WO2010020693A2

WO2010020693A2 - Enzymes for the treatment of obesity

Info

Publication number: WO2010020693A2
Application number: PCT/EP2009/060838
Authority: WO
Inventors: Janne Ejrnaes Toender; Sune Sauer Lobedanz; Carsten Jaeger Johansen
Original assignee: Novozymes A/S
Priority date: 2008-08-21
Filing date: 2009-08-21
Publication date: 2010-02-25
Also published as: WO2010020693A3

Abstract

A method for treatment or prevention of obesity is disclosed where an enzyme capable of transferring an acyl group from a triglyceride to an ingestible polymer is administered to a person in need for such a treatment. During the action of the enzyme in the gastrointestinal tract fatty acids will be transferred to the indigestible polymer and thereby prevented from absorption.

Description

TITLE: ENZYMES FOR THE TREATMENT OF OBESITY

FIELD OF THE INVENTION

The present invention relates to a method for treating or preventing obesity by administering a composition comprising at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer and the indigestible polymer during passage of food through the gastrointestinal system. Further the invention relates to compositions for use in said method.

BACKGROUND OF THE INVENTION

Obesity and treatment thereof

Despite its recognition as a healthcare issue on an epidemic scale, obesity remains largely an unsolved medical problem, and is a major contributor to the global burden of chronic dis- ease and disability. The key cause of this epidemic is the combination of reduced physical activity and the increased consumption of energy-dense, nutrient-poor foods that are high in sugars and saturated fats.

WHO's latest projections (World Health Organization. Obesity and Overweight. Fact sheet No. 311.) Indicates that globally in 2005:

• approximately 1.6 billion adults (age 15+) were overweight;

• at least 400 million adults were obese.

WHO further projects that by 2015, approximately 2.3 billion adults will be overweight and more than 700 million will be obese.

Once considered a problem only in high-income countries, overweight and obesity are now dramatically on the rise in low- and middle-income countries, particularly in urban settings.

The fundamental cause of obesity and overweight is an energy imbalance between calories consumed on one hand, and calories expended on the other hand. Global increases in overweight and obesity are attributable to a number of factors including:

• a global shift in diet towards increased intake of energy-dense foods that are high in fat and sugars but low in vitamins, minerals and other micronutrients; and

• a trend towards decreased physical activity due to the increasingly sedentary nature of many forms of work, changing modes of transportation, and increasing urbanization. Overweight and obesity lead to serious health consequences. Risk increases progressively as BMI increases. Raised body mass index is a major risk factor for chronic diseases such as:

• Cardiovascular disease (mainly heart disease and stroke) - already the world's num- ber one cause of death, killing 17 million people each year.

• Diabetes - which has rapidly become a global epidemic. WHO projects that diabetes deaths will increase by more than 50% worldwide in the next 10 years.

• Musculoskeletal disorders - especially osteoarthritis.

• Some cancers (endometrial, breast, and colon). Obesity also has serious social and psychological consequences, such as low self-esteem and clinical depression, and affects all ages and socioeconomic groups.

Obesity is defined as a body mass index (BMI) of 30 kg per m² or more, where a person's BMI is defined as their weight in kilograms divided by the square of their height in meters.

Similar Overweight is defined as having a BMI of 25 kg per m² or more.

The market for a safe and efficacious drug is therefore potentially huge, but the value of currently approved therapies does not reflect this potential, which is evidence of their limited efficacy and side-effect profiles.

On the market today there are three categories of drugs; the amphetamine-like drugs, the CNS-active drugs, and the lipase-inhibitors. The amphetamine-like drugs are only approved for short-term use (a few weeks) due to their side-effect profiles (cardiovascular and the potential for abuse and dependency). Within the CNS-active drugs Sibutramine is approved in both the US and in Europe, and it works by reduction of energy intake and increased energy expenditure. In Europe another CNS-active drug, Rimonabant, is on the market. However in the US, approval is still pending due to the side effect profile (anxiety/depression). In the last category, lipase-inhibitors, one product is on the market. Xenical (Orlistat) works by inhibition of the pancreas lipase thereby preventing absorption of free fatty acids. As with the other anti-obesity products, Orlistat gives side effects that make people stop the treatment. These side effects are of the socially unacceptable type; with oily spotting, abdominal pain and fecal urgency being some of them.

On the dietary supplements market, the most pronounced trend is an abundance of different products with lack of proven effect.

Overall, there is a need of truly effective anti-obesity treatments without side effects. Lipid digestion

The main component in vegetable and animal oils and fats are triacylglycerols, also called triglycerides. A triglyceride consists of three fatty acid residues esterified to a glycerol backbone. Partial glycerides may also be present as natural constituents. The triglycerides cannot themselves be absorbed by the intestine. They are therefore hydrolyzed into free fatty acids and monoglycerides by pancreatic lipase, which forms an active 1 :1 complex with colipase. The activated complex can only work at a water-lipid interface: it is therefore essential that free fatty acids (FFA) are emulsified by bile salts for optimal activity of these enzymes. Most lipids are absorbed as free fatty acids and 2-monoglycerides, but a small fraction is absorbed as free glycerol and as diglycerides. Once across the intestinal barrier, they reconverted into triglycerides and packaged into chylomicrons or liposomes, which are released into the lymph system and then into the blood.

Transesterification

As mentioned above, side effects such as anal leakage of oil (oily spotting) is occasionally observed in patients treated with lipase inhibitors. This phenomenon reflects physical separation of some liquid unabsorbed dietary fat from the bulk of unabsorbable solids in the lower large intestine.

It has been described that the separation of unabsorbed oil from the feces and thus anal oil leakage can be reduced or prevented, when a lipase inhibitor such as Orlistat is administered in combination with low amounts of one or more substantially non-digestible, substantially non-fermentable, hydrophilic and/or hydrocolloidal food grade thickeners and/or emulsifiers (US6358522 B1 ).

SUMMARY OF THE INVENTION The invention provides a method for treating or preventing obesity comprising oral administering to a person in need for such a treatment an efficient amount of at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer, wherein the at least one enzyme is administered essentially simultaneously with administering or ingestion of the indigestible polymer. The method of the invention may be a therapeutic method intended for the treatment of obesity for health reasons or it may be a method used for cosmetic purposes. In a further aspect the invention relates to a composition, preferably a pharmaceutical composition, for the treatment or prevention of obesity comprising at least one enzyme capable of transferring an acyl group from a triglyceride to an ingestible polymer as well as inert ingredients usually used in pharmaceutical compositions.

In one embodiment the pharmaceutical composition further comprises the indigestible polymer capable of receiving an acyl group by the action of the at least one enzyme.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the realization that the energy absorbed from a diet can be reduced if fatty acids are not adsorbed or are absorbed in a reduced amount. This invention is further based on the realization that absorption of fatty acids can be prevented if the fatty ac- ids in the intestines are bound to an indigestible compound and that enzymes capable of transferring an acyl group or a fatty acid from a triglyceride to an indigestible compound are known.

The present invention has the advantage that it allows the person being treated to have his usual diet and due to the administration of the at least one enzyme and optional an indigesti- ble polymer provided that it is not present in the diet according to the invention, the caloric intake will be reduced and the person being treated will consequently loose weight or at least gain less weight compared with the situation where said person was not administrated said at least one enzyme and optional an ingestible polymer provided that it is not present in the diet.

Since the fatty acids according to the invention are transferred to an indigestible polymer the method of the invention does not have the disadvantages connected to separation of oil in the lower intestine such as oily spotting, abdominal pain and fecal urgency; as observed in connection with known methods of preventing absorption of fatty acids such as administration of lipase inhibitors or have the known disadvantages in a lesser extent.

The present invention relates to the use of an enzyme combined with a indigestible polymer containing a large number of hydroxy groups. When taken in combination with a meal the enzyme will transfer free fatty acids or fatty acids bound to triglycerides to the polymer. This will lower the absorption of dietary lipids, thereby lowering the caloric burden of meals. The enzymatic action will take place in the digestive system.

Transesterification

A general transesterification reaction where the acid moiety is moved from one alcohol moiety to another is shown below.

Enzymatic transesterification is by no means unknown to the scientific society as the examples below will show.

Extensive studies of lipase mediated esterification of mono- and disaccharides with activated acyldonors were carried out in the group of Stanley Roberts (Bashir, N. B.; Phythian, S. J.; Reasonb, A. J.; Roberts, S. M. J.Chem.Soc. Perkin Trans. / 2203-2222 (1995)).

Later esterification of monosaccharides in organic solvents with lauric acid as acyl donor and Novozym 435 (immobilized Candida antarctica lipase B) as catalyst was studied by the group of Matsuno Watanabe, Y.;( Miyawaki, Y.; Adachi, S.; Nakanishi, K.; Matsuno, R. Journal of Molecular Catalysis B: Enzymatic 10, 241-247 (2000)).

Extensive work on esterification of different mono- and disaccharides with different lipases in organic media was performed in European collaboration. (Plou, F. J.; Cruces, M. A.; Ferrer, M.; Fuentes, G.; Pastor, E.; Bernabe, M.; Christensen, M.; Comelles, F.; Parra, J. L.; Balles- teros, A. J. Biotechnol. 96, 55-66 (2002)).

A more focused study on the esterification with te/f-butanol as solvent was reported by the Hayes group, (Zhang, X. and Hayes, D. G. JAOCS 76, 1495-1500 (1999). Dang, H. T.; Obiri, O.; Hayes, D. G. JAOCS 82, 487-493 (2005)).

Just as a very recent paper describes the use of acrylic acid in the esterification (Tsukamoto, J.; Haebel, S.; Valenga, G. P.; Peter, M. G.; Franco, T. T. J. Chem. Technol. Biotechnol. (2008) DOI: 10.1002/jctb).

Even with water as solvent an acetyl esterase from Trichoderma reesei is able to esterify a wide range of saccharides (Kremnicky, L.; Mastihuba, V.; Cote, G. L. J. MoI. Catal. B: Enzymatic 30, 229-239 (2004)).

The importance of solvent choice is underlined by the fact that a recent review has been dedicated to this area (Kennedy, J. F.; Kumar, H.; Panesar, P. S.; Marwaha, S. S.; Goyal, R.; Parmar, A.; Kaur, S. J Chem Technol Biotechnol %λ , 866-876 (2006)).

In addition to all these scientific papers, there are several patents dealing with the subject. (WO98050400, US6528644, US6852852, US5733750 and EP0571421 B1 ). Enzyme classes used according to the invention

According to the invention the at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer may in principle be selected among any such en- zymes.

Whether an enzyme has the property of being capable of transferring an acyl group from a triglyceride to an indigestible polymer can be determined using a simple assay, e.g. consisting of the steps of:

1. Preparing a solution/dispersion of the indigestible polymer, triglyceride and a surfactant in aqueous medium

2. adding the enzyme in question and incubating at a suitable time and temperature for the transfer to take place.

3. determining whether transfer of acyl groups have taken place.

4. based on the determinations in step 3 deciding whether the enzyme in question is capable of transferring an acyl group from triglyceride to the indigestible polymer.

It is within the capabilities of the skilled person to adapt this simple assay to a given indigestible polymer and a given enzyme.

For step 1 the aqueous medium is preferably selected so that it mimics the conditions found in the stomach or intestine where the enzyme is intended to act according to the invention. Thus a preferred pH is in the range of 1-10, more preferred 2-9 and most preferred 3-8. The surfactant may in principle be any surfactant that is known not to inhibit enzyme action. Preferred surfactants include bile salts.

For step 2 a suitable temperature is in the range of 25-40⁰C, most preferred around 37°C and a suitable incubation period is in the range of 10 minutes to 3 hours, even though the incubation period is not critical for the final result.

For step 3 the determination of whether transfer of acyl groups has taken place may be determined using known methods such as determining the amount of generated free fatty acids, mono- and diglycerides. In addition the amount of fatty acids bound to the indigestible polymer can be determined. As an example of a method for determining whether an enzyme has the property of being capable of transferring an acyl group from a triglyceride to an indigestible polymer can be mentioned the assays 1 and 2 below.

Enzymes according to the invention may be selected among: hydrolases including but not limited to lipases, acetyl esterases, ferulic acid esterases, acetyl transferases, esterases, cu- tinases and phospholipases, where lipases are preferred.

The enzyme in the invention may be a hydrolase, more specifically but not limited to a car- boxylic ester hydrolase (generally classified as E. C. 3.1.1.x in accordance with the Enzyme Nomenclature Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology), or acyltransferases (generally classified as E. C. 2.3.1.x) having lipase and/or acyltransferase activity, where the enzyme is capable of transferring an acyl group from a lipid from glycerides to one or more acceptor substrates (described below). As examples of suitable enzymes according to the invention can be mentioned lipases, acetyl esterases, acetyl transferases, esterases, cutinases and phospholipas- es.

The enzyme according to the present invention may exhibit one or more of the following lipase or acyl transfer activities: Carboxylesterase activity (E. C. 3.1.1.1 ), arylesterase activity (E. C. 3.1.1.2), triacylglycerol lipase activity (E. C. 3.1.1.3), phospholipase A2 activity (E. C. 3.1.1.4), lysophospholipase activity (E. C. 3.1.1.5), acetyl esterase activity (E. C. 3.1.1.6), ste- rol esterase activity (E. C. 3.1.1.13), acylglycerol lipase activity (E. C. 3.1.1.23), galactolipase activity (E. C. 3.1.1.26), phospholipase A1 activity (E. C. 3.1.1.32), lipoprotein lipase activity (E. C. 3.1.1.34), wax-ester hydrolase activity (E. C. 3.1.1.50), 1 1-cis-retinyl-palmitate hydrolase activity (E. C. 3.1.1.63), all-trans-retinyl-palmitate hydrolase activity (E. C. 3.1.1.64), cuti- nase activity (E. C. 3.1.1.74), acyloxyacyl hydrolase activity (E. C. 3.1.1.77), hormone- sensitive lipase activity (E. C. 3.1.1.79), phosphatidylcholine-sterol O-acyltransferase activity (Glomset, J.A.J. Lipid Res. 9 (1968) 155-167. [PMID: 4868699].) (E.C. 3.2.1.43), phospholi- pid:diacylglycerol acyltransferase activity (Disclosed in WO2003097825A2) (E.C. 3.2.1.158).

Lipases

A lipase, contained in a composition of the invention, may be obtained from a microorganism, preferably a filamentous fungus, yeast, or a bacterium.

The lipase may be a non-specific lipase capable of releasing or binding any fatty acid group from or to any glyceride position. Such lipases have been obtained from Candida cylindra- cae, Corynebacterium acnes and Staphylococcus aureus. (Macrae, A. R. JA. O. C. S. 60, 291-294 (1983). US5,128,251 ). The lipase may also be of the type that only adds or removes specific fatty acid groups to or from specific glycerides. Such lipases have been obtained from Geotrichum candidium and Rhizopus, Aspergillus, and Mucor genera. The lipase may also be a 1 ,3-specific lipase. Such lipases have been obtained from Thermomyces lanugino- sa, Rhizomucor miehei, Aspergillus niger, Mucor javanicus, Rhizopus delemar, and Rhizopus arrhizus. The lipase may furthermore be a 2-specific lipase such as the enzyme obtained from Pseudozyme sp. (described in WO200504334), or C. paralopsis (Neugnot et al, Eur J Biochem. 2002 Mar;269(6):1734-45.).

In addition, the lipase can be obtained form genus Fusarium, such as a strain of the species Fusarium culmorum, F. heterosporum, F. solani, or F. oxysporum. Or from yeast such as Candida antarctica, C. albicans or C. rugosa.

Sources of enzymes

Enzymes may be provided from any suitable source known to the skilled person. It is known that enzymes are found ubiquitously in all organisms and it is known to obtain enzymes from various sources such as plants, animals, mammals, and microorganisms. Preferably the enzymes according to the invention are obtained from microorganisms such as bacteria, fungi and yeasts.

In the present specification and claims the term "obtained from" as used herein in connection with a specific microbial source, means that the enzyme and consequently the DNA se- quence encoding said enzyme is produced by the specific source. The enzyme is then obtained from said specific source by standard known methods enabling the skilled person to obtain a sample comprising the enzyme and capable of being used in the invention. Said standard methods may be direct purification from said specific source or cloning of a DNA sequence encoding the enzyme followed by recombinant expression either in the same source (homologous recombinant expression) or in a different source (heterologous recombinant expression).

Indigestible Polymers

The indigestible polymer may according to the invention be any polymer that is indigestible capable of acting as an acyl acceptor in a reaction mediated by an enzyme.

In the present application and claims the term "indigestible polymer" is intended to mean a polymer that passes through the Gastro-intestinal tract of a normal healthy human being without being substantially degraded. It should be understood that even though a polymer is considered an indigestible polymer according to the invention some linkages in the polymer may be broken and some material may be lost during passage of the gastro-intestinal tract e.g. due to unspecific enzymatic degradation or hydrolysis caused by the acidic environment in the stomach. Also a polymer where part of the polymer is degraded but the majority of the polymer passes through the gastro-intestinal trace is considered an indigestible polymer according to the invention. For example a branched polymer where the branches are degraded but the backbone remains largely unaffected is considered an ingestible polymer according to the invention. Thus, the amount of a indigestible polymer that leaves the gastro-intestinal tract as feces is more than 50% of the amount that is ingested, preferably more than 60 %, more preferred more than 70 %, more preferred more than 80%, more preferred more than 90%, even more preferred more than 95%, and most preferred more than 98%.

Typically, enzymes suitable for use in the method according to the present invention act by transferring an acyl group from a triglyceride to an OH group of the ingestible polymer. Thus, a preferred ingestible polymer is an ingestible polymer having one or more OH groups, more preferred an ingestible polymer having a large number of OH groups.

As examples of suitable ingestible polymers can be mentioned: inorganic polymers or oligomers such as polymerized silica (SiO₂) such as in Aerosil and Sipernat; or organic polymers or oligomers. These may be water soluble or insoluble natural, synthetic, semisynthetic or modified oligo- or polysaccharides, such as but not limited to cellulose, and cellulose deriva- tives such as methylcellulose, carboxymethyl cellulose and hydroxyethyl cellulose; galacto- mannans such as guar gum and locust bean gum; hemicelluloses such as xylan, arabinoxy- lan, arabinogalactan, araban, glucan; pectin, gum arabic, chitosan, xanthan gum, psyllium seed, ispaghula husk, plantago ovata seeds, karaya gum and mixtures of such compounds.

In one preferred embodiment the indigestible polymer is an indigestible polymer that naturally occurs as part of the diet. Such indigestible polymers include but are not limited to cellulose and hemicelluloses being part of most plant foods; galactomannans such as guar gum and locust bean gum being part of many manufactured foods such as a thickening agent; pectin being part of jams and fruit products and chitin being part of mushrooms. In this embodiment the at least one enzyme capable of transferring an acyl group from a triglyceride to an indi- gestible polymer is administered essentially simultaneously with the meal containing the indigestible polymer and triglycerides.

In another embodiment the indigestible polymer is an indigestible polymer that does not occur as part of the diet. In this embodiment the at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer and the indigestible polymer are both administered essentially simultaneously with the meal containing triglycerides.

Relation between enzyme and polymer

According to the invention the at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer is administered essentially simultaneously with the administering or ingestion of the indigestible polymer. In this connection the term "essentially simultaneously" is intended to mean that the at least one enzyme is administered so the at least one enzyme and the indigestible polymer are present in the stomach together with a triglyceride containing meal. Most efficiently the at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer and the indigestible polymer, if not being part of the meal, are administered less than about 120 minutes before or after the meal, preferably less than 60 minutes before or after the meal, preferably less than 30 minutes before or after the meal more preferably less than 15 minutes after a meal.

Thus, in the embodiment where the meal contains the indigestible polymer capable of acting as an acyl acceptor in a reaction catalysed by the at least one enzyme capable of transferring an acyl group from a triglyceride to the indigestible polymer, the enzyme is administered essentially simultaneously with the triglyceride containing meal.

Composition

The composition of the invention comprises at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer, usual inert excipients known to be included in pharmaceutical compositions and optionally the indigestible polymer.

In this connection the term "inert excipient" is intended to mean that the excipient in question do not have any activity in relation to the treatment of obesity but serves a purpose in the final composition or during the manufacture of the composition. Such inert excipients will be known within the area as water, fillers, diluents, disintegrants, lubricants, colorants, flavours, coatings etc. More teachings relating to the preparation of compositions of the invention may be found in well known textbooks within the pharmaceutical area such as Encyclopedia of Pharmaceutical Technology J. Swarbrick and J. C. Boylan (eds) Marcel Dekker, New York.

In case that the composition comprises at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer, where the indigestible polymer is present in the diet, the composition may not contain the indigestible polymer. In this embodiment the composition will be intended to be administered to a person in need thereof in connection with a meal containing said indigestible polymer.

If the composition comprises at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer where the indigestible polymer is not present in the diet, the composition should also comprise the indigestible polymer. Alternatively the composition may not comprise the indigestible polymer but instead be intended to be administered together with a second composition comprising the indigestible polymer so that the composition comprising the at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer is administered essentially simultaneously with the composi- tion comprising the indigestible polymer.

The compositions according to the invention may in addition to at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer and optionally the indigestible polymer further comprise one or more nutritionally beneficial components such as but not limited to vitamins and minerals.

MATERIALS AND METHODS Enzymes

Aspergillus oryzae ferulic acid esterase variant (disclosed in WO200212472)

Candida antarctica lipase A (disclosed in WO8802775A1 , WO9401541 A1 )

Candida antarctica lipase B (disclosed in WO8802775A1 ) Fusarium oxysporum lipase (Disclosed in WO9826057A1 )

Geotrichum candidum lipase 1 (GCL1 ) (disclosed in EP442558, Schrag, J. D.; Li, Y.; Wu, S.;

Cygler, M. Nature, 351 , 761 (1991 ))

Humicula insolens cutinase variant with the following mutations:

E6Q,G8D,A14P,N15D,E47K,S48E,R51 P,A88H,A91 H,A130V,E179Q,R189V (disclosed in WO0192502A1 )

Hyphozyma lipase (disclosed in WO9324619A1 )

Rhizomucor miehei lipase (disclosed in Boel, E.; Huge-Jensen, B.; Christensen, M.; Thim, L.;

RiI, N. P. Lipids, 23, 701 , (1988)).

Thermomyces lanuginosa lipase (disclosed in WO2005058068A1 ) Thermomyces lanuginosa lipase variant with the following mutations T231 R,N233R

(disclosed in WO0060063A1 ) Polymers

Cellulose (Cellulose powder, Sigma-Aldrich, Product no 22183, fiber length 0.01-0.10 mm) Hydroxyethylcellulose (Sigma-Aldrich, Product no: 434965, Typical Mw: 90,000) Gum guar (Sigma-Aldrich, Product no. G4129) Gum arabic (Bie & Berntsen A/S, Product no 1.04228.1000)

Triglyceride

The triglyceride used as substrate is triolein (Glyceryl trioleate, Sigma-Aldrich, Product no T- 7140).

Assay 1. Primary screen for transesterification activity

In this assay the ability of enzymes to perform transesterification to a polymer containing a large number of hydroxy groups is tested. The assay measures the amount of free fatty acids released in the presence and absence of enzyme and polymer.

3

Acylated carrier

General assay conditions

Triolein (glycerol trioleate) 1 1 μl

Polymer 10 mg

Enzyme (liquid) 0.05 mg (A₂₈₀ = 0.06) Bile salt solution (20 m M in acetate buffer pH 6) 250 μl

Acetonitrile 750 μl

In order to quantify the degree of transesterification the following analyses will be carried out and the amount of Free Fatty Acid formed for each combination will be measured:

a) Enzyme and polymer (shows hydrolysis + transesterification to polymer) b) Enzyme (shows hydrolysis)

The percentage of transesterification = 100x([FFA_b] - [FFA_a])/[FFA_b]

The ingredients are mixed in an eppendorf tube, and the mixture is left shaking overnight (17 h) at 25 ⁰C. To stop the reaction and solubilize the produced free fatty acids, 50 μl 10% Triton X-100 in 1 M phosphoric acid is added, and the ingredients are mixed for 5 min and heated to 50⁰C. Upon this, the concentration of oleic acid (Free Fatty Acid) is measured.

Quantification of free fatty acids:

For quantification of free fatty acids was used a Wako NEFA-C kit (Wako Chemicals GmbH, Neuss, Germany)

Reagent A:

Contents of bottle R1 a (Colour reagent A: 0.3 kU/l acyl-CoA-synthetase, 3.0 kU/l as- corbate oxidase, 0.6 g/l CoA, 5.0 mM ATP, 1.5 mM 4-aminophenazone) dissolved in 10 ml of solution R1 (50 mM phosphate buffer, pH 6.9, 3.0 mM MgCI2, surfactant, stabilizers) (Wako NEFA-C kit). Reagent B:

Contents of bottle R2a (Colour reagent B: 6.6 kU/l acyl-CoA-oxidase, 7.5 kU/l peroxidase) dissolved in 20 ml of solution R2 (1.2 mM MEHA (3-methyl-N-ethyl-N-(β- hydroxyethyl)aniline), surfactant) (Wako NEFA-C kit).

NEFA C Standard:

1 mM oleic acid, surfactant, stabilizers (Wako NEFA-C kit).

Samples from the eppendorf tubes are diluted with 1% Triton X-100 (typically 6.25, 12.5, 25, 50, 100, 200, and 400 times) and the amount of free fatty acids is determined by a modified method of the NEFA C kit (Wako Chemicals GmbH). 25 μl diluted sample is mixed with 50 μl reagent A in the well of a microtiter plate and incubated 15 min at room temperature with shaking. 100 μl reagent B is added and the plate is again incubated 15 min at room temperature with shaking. Absorbance is read at 550 nm. A standard curve is included with 0, 0.0078125, 0.015625, 0.3125, 0.0625, 0.125, 0.25 and 0.5 mM oleic acid.

Assay 2. Transesterification activity in a model of the digestive system

Experimental conditions

Humanized diet:

Poultry meal 73 g/kg (wet weight)

Pea meal 73 g/kg

Casein (precipitated under acidic conditions) 73 g/kg Wheat flour 290 g/kg

Potato starch 290 g/kg

Lard 125 g/kg

Vitamins, minerals, trace elements 76 g/kg

To 200 g of the above mixture add:

Olive oil 25 g

Cream (38% fat) 75 g

Pepsin solution:

700 μg/ml pepsin (Merck 1.07192) dissolved in MiIIi Q water Intestine buffers:

0.8 M MES, 0.8 M sodium acetate, 0.8 M imidazole, adjusted to pH 5 with NaOH 0.8 M MES, 0.8 M sodium acetate, 0.8 M imidazole, adjusted to pH 9 with HCI

Bile salt assay solution:

40 mM bile salts made by dilution 2.5x of bile salt stock solution with MiIIi Q water

Bile salt stock solution:

100 mM bile salts (Galle-Dispert (lipase activating mixture)) made by dissolving 10 g in 200 ml MiIIi Q water (assuming an average molecular weight of the bile salts of 500 g/mol)

In vitro model

Gastric step:

5 g humanized base diet, 625 mg olive oil and 1.875 g cream are mixed with 25 ml 0.1 M HCI. After 15 min stirring pH is adjusted to 3.0 with 6 mM HCI. After further 15 min stirring pH is adjusted to 3.0 with 6 mM HCI. The mixture is heated to 50⁰C and aliquots of 100 μl are transferred to the wells of a microtiter plate containing 20 μl pepsin solution (final concentration of 93 μg/ml in gastric step). 30 μl enzyme sample diluted in 0.01 % Triton X-100 is added, and the plate is incubated 1 hour at 37°C with agitation (e.g. Eppendorf Thermomixer, 700 rpm). To estimate the change in pH during the stomach step, pH is measured in a few selected wells with varying amounts of enzymes added.

Intestinal step:

For the subsequent intestine step, 25 μl intestine buffer mixture (normally 60% of the pH 5 buffer and 40% of the pH 9 buffer, which results in a pH of around 6) is added, the plate is mixed for 2 min and 25 μl bile salt assay solution (normally giving a final concentration of 5 mM) is added. pH is again measured in a few selected wells, and the plate is incubated 2 hours at 37°C. After this incubation pH is again measured in a few selected wells. To stop the reaction and solubilize the produced free fatty acids, 50 μl 10% Triton X-100 in 1 M phosphoric acid is added, and the microtiter plate is mixed for 5 min and heated to 50⁰C.

Quantification of free fatty acids was done as described under Assay 1. Assay 3. Control of enzyme activity in the presence of polymer

In order to verify that the presence of polymer not just simply inhibits enzyme activity, a control experiment can be performed by detecting the amount of monoglyceride formed during the experiments in Assay 1.

TLC in the eluent ethyl acetate:heptane 3:1 was performed with triolein (R_f = 1 ), glyceryl monoglyceride (R_f = 0.2) and oleic acid (R_f = 0.7) as references. The spots were visualized by 1 M H₂SO₄ and heating. Scaling of the amounts were with (-) for nothing, (+) for a trace of compound, (++) for some compound, and (+++) for a lot of compound.

EXAMPLES

Example 1 - Aspergillus oryzae ferulic acid esterase variant

Following the procedure described above (Assay 1 - Primary screen for transesterification activity) the transesterification from triolein to four different polymers was tested.

Example 2 - Candida antarctica lipase A

Example 3 - Candida antarctica lipase B

Example 4 - Fusarium oxysporum lipase Following the procedure described above (Assay 1 - Primary screen for transesterification activity) the transesterification from triolein to four different polymers was tested.

Example 5 - Geotrichum candidum lipase 1 Following the procedure described above (Assay 1 - Primary screen for transesterification activity) the transesterification from triolein to four different polymers was tested.

Example 6 - Humicula insolens cutinase variant

Example 7 - Hyphozyma lipase

Example 8 - Rhizomucor miehei lipase

Example 9 - Thermomyces lanuginosa lipase

Example 10 - Thermomyces lanuginosa lipase variant

Examples 11 -17: Test of enzymes and polymers in the gastrointestinal in vitro model

Example 11 - Thermomyces lanuginosa lipase variant

Following the procedure described above (Assay 2 - Transesterification activity in a gastrointestinal in vitro model) the transesterification from dietary lipids to four different polymers was tested. In the Table below the measured amounts of released free fatty acids (mM) from the in vitro model are given.

Table 1. Released free fatty acids (mM)

It is seen that the release of free fatty acids (FFA) by the Thermomyces lanuginosa lipase variant is reduced by about 40% with the addition of guar gum and hydroxyethyl cellulose (HE cellulose), whereas gum arabic about doubles the activity. Addition of cellulose gives little or no effect with this lipases.

Example 12 - Thermomyces lanuginosa lipase Following the procedure described above (Assay 2 - Transesterification activity in a gastrointestinal in vitro model) the transesterification from dietary lipids to four different polymers was tested. In the Table below the measured amounts of released free fatty acids (mM) from the in vitro model are given.

Table 2. Released free fatty acids (mM)

It is seen that the release of free fatty acids (FFA) by Thermomyces lanuginosa lipase is reduced by about 40% with the addition of guar gum and hydroxyethyl cellulose (HE cellulose), whereas gum arabic about doubles the activity. Addition of cellulose gives little or no effect with this lipases.

Example 13 - Fusarium oxysporum lipase

Following the procedure described above (Assay 2 - Transesterification activity in a gastroin- testinal in vitro model) the transesterification from dietary lipids to four different polymers was tested. In the Table below the measured amounts of released free fatty acids (mM) from the in vitro model are given.

Table 3. Released free fatty acids (mM)

None of the carriers seem to significantly affect the amount of released FFA with Fusarium oxysporum lipase. Example 14 - Humicula insolens cutinase variant

Table 4. Released free fatty acids (mM)

With Humicula insolens cutinase variant the average reduction in measured FFA is about 30% with guar gum and HE cellulose and about 20% with cellulose and gum arabic.

Example 15 - Rhizomucor miehei lipase

Table 5. Released free fatty acids (mM)

The addition of guar gum together with Rhizomucor miehei lipase reduces the released FFA by on average 70%, whereas HE cellulose gives about 60% reduction. Addition of cellulose and gum Arabic give 20-30% decrease in FFA.

Example 16 - Candida antarctica lipase A

Table 6. Released free fatty acids (mM)

None of the carriers seem to significantly affect the amount of released FFA with Candida antarctica lipase A.

Example 17 - Candida antarctica lipase B

Table 7. Released free fatty acids (mM)

Candida antarctica lipase B is seen to release little FFA compared to the other enzymes tested. Still guar gum and HE cellulose reduce the released FFA by on average about 40- 50%, whereas smaller reductions (20-30%) are seen with cellulose and gum arabic.

Example 18 - Humicula insolens cutinase variant and cellulose

Following the procedure described above (Assay 3. Control of enzyme activity in the presence of polymer), the reaction mixture from the finished Example 6 was applied to a TLC plate.

From the TLC plate it was evident that the amount of monoglyceride formed was similar both with and without polymer present, while there was clearly more free fatty acid formed without polymer present.

Example 19 - Preliminary rat study

A preliminary study was carried out to establish if a rise in blood triglycerides can be measured after ingestion of a high-lipid meal. In addition, it was observed whether the animals displayed diarrhea after repeated administration of the high-lipid diet.

The study was performed by BioAdvice in ølstykke, Denmark.

High Lipid diet:

741 ,6 g milk (1.5% fat, UHT milk (ArIa, Denmark)

3X87G sachet Calshake (2077 KJ/100g, Fresenius Kabi)

89.7 g Olive oilm Fluka 75348

29,64 g Methocel, Food grade (DOW Chemical company) Milk and olive oil was mixed using high speed mixer (Ultraturrex), Calshake was added and mixing was continued. Finally, Methocel was slowly added and mixing was continued for 2 minutes.

The diet was heated to 50⁰C with stirring and pH was adjusted to 8 using 2M NaOH. 1050 μl Savinase® 16L (Novozymes A/S, Bagsvaerd, Denmark) diluted 50 fold in water was added and stirring continued for 4 hours whereafter pH was adjusted to 3 using 4M HCI. Finally the mixture was stirred 1 /4 hours before use.

Conclusion Administration of both 10 ml/kg and 5 ml/kg of a high lipid diet (milkshake-like fat emulsion) gave rise to significant increases in plasma TG after 2h. The animals did not gain weight during the study, nor did they appear to be ill or show any tendency to oily stools after repeated dosing of the high lipid diet.

For the effect studies, 5 ml/kg of lipid diet will be used, and the plasma TG cone, will be measured 2h after administration of the fat emulsion.

Example 20

For the study, two enzymes {H. insolens cutinase variant and R. miehei lipase) and one polysaccharide (guar gum) were selected. The treated groups were to be compared with three other animal groups in which only the polysaccharide, the enzyme, and high lipid diet, prepared as described in example 19, was administered (see below). The study was conducted using Wistar rats.

Test groups

The administered doses and sampling frequency are shown below. Both the enzymes and the polysaccharide were administered in one dose. For the enzymes, the dose is very close to the maximum tolerable level, based on already existing in-house toxicology data.

Dosing

Sampling frequencies

a Treatment orally by gavage. When not fasted, the animals were offered food ad libitum.

Feces samples were collected in the morning before administration of fat emulsion and blood sampling. Feces samples were pooled for each treatment group, freeze dried and the amount of dry matter determined.

Claims

1. A method for treating or preventing obesity, comprising oral administering to a person in need for such a treatment an efficient amount of at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer, wherein the at least one polymer is administered essentially simultaneously with administering or ingestion of the indigestible polymer.

2. The method of claim 1 , wherein the at least one enzyme is selected among: hydrolases such as lipases, acetyl esterases, acetyl transferases, esterases, cutinases, phospholipases and proteases.

3. The method of claim 2, wherein the at least one enzyme is selected among: Aspergillus oryzae ferulic acid esterase and variants thereof; Candida antarctica lipase A;

Candida antarctica lipase B;

Fusarium oxysporum lipase;

Geotrichum candidum lipase 1 ;

Humicula insolens cutinase and variants thereof; Hyphozyma lipase;

Rhizomucor miehei lipase;

Thermomyces lanuginosa lipase and variants thereof.

4. The method of any of the claims 1-3, wherein the indigestible polymer is a polymer having one or more OH groups, preferably having several OH groups.

5. The method of claim 4, wherein the indigestible polymer is selected among: inorganic polymers or oligomers such as polymerized silica (SiO₂) such as in Aerosil, Sipernat; organic polymers or oligomers such as carbohydrates.

6. The method of claim 5, wherein the indigestible polymer is selected among water soluble or insoluble natural, synthetic, semisynthetic or modified oligo- or polysaccharides, such as but not limited to cellulose, and cellulose derivatives such as methylcellulose, carboxymethyl cellulose and hydroxyethyl cellulose; galactomammans such as guar gum and locust bean gum; hemicelluloses such as xylan, arabinoxylan, arabinogalactan, araban, glucan; pectin, gum arabic, chitosan, xanthan gum, psyllium seed, ispaghula husk, plantago ovata seeds, karaya gum and mixtures of such compounds.

7. The method of any of the claims 1 -6, wherein the at least one enzyme is: Aspergillus oryzae ferulic acid esterase variant and the polymer is selected among cellulose, hydroxyethyl cellulose and gum arabic;

Candida antarctica lipase A or B and the polymer is hydroxyethyl cellulose, guar gum or gum

Arabic;

Fusaήum oxysporum lipase and the polymer is hydroxyethyl cellulose, guar gum or gum arabic;

Geotrichum candidum lipase 1 and the polymer is cellulose or hydroxyethyl cellulose;

Humicula insolens cutinase variant and the polymer is cellulose, hydroxyethyl cellulose, guar gum or gum arabic;

Hyphozyma lipase and the polymer is hydroxyethyl cellulose, Guar gum or gum Arabic; Rhizomucor miehei lipase and the polymer is cellulose, hydroxy ethyl cellulose or guar gum; or

Thermomyces lanuginosa lipase or Thermomyces lanuginosa lipase variant and the polymer is gum Arabic.

8. A composition comprising an efficient amount of at least one enzyme capable of transferring an acyl group from a triglyceride to an indigestible polymer, optionally the indigestible polymer and at least one inert excipient.

9. The composition of claim 8, wherein the at least one enzyme is selected among: hydrolases such as lipases, acetyl esterases, acetyl transferases, esterases, cutinases, phospholipases and proteases.

10. The composition of claim 9, where the at least one enzyme is selected among: Aspergillus oryzae ferulic acid esterase and variants thereof; Candida antarctica lipase A;

Candida antarctica lipase B;

Fusarium oxysporum lipase;

Geotrichum candidum lipase 1 ;

Humicula insolens cutinase and variants thereof; Hyphozyma lipase;

Rhizomucor miehei lipase; and

Thermomyces lanuginosa lipase and variants thereof.

1 1. The composition of any of the claims 8-10, further comprising the ingestible polymer.

12. The composition of any of the claims 8-1 1 , further comprising one or more nutritionally beneficial components such as vitamins and minerals.

13. Use of a composition of claims 8-12 for the treatment or prevention of obesity.

14. The use of claim 13, wherein the composition is administered less than 60 minutes before or after a triglyceride containing meal, preferably less than 30 minutes before or after a triglyceride containing meal and most preferred less than 15 minutes after a triglyceride containing meal.

15. The use of claim 13 or 14, where the treatment or prevention of obesity is a cosmetic treatment.