WO2005087825A2 - Temperature sensitive polymers - Google Patents
Temperature sensitive polymers Download PDFInfo
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- WO2005087825A2 WO2005087825A2 PCT/NL2005/000203 NL2005000203W WO2005087825A2 WO 2005087825 A2 WO2005087825 A2 WO 2005087825A2 NL 2005000203 W NL2005000203 W NL 2005000203W WO 2005087825 A2 WO2005087825 A2 WO 2005087825A2
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
- C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
- C08F20/52—Amides or imides
- C08F20/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F20/56—Acrylamide; Methacrylamide
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- 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
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- 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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
- C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
- C08F20/52—Amides or imides
- C08F20/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F20/58—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-acryloylmorpholine
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/56—Acrylamide; Methacrylamide
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/58—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/44—Preparation of metal salts or ammonium salts
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
- C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
Definitions
- the invention relates to compositions comprising polymers whose solubility characteristics can be changed by incubation. Another aspect of this invention is the application of such temperature sensitive polymers as release systems of biologically active compounds.
- the invention relates to a novel class of polymers with tuneable thermosensitivity, which polymers are biodegradable forming degradation products that are either endogenous or non- toxic in the human or animal system.
- the fast developments in the field of molecular biology and biotechnology have made it possible to produce a large number of pharmaceutically interesting products in large quantities.
- pharmaceutically active peptides and proteins can suitably be used as drugs in the treatment of life-threatening diseases, e.g.
- the protein drugs should be directed to the sites where their activity is needed during a prolonged period of time. More generally, at present a large number of low molecular weight therapeutics are becoming available which may have unfavorable biopharmaceutical characteristics (low bioavailability; low dissolution characteristics; severe side effects; etc. Therefore new solubilization systems are urgently needed. Moreover, low molecular weight drugs, e.g. cytostatica such as paclitaxel, should be targeted towards specific sites in a body. Suitable drug targeting systems for targeted release are, e.g. micellar structures for release of low molecular weight drugs. Hence, there is a need for delivery systems which have the capacity for sustained, controlled and/or targetted release.
- cytostatica such as paclitaxel
- delivery systems comprising soluble polymers have been proposed.
- Such delivery systems can be obtained by using such soluble polymers for example in the form of microp articles in which the drug is encapsulated.
- the polymer can be present throughout each microparticle, with the drug captured within the different polymer molecules.
- the polymer forms the outer membrane of the microparticle which contains the drug.
- organic solvents have to be used to encapsulate drugs in the microp articles.
- acidic products are frequently formed during degradation, which might result in a lowering of the pH.
- Both a low pH and organic solvents can affect drug stability, especially if the drug is a protein.
- Temperature sensitive (or thermosensitive) polymers with a lower critical solution temperature are presently under investigation for biomedical and pharmaceutical applications.
- Thermosensitive polymers having a LCST are remarkable materials, in that below this temperature such polymers are soluble, and above it they precipitate.
- the lower critical solution temperature can be defined as the temperature at the point of inflection in a graph representing the amount of solids in the sample (for example as measured using light scattering techniques) vs. temperature.
- the LCST can be defined as the lowest temperature where precipitated polymer particles are detected (the 'onset' temperature).
- LCST is defined by the onset.
- Thermosensitive polymers with LCST are soluble in aqueous solutions below the cloud point (CP), but precipitate above this temperature due to the dehydration of the polymer chains.
- LCST-polymers can be used advantageously as drug release systems, because their preparation can be carried out at a temperature which is lower than the temperature at which the release is to be effected, for example the body temperature. Since the temperature can be kept low, there is little risk of denaturation or degradation of the (protein) drug to be released.
- LCST-polymers in drug release systems are used in drug release systems.
- the loading of the drug delivery system can be accomplished in an aqueous system, avoiding the use of toxic organic solvents.
- the LCST-polymers can be chosen such that they are degradable and/or can easily be excreted by the kidneys, once in soluble form.
- BACKGROUND OF THE STATE OF THE ART The use of LCST polymers as controlled release systems is e.g. known from US-A-5, 720,976. In this publication release systems are disclosed, wherein an active ingredient is encapsulated in liposomes. LCST polymers are grafted to the surface of liposomes.
- LCST value of the polymers can be adjusted.
- WO-A-92/07881 discloses that the solubility of polyacrylamide changes as a result of the presence of amide groups, which groups have a buffering effect. This pertains to the solubility per se, not to the LCST, which is not mentioned in this publication. Also in EP-A-0 693 508 and in DE-A-4 023 578, it is described that the temperature sensitivity of certain polymers can be influenced by varying the ratio of the comonomers present in these certain polymers.
- LCST polymer systems can be modified, as is done in accordance with the present invention in such a way, that the LCST value of the polymers changes during incubation and as a result of incubation, and by which the above mentioned advantages of the present invention can be obtained.
- a temperature sensitive polymer can be obtained by choosing a monomer that is suitable for the envisaged application, e.g. a monomer that forms a pharmaceutically acceptable polymer. Suitable monomers are the monomers selected from the group comprising ethylene glycol, lactic acid, acrylamide, methacrylamide, acrylic acid, and derivates and substituted species thereof.
- the change of solubility characteristics is effected by hydrolysis of a group, such as a lactate, present on at least one of the monomers that form the polymer.
- a group such as a lactate
- such a group can advantageously be an enzymatically or chemically hydrolyzable group.
- the ester groups are introduced in the polymer by choosing suitable monomers as a starting material.
- the monomers can be provided with ester groups by techniques known to the person skilled in the art.
- the preferred embodiment is based on poly(N- isopropylacrylamide) (P ⁇ IPAAm), which has its CP (in water) around 32 °C. It is the most extensively studied thermosensitive polymer and is used for the design of thermosensitive drug delivery systems such as polymeric micelles and hydrogels. This polymer has also been used to modify the surface properties of liposomes.
- the CP of P ⁇ IPAAm can be modulated by copolymerising with hydrophobic or hydrophilic comonomers: hydrophobic comonomers decrease the CP whereas hydrophilic comonomers have the opposite effect.
- thermosensitive polymers of WO 01/09198 are thermosensitive copolymers of ⁇ IPAAm and N-(2-hydroxypropyl) methacrylamide lactate (poly( ⁇ IPAAm-co-HPMAm-lactate)) and their block copolymers with polyethylene glycol) (poly(NIPAAm-co-HPMAm-lactate)-b-PEG).
- poly( ⁇ IPAAm-co-HPMAm-lactate) poly( ⁇ IPAAm-co-HPMAm-lactate)
- poly(NIPAAm-co-HPMAm-lactate)-b-PEG block copolymers with polyethylene glycol)
- the invention relates to a temperature sensitive polymer having a lower critical solution temperature that changes during incubation in an aqueous solution or medium, which polymer is a homo or interpolymer of a hydrophobically modified hydroxyalkyl (meth)acrylamide.
- the hydrophobical modification may in particular be effected by a hydrophobic unit, bound to the hydroxyalkyl (meth)acrylamide via a degradable bond (such as a lactate ester).
- a degradable bond such as a lactate ester
- P can be determined by mixing an amount of the polymer in a two- phase system of equal amounts of water and octanol, letting the system phase separate, measuring the equilibrium concentrations of the polymer in the water and the octanol and divide the concentration in water by de concentration in octanol.
- log P is reduced by at least 0.1.
- hydrophobic modification results in a reduction of the cloud point of the polymer, compared to the unmodified hydroxyalkyl (meth)acrylamide, to a cloud point of 37 °C or less.
- Suitable hydrophobic units include lactates, alkyl groups and aryl groups.
- the alkyl may be a linear, branched or cyclic alkyl.
- alkyl groups may have from 1 to 40 carbon atoms, in particular from 2 to 18 carbon atoms.
- alkyl groups include fatty acid ester residues.
- Suitable aryl groups include arylgroups having from 4-40 carbon atomes, in particular from 6 to 18 carbon atoms.
- the lactate may be a monolactate or an oligolactate.
- oligolactate in particular encompasses oligomers of lactic acid comprising 2-10 lactic acid residues.
- the alkyl in hydroxyalkyl (meth)acrylamide is preferably selected from the group consisting of methyl, ethyl, propyl, butyl, pentyl and hexyl.
- alkyls include all constitutional isomers of said alkyls (such as the n-alkyl and the isoalkyl).
- a relatively small alkyl, such as methyl or ethyl is in particular considered suitable for imparting a relatively low hydrophobicity of the polymer, and or relatively high cloud point (CP) (compared to propyl), whereas a relatively large alkyl (such as butyl, pentyl or hexyl) may have the opposite effect.
- the hydroxy alkyl may be a primary hydroxyl alkyl, a secondary hydroxyl alkyl or a tertiary hydroxyl alkyl.
- a hydrophobically modified polymer of primary hydroxyl alkyl usually has a higher hydrolysis rate than a comparable polymer of a secondary hydroxyl alkyl, which in turn usually has a higher hydrolysis rate than a comparable polymer of a tertiary hydroxyl alkyl.
- a polymer derived from a primary, secondary respectively tertiary hydroxyl alkyl may be preferred.
- the polymer is a homo or interpolymer of a N-(2-hydroxyalkyl) (meth)acrylamide modified with a hydrophobic unit, such as a lactate, an alkyl or an aryl.
- said polymer is selected from the group consisting of homopolymers and interpolymers of (hydrophobically modified N-(2-hydroxyethyl) methacrylamide), (hydrophobically modified N-(2-hydroxyethyl) acrylamide), (hydrophobically modified N-(2-hydroxypropyl) methacrylamide) and (hydrophobically modified N-(2-hydroxypropyl) acrylamide).
- the polymer is hydrophobically modified by a monolactate, a dilactate, a trilactate or a tetralactate, preferably a monolactate or a dilactate of the said hydroxyalkyl (meth)acrylamides.
- the polymer may be a copolymer or a blend of different hydrophobically modified hydroxyalkyl (meth)acrylamide polymers.
- the blend or copolymer is a blend respectively copolymer of at least two polymers respectively chemically bound polymer units according to the invention having a different number of lactate moieties.
- the presence of a lactate polymer other than the monolactate and dilactate i.e. the presence of trilactate, tetralactate or higher
- CP cloud point
- a tetralactate polymer according to the invention is considered particularly suitable.
- the amount of higher lactates may be chosen within wide limits. Good results have been achieved with a copolymer, in particular a pHEMAm-dilactaat, wherein at least 5 % of the monomers are other than mono- and dilactate. For good water solubility not more than 22 % of the monomers are other than mono- and dilactate.
- a polymer according to the invention with one or more lactate side chains of at least three lactic acid units (trilactate) , in particular of at least four lactic acid units (tetralactate) has been found suitable to provide micelles of the polymer with improved stability, compared to the analogous polymer comprising only mono-, or dilactate units.
- trimertate lactic acid units
- trimerate lactic acid units
- trimerate lactic acid units
- tetralactate lactic acid units
- a polymer according to the invention preferably has a lower critical solution temperature before incubation below mammalian body (i.e. core) temperature, more preferably below ambient temperature (in particular below 20 °C).
- the lower critical solution temperature after incubation is preferably above mammalian body temperature (i.e. core temperature).
- mammalian body temperature is human body temperature, i.e. above about 37 °C.
- the invention relates to a blend of polymers comprising one or more polymers according to the invention.
- Particularly suitable blends include a blend of hydroxyethyl (meth)acrylamide lactate and at least one other hydroxyalkyl (meth)acrylamide lactate respectively a blend of hydroxypropyl (meth)acrylamide lactate and at least one other hydroxyalkyl (meth)acrylamide lactate.
- the hydroxyethyl/propyl and/or alkyl moieties are N-(2- hydroxyethyl), N-(2-hydroxypropyl), respectively N-(2-hydroxy alkyl).
- the invention further provides a controlled release system comprising a temperature sensitive polymer according to any one of the preceding claims and an active ingredient, such as a drug.
- a drug include hydrophobic drugs with a low water-solubility.
- drugs include paclitaxel and other cytostatics, amphoteracin, corticosteroids, and photosensitizers.
- the controlled release system comprises the polymer according to the invention in the form of a polymeric micelle.
- the polymer usually comprises a hydrophilic block which preferably comprises a polyalkyleneglycol, more preferably a poly(ethyleneglycol).
- the number average molecular weight of the hydrophilic block is preferably in the range of about 500-10000 g/mol.
- the polymer capable of forming the micelle may be of the type AB, ABA or BAB (wherein A and B are respectively the hydrophilic and hydrophobic block)
- the controlled release system is in the form of a hydrogel.
- the polymer according to the invention is an ABA block copolymer or a BAB block copolymer, wherein block A is the temperature sensitive hydrophobically modified poly(hydroxyalkyl
- the invention further relates to a targeting drug composition, comprising a drug and particles of a controlled release system according to the invention, which particles preferably have a weight average diameter of less than 200 nm, more preferably in the range of 10 to 100 nm (as determined by dynamic light scattering)
- the targeting drug composition comprises a homing device.
- Fig. 1 shows a light scattering curve, wherein both the temperature at the point of inflection and the onset temperature are marked.
- Fig. 3 shows a light scattering intensity temperature curve for poly(HPMAm- monolactate-co-HPMAm-dilactate) in isotonic 120 mM ammonium acetate buffer
- Fig. 4 shows the Cloud Point (CP) of poly (HPMAm-monolactate-co-HPMAm- dilactate) as a function of the mole-% HPMAm-monolactate in the copolymer. • is
- Fig. 7 shows the CP of a copolymer of the invention (pHEMAm-lactate) as a function of the tetralactate content in the polymer
- Fig. 8 shows a CryoTEM picture of a micellar solution of a polymer according to the invention.
- Fig. 9 shows a plot for determining the cmc of a polymer according to the invention.
- Fig. 10 shows the effect of the concentration of a polymer according to the invention on the particle size of the micelles.
- Fig. 11 shows the particle size stability of PEG-b-p(HEMAm-dilactate) versus time
- Fig. 12 shows the particle size stability of another polymer according to the invention
- the drug delivery systems based on LCST-polymers can be prepared conveniently by introduction of the drug (such as a protein, a low molecular weight drug or another biologically active agent) into the polymer matrix. This is obtained by mixing the drug with the polymer, which is in dissolved state, for example because it is below its LCST. Subsequently, the mixture is brought in a state in which the polymer precipitates, for example by bringing it above its LCST, by which process the protein drug is captured within the precipitating polymer matrix, thus yielding a drug delivery system.
- the LCST-polymer to be applied is above its critical solubility temperature.
- polymers of which the CP's increase from below to above body temperature in time are very attractive materials, because e.g. the controlled release of drugs without thermal treatment is feasible using such polymers.
- the present invention provides a polymer that is suitable for use in a controlled release system. Consequently, this polymer can be applied as a controlled release system having all the aforementioned advantages.
- the present inventors have found that when certain water soluble polymers are chemically modified, their critical solution temperature will vary in situ, ⁇ iz. upon in ⁇ ivo or in vitro application in an aqueous environment. These changes are time dependent.
- incubation application in an aqueous environment, under conditions enabling the reactions that result in the change of critical temperature, for example as a result of hydrolysis, is referred to as incubation. It is also possible that the incubation is effected by enzymes present in the aqueous environment.
- the polymer of the present invention comprises monomers which have modifiable functionality. The functionality of the monomers can for example be modified by the presence of hydrolysable groups. The modification is effected by the incubation, leading to a change of the water solubility characteristics of the polymer.
- copolymers and terpolymers have the additional advantage that they provide an extra parameter affecting the final result, since different monomers, having different solubility characteristics, can be incorporated in one polymer, as to adjust the solubility characteristics (such as the solubility itself or the temperature dependency of the solubility) of the resulting copolymer.
- Copolymers and terpolymers thus form a preferred embodiment of the present invention.
- the polymer according to the present invention is suitably obtained by choosing the properties of the monomers such that upon incubation the functionality of the monomers changes and as a result the solubility and/or the temperature dependency of the solubility of the entire polymer, changes.
- the monomers are chosen so that their hydrophilicity changes upon incubation. As a result, the hydrophilicity of the entire polymer will change upon incubation. This will lead to a polymer with a different solubility and/or temperature dependency of the solubility.
- poly(NIPAAm-co-HPMAm-lactate)-6-PEGm as described in WO 01/09198 may show controlled instability at body temperature.
- PNIPAAm hydroxyalkyl (meth)acrylamide based polymers
- hydrolysable group may for instance be bound by a bond selected from esters, orthoesters, amides, carbonates, carbamates, anhydrides, ketals, and acetals, preferably by an ester bond.
- thermosensitive and biodegradable polymers which may be described as poly(hydroxyalkyl (meth)acrylamide lactate), wherein the number of lactates per hydroxyalkyl (meth) acrylamide is generally 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
- Such a polymer may be represented by the following formula of the monomeric units constituting the polymer:
- R is the alkyl (which may be linear or branched) and n the number of lactate moieties.
- the ester moiety may be coupled to the R at any position of the alkyl chain, Thus the position of the ester relative to the amide may be ⁇ , ⁇ , ⁇ , ⁇ , ⁇ (etc), or ⁇ position.
- polymers according to the invention are (homo)polymers of a (N-2-hydroxypropyl) methacrylamide lactate, (N-2- hydroxyethyl) methacrylamide lactate, (N-hydroxymethyl) methacrylamide lactate, (N-2-hydroxybutyl) methacrylamide lactate, (N-2-hydroxypentyl) methacrylamide lactate or (N-2-hydroxyhexyl) methacrylamide lactate.
- poly(N-(2-hydroxypropyl) methacrylamide mono/di lactate) poly(HPMAm-mono/di lactate)
- poly(N-(2-hydroxyethyl) methacrylamide mono/di lactate) poly(HEMAm-mono/di lactate))
- poly(N-(hydroxy methyl) methacrylamide mono/di lactate) poly(HMMAm-mono/di lactate)
- poly (N- (2- hydroxybutyl) methacrylamide mono/di lactate) poly(HBMAm-mono/di lactate)
- poly(N-(2-hydroxypentyl) methacrylamide mono/di lactate) poly(HPeMAm- mono/di lactate)) respectively poly(N-(2-hydroxyhexyl) methacrylamide mono/di lactate) (poly(HHMAm-mono/di lactate)).
- the term "interpolymer” refers to a polymer comprising at least two types of monomers, and hence encompasses copolymers, terpolymers, etc.
- the invention relates to homopolymers of HPMAm-dilactate, HEMAm- dilactate and other hydrophilic monomers such as HEMAm- monolactate, HPMAm-monolactate, HEMAm -(lactate) n , HPMAm- (lactate) n , wherein n is an integer from 3 to 10, in particular from 3 to 5; HPMAm; or hydroxy(C 1 - ⁇ alky l)methacry late.
- (meth)acrylamide-mono/dilactate such as poly(HPMAm-mono/dilactate) or poly(HEMAm-mono/dilactate), and a further hydrophilic monomer are suitable to be used.
- the cloud points (CP) of a poly(HPMAm-monolactate) and poly(HPMAm- dilactate) in water were 65 °C and 13 °C, respectively.
- the lower CP for poly(HPMAm-dilactate) is likely due the greater hydrophobicity of the dilactate side group over the monolactate side group.
- the present invention relates to a temperature sensitive polymer having a lower critical solution temperature that changes during incubation in an aqueous solution or medium, which polymer is a homo- or interpolymer of a (N-(hydroxyalkyl) methacrylamide lactate).
- said N- (hydroxyalkyl) methacrylamide lactate is the mono- or dilactate, more preferably the dilactate.
- the alkyl is preferably propyl or ethyl.
- the term "mono/dilactate" means that part of the monomers used in the polymers of the invention are in the monolactate form, and part or all of the monomers used in the polymers of the invention are in the dilactate form. Due to the hydrolysable lactic acid side groups the CP will increase in time with lactic acid, an endogenous compound, and the water-soluble poly(hydroxyalkyl (meth)acrylamide) (such as pHPMAm, pHEMAm) as degradation products.
- pHPMAm is a well-known non-toxic macromolecular carrier which is, among others, used for the development of polymeric prodrugs of cytostatic agents.
- a good biocompatibility of a polymer according to the invention is expected, in particular for poly(HPMAm-lactate) , especially because pHPMAm systems have recently entered into clinical trials.
- the polymer can be synthesized by starting from a mixture of the monomers and carrying out the polymerization reaction. It is also possible to first produce the polymer and subsequently functionalize it by coupling suitable groups.
- compositions according to the present invention comprise block copolymers or terpolymers, random copolymers or terpolymers, random copolymers and polymeric networks, all of which polymers can be grafted, and mixtures (blends) thereof.
- the solubility characteristics of the compositions according to the present invention will change upon incubation, for example when contacted with aqueous media, such as will be the case in in ⁇ i ⁇ o application.
- the polymers according to the present invention have a critical temperature for the composition as synthesized which is below body temperature and preferably below ambient temperature, ⁇ iz. between about 0 to 36°C, preferably between 0 and 20°C, and most preferably between 5 and 10°C.
- LCST crosses the normal human body temperature (which is typically 37°C) upon incubation so that the LCST before incubation is below 37°C, preferably below 20°C, and LCST after incubation is above 37°C, preferably above 38°C.
- a preferred embodiment of the present invention is the use of the temperature sensitive polymer in or as a controlled release system which further comprises an active ingredient. Such systems are for example suitable for the controlled administration of drugs, such as protein drugs.
- the controlled release system of the present invention can be used for the release of biologically active compounds, such as pharmaceutic compounds, e.g. pharmaceutically active peptides and proteins, genetic material e.g.
- Polymeric micelles can be formed by the synthesis of amphiphilic blockcopolymers, e.g. AB block copolymers of a polyalkylene glycol, such as PEG, and a hydrophobic or thermosensitive block. In aqueous solutions, these polymers form micelles with a size of around 20-100 nm similar to those of the method of G.S. Kwon, et al. Langmuir, 9 (1993), 945-949).
- the hydrophobic core of these micelles can be loaded with drugs, e.g. an anti-cancer agent, such as adriamycin or paclitaxel).
- thermosensitive polymer acts as hydrophilic part of the system (e.g. in AB blockcopolymers of NIPAA and styrene; cfr. S. Cammas, et al. Journal of Controlled Release, 48 (1997) 157-164).
- PNIPAA forms the hydrophobic part of the polymeric micelle (in block copolymers of poly(ethylene glycol) and poly(N-isopropylacrylamide); M.D.C. Topp, et al. Macromolecules, 30 (1997) 8518-8520).
- drug release can then be triggered by local hypothermia. Hypothermia is, however, not easily done or technically feasible for all tissues and organs, which limits the applicability of these systems.
- Such a hydrophilic block preferably comprises a polyalkylene glycol, in particular a poly(ethyleneglycol) (PEG).
- PEG poly(ethyleneglycol)
- the polymers of the present invention comprise all possible polymer architectures, such as (multi-)block copolymers (such as AB, ABA, ABAB, etc.) or graft copolymers, random copolymers or terpolymers, or polymeric networks; all of which may be grafted.
- AB blockcopolymers with a thermosensitive block A i.e. the block of the hydrophobically modified hydroxyalkyl (meth)acrylamide) polymer of the invention
- a water-soluble B block e.g. PEG or pHPMAm
- PEG with a Mw of about 500-10000 g/mol, in particular of about 1500-10000 g/mol is used for this purpose.
- PEG with a Mw of about 5000 g/mol is used to form a (PEG 5000) 2 -ABCPA macroinitiator.
- this initiator decomposes by heat, a PEG chain with one radical is formed.
- This radical subsequently initiates the polymerization of monomers (such as HPMAm-mono- and dilactate, as described hereinbelow), by which an AB block copolymer is formed.
- monomers such as HPMAm-mono- and dilactate, as described hereinbelow
- the block copolymers can be prepared by controlled radical polymerization techniques such as atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT) polymerization using macroinitiators, macro-RAFT-agents, or sequential monomer addition.
- ATRP atom transfer radical polymerization
- RAFT reversible addition-fragmentation chain transfer
- ABA block copolymers may be synthesized via any of the above mentioned techniques, e.g. the macroinitiator route by using instead of a monofunctional (i.e. ⁇ -methoxy) PEG or equivalent thereof, an ⁇ - ⁇ -hydroxyl derived macroinitiator, ⁇ iz.
- a polyester macroinitiator which has the ABCPA-groups alternating with PEG groups.
- this initiator decomposes by heat, PEG chains with two radicals are formed. These radicals subsequently initiate the polymerization of monomers (such as HPMAm-mono- and dilactate), by which an ABA block copolymer is formed.
- monomers such as HPMAm-mono- and dilactate
- the ABA block copolymers formed by this route will be soluble in water below the LCST.
- a phase separated system will be formed, wherein as a result of the choice of block copolymer architecture, a hydrogel will be obtained.
- This hydrogel will dissolve gradually when the LCST of block A increases to above 37°C, due to the hydrolysis of the groups present on the monomers of this block.
- These systems are especially suitable for immobilizing cells, which can be employed in biotechnology and tissue engineering. Like the other systems mentioned hereinabove, these hydrogel systems can also be used as matrix for controlled release of active ingredients, in particular pharmaceutical proteins.
- the ABA block copolymers - like the AB block copolymers — may also be prepared by other, conventional synthesis routes, as indicated above (e.g. by RAFT polymerization). In Examples 3 and 4 herein-below, the synthesis of AB and ABA block copolymers are illustrated.
- the controlled release system of the present invention may be in the form of a hydrogel.
- the hydrogel may comprise an ABA block copolymer wherein block A is a temperature sensitive polymer according to the invention and B is a hydrophilic polymer and preferably it is PEG.
- block A is a temperature sensitive polymer according to the invention
- B is a hydrophilic polymer and preferably it is PEG.
- Such ABA block copolymers and hydrogels have the advantages described above.
- the release system is made of particles, which particles have an average diameter of less than 1 ⁇ m, preferably less than 100 nm. To be of practical value, these particles will usually have to be larger than several nm, e.g. greater than 10 nm as determined by light scattering.
- the ratio of different monomers, and especially the mono/dilactate ratio which constitute the interpolymer of the invention, will influence the LCST and its development upon incubation.
- the optimal ratio of each of the monomers will consequently depend strongly on the materials used and the envisaged application. The optimal values can be determined experimentally, as will be illustrated in the Examples hereinafter.
- An important aspect of the present invention is the use of hydrolysable chemical groups in a temperature sensitive polymer in order to change said polymer's solution characteristics, specifically its critical solution temperature, more specifically its lower critical solution temperature (LCST).
- the controlled release systems of the present invention can be prepared by the synthesis of a water soluble polymer. This is e.g.
- step a) functionalizing a monomer with hydrolysable groups, b) optionally mixing of said monomer with at least one monomer of a different type in a suitable ratio using a suitable solvent in the presence of an initiator and/or a catalyst to form said polymer c) removing said solvent and dissolving the polymer, and d) optionally purify said polymer, such as by precipitation; in which process the functionalizing of the monomers of step a) is optionally carried out after step b) on the monomers as they are present in the polymer; and subsequently mixing said water soluble polymer with a releasable compound.
- Suitable initiators and catalysts are known in the art.
- a suitable initiator for step b) is ⁇ , ⁇ '-azoisobutyronitrile (AIBN).
- An example of a suitable catalyst for step a), (e.g. the grafting of HPMAm, HEMam or the like with lactide), is stannous octoate (SnOct 2 ).
- the polymer of the present invention comprises one or more hydrophobically modified hydroxyalkyl methacrylamide monomers.
- the monomers may be selected from monolactate, dilactate or higher lactate esters of the said monomers. With respect to the higher lactate, this is usually chosen in the range of 3 (trilactate) to 10 (decalactate).
- hydroxyalkyl methacrylamide such as HPMAm, HEMAm etc.
- the hydroxyalkyl methacrylamide can be synthesized based upon the technology as described by D. Oupicky et al. (DNA complexes with block and graft polymers of N-2-hydroxypropyl)methacrylamide and 2-
- All monomers that copolymerize with the hydrophobically modified hydroxyalkyl methacrylamide are suitable.
- these are acrylates, methacrylates, acrylamides, methacrylamides, N-vinyl-pyrrolidone, vinyllactates, vinylethers, etc.
- the amount of these comonomers that can be present will vary upon the specific monomers in question and is from 0-70 mole%, preferably from 1-50 mole%.
- the critical issue is the LCST behaviour, which should be maintained.
- a specific polymerization reaction giving the polymers of the invention is described hereinbelow in Example 1.
- the polymers of the present invention can be applied as release systems for a variety of compounds in different applications, such as enzymes, colorants or other additives in laundry applications, adhesives in glues, insecticides or nutrients in agricultural applications, etc. Also possible is the use for the entrapment of living cells for e.g. tissue engineering (see, in this respect, Lee K.Y. Mooney, D.J. Hydrogels for tissue engineering, Chemical Reviews 2001: 101. 1869-1879) Further possible applications are the topical administration polymers of the present invention loaded with active ingredients, e.g. for the treatment of burn wounds and ulcers. The polymers of the invention can also be used for the delivery of genetic material (DNA delivery).
- DNA delivery genetic material
- Neradovic et al Thermoresponsive polymeric micelles with controlled instability based on hydrolytically sensitive N-isopropylacrylamide copolymers. Macromolecules 34, 7589-7591, 2001) were dissolved at a concentration of 0.1 g/mL in 1,4-dioxane.
- the HPMAm-monolactate/HPMAm-dilactate ratios were 100/0, 75/25, 50/50, 25/75, 0/100 (moUrnd).
- ⁇ , ⁇ '-Azoisobutyronitrile (total amount of monomers/AIBN is around 40/1 (mol/mol)) was added as radical initiator and the polymerization was conducted at 70 °C for 24 h in a nitrogen atmosphere.
- the polymers were collected by centrifugation after precipitation in diethyl ether.
- the polymers were further purified by dissolving them in cold water, followed by filtration through a 0.22 ⁇ m filter. After freeze-drying, the products were characterized by 1 H NMR (solvent: CDC1 3 ) and gel permeation chromatography (GPC).
- GPC was done using Plgel 3 ⁇ m MIXED-D + Plgel 3 ⁇ m MIXED-E columns (Polymer Laboratories) and poly(ethylene glycol) standards.
- the eluent was DMF containing lOmM LiCl, the elution rate was 0.7 mL/min. and the temperature was 40 °C.
- the CP of the polymers was determined with static light scattering (SLS) using a Horiba Fluorolog ® fluorometer (650nm, at a 90 ° angle).
- the polymer concentration was varied between 0.1 mg/mL and 5 mg/mL.
- the scattering intensity was measured every 0.2 °C during heating and cooling (the heating/cooling rate was approximately 1 °C/min).
- Onsets on the X- axis, obtained by extrapolation of the intensity-temperature curves during heating to intensity zero were considered as the CP.
- the CP determinations were done at least two times and the deviations were smaller than 0.5 °C. The results of the Example are discussed herein-below.
- Poly(HPMAm- monolactate) has a rather high CP (65 °C in water, Table 1) whereas poly(HPMAm-dilactate) has a relatively low CP (13 °C in water, Table 1). This can be explained by the greater hydrophobicity of the dilactate side group over the monolactate side group.
- the CP of the copolymers linearly increased with mol % of HPMA-monolactate monomer ( Figure 4), meaning that the CP of the copolymers can be tailored by the copolymer composition.
- molecular weight of the polymers decreased as the ratio of HPMAm- dilactate increased (Table 1), the decrease of molecular weight is not the reason for the decrease of the CP.
- thermosensitive and biodegradable polymers of the invention have attractive features especially as materials for drug delivery and biomedical applications.
- the CP of the polymer can be tailored from 10 °C to 65 °C by the copolymer composition.
- the lactic acid side groups are removed by hydrolysis in time. This means that the polymer becomes more hydrophilic in time, which is associated with an increase in CP. Therefore, polymers can be designed which are initially in their precipitated form but which become soluble in time. Further, it is expected that the polymers possess a good biocompatibility.
- PEG5000-6-p(HPMAm-dilactate) 13600 block copolymers was studied.
- the polymer was dissolved at a concentration of lOmg in 1 ml isotonic 120mM ammonium acetate buffer with a pH of 5.0. The temperature was maintained at 0°C by ice-cooling. 1.8 ml of this polymer solution or of the buffer as a reference was cooled with ice. Next a 0.2 ml PTX solution in ethanol was added meanwhile stirring and ice cooling the solution. To sample A and sample B lOmg/ml PTX solution was added and to sample C 20 mg/ml. The volume ratio of PTX solution and polymer solution is 1:9. Thus 10% ethanol (v/v) is present in the mixture.
- p(HPMAm-dilactate)-6-PEG block copolymers (pHPMAmDL-6-PEG) were synthesized by radical polymerisation using HPMAm-dilactate as monomer and PEG2-ABCPA as macroinitiator essentially as described previously for the synthesis of block-copolymers of PEG 5000 and NIPAAm or NIPAAm-HPMAm(- lactate) (Neradovic D, Van Nostrum CF, and Hennink WE. Thermoresponsive polymeric micelles with controlled instability based on hydrolytically sensitive N- isopropylacrylamide copolymers. Macromolecules 34, 7589-7591, 2001) and schematically shown in scheme 1.
- pHPMAmDL-b-PEG Scheme 1 Synthesis route and structure of pHPMAmDL-b-PEG block copolymer.
- the macroinitiator (PEG 5000)2- ABCPA was synthesized as follows. A 50 mL round bottom flask was loaded with 2 g (0.4 mmol) polyethylene glycol 5000 monomethylether (PEG 5000), 0.056 g (0.2 mmol) 4,4-azobis(4-cyanopentanoic acid) (ABCPA), 0.0189 g (0.06 mmol) 4-(dimethylamino)-pyridinium-4-toluene- sulfonate (DPTS) and 0.125 g (0.6 mmol) N,N'-dicyclohexylcarbodiimide (DCC).
- PEG 5000 polyethylene glycol 5000 monomethylether
- ABSCPA 4,4-azobis(4-cyanopentanoic acid)
- DPTS 4-(dimethylamino)-pyri
- the flask was evacuated and filled with nitrogen. Next, 3 mL of 1:1 mixture of dichloromethane (stabilized with amylene) and dry DMF was added using a syringe. The mixture was stirred at room temperature for 24 hours. Next, the reaction mixture was filtered, the solid was washed with dichloromethane and the combined organic solutions were evaporated. Thereafter, the product was dissolved in toluene, remaining insoluble substances were removed by filtration, and the solvent was evaporated. The obtained dry product was extracted with diethyl ether to remove traces of dicyclohexyl urea (DCU). The product obtained was dissolved in water and the solution was filtered to remove remaining solid. The product was collected after freeze-drying (yield 80%).
- This macroinitiator is used for the synthesis of p(HPMAm-dilactate)-b-PEG block copolymers (pHPMAmDL-b-PEG).
- HPMAm-dilactate and PEG2-ABCPA were dissolved at a total concentration of 0.3 g/mL in acetonitrile.
- the ratio of monomer to macroinitiator was varied between 35/1 to 140/1 (mol/mol).
- the polymerization was conducted at 70 °C for 24 hours in a nitrogen atmosphere. The polymers were collected by centrifugation after precipitation in diethyl ether.
- the polymers were further purified by dissolving these in cold water, followed by filtration through a 0.22 ⁇ m filter and freeze-drying.
- the products were characterized by ⁇ NMR (solvent: CDCI3) with a Gemini 300 MHz spectrometer (Varian Associates Inc. NMR Instruments, Palo Alto, CA) and gel permeation chromatography (GPC).
- GPC gel permeation chromatography
- the eluent was DMF containing lOmM LiCl; the elution rate was 0.7 mL/min; and the temperature was 40 °C.
- the number average molecular weight (M n ) of pHPMAmDL block was determined by ⁇ -NMR as follows: a) the value of the integral of the PEG protons divided by 454 (average number of protons per one PEG 5000 chain) gave the integral value for one PEG proton and b) the number of HPMAmDL units in the polymers was determined from the ratio of the integral of the methine proton
- the CMT of block polymer solution was determined with static light scattering using a Horiba Fluorolog fluorometer (650 nm, at a 90 ° angle).
- the scattering intensity was measured every 0.2 °C during heating and cooling (the heating/cooling rate was approximately 1 °C/min). Onsets on the X-axis, obtained by extrapolation of the intensity-temperature curves during heating to intensity zero were considered as the CMT.
- the CMT determinations were done at least two times and the deviations were smaller than 0.5 °C.
- Micelles of block copolymers were formed by quickly heating an aqueous polymer solution from below to above CMT.
- the polymer solution was quickly brought from 0 °C to 50 °C and was left at 50 °C for 1 minute.
- the micelle solution was incubated at 37 °C. Size measurements of the micelles.
- Dynamic light scattering (DLS) measurements were done to determine the size of the micelles, using Malvern 4700 system (United Kingdom) consisting of an Autosizer 4700 Spectrometer, a pump/filter unit, a Model 2013 air-cooler Argon ion laser (75 mW, 488 nm, equipped with a model 2500 remote interface controller, Uniphase) and a computer with DLS software (PCS, version 3.15, Malvern).
- the measurement temperature was 37 °C and the measurement angle was 90°.
- the change in solvent viscosity with temperature was corrected by the software.
- the critical micelle concentration (cmc) of the different block copolymers was determined using pyrene as a fluorescence probe.
- the micelle solutions with different polymer concentrations ranging from to 0.00001 mg/mL to 1.0 mg/ml were obtained by diluting the polymer solution with the same buffer at room temperature.
- Hydrogel forming ABA block copolymers of p(HPMAm-dilactate) (A-block) and PEG (b-block) were obtained using the same synthetic strategy as described for the synthesis of AB blockcopolymer (scheme 1).
- A-block p(HPMAm-dilactate)
- PEG b-block
- This initiator was synthesized by reaction of normal PEG (instead of monomethoxy PEG) with ABCAPA.
- ABA triblockcopolymers of of p(HPMAm-dilactate) (A-block) and PEG (B-block) were obtained as described for the micelle forming AB block copolymers (p(HPMAm-dilactate)-b-PEG block copolymers).
- the hydrogel forming properties of the different ABA blockcopolymers were studied using rheological analysis.
- thermosensitive polymeric micelles based on PEG-block- poly(2-hydroxyethyl methacrylamide-lactate) were made based on the same methodology as described above.
- the oligolactate esters of 2-hydroxyethyl methacrylamide were obtained by ring-opening oligomerization of L-lactide, using HEMAm as the initiator and stannous octoate as a catalyst, essentially as described by Van Dijk et al. [Polymer 38 (1997), 6235-6242]. Briefly, L-lactide (33.5 g; 0.233 mol) and HEMAm (20 g; 0.155 mol) were stirred at 110°C until the lactide was molten. 4-Methoxyphenol ( ⁇ 0.1 mol % relative to HEMAm) was added as radical scavenger.
- HEMAm-Lact HEMAm-Lac2
- HEMAm-Lac 4 HEMAm- Lac3
- HPLC HPLC
- HEMAm-oligolactates were conducted as described by Neradovic et al [Macromolecules; 2003; 36(20); 7491-7498].
- a 10 mM solution of HEMAm-oligolactate in DMSO was diluted 10 times with 100 mM PBS (pH 7.4) in a glass vial and the pH was adjusted to pH 7.4 with 4 M HC1.
- the resulting solutions of HEMAm-monolactate, -dilactate, -trilactate and - tetralactate were incubated in a shaking water bath at 37 °C.
- HEMAm-Laci Concentrations of HEMAm-Laci to HEMAm- Lac 4 were determined by the HPLC method described above. From the concentration versus time plots, the half-lives (tvs) were determined. Stock solutions in DMSO were diluted ten times in PBS buffer to solubilize the oligolactates. Therefore, the reported half life times are expected to be about twice as high in 100 % water, as discussed by Neradovic et al. [Macromolecules 36 (2003), 7491-7498]. The half life times of the prepared HEMAm-Laci and HEMAm-Lac2 are 58 and 5.6 hours respectively.
- HEMA methacrylate analogue of the HEMAm-lactates i.e. N-(2- hydroxyethyl)methacrylate (HEMA) mono- and dilactate
- HEMA N-(2- hydroxyethyl)methacrylate
- HPMAm-monolactate and HPMAm-dilactate are respectively 87.5 and 15.4 hours.
- the HEMAm-lactate offers the possibility to provide micelles with a shorter half-life than the analogous HPMAm-lactate, but a higher half-life than the analogous HEMA-lactate.
- HEMAm derivatives with three and four lactic acid units (HEMAm-Lac3 and HEMAm-Lac 4 ) display even faster hydrolysis kinetics than HEMAm-Lac ⁇ -2.
- HEMAm-Lac3 and HEMAm-Lac 4 display even faster hydrolysis kinetics than HEMAm-Lac ⁇ -2.
- the (co)polymers were synthesized via free radical polymerization in airtight screw-cap glass vials.
- a 200 mg/ml monomer solution total volume approximately 1 ml dioxane
- Both homopolymers (HEMAm, HEMAm-Lacn) and copolymers made from mixtures of HEMAm-Lac2 and HEMAm-Lac ) were synthesized.
- a nitrogen flow was led through the solution for at least 10 minutes.
- the polymerization was conducted at 70 °C for 24 hours while stirring the solution.
- a Plgel 3 ⁇ m MIXED-D column (Polymer Laboratories) was utilized at a Waters System (Waters Associates Inc., Milford, MA, USA) with a differential refractometer Model 410.
- Poly(ethyleneglycol) of defined molecular weights were used as standards.
- the eluent was DMF containing lOmM LiCl.
- the samples were dissolved overnight at a concentration of 5 mg/ml in the eluent and prior to analysis filtered through a 0,45 ⁇ m filter.
- the elution rate was 0.7 ml/min and the temperature was 40 °C.
- Aqueous GPC was performed on the same system with 5 mM ammonium acetate buffer (pH 5.5), PL 8 ⁇ m aquagel OH column (Polymer Laboratories) and dextran standards. Peak areas were determined with Empower Software Version 1154 (Waters Associates Inc).
- Table 5 summarizes the results of all homopolymerizations.
- HEMAm was almost quantitatively converted.
- the DMF solution for GPC analysis however was slightly cloudy and the filtration through 0.45 ⁇ m filter was difficult.
- the product did however fully dissolve in water and was therefore analyzed by aqueous GPC.
- This analysis gave a monomodal distribution with an average molecular weight of 194000 g/mol and a polydispersity of 22.
- the pHEMAm-oligolactates were obtained in a constant high yield (around 80%).
- the thermosensitive properties of the polymers were investigated by static light scattering. To prevent hydrolysis, a pH 5 buffer was used. pHEMAm-Lac3 and pHEMAm-Lac 4 did not dissolve after overnight incubation at 0°, suggesting a cloud point below 0°C.
- the homopolymers of HEMAm and its monolactate derivative did not show any scattering up to 75°C.
- the homopolymer of pHEMAm-Lac2 displayed its CP at 21.7 °C.
- the copolymers of HEMAm-Lac2 and HEMAm-Lac 4 were synthesized with monomer to AIBN ratio of 100:1. Table 6 summarizes their characteristics. The yields and molecular weights were comparable with the homopolymers.
- the copolymer composition corresponds with the feed ratio.
- the CP behavior of these copolymers (figure 7) showed that the amount of hydrophobic HEMAm-Lac 4 incorporated linearly influenced the CP. From this curve, it was predicted that a copolymer with 22% HEMAm-Lac or more would not dissolve at 0°. This was experimentally confirmed (Table 6).
- Block copolymers with HEMAm-Lacn as thermosensitive block and PEG as hydrophilic block were prepared via the macroinitiator route as described by Neradovic et al. [Macromolecules, 2001, 34; 7589-7591].
- Poly(ethyleneglycol) (PEG) ⁇ ooo was chosen to be the hydrophilic part of the blockcopolymer as this polymer favor longer circulation time of nanoparticles drug carriers and lower uptake by the RES.
- a block copolymer of PEG and HEMAm-Lac2 as well as block copolymers with twenty percent HEMAm-Lac and eighty percent HEMAm-Lac 2 with varying monomer to initiator ratios were synthesized. The latter polymers contained HEMAm-Lac to obtain a polymer with a CP just above 0°C. Table 7 summarizes the characteristics of the obtained blockcopolymers.
- the critical micelle concentration was determined with pyrene as a fluorescent probe [see example 3].
- the cmc was determined from the plot of the intensity ratio I338/T333 as a function of the concentration of block copolymer (figure 9).
- the cmc was determined to be 0.08 mg/ml, which is low enough for systemic administration in vivo.
- the particle sizes of micelles prepared from polymer solutions at various concentrations above the cmc are shown in figure 10. Relative large and polydisperse micelles are formed at concentrations below 0.5mg/ml which is close to the cmc.
- the particle size were relatively small (70 nm) with a low polydispersity. 2mg/ml polymer solutions were used for further measurements as this gave the lowest PD. pH dependent stability of the micelles
- the PEG-b-pHPMAm-dilactate micelles (see example 3) dissolved after approximately one week incubation at physiological conditions (aqueous buffer pH 7.4, 37 °C). The stability of the micelles of PEG-b-pHEMAm-lac 2 was followed by DLS measurements during incubation in buffer pH 5 at 37°C to slow down hydrolysis. Under these conditions, the micellar particle size gradually increased in time (figure 11).
- thermosensitive (block co) polymers of HEMAm-oligolactates were synthesized in high yields by free radical polymerization. It is possible to accurately tailor the cloud point by adjusting the copolymer composition of the poly(HEMAm-oligolactates).
- the increase in hydrophobicity of the thermosensitive block (poly(HEMAm-Lac2)) influenced not only the CP but also the micellar particle size and stability. Twenty percent of HEMAm-Lac was sufficient to increase the hydrophobicity sufficiently to produce highly stable micelles.
- micellar drug carrier An important issue determining the effectiveness of a micellar drug carrier is the ability to control the time over which drug release takes place, which can be done by a (micellar) drug release system according to the invention This is advantageous over nondegradable micellar systems as described in the prior art (e.g. PEG-poly(glutamic acid) [Kataoka J Contr Release 2005, p 223].
- drug release is only mediated by diffusion, which is a slow process and difficult to control.
- the thermosensitive polymeric micelles described here have the advantage over nondegradable micelles or liposomes by their ability to destabilize after an induction period that can be tailored by selecting the building block of the polymer quantitatively and/or qualitatively.
- the HEMAm-lactate polymer is capable of providing a micellar system that is stable for approximately 3 hours and thereby controlling the release of encapsulated drugs.
- the degradation products are expected to be bioresorbable, i.e. degradable with elimination from the human body.
- the remaining polymers Mw ⁇ 50000
- the remaining polymers will usually not exhibit toxicity caused by long-term accumulation because it will be excreted by glomerular filtration [Delgado C, Francis GE, Fisher D 1992, The uses and properties of PEG-linked proteins. Crit Rev Ther Carrier Syst 9: 249-304].
- the unique profile of micelle destabilization may be advantageous for in vivo use because the observed induction period is just long enough to allow accumulation of the micelles at the site of e.g. a tumor.
Abstract
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- 2005-03-17 WO PCT/NL2005/000203 patent/WO2005087825A2/en active Application Filing
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WO2001009198A1 (en) * | 1999-07-30 | 2001-02-08 | Stichting Voor De Technische Wetenschappen | Temperature sensitive polymers |
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CA2560468C (en) | 2013-01-08 |
ES2338665T3 (en) | 2010-05-11 |
US20120015034A1 (en) | 2012-01-19 |
EP1776400A2 (en) | 2007-04-25 |
WO2005087825A3 (en) | 2008-01-03 |
US7425581B2 (en) | 2008-09-16 |
CA2560468A1 (en) | 2005-09-22 |
US8685382B2 (en) | 2014-04-01 |
ATE452916T1 (en) | 2010-01-15 |
US20080050435A1 (en) | 2008-02-28 |
US20040247670A1 (en) | 2004-12-09 |
DE602005018489D1 (en) | 2010-02-04 |
EP1776400B1 (en) | 2009-12-23 |
US8110220B2 (en) | 2012-02-07 |
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