Isolated Perfused Rat Lung Research Articles

Pulmonary drug absorption and systemic exposure in human: Predictions using physiologically based biopharmaceutics modeling

Systemic exposure of inhaled drugs is used to estimate the local lung exposure and assess systemic side effects for drugs with local pharmacological targets. Predicting systemic exposure is therefore central for successful development of drugs intended to be inhaled. Currently, these predictions are based mainly on data from in vitro experiments, but the accuracy of these predictions might be improved if they were based on data with higher physiological relevance. In this study, systemic exposure was simulated by applying biopharmaceutics input parameters from isolated perfused rat lung (IPL) data to a lung model developed in MoBi® as an extension to the full physiologically-based pharmacokinetic (PBPK) model in PK-Sim®. These simulations were performed for a set of APIs with a variety of physicochemical properties and formulation types. Simulations based on rat IPL data were also compared to simulations based on in vitro data. The predictive performances of the simulations were evaluated by comparing simulated plasma concentration-time profiles to clinical observations after pulmonary administration. Simulations using IPL-based input parameters predicted systemic exposure well, with predicted AUCs within two-fold of the observed value for nine out of ten drug compounds/formulations, and predicted Cmax values within two-fold for eight out of ten drug compounds/formulations. Simulations using input parameters based on IPL data performed generally better than simulations based on in vitro input parameters. These results suggest that the developed model in combination with IPL data can be used to predict human lung absorption for compounds with different physicochemical properties and types of inhalation formulations.

Drug Absorption Parameters Obtained Using the Isolated Perfused Rat Lung Model Are Predictive of Rat In Vivo Lung Absorption

The ex vivo isolated perfused rat lung (IPL) model has been demonstrated to be a useful tool during drug development for studying pulmonary drug absorption. This study aims to investigate the potential use of IPL data to predict rat in vivo lung absorption. Absorption parameters determined from IPL data (ex vivo input parameters) in combination with intravenously determined pharmacokinetic data were used in a biopharmaceutics model to predict experimental rat in vivo plasma concentration-time profiles and lung amount after inhalation of five different inhalation compounds. The performance of simulations using ex vivo input parameters was compared with simulations using in vitro input parameters, to determine whether and to what extent predictability could be improved by using input parameters determined from the more complex ex vivo model. Simulations using ex vivo input parameters were within twofold average difference (AAFE < 2) from experimental in vivo data for all compounds except one. Furthermore, simulations using ex vivo input parameters performed significantly better than simulations using in vitro input parameters in predicting in vivo lung absorption. It could therefore be advantageous to base predictions of drug performance on IPL data rather than on in vitro data during drug development to increase mechanistic understanding of pulmonary drug absorption and to better understand how different substance properties and formulations might affect in vivo behavior of inhalation compounds.

In vitro, ex vivo and in vivo methods of lung absorption for inhaled drugs.

The assessment and prediction of lung absorption and disposition are an increasingly essential preclinical task for successful discovery and product development of inhaled drugs for both local and systemic delivery. Hence, in vitro, ex vivo and in vivo preclinical methods of lung absorption continue to evolve with several technical, methodological and analytical refinements. As in vitro lung epithelial cell monolayer models, the air-liquid interface (ALI)-cultured Calu-3 cells have most frequently been used, but the NCI-H441 and hAELVi cells have now been proposed as the first immortalized human alveolar epithelial cells capable of forming highly-restricted monolayers. The primary ALI-cultured three-dimensional (3D) human lung cell barriers have also become available; efforts to incorporate aerosol drug deposition into the in vitro lung cell models continue; and stem cell-derived lung epithelial cells and "lung-on-a-chip" technology are emerging. The ex vivo isolated perfused rat lung (IPRL) methods have increasing been used, as they enable the kinetic determination of tissue/organ-level diffusive and membrane protein-mediated absorption and competing non-absorptive loss; the assessment of "pre-epithelial" aerosol biopharmaceutical events in the lung, such as dissolution and release; and the ex vivo-to-in vivo extrapolation and prediction. Even so, in vivo small rodent-based methods have been of mainstay use, while large animal-based methods find an additional opportunity to study region-dependent lung absorption and disposition. It is also exciting that human pharmacokinetic (PK) profiles and systemic exposures for inhaled drugs/molecules may be able to be predicted from these in vivo rodent PK data following lung delivery using kinetic modeling approach with allometric scaling. Overall, the value of these preclinical assessments appears to have shifted more to their translational capability of predicting local lung and systemic exposure in humans, in addition to rationalizing optimal inhaled dosage form and delivery system for drugs/molecules in question. It is critically important therefore to make appropriate selection and timely exploitation of the best models at each stage of drug discovery and development program for efficient progress toward product approval and clinical use.

Effect of dexmedetomidine on pyroptosis during lung ischemia-reperfusion in rats: an in vitro experiment

Objective To evaluate the effect of dexmedetomidine on pyroptosis during lung ischemia-reperfusion (I/R) in rats. Methods Adult male Sprague-Dawley rats, weighing 250-320 g, were used in this study.The model of isolated lung perfusion was established using an IL-2 Isolated Perfused Rat or Guinea Pig Lung System after the rats were anesthetized.Thirty lungs in which an ex vivo lung perfusion model was successfully established were divided into 3 groups (n=10 each) by a random number table method: control group (C group), I/R group and dexmedetomidine group (DEX group). The lungs were continuously perfused with K-H solution for 150 min in C group.After 15 min of perfusion, lungs were subjected to 60-min suspension of ventilation and perfusion, followed by 75 min of reperfusion and ventilation to establish the model of lung I/R injury in I/R and DEX groups.In DEX group, dexmedetomidine 10 nmol/L was added to K-H solution at the beginning of reperfusion.Lung tissues were obtained for determination of wet/dry weight ratio (W/D ratio) and for examination of pathological changes (with a light microscope) and ultrastructure (using an electron microscope), and the alveolar damage rate (IAR) was calculated.The expression of pyroptosis-related factors including NOD-like receptor family protein 3 (NLRP3), caspase-1, interleukin-1β (IL-1β), IL-18 and gasdermin D (GSDMD) protein and mRNA was detected by Western blot or by real-time polymerase chain reaction. Results Compared with C group, the W/D ratio and IAR in lung tissues were significantly increased, and the expression of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD protein and mRNA was up-regulated in I/R and DEX groups (P<0.05). Compared with I/R group, the W/D ratio and IAR in lung tissues were significantly decreased, the expression of NLRP3, caspase-1, IL-1β, IL-18 and GSDMD protein and mRNA was down-regulated (P<0.05), and the pathological changes were significantly attenuated in DEX group. Conclusion The mechanism by which dexmedetomidine reduces isolated rat lung I/R injury may be related to inhibiting pyroptosis. Key words: Dexmedetomidine; Reperfusion injury; Lung; Pyroptosis

Pulmonary Dissolution of Poorly Soluble Compounds Studied in an ex Vivo Rat Lung Model.

Many inhaled drugs are poorly water soluble, and the dissolution rate is often the rate-limiting step in the overall absorption process. To improve understanding of pulmonary drug dissolution, four poorly soluble inhalation compounds (AZD5423 (a developmental nonsteroidal glucocorticoid), budesonide, fluticasone furoate (FF), and fluticasone propionate (FP)) were administered as suspensions or dry powders to the well-established isolated perfused rat lung (IPL) model. Two particle size distributions (d50 = 1.2 μm and d50 = 2.8 μm) were investigated for AZD5423. The pulmonary absorption rates of the drugs from the suspensions and dry powders were compared with historical absorption data for solutions to improve understanding of the effects of dissolution on the overall pulmonary absorption process for poorly soluble inhaled drugs. A physiologically based biopharmaceutical in silico model was used to analyze the experimental IPL data and to estimate a dissolution parameter ( kexvivo). A similar in silico approach was applied to in vitro dissolution data from the literature to obtain an in vitro dissolution parameter ( kinvitro). When FF, FP, and the larger particles of AZD5423 were administered as suspensions, drug dissolution was the rate-limiting step in the overall absorption process. However, this was not the case for budesonide, which has the highest aqueous solubility (61 μM), and the smaller particles of AZD5423, probably because of the increased surface area available for dissolution (d50 = 1.2 μm). The estimated dissolution parameters were ranked in accordance with the solubility of the drugs, and there was good agreement between kexvivo and kinvitro. The dry powders of all the compounds were absorbed more slowly than the suspensions, indicating that wetting is an important parameter for the dissolution of dry powders. A wetting factor was introduced to the in silico model to explain the difference in absorption profiles between the suspensions and dry powders where AZD5423 had the poorest wettability followed by FP and FF. The IPL model in combination with an in silico model is a useful tool for investigating pulmonary dissolution and improving understanding of dissolution-related parameters for poorly soluble inhaled compounds.

Screening bioactive components of Glycyrrhiza uralensis Fisch. with isolated perfused lung extraction and HPLC-ESI-MSn analysis

The isolated perfused rat lung (IPL), coupled with high performance liquid chromatography\ andem mass spectrometry analysis (HPLC–ESI-MSn), has been developed as a tool for screening bioactive components in Glycyrrhiza uralensis Fisch. (GU). First, IPL was perfused with the water extract of GU (EGU), the bioactive components in the EGU would selectively combine to the receptors or channels of lung. By changing the pH of perfused solution, the combined components were eluated and then detected by HPLC-ESI-MSn. Four compounds were detected in the desorption eluate of IPL, among these compounds, liquiritin (1), ononin (2) and glycyrrhizic acid (4) were identified by comparing with the chromatography of the standards, while licorice-saponin G2 (3) were determined by analysis of the structure clearage characterization of mass spectrometry. Then, due to the lack of compound 3 sample, compounds 1, 2 and 4 with respective concentrations of 50 μM, 5 μM, 500 nM, 50 nM and 5 nM were applied to evaluate the protective effect of pulmonary epithelial cells (PEC, A549 cell) injury induced by lipopolysaccharide (LPS) for anti-inflammatory activity assessment. The results showed that except the 5 nM group of compound 1, 5 nM and 50 nM groups of compound 2, all other groups could remarkably inhibit the PEC injury (vs LPS group, 2-500 nM groups: p < 0.05; other groups: p < 0.01), all compound showed the dose-dependent effect. In conclusion, IPL coupled with HPLC-ESI-MSn was successfully used to screen the anti-inflammatory components of GU for the first time. The application of IPL coupled with HPLC-ESI-MSn for screening bioactive components of TCMs is rapid, convenient and reliable, and the isolated perfused technology could be extended to isolated heart, liver, kidney, and so on.

Niacin Pretreatment Attenuates Lung Ischemia and Reperfusion-Induced Pulmonary Barrier Function Impairment by Reducing Oxidative Stress and Activating SIRT1 in an Isolated-Perfused Rat Lung Model

PurposeAlveolar-capillary barrier dysfunction, characterized by alveolar protein leak and lung edema, is a common scenario following cardiopulmonary surgery and thoracic organ transplantation. Reactive oxygen species generated through lung ischemia and reperfusion (I/R) injury during surgery plays a crucial role. Niacin, also known as vitamin B3, has been demonstrated to possess antioxidative and anti-inflammatory capacity. In this study, we examine the pulmonary barrier function via capillary filtration coefficient (Kfc) following lung I/R injury with and without niacin treatment. MethodsStudies were conducted on male Sprague-Dawley rats in 3 groups: sham-operated, lung I/R injury, and niacin-pretreated lung I/R injury group. Rats were subjected to isolated perfused lung preparation. Lung ischemia was established by continuous perfusion and stopping ventilation for 60 minutes, followed by 60 minutes of ventilation. We assessed the Kfc, lung water content, and protein concentration in the lung lavage; pulmonary oxidative stress and lung inflammation were assessed by leukocyte counts, tissue level of tumor nercrosis factor alpha (TNF-α), and tissue content of malondialdehyde (MDA), respectively. We also assessed the tissue protein level of sirtuin (silent mating type information regulation 2 homolog) 1 (SIRT1). ResultsLungs subjected to I/R injury significantly increased Kfc, pulmonary oxidative stress, lung water content, and lavage leukocyte count and protein concentration (P < .05). Rats treated with niacin of 100 mg/kg/day for 4 days increased lung SIRT1 (P < .05) and attenuated lung I/R injury-induced pulmonary oxidative stress and inflammation and also improved Kfc. ConclusionsNiacin pretreatment protects lungs against I/R injury-induced barrier function impairment through the activation of SIRT1 and reduced pulmonary oxidative stress and lung inflammation.

Niacin Pretreatment Attenuates Ischemia and Reperfusion of Pancreas-induced Acute Pancreatitis and Remote Lung Injury Through Suppressing Oxidative Stress and Inflammation and Activation of SIRT1

BackgroundLung injury subsequent to pancreatic ischemia and reperfusion (PIR) due to shock, revascularization, and pancreas transplantation is a major clinical problem. In addition to proteases, massive production and release of reactive oxygen species (ROS) and induction of inflammatory cytokines have been implicated in remote lung injury. Niacin, also known as vitamin B3, is both antioxidative and anti-inflammatory. In this study, we examined the protective effectiveness of niacin pretreatment against PIR-induced pancreatic and remote lung injury. MethodsMale Sprague-Dawley rats were divided into a sham-operated group, a PIR group, and a PIR group pretreated with niacin; the niacin (300 mg/kg per day) was given on 4 consecutive days before the study. Pancreatic ischemia was established by occluding both the gastroduodenal and splenic arteries for 120 minutes, followed by 240 minutes of reperfusion. Lung injury was assessed by pulmonary barrier function via pulmonary filtration coefficient, Kfc, using an isolated-perfused rat lung preparation. Alveolar protein leakage was assessed by protein concentration in the bronchoalveolar lavage fluid (PCBAL). Lung water content was assessed by both wet-weight/dry-weight ratio (W/D) and lung-weight/body-weight ratio (LW/BW). Lung inflammation was evaluated by the lavage differential neutrophil cell count and tissue tumor necrosis-alpha (TNF-α) level. Oxidative stress was assessed by tissue malondialdehyde (MDA) level. Serum lactate dehydrogenase (LDH) and amylase were examined for lung and pancreas injury. We also evaluated lung tissue SIRT1 mRNA expression. ResultsCompared with the sham group, the PIR group had increased serum amylase and LDH, and impaired the pulmonary barrier dysfunction with marked increases in Kfc, PCBAL, W/D, and LW/BW, and augumented oxidative stress and inflammation with elevated tissue MDA and TNF-α and lavage neutrophil count, which correlated with decreased SIRT1 mRNA expression. Conversely, niacin pretreatment reduced pancreatic and remote lung injury and attenuated pulmonary oxidative stress and inflammation, and also protected against PIR-induced pulmonary barrier dysfunction while restoring SIRT1 mRNA expression. ConclusionNiacin pretreatment reduced PIR-induced pancreatic and lung injury and protected against pulmonary barrier function impairment, which was associated with niacin's antioxidative and anti-inflammatory activity and its capacity to increase SIRT1 mRNA expression.

Endocytic Uptake, Transport and Macromolecular Interactions of Anionic PAMAM Dendrimers within Lung Tissue

PurposePolyamidoamine (PAMAM) dendrimers are a promising class of nanocarrier with applications in both small and large molecule drug delivery. Here we report a comprehensive evaluation of the uptake and transport pathways that contribute to the lung disposition of dendrimers.MethodsAnionic PAMAM dendrimers and control dextran probes were applied to an isolated perfused rat lung (IPRL) model and lung epithelial monolayers. Endocytosis pathways were examined in primary alveolar epithelial cultures by confocal microscopy. Molecular interactions of dendrimers with protein and lipid lung fluid components were studied using small angle neutron scattering (SANS).ResultsDendrimers were absorbed across the intact lung via a passive, size-dependent transport pathway at rates slower than dextrans of similar molecular sizes. SANS investigations of concentration-dependent PAMAM transport in the IPRL confirmed no aggregation of PAMAMs with either albumin or dipalmitoylphosphatidylcholine lung lining fluid components. Distinct endocytic compartments were identified within primary alveolar epithelial cells and their functionality in the rapid uptake of fluorescent dendrimers and model macromolecular probes was confirmed by co-localisation studies.ConclusionsPAMAM dendrimers display favourable lung biocompatibility but modest lung to blood absorption kinetics. These data support the investigation of dendrimer-based carriers for controlled-release drug delivery to the deep lung.

Liposomal Aerosols of Nitric Oxide (NO) Donor as a Long-Acting Substitute for the Ultra-Short-Acting Inhaled NO in the Treatment of PAH.

This study seeks to develop a liposomal formulation of diethylenetriamine NONOate (DN), a long acting nitric oxide (NO) donor, with a goal to replace inhaled NO (iNO) in the treatment of pulmonary arterial hypertension (PAH). Liposomal formulations were prepared by a lipid film hydration method and modified with a cell penetrating peptide, CAR. The particles were characterized for size, polydispersity index (PDI), zeta potential, entrapment efficiency, storage and nebulization stability, and in-vitro release profiles. The cellular uptake and transport were assessed in rat alveolar macrophages (NR8383) and transforming growth factor β (TGF-β) activated rat pulmonary arterial smooth muscle cells (PASMCs). The fraction of the formulation that enters the systemic circulation, after intratracheal administration, was determined in an Isolated Perfused Rat Lung (IPRL) model. The safety of the formulations were assessed using an MTT assay and by measuring injury markers in the bronchoalveolar lavage (BAL) fluid; the pharmacological efficacy was evaluated by monitoring the changes in the mean pulmonary arterial (mPAP) and systemic pressure (mSAP) in a monocrotaline (MCT) induced-PAH rat model Liposome size, zeta potential, and entrapment efficiency were 171 ± 4nm, -37 ± 3mV, and 46 ± 5%, respectively. The liposomes released 70 ± 5% of the drug in 8h and were stable when stored at 4°C. CAR-conjugated-liposomes were taken up more efficiently by PASMCs than liposomes-without-CAR; the uptake of the formulations by rat alveolar macrophages was minimal. DN-liposomes did not increase lung weight, protein quantity, and levels of injury markers in the BAL fluid. Intratracheal CAR-liposomes reduced the entry of liposomes from the lung to blood; the formulations produced a 40% reduction in mPAP for 180minutes. This study establishes the proof-of-concept that peptide modified liposomal formulations of long-acting NO donor can be an alternative to short-acting iNO.

Tobramycin Disposition in the Rat Lung Following Airway Administration

A realistic ex vivo model, the isolated perfused rat lung (IPRL), was used to investigate tobramycin's pulmonary disposition at typical therapeutic concentrations. Different nominal doses were administered in aqueous solution to the airways alongside nonbinding absorption markers, fluorescein and mannitol. The mean fraction of each administered dose reaching the perfusate (Fp) was determined as a function of time following administration. Dynamic dialysis was also used to quantify the kinetics of tobramycin binding and/or tissue retention in the IPRL immediately after drug administration. Whereas the absorption markers fluorescein and mannitol both showed monoexponential dose-independent increases in Fp with time, tobramycin's pulmonary absorption into the perfusate was biexponential and dose-dependent due to tissue binding or retention. Best estimates for the first-order rate constants of tobramycin absorption appeared dose-independent (0.065-0.070 min(-1)), with values close to the mean for fluorescein (0.076 min(-1)). The rate constant for dissociation from IPRL tissue was also relatively constant (0.018-0.022 min(-1)), whereas that for association decreased from 0.16 to 0.07 min(-1) with increasing airway dose from 0.002 to 2 mg. Dynamic dialysis data from sliced IPRL tissue following identical airway administration were consistent with those from the intact IPRL, confirming tobramycin's "slow on, slow off" binding and sequestration by the rat lung. Overall, tobramycin absorption was fast following airway administration. However, dose- and concentration-dependent slow-onset tissue binding extended the duration of tobramycin's presence in the rat lung. These findings may explain, in part, the apparent success of inhaled tobramycin therapy when treating pulmonary infections.

Maximal extent of translocation of single-walled carbon nanotubes from lung airways of the rat

This study aimed to examine the extent of pulmonary translocation of single-walled carbon nanotubes (SWCNTs) from lung airways of rat. It utilised an ex vivo isolated perfused rat lung (IPRL) model that retains the intact lung architecture while eliminating the confounding issue of systemic pharmacokinetics. Doses (100μg) of SWCNTs were instilled into the airways of the IPRL and the pulmonary translocation of SWCNTs quantified by inductively coupled plasma mass spectroscopy using CNT-associated nickel as the probe. SWCNT translocation from the airways across an intact pulmonary barrier into what would be the systemic circulation was no greater than 0.05% of the instilled dose over 90min. Pharmacokinetic simulation incorporating a term for mucociliary clearance would predict over a 14 day an approximate cumulative pulmonary translocation from rat lung of no greater than 0.15% from a 100μg deposited dose.

Dry Powder Inhalation Exposures of the Endotracheally Intubated Rat Lung, Ex Vivo and In Vivo: The Pulmonary Pharmacokinetics of Fluticasone Furoate

The isolated perfused rat lung (IPL) is a suitable model for studying lung-specific pharmacokinetics (PK) of inhaled drugs. So far, little has been known, however, whether the PK measured in the ex vivo organ corresponds to the PK measured in similarly exposed animals in vivo, in particular the endotracheally intubated rat (EIR). The purpose of the current research was to compare the PK of inhaled corticosteroid fluticasone furoate (FF) in the IPL and the EIR. Aerosols of FF with mass median aerodynamic diameters ranging from 2.2 to 3.2 μm were generated with the DustGun aerosol generator. The IPL, perfused in the single-pass mode, was exposed via inhalation to 5.6 and 46 μg of FF. Following inhalation, the perfusate was repeatedly sampled for 100 min, after which the lungs were recovered for quantitation of remaining FF. Two groups of EIR were also exposed via inhalation to 7 μg of FF. One group was immediately euthanized for determination of the initial deposition of FF in the lungs. From the second group, four venous blood samples were drawn up to 4 hr after exposure. The animals were then sacrificed for determination of FF remaining in the lungs. Following inhalation, FF was slowly disappearing from both the IPL and the lungs of the EIR, with a half-life of pulmonary retention of 4.3-4.9 hr for all three exposure series. For the low exposure levels, the concentration curve of FF in the IPL perfusate was similar in shape to that in venous blood of the EIR, with a Cmax of 1.0 and 0.8 nM for the IPL and the EIR, respectively. The results indicate that the IPL and the EIR, when used jointly in PK studies, can provide a detailed characterization of inhaled drugs or toxicants.

Extraction of Peptidase Substrates by the Isolated Perfused Rat Lung

Peptidases in the lung are well placed to have an important role in regulating levels of circulating endogenous and therapeutic peptides. They also present a first-pass metabolic barrier for peptides delivered to the lung for systemic absorption. The activities of five peptidases were surveyed in the pulmonary circulation of the asanguinous isolated perfused rat lung (IPRL) using synthetic substrates and selective inhibitors. Extraction ratios (ER) were calculated for the substrates of: aminopeptidase N (AMN), ER 0·37 ± 0·04; dipeptidyl peptidase IV (DPP), ER 0·69 ± 0·05; and angiotensin-converting enzyme (ACE), ER 0·40 ± 0·02. These activities were inhibited (> 95%) by the selective inhibitors bestatin, diprotin A, and captopril, respectively. Substrates for neutral endopeptidase 24.11 (NEP) and carboxypeptidase M (CPM) were minimally degraded with ER of 0·02 and 0·00, respectively. The low activity of NEP, a major membrane bound endopeptidase, indicates that the lungs may not contribute greatly to degradation of either systemic NEP substrates, or exopeptidase-resistant peptides absorbed from the peripheral lung. In contrast peptides susceptible to the exopeptidases AMN, DPP, and ACE, will be substantially degraded during passage through the lung. The absence of CPM activity in the pulmonary circulation of the asanguinous IPRL implies that basic carboxypeptidase activity in blood perfused lungs reported in the literature is more likely to be the result of plasma carboxypeptidase N activity.

Enhanced pulmonary absorption of a macromolecule through coupling to a sequence-specific phage display-derived peptide

With the aim of identifying a peptide sequence that promotes pulmonary epithelial transport of macromolecule cargo we used a stringent peptide-phage display library screening protocol against rat lung alveolar epithelial primary cell cultures. We identified a peptide-phage clone (LTP-1) displaying the disulphide-constrained 7-mer peptide sequence, C-TSGTHPR-C, that showed significant pulmonary epithelial translocation across highly restrictive polarised cell monolayers. Cell biological data supported a differential alveolar epithelial cell interaction of the LTP-1 peptide-phage clone and the corresponding free synthetic LTP-1 peptide. Delivering select phage-clones to the intact pulmonary barrier of an isolated perfused rat lung (IPRL) resulted in 8.7% of lung deposited LTP-1 peptide-phage clone transported from the IPRL airways to the vasculature compared (p < 0.05) to the cumulative transport of less than 0.004% for control phage-clone groups. To characterise phage-independent activity of LTP-1 peptide, the LTP-1 peptide was conjugated to a 53 kDa anionic PAMAM dendrimer. Compared to respective peptide-dendrimer control conjugates, the LTP-1–PAMAM conjugate displayed a two-fold (bioavailability up to 31%) greater extent of absorption in the IPRL. The LTP-1 peptide-mediated enhancement of transport, when LTP-1 was either attached to the phage clone or conjugated to dendrimer, was sequence-dependent and could be competitively inhibited by co-instillation of excess synthetic free LTP-1 peptide. The specific nature of the target receptor or mechanism involved in LTP-1 lung transport remains unclear although the enhanced transport is enabled through a mechanism that is non-disruptive with respect to the pulmonary transport of hydrophilic permeability probes. This study shows proof-of principle that array technologies can be effectively exploited to identify peptides mediating enhanced transmucosal delivery of macromolecule therapeutics across an intact organ.

Isolated perfused lung extraction and HPLC–ESI–MS n analysis for predicting bioactive components of Saposhnikoviae Radix

A novel strategy for predicting bioactive components in traditional Chinese material herb was proposed, using isolated perfused rat lung (IPL) extraction and high performance liquid chromatography\ andem mass spectrometry (HPLC–MS n ) analysis. The hypothesis is that when the IPL is perfused with the extract of Saposhnikoviae Radix (ESR), the potential bioactive components in the ESR should selectively combine with the receptor or channel of lung, by changing the pH of perfused liquid, the combining components would be eluated and then detected by HPLC–ESI–MS n . Five compounds were detected in the desorption eluate of IPL; among these compounds, two potential bioactive compounds, prim-O-glucosylcimifugin ( 2) and 4′-O- β- d-glucosyl-5-O-methylvisamminol ( 4) were identified by comparing with the chromatography of the standard sample, and three other compounds, i.e. cimifugin ( 1), 5-O-methylvisamminol ( 3) and sec-O-glucosylhamaudol ( 5) were determined by analysis of the structure clearage characterization of mass spectrometry. The application of IPL extraction coupled with HPLC–ESI–MS n for predicting potential bioactive components of TCMs is rapid, convenient, operational, economic and reliable.

Formation and Distribution of NNK Metabolites in an Isolated Perfused Rat Lung

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung-specific tobacco carcinogen. Metabolism is critical to its elimination given its lipophilic nature. Although NNK can be metabolized through detoxification pathways that safely eliminate it from the body, it can also be bioactivated, resulting in the formation of potentially carcinogenic DNA adducts. The isolated perfused rat lung (IPRL) system was used to determine the effect of NNK perfusate concentration (0.1 and 1.2 microM) on the formation and distribution of metabolites, the level of individual DNA adducts, and total covalent binding in the lung. Coadministration of the chemopreventive agent phenethyl isothiocyanate (PEITC; 20 microM) was also examined to determine its effect on NNK metabolism. NNK was readily metabolized in the IPRL system. In the 0.1 muM perfusions approximately 55% of metabolites formed were through detoxification pathways, whereas roughly 30% were the result of bioactivation pathways. An increase in NNK concentration increased the percentage of unmetabolized NNK and decreased the apparent metabolic clearance in the lung, but the metabolite profiles remained similar between concentrations. The addition of PEITC reduced the formation of oxidative metabolites and increased 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) formation and the percentage of unmetabolized NNK. PEITC also significantly decreased the formation of DNA adducts in the lung tissue. The level of O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dThd) and O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O(6)-POB-dGuo) decreased by 70 to 75%, and that of O(6)-methylguanine (O(6)-methyl-Gua) and 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua) decreased by 40 to 45%. Pyridylhydroxybutyl-DNA adducts were not detected in any of the treatment groups. Thus, the IPRL system is useful in determining pulmonary metabolism and DNA adduct formation separate from other metabolizing organs.

Carbonic Anhydrase II and Alveolar Fluid Reabsorption during Hypercapnia

Carbonic anhydrase II (CAII) plays an important role in carbon dioxide metabolism and intracellular pH regulation. In this study, we provide evidence that CAII is expressed in both type I (AECI) and type II (AECII) alveolar epithelial cells by RT-PCR and Western blotting in freshly isolated rat cells. These results were further confirmed by double immunostaining with CAII antibodies and AECI- or AECII-specific markers in freshly isolated alveolar epithelial cells and rat lung tissues. Inhibition of CAII by acetazolamide or methazolamide delayed the decrease in the intracellular pH observed during hypercapnia in cultured AECI, AECII, and AECI-like cells. In an isolated-perfused rat lung model, alveolar fluid reabsorption significantly decreased during high CO(2) exposure, which was not prevented by carbonic anhydrase inhibition. Thus, we provide evidence that CAII is expressed in rat alveolar epithelial cells and does not regulate lung alveolar fluid reabsorption during hypercapnia.

Insulin Self-association: Effects on Lung Disposition Kinetics in the Airways of the Isolated Perfused Rat Lung (IPRL)

To characterize the kinetic dependence of pulmonary absorption and metabolism of insulin and lispro on the magnitude of their hexameric association. Hexamer content by weight percent (%Hex) in various insulin-zinc and lispro-zinc solutions were determined by quantitative centrifugal ultrafiltration and zinc titration with terpyridine (QCUF-ZTT). Each of the solutions (0.1 ml) was then administered into the airways of the IPRL of normal and experimental diabetic animals. Rate constants were determined for lung absorption (k (a)) and non-absorptive loss (k (nal); comprising mucociliary clearance and metabolism). %Hex in administered solutions ranged from 3.3 to 94.4%. Data analysis showed excellent correlations between the values for k (a) or k (nal) and %Hex, irrespective of insulin type, concentration, solution pH or ionic strength. The values for k (a) decreased (0.22 --> 0.05 h(-1)) with increasing %Hex, as did values for k (nal). At %Hex in administered solutions >/=50%, values for k (nal) approached estimates for the rate constant for mucociliary clearance, implying that lung metabolism occurred primarily with monomeric insulin. There were no differences in insulin disposition kinetics between lungs taken from experimental diabetic and sham-control animals. The kinetics of pulmonary insulin disposition depended on the magnitude of molecular self-association. Dissociated forms of insulin (dimers or monomers) in the dosing solution showed higher rates than hexamers for both lung absorption and metabolism.

Endothelin decreases lung edema clearance and Na,K‐ATPase activity in alveolar epithelial cells via ET‐B receptor and Nitric Oxide generation

RATIONALE ALI is characterized by high levels of endothelin-1 (ET-1). Administration of ET-1 is associated with a rapid increase in edema formation. We hypothesized that ET-1 may impair the ability of the lung to reabsorb fluid from the alveoli by inhibiting the Na,K-ATPase. METHODS A) Isolated-perfused rat lung model: Alveolar fluid clearance was assessed in an isolated-perfused rat lung model by determining the concentration of Evans blue-tagged albumin in the airspace instillate as a function of time. B) Isolated rat alveolar epithelial cells (AEC) were treated with ET-1 to assess Na,K-ATPase activity by a ouabain-sensitive 86Rb+ uptake and protein analysis by Western blotting. RESULTS Isolated rat lungs perfused for 60 min with different concentrations of ET-1 had a dose dependent decrease in alveolar fluid reabsorption. Non-selective ET A/B receptor antagonist blocked the decrease in lung edema clearance, but not by a selective ET-A antagonist. ET-1 decreased alveolar fluid clearance was blocked by a nitric oxide antagonist (L-NAME). Based on ouabain-sensitivity curves, the endothelin-mediated decrease in edema clearance is due predominantly to AEC type I (AECI). CONCLUSIONS These data support the hypothesis that ET-1 impairs the ability of the lungs to clear fluid by decreasing the Na,K-ATPase activity in AECI cells via ET-B receptor activation and nitric oxide production, thus impairing epithelial lung recovery from lung injury. Funding: Supported by HL-48129, T32-HL076139 and 5F32HL071421