WO2009128789A1 - Vésicules pour l'administration intracellulaire de médicaments - Google Patents

Vésicules pour l'administration intracellulaire de médicaments Download PDF

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WO2009128789A1
WO2009128789A1 PCT/SG2009/000142 SG2009000142W WO2009128789A1 WO 2009128789 A1 WO2009128789 A1 WO 2009128789A1 SG 2009000142 W SG2009000142 W SG 2009000142W WO 2009128789 A1 WO2009128789 A1 WO 2009128789A1
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gly
acid
composition
phe
pegylated
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PCT/SG2009/000142
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Zhilian Yue
Hanry Yu
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Agency For Science, Technology And Research
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    • 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
    • A61K47/6919—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 ribbon or a tubule cochleate

Definitions

  • the present invention refers to the field of biochemistry and in particular to the field of chemical compositions for responsive biopolymer targeted drug delivery.
  • the present invention refers to a composition
  • a composition comprising: ⁇ a pegylated amphiphilic polymer, wherein the pegylated amphiphilic polymer is comprised of at least one polymerizable carboxylic acid and at least one amino acid; wherein the degree of pegylation is adapted to cause the amphiphilic polymer to form a vesicle; wherein the polyethyleneglycol moiety is linked to a cleavable linkage; and wherein the cleavable linkage is cleavable by an external stimulus to cause a conformational change of the vesicle form of the polymer into its linear form.
  • the present invention refers to a method of manufacturing a composition, comprising:
  • an amphiphilic polymer comprising at least one polymerizable carboxylic acid and at least one amino acid
  • the present invention refers to a pharmaceutical composition
  • a pharmaceutical composition comprising a composition described herein and encapsulating a hydrophopic medicament or a mixture of different hydrophobic medicaments therein.
  • the present invention refers to a scaffold comprising a composition described herein or a composition obtained by a method described herein.
  • Figure 1 illustrates the triggered drug release from vesicles described herein by cleavage of the cleavable linkage.
  • Figure 2 shows a reaction scheme illustrating the PEGylation of polyaspartic acid with L -leucine side chains (PAIe) and polyaspartic acid with L -phenylalanine side chains
  • Figure 2 illustrates the reaction scheme for the synthesis of polyaspartic acid with L -leucine side chains (PAIe) and polyaspartic acid with L -phenylalanine side chains (PAph) starting from L-aspartic acid.
  • PAIe polyaspartic acid with L -leucine side chains
  • PAph polyaspartic acid with L -phenylalanine side chains
  • the reaction scheme further differentiates PAIe and PAph into PAle-5A and PAph-5A (carrying mPEG-CH 2 CH 2 NH or
  • PAle-5B and PA ⁇ h-5B (carrying mPEG-S-S-CH 2 CH 2 NH or OH as rest R 2 ), respectively.
  • Figure 3 shows a reaction scheme illustrating the PEGylation of poly ( L -lysine wO-phthalamide).
  • Poly( L -lysine ⁇ o-phthalamide) was prepared by hydrolysis of poly( L -lysine methyl ester iso-phthalamide) and converted to the sodium salt form. The polymer in neutral form was used for the PEGylation.
  • FIG 4 illustrates the variation of I 1 ZI 3 as a function of pH in the emission spectra of pyrene in aqueous PAIe ( ⁇ ), PAle+PEG (•), PAle-5B (A) at 1.0 mg mL "1 , respectively.
  • the I 1 ZI 3 values for pyrene in aqueous PAIe are close to those in water, and remain fairly constant over the pH range studied (between pH 3 and 10). This indicates a hydrophilic conformation of the polymer chains where the pyrene is exposed to water. No significant change of the pyrene spectrum was noted when the polymer is physically mixed with mPEG in aqueous solution.
  • FIG. 5 illustrates the variation Of I 1 ZI 3 as a function of pH in the emission spectra of pyrene in aqueous PAph ( ⁇ ), PAph+PEG (•), PAph-5B (A) at 1.0 mg mL "1 , respectively.
  • PAIe- 5B Figure 4
  • the results shown in Figure 5 also demonstrate an enhanced hydrophobic association through PEGylation of PAph (see the data for PAph-5B).
  • Figure 6 illustrates the particle sizes (in nm) of the vesicles of PL-5B, PAIe- 5B and PAph-5B by dynamic light scattering (1.0 mg mL "1 ) and their drug contents (Dox wt%).
  • Figure 7 shows a TEM micrograph of PAph-5B illustrating the nano-spherical morphology of the vesicles formed by PAph-5B.
  • FIG. 8 shows Doxorubicine (Dox) release profiles from (a) PAle-5B, (b) PA ⁇ h-5B and (c) PL-5B in glutathione (GSH) solution (10 mM in PBS) (A) and in PBS ( ⁇ ).
  • GSH glutathione
  • PAle-5B, PAph-5B and PL-5B have been brought in contact with a solution comprising an increased concentration of GSH as it can be found also in cells in which a high concentration of GSH ( ⁇ 1 to 11 mM) keeps thiol groups in their reduced state.
  • Figure 9 shows the cellular uptake of free Dox (A-C) and PAle-5B with entrapped Dox (D-I) in MCF-7 after Ih and 48 h's uptake, respectively.
  • Red fluorescence is for free Dox (A) and PAle-5B with entrapped dox (D, H); green fluorescence is for nuclear (B, E, H).
  • Figure 9 C, F and I show overlays of green and red fluorescence. The oval areas encircled with a broken line show the position of the nuclei.
  • Figure 10 shows the cell growth inhibition of MCF-7 after incubation with DOX (a), PALe-5B (b), PA ⁇ h-5B (c) and PL-5B (d) at various concentrations, hi Figure 10 the cell viability is plotted against the concentration of drug (ng/ml) wherein the cells have been exposed to the free drug (Figure 10 a) or to one of the three systems for 48 and 72 h, respectively.
  • the results shown in Figure 10 demonstrate a reduced in vitro cytotoxicity for all three systems PAle-5B ( Figure 10 b), PAph-5B (Figure 10 c) and PL-5B (Figure 10 d)) compared to free Dox ( Figure 10 a).
  • the present invention provides a composition
  • a composition comprising: a pegylated amphiphilic polymer, wherein the amphiphilic polymer is comprised of at least one polymerizable carboxylic acid and at least one amino acid; wherein the degree of pegylation is adapted to cause the amphiphilic polymer to form a vesicle; and ⁇ wherein the polyethyleneglycol moiety is linked to a cleavable linkage;
  • This composition comprises a pegylated amphiphilic polymer which forms a vesicular structure, i.e. a vesicle with a hydrophobic core and a hydrophilic outer shell.
  • the hydrophobic core is formed by the amphiphilic polymer while the hydrophilic outer shell or corona is formed by the polyethyleneglycol moiety which is linked to the amphiphilic polymer via a cleavable linkage.
  • the vesicle forms through self assembly of the pegylated amphiphilic polymer in a solution.
  • the vesicle structure can be destroyed by subjecting the pegylated amphiphilic molecule to an external stimulus which results in cleavage of the cleavable linkage and thus the separation of the polyethyleneglycol moiety from the polymer backbone (polymer backbone consists of the carboxylic acid and the amino acid).
  • This cleavage results in an alteration of the balance between hydrophilicity and hydrophobicity in the pegylated amphiphilic polymer and thus transforms the vesicle structure back into its linear and open form. In other words, cleavage results in the core destabilization and consequent disintegration of the vesicle.
  • This disintegration transforms results in a nonvesicular form as illustrated in Figure 1 comprising of the encapsulated chemical component, the polymer backbone and the polyethyleneglycol moiety.
  • the above mechanism of triggered disruption of the vesicle structure by an external stimulus allows the encapsulation of hydrophobic chemical compounds into the hydrophobic core of the vesicle and release of the hydrophobic chemical compounds after the external stimulus causes the cleavage of the cleavable linkage and thus disruption of the vesicle structure.
  • hydrophobic chemical compounds are inherently small and can pass through the membrane of an endosome or lysosome after being released from the vesicle and can thus escape degradation.
  • compositions can be used for the controlled release of laden chemical compound into the cytoplasma of cells and thus circumventing the fusion with a lysosome which could disrupt the encapsulated hydrophobic chemical compounds, hi other words, the hydrophobic chemical compound can be released intracellularily (i.e. inside or within the cell).
  • Hydrophobic means "water fearing”. Hydrophobic refers to the tendency of a molecular entity to repel water or to be incapable of completely dissolving in water. Hydrophobic molecular entities are readily soluble in many nonpolar solvents but only sparingly soluble in water, a polar solvent, and are therefore also called nonpolar molecular entities. In general the more hydrophobic a molecular entity is the more likely it is associated with nonpolar organic matter such as humic substances and lipids (fats).
  • hydrophilic means "water loving”. Hydrophilic refers to the tendency of a molecular entity to interact with polar solvents, in particular with water, or with other polar groups.
  • a hydrophilic molecular entity is one that is typically charge- polarized and capable of hydrogen bonding and is therefore also called polar molecule.
  • An amphiphilic molecule refers to a molecule having a hydrophilic (polar), water-soluble group attached to a nonpolar (hydrophobic), water-insoluble group.
  • the pegylated amphiphilic polymer consist of a hydrophobic group, namely the combination of the at least one polymerizable carboxylic acid and the at least one amino acid, and the hydrophilic group, namely the polyethyleneglycol moiety which is linked to the amphiphilic polymer via a cleavable linkage.
  • a vesicle does not refer to a structure comprising fats, oils, waxes, cholesterol, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, and phospholipids, such as a liposomal vesicle consisting of a phospholipid bilayer or a single phospholipid layer.
  • a vesicle or micelle is composed of a pegylated amphiphilic polymer comprised of at least one carboxylic acid and at least one amino acid.
  • the amphiphilic polymer is pegylated.
  • pegylation describes a process in which poly(ethyleneglycol) chains (PEG; H-(O-CH 2 -CH 2 ) J1 -OH), also known as poly(ethylene oxide), are attached to an amino acid of a protein or a peptide drug.
  • PEG is formed by a process of linking repeating units of ethylene glycol to form polymers with linear or branched shapes of different molecular weight, such as PEG-OH, monomethoxy-PEG-OH and branched monomethoxy-PEG 2 (m-PEG 2 ).
  • the polyethylene glycol moiety is PEG-OH or m-PEG-OH or m-PEG 2 .
  • PEG structures can then be chemically attached to the substance of choice in a process called pegylation.
  • pegylation The preparation of polyethylene glycol moieties is known in the art (see, e.g., Harris, J.M., Chess, R.B., 2003, Nature Reviews Drug Discovery; vol.2, ⁇ p.214).
  • a PEG polymer is first chemically activated in order to react with an amino acid of the target molecule, in this case the amphiphilic polymer.
  • a variety of chemical modifications can be used to prepare an active PEG derivative with a functional group, such as active carbonate, active ester, aldehyde or tresylate.
  • the methods used for such a modification are known in the art (Harris, J.M., Chess, R.B., 2003, supra) and can include, but are not limited to cyanuric chloride method, PEG-succinimidyl succinate method (variant: substitution of the succinate residue by glutarate, substitution of the aliphatic ester by an amide bond) or imidazoyl formate method, to name only a few.
  • the polyethyleneglycol moiety is coupled or linked to the amino acid of the amphiphilic polymer via a cleavable linkage.
  • the cleavable linkage comprises a terminal disulfide group which is covalently bound to a carboxylic acid, or an amine, or an alcohol, or derivatives thereof.
  • One end of the terminal disulfide group binds to the carboxylic acid, or the amine or the alcohol whereas the other end binds to the polyethyleneglycol moiety.
  • the functional group of the carboxylic acid (-COOH), or the amine (-NH 2 ), or the alcohol (-OH) binds to the carboxyl group of the amino acid of the amphiphilic polymer.
  • the polyethyleneglycol moiety is bound to one end of the cleavable linkage and the cleavable linkage is covalently bound with its other end to the carboxyl group of the amino acid of the amphiphilic polymer.
  • a cleavable linkage can include, but are not limited to, R 1 -S-S-R 3 , wherein R 1 may be PEG-OH, m-PEG-OH or m-PEG 2 , and R 3 may be -COOH, -NH 2 or -OH. Any combination of R x and R 3 is possible in the present invention.
  • a cleavable linkage comprising a disulfide group is cleaved upon entering the cytosol of a cell via endocytosis.
  • a disulfide bond is stable in extracellular environments and cleavable inside a cell due to elevated levels of GSH.
  • GSH is part of the glutathione system: GSSG/2GSH.
  • Glutathione is considered to be the major thiol-disulfide redox buffer of the cell.
  • the GSH concentration in the cytosol is 1 to 11 mM.
  • the amount of GSH in the extracellular medium is about 0.002 to 0.8 mM.
  • the pegylated amphiphilic polymer includes a cleavable group which is pH sensitive, i.e. which is cleaved upon the change of pH in its environment. In cellular environments that means a shift in the pH value from basic pH to a more acidic pH.
  • Galande, A.K., Weissleder, R. and Tung, C-H. 2006, Bioconjugate Chemistry, vol.17, no.2, pp.255
  • the hydrazone group is hydrolysed at the double bond between the carbon and nitrogen atom.
  • Kakinoki, A., Kaneo, Y., et al. January 2008, Biol. Pharm. Bull., vol.31, no.l, pp.103
  • doxorubicin to include a pH-sensitive a cis-aconityl group.
  • an amino group can be reacted with cis-aconitic anhydride to form an ( ⁇ unsaturated amide bond.
  • An example of an enzyme cleavable linkage includes, but is not limited to a peptide side chain which is cleavable by lysosomal enzymes, such as proteinases.
  • An example for such a cleavable peptide includes, but is not limited to Gly-Gly, Gly-Phe-Gly, Gly-Phe- Phe, Gly-Leu-Gly, Gly-Val-Ala, Gly-Phe-Ala, Gly-Leu-Phe, Gly-Leu-Ala, Ala-Val-Ala, GIy- Phe-Leu-Gly, Gly-Phe-Phe-Leu, Gly-Leu-Leu-Gly, Gly-Phe-Tyr-Ala, Gly-Phe-Gly-Phe, AIa- Gly-Val-Phe, Gly-Phe-Phe-Gly, Gly-Phe-Leu-Gly-Phe, or Gly-Gly
  • the pegylated amphiphilic polymer comprises the following general formula (I):
  • X is the at least one polymerizable carboxylic acid
  • Y or Z is the at least one amino acid
  • R is -OH or the polyethylenglycol moiety which is linked to a cleavable linkage; n is an integer above 100; and one of q and r is 0 whereas the other is 1; and wherein the at least one polymerizable carboxylic acid and the at least one amino acid are covalently bound via a peptide bond and wherein the polyethyleneglycol moiety is bound to the amphiphilic polymer via the cleavable linkage to the carboxyl group of the at least one amino acid.
  • the pegylated amphiphilic polymer comprises the following general formula (II):
  • X is the at least one polymerizable carboxylic acid
  • Z is the at least one amino acid; R is -OH or the polyethylenglycol moiety which is linked to a cleavable linkage; n is an integer above 100; and wherein the at least one polymerizable carboxylic acid and the at least one amino acid are covalently bound via a peptide bond and wherein the polyethyleneglycol moiety is bound to the amphiphilic polymer via the cleavable linkage to the carboxyl group of the at least one amino acid.
  • the pegylated amphiphilic polymer comprises the following general formula (III):
  • X is the at least one polymerizable carboxylic acid
  • Y is the at least one amino acid
  • R is -OH or the polyethylenglycol moiety which is linked to a cleavable linkage; n is an integer above 100; and wherein the at least one polymerizable carboxylic acid and the at least one amino acid are covalently bound via a peptide bond and wherein the polyethyleneglycol moiety is bound to the amphiphilic polymer via the cleavable linkage to the carboxyl group of the at least one amino acid.
  • Polymerizable carboxylic acid means that two or more carboxylic acid monomers can be connected to form a chain of carboxylic acids including identical repeating units.
  • the at least one polymerizable carboxylic acid can include but is not limited to aspartic acid, o-phthalic acid, w ⁇ -phthalic acid, terephthalic acid, adipic acid, aldaric acid (HOOC-(CHOH) m -COOH), wherein m is an integer from 1 to 5, fumaric acid, glutaric acid, maleic acid, malic acid, malonic acid, succinic acid, tartronic acid or derivatives thereof.
  • the amphiphilic polymer comprises one polymerizable carboxylic acid.
  • carboxylic acids maybe optionally substituted.
  • optionally substituted refers to a group in which none, one, or more than one of the hydrogen atoms has been replaced with one or more group(s) that are independently selected from alkyl, heteroalkyl, haloalkyl, heteroholoalkyl, cycloalkyl, aryl, arylalkyl, heteroaryl, non-aromatic heterocycle, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N- amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, isocyanato, and amino, including mono- and di-substitute
  • the at least one amino acid can include, but is not limited to amino acids with basic side chains, amino acids with acidic side chains, amino acids with nonpolar side chains, amino acids with uncharged polar side chains or derivatives thereof.
  • amino acids with basic side chains include lysine, arginine, histidine or derivatives thereof.
  • amino acids with acidic side chains include aspartic acid, glutamic acid or derivatives thereof.
  • amino acids with nonpolar side chains include glycines, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophane, cysteine or derivatives thereof.
  • amino acids with uncharged polar side chains include asparagine, glutamine, serine, threonine, tyrosine or derivatives thereof.
  • the amino acids are naturally occurring amino acids or modified amino acids.
  • Other examples for naturally occurring amino acids besides the amino acids mentioned above could be selenocysteine or pyrrolisine.
  • the amino acid used can be present in its isomeric L-form or D-form.
  • L-amino acids are used.
  • the amino acid is L -leucine or L -phenylalanine or L -lysine.
  • At least one amino acid means one amino acid or multiple amino acids wherein the multiple amino acids are the same or different from each other.
  • 2 or 3 or 4 or 5 or 6 amino acids are bound to each other via a peptide bond.
  • modified amino acids can include, but are not limited to N-acetyl-
  • a vesicle forms or not depends on the degree of pegylation of the amphiphilic polymer.
  • the polyethyleneglycol moiety bound to the cleavable linkage can be introduced into the amphiphilic polymer using standard DCC mediated coupling chemistry (Haslam, E., Tetrahedron, 1980, vol.30, pp.2409). Control over reaction conditions, principally concentration of the polymer, is required to avoid gelation of the system due to the formation of intermolecular anhydrides.
  • the degree of pegylation can be determined by any method known in the art.
  • One example of determining the degree of pegylation would be as follows:
  • the pegylated polymers are white powders in both neutralised and inoised states, and those in the acid form can be characterised with 1 H NMR spectroscopy in a suitable solvent, such as d 6 -DMSO, from which the degrees of pegylation can be determined.
  • the degrees of pegylation can be calculated based on a M n for the polyethyleneglycol moiety including the cleavable linkage without taking into account of its polydispersity.
  • the value of Mn can be determined by light scattering measurements.
  • the degrees of pegylation are expressed as the numbers of PEG grafts per 100 carboxylic acid groups (DS a as determined by 1 H NMR spectroscopy) and the weight percentage of the PEG side chains (PEG wt%).
  • Ds a for PAle-5B, PAph-5B and PL-5B are around 4.2, 4.1 and 4.2, respectively.
  • the degree of pegylation is between about 40 to about 70 wt% or between about 40 to about 50 wt% based on 1 H NMR spectrum of the pegylated amphiphilic polymers in d 6 -DMSO and varies depending on the structure of the amphiphilic polymer used.
  • the degree of pegylation may be about 40, 45, 50, 55, 60, 65 or 70 wt% based on 1 H NMR spectrum of the pegylated amphiphilic polymers in d 6 -DMSO.
  • the degree of pegylation may be controlled by varying the molar ratio of [COOH]/[PEG]. This means, depending on this ratio, more or less PEG is linked to the amphiphilic polymer.
  • the final degree of pegylation can be determined by 1 H NMR spectroscopy as described above.
  • n is an integer above 100.
  • n may be between about 100 and about 1000 or between about 100 to about 500, or between about 500 to about 800, or between about 500 to about 700.
  • n is 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000.
  • the polyethyleneglycol moiety can have a molecular mass between about 1000 g/mol to about 20000 g/mol.
  • pegylated amphiphilic polymers include, but are not limited to, (pegylated poly(lysine zso-phthalamide)),
  • R 2 is both mPEG-S-S-CH 2 CH 2 NH and -OH, PEG-S-S-CH 2 CH 2 NH and -OH and wherein n is as defined above.
  • R 2 is both means that the pegylated amphophilic polymers carry a polyethyleneglycol moiety while the non-pegylated polymers carry -OH.
  • the present invention is directed to a method of manufacturing a composition as described above.
  • a method can comprise: providing an amphiphilic polymer comprising at least one polymerizable carboxylic acid and at least one amino acid; providing a reactive heterobifunctional polyethyleneglycol derivative, wherein the reactive heterobifunctional polyethyleneglycol derivative is at one end covalently bound to a cleavable linkage; and forming a vesicle by reacting the amphiphilic polymer with the reactive heterobifunctional polyethyleneglycol derivative.
  • amphiphilic polymer comprises a compound according to formula IV:
  • X is the at least one polymerizable carboxylic acid
  • Y or Z is the at least one amino acid
  • n is an integer above 100
  • one of q and r is 0 whereas the other is 1
  • the at least one carboxylic acid and the at least one amino acid are covalently bound via a peptide bond.
  • the size of the vesicles formed is selected to ensure a sufficient biodistribution in the targeted sites and cellular uptake.
  • Biodistribution means the distributions of encapsulated chemical compounds in various organs including targeted organ. In cancer therapy, accumulation in tumor sites with minimal uptakes by liver, lung, kidney etc is preferred. Bigger particle sizes show lower endocytic uptake by cells.
  • the vesicle size can, for example, be in a range between about 10 to about 50 nm, or between about 10 to about 200 nm, or between about 10 to about 100 nm, or between about 20 to about 100 nm, or between about 50 to about 100 nm.
  • the method can further comprise dissolving the vesicle together with the hydrophobic chemical compound in an organic solvent to form a mixture followed by neutralizing of the mixture.
  • the method can further comprise subjecting the neutralized mixture to a dialysis process as it is known in the art.
  • encapsulation means to entrap a hydrophobic chemical compound or a mixture of different hydrophobic chemical compounds within the boundaries (confines) of a vesicle which is formed by the pegylated amphiphilic polymer.
  • the hydrophobic chemical compound can be any hydrophobic chemical compound that one wishes to encapsulate in the vesicle formed by the pegylated amphiphilic polymer.
  • the hydrophobic chemical compound is a hydrophobic pharmaceutical .
  • hydrophobic chemical compounds include, but are not limited to (8S, 1 OS)- 10-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8, 11 -trihydroxy- 8-(2-hydroxyacetyl)-l -methoxy-7,8,9, 10-tetrahydrotetracene-5, 12-dione (Doxorubicine; anticancer drug), ⁇ (lZ)-5-fluoro-2-methyl-l-[4-(methylsulfinyl)benzylidene]-lH-indene-3- yl ⁇ acetic acid (sulindac); of (+)-(S)-2-(6-methoxynaphthalen-2-yl)propanoic acid (naproxen; NSAID), 2-(3-benzoylphenyl)propionic acid (ketoprofen; NSAID), 4-chloro-N-(2- furylmethyl)-5-
  • pegylated amphiphilic polymers referred to herein are available of functional groups that allows facile modification with ligands to improve uptake by targeted cells.
  • a recognition ligand can be linked (such as by covalent binding) either to the amphiphilic polymer or to the polyethylenglycol moiety.
  • a recognition ligand is a molecule which can selectively bind to a specific binding partner.
  • the specific binding partner can be located on the surface of the cell or a scaffold and can thus facilitate targeting specific positions in the patient's body or the scaffold.
  • the recognition ligand can identify specific cells by binding to binding partners located at the cell surface of the target cell.
  • a recognition ligand can include, but is not limited to a ligand targeting liver cells, a ligand targeting CD44, a ligand targeting a tumor cell, to name only a few targets.
  • recognition ligands include, but are not limited to sugar moieties (targeting liver), monovalent and multivalent RDG (targeting tumor angiogenesis), antibodies, such as IgG, IgD, IgA, IgM and IgE (monocolonal or polyclonal); lectins, integrins, selectins, monoclonal antibodies against ⁇ v ⁇ 3 (targeting tumor angiogenesis), hyaluronan (targeting CD44, tumor targeting), folic acid (tumor targeting), transferring (tumor targeting), Herceptin (Trastuzumab) (targeting HER-2-overexpressing breast cancer), Erbitus (Erbitux or Cetuximab) (targeting colon cancer), Gefitinib or Iressa (targeting non-small cell lung cancer and metastatic breast cancer), to name only a few.
  • sugar moieties targeting liver
  • monovalent and multivalent RDG targeting tumor angiogenesis
  • antibodies such as IgG, IgD, IgA, I
  • the recognition ligand may be conjugated to the polymer backbone, i.e. the amphiphilic polymer.
  • these one or more ligands may be coupled to the polymer via commonly known techniques.
  • the conjugation to the amphiphilic polymer may be effected via well establish ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDC) mediated coupling.
  • EDC ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride
  • a bifunctional polyethylene glycol such as, but not limited to, polyethylene glycol bis-o-pyridylthioester (OPTE-PEG-OPTE) can be employed as starting point for coupling one or more of the above mentioned additional ligands to the system.
  • OPTE-PEG-OPTE polyethylene glycol bis-o-pyridylthioester
  • the OPTE-PEG-OPTE only one end is modified to form 1 -amino ethyl-2-dithio for subsequent pegylation to the polymer backbone.
  • the other end of such conjugated bifunctional PEG will be used for later modification with ligands.
  • cysteamine hydrochloride (0.167 g, 1.3 mmol) was dissolved in ethanol (20 mL) and the solution was purged with N 2 for 1 A h.
  • OPTE-PEG-OPTE (5g, 2.0 mmol) in ethanol (50 mL) was added dropwise over a period of 1 A h, after which the reaction was stirred under N 2 and at room temperature overnight.
  • the reaction mixture was concentrated and precipitated into diethyl ether.
  • the crude product was collected and dissolved in deionised water, prior to dialysis against deionised water (Spectra/Por 7, molecular weight cut-off (MWCO) 1 kDa) for 48 h.
  • MWCO molecular weight cut-off
  • the lyophilized powder was further purified by recyrstallisation from ethanol to obtain OPTE-PEG-S-SCH 2 CH 2 NH 2 .
  • Pegylation of PAIe, PAph and PL was then conducted using OPTE-PEG-S-SCH 2 CH 2 NH 2 , respectively, according to the similar procedure as describe for PAle-5B.
  • Incorporation of a recognition ligand can be achieved by reacting any recognition ligand containing -SH with the pegylated polymers in aqueous solutions at 4 0 C so that the OPTE may be substituted by the respective recognition ligand.
  • the present invention refers to a pharmaceutical comprising a composition described herein wherein the pegylated amphiphilic polymer comprises a hydrophobic chemical compound.
  • the present invention refers to a three dimensional scaffold comprising a composition described herein or a composition obtained by a method described herein. Incorporation of a composition described herein into a scaffold can provide for sustained release of chemical compounds to the cells growing in the scaffold. A sustained release of chemical compounds allows controlling the behaviour, function and differentiation of the cells embedded in the scaffold.
  • a scaffold is in general understood as a three dimensional matrix that serves as a template for cell proliferation and ultimately tissue formation. Culturing cells in a scaffold typically involves seeding cells throughout the scaffold and allowing the cells to proliferate in the scaffold for a pre-determined amount of time.
  • a scaffold consists in general of a polymer, preferably a biodegradable polymer.
  • L-aspartic acid, sulfolane, cysteamine hydrochloride, O-(2-aminoethyl)-O'-methylpolyethylene glycol (M n 4400 g mol "1 , HiPEG-NH 2 ), 4-dimethylaminopyridine (DMAP), pyrene, 5,5'-dithio-bis(2- nitrobenzoic acid), triethylamine, doxorubicin hydrochloride (Dox ⁇ Cl) and ⁇ -phthaloyl chloride were received from Sigma- Aldrich.
  • N,N-dimethylforamide (DMF) and methyl sulfoxide (DMSO), L -phenylalanine ethyl estetr hydrochloride, N 5 N'- dicyclohexylcarbodiimide (DCC), and glutathione (reduced) were from Acros Organics.
  • L - leucine methyl ester hydrochloride and L -lysine methyl ester dihydrochloride were from Alfa Aesar. Pyrene was recrystallized twice from ethanol and the other agents were used as received.
  • the reaction mixture was concentrated and precipitated into diethyl ether.
  • the crude product was collected and dissolved in deionised water, prior to dialysis against deionised water (Spectra/Por 7, molecular weight cut-off (MWCO) 1 kDa) for 48 h.
  • MWCO molecular weight cut-off
  • the lyophilized powder was further purified by recyrstallisation from ethanol.
  • PAIe and PAph PEGylation of PAIe and PAph. Both PAIe and PAph were modified with mPEG-NH 2 and InPEG-S-S-CH 2 CH 2 NH 2 , respectively, using a similar procedure.
  • the products were designated as PAle-5A (modified with mPEG-NH 2 ), PAle-5B (modified with InPEG-S-S-CH 2 CH 2 NH 2 ), PAph-5A (modified with HiPEG-NH 2 ) and PAph-5B (modified with InPEG-S-S-CH 2 CH 2 NH 2 ), respectively.
  • the polymer was synthesized using a procedure published elsewhere (Yue, Z., Eccleston, M.E., et al., 2005, Polymer, vol.46, pp.2497).
  • PEGylated PL (PL-5B).
  • PL was treated with mPEG-S-S-CH 2 CH 2 NH 2 using a procedure corresponding to the procedure described for PALe-5B.
  • 1 H-NMR (de-DMSO, ⁇ ppm): 0.7-2.0, 2.8-5.0, -NH 2 -CET 2 -, -GET-NH- and all side chain protons; 7.4-9.0, AJ-JET, -CONl?-.
  • polymer was dissolved in DMSO, to which was added equal volume of deionised water. The mixture was dialyzed against deionised water
  • Dox-HCl were dissolved in DMSO and neutralized by TEA, followed by a similar dialysis process as above but conducted at 277 K in darkness. A freeze-dried sample was weighted, re-dissolved in DMSO and its absorbance was measured at 485 nm on a UV-Vis spectrophotometer (UV-1201, Shimadzu). The amount of loaded drug and drug loading efficiency were determined colorimetrically against a standard curve of Dox at 485 nm.
  • the polymer was dissolved in methanol together with appropriate amount of the methanolic solution of pyrene (IxIO "3 M), prior to dilution with deionised water (1:1). The methanol was then removed by dialysis (Spectra/Por 7, MWCO 1 kDa) in darkness and the final concentration was adjusted to 1.0 mg mL "1 for polymer and 6xlO "7 M for pyrene. All stock solutions were allowed to equilibrate for 48 h in darkness, from which sample solutions were prepared with pHs ranging from 3 to 10. All samples were allowed to equilibrate for 1 h before recording spectra at room temperature.
  • the hydrodynamic diameters and ⁇ -potential of the polymeric vesicles were determined on a dynamic light scattering instrument (ZetaPALS, Brookhaven Instruments Corporation) at 298 K. Sample was filtered through 0.20 ⁇ m filter into pre-cleaned cylindrical cuvette before measurement. For transmission electron microscopy (TEM), sample was stained with 2% phosphotungstic acid (100:1), filtered with 0.20 ⁇ m filter, and loaded on carbon-coated copper grids. The grids were air dried and examined on a JOEL JEM-2000 FX electron microscope operating at an acceleration voltage of 80 keV.
  • TEM transmission electron microscopy
  • MCF-7 breast cancer cells MCF-7
  • human hepatocellular carcinoma cells HepG-2
  • Human cervical carcinoma cells HLa
  • DMEM Dulbecco's Modified Eagle's Medium
  • the cells were cultured at 310.15 K (37 ° C) in a humidified environment with 5% CO 2 .
  • Reduction of cellular metabolic activity was measured using MTT (3 -(4,5- Dimemylthiazol-2-yl)-2,5-di ⁇ henyltetrazolium bromide) assay. Exponentially growing cells were dispensed into a 96-well tissue culture plate (200 ⁇ L supplemented medium), and incubated overnight at 310.15 K (37 ° C) in a humid atmosphere containing 5% CO 2 . The growth medium was then replaced with 200 ⁇ L of filter sterilized polymer solutions and free
  • PAIe, PAph and PL were synthesized and PEGylated according to the procedures illustrated in Figures 2 and 3, respectively.
  • the influences of PEGylation on the solution behavior of PAIe and PAph were investigated using a hydrophobic fluorescent probe - pyrene.
  • Pyrene is known to partition preferentially into hydrophobic domains if present, and its photophysical properties are very sensitive to the polarity of its microenvironment. It can serve as a sensitive "reporter" on the hydrophobic associations of polymer chains.
  • the I 1 ZI 3 values for pyrene in aqueous PAIe are close to those in water, and remain fairly constant over the pH range studied.
  • the slow release of drug in PBS can be attributed to a gradient-controlled diffusion process that typically occurs in stable vesicles.
  • the GSH triggered release also depends on the core structure of vesicles.
  • PAle-5B and PAph-5B demonstrate ⁇ 8-9 folds' increase in the amounts of drug released, much higher than PL-5B. This might be due to their bulky side chains ( L -leucine for PAle-5B and ⁇ phenylalanine for PAph-5B) that prevent the formation of tightly aggregated cores.
  • the physicochemical properties of the cores of polymeric vesicles are critical to the kinetics of cleavage of disulfide linkage and subsequent core destabilization.
  • the intracellular uptake of nanocarriers was studied in human MCF-7 breast cancer cells (MCF-7) using confocal laser scanning microscopy. Free Dox is predominantly accumulated in cell nuclei ( Figure 9A-9C); whereas the nano-carrier encapsulated Dox are localized mostly in the perinuclear regions of cells after 1 - 2 h's incubation ( Figure 9D).
  • a bifunctional polyethylene glycol such as, but not limited to, polyethylene glycol bis-o-pyridyltbioester (OPTE-PEG- OPTE) can be employed as starting point for coupling one or more of the above mentioned additional regonition ligands to the system.
  • OPTE-PEG-OPTE polyethylene glycol bis-o-pyridyltbioester
  • only one end is modified to form l-aminoethyl-2-dithio for subsequent pegylation to the polymer backbone.
  • the other end of such conjugated bifunctional PEG is used for modification with a recognition ligand.
  • cysteamine hydrochloride (0.167 g, 1.3 mmol) was dissolved in ethanol (20 niL) and the solution was purged with N 2 for 1 A h.
  • OPTE-PEG-OPTE (5g, 2.0 mmol) in ethanol (50 mL) was added dropwise over a period of Vz h, after which the reaction was stirred under N 2 and at room temperature overnight.
  • the reaction mixture was concentrated and precipitated into diethyl ether.
  • the crude product was collected and dissolved in deionised water, prior to dialysis against deionised water (Spectra/Por 7, molecular weight cut-off (MWCO) 1 kDa) for 48 h.
  • MWCO molecular weight cut-off
  • the lyophilized powder was further purified by recyrstallisation from ethanol to obtain OPTE-PEG-S-SCH 2 CH 2 NH 2 .
  • Pegylation of PAIe, PAph and PL was then conducted using OPTE-PEG-S-SCH 2 CH 2 NH 2 , respectively, according to the similar procedure as describe for PAle-5B.
  • Incorporation of a recognition ligand can be achieved by reacting any recognition ligand containing -SH with the pegylated polymers in aqueous solutions at 4 0 C so that the OPTE may be substituted by the respective recognition ligand.

Abstract

La présente invention concerne une composition comprenant un polymère amphiphile pégylé, le polymère amphiphile pégylé étant constitué d’au moins un acide carboxylique polymérisable et d’au moins un acide aminé; le degré de pégylation étant adapté pour amener le polymère amphiphile forme une vésicule; la fraction polyéthylèneglycol étant liée à une liaison clivable; et la liaison clivable étant clivable par un stimulus externe pour provoquer un changement de conformation de la forme de vésicule du polymère en sa forme linéaire. La présente invention concerne également la fabrication de telles compositions et leur utilisation comme produit pharmaceutique et dans des échafaudages.
PCT/SG2009/000142 2008-04-17 2009-04-17 Vésicules pour l'administration intracellulaire de médicaments WO2009128789A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8993614B2 (en) 2012-03-15 2015-03-31 F. Hoffmann-La Roche Ag Substituted pyrrolidine-2-carboxamides

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060224095A1 (en) * 2005-04-05 2006-10-05 University Of New Hampshire Biocompatible polymeric vesicles self assembled from triblock copolymers
US20060269479A1 (en) * 2005-04-19 2006-11-30 Colton Clark K Amphiphilic polymers and methods of use thereof
EP1738770A1 (fr) * 2005-06-30 2007-01-03 CTT Cancer Targeting Technologies OY Procédé de synthèse de conjugués comprenant phospholipides, peg et une biomolécule

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060224095A1 (en) * 2005-04-05 2006-10-05 University Of New Hampshire Biocompatible polymeric vesicles self assembled from triblock copolymers
US20060269479A1 (en) * 2005-04-19 2006-11-30 Colton Clark K Amphiphilic polymers and methods of use thereof
EP1738770A1 (fr) * 2005-06-30 2007-01-03 CTT Cancer Targeting Technologies OY Procédé de synthèse de conjugués comprenant phospholipides, peg et une biomolécule

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8993614B2 (en) 2012-03-15 2015-03-31 F. Hoffmann-La Roche Ag Substituted pyrrolidine-2-carboxamides

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