Assessment of ameliorative effect of Trigonella foenum-graecum against CuO-NPs induced toxicity in Oreochromis mossambicus.

The current study assessed the ameliorative effect of Trigonella foenum graceum extract against copper oxide nanoparticles (CuO-NPs) induced toxicity in Oreochromis mossambicus. For this purpose 100 healthy fish weighing 20±2.34g were randomly divided into five different groups in duplicates and designated as control (C) no treatment, positive control (G*) treated with 0.12mg/L of CuO-NPs, experimental co-treated groups G1, G2 and G3 were treated with Trigonella foenum-graecum extract @ 18, 26 and 52mg/L along with 0.12 mg/L of CuO-NPs, respectively. In this study significant (P<0.05) changes were observed in the antioxidant activity of enzymes and histological alterations in the liver and intestine of fish in G*, G1 and G2 groups while a good ameliorative response of Trigonella foenum-graecum was observed in G3. Dose dependent alterations in glutathione, lipid peroxides, catalase, and malondialdehyde as well as histological architecture of liver and intestine were observed in treated groups, where more alterations were observed in positive control and low dose treated groups of Trigonella foenum-graecum. Moreover, more ameliorative effect was observed in high dose of Trigonella foenum-graecum treated group (G3). This study is novel as no previous data is available on the amelioration of Trigonella foenum-graecum extract against CuO-NPs induced toxicity in fish.

A Novel Study Design Using Continuous Intravenous and Intraduodenal Infusions of Midazolam and Voriconazole for Mechanistic Quantitative Assessment of Hepatic and Intestinal CYP3A Inhibition.

The extent of a drug-drug interaction (DDI) mediated by cytochrome P450 (CYP) 3A inhibitors is highly variable during a dosing interval, as it depends on the temporal course of victim and perpetrator drug concentrations at intestinal and hepatic CYP3A expression sites. Capturing the time course of inhibition is therefore difficult using standard DDI studies assessing changes in area under the curve; thus, a novel design was developed. In a 4-period changeover pilot study, 6 healthy men received intraduodenal or intravenous infusions of the CYP3A substrate midazolam (MDZ) at a rate of 0.26 mg/h for 24 hours. This was combined with intraduodenal or intravenous infusion of the CYP3A inhibitor voriconazole (VRZ), administered at rates of 7.5 mg/h from 8 to 16 hours and of 15 mg/h from 16 to 24 hours, after starting midazolam administration. Plasma and urine concentrations of VRZ, MDZ, and its major metabolites were quantified by liquid chromatography-tandem mass spectrometry and analyzed by semiphysiological population pharmacokinetic nonlinear mixed-effects modeling. A model including mechanism-based inactivation of the metabolizing enzymes (maximum inactivation rate constant kinact , 2.83 h-1 ; dissociation rate constant KI , 9.33 µM) described the pharmacokinetics of VRZ well. By introducing competitive inhibition by VRZ on primary and secondary MDZ metabolism, concentration-time profiles, MDZ and its metabolites were captured appropriately. The model provides estimates of local concentrations of substrate and inhibitor at the major CYP3A expression sites and thus of the respective dynamic extent of inhibition. A combination of intravenous and intraduodenal infusions of inhibitors and substrates has the potential to provide a more accurate assessment of DDIs occurring in both gut wall and liver.

Feed pellets containing chitosan nanoparticles as plasmid DNA oral delivery system for fish: In vivo assessment in gilthead sea bream (Sparus aurata) juveniles.

The aim of this study was the assessment of preloaded feed pellets as a delivery system for plasmid DNA (pDNA), with the purpose of evaluating the potential administration of DNA vaccines orally in aquacultured fish. Pellets were made up by usual feed ingredients, which were mixed with chitosan nanoparticles entrapping a model plasmid (pCMVß) expressible in eukaryotic cells before being elaborated. The plasmid is characterized by the insertion of the reporter gene lacZ, encoding for the bacterial enzyme ß-galactosidase (ß-gal). The possible in vivo expression of the exogenous gene was measured in different fish tissues of gilthead sea bream (Sparus aurata) juveniles by two different procedures. On the one hand, the activity of the enzyme ß-gal was detected and quantified in muscle, liver and intestine; on the other, specific IgM against ß-gal antigen was titrated in blood samples. Intramuscular (i.m.) injection of equal amounts of plasmid was also carried out for the purpose of comparison with oral administration. The expression of the reporter gene was detected in fish tissues following both oral and i. m. administration of pDNA up to 60 days. However, organ distribution of the gene expression was more evident after oral (ß-gal activity measured in gut, liver and muscle) than after parenteral administration (restricted to adjacent muscle tissues). In agreement, specific IgM titration indicated that humoral immune response was more intense and sustained throughout the experimental period after oral than after i. m. delivery of equal amounts of pDNA. These results suggest that feed pellets containing chitosan nanoparticles might enable efficient oral delivery of pDNA, a fact that might imply valuable applications in terms of on-farm mass immunization purposes, especially with regard to DNA-based vaccines and small size fish, in which i. m. administration remains unfeasible.

Evaluation of drug-drug interactions for oncology therapies: in vitro-in vivo extrapolation model-based risk assessment.

AIMS: Understanding drug-drug interactions (DDI) is a critical part of the drug development process as polypharmacy has become commonplace in many therapeutic areas including the cancer patient population. The objectives of this study were to investigate cytochrome P450 (CYP)-mediated DDI profiles available for therapies used in the oncology setting and evaluate how models based on in vitro-in vivo extrapolation performed in predicting CYP-mediated DDI risk. METHODS: A dataset of 125 oncology therapies was collated using drug label and approval history information, incorporating in vitro and clinical PK data. The predictive accuracy of the basic and net effect mechanistic static models was assessed using this oncology drug dataset, for both victim and perpetrator potential of CYP3A-mediated DDI. RESULTS: The incidence of CYP3A-mediated interaction potential was 47%, 22% and 11% for substrates, inhibitors and inducers, respectively. The basic models for precipitants gave conservative predictions with no false negatives, whilst the mechanistic static models provided reasonable quantitative predictions (2.3-3-fold error). Further analysis revealed that incorporating DDI at the level of the intestine was in most cases over-predicting interaction magnitude due to overestimates of the rate and extent of oral absorption of the precipitant. Quantifying victim DDI potential was also demonstrated using fmCYP3A estimates from ketoconazole clinical DDI studies to predict the magnitude of interaction on co-administration with the CYP3A inducer, rifampicin (1.6-3.3 fold error). CONCLUSIONS: This work illustrates the utility and limitations of current DDI risk assessment approaches applied to a range of contemporary anti-cancer agents, and discusses the implications for therapeutic combination strategies.

Assessment of enzymatic efficiency on protein digestion in the tilapia Oreochromis niloticus.

The present study develops an experimental procedure aimed to estimate the efficiency of protein digestion in fish by measuring both gut transit rate and total amount of the main intestinal proteases (trypsin and chymotrypsin). The selected species was the Nile tilapia (Oreochromis niloticus). Total time for digestion, calculated through the estimation of gut transit rate using differently colored feeds, was 7.15 h. Mean production of trypsin and chymotrypsin was 15.94 and 24.11 mU in the proximal intestine and much lower (2,39, 4.90 mU) in the distal intestine. The enzyme efficiency, calculated from the average enzyme activity and time of residence of the digesta in each intestinal section, points to the major role of proximal intestine in protein digestion for this species. Results are discussed in relation to the main features characterizing digestion in stomachless fish.

Further assessment of 17alpha-ethinyl estradiol as an inhibitor of different human cytochrome P450 forms in vitro.

17alpha-Ethinyl estradiol (EE) was systematically evaluated as a reversible and time-dependent inhibitor of 11 human drug-metabolizing cytochromes P450 (P450s) (CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and CYP3A5) in vitro. When ranked, the lowest IC(50) (concentration of inhibitor required to decrease activity by 50%) values were obtained with recombinant CYP1A1 (rCYP1A1) [IC(50(total)) = IC(50(free)) = 2.7 microM] and CYP2C19 activity in human liver microsomes (HLM) [IC(50(total)) = 4.4 microM; IC(50(free)) = 2.8 microM]. For rCYP1A1, formal inhibition studies revealed that EE was a competitive inhibitor [K(i(free)) = 1.4 microM]. All the other IC(50) values were greater than 8.0 microM, and the weakest inhibition was observed with CYP1A2 activity in HLM (IC(50(free)) > 39 microM). In agreement, the IC(50) characterizing the inhibition of melatonin (MEL) 6-hydroxylation in human intestine microsomes (CYP1A1-catalyzed) was lower than that of HLM (0.91 versus >40 microM). Because EE is known to affect the pharmacokinetics of CYP2C19 probe drugs, this result raises the possibility that the concentration of EE during first pass may exceed 1000 nM, sufficient to affect CYP1A1 and CYP2C19, with less impact on CYP3A4 and other P450s. The results implicate intestinal CYP1A1, and possibly CYP2C19, as the loci of EE drug interactions with highly extracted drugs like MEL. Overall, it is very difficult to rationalize drug interactions involving EE based on direct inhibition of CYP2B6 (e.g., selegiline) and hepatic CYP1A2 (e.g., MEL, tizanidine, caffeine, and theophylline).

Assessment of intestinal permeability: enzymatic determination of urinary mannitol, raffinose, sucrose and lactose on Hitachi analyzer.

The sugar absorption test is the usual test for measurement of intestinal permeability. After intestinal absorption of probe sugars the subsequently excreted sugars are measured in urine. We have developed four enzymatic methods for the measurement of the urinary concentration of the probe sugars mannitol, raffinose, lactose and sucrose. Mannitol, lactose and sucrose are directly measured on Hitachi 917 using mannitol dehydrogenase, beta-galactosidase and invertase, respectively, as enzyme reagents. Raffinose measurement needs a three hours preincubation with alpha-galactosidase, after which the liberated sucrose is measured. The analytical performances such as within- and between-run precision, linearity, lowest detection limit, interference of other sugars and comparison with a gas chromatographic method are described for the four methods. These methods are accurate an can easily be performed in any clinical laboratory.

Quantitative assessment of intestinal eosinophils and mast cells in inflammatory bowel disease.

Previous studies on the frequency of intestinal mast cells and eosinophils in patients with inflammatory bowel disease yielded conflicting results. In the present morphometric study, we quantified mast cells and eosinophils in the lamina propria by histological and immunohistochemical methods in 64 patients suffering from Crohn's disease (33 cases) or ulcerative colitis (31 cases), and in 29 controls. Histological data from 206 biopsies were related to the presence of mucosal inflammation and clinical parameters. The number of eosinophils was increased in patients with inflammatory bowel conditions (mean +/- SE: 331 +/- 44/mm2) as compared to controls (258 +/- 27/mm2), and was dependent on disease activity and drug treatment. Mean mast cell numbers did not differ between patients and controls. However, a reduced mast cell number was found in toluidine blue-stained sections of actively inflamed tissue areas (143 +/- 16/mm2, versus 206 +/- 18/mm2 in non-inflamed tissue). Immunohistochemical studies using antibodies against the granule proteins tryptase and chymase suggest that this decrease in mast cell numbers is due to mast cell degranulation. The present data show that the number of intestinal mast cells and eosinophils is altered in patients with inflammatory bowel diseases, suggesting that both cell types are involved in the pathogenesis of chronic intestinal inflammation.

Different ouabain sensitivities of Na+/K(+)-ATPase from Poekilocerus bufonius tissues and a possible physiological cost.

1. The properties of Na+/K(+)-transporting ATPase in microsomal preparation from mid-gut of the grasshopper, Poekilocerus bufonius, were investigated and compared with the same enzyme from brain and excretory system. 2. Two components of ATPases activity are present in the three tissues studied. 3. The physiochemical properties of Na+/K(+)-transporting ATPase from mid-gut, brain and excretory system (hind-gut plus Malpighian tubules) are essentially the same. 4. The calculated values of PI50 were 2 (I50 = 1 x 10(-2) M), 3.7 (I50 = 2 x 10(-4) M) and 6.4 (I50 = 3.98 x 10(-7)) for Na+/K(+)-ATPase from mid-gut, excretory system and brain, respectively. The mid-gut contains the most ouabain-resistant Na+/K(+)-ATPase. 5. The results suggest that P. bufonius have developed some tolerance to toxic cardiac glycosides (CGS), but there is a possibility of autotoxicity as indicated by the presence of ouabain-sensitive ATPase from brain tissue. 6. It was concluded that the dissimilarities of Na+/K(+)-ATPases from different tissues of P. bufonius are probably due to tissue-dependent differences in ouabain sensitivity (or isoenzymes pattern) available in the same insect. 7. The atrophy of female flight muscle of P. bufonius suggests the possibility of physiological cost inflicted on insects consuming poisonous plants.

Human intestinal diamine oxidase (DAO) activity in Crohn's disease: a new marker for disease assessment?

The key-enzyme for the metabolism of diamines in man is diamine oxidase (DAO). Its highest activities are in the intestinal mucosa, localized in the cytoplasm of the mature enterocytes of the small and large bowel. If the gut is affected by inflammation in Crohn's disease macroscopical changes are observed. This prospective study investigated if these mucosal alterations are also reflected in changes of mucosal diamine oxidase activity and/or mucosal histamine content respectively. Twenty patients (12 female, 8 male; age: means = 31, range 18-49 years) undergoing gut resection because of complications in Crohn's disease (Jan.-Dec. 1988) formed the basis of the study. Tissue samples of the resected material from areas inflamed and histologically not involved in the disease were investigated for diamine oxidase activities and histamine content. Diamine oxidase activities in the mucosa obtained from the macroscopically normal proximal (155.6; (76-393) mU/g (means, range)) and distal (132; (58.5-295) mU/g) resection margins were similar to our previous findings. In all patients, however, samples from the diseased mucosa had significantly (ca. 50%) lower diamine oxidase activities (74.5; (5-262) mU/g) compared to the healthy tissue. Similar differences were found in material obtained either from whole intestinal wall or from the mucosa. The determination of diamine oxidase activity constitutes possibly a more unambiguous and earlier parameter for assessing the extent of the inflamed area than histological disease presentations. Using biopsies the necessary extent of resection could be estimated before operation: this may influence operative strategies and help in the definition of the minimum amount of inflamed gut to be removed.

Error estimation in the quantification of alkaline phosphatase isoenzymes by selective inhibition methods.

A method for calculating, by selective inhibition, the activities in serum of isoenzymes of alkaline phosphatase (EC 3.1.3.1) originating from bone, liver, intestine, and placenta produced results for a sample of patients for which the imprecision was five- to 25-fold higher than that of the alkaline phosphatase method used--too imprecise for routine clinical use. Error analysis by direct calculation and by Monte Carlo estimation revealed that the algorithm used in the method completely accounted for the increase in imprecision of the isoenzyme estimations. I recommend that all methods involving such algorithms or the principle of multicomponent analysis should undergo a thorough error analysis by use of the techniques described here, to obtain an estimate of the increase in imprecision that is ascribable to the particular numerical technique used.

[Assessment of the in vitro digestibility of dietary proteins and of the degree of the breakdown of their antigenic structures by using polyenzyme systems]. / Otsenka perevarivaemosti in vitro pishchevykh belkov i stepeni rasshchepleniia ikh antigennykh struktur s ispol'zovaniem polifermentnykh sistem.

A comparative assessment was made of the digestion of bovine serum albumin (BSA), chicken ovalbumin (OVA), and casein by means of the gastric juice--duodenal contents floccular gel structures (FGS) system and a four-enzymic system including trypsin, chymotrypsin, peptidase, and bacterial protease preparations. Decomposition of the BSA and OVA antigenic structures with the use of the two systems was also studied. Significant differences in BSA and OVA digestion by the gastric juice--FGS system were detected both with respect to amino nitrogen content and to the degree of their antigenic structure decomposition, whereas no such differences were observed when the four-enzymic system was used. The systems most accurately simulating the 'proteolytic conveyer' conditions of the gastrointestinal tract are preferable for the studies. The developed method is recommended for use in comparative assessment of the nutrient protein sensitizing properties.

GI absorption of beta-lactam antibiotics I: kinetic assessment of competing absorption and degradation in GI tract.

An equation was derived for the simultaneous assessment of rate constatns for absorption and nonenzymatic degradation of unstable drugs in in situ absorption experiments. The equation was substantiated by using a variety of beta-lactam antibiotics in the recirculation technique through the rat small intestine. Plots of the apparent first-order rate constant for the disappearance of the drug from the gut lumen versus the reciprocal of the volume of recirculating solution yielded a straight line with a slope equal to the intrinsic absorption rate constant and with an intercept equal to the nonenzymatic degradation rate constant in the GI lumen. The kinetic method for evaluation of the absorption rate constant also was developed for a more complex situation in the GI lumen involving absorption, nonenzymatic degradation, and enzymatic metabolism. The proposed method was confirmed with carbenicillin indanyl, which was metabolized rapidly to carbenicillin by the action of nonspecific esterase in the intestine. In the absence of information of Michaelis--Menten kinetic parameters, the present method is advantageous for evaluation of the intrinsic absorption rate of all unstable drugs.