PHARMACEUTICAL COMPOSITIONS OF LIPASE-CONTAINING PRODUCTS, IN PARTICULAR OF PANCREATIN

Abstract

Orally administrable pharmaceutical compositions of lipase-containing products, particularly pancreatin and pancreatin-containing products, or of enzyme products which contain at least one lipase of non-animal, especially microbial origin, which improve the lipolytic activity and particularly result in stabilization of the lipase in the acidic pH range. These oral pharmaceutical compositions contain a system which includes at least one surfactant and one co-surfactant and optionally a lipophilic phase, and are self-emulsifiable on contact with a hydrophilic and a lipophilic phase. The compositions according to the invention are suitable for treating or inhibiting maldigestion, especially maldigestion due to chronic exocrine pancreatic insufficiency, in mammals and humans.

Claims

1-50. (canceled)

51. A pharmaceutical composition for oral administration, which is self-emulsifiable on contact with a hydrophilic phase and a lipophilic phase, said composition comprising: (i) an enzyme or enzyme mixture comprising pancreatin or a microbial lipase, wherein the enzyme or enzyme mixture has lipolytic activity and exerts the lipolytic activity in the digestive tract upon oral administration, and (ii) a system comprising: (a) at least one surfactant selected from the group consisting of polyethylene glycol fatty acid esters, polyethylene glycol glycerol fatty acid esters, polyethylene glycol alkyl ethers, polyethylene glycol sterol ethers, polyethylene glycol sorbitan fatty acid esters, sugar esters, polyoxyethylene-polyoxypropylene block copolymers, sodium oleate, sodium lauryl sulfate, sodium lauryl sarcosinate, sodium dioctyl sulfosuccinate, sodium cholate, sodium taurocholate, lauroyl carnitine, palmitoyl carnitine, myristoyl carnitine, alginate salts, propylene glycol alginate, alkylsulfates, sodium docusate, carnitines, and a combination thereof; and (b) at least one co-surfactant which has a hydrophilic lipophilic balance value below 10; wherein the pharmaceutical composition does not comprise any active substances to be absorbed into the bloodstream.

52. The pharmaceutical composition of claim 51, wherein the hydrophilic phase used to form the final emulsion after ingestion is supplied by the physiological fluid of the digestive milieu.

53. The pharmaceutical composition of claim 51, wherein the lipophilic phase used to form the final emulsion in the digestive tract after ingestion is at least partially supplied by the lipids present in ingested food.

54. The pharmaceutical composition of claim 51, wherein the system further comprises a lipidic phase.

55. The pharmaceutical composition of claim 54, wherein (a) the surfactant comprises at least one agent having a hydrophilic-lipophilic balance value above 6 and below 18, and (b) the co-surfactant comprises at least one agent having a hydrophilic-lipophilic balance value below 10; and wherein said system comprising surfactant, co-surfactant and lipidic phase has a hydrophilic-lipophilic balance value of about 4 to 16, and a melting point of at least 20 C.

56. The pharmaceutical composition of claim 55, wherein said system has a melting point of at least 25 C.

57. The pharmaceutical composition of claim 54, wherein the system comprises (a) the surfactant is selected from the group consisting of polyethylene glycol fatty acid esters, polyethylene glycol glycerol fatty acid esters, polyethylene glycol alkyl ethers, polyethylene glycol sterol ethers, polyethylene glycol sorbitan fatty acid esters, sugar esters, polyoxyethylene-polyoxypropylene block copolymers, and mixtures thereof; (b) the co-surfactant is selected from the group consisting of monoacylglycerides, mono-ethers of glycerol, partial esters of propylenglycol, partial esters of polyglycerol, partial esters of ethyl diglycol, and mixtures thereof, and (c) the lipidic phase comprises di- and/or triacylglycerides.

58. The pharmaceutical composition of claim 57, wherein (a) the surfactant is selected from the group consisting of polyethylene glycol fatty acid mono- and/or di-esters with aliphatic C.sub.6-C.sub.22 carboxylic acids, polyethylene glycol glycerol fatty acid esters with aliphatic C.sub.6-C.sub.22 carboxylic acids, polyethylene glycol alkyl mono- and/or di-ethers with aliphatic C.sub.12-C.sub.18 alcohols, and mixtures thereof; (b) the co-surfactant is selected from the group consisting of monoacylglycerides with aliphatic C.sub.6-C.sub.22 carboxylic acids, mono-ethers of glycerol ethers with aliphatic C.sub.12-C.sub.18 alcohols, partial esters of propylenglycol with aliphatic C.sub.6-C.sub.22 carboxylic acids, partial esters of polyglycerol with aliphatic C.sub.6-C.sub.22 carboxylic acids, and mixtures thereof; and (c) the lipidic phase comprises di- and/or triacylglycerides with aliphatic C.sub.6-C.sub.22 carboxylic acids.

59. The pharmaceutical composition of claim 58, wherein (a) the surfactant comprises a mixture of polyethylene glycol mono- and di-esters with aliphatic C.sub.6-C.sub.22 carboxylic acids and/or polyethylene glycol mono- and di-ethers with aliphatic C.sub.12-C.sub.18 alcohols, wherein the polyethylene glycol comprises 6 to 60 ethylene oxide units per molecule, (b) the co-surfactant comprises monoacylglycerides of aliphatic C.sub.6-C.sub.22 carboxylic acids and/or monoethers of glycerol with aliphatic C.sub.12-C.sub.22 alcohols, and (c) the lipidic phase comprises di- and triacylglycerides of aliphatic C.sub.6-C.sub.22 carboxylic acids.

60. The pharmaceutical composition of claim 59, wherein the surfactant is a mixture of polyethylene glycol mono- and di-esters with aliphatic C.sub.6-C.sub.22 carboxylic acids, wherein the polyethylene glycol comprises 6 to 40 ethylene oxide units per molecule, and wherein the co-surfactant comprises monoacylglycerides of aliphatic C.sub.6-C.sub.22 carboxylic acids.

61. The pharmaceutical composition of claim 54, wherein the system comprises 2 to 90% by weight surfactants, 5 to 60% by weight co-surfactants, and 0 to 70% by weight of the lipidic phase, wherein the components surfactant, co-surfactant and the lipidic phase together make up to 100% by weight of the system, and the system makes up 10% to 95% by weight of the pharmaceutical composition.

62. The pharmaceutical composition of claim 61, wherein the system consisting of surfactant, co-surfactant and lipidic phase makes up 10 to 70% by weight of the pharmaceutical composition.

63. The pharmaceutical composition of claim 62, wherein the system consisting of surfactant, co-surfactant and lipidic phase makes up 20 to 50% by weight of the pharmaceutical composition.

64. The pharmaceutical composition of claim 54, wherein the composition contains at least one further pharmaceutically compatible auxiliary, carrier or excipient selected from the group consisting of polyethylene glycol, glycerol, C.sub.1-C.sub.4-alcohols, sugars, cellulosics and mixtures thereof.

65. The pharmaceutical composition of claim 64, wherein said at least one further pharmaceutically compatible auxiliary, carrier, or excipient makes up a maximum of 20% by weight of the composition.

66. The pharmaceutical composition of claim 51, wherein said composition is a solid pharmaceutical preparation in the form of a powder, granules, tablets, or pellets.

67. The pharmaceutical composition of claim 66, wherein the enzyme or enzyme mixtures comprise pancreatin.

68. The pharmaceutical composition of claim 67, wherein the pancreatin makes up 65-85% of the pharmaceutical composition.

69. The pharmaceutical composition of claim 66, wherein the enzyme mixture comprises a mixture of at least one microbial lipase and at least one microbial enzyme selected from the group consisting of proteases and amylases.

70. The pharmaceutical composition of claim 69, wherein microbial enzymes make up 5-80% by weight of the pharmaceutical composition.

71. The pharmaceutical composition of claim 51, wherein the enzyme or enzyme mixture retains at least 90% of the lipolytic activity upon exposure to a buffer having pH 5 for 60 to 90 minutes.

72. The pharmaceutical composition of claim 51, wherein the composition does not contain an enteric coating and wherein the enzyme or enzyme mixture retains at least 90% of the lipolytic activity upon exposure to a buffer having pH 5 for 60 to 90 minutes.

73. A pharmaceutical composition for oral administration, which is self-emulsifiable on contact with a hydrophilic phase and a lipophilic phase, said composition comprising: (i) pancreatin, and (ii) a system comprising: (a) at least one surfactant selected from the group consisting of polyethylene glycol sterol ethers, polyethylene glycol sorbitan fatty acid esters, sugar esters, polyoxyethylene-polyoxypropylene block copolymers, sodium oleate, sodium lauryl sulfate, sodium lauryl sarcosinate, sodium dioctyl sulfosuccinate, sodium cholate, sodium taurocholate, lauroyl carnitine, palmitoyl carnitine, myristoyl carnitine, alginate salts, propylene glycol alginate, alkylsulfates, sodium docusate, carnitines, and a combination thereof; and (b) at least one co-surfactant which has a hydrophilic lipophilic balance value below 10; wherein the pharmaceutical composition does not comprise any active substances to be absorbed into the bloodstream.

74. The pharmaceutical composition of claim 73, wherein the hydrophilic phase used to form the final emulsion after ingestion is supplied by the physiological fluid of the digestive milieu.

75. The pharmaceutical composition of claim 73, wherein the lipophilic phase used to form the final emulsion in the digestive tract after ingestion is at least partially supplied by the lipids present in ingested food.

76. The pharmaceutical composition of claim 73, wherein the system further comprises a lipidic phase.

77. The pharmaceutical composition of claim 76, wherein (a) the surfactant comprises at least one agent having a hydrophilic-lipophilic balance value above 6 and below 18, and (b) the co-surfactant comprises at least one agent having a hydrophilic-lipophilic balance value below 10; and wherein said system comprising surfactant, co-surfactant and lipidic phase has a hydrophilic-lipophilic balance value of about 4 to 16, and a melting point of at least 20 C.

78. The pharmaceutical composition of claim 73, wherein said composition is a solid pharmaceutical preparation in the form of a powder, granules, tablets, or pellets.

79. The pharmaceutical composition of claim 73, wherein the pancreatin makes up 65-85% of the pharmaceutical composition.

80. The pharmaceutical composition of claim 73, wherein the composition does not contain an enteric coating and wherein at least 90% of the lipolytic activity of the pancreatin is retained upon exposure of the composition to a buffer having pH 5 for 60 to 90 minutes.

Description

EXAMPLE 1: PREPARATION OF PANCREATIN-CONTAINING COMPOSITIONS ACCORDING TO THE INVENTION AND COMPARISON OF THE LIPOLYTIC ACTIVITY OF A CONVENTIONAL PANCREATIN FORMULATION AND A PANCREATIN FORMULATION ACCORDING TO THE INVENTION COMPRISING A SYSTEM CONSISTING OF A SURFACTANT, CO-SURFACTANT AND LIPOPHILIC PHASE

a) Conventional Preparation (Pellets) not According to the Invention:

[0153] The conventional formulation was prepared according to the process disclosed in U.S. Pat. No. 5,378,462 (=EP 583,726). 120 g pancreatin and 30 g PEG 4000 were initially dry-mixed and then moistened with 20 g isopropanol. The moist mixture was extruded and then rounded in a suitable rounder with the aid of paraffin oil. The resulting pellets were then dried.

b) Preparation According to the Invention (Pellets) (Example 1A)

[0154] 350 g Gelucire 50/13 was melted in a beaker in a water bath at a temperature of 52 C. The molten mass was mixed with 650 g pancreatin in a dual-jacket mixer for 10 min. The homogenous mixture was placed in a melt extruder for extrusion. Then the extrudate was rounded in a suitable rounder or spheroniser. The resulting pellets had a diameter of 1.0-1.6 mm.

c) Preparation According to the Invention (Granules) (Example 1B)

[0155] 300 g Gelucire 44/14 was melted in a beaker in a water bath at a temperature of 48 C. The molten mass was mixed with 700 g pancreatin in a dual-jacket mixer for approximately 15 minutes and then cooled (melt granulation).

[0156] The activity of the lipase as a function of the pH value and the time-dependent change in lipase activity were determined in accordance with the method of the Federation Internationale Pharmaceutique/European Pharmacopeia (abbreviated hereinafter as FIP/Ph.Eur.). In this standard analysis method, the hydrolytic activity of the lipase in the sample to be investigated is determined with the substrate olive oil. The free fatty acids cleaved off from the triglycerides of the olive oil are titrated with sodium hydroxide solution at a constant pH of 9.0. The lipase activity of the sample is determined by comparing the rate at which the sample hydrolyzes an olive oil emulsion with the rate at which a suspension of a standard pancreas reference powder hydrolyzes the same substrate under the same conditions.

[0157] The absolute and relative lipolytic activity of the conventional preparation and the preparation 1A according to the invention (pellets) determined in each case in accordance with FIP/Ph.Eur. are summarized in the following Table 1. The absolute and relative lipolytic activity of the conventional preparation and the preparation 1B according to the invention (granules) determined in each case in accordance with FIP/Ph.Eur. are summarized in the following Table 2:

TABLE-US-00001 TABLE 1 Absolute and relative lipolytic activity of the standard preparation and the preparation according to the invention (pellets with Gelucire 50/13) Theoretically present Lipase lipase Measured activity activity absolute Relative starting in the lipase lipase pancreatin formulation activity activity Sample [U/g] [U/g] [U/g] [%] Example 1A: 92620 60203 63173 104 Pellets Conventional 92620 74096 69650 94 formulation

TABLE-US-00002 TABLE 2 Absolute and relative lipolytic activity of the standard preparation and the preparation according to the invention (granules with Gelucire 44/14) Theoretically present Lipase lipase Determined activity activity absolute Relative starting in the lipase lipase pancreatin formulation activity activity Sample [U/g] [U/g] [U/g] [%] Example 1B: 84787 59351 67653 114 Granules Conventional 84132 67306 64950 97 formulation

[0158] It is apparent from the data that the addition of systems comprising at least one surfactant, at least one co-surfactant and a lipophilic phase to pharmaceutical preparations of enzymes and enzyme mixtures with at least lipolytic activity, preferably pancreatin and/or pancreatin-like mixtures of digestive enzymes, contributes to improved lipolytic activity compared with conventional formulations of pancreatin known in the prior art.

[0159] The absolute lipolytic activity of the respective pharmaceutical preparation determined in accordance with FIP/Ph.Eur. is expressed with reference to the total lipolytic activity theoretically present in the sample in the form of a relative activity, in order to take account of the different concentrations of pancreatin in the formulations. Comparison of the relative lipase activities determined shows that the relative lipase activity of the preparations according to the invention is approximately 10% higher than those of the conventional formulations. Accordingly, the pharmaceutical preparations according to the invention have increased lipolytic activity compared with conventional pancreatin formulations.

[0160] Furthermore, with reference to the value of the relative lipase activity of the preparation according to the invention of more than 100%, it is apparent that the system consisting of surfactant, co-surfactant and optionally lipophilic phase added to the preparations according to the invention exerts an activating effect on the lipase.

EXAMPLE 2: COMPARISON OF THE STABILITY OF LIPOLYTIC ACTIVITY OF A CONVENTIONAL PANCREATIN FORMULATION AND A PANCREATIN FORMULATION ACCORDING TO THE INVENTION COMPRISING A SYSTEM CONSISTING OF A SURFACTANT, CO-SURFACTANT AND LIPOPHILIC PHASE AT DIFFERENT PH VALUES

[0161] In order to compare the stability of lipolytic activity of a conventional pancreatin formulation and a pharmaceutical formulation according to the invention comprising an enzyme mixture with at least lipolytic activity and a system consisting of at least one surfactant, at least one co-surfactant, and a lipophilic phase, the activity of such a conventional pancreatin formulation was compared with the activity of a mixture of Gelucire and pancreatin incubated for up to 2 hours at different pH values (pH 6, pH 5 and pH 4).

a) Standard Preparation (Pellets):

[0162] The conventional formulation was prepared according to the process disclosed in U.S. Pat. No. 5,378,462. 120 g pancreatin and 30 g PEG 4000 were initially dry-mixed and then moistened with 20 g isopropanol. The moist mixture was extruded and then rounded in a suitable rounder with the aid of paraffin oil. The resulting pellets were then dried.

b) Preparation According to the Invention (Pellets)Example 2

[0163] 300 g Gelucire 44/14 was melted in a beaker in a water bath at a temperature of 48 C. The molten mass was mixed with 700 g pancreatin in a dual-jacket high-speed mixer (melt pelletization).

[0164] The activity of the lipase as a function of the pH value and also the time-dependent change in lipase activity were determined in accordance with the method of the FIP/Ph.Eur. as described above.

[0165] To determine the release behavior of the lipase in the conventional preparation and the preparation according to the invention at different pH values, the samples were incubated in a decomposition apparatus for 2 hours at 37 C. in phosphate buffer solution (pH 6, pH 5, pH 4). Samples were taken at intervals of 15 minutes, and the lipolytic activity in the samples was determined in accordance with the FIP/Ph.Eur. method described above.

[0166] 600 ml buffer (67 mM phosphate, 34 mM NaCl, pH 6.0, pH 5.0, pH 4.0) was heated to a constant temperature of 37 C. in a 1 liter beaker in the decomposition tester. Once the constant temperature had been reached, 2 g of sample was added to the beaker and the decomposition tester was set in motion. The pH value of the phosphate buffer was kept constant during the testing time. Samples were taken at intervals of 15 minutes in each case, and the lipolytic activity in the samples was determined in accordance with FIP/Ph.Eur.

[0167] The relative lipolytic activities of the conventional preparation and the preparation according to the invention determined after 15, 30, 45, 60, 75, 90, 105 and 120 minutes in accordance with FIP/Ph.Eur. are summarised in the following Table 3. Details are given in % of the activity of the respective sample compared with a standard pancreas reference powder in accordance with FIP/Ph.Eur.

TABLE-US-00003 TABLE 3 pH-dependency of the relative lipolytic activity of a conventional pancreatin formulation and a pancreatin preparation according to the invention pH 6 5 4 Time Conventional Conventional Conventional [min] formulation Example 2 formulation Example 2 formulation Example 2 15 93 98 89 100 30 55 30 90 101 82 99 19 39 45 84 97 77 95 13 31 60 81 89 72 95 11 27 75 79 82 71 94 8 23 90 76 76 68 92 7 19 105 72 71 65 89 6 18 120 69 66 61 86 5 17

[0168] It can be seen from this data that the addition of systems consisting of at least one surfactant, at least one co-surfactant, and a lipophilic phase to pharmaceutical preparations of enzymes and enzyme mixtures having at least lipolytic activity, preferably pancreatin and/or pancreatin-like mixtures of digestive enzymes, contributes to stabilizing the lipolytic activity in the acidic pH range. At a pH value of 6, comparison of the lipolytic activity of a conventional pancreatin preparation and a pancreatin preparation according to the invention over a time of 120 minutes shows that the lipolytic activity in both preparations over time decreases only relatively slightly, with the lipolytic activity of the preparation according to the invention increased by approximately 10% compared with the conventional formulation again being observed within the first hour. However, a pH value of 6 is known not to have any great influence on the lipolytic activity. On the other hand, at a pH value of 5 the lipolytic activity of the conventional preparation decreases much more quickly compared with the preparation according to the invention. Whereas the preparation according to the invention has lost less than 10% of the lipolytic activity after 90 minutes, the conventional preparation has only a lipolytic activity of less than 70% remaining compared with a pancreas reference powder in accordance with FIP/Ph.Eur. In particular at a pH value of 4, the preparation according to the invention has a markedly greater lipolytic (residual) activity than the conventional preparation. Accordingly, it can be seen that the pharmaceutical preparations according to the invention have a substantially increased lipolytic activity in an acidic pH medium.

EXAMPLE 3: DOSAGE DEPENDENCE OF A PANCREATIN FORMULATION ACCORDING TO THE INVENTION COMPRISING A SYSTEM CONSISTING OF A SURFACTANT, CO-SURFACTANT AND LIPOPHILIC PHASE ON DIGESTIBILITY OF A HIGH FAT DIET IN THE PANCREATIC EXOCRINE DEFICIENT MINIPIG

[0169] The efficacy of a pelleted pharmaceutical formulation according to the invention comprising an enzyme mixture with at least lipolytic activity and a system consisting of at least one surfactant, at least one co-surfactant, and a lipophilic phase to improve digestion and absorption of fat in minipigs, in which the pancreatic duct has been ligated to induce a complete pancreatic exocrine insufficiency, was analyzed in pigs fed a high (32%) fat diet.

a) Preparation According to the Invention (Pellets)

[0170] 250 g Gelucire 44/14 (Gattefosse) was melted in a beaker in a water bath at a temperature of 48 C. The molten mass was mixed with 750 g pancreatin in a dual-jacket high-speed mixer (melt pelletization). The pellet size of this formulation was similar to that of the commercially available pancreatin product.

Determination of the Activity of Lipase

[0171] Studies were performed in 6 minipigs (Ellegaard, female Gttingen minipigs) with induced pancreatic exocrine insufficiency, weighing 20-30 kg at surgery. The pigs were prepared as previously described by Tabeling R., Gregory P., Kamphues J., 1999: Studies on nutrient digestibilities (pre-caecal and total) in pancreatic duct-ligated pigs and the effects of enzyme substitution. J. Anim. Physiol. a. Anim. Nutr. 82, 251-263. The pancreatic duct was ligated under halothane anaesthesia following a mid-line laparotomy; after which the pigs were chronically fitted with an ileo-caecal re-entrant fistula which was exteriorized on the right flank.

[0172] The success of the pancreatic duct ligation was confirmed by a faecal chymotrypsin test before starting the digestibility studies, which began at least 4 weeks after the pigs had recovered from the surgery.

[0173] The pigs were fed two 250 g meals/day (08.00 and 20.00 h) of a high fat diet (containing: 180 g double-milled Altromin 9021 [modified], 70 g soya oil [Roth]; overall contents are 99% dry matter, 4% crude ash, 32% crude fat, 16% crude protein, 28% starch, 3% crude fiber) plus 0.625 g Cr.sub.2O.sub.3 per meal, mixed with 1 liter water. The meals plus enzymes were carefully mixed together immediately before offering to the pigs. The meals were generally consumed within 5 minutes.

[0174] During the study the pigs received zero, 28,000 or 336,000 FIP lipase units per meal as a formulation according to the invention for 14 days, with a complete collection of faeces for the last 5 days. The faeces (and the feed) were frozen at 20 C., freeze dried, and a Weender analysis was performed [Naumann C, Bassler R. 1993: Die chemische Untersuchung von Futtermitteln. 3. Aufl. VDLUFA-Verlag, Darmstadt] to determine content of dry matter (drying at 103 C. for 8 hours), and crude fat (determined gravimetrically after boiling for 30 minutes with conc. HCl, followed by a 6 hours extraction with petroleum ether). Cr.sub.2O.sub.3 was oxidized to chromate, and chromium content was calculated via extinction at 365 nm [Petry H, Rapp W. 1970: Zur Problematik der Chromoxidbestimmung in Verdauungsversuchen. Z. Tierphysiol. Tierernahrung and Futtermitellkunde 27, 181-189].

[0175] Based on the content of fat and chromium determined per 100 g dry matter feed and faeces (see above), the digestibility of fat (CFA) was calculated according to the formula:

[00001]$\%.Math..Math.\mathrm{fat}.Math..Math.\mathrm{digestibility}=100-\left(\frac{\left[\%.Math..Math.{\mathrm{Cr}}_2.Math.O_3.Math..Math.\mathrm{in}.Math..Math.\mathrm{feed}\right]}{\left[\%.Math..Math.{\mathrm{Cr}}_2.Math.O_3.Math..Math.\mathrm{in}.Math..Math.\mathrm{faeces}\right]}\frac{\left[\%.Math..Math.\mathrm{fat}.Math..Math.\mathrm{in}.Math..Math.\mathrm{faeces}\right]}{\left[\%.Math..Math.\mathrm{fat}.Math..Math.\mathrm{in}.Math..Math.\mathrm{feed}\right]}100\right)$

[0176] The efficacy to improve digestion and absorption of fat in minipigs, in which the pancreatic duct has been ligated to induce a complete pancreatic exocrine insufficiency, measured in the % fat digestibility is given for the preparation according to the invention for different amounts of lipase activity added (given in FIP/Ph.Eur. units).

TABLE-US-00004 TABLE 4 % fat digestibility in minipigs receiving a pancreatin preparation according to the invention 28,000 336,000 FIP FIP 0 Lipase lipase lipase Substitution Units U/meal U/meal No enzymes added 31.66 13.78 Formulation 61.98 11.60 * 79.25 7.00 ** according to the invention *; ** Results are mean S.D.

[0177] It can be seen that the formulation according to the invention caused a very strong and dose-dependent improvement in fat digestibility, already showing a highly efficient improvement at the lower dose tested.

EXAMPLE 4: COMPARISON OF THE STABILITY OF LIPOLYTIC ACTIVITY OF A CONVENTIONAL PANCREATIN POWDER AND A PANCREATIN FORMULATION ACCORDING TO THE INVENTION COMPRISING A SYSTEM CONSISTING OF A SURFACTANT, CO-SURFACTANT AND LIPOPHILIC PHASE AT DIFFERENT PH VALUES

[0178] Further preparations according to the invention were prepared and analyzed with regard to their lipolytic activity in comparison to pancreatin powder at different acidic pH values (pH 6, pH 5 and pH 4).

a) Preparation for Comparison not According to the Invention:

[0179] Pancreatin powder

b) Preparation According to the InventionExample 4A

[0180] 700 g Pancreatin powder

[0181] 200 g Gelucire 44/14 (Gattefosse)

[0182] 100 g Labrasol (Gattefosse)

[0183] The Gelucire 44/14 and the Labrasol were mixed and melted in a beaker in a water bath at a temperature of 48 C. The molten mass was mixed with 700 g pancreatin in a dual-jacket high-speed mixer (melt granulation).

c) Preparation According to the InventionExample 4B

[0184] 800 g Pancreatin powder

[0185] 190 g Maisine (Gattefosse)

[0186] 10 g LPC (Lysophosphatidylcholine)

[0187] The Maisine and the Lysophosphatidylcholine were mixed and melted in a beaker in a water bath at a temperature of 48 C. The molten mass was mixed with 800 g pancreatin in a dual-jacket high-speed mixer (melt granulation).

[0188] The activity of the lipase as a function of the pH value and also the time-dependent change in lipase activity were determined as described in Example 2.

[0189] The release behavior of the lipase at different pH values in the pancreatin powder and the preparation according to the invention was measured as described above for Example 2.

[0190] The relative lipolytic activity determined after 15, 30, 45, 60, 75, 90, 105 and 120 minutes of the pancreatin powder and the preparations Example 4A and Example 4B according to the invention in accordance with FIP/Ph.Eur. are summarized in the following Tables 5A and 5B. Details are given in % of the activity of the respective sample compared with a standard pancreas reference powder in accordance with FIP/Ph.Eur.

TABLE-US-00005 TABLE 5A pH-dependency of the relative lipolytic activity of a standard pancreatin powder and the pancreatin preparation 4A according to the invention pH 6 5 4 Time Pancreatin Pancreatin Pancreatin [min] Powder Example 4A Powder Example 4A Powder Example 4A 15 55.7 90.4 61.5 93.8 19.3 49.0 30 54.9 105.1 53.3 99.0 12.5 32.4 45 48.3 102.9 51.8 95.8 8.4 25.5 60 43.1 97.6 48.1 92.7 6.5 23.1 75 39.0 91.7 41.4 93.2 5.6 19.9 90 35.4 87.2 39.9 91.2 4.3 18.8 105 33.0 82.3 44.8 88.1 3.9 17.8 120 30.4 79.2 39.1 85.6 3.6 16.5

TABLE-US-00006 TABLE 5B pH-dependency of the relative lipolytic activity of a standard pancreatin powder and the pancreatin preparation 4B according to the invention pH 6 5 4 Time Pancreatin Pancreatin Pancreatin [min] Powder Example 4B Powder Example 4B Powder Example 4B 15 55.7 91.9 61.5 83.2 19.3 27.1 30 54.9 87.4 53.3 88.0 12.5 16.4 45 48.3 81.2 51.8 86.3 8.4 13.0 60 43.1 73.7 48.1 83.5 6.5 9.9 75 39.0 69.8 41.4 83.8 5.6 8.9 90 35.4 63.8 39.9 80.0 4.3 7.8 105 33 57.1 44.8 78.4 3.9 6.5 120 30.4 53.0 39.1 74.0 3.6 4.9

[0191] From these data it can be concluded that the addition of systems consisting of at least one surfactant, at least one co-surfactant, and a lipophilic phase to pharmaceutical preparations of enzymes and enzyme mixtures with at least lipolytic activity, preferably pancreatin and/or pancreatin-like mixtures of digestive enzymes, contributes to stabilizing the lipolytic activity in the acidic pH range.

EXAMPLE 5: DETERMINATION OF THE LIPOLYTIC ACTIVITY OF A FORMULATION ACCORDING TO THE INVENTION COMPRISING A LIPASE OF MICROBIAL ORIGIN AND A SYSTEM CONSISTING OF A SURFACTANT, CO-SURFACTANT AND LIPOPHILIC PHASE AND DETERMINATION OF STABILITY AT DIFFERENT PH VALUES

[0192] In order to determine the lipolytic activity and to show the improved stability at acidic pH of a pharmaceutical formulation according to the invention comprising an enzyme mixture with at least lipolytic activity, in which the lipolytic activity is provided by a microbial, optionally recombinantly produced lipase, and a system consisting of at least one surfactant, at least one co-surfactant, and a lipophilic phase, the activity of a pharmaceutical formulation consisting of a mixture of Gelucire and a microbial lipase is determined at different pH values (pH 6, pH 5, pH 4 and 3) and compared to a lipase preparation which has not been stabilized.

a) Preparation According to the Invention (Granulate)

[0193] 562.5 g Gelucire 44/14 was melted in a beaker in a water bath at a temperature of 48 C. 937.5 g of a microbial lipase preparation (the active lipase protein representing about 50 to 60% (w/w) of the dry matter of the preparation) were provided in a dual-jacket mixer at 46 C., then the molten Gelucire was added and the compounds were mixed, first at low speed for 3 min, then for approx. 15 min. at high speed, and finally cooled (melt granulation).

b) Comparison Preparation (not According to the Invention)

[0194] A microbial lipase preparation was prepared by using common spray dry technique.

[0195] The activity of the lipase was determined in accordance with the method of the Federation Internationale Pharmaceutique (abbreviated hereafter to FIP) for microbial lipases, except that the concentration of bile salts is 10 mM.

[0196] Using this standard analysis method, the hydrolytic activity of the lipase in the sample to be investigated is determined using olive oil as a substrate. Released free fatty acids are titrated with sodium hydroxide solution at a constant pH of 7.0. The lipase activity of the sample is determined by comparison of the rate at which the sample hydrolyzes an olive oil emulsion with the rate at which a suspension of a microbial lipase reference powder hydrolyzes the same substrate under the same conditions.

[0197] To determine the pH-stability of the lipase at different pH values in an unstabilized preparation and in the preparation according to the invention, the samples were incubated in a decomposition apparatus for 2 hours at 37 C. in buffer solution (pH 5, pH 4 and pH 3). Samples were taken at intervals of 15 minutes, and the lipolytic activity in the samples was determined in accordance with the FIP method.

[0198] 100 mg of lipase were incubated in 100 ml buffer (0.1 M malonic acid buffer, 1 mM calcium chloride pH 3, 4 and 5) at 37 C. Samples were drawn every 15 mM for a total duration of 2 hours and the lipolytic activity of the samples was determined as follows: An olive oil suspension was prepared by mixing 175 g olive oil with 630 ml of a solution of 700 g of gum arabic and 94.4 g calcium chloride di-hydrate in 5,900 ml water for 15 minutes in a food mixer at maximal speed. The emulsion was cooled to 37 C., and the pH was adjusted to pH 6.8 with sodium hydroxide solution. Three reference solutions were prepared by extracting an appropriate amount of FIP microbial lipase standard with an ice-cold 1% (m/v) solution of sodium chloride such that reference solutions with 50 FIP-U/ml, 65 FIP-U/ml and 80 FIP-U/ml were obtained. Sample solutions were prepared by extracting an amount of sample corresponding to app. 6,500 units activity for 15 minutes with a total of 100 ml ice-cold 1% (m/v) solution of sodium chloride. The samples were further diluted in ice-cold 1% (m/v) solution of sodium chloride such that the titration rate was within the range of the titration rates obtained with the reference solutions.

[0199] The titration rates of the reference and sample solutions were determined by combining in a thermostated vessel 19 ml of olive oil suspension with 10 ml of a solution of 492 mg lipase activating mixture (FIP) in 500 ml of water. The combined solutions were thermostated to 37 C., and the pH was adjusted to pH 7.0. One ml of reference solution or sample solution were added, and the released fatty acids titrated under pH stable conditions with 0.1 M sodium hydroxide solution for a duration of 5 minutes. The titration rate was calculated by linear regression from at least 9 measurement points between the 60th and the 300th second of titration.

[0200] From the titration rates of the reference solutions a calibration function was calculated by linear regression. The calibration function takes the form y=mx+b where y: titration rate; m: slope; x: FIP-units of the reference solution; and b: axis intercept. Using the values thus determined for m and b, the lipolytic activity x was calculated for each sample solution using the formula x=(yb)/m.

[0201] The relative lipolytic activities determined after 0, 15, 30, 45, 60, 75, 90, 105 and 120 minutes of an un-stabilized microbial lipase preparation and the preparation according to the invention in accordance with FIP are determined. A comparison of the results obtained can show the improved lipolytic activity and the increased stability within the acidic pH range of the formulation according to the invention comprising a microbial lipase preparation over the un-stabilized lipase preparation.

[0202] The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations within the scope of the appended claims and equivalents thereof.