EP0918508A1 - Procede ameliore permettant de masquer les liposomes pour eviter leur reconnaissance par le systeme reticuloendothelial - Google Patents

Procede ameliore permettant de masquer les liposomes pour eviter leur reconnaissance par le systeme reticuloendothelial

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Publication number
EP0918508A1
EP0918508A1 EP96913099A EP96913099A EP0918508A1 EP 0918508 A1 EP0918508 A1 EP 0918508A1 EP 96913099 A EP96913099 A EP 96913099A EP 96913099 A EP96913099 A EP 96913099A EP 0918508 A1 EP0918508 A1 EP 0918508A1
Authority
EP
European Patent Office
Prior art keywords
liposome
vesicle
neuraminic acid
matter
carbon
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.)
Withdrawn
Application number
EP96913099A
Other languages
German (de)
English (en)
Inventor
John R. Lau
W. Blair Geho
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SDG Technology Inc
Original Assignee
SDG Technology Inc
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Filing date
Publication date
Application filed by SDG Technology Inc filed Critical SDG Technology Inc
Priority claimed from CA002250529A external-priority patent/CA2250529A1/fr
Publication of EP0918508A1 publication Critical patent/EP0918508A1/fr
Withdrawn legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1217—Dispersions, suspensions, colloids, emulsions, e.g. perfluorinated emulsion, sols
    • A61K51/1227—Micelles, e.g. phospholipidic or polymeric micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1217—Dispersions, suspensions, colloids, emulsions, e.g. perfluorinated emulsion, sols
    • A61K51/1234—Liposomes

Definitions

  • SIALIC ACID An N-acyl derivative of neuraminic acid.
  • NEURAMINIC ACID A synthetically-derived sialic acid.
  • the Chemical Abstracts Index names for this molecule are: D-glycero-D-galacto-2-Nonulopyranosidonic acid, methyl 5-amino-3, 5-dideoxy-(9Cl) ; or D-glycero-D-galacto- Nonulopyranosidonic acid, methyl 5-amino-3, 5-dideoxy-(8Cl) .
  • Another name for this molecule is Methoxyneuraminic Acid, or Beta-Methoxy Neuraminic Acid. As ordered by the applicant from Sigma Chemical Company, P.O. Box 14508, St. Louis, MO 63178, it is named Neuraminic Acid Beta-Methyl-Glycoside (pfs) , product number N 5380 (1992 Catalog) .
  • NEURAMINIDASE An enzyme of the hydrolase class that is in blood and which catalyzes the hydrolysis of glucosidic linkages between a sialic acid residue and a hexose or hexosamine residue at the non-reducing terminal of oligosaccharides in glycoproteins, glycolipids and proteoglycans.
  • LIPOSOME A spherical particle in an aqueous medium, formed by a lipid bilayer enclosing an aqueous compartment.
  • Liposomes also known as vesicles, have been designed to encapsulate a cargo of pharmacological agents useful for in vivo purposes such as the diagnosis and treatment of various diseases and conditions. These cargo-carrying liposomes have, experimentally, shown potential for being site-specific carrier systems for a variety of such agents. Agents so delivered to designated sites in vivo demonstrate significantly enhanced therapeutic indices. Concurrently, decrease in unwanted side effects and wasted portions of dosages is achieved. Agents not so couried spread to nonspecific and often undesirable areas of the body, and possibly cause side effects.
  • the advantages of the prior art in vivo carrier system have been offset, however, by the deleterious effects of the body's reticuloendothelial system (the "RES") , mainly the liver and spleen.
  • the RES acts to screen the body's circulation.
  • the RES will gradually scavenge from the circulation all material it considers foreign and unwanted.
  • Liposomes have certain physical characteristics which render them susceptible to removal by the RES. Once recognized, liposomes, whether given a site-specific molecule for so-called “targeted” delivery or not, are quickly phagocytosed by the RES along with their cargo.
  • This prior revolutionary carrier system has, until the present invention, and the processor invention of U.S. Patent 4,501,728, remained hampered by the RES, the body's own defense mechanism.
  • neuraminidase cleaves the chemical bond between sialic acid and its attached sugar (galactose) , thereby allowing the galactose (sugar) on the surface of the liposome to be recognized by the RES's Ashwell receptor of the liver.
  • Enzymatic removal of the sialic acid is time-dependent. Liposomes intended to circulate for longer periods of time in the vascular system or until attracted to a binding-site, are removed by the RES once the sialic acid mask is compromised or lost.
  • the liposome Along with finding a means to mask the liposome from the RES, it is generally an objective in the art of drug delivery to discover an appropriate site-specific binding molecule. Once discovered, the binding molecule can be incorporated into the liposome. Such masked liposome with its binding molecule, when introduced in vivo, will evade the RES and will carry a designated cargo to a predetermined binding site in the body. When delivered, the cargo is then released to perform its designated function.
  • This invention is embodied by the product of a procedure of applying to the external surface of a liposome carrier system, two types of substituent molecules.
  • One such molecule masks the liposome from the RES and is selected from the class consisting of those chemicals which are classed biologically as synthetically- derived molecules that prevent phagocytosis by the body's RES and which cannot be degenerated and separated from the liposome by the action of circulating endogenous enzymes.
  • the other molecule is one which binds to or is bound by a specific site in the body and which is incorporated as part of the liposome carrier.
  • the combined result of the liposome carrier with these two types of substituent molecules, is highly effective delivery of the liposome's therapeutic or diagnostic cargo to a specific body site, having evaded the body's RES.
  • compositions of matter for in vivo administration of therapeutic or diagnostic agents comprising a lipid membrane structure in the form of a liposome or vesicle, a second component which is a syntheticallyderived molecule that prevents recognition of the liposome or vesicle by the RES and which cannot be cleaved from the liposome or vesicle by the action of endogenous enzymes, and a third component which is a molecule that binds the liposome or vesicle to a specific site in the body and which can be present for site-specific delivery or absent for non-specific delivery of the therapeutic or diagnostic agents in vivo.
  • This invention is the discovery that superior site- specific delivery of the liposome's cargo is obtained by the combined effect of using synthetically-derived RES- avoidance molecules that cannot be cleaved by circulating endogenous enzymes, with site-specific binding molecules that find or are found by a receptor at a desired site in the body.
  • This invention is also the discovery that superior systemic circulation of liposomes without site-specific delivery, can be achieved using the aforementioned synthetic masking molecules without the site-binding molecule component.
  • Figure 1 is a representative of a branched glycoprotein that has sialic acid as the terminal units of the glycan chains.
  • Figure 2 represents B-methoxyneuraminic acid structure.
  • Figure 3 illustrates the first example of a synthetic pathway by which a non-hydrolyzable secondary amide bond can be created by using an amide linkage to connect the B-methoxyneuraminic acid conjugate to an appropriate bridging molecule.
  • Figure 4 illustrates another synthetic pathway by which a secondary amide linkage can be formed between B-methoxyneuraminic acid and photoactivatable biotin resulting in a biotin B-methoxyneuraminic acid conjugate that can be attached to a liposome using streptavidin and biotin phosphatidyl ethanol amine.
  • Figure 5 shows the third type of synthetic pathway which would result in a bond formation between B-methoxyneuraminic acid and 2-iminothiolane (Traut's Reagent) , resulting in yet another secondary amine bond that is not hydrolyzable by the circulating endogenous enzyme neuraminidase.
  • Figure 6A and 6B show another synthetic pathway for establishing a non-hydrolyzable bond between neuraminic acid and p-maleimido phenyl butyrate phosphatidyl ethanol amine through the formation of a thio ether bond.
  • Figure 7 is a graph showing the 2-hour effect of neuraminic acid on I.V. liposome distribution.
  • Figure 8 is a graph showing the effect of neuraminic acid on 51 Chromium radiation.
  • This invention embodies a liposomal delivery system consisting essentially of a phospholipid membrane known commonly as vesicles, or liposomes (vesicles and liposomes, as used herein, mean the same thing) , with two types of substituent molecules incorporated in the membrane.
  • One such type of molecule is classed biologically as a synthetic molecule which masks the liposome from the body's RES and which cannot be cleaved from the liposome by circulating endogenous enzymes.
  • the other type of molecule is one which binds to a specific site in the body. The combination allows delivery of a greatly enhanced index of therapeutic or diagnostic cargo to such specific body site.
  • B-methoxyneuraminic acid used herein as a synthetic liposome masking molecule, is used in this description as an example and is preferred because its use is consistent with data shown herein.
  • the first example set forth hereinafter is a liposome with a permeable membrane, which is designed and synthesized to circulate in the blood for an extended per$od of time as a non-binding time-release drug delivery system.
  • the second example set forth hereinafter is a liposome which, in addition to being masked by the incorporation of neuraminic acid or its biologically active derivatives onto the liposome membrane, is also invested with a site-binding molecule.
  • liposomes Use of naturally-occurring sialic acid residues to mask liposomes from the RES enables liposomes to carry therapeutic or diagnostic agents in vivo for extended periods of time.
  • red blood cells which also have sialic acid residues that perform the same masking function, these liposomes circulate systemically, evading the RES.
  • liposomes masked with naturally-occurring sialic acid residues are subject to the action of circulating endogenous enzymes such as neuraminidase, which cleaves the glycosidic bond of the sialic acid, exposing its penultimate sugar to RES recognition. The enzymatic cleavage of this glycosidic bond is by hydrolysis, and occurs at the bond indicated by reference character 10 of Figure 1.
  • the preferred best mode embodiment uses Beta-methoxyneuraminic acid, the systematic name of which is given above in the Definition section. See Figure 2.
  • the sugar of this molecule has a free primary amino group (-NH-) attached to carbon #5 of the nonulopyranose ring structure, along with a non-reactive B-methoxy group (OCH -) attached to carbon #2 of the ring.
  • the bond of this invention is not susceptible to hydrolysis by the enzyme neuraminidase in the plasma of a warm blooded host;
  • the bond can be of a secondary amine type
  • the bond can be of a thio ether type
  • Figure 3 illustrates the first example of a synthetic pathway by which a nonhydrolyzable secondary amide bond is created by using an amide linkage to connect the B-methoxyneuraminic acid sugar to an appropriate bridging molecule.
  • Reference character 11 of Figure -3 points out the amide linkage (-C-N-) resulting in a secondary amine.
  • Figure 4 illustrates another synthetic pathway by which a secondary amide linkage is formed between B-methoxyneuraminic acid and photoactivatable biotin resulting in a biotin B-methoxyneuraminic acid conjugate that can be attached to 8 liposome using streptavidin and biotin phosphatidyl ethanol amine.
  • Figure 5 shows the third type of synthetic pathway resulting in a bond formation between B-methoxyneuraminic acid and 2-iminothiolane (Traut's Reagent) resulting in vet another secondary amine bond that is not hydrolyzable by the circulating endogenous enzyme neuraminidase. While all three examples illustrate the formation of a second amine bond, the -R- group to which the secondary amine is attached is different.
  • Figure 6A and Figure 6B show another synthetic pathway for establishing a non-hydrolyzable bond between neuraminic acid and p-maleimido phenyl butyrate phosphatidyl ethanol amine through the formation of a thio ether bond.
  • the B-methoxy group is removed from B-methoxyneuraminic acid by HCl.
  • the resulting neuraminic acid is acylated with acetic anhydride to form its fully acylated sugar derivative.
  • This sugar derivative is then reacted with the halo acid hydrogen bromide to form the acetohalo saccharide.
  • the acetohalo saccharide is then reacted with thiourea to form the thiourea acetate derivative, which, in turn, is reacted with potassium carbonate and sodium bisulfite to remove the acetyl groups in order to form the sulfhydryl sugar, 2-thioglycose.
  • 2-thioglycose is reacted with sodium methoxide (a base) in the presence of methanol, to generate the thioneuraminic acid derivative.
  • this neuraminic acid sugar has a free sulfhydryl, it reacts with the maleimido group of p-maleimido phenyl butyrate phosphatidyl ethanolamine, which, in turn, is coupled to the vesicle membrane.
  • insulin was administered in vivo as the cargo of a porous liposome which had attached to its phospholipid membrane only the synthetic masking molecule B-methoxyneuraminic acid.
  • the liposome was found to circulate in the body for significantly extended periods of time, releasing insulin, because of the fact that the B-methoxyneuraminic acid's sugar group could not be hydrolyzed and separated from the liposome by the endogenous enzyme neuraminidase.
  • Figure 7 and 8 together with the following text, establish the exceptional performance of a liposomal delivery system consisting essentially of a phospholipid membrane with a B-methoxyneuraminic acid substituent, wherein said acid is attached to the liposome by a bond which is not hydrolyzed by a circulating endogenous enzyme such as neuraminidase in the blood of a warm blooded animal.
  • a liposomal delivery system consisting essentially of a phospholipid membrane with a B-methoxyneuraminic acid substituent, wherein said acid is attached to the liposome by a bond which is not hydrolyzed by a circulating endogenous enzyme such as neuraminidase in the blood of a warm blooded animal.
  • EXAMPLE #1 AVOIDANCE OF LIVER AND SPLEEN IN RATS Liposomes were made with the following compositions:
  • DSL Distearoyl Lecithin
  • MPB-PE p-maleimido phenyl butyrate phosphatidyl ethanol
  • the liposomes were made using the method described in U.S. Patent 4,603,044. Briefly, the crusts, having the above compositions, were solubilized with 10.0ml of lOmM PhosPhate buffer, PH 7.4, to which a lOul aliquot of 123 I-human growth hormone was added. The mixture was sonicated on a Heat Systems Branson Cell Disrupter at setting #4 for 6.0 minutes at 60°C. The sample was then centrifuged for one hour at 20,000 RPM in a Sorvall RC2B centrifuge at 20 * C.
  • the supernatant was decanted and 410ul was chromatographed over a sephadex G-100 column equilibrated with lOmM phosphate buffer, pH 7.4.
  • the derivatized neuraminic acid (DERIV. N.A.) was then added to the liposomes for the DERIV N.A. sample and allowed to react with the MPB-PE that was already in the liposomal membrane.
  • test samples were then tested in normal rats.
  • rats were used for each group.
  • the test samples were administered intravenously to each rat and the rats' livers and spleens were removed after two hours, under seconal anesthesia, and processed for counting the radio-label.
  • the results of the study are shown in Figure 7.
  • the rats that received the liposomes with the synthetically-derived neuraminic acid had significantly fewer liposomes in their livers and spleens than rats receiving the control liposomes without any neuraminic acid.
  • Syntheticallyderived neuraminic acid is B-methoxyneuraminic acid that has been derivatized according to the above specification and does not contain a glycosidic bond that is hydrolyzable by circulating endogenous enzymes, such as neuraminidase.
  • the rats that received the liposomes with the naturally occurring sialic acid ganglioside showed significantly lower levels of liposomes in their livers than the comparable control rats, but not as low a level of liposomes as was observed in rats receiving the liposomes with the synthetic neuraminic acid derivative.
  • the naturally-occurring ganglioside is a molecule that exhibits a terminal sialic acid residue accompanied by a penultimate galactose group.
  • the sialic acid residue of that ganglioside is connected to a penultimate sugar moiety by a glycosidic bond that is hydrolyzable by the enzyme neuraminidase.
  • the spleens of the rats receiving both the synthetic neuraminic acid and the natural ganglioside were lower than the control rats.
  • EXAMPLE #2 AVOIDANCE OF LIVER, SPLEEN AND HEART IN NUDE MICE WITH LIPOSOMES TARGETED TO TUMORS
  • Sample A Tumor specific antibody (SCCL-1) ; no synthetic neuraminic acid
  • Sample B Tumor specific antibody (SCCL-1) ; with synthetic neuraminic acid
  • GM-1 sialic acid and sialic acid derivatives.
  • Sialic acid itself acts as a protective red blood cell component, to prevent the RES from phagocytosing the red blood cells.
  • This invention addresses this cleavage problem by using a synthetic sialic acid that forms a non-hydrolyzable bond with the liposome.
  • the derivatives of natural sialic acid shown above may also be synthetically attached via a non-hydrolyzable bond by removing the acetyl group in examples #1-4 or the glycol group in example #5.
  • the net effect of this removal is to expose a primary amino group which can be derivatized with succiniroedyl acetyl thio acetic acid to form an amide linkage (-CONH-) which is a bond that cannot be hydrolyzed by a circulating indogenous enzyme such as neuraminidan.
  • This new series of synthetically-manipulated neuraminic acid derivatives when attached to liposomes, provide superior masking from the RES.
  • the preferred embodiment is the synthetic, B-methoxy form of neuraminic acid shown in Figure 2.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Dispersion Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Radiology & Medical Imaging (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract

Une membrane biochimique encapsulée par un résidu d'acide neuraminique destiné à masquer la surface de la membrane afin d'éviter qu'elle soit reconnue et éliminée par les cellules d'interception du système réticuloendothélial du corps.
EP96913099A 1996-04-24 1996-04-24 Procede ameliore permettant de masquer les liposomes pour eviter leur reconnaissance par le systeme reticuloendothelial Withdrawn EP0918508A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/US1996/005699 WO1997039735A1 (fr) 1996-04-24 1996-04-24 Procede ameliore permettant de masquer les liposomes pour eviter leur reconnaissance par le systeme reticuloendothelial
CA002250529A CA2250529A1 (fr) 1996-04-24 1996-04-24 Procede ameliore permettant de masquer les liposomes pour eviter leur reconnaissance par le systeme reticuloendothelial

Publications (1)

Publication Number Publication Date
EP0918508A1 true EP0918508A1 (fr) 1999-06-02

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EP (1) EP0918508A1 (fr)
AU (1) AU5571196A (fr)
WO (1) WO1997039735A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU4090299A (en) * 1998-05-20 1999-12-06 Sdg, Inc. Liposomal delivery complex

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Publication number Priority date Publication date Assignee Title
US4920016A (en) * 1986-12-24 1990-04-24 Linear Technology, Inc. Liposomes with enhanced circulation time
US5354853A (en) * 1993-03-12 1994-10-11 Genzyme Corporation Phospholipid-saccharide conjugates

Non-Patent Citations (1)

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Title
See references of WO9739735A1 *

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AU5571196A (en) 1997-11-12
WO1997039735A1 (fr) 1997-10-30

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