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1.
Toxicol Pathol ; 45(2): 321-334, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28013573

RESUMO

Detecting and monitoring exocrine pancreatic damage during nonclinical and clinical testing is challenging because classical biomarkers amylase and lipase have limited sensitivity and specificity. Novel biomarkers for drug-induced pancreatic injury are needed to improve safety assessment and reduce late-stage attrition rates. In a series of studies, miR-216a and miR-217 were evaluated as potential biomarkers of acute exocrine pancreatic toxicity in rats. Our results revealed that miR-216a and miR-217 were almost exclusively expressed in rat pancreas and that circulating miR-216a and miR-217 were significantly increased in rats following administration of established exocrine pancreatic toxicants caerulein (CL) and 1-cyano-2-hydroxy-3-butene (CHB) as well as in rats administered a proprietary molecule known to primarily affect the exocrine pancreas. Conversely, neither microRNA was increased in rats administered a proprietary molecule known to cause a lesion at the pancreatic endocrine-exocrine interface (EEI) or in rats administered an established renal toxicant. Compared with amylase and lipase, increases in miR-216a and miR-217 were of greater magnitude, persisted longer, and/or correlated better with microscopic findings within the exocrine pancreas. Our findings demonstrate that in rats, miR-216a and miR-217 are sensitive and specific biomarkers of acute exocrine pancreatic toxicity that may add value to the measurement of classical pancreatic biomarkers.


Assuntos
Insuficiência Pancreática Exócrina/sangue , MicroRNAs/sangue , Pâncreas Exócrino/efeitos dos fármacos , Doença Aguda , Alcenos/toxicidade , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Ceruletídeo/toxicidade , Insuficiência Pancreática Exócrina/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Nitrilas/toxicidade , Especificidade de Órgãos , Pâncreas Exócrino/metabolismo , Pâncreas Exócrino/patologia , Ratos Sprague-Dawley , Ratos Wistar , Sensibilidade e Especificidade
2.
Toxicol Pathol ; 45(2): 302-320, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27189632

RESUMO

Gastrointestinal toxicity is dose limiting with many therapeutic and anticancer agents. Real-time, noninvasive detection of markers of toxicity in biofluids is advantageous. Ongoing research has revealed microRNAs as potential diagnostic and predictive biomarkers for the detection of select organ toxicities. To study the potential utility of microRNA biomarkers of intestinal injury in a preclinical toxicology species, we evaluated 3 rodent models of drug-induced intestinal toxicity, each with a distinct mechanism of toxicity. MiR-215 and miR-194 were identified as putative intestinal toxicity biomarkers. Both were evaluated in plasma and feces and compared to plasma citrulline, an established intestinal injury biomarker. Following intestinal toxicant dosing, microRNA changes in feces and plasma were detected noninvasively and correlated with histologic evidence of intestinal injury. Fecal miR-215 and miR-194 levels increased, and plasma miR-215 decreased in a dose- and time-dependent manner. Dose-dependent decreases in plasma miR-215 levels also preceded and correlated positively with plasma citrulline modulation, suggesting miR-215 is a more sensitive biomarker. Moreover, during the drug-free recovery phase, plasma miR-215 returned to predose levels, supporting a corresponding recovery of histologic lesions. Despite limitations, this study provides preliminary evidence that select microRNAs have the potential to act as noninvasive, sensitive, and quantitative biomarkers of intestinal injury.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Fezes/química , Mucosa Intestinal/efeitos dos fármacos , MicroRNAs/sangue , Testes de Toxicidade/normas , Animais , Biomarcadores/análise , Biomarcadores/sangue , Relação Dose-Resposta a Droga , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/sangue , Masculino , MicroRNAs/análise , Ratos Wistar , Sensibilidade e Especificidade , Fatores de Tempo
3.
J Appl Toxicol ; 34(6): 695-702, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24114799

RESUMO

Despite of the attrition due to retinal toxicity during drug development there are no early reliable predictive biomarkers of retinal toxicity and this is increasingly becoming a concern. Thus far, in pharmacology and toxicology the technologies for assessing retinal side effects are limited to inconvenient visual behavioral tests, invasive electroretinograms or terminal histopathology. To address the lack of convenient early predictive retinal toxicity biomarkers, we explored a set of potential novel retinal enriched miRNAs in rats ex vivo and in vivo with known retinal toxicant pan-CDK inhibitors to assess circulating plasma miRNAs in rats and non-retinal toxicants as controls. Rats were administered a single intravitreal (IVT) injection and blood samples were collected pre-dose, various time points post-dose and then analyzed for five retinal enriched miRNAs (miR-96, miR-124a, miR181a, miR-182 and miR-183) by qRT-PCR. Ophthalmic exam, electroretinogram and histopathology were performed as confirmatory tests. All five miRNAs tested in retinal explants culture were highly expressed after pan-CDK inhibitor treatment. In vivo the pan-CDK inhibitors caused elevations of miR-96, miR-124a and miR-183 in blood. These results highly correlated with ocular exam, electroretinograms and microscopic findings. Comparatively, there were no changes in miRNA levels, electroretinograms, or histopathology in the negative control treatment groups. Although these miRNAs need additional confirmatory evaluation whether they truly predict retinal toxicity prior to clinical observations and histopathology, these results provide promise for further testing using additional retinal toxicants.


Assuntos
MicroRNAs/sangue , Inibidores de Proteínas Quinases/toxicidade , Retina/efeitos dos fármacos , Animais , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Eletrorretinografia , Marcadores Genéticos , Injeções Intravítreas , Masculino , Técnicas de Cultura de Órgãos , Inibidores de Proteínas Quinases/administração & dosagem , Ratos Wistar , Retina/enzimologia , Retina/patologia , Fatores de Tempo , Regulação para Cima
4.
PLoS One ; 8(6): e67258, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23826249

RESUMO

PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.


Assuntos
Antineoplásicos/farmacologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Neoplasias Colorretais/tratamento farmacológico , Mutação , Células-Tronco Neoplásicas/efeitos dos fármacos , Piridonas/farmacologia , Pirimidinas/farmacologia , Administração Oral , Adulto , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Masculino , Camundongos SCID , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Stem Cells Transl Med ; 2(3): 233-42, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23408105

RESUMO

Notch signaling mediates breast cancer cell survival and chemoresistance. In this report, we aimed to evaluate the antitumor efficacy of PF-03084014 in combination with docetaxel in triple-negative breast cancer models. The mechanism of action was investigated. PF-03084014 significantly enhanced the antitumor activity of docetaxel in multiple xenograft models including HCC1599, MDA-MB-231Luc, and AA1077. Docetaxel activated the Notch pathway by increasing the cleaved Notch1 intracellular domain and suppressing the endogenous Notch inhibitor NUMB. PF-03084014 used in combination with docetaxel reversed these effects and demonstrated early-stage synergistic apoptosis. Docetaxel elicited chemoresistance by elevating cytokine release and expression of survivin and induced an endothelial mesenchymal transition (EMT) phenotype by increasing the expressions of Snail, Slug, and N-cadherin. When reimplanted, the docetaxel-residual cells not only became much more tumorigenic, as evidenced by a higher fraction of tumor-initiating cells (TICs), but also showed higher metastatic potential compared with nontreated cells, leading to significantly shortened survival. In contrast, PF-03084014 was able to suppress expression of survivin and MCL1, reduce ABCB1 and ABCC2, upregulate BIM, reverse the EMT phenotype, and diminish the TICs. Additionally, the changes to the ALDH(+) and CD133(+)/CD44(+) subpopulations following therapy corresponded with the TIC self-renewal assay outcome. In summary, PF-03084014 demonstrated synergistic effects with docetaxel through multiple mechanisms. This work provides a strong preclinical rationale for the clinical utility of PF-03084014 to improve taxane therapy.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Mama/tratamento farmacológico , Antígeno AC133 , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Docetaxel , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Glicoproteínas/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos SCID , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Taxoides/administração & dosagem , Tetra-Hidronaftalenos/administração & dosagem , Fatores de Tempo , Valina/administração & dosagem , Valina/análogos & derivados , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cutan Ocul Toxicol ; 32(1): 46-53, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22867431

RESUMO

CONTEXT: A number of ocular conditions, such as dry eye, are associated with inflammation on the surface of the eye leading to irritation and ocular pain. Many drugs such as chemotherapeutics, beta blockers, angiotensin-converting enzymes and so forth also cause dry eye but currently there are no validated ocular surface biomarkers available. OBJECTIVE: We evaluated sample stability, assay sensitivity, reproducibility and overall performance of impression cytology (IC) utilizing the cellular surface biomarker human leukocyte antigen DR-1 (HLA-DR) as an ocular surface inflammatory biomarker by flow cytometry in a fit-for-purpose validation study. Additionally, subjects classified as normal or having various degrees of dry eye were evaluated to determine if HLA-DR could demonstrate a clear separation between normal and dry eye samples. RESULTS: The assay demonstrated high dynamic range detecting a broad range of fluorescent intensities in healthy donors. Additionally, inter, intra and stability assay results demonstrated strong concordance and low variability. Overall CV% for both assays were less than 25% for all measured parameters. However, high variability was observed for donor samples assayed beyond day 10 post IC sample collection (4.2-110.8 CV%). DISCUSSION: HLA-DR expression demonstrated a progressive increase in patients with mild to severe levels of dry eye disease providing sufficient evidence it is sensitive enough to monitor inflammatory effects of dry eye when coupled with additional biomarkers and/or methodologies such as cytokine analysis or ICAM-1. This biomarker can be used to monitor ocular surface disorders in patients and to evaluate potential treatment options during drug development. Although our results demonstrate this methodology is reproducible for routine evaluation, limitations around sample integrity exist. CONCLUSION: The ocular cell surface inflammatory biomarker, HLA-DR coupled with impression cytology is a simple non-invasive robust, specific and reproducible assay that can be utilized to measure inflammatory infiltrates on the surface of the eye in IC samples less than 10-days old.


Assuntos
Síndromes do Olho Seco/diagnóstico , Antígenos HLA-DR/metabolismo , Adulto , Biomarcadores/metabolismo , Túnica Conjuntiva/citologia , Síndromes do Olho Seco/metabolismo , Células Epiteliais/metabolismo , Citometria de Fluxo/métodos , Humanos , Inflamação/diagnóstico , Inflamação/metabolismo
7.
Toxicol Pathol ; 41(2): 271-9, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23242577

RESUMO

Eplerenone (Inspra) is an aldosterone receptor antagonist approved for the treatment of hypertension and heart failure after a myocardial infarction. In vitro receptor binding and transactivation studies showed eplerenone had high selectivity for the mineralocorticoid receptor over other steroid receptors (glucocorticoid, androgen, and progesterone). The most sensitive off-target effect of orally administered eplerenone preclinically was prostate atrophy in dogs. Dose-related prostate atrophy was observed at eplerenone dosages ≥15 mg/kg/day for 13 weeks or longer. The no observed adverse effect level (NOAEL) for the prostate effect in dogs was 5 mg/kg/day. The maximal effect was seen by 13 weeks and the atrophy was reversible even after 1 year of daily treatment. An additional study demonstrated dogs with eplerenone-induced prostate atrophy (confirmed by intrarectal ultrasound) had slightly decreased semen volume but no compound-related effects on libido, semen protein content, sperm motility, daily sperm production, or epididymal sperm transit time. Four possible mechanisms for prostate effect were investigated: (1) inhibition of testosterone synthesis and secretion; (2) inhibition of 5α-reductase, the enzyme within the prostate that converts testosterone into the more active growth factor dihydrotestosterone (DHT); (3) competitive antagonism of the androgen receptor; and (4) inhibition of 5α-reductase or competitive antagonism of the androgen receptor by aldosterone, which increased in dogs treated with eplerenone. Data from these studies supported blockade of androgen receptors at suprapharmacological concentrations of eplerenone. Another mineralocorticoid blocker, spironolactone, had greater antiandrogenic activity than eplerenone both in vivo and in vitro, and it has well known clinically significant antiandrogenic effects in humans, whereas eplerenone does not.


Assuntos
Antagonistas de Receptores de Mineralocorticoides/toxicidade , Próstata/efeitos dos fármacos , Espironolactona/análogos & derivados , Animais , Atrofia/induzido quimicamente , Di-Hidrotestosterona/metabolismo , Cães , Eplerenona , Finasterida/farmacologia , Histocitoquímica , Masculino , Antagonistas de Receptores de Mineralocorticoides/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Próstata/química , Próstata/metabolismo , Próstata/patologia , Ligação Proteica , Receptores Androgênicos/metabolismo , Espironolactona/metabolismo , Espironolactona/toxicidade
8.
Bioorg Med Chem Lett ; 22(24): 7605-9, 2012 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-23116892

RESUMO

(S)-1-((4-(3-(6-Amino-5-methoxypyridin-3-yl)-1-isopropyl-1H-pyrazol-4-yl)pyrimidin-2-yl)amino)propan-2-ol, 1, was recently identified as a potent inhibitor of the oncogenic kinase bRAF. Compounds containing 3-methoxy-2-aminopyridine, as in 1, comprised a promising lead series because of their high ligand efficiency and excellent ADME profile. However, following metabolic oxidation, compounds in this series also demonstrated two significant safety risks: mutagenic potential and time-dependent drug-drug interaction (TDI). Metabolite identification studies revealed formation of a reactive metabolite. We hypothesized that minimizing or blocking the formation of such a metabolite would mitigate the safety liabilities. Our investigation demonstrated that structural modifications which either reduced the electron density of the 3-methoxy-2-aminopyridine ring or blocked the reactive site following metabolic oxidation were successful in reducing TDI and AMES mutagenicity.


Assuntos
Aminopiridinas/química , Aminopiridinas/metabolismo , Elétrons , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Testes de Mutagenicidade , Oxirredução , Fatores de Tempo
9.
Int J Toxicol ; 31(6): 529-36, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23117654

RESUMO

Kidney injury biomarkers have been utilized by pharmaceutical companies as a means to assess the potential of candidate drugs to induce nephrotoxicity. Multiple platforms and assay methods exist, but the comparison of these methods has not been described. Millipore's Kidney Toxicity panel, EMD/Novagen's Widescreen Kidney Toxicity panel, and Meso Scales Kidney Injury panel were selected based on published information. Kidney injury molecule 1, cystatin C, clusterin, and osteopontin were the 4 biomarkers common among all kits tested and the focus of this study. Rats were treated with a low and high dose of para-aminophenol, a known nephrotoxicant, and urine samples were collected and analyzed on the Bio-Plex 200 or MSD's Sector Imager 6000, according to manufacturers specifications. Comparatively, of the 3 kits, Millipore was the most consistent in detecting elevations of 3 out of the 4 biomarkers at both dose levels and indicated time points.


Assuntos
Injúria Renal Aguda/urina , Moléculas de Adesão Celular/metabolismo , Clusterina/metabolismo , Cistatina C/metabolismo , Osteopontina/metabolismo , Xenobióticos/toxicidade , Injúria Renal Aguda/etiologia , Aminofenóis/toxicidade , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Receptor Celular 1 do Vírus da Hepatite A , Nefropatias , Masculino , Glicoproteínas de Membrana , Mutagênicos/toxicidade , Ratos , Ratos Wistar , Kit de Reagentes para Diagnóstico , Receptores Virais
10.
Mol Cancer Ther ; 11(10): 2274-83, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22752429

RESUMO

Clinical trials of selective RAF inhibitors in patients with melanoma tumors harboring activated BRAFV600E have produced very promising results, and a RAF inhibitor has been approved for treatment of advanced melanoma. However, about a third of patients developed resectable skin tumors during the course of trials. This is likely related to observations that RAF inhibitors activate extracellular signal-regulated kinase (ERK) signaling, stimulate proliferation, and induce epithelial hyperplasia in preclinical models. Because these findings raise safety concerns about RAF inhibitor development, we further investigated the underlying mechanisms. We showed that the RAF inhibitor PF-04880594 induces ERK phosphorylation and RAF dimerization in those epithelial tissues that undergo hyperplasia. Hyperplasia and ERK hyperphosphorylation are prevented by treatment with the mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitor PD-0325901 at exposures that extrapolate to clinically well-tolerated doses. To facilitate mechanistic and toxicologic studies, we developed a three-dimensional cell culture model of epithelial layering that recapitulated the RAF inhibitor-induced hyperplasia and reversal by MEK inhibitor in vitro. We also showed that PF-04880594 stimulates production of the inflammatory cytokine interleukin 8 in HL-60 cells, suggesting a possible mechanism for the skin flushing observed in dogs. The complete inhibition of hyperplasia by MEK inhibitor in epithelial tissues does not seem to reduce RAF inhibitor efficacy and, in fact, allows doubling of the PF-04880594 dose without toxicity usually associated with such doses. These findings indicated that combination treatment with MEK inhibitors might greatly increase the safety and therapeutic index of RAF inhibitors for the treatment of melanoma and other cancers.


Assuntos
Benzamidas/administração & dosagem , Benzamidas/farmacologia , Difenilamina/análogos & derivados , Epitélio/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Benzamidas/química , Difenilamina/administração & dosagem , Difenilamina/química , Difenilamina/farmacologia , Cães , Relação Dose-Resposta a Droga , Epiderme/efeitos dos fármacos , Epiderme/patologia , Epitélio/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HL-60 , Humanos , Hiperplasia , Interleucina-8/metabolismo , Camundongos , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Multimerização Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/metabolismo , Pirazóis/administração & dosagem , Pirazóis/química , Pirimidinas/administração & dosagem , Pirimidinas/química
11.
Toxicol Pathol ; 40(3): 482-90, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22395791

RESUMO

Although gastrointestinal (GI) toxicity is a significant dose-limiting safety concern noted in multiple therapeutic areas, there are no GI biomarkers that can accurately track, precede, or reliably correlate with histologic evidence of injury. While significant efforts have been made within the pharmaceutical industry, academia, and consortia to address the biomarker gaps in other target organs such as liver, kidney, and muscle (cardiac and skeletal), there have been no concerted efforts in the area of GI biomarkers. Using PAK4 inhibitor as a preclinical rat model of gastric toxicity, selected candidate biomarkers from literature were evaluated to test their usefulness as gastric injury biomarkers in this study. Biomarkers selected in this study include plasma diamino oxidase and citrulline, fecal calprotectin, bile acids, and miRNA. Based on the results, L-citrulline and miR-194 results appear to correlate well with histopathology findings. Although these biomarkers will need additional assay validation and qualification to test if they truly predict the injury prior to histopathology, the results provide promise for further testing using additional GI toxicants. In addition, this article highlights important gaps in GI biomarkers and provides substrate and rationale for additional investments either for further testing of already available biomarkers or to pursue extensive biomarker discovery approaches.


Assuntos
Inibidores Enzimáticos/toxicidade , Trato Gastrointestinal/efeitos dos fármacos , Testes de Toxicidade/métodos , Quinases Ativadas por p21/antagonistas & inibidores , Amina Oxidase (contendo Cobre)/sangue , Animais , Ácidos e Sais Biliares/análise , Biomarcadores/análise , Citrulina/sangue , Modelos Animais de Doenças , Fezes/química , Mucosa Gástrica/metabolismo , Trato Gastrointestinal/enzimologia , Trato Gastrointestinal/metabolismo , Histocitoquímica , Jejuno/química , Jejuno/efeitos dos fármacos , Jejuno/enzimologia , Jejuno/metabolismo , Complexo Antígeno L1 Leucocitário/análise , MicroRNAs/análise , Ratos , Ratos Wistar , Estômago/química , Estômago/efeitos dos fármacos , Estômago/enzimologia
12.
Toxicol Lett ; 204(2-3): 141-7, 2011 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-21557990

RESUMO

Checkpoint kinase 1 (Chk1) is required for both intra-S phase and G2/M checkpoints in cell cycle, and plays critical roles in maintaining genomic stability and transducing DNA damage response. Chk1 deficiency has been shown to inhibit T-cell differentiation and resulted in severe anemia in a Chk1 heterozygous mouse model. To date, there has been a good correlation between Chk1 inhibition and in vitro bone marrow toxicity among small molecule inhibitors. To better understand the role of Chk1 in hematopoiesis, we conducted transient Chk1 gene silencing in human bone marrow progenitor cells using siRNA and electroporation. At 48h post electroporation, approximately 70% inhibition of Chk1 was confirmed using real-time RT-PCR and immunoblotting, which resulted in more than 60% reduction in cell count when compared to the non-specific siRNA control on day 6 post-electroporation. This result was confirmed using a colony forming unit assay, where reduced number in both erythroid and granulocyte colonies was observed with Chk1 siRNA treatment. The Chk1 gene inhibition in bone marrow progenitor cells resulted in significant induction of apoptosis, but not cell cycle arrest, as assessed using flow cytometry. In this study an effective method to knock down a gene of interest was established in hard-to-transfect hematopoietic stem cells. Furthermore, our results support a direct role of Chk1 in maintaining normal hematopoiesis in the bone marrow.


Assuntos
Medula Óssea/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Proteínas Quinases/fisiologia , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Quinase 1 do Ponto de Checagem , Eletroporação , Hematopoese , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , RNA Interferente Pequeno/genética
13.
J Cardiovasc Pharmacol ; 53(6): 507-16, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19455053

RESUMO

ILLUMINATE, the phase 3 morbidity and mortality trial of the cholesteryl ester transfer protein (CETP) inhibitor, torcetrapib, plus atorvastatin terminated in 2006. The underlying morbidity and mortality cause remains undetermined. In addition to lipoprotein changes, off-target increases in blood pressure (BP), sodium, bicarbonate, and aldosterone and potassium decreases were described. We report nonclinical and clinical studies using torcetrapib and a closely related CETP inhibitor, CP-532,623, to further characterize this pharmacology. Pressor effects of torcetrapib and CP-532,623 were observed in monkeys and human subjects. CETP inhibition and high-density lipoprotein cholesterol elevation were demonstrated. In humans, high- versus low-dose CP-532,623 produced significantly greater pressor effects despite similar maximal CETP inhibition. Inhibition of CETP was seen 48 hours post dose, whereas BP elevation dissipated by 24 hours, temporally dissociating CETP inhibition from BP changes. These data, and structural similarities between the compounds, support the conclusion that the BP effects are related to chemotype. We also observed an acute aldosterone increase without changes in renin in monkeys. Continuous BP measurements showed persistent elevations, whereas aldosterone changes were transient, suggesting that increases in BP were not directly the result of renin-angiotensin-aldosterone system activation and may, in part, be due to direct effects on blood vessels or other nongenomic effects.


Assuntos
Anticolesterolemiantes/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Quinolinas/farmacologia , Adolescente , Adulto , Aldosterona/sangue , Animais , Anticolesterolemiantes/administração & dosagem , Anticolesterolemiantes/sangue , HDL-Colesterol/sangue , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Quinolinas/administração & dosagem , Quinolinas/sangue , Sistema Renina-Angiotensina/efeitos dos fármacos , Espectrometria de Massas em Tandem , Adulto Jovem
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