Capsules containing high doses of krill phospholipids

Abstract

Oral capsules have been dismissed as a dosage form for delivery high dosages of krill phospholipids, but by purifying these to high levels it is indeed possible to use this dosage form to deliver, for example, 700 mg or more phospholipids per capsule.

Claims

1. A pharmaceutically acceptable capsule including between 700-1200 mg krill phospholipids, wherein the capsule includes liquid contents comprising: (a) 80-95% by weight krill oil, wherein the krill oil includes 85% by weight or more of krill phospholipids; (b) from 2 to 10% of a lower alcohol selected from the group consisting of ethanol, 2-propanol, 1-propanol; and c) an additive selected from the group consisting of glycerol, propylene glycol, and polyethylene glycol.

2. The capsule of claim 1, which is a soft capsule.

3. The capsule of claim 1, wherein the krill phospholipids are a mixture of phospholipid compounds of formula (I) as defined herein: ##STR00002## wherein R.sub.1 and R.sub.2 are each independently selected from a fatty acid moiety of formula COC.sub.nH.sub.m, a fatty acid moiety of formula CH.sub.2C.sub.nH.sub.m, and H, n is an integer in the range of 11-24 and m=2(np)+1, where p is the number of double bonds in the fatty acid moiety, and R.sub.3 is H or is selected from a choline, ethanolamine, N-acetylethanolamine, inositol and serine.

4. The capsule of claim 3, wherein the mixture of phospholipid compounds of formula (I): includes less than 300 g astaxanthins per gram of phospholipid; comprises less than 0.01% by weight trimethylamine N-oxide; comprises less than 0.01% by weight homarine; includes less than 1% by weight water; and has less than about 0.03% by weight PUFA polymers.

5. The capsule of claim 3, wherein the mixture of phospholipid compounds of formula (I): includes at least 85% by weight of phospholipid compounds of formula (I); includes less than 300 g astaxanthins per gram of phospholipid; includes less than 0.01% by weight trimethylamine N-oxide; and is free from chloroform and hexane.

6. The capsule of claim 5, wherein the mixture of phospholipid compounds of formula (I): includes less than 0.01% by weight homarine; includes less than 5% by weight water; and includes less than 5% by weight sphingomyelin.

7. The capsule of claim 6, wherein the mixture of phospholipid compounds of formula (I): includes C16:0 and C14:0 fatty acid moieties, wherein the weight ratio of C16:0/C14:0 fatty acid moieties in the mixture is between 10:1 and 18:1; includes C18:4 n3/C18:3 n3 fatty acid moieties, wherein the weight ratio of C18:4 n3/C18:3 n3 fatty acid moieties is between 1:1 and 3:2; less than 1% by weight free fatty acids; has less than 0.005% by weight trimethylamine; and is free from canthaxanthin and flavonoid.

Description

MODES FOR CARRYING OUT THE INVENTION

Example 1: Krill Phospholipid Preparation

(1) This example describes the extraction of oil from a wet material. A coagulum from krill comprising about 70% water, 15% lipids and about 15% other dry matter, mainly proteins, was obtained as described in reference 18. This material was subjected to an extraction procedure as follows. 3500 grams of pure ethanol was added to 1004 grams of the coagulum and stirred for 45 minutes. The mixture was then filtered through a filter paper applying vacuum on the receiving flask to obtain 3854 gram of filtrate. 1179 gram of the filtrate was subjected to evaporation on a rotary evaporator and the obtained dry matter was washed 4 times with a 60% solution of ethanol and finally the solvent was evaporated in a rotary evaporator. The obtained oil, 23.7 gram, was solid at room temperature and comprised 76.8% phospholipids. The content of EPA was 200 mg/gram and the content of DHA 87 mg/gram oil.

Example 2: Krill Phospholipid Preparation with Higher Purity

(2) This example describes an alternative method for extraction of oil from the krill wet material, starting from a frozen paste from krill, which was subjected to an extraction procedure as described below. Unlike example 1, all steps were performed under a nitrogen atmosphere.

(3) A frozen krill paste was subjected to an extraction procedure under a nitrogen atmosphere. The paste comprises about 65% water (assessed via dry matter), 17% lipids (about equal weights of phospholipids and neutral lipids), and about 18% other dry matter, mainly proteins.

(4) 100 kg of the frozen coagulum (20 C.) was added to a vessel. Based on the water content of the coagulum, 350 kg of pure ethanol (99.8% w/w, room temp) was then added to the vessel, giving a final ethanol concentration in the liquid phase of about 84% w/w (350 kg ethanol in 415 kg liquid solvents).

(5) The mixture was stirred in the vessel for 45 minutes, with gentle heating if required. Four final temperatures were studied in separate batches, namely a) 2 C., b) 10 C., c) 15 C. and d) 20 C. After stirring was complete, the mixtures were allowed to settle, and they each included a red-coloured liquid phase and a wet slurry containing shell fragments and other insoluble materials. To remove the liquid phase from the slurry the mixtures were decanted, and the liquid material was put through a coarse filter and then serial-filtered through a 75 m and 5 m cartridge filter to obtain a) 345 kg, b) 366 kg, c) 372 kg or d) 374 kg of filtrate, with residual material remaining in the filtration cake.

(6) The filtrates were then subjected to a sequence of washes. Firstly, de-ionized water was added to give 60% w/w ethanol solutions (a: 137 kg water; b: 149 kg; c: 152 kg; d: 155 kg) and the mixtures were stirred for 10-15 minutes and left to settle for 12-24 hrs at room temperature (15-20 C.) in vessels having a valve at the base. The bottom phase was isolated by draining the bottom phase through the valve, to give between 5.4-9.0 kg of a lipid-rich fraction. The lipid-rich fraction was re-washed 2 to 5 times with 60% w/w ethanol at room temperature to give a final material which contained about 80% by weight phospholipids and 20% neutral lipids. In even the first wash, 85% of TMAO was removed, and the further washes led to material with undetectable TMAO (less than 1 mg N/100 g i.e. at least 20-fold lower than reported in Table X of reference 20).

(7) This lipid-rich material was treated by cold acetone precipitation. Three parts w/w acetone were added and the lipid rich material was dissolved by gentle heating and slow stirring. The stirring was stopped and the mixture was cooled to 4 C. for precipitation. When the precipitation was complete, the upper solvent phase was removed. This cold precipitation procedure was performed three times in total, after first re-dissolving in fresh acetone each time.

(8) The precipitate was then subjected to evaporation and freeze-drying to remove residual acetone and water. Batch c (i.e. extracted at 15 C., then washed 360% EtOH before cold acetone precipitation) provided 1.9 kg of solid material (an orange wax) consisting of 98% phospholipids/1.7% neutral lipids with a water content of 3%. Astaxanthins were present at <2 mg/kg. Amino acids, TMAO and homarine were all below the limit of quantification by standard analytical methods.

(9) Looking at specific fatty acids, proportions were as follows, measured across several batches:

(10) TABLE-US-00002 16/14 C18:3 C18:4 18:4/18:3 C14:0 C16:0 Ratio n-3 n-3 Ratio Wet 6-10% 15-17% .sup.2-2.5 1.4-3.1% 3.5-7%.sup. 2-3.sup. paste Final 1.0-1.5% 15-17% 12-16 1.0-2.5% 1.0-2.5% 1-1.5 material

(11) The purified phospholipids included both ether-linked and ester-linked fatty acids, but 10% or fewer were ether-linked. NMR showed ether-linked fatty acid moieties at position sn1 but not at sn2, and ether-linked fatty acids were either fully saturated or were monounsaturated. Where a phospholipid was a phosphatidylcholine, about 10% of the molecules included ether-linked fatty acids; where a phospholipid was a phosphatidylethanolamine (with or without N-acetylation), about 40% of the molecules included ether-linked fatty acids. PUFAs were seen only with ester linkages. 30-40% by weight of fatty acids in the purified phospholipids were omega-3, and these were distributed at the sn1 and sn2 positions (mainly at sn2). Most of the omega-3 fatty acids were EPA and/or DHA, with about 2 more EPA than DHA.

(12) The lysophosphatidylcholine content (0.2-0.4 mol %) is very low in the purified phospholipids, when compared to the amount in the starting wet material (about 1.2-1.4 mol %). No molecules were detected where fatty acid chains had been lost at both the sn1 and sn2 positions. Lyso-phosphatidylethanolamine (with or without N-acetylation) and lyso-phosphatidylinositol also were not seen.

(13) Thus the krill phospholipids obtained by this method have a high purity and a low level of specific contaminants. They are thus well-suited to pharmaceutical use, but their physical state (waxy solid, with a high viscosity even when warmed to 70 C.) is inconvenient for pharmaceutical preparation.

Example 3: Liquefaction of Purified Phospholipids

(14) The material purified according to example 2 (or similar methods) is very viscous and sticky, which makes it unsuitable for filling into capsules. Thus purified krill phospholipids were thus combined with various hydrophilic and lipophilic additives as viscosity regulating agent:

(15) TABLE-US-00003 Hydrophilic additives Lipophilic additives Ethanol Medium-chain triglycerides Glycerol Castor oil Propylene glycol Sesame oil PEG 300 Glyceryl Trioctanoate PEG 400

(16) In early experiments, purified krill phospholipids (98% puritysee example 2) were mixed with MCTs and fully evaporated. The mixture was dissolved in excess ethanol and excess ethanol was removed by rotary evaporation. Results were as follows, where % s are expressed by weight:

(17) TABLE-US-00004 Krill PLs MCT EtOH (dry mass) (%) (%) (%) Result Test 1 88.52 8.85 2.63 Fine viscosity Test 2 91.34 4.69 3.97 Slightly higher than Test 1 Test 3 89.26 8.92 1.8 Too viscous for easy handling Test 4 89.37 8.94 2.48 Too viscous for easy handling

(18) Based on these results, 19 further compositions were designed and evaluated for viscosity using a variety of different viscosity-reducing agents. Viscosity was measured at both 25 C. and 40 C., using a shear rate of 100 s.sup.1 using an AG-G2 Rheometer with 40 mm plate/plate and 500 m gap. Viscosity measurements were as follows, along with an observation whether the compositions remained homogeneous:

(19) TABLE-US-00005 Sample KPL Viscosity-reducing Ethanol Viscosity Homog- # (wt %) agent (wt %) (wt %) Temperature (Pa .Math. s) eneous 1 82.9 MCT 9.3 7.8 25 C. 3 No 82.9 9.3 7.8 40 C. 3 No 2 85.5 MCT 9.6 4.9 25 C. 12 No 85.5 9.6 4.9 40 C. 7 No 3 89.1 MCT 4.7 6.2 25 C. 7 No 89.1 4.7 6.2 40 C. 2 No 4 90.8 MCT 5.1 4.1 25 C. 52 No 90.8 5.1 4.1 40 C. 5 No 5 85.6 MCT 12.9 1.5 25 C. 31 No 85.6 12.9 1.5 40 C. 5 No 6 92.3 None 0.0 7.7 25 C. 4 Yes 92.3 0.0 7.7 40 C. 6 Yes 7 83.6 MCT 12.8 3.7 25 C. 7 No 83.6 12.8 3.7 40 C. 12 No 8 84.2 Castor oil 8.5 7.3 25 C. 3 No 84.2 8.5 7.3 40 C. 2 No 9 88.1 Castor oil 8.8 3.1 25 C. 8 No 88.1 8.8 3.1 40 C. 6 No 10 85.5 Glyceryl 8.6 6.0 25 C. 4 No 85.5 Trioctanoate 8.6 6.0 40 C. 5 No 11 83.1 Glyceryl 8.3 8.6 25 C. 1 No 83.1 Trioctanoate 8.3 8.6 40 C. 3 No 12 86.2 Sesame oil 8.8 5.0 25 C. 4 No 86.2 8.8 5.0 40 C. 11 No 13 83.5 Sesame oil 8.5 8.0 25 C. 3 No 83.5 8.5 8.0 40 C. 4 No 14 81.3 Glycerol 8.2 10.5 25 C. 2 Yes 81.3 8.2 10.5 40 C. 2 Yes 15 85.1 Glycerol 8.5 6.5 25 C. 10 Yes 85.1 8.5 6.5 40 C. 8 Yes 16 85.2 Propylene 8.7 6.1 25 C. 2 Yes 85.2 glycol 8.7 6.1 40 C. 2 Yes 17 89.0 Propylene 8.9 2.0 25 C. 7 Yes 89.0 glycol 8.9 2.0 40 C. 4 Yes 18 84.5 PEG 300 8.5 7.0 25 C. 2 Yes 84.5 8.5 7.0 40 C. 1 Yes 19 89.2 PEG 300 9.1 1.8 25 C. 12 Yes 89.2 9.1 1.8 40 C. 4 Yes

(20) Thus, for instance, composition 16 has a suitable viscosity for convenient liquid processing and has a phospholipid concentration of 835 mg/mL, thus permitting a 700 mg dose to be achieved using a volume of around 840 L, which will fit inside a size 00 hard capsule or a 14 minim soft capsule.

(21) Further test compositions were prepared using 80% by weight of the purified phospholipids and a variety of viscosity-reducing agents using ethanol and one further component. The appearance and viscosity of these compositions was evaluated visually at room temperature. The following compositions have been prepared (% s are by weight):

(22) TABLE-US-00006 Viscosity- Krill reducing PL agent EtOH Visual result Test 5 80 Sesame 15 5 Homogenous. Viscous but oil flows OK. Test 6 80 Soybean 15 5 Homogenous. Viscous but oil flows OK. Test 7 80 Propylene 15 5 Flows nicely, but a few lumps glycol were observed indicating inhomogeneity. Test 8 80 PEG 300 15 5 Homogenous, very good flow. Test 9 80 PEG 400 15 5 Homogenous, very good flow (even better than Test 8). Test 10 85 PEG 400 15 0 Homogenous but very viscous. Test 11 85 PEG 400 12.5 2.5 Homogenous. Flows OK. Test 12 90 PEG 400 7.5 2.5 Homogenous but extremely viscous. Test 13 87 PEG 400 10.5 2.5 Homogenous. Flows OK.

(23) Further viscosity-reducing agents were tested as follows:

(24) TABLE-US-00007 Krill Viscosity (Pa .Math. s) PL EtOH PEG400 PEG600 MCT Glycerol 25 C. 30 C. Test 14 82.5 5 12.5 2987 1848 Test 15 82.5 5 12.5 4229 3345 Test 16 80 7.5 7.5 5 1595 Test 17 80 7.5 7.5 5 737 Test 18 82.5 5 12.5 2271 1708

(25) All five of these were clear and homogenous, although Test 15 was not as stable as the others.

(26) In these test compositions, the concentration of phospholipids in the liquid materials comfortably exceeded 680 mg/mL, and generally fell within the range of about 720-850 mg/mL. Thus with a capsule size above 16 minims it is straightforward to achieve a per-capsule dose of >700 mg.

Example 4: Encapsulation Studies

(27) Krill phospholipids of 98% purity (example 2) were formulated according to the Test 9 viscosity-reducing agent, whereas a krill extract with 80% phospholipids purity (example 1) was formulated like the Test 11 viscosity-reducing agent. The flow properties of these liquid materials were suitable for filling into oral capsules (viscosity at 30 C. was 1270 mPa.Math.s for the 98% purity material, and 1870 mPa.Math.s for the 80% purity material), so it was encapsulated into soft gelatin capsules which included both glycerol and sorbitol as plasticisers. Stability was assessed over a 12 week period, with stability parameters including the amount in the capsule contents of phosphatidylcholine, lysophosphatidylcholine, ethanol, water, glycerol, and sorbitol. Results at the start of the study and after 12 weeks at 40 C. were as follows:

(28) TABLE-US-00008 Parameter 80% purity 98% purity Time zero 12 weeks Time zero 12 weeks PC content 59.4% 58.7% 70.4% 70.4% LPC content 0.42% 1.09% 0.25% 0.99% EtOH content 1.3% 1.4% 3.7% 3.5% H.sub.2O content 3.8% 4.2% 5.2% 5.3% Glycerol content 2.8% 6.5% 3.8% 7.8% Sorbitol content 0.3% 0.8% 0.4% 1.0%

(29) Therefore the phospholipids were stable in the capsules, with very low levels of lyso-PC breakdown products being seen even after 12 weeks at 40 C. It was clear, however, that components from the capsule material (water, sorbitol, and particularly glycerol) were entering the contents. The liquid material which was used to fill the capsules contained no glycerol or sorbitol, and 1.2% water.

(30) One way to inhibit migration of glycerol from the capsule into the contents is to include glycerol already within the liquid contents, as part of the viscosity-reducing agent.

(31) Ethanol levels at time zero were 1.3% or 3.7%, whereas in the initial phospholipid mixture which was used to fill the capsules the level was 2.5% or 5%, respectively. Thus from 25-50% of the ethanol escaped from the capsules between filling and the beginning of the stability study i.e. during drying and storage. This loss of solvent is accompanied by an increase in viscosity of the contents.

(32) It will be understood that the invention is described above by way of example only and modifications may be made while remaining within the scope and spirit of the invention.

REFERENCES

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