ABSTRACT
BACKGROUND: Although atorvastatin (ATV) is well-tolerated, patients may report muscle complaints. These are difficult to predict owing to high interindividual variability. Such side effects are linked to intramuscular accumulation of ATV. This study aimed to investigate the relative role of transporters expressed in muscle tissue in promoting or limiting drug access to cells. The impact of common single nucleotide polymorphisms (SNPs) in SLCO2B1 coding for OATP2B1 and ABCC1 coding for MRP1 on ATV transport was also evaluated. METHODS: HEK293 cells were stably transfected with plasmids containing cDNA encoding wild-type or variant SLCO2B1 and/or ABCC1 to generate single and double stable transfectant HEK293 recombinant models overexpressing variant or wild-type OATP2B1 (influx) and/or MRP1 (efflux) proteins. Variant plasmids were generated by site-directed mutagenesis. Expression analyses were performed to validate recombinant models. Accumulation and efflux experiments were performed at different concentrations. ATV was quantified by LC-MS/MS, and kinetic parameters were compared between single and double HEK transfectants expressing wild-type and variant proteins. RESULTS: The results confirm the involvement of OATP2B1 and MRP1 in ATV cellular transport because it was demonstrated that intracellular accumulation of ATV was boosted by OATP2B1 overexpression, whereas ATV accumulation was decreased by MRP1 overexpression. In double transfectants, it was observed that increased ATV intracellular accumulation driven by OATP2B1 influx was partially counteracted by MRP1 efflux. The c.935G > A SNP in SLCO2B1 was associated with decreased ATV OATP2B1-mediated influx, whereas the c.2012G > T SNP in ABCC1 seemed to increase MRP1 efflux activity against ATV. CONCLUSIONS: Intracellular ATV accumulation is regulated by OATP2B1 and MRP1 transporters, whose functionality is modulated by natural genetic variants. This is significant because it may play a role in ATV muscle side-effect susceptibility.
Subject(s)
Drug-Related Side Effects and Adverse Reactions , Organic Anion Transporters , Humans , HEK293 Cells , Atorvastatin , Chromatography, Liquid , Tandem Mass Spectrometry , Polymorphism, Single Nucleotide/genetics , Multidrug Resistance-Associated Proteins/genetics , Organic Anion Transporters/geneticsABSTRACT
Chronic inflammation that progressively disrupts the lung tissue is a hallmark of cystic fibrosis (CF). In mice, vardenafil, an inhibitor of phosphodiesterase type 5 (PDE5), restores transepithelial ion transport and corrects mislocalization of the most common CF mutation, F508del-CFTR. It also reduces lung pro-inflammatory responses in mice and in patients with CF. To test the hypothesis that macrophages are target effector cells of the immunomo-dulatory effect of vardenafil, we isolated lung macrophages from mice homozygous for the F508del mutation or invalidated for the cftr gene and from their corresponding wild-type (WT) littermates. We then evaluated the effect of vardenafil on the classical M1 polarization, mirroring release of pro-inflammatory cytokines. We confirmed that macrophages from different body compartments express CF transmembrane conductance regulator (CFTR) and showed that vardenafil targets the cells through PDE5- and CFTR-dependent mechanisms. In the presence of the F508del mutation, vardenafil down-regulated overresponses of the M1 markers, tumour necrosis factor (TNF)-α and inducible nitric oxide synthase (NOS)-2. Our study identifies lung macrophages as target cells of the anti-inflammatory effect of vardenafil in CF and supports the view that the drug is potentially beneficial for treating CF as it combines rescue of CFTR protein and anti-inflammatory properties.
Subject(s)
Cystic Fibrosis/pathology , Inflammation Mediators/metabolism , Macrophages/drug effects , Phosphodiesterase 5 Inhibitors/pharmacology , Vardenafil Dihydrochloride/pharmacology , Animals , Cell Polarity/drug effects , Cells, Cultured , Cyclic Nucleotide Phosphodiesterases, Type 5/metabolism , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Down-Regulation/drug effects , Drug Evaluation, Preclinical/methods , Macrophages/metabolism , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/metabolism , Mice, Inbred CFTR , Molecular Targeted Therapy/methods , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Macrophages play a central role in immune and tissue responses of granulomatous lung diseases induced by pathogens and foreign bodies. Circulating monocytes are generally viewed as central precursors of these tissue effector macrophages. Here, we provide evidence that granulomas derive from alveolar macrophages serving as a local reservoir for the expansion of activated phagocytic macrophages. By exploring lung granulomatous responses to silica particles in IL-1-deficient mice, we found that the absence of IL-1α, but not IL-1ß, was associated with reduced CD11b(high) phagocytic macrophage accumulation and fewer granulomas. This defect was associated with impaired alveolar clearance and resulted in the development of pulmonary alveolar proteinosis (PAP). Reconstitution of IL-1α(-/-) mice with recombinant IL-1α restored lung clearance functions and the pulmonary accumulation of CD11b(high) phagocytic macrophages. Mechanistically, IL-1α induced the proliferation of CD11b(low) alveolar macrophages and differentiated these cells into CD11b(high) macrophages which perform critical phagocytic functions and organize granuloma. We newly discovered here that IL-1α triggers lung responses requiring macrophage proliferation and maturation from tissue-resident macrophages.
Subject(s)
CD11b Antigen/metabolism , Cell Proliferation , Granuloma/metabolism , Interleukin-1alpha/metabolism , Lung Diseases/metabolism , Macrophage Activation , Macrophages, Alveolar/metabolism , Pulmonary Alveolar Proteinosis/metabolism , Animals , Cells, Cultured , Disease Models, Animal , Granuloma/chemically induced , Granuloma/genetics , Granuloma/pathology , Interleukin-1alpha/deficiency , Interleukin-1alpha/genetics , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , Lung Diseases/chemically induced , Lung Diseases/genetics , Lung Diseases/pathology , Macrophages, Alveolar/pathology , Mice, Knockout , Phagocytosis , Phenotype , Pulmonary Alveolar Proteinosis/chemically induced , Pulmonary Alveolar Proteinosis/genetics , Pulmonary Alveolar Proteinosis/pathology , Silicon Dioxide , Time FactorsABSTRACT
BACKGROUND: The asbestos-like toxicity of some engineered carbon nanotubes (CNT), notably their capacity to induce mesothelioma, is a serious cause of concern for public health. Here we show that carcinogenic CNT induce an early and sustained immunosuppressive response characterized by the accumulation of monocytic Myeloid Derived Suppressor Cells (M-MDSC) that counteract effective immune surveillance of tumor cells. METHODS: Wistar rats and C57BL/6 mice were intraperitoneally injected with carcinogenic multi-walled Mitsui-7 CNT (CNT-7) or crocidolite asbestos. Peritoneal mesothelioma development and immune cell accumulation were assessed until 12 months. Leukocyte sub-populations were identified by recording expression of CD11b/c and His48 by flow cytometry. The immunosuppressive activity on T lymphocytes of purified peritoneal leukocytes was assessed in a co-culture assay with activated spleen cells. RESULTS: We demonstrate that long and short mesotheliomagenic CNT-7 injected in the peritoneal cavity of rats induced, like asbestos, an early and selective accumulation of monocytic cells (CD11b/c(int) and His48(hi)) which possess the ability to suppress polyclonal activation of T lymphocytes and correspond to M-MDSC. Peritoneal M-MDSC persisted during the development of peritoneal mesothelioma in CNT-7-treated rats but were only transiently recruited after non-carcinogenic CNT (CNT-M, CNT-T) injection. Peritoneal M-MDSC did not accumulate in mice which are resistant to mesothelioma development. CONCLUSIONS: Our data provide new insights into the initial pathogenic events induced by CNT, adding a new component to the adverse outcome pathway leading to mesothelioma development. The specificity of the M-MDSC response after carcinogenic CNT exposure highlights the interest of this response for detecting the ability of new nanomaterials to cause cancer.
Subject(s)
Carcinogens/toxicity , Mesothelioma/chemically induced , Monocytes/immunology , Nanotubes, Carbon/toxicity , Animals , Heterografts , Humans , Male , Mesothelioma/immunology , Mice , Mice, Inbred C57BL , Rats , Rats, WistarABSTRACT
Bictegravir, a key second-generation integrase strand transfer inhibitor in the treatment of HIV, is subject to active efflux transport mediated by ABCB1 (P-glycoprotein). Several coding variants of ABCB1 have been described and associated with variable effects on substrate drugs pharmacokinetics. Here, we investigated the effect of the four most common coding ABCB1 single nucleotide polymorphisms (i.e., c.1199G > A, c.1236C > T, c.2677G > T and c.3435C > T) on the intracellular accumulation of bictegravir. Using a previously validated HEK293 recombinant cell line model, we found decreased bictegravir intracellular concentrations in cell lines overexpressing ABCB1 as compared to control cell lines, in line with the known role of ABCB1 in bictegravir transport. However, we were unable to demonstrate any significant difference in bictegravir intracellular accumulation when comparing HEK293 cells overexpressing the wild type (1236C-2677G-3435C, 1199G) or the variant (1236C-2677G-3435T, 1236T-2677T-3435T or 1199A) proteins. These findings suggest that the ABCB1 c.1199G > A and c.1236C > T-c.2677G > T-c.3435C > T variants have no or at least limited impact on the active transport of bictegravir by ABCB1.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B , Piperazines , Polymorphism, Single Nucleotide , Humans , HEK293 Cells , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Piperazines/metabolism , Heterocyclic Compounds, 3-Ring/metabolism , Amides/metabolism , Pyridones/metabolism , Heterocyclic Compounds, 4 or More Rings/metabolismABSTRACT
The purpose of this study was to investigate the potential clinical relevance of estimating the apparent clearance (CL/F) of atorvastatin through population pharmacokinetic (PopPK) modeling with samples collected in a real-life setting in a cohort of ambulatory patients at risk of cardiovascular disease by using an opportunistic sampling strategy easily accessible in clinical routine. A total of 132 pharmacokinetic (PK) samples at a maximum of three visits were collected in the 70 included patients. The effects of demographic, genetic, and clinical covariates were also considered. With the collected data, we developed a two-compartment PopPK model that allowed estimating atorvastatin CL/F relatively precisely and considering the genotype of the patient for SLCO1B1 c.521T>C single-nucleotide polymorphism (SNP). Our results indicate that the estimation of the CL/F of atorvastatin through our PopPK model might help in identifying patients at risk of myalgia. Indeed, we showed that a patient presenting a CL/F lower than 414.67 L h-1 is at risk of suffering from muscle discomfort. We also observed that the CL/F was correlated with the efficacy outcomes, suggesting that a higher CL/F is associated with a better drug efficacy (i.e., a greater decrease in total and LDL-cholesterol levels). In conclusion, our study demonstrates that PopPK modeling can be useful in daily clinics to estimate a patient' atorvastatin clearance. Notifying the clinician with this information can help in identifying patients at risk of myalgia and gives indication about the potential responsiveness to atorvastatin therapy.
Subject(s)
Hydroxymethylglutaryl-CoA Reductase Inhibitors , Atorvastatin/pharmacokinetics , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Liver-Specific Organic Anion Transporter 1/genetics , Myalgia/chemically induced , Myalgia/drug therapy , Polymorphism, Single NucleotideABSTRACT
OBJECTIVES: ABCC2 contributes to the active cellular efflux of several endogenous and exogenous compounds. The 4544G>A (rs8187710) single nucleotide polymorphism (SNP), which is coding for a C1515Y substitution, has been previously associated with susceptibility to cholestatic liver disease, doxorubicin cardiotoxicity, nonalcoholic fatty liver disease, decreased graft function after renal transplantation and tenofovir-induced proximal nephropathy. It is also involved in differential flavopiridol disposition and increased lopinavir (LPV) cellular accumulation in vivo. The aim of the present study was to investigate whether the 4544G>A SNP causes alterations in ABCC2-mediated transport towards different substrates and to explore the underlying molecular mechanisms. METHODS: Human embryonic kidney cells (HEK293) were stably transfected with full-length ABCC2 cDNA coding the wild-type (4544G) or the variant (4544A) ABCC2 allele. Accumulation and efflux assays were carried out with three different substrates and kinetic parameters were then compared between wild-type and variant HEK293-transfected clones. The vanadate-sensitive ATPase activity of the wild-type and the variant ABCC2-transfected clones was assayed in isolated membranes vesicles. RESULTS: At a comparable expression level, the ABCC2 4544A clone showed higher accumulation of LPV, calcein and carboxyfluorescein diacetate as well as reduced ABCC2-mediated efflux of LPV compared with the ABCC2 4544G clone. The 4544G>A SNP impaired ABCC2 ATPase activity, which likely explains the reduced efflux activity of the 4544A clone. CONCLUSION: The C1515Y amino acid substitution caused by the 4544G>A SNP in ABCC2 impairs its ATPase activity and is associated with higher cellular accumulation of ABCC2 substrates.
Subject(s)
Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Polymorphism, Single Nucleotide , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Alleles , Biological Transport , Genotype , HEK293 Cells , Humans , Lopinavir/metabolism , Multidrug Resistance-Associated Protein 2 , TransfectionABSTRACT
The intracellular penetration of darunavir, a second-generation HIV protease inhibitor, is limited by the activity of the efflux P-glycoprotein (ABCB1). ABCB1 expression and/or activity levels can vary between individuals due to genetic polymorphisms including the c.1199G>A, c.1236C>T, c.2677G>T and c.3435C>T variants, which could in part explain why the pharmacokinetics of darunavir are so variable from one individual to another. While a few clinical studies have failed to demonstrate an influence of these polymorphisms on darunavir pharmacokinetics, drug-drug interactions and methodological limitations may have prevented them from revealing the true influence of ABCB1 variants. In this work, we report on the intracellular accumulation of darunavir in recombinant HEK293 cell lines expressing wild-type ABCB1 or one of several variants: ABCB1 1199A, ABCB1 3435T, and ABCB1 1236T/2677T/3435T. We demonstrate that while ABCB1 expression limits intracellular accumulation of darunavir, there is no significant difference in efflux activity between cells expressing wild-type ABCB1 and those that express any of the studied variants.
Subject(s)
Darunavir , Polymorphism, Genetic , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Darunavir/pharmacokinetics , Darunavir/pharmacology , HEK293 Cells , HumansABSTRACT
Cystic fibrosis (CF) is a genetic disease characterized by progressive lung and chronic digestive manifestations. We have shown that therapeutic doses of vardenafil, a phosphodiesterase type 5 (PDE5) inhibitor, corrects CF Transmembrane conductance Regulator (CFTR)-dependent chloride transport in respiratory and intestinal tissues of F508del homozygous mice. Here, we studied the effect of vardenafil on CFTR in 16HBE14o- and CFBE41o- cell lines. First, the expression levels of PDE5 mRNA in these cell lines were monitored. The two cell lines were exposed to different drugs (dimethyl sulfoxide, 8-Br-cGMP, forskolin or vardenafil). The cAMP and cGMP intracellular concentrations were measured. Finally, we localised the CFTR by immunolabelling. PDE5 was similarly expressed in both wild-type and in CF cells. A fast and transient rise in cGMP intracellular contents followed treatment with vardenafil, confirming its PDE5 inhibitory effect. We showed that vardenafil promoted both the early steps of the cellular processing and the trafficking of F508del without fully addressing the protein to the plasma membrane. The effect was not reproduced by the brominated cGMP analogue and it was not prevented by the combination of a protein kinase G (PKG) inhibitor and vardenafil. These findings support the view that vardenafil partially rescues F508del through cGMP/PKG-independent mechanisms.
Subject(s)
Bronchi/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Epithelial Cells/metabolism , Intracellular Space/metabolism , Mutant Proteins/metabolism , Vardenafil Dihydrochloride/pharmacology , Cell Line , Cyclic AMP/metabolism , Cyclic GMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/metabolism , Epithelial Cells/drug effects , Humans , Signal Transduction/drug effectsABSTRACT
Direct oral anticoagulants (DOAC) are substrates for the ABCB1 transporter (also called P-glycoprotein), an active efflux pump. ABCB1 polymorphisms have been previously reported to influence the pharmacokinetics of several drugs such as immunosuppressants and tyrosine kinase inhibitors. Recently, in vivo studies have suggested that genetic variants might contribute to the inter-individual variability in DOAC plasma concentrations. Therefore, we evaluated the in vitro effect of the most common coding ABCB1 single nucleotide polymorphisms (SNP), 1236 C > T-2677G > T-3435C > T, and the coding ABCB1 1199 G > A SNP on the transport activity towards rivaroxaban. HEK293 cells were transfected to overexpress the ABCB1 wild-type (1236C-2677G-3435C, 1199 G) or variant proteins (1236C-2677G-3435T, 1236T-2677T-3435T or 1199 A). ABCB1 expression decreased the intracellular accumulation of rivaroxaban, when compared to control cells. This confirms the involvement of ABCB1 in the active transport of rivaroxaban. However, the ABCB1 1236 C > T-2677G > T-3435C > T and 1199 G > A SNPs had no significant influence on the intracellular accumulation of rivaroxaban when compared to the wild-type protein. These results suggest that the ABCB1 coding SNPs investigated in the present study are unlikely to contribute to the inter-individual variability in rivaroxaban plasma concentrations.
Subject(s)
Biological Variation, Population/genetics , Rivaroxaban/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , HEK293 Cells , Humans , Polymorphism, Single Nucleotide , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
Overexpression of ABCB1 (also called P-glycoprotein) confers resistance to multiple anticancer drugs, including tyrosine kinase inhibitors (TKIs). Several ABCB1 single nucleotide polymorphisms affect the transporter activity. The most common ABCB1 variants are 1236C > T, 2677G > T, 3435C > T and have been associated with clinical response to imatinib in chronic myelogenous leukaemia (CML) in some studies. We evaluated the impact of these polymorphisms on the anti-proliferative effect and the intracellular accumulation of TKIs (imatinib, nilotinib, dasatinib and ponatinib) in transfected HEK293 and K562 cells. ABCB1 overexpression increased the resistance of cells to doxorubicin, vinblastine and TKIs. Imatinib anti-proliferative effect and accumulation were decreased to a larger extent in cells expressing the ABCB1 wild-type protein compared with the 1236T-2677T-3435T variant relatively to control cells. By contrast, ABCB1 polymorphisms influenced the activity of nilotinib, dasatinib and ponatinib to a much lesser extent. In conclusion, our data suggest that wild-type ABCB1 exports imatinib more efficiently than the 1236T-2677T-3435T variant protein, providing a molecular basis for the reported association between ABCB1 polymorphisms and the response to imatinib in CML. Our results also point to a weaker impact of ABCB1 polymorphisms on the activity of nilotinib, dasatinib and ponatinib.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Dasatinib/pharmacology , Imatinib Mesylate/pharmacology , Imidazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Pyridazines/pharmacology , Pyrimidines/pharmacology , ATP Binding Cassette Transporter, Subfamily B/genetics , HEK293 Cells , Humans , K562 Cells , Polymorphism, Single NucleotideABSTRACT
AIM: ABCB1 (or P-glycoprotein) is implicated in the multidrug-resistance phenotype, including the resistance toward anticancer drugs such as tyrosine kinase inhibitors (TKIs). The purpose of this study was to evaluate in vitro the influence of the ABCB1 1199G>A SNP on ABCB1 transport activity toward selected TKIs (imatinib, nilotinib and dasatinib) that are currently used in chronic myelogenous leukemia. MATERIAL & METHODS: Two different cell lines, HEK293 and K562, were stably transfected with ABCB1 1199G wild-type or ABCB1 1199A variant allele. The impact of this polymorphism on accumulation and antiproliferative effects of imatinib, nilotinib and dasatinib was evaluated. RESULTS: In K562 models, the expression of Asn400 variant protein was associated with lower antiproliferative effects of imatinib, nilotinib and dasatinib compared with Ser400 wild-type protein. Moreover, in HEK293 cells, imatinib and nilotinib intracellular accumulation were lower in variant compared with wild-type models. CONCLUSION: Imatinib, nilotinib and dasatinib are transported more efficiently by the ABCB1 variant (Asn400) compared with the wild-type (Ser400) protein. The impact of ABCB1 1199G>A SNP on TKI response should be further investigated in chronic myelogenous leukemia patients.
Subject(s)
Antineoplastic Agents/pharmacokinetics , Dasatinib/pharmacokinetics , Imatinib Mesylate/pharmacokinetics , Polymorphism, Single Nucleotide , Pyrimidines/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily B/genetics , Biological Transport , HEK293 Cells , Humans , K562 CellsABSTRACT
OBJECTIVES: To assess the impact of the loss-of-function CYP3A5*3 allele (rs776746, 6986A>G SNP) on darunavir (DRV) plasma concentrations. METHODS: 135 HIV-1 infected patients treated with DRV-based therapy were included in the study and plasma samples were obtained immediately before drug intake in order to determine DRV trough concentrations using an ultra performance liquid chromatography method (UPLC) with diode-array detection (DAD). Noteworthy is the fact that in 16 (11.9%) patients, etravirine (ETR) was combined with DRV. CYP3A5 genotypes were determined using real time PCR method (TaqMan® genotyping assay). The patients were then classified into CYP3A5 expressors (CYP3A5*1 allele carriers) and non-expressors (CYP3A5*3 homozygous). Subsequently, the association between DRV plasma trough concentration ([DRV]plasma) and CYP3A5 genotype-based expression status was analyzed. RESULTS: 45% of the patients were classified as CYP3A5 expressors. In the whole cohort, mean [DRV]plasma was not different between CYP3A5 expressors and non-expressors (1894ng/ml [CI95%: 1566-2290] versus 1737ng/ml [CI95%: 1468-2057], p = 0.43). However, in the subgroup of the 16 patients receiving DRV combined with ETR, a significantly lower [DRV]plasma was observed for CYP3A5 expressors when compared to non-expressors (1385ng/ml [CI95%:886.3-2165] versus 3141ng/ml [CI95%:2042-4831], p = 0.007). CONCLUSIONS: Interaction between DRV and ETR is partly mediated by CYP3A5 polymorphism with lower DRV plasma trough concentrations in CYP3A5 expressors suggesting a specific ETR-driven CYP3A5 activation only in CYP3A5 expressors. Consequently, these patients might be more at risk of infra-therapeutic [DRV]plasma. This potentially important observation is a good illustration of a genotype-based drug interaction, which could also have considerable consequences if translated to other CYP3A5-metabolized drugs. Further investigations are thus needed to confirm this association and to explore its clinical impact, mainly in the African population among whom CYP3A5 expressors are more frequent, before recommending systematic CYP3A5 pre-emptive genotyping for DRV-ETR co-administration.
Subject(s)
Cytochrome P-450 CYP3A/genetics , Darunavir/pharmacology , HIV Protease Inhibitors/pharmacology , Polymorphism, Genetic , Pyridazines/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Adult , Darunavir/administration & dosage , Darunavir/therapeutic use , Drug Interactions , Female , HIV Infections/drug therapy , HIV Protease Inhibitors/administration & dosage , HIV Protease Inhibitors/therapeutic use , Humans , Male , Middle Aged , Nitriles , Pyridazines/administration & dosage , Pyridazines/therapeutic use , Pyrimidines , Reverse Transcriptase Inhibitors/administration & dosage , Reverse Transcriptase Inhibitors/therapeutic useABSTRACT
BACKGROUND: Pseudomonas aeruginosa airway infections are a major cause of morbidity and mortality in patients with cystic fibrosis (CF). Azithromycin improves the related clinical outcomes, but its mechanisms of action remain poorly understood. We tested the hypothesis that azithromycin downregulates P. aeruginosa-induced pro-inflammatory responses by modifying release of bacterial proteins. METHODS: We monitored inflammatory markers in lungs of CF mutant mice and their littermate controls in response to conditioned media (CM) collected from the reference P. aeruginosa PAO1 strain cultured in the presence or in the absence of azithromycin. A mass spectrometry-based proteomic approach was applied to examine whether the macrolide elicits a differential release of bacterial proteins. RESULTS: CM collected from azithromycin-untreated PAO1 cultures induced powerful pro-inflammatory neutrophil-dominated responses. Azithromycin attenuated the responses, mainly of macrophage chemoattractant protein-1, tumor necrosis factor-α, and interferon-γ, in CF but not in wild-type mice. Proteomic analysis showed that azithromycin upregulated an array of bacterial proteins including those associated with regulation of immune functions and with repair and resolution of inflammatory responses like the chaperone DnaK and the S-adenosylmethionine synthase, while it downregulated the extracellular heme acquisition protein HasA and the catalytic enzyme lysylendopeptidase. CONCLUSION: Supernatants collected from cultures of the bacterial strain PAO1 represent a novel experimental model to trigger in vivo lung inflammatory responses that should be closer to those obtained with live bacteria, but without bacterial infection. Combined with a bactericidal effect, complex regulation of bacterial innate immune and metabolic factors released in the cultured medium by the action of the macrolide can contribute to its anti-inflammatory effects.
ABSTRACT
OBJECTIVE: ATP-binding cassette, subfamily B, member 1 (ABCB1) transporter, or P-glycoprotein, is an efflux protein implicated in the absorption and the distribution of various compounds, including tacrolimus and cyclosporine A. In vivo studies suggest an association between the ABCB1 1199G>A single nucleotide polymorphism (SNP) and tacrolimus intracellular accumulation. The aim of the present experimental study was to clarify in vitro the impact of the coding ABCB1 1199G>A SNP on ABCB1 transport activity towards both immunosuppressive drugs. METHOD: Two recombinant cell lines, i.e. Human Embryonic Kidney (HEK293) and Human Myelogenous Leukemia (K562) cells, overexpressing ABCB1 carrying either the wild-type allele (1199G) or its mutated counterpart (1199A), were generated. The impact of the 1199G>A SNP on ABCB1 activity towards rhodamine (Rh123), doxorubicin, vinblastine, tacrolimus and cyclosporine A was assessed by accumulation, cytotoxicity and/or kinetic experiments. RESULTS: Tacrolimus accumulation was strongly decreased in cells overexpressing the wild-type protein (1199G) compared to control cells, confirming the ability of ABCB1 to transport tacrolimus. By contrast, overexpression of the variant protein (1199A) had nearly no effect on tacrolimus intracellular accumulation whatever the model used and the concentration tested. Unlike tacrolimus, our results also indicate that cyclosporine A, Rh123 and doxorubicin are transported in a similar extent by the wild-type and variant ABCB1 proteins while the variant protein seems to be more efficient for the transport of vinblastine. CONCLUSION: ABCB1 encoded by the 1199G wild-type allele transports more efficiently tacrolimus in comparison to the 1199A variant protein. This observation indicates that the amino-acid substitution (Ser400Asn) encoded by the 1199A allele drastically decreases the ability of ABCB1 to drive the efflux of tacrolimus in a substrate-specific manner, in agreement with our previously published clinical data. Our study emphasizes the importance of the ABCB1 1199G>A polymorphism for ABCB1 activity and its potential to explain differences in drug response.
Subject(s)
DNA, Recombinant/genetics , Intracellular Space/metabolism , Polymorphism, Single Nucleotide , Tacrolimus/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Biological Transport/drug effects , Biological Transport/genetics , Cyclosporine/metabolism , Doxorubicin/pharmacology , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , K562 Cells , Transfection , Vinblastine/pharmacologyABSTRACT
Morbi-mortality in cystic fibrosis (CF) is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Fibroblasts/metabolism , Pulmonary Fibrosis/metabolism , Animals , Bleomycin , Cell Line , Cells, Cultured , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cytokines/genetics , Cytokines/metabolism , Female , Fibroblasts/drug effects , Fibroblasts/pathology , Gene Expression/drug effects , Imidazoles/pharmacology , Immunohistochemistry , Inflammation Mediators/metabolism , Lipopolysaccharides/pharmacology , Lung/drug effects , Lung/metabolism , Lung/pathology , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Inbred CFTR , Phenotype , Piperazines/pharmacology , Pseudomonas aeruginosa/chemistry , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/genetics , Reverse Transcriptase Polymerase Chain Reaction , Skin/drug effects , Skin/metabolism , Skin/pathology , Sulfones/pharmacology , Triazines/pharmacology , Vardenafil Dihydrochloride , Vasodilator Agents/pharmacologyABSTRACT
BACKGROUND: We tested the hypothesis that vardenafil, a common drug used for improving erectile dysfunction and able to partially normalize transepithelial chloride transport in cystic fibrosis (CF), modulates CF lung inflammation. METHODS: Inflammatory markers in lungs of F508del-CF and wild-type mice were monitored in response to lipopolysaccharide from Pseudomonas aeruginosa (LPS). The effect of pretreatment with vardenafil (0.14 mg/kg) was evaluated. RESULTS: A latent inflammatory status, characterized by neutrophil infiltrate, mouse macrophage inflammatory protein (MIP)-2 and tumor necrosis factor (TNF)-α, was found in baseline conditions in F508del-CF mice. Inflammatory markers were increased after LPS with higher responses in CF. Vardenafil globally attenuated inflammatory responses in both genotypes however reduction of macrophage infiltration, macrophage chemoattractant chemokine and interleukin-1ß was observed in the CF group only. CONCLUSION: Vardenafil reduces lung inflammation with a more pronounced effect in F508del-CF mice, particularly on macrophage cell markers.
Subject(s)
Cystic Fibrosis/drug therapy , Cystic Fibrosis/immunology , Imidazoles/pharmacology , Piperazines/pharmacology , Pneumonia/drug therapy , Pneumonia/immunology , Animals , Animals, Outbred Strains , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Disease Models, Animal , Female , Immunomodulation/drug effects , Immunomodulation/immunology , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/immunology , Macrophages/microbiology , Male , Mice , Mice, 129 Strain , Mice, Inbred CFTR , Phosphodiesterase 5 Inhibitors/pharmacology , Pneumonia/chemically induced , Pseudomonas Infections/drug therapy , Pseudomonas Infections/immunology , Sulfones/pharmacology , Triazines/pharmacology , Vardenafil DihydrochlorideABSTRACT
AIMS: CYP3A4 is involved in the oxidative metabolism of many drugs and xenobiotics including the immunosuppressants tacrolimus (Tac) and cyclosporine (CsA). The objective of the study was to assess the potential influence of a new functional SNP in CYP3A4 on the pharmacokinetic parameters assessed by dose requirements and trough blood levels of both calcineurin inhibitors (CNI) in stable renal transplant patients. PATIENTS & METHODS: A total of 99 stable renal transplant patients receiving either Tac (n = 49) or CsA (n = 50) were genotyped for the CYP3A4 intron 6 C>T (rs35599367) and CYP3A5*3 SNPs. Trough blood levels ([Tac](0) or [CsA](0) in ng/ml), dose-adjusted [Tac](0) or [CsA](0) (ng/ml per mg/kg bodyweight) as well as doses (mg/kg bodyweight) required to achieve target concentrations were compared among patients according to allelic status for CYP3A4 and CYP3A5. RESULTS: Dose-adjusted concentrations were 2.0- and 1.6-fold higher in T-variant allele carriers for the CYP3A4 intron 6 C>T SNP compared with homozygous CC for Tac and CsA, respectively. When CYP3A4/CYP3A5 genotypes were combined, the difference was even more striking as the so-defined CYP3A poor metabolizer group presented dose-adjusted concentration 1.6- and 4.1-fold higher for Tac, and 1.5- and 2.2-fold higher for CsA than the intermediate metabolizer and extensive metabolizer groups, respectively. Multiple linear regression analysis revealed that, taken together, both CYP3A4 intron 6 and CYP3A5*3 SNPs explained more than 60 and 20% of the variability observed in dose-adjusted [Tac](0) and [CsA](0), respectively. CONCLUSION: The CYP3A4 intron 6 C>T polymorphism is associated with altered Tac and CsA metabolism. CYP3A4 intron 6 C>T along with CYP3A5*3 (especially for Tac) pharmacogenetic testing performed just before transplantation may help identifying patients at risk of CNI overexposure and contribute to limit CNI-related nephrotoxicity by refining the starting dose according to their genotype. Original submitted 5 May 2011; Revision submitted 29 June 2011.
Subject(s)
Calcineurin Inhibitors , Cyclosporine/adverse effects , Cytochrome P-450 CYP3A/genetics , Immunosuppressive Agents/adverse effects , Kidney Transplantation , Tacrolimus/adverse effects , Cyclosporine/administration & dosage , Cyclosporine/pharmacokinetics , Dose-Response Relationship, Drug , Genetic Association Studies , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/pharmacokinetics , Polymorphism, Single Nucleotide/genetics , Tacrolimus/administration & dosage , Tacrolimus/pharmacokineticsABSTRACT
Toxicological investigations of carbon nanotubes have shown that they can induce pulmonary toxicity, and similarities with asbestos fibers have been suggested. We previously reported that multiwall carbon nanotubes (MWCNT) induced lung inflammation, granulomas and fibrotic reactions. The same MWCNT also caused mutations in epithelial cells in vitro and in vivo. These inflammatory and genotoxic activities were related to the presence of defects in the structure of the nanotubes. In view of the strong links between inflammation, mutations and cancer, these observations prompted us to explore the carcinogenic potential of these MWCNT in the peritoneal cavity of rats. The incidence of mesothelioma and other tumors was recorded in three groups of 50 male Wistar rats injected intraperitoneally with a single dose of MWCNT with defects (2 or 20 mg/animal) and MWCNT without defects (20 mg/animal). Two additional groups of 26 rats were used as positive (2 mg UICC crocidolite/animal) and vehicle controls. After 24 months, although crocidolite induced a clear carcinogenic response (34.6% animals with mesothelioma vs. 3.8% in vehicle controls), MWCNT with or without structural defects did not induce mesothelioma in this bioassay (4, 0, or 6%, respectively). The incidence of tumors other than mesothelioma was not significantly increased across the groups. The initial hypothesis of a contrasting carcinogenic activity between MWCNT with and without defects could not be verified in this bioassay. We discuss the possible reasons for this absence of carcinogenic response, including the length of the MWCNT tested (< 1 mum on average), the absence of a sustained inflammatory reaction to MWCNT, and the capacity of these MWCNT to quench free radicals.
Subject(s)
Abdominal Neoplasms/chemically induced , Biological Assay , Carcinogenicity Tests/methods , Carcinogens/toxicity , Cell Transformation, Neoplastic/chemically induced , Mesothelioma/chemically induced , Nanotubes, Carbon/toxicity , Animals , Asbestos, Crocidolite/toxicity , Biological Assay/standards , Carcinogenicity Tests/standards , Injections, Intraperitoneal , Male , Nanotubes, Carbon/chemistry , Peritoneal Cavity , Rats , Rats, Wistar , Reference Standards , Risk Assessment , Surface Properties , Time FactorsABSTRACT
Indium-Tin-Oxide (ITO) is a sintered mixture of indium- (In(2)O(3)) and tin-oxide (SnO(2)) in a ratio of 90:10 (wt:wt) that is used for the manufacture of LCD screens and related high technology applications. Interstitial pulmonary diseases have recently been reported in workers from ITO producing plants. The present study was conducted to identify experimentally the exact chemical component responsible for this toxicity and to address possible mechanisms of action. The reactivity of respirable ITO particles was compared with that of its single components alone or their unsintered 90:10 mixture (MIX) both in vivo and in vitro. For all endpoints considered, ITO particles behaved as a specific toxic entity. In vivo, after a single pharyngeal administration (2-20 mg per rat), ITO particles induced a strong inflammatory reaction. At day 3, the inflammatory reaction (cell accumulation, LDH and protein in bronchoalveolar lavage fluid) appeared more marked with ITO particles than with each oxide separately or the MIX. This inflammatory reaction persisted and even worsened after 15 days. After 60 days, this inflammation was still present but no significant fibrotic response was observed. The cytotoxicity of ITO was assessed in vitro in lung epithelial cells (RLE) and macrophages (NR8383 cell line). While ITO particles (up to 200 microg/ml) did not affect epithelial cell integrity (LDH release), a strong cytotoxic response was found in macrophages exposed to ITO, but not to its components alone or mixed. ITO particles also induced an increased frequency of micronuclei in type II pneumocytes in vivo but not in RLE in vitro, suggesting the preponderance of a secondary genotoxic mechanism. To address the possible mechanism of ITO toxicity, reactive oxygen species production was assessed by electron paramagnetic resonance spectrometry in an acellular system. Carbon centered radicals (COO-.) and Fenton-like activity were detected in the presence of ITO particles, not with In(2)O(3), SnO(2) alone, or the MIX. Because the unsintered mixture of SnO(2) and In(2)O(3) particles was unable to reproduce the reactivity/toxicity of ITO particles, the sintering process through which SnO(2) molecules are introduced within the crystal structure of In(2)O(3) appears critical to explain the unique toxicological properties of ITO. The inflammatory and genotoxic activities of ITO dust indicate that a strict control of exposure is needed in industrial settings.