SUBSTANCE FOR REDUCTION OF CHOLESTEROL AS WELL AS LIPIDS CONTENT

Abstract: The invention relates to a substance for reduction of cholesterol as well as lipids content based on the microcrystalline chitosan in a form of solid or gel-like dispersion. The microcrystalline chitosan in a form of powder and/or gel-like dispersion contains a blend of two fractions having differing molecular weights, one reducing the cholesterol (LDL) content and the other reducing the lipids content. The microcrystalline chitosan is further combined with synergistic active substances such as organic acids like ascorbic acid and/or carbonates like sodium carbonate, calcium carbonate and/or magnesium carbonate to increase the cholesterol and lipids reduction.

Description: Substance for reduction of cholesterol as well as lipids content The invention relates to a substance for reduction of serum cholesterol as well as lipids content in mammals, especially humans. The problem caused by lipids and chloesterol to human health are known, such as overweight problems and cardiovascular diseases. Many attempts have been made to reduce the negative effect of lipids and cholesterol contained in the food. One alternative is a special low- fat or fat-free diet. Another solution has been the administration of special drugs or use of special food additives. US-patent 4223023 discloses the use of standard chitosan as a food additive and an orally administrable preparation to reduce the absorption of lipids. The reduction of lipids and cholesterol by microcrystalline chitosan in a form of gel-like dispersion and/or powder characterized by average molecular weight higher than 1 ,000 daltons, preferably 10,000 to 300,000 daltons for reducing cholesterol and higher than 10,000 daltons, preferably 50,000 to 700,000 daltons for reducing lipids, and the deacetylation degree higher than 60 % is described in WO 98/34625. This publication describes the general activity of microcrystalline chitosan for reduction of content of cholesterol and lipids. All well-known slimming or cholesterol reducing agents based on chitosan are non-selective for their activity as well as efficiency of action. The object of this invention is to effectively, specially and selectively reduce cholesterol as well as lipids content using microcrystalline chitosan. For achieving the objects the substance, in a suitable form to be administered orally, is mainly characterized by two fractions of microcrystalline chitosan differing in average molecular weights. These fractions are present simultaneously in the same product which has been prepared by blending the two fractions, each having a characteristic molecular weight distribution and average value of its own. The fraction with lower average molecular weight is responsible for the reduction of cholesterol level, especially the harmful LDL cholesterol, and the fraction with higher average molecular weight incorporated in the same formulation is responsible for the reduction of lipids. It is also possible that the fractions differ in other properties as well. According to an advantageous embodiment the microcrystalline chitosan in the product is combined with synergistic active substances to increase the cholesterol and lipids reduction. The microcrystalline chitosan present in at least two fractions is in a form of powder and/or gel-like dispersion and it is combined with synergistic active substances such as organic acids like ascorbic acid and/or carbonates like sodium carbonate, calcium carbonate and/or magnesium carbonate to improve the efficiency of substance in the body and to increase the cholesterol and lipids reduction. According to a preferred embodiment of the invention the microcrystalline chitosan is combined with synergistic active substances in a weight ratio ranged from 1 :0.001 to 0.001 :1 , preferably 1 :0.01 to 0.01 :1. According to a preferred embodiment of the invention the polydispersity of the blended selected polymer fractions is lower than 4.0. The first fraction has average molecular weight below 140 kD, preferably ranging from 20 to 140 kD for reduction of cholesterol content. The second fraction has average molecular weight higher than 140 kD, preferably in the range of 160-400 kD and most preferably in the range of 200-400 kD for reduction of lipids content and is thus responsible for slimming effect. The weight ratio of the two microcrystalline chitosan fractions ranges from 1 :99999 to 99999:1 , preferably from 1 :4999 to 4999:1. The most practical embodiments have the ratios between 1:19 and 19:1 , preferably between 1 :9 and 9:1. The microcrystalline chitosan is combined with additives characterized by high synergistic action with this natural polymeric material that is able to form complexes with cholesterol and lipids. The synergistic additives consisting of organic acids like ascorbic acid and/or suitable carbonates like sodium carbonate, calcium carbonate and/or magnesium carbonate in a presence of microcrystalline chitosan improve the special form of chitosan activity, protect the mammal organism system as well as protect against removal of the fat-soluble vitamins A and E. Microcrystalline chitosan has a highly developed intrinsic surface, high positive charge and good ability to create hydrogen and ionic bonds and can form a complex with the synergistic active substances. The carbonates contribute to the advantageous behaviour of the administered preparation in the gastrointestinal tract and keep the microcrystalline chitosan well dispersed in the stomach by releasing carbon dioxide upon contact with the acidic environment of the stomach. Further, by suitably choosing the balance between the various carbonates the pH of the preparation can be adjusted. The selectivity of the rate of microcrystalline chitosan action for reduction of cholesterol and/or lipids according to the invention is connected with its fractional content as well as polydispersity degree lower than 4.0. The first fraction of microcrystalline chitosan, defined with viscometric average molecular weight as lying below 140 kD, preferably in a range of 20-140 kD, is subjected to faster enzymatic degradation producing suitable amount of oligoaminosaccharides with assumed structure and ability to combine at high rate with cholesterol, especially its low density fraction LDL (low density lipoprotein). The second fraction of microcrystalline chitosan, defined with viscometric average molecular weight to lie higher than 140 kD, preferably from 160 to 400 kD and most preferably in a range of 200-400 kD, is responsible for reduction of lipids content. Suitable ratio of above fractions in the microcrystalline chitosan used makes possible the selective, specific action against cholesterol on one hand and lipids on the other hand. The substance according to the invention containing microcrystalline chitosan after 4 weeks of patient treatment has reduced the total cholesterol content more than 6 % and the LDL cholesterol more than 15 % in comparison to the initial levels. The advantage of substance containing microcrystalline chitosan is connected with its variety of possible forms from powder to gel-like dispersion. It is not necessary to use a special diet together with the substance according to the invention. The essential components of the substance can be included in an orally administrable unit together with a pharmaceutically accepetable carrier. The substance can thus be in the form of a capsule, tablet or pill. The following methods were used for the determination of microcrystalline chitosan properties: viscometric average molecular weight according to the method described in "Chitin", Pergamon Press, Oxford, 1977 - deacetylation degree according to the method described in the "Cellulose Chemistry and Technology", vol. 11 , p. 633, 1977 polydispersity as well as fraction content according to the Gel Permeation Chromatography method. The contents of total cholesterol, LDL as well as HDL cholesterol triglycerides as well as vitamin A and E were determined according to the standards. The invention is explained further in the following examples which do not restrict the scope of claims. Example 1. The capsules based on hard gelatin with 400 mg of microcrystalline chitosan powder characterized by viscometric average molecular weight of 137 kD, deacetylation degree of 76 % and polydispersity degree of 3.84 and 70 mg of active synergistic agent including 3.8 mg of ascorbic acid, 18.8 mg of sodium carbonate, 25.8 mg of calcium carbonate and 21.6 mg of magnesium carbonate were used for reduction of cholesterol and lipids content. 51 healthy females with body mass index ranging from 28.0 to 34.99 were subjected to in vivo test conducted according to the Declaration of Helsinki and Tokyo with trial plan reviewed by the local ethics committee. The trial was also conducted according to the Good Clinical Practice rules. The patients were asked to maintain their daily living routines including eating habits. The identical capsules containing starch as placebo were used for comparison. The procedure produced a daily dose of 2.4 g of microcrystalline chitosan to be taken in three capsules twice a day. The same system was used for placebo. The patient characteristic is presented in Table 1. Table 1. Patients characteristic before treatment (data given as means ± SD) Patient Treatment group Chitosan/range Placebo/range Totals Number 24 27 51 Age (year) 41.4 ± 7.8 / 24-57 41.3 ± 7.1 / 19-55 41.3 ± 7.3 Height (cm) 165.1 165.2 165.1 Weight (kg) 83.33 ± 9.2 / 71-105 84.63 ± 7.8 / 71 -101 84 ± 8.4 Body mass index 30.39 ± 2.1 / 28-35 30.81 ± 2.2 / 28-35 30.61 ± 2 fs-chol.tot. (mmol/l) 5.55 ± 0.9 5.48 ± 0.8 5.51 ± 0.9 fs-HDL (mmol l) 1.33 ± 0.3 1.34 ± 0.4 1.37 ± 0.4 fs-Trigl. (mmol/l) 1.24 ± 0.6 1.40 ± 0.6 1.33 ± 0.6 fs-LDL (mmol/l) 3.65 ± 0.9 3.45 ± 0.7 3.55 ± 0.8 The treatment parameters after 4 weeks are presented in Table 2. Table 2. Treatment parameters after 4 weeks Parameter Chitosan Placebo Number of patients 24 27 fs-chol.tot. (mmol/l) 5.23 + 0.8 5.41 + 0.8 difference - 0.32 - 0.07 fs-HDL (mmol/l) 1.48 + 0.4 1.54 + 0.5 difference + 0.15 + 0.14 fs-LDL (mmol/l) 3.18 ± 0.8 3.27 ± 0.7 difference - 0.47 - 0.18 fs-Trigl. (mmol/l) 1.26 + 0.6 1.34 + 0.8 difference + 0.2 - 0.06 As the result of 4 weeks long test the serum mean total cholesterol decreased for the patients treated by microcrystalline chitosan by 0.32 mmol/l, whereas with the placebo group only by 0.07 mmol/l. The low density cholesterol (LDL) for microcrystalline chitosan treated group was reduced by 0.48 mmol/l, whereas for the placebo group by 0.18 mmol/l only. Example 2. 0.1 weight parts of microcrystalline chitosan powder characterized by viscometric average molecular weight of 300 kD, deacetylation degree of 84.7 % and polydispersity degree of 3.42 was blended with 99.9 weight parts of fraction of microcrystalline chitosan characterized by viscometric average molecular weight of 123 kD, deacetylation degree of 84.8. % and polydispersity degree of 3.67. The blended microcrystalline chitosan powder was characterized by viscometric average molecular weight of 122 kD, deacetylation degree of 84,8 % and polydispersity degree of 3.65. 400 mg of above blend with addition of 70 mg of active synergistic agents as in Example 1 were used in a form of capsules for reduction of cholesterol and lipids content. The procedure was as in Example 1. The patient characteristics as well as results of treatment after 8 weeks are presented in Table 3. Table 3. The patient characteristics and treatment parameters after 8 weeks Parameter Chitosan Placebo before after before after Number of patients 11 11 14 14 Age, years 38 ± 8 - 44 ± 5 - Height, cm 165 ± 7 - 165 ± 6 - Body mass index 32 ± 2 32 ± 2 33 ± 2 33 ± 2 fs-chol.tot. (mmol/l) 5.75 ± 1.0 5.37 ± 0.9 5.41 ± 0.6 5.19 ± 0.8 difference - - 0.38 - - 0.22 fs-HDL (mmol/l) 1.43 ± 0.29 1.45 ± 0.32 1.27 ± 0.43 1.36 ± 0.51 difference - + 0.02 - + 0.09 fs-LDL (mmol/l) 3.79 ± 0.94 3.49 ± 0.64 3.31 ± 0.82 3.23 ± 0.79 difference - - 0.30 - - 0.08 fs-Trigl. (mmol/l) 1.20 ± 0.45 1.40 ± 0.39 1.33 ± 0.49 1.33 ± 0.43 difference - + 0.2 - 0 As the result of 8 weeks long test, the serum total cholesterol decreased for the patients treated with microcrystalline chitosan by 0.38 mmol/l whereas with the placebo group only by 0.22 mmol/l. The low density cholesterol of LDL for the first group decreased by 0.30 mmol/l whereas for the second group by 0.08 mmol/l only. At the same time the fat soluble vitamins A and E were analyzed before trial as well as after 8 weeks of trial. The results of this test are presented in Table 4. Table 4. The fat soluble vitamins A and E Type of vitamins Chitosan treatment Placebo treatment Vitamin A (μM/l) before 2.44 ± 0.4 2.31 ± 0.3 after 2.41 ± 0.3 2.29 ± 0.3 Vitamin E (μM/l) before 24.80 ± 5.4 24.00 ± 3.8 after 24.60 ± 5.4 24.91 ± 4.1 The results of fat soluble vitamin tests confirm that the used microcrystalline chitosan is fully safe as for their contents. Example 3. Several samples of microcrystalline chitosan (MCCh) powder with a dry weight of 0.15 g and with similar deacetylation degree of DD = 80- 85 %, characterized by viscometric average molecular weight in a range of Mw = 20-180 kD as well as the MCCh formulation with composition presented in Example 1 with viscometric average molecular weight of 130 kD and with a dry MCCh weight of 0.15 g, and a commercially available "fat blocker" containing standard chitosan with DD = 74 % and Mw = 125 kD were used for a test of the fat binding effectivity according to the modified method described by S. Demarger- Andre and A. Domard in Carbohydrate Polymers 22 (1993) 117-126. The fat binding effectivity calculated as the percentage of total fat bound in comparison to initial fatty acids, is presented in following Table 5. Table 5. Fat binding effectivity for different chitosan forms Type of chitosan Mw Fatty acids binding effectivity kD % MCCh 20 44.4 MCCh 165 54.2 MCCh 145 57.0 MCCh 180 52.6 MCCh formulation 130 31.8 Commercial "fat blocker" with 125 17.6 standard chitosan The presented results of the test show that the total fat binding effectivity expresses the action of the MCCh in relation to its average molecular weight. MCCh formulation with composition as in Example 1 has presented the total fat binding effectivity higher than 81 %, in comparison to the commercially available "fat blocker" containing standard chitosan. Raising the average molecular weight will result in still more lipids being absorbed, in the range 75 to 87 %, and the optimal range of Mw is between 200 and 400 kD.

Claims: Claims: 1. Substance for reduction of cholesterol as well as lipids content based on the microcrystalline chitosan in a form of solid or gel-like dispersion, characterized in that the microcrystalline chitosan used in a form of powder and/or gel-like dispersion contains a blend of at least two fractions having differing average molecular weights. 2. Substance as claimed in claim 1 , characterized in that the blended selected polymer fractions have a polydispersity degree lower than 4.0. 3. Substance as claimed in claim 1 or 2, characterized in that the first fraction has average molecular weight below 140 kD, preferably ranging from 20 to 140 kD, to create the reduction of cholesterol content, and the second fraction has average molecular weight above 140 kD, preferably between 160 and 400 kD and most preferably in a range of 200-400 kD to reduce lipids content responsible for slimming effect. 4. Substance as claimed in any of claims 1 to 3, characterized in that the microcrystalline chitosan in a form of powder and/or gel-like dispersion is combined with synergistic active substances such as organic acids like ascorbic acid and/or carbonates like sodium carbonate, calcium carbonate and/or magnesium carbonate to increase the cholesterol and lipids reduction. 5. Substance as claimed in claim 4, characterized in that the microcrystalline chitosan is combined with sodium carbonate, calcium carbonate and magnesium carbonate. 6. Substance as claimed in any of claims 1 to 5, characterized in that the weight ratio of the two microcrystalline chitosan fractions ranges from 1 :99999 to 99999:1. 7. Substance as claimed in claim 6, characterized in that the weight ratio of the two microcrystalline chitosan fractions ranges from 1 :4999 to 4999:1. 8. Substance as claimed in claim 7, characterized in that the weight ratio of the two microcrystalline chitosan fractions ranges from 1 :19 to 19:1 , preferably from 1 :9 to 9:1. 9. Substance as claimed in any of the preceding claims, characterized in that the microcrystalline chitosan is combined with synergistic active substance in a weight ration ranging from 1 :0.001 to 0.001 :1 , preferably 1 :0.01 to 0.01 :1. 10. Substance as claimed in any of the preceding claims, characterized in that it is in form of an orally administrable capsule, tablet or pill.

Inventors: HAEKLI HARRI, POMOELL HARRI, SAEYNAETJOKI ELINA, STRUSZCZYK HENRYK, WULFF MARKETTA, WUOLIJOKI ERKKI, YLITALO PAULI, HAEKLI, HARRI, POMOELL, HARRI, SAEYNAETJOKI, ELINA, STRUSZCZYK, HENRYK, WULFF, MARKETTA, WUOLIJOKI, ERKKI, YLITALO, PAULI
Applicants: HAEKLI HARRI, NOVASSO OY, POMOELL HARRI, SAEYNAETJOKI ELINA, STRUSZCZYK HENRYK, WULFF MARKETTA, WUOLIJOKI ERKKI, YLITALO PAULI, HAEKLI, HARRI, POMOELL, HARRI, SAEYNAETJOKI, ELINA, STRUSZCZYK, HENRYK, WULFF, MARKETTA, WUOLIJOKI, ERKKI, YLITALO, PAULI
Application year: 1999
Main classification: C08B37
IPC codes: A23L1, A61K9, A61K31, A61K33, A61P3, C08B37