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1.
Bioanalysis ; 8(15): 1611-1622, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27397670

RESUMO

BACKGROUND: Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern if located within the complementarity-determining region of therapeutic antibodies. Questions arise as to the extent of modification occurring in circulation due to potential exposure of the therapeutic antibody to different pH regimes. RESULTS: To enable evaluation of site-specific isomerization and deamidation of human mAbs in vivo, immunoprecipitation (IP) has been combined with LC-MS providing selective enrichment, separation and detection of naive and modified forms of tryptic peptides comprising complementarity-determining region sequences. CONCLUSION: IP-LC-MS can be applied to simultaneously quantify in vivo drug concentrations and measure the extent of isomerization or deamidation in PK studies conducted during the drug discovery stage.


Assuntos
Anticorpos Monoclonais/química , Asparagina/análise , Ácido Aspártico/análise , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Cromatografia Líquida/métodos , Humanos , Imunoprecipitação/métodos , Isomerismo , Macaca fascicularis , Masculino , Espectrometria de Massas em Tandem/métodos
2.
Bioanalysis ; 8(4): 265-74, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26807991

RESUMO

BACKGROUND: A target protein-based affinity extraction LC-MS/MS method was developed to enable plasma level determination following ultralow dosing (0.1-3 µg/kg) of an inhibitor of apoptosis proteins molecule. Methodology & results: Affinity extraction (AE) utilizing immobilized target protein BIR2/BIR3 was used to selectively capture the inhibitor of apoptosis proteins molecule from dog plasma and enable removal of background matrix components. Pretreatment of plasma samples using protein precipitation was found to provide an additional sensitivity gain. A LLOQ of 7.8 pM was achieved by combining protein precipitation with AE. The method was used to support an ultralow dose dog toxicity study. CONCLUSION: AE-LC-MS/MS, utilizing target protein, is a highly sensitive methodology for small molecule quantification with potential for broader applicability.


Assuntos
Análise Química do Sangue/métodos , Fracionamento Químico/métodos , Cromatografia Líquida/métodos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Isoquinolinas/análise , Limite de Detecção , Oligopeptídeos/análise , Bibliotecas de Moléculas Pequenas/análise , Espectrometria de Massas em Tandem/métodos , Animais , Cães , Feminino , Humanos , Proteínas Imobilizadas/antagonistas & inibidores , Proteínas Imobilizadas/química , Proteínas Inibidoras de Apoptose/química , Isoquinolinas/química , Isoquinolinas/farmacologia , Masculino , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
3.
Exp Cell Res ; 338(2): 251-60, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26302264

RESUMO

Cellular levels of inhibitor of apoptosis (IAP) proteins are elevated in multiple human cancers and their activities often play a part in promoting cancer cell survival by blocking apoptotic pathways, controlling signal transduction pathways and contributing to resistance. These proteins function through interactions of their BIR (baculoviral IAP repeat) protein domains with pathway components and these interactions are endogenously antagonized by Smac/Diablo (second mitochondrial activator of caspases/direct IAP binding protein with low isoelectric point). This report describes development of synthetic smac mimetics (SM) and compares their binding, antiproliferative and anti-tumor activities. All dimeric antagonists inhibit in vitro smac tetrapeptide binding to recombinant IAP proteins, rescue IAP-bound caspase-3 activity and show anti-proliferative activity against human A875 melanoma cells. One heterodimeric SM, SM3, binds tightly to IAP proteins in vitro and slowly dissociates (greater than two hours) from these protein complexes compared to the other antagonists. In addition, in vitro SM anti-proliferation potency is influenced by ABCB1 transporter (ATP-binding cassette, sub-family B; MDR1, P-gp) activities and one antagonist, SM5, does not appear to be an ABCB1 efflux pump substrate. All dimeric smac mimetics inhibit the growth of human melanoma A875 tumors implanted in athymic mice at well-tolerated doses. One antagonist, SM4, shows broad spectrum in vivo anti-tumor activity and modulates known pharmacodynamic markers of IAP antagonism. These data taken together demonstrate the range of diverse dimeric IAP antagonist activities and supports their potential as anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Caspase 3/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas Mitocondriais/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Apoptose/efeitos dos fármacos , Biomimética/métodos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Células HCT116 , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos
4.
ACS Med Chem Lett ; 6(8): 845-9, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26288682

RESUMO

Early hit to lead work on a pyrrolopyridine chemotype provided access to compounds with biochemical and cellular potency against Janus kinase 2 (JAK2). Structure-based drug design along the extended hinge region of JAK2 led to the identification of an important H-bond interaction with the side chain of Tyr 931, which improved JAK family selectivity. The 4,5-dimethyl thiazole analogue 18 demonstrated high levels of JAK family selectivity and was identified as a promising lead for the program.

5.
ACS Med Chem Lett ; 6(8): 850-5, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26288683

RESUMO

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile.

6.
ACS Med Chem Lett ; 6(7): 770-5, 2015 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-26191364

RESUMO

A series of dimeric macrocyclic compounds were prepared and evaluated as antagonists for inhibitor of apoptosis proteins. The most potent analogue 11, which binds to XIAP and c-IAP proteins with high affinity and induces caspase-3 activation and ultimately cell apoptosis, inhibits growth of human melanoma and colorectal cell lines at low nanomolar concentrations. Furthermore, compound 11 demonstrated significant antitumor activity in the A875 human melanoma xenograft model at doses as low as 2 mg/kg on a q3d schedule.

7.
Regul Toxicol Pharmacol ; 73(1): 27-42, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26111605

RESUMO

This comparative study was conducted to assess background physiologic and pharmacologic parameters of cynomolgus macaques (Macaca fascicularis) from Cambodia, from a mixed Asian source (Cambodia, Vietnam and Indonesia), and from Mauritius. This evaluation provides a comprehensive assessment of several of these parameters in a single study. Ten male and 10 female captive-bred, age-matched macaques from each source were evaluated. Criteria for evaluation included weight gain, assessment of drug metabolizing enzyme activity, metabolomic analysis, immunologic assessments (lymphocyte subsets, TDAR, and serum Ig isotyping), clinical pathology evaluations, physical (respiratory, neurologic, cardiovascular, and ophthalmologic) examinations, pathogen screening, organ weights, and gross and microscopic pathology analyses. The results of this evaluation indicate that, compared to macaques of Asian origin, macaques from Mauritius had the lowest incidence and/or severity of spontaneous pathologic findings in several organs and tissues (lymphoid organs, stomach, kidney, urothelium, heart, arteries and lung) and better testicular maturity at a given age with minimal variability in organ weights. Although slight differences were observed in other parameters, none were considered detrimental to the use of macaques of Asian or Mauritius origin in pharmaceutical candidate safety studies with the use of a consistent source, concomitant controls, and appropriate background knowledge and screening.


Assuntos
Macaca fascicularis/fisiologia , Tamanho do Órgão/fisiologia , Animais , Grupo com Ancestrais do Continente Asiático , Feminino , Humanos , Masculino , Maurício
8.
J Med Chem ; 58(3): 1556-62, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25584393

RESUMO

The prominent role of IAPs in controlling cell death and their overexpression in a variety of cancers has prompted the development of IAP antagonists as potential antitumor therapies. We describe the identification of a series of heterodimeric antagonists with highly potent antiproliferative activities in cIAP- and XIAP-dependent cell lines. Compounds 15 and 17 further demonstrate curative efficacy in human melanoma and lung cancer xenograft models and are promising candidates for advanced studies.


Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Prolina/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Estrutura Molecular , Neoplasias Experimentais/patologia , Prolina/síntese química , Prolina/química , Relação Estrutura-Atividade
9.
Bioanalysis ; 6(13): 1795-811, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25157486

RESUMO

BACKGROUND: The disease state can modulate the penetration of large antibody-sized therapeutic molecules into affected tissues. Suitable bioanalytical methods are required for the quantitative analysis of drug tissue levels to enable a better understanding of the parameters influencing drug penetration and target engagement. RESULTS: Described is a sensitive and selective LC-MS/MS assay for the quantification of human mAb molecules in mouse tissues. By homogenizing tissues directly into serum, a common serum calibration curve can be used for multiple tissues. A generic procedure was used for affinity enrichment. An analytical range of 20 - 20,000 ng/ml was achieved in serum. CONCLUSION: The method described here can be applied for the quantitative analysis of mAb and Fc-fusion therapeutic molecules in a variety of animal tissue matrices.


Assuntos
Anticorpos Monoclonais/análise , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados/análise , Anticorpos Monoclonais Humanizados/sangue , Anticorpos Monoclonais Humanizados/metabolismo , Cromatografia de Afinidade , Feminino , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Peptídeos/análise , Análise de Regressão , Pele/metabolismo , Tripsina/metabolismo , Ustekinumab
10.
Drug Metab Dispos ; 40(9): 1698-711, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22648560

RESUMO

Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -2B6, -2D6, -2C9, -2C8, and -1A2) in human liver microsomes. In most cases, IC50 values were greater than 40 µM, except for dexlansoprazole and lansoprazole with CYP1A2 (IC50 = ∼8 µM) and esomeprazole with CYP2C8 (IC50 = 31 µM). With the exception of CYP2C19 inhibition by omeprazole and esomeprazole (IC50 ratio, 2.5 to 5.9), there was no evidence for a marked time-dependent shift in IC50 (IC50 ratio, ≤ 2) after a 30-min preincubation with NADPH. In the absence of preincubation, lansoprazole (IC50 = 0.73 µM) and esomeprazole (IC50 = 3.7 µM) were the most potent CYP2C19 inhibitors, followed by dexlansoprazole and omeprazole (IC50 = ∼7.0 µM). Rabeprazole and pantoprazole (IC50 = ≥ 25 µM) were the weakest. A similar ranking was obtained with recombinant CYP2C19. Despite the IC50 ranking, after consideration of plasma levels (static and dynamic), protein binding, and metabolism-dependent inhibition, it is concluded that omeprazole and esomeprazole are the most potent CYP2C19 inhibitors. This was confirmed after the incubation of the individual PPIs with human primary hepatocytes (in the presence of human serum) and by monitoring their impact on diazepam N-demethylase activity at a low concentration of diazepam (2 µM). Data described herein are consistent with reports that PPIs are mostly weak inhibitors of cytochromes P450 in vivo. However, two members of the PPI class (esomeprazole and omeprazole) are more likely to serve as clinically relevant inhibitors of CYP2C19.


Assuntos
Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Esomeprazol/farmacologia , Fígado/efeitos dos fármacos , Omeprazol/farmacologia , Inibidores da Bomba de Prótons/farmacologia , 2-Piridinilmetilsulfinilbenzimidazóis/farmacologia , Hidrocarboneto de Aril Hidroxilases/metabolismo , Biotransformação , Células Cultivadas , Simulação por Computador , Citocromo P-450 CYP2C19 , Remoção de Radical Alquila , Dexlansoprazol , Diazepam/metabolismo , Relação Dose-Resposta a Droga , Interações de Medicamentos , Esomeprazol/farmacocinética , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Cinética , Lansoprazol , Fígado/enzimologia , Microssomos Hepáticos/enzimologia , Modelos Biológicos , NADP/metabolismo , Omeprazol/farmacocinética , Pantoprazol , Inibidores da Bomba de Prótons/farmacocinética , Rabeprazol , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
11.
Xenobiotica ; 42(11): 1058-68, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22574883

RESUMO

Numerous groups have described the rat as an in vivo model for the assessment and prediction of drug-drug interactions (DDIs) in humans involving the inhibition of cytochrome P450 3A forms. Even for a well-established substrate-inhibitor pair like midazolam-ketoconazole, however, the magnitude of the DDI in rats (e.g. 1.5- to 5-fold) does not relate to what is observed clinically (e.g. 5- to 16-fold). Because nonlinear substrate pharmacokinetics (PK) may result in a weaker interaction, it was hypothesized that the lower magnitude of interaction observed in rats was due to the saturation of metabolic pathway(s) of midazolam at the doses used (10-20 mg/kg). Therefore, the inhibitory effects of ketoconazole were reevaluated at lower oral (1 and 5 mg/kg) and intravenous (IV) (1 mg/kg) doses of midazolam. In support of the hypothesis, oral exposure at 5 mg/kg dose of midazolam was 18-fold higher compared to that at 1 mg/kg. Furthermore, when the interaction was investigated at the lower midazolam dose (1 mg/kg), ketoconazole increased the IV and oral exposure of midazolam by 7-fold and 11-fold, respectively. A weaker DDI (1.5- to 1.8-fold) was observed at the higher oral midazolam dose. Collectively, these results suggest that the lower reported interaction in rats is likely due to saturation of midazolam clearance at the doses used. Therefore, when the rat is used as a DDI model to screen and differentiate compounds, or predict CYP3A inhibition in humans, it is important to use low doses of midazolam and ensure linear PK.


Assuntos
Antifúngicos/farmacologia , Hipnóticos e Sedativos/farmacocinética , Cetoconazol/farmacologia , Midazolam/farmacocinética , Administração Intravenosa , Administração Oral , Animais , Proteínas Sanguíneas/metabolismo , Cromatografia Líquida , Simulação por Computador , Interações de Medicamentos , Humanos , Hipnóticos e Sedativos/administração & dosagem , Masculino , Espectrometria de Massas , Microssomos Hepáticos/metabolismo , Midazolam/administração & dosagem , Modelos Químicos , Ratos , Ratos Sprague-Dawley
12.
J Pharm Sci ; 100(10): 4127-57, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21541937

RESUMO

The objective of this study is to assess the effectiveness of physiologically based pharmacokinetic (PBPK) models for simulating human plasma concentration-time profiles for the unique drug dataset of blinded data that has been assembled as part of a Pharmaceutical Research and Manufacturers of America initiative. Combinations of absorption, distribution, and clearance models were tested with a PBPK approach that has been developed from published equations. An assessment of the quality of the model predictions was made on the basis of the shape of the plasma time courses and related parameters. Up to 69% of the simulations of plasma time courses made in human demonstrated a medium to high degree of accuracy for intravenous pharmacokinetics, whereas this number decreased to 23% after oral administration based on the selected criteria. The simulations resulted in a general underestimation of drug exposure (Cmax and AUC0- t ). The explanations for this underestimation are diverse. Therefore, in general it may be due to underprediction of absorption parameters and/or overprediction of distribution or oral first-pass. The implications of compound properties are demonstrated. The PBPK approach based on in vitro-input data was as accurate as the approach based on in vivo data. Overall, the scientific benefit of this modeling study was to obtain more extensive characterization of predictions of human PK from PBPK methods.


Assuntos
Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas/métodos , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Farmacocinética , Acesso à Informação , Administração Intravenosa , Administração Oral , Animais , Simulação por Computador , Comportamento Cooperativo , Avaliação Pré-Clínica de Medicamentos , Absorção Gastrointestinal , Humanos , Comunicação Interdisciplinar , Taxa de Depuração Metabólica , Modelos Estatísticos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/sangue , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Reprodutibilidade dos Testes , Especificidade da Espécie
13.
J Pharm Sci ; 100(10): 4090-110, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21541938

RESUMO

The objective of this study was to evaluate the performance of various allometric and in vitro-in vivo extrapolation (IVIVE) methodologies with and without plasma protein binding corrections for the prediction of human intravenous (i.v.) clearance (CL). The objective was also to evaluate the IVIVE prediction methods with animal data. Methodologies were selected from the literature. Pharmaceutical Research and Manufacturers of America member companies contributed blinded datasets from preclinical and clinical studies for 108 compounds, among which 19 drugs had i.v. clinical pharmacokinetics data and were used in the analysis. In vivo and in vitro preclinical data were used to predict CL by 29 different methods. For many compounds, in vivo data from only two species (generally rat and dog) were available and/or the required in vitro data were missing, which meant some methods could not be properly evaluated. In addition, 66 methods of predicting oral (p.o.) area under the curve (AUCp.o. ) were evaluated for 107 compounds using rational combinations of i.v. CL and bioavailability (F), and direct scaling of observed p.o. CL from preclinical species. Various statistical and outlier techniques were employed to assess the predictability of each method. Across methods, the maximum success rate in predicting human CL for the 19 drugs was 100%, 94%, and 78% of the compounds with predictions falling within 10-fold, threefold, and twofold error, respectively, of the observed CL. In general, in vivo methods performed slightly better than IVIVE methods (at least in terms of measures of correlation and global concordance), with the fu intercept method and two-species-based allometry (rat-dog) being the best performing methods. IVIVE methods using microsomes (incorporating both plasma and microsomal binding) and hepatocytes (not incorporating binding) resulted in 75% and 78%, respectively, of the predictions falling within twofold error. IVIVE methods using other combinations of binding assumptions were much less accurate. The results for prediction of AUCp.o. were consistent with i.v. CL. However, the greatest challenge to successful prediction of human p.o. CL is the estimate of F in human. Overall, the results of this initiative confirmed predictive performance of common methodologies used to predict human CL.


Assuntos
Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas/métodos , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Farmacocinética , Acesso à Informação , Administração Intravenosa , Animais , Área Sob a Curva , Simulação por Computador , Comportamento Cooperativo , Cães , Avaliação Pré-Clínica de Medicamentos , Humanos , Comunicação Interdisciplinar , Taxa de Depuração Metabólica , Modelos Estatísticos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/sangue , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Ligação Proteica , Ratos , Reprodutibilidade dos Testes , Especificidade da Espécie
14.
J Pharm Sci ; 100(10): 4074-89, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21452299

RESUMO

The objective of this study was to evaluate the performance of various empirical, semimechanistic and mechanistic methodologies with and without protein binding corrections for the prediction of human volume of distribution at steady state (Vss ). PhRMA member companies contributed a set of blinded data from preclinical and clinical studies, and 18 drugs with intravenous clinical pharmacokinetics (PK) data were available for the analysis. In vivo and in vitro preclinical data were used to predict Vss by 24 different methods. Various statistical and outlier techniques were employed to assess the predictability of each method. There was not simply one method that predicts Vss accurately for all compounds. Across methods, the maximum success rate in predicting human Vss was 100%, 94%, and 78% of the compounds with predictions falling within tenfold, threefold, and twofold error, respectively, of the observed Vss . Generally, the methods that made use of in vivo preclinical data were more predictive than those methods that relied solely on in vitro data. However, for many compounds, in vivo data from only two species (generally rat and dog) were available and/or the required in vitro data were missing, which meant some methods could not be properly evaluated. It is recommended to initially use the in vitro tissue composition-based equations to predict Vss in preclinical species and humans, putting the assumptions and compound properties into context. As in vivo data become available, these predictions should be reassessed and rationalized to indicate the level of confidence (uncertainty) in the human Vss prediction. The top three methods that perform strongly at integrating in vivo data in this way were the Øie-Tozer, the rat -dog-human proportionality equation, and the lumped-PBPK approach. Overall, the scientific benefit of this study was to obtain greater characterization of predictions of human Vss from several methods available in the literature.


Assuntos
Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas/métodos , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Farmacocinética , Acesso à Informação , Administração Intravenosa , Animais , Simulação por Computador , Comportamento Cooperativo , Cães , Avaliação Pré-Clínica de Medicamentos , Humanos , Comunicação Interdisciplinar , Modelos Estatísticos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/sangue , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Ligação Proteica , Ratos , Reprodutibilidade dos Testes , Especificidade da Espécie
15.
J Pharm Sci ; 100(10): 4111-26, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21480234

RESUMO

The objective of this study was to evaluate the performance of the Wajima allometry (Css -MRT) approach published in the literature, which is used to predict the human plasma concentration-time profiles from a scaling of preclinical species data. A diverse and blinded dataset of 108 compounds from PhRMA member companies was used in this evaluation. The human intravenous (i.v.) and oral (p.o.) pharmacokinetics (PK) data were available for 18 and 107 drugs, respectively. Three different scenarios were adopted for prediction of human PK profiles. In the first scenario, human clearance (CL) and steady-state volume of distribution (Vss ) were predicted by unbound fraction corrected intercept method (FCIM) and Øie-Tozer (OT) approaches, respectively. Quantitative structure activity relationship (QSAR)-based approaches (TSrat-dog ) based on compound descriptors together with rat and dog data were utilized in the second scenario. Finally, in the third scenario, CL and Vss were predicted using the FCIM and Jansson approaches, respectively. For the prediction of oral pharmacokinetics, the human bioavailability and absorption rate constant were assumed as the average of preclinical species. Various statistical techniques were used for assessing the accuracy of the simulation scenarios. The human CL and Vss were predicted within a threefold error range for about 75% of the i.v. drugs. However, the accuracy in predicting key p.o. PK parameters appeared to be lower with only 58% of simulations falling within threefold of observed parameters. The overall ability of the Css -MRT approach to predict the curve shape of the profile was in general poor and ranged between low to medium level of confidence for most of the predictions based on the selected criteria.


Assuntos
Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas/métodos , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Farmacocinética , Acesso à Informação , Administração Intravenosa , Administração Oral , Animais , Disponibilidade Biológica , Simulação por Computador , Comportamento Cooperativo , Cães , Avaliação Pré-Clínica de Medicamentos , Absorção Gastrointestinal , Humanos , Comunicação Interdisciplinar , Taxa de Depuração Metabólica , Modelos Estatísticos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/sangue , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Ratos , Reprodutibilidade dos Testes , Especificidade da Espécie
16.
J Pharm Sci ; 100(10): 4050-73, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21523782

RESUMO

This study is part of the Pharmaceutical Research and Manufacturers of America (PhRMA) initiative on predictive models of efficacy, safety, and compound properties. The overall goal of this part was to assess the predictability of human pharmacokinetics (PK) from preclinical data and to provide comparisons of available prediction methods from the literature, as appropriate, using a representative blinded dataset of drug candidates. The key objectives were to (i) appropriately assemble and blind a diverse dataset of in vitro, preclinical in vivo, and clinical data for multiple drug candidates, (ii) evaluate the dataset with empirical and physiological methodologies from the literature used to predict human PK properties and plasma concentration-time profiles, (iii) compare the predicted properties with the observed clinical data to assess the prediction accuracy using routine statistical techniques and to evaluate prediction method(s) based on the degree of accuracy of each prediction method, and (iv) compile and summarize results for publication. Another objective was to provide a mechanistic understanding as to why one methodology provided better predictions than another, after analyzing the poor predictions. A total of 108 clinical lead compounds were collected from 12 PhRMA member companies. This dataset contains intravenous (n = 19) and oral pharmacokinetic data (n = 107) in humans as well as the corresponding preclinical in vitro, in vivo, and physicochemical data. All data were blinded to protect the anonymity of both the data and the company submitting the data. This manuscript, which is the first of a series of manuscripts, summarizes the PhRMA initiative and the 108 compound dataset. More details on the predictability of each method are reported in companion manuscripts.


Assuntos
Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas/métodos , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Farmacocinética , Acesso à Informação , Administração Intravenosa , Administração Oral , Animais , Simulação por Computador , Comportamento Cooperativo , Avaliação Pré-Clínica de Medicamentos , Humanos , Comunicação Interdisciplinar , Modelos Estatísticos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/sangue , Preparações Farmacêuticas/química , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco , Especificidade da Espécie
17.
Drug Metab Dispos ; 38(1): 16-24, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19833845

RESUMO

Monkeys have been proposed as an animal model to predict the magnitude of human clinical drug-drug interactions caused by CYP3A4 enzyme induction. To evaluate whether the cynomolgus monkey can be an effective in vivo model, human CYP3A4 inducers were evaluated both in vitro and in vivo. First, a full-length pregnane X receptor (PXR) was cloned from the cynomolgus monkey, and the sequence was compared with those of rhesus monkey and human PXR. Cynomolgus and rhesus monkey PXR differed by only one amino acid (A68V), and both were highly homologous to human PXR (approximately 96%). When the transactivation profiles of 30 compounds, including known inducers of CYP3A4, were compared between cynomolgus and human PXR, a high degree of correlation with EC(50) values was observed. These results suggest that cynomolgus and human PXR respond in a similar fashion to these ligands. Second, two known human CYP3A4 inducers, rifampicin and hyperforin, were tested in monkey and human primary hepatocytes for induction of CYP3A enzymes. Both monkey and human hepatocytes responded similarly to the inducers and resulted in increased RNA and enzyme activity changes of CYP3A8 and CYP3A4, respectively. Lastly, in vivo induction of CYP3A8 by rifampicin and hyperforin was shown by significant reductions of midazolam exposure that were comparable with those in humans. These results show that the cynomolgus monkey can be a predictive in vivo animal model of PXR-mediated induction of human CYP3A4 and can provide a useful assessment of the resulting pharmacokinetic changes of affected drugs.


Assuntos
Citocromo P-450 CYP3A/biossíntese , Hepatócitos/metabolismo , Macaca fascicularis , Receptores de Esteroides/metabolismo , Xenobióticos/farmacocinética , Adulto , Sequência de Aminoácidos , Animais , Compostos Bicíclicos com Pontes/sangue , Compostos Bicíclicos com Pontes/farmacocinética , Compostos Bicíclicos com Pontes/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Clonagem Molecular , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Interações de Medicamentos/genética , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Hypericum/química , Macaca mulatta , Masculino , Midazolam/sangue , Midazolam/metabolismo , Midazolam/farmacocinética , Pessoa de Meia-Idade , Modelos Animais , Dados de Sequência Molecular , Floroglucinol/análogos & derivados , Floroglucinol/sangue , Floroglucinol/farmacocinética , Floroglucinol/farmacologia , Extratos Vegetais/sangue , Extratos Vegetais/farmacocinética , Receptor de Pregnano X , Receptores de Esteroides/genética , Rifampina/sangue , Rifampina/farmacocinética , Rifampina/farmacologia , Homologia de Sequência de Aminoácidos , Terpenos/sangue , Terpenos/farmacocinética , Terpenos/farmacologia , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Transfecção
18.
Drug Metab Dispos ; 35(11): 1990-5, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17709370

RESUMO

The effect of common organic solvents on the activities of various human cytochromes P450 has been reported. However, very little is known about their influence on CYP2B6 and CYP2C8 enzymes. The purpose of this study was to investigate the effect of solvents on the kinetics of representative CYP2B6 (bupropion hydroxylase) and CYP2C8 (paclitaxel hydroxylase) reactions in human liver microsomes. Methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied at increasing volumes (v/v). Acetonitrile, DMSO, and ethanol were shown to increase the Km and decrease the intrinsic clearance (CLint) of CYP2B6-mediated bupropion hydroxylation in a concentration-dependent manner. These solvents did not noticeably alter the Vmax at concentrations of < or =1% (v/v). Unlike the other solvents studied, the effect of methanol (< or =0.5%, v/v) on CYP2B6 kinetics was negligible. Both DMSO and ethanol increased the Km and decreased the CL(int) of CYP2C8-mediated paclitaxel hydroxylation in a concentration-dependent manner. Acetonitrile had minimal influence on CYP2C8 enzyme kinetics at concentrations of < or =1% (v/v). Methanol decreased the Km of paclitaxel at low concentrations followed by an increase at concentrations of > or =2% (v/v). This differential influence on Km resulted in an increased CLint at low concentrations followed by a decrease at high concentrations. The studied solvents had minimal influence on Vmax of paclitaxel. Collectively, DMSO and ethanol were not suitable for characterizing CYP2B6- and CYP2C8-mediated reactions because they showed concentration-dependent inhibition. Methanol and acetonitrile at concentrations of < or =0.5% and < or =1% (v/v) appeared to be suitable for the measurement of CYP2B6- and CYP2C8-mediated activities, respectively.


Assuntos
Hidrocarboneto de Aril Hidroxilases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/efeitos dos fármacos , Solventes/farmacologia , Acetonitrilos/farmacologia , Bupropiona/análogos & derivados , Bupropiona/metabolismo , Citocromo P-450 CYP2B6 , Citocromo P-450 CYP2C8 , Dimetil Sulfóxido/farmacologia , Etanol/farmacologia , Humanos , Hidroxilação/efeitos dos fármacos , Cinética , Metanol/farmacologia , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Paclitaxel/metabolismo , Taxoides/metabolismo
19.
Chirality ; 19(3): 190-6, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17192836

RESUMO

The effects of route of administration on the stereoselective pharmacokinetics of tramadol (T) and its active metabolite (M1) were studied in rats. A single 20 mg/kg dose of racemic T was administered through intravenous, intraperitoneal, or oral route to different groups of rats, and blood and urine samples were collected. Samples were analyzed using chiral chromatography, and pharmacokinetic parameters (mean +/- SD) were estimated by noncompartmental methods. Following intravenous injection, there was no stereoselectivity in the pharmacokinetics of T. Both enantiomers showed clearance values (62.5 +/- 27.2 and 64.4 +/- 39.0 ml/min/kg for (+)- and (-)-T, respectively) that were equal or higher than the reported liver blood flow in rats. Similar to T, the area under the plasma concentration-time curves (AUCs) of M1 did not exhibit stereoselectivity after intravenous administration of the parent drug. However, the systemic availability of (+)-T was significantly (P < 0.05) higher than that of its antipode following intraperitoneal (0.527 +/- 0.240 vs. 0.373 +/- 0.189) and oral (0.307 +/- 0.136 vs. 0.159 +/- 0.115) administrations. The AUC of the M1 enantiomers, on the other hand, remained mostly nonstereoselective regardless of the route of administration. Pharmacokinetic analysis indicated that the stereoselectivity in the pharmacokinetics of oral T is due to stereoselective first pass metabolism in the liver and, possibly, in the gastrointestinal tract. The direction and extent of stereoselectivity in the pharmacokinetics of T and M1 in rats were in agreement with those previously reported in humans, suggesting that the rat may be a suitable model for enantioselective studies of T pharmacokinetics.


Assuntos
Analgésicos Opioides/farmacocinética , Tramadol/farmacocinética , Analgésicos Opioides/administração & dosagem , Animais , Trato Gastrointestinal/metabolismo , Fígado/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Tramadol/administração & dosagem
20.
J Pharm Sci ; 95(7): 1414-24, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16724323

RESUMO

The steady-state disposition of chlorzoxazone (CZX) and its hydroxylated metabolite 6-hydroxychlorzoxazone (HCZX) was determined in a single-pass isolated perfused rat liver (IPRL) model using constant CZX concentrations of 10-200 microM. The concentrations of CZX, HCZX, and/or HCZX glucuronide in the perfusate, bile, and liver tissues were measured and kinetic parameters calculated. Upon an increase in CZX inlet concentrations from 10 to 200 microM, its extraction ratio sharply declined from 0.681 to 0.087. This was associated with a saturable formation of HCZX, which was rapidly and completely metabolized to its glucuronide conjugate. Whereas the biliary excretion of CZX was negligible, that of HCZX was substantial (up to 40% of the generated metabolite). Overall, 79-93% of the CZX dose (10-200 microM) was recovered in our model as CZX and HCZX. Additionally, HCZX accounted for 56% (200 microM) to 71% (10 microM) of the extracted CZX dose. Further, a preliminary study using the preformed HCZX showed a complete (100%) recovery of the metabolite as its conjugate. Therefore, the unrecovered portion of CZX dose in our study (7-21% of the administered dose or 29-44% of the extracted dose at inlet CZX concentrations of 10-200 microM) is most likely due to parallel metabolism of CZX to other metabolites.


Assuntos
Clorzoxazona/análogos & derivados , Clorzoxazona/metabolismo , Fígado/metabolismo , Animais , Bile/química , Biomarcadores , Citocromo P-450 CYP2E1 , Técnicas In Vitro , Masculino , Relaxantes Musculares Centrais/metabolismo , Perfusão , Ratos , Ratos Sprague-Dawley
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