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1.
Drug Metab Lett ; 11(2): 119-127, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29189189

RESUMO

BACKGROUND: Epidermal Growth Factor (EGF) is a well-known mitogen that has importance in cell proliferation and differentiation. This property has led to the common use of EGF as an additive to some cell culture media. EGF has been previously shown to modulate constitutive Cytochrome P450 (CYP) expression in vitro. OBJECTIVES: To assess the influence of EGF on the basal and induced expression of CYP3A4, CYP1A2 and CYP2B6 in plated human hepatocytes. METHODS: Human hepatocytes were treated with EGF with and without in the presence of positive control inducers. After treatment, CYP isoform mRNA expression and enzyme activity were measured. RESULTS: EGF at concentrations ranging from 0.001-500 ng/mL resulted in a concentration-dependent decrease in basal CYP3A4 catalytic activity by up to 92%. In contrast, rifampicin (RIF)-induced activity was decreased only slightly (up to 23%). CYP3A4 mRNA also decreased in an EGF concentrationdependent manner. In contrast to CYP3A4, CYP1A2 and CYP2B6 activity and mRNA were either not suppressed or suppressed to a lower extent. The preferential effect with CYP3A4 was confirmed in 4 additional donors using a single concentration of EGF (10 ng/mL) and time-dependence experiments revealed that suppression appeared after only 24h of treatment. CONCLUSION: Because of the larger effect on the basal CYP3A4 compared to the induced response, EGF as a media additive enables a higher dynamic range in a CYP3A4 induction assay, potentially expanding the range of donor hepatocytes suitable for use in induction studies. These findings also suggest that EGF may be an important regulator of CYP3A4 expression in vivo.


Assuntos
Meios de Cultura/metabolismo , Inibidores do Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP3A/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Hepatócitos/enzimologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/genética , Indutores do Citocromo P-450 CYP3A/farmacologia , Descoberta de Drogas/métodos , Humanos , RNA Mensageiro/metabolismo , Rifampina/farmacologia
2.
Drug Metab Dispos ; 42(9): 1379-91, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24924386

RESUMO

Cytochrome P450 (P450) induction is often considered a liability in drug development. Using calibration curve-based approaches, we assessed the induction parameters R3 (a term indicating the amount of P450 induction in the liver, expressed as a ratio between 0 and 1), relative induction score, Cmax/EC50, and area under the curve (AUC)/F2 (the concentration causing 2-fold increase from baseline of the dose-response curve), derived from concentration-response curves of CYP3A4 mRNA and enzyme activity data in vitro, as predictors of CYP3A4 induction potential in vivo. Plated cryopreserved human hepatocytes from three donors were treated with 20 test compounds, including several clinical inducers and noninducers of CYP3A4. After the 2-day treatment, CYP3A4 mRNA levels and testosterone 6ß-hydroxylase activity were determined by real-time reverse transcription polymerase chain reaction and liquid chromatography-tandem mass spectrometry analysis, respectively. Our results demonstrated a strong and predictive relationship between the extent of midazolam AUC change in humans and the various parameters calculated from both CYP3A4 mRNA and enzyme activity. The relationships exhibited with non-midazolam in vivo probes, in aggregate, were unsatisfactory. In general, the models yielded better fits when unbound rather than total plasma Cmax was used to calculate the induction parameters, as evidenced by higher R(2) and lower root mean square error (RMSE) and geometric mean fold error. With midazolam, the R3 cut-off value of 0.9, as suggested by US Food and Drug Administration guidance, effectively categorized strong inducers but was less effective in classifying midrange or weak inducers. This study supports the use of calibration curves generated from in vitro mRNA induction response curves to predict CYP3A4 induction potential in human. With the caveat that most compounds evaluated here were not strong inhibitors of enzyme activity, testosterone 6ß-hydroxylase activity was also demonstrated to be a strong predictor of CYP3A4 induction potential in this assay model.


Assuntos
Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Indução Enzimática/genética , Área Sob a Curva , Calibragem , Células Cultivadas , Criopreservação/métodos , Hepatócitos/metabolismo , Humanos , Oxigenases de Função Mista/metabolismo , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/metabolismo , RNA Mensageiro/genética , Testosterona/metabolismo , Estados Unidos , United States Food and Drug Administration
3.
Cancers (Basel) ; 3(1): 478-93, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21603150

RESUMO

Bone is the most common site for metastasis in human prostate cancer patients. Skeletal metastases are a significant cause of morbidity and mortality and overall greatly affect the quality of life of prostate cancer patients. Despite advances in our understanding of the biology of primary prostate tumors, our knowledge of how and why secondary tumors derived from prostate cancer cells preferentially localize bone remains limited. The physiochemical properties of bone, and signaling molecules including specific chemokines and their receptors, are distinct in nature and function, yet play intricate and significant roles in prostate cancer bone metastasis. Examining the impact of these facets of bone metastasis in vivo remains a significant challenge, as animal models that mimic the natural history and malignant progression clinical prostate cancer are rare. The goals of this article are to discuss (1) characteristics of bone that most likely render it a favorable environment for prostate tumor cell growth, (2) chemokine signaling that is critical in the recruitment and migration of prostate cancer cells to the bone, and (3) current animal models utilized in studying prostate cancer bone metastasis. Further research is necessary to elucidate the mechanisms underlying the extravasation of disseminated prostate cancer cells into the bone and to provide a better understanding of the basis of cancer cell survival within the bone microenvironment. The development of animal models that recapitulate more closely the human clinical scenario of prostate cancer will greatly benefit the generation of better therapies.

4.
Phys Rev Lett ; 97(5): 050504, 2006 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-17026087

RESUMO

Quantum simulation uses a well-known quantum system to predict the behavior of another quantum system. Certain limitations in this technique arise, however, when applied to specific problems, as we demonstrate with a theoretical and experimental study of an algorithm proposed by Wu, Byrd, and Lidar [Phys. Rev. Lett. 89, 057904 (2002).10.1103/PhysRevLett.89.057904] to find the low-lying spectrum of a pairing Hamiltonian. While the number of elementary quantum gates required scales polynomially with the size of the system, it increases inversely to the desired error bound E. Making such simulations robust to decoherence using fault tolerance requires an additional factor of approximately 1/E gates. These constraints, along with the effects of control errors, are illustrated using a three qubit NMR system.

5.
Drug Metab Dispos ; 34(5): 734-7, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16501008

RESUMO

Clevidipine is a short-acting dihydropyridine calcium channel antagonist under development for treatment of perioperative hypertension. Patients treated with clevidipine are likely to be comedicated. Therefore, the potential for clevidipine and its major metabolite H152/81 to elicit drug interactions by induction or inhibition of cytochrome P450 was investigated. Induction of CYP1A2, CYP2C9, and CYP3A4 was examined in primary human hepatocytes treated with clevidipine at 1, 10, and 100 microM. Clevidipine was found to be an inducer of CYP3A4, but not of CYP1A2 or CYP2C9, at the 10 microM and 100 microM concentrations of clevidipine tested. Induction response for CYP3A4 to 100 microM clevidipine was approximately 20% of that of the positive control inducer rifampicin. The response of H152/81 was similar. Using cDNA-expressed enzymes, clevidipine inhibited CYP2C9, CYP2C19, and CYP3A4 activities with IC(50) values below 10 microM, whereas CYP1A2, CYP2D6, and CYP2E1 activities were not substantially inhibited (IC(50) values >70 microM). The K(i) values for CYP2C9 and CYP2C19 were 1.7 and 3.3 microM, respectively, and those for CYP3A4 were 8.3 and 2.9 microM, using two substrates, testosterone and midazolam, respectively. These values are at least 10 times higher than the highest clevidipine concentration typically seen in the clinic. Little or no inhibition by H152/81 was found for the enzyme activities mentioned above (IC(50) values >or= 69 microM). The present study demonstrates that it is highly unlikely for clevidipine or its major metabolite to cause cytochrome P450-related drug interactions when used in the dose range required to manage hypertension in humans.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Sistema Enzimático do Citocromo P-450/biossíntese , Di-Hidropiridinas/farmacologia , Piridinas/farmacologia , Células Cultivadas , Criança , Inibidores das Enzimas do Citocromo P-450 , DNA Complementar/biossíntese , DNA Complementar/genética , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Isoenzimas/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade
6.
J Biol Chem ; 279(36): 37499-504, 2004 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-15247288

RESUMO

Increasing evidence suggests that IscS, a cysteine desulfurase, provides sulfur for assembly of transient iron-sulfur clusters in IscU. IscU appears to act as a scaffold and eventually transfers the assembled clusters to target proteins. However, the iron donor for the iron-sulfur cluster assembly largely remains elusive. Here we find that Escherichia coli IscU fails to assemble iron-sulfur clusters when the accessible "free" iron in solution is limited by an iron chelator sodium citrate. Remarkably, IscA, an iron-sulfur cluster assembly protein with an iron association constant of 3.0 x 10(19) m(-1), is able to overcome the iron limitation due to sodium citrate and deliver iron for the IscS-mediated iron-sulfur cluster assembly in IscU. Substitution of the invariant cysteine residues Cys-99 or Cys-101 in IscA with serine completely abolishes the iron binding activity of the protein. The IscA mutants that fail to bind iron are unable to mediate iron delivery for the iron-sulfur cluster assembly in IscU under the limited accessible "free" iron conditions. The results suggest that IscA is capable of recruiting intracellular iron and providing iron for the iron-sulfur cluster assembly in IscU in cells in which the accessible "free" iron content is probably restricted.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Escherichia coli/fisiologia , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Proteínas de Transporte/genética , Citratos/farmacologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Mutagênese Sítio-Dirigida , Citrato de Sódio
7.
Biochem J ; 379(Pt 2): 433-40, 2004 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-14720122

RESUMO

Iron-sulphur clusters are one of the most common types of redox centre in biology. At least six proteins (IscS, IscU, IscA, HscB, HscA and ferredoxin) have been identified as being essential for the biogenesis of iron-sulphur proteins in bacteria. It has been shown that IscS is a cysteine desulphurase that provides sulphur for iron-sulphur clusters, and that IscU is a scaffold for the IscS-mediated assembly of iron-sulphur clusters. The iron donor for iron-sulphur clusters, however, remains elusive. Here we show that IscA is an iron binding protein with an apparent iron association constant of 3.0x10(19) M(-1), and that iron-loaded IscA can provide iron for the assembly of transient iron-sulphur clusters in IscU in the presence of IscS and L-cysteine in vitro. The results suggest that IscA is capable of recruiting intracellular iron and delivering iron for iron-sulphur clusters in proteins.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Ferro/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Espectroscopia de Ressonância de Spin Eletrônica , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Ferro/análise , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Cinética , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
8.
Med Sci Sports Exerc ; 35(8): 1341-7, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12900688

RESUMO

PURPOSE: Based on persistent reports of low oxygen consumption VO(2) from Medical Graphics CPX/D metabolic carts, we compared the CPX/D against an automated Douglas bag system. METHODS: Twelve male athletes completed three, randomized 25-min bouts (5 min at 100, 150, 200, 250, and 300 W) on a cycle ergometer with intervening 30-min rests. One bout was measured on each of the CPX/D, the CPX/D with altered software (CPX/DDelta), and an automated Douglas bag system at Flinders University (FU). The CPX/DDelta software alteration was an apparent lag time correction factor of 60 ms. RESULTS: For the CPX/D, both VO(2) and VCO(2) were significantly lower than the FU system at 100-300 W, and the relative differences ranged -10.7 to -12.0% and -7.7 to -8.2%, respectively. Altering the software approximately halved the VO(2) discrepancy between the CPX/DDelta and FU systems. When data from all five workloads were pooled, V(E) of the CPX/D (67.2 +/- 26.4 L x min-1) and CPX/DDelta (67.5 +/- 26.9 L x min-1) were significantly lower than for the FU system (70.5 +/- 27.1 L x min-1); and at 300 W, the relative differences were -4.0% and -3.4% for the CPX/D and CPX/DDelta, respectively. Altering the software changed the pooled %O(2) from 16.24 +/- 0.40% for the CPX/D to 16.04 +/- 0.39% for the CPX/DDelta, and these were significantly different than pooled data for the FU system (16.15 +/- 0.39%). CONCLUSIONS: During submaximal exercise, the CPX/D yields VO(2) values that are approximately 11% lower than the criterion system, and the source of the discrepancy does not appear to be primarily related to volume measurement. A disturbing observation is that factory defaults for the lag time use different correction factors, which vary by 60 ms and this significantly alters VO(2) and VCO(2).


Assuntos
Exercício Físico/fisiologia , Consumo de Oxigênio/fisiologia , Fisiologia/instrumentação , Medicina Esportiva/instrumentação , Adolescente , Adulto , Dióxido de Carbono/análise , Frequência Cardíaca/fisiologia , Humanos , Masculino , Troca Gasosa Pulmonar/fisiologia , Sensibilidade e Especificidade , Software , Espirometria/instrumentação
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