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(11) EP 2 124 640 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publication and mention (51) Int Cl.: of the grant of the patent: A61K 38/44 (2006.01) A61K 38/52 (2006.01) 27.10.2010 Bulletin 2010/43 A23L 1/03 (2006.01) A23L 1/30 (2006.01) A61K 9/48 (2006.01) A61P 1/00 (2006.01) (2006.01) (21) Application number: 08707777.2 A61P 3/00

(22) Date of filing: 20.02.2008 (86) International application number: PCT/EP2008/001294

(87) International publication number: WO 2008/101672 (28.08.2008 Gazette 2008/35)

(54) FOR USE IN THE TREATMENT OF INTOLERANCE GLUCOSEISOMERASE ZUR BEHANDLUNG VON FRUCTOSE-UNVERTRÄGLICHKEIT GLUCOSE ISOMÉRASE À UTILISER DANS LE TRAITEMENT D’INTOLÉRANCE AU FRUCTOSE

(84) Designated Contracting States: (74) Representative: Hutchins, Michael Richard AT BE BG CH CY CZ DE DK EE ES FI FR GB GR M.R. Hutchins & Co HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT 33 Connaught Way RO SE SI SK TR Tunbridge Wells Designated Extension States: Kent TN4 9QP (GB) AL BA MK RS (56) References cited: (30) Priority: 20.02.2007 DE 102007008664 WO-A-2007/057749 WO-A-2007/059955 WO-A-2007/059956 US-A1- 2003 113 310 (43) Date of publication of application: 02.12.2009 Bulletin 2009/49 • DATABASE EPODOC EUROPEAN PATENT OFFICE, THE HAGUE, NL; JP2000125883 9 May (73) Proprietor: Vitacare Gmbh & Co. Kg 2000 (2000-05-09), INT REAGANTS CORP: 60318 Frankfurt (DE) "fructokinase and its gene" XP002512117 & JP 2000125883 A (INT REAGENTS CORP) 9 May 2000 (72) Inventors: (2000-05-09) • WYROBNIK, Daniel, Henry 60322 Frankfurt (DE) Remarks: • WYROBNIK, Isaac, Harry Thefile contains technical information submitted after 60431 Frankfurt (DE) the application was filed and not included in this specification

Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 124 640 B1

Printed by Jouve, 75001 PARIS (FR) 1 EP 2 124 640 B1 2

Description all the fructose in a fructose containing food or substance in whatever form (e.g. also as part of sucrose) it is con- Field of the Invention tained in such a food or substance. [0011] In contrast to glucose, fructose is assimilated [0001] This invention relates to agents for use in the 5 into the mucosa cells of the small intestine by eased car- treatment or diagnosis of fructose intolerance. rier-mediated diffusion. The enzymatic degradation starts in the liver by the action of the adenosine triphos- Background of the Invention phate (ATP) dependent fructokinase, whereby fructose is converted to fructose-1-phosphate. In the liver and in [0002] US2003/113310 discloses the use of glucose 10 the kidneys, fructose- 1-phosphate is cleaved to glycerine isomerase for treating or preventing obesity and diabe- aldehyde and dihydroxyacetone phosphate by aldolase tes. B. [0003] JP2000125883 discloses a new form of fruc- [0012] Three different types of fructose metabolism tokinase and suggests that the fructokinase can be used disorder are known in humans, namely hereditary fruc- for inter alia the treatment of diagnosis of hereditary fruc- 15 tose intolerance, intestinal fructose intolerance, and fruc- tose intolerance. tose-1,6-diphosphatase deficiency. In addition, there is [0004] WO2007/057749 discloses the use of 5- D-fruc- fructosuria, which generally does not require treatment tose dehydrogenase, optionally in combination with one according to current scientific thinking. or more other such as glucose isomerase, for [0013] Hereditary fructose intolerance (HFI) results the treatment of fructose intolerance. 20 from a deficiency of aldolase B, an that occurs [0005] WO2007/059955 discloses the use of 5- D-fruc- in the intestinal mucosa, in the liver, in lymphocytes and tose dehydrogenase, optionally in combination with one in the kidneys. This enzyme usually breaks down fruc- or more other enzymes such as glucose isomerase, for tose-1-phosphate to fructose-1,6-biphosphate via inter- the treatment of adiposity. mediate stages. If an aldolase B deficiency is present, [0006] WO2007/059956 discloses the use of 5- D-fruc- 25 an excess of fructose-1- phosphate occurs, leading to an tose dehydrogenase in combination with glucose isomer- inhibition of glycogen breakdown and of gluconeogene- ase for the treatment of diabetes. sis and, in turn, to severe hypoglycemia with outbreaks [0007] US6372476 and WO2004/044129 both dis- of sweating, tremor, vomiting and cramps after the intake close novel glucose that may be used in food of fructose. Acidosis, kidney damage and aminoaciduria processing. 30 can occur if this remains undetected. In infants, the risk ranges from hemorrhages to sudden infant death syn- The Invention drome. [0014] The symptoms of the widespread intestinal [0008] 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 35 showing an increasing trend, especially in the western a compound that effects the conversion of fructose to 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- 40 as a result of the bacterial breakdown of the carbohy- ment and complaints that occur as a result of the intake 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, colio- like stomachache, watery di- or animals from other substances, such as sucrose. arrhea, and bowel sounds. This is often incorrectly diag- [0009] According to the present invention, the terms 45 nosed as irritable colon. "food" and "foodstuff" are used as synonyms. They mean [0015] Fructose-1,6-diphosphatase deficiency in- to also include feed in the sense of animal feed. volves a defect of this key enzyme in gluconeogenesis. [0010] 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 50 lactacidosis, seizures, muscular hypotension, and coma. also as free fructose in numerous foodstuffs. Food such 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 [0016] 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- 55 in mild disorders of fructose metabolism, health impair- stuffs that either contain fructose in pure form or from 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

2 3 EP 2 124 640 B1 4 to health impairments. above. The agent contains glucose isomerase. A glucose [0017] The above-mentioned symptoms and com- isomerase in the sense of this invention is an enzyme plaints could only be avoided up to now by maintaining that is able to convert fructose into glucose. This conver- a fructose-, sucrose- and sorbitol-free diet. However, it sion can also be achieved by a isomerase. In the is very difficult for those affected to keep to such a diet, 5 sense of this invention, a xylose isomerase is thus also since fructose is contained in all fruits and many vegeta- a glucose isomerase. A possible method for producing bles, and is widely used as a sweetener by the foodstuffs a xylose isomerase is described in Yamanaka, Biochim- industry. All foods that contain, e.g. sucrose (household ica et Biophysika Acta, issue 151 (3), 1968, 670-680, sugar) also have to be avoided. Such a diet, which is "Purification, Crystallization and Properties of the D-Xy- indeed very strict in the case of hereditary fructose intol- 10 lose-Isomerase from Lactobacillus brevis" and in Ya- erance, is not only difficult to keep to, it is also extremely manaka, Methods in Enzymology, issue 41, 1971, unfavorable from a nutritional physiological point of view, 466-471, "D-Xylose Isomerase from Lactobacillus and considerably impairs the quality of life of those af- brevis". fected. Not only those affected, but also the specialist [0021] The agent according to the present invention community, consisting of doctors, specialists, nutritional 15 can bring about the conversion of the fructose in food or scientists, nutritional advisers, specialist journalists, etc., in food pulp into glucose. The fructose is thus no longer have assumed for decades that there is no alternative to available for the bacterial metabolism in the intestines maintaining the diet described above. Research focused characterized by fermentation, and an excess of fruc- on an alternative to this diet has remained unsuccessful tose-1-phosphate in the liver or elsewhere can no longer to date. An agent that would make it possible to do without 20 occur. This can also prevent an increase in lactate levels maintaining such a diet and would allow the intake of in the blood. fructose containing food would thus satisfy an urgent [0022] A subject matter of the invention is therefore an need for the many people affected that has existed for agent for use in reducing the bioavailability of fructose in decades. It would overcome a prejudice that has been the human or animal body with the help of glucose iso- established in the specialist world and among those af- 25 merase. fected and mean a very considerable improvement and [0023] A subject matter of the invention is also an agent a dramatic step forward in the therapeutic and nutritional that, with the help of glucose isomerase, reduces the options in fructose intolerance, since, apart from main- amount of fructose available to the human or animal body taining a diet, there has simply been no therapy available or to intestinal bacteria colonizing therein. up to now. Such an agent would also put an end to the 30 [0024] A subject matter of the invention is also an agent as yet unsuccessful efforts of the specialist world to en- for use in the case of fructose intolerance, which contains able those affected to eat normally and to consume fruc- glucose isomerase. tose containing meals without suffering side effects. The [0025] A further subject matter of the invention is the importance of such an agent becomes very clear if one use of glucose isomerase in the case of fructose intoler- also takes into consideration that those affected by he- 35 ance. reditary fructose intolerance are threatened by the se- [0026] A further subject matter of the invention is the verest and most dangerous consequences for their use of glucose isomerase for the production of a product health if they, e.g. unknowingly, inadvertently or uninten- for the use in the case of fructose intolerance. tionally consume fructose. All of this would apply all the [0027] Glucose isomerase, which belongs to the group more to an agent that additionally had no negative effects 40 of isomerases, is an enzyme that has the property of on health. converting D-fructose into D-glucose and vice versa. [0018] Thus, it is an object of the present invention to Here, an equilibrium of approximately 50 % glucose and provide an effective agent that can be used not only in 50 % fructose is established, depending on ambient tem- milder disorders of fructose metabolism, but also in he- perature. Whereas fructose is only absorbed slowly from reditary and intestinal fructose intolerance and in fruc- 45 the small intestine, glucose is a sugar that is easily di- tose-1,6-diphosphatase deficiency, especially in order to gested and rapidly absorbed. enable the consumption of normally fructose containing [0028] The invention is thus based on ingested fruc- foodstuffs even if fructose intolerance is present. Further, tose being converted into glucose in vivo by glucose iso- it is an object of the invention to make it possible for those merase that is consumed simultaneously or at least affected by fructose intolerance to eat foodstuffs that they 50 shortly before or thereafter. The enzyme then aims at were not allowed to eat up to now, due to their fructose achieving the above-mentioned equilibrium by convert- content. Moreover, the objective is to provide an agent ing fructose to glucose. However, glucose is absorbed that can reduce or prevent the occurrence of fructose very rapidly, so that the equilibrium cannot be achieved. intolerance symptoms after the intake of fructose. The enzyme continues to convert fructose still available [0019] These objects are solved by the subject matter 55 in the food pulp into glucose until no further fructose is as described in claims 1 to 17 appended hereto. left. The dose of glucose isomerase may be selected in [0020] Therefore, the subject matter of the invention such a way that, even if larger amounts of fructose are is an agent for use in solving the problems described consumed, the reaction can take place quickly enough

3 5 EP 2 124 640 B1 6 that no fructose is absorbed or that the amount of fructose ase has the same taste as an untreated foodstuff and is, absorbed in the meantime is too small to cause the known for the first time, suitable to be consumed by persons gastrointestinal complaints in the case of mild fructose suffering from fructose intolerance, due to the reduced metabolism disorders and intestinal fructose intolerance fructose content which is established after eating. and the known systemic complaints in the case of hered- 5 [0034] According to a further aspect, according to the itary fructose intolerance and fructose-1.6-diphosphate present invention, a medical device can contain the glu- deficiency. cose isomerase. Accordingly, a subject matter of the in- [0029] According to the present invention, a glucose vention is also the use of a medical device which consists isomerase can also be used to reduce the fructose con- of glucose isomerase or contains it along with one or tent in a foodstuff. 10 more other active ingredients. In the sense of this inven- [0030] Foodstuffs in the sense of this invention also tion a "medical device" means any instrument, appara- comprise, among other things, foodstuffs for particular tus, appliance, material or other article, whether used nutritional uses, foods for special medical purposes, aloneor in combination, including the software necessary medical foods, food supplements, dietary supplements, for its proper application intended by the manufacturer dietetic food supplements, health foods, nutraceuticals, 15 to be used for human beings for the purpose of: and food additives. [0031] In a particularly easy way, the invention facili- - diagnosis, prevention, monitoring, treatment or alle- tates the transformation of fructose in a foodstuff into a viation of disease, form that avoids the problems that accompany fructose intolerance. Thus, the invention also makes it possible 20 - diagnosis, monitoring, treatment, alleviation of or for people affected by fructose intolerance to consume compensation for an injury or handicap, such foodstuffs, which had to be avoided up to now be- cause of their fructose content. - investigation, replacement or modification of the [0032] According to the present invention, glucose iso- anatomy or of a physiological process, merase is further mentioned for use in medicine for cur- 25 ing, alleviating, preventing or determining fructose intol- - control of conception, erance, for example as a pharmaceutical composition. Accordingly, a subject matter of the invention is also a and which does not achieve its principal intended action product which consists of glucose isomerase - or con- in or on the human body by pharmacological, immuno- tains it along with one or more other active ingredients, 30 logical or metabolic means, but which may be assisted for a medical use as defined herein. In the sense of this in its function by such means; invention, a pharmaceutical composition is a product, in [0035] Any instrument, apparatus, appliance, material particular a substance or a substance mixture, for use in or other article that does not achieve its intended action a method for surgical or therapeutic treatment of the hu- in or on the human body is not a medical device in the man or animal body and in diagnostic methods that are 35 sense of this application. performed on the human or animal body. Thus, in the [0036] In the following, the invention will be described sense of the invention, pharmaceutical compositions are further in its various aspects. If the term agent is used also products, in particular substances or substance mix- below, this always also stands for a foodstuff, a medical tures, that are intended or suitable for curing, alleviating, device or a pharmaceutical composition. preventing or determining fructose intolerance. 40 [0037] Glucose isomerase is a compound that has [0033] According to a further aspect of the present in- been known for more than 40 years and has only been vention, a foodstuff contains the glucose isomerase. Fur- used for saccharification to date. In the industry, ther, according to the present invention, a foodstuff is it is used for the conversion of glucose into fructose as used that contains glucose isomerase in an amount well as for the conversion of fructose into glucose. which is sufficient to convert fructose into glucose. Such 45 [0038] The agents according to the present invention a foodstuff may be produced advantageously using a can be taken orally before meals, with meals or immedi- method for treating a foodstuff in which the foodstuff is ately after meals, so that they can exert their converting placed in contact with a glucose isomerase under such or also dehydrogenating effect on fructose in the food conditions under which the glucose isomerase can con- pulp. Preferably, the agents according to the present in- vert fructose to glucose, In contrast to otherwise untreat- 50 vention are taken just before meals, during meals or im- ed foodstuffs, such a foodstuff has a reduced fructose mediately after meals. The agents according to the content and therefore, for the first time, is suitable to be present invention may contain the enzyme without further consumed by persons suffering from fructose intoler- additives. However, it is preferable that the agents ac- ance. Particularly advantageously, a foodstuff can be cording to the present invention further contain additives prepared by a method in which a glucose isomerase is 55 that are pharmaceutically acceptable and/or acceptable added to the foodstuff in a manner in which the action of for foodstuffs, such as extenders, binders, stabilizers, the glucose isomerase only starts after the intake of the preservatives, flavorings, etc. Such additives are com- foodstuff. Such a foodstuff that contains glucose isomer- monly used and well known for the production of phar-

4 7 EP 2 124 640 B1 8 maceutical compositions, medical devices, foodstuffs, present invention are preferably formulated in the form foodstuffs for particular nutritional uses, foods for special of capsules (coated or non-coated), tablets (coated or medical purposes, medical foods, food supplements, di- non-coated), capsules containing coated or non-coated etary supplements, dietetic food supplements, health pellets, granules, or micro- or mini-tablets, tablets foods, nutraceuticals, and food additives and specialists 5 pressed from coated or non-coated pellets, dragees, or in this field know which additives in which amounts are micro- or mini-tablets, gel caps or, in liquid form, as a suitable for particular presentation forms. Particularly solution, drops, suspension or gel. The formulation of the preferably, the agents according to the present invention agent according to the present invention as a powder is contain as additives dicalcium phosphate, lactose, mod- particularly suitable for an admixture to a foodstuff. The ified starch, microcrystalline , maltodextrin10 powder may be sprinkled onto a meal or it may be mixed and/or fibersol. into a pulp or a beverage. It is particularly suitable if the [0039] The agents according to the present invention agent offered as bulk powder is packed in single dosage can also be added to a foodstuff before eating. They can amounts, such as in single bags or capsules, or if it is even be added to the foodstuff at the production stage, provided in a dosing apparatus. It is especially preferable with the aim of developing their effect only after consum- 15 if the agents according to the present invention are for- ing the foodstuff. This could possibly be achieved by mi- mulated as a powder or as granules in capsules or as a croencapsulation, for example. In this way, the useable tablet that are administered orally. fructose content of the foodstuff would be reduced in a [0044] For oral administration, the active ingredient particularly advantageous way, without negatively affect- glucose isomerase may be contained in acceptable ex- ing its taste. Therefore, preparations containing glucose 20 cipients and/or carriers. The term "acceptable carrier" re- isomerase are preferred that do not release this enzyme lates to a carrier for pharmaceutical use which directs or these enzymes until they reach the digestive tract of the active ingredient to its target site and which does not a human or animal or let them become effective in an- have negative effects for the recipient, human or animal. other way, especially in the stomach or small intestine. However, the exact form of the carrier is not decisive. Therefore, the invention could be used for example in 25 [0045] The total amount of the carrier and/or excipients the production of sweets, fruit preparations (e.g. apple of an agent containing glucose isomerase is preferably sauce), jam, honey, chocolate and chocolate products, between 5 and 99.9 % by weight, more preferably be- bakery products (e.g. biscuits and cakes), breads, pas- tween 10 and 95 % by weight and even more preferably tas, vegetable dishes, potato dishes, ice cream, cereals, between 25 and 90 % by weight of the composition. dairy products (e.g. fruit yogurt and pudding), fructose 30 [0046] Suitable excipients and/or carriers include mal- containing beverages, fructose containing sauces (e.g. todextrin, calcium carbonate, dicalcium phosphate, tri- tomato ketchup) and fructose containing sweeteners. For calcium phosphate, microcrystalline cellulose, dextrose, dishes that are boiled or baked, the agents according to rice flour, magnesium stearate, stearic acid, croscarmel- the present invention could, e.g. be mixed into or sprin- lose sodium, sodium starch glycolate, crospovidone, su- kled onto them after cooling. 35 crose, vegetable gums, lactose, methylcellulose, povi- [0040] Since fructose is widely used as a sweetener done, carboxymethyl cellulose, corn starch, modified in foodstuffs that are especially produced for diabetics, starch, fibersol, gelatine, hydroxypropylmethyl cellulose the addition of the agents according to the present inven- and the like (including mixtures thereof). Preferable car- tion to diabetic food before eating or the addition of the riers include calcium carbonate, magnesium stearate, agents according to the present invention during the pro- 40 maltodextrin, dicalcium phosphate, modified starch, mi- duction of diabetic food is especially advantageous, to crocrystalline cellulose, fibersol, gelatine, hydroxypropyl- allow diabetics who suffer from fructose intolerance to methyl cellulose and mixtures thereof. The various ingre- eat diabetic food, such as the above mentioned food- dients and the excipient and/or carrier are mixed and stuffs in their respective form as diabetic foods. formed into the desired form using common methods. [0041] The agents according to the present invention 45 The presentation form which is intended for oral admin- can also be added to a foodstuff to exert their effect on istration according to the present invention, such as a the fructose originating from another foodstuff. An exam- tablet or capsule, may be coated with a coating that is ple of this would be the addition of the agents according resistant to low pH values. This makes it possible for the to the present invention to a spread so that the reduction enzyme or enzymes to be released only when they reach of the utilizable fructose contained in the bread occurs 50 the small intestine. Also a coating may be used which is after the intake of the bread, without impairing the taste not resistant against low pH values but which provides of the bread. Another example would be mixed spices. delayed release of the respective enzyme at low pH val- [0042] Another subject matter of the present invention ues. It is also possible to prepare the agent according to comprises the use of agents that in addition to other ac- the present inventionas coated (seeabove) pellets, gran- tive ingredients also contain glucose isomerase. 55 ules, or micro- or mini-tablets which can be filled into non- [0043] The invention may be formulated in any form coated capsulesor which canbe pressed into non-coated which is suitable for the intended route of administration. tablets. Suitable coatings are, for example, cellulose ac- For oral administration, the agents according to the etate phthalate, cellulose derivatives, shellac, polyvi-

5 9 EP 2 124 640 B1 10 nylpyrrolidone derivatives, acrylic acid, polyacrylic acid ing methods to be developed in the future. derivatives and polymethyl methacrylate (PMMA), such [0051] Glucose isomerase is commercially available as Eudragit® (from Röhm GmbH, Darmstadt), in partic- (e.g. Novozymes A/S, Denmark and Danisco, Denmark) ular Eudragit® FS30D (releases the active constituent or and is usually prepared in a microbiological way with the constituents starting at a pH of around 6.8) and Eudragit® 5 help of the microorganism Streptomyces murinus. 5-D- L30D-55 (releases the active constituent or constituents fructose dehydrogenase is also commercially available starting at a pH of around 5.5). If it is desired to release (e.g. Sigma-Aldrich and Toyobo, Japan) and is conven- the enzyme(s) already at a lower pH value, this may be tionally prepared in a microbiological way with the help achieved e.g. by the addition of sodium hydroxide solu- of the microorganism Gluconobacter industrius. Howev- tion to the coating agent Eudragit® L30D- 55, because in 10 er, the invention is not limited to the enzymes that are this case carboxyl groups of the methacrylate would be commercially available at the moment, but generally re- neutralised. Therefore, this coating will be dissolved, for lates to enzymes that can bring about the conversion of example, already at a pH value of 4.0 provided that 5 % fructose - specifically or non-specifically - to glucose or of the carboxyl groups are neutralised. The addition of 5-keto-D-fructose. A person skilled in the art can prepare about 100 g of 4 % sodium hydroxide solution to 1 kg of 15 suitable further enzymes by conventional methods, for Eudragit® L30D-55 would result in a neutralisation of example by mutagenesis of the gene encoding glucose about 6 % of the carboxyl groups. Further details about isomerase which is present in Streptomyces murinus or formulation methods and administration methods can be by mutagenesis of the gene encoding 5-D-fructose de- found in the 21st edition of "Remington: The Science & hydrogenase which is present in Gluconobacter indus- Practice of Pharmacy", published 2005 by Lippincott, Wil- 20 trius. The enzymes may also be prepared with the help liams & Wilkins, Baltimore, USA, in the Encyclopedia of of other microorganisms, such as fungi, in sufficient Pharmaceutical Technology (Editor James Swarbrick) amounts and the required purities, also by the use of and in Prof. Bauer "Lehrbuch der Pharmazeutischen genetic engineering methods which are common today. Technologie", 18th edition, published 2006 by Wissen- If it is desired e.g. to produce the enzymes with other schaftliche Verlagsgesellschaft (ISBN 3804-72222-9). 25 microorganisms, the genetic information of a microor- [0047] Other suitable pharmaceutically acceptable ganism which has been found initially by extensive carriers or excipients for use in the present invention in- screening and which has also been proven as a suitable clude water, mineral oil, ethylene glycol, propylene gly- source of the enzyme with the desired properties can be col, lanolin, glyceryl stearate, sorbitan stearate, isopropyl transferred to a microorganism which is normally used myristate, isopropyl palmitate, acetone, glycerine, phos- 30 for the production of enzymes. Also the modification of phatidylcholine, sodium cholate or ethanol. the enzyme(s) and the production of the enzyme(s) by [0048] The compositions for use in the present inven- means of methods which are presently known or may be tion may also comprise at least one coemulsifying agent, developed in the future in the area of industrial enzyme which includes oxyethylenated sorbitan monostearate, development and enzyme production, such as genetic fatty alcohols, such as stearyl alcohol or cetyl alcohol, or 35 engineering, is possible. The use and the manner of per- esters of fatty acids and polyols, such as glyceryl stear- forming all these methods for developing and producing ate. the enzyme(s) with the desired purities and activities and [0049] Preferably, the agents to be used in the present with the desired properties, in particular with respect to invention are provided in a stabilized form. Generally, the stability of the enzyme(s) at various pH values, re- stabilizationmethods and procedures which may be used 40 garding the optimum of the pH value, the stability at var- according to the present invention include any and all ious temperatures and temperature optimum, are well methods for the stabilization of chemical or biological ma- known to a person skilled in the art. The explanations in terial which are known in the art, comprising e.g. the ad- chapter 2 (page 82 to page 130) of the textbook "Leb- dition of chemical agents, methods which are based on ensmittel-Biotechnologie und Ernährung" of Heinz Rutt- temperature modulation; methods which are based on 45 loff, Jürgen Proll and Andreas Leuchtenberger, pub- irradiation or combinations thereof. Chemical agents that lished by Springer Verlag 1997 (ISBN 3-540-61135-5) may be used according to the present invention include, describe these methods in detail. These methods are among others, preservatives, acids, bases, salts, anti- also described in "Advances in Fungal Biotechnology for oxidants, viscosity enhancers, emulsifying agents, gelat- Industry, Agriculture, and Medicine" by Jan S. Tkacz, inizers, and mixtures thereof. 50 Lene Langeand (published in 2004, ISBN [0050] Conventionally, the industrial production of en- 0-306-47866-8), in "Enzymes in Industry: Production and zymes is performed in a technical fermentation way using Applications" by Wolfgang Aehle (Editor), published in suitable microorganisms (bacteria, moulds, yeasts). The 2004, ISBN 3527295925 and in "Microbial Enzymes and strains are recovered from natural ecosystems according Biotransformations" by Jose-Luis Barredo (Humana to a special screening protocol, isolated as pure cultures 55 Press 2005, ISBN 1588292533). All this also applies to as well as improved in their properties with respect to the the enzymes mentioned below thatcan optionally be add- enzyme spectrum and biosynthesis performance (vol- ed to the agent according to the present invention. ume/time yield). The enzymes can also be produced us- [0052] The activity of glucose isomerase is defined in

6 11 EP 2 124 640 B1 12 units according to the present invention, whereby one stances or foodstuffs, in particular from sucrose, may al- unit is the amount of glucose isomerase that converts 1 so be promoted and accelerated, so that the conversion g of fructose to glucose in 5 minutes at a pH of 7.5 and of fructose into Glucose may occur earlier. Therefore, a temperature of 37°C from an initially 10% solution by the addition of one or more of these enzymes to the agent weight (i.e. 10 g fructose + 90 g water). 5 according to the present invention may have the benefit [0053] At an enzyme activity determined according to of reducing the required amount of glucose isomerase. this definition, the agent according to the present inven- [0059] The activity of is measured in Sumner tion should contain glucose isomerase in an amount or units (SU, assay available e.g. from Bio-Cat Inc., Troy, activity of 0.01 to 100,000 GIU (= glucose isomerase Virginia, USA). An SU is defined as the amount of the units), preferably 0.05 to 10,000 GIU and particularly10 enzyme which converts 1 mg of sucrose into glucose and preferably 0.1 to 1,000 GIU per dose unit. fructose under standard test conditions within 5 minutes [0054] The wide range of the above mentioned dos- at 20°C and a pH value of 4.5. If the agent according to ages may be explained by the fact that the agent(s) ac- the present invention also contains invertase, the activity cording to the present invention can be applied in three of the invertase per dose unit should be between 50 and very different types of fructose intolerance, namely in he- 15 250,000 SU, preferably between 100 and 150.000 SU reditary fructose intolerance, intestinal fructose intoler- and particularly preferably between 150 and 100,000 SU ance, and fructose- 1,6-diphosphatase deficiency, in their per dose unit. range of different severities, and also in milder fructose [0060] The activity of maltase is defined in units, metabolism disorders. Furthermore, the different dosag- wherein one unit is the amount of maltase which will con- es also result from the fact that strongly varying amounts 20 vert maltose to D-glucose at a rate of one milligram per of fructose are administered to the body, depending on minute at 37°C and a pH of 4.0 in a 10% maltose solution the respective food. The enzyme or the enzymes should by weight. be used in a sufficient quantity so that it or they develop [0061] Where the agent according to the present in- a sufficiently high enzyme activity, in other words suffi- vention also contains maltase, the activity per dose unit cient glucose isomerase to convert an amount of fructose 25 should be between 100 and 100,000 units, preferably consumed in a normal meal (e.g. 10 - 50 g) - in free or between 200 and 50,000 units and particularly preferably bound form - into glucose. between 500 and 20,000 units. [0055] The agent according to the present invention [0062] In the case of hereditary fructose intolerance, may comprise one or more additional enzymes, such as it is particularly preferable if, in addition to glucose iso- invertase (syn. beta-fructofuranosidase or beta-fructos- 30 merase, folic acid in an amount of 1 mg to 100 mg, pref- idase), lactase (syn. beta-galactosidase), maltase (syn. erably 2 mg to 50 mg and particularly preferably 3 mg to alpha-glucosidase), alpha-, beta-amylase, glu- 10 mg per dose unit are added to the agent (s) according coamylase, pullulanase, isoamylase, amyloglucosidase, to the present invention, as folic acid increases aldolase cyclomaltodextrin glucantransferase (CGTase). These B activity. enzymes have the property of releasing fructose and/or 35 [0063] It may be advantageous to add an acceptor to glucose from fructose and/or glucose containing sub- the agent according to the present invention, for example stances and foodstuffs - alone or in combination with one at a ratio (acceptor: substrate) of 1:1 to 1:1,000, prefer- or more of these enzymes -, whereby the enzymes pul- ably at a ratio of 1:2 to 1:200 and particularly preferably lulanase and isoamylase also increase the efficiency of at a ratio of 1: 10 to 1:50. Examples of suitable acceptors glucoamylase and beta-amylase. All these enzymes are 40 which may be used include NAD+, NADP+, FAD+, vita- commercially available (e.g. BioCat Inc., Troy, USA or mins, such as vitamin C, vitamin E or vitamin A, ferric Novozymes A/S, Denmark or Amano Enzymes Inc., Ja- cyanide, ketones, aldehydes, 2,6-dichlorophenol-indo- pan or Sigma-Aldrich) and, up to now, have never been phenol, phenazine methosulfate and nitroblue tetrazo- used in combination with glucose isomerase in the med- lium (including mixtures thereof). ical/pharmaceutical field, in particular not in the case of 45 [0064] It may also be advantageous to add metal ions fructose intolerance. Thus this application discloses the to the agent according tothe present invention,especially first medical indication for glucose isomerase in combi- cations such as Mn2+, Mg2+, Ca2+, Zn2+, Fe2+, Co2+ or nation with any or all of these enzymes. Examples for Cu2+, including mixtures thereof, preferably at a molar agents according to the present invention include: ratio of 10-6 to 10-2. For the (xylose) glucose isomerase [0056] Glucose isomerase in combination with inver- 50 from Yamanaka described above, a particularly suitable tase, or Glucose isomerase in combination with maltase, cation is Mn2+. or Glucose isomerase in combination with Invertase and [0065] If the agent according to the present invention maltase. is added to a foodstuff before eating or during production, [0057] For example, said invertase can release fruc- the activity of glucose isomerase should be between 0.01 tose from e.g. sucrose. 55 and 20,000 units, preferably between 0.05 and 10,000 [0058] By the addition of one or more of these enzymes units and particularly preferably between 0.1 and 1,000 to the agent according to the present invention, the en- units per gram fructose in the foodstuff. dogenic release offructose from fructose- containing sub- [0066] The capsule sizes mentioned below refer to the

7 13 EP 2 124 640 B1 14 sizes defined by Capsugel Belgium BVBA, Bomem, Bel- 4. Glucose isomerase for use according to any one of gium. The size of capsules should be chosen in accord- claims 1 to 3 wherein the fructose intolerance is in- ance withthe specified formulation of the agentaccording testinal fructose intolerance. to the present invention. [0067] An agent according to the present invention in 5 5. Glucose isomerase for use according to claim 4 capsules (e.g. of size 3) could contain, e.g., 15 mg glu- wherein the intestinal fructose intolerance is caused cose isomerase (activity of glucose isomerase 1 GIU/mg) by a disorder of fructose absorption. and 135 mg dicalcium phosphate per capsule. [0068] If capsules, e.g. of size 1, are used, they may 6. Glucose isomerase for use according to claim 4 or contain 50 mg glucose isomerase (activity of glucose iso- 10 claim 5 wherein the therapy comprises reducing or merase 1 GIU/mg), 5 mg folic acid and 150 mg malto- preventing the occurrence of fructose intolerance dextrin per capsule. symptoms selected from a feeling of bloating, flatu- [0069] A further example of a composition for the pro- lence, colic-like stomach ache, watery diarrhea and duction of capsules consists of capsules of size 00 that bowel sounds. contain 300 mg glucose isomerase (activity of glucose 15 isomerase 1 GIU/mg) and 170 mg dicalcium phosphate 7. Glucose isomerase for use according to claim 4 or per capsule. claim 5 wherein the therapy comprises reducing or [0070] The invention may for example contain be- preventing the occurrence of an increase in the pro- tween 0.01 and 100,000 GIU (= glucose isomerase units) duction of intestinal gases. and between 1 mg and 100 mg folic acid per dose unit. 20 In addition, suitable additives in the required amount may 8. Glucose isomerase for use according to any one of be used. claims 1 to 7 wherein the glucose isomerase is con- [0071] The invention can be made available, for exam- tained in a pharmaceutical composition. ple as a pharmaceutical composition, medical device, foodstuff, foodstuff for particular nutritional uses, food for 25 9. Glucose isomerase for use according to any one of special medical purposes, medical food, food supple- claims 1 to 7 wherein the glucose isomerase is con- ment, dietary supplement, dietetic food supplement, tained in a foodstuff. health food, nutraceutical, and food additive. [0072] The agents according to the present invention 10. Glucose isomerase for use according to claim 9 can be used to considerably alleviate or eliminate the 30 wherein the foodstuff is selected from (a) foodstuffs symptoms and health impairments caused by fructose for particular nutritional uses; (b) foods for special intolerance. The invention presented here is suitable for medical purposes; (c) medical foods; (d) food sup- use in the case of fructose intolerance and for the ther- plements; (e) dietary supplements; (f) dietetic food apeutic treatment of fructose intolerance. supplements; (g) health foods; (h) nutraceuticals; 35 and (i) food additives.

Claims 11. Glucose isomerase for use according to any one of claims 1 to 10, wherein the Glucose isomerase is 1. Glucose isomerase for use in the therapy or diagno- administered orally. sis of fructose intolerance, provided that the glucose 40 isomerase is not used in combination with 5- D-fruc- 12. Glucose isomerase for use according to claim 11, tose dehydrogenase. wherein the glucose isomerase is formulated in the form of (a) coated or non- coated capsules or (b) coat- 2. Glucose isomerase for use according to claim 1 ed or non-coated tablets or (c) capsules containing wherein the therapy comprises (a) reducing the bi- 45 coated or non-coated pellets, granules, or micro- or oavailability of fructose in the body of a human or mini-tablets or (d) tablets pressed from coated or animal or (b) reducing the amount of fructose avail- non-coated pellets, dragees, or micro- or mini-tab- able to the body of a human or animal or to intestinal lets or (e) gel caps; or, in liquid form as (f) a solution, bacteria colonizing therein. (g) drops, (h) a suspension or (i) gel. 50 3. Glucose isomerase for use according to claim 1, 13. Glucose isomerase for use according to any one of wherein the therapy comprises reducing or prevent- claims 1 to 11, wherein the Glucose isomerase is ing the occurrence of fructose intolerance symptoms formulated as a powder. in a human or animal subject after the intake by the said subject of fructose or a substance or foodstuff 55 14. Glucose isomerase for use according to claim 12, containing fructose in pure form or from which fruc- wherein the coating of the capsules, tablets, pellets, tose can be released in the digestive tract. granules, micro- or mini-tablets and dragees is se- lected from cellulose acetate phthalate, cellulose de-

8 15 EP 2 124 640 B1 16

rivatives, shellac, polyvinylpyrrolidone derivatives, hinderung des Auftretens von Fructoseintoleranz- acrylic acid, polyacrylic acid derivatives and polyme- symptomen umfasst, ausgewählt aus Völlegefühl, thyl methacrylate. Blähungen, kolikartigen Dauchschmerzen, wässri- gem Durchfall und Darmgeräuchen. 15. Glucose isomerase for use according to any one of 5 claims 1 to 14 wherein the composition comprises 7. Glucose-Isomerase zur Verwendung nach An- metal ions. spruch 4 oder Anspruch 5, dadurch gekennzeich- net, dass die Therapie die Reduzierung oder Ver- 16. Glucose isomerase for use according to claim 15, hinderung des Auftretens einer Zunahme der Pro- wherein the metal ions are selected from 2+ Mn, 10 duktion von Darmgasen umfasst. Mg2+, Ca2+, Zn2+, Fe2+, Co2+, Cu2+ or mixtures thereof. 8. Glucose-Isomerase zur Verwendung nach einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, 17. Glucose isomerase for use according to any one of dass die Glucose-Isomerase in einer pharmazeuti- claims 1 to 16 wherein the glucose isomerase is a 15 schen Zusammensetzung enthalten ist. xylose isomerase. 9. Glucose-Isomerase zur Verwendung nach einem der Ansprüche 1 bis 7. dadurch gekennzeichnet, Patentansprüche dass die Glucose- Isomerase in einem Lebensmittel 20 enthalten ist. 1. Glucose-Isomerase zur Verwendung bei der Thera- pie oder Diagnose von Fructoseintoleranz, voraus- 10. Glucose-Isomerase zur Verwendung nach An- gesetzt, die Glucose-Isomerase wird nicht in Kom- spruch 9, dadurch gekennzeichnet, dass das Le- bination mit 5-D-Fructose-Dehydrogenase verwen- bensmittel ausgewahlt ist aus (a) Lebensmitteln, die det. 25 für eine besondere Ernähung bestimmt sind; (b) Le- bensmitteln für besondere medizinische Zwecke; (c) 2. Glucose-Isomerase zur Verwendung nach An- medizinischen Lebensmitteln; (d) Nahrungsergän- spruch 1, dadurch gekennzeichnet, dass die The- zungsmitteln; (e) Diätergänzungen; (f) diätetischen rapie (a) die Reduzierung der Bioverfügbarkeit von Nahrungsergänzungsmitteln; (g) Gesundheitsle- Fructose im Körper eines Menschen oder Tieres30 bensmitteln; (h) Funktionelle Lebensmittel und (i) Le- oder (b) die Reduzierung der Fructosemenge, die bensmittelzusätze. dem Körper eines Menschen oder Tieres oder darin siedelnden Darmbakterien zur Verfügung steht, um- 11. Glucose-Isomerase zur Verwendung nach einem fasst. der Ansprüche 1 bis 10, dadurch gekennzeichnet, 35 dass die Glucose-Isomerase oral zugeführt wird. 3. Glucose-Isomerase zur Verwendung nach An- spruch 1, dadurch gekennzeichnet, dass die The- 12. Glucose-Isomerase zur Verwendung nach An- rapie die Reduzierung oder Verhinderung des Auf- spruch 11, dadurch gekennzeichnet, dass die tretens von Fructoseintoleranzsymptomen bei ei- Glucose-Isomerase in Form von (a) überzogenen nem Menschen oder einem Tier umfasst, nachdem 40 oder nicht überzogenen Kapseln oder (b) überzoge- der Mensch oder das Tier Fructose oder einen Stoff nen oder nicht überzogenen Tabletten oder (c) Kap- oder ein Lebensmittel, der/das Fructose in Reinform seln, die überzogene oder nicht überzogene Pellets, enthält oder aus dem Fructose im Verdauungstrakt Granulate oder Mikro- oder Minitabletten enthalten, freigesetzt werden kann, aufgenommen hat. oder (d) Tabletten, die aus überzogenen oder nicht 45 überzogenen Pellets, Dragees oder Mikro- oder Mi- 4. Glucose-Isomerase zur Verwendung nach einem nitabletten gepresst sind, oder (e) Gelkapseln oder der Ansprüche 1 bis 3, dadurch gekennzeichnet, in flüssiger Form als (f) eine Lösung, (g) Tropfen, (h) dass die Fructoseintoleranz intestinale Fructosein- eine Suspension oder (i) Gel formuliert ist. toleranz ist. 50 13. Glucose-Isomerase zur Verwendung nach einem 5. Glucose-Isomerase zur Verwendung nach An- der Ansprüche 1 bis 11, dadurch gekennzeichnet, spruch 4, dadurch gekennzeichnet, dass die inte- dass die Glucose-Isomerase als ein Pulver formu- stinale Fructoseintoleranz durch eine Störung der liert ist. Fructoseresorption verursacht wird. 55 14. Glucose-Isomerase zur Verwendung nach An- 6. Glucose-Isomerase zur Verwendung nach An- spruch 12, dadurch gekennzeichnet, dass der spruch 4 oder Anspruch 5, dadurch gekennzeich- Überzug der Kapseln, Tabletten, Pellets, Granulate, net, dass die Therapie die Reduzierung oder Ver- Mikro- oder Minitabletten und Dragees ausgewählt

9 17 EP 2 124 640 B1 18

ist aus Celluloseacetatphthalat, Cellulosederivaten, de symptômes d’intolérance au fructose sélection- Schellack, Polyvinylpyrrolidonderivaten, Acrylsäu- nés parmi un sentiment de ballonnement, des flatu- re, Polyacrylsäurederivaten und Polymethylme- lences, des maux d’estomac de type colique, une thacrylat. diarrhée aqueuse et des bruits intestinaux. 5 15. Glucose-Isomerase zur Verwendung nach einem 7. Glucose isomérase pour une utilisation selon la re- der Ansprüche 1 bis 14. dadurch gekennzeichnet, vendication 4 ou la revendication 5, la thérapie com- dass die Zusammensetzung Metallionen umfasst. prenant la réduction ou la prévention de la survenue d’une augmentation de la production de gaz intesti- 16. Glucose-Isomerase zur Verwendung nach An-10 naux. spruch 15, dadurch gekennzeichnet, dass die Me- tallionen ausgewählt sind aus Mn2+, Mg2+, Ca2+, 8. Glucose isomérase pour une utilisation selon l’une Zn2+, Fe2+, Co2+, Cu2+ oder Gemischen daraus. quelconque des revendications 1 à 7, la glucose iso- mérase étant contenue dans une composition phar- 17. Glucose-Isomerase zur Verwendung nach einem 15 maceutique. der Ansprüche 1 bis 16, dadurch gekennzeichnet, dass die Glucose-Isomerase eine Xylose-Isomera- 9. Glucose isomérase pour une utilisation selon l’une se ist. quelconque des revendications 1 à 7, la glucose iso- mérase étant contenue dans un aliment. 20 Revendications 10. Glucose isomérase pour une utilisation selon la re- vendication 9, l’aliment étant sélectionné parmi (a) 1. Glucose isomérase pour une utilisation dans la thé- les denrées alimentaires destinées à une alimenta- rapie ou le diagnostic de l’intolérance au fructose, tion particulière; (b) des aliments diététiques desti- dans la mesure où la glucose isomérase n’est pas 25 nés à des fins médicales spéciales (c) des aliments utilisée en combinaison avec de la 5- D-fructose dés- médicaux; (d) des compléments alimentaires ; (e) hydrogénase. des compléments de régime ; (f) des compléments alimentaires diététiques ; (g) des aliments santé ; (h) 2. Glucose isomérase peur une utilisation selon la re- des alicaments; et (i) des additifs alimentaires. vendication 1, la thérapie comprenant l’étape con- 30 sistant à: (a) réduire la biodisponibilité du fructose 11. Glucose isomérase pour une utilisation selon l’une dans le corps d’un humain ou d’un animal ou (b) quelconque des revendications 1 à 10, la glucose réduire la quantité de fructose disponible pour le isomérase étant administrée par voie orale. corps d’un humain ou d’un animal ou pour les bac- téries intestinales le colonisant. 35 12. Glucose isomérase pour une utilisation selon la re- vendication 11, la glucose isomérase étant formulée 3. Glucose isomérase pour une utilisation selon la re- sous la forme de (a) capsules revêtues ou non re- vendication 1, la thérapie comprenant la réduction vêtues ou de (b) comprimés revêtus ou non revêtus ou la prévention de la survenue de symptômes d’in- ou de (c) capsules contenant des pastilles, granules, tolérance au fructose chez un sujet humain ou ani- 40 ou microcomprimés ou minicomprimés revêtus ou mal après l’ingestion par ledit sujet de fructose ou non revêtus ou de (d) comprimés pressés à partir de d’une substance ou d’un aliment contenant du fruc- pastilles, granules, ou microcomprimés ou minicom- tose sous une forme pure ou à partir de laquelle du primés revêtus ou non revêtus ou de (e) capsules fructose peut être libéré dans le tube digestif. de gel ; ou, sous une forme liquide en tant que (f) 45 solution, (g) gouttes, (h) suspension ou (i) gel. 4. Glucose isomérase pour une utilisation selon l’une quelconque des revendications 1 à 3, l’intolérance 13. Glucose isomérase pour une utilisation selon l’une au fructose étant une intolérance intestinale au fruc- quelconque des revendications 1 à 11, la glucose tose. isomérase étant formulée sous la forme d’une pou- 50 dre. 5. Glucose isomérase pour une utilisation selon la re- vendication 4, l’intolérance intestinale au fructose 14. Glucose isomérase pour une utilisation selon la re- étant provoquée par un trouble de l’absorption du vendication 12, le revêtement des capsules, compri- fructose. més, pastilles, granules, microcomprimés ou mini- 55 comprimés et dragées étant sélectionné parmi l’acé- 6. Glucose isomérase pour une utilisation selon la re- tate phtalate de cellulose, les dérivés de cellulose, vendication 4 ou la revendication 5, la thérapie com- la gomme laque, les dérivés de polyvinylpyrrolidone, prenant la réduction ou la prévention de la survenue l’acide acrylique, les dérivés d’acide polyacrylique

10 19 EP 2 124 640 B1 20

et le méthacrylate de polyméthyle.

15. Glucose isomérase pour une utilisation selon l’une quelconque des revendications 1 à 14, la composi- tion comprenant des ions métalliques. 5

16. Glucose isomérase pour une utilisation selon la re- vendication 15, les ions métalliques étant sélection- nés parmi Mn 2+, Mg2+, Ca2+, Zn2+, Fe2+, Co2+, Cu2+ ou des mélanges de ceux-ci. 10

17. Glucose isomérase pour une utilisation selon l’une quelconque des revendications 1 à 16, la glucose isomérase étant une xylose isomérase. 15

20

25

30

35

40

45

50

55

11 EP 2 124 640 B1

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.

Patent documents cited in the description

• US 2003113310 A [0002] • WO 2007059956 A [0006] • JP 2000125883 B [0003] • US 6372476 B [0007] • WO 2007057749 A [0004] • WO 2004044129 A [0007] • WO 2007059955 A [0005]

Non-patent literature cited in the description

• Yamanaka. Purification, Crystallization and Proper- • Heinz Ruttloff ; Jürgen Proll ; Andreas Leuchten- ties of the D-Xylose-Isomerase from Lactobacillus berger. Lebensmittel-Biotechnologie und brevis. Biochimica et Biophysika Acta, 1968, vol. 151 Ernährung. Springer Verlag, 1997 [0051] (3), 670-680 [0020] • Jan S. Tkacz ; Lene Langeand. Advances in Fungal • Yamanaka. D-Xylose Isomerase from Lactobacillus Biotechnology for Industry, Agriculture, and Medi- brevis. Methods in Enzymology, 1971, vol. 41, cine, 2004 [0051] 466-471 [0020] • Enzymes in Industry: Production and Applications. • Remington: The Science & Practice of Pharmacy. En- 2004 [0051] cyclopedia of Pharmaceutical Technology. Lippin- • Jose-Luis Barredo. Microbial Enzymes and Bi- cott, Williams & Wilkins, 2005 [0046] otransformations. Humana Press, 2005 [0051] • Prof. Bauer. Lehrbuch der Pharmazeutischen Tech- nologie. Wissenschaftliche Verlagsgesellschaft, 2006 [0046]

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