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1.
Cardiovasc Intervent Radiol ; 41(1): 153-162, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29090347

RESUMO

PURPOSE: Gene-directed enzyme prodrug therapy (GDEPT) is a "Trojan-horses" suicide gene therapy that consists of tumor-targeted gene delivery (vectorized by mesenchymal stem cells MSCs) encoding an enzyme that converts a harmless prodrug into cytotoxic metabolites in situ. Then, cytotoxic metabolites passively diffuse in the neighboring tumor cells and kill them (bystander effect). The goal of our study was to assess the feasibility and efficacy of intra-arterial administration of MSCs transduced with an optimized gene (MSC-CYP2B6TM-RED) followed by intravenous administration of cyclophosphamide (CPA) into the VX2 rabbit liver tumor. MATERIALS AND METHODS: Nine rabbits with a VX2 liver tumor were randomly assigned into three groups: Control group A (one rabbit) free of any treatment; Control group B (two rabbits) receiving intravenous injection of cyclophosphamide at day 3 and CPA at day 14; and Group C (six rabbits) receiving the GDEPT treatment, consisting of successive intra-arterial injection of transduced-MSCs at days 0 (n = 6) and 11 (n = 3), followed by injection of CPA at days 3 (n = 6) and 14 (n = 3). The tumor response was assessed by ultrasound scan every 7 days and histopathological analysis at sacrifice (D25). RESULTS: There was a significant difference in the tumor volume between control groups (A + B) and group C at D7: 38/19 cm3 (p = 0.024); D11: 51/20 cm3 (p = 0.024), and D25: 121/37 cm3 (p = 0.048). Tumor necrosis was significantly greater and metastatic spread was lower for rabbits who received GDEPT (78% of total tumor surface) than for control animals (A + B) (22% of total tumor surface (p = 0.006). CONCLUSION: Intra-arterial delivery of transduced-MSCs is feasible and, after CPA injection, resulted in 78% tumor necrosis (p = 0.006) and less metastasis in a VX2 liver tumor model.


Assuntos
Terapia Genética/métodos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Animais , Antineoplásicos Alquilantes/administração & dosagem , Antineoplásicos Alquilantes/uso terapêutico , Efeito Espectador , Ciclofosfamida/administração & dosagem , Ciclofosfamida/uso terapêutico , Modelos Animais de Doenças , Estudos de Viabilidade , Injeções Intra-Arteriais , Projetos Piloto , Coelhos
2.
J Control Release ; 239: 82-91, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27565211

RESUMO

Gene-directed enzyme pro-drug therapy (GDEPT) consists of expressing, in tumor cells, a suicide gene which converts a pro-drug into cytotoxic metabolites, in situ. In a previous work, we demonstrated that the combination of the suicide gene CYP2B6TM-RED (a fusion of a triple mutant of CYP2B6 with NADPH cytochrome P450 reductase) and cyclophosphamide (CPA) constituted a powerful treatment for solid tumors. In this work, we investigated the use of mesenchymal stem cells (MSCs) as cellular vehicles for the delivery of our suicide gene. MSCs were genetically engineered ex-vivo to stably express CYP2B6TM-RED. Ex vivo and in vivo investigations showed that MSCs expressing CYP2B6TM-RED were able 1) to bioactivate CPA and produce local cytotoxic metabolites in tumor sites and 2) to destroy neighboring tumor cells through a bystander effect. Intratumoral injections of CYP2B6TM-RED-MSCs and CPA completely eradicated tumors in 33% of mice without recurrence after 6months. Rechallenge experiments demonstrated an efficient immune response. These data suggest that MSCs expressing CYP2B6TM-RED with CPA could represent a promising treatment for solid tumors to test in future clinical trials.


Assuntos
Genes Transgênicos Suicidas/genética , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , Células-Tronco Mesenquimais/fisiologia , Neoplasias/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/terapia
3.
Ther Drug Monit ; 38(2): 223-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26829596

RESUMO

BACKGROUND: POR*28 is a recently newly described allelic variant of the cytochrome P450 oxidoreductase (POR), which might be associated with an increased metabolic activity of P450 cytochromes (CYP) 3A5 and 3A4. Consequently, carriers of at least 1 allele of this polymorphism could require increased calcineurin inhibitors doses to reach the target residual concentrations (C0). The objective of this study was to test whether the allelic variant of POR, which is associated with an increased metabolic activity of CYP3A, impacts tacrolimus (Tac) pharmacokinetics. METHODS: We tested this hypothesis in a population of 229 kidney transplant recipients (KTR) from a large, multicenter, prospective and randomized study. We have analyzed the association between POR*28 genotype and the proportion of individuals reaching the target Tac residual concentration (Tac C0) 10 days after transplantation. We have also measured the association between POR*28 and the Tac C0, and adjusted Tac C0 (Tac C0/Tac dose) over time using generalized mixed linear models. RESULTS: Ten days after transplantation, there was no difference of frequencies of KTR within the target range of Tac C0 (C0 10-15 ng/mL) according to the POR*28 genotype (P = 0.8). The mean Tac C0 at day 10 in the POR*1/*1 group was 15.3 ± 9.7 ng/mL compared with 15.7 ± 7.8 ng/mL in the POR*1/*28 group and 14.2 ± 6.8 ng/mL, in the POR*28/*28 group, P = 0.8. The adjusted Tac C0 was not associated with POR*28 genotype over time (random effects model, P = 0.9). When restricted to KTR expressing CYP3A5, POR*28 genotype did not impact the proportion of individuals within the Tac C0 target range neither the adjusted Tac C0 (random effects model, P = 0.1). CONCLUSIONS: POR*28 does not significantly influence Tac pharmacokinetic parameters in a large cohort of KTR. This study does not confirm recent findings indicating that POR*28 carriers require more Tac to reach target C0.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Variação Genética/genética , Imunossupressores/farmacocinética , Imunossupressores/uso terapêutico , Tacrolimo/farmacocinética , Tacrolimo/uso terapêutico , Adulto , Alelos , Citocromo P-450 CYP3A/genética , Feminino , Genótipo , Rejeição de Enxerto/genética , Rejeição de Enxerto/metabolismo , Rejeição de Enxerto/prevenção & controle , Humanos , Transplante de Rim/métodos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Transplantados
4.
J Am Soc Nephrol ; 27(9): 2670-83, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-26823555

RESUMO

The ribonuclease angiogenin is a component of the mammalian stress response, and functions in both cell-autonomous and non-cell-autonomous ways to promote tissue adaptation to injury. We recently showed that angiogenin regulates tissue homeostasis during AKI associated with endoplasmic reticulum (ER) stress through the production of transfer RNA fragments that interfere with translation initiation and thereby alleviate ER stress. However, whether the paracrine signaling mediated by angiogenin secretion is a genuine component of the ER stress response to kidney injury is unknown. Here, we explored the molecular mechanisms by which angiogenin is secreted upon ER stress, and determined how it modulates the inflammatory microenvironment. In cultured renal epithelial cells, ER stress specifically induced angiogenin secretion under the selective control of inositol-requiring enzyme 1α, a key activator of the unfolded protein response. The transcription factors spliced X-box-binding protein 1 and p65, which are activated by inositol-requiring enzyme 1α upon ER stress, each bound the angiogenin promoter and controlled the amount of angiogenin secreted. Furthermore, p65 promoted angiogenin transcription in an ER stress-dependent manner. Similar to secretion of the ER stress-induced proinflammatory cytokine IL-6, secretion of angiogenin required the ER-Golgi pathway. Notably, incubation of human macrophages with angiogenin promoted macrophage reprogramming toward an activated and proinflammatory phenotype. In patients, angiogenin expression increased upon renal inflammation, and the urinary concentration of angiogenin correlated with the extent of immune-mediated kidney injury. Collectively, our data identify angiogenin as a mediator of the ER stress-dependent inflammatory response and as a potential noninvasive biomarker of AKI.


Assuntos
Rim/metabolismo , Transdução de Sinais , Resposta a Proteínas não Dobradas/fisiologia , Animais , Células Cultivadas , Estresse do Retículo Endoplasmático/fisiologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Ribonuclease Pancreático/metabolismo , Ribonuclease Pancreático/fisiologia
5.
Eur J Drug Metab Pharmacokinet ; 41(2): 125-38, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25465228

RESUMO

This phase I, pilot clinical study was designed to evaluate the safety and the pharmacokinetic (PK) profiles of the CIME (Metabolic Identity Card) combination of ten drugs, with a view to its use as a phenotyping cocktail. Ten healthy Caucasian subjects were orally dosed with the CIME combination (caffeine-CYP1A2, repaglinide-CYP2C8, tolbutamide-CYP2C9, omeprazole-CYP2C19, dextromethorphan-CYP2D6, midazolam-CYP3A, acetaminophen-UGT1A1, 6&9 and 2B15, digoxin-P-gp, rosuvastatin-OATP1B1&3 and memantine-active renal transport). Blood was collected over 3 days and on day 7. CIME probes and relevant metabolites were assayed by LC-MS/MS and PK parameters were calculated. Main results were: (1) good safety with reversible mild or moderate adverse effects, (2) an analytical method able to quantify simultaneously the 10 probes and the major metabolites, (3) calculation of PK parameters for all probes in general agreed with published values, and (4) identification of the low CYP2D6 metabolizer. This pilot study showed that the CIME combination was well tolerated and that its pharmacokinetics could be accurately measured in healthy volunteers. This combination can now confidently be checked for sensitivity and specificity and for lack of interaction to be validated as a phenotyping cocktail.


Assuntos
Interações de Medicamentos , Preparações Farmacêuticas/metabolismo , Administração Oral , Adulto , Sistema Enzimático do Citocromo P-450/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/etiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Masculino , Projetos Piloto , Adulto Jovem
6.
J Am Soc Nephrol ; 27(3): 863-76, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26195817

RESUMO

Endoplasmic reticulum (ER) stress is involved in the pathophysiology of kidney disease and aging, but the molecular bases underlying the biologic outcomes on the evolution of renal disease remain mostly unknown. Angiogenin (ANG) is a ribonuclease that promotes cellular adaptation under stress but its contribution to ER stress signaling remains elusive. In this study, we investigated the ANG-mediated contribution to the signaling and biologic outcomes of ER stress in kidney injury. ANG expression was significantly higher in samples from injured human kidneys than in samples from normal human kidneys, and in mouse and rat kidneys, ANG expression was specifically induced under ER stress. In human renal epithelial cells, ER stress induced ANG expression in a manner dependent on the activity of transcription factor XBP1, and ANG promoted cellular adaptation to ER stress through induction of stress granules and inhibition of translation. Moreover, the severity of renal lesions induced by ER stress was dramatically greater in ANG knockout mice (Ang(-/-)) mice than in wild-type mice. These results indicate that ANG is a critical mediator of tissue adaptation to kidney injury and reveal a physiologically relevant ER stress-mediated adaptive translational control mechanism.


Assuntos
Lesão Renal Aguda/fisiopatologia , Estresse do Retículo Endoplasmático/fisiologia , Rim/patologia , Biossíntese de Proteínas/fisiologia , Ribonuclease Pancreático/metabolismo , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/patologia , Adaptação Fisiológica , Animais , Apoptose , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Células Epiteliais , Inativação Gênica , Humanos , Rim/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Fatores de Transcrição de Fator Regulador X , Ribonuclease Pancreático/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Genética , Tunicamicina , Proteína 1 de Ligação a X-Box
7.
Xenobiotica ; 45(12): 1129-37, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26095139

RESUMO

1. Ethanol consumption and smoking alter the expression of certain drug-metabolizing enzymes and transporters, potentially influencing the tissue-specific effects of xenobiotics. 2. Amygdala (AMG) and prefrontal cortex (PFC) are brain regions that modulate the effects of alcohol and smoking, yet little is known about the expression of cytochrome P450 enzymes (P450s) and ATP-binding cassette (ABC) transporters in these tissues. 3. Here, we describe the first study on the expression of 19 P450s, their redox partners, three ABC transporters and four related transcription factors in the AMG and PFC of smokers and alcoholics by quantitative RT-PCR. 4. CYP1A1, CYP1B1, CYP2B6, CYP2C8, CYP2C18, CYP2D6, CYP2E1, CYP2J2, CYP2S1, CYP2U1, CYP4X1, CYP46, adrenodoxin and NADPH-P450 reductase, ABCB1, ABCG2, ABCA1, and transcription factors aryl hydrocarbon receptor AhR and proliferator-activated receptor α were quantified in both areas. CYP2A6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, adrenodoxin reductase and the nuclear receptors pregnane X receptor and constitutive androstane receptor were detected but below the limit of quantification. CYP1A2 and CYP2W1 were not detected. 5. Adrenodoxin expression was elevated in all case groups over controls, and smokers showed a trend toward higher CYP1A1 and CYP1B1 expression. 6. Our study shows that most xenobiotic-metabolizing P450s and associated redox partners, transporters and transcription factors are expressed in human AMG and PFC.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Alcoolismo/genética , Tonsila do Cerebelo/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Córtex Pré-Frontal/metabolismo , Fumar/genética , Fatores de Transcrição/genética , Adrenodoxina/biossíntese , Adrenodoxina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alcoolismo/enzimologia , Alcoolismo/metabolismo , Tonsila do Cerebelo/enzimologia , Feminino , Perfilação da Expressão Gênica , Genótipo , Humanos , Isoenzimas/genética , Masculino , Pessoa de Meia-Idade , Córtex Pré-Frontal/enzimologia , Valores de Referência , Fumar/metabolismo
8.
Genome Med ; 7(1): 37, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26015807

RESUMO

BACKGROUND: There has been considerable progress in the management of acute lymphoblastic leukemia (ALL) but further improvement is needed to increase long-term survival. The thiopurine agent 6-mercaptopurine (6-MP) used for ALL maintenance therapy has a key influence on clinical outcomes and relapse prevention. Genetic inheritance in thiopurine metabolism plays a major role in interindividual clinical response variability to thiopurines; however, most cases of thiopurine resistance remain unexplained. METHODS: We used lymphoblastoid cell lines (LCLs) from healthy donors, selected for their extreme thiopurine susceptibility. Thiopurine metabolism was characterized by the determination of TPMT and HPRT activity. We performed genome-wide expression profiling in resistant and sensitive cell lines with the goal of elucidating the mechanisms of thiopurine resistance. RESULTS: We determined a higher TPMT activity (+44%; P = 0.024) in resistant compared to sensitive cell lines, although there was no difference in HPRT activity. We identified a 32-gene transcriptomic signature that predicts thiopurine resistance. This signature includes the GTPBP4 gene coding for a GTP-binding protein that interacts with p53. A comprehensive pathway analysis of the genes differentially expressed between resistant and sensitive cell lines indicated a role for cell cycle and DNA mismatch repair system in thiopurine resistance. It also revealed overexpression of the ATM/p53/p21 pathway, which is activated in response to DNA damage and induces cell cycle arrest in thiopurine resistant LCLs. Furthermore, overexpression of the p53 target gene TNFRSF10D or the negative cell cycle regulator CCNG2 induces cell cycle arrest and may also contribute to thiopurine resistance. ARHGDIA under-expression in resistant cell lines may constitute a novel molecular mechanism contributing to thiopurine resistance based on Rac1 inhibition induced apoptosis and in relation with thiopurine pharmacodynamics. CONCLUSION: Our study provides new insights into the molecular mechanisms underlying thiopurine resistance and suggests a potential research focus for developing tailored medicine.

9.
Biochim Biophys Acta ; 1850(7): 1426-37, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25857771

RESUMO

BACKGROUND: Cytochrome P450 2U1 (CYP2U1) has been identified from the human genome and is highly conserved in the living kingdom. In humans, it has been found to be predominantly expressed in the thymus and in the brain. CYP2U1 is considered as an "orphan" enzyme as few data are available on its physiological function(s) and active site topology. Its only substrates reported so far were unsaturated fatty acids such as arachidonic acid, and, much more recently, N-arachidonoylserotonin. METHODS: We expressed CYP2U1 in yeast Saccharomyces cerevisiae, built a 3D homology model of CYP2U1, screened a library of compounds known to be substrates of CYP2 family with metabolite detection by high performance liquid chromatography-mass spectrometry, and performed docking experiments to explain the observed regioselectivity of the reactions. RESULTS: We show that drug-related compounds, debrisoquine and terfenadine derivatives, subtrates of CYP2D6 and CYP2J2, are hydroxylated by recombinant CYP2U1 with regioselectivities different from those reported for CYP2D6 and 2J2. Docking experiments of those compounds and of arachidonic acid allow us to explain the regioselectivity of the hydroxylations on the basis of their interactions with key residues of CYP2U1 active site. MAJOR CONCLUSION: Our results show for the first time that human orphan CYP2U1 can oxidize several exogenous molecules including drugs, and describe a first CYP2U1 3D model. GENERAL SIGNIFICANCE: These results could have consequences for the metabolism of drugs particularly in the brain. The described 3D model should be useful to identify other substrates of CYP2U1 and help in understanding its physiologic roles.


Assuntos
Sistema Enzimático do Citocromo P-450/química , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Western Blotting , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Simulação por Computador , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Família 2 do Citocromo P450 , Debrisoquina/química , Debrisoquina/metabolismo , Cinética , Espectrometria de Massas , Estrutura Molecular , Oxirredução , Ligação Proteica , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Especificidade por Substrato
11.
Eur J Clin Pharmacol ; 71(2): 173-81, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25519826

RESUMO

OBJECTIVE: The objective of this study was to determine the influence of CYP2C9, VKORC1, CYP4F2, and GGCX genetic polymorphisms on mean daily dose of acenocoumarol in South Indian patients and to develop a new pharmacogenetic algorithm based on clinical and genetic factors. METHODS: Patients receiving acenocoumarol maintenance therapy (n = 230) were included in the study. Single nucleotide polymorphisms (SNP) of CYP2C9, VKORC1, CYP4F2, and GGCX were genotyped by real-time polymerase chain reaction (RT-PCR) method. RESULTS: The mean daily acenocoumarol maintenance dose was found to be 3.7 ± 2.3 (SD) mg/day. The CYP2C9 *1*2, CYP2C9 *1*3, and CYP2C9 *2*3 variant genotypes significantly reduced the dose by 56.7 % (2.0 mg), 67.6 % (1.6 mg), and 70.3 % (1.5 mg) than wild-type carriers 4.1 mg, p < 0.0001. The genetic variants of CYP2C9 and GGCX (rs11676382) were found to be associated with lower acenocoumarol dose, whereas CYP4F2 (rs2108622) was associated with higher doses. Age, body mass index (BMI), variation of CYP2C9, VKORC1, CYP4F2, and GGCX were the major determinants of acenocoumarol maintenance dose, accounting for 61.8 % of its variability (adjusted r (2) = 0.615, p < 0.0001). Among the VKORC1 variants, rs9923231 alone contributed up to 28.6 % of the acenocoumarol dose variation. CONCLUSION: VKORC1 rs9923231 polymorphism had the highest impact on acenocoumarol daily dose. A new pharmacogenetic algorithm was established to determine the acenocoumarol dose in South Indian population.


Assuntos
Acenocumarol/administração & dosagem , Algoritmos , Anticoagulantes/administração & dosagem , Vitamina K Epóxido Redutases/genética , Adulto , Grupo com Ancestrais do Continente Asiático/genética , Carbono-Carbono Ligases/genética , Citocromo P-450 CYP2C9/genética , Sistema Enzimático do Citocromo P-450/genética , Família 4 do Citocromo P450 , Feminino , Genótipo , Humanos , Índia , Masculino , Polimorfismo de Nucleotídeo Único
12.
Ann Biol Clin (Paris) ; 72(6): 689-704, 2014 Nov-Dec.
Artigo em Francês | MEDLINE | ID: mdl-25486665

RESUMO

Alpha- 1-antitrypsin (A1AT) deficiency is a hereditary autosomal codominant genetic disorder resulting in low circulating levels of A1AT and leading to lung and/or liver disease. It remains underdiagnosed and only 5 to 10% of PIZZ patients, the most common form of severe A1AT deficiency, would be actually identified in France. Facilitating early diagnosis of A1AT deficiency would allow a better management of this disease; therefore we have developed and standardized in three laboratories involved in this study, a diagnostic test on dried blood spots (DBS) including quantitative A1AT measurement, phenotyping by IEF electrophoresis and, if necessary, genotyping by SERPINA1 gene sequencing. We performed a quantitative assay on 90 DBS samples by immunoturbidimetric or immunonephelometric methods. We demonstrated that both methods were suitable for this type of sampling and the results obtained were highly correlated (R(2)>0.9) between the three laboratories: for a target value of 1.00 g/L, the results obtained from the three laboratories were between 1.00 and 1.02 g/L. Phenotyping and genotyping were performed under redefined operating conditions and adapted to the analysis of DBS samples. The results were comparable with those obtained for venous blood samples. Following this work, it becomes possible to provide pulmonologists with a reliable kit to perform a capillary blood sampling on filter paper which would allow a large-scale screening of A1AT deficiency in the population particularly affected by this genetic condition.


Assuntos
Teste em Amostras de Sangue Seco/métodos , Deficiência de alfa 1-Antitripsina/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Análise Mutacional de DNA , Diagnóstico Precoce , Genótipo , Humanos , Imunoensaio , Pessoa de Meia-Idade , Nefelometria e Turbidimetria , Fenótipo , Adulto Jovem , alfa 1-Antitripsina/sangue , alfa 1-Antitripsina/genética , Deficiência de alfa 1-Antitripsina/sangue
13.
Biochimie ; 105: 4-11, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24977933

RESUMO

Gene-directed enzyme prodrug therapy (GDEPT) consists of targeted delivery to tumor cells of a suicide gene responsible for the in situ conversion of a prodrug into cytotoxic metabolites. One of the major impediments of GDEPT is to target specifically the tumor cells with the suicide gene. Among gene delivery methods, mesenchymal stem cells (MSCs) have emerged recently as potential cellular vehicles for gene delivery. MSCs are particularly suited for gene transduction. They exhibit remarkable migratory property towards tumors and their metastases and they are weakly immunogenic. This review will summarize the current knowledge about MSCs engineered to express different suicide genes (cytosine deaminase, thymidine kinase, carboxylesterase, cytochrome P450) to elicit a significant antitumor response against brain tumors, ovarian, hepatocellular, pancreatic, renal or medullary thyroid carcinomas, breast or prostate cancer and pulmonary metastases. The potential side effects of these MSC-based tumor therapies will also be considered to highlight certain aspects that need to be improved prior to clinical use.


Assuntos
Terapia Genética , Células-Tronco Mesenquimais , Neoplasias/genética , Neoplasias/terapia , Técnicas de Transferência de Genes , Genes Transgênicos Suicidas/genética , Humanos , Neoplasias/patologia , Veículos Farmacêuticos , Pró-Fármacos/uso terapêutico
14.
Anal Bioanal Chem ; 406(20): 4861-74, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24952904

RESUMO

Cytochromes P450 (CYPs) play critical roles in oxidative metabolism of many endogenous and exogenous compounds. Protein expression levels of CYPs in liver provide relevant information for a better understanding of the importance of CYPs in pharmacology and toxicology. This work aimed at establishing a simple method to quantify six CYPs (CYP3A4, CYP3A5, CYP1A2, CYP2D6, CYP2C9, and CYP2J2) in various biological samples without isotopic labeling. The biological matrix was spiked with the standard peptides prior to the digestion step to realize a label-free quantification by mass spectrometry. The method was validated and applied to quantify these six isoforms in both human liver microsomes and mitochondria, but also in recombinant expression systems such as baculosomes and the HepG2 cell line. The results showed intra-assay and interassay accuracy and precision within 16 % and 5 %, respectively, at the low quality control level, and demonstrated the advantages of the method in terms of reproducibility and cost.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/enzimologia , Fragmentos de Peptídeos/análise , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Cisteína/química , Células Hep G2 , Humanos , Isoenzimas
15.
Pharmacogenomics ; 15(6): 745-57, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24897283

RESUMO

BACKGROUND & AIMS: TPMT activity and metabolite determination (6-thioguanine nucleotides [6-TGN] and 6-methylmercaptopurine nucleotides [6-MMPN]) remain controversial during thiopurine management. This study assessed associations between patient characteristics and TPMT activity, and their impact on metabolite levels. PATIENTS & METHODS: A retrospective review of the laboratory database from a French university hospital identified 7360 patients referred for TPMT phenotype/genotype determination, and/or for 6-TGN/6-MMPN monitoring. RESULTS: Four TPMT phenotypes were identified according to TPMT activity distribution: low, intermediate, normal/high and very high. Based on 6775 assays, 6-TGN concentrations were 1.6-fold higher in TPMT-deficient patients compared with TPMT-normal patients. Azathioprine dose and TPMT genotype were significant predictors of metabolite levels. Furthermore, 6-MMPN and 6-MMPN: 6-TGN ratios were, respectively, 1.6- and 2.2-fold higher in females than in males, despite similar TPMT, 6-TGN and azathioprine doses. An unfavorable ratio (≥20) was associated with a slightly higher TPMT activity. CONCLUSION: These results illustrate the usefulness of pharmacogenomics and metabolite measurement to improve the identification of noncompliance and patients at high risk for toxicity or therapeutic resistance. Original submitted 13 November 2013; Revision submitted 30 January 2014.


Assuntos
Individualidade , Metiltransferases/genética , Metiltransferases/metabolismo , Tioguanina/administração & dosagem , Adulto , Azatioprina/administração & dosagem , Bases de Dados Factuais , Monitoramento de Medicamentos/métodos , Feminino , Genótipo , Nucleotídeos de Guanina/administração & dosagem , Nucleotídeos de Guanina/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Farmacogenética/métodos , Fenótipo , Estudos Retrospectivos , Tioguanina/metabolismo , Tioinosina/administração & dosagem , Tioinosina/análogos & derivados , Tioinosina/metabolismo , Tionucleotídeos/administração & dosagem , Tionucleotídeos/metabolismo , Adulto Jovem
17.
PLoS One ; 9(5): e95532, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24819355

RESUMO

We have investigated in vitro the metabolic capability of 3 extrahepatic cytochromes P-450, CYP1A1, 1B1 and 2J2, known to be over-expressed in various tumors, to biotransform 5 tyrosine kinase inhibitors (TKI): dasatinib, imatinib, nilotinib, sorafenib and sunitinib. Moreover, mRNA expression of CYP1A1, 1B1, 2J2 and 3A4 in 6 hepatocellular and 14 renal cell carcinoma tumor tissues and their surrounding healthy tissues, was determined. Our results show that CYP1A1, 1B1 and especially 2J2 can rapidly biotransform the studied TKIs with a metabolic efficiency similar to that of CYP3A4. The mRNA expression of CYP1A1, 1B1, 2J2 and 3A4 in tumor biopsies has shown i) the strong variability of CYP expression and ii) distinct outliers showing high expression levels (esp. CYP2J2) that are compatible with high intratumoral CYP activity and tumor-specific TKI degradation. CYP2J2 inhibition could be a novel clinical strategy to specifically increase the intratumoral rather than plasma TKI levels, improving TKI efficacy and extending the duration before relapse. Such an approach would be akin to beta-lactamase inhibition, a classical strategy to avoid antibiotic degradation and resistance.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Benzamidas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Citocromo P-450 CYP3A/genética , Sistema Enzimático do Citocromo P-450/genética , Dasatinibe , Células Hep G2 , Humanos , Mesilato de Imatinib , Indóis/metabolismo , Neoplasias Hepáticas/genética , Niacinamida/análogos & derivados , Niacinamida/metabolismo , Compostos de Fenilureia/metabolismo , Piperazinas/metabolismo , Pirimidinas/metabolismo , Pirróis/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sorafenibe , Sunitinibe , Tiazóis/metabolismo
18.
Curr Gene Ther ; 14(3): 236-46, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24766134

RESUMO

Gene-directed enzyme prodrug therapy (GDEPT) consists in targeted delivery to tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. One of the major limitations of this strategy in clinical application was the poor prodrug activation capacity of suicide gene. We built a highly efficient suicide gene capable of bioactivating the prodrug cyclophosphamide (CPA) by fusing a CYP2B6 triple mutant with NADPH cytochrome P450 reductase (CYP2B6TM-RED). Expression of this fusion gene via a recombinant lentivirus (LV) vector converted resistant human (A549) and murine (TC1) pulmonary cell lines into CPA-susceptible cell lines. We tested the efficiency of our GDEPT strategy in C57Bl/6 immunocompetent mice, using TC1 cells expressing the HPV-16 E6/E7 oncoproteins. In mice bearing tumors composed only of TC1-CYP2B6TM-RED cells, four CPA injections (140 mg/Kg once a week) completely eradicated the tumors for more than two months. Tumors having only 25% of TC1-CYP2B6TM-RED cells were also completely eradicated by five CPA injections, demonstrating a major in vivo bystander effect. Moreover, surviving mice were rechallenged with parental TC1 cells. The tumors regressed spontaneously 7 days after cell inoculation or grew more slowly than in control naive mice due to a strong immune response mediated by anti-E7CD8(+)T cells. These data suggest that combining the CYPB6TM-RED gene with CPA may hold promise as a highly effective treatment for solid tumors in humans.


Assuntos
Antineoplásicos/farmacologia , Ciclofosfamida/farmacologia , Genes Transgênicos Suicidas , Terapia Genética/métodos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Linhagem Celular Tumoral , Feminino , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Endogâmicos C57BL , NADPH-Ferri-Hemoproteína Redutase/genética , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Pró-Fármacos/farmacologia
19.
PLoS One ; 9(1): e84708, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24416268

RESUMO

Currently, a non-invasive method to estimate the degree of interstitial fibrosis (IF) in chronic kidney disease is not available in routine. The aim of our study was to evaluate the diagnostic performance of the measurement of urinary low molecular weight (LMW) protein concentrations as a method to determine the extent of IF. The urines specimen from 162 consecutive patients who underwent renal biopsy were used in the analysis. Numerical quantification software based on the colorimetric analysis of fibrous areas was used to assess the percentage IF. Total proteinuria, albuminuria, and the urinary levels of retinol binding protein (RBP), alpha1-microglobulin (α1MG), beta 2-microglobulin (ß2MG), transferrin, and IgG immunoglobulins were measured. There was a significant correlation between the degree of IF and the RBP/creatinine (creat) ratio (R2: 0.11, p<0.0001). IF was associated to a lesser extent with urinary ß2MG and α1MG; however, there was no association with total proteinuria or high molecular weight (HMW) proteinuria. The correlation between IF and RBP/creat remained significant after adjustment to the estimated glomerular filtration rate, age, body mass index, α1MG, and ß2MG. The specificity of the test for diagnosing a fibrosis score of >25% of the parenchyma was 95% when using a threshold of 20 mg/g creat. In conclusion, RBP appears to be a quantitative and non-invasive marker for the independent prediction of the extent of kidney IF. Because methods for the measurement of urinary RBP are available in most clinical chemistry departments, RBP measurement is appealing for implementation in the routine care of patients with chronic kidney disease.


Assuntos
Rim/patologia , Proteínas de Ligação ao Retinol/urina , Biomarcadores/urina , Feminino , Fibrose , Taxa de Filtração Glomerular , Humanos , Rim/fisiopatologia , Masculino , Pessoa de Meia-Idade , Peso Molecular , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/fisiopatologia , Insuficiência Renal Crônica/urina , Proteínas de Ligação ao Retinol/química
20.
Anal Chem ; 86(4): 2166-74, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24437734

RESUMO

(1)H NMR is a nonbiased technique for the quantification of small molecules that could result in the identification and characterization of potential biomarkers with prognostic value and contribute to better understand pathophysiology of diseases. In this study, we used (1)H NMR spectroscopy to analyze the urinary metabolome of patients with acute intermittent porphyria (AIP), an inherited metabolic disorder of heme biosynthesis in which an accumulation of the heme precursors 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) promotes sudden neurovisceral attacks, which can be life-threatening. Our objectives were (1) to demonstrate the usefulness of (1)H NMR to identify and quantify ALA and PBG in urines from AIP patients and (2) to identify metabolites that would predict the response to AIP crisis treatment and reflect differential metabolic reprogramming. Our results indicate that (1)H NMR can help to diagnose AIP attacks based on the identification of ALA and PBG. We also show that glycin concentration increases in urines from patients with frequent recurrences at the end of the treatment, after an initial decrease, whereas PBG concentration remains low. Although the reasons for this altered are elusive, these findings indicate that a glycin metabolic reprogramming occurs in AIPr patients and is associated with recurrence. Our results validate the proof of concept of the usefulness of (1)H NMR spectroscopy in clinical chemistry for the diagnosis of acute attack of AIP and identify urinary glycin as a potential marker of recurrence of AIP acute attacks.


Assuntos
Espectroscopia de Ressonância Magnética/métodos , Porfiria Aguda Intermitente/diagnóstico , Porfiria Aguda Intermitente/urina , Adulto , Seguimentos , Humanos , Hidrogênio , Masculino , Redes e Vias Metabólicas/fisiologia , Pessoa de Meia-Idade , Porfiria Aguda Intermitente/metabolismo
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