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1.
Drug Metab Dispos ; 2023 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-38050097

RESUMO

Drug-drug interaction (DDI) assessment of therapeutic peptides is an evolving area. The industry generally follows DDI guidelines for small molecules, but the translation of data generated with commonly used in vitro systems to in vivo is sparse. In the current study, we investigated the ability of advanced human hepatocyte in vitro systems namely HepatoPac, spheroids, and Liver-on-a-chip to assess potential changes in regulation of CYP1A2, CYP2B6, CYP3A4, SLCO1B1 and ABCC2 in the presence of selected therapeutic peptides, proteins, and small molecules. The peptide NN1177, a glucagon and GLP-1 receptor co-agonist, did not suppress mRNA expression or activity of CYP1A2, CYP2B6, and CYP3A4 in HepatoPac, spheroids, or Liver-on-a-chip; these findings were in contrast to the data obtained in sandwich cultured hepatocytes. No effect of NN1177 on SLCO1B1 and ABCC2 mRNA was observed in any of the complex systems. The induction magnitude differed across the systems (e.g., rifampicin induction of CYP3A4 mRNA ranged from 2.8-fold in spheroids to 81.2-fold in Liver-on-a-chip). Small molecules, obeticholic acid and abemaciclib, showed varying responses in HepatoPac, spheroids and Liver-on-a-chip, indicating a need for EC50 determinations to fully assess translatability data. HepatoPac, the most extensively investigated in this study (3 donors), showed high potential to investigate DDIs associated with CYP regulation by therapeutic peptides. Spheroids and Liver-on-a-chip were only assessed in one hepatocyte donor and further evaluations are required to confirm their potential. This study establishes an excellent foundation towards the establishment of more clinically-relevant in vitro tools for evaluation of potential DDIs with therapeutic peptides. Significance Statement At present, there are no guidelines for drug-drug interaction (DDI) assessment of therapeutic peptides. Existing in vitro methods recommended for assessing small molecule DDIs do not appear to translate well for peptide drugs, complicating drug development for these moieties. Here, we establish evidence that complex cellular systems have potential to be used as more clinically-relevant tools for the in vitro DDI evaluation of therapeutic peptides.

2.
BioDrugs ; 33(6): 673-681, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31549312

RESUMO

BACKGROUND: Nonacog beta pegol (N9-GP, Refixia®, Rebinyn®) is a human recombinant coagulation factor IX (rFIX) conjugated to a 40-kDa polyethylene glycol (PEG) moiety. PEGylation significantly prolongs the circulation half-life compared with conventional FIX replacement treatments, resulting in higher FIX levels. Although there is extensive clinical experience with PEGylated molecules, the potential for abnormal and/or indefinite PEG accumulation during long-term treatment and the hypothetical impact on long-term safety is still under discussion. AIM: The aim of this study was to examine plasma PEG concentrations in children, adolescents and adults undergoing once-weekly intravenous prophylactic treatment with N9-GP for up to 6.5 years. METHODS: Plasma samples were collected as part of the PARADIGM clinical development programme (PARADIGM 2/4 [NCT01333111 and NCT01395810] and PARADIGM 5 [NCT01467427]). Proton nuclear magnetic resonance (1H-NMR) was used to measure plasma PEG concentrations. RESULTS: Steady-state plasma PEG concentrations were reached approximately 6 months after initiation of weekly prophylactic treatment with 40 IU/kg N9-GP. Mean steady-state plasma PEG concentrations were 5.6 µg/mL in children ≤ 12 years old at enrolment (PARADIGM 5) and 5.3 µg/mL in adolescents/adults > 12 years old (PARADIGM 2/4). Plasma PEG concentrations tended to be lower in younger children < 7 years old (mean 4.6 µg/mL). There was a correlation between plasma PEG and FIX activity levels in all age groups. CONCLUSION: PEG steady-state plasma levels were maintained for up to 6.5 years during continuous prophylactic treatment and PEG levels correlated with FIX activity. Apart from the initial increase to steady state, no further systemic PEG accumulation was observed.


Assuntos
Fator IX/uso terapêutico , Plasma/química , Polietilenoglicóis/química , Adolescente , Adulto , Criança , Fator IX/química , Humanos , Masculino , Polietilenoglicóis/uso terapêutico , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico
3.
Sci Rep ; 9(1): 7399, 2019 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-31068648

RESUMO

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

4.
AAPS J ; 19(6): 1804-1813, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28853055

RESUMO

The combination of morphine and gabapentin has shown to be promising for managing postoperative pain but finding the right dose for the combination has proven to be a challenge. The purpose of this study was to quantitatively characterize the pharmacodynamic interaction between the two drugs and to identify the optimal concentration-effect relationship of the combination. Information regarding plasma concentrations and von Frey withdrawal thresholds following incisional surgery on Sprague Dawley rats, after administration of morphine, gabapentin, or their combination was available from published studies. The combined pharmacodynamic effect of morphine and gabapentin was analyzed and linked to drug plasma concentrations via a response surface approach using non-linear mixed-effect modeling. Full reversal of withdrawal thresholds for the pain stimulation to presurgery values was estimated at morphine plasma concentration of 435.1 ng/mL. Co-administration of up to 40 µg/mL of gabapentin led to a reduction of the needed morphine concentration down to 307.5 ng/mL (~ 29% reduction). Combination of concentration ranges of gabapentin between 20 and 40 µg/mL with any morphine concentrations between 100 and 600 ng/mL were found to lead up to 50% increased effect relatively to the effect attained by morphine alone. This study highlights the importance of finding the right combination in multimodal analgesia and demonstrates the usefulness of the response surface approach for the study of pharmacodynamic interactions. The proposed pharmacokinetic-pharmacodynamic model may provide the basis for a rational clinical trial design with the aim to identify the optimal dose combination ratios in humans.


Assuntos
Aminas/farmacologia , Analgésicos/farmacologia , Ácidos Cicloexanocarboxílicos/farmacologia , Morfina/farmacologia , Ácido gama-Aminobutírico/farmacologia , Animais , Relação Dose-Resposta a Droga , Interações Medicamentosas , Gabapentina , Dor Pós-Operatória/tratamento farmacológico , Ratos , Ratos Sprague-Dawley
5.
Sci Rep ; 7(1): 4792, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28684771

RESUMO

Pain is a severe and debilitating complication of metastatic bone cancer. Current analgesics do not provide sufficient pain relief for all patients, creating a great need for new treatment options. The Src kinase, a non-receptor protein tyrosine kinase, is implicated in processes involved in cancer-induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p.o.) from day 7 after inoculation of MRMT-1 mammary carcinoma cells significantly attenuated movement-evoked and non-evoked pain behaviour in cancer-bearing rats. Radiographic - and microcomputed tomographic analyses showed significantly higher relative bone density and considerably preserved bone micro-architecture in the dasatinib treated groups, suggesting a bone-preserving effect. This was supported by a significant reduction of serum TRACP 5b levels in cancer-bearing rats treated with 15 mg/kg dasatinib. Furthermore, immunoblotting of lumbar spinal segments showed an increased activation of Src but not the NMDA receptor subunit 2B. These findings support a role of dasatinib as a disease modifying drug in pain pathologies characterized by increased osteoclast activity, such as bone metastases.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Dasatinibe/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Dor/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Neoplasias Ósseas/complicações , Neoplasias Ósseas/genética , Neoplasias Ósseas/secundário , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Linhagem Celular Tumoral , Feminino , Genes Reporter , Luciferases/genética , Luciferases/metabolismo , Neoplasias Mamárias Animais/complicações , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteossarcoma/complicações , Osteossarcoma/genética , Osteossarcoma/secundário , Dor/etiologia , Dor/genética , Dor/patologia , Manejo da Dor/métodos , Medição da Dor/métodos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Fosfatase Ácida Resistente a Tartarato/antagonistas & inibidores , Fosfatase Ácida Resistente a Tartarato/sangue , Fosfatase Ácida Resistente a Tartarato/genética , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismo
6.
Basic Clin Pharmacol Toxicol ; 120(5): 491-497, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27863000

RESUMO

Coated activated charcoal haemoperfusion (CAC-HP) is a well-known treatment modality. Case reports have revealed conflicting results about the efficacy of CAC-HP in the treatment of amitriptyline (AT) poisoning, and no randomized clinical trials have been identified in the literature. This study aimed at quantifying the efficacy of modern CAC-HP as an adjunctive treatment of AT intoxication compared with standard care alone. Fourteen female Danish landrace pigs were randomized to either standard care or standard care plus 4 hr of CAC-HP. The pigs were anaesthetized, and vital parameters were continuously recorded. Amitriptyline infusion (7.5 mg/kg) was completed in 20 min. Thirty minutes after AT infusion, activated charcoal was instilled orally in both groups. In the intervention group, CAC-HP was initiated 60 min. after AT infusion. Blood and urine samples were collected as were vital parameters at specific time intervals. The protocol was approved by the Danish Experimental Animal Expectorate and complied with the NIH guide for care and use of laboratory animals. Data were managed according to the ARRIVE guidelines. No statistical significant differences between intervention and control groups were found when analysing for differences in AT levels in plasma at any time-point. Furthermore, significant differences between the control and intervention groups in regard to vital parameters could not be found either. In our animal model, the addition of CAC-HP did not improve the clearance of AT compared with standard treatment alone. We suggest that the effect of modern CAC-HP as a treatment modality in AT-poisoned human patients may be inadequate.


Assuntos
Amitriptilina/intoxicação , Antídotos/administração & dosagem , Carvão Vegetal/administração & dosagem , Hemoperfusão/métodos , Amitriptilina/farmacocinética , Animais , Antidepressivos Tricíclicos/farmacocinética , Antidepressivos Tricíclicos/intoxicação , Feminino , Hemodinâmica/efeitos dos fármacos , Distribuição Aleatória , Suínos , Fatores de Tempo , Resultado do Tratamento
7.
Pharm Res ; 33(11): 2630-43, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27380190

RESUMO

PURPOSE: The combination of morphine and gabapentin seems promising for the treatment of postoperative and neuropathic pain. Despite the well characterised pharmacodynamic interaction, little is known about possible pharmacokinetic interactions. The aim of this study was to evaluate whether co-administration of the two drugs leads to modifications of their pharmacokinetic profiles. METHODS: The pharmacokinetics of morphine, morphine-3-glucuronide and gabapentin were characterised in rats following subcutaneous injections of morphine, gabapentin or their combination. Non-linear mixed effects modelling was applied to describe the pharmacokinetics of the compounds and possible interactions. RESULTS: The plasma-concentration-time profiles of morphine and gabapentin were best described using a three- and a one-compartment disposition model respectively. Dose dependencies were found for morphine absorption rate and gabapentin bioavailability. Enterohepatic circulation of morphine-3-glucuronide was modelled using an oscillatory model. The combination did not lead to pharmacokinetic interactions for morphine or gabapentin but resulted in an estimated ~33% diminished morphine-3-glucuronide formation. CONCLUSIONS: The finding of a lack of pharmacokinetic interaction strengthens the notion that the combination of the two drugs leads to better efficacy in pain treatment due to interaction at the pharmacodynamic level. The interaction found between gabapentin and morphine-3-glucuronide, the latter being inactive, might not have any clinical relevance.


Assuntos
Aminas/química , Aminas/farmacocinética , Analgésicos/química , Ácidos Cicloexanocarboxílicos/química , Ácidos Cicloexanocarboxílicos/farmacocinética , Modelos Biológicos , Morfina/química , Morfina/farmacocinética , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/farmacocinética , Aminas/metabolismo , Analgésicos/metabolismo , Analgésicos/farmacocinética , Animais , Ácidos Cicloexanocarboxílicos/metabolismo , Interações Medicamentosas , Circulação Êntero-Hepática , Gabapentina , Glucuronídeos/metabolismo , Morfina/metabolismo , Dinâmica não Linear , Ratos Sprague-Dawley , Espectrometria de Massas em Tandem , Ácido gama-Aminobutírico/metabolismo
8.
Anal Bioanal Chem ; 408(9): 2293-301, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26832729

RESUMO

The paper presents an analytical method for quantification of low molecular weight (LMW) selenium compounds in human plasma based on liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) and post column isotope dilution-based quantification. Prior to analysis, samples were ultrafiltrated using a cut-off value of 3000 Da. The method was validated in aqueous solution as well as plasma using standards of selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenite, and the selenosugar Se-methylseleno-N-acetylgalactosamine (SeGal) for linearity, precision, recoveries, and limits of detection and quantitation with satisfactory results. The method was applied for analysis of a set of plasma samples from cancer patients receiving selenite treatment in a clinical trial. Three LMW selenium compounds were observed. The main compounds, SeGal and selenite were tentatively identified by retention time matching with standards in different chromatographic systems, while the third minor compound was not identified. The identity of the selenosugar was verified by ESI-MS-MS product ion scanning, while selenite was identified indirectly as the glutathione (GSH) reaction product, GS-Se-SG.


Assuntos
Antineoplásicos/administração & dosagem , Ácido Selenioso/administração & dosagem , Selênio/sangue , Antineoplásicos/uso terapêutico , Cromatografia Líquida , Humanos , Limite de Detecção , Espectrometria de Massas , Peso Molecular , Neoplasias/tratamento farmacológico , Padrões de Referência , Ácido Selenioso/uso terapêutico
9.
Anal Chim Acta ; 905: 93-9, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26755142

RESUMO

A fully integrated and automated electromembrane extraction LC-MS (EME-LC-MS) system has been developed and characterized. Hyphenation of a flow-flow EME probe to LC-MS was accomplished by using an in-built 10-port switching valve of the LC-MS system. The 10-port switching valve decoupled the high pressure of the UHPLC-system from the low pressure required for operation of the EME-probe by automated switching between a sample extraction/analysis and a sample load position. In the sample load position the extracted analytes were loaded into a HPLC sample loop. By switching the valve to the sample extraction/analysis position the setup allowed simultaneous analysis of previously loaded analytes while extracting a new sample. Performance of the system was characterized with respect to precision and linearity (RSD < 2.5%, R(2): 0.998) and the setup was applied for studying the in-vitro metabolism of methadone by rat liver microsomes. As the metabolic reaction proceeded, methadone and its metabolites were extracted and analyzed in parallel by LC-MS using either isocratic or gradient elution. Compared to a conventional in-vitro metabolism analysis based on protein precipitation followed by LC-MS analysis the fully automated EME-LC-MS system offers a significant time saving and in addition demonstrates increased sensitivity as the analytes were automatically enriched during the extraction process. The experiment revealed 6 to 16 times higher S/N ratios of the EME-LC-MS method compared to protein precipitation followed by LC-MS and thus concomitantly lower LOD and LOQ. The setup integrates a complete analytical workflow of rapid extraction, enrichment, separation and detection of analytes in a fully automated manner. These attributes make the developed system a powerful alternative approach for a wide range of analytical applications.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas/métodos , Membranas Artificiais
10.
Chem Res Toxicol ; 29(1): 96-100, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26669514

RESUMO

2,5-Dimethoxy-N-benzylphenethylamines (NBOMes) are very potent 5-HT2AR agonists. Illicit use of these psychedelic compounds has emerged in recent years, and several fatalities have been linked to their recreational use. In its [(11)C]-labeled form, one NBOMe (25B-NBOMe) was recently developed as a PET-ligand for clinical investigations of 5HT2AR ([(11)C]Cimbi-36). Herein, we have identified the phase I and phase II metabolites of 25B-NBOMe in pigs as well as in humans. We find that the primary route of metabolism is 5'-demethylation, followed by conjugation to glucuronic acid. Carbon-11 labeling of 25B-NBOMe in three different positions followed by in vivo evaluation in pigs and humans corroborated these findings.


Assuntos
Alucinógenos/metabolismo , Fenetilaminas/metabolismo , Suínos/metabolismo , Animais , Alucinógenos/química , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Fenetilaminas/química , Tomografia por Emissão de Pósitrons
11.
Xenobiotica ; 46(2): 99-107, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26207435

RESUMO

1. The metabolism of midazolam was investigated in vivo in locusts in order to evaluate the presence of an enzyme with functionality similar to human CYP3A4/5. 2. Hydroxylated metabolites of midazolam identical to human metabolites were detected in locusts and the apparent affinities (Km values) were in the same range as reported in humans (in locusts: 7-23 and 33-85 µM for the formation of the 1'-OH and 4-OH metabolites, respectively). 3. The formation of hydroxylated metabolites could successfully be inhibited by co-administration of ketoconazole, a known CYP3A4/5 inhibitor. 4. Besides phase I metabolites, a number of conjugated metabolites were detected using high-resolution mass spectrometry. The most abundant metabolites detected were structurally identified by (1)H NMR as two N-glucosides. NMR analysis strongly suggested that the glycosylation occurred at the two nitrogens (either one in each case) of the imidazole ring. 5. Distribution of midazolam and the glucose conjugates were successfully measured using desorption electrospray mass spectrometry imaging revealing time-dependent changes in distribution over time. 6. In conclusion, it appears that an enzyme with functionality similar to human CYP3A4/5 is present in locusts. However, it appears that conjugation with glucose is the main detoxification pathway of midazolam in locusts.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Gafanhotos/metabolismo , Proteínas de Insetos/metabolismo , Midazolam/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Inibidores do Citocromo P-450 CYP3A/administração & dosagem , Glicosilação , Cetoconazol/administração & dosagem , Imageamento por Ressonância Magnética , Masculino
12.
Anal Chem ; 87(11): 5774-81, 2015 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-25920035

RESUMO

A simple to construct and operate, "dip-in" electromembrane extraction (EME) probe directly coupled to electrospray ionization-mass spectrometry (ESI-MS) for rapid extraction and real time analysis of various analytes was developed. The setup demonstrated that EME-MS can be used as a viable alternative to conventional protein precipitation followed by liquid chromatography-mass spectrometry (LC-MS) for studying drug metabolism. Comparison of EME-MS with LC-MS for drug metabolism analysis demonstrated for the first time that real time extraction of analytes by EME is possible. Metabolism kinetics were investigated for three different drugs: amitriptyline, promethazine, and methadone. By comparing the EME-MS extraction profiles of the drug substances and formed drug metabolites with the metabolism profiles obtained by conventional protein precipitation followed by LC-MS good correlation was obtained with only very limited time delay in the extraction. The results indicate that, by tuning the electromembrane properties, for example, by optimizing the extraction voltage, extremely fast extraction kinetics can be obtained. A metabolic profile could be generated while the drug was metabolized offering a significant time saving as compared to conventional LC-MS where laborious protein precipitation or other sample pretreatments are required before analysis. This makes the developed EME-MS setup a highly promising sample preparation method for various kinds of applications where fast and real-time analysis of analytes is of interest.


Assuntos
Química Farmacêutica/métodos , Preparações Farmacêuticas/análise , Espectrometria de Massas por Ionização por Electrospray , Cinética
13.
Eur J Pharm Sci ; 66: 50-8, 2015 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-25315409

RESUMO

The aim of this study was to develop population pharmacokinetic-pharmacodynamic models for morphine in experimental pain induced by skin heat and muscle pressure, and to evaluate the experimental pain models with regard to assessment of morphine pharmacodynamics. In a randomised, double-blind, placebo-controlled, crossover study, 39 healthy volunteers received an oral dose of 30mg morphine hydrochloride or placebo. Non-linear mixed effects modelling was used to describe the plasma concentrations of morphine and metabolites, and the analgesic effect of morphine on experimental pain in skin and muscle. Baseline pain metrics varied between individuals and occasions, and were described with interindividual and interoccasion variability. Placebo-response did not change with time. For both pain metrics, morphine effect was proportional to baseline pain and was described with a linear model with interindividual variability on drug effect slope and linked to an effect compartment for muscle pressure. The models indicate that a steady-state morphine concentration of 21ng/ml causes 33% and 0.84% increases in stimulus intensity from baseline for muscle pressure and skin heat, respectively. The population pharmacokinetic-pharmacodynamic models developed in this study indicate that mechanical stimulation of muscle is a more clinically relevant pain stimulus for the assessment of morphine pharmacodynamics than thermal stimulation of skin.


Assuntos
Analgésicos Opioides/farmacologia , Analgésicos Opioides/farmacocinética , Modelos Biológicos , Morfina/farmacologia , Morfina/farmacocinética , Dor/tratamento farmacológico , Analgésicos Opioides/sangue , Analgésicos Opioides/metabolismo , Estudos Cross-Over , Método Duplo-Cego , Humanos , Morfina/sangue , Morfina/metabolismo , Pressão
14.
J Biol Chem ; 289(45): 31576-90, 2014 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-25258323

RESUMO

For decades, selenium research has been focused on the identification of active metabolites, which are crucial for selenium chemoprevention of cancer. In this context, the metabolite methylselenol (CH3SeH) is known for its action to selectively kill transformed cells through mechanisms that include increased formation of reactive oxygen species, induction of DNA damage, triggering of apoptosis, and inhibition of angiogenesis. Here we reveal that CH3SeH modulates the cell surface expression of NKG2D ligands. The expression of NKG2D ligands is induced by stress-associated pathways that occur early during malignant transformation and enable the recognition and elimination of tumors by activating the lymphocyte receptor NKG2D. CH3SeH regulated NKG2D ligands both on the transcriptional and the posttranscriptional levels. CH3SeH induced the transcription of MHC class I polypeptide-related sequence MICA/B and ULBP2 mRNA. However, the induction of cell surface expression was restricted to the ligands MICA/B. Remarkably, our studies showed that CH3SeH inhibited ULBP2 surface transport through inhibition of the autophagic transport pathway. Finally, we identified extracellular calcium as being essential for CH3SeH regulation of NKG2D ligands. A balanced cell surface expression of NKG2D ligands is considered to be an innate barrier against tumor development. Therefore, our work indicates that the application of selenium compounds that are metabolized to CH3SeH could improve NKG2D-based immune therapy.


Assuntos
Regulação da Expressão Gênica , Linfócitos/citologia , Metanol/análogos & derivados , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Compostos Organosselênicos/química , Selênio/química , Autofagia , Cálcio/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citotoxicidade Imunológica/imunologia , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Inibidores de Histona Desacetilases/química , Humanos , Imunidade Inata , Imunoterapia/métodos , Células Jurkat , Células Matadoras Naturais/metabolismo , Ligantes , Espectrometria de Massas , Metanol/química , Processamento Pós-Transcricional do RNA
15.
Metallomics ; 6(9): 1639-47, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25027387

RESUMO

The aim of the present work was to demonstrate how selenium labelling of a synthetic cell-penetrating peptide may be employed in evaluation of stability and quantitative estimation of cellular uptake by inductively coupled plasma mass spectrometry (ICP-MS). Two analogues of the cell-penetrating peptide, penetratin, were synthesized, one with selenomethionine (SeMet) added at the N-terminal of the peptide (N-PenM(Se)) and the other with the internal methionine (Met) replaced with SeMet (i-PenM(Se)). The purity of the synthesized peptides was 92% for N-PenM(Se) and 89% for i-PenM(Se) as determined by liquid chromatography (LC)-ICP-MS. The selenium-labelled peptides were investigated by cell uptake studies in HeLa WT cells. The stability of the peptides was monitored in water, cell medium and during cell uptake studies. Total uptake of selenium was quantified by flow injection (FI)-ICP-MS. Speciation analysis of cell samples by LC-ICP-MS showed mainly uptake of the intact peptides, while the amount of intact peptides in cell lysates was semi-quantitatively determined. The selenium-containing penetratin analogues were to some extent degraded in pure cell medium, while an extensive degradation was observed during cell uptake studies. The major degradation products were determined by LC-electrospray ionization mass spectrometry (ESI-MS). The labelling method in combination with FI-ICP-MS, LC-ICP-MS and LC-ESI-MS techniques provided detailed information on the fate of penetratin in cellular uptake studies. Most pharmaceutical peptides, including penetratin, are synthetic analogues of endogenous peptides, and incorporation of selenium may improve the critical assessment of the native drug or drug delivery candidate early in the drug development process.


Assuntos
Peptídeos/análise , Preparações Farmacêuticas/análise , Selênio/análise , Coloração e Rotulagem , Sequência de Aminoácidos , Extratos Celulares , Endocitose , Células HeLa , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/química
16.
Drug Metab Dispos ; 42(7): 1153-62, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24778367

RESUMO

Insects have been proposed as a new tool in early drug development. It was recently demonstrated that locusts have an efflux transporter localized in the blood-brain barrier (BBB) that is functionally similar to the mammalian P-glycoprotein efflux transporter. Two insect BBB models have been put forward, an ex vivo model and an in vivo model. To use the in vivo model it is necessary to fully characterize the locust as an entire organism with regards to metabolic pathways and excretion rate. In the present study, we have characterized the locust metabolism of terfenadine, a compound that in humans is specific to the cytochrome P450 enzyme 3A4. Using high-resolution mass spectrometry coupled to ultra-high-performance liquid chromatography, we have detected metabolites identical to human metabolites of terfenadine. The formation of human metabolites in locusts was inhibited by ketoconazole, a mammalian CYP3A4 inhibitor, suggesting that the enzyme responsible for the human metabolite formation in locusts is functionally similar to human CYP3A4. Besides the human metabolites of terfenadine, additional metabolites were formed in locusts. These were tentatively identified as phosphate and glucose conjugates. In conclusion, not only may locusts be a model useful for determining BBB permeation, but possibly insects could be used in metabolism investigation. However, extensive characterization of the insect model is necessary to determine its applicability.


Assuntos
Citocromo P-450 CYP3A/metabolismo , Gafanhotos/enzimologia , Animais , Humanos , Masculino , Especificidade por Substrato
17.
Anal Bioanal Chem ; 406(2): 421-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24196121

RESUMO

A small and very simple electromembrane extraction probe (EME-probe) was developed and coupled directly to electrospray ionization mass spectrometry (ESI-MS), and this system was used to monitor in real time in vitro metabolism by rat liver microsomes of drug substances from a small reaction (incubation) chamber (37 °C). The drug-related substances were continuously extracted from the 1.0 mL metabolic reaction mixture and into the EME-probe by an electrical potential of 2.5 V. The extraction probe consisted of a 1-mm long and 350-µm ID thin supported liquid membrane (SLM) of 2-nitrophenyl octyl ether. The drugs and formed metabolites where extracted through the SLM and directly into a 3 µL min(-1) flow of 60 mM HCOOH inside the probe serving as the acceptor solution. The acceptor solution was directed into the ESI-MS-system, and the MS continuously monitored the drug-related substances extracted by the EME-probe. The extraction efficiency of the EME-probe was dependant on the applied electrical potential and the length of the SLM, and these parameters as well as the volume of the reaction chamber were set to the values mentioned above to avoid serious depletion from the reaction chamber (soft extraction). Soft extraction was mandatory in order not to affect the reaction kinetics by sample composition changes induced by the EME-probe. The EME-probe/MS-system was used to establish kinetic profiles for the in vitro metabolism of promethazine, amitriptyline and imipramine as model substances.


Assuntos
Técnicas Eletroquímicas/métodos , Éteres/química , Membranas Artificiais , Extração em Fase Sólida/métodos , Amitriptilina/isolamento & purificação , Amitriptilina/metabolismo , Animais , Biotransformação , Imipramina/isolamento & purificação , Imipramina/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Prometazina/isolamento & purificação , Prometazina/metabolismo , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização por Electrospray , Fatores de Tempo
18.
J Nutr Biochem ; 24(12): 2023-30, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24139672

RESUMO

Impaired S-adenosylmethionine (SAM)-dependent transmethylation and methylation capacity feature in diseases related to obesity or aging, and selenium (Se) metabolism is altered in these states. We tested the hypothesis that SAM metabolism is required for methylation and excretion of Se in a rat model. Four hours after selenite and periodate-oxidized adenosine (POA; an inhibitor of SAM metabolism) were administered, circulating markers of single-carbon status were unchanged, except for decreased circulating phosphatidylcholine (P<.05). In contrast, liver and kidney SAM and S-adenosylhomocysteine were elevated (P<.05 for all). Concentrations of total Se were significantly elevated in both liver (P<.001) and kidney (P<.01), however the degree of accumulation in liver was significantly greater than that of kidney (P<.05). Red blood cell Se levels were decreased (P=.01). Trimethylselenonium levels were decreased in liver and kidney (P=.001 for both tissues) and Se-methyl-N-acetylselenohexosamine selenosugar was decreased in liver (P=.001). Urinary output of both trimethylselenonium (P=.001) and selenosugar (P=.01) was decreased as well. Trimethylselenonium production is more inhibited by POA than is selenosugar production (P<.05). This work indicates that low molecular weight Se metabolism requires SAM-dependent methylation, and disrupting the conversion of SAM to S-adenosylhomocysteine prevents conversion of selenite and intermediate metabolites to final excretory forms, suggesting implications for selenium supplementation under conditions where transmethylation is suboptimal, such as in the case of obese or aging individuals.


Assuntos
Compostos Organosselênicos/metabolismo , S-Adenosilmetionina/metabolismo , Ácido Selenioso/metabolismo , Compostos de Selênio/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Suplementos Nutricionais , Eritrócitos/metabolismo , Rim/metabolismo , Fígado/metabolismo , Masculino , Metilação , Peso Molecular , Compostos Organosselênicos/urina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , S-Adenosil-Homocisteína/metabolismo , Selênio/administração & dosagem , Selênio/farmacocinética , Compostos de Selênio/urina , Selenoproteínas/genética , Selenoproteínas/metabolismo
19.
Biol Trace Elem Res ; 145(2): 248-56, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21863324

RESUMO

The aim of the present work was to compare and estimate absorption and biotransformation of selected selenium compounds by studying their fluxes across Caco-2 cells. Five different selenium compounds, selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenate, selenite, and methylseleninic acid (MeSeA), were applied to Caco-2 cells in a concentration of 10 µM, and fluxes in both directions were studied for 2 h. Fluxes of selenite and MeSeA in the presence of excess reduced glutathione (selenite + GSH and MeSeA + GSH) and flux of MeSeA in the presence of excess cysteine (MeSeA + Cys) were also studied. Selenium absorptive and exsorptive fluxes and accumulation in cell cytosol were analyzed by means of flow injection inductively coupled plasma mass spectrometry (ICP-MS). Absorptive flux of SeMet, MeSeCys, and selenate showed values correlating to complete in vivo absorption, while selenite and MeSeA fluxes correlated to poor in vivo absorption. Speciation analysis of cell lysate and donor and receptor solutions by LC-ICP-MS showed limited transformation of all selenium compounds. Extensive transformation as well as significantly increased absorptive flux was observed when co-administering selenite with glutathione compared to administering selenite alone. These observations are possibly due to formation of selenodiglutathione (GS-Se-SG) which may be absorbed differently than selenite. Concomitant application of GSH or cysteine with MeSeA resulted in extensive transformation of MeSeA, including volatile species, whereas no significant increases in fluxes were observed. In summary, the absorption of selenite selenate and the selenoamino acids is considered complete under physiological conditions, but the absorption mechanisms and metabolism of the compounds are different.


Assuntos
Absorção Intestinal/fisiologia , Modelos Biológicos , Compostos Organosselênicos/metabolismo , Compostos de Selênio/metabolismo , Células CACO-2 , Humanos , Absorção Intestinal/efeitos dos fármacos , Espectrometria de Massas/métodos , Compostos Organosselênicos/farmacologia , Compostos de Selênio/farmacologia
20.
Anal Bioanal Chem ; 399(5): 1743-63, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20953781

RESUMO

The aim of this review is to present and evaluate the present knowledge of which selenium species are available to the general population in the form of food and common supplements and how these species are metabolized in mammals. The overview of the selenium sources takes a horizontal approach, which encompasses identification of new metabolites in yeast and food of plant and animal origin, whereas the survey of the mammalian metabolism takes a horizontal as well as a vertical approach. The vertical approach encompasses studies on dynamic conversions of selenium compounds within cells, tissues or whole organisms. New and improved sample preparation, separation and detection methods are evaluated from an analytical chemical perspective to cover the progress in horizontal speciation, whereas the analytical methods for the vertical speciation and the interpretations of the results are evaluated from a biological angle as well.


Assuntos
Células/metabolismo , Compostos Organosselênicos/metabolismo , Compostos de Selênio/metabolismo , Solo/química , Animais , Células/química , Alimentos , Humanos , Compostos Organosselênicos/análise , Compostos de Selênio/análise , Solo/análise
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