US 20080268073A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0268073 A1 SANO et al. (43) Pub. Date: Oct. 30, 2008 (54) CERAMIDASE INHIBITOR (30) Foreign Application Priority Data (75) Inventors: Mutsumi SANO, Otsu-shi (JP): Nov. 28, 2003 (JP) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 2003-4OOO23 Hiroyuki IZu, Kusatsu-shi (JP): Nov. 26, 2004 (JP) - - - - - - - - - - - - - - - - - - - - - PCT/JP2004/017.609 Mitsuo Imamura, Otsu-shi (JP); Motoko Okamoto, Numazu-shi Publication Classification (JP); Toyohisa Kurita, Muko-shi (51) Int. Cl. (JP); Ikunoshin Kato, Koka-shi A636/00 (2006.01) (JP) CD7C 23.3/02 (2006.01) CI2N 9/99 (2006.01) Correspondence Address: BRCH STEWARTKOLASCH & BRCH (52) U.S. Cl. .......................... 424/725; 564/123; 435/184 PO BOX 747 (57) ABSTRACT FALLS CHURCH, VA 22040-0747 (US) The present invention provides a ceramidase activity inhibitor (73) Assignee: TAKARA BIO INC., Otsu-shi (JP) which inhibits ceramidase activity, specifically neutral/alka line ceramidase activity, characterized in that the inhibitor (21) Appl. No.: 12/147,798 comprises, as an active ingredient, a processed product derived from at least one plant selected from the group con sisting of plants belonging to Ginkgoaceae, plants belonging (22) Filed: Jun. 27, 2008 to Cucurbitaceae, plants belonging to Rutaceae, plants belonging to Laminariaceae, plants belonging to Myrtaceae Related U.S. Application Data and plants belonging to Compositae, and a medicament, a (62) Division of application No. 10/580,604, filed on May quasi-drug, cosmetics and a food, each comprising the inhibi 25, 2006. tOr. 2.O 5 1) 0.5 O.O. Wax gourd Bitter cucumber Mugwort Gourd Cucumber Sporophyll of Control wakame seaweed Patent Application Publication Oct. 30, 2008 Sheet 1 of 8 US 2008/0268073 A1 2.O 5 1) 0.5 0.0 Wax gourd Bitter cucumber Mugwort Gourd Cucumber Sporophyll of Control s wakame seaweed Patent Application Publication Oct. 30, 2008 Sheet 2 of 8 US 2008/0268073 A1 Fig. 2) A C18-SM B C6-Cer 8 7 m 6 5 OO S. 34 150 > 3 o? 100 a 2 Cld 50 O 0 O 2 3 O 2 3 4. Incubation time (h) Incubation time (h) C Cl8-Cer D Sph 20 6 18 2 16 5 814 i4 is 12 S S 0 s 3. S. 5 8 2 6 l 4 2 F O O O 1 2 3 O 1. 2. 3 4. Incubation time (h) Incubation time (h) E. SPP F Number of HL60 cells p gd s | U O 2 3 0 2 3 4 Incubation time (h) Incubation time (h) Patent Application Publication Oct. 30, 2008 Sheet 3 of 8 US 2008/0268073 A1 Fig. 3) 16 4 2 O 0.05% O. O.96 0.001% Control Fig. 4 1 O24. 8 6 4 2 O 0.05% 0.01% 0.001% Control Patent Application Publication Oct. 30, 2008 Sheet 4 of 8 US 2008/0268073 A1 Fig. 5) : Patent Application Publication Oct. 30, 2008 Sheet 5 of 8 US 2008/0268073 A1 098 008 ||'|93 ‘l992 º?900 '8990 °C900 900º? sdo "A Sueu Patent Application Publication Oct. 30, 2008 Sheet 7 of 8 US 2008/0268073 A1 Fig. 8) pp. 8 7 6 5 4 3 2 O Patent Application Publication Oct. 30, 2008 Sheet 8 of 8 US 2008/0268073 A1 Fig.9) ppg) 200 180 160 140 120 100 80 60 40 2O O US 2008/0268073 A1 Oct. 30, 2008 CERAMIDASE INHIBITOR to increase and allows sphingosine and sphingosine-1-phos phate level to lower is very useful as a medicine which regu lates cellular growth, differentiation and cell death. 0001. This application is a Divisional of co-pending appli 0005 Ceramide is synthesized de novo from L-serine and cation Ser. No. 10/580,604 filed on May 25, 2006 and for palmitoyl-CoA, and as a substance which inhibits a palmi which priority is claimed under 35 U.S.C. S 120. Application toyl-CoA: Serinepalmitoyl transferase, which is a starting Ser. No. 10/580,604 is the national phase of PCT Interna enzyme in this synthesis system, ISP-I (for example, Non tional Application No. PCT/JP2004/017609 filed on Nov. 26, Patent Publication 1) and sphingofungin B (for example, NPP 2004 under 35 U.S.C. S 371. This application also claims 2) have been known. As an inhibitor of acyl-CoA: Sphinga priority to JP 2003-400023, filed Nov. 28, 2003 in Japan. The nine N-acyltransferase, which is an enzyme for converting entire contents of each of the above-identified applications dihydrosphingosine into dihydroceramide, fumonisin B1 (for are hereby incorporated by reference. example, NPP 3) has been known. Any of these substances have been known to inhibit de novo synthesis of ceramide, TECHNICAL FIELD thereby reducing intracellular ceramide level. 0006. As a substance which increases ceramide level, on 0002 The present invention relates to a ceramidase activ the other hand, D-threo PDMP has been known as a substance ity inhibitor comprising a plant-derived processed product which inhibits the activity of UDP-glucose: ceramide gluco Such as an extract, and a medicament, a quasi-drug, cosmetics Syltransferase for transferring glucose to ceramide to synthe and a food, each comprising the inhibitor. size glucosylceramide (for example, NPP 4), and this sub stance has been known to cause an increase in intracellular BACKGROUND ART ceramide. However, this enzyme is a starting enzyme in bio 0003. In recent years, sphingolipid (sphingoglycolipid, synthesis of sphingoglycolipid, so that synthesis of sphin sphingomyelin and a metabolite thereof Such as ceramide) goglycolipid is also undesirably Supperssed. has begun to attract attention rapidly as a signal molecule 0007 As a metabolic enzyme which regulates the balance which regulates cellular growth, differentiation, apoptosis between ceramide and sphingosine or sphingosine-1-phos etc. In a disease Such as cancer, autoimmune disease or infec phate, the importance of neutral/alkaline ceramidase (for tion, sphingolipid metabolism is significantly changed, example, NPPs 5, 6 and 7) and alkaline ceramidase (for thereby attracting strong interest as a target of drug design. In example, NPP 8) have been remarked, and substances known particular, ceramide is a lipid serving as a basic structure of to inhibit the activity of these enzymes are few. Only sphingolipid and has been considered as an important factor D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol which regulates life and death of a cell. At least three path (D-e-MAPP), a ceramide analogue, has been known to inhibit ways for production of ceramide have been known at present. the activity of alkaline ceramidase to increase intracellular The three pathways are 1) de novo synthesis pathway begin ceramide level (see, for example, Patent Publication 1 and ning with condensation reaction of serine with palmitoyl NPP9). However, the inhibitory activity of this substance on CoA, 2) degradation system of sphingomyelin and 3) degra neutral/alkaline ceramidase is not strong and cannot be satis dation system of glucosylceramide. Among these pathways, factory. NPP 10 describes that another ceramide analogue, the degradation system of sphingomyelin is activated by a cell (1R,2R)-2-N-myristoylamino-1-(4-nitrophenyl)-1,3-pro death-inducing cytokine Such as TNF-C. and Fas, serum pandiol (D-NMAPPD, or B 13) efficiently inhibits acidic depletion, irradiation with ultraviolet rays or radioactive rays, ceramidase rather than alkaline ceramidase, and there is no or oxidation stress. In addition, production of ceramide via report on an effective inhibitor of neutral/alkaline cerami degradation of sphingomyelin is also promoted by a differ dase. NPP 9 describes that N-oleoylethanolamine has an entiation factor such as vitamin D3, interferon-Y or interleu inhibitory effect on acidic ceramidase, but this substance kin-1B. When a ceramide analogue C2-ceramide (D-erythro hardly exhibits an inhibitory effect on neutral/alkaline cera N-acetylsphingosine) is exogenously added to a cell, midase. PP2 discloses a ceramidase activity inhibitor com phenomena Such as apoptosis, differentiation induction and prising an extract of turmeric, Strawberry geranium, Gotukola growth Suppression are induced, or when a cell is treated with (Centella asiantica) or sea onion which Suppresses degrada bacterial sphingomyelinase, ceramide is accumulated in the tion of ceramide in a horny layer of the skin, and a skin cell by degradation of sphingomyelin, and cellular growth medicine for external application comprising these, but there Suppression and apoptosis are induced in the same way as is no description about the inhibitory effect of these plant when C2-ceramide is added. Therefore, intracellular ceram extracts on neutral/alkaline ceramidase, and the effect of ide is considered to function as an important intracellular increasing ceramide level in the skin is not sufficient and signal. cannot be satisfactory. 0004 Ceramide is degraded by the action of ceramidase 0008. On the other hand, ceramide is a major intercellular into a free fatty acid and a long-chain base sphingosine. lipid component in the skin and plays an important role in a Sphingosine is metabolized into sphingosine-1-phosphate moisture retention ability and barrier mechanism of the skin. through phosphorylation of a hydroxyl group at position 1. It In the skin horny layer of atopic dermatitis patients increasing has been known that sphingosine-1-phosphate, contrary to in number in recent years, the ceramide content has been ceramide, exhibits a cellular growth promoting action and reduced, which has been considered as one of the causes of Suppresses apoptosis induced by ceramide. As described dry skin and abnormality in barrier function of a horny layer above, in regulation of cellular growth, differentiation, cell considered characteristic of atopic dermatitis. It has been death etc., the balance between intracellular ceramide and a recently reported that ceramidase-producing bacteria are metabolite thereof Such as sphingosine or sphingosine-1- detected more frequently in a patient with atopic dermatitis phosphate has been considered very important.