RESUMO
Aripiprazole lauroxil (AL) is a long-acting atypical antipsychotic approved for the treatment of schizophrenia in adults. AL has five regimen options that offer three different injection intervals using four different dosage strengths. The relationship between dosage strength (milligram injected), injection interval (time between injection visits), and expected steady-state plasma aripiprazole concentrations may not be readily apparent. This article illustrates the relationship by providing visual scenarios of steady-state plasma aripiprazole concentrations for the five AL regimens. The efficacy of AL was originally demonstrated in a pivotal study of two AL regimens (approved as 441 mg monthly and 882 mg monthly). The three additional regimens (662 mg monthly, 882 mg every 6 weeks, and 1064 mg every 2 months) were approved based on pharmacokinetic bridging studies and population pharmacokinetic models. For this paper, expected steady-state concentrations for each AL regimen were derived from the published population pharmacokinetic models and compared using median values and ranges. The five labeled AL regimens differ in dosage strength and injection interval; however, model-simulated concentrations illustrate that each regimen produces steady-state plasma aripiprazole concentrations within the upper and lower bounds associated with known efficacy for AL 441 mg and 882 mg administered monthly. This visual presentation of the relationship between dosage strength of the AL injection, the interval between successive injections, and steady-state aripiprazole plasma concentrations may demonstrate for clinicians how dosage strength and injection interval can be considered in selecting the AL regimen option that best fits the clinical circumstances of the individual patient.
Assuntos
Antipsicóticos , Adulto , Antipsicóticos/uso terapêutico , Aripiprazol/uso terapêutico , Preparações de Ação Retardada , Humanos , Injeções IntramuscularesRESUMO
One of the challenges with initiating long-acting injectable (LAI) antipsychotic regimens is achieving relevant drug levels quickly. After first injection of the LAI antipsychotic aripiprazole lauroxil (AL), the lag to reaching relevant plasma aripiprazole levels was initially addressed using supplemental oral aripiprazole for 21 days. A 1-day AL initiation regimen using a NanoCrystal® Dispersion formulation of AL (ALNCD; Aristada Initio®) combined with a single 30 mg dose of oral aripiprazole has been developed as an alternative approach. We compared the 1-day AL initiation regimen (ALNCD + 30 mg oral aripiprazole for 1 day) with the 21-day AL initiation regimen (AL + 15 mg/day of oral aripiprazole for 21 days) using kinetic modeling. Observed and modeled data demonstrate that the 1-day AL initiation regimen provides continuous aripiprazole exposure comparable to the 21-day AL initiation regimen. Each component of the 1-day AL initiation regimen (30 mg oral aripiprazole, ALNCD, and AL) contributes to aripiprazole plasma levels at different times, with oral aripiprazole predominating in the first week, then ALNCD and AL over time. In a double-blind, placebo-controlled, phase 1 study in patients with schizophrenia, the 1-day initiation regimen resulted in rapid achievement of relevant plasma aripiprazole levels comparable to those from the 21-day initiation regimen. Safety and tolerability of the 1-day regimen were consistent with the known profile of aripiprazole. Each part of the 1-day initiation regimen, together with AL, is necessary for continuous aripiprazole exposure from treatment initiation until the next regularly scheduled AL injection is administered.
Assuntos
Antipsicóticos/administração & dosagem , Aripiprazol/administração & dosagem , Nanopartículas/normas , Esquizofrenia/tratamento farmacológico , Antipsicóticos/sangue , Antipsicóticos/uso terapêutico , Aripiprazol/sangue , Aripiprazol/uso terapêutico , Humanos , Injeções/métodosRESUMO
BACKGROUND: Aripiprazole lauroxil (AL), a long-acting injectable antipsychotic for the treatment of schizophrenia, requires 21 days of oral aripiprazole supplementation upon initiation. We report findings from a phase 1 study investigating a nanocrystalline milled dispersion of AL (ALNCD) as a potential 1-day initiation regimen. The 1-day initiation regimen is designed to enable rapid achievement of plasma aripiprazole concentrations that are comparable with the 21-day oral initiation regimen. Here, a 6-month pharmacokinetic study compared 2 different initiation regimens for starting AL. METHODS: Patients were randomized 1:1:1:1 to receive 1 of 4 treatments consisting of the 1-day (single ALNCD injection + one 30-mg dose of oral aripiprazole on day 1 only) or the 21-day (15-mg daily dose of oral aripiprazole for 21 days) initiation regimen, each combined with a starting AL dose of either 441 mg or 882 mg. RESULTS: In total, 133/161 patients completed the study. The pharmacokinetic profile of the 1-day initiation regimen was comparable to the 21-day initiation regimen; both achieved aripiprazole concentrations in the therapeutic range within 4 days and remained in a comparable concentration range during treatment initiation. Common adverse events (≥5.0%) were injection-site pain, headache, increased weight, insomnia, dyspepsia, and anxiety. Nine akathisia events occurred (4 events in 4 patients and 5 events in 2 patients in the 1-day and 21-day initiation regimen groups, respectively). CONCLUSIONS: The 1-day initiation regimen resulted in plasma aripiprazole concentrations consistent with the 21-day initiation regimen. Therefore, a single dose of ALNCD with a single 30-mg oral dose of aripiprazole provides an alternative initiation regimen for starting AL.
Assuntos
Antipsicóticos/administração & dosagem , Antipsicóticos/farmacocinética , Aripiprazol/administração & dosagem , Aripiprazol/farmacocinética , Esquizofrenia/sangue , Esquizofrenia/tratamento farmacológico , Administração Oral , Adulto , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacocinética , Método Duplo-Cego , Esquema de Medicação , Feminino , Humanos , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
Plasmodium falciparum is an obligate intracellular pathogen responsible for worldwide morbidity and mortality. This parasite establishes a parasitophorous vacuole within infected red blood cells wherein it differentiates into multiple daughter cells that must rupture their host cells to continue another infectious cycle. Using atomic force microscopy, we establish that progressive macrostructural changes occur to the host cell cytoskeleton during the last 15 h of the erythrocytic life cycle. We used a comparative proteomics approach to determine changes in the membrane proteome of infected red blood cells during the final steps of parasite development that lead to egress. Mass spectrometry-based analysis comparing the red blood cell membrane proteome in uninfected red blood cells to that of infected red blood cells and postrupture vesicles highlighted two temporally distinct events; (Hay, S. I., et al. (2009). A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med. 6, e1000048) the striking loss of cytoskeletal adaptor proteins that are part of the junctional complex, including α/ß-adducin and tropomyosin, correlating temporally with the emergence of large holes in the cytoskeleton seen by AFM as early ~35 h postinvasion, and (Maier, A. G., et al. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134, 48-61) large-scale proteolysis of the cytoskeleton during rupture ~48 h postinvasion, mediated by host calpain-1. We thus propose a sequential mechanism whereby parasites first remove a selected set of cytoskeletal adaptor proteins to weaken the host membrane and then use host calpain-1 to dismantle the remaining cytoskeleton, leading to red blood cell membrane collapse and parasite release.
Assuntos
Citoesqueleto/metabolismo , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Plasmodium falciparum/fisiologia , Proteínas de Ligação a Calmodulina/metabolismo , Calpaína/metabolismo , Membrana Celular/metabolismo , Membrana Celular/parasitologia , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Eritrócitos/metabolismo , Eritrócitos/ultraestrutura , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Merozoítos , Microscopia de Força Atômica , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteólise , Espectrometria de Massas em TandemRESUMO
We report an innovative multiplexed liquidchromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS)-based assay for rapidly measuring a large number of disease specific protein biomarkers in human serum. Furthermore, this approach uses stable isotope dilution methodology to reliably quantify candidate protein biomarkers. Human serum was diluted using a stable isotope labeled proteome (SILAP) standard prepared from the secretome of pancreatic cell lines, subjected to immunoaffinity removal of the most highly abundant proteins, trypsin digested, and analyzed by LC-MRM/MS. The method was found to be precise, linear, and specific for the relative quantification of 72 proteins when analyte response was normalized to the relevant internal standard (IS) from the SILAP. The method made it possible to determine statistically different concentrations for three proteins (cystatin M, IGF binding protein 7, and villin 2) in control and pancreatic cancer patient samples. This method proves the feasibility of using a SILAP standard in combination with stable isotope dilution LC-MRM/MS analysis of tryptic peptides to compare changes in the concentration of candidate protein biomarkers in human serum.
Assuntos
Biomarcadores Tumorais/sangue , Proteínas Sanguíneas/metabolismo , Carcinoma Ductal Pancreático/sangue , Neoplasias Pancreáticas/sangue , Sequência de Aminoácidos , Biomarcadores Tumorais/química , Biomarcadores Tumorais/isolamento & purificação , Proteínas Sanguíneas/química , Proteínas Sanguíneas/isolamento & purificação , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cromatografia Líquida/normas , Humanos , Marcação por Isótopo/métodos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteólise , Padrões de Referência , Espectrometria de Massas em Tandem/normas , Tripsina/químicaRESUMO
Autotaxin (ATX) is a lysophospholipase D that is the main enzyme responsible for generating LPA in body fluids. Although ATX was isolated from a conditioned medium of melanoma cells, later it was discovered to play a critical role in vascular and neuronal development. ATX has also been implicated in primary brain tumor, fibrosis, and rheumatoid arthritis, as well as neurological diseases such as multiple sclerosis, Alzheimer's disease, and neuropathic pain. As ATX and LPA levels are increased upon neuronal injury, a selective ATX inhibitor could provide a new approach to treat neuropathic pain. Herein we describe the discovery of a novel series of nonzinc binding reversible ATX inhibitors, particularly a potent, selective, orally bioavailable, brain-penetrable tool compound BIO-32546, as well as its synthesis, X-ray cocrystal structure, pharmacokinetics, and in vivo efficacy.
RESUMO
BACKGROUND: Aripiprazole lauroxil (AL) is a long-acting injectable antipsychotic approved for treatment of schizophrenia in adults. Approved AL doses and dosing regimens include 441 mg monthly, 662 mg monthly, and 882 mg monthly or every 6 weeks (q6wk), as well as the most recently approved dose, 1064 mg, administered every 2 months. OBJECTIVE: Pharmacokinetics, safety, and tolerability of an AL regimen with a dose interval of every 2 months (1064 mg) were compared with two other regimens available as monthly and q6wk options. METHODS: This study evaluated pharmacokinetics of AL given at a higher dosage strength (1064 mg) and at a longer dose interval (every 8 weeks [q8wk]) than previously studied. Patients with schizophrenia or schizoaffective disorder entering this 44-week, phase I, open-label, multicenter study had no recent exposure to aripiprazole and were maintained on other oral antipsychotics throughout the study. Patients were randomized to one of three AL dose regimens for 24 weeks (four 1064-mg injections [q8wk], five 882-mg injections [q6wk], or seven 441-mg injections [q4wk], with the last AL exposure at week 24). Oral aripiprazole was prohibited. Patients were followed for an additional 20 weeks to assess terminal aripiprazole plasma concentrations and ongoing safety. Plasma concentration samples were obtained at regular intervals to provide pharmacokinetic data for the duration of AL exposure and to measure persistence of plasma aripiprazole concentrations after AL discontinuation. RESULTS: Eligible patients received AL 1064 mg q8wk (n = 35), 882 mg q6wk (n = 34), or 441 mg q4wk (n = 35). Overall, 103/104 (99.0%) patients were taking concomitant non-aripiprazole oral antipsychotic medications during the study. All three AL dose regimens provided continuous exposure to aripiprazole. Mean aripiprazole concentrations from the 1064-mg q8wk regimen were comparable to the 882-mg q6wk regimen and higher than the 441-mg q4wk regimen. Overall incidence by group of any adverse events (AEs) throughout the study was 68.6% (1064 mg q8wk), 50.0% (882 mg q6wk), and 65.7% (441 mg q4wk). The most common AE across regimens was injection-site pain (range 8.6%-11.4%). Serious AEs were reported by eight patients (all but one [increased psychosis in one patient, 441-mg q4wk group] considered unrelated to study drug). Discontinuations due to AEs were reported for 2.9%, 11.8%, and 5.7% of patients receiving the 8-, 6-, and 4-week regimens, respectively. AEs of akathisia, dyskinesia, and dystonia occurred in 2.9%, 8.6%, and 5.7% of patients in the 1064-mg q8wk group, 8.8%, 0%, and 2.9% in the 882-mg q6wk group, and 8.6%, 0%, and 0% in the 441-mg q4wk group, respectively. CONCLUSIONS: AL 1064 mg q8wk provided continuous exposure to aripiprazole throughout the 8-week dosing interval and had a safety profile consistent with the 4- and 6-week regimens. These findings were used to support FDA approval of the 1064-mg dose administered every 2 months. REGISTRATION: Clinicaltrials.gov: NCT02320032.
Assuntos
Antipsicóticos/administração & dosagem , Aripiprazol/administração & dosagem , Transtornos Psicóticos/tratamento farmacológico , Esquizofrenia/tratamento farmacológico , Adulto , Antipsicóticos/efeitos adversos , Antipsicóticos/farmacocinética , Aripiprazol/efeitos adversos , Aripiprazol/farmacocinética , Preparações de Ação Retardada , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Humanos , Injeções Intramusculares , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
BACKGROUND: Aripiprazole lauroxil (AL), a long-acting injectable antipsychotic for the treatment of schizophrenia in adults, can be started with either 21 days of daily oral aripiprazole supplementation or a 1-day initiation regimen consisting of a single injection of a NanoCrystal® Dispersion formulation of AL (ALNCD) and a single dose of 30 mg oral aripiprazole. This phase I study assessed the pharmacokinetics and safety of deltoid versus gluteal ALNCD injections. METHODS: Patients with schizophrenia or schizoaffective disorder (N = 47) were randomized 1:1 to receive a single intramuscular dose of ALNCD in the deltoid or gluteal muscle. Plasma samples were collected over 85 days to measure ALNCD concentration by injection site. Relative aripiprazole bioavailability for deltoid versus gluteal injection was assessed based on area under the curve (AUC∞ and AUClast) and maximum concentration (Cmax) values. Adverse events were monitored throughout the study. RESULTS: Plasma aripiprazole concentrations after a single ALNCD injection were comparable between deltoid and gluteal administration. Mean maximum plasma aripiprazole concentrations were 196.1 ng/ml (deltoid) and 175.0 ng/ml (gluteal). Exposure to aripiprazole was similar, with mean AUC∞ values of 6591 day × ng/ml for deltoid and 6437 day × ng/ml for gluteal. Aripiprazole bioavailability was not significantly different between injection sites. ALNCD administration in the deltoid or gluteal muscle was well tolerated, with similar safety profiles at both sites. CONCLUSION: ALNCD demonstrated similar exposure and safety profiles between the two administration sites, suggesting that ALNCD can be given in either the gluteal or the deltoid muscles as a component of the 1-day initiation regimen for AL.
RESUMO
INTRODUCTION: Diroximel fumarate (DRF) is a novel oral fumarate in development for patients with relapsing forms of multiple sclerosis (MS). Clinical findings from the DRF development program suggest that rates of gastrointestinal (GI) treatment-emergent adverse events (TEAEs) and discontinuation due to GI TEAEs are low, based on clinical and real-world observations of other fumaric acid esters, including dimethyl fumarate (DMF). The incidence of GI TEAEs varies from 40 to 88% in clinical and real-world studies of DMF. The objective of this study is to present GI tolerability findings from the EVOLVE-MS-1 study and present biologic hypotheses for the improved GI properties of DRF. METHODS: GI TEAEs and treatment discontinuation because of GI TEAEs were assessed in DRF-treated patients with relapsing-remitting MS who were participating in the ongoing, 96-week, open-label, phase 3 EVOLVE-MS-1 study. RESULTS: As of March 30, 2018, a total of 696 patients were enrolled in EVOLVE-MS-1. GI TEAEs were reported in 30.9% (215/696) of patients; the vast majority (96%; 207/215) experienced events that were mild or moderate in severity. When GI AEs did occur, they occurred early in treatment, resolved (88.8%; 191/215), and were of short duration [median 7.5 (range 1-87) days] in most patients. GI TEAEs led to < 1% of patients discontinuing treatment. CONCLUSIONS: We suggest that the distinct chemical structure of DRF contributes to the observed low rates of GI TEAEs and GI-associated treatment discontinuations, possibly due to a combination of several factors. We hypothesize that these factors may include less reactivity with off-target proteins and/or lower production of a methanol leaving group that may contribute to GI irritation. A direct comparison of GI tolerability with DRF versus DMF is being evaluated in the EVOLVE-MS-2 study. TRIAL REGISTRATION: ClinicalTrials.gov number NCT02634307. FUNDING: Alkermes Inc. (Waltham, MA, USA) and Biogen (Cambridge, MA, USA).
Assuntos
Fumarato de Dimetilo/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Fumaratos/efeitos adversos , Gastroenteropatias/induzido quimicamente , Gastroenteropatias/terapia , Imunossupressores/efeitos adversos , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Adulto , Fumarato de Dimetilo/uso terapêutico , Feminino , Fumaratos/uso terapêutico , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND AND OBJECTIVES: Aripiprazole lauroxil (AL), a long-acting injectable antipsychotic for the treatment of schizophrenia, requires 21 days of oral aripiprazole supplementation upon initiation (21-day initiation regimen). An alternative 1-day initiation regimen utilizing a nano-crystalline milled dispersion of AL (ALNCD) plus a single 30 mg oral aripiprazole dose achieved aripiprazole concentrations associated with therapeutic doses of aripiprazole in the same time frame as the 21-day initiation regimen when starting AL (441 or 882 mg). A population pharmacokinetic (PopPK) model was developed to describe aripiprazole pharmacokinetics following administration of ALNCD, AL and oral aripiprazole, and evaluate dosing scenarios likely to be encountered in clinical practice. METHODS: In total, 12,768 plasma aripiprazole concentrations from 343 patients (from 4 clinical studies) were included in the PopPK analysis and used to construct the model. RESULTS: Concomitant administration of the 1-day initiation regimen with all approved AL dosing regimens (441, 662, or 882 mg monthly, 882 mg every 6 weeks, or 1064 mg every 2 months) is predicted to achieve aripiprazole concentrations associated with therapeutic doses of AL using the 21-day initiation regimen within 4 days, maintaining these concentrations until the next AL dose. Administration of the first AL injection 10 days after the 1-day initiation regimen resulted in median aripiprazole concentrations just before the second dose of AL ≥ 77% of that when coadministered on the same day. Coadministration of AL with a single ALNCD injection was predicted to be effective in rapidly re-establishing concentrations associated with therapeutic doses of AL following dose delay. CONCLUSIONS: Model-based simulations demonstrate that the 1-day initiation regimen is suitable for starting treatment with all AL doses, allowing a window of ≤ 10 days between initiation and AL administration. ALNCD may also be used to re-establish concentrations associated with therapeutic doses of AL in conjunction with a delayed AL dose.
Assuntos
Aripiprazol/farmacocinética , Simulação por Computador , Modelos Biológicos , Administração Oral , Antipsicóticos/sangue , Antipsicóticos/farmacocinética , Aripiprazol/administração & dosagem , Aripiprazol/sangue , Aripiprazol/química , Preparações de Ação Retardada/farmacocinética , Esquema de Medicação , Humanos , Injeções Intramusculares , Nanopartículas/químicaRESUMO
BACKGROUND: Aripiprazole lauroxil (AL) is a long-acting injectable medication approved for the treatment of schizophrenia. Current AL regimens are 441 mg, 662 mg, and 882 mg administered monthly (every 4 weeks [q4wk]), or 882 mg administered every 6 weeks (q6wk). OBJECTIVE: We examined the feasibility of a 2-month (every 8 weeks [q8wk]) dosing interval of AL in a phase I open-label pharmacokinetic study investigating AL 1064 mg administered q8wk for 24 weeks, followed by 20 weeks of safety and pharmacokinetic measurements (ClinicalTrials.gov ID: NCT02320032). Second, a population pharmacokinetic model (referred to as the 2MPopPK model) was generated using data collected from the present trial, as well as data obtained from earlier studies. METHODS: The phase I study included patients with schizophrenia or schizoaffective disorder maintained on an oral antipsychotic (n = 140) who were assigned to one of three groups: AL 441 mg q4wk, AL 882 mg q6wk, or AL 1064 mg q8wk, with a total of seven, five, or four injections administered, respectively. No oral aripiprazole lead-in supplementation was administered and patients continued on maintenance oral antipsychotics. Pharmacokinetic samples were collected at various time points during the 24-week study period and the 20-week follow-up period. Plasma concentrations obtained from the phase I study were analyzed using non-compartmental methods. Additionally, the data were combined with data collected from prior studies to develop the 2MPopPK model. RESULTS: Following the final injection of AL in the phase I study, maximum aripiprazole concentrations were achieved 24.4-35.2 days after the last dose and persisted for the duration of the study. The mean C avg,ss values were 125.8 ng/ml, 131.1 ng/ml, and 140.7 ng/ml for the 441 mg q4wk, 882 mg q6wk, and 1064 mg q8wk doses, respectively. The mean elimination half-life of aripiprazole following the last dose was 53.9 days for the 1064 mg dose, 55.1 days for the 882 mg dose, and 57.2 days for the 441 mg dose. The 2MPopPK dataset included 14,524 aripiprazole concentrations from 700 patients with schizophrenia. The duration of absorption of aripiprazole was estimated as 43 days (95% confidence interval [CI] 42-45 days), which was preceded by a 3.2-day lag time (95% CI 3.0-3.5 days) for a total duration of input into the systemic circulation of 46 days following intramuscular administration of AL. Multiple-dose simulations showed that the 1064 mg q8wk regimen provides aripiprazole concentrations within the concentration range associated with 441 mg and 882 mg q4wk doses previously demonstrated to be efficacious in a phase III study. CONCLUSION: These data from the phase I study and the 2MPopPK model support the suitability of using the AL 1064 mg dose as a 2-month (q8wk) dose interval option for the treatment of schizophrenia.
Assuntos
Antipsicóticos/administração & dosagem , Antipsicóticos/farmacocinética , Aripiprazol/administração & dosagem , Aripiprazol/farmacocinética , Transtornos Psicóticos/tratamento farmacológico , Esquizofrenia/tratamento farmacológico , Administração Oral , Adulto , Antipsicóticos/efeitos adversos , Aripiprazol/efeitos adversos , Esquema de Medicação , Quimioterapia Combinada , Estudos de Viabilidade , Feminino , Seguimentos , Humanos , Injeções , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Transtornos Psicóticos/sangue , Esquizofrenia/sangueRESUMO
OBJECTIVE: Pancreatic stellate cells (PSCs) are important players in pancreatic fibrosis and are major contributors to the extracellular matrix proteins observed with the stromal response characteristic of pancreatic ductal adenocarcinoma (PDAC). Pancreatic stellate cells are also believed to secrete soluble factors that promote tumor progression; however, no comprehensive analysis of the PSC proteome in either the quiescent or the activated state has been reported. METHODS: Using 2-dimensional tandem mass spectrometry and the RLT-PSC cell line, we present the first comprehensive study describing and comparing the quiescent and activated human PSC-secreted proteomes. RESULTS: Very few proteins are secreted in the quiescent state. In stark contrast, activated PSCs secreted a vast array of proteins. Many of these proteins differed from those secreted by PDAC-derived cell lines. Proteins associated with wound healing, proliferation, apoptosis, fibrosis, and invasion were characterized. Selected proteins were verified in human tissue samples from PDAC, dysplastic pancreas, and normal pancreas using Western blot analysis and immunohistochemical staining. CONCLUSIONS: Our study represents the first comprehensive analysis of proteins secreted by PSCs. These findings lay the foundation for characterizing PSC-derived proteins involved in stroma-tumor interactions and the promotion of pancreatitis and PDAC.
Assuntos
Pâncreas/metabolismo , Proteoma/análise , Proteômica/métodos , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Humanos , Imuno-Histoquímica , Pâncreas/citologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteoma/metabolismo , Espectrometria de Massas em TandemRESUMO
Rat intestinal epithelial cells that permanently express the cyclooxygenase-2 (COX-2) gene (RIES cells) were used to investigate COX-2-mediated arachidonic acid (AA) metabolism. A targeted chiral lipidomics approach was employed to quantify AA metabolites that were secreted by the cells into the culture media. When intact RIES cells were treated with calcium ionophore A-23187 (1 microM) for 1 h, 11-(R)-hydroxyeicosatetraenoic acid (HETE) was the most abundant metabolite, followed by prostaglandin (PG) E 2, 15-(S)-HETE, 15-oxo-eicosatetraenoic acid (ETE), and 15-(R)-HETE. Incubation for a further 23 h after the calcium ionophore was removed resulted in a substantial increase in PGE 2 concentrations while HETE and 15-oxo-ETE concentrations decreased to almost undetectable levels. A similar metabolic profile was observed when RIES cells were treated with increasing concentrations of AA for 24 h. Incubation of the RIES cells with 10 microM AA revealed that maximal concentrations of 11-(R)-HETE, 15-(S)-HETE, and 15-oxo-ETE occurred after 10 min of incubation when the 15-( S)-HETE concentrations were approximately twice that of PGE 2. There was a gradual decrease in the concentrations of HETE and 15-oxo-ETE over time, whereas PGE 2 concentrations increased steadily until they reached a maximum after 24 h of incubation. The ratio of PGE 2 to 15-(S)-HETE was then approximately 20:1. 15-(S)-HETE and 15-oxo-ETE concentrations declined in the cell media during prolonged incubations with pseudo-first-order rate constants of 0.0121 and 0.0073 min(-1), respectively. 15-(S)-HETE was shown to undergo metabolism primarily to 15-oxo-ETE, which was further metabolized to a glutathione (GSH) adduct. The GSH adduct of 15-oxo-ETE was further metabolized in the extracellular milieu to a cysteinylglycine adduct. Thus, we have established for the first time that 15-oxo-ETE can be formed biosynthetically from AA, that 15-(S)-HETE is its immediate precursor, and that 15-oxo-ETE forms a GSH adduct. For ionophore-A-23187-stimulated cells and at early time points for AA-stimulated cells, 11-(R)-HETE was the major eicosanoid to be secreted into the media. Adding increasing concentrations of AA to cells in culture made it possible to estimate with surprising accuracy endogenous eicosanoid production using regression analyses. Thus, after 24 h in the absence of added AA, 11-(R)-HETE and 15-(R)-HETE were estimated to be present at concentrations close to the detection limit of our very sensitive assay. These data further highlight the importance of endogenous COX-2-mediated lipid peroxidation and illustrate the necessity to monitor eicosanoid formation from endogenous stores of AA in cell culture experiments.