was primarily through hepatic metabolism. Additionally, the lysosomal capture of basic TET was identified as a pivotal factor in its vast distribution volume and heterogeneous tissue distribution, which could modulate the absorption dynamics of TET in the gastrointestinal tract. Notably, the PBPK-model-based unbound lung concentration of TET (1.67–1.74 μg/mL) at the recommended clinical dosage surpassed the in vitro threshold for anti-SARS-CoV-2 activity (EC90 = 1.52 μg/mL). Thus, a PBPK model was successfully developed to bridge the in vitro activity and in vivo target exposure of TET to facilitate its repurposing.

Objective: This study aimed to develop a quantitative analytical method for the simultaneous determination of cannabidiol (CBD) and melatonin (MT) in mouse plasma using the protein precipitation method coupled with LC-MS/MS. Additionally, this study sought to investigate the impact of CBD on the pharmacokinetics of MT in mice using this method. Methods: Mouse plasma samples were precipitated with acetonitrile and analyzed using a Kromasil 100-5-C8 (2.1 × 50 mm) column. Following a single administration, thirty male ICR mice were randomly assigned to five groups: MT 2 mg/kg intravenously (i.v.), MT 10 mg/kg orally (p.o.), MT + CBD (10 + 10) mg/kg p.o., MT + CBD (10 + 40) mg/kg p.o., and MT 10 mg/kg p.o. followed by CBD 2 mg/kg i.v. Pharmacokinetic parameters were calculated using a non-compartmental model and analyzed to investigate the interactions of CBD with MT. Results: The calibration curves for CBD and MT were linear over the range of 2 to 1000 ng/mL. Co-administration of a high dose of CBD (40 mg/kg) orally reduced the Cmax of MT (10 mg/kg) to 57% of the control, while the area under the curve from 0.5 to 8 h (AUC(0.5–8h)) was 2.85-fold that of the MT-only group. When CBD (2 mg/kg) was administered intravenously alongside MT orally, the AUC(0.5–8h) was 1.54 times that of MT given orally alone. The AUC of CBD was positively correlated with the AUC of the distribution and elimination phases of MT, while the Cmax of CBD negatively correlated with the Cmax of MT. Conclusions: The developed LC-MS/MS method is robust and suitable for pharmacokinetic studies involving CBD and MT. The in vivo effects of CBD on MT pharmacokinetics are complex. High oral doses of CBD inhibit both the intestinal absorption and metabolic clearance of MT, resulting in a more smooth PK profile.

An LC-MS/MS method was developed and validated for the simultaneous determination of the carboxylic acid ester precursor HD56 and the active product HD561 in cynomolgus monkey plasma. Then, the pharmacokinetic characteristics of both compounds following single and multiple i.g. administrations in cynomolgus monkeys were elucidated. In the method, chromatographic separation was achieved with a C18 reversed-phase column and the target quantification was carried out by an electrospray ionization (ESI) source coupled with triple quadrupole mess detector in positive ionization mode with multiple reaction monitoring (MRM) approach. Using the quantification method, the in vitro stability of HD56 in plasma and HD56 pharmacokinetic behavior after i.g. administration in cynomolgus monkey were investigated. It was approved that HD56 did convert into HD561 post-administration. The overall systemic exposure of HD561 post-conversion from HD56 accounted for only about 17% of HD56. After repeated administration at the same dose, there was no significant difference in exposure levels of both HD56 and HD561. However, after multiple dosing, the exposure of HD56 tended to decrease while that of HD561 tended to increase, resulting in a 30% in the exposure ratio. Remarkably, with a carboxylesterase (CES) activity profile akin to humans, the observed in vivo pharmacokinetic profile in cynomolgus monkeys holds promise for predicting HD56/HD561 PK profiles in humans.

As a novel guanylate cyclase stimulator, SGC003F is being developed for the treatment of heart failure with a reduced ejection fraction (HFrEF). This study aimed to assess the effect of P-glycoprotein (P-gp) inhibition on SGC003F exposure in vivo, comparing plasma and tissue levels, and evaluating the role of P-gp in the small intestine, blood–brain barrier (BBB), and kidney in impacting the tissue exposure. Tariquidar, a P-gp inhibitor, was added to monolayer transport assays to observe the changes in the transmembrane characteristics of SGC003F. Rats were given SGC003F with tariquidar via various routes to measure plasma, tissue, urine, and fecal concentrations. The inclusion of tariquidar significantly altered the pharmacokinetics of SGC003F. In LLC-PK1-MDR1 cells, tariquidar reduced the efflux ratio of SGC003F from 6.56 to 1.28. In rats, it enhanced the plasma AUC by 3.05 or 1.61 times, increased the Cmax by 2.13 or 1.07 times, and notably improved bioavailability from 46.4% to 95%. Additionally, co-administration with tariquidar led to a decrease in fecal excretion and an increase in tissue exposure, with only a moderate effect on the partition ratios in the small intestine and brain. P-gp inhibition impacts SGC003F exposure, with plasma levels not fully reflecting tissue levels. P-gp in the small intestine and BBB affects SGC003F’s pharmacokinetics, warranting further clinical drug–drug interaction (DDI) studies.

Background:Despite the availability of various tools of modeling and simulation, clinical pediatric pharmacokinetic (PK) studies remain far less efficient than those on adults due to ethical constraints. One of the optimal solutions is to substitute urine to blood sampling based on explicit mathematic relationships between them. However, this idea is limited by three main knowledge gaps associated with urine data, i.e., complicated excretion equations with excessive parameters, insufficient frequency that is hard to fit, and the mere expression of amounts with no in vivo distribution volume information involved.Methods:To overcome these obstacles, we sacrificed the precision from mechanistic PK models with complex excretion equations to expediency of compartmental model in which a constant ke is used to cover all the internal parameters. And the total cumulative amounts of urinary drug excretion (Xu∞) were estimated and introduced to the excretion equation so that urine data were likely to be fitted using a semi-log-terminal linear regression method. In addition, urinary excretion clearance (CLr) could be calculated by single point plasma data to anchor the plasma concentration-time (C-t) curve based on the assumption that CLr was kept constant throughout the PK process.Results:After sensitivity analysis of two subjective judgements (the selection of the compartmental model and the selection of plasma time point to calculate CLr), the performance of the optimized models was assessed using desloratadine or busulfan as model drugs in a variety of PK scenarios, from i.v. bolus/infusion to p.o. administration, from a single dose to multiple doses, and from rats to children. The fitting plasma drug concentrations of the optimal model were close to the observed value. Meanwhile, the drawbacks inherent to the simplified and idealized modeling strategy were fully identified.Conclusions:The method proposed by this tentative proof-of-principle study was able to deliver acceptable plasma exposure curves and shed light on the future refinements.

By sorting out the scale, function and personnel allocation of designated hospitals for treating severe COVID-19 patients, combining the intensive care management experience of designated hospitals in Wuhan and Shanghai under different scenarios, and rapidly integrating limited intensive care resources according to local conditions, the stepped intensive care extension working mode of "Critical Pass Forward-Homogeneous Critical Care-Advanced Care Ward" was summarized. It provides reference for the treatment of severe COVID-19 patients in relevant hospitals.

In prolonged liver injury, hepatocytes undergo partial identity loss with decreased regenerative capacity, resulting in liver failure. Here, we identified a five compound (5C) combination that could restore hepatocyte identity and reverse the damage-associated phenotype (e.g., dysfunction, senescence, epithelial to mesenchymal transition, growth arrest, and pro-inflammatory gene expression) in damaged hepatocytes (dHeps) from CCl4-induced mice with chronic liver injury, resembling a direct chemical reprogramming approach. Systemic administration of 5C in mice with chronic liver injury promoted hepatocyte regeneration, improved liver function, and ameliorated liver fibrosis. The hepatocyte-associated transcriptional networks were reestablished with chemical treatment as revealed by motif analysis of ATAC-seq, and a hepatocyte-enriched transcription factor, Foxa2, was found to be essential for hepatocyte revitalization. Overall, our findings indicate that the phenotype and transcriptional program of dHeps can be reprogrammed to generate functional and regenerative hepatocytes by using only small molecules, as an alternative approach to liver repair and regeneration.

Tetrandrine (TET) has been used to treat silicosis in China for decades. The aim of this study was to facilitate rational repurposing of TET against SARS-CoV-2 infection. In this study, we confirmed that TET exhibited antiviral potency against SARS-CoV-2 in the African green monkey kidney (Vero E6), human hepatocarcinoma (Huh7), and human lung adenocarcinoma epithelial (Calu-3) cell lines. TET functioned during the early-entry stage of SARS-CoV-2 and impeded intracellular trafficking of the virus from early endosomes to endolysosomes. An in vivo study that used adenovirus (AdV) 5-human angiotensin-converting enzyme 2 (hACE2)-transduced mice showed that although TET did not reduce pulmonary viral load, it significantly alleviated pathological damage in SARS-CoV-2-infected murine lungs. The systemic preclinical pharmacokinetics were investigated based on in vivo and in vitro models, and the route-dependent biodistribution of TET was explored. TET had a large volume of distribution, which contributed to its high tissue accumulation. Inhaled administration helped TET target the lung and reduced its exposure to other tissues, which mitigated its off-target toxicity. Based on the available human pharmacokinetic data, it appeared feasible to achieve an unbound TET 90% maximal effective concentration (EC90) in human lungs. This study provides insights into the route-dependent pulmonary biodistribution of TET associated with its efficacy.