US 2013 0035635A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0035635 A1 Rau et al. (43) Pub. Date: Feb. 7, 2013

(54) CARRIER-LINKED CARBAMATE PRODRUG Publication Classification LINKERS (51) Int. Cl. A63L/27 (2006.01) (75) Inventors: Harald Rau, Dossenheim (DE); Torben A6M 5/3 (2006.01) Lessmann, Neustadt an der Weinstrasse A613 L/405 (2006.01) (DE) A6IP 29/00 (2006.01) C07C 2.7L/52 (2006.01) (73) Assignee: ASCENDIS PHARMA A/S, Hellerup C07D 207/216 (2006.01) (DK) (52) U.S. Cl...... 604/92; 560/159; 514/.490. 548/544; 51474.25 (21) Appl. No.: 13/574,120 (57) ABSTRACT (22) PCT Filed: Jan. 21, 2011 The present invention relates to a prodrug or a pharmaceuti cally acceptable salt thereof, comprising a drug linker conju (86). PCT No.: PCT/EP2011/050819 gate D-L, wherein D being a biologically active moiety con S371 (c)(1), taining an aromatic hydroxyl group is conjugated to one or (2), (4) Date: Oct. 15, 2012 more polymeric carriers via secondary carbamate-containing linkers L. Such carrier-linked prodrugs achieve drug releases (30) Foreign Application Priority Data with therapeutically useful half-lives. The invention also relates to pharmaceutical compositions comprising said pro Jan. 22, 2010 (EP) ...... 1O151470.1 drugs and their use as medicaments. US 2013/0035635 A1 Feb. 7, 2013

CARRIER-LINKED CARBAMATE PRODRUG 0007 Alternatively, the drugs may be conjugated to a car LINKERS rier through permanent covalent bonds. This approach is applied to various classes of molecules, from So-called Small 0001. The present application claims priority from PCT molecules, through natural products up to larger proteins. Patent Application No. PCT/EP2011/050819 filed on Jan. 21, 2011, which claims priority from European Patent Applica 0008 Liraglutide is an example of a peptide drug that tion No. EP 10 151 470.1 filed on Jan. 22, 2010, the disclo achieves an extended half-life by permanent covalent modi sures of which are incorporated herein by reference in their fication with a palmitoyl moiety. The fatty acid alkyl chain entirety. serves to provide albumin binding in Vivo and the palmitoy lated peptide forms an albumin complex that acts as a drug BACKGROUND OF THE INVENTION reservoir in the blood stream. 0009 Albuferon is an example of a protein drug that 1. Field of the Invention achieves an extended half-life by permanent covalent modi fication with another protein that in itself has a long half-life. 0002 The present invention is directed to carrier-linked The corresponding fusion protein of albumin and interferon prodrugs having temporary carbamate linkages between alpha, Albuferon, exhibits a significantly extended half-life as amine-containing moieties and phenolic hydroxyl groups of compared to interferon alpha. biologically active entities such as peptides, proteins, natural 0010 Many small molecule medicinal agents, like alka products or synthetic chemical compounds. Such carrier loids and anti-tumor agents, show low solubility in aqueous linked prodrugs are characterized by slow release of unmodi fluids. One way to solubilize these small molecule com fied biologically active entity. pounds is to conjugate the Small molecule compounds to 0003. It is noted that citation or identification of any docu hydrophilic (water-soluble) polymers. A variety of water ment in this application is not an admission that such docu soluble polymers, such as human serum albumin, dextran, ment is available as prior art to the present invention. lectins, poly(ethylene glycol) (PEG), poly(styrene-co-maleic 0004 Typically, carriers employed for extended time-ac anhydride), poly(N-hydroxypropylmethacrylamide), poly tion engineering in drug delivery are either used in a non (divinyl ether-co-maleic anhydride), hyaluronic acid have covalent fashion, with the drug physicochemically formu been described for this purpose (R. Duncan, Nature Rev. Drug lated into a solvent-carrier mixture, or by permanent covalent Disc., 2003, 2, 347-360). attachment of a carrier reagent to one of the drug's functional 0011 Covalent modification of biological molecules with groups. poly(ethylene glycol) has been extensively studied since the 0005. Non-covalent drug encapsulation into polymeric late 1970s. So-called PEGylated proteins have shown carriers has been applied to depot formulations for long improved therapeutic efficacy by increasing solubility, reduc acting release profiles. Typically, the drug is mixed with car ing immunogenicity, and increasing circulation half-live in rier material and processed in Such fashion, that the drug Vivo due to reduced renal clearance and proteolysis by becomes distributed inside the bulk carrier. For instance poly mer-drug aggregates may be shaped as microparticles which enzymes (see, for example, Caliceti P., Veronese F. M., Adv. are administered as an injectable Suspension or the polymer Drug Deliv. Rev. 2003, 55, 1261-1277). drug aggregates are formulated as gels which are adminis 0012 However, many biological molecules such as tered in a single bolus injection. Known in the art are also IFNalfa2, saquinavir or somatostatin are inactive or show liposomal formulations, where the carrier may be a polymeric decreased biological activity when a carrier is covalently or non-polymeric entity capable of Solubilizing the drug. conjugated to the drug (T. Peleg-Shulman et al., J. Med. Drug release occurs when the carrier Swells or physically Chem., 2004, 47, 4897-4904). deteriorates or chemical degradation allows diffusion of the 0013. In order to avoid shortcomings imposed by either drug to the exterior and Subsequently into the biological envi the non-covalent polymer mixtures or the permanent covalent ronment. Such chemical degradation processes may be auto attachment, it may be preferable to employ a prodrug hydrolytic or enzyme-catalyzed. An example for a marketed approach for chemical conjugation of the drug to the polymer drug based on bolus administration of a drug-polymer gel is carrier. In Such polymeric prodrugs, the biologically active Lupron Depot. An example for a marketed drug based on moieties (drugs, therapeutic, biological molecule, etc.) are Suspended microparticles is Nutropin Depot. An example for typically linked to the polymeric carrier moiety by a tempo a marketed drug based on a liposomal formulation is DOXil. rary bond formed between the carrier moiety and a hydroxy, 0006. A disadvantage of the non-covalent approach is that amino or carboxy group of the drug molecule. in order to prevent uncontrolled, burst-type release of the 0014 Prodrugs are therapeutic agents that are almost inac drug, encapsulation of the drug has to be highly efficient by tive perse but are predictably transformed into active molecu creating a sterically highly crowded environment. Restrain lar entities (see B. Testa, J. M: Mayer in Hydrolysis in Drug ing the diffusion of an unbound, water Soluble drug molecule and Prodrug Metabolism, Wiley-VCH, 2003, page 4). The requires strong van der Waals contacts, frequently mediated carrier prodrug approach may be applied in Such a fashion through hydrophobic moieties. Many conformationally sen that the drug is released in vivo from the polymer in order to sitive drugs, such as proteins or peptides, are rendered dys regain its biological activity. The reduced biological activity functional during the encapsulation process and/or during of the prodrug as compared to the released drug is of advan Subsequent storage of the encapsulated drug. In addition, tage if a slow or controlled release of the drug is desired. In Such amino-containing drugs readily undergo side reactions this case, a relatively large amount of prodrug may be admin with carrier degradation products (see, for example, D. H. Lee istered without concomitant side effects and the risk of over et al., J. Contr. Rel., 2003, 92,291-299). Furthermore, depen dosing. Release of the drug occurs over time, thereby reduc dence of the release mechanism of the drug upon biodegra ing the necessity of repeated and frequent administration of dation may cause interpatient variability. the drug. US 2013/0035635 A1 Feb. 7, 2013

00.15 Prodrug activation may occur by enzymatic or non conversion occurs in an organ other than the intestine or liver. enzymatic cleavage of the temporary bond between the car If the enzymatic conversion of the prodrug to the drug occurs rier and the drug molecule, or a sequential combination of in the same organ as the metabolic inactivation, the overall both, i.e. an enzymatic step followed by a non-enzymatic metabolism inactivation of the prodrug is more efficient than rearrangement. In an enzyme-free in-vitro environment Such that of the parent drug. This phenomenon is described for as an aqueous buffer Solution, a temporary bond such as an example for paracetamol and its prodrug phenacetin by Pang ester oramide may undergo hydrolysis, but the corresponding & Gillette (J. Pharmacol. Exp. Ther. 207:178, 1978). rate of hydrolysis may be much too slow and thus outside the 0022. In general, protection of a phenolic function in the therapeutically useful range. In an in vivo environment, form of a prodrug requiring enzymatic cleavage may often be esterases or amidases are typically present and the esterases ineffective, since the processing may occur before the drug and amidases may cause significant catalytic acceleration of reaches the side of metabolism. the kinetics of hydrolysis from twofold up to several orders of 0023. Another major drawback of predominantly enzy magnitude (see, for example, R. B. Greenwald et al. J. Med. matic cleavage is interpatient variability. Enzyme levels may Chem. 1999, 42 (18), 3857-3867). differ significantly between individuals resulting in biologi 0016 Prodrugs fall in two classes, bioprecursors and car cal variation of prodrug activation by the enzymatic cleavage. rier-linked prodrugs. Bioprecursors do not contain a carrier The enzyme levels may also vary depending on the site of group and are activated by the metabolic creation of a func administration. For instance, it is known that in the case of tional group. In carrier-linked prodrugs the active Substance is Subcutaneous injection, certain areas of the body yield more linked to a carrier moiety by a temporary linkage. The carrier predictable therapeutic effects than others. To reduce this may be biologically inert, Such as, for instance, PEG or may unpredictable effect, non-enzymatic cleavage or intramo have targeting properties, conferred by antibodies, for lecular catalysis is of particular interest (see, for example, B. example. This invention is concerned with polymeric carrier Testa, J. M: Mayer in Hydrolysis in Drug and Prodrug linked or macromolecular prodrugs, where the carrier itself is Metabolism, Wiley-VCH, 2003, page 5). a macromolecule such as a carrier protein or polysaccharide 0024. Furthermore, it is difficult to establish an in vivo-in or poly(ethylene glycol). vitro correlation of the pharmacokinetic properties for 0017 Cleavage of a carrier prodrug generates a molecular enzyme-dependent carrier-linked prodrugs. In the absence of entity (drug) of increased bioactivity and at least one side a reliable in vivo-invitro correlation optimization of a release product, the carrier. After cleavage, the bioactive entity will profile becomes a cumbersome task. reveal at least one previously conjugated and thereby pro 0025 Several non-enzymatically cleavable prodrugs of tected functional group, and the presence of this group typi aromatic hydroxyl group containing drugs have been cally contributes to the drug's bioactivity. attempted. Hussain and Shefter (Pharm. Res. 5:113-115) 0018. In order to implementaprodrug strategy, at least one describe ester prodrugs of naltrexone, using salicylate. Simi selected functional group in the drug molecule is employed lar results were obtained with prodrugs of beta- for attachment of the carrier polymer. Preferred functional (Hussain et al., Pharm. Res. 5: 44-47). The reported half-life groups are hydroxyl oramino groups. Consequently, both the of the prodrug was around one hour, indicating rapid turn attachment chemistry and hydrolysis conditions depend on over, consequently limiting the usefulness of Such prodrugs. the type of functional group employed. (0026. The international application WO-A 2001/47562 0019. Numerous macromolecular prodrugs are described describes polymeric prodrugs containing phenolic drug moi in the literature where the temporary linkage is a labile ester eties linked to primary amines by means of a hydrolyzable bond. In these cases, the functional group provided by the carbamate group. According to the publication, hydrolysis of bioactive entity is eithera hydroxyl group or a carboxylic acid Such prodrugs yielded the degradation products Y—Ar—OH (e.g. Y Luo, M R Ziebell, G D Prestwich, “A Hyaluronic (phenolic drug moiety)+POLY-NH. (a polymeric carrier car Acid Taxol Antitumor Bioconjugate Targeted to Cancer rying a primary amino group) and hydrogencarbonate. Cells’, Biomacromolecules 2000, 1, 208-218, J Cheng et al. 0027. The use of primary amines in the formation of car Synthesis of Linear, beta-Cyclodextrin Based Polymers and bamate prodrugs of phenolic drug moieties as in WO-A2001/ Their Camptothecin Conjugates, Bioconjugate Chem. 2003, 47562 raises concerns with respect to the safety of the deg 14, 1007-1017, R. Bhatt et al. Synthesis and in Vivo Antitu radation products. It is well known that hydrolysis of mor Activity of Poly(L-glutamic acid) Conjugates of 20OS)- secondary carbamates proceeds through nucleophilic attack Camptothecin, J. Med. Chem. 2003, 46, 190-193; R. B. of hydroxide at the carbonyl moiety (B2 mechanism) to Greenwald, A. Pendri, C. D. Conover, H. Zhao, Y. H. Choe, A. form the tetrahedral intermediate that breaks down by loss of Martinez, K. Shum, S. Guan, J.Med. Chem., 1999, 42,3657 alkoxide ion to give the carboxylated amine, which loses CO 3667; B. Testa, J. M: Mayer in Hydrolysis in Drug and Pro rapidly to give the free amine. On the other hand, hydrolysis drug Metabolism, Wiley-VCH, 2003, Chapter 8). of the primary carbamates proceeds through an elimination 0020 Especially for therapeutic biomacromolecules, but mechanism (E1cB) involving facile loss of a proton on the also for certain Small molecule drugs, it may be desirable to carbamate nitrogen and Subsequent formation of the isocyan link the carrier to aromatic (phenolic) hydroxyl groups of the ate. Depending on the reaction conditions and structure of the bioactive entity. reaction products, such isocyanates may be isolated in good 0021 Drugs containing phenolic hydroxyl groups are yield. For instance Uriz et al (Tetrahedron Letters 43 (2002) known to undergo extensive first-pass metabolism. The rapid 1673-6) detail an efficient method of synthesizing isocyan inactivation is mainly due to Sulfation, glucuronidation and ates from carbamates. In an aqueous environment, isocyan methylation of the phenolic moieties, catalyzed by enzymes ates typically do not persist but still may be remarkably stable in the gut and liver. Therefore, the metabolizable moiety of for hours and may—in the presence of other reactive moi phenolic drugs has been traditionally masked to yield ester eties—undergo side reactions. For instance, Broxton (Austr. prodrugs. This approach is only useful, if the prodrug-to-drug J. Chem. 38 (1985) 77 ff.) describes how during primary US 2013/0035635 A1 Feb. 7, 2013 carbamate hydrolysis in micelles, the intermediate p-nitro phenyl isocyanate was trapped by the hydroxyl group of the (I) functional micelle to form a new carbamate directly bound to R3 R2 V the detergent molecules of the micelle. N s 0028. Furthermore, primary carbamates may exhibit A hydrolysis rates that are not therapeutically useful. For R3a R2a instance, a carbamate of p-nitrophenyl-O CO. NH-phenyl was shown to hydrolyze with a half-life of 26 seconds at pH 7 (Mabey, W. and Mill, T. J. Phys. Chem. Ref. Data 7 (1978) 0.036 R. R. R. Rare independently selected from 383-415). Due to the limitations imposed by the drug moiety hydrogen, Substituted or non-substituted linear, in a corresponding prodrug it may not be possible to slow branched or cyclic C alkyl or heteroalkyl, down a primary carbamate's rate of hydrolysis by several 0037 m is independently 2, 3 or 4: orders of magnitude to achieve release rates compatible with I0038 ii) a moiety L, which is a chemical bond or a spacer, once-daily or once-weekly prodrug administration. and L is bound to a carrier group Z. wherein L' is substituted with one to four Li moieties, 0029. It is noted that in this disclosure and particularly in optionally, L is further substituted. the claims and/or paragraphs, terms such as "comprises'. 0039 Suitable substituents are alkyl (such as C. alkyl). “comprised', 'comprising and the like can have the meaning alkenyl (such as Calkenyl), alkynyl (such as C-alkynyl). attributed to it in U.S. patent law; e.g., they can mean aryl (such as phenyl), heteroalkyl, heteroalkenyl, heteroalky “includes”, “included”, “including, and the like; and that nyl, heteroaryl (such as aromatic 4 to 7 membered hetero terms such as "consisting essentially of and "consists essen cycle) or halogen moieties. tially of have the meaning ascribed to them in U.S. patent 0040. In the present application the following terms are law, e.g., they allow for elements not explicitly recited, but used as described below. exclude elements that are found in the prior art or that affect 0041) “Prodrug: A prodrug is any compound that under a basic or novel characteristic of the invention. goes biotransformation before exhibiting its pharmacological 0030. It is further noted that the invention does not intend effects. Prodrugs can thus be viewed as drugs containing to encompass within the scope of the invention any previously specialized non-toxic protective groups used in a transient disclosed product, process of making the product or method manner to alter or to eliminate undesirable properties in the ofusing the product, which meets the written description and parent molecule. enablement requirements of the USPTO (35 U.S.C. 112, first 0042 “Promoiety” refers to the part of the prodrug which paragraph) or the EPO (Article 83 of the EPC), such that is not the biologically active moiety. Promoiety thus refers to applicant(s) reserve the right to disclaim, and hereby disclose the linker and the carrier, if a carrier is present. a disclaimer of any previously described product, method of 0043 “Carrier-linked prodrug or “carrier prodrug: A making the product, or process of using the product. carrier-linked prodrug is a prodrug that contains a temporary linkage of a given active Substance with a transient carrier SUMMARY OF THE INVENTION group that produces improved physicochemical or pharma cokinetic properties and that can be easily removed in vivo, 0031. Therefore, an object of the present invention is to usually by a hydrolytic cleavage. provide carrier-linked prodrug linkers suitable for drugs con 0044) “Cascade prodrug: A cascade prodrug is a carrier taining aromatic hydroxyl groups from which free drug is prodrug for which the cleavage of the carrier group becomes released with therapeutically useful half-lives. effective only after unmasking an activating group. 0032. This object is achieved by a prodrug or a pharma 0045 “Polymeric cascade prodrug': A polymeric cascade ceutically acceptable salt thereof comprising a drug linker prodrug is a carrier prodrug that contains a temporary linkage conjugate D-L, wherein of a given active Substance with a transient polymeric carrier D is a biologically active moiety containing an aromatic group for which the cleavage of the carrier becomes effective hydroxyl group; and only after unmasking an activating group. 0046) “Bioprecursor prodrug': A bioprecursor prodrug is L is a non-biologically active linker containing a prodrug that does not imply the linkage to a carrier group, 0033 i) a moiety L' represented by formula (I), but results from a molecular modification of the active prin ciple itself. This modification generates a new compound, able to be transformed metabolically or chemically, the (I) O resulting compound being the active principle. 0047 "Biotransformation”: Biotransformation is the R3V R2 ) - chemical conversion of Substances by living organisms or N N enzyme preparations. A. 0048. The previous definitions are based on IUPAC, as R3a R2-J R! given under http://www.chem.qmul.ac.uk/iupac/medchem/ (accessed on 8 Mar. 2004) 0049) “Linker: Cleavage-controlling chemical structures 0034 wherein the dashed line indicates the attachment or groups present in carrier prodrugs that are not provided by of L' to the aromatic hydroxyl group of D by forming a either the carrier entity or by the drug. carbamate group; 0050 “Sustained release' or “sustained release rate' I0035) R' is selected from the group consisting of Ca means that the administration intervals of the respective pro alkyl; heteroalkyl, C-7 cycloalkyl, and drug are expanded. Drugs with a daily dosage may for US 2013/0035635 A1 Feb. 7, 2013

example be turned into a Sustained release form with a week 0057 The term “poly(ethylene glycol) based” or “PEG long or even longer interval between two administrations. based as understood herein means that the mass proportion 0051. Aromatic hydroxyl group containing biologically of PEG chains in the hydrogel is at least 10% by weight, active moiety D' means the part, e.g. the moiety or fragment, preferably at least 25%, based on the total weight of the of the prodrug conjugate D-L, which results after cleavage in hydrogel. The remainder can be made up of other polymers. the drug D-H, the active agent, of known biological activity. Moreover the term “water-insoluble” refers to a Swellable “L” in D-L refers to the promoiety. In addition, the subterm three-dimensionally crosslinked molecular network forming 'aromatic hydroxyl group containing” means that the respec the hydrogel. The hydrogel if suspended in a large Surplus of tive moiety D and analogously the corresponding drug D-H water or aqueous buffer of physiological osmolality may take contain at least one aromatic fragment, and which at least one up a substantial amount of water, e.g. up to 10-fold on a aromatic fragment is Substituted with at least one hydroxyl weight per weight basis, and is therefore swellable but after group. To be aromatic, the number of pi electrons must satisfy removing excess water still retains the physical stability of a the Hikkel rule (4n+2) and cycle has to be planar. Within the gel and a shape. Such shape may be of any geometry and it is Scope of the invention, 'aromatic hydroxyl group-contain understood that such an individual hydrogel object is to be ing” and “phenolic are used synonymously. considered as a single molecule consisting of components 0052 “Non-biologically active linker” means a linker wherein each component is connected to each other compo which does not show pharmacological effects. nent through chemical bonds. 0058. The term “PEG or “PEGylation residue” is used 0053 Suitable carriers are polymers and can either be herein exemplary for suitable water-soluble polymers char directly conjugated to the linker or via a non-cleavable spacer. acterized by repeating units. Suitable polymers may be The term “prodrug according to the invention” refers to car selected from the group consisting of polyalkyloxy polymers, rier-linked prodrugs of biologically active agent, wherein the hyaluronic acid and derivatives thereof, polyvinyl alcohols, carrier is PEG or a hydrogel. The terms “PEG prodrug’. polyoxazolines, polyanhydrides, poly(ortho esters), polycar “PEG-linked prodrug”, “hydrogel prodrug and “hydrogel bonates, polyurethanes, polyacrylic acids, polyacrylamides, linked prodrug” refer to prodrugs of biologically active polyacry-lates, polymethacrylates, polyorganophosp agents transiently linked to a PEG or to a hydrogel, respec haZenes, polysiloxanes, polyvinylpyrrolidone, polycy tively, and are used synonymously. The term “polymer pro anoacrylates, and polyesters. Preferred are polyalkyloxy drug” refers to carrier-linked prodrugs of a biologically active polymers, especially poly(ethylene glycol) polymers con agent, wherein the carrier is a polymer. taining at least 10% by weight ethylene oxide units, more 0054 The term polymer describes a molecule comprised preferably at least 25% by weight, even more preferably at of repeating structural units connected by chemical bonds in least 50% by weight a linear, circular, branched, crosslinked or dendrimeric way 0059 A “hydrogel' may be defined as a three-dimen or a combination thereof, which can be of synthetic or bio sional, hydrophilic or amphiphilic polymeric network logical origin or a combination of both. Typically, a polymer capable of taking up large quantities of water. The networks has a molecular weight of at least 1 kDa. are composed of homopolymers or copolymers, are insoluble 0055 Polymers are preferably selected from the group due to the presence of covalent chemical or physical (ionic, consisting of for example, 2-methacryloyl-oxyethyl phos hydrophobic interactions, entanglements) crosslinks. The phoyl cholins, hydrogels, PEG based hydrogels, poly(acrylic crosslinks provide the network structure and physical integ acids), poly(acrylates), poly(acrylamides), poly(alkyloxy) rity. Hydrogels exhibit a thermodynamic compatibility with polymers, poly(amides), poly(amidoamines), poly(amino water which allows them to swell in aqueous media. The acids), poly(anhydrides), poly(aspartamides), poly(butyric chains of the network are connected in Such a fashion that acids), poly(glycolic acids), polybutylene terephthalates, pores exist and that a Substantial fraction of these pores are of poly(caprolactones), poly(carbonates), poly(cyanoacry dimensions between 1 nm and 1000 nm. lates), poly(dimethylacrylamides), poly(esters), poly(ethyl enes), poly(ethyleneglycols), poly(ethylene oxides), poly 0060) “Free form” of a drug refers to the drug in its (ethyl phosphates), poly(ethyloxazolines), poly(glycolic unmodified, pharmacologically active form, such as after acids), poly(hydroxyethyl acrylates), poly(hydroxyethylox being released from a polymer conjugate. aZolines), poly(hydroxymethacrylates), poly(hydroxypropy 0061. A strong in vivo/in vitro correlation is observed, if lmethacrylamides), poly(hydroxypropyl methacrylates), the release kinetics exhibited by a carrier-linked prodrug con poly(hydroxypropyloxazolines), poly(iminocarbonates), jugate according to the invention in Vivo has a half-life that is polylactic acids), polylactic-co-glycolic acids), poly(meth not smaller than half the value exhibited by the same carrier acrylamides), poly(methacrylates), poly(methyloxazolines), linked prodrug conjugate in aqueous buffer of pH 7.4 at 37°C. poly(organophosphaZenes), poly(ortho esters), poly(oxazo It is understood that in the case of soluble carriers, release lines), poly(propylene glycols), poly(siloxanes), poly(ure kinetics may be recorded as hydrolysis kinetics. thanes), poly(vinyl alcohols), poly(Vinyl amines), poly(vi 0062. The terms “drug”, “biologically active molecule'. nylmethylethers), poly(vinylpyrrolidones), silicones, “biologically active moiety”, “biologically active agent'. celluloses, carbomethyl celluloses, hydroxypropyl methyl “active agent, and the like mean any Substance which can celluloses, chitins, chitosans, dextrans, dextrins, gelatins, affect any physical or biochemical properties of a biological hyaluronic acids and derivatives, mannans, pectins, rham organism, including but not limited to viruses, bacteria, fungi, nogalacturonans, starches, hydroxyalkyl starches, hydroxy plants, animals, and humans. In particular, as used herein, ethyl starches and other carbohydrate-based polymers, biologically active molecules include any Substance intended Xylans, and copolymers thereof. for diagnosis, cure, mitigation, treatment, or prevention of 0056 More preferably, Z is a biodegradable poly(ethylene disease in humans or other animals, or to otherwise enhance glycol) based water-insoluble hydrogel. physical or mental well-being of humans or animals. US 2013/0035635 A1 Feb. 7, 2013

0063 “Spacer” refers to any moiety suitable for connect Clso alkynyl), e.g. if present at the end of a molecule: ing two moieties, such as Clso alkyl, C2 so alkenyl or C2-so - C=CH, -CH C=CH, CH, CH, C=CH, CH alkinyl, which fragment is optionally interrupted by one or C=C CH, or e.g. —C=C when two moieties of a mol ecule are linked by the alkynyl group. Optionally, each hydro gen of a C-so alkynyl carbon may be replaced by a Substituent as further specified. Accordingly, the term “alkynyl relates to bered heterocyclyl phenyl or naphthyl. a carbon chain with at least one carbon carbon triple bond. 0064 “Functional groups' mean groups of atoms within Optionally, one or more double bonds may occur. The term molecules that exhibit a specific chemical activity. Examples C- alkynyl is defined accordingly. are amides, amines, alcohols, carbonyls, carboxylic acids, 0073 “C., cycloalkyl or “C., cycloalkyl ring” means a thiols. cyclic alkyl chain having 3 to 7 carbon atoms, which may 0065 “Protective groups' refers to a moiety which tem have carbon-carbon double bonds being at least partially satu porarily protects a functional group of a molecule during rated (unsubstituted C-7 cycloalkyl), e.g. cyclopropyl. synthesis to obtain chemoselectivity in Subsequent chemical cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohep reactions. Protective groups for alcohols are, for example, tyl. Optionally, each hydrogen of a cycloalkyl carbon may be benzyl and trityl, protective groups for amines are, for replaced by a substituent. The term "C., cycloalkyl” or “C. example, tert-butyloxycarbonyl, 9-fluorenylmethyloxycar 7cycloalkyl ring also includes bridged bicycles like norbo bonyl and benzyl and for thiols examples of protective groups nane (norbonanyl) or norbonene (norbonenyl). Accordingly, are 2,4,6-trimethoxybenzyl, phenylthiomethyl, acetamidom "Cs cycloalkyl means a cycloalkyl having 3 to 5 carbon ethyl, p-methoxybenzyloxycarbonyl, tert-butylthio, triph atOmS. enylmethyl, 3-nitro-2-pyridylthio. 4-methyltrity1. 0074 “Halogen' means fluoro, chloro, bromo or iodo. It is 0066. The term “Protected functional groups' means a generally preferred that halogen is fluoro or chloro. functional group protected by a protective group. (0075 “4 to 7 membered heterocyclyl or “4 to 7 mem 0067. Acylating agent’ means a moiety of the structure bered heterocycle” means a ring with 4, 5, 6 or 7 ring atoms R—(C=O)—, providing the acyl group in an acylation reac that may contain up to the maximum number of double bonds tion, optionally connected to a leaving group, such as acid (aromatic or non-aromatic ring which is fully, partially or chloride, N-hydroxy succinimide, pentafluorphenol and un-saturated) wherein at least one ring atom up to 4 ring para-nitrophenol. atoms are replaced by a heteroatom selected from the group 0068 Alkyl means a straight-chain or branched carbon consisting of sulfur (including —S(O)— —S(O) ), oxy chain (unsubstituted alkyl). Optionally, each hydrogen of an gen and nitrogen (including=N(O)—) and wherein the ring alkyl carbon may be replaced by a substituent. is linked to the rest of the molecule via a carbon or nitrogen 0069) “Heteroalkyl refers to analogs of alkyls in which atom (unsubstituted 4 to 7 membered heterocyclyl). one or more than one methylene group is replaced by a het 0076 Examples for a 4 to 7 membered heterocycles are eroatom, such as nitrogen, oxygen, Sulfur, phosphorus, or aZetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrro boron. If the methylene group is replaced by nitrogen, phos line, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, phorous or boron, these heteroatoms may be further substi oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, tuted. Suitable substituents are alkyl, alkenyl, alkynyl, aryl, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahy heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl or halo drofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, gen moieties (such as those described above). The terms pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, heteroalkenyl and heteroalkynyl are defined accordingly. isothiazolidine, thiadiazolidine, Sulfolane, pyran, dihydropy 0070 "Ca alkyl” means an alkyl chain having 1 to 4 ran, tetrahydropyran, imidazolidine, pyridine, pyridazine, carbon atoms (unsubstituted C. alkyl), e.g. if present at the pyrazine, pyrimidine, piperazine, piperidine, morpholine, tet end of a molecule: methyl, ethyl, n-propyl, isopropyl. n-butyl, razole, triazole, triazolidine, tetrazolidine, diazepane, isobutyl, sec-butyl tert-butyl, or e.g. —CH2—, —CH2— azepine or homopiperazine. Optionally, each hydrogen of a 4 CH-, —CH(CH) , CH2—CH2—CH2—, CH to 7 membered heterocyclyl may be replaced by a substituent. (CHS)— —C(CH) , when two moieties of a molecule (0077) “9 to 11 membered heterobicyclyl” or “9 to 11 mem are linked by the alkyl group. Optionally, each hydrogen of a bered heterobicycle” means a heterocyclic system of two C. alkyl carbon may be replaced by a substituent. Accord rings with 9 to 11 ring atoms, where at least one ring atom is ingly, "Clso alkyl means an alkyl chain having 1 to 50 shared by both rings and that may contain up to the maximum carbon atoms. The term C is defined accordingly. number of double bonds (aromatic or non-aromatic ring 0071 "Clso alkenyl' means a branched or unbranched which is fully, partially or un-saturated) wherein at least one alkenyl chain having 2 to 50 carbon atoms (unsubstituted ring atom up to 6 ring atoms are replaced by a heteroatom Clso alkenyl), e.g. if present at the end of a molecule: selected from the group consisting of Sulfur (including -CH=CH, -CH=CH-CH, CH-CH=CH, —S(O)— —S(O) ), oxygen and nitrogen (including -CH=CH-CH CH-CH=CH-CH=CH, or e.g. —N(O)—) and wherein the ring is linked to the rest of the —CH=CH , when two moieties of a molecule are linked molecule via a carbon or nitrogenatom (unsubstituted 9 to 11 by the alkenyl group. Optionally, each hydrogen of a C-so membered heterobicyclyl). alkenyl carbon may be replaced by a substituent as further (0078 Examples for a 9 to 11 membered heterobicycle are specified. Accordingly, the term “alkenyl relates to a carbon indole, indoline, benzofuran, benzothiophene, benzoxazole, chain with at least one carbon carbon double bond. Option benzisoxazole, benzothiazole, benzisothiazole, benzimida ally, one or more triple bonds may occur. The term C Zole, benzimidazoline, quinoline, quinazoline, dihydro alkenyl is defined accordingly. quinazoline, quinoline, dihydroquinoline, tetrahydroquino 0072 "Clso alkynyl means a branched or unbranched line, decahydroquinoline, isoquinoline, alkynyl chain having 2 to 50 carbon atoms (unsubstituted decahydroisoquinoline, tetrahydroisoquinoline, dihydroiso US 2013/0035635 A1 Feb. 7, 2013 quinoline, benzazepine, purine or pteridine. The term 9 to 11 agent. To be considered stable, the composition contains less membered heterobicycle also includes spiro structures of two than 10%, preferably less than 5% of the drug in its free form. rings like 1,4-dioxa-8-azaspiro4.5 decane orbridged hetero I0083. “Therapeutically effective amount’ means an cycles like 8-aza-bicyclo[3.2.1]octane. Optionally, each amount sufficient to cure, alleviate or partially arrest the hydrogen of a 9 to 11 membered heterobicyclyl may be clinical manifestations of a given disease and its complica replaced by a substituent. tions. An amount adequate to accomplish this is defined as 0079. In case the prodrugs according to the present inven “therapeutically effective amount’. Effective amounts for tion contain one or more acidic or basic groups, the invention each purpose will depend on the severity of the disease or also comprises their corresponding pharmaceutically or toxi injury as well as the weight and general state of the Subject. It cologically acceptable salts, in particular their pharmaceuti will be understood that determining an appropriate dosage cally utilizable salts. Thus, the prodrugs which contain acidic may beachieved using routine experimentation, by construct groups can be used according to the invention, for example, as ing a matrix of values and testing different points in the alkali metal salts, alkaline earth metal salts or as ammonium matrix, which is all within the ordinary skills of a trained salts. More precise examples of Such salts include sodium physician. Within the scope of this invention, therapeutically salts, potassium salts, calcium salts, magnesium salts or salts effective amount relates to dosages that aim to achieve thera with ammonia or organic amines such as, for example, ethy peutic effect for an extended period of time, i.e. for 12 hours, lamine, ethanolamine, triethanolamine or amino acids. Pro or 24 hours, or three days or longer, for instance one week or drugs which contain one or more basic groups, i.e. groups two weeks. which can be protonated, can be present and can be used I0084) “Excipients’ refers to compounds administered according to the invention in the form of their addition salts together with the therapeutic agent, for example, buffering with inorganic or organic acids. Examples for Suitable acids agents, isotonicity modifiers, preservatives, stabilizers, anti include hydrogen chloride, hydrogen bromide, phosphoric adsorption agents, oxidation protection agents, or other aux acid, Sulfuric acid, nitric acid, methanesulfonic acid, p-tolu iliary agents. However, in some cases, one excipient may have enesulfonic acid, naphthalenedisulfonic acids, oxalic acid, dual or triple functions. acetic acid, tartaric acid, lactic acid, Salicylic acid, benzoic I0085 “Dry composition” means that the prodrug compo acid, formic acid, propionic acid, pivalic acid, diethylacetic sition is provided in a dry form in a container. Suitable meth acid, malonic acid. Succinic acid, pimelic acid, fumaric acid, ods for drying are spray-drying and lyophilization (freeze maleic acid, malic acid, Sulfaminic acid, phenylpropionic drying). Such dry composition of prodrug has a residual water acid, gluconic acid, ascorbic acid, isonicotinic acid, citric content of a maximum of 10%, preferably less than 5% and acid, adipic acid, and other acids known to the person skilled more preferably less than 2% (determined according to Karl in the art. If the prodrugs simultaneously contain acidic and Fischer). The preferred method of drying is lyophilization. basic groups in the molecule, the invention also includes, in I0086 “Lyophilized composition” means that the prodrug addition to the salt forms mentioned, inner salts or betaines composition was first frozen and Subsequently subjected to (Zwitterions). The respective salts of the prodrugs of the water reduction by means of reduced pressure. This terminol present invention can be obtained by customary methods ogy does not exclude additional drying steps which occur in which are known to the person skilled in the art like, for the manufacturing process prior to filling the composition example by contacting these with an organic or inorganic acid into the final container. or base in a solvent or dispersant, or by anion exchange or I0087 “Lyophilization' (freeze-drying) is a dehydration cation exchange with other salts. The present invention also process, characterized by freezing a composition and then includes all salts of the prodrugs which, owing to low physi reducing the Surrounding pressure and, optionally, adding ological compatibility, are not directly Suitable for use in heat to allow the frozen water in the composition to sublime pharmaceuticals but which can be used, for example, as inter directly from the solid phase to gas. Typically, the sublimed mediates for chemical reactions or for the preparation of water is collected by desublimation. pharmaceutically acceptable salts. I0088 “Reconstitution” means the addition of a liquid to 0080. The term “pharmaceutically acceptable” means bring back the original form of a composition. approved by a regulatory agency, Such as the EMEA (Europe) I0089 "Reconstitution solution” refers to the liquid used to and/or the FDA (US) and/or any other national regulatory reconstitute the dry composition of a prodrug prior to admin agency for use in animals, preferably in humans. istration to a patient in need thereof. 0081 "Pharmaceutical composition” or “composition' 0090 “Container” means any container in which the pro means a composition containing one or more active ingredi drug composition is comprised and can be stored until recon ents, for example a drug or a prodrug, and one or more inert stitution. ingredients, as well as any product which results, directly or (0091) “Buffer” or “buffering agent” refers to chemical indirectly, from combination, complexation or aggregation of compounds that maintain the pH in a desired range. Physi any two or more of the ingredients, or from dissociation of ologically tolerated buffers are, for example, Sodium phos one or more of the ingredients, or from other types of reac phate. Succinate, histidine, bicarbonate, citrate and acetate, tions or interactions of one or more of the ingredients. Sulphate, nitrate, chloride, pyruvate. Antacids such as Accordingly, the pharmaceutical compositions of the present Mg(OH) or ZnCO may be also used. Buffering capacity invention encompass any composition made by admixing a may be adjusted to match the conditions most sensitive to pH prodrug of the present invention and a pharmaceutically stability. acceptable excipient. 0092. A “lyoprotectant” is a molecule which, when com I0082 “Stable” and “stability” means that within the indi bined with a protein of interest, significantly prevents or cated storage time the polymer conjugates remain conjugated reduces chemical and/or physical instability of the protein and do not hydrolyze to a substantial extent and exhibit an upon drying in general and especially during lyophilization acceptable impurity profile relating to the biologically active and Subsequent storage. Exemplary lyoprotectants include US 2013/0035635 A1 Feb. 7, 2013

Sugars, such as Sucrose or trehalose; amino acids such as because they do not facilitate a better understanding of the monosodium glutamate or histidine; methylamines such as present invention, a discussion of Such elements is not pro betaine; lyotropic salts such as magnesium sulfate; polyols vided herein. Such as trihydric or higher Sugar alcohols, e.g. glycerin, eryth ritol, glycerol, arabitol. Xylitol, Sorbitol, and mannitol; ethyl 0102 The present invention will now be described in ene glycol; propylene glycol; poly(ethylene glycol); pluron detail on the basis of exemplary embodiments. ics: hydroxyalkyl starches, e.g. hydroxyethyl starch (HES), 0103) The present invention addresses disadvantages of and combinations thereof. previous carbamate prodrugs, such as, for example, safety 0093 "Surfactant refers to wetting agents that lower the issues of hydrolysis products and therapeutically unsuitable Surface tension of a liquid. hydrolysis rates. As there is a need for advanced carrier 0094) “Isotonicity modifiers’ refer to compounds which linked prodrugs of drugs containing aromatic hydroxyl minimize pain that can result from cell damage due to osmotic groups, it was now Surprisingly found that drugs containing pressure differences at the injection depot. aromatic hydroxyl groups can be conjugated to polymeric 0095. The term “stabilizers” refers to compounds used to carriers via secondary carbamate-containing linkers and that stabilize the polymer prodrug. Stabilisation is achieved by Such carrier-linked prodrugs achieve drug releases with thera strengthening of the protein-stabilising forces, by destabili peutically useful half-lives. sation of the denatured state, or by direct binding of excipi 0104 Such polymeric secondary carbamate prodrugs ents to the protein. allow for an extended, controlled release mechanism, which 0096 “Anti-adsorption agents’ refers to mainly ionic or reduces the frequency with which a drug has to be taken. non-ionic Surfactants or other proteins or Soluble polymers Furthermore, as isocyanate formation is not observed for used to coat or adsorb competitively to the inner surface of the secondary carbamate hydrolysis, such groups offer more pre composition’s container. Chosen concentration and type of dictable release rates and predictable hydrolysis products excipient depends on the effect to be avoided but typically a with a greatly improved safety profile compared to primary monolayer of surfactant is formed at the interface just above carbamates. the CMC value. 0097 “Oxidation protection agents' refers to antioxidants 0105. The invention provides for carrier-linked carbamate Such as ascorbic acid, ectoine, glutathione, methionine, prodrugs which are characterized by a carrier connected to a monothioglycerol, morin, polyethylenimine (PEI), propyl linker, wherein the linker has a first and a second amine group gallate, Vitamin E, chelating agents such aus citric acid, and is connected to the drug moiety through a carbamate bond EDTA, hexaphosphate, thioglycolic acid. connecting the second amine group and the drug moiety’s 0.098 “Antimicrobial refers to a chemical substance that aromatic hydroxyl group. Such carbamate prodrugs are fur kills or inhibits the growth of microorganisms, such as bac ther characterized by the linker's second amine group being a teria, fungi, yeasts, protozoans and/or destroys viruses. secondary amine group. The carrier is connected to the 0099. “PEG based as understood herein means that the amine-containing linker via a permanent linkage, and the mass proportion of PEG chains in the hydrogel is at least 10% carbamate bond between the promoiety and the drug moiety by weight, preferably at least 25%, based on the total weight is a temporary linkage exhibiting extended autohydrolysis at of the hydrogel. The remainder can be made up of other a therapeutically useful rate at pH 7.4 and 37° C. spacers and/or oligomers or polymers, such as oligo- or polyl 0106 Due to the presence of a permanent bond between ysines. the carrier and the carbamate linker, the prodrugs according to 0100. The term “hydrolytically degradable' or “biode the present invention ensure release of unmodified native gradable' refers within the context of the present invention to drug molecules from a stable conjugate comprising carrier linkages which are non-enzymatically hydrolytically degrad and linker moiety. Furthermore, in these linker structures, the able under physiological conditions (aqueous buffer at pH presence of the carrier entity still allows for therapeutically 7.4, 37° C.) with half-lives ranging from one hour to three useful autohydrolysis rates, an essential prerequisite for pro months, include, but are not limited to, aconityls, acetals, drug applications. carboxylic anhydrides, esters, imines, hydrazones, maleamic 0107. In this invention, the hydrolytic lability required for acid amides, ortho esters, phosphamides, phosphoesters, a temporary linkage is introduced into the prodrug carbamate phosphosilyl esters, silyl esters, Sulfonic esters, aromatic car bond by selecting the structural properties of the linker for bamates, combinations thereof, and the like. Preferred biode neighboring group participation. In carbamate bond cleavage gradable linkages are esters, carbonates, phosphoesters and under physiological conditions, the cleavage products are a Sulfonic acid esters and most preferred are esters or carbon free aromatic hydroxyl group containing moiety, hydrogen ates. It is understood that for in vitro studies accelerated carbonate and an amine-containing residue. For a given phe conditions like, for example, pH 9, 37° C., aqueous buffer, nolic moiety, a corresponding aliphatic promoiety linked may be used for practical purposes. through a carbamate is highly likely to exhibit significant hydrolytic stability and is therefore unlikely to release the DETAILED DESCRIPTION OF EMBODIMENTS phenolic drug moiety in a therapeutically useful time frame. 0101. It is to be understood that the figures and descrip For this reason, neighboring group participation is desirable tions of the present invention have been simplified to illustrate to introduce enzyme-independent lability into the carbamate elements that are relevant for a clear understanding of the bond. Such neighboring group may be a first amine group. If present invention, while eliminating, for purposes of clarity, the linker structure is designed Such that the first amine may many other elements which are conventional in this art. Those intramolecularly catalyze the hydrolysis of the carbamate of ordinary skill in the art will recognize that other elements bond to the drug moiety, the hydrolysis of the carbamate bond are desirable for implementing the present invention. How may be facilitated to the extent that hydrolysis under physi ever, because such elements are well known in the art, and ological conditions in a time range useful for drug delivery is US 2013/0035635 A1 Feb. 7, 2013

possible. Such preferred first amine groups may be primary, oxidase, urokinase, vaccines, vascular endothelial growth secondary or tertiary amine or quaternary ammonium cat factor (VEGF), vasoactive intestinal peptide, vasopressin, ions. Ziconotide, lectin and ricin. 0113 Preferably, D-H is a protein prepared by recombi 0108 Corresponding prodrugs are composed of an ali nant DNA technologies. phatic linker containing a first amine, a permanent linkage to 0114 Preferably, D-H is a protein selected from the group a polymer carrier, wherein the linker is conjugated to an of proteins consisting of antibodies, antibody fragments, aromatic hydroxyl group-containing drug by means of a tem single chain antigen binding proteins, catalytic antibodies porary carbamate bond involving a second amine provided by and fusion proteins. the aliphatic linker, and wherein the second amine forming 0115 More preferably, D-H is a protein selected from the the carbamate bond is a secondary amine. group of proteins consisting of antibody fragments, single 0109 Preferably, linkers of the present invention have a chain antigen binding proteins, catalytic antibodies and hydrolysis rate between 1 h and 2 years at pH 7.4 and 37° C. fusion proteins. and hydrolysis rates in buffer and plasma are essentially iden 0116 Preferably, D-H is a small molecule bioactive agent tical, i.e. the hydrolysis rates exhibit a strong in vivo/in vitro selected from the group of agents consisting of central ner correlation. Vous system-active agents, anti-infective, anti-allergic, immunomodulating, anti-obesity, anticoagulants, antidia 0110 Preferably, D-His a small molecule bioactive agent betic, anti-neoplastic, antibacterial, anti-fungal, analgesic, or a biopolymer. contraceptive, anti-inflammatory, steroidal, vasodilating, 0111 Preferably, D-His a biopolymer selected from the vasoconstricting, and cardiovascular agents with at least one group of biopolymers consisting of proteins, or polypeptides. aromatic hydroxyl group. 0112 Preferably, D-His a polypeptide containing a phe 0117 D may be any aromatic hydroxyl-containing bio nolic moiety selected from the group of polypeptides consist logically active moiety known to a person skilled in the art, ing of ACTH, adenosine deaminase, agallsidase, alfa-1 antit which is derived from the corresponding biologically active rypsin (AAT), alfa-1 proteinase inhibitor (API), alteplase, drug D-H obtained after cleavage of D from the drug linker amylins (amylin, symlin), anistreplase, ancrod serine pro conjugate D-L. As indicated above, D contains at least one tease, antibodies (monoclonal or polyclonal, and fragments aromatic fragment, which at least one aromatic fragment is or fusions), antithrombin III, antitrypsins, aprotinin, aspara Substituted with at least one hydroxyl group and said ginases, atosiban, biphalin, bivalirudin, bone-morphogenic hydroxyl group is in turn connected to the amine-containing proteins, bovine pancreatic trypsin inhibitor (BPTI), cadherin moiety L' by forming a carbamate bond. fragments, calcitonin (salmon), collagenase, complement C1 0118. To be aromatic, the number of pi electrons must esterase inhibitor, conotoxins, cytokine receptor fragments, satisfy the Hickel rule (4n+2) and the cycle has to be planar. DNase, dynorphine A, endorphins, enfuVirtide, enkephalins, D may contain further substituents besides at least one aro erythropoietins, exendins, factor VII, factor VIIa, factor VIII, matic hydroxyl group. Such as further aliphatic or aromatic factor VIIIa, factor IX, fibrinolysin, fibroblast growth factor hydroxyl groups, amines, amides, carbonyls, carboxylic (FGF), growth hormone releasing peptide 2 (GHRP2), fusion acids, thiols or halogens. The term “aromatic' or 'aromatic proteins, follicle-stimulating hormones, gramicidin, ghrelin, fragment’ means any aromatic fragment known to a person desacyl-ghrelin, granulocyte colony stimulating factor skilled in the art Such as aryl, e.g. phenyl or naphthyl, or (G-CSF), galactosidase, glucagon, glucagon-like peptides, heteroaryl, such as aromatic 4 to 7 membered heterocyclyls or glucocerebrosidase, granulocyte macrophage colony stimu aromatic 9 to 11 membered heterobicyclyls. Such aromatic lating factor (GM-CSF), human heat shock proteins (HSP), heterocyclyls are, for example, furans, thiophenes, pyrroles, phospholipase-activating protein (PLAP), gonadotropin imidazoles, pyrazoles, oxazoles, isoxazoles, thiazoles, chorionic (hCG), hemoglobins, hepatitis B vaccines, hirudin, isothiazoles, thiadiazoles, thiadiazolines, pyridines, human serine protease inhibitor, hyaluronidases, idurnon pyridazines, pyrazines, pyrimidines, tetrazoles and thriaz idase, immune globulins, influenza vaccines, interleukins (1 oles. alfa, 1 beta, 2, 3, 4, 6, 10, 11, 12, 13, 21), IL-1 receptor 0119 The aromatic fragment comprises mono-, bi- or antagonist (rhIL-1 ra), insulins, insulin like growth factors, polycyclic fragments. In case of bi- or polycyclic fragments it insulin-like growth factor binding protein (rhIGFBP), inter is Sufficient that only one of said cycles is aromatic. D may ferons (alfa 2a, alfa 2b, alfa 2c, beta 1 a, beta 1b, gamma 1a, contain two or more further aromatic fragments as defined gamma 1b), intracellular adhesion molecule, keratinocyte before, which are bound to the first aromatic fragment, which growth factor (KGF), P-selectinglycoprotein ligand (PSGL), is Substituted with at least one amino group, either directly by transforming growth factors, lactase, leptin, leuprolide, a chemical bound or by a spacer. Said two or more additional levothyroxine, luteinizing hormone, lyme vaccine, natriuretic aromatic fragments may also contain at least one amino peptides (ANP, BNP. CNP and fragments), neuropeptide Y. group. In the following, D is defined by the corresponding pancrelipase, pancreatic polypeptide, papain, parathyroid aromatic hydroxyl containing biologically active drug D-H. hormone, PDGF, pepsin, peptide YY. platelet activating fac I0120 In one embodiment, D-H may be selected from the tor acetylhydrolase (PAF-AH), prolactin, protein C, thymal group of aromatic hydroxyl containing biologically active fasin, octreotide, Secretin, sermorelin, Soluble tumor necrosis agents consisting of (-)-Chicoric acid, (-)-cis-Resorcylide, factor receptor (TNFR), superoxide dismutase (SOD), soma (-)-Conophylline, (-)-, (-)-Gossy tropins (growth hormone), Somatoprim. Somatostatin, Strep pol, (-)-Indocarbazostatin B, (-)-Salbutamol hydrochloride, tokinase, Sucrase, terlipressin, tetanus toxin fragment, tilac (-)-Salmeterol, (-)-Subersic acid, (-)-Vexibinol, (+)-alpha tase, thrombins, thymosin, thyroid stimulating hormone, Viniferin, (+)-Etorphine, (+)-Indocarbazostatin, (+)- SCH thyrotropin, tumor necrosis factor (TNF). TNF receptor-IgG 351448, (R)-Albuterol hydrochloride, (R)-Gossypol, (R)-Tu Fc, tissue plasminogen activator (tPA), TSH, urodilatin, urate lobuterol, (R,R)-Formoterol tartrate, (S)-(+)-Curcuphenol, US 2013/0035635 A1 Feb. 7, 2013

(S)-Methylmaltrexone bromide, 111 In-DTPA-Pro1,Tyr4 nidin, , Bioxalomycin alpha 1, Bioxalomycin bombesin, 8-Gingerol, 99TcDemobesin 3, 99TcDe alpha2, Bismuth subsalicylate, Bisphenol, Bivalirudin, Bix, mobesin 4, Arg(Me)9] MS-10, D11G.K26R.Y40YR Bizelesin, Bogorol A, Brandisianin A, Brandisianin B. Bran Plectasin, D11G.M13K.K26R,Y40YR-Plectasin, D9N, disianin C, Brasilicardin A, Brevifolin carboxylic acid, Brey M13L.Q14R)-Plectasin, D9SQ14K.V36L-Plectasin, nin A, Breynin B. Bromotopsentin, Buflomedil pyridoxal D-Tyr1Arg(Me)9] MS-10, D-Tyr1AzaGly7. Arg(Me)9] phosphate, Buprenorphine hydrochloride, Burefrine, MS-10, D-Tyr1 MS-10, Gln20-Pancreatic polypeptide(2- Buserelin acetate. Butein, Buteranol, Butorphan, Butorpha 36), Glu10.Nle17.Nle30-Pancreatic polypeptide (2-36), nol tartrate, Calebin A, Calocoumarin A, Caloporoside D, Glu10-Pancreatic polypeptide(2-36), Leu13-Motilin, Caloporoside E. Caloporoside F, Calphostin A, Calphostin B, N5R,M13Y,N17R-Plectasin, Niel 7.Nle30-Pancreatic Calphostin C, Calphostin D. Calphostin I, Capillarisin, Cap polypeptide(2-36), psiCH2NHITpg4Vancomycin agly sazepine, Carbazomadurin A, Carbazomadurin B, Carbeto con, Trp 19 MS-10, 13-Deoxyadriamycin hydrochloride, cin, Carbidopa, Carmoterol hydrochloride, Carperitide, 13-Deoxydoxorubicin hydrochloride, 14-Methoxymetopon, Caspofungin acetate, Cassigalol A, Catechaldehyde, Cavtra 14-Phenylpropoxymetopon, 17beta-Estradiol. 18, 19-Dehy tin, Cefetecol, CefoperaZone sodium, Ce?piramide Sodium, drobuprenorphine hydrochloride, 2,12-Dimethyleurotinone, Cefprozil, Cefprozil monohydrate, Cetrorelix acetate, Cha 2'-Hydroxymatteucinol, 2-Methoxyestradiol, 2-Methyleuro etoatrosin A, Chafuroside, Chicoric acid-(-), Chloroorienti tinone, 3.5-Dicaffeoylquinic acid, 3-Bromodiosmetime, cin A, Chloroorienticin B, Chloropeptin II, Chondramide A, 3-Bromodiosmine, 3-Chlorodiosmetine, 3-Chlorodiosmine, Chondramide B, Chondramide C, Chondramide D, Cicleta 3-O-Methylguercetin, 4',7,8-Trihydroxyisoflavone, 4-Ami nine, Cinnatriacetin A, Cinnatriacetin B, cis-6-Shogaol, Cit nosalicylic acid, 4'-Dehydrodeacetylgriseusin A, 4-Demeth pressine I, Citreamicin-alpha, Citreamicin-eta, Citrusinine-I, ylpenclomedine, 4'-epi-Adriamycin, 4-epi-Doxorubicin, Clausenamine A, Clioquinol, Clostomycin B1, Combretasta 4-Hydroxyatomoxetine, 4-Iodopropofol, 5-Aminosalicylic tin A-1, Combretastatin A-2, Combretastatin A-3, Combret acid, 5-Iodofredericamycin A, 5-O-(E)-p-Coumaroylquinic astatin B-1, Combretastatin B-2, Combretastatin B-3, Com acid, 5Z-7-Oxozeaenol, 6-Carboxygenistein, 6-O-mPEG4 bretastatin B-4, Combretastatin D-1, Combretastatin D-2, Nalbupine, 6-O-mPEG5-Nalbuphine, 7-Chlorokynurenic Complestatin, Conantokin-R, coniferol alcohol, Conophyl acid, 7-Methylcapillarisin, 7-Monohydroxyethylrutoside, line, Contulakin G, Coproverdine, Corylifolinin, Corynecan 8(R)-Fluoroidarubicin hydrochloride, 8.9-Dehydroasco din, Cosalane, Crisamicin C, Crobenetine, Crobenetine chlorin, 8-Carboxy-iso-iantheran A, 8-Paradol, 8-Prenylapi hydrochloride, Cucoline, Curtisian A. Curtisian B, Curtisian genin, 8-Prenylmaringenin, 9-HydroxycrisamicinA, A-42867 D. Cyanidin, Cyanidin chloride monohydrate, Cyanidol chlo pseudoaglycone, Abarelix, Acacetin, Acetaminophen, Acla ride, Cyclocommunol, Cycloproparadicicol, Cyclotheona cinomycin A, Aclacinomycin-X, Aclarubicin, mide A, Cyclothialidine, Cycloviolin A, Cycloviolin B, hydrochloride, Acotiamide hydrochloride hydrate, Acrove Cycloviolin C, Cycloviolin D, Cyrtominetin, Cytogenin, stone, Actinoplanone A, Actinoplanone B. Aculeacin Cytosporone B, Cytotrienin A, Cytotrienin I, Cytotrienin II, Agamma, Adaphostin, Adarotene, AdXanthromycin A, Aero Dactylocycline A, Dactylocycline B, DADMe-Immucillin thricin 1, Aerothricin 16, Aerothricin 41, Aerothricin 45, G, DADMe-Immucillin-H. Dalargin, Dalbavancin, D-allo Aerothricin 50, Aerothricin 55, Afamelanotide, Ajulemic Ileu3 PYY(3-36), Damnacanthal, Damunacantal, Daph acid, Albolabrin, Albuterol nitrate, Albuterol sulfate, Alche nodorin A, Daphnodorin B. Daphnodorin C ((-)- mix, Aldifen, alpha-Human atrial natriuretic polypeptide, enantiomer), Darbufelone, Darbufelone mesilate, alpha-Mangostin, alpha-Methylepinephrine, alpha-Methyl Daunomycin, Daunorubicin, Daurichromenic acid, Davidi norepinephrine, alpha-Peltatin ((-)-enantiomer). Altromycin genin, Deacetyl moxisylyte hydrochloride, Decaplanin, A. Altromycin B. Altromycin C. Altromycin D. Altromycins, Decyl gallate, Deferasirox, Dehydroequol, Dehydroz Alvimopan hydrate, Alvocidib hydrochloride, Amamistatin ingerone, Dekafin 10, Delphinidin, Delphinidin chloride, A, Amamistatin B, Amarogentin, Amelubant, Amidox, Ami Delphinidol, DELTA9-Tetrahydrocannabinol, Deltorphin E, nocandin, Amodiaquine, Amoxicillin, Amoxicillin trihy Denopamine, Deoxymulundocandin, Dersalazine, drate, Amoxycillin trihydrate, Amrubicin hydrochloride, Desacetylravidomycin N-Oxide, Desglugastrin Amurensin H. Angiopeptin acetate, Angiotensin II (human), tromethamine, Deslorelin, Desmopressin acetate, Desulfated Anguillosporal, Anidulafungin, Ankinomycin, Annamycin, hirudin (54-65), Desulfated hirugen, Desvenlafaxine succi Annulin C, Annamycin, Antimycin A11, Antimycin A12, nate, Dexanabinol, Dexanabinone, Dextronatrin, Dextror Antimycin A13, Antimycin A14, Antimycin A15, Antimycin phan, Dexylosylbenanomycin A, D-Fluviabactin, Diaza A16, Apalcillin sodium, Apcitide technetium (99mTc). Api philonic acid, Diazepinomicin, Didehydro-epsilon-viniferin, cularen A, Apicularen B, , Apomine, Apomorphine Dieckol, Difimicin, Diflunisal, Dihydrexidine, Dihydroav hydrochloride, Arbidol, Arbutamine hydrochloride, Areni enanthramide D, Dihydrogranaticin B, Dihydrohonokiol B, cin, Arenicin-1, Arenicin-2, Arformoterol tartrate, Argiopine, Dihydroraloxifene, Dilevalol, Dilevalol hydrochloride, Argiotoxin-636, Artepillin C, hydrochloride, Dinapsoline, Dinofan, Dinoxyline, Dipotassium gossypolate, Aspoxicillin, Astringinine, Atalaphillidine, Atalaphillinine, Disagregin, Disoprofol, Dobutamine hydrochloride, Dob Atraric acid, Atrial natriuretic factor (99-126), AVorelin, utamine phosphate, Dopexamine, Dopexamine hydrochlo Axitirome, AZaresveratrol, Anatoxin, AZelastine embonate, ride, Dosmalfate, Doxorubicin hydrochloride, Doxorubicin, AZepinostatin, AZOdisal disodium, , , Bal morpholinyl, DoxoTam 12, Doxycycline hyclate, Doxycy , Balhimycin, Balsalazide disodium, Banoxantrone, cline hydrochloride ethanol hydrate, Dronabinol, Droxidopa, acetate, BaZedoxifene hydrochloride, Bedo Dumorelin, Duocarmycin B1, Duocarmycin B2, Duocarmy radrine Sulfate, Benadrostin, Benanomicin A, Benanomicin cin C1, Duocarmycin C2, Dutomycin, Dynemicin A, Dyne B, Benastatin A, Benastatin B, Benastatin C, Benastatin D, micin C, Dyofin-1, Dyofin-2, Dyofin-9, Echistatin, Econa Benzalazine, Benzbromaron, Benzbromarone, Berefrine, Zole sulfosalicylate, Ecopipam, Ecteinascidin 1560, Berupipam maleate, beta-Guttiferin, beta-Mangostin, Biem Ecteinascidin 722, Ecteinascidin 729, Ecteinascidin 736, Ect US 2013/0035635 A1 Feb. 7, 2013

einascidin 743, Ecteinascidin 745, Ecteinascidin 757, Ectein reotide acetate, , Lasofoxifene tartrate, Lata ascidin 770, Ecteinascidin 875, Edotecarin, Edotreotide moxef sodium, L-Chicoric acid, L-DOPA 3-O-glucoside, yttrium, Efepristin, Eflucimibe, Eflumast, Eformoterol fuma L-DOPA 4-O-glucoside, L-Dopa methyl ester hydrochloride, rate, Eldacimibe, Ellagic acid-4-gallate, Elliptinium acetate, L-Dopamide, Lecirelin, Leconotide, LedaZerol, Leuprolide Elsibucol, Eltrombopagolamine, Emodin, Emodol, Emoxyl, acetate, Leuprorelin acetate, Leurubicin, Levalbuterol hydro Enazadrem, Endothelin, Endothelin 1, Enfluvirtide, chloride, Levodopa, Levodopa 3-O-glucoside, Levodopa Enofelast, Entacapone, Enteric-Coated Mycophenolate 4-O-glucoside, Levodopa methyl ester hydrochloride, Levor Sodium, ent-, ent-Etorphine, Ephdine, Epidoxoform, phanol tartrate, Levosalbutamol hydrochloride, L-Fluviabac Epidoxorubicin, Epigallocatechin gallate, Epigallocatechin tin, L-Fluvibactin, L-Fluvibactine, Linderol A, Linopristin, 3-gallate, Epirubicin hydrochloride, Eplivanserin, Eplivan Linopristine, Lipiarmycin, Lipiarmycin A3. Lipiarmycin B3. serin fumarate, Eplivanserin mesilate, Epocarbazolin A, Lipiarmycin B4, Liquiritin apioside, Liraglutide, Lithosper Epocarbazolin B, Eprotirome, Eptazocine hydrobromide, mic acid B magnesium salt, Lobatamide C. Lobatamide F. Erabulenol A, Erabulenol B, Eremomycin, Erythromycin sal Loloatin B. Luminacin D. Luteolin, Macrocarpin A, Macro nacedin, Eserine salicylate, , Estradiol, Estriol, Eta carpin B. Magnesium lithospermate B. Magnesium salviano locib sodium, Etamsylate, Ethamsylate, Ethinyl estradiol, late B, Magnesium tanshinoate B, Makaluvamine D. Makalu , Ethinyloestradiol, Ethyl gallate, Etoposide, vamine E. Makaluvamine F, Malonoben, Maltolyl Eurocin, Eurotinone, Euxanthone, Evernimicin, Exifone, p-coumarate, Mannopeptimycin beta, Manzamine F. Marme EZetimibe, Factor P-Zyma, Fadolmidine hydrochloride, lin, Masoprocol, MASTPROM, Matteuorienate A. Matteuo Favipiravir, Feglymycin, Feglymycine, Fenoldopam mesi rienate B, Matteuorienate C, Medicarpin, Melanotan, Mel late, Fenoterol hydrobromide, Ferpifosate sodium, Fidaxo anotan I, Melevodopa hydrochloride, Mellein, Meluadrine, micin, Fidexaban, Flavalfate, Flavopiridol, Fluostatin A. Flu Meluadrine tartrate, Memno-peptide A, Meptazinol hydro ostatin B, Foetidine 1, Foetidine2, Folipastatin, Formobactin, chloride, Mesalamine, Mesalazine, Metaproterenol sulfate, Formoterol fumarate, Fosopamine, Frederine. Frog neurome Metaraminol, Meterelin, Methanobactin, Methyl gallate, din U. , Furaquinocin A, Furaquinocin B, Fusa Methyldopa, Methylmaltrexone bromide, Metirosine, Mety candin A, Fusacandin B, Fusidienol, Gaboxadol, Galactomy rosine, Micafungin sodium, Michellamine B, Miconazole cin I, Galactomycin II, Galarubicin hydrochloride, Gallinacin sulfosalicylate, Microcin 25, Microcin J25, Mideplanin, 1, Gallinacin 1alpha, Galparan, Gambogic acid, gamma Mimopezil, Minocycline hydrochloride, , Mangostin, gamma-Tocotrienol, Ganirelix, Ganirelix Mitoxantrone hydrochloride, Mitoxantrone hydrochloride, acetate, Garvalone C, Garveatin E, Garveatin F. -7- MivaZerol, Modecamide, Mollugin, Morphine 6-O-glucu phosphate, Gibbosin, Gigantol, Gilvusmycin, Gimeracil, ronide, Morphine glucuronide, Morphine hydrochloride, Gimestat, Glucagon-like peptide I (7-37), Glucopiericidinol Morphine sulfate, Morphine sulphate, Moxalactam diso A1, Glucopiericidinol A2, Gludopa, Glycothiohexide alpha, dium, Moxifetin hydrogen maleate, Multiple sclerosis vac Goserelin, Granaticin B. Griseusin C, Hatomarubigin A, cine, Mumbaistatin, muC-Conotoxin MrVIB, Mureidomy Hatomarubigin B. Hatomarubigin C, Hatomarubigin D. Heli cinA, Mureidomycin B. Mureidomycin C, Mureidomycin D, quinomycin, Helvecardin A, Helvecardin B. Hericenal A. Mureidomycin E, Mureidomycin F. Mureidomycins, Myco Hericenal B. Hericenal C. Herpefungin, Hidrosmin, phenolate mofetil, Mycophenolate sodium, Mycophenolic Hidrosmina, Hirudin desulfated, Hirulog-1. Histrelin, Histre acid sodium salt, Myrciacitrin I, Myrciacitrin II, Myrciaphe lin acetate, Hongoquercin A, Hongoquercin B, Honokiol none B. Myriceric acid A. Mytolbilin, Mytolbilin acid, diepoxide, Honokiol diepoxide, Human adrenomedullin, Mytolbilin acid methyl ester, Mytolbillinol, Naamidine A, Human adrenomedullin (22-52), Human angiotensin II, Nabilone, N-Acetylcolchinol, Nafarelin acetate, Nagresti Hydromorphone methiodide, Hydroxyakalone, Hymenista pen, Nalbuphine hydrochloride, Nalfurafine hydrochloride, tin 1, Hypeptin, Hypericin, Hyperin, Hyperoside, Icariin, Ida N-Allylsecoboldine, Nalmefene, Nalmetrene, Naloxone rubicin hydrochloride, Idronoxil, Ifenprodil, Imidacrine, Imi hydrochloride, Naltrexone hydrochloride, Naltrindole, Nap daZoacridinone, Incyclinide, Indacaterol, Indanocine, Insulin samycin A, Napsamycin B, Napsamycin C, Napsamycin D, chain B (9-23) peptide, Integracin A, Integracin B, Integracin Nardeterol, NCP-tazop sine, N-Cyclopentyl-tazop sine, C, Integramycin, Integrastatin A, Integrastatin B, Intoplicine, Nebicapone, Nelfinavir mesilate, Nemorubicin, Neparensi Iodochlorhydroxyquin, Iododiflunisal, Iodorubidazone (p), nol A, Neparensinol B, Neparensinol C, Nerfilin I, Neurome Iolopride (1231), Ioxipride, Iralukast, Iralukast sodium, din U-25, Neutrophil-activating factor, Nicanartine, Niteca Irciniastatin A, Irciniastatin B, Isalmadol, Iseganan hydro pone, Nitrofen, Nitrophene, N-Methylhydromorphonium chloride, Isobavachalcone, Isochlorogenic acida, Isodoxoru iodide, Nocardione A, Nocathiacin I, Nocathiacin II, bicin, Iso-iantheran A, , Isomolpan hydro Nocathiacin III, Nocathiacin IV, NO-Mesalamine, Nordam chloride, Isoquine, Isovanihuperzine A, Jadomycin B, nacanthal, Nordamunacantal, Nordihydroguaiaretic acid, Jaspamide, Jasplakinolide, Kadsuphilin C, Kaitocephalin, Norkurarinone, Nostocyclopeptide M1, Nothramicin, N-tert Kampanol A, Kampanol B, Kanglemycin A, Kapurimycin butyl isoquine, Obelmycin H, Ochracin, Ochromycinone, A1, Kapurimycin A2, Kapurimycin A3. Karnamicin B1, Kas Octreother, Octyl gallate, Odapipam acetate, O-Demethyl sinatuerin-1, Kehokorin D. Kehokorin E. Keoxifene hydro chlorothricin, O-Demethylmurrayafoline A, O-Desmethyl chloride, Kigamicin A, Kigamicin B. Kigamicin C, Kigami Venlafaxine Succinate, Oenothein B, Okicenone, Olanzapine cin D. Kigamicin E. Kigamicinone, Kistamicin A, Klainetin pamoate, Olcegepant, Olsalazine sodium, omega-Conotoxin A, Klainetin B, Kodaistatin A, Kodaistatin B, Kodaistatin C, CVID, omega-Conotoxin MVIIA, Onjixanthone I, Onjixan Kodaistatin D. Korupensamine A, Korupensamine B, Koru thone II, Oolonghomobisflavan A, Oolonghomobisflavan C, pensamine C. Korupensamine D. Kosinostatin, Kukoline, Orciprenaline sulphate, Orienticin A. Orienticin B, Orienticin Kushenol F. Kushnol F. Labetalol hydrochloride, Laccaridi C, Orienticin D. Oritavancin, Orniplabin, Orthosomycin A, one A, Lactonamycin, Lactosylphenyl trolox, Ladirubicin, Orthosomycin B, Orthosomycin C, Orthosomycin D, Ortho Lamellarin alpha 20-sulfate Sodium salt, Lamifiban, Lan somycin E, Orthosomycin F. Orthosomycin G, Orthosomy US 2013/0035635 A1 Feb. 7, 2013 cin H, Osutidine, OTAC. Oximidine III, Oxymetazoline Synthetic neutrophil inhibitor peptide, Syriacusin A, Syri hydrochloride, Oxymorphazole dihydrochloride, Oxymor acusin B, Syriacusin C, Tageflar, Taiwanhomoflavone A, Tal phone hydrochloride, Oxyphenarsine, Oystrisin, Ozarelix, netant, TAP-doxorubicin, Tapentadol hydrochloride, Tara Paeciloquinone A, Paeciloquinone B, Paeciloquinone D. Pan manon A, Tazofelone, TaZopsine, Tebufelone, Technetium cratistatin-3,4-cyclic phosphate Sodium salt, Pannorin, Pap (99mTc) apcitide, Technetium (99mTc) depreotide, Tech uamide A, Papuamide B, Papuamide C, Papuamide D, Parac netium Tc 99m depreotide, Teicoplanin-A2-1, Teicoplanin etamol, Paradol-8, Parvisporin B, PEG-Vancomycin, A2-2, Teicoplanin-A2-3, Teicoplanin-A2-3, Teicoplanin-A2 Penicillide, Pentafuside, Pentazocine hydrochloride, Pepti 5, Telavancin hydrochloride, Temoporfin, Teniposide, Tenuifoliside A, Tenuifoliside B, Tenuifoliside C, Terbutaline cinnamin E. Peptide Leucine Arginine, Phaffiaol, Phakellista sulfate, Terlipressin, Terprenin, Tetracycline hydrochloride, tin 7, Phakellistatin 8, Phakellistatin 9, Phenochalasin A, Tetragalloylquinic acid, Tetrahydrocurcumin, Tetrahydro Phenoxodiol, Phentolamine mesilate, Phlorofucofuroeckol, echinocandin B, Tetrahydroswertianolin, Textilinin-1, Tha Phlorofucofuroeckol A, Phomopsichalasin, Phthalascidin, natin, Thenorphine. Theophylline rutoside. Theprubicin, Thi Physostigmine salicylate, Piceasin, Piceatannol, Pidoben aZinotrienomycin B, Thiazinotrienomycin F, Zone, Pidorubicin, , Pipendoxifene, Pirarubicin, Thiazinotrienomycin G. Thielavin G. Thielocin B3. Thienor Pirbuterol hydrochloride, Pirenoxine, Pirfenoxone, Pitts phine. Thiocoraline. Thiocoraline A. Thiocoraline NF, burgh Compound B, Plantagoside, Platencin, Platensimycin, Thr10-Contulakin G, Thymopentin, Tiacumicin B, Tifu Pluraflavin A, Pluraflavin B, Pluraflavin E, Pneumocandin Virtide, Tigecycline, Tigilcycline, Tipelukast, Tocotrienol, A0, Pneumocandin B0, Pneumocandin B0 2-phosphate, Tokaramide A, Tolcapone, Tolterodine tartrate, Topotecan Pneumocandin DO, , Polyketomy acetate, Topotecan hydrochloride, Top sentin, Top sentine B1, cin, Popolohuanone E. Pradimicin A, Pradimicin B. Pradimi Trabectedin, trans-, Traxoprodil, Traxoprodil cin C, Pradimicin D, Pradimicin E, Pradimicin FA-1, mesylate, Trimidox, Triphendiol, Triproamylin acetate, Tro Pradimicin FA-2, Pradimicin FL, Pradimicin FS ((+)-enanti glitaZone, Troxerutin, Tubastrine ((+)-enantiomer), Tubul omer), Pradimicin L. Pradimicin Q, Pradimicin S. Pradimicin ysin A, Tubulysin B, Tubulysin C, Tucaresol, Tulobuterol T1, Pradimicin T2, Pramlintide acetate, , Prisoti (R), Tyropeptin A10, Tyropeptin A6, Tyropeptin A9, nol, Pristinamycin IA, Pristinamycin IB. Probucol, Pro Tyroservaltide, Tyroservatide, Tyrphostin 47, Tyrphostin A9, caterol hydrochloride hemihydrate, Propeptin, Propeptin T. Tyrphostin AG-213, Uncarinic acid A, Uncarinic acid B, Propofol, Propyl gallate, Protegrin IB-367, Protocatechuic Uncialamycin, Valrubicin, Vancomycin hydrochloride, Vas acid, Protocatechuic aldehyde, Pseudohypericin, Psymberin, tribil. V-Echinocandin, Veinamitol, Venorphin, Verticillatine, Purpuromycin, Pyridavone, Pyrindamycin A. Pyrindamycin Vesiculin, Vexibinol. V-Glycopeptide, Vialinin B, Vinaxan B, Pyrphenoxone, 3-galactoside, Quercetin 3-O- thone, Viniferifuran, Virgisin-1, Virgisin-2, Vitamin-P4, W beta-D-galactopyranoside, Quercetin-3-O-methyl ether, Peptide, Wiedendiol A, Wiedendiol B, Woodorien, Xamot Quinagolide hydrochloride, Quinobene, Quinupristin mesi erol fumarate, Xanthoangelol E. Xanthofulvin, Xanthomeg late, rac-Apogossypolone, rac-Tolterodine, Radolmidine nin, Xipamide, Yatakemycin, Yttrium-90 edotreotide, hydrochloride, hydrochloride, Ramoplanin A1, Zelandopam hydrochloride, Ziconotide, Zorubicin hydro Ramoplanin A2, Ramoplanin A3. Ramorelix, Rancinamycin chloride. IV. Rat adrenomedullin, Ravidomycin N-Oxide, Rawsonol, Reblastatin, Recombinant Jerdostatin, Relaxin-3/INSL5 chi I0121 Preferably, the carrier group Z is a polymer with a meric peptide, Reproterol hydrochloride, Resobene, Resor molecular weighte500 g/mol. thiomycin, Resveratrol, Retaspimycin hydrochloride, Rhodi I0122. In one embodiment, the carrier Z may be a PEG ocyanoside B, Rhododaurichromanic acid A. moiety. Such PEG moiety may be attached to the biologically Rhododaurichromenic acid, Rifabutin, Rifalazil, Rifamexil, active agent through one or more linkages. In case of one Rifampicin, Rifampin, Rifapentine, Rifaximin, Rimoterol linkage, the corresponding PEG in the PEG prodrug mono hydrobromide, Riodoxol, r-Jerdostatin, Rohitukine, Rotigap conjugate may be linear or branched. In case of more than one tide, Rotigotine, Roxindole mesilate, Ruboxyl, Rufigallol, linkage. Such as in a bisconjugate, the corresponding PEG in Rumycin 1, Rumycin 2, Russuphelin A, Sabarubicin hydro the PEG prodrug may be linear or branched. Bisconjugates chloride, Saintopin, Saintopin E. Sakyomicin A, Sakyomicin may contain one or two transient linkages, and PEG may be E. Salazodine, Salazopyridazin, Salazosulfapyridine, Salb linear or branched or may contain a mixture of one linear and utamol nitrate, Salbutamol Sulfate, Salcaprozic acid sodium one branched chain. In case the bisconjugate contains one salt, Salicylazobenzoic acid, Salicylihalamide A, Salicyli transient linkage and one linear and one branched chain the halamide B, Saliphenylhalamide, Salmaterol, Salmeterol, transient linkage may be on either chain. In case a branched Salmeterol Xinafoate, Saloxin, Salvianolic acid L. Sampatri PEG chain is used, there may be one or more branching units. lat, Sanglifehrin A, Sanglifehrin B, Sanglifehrin C, Sanglife I0123. A branched PEG is a PEG molecule consisting of a hrin D. Saptomycin D. Sapurimycin, Saricandin, Secoisola branching point connecting two or more PEG chains, to form riciresinol diglucoside, Seglitide, Semorphone a molecule with one anchoring point for attachment to the hydrochloride, Sermorelin, Shiga vaccine, Shishijimicin A, biologically active agent. This could be two 20 kDa PEG Shishijimicin B, Shishijimicin C, Siamycin I, Siamycin II, chains joined together to form one branched 40 kDa PEG Sibenadet hydrochloride, Sifuvirtide, Silychristin, Sinome molecule. In the case where the molecule contains two or nine, Sivifene, Siwenmycin, Sodium azodisalicylate. Soote three branching points, the molecule is referred to 3- and penseone, Sophoraflavanone G. Spinorphin, Spinosulfate A, 4-armed PEG, respectively. Spinosulfate B, Spiroximicin, Stachybocin A, Stachybocin B, 0.124. In summary and within the restrictions mentioned Stachybocin C, Stachybotrin C, Stachybotrydial, Staplabin, above, the PEG polymer is not limited to a particular structure Stearyl-norleucine-VIP, Sterenin A, Sterenin C, Sterenin D, and can be linear, branched, or multi-branched. Streptopyrrole. Succinobucol, Sulfasalazine, Sulphasalazine, 0.125 Preferably, Z is a hydrogel and more preferably a Sulphazocine, Super-Leu-Dox, Susalimod, Symbioimine, PEG-based hydrogel. Preferably, the covalent attachment US 2013/0035635 A1 Feb. 7, 2013 formed between the linker and the hydrogel Z is a permanent interconnected biodegradable functional groups and drug bond. The term "PEG based as understood herein means that linker conjugates and capping groups is 16-128, preferred the mass proportion of PEG chains in the hydrogel is at least 20-100, more preferred 24-80 and most preferred 30-60. 10% by weight, preferably at least 25%, based on the total 0.136 Preferably, the sum of interconnected functional weight of the hydrogel. The remainder can be made up of groups and hydrogel-connected drug-linker conjugates and spacers and/or oligomers or polymers, such as oligo- or polyl capping groups of a backbone moiety is equally divided by ysines. the number of PEG-based polymeric chains extending from 0126 Preferably, L' is substituted with one L. moiety. the branching core. For instance, if there are 32 intercon 0127. Moreover, the term “water-insoluble' refers to a nected functional groups and hydrogel-connected drug swellable three-dimensionally crosslinked molecular net linker conjugates and capping groups, eight groups may be work froming the hydrogel. If suspended in a large Surplus of provided by each of the four PEG-based polymeric chains water or aqueous buffer of physiological osmolality the extending from the core, preferably by means of dendritic hydrogel may take up a substantial amount of water, e.g. up to moieties attached to the terminus of each PEG-based poly 10-fold on a weight per weight basis, and is therefore meric chain. Alternatively, four groups may be provided by swellable but after removing excess water still retains the each of eight PEG-based polymeric chains extending from physical stability of a gel and a shape. Such shape may be of the core or two groups by each of sixteen PEG-based poly any geometry and it is understood that such an individual meric chains. If the number of PEG-based polymeric chains hydrogel object is to be considered as a single molecule extending from the branching core does not allow for an equal consisting of components wherein each component is con distribution, it is preferred that the deviation from the mean nected to each other component through chemical bonds. number of the sum of interconnected functional groups and 0128. According to this invention, the hydrogel may be hydrogel-connected drug-linker conjugates and capping composed of backbone moieties interconnected by hydrolyti groups per PEG-based polymeric chain is kept to a minimum. cally degradable bonds. 0.137 Preferably, a backbone moiety is further character 0129. Preferably, the backbone moiety has a molecular ized by having a branching core, from which at least three weight in the range of from 1 kDa to 20 kDa, more preferably PEG-based polymeric chains extend. Accordingly, in a pre from 1 kDa to 15 kDa and even more preferably from 1 kDa ferred aspect the backbone reagent comprises a branching to 10 kDa. The backbone moieties are preferably also PEG core, from which at least three PEG-based polymeric chains based, comprising one or more PEG chains. extend. Such branching cores may be comprised of Suitably 0130. A preferred aspect of the present invention is a car substituted derivatives of pentaerythritol, tripentaerythritol, rier-linked prodrug comprising a biodegradable hydrogel as hexaglycerine, Sucrose, Sorbitol, fructose, mannitol, glucose, carrier, wherein a number of permanent linkages of the back cellulose, amylases, starches, hydroxyalkyl starches, polyvi bone moieties exist with the linker L to which the biologically nylalcohols, dextranes, hyaluronans, or branching cores may active moiety is covalently attached. be comprised of poly- or oligoamines such as ornithine, 0131) Ideally, the hydrogel-connected drug-linker conju diaminobutyric acid, trilysine, tetralysine, pentalysine, hexy gates are dispersed homogeneously throughout the hydrogel lysine, heptalysine or oligolysine, nonalysine, decalysine, according to the invention, and may or may not be present on undecalysine, dodecalysine, tridecalysine, tetradecalysine, the Surface of the hydrogel according to the invention. pentadecalysine or oligolysines, low-molecular weight PEI. 0132) The functional groups may be attached to a linear polyvinylamines, low-molecular weight PEI, polyvin chain. In this case, the functional groups may be spaced lyamines, hexaglycerine, tripentaerythritol, in bound form. regularly or irregularly across the chain, or alternatively, the 0.138 Preferably, the branching core extends three to six chain may be terminated by two dendritic moieties, providing teen PEG-based polymeric chains, more preferably four to for the total of functional groups. eight. Preferred branching cores may be comprised of pen 0.133 Remaining reactive functional groups which are not taerythritol, ornithine, diaminobutyric acid, trilysine, tetral connected to a transient prodrug linker or to a spacer con ysine, pentalysine, hexylysine, heptalysine or oligolysine, nected to a transient prodrug linker may be capped with low-molecular weight PEI, hexaglycerine, tripentaerythritol Suitable blocking reagents. in bound form. Preferably, the branching core extends three to 0134 Preferably, the covalent attachment formed between sixteen PEG-based polymeric chains, more preferably four to the reactive functional groups provided by the backbone moi eight. Preferably, a PEG-based polymeric chain is a linear eties and the prodrug linker are permanent bonds. Suitable poly(ethylene glycol) chain, of which one end is connected to functional groups for attachment of the prodrug linker to the the branching core and the other to a hyperbranched dendritic hydrogel according to the invention include but are not lim moiety. It is understood that a polymeric PEG-based chain ited to carboxylic acid and derivatives, carbonate and deriva may be terminated or interrupted by alkyl or aryl groups tives, hydroxyl, hydrazine, hydroxylamine, maleamic acid optionally substituted with heteroatoms and chemical func and derivatives, ketone, amino, aldehyde, thiol and disulfide. tional groups. 0135 Such biodegradable hydrogel may be composed of 0.139 Preferentially, a backbone moiety is characterized backbone moieties interconnected by hydrolytically degrad by having a branching core, from which at least three chains able bonds. The backbone moiety is characterized by a num extend. Such branching cores may be provided by suitably ber of functional groups, comprising interconnected biode substituted derivatives of poly- or oligoalcohols, preferably gradable functional groups and hydrogel-connected drug pentaerythritol, tripentaerythritol, hexaglycerine, Sucrose, linker conjugates, and optionally capping groups. This means Sorbitol, fructose, mannitol, glucose, cellulose, amyloses, that a backbone moiety is characterized by a number of starches, hydroxyalkyl starches, polyvinylalcohols, dex hydrogel-connected drug-linker conjugates; functional tranes, hyualuronans, or branching cores may be provided by groups, comprising biodegradable interconnected functional Suitably Substituted derivatives of poly- or oligoamines Such groups; and optionally capping groups. Preferably, the Sum of as trilysine, tetralysine, pentalysine, hexylysine, heptalysine, US 2013/0035635 A1 Feb. 7, 2013 octalysine, nonalysine, decalysine, undecalysine, dedecal to 2 kDa. Preferably, each dendritic moiety has at least 3 ysine, tridecalysine, tetradecalysine, pentadecalysine or oli branchings and at least 4 reactive functional groups, and at golysines, polyethyleneimines, polyvinylamines. Preferably, most 63 branchings and 64 reactive functional groups, pre the branching core extends three to sixteen chains, more ferred at least 7 branchings and at least 8 reactive functional preferably four to eight. Preferably, such chain is a linear groups and at most 31 branchings and 32 reactive functional polyethylene glycol chain, of which one end is connected to the branching core and the other to a hyperbranched dendritic groups. moiety. 0145 Examples for such dendritic moieties are comprised 0140 Preferably, a PEG-based polymeric chain is a suit of trilysine, tetralysine, pentalysine, hexylysine, heptalysine, ably substituted poly(ethylene glycol) derivative (PEG octadecalysine, nonadecalysine in bound form. Examples for based). such preferred dendritic moieties are comprised of trilysine, tetralysine, pentalysine, hexylysine, heptalysine in bound 0141 Preferred structures for corresponding PEG-based bound form, most preferred trilysine, pentalysine or heptal polymeric chains extending from a branching core contained ysine. Preferably, such dendritic moieties are comprised of in a backbone moiety are multi-arm PEG derivatives as, for lysine, dilysine, trilysine, tetralysine, pentalysine, hexyl instance, detailed in the products list of Jenkem Technology, ysine, heptalysine, most preferred trilysine, pentalysine or USA (accessed by download from www.jenkemusa.com on heptalyine, in bound form. Jul. 28, 2009), 4ARM-PEG Derivatives (pentaerythritol core), 8ARM-PEG Derivatives (hexaglycerin core) and 0146 Most preferably, the hydrogel of the prodrugs of the 8ARM-PEG Derivatives (tripentaerythritol core). Most pre present invention is characterized in that the backbone moiety ferred are 4arm PEG Amine (pentaerythritol core) and 4arm has a quarternary carbon of formula C(A-Hyp), wherein PEG Carboxyl (pentaerythritol core), 8arm PEG Amine each A is independently a poly(ethylene glycol) based poly (hexaglycerin core), 8arm PEG Carboxyl (hexaglycerin meric chain terminally attached to the quarternary carbon by core), 8arm PEG Amine (tripentaerythritol core) and 8arm a permanent covalent bond and the distal end of the PEG PEG Carboxyl (tripentaerythritol core). Preferred molecular based polymeric chain is covalently bound to a dendritic weights for such multi-arm PEG-derivatives in a backbone moiety Hyp, each dendritic moiety Hyp having at least four moiety are 1 kDa to 20 kDa, more preferably 2.5 kDa to 15 groups representing the interconnected functional groups and kDa and even more preferably 5 kDa to 10 kDa. It is under biodegradable and permanent linkages. stood that the terminal amine groups of the above mentioned 0147 Preferably, each A is independently selected from multi-arm molecules are present in bound form in the back the formula—(CH), (OCH2CH2)X , wherein n1 is 1 or bone moiety to provide further interconnected functional 2; n is an integer in the range of from 5 to 50; and X is a groups and reactive functional groups of a backbone moiety. functional group covalently linking A and Hyp. 0142. It is preferred that the sum of interconnected func 0148 Preferably, A and Hyp are covalently linked by an tional groups and reactive functional groups of a backbone amide functional group. moiety is equally divided by the number of PEG-based poly meric chains extending from the branching core. If the num 0149 Preferably, the dendritic moiety Hyp is a hyper ber of PEG-based polymeric chains extending from the branched polypeptide. Preferably, the hyperbranched branching core does not allow for an equal distribution, it is polypeptide comprises lysine in bound form, most preferably preferred that the deviation from the mean number of the sum Hyp is heptalysinyl in bound form. Preferably, each dendritic of interconnected and reactive functional groups per PEG moiety Hyp has a molecular weight in the range of from 0.4 based polymeric chain is kept to a minimum. kDa to 4 kDa, more preferably in the range of from 0.4kDa to 0143 More preferably, the sum of interconnected and 2 kDa. It is understood that a backbone moiety C(A-Hyp) reactive functional groups of a backbone moiety is equally can consist of the same or different dendritic moieties Hyp divided by the number of PEG-based polymeric chains and that each Hyp can be chosen independently. Each moiety extending from the branching core. For instance, if there are Hyp consists of between 5 and 32 lysines, preferably of at 32 interconnected functional groups and reactive functional least 7 lysines, i.e. each moiety Hyp is comprised of between groups, eight groups may be provided by each of the four 5 and 32 lysines in bound form, preferably of at least 7 lysines PEG-based polymeric chains extending from the core, pref in bound form. Most preferably Hyp is comprised of heptal erably by means of dendritic moieties attached to the termi ysinyl. nus of each PEG-based polymeric chain. Alternatively, four groups may be provided by each of eight PEG-based poly 0150. The reaction of polymerizable functional groups a meric chains extending from the core or two groups by each backbone reagent, more specifically of Hyp with the poly of sixteen PEG-based polymeric chains. merizable functional groups of polyethyleneglycol based 0144. Such multi-arm PEG derivatives may be connected crosslinker reagents results in a permanent amide bond. to dendritic moieties to obtain additional functional groups. 0151. One preferred backbone moiety is shown below, Preferably, each dendritic moiety has a molecular weight in dashed lines indicate interconnecting biodegradable linkages the range of from 0.4 kDa to 4 kDa, more preferably 0.4kDa to crosslinker moieties and n is an integer of from 5 to 50: US 2013/0035635 A1 Feb. 7, 2013 14

H N

0152 Preferably, C(A-Hyp) has a molecular weight in conditions like, for example, pH 9, 37° C., aqueous buffer, the range of from 1 kDa to 20 kDa, more preferably 2.5 kDa may be used for practical purposes. to 15 kDa and even more preferably 5 kDa to 10 kDa. 0157 Permanent linkages are non-enzymatically hydro I0153. Preferably, L is attached to Z through a thiosuccin lytically degradable under physiological conditions (aqueous imide group which in turn is attached to the hydrogel's back buffer at pH 7.4, 37° C.) with half-lives of six months or bone moiety through a spacer, Such as an oligoethylene glycol longer, Such as, for example, amides. chain. Preferably, the linkage of this spacer chain to the back 0158. The degradation of the hydrogel is a multi-step reac bone moiety is a permanent bond, preferably an amide bond. tion where a multitude of degradable bonds is cleaved result ing in degradation products which may be water-soluble or 0154) Preferably, L is a chemical bond. water-insoluble. However each water-insoluble degradation 0155 Biodegradability of the hydrogels according to the product further comprises degradable bonds so that it can be present invention is achieved by introduction of hydrolyti cleaved in that water-soluble degradation products are cally degradable bonds. obtained. These water-soluble degradation products may 0156 The terms “hydrolytically degradable’ “biodegrad comprise one or more backbone moieties. It is understood able' or “hydrolytically cleavable”, “auto-cleavable', or that released backbone moieties may, for instance, be perma “self-cleavage”, “self-cleavable', “transient’ or “temporary’ nently linked to spacer or blocking groups and/or prodrug refer within the context of the present invention to bonds and linker degradation products. linkages which are non-enzymatically hydrolytically degrad 0159. In such hydrogel-linked prodrugs according to the able under physiological conditions (aqueous buffer at pH invention, it is desirable that almost all release of biologically 7.4, 37° C.) with half-lives ranging from one hour to three active agent (>90%) has occurred before a significant amount months, include, but are not limited to, aconityls, acetals, of release of the backbone degradation products (<10%) has carboxylic anhydrides, esters, imines, hydrazones, maleamic taken place. This can be achieved by adjusting the hydrogel acid amides, ortho esters, phosphamides, phosphoesters, linked prodrugs half-life Versus the hydrogel degradation phosphosilyl esters, silyl esters, Sulfonic esters, aromatic car kinetics. bamates, combinations thereof, and the like. Preferred biode 0160 To introduce the hydrolytically cleavable bonds into gradable linkages are esters, carbonates, phosphoesters and the hydrogel carrier of the invention, the backbone moieties Sulfonic acid esters and most preferred are esters or carbon can be directly linked to each other by means of biodegrad ates. It is understood that for in vitro studies accelerated able bonds. US 2013/0035635 A1 Feb. 7, 2013

0161. In one embodiment, the backbone moieties of the that such polylactic acid) or poly(glycolic acid) chain may be biodegradable hydrogel carrier may be linked together terminated or interrupted by alkyl oraryl groups and that they directly, i.e. without crosslinker moieties. The hyperbranched may optionally be substituted with heteroatoms and chemical dendritic moieties of two backbone moieties of such biode functional groups. gradable hydrogel may either be directly linked through an (0170 Preferably, the crosslinker moieties are PEG based, interconnected functional group that connects the two hyper preferably represented by only one PEG based molecular chain. Preferably, the poly(ethylene glycol) based crosslink branched dendritic moieties. Alternatively, two hyper ers are hydrocarbon chains comprising ethylene glycol units, branched dendritic moieties of two different backbone moi optionally comprising further functional groups, wherein the eties may be interconnected through two spacer moieties poly(ethylene glycol) based crosslinker moieties comprise at connected to a backbone moiety and on the other side con least each methylene glycol units, wherein m is an integer in nected to a crosslinking moiety separated by an intercon the range of from 3 to 100, preferably from 10 to 70. Prefer nected functional group. ably, the poly(ethylene glycol) based crosslinkers have a 0162 Preferably, backbone moieties may be linked molecular weight in the range of from 0.5 kDa to 5 kDa. together through crosslinker moieties, each crosslinker moi 0171 In a preferred embodiment of the present invention ety being terminated by at least two of the hydrolytically the crosslinker moiety consists of a PEG chain, which is degradable bonds. In addition to the terminating degradable symmetrically connected through ester bonds to two alpha, bonds, the crosslinker moieties may contain further biode omega-aliphatic dicarboxylic spacers provided by backbone gradable bonds. Thus, each end of the crosslinker moiety moieties through permanent amide bonds. linked to a backbone moiety shows a hydrolytically degrad 0172. The dicarboxylic acids of the spacer moieties con able bond, and additional biodegradable bonds may option nected to the backbone moiety and on the other side con ally be present in the crosslinker moiety. nected to a crosslinking moiety consists of 3 to 12 carbon 0163 The hydrogel may contain one or more different atoms, most preferably between 5 and 8 carbon atoms and types of crosslinker moieties, preferably one. The crosslinker may be substituted at one or more carbon atom. Preferred moiety may be a linear or branched molecule and preferably Substituents are alkyl groups, hydroxy groups or amido groups or Substituted amino groups. One or more of the is a linear molecule. In a preferred embodiment of the inven aliphatic dicarboxylic acids methylene groups may option tion, the crosslinker moiety is connected to backbone moi ally be substituted by O or NH or alkyl-substituted N. Pre eties by at least two biodegradable bonds. ferred alkyl is linear or branched alkyl with 1 to 6 carbon 0164. If present in a hydrogel according to the invention as atOmS. degradable interconnected functional group, preferred biode (0173 Preferably, there is a permanent amide bond gradable linkages are carboxylic esters, carboxylic anhy between the hyperbranched dendritic moiety and the spacer drides, carbonates, phosphoesters and Sulfonic acid esters; moiety connected to a backbone moiety and on the other side more preferably carboxylic esters, carbonates, phosphoesters is connected to a crosslinking moiety. and Sulfonic acid esters and most preferred are carboxylic 0.174. One preferred crosslinker moiety is shown below: esters or carbonates. dashed lines indicate interconnecting biodegradable linkages 0.165. In one embodiment, a crosslinker moiety consists of to backbone moieties: a polymer. Preferably, crosslinker moieties have a molecular weight in the range of from 0.5kDa to 5 kDa, more preferably, from 1 kDa to 4 kDa, even more preferably from 1 kDa to 3 kDa. O 0166 In addition to oligomeric or polymeric crosslinking Y&F 1-N-1Ng O y1 s moieties, low-molecular weight crosslinking moieties may be used, especially when hydrophilic high-molecular weight backbone moieties are used for the hydrogel formation. wherein q is an integer of from 5 to 50. 0167 Preferably, the poly(ethylene glycol) based 0.175 Preferably, the hydrogel carrier is composed of crosslinker moieties are hydrocarbon chains comprising eth backbone moieties interconnected by hydrolytically degrad ylene glycol units, optionally comprising further functional able bonds. groups, wherein the poly(ethylene glycol) based crosslinker 0176 More preferably, the backbone moieties comprise a moieties comprise at least each methylene glycol units, branching core of the following formula: wherein m is an integer in the range of from 3 to 100, prefer ably from 10 to 70. Preferably, the poly(ethylene glycol) based crosslinker moieties have a molecular weight in the range of from 0.5 kDa to 5 kDa. 0168 If used in reference to a crosslinker moiety or a PEG-based polymeric chain connected to a branching core, the term "PEG-based refers to a crosslinker moiety or PEG wherein the dashed line indicates attachment to the remainder based polymeric chain comprising at least 20 weight 96 eth of the backbone moiety. ylene glycol moieties. 0177 More preferably, the backbone moieties comprise a 0169. In one embodiment, monomers constituting the structure of the following formula: polymeric crosslinker moieties are connected by biodegrad able bonds. Such polymeric crosslinkers may contain up to 100 biodegradable bonds or more, depending on the molecu lar weight of the crosslinker moiety and the molecular weight of the monomer units. Examples for Such crosslinkers are C O1N-N-n pi N 4w w y polylactic acid or polyglycolic acid based. It is understood US 2013/0035635 A1 Feb. 7, 2013

wherein n is an integer of from 5 to 50 and the dashed line wherein the dashed lines indicate attachment to the rest of the indicates attachment to the remainder of the backbone moi molecule and carbon atoms marked with asterisks indicate in ety. a preferred embodiment S-configuration. However, it is 0.178 Preferably, backbone moiety comprises a hyper understood that hyperbranched moieties Hyp as shown above branched moiety Hyp. may also be in R-confirmation or may be racemic. 0179 More preferably, the backbone moiety comprises a hyperbranched moiety Hyp of the following formula: 0180 Preferably, the backbone moieties are attached to at least one spacer of the following formula:

wherein one of the dashed lines indicates attachment to the hyperbranched moiety Hyp and the second dashed line indi cates attachment to the rest of the molecule; and wherein mis an integer of from 2 to 4. 0181 Preferably, the backbone moieties are linked together through crosslinker moieties having the following Structure

Y{ O 1n-N-1NO c1,

wherein q is an integer from 3 to 100. 0182 More preferably, the backbone moieties of the hydrogel are linked together through moieties of the follow ing formula:

O O O O

y w O1-N-N O F

wherein the dashed lines indicate attachment to a backbone moiety, respectively, and wherein n is 45. 0183 In an alternative preferred embodiment, the back bone moieties of the hydrogel are linked together through moieties of the following formula:

US 2013/0035635 A1 Feb. 7, 2013

wherein the dashed lines indicate attachment to a backbone 0188 Optionally, the prodrug linker may be first conju moiety, respectively, and wherein n is 22. gated to the biologically active agent and the resulting pro 0184 The hydrolysis rate of the biodegradable bonds drug linker conjugate may then react with the hydrogels between backbone and crosslinker moieties is influenced or reactive functional groups. Alternatively, after activation of determined by the number and type of connected atoms adja one of the functional groups of the prodrug linker, the linker cent to the PEG-ester carboxy group. For instance by select hydrogel conjugate may be contacted with biologically active ing from Succinic, adipic or glutaric acid for PEG ester for agent in the second reaction step and excess biologically mation it is possible to vary the degradation half-lives of the active agent may be removed by filtration after conjugation of biodegradable hydrogel carrier. the biologically active agent to the hydrogel-bound prodrug 0185. The hydrogel-linked prodrug of the present inven linker. tion can be prepared starting from the hydrogel of the present 0189 A preferred process for the preparation of a prodrug invention by convenient methods known in the art. It is clear according to the present invention is as follows: to a practitioner in the art that several routes exist. For 0190. A preferred starting material for the backbone example, the prodrug linker mentioned above to which the reagent synthesis is a 4-arm PEG tetra amine or 8-arm PEG biologically active moiety is covalently attached can be octa amine, with the PEG reagent having a molecular weight reacted with the reactive functional groups of the hydrogel of ranging from 2000 to 10000 Dalton, most preferably from the present invention with or with the prodrug linker already 2000 to 5000 Da. To such multi-arm PEG-derivatives, lysine bearing the active moiety in part or as whole. residues are coupled sequentially to form the hyperbranched 0186. In a preferable method of preparation, the hydrogel backbone reagent. It is understood that the lysines can be is generated through chemical ligation reactions. The hydro partially or fully protected by protective groups during the gel may be formed from two macromolecular educts with coupling steps and that also the final backbone reagent may complementary functionalities which undergo a reaction contain protective groups. A preferred building block is bis Such as a condensation or addition. One of these starting boc lysine. Alternatively, instead of sequential additions of materials is a crosslinker reagent with at least two identical lysine residues, a dendritic poly-lysine moiety may be functional groups and the other starting material is a homo assembled first and subsequently coupled to the 4-arm PEG multifunctional backbone reagent. Suitable functional groups tetra amine or 8-arm PEG octa amine. It is desirable to obtain present on the crosslinker reagent include terminal amino, backbone reagent carrying 32 amino groups, consequently carboxylic acid and derivatives, maleimide and other alpha, seven lysines would be attached to each arm of a 4-arm PEG, beta unsaturated Michael acceptors like vinylsulfone, thiol, or three lysines would be attached to each arm of an 8-arm hydroxyl groups. Suitable functional groups present in the PEG. In another embodiment, the multi-arm PEG derivative is a tetra- or octa carboxy PEG. In this case, the dendritic backbone reagent include but are not limited to amino, car moieties may be generated from glutaric or aspartic acid, and boxylic acid and derivatives, maleimide and other alphabeta the resulting backbone reagent would carry 32 carboxy unsaturated Michael acceptors like vinylsulfone, thiol, groups. It is understood that all or a fraction of the backbone hydroxyl groups. reagents functional groups may be present in a free form, as 0187. If the crosslinker reagent's reactive functional salts or conjugated to protecting groups. It is understood that groups are used substoichiometrically with respect to back due to practical reasons the backbone reagent's number of bone reactive functional groups, the resulting hydrogel will lysines per PEG-arm will be between six and seven, more be a reactive hydrogel with free reactive functional groups preferably approximately seven. attached to the backbone structure. 0191) A preferred backbone reagent is shown below: is- NH2 O NH NH2 NH2

O O O HN N H C O NH2 N N-1-on- H NH2 O O

N H HN NH2

O US 2013/0035635 A1 Feb. 7, 2013

0.192 Synthesis of the crosslinker reagent starts from a may be provided by suitably substituted derivatives of lysine, linear PEG chain with a molecular weight ranging from 0.2 to dilysine, trilysine, tetralysine, pentalysine, hexylysine, hep 5 kDa, more preferably from 0.6 to 2 kDa, which is esterified talysine, or oligolysine, low-molecular weight PEI. Prefer with a half ester of a dicarboxylic acid, preferably adipic acid ably, the multi-functional moiety is lysine. or glutaric acid. A preferred protecting group for half ester formation is the benzylic group. The resulting bis dicarboxy 0198 Further, such hydrogel according to the invention lic acid PEG half esters are converted into more reactive may be functionalized with a spacer carrying a different reac carboxy compounds, such as acyl chlorides or active esters, tive functional group than provided by the hydrogel. For e.g. pentafluorophenyl or N-hydroxysuccinimide esters, most instance maleimide reactive functional groups may be intro preferred N-hydroxysuccinimde esters, of which a preferred duced into the hydrogel by coupling a suitable heterobifunc selected structure is shown below. tional spacer such as Mal-PEG6-NHS to the hydrogel. Such

O O O O O O

N-O --- O 1-N-1NO --- O-N

O O wherein each mindependently is an integer ranging from 2 to functionalized hydrogel can be further conjugated to drug 4, and linker reagents, carrying a reactive thiol group on the linker q is an integer of from 3 to 100. moiety to form hydrogel-linked prodrugs according to the (0193 More preferred is the following structure: present invention.

O O O O O O

N - O ---* O 1N-9-ng O ---* O-N

O wherein r is either 1 or 2, preferably 1. 0199 After loading the drug-linker conjugate to the func (0194 Alternatively, the bis dicarboxylic acid PEG half tionalized maleimido group-containing hydrogel, all remain esters may be activated in the presence of a coupling agent ing functional groups are capped with a suitable blocking reagent, such as mercaptoethanol, to prevent undesired side such as DCC or HOBt or PyBOP. reactions. 0.195. In an alternative embodiment, the backbone reagent 0200. A particularly preferred method for the preparation carries carboxy groups and the corresponding crosslinker of a prodrug of the present invention comprises the steps of reagent would be selected from ester-containing amino-ter 0201 (a) reacting a compound of formula C(A-X1), minated PEG-chains. wherein A-X1 represents Abefore its binding to Hyp or 0196. Backbone reagent and crosslinker reagent may be a precursor of Hyp and X1 is a Suitable functional group, polymerized to form the hydrogel according to the invention with a compound of formula Hyp'-X2, wherein Hyp'-X2 using inverse emulsion polymerization. After selecting the represents Hyp before its binding to A or a precursor of desired stoichiometry between backbone and crosslinker Hyp and X2 is a suitable functional group to react with functional groups, backbone and crosslinker are dissolved in X1; DMSO and a suitable emulgator with an appropriately 0202 (b) optionally reacting the resulting compound selected HLB value, preferably Arlacel P135, is employed to from step (a) in one or more further steps to yield a forman inverse emulsion using a mechanical stirrer and con compound of formula C(A-Hyp) having at least four trolling the stirring speed. Polymerization is initiated by the functional groups; addition of a suitable base, preferably by N.N.N',N'-tetram 0203 (c) reacting the at least four functional groups of ethylene diamine. After stirring for an appropriate amount of the resulting compound from Step (b) with a poly(ethyl time, the reaction is quenched by the addition of an acid, Such ene glycol) based crosslinker precursor, wherein the as acetic acid and water. The beads are harvested, washed, and crosslinker precursor is used in a Sub-stoichiometric fractionated according to particle size by mechanical sieving. amount compared to the total number of functional Optionally, protecting groups may be removed at this stage. groups of C(A-Hyp) to yield a hydrogel; 0197). In an alternative embodiment of this invention, 0204 (d) reacting remaining un-reacted functional multi-functional moieties are coupled to the reactive func groups (representing the reactive functional groups of tional groups of the polymerized reactive hydrogel to increase the backbone comprised in the hydrogel) in the hydrogel the number of functional groups which allows increasing the backbone of step (c) with a covalent conjugate of bio drug load of the hydrogel. Such multi-functional moieties logically active moiety and transient prodrug linker or US 2013/0035635 A1 Feb. 7, 2013 19

first reacting the un-reacted functional groups with the N',N'-tertramethylethylene diamine (TMEDA). Stirring in transient prodrug linker and Subsequently with the bio the presence of base is continued for 1 to 16 h. logically active moiety; 0213 During stirring, droplets of dispersed phase are 0205 (e) optionally capping remaining un-reacted hardened to become crosslinked hydrogel beads according to functional groups to yield a prodrug of the present inven the invention which can be collected and fractionation tion. according to size is performed on a vibrational continuous 0206 Such hydrogel is preferably comminuted by sieving machine with a 75 um and a 32 um deck to give mechanical processes Such as stirring, crushing, cutting hydrogel microparticles according to the invention. pressing, or milling, and optionally sieving. For emulsion 0214. The hydrogel for the prodrug of the present inven polymerization, the reaction mixture is comprised of the dis tion can be obtained from the preparation methods inform of persed phase and the continuous phase. micro-particles. In a preferred embodiment of the invention, 0207 For the dispersed phase, backbone reagent and the reactive hydrogel is a shaped article Such as a mesh or a crosslinker reagent are mixed in a ratio aminefactive ester of stent. Most preferably, the hydrogel is formed into micropar 5:1 to 1.05:1, preferably of 2:1 to 1.05:1 and are dissolved in ticulate beads which can be administered as Subcutaneous or DMSO to give a to give a solution with a concentration of 5 to intramuscular injectably by means of a standard Syringe. 50 g per 100 mL, preferably 7 to 30 g per 100 ml, more Such soft beads may have a diameter of between 1 and 500 preferably 7.5 to 20g per 100 ml and most preferably 10 to 20 micrometer. g per 100 ml. 0215 Preferably, such beaded carrier-linked hydrogel 0208. The continuous phase is any solvent, that is not prodrugs have a diameter of between 10 and 100 micrometer miscible with DMSO, not basic, aprotic and shows a viscosity if suspended in an isotonic aqueous formulation buffer, most lower than 10 Pas. Preferably, the solvent is not miscible preferably a diameter of between 20 and 100 micrometer, with DMSO, not basic, aprotic, shows a viscosity lower than most preferably a diameter of between 25 and 80 micrometer. 2 Pas and is non-toxic. More preferably, the solvent is a 0216 Preferably, such beaded carrier-linked hydrogel saturated linear or branched hydrocarbon with 5 to 10 carbon prodrugs can be administered by injection through a needle atoms. Most preferably, the solvent is n-heptane. smaller than 0.6 mm inner diameter, preferably through a 0209 To form an emulsion of the dispersed phase in the needle smaller than 0.3 mm inner diameter, more preferably continuous phase, an emulsifier is added to the continuous through a needle small than 0.25 mm inner diameter, even phase before adding the dispersed phase. The amount of more preferably through a needle smaller than 0.2 mm inner emulsifier is 2 to 50 mg per mL dispersed phase, more pref diameter, and most preferably through a needle Small than erably 5 to 20 mg per mL dispersed phase, most preferably 10 0.16 mm inner diameter. mg per mL dispersed phase. 0217. It is understood that the terms “can be administered 0210. The emulsifier has an HLB-value of 3 to 8. Prefer by injection”, “injectable' or “injectability” refer to a com ably, the emulsifier is a triester of sorbitol and a fatty acid or bination of factors such as a certain force applied to a plunger an poly(hydroxyl fatty acid)-poly(ethylene glycol) conju of a syringe containing the carrier-linked hydrogel prodrugs gate. More preferably, the emulsifier is an poly(hydroxy-fatty according to the invention Swollen in a liquid at a certain acid)-polyethylene glycol conjugate, with a linear poly(eth concentration (w/v) and at a certain temperature, a needle of ylene glycol) of a molecular weight in the range of from 0.5 a given inner diameter connected to the outlet of such syringe, kDa to 5 kDa and poly(hydroxy-fatty acid) units of a molecu and the time required to extrude a certain volume of the lar weight in the range of from 0.5 kDa to 3 kDa on each end carrier-linked hydrogel prodrugs according to the invention of the chain. Most preferably, the emulsifier is poly(ethylene from the Syringe through the needle. glycol) dipolyhydroxy stearate, Cithrol DPHS (Cithrol 0218. In order to provide for injectability, a volume of 1 DPHS, former Arlacel P135, Croda International Plc). mL of the carrier-linked hydrogel prodrugs swollen in water 0211 Droplets of the dispersed phase are generated by to a concentration of at least 5% (w/v) and contained in a stirring with an axial flow impeller with a geometry similar to Syringe holding a plunger of a diameter of 4.7 mm can be stirrers such as Isojet, Intermig, Propeller (EKATO Ruhr- and extruded at room temperature within 10 seconds by applying Mischtechnik GmbH, Germany), most preferably similar to a force of less than 60 Newton, such as less than 50 Newton, Isojet or Propeller with a diameter of 50 to 90% of the reactor preferably by applying a force of less than 40 Newton. diameter. Preferably, stirring is initated before addition of the 0219 Preferably injectability measurement is carried out dispersed phase. Stirrer speed is set to 0.6 to 2.4 m/s, such as for the carrier-linked hydrogel prodrugs of the present inven 0.8 to 2.3 m/s, preferably to 0.6 to 1.7 m/s. The dispersed tion swollen in water to a concentration of ca. 15% (w/v). phase is added at room temperature, and the concentration of 0220 By consequence, the prodrugs according to the the disperse phase is 2% to 70%, preferably 5 to 50%, more present invention show the beneficial effect of a controlled preferably 10 to 40%, and most preferably 20 to 35% of the release rate in respect of the released drug D-H. Preferably, a total reaction Volume. The mixture of dispersed phase, emul Sustained release rate is obtained. Sustained release means sifier and continuous phase is stirred for 5 to 60 min before that the administration intervals of the respective prodrug are adding the base to the effect polymerization. expanded. For example, prodrugs according to the present 0212 5 to 10 equivalents (referred to each amide bond to invention which are based on drugs commonly applied once beformed) of a base are added to the mixture of dispersed and or several times a day provide therapeutically effective levels continuous phase. The base is aprotic, non nucleophilic and for at least three days, more preferably for at least one week soluble in the disperse phase. Preferably, the base is aprotic, and even more preferably for at least one month. non nucleophilic, well soluble in both disperse phase and 0221) The prodrug according to the present invention DMSO. More preferably, the base is aprotic, non nucleo show excellent in vivo/invitro correlation of linker cleavage, philic, well soluble in both disperse phase and DMSO, an a high degree of enzyme independence and show a higher amine base and non-toxic. Most preferably, the base is N.N. stability at lower pH, resulting in a pH dependent cleavage. US 2013/0035635 A1 Feb. 7, 2013 20

0222. A strong in vivo/in vitro correlation is observed, if water removal. For this purpose, Sugars and polyols may the release kinetics exhibited by a carrier-linked prodrug con be used, but corresponding positive effects have also jugate according to the invention in Vivo has a half-life that is been observed for Surfactants, amino acids, non-aque not smaller than half the value exhibited by the same carrier ous solvents, and other peptides. Trehalose is particulary linked prodrug conjugate in aqueous buffer of pH 7.4 at 37° efficient at reducing moisture-induced aggregation and C., wherein the release kinetics in vivo is measured as plasma also improves thermal stability potentially caused by levels of free drug. It is understood that in the case of soluble exposure of protein hydrophobic groups to water. Man carriers, release kinetics may be recorded as hydrolysis kinet nitol and Sucrose may also be used, either as solely of 1CS cryoprotectant or in combination with each other where 0223) Another aspect of the present invention are pharma higher ratios of mannitol:Sucrose are known to enhance ceutical compositions of the carrier-linked prodrugs physical stability of a lyophilized cake. Mannitol may described before. Such pharmaceutical compositions contain also be combined with trehalose. Trehalose may also be one or more excipients, selected from the groups consisting combined with sorbitol or sorbitol may be used as the of: sole protectant. Starch or starch derivatives may also be 0224 (i) Buffering agents: physiologically tolerated used. buffers to maintain pH in a desired range, such as sodium 0230 (vii) Oxidation protection agents: antioxidants phosphate, bicarbonate. Succinate, histidine, citrate and Such as ascorbic acid, ectoine, methionine, glutathione, acetate, Sulphate, nitrate, chloride, pyruvate. Antacids monothioglycerol, morin, polyethylenimine (PEI), pro such as Mg(OH) or ZnCO may be also used. Buffering pyl gallate, Vitamin E, chelating agents such aus citric capacity may be adjusted to match the conditions most acid, EDTA, hexaphosphate, thioglycolic acid. sensitive to pH stability. 0231 (viii) Viscosifiers or viscosity enhancers: retard 0225 (ii) Isotonicity modifiers: to minimize pain that settling of the particles in the vial and Syringe and are can result from cell damage due to osmotic pressure used in order to facilitate mixing and resuspension of the differences at the injection depot. Glycerin and sodium particles and to make the Suspension easier to inject (i.e., chloride are examples. Effective concentrations can be low force on the Syringe plunger). Suitable viscosifiers determined by osmometry using an assumed osmolality or viscosity enhancers are, for example, carbomer vis of 285-315 mCSmol/kg for serum. cosifiers like Carbopol 940, Carbopol Ultrez, 10, cellu 0226 (iii) Preservatives and/or antimicrobials: multi lose derivatives like hydroxypropylmethylcellulose dose parenteral preparations may require the addition of (hypromellose, HPMC) or diethylaminoethyl cellulose preservatives at a Sufficient concentration to minimize (DEAE or DEAE-C), colloidal magnesium silicate the risk of patients becoming infected upon injection and (Veegum) or sodium silicate, hydroxyapatite gel, trical corresponding regulatory requirements have been estab cium phosphate gel, Xanthans, carrageenans like Satia lished. Typical preservatives include m-cresol, phenol, gum UTC 30, aliphatic poly(hydroxy acids). Such as methylparaben, ethylparaben, propylparaben, butylpa poly(D.L- or L-lactic acid) (PLA) and poly(glycolic raben, chlorobutanol, benzyl alcohol, phenylmercuric acid) (PGA) and their copolymers (PLGA), terpolymers nitrate, thimerosol, Sorbic acid, potassium Sorbate, ben of D.L-lactide, glycolide and caprolactone, poloxamers, Zoic acid, chlorocresol, and benzalkonium chloride. hydrophilic poly(oxyethylene) blocks and hydrophobic 0227 (iv) Stabilizers: Stabilisation is achieved by poly(oxypropylene) blocks to make up a triblock of strengthening of the protein-Stabilising forces, by desta poly(Oxyethylene)-poly(oxypropylene)-poly(Oxyethyl bilisation of the denatured stater, or by direct binding of ene) (e.g. Pluronic R), polyetherester copolymer, Such as excipients to the protein. Stabilizers may be amino acids a poly(ethylene glycol) terephthalate/polybutylene Such as alanine, arginine, aspartic acid, glycine, histi terephthalate copolymer, Sucrose acetate isobutyrate dine, lysine, proline, Sugars such as glucose, Sucrose, (SAIB), dextran or derivatives thereof, combinations of trehalose, polyols such as glycerol, mannitol, Sorbitol, dextrans and PEG, polydimethylsiloxane, collagen, chi salts such as potassium phosphate, Sodium Sulphate, tosan, polyvinyl alcohol (PVA) and derivatives, poly chelating agents such as EDTA, hexaphosphate, ligands alkylimides, poly (acrylamide-co-diallyldimethyl Such as divalent metal ions (Zinc, calcium, etc.), other ammonium (DADMA)), polyvinylpyrrolidone (PVP), salts or organic molecules such as phenolic derivatives. glycosaminoglycans (GAGs) Such as dermatan Sulfate, In addition, oligomers or polymers such as cyclodex chondroitin Sulfate, keratan Sulfate, heparin, heparan trins, dextran, dendrimers, PEG or PVP or protamine or sulfate, hyaluronan, ABA triblock or AB block copoly HSA may be used. mers composed of hydrophobic A-blocks, such as poly 0228 (v) Anti-adsorption agents: Mainly ionic or iron lactide (PLA) or poly(lactide-co-glycolide) (PLGA), ionic Surfactants or other proteins or Soluble polymers and hydrophilic B-blocks, such as poly(ethylene glycol) are used to coat or adsorb competitively to the inner (PEG) or polyvinyl pyrrolidone. Such block copolymers Surface of the composition’s container, e.g. poloxamer as well as the abovementioned poloxamers may exhibit (Pluronic(RF-68), PEG dodecyl ether (Bri35), polysor reverse thermal gelation behavior (fluid state at room bate 20 and 80, dextran, poly(ethylene glycol), PEG temperature to facilitate administration and gel state polyhistidine, BSA and HSA and gelatines. Chosen con above sol-gel transition temperature at body tempera centration and type of excipient depends on the effect to ture after injection). be avoided but typically a monolayer of surfactant is 0232 (ix) Spreading or diffusing agent: modifies the formed at the interface just above the CMC value. permeability of connective tissue through the hydrolysis 0229 (vi) Lyo- and/or cryoprotectants: During freeze of components of the extracellular matrix in the intras or spray drying, excipients may counteract the destabi titial space Such as, but not limited to, hyaluronic acid, a lising effects caused by hydrogen bond breaking and polysaccharide found in the intercellular space of con US 2013/0035635 A1 Feb. 7, 2013 21

nective tissue. A spreading agent such as, but not limited 0243 Another aspect is a reconstituted composition com to, hyaluronidase temporarily decreases the Viscosity of prising a therapeutically effective amount of a prodrug the extracellular matrix and promotes diffusion of according to the invention, and optionally one or more phar injected drugs. maceutically acceptable excipients. 0233 (x) Other auxiliary agents: such as wetting 0244 Another aspect of the present invention is the agents, viscosity modifiers, antibiotics, hyaluronidase. method of manufacturing a liquid or Suspension composition Acids and bases such as hydrochloric acid and sodium of carrier-linked prodrug. In one embodiment, such compo hydroxide are auxiliary agents necessary for pH adjust sition is made by ment during manufacture. 0245 (i) admixing the carrier-linked prodrug with one 0234. The composition of a prodrug according to the or more excipients, invention may be provided as a liquid composition, a suspen 0246 (ii) transferring amounts of the liquid or Suspen sion composition or as a dry composition. sion composition equivalent to single or multiple doses 0235. In one embodiment, the pharmaceutical composi into Suitable containers, and tion of a prodrug according to the invention is a dry compo 0247 (iii) sealing the containers. sition. Suitable methods of drying are, for example, spray 0248. Another aspect of the present invention is the drying and lyophilization (freeze-drying). Preferably, the method of manufacturing a dry composition of carrier-linked pharmaceutical composition of prodrug is dried by lyo prodrug. In one embodiment, Such composition is made by philization. 0249 (i) admixing the carrier-linked prodrug with one 0236 Preferably, the prodrug is sufficiently dosed in the or more excipients, composition to provide therapeutically effective amounts of 0250 (ii) transferring amounts equivalent to single or biologically active agent for at least 12 hours in one applica multiple doses into Suitable containers, tion. More preferably, one application of prodrug is sufficient 0251 (iii) drying the composition in said containers, for at least one day, more preferably for at least 3 days, more and preferably for at least 1 week and most preferably for at least 0252 (iv) sealing the containers. 4 weeks. 0253) Suitable containers are vials, syringes, dual-cham 0237. In one embodiment of the present invention, the ber Syringes, ampoules, and cartridges. composition of prodrug is provided as a single dose, meaning 0254 Another aspect is a kit of parts. For liquid and sus that the container in which it is Supplied contains one phar pension compositions, and when the administration device is maceutical dose. simply a hypodermic syringe, the kit may comprise the Syringe, a needle and a container comprising the carrier 0238. In another embodiment, the composition is pro linked prodrug composition for use with the Syringe. In case vided as a multiple dose composition, meaning that it con of a dry composition, the container may have one chamber tains more than one therapeutic dose. Preferably, a multiple containing the dry carrier-linked prodrug composition, and a dose composition contains at least 2 doses. Such multiple second chamber comprising a reconstitution solution. In dose composition of prodrug can either be used for different more preferred embodiments, the injection device is other patients in need thereof or is intendend for use in one patient, than a simple hypodermic syringe and so the separate con wherein the remaining doses are stored after the application tainer with carrier-linked prodrug composition is adapted to of the first dose until needed. engage with the injection device Such that in use the liquid or 0239. In another aspect of the present invention the pro Suspension or reconstituted dry composition in the container drug composition is comprised in a container. For liquid or is in fluid connection with the outlet of the injection device. Suspension compositions, the container is preferably a single Examples of administration devices include but are not lim chamber Syringe. For dry compositions, preferably the con ited to hypodermic syringes and pen injector devices. Particu tainer is a dual-chamber Syringe. The dry composition larly preferred injection devices are the pen injectors, in according to the present invention is provided in a first cham which case the container is a cartridge, preferably a dispos ber of the dual-chamber Syringe and reconstitution solution is able cartridge. provided in a second chamber of the dual-chamber syringe. 0255. A preferred kit of parts comprises a needle and a 0240 Prior to applying the dry composition of prodrug to container containing the dry carrier-linked prodrug composi a patient in need thereof, the dry composition is reconstituted. tion according to the present invention and optionally further Reconstitution can take place in the containerin which the dry containing a reconstitution solution, the container being composition of prodrug is provided, such as in a vial, Syringe, adapted for use with the needle. Preferably, the container is a dual-chamber Syringe, ampoule, and cartridge. Reconstitu dual-chamber syringe. tion is done by adding a predefined amount of reconstitution 0256 In another aspect, the invention provides a cartridge Solution to the dry composition. Reconstitution solutions are containing a composition of carrier-linked prodrug as here sterile liquids, such as water or buffer, which may contain inbefore described for use with a pen injector device. The further additives, such as preservatives and/orantimicrobials, cartridge may contain a single dose or multiplicity of doses of such as, for example, benzylalcohol and cresol. Preferably, carrier-linked prodrug. the reconstitution solution is sterile water. 0257. In one embodiment of the present invention the sus 0241. A further aspect is a method of preparing a recon pension composition of carrier-linked prodrug does not only stituted composition comprising a therapeutically effective comprise a carrier-linked prodrug and one or more excipients, amount of prodrug, and optionally one or more pharmaceu but also other biologically active agents, either in their free tically acceptable excipients, the method comprising the step form or as prodrugs or carrier-linked prodrugs such as PEG of prodrugs or hydrogel prodrugs. Preferably, Such additional 0242 contacting the composition of the present inven one or more biologically active agent is a prodrug, more tion with a reconstitution Solution. preferably a PEG or hydrogel prodrug. US 2013/0035635 A1 Feb. 7, 2013 22

0258. In an alternative embodiment, the carrier-linked cursor L-Y with a biologically active drug D-H to obtain the prodrug composition according to the present invention is drug linker conjugate D-L by forming an carbamate bond, combined with a second biologically active compound in wherein Y is a leaving group. Such way that the carrier-linked prodrug composition accord ing to the invention is administered to a patient in need thereof 0265. In respect of the prodrug precursor L-Y, L has the first, followed by the administration of the second compound. same meaning as indicated above in connection with the drug Alternatively, the carrier-linked prodrug composition is linker conjugate D-L. The same holds true for the analogous administered to a patient in need thereof after another com employment of the prodrug precursor L1-Y in respect of the pound has been administered to the same patient. moiety L1 represented by formula (I). 0259 Another subject of the present invention is the use of 0266 Y is a leaving group. Such leaving groups are known prodrugs or a pharmaceutically acceptable salt thereof com to a person skilled in the art. Preferably, Y is chloride, bro prising a drug linker conjugate D-L as pharmaceuticals or mide, fluoride, nitrophenoxy, imidazolyl. N-hydroxysuccin medicaments, respectively. With respect of the definitions of imidyl, N-hydroxybenzotriazolyl, N-hydroxyazobenzotriaz the drug linker conjugate D-L as well as further Substituents olyl, pentafluorophenoxy, 2-thiooxo-thiazolidinyl, or Such as L' the same explanations as laid out above in the N-hydroxysulfosuccinimidyl. context of the prodrug as such apply. 0267 In case the synthesis of a carrier-linked prodrug 0260 Yet another aspect of the present invention is a car according to the present invention is carried out by employing rier-linked prodrug of the present invention or a pharmaceu a precursor L'-Y, a drug linker intermediate (L-D) is tical composition of the present invention for use in a method obtained by reacting L'-Y with the biologically active drug of treating or preventing diseases or disorders which can be D-H by forming a carbamate bond. In Such case, said drug treated by the biologically active moiety released from the linker intermediate L'-D is reacted further to obtain the drug carrier-linked prodrug according to the present invention. linker conjugate D-L by adding the moiety L and the carrier 0261) Another subject of the present invention is a phar group Z to said drug linker intermediate L'-D. It has to be maceutical composition comprising an effective dose of at least one prodrug or a pharmaceutically acceptable salt indicated that the addition of L and/or Z to L'-D may be thereofas defined above and a pharmaceutically acceptable performed in several steps by preparing further intermediate excipient. Furthermore, the present invention also comprises compounds prior to obtaining the drug linker conjugate D-L. the use of Such pharmaceutical compositions as pharmaceu 0268 Alternatively, a prodrug precursor L*-Y may be ticals or medicaments, respectively. employed instead of L'-Y, wherein L* is selected from a 0262 Another subject of the present invention is a method fragment of L', L' containing at least one protecting group or for the synthesis of a prodrug or a pharmaceutically accept L' additionally containing precursors of L and/or Z. able salt thereofas defined above. Prodrugs or precursors of prodrugs according to the present invention may be prepared EXAMPLES by known methods or in accordance with the reaction sequences described below. The starting materials used in the preparation (synthesis) of prodrugs of the invention or pre Example 1 cursors thereof are known or commercially available, or can be prepared by known methods or as described below. Synthesis of Paracetamol-Linker Conjugate 1 0263 All reactions for the synthesis of the prodrugs according to the present invention including precursors such 0269 as the moiety L' according to the formula (I) are per se well-known to the skilled person and can be carried out under standard conditions according to or analogously to proce dures described in the literature, for example in Houben 1 Weyl, Methoden der Organischen Chemie (Methods of Organic Chemistry). Thieme-Verlag, Stuttgart, or Organic H Reactions, John Wiley & Sons, New York. Depending on the circumstances of the individual case, in order to avoid side O C N reactions during the synthesis of a prodrug or a precursor --~~~~ O thereof, it can be necessary or advantageous to temporarily block functional groups by introducing protective groups and to deprotect them in a later stage of the synthesis, or introduce functional groups in the form of precursor groups which in a later reaction step are converted into the desired functional 0270 Paracetamol (1 mmol) was dissolved in 10 ml of groups. Such synthesis strategies and protective groups and THF and nitrophenyl-chloroformate (1.1 mmol) and DIPEA precursor groups which are suitable in an individual case are (1.1 mmol) were added. After 30 min, N,N-bis3-(methy known to the skilled person. If desired, the prodrugs or pre lamino)propylmethylamine (2 mmol) was added and reac cursors can be purified by customary purification procedures, tion mixture was stirred at room temperature for 30 min. 1 for example by recrystallization or chromatography. was purified by RP-HPLC. 0264. The prodrugs according to the present invention (or a pharmaceutically acceptable salt thereof) may be prepared (0271. Yield 83 mg (14%). by a method comprising the step of reacting a prodrug pre 0272. MS: m/=351.26 M+H". US 2013/0035635 A1 Feb. 7, 2013

Example 2 Synthesis of PEG-Linker-Paracetamol Conjugate 3 0273

H O O N - 1-1a N 1n 1a u-C O

(0274) 1 (31 umol) was dissolved in DMSO (250 uL). 5 -continued kDa methoxy poly(ethylene glycol) carboxylic acid NHS ester (15umol) was dissolved in DMSO (480 uL) and added f to the solution of the amine. DIPEA (124 umol) was added s1N1\-1N1 N1)NHE, and the mixture solution was stirred at room temperature for 30 min. 2 was purified by RP-HPLC. 3 0275 Yield 30 mg (41%). 0280 Triphenylmethane thiol (247 mg, 0.89 mmol) was Example 3 suspended in DMSO (1 mL) and DBU (152 uL, 1.02 mmol) was added. After stirring at room temperature for 5 min, 4-Bromohexanol (173 mg, 0.96 mmol) was added, and the Release of Paracetamol In Vitro mixture was stirred for 10 min. The reaction mixture was diluted with ethyl acetate, quenched with 0.1 M HCl and 0276) 2 was dissolved in 60 mM sodium phosphate, pH extracted with ethyl acetate. The crude product was purified 7.4, and incubated at 37° C. Aliquots were analyzed by RP by flash chromatography. HPLC at 242 nm and MS for released paracetamol. MS showed release of unmodified paracetamol. (0281. Yield 283 mg (85%). 0282. MS: m/=375.17 M+H". (0277 t—28.8 d. 0283 6-Triphenylmercapto-hexane-1-ol (466 mg, 1.24 mmol) and triethylamine (600 uL, 4.34 mmol) were dissolved Example 4 in dichloromethane (3.5 mL) and DMSO (500 uL), cooled to 0° C. and added to a 0° C. suspension of sulfur trioxide Synthesis of Paracetamol Linker Conjugate 4 pyridine complex (408 mg, 2.57 mmol) in DMSO (400 uL). The mixture was stirred for 20 min. The mixture was diluted 0278 with dichloromethane, quenched with 0.1 m HCl and brine and extracted with dichloromethane. The crude product was purified by flash chromatography. 4 (0284. Yield 329 mg (71%). O 0285 MS: m/z =397 M+Na". 0286 To a 0°C. solution of 6-triphenylmercaptohexanal ls 1N-N (329 mg, 0.88 mmol), N,N'-diethyl ethylene diamine (630 O STrt uL, 4.4 mmol) and acetic acid (700 u, 12.3 mmol) in dichlo Et romethane (5.5 mL) was added sodium triacetoxy borohy dride (556 mg, 2.64 mmol), and the mixture was allowed to warm to room temperature over the course of 45 min. The mixture was diluted with dichloromethane and washed with aqueous NaOH and brine. The crude product was purified by NHAC RP-HPLC. (0287 Yield 141 mg (34%). 0288 MS: m/z =475.33 M+H". (0279. Synthesis scheme: 0289. To a solution of paracetamol (24 mg., 0.16 mmol) in tetrahydrofurane (1 mL) were added p-nitrophenyl chlorofor OH DBU mate (35 mg, 0.17 mmol) and DIPEA (68 uL, 0.39 mmol), Trit-SHo -- Br HeDMSO and the mixture was stirred at room temperature for 2 h. A solution of amine 3 (70 mg, 0.10 mmol) and DIPEA (40 uL. 1N1-1a-' SO3*pyridine 0.23 mmol) in tetrahydrofurane (0.5 mL) was added, and after TrtS DMSO, CH2Cl2 1 h the reaction mixture was quenched with acetic acid. The NHEt crude product was purified by RP-HPLC. TrtS 1n-1-14' Ellin?NaBH(OAc)3 1 "- 0290. Yield 38 mg (50%). 0291 MS: m/=652.36 M+H". US 2013/0035635 A1 Feb. 7, 2013 24

Example 5 0299 While this invention has been described in conjunc tion with the specific embodiments outlined above, it is evi Synthesis of PEG-Linker-Paracetamol Conjugate 6 dent that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, the pre 0292 ferred embodiments of the invention as set forth above are intended to be illustrative, not limiting. Various changes may be made without departing from the spirit and scope of the O Et inventions as defined in the following claims. O - -^- - n-1N1 S-1Ns. ABBREVIATIONS (0300 d days Et TFA DBU 1,3-diazabicyclo5.4.0]undecene CHCl2 DIEPA diisopropylethylamine DMSO dimethylsulfoxide Et ethyl NHAC MeCN acetonitrile MS mass spectrometry 4 NHS N-hydroxy succinimide O Et PEG poly(ethylene glycol) O ul N1 a- n-1\-1N1as RP-HPLC reversed-phase high performance liquid chroma l PEG-5k tography t Dai-maleimide thalf-life -e- TFA trifluoro acetic acid THF tetrahydrofuran Trt triphenylmethyl, trityl NHAC 1. A prodrug or a pharmaceutically acceptable salt thereof 5 comprising: O a drug linker conjugate D-L: wherein D is a biologically active moiety containing an lO Et PEG5kDA aromatic hydroxyl group; and O ^- N-1-1-1s wherein L is a non-biologically active linker containing, Et O i) a moiety L' represented by formula (I);

(I) NHAC R3 R2 6

0293 At 0°C., solid 4 (38 mg, 50 umol) was treated with iii. a solution of trifluoroacetic acid in dichloromethane (1:1, V/V, 2 mL). After 1 min, triethylsilane (100LL) was added, and the wherein the dashed line indicates the attachment of L' reaction mixture was quenched with water after 15 min. The to the aromatic hydroxyl group of D by forming a crude product was purified by PR-HPLC. carbamate group; wherein R' is selected from the group consisting of: 0294. Yield 22 mg (84%). Calkyl, 0295 MS: m/z-410.25 M+H". heteroalkyl; 0296 Linker thiol 5 (5 mg, 10 dissolved in 338 uL MeCN/ C-7 cycloalkyl; and water 9:1 V/V, 0.05%TFA) was added to a solution of 5 kDa methoxy poly(ethylene glycol) maleimide (73 mg, 14.6 umol) in MecN/water 1:1 (v/v, 2 mL). phosphate buffer (0.5 M, pH7.40, 200 uL) was added, and the mixture was incu bated at room temperature for 15 min. The reaction mixture was quenched with acetic acid and the crude product was purified by RP-HPLC. iii. 0297. Yield 6.3 mg (8%). wherein R. R. R. and R are independently Example 6 Selected from hydrogen, Substituted or non-substi tuted linear, branched or cyclic C alkyl, and het Release of Paracetamol In Vitro eroalkyl, and wherein m is independently 2, 3 or 4; and 0298 6 was dissolved in 60 mM sodium phosphate, pH ii) a moiety L, which is a chemical bondora spacer, and 7.4, and incubated at 37° C. Aliquots were analyzed by RP L is bound to a carrier group Z: HPLC at 242 nm and MS for released paracetamol. MS wherein L' is substituted with one to four Li moieties; showed release of unmodified paracetamol. and t12-2.1d. wherein optionally, L is further substituted. US 2013/0035635 A1 Feb. 7, 2013 25

2. The prodrug according to claim 1: biologically active agent for at least 12 hours in one wherein L is a chemical bond. application. 3. The prodrug according to claim 1: 12. A kit of parts comprising: wherein the carrier group Z is a polymer with a molecular a needle; and weight:2500 g/mol. a container containing: 4. The prodrug according to claim 1: reconstitution Solution; and the dry composition according to claim 11 configured wherein the carrier group Z is a hydrogel. for use with the needle. 5. The prodrug of claim 4: 13. The kit of parts according to claim 12: wherein the hydrogel is composed of backbone moieties wherein the container is a dual-chamber Syringe, and interconnected by hydrolytically degradable bonds. wherein one of the two chambers of the dual-chamber 6. The prodrug of claim 5: Syringe contains the dry pharmaceutical composition wherein the backbone moieties have a molecular weight in and the second chamber of said dual-chamber Syringe the range of from 1 kDa to 20 kDa. contains the reconstitution solution. 7. The prodrug of claim 5: 14. The prodrug according to claim 1: wherein backbone moieties are linked together through wherein the prodrug is configured for use as pharmaceuti crosslinker moieties, each crosslinker moiety being ter cal. minated by at least two of the hydrolytically degradable 15. A method for the synthesis of a prodrug or a pharma bonds. ceutically acceptable salt thereof according to claim 1, com 8. The prodrug of claim 7: prising: wherein the crosslinker moieties have a molecular weight a step of reacting a prodrug precursor L-Y or L'-Y with a in the range of from 0.5 kDa to 5 kDa. biologically active drug D-H, to obtain the drug linker 9. A pharmaceutical composition comprising: conjugate D-L or a drug linker intermediate D-L' by a prodrug of claim 1 or a pharmaceutically acceptable salt forming a carbamate bond; thereof, and wherein Y is a leaving group. a pharmaceutically acceptable excipient. 16. The prodrug according to claim 1: wherein the carrier group Z is a PEG based hydrogel. 10. A pharmaceutical composition according to claim 9: 17. The prodrug according to claim 1: wherein the pharmaceutical composition is dry. wherein the carrier group Z is a biodegradable poly(ethyl 11. A pharmaceutical composition according to claim 9: ene glycol) based water-insoluble hydrogel. wherein the prodrug is sufficiently dosed in the composi tion to provide a therapeutically effective amount of k k k k k