EP0548210A1 - Liposomes - Google Patents

Liposomes

Info

Publication number
EP0548210A1
EP0548210A1 EP91916721A EP91916721A EP0548210A1 EP 0548210 A1 EP0548210 A1 EP 0548210A1 EP 91916721 A EP91916721 A EP 91916721A EP 91916721 A EP91916721 A EP 91916721A EP 0548210 A1 EP0548210 A1 EP 0548210A1
Authority
EP
European Patent Office
Prior art keywords
liposomes
peptide
substances
liposomes according
adjuvant
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Withdrawn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
EP91916721A
Other languages
German (de)
English (en)
Inventor
Gregory Gregoriadis
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.)
School of Pharmacy University of London
Original Assignee
School of Pharmacy University of London
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
Priority claimed from GB909019726A external-priority patent/GB9019726D0/en
Priority claimed from GB909019729A external-priority patent/GB9019729D0/en
Application filed by School of Pharmacy University of London filed Critical School of Pharmacy University of London
Publication of EP0548210A1 publication Critical patent/EP0548210A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • 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

Definitions

  • Iiposomes on intravenous injection, exhibit short half-lives in blood circulation; see Senior et al, Biochim. Biophys. Acta 839 (1985) 1-8. They are therefore unsuitable for maintaining increased concentrations of drugs and other substances within the vascular system. In addition, such large Iiposomes are unsuitable for use as delivery systems for targeting of drugs or other substances to specific sites, for example to diseased cells in the body.
  • a synthetic peptide In order to be effective, a synthetic peptide must possess a high level of immunogenicity and induce antibodies which cross-react extensively with the target organism or cell; see Van Regenmortel, Immunology Today 10 (1989) 266-272. In practice, peptides alone tend to possess weak immunogenicity, and anti-peptide antibodies often do not recognise the native protein antigen; see Jemmerson et al, Molecular Immunology 26 (1989) 301-307.
  • novel synthetic peptide vaccine preparations comprise Iiposomes containing entrapped or covalently- linked peptides together with molecules which augment the immune response to the peptides.
  • Iiposomes of the invention examples include pharmaceuticals, vaccines, genetic materials, enzymes, hormones, vitamins, carbohydrates, proteins/peptides, lipids, organic molecules, and inorganic molecules or atoms. More specific examples are anti-tumour and anti-microbial agents, enzymes, hormones, vitamins, metal chelators and genetic material, preferably carbohydrates or proteins/peptides.
  • the Iiposomes may be incorporated into vaccines.
  • microfluidisation distorts large and flexible vesicles by extrusion through a capillary, thereby increasing the number of vesicles of similar size which "bud off” from simple parent vesicles.
  • the minimum amount of retained solute is 35%, i.e. 2.5 mg with respect to a starting amount of 7.2 mg of the relevant substance. This is for up to 66 ⁇ moles, i.e. up to 50 mg, phosphatidylcholine (approximate molecular weight 760) up to 25 mg cholesterol (molecular weight 387). Therefore, by means of the present invention, it is possible to produce Iiposomes having mean diameters of less than 200 nm which contain at least 50 mg of the entrapped substance per g phospholipid or at least 33 mg of the substance per g of liposome-forming materials.
  • liposome preparations are made by standard methods for producing Iiposomes, for example as described by Gregoriadis, ed., Liposome Technology (1984) vols. 1 to 3, CRC Press Inc. In such methods, liposome components, peptides and adjuvant molecules are either mixed or covalently linked prior to formation of Iiposomes.
  • Solute-containing DRV Iiposomes were prepared by the procedure discussed elsewhere (Kirby and Gregoriadis, 1984; Gregoriadis et al, 1987). Briefly, small
  • unilamellar vesicles made in distilled water from equimolar PC (16.5, 33 66 or 132 ⁇ spl) and cholesterol were mixed with an equal volume (2.0 ml) of maltose (7.2 mg) or tetanus toxoid (72 mg) dissolved in PBS (0.44mM sodium phosphate, 2.7mM potassium chloride and 0.14 M sodium chloride, pH 7.4) and supplemented with traces (4.9 x 10 5 - 8.7 x 10 5 dpm) of the respective radiolabelled solutes.
  • SUV unilamellar vesicles
  • the suspensions (2.0 ml) were each divided into two equal portions. One portion was centrifuged at 20,000g for 30 min, the pellet washed three times with PBS and suspended in 20 ml of the same buffer (washed DRV) for microfluidization.
  • Washed and unwashed DRV (20 ml each) were passed for 1.8, 35, 5.2, 7.1 and 10.6 full cycles through a Microfluidizer 110TM kindly provided by Microfluidics Corp., Newton, MA, USA.
  • the pressure gauge was set at 60 psi throughout the procedure to give a flow rate of 35 ml per minute.
  • samples containing maltose were dialysed exhaustively against distilled water or PBS, and the toxoid containing vesicles or centrifuged for 30 min at 20,000g (1.8 and 35 cycles) or 35,000g (52, 7.1 and 10.6 cycles), the pellets washed twice in PBS or water and resuspended in 1.0 ml of the respective solvents.
  • the supernatants obtained on oentrifugation were passed (Senior et al, 1985) through Sepharose 4B columns; very little (less than.3%) of the solute in the centrifuged samples eluted in the liposome form.
  • Particle size distributions were measured by photon correlation spectroscopy of samples diluted in either water or PBS, using a Malvern Model 4700 apparatus (Malvem Instruments Ltd., Malvern, UK) equipped with a 25mW helium/neon laser. Mean diameters and size distributions are obtained: the z-average mean diameter, polydispersity factor and cumulative percentage mass and number undersize data were recorded as a function of the number of microfluidization cycles. The performance of the instrument was checked with monodisperse polystyrene latex suspensions (Polysciences, UK) and mixtures of such latex samples to verify the ability of the system to accurately measure polydisperse or bidisperse systems.
  • solute occurs, large molecules such as proteins leak at slower rates than smaller molecules. Furthermore, no apparent difference in solute retention was observed for preparations containing varying amounts of PC (8.5-66 ⁇ moles) (Figs.
  • Figs. 1 and 2 also indicate that, generally, a greater proportion of solute (maltose or toxoid) is retained by unwashed vesicles (ie. preparations microfluidized in the presence of unentrapped solute) than by washed DRV.
  • solute maltose or toxoid
  • a greater proportion of solute is retained by unwashed vesicles (ie. preparations microfluidized in the presence of unentrapped solute) than by washed DRV.
  • the presence of unentrapped solute during microfluidization diminishes solute leakage perhaps by reducing the osmotic rupture of the vesicles and by reducing initial concentration gradients across the membrane.
  • Fig. 6 illustrates the distribution of diameters by mass.
  • Table 1 shows the average mean size (nm) of microfluidised DRV, i.e. maltose-containing washed or unwashed DRV (33 ⁇ moles) PC were microfluidised in the presence of water or PBS for up to 10.6 cycles and samples measured for vesicle size (diameter in nm) by dynamic light scattering (Photon correlation spectroscopy). Polydispersity indexes ranging from 0.503 to 0.653 (water) and 0.517 to 0.653 (PBS) were similar to those obtained with some of the lipid compositions of Iiposomes employed by Talsma et al (1987).
  • Figure 1 shows entrapment values (on a scale of 0-100% of originally entrapped starting material) for 14 C-labelled maltose by DRVs following microfluidisation, with respect to cycles on the abscissa.
  • 14 C-maltose containing DRVs composed of eguimolar phosphatidylcholine (PC) and cholesterol were microfucidised in the washed (filled bars) or unwashed (shaded bars) forms in the presence of PBS for up to 5.2 cycles.
  • Amounts of liposomal phospholipid passed through the microfluidiser were 8.25 (A), 16.5 (B), 33 (C) and 66 (D) ⁇ mols. Values denote % retention of the originally entrapped C-maltose.
  • Original entrapment values were 27.1 (A), 33.6 (B), 66.1 (C) and 65.7 (D).
  • Figure 2 shows entrapment values for 125 I-labelled tetanus toxoid by DRVs following microfluidisation. Details are as in Figure 1 except that amounts of liposomal phospholipid passed through the microfluidiser were 8.25 (A) and 33 (B) ⁇ mols and original entrapment values were 19.2 (A) and 56 (B).
  • Figure 3 shows the effect of the liquid medium on 14 C- maltose retention by DRVs during microfluidisation. Details are as in Figure 1 except that DRVs were microfluidised is the presence of distilled water (A) or PBS (B) for up to 10.6 cycles, the amount of liposomal phospholipid passed through the microfluidiser was 33 ⁇ mols and original entrapment values were 54.3 (A) and 55.2 (B).
  • Figure 5 is on the same basis as Fig. 4. It shows the size distribution of vesicles containing maltose prepared in the presence of PBS (50 mg maltose/PBS) as a function of the number of cycles through the microfluidiser, showing that, after 10.6 cycles, the number of vesicles with diameters greater than 300 nm is decreased but a biphasic distribution is still maintained.
  • PBS 50 mg maltose/PBS
  • Figure 6 shows the mass distribution of vesicles containing maltose (50 mg maltose/water) as a function of cycles through the microfluidiser as in Figure 4, showing the narrow distribution size after 10.6 cycles.
  • Liposomes were formed from equimolar distearoyl phosphatidylcholine and cholesterol using the dehydration-rehydration procedure (see Kirby and Gregoriadis, 1984).
  • serum IgG antibodies specific for 3-VP2 peptide were estimated by ELISA assay (see Davis and Gregoriadis, 1987) whereby microtitre wells were coated with 3-VP2 peptide, diluted sera samples (1:1280 for IgGl, 1:160 for other subclasses) were added, and bound IgG's were measured through binding of IgG subclass specific antibodies.
  • the ELISA signal measured as optical density at 492 nm was produced using horseradish peroxidase- labelledsecond-antibody and 3,3',5,5'-tetramethylbenzidine (Miles) as substrate.
  • Table 2 gives the results of the IgG subclass ELISA (median values underlined).
  • mice were injected intramuscularly in groups of five on days 0 and 28 with either 20 ⁇ g free pre-S peptide or with Iiposomes containing 20 ⁇ g pre-S peptide or with Iiposomes containing 20 ⁇ g S peptide and 20 ⁇ g pre-S peptide.
  • Liposomes were formed from equimolar egg phosphatidylcholine and cholesterol using the dehydration- rehydration procedure (see Kirby and Gregoriadis, 1984).

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Dispersion Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

De nouveaux liposomes présentent un diamètre moyen ne dépassant pas 200 nm et contenant au moins 33 mg d'une ou de plusieurs substances emprisonnées par g de matière formant les liposomes. Ils peuvent aussi comprendre, emprisonnés ensemble ou liés de manière covalente dans le même liposome, un peptide synthétique et un adjuvant qui potentialise l'immunogénicité du peptide. Les liposomes sont utiles dans des vaccins.
EP91916721A 1990-09-10 1991-09-10 Liposomes Withdrawn EP0548210A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9019726 1990-09-10
GB909019726A GB9019726D0 (en) 1990-09-10 1990-09-10 Synthetic peptide vaccines
GB909019729A GB9019729D0 (en) 1990-09-10 1990-09-10 Liposome preparations
GB9019729 1990-09-10

Publications (1)

Publication Number Publication Date
EP0548210A1 true EP0548210A1 (fr) 1993-06-30

Family

ID=26297632

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91916721A Withdrawn EP0548210A1 (fr) 1990-09-10 1991-09-10 Liposomes

Country Status (3)

Country Link
EP (1) EP0548210A1 (fr)
JP (1) JPH06505701A (fr)
WO (1) WO1992004009A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6004534A (en) * 1993-07-23 1999-12-21 Massachusetts Institute Of Technology Targeted polymerized liposomes for improved drug delivery
WO1995003035A1 (fr) * 1993-07-23 1995-02-02 Massachusetts Institute Of Technology Liposomes polymerises presentant une stabilite amelioree et s'administrant oralement
US6060082A (en) 1997-04-18 2000-05-09 Massachusetts Institute Of Technology Polymerized liposomes targeted to M cells and useful for oral or mucosal drug delivery
SI1031347T1 (en) * 1999-01-27 2002-10-31 Idea Ag Transnasal transport/immunisation with highly adaptable carriers
SI1031346T1 (en) * 1999-01-27 2002-08-31 Idea Ag Noninvasive vaccination through the skin
US7902329B2 (en) * 2007-06-27 2011-03-08 The Board Of Trustees Of The Leland Stanford Junior University Oligopeptide tyrosinase inhibitors and uses thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235877A (en) * 1979-06-27 1980-11-25 Merck & Co., Inc. Liposome particle containing viral or bacterial antigenic subunit
CA1338702C (fr) * 1987-03-05 1996-11-12 Lawrence D. Mayer Formulations d'agents liposomiques-antineoplasiques a faible teneur en medicaments-lipides
FR2624741B1 (fr) * 1987-12-21 1991-06-28 Pasteur Institut Compositions a base d'une combinaison de liposomes et de lymphokine presentant des proprietes immunostimulantes et leurs applications en medecine humaine et veterinaire
AU4525589A (en) * 1988-10-27 1990-05-14 Regents Of The University Of Minnesota Liposome immunoadjuvants containing il-2
WO1990004943A1 (fr) * 1988-11-09 1990-05-17 Unger Evan C Agents liposomiques de contraste radiologique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9204009A1 *

Also Published As

Publication number Publication date
JPH06505701A (ja) 1994-06-30
WO1992004009A1 (fr) 1992-03-19

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