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1.
Sci Transl Med ; 11(502)2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31341059

RESUMO

TYK2 is a nonreceptor tyrosine kinase involved in adaptive and innate immune responses. A deactivating coding variant has previously been shown to prevent receptor-stimulated activation of this kinase and provides high protection from several common autoimmune diseases but without immunodeficiency. An agent that recapitulates the phenotype of this deactivating coding variant may therefore represent an important advancement in the treatment of autoimmunity. BMS-986165 is a potent oral agent that similarly blocks receptor-stimulated activation of TYK2 allosterically and with high selectivity and potency afforded through optimized binding to a regulatory domain of the protein. Signaling and functional responses in human TH17, TH1, B cells, and myeloid cells integral to autoimmunity were blocked by BMS-986165, both in vitro and in vivo in a phase 1 clinical trial. BMS-986165 demonstrated robust efficacy, consistent with blockade of multiple autoimmune pathways, in murine models of lupus nephritis and inflammatory bowel disease, supporting its therapeutic potential for multiple immune-mediated diseases.

2.
J Med Chem ; 62(20): 8973-8995, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31318208

RESUMO

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

3.
J Pharm Biomed Anal ; 171: 30-34, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30959317

RESUMO

Conjugation of macromolecular drugs to polyethylene glycol (PEG) improves their therapeutic potential by reducing their rate of degradation, thereby extending the drugs half life. As a substantial component of the drug, it is necessary to measure the pharmacokinetic (PK) characteristics of PEG in vivo. A quantitative NMR-based method was developed and successfully applied to measuring double-branched polyethylene glycol 40 kDa (PEG40) in serum samples, enabling determination of PK parameters of PEG40 in preclinical species. NMR is ideal for measuring such polymers because a single, sharp peak is obtained for all the equivalent methylene protons; this amplifies the signal and makes the method insensitive to polymeric heterogeneity. High field NMR (600 MHz) with proton-observe cryoprobe technology allowed for analysis of samples in 300 nM range. Mice received 50 mg/kg of PEG40 intravenously (IV) and serum samples were collected at regular intervals for up to 72 h after dosing. The serum samples were analyzed for PEG40 using the NMR method and PK parameters were calculated using non-compartmental analysis. The volume of distribution was determined to be 0.17 L/kg for IV dosing, indicating limited distribution to interstitial space. A low clearance and observed half life of 18 h is consistent with previous reports on the PK properties of a variety of different PEG molecules ranging from 3 kDa to 190 kDa using 125I-labeled PEG in mice. The current NMR technique is easy to implement and does not require labeling of the PEG. Additionally, this is the first report, to our knowledge, of NMR spectroscopy application to PK profiling in serum.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Veículos Farmacêuticos/farmacocinética , Polietilenoglicóis/farmacocinética , Animais , Meia-Vida , Injeções Intravenosas , Limite de Detecção , Masculino , Camundongos , Veículos Farmacêuticos/administração & dosagem , Polietilenoglicóis/administração & dosagem
4.
CPT Pharmacometrics Syst Pharmacol ; 7(3): 135-146, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29349875

RESUMO

A cross-industry survey was conducted to assess the landscape of preclinical quantitative systems pharmacology (QSP) modeling within pharmaceutical companies. This article presents the survey results, which provide insights on the current state of preclinical QSP modeling in addition to future opportunities. Our results call attention to the need for an aligned definition and consistent terminology around QSP, yet highlight the broad applicability and benefits preclinical QSP modeling is currently delivering.


Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/normas , Farmacologia Clínica/métodos , Desenho de Drogas , Descoberta de Drogas/normas , Indústria Farmacêutica , Humanos , Modelos Biológicos , Farmacologia Clínica/normas , Inquéritos e Questionários
5.
Anal Chem ; 89(9): 5115-5123, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28383906

RESUMO

We demonstrate a novel strategy using affinity extraction (AE) LC-MS to directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay. The assay measures total drug and target concentration at the site of therapeutic action, as well as the amount of target bound to drug. The case study presented applies the strategy to measure drug engagement of a membrane bound receptor (CD40) that is critical to immune regulation in colon biopsies collected from monkey dosed with an anti-CD40 antibody. Unlike other techniques that measure receptor occupancy, such as flow cytometry, this technique does not rely on viable cells allowing measurement of frozen samples in a remote setting from the clinic.


Assuntos
Anticorpos/análise , Antígenos CD40/análise , Colo/química , Membrana Mucosa/química , Animais , Anticorpos/imunologia , Antígenos CD40/imunologia , Cromatografia de Afinidade/métodos , Humanos , Macaca fascicularis , Ratos , Espectrometria de Massas em Tandem/métodos
6.
J Med Chem ; 60(9): 3795-3803, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28418664

RESUMO

We have recently disclosed 5-phenyl-N-(pyridin-2-ylmethyl)-2-(pyrimidin-5-yl)quinazolin-4-amine 1 as a potent IKur current blocker with selectivity versus hERG, Na and Ca channels, and an acceptable preclinical PK profile. Upon further characterization in vivo, compound 1 demonstrated an unacceptable level of brain penetration. In an effort to reduce the level of brain penetration while maintaining the overall profile, SAR was developed at the C2' position for a series of close analogues by employing hydrogen bond donors. As a result, 5-[5-phenyl-4-(pyridin-2-ylmethylamino)quinazolin-2-yl]pyridine-3-sulfonamide (25) was identified as the lead compound in this series. Compound 25 showed robust effects in rabbit and canine pharmacodynamic models and an acceptable cross-species pharmacokinetic profile and was advanced as the clinical candidate. Further optimization of 25 to mitigate pH-dependent absorption resulted in identification of the corresponding phosphoramide prodrug (29) with an improved solubility and pharmacokinetic profile.


Assuntos
Fibrilação Atrial/tratamento farmacológico , Bloqueadores dos Canais de Potássio/uso terapêutico , Quinazolinas/uso terapêutico , Bloqueadores dos Canais de Sódio/uso terapêutico , Sulfonamidas/uso terapêutico , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Cães , Espectrometria de Massas , Bloqueadores dos Canais de Potássio/farmacologia , Espectroscopia de Prótons por Ressonância Magnética , Quinazolinas/química , Quinazolinas/farmacologia , Coelhos , Bloqueadores dos Canais de Sódio/farmacologia , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia
7.
ACS Med Chem Lett ; 7(9): 831-4, 2016 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-27660686

RESUMO

A new series of phenylquinazoline inhibitors of Kv 1.5 is disclosed. The series was optimized for Kv 1.5 potency, selectivity versus hERG, pharmacokinetic exposure, and pharmacodynamic potency. 5-Phenyl-N-(pyridin-2-ylmethyl)-2-(pyrimidin-5-yl)quinazolin-4-amine (13k) was identified as a potent and ion channel selective inhibitor with robust efficacy in the preclinical rat ventricular effective refractory period (VERP) model and the rabbit atrial effective refractory period (AERP) model.

8.
Bioanalysis ; 8(3): 193-204, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26811930

RESUMO

BACKGROUND: Therapeutic protein discovery study highlights the need for the development of quantitative bioanalytical methods for determining the levels of both the therapeutic protein and the target protein, as well. RESULTS: For the quantitation of BMS-986089, both accuracy (99-103%) and precision (2.4-12%) were obtained for the analysis of the surrogate peptide (ITYGGNSPVQEFTVPGR), in addition to the accuracy (100-108%) and precision (0.7-18%) that were obtained for the analysis of the surrogate peptide (VVSVLTVLHQDWLNGK). For Myostatin, accuracy (94-103%) and precision (2.4-14.9%) were obtained for the analysis of the surrogate peptide (IPAMVVDR). CONCLUSION: The developed method was applied to the analysis of samples following dosing of BMS-986089 to mice. This method highlights the potential of LC-MS/MS-based methods to eventually assess in vivo drug-target engagement.


Assuntos
Cromatografia Líquida/métodos , Miostatina/análise , Proteínas Recombinantes de Fusão/análise , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Animais , Cromatografia Líquida/economia , Análise Custo-Benefício , Humanos , Imunoglobulina G/análise , Imunoglobulina G/metabolismo , Imunoglobulina G/farmacologia , Imunoglobulina G/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Miostatina/metabolismo , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Ratos , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Espectrometria de Massas em Tandem/economia , Tripsina/metabolismo
9.
Bioorg Med Chem Lett ; 25(21): 4983-4986, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-25801931

RESUMO

Phenethyl aminoheterocycles like compound 1 were known to be potent I(Kur) blockers although they lacked potency in vivo. Modification of the heterocycle led to the design and synthesis of pseudosaccharin amines. Compounds such as 14, 17d and 21c were found to be potent K(V)1.5 blockers and selective over other cardiac ion channels. These compounds had potent pharmacodynamic activity, however, they also showed off-target activities such as hemodynamic effects.


Assuntos
Aminas/farmacologia , Canal de Potássio Kv1.5/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Aminas/síntese química , Aminas/química , Animais , Pressão Sanguínea/efeitos dos fármacos , Cicloexanos/química , Cicloexanos/farmacologia , Relação Dose-Resposta a Droga , Humanos , Canal de Potássio Kv1.5/metabolismo , Camundongos , Estrutura Molecular , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/química , Coelhos , Ratos , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade
10.
J Immunol Methods ; 419: 18-24, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25728474

RESUMO

Bioanalytical data from early human studies conducted in normal volunteers are often used for building pharmacokinetic/pharmacodynamic models that can predict outcomes of future studies in diseased patients. Thus, it is important to develop and validate reliable and accurate bioanalytical assays that instill confidence that the intended therapeutic species (total or free) are being measured. Assays quantifying the free therapeutic species, the partially bound (for multivalent therapeutics) and unbound species, require much more characterization than assays that quantify the total therapeutic species. We have developed an immunoassay to measure free BMS-962476, an Adnectin protein therapeutic against soluble proprotein convertase subtilisin kexin (PCSK)-9, and performed an in-depth characterization of the accuracy of this assay with the assistance of modeling. The experimental data correlates with modeled data within 15% at all clinically relevant levels of PCSK9 in normal and diseased populations.


Assuntos
Anticorpos Anti-Idiotípicos/imunologia , Anticorpos Monoclonais/imunologia , Imunoensaio/métodos , Modelos Imunológicos , Algoritmos , Anticorpos Anti-Idiotípicos/metabolismo , Anticorpos Monoclonais/metabolismo , Cinética , Ligantes , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacologia , Pró-Proteína Convertase 9 , Pró-Proteína Convertases/antagonistas & inibidores , Pró-Proteína Convertases/imunologia , Pró-Proteína Convertases/metabolismo , Ligação Proteica , Proteínas/imunologia , Proteínas/metabolismo , Proteínas/farmacologia , Reprodutibilidade dos Testes , Serina Endopeptidases/imunologia , Serina Endopeptidases/metabolismo
11.
J Med Chem ; 57(18): 7499-508, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25208139

RESUMO

G-protein-coupled receptor 119 (GPR119) is expressed predominantly in pancreatic ß-cells and in enteroendocrine cells in the gastrointestinal tract. GPR119 agonists have been shown to stimulate glucose-dependent insulin release by direct action in the pancreas and to promote secretion of the incretin GLP-1 by action in the gastrointestinal tract. This dual mechanism of action has generated significant interest in the discovery of small molecule GPR119 agonists as a potential new treatment for type 2 diabetes. Herein, we describe the discovery and optimization of a new class of pyridone containing GPR119 agonists. The potent and selective BMS-903452 (42) was efficacious in both acute and chronic in vivo rodent models of diabetes. Dosing of 42 in a single ascending dose study in normal healthy humans showed a dose dependent increase in exposure and a trend toward increased total GLP-1 plasma levels.


Assuntos
Descoberta de Drogas , Hipoglicemiantes/farmacologia , Terapia de Alvo Molecular , Piridonas/farmacologia , Receptores Acoplados a Proteínas-G/metabolismo , Sulfonas/farmacologia , Animais , Ensaios Clínicos como Assunto , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Desenho de Drogas , Hipoglicemiantes/química , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/uso terapêutico , Masculino , Camundongos , Modelos Moleculares , Conformação Proteica , Piridonas/química , Piridonas/farmacocinética , Piridonas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/química , Sulfonas/química , Sulfonas/farmacocinética , Sulfonas/uso terapêutico
12.
J Med Chem ; 57(5): 2013-32, 2014 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-24521299

RESUMO

The biphenyl derivatives 2 and 3 are prototypes of a novel class of NS5A replication complex inhibitors that demonstrate high inhibitory potency toward a panel of clinically relevant HCV strains encompassing genotypes 1-6. However, these compounds exhibit poor systemic exposure in rat pharmacokinetic studies after oral dosing. The structure-activity relationship investigations that improved the exposure properties of the parent bis-phenylimidazole chemotype, culminating in the identification of the highly potent NS5A replication complex inhibitor daclatasvir (33) are described. An element critical to success was the realization that the arylglycine cap of 2 could be replaced with an alkylglycine derivative and still maintain the high inhibitory potency of the series if accompanied with a stereoinversion, a finding that enabled a rapid optimization of exposure properties. Compound 33 had EC50 values of 50 and 9 pM toward genotype-1a and -1b replicons, respectively, and oral bioavailabilities of 38-108% in preclinical species. Compound 33 provided clinical proof-of-concept for the NS5A replication complex inhibitor class, and regulatory approval to market it with the NS3/4A protease inhibitor asunaprevir for the treatment of HCV genotype-1b infection has recently been sought in Japan.


Assuntos
Antivirais/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Imidazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/química , Antivirais/farmacocinética , Área Sob a Curva , Cães , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Hepacivirus/enzimologia , Hepacivirus/fisiologia , Imidazóis/química , Imidazóis/farmacocinética , Espectroscopia de Ressonância Magnética , Ratos , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-Atividade
13.
AAPS J ; 15(3): 717-27, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23588584

RESUMO

Antibodies or antibody-related fusion proteins binding to soluble antigens in plasma form an important subclass of approved therapeutics. Pharmaceutical companies are constantly trying to accelerate the pace of drug discovery and development of these antibodies and identify superior candidates in face of significant attrition rates. Understanding the interplay between drug- and target-related factors on magnitude and duration of target inhibition is imperative for successful advancement of these therapeutics. Simulations using a target-mediated drug disposition model were performed to evaluate the influence of antibody-target binding affinity, baseline target concentration, and target turnover on magnitude and duration of soluble target inhibition. These simulations assumed intravenous dosing of the antibody and evaluated multiple parameters over a wide range. These simulations reveal that improvement in affinity reaches a point of diminishing returns following which further improvement in affinity does not alter the magnitude and more importantly the duration of target inhibition. Evaluation of unbound antibody and target kinetics indicated that point of diminishing returns in duration of inhibition was due to target-mediated binding and subsequent elimination of antibody at later time points. Similarly, influence of baseline target concentration and target turnover on magnitude and duration of target inhibition in plasma is shown. Additionally, the fraction of dose eliminated via target mediated elimination (Fel(™)) can be a useful tool to enable selection of strategies to increase duration of target inhibition. The implications of these simulations in drug discovery and development with regard to target identification, antibody optimization, and backup candidate selection are discussed.


Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos/metabolismo , Simulação por Computador , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Afinidade de Anticorpos/fisiologia , Antígenos/imunologia , Modelos Imunológicos , Ligação Proteica/fisiologia
14.
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
15.
Drug Metab Lett ; 2(3): 169-75, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19356089

RESUMO

The SXR humanized mouse model was used to quantitatively assess an in vivo induction response of the human PXR agonist, rifampicin. Three days of rifampicin treatment increased RNA expression and microsomal enzyme activity of CYP3A11, as well as significantly reduced triazolam plasma exposure. These results indicate that the humanized SXR mouse can be used as a model to predict human CYP3A4 induction and the resulting pharmacokinetic changes of CYP3A4 substrates in humans.


Assuntos
Citocromo P-450 CYP3A/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Receptores de Esteroides/agonistas , Rifampina/farmacologia , Animais , Antibióticos Antituberculose/administração & dosagem , Antibióticos Antituberculose/farmacologia , Citocromo P-450 CYP3A/metabolismo , Relação Dose-Resposta a Droga , Interações de Medicamentos , Indução Enzimática/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Modelos Animais , Receptor de Pregnano X , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Rifampina/administração & dosagem , Especificidade da Espécie , Triazolam/farmacocinética
16.
Pharm Res ; 24(12): 2231-8, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17922174

RESUMO

PURPOSE: To evaluate the effectiveness of a liver-targeted dextran prodrug (DMP) of methylprednisolone (MP) in cold preservation-warm reperfusion injury associated with liver transplantation. METHODS: The effects of donor pretreatment with single 5 mg/kg doses of MP or DMP on ischemia-reperfusion damage to the liver were studied after 8 or 24 h of cold preservation in both isolated perfused rat liver (IPRL) and syngeneic orthotopic rat liver transplantation (OLT) models. RESULTS: In IPRL studies, donor pretreatment with DMP, and to a lesser degree MP, significantly improved the uptake of hyaluronic acid (HA), a marker of endothelial cell function, following 8 h of cold preservation. However, neither pretreatment was protective after 24 h of preservation. In the OLT model using 24-h-preserved livers, the seven-day survival of untreated grafts was 50%. DMP pretreatment of donors significantly improved graft survival to 100%, whereas MP pretreatment was ineffective. Additionally, only DMP significantly increased the blood glucose concentrations and decreased the plasma concentrations of tumor necrosis factor-alpha after OLT. Other measured markers of liver injury were not affected by either pretreatment. CONCLUSIONS: Selective delivery of methylprednisolone to the liver as a donor pretreatment strategy improves 24-h preserved graft survival in the OLT model.


Assuntos
Isquemia Fria/efeitos adversos , Dextranos/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Transplante de Fígado/efeitos adversos , Fígado/efeitos dos fármacos , Metilprednisolona/farmacologia , Pró-Fármacos/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Glicemia/efeitos dos fármacos , Dextranos/uso terapêutico , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ácido Hialurônico/metabolismo , Fígado/irrigação sanguínea , Fígado/metabolismo , Fígado/cirurgia , Masculino , Metilprednisolona/análogos & derivados , Metilprednisolona/uso terapêutico , Pró-Fármacos/uso terapêutico , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Fator de Necrose Tumoral alfa/sangue
17.
Transplantation ; 81(5): 678-85, 2006 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-16534468

RESUMO

BACKGROUND: The use of methylprednisolone (MP) and other corticosteroids for the treatment of acute liver allograft rejection is associated with severe toxicities in nontarget tissues. Therefore, selective delivery of MP to the liver may improve its efficacy and alleviate its side effects. We investigated the effects of a novel liver-targeted dextran prodrug of MP (DMP) in an orthotopic rat liver transplantation (OLT) model. METHODS: The model consisted of a high responder rejection strain combination (Dark Agouti donors and Lewis recipients). Liver recipients were intravenously administered saline or a single subtherapeutic dose of MP (5 mg/kg) as the parent drug (MP) or its prodrug (DMP). Different groups were then monitored for graft survival or euthanized 5 or 9 days posttransplantation. Plasma chemistry, including alkaline phosphatase and bilirubin, allograft histology, and survival duration were determined. RESULTS: Untreated recipients exhibited elevated plasma levels of liver injury markers, progressive portal and venous inflammation and cellular infiltration in liver allografts, and a mean graft survival time (MST) of 10.5 days. MP treatment did not alter any of these parameters. In contrast, a single dose of DMP resulted in a decrease in plasma levels of liver injury markers, a decrease in histological grade of rejection on day 5, and a substantial increase in MST (27.5 days). CONCLUSIONS: These results demonstrate attenuation of acute rejection following local (allograft) immunosuppression with a single subtherapeutic dose of MP delivered as a liver-targeted prodrug. Dextran prodrugs may be useful for selective delivery of immunosuppressants to the liver following liver transplantation.


Assuntos
Dextranos/administração & dosagem , Rejeição de Enxerto/prevenção & controle , Imunossupressores/administração & dosagem , Transplante de Fígado , Metilprednisolona/administração & dosagem , Pró-Fármacos/administração & dosagem , Doença Aguda , Fosfatase Alcalina/sangue , Animais , Bilirrubina/sangue , Proliferação de Células , Dextranos/uso terapêutico , Modelos Animais de Doenças , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/efeitos dos fármacos , Imunoglobulina M/sangue , Imunossupressores/uso terapêutico , Interleucina-2/análise , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Metilprednisolona/uso terapêutico , Pró-Fármacos/uso terapêutico , Ratos , Ratos Endogâmicos Lew , Baço/citologia , Baço/efeitos dos fármacos , Fator de Necrose Tumoral alfa/análise
18.
Pharm Res ; 21(6): 1000-8, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15212166

RESUMO

PURPOSE: Cold preservation of the liver before transplantation may change uptake and excretory functions of hepatocytes. We hypothesized that an increase in the duration of preservation would result in a progressive decrease in the hepatic uptake and/or biliary excretion of indocyanine green (ICG), which would be attenuated by pharmacologic interventions. METHODS: Donor rats (n = 40) were administered saline (control) or single 5 mg/kg doses of methylprednisolone (MP) or its liver-targeted prodrug (DMP) 2 h prior to liver harvest. Following preservation in cold University of Wisconsin solution for 0, 24, 48, or 72 h, livers were reperfused in a single-pass manner for 30 min in the presence of ICG (approximately 4 microg/ml), followed by 60 min of ICG-free perfusion. The inlet, outlet, and bile concentrations of ICG were measured periodically by high performance liquid chromatography (HPLC), and kinetic parameters were estimated. RESULTS: Effects of duration of preservation: In unpreserved livers, a significant portion of ICG dose (16%) was effluxed from the liver during the washout period. Cold preservation for 24-72 h progressively increased (p < 0.05) the efflux of ICG (>2-fold at 72 h). Similarly, average extraction ratio showed a modest (30-40%) decrease with increasing preservation time (p < 0.05). However, biliary excretion of ICG showed the most sensitivity to the preservation time (14 to >800-fold decline). Effects of pretreatment: DMP caused significant (p < 0.05) increases in biliary ICG levels (>12-fold) and bile flow rates (6-15-fold) of preserved livers. Although MP pretreatment significantly (p < 0.05) increased (6-fold) bile flow rates in 48-h preserved livers, its effects on biliary ICG levels were not significant (p > 0.05). CONCLUSIONS: Biliary excretion of ICG is the most sensitive kinetic parameter to prolonged cold ischemia-reperfusion injury in a rat liver perfusion model. The injury may be significantly attenuated by pharmacologic pretreatment of the liver donors.


Assuntos
Criopreservação , Dextranos/farmacocinética , Verde de Indocianina/metabolismo , Isquemia , Fígado , Hemissuccinato de Metilprednisolona/farmacocinética , Pró-Fármacos/farmacocinética , Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Alanina Transaminase/química , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/química , Aspartato Aminotransferases/metabolismo , Bile/química , Bile/efeitos dos fármacos , Bile/metabolismo , Dextranos/administração & dosagem , Dextranos/síntese química , Combinação de Medicamentos , Verde de Indocianina/farmacologia , L-Lactato Desidrogenase/química , L-Lactato Desidrogenase/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Falência Hepática , Transplante de Fígado/métodos , Hemissuccinato de Metilprednisolona/administração & dosagem , Hemissuccinato de Metilprednisolona/síntese química , Preservação de Órgãos/métodos , Soluções para Preservação de Órgãos , Perfusão/métodos , Pró-Fármacos/administração & dosagem , Pró-Fármacos/síntese química , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão , Fatores de Tempo , Doadores de Tecidos
19.
J Pharm Pharm Sci ; 7(1): 47-54, 2004 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-15144734

RESUMO

PURPOSE: To develop an isolated perfused rat liver model to study the hepatic disposition of cyclosporine A (CyA) in both sexes. METHODS: Livers were isolated from male (n = 6) and female (n = 7) rats and perfused with a physiological buffer in a single-pass manner. A bolus 1-mg dose of CyA was injected into the inlet catheter and periodical samples (0-15 min) were collected from the outlet perfusate. The concentrations of CyA in the outlet perfusate, collected bile (0-15 min), and liver tissue (at the end of perfusion) were quantitated by HPLC and subjected to statistical moment analysis. RESULTS: The dilution curves of CyA in the outlet perfusate exhibited unusually long terminal phases due to large volume of distribution of the drug (approximately 100 mL/g) and its slow release from binding sites in the liver (net release rate constant of approximately 0.020 min(-1)). This was in contrast to the rapid uptake of the drug, indicated by significant amounts of the intact drug (>40%) taken up during one single pass through the liver. Consequently, the liver tissue:perfusate distribution ratio of CyA was very high (approximately 220). No significant differences were found between the male and female livers in any of the estimated parameters. CONCLUSIONS: The tissue binding of cyclosporine A is substantial, slowly reversible, and gender-independent in isolated perfused rat livers.


Assuntos
Ciclosporina/farmacocinética , Fígado/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Imunossupressores/farmacocinética , Técnicas In Vitro , Masculino , Perfusão , Ratos , Ratos Sprague-Dawley , Fatores Sexuais
20.
Pharm Res ; 20(7): 1001-8, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12880285

RESUMO

PURPOSE: Activation of hepatic Kupffer cells (KCs) during organ preservation and subsequent reperfusion causes release of proinflammatory mediators and is responsible, at least in part, for rejection of transplanted livers. Our hypothesis was that donor pretreatment, before liver harvest, with methylprednisolone (MP) or its dextran prodrug (DMP) would reduce KC activation. METHODS: Adult donor rats were administered a single 5-mg/kg (MP equivalent) IV dose of MP or DMP or saline 2 h before liver harvest. The livers were then stored in University of Wisconsin solution for 24, 48, or 96 h (n = 4/treatment/time). A recirculating perfusion model was used to study, for 180 min, the release of KC activation markers, tumor necrosis factor (TNF)-alpha and acid phosphatase, and other biochemical indices from the cold-preserved livers. RESULTS: Cold ischemia-reperfusion resulted in release of substantial levels of TNF-alpha in untreated groups. Pretreatment of rats with MP or DMP caused a significant (p < 0.0001) reduction in TNF-alpha AUC in the perfusate, with no significant differences between MP and DMP. The maximum inhibitory effect of MP (77.5 +/- 10.2%) was observed after 48 h of preservation, whereas DMP showed maximal inhibition of TNF-alpha AUC at both 24 (74.5 +/- 15.8%) and 48 (74.8 +/- 12.6%) h of preservation. Similarly, both MP and DMP resulted in a significant (p < 0.0004) decrease in acid phosphatase levels of cold-preserved livers. However, neither pretreatment had any substantial effect on the levels of other biochemical markers. CONCLUSIONS: Both MP and DMP pretreatments decreased the release of TNF-alpha and acid phosphatase from livers subjected to cold ischemia preservation. Therefore, pretreatment of liver donors with MP or its prodrug decreases KC activation by cold ischemia-reperfusion.


Assuntos
Criopreservação/métodos , Macrófagos do Fígado/efeitos dos fármacos , Fígado , Metilprednisolona/farmacologia , Preservação de Órgãos/métodos , Pró-Fármacos/farmacologia , Animais , Macrófagos do Fígado/metabolismo , Substâncias Macromoleculares , Masculino , Ratos , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
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