RESUMO
Antagonism of the calcitonin gene-related peptide (CGRP) receptor may be a useful approach for migraine treatment. Selective PEGylated peptide antagonists to the CGRP receptor are described, derived from CGRP(8-37) with polymer derivatization at an engineered lysine-25 residue. Potent PEGylated peptides with improved pharmacokinetics were identified through peptide side-chain modification to mitigate metabolic liabilities. PEGylated Ac-Trp-[Cit(11,18),hArg(24),Lys(25),Asp(31),Pro(34),1-Nal(35)]CGRP(8-37)-NH2, 9, elicits a dose-dependent reduction of intradermal CGRP-induced local blood flow in rodents with an ED50 of 0.52 mg kg(-1) without any overt adverse effects.
Assuntos
Peptídeo Relacionado com Gene de Calcitonina , Receptores de Peptídeo Relacionado com o Gene de CalcitoninaRESUMO
Cynomolgus monkeys dosed with a therapeutic monoclonal antibody (mAbY.1) at ≥ 50 mg/kg had unexpected acute thrombocytopenia (nadir ~3,000 platelets/µl), sometimes with decreases in red cell mass. Increased activated macrophages, mitotic figures, and erythrophagocytosis were observed in the spleen. Binding of mAbY.1 to cynomolgus peripheral blood cells could not be detected in vitro. mAbY.1 induced phagocytosis of platelets by peripheral blood monocytes from cynomolgus monkeys, but not from humans. mAbs sharing the same constant domain (Fc) sequences, but differing from mAbY.1 in their variable domains, bound competitively to and had similar biological activity against the intended target. None of these antibodies had hematologic liabilities in vitro or in vivo. Neither the F(ab')2 portion of mAbY.1 nor the F(ab')2 portion on an aglycosylated Fc (IgG1) framework caused phagocytosis of platelets in vitro. These data suggest that the hematologic effects of mAbY.1 in cynomolgus monkeys likely occurred through an off-target mechanism, shown to be driven by 1 to 3 amino acid differences in the light chain. The hematologic effects made mAbY.1 an unsuitable candidate for further development as a therapeutic agent. This example demonstrates that nonclinical safety studies may be essential for understanding off-target effects of mAbs prior to clinical trials.
Assuntos
Anemia/induzido quimicamente , Anticorpos Monoclonais/toxicidade , Trombocitopenia/induzido quimicamente , Anemia/sangue , Animais , Anticorpos Monoclonais/administração & dosagem , Plaquetas/patologia , Feminino , Humanos , Macaca fascicularis , Ativação de Macrófagos , Masculino , Fagocitose , Reticulócitos/patologia , Baço/efeitos dos fármacos , Baço/patologia , Trombocitopenia/sangueRESUMO
Human murine double minute 2 (MDM2) is a negative regulator of p53, which plays an important role in cell cycle and apoptosis. We report several optimizations to the synthesis of the chromenotriazolopyrimidine series of MDM2-p53 protein-protein interaction inhibitors. Additionally, the in vitro and in vivo stability, pharmacokinetic properties and solubility were improved through N-substitution.
Assuntos
Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Pirimidinas/síntese química , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Estabilidade de Medicamentos , Humanos , Bombas de Infusão , Concentração Inibidora 50 , Estrutura Molecular , Pirimidinas/farmacocinética , Ratos , Solubilidade , Relação Estrutura-AtividadeRESUMO
SAR exploration of the central diamine, benzyl, and terminal aminoalkoxy regions of the N-cyclic azaalkyl benzamide series led to the identification of very potent human urotensin-II receptor antagonists such as 1a with a K(i) of 4 nM. The synthesis and structure-activity relationships (SAR) of N-cyclic azaalkyl benzamides are described.
Assuntos
Benzamidas/química , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Sítios de Ligação , Química Farmacêutica/métodos , Diaminas/química , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Cinética , Modelos Químicos , Relação Estrutura-AtividadeRESUMO
To realize the medicinal potential of peptide toxins, naturally occurring disulfide-rich peptides, as ion channel antagonists, more efficient pharmaceutical optimization technologies must be developed. Here, we show that the therapeutic properties of multiple cysteine toxin peptides can be rapidly and substantially improved by combining direct chemical strategies with high-throughput electrophysiology. We applied whole-molecule, brute-force, structure-activity analoging to ShK, a peptide toxin from the sea anemone Stichodactyla helianthus that inhibits the voltage-gated potassium ion channel Kv1.3, to effectively discover critical structural changes for 15× selectivity against the closely related neuronal ion channel Kv1.1. Subsequent site-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000× over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmacological potential of the optimized toxin peptide was demonstrated by potent and sustained inhibition of cytokine secretion from T cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys.
Assuntos
Venenos de Cnidários/química , Canal de Potássio Kv1.3/antagonistas & inibidores , Peptídeos/química , Polietilenoglicóis/química , Animais , Células CHO , Venenos de Cnidários/farmacocinética , Venenos de Cnidários/farmacologia , Cricetulus , Cristalografia por Raios X , Cães , Células HEK293 , Humanos , Interferon gama/sangue , Interferon gama/metabolismo , Interleucina-17/sangue , Interleucina-17/metabolismo , Interleucina-2/sangue , Interleucina-2/metabolismo , Canal de Potássio Kv1.1/antagonistas & inibidores , Macaca fascicularis , Masculino , Camundongos , Simulação de Acoplamento Molecular , Técnicas de Patch-Clamp , Peptídeos/farmacocinética , Peptídeos/farmacologia , Ratos Sprague-Dawley , Especificidade da Espécie , Estereoisomerismo , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
BACKGROUND: AMG 517 or 1-aminobenzotriazole were quantified by LC-MS/MS from low blood/plasma volumes for rat pharmacokinetic (PK) characterization in order to qualify manual/automated dried blood spot (DBS) sampling and plasma separation capillary sampling. In addition, mouse serial automated blood sampling was compared with standard composite sampling. MATERIALS & METHODS: AMG 517 or 1-aminobenzotriazole was administered to rats or mice and multiple microsampling techniques were used to obtain blood or plasma. RESULTS: PK parameters derived from DBS and whole blood-obtained drug concentrations were within 7% for manual DBS and 20% for automated DBS. Plasma PK parameters derived from capillary or standard plasma-obtained drug concentrations differed by 6%. Plasma PK parameters obtained from serial automated blood sampling or manual composite sampling were within 20%. CONCLUSION: Collectively, these results suggest that the microsampling applications that were investigated are attractive approaches for quantifying drug candidates in low matrix volumes that can be successfully employed within discovery-stage rodent PK studies.
Assuntos
Benzotiazóis/sangue , Cromatografia Líquida de Alta Pressão/métodos , Pirimidinas/sangue , Espectrometria de Massas em Tandem/métodos , Triazóis/sangue , Animais , Benzotiazóis/farmacocinética , Coleta de Amostras Sanguíneas/métodos , Descoberta de Drogas , Masculino , Camundongos , Farmacocinética , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Triazóis/farmacocinéticaRESUMO
Acetyl-CoA carboxylase (ACC) is a target of interest for the treatment of metabolic syndrome. Starting from a biphenyloxadiazole screening hit, a series of piperazine oxadiazole ACC inhibitors was developed. Initial pharmacokinetic liabilities of the piperazine oxadiazoles were overcome by blocking predicted sites of metabolism, resulting in compounds with suitable properties for further in vivo studies. Compound 26 was shown to inhibit malonyl-CoA production in an in vivo pharmacodynamic assay and was advanced to a long-term efficacy study. Prolonged dosing with compound 26 resulted in impaired glucose tolerance in diet-induced obese (DIO) C57BL6 mice, an unexpected finding.
Assuntos
Acetil-CoA Carboxilase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Oxidiazóis/farmacologia , Piperazinas/farmacologia , Acetil-CoA Carboxilase/metabolismo , Animais , Dieta/efeitos adversos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Estrutura Molecular , Oxidiazóis/síntese química , Oxidiazóis/química , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-AtividadeRESUMO
The goal of this work was to obtain full pharmacokinetic profiles from individual mice with the use of an automated blood sampling system and dried blood spot (DBS) technique. AMG 517, a potent and selective vanilloid receptor (VR1) antagonist, was dosed to mice (n=3) intravenously and blood samples were collected using the automated blood sampling system with the "no blood waste" method. The collected blood samples were a mixture of 25 µL blood and 50 µL of heparinized saline solution. Two 15 µL aliquots were manually spotted onto a DBS card and dried at room temperature for at least 2h before being stored in zip bags with desiccant. The remaining samples (45 µL) were stored at -70°C until analysis. Both the DBS and the whole blood samples (diluted with saline (1:2, v/v)) were extracted and analyzed by liquid chromatography-tandem mass spectrometry. The overall extraction recovery of the analyte from the dried blood spots was determined to be about 90%. The pharmacokinetic parameters calculated using the whole blood or the DBS concentration data were comparable, and were obtained from only 3 mice, whereas conventional sampling and analysis would have required up to 27 mice to achieve the same result. The analyte was shown to be stable in the diluted whole blood (blood:saline 1:2) at room temperature for at least 4h and in the DBS for at least 34 days when stored at room temperature. These results indicated that the automated blood sampling system and DBS collection are promising techniques to obtain full pharmacokinetic profiles from individual mice and reduce the use of animals.
Assuntos
Benzotiazóis/sangue , Coleta de Amostras Sanguíneas/métodos , Cromatografia Líquida/métodos , Teste em Amostras de Sangue Seco/métodos , Pirimidinas/sangue , Espectrometria de Massas em Tandem/métodos , Animais , Benzotiazóis/administração & dosagem , Benzotiazóis/farmacocinética , Calibragem , Técnicas de Laboratório Clínico/métodos , Monitoramento de Medicamentos/métodos , Masculino , Camundongos , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
We have discovered novel benzofuran-based S1P1 agonists with excellent in vitro potency and selectivity. 1-((4-(5-Benzylbenzofuran-2-yl)-3-fluorophenyl)methyl) azetidine-3-carboxylic acid (18) is a potent S1P1 agonist with >1000× selectivity over S1P3. It demonstrated a good in vitro ADME profile and excellent oral bioavailability across species. Dosed orally at 0.3 mg/kg, 18 significantly reduced blood lymphocyte counts 24 h postdose and demonstrated efficacy in a mouse EAE model of relapsing MS.
RESUMO
Calcitonin gene-related peptide (CGRP) is a 37-residue neuropeptide that can be converted to a CGRP(1) receptor antagonist by the truncation of its first seven residues. CGRP(8-37), 1, has a CGRP(1) receptor K(i) = 3.2 nM but is rapidly degraded in human plasma (t(1/2) = 20 min). As part of an effort to identify a prolonged in vivo circulating CGRP peptide antagonist, we found that the substitution of multiple residues in the CGRP peptide increased CGRP(1) receptor affinity >50-fold. Ac-Trp-[Arg(24),Lys(25),Asp(31),Pro(34),Phe(35)]CGRP(8-37)-NH(2), 5 (K(i) = 0.06 nM) had the highest CGRP(1) receptor affinity. Using complimentary in vitro and in vivo metabolic studies, we iteratively identified degradation sites and prepared high affinity analogues with significantly improved plasma stability. Ac-Trp-[Cit(11,18),hArg(24),Lys(25),2-Nal(27,37),Asp(31),Oic(29,34),Phe(35)]CGRP(8-37)-NH(2), 32 (K(i) = 3.3 nM), had significantly increased (>100-fold) stability over 1 or 5, with a cynomolgus monkey and human in vitro plasma half-life of 38 and 68 h, respectively.
Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Química Farmacêutica/métodos , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Cinética , Macaca fascicularis , Masculino , Conformação Molecular , Dados de Sequência Molecular , Ligação ProteicaRESUMO
N,N'-diarylsquaramides were prepared and evaluated as antagonists of CXCR2. The compounds were found to be potent and selective antagonists of CXCR2. Significant differences in SAR was observed relative to the previously described N,N'-diarylurea series. As was the case in the N,N'-diarylurea series, placing sulfonamide substituent adjacent to the acidic phenol significantly reduced the clearance in rat pharmacokinetic studies.
Assuntos
Ciclobutanos/síntese química , Ciclobutanos/farmacologia , Ciclobutanos/farmacocinética , Receptores de Interleucina-8B/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/síntese química , Ureia/farmacologia , Ureia/farmacocinética , Animais , Células CHO , Fenômenos Químicos , Físico-Química , Cricetinae , Cricetulus , Indicadores e Reagentes , Espectrometria de Massas , Fenóis/química , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.
Assuntos
Integrina alfaVbeta3/antagonistas & inibidores , Fenilbutiratos/farmacologia , Fenilbutiratos/farmacocinética , Acetatos/química , Administração Oral , Aminopiridinas/química , Animais , Disponibilidade Biológica , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Meia-Vida , Humanos , Fenilbutiratos/química , RatosRESUMO
Bacterial enoyl-acyl carrier protein (ACP) reductase (FabI) catalyzes the final step in each elongation cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. High-throughput screening of the Staphylococcus aureus FabI enzyme identified a novel, weak inhibitor with no detectable antibacterial activity against S. aureus. Iterative medicinal chemistry and X-ray crystal structure-based design led to the identification of compound 4 [(E)-N-methyl-N-(2-methyl-1H-indol-3-ylmethyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide], which is 350-fold more potent than the original lead compound obtained by high-throughput screening in the FabI inhibition assay. Compound 4 has exquisite antistaphylococci activity, achieving MICs at which 90% of isolates are inhibited more than 500 times lower than those of nine currently available antibiotics against a panel of multidrug-resistant strains of S. aureus and Staphylococcus epidermidis. Furthermore, compound 4 exhibits excellent in vivo efficacy in an S. aureus infection model in rats. Biochemical and genetic approaches have confirmed that the mode of antibacterial action of compound 4 and related compounds is via inhibition of FabI. Compound 4 also exhibits weak FabK inhibitory activity, which may explain its antibacterial activity against Streptococcus pneumoniae and Enterococcus faecalis, which depend on FabK and both FabK and FabI, respectively, for their enoyl-ACP reductase function. These results show that compound 4 is representative of a new, totally synthetic series of antibacterial agents that has the potential to provide novel alternatives for the treatment of S. aureus infections that are resistant to our present armory of antibiotics.