Publications by authors named "Riku Niemi"

14 Publications

  • Page 1 of 1

Synthesis, cannabinoid receptor activity, and enzymatic stability of reversed amide derivatives of arachidonoyl ethanolamide.

Bioorg Med Chem 2006 Aug 27;14(15):5252-8. Epub 2006 Apr 27.

Department of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland.

Retroanandamide (2f) and its 10 analogues (1a-e, 2a-e) were synthesized and evaluated for the cannabinoid receptor activation by a [35S]GTPgammaS binding assay using rat cerebellar membranes, and Chinese hamster ovary cell membranes expressing human CB2 receptors. The primary goal of the study was to develop cannabinoid receptor agonists having improved enzymatic stability compared to endogenous N-arachidonoyl ethanolamide (AEA). Furthermore, by reversing the amide bond of AEA, the formation of arachidonic acid would be prevented. Finally, an effect of the carbonyl carbon position on the cannabinoid receptor activity was explored by synthesizing retroanandamide analogues having different chain lengths (1a-e, C19; 2a-f, C20). All the synthesized compounds, except 2c, behaved as partial agonists for the both cannabinoid receptors. In rat brain homogenate, the reversed amides possessed significantly higher stability against FAAH induced degradation than AEA. Therefore, the reversed amide analogues of AEA may serve as enzymatically stable structural basis for the drug design based on the endogenous cannabinoids.
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http://dx.doi.org/10.1016/j.bmc.2006.03.051DOI Listing
August 2006

Controlled release of saccharides from matrix tablets.

Eur J Pharm Biopharm 2006 Feb 15;62(2):163-70. Epub 2005 Dec 15.

Department of Pharmaceutics, University of Kuopio, Kuopio, Finland.

The aim of this study was to design site specific, controlled release tablets of N-acetyl-d-glucosamine (NAG), maltose monohydrate and maltopentaose by using hydrophobic matrix formers starch acetate (SA) and ethyl cellulose (EC). The optimized matrices, which had either low porosity and high drug load or high porosity and low drug load, released the saccharides within the desired 2-4 h. In general, it was possible to control the release rate of saccharides by altering the relative amount of hydrophobic matrix former in the tablet and tablet porosity. The release type of saccharides from these formulations varied from immediate release to sustained release. In the case of sustained release formulations, it was found that the release of maltose monohydrate and maltopentaose was biphasic and slower than the release rate of NAG from similar tablets. NAG release kinetics followed square root of time kinetics, while in the case of maltose monohydrate and maltopentaose, the release kinetics were zero order in both phases. The biphasic dissolution profile was proposed to be caused by water mediated recrystallisation of the disordered material formed during the dissolution. Both SA and EC matrices were found to represent suitable controlled oral delivery vehicles for saccharides.
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http://dx.doi.org/10.1016/j.ejpb.2005.07.009DOI Listing
February 2006

In-vitro corneal permeation of cannabinoids and their water-soluble phosphate ester prodrugs.

J Pharm Pharmacol 2005 Sep;57(9):1153-7

Department of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland.

Topically administered cannabinoids have been shown to reduce intraocular pressure by interacting with the ocular cannabinoid receptor. Most cannabinoids have very poor aqueous solubility, which limits their pharmaceutical development and usefulness. In this study, permeation of three cannabinoids (arachidonylethanolamide, R-methanandamide and noladin ether) and their water-soluble phosphate ester prodrugs across isolated rabbit cornea was investigated in vitro. Hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was used to solubilize the parent cannabinoids in permeation studies to achieve the required concentration in donor and receiving cells. Highest fluxes were obtained with lipophilic parent compounds administered with HP-beta-CD, and the fluxes of phosphate esters were 45-70% that of their corresponding parent compounds. Phosphate esters hydrolysed on the surface of the cornea or during the permeation to release the lipophilic parent compound, which further permeated the cornea. No phosphate esters were detected on the endothelial side of the cornea. Although the phosphate esters had lower fluxes than their corresponding parent compounds in these HP-beta-CD formulations, the results are promising and the fluxes of phosphate esters are significantly higher than the fluxes of parent compounds administered as a suspension (due to their low aqueous solubility) without HP-beta-CD.
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http://dx.doi.org/10.1211/jpp.57.9.0009DOI Listing
September 2005

Characterization of the sulfhydryl-sensitive site in the enzyme responsible for hydrolysis of 2-arachidonoyl-glycerol in rat cerebellar membranes.

Chem Biol 2005 Jun;12(6):649-56

Department of Pharmaceutical Chemistry, P.O. Box 1627, FIN-70211 Kuopio, Finland.

We have previously reported that the endocannabinoid, 2-arachidonoyl-glycerol (2-AG), is hydrolyzed in rat cerebellar membranes by monoglyceride lipase (MGL)-like enzymatic activity. The present study shows that, like MGL, 2-AG-degrading enzymatic activity is sensitive to inhibition by sulfhydryl-specific reagents. Inhibition studies of this enzymatic activity by N-ethylmaleimide analogs revealed that analogs with bulky hydrophobic N-substitution were more potent inhibitors than hydrophilic or less bulky agents. Interestingly, the substrate analog N-arachidonylmaleimide was found to be the most potent inhibitor. A comparison model of MGL was constructed to get a view on the cysteine residues located near the binding site. These findings support our previous conclusion that the 2-AG-degrading enzymatic activity in rat cerebellar membranes corresponds to MGL or MGL-like enzyme and should facilitate further efforts to develop potent and more selective MGL inhibitors.
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http://dx.doi.org/10.1016/j.chembiol.2005.04.013DOI Listing
June 2005

Synthesis, hydrolysis, and intraocular pressure lowering effects of fadolmidine prodrugs.

Int J Pharm 2005 May;295(1-2):121-7

Department of Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland.

The objective of this study was to synthesize and evaluate various esters of fadolmidine, a novel alpha2-adrenergic agonist, as potential ophthalmic prodrugs. All studied prodrugs released the parent drug (i.e., fadolmidine) quantitatively via enzymatic hydrolysis in 80% human serum. The pivalyl ester was considered to be the most promising prodrug in this series, due to its good chemical stability (pH 5.0; 37 degrees C; t(1/2)=310 days) and optimal lipophilicity (logP(app)=1.8; 1-octanol/phosphate buffer, pH 5.0), and was selected for further evaluation of its intraocular pressure (IOP) lowering effects in normotensive rabbits. The pivalyl ester showed increased IOP lowering ability when compared to an equimolar dose of fadolmidine, which was probably due to its increased lipophilicity and subsequent enhanced corneal penetration. The duration of action for the pivalyl ester was also longer than that of fadolmidine.
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http://dx.doi.org/10.1016/j.ijpharm.2005.02.002DOI Listing
May 2005

Fadolmidine-induced ocular hypotension in normotensive rabbits.

J Pharm Pharmacol 2005 Feb;57(2):191-5

Department of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN 70211, Kuopio, Finland.

Fadolmidine, a novel selective alpha2-adrenoceptor agonist, was evaluated for its efficacy to lower intraocular pressure in normotensive rabbits (n=5-6). The dose-response profile between 0.004 microg and 12.5 microg of fadolmidine was determined. The effect of pH on the partition of fadolmidine was studied in order to select an optimal pH for topical fadolmidine administration. After topical administration, fadolmidine significantly lowered the intraocular pressure in normotensive rabbits. The onset of action was immediate, with no initial increase in intraocular pressure. A significant decrease in intraocular pressure was already observed at 1 h after dosing. The maximum decrease in intraocular pressure was observed after a 2.5 microg dose of fadolmidine in both eyes at 2 h after dosing. The mean maximum decrease in the treated and untreated eye was 6.4 mmHg and 3.9 mmHg, respectively. In conclusion, fadolmidine is a potent intraocular pressure lowering agent. In addition, fadolmidine does not cause a significant initial increase in intraocular pressure. Because of the strong dependence of the distribution coefficient on pH, the pH of the administered solution is important, with physiological pH being optimal in this respect.
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http://dx.doi.org/10.1211/0022357055308DOI Listing
February 2005

Monoglyceride lipase-like enzymatic activity is responsible for hydrolysis of 2-arachidonoylglycerol in rat cerebellar membranes.

Biochem Pharmacol 2004 Apr;67(7):1381-7

Department of Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland.

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid that binds to CB1 and CB2 cannabinoid receptors, inducing cannabimimetic effects. However, the cannabimimetic effects of 2-AG are weak in vivo due to its rapid enzymatic hydrolysis. The enzymatic hydrolysis of 2-AG has been proposed to mainly occur by monoglyceride lipase (monoacylglycerol lipase). Fatty acid amide hydrolase (FAAH), the enzyme responsible for the hydrolysis of N-arachidonoylethanolamide (AEA), is also able to hydrolyse 2-AG. In the present study, we investigated the hydrolysis of endocannabinoids in rat cerebellar membranes and observed that enzymatic activity towards 2-AG was 50-fold higher than that towards AEA. Furthermore, various inhibitors for 2-AG hydrolase activity were studied in rat cerebellar membranes. 2-AG hydrolysis was inhibited by methyl arachidonylfluorophosphonate, hexadecylsulphonyl fluoride and phenylmethylsulphonyl fluoride with ic(50) values of 2.2 nM, 241 nM and 155 microM, respectively. Potent FAAH inhibitors, such as OL-53 and URB597, did not inhibit the hydrolysis of 2-AG, suggesting that 2-AG is inactivated in rat cerebellar membranes by an enzyme distinct of FAAH. The observation that the hydrolysis of 1(3)-AG and 2-AG occurred at equal rates supports the role of MGL in 2-AG inactivation. This enzyme assay provides a useful method for future inhibition studies of 2-AG degrading enzyme(s) in brain membrane preparation having considerably higher MGL-like activity when compared to FAAH activity.
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http://dx.doi.org/10.1016/j.bcp.2003.12.003DOI Listing
April 2004

An optimized approach to study endocannabinoid signaling: evidence against constitutive activity of rat brain adenosine A1 and cannabinoid CB1 receptors.

Br J Pharmacol 2003 Dec 17;140(8):1451-9. Epub 2003 Nov 17.

Department of Physiology, University of Kuopio, PO Box 1627, Kuopio FIN-70211, Finland.

At nanomolar concentrations, SR141716 and AM251 act as specific and selective antagonists of the cannabinoid CB1 receptor. In the micromolar range, these compounds were shown to inhibit basal G-protein activity, and this is often interpreted to implicate constitutive activity of the CB1 receptors in native tissue. We show here, using [35S]GTPgammaS binding techniques, that micromolar concentrations of SR141716 and AM251 inhibit basal G-protein activity in rat cerebellar membranes, but only in conditions where tonic adenosine A1 receptor signaling is not eliminated. Unlike lipophilic A1 receptor antagonists (potency order DPCPX>>N-0840 approximately cirsimarin>caffeine), adenosine deaminase (ADA) was not fully capable in eliminating basal A1 receptor-dependent G-protein activity. Importantly, all antagonists reduced basal signal to the same extent (20%), and the response evoked by the inverse agonist DPCPX was not reversed by the neutral antagonist N-0840. These data indicate that rat brain A1 receptors are not constitutively active, but that an ADA-resistant adenosine pool is responsible for tonic A1 receptor activity in brain membranes. SR141716 and AM251, at concentrations fully effective in reversing CB1-mediated responses (10-6 m), did not reduce basal G-protein activity, indicating that CB1 receptors are not constitutively active in these preparations.4 At higher concentrations (1-2.5 x 10-5 m), both antagonists reduced basal G-protein activity in control and ADA-treated membranes, but had no effect when A1 receptor signaling was blocked with DPCPX. Moreover, the CB1 antagonists right-shifted A1 agonist dose-response curves without affecting maximal responses, suggesting competitive mode of antagonist action. The CB1 antagonists did not affect muscarinic acetylcholine or GABAB receptor signaling. When further optimizing G-protein activation assay for the labile endocannabinoid 2-arachidonoylglycerol (2-AG), we show, by using HPLC, that pretreatment of cerebellar membranes with methyl arachidonoyl fluorophosphonate (MAFP) fully prevented enzymatic degradation of 2-AG and concomitantly enhanced the potency of 2-AG. In contrast to previous claims, MAFP exhibited no antagonist activity at the CB1 receptor.6 The findings establish an optimized method with improved signal-to-noise ratio to assess endocannabinoid-dependent G-protein activity in brain membranes, under assay conditions where basal adenosinergic tone and enzymatic degradation of 2-AG are fully eliminated.
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http://dx.doi.org/10.1038/sj.bjp.0705577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1574161PMC
December 2003

Synthesis, in vitro evaluation, and intraocular pressure effects of water-soluble prodrugs of endocannabinoid noladin ether.

J Med Chem 2003 Nov;46(23):5083-6

Department of Pharmaceutical Chemistry, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland.

The poor aqueous solubility of 2-arachidonyl glyceryl ether (noladin ether) 2 hinders both pharmacological studies and pharmaceutical development. The synthesized mono- and diphosphate esters of noladin ether (4 and 6) considerably increased the aqueous solubility of noladin ether (>40000-fold), showed high stability against chemical hydrolysis in buffer solutions, and were rapidly converted to the parent drug via enzymatic hydrolysis. The monophosphate ester of noladin ether reduced intraocular pressure in normotensive rabbits.
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http://dx.doi.org/10.1021/jm030877jDOI Listing
November 2003

First synthesis of etidronate partial amides starting from PCl3.

Org Biomol Chem 2003 Sep;1(18):3223-6

University of Kuopio, Department of Chemistry, Finland.

Methods for the preparation of mixed tetra-amide esters 1 and 2, the partial amide ester 3, and tri- and P,P-diamides 4 and 5 from monophosphorus spieces 12, 8 and 9, respectively, were developed. Compounds 8 and 9 were obtained from phosphorus trichloride via MeOPCl2, which was treated with 2 eq. and 4 eq. of piperidine, followed by water or acetyl chloride, respectively. Tetrasubstituted amide bisphosphonates 1 and 2 were selectively dealkylated with lithium or silyl halide to achieve target compounds 3-5. Piperidine was found to be a good desilylation reagent. Quantum mechanical calculations illustrate why derivative 2 was produced in low yield. The usefulness of compounds 1, 3 and 4 as prodrugs of etidronate was determined in aqueous buffer and human serum.
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http://dx.doi.org/10.1039/b305979kDOI Listing
September 2003

Epimer interconversion, isomerization, and hydrolysis of tetrahydrouridine: implications for cytidine deaminase inhibition.

J Pharm Sci 2003 Oct;92(10):2027-39

Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536, USA.

Tetrahydrouridine (THU) is an inhibitor of cytidine deaminase (CDA), the enzyme responsible for the deactivation of ara-C and other cytidine analogues in vivo, and therefore is capable of improving the therapeutic efficacy of these antitumor agents. In aqueous solution formulations, THU exists as a mixture of epimers differing in stereochemistry of the 4-OH substituent. The aims of this study were to investigate the interconversion kinetics of the epimers of THU, the CDA inhibitory effects of these epimers, and the stability and degradation mechanisms of THU epimer mixtures in aqueous solution with the ultimate goal of developing optimal conditions for a parenteral formulation of THU. A stability indicating HPLC assay utilizing a derivatized beta-cyclodextrin column was developed to separate the two epimers of THU and to monitor their reversible isomerization to their beta-ribopyranosyl counterparts and their hydrolysis to form N-glycosidic bond cleavage products. MS and one- and two-dimensional (1)H- and (13)C-NMR measurements were conducted to identify THU epimers and degradation products and to quantitatively model the degradation kinetics. The interconversion reaction between the two THU epimers is acid catalyzed with a first-order rate constant for conversion of epimer 1(1) to epimer 1(2) of (7.4 +/- 0.3) x 10(-3) h(-1) and an equilibrium constant ([1(2)]/[1(1)] of 1.7 +/- 0.1 at pH 7.4 and 25 degrees C. Epimer interconversion was therefore sufficiently slow at pH 7.4 to allow the isolation of each and evaluation of their CDA inhibitory activities utilizing 1% (w/v) mouse kidney homogenates as a source for cytidine deaminase and cytidine as a substrate. Inhibition constants for the two THU epimers (1(1) and 1(2)) were determined to be 8 +/- 1 x 10(-7) M and 6.2 +/- 0.2 x 10(-8) M, respectively. Studies at elevated temperature suggested that THU degradation from epimer mixtures is biphasic with the initial rate of disappearance being acid catalyzed and first order in initial THU concentration, thus ruling out dimerization as a potential reaction mechanism. NMR/MS analyses revealed that the major degradation products included the beta-ribopyranosyl THU isomers (two epimers), the reduced pyrimidinone base (tetrahydrouracil), and various anomers of D-ribose formed through N-glycosidic bond cleavage, and the products of subsequent reactions of the base. Kinetic modeling of the data obtained from both HPLC and NMR measurements indicated that in an acidic solution THU beta-ribofuranosyl --> beta-ribopyranosyl isomerization is a rapid equilibrium reaction, which proceeds through an intermediate observable in 1H-NMR, and is followed by slower N-glycosidic bond hydrolysis. All the reactions between THU, its ribopyranosyl isomers, the intermediate, and the base are acid catalyzed and appear to proceed through the same sugar ring-opened intermediate (carbinolamine), consistent with previous literature.
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http://dx.doi.org/10.1002/jps.10447DOI Listing
October 2003

Anandamide prodrugs. 1. Water-soluble phosphate esters of arachidonylethanolamide and R-methanandamide.

Eur J Pharm Sci 2003 May;19(1):37-43

Department of Pharmaceutical Chemistry, University of Kuopio, PO Box 1627, FIN-70211, Kuopio, Finland

Phosphate esters of arachidonylethanolamide (AEA) and R-methanandamide were synthesized and evaluated as water-soluble prodrugs. Various physicochemical properties (pK(a), partition coefficient, aqueous solubility) were determined for the synthesized phosphate esters. The chemical stability of phosphate esters was determined at pH 7.4. In vitro enzymatic hydrolysis rates were determined in 10% liver homogenate, and in a pure enzyme-containing (alkaline phosphatase) solution at pH 7.4. The intraocular pressure (IOP) lowering properties of R-methanandamide phosphate ester were tested on normotensive rabbits. The phosphate promoiety increased the aqueous solubility of the parent compounds by more than 16500-fold at pH 7.4. Phosphate esters were stable in buffer solutions, but rapidly hydrolyzed to their parent compounds in alkaline phosphatase solution (t(1/2)<<15 s) and liver homogenate (t(1/2)=8-9 min). The phosphate ester of R-methanandamide reduced IOP in rabbits. These results indicate that the phosphate esters of AEA and R-methanandamide are useful water-soluble prodrugs.
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http://dx.doi.org/10.1016/s0928-0987(03)00044-7DOI Listing
May 2003

Cellular uptake and metabolism of clodronate and its derivatives in Caco-2 cells: a possible correlation with bisphosphonate-induced gastrointestinal side-effects.

Eur J Pharm Sci 2003 May;19(1):23-9

Department of Pharmaceutics, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland.

Purpose: To investigate possible reasons for the low frequency of GI side-effects of clodronate, even though clodronate is known to be metabolised into a cytotoxic nucleotide analogue (AppCCl(2)p) by many cell types. The effects of some lipophilic prodrugs of clodronate were also studied.

Methods: The effects of clodronate and its lipophilic derivatives on the proliferation and viability of Caco-2 cells were examined using an MTT assay. The intracellular uptake of 14C-clodronate and the accumulation of a clodronate metabolite (AppCCl(2)p) in Caco-2 cells were evaluated using ion-pairing HPLC-ESI-MS.

Results: Clodronate had little effect on growth of proliferating, or the viability of confluent, Caco-2 cells. The uptake of clodronate by Caco-2 cells was only about 0.04% of total clodronate. The potentially cytotoxic clodronate metabolite, AppCCl(2)p, was detected in Caco-2 cell extracts after 3 h of exposure. Dianhydride- and triPOM-clodronate were metabolised to AppCCl(2)p more efficiently and also affected the viability of Caco-2 cells more than clodronate.

Conclusions: Clodronate appears to be metabolised into a cytotoxic ATP-analogue (AppCCl(2)p) by any cell type capable of internalising the drug. However, the cytotoxicity depends on the degree of uptake of clodronate. Due to the very low initial uptake of clodronate by epithelial Caco-2 cells, they do not accumulate sufficient intracellular concentrations of AppCCl(2)p to affect cell function. This explains the low frequency of gastrointestinal side-effects caused by oral clodronate therapy.
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http://dx.doi.org/10.1016/s0928-0987(03)00039-3DOI Listing
May 2003

A hydrophobicity scale for the lipid bilayer barrier domain from peptide permeabilities: nonadditivities in residue contributions.

Biochemistry 2003 Feb;42(6):1624-36

Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84108, USA.

Passive peptide transport across lipid membranes is governed by the energetics of partitioning into the ordered chain interior coupled with the rate of diffusion across this region. A hydrophobicity scale for peptide transfer into the barrier region of membranes derived from permeability coefficients would be useful to predict passive permeation of peptides across biomembranes and for determining the thermodynamics of peptide/protein insertion into the membrane interior. This study reports transport rates across large unilamellar vesicles (LUVs) composed of egg lecithin at 25 degrees C for a series of peptides having the general structure N-p-toluyl-(X)(n) (n =1-3), where X is glycine, alanine, or sarcosine. Apparent residue group contributions were calculated from permeability coefficients, P(RX), using the equation Delta(Delta G degrees )(X) = -RT ln(P(RX)/P(RH)). Multiple linear least-squares regression analysis performed for the set of 14 permeants yielded the best correlation (r(2) = 0.9993) when the following permeant descriptors were utilized: side-chain nonpolar surface area, number of -CONH- residues, number of toluyl-CON(Me)- residues, and number of other -CON(Me)- residues. The backbone -CONH- residue contribution in peptides, 4.6 kcal/mol, is significantly lower than that obtained for a single isolated -CONH- (>6 kcal/mol), suggesting a possible influence of intramolecular hydrogen bonding. Under closer scrutiny, Delta(Delta G degrees )(X) for the Ala and Gly residues decrease with increasing peptide length. The effect of N-methylation is also highly dependent on position and number of N-methyl groups on the molecule (Delta(Delta G degrees )(X) = -0.5 to -2.2 kcal/mol). These nonadditivities may be rationalized by considering the effects of peptide length and N-methylation on membrane-induced intramolecular hydrogen bonding leading to various folded conformations.
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http://dx.doi.org/10.1021/bi026701lDOI Listing
February 2003
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