CA1275248C - Method for preserving liposomes - Google Patents
Method for preserving liposomesInfo
- Publication number
- CA1275248C CA1275248C CA000499252A CA499252A CA1275248C CA 1275248 C CA1275248 C CA 1275248C CA 000499252 A CA000499252 A CA 000499252A CA 499252 A CA499252 A CA 499252A CA 1275248 C CA1275248 C CA 1275248C
- Authority
- CA
- Canada
- Prior art keywords
- liposomes
- trehalose
- preserving
- initial
- encapsulated
- 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 - Lifetime
Links
- 239000002502 liposome Substances 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims abstract description 46
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims abstract description 51
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims abstract description 51
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims abstract description 50
- 239000003755 preservative agent Substances 0.000 claims abstract description 26
- 239000000463 material Substances 0.000 claims abstract description 22
- 239000000203 mixture Substances 0.000 claims abstract description 12
- 150000002632 lipids Chemical class 0.000 claims description 14
- 239000003814 drug Substances 0.000 claims description 10
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 7
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 150000001875 compounds Chemical class 0.000 claims description 5
- 150000002772 monosaccharides Chemical group 0.000 claims description 5
- 229940124597 therapeutic agent Drugs 0.000 claims description 5
- 239000002246 antineoplastic agent Substances 0.000 claims description 4
- 229940041181 antineoplastic drug Drugs 0.000 claims description 4
- 150000001720 carbohydrates Chemical class 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 108060003951 Immunoglobulin Proteins 0.000 claims description 2
- 102000018358 immunoglobulin Human genes 0.000 claims description 2
- 229920002521 macromolecule Polymers 0.000 claims description 2
- 239000002195 soluble material Substances 0.000 claims description 2
- 229940124549 vasodilator Drugs 0.000 claims description 2
- 239000003071 vasodilator agent Substances 0.000 claims description 2
- 230000002921 anti-spasmodic effect Effects 0.000 claims 1
- 239000000812 cholinergic antagonist Substances 0.000 claims 1
- 229940127230 sympathomimetic drug Drugs 0.000 claims 1
- 125000000647 trehalose group Chemical group 0.000 claims 1
- 238000004108 freeze drying Methods 0.000 abstract description 16
- 229940074410 trehalose Drugs 0.000 description 44
- 239000002691 unilamellar liposome Substances 0.000 description 19
- ODBLHEXUDAPZAU-UHFFFAOYSA-N threo-D-isocitric acid Natural products OC(=O)C(O)C(C(O)=O)CC(O)=O ODBLHEXUDAPZAU-UHFFFAOYSA-N 0.000 description 17
- 239000000523 sample Substances 0.000 description 15
- ODBLHEXUDAPZAU-ZAFYKAAXSA-N D-threo-isocitric acid Chemical compound OC(=O)[C@H](O)[C@@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-ZAFYKAAXSA-N 0.000 description 14
- ODBLHEXUDAPZAU-FONMRSAGSA-N Isocitric acid Natural products OC(=O)[C@@H](O)[C@H](C(O)=O)CC(O)=O ODBLHEXUDAPZAU-FONMRSAGSA-N 0.000 description 14
- 229940067631 phospholipid Drugs 0.000 description 9
- 150000003904 phospholipids Chemical class 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 230000014759 maintenance of location Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 238000005538 encapsulation Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 210000004379 membrane Anatomy 0.000 description 5
- 235000014633 carbohydrates Nutrition 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 4
- 230000000717 retained effect Effects 0.000 description 4
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000000527 sonication Methods 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 2
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 2
- 102000012011 Isocitrate Dehydrogenase Human genes 0.000 description 2
- 108010075869 Isocitrate Dehydrogenase Proteins 0.000 description 2
- -1 carbohydrate compound Chemical class 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229940039227 diagnostic agent Drugs 0.000 description 2
- 239000000032 diagnostic agent Substances 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001952 enzyme assay Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 238000002165 resonance energy transfer Methods 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 1
- 229930003347 Atropine Natural products 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- BALXUFOVQVENIU-GNAZCLTHSA-N Ephedrine hydrochloride Chemical compound Cl.CN[C@@H](C)[C@H](O)C1=CC=CC=C1 BALXUFOVQVENIU-GNAZCLTHSA-N 0.000 description 1
- RKUNBYITZUJHSG-UHFFFAOYSA-N Hyosciamin-hydrochlorid Natural products CN1C(C2)CCC1CC2OC(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-UHFFFAOYSA-N 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 241001077660 Molo Species 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 229940008238 amphetamine sulfate Drugs 0.000 description 1
- PYHRZPFZZDCOPH-UHFFFAOYSA-N amphetamine sulfate Chemical compound OS(O)(=O)=O.CC(N)CC1=CC=CC=C1.CC(N)CC1=CC=CC=C1 PYHRZPFZZDCOPH-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- RKUNBYITZUJHSG-SPUOUPEWSA-N atropine Chemical compound O([C@H]1C[C@H]2CC[C@@H](C1)N2C)C(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-SPUOUPEWSA-N 0.000 description 1
- 229960000396 atropine Drugs 0.000 description 1
- STECJAGHUSJQJN-QBMZJCKTSA-N atroscine Chemical compound C([C@@H]1N([C@H](C2)[C@@H]3[C@H]1O3)C)C2OC(=O)C(CO)C1=CC=CC=C1 STECJAGHUSJQJN-QBMZJCKTSA-N 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- 229940124630 bronchodilator Drugs 0.000 description 1
- 239000000168 bronchodilator agent Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 229960002534 ephedrine hydrochloride Drugs 0.000 description 1
- 229960003072 epinephrine hydrochloride Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- UYDLBVPAAFVANX-UHFFFAOYSA-N octylphenoxy polyethoxyethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCOCCOCCO)C=C1 UYDLBVPAAFVANX-UHFFFAOYSA-N 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/227—Liposomes, lipoprotein vesicles, e.g. LDL or HDL lipoproteins, micelles, e.g. phospholipidic or polymeric
Landscapes
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Acoustics & Sound (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Radiology & Medical Imaging (AREA)
- Medicinal Preparation (AREA)
Abstract
Abstract A novel method is provided for preserving liposomes containing biologically active molecules, such that when rehydrated, liposome structures retain substan-tially all material originally encapsulated. A pre-serving agent having at least two nonosaccharide units is used either internally or externally or both. In a preferred embodiment, trehalose is used as a preserving agent, both inside the liposomes as an encapsulate material and externally, in solution, during freeze-drying. The invention also includes a lyophilized com-position prepared by the disclosed method.
Description
~;~'75;~
~ETHOD FOR PRE6ERVING LIPOSOMES
Field of the Invent on The present invention relates generally to liposomes, and more particularly relates to a method of preserviny liposomes containing biologically acti~e mol-ecules. This process is useful in applications such as in vivo drug delivery and preservation of diagnostic agents.
This invention was made with U.S. Government support under Grant No.~PCM 82-17538 with the National Science Foundation and the University of CaIifornia. The U~S.~Government has certain rights in this invention.
Backqround of th`e Invention Lipo~somes are unilamel:Lar or multilamellar ~lipid vesicles which~enclose~a flu~id space. The walls o~
the vesicles are formed by a bimolecular layer of one or more lipid components having polar heads and non-polar tails. In~an aqueous (or polar) solution, the polar heads of one layer orient outwardly ~o extend into the surrounding medium, and the non-polar tail portions of the~lipids associate with each other, thus providing a polar~surface and a non-polar core in the wall of the vesic~e. Unilamellar liposomes have one such bimolecu-lar layer, whereas multilamellar liposomes generally have a plurality oE substantially concentric bimolecular layers.
Liposomes are well recognized as use~ul for encapsulation Oe drugs and other therapeutic agents and or carrying these agents to in vivo sites. For example, ~ :~. .
:: , . : :
- .. . . .
- , ~ : ,. .
'. , ' .
~L~'7S~
U.S. Patent 3,g93,754, inventors Rahman et al., issued November 23, 1976, discloses an improved chemotherapy method in which an anti-tumor drug is encapsulated within liposomes and then injected. U.S. Patent 4,263,42~, inventors Apple et al., issued April 21, 1981, discloses an antitumor drug which may be more effectively delivered to selective cell sites in a mammalian organism by incorporating the drug within uniformly sized liposomes.
Drug administration via liposomes can have reduced toxicity, altered tissue distribution, increased drug effectiveness, and an improved therapeutic indexO
Liposomes have also been used successfully for introducing various chemicals~ biochemicals, genetic material and the like into viable cells ln vitro, and as carriers for diagnostic agents.
A variety of methods for preparing liposomes are known, many of which have been described by Szoka and Papahadjopoulos, Ann. Rev. BiophYsics Bioeng. 9: 467-508 (1~80). Also, several liposome encapsulation methods are disclosed in the patent literature, notably in U.S.
Z- patent 4,235,871, to Papahadjopoulos et al., issued November 25, 19~0, and in U~S. patent 4,016,100 to Suzuki et al., issued April 5, 1977.
Although encapsulation of therapeutic agents and biologically active compounds in liposomes bas sig-nificant commercial potential, a major dif~iculty that has been encountered in the commercial use of liposome encapsulates is with their long term stability. Although liposome structures may be maintained intact under certain storage conditions, such conditions are often 3a. inconvenient or unavailable. It is as a solution to this problem that the method of this invention is presented.
~, , : -. .:
~.~75~4~
Summary of the In ention Accordingly, it is an object o~ the present invention t~ provide a commercially feasible method of preserving liposomes.
It is another object of the present invention to provide a commercially feasible method o~ preserving liposomes by means of freeze-drying, wherein upon rehy~
dration, resultant liposomes can retain as much as lOU~
of their original encapsulated material.
It is still another object of the present invention to provide a method oE preserving liposomes by means of a carbohydrate compound capable of preserving structure and function in biological membranes.
It is a further object o~ the present invention to provide a method of preserving liposomes by means o~ a preserving agent such as trehalose, present either as encapsulated material inside the liposome or externally in solution during freeze-drying, or both.
It is yet another object of the present invention to provide a lyophilized composition which upon rehydration retains up to 100% of original encapsulated material.
Further objects an~ advantayes oE this inven-tion will become apparent from the study of the following portion of the specification, the claims and the attached drawings~
In one aspect the present invention is directed to a method for preserving liposomes, comprising:
providing i-nitial liposomes, said liposomes having an initial quantity of water-soluble material encapsulated therein, said material including a first preserving agent;
contacting said initial liposomes with a second preserving agent in an aqueous solution; and, lyophilizing said c~
.~ :
. , . -. . :
, ~ ' . . - ' ' , ':
. ., -,' : ' ~ . ... .
~S;~4~
3a initial liposomes in the presence of said second preserving agent to form lyophilizates.
In a further aspect of the present invention, a method for preserving liposomes inc1udes freeze-drying liposomes in the presence of a preserving agent capable of preserving structure and function in biological membranes.
Preferred preserving agents include carbohydrates having at least two monosaccharide units, and especially preferred compounds include the disaccharides . . - -~: : . . . .
sucrose, maltose, and trehalose.
In another aspect of the present invention, the method comprises freeze-drying liposomes which in addi-~ion to containing biologically active molecules or therapeutic agents contain a preserving agent such as trehalose internally. In a preferred embodiment of the inventive method, an appropriate compound such as trehal-ose is present both inside and outside the lipid membrane;
preferred weight ratios of total preserving agent to lipid range from about 0.1:1 to 3.0:1~ An especially preferred weight ratio is about 1~:1Ø
The invention also embodies a lyophiliæed composition such that when reconstituted by rehydration, resultant liposomes retain substantially all of their originally encapsulated material. Such a lyophilized composition may be prepared by the method as outlined above.
Detailed Description of the Invention The invention comprises a method for preserving liposomes containing biologically active molecules using a preserving agent. The method involves either freeze-drying liposomes in the presence of a preserving agent, or - freeze-drying liposomes which contain a preserving agent internally in addition to encapsulated medicaments, or both. Preferred preserving agents are carbohydrates having at least two monosaccharide units joined in glycosidic linkage, and particularly preferred pre-serving agents include sucrose, maltose and trehalose.
Of these, trehalose has been found to be the most effective preserving agent for use with the inventive method.
Trehalose is a naturally occurring sugar found .: ~, . . . . - . ..................... . - .
.. : . ~ .. . .
.: , :. .
7S~8 at high concentrations in organisms capable of surviving dehydrationO Trehalose is especially effective in pre-serving structure and function in dry biological mem-branes. Liposomes which are freeze-dried in the presence of trehalose and which additionally contain encapsulated trehalose, exhibit particularly good retention of encap~
sulates. That is, when liposomes are exposed to tre-halose both internally and externally during freeze-drying, they can retain as much as 100% of their original encapsulated contents upon rehydrakion. This is in sharp contrast to liposomes which are freeze-dried without any preserving agent, which show extensive fusion between liposomes and loss of contents to the surrounding medium.
Representative phospholipids used in forming liposomes which may be used in this process include phos phatidylcholine, phosphatidylserine, phosphatidic acid and mixtures thereof. Both natural and synthetic phos-pholipids may be successfully usedO
The biologically active or therapeutic encap-sulated material is preferably water soluble. Examples of suitable therapeutic agents with which this preserva-tion method can successfully be carried out include sympa~homimetic drugs such as amphetamine sulfate, epi-nephrine hydrochloride, or ephedrine hydrochloride; an-tispasmodics such as atropine or scopalamine; broncho-dilators such as isoproternol; vasodilators such as dilthiazen; hormones such as insulin: and antineoplastic drugs such as adriamycin. Suitable biologically active molecules include, for example, RNA, DNA, enzymes and immunoglobulins.
Small unilamellar vesicles (SUV's) are pre-pared as starting materials prior to encapsulation of trehalose, and may be prepared by any of the available ... . , : :
5~4~3 techniques. Suitable techniques include injection of the lipid in an organic solvent into water, extrusion from a French pressure cell, and sonication. The material to be trapped may be added at any s~age during preparation of the small unilamellar vesicles, but in practice it is most convenient to mix the small unilamellar vesicles with an aqueous solution of the material to be trapped immedi-ately be~ore preparation of large unilamellar vesicles.
Preferred weight ratios of encapsulate to lipid are about l.li:l.O.
Large unilamellar vesicles (LUV's) with in-creased trapping e~ficiency may tben be prepared by either freeze-thawing or rotary evaporation. An exemplary rotary evaporation method and one which is especially effective in conjunction with the method disclosed herein is illustrated in Deamer, D.W., "A Novel Method for Encapsulation of Macromolecules in Liposomes" in Gregori-adis, G. (ed.) Liposome Technolo~Y (19~4). The method comprises providing a polar solution having initial liposomes and a quantity of material to be encapsulated.
Substantially~all of the solution is removed, and the resultant liposomes are then recove~ed by hydration o~
the concentrated admixture. ThiS method is also the subject of U.S. Patent No. 4,515,736, inventor Deamer, et al., issued May 7, 1985. The resulting vesicles may then be made more uniform by filtration, centrifugation or gel permeation chromatography.
; Trehalose may be added at any stage during preparation of the large unilamellar vesicles, but greatly improved preservation is attained with trehalose present on both sides of the phospholipid bilayer. ~herefore, trehalose is preferably added be~ore the large ~' ' ' . : ..
. ~ ~
75~
unilamellar vesicles are prepared, so that trehalose is trapped inside. The preferred weight ratio of total trehalose to lipid ranges from about 0.1:1 to about 3.0:1;
a particularly preferred weight ratio is approximately 1.0:1Ø The large unilamellar vesicles are then frozen in liquid nitrogen and lyophilized Under some circum-stances, as when lipids are used which are susceptible to damage due to the presence of oxygen, it may be desirable to seal the dry preparations under vacuum. Rehydration is accomplished simply by adding water to the dry mixture.
Although in a preferred embodiment of the invention, the liposomes are exposed to trehalose, it should be understood that a variety of preseeving agents may be substituted for trehalose, including carbohydrate compounds which are composed of at least two monosac-charide units. In particular, sucrose and maltose are suitable alternatives.
The follo~ing examples illustrate certain as-pects and embodiments oE the present invention, and are not intended to limit the scope of the invention as defined in the appended claims.
Exam~le_l ! A phospholipid mixture consisting of approximately 40 mg. dipalmitoyl phosphatidylcholine and phosphatidic acid in a molar ratio of 95:5 was sonicated to optical clarity in a bath sonicator. Large unilamellar vesicles were prepared by freeze-thawing in a 50 mM solution of isocitric acid in water as the compound to be encap-sulated. Excess isocitric acid was removed by dialysis.
Trehalose (2.0:1.U trehalose:phospholipid weight ratio) was added either after freeze-thawing or beforehand, thus providin~ so~e large unilamellar vesicles with external ~, .
- .
.~: . . .- ' . . ~:
'' ~ '' .' : . ., :
~ - . ,.~ , - .
trehalose only and some vesicles with trehalose both externally and internally. Isocitric acid was assayed by adding isocitrate dehydrogenase and NADP to the outside of the vesicles according to the method of Plaut, et al.
(Eds.), Methods in Enz~molo~y, Volume 5 (New York: Aca-demic Press). Isocitrate external to the vesicles was oxidized by the isocitrate dehydrogenase, resulting in reduction of NADP to NADPH, the rate and amount of which may be recorded fluorometrically. Total isocitric acid in the vesicles was assayed following addition of Triton X-lO0 (octylphenoxy polyethoxyethanol, a detergent and emulsifier manufactured by Rohm ~ Haas Co., Philadelphia, PA; "TRITON" is a registered trademark of Rohm & Haas Co~), which releases the trapped isocitric acid into the surrounding medium. Isocitric acid trapped in the vesi-cles was assayed before and after both lyophilization and rehydration, thus providing an estimate of the efficiency with which the trapped isocitrate was retained. As may be seen in Table 1, the results show that over sixty percent (60%) of the trapped isocitrate was retained when the vesicles were lyophilized with trehalose both inside and outside the vesicles. When trehalose was present externally only, there was still a significant increase in the efficiency of retention, but to a lesser degree than in the case where trehalose was present on both sides of the lipid membrane. Examination of lipid concen-tration at time of freezing showed that such had no significant effect on retention of trapped material folIowing lyophilization.
. :: .:, .: ~ .
~.~'75~4~3 Table 1 Method of .
Preparing Concentration g Trehalose Trehalose %Reten-LUV's of Lip_d /g Lipid External Internal tion (mg/ml) FT* 10.8 0 - - 0 FT 11.1 0.08 - + 0 FT 10.8 1.78 + - 42 FT 11.1 1.78 + ~ 61 :~.
:
*FT = freeze-thaw :`:
, ,:
' :
:
: , . . . ;, . . . . . .
: . : , : ..
- . , . ~ . -'7S~
1~
~e~
Small unilamellar vesicles of were made by sonication of 43 mg egg phosphatidylcholine in 4 ml of water. Large unilamellar vesicles were prepared by rotary drying the phospholipid in the presence of 32 mg of trehalose and 13 mg of isocitric acid. The weight ratios of phospholipid:trehalose:isocitric acid were approxi-mately 4:3:1. Excess isocitric acid and trehalose were removed by dialysis a~ainst distilled water, and the amount oE isocitric acid trapped in the vesicles was determined by the enzyme assay described in Example 1.
Trehalose was added to the dialyzed liposomes to give a final weight ratio of phospholipid:trehalose of 1.0:1.4, and the sample was lyophilized. The sample was then rehydrated with distilled water, and the amount of isocitric acid remaining in the liposomes was determined by enzyme assay. The lyophilized vesicles retained 75%
of their original contents.
Example 3 A phospholipid mixture of palmitoyloleoyl phosphatidylcholine (9U~) and phosphatidylserine (10~) was hydrated to 10 mg./ml., and small unilamellar vesicles were then prepared by sonication. Large uni-lamellar vesicles were prepared by rotary drying in the presence of isocitric acid, which served as the encap-sulated molecule. Essentially the same techniques as previously described in Examples 1 and 2 were used.
Efficiency of retention o isocitric acid following lyophilization and rehydration was recorded as before, with large unilamellar vesicles lyophili~ed first in the presence and then in the absence of trehalose. As may be seen in Table 2, the results show that 100~ of the trapped ..
.
~ ~ 7~j~ 4~
isocitric acid is retained when the large unilamellar vesicles are lyophilized and rehydrated under the stated conditions. As the previous examples demonstrated, tre-halose is preferably present both externally and inter-nally to optimize retention of the encapsulate.
~ 4 One of the damaging events presumed to be occurring during lyophilization is close approach of the large unilamellar vesicles to each other, leading to -fusion and leakage of the vesicular contents. Fusion has been assayed by resonance energy transfer, a fluorescence method which depends upon energy trans~er from an excited probe (the "donor probe") to a second probe (the "acceptor probe"). The acceptor probe fluoresces when the energy transfer occurs. In order for the transfer to occur the two probes must be in close proximity. Thus probe intermixing can be used as an assay for fusion between vesicles during lyophilization. Large unilamellar vesi-cles were prépared with donor probe in one preparation and acceptor probe in anotherj and the two preparations were mixed be~ore lyophilization. Following lyophilization and rehydration, probe intermixing was measured, with the results listed in Table 3~ The results show that with increasing trehalose concentration there is a decrease in probe intermixing. Furthermore, the presence of tre-halose inside the liposomes alone significantly reduce probe mixing. Thus, use of trehalose tends to reduce fusion of the vesicles.
, . ~ . . , ~ , - : . : ,, , : `: ~, : ~ ' ' ` :
:. : . : ' ' ' , ' .
1 ~ 7~3~ 4 Table 2 Method of Preparing g Trehalose Trehalose ~Reten-LUV's ~ External Internal tion _ RD* 0.06 - + 0 RD 3.2 + + 1~0 RD O -- -- O
RD 3.9 + - 26 RD 0.11 + + 22 RD 0.19 + + 49 RD 0.33 + + 69 RD ~.63 + + 76 RD 0.91 + + 86 RD 1.76 + + 99 ; 15 ....
*RD = rotary drying . "
.:
- ' , . ' ` ` ' ', . . .
~-~'75~ 8 Table 3 Method of Preparing g ~rrehalose Trehalose ~ Probe LUV's /~ L~pid External Internal RD* 0.05 - + 72 RD 0.15 + + 39 RD 0.25 + + 29 RD 0.50 + + 12 ~D 0.95 + + 8 FT** 0. - _ 93 ~
FT 0.4 + + 79 0 FT 0.8 + + 59 FT 1.2 + + 54,0 FT 1.6 ~ + 38.0 FT ~2.0 + + 15.0 .
*RD = rotary drying ~ ~*FT = fre~eze-thaw : -FU9ION BETWEEN PALMITOYLOLEOYL PHOSPHATIDYLCHOLINE: -PHOSPHATIDYLSERINE (90:10) LARGE UNILAMELLAR VESICLES, AS ASSAYED BY RESONANCE ENERGY TRANSFER BETWEEN FL~ORES-CENT PROBES
' ' ' '. ': ' ". ;':
75~
Example ~
A further experiment was carried out identical to that set forth in Example 3, with first maltose and then sucrose as the preserving agent. Results are set forth in Tables 4 and 5. As may be concluded from those tables, both maltose and sucrose provide good retention of encapsulated material following lyophilization.
Table 4 .
Method of Preparing g Trehalose Trehalo e ~Reten-LUV's /g Li~id Externel Internal tion RD 0.05 - + ~ 3 RD U.lS + + 41 RD Q.25 + + 88 : RD 0 49 RD : ~.64 ~ + + lO0 , Table 5 ':
. . .
~: : Method of ~ Preparing g Trehalose Trehalose %Reten- -~
: 2:5 LUV'~ ~LipidExternal Internal tion :
RD 0.07 - + 20 RD 0.3S + + 57 RD 0.49 + + ~9 RD 0,83 + + 86 RD l.lS + + 91 .
~ETHOD FOR PRE6ERVING LIPOSOMES
Field of the Invent on The present invention relates generally to liposomes, and more particularly relates to a method of preserviny liposomes containing biologically acti~e mol-ecules. This process is useful in applications such as in vivo drug delivery and preservation of diagnostic agents.
This invention was made with U.S. Government support under Grant No.~PCM 82-17538 with the National Science Foundation and the University of CaIifornia. The U~S.~Government has certain rights in this invention.
Backqround of th`e Invention Lipo~somes are unilamel:Lar or multilamellar ~lipid vesicles which~enclose~a flu~id space. The walls o~
the vesicles are formed by a bimolecular layer of one or more lipid components having polar heads and non-polar tails. In~an aqueous (or polar) solution, the polar heads of one layer orient outwardly ~o extend into the surrounding medium, and the non-polar tail portions of the~lipids associate with each other, thus providing a polar~surface and a non-polar core in the wall of the vesic~e. Unilamellar liposomes have one such bimolecu-lar layer, whereas multilamellar liposomes generally have a plurality oE substantially concentric bimolecular layers.
Liposomes are well recognized as use~ul for encapsulation Oe drugs and other therapeutic agents and or carrying these agents to in vivo sites. For example, ~ :~. .
:: , . : :
- .. . . .
- , ~ : ,. .
'. , ' .
~L~'7S~
U.S. Patent 3,g93,754, inventors Rahman et al., issued November 23, 1976, discloses an improved chemotherapy method in which an anti-tumor drug is encapsulated within liposomes and then injected. U.S. Patent 4,263,42~, inventors Apple et al., issued April 21, 1981, discloses an antitumor drug which may be more effectively delivered to selective cell sites in a mammalian organism by incorporating the drug within uniformly sized liposomes.
Drug administration via liposomes can have reduced toxicity, altered tissue distribution, increased drug effectiveness, and an improved therapeutic indexO
Liposomes have also been used successfully for introducing various chemicals~ biochemicals, genetic material and the like into viable cells ln vitro, and as carriers for diagnostic agents.
A variety of methods for preparing liposomes are known, many of which have been described by Szoka and Papahadjopoulos, Ann. Rev. BiophYsics Bioeng. 9: 467-508 (1~80). Also, several liposome encapsulation methods are disclosed in the patent literature, notably in U.S.
Z- patent 4,235,871, to Papahadjopoulos et al., issued November 25, 19~0, and in U~S. patent 4,016,100 to Suzuki et al., issued April 5, 1977.
Although encapsulation of therapeutic agents and biologically active compounds in liposomes bas sig-nificant commercial potential, a major dif~iculty that has been encountered in the commercial use of liposome encapsulates is with their long term stability. Although liposome structures may be maintained intact under certain storage conditions, such conditions are often 3a. inconvenient or unavailable. It is as a solution to this problem that the method of this invention is presented.
~, , : -. .:
~.~75~4~
Summary of the In ention Accordingly, it is an object o~ the present invention t~ provide a commercially feasible method of preserving liposomes.
It is another object of the present invention to provide a commercially feasible method o~ preserving liposomes by means of freeze-drying, wherein upon rehy~
dration, resultant liposomes can retain as much as lOU~
of their original encapsulated material.
It is still another object of the present invention to provide a method oE preserving liposomes by means of a carbohydrate compound capable of preserving structure and function in biological membranes.
It is a further object o~ the present invention to provide a method of preserving liposomes by means o~ a preserving agent such as trehalose, present either as encapsulated material inside the liposome or externally in solution during freeze-drying, or both.
It is yet another object of the present invention to provide a lyophilized composition which upon rehydration retains up to 100% of original encapsulated material.
Further objects an~ advantayes oE this inven-tion will become apparent from the study of the following portion of the specification, the claims and the attached drawings~
In one aspect the present invention is directed to a method for preserving liposomes, comprising:
providing i-nitial liposomes, said liposomes having an initial quantity of water-soluble material encapsulated therein, said material including a first preserving agent;
contacting said initial liposomes with a second preserving agent in an aqueous solution; and, lyophilizing said c~
.~ :
. , . -. . :
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. ., -,' : ' ~ . ... .
~S;~4~
3a initial liposomes in the presence of said second preserving agent to form lyophilizates.
In a further aspect of the present invention, a method for preserving liposomes inc1udes freeze-drying liposomes in the presence of a preserving agent capable of preserving structure and function in biological membranes.
Preferred preserving agents include carbohydrates having at least two monosaccharide units, and especially preferred compounds include the disaccharides . . - -~: : . . . .
sucrose, maltose, and trehalose.
In another aspect of the present invention, the method comprises freeze-drying liposomes which in addi-~ion to containing biologically active molecules or therapeutic agents contain a preserving agent such as trehalose internally. In a preferred embodiment of the inventive method, an appropriate compound such as trehal-ose is present both inside and outside the lipid membrane;
preferred weight ratios of total preserving agent to lipid range from about 0.1:1 to 3.0:1~ An especially preferred weight ratio is about 1~:1Ø
The invention also embodies a lyophiliæed composition such that when reconstituted by rehydration, resultant liposomes retain substantially all of their originally encapsulated material. Such a lyophilized composition may be prepared by the method as outlined above.
Detailed Description of the Invention The invention comprises a method for preserving liposomes containing biologically active molecules using a preserving agent. The method involves either freeze-drying liposomes in the presence of a preserving agent, or - freeze-drying liposomes which contain a preserving agent internally in addition to encapsulated medicaments, or both. Preferred preserving agents are carbohydrates having at least two monosaccharide units joined in glycosidic linkage, and particularly preferred pre-serving agents include sucrose, maltose and trehalose.
Of these, trehalose has been found to be the most effective preserving agent for use with the inventive method.
Trehalose is a naturally occurring sugar found .: ~, . . . . - . ..................... . - .
.. : . ~ .. . .
.: , :. .
7S~8 at high concentrations in organisms capable of surviving dehydrationO Trehalose is especially effective in pre-serving structure and function in dry biological mem-branes. Liposomes which are freeze-dried in the presence of trehalose and which additionally contain encapsulated trehalose, exhibit particularly good retention of encap~
sulates. That is, when liposomes are exposed to tre-halose both internally and externally during freeze-drying, they can retain as much as 100% of their original encapsulated contents upon rehydrakion. This is in sharp contrast to liposomes which are freeze-dried without any preserving agent, which show extensive fusion between liposomes and loss of contents to the surrounding medium.
Representative phospholipids used in forming liposomes which may be used in this process include phos phatidylcholine, phosphatidylserine, phosphatidic acid and mixtures thereof. Both natural and synthetic phos-pholipids may be successfully usedO
The biologically active or therapeutic encap-sulated material is preferably water soluble. Examples of suitable therapeutic agents with which this preserva-tion method can successfully be carried out include sympa~homimetic drugs such as amphetamine sulfate, epi-nephrine hydrochloride, or ephedrine hydrochloride; an-tispasmodics such as atropine or scopalamine; broncho-dilators such as isoproternol; vasodilators such as dilthiazen; hormones such as insulin: and antineoplastic drugs such as adriamycin. Suitable biologically active molecules include, for example, RNA, DNA, enzymes and immunoglobulins.
Small unilamellar vesicles (SUV's) are pre-pared as starting materials prior to encapsulation of trehalose, and may be prepared by any of the available ... . , : :
5~4~3 techniques. Suitable techniques include injection of the lipid in an organic solvent into water, extrusion from a French pressure cell, and sonication. The material to be trapped may be added at any s~age during preparation of the small unilamellar vesicles, but in practice it is most convenient to mix the small unilamellar vesicles with an aqueous solution of the material to be trapped immedi-ately be~ore preparation of large unilamellar vesicles.
Preferred weight ratios of encapsulate to lipid are about l.li:l.O.
Large unilamellar vesicles (LUV's) with in-creased trapping e~ficiency may tben be prepared by either freeze-thawing or rotary evaporation. An exemplary rotary evaporation method and one which is especially effective in conjunction with the method disclosed herein is illustrated in Deamer, D.W., "A Novel Method for Encapsulation of Macromolecules in Liposomes" in Gregori-adis, G. (ed.) Liposome Technolo~Y (19~4). The method comprises providing a polar solution having initial liposomes and a quantity of material to be encapsulated.
Substantially~all of the solution is removed, and the resultant liposomes are then recove~ed by hydration o~
the concentrated admixture. ThiS method is also the subject of U.S. Patent No. 4,515,736, inventor Deamer, et al., issued May 7, 1985. The resulting vesicles may then be made more uniform by filtration, centrifugation or gel permeation chromatography.
; Trehalose may be added at any stage during preparation of the large unilamellar vesicles, but greatly improved preservation is attained with trehalose present on both sides of the phospholipid bilayer. ~herefore, trehalose is preferably added be~ore the large ~' ' ' . : ..
. ~ ~
75~
unilamellar vesicles are prepared, so that trehalose is trapped inside. The preferred weight ratio of total trehalose to lipid ranges from about 0.1:1 to about 3.0:1;
a particularly preferred weight ratio is approximately 1.0:1Ø The large unilamellar vesicles are then frozen in liquid nitrogen and lyophilized Under some circum-stances, as when lipids are used which are susceptible to damage due to the presence of oxygen, it may be desirable to seal the dry preparations under vacuum. Rehydration is accomplished simply by adding water to the dry mixture.
Although in a preferred embodiment of the invention, the liposomes are exposed to trehalose, it should be understood that a variety of preseeving agents may be substituted for trehalose, including carbohydrate compounds which are composed of at least two monosac-charide units. In particular, sucrose and maltose are suitable alternatives.
The follo~ing examples illustrate certain as-pects and embodiments oE the present invention, and are not intended to limit the scope of the invention as defined in the appended claims.
Exam~le_l ! A phospholipid mixture consisting of approximately 40 mg. dipalmitoyl phosphatidylcholine and phosphatidic acid in a molar ratio of 95:5 was sonicated to optical clarity in a bath sonicator. Large unilamellar vesicles were prepared by freeze-thawing in a 50 mM solution of isocitric acid in water as the compound to be encap-sulated. Excess isocitric acid was removed by dialysis.
Trehalose (2.0:1.U trehalose:phospholipid weight ratio) was added either after freeze-thawing or beforehand, thus providin~ so~e large unilamellar vesicles with external ~, .
- .
.~: . . .- ' . . ~:
'' ~ '' .' : . ., :
~ - . ,.~ , - .
trehalose only and some vesicles with trehalose both externally and internally. Isocitric acid was assayed by adding isocitrate dehydrogenase and NADP to the outside of the vesicles according to the method of Plaut, et al.
(Eds.), Methods in Enz~molo~y, Volume 5 (New York: Aca-demic Press). Isocitrate external to the vesicles was oxidized by the isocitrate dehydrogenase, resulting in reduction of NADP to NADPH, the rate and amount of which may be recorded fluorometrically. Total isocitric acid in the vesicles was assayed following addition of Triton X-lO0 (octylphenoxy polyethoxyethanol, a detergent and emulsifier manufactured by Rohm ~ Haas Co., Philadelphia, PA; "TRITON" is a registered trademark of Rohm & Haas Co~), which releases the trapped isocitric acid into the surrounding medium. Isocitric acid trapped in the vesi-cles was assayed before and after both lyophilization and rehydration, thus providing an estimate of the efficiency with which the trapped isocitrate was retained. As may be seen in Table 1, the results show that over sixty percent (60%) of the trapped isocitrate was retained when the vesicles were lyophilized with trehalose both inside and outside the vesicles. When trehalose was present externally only, there was still a significant increase in the efficiency of retention, but to a lesser degree than in the case where trehalose was present on both sides of the lipid membrane. Examination of lipid concen-tration at time of freezing showed that such had no significant effect on retention of trapped material folIowing lyophilization.
. :: .:, .: ~ .
~.~'75~4~3 Table 1 Method of .
Preparing Concentration g Trehalose Trehalose %Reten-LUV's of Lip_d /g Lipid External Internal tion (mg/ml) FT* 10.8 0 - - 0 FT 11.1 0.08 - + 0 FT 10.8 1.78 + - 42 FT 11.1 1.78 + ~ 61 :~.
:
*FT = freeze-thaw :`:
, ,:
' :
:
: , . . . ;, . . . . . .
: . : , : ..
- . , . ~ . -'7S~
1~
~e~
Small unilamellar vesicles of were made by sonication of 43 mg egg phosphatidylcholine in 4 ml of water. Large unilamellar vesicles were prepared by rotary drying the phospholipid in the presence of 32 mg of trehalose and 13 mg of isocitric acid. The weight ratios of phospholipid:trehalose:isocitric acid were approxi-mately 4:3:1. Excess isocitric acid and trehalose were removed by dialysis a~ainst distilled water, and the amount oE isocitric acid trapped in the vesicles was determined by the enzyme assay described in Example 1.
Trehalose was added to the dialyzed liposomes to give a final weight ratio of phospholipid:trehalose of 1.0:1.4, and the sample was lyophilized. The sample was then rehydrated with distilled water, and the amount of isocitric acid remaining in the liposomes was determined by enzyme assay. The lyophilized vesicles retained 75%
of their original contents.
Example 3 A phospholipid mixture of palmitoyloleoyl phosphatidylcholine (9U~) and phosphatidylserine (10~) was hydrated to 10 mg./ml., and small unilamellar vesicles were then prepared by sonication. Large uni-lamellar vesicles were prepared by rotary drying in the presence of isocitric acid, which served as the encap-sulated molecule. Essentially the same techniques as previously described in Examples 1 and 2 were used.
Efficiency of retention o isocitric acid following lyophilization and rehydration was recorded as before, with large unilamellar vesicles lyophili~ed first in the presence and then in the absence of trehalose. As may be seen in Table 2, the results show that 100~ of the trapped ..
.
~ ~ 7~j~ 4~
isocitric acid is retained when the large unilamellar vesicles are lyophilized and rehydrated under the stated conditions. As the previous examples demonstrated, tre-halose is preferably present both externally and inter-nally to optimize retention of the encapsulate.
~ 4 One of the damaging events presumed to be occurring during lyophilization is close approach of the large unilamellar vesicles to each other, leading to -fusion and leakage of the vesicular contents. Fusion has been assayed by resonance energy transfer, a fluorescence method which depends upon energy trans~er from an excited probe (the "donor probe") to a second probe (the "acceptor probe"). The acceptor probe fluoresces when the energy transfer occurs. In order for the transfer to occur the two probes must be in close proximity. Thus probe intermixing can be used as an assay for fusion between vesicles during lyophilization. Large unilamellar vesi-cles were prépared with donor probe in one preparation and acceptor probe in anotherj and the two preparations were mixed be~ore lyophilization. Following lyophilization and rehydration, probe intermixing was measured, with the results listed in Table 3~ The results show that with increasing trehalose concentration there is a decrease in probe intermixing. Furthermore, the presence of tre-halose inside the liposomes alone significantly reduce probe mixing. Thus, use of trehalose tends to reduce fusion of the vesicles.
, . ~ . . , ~ , - : . : ,, , : `: ~, : ~ ' ' ` :
:. : . : ' ' ' , ' .
1 ~ 7~3~ 4 Table 2 Method of Preparing g Trehalose Trehalose ~Reten-LUV's ~ External Internal tion _ RD* 0.06 - + 0 RD 3.2 + + 1~0 RD O -- -- O
RD 3.9 + - 26 RD 0.11 + + 22 RD 0.19 + + 49 RD 0.33 + + 69 RD ~.63 + + 76 RD 0.91 + + 86 RD 1.76 + + 99 ; 15 ....
*RD = rotary drying . "
.:
- ' , . ' ` ` ' ', . . .
~-~'75~ 8 Table 3 Method of Preparing g ~rrehalose Trehalose ~ Probe LUV's /~ L~pid External Internal RD* 0.05 - + 72 RD 0.15 + + 39 RD 0.25 + + 29 RD 0.50 + + 12 ~D 0.95 + + 8 FT** 0. - _ 93 ~
FT 0.4 + + 79 0 FT 0.8 + + 59 FT 1.2 + + 54,0 FT 1.6 ~ + 38.0 FT ~2.0 + + 15.0 .
*RD = rotary drying ~ ~*FT = fre~eze-thaw : -FU9ION BETWEEN PALMITOYLOLEOYL PHOSPHATIDYLCHOLINE: -PHOSPHATIDYLSERINE (90:10) LARGE UNILAMELLAR VESICLES, AS ASSAYED BY RESONANCE ENERGY TRANSFER BETWEEN FL~ORES-CENT PROBES
' ' ' '. ': ' ". ;':
75~
Example ~
A further experiment was carried out identical to that set forth in Example 3, with first maltose and then sucrose as the preserving agent. Results are set forth in Tables 4 and 5. As may be concluded from those tables, both maltose and sucrose provide good retention of encapsulated material following lyophilization.
Table 4 .
Method of Preparing g Trehalose Trehalo e ~Reten-LUV's /g Li~id Externel Internal tion RD 0.05 - + ~ 3 RD U.lS + + 41 RD Q.25 + + 88 : RD 0 49 RD : ~.64 ~ + + lO0 , Table 5 ':
. . .
~: : Method of ~ Preparing g Trehalose Trehalose %Reten- -~
: 2:5 LUV'~ ~LipidExternal Internal tion :
RD 0.07 - + 20 RD 0.3S + + 57 RD 0.49 + + ~9 RD 0,83 + + 86 RD l.lS + + 91 .
Claims (11)
1. A method for preserving liposomes, comprising:
providing initial liposomes, said liposomes having an initial quantity of water-soluble material encapsulated therein, said material including a first preserving agent which is a carbohydrate having at least two monosaccharide units joined in glycosidic linkage contacting said initial liposomes with a second preserving agent which is a carbohydrate having at least two monosaccharide units joined in glycosidic linkage in an aqueous solution; and, lyophilizing said initial liposomes in the presence of said second preserving agent to form lyophilizates.
providing initial liposomes, said liposomes having an initial quantity of water-soluble material encapsulated therein, said material including a first preserving agent which is a carbohydrate having at least two monosaccharide units joined in glycosidic linkage contacting said initial liposomes with a second preserving agent which is a carbohydrate having at least two monosaccharide units joined in glycosidic linkage in an aqueous solution; and, lyophilizing said initial liposomes in the presence of said second preserving agent to form lyophilizates.
2. The method as in claim 1, further com-prising recovering resultant liposomes from said lyo-philizates by admixing said lyophilizates with an aqueous solution, wherein the resultant liposomes encapsulate at least about 25 wt.% of said initial quantity of encap-sulated material.
3. The method as in claim 2, wherein said first preserving agent is trehalose, and said resultant liposomes encapsulate at least about 60% of said initial quantity of encapsulated material.
4. The method as in claim 1 or 2, wherein the weight ratio of said first and second preserving agents, combined, to lipid, ranges from about 0.1:1 to about 3.0:1.
5. The method as in claim l or 2, wherein the weight ratio of said first and second preserving agents, combined, to lipid, is approximately 1.0:1Ø
6. The method as in claim 1 or 2, wherein said first preserving agent is selected from the group consisting of trehalose, maltose and sucrose.
7. The method as in claim 1 or 2, wherein said second preserving agent is selected from the group consisting of trehalose, maltose and sucrose.
8. A lyophilized composition useful in storing encapsulated material, prepared by the process comprising:
providing initial liposomes, said liposomes having an initial quantity of material encapsulated therein, said material including a quantity of trehalose;
contacting said initial liposomes with a quantity of trehalose in aqueous solution;
lyophilizing said initial liposomes in the presence of trehalose to form lyophilizates.
providing initial liposomes, said liposomes having an initial quantity of material encapsulated therein, said material including a quantity of trehalose;
contacting said initial liposomes with a quantity of trehalose in aqueous solution;
lyophilizing said initial liposomes in the presence of trehalose to form lyophilizates.
9. The composition of claim 8 , wherein said encapsulated material includes a water-soluble therapeutic agent or bioloaically active compound.
10. The composition of claim 9, wherein said encapsulated material includes a macromolecule.
11. The composition of claim 10, wherein said encapsulated material includes a sympathomimetic drug, an antispasmodic, a vasodilator, an antineoplastic drug, RNA, DNA, an enzyme, or an immunoglobulin.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US69067985A | 1985-01-11 | 1985-01-11 | |
US690,679 | 1985-01-11 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA1275248C true CA1275248C (en) | 1990-10-16 |
Family
ID=24773483
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA000499252A Expired - Lifetime CA1275248C (en) | 1985-01-11 | 1986-01-09 | Method for preserving liposomes |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0208764A4 (en) |
JP (1) | JPS62501631A (en) |
AU (1) | AU587600B2 (en) |
CA (1) | CA1275248C (en) |
DK (1) | DK175799B1 (en) |
WO (1) | WO1986003938A1 (en) |
Families Citing this family (24)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1260393A (en) * | 1984-10-16 | 1989-09-26 | Lajos Tarcsay | Liposomes of synthetic lipids |
US5484432A (en) * | 1985-09-27 | 1996-01-16 | Laser Biotech, Inc. | Collagen treatment apparatus |
GB8604983D0 (en) * | 1986-02-28 | 1986-04-09 | Biocompatibles Ltd | Protein preservation |
WO1988006441A1 (en) * | 1987-02-23 | 1988-09-07 | Vestar, Inc. | Dehydrating vesicule preparations for long-term storage |
ATE107502T1 (en) * | 1988-10-05 | 1994-07-15 | Vestar Inc | METHOD OF PREPARING LIPOSOMES WITH IMPROVED STABILITY DURING DRYING. |
US6007817A (en) | 1988-10-11 | 1999-12-28 | University Of Southern California | Vasopermeability enhancing immunoconjugates |
IL91933A (en) * | 1988-10-11 | 1994-12-29 | Univ Southern California | Vasoactive immunoconjugates useful for increasing the vascular permeability or the blood supply to neoplastic or otherwise diseased tissues |
US5059518A (en) * | 1988-10-20 | 1991-10-22 | Coulter Corporation | Stabilized lyophilized mammalian cells and method of making same |
GB9111611D0 (en) | 1991-05-30 | 1991-07-24 | Sandoz Ltd | Liposomes |
DE69233119T2 (en) * | 1991-06-18 | 2004-05-13 | Imarx Pharmaceutical Corp., Tucson | NEW LIPOSOMAL DRUG RELEASE SYSTEMS |
JP3187622B2 (en) | 1993-10-07 | 2001-07-11 | カネボウ株式会社 | Liposome |
AU2215995A (en) * | 1994-04-07 | 1995-10-30 | Akzo Nobel N.V. | Freeze-dried compositions comprising rna |
DK0967862T3 (en) * | 1997-02-07 | 2003-05-12 | Elan Drug Delivery Ltd | Methods and compositions for preparing dried, storage-stable platelets |
GB9813100D0 (en) * | 1998-06-18 | 1998-08-19 | Secr Defence | Method of forming liposomes |
US6710038B1 (en) | 1999-12-14 | 2004-03-23 | Kibun Food Chemifa Co., Ltd. | Emulsification method using propylene glycol hyaluronate |
EP1255439A4 (en) | 2000-02-10 | 2007-01-03 | Univ California | Therapeutic platelets and methods |
US6770478B2 (en) | 2000-02-10 | 2004-08-03 | The Regents Of The University Of California | Erythrocytic cells and method for preserving cells |
PL363618A1 (en) * | 2000-11-09 | 2004-11-29 | Neopharm, Inc. | Sn-38 lipid complexes and methods of use |
WO2005002546A1 (en) * | 2003-06-27 | 2005-01-13 | Smithkline Beecham Corporation | Stabilized topotecan liposomal composition and methods |
JPWO2008026310A1 (en) * | 2006-08-29 | 2010-01-14 | 株式会社セレックス | Trehalose-containing oral mucosa protective agent |
ES2657686T3 (en) | 2009-06-24 | 2018-03-06 | Lipoid Gmbh | Composition for cosmetic, pharmaceutical or dietary applications |
SI2768484T1 (en) | 2011-10-21 | 2019-12-31 | Jazz Pharmaceuticals Research Llc | Lyophilized liposomes |
US10448631B2 (en) | 2015-09-22 | 2019-10-22 | East Carolina University | Cryopreservation using sucralose |
EP3813788A1 (en) | 2018-06-27 | 2021-05-05 | Breath Therapeutics GmbH | Pharmaceutical compositions in lyophilized form |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CH621479A5 (en) * | 1977-08-05 | 1981-02-13 | Battelle Memorial Institute | |
CA1173360A (en) * | 1979-06-22 | 1984-08-28 | Jurg Schrank | Pharmaceutical preparations |
DE3374837D1 (en) * | 1982-02-17 | 1988-01-21 | Ciba Geigy Ag | Lipids in the aqueous phase |
US4515736A (en) * | 1983-05-12 | 1985-05-07 | The Regents Of The University Of California | Method for encapsulating materials into liposomes |
GB8407557D0 (en) * | 1984-03-23 | 1984-05-02 | Hayward J A | Polymeric lipsomes |
US4880635B1 (en) * | 1984-08-08 | 1996-07-02 | Liposome Company | Dehydrated liposomes |
-
1986
- 1986-01-08 EP EP19860900891 patent/EP0208764A4/en not_active Withdrawn
- 1986-01-08 WO PCT/US1986/000016 patent/WO1986003938A1/en not_active Application Discontinuation
- 1986-01-08 AU AU53183/86A patent/AU587600B2/en not_active Expired
- 1986-01-08 JP JP61500642A patent/JPS62501631A/en active Pending
- 1986-01-09 CA CA000499252A patent/CA1275248C/en not_active Expired - Lifetime
- 1986-09-08 DK DK198604283A patent/DK175799B1/en not_active IP Right Cessation
Also Published As
Publication number | Publication date |
---|---|
JPS62501631A (en) | 1987-07-02 |
AU5318386A (en) | 1986-07-29 |
DK428386A (en) | 1986-11-11 |
AU587600B2 (en) | 1989-08-24 |
EP0208764A1 (en) | 1987-01-21 |
DK175799B1 (en) | 2005-02-28 |
DK428386D0 (en) | 1986-09-08 |
EP0208764A4 (en) | 1987-10-08 |
WO1986003938A1 (en) | 1986-07-17 |
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