EP1214361A1 - Polymeres sensibles a la temperature - Google Patents

Polymeres sensibles a la temperature

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Publication number
EP1214361A1
EP1214361A1 EP00950086A EP00950086A EP1214361A1 EP 1214361 A1 EP1214361 A1 EP 1214361A1 EP 00950086 A EP00950086 A EP 00950086A EP 00950086 A EP00950086 A EP 00950086A EP 1214361 A1 EP1214361 A1 EP 1214361A1
Authority
EP
European Patent Office
Prior art keywords
polymer
lactate
temperature
polymers
lcst
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00950086A
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German (de)
English (en)
Inventor
Wilhelmus Everardus Hennink
Dragana Mulders-Neradovic
Cornelis Franciscus Van Nostrum
Wouter Leonardus Joseph Hinrichs
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Rijksuniversiteit Utrecht
Stichting voor de Technische Wetenschappen STW
Original Assignee
Rijksuniversiteit Utrecht
Stichting voor de Technische Wetenschappen STW
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Priority to EP00950086A priority Critical patent/EP1214361A1/fr
Publication of EP1214361A1 publication Critical patent/EP1214361A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/44—Preparation of metal salts or ammonium salts
    • 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
    • C08F220/10—Esters
    • C08F220/20—Esters of polyhydric alcohols or phenols, e.g. 2-hydroxyethyl (meth)acrylate or glycerol mono-(meth)acrylate
    • 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
    • C08F220/10—Esters
    • C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
    • C08F220/28—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety
    • C08F220/283—Esters containing oxygen in addition to the carboxy oxygen containing no aromatic rings in the alcohol moiety and containing one or more carboxylic moiety in the chain, e.g. acetoacetoxyethyl(meth)acrylate
    • 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

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.
  • pharmaceutically active peptides and proteins can suitably be used as drugs in the treatment of life-threatening diseases, e.g. cancer, and of several types of viral, bacterial and parasital diseases; in the treatment of e.g. diabetes; in vaccines, e.g. for prophylactic aims; and for anti-conception purposes.
  • Especially the specialized biological activities of these types of drugs provide tremendous advantages over other types of pharmaceutics.
  • low molecular weight pharmaceuticals such as cytostatics, antibiotics, etc., can be produced in large amounts.
  • cytokines such as interleukines, interferons, tumor necrosis factor (TNF), insulin, proteins for use in vaccines, and growth hormones.
  • proteins and proteinaceous products including peptides, which group of products will be referred to as protein drugs herein- below. cannot be administered orally. These products tend to degrade rapidly in the gastro-intestinal tract, in particular because of the acidic environment and the presence of proteolytic enzymes therein.
  • protein drugs are not able to pass endothelial and epithelial barriers, due to their size and, generally, polar character.
  • protein drugs have to be brought in the system parenterally, i.e. by injection, however, the pharmacokinetical profile of these products is such that injection of the product per se requires a frequent administration.
  • proteinaceous material is eliminated from the blood circulation within minutes.
  • the therapeutic efficacy is strongly dependent on effective delivery, e.g. intra- or peritumoral.
  • the protein drugs should be directed to the sites where their activity is needed during a prolonged period of time.
  • the present inventors have now found that the use of temperature sensitive polymers, and especially those with a lower critical solution temperature, have a number of advantages.
  • Temperature sensitive polymers with a lower critical solution temperature 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).
  • An example of a hght scattering curve is shown in Figure 1. Both the temperature at the point of inflection and the onset temperature are marked.
  • 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 httle risk of denaturation or degradation of the (protein) drug to be released.
  • Another important advantage of the use of LCST-polymers in drug release systems is that 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.
  • the use of LCST polymers as controlled release systems is e.g.
  • 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.
  • the LCST-polymer systems of the present invention can be used for drugs-targeting by incorporation into the matrix of compounds which make the system suitable for physico-chemical or physical homing strategies.
  • Such strategies employ a homing device, which is a characteristic protrusion on a particle, capable of recognizing the target cell or tissue.
  • homing devices are monoclonal antibodies or fragments thereof, growth factor, insuline, sugar moieties, transferin, etc.
  • homing devices are designed such that they only recognize and interact with specific structures on the surface of target cells or tissues.
  • homing devices are designed to accumulate at target sites by physical means, such as a local magnetic field or heat. See for example D.J.A. Crommelin et al, Adv. Drug. Deliv. Rev. 17 (1995) pp. 49-60.
  • the protein drug delivery systems based on LCST-polymers can be prepared conveniently by introduction of the protein drug into the polymer matrix. This is obtained by mixing the protein 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.
  • it is essential that the LCST-polymer to be apphed is not below or above its critical solubility temperature. Effective apphcation as controlled release system can only be obtained when the in vivo temperature is just below the critical solution temperature.
  • 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, viz. upon in vivo or in vitro apphcation in an aqueous environment. These changes are time dependent.
  • apphcation 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 functionahty.
  • 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, terpolymers and other interpolymers are to be understood.
  • 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 obtained by choosing the properties of the monomers such that upon incubation the functionahty 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.
  • the hydrophilicity of the entire polymer will change upon incubation.
  • a temperature sensitive polymer can be obtained according to the present invention 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.
  • Preferred monomers are iV-isopropyl acrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), 2-hydroxyethyl aery late (HEA), acrylamide (AAm), glyceryl methacrylate or glycidyl methacrylate (GMA), glyceryl aery late or glycidyl acrylate (GA), hydroxypropyl methacrylamide (HPMAAm), dimethyl-aminoethyl methacrylate (DMAEMA) and dimethylaminoethyl acrylate (DMAEA).
  • NIPAAm 2-hydroxyethyl methacrylate
  • HEMA 2-hydroxyethyl methacrylate
  • HOA 2-hydroxyethyl aery late
  • AAm acrylamide
  • GMA glyceryl methacrylate or glycidyl methacrylate
  • GMA glyceryl aery late or glycidyl acrylate
  • the change of solubility characteristics is effected by hydrolysis of a group present on at least one of the monomers that form the polymer.
  • a group is preferably chosen from ester, amide, carbonate, carbamate, and anhydride groups.
  • a group comprises a lactate unit, such as a monolactate, a dilactate or an oligolactate group.
  • a group can advantageously be an enzymatically or chemically hydrolizable group.
  • the ester groups are introduced in the polymer by choosing suitable monomers as a starting material, such as 2-hydroxyethyl methacrylate-monolactate.
  • the monomers can be provided with ester groups by techniques known to the person skilled in the art.
  • 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 adding 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 thereof.
  • 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 vivo apphcation.
  • this critical temperature is preferably between 0 to 100°C before incubation to form a polymer having a critical solution temperature that is within the same range.
  • the polymers according to the present invention have a critical temperature for both the composition as synthesized and the composition after incubation which is around body temperature, viz. between about 20 to 45°C, preferably between 30 and 42°C, and most preferably between 36 and 38°C.
  • body temperature viz. between about 20 to 45°C, preferably between 30 and 42°C, and most preferably between 36 and 38°C.
  • the value of 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 35°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 polymer in or as a controlled release system. For example 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. nucleotides, plasmid DNA. anti-sense ohgonucleotides, nutrients, etc.
  • biologically active compounds such as pharmaceutic compounds, e.g. pharmaceutically active peptides and proteins, genetic material e.g. nucleotides, plasmid DNA. anti-sense ohgonucleotides, nutrients, etc.
  • the LCST polymer preferably comprises a cationic group, such as DMAEMA.
  • Polymeric micelles can be formed by the synthesis of amphiphilic blockcopolymers, e.g. AB block copolymers of PEG and poly( ⁇ -benzyl-L-aspartic acid) (G.S. Kwon, M. Naito, M. Yokoyama, T. Okana, Y. Sakurai and K. Kataoka, Pharm. Res. 12 (1995) pp. 192-195). In aqueous solutions, these polymers form micelles with a size of around 20 nm (G.S. Kwon, M. Naito, M. Yokoyama, T.
  • the hydrophobic core of these micelles can be loaded with drugs, e.g. the anti-cancer agent adriamycin. After in vivo administration of the these systems the adriamycin loaded micelles selectively accumulate in certain tumors, simultaneously releasing the drug, which results in killing of tumor cells (M. Yokoyama, S. Fukushima, R. Uehara, K. Okamoto, K. Kataoka, Y. Sakurai and T. Okano, Journal of Controlled Release, 50 (1998) pp. 79-92).
  • drugs e.g. the anti-cancer agent adriamycin
  • thermosensitive polymer acts as hydrophilic part of the system (e.g. in AB blockcopolymers of V-isopropyl acrylamide and styrene; S. Cammas, K. Suzuki, C. Sone, Y. Sakurai, K. Kataoka, and T. Okano, Journal of Controlled Release, 48 (1997) pp. 157-164).
  • PNIPAAm forms the hydrophobic part of the polymeric micelle (in block copolymers of poly (ethylene glycol)) and poly(N- isopropylacrylamide; M.D.C. Topp, P.J.
  • 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 poly(ethyleneglycol) (PEG).
  • PEG poly(ethyleneglycol)
  • the LCST of the thermosenstitive block is initially below body temperature, polymeric micelles are formed at 37°C. Due to hydrolysis of the side groups present in the thermosensitive block of the system, the LCST will increase, resulting in destabilization of the micelle when the LCST passes 37°C.
  • a drug is incorporated in the hydrophobic core, its release will be affected by this process.
  • These systems can be favorably applied in e.g. cancer treatment, treatment of rheumatism, arthritis, infections and/or inflammations.
  • 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 a polymeric networks; all of which may be grafted.
  • polymer architectures such as (multi-)block copolymers (such as AB, ABA, ABAB, etc.) or graft copolymers, random copolymers or terpolymers, or a polymeric networks; all of which may be grafted.
  • thermosensitive block A e.g. NIPAAMm copolymerized with a comonomer with hydrolyzable side groups
  • watersoluble B block e.g. PEG
  • these polymers are prepared using a so called macroinitiator.
  • PEG with a Mw of about 1500-6000, is used for this purpose.
  • PEG with a Mw of about 5000 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 NIPAAm/HPMA-lactate, as described hereinbelow), by which an AB block copolymer is formed.
  • monomers such as NIPAAm/HPMA-lactate, as described hereinbelow
  • Such polymers form a micellar structure when the temperature rises above its LCST.
  • These micelles destabilize when the hydrolysis results in a A block with an increased LCST (above the temperature at which the micelles are apphed, preferably at body temperature).
  • the ratio of NIPAAm / HPMAm-lactate is preferably from 5-80, most preferably from 20-50.
  • ABA block copolymers may be synthesized via the macroinitiator route by using instead of a monofunctional (i.e. -methoxy) PEG or equivalent thereof, an ⁇ - ⁇ -hydroxyl derived macroinitiator, viz. a macroinitiator which has the ABCPA-groups at both ends of the molecule.
  • an ⁇ - ⁇ -hydroxyl derived macroinitiator viz. a macroinitiator which has the ABCPA-groups at both ends of the molecule.
  • this initiator decomposes by heat, a PEG chain with two radicals is formed. These radicals subsequently initiate the polymerization of monomers (such as NIPAAm/HPMA-lactate), by which an ABA block copolymer is formed.
  • monomers such as NIPAAm/HPMA-lactate
  • ABA block copolymers like the AB block copolymers - may be prepared by other, conventional synthesis routes as well.
  • 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.
  • the polymer used in the present invention is for example a terpolymer of N-isopropylacrylamide (NIPAAm), HEMA-monolactate and acrylamide (AAm) in which the respective NIPAAm/HEMA-monolacatate and AAm monomer ratios are chosen to be for example 50/20/30. It will be understood that the ratio of different monomers which constitute the copolymer or terpolymer. will influence the LCST and its development upon incubation. Generally for practical apphcation, e.g. apphcation in mammals, it is desirable to choose the ratios such that the LCST before incubation is below body temperature and after incubation above body temperature. 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).
  • LCST critical solution temperature
  • the effect of the incubation can be an increase as well as a decrease of the critical temperature upon incubation.
  • the controlled release systems of the present invention can be prepared by the synthesis of a water soluble polymer. This is done by a) functionalizing a monomer with hydrolysable groups, b) 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) precipitating said polymer; 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 as well as the catalysts for step a) are known in the art.
  • An example of a suitable initiator is ⁇ , ⁇ '-azoisobutyronitrile (AIBN).
  • An example of a suitable catalyst is stannous octoate (SnOct 2 ).
  • the polymers of the present invention can be applied as release systems for a variety of compounds in different apphcations, such as enzymes, colorants or other additives in laundry applications, adhesives in glues, insecticides or nutrients in agricultural applications, etc. Further possible applications are the topical administration polymers of the present invention loaded with active ingredients, e.g. for the treatment of burns.
  • the polymers of the invention can also be used for the delivery of genetic material (DNA delivery).
  • 2-(Methacryloyloxy)ethyl-mono- or -oligolactate which is HEMA esterified with one or more lactic acid groups, was synthesized using HEMA and L-lactide at a molar ratio of 2 to 1, essentially as described in Van Dijk- Wolthuis, W.N.E., Tsang, S.K.Y., Kettenes-van den Bosch, J.J. and Hennink W.E., A New Class of Polymerizable Dextrans with Hydrolyzable Groups: Hydroxyethyl Methacrylated Dextran With and Without Ohgolactate Spacer', Polymer, 38, 6235-6242, (1997).
  • NIPAAm iV-isopropylacrylamide
  • HEMA- monolactate iV-isopropylacrylamide
  • NIPAAm/HEMA-monolactate ratios 100/0, 95/5, 90/10, 80/20, 65/35 and 50/50 (mol/mol), the total monomer concentration being 0.1 g/cm 3 in 1,4-dioxane.
  • the copolymerization was conducted at 60°C for 20 hours in a nitrogen atmosphere.
  • Polymers without the monolactate group on the HEMA monomer i.e. poly(7V-isopropylacrylamide-co-2-hydroxyethyl methacrylate), were prepared to be used as a reference, starting from NIPAAm and 2-hydroxyethyl methacrylate (HEMA) in ratios NIPAAm/HEMA of 100/0, 95/5, 90/10, 80/20 and 60/40.
  • NIPAAm and 2-hydroxyethyl methacrylate HEMA
  • PBS phosphate buffered saline
  • the samples were filtrated (FP 030/3, Disposable Filter Holder, 0.2 mm; Schleicher & Schuell GmbH, Dassel, Germany) and then dialyzed against water at 4°C (Dialysis Tubing- Visking, Size 9 Inf Dia 36/32"-28.6 mm: 30 M, MWCO-12-14000 Daltons; MEDICELL International Ltd., London, Great Britain).
  • the hydrolyzed polymers were collected after freeze-drying.
  • NIPAAm V-isopropylacrylamide
  • HEMA-monolactate HEMA-monolactate
  • acrylamide AAm
  • NIPAAm NIPAAm/HEMA-monolactate
  • AAm AAm ratios of 70/20/10, 60/20/10/ and 50/20/30 (mol/mol/mol), the total monomer concentration being 0.1 g/cm 3 in 1,4-dioxane (volume 5 ml).
  • AIBN was used as initiator (250 mol monomers/1 mol initiator).
  • the copolymerization was conducted at 60°C for 20 hours in a nitrogen atmosphere.
  • the terpolymer prepared with the 70/20/10 comonomer ratio was isolated as described for the NIPAAm/HEMA-monolactate polymers (Example 1).
  • the other terpolymers were isolated as follows. To the polymer solution, 20 ml water was added and the resulting mixture was dialyzed against water at 4°C. The polymers were collected after freeze-drying.
  • NIPAAm/HEMA AAm terpolymers Polymers without the monolactate group on the HEMA monomer, i.e. NIPAAm/HEMA AAm terpolymers, were prepared to be used as a reference, starting from NIPAAm, HEMA and AAm in ratios NIPAAm/HEMA/AAm of 70/20/10, 60/20/20 and 50/20/30 (mol/mol/mol). The synthesis was carried out as in Example 3 with a total monomer/AIBN ratio of 125/1 mol mol). The polymers were isolated after dialysis and freeze drying as described for the polymers in Example 3.
  • Table 1 shows that it is possible to synthesize polymers that have an LCST below body temperature, but have an LCST above this temperature after hydrolysis of the side groups. This means that the polymer is initially insoluble in water at 37°C, however, the polymer gradually start to dissolve once (part of) the side groups are hydrolyzed.
  • Poly(NIPAAm-co-glycidylmethacrylate) was synthesized as in the previous Examples using different ratios of the NIPAAm and glycidylmethacrylate monomer.
  • the epoxy group of the glycidylmethacrylate can be hydrolized to the corresponding diol group, yielding a glycerylmethacrylate functional unit. Hydrolysis was performed using an aqueous solution of sulfuric acid. The results are summarized in Table 2.
  • HPMAm N-(2-hydroxypropyl) methacrylamide esterified with lactate groups (HPAAm-lactate) was synthesized essentially as HEMA-lactate described in Example 1.
  • a polymerization inhibitor (4 methoxyphenol, 1 mol-% with respect to HPMAm) was added. The resulting mixture was stirred for one hour at and 110°C and thereafter allowed to cool to room temperature.
  • HPMAm Essentially pure HPMAm with one lactate group (HPMAm - (lactate)i) and two lactate groups (HPMAm-(lactate)2) were obtained from the crude reaction mixture by preparative column chromatography (Econosphere C8, 10 ⁇ m, 250x22 mm, Alltech, IL) essentially as described for the purification HEMA-lactate using an ActaTM purifier system in Example 1. Samples enriched in either HPMAm-(lactate) 1 or HPMAm- (lactate) 2 were obtained using column chromatography (straight phase; silica 60H). The polydisperse product was dissolved in dichloromethane and apphed onto the column and the column was developed using dichloromethane with 2 % methanol to obtain the enriched fractions.
  • Copolymers of N-isopropylacrylamide (NIPAAm) and N-(2- hydroxypropyl) methacrylamide-lactate (HPMAm-lactate) were prepared by radical polymerization.
  • HPMAm-lactate was synthesized as described in Example 6 and purified by column chromatography.
  • the NIPAAm / HPMAm- lactate ratios were 95/5, 90/10, 80/20, 65/35 and 50/50 (mol mol).
  • the copolymerization was conducted at 60°C for 20 h in a nitrogen atmosphere. Subsequently, the solvent was removed under reduced pressure and the copolymers were dissolved in acetone (around 20% (w/v)) and precipitated in an excess of diethyl ether. The precipitated polymers were isolated by filtration and dried in a vacuum oven at 40°C.
  • Poly(N-isopropylacrylamide-co-N-(2-hydroxypropyl) methacrylamide) copolymers were prepared starting from NIPAAm and N-(2- hydroxypropyl) methacrylamide (HPMAm) in ratios NIPAAm/HPMAm of 95/5, 90/10, 80/20, 65/35 and 50/50 (mol/mol).
  • the copolymerization was conducted at 60°C for 20 h in a nitrogen atmosphere. Polymers with the monomer ratio 95/5 and 90/10 were isolated as follows.
  • the pH of the homogenous solutions was adjusted to « 11 by addition of IN NaOH.
  • the solutions were incubated at 37°C for 4-7 days. After 1 day a drop in pH was observed, indicating that lactic acid was spht off from the polymer backbone.
  • the samples were cooled to « 5°C and then dialyzed against water at 4°C (Dialysis Tubing- Visking, Size 9 Inf Dia 36/32"- 28.6 mm: 30M (Approx), MWCO-12-14000 Daltons; MEDICELL International LTD., London, Great Britain).
  • the hydrolysed polymers were collected after freeze-drying.
  • the different copolymers of reference Example 7 were treated in the same way. The results are given in Table 3.
  • Table 3 shows that it is possible to synthesize polymers that have an
  • Stock solutions (10 mM, 10 ml) were prepared by dissolving HEMA- monolactate and HEMA-dilactate separately in DMSO. Next, 1 ml of stock solution was diluted with 9 ml 100 mM PBS (pH 7.5). The degradation was carried out in glass bottles (20 ml) in a water-bath at 37°C. Samples (300 ⁇ l) of these solutions were periodically drawn and diluted with 700 ⁇ l of 1M acetic buffer (pH 3.4) to stop further degradation. The degradation of HEMA- monolactate was followed for 5 days and the degradation of HEMA-dilactate was followed for 10 h. The different samples were analyzed by HPLC (LiChrosphere 100
  • HEMA-dilactate has a circa 10 fold lower stability than HEMA- monolactate.
  • the fact that the degradation product of HEMA-dilactate is almost exclusively HEMA (and not a mixture of HEMA-monolactate and HEMA) demonstrates that the ester bonds in HEMA-dilactate do not have the same susceptibility for hydrolysis.
  • a possible explanation is that the OH-end group participates via a so called back-biting mechanism in the degradation of HEMA-dilactate yielding HEMA and lactide. The latter compound is then in a two step process rapidly converted into lactic acid.
  • the present invention provides a method for controlling the kinetics of the insoluble/soluble conversion of polymers which are grafted with lactate groups, which method comprises changing the DS of said polymer. For example, when the DS is changed from 1 to 2, the rate of degradation of the polymer decreases dramatically, i.e. with about a factor ten.
  • HPMAAm-monolactate and HPMAAm-dilactate were evaluated essentially as described for HEMA- monolactate and HEMA-dilactate (Example 9).
  • the samples were incubated in lOOmM carbonate buffer, pH 9.0 at 37°C.
  • the degradation of HPMAAm-monolactate was followed for 13 hours and the degradation of HEMA-dilactate was followed for 90 minutes.
  • the different samples were analysed by HPLC (LiChrosphere 100 RP-18 (5 ⁇ m, 125 x 4 mm i.d.) to monitor the concentrations of HPMAAm-dilactate, HPMAAm- monolactate.
  • HPMAAm and MMAc metalhacry c acid
  • the pH of both eluents was adjusted with perchloric acid.
  • the gradient was run from 100 % A to 100 % B in 26 minutes, with the flow rate 1 ml min.
  • For detection was used UV-detector at the wavelength 210 nm.
  • Example 9 HPMAm-dilactate is more susceptible to hydrolysis. However, the ester bond which is preferentially cleaved differs. In HEMA-dilactate lactoyl lactate is split off in one step, whereas in HPMAm-dilactate both lactate and lactoyl lactate are spht of.
  • the macroinitiator was synthesized as follows. All glassware was dried in an oven at 150°C for at least one hour. A 50 ml round bottom flask was loaded with 0.4 mmol polyethylene glycol 5000 monomethylether (PEG 5000), 0.2 mmol of 4,4-azobis(4-cyanopentanoic acid) (ABCPA), 0.06 mmol 4- (dimethylamino)pyridinium-4-p-toluenesulfonate (DPTS) and 0.6 mmol N,N'- dicyclohexylcarbodiimide (DCC). The flask was evacuated three times and filled with nitrogen.
  • PEG 5000 polyethylene glycol 5000 monomethylether
  • ABSCPA 4,4-azobis(4-cyanopentanoic acid)
  • DPTS 4- (dimethylamino)pyridinium-4-p-toluenesulfonate
  • DCC N,N'- dicyclohexylcarbod
  • the product was analysed by ⁇ -NMR in CDCI3 and the ratio of PEG 5000 to ABCPA was about 2:1.
  • the macroinitiator was further characterized by GPC. To this end, 0.144 g of the PEGylated macroinitiator and 0.001 g of 4-methoxyphenol were dissolved in 5 ml of freshly distilled 1,4-dioxane. 50 ⁇ l was diluted with 9.95 ml dichloromethane (HPLC-grade). The solution of the macroinitiator was heated for at 80°C for 24 hours. Next, 50 ⁇ l was diluted with 9.95 ml dichloromethane. The samples were analysed by GPC (Waters 60F model gradient pump, Waters 410 differential refractometer, Shodex KF 80M column with Shodex KF-G precolumn).
  • M n number average molecular weight
  • Mw weight average molecular weight * after 24 hrs at 80°C
  • the micelle forming properties the obtained blockcopolymer were investigated using DLS (Dynamic Light Scattering). At room temperature a mixture of water and blockcopolymer (1 mg/ml) gave a clear and homogeneous solution. After incubation at 37°C (above the LCST of the NIPAAm block), a shghtly turbid solution was obtained. DLS measurements revealed that the sizes of the formed micelles were about 150 nm. By lowering the temperature to 25°C, the solution became clear again, demonstrating the reversibihty of the micelles. When a control experiment with PNIPAAm was carried out, visible inspection as well as DLS measurements showed the presence of large aggregates at 37°C.
  • DLS Dynamic Light Scattering
  • This Example demonstrates the synthesis route via a macroinitiator by the copolymerization reaction with NIPAAm.
  • This route is not limited to NIPAAm, but can easily be extended to different copolymerizations (e.g. copolymerization of NIPAAm and HEMA-mono/dilactate (see e.g. Example 1) or HPMAm-mono/dilactate (see e.g. Example 6)).

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Abstract

L'invention concerne des compositions renfermant des polymères dont les caractéristiques de solubilité peuvent être modifiées par incubation. Un autre aspect de la présente invention est l'application de ces polymères sensibles à la température comme systèmes de libération de composés biologiquement actifs. Ces polymères comprennent des monomères dont la fonctionnalité peut être modifiée par la présence de groupes hydrolysables. La modification est obtenue par incubation, entraînant un changement des caractéristiques d'hydrosolubilité des polymères. Les polymères utilisés dans la présente invention contiennent des groupes chimiques hydrolysables. Par conséquent, les caractéristiques de solution des polymères, notamment leur température inférieure critique de solution (TCIS) est modifiée lors de l'incubation.
EP00950086A 1999-07-30 2000-07-28 Polymeres sensibles a la temperature Withdrawn EP1214361A1 (fr)

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EP99202523 1999-07-30
EP99202523A EP1072617A1 (fr) 1999-07-30 1999-07-30 Polymères sensibles à la température
EP00950086A EP1214361A1 (fr) 1999-07-30 2000-07-28 Polymeres sensibles a la temperature
PCT/NL2000/000542 WO2001009198A1 (fr) 1999-07-30 2000-07-28 Polymeres sensibles a la temperature

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US9645149B2 (en) 2011-09-30 2017-05-09 The Regents Of The University Of Michigan System for detecting rare cells
US10073024B2 (en) 2012-10-29 2018-09-11 The Regents Of The University Of Michigan Microfluidic device and method for detecting rare cells
US10130946B2 (en) 2011-09-30 2018-11-20 The Regents Of The University Of Michigan System for detecting rare cells
US10317406B2 (en) 2015-04-06 2019-06-11 The Regents Of The University Of Michigan System for detecting rare cells

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US10130946B2 (en) 2011-09-30 2018-11-20 The Regents Of The University Of Michigan System for detecting rare cells
US10935550B2 (en) 2011-09-30 2021-03-02 The Regents Of The University Of Michigan Functionalized graphene oxide system for detecting rare cells
US10073024B2 (en) 2012-10-29 2018-09-11 The Regents Of The University Of Michigan Microfluidic device and method for detecting rare cells
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US10317406B2 (en) 2015-04-06 2019-06-11 The Regents Of The University Of Michigan System for detecting rare cells

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