Nanomedicine : nanotechnology, biology, and medicine, Jan 22, 2017
Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is kn... more Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is known about their interaction with the respiratory tract lining fluids (RTLFs). Here we combined the use of nano-silica, as a protein concentrator, with label-free snapshot proteomics (LC-MS/MS; key findings confirmed by ELISA) to generate a quantitative profile of the RTLF proteome and provided insight into the evolved corona; information that may be used in future to improve drug targeting to the lungs by inhaled medicines. The asthmatic coronal proteome displayed a reduced contribution of surfactant proteins (SP-A and B) and a higher contribution of α1-antitrypsin. Pathway analysis suggested that asthmatic RTLFs may also be deficient in proteins related to metal handling (e.g. lactoferrin). This study demonstrates how the composition of the corona acquired by inhaled nanoparticles is modified in asthma and suggests depressed mucosal immunity even in mild airway disease.
A series of novel, potent and selective human β2 adrenoceptor agonists incorporating a urea moiet... more A series of novel, potent and selective human β2 adrenoceptor agonists incorporating a urea moiety on the terminal right-hand side phenyl ring of (R)-salmeterol is presented. Urea 9j had long duration of action in vitro on guinea pig trachea, and also in vivo similar to that of salmeterol. It had lower oral absorption and bioavailability than salmeterol in both rat and dog. It had a turnover ratio similar to salmeterol, with no evidence for formation of any aniline metabolites in human liver microsomes and hepatocytes. However no crystalline salts suitable for inhaled delivery were identified.
Nanomedicine: Nanotechnology, Biology and Medicine, 2016
When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fl... more When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fluid (RTLF) acquiring a biomolecular corona reflecting the interaction of the RTLF with the nanomaterial surface. Label-free snapshot proteomics was used to generate quantitative profiles of the proteins within the corona formed around silica (SiO2) and poly(vinyl) acetate (PVAc) nanoparticles in RTLF, the latter employed as an archetype drug delivery vehicle. The evolved PVAc corona was significantly enriched compared to that observed on SiO2 nanoparticles (698 vs. 429 proteins identified); however both coronas contained a substantial contribution from innate immunity proteins, including surfactant protein A, napsin A and complement (C1q and C3) proteins. Functional protein classification supports the hypothesis that corona formation in RTLF constitutes opsonisation, preparing particles for phagocytosis and clearance from the lungs. These data highlight how an understanding of the evolved corona is necessary for the design of inhaled nanomedicines with acceptable safety and tailored clearance profiles.
Inhaled drugs are frontline therapies for respiratory diseases including asthma and COPD. Althoug... more Inhaled drugs are frontline therapies for respiratory diseases including asthma and COPD. Although the overall contribution of pulmonary drug metabolism to the disposition of inhaled drugs is often considered to be minor compared to that of the liver, the drug metabolising enzyme (DME) capacity of the human lung has not been studied in detail and in vitro pulmonary models that utilise both phase I and phase II metabolism are poorly characterised. We report here the development of an in vitro model for the characterisation of pulmonary drug metabolism. Isolated lung cells were prepared from fresh human lung parenchyma and incubated for up to 12 hours with a range of phase I and phase II probe substrates to determine their rates and routes of metabolism. For comparison, the same substrates were incubated with pooled, cryopreserved human hepatocytes. Sample analyses were performed by LCMS. Depletion of probe substrates was observed in isolated lung cells, although at generally <10% ...
ABSTRACT The liver performs a wide range of physiologically important functions, including the sy... more ABSTRACT The liver performs a wide range of physiologically important functions, including the synthesis and secretion of albumin, fibrinogen, and other plasma proteins; the synthesis of cholesterol and bile acids, and the metabolism of drugs, steroids, and amino acids. The liver has a central role in energy metabolism as the major store of glycogen, as the site of gluconeogenesis, and in the synthesis of fatty acids and triglycerides. The liver is, therefore, a vital organ, but it is difficult to study specific liver functions in vivo owing to interfering influences from other organs, e g., the kidney, gut, and lungs, which metabolize drugs and the muscle involvement in glucose homeostasis. An isolated liver preparation seems necessary, and the isolated human hepatocyte appears to be a suitable experimental model for the study of liver-specific functions.
The pulmonary and hepatic expression and catalytic activities of phase I and II drug-metabolizing... more The pulmonary and hepatic expression and catalytic activities of phase I and II drug-metabolizing enzymes were compared using human lung and liver tissue, and lung parenchymal cells (LPCs) and cryopreserved hepatocytes. Cytochrome P450 gene expression was generally lower in lung than in liver and CYP3A4 expression in lung was negligible. Esterase gene expression was similar in lung and liver. Expression of all sulfotransferase isoforms in lung was similar to or higher than that in liver. Lung tissue expressed low levels of UGT. However, the expression of UGT2A1 in lung was higher than that in liver. There was a range of catalytic activities in LPCs, including cytochrome P450, esterase, and sulfation pathways. Phase I activities were generally less than 10% of those determined in hepatocytes. Rates of ester hydrolysis and sulfation in LPCs were similar to those in hepatocytes. When measurable, glucuronidation in LPCs was present at very low levels, reflecting the gene expression data. The metabolism of salbutamol, formoterol, and budesonide was also investigated. Production of salbutamol-4-O-sulfate and budesonide oleate was observed in LPCs from at least two of three donor preparations studied. Formoterol sulfate and low levels of formoterol glucuronide were detected in one of three donors. In general, drug-metabolizing capability of LPCs is low compared with liver, although some evidence for substantial sulfation and deesterification capacity was observed. Therefore, these data support the use of this cell-based system for the investigation of key routes of xenobiotic metabolism in human lung parenchyma.
This paper is the first report of a P450-electrode in a microfluidic format. A 30 μL microfluidic... more This paper is the first report of a P450-electrode in a microfluidic format. A 30 μL microfluidic cell was made in poly(methyl methacrylate) containing the inlet, outlet, and reaction chamber with two electrode strips, one of which contains the human cytochrome P450 3A4 covalently bound to gold via a 6-hexanethiol and 7-mercaptoheptanoic acid (1:1) self-assembled monolayer. The electrochemical response of the P450-electrode in the microfluidic cell was tested using four drugs that are known substrates of P450 3A4: quinidine, nifedipine, alosetron and ondansetron. Titration experiments allowed the electrochemical measurements of K(M) for the four drugs, with values of 2.9, 29.1, 113.4, and 114.1 mM, respectively. The K(M) values are found to be in good agreement and correctly ranked with respect to the published literature on human liver microsomes and baculosomes: [ondansetron ≈ alosetron &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; nifedipine &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; quinidine]. The results presented in this paper represent a step forward for a rapid evaluation of the interaction of P450 and drug, requiring small volumes of new chemical entities to be tested.
Nanomedicine : nanotechnology, biology, and medicine, Jan 22, 2017
Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is kn... more Most inhaled nanomedicines in development are for the treatment of lung disease, yet little is known about their interaction with the respiratory tract lining fluids (RTLFs). Here we combined the use of nano-silica, as a protein concentrator, with label-free snapshot proteomics (LC-MS/MS; key findings confirmed by ELISA) to generate a quantitative profile of the RTLF proteome and provided insight into the evolved corona; information that may be used in future to improve drug targeting to the lungs by inhaled medicines. The asthmatic coronal proteome displayed a reduced contribution of surfactant proteins (SP-A and B) and a higher contribution of α1-antitrypsin. Pathway analysis suggested that asthmatic RTLFs may also be deficient in proteins related to metal handling (e.g. lactoferrin). This study demonstrates how the composition of the corona acquired by inhaled nanoparticles is modified in asthma and suggests depressed mucosal immunity even in mild airway disease.
A series of novel, potent and selective human β2 adrenoceptor agonists incorporating a urea moiet... more A series of novel, potent and selective human β2 adrenoceptor agonists incorporating a urea moiety on the terminal right-hand side phenyl ring of (R)-salmeterol is presented. Urea 9j had long duration of action in vitro on guinea pig trachea, and also in vivo similar to that of salmeterol. It had lower oral absorption and bioavailability than salmeterol in both rat and dog. It had a turnover ratio similar to salmeterol, with no evidence for formation of any aniline metabolites in human liver microsomes and hepatocytes. However no crystalline salts suitable for inhaled delivery were identified.
Nanomedicine: Nanotechnology, Biology and Medicine, 2016
When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fl... more When inhaled nanoparticles deposit in the lungs, they transit through respiratory tract lining fluid (RTLF) acquiring a biomolecular corona reflecting the interaction of the RTLF with the nanomaterial surface. Label-free snapshot proteomics was used to generate quantitative profiles of the proteins within the corona formed around silica (SiO2) and poly(vinyl) acetate (PVAc) nanoparticles in RTLF, the latter employed as an archetype drug delivery vehicle. The evolved PVAc corona was significantly enriched compared to that observed on SiO2 nanoparticles (698 vs. 429 proteins identified); however both coronas contained a substantial contribution from innate immunity proteins, including surfactant protein A, napsin A and complement (C1q and C3) proteins. Functional protein classification supports the hypothesis that corona formation in RTLF constitutes opsonisation, preparing particles for phagocytosis and clearance from the lungs. These data highlight how an understanding of the evolved corona is necessary for the design of inhaled nanomedicines with acceptable safety and tailored clearance profiles.
Inhaled drugs are frontline therapies for respiratory diseases including asthma and COPD. Althoug... more Inhaled drugs are frontline therapies for respiratory diseases including asthma and COPD. Although the overall contribution of pulmonary drug metabolism to the disposition of inhaled drugs is often considered to be minor compared to that of the liver, the drug metabolising enzyme (DME) capacity of the human lung has not been studied in detail and in vitro pulmonary models that utilise both phase I and phase II metabolism are poorly characterised. We report here the development of an in vitro model for the characterisation of pulmonary drug metabolism. Isolated lung cells were prepared from fresh human lung parenchyma and incubated for up to 12 hours with a range of phase I and phase II probe substrates to determine their rates and routes of metabolism. For comparison, the same substrates were incubated with pooled, cryopreserved human hepatocytes. Sample analyses were performed by LCMS. Depletion of probe substrates was observed in isolated lung cells, although at generally <10% ...
ABSTRACT The liver performs a wide range of physiologically important functions, including the sy... more ABSTRACT The liver performs a wide range of physiologically important functions, including the synthesis and secretion of albumin, fibrinogen, and other plasma proteins; the synthesis of cholesterol and bile acids, and the metabolism of drugs, steroids, and amino acids. The liver has a central role in energy metabolism as the major store of glycogen, as the site of gluconeogenesis, and in the synthesis of fatty acids and triglycerides. The liver is, therefore, a vital organ, but it is difficult to study specific liver functions in vivo owing to interfering influences from other organs, e g., the kidney, gut, and lungs, which metabolize drugs and the muscle involvement in glucose homeostasis. An isolated liver preparation seems necessary, and the isolated human hepatocyte appears to be a suitable experimental model for the study of liver-specific functions.
The pulmonary and hepatic expression and catalytic activities of phase I and II drug-metabolizing... more The pulmonary and hepatic expression and catalytic activities of phase I and II drug-metabolizing enzymes were compared using human lung and liver tissue, and lung parenchymal cells (LPCs) and cryopreserved hepatocytes. Cytochrome P450 gene expression was generally lower in lung than in liver and CYP3A4 expression in lung was negligible. Esterase gene expression was similar in lung and liver. Expression of all sulfotransferase isoforms in lung was similar to or higher than that in liver. Lung tissue expressed low levels of UGT. However, the expression of UGT2A1 in lung was higher than that in liver. There was a range of catalytic activities in LPCs, including cytochrome P450, esterase, and sulfation pathways. Phase I activities were generally less than 10% of those determined in hepatocytes. Rates of ester hydrolysis and sulfation in LPCs were similar to those in hepatocytes. When measurable, glucuronidation in LPCs was present at very low levels, reflecting the gene expression data. The metabolism of salbutamol, formoterol, and budesonide was also investigated. Production of salbutamol-4-O-sulfate and budesonide oleate was observed in LPCs from at least two of three donor preparations studied. Formoterol sulfate and low levels of formoterol glucuronide were detected in one of three donors. In general, drug-metabolizing capability of LPCs is low compared with liver, although some evidence for substantial sulfation and deesterification capacity was observed. Therefore, these data support the use of this cell-based system for the investigation of key routes of xenobiotic metabolism in human lung parenchyma.
This paper is the first report of a P450-electrode in a microfluidic format. A 30 μL microfluidic... more This paper is the first report of a P450-electrode in a microfluidic format. A 30 μL microfluidic cell was made in poly(methyl methacrylate) containing the inlet, outlet, and reaction chamber with two electrode strips, one of which contains the human cytochrome P450 3A4 covalently bound to gold via a 6-hexanethiol and 7-mercaptoheptanoic acid (1:1) self-assembled monolayer. The electrochemical response of the P450-electrode in the microfluidic cell was tested using four drugs that are known substrates of P450 3A4: quinidine, nifedipine, alosetron and ondansetron. Titration experiments allowed the electrochemical measurements of K(M) for the four drugs, with values of 2.9, 29.1, 113.4, and 114.1 mM, respectively. The K(M) values are found to be in good agreement and correctly ranked with respect to the published literature on human liver microsomes and baculosomes: [ondansetron ≈ alosetron &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; nifedipine &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; quinidine]. The results presented in this paper represent a step forward for a rapid evaluation of the interaction of P450 and drug, requiring small volumes of new chemical entities to be tested.
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