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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances

Definitions

• the present invention regards new polymeric conjugates of phospholipids comprehending at least a polymer conjugated, through a branched molecule, at least to two phospholipids, and their use, as such or in combination, with other amphypatic substances to deliver biologically active agents.
• the above reported new polymer-phospholipids conjugates can be used in preparations, 'kits', formulation of liposomes, for diagnostic application or in therapy.
• liposomes possess increased plasma residence time, as compared to the same drug but present in the free state, a further increase is often needed to reach wanted tissues, cells or organs that reside far from the administration site.
• a procedure already established is represented by the covering of the liposomal surface by an hydrophilic agent, as for instance the chains of an hydrophilic polymer such as poly (ethylene glycol) (PEG).
• PEG poly (ethylene glycol)
• modified liposomes are also called ⁇ long circulation' or ⁇ sterically stabilized' liposomes.
• the most followed of this strategy consists in the coupling of PEG chains of about 1-5 kDa molecular weight.
• PEG-phospholipids are used in the preparation, they are added at a percentage of about 5% (as phospholipids equivalents) over all of the lipids present in the liposomal formulation ( see for instance, Stealth Liposomes, CRC Press, Lasic, D. and Martin., eds . , Boca Raton, FIa., (1995) and the here reported references).
• the pharmacokinetics of these liposomes is characterized by a dose dependent captation by liver, spleen, by mononuclear fagocites, on the contrary of the conventional liposomes which are more rapidly removed from blood and accumulated on such organs.
• PEGylated phospholipids The most common and commercially available PEGylated phospholipids is distearoyl phosphatidil ethanolamine (DSPE), PEG-DSPE (5000 Da), a compound already employed in doxorubicin liposome entrapment (Doxil, Caelix®) . It was demonstrated that PEGylated liposomes undergo unexpected rapid elimination, called decelerated blood clearence' (ABC) , mainly observed following repeated administrations, probably due to the complement activation. As a reason of such a phenomenon it was advocated a premature loss of the protective PEG layer due to the release of the PEG-DSPE chains, (see for instance, Parr M. J., et al Biochim. Biophys. Acta, 1195: 21-30 1994).
• ABS decelerated blood clearence'
• the phospholipids of one conjugate molecule increase the interactions with other molecules resulting in an overall enhanced stability of the system. Furthermore, the increased dimension of such hydrophobic moiety in the double layer of the membrane will allow for a reduction of release rate of the entrapped drugs.
• the present invention describes in particular conjugates that comprehend an hydrophilic polymer bound, directly or through other moieties to at least two phospholipids molecules. Furthermore such phospholipids can be bound to the hydrophilic polymer, directly or through one or more then one spacer groups, and/or one or more then one other polymers .
• the use of different polymers or of dendrimeric structures allows for the linking of an increased number of phospholipids molecules to the hydrophilic polymer.
• the spacers are preferentially chosen among alkyl, or aromatic groups, or cleavable peptides or other biodegradable sequences.
• one, or more then one targeting molecule such as antibodies, hormones, sugars, can be bound to the opposite site of the polymer chain with respect to the one where the phospholipids are bound, directly or through suitable spacers and/or other polymers.
• the amphypatic nature of the conjugates reported in the present invention allows for a stable localization at the liposome surface.
• the hydrophobic phospholipids moiety of such conjugates will intercalate in the phospholipids double layer of the liposome, whereas the hydrophilic polymer will be extended towards the aqueous milieu where the liposomes are dispersed.
• the polymeric chains will therefore form an hydrophilic cloud that protect the liposomes from opsonization.
• the novelty and the advantage of such new polymer-phospholipides conjugates resides in the yielding increased stability of the hydrophilic moiety, as compared to the already known long circulating liposomes, where the hydrophilic PEG-DSPE moiety undergoes continuous detachment from the liposome surface.
• the inventors have also observed that the liposomes obtained with the phospholipids-polymer conjugates described in the present invention, also release the entrapped therapeutic agents more slowly and at a sufficiently longer time as requested in several diagnostic or therapeutic applications.
• the present invention provides new polymer-conjugated phospholipids that can be used in the preparation of liposomes as long circulation carrier for biologically active agents to be used both in diagnostic and therapeutic.
• the polymer-phospholipids object of this invention (termed here also as “conjugates”) include a hydrophilic polymer linked through a branching unit to two or more phospholipids, the hydrophilic polymer may also possibly be linked, directly or through a branching unit to one or more targeting residues.
• the conjugates preferably have the following formula:
• PL is a phospholipid
• MF is a branching unit having functionalizable residues to bind directly or by means of other portions PL and Poll,
• Poll is a water-soluble hydrophilic polymer, of synthetic or natural origin
• Pol2 and BM may be present or absent.
• BM and Pol2 are absent, Pol2, if present, is a branching unit that has functionalizable residues to bind directly or by means of other portions Poll and BM,
• BM if present, is a targeting residue
• aromatic groups or biodegradable sequence or peptides i.e. Gly-Phe-Leu-Gly and Gly-Leu-Phe-Gly
• the phospholipid PL has the following general formula :
• Rl and R2 are independently an alkyl group, saturated or unsaturated, preferably comprising from 1 to 22 carbons, eventually substituted with one or more groups, preferably chosen among oxi, hydroxyl, or amino groups,
• R3 is a bond or any group that allows the coupling of PL with MF, as for example, but not exclusively, ethanolamine, serine and glycerol.
• the phospholipids (PL) used for the present invention are amphiphilic molecules containing a phosphoric group; phospholipids can be natural or of synthetic origin (Berg JM, Tymoczko JL, and L. Stryer, Biochemistry. 5th ed. 2002, New York : WH Freeman. VIII, 974, (and following pages), J. Lindberg, et al. J. Org. Chem. 67 (1): 194-199 (2002)).
• the phospholipids present in the conjugate are capable of interacting with the phospholipid bilayer of liposomes or promote the formation of micelles.
• Preferred phospholipids are, but not exclusively, distearoyl-phosphatidylethanolamine (DSPE) , dipalmitoyl- phosphatidylethanolamine (DPPE) , dimyristoyl- phosphatidylethanolamine (DMPE) , dioleoyl- phosphatidylethanolamine (DOPE) , dielaidoyl- phosphatidylethanolamine (DEPE) , distearoyl- phosphatidylethanolamine (DSPS), dipalmitoyl-phosphatidyl- serine (DPPS) , dimyristoyl-phosphatidyl-serine (DMPS) , dioleoyl-phosphatidyl-serine (DOPS) , dimyristoyl- phosphatidylethanolamine (DMPE) , distearoyl-phosphatidyl- glycerol (DSPG) , dipalmitoyl-phosphatidyl-gly
• BM may be present or not, and if this is a targeting residue for selective recognition of cancer tissues, inflamed or infected sites and useful for diagnosis or therapy.
• targeting residues for the invention are peptides, monoclonal antibodies, antibody fragments, biotin, hormones and sugars.
• n indicating the number of molecules of phospholipid coupled via the branching unit to Poll, and it is a number greater than 1, for example, but not exclusively, from 2 to 20, and preferably from 4 to 8, to obtain a compound with a hydrophobic moiety greater than the conjugate PEG-DSPE.
• MF is a tri- or tetra-functional molecule, or a multifunctional polymer or a dendrimeric structure; MF has functional groups useful to bind directly or by means of other portions PL and Poll.
• Preferred MF molecules are selected among glutamic acid, aspartic acid, beta-glutamic acid, homoglutamic acid, aminoadipic acid, lysine or 3-hydroxyl 2 amine propanol, or any other amino dicarboxylic amino acid or amino tricarboxylic amino acid.
• Preferred MF multifunctional polymers are selected among polyaspartic acid, polyglutamic acid, poly (hydroxy aspartamide) , poly (hydroxy glutamide) , poly (hydroxy propylmethacrylamide) and polylysine, and in preferred embodiments the polymer has a molecular weight ranging from 500 and 5000 Da.
• MF is a dendrimer, this is preferably based on glutamic acid, aspartic acid, beta- glutamic acid, homoglutamic acid, aminoadipic acid, lysine or 3-hydroxyl 2 amine propanol, or any other amino dicarboxylic amino acid or amino tricarboxylic amino acid.
• the preferred functional groups of MF are included amino and carboxyl groups.
• the conjugation can be mediated by an ester, amide, carbamate or other covalent bonds.
• Pol2 if present, is a branching unit and in some preferred embodiments of the invention Pol2 is tri- or tetra- functional molecule, a multifunctional polymer or a dendrimeric structure. Pol2 has residues functionalized for covalent binding of several BM residues (in this case m ⁇ l, for example, from 1 to 10, and preferably from 1 to 4) to obtain a more selectively targeted conjugate.
• the branching unit can be preferably represented by glutamic acid, aspartic acid, beta-glutamic acid, homoglutamic acid, aminoadipic acid, 3 hydroxy 2-amino propanol or another bicarboxylic or tricarboxylic amino acid.
• Pol2 also has a functional group for conjugation to Poll directly or by means of other portions.
• Among the preferred functional groups of Pol2 are included amino and carboxyl groups.
• the conjugation can be mediated by an ester, amide, carbamate or other covalent bonds .
• Poll is a water soluble hydrophilic polymer, of natural or synthetic origin, which possesses at least one functional group for conjugation to the MF or at least two functional groups if Pol2 and/or BM are present. Poll is coupled to at least two PL molecule by means of MF. When Poll is a monofunctional polymer the only functional group must be coupled to the block including the PL molecules. Such conjugation may be direct or mediated by other portions. When Poll has two end groups (Poll bifunctional or heterobifunctional) one must be involved in conjugation with PL block, this including at least two molecules of PL (PL molecole can be also different on the same block) , and the other may be either conjugated directly or by means of other portions to Pol2 and/or BM.
• the Poll mono-, bifunctional or heterobifunctional preferred polymers there are poly (ethylene glycol), polyvinylpirrolidone, poly (2- ethyl-2-oxazolyne) , poly (N-acryloylmorpholine) .
• the group/groups of Poll can be activated for example according to known procedures (eg as reported for PEG-COOH and PEG- NH2) and others (Sabine Herman, et al. J. of Bioactive and Compatible Polymers, 10: 145-187 (1995)).
• the polymer Poll has an preferred average molecular weight between 1000 and 40000 Da, preferably at least 1500 Da, more preferably at least 2000 Da, and even more preferably at least 5000 Da.
• Poll is a derivative of poly (ethylene glycol) (PEG), of linear or branched structure, mono-, bi-functional or heterobifunctional, with an average molecular weight between 1000 and 40000 Da.
• PEG poly (ethylene glycol)
• Some preferred Poll are mPEG-OH 2000 Da, mPEG-COOH 2000 Da, mPEG-OH 3400 Da, mPEG-COOH 3400 Da, mPEG-OH 5000 Da and mPEG-COOH 5000 Da.
• the preferred conjugates according to the present invention there are the conjugates having the following general formula:
• PEG poly (ethylene glycol)
• monofunctional, bifunctional or heterobifunctional with mean molecular weight between 1000 and 40000 Da, and preferably mPEG-COOH 2000 Da, mPEG- COOH 3400 Da or mPEG-COOH 5000 Da.
• MF is a branching unit that can be a dendrimer; for example, MF can comprise one or more beta-glutamic acid residues (BG) as branching molecule, or MF can be an homopolymer, based on glutamic acid (AG) .
• the conjugates according to this invention are suitable either for a direct use, as amphiphilic compounds to solubilize hydrophobic molecules or for the preparation of long circulating liposomes.
• the conjugate object of this invention can be used in molar concentration ranging from 1 to 100% of the total lipid composition.
• the liposomes comprising one of the conjugates here presented can be useful for the delivery, the storage and the formulation of drugs (either hydrophobic or hydrophilic) , peptides, proteins and diagnostic agents.
• Step 1 Synthesis (COOH) 2 -BG-mPEG 5000 Da.
• Step 2 Synthesis (DSPE) 2 -BG-mPEG 5000 Da.
• Carboxylic groups of (COOH) 2 -BG-mPEG were activated by DCCI/NHS and coupled to amino group of DSPE, yielding: (DSPE) 2 -BG-mPEG 5000 Da.
• Example 2 Preparation of (DSPE) 4 - (BG) 2 -BG-mPEG 5000 Da Step 1. Synthesis of (COOH) 4 - (BG) 2 -BG-mPEG 5000 Da. The activated carboxylic groups of (NHS) 2 -BG-mPEG (5400 Da) , obtained as described in step 2 of example 1, were conjugated to the amino group of the ⁇ -glutamic acid, yielding the product: (COOH) 4 - (BG) 2 -BG-mPEG 5000 Da.
• Step 2 Synthesis and purification of (DSPE) 4 - (BG) 2 -BG-mPEG 5000 Da.
• the carboxylic groups of (COOH) 4 - (BG) 2 -BG-mPEG 5000 Da were activated via DCCI/NHS and reacted with the amino group of DSPE to obtain: (DSPE) 4 - (BG) 2 -BG-mPEG 5000 Da.
• the coupling and the purification of the product were conducted as reported for the step 2 and 3 of example 1. In this case the excesses of DSPE and EtN 3 were adjusted to the number of activated carboxylic groups of (NHS) 4 -(BG) 2 - BG-mPEG. Overall yield: 65%
• Example 4 Preparation of a liposomal formulation containing one of the conjugate object of the present invention Liposome can be prepared by several methods. Here, as an example, is reported the method of "thin layer evaporation".
• Epirubicin a well known anticancer drug, as been chosen as model drug to be incorporated into the liposomes with the aim to study its kinetic release rate. Furthermore, the pharmacokinetic of these new liposomal formulation of epirubicin were studied in mice.
• long circulating liposomes comprising phosphatidylcholine / cholesterol / DSPE-mPEG 5000 Da (commercial conjugate for the preparation of long circulating liposomes) , liposomi
• Each liposomal formulation was separately prepared by dissolving in 3 ml of CHCI 3 in round bottom flask the amounts of phosphatidylcholine, cholesterol and, according to the schedule, the amphiphilic conjugate if present.
• the mixture was dried in rotavapor and if necessary in oven at 105 0 C.
• the obtained thin layer was hydrated with 2 ml of PBS containing epirubicin.
• the mixture was heated at 6O 0 C (3 min) and mixed (3 min) for 3 times, and then left at 4 0 C for 1 hour.
• the liposome suspension was sonicated for 5 min in ice-bath and then filtered with a 0.22 ⁇ m cut-off filter.
• the filtered solution was purified by gelpermeation chromatography (GPC) using a sephadex G-50 resin with 15 x 1 cm column; elution was performed with PBS.
• the purified liposomes suspension was following used for the drug release and pharmacokinetic studies
• Example 5 Study of epirubicin release from liposomes prepared as described in the example 4.
• each liposomal formulation prepared in the example 4 was performed by adding 2 ml of one formulation in a dialysis tube (cut-off 10000 Da) .
• the tube was put in 100 ml of PBS.
• the amount of released drug in the receiving solution was evaluated by RP-HPLC.
• the kinetic release half-life are reported in table 2.
• the liposomal formulation LIPO 4 had the slower drug rate release.
• Table 2 Half-life of epirubicin release from the different liposomal formulation prepared in the example 4
• Example 6 Pharmacokinetic study of liposomal formulations prepared as described in the example 4.
• the liposomal formulations were tested directly as prepared in the example 4 or after reduction to small volume, to invrease the liposome concentration, by ultracentrifugation using a membrane with 60000 Da cut-off.
• the pharmacokinetics were evaluated in Balb-c mice weighting about 23 g.
• the animals were treated with a 3 mg/kg dose (epirubicin equiv. ) injected through the tail vein 180 ⁇ l of liposomal formulation.
• 150 ⁇ l of blood were withdrawn from the rear plexus of the eyeball.
• the blood was treated to determine the amount of epirubicin.
• Table 3 are reported the main pharmacokinetic parameters.
• the LIPO 4 formulation demonstrated the most prolonged in vivo half- life among the tested formulation.

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