Rat Lung Homogenates Research Articles

Development and validation of a bioanalytical method for the quantification of CHF6550 and its metabolite (CHF6671) in rat plasma and lung homogenate using LC–MS/MS

A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for accurate determination of CHF6550 and its main metabolite in rat plasma and lung homogenate samples. All biological samples were prepared by simple protein precipitation method using deuterated internal standards. The analytes were separated on a HSS T3 analytical column with 3.2 min run time at flow rate of 0.5 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by selected-reaction monitoring of the transitions at m/z 735.3 → 98.0 for CHF6550 and m/z 638.3 → 319.2 and 638.3 → 376.2 for CHF6671. The calibration curves for plasma samples were linear between 50 and 50000 pg/mL for both analytes. The calibration curves for lung homogenate samples were linear within 0.1–100 ng/mL for CHF6550 and 0.3–300 ng/mL for CHF6671. The method was successfully applied to a 4-week toxicity study.

Development and Validation of a Bioanalytical Method for the Quantification of CHF6550 and its Metabolite (CHF6671) in Rat Plasma and Lung Homogenate Using LC–MS/MS

Development and Validation of a Bioanalytical Method for the Quantification of CHF6550 and its Metabolite (CHF6671) in Rat Plasma and Lung Homogenate Using LC–MS/MS

Interaction between green tea and perindopril reduces inhibition of angiotensin-converting enzyme activity

Purpose: To investigate the blood pressure-lowering effect of green tea (GT) extract alone and in combination with an angiotensin converting enzyme (ACE) inhibitor, perindopril, on rats.
 Methods: The study consisted of four groups of five spontaneously hypertensive rats (SHR): negative control (2 % tragacanth mucilage), positive control group (perindopril, 0.36 mg/kg/day) and two treatment groups (green tea, 25 mg/kg/day; and combined green tea/perindopril). The treatments were given orally for 14 days. Systolic blood pressure was measured before and after treatment using the tail cuff technique. Angiotensin converting enzyme activity in the lung homogenate of the hypertensive rats was determined spectrophotometrically.
 Results: Green tea extract significantly reduced the rats’ systolic blood pressure (p < 0.05) but did not inhibit the angiotensin-converting enzyme. The combination of green tea extract with perindopril also caused a significant decline in blood pressure (p < 0.001). However, the green tea extract attenuated the inhibition of the angiotensin-converting enzyme activity by perindopril.
 Conclusion: Green tea extract produces anti-hypertensive activity in rats, but its mechanism of action is not via inhibition of angiotensin-converting enzyme. The interaction of GT extract with perindopril results in a reduction of ACE inhibitory activity.

Repetitive diacetyl vapor exposure promotes ubiquitin proteasome stress and precedes bronchiolitis obliterans pathology.

Bronchiolitis obliterans (BO) is a devastating lung disease seen commonly after lung transplant, following severe respiratory tract infection or chemical inhalation exposure. Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha-diketone known to cause BO when inhaled, however, the mechanisms of how inhalation exposure leads to BO development remains poorly understood. In the current work, we combined two clinically relevant models for studying the pathogenesis of DA-induced BO: (1) an in vivo rat model of repetitive DA vapor exposures with recovery and (2) an in vitro model of primary human airway epithelial cells exposed to pure DA vapors. Rats exposed to 5 consecutive days 200 parts-per-million DA 6h per day had worsening survival, persistent hypoxemia, poor weight gain, and histologic evidence of BO 14days after DA exposure cessation. At the end of exposure, increased expression of the ubiquitin stress protein ubiquitin-C accumulated within DA-exposed rat lung homogenates and localized primarily to the airway epithelium, the primary site of BO development. Lung proteasome activity increased concurrently with ubiquitin-C expression after DA exposure, supportive of significant proteasome stress. In primary human airway cultures, global proteomics identified 519 significantly modified proteins in DA-exposed samples relative to controls with common pathways of the ubiquitin proteasome system, endosomal reticulum transport, and response to unfolded protein pathways being upregulated and cell-cell adhesion and oxidation-reduction pathways being downregulated. Collectively, these two models suggest that diacetyl inhalation exposure causes abundant protein damage and subsequent ubiquitin proteasome stress prior to the development of chemical-induced BO pathology.

Ambient particulate matter-associated autophagy alleviates pulmonary inflammation induced by Platanus pollen protein 3 (Pla3)

Subpollen particles (SPPs) with diameter less than 1 mm released from allergenic pollen grains contain allergens could trigger asthma and lung inflammation after being inhaled. In the meaning time, ambient fine particles attached on the pollen grains could have further effects on the inflammation. However, the mechanisms underlying these phenomena have not been fully elucidated. In this study, the effects of autophagy triggered by PM2.5 and Platanus SPPs were evaluated by using the A549 cell lines and a pollen sensitized rat model. First, autophagy in A549 cells was analyzed after exposure to PM2.5 using acridine orange staining, real-time quantitative PCR (qRT-PCR), and western blot (WB) assays. The increased levels of ROS, superoxide dismutase, and malonaldehyde in the lung homogenates of rats exposed to SPPs indicated that inflammatory response was triggered in the lungs. Treatment with autophagy-inhibiting drugs showed that autophagy suppressed ROS formation and decreased the production of thymic stromal lymphopoietin (TSLP), a critical pathway altering the inflammatory response. Although the effect was indirect, autophagy appeared to negatively regulate TSLP levels, resulting in a compromised immune response. These results suggested that SPPs promote ROS generation and increase TSLP levels, triggering downstream inflammation reactions. However, ambient PM2.5 could aggravate autophagy, which in turn effectively suppressed ROS and TSLP levels, leading to the alleviation of the immune response and pulmonary inflammation.

Recombinant adiponectin protects the newborn rat lung from lipopolysaccharide-induced inflammatory injury.

Preterm infants are at high risk for developing bronchopulmonary dysplasia and pulmonary hypertension from inflammatory lung injury. In adult models, adiponectin (APN)—an adipocyte‐derived hormone—protects the lung from inflammatory injury and pulmonary vascular remodeling. Cord blood APN levels in premature infants born < 26 weeks gestation are 5% of the level in infants born at term. We previously reported the expression profile of APN and its receptors in neonatal rat lung homogenates during the first 3 weeks of postnatal development. Here, we characterize the expression profile of APN and its receptors in specific lung cells and the effects of exogenous recombinant APN (rAPN) on lipopolysaccharide‐(LPS)‐induced cytokine and chemokine production in total lung homogenates and specific lung cells. In vitro, rAPN added to primary cultures of pulmonary artery smooth muscle cells attenuated the expression of LPS‐induced pro‐inflammatory cytokines while increasing the expression of anti‐inflammatory cytokines. In vivo, intraperitoneal rAPN (2 mg/kg), given 4 hr prior to intrapharyngeal administration of LPS (5 mg/kg) to newborn rats at postnatal day 4, significantly reduced gene and protein expression of the pro‐inflammatory cytokine IL‐1ß and reduced protein expression of the chemokines monocyte chemoattractant protein (MCP‐1) and macrophage inflammatory protein‐1 alpha (MIP‐1α) in the lung. LPS‐induced histopathological changes in the lung were also decreased. Moreover, rAPN given 20 hr after intrapharyngeal LPS had a similar effect on lung inflammation. These findings suggest a role for APN in protecting the lung from inflammation during early stages of lung development.

Pulmonary delivery of a p38 α/β MAP kinase inhibitor: bioanalytical method validation and biodistribution in rat plasma and respiratory tissues

PF-03715455, an inhaled p38 α/β mitogen-activated protein (MAP) kinase inhibitor (MAPK), has being identified as an agent with potential therapeutic action on lung diseases such as COPD and severe asthma. However, little is known about this MAPKs local and systemic pharmacokinetics after pulmonary delivery. Consequently, the aim of the present work was to develop and validate a method of extraction and quantification of PF-03715455 in rat plasma and lung tissues and to determine the drug biodistribution in plasma and respiratory tissues after intratracheal administration of the drug solution in rats. The method was validated in rat plasma samples and resulted selective and linear in the concentration range of 0.08-100 ng/ml. Then a partial validation was carried out on samples obtained by the extraction and quantification of PF-03715455 from rat lung homogenate in order to ascertain method applicability on lung tissue samples. The intratracheal administration of drug in solution to rats evidenced a rapid elimination from the plasma, while on the contrary a prolonged residence time in lung tissue was evidenced. In conclusion, a linear, accurate, precise and reproducible method has been developed and validated according to FDA and EMA guidelines to quantify plasmatic and tissue-associated concentrations of PF-03715455 in order to investigate this compound in pharmacokinetics pre-clinical studies in rats. The administration of drug solution evidenced a prolonged permanence of the drug in the lungs that could be related to a slow absorption/poor permeability of the drug across airways epithelia.

Recombinant Measles AIK-C Vaccine Strain Expressing Influenza HA Protein.

Recombinant measles AIK-C vaccine expressing the hemagglutinin (HA) protein of influenza A/Sapporo/107/2013(H1N1pdm) (MVAIK/PdmHA) was constructed. Measles particle agglutination (PA) and influenza hemagglutinin inhibition (HI) antibodies were induced in cotton rats immunized with MVAIK/PdmHA. Cotton rats immunized with two doses of the HA split vaccine were used as positive controls, and higher HI antibodies were detected 3 weeks after the first dose. Following the challenge of A/California/07/2009(H1N1pdm), higher viral loads (107 TCID50/g) were detected in the lung homogenates of cotton rats immunized with the empty vector (MVAIK) or control groups than those immunized with MVAIK/Pdm HA (103 TCID50/g) or the group immunized with HA split vaccine (105 TCID50/g). Histopathologically, destruction of the alveolar structure, swelling of broncho-epithelial cells, and thickening of the alveolar wall with infiltration of inflammatory cells and HA antigens were detected in lung tissues obtained from non-immunized rats and those immunized with the empty vector after the challenge, but not in those immunized with the HA spilt or MVAIK/PdmHA vaccine. Lower levels of IFN-α, IL-1β, and TNF-α mRNA, and higher levels of IFN-γ mRNA were found in the lung homogenates of the MVAIK/PdmHA group. Higher levels of IFN-γ mRNA were detected in spleen cell culture from the MVAIK/PdmHA group stimulated with UV-inactivated A/California/07/2009(H1N1pdm). In conclusion, the recombinant MVAIK vaccine expressing influenza HA protein induced protective immune responses in cotton rats.

Presence of sialic acids in bronchioloalveolar cells and identification and quantification of N-acetylneuraminic and N-glycolylneuraminic acids in the lung

The lung, in air-breathing vertebrates, is a tree-like structure composed of branching tubes ending in alveoli and lined by diverse and highly specialized epithelial cells.A dense array of complex and diverse glycoconjugates is present on essentially all animal cell surfaces. Sialic acids are widely allocated at the outermost ends of glycan chains, attached to membrane proteins and lipids below. Due to their abundance and their terminal position in glycans, sialic acids are implicated in many physiological and pathological functions. Although the composition of lung epithelial cell-surface glycans has been studied over the years, it is not yet completely understood. In the present work, we aimed to histochemically localize N-acetylneuraminic acid (Neu5Ac)>N-glycolylneuraminic acid (Neu5Gc) residues on rat bronchioloalveolar epithelial cell surfaces using light microscopy (LM) methods. In lung membranes isolated from adult rat lung homogenates, we also separated, identified and quantified Neu5Ac and Neu5Gc by means of high-performance liquid chromatography (HPLC), and systematically described the optimized HPLC methods used. Sialic acid residues were localized on the surface coat of bronchioloalveolar cells, and the mean quantification of Neu5Ac and Neu5Gc in the adult rat lung homogenates was 12,26 and 2,73 μg/mg prot., respectively, revealing a manifest preponderance of Neu5Ac. A coefficient of variation (CV) of 4,98% and 4,40%, respectively was obtained and an optimal dispersion variability expressed by the SD and the CV was also reported, confirming the efficiency of the methodology.To the best of our knowledge, our group was the first to identify, separate and quantify sialic acids in purified lung membranes.The presence of these residues contributes to a strong anionic shield and may provide an hydrating and protective barrier as well as a repulsive structure that is crucial to lung physiology.

Influence of Moringa (Moringa oleifera) leaf extracts on the antioxidant and angiotensin-1 converting enzyme inhibitory properties of lisinopril

This study investigated the in vitro effect of various combinations of aqueous extracts of moringa (Moringa oleifera) leaves on the ACE inhibitory and antioxidant properties of lisinopril, which is a popular synthetic ACE inhibitor. Moringa leaves were air-dried and blended into powdery form. Thereafter, the aqueous extract was prepared and freeze-dried. The following sample mixtures were prepared: L = 100% lisinopril (1 mg/ml); M = 100% moringa extract (1 mg/ml); 1L + 3M = 25% lisinopril + 75% moringa extract; L + M = 50% lisinopril + 50% moringa extract; 3L + 1M = 75% lisinopril + 25% moringa extract. The results revealed that 100% moringa and the various combinations showed higher ACE inhibition than 100% lisinopril in both rat heart and lung homogenates. Furthermore, all the samples caused the inhibition of Fe2+-induced lipid peroxidation in rat heart and lung tissue homogenates, with moringa leaf extract having the highest inhibitory ability. All the samples also showed antioxidant properties (ferric reducing antioxidant property, 2,2’-azino-bis (-3-ethylbenzthiazoline-6-sulphonate [ABTS*] and 1,1-diphenyl-2-picrylhydrazyl [DPPH] free radicals scavenging abilities, and Fe2+ chelating ability). This study revealed that moringa leaf extracts could improve the antioxidant and ACE inhibitory properties of lisinopril. However, further in vivo experiments and clinical trials are recommended.

Lansoprazole-sulfide, pharmacokinetics of this promising anti-tuberculous agent.

Lansoprazole (LPZ) is a commercially available proton-pump inhibitor whose primary metabolite, lansoprazole sulfide (LPZS) was recently reported to have in vitro and in vivo activity against Mycobacterium tuberculosis. It was also reported that a 300 mg kg-1 oral administration of LPZS was necessary to reach therapeutic levels in the lung, with the equivalent human dose being unrealistic. A validated liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for the simultaneous quantification LPZ and LPZS in rat plasma and lung homogenates was developed. We administered 15 mg kg-1 oral doses of LPZ to a healthy rat model to determine the pharmacokinetics of its active metabolite, LPZS, in plasma and lung tissue. We found that the LPZS was present in amounts that were below the limit of quantification. This prompted us to administer the same dose of LPZS to the experimental animals intraperitoneally (i.p.). Using this approach, we found high concentrations of LPZS in plasma and lung, 7841.1 and 9761.2 ng mL-1 , respectively, which were significantly greater than the minimum inhibitory concentration (MIC) for Mycobacterium tuberculosis. While oral and i.p. administration of LPZ resulted in significant concentrations in the lung, it did not undergo sufficient cellular conversion to its anti-TB metabolite. However, when LPZS itself was administered i.p., significant amounts penetrated the tissue. These results have implications for future in vivo studies exploring the potential of LPZS as an anti-TB compound.

Role of vitamin C against bifenthrin induced oxidative damage in lungs of Wistar rats

The aim of present study was to unravel the protective role of vitamin C on oxidative stress parameters in lung homogenates of bifenthrin intoxicated rats. Rats were divided into four groups. Group I served as control while group II animals were treated with bifenthrin @ 5.8mg/Kg/day. In group III, vitamin C was orally administered @ 60mg/Kg/day where as group IV received both vitamin C and bifenthrin @ 60mg/Kg/day and 5.8mg/Kg/day respectively. After 30th day of treatment, lung samples were taken and analysed for oxidative stress parameters. Significant (P&lt;0.05) increase in lipid peroxidation was observed from control value of 4.80±0.39 to 7.90±0.50 in bifenthrin treated animals. Mean control values of SOD, GSH-Px and CAT were 0.55±0.05, 0.98±0.03 and 138.70±6.01 which were significantly (P&lt;0.05) decreased to values of 0.27±0.0, 0.53±0.05 and 91.10±9.70 respectively in bifenthrin treated animals. The value of GST increased significantly (p&lt;0.05) to 1.05±0.06 in bifenthrin administered animals from control value of 0.70±0.08. Pre-treatment with vitamin C in ameliorative group IV significantly restored the normal values of lipid peroxidation, SOD, GST and CAT but could not reverse the decreased values of GSH-Px. The present research is first of its type where in free radical generation due to bifenthrin –a commonly used insecticide was evaluated in lung homogenates when given orally which might be due to residues present in the lung. Besides it will be helpful in better understanding of toxicological profile of pyrethoids, the most commonly used insecticides.

Isoforskolin from Native Plant &amp;lt;i&amp;gt;Coleus forskohlii&amp;lt;/i&amp;gt; of Yunnan, China Plays Multiple Biological Roles

Isoforskolin (ISOF) was isolated from Coleus forskohlii native to Yunnan in China. It is identified as one analog of diterpene forskolin (FSK) which comes from the Indian plant Coleus forskohlii. Researches found that the Yunnan native plant Coleus forskohlii contained rich ISOF but not FSK. ISOF was reported to activate adenylyl cyclase (AC) isoforms. The ophthalmologic and cardiovascular effects of ISOF were firstly reported in the 1990s. The researchers in Kunming, China, found ISOF could lower blood pressure and intraocular pressure. Recent researches found the respiratory tract effects of ISOF, and showed that ISOF significantly elevated cyclic adenosine monophosphate (cAMP) levels in rat lung homogenate, and relaxed the histamine-induced contraction of isolated guinea pig trachea and lung smooth muscle, isoforskolin pretreatment attenuates acute lung injury induced by lipopolysaccharide (LPS) in several models, lowered LPS-induced secretion of the inflammatory cytokine in human monocytes.

Pulmonary instillation of MWCNT increases lung permeability, decreases gp130 expression in the lungs, and initiates cardiovascular IL-6 transsignaling.

Pulmonary instillation of multiwalled carbon nanotubes (MWCNT) has the potential to promote cardiovascular derangements, but the mechanisms responsible are currently unclear. We hypothesized that exposure to MWCNT would result in increased epithelial barrier permeability by 24 h postexposure and initiate a signaling process involving IL-6/gp130 transsignaling in peripheral vascular tissue. To test this hypothesis we assessed the impact of 1 and 10 μg/cm(2) MWCNT on transepithelial electrical resistance (TEER) and expression of barrier proteins and cell activation in vitro using normal human bronchial epithelial primary cells. Parallel studies using male Sprague-Dawley rats instilled with 100 μg MWCNT measured bronchoalveolar lavage (BAL) differential cell counts, BAL fluid total protein, and lung water-to-tissue weight ratios 24 h postexposure and quantified serum concentrations of IL-6, soluble IL-6r, and soluble gp130. Aortic sections were examined immunohistochemically for gp130 expression, and gp130 mRNA/protein expression was evaluated in rat lung, heart, and aortic tissue homogenates. Our in vitro findings indicate that 10 μg/cm(2) MWCNT decreased the development of TEER and zonula occludens-1 expression relative to the vehicle. In rats MWCNT instillation increased BAL protein, lung water, and induced pulmonary eosinophilia. Serum concentrations of soluble gp130 decreased, aortic endothelial expression of gp130 increased, and expression of gp130 in the lung was downregulated in the MWCNT-exposed group. We propose that pulmonary exposure to MWCNT can manifest as a reduced epithelial barrier and activator of vascular gp130-associated transsignaling that may promote susceptibility to cardiovascular derangements.

Protective effects of hydrogen sulfide inhalation on oxidative stress in rats with cotton smoke inhalation-induced lung injury.

The aim of the present study was to investigate the mechanism by which hydrogen sulfide (H2S) inhalation protects against oxidative stress in rats with cotton smoke inhalation-induced lung injury. A total of 24 male Sprague-Dawley rats were separated randomly into four groups, which included the control, H2S, smoke and smoke + H2S groups. A rat model of cotton smoke inhalation-induced lung injury was established following inhalation of 30% oxygen for 6 h. In addition, H2S (80 ppm) was inhaled by the rats in the H2S and smoke + H2S groups for 6 h following smoke or sham-smoke inhalation. Enzyme-linked immunosorbent assays were performed to measure various indices in the rat lung homogenate, while the levels of nuclear factor (NF)-κBp65 in the lung tissue of the rats were determined and semiquantitatively analyzed using immunohistochemistry. In addition, quantitative fluorescence polymerase chain reaction was employed to detect the mRNA expression of inducible nitric oxide synthase (iNOS) in the rat lung tissue. The concentrations of malondialdehyde (MDA), nitric oxide (NO), inducible iNOS and NF-κBp65, as well as the sum-integrated optical density of NF-κBp65 and the relative mRNA expression of iNOS, in the rat lung tissue from the smoke + H2S group were significantly lower when compared with the smoke group. The concentrations of MDA, NO, iNOS and NF-κBp65 in the H2S group were comparable to that of the control group. Therefore, inhalation of 80 ppm H2S may reduce iNOS mRNA transcription and the production of iNOS and NO in rats by inhibiting NF-κBp65 activation, subsequently decreasing oxidative stress and cotton smoke inhalation-induced lung injury.

Differential effects of dexamethasone and rosiglitazone in a sephadex-induced model of lung inflammation in rats: possible role of tissue inhibitor of metalloproteinase-3.

Objectives:To study the effects of two different classes of drugs in sephadex-induced lung inflammation using rats and explore the potential mechanism (s).Materials and Methods:Effects of dexamethasone (0.3 mg/kg, p.o.) and rosiglitazone (10 mg/kg, p.o.) treatments were evaluated up to 3 days in sephadex challenged rats. 72 h postsephadex administration, broncho-alveolar lavage fluid (BALF) was collected for cell count and cytokine estimation. Lung tissues were harvested for gene expression and histopathology.Results:Dexamethasone treatment resulted in significant inhibition of lymphocytes, monocytes, eosinophils and neutrophils, whereas rosiglitazone inhibited eosinophils and neutrophils only. Further, dexamethasone reduced the elevated levels of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) after sephadex challenge while rosiglitazone significantly reduced the PGE2 levels without altering LTB4 in the BALF. Hydroxyproline content in rat lung homogenate was significantly reduced with dexamethasone treatment but not with rosiglitazone. Both the drugs were found to suppress matrix metallo proteinase 9, whereas only dexamethasone showed inhibition of tumor necrosis factor-alpha and up-regulation of tissue inhibitor of metalloproteinase 3 (TIMP-3) expression and preserved the broncho-alveolar microstructure.Conclusions:Our results revealed that up-regulation of TIMP-3 corroborated well with dexamethasone mediated inhibition of collagen degradation and restoration of alveolar micro-architecture.

Expression of microRNAs in lung homogenates in rats with chronic obstructive pulmonary disease

To determine the expression spectrum of microRNA (miRNA) in the lung homogenate of rats with chronic obstructive pulmonary disease(COPD) or healthy rats, to look for differentially expressed miRNA, and to explore their potential role in pathogenesis of COPD. Twenty male Sprague-Dawley (SD) rats were randomly divided into normal control group and COPD model group, with 10 rats in each group. COPD rat models were reproduced by smoke inhalation as well as intratracheal instillation of lipopolysaccharide (LPS). The samples of the lung were harvested, and the histopathological examination of the right lung was carried out to evaluate the degree of lung injury. Total RNAs were isolated from the left lung. The miRNA expressions in lung tissue of rats with COPD or normal rats were determined by miRNA chip eichnology to screen the miRNA with differential expression, and hierarchical clustering analysis was made. The data were analyzed to study the expression difference of miRNAs between the two groups, and to construct the miRNA-Target network. Compared with normal control group, 20 miRNA with increased expression were found in COPD model group, the top 10 were miR-30c-2, miR-199a-5p, miR-30a, miR-145, miR-151, miR-674-5p, miR-214, miR-423, miR-28 and miR-181b, and only miR-376b-3p showed down-regulation. Hierarchical clustering analysis showed that significant differences in individual miRNA in lung tissue between rats of two groups were found. However, similar samples could not be gathered well, thus it may give rise to inconsistency during the sample preparation. Many miRNAs had multiple target genes in miRNA-Target network, such as miR-30c-2, miR-145, miR-181b, miR-181a, miR-181d, miR-199. The study reveals that there is a variance of miRNAs expression profile in lung homogenates between COPD group and the normal control group of rats, and many miRNAs have multiple target genes, such as miR-30c-2, miR-145, miR-181b, miR-181a, miR-181d, and miR-199. Therefore, the results may be valuable to centain extent for the interpretation of COPD pathogenesis.

Effect of protease inhibitors on pulmonary bioavailability of therapeutic proteins and peptides in the rat

The objective of the present study was to evaluate the effect of protease inhibitors on the pulmonary absorption of therapeutic peptides and proteins with varying molecular weights. Dry powder formulations of leuprolide (1.2kD), salmon calcitonin (3.4kD), human insulin (5.8kD), human leptin (16.0kD), and human chorionic gonadotropin (HCG) (36.5kD) were prepared with or without protease inhibitors; aprotinin and bestatin. The formulations were administered intrapulmonary to anesthetized rats. The pharmacokinetics of these proteins were assessed by measuring serum drug concentrations. In addition, in vitro stability of these proteins in rat lung homogenate was assessed using the trifluoroacetic acid method. Bioavailability of leuprolide following pulmonary administration was 75% higher compared to subcutaneously administered leuprolide. Protease inhibitors had little or no effect on the pulmonary bioavailability of leuprolide. However, protease inhibitors (1mg/kg) increased the bioavailability of calcitonin by more than 50%. Similarly, the bioavailabilities of leptin and HCG in the presence of bestatin were increased by 1.9 and 2.1-fold, respectively. Leuprolide was stable both in the lung cytosol and subcellular pellets with about 10% degradation at the end of the study period (4h). In contrast, calcitonin, insulin, leptin and HCG were significantly degraded in the lung cytosol and subcellular pellets. Presence of protease inhibitors in formulation could improve the stability of protein drugs. The results of this study demonstrate that the pulmonary absorption of proteins may be enhanced by the selection of optimal concentration and type of protease inhibitor.

Effect of Dermal Application of Bifenthrin on Acetylcholinesterase and Oxidative Stress Indices in Rat Blood, Lung and Kidney

The oxidative stress inducing potential of bifenthrin was evaluated in the blood, lung and kidney homogenates of rats following its repeated dermal application for a period of 30 days. Bifenthrin treated animals showed significant increase in lipid peroxidation after 20 and 30 days of treatment in erythrocytes, lung and kidney. Blood glutathione decreased significantly both after 20 and 30 days of exposure. Superoxide dismutase increased significantly after 20th day of application of bifenthrin followed by significant decrease after 30th day. In lung superoxide dismutase activity decreased non-significantly after 20th day which decreased significantly after 30th day of application. Superoxide dismutase in kidney decreased significantly both after 20th and 30th day of treatment. The activity of glutathione peroxidase significantly decreased in erythrocytes, lung and kidney both after 20 and 30 days of exposure. No significant change in the activity of glutathione–S-transferase was observed after 20th day in blood and a significant decrease was observed after 30th day. Significant increase of glutathione–S-transferase was observed in lung both after 20 and 30 days of treatment where as in kidney significant increase was observed only after 30 days. There was a significant decrease of catalase in erythrocytes, lung and kidney both after 20th and 30th day of application. Non-significant changes were observed in acetylcholinesterase activities in treatment group both after 20th and 30th day. In conclusion bifenthrin treated animals showed alteration in antioxidant enzyme system suggesting its oxidative stress potential.

The involvement of serotonin metabolism in cigarette smoke-induced oxidative stress in rat lung in vivo

Recently, we have reported the dysregulation of circulating serotonin (5-hydroxytryptamine, 5-HT) homeostasis in patients with chronic obstructive pulmonary disease (COPD). An increase in metabolism of 5-HT has been reported to induce oxidative stress via monoamine oxidase (MAO)-dependent pathway. The present study aimed at investigating the effect of cigarette smoke exposure on the systemic circulation and local airway 5-HT levels as well as MAO-mediated oxidative pathway using a cigarette smoke-exposed rat model. Male Sprague-Dawley rats (150–200 g) were exposed to either sham air or 4% (v/v, smoke/air) cigarette smoke for 1 hour daily for 56 consecutive days. Sera, bronchoalveolar larvage (BAL) and lung tissues were collected 24 hours after the last exposure. We found a significant reduction in the reduced glutathione (rGSH) and an elevation in advanced oxidation protein products (AOPP), a protein oxidation marker, in the lung of cigarette smoke-exposed group (p < 0.05). A significant increase in 5-HT was found in serum (p < 0.05), but not in the BAL or lung, after cigarette smoke exposure. MAO-A activity was significantly elevated in the lung of cigarette smoke-exposed group (p < 0.05). Furthermore, increased superoxide anion levels were found in lung homogenates of cigarette smoke-exposed rats after incubation with 5-HT (p < 0.05), which was positively associated with the increase in MAO-A activity (r = 0.639, p < 0.05). Our findings supported the presence of GSH disruption and protein oxidation in the lung after cigarette smoke exposure. The metabolism of 5-HT by MAO-A in the lung enhanced cigarette smoke-induced superoxides, which might contribute to the pathogenesis of COPD.