21.
Göransson, Ulf, et al.
(författare)
Expression of Viola cyclotides by liquid chromatography-mass spectrometry and tandem mass spectrometry sequencing of intercysteine loops after introduction of charges and cleavage sites by aminoethylation
2003
Ingår i: Analytical Biochemistry. - 0003-2697 .- 1096-0309. ; 318:1, s. 107-117
Tidskriftsartikel (refereegranskat) abstract
The expression of cyclotides—macrocyclic plant peptides—was profiled in six violets, Viola cotyledon, V. biflora, V. arvensis,V. tricolor, V. riviniana, and V. odorata, by LC-MS. All were found to express notably complex mixtures, with single speciescontaining >50 cyclotides. To facilitate their sequencing by MS-MS, an analytical strategy is presented involving aminoethylation ofcysteines. This overcomes a number of problems intimately associated with the cyclotide core structure—that is, their joined N and Ctermini, disulfide knot, and low or clustered content of positively charged amino acids and enzymatic cleavage sites. As a result,charges as well as cleavage sites are introduced at the most conserved part of their sequence, the cysteines. Combined with trypticdigestion, all intercysteine loops are then of suitable size and charge for MS-MS sequencing. The utility of this strategy is shown bythe sequencing of two novel cyclotides isolated from V. cotyledon; vico A (cyclo-(AESCVYIPCFTGIAGCSCKNKVCYYNGSIPC)) and vico B(cyclo-(AESCVYIPCITGIAGCSCKNKVCYYNGSIPC)); their complete sequence could be determined bynanospray MS-MS. The strategy for converting conserved cysteines to enzymatic cleavage sites might also benefit the study of otherpeptides and proteins displaying similar structural problems for MS analysis.
22.
Hallberg, Mathias, et al.
(författare)
Neuropeptide conversion to bioactive fragments : an important pathway in neuromodulation
2003
Ingår i: Current protein and peptide science. - : Bentham Science Publishers Ltd.. - 1389-2037 .- 1875-5550. ; 4:1, s. 31-44
Tidskriftsartikel (refereegranskat) abstract
Biosynthetic pathways for the formation of neuroactive peptides and the processes for their inactivation include several enzymatic steps. In addition to enzymatic processing and degradation, several neuropeptides have been shown to undergo enzymatic conversion to fragments with retained or modified biological activity. This has most clearly been demonstrated for e.g. opioid peptides, tachykinins, calcitonin gene-related peptide (CGRP) as well as for peptides belonging to the renin-angiotensin system. Sometimes the released fragment shares the activity of the parent compound. However, in many cases the conversion reaction is linked to a change in the receptor activation profile, i.e. the generated fragment acts on and stimulates a receptor not recognized by the parent peptide. This review will describe the characteristics of certain neuropeptide fragments having the ability to modify the biological action of the peptide from which they are derived. Focus will be directed to the tachykinins, the opioid peptides, angiotensins as well as to CGRP, bradykinin and nociceptin. The κ opioid receptor selective opioid peptide, dynorphin, recognized for its ability to produce dysphoria, is converted to the δ opioid receptor agonist Leu-enkephalin, with euphoric properties. The tachykinins, typified by substance P (SP), is converted to the bioactive fragment SP(1-7), a heptapeptide mimicking some but opposing other effects of the parent peptide. The bioactive angiotensin II, known to bind to and stimulate the AT-1 and AT-2 receptors, is converted to angiotensin IV (i.e. angiotensin 3-8) with preference for the AT-4 sites or to angiotensin (1-7), not recognized by any of these receptors. Both angiotensin IV and angiotensin (1-7) are biologically active. For example angiotensin (1-7) retains some of the actions ascribed for angiotensin II but is shown to counteract others. Thus, it is obvious that the activity of many neuroactive peptides is modulated by bioactive fragments, which are formed by the action of a variety of peptidases. This phenomenon appears to represent an important regulatory mechanism that modulates many neuropeptide systems but is generally not acknowledged.
23.
Hornestam, Björn, et al.
(författare)
Intravenously administered digoxin in patients with acute atrial fibrillation : a population pharmacokinetic/pharmacodynamic analysis based on the Digitalis in Acute Atrial Fibrillation trial
2003
Ingår i: European Journal of Clinical Pharmacology. - : Springer Science and Business Media LLC. - 0031-6970 .- 1432-1041. ; 58:11, s. 747-755
Tidskriftsartikel (refereegranskat) abstract
OBJECTIVE: Atrial fibrillation is commonly treated with intravenously administered digoxin. The main objective of this study was to investigate the relationship between plasma concentration of digoxin and heart rate. SUBJECTS AND METHODS: Plasma concentrations of digoxin were analysed in 105 patients allocated to digoxin therapy in the Digitalis in Acute Atrial Fibrillation (DAAF) trial. A pharmacokinetic/pharmacodynamic (PK/PD) model for the relationship among digoxin dose, plasma concentration and heart rate in patients remaining in atrial fibrillation was constructed using non-linear, mixed-effect modelling. One hundred and twenty-two placebo-treated patients were included as a control group. In 56 patients, one late sample at 16 h after the first dose of digoxin was obtained while in 49 patients an early sample at 0.25-0.5 h and a late sample 16 h after the first dose were obtained. Heart rate was measured at 0, 2, 6, 12 and 16 h after inclusion, with data from 98, 89, 67, 56 and 53 patients available at each time point, respectively. RESULTS: A two-compartment model best described the time course of digoxin concentrations in plasma. Digoxin and creatinine clearance correlated strongly and mean plasma concentration of digoxin at 16 h was within recommended levels (1.6+/-1.0 nM). The decrease in heart rate in placebo-treated patients was, on average, 0.5 beats/min (bpm) per hour. In patients on digoxin, a linear relationship between the estimated digoxin concentration at the effect site and the drop-in heart rate was found. The half-life for the digoxin distribution to the effect compartment was approximately 3.8 h. The degree of reduction was related to the initial heart rate and patients with higher heart rate had a more pronounced decrease. The model predicted that a digoxin concentration of 1 nM at the effect site reduces heart rate by 9.4%. CONCLUSION: A PK/PD model for the relationship between the plasma concentration of digoxin, the estimated concentration at the effect site and the reduction in heart rate during atrial fibrillation could be defined using a population pharmacokinetic approach. Our data indicate that a more aggressive dosing regimen of digoxin may be more effective in terms of heart rate reduction.
24.
Hosseinpour, Fardin, et al.
(författare)
25-Hydroxylation of vitamin D3 in primary cultures of pig hepatocytes:evidence for a role of both CYP2D25 and CYP27A1
2003
Ingår i: Biochemical and Biophysical Research Communications - BBRC. - 0006-291X .- 1090-2104. ; 303:3, s. 877-883
Tidskriftsartikel (refereegranskat) abstract
There has been some controversy over whether the 25-hydroxylation of vitamin D(3) is carried out by one enzyme or two and whether this cytochrome P450 enzyme is found in the mitochondrial or microsomal fractions of liver. The pig is currently the only species in which both the microsomal 25-hydroxylase (CYP2D25) and the mitochondrial 25-hydroxylase (CYP27A1) have been cloned and characterized. In this paper, the roles of the two enzymes in 25-hydroxylation of vitamin D(3) are examined in primary cultures of hepatocytes. Inhibition experiments indicated that tolterodine and 7 alpha-hydroxy-4-cholesten-3-one were selective inhibitors of the CYP2D25- and CYP27A-mediated 25-hydroxylation of vitamin D(3), respectively. Addition of each inhibitor to primary hepatocytes decreased the total 25-hydroxylation of vitamin D(3) to about the same extent. No inhibition of other hydroxylase activities tested was found. Phorbol 12-myristate 13-acetate down-regulated the expression of both CYP2D25 and CYP27A1 as well as the 25-hydroxylase activity of the hepatocytes. The results implicate that both CYP2D25 and CYP27A1 contribute to the 25-hydroxylation in hepatocytes and are important in the bioactivation of vitamin D(3).
25.
Huss, Ulrika, 1973-
(författare)
Studies on the Effects of Plant and Food Constituents on Cyclooxygenase-2 : Aspects in Inflammation and Cancer
2003
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
In inflammatory events, the cyclooxygenase-2 enzyme (COX-2) catalyses prostaglandin biosynthesis. This is a process, which could possibly be affected by natural compounds including those abundant in food. Two systems have been established to enable investigations of the effects of natural compounds on COX-2. The first method developed was an in vitro method suitable for measuring inhibition of COX-2 catalysed prostaglandin E2 biosynthesis, based on scintillation proximity assay technology. The second system established, comprises a cell model, suitable for studying the effects of compounds on COX-2 and inducible nitric oxide synthase (iNOS) at different cellular levels, including the effects on mRNA, protein, prostaglandin E2 and nitrite levels.The plants, Plantago major L. and Urtica dioica L., were subjected to bioassay-guided isolation and fatty acids, e.g. α-linolenic acid and linoleic acid, were isolated as COX-2 inhibitory principles. The effects of structurally related fatty acids were also studied and the most potent COX-2 inhibitors were eicosapentaenoic acid and its synthetic derivative all-(Z) -5-thia-8,11,14,17-eicosatetraenoic acid. The latter was also found to decrease COX-2 protein levels assessed by the cell model. The inhibitory effects on COX-2 catalysed prostaglandin biosynthesis of 49 plant metabolites with different biosynthetic origin were studied. The phenolic compounds, eugenol, pyrogallol, but also cinnamaldehyde, were found to inhibit COX-2.Recently, COX-2 has attracted attention because of its involvement in cancer. The compounds in our diet are suggested to play an important role in the aetiology of colon cancer. To investigate the influence of diet on COX-2 in relation to colon cancer cells, a number of human faecal water samples were assessed for their effects on COX-2 enzymatic activity and protein synthesis. Of the faecal waters tested, two samples exhibited a weak inhibitory effect on COX-2 enzymatic activity, and one sample decreased COX-2 protein levels in a colon cancer cell line. The chemical content of faecal water was analysed and a variety of phenolic compounds were identified, including flavonoids and phenolic acids. Hitherto, a number of the identified compounds in faecal water have been assessed for the effects on COX-2, but none of the tested compounds were found to be responsible for the observed effects of faecal water on COX-2. Moreover, the cytotoxic effects of natural COX-2 inhibitors, traditional non-steroidal anti-inflammatory drugs, and the selective COX-2 inhibitor rofecoxib were evaluated using a human cancer cell line panel. Of the compounds tested, curcumin was found to be most potent.This thesis contributes to increase the general knowledge of the effects on COX-2 of commonly occurring plant constituents, including fatty acids and phenolic compounds. As natural parts of our diet, these compounds, abundant in fruits and vegetables, may affect different targets in our bodies, including COX-2.
26.
Hägerström, Helene, 1974-
(författare)
Polymer Gels as Pharmaceutical Dosage Forms : Rheological Performance and Physicochemical Interactions at the Gel-Mucus Interface for Formulations Intended for Mucosal Drug Delivery
2003
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Drug delivery to the nasal and ocular mucosa faces several obstacles. One of these is from the effective clearance mechanisms present in the nose and eye. Polymer gels with suitable rheological properties can facilitate the absorption of poorly absorbed drugs by increasing the contact time of the drug with the mucosa. This has been attributed to the rheological and mucoadhesive properties of the gel. The main objective of this thesis was to investigate the importance of these features for the anticipated in vivo contact time, here exemplified by the ocular and nasal routes of administration.The in situ gelling polymer gellan gum was found to have a favourable rheological and in vivo performance. When administered in the nasal cavity of rats, a gel was formed that could remain at the site of administration for up to 4 hours. In addition, the epithelial uptake and transfer of a 3 kDa fluorescein dextran was higher than for a mannitol solution. Therefore, it was concluded that a gellan gum formulation should be a promising strategy for nasal drug delivery.The potential mucoadhesive properties of a variety of polymer gels were investigated using a rheological method and by measuring the tensile force required to detach the gel from a mucosa. With both methods the rheological properties of the gel were a determining factor for the results obtained. The rheological method was found to have several limitations. One of these was that a positive response, interpreted as mucoadhesion, was only seen with weak gels. The tensile method could, in contrast, detect strengthening of the mucus only for strong gels. However, this method reflects the in vivo performance of the gel better than the rheological method.Finally, dielectric spectroscopy was explored as a tool for investigating the likelihood of intimate surface contact between the gel and the mucus layer. This novel approach involved determining the ease with which a charged particle can pass the gel-mucus interface layer, and may enable the study of the events at the interface closer to the molecular level, than is possible with the rheological and tensile strength methods.
27.
Jansson, Britt, et al.
(författare)
Simultaneous enantiospecific separation and quantitation of mephenytoin and its metabolites nirvanol and 4'-hydroxymephenytoin in human plasma by liquid chromatography.
2003
Ingår i: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences. - 1570-0232 .- 1873-376X. ; 791:1-2, s. 179-91
Tidskriftsartikel (refereegranskat) abstract
A high-performance liquid chromatographic method for the enantiospecific quantitation of S- and R-mephenytoin and its metabolites S- and R-nirvanol and S- and R-4'-hydroxymephenytoin in plasma is described. The compounds were separated using a reversed-phase C(2) column in tandem with a chiral alpha(1)-acid glycoprotein column and were detected using ultraviolet detection at 205 nm. The lower limit of quantification was 10 ng/ml for all compounds using 0.5 ml human plasma (intra-day coefficient of variation <13%, accuracy <+/-20%). The method was validated for human plasma in the concentration range 10-2000 ng/ml for each of the six compounds. The method allows for the simultaneous characterisation of the metabolic capacity of two human drug-metabolising enzymes, CYP2C19 and CYP2B6, and may be used when investigating polymorphisms or changes in activity of these two enzymes.
28.
29.
Jonsson, E Niclas, et al.
(författare)
Population pharmacokinetics of levosimendan in patients with congestive heart failure
2003
Ingår i: British Journal of Clinical Pharmacology. - : Wiley. - 0306-5251 .- 1365-2125. ; 55:6, s. 544-551
Tidskriftsartikel (refereegranskat) abstract
AIMS: The aim of this study was to characterize the population pharmacokinetics of levosimendan in patients with heart failure (NYHA grades III and IV) and its relationship to demographic factors, disease severity and concomitant use of digoxin and beta-blocking agents. METHODS: Data from two efficacy studies with levosimendan administered by intravenous infusion were combined (190 patients in total). The data were analysed using a nonlinear mixed-effects modelling approach as implemented in the NONMEM program. The model development was done in three sequential steps. First the best structural model was determined (e.g. a one-, two- or three-compartment pharmacokinetic model). This was followed by the identification and incorporation of important covariates into the model. Lastly the stochastic part of the model was refined. RESULTS: A two-compartment model best described levosimendan pharmacokinetics. Clearance and the central volume of distribution were found to increase linearly with bodyweight. No other covariates, including concomitant use of digoxin and beta-blocking agents, influenced the pharmacokinetics. In the final model, a 76-kg patient was estimated to have a clearance +/- s.e. of 13.3 +/- 0.4 l h-1 and a central volume of distribution of 16.8 +/- 0.79 l. The interindividual variability was estimated to be 39% and 60% for clearance and central volume of distribution, respectively. Weight changed clearance by 1.5% [95% confidence interval (CI) 0.9%, 2.1%] and the central volume of distribution by 0.9% (95% CI 0.5%, 1.3%) per kg. CONCLUSIONS: The population pharmacokinetics parameters of levosimendan in this patient group were comparable to those obtained by traditional methods in healthy volunteers and patients with mild heart failure. Bodyweight influenced the clearance and the central volume of distribution, which in practice is accounted for by weight adjusting doses. None of the other covariates, including digoxin and beta-blocking agents, significantly influenced the pharmacokinetics of levosimendan.
30.
Jönsson, Siv, et al.
(författare)
A rational approach for selection of optimal covariate-based dosing strategies
2003
Ingår i: Clinical Pharmacology and Therapeutics. - : Springer Science and Business Media LLC. - 0009-9236 .- 1532-6535. ; 73:1, s. 7-19
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt) abstract
BACKGROUND: At present, there is no rational approach for choosing a dosing strategy for individualization based on a covariate. An approach to use in establishment of an a priori dosing strategy for individualization is presented. Factors influencing the choice of such a dosing strategy are identified. METHODS: The approach requires definition of the following: target variable, seriousness of deviations from the target (ie, risk function), population model, covariate distributions, and constraints. Minimizing the total risk yields an optimal dosing strategy, estimated as dose sizes for different subpopulations and covariate cutoff values at which doses are increased or decreased. The method was illustrated with the use of simulated and real drug examples for the situation in which clearance is related to creatinine clearance. RESULTS: The estimated optimal cutoff(s) paralleled the median creatinine clearance in the population. The extent of variability in clearance explained by creatinine clearance was the main factor influencing the optimal ratios between adjacent dose sizes. An optimal dosing strategy was possible to estimate for the real drug. CONCLUSIONS: The method is simple to perform, although one difficulty lies in defining the target variable and risk function. Our results imply that commonly used constraints in dosing strategies based on renal function (ie, dose ratio of 2 and predetermined cutoffs) are nonoptimal in the sense we propose. Because an optimal dosing strategy may not be practical to use, the therapeutic cost that would result with any constraint can be assessed by comparison of the outcome after the desired and the optimal strategy.
