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WO1994013323A1 - Methode d'inhibition de la clairance des liposomes contenus dans la circulation - Google Patents

Methode d'inhibition de la clairance des liposomes contenus dans la circulation Download PDF

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Publication number
WO1994013323A1
WO1994013323A1 PCT/US1993/011729 US9311729W WO9413323A1 WO 1994013323 A1 WO1994013323 A1 WO 1994013323A1 US 9311729 W US9311729 W US 9311729W WO 9413323 A1 WO9413323 A1 WO 9413323A1
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Prior art keywords
animal
liposome
apolipoprotein
liposomes
active ingredient
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Application number
PCT/US1993/011729
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English (en)
Inventor
Arcadio Chonn
Sean C. Semple
Pieter R. Cullis
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The Liposome Company, Inc.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by The Liposome Company, Inc. filed Critical The Liposome Company, Inc.
Publication of WO1994013323A1 publication Critical patent/WO1994013323A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides

Definitions

  • This invention relates to a method of inhibiting the clearance of liposomes from the circulation, specifically to the use of anti-apolipoprotein
  • Liposomes are completely closed bilayer membranes containing an entrapped aqueous phase. Liposomes may be any of a variety of unilamellar (single membrane) or multilamellar (multiple membrane with an "onion-like" structure) vesicles.
  • the original liposome preparations of Bangham et al. involved suspending phospholipids in an organic solvent which was the evaporated to dryness, leaving a waxy deposit of phospholipid on the reaction vessel. Then, an appropriate amount of aqueous phase was added, the mixture was allowed to "swell” and the resulting liposomes, consisting of a mixture of multilamellar vesicles, were mechanically dispersed.
  • the structure of the resulting membrane bilayer is such that the hydrophobic, non-polar "tails” of the lipid molecules orient toward the center of the bilayer while the hydrophilic, polar "heads” of the molecules orient towards the aqueous phase.
  • Occluded by the bilayer is an aqueous compartment, some of which makes up the lumen of the vesicle and the other part of which (in multilamellar vesicles) is between the adjacent layers of the lipid membrane.
  • Bangham's technique provided the basis for the development of small unilamellar vesicles (SUVs) by Paphadjopoulos and Miller (Biochim. Biophys. Acta. 135: 624 (1967)).
  • SPLVs stable plurilamellar vesicles
  • SPLVs are lipid vesicles possessing from a few to over 100 lipid bilayers. There is a substantially equal distribution of solute in the compartments enclosed by each of the these bilayers. SPLVs are prepared by dissolving an amphipathic lipid or mixture of such lipids in an organic solvent. Most amphipathic lipids may be constituents of SPLVs.
  • aqueous phase containing the molecules to be entrapped in the liposome is added to the lipid solution.
  • This biphasic mixture is then converted to SPLVs by emulsifying the aqueous material within the solvent while evaporating the solvent.
  • SPLVs offer increased stability during storage in buffer and the ability to be maintained in harsh physiological environments. The disclosures of these references are incorporated herein by reference to indicate the state of the art with respect to the preparation of liposomes.
  • liposomes are as a carrier for a variety of materials, such as drugs, diagnostic agents and cosmetic agents (see, e,g, Fountain et al., U.S. Patent No. 4,588,578; Fountain et al., U.S. Patent No. 5,000,958; Bally et al., U.S. Patent No. 5,047,245; Janoff et al., U.S. Patent No. 5,059,591).
  • one of the problems associated with the use of. liposomes is their clearance from the circulation of the animals to which they have been administered.
  • Liposomes are internalized by phagocytic cells of the reticuloendothelial system (RES), and are cleared from the system rapidly, thereby inhibiting the effectiveness of the entrapped drug against diseases involving cells other than the RES. Even in the RES. where liposomal drugs are particularly effective because of their internalization by phagocytic cells, their utility is inhibited by the degradation of the entrapped drugs by enzymes contained in lysosomes of the phagocytic cells. Therefore, a method of inhibiting the recogntion and clearance of liposomes by the RES would increase the utility of liposomal formulations to be administered to animals.
  • RES reticuloendothelial system
  • This invention provides a method of reducing the rate of clearance of a liposome from the plasma of an animal, e.g., a mammal, which comprises administering to the animal the liposome and a composition comprising a carrier and an amount of an active ingredient effective to bind to apolipoprotein H and decrease the plasma concentration of apoH, wherein the liposome comprises an anionic lipid and the plasma concentration of apoH is decreased to a level at which binding to the anionic lipid is reduced.
  • the liposome can have one or more bilayers.
  • the active ingredient is a substance which preferentially binds to apoH in the plasma, e.g., an anti-apolipoprotein H antibody.
  • the anionic lipid is selected from the group consisting of cardiolipin, phosphatidic acid and phosphatidylserine.
  • composition is by intravenous injection.
  • the composition may be administered prior to administration of the liposome to the animal or concurrently with such administration.
  • a method of administering a chemical species to an animal preferably a mammal, more preferably, a human, which comprises: encapsulating the chemical in a liposome having a bilayer which comprises an anionic lipid; administering the liposome to the animal; and administering to the animal a composition comprising a carrier and an amount of an active ingredient effective to bind to apolipoprotein H and decrease the plasma concentration of apolipoprotein H, wherein the plasma concentration of apolipoprotein H is reduced to a level at which binding to the anionic lipid is reduced.
  • the chemical species may be a drug, for example, a therapeutic agent, an imaging agent or a nucleic acid.
  • FIG. 1 Non-reducing SDS-PAGE Analysis of Proteins Associated With Liposomes Recovered From the Circulation of Mice.
  • FIG. 1 Apolipoprotein H Immunoblot Analysis of Proteins Associated With Liposomes In Vivo.
  • This invention provides a method of reducing the rate of clearance of a liposome from the plasma of an animal, e.g., a mammal, which comprises administering to the animal the liposome and a composition which comprises a carrier and an active ingredient in an amount effective to bind to apoH and decrease the plasma concentration of apolipoprotein H, wherein the liposome comprises an anionic lipid and the plasma concentration of apolipoprotein H is decreased to a level at which binding to the anionic lipid is reduced.
  • the anionic lipid may, but is not required, to be cardiolipin (CL), phosphatidic acid (PA) or phosphatidylserine (PS).
  • the liposome may be any lipid vesicle which can incorporate an anionic lipid and be administered to an animal. Such liposomes can have one or more bilayers.
  • the method provided by this invention involves the use of a composition comprising a carrier and an active ingredient.
  • the carrier is any pharmaceutically accepatable carrier suitable for administration to an animal and includes, but is not limited to, aqueous buffers and physiological saline. Selection of a carrier, which will depend upon a number of factors such as the active ingredient selected, the size of the dose, the intended route of administration and the type of animal being treated, which are well known to those of ordinary skill in the art.
  • Presently preferred active ingredients are anti-apolipoprotein H antibodies, polyethylene glycol and ganglioside GM1. However, the practice of this invention is not limited to these specific substances. Rather, anything which preferentially binds to apoH and which can be administered to an animal is contemplated.
  • the effective amount of the active ingredient is any amount which will bind to apoH in the plasma and reduce the plasma levels of the lipoprotein to a level at which its binding to liposomes in circulation is reduced.
  • the effective amount of the active ingredient will depend upon a number of factors, such as the particular active ingredient chosen, the route of administration, and the type and size of the animal, which are well known to those of ordinary skill in the art.
  • the effective amount of the anti-apolipoprotein H antibody is typically at least about 1 microgram of the antibody per kg of the animal's body weight. Desirably, the effective amount of the anti-apolipoprotein H antibody is from about 1 microgram of the antibody per kg of the animal's body weight to about 1 milligram of the antibody per kg of body weight.
  • Administration of the composition may be prior to administration of the liposome or concurrently with administration of the liposome.
  • This invention also provides a method of administering a chemical species to an animal which comprises: encapsulating the chemical species in a liposome having a bilayer which comprises an anionic lipid; administering the liposome to the animal; and administering to the animal an amount of a composition comprising a cerrier and and an amount of an active ingredient effective to bind to apolipoprotein H and decrease the plasma concentration of apolipoprotein H, wherein the plasma concentration of apolipoprotein H is reduced to a level at which binding to the anionic lipid is reduced.
  • the chemical species is a drug, for example, a therepeutic agent, an imaging agent or a nucleic acid.
  • the method provided herein may also be used to administer viruses and other substances capable of being incorporated into liposomes to animals.
  • Liposomes are known to be effective for the administration of drugs to animals (see, e.g., Fountain et al., U.S. Patent No. 4,588,578; Fountain et al., U.S. Patent No. 5,000,958; Bally et al., U.S. Patent No. 5,047,245; Janoff et al., U.S. Patent No. 5,059,591).
  • Liposomal drugs are less toxic than their free counterparts. Encapsulation of drugs in liposomes leads to their gradual release; accordingly, less of the liposomal drug than the free drug need be administered to maintain an effective therapeutic level in serum.
  • liposomal drugs offer distinct therapeutic advantages over their free counterparts.
  • a limitation of their use is the rapid clearance of liposomes from the circulation. A method of decreasing the liposomal clearance rate would therefore enhance the therapeutic benefits of liposomal drugs.
  • the animal may be a mammal, e.g., a rodent or human, or any other animal to which liposomes may be administered.
  • the chemical species may be encapsulated in the liposomes when they are formed by adding the chemical to the original medium in which the liposomes are formed.
  • the chemical species may be encapsulated in the liposomes after they are formed, e.g., by using a transmembrane potential to transport a charged species across the liposomal bilayer according to the method described by Bally et al., U.S. Patent No. (5,077,056)
  • the liposome is any liposome which can encapsulate a chemical and be administered to an animal, including, but not limited to, unilamellar, multilamellar or stable plurilamellar vesicles.
  • Anionic lipids suitable for incorporating in liposomes are well known to those skilled in the art and include, but are not limited to, cardiolipin, phosphatidic acid and phosphatidylserine.
  • an "effective amount" of an anti-lipoprotein H antibody is any amount of the antibody which, when administered to an animal either alone or as a constituent of a pharmaceutical composition, is effective to bind to apolipoprotein H (apoH) in the animal's plasma and reduce the amount of apoH which will bind to liposomes.
  • apoH apolipoprotein H
  • LUVs Large unilamellar vesicles composed of phosphatidylcholine (PC) /Cholesterol (CH) (55:45), PC:CH:dioleoyl phosphatidylserine (DOPS) (35:45:20), PC: CH: Cardiolipin (CL) (35:35: 10), or PC:CH:dioleoyl phosphatidic acid (DOPA) (35:45:20) were prepared by a previously described extrusion procedure (Chonn et al., 1991 ; Hope et al., 1985).
  • Liposome preparations were 20 mM (for in vivo experiments) or 50 mM of total lipid (for in vitro experiments) in isotonic Hepes-buffered saline (HBS: 20 mM Hepes, pH 7.4, 145 mM NaCl) sterilized using Syrfil 0.22 m filters (Nucleopore, Pleasonton, CA).
  • HBS isotonic Hepes-buffered saline
  • Syrfil 0.22 m filters Nucleopore, Pleasonton, CA.
  • the liposomes were radiolabelled by incorporating the lipid marker [3H]cholesterylhexadecyl ether (10 Ci/30 mole total lipid) to quantitate the concentration of the recovered liposome suspensions (Stein et al., FEBS Lett, i ⁇ : 104 (1980)).
  • CD 1 mice (23-25 g females from Charles River) were treated with 200 microliters of a liposome suspension. After 2 minutes, the mice were sacrificed by overexposure to carbon dioxide, and blood was collected via cardiac puncture into ice cold 1.5 ml polypropylene micro test tubes. The blood was immediately cooled to 0 degrees Celsius, using an ice water bath, to prevent coagulation. It was then centrifuged at 12,000 rpm and 4 degrees Celsius for 5 minutes. Alternatively, 120 ⁇ l of an LUV (large unilamellar vesicle) suspension was incubated with 480 ⁇ l of human serum for 30 minutes at 37 degrees C. The incubation mixture was then cooled to 0 degrees C using an ice water bath.
  • LUV large unilamellar vesicle
  • Protein separation was performed by SDS-polyacrylamide gel electrophoresis using the mini Protean-II electrophoresis apparatus (Bio-
  • Mini Protean II gradient gels were transferred onto nitrocellulose filters (Bio Rad) using a Mini Trans Blot Electrophoresis Transfer Cell (Bio Rad) at a constant current of 350 mA for 60 min., followed by immunoblot analysis employing the Enhanced Chemiluminscence western blotting detection system (Amersham).
  • the blocking buffer consisted of PBS, pH 7.5, 5 % dried skim milk powder and 1% Tween 20 detergent (Sigma). Rabbit antisera to human apoH (Behring, U.K.) was used at a dilution of 1 :5,000. Peroxidase-coated goat antirabbit IgG was used at a dilution of 1 :5,000. Blocking buffer was used as the diluent. Immunoblot analysis was repeated twice.
  • ApoH was purified by a modification of the method of Polz et al. (Int. J. Biochem 1_1_: 265 (1980)). Initially, 40 ml of normal mouse serum (Cedar Lane Laboratories, Hornby, Canada) was acidified by the addition of 0.6 ml of 70% (v/v) perchloric acid at 0 degrees C. The resulting solution was stirred for 15 min., neutralized with 12.5% sodium bicarbonate and centrifuged to remove precipitated proteins. The supernatant was dialyzed against 50 mM Tris, pH 7.4, 30 mM NaCl. Proteins were eluted with a step gradient of NaCl in 50 mM Tris, pH 7.4..
  • the protein content of the 1-ml fractions was determined spectrophotometrically by measuring absorbance at 280 nm; furthermore, column fractions from this and subsequent columns were screened immunologically for apoH using a dot blot apparatus (Bio Rad). Briefly, ten ml of the column fractions was applied onto a nitrocellulose membrane (Bio Rad) under vacuum. The membrane was then blocked and probed for apoH as described above. Fractions positive for apoH were pooled and dialyzed against 50 mM sodium acetate buffer, pH 4.8, 50 mM NaCl.
  • the dialyzed sample was concentrated, as described above, and further purified using an FPLC Mono-S column (Pharmacia).
  • One-ml samples were applied to the column, and eluted with a linear NaCl gradient in 50 mM sodium acetate buffer, pH 4.8.
  • Fractions were analysed by SDS- PAGE and dot-blot analysis to determine which contained apoH and to monitor the extent of the purification. ApoH-containing fractions were pooled, concentrated and stored at minus 20 degrees C.
  • Figure 1 shows the major proteins recovered from the blood of CD1 mice 2 minutes post injection with the liposomes. It is clear that there are two major proteins having electrophoretic mobilities corresponding to 66,000 daltons and 50-55 kD that are associated with the liposomal membranes. Based on the apparent molecular weight, and the fact that albumin is the most abundant plasma protein (approximately 40 mg/ml), we suspected that the 66 kD band was albumin. This was confirmed by immunoblot analysis.
  • the levels of apolipoprotein H binding to anionic vesicles corresponds to similar, and even greater, levels than those for albumin. This is significant, as the reported values for the concentration of apoH in rats and humans is approximately 0.2 mg/ml plasma (60% is found in the lipoprotein-free bottom fraction after centrifugation and the remaining 40% is associated with triglyceride-rich lipoproteins; Polz and Kostner, 1979; Lee et al., 1983). By direct comparison, the reported values for concentration of albumin in rats is approximately 40 mg/ml, 200-fold greater than the plasma concentration of apoH.
  • apoH may play a significant role in immune detection of foreign particles, as it is associated in relatively low amounts with phosphatidylglycerol, phosphatidylinositol vesicles or phosphatidylcholine- cholesterol (55:45) vesicles, which possess moderately slow clearance kinetics and at very low levels with ganglioside GM1 -containing liposomes, which are capable of extended circulation times. Furthermore, apoH has been found to exert a significant effect on triglyceride clearance in rats (Wurm et al., Metabolism, 31: 484 (1982)).
  • apoH has been shown to function as a cofactor for the binding of anti-phospholipid antibodies to membranes containing anionic phospholipids (McNeil et al., Proc. Natl. Acad. Sci. USA 87: 4120 (1990); Galli et al., Lancet 335: 1544 (1990)).

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Abstract

Méthode permettant d'abaisser la vitesse de clairance, d'un liposome, contenant un lipide anionique, du plasma d'un animal. Une composition constituée d'un excipient et d'un ingrédient actif sous une dose appropriée pour se lier à l'apolipoprotéine H et abaisser sa concentration dans le plasma est administrée au sujet afin de réduire la concentration dans le plasma de l'apoH à un niveau auquel la liaison au lipide anionique est réduite.
PCT/US1993/011729 1992-12-04 1993-12-03 Methode d'inhibition de la clairance des liposomes contenus dans la circulation WO1994013323A1 (fr)

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US98492992A 1992-12-04 1992-12-04
US07/984,929 1992-12-04

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WO1994013323A1 true WO1994013323A1 (fr) 1994-06-23

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8956616B2 (en) 2005-01-24 2015-02-17 Board Of Regents, The University Of Texas System Constructs binding to phosphatidylserine and their use in disease treatment
CN108404139A (zh) * 2018-01-29 2018-08-17 南昌大学 低浓度单唾液酸四己糖神经节苷脂修饰的重组脂蛋白的制备方法及其应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992009893A1 (fr) * 1990-11-29 1992-06-11 Carbaugh John E COMPOSITIONS ET PROCEDES DIAGNOSTIQUES ET THERAPEUTIQUES POUR DETERMINER LA PRESENCE DE LA LIPOPROTEINE(a)

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992009893A1 (fr) * 1990-11-29 1992-06-11 Carbaugh John E COMPOSITIONS ET PROCEDES DIAGNOSTIQUES ET THERAPEUTIQUES POUR DETERMINER LA PRESENCE DE LA LIPOPROTEINE(a)

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ARCADIO CHONN ET AL.: "ASSOCIATION OF BLOOD PROTEINS WITH LARGE UNILAMELLAR LIPOSOMES IN VIVO.", JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 267, no. 26, September 1992 (1992-09-01), BALTIMORE, MD US, pages 18759 - 18765 *
CHEMICAL ABSTRACTS, vol. 116, no. 12, 23 March 1992, Columbus, Ohio, US; abstract no. 113450d *
H. PATRICK MCNEIL ET AL.: "ANTI-PHOSPHOLIPID ANTIBODIES ARE DIRECTED AGAINST A COMPLEX ANTIGEN THAT INCLUDES A LIPID-BINDING INHIBITOR OF COAGULATION: BETA2-GLYCOPRTEIN I (APOLIPOPROTEIN H)", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 87, June 1990 (1990-06-01), WASHINGTON US, pages 4120 - 4124 *
M. GALLI ET AL.: "ANTICARDIOLIPIN ANTIBODIES (ACA) DIRECTED NOT TO CARDIOLIPIN BUT TO A PLASMA PROTEIN COFACTOR.", THE LANCET, vol. 335, 30 June 1990 (1990-06-30), pages 1544 - 1547 *
MARTIN C. WOODLE ET AL.: "PROLONGED SYSTEMIC DELIVERY OF PEPTIDE DRUGS BY LONG-CIRCULATING LIPOSOMES: ILLUSTRATION WITH VASOPRESSIN IN THE BRATTLEBORO RAT.", PHARM. RES., vol. 9, no. 2, 1992, pages 260 - 265 *
T. M. ALLEN ET AL.: "PHOSPHATIDYLSERINE AS A DETERMINANT OF RETICULOENDOTHELIAL RECOGNITION OF THE ERYTHROCYTE SURFACE.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 85, November 1988 (1988-11-01), WASHINGTON US, pages 8067 - 8071 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8956616B2 (en) 2005-01-24 2015-02-17 Board Of Regents, The University Of Texas System Constructs binding to phosphatidylserine and their use in disease treatment
CN108404139A (zh) * 2018-01-29 2018-08-17 南昌大学 低浓度单唾液酸四己糖神经节苷脂修饰的重组脂蛋白的制备方法及其应用

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