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Home , Xylose isomerase

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(11) EP 2 289 352 A2

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication: (51) Int Cl.: 02.03.2011 Bulletin 2011/09 A23L 1/308 (2006.01) A61K 38/44 (2006.01)

(21) Application number: 10010195.5

(22) Date of filing: 20.02.2008

(84) Designated Contracting States: (72) Inventors: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR • Wyrobnik, Daniel Henry HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT 60322 Frankfurt (DE) RO SE SI SK TR • Wyrobnik, Isaak Harry Designated Extension States: 60431 Frankfurt (DE) AL BA MK RS (74) Representative: Hutchins, Michael Richard (30) Priority: 20.02.2007 DE 102007008664 M.R. Hutchins & Co 33 Connaught Way (62) Document number(s) of the earlier application(s) in Tunbridge Wells accordance with Art. 76 EPC: Kent TN4 9QP (GB) 08707777.2 / 2 124 640 Remarks: (71) Applicant: Vitacare GmbH & Co. KG This application was filed on 22-09-2010 as a 60318 Frankfurt (DE) divisional application to the application mentioned under INID code 62.

(54) Agent for use in the case of intolerance

(57) The invention provides the use of an agent com- after the ingestion of a fructose-containing food or sub- prising for (a) reducing the bioavail- stance, but excluding the use of an agent containing 5- ability of fructose in the body of a human or animal or (b) D-fructose dehydrogenase. reducing the amount of fructose available to the human or animal body or to intestinal bacteria colonizing therein EP 2 289 352 A2

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 289 352 A2 2

Description drome. [0007] The symptoms of the widespread intestinal [0001] The subject matter of the invention is an agent fructose intolerance are different, and its incidence is for use in the case of fructose intolerance, which contains showing an increasing trend, especially in the western a compound that effects the conversion of fructose to 5 industrialized nations. It is caused by a disorder of fruc- glucose. The term fructose intolerance is used in the con- toseabsorption resultingfrom the impairment of transport text of this patent application to mean not only the med- processes in the mucosa of the small intestine. Those ically defined fructose intolerance and fructose metabo- affected suffer from unclear abdominal symptoms and, lism disorder (see below), but any form of health impair- as a result of the bacterial breakdown of the carbohy- ment and complaints that occur as a result of the intake 10 drates passing into the colon, the production of intestinal of fructose or fructose containing foodstuffs, or due to gases is increased. The symptoms include, e.g. a feeling the release of fructose in the digestive tract of humans of bloating, flatulence, colic- like stomachache, watery di- or animals from other substances, such as sucrose. arrhea, and bowel sounds. This is often incorrectly diag- [0002] According to the present invention, the terms nosed as irritable colon. "food" and "foodstuff" are used as synonyms. They mean 15 [0008] Fructose-1,6-diphosphatase deficiency in- to also include feed in the sense of animal feed. volves a defect of this key in gluconeogenesis. [0003] Fructose is a ketohexose, of course, and is an This causes an increase in lactate levels in the blood important energy providing ingredient of food. It is after fructose exposure and fasting hypoglycemia, with present as a component of di- and oligosaccharides, but lactacidosis, seizures, muscular hypotension, and coma. also as free fructose in numerous foodstuffs. Food such 20 The development of fatty liver also leads to hepatomeg- as fruit and fruit juices contains large amounts of fructose, aly. but in particular also sucrose, which is cleaved to fructose [0009] Not all disorders of fructose metabolism neces- and glucose in the body. In the following, the term ’fruc- sarily lead to severe fructose intolerance. However, even tose containing’ is used to mean all substances and food- in mild disorders of fructose metabolism, health impair- stuffs that either contain fructose in pure form or from 25 ments are often to be observed, which could only be in- which fructose can be released in the digestive tract. The fluenced by a change of diet up to now. Excessive con- ’fructose content’ of substances and foodstuffs refers to sumption of fructose containing foodstuffs can also lead all the fructose in a fructose containing food or substance to health impairments. in whatever form (e.g. also as part of sucrose) it is con- [0010] The above-mentioned symptoms and com- tained in such a food or substance. 30 plaints could only be avoided up to now by maintaining [0004] In contrast to glucose, fructose is assimilated a fructose-, sucrose- and sorbitol-free diet. However, it into the mucosa cells of the small intestine by eased car- is very difficult for those affected to keep to such a diet, rier-mediated diffusion. The enzymatic degradation since fructose is contained in all fruits and many vegeta- starts in the liver by the action of the adenosine triphos- bles, and is widely used as a sweetener by the foodstuffs phate (ATP) dependent fructokinase, whereby fructose 35 industry. All foods that contain, e.g. sucrose (household is converted to fructose-1-phosphate. In the liver and in sugar) also have to be avoided. Such a diet, which is the kidneys, fructose- 1-phosphate is cleaved to glycerine indeed very strict in the case of hereditary fructose intol- aldehyde and dihydroxyacetone phosphate by aldolase erance, is not only difficult to keep to, it is also extremely B. unfavorable from a nutritional physiological point of view, [0005] Three different types of fructose metabolism 40 and considerably impairs the quality of life of those af- disorder are known in humans, namely hereditary fruc- fected. Not only those affected, but also the specialist tose intolerance, intestinal fructose intolerance, and fruc- community, consisting of doctors, specialists, nutritional tose-1,6-diphosphatase deficiency. In addition, there is scientists, nutritional advisers, specialist journalists, etc., fructosuria, which generally does not require treatment have assumed for decades that there is no alternative to according to current scientific thinking. 45 maintaining the diet described above. Research focused [0006] Hereditary fructose intolerance (HFI) results on an alternative to this diet has remained unsuccessful from a deficiency of aldolase B, an enzyme that occurs to date. An agent that would make it possible to do without in the intestinal mucosa, in the liver, in lymphocytes and maintaining such a diet and would allow the intake of in the kidneys. This enzyme usually breaks down fruc- fructose containing food would thus satisfy an urgent tose-1-phosphate to fructose-1,6-biphosphate via inter- 50 need for the many people affected that has existed for mediate stages. If an aldolase B deficiency is present, decades. It would overcome a prejudice that has been an excess of fructose-1- phosphate occurs, leading to an established in the specialist world and among those af- inhibition of glycogen breakdown and of gluconeogene- fected and mean a very considerable improvement and sis and, in turn, to severe hypoglycemia with outbreaks a dramatic step forward in the therapeutic and nutritional of sweating, tremor, vomiting and cramps after the intake 55 options in fructose intolerance, since, apart from main- of fructose. Acidosis, kidney damage and aminoaciduria taining a diet, there has simply been no therapy available can occur if this remains undetected. In infants, the risk up to now. Such an agent would also put an end to the ranges from hemorrhages to sudden infant death syn- as yet unsuccessful efforts of the specialist world to en-

2 3 EP 2 289 352 A2 4 able those affected to eat normally and to consume fruc- for use in the case of fructose intolerance, which contains tose containing meals without suffering side effects. The glucose isomerase. importance of such an agent becomes very clear if one [0018] A further subject matter of the invention is the also takes into consideration that those affected by he- use of glucose isomerase in the case of fructose intoler- reditary fructose intolerance are threatened by the se- 5 ance. verest and most dangerous consequences for their [0019] A further subject matter of the invention is the health if they, e.g. unknowingly, inadvertently or uninten- use of glucose isomerase for the production of a product tionally consume fructose. All of this would apply all the for the use in the case of fructose intolerance. more to an agent that additionally had no negative effects [0020] Glucose isomerase, which belongs to the group on health. 10 of , is an enzyme that has the property of [0011] Thus, it is an object of the present invention to converting D-fructose into D-glucose and vice versa. provide an effective agent that can be used not only in Here, an equilibrium of approximately 50 % glucose and milder disorders of fructose metabolism, but also in he- 50 % fructose is established, depending on ambient tem- reditary and intestinal fructose intolerance and in fruc- perature. Whereas fructose is only absorbed slowly from tose-1,6-diphosphatase deficiency, especially in order to 15 the small intestine, glucose is a sugar that is easily di- enable the consumption of normally fructose containing gested and rapidly absorbed. foodstuffs even if fructose intolerance is present. Further, [0021] The invention is thus based on ingested fruc- it is an object of the invention to make it possible for those tose being converted into glucose in vivo by glucose iso- affected by fructose intolerance to eat foodstuffs that they merase that is consumed simultaneously or at least were not allowed to eat up to now, due to their fructose 20 shortly before or thereafter. The enzyme then aims at content. Moreover, the objective is to provide an agent achieving the above-mentioned equilibrium by convert- that can reduce or prevent the occurrence of fructose ing fructose to glucose. However, glucose is absorbed intolerance symptoms after the intake of fructose. very rapidly, so that the equilibrium cannot be achieved. [0012] These objects are solved by the subject matter The enzyme continues to convert fructose still available as described in claims 1 to 40. 25 in the food pulp into glucose until no further fructose is [0013] Therefore, the subject matter of the invention left. The dose of glucose isomerase may be selected in is an agent that can solve all of the problems described such a way that, even if larger amounts of fructose are above. The agent contains glucose isomerase. A glucose consumed, the reaction can take place quickly enough isomerase in the sense of this invention is an enzyme that no fructose is absorbed or that the amount of fructose that is able to convert fructose into glucose. This conver- 30 absorbed in the meantime is too small to cause the known sion can also be achieved by a isomerase. In the gastrointestinal complaints in the case of mild fructose sense of this invention, a xylose isomerase is thus also metabolism disorders and intestinal fructose intolerance a glucose isomerase. A possible method for producing and the known systemic complaints in the case of hered- a xylose isomerase is described in Yamanaka, Biochim- itary fructose intolerance and fructose-1.6-diphosphate ica et Biophysika Acta, issue 151 (3), 1968, 670-680,35 deficiency. "Purification, Crystallization and Properties of the D-Xy- [0022] The enzyme 5- D-fructose dehydrogenase (syn. lose-Isomerase from Lactobacillus brevis" and in Ya- fructose 5-dehydrogenase) can bring about the conver- manaka, Methods in Enzymology, issue 41, 1971, sion of the fructose in food to 5-keto- D-fructose by dehy- 466-471, "D-Xylose Isomerase from Lactobacillus drogenation. Thus, the fructose is changed in such a brevis", whereby these documents are hereby incorpo- 40 manner that it is no longer available to the metabolism rated by reference. of the human or animal body. A 5-D-fructose dehydro- [0014] The agent according to the present invention genase in the sense of this invention is an enzyme that can bring about the conversion of the fructose in food or can bring about the dehydrogenation of fructose to 5- in food pulp into glucose. The fructose is thus no longer keto-D-fructose. Particularly favorable effects can there- available for the bacterial metabolism in the intestines 45 fore be achieved if glucose isomerase and 5-D-fructose characterized by fermentation, and an excess of fruc- dehydrogenase are used in combination. tose-1-phosphate in the liver or elsewhere can no longer A possible method for the production of a 5-D-fructose occur. This can also prevent an increase in lactate levels dehydrogenase is, for example, described in Ameyama in the blood. et al., Journal of Bacteriology 1981, 814-823, "D-Fruc- [0015] A subject matter of the invention is therefore an 50 tose Dehydrogenase of Gluconobacter industrius: Puri- agent that reduces the bioavailability of fructose in the fication, Characterization and Application of Enzymatic human or animal body with the help of glucose isom. Microdetermination of D-Fructose", the content of which erase. is incorporated herein by reference. [0016] A subject matter of the invention is also an agent [0023] A further subject matter of the present invention that, with the help of glucose isomerase, reduces the55 is therefore an agent that, beside glucose isomerase, amount of fructose available to the human or animal body additionally contains 5- D-fructose dehydrogenase. Such or to intestinal bacteria colonizing therein. a combination agent can also be used in the form of two [0017] A subject matter of the invention is also an agent separate dose units, e.g. two separate tablets, one of

3 5 EP 2 289 352 A2 6 which contains glucose isomerase and the other 5-D- erance. Particularly advantageously, a foodstuff can be fructose dehydrogenase. prepared by a method in which a glucose isomerase - [0024] According to the present invention, a glucose alone or in combination with 5-D-fructose dehydroge- isomerase alone or in combination with 5- D-fructose de- nase - is added to the foodstuff in a manner in which the hydrogenase can also be used to reduce the fructose 5 action of the glucose isomerase - alone or in combination content in a foodstuff. with 5-D-fructose dehydrogenase - only starts after the [0025] Foodstuffs in the sense of this invention also intake of the foodstuff. Such a foodstuff that contains glu- comprise, among other things, foodstuffs for particular cose isomerase or glucose isomerase and 5-D-fructose nutritional uses, foods for special medical purposes, dehydrogenase has the same taste as an untreated food- medical foods, food supplements, dietary supplements, 10 stuff and is, for the first time, suitable to be consumed by dietetic food supplements, health foods, nutraceuticals, persons suffering from fructose intolerance, due to the and food additives. reduced fructose content which is established after eat- [0026] In a particularly easy way, the invention facili- ing. tates the transformation of fructose in a foodstuff into a [0029] According to a further aspect, according to the form that avoids the problems that accompany fructose 15 present invention, a medical device is provided that con- intolerance. Thus, the invention also makes it possible tains glucose isomerase - alone or in combination with for people affected by fructose intolerance to consume 5-D-fructose dehydrogenase. Accordingly, a subject such foodstuffs, which had to be avoided up to now be- matter of the invention is also a medical device which cause of their fructose content. consists of glucose isomerase - alone or in combination [0027] According to the present invention, glucose iso- 20 with 5-D-fructose dehydrogenase - or contains it along merase - alone or in combination with 5-D-fructose de- with one or more other active ingredients - alone or in hydrogenase - is further mentioned for use in medicine, combination with 5-D-fructose dehydrogenase. In the for example as a pharmaceutical composition. Accord- sense of this invention a "medical device" means any ingly, a subject matter of the invention is also a product instrument, apparatus, appliance, material or other arti- which consists of glucose isomerase - alone or in com- 25 cle, whether used alone or in combination, including the bination with 5-D-fructose dehydrogenase - or contains software necessary for its proper application intended by it - alone or in combination with 5- D-fructose dehydroge- the manufacturer to be used for human beings for the nase - along with one or more other active ingredients, purpose of: for use in a medical method, in particular in a method for the therapeutic treatment of the human or animal body. 30 - diagnosis, prevention, monitoring, treatment or alle- In the sense of this invention, a pharmaceutical compo- viation of disease, sition is a product, in particular a substance or a sub- - diagnosis, monitoring, treatment, alleviation of or stance mixture, for use in a method for surgical or ther- compensation for an injury or handicap, apeutic treatment of the human or animal body and in - investigation, replacement or modification of the diagnostic methods that are performed on the human or 35 anatomy or of a physiological process, animal body. Thus, in the sense of the invention, phar- - control of conception, maceutical compositions are also products, in particular substances or substance mixtures, that are intended or and which does not achieve its principal intended action suitable for curing, alleviating, preventing or determining in or on the human body by pharmacological, immuno- fructose intolerance. 40 logical or metabolic means, but which may be assisted [0028] According to a further aspect of the present in- in its function by such means; vention, a foodstuff is provided which contains glucose Any instrument, apparatus, appliance, material or other isomerase - alone or in combination with 5-D-fructose article that does not achieve its intended action in or on dehydrogenase. Further, according to the present inven- the human body is not a medical device in the sense of tion, a foodstuff is provided that contains glucose isomer- 45 this application. ase - alone or in combination with 5- D-fructose dehydro- [0030] In the following, the invention will be described genase - in an amount which is sufficient to convert fruc- further in its various aspects. If the term agent is used tose into glucose. Such a foodstuff may be produced ad- below, this always also stands for a foodstuff, a medical vantageously using a method for treating a foodstuff in device or a pharmaceutical composition. which the foodstuff is placed in contact with a glucose 50 [0031] 5-D-Fructose dehydrogenase is a compound isomerase - or with a glucose isomerase in combination that has been known for nearly 40 years, but has only with a 5-D-fructose dehydrogenase - under such condi- been used for analytical purposes to date. Glucose iso- tions under which the glucose isomerase - alone or in merase is a compound that has been known for more combination with the 5-D-fructose dehydrogenase - can than 40 years and has only been used for saccha- convert fructose to glucose. In contrast to otherwise un- 55 rification to date. In the industry, it is used for the conver- treated foodstuffs, such a foodstuff has a reduced fruc- sion of glucose into fructose as well as for the conversion tose content and therefore, for the first time, is suitable of fructose into glucose. to be consumed by persons suffering from fructose intol- [0032] Until now, glucose isomerase has not been

4 7 EP 2 289 352 A2 8 used and particularly not in combination with 5-D-fruc- the present invention could, e.g. be mixed into or sprin- tose dehydrogenase in the medical/pharmaceutical field, kled onto them after cooling. in particular in the case of fructose intolerance in humans [0035] Since fructose is widely used as a sweetener or animals. Thus, the invention disclosed herein is the in foodstuffs that are especially produced for diabetics, first medical indication for glucose isomerase and the 5 the addition of the agents according to the present inven- first medical indication for the combination of the two en- tion to diabetic food before eating or the addition of the zymes. Until now, the two have not been used agents according to the present invention during the pro- for the therapeutic treatment of the human or animal body duction of diabetic food is especially advantageous, to or for diagnostic purposes on the human or animal body. allow diabetics who suffer from fructose intolerance to [0033] The agents according to the present invention 10 eat diabetic food, such as the above mentioned food- can be taken orally before meals, with meals or immedi- stuffs in their respective form as diabetic foods. ately after meals, so that they can exert their converting [0036] The agents according to the present invention or also dehydrogenating effect on fructose in the food can also be added to a foodstuff to exert their effect on pulp. Preferably, the agents according to the present in- the fructose originating from another foodstuff. An exam- vention are taken just before meals, during meals or im- 15 ple of this would be the addition of the agents according mediately after meals. The agents according to the to the present invention to a spread so that the reduction present invention may contain the enzyme without further of the utilizable fructose contained in the bread occurs additives. However, it is preferable that the agents ac- after the intake of the bread, without impairing the taste cording to the present invention further contain additives of the bread. Another example would be mixed spices. that are pharmaceutically acceptable and/or acceptable 20 [0037] A subject matter of the present invention are for foodstuffs, such as extenders, binders, stabilizers, also agents that in addition to other active ingredients preservatives, flavorings, etc. Such additives are com- also contain glucose isomerase, either alone or in com- monly used and well known for the production of phar- bination with 5-D-fructose dehydrogenase. maceutical compositions, medical devices, foodstuffs, [0038] The invention may be formulated in any form foodstuffs for particular nutritional uses, foods for special 25 which is suitable for the intended route of administration. medical purposes, medical foods, food supplements, di- For oral administration, the agents according to the etary supplements, dietetic food supplements, health present invention are preferably formulated in the form foods, nutraceuticals, and food additives and specialists of capsules (coated or non-coated), tablets (coated or in this field know which additives in which amounts are non-coated), capsules containing coated or non-coated suitable for particular presentation forms. Particularly30 pellets, granules, or micro- or mini-tablets, tablets preferably, the agents according to the present invention pressed from coated or non-coated pellets, dragees, or contain as additives dicalcium phosphate, lactose, mod- micro- or mini-tablets, gel caps or, in liquid form, as a ified starch, microcrystalline , maltodextrin solution, drops, suspension or gel. The formulation of the and/or fibersol. agent according to the present invention as a powder is [0034] The agents according to the present invention 35 particularly suitable for an admixture to a foodstuff. The can also be added to a foodstuff before eating. They can powder may be sprinkled onto a meal or it may be mixed even be added to the foodstuff at the production stage, into a pulp or a beverage. It is particularly suitable if the with the aim of developing their effect only after consum- agent offered as bulk powder is packed in single dosage ing the foodstuff. This could possibly be achieved by mi- amounts, such as in single bags or capsules, or if it is croencapsulation, for example. In this way, the useable 40 provided in a dosing apparatus. It is especially preferable fructose content of the foodstuff would be reduced in a if the agents according to the present invention are for- particularly advantageous way, without negatively affect- mulated as a powder or as granules in capsules or as a ing its taste. Therefore, preparations containing glucose tablet that are administered orally. isomerase - alone or in combination with 5-D-fructose [0039] For oral administration, the active ingredient dehydrogenase - are preferred that do not release this 45 glucose isomerase alone or in combination with 5- D-fruc- enzyme or these enzymes until they reach the digestive tose dehydrogenase may be contained in acceptable ex- tract of a human or animal or let them become effective cipients and/or carriers. The term "acceptable carrier" re- in another way, especially in the stomach or small intes- lates to a carrier for pharmaceutical use which directs tine. Therefore, the invention could be used for example the active ingredient to its target site and which does not in the production of sweets, fruit preparations (e.g. apple 50 have negative effects for the recipient, human or animal. sauce), jam, honey, chocolate and rations (e.g. apple However, the exact form of the carrier is not decisive. sauce), jam, honey, chocolate and chocolate products, [0040] The total amount of the carrier and/or excipients bakery products (e.g. biscuits and cakes), breads, pas- of an agent containing glucose isomerase or glucose iso- tas, vegetable dishes, potato dishes, ice cream, cereals, merase and 5-D-fructose dehydrogenase is preferably dairy products (e.g. fruit yogurt and pudding), fructose 55 between 5 and 99.9 % by weight, more preferably be- containing beverages, fructose containing sauces (e.g. tween 10 and 95 % by weight and even more preferably tomato ketchup) and fructose containing sweeteners. For between 25 and 90 % by weight of the composition. dishes that are boiled or baked, the agents according to [0041] Suitable excipients and/or carriers include mal-

5 9 EP 2 289 352 A2 10 todextrin, calcium carbonate, dicalcium phosphate, tri- col, lanolin, glyceryl stearate, sorbitan stearate, isopropyl calcium phosphate, microcrystalline cellulose, dextrose, myristate, isopropyl palmilate, acetone, glycerine, phos- rice flour, magnesium stearate, stearic acid, croscarmel- phatidylcholine, sodium cholate or ethanol, but are not lose sodium, sodium starch glycolate, crospovidone, su- limited thereto. crose, vegetable gums, lactose, methyleellulose, povi- 5 [0043] The compositions for use in the present inven- done, carboxymethyl cellulose, corn starch, modified tion may also comprise at least one coemulsifying agent, starch, fibersol, gelatine, hydroxypropylmethyl cellulose which includes oxyethylenated sorbitan monostearate, and the like (including mixtures thereof). Preferable car- fatty alcohols, such as stearyl alcohol or cetyl alcohol, or riers include calcium carbonate, magnesium stearate, esters of fatty acids and polyols, such as glyceryl stear- maltodextrin, dicalcium phosphate, modified starch, mi- 10 ate, but is not limited thereto. crocrystalline cellulose, fibersol, gelatine, hydroxypropyl- [0044] Preferably, the agents to be used in the present methyl cellulose and mixtures thereof. The various ingre- invention are provided in a stabilized form. Generally, dients and the excipient and/or carrier are mixed and stabilizationmethods and procedures which may be used formed into the desired form using common methods. according to the present invention include any and all The presentation form which is intended for oral admin- 15 methods for the stabilization of chemical or biological ma- istration according to the present invention, such as a terial which are known in the art, comprising e.g. the ad- tablet or capsule, may be coated with a coating that is dition of chemical agents, methods which are based on resistant to low pH values. This makes it possible for the temperature modulation; methods which are based on enzyme or enzymes to be released only when they reach irradiation or combinations thereof. Chemical agents that the small intestine. Also a coating may be used which is 20 may be used according to the present invention include, not resistant against low pH values but which provides among others, preservatives, acids, bases, salts, anti- delayed release of the respective enzyme at low pH val- oxidants, viscosity enhancers, emulsifying agents, gelat- ues. It is also possible to prepare the agent according to inizers, and mixtures thereof. the present inventionas coated (seeabove) pellets, gran- [0045] Conventionally, the industrial production of en- ules, or micro- or mini-tablets which can be filled into non- 25 zymes is performed in a technical fermentation way using coated capsulesor which canbe pressed into non-coated suitable microorganisms (bacteria, moulds, yeasts). The tablets. Suitable coatings are, for example, cellulose ac- strains are recovered from natural ecosystems according etate phthalate, cellulose derivatives, shellac, polyvi- to a special screening protocol, isolated as pure cultures nylpyrrolidone derivatives, acrylic acid, polyacrylic acid as well as improved in their properties with respect to the derivatives and polymethyl methacrylate (PMMA), such 30 enzyme spectrum and biosynthesis performance (vol- as Eudragit® (from Röhm GmbH, Darmstadt), in partic- umeltime yield). The enzymes can also be produced us- ular Eudragit® FS30D (releases the active constituent or ing methods to be developed in the future. constituents starting at a pH of around 6.8) and Eudragit® [0046] Glucose isomerase is commercially available L30D-55 (releases the active constituent or constituents (e.g. Novozymes A/S, Denmark and Danisco, Denmark) starting at a pH of around 5.5). If it is desired to release 35 and is usually prepared in a microbiological way with the the enzyme(s) already at a lower pH value, this may be help of the microorganism Streptomyces murinus. 5-D- achieved e.g. by the addition of sodium hydroxide solu- fructose dehydrogenase is also commercially available tion to the coating agent Eudragit® L30D- 55, because in (e.g. Sigma-Aldrich and Toyobo, Japan) and is conven- this case carboxyl groups of the methacrylate would be tionally prepared in a microbiological way with the help neutralised. Therefore, this coating will be dissolved, for 40 of the microorganism Gluconobacter industrius. Howev- example, already at a pH value of 4.0 provided that 5 % er, the invention is not limited to the enzymes that are of the carboxyl groups are neutralised. The addition of commercially available at the moment, but generally re- about 100 g of 4 % sodium hydroxide solution to 1 kg of lates to enzymes that can bring about the conversion of Eudragit® L30D-55 would result in a neutralisation of fructose - specifically or non-specifically - to glucose or about 6 % of the carboxyl groups. Further details about 45 5-keto-D-fructose. A person skilled in the art can prepare formulation methods and administration methods can be suitable further enzymes by conventional methods, for found in the 21st edition of "Remington: The Science & example by mutagenesis of the gene encoding glucose Practice of Pharmacy", published 2005 by Lippincott, Wil- isomerase which is present in Streptomyces murinus or liams & Wilkins, Baltimore, USA, in the Encyclopedia of by mutagenesis of the gene encoding 5-D-fructose de- Pharmaceutical Technology (Editor James Swarbrick) 50 hydrogenase which is present in Gluconobacter indus- and in Prof. Bauer "Lehrbuch der Pharmazeutischen trius. The enzymes may also be prepared with the help Technologie", 18th edition, published 2006 by Wissen- of other microorganisms, such as fungi, in sufficient schaftliche Verlagsgesellschaft (ISBN 3804-72222-9). amounts and the required purities, also by the use of The contents of thesedocuments are incorporated herein genetic engineering methods which are common today. by reference. 55 If it is desired e.g. to produce the enzymes with other [0042] Other suitable pharmaceutically acceptable microorganisms, the genetic information of a microor- carriers or excipients for use in the present invention in- ganism which has been found initially by extensive clude water, mineral oil, ethylene glycol, propylene gly- screening and which has also been proven as a suitable

6 11 EP 2 289 352 A2 12 source of the enzyme with the desired properties can be ance, and fructose- 1,6-diphosphatase deficiency, in their transferred to a microorganism which is normally used range of different severities, and also in milder fructose for the production of enzymes. Also the modification of metabolism disorders. Furthermore, the different dosag- the enzyme(s) and the production of the enzyme(s) by es also result from the fact that strongly varying amounts means of methods which are presently known or may be 5 of fructose are administered to the body, depending on developed in the future in the area of industrial enzyme the respective food. The enzyme or the enzymes should development and enzyme production, such as genetic be used in a sufficient quantity so that it or they develop engineering, is possible, The use and the manner of per- a sufficiently high enzyme activity, in other words suffi- forming all these methods for developing and producing cient glucose isomerase to convert an amount of fructose the enzyme(s) with the desired purities and activities and 10 consumed in a normal meal (e.g. 10 - 50 g) - in free or with the desired properties, in particular with respect to bound form - into glucose. the stability of the enzyme(s) at various pH values, re- [0051] The agent according to the present invention garding the optimum of the pH value, the stability at var- may comprise one or more additional enzymes, such as ious temperatures and temperature optimum, are well (syn, beta-fructofuranosidase or beta-fructos- known to a person skilled in the art. The explanations in 15 idase), lactase (syn. beta-galactosidase), maltase (syn. chapter 2 (page 82 to page 130) of the textbook "Leb- alpha-glucosidase), alpha-, beta-amylase, glu- ensmittel-Biotechnologie und Ernährung" of Heinz Rutt- coamylase, pullulanase, isoamylase, amyloglucosidase, loff, Jürgen Proll and Andreas Leuchtenberger, pub- cyclomaltodextrin glucantransferase (CGTasey. These lished by Springer Verlag 1997 (ISBN 3-540-61135-5) enzymes have the property of releasing fructose and/or describe these methods in detail. These methods are 20 glucose from fructose and/or glucose containing sub- also described in "Advances in Fungal Biotechnology for stances and foodstuffs - alone or in combination with one Industry, Agriculture, and Medicine" by Jan S. Tkacz, or more of these enzymes -, whereby the enzymes pul- Lene Langeand try, Agriculture, and Medicine" by Jan S. lulanase and isoamylase also increase the efficiency of Tkacz, Lene Langeand (published in 2004, ISBN glucoamylase and beta-amylase. All these enzymes are 0-306-47866-8), in "Enzymes in industry: Production and 25 commercially available (e.g. BioCat Inc., Troy, USA or Applications" by Wolfgang Aehle (Editor), published in Novozymes A/S, Denmark or Amano Enzymes Inc., Ja- 2004, ISBN 3527295925 and in "Microbial Enzymes and pan or Sigma-Aldrich) and, up to now, have never been Biotransformations" by Jose-Luis Barredo (Humana used in combination with glucose isomerase - alone or Press 2005, ISBN 1588292533). These documents are in combination with 5- D-fructose dehydrogenase - in the herewith incorporated into the patent application by ref- 30 medical/pharmaceutical field, in particular not in the case erence. All this also applies to the enzymes mentioned of fructose intolerance. Thus this application discloses below that can optionally be added to the agent according the first medical indication for glucose isomerase - alone to the present invention. or in combination with 5-D-fructose dehydrogenase - in [0047] The activity of glucose isomerase is defined in combination with any or all of these enzymes. Examples units according to the present invention, whereby one 35 for agents according to the present invention include: unit is the amount of glucose isomerase that converts 1 g of fructose to glucose in 5 minutes at a pH of 7.5 and Glucose isomerase in combination with invertase, or a temperature of 37°C from an initially 10% solution by Glucose isomerase in combination with maltase, or weight (i.e. 10 g fructose + 90 g water). Glucose isomerase in combination with Invertase [0048] At an enzyme activity determined according to 40 and maltase, or glucose isomerase in combination this definition, the agent according to the present inven- with 5-D-fructose dehydrogenase and invertase, or tion should contain glucose isomerase in an amount or glucose isomerase in combination with 5- D-fructose activity of 0.01 to 100,000 GIU (= glucose isomerase dehydrogenase and maltase, or glucose isomerase units), preferably 0.05 to 10,000 GIU and particularly in combination with 5-D-fructose dehydrogenase preferably 0.1 to 1,000 GIU per dose unit. 45 and invertase and maltase. [0049] If the agent according to the present invention also contains 5-D-fructose dehydrogenase, this enzyme [0052] For example, said invertase can release fruc- should be present in an amount or activity of 10 to 5 tose from e.g. sucrose. million units, preferably 25 to 2.5 million units and partic- [0053] By the addition of one or more of these enzymes ularly preferably 50 to 1 million units per dose unit. One 50 to the agent according to the present invention, the en- unit of this enzyme is defined as the amount that converts dogenic release offructose from fructose- containingsub- one micromole of D-fructose into 5-keto-D-fructose per stances or foodstuffs, in particular from sucrose, may al- minute at pH 4.5 and 37°C. so be promoted and accelerated, so that the conversion [0050] The wide range of the above mentioned dos- of fructose into Glucose or into Glucose and 5-keto-D- ages may be explained by the fact that the agent(s) ac- 55 fructose may occur earlier. Therefore, the addition of one cording to the present invention can be applied in three or more of these enzymes to the agent according to the very different types of fructose intolerance, namely in he- present invention may have the benefit of reducing the reditary fructose intolerance, intestinal fructose intoler- required amount of glucose isomerase or glucose iso-

7 13 EP 2 289 352 A2 14 merase and 5-D-fructose dehydrogenase. units and particularly preferably between 50 and 25,000 [0054] The activity of invertase is measured in Sumner units per gram fructose in the foodstuff. units (SU, assay available e.g. from Bio-Cat Inc., Troy, [0062] The capsule sizes mentioned below refer to the Virginia, USA). An SU is defined as the amount of the sizes defined by Capsugel Belgium BVBA, Bomem, Bel- enzyme which converts 1 mg of sucrose into glucose and 5 gium. The size of capsules should be chosen in accord- fructose under standard test conditions within 5 minutes ance withthe specified formulation of the agentaccording at 20°C and a pH value of 4.5. If the agent according to to the present invention. the present invention also contains invertase, the activity [0063] An agent according to the present invention in of the invertase per dose unit should be between 50 and capsules (e.g. of size 3) could contain, e.g., 15 mg glu- 250,000 SU, preferably between 100 and 150.000 SU 10 cose isomerase (activity of glucose isomerase 1 GIU/mg) and particularly preferably between 150 and 100,000 SU and 135 mg dicalcium phosphate per capsule. per dose unit. [0064] If capsules, e.g. of size 1, are used, they may [0055] The activity of maltase is defined in units, contain 50 mg glucose isomerase (activity of glucose iso- wherein one unit is the amount of maltase which will con- merase 1 GIU/mg), 5 mg folic acid and 150 mg malto- vert maltose to D-glucose at a rate of one milligram per 15 dextrin per capsule. minute at 37°C and a pH of 4.0 in a 10% maltose solution [0065] A further example of a composition for the pro- by weight. duction of capsules consists of capsules of size 3 that [0056] Where the agent according to the present in- contain 15 mg glucose isomerase (activity of glucose iso- vention also contains maltase, the activity per dose unit merase 1 GIU/mg), 75 mg 5-D-fructose dehydrogenase should be between 100 and 100,000 units, preferably 20 (activity of 5- D-fructose dehydrogenase 90 units/mg) and between 200 and 50,000 units and particularly preferably 60 mg dicalcium phosphate per capsule. between 500 and 20,000 units. [0066] A further example of a composition for the pro- [0057] In the case of hereditary fructose intolerance, duction of capsules consists of capsules of size 00 that it is particularly preferable if, in addition to glucose iso- contain 300 mg glucose isomerase (activity of glucose merase and possibly also 5-D-fructose dehydrogenase, 25 isomerase 1 GIU/mg) and 170 mg dicalcium phosphate folic acid in an amount of 1 mg to 100 mg, preferably 2 per capsule. mg to 50 mg and particularly preferably 3 mg to 10 mg [0067] The invention may for example contain be- per dose unit are added to the agent (s) according to the tween 0.01 and 100,000 GIU (= glucose isomerase units) present invention, as folic acid increases aldolase B ac- and between 1 mg and 100 mg folic acid per dose unit. tivity. 30 In addition, suitable additives in the required amount may [0058] It may be advantageous to add an acceptor to be used. The invention may additionally contain between the agent according to the present invention, for example 10 and 5 million units of 5- D-fructose dehydrogenase per at a ratio (acceptor : substrate) of 1:1 to 1: 1,000, prefer- dose unit. ably at a ratio of 1:2 to 1:200 and particularly preferably [0068] The invention can be made available for med- at a ratio of 1: 10 to 1:50. Examples of suitable acceptors 35 ical purposes and non-medical purposes, e.g. as a phar- which may be used include NAD+, NADP+, FAD+, vita- maceutical composition, medical device, foodstuff, food- mins, such as vitamin C, vitamin E or vitamin A, ferric stuff for particular nutritional uses, food for special med- cyanide, ketones, aldehydes, 2,6-dichlorophenol-indo- ical purposes, medical food, food supplement, dietary phenol, phenazine methosulfate, nitroblue tetrazolium supplement, dietetic food supplement, health food, nu- (including mixtures thereof), but are not limited thereto. 40 traceutical, and food additive. [0059] It may also be advantageous to add metal ions [0069] The agents according to the present invention to theagent according tothe present invention, especially can be used to considerably alleviate or eliminate the cations such as Mn2+, Mg2+, Ca2+, Zn2+, Fe2+, Co2+ or symptoms and health impairments caused by fructose Cu2+, including mixtures thereof, preferably at a molar intolerance. The invention presented here is suitable for ratio of 10-6 to 10-2. For the (xylose) glucose isomerase 45 use in the case of fructose intolerance and for the ther- from Yamanaka described above, a particularly suitable apeutic treatment of fructose intolerance. cation is Mn2+. [0060] If the agent according to the present invention is added to a foodstuff before eating or during production, Claims the activity of glucose isomerase should be between 0.01 50 and 20,000 units, preferably between 0.05 and 10,000 1. The use of an agent comprising glucose isomerase units and particularly preferably between 0.1 and 1,000 for (a) reducing the bioavailability of fructose in the units per gram fructose in the foodstuff. body ofa humanor animal or(b) reducingthe amount [0061] If the agent according to the present invention of fructose available to the human or animal body or also contains 5- D-fructose dehydrogenase and is added 55 to intestinal bacteria colonizing therein after the in- to a foodstuff before eating or during production, the ac- gestion of a fructose-containing food or substance, tivity of 5-D-fructose dehydrogenase should be between but excluding the use of an agent containing 5-D- 10 and 100,000 units, preferably between 25 and 60,000 fructose dehydrogenase.

8 15 EP 2 289 352 A2 16

2. The use according to claim 1 wherein the agent is ducing the amount of fructose available to the human taken orally, for example before meals, with meals or animal body or to intestinal bacteria colonizing or immediately after meals, so that it can exert its therein after the ingestion of a fructose-containing effect on fructose in the ingested food. food or substance, the agent comprising glucose iso- 5 merase, wherein the agent is as defined in any one 3. The use according to claim 1 or claim 2 wherein the claims 1 to 9, but excluding the use of an agent con- agent is formulated in the form of capsules (coated taining 5-D-fructose dehydrogenase. or non-coated), tablets (coated or non- coated), cap- sules containing coated or non- coated pellets, gran- 11. Glucose isomerase for use in the therapy or diagno- ules, or micro- or mini-tablets, tablets pressed from 10 sis of fructose intolerance wherein the glucose iso- coated or non-coated pellets, dragees, or micro-or merase is contained in a medical device, provided mini-tablets, gel caps or, in liquid form, as a solution, that the glucose isomerase is not used in combina- drops, suspension or gel. tion with 5-D-fructose-dehydrogenase.

4. The use according to any one of claims 1 to 3 wherein 15 12. Glucose isomerase for use according to claim 11 the agent is in the form of a medical device. wherein:

5. An agent comprising glucose isomerase and one or (a) the glucose isomerase is taken orally, for ex- more additional enzymes selected from invertase ample before meals, with meals or immediately (syn. beta-fructofuranosidase or beta- fructosidase), 20 after meals, so that it can exert its effect on fruc- lactase (syn. beta-galactosidase), maltase (syn. al- tose in the ingested food; and/or pha-glucosidase), alpha-amylase, beta-amylase, (b) the glucose isomerase is formulated in the glucoamylase, pullulanase, iso-amylase, amyloglu- form of capsules (coated or non-coated), tablets cosidase and cyclomaltodextrin glucantransferase (coated or non-coated), capsules containing (CGTase), but excluding agents comprising 5-D-25 coated or non-coated pellets, granules, or mi- fructose dehydrogenase. cro- or mini-tablets, tablets pressed from coated or non-coatedpellets, dragees, or micro- or mini- 6. An agent according to claim 5 comprising (i) glucose tablets, gel caps or, in liquid form, as a solution, isomerase in combination with invertase, or (ii) glu- drops, suspension or gel. cose isomerase in combination with maltase, or (iii) 30 glucose isomerase in combination with Invertase 13. Glucose isomerase for use according to claim 11 or and maltase. claim 12 wherein the therapy comprises:

7. An agent according to claim 5 or claim 6 which is (i) (a) reducing the bioavailability of fructose in the a foodstuff, (ii) a medical device or (iii) a pharmaceu- 35 body of a human or animal; and/or tical composition. (b) reducing the amount of fructose available to the body of a human or animal or to intestinal 8. An agent according to any one of claims 5 to 7 where- bacteria colonizing therein; and/or in the agent is formulated in the form of capsules (c) reducing or preventing the occurrence of (coated or non- coated), tablets (coated or non- coat- 40 fructose intolerance symptoms in a human or ed), capsules containing coated or non- coated pel- animal subject after the intake by the said sub- lets, granules, or micro- or mini-tablets, tablets ject of fructose or a substance or foodstuff con- pressed from coated or non- coated pellets, dragees, taining fructose in pure form or from which fruc- or micro- or mini-tablets, gel caps or, in liquid form, tose can be released in the digestive tract. as a solution, drops, suspension or gel. 45 14. Glucose isomerase for use according to any one of 9. Glucose isomerase in combination with one or more claims 11 to 13 wherein the fructose intolerance is additional enzymes selected from invertase (syn. be- intestinal fructose intolerance; and optionally where- ta-fructofuranosidase or beta- fructosidase), lactase in the intestinal fructose intolerance is caused by a (syn. beta-galactosidase), maltase (syn. alpha- glu- 50 disorder of fructose absorption. cosidase), alpha-amylase, beta-amylase, glu- coamylase, pullulanase, iso- amylase, amyloglu- 15. Glucose isomerase for use according to claim 14 cosidase and cyclomaltodextrin glucantransferase wherein the therapy comprises (a) reducing or pre- (CGTase), for use in medicine, but excluding com- venting the occurrence of fructose intolerance symp- binations comprising 5-D- fructose dehydrogenase. 55 toms selected from a feeling of bloating, flatulence, colic-like stomach ache, watery diarrhea and bowel 10. An agent for use in (a) reducing the bioavailability of sounds and/or (b) comprises reducing or preventing fructose in the body of a human or animal or (b) re- the occurrence of an increase in the production of

9 17 EP 2 289 352 A2 18 intestinal gases.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description

• Yamanaka. Purification, Crystallization and Proper- • Encyclopedia of Pharmaceutical Technology [0041] ties of the D-Xylose-Isomerase from Lactobacillus • Lehrbuch der Pharmazeutischen Technologie. Wis- brevis. Biochimica et Biophysika Acta, 1968, vol. 151 senschaftliche Verlagsgesellschaft, 2006 [0041] (3), 670-680 [0013] • Lebensmittel-Biotechnologie und Ernährung. Spring- • Yamanaka. D-Xylose Isomerase from Lactobacillus er Verlag, 1997, 82-130 [0046] brevis. Methods in Enzymology, 1971, vol. 41, • Jan S. Tkacz ; Lene Lange. Advances in Fungal 466-471 [0013] Biotechnology for Industry, Agriculture, and Medi- • Ameyama et al. D-Fructose Dehydrogenase of Glu- cine [0046] conobacter industrius: Purification, Characterization • Jan S. Tkacz ; Lene Langeand. Agriculture, and and Application of Enzymatic Microdetermination of Medicine, 2004 [0046] D-Fructose. Journal of Bacteriology, 1981, 814-823 • Enzymes in industry: Production and Applications. [0022] 2004 [0046] • Remington: The Science & Practice of Pharmacy. • Jose-Luis Barredo. Microbial Enzymes and Bi- Lippincott, Williams & Wilkins [0041] otransformations. Humana Press, 2005 [0046]

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