ABSTRACT
INTRODUCTION: There is an urgent need to identify therapies that prevent SARS-CoV-2 infection and improve the outcome of COVID-19 patients. OBJECTIVE: Based upon clinical observations, we proposed that some psychotropic and antihistaminic drugs could protect psychiatric patients from SARS-CoV-2 infection. This observation is investigated in the light of experimental in vitro data on SARS-CoV-2. METHODS: SARS-CoV-2 high-throughput screening results are available at the NCATS COVID-19 portal. We investigated the in vitro anti-viral activity of many psychotropic and antihistaminic drugs using chemoinformatics approaches. RESULTS AND DISCUSSION: We analyze our clinical observations in the light of SARS-CoV-2 experimental screening results and propose that several cationic amphiphilic psychotropic and antihistaminic drugs could protect people from SARS-CoV-2 infection; some of these molecules have very limited adverse effects and could be used as prophylactic drugs. Other cationic amphiphilic drugs used in other disease areas are also highlighted. Recent analyses of patient electronic health records reported by several research groups indicate that some of these molecules could be of interest at different stages of the disease progression. In addition, recently reported drug combination studies further suggest that it might be valuable to associate several cationic amphiphilic drugs. Taken together, these observations underline the need for clinical trials to fully evaluate the potentials of these molecules, some fitting in the so-called category of broad-spectrum antiviral agents. Repositioning orally available drugs that have moderate side effects and should act on molecular mechanisms less prone to drug resistance would indeed be of utmost importance to deal with COVID-19.
ABSTRACT
Several studies have reported that certain psychoactive drugs could have a protective effect against SARS-CoV-2. Herein, we propose that antihistamines (anti-H1) and cationic amphiphilic drugs (CAD), specifically, have the capacity to disrupt virus entry and replication. In addition, several of these molecules have limited side effects and as such could be promising prophylactic candidates against SARS-CoV-2 infection.
Subject(s)
COVID-19 Drug Treatment , Histamine H1 Antagonists/pharmacology , SARS-CoV-2 , Surface-Active Agents/pharmacology , COVID-19/virology , Drug Repositioning , Humans , Models, Biological , Pandemics , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Internalization/drug effects , Virus Replication/drug effectsSubject(s)
Coronavirus Infections/prevention & control , Drug Repositioning , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Psychotropic Drugs/therapeutic use , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Humans , Mental Disorders/drug therapy , Pneumonia, Viral/virology , Psychotropic Drugs/pharmacology , COVID-19 Drug TreatmentABSTRACT
It was previously demonstrated that engineered mesenchymal stem cells (MSCs) which express a high level of a very efficient modified gene CYP2B6* (CYP2B6TM-RED) acting as a suicide gene (MSC-2B6*) in combination with cyclophosphamide (CPA) constitute a powerful cell/gene therapy approach for solid tumors. In murine models, this combination led to tumor eradication and triggered a durable immune response against tumoral cells, which prevented recurrence and metastasis. The first goal, in this work, was to determine whether the mechanism of tumor cell death caused by CPA metabolites could explain the appearance of this anti-tumor immune response. In vitro, CPA metabolites produced by MSC-2B6* were able to induce immunogenic cell death (ICD) of tumor cells. Indeed, all ICD characteristic events were clearly identified: calreticulin translocation, LC3II expression and release of ATP and HMGB1. The second goal was to determine the respective roles of the direct cytotoxicity of CPA metabolites and the immune anti-tumor response due to ICD of tumor cells during tumor eradication. In vivo, the early inhibition of ICD (with anti-HMGB1 antibodies) or the depletion of CD8+T lymphocytes (with anti-CD8 antibodies) prevented tumor eradication by CPA metabolites and tumor regrowth occurred, despite CPA treatment. In conclusion, the full eradication of the tumors depends on the association of cytotoxic CPA metabolites triggering the ICD of tumor cells and an anti-tumor immune response. The absence of one or the other of these effects prevents the complete eradication of tumors.
ABSTRACT
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.
Subject(s)
Genetic Therapy/methods , Liver Neoplasms, Experimental/drug therapy , Animals , Antineoplastic Agents, Alkylating/administration & dosage , Antineoplastic Agents, Alkylating/therapeutic use , Bystander Effect , Cyclophosphamide/administration & dosage , Cyclophosphamide/therapeutic use , Disease Models, Animal , Feasibility Studies , Injections, Intra-Arterial , Pilot Projects , RabbitsABSTRACT
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.
Subject(s)
Genes, Transgenic, Suicide/genetics , Genetic Engineering/methods , Genetic Therapy/methods , Genetic Vectors/genetics , Mesenchymal Stem Cells/physiology , Neoplasms/genetics , Animals , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Disease Models, Animal , Female , Genetic Vectors/administration & dosage , Humans , Mice , Mice, Inbred C57BL , Neoplasms/therapyABSTRACT
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.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Genetic Variation/genetics , Immunosuppressive Agents/pharmacokinetics , Immunosuppressive Agents/therapeutic use , Tacrolimus/pharmacokinetics , Tacrolimus/therapeutic use , Adult , Alleles , Cytochrome P-450 CYP3A/genetics , Female , Genotype , Graft Rejection/genetics , Graft Rejection/metabolism , Graft Rejection/prevention & control , Humans , Kidney Transplantation/methods , Male , Middle Aged , Prospective Studies , Transplant RecipientsABSTRACT
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.
Subject(s)
Kidney/metabolism , Signal Transduction , Unfolded Protein Response/physiology , Animals , Cells, Cultured , Endoplasmic Reticulum Stress/physiology , Macrophages/physiology , Mice , Mice, Inbred C57BL , Ribonuclease, Pancreatic/metabolism , Ribonuclease, Pancreatic/physiologyABSTRACT
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.
Subject(s)
Acute Kidney Injury/physiopathology , Endoplasmic Reticulum Stress/physiology , Kidney/pathology , Protein Biosynthesis/physiology , Ribonuclease, Pancreatic/metabolism , Acute Kidney Injury/chemically induced , Acute Kidney Injury/pathology , Adaptation, Physiological , Animals , Apoptosis , Cells, Cultured , DNA-Binding Proteins/metabolism , Endoribonucleases/genetics , Endoribonucleases/metabolism , Epithelial Cells , Gene Silencing , Humans , Kidney/physiopathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Poly(ADP-ribose) Polymerases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, Sprague-Dawley , Regulatory Factor X Transcription Factors , Ribonuclease, Pancreatic/genetics , Signal Transduction , Transcription Factors/metabolism , Transcription, Genetic , Tunicamycin , X-Box Binding Protein 1ABSTRACT
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.
Subject(s)
Drug Interactions , Pharmaceutical Preparations/metabolism , Administration, Oral , Adult , Cytochrome P-450 Enzyme System/metabolism , Drug-Related Side Effects and Adverse Reactions/etiology , Drug-Related Side Effects and Adverse Reactions/metabolism , Female , Healthy Volunteers , Humans , Male , Pilot Projects , Young AdultABSTRACT
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.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Alcoholism/genetics , Amygdala/metabolism , Cytochrome P-450 Enzyme System/genetics , Prefrontal Cortex/metabolism , Smoking/genetics , Transcription Factors/genetics , Adrenodoxin/biosynthesis , Adrenodoxin/genetics , Adult , Aged , Aged, 80 and over , Alcoholism/enzymology , Alcoholism/metabolism , Amygdala/enzymology , Female , Gene Expression Profiling , Genotype , Humans , Isoenzymes/genetics , Male , Middle Aged , Prefrontal Cortex/enzymology , Reference Values , Smoking/metabolismABSTRACT
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.
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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.
Subject(s)
Cytochrome P-450 Enzyme System/chemistry , Models, Molecular , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Blotting, Western , Catalytic Domain , Chromatography, High Pressure Liquid , Computer Simulation , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P450 Family 2 , Debrisoquin/chemistry , Debrisoquin/metabolism , Kinetics , Mass Spectrometry , Molecular Structure , Oxidation-Reduction , Protein Binding , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Substrate SpecificityABSTRACT
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.
Subject(s)
Acenocoumarol/administration & dosage , Algorithms , Anticoagulants/administration & dosage , Vitamin K Epoxide Reductases/genetics , Adult , Asian People/genetics , Carbon-Carbon Ligases/genetics , Cytochrome P-450 CYP2C9/genetics , Cytochrome P-450 Enzyme System/genetics , Cytochrome P450 Family 4 , Female , Genotype , Humans , India , Male , Polymorphism, Single NucleotideABSTRACT
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.
Subject(s)
Dried Blood Spot Testing/methods , alpha 1-Antitrypsin Deficiency/diagnosis , Adult , Aged , Aged, 80 and over , Cohort Studies , DNA Mutational Analysis , Early Diagnosis , Genotype , Humans , Immunoassay , Middle Aged , Nephelometry and Turbidimetry , Phenotype , Young Adult , alpha 1-Antitrypsin/blood , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin Deficiency/bloodABSTRACT
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.
Subject(s)
Genetic Therapy , Mesenchymal Stem Cells , Neoplasms/genetics , Neoplasms/therapy , Gene Transfer Techniques , Genes, Transgenic, Suicide/genetics , Humans , Neoplasms/pathology , Pharmaceutical Vehicles , Prodrugs/therapeutic useABSTRACT
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.
Subject(s)
Individuality , Methyltransferases/genetics , Methyltransferases/metabolism , Thioguanine/administration & dosage , Adult , Azathioprine/administration & dosage , Databases, Factual , Drug Monitoring/methods , Female , Genotype , Guanine Nucleotides/administration & dosage , Guanine Nucleotides/metabolism , Humans , Male , Middle Aged , Pharmacogenetics/methods , Phenotype , Retrospective Studies , Thioguanine/metabolism , Thioinosine/administration & dosage , Thioinosine/analogs & derivatives , Thioinosine/metabolism , Thionucleotides/administration & dosage , Thionucleotides/metabolism , Young AdultABSTRACT
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.