CA2023404C - Phosphorylated glycosidase inhibitor prodrugs - Google Patents
Phosphorylated glycosidase inhibitor prodrugs Download PDFInfo
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- CA2023404C CA2023404C CA002023404A CA2023404A CA2023404C CA 2023404 C CA2023404 C CA 2023404C CA 002023404 A CA002023404 A CA 002023404A CA 2023404 A CA2023404 A CA 2023404A CA 2023404 C CA2023404 C CA 2023404C
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- deoxynojirimycin
- phosphate
- glycosidase
- dnj
- butyl
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
- C07F9/576—Six-membered rings
- C07F9/59—Hydrogenated pyridine rings
Abstract
A method is provided for converting therapeutic glycosidase inhibitors to novel prodrugs by phosphorylation of a free hydroxyl group on the molecule to substantially reduce the glycosidase inhibitory activity without thereby substantially reducing the therapeutic activity.
Description
-1- 07-27(611)A
PHOSPHORYLATED GLYCOSIDASE INHIBITOR PRODRUGS
Background of the Invention This invention relates to novel prodrugs and, more particularly, to phosphorylated derivatives of glycosidase inhibitors.
It is known that polyhydroxylated pyrrolidines and piperidines provide an extensive class of powerful and specific glycosidase inhibitors. See, far example, Scofield et al., Life Sci. 39, 645-650 (1986); Elbein, Ann. Rev.
Biochem. 56, 497-534 (1987); and Fleet et al., FEBS Lett. 237, 128-132 (1988). Several of these glycosidase inhibitors have been found to inhibit human immunodeficiency virus (HIV) syncytium formation and virus replication, thereby indicating their potential use as antiretroviral agents. Three such compounds 'thus suggested as potential anti-AIDS drugs are castanospermine, 1-deoxynojirimycin (DNJ) and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DNmP).
See, for example, Sunkara et al., Biochem. Biophys.
Res. Commun. 148(1), 206-210 (1987); Tyms et al., Lancet, Oct. 31, 1987, pp. 1025-1026; Walker et al., Proc. Natl. Acad. Sci. USA 84, 8120-8124 (1987); and Gruters et al., Nature 330, 74-77 (1987).
N-alkylated derivatives of these compounds also have been suggested as potential antiviral agents and, in particular, the n-butyl derivative of 1,5-dideoxy-1,5-imino-D-glucitol, also referred to as N-butyl-deoxy-nojirimycin, has been shown to reduce the virus titer by an order of greater than five logarithms at noncytotoxic concentrations by Karpas et al., _2_ 07-27(611)A
Proc. Natl. Acad. Sci. USA 85, 92299233 (1988). See, also European Patent Application EP 375,651, published June 27, 1990, and U.S. Patent 4,849,430.
Some of the glycosidase inhibitors which are potent inhibitors of a-glucosidases, particularly disaccharidases, are suggested as useful agents .for treatment of hyperglycemia, hyperlipoproteinaemia, and various gastrointestinal problems. See, e.g., U.S. Patents 4,065,562: 4,182,767; 4,278,683;
4,533,668; and 4,639,436.
A problem that arises in the oral administration of the glycosidase inhibitors for therapeutic use is that the concomitant inhibition of the enzymatic splitting of dietary disaccharides can cause undesirable gastrointestinal problems such as diarrhea, digestive flatulence and the like. A means of overcoming these problems without loss of the desired therapeutic benefit of the drug would have significant use.
Brief Description of the Invention In accordance with the present invention, inhibitors of glycosidase enzymes are converted to novel prodrugs by phosphorylation of a free hydroxyl group on the molecule. As a result, glycosidase inhibition in the gastrointestinal tract is substantially reduced or eliminated upon oral administration of the prodrug. The prodrug is then absorbed in the intestines and metabolized to the active drug by reaction with phosphatase enzymes present in normal tissue and serum, e.g., alkaline phosphatase and glucose-6-phosphatase, which readily remove the labile phosphate group from the prodrug.
PHOSPHORYLATED GLYCOSIDASE INHIBITOR PRODRUGS
Background of the Invention This invention relates to novel prodrugs and, more particularly, to phosphorylated derivatives of glycosidase inhibitors.
It is known that polyhydroxylated pyrrolidines and piperidines provide an extensive class of powerful and specific glycosidase inhibitors. See, far example, Scofield et al., Life Sci. 39, 645-650 (1986); Elbein, Ann. Rev.
Biochem. 56, 497-534 (1987); and Fleet et al., FEBS Lett. 237, 128-132 (1988). Several of these glycosidase inhibitors have been found to inhibit human immunodeficiency virus (HIV) syncytium formation and virus replication, thereby indicating their potential use as antiretroviral agents. Three such compounds 'thus suggested as potential anti-AIDS drugs are castanospermine, 1-deoxynojirimycin (DNJ) and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DNmP).
See, for example, Sunkara et al., Biochem. Biophys.
Res. Commun. 148(1), 206-210 (1987); Tyms et al., Lancet, Oct. 31, 1987, pp. 1025-1026; Walker et al., Proc. Natl. Acad. Sci. USA 84, 8120-8124 (1987); and Gruters et al., Nature 330, 74-77 (1987).
N-alkylated derivatives of these compounds also have been suggested as potential antiviral agents and, in particular, the n-butyl derivative of 1,5-dideoxy-1,5-imino-D-glucitol, also referred to as N-butyl-deoxy-nojirimycin, has been shown to reduce the virus titer by an order of greater than five logarithms at noncytotoxic concentrations by Karpas et al., _2_ 07-27(611)A
Proc. Natl. Acad. Sci. USA 85, 92299233 (1988). See, also European Patent Application EP 375,651, published June 27, 1990, and U.S. Patent 4,849,430.
Some of the glycosidase inhibitors which are potent inhibitors of a-glucosidases, particularly disaccharidases, are suggested as useful agents .for treatment of hyperglycemia, hyperlipoproteinaemia, and various gastrointestinal problems. See, e.g., U.S. Patents 4,065,562: 4,182,767; 4,278,683;
4,533,668; and 4,639,436.
A problem that arises in the oral administration of the glycosidase inhibitors for therapeutic use is that the concomitant inhibition of the enzymatic splitting of dietary disaccharides can cause undesirable gastrointestinal problems such as diarrhea, digestive flatulence and the like. A means of overcoming these problems without loss of the desired therapeutic benefit of the drug would have significant use.
Brief Description of the Invention In accordance with the present invention, inhibitors of glycosidase enzymes are converted to novel prodrugs by phosphorylation of a free hydroxyl group on the molecule. As a result, glycosidase inhibition in the gastrointestinal tract is substantially reduced or eliminated upon oral administration of the prodrug. The prodrug is then absorbed in the intestines and metabolized to the active drug by reaction with phosphatase enzymes present in normal tissue and serum, e.g., alkaline phosphatase and glucose-6-phosphatase, which readily remove the labile phosphate group from the prodrug.
-3- 07-27(611)A
The preferred glycosidase inhibitors which are phosphorylated in accordance with the present invention are the polyhydroxylated pyrrolidines and piperidines, especially those illustrated by Fleet et al., FEBS Lett. 237, 128-132 (1988), such as castanospermine, deoxynojirimycin, deoxymannojirimycin and their N-alkylated derivatives, e.g., N-butyl-deoxynojirimycin, and others disclosed in Fleet et al., European Patent Application EP 332,395, published June 28, 1989.
Various of the latter type compounds having a pyranose ring are preferably phosphorylated at the C-6 hydroxyl group. This phosphoryl.ation is conveniently carried out enzymatically by reaction of the polyhydroxylated piperidine with yeast hexokinase (HK). This enzyme transfers a phosphate group from adenosine triphosphate (ATP) to D-glucose but also is known to phosphorylate D-fructose, D-mannose and D-2-deoxyglucose at almost equivalent rates. The general reaction proceeds as follows:
Glucose + ATP, ~~..---~~ Glucose-6-phosphate + ADP
The polyhydroxylated piperidines, e.g., deoxyno-jirimycin, having a C-6 hydroxyl group are similarly phosphorylated. A typical such enzymic method of preparation of the analogous nojirimycin 6-phosphate is described by Drueckhammer and Wong, J. Org. Chem.
50, 5912-5913 (1985).
The polyhydroxylated piperidines and pyrrolidines can also be chemically phosphorylated.
The similarity of the hydroxyl functional groups on these compounds requires selective masking or -4- 07-27(611)A
blocking of several of these hydroxyls so that the desired phosphorylation can be carried out at a specific locus. For example, the selective blocking of a primary hydroxyl group can be carried out by introducing a silyl protecting group at C6 by reaction with t-butyldimethylsilyl chloride as described by Ogilvie and Hakimelahi, Carbohydr. Res. 15, 234-239 (1983). Another convenient blocking group can be introduced to form a ylide such as, for example, by acetonation to form a 2,3-O-isopropylidene derivative of the piperidine.
Following phosphorylation of the unprotected hydroxyl groups, the protecting groups can be removed by acid cleavage, for example, by treatment with aqueous trifluoracetic acid. Methods of chemically phosphorylating appropriately blocked polyhydroxylated piperidines and pyrrolidines can be analogous to conventional methods known for preparing sugar phosphates, as described, e.g., by Stanek et al, The Monosaccharides, pp 229-236, Academic Press, New York and London, 1963.
Although methods of making the phosphorylated glycosidase inhibitor prodrugs are described herein, it will be appreciated that these drugs and their method of administration as claimed herein are not limited to any particular method of their preparation.
Detailed Description of the Invention In order to illustrate the invention in greater detail, the invention will be described with particular reference to phosphorylation of the antiviral drug, N-butyl-deoxynojirimycin, to form a useful prodrug. It will be understood that similar _5_ 07-27(611)A
prodrugs can be made by analogous phosphorylation of other polyhydroxylated pyrrolidines and piperidines.
N-alkylation of the polyhydroxylated pyrrolidines and piperidines can be conveniently carried out by hydrogenation of the amine together with an appropriate aldehyde in suitable solvent media in the presence of palladium black catalyst as described, e.g., by Fleet et al. in European Patent Application EP 332,395, published June 28, 1989.
In particular, the antiviral drug, N-butyl-deoxynojirimycin, is usually produced by appropriate alkylation of deoxynojirimycin such as by reaction with butyraldehyde. Typical alkylation procedures are also described, e.g., in U.S. Patents 4,182,787 and 4,639,436. In order to enzymatically phosphorylate this drug it is necessary to first phosphorylate the deoxynojirimycin and then alkylate with the butyraldehyde. The reaction sequence can be illustrated as follows:
-6- 97-27(611)A
PROTOCOL FOR THE SYNTHESIS OF
N-BUTYLDEOXYNOJiRIMYCIN-6-PHOSPHATE
OH
HO,,, ,,.OH
N~CH20H
H
Deoxynojirimycin (DNJ) ATP creatine yeast creatine hexokinase phosphokinase ~'-ADP ~ phosphocreatine OH
HO.,, ,,.OH
N~CH20P03H
H
Deoxynojirimycin-6-phosphate chemical alkylation OH
HO,,, ,,OOH
N ~CH20P03H
(CH2)~CH3 N-Butyldeoxynojirimycin-6-phosphate -7- 07-27(611)A
The resulting N-butyl-deoxynojirimycin-6-phosphate can then be administered orally as a prodrug. Although the N-butyl-deoxynojirimycin is a potent inhibitor of a-glucosidase 1 and gut disaccharidases, the 6-phosphorylated derivative is a very weak inhibitor of these enzymes. The reduction of disaccharidase enzyme inhibitory activity by converting the N-butyl-deoxynojirimycin to the 6-phosphorylated derivative is illustrated hereinafter in vitro against the porcine disaccharidases, sucrase and maltase. Reduction of enzyme inhibitory activity is also illustrated against a cellular-derived porcine a-glycosidase.
Upon oral administration of the N-butyl-deoxynojirimycin-6-phosphate, metabolism of the prodrug to the effective antiviral N-butyl-deoxynojirimycin can proceed according to one or more of the following schemes:
2~23~~4 -8- 07-27(611)A
INTESTINAL LUMEN ~ SERUM ; HIV-INFECTED CELL
i N-Bu-DNJ-6-P
OH ~ OH ~ OH
HOB,, ,,OOH ~HO,,. ,~~OH ~ HO,,, ,~~OH
i -CH20P03H; CH OPO H ~ °CH20P03H
N 2 3~ I
CH CH
(CH2)3CH3 ~ ~CH2)3CH3 i ~ 2)3 3 i 1 1 i i 1 gut ~ serum ; cellular pi phosphatases ~ pi and/or ; Pi phosphatases tissue ' phosphatases ' i 1 OH ~ H ~ H
HO,,, ,,,OH ;HO,,, ,,OOH ~ HO~,, ,,OOH
CHZOH ~ N CH20H ~ N~CH20H
I i I 1 I
OH2)sCHs ; (CH2)3~',H3 ~ tC~"~2)3CH3 1 i i N-Bu-DNJ
~~2~~~~
-a- o~-z~~sy A
Biolability of the phosphate group of N-butyl-deoxynojirimycin-6-phosphate was demonstrated in vitro by the following reaction in which the compound is completely dephosphorylated.
OH OH
HO~,, ,,vOH HO~., ,,vOH
alkaline phosphatase Pi I
UH2)3CH3 ~CHz)3CH3 N-Bu-DNJ-6-P N-Bu-DNJ
Since alkaline phosphatase exists on the surface of many cells, surface binding of the phosphorylated prodrug to the alkaline phosphatase may be useful for enhancing the targeting of the drug to certain tissues.
2023409:
-10- 07-27(611)A
The following examples will illustrate the invention in greater detail although it will be appreciated that the invention is not limited to these specific examples.
Example 1 The synthesis of N-butyldeoxynojirimycin-6-phosphate (N-BuDNJ-6-P) was achieved by enzymic phosphorylation of the parent compound, deoxynojirimycin (DNJ), and subsequent chemical alkylation of deoxynojirimycin-6-phosphate (DNJ-6-P) by a procedure similar to that used to synthesize N-butyldeoxynojirimycin (N-BuDNJ) from DNJ. It was found necessary to enzymatically phosphorylate DNJ prior to chemical alkylation since N-BuDNJ is not an efficient enzyme substrate.
Synthesis and Isolation of Deoxynojirimycin-6-Phosphate. The following are incubated in 50 mM
sodium phosphate buffer, pH 8.0, at 30°C: DNJ, 12 mM; adenosine triphosphate, 30 mM; phosphocreatine, 50 mM; magnesium chloride, 60 mM; 150 units/ml yeast hexokinase (Sigma Type VII) and 20 units/ml bovine heart creatine phosphokinase (Sigma Type III). After 20 h, half the original amount of fresh creatine phosphokinase is added, and incubation continued for a further 20 h. Conversion of DNJ to DNJ-6-P is monitored by high performance anion exchange chromatography, (HPAEC) and typically there is 75%
phosphorylation of the substrate, DNJ. Protein is removed by ultrafiltration, and the resulting material subjected to sequential gel-permeation chromatography using a column of TSK-HW40(S) equilibrated and eluted with water and then a similar -11- 07-27(611)A
column equilibrated and eluted with O.1M sodium acetate buffer, pH 6Ø Purity of the DNJ-6-P is typically greater than 99% pure as judged by 500 MHz proton NMR spectroscopy.
Alkvlation of Deoxvnoiirimvcin-6-phosphate to Give N_-Butyldeoxynojirimycin-6-phosphate. The chemical alkylation of DNJ-6-P was analogous to that previously used for the alkylation of DNJ, namely reaction of DNJ-6-P with butyraldehyde in appropriate solvent media under a hydrogen atmosphere in the presence of palladium black catalyst. The resulting product, N-butyldeoxynojirimycin-6-phosphate, was then recovered by chromatographic procedures. The detailed preparation was as follows:
The following were incubated in O.1M sodium acetate buffer, pH 5.0, at room temperature under an atmosphere of hydrogen: DNJ-6-P, 65 mM; butyraldehyde, 190 mM; and palladium black {catalyst), 20 mg/ml. At the end of the reaction (monitored by high performance anion exchange chromatography), the catalyst was removed by filtration, the products were isolated on a column of Amberlite~ CG-400 (OH form) resin and subsequently recovered by elution with 10%
acetic acid. The N-BuDNJ-6-P was purified by chromatography on a column of TSK-HW40(S) equilibrated and eluted with 50 mM sodium acetate buffer, pH 6Ø
~0~3404 -12- 07-27(611)A
Example 2 INHIBITION OF a-GLUCOSIDASE I, SUCRASE AND
MALTASE BY N-BUTYLDEOXYNOJIRIMYCIN AND N-BUTYL
Inhibition constants were determined for N-BuDNJ and N-BuDNJ-6-P against a-glucosidase I, sucrase and maltase. For comparative purposes similar measurements were made for deoxynojirimycin, deoxynojirimycin-6-phosphate and castanospermine.
N-BuDNJ is a potent inhibitor of sucrase and maltase whereas N-BuDNJ-6-P is only a very weak inhibitor of these enzymes and also a-glucosidase I. Methods for the isolation of these enzymes and for deterrning the inhibition constants are given below.
Porcine Microsomal Alpha-Glucosidase I.
Alpha-glucosidase I was isolated from porcine liver microsomes by an affinity chromatographic method similar to that described by Hettkamp et al., Eur. J. Biochem. 142, 85-90 (1981). The purified preparation had a specific activity of 10000 units/mg protein as assayed against [14C-Glc]Glc3Man9GlcNAc2, a biosynthetically-labelled substrate isolated from porcine thyroid microsomes.
Inhibition constants for the various DNJ
derivatives and castanospermine were determined assuming competitive inhibition and were derived from the initial rates for the hydrolysis of [i4C-Glc]Glc3MangGlcNAc2 (at a concentration of 0.044 NM), at pH 7.0 and 37°C in the presence and absence of several fixed concentrations of each inhibitor.
Porcine Intestinal Sucrase and Maltase. A
sucrase-isomaltase complex was extracted from pig -13- 07-27(611)A
small intestine by treatment with urea and then solubilized by digestion with papain. The enzyme-complex was recovered by ethanol precipitation and then further purified by cellulose DE-52 anion exchange chromatography. The activity of sucrase was assayed by incubating an appropriate amount of enzyme at 37°C in a final volume of 150 ~1 containing 25 mM
sodium citrate buffer, pH 6.0, and 30 mM sucrose as substrate. After 30 min. the enzyme was inactivated by heating at 100°C for 3 min, the reaction mixture centrifuged at 15000 x g for 15 min and a 50 Nl aliquot of the supernatant removed and the enzyme-released glucose determined by the glucose oxidase-peroxidase method described by Trinder, Ann. Clin. Biochem. 6, 24 (1969). Under the standard assay conditions, the release of glucose was linear up to 10% hydrolysis of the substrate. The purified enzyme preparation had a specific activity of 4.08 units sucrase/mg protein. Maltase activity associated with the sucrase-isomaltase complex [Kolinska and Semenze, Biochim. Biophys. Acta 146, 181 (1967)] was assayed in a manner similar to sucrase except that the substrate was 30 mM maltose. Under the standard assay conditions release of glucose was linear up to 8% hydrolysis of the substrate. The specific activity of maltase was 15.2 units/mg protein.
Inhibition constants for the various DNJ
derivatives and castanospermine were determined from Lineweaver-Burk plots of the enzyme-catalyzed reaction in the presence and absence of different fixed concentrations of each inhibitor. Incubations using sucrase were for 30 min at a concentration of 90 milliunits/ml and for maltase were for 12 min at a concentration of 80 milliunits/ml.
20234~~~
-14- 07-27(611)A
Table I
Activity of Amin o-sugar Against PorcineGlucosidase Derivatives I, Maltase Sucrase and K (~M) i Compound Microsomal Intestinal Intestinal a-GlucosidaseSucrase Maltase I
Castanospermine 0.04 x'0.064 0.34 N-BuDNJ 0.22 0.18 0.92 N-BuDNJ-6-P 4000 1500 330 DNJ 0.83 0.050 0.09 DNJ-6-P 6600 520 1.0 *Non-competitive inhibitor, no pre-incubation Example 3 N-Butyldeoxynojirimycin-6-phosphate (1 mM) was incubated with bovine intestinal alkaline phosphatase (Sigma Type VII, 130 U/ml) at 37°C in 0.1 M Na2CO3/NaHCO3 buffer, pH 9.5. After incubation for 16 h the reaction products were assayed by high performance anion exchange chromatography which established that the N-butyldeoxynojirimycin-6-phosphate had been completely dephosphorylated.
2~234~4 -15- 07-27(611)A
The prodrug antiviral agents described herein can be used for administration to patients infected with the viruses by conventional means, preferably in formulations with pharmaceutically acceptable diluents and carriers. These agents can be used in the free amine form or in the salt form.
Pharmaceutically acceptable salt derivatives are illustrated, far example, by the HC1 salt. The amount of the active agent to be administered must be an effective amount, that is, an amount which is medically beneficial but does riot present toxic affects which overweigh the advantages which accompany its use. It would be expected that the adult human dosage would normally range upward from about one milligram of the prodrug. A suitable route of administration is orally in the form of capsules, tablets, syrups, elixirs and the like, although parenteral administration also can be used.
Appropriate formulations of the prodrug in pharmaceutically acceptable diluents and carriers in therapeutic dosage form can be prepared by reference to general texts in the field such as, for example, Remington's Pharmaceutical Sciences, Ed. Arthur Osol, 16th ed., 1980, Mack Publishing Co., Easton, PA.
Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such examples be included within the scope of the appended claims.
The preferred glycosidase inhibitors which are phosphorylated in accordance with the present invention are the polyhydroxylated pyrrolidines and piperidines, especially those illustrated by Fleet et al., FEBS Lett. 237, 128-132 (1988), such as castanospermine, deoxynojirimycin, deoxymannojirimycin and their N-alkylated derivatives, e.g., N-butyl-deoxynojirimycin, and others disclosed in Fleet et al., European Patent Application EP 332,395, published June 28, 1989.
Various of the latter type compounds having a pyranose ring are preferably phosphorylated at the C-6 hydroxyl group. This phosphoryl.ation is conveniently carried out enzymatically by reaction of the polyhydroxylated piperidine with yeast hexokinase (HK). This enzyme transfers a phosphate group from adenosine triphosphate (ATP) to D-glucose but also is known to phosphorylate D-fructose, D-mannose and D-2-deoxyglucose at almost equivalent rates. The general reaction proceeds as follows:
Glucose + ATP, ~~..---~~ Glucose-6-phosphate + ADP
The polyhydroxylated piperidines, e.g., deoxyno-jirimycin, having a C-6 hydroxyl group are similarly phosphorylated. A typical such enzymic method of preparation of the analogous nojirimycin 6-phosphate is described by Drueckhammer and Wong, J. Org. Chem.
50, 5912-5913 (1985).
The polyhydroxylated piperidines and pyrrolidines can also be chemically phosphorylated.
The similarity of the hydroxyl functional groups on these compounds requires selective masking or -4- 07-27(611)A
blocking of several of these hydroxyls so that the desired phosphorylation can be carried out at a specific locus. For example, the selective blocking of a primary hydroxyl group can be carried out by introducing a silyl protecting group at C6 by reaction with t-butyldimethylsilyl chloride as described by Ogilvie and Hakimelahi, Carbohydr. Res. 15, 234-239 (1983). Another convenient blocking group can be introduced to form a ylide such as, for example, by acetonation to form a 2,3-O-isopropylidene derivative of the piperidine.
Following phosphorylation of the unprotected hydroxyl groups, the protecting groups can be removed by acid cleavage, for example, by treatment with aqueous trifluoracetic acid. Methods of chemically phosphorylating appropriately blocked polyhydroxylated piperidines and pyrrolidines can be analogous to conventional methods known for preparing sugar phosphates, as described, e.g., by Stanek et al, The Monosaccharides, pp 229-236, Academic Press, New York and London, 1963.
Although methods of making the phosphorylated glycosidase inhibitor prodrugs are described herein, it will be appreciated that these drugs and their method of administration as claimed herein are not limited to any particular method of their preparation.
Detailed Description of the Invention In order to illustrate the invention in greater detail, the invention will be described with particular reference to phosphorylation of the antiviral drug, N-butyl-deoxynojirimycin, to form a useful prodrug. It will be understood that similar _5_ 07-27(611)A
prodrugs can be made by analogous phosphorylation of other polyhydroxylated pyrrolidines and piperidines.
N-alkylation of the polyhydroxylated pyrrolidines and piperidines can be conveniently carried out by hydrogenation of the amine together with an appropriate aldehyde in suitable solvent media in the presence of palladium black catalyst as described, e.g., by Fleet et al. in European Patent Application EP 332,395, published June 28, 1989.
In particular, the antiviral drug, N-butyl-deoxynojirimycin, is usually produced by appropriate alkylation of deoxynojirimycin such as by reaction with butyraldehyde. Typical alkylation procedures are also described, e.g., in U.S. Patents 4,182,787 and 4,639,436. In order to enzymatically phosphorylate this drug it is necessary to first phosphorylate the deoxynojirimycin and then alkylate with the butyraldehyde. The reaction sequence can be illustrated as follows:
-6- 97-27(611)A
PROTOCOL FOR THE SYNTHESIS OF
N-BUTYLDEOXYNOJiRIMYCIN-6-PHOSPHATE
OH
HO,,, ,,.OH
N~CH20H
H
Deoxynojirimycin (DNJ) ATP creatine yeast creatine hexokinase phosphokinase ~'-ADP ~ phosphocreatine OH
HO.,, ,,.OH
N~CH20P03H
H
Deoxynojirimycin-6-phosphate chemical alkylation OH
HO,,, ,,OOH
N ~CH20P03H
(CH2)~CH3 N-Butyldeoxynojirimycin-6-phosphate -7- 07-27(611)A
The resulting N-butyl-deoxynojirimycin-6-phosphate can then be administered orally as a prodrug. Although the N-butyl-deoxynojirimycin is a potent inhibitor of a-glucosidase 1 and gut disaccharidases, the 6-phosphorylated derivative is a very weak inhibitor of these enzymes. The reduction of disaccharidase enzyme inhibitory activity by converting the N-butyl-deoxynojirimycin to the 6-phosphorylated derivative is illustrated hereinafter in vitro against the porcine disaccharidases, sucrase and maltase. Reduction of enzyme inhibitory activity is also illustrated against a cellular-derived porcine a-glycosidase.
Upon oral administration of the N-butyl-deoxynojirimycin-6-phosphate, metabolism of the prodrug to the effective antiviral N-butyl-deoxynojirimycin can proceed according to one or more of the following schemes:
2~23~~4 -8- 07-27(611)A
INTESTINAL LUMEN ~ SERUM ; HIV-INFECTED CELL
i N-Bu-DNJ-6-P
OH ~ OH ~ OH
HOB,, ,,OOH ~HO,,. ,~~OH ~ HO,,, ,~~OH
i -CH20P03H; CH OPO H ~ °CH20P03H
N 2 3~ I
CH CH
(CH2)3CH3 ~ ~CH2)3CH3 i ~ 2)3 3 i 1 1 i i 1 gut ~ serum ; cellular pi phosphatases ~ pi and/or ; Pi phosphatases tissue ' phosphatases ' i 1 OH ~ H ~ H
HO,,, ,,,OH ;HO,,, ,,OOH ~ HO~,, ,,OOH
CHZOH ~ N CH20H ~ N~CH20H
I i I 1 I
OH2)sCHs ; (CH2)3~',H3 ~ tC~"~2)3CH3 1 i i N-Bu-DNJ
~~2~~~~
-a- o~-z~~sy A
Biolability of the phosphate group of N-butyl-deoxynojirimycin-6-phosphate was demonstrated in vitro by the following reaction in which the compound is completely dephosphorylated.
OH OH
HO~,, ,,vOH HO~., ,,vOH
alkaline phosphatase Pi I
UH2)3CH3 ~CHz)3CH3 N-Bu-DNJ-6-P N-Bu-DNJ
Since alkaline phosphatase exists on the surface of many cells, surface binding of the phosphorylated prodrug to the alkaline phosphatase may be useful for enhancing the targeting of the drug to certain tissues.
2023409:
-10- 07-27(611)A
The following examples will illustrate the invention in greater detail although it will be appreciated that the invention is not limited to these specific examples.
Example 1 The synthesis of N-butyldeoxynojirimycin-6-phosphate (N-BuDNJ-6-P) was achieved by enzymic phosphorylation of the parent compound, deoxynojirimycin (DNJ), and subsequent chemical alkylation of deoxynojirimycin-6-phosphate (DNJ-6-P) by a procedure similar to that used to synthesize N-butyldeoxynojirimycin (N-BuDNJ) from DNJ. It was found necessary to enzymatically phosphorylate DNJ prior to chemical alkylation since N-BuDNJ is not an efficient enzyme substrate.
Synthesis and Isolation of Deoxynojirimycin-6-Phosphate. The following are incubated in 50 mM
sodium phosphate buffer, pH 8.0, at 30°C: DNJ, 12 mM; adenosine triphosphate, 30 mM; phosphocreatine, 50 mM; magnesium chloride, 60 mM; 150 units/ml yeast hexokinase (Sigma Type VII) and 20 units/ml bovine heart creatine phosphokinase (Sigma Type III). After 20 h, half the original amount of fresh creatine phosphokinase is added, and incubation continued for a further 20 h. Conversion of DNJ to DNJ-6-P is monitored by high performance anion exchange chromatography, (HPAEC) and typically there is 75%
phosphorylation of the substrate, DNJ. Protein is removed by ultrafiltration, and the resulting material subjected to sequential gel-permeation chromatography using a column of TSK-HW40(S) equilibrated and eluted with water and then a similar -11- 07-27(611)A
column equilibrated and eluted with O.1M sodium acetate buffer, pH 6Ø Purity of the DNJ-6-P is typically greater than 99% pure as judged by 500 MHz proton NMR spectroscopy.
Alkvlation of Deoxvnoiirimvcin-6-phosphate to Give N_-Butyldeoxynojirimycin-6-phosphate. The chemical alkylation of DNJ-6-P was analogous to that previously used for the alkylation of DNJ, namely reaction of DNJ-6-P with butyraldehyde in appropriate solvent media under a hydrogen atmosphere in the presence of palladium black catalyst. The resulting product, N-butyldeoxynojirimycin-6-phosphate, was then recovered by chromatographic procedures. The detailed preparation was as follows:
The following were incubated in O.1M sodium acetate buffer, pH 5.0, at room temperature under an atmosphere of hydrogen: DNJ-6-P, 65 mM; butyraldehyde, 190 mM; and palladium black {catalyst), 20 mg/ml. At the end of the reaction (monitored by high performance anion exchange chromatography), the catalyst was removed by filtration, the products were isolated on a column of Amberlite~ CG-400 (OH form) resin and subsequently recovered by elution with 10%
acetic acid. The N-BuDNJ-6-P was purified by chromatography on a column of TSK-HW40(S) equilibrated and eluted with 50 mM sodium acetate buffer, pH 6Ø
~0~3404 -12- 07-27(611)A
Example 2 INHIBITION OF a-GLUCOSIDASE I, SUCRASE AND
MALTASE BY N-BUTYLDEOXYNOJIRIMYCIN AND N-BUTYL
Inhibition constants were determined for N-BuDNJ and N-BuDNJ-6-P against a-glucosidase I, sucrase and maltase. For comparative purposes similar measurements were made for deoxynojirimycin, deoxynojirimycin-6-phosphate and castanospermine.
N-BuDNJ is a potent inhibitor of sucrase and maltase whereas N-BuDNJ-6-P is only a very weak inhibitor of these enzymes and also a-glucosidase I. Methods for the isolation of these enzymes and for deterrning the inhibition constants are given below.
Porcine Microsomal Alpha-Glucosidase I.
Alpha-glucosidase I was isolated from porcine liver microsomes by an affinity chromatographic method similar to that described by Hettkamp et al., Eur. J. Biochem. 142, 85-90 (1981). The purified preparation had a specific activity of 10000 units/mg protein as assayed against [14C-Glc]Glc3Man9GlcNAc2, a biosynthetically-labelled substrate isolated from porcine thyroid microsomes.
Inhibition constants for the various DNJ
derivatives and castanospermine were determined assuming competitive inhibition and were derived from the initial rates for the hydrolysis of [i4C-Glc]Glc3MangGlcNAc2 (at a concentration of 0.044 NM), at pH 7.0 and 37°C in the presence and absence of several fixed concentrations of each inhibitor.
Porcine Intestinal Sucrase and Maltase. A
sucrase-isomaltase complex was extracted from pig -13- 07-27(611)A
small intestine by treatment with urea and then solubilized by digestion with papain. The enzyme-complex was recovered by ethanol precipitation and then further purified by cellulose DE-52 anion exchange chromatography. The activity of sucrase was assayed by incubating an appropriate amount of enzyme at 37°C in a final volume of 150 ~1 containing 25 mM
sodium citrate buffer, pH 6.0, and 30 mM sucrose as substrate. After 30 min. the enzyme was inactivated by heating at 100°C for 3 min, the reaction mixture centrifuged at 15000 x g for 15 min and a 50 Nl aliquot of the supernatant removed and the enzyme-released glucose determined by the glucose oxidase-peroxidase method described by Trinder, Ann. Clin. Biochem. 6, 24 (1969). Under the standard assay conditions, the release of glucose was linear up to 10% hydrolysis of the substrate. The purified enzyme preparation had a specific activity of 4.08 units sucrase/mg protein. Maltase activity associated with the sucrase-isomaltase complex [Kolinska and Semenze, Biochim. Biophys. Acta 146, 181 (1967)] was assayed in a manner similar to sucrase except that the substrate was 30 mM maltose. Under the standard assay conditions release of glucose was linear up to 8% hydrolysis of the substrate. The specific activity of maltase was 15.2 units/mg protein.
Inhibition constants for the various DNJ
derivatives and castanospermine were determined from Lineweaver-Burk plots of the enzyme-catalyzed reaction in the presence and absence of different fixed concentrations of each inhibitor. Incubations using sucrase were for 30 min at a concentration of 90 milliunits/ml and for maltase were for 12 min at a concentration of 80 milliunits/ml.
20234~~~
-14- 07-27(611)A
Table I
Activity of Amin o-sugar Against PorcineGlucosidase Derivatives I, Maltase Sucrase and K (~M) i Compound Microsomal Intestinal Intestinal a-GlucosidaseSucrase Maltase I
Castanospermine 0.04 x'0.064 0.34 N-BuDNJ 0.22 0.18 0.92 N-BuDNJ-6-P 4000 1500 330 DNJ 0.83 0.050 0.09 DNJ-6-P 6600 520 1.0 *Non-competitive inhibitor, no pre-incubation Example 3 N-Butyldeoxynojirimycin-6-phosphate (1 mM) was incubated with bovine intestinal alkaline phosphatase (Sigma Type VII, 130 U/ml) at 37°C in 0.1 M Na2CO3/NaHCO3 buffer, pH 9.5. After incubation for 16 h the reaction products were assayed by high performance anion exchange chromatography which established that the N-butyldeoxynojirimycin-6-phosphate had been completely dephosphorylated.
2~234~4 -15- 07-27(611)A
The prodrug antiviral agents described herein can be used for administration to patients infected with the viruses by conventional means, preferably in formulations with pharmaceutically acceptable diluents and carriers. These agents can be used in the free amine form or in the salt form.
Pharmaceutically acceptable salt derivatives are illustrated, far example, by the HC1 salt. The amount of the active agent to be administered must be an effective amount, that is, an amount which is medically beneficial but does riot present toxic affects which overweigh the advantages which accompany its use. It would be expected that the adult human dosage would normally range upward from about one milligram of the prodrug. A suitable route of administration is orally in the form of capsules, tablets, syrups, elixirs and the like, although parenteral administration also can be used.
Appropriate formulations of the prodrug in pharmaceutically acceptable diluents and carriers in therapeutic dosage form can be prepared by reference to general texts in the field such as, for example, Remington's Pharmaceutical Sciences, Ed. Arthur Osol, 16th ed., 1980, Mack Publishing Co., Easton, PA.
Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such examples be included within the scope of the appended claims.
Claims (6)
1. A method of substantially reducing the glycosidase inhibitory activity of a therapeutic deoxynojirimycin-based glycosidase inhibitor having a free hydroxyl group on the molecule without thereby substantially reducing its potential therapeutic activity which consists in converting said deoxynojirimycin-based glycosidase inhibitor to a phosphorylated prodrug derivative which is amenable upon oral administration to dephosphorylation by normal tissue or serum phosphatases.
2. The method of Claim 1 in which the glycosidase inhibitor is selected from the group consisting of polyhydroxylated pyrrolidines and piperidines.
3. The method of Claim 1 in which the glycosidase inhibitor is N-butyl-deoxynojirimycin.
4. The method of Claim 2 in which the phosphorylation of the glycosidase inhibitor is carried out by reaction with yeast hexokinase.
5. The method of Claim 3 in which the prodrug derivative is N-butyl-deoxynojirimycin-6-phosphate.
6. N-Butyl-deoxynojirimycin-6-phosphate.
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US07/394,914 US5043273A (en) | 1989-08-17 | 1989-08-17 | Phosphorylated glycosidase inhibitor prodrugs |
US394,914 | 1989-08-17 |
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CA2023404A1 CA2023404A1 (en) | 1991-02-18 |
CA2023404C true CA2023404C (en) | 2001-10-23 |
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US (1) | US5043273A (en) |
EP (1) | EP0413674B1 (en) |
JP (1) | JPH0791309B2 (en) |
AT (1) | ATE134639T1 (en) |
CA (1) | CA2023404C (en) |
DE (1) | DE69025515T2 (en) |
DK (1) | DK0413674T3 (en) |
ES (1) | ES2084019T3 (en) |
GR (1) | GR3019753T3 (en) |
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US5536732A (en) * | 1990-04-27 | 1996-07-16 | Merrell Pharmaceuticals Inc. | N-derivatives of 1-deoxy nojirimycin |
US5252587A (en) * | 1990-04-27 | 1993-10-12 | Merrell Dow Pharmaceuticals, Inc. | N-derivatives of 1-deoxy nojirimycin |
EP0453692A1 (en) * | 1990-04-27 | 1991-10-30 | Merrell Dow Pharmaceuticals Inc. | Novel nojirimycin derivatives |
GB9602080D0 (en) * | 1996-02-02 | 1996-04-03 | Pfizer Ltd | Pharmaceutical compounds |
BR9813508A (en) | 1997-12-11 | 2000-10-03 | Univ Oxford | Membrane-associated viral replication inhibition |
US6465488B1 (en) | 1997-12-11 | 2002-10-15 | Chancellor, Masters & Scholars Of The University Of Oxford | Inhibition of glycolipid biosynthesis |
US6274597B1 (en) * | 1998-06-01 | 2001-08-14 | Mount Sinai School Of Medicine Of New York University | Method of enhancing lysosomal α-Galactosidase A |
US7816560B1 (en) | 1999-08-10 | 2010-10-19 | Thomas Jefferson University | Long chain n-alkyl compounds and oxa-derivatives thereof |
US7256005B2 (en) | 1999-08-10 | 2007-08-14 | The Chancellor, Masters And Scholars Of The University Of Oxford | Methods for identifying iminosugar derivatives that inhibit HCV p7 ion channel activity |
EP2027137B1 (en) | 2005-06-08 | 2015-02-18 | Amicus Therapeutics, Inc. | Imino and amino sugar purification |
CA2616537A1 (en) | 2005-07-27 | 2007-02-01 | University Of Florida Research Foundation, Inc. | Small compounds that correct protein misfolding and uses thereof |
PT1968990E (en) | 2005-12-27 | 2015-03-16 | Otsuka Pharma Co Ltd | Water-soluble benzoazepine compound and its pharmaceutical composition |
EP2356990A3 (en) | 2006-08-02 | 2011-10-19 | University of Oxford | Liposome treatment of viral infections |
US20080131398A1 (en) * | 2006-08-21 | 2008-06-05 | United Therapeutics Corporation | Combination therapy for treatment of viral infections |
EP1995323A1 (en) * | 2007-05-24 | 2008-11-26 | Libragen | Method for preparing C-6 phosphorylated D-aldohexoses and C-6 phosphorylated D-aldohexose derivatives |
CN102046151A (en) * | 2008-03-26 | 2011-05-04 | 牛津大学 | Endoplasmic reticulum targeting liposomes |
US8450345B2 (en) | 2009-02-23 | 2013-05-28 | The Chancellor, Masters And Scholars Of The University Of Oxford | Iminosugars and methods of treating viral diseases |
EP2400843B1 (en) * | 2009-02-24 | 2015-11-11 | United Therapeutics Corporation | Iminosugars and methods of treating arenaviral infections |
US8703744B2 (en) * | 2009-03-27 | 2014-04-22 | The Chancellor, Masters And Scholars Of The University Of Oxford | Cholesterol level lowering liposomes |
WO2010135731A1 (en) * | 2009-05-22 | 2010-11-25 | Kaufman Herbert E | Preparations and methods for ameliorating or reducing presbyopia |
EP2440205B1 (en) * | 2009-06-12 | 2014-08-27 | United Therapeutics Corporation | Iminosugars for use in the treatment of bunyaviral and togaviral diseases |
IN2012DN00352A (en) | 2009-06-16 | 2015-08-21 | Bikam Pharmaceuticals Inc | |
CA2772875A1 (en) * | 2009-09-04 | 2011-03-10 | United Therapeutics Corporation | Methods of treating orthomyxoviral infections |
WO2011028779A1 (en) | 2009-09-04 | 2011-03-10 | United Therapeutics Corporation | Iminosugars and methods of treating filoviral diseases |
ES2485623T3 (en) * | 2009-09-04 | 2014-08-13 | United Therapeutics Corporation | Procedure for the treatment of poxvirus infections |
EP2509598A1 (en) * | 2009-12-07 | 2012-10-17 | The Chancellor, Masters And Scholars Of The University Of Oxford | N-substituted deoxynojirimycin compounds for use in inhibiting osteoclastogenesis and/or osteoclast activation |
CN103917093B (en) | 2011-06-14 | 2017-08-11 | 比卡姆药品公司 | Opsin binding partner, composition and application method |
EP2768307B1 (en) | 2011-10-19 | 2020-03-18 | Bikam Pharmaceuticals, Inc. | Opsin-binding ligands, compositions and methods of use |
CA2862807A1 (en) | 2011-11-30 | 2013-06-06 | Bikam Pharmaceuticals, Inc. | Opsin-binding ligands, compositions and methods of use |
CA2862746A1 (en) | 2011-12-01 | 2013-06-06 | Bikam Pharmaceuticals, Inc. | Opsin-binding ligands, compositions and methods of use |
JP5876856B2 (en) * | 2013-08-02 | 2016-03-02 | 中国医学科学院薬物研究所 | Use of an effective fraction of alkaloids derived from mulberry branches to prepare hypoglycemic agents |
CN105636589B (en) | 2013-09-16 | 2019-07-23 | 伊美根特病毒学公司 | Deoxidization nojirimycin derivative and its application method |
SG10201903630UA (en) | 2014-11-05 | 2019-05-30 | Emergent Virology Llc | Iminosugars useful for the treatment of viral diseases |
WO2017192599A1 (en) | 2016-05-02 | 2017-11-09 | Florida State University Research Foundation, Inc. | Treatment of zika virus infections using alpha-glucosidase inhibitors |
WO2017220298A1 (en) * | 2016-06-22 | 2017-12-28 | Unilever N.V. | Edible fat-continuous emulsion comprising 1-deoxynojirimycin and lecithin |
EP3318277A1 (en) | 2016-11-04 | 2018-05-09 | Institut du Cerveau et de la Moelle Epiniere-ICM | Inhibitors of glucosylceramide synthase for the treatment of motor neuron diseases |
WO2021168483A2 (en) | 2020-02-21 | 2021-08-26 | Florida State University Research Foundation, Inc. | Treatment of human coronavirus infections using alpha-glucosidase glycoprotein processing inhibitors |
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US4847244A (en) * | 1985-09-17 | 1989-07-11 | Burroughs Wellcome Co. | Treatment of human viral infections |
GB8716918D0 (en) * | 1987-07-17 | 1987-08-26 | Inverni Della Beffa Spa | Soluble derivatives of silybin |
US4999360A (en) * | 1987-12-21 | 1991-03-12 | Monsanto Company | Method of inhibiting virus |
US4849430A (en) * | 1988-03-09 | 1989-07-18 | Monsanto Company | Method of inhibiting virus |
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- 1989-08-17 US US07/394,914 patent/US5043273A/en not_active Expired - Lifetime
-
1990
- 1990-08-13 AT AT90870126T patent/ATE134639T1/en not_active IP Right Cessation
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JPH03118327A (en) | 1991-05-20 |
JPH0791309B2 (en) | 1995-10-04 |
ATE134639T1 (en) | 1996-03-15 |
US5043273A (en) | 1991-08-27 |
GR3019753T3 (en) | 1996-07-31 |
ES2084019T3 (en) | 1996-05-01 |
EP0413674B1 (en) | 1996-02-28 |
DK0413674T3 (en) | 1996-06-10 |
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