CA1111347A - Liposome delivery systems - Google Patents
Liposome delivery systemsInfo
- Publication number
- CA1111347A CA1111347A CA320,271A CA320271A CA1111347A CA 1111347 A CA1111347 A CA 1111347A CA 320271 A CA320271 A CA 320271A CA 1111347 A CA1111347 A CA 1111347A
- Authority
- CA
- Canada
- Prior art keywords
- delivery system
- sterol
- medicament
- water
- aliphatic lipid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
Abstract
Abstract of the Disclosure liposomes formed from liquid crystals of a sterol, for example cholesterol, and an aliphatic lipid capable of forming micelles in water, for example sodium oleate may be used to encapsulate medicaments for oral or parenteral administration.
Description
Case 600-6802 34~7 Liposome Delivery Systems . . . _ . . .
This invention relates to liposome delivery systems for medicaments.
Liposomes may be formed when liquid crystals of certain lipids are agitated, for example by ultrasonic radiation.
They are microvesicles generally from half a micron to several microns diameter, which normall~ have a multilamellar structure in which the liposome walls consist of several layers of oriented lipid molecules, giving an "onion-like"
structure. Larger, unilamellar liposomes may also be preparea.
It is known that medicaments may be entrapped within, or between the layers of, liposomes at the time they are formed. In all cases which have been described, the liposomes are phospholipid-cholesterol systems, which are prepared by dissolving the phospholipid and cholesterol in a solvent which is evaporated to leave a thin film of lipid. An aqueous solution of medicament is added, which swells the phospho-lipid/cholesterol mixture to give a li~uid crystalline system. Ultrasonic treatment then gives liposom.es which en-capsulate the medicament.
It has now been found that liposome~ capable of en-capsulating drugs may be prepared without the use of complex -, ~ ' , .
. ~
. ~ .
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This invention relates to liposome delivery systems for medicaments.
Liposomes may be formed when liquid crystals of certain lipids are agitated, for example by ultrasonic radiation.
They are microvesicles generally from half a micron to several microns diameter, which normall~ have a multilamellar structure in which the liposome walls consist of several layers of oriented lipid molecules, giving an "onion-like"
structure. Larger, unilamellar liposomes may also be preparea.
It is known that medicaments may be entrapped within, or between the layers of, liposomes at the time they are formed. In all cases which have been described, the liposomes are phospholipid-cholesterol systems, which are prepared by dissolving the phospholipid and cholesterol in a solvent which is evaporated to leave a thin film of lipid. An aqueous solution of medicament is added, which swells the phospho-lipid/cholesterol mixture to give a li~uid crystalline system. Ultrasonic treatment then gives liposom.es which en-capsulate the medicament.
It has now been found that liposome~ capable of en-capsulating drugs may be prepared without the use of complex -, ~ ' , .
. ~
. ~ .
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- 2 - 600-68~2 phospholiplds. In place of a phospholipid is used an ali-phatic lipid capable of forming micelles in water.
Accordin~ly, the present invention provides a liposome medicament delivery system in which a medicament is encap-sulated within liposomes comprising a sterol and analiphatic lipid capable of formin~ micelles in water.
The aliphatic lipid may be any pharmacologically acceptable aliphatic surface-active compound which forms micelles in aqueous media when present in concentrations above the critical micelle concentration (CMC). A micelle is a colloidal-sized aggregation of surfactant molecules in an aqueous medium, in which the molecules are oriented with their hydrophilic ends outwards and their lipophilic ends inwards. Preferably the aliphatic lipid is a sodium or potassium salt of a C4 to C18 C~turate~ or unsaturated fatty acid. Suitable acids include butyric, isovaleric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, oleic, linoleic and linolenic acids. The salt is more preferably the salt of an unsaturated fatty acid, particularly of 14 to 18 carbon atoms. Particularly preferred are sodium and potassium oleate.
The sterol may be any pharmacologically acceptable sterol capable of forming liposomes with the above aliphatic lipids. Preferred sterols are cholesterol, ~-sitosterol, 25 desmosterol, 7-keto-cholesterol, ~-cholestanol and estradiol, particularly cholesterol and ~-sitosterol.
Accordin~ly, the present invention provides a liposome medicament delivery system in which a medicament is encap-sulated within liposomes comprising a sterol and analiphatic lipid capable of formin~ micelles in water.
The aliphatic lipid may be any pharmacologically acceptable aliphatic surface-active compound which forms micelles in aqueous media when present in concentrations above the critical micelle concentration (CMC). A micelle is a colloidal-sized aggregation of surfactant molecules in an aqueous medium, in which the molecules are oriented with their hydrophilic ends outwards and their lipophilic ends inwards. Preferably the aliphatic lipid is a sodium or potassium salt of a C4 to C18 C~turate~ or unsaturated fatty acid. Suitable acids include butyric, isovaleric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, oleic, linoleic and linolenic acids. The salt is more preferably the salt of an unsaturated fatty acid, particularly of 14 to 18 carbon atoms. Particularly preferred are sodium and potassium oleate.
The sterol may be any pharmacologically acceptable sterol capable of forming liposomes with the above aliphatic lipids. Preferred sterols are cholesterol, ~-sitosterol, 25 desmosterol, 7-keto-cholesterol, ~-cholestanol and estradiol, particularly cholesterol and ~-sitosterol.
3~ ~
- 3 - 600-680~
The nature of the medicament to be encapsulated is not critical. Suitable medicaments include vaccines and antigens, as well as drugs. Suitable dru~s include hormones, e.g.
insulin; ergot alkaloids, e.g. dihydroergotoxin, dihydro-ergotamine and bromocryptine and anoretics, e.g. mazindol.
The invention also provides a process for the prepara-tion of the liposomal delivery system, in which either a) solid crystals of the sterol are contacted with an aqueous micellar solution of the aliphatic lipid and the medicament, and the resulting liquid crystals are converted to liposomes by ultrasonic irradiation, b) the sterol is dissolved in a mixture of an aqueous medium containing the aliphatic lipid and medicament with a water-miscible solvent, and the solvent is evaporated, or c) the sterol and medicament are dissolved in a common solvent, the solvent is evaporated, the residue is contacted with an aqueous micellar solution of the aliphatic lipid and the resulting liquid crystals are converted to liposomes by ultrasonic irradiation.
In process variant a), the sterol crystals are con-tacted with the micellar solution at a temperature of up to 60C, preferably 20-50C, more preferably 25-45C.
Penetration of the sterol crystals by the micelles requires from 2-60 minutes, and sonication should be carried out only after penetration is complete and a liquid crystal system has been formed.
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- 3 - 600-680~
The nature of the medicament to be encapsulated is not critical. Suitable medicaments include vaccines and antigens, as well as drugs. Suitable dru~s include hormones, e.g.
insulin; ergot alkaloids, e.g. dihydroergotoxin, dihydro-ergotamine and bromocryptine and anoretics, e.g. mazindol.
The invention also provides a process for the prepara-tion of the liposomal delivery system, in which either a) solid crystals of the sterol are contacted with an aqueous micellar solution of the aliphatic lipid and the medicament, and the resulting liquid crystals are converted to liposomes by ultrasonic irradiation, b) the sterol is dissolved in a mixture of an aqueous medium containing the aliphatic lipid and medicament with a water-miscible solvent, and the solvent is evaporated, or c) the sterol and medicament are dissolved in a common solvent, the solvent is evaporated, the residue is contacted with an aqueous micellar solution of the aliphatic lipid and the resulting liquid crystals are converted to liposomes by ultrasonic irradiation.
In process variant a), the sterol crystals are con-tacted with the micellar solution at a temperature of up to 60C, preferably 20-50C, more preferably 25-45C.
Penetration of the sterol crystals by the micelles requires from 2-60 minutes, and sonication should be carried out only after penetration is complete and a liquid crystal system has been formed.
, , , - : - ~ ~ . I .
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- 4 - 600-6802 In process variant b), the water-miscible solvent is suitably acetone, dioxane, or a Cl ~alcohol. The sterol is dissolved with stirring at a temperature of up to 60C, preferably 20-50~c. Evaporation of the water-miscible solvent reduces the liquid crystals to liposomes without ultrasonic irradiation being required.
Process variant c) is suitable where the medicament to be encapsulated is not soluble in the aqueous micellar solution of the aliphatic lipid. A suitable common solvent for the sterol and the medicament may for example be chloro-form, benzene or petroleum ether. The residue after evapor-ation is contacted with the a~ueous micellar solution and subjected to ultrasonic irradiation under the same preferred conditions as in variant a).
In all three process variants, the process is pre-ferably carried out under an inert atmosphere, for example nitrogen, to prevent autooxidation of the lipid and/or the sterol.
The proportions of aliphatic lipid, st-erol and water in the liposome delivery systems of the invention may be:
aliphatic lipid 0.03%-20%, sterol 1.0%-55%, water 45%-97%.
Preferably the proportions are: aliphatic lipid 1.0%-15%, sterol 1~-40~, water 50~-97%; more preferably aliphatic lipid 5%-10~, sterol 1.0%-10%, water 75%-95~ (by weight).
In general, suitable proportions are those in which liquid crystals are seen to be formed on contacting the sterol with a micellar solution of the aliphatic lipid in , . ::; :.
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water. For the system sodium oleate/cholesterol/water, these proportions have been determined experimentally by adding aqueous solutions of sodium oleate to solid cholesterol in the proportions shown in Table I, equilib-rating for 48 hours and examining for the presence of liquid crystals.
T a b 1 e Sample ~ ~ % liq. crystals No. oleate cholesterol water formed ?
1 5 5 90 yes 2 5 15 80 yes 3 5 25 70 yes 4 5 35 60 yes yes 6 5 55 40 no 7 10 5 85 yes 8 10 15 75 yes 9 10 25 65 yes yes 11 10 45 45 yes 12 10 55 35 no 13 15 5 80 yes 14 15 15 70 yes yes 16 15 35 50 yes 17 20 15 65 yes 18 20 5 75 yes Figure 1 shows a phase diagram of the three-component system sodium oleate/cholesterol/water. Liquid crystal-line phases were found within the area ABCDE on the phase diagram. Similar phase diagrams may be constructed for other systems according to the invention.
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3~7 - ~ - 600-6802 The composition of the total delivery system is not of course the same as that of the liposomes themselves. Thus the liposomes will contain essentially all the sterol and much of the aliphatic lipid present in the system, but will
Process variant c) is suitable where the medicament to be encapsulated is not soluble in the aqueous micellar solution of the aliphatic lipid. A suitable common solvent for the sterol and the medicament may for example be chloro-form, benzene or petroleum ether. The residue after evapor-ation is contacted with the a~ueous micellar solution and subjected to ultrasonic irradiation under the same preferred conditions as in variant a).
In all three process variants, the process is pre-ferably carried out under an inert atmosphere, for example nitrogen, to prevent autooxidation of the lipid and/or the sterol.
The proportions of aliphatic lipid, st-erol and water in the liposome delivery systems of the invention may be:
aliphatic lipid 0.03%-20%, sterol 1.0%-55%, water 45%-97%.
Preferably the proportions are: aliphatic lipid 1.0%-15%, sterol 1~-40~, water 50~-97%; more preferably aliphatic lipid 5%-10~, sterol 1.0%-10%, water 75%-95~ (by weight).
In general, suitable proportions are those in which liquid crystals are seen to be formed on contacting the sterol with a micellar solution of the aliphatic lipid in , . ::; :.
': . ' ' , : , :,: ~ . : :
. ,: ; ,:
34'~
water. For the system sodium oleate/cholesterol/water, these proportions have been determined experimentally by adding aqueous solutions of sodium oleate to solid cholesterol in the proportions shown in Table I, equilib-rating for 48 hours and examining for the presence of liquid crystals.
T a b 1 e Sample ~ ~ % liq. crystals No. oleate cholesterol water formed ?
1 5 5 90 yes 2 5 15 80 yes 3 5 25 70 yes 4 5 35 60 yes yes 6 5 55 40 no 7 10 5 85 yes 8 10 15 75 yes 9 10 25 65 yes yes 11 10 45 45 yes 12 10 55 35 no 13 15 5 80 yes 14 15 15 70 yes yes 16 15 35 50 yes 17 20 15 65 yes 18 20 5 75 yes Figure 1 shows a phase diagram of the three-component system sodium oleate/cholesterol/water. Liquid crystal-line phases were found within the area ABCDE on the phase diagram. Similar phase diagrams may be constructed for other systems according to the invention.
,.. ;~
, - ~ ,,: .
~ : :
3~7 - ~ - 600-6802 The composition of the total delivery system is not of course the same as that of the liposomes themselves. Thus the liposomes will contain essentially all the sterol and much of the aliphatic lipid present in the system, but will
5 be suspended in a continuous aqueous phase, which may also contain micelles of the aliphatic lipid, or molecules of the aliphatic lipid at concentrations below the CMC.
The liposomes prepared according to the invention are from 10 to 600 nm in diameter.
The liposome delivery system of the invention may be used both for oral and for parenteral administration of medicaments, optionally after concentration or isolation of the lipsomes for example by ultracentrifugation. Oral ad-ministration is preferred, ho~ever, as the lipo~ome encapsu-lation may serve to protect drugs such as insulin which are labile in the digestive system. For oral administration the liposome suspension may be admixed with pharmacologically acceptable diluents or carriers and with conventional adjuvants such as flavourings and colourings, and administered in such forms as syrups, elixirs, capsules etc. For parenteral ad-ministration, the concentrated or isolated liposomes may be suspended in a suitable carrier liquid, for example sterile distilled ~tater or physiological saline solution. Supposi-torial administration may also be utilised.
The following Examples illustrate the invention:
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EXP*~PLE 1:
_ I~o concentrations of bovine insulin were prepared:
1) 2500 international units (IU) or 102.9 mg (Specific Acti~ity = 24.3 units/mg) was dissolved in 5 ml of a 16 g/l aqueous sodium oleate solution, 2) 1250 international units o~ 51.45 mg (Specific Activity = 24.3 units/mg) was also dissolved in 5 ml of a 16 g/l aqueous sodium oleate solution.
Each of insulin preparations (1) and t2) were trans-ferred to a 10 ml beaker containing 20 mg of cholesterol.
The cholesterol was prepared by dissolving 200 mg of cho-lesterol in 10 ml of chloroform, then placing one ml of the solution in a 10 ml beaker and removing the solvent under nitrogen at room temperature. Each beaker was flushed with nitrogen, covered with plastic film and placed in a 37C
water bath with mild oscillation for 1 hour to allow penetration of the cholesterol crystals. The liquid crystal suspensions were then sonicated for 2 one minute periods with a Biosonik IV Ultrasonic Generator (Brownwill) with a 4 mm diameter probe. The beakers were placed in an ice bucket during the sonication. After sonication, the final composi-tions of the liposome-containing systems were:
Composition A) 500 IU/ml insulin = 20.9 mg/ml cholesterol = 4 mg/ml sodium oleate = 16 mg/ml Composition B) 250 IU/ml insulin = 10.4 mg/ml cholesterol = 4 mg/ml sodium oleate = 16 mg/ml.
.
- 8 ~$~3~7 600-6802 Suspensions of the liposome compositions A) and B) were given orally to mice, 0.1 ml of the suspension per 10 g body weight being administered. This corresponds to 5000 IU/kg of composition A) or 2500 IU/kg of composition B).
Composition A) was diluted 1:10 for injection i.m. and administered at 10 IU/kg, as was bovine pancreatic insulin (Sigma Chemical).
After two and four hours the animals were sacrificed by anethetizing with 85 mg/]cg of sodium hexobarbital i.p.
and collecting blood via cardiac puncture. The collected blood was placed in an Auto Analyzer Cup containing 0.025 ml of heparin, 1000 units/ml. The blood samples were capped, shaken, and kept in an ice bucket. Glucose was determined by the Auto Analyzer potassium ferricyanide method No. N-2b.
Carboxymethyl cellulose has no effect on blood glucose whether given either by the p.o. or the i.m. route. Therefore, the test materials whether given p.o. or i.m. can be related to the carboxymethyl cellulose control.
Normal fasting blood glucose levels are from 70 to 100 mgt 20 100 ml.
The results are shown in Table II.
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- 10 - 600 6~02 Significant reduction of the blood glucose level was seen 4 hours after oral dosing with 5000 IU/kg insulin in the liposomes of this invention. In the i.m. route, liposome-encapsulated insulin had equivalent activity to unencapsul-ated insulin at 10 IU/kg, indicatin~ that the insulinactivity was no~ effected by incorporation into the lipo-somes.
The liposomes of composition A) were also compared with both positively and negatively charged lecithin-cholesterol liposomes, prepared as described in ~eissmann, G., et al., Proc. i~at. Acad. Sci. USA 72.88-92 (1975); Sessa, G. &
Weissmann, G., J. Biol. Chem. 245. 3295-3301 (1970~;
Weissmann, G., Brand, A. & Franklin, E.C., J. Clin.Invest.
53. 536-543 (1974); and Weissmann, G. & Rita, G.A., ~ature 240. 167-172 (1972), in the presence of 500 IU/ml of hovine insulin.
The liposomes of this invention and the lecithin liposomes were both tested in mice and analyzed as described above.
The results are shown in Table III.
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- 12 ~ 3~'7 600-6802 The results show that at both time periods the liposomes of this invention were superior to the lecithin phospholipid liposomes when given p.o, All preparations were equally effective when given intramuscularly.
EXAMPLE 2:
Liposomes were prepared by dissolving 20 mg (25.3 I~/
mg) of bovine insulin in a solution of 0.5 g sodium oleate in 9 g of water, and then adding O.S cholesterol crystals. The composition was allowed to equilibrate and was sonicated as described in Example 1.
The liposomes were isolated by ultrafiltration in a centrifuge rotated at 20,000 RPM for 2 hours. A 40~ sucrose underlayer was used to fill the tuhe.
Three fractions were isolated;
1) a clear top layer, 2) the liposome layer, and 3) an infranatent above the sucrose layer.
The three fractions were tested by administration to mice at a dose of 0.1 ml of each fraction/lO g of body weight.
The animals weighed from 20-30 g and were fasted overnight prior to testing.
Each fraction had a separate control:
1st fraction - insulin in water (20 mg insulin/9 ml water);
2nd fraction - liposomes previously prepared withouk insulin to which insulin was added prior to admin-istration to the mice ~20 mg insulin/9 ml liposome mixture);
.
l3~'7 - 13 - 600-6~02 rd fraction - 20 mg insulin/9 ml water and included the sucrose cushion to allow for the e~ect on blood glucose.
The animals were dos~d orally as described in Example and the results analyzed as described in Example 1.
The results were as follows:
. . .~ .
BLOOD GLUCOSE M5/100 ~IL
._ ..
Control Fraction ~
Top ~raction 152 + 6 206 + 2 35 .
Liposomes 164 + - 8 136 + 14 17 Infranatant 151 + 16 173 + 20 15 _ . .. __ _ I , Only the liposome entrapped insulin decreases blood glucose. The other fractions tend to elevate blood glucose.
EXAMPLE 3:
_ A slurry of 7 . 5 g cholesterol in 100 ml of 99~ pure acetone is poured into a 2 1 beaker and evaporated so that the cholesterol is dispersed evenly over the botto~ of the beaker.
A 5% aqueous micellar solution of sodium oleate is prepared by dissolving 10 g purified sodium oleate in 200 ml distilled water, and 400 mg dihydroergotoxlne Inethanesulphate is dissolved in this solution. 150 ml of this sodium oleate /
dihydroergo'oxine solution is added to the cholesterol, and the mixture is stirred under nitrogen at roo~ temperature , 3~ ~
- 14 - 600~6802 for 1 hour, then ultracentrifuged at 20,000 rpm for 24 hours.
~ hree fractions were isolated: a) an upper clear yellow layer (40~ vol) containing micelles of sodium oleate, b) a middle viscous yellow-white layer (20% vol) and c) a lower opaque white lower layer (20% vol) containing liposomes.
Spectrophotometric assay for dihydroergotoxin was carried out by the van Urk method, in which equal quantities of the dihydroergotoxln-containing solution and the van Urk reagent are mixed, left to stand for 30 rrinutes, filtered and the absorbance of the solution at 550 nm corrpared with that of a mixture of a standard dihydroergotoxin solution and the van Urk reagent. The van Urk reasent i5 prepared by dissolving 2.5 g p-dimethylaminobenzaldehyde in a mixture of 700 ml distilled water with 1300 ml conc. sulphuric acid, adding 4 ml of 5~ aqueous ferric chloride solution, and making up to 2 litres with distilled water.
The results showed no variation in dihydroergotoxin concentration in the free fractions, indicating that at least sorne of the dihydroergotoxin had become encapsulated in the liposomes.
- ... .
The liposomes prepared according to the invention are from 10 to 600 nm in diameter.
The liposome delivery system of the invention may be used both for oral and for parenteral administration of medicaments, optionally after concentration or isolation of the lipsomes for example by ultracentrifugation. Oral ad-ministration is preferred, ho~ever, as the lipo~ome encapsu-lation may serve to protect drugs such as insulin which are labile in the digestive system. For oral administration the liposome suspension may be admixed with pharmacologically acceptable diluents or carriers and with conventional adjuvants such as flavourings and colourings, and administered in such forms as syrups, elixirs, capsules etc. For parenteral ad-ministration, the concentrated or isolated liposomes may be suspended in a suitable carrier liquid, for example sterile distilled ~tater or physiological saline solution. Supposi-torial administration may also be utilised.
The following Examples illustrate the invention:
- . ~ .' ',' :-' : .
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3~
EXP*~PLE 1:
_ I~o concentrations of bovine insulin were prepared:
1) 2500 international units (IU) or 102.9 mg (Specific Acti~ity = 24.3 units/mg) was dissolved in 5 ml of a 16 g/l aqueous sodium oleate solution, 2) 1250 international units o~ 51.45 mg (Specific Activity = 24.3 units/mg) was also dissolved in 5 ml of a 16 g/l aqueous sodium oleate solution.
Each of insulin preparations (1) and t2) were trans-ferred to a 10 ml beaker containing 20 mg of cholesterol.
The cholesterol was prepared by dissolving 200 mg of cho-lesterol in 10 ml of chloroform, then placing one ml of the solution in a 10 ml beaker and removing the solvent under nitrogen at room temperature. Each beaker was flushed with nitrogen, covered with plastic film and placed in a 37C
water bath with mild oscillation for 1 hour to allow penetration of the cholesterol crystals. The liquid crystal suspensions were then sonicated for 2 one minute periods with a Biosonik IV Ultrasonic Generator (Brownwill) with a 4 mm diameter probe. The beakers were placed in an ice bucket during the sonication. After sonication, the final composi-tions of the liposome-containing systems were:
Composition A) 500 IU/ml insulin = 20.9 mg/ml cholesterol = 4 mg/ml sodium oleate = 16 mg/ml Composition B) 250 IU/ml insulin = 10.4 mg/ml cholesterol = 4 mg/ml sodium oleate = 16 mg/ml.
.
- 8 ~$~3~7 600-6802 Suspensions of the liposome compositions A) and B) were given orally to mice, 0.1 ml of the suspension per 10 g body weight being administered. This corresponds to 5000 IU/kg of composition A) or 2500 IU/kg of composition B).
Composition A) was diluted 1:10 for injection i.m. and administered at 10 IU/kg, as was bovine pancreatic insulin (Sigma Chemical).
After two and four hours the animals were sacrificed by anethetizing with 85 mg/]cg of sodium hexobarbital i.p.
and collecting blood via cardiac puncture. The collected blood was placed in an Auto Analyzer Cup containing 0.025 ml of heparin, 1000 units/ml. The blood samples were capped, shaken, and kept in an ice bucket. Glucose was determined by the Auto Analyzer potassium ferricyanide method No. N-2b.
Carboxymethyl cellulose has no effect on blood glucose whether given either by the p.o. or the i.m. route. Therefore, the test materials whether given p.o. or i.m. can be related to the carboxymethyl cellulose control.
Normal fasting blood glucose levels are from 70 to 100 mgt 20 100 ml.
The results are shown in Table II.
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- 10 - 600 6~02 Significant reduction of the blood glucose level was seen 4 hours after oral dosing with 5000 IU/kg insulin in the liposomes of this invention. In the i.m. route, liposome-encapsulated insulin had equivalent activity to unencapsul-ated insulin at 10 IU/kg, indicatin~ that the insulinactivity was no~ effected by incorporation into the lipo-somes.
The liposomes of composition A) were also compared with both positively and negatively charged lecithin-cholesterol liposomes, prepared as described in ~eissmann, G., et al., Proc. i~at. Acad. Sci. USA 72.88-92 (1975); Sessa, G. &
Weissmann, G., J. Biol. Chem. 245. 3295-3301 (1970~;
Weissmann, G., Brand, A. & Franklin, E.C., J. Clin.Invest.
53. 536-543 (1974); and Weissmann, G. & Rita, G.A., ~ature 240. 167-172 (1972), in the presence of 500 IU/ml of hovine insulin.
The liposomes of this invention and the lecithin liposomes were both tested in mice and analyzed as described above.
The results are shown in Table III.
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- 12 ~ 3~'7 600-6802 The results show that at both time periods the liposomes of this invention were superior to the lecithin phospholipid liposomes when given p.o, All preparations were equally effective when given intramuscularly.
EXAMPLE 2:
Liposomes were prepared by dissolving 20 mg (25.3 I~/
mg) of bovine insulin in a solution of 0.5 g sodium oleate in 9 g of water, and then adding O.S cholesterol crystals. The composition was allowed to equilibrate and was sonicated as described in Example 1.
The liposomes were isolated by ultrafiltration in a centrifuge rotated at 20,000 RPM for 2 hours. A 40~ sucrose underlayer was used to fill the tuhe.
Three fractions were isolated;
1) a clear top layer, 2) the liposome layer, and 3) an infranatent above the sucrose layer.
The three fractions were tested by administration to mice at a dose of 0.1 ml of each fraction/lO g of body weight.
The animals weighed from 20-30 g and were fasted overnight prior to testing.
Each fraction had a separate control:
1st fraction - insulin in water (20 mg insulin/9 ml water);
2nd fraction - liposomes previously prepared withouk insulin to which insulin was added prior to admin-istration to the mice ~20 mg insulin/9 ml liposome mixture);
.
l3~'7 - 13 - 600-6~02 rd fraction - 20 mg insulin/9 ml water and included the sucrose cushion to allow for the e~ect on blood glucose.
The animals were dos~d orally as described in Example and the results analyzed as described in Example 1.
The results were as follows:
. . .~ .
BLOOD GLUCOSE M5/100 ~IL
._ ..
Control Fraction ~
Top ~raction 152 + 6 206 + 2 35 .
Liposomes 164 + - 8 136 + 14 17 Infranatant 151 + 16 173 + 20 15 _ . .. __ _ I , Only the liposome entrapped insulin decreases blood glucose. The other fractions tend to elevate blood glucose.
EXAMPLE 3:
_ A slurry of 7 . 5 g cholesterol in 100 ml of 99~ pure acetone is poured into a 2 1 beaker and evaporated so that the cholesterol is dispersed evenly over the botto~ of the beaker.
A 5% aqueous micellar solution of sodium oleate is prepared by dissolving 10 g purified sodium oleate in 200 ml distilled water, and 400 mg dihydroergotoxlne Inethanesulphate is dissolved in this solution. 150 ml of this sodium oleate /
dihydroergo'oxine solution is added to the cholesterol, and the mixture is stirred under nitrogen at roo~ temperature , 3~ ~
- 14 - 600~6802 for 1 hour, then ultracentrifuged at 20,000 rpm for 24 hours.
~ hree fractions were isolated: a) an upper clear yellow layer (40~ vol) containing micelles of sodium oleate, b) a middle viscous yellow-white layer (20% vol) and c) a lower opaque white lower layer (20% vol) containing liposomes.
Spectrophotometric assay for dihydroergotoxin was carried out by the van Urk method, in which equal quantities of the dihydroergotoxln-containing solution and the van Urk reagent are mixed, left to stand for 30 rrinutes, filtered and the absorbance of the solution at 550 nm corrpared with that of a mixture of a standard dihydroergotoxin solution and the van Urk reagent. The van Urk reasent i5 prepared by dissolving 2.5 g p-dimethylaminobenzaldehyde in a mixture of 700 ml distilled water with 1300 ml conc. sulphuric acid, adding 4 ml of 5~ aqueous ferric chloride solution, and making up to 2 litres with distilled water.
The results showed no variation in dihydroergotoxin concentration in the free fractions, indicating that at least sorne of the dihydroergotoxin had become encapsulated in the liposomes.
- ... .
Claims (17)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A liposome medicament delivery system in which a medicament is encapsulated within liposomes comprising a sterol and an aliphatic lipid capable of forming micelles in water.
2. A delivery system according to Claim 1 in which the sterol is cholesterol, B-sitosterol, desmosterol, 7-keto-cholesterol, B-cholestanol or estradiol.
3. A delivery system according to Claim 2 in which the sterol is cholesterol or B-sitosterol.
4. A delivery system according to Claim 1 in which the aliphatic lipid is a sodium or potassium salt of a C4 to C18 saturated or unsaturated fatty acid.
5. A delivery system according to Claim 4 in which the aliphatic lipid is a sodium or potassium salt of a C14 to C18 unsaturated fatty acid.
6. A delivery system according to Claim 1 in which the sterol is cholesterol and the aliphatic lipid is sodium or potassium oleate.
7. A delivery system according to Claim 1 in which the medicament is insulin.
8. A delivery system according to Claim 1 in which the medicament is an ergot alkaloid.
9. A delivery system according to Claim 1 in which the proportions by weight of aliphatic lipid, sterol and water are 0.03% - 20%, 1.0% - 55% and 45% - 97%
respectively.
respectively.
10. A delivery system according to Claim 9 in which the proportions by weight of aliphatic lipid, sterol and water are 1.0% - 15%, 1.0% - 40% and 50% - 97%
respectively.
respectively.
11. A delivery system according to Claim 10 in which the proportions by weight of aliphatic lipid, sterol and water are 5% - 10%, 1.0% - 10% and 75% - 95 respectively.
12. A delivery system according to Claim 8 in which the aliphatic lipid is sodium oleate and the proportions by weight of sodium oleate, cholesterol and water are such as lie within the area ABCDE on the triangular graph :
13. A process for the preparation of a liposome medicament delivery system according to Claim 1 in which either a) solid crystals of the sterol are contacted with an aqueous micellar solution of the aliphatic lipid and the medicament, and the resulting liquid crystals are converted to liposomes by ultrasonic irradiation, b) the sterol is dissolved in a mixture of an aqueous medium containing the aliphatic lipid and medicament with a water-miscible solvent, and the solvent is evaporated, or c) the sterol and medicament are dissolved in a common solvent, the solvent is evaporated, the residue is contacted with an aqueous micellar solution of the aliphatic lipid and the resulting liquid crystals are converted to liposomes by ultrasonic irradiation.
14. A process according to Claim 13, carried out at 20° - 50°C in an inert gas atmosphere.
15. A pharmaceutical composition comprising a liposome medicament delivery system according to Claim 1 in admixture with a pharmaceutically acceptable diluent or carrier.
16. A pharmaceutical composition according to Claim 15 for parenteral administration, in which the liposomes are suspended in sterile distilled water or physiological saline solution.
17. A pharmaceutical composition according to Claim 15 for oral administration, in the form of a syrup, elixir or capsule.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US87283578A | 1978-01-27 | 1978-01-27 | |
| US872,835 | 1978-01-27 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1111347A true CA1111347A (en) | 1981-10-27 |
Family
ID=25360392
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA320,271A Expired CA1111347A (en) | 1978-01-27 | 1979-01-25 | Liposome delivery systems |
Country Status (19)
| Country | Link |
|---|---|
| JP (1) | JPS54113417A (en) |
| AT (1) | AT368888B (en) |
| AU (1) | AU524529B2 (en) |
| BE (1) | BE873715A (en) |
| CA (1) | CA1111347A (en) |
| DE (1) | DE2902672A1 (en) |
| DK (1) | DK22879A (en) |
| ES (1) | ES477217A1 (en) |
| FI (1) | FI790164A (en) |
| FR (1) | FR2415460A1 (en) |
| GB (1) | GB2013087B (en) |
| IE (1) | IE790144L (en) |
| IL (1) | IL56499A (en) |
| IT (1) | IT1109867B (en) |
| NL (1) | NL7900545A (en) |
| NZ (1) | NZ189477A (en) |
| PT (1) | PT69132A (en) |
| SE (1) | SE7900458L (en) |
| ZA (1) | ZA79349B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113967192A (en) * | 2021-11-09 | 2022-01-25 | 陕西海斯夫生物工程有限公司 | Pharmaceutical composition for accelerating wound healing, preparation method and application thereof |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2026340B (en) * | 1978-07-03 | 1982-12-22 | Ash P | Stabilising microvesicles |
| JPS5874619A (en) * | 1981-10-29 | 1983-05-06 | Nippon Shinyaku Co Ltd | Liposome and its preparation |
| DE3374837D1 (en) * | 1982-02-17 | 1988-01-21 | Ciba Geigy Ag | Lipids in the aqueous phase |
| EP0102324A3 (en) * | 1982-07-29 | 1984-11-07 | Ciba-Geigy Ag | Lipids and surfactants in an aqueous medium |
| DE3421468A1 (en) * | 1984-06-08 | 1985-12-19 | Dr. Rentschler Arzneimittel Gmbh & Co, 7958 Laupheim | LIPID NANOPELLETS AS A CARRIER SYSTEM FOR MEDICINAL PRODUCTS FOR PERORAL USE |
| US4849227A (en) * | 1986-03-21 | 1989-07-18 | Eurasiam Laboratories, Inc. | Pharmaceutical compositions |
| US5077211A (en) * | 1988-07-06 | 1991-12-31 | Applied Genetics, Inc. | Purification and administration of dna repair enzymes |
| WO1992003123A1 (en) * | 1990-08-28 | 1992-03-05 | Liposome Technology, Inc. | Liposome alternative bilayer formulations |
| FR2714621B1 (en) * | 1994-01-06 | 1996-02-23 | Centre Nat Rech Scient | Process for the preparation of liposomes without using an organic solvent. |
| CA2347094C (en) | 1999-08-13 | 2015-02-03 | Suntory Limited | Microorganisms that extracellularly secrete lipids and methods of producing lipid and lipid vesicles encapsulating lipids using said microorganisms |
| JP4088097B2 (en) | 2002-04-26 | 2008-05-21 | サントリー株式会社 | Method for producing highly unsaturated fatty acid-containing lipid |
| FR2979239A1 (en) * | 2011-08-25 | 2013-03-01 | Trophos | LIPOSOME COMPRISING AT LEAST ONE CHOLESTEROL DERIVATIVE |
| AU2013203682B2 (en) * | 2011-08-25 | 2016-03-31 | Trophos | Liposome comprising at least one cholesterol derivative |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3957951A (en) * | 1974-08-23 | 1976-05-18 | International Telephone And Telegraph Corporation | Process for removing contaminants from hot waste gas streams |
| FR2315991A1 (en) * | 1975-06-30 | 1977-01-28 | Oreal | METHOD OF MANUFACTURING AQUEOUS DISPERSIONS OF LIPID SPHERULES AND CORRESPONDING NEW COMPOSITIONS |
| FR2408387A2 (en) * | 1975-06-30 | 1979-06-08 | Oreal | COMPOSITIONS BASED ON AQUEOUS DISPERSIONS OF LIPID SPHERULES |
| FR2374910A1 (en) * | 1976-10-23 | 1978-07-21 | Choay Sa | PREPARATION BASED ON HEPARIN, INCLUDING LIPOSOMES, PROCESS FOR OBTAINING IT AND MEDICINAL PRODUCTS CONTAINING SUCH PREPARATIONS |
-
1979
- 1979-01-18 DK DK22879A patent/DK22879A/en not_active Application Discontinuation
- 1979-01-18 FI FI790164A patent/FI790164A/en unknown
- 1979-01-18 SE SE7900458A patent/SE7900458L/en not_active Application Discontinuation
- 1979-01-24 NL NL7900545A patent/NL7900545A/en not_active Application Discontinuation
- 1979-01-24 DE DE19792902672 patent/DE2902672A1/en not_active Withdrawn
- 1979-01-25 CA CA320,271A patent/CA1111347A/en not_active Expired
- 1979-01-25 AU AU43678/79A patent/AU524529B2/en not_active Ceased
- 1979-01-25 BE BE0/193081A patent/BE873715A/en not_active IP Right Cessation
- 1979-01-25 NZ NZ189477A patent/NZ189477A/en unknown
- 1979-01-25 IL IL56499A patent/IL56499A/en unknown
- 1979-01-26 ES ES477217A patent/ES477217A1/en not_active Expired
- 1979-01-26 PT PT7969132A patent/PT69132A/en unknown
- 1979-01-26 ZA ZA79349A patent/ZA79349B/en unknown
- 1979-01-26 AT AT0058979A patent/AT368888B/en not_active IP Right Cessation
- 1979-01-26 IT IT19649/79A patent/IT1109867B/en active
- 1979-01-26 FR FR7901999A patent/FR2415460A1/en active Granted
- 1979-01-26 GB GB7902881A patent/GB2013087B/en not_active Expired
- 1979-01-26 JP JP730779A patent/JPS54113417A/en active Pending
- 1979-01-30 IE IE790144A patent/IE790144L/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113967192A (en) * | 2021-11-09 | 2022-01-25 | 陕西海斯夫生物工程有限公司 | Pharmaceutical composition for accelerating wound healing, preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2013087B (en) | 1982-07-21 |
| DE2902672A1 (en) | 1979-08-02 |
| GB2013087A (en) | 1979-08-08 |
| ES477217A1 (en) | 1980-04-01 |
| AU524529B2 (en) | 1982-09-23 |
| PT69132A (en) | 1979-02-01 |
| NL7900545A (en) | 1979-07-31 |
| BE873715A (en) | 1979-07-25 |
| ATA58979A (en) | 1982-04-15 |
| AT368888B (en) | 1982-11-25 |
| IE790144L (en) | 1979-07-27 |
| IT7919649A0 (en) | 1979-01-26 |
| ZA79349B (en) | 1980-09-24 |
| DK22879A (en) | 1979-07-28 |
| IT1109867B (en) | 1985-12-23 |
| SE7900458L (en) | 1979-07-28 |
| FR2415460B1 (en) | 1983-05-20 |
| FI790164A (en) | 1979-07-28 |
| JPS54113417A (en) | 1979-09-05 |
| AU4367879A (en) | 1979-08-02 |
| IL56499A (en) | 1981-09-13 |
| IL56499A0 (en) | 1979-03-12 |
| NZ189477A (en) | 1981-05-01 |
| FR2415460A1 (en) | 1979-08-24 |
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