US 2013 0089535A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0089535 A1 Yamashiro et al. (43) Pub. Date: Apr. 11, 2013 (54) AGENT FOR REDUCING ACETALDEHYDE Publication Classification NORAL CAVITY (51) Int. Cl. (75) Inventors: Kan Yamashiro, Kakamigahara-shi (JP); A68/66 (2006.01) Takahumi Koyama, Kakamigahara-shi A638/51 (2006.01) (JP) A61O 11/00 (2006.01) A638/44 (2006.01) Assignee: AMANOENZYME INC., Nagoya-shi (52) U.S. Cl. (73) CPC. A61K 8/66 (2013.01); A61K 38/44 (2013.01); (JP) A61 K38/51 (2013.01); A61O II/00 (2013.01) (21) Appl. No.: 13/703,451 USPC .......... 424/94.4; 424/94.5; 435/191: 435/232 (22) PCT Fled: Jun. 7, 2011 (57) ABSTRACT Disclosed herein is a novel enzymatic agent effective in (86) PCT NO.: PCT/UP2011/062991 reducing acetaldehyde in the oral cavity. It has been found S371 (c)(1), that an aldehyde dehydrogenase derived from a microorgan (2), (4) Date: Dec. 11, 2012 ism belonging to the genus Saccharomyces and a threonine aldolase derived from Escherichia coli are effective in reduc (30) Foreign Application Priority Data ing low concentrations of acetaldehyde. Therefore, an agent for reducing acetaldehyde in the oral cavity is provided, Jun. 19, 2010 (JP) ................................. 2010-140O26 which contains these enzymes as active ingredients. Patent Application Publication Apr. 11, 2013 Sheet 1 of 2 US 2013/0089535 A1 FIG 1) 10.5 1 0 9.9.5 8. 5 CONTROL TA AD (BSA) ENZYME Patent Application Publication Apr. 11, 2013 Sheet 2 of 2 US 2013/0089535 A1 FIG 2) 110 the CONTROL (BSA) 100 354. 0.0O O 2 4 6 8 10 2 14 16 18 20 TIME (min) US 2013/0O89535 A1 Apr. 11, 2013 AGENT FOR REDUCING ACETALDEHYDE lizes the property of L-cysteine to bind to acetaldehyde. How NORAL CAVITY ever, this reaction is a reversible reaction, and therefore there is a possibility that acetaldehyde is again liberated. Further, TECHNICAL FIELD L-cysteine itself has a distinctive smell, which makes it dif ficult to apply L-cysteine to oral care products. On the other 0001. The present invention relates to an agent for reduc hand, the aldehyde oxidase derived from a microorganism is ing acetaldehyde in the oral cavity. More specifically, the advantageous in that it does not require a coenzyme, but there present invention relates to an agent that reduces acetalde is a problem that hazardous hydrogen peroxide is generated hyde in the oral cavity by an acetaldehyde-degrading enzyme by a reaction involving the aldehyde oxidase. Further, the and a use thereof. This application is based upon and claims reported aldehyde oxidase derived from a microorganism the benefit of priority from prior Japanese Patent Application exhibits activity under acidic conditions (at around pH 3.5), No. 2010-140026 filed on Jun. 19, 2010 in Japan, the entire but its reactivity under physiological conditions is unknown. contents of which are incorporated herein by reference. Furthermore, the confirmed reactivity of the enzyme is noth ing more than reactivity only toward relatively high concen BACKGROUND ART trations (in the order of mM) of substrate. The concentration 0002. In recent years, it has been reported that acetalde of acetaldehyde contained in saliva is usually in the order of hyde in saliva not only causes bad breath but also increases uM, but the reactivity of an enzymatic reaction is lowered the risk of diseases Such as upper digestive system cancers. when the concentration of a substrate is Kim or less. There Acetaldehyde in saliva is derived from various sources, but in fore, there is a demand for development of an enzyme that has recent years, acetaldehyde resulting from alcohol metabolism Sufficientactivity against low concentrations of substrate. It is during alcohol drinking or from Smoking or acetaldehyde to be noted that it has been reported that a superoxide dismu derived from periodontal pathogens in the mouth has been tase derived from a microorganism belonging to the genus known as a typical example and regarded as a problem. Gluconobacter exhibits acetaldehyde-degrading activity 0003. Among them, acetaldehyde produced as a metabolic (Patent Document 2). product in the course of alcoholysis accumulates in Saliva during alcohol drinking, which constitutes a risk factor for PRIOR ART DOCUMENTS esophageal cancer and stomach cancer. Particularly, it has been reported that in the case of flushers (people whose Patent Documents genome type of aldehyde dehydrogenase is AG or GG), the 0008 Patent Document 1: JP 2010-57482 A concentration of acetaldehyde in Saliva is increased up to almost 100 uM by alcohol drinking. 0009 Patent Document 2: JP 2010-110248 A 0004. It is also known that the concentration of acetalde Non-Patent Document hyde in saliva is increased by Smoking. It is considered that one of its causes is acetaldehyde contained in tobacco Smoke 0.010 Non-Patent Document 1: Salaspuro V. Hietala J, (Filtration of tobacco smoke (written by OtaniY), The history Kaihovaara P. et al., Int J. Cancer. 2002 Jan. 20; 97(3): of smoking research: 1996-2005, edited by Misu Y. Agari I, 361-4. Ohwada H. Nakao K, and Itani S, Smoking Research Foun dation, 2007). SUMMARY OF THE INVENTION 0005. A research group of Tokyo University has reported that drinking of one or more cans of beer per day and Smoking Problems to be Solved by the Invention makes the risk of esophageal cancer for flushers up to 190 0011. As has been described above, it has been found that times higher than that for people who are not flushers and do acetaldehyde in the oral cavity (in the mouth) poses a risk for not drink alcohol and smoke (Cui R, Kamatani Y, et. al. health problems, and therefore there is a demand for devel Functional variants in ADH1B and ALDH2 coupled with opment of oral care products that can degrade and remove alcohol and Smoking synergistically enhance esophageal can such acetaldehyde. It is therefore an object of the present cer risk. Gastroenterology 137:1768-1775 (2009)). invention to provide a novel enzyme preparation effective in 0006. It has been found by epidemiological studies that reducing acetaldehyde in the oral cavity. Some bacteria present in the mouth synthesize acetaldehyde and acetaldehyde derived from such bacteria is a cause of bad Means for Solving the Problems breath or a risk factor for upper digestive system cancers such as esophageal cancer and stomach cancer (which was 0012. The present inventors have intensively studied using reported at the annual meeting of the Japanese Cancer Asso artificial saliva to find enzymes effective in reducing acetal ciation in 2009). According to the results of an epidemiologi dehyde in the oral cavity. As a result, the present inventors cal study performed by Aichi Cancer Center Research Insti have found that an aldehyde dehydrogenase and a threonine tute (in Nagoya City) on about 3,800 people, people who aldolase exhibit excellent activity at low substrate concentra brush teeth twice or more a day have a lower risk for oral tions, which has led to the completion of the present inven cancer or esophageal cancer by 30% than people who brush tion. The present invention includes the following (1) to (10). teeth once a day. It is considered that the reason for this is that 0013 (1) An agent for reducing acetaldehyde in an oral oral bacteria that produce acetaldehyde and produced acetal cavity, including an aldehyde dehydrogenase or a threonine dehyde are washed away by tooth brushing. aldolase. 0007 Meanwhile, as means for removing acetaldehyde in 0014 (2) The agent for reducing acetaldehyde in an oral saliva or foods, one using L-cysteine (Non-Patent Document cavity according to (1), wherein the aldehyde dehydrogenase 1) and one using an aldehyde oxidase derived from a micro is derived from a microorganism belonging to the genus Sac organism (Patent Document 1) are known. The former uti charomyces. US 2013/0O89535 A1 Apr. 11, 2013 00.15 (3) The agent for reducing acetaldehyde in an oral Escherichia coli (see, for example, Eur. J. Biochem, 255, cavity according to (2), wherein the microorganism belong 220-226 (1998)) is preferably used. ing to the genus Saccharomyces is Saccharomyces cerevisiae. 0028. The concentration of acetaldehyde (substrate) at 0016 (4) The agent for reducing acetaldehyde in an oral which the acetaldehyde-reducing agent according to the cavity according to (1), wherein the threonine aldolase is present invention can act is not particularly limited, but is derived from Escherichia coli. preferably in the range of 1 uM to 1 mM, more preferably in 0017 (5) The agent for reducing acetaldehyde in an oral the range of 1 uM to 100 uM. It is to be noted that the strength cavity according to any one of (1) to (4), which exhibits (degree) of activity of the acetaldehyde-reducing agent degradation activity against acetaldehyde at a substrate con according to the present invention is not particularly limited. centration of 1 uM to 1 mM. 0029. The acetaldehyde-reducing agent according to the 0018 (6) The agent for reducing acetaldehyde in an oral present invention may contain, in addition to the active ingre cavity according to any one of (1) to (4), which exhibits dient (the aldehyde dehydrogenase or the threonine aldolase), degradation activity against acetaldehyde at a substrate con an excipient, a buffering agent, a suspension agent, a stabi centration of 1 uM to 100 uM. lizer, a pH adjuster, a preservative, an antiseptic, a perfume, a 0019 (7) An oral composition including the agent for thickener, fat and oil, a gloss agent, a binder, a binder rein reducing acetaldehyde in an oral cavity according to any one forcement, an emulsion stabilizer, a normal Saline solution, of (1) to (6).