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1.
Bioanalysis ; 10(9): 633-644, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29749254

RESUMO

AIM: Coproporphyrins (CP-I and CP-III) have been identified as possible biomarkers to predict human hepatic organic anion-transporting polypeptides-mediated-drug-interactions for a new drug entering clinical development. RESULTS: The method is applicable to quantify plasma CP-I and CP-III within 0.078-15.0 nM. The results identify and address a number of challenges encountered with porphyrin assays such as photodegradation and interferences. To overcome interferences from ubiquitous porphyrins, a surrogate matrix was used to prepare calibration standards. Quality controls were prepared in plasma and surrogate matrix to ensure parallelism between surrogate matrix and plasma. CONCLUSION: A robust UHPLC-MS/MS assay was developed and validated for CP-I and CP-III in plasma, and is currently applied to clinical studies to confirm suitability of Coproporphyrins as a potential substitute for drug-drug interaction study.


Assuntos
Biomarcadores Farmacológicos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Coproporfirinas/sangue , Transportadores de Ânions Orgânicos/metabolismo , Espectrometria de Massas em Tandem/métodos , Biomarcadores Farmacológicos/química , Coproporfirinas/química , Desenho de Drogas , Interações de Medicamentos , Humanos , Transportadores de Ânions Orgânicos/química , Rifampina/sangue , Rifampina/química , Rosuvastatina Cálcica/sangue , Rosuvastatina Cálcica/química
2.
Drug Metab Dispos ; 45(8): 908-919, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28576766

RESUMO

Multiple endogenous compounds have been proposed as candidate biomarkers to monitor organic anion transporting polypeptide (OATP) function in preclinical species or humans. Previously, we demonstrated that coproporphyrins (CPs) I and III are appropriate clinical markers to evaluate OATP inhibition and recapitulate clinical drug-drug interactions (DDIs). In the present study, we investigated bile acids (BAs) dehydroepiandrosterone sulfate (DHEAS), hexadecanedioate (HDA), and tetradecanedioate (TDA) in plasma as endogenous probes for OATP inhibition and compared these candidate probes to CPs. All probes were determined in samples from a single study that examined their behavior and their association with rosuvastatin (RSV) pharmacokinetics after administration of an OATP inhibitor rifampin (RIF) in healthy subjects. Among endogenous probes examined, RIF significantly increased maximum plasma concentration (Cmax) and area under the concentration-time curve (AUC)(0-24h) of fatty acids HDA and TDA by 2.2- to 3.2-fold. For the 13 bile acids in plasma examined, no statistically significant changes were detected between treatments. Changes in plasma DHEAS did not correlate with OATP1B inhibition by RIF. On the basis of the magnitude of effects for the endogenous compounds that demonstrated significant changes from baseline over interindividual variations, the overall rank order for the AUC change was found to be CP I > CP III > HDA ≈ TDA ≈ RSV > > BAs. Collectively, these results reconfirmed that CPs are novel biomarkers suitable for clinical use. In addition, HDA and TDA are useful for OATP functional assessment. Since these endogenous markers can be monitored in conjunction with pharmacokinetics analysis, the CPs and fatty acid dicarboxylates, either alone or in combination, offer promise of earlier diagnosis and risk stratification for OATP-mediated DDIs.


Assuntos
Ácidos e Sais Biliares/sangue , Biomarcadores/sangue , Coproporfirinas/sangue , Sulfato de Desidroepiandrosterona/sangue , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Ácidos Palmíticos/sangue , Adolescente , Adulto , Área Sob a Curva , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Interações de Medicamentos/fisiologia , Células HEK293 , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Rifampina/farmacologia , Rosuvastatina Cálcica/farmacologia , Adulto Jovem
3.
J Pharmacol Exp Ther ; 358(3): 397-404, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27317801

RESUMO

In the present study, an open-label, three-treatment, three-period clinical study of rosuvastatin (RSV) and rifampicin (RIF) when administered alone and in combination was conducted in 12 male healthy subjects to determine if coproporphyrin I (CP-I) and coproporphyrin III (CP-III) could serve as clinical biomarkers for organic anion transporting polypeptide 1B1 (OATP1B1) and 1B3 that belong to the solute carrier organic anion gene subfamily. Genotyping of the human OATP1B1 gene was performed in all 12 subjects and confirmed absence of OATP1B1*5 and OATP1B1*15 mutations. Average plasma concentrations of CP-I and CP-III prior to drug administration were 0.91 ± 0.21 and 0.15 ± 0.04 nM, respectively, with minimum fluctuation over the three periods. CP-I was passively eliminated, whereas CP-III was actively secreted from urine. Administration of RSV caused no significant changes in the plasma and urinary profiles of CP-I and CP-III. RIF markedly increased the maximum plasma concentration (Cmax) of CP-I and CP-III by 5.7- and 5.4-fold (RIF) or 5.7- and 6.5-fold (RIF+RSV), respectively, as compared with the predose values. The area under the plasma concentration curves from time 0 to 24 h (AUC0-24h) of CP-I and CP-III with RIF and RSV increased by 4.0- and 3.3-fold, respectively, when compared with RSV alone. In agreement with this finding, Cmax and AUC0-24h of RSV increased by 13.2- and 5.0-fold, respectively, when RIF was coadministered. Collectively, we conclude that CP-I and CP-III in plasma and urine can be appropriate endogenous biomarkers specifically and reliably reflecting OATP inhibition, and thus the measurement of these molecules can serve as a useful tool to assess OATP drug-drug interaction liabilities in early clinical studies.


Assuntos
Coproporfirinas/sangue , Coproporfirinas/urina , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Rifampina/farmacologia , Rosuvastatina Cálcica/farmacologia , Adulto , Biomarcadores/sangue , Biomarcadores/urina , Interações de Medicamentos , Humanos , Masculino , Pessoa de Meia-Idade , Rifampina/farmacocinética , Rosuvastatina Cálcica/farmacocinética , Adulto Jovem
4.
PLoS One ; 8(2): e53192, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23383297

RESUMO

BACKGROUND: Chronic glucocorticoid excess has been linked to increased atherosclerosis and general cardiovascular risk in humans. The enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ßHSD1) increases active glucocorticoid levels within tissues by catalyzing the conversion of cortisone to cortisol. Pharmacological inhibition of 11ßHSD1 has been shown to reduce atherosclerosis in murine models. However, the cellular and molecular details for this effect have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: To examine the role of 11ßHSD1 in atherogenesis, 11ßHSD1 knockout mice were created on the pro-atherogenic apoE⁻/⁻ background. Following 14 weeks of Western diet, aortic cholesterol levels were reduced 50% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. 11ßHSD1⁺/⁺/apoE⁻/⁻ mice without changes in plasma cholesterol. Aortic 7-ketocholesterol content was reduced 40% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. In the aortic root, plaque size, necrotic core area and macrophage content were reduced ∼30% in 11ßHSD1⁻/⁻/apoE⁻/⁻mice. Bone marrow transplantation from 11ßHSD1⁻/⁻/apoE⁻/⁻ mice into apoE⁻/⁻ recipients reduced plaque area 39-46% in the thoracic aorta. In vivo foam cell formation was evaluated in thioglycollate-elicited peritoneal macrophages from 11ßHSD1⁺/⁺/apoE⁻/⁻ and 11ßHSD1⁻/⁻/apoE⁻/⁻ mice fed a Western diet for ∼5 weeks. Foam cell cholesterol levels were reduced 48% in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice vs. control. Microarray profiling of peritoneal macrophages revealed differential expression of genes involved in inflammation, stress response and energy metabolism. Several toll-like receptors (TLRs) were downregulated in 11ßHSD1⁻/⁻/apoE⁻/⁻ mice including TLR 1, 3 and 4. Cytokine release from 11ßHSD1⁻/⁻/apoE⁻/⁻-derived peritoneal foam cells was attenuated following challenge with oxidized LDL. CONCLUSIONS: These findings suggest that 11ßHSD1 inhibition may have the potential to limit plaque development at the vessel wall and regulate foam cell formation independent of changes in plasma lipids. The diminished cytokine response to oxidized LDL stimulation is consistent with the reduction in TLR expression and suggests involvement of 11ßHSD1 in modulating binding of pro-atherogenic TLR ligands.


Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Células Espumosas/metabolismo , Glucocorticoides/metabolismo , Análise de Variância , Animais , Aterosclerose/prevenção & controle , Pressão Sanguínea , Transplante de Medula Óssea , Colesterol/metabolismo , Dieta Aterogênica , Cetocolesteróis/metabolismo , Lipídeos/sangue , Masculino , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Toll-Like/metabolismo
5.
BMC Pharmacol ; 12: 2, 2012 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-22475049

RESUMO

BACKGROUND: Dipeptidylpeptidase 4 (DPP4) inhibitors have clinical benefit in patients with type 2 diabetes mellitus by increasing levels of glucose-lowering incretin hormones, such as glucagon-like peptide -1 (GLP-1), a peptide with a short half life that is secreted for approximately 1 hour following a meal. Since drugs with prolonged binding to their target have been shown to maximize pharmacodynamic effects while minimizing drug levels, we developed a time-dependent inhibitor that has a half-life for dissociation from DPP4 close to the duration of the first phase of GLP-1 release. RESULTS: Saxagliptin and its active metabolite (5-hydroxysaxagliptin) are potent inhibitors of human DPP4 with prolonged dissociation from its active site (Ki = 1.3 nM and 2.6 nM, t1/2 = 50 and 23 minutes respectively at 37°C). In comparison, both vildagliptin (3.5 minutes) and sitagliptin ( < 2 minutes) rapidly dissociated from DPP4 at 37°C. Saxagliptin and 5-hydroxysaxagliptin are selective for inhibition of DPP4 versus other DPP family members and a large panel of other proteases, and have similar potency and efficacy across multiple species.Inhibition of plasma DPP activity is used as a biomarker in animal models and clinical trials. However, most DPP4 inhibitors are competitive with substrate and rapidly dissociate from DPP4; therefore, the type of substrate, volume of addition and final concentration of substrate in these assays can change measured inhibition. We show that unlike a rapidly dissociating DPP4 inhibitor, inhibition of plasma DPP activity by saxagliptin and 5-hydroxysaxagliptin in an ex vivo assay was not dependent on substrate concentration when substrate was added rapidly because saxagliptin and 5-hydroxysaxagliptin dissociate slowly from DPP4, once bound. We also show that substrate concentration was important for rapidly dissociating DPP4 inhibitors. CONCLUSIONS: Saxagliptin and its active metabolite are potent, selective inhibitors of DPP4, with prolonged dissociation from its active site. They also demonstrate prolonged inhibition of plasma DPP4 ex vivo in animal models, which implies that saxagliptin and 5-hydroxysaxagliptin would continue to inhibit DPP4 during rapid increases in substrates in vivo.


Assuntos
Adamantano/análogos & derivados , Dipeptídeos/metabolismo , Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/metabolismo , Hipoglicemiantes/metabolismo , Adamantano/metabolismo , Algoritmos , Animais , Artefatos , Clonagem Molecular , Dipeptidases/metabolismo , Dipeptidil Peptidase 4/sangue , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Indicadores e Reagentes , Cinética , Macaca fascicularis , Nitrilos/metabolismo , Ligação Proteica , Pirazinas/metabolismo , Pirrolidinas/metabolismo , Fosfato de Sitagliptina , Especificidade da Espécie , Triazóis/metabolismo , Vildagliptina
6.
J Sep Sci ; 33(6-7): 923-9, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20127913

RESUMO

RP-HPLC has largely been the analytical method of choice in the pharmaceutical industry for many years because of the poor aqueous solubility and hydrophobicity of most small molecule drug candidates. RP-HPLC coupled to MS has provided an excellent analytical tool for qualitatively and quantitatively determining levels of drug molecules or drug metabolites for studies such as purity assessment and pharmacokinetic profiling. Quantitation of endogenous metabolites is an emerging field in drug discovery, gaining popularity for evaluating pharmacokinetic-pharmacodynamic relationships and targeting activity and efficacy. While RP-HPLC-MS is suitable for a range of applications, many endogenous molecules, especially those found in urine, are small polar compounds that do not retain well by RP-HPLC. This has made hydrophilic interaction LC (HILIC) an attractive alternative and useful approach to polar molecule analysis. Additionally, because HILIC is routinely used with traditional RP organic solvents such as ACN and methanol, it can be easily coupled to MS. This paper will review selected examples from the current literature as well as discuss some new applications from the author's own laboratory focusing on the applicability of HILIC to quantitative and qualitative profiling of endogenous metabolites in drug discovery.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Descoberta de Drogas , Espectrometria de Massas/métodos , Farmacocinética
7.
Artigo em Inglês | MEDLINE | ID: mdl-18760978

RESUMO

Plasma levels of 1,5-anhydroglucitol (1-deoxyglucose), a short-term marker of glycemic control, have been measured and used clinically in Japan since the early 1990s. Plasma levels of 1,5-anhydroglucitol are typically measured using either a commercially available enzymatic kit or GC/MS. A more sensitive method is needed for the analysis of 1,5-anhydroglucitol in urine, where levels are significantly lower than in plasma. We have developed a sensitive and selective LC/MS(3) assay utilizing hydrophilic interaction liquid chromatography and ion trap mass spectrometry for the quantitative determination of 1,5-anhydroglucitol in human urine. Diluted human urine samples were analyzed by LC/MS(3) using an APCI source operated in the negative ionization mode. Use of an ion trap allowed monitoring of MS(3) transitions for both 1,5-anhydroglucitol and the internal standard which provided sufficient selectivity and sensitivity for analysis from 50 microL of human urine. Quantitation of 1,5-anhydroglucitol levels in urine was accomplished using a calibration curve generated in water (calibration range 50 ng/mL to 10 microg/mL). Method ruggedness and reproducibility were evaluated by determining the intra- and inter-day accuracies and precision of the assay, as well as the bench-top and freeze-thaw stability. For both inter- and intra-assay evaluations, the accuracy of the assay was found to be acceptable, with the concentrations of all QCs tested not deviating more than 8% from theoretical. Four-hour bench-top and freeze-thaw stabilities were also evaluated; 1,5-anhydroglucitol was found to be stable at room temperature (<18% deviation from theoretical) and during 3 freeze-thaw cycles (<1% deviation from theoretical, except at the lowest QC level). The LC/MS(3) assay was then used to successfully determine the concentration of 1,5-AG in more than 200 urine samples from diabetic patients enrolled in a clinical study.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Desoxiglucose/urina , Diabetes Mellitus/diagnóstico , Espectrometria de Massas/métodos , Biomarcadores , Diabetes Mellitus/urina , Feminino , Humanos , Masculino , Sensibilidade e Especificidade
8.
Rapid Commun Mass Spectrom ; 22(9): 1359-66, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18381620

RESUMO

Triple quadrupole mass spectrometers are generally considered the instrument of choice for quantitative analysis. However, for the analysis of large peptides we have encountered some cases where, as the data presented here would indicate, ion trap mass spectrometers may be a good alternative. In general, specificity and sensitivity in bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays are achieved via tandem MS (MS/MS) utilizing collision-induced dissociation (CID) while monitoring unique precursor to product ion transitions (i.e. selected reaction monitoring, SRM). Due to the difference in CID processes, triple quadrupoles and ion traps often generate significantly different fragmentation spectra of product ion species and intensities. The large peptidic analytes investigated here generated fewer fragments with higher relative abundance on the ion trap as compared to those generated on the triple quadrupole, resulting in lower limits of detection on the ion trap.


Assuntos
Peptídeos/análise , Calibragem , Cromatografia Líquida , Desenho de Equipamento , Peptídeo 1 Semelhante ao Glucagon/química , Indicadores e Reagentes , Espectrometria de Massas , Peptídeos/química , Padrões de Referência , Vasopressinas/química
9.
Assay Drug Dev Technol ; 5(2): 247-64, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17477833

RESUMO

An automated high throughput process, termed the MetFast assay, is described to assess in vitro the general microsomal cytochrome P450 beta-nicotinamide adenine dinucleotide phosphate-mediated first-pass metabolic stability of potential drug candidates as a utility for pharmaceutical profiling. Utilizing robotic protocols with a multiprobe liquid handler, compounds are incubated with liver microsomes from different species. Samples are then analyzed by in-line liquid chromatography (LC)-mass spectrometry (MS) to determine the amount of compound remaining after a certain time, which allows calculation of metabolism rates. To quantitatively assess large numbers of structurally diverse compounds by LC-MS, a strategy based on an iterative two-step process was devised. Initially compounds are qualitatively analyzed by LC-ultraviolet (UV)/MS (step 1) to determine purity (UV detection) and structural integrity (MS detection). This step ensures that only correct and verified compounds with sufficient purity are being assayed to obtain reproducible high data quality. In addition, all necessary information is gathered to automatically generate specific quantitative methods for the subsequent bioanalytical analysis of metabolic stability samples by LC-UV/MS (step 2). In-house-developed, highly flexible and sophisticated data management software, termed SmartReport, is utilized for automated qualitative and quantitative LC-MS analysis set-up, data processing, and results reporting. The integration of key aspects, inherent "universal" collision-induced dissociation settings of ion trap mass spectrometers for tandem mass spectrometric scan functions utilized for compound-specific and sensitive quantitative MS methods, generic fast-LC conditions, generic MS instrument settings, and the functionality of SmartReport software resulted in an analytical process that routinely provides reproducible high-quality metabolic stability data on structurally diverse compounds. Described here is the setup of the MetFast assay, and metabolic stability data from assay validation compounds are given.


Assuntos
Preparações Farmacêuticas/metabolismo , Cromatografia Líquida , Interpretação Estatística de Dados , Avaliação Pré-Clínica de Medicamentos , Indicadores e Reagentes , Espectrometria de Massas , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Controle de Qualidade , Reprodutibilidade dos Testes , Robótica , Software , Solventes , Espectrofotometria Ultravioleta
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