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1.
Front Pharmacol ; 10: 1272, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31736754

RESUMO

Hypericum perforatum L., also known as Saint John's Wort, has been well studied for its chemical composition and pharmacological activity. In this study, the antiviral activities of H. perforatum on infectious bronchitis virus (IBV) were evaluated in vitro and in vivo for the first time. The results of in vitro experiments confirmed that the antiviral component of H. perforatum was ethyl acetate extraction section (HPE), and results showed that treatment with HPE significantly reduced the relative messenger ribonucleic acid (mRNA) expression and virus titer of IBV, and reduced positive green immunofluorescence signal of IBV in chicken embryo kidney (CEK) cells. HPE treatment at doses of 480-120 mg/kg for 5 days, reduced IBV induced injury in the trachea and kidney, moreover, reduced the mRNA expression level of IBV in the trachea and kidney in vivo. The mRNA expression levels of IL-6, tumor necrosis factor alpha (TNF-α), and nuclear factor kappa beta (NF-κB) significantly decreased, but melanoma differentiation-associated protein 5 (MDA5), mitochondrial antiviral signaling gene, interferon alpha (IFN-α), and interferon beta (IFN-ß) mRNA levels significantly increased in vitro and in vivo. Our findings demonstrated that HPE had significant anti-IBV effects in vitro and in vivo, respectively. In addition, it is possible owing to up-regulate mRNA expression of type I interferon through the MDA5 signaling pathway and down-regulate mRNA expression of IL-6 and TNF-α via the NF-κB signaling pathway. Moreover, the mainly active compositions of HPE analyzed by high-performance liquid chromatography/electrospray ionization-mass spectroscopy (ESI-MS) are hyperoside, quercitrin, quercetin, pseudohypericin, and hypericin, and a combination of these compounds could mediate the antiviral activities. This might accelerate our understanding of the antiviral effect of H. perforatum and provide new insights into the development of effective therapeutic strategies.

2.
J Cell Biochem ; 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31692102

RESUMO

Amplified in breast cancer 1 (AIB1) is overexpression in various cancers and promotes tumor cell proliferation, survival, and invasiveness. However, the role of AIB1 in the regulation of gastric cancer (GC) cell epithelial-mesenchymal transition (EMT) is still largely unclear. In the present study, immunohistochemistry showed that AIB1 was upregulated in our cohort of patients with GC and correlated with poor survival. Knockdown of AIB1 reduced the invasive ability of GC cells, downregulated the expression of epithelial cell marker E-cadherin, and upregulated mesenchymal cell marker vimentin. AIB1 overexpression elicited the opposite effect. PI-103, the inhibitor of the PI3K/AKT signaling, partially reversed AIB1 overexpression mediated a decrease in E-cadherin and an increase in vimentin. The present data demonstrated that AIB1 augmented the EMT via activation of PI3K/AKT signaling. In conclusion, our results suggested a novel role of AIB1 in GC invasion and EMT and raised the possibility of using this molecule as an indicator for GC treatment.

3.
Poult Sci ; 98(12): 6367-6377, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31399732

RESUMO

Avian infectious bronchitis virus (IBV), a coronavirus, causes infectious bronchitis leading to enormous economic loss in the poultry industry worldwide. Hypericin (HY) is an excellent compound that has been investigated in antiviral, antineoplastic, and antidepressant. To investigate the inhibition effect of HY on IBV infection in chicken embryo kidney (CEK) cells, 3 different experimental designs: pre-treatment of cells prior to IBV infection, direct treatment of IBV-infected cells, and pre-treatment of IBV prior to cell infection were used. Quantitative real-time PCR (qRT-PCR), immunofluorescence assay (IFA), flow cytometry, and fluorescence microscopy were performed and virus titer was determined by TCID50. The results revealed that HY had a good anti-IBV effect when HY directly treated the IBV-infected cells, and virus infectivity decreased in a dose-dependent manner. Furthermore, HY inhibited IBV-induced apoptosis in CEK cells, and significantly reduced the mRNA expression levels of Fas, FasL, JNK, Bax, Caspase 3, and Caspase 8, and significantly increased Bcl-2 mRNA expression level in CEK cells. In addition, HY treatment could decrease IBV-induced reactive oxygen species (ROS) generation in CEK cells. These results suggested that HY showed potential antiviral activities against IBV infection involving the inhibition of apoptosis and ROS generation in CEK cells.

4.
Curr Pharmacol Rep ; 4(4): 285-291, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30319936

RESUMO

Purpose of review: Monoclonal antibodies targeting key checkpoints in immune stimulatory pathways have over the last years become the mainstay of cancer immunotherapy. This article provides a brief review of the application and key impact of pharmacometrics and quantitative clinical pharmacology approaches in the development of these novel biologics. Recent findings: The clinical development and selection of optimal dosing regimens for monoclonal antibodies used in immune-oncology has been facilitated by an extensive application of pharmacometric approaches to characterize the exposure-response relationship for major efficacy and safety endpoints. These analysis techniques were applied for the anti CTLA-4 antibody ipilimumab, as well as the anti PD1/PD-L1 antibodies nivolumab, pembrolizumab, avelumab, atezolizumab and durvalumab. The utilization of quantitative clinical pharmacology, including model-based analyses, did not only support the identification of efficacious doses with acceptable safety limits, but was also able to address complicating challenges such as time- and response-dependent changes in antibody clearance as observed for most compounds. Summary: A widespread and systematic application of pharmacometric approaches has provided key aspects in elucidating, interpreting and integrating preclinical, biochemical and clinical data in support of the development of safe and efficacious dosing regimens of monoclonal antibodies used in immuno-oncology, thereby facilitating the clinical use of this promising new class of biologics in cancer patients with unmet medical needs.

5.
Nat Commun ; 9(1): 3492, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30154410

RESUMO

The Unc-51 like autophagy activating kinase 1 (ULK1) complex plays a central role in the initiation stage of autophagy. However, the function of ULK1 in the late stage of autophagy is unknown. Here, we report that ULK1, a central kinase of the ULK1 complex involved in autophagy initiation, promotes autophagosome-lysosome fusion. PKCα phosphorylates ULK1 and prevents autolysosome formation. PKCα phosphorylation of ULK1 does not change its kinase activity; however, it decreases autophagosome-lysosome fusion by reducing the affinity of ULK1 for syntaxin 17 (STX17). Unphosphorylated ULK1 recruited STX17 and increased STX17's affinity towards synaptosomal-associated protein 29 (SNAP29). Additionally, phosphorylation of ULK1 enhances its interaction with heat shock cognate 70 kDa protein (HSC70) and increases its degradation through chaperone-mediated autophagy (CMA). Our study unearths a key mechanism underlying autolysosome formation, a process in which the kinase activity of PKCα plays an instrumental role, and reveals the significance of the mutual regulation of macroautophagy and CMA in maintaining the balance of autophagy.


Assuntos
Autofagossomos/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Western Blotting , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia Eletrônica de Transmissão , Fosforilação , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Sinaptossomos/metabolismo
6.
Cereb Cortex ; 28(9): 3332-3346, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28968698

RESUMO

Axon growth is tightly controlled to establish functional neural circuits during brain development. Despite the belief that cytoskeletal dynamics is critical for cell morphology, how microtubule acetylation regulates axon development in the mammalian central nervous system remains unclear. Here, we report that loss of α-tubulin acetylation by ablation of MEC-17 in mice predisposes neurons to axon overbranching and overgrowth. Introduction of MEC-17F183A lacking α-tubulin acetyltransferase activity into MEC-17-deficient neurons failed to rescue axon defects. Moreover, loss of α-tubulin acetylation led to increases in microtubule debundling, microtubule invasion into filopodia and growth cones, and microtubule plus-end dynamics along the axon. Taxol application dampened microtubule hyperdynamics and suppressed axon overbranching and overgrowth in MEC-17-deficient neurons. Thus, our study reveals that α-tubulin acetylation acts as a brake for axon overbranching and overgrowth by dampening microtubule dynamics, providing insight into the role of microtubule post-translational modifications in regulating neural development.


Assuntos
Axônios/fisiologia , Microtúbulos/metabolismo , Neurogênese/fisiologia , Crescimento Neuronal/fisiologia , Tubulina (Proteína)/metabolismo , Acetilação , Acetiltransferases/deficiência , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microtúbulos/deficiência , Neurônios/metabolismo
7.
Zhongguo Zhen Jiu ; 37(8): 840-844, 2017 Aug 12.
Artigo em Chinês | MEDLINE | ID: mdl-29231344

RESUMO

OBJECTIVE: To observe the clinical effects differences and partial mechanism for chronic nonbacterial prostatitis (CNP) among drug oil moxibustion, simple moxibustion, and conventional western medicine. METHODS: A total of 120 patients who met the criteria of inclusion were randomly assigned into a drug oil moxibustion group, a moxibustion group and a western medication group, 40 cases in each one. Moxibustion was used at Guanyuan (CV 4), Zhongji (CV 3), Qihai (CV 6) and bilateral Yinlingquan (SP 9), Sanyinjiao (SP 6), Shenshu (BL 23), Mingmen (GV 4), Pangguangshu (BL 28), Ciliao (BL 32), and Zhibian (BL 54), etc. The same moxibustion was used at the same acupoints in the drug oil moxibustion group after external application of medicated oil. Thirty min treatment was used once a day in alternated abdomen and back. In the western medication group, oral tamsulosin hydrochloride capsules were applied once a day, one capsule at a time. All the treatment was given for 30 days. Chronic prostatitis symptom index from National Institutes for Health (NIH-CPSI), the contents of Zinc (Zn) and C-reactive protein (CRP), as well as the number of white blood cells (WBC) and density of lecithin bodies were observed before and after treatment and 1 month after treatment. The effects were evaluated after treatment. RESULTS: After treatment, the total effective rate of the drug oil moxibustion group was 90.0% (36/40), which was significantly higher than 72.5% (29/40) of the moxibustion group and 62.5% (25/40) of the western medication group (both P<0.05). After treatment and at follow-up in the three groups, the NIH-CPSI scores were lower than those before treatment (all P<0.05), and those in the drug oil moxibustion group were lower than the results in the moxibustion group and the western medication group (all P<0.05). The contents of Zn in the three groups were higher than those before treatment (all P<0.05), with better results in the drug oil moxibustion group (all P<0.05), and higher Zn contents in the moxibustion group compared with those in the western medication group (both P<0.05). The CRP levels were lower than those before treatment (all P<0.05), and those in the drug oil moxibustion group were better than those in the moxibustion group and western medication group (all P<0.05). The CRP contents in the moxibustion group were lower than those in the western medication group (both P<0.05). The number of WBC were lower than those before treatment (all P<0.05), with better results in the drug oil moxibustion group (all P<0.05). The concentrations of lecithin were higher than those before treatment (all P<0.05), with better results in the drug oil moxibustion group (all P<0.05). CONCLUSIONS: The clinical effect of drug oil moxibustion is better than those of simple moxibustion and western medicine, which has advantages in improving clinical symptoms, Zn, the density of lecithin body and decreasing CRP content and the number of WBC.


Assuntos
Proteína C-Reativa/análise , Moxibustão/métodos , Próstata/química , Prostatite/terapia , Sulfonamidas/administração & dosagem , Agentes Urológicos/administração & dosagem , Zinco/análise , Pontos de Acupuntura , Doença Crônica , Humanos , Contagem de Leucócitos , Masculino , Próstata/patologia , Prostatite/metabolismo , Prostatite/patologia , Tansulosina
8.
Lancet Haematol ; 4(2): e75-e82, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28159192

RESUMO

BACKGROUND: Kids B-LONG was a multicentre, open-label, phase 3 study assessing the safety, efficacy, and pharmacokinetics of recombinant factor IX Fc fusion protein (rFIXFc) in previously treated paediatric patients younger than 12 years with severe haemophilia B. METHODS: The study enrolled 30 previously treated boys younger than 12 years with haemophilia B (≤2 IU/dL [≤2%] endogenous coagulation factor IX [FIX] activity). All patients were initially given rFIXFc prophylaxis (50-60 IU/kg) once per week with adjustments to dose (≤100 IU/kg per infusion) or dosing frequency (up to two times per week) as needed. The primary outcome measure was development of inhibitors (neutralising antibodies). Secondary outcomes were pharmacokinetics, annual bleeding rate (ABR), spontaneous joint ABR, the number of infusions and dose required to resolve a bleed, time from last infusion of rFIXFc to a bleeding episode, assessment of response to treatment, and total annualised rFIXFc consumption for prevention and treatment of bleeding episodes. All patients underwent sequential pharmacokinetic evaluations of their prestudy FIX and rFIXFc. The completed trial is registered with ClinicalTrials.gov, number NCT01440946. FINDINGS: No patients developed inhibitors to rFIXFc; in the 30 enrolled patients the most common adverse events were nasopharyngitis (n=7; 23%) and fall (n=6; 20%); four patients (13%) had serious adverse events. Overall, rFIXFc exhibited a prolonged half-life of 68·6 h (95% CI 61·8-76·0), reduced clearance, and similar recovery compared with prestudy FIX. The median ABR was 2·0 (0·0-3·1) overall and 0·0 (0·0-0·0) for spontaneous joint bleeds; ten (33%) of 30 patients reported no bleeding, and 19 (63%) reported no joint bleeding on-study. The median average prophylactic dose of rFIXFc was 58·6 IU/kg (IQR 52·3-64·8) per week. Throughout the study, 29 (97%) of 30 patients remained on once per week infusions. INTERPRETATION: Weekly infusions of rFIXFc were well tolerated and resulted in low bleeding rates in children with severe haemophilia B. FUNDING: Biogen, Sobi.


Assuntos
Fator IX/uso terapêutico , Hemofilia B/tratamento farmacológico , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Criança , Pré-Escolar , Hemartrose , Hemorragia , Humanos , Masculino , Resultado do Tratamento
9.
Br J Clin Pharmacol ; 82(5): 1333-1342, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27333593

RESUMO

AIM: Daclizumab high yield process (HYP) is a humanized IgG1 monoclonal antibody that binds to the α-subunit of the interleukin-2 receptor and is being developed for treatment of multiple sclerosis (MS). This manuscript characterized the pharmacokinetic-pharmacodynamic (PK-PD) relationships of daclizumab HYP in subjects with MS. METHODS: Approximately 1400 subjects and 7000 PD measurements for each of three biomarkers [CD25 occupancy, CD56bright natural killer (NK) cell count, regulatory T cell (Treg) count] from four clinical trials were analyzed using non-linear mixed effects modelling. Evaluated regimens included 150 or 300 mg subcutaneous (s.c.) every 4 weeks. RESULTS: CD25 occupancy was characterized using a sigmoidal maximum response (Emax ) model. Upon daclizumab HYP treatment, CD25 saturation was rapid with complete saturation occurring after approximately 7 h and maintained when daclizumab HYP serum concentration was ≥5 mg l-1 . After the last 150 mg s.c. dose, unoccupied CD25 returned to baseline levels in approximately 24 weeks, with daclizumab HYP serum concentration approximately ≤1 mgl-1 1L. CD56bright NK cell expansion was characterized using an indirect response model. Following daclizumab HYP 150 mg s.c. every 4 weeks, expansion plateaus approximately at week 36, at which the average maximum expansion ratio is 5.2. After the last dose, CD56bright NK cells gradually declined to baseline levels within 24 weeks. Treg reduction was characterized by a sigmoidal Emax model. Average maximum reduction of 60% occurred approximately 4 days post 150 mg s.c. dose. After the last dose, Tregs were projected to return to baseline levels in approximately 20 weeks. CONCLUSIONS: Robust PK-PD models of CD25 occupancy, CD56bright NK cell expansion and Treg reduction by daclizumab HYP were developed to characterize its key pharmacodynamic effects in the target patient population.


Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/farmacocinética , Antígeno CD56/efeitos dos fármacos , Subunidade alfa de Receptor de Interleucina-2/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Esclerose Múltipla/sangue , Linfócitos T Reguladores/efeitos dos fármacos , Anticorpos Monoclonais Humanizados/sangue , Ensaios Clínicos como Assunto , Daclizumabe , Humanos , Células Matadoras Naturais/citologia , Contagem de Linfócitos , Dinâmica não Linear , Linfócitos T Reguladores/citologia
10.
Clin Pharmacokinet ; 55(8): 943-55, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26873229

RESUMO

BACKGROUND AND OBJECTIVES: Daclizumab high-yield process (HYP) is a humanized IgG1 monoclonal antibody that binds to the α-subunit (CD25) of the interleukin-2 receptor. The present work characterized the population pharmacokinetics of daclizumab HYP in healthy volunteers (HVs) and subjects with relapsing-remitting multiple sclerosis (RRMS) and evaluated the effects of covariates on daclizumab HYP exposure. METHODS: Measurable serum concentrations (n = 17,139) from 1670 subjects in seven clinical studies (three phase I, one immunogenicity, one phase II with extension, and one phase III) were included in this pharmacokinetic analysis using non-linear mixed-effects modeling. The three phase I studies evaluated single or multiple doses that ranged from 50 to 400 mg with either intravenous or subcutaneous (SC) administration in HVs (n = 71). The phase II with extension studies evaluated doses of 150 or 300 mg SC every 4 weeks (n = 567), and the immunogenicity (n = 113) and the phase III (n = 919) studies evaluated 150 mg SC every 4 weeks, all in RRMS patients. RESULTS: A two-compartment model with first-order absorption and elimination adequately described daclizumab HYP pharmacokinetics. Clearance (CL) was 0.212 L/day and the central volume of distribution was 3.92 L, scaled by [body weight (kg)/68] with exponents of 0.87 and 1.12, respectively. The peripheral volume of distribution was 2.42 L. Absorption lag time, mean absorption time, and absolute bioavailability (100-300 mg SC) were 1.61 h, 7.2 days, and 88 %, respectively. The daclizumab HYP terminal half-life was 21 days. Baseline CD25, age, and sex did not influence daclizumab HYP pharmacokinetics. Body weight explained 37 and 27 % of the inter-individual variability for CL and central volume of distribution, respectively. Neutralizing antibody (NAb)-positive status (included as a time-varying covariate) increased daclizumab HYP CL by 19 %. CONCLUSIONS: Consistent with previous findings in HVs, this analysis including extensive data from RRMS patients demonstrates that daclizumab HYP is characterized by slow CL, linear pharmacokinetics at doses above 100 mg, and high SC bioavailability. The pharmacokinetics of daclizumab HYP were not influenced by age (range 18-66 years), the sex of adult subjects, or the baseline CD4+CD25+ T cells (target level). The impact of covariates (body weight, NAb) on daclizumab HYP pharmacokinetics is unlikely to be clinically relevant.


Assuntos
Administração Intravenosa/métodos , Anticorpos Monoclonais Humanizados/farmacocinética , Imunossupressores/farmacocinética , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Receptores de Interleucina-2/efeitos dos fármacos , Adolescente , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/sangue , Disponibilidade Biológica , Daclizumabe , Relação Dose-Resposta a Droga , Feminino , Voluntários Saudáveis , Humanos , Imunoglobulina G , Imunossupressores/administração & dosagem , Imunossupressores/sangue , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Adulto Jovem
11.
Asian J Androl ; 17(6): 985-90, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26262775

RESUMO

Although paternal ethanol (EtOH) abuse has been shown to affect the growth and behavior of offspring, the exact molecular and mechanistic basis remains largely unclear. Methylation alterations in imprinted genes may be related to well-documented teratogenic effects of ethanol. Here we show that chronic paternal ethanol exposure increases the susceptibility to abnormal behavior in offspring through male game epigenetic alteration. In our study, different doses of ethanol (0, 1.1, 3.3 g kg-1 ) were administered intra-gastrically to male mice and decreased sperm motility was found in the highest ethanol-exposed group compared with the controls. Data also showed a dose-dependent increase in deaf mice of the paternally ethanol-exposed groups. The methylation of H19, Peg3, Ndn and Snrpn was assessed in paternal spermatozoa and in the cerebral cortices of deaf mice. EtOH affected methylation of Peg3 (CpG 3, 7 and 9) in paternal spermatozoa and in the cerebral cortices of deaf mice, but the level of mRNA expression did not change, suggesting that other gene regulation may be involved in these processes. Overall, chronic paternal ethanol exposure could alter the methylation of imprinted genes in sire spermatozoa that could also be passed on to offspring, giving rise to developmental disorders. Our results provide possible epigenetic evidence for a paternal ethanol exposure contribution to Fetal Alcohol Syndrome (FAS).


Assuntos
Anti-Infecciosos Locais/farmacologia , Córtex Cerebral/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Etanol/farmacologia , Impressão Genômica/efeitos dos fármacos , Audição/efeitos dos fármacos , Exposição Paterna , RNA Mensageiro/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Córtex Cerebral/metabolismo , Surdez/genética , Transtornos do Espectro Alcoólico Fetal/genética , Expressão Gênica/efeitos dos fármacos , Audição/genética , Fatores de Transcrição Kruppel-Like/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/genética , Masculino , Camundongos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/efeitos dos fármacos , Proteínas Nucleares/genética , RNA Longo não Codificante/efeitos dos fármacos , RNA Longo não Codificante/genética , RNA Mensageiro/metabolismo , Espermatozoides/metabolismo , Proteínas Centrais de snRNP/efeitos dos fármacos , Proteínas Centrais de snRNP/genética
12.
Expert Opin Drug Metab Toxicol ; 11(7): 1115-25, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25936400

RESUMO

INTRODUCTION: Assessments of the pharmacokinetic/pharmacodynamic (PK/PD) characteristics are an integral part in the development of novel therapeutic agents. Compared with traditional small molecule drugs, therapeutic proteins possess many distinct PK/PD features that necessitate the application of modified or separate approaches for assessing their PK/PD relationships. AREAS COVERED: In this review, the authors discuss tools that are utilized to describe and predict the PK/PD features of therapeutic proteins and that are valuable additions in the armamentarium of drug development approaches to facilitate and accelerate their successful preclinical and clinical development. EXPERT OPINION: A variety of state-of-the-art PK/PD tools is currently being applied and has been adjusted to support the development of proteins as therapeutics, including allometric scaling approaches, target-mediated disposition models, first-in-man dose calculations, physiologically based PK models and empirical and semi-mechanistic PK/PD modeling. With the advent of the next generation of biologics including bioengineered antibody constructs being developed, these tools will need to be further refined and adapted to ensure their applicability and successful facilitation of the drug development process for these novel scaffolds.


Assuntos
Desenho de Drogas , Modelos Biológicos , Proteínas/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Proteínas/farmacocinética , Proteínas/farmacologia
13.
Biomed Res Int ; 2015: 503692, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25629048

RESUMO

BACKGROUND: Aldehyde dehydrogenase 2 (ALDH2) plays a crucial role in myocardial protection against ischemia. Downregulation of ALDH2 was evidenced after myocardial infarction and the underlying mechanism is not fully understood. DNA methylation can regulate gene transcription in epigenetic level. We thus hypothesized that DNA methylation may affect ALDH2 expression in myocardial infarction (MI). METHODS: MI was induced in Sprague-Dawley rats. MI border zone tissues were harvested at 1st week, 2nd week, and 3rd week after MI. Bisulfite sequencing PCR (BSP) was performed to detect the methylation levels of ALDH2 core promoter. Sequenom MassARRAY platform (MassARRAY) was used to examine the methylation levels of ALDH2 promoter upstream sequence. ALDH2 protein and mRNA expression were assayed by Western blot and real-time PCR, respectively. RESULTS: Compared with Sham group, ALDH2 protein and mRNA expression of MI groups was significantly downregulated. Compared with Sham group, DNA methylation level of CpG sites in ALDH2 promoter upstream sequence was significantly higher in MI groups in a time-dependent manner (CpG1, CpG2, and CpG7, P < 0.01). DNA methylation did not affect ALDH2 core promoter sequence during the progress of MI. No significant difference was detected in DNA methylation level of ALDH2 promoter upstream sequence among MI groups. CONCLUSION: Aberrant hypermethylation of CpG sites in ALDH2 promoter upstream sequence is associated with myocardial ischemia injury and may partly result in ALDH2 downregulation after MI.


Assuntos
Aldeído Desidrogenase/genética , Metilação de DNA/genética , Proteínas Mitocondriais/genética , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Regiões Promotoras Genéticas , Aldeído-Desidrogenase Mitocondrial , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Western Blotting , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Decitabina , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo
14.
Zhonghua Yi Xue Za Zhi ; 94(16): 1223-6, 2014 Apr 29.
Artigo em Chinês | MEDLINE | ID: mdl-24924885

RESUMO

OBJECTIVE: To explore the HIRA gene sequences of 3'UTR region and elucidate the role of 3'UTR region of HIRA gene in the pathogenesis of tetralogy of Fallot (TOF). METHODS: Patients of TOF were confirmed by cardiac catheterization or surgery between April 2007 and December 2012 at our hospital. Mutations and single nucleotide polymorphisms (SNPs) were screened in 278 unrelated probands with isolated TOF and 515 controls. Target Scan was used to predict micro RNAs with possible combinations with 3'UTR region of HIRA gene. Dual-luciferase assay and real-time PCR were performed to detect the inhibition activity of micro RNAs on target genes. And χ(2) and t tests were used to analyze the results. RESULTS: Statistically significant change occurred in the alleleic frequencies of existing SNPs (rs:117447448) between TOF patients and control group (11.5% (32/278) vs 4.9% (25/515), P = 0.001) . The combining site of miR328 was predicted to be 10 bp upstream of SNP site. MiR328 was expressed in heart and it was related with myocardial infarction and atrial fibrillation. Dual-luciferase assay showed a decreased level of luciferase after co-transfection with miR328 (0.012 5 ± 0.000 6 vs 0.019 6 ± 0.003 8, P = 0.034). So was the expression of HIRA (1.039 6 ± 0.077 2 vs 1.608 7 ± 0.274 9, P = 0.037). However, the luciferase level was not affected by SNP (rs:117447448) (P = 0.380). CONCLUSIONS: The SNP (rs:117447448) of 3'UTR region of HIRA gene is related with TOF. HIRA is the target gene of miR328. Although SNP (rs:117447448) is not a major site of target gene HIRA for micro RNA328, it provides an important clue to in-depth studies of 3'UTR region of HIRA gene in the pathogenesis of TOF.


Assuntos
Proteínas de Ciclo Celular/genética , Chaperonas de Histonas/genética , MicroRNAs/genética , Tetralogia de Fallot/genética , Fatores de Transcrição/genética , Regiões 3' não Traduzidas/genética , Estudos de Casos e Controles , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Polimorfismo de Nucleotídeo Único
15.
Neuropharmacology ; 81: 126-33, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24486713

RESUMO

Research confirms that maternal ethanol (EtOH) exposure can induce physical and mental disorders in offspring, yet the effect of paternal ethanol exposure on offspring is unclear. Methylation alterations in imprinted genes may be related to the well-documented teratogenic effects of ethanol. Here, we report that ethanol (0, 1.1, 3.3 g/kg) was administered intragastrically to male mice and a behavioral study was performed on their F1 generation. Data show that F1 mice with fathers exposed to the highest dose of ethanol had delayed cognitive performance and increased anxiety and depression. A specific circling behavior was observed in the offspring of the paternally ethanol-exposed group. The degree of methylation and mRNA expression of H19, Peg3, Ndn and Snrpn were assessed in paternal sperm and in the cerebral cortices of each offspring. It did affect methylation in paternal sperm (H19 and Peg3) and in the offspring's cerebral cortices (CpG7 and CpG11 in Peg3 and Snrpn), but the level of mRNA expression has not changed. In the circling mice, the highest ethanol exposure increase in methylation (CpG 1, 2, 7 and 11) and decreases in mRNA of Peg3.Thus, chronic paternal ethanol exposure can affect the methylation of imprinted genes in sire sperm that may be passed on to offspring, giving rise to mental deficits.


Assuntos
Depressores do Sistema Nervoso Central/toxicidade , Deficiências do Desenvolvimento/induzido quimicamente , Etanol/toxicidade , Transtornos Mentais/induzido quimicamente , Exposição Paterna , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Animais , Córtex Cerebral , Ilhas de CpG/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Gravidez , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Espermatozoides/metabolismo , Espermatozoides/patologia
16.
Clin Pharmacokinet ; 53(5): 467-77, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24452809

RESUMO

BACKGROUND AND OBJECTIVES: Recombinant factor IX Fc fusion protein (rFIXFc) is a clotting factor developed using monomeric Fc fusion technology to prolong the circulating half-life of factor IX. The objective of this analysis was to elucidate the pharmacokinetic characteristics of rFIXFc in patients with haemophilia B and identify covariates that affect rFIXFc disposition. METHODS: Population pharmacokinetic analysis using NONMEM(®) was performed with clinical data from two completed trials in previously treated patients with severe to moderate haemophilia B. Twelve patients from a phase 1/2a study and 123 patients from a registrational phase 3 study were included in this population analysis. RESULTS: A three-compartment model was found to best describe the pharmacokinetics of rFIXFc. For a typical 73 kg patient, the clearance (CL), volume of the central compartment (V 1) and volume of distribution at steady state (V ss) were 2.39 dL/h, 71.4 dL and 198 dL, respectively. Because of repeat pharmacokinetic profiles at week 26 for patients in a subgroup, inclusion of inter-occasion variability (IOV) on CL and V 1 were evaluated and significantly improved the model. The magnitude of IOV on CL and V 1 were both low to moderate (<20 %) and less than the corresponding inter-individual variability. Body weight (BW) was found to be the only significant covariate for rFIXFc disposition. However, the impact of BW was limited, as the BW power exponents on CL and V 1 were 0.436 and 0.396, respectively. CONCLUSION: This is the first population pharmacokinetic analysis that systematically characterized the pharmacokinetics of long-lasting rFIXFc in patients with haemophilia B. The population pharmacokinetic model for rFIXFc can be utilized to evaluate and optimize dosing regimens for the treatment of patients with haemophilia B.


Assuntos
Fator IX/farmacocinética , Hemofilia B/metabolismo , Modelos Biológicos , Proteínas Recombinantes de Fusão/farmacocinética , Adolescente , Adulto , Idoso , Criança , Humanos , Fragmentos Fc das Imunoglobulinas , Pessoa de Meia-Idade , Proteínas Recombinantes de Fusão/sangue , Adulto Jovem
17.
Biochem J ; 458(1): 159-69, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24180524

RESUMO

The Hippo signalling pathway can suppress the Wnt/ß-catenin signalling pathway through the last downstream effectors YAP (Yes-associated protein)/TAZ (tafazzin). MST (mammalian sterile 20-like kinase) 1 functions as the upstream kinase of the Hippo pathway, and CK1ε (casein kinase 1ε) plays roles in the up-stream signal transduction of the Wnt/ß-catenin pathway. In the present study, using tandem affinity purification and MS analysis, CK1ε was identified as a novel partner of MST1. Further analysis showed that the interaction between MST1 and CK1ε was mediated by their kinase domains and enhanced by the activation of MST1. To exclude the interference of the phosphorylated YAP/TAZ, the transduction from MST1 to YAP/TAZ was blocked using anti-WW45 shRNA. In the sh-WW45 cells, MST1 still inhibited the Wnt3A-induced phosphorylation of DVL2 (dishevelled 2) and Wnt/ß-catenin signalling by disturbing the interaction of DVL2 and CK1ε. The growth-suppressive effect of MST1 in the presence of Wnt3A was effectively relieved by the downstream activation of the Wnt/ß-catenin pathway. Moreover, MST2, the close homologue of MST1, also displayed the similar function in suppressing the Wnt/ß-catenin pathway. Therefore the results of the present study revealed that, in addition to the phosphorylated YAP/TAZ, the Hippo pathway can suppress the Wnt/ß-catenin pathway directly through MST1/2.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Linhagem Celular , Cromatografia Líquida , Humanos , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem
18.
BJU Int ; 112(4): E415-23, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23879920

RESUMO

OBJECTIVE: To examine the prognostic significance of the expression of platelet-derived growth factor-BB (PDGF-BB) and differentiated microvascular density (MVD) in patients with clear cell renal cell carcinoma (ccRCC). PATIENTS AND METHODS: We used the vascular marker cluster of differentiation 34 (CD34) to identify tumour blood vessels. The expression of PDGF-BB and CD34 was detected by immunohistochemistry (IHC) in tissue microarrays (TMAs) from 100 ccRCCs. Prognostic effects of individual parameters were calculated using Cox regression models and Harrell's concordance index (c-index). RESULTS: Higher grade and more advanced stage ccRCCs had significantly less PDGF-BB expression and differentiated MVD (P < 0.05). Higher PDGF-BB expression was an independent prognostic factor for longer survival, and moreover, the final model built by the addition of PDGF-BB expression improved the predictive accuracy for disease-free survival (c-index 0.707) compared with the clinicopathological-based model (c-index 0.695). PDGF-BB expression was positively associated with differentiated MVD assessed by Spearman correlation and factor analysis (r = 0.634, P < 0.001). CONCLUSION: PDGF-BB is as a novel and promising prognostic marker and antiangiogenic therapeutic target for the treatment of ccRCC.


Assuntos
Carcinoma de Células Renais/irrigação sanguínea , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/irrigação sanguínea , Neoplasias Renais/metabolismo , Proteínas Proto-Oncogênicas c-sis/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Becaplermina , Carcinoma de Células Renais/patologia , Feminino , Humanos , Neoplasias Renais/patologia , Masculino , Microvasos , Pessoa de Meia-Idade , Metástase Neoplásica , Neovascularização Patológica , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Adulto Jovem
19.
Clin Pharmacokinet ; 52(10): 855-68, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23719681

RESUMO

Peptides, defined as polymers of less than 50 amino acids with a molecular weight of less than 10 kDa, represent a fast-growing class of new therapeutics which has unique pharmacokinetic characteristics compared to large proteins or small molecule drugs. Unmodified peptides usually undergo extensive proteolytic cleavage, resulting in short plasma half-lives. As a result of their low permeability and susceptibility to catabolic degradation, therapeutic peptides usually have very limited oral bioavailability and are administered either by the intravenous, subcutaneous, or intramuscular route, although other routes such as nasal delivery are utilized as well. Distribution processes are mainly driven by a combination of diffusion and to a lesser degree convective extravasation dependent on the size of the peptide, with volumes of distribution frequently not larger than the volume of the extracellular body fluid. Owing to the ubiquitous availability of proteases and peptidases throughout the body, proteolytic degradation is not limited to classic elimination organs. Since peptides are generally freely filtered by the kidneys, glomerular filtration and subsequent renal metabolism by proteolysis contribute to the elimination of many therapeutic peptides. Although small peptides have usually limited immunogenicity, formation of anti-drug antibodies with subsequent hypersensitivity reactions has been described for some peptide therapeutics. Numerous strategies have been applied to improve the pharmacokinetic properties of therapeutic peptides, especially to overcome their metabolic instability, low permeability, and limited tissue residence time. Applied techniques include amino acid substitutions, modification of the peptide terminus, inclusion of disulfide bonds, and conjugation with polymers or macromolecules such as antibody fragments or albumin. Application of model-based pharmacokinetic-pharmacodynamic correlations has been widely used for therapeutic peptides in support of drug development and dosage regimen design, especially because their targets are often well-described endogenous regulatory pathways and processes.


Assuntos
Peptídeos/farmacologia , Animais , Humanos , Imunomodulação , Peptídeos/farmacocinética , Peptídeos/uso terapêutico
20.
Mol Biol Rep ; 40(8): 5115-22, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23700295

RESUMO

Renal cell carcinoma (RCC) is the most common tumor arising from the cells in the lining of tubules in the kidney. Some members of the Ca2+-permeable transient receptor potential canonical (TRPC) family of channel proteins have demonstrated a role in the proliferation of some types of cancer cells. In this study, we investigated the role of TRPC6 in the development of human RCC. RT-PCR and Western blotting were used to investigate TRPC6 expression in 1932 and ACHN cells. Immunohistochemical techniques were applied to study TRPC6 expression in 60 cases of RCC primary tissue samples and 10 cases of corresponding normal renal tissues. To inhibit TRPC6 activity or expression, RNA interference was used. The effects of TRPC6 channels on RCC cell viability and cell cycle progression were investigated by MTT and flow cytometry. TRPC6 was expressed in 1932 and ACHN cells. TRPC6 protein was detected in 73.3% of RCC samples, and there was a significant difference compared with the normal renal samples (30%) (p<0.05). Moreover the level of TRPC6 expression was associated with RCC Fuhrman grade (p<0.01). Blockade of TRPC6 channels in ACHN cells suppressed basal cell proliferation and partially inhibited HGF-induced cell proliferation. Furthermore, inhibition of TRPC6 channels expression prolonged the transition through G2/M phase in ACHN cells. In summary, expression of TRPC6 is markedly increased in RCC specimens and associated with RCC histological grade. TRPC6 plays an important role in ACHN cells proliferation.


Assuntos
Carcinoma de Células Renais/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Canais de Cátion TRPC/metabolismo , Western Blotting , Carcinoma de Células Renais/fisiopatologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Primers do DNA/genética , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imuno-Histoquímica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canal de Cátion TRPC6 , Sais de Tetrazólio , Tiazóis
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