Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
  • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
  • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/47—Quinolines; Isoquinolines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
  • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
  • A61K38/13—Cyclosporins
• • 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
  • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
• • 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 a novel class of polymers for
• Another aspect of the invention is a controlled release system for active ingredients (active agents) comprising such polymers.
• Amphiphilic polymers are known to be used in encapsulation systems in the form micelles in an aqueous environment with a hydrophobic core, in which the hydrophobic drug can be captured.
• Desired characteristics of the micelles carrying a drug are a high stability of the loaded micelle and good retention of the drug (prolonged circulation in blood), when administered in a body after, e.g., intravenous administration.
• Another important property is a sufficient retention of the drug in the micelle after administration, which corresponds to a prolonged circulation of the encapsulated drug in blood.
• Many drugs generally have a short half-life in the human or animal body. Therefore, multiple daily injections or continuous infusions would be required to have a desired therapeutic effect. This is why systems for controlled or sustained release are very desirable for such purposes.
• the present invention provides, in one aspect, a block copolymer comprising at least one hydrophilic block and at least one hydrophobic block, wherein the
• hydrophobic block is selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, vinylethers and their aromatic derivatives, wherein at least one hydrophobic block contains an aromatic side group, which is bound to the hydrophobic block with or through a degradable linker, wherein the aromatic side group is an aryl.
• the present invention provides a controlled release system comprising the block copolymer according to the invention and a physically entrapped active ingredient.
• Figure 1 shows the encapsulation efficiency (%) and loading capacity (%) of the loaded micelles according to the invention plotted against the feed PTX concentration (mg/mL).
• PTX is paclitaxel, which is used as a model drug.
• Figure 2 shows Z-average hydrodynamic diameter (Z av e, nm) and polydispersity index (PDI) of the loaded micelles according to the invention as measured by dynamic light scattering plotted against the loaded PTX concentration (mg/mL).
• Figure 3 shows plasma concentrations of PTX after i.v. injections of four different PTX formulations in A431 human tumor-bearing female Crl:NU-Fox nu lnu mice, measured in time.
• Figure 4 shows the injected dose (ID) in %/g organ in different organs of the mice 24 hours after i.v. injection of PTX-loaded mPEG-b- p(HPMAm-Bz) micelles.
• Figure 5A shows the injected dose (%ID in blood) plotted against the time pi. (post injection) in hours.
• Figure 5D shows the injected dose to the volume of the tumor (%ID / cm 3 of the tumor) plotted against the post injection time (h).
• Figure 5E shows the injected dose to the volume of the organ (%ID / cm 3 organ) for different organs of the mice.
• Model drug is PTX.
• Figure 6 shows the development of the tumor size in mm 3 in the days after initial treatment for the micelles of the invention and for comparative systems being Taxol®, empty micelles and phosphate-buffered saline PBS solution.
• Figure 7 shows % survival plotted against the days after initial treatment with the loaded micelles with PTX according to the invention and comparative systems being Taxol®, empty micelles and phosphate-buffered saline PBS solution.
• Figure 8 shows the development of the tumor size MDA-MB-468 xenografts in mm 3 in the days after initial treatment for the micelles of the invention with PTX (15 mg/kg and 30 mg/kg) and for comparative systems being Taxol®, empty micelles and phosphate-buffered saline PBS solution.
• the arrows show the i.v. injections.
• Figure 9 shows chemical formulae of encapsulated active agents.
• Figure 10 shows the chemical formulae of mPEG-p(HPMAm-Bz) and mPEG-b-p(BHMPO).
• Figure 11 shows the size and light scattering intensity measured by DLS as a function of time, for empty micelles of mPEG-b-p(BHMPO) at pH 7.4, 6.5 and 5.0.
• the present invention is based on the judicious insight that the stability of polymer micelles and retention of a hydrophobic drug physically entrapped in the micelles can be increased when using a particular polymer with aromatic side groups. It is surprising to obtain highly stable micelles with long circulation in blood for drugs that only physically entrapped in the micelles. Without wishing to be bound by any theory, it is believed by the inventors that the aromatic side groups on the polymeric chains provide for increased physical binding with the drug molecules, especially when the latter contain themselves aromatic groups so that aromatic interactions between the polymer chains and the drug molecules are possible. Also aromatic interactions between the polymer chains aid to their stability in the circulation.
• copolymer means a polymer wherein at least two types of monomers are present.
• Amphiphilic means possessing both hydrophobic and hydrophilic properties.
• active ingredient includes therapeutic and diagnostic agents.
• Therapeutic agents are compounds capable of exerting a preventive, therapeutic or pharmacological action in a human or animal body. Under preventive or therapeutic actions are usually understood beneficial effects of interactions with or effect on a cell tissue in a human or animal body.
• diagnostics-related active ingredients include those useful for angiography, MRI, CT scan imaging, nuclear magnetic resonance imaging, radiography, X-ray contrast imaging, ultrasound, etc.
• diagnostic active ingredients are, for example, photosensitizers, imaging agents (such as MRI contrast agents), radioisotope compounds, fluorescent compounds, dyes.
• “Hydrophobic” means having a partition coefficient logP in octane- water higher than 1.
• Non-covalent interaction means any interaction which is not covalent, i.e. any weak bonding between atoms or bonds which bonding does not involve the sharing of electron pairs. Examples of non-covalent interaction are hydrophobic, aromatic ( ⁇ - ⁇ ), hydrogen bonding, electrostatic, stereo complex, and metal-ion interactions. Entrapment is also used as a synonym for binding, loading or encapsulation of the active ingredient.
• Linker means a chemical group having functionality to connect different parts (or building blocks) of a molecule, typically two parts.
• Degradable means in this context biodegradable.
• “Hydrolysable” means degradable by means of hydrolysis.
• “Physiological conditions” are the conditions present in an organism to be treated with the controlled release system of the invention; for humans these conditions encompass a pH of about 7.4 and a temperature of about 37°C.
• the copolymer according to the present invention is a block copolymer comprising at least one hydrophilic block and at least one hydrophobic block. The copolymer is therefore an amphiphilic block copolymer. In aqueous solutions, these polymers form micelles with a hydrophobic core, which can be loaded, particularly with hydrophobic drugs.
• the copolymer of the invention is suitable for use in controlled release systems, target delivery systems and others used inside a human or animal body. In some embodiments, this may mean that the copolymer of the invention is a bioresorbable polymer or it converts to a bioresorbable polymer after hydrolysis, which means that the polymer material is safely absorbed by the body and it disappears from the body over time.
• the polymers of the present invention can have 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.
• (multi-)block copolymers such as AB, ABA, ABAB, etc.
• graft copolymers random copolymers or terpolymers, or a polymeric networks; all of which may be grafted.
• the hydrophobic block contains monomers that are preferably selected from the group of acrylates, methacrylates, acrylamides,
• the hydrophobic block contains methacrylamide or its derivatives, more preferably hydroxyalkyl methacrylamide.
• methacrylamide or its derivatives particularly aromatic derivatives are preferred, which means that the derivative contains an aromatic group, more preferably an aryl or a substituted aryl.
• preferred monomers include 2 -hydroxy ethyl methacrylate (HEMA), 2 -hydroxy ethyl acrylate (HE A), glyceryl methacrylate or glycidyl methacrylate (GMA), glyceryl acrylate or glycidyl acrylate (GA),
• HPMAAm hydroxypropyl methacrylamide
• At least one hydrophobic block of the copolymer of the invention contains an aromatic side group, coupled through a degradable linker.
• Aromatic means that the side group contains at least one aromatic ring.
• Aromatic includes heteroaryl and aryl groups. Heteroaryls contain heteroatoms such as N or S in at least one ring and are optionally
• Aryl means throughout this description and the claims a monocyclic or polycyclic aromatic hydrocarbon group derived from the corresponding arene by removal of a hydrogen atom from a ring carbon atom, which is optionally substituted.
• suitable aromatic groups are phenyl, naphthyl, indoyl and their derivatives.
• particularly suitable aryl groups are phenyl and naphthyl.
• arylalkyl groups are suitable, such as arylmethyl (particularly, phenyl) and other arylalkyl groups with C1-C4 carbon atoms in the alkyl moiety.
• the aromatic side group is preferably an aryl or an arylalkyl group.
• the aromatic group is benzyl, phenyl or naphthyl or derivatives thereof, more preferably phenyl or naphthyl.
• the aromatic group is present in more than 25 mol.% of the side groups of the monomers that form the hydrophobic blocks, more preferably at least 50 mol.%. Yet more preferably, at least 75 mol.% of monomers of the hydrophobic blocks contain the aromatic group and most preferably, 100 mol.% of monomers of the hydrophobic blocks.
• the hydrophobic block of the copolymer of the invention does not contain lactate groups.
• Lactate groups mean a monolactate, a dilactate or an oligolactate group.
• a lactate can be used to provide the copolymer with thermosensitive properties.
• Thermosensitive copolymers with N-(2-hydroxypropyl)methacrylamide monolactate as a part of the hydrophobic block were reported in Y. Shi et al., Biomacromolecules 2013, 14, 1826-1837. However, when the present inventors tested these paclitaxel loaded micelles in vivo they observed a half-life of the drug of less than 2 minutes pointing to a rapid destabilization of these thermosensitive micelles and/or a rapid release of the drug from the micelles. In contrast, the half-life of the micelles according to the invention loaded with the same drug paclitaxel is about 8 hours.
• Example 3 and Figure 3 in the present application use is made of a degradable linker for the coupling of the aromatic side group to the polymer backbone of the
• the hnker is preferably degradable under physiological conditions, and more preferably it is hydrolysable under physiological conditions.
• the linkers might also be hydrolyzed by the actions of enzymes. Generally, known linkers can be used, selected from esters, orthoesters, amides, carbonates, carbamates,
• anhydrides, ketals, acetals, hydrazone and derivatives thereof an ester linker is used, preferably of formula— C(0)-0-.
• the phenyl group together with the ester linker forms a benzoyl group
• the naphthyl group together with the ester linker - a naphthoyl group is used.
• a pH-sensitive hnker is used.
• such linker hydrolyzes at a pH less than 7, more preferably at a pH of 6.5 or less.
• An advantage of a pH-sensitive hnker is that it can selectively release the entrapped drug at areas with an increased acidity, such as some tumors or inflammation sites. Also degradation of the micelles and release of entrapped drug might occur in endosomes or lysosomes having a lower pH, after cellular binding and intern alization of the drug-loaded micelles.
• hydrazone linkers with the following general formula:
• Ri, R2 can be independently of each other H or alkyl groups, and wherein R3 is an alkyl group or a ketone group -C(O)-.
• the alkyl group is a C1-C3 alkyl. More preferably, Ri or R2 is a methyl group. In a preferred embodiment, R3 is a ketone group -C(O)-.
• hydrophilic block of the copolymer of the invention any suitable polymer can be used.
• the hydrophilic block comprises a polyalkylene glycol, more preferably a polyethylene glycol (PEG).
• PEG polyethylene glycol
• the weight ratio of the hydrophobic to hydrophilic block is preferably in the range from 10:90 to 90: 10, more preferably from 40:60 to 60:40.
• the copolymer can be prepared by starting from a mixture of the monomers and carrying out the polymerization reaction. Preferably, the copolymer is obtained by radical polymerization. It is also possible to first produce the copolymer and subsequently functionalize it by coupling suitable groups. Methods of synthesis of copolymers are known to a skilled person. An example of a suitable method to synthesize monomers is described in Y. Shi et al, Biomacromolecules 2013, 14, 1826-1837. An example of a synthesis of macroinitiator that can be used in the
• a preferred embodiment of the present invention is the use of the copolymer of the invention in or as a controlled release system which system further comprises at least one physically entrapped active ingredient.
• the invention provides a controlled release system comprising the block copolymer according to any one of the preceding claims and at least one physically entrapped active ingredient.
• the polymer is in the form of a polymeric micelle having a hydrophobic core and the active ingredient is physically entrapped in the hydrophobic core of the micelle.
• the amphiphilic block copolymers of the present invention are particularly suitable to form a controlled release system in the form of polymeric micelles.
• Polymeric micelles are, in turn, particularly suitable for encapsulation and controlled and targeted release of hydrophobic active ingredients because they can be entrapped in the hydrophobic core of the micelle.
• the micelles of the present invention are also particularly suitable for targeted drug delivery; that is, they can selectively accumulate at the site of action such as inflamed tissue due to the EPR effect (Enhanced Permeability and Retention effect; reference is made to Maeda, H.;
• the micelles according to the invention loaded with paclitaxel show slow release from the micelles (50% of loading in about 10 days) demonstrating a good retention of the drug in the micelles.
• the micelles also show long blood circulation time (half-time of around 8 hours).
• the PTX-loaded micelles showed full inhibition of the tumor growth in mice, whereas at an equal dose the commercially used Taxol® formulation of PTX was much less effective.
• the polymeric micelles of the invention are rather monodisperse.
• the micelles have a size 20- 100 nm. This refers to Z- average hydrodynamic diameter, as measured by dynamic light scattering.
• the polydispersity index PDI is preferably less than 0.3, more preferably less than 0.15, as measured by dynamic light scattering.
• the active ingredient is preferably a therapeutic or diagnostic agent. As mentioned above, more active ingredients can be loaded
• the controlled release system comprises two active ingredients. These can be for example two therapeutic agents, which is particularly useful in combination therapy where several drugs are administered simultaneously.
• a system comprising a therapeutic agent and a diagnostic agent such as an imaging agent (e.g. MRI contrast agent).
• the active ingredient molecule contains at least one aromatic group.
• the loaded micelles are particularly stable and have high retention of the drug in vivo. Without wishing to be bound by any theory, it is believed that this for a part could be explained by enhanced aromatic interaction between the aromatic side groups of the polymer chains and of the drug molecules.
• the type of active ingredient to be entrapped includes but is not limited to, drug molecules, for example of the (gluco)corticosteroid type or of the chemostatic type, peptides/proteins, imaging agents, genetic material or a combination of these.
• the drugs preferably include anticancer or cytostatic drugs, corticosteroids, antibiotics, anti viral drugs. Active ingredients of different chemical origin can be entrapped in the polymeric micelles of the present invention.
• the present invention is beneficial for entrapping hydrophobic active ingredients, with or without aromatic groups, which usually have significant problems with delivery due to the poor aqueous solubility.
• An example of a hydrophobic active ingredient without aromatic groups is dexamethasone.
• Examples of hydrophobic active ingredients with aromatic groups are bedaquiline and paclitaxel.
• the hydrophobic active ingredients to be encapsulated have preferably a log P of at least 1, more preferably at least 1.5.
• the aqueous solubility of the active ingredient is preferably less than 5 mg/ml.
• non-hydrophobic active ingredients can successfully be encapsulated, e.g. amphiphilic compounds carrying a charge such as salts.
• An example of this is doxorubicin hydrochloride.
• active ingredients that were found to be successfully entrapped in the micelles of the present invention include DAPT (notch inhibitor) and cyclosporin A (immunosuppressant).
• the controlled release system releases paclitaxel as active ingredient.
• the controlled release system of the present invention is suitable for treatment of diseases characterized by inflammation and increased vascular permeability.
• diseases include but are not limited to the diseases selected from the group consisting of cancer, infection,
• ophthalmological diseases viral infection, bacterial infection, fungal infection, mucoplasma infection, parasite infection, inflammation, dermatological diseases, cardiovascular diseases, diseases of the central nerve system, auto-immune diseases, proliferative diseases, arthritis, psychotic diseases, psoriasis, diabetes, metabolic disorders, lung diseases, respiratory diseases, pulmonary diseases, COPD, diseases of the
• the controlled release system of the present invention is also suitable for delivery of anesthetics, to be used in vaccination, being either therapeutic or preventive.
• the mean particle size (Z av e) and polydispersity index (PDI) of the micelles was determined with dynamic light scattering (DLS) using a Malvern ALV/CGS-3 Goniometer. ⁇ - ⁇ , spectra were recorded with a Gemini 300 MHz spectrometer (Varian Associates Inc., NMR
• Example 1 Copolymer synthesis Copolymer poly(ethylene glycol)-b-(N-(2-benzoyloxypropyl) methacrylamide)) (mPEG-b-p(HPMAm-Bz)) was synthesized using mPEG2- ABCPA as macroinitiator and HPMAm-Bz as monomer. The monomer has been synthesized following the procedure described in Y. Shi et al.,
• the monomer was dissolved at a concentration of 0.3 g/mL in acetonitrile (dried on A4 molecular sieves) and the molar ratio of monomer- to-macroinitiator was 200/1.
• the solution was degassed by flushing with nitrogen for 30 minutes.
• the reaction was conducted at 70 °C for 24 hours under a nitrogen atmosphere.
• the polymer was purified by precipitation in diethyl ether and this dissolution/precipitation procedure was repeated twice.
• the polymer was dried under vacuum at room temperature for 24 hours and collected as a white powder.
• the number average molecular weight (Mn ) of the synthesized polymers was 20,000 Da by gel permeation chromatography (GPC) analysis (PDI being Mw/Mn was 1.7) and 22,000 Da by ⁇ NMR analysis.
• the content of PEG in the copolymer is -23 wt.% (5,000 Da of PEG and 17,000 Da of the hydrophobic block based on the NMR analysis).
• the critical micelle concentration (CMC) of the polymer was 1.3 ⁇ g/mL as measured using pyrene as the probe according to a method reported in Langmuir, 2004, 20 (21), pp 9388-9395.
• mPEG-b-p(HPMAm-Bz) micelles were prepared as follows. mPEG-b-p(HPMAm-Bz) was dissolved in THF at a concentration of 27 mg/mL and subsequently, 1 mL of the polymer solution was added drop wise to 1 mL of reverse osmosis (RO) water while stirring. The mixture was incubated at room temperature for 48 hours to allow evaporation of THF. The resulting micellar dispersion was filtered through 0.45 ⁇ nylon membrane (Acrodisc®).
• RO reverse osmosis
• Paclitaxel (PTX)-loaded micelles were prepared as follows. PTX and mPEG-b-p(HPMAm-Bz) (structure shown in Fig. 10) were dissolved in THF with PTX concentration ranging from 3 to 12 mg/mL and 27 mg/mL for the polymer. 1 mL of the polymer solution was added dropwise to 1 mL of reverse osmosis (RO) water while stirring. The mixture was incubated at room temperature for 48 hours to allow evaporation of THF. The resulting micellar dispersion was filtered through 0.45 ⁇ nylon membrane
• Figure 1 shows the encapsulation efficiency (%) and loading capacity (%) of the loaded micelles against the feed PTX concentration (mg/mL).
• Figure 2 shows Z-average hydrodynamic diameter (Z av e, nm) and polydispersity index (PDI) of the loaded micelles against the loaded PTX concentration (mg/mL) measured by dynamic light scattering.
• the polymeric micelles of the invention are rather monodisperse and preferably have a size of - 70-100 nm with a
• polydispersity index PDI less than 0.15.
• Plasma concentrations of PTX after i.v. injections of different PTX formulations in A431 human tumor-bearing female Crl:NU-Fox nu lnu mice (22.5 ⁇ 2.5 g) mice (n 7-8) were measured in time.
• the four formulations included PTX loaded micelles of the invention mPEG-b-p(HPMAm-Bz) obtained according to Example 2 and three comparative formulations, being PTX loaded in thermosensitive micelles mPEG-b-p(HPMAm-Bz 3 o-co-HPMAm-Lac7o) and mPEG-b- p(HPMAm-Nt25-co-HPMAm-Lac75), and commercially available Taxol®.
• the results are shown in Figure 3.
• thermosensitive mPEG-b-p(HPMAm-Bz/Nt-co- HPMAm-Lac) micelles were below detection limit ( ⁇ 0.6 ⁇ g/mL plasma) 24 hours post injection. It has been estimated that the half-life of drug administered as formulation of thermosensitive micelles was less than 2 minutes.
• amphiphilic block copolymer according to the invention forms stable micelles with excellent retention of the loaded PTX.
• Figure 4 shows the injected dose (ID) in %/g organ in different organs of the mice 24 hours after i.v. injection of PTX-loaded mPEG-b- p(HPMAm-Bz) micelles.
• ID injected dose
• mPEG-b-p(HPMAm-Bz-co-AEMAm) was synthesized according to Example 1 with 2 mol.% of AEMAm (N-(2-aminoethyl)methacrylamide hydrochloride, relative to HPMAm-Bz copolymerized in the hydrophobic block of the polymer.
• AEMAm N-(2-aminoethyl)methacrylamide hydrochloride, relative to HPMAm-Bz copolymerized in the hydrophobic block of the polymer.
• the primary amine side groups of mPEG-b-p(HPMAm- Bz-co-AEMAm) were subsequently reacted with Cy7 NHS ester.
• Cy7 NHS ester was dissolved in DMSO (dried on 4 angstrom molecular sieves) at a concentration of 10 mg/mL.
• the polymer (31 mg) was
• mPEG-b-p(HPMAm-Bz) micelles chemically labeled with Cy7 and physically loaded with Cy5.5 were prepared similarly to the PTX-loaded mPEG-b-p(HPMAm-Bz) micelles described in Example 2. Briefly, 1 mL THF solution of 26.6 mg of non-labeled mPEG-b-p(HPMAm-Bz), 0.4 mg of Cy7 labeled mPEG-b-p(HPMAm-Bz) and 0.02 mg of Cy5.5 (as a physically loaded modal drug) was added dropwise to 1 mL of water while stirring. The micellar dispersion was incubated at room temperature for 48 hours to allow evaporation of THF. Next, the resulting micellar dispersion was filtered through 0.45 ⁇ nylon membrane (Acrodisc ® ).
• the injected dose (%ID in blood) is shown against the time pi. (post injection) in hours.
• the injected dose to the volume of the tumor (%ID / cm 3 of the tumor) is shown against the post injection time (h).
• Figure 5E shows the injected dose to the volume of the organ (%ID / cm 3 organ) for different organs of the mice.
• Model drug is Cy5.5. Since the Cy7 label is covalently linked to the polymer, it reports about the fate of the injected micelles.
• Figure 6 shows the development of the tumor size in mm 3 in the days after initial treatment.
• the lower two curves represent the data of the micelles of the invention loaded with PTX.
• the three other curves provide the data obtained with commercially available Taxol®, empty micelles and PBS as comparative data.
• Figure 7 shows % survival plotted against the days after initial treatment with the loaded micelles with PTX according to the invention and comparative systems.
• micellar formulations are impressive. Complete tumor regression was observed at 36 day for the animals that received the high dose of micellar formulations (30 mg PTX/kg). Also the low dose micellar formulation (15 mg PTX/kg) show a substantial and better antitumor effect than an equal dose of the commercial Taxol® formulation.
• mice bearing MDA-MB-468 breast carcinoma xenografts Similar to Example 4, the therapeutic efficacy of the PTX-loaded mPEG-b-p(HPMAm-Bz) micelles was studied in mice bearing MDA-MB-468 breast carcinoma xenografts. In this study mice received similar treatments as in the A431 study (Example 4) but i.v. injections were administered once a week rather than every other day. Treatment was initiated 4 weeks after inoculation with the tumor cells, when tumors had reached a volume of ⁇ 100 mm 3 .
• Figure 8 shows the development of the tumor size in mm 3 in the days after initial treatment.
• the lower two curves represent the data of the micelles of the invention loaded with PTX.
• the three other curves provide the data obtained with commercially available Taxol®, empty micelles and PBS as comparative data. Arrows represent i.v. injections.
• Figure 8 shows that Taxol ® (15 mg/kg PTX) only slightly inhibited tumor growth, while the tumors of mice that received the PTX-loaded micelles (15 and 30 mg/kg PTX) decreased in volume and completely regressed after 60 days.
• micellar formulation Repeated injections of PTX-loaded polymeric micelles were generally well tolerated and mice did not suffer from significant weight loss throughout the course of the therapeutic efficacy studies. Furthermore, no organ toxicities were observed for the empty or PTX-loaded polymeric micelles, as representatively assessed for liver, spleen and kidney by histopathological analysis. These observations suggest a favorable toxicity profile for the micellar formulation.
• the controlled release system of the invention is an attractive carrier system for the development of PTX- nanomedicines.
• the polymeric micelles are produced in a straightforward and cost-effective manner, without the need for chemical crosslinking or covalent drug conjugation, and are characterized by excellent loading capacity, enhanced stability and strong PTX retention.
• the IC50 values of micellar PTX were comparable to those of the free drug (i.e. Taxol ® ), for both A431 and MDA-MB-468 cells, indicating that the potency of PTX was not compromised by encapsulation.
• the micellar characteristics ensure prolonged circulation kinetics, substantial tumor accumulation and efficient tumor regression in two well-known and routinely used xenograft models, i.e. A431 and MDA-MB-468, without inducing significant toxicity,
• Example 6 Encapsulation of different active agents The encapsulation procedure was followed to encapsulate different drugs from the list: dexamethasone, doxorubicin hydrochloride (DOX.HC1), bedaquiline, DAPT and cyclosporin A.
• the polymers used are mPE G-b -p (HPMAm -Bz) with batch numbers J01 (M n 48.000,
• micellar formulations 1 mL of tetrahydrofuran in which 5 mg of polymer and 1 mg of drug/drug candidate were dissolved was added dropwise to 1 mL reverse osmosis (RO) water with stirring and the mixture was kept stirring for 1 more minute. The mixtures were placed in fume hood overnight to evaporate tetrahydrofuran.
• RO reverse osmosis
• micellar dispersions except the dox formulations
• DLS size of the formed micelles
• acetonitrile 1 volume of micellar dispersion was added to 19 volumes of acetonitrile
• UV- Visible spectroscopy all drugs except for paclitaxel that was analyzed by HPCL.
• DOX.HCl- loaded micelles the formulations were centrifuged using vivaspin tubes at 10000 g for 5 minutes and the non-loaded drug in the filtrate was quantified by UV- Visible spectroscopy to calculate the EE and LC. The results are presented in Table 1.
• the table shows that drugs and drug candidates with log P > 1 can successfully be loaded in the polymeric micelles with high EE and LC.
• drugs having aromatic groups include drugs having aromatic groups (PTX, bedaquiline, DOX.HCl, DAPT and imatinib free base), hydrophobic drugs without aromatic groups (dexamethasone and cyclosporine A), drug (candidates) with aromatic groups and also with charged groups (DOX.HCl, imatinib free base and bedaquiline).
• this table shows a good EE and LC for a polymer modified with naphthyl groups (PEG-p(HPMAm-Nt)25o) for paclitaxel.
• Example 7 pH-sensitive micelles
• a block copolymer of mPEG-b-p(BHMPO) was synthesized via a macroinitiator route using mPEG2-ABCPA as macroinitiator and BHMPO as monomer.
• the monomer was dissolved at a concentration of 0.3 g/mL in DMSO (dried on A4 molecular sieves) and the molar ratio of monomer-to- macroinitiator was 200/1.
• the solution was degassed by flushing with nitrogen for 30 minutes.
• the reaction was conducted at 70 °C for 24 hours under a nitrogen atmosphere.
• the polymer was purified by precipitation in diethyl ether and this dissolution/precipitation procedure was repeated twice.
• the polymer was dried under vacuum at room temperature for 24 hours and collected as a pale-yellow powder.
• PTX Paclitaxel
• BHMPO mPEG-b-p
• pH-dependent stability of the empty mPEG-b-p(BHMPO) micelles The empty mPEG-b-p(BHMPO) micelles were prepared using the above method, without adding PTX. The pH of the micellar dispersion was adjusted to 5.0 or 6.5 by diluting the micelles 5 times in 150 mM ammonium acetate buffer at pH 5.0 or NaH2P0 4 buffer at pH 6.5, respectively. The pH dependent stability of the empty mPEG-b-p(BHMPO) micelles at different pH values and 37 °C was studied by monitoring the size and the light scattering intensity of the micelles by DLS. The results are shown in Figure 11.
• the hydrazone bonds between the benzoyl groups and the polymer backbone are sensitive to acidic pH values (6.5 and 5.0), while stable at neutral pH (7.4). Therefore the micelles are expected to be stable in the blood circulation and degrade specifically in tumor tissues and cells, by which PTX loaded in the micelles can be released to kill the tumor cells.

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