US009078929B2

(12) United States Patent (10) Patent No.: US 9,078,929 B2 Kuebellbeck et al. (45) Date of Patent: Jul. 14, 2015

(54) e-POLYLYSINE CONUGATES AND THE USE (56) References Cited THEREOF U.S. PATENT DOCUMENTS (75) Inventors: Armin Kuebelbeck, Bensheim (DE); 2009/0209508 A1 8/2009 Lange et al. Gregor Larbig. Gelnhausen (DE); 2009/0287.005 A1* 11/2009 Baker et al...... 549/510 Walter Mier, Bensheim (DE); Barbro Beijer, Nussloch (DE); Uwe FOREIGN PATENT DOCUMENTS Haberkorn, Schwetzingen (DE) WO WO90, 13256 * 11/1990 WO WO90, 1325.6 A1 11, 1990 (73) Assignee: Merck Patent GmbH, Darmstadt (DE) WO WOO3,O86293 A2 10, 2003 WO WO 2007/023398 A2 3, 2007 (*) Notice: Subject to any disclaimer, the term of this WO WO 2008/O17122 * 2/2008 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 161 days. Matsusaki et al. Self-assembling bionanoparticles of poly(epsilon (21) Appl. No.: 13/384,789 lysine) bearing cholesterol as a biomesogen, Biomacromolecules. Jul.-Aug. 2005;6(4):2374-9.* (22) PCT Fled: Jul. 9, 2010 UZgiris et al. Conformation and structure of polymeric contrast agents for medical imaging, Biomacromolecules. Jan.-Feb. PCT NO.: PCT/EP201 O/OO4198 2004:5(1):54-61.* (86) Steeghs et al. Small Molecule Tyrosine Kinase Inhibitors in the S371 (c)(1), Treatment of Solid Tumors: An Update of Recent Developments, (2), (4) Date: Jan. 19, 2012 Ann Surg. Oncol. Feb. 2007;14(2):942-53. Epub Nov. 14, 2006.* Zahid et al. Identification of a Cardiac Specific Protein Transduction PCT Pub. No.: WO2O11AOO9539 Domain by InVivo Biopanning Using a M13 Phage Peptide Display (87) Library in Mice, PLoS ONE, Aug. 2010, vol. 5, Issue 8, e12252).* PCT Pub. Date: Jan. 27, 2011 Szende et al. Antitumor effect of lysine-isopeptides, Cancer Cell International 2002, 2:4. (65) Prior Publication Data International Search Report of PCT/EP2010/004 198 (Sep. 3, 2010). I.L. Shih et al., “Microbial Synthesis of Poly(e-lysine) and Its Various US 2012/O122788A1 May 17, 2012 Applications'. Bioresource Technology, vol. 97. No. 9 (2006) pp. 1148-1159. (30) Foreign Application Priority Data D. Huang et al., “Design and Biophysical Characterization of Novel Polycationic e-Peptides for DNA Compaction and Delivery'. Jul. 20, 2009 (EP) ...... O90093.93 Biomacromolecules, vol. 9, No. 1 (2008) pp. 321-330. M. Matsusaki et al., “Self-Assembling Bionanoparticles of Poly(e- (51) Int. C. Lysine) Bearing Cholesterol as a Biomesogen'. Biomacromolecules, A6 IK38/00 (2006.01) vol. 6, No. 4 (2005) pp. 2374-2379. A6IPI3/2 M.V. Pimm et al., “In Labelling of a Branched Polypeptide Drug (2006.01) Carrier with a Poly(L-lysine) Backbone'. International Journal of C07K5/00 (2006.01) Pharmaceuticals, vol. 79, No. 1-3 (1992) pp. 77-80. C07K 700 (2006.01) Kornguth, S.E. et al. “Preferential Binding of Radiolabeled Poly-L- C07K 16/00 (2006.01) lysines to C6 and U87 MG Glioblastomas Compared with C07K 17/00 (2006.01) Endothelial Cells in Vitro Cancer Research, 49:6390-6395, Nov. 15, A6 IK38/04 (2006.01) 1989. A6 IK 47/48 (2006.01) A6 IK 49/08 (2006.01) * cited by examiner A6 IK5I/04 (2006.01) CO7K 7/06 (2006.01) Primary Examiner — Karheinz R Skowronek CO7K (4/OO (2006.01) Assistant Examiner — Sergio Coffa CO7K 7/08 (2006.01) (74) Attorney, Agent, or Firm — Millen, White, Zelano & (52) U.S. C. Branigan, P.C. CPC ...... A61K 47/48315 (2013.01); A61K 49/085 (2013.01); A61K5I/0497 (2013.01); C07K (57) ABSTRACT 7/00 (2013.01); C07K 7/06 (2013.01); C07K The present invention relates to e-polylysine conjugates, in 7/08 (2013.01); C07K 14/00 (2013.01) particular conjugates of e-polylysine with compounds carry (58) Field of Classification Search ing carboxyl groups, and to the preparation and use thereof CPC. A61K47/48: A61K47/48315; C07K 7/00; for targeting of the kidney. C07K 7/06; C07K 7/08; C07K 14/00 See application file for complete search history. 17 Claims, 4 Drawing Sheets U.S. Patent Jul. 14, 2015 Sheet 1 of 4 US 9,078,929 B2

Fig. 1

U.S. Patent Jul. 14, 2015 Sheet 2 of 4 US 9,078,929 B2

Fig. 2 200,0

180,0

160,0

140,0 2E P1 10' 2NENE 120,0 P1 1 h 2.2NE P1 6h 3. 100,0 P2 10' 3. P2 1 h 2NE P26 h 2. 80,0 P3 O' 2NE N P3 1 h 2NE 60,0 EP3 6h 3. 2NEYNE 40,0 2NE NE 2NENE 20,0 2NE 2NENE OO s 8% u Y a Er O 2NE see E Blood Heart Lung Spleen Liver Kidney Muscle U.S. Patent Jul. 14, 2015 Sheet 3 of 4 US 9,078,929 B2

Fig. 3

DTPA

OH MAG3

O HO SH

HO SH O

DMSA U.S. Patent Jul. 14, 2015 Sheet 4 of 4 US 9,078,929 B2

Fig. 4

-- eanalapril derivativeas 1 eanalapril derivative 2

O- at eanalapril derivative 3

eanalapril derivative 4 US 9,078,929 B2 1. 2 e-POLYLYSINE CONUGATES AND THE USE molecules for angiography. As a further development, radi THEREOF ography supplies cross-sectional images. In CT, radiographs are recorded from various directions by sensors (detectors) The present invention relates to e(epsilon)-polylysine con and reproduced by computer as three-dimensional radiogra jugates, in particular conjugates of e-polylysine with com phy. Owing to the broad applicability of CT, this method is pounds carrying carboxyl groups, and to the preparation and known as the “work-horse of classical radiology”. However, use thereof for targeting of the kidney. the low sensitivity of the method limits its use as a method for The kidney is an organ which is of importance, in particu molecular imaging. lar, for the transport and excretion of various Substances and Single Photon Emission Computed Tomography (SPECT) in the production of hormones. 10 One function of the kidneys is the excretion of end products Scintigraphy uses the gamma radiation of radioactively of metabolism, the so-called urophanic Substances, and tox labelled substances (radiotracers) emitted by short-lived ins from the body through the formation of urine, which is radionuclides. These accumulate specifically in the target finally excreted from the body via the urinary tract. The tissue in the body. With the aid of a gamma camera, the kidney regulates the water balance and thus serves for long 15 emitted radiation is recorded and converted into an image. term regulation of blood pressure. It regulates the electrolyte Single Photon Emission Computed Tomography (SPECT) is balance and the acid-base balance by control of the compo the three-dimensional variant of scintigraphy. In SPECT, the sition of urine. Furthermore, the kidney is an important organ radiation is recorded from various angles as in CT, and a for intermediary metabolism in the body (it effects gluconeo three-dimensional image is obtained in the computer. In static genesis). The kidney produces hormones, such as, for SPECT, the concentration of the radiotracer at a certain point example, erythropoietin, for blood formation and is the site of in time is measured. In dynamic SPECT, the measurement is degradation of peptide hormones. However, many functions repeated at certain time intervals. In this way, the change in of the kidney itself are also controlled by hormones. accumulation can be investigated. The kidney is therefore an organ which is important to life, Positron Emission Tomography (PET) for which many diagnostic and therapeutic methods have 25 In clinical application, PET supplements the more struc already been developed. For example, immunosuppressants, turally oriented imaging methods of diagnostic radiology. cytostatics, immunotherapeutic agents, antiphlogistics, anti Positron emission tomography (PET) is a modern function biotics, Virostatics, antihypertensives, uricosurics, or diuret ally imaging method. With the aid of positron-emitting ics are employed for the treatment of the kidney or for influ atoms, it enables excellent resolution in the detection of encing kidney function. It is particularly important here that 30 radioisotopes (a resolution of 2-3 mm is achieved in modern the medicaments reach the kidney in as targeted a manner as PET instruments, even in the case of full-body tomographs). possible. If these radioisotopes, for example Ga, are employed for the Equally, representation of the kidney in imaging methods labelling of biomolecules, the biochemical processes in indi is also of major importance. vidual organs of the organism can be imaged. An essential With the aid of established nuclear-medical and radiologi 35 advantage of nuclear-medical methods is the high sensitivity, cal methods, such as SPECT, PET, ultrasound and MRT, owing to which the tracers can only be employed in tiny enzymatic processes, metabolic processes, the expression of amounts (nanogram quantinties). Today, the PET camera is certain genes and molecular reactions, besides morphological integrated with a CT instrument (this offers high local reso structures, can be depicted by So-called molecular imaging. lution of about <1 mm). PET/CT technology has introduced The imaging modalities mentioned above can, if necessary, 40 revolutionary results into diagnostics in recent years. The be further Supplemented by computer tomographic and opti functional images of PET and the morphological information cal imaging methods (near-infrared imaging, fluorescence of CT in the same anatomical structures can thus be obtained tomography). The focus of "molecular imaging is at present in a single representation. still on the diagnosis of cancer diseases, neurological ques An organ which is very frequently and very intensively tions and the monitoring of gene therapies, but in the future 45 investigated in medical diagnostics is the kidney. The most will be extended to all areas in which cellular changes have to frequent investigation method here is renal Scintigraphy. be discovered as early as possible. Renal Scintigraphy As signal Source for the imaging methods, a 'signal mol Renal Scintigraphy is a nuclear-medical investigation ecule' is generally coupled to a “carrier molecule'. The “car method which allows the assessment of renal function from rier molecule' ensures highly specific targeting by, for 50 static and dynamic points of view. The blood Supply, function example, binding specifically to the target cells or becoming and excretion of each individual kidney are assessed here. It is trapped therein. For example, the carrier molecule can be the an established method for the recognition of parenchymal ligand of a receptor or the Substrate of an enzyme. The “signal scarring, in particular in children, and furthermore serves for molecule' can be rendered visible by means of one or more the assessment of regional and side-separated renal function. imaging techniques. Examples of signal molecules are, for 55 A distinction is made between two forms of renal Scintig example, complexing agents or chelating agents whose metal raphy: ions can be detected via imaging techniques. The compound Static Renal Scintigraphy or conjugate comprising signal molecule and carrier mol In static renal Scintigraphy, the functional kidney tissue is ecule is called the "diagnostic agent'. The various imaging represented using the radionuclide "Tc. The technetium techniques will be discussed in detail below. 60 here is bound in complex form to, for example, 2,3-dimer Computer Tomography (CT) captosuccinic acid (DMSA). Static renal Scintigraphy is In classical radiography, the tissue-specific attenuation of therefore principally suitable for the representation of kid X-rays is depicted on an X-ray film. “Hard tissues, for neys having anomalies (dystrophy, horseshoe kidney, etc.) or example bones, absorb a large amount of radiation here in state after inflammation. contrast to 'soft tissue. Such as fat and muscle. X-ray con 65 Dynamic Renal Scintigraphy trast agents used are substances which contain elements hav By contrast, dynamic renal Scintigraphy investigates renal ing a high atomic number, for example iodine-containing function. Thus, the glomerular filtration rate, renal blood flow US 9,078,929 B2 3 4 (RBF) and tubular secretion can be investigated with the carboxyl groups have extremely high selectivity for the kid question of renal function and clearance thereof. ney. This means that these conjugates are taken up virtually Radiopharmaceuticals which are currently used are the exclusively by the kidney tissue. These conjugates, coupled to following Substances: signal molecules, such as, for example, radioisotopes and/or "Tc-MAG3 mercaptoacetyltriglycine active compounds, can be employed for the diagnostic and/or "Tc-DMSA 2,3-dimercaptosuccinic acid therapeutic treatment of the kidney. "Tc-DTPA diethylenetriaminepentaacetic acid The present invention therefore relates to a conjugate com 'I-OIH hippuran (ortho-iodohippuric acid) prising at least one compound carrying carboxyl groups and a FIG.3 shows the chemical structure of MAG3, DMSA and linear or branched oligomer which consists of peptidically DTPA. 10 linked monomer units and which is either built up in total Renal function Scintigraphy (dynamic renal Scintigraphy) from more than 50% (based on the number of monomer units) is employed in the following indications: ofe-lysine monomer units or comprises at least 10 Successive for clarification of side-separated renal function in kidney monomer units which are built up from at least 70% (based on diseases, such as, for example, kidney Stones (nephroli the number of monomer units) of e-lysine monomer units. thiasis), renal tumours, dystrophic (incorrectly located) 15 Preference is given to the use of compounds carrying car kidneys or dysplastic (malformed) kidneys boxyl groups in which the proportion of the carboxyl groups for the investigation of part-function in the case of double in the molar mass of the compound carrying carboxyl groups kidneys is greater than 30%, particularly preferably greater than 40%. for the investigation of urinary flow disorders In a preferred embodiment, in particular for therapeutic for clarification of vesicorenal reflux (an anomaly of the applications, the conjugate additionally comprises at least urinary tract) one active compound, preferably covalently bonded. if renovascular hypertonia is suspected In a preferred embodiment, the oligomer has a chain length for testing renal function before living kidney donation of 10 to 50 monomer units. for progress control of Surgically repaired vascular con In a particularly preferred embodiment, the oligomer con strictions or obstructions 25 sists only of e-lysine monomer units, in particular only of for the assessment of transplanted kidneys e-lysine units. in emergency diagnosis if kidney injury (kidney trauma) is In a preferred embodiment, at least one compound carrying Suspected carboxyl groups is bonded via the amino group of an e-lysine in the case of Sudden greatly reduced urine excretion monomer unit, i.e. one or more e-lysine monomer units carry (anuria) in order to exclude a renal embolism oran acute 30 on their amino group a compound carrying carboxyl groups urinary obstruction which is conjugated directly or via a spacer. in order to determine total clearance In an embodiment, which is particularly preferred for diag in order to detect or exclude urine leakage nostic applications, the compound carrying carboxyl groups To date, renal function Scintigraphy has generally been is a complexing agent, particularly preferably DOTA (=14, carried out by means of SPECT, since the approved tracers 35 7,10-traazacyclododecane-N, -N', -N", -N"-tetraacetic acid) MAG3 and DMSA can only be labelled with the SPECT or DTPA (diethylenetriaminepentaacetic acid). nuclide "Tc. Consequently, it has to date not been possible The present invention also relates to a process for the to use the considerably improved possibilities today due to preparation of the compounds according to the invention PET and PET/CT for renal function diagnosis. which comprises at least the following process steps: It would also be desirable for therapeutic purposes if tar 40 a) provision of an oligomer according to the invention which geting of the kidney could be improved. Today, about 280 contains at least one reactive group, million people suffer from chronic kidney diseases. The use b) conjugation of at least one optionally activated compound or dosage of medicaments for the treatment of kidney dis carrying carboxyl groups to the oligomer from step a) eases is frequently restricted by side effects of the medica In an embodiment of the process according to the inven ments. If it were possible to develop a drug targeting concept, 45 tion, if the conjugate comprises a complexing agent, the com by means of which known or also new medicaments reach the pound obtained in step b) is brought into contact with metal kidney in a targeted manner, the therapeutic treatment of salts in a further step c), so that metal ions are complexed by kidney diseases would be greatly improved. the complexing agents. WO 03/086293 describes an attempt to improve the flavour The present invention additionally relates to a conjugate of medicaments by them forming a complex with polylysine 50 according to the invention as medicament, such as, in particu or polyarginine. The complex here consists of an organic salt, lar, a therapeutic composition or an image-enhancing com i.e. there is no covalent bond, but instead an ionic bond, position. between polylysine?polyarginine and the medicament. No The present invention also relates to a medicament orphar thing is disclosed regarding the possibility of the use of polyl maceutical composition, in particularatherapeutic or image ysine conjugates in the drug targeting of the kidney. 55 enhancing composition, at least comprising a conjugate Ing-Lung Shih et al., Bioresource Technology 97 (2006) according to the invention. 1148-1159, disclose the use of e-polylysine in drug targeting. The present invention also relates to a kit for the prepara It is proposed that e-polylysine be covalently bonded to active tion of a medicament or pharmaceutical composition, in par compounds. The aim of the approach described therein is an ticularatherapeutic or image-enhancing composition, at least increase in the uptake rate in cells. Specificity for specific 60 comprising a conjugate according to the invention. This con tissue is neither aimed at nor achieved. jugate can then be reacted, depending on the application, for The object of the present invention was therefore to provide example, with a suitable active compound for the preparation a carrier or carrier molecule for a therapeutic agent or diag of a therapeutic composition or, if a complexing agent is nostic agent which has the highest possible affinity and selec present, with metal ions which have an image-enhancing tivity for the kidney. 65 and/or therapeutic action. Surprisingly, it has been found that conjugates of e-polyl The present invention also relates to the use of the conju ysine or e-polylysine derivatives with compounds carrying gate according to the invention for the preparation of macro US 9,078,929 B2 5 6 molecule conjugates, where two or more conjugates accord carboxyl groups, and at least one group or functionality for ing to the invention are bound to a macromolecule, and a bonding to the oligomer of the conjugate according to the macromolecule conjugate composed of at least two or more invention. Examples thereof are NOTA, TETA, EDTA or conjugates according to the invention which are covalently preferably DOTA or DTPA. In this case, the compounds bonded to a macromolecule. carrying carboxyl groups simultaneously also fulfil the func The present invention also relates to the use of a conjugate tion of a complexing agent, which is particularly advanta according to the invention for targeting of the kidney. The geous for diagnostic applications. targeting of the kidney here preferably serves for enriching Preferred compounds carrying carboxyl groups are those medicaments for pharmaceutical or diagnostic applications in which contain two or more free carboxyl groups after conju the kidney, i.e. generating increased uptake in the kidney in 10 gation to the oligomer. relation to the remainder of the body. FIG. 1 shows a diagrammatic representation of the com It has been found that the specificity achieved in the target pound according to the invention obtained in Synthesis ing of the kidney is particularly high if the carboxyl groups of Example 1. the compound carrying carboxyl groups make up a large FIG. 2 shows the organ distribution of ''In-loaded 15 proportion of the molar mass of the compound carrying car e-polylysine-DOTA. Further details are given in Use boxyl groups. Preference is therefore given to compounds Example 1. carrying carboxyl groups in which the proportion of the car FIG.3 shows the chemical structure of MAG3, DMSA and boxyl groups in the molar mass is greater than 30%, prefer DTPA. ably greater than 40%. FIG. 4 shows enalapril derivatives corresponding to For example, the molar mass of DOTA is 404 g/mol. The 4 Example 7. carboxyl groups thereof make up a proportion of 180 g/mol Substances which improve the representation of the target (4xCOOH=4x45 g/mol). This gives a proportion of carboxyl organ in certain diagnostic methods act as image-enhancing groups in the molar mass of DOTA of about 44%. composition or contrast medium or image-enhancing, gener Citric acid has a molar mass of 192 g/mol. The carboxyl ally by increasing the contrast to the environment or the signal 25 groups (3x45 g/mol) make up 135 g/mol thereof. This gives a of the target organ in relation to the environment. proportion of carboxyl groups in the molar mass of citric acid A compound carrying carboxyl groups in accordance with of about 70%. the present invention is a chemical compound which contains Compounds carrying carboxyl groups which are particu at least one carboxyl group (-COOH) and at least one group larly preferred in accordance with the invention are therefore or functionality for bonding to the oligomer of the conjugate 30 those which contain two or more free carboxyl groups after according to the invention. The bonding to the oligomer can conjugation to the oligomerand in which the proportion of the take place in any known manner which results in covalent carboxyl groups in the molar mass is greater than 30%, pref bonding of the oligomer and compound carrying carboxyl erably greater than 40%, such as, for example, DOTA, DTPA groups. Examples of functional groups via which bonding and citric acid. can take place are —NH, -SH, -OH,-Hal (for example 35 A spacer, often also called a linker, effects a covalent bond —Cl, —Br. —I), -alkyne. —NCS, NCO. —SOCl, -azide, between two parts of a molecule, in the present case, for -carbonate, -aldehyde, -epoxide, —COOH, -COOR, where example, between the oligomer according to the invention R in this case is preferably a halogen or preferably an activa and a compound carrying carboxyl groups or an active com tor, i.e. a good leaving group. Such as, for example, N-hy pound. A spacer will generally be introduced if the connec droxysuccinimide, pentafluorophenyl or para-nitrophenyl. 40 tion between two moieties is not to take place only via a direct An overview of possible covalent types of coupling is found, chemical bond, but instead a certain separation is to be gen for example, in "Bioconjugate Techniques'. Greg T. Herman erated between two moieties. Equally, a spacer can provide son, Academic Press, 1996, on pages 137 to 165. the chemical functionalities which are necessary in order to The compound carrying carboxyl groups preferably con connect two parts of a molecule which would otherwise not tains two or more carboxyl groups. Examples of compounds 45 react with one another. The conjugation of a spacer to the carrying carboxyl groups which are Suitable in accordance oligomer, the compound carrying carboxyl groups or an with the invention are: citric acid, Succinic acid, fumaric acid, active compound preferably takes place via an amide or ester maleic acid, glutaric acid, adipic acid, tartaric acid, oxalic bond. Spacers can be, for example, aliphatic hydrocarbons, acid, malonic acid, Succinic acid, glutaric acid, adipic acid, polyethers (such as polyethylene glycols), oligo-peptides or Suberic acid, azelaic acid, sebacic acid, the corresponding 50 similar elements having a chain structure. The spacer may be branched fatty acids, maleic acid, fumaric acid, cyclohex stable, i.e. it cannot be cleaved under physiological condi anedicarboxylic acid and the corresponding position isomers tions or can only be cleaved to a slight extent, or it may be and similar aliphatic dibasic acids; tetrahydrophthalic acid, unstable, i.e. it can be cleaved, at least under certain physi 5-norbornene-2,3-dicarboxylic acid and similar alicyclic ological conditions. dibasic acids; tricarbal lytic acid, aconitic acid, trimesic acid 55 Active compounds, peptides, complexing agents or other and similar tribasic acids; adamantanetetracarboxylic acid, functionalities can be bonded to the oligomer directly or by butanetetracarboxylic acid, cyclopentanetetracarboxylic means of a spacer. acid, tetrahydrofurantetracarboxylic acid and similar tetraba Examples of functional groups via which direct bonding sic acids; Sugar acids, in particular aldaric acids, such as, for can take place are —NH, SH, —OH, -Hal (for example example, glucaric acid, galactaric acid; malic acid, tartaric 60 —C1, —Br. —I), -alkyne, NCS, NCO. —SOCl, -azide, acid, citric acid and similar hydroxy-fatty acids; trimellitic -carbonate, -aldehyde, -epoxide. —COOH, -COOR, where acid, pyromellitic acid, biphenyltetracarboxylic acid, ben R in this case is preferably a halogen or preferably an activa Zophenonetetracarboxylic acid, diphenylsulfonetetracar tor, i.e. a good leaving group. Such as, for example, N-hy boxylic acid and similar aromatic polycarboxylic acids. droxysuccinimide, pentafluorophenyl or para-nitrophenyl. In accordance with the invention, the compound carrying 65 An overview of possible covalent types of coupling is found, carboxyl groups can also be complexing agents which con for example, in "Bioconjugate Techniques'. Greg T. Herman tain at least one carboxyl group, preferably two or more son, Academic Press, 1996, on pages 137 to 165. US 9,078,929 B2 7 8 For example, active compounds can be bonded to the con pemetrexed, raltitrexed, fluorouracil, capecitabine, cytosi jugate according to the invention via a cleavable linker. This narabinoside, gemcitabine, tioguanine, pentostatin, mercap linker is then cleaved in vivo under certain conditions, for topurine, fludarabine, caldribine, hydroxycarbamide, mito example by enzymatic or chemical cleavage, and liberates the tane, azacitidine, cytarabine, nelarabine, bortezomib, active compound. Suitable for this purpose are linkers which anagrelide, in particular the protein kinase inhibitors, such as, contain carboxylate and disulfide bonds, in which the former for example, imatinib, erlotinib, Sunitinib, Sorafenib, dasat groups are hydrolysed enzymatically or chemically and the inib, lapatinib or nilotinib, immunotherapeutic agents, for latter are separated off by disulfide exchange, for example in example cetuximab, alemtuzumab and bevacizumab, the presence of glutathione. antiphlogistics, for example naproxen, ibuprofen, indometa An example of a cleavable spacer is also an oligopeptide 10 cin, prednisolone, prednisone, hydrocortisone or budesonide, which can be cleaved specifically with the aid of specific, antibiotics, in particular the penicillins, such as, for example, endogenous or exogenous enzymes. Thus, for example, the benzylpenicillin, methicillin or amoxicillin, the cephalospor peptide sequence DEVD (Asp-Glu-Val-Asp) is cleaved after ins, such as, for example, cefuroxim, cefotaxim, cefadroxil or apoptosis induction by Caspase-3. Thus, for example, an cefixim, the B-lactamase inhibitors, such as, for example, active compound or a compound carrying carboxyl groups 15 clavulanic acid, Sulbactam or taZobactam, the carbapenems, which is bonded via a spacer of this type can be removed from Such as, for example, imipenem or meropenem, the mono the kidney after a certain residence time therein, or alterna bactams, such as, for example, aztreonam, the tetracyclines, tively a corresponding functionality (presence or absence of a Such as, for example, tetracycline, chlortetracycline, oxytet certain enzyme) of the kidney can also be checked. Further racycline, doxycycline, minocycline or tigecycline, the mac examples are the peptide sequences CPEN FFWGGGG or rolide antibiotics, such as, for example, erythromycin A, the PENFF, which can be cleaved by matrix metalloprotease-13. glycopeptide antibiotics, such as, for example, Vancomycin, A simple embodiment of a cleavable spacer is the formation the enediynes, such as, for example, calicheamicin, Virostat of a carboxylate, which can easily be cleaved by esterases. ics, for example aciclovir, Valaciclovir, ganciclovir, Valgan Alternatively, the spacer may contain an acid-labile struc ciclovir, penciclovir, famciclovir, brivudine, cidofovir, fos ture, for example a hydrazone, an imine, a carboxyhydraZone, 25 carnet, idoxuridine or tromantadine, antihypertensives, in an acetal or ketal (see, for example, Haag-R, Kratz-F, particular the ACE inhibitors, such as, for example, Angewandte Chemie page 1218 (2006)). benazepril, captopril, cilaZapril, enalapril, fosinopril, lisino In accordance with the invention, amino acids are com pril, perindopril, quinapril, ramipril, trandolapril or Zofeno pounds which carry at least one amino group and at least one pril, the Sartans, such as, for example, losartan, balsartan, carboxyl group. Examples are natural, proteinogenic amino 30 irbesartan, candesartan, eprosartan, olmesartan or telmisar acids or non-proteinogenic amino acids which occur in the tan, the renin inhibitors, such as, for example, alliskiren, and organism or are prepared synthetically. the beta blockers, such as, for example, proproanolol, pin A peptide is a compound which is formed from the linking dolol, Sotalol, bopindolol, atenolol, bisorpolol, celiprolol, of two or more amino acids. The individual amino acids here esmolol, metoprolol, nebivolol, oxprenolol, carvedilol or are connected in a defined sequence to form a chain, usually 35 labetalol, uricosurics, for example probenecid or benzbroma unbranched. The amino acids in peptides and in the larger rone, or diuretics, for example acetazolamide, furosemide, proteins are connected to one another via amide bonds. torasemide, bumetanide, piretanide, azosemide, etacrynic In accordance with the invention, a Solid phase is an acid, etozoline, hydrochlorothiazide, benzthiazide, chlorothi organic, inorganic or organic/inorganic composite material azide, chlorthalidone, indapamide, mefruside, metolaZone, which can be employed as resin or Support in Solid-phase 40 clopamide, Xipamide, hydroflumethiazide, methyclothiazide, synthesis. Furthermore, Surfaces of mouldings, such as, for polythiazide, amiloride, triameterene, Spironolactone, can example, microtitre plates or particulate materials, such as, renone, eplerenone or spironolactone. for example, organic or inorganic nanoparticles, metal par Further antitumour agents, for example agents which are ticles or the like, are also regarded as solid phase in accor effective against proliferating cells, are in accordance with dance with the invention. 45 the invention likewise active compounds. Illustrative antitu In accordance with the invention, active compounds or mour agents include cytokines, such as, for example, inter active-compound molecules in accordance with the German leukin-2 (IL-2), tumour necrosis factor or the like, lectin Medicines Act are substances which are intended to be used inflammation reaction promoters (selectins), Such as, for as pharmaceutically active constituents in the preparation of example, L-selectin, E-selectin, P-selectin or the like, and medicaments or to become pharmaceutically active constitu 50 similar molecules. ents on use in the preparation of medicaments (German Medi In accordance with the invention, one or more identical or cines Act S4 (19)). Active compounds generally cause a spe different active-compound molecules can be bonded per con cific effect in an organism. An active compound according to jugate according to the invention. the invention is typically a pharmaceutically active molecule Equally, in particular in the case of macromolecules, such or medicament, Such as, for example, immunosuppressants, 55 as, for example, relatively large active-compound molecules, for example azathioprine, mycophenolate-mofetil, for example proteins, it is conversely possible for two or ciclosporin, tacrolimus, sirolimus, fingolimod or triptolide, more, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10, conjugates accord cytostatics, for example bleomycin, dactinomycin, mitomy ing to the invention to be bonded to one active-compound cin, daunorubicin, doxorubicin, epirubicin, idarubicin, molecule in order to enable kidney-specific accumulation of mitoxantron, doxofluridine, cisplatin, carboplatin, oxalipl 60 the active compound. Covalent bonding of the conjugates atin, satraplatin, camptothecin, toptecan, irinotecan, amsa according to the invention to the macromolecule typically crine, etoposide, teniposide, cyclophosphamide, trofosfa also takes place here. In accordance with the invention, mac mide, melphalan, chlorambucil, estramustine, buSulfan, romolecules are taken to be not only large molecules, such as chlorambucil, chlormethine, treosulfan, carmustine, lomus proteins, but also any form of particles (for example nanopar tine, nimustine, procarbazine, streptozocine, dacarbazine, 65 ticles), liposomes or other systems by means of which active ifosfamide, temozolomide, thiotepa, Vinorelbine, Vincristine, compounds can be transported or to which active compounds vinblastine, Vindesine, paclitaxel, docetaxel, methotrexate, can be bonded. US 9,078,929 B2 9 10 In addition to the active-compound molecules, or instead SPECT, PET, CT or MRT measurements. Preferred complex of the active-compound molecules, other functionalities, ing agents are DOTA or DTPA or derivatives thereof. In Such as, for example, functionalities for diagnostic or imag accordance with the invention, complexing agents are both ing methods, may also be bonded to the conjugate according molecules to which the metal ions are already bonded and to the invention. also molecules to which metalions can be bonded, but are not Equally, fluorine-containing side chains can be incorpo bonded at the present stage. rated as functionality via optional spacers. The accumulation Metalions which are suitable inaccordance with the inven of the corresponding molecules in the kidneys can thus be tion for bonding to complexing agents are, for example, Fe", represented with the aid of F nuclear resonance tomogra Fe", Cu?", Cr', Gd", Eu", Dy", La", Yb and/or Mn." phy. Highly symmetrically arranged fluorine atoms, which 10 have a uniform resonance frequency, are particularly advan or also the ions of radionuclides, such as gamma emitters, tageous here. In order to improve the 'F signal, a contrast positron emitters, Auger electron emitters, alpha emitters and agent which is usual in nuclear spintomography, Such as, for fluorescence emitters, for example 'Cr, Ga, Ga, '''In, example, gadobutrol (Magnevist(R), can be used. 99mTc, 140La, 175Yb. Sm, 16Ho, 88y. 90Y. 14°Pm, 77Lu, If the conjugate according to the invention comprises com 15 *7Sc, 142 Pr. 159Gd, 212Bi, 7°As, 72Se, 7Ru, 109Pd, 105Rh, plexing agents, it is particularly advantageous to integrate 10 Rh. Sb, 12 Ba, '7Hg, 2. At, 16°Eu, 208Pb, 212Pb, gadolinium or manganese or another strongly paramagnetic Cu,7Cu, Re, Re, Au and/or 'Ag. metalion which is known to the person skilled in the art with Examples of suitable metal ions and their respective use the aid of a complexing agent located on the conjugate a. according to the invention. Suitable complexing agents here In for SPECT are, for example, DOTA and DTPA. Ga for PET Furthermore, complexing agents which do not belong to 'Y for therapy the group of compounds carrying carboxyl groups can be Gd, Eu, Mn for MRT conjugated—also optionally via spacers. Examples are tantalum, tungsten or other elements having a high atomic hydroxyquinoline, thiourea, guanidine, dithiocarbamate, 25 number for computer tomography hydroxamic acid, amide oxime, aminophosphoric acid, (cy In accordance with the invention, the term oligomer is clic) polyamino, mercapto, 1.3-dicarbonyl and crown ether applied to the part of the conjugate that consists of an oligo radicals with various metals having in some cases very spe mer which consists of peptidically linked monomer units. The cific activities with respect to ions. oligomer typically consists of 5 to 1000, preferably 8 to 100, Functionalities for cell-specific targeting, Such as, for 30 particularly preferably 10 to 50, monomer units. In a particu example, antibodies, antibody fragments or aptamers, may larly preferred embodiment, the oligomer consists of e-polyl also be bonded to the conjugate according to the invention. ysine which has between 8 and 100 monomer units, particu Fluorescent dyes or interleukins, such as IL-2, may also be larly preferably between 10 and 50 monomer units. bonded. In other embodiments, however, up to 50% of the e-lysine “Peptidically linked in accordance with the invention 35 monomer units may be replaced by other monomer units means that an —NH CO bond is present between two and/or up to 50% of the e-lysine monomer units may be monomer units, as is also present in peptides or proteins derivatised or modified by the introduction of further func between two amino acids as monomer units. This means that tionalities. Likewise, the oligomer which consists of peptidi two monomer units are linked in Such a way that an —NH cally linked monomer units may comprise a plurality of Suc group of the one monomer is linked to a C=O group of the 40 cessive monomer units which are note-lysine monomer units other monomer. The following bonding structure accordingly if it comprises at least 10 Successive monomer units which arises: M-NH CO-M-NH CO-M-NH CO-M, where M consist of at least 70% (based on the number of monomer is the part of the monomer that is not involved in the bonding. units), preferably at least 80%, of e-lysine units. This is the The conjugate of the present invention consists at least of case, for example, if a chain of 10 to 20 monomer units (for two covalently bonded parts—a compound carrying carboxyl 45 example comprisingamino acids) in which no e-lysine mono groups and an oligomer. In a preferred embodiment, the con mer unit is present and Subsequently, for example, ten mono jugate comprises an oligomer, one or more compounds car merunits, of which eight are e-lysine monomer units and two rying carboxyl groups and one or more active-compound consist of other amino acids, is located at one end of the molecules. In a particularly preferred embodiment, the con oligomer. jugate according to the invention comprises a plurality of, i.e., 50 In accordance with the invention, the term monomer unit is for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10, identical or different applied to any part of the oligomer that is peptidically linked compounds carrying carboxyl groups and a plurality of, i.e., to at least one further part of the oligomer. Terminal monomer for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10, identical or different units here are generally only peptidically linked to one further active-compound molecules. monomer unit. Monomer units in the middle of the oligomer It has been found that the conjugates according to the 55 are peptidically linked to two further monomer units. Mono invention accumulate particularly specifically in the kidney if merunits which are peptidically linked to three further mono a compound carrying carboxyl groups is covalently bonded to mers are located at branching points. In the case of monomer 10 to 80% of the monomer units. units in the middle of the oligomer, the monomer unit typi In accordance with the invention, a complexing agent is cally provides on the one hand the NH part of the peptidic any molecular structure which is capable of complexing 60 bond and on the other hand the CO part. metal ions, i.e. of forming a metalchelate complex with the In accordance with the invention, an e-lysine monomerunit metal ions. Complexing agents are frequently also known as is an e-lysine unit, an ornithine unit, a 2,3-diaminopropionic chelating agents. Examples of complexing agents which are acid unit or a 2,4-diaminobutyric acid unit. The e-lysine suitable in accordance with the invention are EDTA, NOTA, monomer unit preferably consists of an e-lysine unit. The TETA, iminodiacetic acid, DOTA or DTPA. Particular pref 65 term unit here is in each case intended to show that it is a unit erence is given in accordance with the invention to complex or monomer in a peptidically linked oligomer and not the free ing agents which bind metal ions which can be detected in amino acid.

US 9,078,929 B2 13 also mixtures of e-polylysine of various chain length or mix tures comprising various e-polylysine derivatives therefore fall under the term e-polylysine and e-polylysine derivatives. It has furthermore been found that not only conjugates of e-polylysine, but also conjugates of e-polylysine derivatives, 5 R'O -P(O)(NR')NR - PR=N-or-P(O)R’ exhibit excellent accumulation in the kidney. and one or more methylene groups present in M may be Typical conjugates which are suitable in accordance with mono- or disubstituted, independently of one another, by the invention can also be represented by formula III R", where A-(Lys),-E III R" is linear or branched C- to Cs-alkyl, C- to C7-cycloalkyl, 10 in which unsubstituted or substituted phenyl and n is a number between 5 and 1000, R" is linear or branched C- to C-alkyl, C- to C-cycloalkyl, A is unsubstituted or substituted phenyl one or more charged or uncharged end groups bonded or CN, OR, NH, NHR', NR', P(O)R’ - P(O) directly or via a spacer or a dendritic functionality, for 15 (OR), -P(O)(NR'), C(O)R', O(O)OR', O(O) example hydrogen, —CN, —OR', NR', -P(O)R', OH, -C(O)NR', -SONR', C(O)Hal, SO,OH, -P(O)(OR), -P(O) (NR), C(O)R', C(O)OR', —SOHal, NO, -Hal and - C(O)OH, -C(O)NR', -SO2NR', C(O)Hal, Hal= F. —Cl, —Br or —I, SO,OH, -SOHal, NO,-Hal or where compounds carrying carboxyl groups, active com one or more groups bonded directly or via a spacer or a pounds, complexing agents, peptides, solubilisers, protect dendritic functionality, such as, for example, com ing groups, a Solid phase, dyes or similar components may pounds carrying carboxyl groups, active-compound be bonded, directly or via a spacer, to all functional groups molecules, complexing agents, dyes, one or more iden which are suitable for conjugation (for example NH, NH, tical or different amino acids, peptides, proteins, solubi COOH, OH, -SH, -Hal (for example - C1, Br, I), lisers, protecting groups or a solid phase 25 -alkyne, -azide, -aldehyde) of the monomer units Lys, inde E is pendently of one another, with the proviso that the com one or more charged or uncharged end groups bonded pound according to the invention contains at least one directly or via a spacer or a dendritic functionality, for compound carrying carboxyl groups and either more than example hydrogen, —CN, —OR', —NH2, NHR'. 50% of the monomer units (based on the number of mono 30 mer units) are e-lysine monomer units or at least 70% (based on the number of monomer units) of at least 10 Successive monomer units are e-lysine monomer units. one or more groups bonded directly or via a spacer or a In a preferred embodiment of the compound according to dendritic functionality, Such as, for example, compounds 35 the invention, A is –C(O)OR', -C(O)OH, -C(O)NR' or carrying carboxyl groups, active-compound molecules, —C(O)Hal or one or more components bonded directly or via complexing agents, dyes, one or more identical or different a spacer or a dendritic functionality, Such as compounds car amino acids, peptides, proteins, Solubilisers, protecting rying carboxyl groups, active compounds, complexing groups or a solid phase, agents, peptides, solubilisers, protecting groups, a solid phase Lysis, independently of one another, 40 or dyes. an e-lysine monomer unit corresponding to the definition A is particularly preferably —OH, —OCH, OCHCH already given or one or more active-compound molecules bonded directly a monomer unit conforming to formula I or II or via a spacer or a dendritic functionality. a group conforming to formula IV In a preferred embodiment, E is 45 —H, —CH, -CH2CH or (NH-M-CO) IV O where M can denote, independently of one another, one or more components bonded directly or via a spacer or a O—, S—, S(O) , SO , SOO . dendritic functionality, Such as compounds carrying car C(O)—, C(O)O , CH , CHR , boxyl groups, active compounds, complexing agents, pep —CR' , CR'—CH , - CH-CR' , 50 tides, solubilisers, protecting groups, a Solid phase or dyes. -CH=CH-, -CR'—CR -C=C - N'R' , E is particularly preferably - P(O)R'O C(O)NR' , SONR' OP(O) one or more active-compound molecules bonded directly or RO , -P(O)(NR')NR' PR=N or P(O) via a spacer or R" - or a dendritic functionality. straight-chain or branched alkyl having 1 to 20 C atoms, 55 If the compound according to the invention contains one or straight-chain or branched alkenyl having 2-20 C atoms more groups Lys which contain an NH or COOH group. and one or more double bonds, further monomer units may be bonded via them via a peptidic straight-chain or branched alkynyl having 2-20 C atoms bond, and singly or multiply branched compounds arise. The and one or more triple bonds, compounds according to the invention are preferably Saturated, partially or fully unsaturated cycloalkyl having 60 unbranched. 3-7 C atoms, which may be substituted by alkyl groups n is preferably a number between 8 and 100, particularly having 1-6 C atoms, preferably between 10 and 50. where one or more non-adjacent methylene groups may be The conjugates according to the invention preferably com replaced by —O— or —S— and adjacent methylene prise not only one, but instead a plurality of compounds groups may be replaced by alkenyl or alkynyl groups, 65 carrying carboxyl groups. These can be bonded directly or via where one or more methylene groups may be replaced, inde a spacer to the carboxyl- and/or amino-terminal end of the pendently of one another, by —O— —S— —S(O)—, oligomer and/or to functional group of the monomer units US 9,078,929 B2 15 16 which is suitable for conjugation (for example NH, -NH2, peptide synthesis (for example Fmoc peptide synthesis or —COOH, -OH, -SH, -Hal (for example—Cl. —Br. —I), Boc peptide synthesis). Solid-phase syntheses of this type are -alkyne, -azide, -aldehyde). known to the person skilled in the art. Suitable textbooks for In a preferred embodiment, the bonding of the compounds peptide synthesis are Solid-Phase Peptide Synthesis: 289 carrying carboxyl groups takes place via amino groups of the (Methods in Enzymology) by Sidney P. Colowick (author), monomer units, for example the free amino group of e-lysine. Gregg B. Fields (publisher), Melvin I. Simon (publisher) The conjugate according to the invention should preferably Academic Press Inc (November 1997) or Fmoc Solid Phase contain at least one compound carrying carboxyl groups per Peptide Synthesis: A Practical Approach by W. Chan (au 10 monomer units, particularly preferably between 3 and 6 thor), W. C. Chan (publisher), Peter D. White (publisher) compounds carrying carboxyl groups per 10 monomer units. 10 Equally, however, it is also possible for one compound car “Oxford Univ Pr(2 Mar. 2000). The monomers employed in rying carboxyl groups to be bonded to more than 9 of 10 each case are selected here in Such a way that an oligomer or monomerunits or to all monomerunits. The optimum number conjugate corresponding to the present invention is formed. of compounds carrying carboxyl groups per 10 monomer Depending on the type of monomer unit, the synthesis can be units depends on the type of the compound carrying carboxyl 15 carried out using a derivatised monomer unit directly or a groups and the type of the monomer units. The above-men monomer unit which is firstly protected at the site intended for tioned preferred number of compounds carrying carboxyl the derivatisation. When the synthesis of the oligomer is groups per monomer unit apply, in particular, to oligomers complete, the final derivatisation with the compounds carry which are built up entirely from e-lysine monomer units. The ing carboxyl groups, active compounds, etc., can then be distribution of the compounds carrying carboxyl groups in carried out either on the solid phase or after cleaving-off from the conjugate according to the invention can be random, the Solid phase in solution. meaning that, for example, the first monomer units contain The bonding of the compounds carrying carboxyl groups —NH, followed by a monomer unit with a compound car in this case preferably takes place to the finished oligomer, i.e. rying carboxyl groups, then again one containing —NH2, either still on the solid phase when the solid-phase synthesis then two times a monomer unit which carries a compound 25 of the oligomer is complete or after the latter has been cleaved carrying carboxyl groups, then again twice one containing offin solution. —NH2, etc. If the compound carrying carboxyl groups or an active Equally, the distribution of the complexing agents in the compound or similar components (the process is described compound according to the invention may also be ordered in below by way of example for a complexing agent) is to be Such a way that, for example, each second monomer unit has 30 bonded, for example, to the N-terminal end of the oligomer, a compound carrying carboxyl groups bonded thereto. The the oligomers are typically generated with an amino-terminal ordering may also take place in such a way that all monomer protecting group, such as, for example, Fmoc. If the complex units at one end of the conjugate according to the invention ing agent is able to withstand the conditions used for cleaving have a conjugated compound carrying carboxyl groups, while off the oligomer from the synthesis resin and for deprotecting the remainder of the monomers contain free NH functions. 35 the side chains, the Fmoc can be cleaved off from the N The compounds carrying carboxyl groups are preferably terminal of the complete resin-bound polypeptide, enabling randomly distributed in the conjugates according to the inven the complexing agent to be bonded to the free N-terminal tion. amine. In Such cases, the complexing agent is typically acti The conjugates according to the invention can be prepared, vated by processes which are generally known in the art for in particular, by various processes knownto the person skilled 40 producing an active ester or active carbonate group which is in the art in the area of peptide synthesis. effective for forming an amide or carbamate bond to the 1. Synthesis Starting from e-polylysine oligomeramino group. It is of course also possible to use a Here, synthetic or natural e-polylysine of uniform or vari different linking chemistry. ous chain length is typically reacted with the corresponding In order to help to minimise side reactions here, guanidino compounds carrying carboxyl groups in Solution. To this end, 45 and amidino groups may be blocked using conventional pro for example, firstly the compounds carrying carboxyl groups tecting groups, such as, for example, carbobenzyloxy groups can be activated. This can be carried out, for example, by (CBZ), di-t-BOC, PMC, Pbf, N NO, and the like. activation of one or more of their carboxyl groups by convert Coupling reactions are carried out by known coupling pro ing them into the active ester or acid chloride. This is followed cesses in solvents, such as, for example, N,N-dimethylforma by the reaction with e-polylysine, where the conjugation pref 50 mide (DMF), N-methyl-pyrrolidone, dichloromethane and/ erably takes place at the free amino groups. Alternatively, for or water. Illustrative coupling reagents include example, one or more carboxyl groups of the compound O-benzotriazolyloxytetramethyluronium hexafluorophos carrying carboxyl groups can be activated using a coupling phate (HATU), dicyclohexylcarbodiimide, bromo-tris(pyrro reagent, such as dicyclohexylcarbodiimide or HATU, and lidino)phosphonium bromide (PyBroP), etc. Other reagents reacted with the e-polylysine, where the conjugation prefer 55 may be present, Such as, for example, N,N-dimethylaminopy ably takes place at the free amino groups. Reaction conditions ridine (DMAP), 4-pyrrolidinopyridine, N-hydroxysuccinim for reactions of this type are known to the person skilled in the ide or N-hydroxybenzotriazole. art. Suitable solvents are, for example, water, acetonitrile, Correspondingly, the bonding of compounds carrying car DMSO, DMF, dioxane, THF, methanol or mixtures of two or boxyl groups, active compounds, complexing agents or simi more of the said solvents. 60 lar components can also take place to the amino groups of the 2. Solid-Phase Synthesis non-terminal e-lysine monomer units. In particular if the conjugate is to comprise one or more If the molecule contains complexing agents, the metal ions monomer units which do not consist of e-lysine or which can be complexed by known methods. consist of derivatised e-lysine monomer units, a Solid-phase The present invention additionally relates to the use of the synthesis for the preparation of the conjugates according to 65 conjugates according to the invention for the preparation of a the invention may be advantageous. This solid-phase synthe pharmaceutical composition or a medicament, in particular a sis is carried out in a corresponding manner to a conventional therapeutic composition, and/oran image-enhancing compo US 9,078,929 B2 17 18 sition (for example a contrast agent) and/or a radiolabelled cally acceptable inert excipient, such as, for example, ethanol, tracer for nuclear-medical imaging. glycerol, water and the like. Powders are prepared by com The present invention additionally relates to the conjugates minuting the compound to a Suitable fine size and mixing it according to the invention to which one or more active com with a pharmaceutical excipient comminuted in a similar pounds are preferably covalently bonded, and/or pharmaceu- 5 manner, such as, for example, an edible carbohydrate. Such tically usable salts and stereoisomers thereof, including mix as, for example, starch or mannitol. A flavour, preservative, tures thereof in all ratios, and optionally excipients and/or dispersant and dye may likewise be present. adjuvants Capsules are produced by preparing a powder mixture as as medicament described above and filling shaped gelatine shells therewith. for use as medicament 10 Glidants and lubricants. Such as, for example, highly disperse as active compound or active component in a medicament silicic acid, talc, magnesium Stearate, calcium Stearate or as diagnostic agent polyethylene glycol in solid form, can be added to the powder for use as diagnostic agent mixture before the filling operation. A disintegrant or solubi for use in the targeting of the kidney liser, Such as, for example, agar-agar, calcium carbonate or and, in particular, as medicament for the treatment of dis- 15 Sodium carbonate, can likewise be added in order to improve eases of the kidney. the availability of the medicament after the capsule has been Atherapeutic composition generally consists at least of the taken. active compound—in this case the conjugate according to the In addition, if desired or necessary, suitable binders, lubri invention with active compound preferably covalently cants and disintegrants as well as dyes can likewise be incor bonded—and one or more Suitable solvents and/or excipients 20 porated into the mixture. Suitable binders include starch, which allow application of the therapeutic composition. gelatine, natural Sugars, such as, for example, glucose or A diagnostic composition or diagnostic agent serves as B-lactose, Sweeteners made from maize, natural and synthetic image-enhancing or imaging composition in diagnostic rubber, Such as, for example, acacia, tragacanth or sodium methods. A diagnostic agent generally consists at least of the alginate, carboxymethylcellulose, polyethylene glycol, signal Source, i.e. the imaging and/or image-enhancing com- 25 waxes, and the like. The lubricants used in these dosage forms ponent in this case the conjugate according to the invention, include Sodium oleate, Sodium Stearate, magnesium Stearate, where in this case at least one compound carrying carboxyl Sodium benzoate, Sodium acetate, sodium chloride and the groups in the conjugate is preferably a complexing agent— like. The disintegrants include, without being restricted and one or more Suitable solvents and/or excipients which thereto, starch, methylcellulose, agar, bentonite, Xanthan gum allow application of the diagnostic composition. 30 and the like. The tablets are formulated by, for example, For diagnostic applications, the conjugate according to the preparing a powder mixture, granulating or dry-pressing the invention preferably serves as signal source in an image mixture, adding a lubricant and a disintegrant and pressing enhancing contrast medium, enabling the latter to be detected the entire mixture to give tablets. A powder mixture is pre by means of nuclear-medical and/or radiological methods, pared by mixing the compound comminuted in a Suitable such as SPECT, PET, ultrasound, and or also by magnetic 35 manner with a diluent or a base, as described above, and resonance tomography, computer-tomographic and optical optionally with a binder, such as, for example, carboxymeth imaging methods (near-infrared imaging). Detection meth ylcellulose, an alginate, gelatine or polyvinylpyrrolidone, a ods and applications of image-enhancing contrast media are dissolution retardant, such as, for example, paraffin, an known to the person skilled in the art. Examples of suitable absorption accelerator, such as, for example, a quaternary applications are the diagnosis of cancer diseases, neurologi- 40 salt, and/or an absorbent, such as, for example, bentonite, cal questions, checking the response to a therapy, checking of kaolin or dicalcium phosphate. The powder mixture can be the degree of damage of a kidney in the case of for example, granulated by wetting it with a binder, such as, for example, autoimmune diseases, and monitoring of gene therapies, but syrup, starch paste, acadia mucilage or solutions of cellulose also the recognition of cellular changes. or polymer materials and pressing it through a sieve. As an The invention furthermore relates to medicaments or phar- 45 alternative to granulation, the powder mixture can be run maceutical compositions comprising at least one conjugate through a tableting machine, giving lumps of non-uniform according to the invention and/or pharmaceutically usable shape, which are broken up to form granules. The granules salts and stereoisomers thereof, including mixtures thereof in can be lubricated by addition of Stearic acid, a stearate salt, all ratios, and optionally excipients and/or adjuvants. talc or mineral oil in order to prevent sticking to the tablet Pharmaceutical compositions or medicaments can be 50 casting moulds. The lubricated mixture is then pressed to give adapted for administration via any desired suitable method, tablets. The compounds according to the invention can also be for example by oral (including buccal or Sublingual), rectal, combined with a free-flowing inert excipient and then pressed nasal, topical (including buccal, Sublingual or transdermal), directly to give tablets without carrying out the granulation or vaginal or parenteral (including Subcutaneous, intramuscular, dry-pressing steps. A transparent or opaque protective layer intravenous or intradermal) methods. Such formulations can 55 consisting of a shellac sealing layer, a layer of Sugar or poly be prepared using all processes known in the pharmaceutical mer material and a gloss layer of wax may be present. Dyes art by, for example, combining the active ingredient with the can be added to these coatings in order to be able to differen excipient(s) or adjuvant(s). tiate between different dosage units. Pharmaceutical formulations adapted for oral administra Oral liquids, such as, for example, Solution, syrups and tion can be administered as separate units, such as, for 60 elixirs, can be prepared in the form of dosage units so that a example, capsules or tablets; powders or granules; Solutions given quantity comprises a prespecified amount of the com or Suspensions in aqueous or non-aqueous liquids; edible pound. Syrups can be prepared by dissolving the compound foams or foam foods; or oil-in-water liquid emulsions or in an aqueous Solution with a suitable flavour, while elixirs are water-in-oil liquid emulsions. prepared using a non-toxic alcoholic vehicle. Suspensions Thus, for example, in the case of oral administration in the 65 can be formulated by dispersion of the compound in a non form of a tablet or capsule, the active-ingredient component toxic vehicle. Solubilisers and emulsifiers, such as, for can be combined with an oral, non-toxic and pharmaceuti example, ethoxylated isostearyl alcohols and polyoxyethyl US 9,078,929 B2 19 20 ene Sorbitol ethers, preservatives, flavour additives, such as, immediately before use is necessary. Injection Solutions and for example, peppermint oil or natural Sweeteners or saccha Suspensions prepared in accordance with the recipe can be rin, or other artificial Sweeteners and the like, can likewise be prepared from Sterile powders, granules and tablets. added. The conjugates according to the invention are preferably The dosage unit formulations for oral administration can, if 5 administered parenterally. desired, be encapsulated in microcapsules. The formulation It goes without saying that, in addition to the above par can also be prepared in Such away that the release is extended ticularly mentioned constituents, the formulations may also or retarded, such as, for example, by coating or embedding of comprise other agents usual in the art with respect to the particulate material in polymers, wax and the like. particular type of formulation; thus, for example, formula The conjugates according to the invention can also be 10 administered in the form of liposome delivery systems, such tions which are suitable for oral administration may comprise as, for example, Small unilamellar vesicles, large unilamellar flavours. vesicles and multilamellar vesicles. Liposomes can be A therapeutically effective amount of the conjugate formed from various phospholipids, such as, for example, according to the invention depends on a number of factors, cholesterol, Stearylamine or phosphatidylcholines. 15 including the type of coupled active compound, the age and The conjugates according to the invention can also be weight of the patient, the precise condition that requires treat delivered using monoclonal antibodies as individual carriers ment, and its severity, the nature of the formulation and the to which the conjugates are coupled. The conjugates can also method of administration. be coupled to Soluble polymers as targeted medicament car The present invention also relates to a kit for the prepara riers. Such polymers may encompass polyvinylpyrrolidone, tion of a pharmaceutical composition, in particular an image pyran copolymer, polyhydroxypropylmethacryla enhancing or therapeutic composition, at least comprising a midophenol, polyhydroxyethylaspartamidophenol or poly conjugate according to the invention. If this conjugate con ethylene oxide polylysine, substituted by palmitoyl radicals. tains a complexing agent, this has preferably still not com The compounds may furthermore be coupled to a class of plexed any metal ions having an image-enhancing or thera biodegradable polymers which are suitable for achieving con 25 peutic action. trolled release of a medicament, for example polylactic acid, The conjugate according to the invention can be in dis poly-e-caprolactone, polyhydroxybutyric acid, polyorthoe Solved form in the kit in a solvent (for example an aqueous sters, polyacetals, polydihydroxypyrans, polycyanoacrylates buffer) or preferably in the form of the lyophilisate. and crosslinked or amphipathic block copolymers of hydro Since the metalions that are complexed by the complexing gels. 30 agent of the conjugate according to the invention are radio Pharmaceutical formulations adapted for transdermal active for many applications, pharmaceutical compositions administration can be administered as independent plasters which comprise the conjugate cannot be prepared as far in for extended, close contact with the epidermis of the recipi advance as desired. Furthermore, owing to the radioactivity, ent. Thus, for example, the active ingredient can be delivered certain procedures regarding occupational safety must befol from the plaster by iontophoresis, as described in general 35 lowed during the preparation. For this reason, it is preferred in terms in Pharmaceutical Research, 3(6), 318 (1986). accordance with the invention to provide a kit which com Pharmaceutical compounds adapted for topical adminis prises the conjugate according to the invention, where the tration can be formulated as ointments, creams, Suspensions, complexing agent has not yet complexed the metal ions nec lotions, powders, Solutions, pastes, gels, sprays, aerosols or essary for the final application. oils. 40 It has been found that the conjugates according to the Pharmaceutical formulations adapted for rectal adminis invention have already accumulated specifically, i.e. exclu tration can be administered in the form of Suppositories or sively or virtually exclusively, in the kidney a short time after CCS. application. In the case of the preferred intravenous adminis Pharmaceutical formulations adapted for nasal administra tration of the conjugates according to the invention, accumu tion in which the carrier Substance is a Solid comprise a coarse 45 lation in the kidney is observed after only 5 minutes. After one powder having a particle size, for example, in the range hour, more than 30%, preferably more than 50%, particularly 20-500 microns, which is administered in the manner in preferably more than 70%, very particularly preferably more which Snuff is taken, i.e. by rapid inhalation via the nasal than 80%, of the injected dose is located in the kidney (% data passages from a container containing the powder held close to based on measurement of the radioactivity). the nose. Suitable formulations for administration as nasal 50 In organ distribution studies with radiolabelled conjugates spray or nose drops with a liquid as carrier Substance encom according to the invention (for example PET measurements pass active-ingredient solutions in water or oil. or non-invasive imaging), the conjugates according to the Pharmaceutical formulations adapted for administration invention typically exhibit at least a two-fold, preferably at by inhalation encompass finely particulate dusts or mists, least a five-fold, particularly preferably at least a ten-fold which can be generated by various types of pressurised dis 55 enrichment in the kidney in relation to the remainder of the pensers with aerosols, nebulisers or insufflators. body (blood, heart, lung, spleen, liver, muscle, brain) one Pharmaceutical formulations adapted for parenteral hour after application. This means that the signal, which administration include aqueous and non-aqueous sterile correlates directly with the amount of radiolabelled com injection solutions comprising antioxidants, buffers, bacte pound, in the kidney is at least twice as strong as the sum of riostatics and Solutes, by means of which the formulation is 60 the signals obtained from blood, heart, lung, spleen, liver, rendered isotonic with the blood of the recipient to be treated: muscle and brain together. and aqueous and non-aqueous sterile Suspensions, which may The conjugates according to the invention can therefore be comprise Suspension media and thickeners. The formulations employed extremely well for diagnostic applications, such as can be administered in single-dose or multidose containers, renal scintigraphy, renal PET and renal MRT functional test for example sealed ampoules and vials, and stored in freeze 65 ing of the kidney in general, for the therapy and diagnosis of dried (lyophilised) state, so that only the addition of the sterile renal cancer and, if desired, metastases of renal cancer, CT of carrier liquid, for example water for injection purposes, the kidney and/or ultrasound of the kidney. US 9,078,929 B2 21 22 The therapeutic application is, in particular, in the drug FIG. 2 shows a diagrammatic representation of the com targeting for the organ the kidney. In particular, the conju pound obtained in Example 1, where the DOTA modifica gates according to the invention can serve as medicaments for tions, in contrast to the simplified representation in FIG.2, are the treatment of diseases of the kidney or of diseases in the of course randomly distributed in the molecule. treatment of which medicaments are employed whose site of 5 action is the kidney. One or more active compounds. Such as Example 2 antibiotics, inflammation inhibitors, ACE inhibitors, diuret ics, immunosuppressants or chemotherapeutic agents, are e-L-polylysine-DTPA preferably bonded to the conjugates according to the inven tion, for example via cleavable spacer sequences. 10 75 mg of e-L-polylysine and 310 mg of DTPA difluorophe The use of the conjugates according to the invention for nyl ester tetra-t-butyl ester are dissolved in 4 ml of methanol blocking the resorption of kidney-toxic Substances is also and stirred at RT for 20h. The reaction solution is evaporated, possible. and 4 ml of TFA+100 ul of water are added to the residue and In accordance with the invention, targeting of the kidney 15 left to stand for 20h. The product is precipitated using diethyl means the achievement of increased uptake of the applied ether. Purification by HPLC and lyophilisation gives 150 mg substance in the kidney in relation to the remainder of the as colourless Solid. body. During targeting of the kidney with the conjugate according to the invention, at least a two-fold, preferably at Example 3 least a five-fold, particularly preferably at least a ten-fold enrichment is preferably achieved in the kidney in relation to Loading with '''In the remainder of the body (blood, heart, lung, spleen, liver, 1.5 mg of e-L-polylysine-DOTA are dissolved in 500 ul of muscle, brain) by administration of a conjugate according to ethanol, 100 ul of DMSO and 500 ul of 0.4M sodium acetate the invention. These values are determined by means of organ buffer, pH 5.30 ul of this solution are diluted with 30 ul of distribution studies with radiolabelled conjugates according 25 buffer, '''InCls (20 MBq) is added, and the mixture is heated to the invention (for example PET measurements or non at 70° C. for 8 min. Radio-HPLC shows complete complex invasive imaging). The enrichment in the kidney typically 1ng. takes place after 30 minutes to 8 hours, depending on the type 1.2 mg of e-L-polylysine-DTPA are dissolved in 400 ul of of application. 0.4 M sodium acetate buffer, pH 5. 20 MBq of '''InCls are Even without further comments, it is assumed that a person 30 added to 80 ul of this solution, and the mixture is heated at 70° skilled in the art will be able to utilise the above description in C. for 10 min. Radio-HPLC shows complete complexing. the broadest scope. The preferred embodiments and examples should therefore merely be regarded as descriptive disclosure Example 4 which is absolutely not limiting in any way. The complete disclosure content of all applications, pat 35 Loading with Ga ents and publications mentioned above and below, in particu lar the corresponding application EP 09009393.1-20, filed on 50 ul of the solution of e-L-polylysine-DOTA from 20 Jul. 2009, is incorporated into this application by way of Example 3 are added to 100 ul of a solution of 50 MBq of reference. GaCl (pH3), and the mixture is heated at 95°C. for 10 min. 40 HPLC shows complete complexing. EXAMPLES Example 5 1. Material Syntheses Loading with Gd Example 1 45 10 mg of e-L-polylysine-DOTA and 50 mg of GdCl e-L-polylysine-DOTA hexahydrate are heated at 60° C. for 30 min in 600 ul of 0.4M sodium acetate buffer pH 5. Purification by HPLC and lyo DOTA 2,6-difluorophenyl ester: From DOTA and 2,6-dif philisation gives 10 mg as colourless Solid. luorophenol with DCC (Mier et al. Bioconjugate Chem. 50 2005, 16, 237) Example 6 e-L-Polylysine, average molar mass about 4000 (princi pally consisting of 29-34 lysine units), is purchased as 25% EPL-DOTA Modification Using 'F aqueous Solution from Chisso Corp. (Japan) and lyophilised. e-Polylysine (30 mg) is dissolved in water (2000), and a 55 50 mg of e-L-polylysine-DOTA are dissolved in 3 ml of solution of DOTA 2,6-difluorophenyl ester (100 mg) in methanol, and 200 ul of triethylamine and 200 ul of ethyl methanol (1 ml) is added, and 100 ul of N,N-diisopropylethy trifluoroacetate are added, and the mixture is stirred at RT for lamine are added, and the mixture is stirred at RT for 2 days. 1 h. Purification by RP-HPLC using an elution gradient com DOTA 2,6-difluorophenyl ester (100 mg) was then again prising water and acetonitrile and lyophilisation gives 35 mg added, and the mixture was stirred overnight at RT. The 60 as colourless Solid. mixture is then diluted with water and purified preparatively by HPLC. Clean fractions are lyophilised together. DOTA-e- Example 7 polylysine (98 mg) is obtained as colourless Solid Substance. The number of DOTA units per molecule of e-polylysine is Enalapril Derivative of e-polylysine determined by loading with Gd and by MS as being about 10 65 DOTA units per molecule of e-polylysine; i.e. about 30% of Enalapril: Enalapril maleate (Bosche Scientific) is applied the amino groups of the e-polylysine have reacted. to a column containing DoweX 50 Wx8 and washed with US 9,078,929 B2 23 24 water until neutral. Enalapril is then eluted with 2% pyridine Solution B: 100 mg of e-polylysine in 650 ul of water and in water, the solvents are evaporated, and the residue is evapo 350 ul of acetonitrile rated a number of times with acetonitrile. Enalapril is 5 ul of N,N-diisopropylethylamine is added to 100 ul of obtained as colourless oil and is used further directly. solution B and mixed. 25ul of solution A is then added and mixed as quickly as possible. After a few minutes, HPLC Enalapril t-butyl ester: Synthesis is carried out from enala shows a peak of unreacted e-polylysine and a new main peak, pril and N,N'-dicyclohexyl-O-t-butylisourea (Chadran, Ravi but no longer a peak for enalapril derivative 4. The newly US 200624 1017) formed product is isolated by preparative HPLC and charac Enalaprilate t-butyl ester: Synthesis is carried out from terised by MS as the desired mono-enalapril-e-polylysine enalapril t-butyl ester using NaOH (Iwasaki et al. Chem. derivative. Pharm. Bull. 37(2) 280 (1989) 10 DOTA-e-polylysine-enalapril derivative: An excess of N,N'-Diisopropyl-O-benzylisourea: Synthesis is carried DOTA 2,6-difluorophenyl ester in methanol is added to out from benzyl alcohol and N,N'-dicyclohexylcarbodiimide e-polylysine-enalapril derivative in water N,N-diisopropyl (Mathias, Synthesis 1979,561.) ethylamine is added. Purification by preparative HPLC gives the desired compound. Benzyl 6-hydroxyhexanoate: A solution of N,N'-diisopro 15 pyl-abenzyliso-urea (2.34 g; 10 mmol) in THF (8 ml) is added Example 7 to 6-hydroxyhexanoic acid (ABCR) (1.32 g; 10 mmol), and the mixture is stirred at RT for 2 days. The solid material is Rapamycin Derivative of e-Polylysine filtered off, and the filtrate is evaporated. The residue is eluted on silica gel with ethyl acetate:hexane 1:2 to 1:1. Yield 90%. Rapamycin 42-hemisuccinate: is prepared from rapamycin Benzyl hexanoate 6-O N,N'-diisopropylisourea: N,N'- (LC-Laboratories) succinic anhydride and lipase acrylic resin Diisopropylcarbodiimide (1.14g, 9 mmol) is added to benzyl from Candida antarictica (Sigma). (Guetal. Org. Lett. 2005, 6-hydroxyhexanoate (2.0 g; 9 mmol), CuCl (30 mg) and THF 7,3945-3948) (6 ml) are added, and the mixture is stirred overnight at RT. Rapamycin 42-hemisuccinyl-NHS ester: Rapamycin The mixture is then diluted with THF and filtered through 25 42-hemisuccinate (180 mg; 0.177 mmol) is dissolved in neutral aluminium oxide in a frit and rinsed with THF. The acetonitrile (1.5 ml), and N.N.N',N'-tetramethyl-O-(N-suc filtrate is evaporated, the residue is taken up in diethyl ether, cimidyl)uronium tetrafluoroborate (TSTU) (56 mg; 0.186 the solid material is filtered off, and the filtrate is evaporated. mmol) in acetonitrile (0.5 ml) is added, and N,N-diisopropy The product is used without further purification. lethylamine (36 ul; 0.20 mmol) is added, and the mixture is Enalapril derivative 1 (corresponding to FIG.4): A solution 30 stirred at RT. HPLC shows complete reaction after 30 min. ofenalapril t-butyl ester (2.87 g7.09 mmol) in THF (20 ml) is The product is purified by preparative HPLC. After lyophili added to benzyl hexanoate 6-O-N,N'-diisopropylisourea sation, the product (125 mg) is obtained as colourless solid (2.47 g, 7.09 mmol). 20 mg of DMAP are added, and the compound. MS shows the exact mass expected. mixture is heated under reflux for 7h, then stirred overnight at e-Polylysine-Rapamycin Derivative: 60° C. The reaction solution is cooled in ice, and the solid 35 e-Polylysine (3 ml of 25% aqueous solution; Chisso) is material is filtered off. The filtrate is evaporated, and the diluted with water (30 ml) and adjusted to pH about 3 using residue is eluted on silica gel with ethyl acetate:hexane 1:2 to TFA and lyophilised. 2:1. Clean fractions are evaporated together. Yield 85%. Solution A: 30 mg of rapamycin 42-hemisuccinyl-NHS 'C and H-NMR confirm the structure indicated. ester in 500 ul of acetonitrile Enalapril derivative 2 (corresponding to FIG. 4): Enalapril 40 Solution B: 500 mg of e-polylysine (acidic, see above) is derivative 1 (400 mg) is dissolved in methanol (50 ml), 10% dissolved in 5 ml of water:acetonitrile (2:1) and adjusted to Pd/C (70mg) is added, and the mixture is hydrogenated using pH 7-7.5 by addition of triethylamine. hydrogen at atmospheric pressure. After 40 min, HPLC Solution A is added to solution B with stirring. A cloudy shows complete reaction. The catalyst is filtered off, and the mixture forms. Addition of acetonitrile (1 ml) causes the solvent is evaporated. The product is used without further 45 formation of a clear solution. HPLC after about 15 min shows purification. 'C and H-NMR confirm the structure indi complete reaction of the rapamycin 42-hemi-succinyl-NHS cated. ester and formation of a new product besides unreacted Enalapril derivative 3 (corresponding to FIG. 4): Enalapril e-polylysine. The newly formed product is isolated by pre derivative 2 (183 mg 0.35 mmol) is dissolved in acetonitrile parative HPLC and characterised by MS as the desired mono (2 ml), and N.N.N',N'-tetramethyl-O-(N-succimidyl)uro 50 rapamycin-e-polylysine derivative. nium tetrafluoroborate (TSTU) (106 mg; 0.35 mmol) in DOTA-e-polylysine-rapamycin derivative: DOTA 2,6-dif acetonitrile (2 ml) is added, and N,N-diisopropylethylamine luorophenyl ester (20 mg; 40 umol) in acetonitrile (200 ul is (63 ul; 0.35 mmol) is added, and the mixture is stirred at RT. added to the e-polylysine-rapamycin derivative (10 mg; about HPLC shows complete reaction after 5 min. The product is 2 umol) dissolved in water:acetonitrile 2:1 (5000, and the purified by means of preparative HPLC. After lyophilisation, 55 reaction solution is adjusted to pH 7-7.5 by addition of tri the product is obtained as colourless semi-solid compound. ethylamine and reacted overnight at RT. Purification by pre MS (mass spectrometry) shows the exact mass expected. parative HPLC gives, after lyophilisation, the desired com Enalapril derivative 4 (corresponding to FIG. 4): Enalapril pound as colourless solid substance. The number of DOTA derivative 3 (8 mg) is left to stand at RT dissolved in a mixture groups per molecule is determined by MS. The proportion is of TFA (200 ul) and triisopropylsilane (5ul). HPLC shows 60 between 15 and 18 DOTA units per molecule. complete reaction after 1 hour. Acetonitrile (1 ml) is added, and the mixture is then evaporated. The residue is dissolved in Example 8 1 ml of acetonitrile:water 1:1 and lyophilised. MS shows the exact mass expected. Sorafenib Derivative of e-Polylysine e-Polylysine-Enalapril Derivative: 65 Solution A: 8 mg of enalapril derivative 4 in 100 ul of 4-Chloro(2-pyridyl))N-2-hydroxyethylcarboxamide acetonitrile (S-2): Methyl 4-chloropyridine-2-carboxylate hydrochloride US 9,078,929 B2 25 26 (S-1)(2.08 g; 10 mmol) (Bankston et al. Organic Process Solution B: 500 mg of e-polylysine (acidic, see above) is Research & Development 2002, 6,777-781) is initially intro dissolved in 5 ml of water:acetonitrile (2:1) and adjusted to duced in methanol (5 ml) and cooled in ice. Ethanolamine (3 pH7-7.5 by addition of triethylamine. ml; 50 mmol) in THF (50 ml) added dropwise over the course Solution A is added to solution B with stirring. A cloudy of 10 min, the mixture is then stirred at 0° for a further 3 hand 5 solution forms. Addition of acetonitrile (1 ml) causes the subsequently at RT overnight. After evaporation, the residue formation of a clear solution. HPLC after about 15 min shows is taken up in ethylacetate and washed with 5% NaHCO3, the complete reaction of the Sorafenib derivative S-6 formation of organic solution is dried and evaporated. The product, S-2, a new product besides unreacted e-polylysine. The newly (1.84 g 91%) is obtained as pale-yellow solid substance in formed product (71 mg) is isolated by preparative HPLC and high purity (HPLC). ''C NMR (DMSO-d): 8 42.15, 59.98, 10 characterised by MS as the desired mono-Sorafenib-e-polyl 122.30, 126.82, 145.01, 150.43, 152.09, 163.20 p.p.m. (4-(4-Aminophenoxy)(2-pyridyl)-N-2-hydroxyethylcar ysine derivative. boxamide (S-3): K tert-butoxide (3.17 g; 28.24 mmol) is DOTA-e-polylysine-Sorafenib derivative: DOTA 2.6-fluo added in portions to a solution of 4-aminophenol (2.96 g; 27.1 rophenyl ester (197 mg: 360 umol) in acetonitrile (1.5 ml) is mmol) in DMF (45 ml) under argon, and the mixture is then 15 added to e-polylysine-Sorafenib derivative (71 mg; about 15 stirred at RT for 1.5 h. A solution of S-2 (5.43 g; 27 mmol) in umol) dissolved in water:acetonitrile 2:1 (1.5 ml), and the DMF (10 ml) added, and KCO (2.0 g) subsequently added. reaction solution is adjusted to pH 7-7.5 by addition of tri The reaction is heated at 70° in an oil bath for 6h then cooled, ethylamine and reacted overnight at RT. Purification by pre and ethyl acetate (400 ml) and saturated NaCl (400 ml) are parative HPLC gives, after lyophilisation, the desired com added. The aqueous phase is extracted again with ethyl pound as colourless solid substance (90 mg). The number of acetate, and the combined organic phases are washed with DOTA groups per molecule is determined by MS as 16-20. 4x100 ml of saturated NaCl, dried and evaporated. The dark residue is purified firstly on silica gel with ethyl acetate/ 2. Use Examples hexane (3:1)-ethyl acetate/methanol (10:1). Fractions con taining product are evaporated together and then eluted on 25 1. Organ Distribution Study alumina with ethyl acetate/hexane (3:1)-ethyl acetate/metha nol (10:1). The product as pale-yellow solid substance (3.20 In order to determine the pharmacokinetics, e-polylysine g; 43%) in high purity (HPLC) MS: M+Na=296.100; calcu DOTA is labelled with the radioactive nuclide '''In in accor lated for CHNNaO-296.1011. dance with Synthesis Example 2/3 and NMRI mice are N-(4-Chloro-3-(trifluoromethyl)phenyl)-((4-(2-(N-hy 30 injected in groups of three animals each via the tail vein. The droxyethyl-carbamoyl)(4-pyridyloxy))phenyl)amino)car amount used is about 5ug of e-polylysine-DOTA per animal, boxamide (S-4): A solution of S-3 (1.50 g; 5.49 mmol) in the dose is about 1 MBq per animal. dichloromethane (20 ml) is cooled in ice, and a solution of The animals are Subsequently dissected, and the isolated 4-chloro-3-trifluoromethylphenyl isocyanate (1.26 g; 5.70 organs (blood, heart, lung, spleen, liver, kidney, muscle, mmol) in dichloromethane (20 ml) is added. After 1 h, the 35 brain) are investigated with respect to their radioactivity in a precipitated solid substance is filtered off and rinsed with gamma counter. The amount of radioactivity in the corre dichloromethane and dried. The product (S-4) (2.34 g; 4.73 sponding organ represents directly the amount of e-polyl mmol: 86%) is obtained as white solid substance. MS: ysine-DOTA taken up. The animals are killed after various M+H=495.1037 calculated for CHCIFNO. 495.1047. times. This is carried out, like the entire performance of the Sorafenib derivative S-5: S-4 (2.30 g; 4.65 mmol) is dis 40 experiment, in accordance with the ethical principles for sci Solved in pyridine (15 ml), and a solution of Succinic anhy entific performance of animal experiments. Surprisingly, it is dride (581 mg, 5.81 mmol) in pyridine (5 ml) is added, N.N- observed in the measurements that over 150 per cent of the dimethylaminopyridine (284 mg, 2.33 mmol) is added, and injected dose per gram of kidney tissue accumulates in the the mixture is stirred overnight at RT. The reaction mixture kidneys of the experimental animals only 10 minutes after applied to a column containing about 20 ml of pyridinium 45 injection of the '''In-loaded e-polylysine-DOTA. A small DoweX in pyridine:methanol: water (8:1:1) and eluted with proportion is excreted via the urine. the same mixture. The eluate is evaporated, the residue is The graphical representation of the accumulation in the evaporated a number of times with toluene finally with aceto various organs investigated is shown in FIG. 2. The Y axis nitrile. The product (S-5) is obtained as white foam (2.65 g; represents the specific accumulation in the particular 4.45 mmol; 95%) MS: M+H=595.1115; calculated for 50 organs—indicated as percent of the injected dose per gram of CHCIFNO. 595.1207. tissue. P1 to P3 represent the results for conjugates with Sorafenib derivative S-6: S-5 (180 mg; 0.30 mmol) was various degree of loading: P1: about 10% of the lysine mono dissolved in acetonitrile (3 ml), and a solution of N.N.N',N'- mers carry a DOTA, P2: about 30% of the lysine monomers tetramethyl-O-(N-succimidyl)uronium tetrafluoroborate carry a DOTA and P3: about 50% of the lysine monomers (TSTU) (99mg; 0.32 mmol) in acetonitrile (3 ml) was added, 55 carry a DOTA. and N,N-diisopropylethylamine (67 ul; 0.37 mmol) was added. HPLC shows complete reaction after 10 min. 0.1% 2. PET Measurement TFA in water (2 ml) is added, and the mixture is purified preparatively HLPC. S-6 is obtained as “off-white solid For PET measurements, labelling is carried out with substance (163 mg 0.24 mmol; 78%). MS: M+H=692.1368: 60 68-gallium, in accordance with Synthesis Example 4, and the calculated for CHCIFNO: 692.1371. kidney accumulation is investigated dynamically in a small e-Polylysine-Sorafenib Derivative: animal PET scanner (Siemens). It is found here that the com e-Polylysine (3 ml of 25% aqueous solution; Chisso) is pound (e-L-polylysine-DOTA, loaded with Ga)achieves an diluted with water (30 ml) and adjusted to pH about 3 using extraordinarily good organ to background ratio and accumu TFA and lyophilised. 65 lates virtually exclusively in the kidney. Typical values here Solution A: 17 mg of Sorafenib derivative S-6 in 500 ul of are a 10-fold accumulation in the kidney in relation to the acetonitrile remainder of the body. US 9,078,929 B2 27 28 3. Comparative Experiments 70% (based on the number of such successive monomer units) of e-lysine monomer units, The uptake of "Tc-MAG3 and of "Tc-DMSA are com wherein the conjugate contains from 3 to 6 compounds pared with the uptake of (e-L-polylysine-DOTA, loaded with carrying carboxyl groups per 10 monomer units, '''In) in the rat and mouse model in planar Scintigraphs. Both 5 wherein, in the oligomer, said e-lysine monomer units are known tracers exhibit a worse kidney to background ratio bonded via the e-amino group of their side chain, Such than the Substance according to the invention. that the oligomer is unbranched, wherein, in the compound carrying carboxyl groups, the 3. Conjugation of Various Compounds Carrying proportion of carboxyl groups by molecular weight is Carboxyl Groups 10 greater than 30% and wherein at least one compound carrying carboxyl groups In order to investigate the effect of various compounds which is conjugated to the oligomer contains two or carrying carboxyl groups on the accumulation in the kidney, more free carboxyl groups. e-polylysine is conjugated with a DOTA unit in order to 2. The conjugate of claim 1, wherein, in addition, at least facilitate labelling with radionuclides for animal experi 15 one active compound is covalently bonded to the conjugate. ments. In addition to the one DOTA unit, a plurality of units of 3. The conjugate of claim 1, wherein the oligomer has a another compound carrying carboxyl groups or of a com chain length of 10 to 50 monomer units. pound which carries no carboxyl groups are conjugated on. 4. The conjugate of claim 1, wherein the oligomer consists Organ distribution studies of these conjugates show that con only of e-lysine monomer units. jugates with DO-3AM (1,4,7,10-tetraazacyclo-dodecane-N, 5. The conjugate of claim 1, wherein the oligomer consists -N', -N" -triamide, N'-monoacetic acid, Macrocyclics), of e-polylysine. which contains no carboxyl groups after conjugation, exhibit 6. The conjugate of claim 1, wherein at least one compound an accumulation in the kidney which do not exceed the accu carrying carboxyl groups is bonded, directly or via a spacer, mulation already achieved with a DOTA group, while the to the amino group of an e-lysine monomer unit. conjugates with citric acid exhibit excellent accumulation 25 7. The conjugate of claim 1, wherein at least one compound and the conjugates with Succinic acid exhibit good accumu carrying carboxyl groups is a complexing agent. lation in the kidney. 8. A process for the preparation of a conjugate of claim 1 Synthesis of an e-polylysine with only one DOTA unit per which comprises at least the following process steps: molecule (Ref. 1): e-Polylysine (780 mg) is dissolved in a) providing an oligomer which consists of peptidically water:methanol 1:1, and DOTA 2,6-difluorophenyl ester (103 30 linked monomer units and which is either built up from mg) in methanol is added, diisopropylethylamine (100 ul) is more than 50% (based on the total number of monomer added, and the mixture is stirred overnight at RT. The product units) of e-lysine monomer units or comprises at least 10 is purified by HPLC and lyophilised. MS shows a mixture of Successive monomer units which are built up from at e-polylysine and e-polylysine-1xDOTA in the ratio about 2:1. least 70% (based on the number of such successive This compound, Ref. 1, is used for the preparation of all 35 monomerunits) of e-lysine monomerunits, such that the following derivatives. oligomer is unbranched, and DO-3AM-acetic acid derivative of Ref. 1: DO-3AM-acetic b) conjugating with the oligomer at least one compound acid (42 mg) and TSTU (31 mg) are dissolved in DMF (2 ml), carrying two or more free carboxyl groups to the oligo and diisopropylethylamine (18 Jul) is added. After 1 min, this mer from step a), wherein, optionally, at least one active solution is added to Ref. 1 (16 mg) in water:dioxane 1:1 (4000 40 compound is covalently bonded to the conjugate such and left to react at RT overnight. After dilution with water, the that the conjugate contains from 3 to 6 compounds car product is purified HPLC and lyophilised. rying carboxyl groups per 10 monomer units. Citric acid derivative of Ref. 1: Citric anhydride is prepared 9. A medicament composition comprising a conjugate of from citric acid (2 mmol) and dicyclohexylcarbodiimide (1 claim 1. mmol) in THF (5 ml). After 30 min, the dicyclohexylurea 45 10. A method for targeting of the kidney with a drug com formed is filtered off and rinsed with THF. The filtrate is prising administering the drug together with a conjugate of evaporated and used further directly. Citric anhydride (80 mg) claim 1. in pyridine (200 ul) is added to a solution of Ref. 1 (38 mg) 11. A macromolecule conjugate composed of at least two and dimethylaminopyridine (5 mg) in pyridine:Water 1:1 conjugates according to claim 1 which are covalently bonded (2000, and the mixture is stirred at RT overnight. After dilu 50 to a macromolecule. tion with water, the product is purified HPLC and lyophilised. 12. A therapeutic or image-enhancing composition, com The number of citric acid units per molecule of e-polylysine prising a conjugate corresponding to claim 1. 1xDOTA can be determined by MS as 4-8. 13. A kit for the preparation of a medicament comprising a Succinic acid derivative of Ref. 1: Succinic anhydride (100 conjugate corresponding to claim 1. mg) in pyridine:dioxane 1:1 (2000 ul) is added to a solution of 55 14. A kit according to claim 13, for the preparation of a Ref. 1 (38 mg) and dimethylaminopyridine (5 mg) in pyridin therapeutic or image-enhancing composition. e:water 1:1 (10000, and the mixture is stirred at RT overnight. 15. The conjugate of claim 2, wherein the active compound After dilution with water, the product is purified by HPLC and is selected from the group consisting of immunosuppres lyophilised. sants, cytostatics, protein kinase inhibitors, immunothera 60 peutic agents, antiphlogistics, antibiotics, cephalosporins, The invention claimed is: B-lactamase inhibitors, carbapenems, monobactams, tetracy 1. A conjugate comprising at least one compound carrying clines, macrollide antibiotics, glycopeptide antibiotics, ene carboxyl groups and an oligomer which consists of 10 to 1000 diynes, Virostatics, antihypertensives, Sartans, renin inhibi peptidically linked monomerunits and which is either built up tors, beta blockers, uricoSurics, and diuretics. from more than 50% (based on the total number of monomer 65 16. The conjugate of claim 2, wherein the active compound units) of e-lysine monomer units or comprises at least 10 is selected from the group consisting of azathioprine, myco Successive monomer units which are built up from at least phenolate-mofetil, ciclosporin, tacrolimus, sirolimus, fin US 9,078,929 B2 29 30 golimod, triptolide, bleomycin, dactinomycin, mitomycin, cin, calicheamicin, aciclovir, Valaciclovir, ganciclovir, daunorubicin, doxorubicin, epirubicin, idarubicin, mitox Valganciclovir, penciclovir, famciclovir, brivudine, cidofovir, antron, amsacrine, doxofluridine, cisplatin, carboplatin, foscarnet, idoxuridine, tromantadine, benazepril, captopril, oxaliplatin, Satraplatin, camptothecin, toptecan, irinotecan, cilaZapril, enalapril, fosinopril, lisinopril, perindopril, etoposide, teniposide, cyclophosphamide, trofosfamide, mel quinapril, ramipril, trandolapril, Zofenopril, losartan, balsar phalan, chlorambucil, estramustine, buSulfan, chlorambucil, tan, irbesartan, candesartan, eprosartan, olmesartan, telmis chlormethine, treosulfan, carmustine, lomustine, nimustine, artan, alliskiren, proproanolol, pindolol, Sotalol, bopindolol. procarbazine, Streptozocine, dacarbazine, ifosfamide, temo atenolol, bisorpolol, celiprolol, esmolol, metoprolol, nebiv Zolomide, thiotepa, Vinorelbine, Vincristine, vinblastine, Vin olol, oxprenolol, carvedilol, labetalol, probenecid, benzbro desine, paclitaxel, docetaxel, methotrexate, pemetrexed, 10 raltitrexed, fluorouracil, capecitabine, cytosinarabinoside, marone, acetazolamide, furosemide, torasemide, bumet gemcitabine, tioguanine, pentostatin, mercaptopurine, flu anide, piretanide, aZosemide, etacrynic acid, etoZoline, hydrochlorothiazide, benzthiazide, chlorothiazide, chlortha darabine, caldribine, hydroxycarbamide, mitotane, azaciti lidone, indapamide, mefruside, metolaZone, clopamide, Xipa dine, cytarabine, nelarabine, bortezomib, anagrelide, ima mide, hydroflumethiazide, methyclothiazide, polythiazide, tinib, erlotinib, Sunitinib, Sorafenib, dasatinib, lapatinib or 15 nilotinib, cetuximab, alemtuzumab, bevacizumab, naproxen, amiloride, triameterene, Spironolactone, canrenone, ibuprofen, indometacin, prednisolone, prednisone, hydrocor eplerenone and Spironolactone. tisone, budesonide, benzylpenicillin, methicillin, amoxicil 17. The conjugate of claim 1, wherein the compound car lin, cefuroxim, cefotaxim, cefadroxil, cefixim, clavulanic rying carboxyl groups is 1,4,7,10-tetraazacyclododecane-N, acid, Sulbactam, taZobactam, imipenem, meropenem, aztre -N'-N", -N"-tetraacetic acid or diethylenetriaminepentaace onam, tetracycline, chlortetracycline, Oxytetracycline, doxy tic acid. cycline, minocycline, tigecycline, erythromycin A, Vancomy