WO2006003014A2 - Hydrogel polymeric conjugates of a prodrug - Google Patents

Hydrogel polymeric conjugates of a prodrug Download PDF

Info

Publication number
WO2006003014A2
WO2006003014A2 PCT/EP2005/007316 EP2005007316W WO2006003014A2 WO 2006003014 A2 WO2006003014 A2 WO 2006003014A2 EP 2005007316 W EP2005007316 W EP 2005007316W WO 2006003014 A2 WO2006003014 A2 WO 2006003014A2
Authority
WO
WIPO (PCT)
Prior art keywords
hydrogel
poly
biologically active
active moiety
derivatives
Prior art date
Application number
PCT/EP2005/007316
Other languages
French (fr)
Other versions
WO2006003014A3 (en
Inventor
Ulrich Hersel
Harald Rau
Robert Schnepf
Dirk Vetter
Thomas Wegge
Original Assignee
Complex Biosystems Gmbh
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from GB0415041A external-priority patent/GB0415041D0/en
Priority claimed from EP04019303A external-priority patent/EP1625856A1/en
Application filed by Complex Biosystems Gmbh filed Critical Complex Biosystems Gmbh
Priority to EP09161154.1A priority Critical patent/EP2111872B1/en
Priority to EP05767518.3A priority patent/EP1781335B1/en
Publication of WO2006003014A2 publication Critical patent/WO2006003014A2/en
Publication of WO2006003014A3 publication Critical patent/WO2006003014A3/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • 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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • 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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/795—Polymers containing sulfur
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention is directed to hydrogel depot formulations of biologically active moieties.
  • the hydrogel depot formulations comprise biologically active moieties such as peptides, proteins, oligo- or polynucleotides, natural products or synthetic chemical compounds linked reversibly to mesoporous hydrogels.
  • Pore in a three-dimensional polymer network that is larger than a given biologically active moiety (dependent upon the size of the biologically active moiety but usually larger than 1 nm but smaller than 100 nm)
  • a polymer or oligomer whose molecules each have at least one polymerizable functional group, often at the end or at the ends, that enables it to act as a monomer. After polymerization, the groups are part of the main chain of the final polymer. Homopolymerization or copolymerization of a macromonomer yields comb, graft, or cross- linked polymers.
  • a reaction involving pairs of polymer chains that results in the formation of regions in a polymer from which at least four chains emanate.
  • the region may be an atom, a group of atoms, or a number of branch points connected by bonds, groups of atoms, oligomeric, or polymeric chains.
  • Biodegradable polymer
  • a polymer susceptible to degradation under in vivo conditions include but are not limited to degradation by enzymatic or chemical means under conditions present in a living body.
  • Degradation is defined as a chemical change in a polymeric material, accompanied by cleavage of chemical bonds in the polymer and a lowering of its molar mass.
  • a polymer having reactive functional groups that can be transformed under the conditions required for a given reaction or application.
  • a hydrogel may be defined as a three-dimensional, hydrophilic or amphiphilic polymeric network capable of taking up large quantities of water.
  • the networks are composed of homopolymers or copolymers, are insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks.
  • the crosslinks provide the network structure and physical integrity.
  • Hydrogels exhibit a thermodynamic compatibility with water which allow them to swell in aqueous media.
  • the chains of the network are connected in such a fashion that pores exist and that a substantial fraction of these pores are of dimensions between 1 nm and 1000 nm.
  • a prodrug is any compound that undergoes biotransformation before exhibiting its pharmacological effects.
  • Prodrugs can thus be viewed as biologically active moieties (such as drugs) containing specialized non-toxic protective groups used in a transient manner to alter or to eliminate undesirable properties in the parent molecule.
  • Carrier-linked prodrug (Carrier prodrug):
  • a carrier-linked prodrug is a prodrug that contains a temporary linkage of a given active substance with a transient carrier group that produces improved physicochemical or pharmacokinetic properties and that can be easily removed in vivo, usually by a hydrolytic cleavage.
  • a cascade prodrug is a prodrug for which the cleavage of the carrier group becomes effective only after unmasking an activating group.
  • PLGA polylactide- coglycolides
  • these materials are rather hydrophobic, and only few protein and peptide drugs could be formulated into delivery systems (e.g somatropin, Nutropin Depot; triptorelin, TrelstarTM Depot; octreotide, Sandostatin ® LAR 9 ; leuprolide, Lupron Depot* ).
  • somatropin Nutropin Depot
  • triptorelin TrelstarTM Depot
  • octreotide Sandostatin ® LAR 9
  • leuprolide Lupron Depot*
  • the hydrophobic nature of PLGA is exploited in the production process of such PLGA- protein formulations.
  • PLGA is provided as a solution in a water-miscible organic solvent, protein is dissolved in water, and the two solutions are mixed in a mixing step.
  • PLG precipitates and physically entraps the protein in a precipitate.
  • the precipitate has a low water content and pore sizes are smaller than 1 nm and do not exhibit hydrogel-like properties.
  • Dependent on the conditions of the mixing step, the water-miscible organic solvent used and the physicochemical properties of the protein, the loading of the precipitate of the PLGA and the protein may vary greatly.
  • Dong Hee Na et al. showed that upon degradation of the precipitates the encapsulated protein and peptide drags are chemically modified by acylation resulting in the release of modified drag moieties (see Dong Hee Na et al.2003, J.Contr. Release 92, 291- 299).
  • hydrogels are promising materials for drug delivery applications, in particular for the delivery of peptide, protein, oligonucleotide or polynucleotide drugs ("biotherapeutics"). These biotherapeutics are fragile macromolecules which often require a well hydrated environment for activity and structural integrity. The high water content of the hydrogels render the material biocompatible and minimize inflammation reactions of tissue in contact with the hydrogel. Especially for the delivery of protein therapeutics, the high degree of hydration may help to preserve the folding of the protein which is a prerequisite for its bioactivity . In hydrophobic environments, proteins tend to denature and aggregate and lose activity. Two different approaches for the preparation of hydrogel-based depots are known in the art, non-covalent and covalent depots.
  • biologically active moieties such as drugs are encapsulated physically without chemical linkage to the hydrogel.
  • the average pore size in the three-dimensional network of the hydrogel has to be smaller than the size of the biologically active moiety for efficient encapsulation by the hydrogel. Therefore, the biologically active moiety can not be incorporated into the hydrogel after hydrogel formation.
  • the hydrogels have to be chemically crosslinked in the presence of the biologically active moiety or pores have to be formed through physical crosslinks in a self-assembly process, also in the presence of the biologically active moiety.
  • the size of the pore size is the key factor governing the encapsulation of the biologically active moiety.
  • the biologically active moiety will rapidly effuse out of the interior of the hydrogel (so-called ,,burst" release). Therefore the crosslinking is allowed to proceed to such extent that a hydrogel with pores is formed, and the biologically active moiety is physically entrapped inside the pores.
  • the size of the pore in a chemically crosslinked hydrogel may be determined by the measurement of the diffusion of different molecules with known sizes (for example a set of different proteins) into the hydrogels. For example, this can be done experimentally by size exclusion chromatography in which the hydrogel is shaped in bead form and packed into a size exclusion chromatography column. Once the hydrodynamic diameter of the protein is larger than the pores in the hydrogel, no diffusion of the protein into the beads made of the hydrogel can take place and the protein elutes in the exclusion volume of the exclusion chromatography column.
  • the size of the pores in self-assembled networks is difficult to measure due to the structural instability of self-assemble networks which is due to the usually weak physical interactions within the self-assembled network.
  • the hydrogels can be prepared by crosslinking hydrophilic biopolymers or synthetic polymers.
  • hydrogels formed from physical or chemical crosslinking of hydrophilic biopolymers include but are not limited to hyaluronans, chitosans, alginates, collagen, dextran, pectin, carrageenan, polylysine, gelatin or agarose, (see.: W.E. Hennink and CF. van Nostrum, Adv. Drug Del. Rev.2002, 54, 13-36 and A.S. Hoffman, Adv. Drag Del. Rev.2002, 43, 3-12). These materials consist of high-molecular weight backbone chains made of linear or branched polysaccharides or polypeptides.
  • Examples for drag-biopolymer hydrogel encapsulation include the encapsulation of recombinant human interleukin-2 from in chemically crosslinked dextran-based hydrogels (JA. Cadee et al., J Control. Release.2002, 78,1-13) and the encapsulation of insulin in an ionically chrosslinked chitosan/hyaluronan complex (S. Surini et al., J.Control. Release 2003,90, 291-301)
  • hydrogels based on chemical or physical crosslinking synthetic polymers include but are not limited to (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, polyethylene glycol) (PEO), poly (propylene glycol) (PPO), PEO-PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A- PEO-PL(G)A copolymers, poly(ethylene imine), etc. (see A.S Hoffman, Adv. Drag Del. Rev 2002, 43, 3-12).
  • protein-polymer encapsulation examples include the encapsulation of insulin in physically crosslinked PEG-g-PLGA and PLGA-g-PEG copolymers (see B. Jeong et al. Biomacromolecules 2002, 3, 865-868) and the encapsulation of bovine serum albumin in chemically crosslinked acrylate-PGA-PEO-PGA-acrylate macromonomers (see A.S. Sawhney et al. Macromolecules 1993, 26, 581-587)
  • This first non-covalent approach has several drawbacks: As the polymerization or crosslinking step to form the hydrogel has to be carried out in the presence of the biologically active moiety (i.e. a protein), the biologically active moiety is exposed to solvents and polymerization reaction conditions which may cause denaturation or chemical modification of the biologically active moiety. Furthermore, the quality of the end product is difficult to control and batch to batch variations may occur. Additionally, the loading of the hydrogels with the biologically active moiety is usually rather low ( ⁇ 15 % protein) and is difficult to control.
  • the biologically active moiety i.e. a protein
  • a further drawback of the non-covalent type of the hydrogels is the so-called burst effect.
  • the burst effect is characterized by a fast and uncontrolled initial release of a weakly bound biologically active moiety from the hydrogel.
  • the initial burst release can account for up to 20 % of the encapsulated biologically active moiety.
  • degradation of the hydrogels is required for release of the biologically active moiety from the crosslinked hydrogels.
  • Self-assembled systems may also depend on degradation or desaggregation for release of the biologically active moiety.
  • Degradation of the hydrogel increases the size of the pore to the extent that the biologically active moiety may diffuse out of the interior of the hydrogel into surrounding body fluids to exert its bioactivity. Degradation of the hydrogel is a process which is dependent on a number of parameters, some of which are not well understood. As the degradation of the hydrogel is dependent upon in vivo conditions, there may be significant contribution of complex biodegradation processes to the overall degradation of the hydrogel.
  • the small size of the pore may reduce the water content of the hydrogel and therefore its compatibility with fragile biomolecules.
  • ester bonds typically employed as biodegradable bonds may spontaneously hydrolyze at the biological pH of 7.4 in plain buffer in the absence of ester-cleaving proteins.
  • microporous hydrogels require a high amount of ester bonds in order to effect efficient release of the biologically active moiety. Both the high local concentration of the ester bonds and the tight encapsulation of the biologically active moiety may lead to side reactions.
  • an amino group present in the biologically active moiety may be positioned in proximity to an ester group, with the amino group providing a nucleophile effecting ester cleavage and subsequent amidation.
  • This process results in a very stable amide linkage between the biologically active moiety and the polymer.
  • the biologically active moiety will not be released until the polymer chain to which the biologically active moiety is attached to is degraded, and the biologically active moiety will be permanently modified.
  • Such modifications are known to reduce bioactivity of the biologically active moiety and may also cause side effects, such as immunogenicity or cancerogenicity.
  • this undesirable modification process is largely uncontrolled and gives rise to a variety of molecular species. This type of side-reaction is described in Dong Hee Na et al.2003, J.Contr. Release 92, 291-299.
  • the biologically active moiety such as a drag molecule
  • a hydrogel with mesopores a so-called mesoporic hydrogel
  • the release of the biologically active moiety from the mesopores in the hydrogels is prevented by the attachment of the biologically active moiety.
  • J. Harris and X. Zhao describe the reversible covalent attachment of a Iy sozyme protein to a hydrogel prepared by radical copolymerization of a PEO-diacrylate and a lysozyme-modified PEO-monoacrylate.
  • the lysozyme was coupled to the PEO-monoacrylate via a thiourea group and a short biodegradable ester linker. Release of the lysozyme from the hydrogel was effected by incubation in pH 7 buffer.
  • modified protein moieties are released as the cleaved linker moiety is still attached to the protein via the stable thiourea group. Furthermore, as the reaction of the activated linker-PEO- monoacrylate with the amino groups of the lysozyme is not regioselective, a variety of differently modified regioisomers are released, which is undesirable. Furthermore, as the encapsulation of the protein is upon hydrogel formation by radical polymerization, the protein can, in addition to the covalent attachment, also be encapsulated in pores of the formed three- dimensional network that are smaller than the diameter of the protein. Therefore, the release of the protein is not solely governed by cleavage of the linker but can also be influenced by the structure of the hydrogel.
  • Hubbell and coworkers (US Patent 23220245 Al) described in a similar approach the reversible attachment of a small synthetic peptide via a cysteine residue to a non ⁇ biodegradable hydrogel.
  • the peptide was coupled to PEO-diacrylate by a Michael addition reaction.
  • the peptide modified PEO-monacrylate was radically crosslinked with a PEG- diacrylamide to form a peptide modified hydrogel. Release of the propionyl-modified peptide was effected by incubation of the hydrogel in pH 7.4 buffer at 37 0 C.
  • mesoporous hydrogels can be used as polymer carriers for drug depots if provided as a carrier in a prodrug system.
  • the invention is directed towards mesoporous hydrogel prodrugs (MHP) of biologically active moieties, such as drag molecules.
  • MHP mesoporous hydrogel prodrugs
  • the MHP may be administered to a patient to form a depot inside the patient which provides for a sustained release of the biologically active moiety over a desired period of time.
  • a main advantage of the MHPs is to provide depot formulations without the need for encapsulation.
  • many biocompatible hydrogel materials with large pore sizes could not be used for drag formulation due to their lack of encapsulating properties.
  • biologically active moiety would be released too fast for most therapeutic applications from such well-hydrated and mechanically soft materials.
  • the provision of a hydrogel as prodrug carrier according to the invention allows the development of superior drug delivery systems.
  • Carrier material properties such as biocompatibility (minimal irritation, immunogenicity, toxicity) may be optimized independently from the release properties of the biologically active moiety as the release properties are solely governed by the prodrug linker cleavage kinetics.
  • the release of the biologically active moiety is therefore largely independent from the carrier material (i.e. the hydrogel) and does not require chemical or enzymatic degradation of the hydrogel.
  • the MHP system consists of three parts, a mesoporous hydrogel carrier, a prodrug linker and a biologically active moiety, such as a drug molecule.
  • the prodrug linker is covalently bonded to the mesoporous hydrogel carrier and to the biologically active moiety in such a fashion that the biologically active moiety-linker-hydrogel conjugate is a carrier prodrug.
  • Suitable biologically active moieties include but are not limited to small organic molecule bioactive agents, biopolymers like proteins, polypeptides and oligo- or poly-nucleotides (RNA, DNA), and peptide nucleic acids (PNA).
  • Suitable organic small molecule bioactive drugs include, without limitation, moieties such as central nervous system-active agents, anti-infective, anti-neoplastic, antibacterial, anti-fungal, analgesic, contraceptive, anti-inflammatory, steroidal, vasodilating, vasoconstricting, and cardiovascular agents.
  • Non-exclusive examples of such compounds are daunorubicin, doxorubicin, idarubicin, mitoxantron, aminoglutethimide, amantadine, diaphenylsulfon, ethambutol, sulfadiazin, sulfamerazin, sulfamethoxazol, sulfalen, clinafloxacin, paclitaxel, moxifloxacin, ciprofloxaxin, enoxacin, norfloxacin, neomycin B, sprectinomycin, kanamycin A, meropenem, dopamin, dobutamin, lisinopril, serotonin, carbutamid, acivicin, etc.
  • Suitable proteins and polypeptides include but are not limited to ACTH, adenosine deaminase, agalsidase, albumin, alfa-1 antitrypsin (AAT), alfa-1 proteinase inhibitor (API),reteplase, anistreplase, ancrod serine protease, antibodies (monoclonal or polyclonal, and fragments or fusions), antithrombin IH, antitrypsins, aprotinin, asparaginases, biphalin, bone- morphogenic proteins, calcitonin (salmon), collagenase, DNase, endorphins, enfuvirtide, enkephalins, erythropoietins, factor VTfa, factor VDI, factor VHIa, factor IX, fibrinolysin, fusion proteins, follicle-stimulating hormones, granulocyte colony stimulating factor (G- CSF), galact
  • any synthetic polypeptide or any portion of a polypeptide with in vivo bioactivity is included herein.
  • proteins prepared by recombinant DNA methodologies including mutant versions of aforementioned proteins, antibody fragments, single chain binding proteins, catalytic antibodies and fusion proteins are included.
  • linking agent it is preferred for the linking agent to form a reversible linkage to the biologically active moiety, preferably in such a fashion that after cleavage of the linker, the biologically active moiety is released in an unmodified form.
  • a variety of different linking agents or linking groups may be applied for this purpose as described by B. Testa et al. ( B . Testa, J. Mayer, Hydrolysis in Drag and Prodrug Metabolism, Wiley-VCH, 2003).
  • the linker is a cascade prodrug linker constituted of a masking group and an activating group.
  • the biologically active moiety is bound to the activating group, preferably through a carbamate bond.
  • the release of the biologically active moiety is effected by a two-step mechanism.
  • the masking group is detached from the linker by cleavage of the bond connecting the masking group and activating group.
  • the bond connecting the masking group and the activating group may also be a carbamate bond.
  • the bond between biologically active moiety and the activating group is cleaved, and the biologically active moiety is released.
  • the cleavage of the masking group is the rate-limiting step of the release of the biologically active moiety.
  • the cleavage of the masking group is preferably based on a hydrolytic process, most preferably catalyzed by a nucleophile present in the masking group.
  • this nucleophile attacks in an intramolecular fashion the carbon of the carbamate group constituting the linkage between the masking group and the activating group.
  • the presence of the nucleophile in the vicinity of the carbamate group enhances the susceptibility of the carbamate group to hydrolysis.
  • the nucleophile is a tertiary amine which does not undergo a reaction with the carbamate carbonyl and does not lead to a cyclization product.
  • linker it is also preferred that at least part of the linker remains attached to the hydrogel after cleavage of the bond with the biologically active moiety. If the linker is a cascade prodrug linker, it is preferred for the activating group to remain stably bound to the hydrogel.
  • Hydrogels are three-dimensional, hydrophilic or amphiphilic polymeric networks capable of taking up large quantities of water.
  • the networks are composed of homopolymers or copolymers and are insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks.
  • the crosslinks provide the network with structure and physical integrity.
  • Such reactive mesoporous hydrogels are characterized by the following structural components: crosslinking moiety, backbone moiety, reactive functional groups, pores, and optionally biodegradable bonds.
  • Non-limiting examples for suitable polymers for the synthesis of hydrogels are chemically or physically crosslinked functionalized or non-functionalized polyalkyloxy-based polymers like poly ⁇ ropylene glycol) or poly(ethylene glycol), dextran, chitosan, hyaluronic acid and derivatives, alginate, xylan, mannan, ca ⁇ ageenan, agarose, cellulose, starch, hydroxyethyl starch (HES) and other carbohydrate-based polmers, polyvinyl alcohols), poly(oxazolines), poly (anhydrides), poly(ortho esters), polycarbonates), poly(urethanes), poly(acrylic acids), poly(acrylamides) such as poly(hydroxypropyhnethacrylamide) (HMPA), poly(acrylates), poly(methacrylates) like poly(hydroxyethylmethacrylate), poly(organophosphazenes), poly(siloxanes), polyvinylpyrrolidone), poly(cyanoacryl
  • polymers may serve as backbone moieties or crosslinking moieties.
  • backbone moieties or crosslinking moieties.
  • low-molecular crosslinking moieties may be used, especially when hydrophilic high-molecular weight backbone moieties are used for the hydrogel formation.
  • the chains of the network in a mesoporous hydrogel are connected in such a fashion that pores exist in the hydrated state and that a substantial fraction of these pores are of dimensions between 1 and 100 nm.
  • the hydrogel is mesoporous with respect to the biologically active moiety (e.g. drug molecule) to be carried, Le. the average size of the pore of the hydrogel is larger than the diameter of the biologically active moiety.
  • a hydrogel which is mesoporous with respect to an insulin molecules has pores of more than 4 nm in size (in the hydrated state).
  • the dimensions of the pores may be controlled by adjusting both length of crosslinker and degree of crosslinking.
  • crosslinker For example, if small molecule crosslinkers are used for instance on a biopolymer such as dextran, porosity may be controlled through the degree of crosslinking. Usually, the lower the degree of crosslinking is the larger the size of the pores.
  • Crosslinker length refers to the spacer length between the two reactive groups used for the crosslinking of the backbone moiety.
  • a typical polymeric crosslinker for the mesoporous hydrogel has at least two functional groups usually at the ends of the polymeric chain. The functional groups are usually connected by a linear or branched chain of MW between 500 and 50000.
  • Such crosslinkers may be macromonomers in which case, the macromonomers are characterized by having at least two polymerizable functional groups.
  • the hydrogel is a functionalized material.
  • the reactive functional groups serve as conjugation sites for the linker.
  • the reactive functional groups are dispersed homogeneously throughout the hydrogel, and may or may not be present on the surface of the hydrogel.
  • Non-limiting examples of such reactive functional groups include but are not limited to carboxylic acid and activated derivatives, amino, maleimide, thiol, sulfonic acid and derivatives, carbonate and derivatives, carbamate and derivatives, hydroxyl , aldehyde, ketone, hydrazine , isocyanate, isothiocyanate, phosphoric acid and derivatives, phosphonic acid and derivatives, haloacetyl, alkyl halides, acryloyl and other alpha-beta unsaturated michael acceptors, arylating agents like aryl fluorides, hydroxylamine, disulfides like pyridyl disulfide, vinyl sulfone, vinyl ket
  • Preferred functional groups for the polymer include but are not limited to thiol, maleimide, amino, carboxylic acid and derivatives, carbonate and derivatives, carbamate and derivatives, aldehyde, and haloacetyl.
  • the reactive mesoporous hydrogel is a shaped article such as a mesh or a stent.
  • the hydrogel is formed into microparticulate beads which can be administered as subcutaneous or intramuscular injectably by means of a standard syringe.
  • Such soft beads may have a diameter of between 1 and 500 micrometer.
  • Biodegradability of the mesoporous hydrogel is of importance if the hydrogel is to be used for medical applications such as wound healing, wound sealing or for drug delivery (or, indeed, delivery of any type of biologically active moieties).
  • the hydrogel is administered by a subcutaneous or intramuscular injection or applied topically to a wound and left in the organism to be degraded in vivo and resorbed or excreted.
  • biodegradable bonds For biodegradability of the hydrogel, biodegradable bonds have to be incorporated into the backbone and/or crosslinking moieties.
  • the susceptibility of these biodegradable bonds to cleavage under in vivo conditions may cause complete degradation of the hydrogel after a certain time period, which is desirable for the abovementioned applications in the medical field
  • Cleavage of these biodegradable bonds may be enzymatically or chemically triggered or be a combination of both.
  • Biodegradable bonds which may be cleaved chemically under in vivo conditions include but are not limited to phosphate, phosphonate, carbonate, carbamate, disulfide and ester bonds.
  • Hydrogels with biopolymer backbones or biopolymer crosslinkers are per se biodegradable on the surface of the hydrogel article if enzymes are present for which the backbone chains are substrates.
  • the rate of degradation under in vivo conditions is different for every different type of hydrogel.
  • the degradation rate is a function of the degradability of the backbone (number of cleavable bonds, dependence of bond cleavage upon autohydrolysis or enzymatic catalysis) and the degree of crosslinking.
  • Suitable biopolymers include but are not limited to carbohydrate- based polymers like dextran, chitosan, hyaluronic acid and derivatives, alginate, xylan, mannan, carrageenan, agarose, cellulose, starch, and hydroxyethyl starch and poly- or oligopeptide based oligomers or polymers like synthetic peptide sequences, collagen and gelatin.
  • the biologically active moiety has to be reacted with the hydrogel after the hydrogel has been synthesized. This ensures that the release of the biologically active moiety from the hydrogel is governed by the prodrug linker and is independent from the optional hydrogel degradation. This is because after cleavage of the prodrug linker the biologically active moiety can freely diffuse out of the mesopores of the hydrogel.
  • a further advantage of this method of preparation is that reagents and solvents contacted with the hydrogel during the preparation of the hydrogel may be removed from the hydrogel after completion of the preparation by a filtration step. Efficient reagent and solvent removal avoid denaturation or modification of the biologically active moiety added to the hydrogel. Efficient reagent and solvent removal also avoids leakage of potentially toxic substances after administration to an organism.
  • MHPs can also be prepared by several other methods.
  • a prodrug linker agent can be coupled to the reactive mesoporous hydrogel in a first reaction step.
  • a suitable prodrug linking agent would carry two functional groups. The first one of the functional groups would serve as the attachment of the prodrug linker to the hydrogel, and the second one of the functional groups would subsequently be conjugated to the biologically active moiety through a suitable functional group present in the biologically active moiety.
  • Such first reactive functional groups should be complementary to a functional group present in the reactive mesoporous hydrogel.
  • Non-limiting examples of such first reactive functional groups include but are not limited to carboxylic acid and activated derivatives, amino, maleimide, thiol, sulfonic acid and derivatives, carbonate and derivatives, carbamate and derivatives, hydroxyl , aldehyde, ketone, hydrazine , isocyanate, isothiocyanate, phosphoric acid and derivatives, phosphonic acid and derivatives, haloacetyl, alkyl halides, acryloyl and other alpha-beta unsaturated Michael acceptors, arylating agents like aryl fluorides, hydroxylamine, disulfides like pyridyl disulfide, vinyl sulfone, vinyl ketone, diazoalkanes, diazoacetyl compounds, epoxide, oxirane, and aziridine.
  • the linker- hydrogel conjugate may be contacted with the biologically active moiety in the second reaction step and excess biologically active moiety (e.g. excess drug) may be removed by filtration after conjugation of the biologically active moiety to the hydrogel-bound prodrug linker.
  • excess biologically active moiety e.g. excess drug
  • the biologically active moiety remains bound inside the hydrogel by the covalent attachment of a suitable functional group present on the biologically active moiety to the second functional group of the prodrug linker.
  • Suitable second functional groups of the prodrug linker include but are not limited to carboxylic acid and derivatives, carbonate and derivatives, hydroxyl, hydrazine, hydroxylamine, maleamic acid and derivatives, ketone, amino, aldehyde, thiol and disulfide.
  • Suitable functional groups present on the biologically active moiety include but are not limited to thiol, carboxylic acid, amino, hydroxyl, ketone, imidazole.
  • this reaction sequence may be inverted, and the prodrug linker may be first conjugated to the biologically active moiety and the resulting biologically active moiety - prodrug linker conjugate may then react with the reactive mesoporous hydrogel (Method B in figure 1).
  • Hydrogels which are reactive and mesoporous may be prepared by a variety of different methods.
  • One particular synthesis process is based on using a crosslinking macromonomer carrying at least two polymerizable functional groups and a non-crosslinking macromonomer or monomer carrying one polymerizable functional group and at least one functional group which is not intended to participate in the polymerization step. Additional diluent monomers may or may not be present. Copolymerization of these components results in a hydrogel containing functional groups provided by the non-crosslinking macromonomer.
  • the conditions for polymerization are chosen such that the functional group is not modified.
  • the functional group may be protected by use of a reversible protecting group known to the person skilled in the art, which is removed after the polymerization.
  • Useful polymerizable functional groups include but are not limited to radically polymerizable groups like vinyl, vinyl-benzene, acrylate, acrylamide, methacylate, methacrylamide and ionically polymerizable groups like oxetane, aziridine,and oxirane.
  • the hydrogel is generated through chemical ligation reactions.
  • the hydrogel may be formed from two macromolecular educts with complementary functionalities which undergo a reaction such as a condensation or addition.
  • One of these starting materials is a crosslinker with at least two identical functional groups and the other starting material is a homomultifunctional backbone structure.
  • Suitable functional groups present on the crosslinker include terminal amino, carboxylic acid and derivatives, maleimide and other alpha,beta unsaturated Michael acceptors like vinylsulfone, thiol, hydroxyl groups
  • Suitable functional groups present in the backbone structure include but are not limited to amino, carboxylic acid and derivatives, maleimide and other alpha,beta unsaturated Michael acceptors like vinylsulfone, thiol, hydroxyl groups.
  • the resulting hydrogel will be a reactive hydrogel with free reactive functional groups attached to the backbone structure.
  • This invention also includes degradable mesoporous hydrogel prodrugs exhibiting minimal release of biologically active moiety conjugated to hydrogel degradation products.
  • the degradable bonds are exclusively located in the crosslinking chains.
  • the crosslinker carrying the degradable bonds is symmetrical with respect to the degradable bonds, rendering the bonds chemically identical.
  • Such a biodegradable mesoporous hydrogel prodrug is shown schematically in figure 2.
  • Functional groups for attachment of the prodrug linker are positioned on side chains emanating from the backbone polymer. There are no degradable bonds between these functional groups and the non-biodegradable backbone or in the non-biodegradable backbone itself.
  • Cleavage of the degradable bonds of the crosslinker results in a two-stage process. Shortly after initial time only cleavage products containing crosslinker units are released from the hydrogel when each non-biodegradable backbone is linked with at least one other non ⁇ biodegradable backbone via several crosslinkers. If, for instance, esters of PEG have been used, the released compound is PEG. The release of PEG from this type of hydrogel follows approximately first order kinetics.
  • FIG. 3 shows schematically a partially degraded (figure 3a) and a fully degraded (figure 3b) MHP.
  • This lag time can be controlled by adjusting the number of crosslinks and the halflife of the biodegradable bond.
  • the lag time can be increased by incorporating more crosslinks per backbone and increasing the half-life of the biodegradable bond. The effect of an increased lag time by increasing the number of crosslinks is shown in figure 10.
  • Fig. 1 shows the preparation process for mesoporous hydrogel prodrugs
  • Fig.2 shows schematically the structure of biodegradable mesoporous hydrogel prodrugs
  • Fig. 3 shows the degradation process of biodegradable mesoporous hydrogel prodrugs
  • Fig.4 shows schematically anundesired polymer modified prodrug
  • Fig.5 shows the insulin release from a polyacrylamide-based mesoporous hydrogel prodrug
  • Fig.6 shows the insulin release from a carbohydrate-based mesoporous hydrogel prodrug
  • Fig.7 shows the in vivo release of insulin from mesoporous hydrogel prodrugs
  • Fig. 8 shows the insulin release from a biodegradable mesoporous hydrogel prodrug
  • Fig.9 shows the LCMS characterization of released insulin after explantation
  • Fig. 10 shows the degradation of biodegradable hydrogels
  • Non-biodegradable mesopor ⁇ us NH2-PEGA hydrogel beads with 0.4 mmol/g loading and 150-300 ⁇ m bead size were purchased from Novabiochem.
  • NH 2 -PEGA Versamatrix-800 hydrogel beads with 0.31 mmol/g loading and 80-100 ⁇ m bead size were obtained from Versamatrix (Denmark).
  • Hydrogel Ib was synthesized following the protocol above with the following modification.
  • 2.5 g methanol-wet NH 2 -PEGA-hydrogel ( ⁇ 250 mg dry resin) was reacted with 6.8 mg (0.04 mmol) 3-maleimidopropionic acid, 117.6 ⁇ l (1.96 mmol) acetic acid and 313 ⁇ l (2 mmol) DIC in 4 ml DMF for 30 min. Finally the hydrogel was washed as described.
  • Hydrogel Ic was synthesized following the protocol above with the following modification.
  • the rats weighing 200-300 g were kept under standard conditions and fed ad libitum.
  • Blood samples 150 ⁇ l were collected from the retro-orbital plexus and a suspension of rh-insulin loaded hydrogel in 300-400 ⁇ l PBS was subcutaneously administered in the upper hind leg area. Blood was drawn at different times after administration. Animals were lightly anesthetized by inhaled isoflurane during all blood draws and injections. All blood samples were collected into tubes containing EDTA and centrifuged. Plasma was separated and stored at -18 0 C until assayed, rh-insulin concentration was determined from plasma samples using the species specific sandwich human insulin ELISA kit (Mercodia, Sweden). The results were statistically analyzed and plasma rh-insulin concentrations were plotted over time after administration (figure 7).
  • Hydrogel Sample Hydrogel dose Total rh-insulin dose / rat N rats sample volume (calculated from in vitro release) 5b 400 ⁇ l 16 mg -138 nmol 1 After 6 days the rat was sacrificed. The hydrogel was explanted and washed 10 times with water, 10 times with 50/50 (v/v) acetonitrile/water and dried under vacuum. About 1 mg of hydrogel was weighed into a test tube and incubated with 400 ⁇ l 100 mM Tris/HCl buffer pH 9, at 37 0 C. After 24 h the solution was separated from the hydrogel and analyzed for rh- insulin and enzymatic degradation products thereof by LC/MS (figure 9).
  • the strong peak at 4.2 min elution time can be assigned to rh-insulin according to the mass spectrum. Smaller peaks before and after the rh-insulin peak correspond to the A- and B-chain of rh-insulin, respectively. No rh-insulin related degradation products were detected. A- and B-chain were also detected in in vitro release experiments at pH 9 and thus do not indicate enzymatic degradation.
  • Compound 10 was synthesized as described in co-pending UK patent application No. 0415043.9, the disclosure of which is incorporated by reference. 025 mg of compound 10 (35 nmol) was dissolved in 100 ⁇ l 40/40/20 (v/v/v) acetonitrile/water/0.5 M phosphate buffer pH 7.0. The solution was incubated for 3 min with 5.6 mg of maleimide derivatized biodegradable hydrogel 9 to give fluorescein-carboxamido- Lys(B29)-rh-insulin loaded hydrogel 11. The hydrogel 11 was washed five times with 50/50 (v/v) acetonitrile/water, three times with acetonitrile and dried under vaccuum.
  • Fmoc-Ado-OH was coupled by incubation of 156 mg (0.45 mmol) Fmoc-Ado-OH 173 mg (0.45 mmol) TBTU and 157 ⁇ l DIEA (0.9 mmol) in 3 ml DMF according to the procedure above. Fmoc was removed and the resin was reacted with 338 mg (0.9 mmol) 5,6- carboxyfluorescein (isomeric mixture), 140 mg (0.9 mmol) HOBt and 141 ⁇ l (0.9 mmol) DIC in 3 ml DMF for 2 h. Finally the resin was incubated in 2/2/96 (v/v/v) piperidine/DBU/DMF for 10 min, washed 10 times in DCM and dried under vacuum.
  • Hydrogel degradation experiment was performed at pH 9 to reduce the time of degradation.
  • Degradation time at physiological pH 7.4 was estimated by a scaling factor 40 that accounts for the approx.40 fold increased hydroxide ion concentration at pH 9 compared to pH 7.4.
  • the hydrogel was suspended in 1 ml 50 mM borate buffer (pH 9.0), 150 mM NaCl, 0.005 % Tween and incubated at 37 0 C.60 ⁇ l samples were taken at different time intervals and quantitatively analyzed for polymer backbone coupled fluorescein 14 by photometry at 500 nm. The data show a delayed and sigmoidal release of 14 (data not shown).
  • the hydrogel 15 was washed 10 times with DMF and finally with acetonitrile. 1.5 mg 3 was dissolved in 25/75 (v/v) acetonitrile/50 mM phosphate buffer pH 7.4 and reacted with 10.8 mg maleimide derivatized hydrogel 15 for 10 min.
  • the rh-insulin loaded hydrogel 16 was washed five times with 50/50 (v/v) acetonitrile/water and three times with acetonitrile and dried under vacuum.
  • 3-maleimidopropionic acid was coupled by DIC activation using 3 equivalents 3- maleimidopropionic acid in relation to free amino groups on the resin.
  • Compound 18 was cleaved from the resin by incubation with 94/3/3 (v/v/v) DCM/triethylsilane/TFA for 60 min. After evaporation of the solvent, 18 was purified by RP-HPLC and lyophilized.
  • the resulting hydrogel 20 was ground to particles ⁇ lmm and transferred into a syringe equipped with a polypropylene frit. The hydrogel particles were washed five times each with
  • Hydrogel 21 was synthesized as described above using 23.3 mg (20 ⁇ mol) 17 and 17.9 mg (5 ⁇ mol) 19.
  • Hydrogel degradation experiments were performed at pH 9 to reduce the time of degradation.
  • Degradation time at physiological pH 7.4 was estimated by a scaling factor 40 that accounts for the AO fold increased hydroxide ion concentration at pH 9 compared to pH 7.4.
  • 50 mg of hydrogel 20 or 21 was suspended in 1 ml 50 mM sodium borate buffer (pH 9.0), 150 mM NaCl, 0.005 % Tween and incubated at 37°C.30 ⁇ l samples were taken at regular intervals and quantitatively analyzed for polymer backbone coupled naphthyl 22 or 23 by photometry at 280 run (figure 10).
  • the degradation kinetic shows a sigmoidal curve.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Nanotechnology (AREA)
  • Diabetes (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Endocrinology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Medical Informatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The application relates to a polymeric prodrug which comprises a hydrogel, a biologically active moiety and a reversible prodrug linker. The prodrug linker covalently links the hydrogel and the biologically active moiety at a position and the hydrogel has a plurality of pores with openings on the surface of the hydrogel. The diameter of the pores is larger than that of the biologically active moiety at least at all points of the pore between at least one of the openings and the position of the biologically active moiety.

Description

Hydrogel
Field
The present invention is directed to hydrogel depot formulations of biologically active moieties. The hydrogel depot formulations comprise biologically active moieties such as peptides, proteins, oligo- or polynucleotides, natural products or synthetic chemical compounds linked reversibly to mesoporous hydrogels.
Background of the invention
Definitions
Micropore:
Pore in a three-dimensional polymer network that is smaller than a given biologically active moiety (smaller than 1 nm)
Mesopore:
Pore in a three-dimensional polymer network that is larger than a given biologically active moiety (dependent upon the size of the biologically active moiety but usually larger than 1 nm but smaller than 100 nm)
Macromonomer:
A polymer or oligomer whose molecules each have at least one polymerizable functional group, often at the end or at the ends, that enables it to act as a monomer. After polymerization, the groups are part of the main chain of the final polymer. Homopolymerization or copolymerization of a macromonomer yields comb, graft, or cross- linked polymers.
Crosslinking:
A reaction involving pairs of polymer chains that results in the formation of regions in a polymer from which at least four chains emanate.
The region may be an atom, a group of atoms, or a number of branch points connected by bonds, groups of atoms, oligomeric, or polymeric chains. Biodegradable polymer:
A polymer susceptible to degradation under in vivo conditions. In vivo conditions include but are not limited to degradation by enzymatic or chemical means under conditions present in a living body. Degradation is defined as a chemical change in a polymeric material, accompanied by cleavage of chemical bonds in the polymer and a lowering of its molar mass.
Reactive polymer:
A polymer having reactive functional groups that can be transformed under the conditions required for a given reaction or application.
Hydrogel:
A hydrogel may be defined as a three-dimensional, hydrophilic or amphiphilic polymeric network capable of taking up large quantities of water. The networks are composed of homopolymers or copolymers, are insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks. The crosslinks provide the network structure and physical integrity. Hydrogels exhibit a thermodynamic compatibility with water which allow them to swell in aqueous media. The chains of the network are connected in such a fashion that pores exist and that a substantial fraction of these pores are of dimensions between 1 nm and 1000 nm.
Prodrug:
A prodrug is any compound that undergoes biotransformation before exhibiting its pharmacological effects. Prodrugs can thus be viewed as biologically active moieties (such as drugs) containing specialized non-toxic protective groups used in a transient manner to alter or to eliminate undesirable properties in the parent molecule.
Carrier-linked prodrug (Carrier prodrug):
A carrier-linked prodrug is a prodrug that contains a temporary linkage of a given active substance with a transient carrier group that produces improved physicochemical or pharmacokinetic properties and that can be easily removed in vivo, usually by a hydrolytic cleavage.
Cascade prodrug
A cascade prodrug is a prodrug for which the cleavage of the carrier group becomes effective only after unmasking an activating group. Protein depots
Among the first polymers employed for protein drag delivery applications were polylactide- coglycolides (PLGA). These materials are rather hydrophobic, and only few protein and peptide drugs could be formulated into delivery systems (e.g somatropin, Nutropin Depot; triptorelin, Trelstar™ Depot; octreotide, Sandostatin® LAR9 ; leuprolide, Lupron Depot* ). The hydrophobic nature of PLGA is exploited in the production process of such PLGA- protein formulations. PLGA is provided as a solution in a water-miscible organic solvent, protein is dissolved in water, and the two solutions are mixed in a mixing step. As a consequence, PLG precipitates and physically entraps the protein in a precipitate. The precipitate has a low water content and pore sizes are smaller than 1 nm and do not exhibit hydrogel-like properties. Dependent on the conditions of the mixing step, the water-miscible organic solvent used and the physicochemical properties of the protein, the loading of the precipitate of the PLGA and the protein may vary greatly.
Furthermore, Dong Hee Na et al. showed that upon degradation of the precipitates the encapsulated protein and peptide drags are chemically modified by acylation resulting in the release of modified drag moieties (see Dong Hee Na et al.2003, J.Contr. Release 92, 291- 299).
In order to address a fundamental shortcoming of PLGA-precipitates, recent developments focused on the use of hydrogels for protein delivery. Hydrogels are promising materials for drug delivery applications, in particular for the delivery of peptide, protein, oligonucleotide or polynucleotide drugs ("biotherapeutics"). These biotherapeutics are fragile macromolecules which often require a well hydrated environment for activity and structural integrity. The high water content of the hydrogels render the material biocompatible and minimize inflammation reactions of tissue in contact with the hydrogel. Especially for the delivery of protein therapeutics, the high degree of hydration may help to preserve the folding of the protein which is a prerequisite for its bioactivity . In hydrophobic environments, proteins tend to denature and aggregate and lose activity. Two different approaches for the preparation of hydrogel-based depots are known in the art, non-covalent and covalent depots.
In the non-covalent approach, biologically active moieties such as drugs are encapsulated physically without chemical linkage to the hydrogel. For this approach, the average pore size in the three-dimensional network of the hydrogel has to be smaller than the size of the biologically active moiety for efficient encapsulation by the hydrogel. Therefore, the biologically active moiety can not be incorporated into the hydrogel after hydrogel formation. In the non-covalent approach, the hydrogels have to be chemically crosslinked in the presence of the biologically active moiety or pores have to be formed through physical crosslinks in a self-assembly process, also in the presence of the biologically active moiety. The size of the pore size is the key factor governing the encapsulation of the biologically active moiety. If the pores are larger than the biologically active moiety, the biologically active moiety will rapidly effuse out of the interior of the hydrogel (so-called ,,burst" release). Therefore the crosslinking is allowed to proceed to such extent that a hydrogel with pores is formed, and the biologically active moiety is physically entrapped inside the pores.
The size of the pore in a chemically crosslinked hydrogel may be determined by the measurement of the diffusion of different molecules with known sizes (for example a set of different proteins) into the hydrogels. For example, this can be done experimentally by size exclusion chromatography in which the hydrogel is shaped in bead form and packed into a size exclusion chromatography column. Once the hydrodynamic diameter of the protein is larger than the pores in the hydrogel, no diffusion of the protein into the beads made of the hydrogel can take place and the protein elutes in the exclusion volume of the exclusion chromatography column.
The size of the pores in self-assembled networks is difficult to measure due to the structural instability of self-assemble networks which is due to the usually weak physical interactions within the self-assembled network.
The hydrogels can be prepared by crosslinking hydrophilic biopolymers or synthetic polymers. Examples of the hydrogels formed from physical or chemical crosslinking of hydrophilic biopolymers include but are not limited to hyaluronans, chitosans, alginates, collagen, dextran, pectin, carrageenan, polylysine, gelatin or agarose, (see.: W.E. Hennink and CF. van Nostrum, Adv. Drug Del. Rev.2002, 54, 13-36 and A.S. Hoffman, Adv. Drag Del. Rev.2002, 43, 3-12). These materials consist of high-molecular weight backbone chains made of linear or branched polysaccharides or polypeptides.
Examples for drag-biopolymer hydrogel encapsulation include the encapsulation of recombinant human interleukin-2 from in chemically crosslinked dextran-based hydrogels (JA. Cadee et al., J Control. Release.2002, 78,1-13) and the encapsulation of insulin in an ionically chrosslinked chitosan/hyaluronan complex (S. Surini et al., J.Control. Release 2003,90, 291-301)
Examples of hydrogels based on chemical or physical crosslinking synthetic polymers include but are not limited to (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, polyethylene glycol) (PEO), poly (propylene glycol) (PPO), PEO-PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A- PEO-PL(G)A copolymers, poly(ethylene imine), etc. (see A.S Hoffman, Adv. Drag Del. Rev 2002, 43, 3-12).
Examples for protein-polymer encapsulation include the encapsulation of insulin in physically crosslinked PEG-g-PLGA and PLGA-g-PEG copolymers (see B. Jeong et al. Biomacromolecules 2002, 3, 865-868) and the encapsulation of bovine serum albumin in chemically crosslinked acrylate-PGA-PEO-PGA-acrylate macromonomers (see A.S. Sawhney et al. Macromolecules 1993, 26, 581-587)
This first non-covalent approach has several drawbacks: As the polymerization or crosslinking step to form the hydrogel has to be carried out in the presence of the biologically active moiety (i.e. a protein), the biologically active moiety is exposed to solvents and polymerization reaction conditions which may cause denaturation or chemical modification of the biologically active moiety. Furthermore, the quality of the end product is difficult to control and batch to batch variations may occur. Additionally, the loading of the hydrogels with the biologically active moiety is usually rather low ( <15 % protein) and is difficult to control.
A further drawback of the non-covalent type of the hydrogels is the so-called burst effect. The burst effect is characterized by a fast and uncontrolled initial release of a weakly bound biologically active moiety from the hydrogel. The initial burst release can account for up to 20 % of the encapsulated biologically active moiety.
As in PLGA precipitates, degradation of the hydrogels is required for release of the biologically active moiety from the crosslinked hydrogels. Self-assembled systems may also depend on degradation or desaggregation for release of the biologically active moiety. Degradation of the hydrogel increases the size of the pore to the extent that the biologically active moiety may diffuse out of the interior of the hydrogel into surrounding body fluids to exert its bioactivity. Degradation of the hydrogel is a process which is dependent on a number of parameters, some of which are not well understood. As the degradation of the hydrogel is dependent upon in vivo conditions, there may be significant contribution of complex biodegradation processes to the overall degradation of the hydrogel.
The small size of the pore may reduce the water content of the hydrogel and therefore its compatibility with fragile biomolecules.
It is difficult to optimize release kinetics in vivo which are in turn dependent on the conditions of the copolymerization process carried out in presence of the biologically active moiety. This poses altogether a significant obstacle for the successful development of these types of drug delivery systems.
Other inherent drawbacks of degradation-dependent release of the biologically active moiety are interpatient and injection site variability when the degradation is catalyzed by enzymes. Enzyme levels and specificities vary greatly between patients and also in dependence upon the tissue chosen for injection and other difficult to control parameters such as the depth of needle insertion. Furthermore, lack of control over degradation typically may lead to burst effects
Another complication lies in the fact that polymer degradation under in vivo conditions may also occur chemically, without the contribution of biological factors such as enzymes. For instance ester bonds typically employed as biodegradable bonds (cleavable by esterases but also certain proteases) may spontaneously hydrolyze at the biological pH of 7.4 in plain buffer in the absence of ester-cleaving proteins. Typically, microporous hydrogels require a high amount of ester bonds in order to effect efficient release of the biologically active moiety. Both the high local concentration of the ester bonds and the tight encapsulation of the biologically active moiety may lead to side reactions. It may be possible that an amino group present in the biologically active moiety may be positioned in proximity to an ester group, with the amino group providing a nucleophile effecting ester cleavage and subsequent amidation. This process results in a very stable amide linkage between the biologically active moiety and the polymer. The biologically active moiety will not be released until the polymer chain to which the biologically active moiety is attached to is degraded, and the biologically active moiety will be permanently modified. Such modifications are known to reduce bioactivity of the biologically active moiety and may also cause side effects, such as immunogenicity or cancerogenicity. In addition this undesirable modification process is largely uncontrolled and gives rise to a variety of molecular species. This type of side-reaction is described in Dong Hee Na et al.2003, J.Contr. Release 92, 291-299.
In the alternative covalent depot approach the biologically active moiety (such as a drag molecule) is reversibly attached to the hydrogel by a covalent or ionic linkage. In this case a hydrogel with mesopores (a so-called mesoporic hydrogel) can be used. The release of the biologically active moiety from the mesopores in the hydrogels is prevented by the attachment of the biologically active moiety.
There are only few examples describing this second approach of reversibly linking the biologically active moiety to the hydrogel.
J. Harris and X. Zhao (in European Patent No. EP 1 053 019 Bl) describe the reversible covalent attachment of a Iy sozyme protein to a hydrogel prepared by radical copolymerization of a PEO-diacrylate and a lysozyme-modified PEO-monoacrylate. The lysozyme was coupled to the PEO-monoacrylate via a thiourea group and a short biodegradable ester linker. Release of the lysozyme from the hydrogel was effected by incubation in pH 7 buffer.
Upon cleavage of the ester bond in the approach described in the '019 patent application, modified protein moieties are released as the cleaved linker moiety is still attached to the protein via the stable thiourea group. Furthermore, as the reaction of the activated linker-PEO- monoacrylate with the amino groups of the lysozyme is not regioselective, a variety of differently modified regioisomers are released, which is undesirable. Furthermore, as the encapsulation of the protein is upon hydrogel formation by radical polymerization, the protein can, in addition to the covalent attachment, also be encapsulated in pores of the formed three- dimensional network that are smaller than the diameter of the protein. Therefore, the release of the protein is not solely governed by cleavage of the linker but can also be influenced by the structure of the hydrogel.
Hubbell and coworkers (US Patent 23220245 Al) described in a similar approach the reversible attachment of a small synthetic peptide via a cysteine residue to a non¬ biodegradable hydrogel. The peptide was coupled to PEO-diacrylate by a Michael addition reaction. The peptide modified PEO-monacrylate was radically crosslinked with a PEG- diacrylamide to form a peptide modified hydrogel. Release of the propionyl-modified peptide was effected by incubation of the hydrogel in pH 7.4 buffer at 370C.
Due to the current use of microporous hydrogel materials in both non-covalent and covalent drug depots, numerous problems have hindered the development of a robust and reliable system for sustained drug delivery from a depot formed from a hydrogel.
Detailed Description of the Invention
It has now been surprisingly discovered, that mesoporous hydrogels can be used as polymer carriers for drug depots if provided as a carrier in a prodrug system.
The invention is directed towards mesoporous hydrogel prodrugs (MHP) of biologically active moieties, such as drag molecules. The MHP may be administered to a patient to form a depot inside the patient which provides for a sustained release of the biologically active moiety over a desired period of time.
A main advantage of the MHPs is to provide depot formulations without the need for encapsulation. Until now, many biocompatible hydrogel materials with large pore sizes could not be used for drag formulation due to their lack of encapsulating properties. In such biocompatible hydrogel materials, biologically active moiety would be released too fast for most therapeutic applications from such well-hydrated and mechanically soft materials. The provision of a hydrogel as prodrug carrier according to the invention allows the development of superior drug delivery systems. Carrier material properties such as biocompatibility (minimal irritation, immunogenicity, toxicity) may be optimized independently from the release properties of the biologically active moiety as the release properties are solely governed by the prodrug linker cleavage kinetics. The release of the biologically active moiety is therefore largely independent from the carrier material (i.e. the hydrogel) and does not require chemical or enzymatic degradation of the hydrogel.
The MHP system consists of three parts, a mesoporous hydrogel carrier, a prodrug linker and a biologically active moiety, such as a drug molecule. The prodrug linker is covalently bonded to the mesoporous hydrogel carrier and to the biologically active moiety in such a fashion that the biologically active moiety-linker-hydrogel conjugate is a carrier prodrug.
Biologically active moiety
Suitable biologically active moieties include but are not limited to small organic molecule bioactive agents, biopolymers like proteins, polypeptides and oligo- or poly-nucleotides (RNA, DNA), and peptide nucleic acids (PNA).
Suitable organic small molecule bioactive drugs include, without limitation, moieties such as central nervous system-active agents, anti-infective, anti-neoplastic, antibacterial, anti-fungal, analgesic, contraceptive, anti-inflammatory, steroidal, vasodilating, vasoconstricting, and cardiovascular agents. Non-exclusive examples of such compounds are daunorubicin, doxorubicin, idarubicin, mitoxantron, aminoglutethimide, amantadine, diaphenylsulfon, ethambutol, sulfadiazin, sulfamerazin, sulfamethoxazol, sulfalen, clinafloxacin, paclitaxel, moxifloxacin, ciprofloxaxin, enoxacin, norfloxacin, neomycin B, sprectinomycin, kanamycin A, meropenem, dopamin, dobutamin, lisinopril, serotonin, carbutamid, acivicin, etc.
Suitable proteins and polypeptides include but are not limited to ACTH, adenosine deaminase, agalsidase, albumin, alfa-1 antitrypsin (AAT), alfa-1 proteinase inhibitor (API), alteplase, anistreplase, ancrod serine protease, antibodies (monoclonal or polyclonal, and fragments or fusions), antithrombin IH, antitrypsins, aprotinin, asparaginases, biphalin, bone- morphogenic proteins, calcitonin (salmon), collagenase, DNase, endorphins, enfuvirtide, enkephalins, erythropoietins, factor VTfa, factor VDI, factor VHIa, factor IX, fibrinolysin, fusion proteins, follicle-stimulating hormones, granulocyte colony stimulating factor (G- CSF), galactosidase, glucagon, glucocerebrosidase, granulocyte macrophage colony stimulating factor (GM-CSF), phospholipase-activating protein (PLAP), gonadotropin chorionic (hCG), hemoglobins, hepatitis B vaccines, hirudin, hyaluronidases, idurnonidase, immune globulins, influenza vaccines, interleukins (1 alfa, 1 beta, 2, 3, 4, 6, 10, 11, 12), IL-I receptor antagonist (rhlL-lra), insulins, interferons (alfa 2a, alfa 2b, alfa 2c, beta Ia, beta Ib, gamma Ia, gamma Ib), keratinocyte growth factor (KGF), transforming growth factors, lactase, leuprolide, levothyroxine, luteinizing hormone, lyme vaccine, natriuretic peptide, pancrelipase, papain, parathyroid hormone, PDGF, pepsin, platelet activating factor acetylhydrolase (PAF-AH), prolactin, protein C, octreotide, secretin, sermorelin, superoxide dismutase (SOD), somatropins (growth hormone), somatostatin, streptokinase, sucrase, tetanus toxin fragment, tilactase, thrombins, thymosin, thyroid stimulating hormone, thyrotropin, tumor necrosis factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator (tPA), TSH, urate oxidase, urokinase, vaccines, plant proteins such as lectins and ricins.
Also included herein is any synthetic polypeptide or any portion of a polypeptide with in vivo bioactivity. Furthermore, proteins prepared by recombinant DNA methodologies including mutant versions of aforementioned proteins, antibody fragments, single chain binding proteins, catalytic antibodies and fusion proteins are included.
Linker
It is preferred for the linking agent to form a reversible linkage to the biologically active moiety, preferably in such a fashion that after cleavage of the linker, the biologically active moiety is released in an unmodified form. A variety of different linking agents or linking groups may be applied for this purpose as described by B. Testa et al. ( B . Testa, J. Mayer, Hydrolysis in Drag and Prodrug Metabolism, Wiley-VCH, 2003).
In a preferred embodiment the linker is a cascade prodrug linker constituted of a masking group and an activating group. The biologically active moiety is bound to the activating group, preferably through a carbamate bond. The release of the biologically active moiety is effected by a two-step mechanism. In the first step, the masking group is detached from the linker by cleavage of the bond connecting the masking group and activating group. The bond connecting the masking group and the activating group may also be a carbamate bond. Subsequently, in a second step, the bond between biologically active moiety and the activating group is cleaved, and the biologically active moiety is released. As this second step is faster than the first step , the cleavage of the masking group is the rate-limiting step of the release of the biologically active moiety.
The cleavage of the masking group is preferably based on a hydrolytic process, most preferably catalyzed by a nucleophile present in the masking group. In an autocatalytic fashion, this nucleophile attacks in an intramolecular fashion the carbon of the carbamate group constituting the linkage between the masking group and the activating group. The presence of the nucleophile in the vicinity of the carbamate group enhances the susceptibility of the carbamate group to hydrolysis. In a preferred embodiment, the nucleophile is a tertiary amine which does not undergo a reaction with the carbamate carbonyl and does not lead to a cyclization product.
Release of the biologically active moiety is initiated by an intramolecular rearrangement of the 1,6-elimination type, followed by autohydrolysis.
It is also preferred that at least part of the linker remains attached to the hydrogel after cleavage of the bond with the biologically active moiety. If the linker is a cascade prodrug linker, it is preferred for the activating group to remain stably bound to the hydrogel.
Reactive mesoporous hydrogel
Hydrogels are three-dimensional, hydrophilic or amphiphilic polymeric networks capable of taking up large quantities of water. The networks are composed of homopolymers or copolymers and are insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks. The crosslinks provide the network with structure and physical integrity.
Such reactive mesoporous hydrogels are characterized by the following structural components: crosslinking moiety, backbone moiety, reactive functional groups, pores, and optionally biodegradable bonds.
Backbone and crosslinking moieties
Non-limiting examples for suitable polymers for the synthesis of hydrogels are chemically or physically crosslinked functionalized or non-functionalized polyalkyloxy-based polymers like polyφropylene glycol) or poly(ethylene glycol), dextran, chitosan, hyaluronic acid and derivatives, alginate, xylan, mannan, caπageenan, agarose, cellulose, starch, hydroxyethyl starch (HES) and other carbohydrate-based polmers, polyvinyl alcohols), poly(oxazolines), poly (anhydrides), poly(ortho esters), polycarbonates), poly(urethanes), poly(acrylic acids), poly(acrylamides) such as poly(hydroxypropyhnethacrylamide) (HMPA), poly(acrylates), poly(methacrylates) like poly(hydroxyethylmethacrylate), poly(organophosphazenes), poly(siloxanes), polyvinylpyrrolidone), poly(cyanoacrylates), poly(esters) such as poly(lactic acid) or poly(glycolic acids), poly(iminocarbonates), poly(amino acids) such as poly(glutamic acid) or poly lysine, collagen, gelatin, copolymers, grafted copolymers, cross-linked polymers, hydrogels, and block copolymers from the above listed polymers.
These polymers may serve as backbone moieties or crosslinking moieties. In addition to oligomeric or polymeric crosslinking moieties, low-molecular crosslinking moieties may be used, especially when hydrophilic high-molecular weight backbone moieties are used for the hydrogel formation.
Suitable physical or chemical crosslinking methods are known to the person skilled in the art and are described in W.E. Hennink and CI7. van Nostrum, Adv. Drug Del. Rev.2002, 54, 13- 36.
Pores
The chains of the network in a mesoporous hydrogel are connected in such a fashion that pores exist in the hydrated state and that a substantial fraction of these pores are of dimensions between 1 and 100 nm.
The hydrogel is mesoporous with respect to the biologically active moiety (e.g. drug molecule) to be carried, Le. the average size of the pore of the hydrogel is larger than the diameter of the biologically active moiety. For instance a hydrogel which is mesoporous with respect to an insulin molecules has pores of more than 4 nm in size (in the hydrated state).
The dimensions of the pores may be controlled by adjusting both length of crosslinker and degree of crosslinking. For example, if small molecule crosslinkers are used for instance on a biopolymer such as dextran, porosity may be controlled through the degree of crosslinking. Usually, the lower the degree of crosslinking is the larger the size of the pores.
The size of the pore increases with crosslinker length. Crosslinker length refers to the spacer length between the two reactive groups used for the crosslinking of the backbone moiety. A typical polymeric crosslinker for the mesoporous hydrogel has at least two functional groups usually at the ends of the polymeric chain. The functional groups are usually connected by a linear or branched chain of MW between 500 and 50000. Such crosslinkers may be macromonomers in which case, the macromonomers are characterized by having at least two polymerizable functional groups.
Functional groups
The hydrogel is a functionalized material. The reactive functional groups serve as conjugation sites for the linker. Ideally, the reactive functional groups are dispersed homogeneously throughout the hydrogel, and may or may not be present on the surface of the hydrogel. Non-limiting examples of such reactive functional groups include but are not limited to carboxylic acid and activated derivatives, amino, maleimide, thiol, sulfonic acid and derivatives, carbonate and derivatives, carbamate and derivatives, hydroxyl , aldehyde, ketone, hydrazine , isocyanate, isothiocyanate, phosphoric acid and derivatives, phosphonic acid and derivatives, haloacetyl, alkyl halides, acryloyl and other alpha-beta unsaturated michael acceptors, arylating agents like aryl fluorides, hydroxylamine, disulfides like pyridyl disulfide, vinyl sulfone, vinyl ketone, diazoalkanes, diazoacetyl compounds, epoxide, oxirane, and aziridine.
Preferred functional groups for the polymer include but are not limited to thiol, maleimide, amino, carboxylic acid and derivatives, carbonate and derivatives, carbamate and derivatives, aldehyde, and haloacetyl.
In a preferred embodiment of the invention, the reactive mesoporous hydrogel is a shaped article such as a mesh or a stent. Most preferably, the hydrogel is formed into microparticulate beads which can be administered as subcutaneous or intramuscular injectably by means of a standard syringe. Such soft beads may have a diameter of between 1 and 500 micrometer. Biodegradable bonds
Biodegradability of the mesoporous hydrogel is of importance if the hydrogel is to be used for medical applications such as wound healing, wound sealing or for drug delivery (or, indeed, delivery of any type of biologically active moieties). In such applications, the hydrogel is administered by a subcutaneous or intramuscular injection or applied topically to a wound and left in the organism to be degraded in vivo and resorbed or excreted.
For biodegradability of the hydrogel, biodegradable bonds have to be incorporated into the backbone and/or crosslinking moieties. The susceptibility of these biodegradable bonds to cleavage under in vivo conditions may cause complete degradation of the hydrogel after a certain time period, which is desirable for the abovementioned applications in the medical field Cleavage of these biodegradable bonds may be enzymatically or chemically triggered or be a combination of both.
Biodegradable bonds which may be cleaved chemically under in vivo conditions include but are not limited to phosphate, phosphonate, carbonate, carbamate, disulfide and ester bonds.
There exists a huge variety of bonds which may be cleaved enzymatically. Hydrogels with biopolymer backbones or biopolymer crosslinkers are per se biodegradable on the surface of the hydrogel article if enzymes are present for which the backbone chains are substrates. The rate of degradation under in vivo conditions is different for every different type of hydrogel. In general, the degradation rate is a function of the degradability of the backbone (number of cleavable bonds, dependence of bond cleavage upon autohydrolysis or enzymatic catalysis) and the degree of crosslinking. Even though crosslinks do not directly contribute to the degradability of the hydrogel, the crosslinks can enable enzyme access into the hydrogel if the degree of crosslinking is small enough that the pores are large enough for the enzymes to penetrate into the hydrogel. Suitable biopolymers include but are not limited to carbohydrate- based polymers like dextran, chitosan, hyaluronic acid and derivatives, alginate, xylan, mannan, carrageenan, agarose, cellulose, starch, and hydroxyethyl starch and poly- or oligopeptide based oligomers or polymers like synthetic peptide sequences, collagen and gelatin.
MHP Preparation process
In order to guarantee that the prodrug of the biologically active moiety is only coupled to mesopores of the hydrogel, the biologically active moiety has to be reacted with the hydrogel after the hydrogel has been synthesized. This ensures that the release of the biologically active moiety from the hydrogel is governed by the prodrug linker and is independent from the optional hydrogel degradation. This is because after cleavage of the prodrug linker the biologically active moiety can freely diffuse out of the mesopores of the hydrogel.
A further advantage of this method of preparation is that reagents and solvents contacted with the hydrogel during the preparation of the hydrogel may be removed from the hydrogel after completion of the preparation by a filtration step. Efficient reagent and solvent removal avoid denaturation or modification of the biologically active moiety added to the hydrogel. Efficient reagent and solvent removal also avoids leakage of potentially toxic substances after administration to an organism.
Representative examples for the preparation of MHP are given in the expamples section. MHPs can also be prepared by several other methods.
To prepare a MHP (Method A in figure 1), a prodrug linker agent can be coupled to the reactive mesoporous hydrogel in a first reaction step. Such a suitable prodrug linking agent would carry two functional groups. The first one of the functional groups would serve as the attachment of the prodrug linker to the hydrogel, and the second one of the functional groups would subsequently be conjugated to the biologically active moiety through a suitable functional group present in the biologically active moiety.
Such first reactive functional groups should be complementary to a functional group present in the reactive mesoporous hydrogel. Non-limiting examples of such first reactive functional groups include but are not limited to carboxylic acid and activated derivatives, amino, maleimide, thiol, sulfonic acid and derivatives, carbonate and derivatives, carbamate and derivatives, hydroxyl , aldehyde, ketone, hydrazine , isocyanate, isothiocyanate, phosphoric acid and derivatives, phosphonic acid and derivatives, haloacetyl, alkyl halides, acryloyl and other alpha-beta unsaturated Michael acceptors, arylating agents like aryl fluorides, hydroxylamine, disulfides like pyridyl disulfide, vinyl sulfone, vinyl ketone, diazoalkanes, diazoacetyl compounds, epoxide, oxirane, and aziridine. Preferred first functional groups of the prodrug linker include thiol, maleimide, amino, carboxylic acid and derivatives, carbonate and derivatives, carbamate and derivatives, aldehyde, and haloacetyl.
After activation of the second one of the functional groups of the prodrug linker, the linker- hydrogel conjugate may be contacted with the biologically active moiety in the second reaction step and excess biologically active moiety (e.g. excess drug) may be removed by filtration after conjugation of the biologically active moiety to the hydrogel-bound prodrug linker. Despite the large size of the pore of the hydrogel, the biologically active moiety remains bound inside the hydrogel by the covalent attachment of a suitable functional group present on the biologically active moiety to the second functional group of the prodrug linker.
Suitable second functional groups of the prodrug linker include but are not limited to carboxylic acid and derivatives, carbonate and derivatives, hydroxyl, hydrazine, hydroxylamine, maleamic acid and derivatives, ketone, amino, aldehyde, thiol and disulfide.
Suitable functional groups present on the biologically active moiety include but are not limited to thiol, carboxylic acid, amino, hydroxyl, ketone, imidazole.
Optionally this reaction sequence may be inverted, and the prodrug linker may be first conjugated to the biologically active moiety and the resulting biologically active moiety - prodrug linker conjugate may then react with the reactive mesoporous hydrogel (Method B in figure 1).
These preparation methods are shown schematically in figure 1.
Reactive mesoporous hydrogel synthesis process
Hydrogels which are reactive and mesoporous may be prepared by a variety of different methods. One particular synthesis process is based on using a crosslinking macromonomer carrying at least two polymerizable functional groups and a non-crosslinking macromonomer or monomer carrying one polymerizable functional group and at least one functional group which is not intended to participate in the polymerization step. Additional diluent monomers may or may not be present. Copolymerization of these components results in a hydrogel containing functional groups provided by the non-crosslinking macromonomer. In order to ensure that the functional group is available for reactions after completion of the polymerization, the conditions for polymerization are chosen such that the functional group is not modified. Alternatively, the functional group may be protected by use of a reversible protecting group known to the person skilled in the art, which is removed after the polymerization.
Useful polymerizable functional groups include but are not limited to radically polymerizable groups like vinyl, vinyl-benzene, acrylate, acrylamide, methacylate, methacrylamide and ionically polymerizable groups like oxetane, aziridine,and oxirane.
In an alternative method of preparation, the hydrogel is generated through chemical ligation reactions. The hydrogel may be formed from two macromolecular educts with complementary functionalities which undergo a reaction such as a condensation or addition. One of these starting materials is a crosslinker with at least two identical functional groups and the other starting material is a homomultifunctional backbone structure. Suitable functional groups present on the crosslinker include terminal amino, carboxylic acid and derivatives, maleimide and other alpha,beta unsaturated Michael acceptors like vinylsulfone, thiol, hydroxyl groups Suitable functional groups present in the backbone structure include but are not limited to amino, carboxylic acid and derivatives, maleimide and other alpha,beta unsaturated Michael acceptors like vinylsulfone, thiol, hydroxyl groups.
If the crosslinker reactive functional groups are used substoichiometrically with respect to backbone reactive functional groups, the resulting hydrogel will be a reactive hydrogel with free reactive functional groups attached to the backbone structure.
MHP with staged release and degradation kinetics
This invention also includes degradable mesoporous hydrogel prodrugs exhibiting minimal release of biologically active moiety conjugated to hydrogel degradation products.
In general it is of advantage to limit the structural diversity of degradation products of a polymeric drug formulation with respect to chain lengths, substitutions or modifications. Specifically, the release of biologically active moiety-polymer conjugates should be avoided. Hydrogel degradation may result in the release of degradation product conjugates of the biologically active moiety tethered to degradation products by means of the prodrug linker if degradation kinetics of the hydiogel are of a similar order as prodrug cleavage kinetics. The degradation product conjugates (shown schematically in figure 4) are undesired modifications of the biologically active moieties. Only few of these degradation product conjugates will appear if the cleavage of degradable hydrogel is at least one order of magnitude slower than the release of the biologically active moeitey. There may, however, be applications, such as weekly injections of MHP in which a more rapid disintegration of the hydrogel is desirable.
It has now been surprisingly found that through controlling the degree of crosslinking, the degree of chain length of the backbone polymer, the positioning of degradable bonds and the site of the prodrug linker, the release kinetics of the resulting degradation products from the hydrogel can be fine-tuned and the heterogeneity of degradation products can be minimized.
In such a hydrogel with controlled degradation properties, the degradable bonds are exclusively located in the crosslinking chains. In a most preferred embodiment there are precisely two degradable bonds per linear crosslinking chain and they are positioned between the chain and backbone attachment site. The crosslinker carrying the degradable bonds is symmetrical with respect to the degradable bonds, rendering the bonds chemically identical. Such a biodegradable mesoporous hydrogel prodrug is shown schematically in figure 2.
Functional groups for attachment of the prodrug linker are positioned on side chains emanating from the backbone polymer. There are no degradable bonds between these functional groups and the non-biodegradable backbone or in the non-biodegradable backbone itself.
Cleavage of the degradable bonds of the crosslinker results in a two-stage process. Shortly after initial time only cleavage products containing crosslinker units are released from the hydrogel when each non-biodegradable backbone is linked with at least one other non¬ biodegradable backbone via several crosslinkers. If, for instance, esters of PEG have been used, the released compound is PEG. The release of PEG from this type of hydrogel follows approximately first order kinetics.
After a certain lag time, backbone structures are released. Unlike the release of crosslinker cleavage product, the release of the backbone structures follows sigmoidal kinetics. The released backbone is a linear chain substituted with residues formerly connected to crosslinking moieties (for instance side chains terminating with carboxylic acids) and the functional group moiety. Figure 3 shows schematically a partially degraded (figure 3a) and a fully degraded (figure 3b) MHP.
It is desirable to prolong the lag time to such an extent, that almost all drug release ( >90 %) has occurred before a significant amount of release of the backbone ( <10 %) has taken place. This lag time can be controlled by adjusting the number of crosslinks and the halflife of the biodegradable bond. The lag time can be increased by incorporating more crosslinks per backbone and increasing the half-life of the biodegradable bond. The effect of an increased lag time by increasing the number of crosslinks is shown in figure 10.
Brief description of the figures:
Fig. 1 shows the preparation process for mesoporous hydrogel prodrugs
Fig.2 shows schematically the structure of biodegradable mesoporous hydrogel prodrugs
Fig. 3 shows the degradation process of biodegradable mesoporous hydrogel prodrugs
Fig.4 shows schematically anundesired polymer modified prodrug
Fig.5 shows the insulin release from a polyacrylamide-based mesoporous hydrogel prodrug
Fig.6 shows the insulin release from a carbohydrate-based mesoporous hydrogel prodrug
Fig.7 shows the in vivo release of insulin from mesoporous hydrogel prodrugs
Fig. 8 shows the insulin release from a biodegradable mesoporous hydrogel prodrug
Fig.9 shows the LCMS characterization of released insulin after explantation
Fig. 10 shows the degradation of biodegradable hydrogels
EXAMPLES
Maleimide derivatization of polyacrylamide based non-biodegradable reactive mesoporous hydrogel (amino-PEGA)
Figure imgf000021_0001
Non-biodegradable mesoporøus NH2-PEGA hydrogel beads with 0.4 mmol/g loading and 150-300 μm bead size were purchased from Novabiochem. NH2-PEGA Versamatrix-800 hydrogel beads with 0.31 mmol/g loading and 80-100 μm bead size were obtained from Versamatrix (Denmark).
2.5 g methanol-wet NH2-PEGA-hydrogel (0.4 mmol/g NH2-loading) was weighed into a syringe equipped with a polypropylene frit. Maleimide loading was adjusted by acylation employing a mixture of activated maleimidopropionic acid and acetic acid as described in the following. The hydrogel was washed 5 times with DMF and reacted with 13.5 mg (0.08 mmol) 3-maleimidopropionic acid, 115.2 μl (1.92 mmol) acetic acid and 313 μl (2 mmol) DIC in 4 ml DMF for 30 min. The maleimide derivatized hydrogel Ia was washed 10 times with DMF and DCM and finally with acetonitrile.
Hydrogel Ib was synthesized following the protocol above with the following modification. 2.5 g methanol-wet NH2-PEGA-hydrogel (~250 mg dry resin) was reacted with 6.8 mg (0.04 mmol) 3-maleimidopropionic acid, 117.6 μl (1.96 mmol) acetic acid and 313 μl (2 mmol) DIC in 4 ml DMF for 30 min. Finally the hydrogel was washed as described. Hydrogel Ic was synthesized following the protocol above with the following modification. 2 g methanol-wet NH2-PEGA Versamatrix-800 hydrogel (0.31 mmol/g NH2-loading, dry resin) was washed as described and reacted with 25.3 mg (0.15 mmol) 3-maleimidoproρionic acid, 115.2 μl (1.85 mmol) acetic acid and 313 μl (2 mmol) DIC in 4 ml DMF for 30 min. Finally the hydrogel was washed as described.
Synthesis of rh-insulin loaded PEGA hydrogel 4 and 5a,b
Figure imgf000022_0001
Figure imgf000022_0002
4 5a,b
2 and 3 were synthesized as described in co-pending UK patent application No.0415043.9 30 mg of maleimide derivatized resin Ib (loading 15 μmol/g, 450 nmol) was reacted with 3 mg of compound 2 (480 nmol, 1.06 eq) in 600μl 20/80 (v/v) acetonitrile/50 mM phosphate buffer (pH 7.4) for lOmin to give rh-insulin loaded hydrogel 4. The hydrogel 4 was washed 5 times with 50/50 (v/v) acetonitrile/water and three times with acetonitrile and dried under vacuum.
Synthesis of hydiogel 5a or 5b by reacting 1 equivalent compound 3 in relation to the theoretical amount of maleimide groups on the hydrogel with hydrogel Ib or Ic, respectively, followed the synthesis protocol above.
In vitro rh-insulin release experiments from non-biodegradable mesoporous hydrogel prodrug 4
hydrogel
Figure imgf000023_0001
Figure imgf000023_0002
4 mg of 4 was weighed into a test tube and incubated with 1000 μl 10 mM HEPES buffer pH 7.4, 150 mM NaCl, 0.005 % Tween at 370C. 45μl samples were taken at regular intervals and quantitatively analyzed for rh-insulin by a RP-HPLC assay. The rh-insulin peak was integrated and rh-insulin concentration was obtained from a standard curve. A first order release kinetic was fitted to the data points to give the linker half life and maximal rh-insulin release at t =∞ (figure 5). Release of rh-insulin from PEGA hydrogel 5a or 5b was carried out as described above. In vivo release experiments of rh-insulin from non-biodegradable mesoporous hydrogel prodrugs 4 and 5a
In vivo studies were conducted at the ,,Steinbeis-Transfer-Zentrum Biopharmazie und Analytik - Heidelberg" in male and female Wistar rats.
The rats weighing 200-300 g were kept under standard conditions and fed ad libitum. Blood samples (150μl) were collected from the retro-orbital plexus and a suspension of rh-insulin loaded hydrogel in 300-400μl PBS was subcutaneously administered in the upper hind leg area. Blood was drawn at different times after administration. Animals were lightly anesthetized by inhaled isoflurane during all blood draws and injections. All blood samples were collected into tubes containing EDTA and centrifuged. Plasma was separated and stored at -180C until assayed, rh-insulin concentration was determined from plasma samples using the species specific sandwich human insulin ELISA kit (Mercodia, Sweden). The results were statistically analyzed and plasma rh-insulin concentrations were plotted over time after administration (figure 7).
Hydrogel Sample Hydrogel dose /rat Total rh-insulin dose /rat N rats volume (calculated from in vitro release)
5a 400 μl 9 mg -26 nmol 3
4 300 ul 10.7 mg -38 nmol 2
Explantation of administered mesoporous hydrogel prodrug 5b and investigation of rh- insulin integrity
Hydrogel administration, blood sampling and determination of the rh-insulin concentration followed the protocol described above.
Hydrogel Sample Hydrogel dose Total rh-insulin dose / rat N rats sample volume (calculated from in vitro release) 5b 400 μl 16 mg -138 nmol 1 After 6 days the rat was sacrificed. The hydrogel was explanted and washed 10 times with water, 10 times with 50/50 (v/v) acetonitrile/water and dried under vacuum. About 1 mg of hydrogel was weighed into a test tube and incubated with 400 μl 100 mM Tris/HCl buffer pH 9, at 370C. After 24 h the solution was separated from the hydrogel and analyzed for rh- insulin and enzymatic degradation products thereof by LC/MS (figure 9). The strong peak at 4.2 min elution time can be assigned to rh-insulin according to the mass spectrum. Smaller peaks before and after the rh-insulin peak correspond to the A- and B-chain of rh-insulin, respectively. No rh-insulin related degradation products were detected. A- and B-chain were also detected in in vitro release experiments at pH 9 and thus do not indicate enzymatic degradation.
Synthesis of biodegradable reactive mesoporous hydrogel 8
Synthesis of macromonomerbis-acryl-glvcyl-PEGgOO (6)
HO °v O- OH PEG900
Gly-OH / DCC / DMAP
Figure imgf000025_0001
Figure imgf000025_0002
acryl chloride
Figure imgf000025_0003
Figure imgf000025_0004
10 g PEG900 (11.1 mmol) were dissolved in 50 ml DCM and the solution was cooled to 00C. A solution of 3.9 g (23.3 mmol) boc-glycine, 5.67 g (46.6 mmol) DMAP, and 950 mg DCC (4.6 mmol) in 50 ml DCM was added and the mixture was stirred over night at room temperature. The solid byproduct dicyclohexyl urea was filtered off and the filtrate was washed two times with 1 M HCl and two times with water. The organic phase was dried over sodium sulfate and was concentrated in vacuo to approximately 50 ml.50 ml TFA were added at 0°C and the solution was stirred at room temperature for 30 min to remove the boc protecting groups. The solvent was removed under reduced pressure and the resulting oil was redissolved in 50 ml DCM.5 ml 2 M HCl in diethyl ether was added and the product precipitated by the addition of 400 ml diethyl ether and collected by centrifugation. The resulting oil was dissolved in 100 ml 0.1 M aqueous HCl and lyophilized (yield: 9.5 g) LC/MS:
[M-IHJ+ =882.3, 926.4, 970.4, 1014.4, 1058.4, 1102.5, 1146.6 (MW +H calculated =1014.2 -y- x*44.05 g/mol)
1.4 g bis-glycyl-PEG900 were dissolved in 15 ml DCM and 750 μl (5.32 mmol) triethylamin and 220 μl (2.66 mmol) acryl chloride were added at 00C. The mixture was stirred at room temperature for 30 min and 35 ml DCM were added. The organic phase was washed with 1 M aqueous HCl and 5 times with water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure to approximately 10 ml. The product 6 was precipitated by the addition of 60 ml 1/1 (v/v) diethyl ether/heptane and collected by centrifucation (yield
1.05 g).
LC/MS:
[MHNa]+ =1012.0, 1056.6, 1100.5, 1144.5, 1188.6, 1232.5, 1275.6, 1319.7 (MW +Na calculated =1144.2 4/- x*44.05 g/mol) Synthesis of N-bocJST "-acrvlovl-O.lO-trioxatridecane-lΛS-diamine
Figure imgf000027_0001
acryl chloride / triethyl amine
Figure imgf000027_0002
Figure imgf000027_0003
2 g (9.1 mmol) 4,7,10-trioxatridecane-l,13-diamine were dissolved in 15 ml DCM and 1 g (4.6 mmol) di-tert.-butyl-dicarbonate in 10 ml DCM was added dropwise at 00C. The solution was stirred for 2 h at room temperature and the organic phase was washed five times with water. The organic phase was dried over sodium sulfate and concentrated under reduced pressure to approximately 10 ml. Mono-boc-4,7,10-trioxatridecane-l,13-diamine was precipitated as hydrochloride salt by the addition of 2 M HCl in diethyl ether (yield: 1.1 g, 3.1 mmol, 67 %).
1.1 g (3.1 mmol) mono-boc-4,7,10-trioxatridecane-l,13-diamine was dissolved in 10 ml DCM and 900 μl (6.2 mmol) triethylamine was added. The mixture was cooled to 00C and 260 μl (3.2 mmol) acryl chloride in 10 ml DCM was added dropwise. The solution was stirred at room temperature for 30 min and the organic phase was washed two times with 0.1 M aqueous HCl. The organic phase was dried over sodium sulfate and concentrated to 7 ml under reduced pressure. The product 7 was precipitated by the addition of 50 ml 1/1 (v/v) diethyl ether/heptane and collected by centrifugation. (yield: 940 mg, 2.5 mmol, 78 %). LC/MS: [M-INa]+ =398.1 (MW +Na calculated =397.4 g/mol) Biodegradable reactive mesoporous hvdrogel formation
biodegradable NH, hydrogel
Figure imgf000028_0001
100 mg (100 μmol) compound 6, 6 mg (15 μmol) compound 7 and 21 mg (212 μmol) NJST- dimethylacrylamide were dissolved in 500 μl 50 mM phosphate buffer (pH 7.0) in a test tube. After addition of 30 μl 1 M ammonium peroxodisulfate (APS), the solution was vortexed and polymerization was initiated by addition of 80 μl 2 M N,N,N',N'-tetra- methylethylenediamine (TEMED) / HCl, pH 7.0. The spontaneously formed hydrogel was incubated for further 30 min, ground to particles <lmm and transferred into a syringe equipped with a polypropylene frit. After extensive washing of the hydrogel with water, DMF and DCM (5 times each), boc-protecting groups were cleaved by incubation with (v/v) 50/50 TFA/DCM for lOmin. Finally, the hydrogel 8 was washed five times with DCM, five times with DMF, once with 1/99 (v/v) DIEA/DMF and five times with DMF. Synthesis of maleimide derivatized biodegradable polvacrylamide based mesoporous hvdrogel 9
biodegradable hydrogel
Figure imgf000029_0001
9
100 mg ground biodegradable hydrogel 8 was weighed into a syringe equipped with a polypropylene frit. Maleimide derivatization was achieved by acylation with 101 mg (0.6 mmol) maleimidopropionic acid, 94 μl (0.6 mmol) DIC in 2 ml DMF for 30min to give maleimide derivatized hydrogel 9. The hydrogel was washed 10 times with DMF and DCM.
Synthesis of fluorescein-carboxamido-Lvs(B29)-rh-insulin loaded biodegradable polyacrylamide based mesoporous hydrogel prodrug 11
biodegradable hydrogel JL-*
Figure imgf000029_0002
pH 7.0
Figure imgf000029_0003
Compound 10 was synthesized as described in co-pending UK patent application No. 0415043.9, the disclosure of which is incorporated by reference. 025 mg of compound 10 (35 nmol) was dissolved in 100 μl 40/40/20 (v/v/v) acetonitrile/water/0.5 M phosphate buffer pH 7.0. The solution was incubated for 3 min with 5.6 mg of maleimide derivatized biodegradable hydrogel 9 to give fluorescein-carboxamido- Lys(B29)-rh-insulin loaded hydrogel 11. The hydrogel 11 was washed five times with 50/50 (v/v) acetonitrile/water, three times with acetonitrile and dried under vaccuum.
In vitro fluorescein-carboxamido-Lvs(B29)-rh-insulin release experiments from biodegradable polyacrylamide based mesoporous hvdrogel prodrug 11
rh
Figure imgf000030_0001
The fluorescem-carboxamido~Lys(B29)-m-msulin loaded biodegradable hydrogel 11 was incubated in 100 μl 10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 0.005 % Tween at 37°C. 80 jtil samples were taken at regular intervals and quantitatively analyzed for fluorescein- carboxamido-Lys(B29)-rh-insulin by a RP-HPLC assay. A first order release kinetic was fitted to the data points to give the half life of the prodrug linker and maximal fluorescein- carboxamido-Lys(B29)-rh-insulin concentration at t =∞ (figure 8). The integrity of the released fluorescein-carboxamido-Lys(B29)-rh-insulin 12 was confirmed by RP-LCMS and SEC (data not shown).
Synthesis of fluorescein labeled compound 12
Figure imgf000031_0001
03 g Sieber amide resin (loading 0.5 mmol/g) was weighed into a syringe equipped with a polypropylene frit. Fmoc-removal was achieved by incubation in 2/2/96 (v/v/v) piperidin/DBU/DMF for 10 min and the resin was washed 5 times with DMF. The resin was incubated for 1 h with 264 mg (0.45 mmol) Fmoc-Cys(Trt)-OH, 144 mg (0.45 mmol) TBTU and 157 μl DIEA (0.9 mmol) in 3 ml DMF and washed 5 times with DMF. After fmoc- removal, Fmoc-Ado-OH was coupled by incubation of 156 mg (0.45 mmol) Fmoc-Ado-OH 173 mg (0.45 mmol) TBTU and 157 μl DIEA (0.9 mmol) in 3 ml DMF according to the procedure above. Fmoc was removed and the resin was reacted with 338 mg (0.9 mmol) 5,6- carboxyfluorescein (isomeric mixture), 140 mg (0.9 mmol) HOBt and 141 μl (0.9 mmol) DIC in 3 ml DMF for 2 h. Finally the resin was incubated in 2/2/96 (v/v/v) piperidine/DBU/DMF for 10 min, washed 10 times in DCM and dried under vacuum.
12 was cleaved from the resin with 50/5/45 (v/v/v) TFA/TES/DCM for 30 min and purified by RP-HPLC.
MS: [MH]+ =625 g/mol (MW calculated =624 g/mol) Degradation of biodegradable polvacrylamide-based mesoporous hvdrogel 13 in vitro
biodegradable + HS-R1 hydrogel
Figure imgf000032_0001
Figure imgf000032_0002
biodegradable hydrogel
Figure imgf000032_0003
13
pH 9.0, 37 0C
Figure imgf000032_0004
A 100 μM solution of compound 12 (35 nmol) in 500 μ\ 40/40/20 (v/v/v) acetonitrile/water/0.5 M phosphate buffer pH 7.0 was reacted with 5 mg of maleimide derivatized biodegradable hydrogel 9 for 5min to give the fluorescein labeled hydrogel 13. The hydrogel 13 was washed 5 times with 50/50 (v/v) acetonitiile/water and three times with acetonitrile and dried under vaccuum.
Hydrogel degradation experiment was performed at pH 9 to reduce the time of degradation. Degradation time at physiological pH 7.4 was estimated by a scaling factor 40 that accounts for the approx.40 fold increased hydroxide ion concentration at pH 9 compared to pH 7.4. The hydrogel was suspended in 1 ml 50 mM borate buffer (pH 9.0), 150 mM NaCl, 0.005 % Tween and incubated at 370C.60 μl samples were taken at different time intervals and quantitatively analyzed for polymer backbone coupled fluorescein 14 by photometry at 500 nm. The data show a delayed and sigmoidal release of 14 (data not shown).
Synthesis of rh-insulin loaded carbohydrate-based mesoporous hydrogel prodrug 16 and in vitro release
NHS-activated "Sepharose 4 Fast Flow" hydrogel beads (chemically crosslinked agarose, crosslinker epichlorhydrin) were purchased from Amersham.
1.5 g ethanol-wet Sepharose hydrogel (150 mg dry hydrogel) was weighed into a syringe equipped with a polypropylene frit and reacted with 1 M 4,7,10-trioxatridecan-l,13-diamin in DMF for 30 min. After 5 washing steps with DMF, hydrogel was reacted with 8.5 mg (0.05 mmol) 3-maleimidopropionic acid, 57 μl (0.95 mmol) acetic acid, 151 mg (1 mmol) HOBt and 158 μl (1 mmol) DIC in 4 ml DMF for 30minto give maleimide derivatized hydrogel 15. The hydrogel 15 was washed 10 times with DMF and finally with acetonitrile. 1.5 mg 3 was dissolved in 25/75 (v/v) acetonitrile/50 mM phosphate buffer pH 7.4 and reacted with 10.8 mg maleimide derivatized hydrogel 15 for 10 min. The rh-insulin loaded hydrogel 16 was washed five times with 50/50 (v/v) acetonitrile/water and three times with acetonitrile and dried under vacuum.
2 mg rh-insulin-loaded hydrogel 16 was suspended in 1000 μl 10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 0.005 % Tween and incubated at 370C. 60 μl samples were taken at regular intervals and quantitatively analyzed for rh-insulin by an RP-HPLC assay. A first order release kinetic was fitted to the data points to give the half life of the prodrug linker and maximal rh-insulin release at t =∞ (figure 6). The integrity of the released rh-insulin was confirmed by LCMS (data not shown). Synthesis of biodegradable mesoporous hydrogel 20 and 21
Synthesis of bis-mercaptoacetyl-glvcyl-PEGgOO (17)
Figure imgf000034_0001
17
1 g (0.92 mmol) bis-glycyl-PEG900 x 2HCl was dissolved in 7 ml DMF and 600 mg (2 mmol) SAMA-OPfp ester was added. The mixture was stirred at room temperature for 60 min.200 μl hydrazin hydrate was added and the mixture stirred for 5 min at room temperature to remove the acetyl group. After addition of 400 μl acetic acid the product 17 was purified by RP-HPLC and lyophilized (yield 740 mg).
LC/MS:
[MHH]+ =1030.2, 1074.2; 1118.3; 1162.2; 1206.3; 1250.3; 1294.4, 1338.4 (MW +H calculated =1161.4 H/- x*44.05 g/mol)
Synthesis of l-røphtylacetyl-tetra-Lvs(Ado-mp)-amide (18) and 1-naphtylacetyl-octa- Lvs(Ado-mp)-amide (19)
Figure imgf000035_0001
18 was synthesized by standard solid-phase organic synthesis using TBTU/DIEA activation as described for compound 12.
To 1 g TentaGel Sieber amide resin (0.17 mmol/g loading) four times Fmoc-Lys(ivDde)-OH and 1-naphthyl acetic acid was coupled by TBTU/DIEA activation using 3 equivalents amino acid in relation to free amino groups on the resin. To remove the ivDde protecting group Hie resin was incubated three times with 4 % hydrazine in DMF for 7 min. After washing of the resin with DMF, Fmoc-Ado-OH was coupled to the amino groups by TBTU/DIEA activation. 3-maleimidopropionic acid was coupled by DIC activation using 3 equivalents 3- maleimidopropionic acid in relation to free amino groups on the resin. Compound 18 was cleaved from the resin by incubation with 94/3/3 (v/v/v) DCM/triethylsilane/TFA for 60 min. After evaporation of the solvent, 18 was purified by RP-HPLC and lyophilized.
19 was synthesized as described above by coupling eight Fmoc-Lys(ivDde)-OH residues instead of four residues.
LC/MS: n =4:
[M-IHl+ =1883.9; [MHNa]+ =1905.2 (MW calculated =1879.1 g/mol)
n =8:
[M-SNa]2+ =1812; [MHH-INa]2+ =1800; [M +2H]2+ =1790 (MW calculated =3573.0 g/mol) Biodegradable hydrogel 20 and 21 formation
Figure imgf000036_0001
232 mg (20 μmol) 17 and 18.8 mg (10 μmol) 18 were dissolved, in 150 μl water and 50 μl 0.5
M sodium acetate buffer pH 5.5 were added. The solution was incubated at room temperature for 60 min.
The resulting hydrogel 20 was ground to particles <lmm and transferred into a syringe equipped with a polypropylene frit. The hydrogel particles were washed five times each with
1/1 acetonitrile/water, water, and methanol and then dried under vacuum.
Hydrogel 21 was synthesized as described above using 23.3 mg (20 μmol) 17 and 17.9 mg (5 μmol) 19.
Degradation study
20 or 21
Figure imgf000037_0001
Hydrogel degradation experiments were performed at pH 9 to reduce the time of degradation. Degradation time at physiological pH 7.4 was estimated by a scaling factor 40 that accounts for the AO fold increased hydroxide ion concentration at pH 9 compared to pH 7.4. 50 mg of hydrogel 20 or 21 was suspended in 1 ml 50 mM sodium borate buffer (pH 9.0), 150 mM NaCl, 0.005 % Tween and incubated at 37°C.30 μl samples were taken at regular intervals and quantitatively analyzed for polymer backbone coupled naphthyl 22 or 23 by photometry at 280 run (figure 10). The degradation kinetic shows a sigmoidal curve.
Abbreviation:
Ado 8-amino-3 ,6-dioxa-octanoyl
Boc t-butyloxycarbonyl
DBU 1 ,3-diazabicyclo[5.4.0]undecene
DCM dichloromethane
(iv)Dde l-(4,4-dimethyl-2,6-dioxo-cyclohexyliden)3-methyl-butyl
DIC diisopropylcarbodiimide
DIEA diisopropylethylamine
DMF NJST-dimethylformamide
EDTA ethylenediaminetetraacetic acid fmoc 9-fluorenyhnethoxycarbonyl
Fmoc-Ado-OH Fmoc-8-amino-3,6-dioxaoctanoic acid
HEPES N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid)
HOBt N-hydroxybenzotriazole
LCMS mass spectrometry-coupled liquid chromatography mp 3-maleimidopropionyl
MS mass spectrum
MW molecular mass
PEG poly(ethylene glycol)
RP-HPLC reversed-phase high performance liquid chromatography
RT room temperature
SAMA-OPfp S-acetyl-mercaptoacetic acid pentafluorophenyl ester
SEC size exclusion chromatography
TBTU O-CBenzotriazol-l-yO-NJISrjSr'JSf -tetramethyluronium tetrafluoroborate
TES triethylsilane
TFA trifluoroacetic acid

Claims

Claims
1. A polymeric prodrug comprising a hydrogel, a biologically active moiety and a reversible prodrug linker, wherein - the prodrug linker covalently links the hydrogel and the biologically active moiety at a position; and
- the hydrogel has a plurality of pores with openings on the surface of the hydrogel, wherein the diameter of the pores is larger than the biologically active moiety at least at all points of the pore between at least one of the openings and the position of the biologically active moiety.
2. The polymeric prodrug according to claim 1 , wherein the biologically active moiety is a biopolymer.
3. The polymeric prodrug according to claim 1, wherein the biologically active moiety is selected from the group of proteins or polypeptides consisting of ACTH, adenosine deaminase, agalsidase, albumin, alfa-1 antitrypsin (AAT), alfa-1 proteinase inhibitor (API), alteplase, anistreplase, ancrod serine protease, antibodies (monoclonal or polyclonal, and fragments or fusions), antithrombin III, antitrypsins, aprotinin, asparaginases, biphalin, bone-morphogenic proteins, calcitonin (salmon), collagenase, DNase, endorphins, enfuvirtide, enkephalins, erythropoietins, factor Vila, factor VIII, factor Villa, factor DC, fibrinolysin, fusion proteins, follicle-stimulating hormones, granulocyte colony stimulating factor (G-CSF), galactosidase, glucagon, glucocerebrosidase, granulocyte macrophage colony stimulating factor (GM-CSF), phospholipase-activating protein (PLAP), gonadotropin chorionic (hCG), hemoglobins, hepatitis B vaccines, hirudin, hyaluronidases, idurnonidase, immune globulins, influenza vaccines, interleukins (1 alfa, 1 beta, 2, 3, 4, 6, 10, 11, 12), IL-I receptor antagonist (rhlL-lra), insulins, interferons (alfa 2a, alfa 2b, alfa 2c, beta Ia, beta Ib, gamma Ia, gamma Ib), keratinocyte growth factor (KGF)5 transforming growth factors, lactase, leuprolide, levothyroxine, luteinizing hormone, lyme vaccine, natriuretic peptide, pancrelipase, papain, parathyroid hormone, PDGF, pepsin, platelet activating factor acetylhydrolase (PAF-AH), prolactin, protein C, octreotide, secretin, sermorelin, superoxide dismutase (SOD), somatropins (growth hormone), somatostatin, streptokinase, sucrase, tetanus toxin fragment, tilactase, thrombins, thymosin, thyroid stimulating hormone, thyrotropin, tumor necrosis factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator (tPA), TSH, urate oxidase, urokinase, vaccines, and plant proteins such as lectins and ricins.
4. The polymeric prodrug of claim 1 , wherein the biologically active moiety is insulin.
5. The polymeric prodrug according to claim 1, wherein the biologically active moiety is an organic small molecule bioactive agent.
6. The polymeric prodrug according to claim 5, wherein the biologically active moiety is selected from the group of organic small molecule bioactive agents consisting of central nervous system-active agents, anti-infective, anti-neoplastic, antibacterial, anti-fungal, analgesic, contraceptive, anti-inflammatory, steroidal, vasodilating, vasoconstricting, and cardiovascular agents.
7. The polymeric prodrug of claim 1 , wherein the biologically active moiety is a an anti-sense or interfering oligonucleotide.
8. The polymeric prodrug of any of the above claims, wherein the polymeric prodrug is a cascade prodrug.
9. The polymeric prodrug of any of the above claims wherein the prodrug linker comprises a carbamate group.
10. The polymeric prodrug of any of the above claims, wherein the hydrogel is synthesised from the group of polymers consisting of polyalkyloxy-based polymers like poly(propylene glycol) or poly(ethylene glycol), dextran, chitosan, hyaluronic acid and derivatives, alginate, xylan, mannan, carrageenan, agarose, cellulose, starch, hydroxyethyl starch (HES) and other carbohydrate-based polmers, poly(vinyl alcohols), poly(oxazolines), poly(anhydrides), poly(ortho esters), poly(carbonates), poly(urethanes), poly(acrylic acids), poly(acrylamides) such as poly(hydroxypropylmethacrylatnide) (HMPA), poly(acrylates), poly(methacrylates) like poly(hydroxyethylmethacrylate), poly(organophosphazenes), poly(siloxanes), poly(vinylpyrrolidone), poly(cyanoacrylates), poly(esters) such as poly(lactic acid) or poly(glycolic 5 acids), poly(iminocarbonates), poly(amino acids) such as poly(glutamic acid) or poly lysine, collagen, and gelatin, copolymers, grafted copolymers, cross-linked polymers, and block copolymers from the above listed polymers.
11. The polymeric prodrug of any of the above claims, wherein the hydrogel is 10 polyacrylamide or a derivate thereof.
12. The polymeric prodrug of any of the above claims, wherein the hydrogel is poly(ethylene glycol acrylamide) or a derivate thereof.
15 13. The polymeric prodrug of any of the above claims, wherein the hydrogel further comprises biodegradable bonds selected from the group of chemically-cleavable bonds consisting of phosphate, phosphonate, carbonate, carbamate, disulfide and ester bonds.
0 14. The polymeric prodrug of any one of claims 1 to 13, wherein the hydrogel further comprises biodegradable bonds which are enzymatically cleavable.
15. The polymeric prodrug of any of the above claims, wherein the prodrug linker is attached to a non-biodegradable backbone of the hydrogel. 5
16. The polymeric prodrug of claim 15, wherein cross-linkers of the hydrogel further comprises biodegradable bonds selected from the group of chemically- cleavable bonds consisting of phosphate, phosphonate, carbonate, carbamate, disulfide and ester bonds. 0
17. The polymeric prodrug of any of the above claims being formed as a medical implant.
18. The polymeric prodrug of any one of claims 1 to 17 being in substantially bead- shaped form.
19. Hydrogel having a plurality of pores with openings, the pores comprising
5 reversible prodrug linkers for covalently linking the hydrogel with a biologically active moiety at a position within the pore, and wherein such pores are of such a size that they are accessible to the biologically active moiety between the opening and the position.
10 20. The hydrogel according to claim 19, wherein the biologically active moiety is a biopolymer.
21. The hydrogel according to claim 19, wherein the biologically active moiety is selected from the group of proteins or polypeptides consisting of ACTH,
15 adenosine deaminase, agalsidase, albumin, alfa-1 antitrypsin (AAT), alfa-1 proteinase inhibitor (API), alteplase, anistreplase, ancrod serine protease, antibodies (monoclonal or polyclonal, and fragments or fusions), antithrombin III, antitrypsins, aprotinin, asparaginases, biphalin, bone-morphogenic proteins, calcitonin (salmon), collagenase, DNase, endorphins, enfuvirtide, enkephalins, 0 erythropoietins, factor Vila, factor VIII, factor Villa, factor IX, fibrinolysin, fusion proteins, follicle-stimulating hormones, granulocyte colony stimulating factor (G-CSF), galactosidase, glucagon, glucocerebrosidase, granulocyte macrophage colony stimulating factor (GM-CSF), phospholipase-activating protein (PLAP), gonadotropin chorionic (hCG), hemoglobins, hepatitis B 5 vaccines, hirudin, hyaluronidases, idurnonidase, immune globulins, influenza vaccines, interleukins (1 alfa, 1 beta, 2, 3, 4, 6, 10, 11, 12), IL-I receptor antagonist (rhlL-lra), insulins, interferons (alfa 2a, alfa 2b, alfa 2c, beta Ia, beta Ib, gamma Ia, gamma Ib), keratinocyte growth factor (KGF), transforming growth factors, lactase, leuprolide, levothyroxine, luteinizing hormone, lyme 0 vaccine, natriuretic peptide, pancrelipase, papain, parathyroid hormone, PDGF5 pepsin, platelet activating factor acetylhydrolase (PAF-AH), prolactin, protein C, octreotide, secretin, sermorelin, superoxide dismutase (SOD), somatropins (growth hormone), somatostatin, streptokinase, sucrase, tetanus toxin fragment, tilactase, thrombins, thymosin, thyroid stimulating hormone, thyrotropin, tumor necrosis factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator (tPA), TSH, urate oxidase, urokinase, vaccines, and plant proteins such as lectins and ricins.
5 22. The hydrogel of claim 19, wherein the biologically active moiety is insulin.
23. The hydrogel according to claim 19, wherein the biologically active moiety is an organic small molecule bioactive agent.
10 24. The hydrogel according to claim 23, wherein the biologically active moiety is selected from the group of organic small molecule bioactive agents consisting of central nervous system-active agents, anti-infective, anti-neoplastic, antibacterial, anti-fungal, analgesic, contraceptive, anti-inflammatory, steroidal, vasodilating, vasoconstricting, and cardiovascular agents.
15
25. The hydrogel of claim 19, wherein the biologically active moiety is an anti- sense or interfering olignucleotide.
26. The hydrogel of any of claims 19 to 25, wherein the reversible prodrug linker 20 comprises a masking group and an activating group.
27. The hydrogel of any of claims 19 to 26, wherein the reversible prodrug linker comprises a carbamate bond.
25 28. The hydrogel of any one of claims 19 to 27, wherein the reversible prodrug linker has a functional group.
29. The hydrogel of claim 28, wherein the functional group is selected from the goup of functional groups consisting of carboxylic acid and derivatives, 30 carbonate and derivatives, hydroxyl, hydrazine, hydroxylamine, maleamic acid and derivatives, ketone, amino, aldehyde, thiol and disulfide groups.
30. The hydrogel of any of claims 19 to 29, wherein the hydrogel is synthesised from the group of polymers consisting of polyalkyloxy-based polymers like poly(propylene glycol) or poly(ethylene glycol), dextran, chitosan, hyaluronic acid and derivatives, alginate, xylan, mannan, carrageenan, agarose, cellulose, 5 starch, hydroxyethyl starch (HES) and other carbohydrate-based polmers, poly(vinyl alcohols), poly(oxazolines), poly(anhydrides), poly(ortho esters), polycarbonates), ρoly(urethanes), poly(acrylic acids), ρoly(acrylamides) such as poly(hydroxypropyhnethacrylamide) (HMPA), poly(acrylates), poly(methacrylates) like poly(hydroxyethylmethacrylate), 10 poly(organophosphazenes), poly(siloxanes), ρoly(vinylpyrrolidone), poly(cyanoacrylates), poly(esters) such as poly(lactic acid) or poly(glycolic acids), poly(iminocarbonates), poly(amino acids) such as poly(glutamic acid), collagen, and gelatin.
15 31. The hydrogel of any of claims 19 to 30, wherein the hydrogel is polyacrylamide or a derivate thereof.
32. The hydrogel of any of claims 19 to 30, wherein the hydrogel is poly(ethylene glycol acrylamide) or a derivate thereof. 0
33. The hydrogel of any one of claims 19 to 33, wherein the prodrug linker is attached to a non-biodegradable backbone of the hydrogel.
34. The hydrogel of claim 33, wherein cross-linkers of the hydrogel further 5 comprises biodegradable bonds selected from the group of chemically- cleavable bonds consisting of phosphate, phosphonate, carbonate, carbamate, disulfide and ester bonds.
35. The hydrogel of any of claims 19 to 33, further comprising biodegradable bonds 0 selected from the group of chemically cleavable bonds consisting of phosphate, phosphonate, carbonate, carbamate, disulfide and ester bonds.
36. The hydrogel of any of claims 19 to 35, further comprising biodegradable bonds which are enzymatically cleavable.
37. Method for the manufacture of a mesoporous hydrogel-biologically active moiety conjugate comprising:
- a first step of synthesizing the mesoporous hydrogel; followed by
- a step of connecting a prodrug linker to the mesoporous hydrogel; and
- a step of conjugating a biologically active moiety to the prodrug linker; wherein the last steps can be carried out in either order.
38. The method of claim 37, wherein the prodrug linker has two functional groups, a first one of the two functional groups being complementary to a functional group attached to the mesoporous hydrogel and a second one of the two functional groups being conjugable to the biologically active moiety.
39. The method of claim 38, wherein the first one of the two functional groups is selected from the group of functional groups consisting of carboxylic acid and activated derivatives, amino, maleimide, thiol, sulfonic acid and derivatives, carbonate and derivatives, carbamate and derivatives, hydroxyl , aldehyde, ketone, hydrazine , isocyanate, isothiocyanate, phosphoric acid and derivatives, phosphonic acid and derivatives, haloacetyl, alkyl halides, acryloyl and other alpha-beta unsaturated michael acceptors, arylating agents like aryl fluorides, hydroxylamine, disulfides like pyridyl disulfide, vinyl sulfone, vinyl ketone, diazoalkanes, diazoacetyl compounds, epoxide, oxirane, and aziridine.
40. The method of claim 38, wherein the first one of the two functional groups is selected from the group of functional groups consisting of thiol, maleimide, amino, carboxylic acid and derivatives, carbonate and derivatives, carbamate and derivatives, aldehyde, and haloacetyl.
41. The method of claim 38, wherein the first one of the two functional groups is a thiol or maleimide group.
42. The method of claim 38, wherein the second one of the two functional groups is selected from the group of functional groups consisting of carboxylic acid and derivatives, carbonate and derivatives, hydroxyl, hydrazine, hydroxylamine, maleamic acid and derivatives, ketone, amino, aldehyde, thiol and disulfide
5 groups.
43. The method of any one of claims 37 to 42, wherein the biologically active moiety has a moiety functional group complimentary to the second one of the two functional groups.
10
44. The method of claim 43, wherein the moiety functional group is selected from the group of functional groups consisting of thiol, carboxylic acid, amino, hydroxyl, ketone and imidazole.
15 45. The method of any one of claims 37 to 44, wherein the biologically active moiety is a biopolymer.
46. The method of any one of claims 37 to 44, wherein the biologically active moiety is selected from the group of proteins or polypeptides consisting of 0 ACTH, adenosine deaminase, agalsidase, albumin, alfa-1 antitrypsin (AAT), alfa-1 proteinase inhibitor (API), alteplase, anistreplase, ancrod serine protease, antibodies (monoclonal or polyclonal, and fragments or fusions), antithrombin III, antitrypsins, aprorinin, asparaginases, biphalin, bone-morphogenic proteins, calcitonin (salmon), collagenase, DNase, endorphins, enfuvirtide, enkephalins, 5 erythropoietins, factor Vila, factor VIII, factor Villa, factor DC, fibrinolysin, fusion proteins, follicle-stimulating hormones, granulocyte colony stimulating factor (G-CSF), galactosidase, glucagon, glucocerebrosidase, granulocyte macrophage colony stimulating factor (GM-CSF), phospholipase-activating protein (PLAP), gonadotropin chorionic (hCG), hemoglobins, hepatitis B 0 vaccines, hirudin, hyaluronidases, idurnonidase, immune globulins, influenza vaccines, interleukins (1 alfa, 1 beta, 2, 3, 4, 6, 10, 11, 12), IL-I receptor antagonist (rhlL-lra), insulins, interferons (alfa 2a, alfa 2b, alfa 2c, beta Ia, beta Ib, gamma Ia, gamma Ib), keratinocyte growth factor (KGF), transforming growth factors, lactase, leuprolide, levothyroxine, luteinizing hormone, lyme vaccine, natriuretic peptide, pancrelipase, papain, parathyroid hormone, PDGF, pepsin, platelet activating factor acetylhydrolase (PAF-AH), prolactin, protein C, octreotide, secretin, sermorelin, superoxide dismutase (SOD), somatropins (growth hormone), somatostatin, streptokinase, sucrase, tetanus toxin fragment, 5 tilactase, thrombins, thymosin, thyroid stimulating hormone, thyrotropin, tumor necrosis factor (TNF), TNF receptor-IgG Fc, tissue plasminogen activator (tPA), TSH, urate oxidase, urokinase, vaccines, and plant proteins such as lectins and ricins.
10 47. The method of any one of claims 37 to 44, wherein the biologically active moiety is insulin.
48. The method of any of claims 37 to 44, wherein the biologically active moiety is an organic small molecule bioactive agent.
15
49. The method of claim 37 to 44, wherein the biologically active moiety is selected from the group of moieties consisting of central nervous system-active agents, anti-infective, anti-neoplastic, antibacterial, anti-fungal, analgesic, contraceptive, anti-inflammatory, steroidal, vasodilating, vasoconstricting, and cardiovascular
20 agents with at least one primary or secondary amino group.
50. The method of any one of claims 37 to 44, wherein the biologically active moiety is an anti-sense or interfering nucleic acid.
25 51. The method of any one of claims 37 to 50, wherein the hydrogel is synthesised from the group of polymers consisting of polyalkyloxy-based polymers like poly(propylene glycol) or poly(ethylene glycol), dextran, chitosan, hyaluronic acid and derivatives, alginate, xylan, mannan, carrageenan, agarose, cellulose, starch, hydroxyethyl starch (HES) and other carbohydrate-based polmers,
30 poly(vinyl alcohols), poly(oxazolines), poly(anhydrides), poly(ortho esters), polycarbonates), poly(urethanes), poly(acrylic acids), poly(acrylamides) such as poly(hydroxypropyhnethacrylamide) (HMPA), poly(acrylates), poly(methacrylates) like poly(hydroxyethyhnethacrylate), poly(organophosphazenes), poly(siloxanes), poly(vinylpyrrolidone), poly(cyanoacrylates), poly(esters) such as poly(lactic acid) or poly(glycolic acids), poly(iminocarbonates), ρoly(amino acids) such as poly(glutamic acid), collagen, and gelatin.
5 52. The method of any one of claims 37 to 51, wherein the hydrogel is polyacrylamide or a derivate thereof.
53. The method of any one of claims 37 to 51, wherein the hydrogel is poly(ethylene glycol acrylamide) or a derivate thereof.
10
54. The method of any one of claims 37 to 53, wherein the hydrogel is functionalized with a functional group selected from the group of reactive functional groups consisting of carboxylic acid and activated derivatives, amino, maleimide, thiol, sulfonic acid and derivatives, carbonate and derivatives,
15 carbamate and derivatives, hydroxyl , aldehyde, ketone, hydrazine , isocyanate, isothiocyanate, phosphoric acid and derivatives, phosphonic acid and derivatives, haloacetyl, alkyl halides, acryloyl and other alpha-beta unsaturated michael acceptors, arylating agents like aryl fluorides, hydroxylamine, disulfides like pyridyl disulfide, vinyl sulfone, vinyl ketone, diazoalkanes, diazoacetyl
20 compounds, epoxide, oxirane, and aziridine.
55. The method of any one of claims 37 to 53, wherein the hydrogel is functionalized with a functional group selected from the group of functional groups consisting of thiol, maleimide, amino, carboxylic acid and derivatives,
25 carbonate and derivatives, carbamate and derivatives, aldehyde, and haloacetyl.
56. The method of any one of claims 37 to 53, wherein the hydrogel is functionalized with maleimide.
30 57. The method of any one of claims 37 to 56, wherein the prodrug linker is attached to a non-degradable backbone of the mesoporous hydrogel.
58. The method of claim 57 wherein crosslinkers of the mesoporous hydrogel further comprise biodegradable bonds selected from the group of chemically- cleavable bonds consisting of phosphate, phosphonate, carbonate, carbamate, disulfide and ester bonds.
59. The method of any one of claims 37 to 57, wherein the mesoporous hydrogel 5 further comprises biodegradable bonds that are enzymatically cleavable.
60. Method of treatment comprising:
- a first step of applying a polymeric prodrug of any of claims 1 to 20;
- a second step of cleavage of the prodrug linkers; and 10 - a third step of release of the biologically active moiety.
61. The method of treatment of claim 60 wherein the rate of the cleavage in the second step is governed by pH.
15 62. The method of treatment of claim 60 or claim 61, wherein the cleavage of the prodrug linker occurs substantially chemically.
63. The method of treatment of claim 60, wherein the cleavage of the prodrug linker occurs substantially enzymatically.
20
64. The method of treatment of any of claims 60 to 63, wherein the release of the biologically active moiety is independent of a degradation of the biodegradable hydrogel.
25 65. The method of treatment of any one of the claims 60 to 64, wherein the hydrogel degradates into products having a molecular weight of less than 5OkDa.
66. The method of treatment of claim 65, wherein the molecular weight is less than 3OkDa.
PCT/EP2005/007316 2004-07-05 2005-07-05 Hydrogel polymeric conjugates of a prodrug WO2006003014A2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP09161154.1A EP2111872B1 (en) 2004-07-05 2005-07-05 Hydrogel conjugates
EP05767518.3A EP1781335B1 (en) 2004-07-05 2005-07-05 Hydrogel conjugates

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
GB0415041.3 2004-07-05
GB0415041A GB0415041D0 (en) 2004-07-05 2004-07-05 Hydrogel formulations
EP04019303.9 2004-08-13
EP04019303A EP1625856A1 (en) 2004-08-13 2004-08-13 Hydrogel polymeric conjugates of a prodrug
GBGB0505250.1A GB0505250D0 (en) 2004-07-05 2005-03-15 Hydrogel formulations
GB0505250.1 2005-03-15

Publications (2)

Publication Number Publication Date
WO2006003014A2 true WO2006003014A2 (en) 2006-01-12
WO2006003014A3 WO2006003014A3 (en) 2007-01-11

Family

ID=34524794

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2005/007316 WO2006003014A2 (en) 2004-07-05 2005-07-05 Hydrogel polymeric conjugates of a prodrug

Country Status (5)

Country Link
US (3) US7968085B2 (en)
EP (1) EP1781335B1 (en)
ES (1) ES2858460T3 (en)
GB (1) GB0505250D0 (en)
WO (1) WO2006003014A2 (en)

Cited By (86)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007028347A2 (en) * 2005-09-05 2007-03-15 Zentiva, A.S. Method for the preparation of polymeric conjugates of doxorubicin with ph- controlled release of the drug
WO2008051291A2 (en) * 2006-04-11 2008-05-02 Ordway Research Institute Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations thereof
EP2067494A1 (en) 2007-12-04 2009-06-10 Charité-Universitätsmedizin Berlin Sheet or tubular structure consisting of elastic biocompatible material and its use
EP2113256A1 (en) 2008-04-29 2009-11-04 Ascendis Pharma AS PEGylated rhGH compounds
WO2010100220A1 (en) 2009-03-05 2010-09-10 Ascendis Pharma As Interferon alpha carrier prodrugs
WO2011012718A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Prodrugs comprising an insulin linker conjugate
WO2011012715A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Biodegradable polyethylene glycol based water-insoluble hydrogels
WO2011012723A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Injectable sustained release compositions comprising a pramipexole prodrug
WO2011012722A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Prodrugs containing an aromatic amine connected by an amido bond to a linker
WO2011012721A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Carrier linked pramipexole prodrugs
WO2011012719A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Long acting insulin composition
US20110053848A1 (en) * 2008-02-01 2011-03-03 Ascendis Pharma As Prodrug comprising a drug linker conjugate
WO2011042450A1 (en) 2009-10-06 2011-04-14 Ascendis Pharma As Carrier linked paliperidone prodrugs
WO2011042453A1 (en) 2009-10-06 2011-04-14 Ascendis Pharma As Subcutaneous paliperidone composition
WO2011051406A1 (en) 2009-10-29 2011-05-05 Ascendis Pharma As Sterilization of biodegradable hydrogels
WO2011073234A2 (en) 2009-12-15 2011-06-23 Ascendis Pharma As Growth hormone composition
US8071134B2 (en) 2003-09-15 2011-12-06 Ordway Research Institute, Inc. Thyroid hormone analogs and methods of use
WO2013024051A1 (en) 2011-08-12 2013-02-21 Ascendis Pharma A/S Sustained release composition of prostacyclin
WO2013024053A1 (en) 2011-08-12 2013-02-21 Ascendis Pharma A/S Carrier-linked prodrugs having reversible carboxylic ester linkages
WO2013024052A1 (en) 2011-08-12 2013-02-21 Ascendis Pharma A/S Carrier-linked treprostinil prodrugs
WO2013036847A1 (en) * 2011-09-07 2013-03-14 Prolynx Llc Hydrogels with biodegradable crosslinking
WO2013053856A1 (en) 2011-10-12 2013-04-18 Ascendis Pharma A/S Prevention and treatment of ocular conditions
EP2087910A3 (en) * 2004-03-23 2013-05-15 Complex Biosystems GmbH Polymeric prodrugs
US8518451B2 (en) 2003-09-15 2013-08-27 Albany College of Pharmacy and Health Services Thyroid hormone analogs and methods of use
US20130253083A1 (en) * 2011-03-18 2013-09-26 Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) Polymer supported reagents and methods or reducing aromatic nitro compounds by using the same
WO2013160340A1 (en) 2012-04-25 2013-10-31 Ascendis Pharma A/S Prodrugs of hydroxyl-comprising drugs
US8668926B1 (en) 2003-09-15 2014-03-11 Shaker A. Mousa Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations thereof
WO2014056915A1 (en) 2012-10-11 2014-04-17 Ascendis Pharma A/S Diagnosis, prevention and treatment of diseases of the joint
WO2014056923A1 (en) 2012-10-11 2014-04-17 Ascendis Pharma Ophthalmology Division A/S Vegf neutralizing prodrugs for the treatment of ocular conditions
WO2014056926A1 (en) 2012-10-11 2014-04-17 Ascendis Pharma A/S Hydrogel prodrugs
WO2014086961A1 (en) 2012-12-07 2014-06-12 Ascendis Pharma A/S Carrier-linked prostanoid prodrugs
US8802240B2 (en) 2011-01-06 2014-08-12 Nanopharmaceuticals Llc Uses of formulations of thyroid hormone analogs and nanoparticulate forms thereof to increase chemosensitivity and radiosensitivity in tumor or cancer cells
WO2014173762A1 (en) * 2013-04-22 2014-10-30 Ascendis Pharma A/S Modified hydrogels
WO2014173759A1 (en) 2013-04-22 2014-10-30 Ascendis Pharma A/S Hydrogel-linked prodrugs releasing modified drugs
US20150030626A1 (en) * 2011-11-09 2015-01-29 Ascend Biopharamaceuticals Ltd Immunomodulatory conjugates
WO2015067791A1 (en) 2013-11-11 2015-05-14 Ascendis Pharma Relaxin Division A/S Relaxin prodrugs
US9133276B2 (en) 2010-09-17 2015-09-15 Sanofi-Aventis Deutschland Gmbh Prodrugs comprising an exendin linker conjugate
US9180107B2 (en) 2009-03-31 2015-11-10 Nanopharmaceuticals Llc Combination treatment of cancer with cetuximab and tetrac
US9198887B2 (en) 2003-09-15 2015-12-01 Nanopharmaceuticals Llc Thyroid hormone analogs and methods of use
US9220788B2 (en) 2009-06-17 2015-12-29 Nanopharmaceuticals Llc Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations and uses thereof
US9272049B2 (en) 2005-09-16 2016-03-01 Nanopharmaceuticals Llc Methods of stimulating fat mobilization using a polymer conjugated polyphenol
US9272048B2 (en) 2008-04-29 2016-03-01 Ascendis Pharma Growth Disorders Division A/S PEGylated recombinant human growth hormone compounds
US9289395B2 (en) 2006-12-22 2016-03-22 Nanopharmaceuticals Llc Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations and uses thereof
US9353170B2 (en) 2007-06-08 2016-05-31 Sanofi-Aventis Deutschland Gmbh Long-acting transient polymer conjugates of exendin
US9498536B2 (en) 2005-09-15 2016-11-22 Nanopharmaceuticals Llc Method and composition of thyroid hormone analogues and nanoformulations thereof for treating anti-inflammatory disorders
WO2016196124A2 (en) 2015-05-29 2016-12-08 Ascendis Pharma Inc. Prodrugs comprising a pyroglutamate linker
WO2017053807A2 (en) 2015-09-23 2017-03-30 Genentech, Inc. Optimized variants of anti-vegf antibodies
WO2017148883A1 (en) 2016-03-01 2017-09-08 Ascendis Pharma Bone Diseases A/S Pth prodrugs
WO2018011266A1 (en) 2016-07-13 2018-01-18 Ascendis Pharma A/S Conjugation method for carrier-linked prodrugs
WO2018060314A1 (en) 2016-09-29 2018-04-05 Ascendis Pharma Growth Disorders A/S Combination therapy with controlled-release cnp agonists
WO2018060312A1 (en) 2016-09-29 2018-04-05 Ascendis Pharma Bone Diseases A/S Pth compounds with low peak-to-trough ratios
WO2018060310A1 (en) 2016-09-29 2018-04-05 Ascendis Pharma Bone Diseases A/S Dosage regimen for a controlled-release pth compound
WO2018060311A1 (en) 2016-09-29 2018-04-05 Ascendis Pharma Bone Diseases A/S Incremental dose finding in controlled-release pth compounds
CN107998099A (en) * 2017-12-08 2018-05-08 江苏力凡胶囊有限公司 A kind of enteric capsulation sealing compound
US10130686B2 (en) 2005-09-15 2018-11-20 Nanopharmaceuticals Llc Method and composition of thyroid hormone analogues and nanoformulations thereof for treating inflammatory disorders
US10201616B2 (en) 2016-06-07 2019-02-12 Nanopharmaceuticals, Llc Non-cleavable polymer conjugated with αVβ3 integrin thyroid antagonists
US20190111140A1 (en) * 2013-06-19 2019-04-18 The Regents Of The University Of California Chemical structures for localized delivery of therapeutic agents
US10328043B1 (en) 2018-04-11 2019-06-25 Nanopharmaceuticals, Llc. Composition and method for dual targeting in treatment of neuroendocrine tumors
WO2019189330A1 (en) * 2018-03-28 2019-10-03 持田製薬株式会社 Novel reactive alginic acid derivatives
WO2020141221A1 (en) 2019-01-04 2020-07-09 Ascendis Pharma A/S Conjugates of pattern recognition receptor agonists
WO2020141225A1 (en) 2019-01-04 2020-07-09 Ascendis Pharma A/S Minimization of systemic inflammation
WO2020141222A1 (en) 2019-01-04 2020-07-09 Ascendis Pharma A/S Sustained local drug levels for innate immune agonists
WO2020141223A1 (en) 2019-01-04 2020-07-09 Ascendis Pharma A/S Induction of sustained local inflammation
US10806807B2 (en) 2014-03-14 2020-10-20 The Regents Of The University Of California TCO conjugates and methods for delivery of therapeutic agents
WO2020254606A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma A/S Conjugates of heteroaromatic nitrogen-comprising compounds
WO2020254617A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Anti-ctla4 compounds with localized pk properties
WO2020254612A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Controlled-release tyrosine kinase inhibitor compounds with localized pd properties
WO2020254613A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Controlled-release tyrosine kinase inhibitor compounds with localized pk properties
WO2020254611A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Anti-ctla4 conjugates
WO2020254607A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Anti-ctla4 compounds with localized pd properties
WO2020254602A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma A/S Conjugates of an electron-donating nitrogen or tertiary amine comprising compounds
WO2020254609A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Tyrosine kinase inhibitor conjugates
WO2020254603A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma A/S CONJUGATES OF π-ELECTRON-PAIR-DONATING HETEROAROMATIC NITROGEN-COMPRISING COMPOUNDS
US10961204B1 (en) 2020-04-29 2021-03-30 Nanopharmaceuticals Llc Composition of scalable thyrointegrin antagonists with improved blood brain barrier penetration and retention into brain tumors
WO2021136808A1 (en) 2020-01-03 2021-07-08 Ascendis Pharma A/S Conjugates undergoing intramolecular rearrangements
WO2021224169A1 (en) 2020-05-04 2021-11-11 Ascendis Pharma A/S Hydrogel irradiation
WO2022029178A1 (en) 2020-08-05 2022-02-10 Ascendis Pharma A/S Conjugates comprising reversible linkers and uses thereof
WO2022064035A1 (en) 2020-09-28 2022-03-31 Ascendis Pharma Bone Diseases A/S Improvement of physical and mental well-being of patients with hypoparathyroidism
US11351137B2 (en) 2018-04-11 2022-06-07 Nanopharmaceuticals Llc Composition and method for dual targeting in treatment of neuroendocrine tumors
WO2022207798A1 (en) 2021-04-01 2022-10-06 Ascendis Pharma A/S Use of long-acting growth hormone for treating inflammation-induced diseases
US11633487B2 (en) 2014-08-06 2023-04-25 Ascendis Pharma A/S Prodrugs comprising an aminoalkyl glycine linker
US11642415B2 (en) 2017-03-22 2023-05-09 Ascendis Pharma A/S Hydrogel cross-linked hyaluronic acid prodrug compositions and methods
WO2023110727A2 (en) 2021-12-13 2023-06-22 Ascendis Pharma Oncology Division A/S Novel cancer treatments with tlr7/8 agonists
US11723888B2 (en) 2021-12-09 2023-08-15 Nanopharmaceuticals Llc Polymer conjugated thyrointegrin antagonists
WO2024094673A1 (en) 2022-11-02 2024-05-10 Ascendis Pharma Bone Diseases A/S Pth treatment regimen comprising two pth compounds
US12016903B2 (en) 2014-11-21 2024-06-25 Ascendis Pharma Endocrinology Division A/S Long-acting growth hormone treatment

Families Citing this family (137)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030008396A1 (en) * 1999-03-17 2003-01-09 Ku David N. Poly(vinyl alcohol) hydrogel
US7923431B2 (en) 2001-12-21 2011-04-12 Ferrosan Medical Devices A/S Haemostatic kit, a method of preparing a haemostatic agent and a method of promoting haemostatis
US7955288B2 (en) * 2002-12-11 2011-06-07 Ferrosan Medical Devices A/S Gelatine-based materials as swabs
CA2554994C (en) * 2004-01-30 2015-05-19 Ferrosan A/S Haemostatic sprays and compositions
WO2005077013A2 (en) 2004-02-06 2005-08-25 Georgia Tech Research Corporation Surface directed cellular attachment
CA2558661C (en) * 2004-02-06 2012-09-04 Georgia Tech Research Corporation Load bearing biocompatible device
US20050278025A1 (en) * 2004-06-10 2005-12-15 Salumedica Llc Meniscus prosthesis
US7968085B2 (en) * 2004-07-05 2011-06-28 Ascendis Pharma A/S Hydrogel formulations
CN101001649B (en) * 2004-07-09 2011-08-31 弗罗桑医疗设备公司 Hemostatic compositions comprising hyaluronic acid and methods of making the same
US8222015B2 (en) * 2007-05-11 2012-07-17 Toyota Motor Engineering & Manufacturing North America, Inc. Heat resistant bioactive composition
CN102014973A (en) * 2008-02-29 2011-04-13 弗罗桑医疗设备公司 Device for promotion of hemostasis and/or wound healing
US9345662B2 (en) 2008-11-19 2016-05-24 Rutgers, The State University Of New Jersey Degradable hydrogel compositions and methods
DK3566713T3 (en) 2009-03-10 2021-04-06 Allergan Pharmaceuticals Int Ltd Injectable biomaterials
RU2573911C2 (en) 2009-05-20 2016-01-27 Байомарин Фармасьютикал Инк. Versions of c-type natriuretic peptide
US20120108514A1 (en) 2009-07-09 2012-05-03 University Of Iowa Research Foundation Long acting atrial natriuretic peptide (la-anp) and methods for use thereof
WO2011068774A2 (en) * 2009-12-02 2011-06-09 University Of Virginia Patent Foundation Methods for regulating gelation of hydrogel solutions and uses thereof
EP2525830B1 (en) 2010-01-22 2016-05-11 Ascendis Pharma A/S Dipeptide-based prodrug linkers for aliphatic amine-containing drugs
WO2011089215A1 (en) * 2010-01-22 2011-07-28 Ascendis Pharma As Dipeptide-based prodrug linkers for aromatic amine-containing drugs
US9561285B2 (en) 2010-01-22 2017-02-07 Ascendis Pharma As Carrier-linked carbamate prodrug linkers
US10010272B2 (en) 2010-05-27 2018-07-03 Profusa, Inc. Tissue-integrating electronic apparatus
JP6335509B2 (en) 2010-10-01 2018-05-30 アプライド メディカル リソーシーズ コーポレイション Portable laparoscopic trainer
WO2012048150A1 (en) * 2010-10-06 2012-04-12 Profusa, Inc. Tissue-integrating sensors
US9644042B2 (en) 2010-12-17 2017-05-09 Carnegie Mellon University Electrochemically mediated atom transfer radical polymerization
CN102764198A (en) * 2011-05-06 2012-11-07 强生消费者公司 Composition containing hydrogel particles
CA2837303C (en) 2011-05-26 2019-08-20 Cartiva, Inc. Tapered joint implant and related tools
US20140275420A1 (en) 2011-08-22 2014-09-18 Carnegie Mellon University Atom transfer radical polymerization under biologically compatible conditions
JP2014525439A (en) 2011-08-30 2014-09-29 メイヨ・ファウンデーション・フォー・メディカル・エデュケーション・アンド・リサーチ Natriuretic polypeptide
WO2013091001A1 (en) * 2011-12-19 2013-06-27 The University Of Sydney A peptide-hydrogel composite
WO2013096632A1 (en) 2011-12-20 2013-06-27 Applied Medical Resources Corporation Advanced surgical simulation
US9611305B2 (en) 2012-01-06 2017-04-04 Mayo Foundation For Medical Education And Research Treating cardiovascular or renal diseases
CA2865349C (en) 2012-03-06 2021-07-06 Ferrosan Medical Devices A/S Pressurized container containing haemostatic paste
US10350072B2 (en) 2012-05-24 2019-07-16 Cartiva, Inc. Tooling for creating tapered opening in tissue and related methods
RU2636240C2 (en) 2012-06-12 2017-11-21 Ферросан Медикал Дивайсиз А/С Dry haemostatic composition
CA2880277A1 (en) 2012-08-03 2014-02-06 Applied Medical Resources Corporation Simulated stapling and energy based ligation for surgical training
US10400280B2 (en) 2012-08-14 2019-09-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10323279B2 (en) 2012-08-14 2019-06-18 10X Genomics, Inc. Methods and systems for processing polynucleotides
US9701998B2 (en) 2012-12-14 2017-07-11 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10752949B2 (en) 2012-08-14 2020-08-25 10X Genomics, Inc. Methods and systems for processing polynucleotides
JP6367196B2 (en) 2012-08-14 2018-08-01 テンエックス・ジェノミクス・インコーポレイテッド Microcapsule composition and method
US9951386B2 (en) 2014-06-26 2018-04-24 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10221442B2 (en) 2012-08-14 2019-03-05 10X Genomics, Inc. Compositions and methods for sample processing
US10273541B2 (en) 2012-08-14 2019-04-30 10X Genomics, Inc. Methods and systems for processing polynucleotides
US11591637B2 (en) 2012-08-14 2023-02-28 10X Genomics, Inc. Compositions and methods for sample processing
US20140066367A1 (en) * 2012-08-31 2014-03-06 Mayo Foundation For Medical Education And Research Subcutaneous delivery of a long-acting natriuretic peptide
WO2014039245A1 (en) * 2012-09-07 2014-03-13 The Regents Of The University Of California Method of creating hydrogels through oxime bond formation
EP2897625B1 (en) * 2012-09-10 2019-10-23 Wake Forest University Health Sciences Amniotic membrane and its use in wound healing and tissue engineering constructs
ES2813340T3 (en) 2012-09-21 2021-03-23 Intensity Therapeutics Inc Cancer treatment method
KR102105979B1 (en) 2012-09-26 2020-05-04 어플라이드 메디컬 리소시스 코포레이션 Surgical training model for laparoscopic procedures
KR102104984B1 (en) 2012-09-27 2020-04-27 어플라이드 메디컬 리소시스 코포레이션 Surgical training model for laparoscopic procedures
US10679520B2 (en) 2012-09-27 2020-06-09 Applied Medical Resources Corporation Surgical training model for laparoscopic procedures
EP2901437B1 (en) 2012-09-27 2019-02-27 Applied Medical Resources Corporation Surgical training model for laparoscopic procedures
EP2901439A1 (en) 2012-09-28 2015-08-05 Applied Medical Resources Corporation Surgical training model for laparoscopic procedures
EP3467805B1 (en) 2012-09-28 2020-07-08 Applied Medical Resources Corporation Surgical training model for transluminal laparoscopic procedures
WO2014063194A1 (en) 2012-10-23 2014-05-01 The University Of Sydney Elastic hydrogel
RU2668877C2 (en) 2012-12-10 2018-10-04 Эластаджен Пти Лтд, Scalable producing of three-dimensional elastic structures
US10533221B2 (en) 2012-12-14 2020-01-14 10X Genomics, Inc. Methods and systems for processing polynucleotides
EP3567116A1 (en) 2012-12-14 2019-11-13 10X Genomics, Inc. Methods and systems for processing polynucleotides
EP2925256A4 (en) * 2013-01-22 2016-08-17 Prolynx Llc Sealants having controlled degradation
US9644204B2 (en) 2013-02-08 2017-05-09 10X Genomics, Inc. Partitioning and processing of analytes and other species
EP3660816B1 (en) 2013-03-01 2021-10-13 Applied Medical Resources Corporation Advanced surgical simulation constructions and methods
CA2904031A1 (en) 2013-03-14 2014-10-02 Profusa, Inc. Method and device for correcting optical signals
EP2997562B1 (en) 2013-05-15 2019-10-30 Applied Medical Resources Corporation Hernia model
US10219729B2 (en) 2013-06-06 2019-03-05 Profusa, Inc. Apparatus and methods for detecting optical signals from implanted sensors
EP3011550B1 (en) 2013-06-18 2018-01-03 Applied Medical Resources Corporation Gallbladder model
AU2014283170B2 (en) 2013-06-21 2017-11-02 Ferrosan Medical Devices A/S Vacuum expanded dry composition and syringe for retaining same
JP6517201B2 (en) 2013-07-24 2019-05-22 アプライド メディカル リソーシーズ コーポレイション First entry model
US10198966B2 (en) 2013-07-24 2019-02-05 Applied Medical Resources Corporation Advanced first entry model for surgical simulation
CA2920312C (en) 2013-08-13 2022-07-19 Elastagen Pty Ltd Regeneration of damaged tissue
US20170158836A1 (en) * 2013-11-25 2017-06-08 Carnegie Mellon University Ordered macroporous hydrogels for bioresponsive processes
RU2678592C1 (en) 2013-12-11 2019-01-30 Ферросан Медикал Дивайсиз А/С Dry composition comprising extrusion enhancer
ES2891756T3 (en) 2014-03-26 2022-01-31 Applied Med Resources Simulated dissectable tissue
WO2015157567A1 (en) 2014-04-10 2015-10-15 10X Genomics, Inc. Fluidic devices, systems, and methods for encapsulating and partitioning reagents, and applications of same
US10328149B2 (en) 2014-06-13 2019-06-25 Tenboron Oy Conjugates comprising an anti-EGFR1 antibody
KR102531677B1 (en) 2014-06-26 2023-05-10 10엑스 제노믹스, 인크. Methods of analyzing nucleic acids from individual cells or cell populations
DK3200809T3 (en) 2014-10-02 2023-12-04 Wake Forest Univ Health Sciences Inc Amniotic membrane powder and its use in wound healing and tissue engineering constructs
WO2016058612A1 (en) 2014-10-13 2016-04-21 Ferrosan Medical Devices A/S Dry composition for use in haemostasis and wound healing
AU2015339148B2 (en) 2014-10-29 2022-03-10 10X Genomics, Inc. Methods and compositions for targeted nucleic acid sequencing
US9975122B2 (en) 2014-11-05 2018-05-22 10X Genomics, Inc. Instrument systems for integrated sample processing
ES2765731T3 (en) 2014-11-13 2020-06-10 Applied Med Resources Tissue simulation models and methods
AU2015371184B2 (en) 2014-12-24 2020-06-25 Ferrosan Medical Devices A/S Syringe for retaining and mixing first and second substances
EP3244992B1 (en) 2015-01-12 2023-03-08 10X Genomics, Inc. Processes for barcoding nucleic acids
US9982070B2 (en) 2015-01-12 2018-05-29 Carnegie Mellon University Aqueous ATRP in the presence of an activator regenerator
CA2970367A1 (en) 2015-02-19 2016-08-25 Applied Medical Resources Corporation Simulated tissue structures and methods
US11274343B2 (en) 2015-02-24 2022-03-15 10X Genomics, Inc. Methods and compositions for targeted nucleic acid sequence coverage
EP4286516A3 (en) 2015-02-24 2024-03-06 10X Genomics, Inc. Partition processing methods and systems
AU2016243659B2 (en) 2015-03-31 2020-04-23 Cartiva, Inc. Hydrogel implants with porous materials and methods
EP3277228B1 (en) 2015-03-31 2020-01-15 Cartiva, Inc. Carpometacarpal (cmc) implants
ES2716924T3 (en) 2015-05-14 2019-06-18 Applied Med Resources Synthetic tissue structures for training and electrosurgical stimulation
US20160348093A1 (en) * 2015-05-18 2016-12-01 10X Genomics, Inc. Mobile Solid Phase Compositions for Use in Biochemical Reactions and Analyses
CA2988767A1 (en) 2015-06-09 2016-12-15 Applied Medical Resources Corporation Hysterectomy model
CN104841008B (en) * 2015-06-11 2016-10-12 新乡医学院第一附属医院 A kind of aerogel dressing and preparation method thereof
EP3316930B1 (en) 2015-07-03 2019-07-31 Ferrosan Medical Devices A/S Syringe for mixing two components and for retaining a vacuum in a storage condition
ES2824529T3 (en) 2015-07-16 2021-05-12 Applied Med Resources Simulated dissectable tissue
JP6862413B2 (en) 2015-07-22 2021-04-21 アプライド メディカル リソーシーズ コーポレイション Appendectomy model
JP6952348B2 (en) 2015-07-24 2021-10-20 トリンプ アイピー ピーティーワイ リミテッド Sterilizing polymers and composites of polymers and cells and their synthesis
KR102649261B1 (en) 2015-10-02 2024-03-20 어플라이드 메디컬 리소시스 코포레이션 Hysterectomy Model
CA3005880A1 (en) 2015-11-20 2017-05-26 Applied Medical Resources Corporation Simulated dissectible tissue
DK3882357T3 (en) 2015-12-04 2022-08-29 10X Genomics Inc Methods and compositions for the analysis of nucleic acids
JP6033392B2 (en) * 2015-12-17 2016-11-30 クリスタル・デリバリー・ビー・ブイ Method for preparing controlled release system
CA3009317A1 (en) 2015-12-23 2017-06-29 Viking Scientific, Inc. Hydrogel prodrug for treatment
US20170360815A1 (en) 2016-02-25 2017-12-21 Applied Biological Laboratories, Inc. Compositions and methods for protecting against airborne pathogens and irritants
WO2017147540A1 (en) 2016-02-25 2017-08-31 Applied Biological Laboratories, Inc. Compositions and methods for protecting against airborne pathogens and irritants
WO2017197338A1 (en) 2016-05-13 2017-11-16 10X Genomics, Inc. Microfluidic systems and methods of use
GB201609983D0 (en) * 2016-06-08 2016-07-20 Thermo Fisher Scient Geneart Gmbh And Life Technologies As Solid support
JP7063892B2 (en) 2016-06-27 2022-05-09 アプライド メディカル リソーシーズ コーポレイション Simulated abdominal wall
US20200188524A1 (en) * 2016-09-22 2020-06-18 University Of Washington Molecular logic gates for controlled material degradation
US10550429B2 (en) 2016-12-22 2020-02-04 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10011872B1 (en) 2016-12-22 2018-07-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
WO2018119400A1 (en) 2016-12-22 2018-06-28 Profusa, Inc. System and single-channel luminescent sensor for and method of determining analyte value
US10815525B2 (en) 2016-12-22 2020-10-27 10X Genomics, Inc. Methods and systems for processing polynucleotides
WO2018132582A1 (en) 2017-01-12 2018-07-19 Carnegie Mellon University Surfactant assisted formation of a catalyst complex for emulsion atom transfer radical polymerization processes
WO2018140966A1 (en) 2017-01-30 2018-08-02 10X Genomics, Inc. Methods and systems for droplet-based single cell barcoding
CA3053498A1 (en) 2017-02-14 2018-08-23 Applied Medical Resources Corporation Laparoscopic training system
US10847057B2 (en) 2017-02-23 2020-11-24 Applied Medical Resources Corporation Synthetic tissue structures for electrosurgical training and simulation
CN110678117A (en) * 2017-03-01 2020-01-10 梅特罗诺姆保健公司 Analyte sensor and method of manufacturing an analyte sensor
CN108732296B (en) * 2017-04-14 2020-12-04 常州药物研究所有限公司 Method for detecting crosslinking degree of crosslinked glucan microspheres
JOP20190245A1 (en) 2017-04-20 2019-10-15 Novartis Ag Sustained release delivery systems comprising traceless linkers
CN109526228B (en) 2017-05-26 2022-11-25 10X基因组学有限公司 Single cell analysis of transposase accessible chromatin
US10844372B2 (en) 2017-05-26 2020-11-24 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
US11034950B2 (en) * 2017-11-02 2021-06-15 Saint Louis University Delivering enzyme using an injectable hydrogel depot
EP3625361A1 (en) 2017-11-15 2020-03-25 10X Genomics, Inc. Functionalized gel beads
US10829815B2 (en) 2017-11-17 2020-11-10 10X Genomics, Inc. Methods and systems for associating physical and genetic properties of biological particles
WO2019195166A1 (en) 2018-04-06 2019-10-10 10X Genomics, Inc. Systems and methods for quality control in single cell processing
BR112020021998A2 (en) 2018-05-09 2021-01-26 Ferrosan Medical Devices A/S methods for preparing a hemostatic composition and for reconstituting a dry thrombin composition, hemostatic composition, and, kit.
CN108753202B (en) * 2018-05-31 2021-04-02 东华大学 Double-component hydrogel adhesive and preparation and application thereof
TW202027794A (en) 2018-10-03 2020-08-01 瑞士商諾華公司 Sustained delivery of angiopoetin-like 3 polypeptides
CN110075348B (en) * 2019-04-11 2021-10-22 温州医科大学 Sol system for preparing pH-sensitive double-network hydrogel, hydrogel and application
US10758329B1 (en) 2019-08-20 2020-09-01 Raymond L. Wright, III Hydrating mouth guard
GB202001439D0 (en) * 2020-02-03 2020-03-18 Univ Nottingham Drug formulations
CN114716662A (en) * 2021-01-06 2022-07-08 北京键凯科技股份有限公司 Acrylic acid and derivative functionalized water-soluble polymer thereof and application thereof
WO2022204722A2 (en) * 2021-03-26 2022-09-29 The Board Of Trustees Of The Leland Stanford Junior University Controlled release of bacteriophage to treat implant infections
CN113336972B (en) * 2021-06-21 2022-08-30 陕西科技大学 Multifunctional collagen-based nano composite hydrogel and preparation method thereof
WO2023018232A1 (en) * 2021-08-10 2023-02-16 주식회사 에스엠엘제니트리 Bead complex for detecting nucleic acid molecule in biological sample and method for detecting nucleic acid using same
KR102448191B1 (en) * 2021-08-10 2022-09-28 주식회사 에스엠엘제니트리 Conjugated bead, process thereof and biological assay system and assay process inlcuidg thereof
CN114306286B (en) * 2021-10-30 2023-08-18 钱峙涛 Hydrogel patch and preparation method thereof
CN114652892B (en) * 2022-05-20 2022-09-16 上海明悦医疗科技有限公司 Medical membrane material and preparation method thereof, uterine cavity anti-adhesion device and medical device
KR20240041389A (en) * 2022-09-22 2024-04-01 주식회사 에스엠엘제니트리 Maleimide group-modified bead complex and thiol group-modifed nucleotide molecule for detection of nucleic acid molecules in biological samples and method of detecting nucleic acid using the same

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999034833A1 (en) * 1998-01-07 1999-07-15 Shearwater Polymers, Incorporated Degradable heterobifunctional poly(ethylene glycol) acrylates and gels and conjugates derived therefrom
WO2000044808A1 (en) * 1999-02-01 2000-08-03 Hubbell Jeffrey A Biomaterials formed by nucleophilic addition reaction to conjugated unsaturated groups
WO2002083180A1 (en) * 2001-03-23 2002-10-24 Syntarga B.V. Elongated and multiple spacers in activatible prodrugs
WO2003049677A2 (en) * 2001-12-10 2003-06-19 Nanogen, Inc. Mesoporous permeation layers for use on active electronic matrix devices
WO2004043493A1 (en) * 2002-11-14 2004-05-27 Syntarga B.V. Prodrugs built as multiple self-elimination-release spacers
EP1525890A1 (en) * 2003-10-02 2005-04-27 Complex Biosystems GmbH Protein-Proteophore complexes
EP1579873A1 (en) * 2004-03-23 2005-09-28 Complex Biosystems GmbH Polymeric prodrugs

Family Cites Families (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US220245A (en) * 1879-10-07 Improvement in provision-safes
US2322245A (en) 1943-05-20 1943-06-22 Marshall L Lockhart Ypodermic injector and method of use thereof
US4024073A (en) * 1972-01-08 1977-05-17 Toray Industries, Inc. Hydrogel and production thereof
US5616568A (en) * 1993-11-30 1997-04-01 The Research Foundation Of State University Of New York Functionalized derivatives of hyaluronic acid
PT910412E (en) * 1996-07-01 2003-09-30 Univ Utrecht HYDROLOGICAL HYDROGEES FOR CONTROLLED LIBERATION
DE19626762A1 (en) * 1996-07-03 1998-01-08 Basf Ag Enzymatically cleavable linker for solid phase synthesis
AU1825299A (en) 1997-12-17 1999-07-05 Enzon, Inc. Polymeric prodrugs of amino- and hydroxyl-containing bioactive agents
US6624142B2 (en) 1997-12-30 2003-09-23 Enzon, Inc. Trimethyl lock based tetrapartate prodrugs
WO2000021572A2 (en) 1998-10-09 2000-04-20 The University Of Michigan Hydrogels and water soluble polymeric carriers for drug delivery
US6372813B1 (en) * 1999-06-25 2002-04-16 Motorola Methods and compositions for attachment of biomolecules to solid supports, hydrogels, and hydrogel arrays
BR0107942A (en) * 2000-01-28 2004-01-06 Infimed Therapeutics Inc Slow Release Protein Polymers
US6884778B2 (en) 2000-04-14 2005-04-26 William Marsh Rice University Biocompatible macromers
US7291673B2 (en) * 2000-06-02 2007-11-06 Eidgenossiche Technische Hochschule Zurich Conjugate addition reactions for the controlled delivery of pharmaceutically active compounds
JP4841066B2 (en) 2000-09-01 2011-12-21 ライスユニバーシティ Nitric oxide-forming hydrogel materials
US7172770B2 (en) * 2001-02-19 2007-02-06 Board Of Regents, The University Of Texas System Mesoporous compositions for use in drug delivery
US7615593B2 (en) * 2001-04-23 2009-11-10 Wisconsin Alumni Research Foundation Bifunctional-modified hydrogels
MXPA03009727A (en) * 2001-04-26 2004-01-29 Control Delivery Sys Inc Sustained release drug delivery system containing codrugs.
US7247609B2 (en) * 2001-12-18 2007-07-24 Universitat Zurich Growth factor modified protein matrices for tissue engineering
EP1531868A2 (en) 2002-06-03 2005-05-25 Alnis Biosciences, Inc. Therapeutic agent-containing polymeric nanoarticles
US7968085B2 (en) 2004-07-05 2011-06-28 Ascendis Pharma A/S Hydrogel formulations

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999034833A1 (en) * 1998-01-07 1999-07-15 Shearwater Polymers, Incorporated Degradable heterobifunctional poly(ethylene glycol) acrylates and gels and conjugates derived therefrom
WO2000044808A1 (en) * 1999-02-01 2000-08-03 Hubbell Jeffrey A Biomaterials formed by nucleophilic addition reaction to conjugated unsaturated groups
WO2002083180A1 (en) * 2001-03-23 2002-10-24 Syntarga B.V. Elongated and multiple spacers in activatible prodrugs
WO2003049677A2 (en) * 2001-12-10 2003-06-19 Nanogen, Inc. Mesoporous permeation layers for use on active electronic matrix devices
WO2004043493A1 (en) * 2002-11-14 2004-05-27 Syntarga B.V. Prodrugs built as multiple self-elimination-release spacers
EP1525890A1 (en) * 2003-10-02 2005-04-27 Complex Biosystems GmbH Protein-Proteophore complexes
EP1579873A1 (en) * 2004-03-23 2005-09-28 Complex Biosystems GmbH Polymeric prodrugs

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AUZANNEAU F.I. ET AL: "Synthesis, characterization and biocompatibility of PEGA resins" JOURNAL OF PEPTIDE SCIENCE, vol. 1, 1995, pages 31-44, XP008047545 *
FRANSSEN O ET AL: "Controlled release of a model protein from enzymatically degrading dextran microspheres" JOURNAL OF CONTROLLED RELEASE, ELSEVIER SCIENCE PUBLISHERS B.V., AMSTERDAM, NL, vol. 59, no. 2, 20 May 1999 (1999-05-20), pages 219-228, XP004169751 ISSN: 0168-3659 *

Cited By (153)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9750709B2 (en) 2003-09-15 2017-09-05 Nanopharmaceuticals Llc Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations thereof
US8668926B1 (en) 2003-09-15 2014-03-11 Shaker A. Mousa Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations thereof
US9980933B2 (en) 2003-09-15 2018-05-29 Nanopharmaceuticals Llc Thyroid hormone analogs and methods of use
US8071134B2 (en) 2003-09-15 2011-12-06 Ordway Research Institute, Inc. Thyroid hormone analogs and methods of use
US9198887B2 (en) 2003-09-15 2015-12-01 Nanopharmaceuticals Llc Thyroid hormone analogs and methods of use
US9579300B2 (en) 2003-09-15 2017-02-28 Nanopharmaceuticals Llc Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations thereof
US8518451B2 (en) 2003-09-15 2013-08-27 Albany College of Pharmacy and Health Services Thyroid hormone analogs and methods of use
EP2087910A3 (en) * 2004-03-23 2013-05-15 Complex Biosystems GmbH Polymeric prodrugs
EP1732607B1 (en) * 2004-03-23 2019-05-15 Ascendis Pharma GmbH Polymeric prodrug with a self-immolative linker
WO2007028347A3 (en) * 2005-09-05 2007-05-10 Zentiva As Method for the preparation of polymeric conjugates of doxorubicin with ph- controlled release of the drug
EA015091B1 (en) * 2005-09-05 2011-06-30 ЗЕНТИВА, а.с. A method for the preparation of polymeric conjugates of doxorubicin with ph-controlled release of the drug
WO2007028347A2 (en) * 2005-09-05 2007-03-15 Zentiva, A.S. Method for the preparation of polymeric conjugates of doxorubicin with ph- controlled release of the drug
US10130686B2 (en) 2005-09-15 2018-11-20 Nanopharmaceuticals Llc Method and composition of thyroid hormone analogues and nanoformulations thereof for treating inflammatory disorders
US9498536B2 (en) 2005-09-15 2016-11-22 Nanopharmaceuticals Llc Method and composition of thyroid hormone analogues and nanoformulations thereof for treating anti-inflammatory disorders
US9272049B2 (en) 2005-09-16 2016-03-01 Nanopharmaceuticals Llc Methods of stimulating fat mobilization using a polymer conjugated polyphenol
WO2008051291A3 (en) * 2006-04-11 2008-12-24 Ordway Res Inst Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations thereof
WO2008051291A2 (en) * 2006-04-11 2008-05-02 Ordway Research Institute Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations thereof
US9289395B2 (en) 2006-12-22 2016-03-22 Nanopharmaceuticals Llc Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations and uses thereof
US9353170B2 (en) 2007-06-08 2016-05-31 Sanofi-Aventis Deutschland Gmbh Long-acting transient polymer conjugates of exendin
EP2067494A1 (en) 2007-12-04 2009-06-10 Charité-Universitätsmedizin Berlin Sheet or tubular structure consisting of elastic biocompatible material and its use
US20110053848A1 (en) * 2008-02-01 2011-03-03 Ascendis Pharma As Prodrug comprising a drug linker conjugate
US8906847B2 (en) 2008-02-01 2014-12-09 Ascendis Pharma A/S Prodrug comprising a drug linker conjugate
EP2596805A1 (en) 2008-02-01 2013-05-29 Ascendis Pharma A/S Prodrug comprising a drug linker conjugate
US20150057221A1 (en) * 2008-02-01 2015-02-26 Ascendis Pharma A/S Prodrug comprising a drug linker conjugate
EP3050576A1 (en) 2008-04-29 2016-08-03 Ascendis Pharma Growth Disorders Division A/S Pegylated recombinant human growth hormone compounds
US9272048B2 (en) 2008-04-29 2016-03-01 Ascendis Pharma Growth Disorders Division A/S PEGylated recombinant human growth hormone compounds
US10098930B2 (en) 2008-04-29 2018-10-16 Ascendis Pharma Endocrinology Division A/S PEGylated Recombinant Human Growth Hormone Compounds
EP2113256A1 (en) 2008-04-29 2009-11-04 Ascendis Pharma AS PEGylated rhGH compounds
US10960053B2 (en) 2008-04-29 2021-03-30 Ascendis Pharma Endocrinology Division A/S PEGylated recombinant human growth hormone compounds
WO2010100220A1 (en) 2009-03-05 2010-09-10 Ascendis Pharma As Interferon alpha carrier prodrugs
US9180107B2 (en) 2009-03-31 2015-11-10 Nanopharmaceuticals Llc Combination treatment of cancer with cetuximab and tetrac
US9220788B2 (en) 2009-06-17 2015-12-29 Nanopharmaceuticals Llc Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations and uses thereof
US9839614B2 (en) 2009-06-17 2017-12-12 Nanopharmaceuticals, Llc Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations and uses thereof
CN102548583A (en) * 2009-07-31 2012-07-04 赛诺菲-安万特德国有限公司 Prodrugs comprising an insulin linker conjugate
CN102548583B (en) * 2009-07-31 2015-04-22 赛诺菲-安万特德国有限公司 Prodrugs comprising an insulin linker conjugate
WO2011012718A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Prodrugs comprising an insulin linker conjugate
WO2011012715A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Biodegradable polyethylene glycol based water-insoluble hydrogels
US9457066B2 (en) 2009-07-31 2016-10-04 Sanofi-Aventis Deutschland Gmbh Prodrugs comprising an insulin linker conjugate
CN102573913B (en) * 2009-07-31 2014-06-18 阿森迪斯药物股份有限公司 Biodegradable polyethylene glycol based water-insoluble hydrogels
CN102573913A (en) * 2009-07-31 2012-07-11 阿森迪斯药物股份有限公司 Biodegradable polyethylene glycol based water-insoluble hydrogels
JP2013500950A (en) * 2009-07-31 2013-01-10 アセンディス ファーマ エー／エス Biodegradable polyethylene glycol water-insoluble hydrogel
AU2010277556B2 (en) * 2009-07-31 2014-10-09 Ascendis Pharma As Biodegradable polyethylene glycol based water-insoluble hydrogels
WO2011012723A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Injectable sustained release compositions comprising a pramipexole prodrug
EP3782649A1 (en) * 2009-07-31 2021-02-24 Ascendis Pharma A/S Biodegradable polyethylene glycol based water-insoluble hydrogels
US11559482B2 (en) 2009-07-31 2023-01-24 Ascendis Pharma A/S Biodegradable polyethylene glycol based water-insoluble hydrogels
WO2011012722A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Prodrugs containing an aromatic amine connected by an amido bond to a linker
WO2011012721A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Carrier linked pramipexole prodrugs
US9265723B2 (en) 2009-07-31 2016-02-23 Sanofi-Aventis Deutschland Gmbh Long acting insulin composition
RU2554854C9 (en) * 2009-07-31 2017-02-03 Асцендис Фарма Ас Biodegradable water-insoluble polyethyleneglycol-based hydrogels
WO2011012719A1 (en) 2009-07-31 2011-02-03 Ascendis Pharma As Long acting insulin composition
RU2554854C2 (en) * 2009-07-31 2015-06-27 Асцендис Фарма Ас Biodegradable water-insoluble polyethyleneglycol-based hydrogels
US9173953B2 (en) 2009-07-31 2015-11-03 Ascendis Pharma As Prodrugs containing an aromatic amine connected by an amido bond to a linker
US9138462B2 (en) 2009-07-31 2015-09-22 Sanofi-Aventis Deutschland Gmbh Prodrugs comprising an insulin linker conjugate
WO2011042450A1 (en) 2009-10-06 2011-04-14 Ascendis Pharma As Carrier linked paliperidone prodrugs
WO2011042453A1 (en) 2009-10-06 2011-04-14 Ascendis Pharma As Subcutaneous paliperidone composition
US8758780B2 (en) 2009-10-06 2014-06-24 Ascendis Pharma As Subcutaneous paliperidone composition
US8986609B2 (en) 2009-10-29 2015-03-24 Ascendis Pharma A/S Sterilization of biodegradable hydrogels
WO2011051406A1 (en) 2009-10-29 2011-05-05 Ascendis Pharma As Sterilization of biodegradable hydrogels
US10682395B2 (en) 2009-12-15 2020-06-16 Ascendis Pharma Endocrinology Division A/S Dry growth hormone composition transiently linked to a polymer carrier
WO2011073234A2 (en) 2009-12-15 2011-06-23 Ascendis Pharma As Growth hormone composition
US9919033B2 (en) 2009-12-15 2018-03-20 Ascendis Pharma Endocrinology Division A/S Dry growth hormone composition transiently linked to a polymer carrier
US9511122B2 (en) 2009-12-15 2016-12-06 Ascendis Pharma Growth Disorders Division A/S Dry growth hormone composition transiently linked to a polymer carrier
US9133276B2 (en) 2010-09-17 2015-09-15 Sanofi-Aventis Deutschland Gmbh Prodrugs comprising an exendin linker conjugate
US8802240B2 (en) 2011-01-06 2014-08-12 Nanopharmaceuticals Llc Uses of formulations of thyroid hormone analogs and nanoparticulate forms thereof to increase chemosensitivity and radiosensitivity in tumor or cancer cells
US20130253083A1 (en) * 2011-03-18 2013-09-26 Iucf-Hyu (Industry-University Cooperation Foundation Hanyang University) Polymer supported reagents and methods or reducing aromatic nitro compounds by using the same
WO2013024051A1 (en) 2011-08-12 2013-02-21 Ascendis Pharma A/S Sustained release composition of prostacyclin
US10729778B2 (en) 2011-08-12 2020-08-04 Ascendis Pharma A/S Carrier-linked treprostinil prodrugs
WO2013024053A1 (en) 2011-08-12 2013-02-21 Ascendis Pharma A/S Carrier-linked prodrugs having reversible carboxylic ester linkages
WO2013024052A1 (en) 2011-08-12 2013-02-21 Ascendis Pharma A/S Carrier-linked treprostinil prodrugs
US9561287B2 (en) 2011-08-12 2017-02-07 Ascendis Pharma A/S Carrier-linked treprostinil prodrugs
US9872864B2 (en) 2011-08-12 2018-01-23 Ascendis Pharma A/S Sustained release composition of prostacyclin
EP3643306A2 (en) 2011-08-12 2020-04-29 Ascendis Pharma A/S Carrier-linked prodrugs having reversible carboxylic ester linkages
WO2013036847A1 (en) * 2011-09-07 2013-03-14 Prolynx Llc Hydrogels with biodegradable crosslinking
US9649385B2 (en) 2011-09-07 2017-05-16 Prolynx Llc Hydrogels with biodegradable crosslinking
US10398779B2 (en) 2011-09-07 2019-09-03 Prolynx Llc Hydrogels with biodegradable crosslinking
US11179470B2 (en) 2011-09-07 2021-11-23 Prolynx Llc Hydrogels with biodegradable crosslinking
US11181803B2 (en) 2011-09-07 2021-11-23 Prolynx Llc Hydrogels with biodegradable crosslinking
US11454861B2 (en) 2011-09-07 2022-09-27 Prolynx Llc Hydrogels with biodegradable crosslinking
WO2013053856A1 (en) 2011-10-12 2013-04-18 Ascendis Pharma A/S Prevention and treatment of ocular conditions
AU2012322917B2 (en) * 2011-10-12 2016-11-03 Ascendis Pharma Ophthalmology Division A/S Prevention and treatment of ocular conditions
US20150030626A1 (en) * 2011-11-09 2015-01-29 Ascend Biopharamaceuticals Ltd Immunomodulatory conjugates
US10344261B2 (en) * 2011-11-09 2019-07-09 Ascend Biopharmaceuticals Ltd Immunomodulatory conjugates
WO2013160340A1 (en) 2012-04-25 2013-10-31 Ascendis Pharma A/S Prodrugs of hydroxyl-comprising drugs
US9855340B2 (en) 2012-10-11 2018-01-02 Ascendis Pharma A/S Hydrogel prodrugs
US10519226B2 (en) 2012-10-11 2019-12-31 Ascendis Pharma Opthalmology Division A/S VEGF neutralizing prodrugs for the treatment of ocular conditions
US10980860B2 (en) 2012-10-11 2021-04-20 Ascendis Pharma A/S Diagnosis, prevention and treatment of diseases of the joint
WO2014056926A1 (en) 2012-10-11 2014-04-17 Ascendis Pharma A/S Hydrogel prodrugs
WO2014056915A1 (en) 2012-10-11 2014-04-17 Ascendis Pharma A/S Diagnosis, prevention and treatment of diseases of the joint
WO2014056923A1 (en) 2012-10-11 2014-04-17 Ascendis Pharma Ophthalmology Division A/S Vegf neutralizing prodrugs for the treatment of ocular conditions
US9913912B2 (en) 2012-12-07 2018-03-13 Ascendis Pharma A/S Carrier-linked prostanoid prodrugs
WO2014086961A1 (en) 2012-12-07 2014-06-12 Ascendis Pharma A/S Carrier-linked prostanoid prodrugs
WO2014173759A1 (en) 2013-04-22 2014-10-30 Ascendis Pharma A/S Hydrogel-linked prodrugs releasing modified drugs
AU2014257748C1 (en) * 2013-04-22 2019-03-07 Ascendis Pharma A/S Modified hydrogels
AU2014257748B2 (en) * 2013-04-22 2018-11-01 Ascendis Pharma A/S Modified hydrogels
WO2014173762A1 (en) * 2013-04-22 2014-10-30 Ascendis Pharma A/S Modified hydrogels
US11116849B2 (en) 2013-04-22 2021-09-14 Ascendis Pharma A/S Hydrogel-linked prodrugs releasing tagged drugs
US20190111140A1 (en) * 2013-06-19 2019-04-18 The Regents Of The University Of California Chemical structures for localized delivery of therapeutic agents
US10953098B2 (en) * 2013-06-19 2021-03-23 The Regents Of The University Of California Chemical structures for localized delivery of therapeutic agents
WO2015067791A1 (en) 2013-11-11 2015-05-14 Ascendis Pharma Relaxin Division A/S Relaxin prodrugs
US10806807B2 (en) 2014-03-14 2020-10-20 The Regents Of The University Of California TCO conjugates and methods for delivery of therapeutic agents
US11633487B2 (en) 2014-08-06 2023-04-25 Ascendis Pharma A/S Prodrugs comprising an aminoalkyl glycine linker
US12016903B2 (en) 2014-11-21 2024-06-25 Ascendis Pharma Endocrinology Division A/S Long-acting growth hormone treatment
US11298427B2 (en) 2015-05-29 2022-04-12 Ascendis Pharma A/S Prodrugs comprising a pyroglutamate linker
WO2016196124A2 (en) 2015-05-29 2016-12-08 Ascendis Pharma Inc. Prodrugs comprising a pyroglutamate linker
WO2017053807A2 (en) 2015-09-23 2017-03-30 Genentech, Inc. Optimized variants of anti-vegf antibodies
WO2017148883A1 (en) 2016-03-01 2017-09-08 Ascendis Pharma Bone Diseases A/S Pth prodrugs
US11793861B2 (en) 2016-03-01 2023-10-24 Ascendis Pharma Bone Diseases A/S PTH prodrugs
US10695436B2 (en) 2016-06-07 2020-06-30 Nanopharmaceuticals, Llc Non-cleavable polymer conjugated with alpha V beta 3 integrin thyroid antagonists
US10201616B2 (en) 2016-06-07 2019-02-12 Nanopharmaceuticals, Llc Non-cleavable polymer conjugated with αVβ3 integrin thyroid antagonists
US11896671B2 (en) 2016-07-13 2024-02-13 Ascendis Pharma A/S Conjugation method for carrier-linked prodrugs
WO2018011266A1 (en) 2016-07-13 2018-01-18 Ascendis Pharma A/S Conjugation method for carrier-linked prodrugs
US11759504B2 (en) 2016-09-29 2023-09-19 Ascendis Pharma Bone Diseases A/S PTH compounds with low peak-to-trough ratios
US11890326B2 (en) 2016-09-29 2024-02-06 Ascendis Pharma Bone Diseases A/S Controlled-release PTH compound
US11857603B2 (en) 2016-09-29 2024-01-02 Ascendis Pharma Bone Diseases A/S PTH compounds with low peak-to-trough ratios
EP4275677A2 (en) 2016-09-29 2023-11-15 Ascendis Pharma Bone Diseases A/S Dosage regimen for a controlled-release pth compound
WO2018060310A1 (en) 2016-09-29 2018-04-05 Ascendis Pharma Bone Diseases A/S Dosage regimen for a controlled-release pth compound
WO2018060311A1 (en) 2016-09-29 2018-04-05 Ascendis Pharma Bone Diseases A/S Incremental dose finding in controlled-release pth compounds
US11918628B2 (en) 2016-09-29 2024-03-05 Ascendis Pharma Bone Diseases A/S Controlled-release PTH compound
WO2018060312A1 (en) 2016-09-29 2018-04-05 Ascendis Pharma Bone Diseases A/S Pth compounds with low peak-to-trough ratios
US11590207B2 (en) 2016-09-29 2023-02-28 Ascendis Pharma Bone Diseases A/S Dosage regimen for a controlled-release PTH compound
US11564974B2 (en) 2016-09-29 2023-01-31 Ascendis Pharma Growth Disorders A/S Combination therapy with controlled-release CNP agonists
WO2018060314A1 (en) 2016-09-29 2018-04-05 Ascendis Pharma Growth Disorders A/S Combination therapy with controlled-release cnp agonists
US11642415B2 (en) 2017-03-22 2023-05-09 Ascendis Pharma A/S Hydrogel cross-linked hyaluronic acid prodrug compositions and methods
CN107998099A (en) * 2017-12-08 2018-05-08 江苏力凡胶囊有限公司 A kind of enteric capsulation sealing compound
JPWO2019189330A1 (en) * 2018-03-28 2021-03-11 持田製薬株式会社 New reactive alginic acid derivative
US11472892B2 (en) 2018-03-28 2022-10-18 Mochida Pharmaceutical Co., Ltd. Reactive alginic acid derivatives
WO2019189330A1 (en) * 2018-03-28 2019-10-03 持田製薬株式会社 Novel reactive alginic acid derivatives
US11077082B2 (en) 2018-04-11 2021-08-03 Nanopharmaceuticals, Llc Composition and method for dual targeting in treatment of neuroendocrine tumors
US10328043B1 (en) 2018-04-11 2019-06-25 Nanopharmaceuticals, Llc. Composition and method for dual targeting in treatment of neuroendocrine tumors
US11351137B2 (en) 2018-04-11 2022-06-07 Nanopharmaceuticals Llc Composition and method for dual targeting in treatment of neuroendocrine tumors
WO2020141222A1 (en) 2019-01-04 2020-07-09 Ascendis Pharma A/S Sustained local drug levels for innate immune agonists
WO2020141221A1 (en) 2019-01-04 2020-07-09 Ascendis Pharma A/S Conjugates of pattern recognition receptor agonists
WO2020141223A1 (en) 2019-01-04 2020-07-09 Ascendis Pharma A/S Induction of sustained local inflammation
WO2020141225A1 (en) 2019-01-04 2020-07-09 Ascendis Pharma A/S Minimization of systemic inflammation
WO2020254602A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma A/S Conjugates of an electron-donating nitrogen or tertiary amine comprising compounds
WO2020254603A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma A/S CONJUGATES OF π-ELECTRON-PAIR-DONATING HETEROAROMATIC NITROGEN-COMPRISING COMPOUNDS
WO2020254617A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Anti-ctla4 compounds with localized pk properties
WO2020254612A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Controlled-release tyrosine kinase inhibitor compounds with localized pd properties
WO2020254613A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Controlled-release tyrosine kinase inhibitor compounds with localized pk properties
WO2020254606A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma A/S Conjugates of heteroaromatic nitrogen-comprising compounds
WO2020254611A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Anti-ctla4 conjugates
WO2020254607A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Anti-ctla4 compounds with localized pd properties
WO2020254609A1 (en) 2019-06-21 2020-12-24 Ascendis Pharma Oncology Division A/S Tyrosine kinase inhibitor conjugates
WO2021136808A1 (en) 2020-01-03 2021-07-08 Ascendis Pharma A/S Conjugates undergoing intramolecular rearrangements
US11186551B2 (en) 2020-04-29 2021-11-30 Nanopharmaceuticals Llc Composition of scalable thyrointegrin antagonists with improved retention in tumors
US10961204B1 (en) 2020-04-29 2021-03-30 Nanopharmaceuticals Llc Composition of scalable thyrointegrin antagonists with improved blood brain barrier penetration and retention into brain tumors
WO2021224169A1 (en) 2020-05-04 2021-11-11 Ascendis Pharma A/S Hydrogel irradiation
WO2022029178A1 (en) 2020-08-05 2022-02-10 Ascendis Pharma A/S Conjugates comprising reversible linkers and uses thereof
WO2022064035A1 (en) 2020-09-28 2022-03-31 Ascendis Pharma Bone Diseases A/S Improvement of physical and mental well-being of patients with hypoparathyroidism
WO2022207798A1 (en) 2021-04-01 2022-10-06 Ascendis Pharma A/S Use of long-acting growth hormone for treating inflammation-induced diseases
US11723888B2 (en) 2021-12-09 2023-08-15 Nanopharmaceuticals Llc Polymer conjugated thyrointegrin antagonists
WO2023110727A2 (en) 2021-12-13 2023-06-22 Ascendis Pharma Oncology Division A/S Novel cancer treatments with tlr7/8 agonists
WO2024094673A1 (en) 2022-11-02 2024-05-10 Ascendis Pharma Bone Diseases A/S Pth treatment regimen comprising two pth compounds

Also Published As

Publication number Publication date
EP1781335A2 (en) 2007-05-09
EP1781335B1 (en) 2021-01-13
US9056126B2 (en) 2015-06-16
US20110223230A1 (en) 2011-09-15
ES2858460T3 (en) 2021-09-30
US20060002890A1 (en) 2006-01-05
US7968085B2 (en) 2011-06-28
WO2006003014A3 (en) 2007-01-11
GB0505250D0 (en) 2005-04-20
US10029015B2 (en) 2018-07-24
US20150238625A1 (en) 2015-08-27

Similar Documents

Publication Publication Date Title
US10029015B2 (en) Hydrogel formulations
US20210283264A1 (en) Polymeric Prodrugs with a Self-Immolative Linker
US8980242B2 (en) Aliphatic prodrug linker
US7291673B2 (en) Conjugate addition reactions for the controlled delivery of pharmaceutically active compounds
EP1292709B1 (en) Conjugate addition reactions for the controlled delivery of pharmaceutically active compounds
EP1625856A1 (en) Hydrogel polymeric conjugates of a prodrug
EP2111872B1 (en) Hydrogel conjugates
EP1625855A1 (en) Polymeric prodrug with a self-immolative linker

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

WWE Wipo information: entry into national phase

Ref document number: 2005767518

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2005767518

Country of ref document: EP