CYP2J2/EET reduces vulnerability to atrial fibrillation in chronic pressure overload mice.

Growing evidence has well established the protective effects of CYP2J2/EET on the cardiovascular system. The aim of the present study was to determine whether CYP2J2/EET has a preventive effect on atrial fibrillation (AF) and to investigate the underlying mechanisms. Wild-type mice were injected with or without AAV9-CYP2J2 before abdominal aortic constriction (AAC) operation. After 8 weeks, compared with wild-type mice, AAC mice display higher AF inducibility and longer AF durations, which were remarkably attenuated with AAV9-CYP2J2. Also, AAV9-CYP2J2 reduced atrial fibrosis area and the deposit of collagen-I/III in AAC mice, accompanied by the blockade of TGF-ß/Smad-2/3 signalling pathways, as well as the recovery in Smad-7 expression. In vitro, isolated atrial fibroblasts were administrated with TGF-ß1, EET, EEZE, GW9662, SiRNA Smad-7 and pre-MiR-21, and EET was demonstrated to restrain the differentiation of atrial fibroblasts largely dependent on Smad-7, due to the inhibition of EET on MiR-21. In addition, increased inflammatory cytokines, as well as activated NF-κB pathways induced by AAC surgery, were also significantly blunted by AAV9-CYP2J2 treatment. These effects of CYP2J2/EET were partially blocked by GW9662, the antagonist of PPAR-γ. In conclusion, this study revealed that CYP2J2/EET ameliorates atrial fibrosis through modulating atrial fibroblasts activation by disinhibition of MiR-21 on Smad-7, and attenuates atrial inflammatory response by repressing NF-κB pathways, reducing the vulnerability to AF, and CYP2J2/EET exerts its role at least partially through PPAR-γ activation. Our findings might provide a novel upstream therapeutic strategy for AF.

Multifunctionalized biocatalytic P22 nanoreactor for combinatory treatment of ER+ breast cancer.

BACKGROUND: Tamoxifen is the standard endocrine therapy for breast cancers, which require metabolic activation by cytochrome P450 enzymes (CYP). However, the lower and variable concentrations of CYP activity at the tumor remain major bottlenecks for the efficient treatment, causing severe side-effects. Combination nanotherapy has gained much recent attention for cancer treatment as it reduces the drug-associated toxicity without affecting the therapeutic response. RESULTS: Here we show the modular design of P22 bacteriophage virus-like particles for nanoscale integration of virus-driven enzyme prodrug therapy and photodynamic therapy. These virus capsids carrying CYP activity at the core are decorated with photosensitizer and targeting moiety at the surface for effective combinatory treatment. The estradiol-functionalized nanoparticles are recognized and internalized into ER+ breast tumor cells increasing the intracellular CYP activity and showing the ability to produce reactive oxygen species (ROS) upon UV365 nm irradiation. The generated ROS in synergy with enzymatic activity drastically enhanced the tamoxifen sensitivity in vitro, strongly inhibiting tumor cells. CONCLUSIONS: This work clearly demonstrated that the targeted combinatory treatment using multifunctional biocatalytic P22 represents the effective nanotherapeutics for ER+ breast cancer.

Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed.

Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery.

BACKGROUND: The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. RESULTS: We have designed a nano-bioreactor based on the encapsulation of a cytochrome P450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degradation and acidic pH was increased. Cytochrome P450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine. CONCLUSION: This work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymatic delivery vehicles for clinical applications.

Using Simcyp to project human oral pharmacokinetic variability in early drug research to mitigate mechanism-based adverse events.

Positive allosteric modulators ('potentiators') of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) have been shown to display a mechanism-based exposure-response continuum in preclinical species with procognitive electrophysiological and behavioral effects ('efficacy') at low exposures and motor coordination disruptions at progressively higher exposures. Due to the dose-capping nature of such motor coordination deficits, an exposure threshold-mediated adverse event (C(AE) ), the adequacy of separation between the maximal total plasma compound concentration (C(max) ) at a predicted clinically efficacious oral dose and this adverse event (AE) was explored in early drug research with three AMPAR potentiators considered potential candidates for clinical trials. In vitro metabolism studies in human liver microsomes and human hepatocytes demonstrated the metabolic clearance for each compound was predominately due to cytochromes P450 (CYP). Thus, for each compound's anticipated clinically efficacious dose, human C(max) variability following oral administration was assessed using Simcyp software, which combines its virtual human populations database using extensive demographic, physiological and genomic information with routinely collected compound-specific in vitro biochemical data to simulate and predict drug disposition. Using a combination of experimentally determined recombinant human CYP intrinsic clearances for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4, human binding factors, expected fraction absorbed and estimated steady-state volume of distribution, Simcyp simulations demonstrated that two of the three potentiators had acceptable projected C(max) variability (i.e. the 95th percentile C(max) did not breach C(AE) ). This evaluation aided in the selection of compounds for preclinical progression, and represents a novel application of pharmacologically based pharmacokinetic (PBPK) software approaches to predict interpatient variability.

Expresion del receptor de esteroides y xenobioticos (SXR) y del gen de multirresistencia drogas (MDR1) y de los polimorfismos de las enzimas GSTs, SULTs y CYP en tumores vesicales profundos, analisis de su expresion y correlación con otros factores pronósticos / Steroid and xenobiotic receptor (SXR), multidrug resistance gene (MDR1) and GSTs, SULTs and CYP polymorphism expression in invasive bladder cancer, analysis of their expression and correlation with other prognostic factors

Introducción: El receptor de esteroides y xenobióticos SXR se ha demostrado su activación por parte de numerosos medicamentos, incluidos potentes inductores del citocromo P450, como la rifampicina y el cotrimazol. La función del SXR es bien conocida, y consiste en regular de manera positiva la trascripción del citocromo P450 3A4 (CYP3A4) y el gen de multirresistencia a drogas (multidrug resistance gene) MDR1, se considera una llave clave en el mecanismo regulador del metabolismo de los xenobióticos encontrándose involucrado en todas las fases de detoxificación Múltiples enzimas involucradas en el metabolismo y la degradación de hidrocarburos policíclicos aromáticos (PAH) son polimórficas en humanos, incluyendo la glutation S-transferasa (GSTs), N-acetiltransferasa (NATs), sulfotransferas (SULTs)1A1 y el citocromo p450 (CYP)1B1. Objetivos: Los objetivos que nos hemos planteado son los siguientes: 1. Analizar la expresión del factor de trascripción SXR y del MDR1 en vejiga mediante RT-PCR en tiempo real, tanto en vejiga tumoral como vejiga normal. 2. Analizar la relación de los factores clínicos y patológicos con la expresión del SXR y del MDR1. 3. Analizar la expresión de los polimorfismos de CYP1B1, GSTM1 GSTT1 y SULT1A1, y su correlación con distintos factores clínico patológicos y moleculares. Material y Métodos: De manera prospectiva se calculó un tamaño muestral necesario para este estudio. Se incluyeron 67 pacientes de dos instituciones distintas (Hospital Universitario Miguel Servet (49 HUMS) y Clínica Universitaria de Navarra (18 CUN)), diagnosticados de cáncer vesical infiltrante y tratados mediante cistectomía radical, se le realizó la determinación de la expresión de SXR y MDR1 mediante PCR cuantitativa en tiempo real, así como de los polimorfismos CYP1B1, GSTM1 GSTT1 y SULT1A1 mediante RFLP (restricción de la longitud del fragmento del polimorfismo). Se correlaciona mediante tablas de contingencia la correlación con el resto de los factores pronósticos. Resultados: La media de seguimiento de los pacientes fue de 23,7 meses, con una mediana de 28,26 meses. De los 67 pacientes estudiados, 31 pacientes (46,3%) presentaron progresión de la enfermedad, bien en forma de recidiva local, metástasis a distancia o ambos, con un tiempo medio a recidiva de 12,4 meses, mediana de 10 meses, con un rango de 1,1 mes a 31,9 meses. 36 pacientes (53,7%) no presentaron evidencia de progresión de la enfermedad. El receptor de esteroides y xenobióticos SXR así como el gen de multirresistenia a drogas (Multidrug resistance gene (MDR1)), se expresan en vejiga normal (0,94ΔCt y 0,94ΔCt) y en vejiga tumoral de la pieza de cistectomía (1,09 ΔCt y 0,45 ΔCt). Hemos analizado su expresión de manera cuantitativa y de manera cualitativa. La expresión de SXR se correlaciona con la presencia de carcinoma in situ (p=0,024), infiltración vasculo-linfática (p=0,05) mientras que MDR1 se correlaciona con la presencia de infiltración vasculo linfática (p=0,05) A su vez ambos la presencia de ambos factores se correlaciona entre ellos (p=0,011) Los polimorfismos: CYP1B1, GSTM1, GSTT1 y SULT1A1, se expresan en vejiga pero su expresión no guarda correlación con ningún factor pronóstico Conclusiones: El SXR y el MDR1 se expresan tanto en vejiga normal y tumoral. Y que dicha expresión guarda una correlación con factores pronósticos con influencia en la supervivencia descritas en la literatura

Introduction: Steroid and Xenobiotic Receptor (SXR) has demonstrated its activation by numerous drugs, including cytochrome P450 potent inducers like rifampicina or cotrimazol. The role of SXR is well known, and lies regulating in a positive manner cytochrome P450 3A4 (CYP3A4) transcription and the multidrug resistance gene (MDR1), it’s considered a key in the xenobiotic detoxification mechanism, being involved in all phases of the detoxification process. Enzymes involved in Policyclic Aromatic hidrocarbures (PAH) metabolism and degradation are polymorphic in humans, including glutation S-transferases (GSTs), N-acetiltransferases (NATs), sulfotransferases (SULTs)1A1 and cytochrome p450 (CYP)1B1. Objectives: The objectives we’ve planned are: 1. Analyze the expression of the transcription factor SXR and MDR1 in bladder by means of RT-PCR real time, both in normal bladder and in tumoral bladder. 2. Analyze the relation between clinical and pathological factors with the expression of SXR and MDR1. 3. Analyze the expression of the polymorphims CYP1B1, GSTM1 GSTT1 and SULT1A1 and their correlation with different clinic-pathological and molecular factors. Material and Methods: In a prospective way the size of the sample was estimated. In 67 patients from two institutions (Hospital Universitario Miguel Servet (49 HUMS) and Clinica Universitaria de Navarra (18 CUN)), diagnosed of invasive bladder cancer and treated by means of radical cystectomy, were determined the expression of both SXR and MDR1 by means of real time PCR, as well as the polymorphisms CYP1B1, GSTM1 GSTT1 y SULT1A1 by means of RFLP (Restriction fragment length polymorphism). Correlations with other prognostic factors by contingency tables were performed. Results: Average follow up was 23,7 months with a median of 28,26 months. Of the 67 patients studied, 31 patients (46,3) presented disease progression, in form of local recurrence or in distant metastasis or both. With a average time to progression of 12,4 months and a median of 10 months, with a range of 1,1 month to 31,9 month. 36 patients (53,7%) did not have any evidence of disease progression during follow up. The Steroid and Xenobiotic Receptor as well as the Multidrug Resistance Gene (MDR1) are expressed in both normal bladder (0,94ΔCt y 0,94ΔCt) and tumoral bladder in the cystectomy specimen(1,09 ΔCt y 0,45 ΔCt). We’ve analyzed their expression in a quantitative manner and in a qualitative manner. The expression of SXR correlates with the presence of ca. in situ (p=0,024), vasculo-lymphatic invasion (p=0,05) mean while MDR1 correlates with presence of vasculo-lymphatic invasion (p=0,05) Both factors are correlate between each others (p=0,011). Polymorphisms: CYP1B1, GSTM1, GSTT1 and SULT1A1, are expressed in these patients but their expression doesn’t correlates with any prognostic factor Conclusions: Both SXR and MDR1 are expressed in normal bladder as well as in tumoral bladder. And their expression correlates with different prognostic factors with influence in the survival described in the literature

Adverse effects by artificial grapefruit seed extract products in patients on warfarin therapy.

OBJECTIVE: Grapefruit seed extract (GSE) is promoted as a natural product with antibacterial and antiviral properties. The aim of this study was to investigate the composition of some commercially available GSE products and evaluate their effect in vitro on two cytochrome P450 enzymes, CYP2C9 and CYP3A4. METHODS: A couple on lifelong treatment with warfarin and continuous regular follow-ups took some drops of a GSE product for 3 days. The female patient experienced a minor subcutaneous haematoma 3 days later, and her international normalised ratio (INR) value was 7.9. This was reported to the Swedish Medical Products Agency (MPA) as a spontaneous post-marketing report concerning adverse drug reactions/interactions. The composition of the GSE products was determined by proton and carbon-13 nuclear magnetic resonance spectroscopy (NMR). The inhibitory effect of the GSE products on the cytochrome P450 enzymes was tested in an in vitro baculosome assay. RESULTS: The NMR analysis showed that all three investigated GSE products contained the synthetic preservative benzethonium chloride (BTC) in addition to glycerol and water. No authentic GSE extract was found in any of the three GSE products analysed. Furthermore, BTC was found to be a potent inhibitor of CYP3A4 and CYP2C9 activity in vitro. CONCLUSION: Our results suggest that BTC in the GSE products is responsible for the increase in the INR value in a patient on warfarin treatment.

Central administration of a cytochrome P450-7B product 7 alpha-hydroxypregnenolone improves spatial memory retention in cognitively impaired aged rats.

Pregnenolone (PREG) and dehydroepiandrosterone (DHEA) have been reported to improve memory in aged rodents. In brain, these neurosteroids are transformed predominantly into 7alpha-hydroxylated metabolites by the cytochrome P450-7B1 (CYP7B). The biological role of steroid B-ring hydroxylation is unclear. It has been proposed to generate bioactive derivatives that enhance cognition, immune, and other physiological processes. In support, 7alpha-hydroxylated DHEA increases the immune response in mice with greater potency than the parent steroid. Whether the memory-enhancing effects of PREG in rats is mediated via its 7alpha-hydroxylated metabolite 7alpha-hydroxyPREG is not known. We investigated this by treating memory-impaired aged rats (identified by their spatial memory performances in the Morris water maze task compared with young controls) with 7alpha-hydroxyPREG or PREG administered intracerebroventricularly using osmotic minipumps and then tested the rats during week 2 of steroid treatment in the eight-arm radial-arm version of the water maze (RAWM) that allows repeated assessment of learning. CYP7B bioactivity in hippocampal tissue (percentage conversion of [14C]DHEA to [14C]7alpha-hydroxyDHEA) was decreased selectively in memory-impaired aged rats compared with both young and memory-intact aged rats. 7alpha-hydroxyPREG (100 ng/h) but not PREG (100 ng/h) administration to memory-impaired aged rats for 11 d enhanced spatial memory retention (after a 30 min delay between an exposure trial 1 and test trial 2) in the RAWM. These data provide evidence for a biologically active enzyme product 7alpha-hydroxyPREG and suggests that reduced CYP7B function in the hippocampus of memory-impaired aged rats may, in part, be overcome by administration of 7alpha-hydroxyPREG.

Oxygen-induced reperfusion-injury is caused by ROS: Amelioration is possible by recombinant-DNA antioxidant enzymes and mimics in selected tissues.

Billions of years of photolytic cleavage of the water molecule has led to a build-up at sea-level of a "toxic" oxygen concentration in the atmosphere (to 20%). Unfortunately, this dioxygen is likely to be converted in the mitochondria (cell organelles) during cellular respiration to generate reactive oxygen species (ROS); including free-radicals such as superoxide anion (.O2') and hydroxyl radical (.OH) (peroxide O2") a ROS is not a free radical because its electrons are paired). Development of recombinant-DNA improved isoenzyme forms (or mimics) of antioxidant enzymes such as superoxide dismutases is predicted in this hypothesis to be utilised to ameliorate reperfusion-injury (and other oxygen-induced molecular pathology). Introduction into the human genome of the genes for expression of antioxidant enzymes, to order in particular tissues, is imminent. Furthermore, O2 itself will be recognised universally as a harmful gas that can subject the cell to oxidative stress; because it produces ROS, such as the superoxide anion when it acts as the terminal electron acceptor in cellular respiration in the production of water from hydrogen ions in mitochondria. In conclusion, therefore, oxygen-induced injury in humans can no longer be accepted during medical techniques such as reperfusion procedures, because this is associated with reperfusion-injury that can be the cause of several serious medical conditions arising from biomolecular pathology. Some of this is caused by isoforms of cytochromes P450 (CYP; EC 1.14.14.1) such as 3A4, 2D6 and 2C19. These can generate ROS in the liver at low substrate concentrations by futile recycling of oxygen.

Metabolizing enzyme toxicology assay chip (MetaChip) for high-throughput microscale toxicity analyses.

The clinical progression of new chemical entities to pharmaceuticals remains hindered by the relatively slow pace of technology development in toxicology and clinical safety evaluation, particularly in vitro approaches, that can be used in the preclinical and early clinical phases of drug development. To alleviate this bottle-neck, we have developed a metabolizing enzyme toxicology assay chip (MetaChip) that combines high-throughput P450 catalysis with cell-based screening on a microscale platform. The MetaChip concept is demonstrated by using sol-gel encapsulated P450s to activate the prodrug cyclophosphamide, which is the major constituent of the anticancer drug Cytoxan, as well as other compounds that are activated by P450 metabolism. The MetaChip provides a high-throughput microscale alternative to currently used in vitro methods for human metabolism and toxicology screening based on liver slices, cultured human hepatocytes, purified microsomal preparations, or isolated and purified P450s. This technology creates opportunities for rapid and inexpensive assessment of ADME/Tox (absorption, distribution, metabolism, excretion/toxicology) at very early phases of drug development, thereby enabling unsuitable candidates to be eliminated from consideration much earlier in the drug discovery process.

Comparative pharmacokinetic interaction profiles of pravastatin, simvastatin, and atorvastatin when coadministered with cytochrome P450 inhibitors.

Three-hydroxy-3-methylglutaryl coenzyme-A reductase inhibitors (statins) are first-line treatments for hypercholesterolemia. Although exceedingly well tolerated, treatment with statins incurs a small risk of myopathy or potentially fatal rhabdomyolysis, particularly when coadministered with medications that increase their systemic exposure. Studies compared the multiple-dose pharmacokinetic interaction profiles of pravastatin, simvastatin, and atorvastatin when coadministered with 4 inhibitors of cytochrome P450-3A4 isoenzymes in healthy subjects. Compared with pravastatin alone, coadministration of verapamil, mibefradil, or itraconazole with pravastatin was associated with no significant changes in pravastatin pharmacokinetics. However, concomitant verapamil increased the simvastatin area under the concentration:time curve (AUC) approximately fourfold, the maximum serum concentration (C(max)) fivefold, and the active metabolite simvastatin acid AUC and C(max) approximately four- and threefold, respectively (all comparisons p <0.001). Similar (greater than fourfold) important increases in these parameters and a >60% increase in the serum half-life (p = 0.03) of atorvastatin were observed when coadministered with mibefradil. The half-life of atorvastatin also increased by approximately 60% (p = 0.052) when coadministered with itraconazole, which elicited a 2.4-fold increase in the C(max) of atorvastatin and a 47% increase in the AUC (p <0.001 for C(max) and AUC). Clarithromycin significantly (p <0.001) increased the AUC (and C(max)) of all 3 statins, most markedly simvastatin ( approximately 10-fold increase in AUC) and simvastatin acid (12-fold), followed by atorvastatin (greater than fourfold) and then pravastatin (almost twofold). Pravastatin has a neutral drug interaction profile relative to cytochrome P450-3A4 inhibitors, but these substrates markedly increase systemic exposure to simvastatin and atorvastatin.

Hepatobiliary excretion of berberine.

Berberine is a bioactive herbal ingredient isolated from the roots and bark of Berberis aristata or Coptis chinensis. To investigate the detailed pharmacokinetics of berberine and its mechanisms of hepatobiliary excretion, an in vivo microdialysis coupled with high-performance liquid chromatography was performed. In the control group, rats received berberine alone; in the drug-treated group, 10 min before berberine administration, the rats were injected with cyclosporin A (CsA), a P-glycoprotein (P-gp) inhibitor; quinidine, both organic cation transport (OCT) and P-gp inhibitors; SKF-525A (proadifen), a cytochrome P450 inhibitor; and probenecid to inhibit the glucuronidation. The results indicate that berberine displays a linear pharmacokinetic phenomenon in the dosage range from 10 to 20 mg kg(-1), since a proportional increase in the area under the concentration-time curve (AUC) of berberine was observed in this dosage range. Moreover, berberine was processed through hepatobiliary excretion against a concentration gradient based on the bile-to-blood distribution ratio (AUC(bile)/AUC(blood)); the active berberine efflux might be affected by P-gp and OCT since coadministration of berberine and CsA or quinidine at the same dosage of 10 mg kg(-1) significantly decreased the berberine amount in bile. In addition, berberine was metabolized in the liver with phase I demethylation and phase II glucuronidation, as identified by liquid chromatography/tandem mass spectrometry. Also, the phase I metabolism of berberine was partially reduced by SKF-525A treatment, but the phase II glucuronidation of berberine was not obviously affected by probenecid under the present study design.

The effect of repeat dosing with cimetidine on the pharmacokinetics of intravenous granisetron in healthy volunteers.

The primary route of elimination of granisetron is by oxidative hepatic metabolism, thus its pharmacokinetic profile may be altered by co-administration of other drugs that inhibit or induce hepatic drug metabolizing enzymes. This open-label study investigated the effect of inhibition of cimetidine, a potent inhibitor of CYP1A2, CYP2D6 and CYP3A4, on the pharmacokinetic profile of intravenous granisetron in healthy male volunteers. Subjects (n = 12; 18-60 years) received granisetron (40 microg kg(-1)) infused over 3 min, six days before and on the eighth day of dosing with cimetidine (200 mg, four times a day). Blood samples were taken for pharmacokinetic analysis at intervals over 48 h following the administration of each dose of granisetron. Clinical chemistry, haematology and urinalysis were performed before, and 24 h after, each infusion. Electrocardiogram (ECG), resting blood pressure (BP) and pulse were monitored. There were no significant changes in the ECG, lead II trace or ECG time intervals, pulse or blood pressure on each study day. Minor falls in pulse rate and BP (likely to be related to recumbent posture) were seen during both granisetron dosing days, lasting 2 h after each infusion. No significant changes were apparent in the clinical chemistry, haematology or urinalysis measurements following granisetron dosing. No pharmacokinetic parameters measured after cimetidine administration were significantly different from those taken before. Adverse events were mild-to-moderate in severity and were similar to those reported in other studies with granisetron. The pharmacokinetics of granisetron, when administered as a single dose, appeared to be unaltered by cimetidine, an inhibitor of multiple hepatic enzymes (CYP1A2, CYP2D6 and CYP3A4). Granisetron was equally well tolerated before and after repeated dosing with cimetidine.

Repeated gene transfer of naked prostacyclin synthase plasmid into skeletal muscles attenuates monocrotaline-induced pulmonary hypertension and prolongs survival in rats.

A safer, less invasive method for repeated transgene administration is desirable for clinical application of gene therapy targeting chronic diseases, including pulmonary hypertension (PH). Thus, effects of prostaglandin I2 (prostacyclin) synthase (PGIS) gene transfer by the naked DNA method into skeletal muscle were investigated in monocrotaline (MCT)-induced PH rats. A single injection of rat PGIS cDNA-encoding plasmid into thigh muscle 3 days after bupivacaine pretreatment transiently increased muscle PGIS protein expression and muscle and serum levels of a stable prostacyclin metabolite (6-keto-prostaglandin F1). The muscle 6-keto-prostaglandin F1 level peaked on day 2 but was still elevated on day 7; prostacyclin selectively increased lung cyclic AMP levels as compared with liver and kidney. MCT induced a marked rise in right ventricular (RV) systolic pressure, pulmonary arterial wall thickening, and RV hypertrophy. Repeated PGIS gene transfer every week lowered RV systolic pressure and ameliorated RV and pulmonary artery remodeling in MCT-induced PH rats. Furthermore, repeated PGIS gene transfer significantly improved the survival rate of MCT-induced PH rats. In conclusion, repeated PGIS gene transfer into skeletal muscle not only attenuated the development of PH and cardiovascular remodeling but also improved the prognosis for MCT-induced PH rats. This study may provide insight into a new treatment strategy for PH.

Sustained P450 expression and prodrug activation in bolus cyclophosphamide-treated cultured tumor cells. Impact of prodrug schedule on P450 gene-directed enzyme prodrug therapy.

Cytochrome P450-based gene therapy can substantially increase the sensitivity of tumor cells to P450-activated cancer chemotherapeutic prodrugs such as cyclophosphamide (CPA) without increasing host toxicity. While the role of 4-OH-CPA, the primary active metabolite of CPA, in eliciting tumor cell death is well established, the effect of 4-OH-CPA exposure on the capacity of P450-expressing tumor cells for continued metabolism and activation of CPA has not been investigated. The present study addresses this question and characterizes the impact of CPA dose and treatment schedule on the ability of P450-expressing tumor cells to sustain prodrug activation over time. 9L gliosarcoma cells expressing human P450 2B6 and treated with CPA in a continuous manner exhibited a time- and CPA dose-dependent decrease in P450-catalyzed CPA 4-hydroxylase activity. This decrease reflects a selective, 4-OH-CPA-induced loss of cellular P450 protein content. By contrast, when the P450-expressing tumor cells were treated with CPA as a single 8 hours exposure, cellular CPA 4-hydroxylase activity and P450 protein expression were substantially prolonged when compared to continuous prodrug treatment. This schedule-dependent effect of CPA was influenced by the level of P450 protein expressed in the tumor cells. At high P450 protein and activity levels, which could be achieved by culturing the tumor cells at high cell density, net production and release of 4-OH-CPA into the culture media was increased substantially. This increase fully offset the decline in CPA 4-hydroxylase activity as the tumor cells underwent CPA-induced apoptotic death. These findings demonstrate the impact of CPA dose and treatment schedule on the efficacy of P450 gene-directed enzyme prodrug therapy, with bolus CPA treatment being compatible with sustained expression of P450 protein and maintenance of P450-dependent prodrug activation by the target tumor tissue.

Is cytochrome P450 2C9 genotype associated with NSAID gastric ulceration?

AIMS: The aim of this study was to explore whether genetic variation of cytochrome P450 2C9 (CYP2C9) contributes to NSAID-associated gastric ulceration. The hypothesis tested was that CYP2C9 poor metabolizer genotype would predict higher risk of gastric ulceration in patients on NSAIDs that are metabolized by CYP2C9, due to higher plasma NSAID concentrations. METHODS: Peripheral blood DNA samples from 23 people with a history of gastric ulceration attributed to NSAIDs metabolized by CYP2C9, and from 32 people on NSAIDs without gastropathy, were analysed to determine CYP2C9 genotype. RESULTS: The following genotypes were found: *1/*1 (wild type) in 70% of cases and 58% of controls, *1/*2 in 17% of cases and 29% of controls, *1/*3 in 13% of cases and 13% of controls. The difference between case and control nonwild-type genotype frequency was 11.5% (95% CI -14,37%), with the direction of the difference being against the hypothesis. No individuals with homozygote poor metaboliser genotype were identified. The differences in genotype frequencies between the two groups were not significant and the frequencies were similar to those in a large published population study. Ninety-five percent binomial confidence interval analysis confirms that there is no apparent clinically significant relationship between CYP2C9 genotype and risk of gastric ulceration although a small difference in risk in poor metabolizers cannot be excluded. CONCLUSIONS: These results do not support the hypothesis that gastric ulceration resulting from NSAID usage is linked to the poor metabolizing genotypes of CYP2C9.

Gene transfer of human prostacyclin synthase ameliorates monocrotaline-induced pulmonary hypertension in rats.

BACKGROUND: Prostacyclin is a potent vasodilator that also inhibits platelet adhesion and cell growth. We investigated whether in vivo gene transfer of human prostacyclin synthase (PGIS) ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. METHODS AND RESULTS: The cDNA encoding PGIS was intratracheally transfected into the lungs of rats by the hemagglutinating virus of Japan-liposome method. Rats transfected with control vector lacking the PGIS gene served as controls. Three weeks after MCT injection, mean pulmonary arterial pressure and total pulmonary resistance had increased significantly; the increases were significantly attenuated in PGIS gene-transfected rats compared with controls [mean pulmonary arterial pressure, 31+/-1 versus 35+/-1 mm Hg (-12%); total pulmonary resistance, 0.087+/-0.01 versus 0.113+/-0.01 mm Hg x mL x min(-1) x kg(-1) (-23%), both P:<0.05]. Systemic arterial pressure and heart rate were unaffected. Histologically, PGIS gene transfer inhibited the increase in medial wall thickness of peripheral pulmonary arteries that resulted from MCT injection. PGIS immunoreactivity was intense predominantly in the bronchial epithelium and alveolar cells. Lung tissue levels of 6-keto-PGF(1alpha), a stable metabolite of prostacyclin, were significantly increased for >/=1 week after transfer of PGIS gene. The Kaplan-Meier survival curves demonstrated that repeated transfer of PGIS gene every 2 weeks increased survival rate in MCT rats (log-rank test, P:<0.01). CONCLUSIONS: Intratracheal transfer of the human PGIS gene augmented pulmonary prostacyclin synthesis, ameliorated MCT-induced pulmonary hypertension, and thereby improved survival in MCT rats.

Adrenal cytochrome P-45011 beta-proteoliposomes catalyzing aldosterone synthesis: preparation and characterization.

Purified cytochrome P-45011 beta from bovine adrenocortical mitochondria was successfully incorporated into the liposome membranes composed of phosphatidylcholine, phosphatidylethanolamine and cardiolipin at a molar ratio of 2:2:1. The incorporation of P-45011 beta into the liposome membranes was ascertained by the Ficoll density gradient centrifugation and the protein refractoriness to trypsin digestion. The prepared proteoliposomes containing P-45011 beta and phospholipid at a molar ratio of 1:3000 were unilamellar vesicles of about 40 nm in average diameter. The P-45011 beta embedded in the liposome membranes was found to be more stable than the detergent-solubilized form. The reconstituted system containing the P-45011 beta-proteoliposomes, adrenodoxin and NADPH-adrenodoxin reductase showed catalytic activities not only for the hydroxylation of 11-deoxycorticosterone at 11 beta- and 18-positions but also for its conversion into aldosterone with a turnover number of 2.3 nmol/min per nmol of P-45011 beta. A successive reaction without the intermediates leaving from the enzyme was suggested for the P-45011 beta-mediated conversion of 11-deoxycorticosterone to aldosterone following the result that the formation of aldosterone was linear with respect to time without the lag phase; this was confirmed by the result that radioactivity in aldosterone from 3H-labeled 11-deoxycorticosterone was scarcely decreased by the addition of unlabeled intermediates to the reactions system.