LIPOSOMAL COMPOSITIONS FOR MUCOSAL ADMINISTRATION

专利摘要:
A liposomal composition comprising lipids that form a liposomal lipid bilayer with phospholipid-PEG conjugates incorporated into the liposomal lipid bilayer, and a chitosan or chitosan derived Figure 1 is described and claimed.
公开号:BE1022346A9
申请号:E20155134
申请日:2015-03-12
公开日:2016-10-07
发明作者:Nupur Dutta；Hardeep Oberoi；David Burkhart；Jay T Evans
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

LIPOSOMAL COMPOSITIONS FOR MUCOSAL ADMINISTRATION Statement Regarding Government-Funded Research
Aspects of this invention have been made with the support of the United States Government and under the terms of the NIH NO. HHSN272200900008C, the United States Government may have certain rights in the invention.
Context
It has recently been reported the rapid transport of nanoparticles through the human mucosa for nanoparticles sufficiently coated with short-chain polyethylene glycol (PEG, generally less than 5000 units) or certain Pluronic polymers [Cu Y, Saltzman WM. Mol Pharm. 2009; 6 (1): 173-181; Hanes J, et al. Nanomedicine. 2011; 6 (2): 365-375]. This approach, called mucosal penetration by the authors, is thought to be based on a decrease in mucoadherence (rather than an increase in mucoadhesion), allowing rapid penetration of the nanoparticles through the mucus.
Toll-4 receptor modulators (TLR4) are immunogenic compounds used in pharmaceutical compositions and especially as adjuvants in human vaccines. TLR-4 agonists have been formulated in liposomes for injection administration for vaccines. Aminoalkyl glucosaminide phosphates (AGP) are modulators of TLR4, some of which are particularly potent and potentially reactogenic. In general, there is a need for improved liposomal compositions and particularly improved liposomal compositions for TLR4 modulators for administration of pharmaceutical compositions. Summary of the invention
Methods and compositions for liposomal formulations for mucosal administration are provided.
In one embodiment, the invention provides a liposomal composition comprising lipids that form a liposomal lipid bilayer and that further comprises phospholipid-PEG conjugates incorporated into the liposomal lipid bilayer. In addition, the liposomal composition comprises a TLR4 agonist (e.g., AGP) and suitably comprises HEPES buffer.
In one embodiment, the liposomes of the liposome are DOPC in the presence of cholesterol.
In one embodiment, the invention provides a liposomal composition comprising lipids that form a liposomal lipid bilayer and that further comprises PEG / surfactant copolymers such as poloxamers that are incorporated into the liposomal lipid bilayer. In addition, the liposomal composition comprises a TLR4 agonist (e.g., AGP) and suitably comprises HEPES buffer. In one embodiment, the liposome lipids are DOPC in the absence of cholesterol.
In a suitable embodiment, the liposomal composition comprises chitosan or a derivative of chitosan.
In a suitable embodiment, the invention provides a liposomal formulation comprising a DOPC liposome in the absence of sterol, poloxamers, wherein the poloxamers are incorporated into the DOPC liposome bilayer, an AGP in HEPES buffer, and optionally chitosan or a derivative of chitosan.
In a suitable embodiment, the invention provides a liposomal formulation comprising a DOPC liposome in the presence of sterol, suitably cholesterol, a phospholipid-PEG conjugate where the phospholipid-PEG conjugate is incorporated into the DOPC liposome bilayer. -sterol, a TLR4 agonist (eg, AGP) in HEPES buffer, and optionally chitosan or a chitosan derivative.
In one embodiment, the present invention provides a liposomal composition comprising a phospholipid, a phospholipid-PEG conjugate or a poloxamer and an aminoalkanesulfonic buffer such as HEPES, HEPPS / EPPS, MOPS, MOBS and PIPES.
In one embodiment, the present invention provides a liposomal composition comprising a phospholipid, a phospholipid-PEG conjugate or a poloxamer and an aminoalkyl glucosaminide phosphate (AGP), suitably CRX-601, CRX 6Q2, CRX 527, CRX 547, CRX 526, CRX 529 or CRX 524.
In one embodiment, the present invention provides a liposomal composition comprising a phospholipid, a phospholipid-PEG conjugate or a poloxamer, an AGP, an aminoalkanesulfonic buffer and a chitosan or a chitosan derivative, suitably chitosan oligosaccharide lactate, chitosan glycol , trimethylchitosan or methylglycol chitosan.
In another embodiment, the present invention provides a method of improving the production of a liposomal composition for sublingual administration comprising the steps of: dissolving a lipid, such as dioleoylphosphatidylcholine ("DOPC") , a phospholipid-PEG conjugate (or a poloxamer in the absence of cholesterol), and an AGP in an organic solvent, removing the solvent to produce a phospholipid film, adding the film to HEPES buffer or HEPES buffer in saline, dispersing the film in the solution, and extruding the solution successively through polycarbonate filters to form unilamellar liposomes. The liposomal composition can be further filtered aseptically.
In a suitable embodiment, a liposomal composition has high incorporation of a TLR4 agonist (eg, AGP) when the liposome is formed with cholesterol.
In another embodiment, a liposomal composition exhibits high incorporation of a particular AGP, CRX 601, when the liposome is formed without sterol such as cholesterol, providing benefits for the production and formulation of such liposomal compositions, including liposomal compositions comprising a poloxamer.
The liposomes of the present invention are beneficial in both the production and the use of a pharmaceutical composition. Other embodiments are disclosed in the descriptions, figures and claims provided herein.
Brief description of the drawings
Figure 1 shows the stability of liposome adjuvants in the presence of methylglycol chitosan (MGC). Size / IPD and potential values □ with increasing concentration of MGC for unmodified adjuvanted liposomes, modified with PE-PEG2K and PE-PEG5K (A) and (C); and Pluronic-modified liposomes L64, F68 and F127 (B) and (D). For (A) and (B), the sizes are represented as bars and the IPD values as points. The data are expressed in mean + SD, (n = 3). Particles in the Qn range have tended to precipitate over time.
Figure 2 shows the characterization of liposomes of phospholipid-PEG loaded with adjuvant in the presence of methylglycol chitosan (MGC). Size / IPD and potential values □ with increasing MGC concentration for PE-PEG2K and PE-PEG5K modified 1% (top row) and 25% (bottom row) modified adjuvant liposomes. Sizes are represented as bars and IPD values as dots. The data are expressed as mean ± SD, (n = 2) for the 1% change in mol (top row) and n = 1 for the 25% change in mol (bottom row). Particles in the size range of the order of Qn tended to precipitate over time.
Figure 3 shows the characterization of Pluronic liposomes loaded with adjuvant in the presence of methylglycol chitosan (MGC). Size / IPD and potential values □ with increasing concentration of MGC for Pluronic L64, F68 and F127-modified liposomes at 15% (top row) and 25% (bottom row). Sizes are represented as bars and IPD values as dots. The data are expressed as mean ± SD, (n = 2) for the 1% change in mol (top row) and n = 1 for the 25% change in mol (bottom row). Particles in the size range of the order of Qn tended to precipitate over time.
Figure 4 shows post-secondary serum IgG titers (top), posttrial serum IgG titers (middle), and IGA titers and IGA titers of post-tertiary tracheal / vaginal lavage fluids (bottom). after a study of liposomes modified with phospholipid-PEG. Liposomes with 1, 5 and 25% mole of MPEG-2000-DSPE or MPEG-5000-DPPE substitution were evaluated at a dose of 5 μg of CRX-601 / animal / vaccination. The dose of CRX-601 in the IM control is 1 μg / animal / vaccination.
Figure 5 shows the post-tertiary HI titers (A) and IGA titers of the post-tertiary tracheal / vaginal lavage fluids (B) from a phospholipid-PEG modified liposome study.
Figure 6. Post-secondary serum IgG titers (top), post-tertiary serum IgG titers (in the middle), and IGA titers of posterior tracheal / vaginal lavage fluids (bottom) from a modified liposome study by poloxamer 407. Liposomes with 5, 10 and 15 mol% of poloxamer 407 substitutions were evaluated at a dose of 1 or 5 μg of CRX-601 / animal / vaccination dose.
Figure 7. Post-secondary serum IgG titers (top), post-tertiary serum IgG titers (middle), and IGA titers of posterior tracheal / vaginal lavage fluids (bottom) from a modified liposome study by poloxamers 407, 188, and 184. Liposomes with 15 and 25 mol% of poloxamer substitutions 407, 188 or 184 were evaluated at a dose of 5 μg of CRX-601 / animal / vaccination dose.
Figure 8 shows the post-tertiary HI titers (A) and IGA titers of tracheal / vaginal washings (B) from a study of liposomes modified with poloxamers 407, 188, and 184. On the panels, the labels in the figure represent poloxamer 407 (F127), poloxamer 188 (F68) and poloxamer 184 (L64).
Figure 9. Post-secondary serum IgG titers (top), post-tertiary serum IgG titers (middle), and IGA titers of posterior tracheal / vaginal lavage fluids (bottom) from a modified liposome study + methylglycol chitosan. Liposomes in combination with methylglycol chitosan were evaluated at a dose of 5 μg of CRX-601 / animal / vaccination. The dose of CRX-601 in the IM control is 1.5 Dg / animal / vaccination.
Figure 10. Titers of post-secondary serum IgG (A), post-tertiary serum IgG titers (B), and post-tertiary HI titres (C) and IGA titers of tracheal / vaginal lavage fluids (D) from one study liposomes modified with phospholipid-PEG + methylglycol chitosan or chitosan oligosaccharide lactate. Liposomes with 5% mole MPEG-2000-DSPE or MPEG-5000-DPPE substitution in combination with chitosan methylglycol or lactose oligosaccharide chitosan were evaluated at a dose of 5 Dg of CRX-601 / animal / vaccination. The dose of CRX-601 in the IM control is 1.5 Dg / animal / vaccination.
Figure 11. Post-secondary serum IgG titers (top), post-tertiary serum IgG titers (middle), and IGA titers of posterior tracheal / vaginal lavage fluids (bottom) from a modified liposome study by poloxamer 407 (on the legends of the figure, indicated by F127) + methylglycol chitosan. Liposomes with 5, 15 or 15 mol% poloxamer 407 addition in combination with methylglycol chitosan were evaluated at a dose of 5 μg of CRX-601 / animal / vaccination. The dose of CRX-601 in the IM control is 1.5 Dg / animal / vaccination.
Figure 12. Titers of post-secondary serum IgG (A), post-tertiary serum IgG titers (B), post-tertiary HI titers (C), and IGA titers of tracheal / vaginal lavage fluids from a modified liposome study + methylglycol chitosan. Liposomes in combination with methylglycol chitosan were evaluated at a dose of 5 Dg of CRX-601 / animal / vaccination. The dose of CRX-601 in the IM control is 1.5 Dg / animal / vaccination.
Detailed Description of the Invention Liposomes
The term "liposome (s)" generally refers to uni- or multilamellar lipid structures (particularly 2, 3, 4, 5, 6, 7, 8, 9, or 10 lamellar depending on the number of lipid membranes formed) containing an aqueous interior. Liposomes and liposomal formulations are well known in the state of the art. Lipids that are capable of forming liposomes include all substances with greasy or greasy properties. The lipids which may constitute the lipids in the liposomes may be chosen from the group comprising glycerides, glycerophospholipids, glycerophosphinolipids, glycerophosphonolipids, sulpholipids, sphingolipids, phospholipids, isoprenolides, steroids, stearines and sterols. archeolipids, synthetic cationic lipids and carbohydrate-containing lipids.
In a particular embodiment of the invention, the liposomes comprise a phospholipid. Suitable phospholipids include (but are not limited to): phosphocholine (PC) which is an intermediate in the synthesis of phosphatidylcholine; natural phospholipid derivatives: egg phosphocholine, soy phosphocholine, hydrogenated soy phosphocholine, sphingomyelin as natural phospholipids; and synthetic phospholipid derivatives: phosphocholine (didecanoyl-La-phosphatidylcholine [DDPC], dilauroylphosphatidylcholine [DLPC], dimyristoyl-phosphatidylcholine [DMPC], dipalmitoyl-phosphatidylcholine [DPPC], distearoyl-phosphatidylcholine [DSPC], dioleoylphosphatidylcholine [ DOPC], 1-palmitoyl, 2-oleoyl-phosphatidylcholine [POPC], diallydoylphosphatidylcholine [DEPC]), phosphoglycerol (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol [DMPG], 1/2-dipalmitoyl- sn-glycero-3-phosphoglycerol [DPPG], 1,2-distearoyl-sn-glycero-3-phosphoglycerol [DSPG], 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol [POPG]), phosphatidic acid (1,2-dimyristoyl-sn-glycero-3-phosphatidic acid [DMPA], dipalmitoylphosphatidic acid [DPPA], distearoylphosphatidic acid [DSPA]), phosphoethanolamine (1,2-dimyristoyl-sn-glycerol) 3-phosphoethanolamine [DMPE], 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine [DPPE], 1,2-distearoyl-sn-glycero-3-phosphoeth anolamine [DSPE], 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [DOPE]), phosphoserine, polyethylene glycol [PEG] -phospholipid (mPEG-phospholipid, polyglycerin-phospholipid, functionalized phospholipid, terminal activated phospholipid) 1,2-Dioleoyl-3- (trimethylammonium) propane (DOTAP) and sphingomyelin (SPNG). In one embodiment, liposomes include 1-palmitoyl-2-oleoyl-glycero-3-phosphoethanolamine. In one embodiment, highly purified phosphatidylcholine is used and may be selected from the group consisting of phosphatidylcholine (egg), hydrogenated phosphatidylcholine (egg), phosphatidylcholine (soy), and hydrogenated phosphatidylcholine ( soya). In another embodiment, the liposomes comprise phosphatidylethanolamine [POPE] or a derivative thereof.
The size of the liposome can vary from 30 nm to 5 μm according to the phospholipid composition and the process used for their preparation. In particular embodiments of the invention, the size of the liposome will be in the range of 30 nm to 500 nm and in other embodiments from 50 nm to 200 nm, suitably less than 200 nm. Dynamic scattering of laser light is a method used to measure the size of liposomes well known to those skilled in the art.
In a suitable liposomal formulation, the lipid comprises dioleoylphosphatidylcholine [DOPC] (2-dioleoyl-sn-glycero-3-phosphocholine) and a sterol, especially cholesterol, and optionally in the absence of sterol.
Liposomal composition
A "liposomal composition" is a prepared composition comprising a liposome and the content within the liposome, particularly including, but not limited to: a) the lipids that form the bilayer (s) of the liposome, b) compounds other than lipids within liposome bilayer (s), (c) within and associated compounds within or within aqueous liposome, and (d) bound compounds or associated with the outer layer of the liposome.
Thus, in addition to the liposome lipids, a liposomal composition of the present invention may conveniently include, but is not limited to, pharmaceutically active components, vaccine antigens and adjuvants, excipients, carriers, agents and the like. mucoadherents, muco-penetrating agents and buffering agents. In a preferred embodiment, such compounds are complementary and / or are not significantly detrimental to the stability or efficiency of incorporation of AGP from the liposomal composition. "Liposomal Formulation" means a liposomal composition, such as those described herein, formulated appropriately with other compounds for storage and / or administration to a subject.
Thus, a "liposomal formulation" of the present invention comprises a liposomal composition as defined herein, and may further include, but is not limited to, liposomal compositions outside the scope of the present invention, as well as pharmaceutically active compounds, vaccine antigens and adjuvants, excipients, carriers and buffering agents. In a preferred embodiment, such compounds are complementary and / or are not significantly detrimental to the stability or efficiency of incorporation of AGP from the liposomal composition of the present invention.
Aminoalkyl glucosaminide phosphate compounds. AGPs are modulators of the Toll-4 receptor (TLR4). The Toll-4 receptor recognizes bacterial LPS (lipopolysaccharide) and when activated, initiates an innate immune response. PFAs are monosaccharide mimetics of bacterial LPS lipid A and have been developed with ether and ester linkages on the "acyl chains" of the compound. Methods of making such compounds are known and disclosed, for example, in WO 2006/016997, US Pat. Nos. 7,288,640 and 6,113,918, and WO 01/90129, which are incorporated herein by reference. in their entirety. Other AGPs and related processes are disclosed in US Patent No. 7,129,219, US Patent No. 6,525,028 and US Patent No. 6,911,434. AGPs with ether linkages on acyl chains employed in the composition of the invention are known and disclosed in WO 2006/016997 which is incorporated herein by reference in its entirety. Of particular interest are the aminoalkyl glucosaminide phosphate compounds shown and described according to formula (III) in paragraphs [0019] to [0021] in WO 2006/016997.
The aminoalkyl glucosaminide phosphate compounds employed in the present invention have the structure represented by the following Formula 1:
(Formula 1) wherein m is 0 to 6 n is 0 to 4; X is 0 or S, preferably 0; Y represents 0 or NH; Z represents 0 or H; each R 1, R 2, R 3 is independently selected from the group consisting of C 1 -C 20 acyl and C 1 -C 20 alkyl; R4 is H or Me; R5 is independently selected from the group consisting of -H, -OH, -C1-C4 alkoxy, -PO3R8R9, -OPO3R8R9, -SO3R8, -OSO3R8, -NR8R9, -SR8, -CN, -NO2, -CHO, -CO2R8, and -CONR8R9, wherein R8 and R9 are each independently selected from H and C1-C4alkyl; and each R6 and R7 is independently H or PO3H2.
In formula 1, the configuration of the 3 stereogenic centers to which normal fatty acyl residues (i.e., acyloxy or secondary alkoxy residues, eg, RiO, R 2 O, and R 3 O) are attached, is R or S, preferably R (as it is designated by the Cahn-Ingold-Prelog priority rules). The configuration of the stereogenic centers of the aglycone to which R4 and R5 are attached may be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to be within the range of the present invention.
The number of carbon atoms between the X heteroatom and the nitrogen atom of the aglycone is determined by the variable "n", which may be an integer from 0 to 4, preferably an integer of 0 to 2.
The length of the normal fatty acid chain R 1, R 2 and R 3 may be from about 6 to about 16 carbons, preferably from about 9 to about 14 carbons. The lengths of the chains may be the same or different. Some preferred embodiments include chain lengths where R 1, R 2 and R 3 are 6 or 10 or 12 or 14.
Formula 1 includes L / D-seryl-, -threonyl-, -cysteinyl-ether and lipid ester AGP, both agonists and antagonists and their homologs (n = 1 to 4), as well as various bio-isosteres. of carboxylic acid (i.e., R5 is an acid group capable of forming a salt, the phosphate may be in the 4 or 6 position of the glucosamine unit, but preferably it is in the 4 position) .
In a preferred embodiment of the invention employing an AGP compound of Formula 1, n is 0, R5 is CO2H, R6 is PO3H2, and R7 is H. This preferred AGP compound is represented by the following structure of formula:
(Formula la) wherein X is 0 or S; Y represents 0 or NH; Z represents 0 or H; each R 1, R 2, R 3 is independently selected from the group consisting of C 1 -C 20 acyl and C 1 -C 20 alkyl and R 4 is H or methyl.
In formula la, the configuration of the 3 stereogenic centers to which normal fatty acyl residues (i.e., secondary acyloxy or alkoxy residues, eg, RiO, R 2 O, and R 3 O) are attached, is R or S, preferably R (as it is designated by the Cahn-Ingold-Prelog priority rules). The configuration of the stereogenic centers of the aglycone to which R4 and CO2H are attached may be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to be within the range of the present invention.
Formula la encompasses AGP, L / D-seryl-, -threonyl-, -cysteinyl ether or lipid ester, both agonists and antagonists.
In both Formula I and Formula Ia, Z is set to 0 by a double bond or two hydrogen atoms which are each attached by a single bond. That is, the compound is bound by an ester when Z = Y = 0; bound by an amide when Z = 0 and Y = NH; and bound by an ether when Z = H / H and Y = 0.
Especially preferred compounds of formula 1 are called CRX-601 and CRX-527. Their structures are represented as follows:
(CRX-527) (CRX-601)
In addition, another preferred embodiment employs CRX-547 having the structure shown.
(CRX 547)
Still other embodiments include AGPs such as CRX 602 or CRX 526 providing increased stability to AGPs with shorter secondary alkyl or acyl chains. (CRX 602)
(CRX-526) Other AGPs suitable for use in the present invention include CRX 524 and CRX 529.
tampons
In one embodiment of the present invention, a liposomal composition is buffered using a zwitterionic buffer. Suitably, the zwitterionic buffer is an aminoalkanesulfonic acid or a suitable salt. Examples of aminoalkanesulfonic buffers include, but are not limited to, HEPES, HEPPS / EPPS, MOPS, MOBS and PIPES. Preferably, the buffer is a pharmaceutically acceptable buffer, suitable for use in humans, as for use in a commercial injection product. Most preferred is the HEPES buffer. The liposomal composition may suitably comprise an AGP.
In suitable embodiments of the present invention, the liposomes are buffered using a buffer selected from the group consisting of: i) HEPES having a pH of about 7, ii) citrate (eg, sodium citrate) having a pH about 5, and iii) acetate (eg, ammonium acetate) having a pH of about 5.
In a preferred embodiment of the present invention, the AGP CRX-601, CRX-527 and CRX-547 are included in a buffered liposomal composition using HEPES having a pH of about 7. The buffers can be used with a appropriate amount of saline or other excipient to obtain the desired isotonicity. In a preferred embodiment, 0.9% saline is used.
HEPES: CAS Registry Number: 7365-45-9 CsHia ^ C ^ S
1-piperazine-ethanesulfonic acid, 4- (2-hydroxyethyl) -HEPES is a zwitterionic buffer designed to buffer the physiological pH range from about 6 to about 8 (e.g., 6.15 to 8.35) and more specifically to a more useful range of about 6.8 to about 8.2 and, as in the present invention, between about 7 and about 8 or between 7 and 8, and preferably between about 7 and less than 8. HEPES is generally a white crystalline powder and has the molecular formula: C8H18N2O4S of following structure:
HEPES is well known and commercially available. (See, for example, Good et al., Biochemistry 1966).
PEG
Polyethylene glycol (PEG), also known as polyethylene oxide (PEO) or polyoxyethylene (POE) is a hydrophilic polymer (polyether) with many applications ranging from industrial manufacturing to medicine. This polymer is inexpensive, has good biocompatibility and has been approved for internal applications in humans by regulatory agencies. PEG chains of molecular weights in the range of 1 to 15 kDa have been widely used as steric protectors in various colloidal systems. Due to its high aqueous solubility, high mobility and high exclusion volume, hydrated PEG forms a dense brush of polymeric chains that unfolds and covers the surface of the particle. This minimizes the interfacial free energy of the particle surface, and prevents its interaction with other particles, providing colloidal stability to the system. The ability of a PEG coating to inhibit interaction with proteins and other biomolecules in blood and cells has been widely used to extend the circulation time of drug carriers in the blood, to reduce the opsonization of particles and make them less recognizable by the reticuloendothelial system (SRE) in the liver and spleen. In particular, for administration by the mucosal route, it has been shown that PEG, according to the length of its chains, has both muco-adherent (long chain) and muco-inert (short chain) properties.
Modification of the surface of colloidal drug carriers, particularly liposomes, with PEG can be achieved in several ways: 1) using PEG-amphiphilic lipid conjugates, PEG copolymers such as poloxamers, or other of these hydrophobic PEG conjugates, either by physically absorbing them on the surface of the vesicles, or by incorporating them during the preparation of the liposomes, or 2) by covalently grafting PEG chains with terminal functional groups to reactive groups on the surface of the preformed liposomes.
Phospholipid-PEG conjugates
PEG conjugates with phospholipids have been widely used to incorporate PEG on liposomes. The phospholipid moiety acts as an anchor by incorporation into the hydrophobic interior of the bilayer and grafts the PEG chain to the aqueous surface of the liposome. These conjugates exhibit excellent biocompatibility. Several different conjugates depending on the length of the PEG chain and the type of phospholipid used are available. Doxil, a clinically approved liposomal formulation of doxorubicin, and many other liposomal formulations in late-stage clinical trials (such as Lipoplatin, SPI-77, Lipoxal, etc.) are based on this concept of PEG-phospholipid incorporation.
Many phospholipid-PEG conjugates are known from the state of the art and many phospholipid-PEG conjugates are commercially available, such as: MPEG-2000-DSPE: sodium salt of N- (carbonyl-methoxypolyethylene) glycol-2000) -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, MPEG-5000-DPPE: sodium salt of N- (carbonyl-methoxy-polyethylene glycol-5000) -1,2-dipalmitoyl-sn -glycerol-3-phosphoethanolamine. Other related phospholipid-PEG conjugates include, but are not limited to: DPPE-mPEG (1000): 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -1000] (ammonium salt); DSPE-mPEG (1000): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -1000] (ammonium salt); DOPE-mPEG (1000): 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -1000] (ammonium salt); DPPE-mPEG (2000): 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt); DOPE-mPEG (2000): 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt); DSPE-mPEG (5000): 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -5000] (ammonium salt); and DOPE-mPEG (5000): 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -5000] (ammonium salt).
poloxamers
The poloxamers are amphiphilic nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly (propylene oxide)) flanked by two hydrophilic PEG chains. Poloxamers are also known by the trade names Synperonics, Pluronics and Kolliphor. The lengths of the polymer sequences can be customized, so there are many different poloxamers, differing in their properties and they can exist in the form of liquids, pastes or solids. Because of their amphiphilic structure, these polymers have surfactant properties that can be used to emulsify water-insoluble substances, form supramolecular associations (micelles or vesicles) in aqueous solutions that can trap various compounds, or they can be incorporated into other colloidal particles such as liposomes. A characteristic feature of these synthetic polymers is relatively low toxicity and biological compatibility. For this reason, these polymers are commonly used in industrial applications, cosmetics and pharmaceuticals. They have also been used in therapy for burns and wounds, in cryoprotectants, drug emulsifiers, vaccine adjuvants, in medical imaging, in the management of vascular diseases and have been shown to sensitize patients. drug-resistant cancers to chemotherapy. The central hydrophobic sequence is essential for the incorporation of poloxamers into bilayers of liposomes and other colloidal drug delivery particles.
Pharmaceutically acceptable poloxamers include, but are not limited to: Poloxamer 407 (Pluronic® F127); poloxamer 184 (Pluronic® L64) and poloxamer 188 (Pluronic® L68)
Pluronic is a registered trademark of BASF.
Many other poloxamers are commercially available.
chitosan
Chitosan is a natural cationic polysaccharide derived from chitin by partial deacetylation of its acetamido groups in strongly alkaline solutions. During the last two decades, chitosan has found widespread use in biomedical and drug delivery applications due to its low toxicity, good biocompatibility and excellent mucoadherent properties (van der Lubben, IM, Verhoef, JC , Borchard, G. & Junginger, HE Chitosan for Mucosal Vaccination, Adv Drug Deliv Rev. 52, 139-144, 2001). The mucosal adhesion of chitosan is thought to involve complex mechanisms, with the electrostatic interaction between cationic chitosan and the anionic mucin coating on the mucosal surface being the main factor, although it is thought that the hydrogen and the hydrophobic effects also play a significant role.
The non-derivatized chitosan has a limited solubility (□! Mg / ml) and is soluble only under acidic conditions (pH <6.5). However, chitosan derivatives such as chitosan glycol, chitosan methylglycol, and chitosan oligosaccharide lactate show significantly improved solubility ([10 mg / ml) at physiological pH.
Commercially available chitosan derivatives include, but are not limited to: chitosan oligosaccharide lactate, chitosan glycol, or methylglycol chitosan (MGC). These derivatives have variable physical properties over chitosan, which may make them more suitable for use with antigens, adjuvants, liposomes and the like.
Preparation of liposomes
Conventional methods for making liposomes include, but are not limited to, methods reported in Liposomes: A Practical Approach, V.P. Torchilin, Volkmar Weissig Oxford University Press, 2003 and are well known in the state of the art.
In an appropriate method of making a liposomal composition of the present invention, an AGP (e.g., CRX-601 (0.2% w / w)) and DOPC (specifically, 1,2-dioleoyl-sn- glycero-3-phosphocholine, 3 to 4% w / v) and optionally a sterol (for example, cholesterol (1% w / v)) are dissolved in an organic phase of chloroform or tetrahydrofuran in a round bottom flask. The organic solvent is removed by evaporation on a rotary evaporator and further with a high pressure under vacuum for 12 hours. To the mixed phospholipidic film thus obtained are added 10 ml of an aminoalkanesulfonic buffer such as 10 mM HEPES or a 10 mM HEPES-physiological saline buffer pH 7.2. The mixture is sonicated on a water bath (20 to 30 ° C) and vortexed intermittently until the film along the walls of the flask is dispersed in the solution (30 min to 1.5 h) . The solution is then successively extruded through polycarbonate filters using a mini-lipid extruder (Lipex ™ Extruder (Northern Lipids Inc., Canada)) to form unilamellar liposomes. The liposomal composition is then aseptically filtered using a 0.22 μm filter in a sterile depyrogenic container. The average particle size of the resulting formulation measured by dynamic light scattering is 80 to 120 nm with a net negative zeta potential. The formulation represents final target concentrations of 2 mg / ml CRX-601, 10 mg / ml cholesterol, and 40 mg / ml total phospholipid.
Suitably, a PEG-phospholipid (eg, MPEG-2000-DSPE (0.1 to 3% w / v) or MPEG-5000-DPPE (0.3 to 6% w / v)) or a poloxamer ( for example, poloxamer 407 (1-16% w / v) is dissolved with AGP and DOPC lipids at the beginning of the process. Suitably, the liposomal composition in the formulations may be mixed with an aseptic solution of chitosan (eg, MGC 200 mg) dissolved in HEPES. The aminoalkyl glucosaminide 4-phosphate (AGP) CRX-601 used in this work can be synthesized as previously described (Bazin, 2008 32447 / id), and purified by chromatography (up to> 95% purity). CRX-601, either in the starting material or in the final product, can be quantified by a conventional reverse phase HPLC analytical method.
In one embodiment during liposome preparation, CRX-601 formulated in HEPES buffer (pH = 7.0) achieves the desired reduction in particle size five times faster, compared to the liposome hydration buffer ( "LHB", phosphate-based, pH = 6.1). Rehydration of CRX-601 lipid films in HEPES buffer requires four times less total pressure and time to formulate liposomes compared to LHB phosphate buffer. This is a significant improvement since it releases both energy and time and exerts far less stress on AGP during liposome treatment.
Suitable ranges (w / v) of the components of a liposomal composition include an embodiment comprising a lipid in a range of about 3 to 4% w / v, a 1% w / v sterol, an active component, as an AGP, in the range of 0.1 to 1% w / v and a 10mM aminoalkanesulfonic buffer. In one embodiment, the sterol is suitably present in a range of 0.5 to 4% w / v. In addition, in one embodiment, the lipid / sterol / active component ratio is about 3 to 4/1 / 0.1 to 1.
Examples
Preparation of DOPC liposomes modified with CRX-601
Example 1 - Liposomes with 1% mole of MPEG-2000-DSPE substitution
The mol% substitution in this example refers to the amount of MPEG-2000-DSPE relative to the total phospholipid content. CRX-601 (20 mg), 1,2-dioleoyl-sn-glycero-3-phosphocholine, abbreviated as DOPC (396 mg), cholesterol (100 mg) and PEG-phospholipid [sodium salt of N- ( carbonyl methoxypolyethylene glycol-2000) -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, abbreviated as MPEG-2000-DSPE (15 mg)] were dissolved in an organic phase of tetrahydrofuran in a round bottom flask. The organic solvent was removed by evaporation on a rotary evaporator and further under high pressure vacuum for 12 h. To the mixed phospholipidic film thus obtained were added 10 ml of 10 mM HEPES buffer or 10 mM physiological saline pH 7.2. The mixture was sonicated on a water bath (20 to 30 ° C) and vortexed intermittently until the film along the walls of the flask was dispersed in the solution (30 min to 1.5 hrs). ). The solution was then extruded successively through polycarbonate filters having a pore size of 600 nm (1 pass), 400 nm (1 pass), and 200 nm (2 to 4 pass) using a mini lipid extruder (Lipex ™ Extruder (Northern Lipids Inc., Canada)) to form unilamellar liposomes. The liposomal composition was then aseptically filtered using a 0.22 μm filter in a sterile depyrogenic container. The average particle size of the resulting formulation measured by dynamic light scattering was 80 to 120 nm with a net negative zeta potential. The formulation represents final target concentrations of 2 mg / ml CRX-601, 10 mg / ml cholesterol, and 40 mg / ml total phospholipids. The aminoalkyl glucosaminide 4-phosphate (AGP) CRX-601 used in these works was synthesized as previously described (Bazin, 2008 32447 / id), and purified by chromatography (up to> 95% purity). CRX-601, either in the starting material or in the final product was quantified by a conventional reverse phase HPLC analytical method.
Example 2 Liposomes with an Upper Molecular% MPEG-2000-DSPE Substitution
The liposomal formulations were prepared as in Example 1 but with varying amounts of DOPC and MPEG-2000-DSPE to obtain the desired% mole of
Substitution. Formulations with targeted substitutions of 5, 10, 15, and 25 mol% were prepared. Representative mean particle sizes and zeta potential measured by dynamic light scattering are shown in Table 1. Formulations with more than 25 mol% of substitutions are difficult to prepare, limited by dissolution of the lipid film in the buffer during sonication, when the solubility limit of the components is approached. At high substitutions, PEG phospholipids are expected to saturate the bilayer of liposomes with excess solution in the form of micelles or unimeros.
Table 1
Representative Parameters of Formulations for MPEG-2000-DSPE Modified DOPC-Cholesterol Liposomes
Example 3 Liposomes with 1% Molar of MPEG-5000-DPPE Substitution
The liposomal formulation was prepared as in Example 1 but with the PEG-phospholipid [sodium salt of N- (carbonyl-methoxypolyethylene glycol-5000) -1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, abbreviated MPEG -5000-DPPE (33 mg)] used in place of MPEG-2000-DSPE. The average particle size of the resulting formulation measured by dynamic light scattering was 80 to 120 nm.
Example 4 - Liposomes with a higher mole% substitution MPEG-5000-DPPE
Liposomal formulations were prepared as in Example 3 but with varying amounts of DOPC and MPEG-5000-DPPE to obtain the desired% mole of substitution. Formulations with targeted substitutions of 5, 10, 15, and 25 mol% were prepared. The representative mean particle sizes and zeta potential measured by dynamic light scattering are shown in Table 2. Formulations with more than 25 mol% of substitutions are difficult to prepare, limited by the dissolution of the lipid film in the buffer during sonication, when the solubility limit of the components is approached. At high substitutions, PEG phospholipids are expected to saturate the bilayer of liposomes with excess solution in the form of micelles or unimeros.
Table 2
Representative Parameters of Formulations for MPEG-5000-DPPE Modified DOPC-Cholesterol Liposomes
Example 5 - Preparation of liposomes with addition of 1 mol% of poloxamer 407
The% by mole of addition in this example refers to the amount of poloxamer relative to the total phospholipid content. CRX-601 (20 mg), DOPC (400 mg), and poloxamer 407 (64 mg) were dissolved in an organic phase of tetrahydrofuran in a round bottom flask and processed as discussed in the example. 1. No cholesterol was included in these preparations as it has been reported to reduce the incorporation of poloxamers into the phospholipid bilayer. The average particle size of the resulting formulation measured by dynamic light scattering was 120 to 180 nm. The formulation represents the final target concentrations of 2 mg / ml of CRX-601, 40 mg / ml of DOPC, and 1 mol% (based on DOPC) of poloxamer 407.
Example 6 - Preparation of liposomes with a higher mole% of poloxamer 407 addition
Liposomal formulations were prepared as in Example 5 but increasing the amounts of Poloxamer 407. Formulations with targeted substitutions of 5, 10, 15 and 25 mol% were prepared. Representative mean particle sizes and zeta potential as measured by dynamic light scattering are shown in Table 3. Formulations with more than 25 mol% of additions are difficult to prepare, limited by the dissolution of the lipid film. poloxamer in the buffer during sonication, when the solubility limit of the components is approached. At high substitutions, poloxamer 407 is expected to saturate the liposome bilayer with excess in solution as micelles or unimeras.
Table 3
Representative Parameters of Formulations for Poloxamer 407 Modified DOPC Liposomes
Example 7 - Preparation of liposomes with addition of poloxamer 188
The liposomal formulations were prepared as in Example 6 but with poloxamer 188 in place of poloxamer 407. Formulations with targeted substitutions of 15 and 25 mol% were prepared. Representative mean particle sizes and zeta potential as measured by dynamic scattering of light are shown in Table 4.
Example 8 - Preparation of liposomes with addition of poloxamer 184
Liposomal formulations were prepared as in Example 6 but with poloxamer 184 in place of poloxamer 407. Formulations with targeted substitutions of 15 and 25 mol% were prepared. Representative mean particle sizes and zeta potential as measured by dynamic scattering of light are shown in Table 4.
Table 4
Representative Parameters of the Formulations for DOPC Liposomes Modified by Poloxamer 188 or Poloxamer 184
Example 9 - Preparation of liposomal formulation with chitosan
Chitosan methylglycol (chitosan glycol trimethylammonium iodide, 200 mg) was dissolved in 10 ml of 10 mM HEPES-physiological saline pH 7.2 buffer to produce a concentration of 20 mg / ml. The solution was aseptically filtered using a 0.22 μm filter in a sterile depyrogenous container. The formulations of Examples 1 to 4 were aseptically mixed with varying volumes of methylglycol chitosan solution to produce concentrations in the range of 1 to 10 mg / ml. While traditional formulations of DOPC-cholesterol liposomes aggregate when mixed with chitosan or its derivatives including methylglycol chitosan (Table 5), the modified liposomes (Examples 1-4) reported here remain stable in suspension (Table 1). 6). The representative mean particle sizes and zeta potential measured by dynamic scattering of light shown in Table 6 indicate some increase in particle size and zeta potential reversal (a net positive potential from a negative potential). net) exceeding approximately 1 mg / ml of methylglycol chitosan, consistent with the coating of the surface with methylglycol chitosan. At concentrations above a certain threshold, it is expected that methylglycol chitosan will saturate the liposome bilayer with an excess in solution.
Table 5
Representative parameters of the formulations indicating aggregation of the unmodified DOPC liposomal formulation with varying concentrations of methylglycol chitosan (MGC). The concentration of CRX-601 is 1 mg / ml in all cases.
Table 6
Representative parameters of formulations for DOPC-cholesterol liposomes modified with MPEG-2000-DSPE and MPEG-5000-DPPE coated with methylglycol chitosan (MGC) at 1, 5, 15 and 30% liposomal modification prepared in 10 mM HEPES- physiological serum. The concentration of CRX-601 is 1 mg / ml in all cases.
Chitosan oligosaccharide lactate. Studies were performed in the same manner as with methylglycol chitosan (MGC) above except that chitosan oligosaccharide lactate was used in place of methylglycol chitosan. All compositions tested with liposomes of Example 1 showed aggregation. All other formulations remained stable in suspension.
Chitosan Glycol. Studies were conducted in the same manner as with chitosan methylglycol (MGC) above except that chitosan glycol was used in place of methylglycol chitosan. All compositions tested with liposomes of Examples 1 and 2 showed aggregation. The liposomes of Examples 3 and 4 remained stable in suspension.
Liposomes coated with chitosan
Chitosan-coated liposome formulations were prepared by mixing unmodified, phospholipid-PEG modified or Pluronic-modified liposomes with the chitosan derivative and evaluated for size and potential changes. When combined with MGC, the unmodified liposomes aggregated, leading to precipitation, from 0.4 to 2 mg / ml MGC, as shown in FIG. size located in the range of about pm. At MGC concentrations exceeding 2 mg / ml, the formulations appeared colloidally stable initially but tended to aggregate over 1 to 4 days. Liposomes modified with PE-PEG2K and PE-PEG5K with 5 mol% modification were colloidally stable in the presence of MGC without any major change in size at any of the concentrations tested (Figure 1Ά). Potential reversal □ from negative to positive potential occurred at approximately 0.5 mg / ml MGC with unmodified liposomes and 1 mg / ml with 5% PE-PEG2K or PE-PEG5K modification. Modification with as little as 1 mol% of PE-PEG2K or PE-PEG5K has been shown to provide sufficient protection against MGC-mediated aggregation at most concentrations (Figure 2A). At 1% modification, liposomes with PE-PEG5K were more resistant to MGC-induced aggregation than PE-PEG2K-modified liposomes. From 0.4 to 0.6 mg / ml MGC, no major change in particle size or IPD with 1% PE-PEG5K modification was observed, but an increase in size to the range of the order of μπι with 1% modification PE-PEG2K. The change up to 25% did not result in any destabilization / aggregation in the presence of MGC (Figure 2B).
Among the Pluronic-modified liposomes, F127-modified liposomes were the most stable, showing no visible aggregation or increase in polydispersity over the full range of assessed MGC concentrations (Figure 1B). The increase in particle size was about 10 to 30 nm, and the potential reversal from a net to positive negative potential occurred at concentrations of 0.4 mg / ml MGC. F127 modified liposomes at 15% and 25% modification were similarly stable in the presence of MGC (Figure 3A), but at 1% modification they aggregated (data not shown). Liposomes with 5 mol% L64 modification when combined with MGC showed an increase in size and polydispersity of 0.2 to 0.8 mg / ml MGC (FIG. 1B), corresponding complete neutralization of the surface charge of the liposomes. At 1 mg / ml MGC, similar to unmodified liposomes, the L64-modified liposomes initially appeared stable but tended to aggregate and precipitate over time (Fig. 1B). F68-modified liposomes were the least stable, and caused instant precipitation with MGC at all concentrations tested. Similar trends were observed with liposomes with a greater Pluronic modification of 15 and 25% (Figure 3). Therefore, the stability order of Pluronic-modified liposomes in the presence of MGC was F127> L64> F68.
The summary of stability assessment for these liposomal formulations in the presence of chitosan derivatives, MGC, GC and CO, is shown in Table 7. Overall, of all the chitosan derivatives tested, the lowest was observed. aggregation with the MGC. Phospholipid-PEG modified liposomes were more stable against chitosan-induced aggregation than Pluronic-modified liposomes. Only formulations that showed a significant reduction in cha-rge (phospholipid-PEG or Pluronic F127-modified liposomes) were resistant against chitosan-induced aggregation. PE-PEG5K-modified liposomes were more stable than PE-PEG2K-modified liposomes, as evidenced by the lack of any change in size / IPD in the presence of 1% modified MGC and increased stability in the presence of GC and CO.
Table 7 Summary of stability of unmodified, phospholipid-PEG and Pluronic modified liposomes with varying degree of modification against chitosanea derivative-induced aggregation
The lowest modification tested was 1 mol%.
Partial aggregation 0.2 to 0.6 mg / ml bMGC: methylglycol chitosan, CGC: chitosan glycol, dCO: chitosan oligosaccharide lactate
Example 10 Pyrogen Test in Rabbits
The pyrogen test is used here as a surrogate for the incorporation of CRX-601 in the modified liposomes of Examples 1 to 6 and as a measure of their stability in a biological medium. The test was performed at Pacific Biolabs (Hercules, CA) according to their SOP of 16E-02, which follows the procedures presented in USP <151>. All formulations of Examples 1 to 6 lacked pyrogenicity up to a concentration of at least 250 ng CRX-601 / kg body weight of the animal, except for the formulations of Example 7 (liposomes modified by poloxamer 188) and of Example 8 (liposomes modified with poloxamer 184). This lack of pyrogenicity up to 250 ng / kg corresponding to a 100-fold improvement over CRX-601-free (maximum non-pyrogenic dose of 2.5 ng / kg), and indicates a> 99% incorporation of CRX-601 in the bilayer liposomes. The individual temperature increases from the three rabbits per test are shown in Table 8.
Table 8
Representative pyrogenic test measures in rabbits for the formulations described in Examples 2, 4, 6, and 9. The values in parentheses represent the maximum temperature change for three animals during the analysis period. A rise in temperature of 0.5 ° C or higher is considered a pyrogenic response. The symbols P and F indicate a "Success" or "Fail" response, respectively.
Abbreviations: methylglycol chitosan (MGC); chitosan oligosaccharide lactate (CL)
Example 11 - Sublingual vaccination of mice and determination of specific antibody responses
Female BALB / c mice (6 to 8 weeks old) obtained from Charles River Laboratories (Wilmington, Mass.) Were used for these studies. In anesthetized mice by intraperitoneal (i.p.) administration of ketamine (100 mg / kg) and xylazine (10 mg / kg), the vaccine was administered sublingually (5-6 μΐ). All mice were vaccinated on days 0, 21 and 42 with 5 μg CRX-601 in the liposomal formulation mixed with 1 or 1.5 μg HA / mouse using influenza A / Victoria / 210/2009 antigen H3N2. The serum was taken at day 36 (14dp2) under anesthesia, at day 56 (14dp3) the mice were sacrificed and a final sample of vaginal lavage, tracheal lavage and serum was collected. All animals were used in accordance with guidelines established by the U.S. Department of Health and Human Services Office of Animal Welfare Laboratory and the Institutional Animal Care and Use Committee at GSK Biologicals, Hamilton, Montana.
Specific antibody responses were measured by two independent immunological tests, the enzyme linked immunosorbent assay (ELISA) and the influenza hemagglutinin (IH) inhibition test.
ELISA was performed using 96-well plates sensitized with fragmented influenza virus (Nunc Maxisorp) and detecting bound immunoglobulins from serum or tracheal wash or vaginal lavage samples using IgG, IgG1. , IgG2a or goat anti-mouse IgA bound to peroxidase. This was followed by the addition of a chromogen specific to the enzyme, which resulted in a color intensity directly proportional to the amount of specific anti-influenza IgG / IGA contained in the serum. The optical density was read at 450 nm.
The HI test was performed by evaluating the inhibition of chicken or cock red blood cells during exposure to the influenza virus in the presence of mouse serum. The reciprocal of the last influenza virus dilution that resulted in complete or partial agglutination of red blood cells was used to calculate the HI titre and expressed in units of HA / 50 μΐ of serum.
Example 12 Sublingual Vaccination of Mice with Liposomes Modified with MPEG-2000-DSPE or MPEG-5000-DPPE (NIH No. 162)
The mice were vaccinated using the procedure set forth in Example 11 with liposomes modified with 1, 5, and 25 mole% MPEG-2000-DSPE or MPEG-5000-DPPE of Examples 1-4. Serum IgG 14 days after secondary vaccinations and tertiary vaccinations are shown in Figure 4 (and also with IH titles in Figure 5). Of the sublingual treatment groups, titers were highest in mice receiving CRX-601 in the 25% MPEG-5000-DPPE liposome treatment group, significantly greater than the CRX liposome treatment groups -601 aqueous or unmodified CRX-601 liposomes.
Example 13 Sublingual Vaccination of Mice with Liposomes Modified with Poloxamer 407 (NIH No. 158)
Mice were vaccinated using the procedure set forth in Example 11 with liposomes modified with 5, 10 and 15 mol% of poloxamer 407 of Examples 5 and 6. Titers of serum IgG 14 days after secondary and tertiary vaccinations are presented in Figure 6. Among the sublingual treatment groups, titers were highest in mice receiving CRX-601 in the liposome treatment group modified with 15% poloxamer 407 (post-secondary), significantly greater than treatment groups with aqueous CRX-601 liposomes or unmodified CRX-601 liposomes.
Example 14 Sublingual Vaccination of Mice with Liposomes Modified with Poloxamers 407, 188, and 184 (NIH No. 167)
The mice were vaccinated using the procedure set forth in Example 11 with liposomes modified with 15 and 25 mol% of poloxamer 407, or 188, or 184 of Examples 5 to 8. Titers of serum IgG 14 days after Secondary and tertiary vaccinations are shown in Figure 7 (and with IH titles in Figure 8). Of the sublingual treatment groups, titers were highest in mice receiving CRX-601 in the treatment group with 15% liposomes of poloxamer 188. Titers in treatment groups with CRX-liposomes Poloxamer-modified 601s were generally better than treatment groups with aqueous CRX-601 liposomes or unmodified CRX-601 liposomes.
Example 15 - Sublingual vaccination of mice with liposomes modified with MPEG-2000-DSPE or MEPEG-5000-SPPE and methylglycol chitosan or chitosan oligosaccharide lactate (NIH No. 163)
Mice were vaccinated using the procedure set forth in Example 11 with liposomes modified with 5 mole% MPEG-2000-DSPE or MPEG-5000-DPPE of Examples 2 and 4 formulated with chitosan methylglycol or chitosan
Oligosaccharide lactate as described in Example 9. The serum IgG titers 14 days after the secondary and tertiary vaccinations are shown in Figure 9 (and also with the HI titles in Figure 10). Of the sublingual treatment groups, titers in the liposome + methylglycol chitosan treatment groups were generally better than in treatment groups with unmodified CRX-601 liposomes.
Example 16 Sublingual Vaccination of Mice with Liposomes Modified with Poloxamers and Methylglycol Chitosan (NIH No. 164)
The mice were vaccinated using the procedure set forth in Example 11 with liposomes modified with 5, 15 and 25 mol% poloxamer 407 (labeled F127 in Figures 11 and 12) of Examples 5 to 8 and 15 or 25 mol% poloxamer 407 formulated with methylglycol chitosan as described in Example 9. Serum IgG titers 14 days after the secondary and tertiary vaccinations are shown in Fig. 11 (and also with the IH titers on the Figure 12).

权利要求:
Claims (24)
[1]
A liposomal composition comprising lipids that form a liposomal lipid bilayer, phospholipid-PEG conjugates incorporated into the liposomal lipid bilayer, and a chitosan or a chitosan derivative.
[2]
2. Liposomal composition according to the preceding claims further comprising an aminoalkyl glucosaminide phosphate (AGP) and an aminoalkanesulfonic buffer.
[3]
A liposomal composition according to any one of the preceding claims wherein the liposome lipids are DOPC.
[4]
A liposomal composition according to any one of the preceding claims wherein the liposomal composition further comprises cholesterol.
[5]
5. A liposomal composition comprising lipids that form a liposomal lipid bilayer, PEG / surfactant copolymers such as poloxamers incorporated into the liposomal lipid bilayer, AGP and aminoalkanesulfonic buffer.
[6]
A liposomal composition according to any one of the preceding claims wherein the liposome lipids are DOPC in the absence of cholesterol.
[7]
A liposomal composition according to any one of the preceding claims, wherein the phospholipid-PEG conjugate is selected from the group consisting of poloxamer 407 (Pluronic® F127), poloxamer 184 (Pluronic® L64), poloxamer 188 (Pluronic® L68) .
[8]
A liposomal composition according to any of the preceding claims, wherein the phospholipid-PEG conjugate is MPEG-2000-DSPE, sodium salt of N- (carbonyl-methoxypolyethylene glycol-2000) -1,2-distearoyl-sn- glycero-3-phosphoethanolamine, or MPEG-5000-DPPE, sodium salt of N- (carbonyl-methoxypolyethylene glycol-5000) -1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine.
[9]
A liposomal composition according to any one of the preceding claims, wherein the liposomal composition further comprises chitosan.
[10]
The liposomal composition according to any one of claims 2 to 9, wherein the aminoalkanesulfonic buffer is selected from the group consisting of HEPES, HEPPS / EPPS, MOPS, MOBS and PIPES.
[11]
A liposomal composition according to any one of claims 2 to 10, wherein the AGP is selected from the group consisting of: CRX-601, CRX 602, CRX 527, CRX 547, CRX 526, CRX 529 or CRX 524.
[12]
A liposomal composition according to any one of the preceding claims, comprising a chitosan, wherein the chitosan is selected from the group consisting of chitosan oligosaccharide lactate, trimethyl chitosan glycol chitosan, and methyl glycol chitosan.
[13]
An improved method of producing a liposomal composition for mucosal administration comprising the steps of: a. dissolving a lipid, such as dioleoylphosphatidylcholine, phospholipid-PEG conjugate, and AGP in an organic solvent, b. removing the solvent to produce a phospholipidic film, c. adding the film to HEPES buffer or HEPES buffer in saline, d. the dispersion of the film in the solution, and e. extruding the solution successively through polycarbonate filters to form unilamellar liposomes.
[14]
The method of claim 13, wherein the AGP is CRX-601.
[15]
A liposomal composition according to any one of claims 2 to 14, wherein the AGP CRX-601 is present in an amount less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2 or less than 1 mg.
[16]
The liposomal composition of claim 15, wherein the AGP CRX-601 is present in an amount of between 30 Dg / ml and 6 mg / ml.
[17]
Liposomal composition according to any one of the preceding claims, wherein the liposome is multilamellar.
[18]
18. A liposomal composition according to any one of the preceding claims, wherein the liposome is 2, 3, 4, 5, 6, 7, 8, 9, or 10 lamellar.
[19]
The lamellar composition of any one of the preceding claims, wherein the liposome is unilamellar.
[20]
A liposomal composition according to any one of the preceding claims, wherein the size of the liposome will be in the range of 50 nm to 500 nm and in other embodiments of 50 nm to 200 nm.
[21]
21. A liposomal composition according to any one of the preceding claims, wherein the size of the liposome will be in the range of about 80 to 120 nm.
[22]
Liposomal composition according to any one of the preceding claims, wherein the liposomal structures contain an aqueous interior.
[23]
Liposomal composition according to any one of the preceding claims, further comprising a lipid A mimetic, a TLR4 ligand, or an AGP.
[24]
A liposomal composition according to any one of the preceding claims, wherein the AGP is a compound having the structure represented by formula 1:

(Formula 1) wherein m is 0 to 6 n is 0 to 4; X is 0 or S, preferably 0; Y represents 0 or NH; Z represents 0 or H; each R 1, R 2, R 3 is independently selected from the group consisting of C 1 -C 20 acyl and C 1 -C 20 alkyl; R4 is H or Me; R 5 is independently selected from the group consisting of -H, -OH, -C 1-4 alkoxy, -PO 3 R 8 R 9, -OPO 3 R 8 R 9, -SO 3 R 8, -OSO 3 R 8, -NR 8 R 9, -SR 8, -CN, NO 2, -CHO, -CO2R8, and -CONR8R9, wherein Re and R9 are each independently selected from H and C1-C4alkyl and each of R6 and R7 independently is H or PO3H2.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US201461952124P| true| 2014-03-12|2014-03-12|

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US61/952,124|2014-03-12|

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