JPS60501105A - Colon-specific drug delivery system - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Colon-specific drug delivery system Background of the invention The present invention relates to a colon-specific drug delivery system. In more detail The present invention shows that when ingested by mammals, colonic microbial agglutination (1ava) produces It reacts with the glycosidic bonds that are absorbed into the intestinal mucosa or is absorbed by Drug precursor Cpro, a synthetic drug glycoside that releases free drug that can be absorbed The present invention relates to a colon-specific drug delivery system based on the use of compositions such as drsg).

In recent years, drugs have been delivered to specific areas to reduce side effects and increase pharmacological reactivity. Increasingly, importance is being placed on methods of activating this area. portable Pumps, drug-impregnated adhesive patches, drugs encapsulated in vesicles, and drug carriers - have been proposed to achieve site specificity. The other way is that physical Pre-drugs that, depending on their properties, can reach specific sites and then be transformed into active drugs on the spot. CV, J., Stella and J., Himmelstein Ng, J., Mgd., Chem.

23:1275-1282 (1980): A, A, C: Y Kura and S, H, Y. lk Kosky, J., Pharrn, Sci. 64:181-210 (1 975)). Systemically in the kidneys, brain, chest or central nervous system, or locally Administered to the eye or skin: Report on site-specific delivery of r'L drug precursors I'm here. In all of these cases, the parent drug 2 The agent is released chemically or by specific enzymes present at the target site.

It is known to confer colonic specificity to bioactive compounds in humans. a Intraquinone laxatives and carcinogenic diazine (cyasi%) are found in plants. It is a natural glycoside. R, R, Sci x line, J, Pharm, Sci, 57:2021-2137 (1968). When ingested, these The substances pass unabated into the large intestine, where they are digested by the enzymatic action of the colonic microbiota. The active part releases tt, le. The azoreductase activity of the colonic microbiota is associated with specific It is possible to activate rufa drugs by reducing the azo bond present in these compounds. currently known. G. L. Mandel and M. A. Sampf, “Pharmacology of Therapeutic Agents. Basics, 6th edition (A.G., Gilman, L.S., Hoodman and A.

df route 7th edition, -v y p mi y an, new york, new york, ( 198G), pp. 1106-1165). Identified as non-absorbable polymer Reduction of the azo bond between aromatic amines of becomes the basis of T., M., Perkinen, J. P., Brown and E. E., Wine. Gard, U.S. Pat. No. 4,190.71'6 (February 26, 1980); and J, P, Plow 7s AppL-Enviro, MicrobioL, 41: 1283-1286 (1981).

The gut microbiota has specific enzymatic activities that can hydrolyze a number of specific chemical bonds. I also know that. B.S.

Dreiser and M. J. Hill, 1 “Human gut microbiota” (Academic Preliminary Research). S., Rondo, N. (1974), 54 colon specific after ntv ingested by mammals. Sex drug glycosides enter the stomach and small intestine into endogenous mucosal enzymes present in recipient mammals. Therefore, it was not known that it could pass without being significantly hydrolyzed. difference Moreover, before the present invention, colonic microorganisms passed through this area! Phase cricosidase activity They were also unaware that it essentially cleaves depending on sex. Also, before the teaching of the present invention If cleavage of such drug precursors occurs, they may be adsorbed to the intestinal mucosa or I also didn't know that the free drug absorbed by the drug was released.

This kind of synthetic drug precursors consisting of drug glycosides has a colon-specific Drug precursors have been synthesized that have advantages over deceptive compounds used in sex-imparting systems. Due to the fact that it is It becomes possible. For example, ulcerative colitis (a condition that increases your risk of colon cancer!) and other inflammation. Described herein are synthetic, drug glycosides that are useful in the treatment of intestinal disease. flame Natural drug glycosides useful in the treatment of intestinal diseases are not found in nature.

The types of synthetic drug glycosides shown here are 1. Transformation! ! Give the drug to the stomach and small intestine It is also useful when packing and transporting the liver in a state with minimal damage, and transferring it to the liver after a long time. Ru. Synthetic drug glycosides found in nature useful in delivering chemotherapy drugs to the colon It has not been.

The drug glycosides of the present invention contain free drug until the drug precursor reaches the colonic region. as a nullifying (d-4 sabling) moiety that substantially prevents the release or absorption of Simple sugars (Simple%gar) are used. Therefore, the colonic microbiota Cosidases act on drug glycosides, releasing the sugar moiety from the drug moiety. this The released sugar residues can be used by the intestinal IIBI garden as an energy source. US Used in the colon-specific drug delivery system shown in Patent No. 4,190゜716. The non-absorbable polymer backbone is non-absorbable to patients who have been exposed to this polymer for a significant period of time. Although they may prove harmful, the long-term effects of these compounds are still being evaluated. It has not been. In contrast, the colon-specific drug delivery system disclosed herein is Sea urchin leaves no unnatural and unusable compounds for the body to fight.

The object of the present invention is to provide a protein that is essentially cleavable by the glycosidase enzymatic activity of the colonic microbiota. but not significantly hydrolyzed by endogenous enzymes produced by the recipient mammal. Therefore, drug glycosylation by glycosidases produced by the colonic microbiota The maximum amount (moat 51gn-4ficIS%t amosst Synthetic drug precursors consisting of drug glycosides whose free drug is released in the colonic region The objective of the present invention is to provide a colon-specific drug delivery system useful for the treatment of diseases using .

pIII can be cleaved by sidase enzyme activity, but within the range produced by the recipient mammal. The live enzyme Li is not significantly hydrolyzed and is therefore not produced by colonic microbial species. After cleavage of drug glycosidase by glycosidase, the largest amount of free drug is released into the colonic region. Disease control using synthetic drug precursors consisting of steroid glycosides released in the The aim is to provide a colon-specific drug delivery system useful in the treatment of disease.

Another object of the invention is to substantially increase the I-galactosidase enzymatic activity of the colonic microbiota. more cleavable but not significantly by endogenous enzymes produced by the recipient mammal. Drugs with glycosidases that are not hydrolyzed and therefore produced by the colonic microflora steroids in which the greatest amount of free drug is released in the colon region after cleavage of the drug glycoside An object of the present invention is to provide a colon-specific synthetic drug precursor composition consisting of glycosides. .

Another object of the invention is to substantially increase the a-galactosidase enzymatic activity of the colonic microbiota. more cleavable but not significantly by endogenous enzymes produced by the recipient mammal. Drugs by glycosidases that are not hydrolyzed and therefore produced by the colonic microflora steroids in which the greatest amount of free drug is released in the colon region after cleavage of the drug glycoside An object of the present invention is to provide a colon-specific synthetic drug precursor composition consisting of glycosides. .

Another object of the invention is to essentially cleavable by lactosidase enzyme activity but produced by the recipient mammal It is not significantly hydrolyzed by endogenous enzymes and is therefore not produced by the colonic microbiota. After cleavage of drug glycosides by glycosidases, the largest amount of free drug is released into the colonic region. Colon-specific synthetic drug precursor suite consisting of steroid glycosides released in the and Jin, who provides the manufacturing method for the product.

Another object of the invention is to essentially inhibit the β-cellobiosidase enzymatic activity of the colonic microbiota. more cleavable but not significantly by endogenous enzymes produced by the recipient mammal. Drugs due to glycosidases that are not hydrolyzed and therefore produced by the colonic microflora steroids in which the greatest amount of free drug is released in the colon region after cleavage of the drug glycoside Provided is a method for producing a colon-specific synthetic drug precursor composition comprising drug glycosides. That's true.

Other objects of the invention will be apparent to those skilled in the art from the following description taken in conjunction with the accompanying drawings. It will be.

drawing Figure 1 is a chemical formula showing the production and enzymatic hydrolysis of glycosides and glycosides. This is a diagram.

Figure showing HPLC chromatogram of Ladd's cecal contents. It is a diagram.

Figure 4 shows intragastric administration of free steroids prednisolone and dexamethazoy. FIG. 3 is a graphical illustration showing the recovery of steroids at various times afterward.

Figure 5 shows Edie Hofstay for determining V and KM (.2.). - (Eadie-Hoffstgg) plot at various concentrations. FIG. 2 is a graphical representation of the hydrolysis of p-nitrophenyl-glucoside.

Figure 6 shows Edie-hora for determining V2 and KM(a p p). p-Nitrophenyl-ga at various concentrations plotted by the Stay method. It is a graph showing hydrolysis of lactoside.

In the system diagram shown in Figure 1, "a" is A9. Co, is the molecular sieve CCl4: W″b ” is 0.01JV-NaOH%MgqH: and “C” is β-galactosylate. 1, 4, 23% and 15 are compounds, 1.2 ．． 31.4.23.14 and 15; the letter R is basic and the letter Ac is means acetyl. Other characters such as 0. H, F and B de respectively! ! ! Represents four atoms, two hydrogen atoms, a fluorine atom, and a bromine atom.

In the chromatogram shown in Figure 2, peak A is glucoside l; peak B is glucoside l. Beak A was added as an internal standard before homogenization. It was prednisolone. Chromatogram, in order to be born, Artex ( Altos) 5 p Ultra xy, x A (Ultra-/dan(, Te, 2, o , q) and prednisolone soil (5-25, da) in the stomach, after administration. Figure 2 shows the recovery of steroids at various times. Data points are intestinal toxins (black circles and The graph in Figure 5 shows that the caecal contents at relatively low concentrations are Figure 2 shows the rate and substrate relationship for the hydrolysis of trophenyl glucoside. Tile X approximation Edie-Hofstay plot to determine VrRtL□ was used. The Edie-Hofstey plot of this relationship deviates from a straight line. This is because β-D-glucosidase activity is naturally This indicates that the enzyme activity is more heterogeneous than the enzyme activity.

The graph in Figure 6 shows the hydrolysis of p-nitrophenyl-galactoside by the cecal contents. The solution will show the relationship between velocity and substrate.

Gastrointestinal bacteria found only in the area.

Colonic specificity - essentially restricted to the colonic region of the mammalian gastrointestinal tract.

PSI - Proximal small intestine.

DSI-Distal small intestine.

Endogenous enzymes - produced by the recipient mammal (by bacteria found in the mammal's intestine) Enzymes secreted into the gastrointestinal tract of mammals (as opposed to enzymes produced by humans).

Naturally produced naturally. Natural, as used here, is produced within living organisms. would represent a compound.

Produced by one of the synthesis methods. Synthesis used here refers to compounds that are not naturally produced. It will represent things.

Simple sugars - monosaccharides, dimonosaccharides or oligosaccharides.

Aglycone - Compounds that do not have sugars attached to them by glycosidic bonds.

10 Cricoside □Contains a sugar moiety and an aglycone moiety linked together through glycosidic bonds. compound.

Glycosidic bond - the bond between an aglycone and the reducing end of a sugar.

Drugs - not used for their mechanical properties, but for the patient (human or animal) Pain relief as an aid in diagnosing, treating, or preventing disease or other abnormal conditions. or for the relief of suffering, or for the control or control of a physiological or pathological condition. Compounds or non-infectious biological substances that can be administered or used to improve.

drug precursor - a substance with specific physicochemical properties that allows it to reach a target organ or tissue , a potential form of the active agent.

Once there, the active agent is formed in situ either chemically or enzymatically.

Hydrophilic - The state of being hydrophilic.

most significant - the largest amount that can be measured. child This phrase, as used here, refers to the glycolysis on drug precursors when cleavage occurs in the colon region. Aglycones released upon cleavage of cid-bonds to other areas of the mammalian gastrointestinal tract. represents the relative amount compared to the release at

Scientific publications and patents cited herein are specifically incorporated by reference.

Compound summary The following compounds are mentioned in the description and claims herein.

Compound 1 is 9α-fluoro-11β, 17α-dihydroxy-16α-methyl- 3,20-dioxopregna-1,4-dien-21-yl β-D-glucopi Ranoside: This is referred to here as ``Compound 1'', ``(1), ``1'', DEXAGLU or It is also called dexamethacin-glucoside.

Compound 12 is 11β,17α-dihydroxy-3,20-dioxopregna-1 , 4-dien-21-yl β-dizolone-glucoside.

Compound 3 is 9α-fluoro-11β, 17α, 21-trihydroxy-16α- Methyl-3,20-dioxopregna-1,4-diene: Compound 4 is 11β, 17α, 214 lyhydroxy-3,20-g oxypre Gunar-1,4-diene.

This is referred to here as one compound 41.1 (4d, 'sat', 'pratL-' or 'pred'). It's called Nizoron.

Compound 5 is 11β,17α-dihydroxy-3,20-dioxypregna-4- En-21-yl β-D-glucopyranoside. This is one compound here. 5-.

It is called ``shisuke'', ``flea'' or ``hydrocortisone glucoside''.

2 Compound 6 is 11β, 17α, 21-trihydroxy-3,20-dioxopreg Na-4-2y. This compound 7 is 9α-fluoro-11β, 17α-dihydrogen. Droxy-3,20-dioxopregn-4-ene-2nyyl β-D-glue Copyranoside. This is referred to here as “Compound 71 # (7) #, II 7 1″ or fludrocortisone-glucoside.

Compound 8 is 9α-fluoro-11β, 17α, 21-drihydroxy-3,20 -Dioxopregna-4-enzyme. This is referred to here as “Compound 8”, ° (mark) , “dan” or fludrocortizo/.

Compound 9 is 9α-fluoro-11β, 17α-dihydroxy-16α-methyl- 3,20-dioxopregna-1,4-dien-21-yl β-D-galacto It is a pyranoside. This is referred to here as “compound 9”, “(9)”, 9” or dex Samethacin-galactoside.

Compound 10 is 11β,17α-dihydroxy-3,20-dioxopregna-4 -en-2niyl β-D-galactopyranoside. Here, we will convert this into Compound 10'', compound 11 is 11β,17α-dihydroxy-3,20-diox Sopregna-4-en-21-yl β-D-″13 Galactopyranoside. Here cn is 1 compound 11”, 1(11)#, @ 11” or hydrocortisone-galactoside.

Compound 12 is 9a-fluoropy-111/, 17g-dihydroxy-3,20- Dioxopregna-4-en-21-yl β-D-galactopyranoside . This is referred to here as one compound 12'', ``(12)'', -rodi or fludrocorti It is called zon-galactoside.

Compound 13 is 11β,17α-dihydroxy-3°20-dioxypregna-1 , 4-diey-21-yl-II-D-cellobioside. rt here 1 compound 13”%@(13d,@13’ or prednisology cellobioside) To tell.

Compound 14 is 9α-fluoro-117,17α-dihydroxy-16σ-methyl -3,20-dioxopregna-1,4-dien-21-yl 2/, s/, 4/, s/-tetra-O-acetyl-11-D-gelcopinoside.

Note 1 Compound 14'', -(14)'', left 4J or DEXATAGLU Say.

Compound 15 is 11β,17α-dihydroxy-3°20-dioxopregna-1 ,4-dien-21-yl 2', 3', 4', 6'-tetra-O- Acetyl-11-D-glucoside. Here, one compound is 15". @(15)”, @15” or PREDTAGLU.

Compound 16 is 11β,17α-dihydroxy-3,20l4 -Dioxopregnar 4-en-21-yl 2/, 3/.

4/, a/-tetra-0-7 cetyl-11-D = glucopyranoside. child In this case, “Compound 16”, “(16)”, @ leaving or hydrocortisone It's called tetraacetyl glucoside.

Compound 17 is 9-a-7#o''-11/t17 a-dihydroxy-3, 20-Dioxopregna-4-en-21-yl 2/, a/, 4/, 6 /-tetra-0-7 cetyl-11-D-glucopyranoside. here is one compound 17”, @(17)’, -17’ or fludrocortisone tet It's called laacetyl glucoside.

Compound 18 is 9α-fluoro-1111,17α-dihydroxy-16ff-methane. Chil-3,20-dioxopregna71, 4-dien-21-yl 2/, a/, 4/, r, /-tetra-O-acetyl-β-D-galactopyranoside It is. rt is herein referred to as “compound 18”, @(18)”, 118” or dex It is called Sametagan tetraacetylgalactoside.

Compound 19 is 11/,17r-dihydroxy-3°20-dioxopregna-1 ,4-dien-21-yl 2',a',4'#6'-tetra-O Chil-β-D-galactopyranoside. Here we refer to one compound 19”,’ (19)”, @19” or prednisolone tetraacetylgalactoside. cormorant.

Compound 20 is 11/,17α-dihydroxy-3,20-dioxopregna-4 -En-21-yl 2't3't41.61-tetra-O-acetyl-β- D-galactopyranoside. Here, one compound 20'', ``(Buchi'', chemical Compound 21 is 9α-fluoro-11β, 17α-dihydroxy-3,20-diox Sopregna-4-en-21-yl 2/, 3/, 4/, 5/-tetra- O-acetyl-β-D-galactopyranoside. This is referred to as “compound 2” here. 1”, “(21)”, “21- or fludrocortisone tetraacetylga It's called lactosid.

Compound 22 is 11β,17α-dihydroxy-3,20-dioxopregna-1 , 4-dien-21-yl hepta-〇-acetyl-β-D-cellobioside. Ru. Strain’l Cocote f’;t” Compound 22”, -(22)”, 2U” or prednisolone heptaacetyl cellobioside.

Compound 23 is 2,3,4,6-Citler O-acetyl-1-yrom-α-D-g It is Rucobiranose. This is referred to here as "-compound 23", "(23)" , or tetraacetyl-1-bromglucose.

Compound 24 is 2,3,4,6-Citler O-acetyl-1-bromo-α-D - Galactopyranose. This is referred to here as “compound 24”, “(24)” , ``color A1 or tetraacetyl-1-bromogalactose.

Compound zs is hebuta〇-acetyl-1-bromo-α6 -D-cellobiose. This is referred to as “compound 25”, “(25)”, “ 25''-or heptaacetyl-1-from-alpha-cellobiost.

All steroids, namely 2,3,4,6-chitler O-acetyl-1-butyl rom-α-D-glucobylanose, 2,3,4.6-chitler O-acetyl-1 -bromo-α-D-galactopyranose, p-nitrophenyl substrate and p-nitrophenyl Trophenol was purchased from Sigma Chemical Company. Octa-O-acetyl- α-D-cellobioside was obtained from Aldrich Chemical Company and was dissolved in acetic acid at 31 -Hydrobromic acid was purchased from Eastman Kodak Company.

Preparative methods and assays employed in the description of this invention include: Ru.

(Aldrich Chemical Co., Ltd.). All solvent evaporation is done by rotating It was carried out under reduced pressure with a water aspirator using an evaporator. The melting point is Buc hi) Melting point determined by instrument and uncorrected. The UV spectrum is based on potassium (C). ar'/) 210 as measured by spectrometer. The IR switch is - Measured using a KinElmer Model 137 Swictrometer. “H-NMR Spectrum is UCB200 or UCB250 (Parkleaf) Homemade 200MHz and 25MHz installed at the University of California Department of Chemistry 0 AtHg measured in dimethyl-d,-sulfoxide using a 7-lier conversion device). established and recorded.

These are ppmcδ) downward from hh, si, and the coupling constants C, Lher (denoted with t). Elemental analysis by University of California, Berkeley Chemistry It was conducted by the Department of Analytical Research Institute. Analytical values are ±0.4 inches of theoretical values unless otherwise noted. It was within

chromatography High pressure liquid chromatography (CHPLC): 2 110A type pumps, 160 type U V detector, 420 type microprocessor/programmer-1 and stainless steel Column (4,6X 2fic!n% 5pm Ultrasphere (:'-18) The analysis was carried out on an Altos analysis system consisting of:

A flow rate of 14 M 7 min was employed to monitor absorption at s 254 nu. every minute The solvent system used for separation was Mg0H70,01M-KHtPCh (5a 5:4 3.5). For low pressure preparation (mudography (flash chromatography) Fee, J.T., Pacer Chemical Co.) is 3.7X of 4011rnRP-18. 223 column with Mg0H/water (68:32) as eluent or 4 CHCl5/95% using a 3.0 x 18 cInO column on 0 μm silica gel The eluent was EtOH (65:35). TLC is aluminum based The experiments were carried out on silica gel 60 plates (E, Merck & Co.). steroids and their glycosides are l8 Toluenesulfonic acid/959bF;tOH (20:80, W/V) and heating at 110° C. for 10 minutes.

11β,17α-dihydroxy-3,20-dioxypregna-1,4-diene -21-yl 2', 3', 4', 6'-tetra O-acetyl-β-D - glucoside prednisolone 4 (120 g, a33 mmol) in dried CHC1s Dissolve in C3owu and boil dry over 4A molecular sieves in a 5001 volume round bottom flask. CBC1sC200m). After distillation of 10 to 201, new Prepared in CC, M., Matsufkloski and G. H., Coleman, “Organic Synthesis. "Collection 3, Wiley Publishing, New York, NY (1955) 434-43. Silver carbonate (14.1 mmol of 190L) was added to the flask. Next 2,3,4,6-Citler O-acetyl in CHCl5C100xi) dried at -1-bromo-α-D-glucobylanose (also 3.3.50 g, 8.50 mmol) A solution of 1) was added dropwise from the dropping funnel. The reaction mixture was protected from light and stirred thoroughly.

The addition of the bromo sugar took approximately 1 hour, during which time the solvent was continuously distilled. Bro After all the sugar has been added, distillation is continued for an additional hour and the mixture is distilled by adding dry CHCl. capacity was maintained. This solution was filtered, washed with cold saturated NaCl, and diluted with sodium sulfate. The solvent was removed. A few milliliters of methanol to remove the oily residue and purified by flash chromatography on RP-18. A fraction was collected and the solvent was removed. Crystallization of PREDGLU from methanol/water 0.87 g (38 g) was obtained. Melting point 119-121℃; TLC: RfO, 36 (ethyl acetate/isooctane 9:1); UV2. ,, :242nm(t13500);IRCKBr):345G (OH), 1760 (acetyl), 1650 (C=0), 1190 (acetyl) , 896 cm -';' H-NMR: δ0,77 (a.

3H, C-IB>, 1.45 (s, 3H, C-19), L99 (J, 9 H, C-2't 3', 4' acetyl), 2.08 (8# 3H, C-5/ Acetyl), 4.20Cd, IH, C-1', J=8), 4.58CAB , q, 2H, C-21°J = 18.5.92 (s, IH, C-4), 6.15 ( d.

IB, C-2,7=11), 'L40Cd, IH, C-1°J-11). elemental content Analysis (C□&a　014　) C, H.

Compound 14: DEXATAGL, U 9α-fluoro-11β, 17α-dihydroxy-16α-methyl-3,20- Dioxopregna-1,4-Scheer 21-yl-'193'e 4'#6' -7 tolar 0-acetyl-β-D-glucopyranoside.

PREDTAGLU15 (as described for prednisolone) EXATAGLU14 was prepared from dexamethacin. from methanol/water Crystallization of DEXATAGLU yielded 0.55 g (25%). Melting point 0 119.5-121°C; TLC: Rf O, 45 (ethyl acetate/isooctane, 9:1);UVλM! :239 nm (g14300), IRCKBr): 3450 C0H), 1760 (acetyl), 1650CC=O), 1190 ( acetyl), 896 cm':'HNMR: δ0.76 (d, 3H, C-1 6αC11s, J=6), 0.88 (a, 3H, C-18), 1.49 ( g, 3H, C-19), 2.00 (g, 9H, C2/, 3/, 41. chill), 2.09 (s, 3H, C-6' acetyl), 4.18 (d, IH , C-1', Jshi8), 4.5'1CABq, 2H, C-21, J=18), 6.02Cs, IH, C-4), 6.23 (d, IH, C-1,, 7; 11), 7-33Cd, IH, C-2J=11). Original cumulative analysis, calculated value (Csa&yOtJ ’): C, 59, 75; H.

6.63° Actual value: C, 59,08H, 6,54° Compound 2: Prednisolone- Glucoside 11β, 17α-dihydroxy-3,20-dioxypvfs-1, 4-dien-21-yl β-D-gluco in methanol (10x/) and ben It was dissolved in Zen (5,0rLe). 0-04 in methanol (s, om) then N.NαOH was added. The reaction was carried out under stirring at room temperature under nitrogen. 30 minutes later The solution was neutralized by adding a few drops of acetic acid. After removing the solvent under reduced pressure, sift the residue. Purified by flash chromatography on silica gel. appropriate 1 The fractions were combined, the solvent was removed under reduced pressure, and the residue was dissolved in t-butyl alcohol/water (15 m, 1:1) solution.

The solution was frozen and the solvent was removed by lyophilization. Yield is 0.11g (73%) there were. TLC: Rf 0.58 (chloroform 795% methanol, 3: 2); UVλrntLZ '242nmCt 13200): IRCKBr): 3450C-OH), 1650CC=O), 896cm-”;”H- NMR: δ 0.77Cs, 3H, C-18), 1.45 (s, 3H, C -19), 4.20 (d, IH, C-1', J=8), 4.58 (A Bq, 2H, C-21, J=18), 5.92C11,1H.

C-4), 6.15 (d, IH, C-1, J=11), 7.40Cd, IH, C -2, J=11). Elemental analysis, calculated value CCHHssOIo・HtO): C, 6 0,00: H, 7,41° Actual value: C, 59,94: H, 7,64° Compound 1 : Dexamethasone-glucoside 9α-fluoro-11β, 17α-dihydroxy -16α-methyl-3,20-dioxopregna-1,4-dien-21-yl -β-D-glucopyranoside.

h). TLC: Rf O, 51 (chloroform/95% ethanol, 3:2)", UV λrruLZ')' 239sm (g 14500); I A’CKBr): 3450 (-QH), 1650CC=O), 896 cm” :”HNMR: δ 0.78 (d, 3H, C-16α-methyl, J=7 ), α88 (s, 3H.

C-18), L49Cs, 3H, C-19), 4.17(d, IH, ni’- 1'; J=8), 4.57CABq, 2H, C-21, J=18), 6. 03 (#, IH, C-4), 6.23 (d, iH, C-1, J=11), 7 ．． 35 (d.

IH, C-2, J=11), elemental analysis, calculated value (CuHs.

01ffiF-H, 0:C, 58,43; H, 7-65° Actual value 20.

5B-58; H, 7, 36° Compound 18: Dexamethacin/tetraacetergalac Shioko 9α-fluoro-11β, 17α-dihydroxy-16α-methyl-3,20- Dioxopregna-1,4-dien-21-yl 2/, a/, 4/ , 61-tetra-0-acetyl-β-D-galactopyranoside Describes PREDTAGLUI5 (steroid 4, from prednisolone) Compound 18 was prepared from dexamethacin 1 as described above. MsOH/water paste Crystallization of gave 0.56 g (26%). Melting point: 133-135℃:T LC: Rf O, 39 (ethyl acetate/isoocta/#9:1); UV λ, z : 239 am (g 15400); IRCKBr): 3450 Con) , 1750 C0Ac), 1660 (C-0'), 162G (C-C), 124 0 (OAa), 985, 960, 895cIIt″-1+5HNMR6 0,78 (d.

3H, C-J-16CHs), O-88 (8, 3H, C-18), 149 (g, 3H, C-19), 1.93 (a, 3H, C-4'0Ac), 2.00 (s , 3H, C-3' 0Ac), 2.06 (s, 3H, C-2', 0Ac), 2.12 (s), 3H.

C-6' OAc), 4.18 (d, IH, C-1', J≠7.2), 4,5 2 CABq, 2H, C-21, J=1B, 3), 6.01 (a, IH, C- 2, J=11), 7.32Cd.

IH, C-1, J-10). Elemental analysis (C5teO凰banF)C.

0 11β,17α-dihydroxy-3,20-dioxopregna-1,4-diene -21-yl 2', 3', 4', 6'-tetra-0-acetyl-β-D - Galactopyranoside PRHDTAGLU15 (steroid (, prednisolone) Compound 19 was prepared from prednisolone as described for . Crystallization of the paste from Mg0H/water gave 0.8277 (37%).

Melting point: 134-136°C: TLC: RfO, 39C ethyl acetate/isooctane, 9:1; UI/λmam: 242 ns (ε14700); IRCKB de) : 3500 C0H), 1760 C0Ac), 1650 (C=0), 1 620 (ni'=C), 1240 C0Ac), 9.0 00II-'; Otori HN ME J O, 78 (s, 3 H, C-18), 1.38i, 3#, C-1 9), 1.91 (a, 3H, C4' 0Aa), 1.99 (s, 3H, C -3' OAc), 2.03 (s, 3H, C-2' 0Ac), 2-0 9 (a, 3H.

C-6'0Ac), 4.23Cd, IH, C-1', J=4 7.2), 4.49 (ABq = 2H-C21p J = 18), 5.9 2 (a, IH, C-4), 6.15 (d, iH, C-1, 7=11), 7. 40 (d, iH, C-2, /=11).

Elemental analysis (C5JuOu　) Calculated value: C, 60, 78:H, 6-66° Actual value: c, ss, sa; H, s, ss.

Compound 16: Hydrocortizo 7/tetraacetyl glucoside 11β, 17α-di Hydroxy-3,20-dioxypregnath-4-eny-21-yl 2',3 ',4',6'-tetra-O-acetyl-β-D-glucopyranoside f As described for Sid 15 (from steroid and bromine sugars), the compound Product 16 was prepared from hydrocortisone cape) and bromosugar 23. Mg0H/water 52g (23g) of (L) was obtained by crystallization from 2°C; TLC: Rf O, 39 (ethyl acetate/inoctane, 9:1); UV λ1□: 242aWL (g 15700); IRCKBr): 34 50 (OH), 1760 C0Ac), 1645 (C=0), 1610 ((1 ’=C), 1240 C0Ae), 950.908.875cIR-”;”HN MRδ 0.80 (#, 3H, ni'-18,1,40(s, 3H, C-1 9), 1.96 (s, 3H, C-4'0Ac), L99 (s, 3H , C-3' 0Ae), 2.04 (Jl 13 Hy C2' ()AC), 2 ．． o5cs, sH, C-6' 0Ac), 4.20Cd, IH, C-1', J= 7.5), 4.60CABq, 2H, C-21, J=18>, 5.60(s, I H, C'-4゜Elemental analysis (C-4-14) H; C: Calculation 5 Value, 60.69; Actual value, 60.02° Compound 20: Hydrocortizoy Tetra Acetylgalactoside 11β,17α-dihydroxy-3,20-dioxopre Gunar-4-en-21°-yl 2'e 3't 4't 6'-tetra-O- Acetyl-β-D-galactopyranoside glucoside 11 (from the steroid tip) Compound 20 was prepared from steroids and bromine sugar moxibustion as described above. did. Crystallization of the paste from MaOH/water gave 0.57 g (26 pieces).

Melting point: 122-124'C: TLC: Rf O, 42 (ethyl acetate/isooc Tan); UV λm5! :1242am(g16700):IRCKBr): 3450 C0H), 1760 C0Ac), 1660 CC=O), 1620 (C=C), t230C0Ac), 950.908.896cm-” ;”HNMRδ 0.8 (i (a, 3H, C-18), 142 (s) , 3H, C-19, 1, 92 (Jl, 3H, C-4' 0Ac), 198 ( s, 3H, C-3' 0Ac), 2.04 (ae 3H, C-2' 0Ac) ), 2-09 (s, 3H, C-6'0Ae), 4.24 (d, IH, C-1', J=7.6), 4.58CABQ, 2H, C-21, J=18), 5.60 (a, IH.

C-4). Elemental analysis (C5JaOu) H, C: Calculated value, 60.69; Actual value, 6 (L27゜ Compound 17: fludrocortizo/tetraacetylglucoside 9α-fluoro- 11β,17α-dihydroxy-3°20-dioxopregn-4-ene-21 -Ile 2'.

a/, 4/, 6/-tetra-O-acetyl-β-D-glucopi26 Compound 4m7 was prepared from fludrocortisone as described above.

Crystallization of 17 from MaOH/water yielded o,4sII (zs%) 0.39 (acetic acid ethyl/inoctane): UV λrrLnZ '239 nWL (g 175 00); IRCKBr): 3450 (OHHI3750 C0Ac), xs6 0 (Cm0), 1625 (C=C), 1250 C0Ac), 898 cIR- ";"HNMR δ0.76 (S, 3H, C-18), 1.49 (a, 3H, C-19), 1.95 (s, 3H, C-4'0Ac), 2.00C8a 6H, C4,'j　Ac), 2.04CB, 3H.

C6' 0Ac), 4.25Cd, IH, C-1', J=718), 4.45C ABq, 2H, C-21, J-18.

5.75Cs, iH, C-4). Elemental decomposition (C or IkrOuF) 9α-Full Or-11β,17α-dihydroxy-3゜20-dioxypregna-4-y -21-il・2′.

a/, 4/, 6/-tetra-O-acetyl-β-D-galactopyranod As described for glucoside 11 (from steroid faeces and bromine sugars) 0.45 g (23%) of Compound 21 was obtained by steroid crystallization. Melting point= 130-132°C; TLC: Rf O, 40 (ethyl acetate/in-octane): U V λyBz: 239 nrnce 17100); IRCKBr): 3500 C0H), 1780 (OAc), 1660CC-Q,), 1620 CC=C), 1250C0Ac),'HNMR:a Oo-78(,3H,C -18), 1.48Cs, 3H, C-19), 1.92 (s, 3H, C- 4'C)Ac), 1-99 (x, 3H, C-3'0Ac), 2.03C 11#3H, C-2' 0Ac), 22-09 (,3H, C-6' 0Ac), 4.25 (d, IH, C-1', J=8), 4.53CABq, 2H, C-2 1, J=18), 5.73 (g, IH, 4) o elemental analysis (CmBuOI4 F) C, H.

25: Hetaaceru 1-brome-α-cellobiose hepta-O-acetyl-1-bromo-α-D-cellobio according to methods described in the literature. manufactured the base. G, N, Borenbatsul, J, W, Long, D, G, Benja Min, J.A., Lindquist, J-kn-Cham, Soc., 77:3. 310 (1955); Freudenberg, W., Nagai, A3 Roux, 494: 63 (1932). Octa-O-acetyl-α-D-cellobiose (7.5 g, 11.1 mmol) dissolved in 31% HBr in acetic acid (35 m) did. The mixture was stirred at 4°C for 24 hours. Ice-cold water (5N) was then added. Afterwards, CHCl* (10'ld) was added. The organic phase is then heated to a low temperature saturated Nα C! Washed several times with solution and dried 8 (A'at S O4). Crystallization of bromo sugar 25 by addition of dry ethyl ether 4.68JiD611) was obtained. Melting point = 181-182℃ (Literature melting point 1 83° C., see Freude/Berck and Nagai, supra), [α]D +93.5 (c 5.4 CHCl5), (Reference [α), +95.8°L, J, Hynes, F, H0 News, Adv, Carboh/dr.

ch-door+j 10:213 (1955).

11β,17α-dihydroxy-3,20-dioxypre/J--1,4-die -21-yl hepta-0-7 cetyl-β-D-cellobioside Pido Kei (0.6 g, 1.6 mol) and bromo sugar u (&7p, 5.2 mmol) Manufactured from From the crystallization of 41 from MeOH/water, 0.42 g (25 )was gotten. Melting point: 135-136°C; TLC: RfO, 39C ethyl acetate/ Isooctay): UV λmaz: 242ss (t16900); IRCK Br): 3500COH), 1750COA, c), 1660CC=O), 16 20CC=C), 1230C0Ac), 912.870.782cm': 'HNMRδ 0.78Cs, 3H, C-18), 1.42 (g.

3H, C-19), 1.91 (s, 3H, 0Ac), 1.97 (8,6H, 0AC), L99 (Jl, 3H, 0Ac), ZOI9 (s, 6H*OAc), 2.07 (#, 3H, 0Ac), 4.28 Cd, LH, C-1', J=7.6), 4.49 CABq, 2H.

C-21, J=18), 5.92 (g, iH, C-4), 615 (d, Iff, C-2, J=11), 7.40 (d.

If, C-1, J = 10° elemental analysis (CuHvOn) C, H.

Compound 5: Hydrocortisone glucoside 11β, 17α-dihydroxy-3, 20-dioxopref+-4-en-21-yl β-D-frucohyranoside It was dissolved in Yohihenzey (5m). Then NaOH in NaOH (0,04N, 5.0 ml) was added. The reaction was carried out with stirring at room temperature under N. 4 After 5 minutes, a few drops of acetic acid were added to neutralize the solution. Remove the solvent and silica the residue. Purified by flash chromatography on a column. Purified glucoside 1 6 was then dissolved in t-butyl alcohol/water (xsm, i:x). This solution was frozen and the solvent was removed by lyophilization to yield 0.121 Ig (77%). R f 0.49 (CHCIJ95%EtOH, 65:35); UV 2t7!5 z" 242rLm (g 15800); IRCKBr): 3450 C0 H), 1650CC=O), 1620CC=C), 945,910.868cm -”;”HNMRδ　0.76Ca, 3H, C-18>, 1.36C8, 3H, C-19), 4.17 (d, IB, C-1'.

30 J-7, 7), 4.5'1CABq, 2H, C-21, J=18.2), 5. 56Ca, IH, C-4). Elemental analysis (etc. Jio□5J20) C, H.

Compound 7: Fludrocortisone glucoside 9α-fluoro-11β, 17α- Dihydroxy-3゜20-dioxopregna-4-en/-21-yl β-D- Glucopyranoside Compound 7 as described for glucoside heather (from acetyl glucoside 16) was produced from acetylglucosytobi. After freeze-drying, 0.11 g (711) Glucoside 7 was obtained. T L C: Rf 0.50 CCHC-1s / 95% EtOH, 7:3)”, UV λrrLtLz: 239 am (C 17800); IRCKBr): 3450 C0H), 1650 (C= 0), 1620 (C=C), 938.895 cm-”; Heavy HNMR δ 0. 78 (s, 3H, C-18), 1.49Cs, 3H, C-19), 4.1 9 (d, IH, (:'-1'.

J=7.6.4.57 (ABf, 2H, C-21, J=18), 55-70 ( , IH, C-4). Elemental analysis C, Ct H4t JIl・F-Hl0) H, C: Total Calculated value, 57.96; Actual value.

5 L53゜ Compound 9: Dexamethazoy galactoside 9α-fluoro-11β, 17α-di Hydroxy-16α-methyl-3,20-dioxopregna-1,4-diene- 21-yl β-D-galactopyranoside glucoside 1 (acetyl glucoside■ Compound 9 was prepared from 7 cetylgalactoside U as described for Ta. After freeze-drying, 95■ (60%) galactosides! was gotten. TL C: Rf O, 46; CCHCl5/95% EtOH, 7: 3): UV λ maw: 239 nm (ε14400): IRCKEr): 3450 COf i), 1680 (C=0), 1625CC=C), 990.891cIn-” t”HNMRδ 0.76 (d, 3H, C16CHs-J-7), 0,85 ( s, 3H, C-18), 1-50 CB.

3H, C-19), 4.19Cd, IH, C-1', 7-7.8), 4.58 CABq, 2h, C-21, J=18), 6.01 (a, IH, C-4), 6. 24Cd, IH, C-1, J = 10.1), 7.39 (d, IH, C-2, J=10.2). Elemental analysis (C-ra01°F-H-,0) Calculated value: C260, 65; H, 7, 1? . Actual value: C, 60-44; H.

6.98° A: Rednisolone galactoside 11β,17α-dihydroxy-3,20-dioxopglucoside i (acetyl Compound 10 was converted to acetylgalactoside as described for glucoside Manufactured from No. 19. After freeze-drying, 0.13g (83%) of galactoside and Liao Obtained. TLC: Rf O, 39CCHCI3/95% EtOH, 7: 3)", UV λmat'242nmCz 14500):IRCKBr): 34502 COH), 1650 ((:’=0). 1615 (C=C), 8993-”; NMR60, 78Cs, 3H, C-18), 1.39 (Jl, 3H, C-1 9), 4.13 (d.

IH, C-1', J=7.3), 4.53CABQ, 2H, C-21, J= 18.1), 6.16Cd, IH, C-1, J=10.1), 7.33 (d , IH, C-2, J=10.1).

Elemental analysis (CttHuOIo-1bO), H0 compound 11: Hydroco Lutisone galactoside 11β,17α-dihydroxy-3,2o-dioxyp The compound Product 11 was prepared from acetylgalactoside color μ. After freeze-drying, 95IR9 ( 60%) of compound 11 was obtained. T L C: Rf O, 42CCHCl5 /95% EtOH, 7: 3); UV 2. rLa,: 242 nm (t1 5700) * IRCIBr)’ 3450 (()Hl 1660CC= O), 1620CC=C), 1080,1033.930.896,870cI m-';"HNMRa O, 79 (s, 3A, C-18), 1.42 (s, 3H, C-19), 2H, C-21, J=18), 5.58 (a, IH, C -4). Elemental analysis (C*rH3Oso・HtO) C, H0 compound 12: Full Drocortizo/galactoside 9α-fluoro-11β, 17α-dihydroxy -3゜3 20-Dioxopregn-4-en-21-yl β-D-galactopyranoside As described for galactoside interactions (from acetyl glucoside 16), the compound Product 12 was prepared from acetylgalactoside υ. After freeze-drying, 0.11g (7 1%) of compound 12 was obtained. TLC:Rf 0049CCHCノ.

/95% EtOH, 7: 3); UV 2rrL, z: 239 nm (ε 17 700); IRCIBr): 3450 (OH), 1660 (C=0), 1620CC=C), 1080.1033.930.896.87 GcIn- ";"HNMRδ 0.78Cs, 3H, C-18), 1.50 (a, 3H, C-19), 4=20 (d, IH, C-1', J=7-8), 4.5 5 (ABq.

2H, C-21, J=18), 5.70Cs, IH, c-4). Elemental analysis ( CrrHs*01oF-HtO) C, HoA: Rednisolo/・Cellobiosi de 11β,17α-dihydrokycu3.20-dioxop-1,+-diene -21-1 β-D-cellobioside As described for the glucoside interactions (from acetyl glucoside 16), the compound 13 was prepared from acetyl cellobioside 22. After freeze-drying, 86 ~ (60% ) Compound 13 was obtained. T L C: Rf O, 33CCHCl5/95 % EtOH, 7: 3); UV λnLcLz: 242xm (g1550 0); IR (KBτ): 3450COB included 1680CC=O), 1620(C =C), 1170.950.898.87534 Pot-”:”IiNMRδ α75 (a, 3H, C-18), 1.28 (g, 3 H, C-19), 4-15 (d, IH, C-1', J-7,6), 4.51CA Bq, 2H, C-21, J=18), 5.91 (x, lH, C-4), 6 ．． 20Cd, IH, C-1, J=10.2), 7.30 (d, IH, C- 2°J-10,1). Elemental analysis (C, s&O, -H, O) C, H.

In vivo test Male Sprague-Dawley C8p Deαg-Dawla’l) rats (approximately 250 g) were fed with laboratory rat chow and ad libitum water. these rats Animals were fasted overnight (16 hours) before administration of glucosides or free steroids.

Water bottles were removed from cages at least 30 minutes before drug administration. Prednisolo Dexamethacin glucoside 2 (7,5111p) or dexamethacin glucoside ↓ (7 , 5 mg) in a solution of water and 795% ethanol (3:1) (0.5 mg) by intragastric intubation. M). Prednisolo/4 (5.25g) or Dexamethazo’: /3 (5,1 ■) is a solution (0,5 sl) of water 795% ethanol (1:1) It was administered as follows. Oxygenate the animal at appropriate intervals (3, 4, 5 or 6 hours) The animals were sacrificed using carbon anesthesia and their thoracotomy was performed. The small intestine and cecum were removed and cut into sections. Ta. Remove the contents by rinsing the sections with cold 0.9% saline (5,011/ml). isolated from tissue. The contents were immediately diluted with methanol to 3011j. set Weave 0.01 M-KH! Suspended in P04 (5,0 penalty).

Then internal standard prednisoloyl or dexamethacin depending on the steroid being tested. and was added to all samples.

Both contents and tissue were then homogenized using a Polytron homogenizer. (Brinkmann Institute) for 1 to 2 minutes at medium speed. I lost it. The contents were then diluted with methanol to a total volume of 4 Qd. organization is meta diluted with ethanol to a total volume of 25-. Centrifuge all samples at 5,000 # 15 minutes), then filter the supernatant through a membrane filter (0.45 μm/(-1 pores). -CVαday 8αpode) 800). Then the sample (1,4m) was 0,0 Diluted with 1 Af-KHtP 04 (0-617) and the resulting solution 20 pi was directly injected onto an HPLC column for analysis.

β-glucosidase (EC3) in M-acetate buffer (pH 5, 0, 10, Om) , 2, 1, 21, from Almant, 1 unit is salicin or 1.0 micromol/ Incube unit with release of glucose at pH 5.0 and 37°C treated with enzyme. Aliquots (0,1 m) were removed at various time points and diluted with methanol ( 9.9m). After centrifugation (5,000 g, 1 o min), the sample was ．． Dilute 0IN- &PO4 1:1) and directly add 20 pl of the resulting solution. was injected onto an HPLC column for analysis.

6 Glucosides 1 and 2 (5,011 g) were similarly homogenized at 37°C. Feces (0,51110, OIM phosphate buffer (f) H7,5) 1 O N). The feces were from rats fed a high cellulose diet. Obtained. S, Y., Shiaw, G., W., Chan, J.

See Ntttr-, 113:136 (1983).

An aliquot (0.2 mg) was removed and quenched in a muffler (3.66 d). centrifugal After separation (5,000 g, 10 minutes), the sample was passed through a men's rain filter (0.4 It was passed through a 5 μm pore, Persabo 450 (Gelman Science).

These were then diluted with 0.01 M-KHtP 04 C1:1).

20 μl of the resulting solution was directly injected onto an 11PLC column for analysis.

in vitro metabolism Three p-nitrophenyl glycoside substrates, namely p-nitrophenyl-β- D-glucoside Cp-NP-ctlc), p-nitrophenyl-β-D-galac toside (p=NP-gag), and p-nitrophenyl-β-D-cellobi The hydrolysis rate of osside (p-NP-tel) was determined by the gastric, proximal small intestine CPSI), distal small intestine CD5I), and in the homogenized contents of the cecum. small The entire intestine was divided into two equal length sections to obtain PSI and DSI). gastrointestinal contents with preserved feed (Purinα, chow for rats) and any amount of water. Breeding male Sprague-Dawley rats (300-4oo, obtained from l) Ta. Intestinal contents of each segment were pooled from 4 animals for each assay. movement of 4 animals By pooling the contents of the gastrointestinal tract, the SD is reduced by a factor of 2. is expected. After taking it out, quickly weigh the contents and then pour it into cold 0.01M phosphorus. 100M in acid salt buffer (pH 7,0) (gastric, PSI and DSI) and 1i20 diluted to 0 dc cecum). Pour the diluted contents into a Polytron homogenizer (Princess). Homogenize for 1 to 2 minutes at medium speed using a TH was measured. The homogenate was then stored on ice (approximately 30 minutes). homo dienate (0.8 sl for stomach and Psi, 0.2 sl for DB I, 0- for cecum) 64m) to the appropriate substrate solution (0,01&’j phosphate buffer, pH 7,0). 1.0 mM substrate was obtained (total volume: 2.25 d). Shake the reaction at 37℃ It was carried out in a water bath. 10. After 20 or 30 minutes, 0.2N-NtLOH (CL The reaction was stopped by the addition of 25 m). 40% of the released p-nitrophenol Measured spectrophotometrically at 3 nm.

Glycosides as drug precursors 1.2. 5. 7, and 10.1 above, ↓ and The hydrolysis rates of L and L were measured in the same manner. However, higher concentrations of each homo I used dienate. Before homogenization, the contents of the stomach and PSI were o,o DSI contents were diluted to 50 d with phosphate buffer (pH 7.0). In 2004, the cecal contents were diluted. These homogenates CL Psi 38 and DSI2Ad, and caecum 1.7 m) in an appropriate substrate solution (0,01Af Phosphate buffer, pH 7,0) to give 11-0 rn in a total volume of 2.5 d. A substrate was obtained. Aliquots (0,3id) were removed at various time points up to 30 min and M It was quenched with ttOH (4.7 ml). Centrifugation (5,000,!i+, 10 minutes) Afterwards, the sample was diluted with 0.01M-KH,PO, 1:1), and the resulting solution 20 PI was directly injected onto the HPLC column for analysis.

p-MP-gl using the pooled cecal homogenization (200 m) Regarding the hydrolysis of c and p-MP-gal, Kt (app) VmcLz' Decided. A range of substrates containing their apparent KM (56-1000 micromoles) A final volume of 2.25 m2) was used for each reaction. Cecal homogenate used The amount was 0.04d. The speed was tested in duplicate at 37°C in a shaking water bath. After 15 minutes, 0.2N-NaOH (0-25wl) was added. It was stopped by the addition of Spectroscopic analysis of the release of p-nitrophenol at 403 rLm Measured by photometry. K for both reactions using the Edie-Hofstey plot. Determine M (αp, ) (micromol) and V engineering 2 (micromol 7 min/l) Ta. Wet weight ■ measured immediately after harvesting and pooling was used throughout.

Vm6Z was determined. Again, cecal contents from four rats were pooled and weighed. Weigh, dilute to 100d, 0.01M phosphate buffer pH 7.0), and homogenize. did. Substrate concentrations in the range containing the apparent KM (0.5-48 μM 5 final volume 2. 5m) was used in each reaction. The amount of cecal homogenate used was 0.8 . Reactions were carried out in duplicate at 37° C. in a shaking water bath. 15 minutes later The reaction was stopped by taking out the aliquot (0,3d), and this was added to MeOH (4 , 7). After centrifugation (5,000g for 110 minutes), the sample was ．． OI M-KHz diluted with PO4 (1:1) and directly analyzed the resulting solution was injected onto an HPLC column. In this case too, the E.D.-Hofstay plot KM Ca, pp) and V4□ were determined using

Determination of apparent partition coefficient Pre-drug between n-octatool and aqueous phase (0,01M phosphate buffer, pH 7,0) The partitioning of precursor substances and free steroids was determined at 37°C. octatool and loose Both solutions were saturated with the relevant aqueous or organic phase before use.

Equal volumes (1.0 m) of both phases were used and stirred for 30 minutes. The initial concentration of glycosides is It was 10mM (dissolved in the aqueous phase).

The initial concentration of steroid was 10 mM (dissolved in the organic phase). in equilibrium The amount of glycosides and free steroids in the aqueous phase was determined spectrophotometrically. At 239 nm for tacine and fludrocortisone compounds, prednisolone 0 for Ron and Hydrocortisone compounds In this case, it was measured at 242 nrn. Glycosides or free esters in the octatool phase The concentration of roids was determined by difference.

description of the invention Most generally, the present invention provides colon-specific drugs based on the use of synthetic drug precursor compositions. Disclose the grant system. Drug precursors are agglomerates linked to sugars by glycosidic bonds. Consists of a combination of recon. According to the invention, the aglycone is a pharmaceutical composition, and the sugar is recognized as a substrate by bacterial glycosidases produced by the colonic microbiota. The glycosidic bonds are formed by the action of glycosidase enzymes in the colonic microbiota. Glue that can be cleaved (after the sugar is recognized as a substrate by bacterial glycosidases) It is a lycosidic bond. Also according to the invention, the combination of drug precursors is leaving the gastrointestinal tract without being absorbed by the gastrointestinal tract or produced by the recipient mammal. of sufficient size and size to pass through without being significantly hydrolyzed by endogenous enzymes. It has 0 and hydrophilic properties. As a result, the drug precursor reaches the colon region of the mammal. Here, the glycosidic bond of the drug precursor is opened by colonic bacterial glycosidase. It will cleave and release free drug in the largest amount into the colonic region of the intestine.

More particularly, the present invention provides colon-specific drugs based on the use of synthetic drug precursor compositions. A drug delivery system in which a drug precursor is linked to a sugar moiety via a glycosidic bond. Discloses a system consisting of recon, which forms a drug glycoside. do.

1 Aglycone moieties of pharmaceutical compositions useful in the colon-specific drug delivery systems disclosed herein. is most likely to occur if its adsorption or absorption is primarily directed to the colonic region. It will consist of drugs that are effective. This type of drug is useful in treating inflammatory bowel diseases. This includes steroids and antibiotics. These drugs can also be absorbed If so, they are generally lipophilic rather than hydrophilic, and the intestinal viscosity It will be small enough to pass through the membrane. As is well known, lipophilic substances are generally They penetrate membranes more quickly than hydrophilic substances, and smaller molecules penetrate membranes faster than larger molecules. This is because it penetrates even more quickly. Steroids, antibiotics, and cancer Chemotherapeutic drugs are preferred acrylph moieties for use in drug precursors of the invention. be. Particularly preferred are steroid drugs, such as prednisolone and dexame. Includes Tacin, Hydrocortisone and Fludrocortisone.

Sugar moieties of drug precursor compositions useful in the colon-specific drug delivery systems disclosed herein. is converted into a substrate by glycosidases produced by bacteria present in the mammalian intestines. It will be recognized as a vine sugar moiety. of these bacterial glycosidases. The hydrophilic effect is a mechanism in which the glycosidic bond that connects the sugar moiety to the aglycone is cleaved. Therefore, the glycoside and/or sugar moiety or part thereof It is essential that it can be recognized as a substrate by bacterial enzymes. Also sugar -Pharmaceutical composition Sugawa 42 Drug glycosides are drug precursors that are introduced into the mammalian gastrointestinal tract by the mammalian gastrointestinal tract. without significant absorption or due to endogenous enzymes produced by the recipient mammal. Therefore, it must have sufficient dimensions and hydrophilicity to pass through without being significantly hydrolyzed. It is also essential that it is sustainable.

The primary glycosidase produced by the colonic microbiota is β-glycosidase. However, α-glycosidase is also produced. Therefore α and β-glycose (sugars) Both can be utilized as sugar moieties in the synthetic drug glycosides disclosed herein.

Since β-glycosidase is the main colonic microbiota enzyme, β-glycosides are It is preferred as a drug precursor for use in the colon-specific drug delivery system of the invention.

However, those skilled in the art will recognize that certain α-glycosides may also be utilized. cormorant.

The sugar moiety on the synthetic drug precursor will preferably be a naturally occurring simple sugar. preferred are the natural monosaccharides, disaccharides and oligosaccharides hequinose and pentose.

Particularly preferred are the natural monosaccharide hexoses D-glucose and D-glucose. lactose, and the disaccharide D-cellobiose.

In the present invention, the sugar moiety on the drug precursor is a substrate for bacterial glycosidase. As long as the substrate specificity of the parent sugar molecule is maintained, these natural It will be recognized by those skilled in the art that the sugars of scales can be modified. For example, sugar + 1 acid It can be replaced with an elementary sulfur atom. This kind of synthetic derivative of natural sugar is are within the scope of the invention as long as the substrate specificity of the parent compound remains intact.

Those skilled in the art will understand that glycons (gLyCon'), aglycones and glycosidic bonds are Possibly all modified to improve or change the rate and site of drug release. It will also be acknowledged that it is a good idea. Such modifications also fall within the scope of the invention. included. For example, CJR-Hots can be modified by modifying the functionality of sugar residues. Whittaker, 1 Principles of Enzymology in Food Science, Marcel Detzker, New - New York, York (1972) pp. 434-442). Or in-vehicle The use of oligosaccharide group carriers to reduce the rate of hydrolysis in I can do it. Refractory (rgfractorl) drug precursors are slowly released in the colon. Hydrolysis may prove to be an effective mechanism for long-lasting effects. here As was proven in 2006, changing the agrico y may also change the status of the grant.

Furthermore, the stereochemistry of glycosidic bonds can be used to alter the rate and site of release. It may also be possible that a specific α-glycoside-based system for the small intestine is Can be cleaved by enzymes. Absorption then depends on the rate of release in the small intestine, and the physicochemical properties of the parent drug.

The drug precursor providing system of the present invention is characterized in that the drug precursor is a specific glycosylation agent produced by intestinal bacteria. Synthetic drug glycos whose active drug is released upon hydrolysis by sidases Based on the use of cid-based drug precursor compositions. This type of intestine in humans C.B., S., Dreser and M.J., since endophytic bacteria are normally found in the colon region. Haile, 'Human Intestinal Bacteria', Academic Press: London (1974). According to the drug precursor delivery system of the present invention, the effective drug is usually delivered to the intestine. will result in release into this area of the body. However, certain disease states (e.g. localized ileitis or Crohn's disease) causes intestinal bacteria to accumulate in normally sterile areas of the small intestine This will also be understood by those skilled in the art. This accumulation is ingested due to knee tension and inflammation. It occurs because the absorbed substances do not pass through the small intestine as quickly as they normally would. child The ingested substances “accumulate” due to a slowdown in the transport of Bacteria not previously found in this area of the intestine will "accumulate".

In such cases, the drug precursor delivery system of the present invention can be used to deliver effective drugs to diseased areas of the small intestine. medicine can be delivered.

This region is an area of the small intestine where colonic bacteria that produce glycosidases normally accumulate. This is because it matches.

The activity of glycosidic enzymes in colonic bacteria can be significantly altered by diet. It has been shown that For example, in the case of test subjects (humans), eating beans is while oat bran diet significantly increased β-glucosidase activity. increase G-W, Chan, HoE.

Tsukumoto, C, P, Jolly, A, P-Block, M-J, Kretsuchi and Tori , H. Calloway, Fgd. Proc. 38: 767 (1979) (abstract ) Please refer to The drug delivery system of the present invention substantially targets specific glycans of the colonic microbiota. It is based on the cosidase activity and also on the glycosidase enzyme of colonic bacteria. colon-specific drug delivery system of the present invention. Physicians using the . Ta For example, treatment of glycosidase activity with food can be done by keeping glycosidase activity at standard. Also, in patients with diseased colons who may have low enzyme levels. may be extremely useful in increasing enzyme activity.

To form the drug precursor used in the colon-specific drug delivery system of the present invention, According to the present invention, sugar residues are linked to drug aglycones to form synthetic drug glycosides. It is taught that These drug glycosides are synthesized by known chemical methods. Can be done. K, Igarashi, Adν.

Carboh/dr-Chern, Biochgrn, 34: 243-28 3 (J977). Manufacture the drug glycosides specified herein. A particularly preferred method for rr) This is a modification of the reaction.

C. Meistle and Meishel, Httlv, Chirn.

Actα, 28:1153-1160 (1944); 14', Könitzs and E, Knorr, Ber, 34:957-981 (1901); C, A, Carboh, 5-dr, Chgrn, Eiochtrn, 34: See 243-283 (2977). Generally, this method is The presence of heavy metal salts of organic bases as acid acceptors for acylated glycosyl halides Involves treatment with alcohol during treatment.

The variant used here is to chlorinate the per-acylated glycosyl halide. Combined with a suitable steroid in the presence of silver carbonate as an acid acceptor in loform It involves something. Please refer to FIG. Next, the conjugate on the sugar residue of this drug glycoside is The cetyl protecting group is removed by treatment with base to yield the synthetic drug glycoside.

This paper describes the use of a modified Koenzi-Knorr reaction to produce drug glycosides. Here, glucoside 2 (and its acetyl precursor PRDTAGLU) and glucoside 2 (and its acetyl precursor PRDTAGLU) Regarding the production of lucoside λ (and its acetyl precursor DEXATAGLU) explain. Please refer to the example. Furthermore, as discussed in Example ■, A modification of the Nor reaction can be used to produce a variety of other drug glycosides useful in the drug delivery system of the present invention. It is also used for hunting. These drug glycosides include: Predoni Zolone-21-β-D-galactoside, 10; Dexamethacin-21-β-D- - Glucoside, Engineering: Fludrocortisone-21-β-D-galactoside, Lb example Side door Furthermore, for the production and use of drug glycosides consisting of trisaccharides bound to drugs. This will be explained in 47 Jisho GG-501105 (14). This type of drug glycosi is represented by prednisolone-21β-D-cellobioside, 13.

Synthetic drug glycosylation as a drug precursor in the colon-specific drug delivery system of the present invention When using drugs, the drug precursor is administered orally or intragastrically to the recipient mammal. do.

The drug precursor is then passed through the recipient mammal's gastrointestinal system. synthetic drug precursors The drug precursor is larger and more hydrophilic than the parent drug; It has lower permeability than body drugs. In addition, glycose (saccharide) is bonded to aglycone. The glycosidic linkages present in the bacterial glycosidic system produced by the colonic microbiota This synthetic drug precursor is a bond that is substantially selectively cleaved by the enzyme. leaves the gastrointestinal tract without being significantly absorbed by the gastrointestinal tract or by the recipient mammal. It will pass without being significantly hydrolyzed by the endogenous enzymes produced.

Once in the colon region, drug precursors are acted upon by bacterial glycosidases and their As a result of adsorption to the colonic mucosa, 1Jc releases free drug which is thereby absorbed. Will.

In a preferred form of the invention, the synthetic drug precursor is dexamethacin, 21yl β-D- of donizolone, hydrocortizo/and fludrocortisone glucoside, as well as 21-yl β-D-cellobioside of prednisolone. Become. Particularly preferred is the steroid glycoside dexamethacin-21. β-D-g8 It is lucoside (1).

Dexamethasaccharide 2,3,4,6-chitra using a modification of the Koenitz-Knorr reaction -O-acetyl-1-bromo-α-D-glucobylanose in chloroform with acid It was conjugated with the appropriate steroid in the presence of silver carbonate as a receptor. acetyl glycol The coside product is isolated and then the acetyl protecting group on the sugar residue of the glycoside is removed with a base. It was removed by treatment.

These two synthetic drug glycosides are then used for colon-specific drug delivery. used in the system. More detailed example! The drug glycosides ↓ and 2 as shown in It was administered intragastrically to host mammal rats. analysis of recipient animal intestinal contents and tissues. In this animal, both drugs glycosides 1 and 2 produced results within 4 to 5 hours. It has been shown that they reach the intestine, where they are rapidly hydrolyzed. Dexa Delivery of metazo/(via glycoside 1) to prednisolone in recipient rats (via glycoside 2), but both steps When administered via the colon-specific drug delivery system shown here, the colon The area has been reached. (Guri 9 Prednisolone and dexamethacin, which are drug-releasing steroids, were orally administered. In most cases, they were absorbed from the small intestine.

For the specificity of glycoside/glycosidase-based colon-specific drug delivery The influence of the structure of drug precursors was further investigated in the production of seven steroid glycosides. (see Example 2), determine their relative lipophilicity (see Example V) and label them. Hydrolyzed with bacterial glycosidase obtained from the intestine of a cat (see examples ■ and ■). transformation The preparation of Compounds 1 and 2 is briefly described in Example 3. Abbreviate the production of additional compounds on the side door . (The nine steroid glycosides are dexamethazo/, prednisone, p-hydrocol, 21-yl β-D-glucoside and gala of tisone and fludrocortisone and 21-yl β-D-cellobioside of prednisolone. Ru. ) As shown in Examples ■ and ■, these nine diacetylated glycosylation drug precursors and β-D-glucosides, galactosides and cellobiosides. from the rat stomach, proximal small intestine (CPSI), distal small intestine (DSI), and cecum. Hydrolyzed by the contents. As the data in Example ■ show, all drug precursors are gradually by PSI and stomach contents, more rapidly by DSI contents; It was most rapidly hydrolyzed by the cecal contents. However, the drug precursor itself They exhibited very different susceptibilities to hydrolysis. catalyzed by DSI contents. 50 The hydrolysis rate decreased in the following order: Prednisologalactoside I It wasn't long. Hydrolysis of drug precursors in the cecal contents is caused by hydrocortisol - hydrolyzed more slowly than other drug precursor glucosides). there were. Use the E.D.-Hofstey plot to determine the KM(app) and and VrrLG□ were determined. Eday 11ho 7stay plot shows that in the colon Bacterial β-D-glucosidase activity is naturally occurring in β-D-galactosidase. This suggests that the activity may be more heterogeneous. Please refer to example ■.

Possibility of physicochemical properties of drug precursors influencing the specificity of the delivery system of the present invention lipophilicity is extremely important in determining the rate of permeation across biological membranes. Therefore, the gastrointestinal absorption predicted by the partition coefficient between octanolu buffer and I also looked into it. More specifically, the relative lipophilicity of drug precursors and free steroids were compared by measuring their octano-buffer partition coefficients (P). free space The logP of the teroid was L54 ~ 1.73.

The logP of the drug precursor is the logP of cellobiocitro (quite low, −0,5 It was 0.11 to 0.84 except for 6). Please refer to example ■.

The specificity of the lower intestine contribution of drug precursor glycosides to DSI contents suggests that It can be estimated from the hydrolysis rate and the partition coefficient between octanolu buffer. That will be recognized by those skilled in the art. These parameters are DSI Determine the amount of drug precursor that can survive premature hydrolysis or absorption in This is because For example, under the assay conditions used in this study, glucoside l is 1 A hydrolytic specific activity of 9 nrnol/min/g was maintained in the DSI content and 0. It had a logP value of 59. When this drug precursor is orally administered to rats , approximately K of the dose reached the cecum. Hydrolysis with DSI content lower than this drug precursors with lower logP values are more effective for both. The fruitful bestowal will be served in due course.

Data obtained from in vivo and in vitro studies indicate that trisaccharides (fc e.g. ose) or even trisaccharides as hydrophilic moieties, in some cases has been shown to produce excellent drug precursors. This kind of oligosaccharide fruit cake Rear is necessary when delivering relatively small or relatively lipophilic drugs to the large intestine. It would be important.

Furthermore, the in vitro data obtained in Examples Water splitting rates and relative lipophilicity, when combined with the in vivo data obtained in Example To estimate the site specificity of drug precursor glycosides before conducting extensive animal experiments. Which indicates that.

As described above, the drug glycoside of the present invention can be used under the conditions found in the bilaminar ducts of mammals. It is recognized that their glycosidic bonds have the property of being cleaved by bacteria. . The free drug released by this cleavage is absorbed by the colonic mucosa. free drug Based on what pharmacology tells us, we can thus treat drugs via their drug precursors. It is shown that it is possible to administer.

3 Akira” Certain embodiments of the invention are outlined in the examples below. These examples are for illustrative purposes only and is not intended to limit the scope of the claims in any way.

Example 1 Various syntheses for use in the colon-specific drug delivery system disclosed herein in accordance with the teachings of the present invention. Pharmaceutical glycosides can be produced. An example of this is this type of steroid glycosylation. two species, namely dexamethacin-21/1-D-glucoside and prednisone. Zolone-21β-D-glucoside layer was used in the colon-specific system disclosed herein. This is a comparison based on the usage.

Dexamethacin and prednisolone are steroids useful in treating inflammatory bowel disease. It is a de-based drug. Drug precursors and two other steroid compounds It is a drug glycoside. The colon-specific drug delivery system disclosed herein has two advantages. To investigate the efficacy of L and S, these drug precursors were administered to rats and to analyze intestinal contents and tissue to determine where steroids are released and absorbed. I judged it. Prednisolone and dexamethacin were also administered to treat the drug glucoside. Absorption of free steroids was compared. For both glycosides L and S, 4-5 hours It was observed that it reached the colon, where it was rapidly hydrolyzed. Dexametac (via glycoside L) to the lower stratum of the rat is prednisolone (glucoside 54 Oral use of glucoside l seems to be more specific than in the case of 60X of the amount and 155X of the oral dose of glucoside 2 reached the cecum. to this In contrast, the free steroids prednisolone 2 and dexamethacin were orally administered. In most cases, they are absorbed almost exclusively from the small intestine, with less than 1% of the oral dose reaching the caecum. reached.

Dexamethacin and prednisolone using a modification of the Koenitz-Knorr reaction Glycosides 1p and 1 were prepared by glycosylation of . C, Meistor and , Meeshel, HaLv, Chim, Acta, 28: 1153-11 60 (1944); and W, Könitzhis and UB, Knorr, Ihr, 34. :957-981 (1901). 2, 3, 4 which are bromo sugars. 6-tetra-0-fucetyl-1-bromo-α-D-glucobylanose Carbon I as an acid receptor in the atmosphere! ! It was combined with the appropriate steroid in the presence of silver.

Please refer to FIG. The product acetyl cricoside was transferred to the reverse phase <c-is) on a packing material. It was isolated from the reaction mixture using a column chroma dog 2 filter.

Accommodation is 38X% for PREDTAGLU -TAGLU It was 253I6. These fairly high yields are typical for this reaction. It is. K, Igarashi Adv, Carbohydr, Chatn, Eio Chatn, 34: 243-283 (1977).

Proton NMR revealed that the glucoside produced was β-linked. became. Anomeric proton (C-1') HA PREDTAGLU, 15 leek It is 4.20 p voice, DEXATAGLU, 14 is 4.18 pp A doublet is shown in m. The cupping constant was 8 Hz for both compounds. . These resonance signals are adjacent c-i'.

This shows that the relationship between the 2' flotons is trans-siaxial. D, G, U Williamnon, D., G., Collins, D., S., Reine and S., Bernstein. Biofustry 8:4299-4304 (1969). The chill protecting group-I was removed by treatment with 0.01 #sodium hydroxide.

The “H-NME” of these compounds also differs from the stereochemistry at their anomeric carbon (β − binding). In addition, the glucoside name and glucoside 1 are By treatment with lucosidase, the glucose moiety is removed in each case Ta. Glucoside 1 was hydrolyzed several orders of magnitude more rapidly than glucoside 1. Sara These glucosides were incubated with homogenized rat feces. They were each significantly hydrolyzed.

Separation of glycosides from free steroids was performed by HPLC. Glucoside Typical chromatograph of the cecal contents of rats administered intragastrically with 1 and sacrificed 6 hours later. The ram is shown in Figure 2. Peak A is glucoside 1; peak B is dexamethacin dan; and peak C homogenizes 6 Prednisolone was previously added as an internal standard.

(Altex 5μ inertia ultrasphere, C-18 column Mg0H70,01 M- to &P04 (56,5:43.5) at a flow rate of 1.255 g/min. eluted. ) Small intestine and caecum at various times after oral administration of glucoside l. The recovery of glucosides and free steroids from the intestine is shown in Fjl above. table As shown by the data, after 3 and 4 hours glucoside l was mainly recovered in the small intestine. Very small amounts of glucosides are found in the small intestine or cecum for up to 5 hours. It was done. (At the same time, a large amount of steroids was recovered from the cecum.) Free drug was rapidly released as Do1 passed from the lower small intestine to the cecum. trial The fact that some free steroids were detected in the small intestine at the time of the This indicates that some hydrolysis occurred before the precursor substance reached the cecum or colon.

Recovery of steroid glucosides and free steroids from intestinal harmful substances This is shown graphically in Figure 3. The glucosides 2 (CPREGLU) sp and Recovery of rednisolone A<PRED) is shown in panels C and D. black circle and three Square symbols indicate intestinal toxins: hollow circles and 31 Square symbols indicate intestinal tissue.

From these results, overall the provision of glycoside 1 to the rat 1 intestine and its effect on It is clear that the subsequent release of steroids is highly specific. 4th hour 59% of the administered glucoside dose was recovered from the lower small intestinal contents. This gluco If all the cid passes into the cecum, almost 60% of the dose will be delivered specifically to the cecum. It should be. At the 5th hour, an average of 2.24 ~ (i.e. equivalent to 44X of the dose) was It was recovered as a free steroid in the cecum.

The difference (59X vs. 44N) is due to the free drug being released after the drug precursor is hydrolyzed in the cecum. This is thought to be due to absorption by the cecal mucosa.

Glucose from the small intestine 2 and cecum at various times after oral administration of glucosides. The recovery of coside 2j- and free steroid skin is shown below. 3 hours and The recovery of glucoside 1 from the small intestine after 4 hours was significantly lower than that of glucoside 1. won. Some free steroids up to 5 hours free steroids (all of the time points examined In this case, glycosidase was detected in the small intestine of rats. It has been shown that there are Glucosides and steroids from intestinal contents2 and tissues The recovery of Id 1 is shown graphically in panels C2 and D of FIG.

58 Only X+, SX was recovered intact from the lower small intestine after 4 hours. therefore administration Only about 1596 of the amount should be applied. Kohei Ban until 5 hours An average of 0.57 IIIi of steroid 4 (i.e. an amount corresponding to dose IIX) is Recovered from the cecum. Again, the difference (14,8,X vs. 11%) is probably due to hydrolysis. This is probably because steroid 4 is later absorbed in the cecum and enters the systemic circulation.

No glucosides or free steroids were recovered from the colons of the test animals. child This is probably due to slow transport times and no release at the time tested after administration. This is likely due to the fact that the steroids were unable to pass into the colon of the rat. child The transport time in this experiment was determined by the time required to pass through the gastrointestinal tract of rats fed preserved diets. Although it is lower than the value of 6.6 ± 0.4 hours that was subsequently reported, it is still close to this. Was. V-J, Williams, W, Señor, A%st, J, Biol, s. ci, 35:373 (1982).

Differences in free steroids recovered in the small intestine and cecum further demonstrate the specificity of drug release. The evaluation was made by comparing the Paired t-tests revealed that free stems in the cecum were It is statistically significant that the release of Roidan is superior to that in the small intestine. It was shown that (t=2.32, p<0.025). similar steroids It is statistically significant that the release of 4 in the p1 caecum is predominant due to the distribution of the collection of doshiba. This was shown to be unexpected (t=1.72, o, os<p<0.10).

The transit time to the intestine varies widely, with pre-dose often reaching the cecum by the time of slaughter. It wasn't. If these animals are excluded from statistical calculations, the specificity of the release is larger for both steroids 2 and 4. 1 out of 16 test animals A measurable amount of glucoside 1 reached the caecum in 2 animals. These 10 movements Analyzing the data obtained only from substances, it is statistically clear that the release of steroids is dominant. was shown to be highly significant (t=3.17, p<0.01). gluco Regarding the cider color, the dose reached the caecum in 2 out of 8 of the 16 test animals. This was recognized. The data obtained from these eight animals indicate that the release of steroid groups The dominance was statistically significant (t=3.94, p<0.0 05). However, 4-hour and 5-hour oral glucosides and steroids (The sum of However, it was extremely slow.

Unmodified steroid 3j? A control experiment in which the drugs were administered showed that they were It was shown that it was completely absorbed from the small intestine. These data are shown in FIG. this In the figure, 5.251119 doses of dexamethacin CDEXA) 3 were administered. Recovery of steroids from objects in panel A; prednisolone (PRED) soil in 5 ．． Recovery of steroids from animals treated with 10II9 is shown in panel B. data point Is it an intestinal poison?0 (n=3) (shown as black circles) and from tissues (hollow (shown as a circle).

Based on the release of anti-inflammatory steroids from new steroid glycosides. laboratory la Despite the obvious anatomical differences between rats and humans, the rat’ti intestine It is considered to be a satisfactory model for the human proximal colon. both organs Receptor for digested material from the small intestine, both with large bacterial populations and a wide range of microorganisms It is an active site. Therefore, here we use the rat cecum and colon combined, or or the human colon with its ill-defined caecal region. The term "Low #r intgs-t$% #)" is used.

Although the rat model is useful, The problem of relatively large populations and subsequent high levels of glycosidase activity There is a problem. For example, an average of 10' in the stomach, upper small intestine, and lower small intestine of rats. °7.106°92 and 1g? , f microorganisms/l wet weight). child In contrast, the bacterial populations in the human stomach and small intestine are much smaller. Human helmet, top The lower and lower small intestines have an average of lO°, 10" and lO" cells/l wetness, respectively. (weight) of microorganisms are present. Dreiser, B.S. and Hill, M.J. 1 Human Intestinal Bacteria” (Academic Press: London (1974), 54- p. 71) and G. Hawksworth 1 S, B, P;, Dreiser and M, J, Hill, J-Mad, Mic-rob. See ioL, 4:451 (1971). In the gastrointestinal system It is a bacterial species that makes up the majority of microorganisms.

Furthermore, both species produce measurable amounts of I-glucosidase in vivo. It has been shown that this occurs. See Hawksworth et al., supra.

Despite high levels of microbial activity in the rat upper intestine, glucoside 1 showed remarkable specificity to the lower small intestine. This drug precursor has no place for human use. It should be even more unique. Microorganisms in the human stomach and small intestine This is because the activity is significantly lower than in rats. Sulfasalazine (for humans) (successful drug precursors) must be activated by the colonic microbiota. . M.A., Peraperkone and Hipp., Goldman, J., P.R.M., Ezp. , Ther, 181: 55 (1972), Narahi, M.A., Perapako. Gastroenterology 64:240 (1973) ) Please refer to Sulfasalazine is conjugated with glycoside/glycosidase of the present invention. It acts in much the same way as the given system. Therefore, between these two drug precursor delivery systems, , and the degree of glucoside 10 specificity shown in the rat model. Based on this, it is possible that certain drugs may be effective in the human colon via drug precursor glycosides. 62 It seems possible.

Several factors contributed to the relatively poor performance of Glycoside I in the rat model. This is probably due to Glycoside 2 has a significantly greater effect on gastric 2 and gastrointestinal It is possible that it was absorbed from the small intestine, but glucoside l was absorbed more than glucoside. There was a strong possibility that it was hydrolyzed in the stomach and small intestine. Commercially available β-D-glucosidase is more sensitive to glucoside expression than to glucoside 1. It was even more active. We believe that similar factors are acting in the gastrointestinal tract of rats. be exposed.

Example　■ This example shows a combination of seven other drug glycosides useful in the colon-specific drug delivery system of the present invention. Indicates completion.

A modification of the Koenitz-Knorr reaction discussed above can be used to react with a number of other drug glycoconjugates. Synthesized Sid. Some of these drug glycosides are listed below. in parentheses The compound is the protected glycoside product produced in the first step of the modified process. Protection The protecting group (acetyl functionality) is removed with a base to form the glycoside in front of the compound in parentheses. I got it. These compounds include: Prednisolone-21β- D-galactoside, xo(y and prednisolone-21-yl 2,3.4, 6-Chitler〇-syn-21-yl 2,3.4.6-Chitler O-acetyl 11-D-galactoside, 18); hydrocortisone-21-β-D-glucosy Do, 5C>YU hydrocortisone-21-yl 2,3,4. ．． 6-Q-tetra -0-7 cetyl-β-D-glucosy)', 16); Hydrocortisone-21-β -D-galactoside, oral (and hydrocortisone-21-yl 2,3,4. 6-Chitler O-acetyl-β-D-galactoside, 20); Fludrocortizo -21-β-D-glucoside, 7 (2 and fludrocortisone-21-i 17): Fludrocortisone-21-β-D--112ctocyto.

12 (and fludrocortisone-21-yl 2,3°4.6-Citler O- Acetyl-11-D-galactoside.

and prednisolone-21-/ID-cellobiacetyl glycoside. Proton NMR spectroscopy shows an I-bond at their heptadonomer carbon. did.

All compounds showed a double bond at about 4.2 ppm next to the 7-nomer proton. (Coupling foot 7.2-8.0Hz).

These resonance signals are due to the trans-siaxial relationship between C-1' and 2' protons. showing,,D.G.,Williamson, D.C.,Collins, D.S.,Reine,E. B, Conrow 2 and S, Bernstein, Biochemistry 8: 429 9 (1969).

4 Removal of acetyl binding groups on sugar residues using 0.01N NaOH in Mg0B Achieved by base-catalyzed hydrolysis. , yield y4 is 60 for this process It was ~83N.

According to 1H-Nuit spectral analysis, the free glycoside was still β-linked. .

Example: rin Glycoside/glycosidase-based delivery system of the invention in laboratory rats Studies have shown that certain anti-inflammatory steroids are It was shown that the protein was distributed to the lower intestine with a degree of specificity. Factors that control specificity To better understand the effects of other drug glycosides in the gastrointestinal tract of mammals. In order to prove the behavior, three kinds of p-nitrophenyl-glycoside and steroid The rate of aglycone release from nine drug precursors, which are isoglycosides, was determined in rats. The proximal small intestine (Psi), the distal small intestine (DSI)'s? and freshly prepared from cecum The measurement was carried out using a homogenate prepared by

In more detail (Engineering, general properties of natural enzymes (micromolar 7 min)'S? and specific activities ( Nanomol 7 min/9) first in the stomach, PSI.

Three p-nitrophenyl groups were found in DSI2 and in the intestinal pathogen homogenate. It was measured using a coside support.

rt is p-nitronenyl β-D-flucosi)’ (p-NP-gl　c　) KaG) β-D-glucosidase (Koyoru and Arnitrophenyl-β-D - cellobioside (p-NP-cgj) to β-D-cellobiosidase and β- D-gu 5 This was achieved with the release of p-nitrophenyl by lucosidase. These measurements The results are shown in Table 1. 2 and 3 m of support in all tested segments of 4 al. Along the entire rat gastrointestinal tract, p-nitrophenol was released from all The presence of cricosidase activity was demonstrated. The overall nature of each glycosidase is It is generally lowest in stomach 2 and PsI, relatively high in DSI, and in caecum. It was the highest. The specific activities of 3m glycosidases followed the same general pattern. The highest activity was observed in the chicken and cecum. Glycosidase activity along the gastrointestinal tract In addition to a sex gradient, a cap on enzyme levels was observed. Cecal β-D-galactosidase The specific activity of β-D-glucosidase in the cecum is approximately four times that of β-D-glucosidase in the cecum. The specific activity was about 16 times that of D-cellobiosidase.

Comprehensive hydrolysis of all nine drug precursor glycosides shown in Example 1 and and specific activity were also measured. The results of these measurements were summarized as NPIC glucoside l, 2.5 , E) Ofubi■ (galactoside 9.10% 11112 and cellobioside 13 ). The clothing is shown below. A comprehensive review of the hydrolysis of all drug precursors. and specific activity were relatively low in stomach 2 and PSI contents. These are DSI Increases in content homogenate, highest in cecal content homogenate Met. This pattern is a promising finding regarding the hydrolysis of p-HP-glycosides. Follow. However, in all homogenates tested, 66 Hydrolysis of each type of drug precursor (glucoside, galactoside 2 and cellobioside) The generality and specific activity for the solutions are better than those of the corresponding p-HP-glycosides. It was quite low.

This data indicates that the hydrolysis of all drug precursors is limited to the contents from the stomach and PSI. It is relatively slow when incubated with L DSI homogenate. It is shown that it is relatively rapid in the nate and most rapid in the cecum. . Loss of drug precursors due to gastric and PS7 hydrolysis is negligible Will. Transport in this part of the gastrointestinal tract is extremely rapid. i.e. during transportation The time is about 40 minutes. Low enzyme activity 2 and kinetics in rat stomach and PSI This combination of kana transport is likely due to the specificity of its contribution to glycosida in DSI. meaning that it will be a function of enzyme activity and residence time. Transport to PSI The rate of drug application to the rat caecum is much slower, increasing the possibility of premature hydrolysis. The specificity of the given will be lower.

Each drug precursor was incubated with a D81 volume of harmful substance modienate. , differences in generality and specific activity were revealed. dexamethacin drug precursor l and 1 was more resistant to hydrolysis than other drug precursors. Tested in vivo did! Among 2M lucosides, 1 glucoside is present in the DSI Uchitanimo homogenate. As shown in Example I, it was found to be hydrolyzed three times more slowly than glucosides. As shown in animal studies, glucoside 1 and glucoside 2 were found in the lower intestine of rats. (59N for 1, 14X' for 2) ). Therefore, there is a rough relationship between generality and specific activity in DSI content harmful substance dienate. The specificity of conferring was observed in vivo. All other drug precursors Because they were more significantly hydrolyzed in the DSI homogenate, the Coside 111 would not be specifically distributed to the lower intestine.

All drug precursors except glucosides and It was hydrolyzed even more rapidly than the harmful substances. Galactoside is a cecal homozygous enates hydrolyze even more rapidly than with their glucoside counterparts. understood. Therefore, any drug precursor that reaches the cecum after oral administration is rapidly would be expected to release the following drugs (with the exception of glucosides and (possibly), galactoside drugs, precursors probably reach the lower intestine will release significant amounts of their R-steroids in the DSI before they are released. Therefore The galactoside drug precursors described here are probably responsible for drug attachment to the rat cecum. He would be an inferior candidate for this. The human intestine has a steeper colonization gradient. It should be noted that Compounds that are not specifically conferred in rats are It may be specifically conferred in the intestine of animals. Furthermore, it is possible to add drugs to DSI. It is desired that 'B There may be other uses.

Compounds 13.102 and 2 were incubated in DSI or cecal homogenate. If the prednisolone level is 10 glucosides or galactosides, The release rate was even slower than that of steroid 4. Therefore, cellobioside L! via The administration of steroid U to the cecum of rats in which the It seems that the addition of xamethacin (2) #1 is not specific. But predoni Zolone-glycosides are the most enzyme-labile drug precursors tested. there were. Cellobioside derivatives of any of the other three steroids probably would improve the given specificity. observed in vivo in rat cecum. almost equal to that. The slow release of steroid↓ from cellobioside IJ is probably due to The rate of hydrolysis by β-D-cellobiosidase or β-D-glucosidase This is probably due to the fact that the hydrolysis is slower than the two-step hydrolysis.

p-nitrophenyl-β-D-glucoside Cp-NP-ylc), p-nitrophenyl phenyl-β-D-galactoside (p-BP-gaL and glucosides and galactoside, 69 One drug precursor for the system is shown in Table ■.

Enzyme reaction pooled homogenate using Eadie-Hofstey plot E.D. on the hydrolysis of p-MP-glc and p-NP-gag in φ The 7 stay plots (relationship between V and V/(S)) are shown in Figures 5 and 6. vinegar. At relatively low substrate concentrations, the rate-substrate for hydrolysis of p-MP-gLa The plot of has deviated from linearity. The hydrolysis of p-MP-gag is essentially It was linear.

9 HP (116# p-NP-gag, and glucoside and gala When calculating the KM and XWS for the hydrolysis of ctoside drug precursors. , II-D-glucosidase activity is naturally was found to be more heterogeneous than gender. This is shown in Figures 5 and 6. Various types of 1fAli found in Diekhofstay plots and growing in the large intestine. It seems to vary depending on the species.

The partition coefficient, often calculated between octanol and the aqueous phase, is This is extremely useful when investigating the correlation between the lipophilicity and absorption pattern of compounds absorbed by diffusion. This has proven to be useful. A, Leo, C6) Nshu and Tori, Elkin Su, ch., Rev. 71: 525 (1971) 70 Please refer to Therefore, the partition coefficients of drug precursors and free steroids can be determined. Ta.

More specifically, the apparent octanol-buffer (0.01 M phosphate buffer) , pH 7,0) partition coefficient CP) was measured at 37°C. The results of these measurements (Lo (expressed as yP) are shown in Table ■. Cellobioside 13 is all test drug precursors It showed the lowest l0QP (-0,56) among them. Galactoside drug precursor All substances exhibited lower LoyP values than the corresponding glucoside drug precursors Ta.

As expected, LoyP values for free steroids are lower than for glycosides. It was even bigger.

Example 1 shows the effect of including a woody moiety (i.e., glucose) in dexamethacin. show. In more detail, the data contained therein are of the glucose/dexamethacin type. After oral administration of the drug glycoside (glucoside 1), the dose of 78N was The wound was recovered from the intestinal lumen of the animal 3 hours later. In contrast, oral administration of steroid 1 Only 3.9X of the dose was recovered after 3 hours. In this way, hydrophilic glucose By conjugating the steroid moiety to steroid 3, its absorption was severely hindered. child This is the difference in the dispersion coefficient of glucoside l(0,59) and steroid (1,72). is reflected. Since the logP of 'glucoside' is lower than that of glucoside l, The relatively poor specificity of the addition of glucoside 2 to rat hedel 2 is mainly due to , the drug is absorbed in the upper intestine rather than being absorbed from the gastrointestinal lumen before entering the cecum. This is thought to be due to early release.

! The table below refers to:

4 5-000JI (LO20,055G, 24 0.12 0.23 2. 24α Values represent the average of 4 animals.

4 1.73 0.18 G, 0 0.05 0.19 0.09 0.0 0 ．． 576 G, 18 0.03 0.06 0.29)2 m; trophenyl glucoside, p-nitrophenyl galactosin F, spz p-nitrophue p-NP-glc p-MP-gag p-IJP-caLTo t, Sp, Tot, Sp, Tot, Sp.

Get-act, act, act, act, act.

Stomach 60.0B '12 ()TOS7.8 0.35342P81' 0-15 4 & 2 α20 58.3 0.05 7.2 DSI O, 658 & 6 to 3 6 425 0.2526.0 Cecum 9.1 454 To 3 8 1620 2J 89 & 5a, Tot, act, : activity of the contents of the entire intestinal segment.

Displayed in micromorph minutes. Sp, act: specific activity of intestinal segment contents.

Expressed in nanomoles 7/9 (wet weight).

b The pH of the 19 homogenate was approximately 5.0.

c, PS I, DB 1. and the pH of the cecal homogenate was 7.0. .

〒4 Table 11 1-MP-gle%p-MP-gaj and drug precursors 1. ! , S, ヱ, Dan, Shi0% Shil and LiKgM KM Capp) (Micromo ) and V spider (micromol 7 min/11)'p-NP-g1gb 36 0 697 p-MP-gag” 166 1233 galactoside 12 33.3 20.6 G. Measurements were performed in pooled cecal contents hosodine.

b, The release of p-dithotetraphenol was determined spectrophotometrically at 403% scratch #IIJ. The reaction was tracked by Rujin.

C. Measured by HPLC tracking of free steroid release.

Flo 6. Stir at 37°C for 30 minutes. Measure the concentration in the aqueous phase.

C6 documents (Leo et al., supra) Number: 1.42) - 0 documents (Leo et al., supra) Value “: 1.68fufu about The above description and examples presented are based on the use of synthetic drug glycoside drug precursors. This demonstrates the effectiveness of a colon-specific drug delivery system, and this system has been shown to be effective in mammals. When ingested, it reacts with the intestinal microflora and enters the mucous membrane.

The free drug may be absorbed or otherwise absorbed.

put out The present invention and drug delivery system combine the convenience of oral administration with the specificity of topical application. Motsu. This medicine is useful in treating inflammatory bowel disease in the buttocks. presented and described here Various other modifications may be made to the foregoing description and figures.

will be clear to those skilled in the art. Such modifications are included in the scope of the patent claims. Monotosu stone.・　.

Brief description of the drawing 0 5 10 15202530 minutes □ν(,uM/ri) ν(JJM/Evening) Top investigation report

Claims (1)

[Claims] L. A synthetic drug precursor composition for use in a colon-specific drug delivery system, , the pharmaceutical composition is a combination of aglycones linked to sugars by glycosidic bonds. then the aglycone is the pharmaceutical composition and the sugar is produced by the colonic microbiota. It is a lactic sugar that is recognized as a substrate by bacterial cricosidases that are The sid bond is formed in the colon after the sugar is recognized as a substrate by the bacterial glycosidase. A glycosidic bond that can be cleaved by the glycosidase enzyme activity of the microbiota; The drug is a combination of a glycone and a sugar linked to it by a glycosidic bond. The precursor further comprises a combination of drug precursors that enters the mammal's gastrointestinal tract. Endogenous enzymes that are not significantly absorbed by the human body or that are produced by the recipient mammal of sufficient size and hydrophilicity to be able to pass through without being significantly hydrolyzed by so that the drug precursor reaches the colon region of the mammal, where the drug precursor The glycosidic bond on the precursor is cleaved by the bacterial glycosidase and released into the colon region. A synthetic drug precursor composition that releases free drug. 2 The aglycone is a steroid drug composition, the sugar is a simple sugar: Glycosides whose lycosidic bonds can be cleaved by the glycosidase activity of the colonic microbiota The combination Jfi according to claim 1, which is a bond, the mulberry agent precursor 6a steroid The drug compositions include prednisolone and Dexaf 9. selected from the group consisting of methacin, hydrocortisone and fludrocortisone a pharmaceutical composition; the simple sugars are D-glucose, D-galactose and D-cello; A simple sugar selected from the group consisting of bioses! ll: Glycosida of the colonic microbiota -ase activity is β-galactosidase, α-galactosidase, β-glucosidase and β-cellobiosidase. The synthetic drug precursor according to claim 2, which is a glycosidase activity generated by quality composition. Can be essentially cleaved by the glycosidase enzymatic activity of the live colonic microbiota, but the receptor Not significantly hydrolyzed by endogenous enzymes produced by mammals, Therefore, the largest amount of free drug is due to glycosidases produced by the colonic microbiota. consisting of a drug glycoside that is released into the colon region after cleavage of the drug glycoside; Colon-specific synthetic drug precursor compositions. 5. The drug according to claim 4, wherein the drug glycoside is drug-β-D-glucoside. Drug glycosides listed. 6 Prednisolone-21-11-D-glucoside CPREDGLU) Synthetic drug precursor compositions. 7 A compound consisting of dexamethacin-21-β-D-glucoside CDEXAGLU) pharmaceutical precursor composition. a Synthetic drug precursor consisting of prednisolone-21-11-D-galactoside Composition. a Dexamethacin-21-β-D-galactoside to 80 A synthetic drug precursor composition. 1α Hydrocortisone-21-11-D-glucoside synthetic drug precursor quality composition.　 it Synthetic drug precursor consisting of hydrocortisone-21-β-D-galactoside quality composition. Synthetic drug precursor consisting of 1z fludrocortisone-21-β-D-glucoside Composition. la Synthetic drug precursor consisting of fludrocortinone-21-β-D-galactoside composition of matter. Synthetic drug precursor set consisting of 14 prednisolone-21-1-D-cellobioside A product. Administering the synthetic drug precursor composition to a 1fL recipient mammal through its gastrointestinal tract. 1. A method of applying a drug compound to the intestine of a mammal, comprising: The precursor composition consists of a combination of aglycones linked to sugars by glycosidic bonds. , where the aglycone is the pharmaceutical composition and the sugar is produced by the mammalian intestinal microflora. It is a sugar that can be recognized as a substrate by bacterial glycosidases produced by The cosidic bond is formed after the sugar is recognized as a substrate by the mvti glycosidase. Glycosyl acids that can be cleaved by the glycosidase enzyme activity of the mammalian intestinal microbiota A combination of an aglycone and a sugar linked to it by a glycosidic bond The drug precursor is further characterized in that the combination of drug precursors is present in the gastrointestinal tract of a mammal. without being significantly absorbed by the gastrointestinal tract or produced by the recipient mammal. of sufficient size to be able to pass through without being significantly hydrolyzed by endogenous enzymes. and hydrophilic, so that the drug precursor reaches the mammalian intestine, where it Glycosidic bonds on drug precursors are cleaved by the bacterial cricosidase and transferred to brain regions. A method consisting of releasing a free agent into the area. 1a administering the synthetic drug precursor composition to a recipient mammal through its gastrointestinal tract. A method of applying a drug compound to the colon region of a mammal, comprising: The precursor composition consists of a combination of aglycones linked to sugars by glycosidic bonds. , in which the aglycone is the pharmaceutical composition and the sugar is a microorganism produced by the colonic microbiota. A sugar that can be recognized as a substrate by fungal glycosidases and forms a glycosidic bond. After the sugar is recognized as a substrate by the bacterial cricosidase, it is transferred to the colonic microbiota. A glycosidic bond that can be cleaved by glycosidase enzyme activity, forming an aglycone The drug precursor, which is a combination of and a sugar linked to it by a glycosidic bond, is Additionally, this combination of drug precursors significantly absorbs the gastrointestinal tract of mammals. or by endogenous enzymes produced by the recipient mammal. be of sufficient size and hydrophilicity to pass through without being hydrolyzed; The drug precursor reaches the colon region of the mammal, where the group on the drug precursor The lycosidic bond is cleaved by the bacterial glycosidase, releasing free drug into the colon region. 2 than giving out How to become.