RESUMO
The synthesis and structure-activity relationship studies on 5-trifluoromethylpyrido[4,3-d]pyrimidin-4(3H)-ones as antagonists of the human calcium receptor (CaSR) have been recently disclosed [ Didiuk et al. ( 2009 ) Bioorg. Med. Chem. Lett. 19 , 4555 - 4559 ). On the basis of its pharmacology and disposition attributes, (R)-2-(2-hydroxyphenyl)-3-(1-phenylpropan-2-yl)-5-(trifluoromethyl)pyrido[4,3-d]pyrimidin-4(3H)-one (1) was considered for rapid advancement to first-in-human (FIH) trials to mitigate uncertainty surrounding the pharmacokinetic/pharmacodynamic (PK/PD) predictions for a short-acting bone anabolic agent. During the course of metabolic profiling, however, glutathione (GSH) conjugates of 1 were detected in human liver microsomes in an NADPH-dependent fashion. Characterization of the GSH conjugate structures allowed insight(s) into the bioactivation pathway, which involved CYP3A4-mediated phenol ring oxidation to the catechol, followed by further oxidation to the electrophilic ortho-quinone species. While the reactive metabolite (RM) liability raised concerns around the likelihood of a potential toxicological outcome, a more immediate program goal was establishing confidence in human PK predictions in the FIH study. Furthermore, the availability of a clinical biomarker (serum parathyroid hormone) meant that PD could be assessed side by side with PK, an ideal scenario for a relatively unprecedented pharmacologic target. Consequently, progressing 1 into the clinic was given a high priority, provided the compound demonstrated an adequate safety profile to support FIH studies. Despite forming identical RMs in rat liver microsomes, no clinical or histopathological signs prototypical of target organ toxicity were observed with 1 in in vivo safety assessments in rats. Compound 1 was also devoid of metabolism-based mutagenicity in in vitro (e.g., Salmonella Ames) and in vivo assessments (micronuclei induction in bone marrow) in rats. Likewise, metabolism-based studies (e.g., evaluation of detoxicating routes of clearance and exhaustive PK/PD studies in animals to prospectively predict the likelihood of a low human efficacious dose) were also conducted, which mitigated the risks of idiosyncratic toxicity to a large degree. In parallel, medicinal chemistry efforts were initiated to identify additional compounds with a complementary range of human PK predictions, which would maximize the likelihood of achieving the desired PD effect in the clinic. The back-up strategy also incorporated an overarching goal of reducing/eliminating reactive metabolite formation observed with 1. Herein, the collective findings from our discovery efforts in the CaSR program, which include the incorporation of appropriate derisking steps when dealing with RM issues are summarized.
Assuntos
Anabolizantes/química , Anabolizantes/metabolismo , Osteoporose/tratamento farmacológico , Piridinas/química , Piridinas/metabolismo , Pirimidinonas/química , Pirimidinonas/metabolismo , Receptores de Detecção de Cálcio/antagonistas & inibidores , Anabolizantes/efeitos adversos , Animais , Cristalografia por Raios X , Humanos , Piridinas/efeitos adversos , Pirimidinonas/efeitos adversos , RatosRESUMO
Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.
Assuntos
Avaliação Pré-Clínica de Medicamentos/normas , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Preparações Farmacêuticas/metabolismo , Animais , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/estatística & dados numéricos , Humanos , Farmacocinética , Controle de QualidadeRESUMO
In vitro covalent binding assessments of drugs have been useful in providing retrospective insights into the association between drug metabolism and a resulting toxicological response. On the basis of these studies, it has been advocated that in vitro covalent binding to liver microsomal proteins in the presence and the absence of NADPH be used routinely to screen drug candidates. However, the utility of this approach in predicting toxicities of drug candidates accurately remains an unanswered question. Importantly, the years of research that have been invested in understanding metabolic bioactivation and covalent binding and its potential role in toxicity have focused only on those compounds that demonstrate toxicity. Investigations have not frequently queried whether in vitro covalent binding could be observed with drugs with good safety records. Eighteen drugs (nine hepatotoxins and nine nonhepatotoxins in humans) were assessed for in vitro covalent binding in NADPH-supplemented human liver microsomes. Of the two sets of nine drugs, seven in each set were shown to undergo some degree of covalent binding. Among hepatotoxic drugs, acetaminophen, carbamazepine, diclofenac, indomethacin, nefazodone, sudoxicam, and tienilic acid demonstrated covalent binding, while benoxaprofen and felbamate did not. Of the nonhepatotoxic drugs evaluated, buspirone, diphenhydramine, meloxicam, paroxetine, propranolol, raloxifene, and simvastatin demonstrated covalent binding, while ibuprofen and theophylline did not. A quantitative comparison of covalent binding in vitro intrinsic clearance did not separate the two groups of compounds, and in fact, paroxetine, a nonhepatotoxin, showed the greatest amount of covalent binding in microsomes. Including factors such as the fraction of total metabolism comprised by covalent binding and the total daily dose of each drug improved the discrimination between hepatotoxic and nontoxic drugs based on in vitro covalent binding data; however, the approach still would falsely identify some agents as potentially hepatotoxic.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Hepatócitos/efeitos dos fármacos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Testes de Toxicidade/métodos , Acetaminofen/química , Acetaminofen/metabolismo , Acetaminofen/farmacologia , Sítios de Ligação , Buspirona/química , Buspirona/metabolismo , Buspirona/farmacologia , Carbamazepina/química , Carbamazepina/metabolismo , Carbamazepina/farmacologia , Diclofenaco/química , Diclofenaco/metabolismo , Diclofenaco/farmacologia , Difenidramina/química , Difenidramina/metabolismo , Difenidramina/farmacologia , Relação Dose-Resposta a Droga , Hepatócitos/metabolismo , Humanos , Indometacina/química , Indometacina/metabolismo , Indometacina/farmacologia , Meloxicam , Microssomos Hepáticos/efeitos dos fármacos , Estrutura Molecular , Paroxetina/química , Paroxetina/metabolismo , Paroxetina/farmacologia , Piperazinas , Propranolol/química , Propranolol/metabolismo , Propranolol/farmacologia , Cloridrato de Raloxifeno/química , Cloridrato de Raloxifeno/metabolismo , Cloridrato de Raloxifeno/farmacologia , Sinvastatina/química , Sinvastatina/metabolismo , Sinvastatina/farmacologia , Relação Estrutura-Atividade , Tiazinas/química , Tiazinas/metabolismo , Tiazinas/farmacologia , Tiazóis/química , Tiazóis/metabolismo , Tiazóis/farmacologia , Ticrinafeno/química , Ticrinafeno/metabolismo , Ticrinafeno/farmacologia , Triazóis/química , Triazóis/metabolismoRESUMO
Higher-throughput ADME programs in early drug discovery are becoming common throughout the pharmaceutical industry as companies strive to reduce their compound attrition in later-stage development. Many of the ADME assays developed into higher-throughput formats rely on LC/MS analyses. Since the biological aspects of the assay are amenable to parallel processes using dense plate formats, the number of samples generated from these assays produce a large analysis load for serial LC/MS. Presented in this report are two novel strategies, including a sample pooling method and a two time-point method, that could be used in drug discovery to reduce the number of samples generated during multiple time-point in-vitro ADME assays. One hundred and sixty-three compounds were subjected to human microsomal incubations with full time-point method samples taken at t = 0, 5, 15, 30, and 45 min. The ER data correlation (R(2)) between the full time-point method and the pooling method and two time-point methods were 0.98 and 0.97, respectively. Both methods have the potential to: 1. produce data of similar quality to traditional high throughput ADME assays, 2. be easily implemented, 3. shorten analytical run times, and 4. be reproducible and robust.