JP3545601B2 - Lactosylceramide synthase inhibitor - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention is considered to be involved in the onset of arteriosclerosis, inflammation, cancer metastasis, adhesion of bacteria, viruses, toxins to cells involved in angiogenesis and infection, genetic diseases causing glycolipid metabolism abnormalities, cataracts, etc. Specific inhibitors of lactosylceramide synthase.
[0002]
[Prior art]
Lactosylceramide synthase acts on glycosphingolipid biosynthesis, which is said to be involved in various recognition mechanisms of cells. That is, it is said that lactosylceramide synthase transfers galactose from UDP-galactose to glucosylceramide to generate lactosylceramide, thereby starting to generate glycosphingolipids. For this reason, search and research for lactosylceramide synthase have recently been conducted.
[0003]
On the other hand, arteriosclerosis is a symptom in which arteries are hardened or contracted due to hypertension, abnormal endocrine function, abnormal lipid metabolism, and the like.Lactosylceramide synthase has recently been involved in the development of this arteriosclerosis. It is becoming clear.
[0004]
Recently, it has been recently revealed that cell surface glycolipids are involved in metastasis of cancer cells. For example, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), which inhibits glucosylceramide synthesis, further inhibits glucosylceramide synthesis in cancer cells. It is known that the biosynthesis of complex glycolipids is inhibited, and as a result, the metastasis of Lewis lung cancer cells (3LL cells) to the lung is inhibited (J. Inokuchi et al Cancer Research, 50, 6731-37, 1990). Furthermore, it is becoming clear that sialyl Lewis X sugar chains derived from the cell membrane of leukocytes are involved in processes such as leukocyte rolling, cell adhesion to endothelial cells, and tissue infiltration. Therefore, by inhibiting the synthesis of sugar chains on these cell surfaces, it is possible to suppress the metastasis of cancer and the early stage of the local tissue inflammatory reaction process, that is, rolling of leukocytes on the vascular endothelial cell membrane and infiltration into adherent extravascular tissues. It is expected to inhibit.
[0005]
For this reason, a substance that inhibits lactosylceramide synthase in the future, in particular, a galactose transferase which is common in transferring galactose from UDP-galactose (transfers galactose to a sugar chain of a glycoprotein. Yadav SP and Research on a substance that does not inhibit the activity of Brew KJ Biol. Chem. Jan. 1991, 266 (2) p. 698-703) but specifically inhibits only the activity of lactosylceramide synthase is needed. However, at present, almost no substance that specifically inhibits lactosylceramide synthase has been found so far.
[0006]
Therefore, an object of the present invention is to provide a specific inhibitor of lactosylceramide synthase.
[0007]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to achieve the above object and surprisingly found that N-dodecyldeoxynojirimycin, nojirimycin sulfite adduct, nojirimycin sulfite adduct which is known to have a glucosidase inhibitory action and the like The present inventors have found that sex derivatives and salts thereof specifically have an inhibitory action on lactosylceramide synthase, and completed the present invention.
[0008]
That is, the present invention provides a lactosylceramide containing as an active ingredient one or more selected from the group consisting of N-dodecyldeoxynojirimycin, nojirimycin sulfite adduct, nojirimycin sulfite adduct hydrophobic derivative and salts thereof. A synthase inhibitor is provided.
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
Nojirimycin has a structure in which an oxygen atom constituting a pyranose ring of D-glucose is substituted with NH. Such nojirimycin and its derivatives are conventionally known as antibiotics (Ishida N., Kumagai K., Niida T., Hakomoto K., Shomura T. J. Antibiot. Mar. 1967, 20 (2) p.62). -65, and Ishida N., Kumagai K., Niida T., Tsuruoka T., Yumoto H. J. Antibiot. Mar. 1967, 20 (2) p. 66-71).
[0010]
Nojirimycin and its derivatives are also known as various glycosidase inhibitors. For example, nojirimycin A is known to inhibit cancer metastasis by inhibiting α, β-glucosidase (Tsuruka T., Fukuyasu H., Ishii M., Usui T., Shibahara S., Inouye S. J. Antibiot. Feb. 1996, 49 (2) 155-161). It is known that castanospermine inhibits angiogenesis and suppresses tumor growth by inhibiting α-glucosidase (Pili R., Chang J., Partis RA, Mueller R.). A., Christ FJ, Paassanti A. Cancer Res. July 1995, 55 (13) p.2920-2926).
[0011]
N-butyldeoxynojirimycin has recently been shown to be an inhibitor of glycolipid biosynthesis (Platt FM, Neises GR, Dwek RA, and Butters TD). J. Biol.Chem.Mar.1994, 269 (11), p.8362-8365). That is, when crushed rat brain was used as the enzyme source of glucosylceramide synthase, 0.45 mM and 4.5 mM N-butyldeoxynojirimycin exhibited 21% and 57% glucosylceramide synthase inhibitory effects, respectively. Was. The application to the treatment of genetic diseases such as Tay-Sachs disease using such an inhibitory action has been reported (Platt FM, Nieses GR, Reinkensmeier G., Townsend MJ, Perry V.). H., Butters TD Science, Apr. 1997, 276 (18) p.428-431).
[0012]
Furthermore, N- (n-butyl) -deoxygalactonojirimycin inhibits α-D-galactosidase, deoxyfuconojirimycin hydrochloride inhibits α-L-fucosidase, and deoxygalactonojirimycin hydrochloride inhibits α-D-galactosidase. N-dodecyldeoxynojirimycin hydrochloride inhibits glucosidase, N-methyldeoxynojirimycin inhibits cleavage of N-linked glycoprotein by inhibiting glucosidase activity, and nojirimycin sulfite adduct inhibits several glucosidases. It is known to have an effect.
[0013]
However, N-dodecyldeoxynojirimycin, nojirimycin sulfite adduct, nojirimycin sulfite adduct hydrophobic derivatives and salts thereof do not inhibit galactose transferase and specifically inhibit lactosylceramide synthase. Not known at all.
[0014]
The nojirimycin sulfite adduct is one in which HSO ₃ — is bonded to the oxygen atom of the hydroxyl group (may be either α or β) at the carbon atom at the 1-position of nojirimycin. The nojirimycin bisulfite adduct hydrophobic derivative, N- alkyl Roh but Jiri mycin sulfite adduct, of which N-C ₁ -C ₁₂ alkyl deoxynojirimycin sulfite adducts are preferred.
[0015]
These salts include, but are not limited to, sodium, potassium, ammonium, triethanolamine, calcium, lithium salts and the like.
[0016]
Such N-dodecyldeoxynojirimycin and nojirimycin sulfite adducts can be synthesized according to a conventional method (for example, Inouye S., Tsuruoka T., Ito T., Niida T. Tetrahedron Mar. 1968, 24 (5)). pp. 215-2214). Also, commercially available products can be used. The hydrophobic derivative of nojirimycin sulfite adduct can be obtained, for example, by adding or substituting a hydrophobic group such as an alkyl halide to a nojirimycin sulfite adduct in a conventional manner.
[0017]
These lactosylceramide synthase inhibitors do not inhibit other galactosyltransferases but specifically inhibit lactosylceramide synthase. Therefore, it is useful as a drug for arteriosclerosis, a cancer metastasis inhibitor, an anti-inflammatory agent, a genetic disease therapeutic agent, a cataract preventive agent and the like, and other reagents.
[0018]
When the lactosylceramide synthase inhibitor of the present invention is used as a medicament, N-dodecyldeoxynojirimycin, nojirimycin sulfite adduct, nojirimycin sulfite adduct hydrophobic derivative or a salt thereof per adult per day The dosage is, for example, preferably 100 to 10,000 mg, particularly preferably 500 to 3000 mg.
[0019]
The medicament containing the lactosylceramide synthase inhibitor of the present invention can be administered either externally or internally, but is preferably used internally. The lactosylceramide synthase inhibitor of the present invention can be formulated into solid preparations such as powders, granules, capsules, pills and tablets, or liquids such as solutions, suspensions and emulsions. In formulating, commonly used excipients, binders, disintegrants, lubricants, coating agents, bases, suspending agents, emulsifiers, humectants, preservatives, stabilizers, surfactants , A flavoring agent, etc., and can be produced according to a conventional method.
[0020]
【Example】
Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.
[0021]
Reference Example 1
Method for producing lactosylceramide synthase (1) Method for preparing rat brain membrane fraction:
All operations were performed at 0-4 ° C. Two- to three-week-old Wistar rats (mixed male and female) were sacrificed by cervical dislocation, and the brain was removed. The excised brain was stored at -80 ° C until use.
To collect the rat brain membrane fraction, the brain was minced with scissors and homogenized by adding 9 volumes of a homogenizing buffer (0.32 M sucrose, 0.25 mM DTT, 1 mM EDTA). The supernatant obtained by centrifuging the homogenate at 800 × g was further centrifuged at 10,000 × g for 30 minutes. The resulting precipitate was added to a suspension buffer (0.25 M sucrose, 1 mM DTT, 0.02% azide). It was suspended in 50 mM Tris-HCl buffer (pH 7.4) containing sodium chloride and protease inhibitor (PMSF) to obtain a rat brain membrane fraction. Stored at -145 ° C until used for experiments.
[0022]
(2) Solubilization of enzyme:
Triton X-100 is added to the suspended brain membrane fraction so that the final concentration of the protein is 4 mg / ml and Triton X-100 is 1%, and the mixture is left at 0 ° C. for 2 hours, and then centrifuged at 100,000 × g. Separated, and the obtained supernatant was used as a solubilized fraction.
[0023]
(3) Affinity chromatography with WGA (Wheat Germ Lectin) -agarose:
Equilibrated with 10 mM Tris-HCl buffer pH 7.4 containing 500 mM sodium chloride, 1% Triton X-100, 1 mM DTT, 0.02% sodium azide, 100 μM pefablock (protease inhibitor). After applying a solubilized fraction prepared from about 150 rat brains to a wheat germ lectin-agarose column (150 ml), the column was washed with about 10 times the column volume of the same buffer and containing 200 mM GlcNAc. The enzyme was eluted with the same buffer.
[0024]
(4) Affinity chromatography with UDP-hexanolamine agarose:
After adding MnCl ₂ to the enzyme sample specifically eluted from WGA-agarose to 10 mM, 500 mM sodium chloride, 10 mM MnCl ₂ , 1% Triton X-100, 1 mM DTT, 0.02 The mixture was applied to a column (40 ml) of UDP-hexanolamine agarose equilibrated with 10 mM Tris-HCl buffer pH 7.4 containing 100% sodium azide, 100 μM pefabloc. After washing away the non-adsorbed fraction, the enzyme was eluted with the same buffer containing 1 mM UDP.
[0025]
(5) Hydroxyapatite column chromatography:
The sample eluted from the UDP-hexanolamine agarose column was replaced with 1% Triton X-100, 1 mM DTT, 0.02% sodium azide to exchange the sample buffer of (4) for a phosphate buffer. The protein was applied to a PD-10 column equilibrated with 10 mM sodium phosphate pH 6.8 containing 100 μM pefablock, and eluted with the same buffer to obtain a protein fraction. This was applied to a hydroxyapatite column (0.5 ml) equilibrated with the same buffer, and the non-adsorbed fraction having enzyme activity was used in the next step.
[0026]
(6) Ion exchange chromatography with Mini Q:
A mini-Q column (Pharmacia) in which the sample of (5) was equilibrated with 10 mM sodium phosphate pH 6.8 containing 1% Triton X-100, 1 mM DTT, 0.02% sodium azide, and 100 μM pefablock. And the enzyme was eluted by a linear concentration gradient method of 0 to 2 M sodium chloride, and a fraction having enzyme activity was collected.
[0027]
The obtained lactosylceramide synthase had the following enzymatic properties. (1) Action Transfer galactose from uridine diphosphate-galactose to glucosylceramide to synthesize lactosylceramide.
(2) Substrate specificity Strongly acts on glucosylceramide, weakly acts on globoside, and hardly acts on ceramide, lactosylceramide, GA2 and GM2.
(3) Metal requirements Require manganese, magnesium and calcium.
(4) Optimum pH
pH around 6.4-7.4 (5) Molecular weight 54,000-68,000 (by SDS-PAGE)
[0028]
Test example 1
The following method of Trincera et al. (Trinchera M. et al .: Biochemistry 30, 2719-2724 (1991)) was modified by the method of nojirimycin sulfite adduct, its acetylated derivative, and N-dodecyldeoxynojirimycin. The lactosylceramide synthase inhibitory effect was measured. That is, the nojirimycin sulfite adduct was dissolved in an organic solvent (chloroform: methanol = 2: 1) in five test tubes in such amounts that the final concentrations were 0 mM, 0.1 mM, 0.2 mM, 1 mM, and 5 mM, respectively. 20 μg of glucosylceramide and Triton X-100 dissolved in methanol were mixed in an amount to give a final concentration of 0.4% and mixed well. Next, the organic solvent was removed under a nitrogen stream to form a lipid membrane, which was then left under vacuum for 1 hour or more. To this, 1.0 μmol of MnCl ₂ , 750 nmol of CDP-choline, and 20.0 μmol of sodium cacodylate (Sodium Cacodylate, pH 7.2) as a buffer were added, followed by stirring and ultrasonic treatment. Furthermore, 0.15 mg of the purified rat brain lactosylceramide synthase (lactosylceramide synthesizing activity of about 0.4 pmol / min), 0.15 mg of DOPC, and 2.1 nmol of UDP- [U- ^14C ] galactose ( 23 kBq) to a final volume of 100 μl. After adding UDP- [U- ^14C ] galactose, the mixture was incubated at 37 ° C for 30 minutes. Thereafter, 1 ml of methanol was added to stop the reaction. 0.3 ml of distilled water and 0.5 ml of chloroform were added thereto, and the total lipid in the reaction solution was extracted by the Bligh and Dyer methods. The amount of radiolabeled lactosylceramide in the total lipid fraction was measured by measuring the radioactivity using a Packard top count, and the lactosylceramide synthase activity was calculated. The results are shown in FIG. FIG. 1 shows lactosimilar concentrations of nojirimycin sulfite adducts and their acetylated derivatives at 0 mM, 0.1 mM, 0.2 mM, 1 mM and 5 mM, and at a N-dodecyldeoxynojirimycin concentration of 5 mM. It shows the activity of silceramide synthase.
[0029]
From FIG. 1, it was confirmed that lactosylceramide synthase activity was inhibited by the addition of the nojirimycin sulfite adduct and its hydrophobic derivative, N-dodecyldeoxynojirimycin.
[0030]
Test example 2
The test that the inhibitory effect of the lactosylceramide synthase inhibitor of the present invention is specific to lactosylceramide synthase was performed according to the method of Hill et al. (Trayer IP and Hill RL). J. Biol. Chem. Nov. 1971, 246 (21), pp. 6666-6675). That is, 0 mM or 5 mM nojirimycin sulfite adduct, 0.2 M cacodylate buffer (pH 7.2), 100 μM glucose, 0.5 mg / ml α-lactalbumin, 0.3 mg / ml bovine Serum albumin, 20 μM MnCl ₂ , 23 kBq ¹⁴ C-UPD-galactose (11.3 GBq / mmol), and 20 units of bovine milk-derived galactose transferase (1 unit corresponds to 1 μmol / min) were added to a final volume of 100 μl at 37 ° C. The reaction was performed for 10 minutes. Unreacted ¹⁴ C-UPD-galactose was removed using a small column of Dowex1-X8, and the radioactivity of the product was measured using a liquid scintillation counter. FIG. 2 shows the results. FIG. 2 shows the activity of galactose transferase when the concentration of the nojirimycin sulfite adduct was 0 mM and 5 mM.
[0031]
As shown in FIG. 2, the nojirimycin sulfite adduct did not inhibit the activity of galactose transferase derived from bovine milk at a concentration of 5 mM, and the lactosylceramide synthase inhibitor of the present invention specifically inhibited lactosylceramide synthase. Was confirmed.
[0032]
【The invention's effect】
The lactosylceramide synthase inhibitor of the present invention can specifically inhibit the activity of lactosylceramide synthase without inhibiting the activity of another enzyme having substrate similarity.
[Brief description of the drawings]
FIG. 1: Lactosyl when the concentration of nojirimycin sulfite adduct and its acetylated derivative is 0 mM, 0.1 mM, 0.2 mM, 1 mM and 5 mM, and when the concentration of N-dodecyldeoxynojirimycin is 5 mM. It shows the activity of ceramide synthase.
FIG. 2 shows the activity of galactosyltransferase when the concentrations of nojirimycin sulfite adducts are 0 mM and 5 mM.

Claims (1)

A lactosylceramide synthase inhibitor comprising, as an active ingredient, one or more selected from the group consisting of N-dodecyldeoxynojirimycin, nojirimycin sulfite adducts, nojirimycin sulfite adduct hydrophobic derivatives and salts thereof.

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