A transcriptomic overview of lung and liver changes one day after pulmonary exposure to graphene and graphene oxide.

Hazard evaluation of graphene-based materials (GBM) is still in its early stage and it is slowed by their large diversity in the physicochemical properties. This study explores transcriptomic differences in the lung and liver after pulmonary exposure to two GBM with similar physical properties, but different surface chemistry. Female C57BL/6 mice were exposed by a single intratracheal instillation of 0, 18, 54 or 162 µg/mouse of graphene oxide (GO) or reduced graphene oxide (rGO). Pulmonary and hepatic changes in the transcriptome were profiled to identify commonly and uniquely perturbed functions and pathways by GO and rGO. These changes were then related to previously analyzed toxicity endpoints. GO exposure induced more differentially expressed genes, affected more functions, and perturbed more pathways compared to rGO, both in lung and liver tissues. The largest differences were observed for the pulmonary innate immune response and acute phase response, and for hepatic lipid homeostasis, which were strongly induced after GO exposure. These changes collective indicate a potential for atherosclerotic changes after GO, but not rGO exposure. As GO and rGO are physically similar, the higher level of hydroxyl groups on the surface of GO is likely the main reason for the observed differences. GO exposure also uniquely induced changes in the transcriptome related to fibrosis, whereas both GBM induced similar changes related to Reactive Oxygen Species production and genotoxicity. The differences in transcriptomic responses between the two GBM types can be used to understand how physicochemical properties influence biological responses and enable hazard evaluation of GBM and hazard ranking of GO and rGO, both in relation to each other and to other nanomaterials.

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.

Caproyl-Modified G2 PAMAM Dendrimer (G2-AC) Nanocomplexes Increases the Pulmonary Absorption of Insulin.

We aimed to investigate the absorption-enhancing effect (AEE) of caproyl-modified G2 PAMAM dendrimer (G2-AC) on peptide and protein drugs via the pulmonary route. In this study, G2 PAMAM dendrimer conjugates modified with caproic acid was synthesized, the pulmonary absorption of insulin as models with or without G2-AC were evaluated. The results indicated that G2-AC6 exhibited a greatest AEE for insulin in various caproylation levels of G2-AC. G2-AC6 could significantly enhance the absorption of insulin, and the AEE of G2-AC6 was concentration-dependent. In toxicity tests, G2-AC6 displayed no measurable cytotoxicity to the pulmonary membranes over a concentration range from 0.1% (w/v) to 1.0% (w/v). Measurements of the TEER and permeability showed that G2-AC6 significantly reduced the TEER value of CF and increased its Papp value. The results suggested that G2-AC6 could cross epithelial cells by means of a combination of paracellular and transcellular pathways. These findings suggested G2-AC6 at lower concentrations (below 1.0%, w/v) might be promising absorption enhancers for increasing the pulmonary absorption of peptide and protein drugs.

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 ( kex vivo). A similar in silico approach was applied to in vitro dissolution data from the literature to obtain an in vitro dissolution parameter ( kin vitro). 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 kex vivo and kin vitro. 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.

A study on reducing the absorption of lidocaine from the airway in cats.

PURPOSE:: To determine if the combination of lidocaine with epinephrine or gamma globulin would decrease the rate or reduce the amount of local absorption of lidocaine through the airway. METHODS:: Twenty adult male cats were randomly and evenly distributed into four groups: 1) Group LG: lidocaine administered with gamma globulin; 2) Group LS: lidocaine administered with physiological saline); 3) Group LE: lidocaine administered with epinephrine; 4) Group C: control group. Invasive blood pressure, heart rate, and concentration of lidocaine were recorded before and after administration. RESULTS:: The peak of plasma concentrations appeared difference (Group LG: 1.39 ± 0.23 mg/L; Group LS: 1.47 ± 0.29 mg/L and Group LE: 0.99 ± 0.08 mg/L). Compared to Group C, there were significant differences in the average heart rate of Groups LG, LS, and LE (P < 0.05). The average systolic blood pressures were significantly different when each group was compared to Group C (P < 0.05). The biological half-life, AUC0-120, peak time, and half-life of absorption among the three groups have not presented statistically significant differences (P > 0.05). CONCLUSION:: Administering lidocaine in combination with gamma globulin through airway causes significant decrease the rate and reduce the amount of local absorption of lidocaine in cats.

A study on reducing the absorption of lidocaine from the airway in cats

Abstract Purpose: To determine if the combination of lidocaine with epinephrine or gamma globulin would decrease the rate or reduce the amount of local absorption of lidocaine through the airway. Methods: Twenty adult male cats were randomly and evenly distributed into four groups: 1) Group LG: lidocaine administered with gamma globulin; 2) Group LS: lidocaine administered with physiological saline); 3) Group LE: lidocaine administered with epinephrine; 4) Group C: control group. Invasive blood pressure, heart rate, and concentration of lidocaine were recorded before and after administration. Results: The peak of plasma concentrations appeared difference (Group LG: 1.39 ± 0.23 mg/L; Group LS: 1.47 ± 0.29 mg/L and Group LE: 0.99 ± 0.08 mg/L). Compared to Group C, there were significant differences in the average heart rate of Groups LG, LS, and LE (P < 0.05). The average systolic blood pressures were significantly different when each group was compared to Group C (P < 0.05). The biological half-life, AUC0-120, peak time, and half-life of absorption among the three groups have not presented statistically significant differences (P > 0.05). Conclusion: Administering lidocaine in combination with gamma globulin through airway causes significant decrease the rate and reduce the amount of local absorption of lidocaine in cats.

Aerosolized liposomes with dipalmitoyl phosphatidylcholine enhance pulmonary absorption of encapsulated insulin compared with co-administered insulin.

OBJECTIVE: We have previously shown that aerosolized liposomes with dipalmitoyl phosphatidylcholine (DPPC) enhance the pulmonary absorption of encapsulated insulin. In this study, we aimed to compare insulin encapsulated into the liposomes versus co-administration of empty liposomes and unencapsulated free insulin, where the DPCC liposomes would serve as absorption enhancer. SIGNIFICANCE: The present study provides the useful information for development of noninvasive treatment of diabetes. METHODS: Co-administration of empty DPPC liposomes and unencapsulated free insulin was investigated in vivo to assess the potential enhancement in protein pulmonary absorption. Co-administration was compared to DPPC liposomes encapsulating insulin, and free insulin. RESULTS: DPPC liposomes enhanced the pulmonary absorption of unencapsulated free insulin; however, the enhancing effect was lower than that of the DPPC liposomes encapsulating insulin. The mechanism of the pulmonary absorption of unencapsulated free insulin by DPPC liposomes involved the opening of epithelial cell space in alveolar mucosa, and not mucosal cell damage, similar to that of the DPPC liposomes encapsulating insulin. In an in vitro stability test, insulin in the alveolar mucus layer that covers epithelial cells was stable. These findings suggest that, although unencapsulated free insulin spreads throughout the alveolar mucus layer, the concentration of insulin released near the absorption surface is increased by the encapsulation of insulin into DPPC liposomes and the absorption efficiency is also increased. CONCLUSION: We revealed that the encapsulation of insulin into DPPC liposomes is more effective for pulmonary insulin absorption than co-administration of DPPC liposomes and unencapsulated free insulin.

Enhanced pulmonary absorption of poorly soluble itraconazole by micronized cocrystal dry powder formulations.

Micronized cocrystal powders and amorphous spray-dried formulations were prepared and evaluated in vivo and in vitro as pulmonary absorption enhancement formulations of poorly soluble itraconazole (ITZ). ITZ cocrystals with succinic acid (SA) or l-tartaric acid (TA) with a particle size diameter of <2µm were successfully micronized using the jet-milling system. The cocrystal crystalline morphologies observed using scanning electron microscopy (SEM) suggested particle shapes that differed from those of the crystalline or spray-dried amorphous ITZ. The micronized ITZ cocrystal powders showed better intrinsic dissolution rate (IDR) and pulmonary absorption profile in rats than that of the amorphous spray-dried formulation and crystalline ITZ with comparable particle sizes. Specifically, in rat pharmacokinetic studies following pulmonary administration, micronized ITZ-SA and ITZ-TA cocrystals showed area under the curve from 0 to 8h (AUC0-8h) values approximately 24- and 19-fold higher than those of the crystalline ITZ and 2.0- and 1.6-fold higher than the spray-dried ITZ amorphous values, respectively. The amorphous formulation appeared physically instable during the studies due to rapid crystallization of ITZ, which was its disadvantage compared to the crystalline formulations. Therefore, this study demonstrated that micronized cocrystals are promising formulations for enhancing the pulmonary absorption of poorly soluble compounds.

Arachidonic acid with taurine enhances pulmonary absorption of macromolecules without any serious histopathological damages.

Therapeutic peptides and protein are being used in several indications; however, their poor permeability still remains to be solved. This study focused on the pulmonary route of macromolecules. First, the effects of arachidonic acid (AA) as an absorption enhancer on drug serum concentration, after intratracheal administration, were investigated in rats. Second, the safety of AA was assessed in rats in an acute toxicity study for 7days. AA enhanced the exposure of both interferon-α (IFN-α) and fluorescein isothiocyanate 4000 (FD-4). In addition, the histopathological analysis indicated that AA caused alveolitis and bronchitis in rats. In combination with Taurine (Tau), these lung injuries were prevented through the histopathological analysis. The combined use of Tau with AA did not show any changes in the pharmacokinetics of FD-4. From these results, we suggest the combined use of AA with Tau as a novel formulation on the pulmonary route of macromolecule drugs. This formulation could improve the bioavailability of macromolecule drugs without any serious local damage to the lungs.

Oral uptake of nanoparticles: human relevance and the role of in vitro systems.

Nanoparticles (NPs) present in environment, consumer and health products, food and medical applications lead to a high degree of human exposure and concerns about potential adverse effects on human health. For the general population, the exposure through contact with the skin, inhalation and oral uptake are most relevant. Since in vivo testing is only partly able to study the effects of human oral exposure, physiologically relevant in vitro systems are being developed. This review compared the three routes taking into account the estimated concentration, size of the exposed area, morphology of the involved barrier and translocation rate. The high amounts of NPs in food, the large absorption area and the relatively high translocation rate identified oral uptake as most important portal of entry for NPs into the body. Changes of NP properties in the physiological fluids, mechanisms to cross mucus and epithelial barrier, and important issues in the use of laboratory animals for oral exposure are mentioned. The ability of in vitro models to address the varying conditions along the oro-gastrointestinal tract is discussed, and requirements for physiologically relevant in vitro testing of orally ingested NPs are listed.

Evaluation of the Transwell System for Characterization of Dissolution Behavior of Inhalation Drugs: Effects of Membrane and Surfactant.

Assessing the dissolution behavior of orally inhaled drug products (OIDs) has been proposed as an additional in vitro test for the characterization of innovator and generic drug development. A number of suggested dissolution methods (e.g., commercially available Transwell or Franz cell systems) have in common a membrane which provides the separation between the donor compartment, containing nondissolved drug particles, and an acceptor (sampling) compartment into which dissolved drug will diffuse. The goal of this study was to identify and overcome potential pitfalls associated with such dissolution systems using the inhaled corticosteroids (ICS), viz., budesonide, ciclesonide, and fluticasone propionate, as model compounds. A respirable fraction (generally stage 4 of a humidity, flow, and temperature controlled Andersen Cascade Impactor (ACI) or a Next Generation Impactor (NGI)) was collected for the tested MDIs. The dissolution behavior of these fractions was assessed employing the original and an adapted Transwell system using dissolution media which did or did not contain surfactant (0.5% sodium dodecyl sulfate). The rate with which the ICS transferred from the donor to the acceptor compartment was assessed by HPLC. Only a modified system that incorporated faster equilibrating membranes instead of the original 0.4 µm Transwell membrane resulted in dissolution and not diffusion being the rate-limiting step for the transfer of drug from the donor to the acceptor compartment. Experiments evaluating the nature of the dissolution media suggested that the presence of a surfactant (e.g., 0.5% SDS) is essential to obtain rank order of dissolution rates (e.g., for budesonide, fluticasone propionate, and ciclesonide) that is in agreement with absorption rates of these ICS obtained in studies of human pharmacokinetics. Using the optimized procedure, the in vitro dissolution behavior of budesonide, ciclesonide, and fluticasone propionate agreed approximately with descriptors of in vivo absorption. The optimized procedure, using membranes with increased permeability and surfactant containing dissolution medium, represents a good starting point to further evaluate in vitro/in vivo correlations.

Improvement of pulmonary absorption of poorly absorbable macromolecules by hydroxypropyl-ß-cyclodextrin grafted polyethylenimine (HP-ß-CD-PEI) in rats.

Effects of hydroxypropyl-ß-cyclodextrin grafted polyethylenimine (HP-ß-CD-PEI) including HP-ß-CD-PEI600, HP-ß-CD-PEI1800, HP-ß-CD-PEI10000 on the pulmonary absorption of insulin, calcitonin, 5(6)-carboxyfluorescein (CF) and fluorescein isothiocyanate dextrans (FDs) with various molecular weights (FD4, FD10 and FD70) were examined by an pulmonary absorption study in rats. Pulmonary absorptions of these poorly absorbable drugs were significantly enhanced by HP-ß-CD-PEI1800 and HP-ß-CD-PEI10000, and HP-ß-CD-PEI1800 with the concentration of 5% (w/v) provided maximal absorption enhancing effect on pulmonary absorption of these model drugs. The toxicity study demonstrated that HP-ß-CD-PEI did not induce any toxic action to rat pulmonary membranes. In addition, zeta potential of insulin solution changed to positive by addition of various HP-ß-CD-PEI, meanwhile, the degree of positive charge was linearly correlated with absorption enhancing effect of HP-ß-CD-PEI, suggesting that positive charge of HP-ß-CD-PEI might be related to their absorption enhancing mechanisms for enhancing pulmonary absorption of insulin in rats. In conclusion, HP-ß-CD-PEI is a potential and safe absorption enhancer for improving absorption of hydrophilic macromolecules especially peptide and protein drugs by pulmonary delivery.

Hydrogen sulfide decreases ß-adrenergic agonist-stimulated lung liquid clearance by inhibiting ENaC-mediated transepithelial sodium absorption.

In pulmonary epithelia, ß-adrenergic agonists regulate the membrane abundance of the epithelial sodium channel (ENaC) and, thereby, control the rate of transepithelial electrolyte absorption. This is a crucial regulatory mechanism for lung liquid clearance at birth and thereafter. This study investigated the influence of the gaseous signaling molecule hydrogen sulfide (H2S) on ß-adrenergic agonist-regulated pulmonary sodium and liquid absorption. Application of the H2S-liberating molecule Na2S (50 µM) to the alveolar compartment of rat lungs in situ decreased baseline liquid absorption and abrogated the stimulation of liquid absorption by the ß-adrenergic agonist terbutaline. There was no additional effect of Na2S over that of the ENaC inhibitor amiloride. In electrophysiological Ussing chamber experiments with native lung epithelia (Xenopus laevis), Na2S inhibited the stimulation of amiloride-sensitive current by terbutaline. ß-adrenergic agonists generally increase ENaC abundance by cAMP formation and activation of PKA. Activation of this pathway by forskolin and 3-isobutyl-1-methylxanthine increased amiloride-sensitive currents in H441 pulmonary epithelial cells. This effect was inhibited by Na2S in a dose-dependent manner (5-50 µM). Na2S had no effect on cellular ATP concentration, cAMP formation, and activation of PKA. By contrast, Na2S prevented the cAMP-induced increase in ENaC activity in the apical membrane of H441 cells. H441 cells expressed the H2S-generating enzymes cystathionine-ß-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, and they produced H2S amounts within the employed concentration range. These data demonstrate that H2S prevents the stimulation of ENaC by cAMP/PKA and, thereby, inhibits the proabsorptive effect of ß-adrenergic agonists on lung liquid clearance.

Paracetamol and ibuprofen block hydrothorax absorption in mice.

OBJECTIVES: Non-steroidal anti-inflammatory agents (NSAIDs) and paracetamol alter pleural permeability, hindering pleural fluid recycling. The aim of this study was to investigate the effect of different analgesic and anti-inflammatory agents on fluid recycling in an induced hydrothorax model in mice. METHODS: Hydrothorax was induced in C57BL/6 mice by injecting 500 µl phosphate-buffered saline-bovine serum albumin 1% isosmotic in the right hemithorax. Paracetamol (1 g/kg), ibuprofen (250 mg/kg) and parecoxib (2 mg/kg) were administered systematically by intraperitoneal injections. Each drug group included eight mice, which were sacrificed at 2 h and 4 h, respectively, after injections. The remaining hydrothorax volume and total cells contained were determined. RESULTS: Regarding the paracetamol and ibuprofen groups, the remaining hydrothorax volume was greater than in the control group (350 ± 61, 348 ± 62 and 270 ± 51 µl, respectively, P = 0.042) when mice were sacrificed within 2 h. Similar observations were made in groups sacrificed after 4 h (202 ± 45 and 198 ± 44 vs 107 ± 56 µl, respectively, P = 0.002). In the parecoxib group, the remaining hydrothorax volume was 122 ± 53 µl (P = 0.038 versus paracetamol and ibuprofen, P > 0.05 versus control group). At the same time, the absorption rate in the paracetamol and ibuprofen groups was lower than in the parecoxib and control groups (P = 0.033). In the parecoxib group, the absorption rate was lower than that in the control group after 2 h (P = 0.042). In the paracetamol and ibuprofen groups, the total cell count and the macrophage and the neutrophils counts were increased, compared with the control and parecoxib groups (P = 0.025, 0.028 and 0.032, respectively). CONCLUSIONS: Paracetamol and ibuprofen acutely hinder pleural fluid recycling by lowering the fluid absorption rate (higher remaining hydrothorax volume), while they increased total white cell counts. COX-2s presented lower remaining hydrothorax volume without acutely increasing the absorption rate. These findings could present some relevance to the administration of painkillers in patients with pleural effusion after thoracotomy.

Improvement of pulmonary absorptions of poorly absorbable drugs using Gelucire 44/14 as an absorption enhancer.

OBJECTIVES: This study aims to evaluate the absorption-enhancing effects of Gelucire 44/14 on the pulmonary absorption of different poorly absorbable drugs and relative mechanism of action. METHODS: Absorption-enhancing effect of Gelucire 44/14 were examined by an in-vivo pulmonary absorption experiment in rats, and the membrane toxicity of Gelucire 44/14 was evaluated by measuring levels of protein and dehydrogenase (LDH) in the bronchoalveolar lavage fluid (BALF) and morphological observation. KEY FINDINGS: Pulmonary absorptions of fluorescein isothiocyanate-dextrans, insulin and calcitonin were enhanced by Gelucire 44/14 (0.1-2.0%, w/v) in a concentration-dependent manner, and the maximal absorption-enhancing effect was obtained when the concentration of Gelucire 44/14 increased to 2.0% (w/v). Furthermore, Gelucire 44/14 neither increase the levels of protein and LDH in BALF nor change morphology of lung compared with control group. In addition, a well correlation between the absorption-enhancing effect and surface tension of insulin solution in the presence of Gelucire 44/14 was observed, suggesting Gelucire 44/14-mediated decrease in the surface tension of the gas-liquid interface in alveolar tissue was possible one of the improving mechanisms of Gelucire 44/14. CONCLUSION: Gelucire 44/14 was a potential and safe absorption enhancer for improving the absorption of poorly absorbable drugs including insulin and calcitonin by pulmonary delivery.

Netrin-1 promotes epithelial sodium channel-mediated alveolar fluid clearance via activation of the adenosine 2B receptor in lipopolysaccharide-induced acute lung injury.

BACKGROUND: The epithelial sodium channel (ENaC) is the driving force for pulmonary edema absorption in acute lung injury (ALI). Netrin-1 is a newly found anti-inflammatory factor that works by activating the adenosine 2B receptor (A2BAR). Meanwhile, activated A2BAR has the potential to enhance ENaC-dependent alveolar fluid clearance (AFC). However, whether netrin-1 can increase ENaC-mediated AFC by activating A2BAR remains unclear. OBJECTIVES: To investigate the effect of netrin-1 on AFC in ALI and clarify the pathway via which netrin-1 regulates the expression of ENaC in vivo and in vitro. METHODS: An ALI model was established by intratracheal instillation of lipopolysaccharide (LPS; 5 mg/kg) in C57BL/J mice, followed by netrin-1 with or without pretreatment with PSB1115, via the caudal vein. Twenty-four hours later, the lungs were isolated for determination of the bronchoalveolar lavage fluid, the lung wet/dry weight (W/D) ratio, AFC, the expressions of α-, ß-, and γ-ENaC, and cyclic adenosine monophosphate (cAMP) levels. LPS-stimulated MLE-12 cells were incubated with netrin-1 with or without preincubation with PSB1115. Twenty-four hours later, the expressions of α-, ß-, and γ-ENaC were detected. RESULTS: In vivo, netrin-1 expression was significantly decreased during ALI. Substituted netrin-1 significantly dampened the lung injury, decreased the W/D ratio, and enhanced AFC, the expressions of α-, ß-, and γ-ENaC, and cAMP levels in ALI, which were abolished by specific A2BAR inhibitor PSB1115. In vitro, netrin-1 increased the expressions of α-, ß-, and γ-ENaC, which were prevented by PSB1115. CONCLUSION: These results indicate that netrin-1 dampens pulmonary inflammation and increases ENaC-mediated AFC to alleviate pulmonary edema in LPS-induced ALI by enhancing cAMP levels through the activation of A2BAR.