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1.
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
2.
J Labelled Comp Radiopharm ; 60(3): 150-159, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27868223

RESUMO

Two regioisomeric glucuronide metabolites of dapagliflozin (BMS-512148) were synthesized and used to elucidate the structures of dapagliflozin metabolites observed in human urine samples. The structures of the synthetic metabolites were assigned by heteronuclear multiple-bond correlation, ROESY, and total correlation spectroscopy experiments. Analogues of these metabolites containing carbon-13 as a stable label were also prepared for use as internal standards for the analysis of urine samples obtained from patients participating in clinical studies.


Assuntos
Compostos Benzidrílicos/química , Compostos Benzidrílicos/síntese química , Glucosídeos/química , Glucosídeos/síntese química , Glucuronídeos/metabolismo , Compostos Benzidrílicos/metabolismo , Técnicas de Química Sintética , Glucosídeos/metabolismo , Marcação por Isótopo , Estereoisomerismo
3.
J Chromatogr A ; 1455: 133-139, 2016 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-27286648

RESUMO

UV spectrophotometry is widely used to determine the molar extinction coefficients (MECs) of cytotoxic drugs as well as the drug antibody ratios (DARs) of antibody drug conjugates (ADCs). However, the unknown purity of a drug due to interfering impurities can lead to erroneous MECs and DARs. Hence, reliable methods to accurately determine purity and the MECs of drugs with limited quantity is urgently needed in Drug Discovery. Such a method has been developed. It achieves absolute purity and accurate MEC determination by a single automated HPLC analysis that uses less than 5µg of material. Specifically, analytical HPLC separation with online UV detection was used to resolve impurities and measure absorbance from only the compound of interest. Simultaneously, an online chemiluminescence nitrogen detector (CLND) was used to determine the concentration of the analyte. The MECs were then calculated from the absorbance and concentration results. The accuracy of the method was demonstrated using caffeine and a commercial cytotoxic drug, DM1. This approach is particularly suited to analyzing mixtures or samples with low purities. Excellent reproducibility was demonstrated by analyzing a proprietary drug with linker synthesized from different batches with very different levels of purity. In addition, the MECs of drug with linker, along with ADC peak areas measured from size exclusion chromatography (SEC), were used to calculate DARs for 21 in-house ADCs. The DAR results were consistent with those obtained by MS analysis.


Assuntos
Anticorpos/química , Antineoplásicos Fitogênicos/química , Imunoconjugados/química , Maitansina/análogos & derivados , Anticorpos/imunologia , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Medições Luminescentes , Espectrometria de Massas , Maitansina/química , Nitrogênio/análise , Espectroscopia de Prótons por Ressonância Magnética , Espectrofotometria Ultravioleta
4.
Drug Metab Dispos ; 44(6): 809-20, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27029743

RESUMO

Daclatasvir is a first-in-class, potent, and selective inhibitor of the hepatitis C virus nonstructural protein 5A replication complex. In support of nonclinical studies during discovery and exploratory development, liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance were used in connection with synthetic and radiosynthetic approaches to investigate the biotransformation of daclatasvir in vitro and in cynomolgus monkeys, dogs, mice, and rats. The results of these studies indicated that disposition of daclatasvir was accomplished mainly by the release of unchanged daclatasvir into bile and feces and, secondarily, by oxidative metabolism. Cytochrome P450s were the main enzymes involved in the metabolism of daclatasvir. Oxidative pathways included δ-oxidation of the pyrrolidine moiety, resulting in ring opening to an aminoaldehyde intermediate followed by an intramolecular reaction between the aldehyde and the proximal imidazole nitrogen atom. Despite robust formation of the resulting metabolite in multiple systems, rates of covalent binding to protein associated with metabolism of daclatasvir were modest (55.2-67.8 pmol/mg/h) in nicotinamide adenine dinucleotide phosphate (reduced form)-supplemented liver microsomes (human, monkey, rat), suggesting that intramolecular rearrangement was favored over intermolecular binding in the formation of this metabolite. This biotransformation profile supported the continued development of daclatasvir, which is now marketed for the treatment of chronic hepatitis C virus infection.


Assuntos
Biotransformação/fisiologia , Imidazóis/metabolismo , Pirrolidinas/metabolismo , Animais , Bile/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Haplorrinos , Hepatócitos/metabolismo , Humanos , Macaca fascicularis , Espectroscopia de Ressonância Magnética/métodos , Masculino , Espectrometria de Massas/métodos , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Oxirredução , Ratos , Ratos Sprague-Dawley
6.
J Med Chem ; 57(18): 7550-64, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25101488
7.
Chem Res Toxicol ; 25(3): 556-71, 2012 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-22295996

RESUMO

A drug candidate, BMS-A ((N-(4-((1H-pyrrolo[2,3-b]pyridin-4-yl)oxy)-3-fluorophenyl)-1-(4-fluorophenyl) 2-oxo-1,2-dihydropyridine- 3-carboxamide)), was associated with dose- and time-dependent vacuolar degeneration and necrosis of the adrenal cortex following oral administration to rats. Pretreatment with 1-aminobenzotriazole (ABT), a nonspecific P450 inhibitor, ameliorated the toxicity. In vivo and in vitro systems, including adrenal cortex-derived cell lines, were used to study the mechanism responsible for the observed toxicity. Following an oral dose of the C-14 labeled compound, two hydroxylated metabolites of the parent (M2 and M3) were identified as prominent species found only in adrenal glands and testes, two steroidogenic organs. In addition, a high level of radioactivity was covalently bound to adrenal tissue proteins, 40% of which was localized in the mitochondrial fraction. ABT pretreatment reduced localization of radioactivity in the adrenal gland. Low levels of radioactivity bound to proteins were also observed in testes. Both M3 and covalent binding to proteins were found in incubations with mitochondrial fraction isolated from adrenal tissue in the presence of NADPH. In vitro formation of M3 and covalent binding to proteins were not affected by addition of GSH or a CYP11B1/2 inhibitor, metyrapone (MTY), but were inhibited by ketoconazole (KTZ) and a CYP11A1 inhibitor, R-(+)-aminoglutethimide (R-AGT). BMS-A induced apoptosis in a mouse adrenocortical cell line (Y-1) but not in a human cell line (H295R). Metabolite M3 and covalent binding to proteins were also produced in Y-1 and to a lesser extent in H295R cells. The cell toxicity, formation of M3, and covalent binding to proteins were all diminished by R-AGT but not by MTY. These results are consistent with a CYP11A1-mediated bioactivation to generate a reactive species, covalent binding to proteins, and subsequently rat adrenal toxicity. The thorough understanding of the metabolism-dependent adrenal toxicity was useful to evaluate cross-species adrenal toxicity potential of this compound and related analogues.


Assuntos
Glândulas Suprarrenais/efeitos dos fármacos , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/toxicidade , Piridinas/farmacocinética , Piridinas/toxicidade , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Animais , Radioisótopos de Carbono/farmacocinética , Radioisótopos de Carbono/toxicidade , Linhagem Celular , Humanos , Masculino , Camundongos , Inibidores de Proteínas Quinases/sangue , Piridinas/sangue , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual
8.
Chem Res Toxicol ; 24(5): 663-9, 2011 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-21417487

RESUMO

The thiophene moiety has been recognized as a toxicophore because of the potential of oxidative bioactivation leading to electrophilic species. The introduction of bulky or electron-withdrawing groups at the α-carbon to the sulfur atom has the potential to reduce or eliminate bioactivation. In this article, we describe the bioactivation of a variety of substituted thiophenes. These compounds were incubated in NADPH-fortified human liver microsomes with or without the addition of reduced glutathione (GSH) as a trapping agent. The resulting GSH adducts were characterized by high performance liquid chromatography/high resolution mass spectrometry with the aid of a background subtraction methodology. Four of the five α-chlorothiophenes tested formed NADPH-dependent GSH adducts. Most adducts had masses consistent with the nominal substitution of chlorine by GSH. LC/MS/MS and proton NMR of the major GSH adduct of 1-(5-chlorothiophen-2-yl)ethanone (1a) confirmed that GSH displaced chlorine. To further explore the effect of different substitutions on the bioactivation potential, a series of 2-acetylthiophenes substituted at the C4 or C5 positions were tested in a quantitative thiol-trapping assay using dansyl glutathione. Substitutions at the C4 or C5 positions gave adduct levels that decreased in the following order: 4-H, 5-H (no substitution) > 4-Br ∼ 4-Cl > 5-Cl > 5-CN > 4-CH(3) > 5-Br > 5-CH(3) (no adduct detected). In conclusion, bioactivation was detected in a series of substituted thiophenes. Although substitutions on the thiophene ring can reduce the formation of reactive metabolites, the degree of reduction is dependent on the substitution position and substituent.


Assuntos
Tiofenos/metabolismo , Cromatografia Líquida de Alta Pressão , Glutationa/química , Glutationa/metabolismo , Halogenação , Humanos , Espectrometria de Massas , Microssomos Hepáticos/metabolismo , Tiofenos/química
9.
Drug Metab Dispos ; 39(5): 891-903, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21289073

RESUMO

Brivanib [(R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[1,2,4]triazin-6-yloxy)propan-2-ol, BMS-540215] is a potent and selective dual inhibitor of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) signaling pathways. Its alanine prodrug, brivanib alaninate [(1R,2S)-2-aminopropionic acid 2-[4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy]-1-methylethyl ester, BMS-582664], is currently under development as an oral agent for the treatment of cancer. This study describes the in vivo biotransformation of brivanib after a single oral dose of [(14)C]brivanib alaninate to intact rats, bile duct-cannulated (BDC) rats, intact monkeys, BDC monkeys, and humans. Fecal excretion was the primary route of elimination of drug-derived radioactivity in animals and humans. In BDC rats and monkeys, the majority of radioactivity was excreted in bile. Brivanib alaninate was rapidly and completely converted via hydrolysis to brivanib in vivo. The area under the curve from zero to infinity of brivanib accounted for 14.2 to 54.3% of circulating radioactivity in plasma in animals and humans, suggesting that metabolites contributed significantly to the total drug-related radioactivity. In plasma from animals and humans, brivanib was a prominent circulating component. All the metabolites that humans were exposed to were also present in toxicological species. On the basis of metabolite exposure and activity against VEGF and FGF receptors of the prominent human circulating metabolites, only brivanib is expected to contribute to the pharmacological effects in humans. Unchanged brivanib was not detected in urine or bile samples, suggesting that metabolic clearance was the primary route of elimination. The primary metabolic pathways were oxidative and conjugative metabolism of brivanib.


Assuntos
Alanina/análogos & derivados , Antineoplásicos/metabolismo , Triazinas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Administração Oral , Alanina/administração & dosagem , Alanina/metabolismo , Alanina/farmacocinética , Alanina/urina , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/urina , Bile/metabolismo , Biotransformação , Fezes , Humanos , Macaca fascicularis , Masculino , Neoplasias/tratamento farmacológico , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Triazinas/administração & dosagem , Triazinas/farmacocinética , Triazinas/urina
10.
Chem Res Toxicol ; 24(1): 125-34, 2011 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-21080678

RESUMO

BMS-690514 ((3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4] triazin-5-yl)methyl)-3-piperidinol) is an oral oncologic agent being developed for the treatment of patients with advanced nonsmall cell lung cancer and breast cancer. The compound is metabolized via multiple metabolic pathways, including P450-mediated oxidation at one of the carbons of its pyrrolotriazine group. Oxidation at this site results in the formation of two metabolites, M1 and M37. Mass spectrometric and NMR analysis revealed that M1 underwent an unusual structural change, where the pyrrolotriazine moiety rearranged to yield a hydroxypyridotriazine group. In contrast, the structure of the pyrrolotriazine moiety remained intact in M37. In vitro experiments with liver microsomes and deuterated or tritiated BMS-690514 containing the isotopic label on the carbon that underwent oxidation indicated that during the formation of M1, the isotope label was retained at the site of hydroxylation, while the label was lost during the formation of M37. On the basis of these results, a mechanism for the formation of M1 was proposed as follows: BMS-690514 was first oxidized by P450 enzymes either via epoxidation or an iron-oxo addition pathway to form a zwitterionic intermediate. This was followed by opening of the pyrrolotriazine ring to form an aldehyde intermediate, which could be partially trapped with methoxyamine. The aldehyde intermediate then reacted with the secondary amine of the methoxyaniline group in the molecule to form the pyridotriazine moiety of M1. This mechanism is consistent with the observed retention of the isotope label in M1. Metabolite M37 may be formed either via a common zwitterionic intermediate, shared with M1, or through a direct insertion pathway. In in vitro human liver microsome incubations, the abundance of M1 was higher than M37, suggesting that breaking of the carbon-nitrogen bond to generate the aldehyde intermediate, a process similar to N-dealkylation, was a preferred pathway.


Assuntos
Antineoplásicos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Piperidinas/metabolismo , Pirróis/metabolismo , Triazinas/química , Antineoplásicos/química , Sistema Enzimático do Citocromo P-450/química , Humanos , Hidroxilação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Microssomos Hepáticos/enzimologia , Oxirredução , Piperidinas/química , Pirróis/química , Triazinas/metabolismo
11.
Bioanalysis ; 2(7): 1263-76, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21083239

RESUMO

Withdrawals from the market due to unforeseen adverse events have triggered changes in the way therapeutics are discovered and developed. This has resulted in an emphasis on truly understanding the efficacy and toxicity profile of new chemical entities (NCE) and the contributions of their metabolites to on-target pharmacology and off-target receptor-mediated toxicology. Members of the pharmaceutical industry, scientific community and regulatory agencies have held dialogues with respect to metabolites in safety testing (MIST); and both the US FDA and International Conference on Harmonisation have issued guidances with respect to when and how to characterize metabolites for human safety testing. This review provides a brief overview of NMR spectroscopy as applied to the structure elucidation and quantification of drug metabolites within the drug discovery and development process. It covers advances in this technique, including cryogenic cooling of detection circuitry for enhanced sensitivity, hyphenated LC-NMR techniques, improved dynamic range through new solvent-suppression pulse sequences and quantitation. These applications add to the already diverse NMR toolkit and further anchor NMR as a technique that is directly applicable to meeting the requirements of MIST guidelines.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Espectroscopia de Ressonância Magnética/métodos , Preparações Farmacêuticas/metabolismo , Testes de Toxicidade/métodos , Animais , Humanos , Espectroscopia de Ressonância Magnética/instrumentação , Preparações Farmacêuticas/análise , Solventes/química
12.
Drug Metab Dispos ; 38(7): 1189-201, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20363952

RESUMO

(3R,4R)-4-Amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f] [1,2,4]triazin-5-yl)methyl)-3-piperidinol (BMS-690514) is a potent inhibitor of human epidermal growth factor receptors 1, 2, and 4 and vascular endothelial growth factor receptors 1 through 3. BMS-690514 is an oral oncologic agent currently being developed for the treatment of patients with advanced non-small cell lung cancer and breast cancer. In this investigation, a series of studies was conducted to determine the biotransformation of [(14)C]BMS-690514 after oral administration to rats, rabbits, and dogs. After administration of a single oral dose of [(14)C]BMS-690514 to rats and dogs, the majority of the radioactive dose (61-71%) was recovered in the feces, whereas 18 to 20% was eliminated in urine. In bile duct-cannulated rats, 83 and 17% of the administered radioactivity was recovered in the bile and urine, respectively, suggesting that biliary secretion was a major route for the elimination of BMS-690514-derived radioactivity in rats. The parent compound underwent extensive metabolism in both species, with <12% of the administered radioactivity recovered as BMS-690514 in the excreta samples. Metabolite profiles in plasma were qualitatively similar in rats, rabbits, and dogs. Unchanged BMS-690514 was a prominent drug-related component in the plasma profiles from all the species. However, multiple metabolites contributed significantly to the circulating radioactivity, particularly for rabbit and dog, in which metabolites comprised 73 to 93% of the area under the time curve (0-8 h). Circulating metabolites included M6, a direct O-glucuronide conjugate; M1, a hydroxylated metabolite; and glucuronide conjugates of hydroxylated and O-demethylated metabolites. Overall, the results from these studies suggested that BMS-690514 was well absorbed and highly metabolized through multiple pathways in these preclinical species.


Assuntos
Antineoplásicos/farmacocinética , Piperidinas/farmacocinética , Pirróis/farmacocinética , Triazinas/farmacocinética , Administração Oral , Animais , Bile/metabolismo , Biotransformação , Radioisótopos de Carbono/metabolismo , Cães , Feminino , Masculino , Redes e Vias Metabólicas , Piperidinas/administração & dosagem , Pirróis/administração & dosagem , Coelhos , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Triazinas/administração & dosagem
13.
Drug Metab Dispos ; 37(5): 1115-28, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19196843

RESUMO

3'-tert-Butyl-3'-N-tert-butyloxycarbonyl-4-deacetyl-3'-dephenyl-3'-N-debenzoyl-4-O-methoxy-paclitaxel (BMS-275183) is a taxane analog that has the potential for oral use in the treatment of various types of cancer. In this study, the metabolism and excretion of [(14)C]BMS-275183 were evaluated after a single oral administration of [(14)C]BMS-275183 to rats and dogs (15 and 1 mg/kg, respectively). To aid metabolite identification by mass spectrometry (MS), a stable labeled (phenyl-(13)C(6)) BMS-275183 was included in 1:1 ratio of (13)C(6)/(12)C in the dose administration. Fecal excretion was the major route of elimination for [(14)C]BMS-275183 in both species (85-86 and <9% of the dose in feces and urine, respectively). The highest radioactivity in plasma was observed at 1 h postdose, suggesting rapid absorption of the drug in both species. The total radioactivity in plasma was measurable up to 24 h postdose. Metabolites were identified by liquid chromatography-MS and/or NMR spectroscopy. [(14)C]BMS-275183 was the prominent component in rat and dog plasma and was detected up to 24 h along with various oxidative and hydrolytic metabolites. [(14)C]BMS-275183 was extensively metabolized in both species, forming mainly oxidative metabolites, and unchanged parent drug accounted for <3.5% of the administered dose in urine and feces. The prominent metabolites resulted from oxidation of the tert-butyl groups on the side chain and further oxidation and cyclization of the tert-butylhydroxylated metabolites. A total of 30 oxidative metabolites including M13, a prominent ester cleavage metabolite, were identified in rat and dog samples.


Assuntos
Antineoplásicos Fitogênicos/farmacocinética , Hidrocarbonetos Aromáticos com Pontes/farmacocinética , Animais , Biotransformação , Cromatografia Líquida de Alta Pressão , Cães , Fezes/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Ratos , Especificidade da Espécie
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