US 20100158814A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0158814 A1 Bussat et al. (43) Pub. Date: Jun. 24, 2010

(54) FIBRIN-BINDING PEPTIDES AND Related U.S. Application Data CONUGATES THEREOF (60) Provisional application No. 60/869,472, filed on Dec. 11, 2006. (75) Inventors: Philippe Bussat, Feigeres (FR); Publication Classification Bernard Lamy, Saint-Julien-en-Genevois (FR); (51) Int. Cl. A6IB 8/00 (2006.01) Edmund R. Marinelli, A638/16 (2006.01) Lawrenceville, NJ (US): Sibylle A638/10 (2006.01) Pochon, Troinex (CH); Bo Song, C07K I4/00 (2006.01) Princeton, NJ (US); Rolf E. C07K 7/08 (2006.01) Swenson, Princeton, NJ (US) A6IP 7/02 (2006.01) A6IP35/04 (2006.01) Correspondence Address: A6IP 29/00 (2006.01) LUCAS & MERCANTI, LLP (52) U.S. Cl...... 424/9.52: 514/12: 514/13: 514/14: 475 PARKAVENUE SOUTH, 15TH FLOOR 530/324; 530/326;977/774 NEW YORK, NY 10016 (US) (57) ABSTRACT Fibrin-binding peptides having high binding affinity and (73) Assignee: BRACCO IMAGING S.P.A., excellent physical characteristics compared to previously Milan (IT) known fibrin-binding peptides are provided. These fibrin binding peptides may be conjugated to a detectable label or a (21) Appl. No.: 12/517,239 therapeutic agent and used to detect and facilitate treatment of pathological conditions associated with the presence offibrin Such as thrombic, angiogenic and neoplastic conditions. (22) PCT Filed: Dec. 11, 2007 These peptides may be used in imaging processes Such as MRI, ultrasound and nuclear medicine imaging (e.g. PET, (86). PCT No.: PCT/US07/25403 Scintigraphic imaging., etc.). The peptides may also be used therapeutically. The present invention also provides pro S371 (c)(1), cesses and methods for making and using Such peptides and (2), (4) Date: Jun. 2, 2009 conjugates thereof.

1. 10% NHNHDMF (2 x 10 min) 2. DMF wash (4X)

1. Fmoc--OHHATADEANMP, 2 hr? 2.20% Piperidine/DMF (2 x 10 min) 3. DMF wash (4x) 4. Repeat steps 1-3

1. Reagent B, 4.5 h, RT 2. Evaporation of volatiles 3, EtO (precipitation of crude linear peptide) 4, DMSO (aq) f N-methylglucamine (pH 8), 48 h, RT

O -wop P- A E swif two P G a g-in-,H

HN1-a or-o-o-o- NH H or Patent Application Publication Jun. 24, 2010 Sheet 1 of 11 US 2010/0158814 A1

2. DMF wash (4X)

1. Fmoc--OHHATADEANMP, 2 h, RT 2.20% Piperidine DMF (2 x 10 min) 3. DMF wash (4x) 4. Repeat steps 1-3

1. Reagent B, 4.5 h, RT 2. Evaporation of volatiles 3. EtO (precipitation of crude linear peptide) 4. DMSO (aq) 1 N-methylglucamine (pH 8), 48 h, RT

O3-wop de A E swif two P G a g-in,H HC

HN2 N11-0 uf n-o-o-NH H O

Figure 1 Patent Application Publication Jun. 24, 2010 Sheet 2 of 11 US 2010/0158814 A1

1. S2 o -R wap P A E swit Fw D PG a cliniH s O 9. 1,45 equiv y- RWW cap C P A eswt two p a gold,

C H, sh, 2 HPLCPurification 2 O NH

H

two plp as swiftwo e o a cle, 1. Pd(PPh), (1 equiv NMMHoAc:MF (1:2:10, whiv) 2. HPCPurification N

Figure 2

Patent Application Publication Jun. 24, 2010 Sheet 5 of 11 US 2010/0158814 A1

1...mocrg2NS (1.28 equiv.) O H war at swt, were go- NH, DEA(316hrt equiv) 1 DMF HC)wap c. PAE swT F & WDP GG 2 Piperidine (80equiv.) Osh, RT

'n-e-or-o-o: 3, PCPurification

— )-wap c. P. A E swT F cWD P G G HC

0.87 equiv O DEA(146 equiv MFloCM1:1 ww 16h, RT 2PCPurification

Figure 5 Patent Application Publication Jun. 24, 2010 Sheet 6 of 11 US 2010/0158814 A1

C. N Ho:c- N COH HOC-1 \ | o C CO2H HO.C-1 \ / C-9-o-cool

3 4

Figure 6A

Patent Application Publication Jun. 24, 2010 Sheet 8 of 11 US 2010/0158814 A1

CO2H HO-C-N f \,-CO2H HO2C r C N t HN CO2H HO.C-1 N\ / Y-COHN 2 COH N1 NNH 15 16

R R R R

Hooc -cool Hooc- N 9-cooh N N Hooc- \- cool Hooc COOHO R go

17 18

Hooc- -COOH Oy C7 N-cooh rock N\ y-cool

19 20

HO,c-N W V 1 CON(CH), N N ) N N .N . C,N D ph Ho,c-/ \ /\-COH HOOC-1 \ J S-Q

21a 21b

Figure 6C Patent Application Publication Jun. 24, 2010 Sheet 9 of 11 US 2010/0158814 A1

H NCS

Figure 7A Patent Application Publication Jun. 24, 2010 Sheet 10 of 11 US 2010/0158814 A1

29 30

N N N bH (bH OH OH

31 32

H O

O" h 1. H c'ch 3 iNNHCOCH, 33

Figure 7B Patent Application Publication Jun. 24, 2010 Sheet 11 of 11 US 2010/0158814 A1

Reference Cheate Compound 2 Complex 1

Figure 8 US 2010/0158814 A1 Jun. 24, 2010

FBRN-BINDING PEPTDES AND tion, hemorrhagic events, and migratory thrombophlebitis. CONUGATES THEREOF Hemostatic complications are a common cause of death in patients with . Many tumor cells possess strong proco RELATED APPLICATIONS agulant activities that promote the local activation of the 0001. This application claims priority to and benefit of coagulation system. Tumor-mediated activation of the coagu U.S. Provisional Application No. 60/869,472, filed Dec. 11, lation cascade has been implicated in both the formation of 2006, the contents of which are hereby incorporated by ref tumor stroma and the promotion of hematogenous metastasis. erence in its entirety. Fibrin matrix, moreover, is known to promote the migration of a substantial number of distinct cell types, including both FIELD OF THE INVENTION transformed cells, macrophages, and fibroblasts. In particu lar, much like in a healing wound, the deposition of fibrin/ 0002 The present invention relates to fibrin-binding pep fibrinogen, along with other adhesive glycoproteins, into the tides, polypeptides and compositions for the detection and extracellular matrix (ECM) have been shown to serve as a treatment of pathological conditions associated with fibrin scaffold to Support binding of growth factors and to promote deposition or accumulation, such as intravascular thrombosis the cellular responses of adhesion, proliferation, and migra and conditions associated with angiogenic processes. The tion during angiogenesis and tumor cell growth (see, for invention includes compounds for diagnostic and/or thera instance: Dvorak H F. Nagy J A, Berse B, et al. Vascular peutic applications comprising fibrin-binding peptides. It permeability factor, fibrin, and the pathogenesis of tumor also includes methods of making and using Such peptides, stroma formation, Ann NYAcadSci, 1992; 667: 101; Rickles compounds and compositions. FR, Patierno S, Fernandez PM. Tissue Factor, Thrombin, and Cancer. Chest. 2003; 124:58S-68S; Brown HF, Vander Water BACKGROUND OF THE INVENTION L. Hervey V. S. Dvorak H F. Fibrinogen influx and accumu 0003. Thrombus associated diseases are vascular condi lation of cross-linked fibrin in healing wounds and in tumor tions that develop due to the presence of a clot. Such diseases stroma. Am J Pathol. 1988: 130:4559: Dvorak HF, Hervey V are a major cause of mortality, and therefore developing S, Estrella P. Brown L. F. Mc-Donagh J, Dvorak AM. Fibrin thrombus-specific diagnosis, treatment, and detection meth containing gels induce angiogenesis: implication for tumor odologies and reagents is of great clinical importance. Pul stroma generation and wound healing. Lab Invest. 1987; monary embolism (PE), deep-vein thrombosis (DVT), stroke, 57:673 and Rickles F R, Patierno S, Fernandez PM. Tissue and atherosclerosis are examples of thrombus-associated dis Factor. Thrombin and Cancer. Cest. 2003: 124:58S-68S). CaSCS. Most solid tumors in contain considerable amounts 0004 DVT is a condition in which blood clots form in the of cross-linked fibrin, Suggesting that it is important in tumor deep blood vessels of the legs and groin. These clots can block stroma formation. Studies indicate that both fibrinogen and the flow of blood from the legs back to the . Sometimes, fibrin localize at the tumor-host cell interface (see, for a piece of a clot is detached and carried by the bloodstream instance: Rickles F R, Patierno S, Fernandez, P M. Tissue through the heart to a blood vessel, where it lodges and Factor, Thrombinand Cancer. Cest. 2003; 124:58S-68S: Cos reduces, or blocks, the flow of blood to a vascular tissue. This tantini V. Zacharski L. R. MemoliVA et al. Fibrinogen depo is called an embolism. If Such a clot lodges in pulmonary sition without thrombin generation in primary breast blood vessel it can be fatal. cancer. Cancer Res. 1991; 51: 349-353 and Simpson-Haid 0005. In the United States alone an estimated 600,000 aris PJ and Rybarczyky B. Tumors and Fibrinogen: The Role patients suffer from PE's each year. In approximately 378, of Fibrinogen as an Extracellular Matrix . Ann. N.Y. 000 of these patients, PE goes undetected, and approximately Acad. Sci., 2001 936(1): 406-425). Fibrin matrices promote 114,000 of these patients later die due to complications asso neovascularization, Supporting the notion that fibrin may ciated with the disease. This high mortality is partly due to the facilitate tumor Stroma formation by mechanisms that are absence of clinical symptoms in many cases and to the sig analogous to wound repair. nificant limitations associated with currently available meth 0007 Moreover, a correlation seems to exist between ods of investigation and detection. plasma fibrinogen levels and tumor size, depth of tumor inva 0006 Fibrin is also associated with various . The sion and metastasis (See, for instance, Lee J. H. Ryu KW. Kim existence of heterogeneous pattern offibrin/fibrinogen depo S. Bae J. M. Preoperative plasma fibrinogen levels in gastric sition in various tumor types is a concept Supported by a cancer patients correlate with extent of tumor. Hepatogastro Substantial body of correlative and indirect evidence Suggest enterology 2004; 51:1860-3). In addition, it is known that ing that fibrin/fibrinogen is important in tumor stoma forma fibrin/platelets are involved in protecting tumor cells from the tion (see, for instance: Costantini V. Zacharski L. R. Fibrin and action of the circulating natural killers units provided by cancer. Thromb Haemost. 1993; 69:406; Dvorak HF. Throm human immune system thus improving the Survival of circu bosis and cancer. Hum Pathol. 1987; 18:275; Dvorak H F, lating tumor (See, for instance, Palumbo J S. et al. platelets Nagy JA, Berse B, et al. Vascular permeability factor, fibrin, and fibrin(ogen) increase metastatic potential by impeding and the pathogenesis of tumor stroma formation, Ann NY natural killer cell-mediated elimination of tumor cells. Blood, AcadSci, 1992: 667: 101; Cavanagh PG, Sloane BF, HonnK 2005: 105:178). This implies, for example, that a conven V. Role of the coagulation system in tumor-cell-induced tional tumor therapy using antibodies that target tumors may platelet aggregation and metastasis. Hemostasis. 1988; 18:37 not effectively treat tumors containing fibrin because these and Bardos H. Molnar P. Csecsei G, Adany R. Fibrin deposi tumor are protected by fibrin. tion in primary and metastatic human brain tumours. Blood 0008 Thus, visualization of fibrin deposition and targeted Coagul Fibrinolysis. 1996; 7:536). Indeed, many significant inhibition/destruction of established vasculature and clotted hemostatic abnormalities have been described in patients fibrin is considered an important tool against malignant dis with cancer, including disseminated intravascular coagula ease progression. Consequently, there remains a need for US 2010/0158814 A1 Jun. 24, 2010 improved fibrin-binding compounds for use in sensitive diag a radiotherapeutic agent, a tumoricidal agent, or a throm nosis and specific therapy of pathological conditions associ bolytic agent. In a preferred embodiment, a peptide is modi ated with fibrin deposition, and, particularly, of solid tumors. fied by conjugation to a radiotherapeutic agent comprising a 0009 Fibrin also has been implicated in angiogenic pro therapeutic radionuclide. cesses. In a developing embryo, the primary vascular network 00.15 Aparticularly advantageous use of the binding moi is established by in situ differentiation of meso-dermal cells eties disclosed herein is in a method of imaging thrombi, and in a process called vasculogenesis. After embryonic vasculo pathological processes associated with fibrin in vivo. Such genesis it is believed that all Subsequent generation of new processes include, for example, pulmonary embolism (PE), blood vessels, in the embryo or in adults, is governed by the deep-vein thrombosis (DVT), stroke, atherosclerosis, and sprouting or splitting of new capillaries from the pre-existing cancer, particularly Solid tumors. The method entails the use vasculature in a process called angiogenesis (Pepper, M. et of fibrin specific binding moieties according to the invention al., 1996. Enzyme Protein, 49:138-162: Risau, W., 1997. for detecting thrombi or fibrin-associated pathological pro Nature, 386:671-674). Angiogenesis is not only involved in cesses, where the binding moieties have been detectably embryonic development and normal tissue growth and repair, labeled for use as imaging agents, including magnetic reso it is also involved in the female reproductive cycle, establish nance imaging (MRI) contrast agents, X-ray imaging agents, ment and maintenance of pregnancy, and in repair of wounds radiopharmaceutical imaging agents, ultrasound imaging and fractures. agents, and optical imaging agents. 0010. In addition to normal angiogenic processes, angio genic events also are involved in a number of important patho 0016. In addition, the newly discovered fibrin binders can logical processes, notably tumor growth and metastasis, and also be used advantageously to detect numerous other patho other conditions in which blood vessel proliferation is physiologies in which fibrin plays a role. In these cases, fibrin increased, such as diabetic retinopathy, psoriasis, arthropa imaging can be a useful direct or Surrogate marker for diag thies and rheumatoid arthritis. Indeed, angiogenesis is so nosis or therapeutic monitoring. For example, peritoneal important in the transition of a tumor from hyperplastic to adhesions often occur after Surgery or inflammatory pro neoplastic growth, that inhibition of angiogenesis has shown cesses, and are comprised of a fibrin network, fibroblasts, promise as a cancer therapy (Kim, K. et al., 1993. Nature, macrophages, and new blood vessels. Patients Suffering from 362:841-844). In these pathological processes, fibrin pro rheumatoid arthritis, lupus, or septic arthritis often have bits vides the structural mesh required for the generation of new of fibrin-containing tissues called rice bodies in the synovial blood vessels. fluid of their joints. In thrombotic thrombocytopenic purpura, 0011. There is a need, therefore, for sensitive and effective a type of anemia, fibrin deposits in arterioles cause turbulent assays to detect the presence of fibrin and fibrin-associated blood flow, resulting in stress and destruction of the red blood diseases. More specifically there is a need for non-invasive cells. The fibrin binding moieties of the instant invention can reagents that can specifically bind fibrin and can be used to be used in the detection and diagnosis of such fibrin-related detect pathological thrombic conditions as well as conditions disorders. associated with pathological angiogenic processes. 0017. The fibrin specific agents can also be used to detect other conditions including but not limited to hypoxia or SUMMARY OF THE INVENTION ischemia of the heart, kidney, liver, lung, brain, or other organs, as well as the detection of tumors, diabetic retinopa 0012. In answer to the need for improved materials and thy, early or high-risk atherosclerosis, and other autoimmune methods for detecting, localizing, measuring and treating and inflammatory disorders. Fibrin specific agents also can fibrin clots, and pathological processes associated with fibrin, provide both director Surrogate markers of disease models in we have now Surprisingly discovered several non-naturally which hypoxia and angiogenesis are expected to play a role. occurring polypeptides that exhibit an unexpectedly high In hypoxic conditions, for example, fibrin(ogen) is expressed degree of fibrin-specific binding. These polypeptides are under the control of hypoxia-inducible factor 1 (HIF-1). capable of Superior fibrin specific binding compared to pre 0018. The fibrin-binding peptides of the invention may viously known peptides and have improved physical proper also be used therapeutically to treat pathophysiologies in ties such as solubility. which fibrin plays a role, including, but not limited to, fibrin 0013 Another aspect of the present invention relates to clots, tumors, hypoxia or ischemia of various organs, patho modifications of the foregoing peptides to provide fibrin spe logical processes associated with angiogenesis, peritoneal cific imaging agents by conjugation to a detectable label. For adhesions, rheumatoid arthritis, lupus, septic arthritis, and example, compounds in which a fibrin-binding peptide is thrombotic thrombocytopenic purpura. For example, the conjugated to a radiolabel, an enzymatic label, a label detect fibrin-binding peptides may be conjugated to an appropriate able by magnetic resonance imaging (MRI) such as MR para therapeutic radionuclide and used for radiotherapy, particu magnetic chelates or microparticles, conjugation to or incor larly to treat tumors. Additionally, the fibrin-binding peptides poration into an ultrasound contrast agent Such as gas-filled may be conjugated to an appropriate therapeutic agent. microvesicles (e.g. microbubbles, microparticles, micro spheres, emulsions, or liposomes), or conjugation to an opti 0019. These and other aspects of the present invention will cal imaging agent, including optical dyes. Binding moieties become apparent with reference to the following detailed according to the present invention are useful in any applica description. tion where binding, detecting or isolating fibrin or its frag ments is advantageous. BRIEF DESCRIPTION OF THE DRAWINGS 0014. The present invention also relates to modifications of the foregoing peptides to provide fibrin specific therapeu 0020 FIG. 1 illustrates a method for the preparation of a tics by conjugation to a therapeutic agent. Such agents may representative linker-functionalized peptide according to the include, for example, a chemotherapeutic, a cytotoxic agent, present invention. US 2010/0158814 A1 Jun. 24, 2010

0021 FIG. 2 illustrates a method for the preparation of a 0033 Specific fibrin-binding peptides are described representative 5-carboxyfluorescein labeled peptide accord herein (including, for example, those included in Tables 1 and ing to the present invention. 2) and hybrid and chimeric peptides incorporating Such pep 0022 FIG. 3 illustrates a method for the preparation of a tides. representative DSPE-PEG200 peptide conjugate according 0034. In addition to the detectable labels described further to the present invention. herein, the binding polypeptides may be linked or conjugated 0023 FIG. 4 illustrates a method for the preparation of a to a therapeutic agent including a radiotherapeutic agent, a representative DSPE-PG4-Glut peptide conjugate according cytotoxic agent, a tumoricidal agent or enzyme, a throm to the present invention. bolytic agent or enzyme (e.g., tRA, plasmin, Streptokinase, 0024 FIG. 5 illustrates a method for the preparation of a urokinase, hirudin), a liposome (e.g., loaded with a therapeu representative DPPE-Glut-PG2-JJ peptide conjugate accord tic agent Such as a thrombolytic, an ultrasound appropriate ing to the present invention. gas, or both). In addition, binding polypeptides of the inven 0025 FIGS. 6a, 6b, 6c, illustrate examples of preferred tion may be bound or linked to a solid Support, well, plate, chelators for either 'In and lanthanides such as paramag bead, tube, slide, filter, or dish. All such modified fibrin netic Gd " or radioactive lanthanides such as, for example, binding moieties are also considered fibrin-binding moieties 77Lu, 90Y. Sm, and Ho. so long as they retain the ability to bind fibrin or fibrin-derived 0026 FIGS. 7a, 7b illustrate examples of preferred chela polypeptides. tors of radioactive metalions such as "Tc, Re and 'Re; 0035. A “labelling group” or “detectable label,” as used 0027 FIG.8 relates to T1 weighted MRI images (Succes herein, is a group or moiety capable of generating a signal that sive slices) acquired 4 h post injection (25 umol/kg of com is detectable. In particular a labeling group or diagnostic label plex) of chelate complex 1 (bottom) and Reference Com may generate a signal for diagnostic imaging, such as mag pound 2 (top) as described in Example 26. netic resonance imaging, radioimaging, ultrasound imaging, X-ray imaging, light imaging, or carry a moiety Such as a DEFINITIONS radioactive metal or other entity that may be used in radio therapy or other forms of therapy. 0028. As used herein, unless otherwise specified, the term 0036. The terms “therapeutic agent” or “therapeutic” refer "polypeptide' is used to refer to a compound of two or more to a compound or an agent having a beneficial, therapeutic or amino acids joined through the main chain (as opposed to side cytotoxic effect in vivo. Therapeutic agents include those chain) by a peptide amide bond ( C(O)NH ). The term compositions referred to as, for example, bioactive agents, "peptide' is used interchangeably herein with “polypeptide' cytotoxic agents, drugs, chemotherapy agents, radiothera but is generally used to refer to polypeptides having fewer peutic agents, genetic material, etc. than 25 amino acids. 0037. The term “patient’ as used herein refers to any mam 0029. The term “fibrin-derived polypeptide” refers to any mal, especially humans. subcomponent of fibrin or fragment of fibrin that is immuno 0038. The term “pharmaceutically acceptable' carrier or logically cross-reactive with fibrin, including immunologi excipient refers to a non-toxic carrier or excipient that can be cally reactive fragments of the protein. administered to a patient, together with a compound of this 0030. The term “binding refers to the determination by invention, such that it does not destroy the biological or standard assays, including those described herein, that a bind pharmacological activity thereof ing polypeptide recognizes and binds reversibly to a given 0039. The following common abbreviations are used target. Such standard assays include, but are not limited to throughout this specification: AcO-acetic anhydride, equilibrium dialysis, gel filtration, and the monitoring of CAN acetonitrile, Ac—acetyl, API-ES Atmospheric spectroscopic changes that result from binding. pressure ionization electrospray, BOP benzotriazol-1- 0031. The term “specificity' refers to a binding polypep yloxy-tris(dimethylamino)-phosphonium hexafluorophos tide having a higher binding affinity for one target over phate, Bn benzyl, Cbz benzyloxycarboxyl, CF5 another. The term “fibrin specificity' refers to a fibrin binding NHS 5 carboxyfluorescein, succinimidyl ester (single moiety having a higher affinity for fibrin than for an irrelevant isomer), Cha-2-Cyclohexyl-L-, DCC—dicyclo target. Binding specificity can be characterized by a dissocia hexylcarbodiimide, DCM Dichloromethane, Ddhh—12, tion equilibrium constant (K) or an association equilibrium 26-diamino-1,11-dioxo-3,6,9,16,19,22-hexaoxahexa constant(K) for the two tested target materials, or can be any cosanoyl, Dga—diglycolyl, 3-oxapentan-1,5-di-oyl, DIC measure of relative binding strength. N,N'-diisopropylcarbodiimide, DIEA N,N- 0032. The term “binding moiety” as used herein refers to Diisopropylethylamine, DMA-dimethylacetamide, any molecule capable of forming a binding complex with DMF Dimethylformamide, DMSO–Dimethyl sulfoxide, another molecule. “Fibrin binding moiety' is a binding moi DPPE—1,2-Dipalmitoyl-sn-glycero-3-phosphoethanola ety that forms a complex with a clot, soluble or insoluble mine, commonly also identified as dipalmitoylphosphati fibrin, or a soluble or insoluble fragment of fibrin having a dylethanolamine, DPPG-1,2-Dipalmitoyl-sn-glycero-3- structure or characteristic exhibited by fibrin but not fibrino phosphor-rac-(1-glycerol) sodium salt, commonly also gen. Included among Such soluble or insoluble fragments of identified as dipalmitoylphosphatidylglycerol, DPPS-1,2- fibrin are fragments defined as “fibrin-derived polypeptides. Dipalmitoyl-sn-glycero-phospho-L-, commonly also Fibrin-derived polypeptides, for the purposes of this inven identified as dipalmitoylphosphatidylserine, DSPA 12 tion will be used as a collective term for the DD, DD-dimer, Distearoyl-sn-glycero-phosphate Sodium salt, commonly and DD(E) polypeptides described herein. Such fibrin-de also identified as distearoylphosphatidic acid, DSPE—1.2- rived polypeptides are typically generated by proteolytic Distearoyl-sn-glycerol-3-phosphoethanolamine, commonly treatment of crosslinked fibrin but retain structural features also identified as distearoylphosphatidylethanolamine, unique to fibrin. DSPE-PG4-NH {1,2-Distearoyl-sn-glycero-3-phospho US 2010/0158814 A1 Jun. 24, 2010 ethanolamine-N-amino(polyethylene glycol)2000, DPPE-PG4-NH {1,2-Dipalmitoyl-sn-glycero-3-phos -continued phoethanolamine-N4amino(polyethylene glycol)2000, DSPE-PEG1000—distearoyl-glycero-phosphoethanola 3-letter 1-letter mine-N-methoxy (polyethylene glycol)1000, DSPS-1,2- histidine his H Distearoyl-sn-glycero-3-(phosphor-L-serine), commonly isoleucine ille I leucine leu L also identified as distearoylphosphatidylserine, DSG—disuc lysine lys K cinimidylglutarate, EDAC, 1-ethyl 3-(3-dimethylaminopro methionine let M pyl)carbodiimide HCl, EtOH ethanol, EtO—diethyl ether, phenylalanine phe F EtOAc—Ethyl acetate, Fmoc 9-Fluoroenylmethoxyloxy pro P carbonyl, Ffe4—L-4-Fluorophenylalanine, F34fe—L-3,4- serine Se S thr T difluorophenylalanine, Glut Glutaryl, pentan-1,5-di-oyl, tryptophan trp W HOAc—acetic acid, HOAt—1-hydroxy-7-azabenzotriazole, tyrosine tyr Y HPLC High performance liquid chromatography, Hypt4— val V trans-4-hydroxy-L-proline, Fmoc-J or Fmoc-Adoa-Fmoc 8-amino-3,6-dioxaoctanoic acid, HATU N-(Dimethy lamino)-1H-1,2,3-triazolo(4,5-b)pyridine-1-ylmethylene N-methylenemethanaminium hexafluorophosphate-N- DETAILED DESCRIPTION OF THE INVENTION oxide, HBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate, HOBt N 0041. The present invention provides novel binding moi Hydroxybenzotriazole, ivDde—(4. 4-dimethyl-2, eties for fibrin. Such binding moieties make possible the 6-dioxocyclohex-1-ylidene)-3-methylbutyl, MALDI-Ma efficient detection, imaging and localization of fibrin or trix Assisted Laser Desorption Ionization, Neg. ion-Nega fibrin-derived peptides in a solution or system that contains tive ion, MeOH methanol, MS mass spectrum, NHS N fibrin or fibrin-derived polypeptides. In particular, the bind hydroxySuccinimide, NMM-N-Methylmorpholine, ing moieties of this invention, when appropriately labeled, are NMP N-Methylpyrrolidone, PEG polyethylene glycol (if useful for detecting, imaging and localizing fibrin-containing followed by a number, e.g. PEG4000, this identifies the thrombi or other fibrin specific pathophysiologies, and can approximate mean molecular weight of the polydispersed thus be used to form a variety of diagnostic and therapeutic PEG polymer, i.e. about 4000 daltons in the example), PFE— agents for diagnosing and treating pathological conditions perfluoroethanol, Pip Piperidine, Pd/C palladium on car associated with, for example, angiogenesis, thrombosis and bon catalyst, Pd(PPh.) Tetrakis(triphenyl-phosphine)pal cancer, particularly Solid tumors. The preferred binding moi ladium(0), Pos. Ion Positive ion, PyBOP benzotriazol-1- eties of the present invention bind fibrin and/or fibrin-derived yl-oxytripyrrolidinophosphonium hexafluorophosphate, polypeptides with high affinity, i.e., acting at low, physiologi Pyr pyridine, to Retention time (minutes), Reagent B cally relevant concentrations, comparable to known anti-fi (TFA:HO:phenol:triisopropylsilane, 88.5:5:2), SATA S brin antibodies and other fibrin-binding and repre Acetylthiolacetyl, S(Galnac)—O-(2-Acetamido-2-deoxy-C.- sent an improvement over previously known fibrin binding D-galactopyranosyl)-L-serine, SPPS solid phase peptide moieties. synthesis, Stearate—sodium Stearate, Su Succinimidyl, 0042 Utilizing the techniques described below (including SuO Succinimidyloxy, t-Bu—tert-Butyl, TEA Triethy techniques described in the Examples section), the polypep lamine. Thf2ca Tetrahydrofuran-2-carboxylic acid, TFA tides shown in Table 1 and in Table 2 were unexpectedly Trifluoroacetic Acid, TIPS Triisopropylsilane, 9-fluorenyl found to have Superior fibrin-specific binding along with methyloxycarbonyl (fmoc or Fmoc). Ttda—4,7,10 excellent physical properties as compared to previously trioXatridecane-1,13-diamino, Tuda—3,6,9- known peptides. In particular, substitution of Ala for Trp at Trioxaundecane-1,11-di-oyl, Alloc-Allyloxycarbonyl, position 6 of a previously known peptide, Boc tert-Butoxycarbonyl, DSG—Di-N-hydroxysuccinim Ac-WQPC*PWESWTFC*WDPGGGK-NH. (SEQ ID NO. idyl-glutarate, PEG3400-NHS Polyethyleneglycol 3400 122), resulted in improved potency as well as a more hydro N-hydroxysuccinimidyl ester, Pmc 2,2,5,7,8-pentamethyl philic peptide as demonstrated by HPLC. The improved prop chroman-6-sulfonyl, Trt Trity1, DMAC dimethylaceta erties of this substitution were unexpected as replacing either mide. of the other Trp residues in the peptide lead to no or reduced fibrin binding. ABBREVIATIONS FOR AMINO ACIDS 0043. Another method that was used to modify the peptide Ac-WQPC*PWESWTF*CWDPGGGK-NH. (SEQ ID NO. 0040 122), was attaching amino acids at the N- or C-terminus. In general attachment of amino acids at the C-terminus did not dramatically change the potency of the peptides. However Amino Acid 3-letter 1-letter when polar amino acids (such as Arg) were added to the N-terminus, improvement in binding was observed. Consid alanine ala A. arginine arg R ering the importance of the Trp at position 1, the introduction asparagine 8Sl N of polarity with beneficial effects was unexpected. aspartic acid asp D cys C 0044 Another modification that led to improvements in glutamic acid glu E potency was the introduction of the unusual amino acid cyclo glutamine gln Q hexylalanine (Cha) for Phe at position 11. Considering that gly G substituted phenylalanine derivatives led to weaker binding, it was not anticipated that changing to the more bulky, less US 2010/0158814 A1 Jun. 24, 2010

aromatic residue would improve potency. The combination of encompasses fibrin-binding moieties with or without the these three modifications led to further improved potency. GGGK linker and/or the Ac-group, as well as such moieties 0045 Table 1 below provides for each of the fibrin-bind with a different or additional linker, such as those described ing peptides of the invention, its sequence, the sequence of the herein. Note that for Seq005 peptides were prepared with fibrin-binding moiety prepared and tested, analytical data for additional linkers: Ac-WQPCPAESWTFCWDPGSAGSK these peptides (including HPLC data and mass spectral data) NH. (Seq005-P2) (SEQ ID NO. 134) (HPLC Data: System and, for most of the peptides, binding affinity measurements D, to 3.53; Mass Spectral Data: Neg. ion: M-H:2377.8, compared to a previously known fibrin-binding peptide hav M-2H/2:1188.4 including linker GSAGSK (SEQID NO. ing the sequence Ac-WQPC*PWESWTFC*WDPGGGK 137) and Ac-WQPC*PAESWTFC*WDPGAGSGK-NH. NH. (SEQ ID NO. 122) (relative ICs-1). A relative ICso (Seq005-P3) (SEQ ID NO. 135) HPLC Data: System D, to lower than 1 indicates better binding than the comparative 3.54; Mass Spectral Data: Neg. ion: M-H:2348.1, M-2H/ peptide. 2:1173.4), including linker GAGSGK (SEQID NO. 138). As 0046) Note that the fibrin-binding moieties prepared and shown in Example 21, the alternative linkers did not compro tested included the linker GGGK at the C terminus and in mise the ability of targeted microvesicles with Seq005 to bind Some cases an Ac-group at the N terminus. The invention to fibrin.

TABLE 1 Fibrin-binding peptides Mass Spectral Rel ICso (n = 2, Prepared Prepared HPLC Data Data Competition FP Seq. ID Sequence Sequence Sequence ID (System, t) (Mode: Ions) assay) Seq005 WQPC*PAES Ac Seq005-P B, 4.93 Neg. ion: M-H: 0.875 WTFC: WDP WQPCPAESW 2189.6; M - 2H/2: (SEQID NO. 1) TFC*WDPGGG 1094.4 K-NH2, (SEQID NO. 2) Seq014-P A, 4.70 Neg. ion: M + Na - 2H): O.235 2636.8; M - 2H/ 2: 1306.2

Seq015-P A, 4.44 Pos. ion: O.370 2M + 3H]/3: 1756.9; M + 2H/2: 1317.3; M+3H]/3: 876.1 Seq016-P B, 4.08 Neg. ion: M-H: 1.04 2160.2: M - 2H/2: 1180.5: M-3H]/3: 786.8 Seq017-P B, 3.96 Neg. ion: M-H: O.714 2738.7; M-2H/2: 1369.1

Seq018-P B, 4.27 Neg. ion: M-H: O494 2311.8; M - 2H/2: 1154.8

Seq019-P B, 4.26 Neg. ion: M-H: O.6O1 2322.6; M - 2H/2: 1160.4

Seq020-P B, 4.33 Neg. ion: M-H: O428 2341.2: M - 2H/2: 1169.8

Seq021-P B, 3.94 Neg. ion: M-H: O622 2319.6; M - 2H/2: 1159.3

Seq022-P B, 3.79 Pos. ion O.S11 IM+ 2H/2: 1239.3: IM+ 3H/3: 826.8

US 2010/0158814 A1 Jun. 24, 2010

TABLE 1-continued Fibrin-binding peptides

Mass Spectral Rel ICso (n = 2, Prepared Prepared HPLC Data Data Competition FP Seq. ID Sequence Sequence Sequence ID (System, t) (Mode: Ions) assay) Seq050 GWQPC*PAE GWQPC*PAE Seq050-P D, 3.92 Neg. ion: M - H: S SWT-Cha- WT-Cha 2294.7, M - 2H/2: C*WDP (SEQ C*WDPGGGK 1146.9 (as Aloc D NO. 71) NH-i (SEQ ID peptide) NO. 72) Seq051 RGWQPC*PW Ac- Seq051-P D, 3.64 Neg. ion: M - H: ESWTFC*WD RGWQPC*PWE 2517.9, M-2H/2: P (SEQID SWTFC*WDPG 1258.8, M + TFA NO. 73) GGK-NH. (SEQ 2H/2: 1315.5 ID NO. 74) Seq052 RGWQPC*PA Ac- Seq052-P D, 3.46 Neg. Ion: M-H): ESWTFC*WD RGWQPC*PAE 2403.3, M - 2H/2: P (SEQID SWTFC*WDPG 1200.9 NO. 75) GGK-NH. (SEQ ID NO. 76) Seq053 RGWQPC*PA Ac- Seq053-P D, 3.48 Neg. ion: M - H: ESWT-Cha- RGWQPC*PAE 2409.0, M - 2H/2: C*WDP (SEQ SWT-Cha 1204.1, M + TFA D NO. 77) C*WDPGGGK 2H/2: 1261.1 NH, (SEQID NO. 78) Seq054 RGWQPC*PA RGWQPC*PAE Seq054-P D, 3.91 Neg. ion: M - H: ESWT-Cha- SWT-Cha 2451.0, M - 2H/2: C*WDP (SEQ C*WDPGGGK 1224.7, M + TFA D NO. 79) NH. (SEQID 2H/2: 1282.2 (as NO.80) Aloc peptide) Seq055 GWQPC*PAE Ac- Seq055-P D, 3.59 Neg. ion: M-H): SWT-Cha- GWQPC*PAES 2253.1, M-2H/2: C*WDP (SEQ WT-Cha 1125.9 ID NO. 81) C*WDPGGGK NH. (SEQID NO. 82) Seq056 RWQPC*PAES RWQPC*PAES Seq056-P D, 3.71 Neg. ion: M - H: WTFC: WDP WTFC*WDPGG 2388.0, M - 2H/2: (SEQID GK-NH-i (SEQ 11934, M+ TFA NO. 83) ID NO. 84) 2H/2: 1250.8 (as Aloc peptide) Seq057 RWQPC*PAES RWQPC*PAES Seq057-P D, 3.71 Neg. ion: M - H: WT-Cha- WT-Cha 2394.0, M - 2H/2: C*WDP (SEQ C*WDPGGGK 1196.4, M+ TFA ID NO. 85) NH-i (SEQID 2H/2: 1252.9 (as NO. 86) Aloc peptide) = Analytical data reported for peptide bearing the ivDde group on N of all lysine groups of the peptide except for the C-terminal lysine. = Analytical data reported for N-terminal Alloc-protected peptide. S = Direct binding assay conducted using CF5 labeled peptide, see table 2

0047. The details of the HPLC systems used are set forthin 0049. As used in Table 1 and elsewhere herein, the desig the Examples section. Details of fibrin-binding assays are nation “C” refers to a cysteine residue that contributes to a also set forth in the Examples section. disulfide bond. 0050. As shown in Table 1, all of the peptides described 0048 Changes from the known peptide, therein have equivalent or far Superior binding than the com Ac-WQPC*PWESWTFC*WDPGGGK-NH. (SEQ ID NO. parative peptide. 122), also referred to herein as Seq000, are underlined, but 0051 Table 2 below provides for each of the 5-carboxy omissions from this sequence (e.g. initial Ac, etc.) are not fluorescein labeled fibrin-binding peptides included therein, its sequence, HPLC data, mass spectral data and, for most of highlighted. Thus, for example, within the peptide of the the peptides, binding affinity measurements compared to the invention coded as Seq005, the amino acid therein defined as previously known fibrin-binding peptide having the sequence 'A' replaces, in that same position, the corresponding amino Ac-WQPC*PWESWTFC*WDPGGGK-NH. (Seq000) acid “W' in the prior art peptide, Seq000. (SEQID NO. 122).

US 2010/0158814 A1 Jun. 24, 2010 12

TABLE 2 - continued Fibrin Binding Peptides - 5 - Carboxyfluorescein Labeled Peptides HPLC Data K (uM) (System, Mass Spectral Data Direct Binding Seq. ID Sequence tR) (Mode; Ions) (n = 2) Seqo 56 - RWQPC*PAESW D, 3.81 Neg. ion; M - H: CFs TEC kWDPGGGK 2663. 1, 2M - 3H/3: (CF5) -NH, 1774. 2, M - 2H/2: (SEQ ID NO. 12O) 1330.3 Seq057 - RWQPC*PAESW D, 3.91 Neg. ion; M - H: CFs T-Cha- 2668. 2, M - 2H/2: CkWDPGGGK 1333.3 (CF5) -NH, (SEQ ID NO. 121)

0052. The details of the HPLC systems used are set forthin equivalentor far Superior binding than the comparative fibrin the Examples section. Details of fibrin-binding assays are binding peptide. also set forth in the Examples section. 0054 Table 3 below, provides residue abbreviations and 0053 As shown in Table 2, all of the 5-carboxyfluorescein the corresponding structure of Some of the commonly used labeled fibrin-binding peptides described therein have residues referenced in Table 1 and Table 2 above.

TABLE 3

Abbreviations for Residues

Residue Residue Abbreviation Structure Abbreviation Structure

Cha O S(Galnac) OH b. O O O NH OH N H. OOH NH

Ffe4 O Thf2ca O O

HN F -

F34fe O HyptA HO

O N N F H

F

Btn CF5 HO O OH

C O O US 2010/0158814 A1 Jun. 24, 2010

0055 Direct synthesis of the fibrin-binding peptides of the polypeptide sequence with the expectation that the resulting invention may be accomplished using conventional tech polypeptides would have a similar or improved profile of the niques, including Solid-phase peptide synthesis, Solution properties described above: phase synthesis, etc. Solid-phase synthesis is preferred. See 0063 Substitution of alkyl-substituted hydrophobic Stewartet al., Solid-Phase Peptide Synthesis (W. H. Freeman amino acids: Including alanine, leucine, isoleucine, Valine, Co., San Francisco, 1989); Merrifield, J. Am. Chem. Soc., norleucine, S-2-aminobutyric acid, S-cyclohexylalanine or 85:2149-2154 (1963); Bodanszky and Bodanszky, The Prac other simple alpha-amino acids Substituted by an aliphatic tice of Peptide Synthesis (Springer-Verlag, New York, 1984), side chain from 1-10 carbons including branched, cyclic and incorporated herein by reference. straight chain alkyl, alkenyl or alkynyl Substitutions. 0056. A fibrin-binding peptide according to the present 0064 Substitution of aromatic-substituted hydrophobic invention is preferably purified once it has been isolated or amino acids: Including phenylalanine, tryptophan, tyrosine, synthesized. For purification purposes, there are many stan biphenylalanine, 1-naphthylalanine, 2-naphthylalanine, dard methods that may be employed, including reversed 2-benzothienylalanine, 3-benzothienylalanine, histidine, phase high-pressure liquid chromatography (RP-HPLC) amino, alkylamino, dialkylamino, aza, halogenated (fluoro, using an alkylated silica column such as C-, C- or Cs chloro, bromo, or iodo) or alkoxy (from C-C)-substituted silica. A gradient mobile phase of increasing organic content forms of the previous listed aromatic amino acids, illustrative is generally used to achieve purification, for example, aceto examples of which are: 2-, 3-, or 4-aminophenylalanine, 2-, nitrile in an aqueous buffer, usually containing a small 3-, or 4-chlorophenylalanine, 2-, 3-, or 4-methylphenylala amount of trifluoroacetic acid. Ion-exchange chromatogra nine, 2-, 3-, or 4-methoxyphenylalanine, 5-amino-, 5-chloro-, phy can also be used to separate peptides based on their 5-methyl- or 5-methoxytryptophan, 2-, 3-, or 4-amino-, 2-, charge. The degree of purity of the polypeptide may be deter 3'-, or 4-chloro-, 2, 3, or 4-biphenylalanine, 2-, 3-, or 4'-me mined by various methods, including identification of a major thyl-2-, 3- or 4-biphenylalanine, and 2- or 3-pyridylalanine. large peak on HPLC. A polypeptide that produces a single 0065. Substitution of amino acids containing basic func peak that is at least 95% of the input material on an HPLC tions: Including arginine, lysine, histidine, ornithine, 2.3- column is preferred. Even more preferable is a polypeptide diaminopropionic acid, homoarginine, alkyl, alkenyl, or aryl that produces a single peak that is at least 97%, at least 98%, Substituted (from C-C branched, linear, or cyclic) at least 99% or even 99.5% or more of the input material on an derivatives of the previous amino acids, whether the substitu HPLC column. ent is on the heteroatoms (such as the alpha nitrogen, or the 0057. In order to ensure that the fibrin-binding peptide distal nitrogen or nitrogens, or on the alpha carbon, in the obtained is the desired peptide for use in compositions of the pro-R position for example. Compounds that serve as illus present invention, analysis of the peptide composition may be trative examples include: N-epsilon-isopropyl-lysine, 3-(4- carried out. Such composition analysis may be conducted tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine, using high resolution mass spectrometry to determine the N.N-gamma, gamma-diethyl-homoarginine. Included also molecular weight of the peptide. Alternatively, the amino acid are compounds such as alpha methyl arginine, alpha methyl content of the peptide can be confirmed by hydrolyzing the 2,3-diaminopropionic acid, alpha methyl histidine, alpha peptide in aqueous acid, and separating, identifying and methyl ornithine where alkyl group occupies the pro-R posi quantifying the components of the mixture using HPLC, oran tion of the alpha carbon. Also included are the amides formed amino acid analyzer. Protein sequencers, which sequentially from alkyl, aromatic, heteroaromatic (where the heteroaro degrade the peptide and identify the amino acids in order, may matic group has one or more nitrogens, oxygens or Sulfur also be used to determine definitely the sequence of the pep atoms singly or in combination) carboxylic acids or any of the tide. many well-known known activated derivatives such as acid chlorides, active esters, active azolides and related deriva 0058. The fibrin binding polypeptides of the invention tives) and lysine, ornithine, or 2,3-diaminopropionic acid. may be conformationally restrained by disulfide linkages 0066. Substitution of acidic amino acids: Including aspar between the two cysteine residues in their sequence. This tic acid, glutamic acid, homoglutamic acid, tyrosine, alkyl, conformational restraint ensures that the peptides have a aryl, aralkyl, and heteroaryl Sulfonamides of 2,3-diaminopro binding structure that contributes to the peptides affinity for pionic acid, ornithine or lysine and tetrazole-substituted alkyl fibrin and their specificity for fibrin over fibrinogen. Other amino acids. methods for constraining peptides which would retain a simi 0067 Substitution of side chain amide residues: Including lar conformation and fibrin specificity for the peptide have asparagine, glutamine, and alkyl or aromatic Substituted been described in the art and may be used herein. derivatives of asparagine or glutamine. 0059 Modification or Optimization of Binding Polypep 0068 Substitution of hydroxyl containing amino acids: tides Including serine, threonine, homoserine, 2,3-diaminopropi 0060 Modification or optimization of the fibrin-binding onic acid, and alkyl or aromatic Substituted derivatives of polypeptides is within the scope of the present invention. serine or threonine. Specifically, a polypeptide sequence of the invention can be 0069. It is also understood that the amino acids within further modified to optimize its potency, pharmacokinetic each of the categories listed above may be substituted for behavior, stability and/or other biological, physical and another of the same group. chemical properties. 0070. Substitution of Amide Bonds 0061 Substitution of Amino Acid Residues 0071 Another type of modification within the scope of the 0062. Substitutions of amino acids within the same class invention is the substitution of amide bonds within the back (e.g., Substituting one basic amino acid for another) are well bone of a binding polypeptide. For example, to reduce or known in the art. For example, one can make the following eliminate undesired proteolysis, or other degradation path isosteric and/or conservative amino acid changes in the parent ways which diminish serum stability, resulting in reduced or US 2010/0158814 A1 Jun. 24, 2010

abolished bioactivity, or to restrict or increase conformational in manual or automated Solution phase peptide synthesis. flexibility, it is common to substitute amide bonds within the Similarly D- or L-N'-(2-acetamido-2-deoxy-3,4,6-tri-O- backbone of the peptides with functionality that mimics the acetyl-B-D-glucopyranosyl)-asparagine can be employed. existing conformation or alters the conformation in the man The use of amino acids glycosylated on a pendant oxygen, ner desired. Such modifications may produce increased bind nitrogen or Sulfur function by the agency of Suitably func ing affinity or improved pharmacokinetic behavior. It is tionalized and activated carbohydrate moieties that can be understood that those knowledgeable in the art of peptide employed in glycosylation is anticipated. Such carbohydrate synthesis can make the following amide bond-changes for functions could be monosaccharides, disaccharides or even any amide bond connecting two amino acids with the expec larger assemblies of oligosaccharides (Kihlberg, January tation that the resulting peptides could have the same or (2000) Glycopeptide synthesis. In: Fmoc Solid Phase Peptide improved activity: insertion of alpha-N-methylamides or Synthesis—A Practical Approach (Chan, W. C. and White, P. peptide amide backbone thioamides, removal of the carbonyl D. Eds) Oxford University Press, New York, N.Y. Chap. 8, pp to produce the cognate secondary amines, replacement of one 195-213). amino acid with an aza-aminoacid to produce semicarbazone 0078. Also anticipated is the appendage of carbohydrate derivatives, and use of E-olefins and substituted E-olefins as functions to amino acids by means other than glycosylation amide bond Surrogates. via activation of a leaving group at the anomeric carbon. 0072 Introduction of D-Amino Acids Linkage of the amino acid to the glycoside is not limited to the 0073. Another approach within the scope of the invention formation of a bond to the anomeric carbon of the carbohy is the introduction of D-alanine, or another D-amino acid, drate function. Instead, linkage of the carbohydrate moiety to distal or proximal to a labile peptide bond. In this case it is the amino acid could be through any Suitable, Sufficiently also understood to those skilled in the art that such D-amino reactive oxygen atom, nitrogen atom, carbon atom or other acid substitutions can, and at times, must be made, with pendant atom of the carbohydrate function via methods D-amino acids whose side chains are not conservative employed for formation of C-heteroatom, C C or heteroa replacements for those of the L-amino acid being replaced. tom-heteroatom (examples are S. S. O. N. N. N. P. O. This is because of the difference in chirality and hence side P N) bonds known in the art. chain orientation, which may result in the accessing of a 007.9 Formation of Salts previously unexplored region of the binding site of the target 0080. It is also within the scope of the invention to form which has moieties of different charge, hydrophobicity, steric different salts that may increase the water solubility or the requirements, etc., than that serviced by the side chain of the ease of formulation of these peptides. These may include, but replaced L-amino acid. are not restricted to, N-methylglucamine (meglumine), 0074) Modifications to Improve Pharmacokinetic or Phar acetate, oxalates, ascorbates etc. macodynamic Properties 0081 Structural Modifications which Retain Structural 0075. It is also understood that use of the binding moieties Features of the invention in a particular application may necessitate I0082. Yet another modification within the scope of the modifications of the peptide or formulations of the peptide to invention is truncation of cyclic polypeptides. The cyclic improve pharmacokinetic and pharmacodynamic behavior. It nature of many polypeptides of the invention limits the con is expected that the properties of the peptide may be changed formational space available to the peptide sequence, particu by attachment of moieties anticipated to bring about the larly within the cycle. Therefore truncation of the peptide by desired physical or chemical properties. Such moieties affect one or more residues distal or even proximal to the cycle, at ing the pharmacokinetic and pharmacodynamic behavior either the N-terminal or C-terminal region may provide trun may be appended to the peptide using acids or amines, via cated peptides with similar or improved biological activity. A amide bonds or urea bonds, respectively, to the N- or C-ter unique sequence of amino acids, even as Small as three amino minus of the peptide, or to the pendant amino group of a acids, which is responsible for the binding activity, may be Suitably located lysine or lysine derivative, diaminopropionic identified, as noted for RGD peptides. See e.g., E. F. Plow et acid, ornithine, or other amino acid in the peptide that pos al., Blood (1987), 70(1), 110-5: A. Oldberg et al., Journal of sesses a pendant amine group or a pendant alkoxyamino or Biological Chemistry (1988), 263(36), 19433-19436; R. hydrazine group. The moieties introduced may be groups that Taub et al., Journal of Biological Chemistry (1989 Jan. 5), are hydrophilic, basic, or nonpolar alkyl or aromatic groups 264(1), 259-65; A. Andrieux et al., Journal of Biological depending on the peptide of interest and the extant require Chemistry (1989 Jun. 5), 264(16), 9258-65; and U.S. Pat. ments for modification of its properties. Nos. 5,773,412 and 5,759,996, each of which is incorporated 0076 Glycosylation of Amino Acid Residues herein by reference in its entirety. 0077. Yet another modification within the scope of the I0083. It has also been shown in the literature that large invention is to employ glycosylated amino acid residues (e.g. peptide cycles can be substantially shortened, eliminating serine, threonine or asparagine residues), singly or in combi extraneous amino acids, but Substantially including the criti nation in either the binding moiety or the linker moiety or cal binding residues. See U.S. Pat. No. 5,556,939, which is both. Glycosylation, which may be carried out using standard incorporated herein by reference in its entirety. Shortened conditions, may be used to enhance solubility, alter pharma cyclic peptides can be formed using disulfide bonds or amide cokinetics and pharmacodynamics or to enhance binding via bonds of Suitably located carboxylic acid groups and amino a specific or non-specific interaction involving the glycosidic groups. moiety. In another approach glycosylated amino acids such as 0084. Furthermore, D-amino acids can be added to the O-(2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-B-D-glucopyra peptide sequence to stabilize turn features (especially in the nosyl) serine or the analogous threonine derivative (either the case of glycine). In another approach alpha, beta, gamma or D- or L-amino acids) may be incorporated into the peptide delta dipeptide or turn mimics (such as C, B, Y, or ö turn during manual or automated Solid phase peptide synthesis, or mimics) some of which are shown in structures 1, 2 and 3. US 2010/0158814 A1 Jun. 24, 2010

below, can be employed to mimic structural motifs and turn retaining the activity and other desired properties of the bind features in a peptide and simultaneously provide stability ing polypeptides used in the invention: from proteolysis and enhance other properties such as, for 0087 Oxime Linker example, conformational stability and solubility (structure 1: I0088. The oxime moiety has been employed as a linker by Hart et al., J. Org. Chem., 64, 2998-2999(1999); structure 2: investigators in a number of contexts. Of the most interest is Hanessian et al., “Synthesis of a Versatile Peptidomimetic the work by Wahl, F and Mutter, M, Tetrahedron Lett. (1996) Scaffold' in Methods in Molecular Medicine, Vol. 23: Pepti 37,6861-6864). The amino acids containing an aminoalcohol domimetics Protocols, W. M. Kazmierski Ed. (Humana Press function (4), and containing analkoxyamino function (5), are Inc. Totowa N.J. 1999), Chapter 10, pp. 161-174; structure 3: incorporated into the peptide chain, not necessarily at the end WO O1 (16135. of the peptide chain. After formation of the peptide, the sidechain protecting groups are removed. The aldehyde group is unmasked and an oxime linkage is formed.

Fmoc-NH COOH

HN1 Trt

Fmoc-NH OCOOH 0085. Substitution of Disulfide Mimetics I0086 Also included within the scope of the invention is the substitution of disulfide mimetics for disulfide bonds 0089 Lanthionine Linker within the binding polypeptides of the invention. For disul 0090 Lanthionines are cyclic sulfides, wherein the disul fide-containing peptides of the invention, the disulfide bonds fide linkage (S S) is replaced by a (C S) linkage. Thus the might need to be replaced to avoid certain difficulties that are lability to reduction is far lower and this linkage should be sometimes posed by the presence of a disulfide bond. For stable to Stannous chloride. Lanthionines may be prepared by example, when generating '"Tc (or other radionuclide)- a number of methods. based radiopharmaceuticals or certain other constructs with 0091 Preparation of Lanthionines Using Bromoacety binding peptides of the invention, the presence of the disulfide lated Peptides bond can be a significant problem. The integrity of the disul 0092 Lanthionines are readily prepared using known fide bond is difficult to maintain during procedures designed methods. See, for example, Robey et al. (Robey, F. A. and to incorporate’"Tc via routes that are reliant upon the reduc Fields, R. L. Anal. Biochem. (1989) 177, 373-377) and tion of pertechnetate ion and Subsequent incorporation of the Inman, et al. (Inman, J. K. Highet, P. F.; Kolodny, N.; and reduced Tc species into Substances bearing Tc-compatible Robey, F. A. Bioconjugate Chem. (1991) 2, 458–463; Ploin chelating groups. This is because the disulfide bond is rather sky, A. Cooney, M. C. Toy-Palmer, A. Osapay, G. and Good easily reduced by the reducing agents commonly used in kits man, M.J. Med. Chem. (1992) 35, 4185-4194; Mayer, J. P.: devised for one-step preparation of radiopharmaceuticals. Zhang, J.; and Liu, C. F. in: Tam, J. P. and Kaumaya, P. T. P. Therefore, the ease with which the disulfide bond can be (eds), “Peptides, Frontiers of Peptide Science.” Proceedings reduced during Tc chelation may require Substitution with of the 15" American Peptide Symposium, June 14-19 Nash mimetics of the disulfide bonds. Accordingly, another modi ville, Tenn. Klumer Academic Pub. Boston. pp. 291-292;. fication within the scope of the invention is to substitute the Wakao, Norihiro; Hino, Yoichi: Ishikawa, Ryuichi. Jpn. disulfide moiety with mimetics, utilizing the methods dis Kokai Tokkyo Koho (1995), 7 pp.JP 07300452A2 19951114 closed herein or known to those skilled in the art, while Heisei; JP 95-49692 19950309; JP 94-41458 1994.0311 have US 2010/0158814 A1 Jun. 24, 2010

published in this area. Preparation of peptides using Boc (eds), Jun. 14-19, 1995, Nashville, Tenn. (Klumer Academic automated peptide synthesis followed by coupling the pep Pub. Boston) pp. 291-292. Conjugate addition of thiols to tide terminus with bromoacetic acid gives bromoacetylated acrylamides has also been amply demonstrated and a refer peptides in good yield. Cleavage and deprotection of the ence to the addition of 2-mercaptoethanol to acrylamide is peptides is accomplished using HF/anisole. If the peptide contains a cysteine group its reactivity can be controlled with provided. Wakao et al., Jpn. Kokai Tokkyo Koho, JP low pH. If the pH of the medium is raised to 6-7, then either 07300452A2 (1995). polymerization or cyclization of the peptide takes place. 0.096 Diaryl Ether or Diarylamine Linkage Polymerization is favored at high (100 mg/mL) concentra 0097 Diaryl Ether Linkage from Intramolecular Cycliza tion, whereas cyclization is favored at lower concentrations (1 tion of Aryl Boronic Acids and Tyrosine mg/mL), e.g., in Scheme 1 below, 6 cyclizes to 7. 0098. The reaction of arylboronic acids with phenols, amines and heterocyclic amines in the presence of cupric acetate, in air, at ambient temperature, in dichloromethane using either pyridine or triethylamine as a base to provide R2 unsymmetrical diaryl ethers and the related amines in good yields (as high as 98%) has been reported. See, Evans et al., R1 O s Tetrahedron Lett., 39:2937-2940 (1998); Chan et al., Tetra hedron Lett., 39:2933-2936 (1998); Lam et al., Tetrahedron N NH Lett., 39:2941-2944 (1998). In the case of N-protected O N tyrosine derivatives as the phenol component the yields were R4 NH w s ~1 mg/mL. also as high as 98%. This demonstrates that amino acid Hess amides (peptides) are expected to be stable to the transforma N SH NH2 tion and that yields are high. Precedent for an intramolecular H H reaction exists in view of the facile intramolecular cycliza N N tions of peptides to lactams, intramolecular biaryl ether for O mation based on the SAr reaction and the generality of O O intramolecular cyclization reactions under high dilution con Br ditions or on resin, wherein the pseudo-dilution effect mimics 6 high dilution conditions. R2 0099 Formation of Cyclic Peptides with a Lactam Link R1 O O age via Intramolecular Native Chemical Ligation

Scheme 2 - Formation of Cyclic Peptides with a Thiazolidine Linkage via I Reaction of Pepti ifth Cysteine Moief

NH2

s1 S O gNg, Ya ON HeNaIO4 N O 0093. Inman et al. demonstrated the use of N'-(Boc)-N'- N-(bromoacetyl)-3-alanyl-L-lysine as a carrier of the bro HN moacetyl group that could be employed in Boc peptide Syn thesis thus allowing placement of a bromoacetyl bearing HO moiety anywhere in a sequence. In preliminary experiments 8 they found that peptides with 4-6 amino acids separating the bromoacetyl-lysine derivative from a cysteine tend to cyclize, indicating the potential utility of this strategy. -( OOC 0094 Preparation of Lanthionines via Cysteine Thiol 1. O n-\, COO Addition to Acrylamides 0095 Several variants of this strategy may be imple HN (< foc1)\/ mented. Resin-bound serine can be employed to prepare the lanthionine ring on resin either using a bromination-dehydro N O bromination-thiol addition sequence or by dehydration with disuccinimidyl carbonate followed by thiol addition. Ploin sky et al., M.J.Med. Chem., 35:4185-4194 (1992); Mayer et O H al., “Peptides, Frontiers of Peptide Science', in Proceedings 9 of the 15" American Peptide Symposium, Tam & Kaumaya US 2010/0158814 A1 Jun. 24, 2010 17

formed by the residues in the main chain, and the second ring -continued being the thiazolidine ring. Scheme 2, above, provides an example. The required aldehyde function can be generated by Sodium metaperiodate cleavage of a Suitably located vicinal aminoalcohol function, which can be present as an unpro tected serine tethered to the chain by appendage to a side chain amino group of a lysine moiety. In some cases, the required aldehyde function is generated by unmasking of a N protected aldehyde derivative at the C-terminus or the N-ter HN O minus of the chain. An example of this strategy is found in: Botti, P.; Pallin, T.D. and Tam, J. P. J. Am. Chem. Soc. 1996, O, N 118, 10018-10034. O 0101 Lactams Based on Intramolecular Cyclization of Pendant Amino Groups with Carboxyl Groups on Resin 10 0102 Macrocyclic peptides can be prepared by lactam formation by either head to tail or by pendant group cycliza H tion. The basic strategy is to prepare a fully protected peptide N wherein it is possible to remove selectively an amine protect H ing group and a carboxy protecting group. Orthogonal pro S tecting schemes have been developed. Of those that have been N developed, the allyl, trity1 and Dde methods have been employed most. See, Mellor et al., “Synthesis of Modified Peptides in Fmoc Solid Phase Synthesis: A Practical Approach, White and Chan (eds) (Oxford University Press, New York, 2000), Chapt. 6, pp. 169-178. The Dde approach is of interest because it utilizes similar protecting groups for both the carboxylic acid function (Dmab ester) and the amino group (Dde group). Both are removed with 2-10% hydrazine 0100 Another approach that may be employed involves in DMF at ambient temperature. Alternatively, the Dde can be intramolecular cyclization of Suitably located vicinal amino used for the amino group and the allyl group can be used for mercaptain functions (usually derived from placement of a the carboxyl. cysteine at a terminus of the linear sequence or tethered to the 0103) A lactam function, available by intramolecular cou sequence via a side-chain nitrogen of a lysine, for example) pling via standard peptide coupling reagents (such as HATU, and aldehyde functions to provide thiazolidines which result PyBOP etc), could act as a surrogate for the disulfide bond. in the formation of a bicyclic peptide, one ring of which is that The Dde/Dmab approach is shown in Scheme 3a, below. US 2010/0158814 A1 Jun. 24, 2010 18

?I wolnol SI OOOOO ±NR-(@)NNNN HH O NH VHL

H N

ZI US 2010/0158814 A1 Jun. 24, 2010 19

0104 Thus, a linear sequence containing, for example, the Dde-protected lysine and Dmab ester may be prepared on a Tentagel-based Rink amide resin at low load (-0.1-0.2 mmol/ g). Deprotection of both functions with hydrazine is then followed by on-resin cyclization to give the desired products.

US 2010/0158814 A1 Jun. 24, 2010

0105. In the allyl approach, shown in Scheme 3b, the (21):5855-5856 (1995). One can prepare either C-allylated pendant carboxyl which is to undergo cyclization is protected amino acids or possibly N-allylated amino acids and employ as an allyl ester and the pendant amino group is protected as them in this reaction in order to prepare carba-bridged cyclic an alloc group. On resin, both are selectively unmasked by peptides as Surrogates for disulfide bond containing peptides. treatment with palladium tris-triphenylphosphine in the pres ence of N-methylmorpholine and acetic acid in DMF. 0110. One may also prepare novel compounds with ole Residual palladium salts are removed using sodium dieth finic groups. Functionalization of the tyrosine hydroxyl with yldithiocarbamate in the presence of DIEA in DMF, followed an olefin-containing tether is one option. The lysine e-amino by subsequent washings with DMF. The lactam ring is then group may be another option with appendage of the olefin formed employing HATU/HOAt in the presence of N-meth containing unit as part of an acylating moiety, for example. If ylmorpholine. Other coupling agents can be employed as instead the lysine side chain amino group is alkylated with an olefin containing tether, it can still function as a point of described above. The processing of the peptide is then carried attachment for a reporter as well. The use of 5-pentenoic acid out as described above to provide the desired peptide lactam. as an acylating agent for the lysine, ornithine, or diaminopro 0106 Subsequently cleavage from resin and purification pionic side chain amino groups is another possibility. The may also be carried out. For functionalization of the N-ter minus of the peptide, it is understood that amino acids, such as length of the olefin-containing tether can also be varied in trans-4-(iV-Dde)methylaminocyclohexane carboxylic acid, order to explore structure activity relationships. trans-4-(iV-Dde)methylaminobenzoic acid, or their alloc 0111 Manipulation of Peptide Sequences congeners could be employed. Yet another approach is to 0112. Other modifications within the scope of the inven employ the safety catch method to intramolecular lactam tion include manipulations of peptide sequences which can be formation during cleavage from the resin. expected to yield peptides with similar or improved biologi 0107 Cyclic Peptides Based on Olefin Metathesis cal properties. These include amino acid translocations (Swapping amino acids in the sequence), use of retro-inverso 0108. The Grubbs reaction (Scheme 4, below) involves the peptides in place of the original sequence or a modified origi metathesis/cyclization of olefin bonds and is illustrated as nal sequence, peptoids, retro-inverso peptoid sequences, and shown below. See, Schuster et al., Angewandte. Chem. Int. synthetic peptides. Structures wherein specific residues are Edn Engl., 36:2036-2056 (1997); Miller et al., J. Am. Chem. peptoid instead of peptidic, which result in hybrid molecules, Soc., 118:9606-9614 (1996). neither completely peptidic nor completely peptoid, are con templated as well. 0113. The peptides or conjugates of the invention may include one or more protecting groups on any appropriate es Ph amino or carboxylic groups or other appropriate functional C groups. Unless otherwise noted, the term “protecting group' c1 refers to a protective group adapted to preserve the character R R2 PCys R R2 istic chemical function of the functional group to which it is -- bound. For example, protecting groups may be used to pre serve amino or carboxyl functions and may include, for example, Fmoc, benzyl, benzyloxycarbonyl or alkyl esters or other groups commonly intended for protection of Such func 16 17 tions. Additional protecting groups are disclosed in, for Basic Grubbs reaction example, T. W. Green, Protective Groups in Organic Synthe /N = Optional tether sis (Wiley, N.Y. 1981). 0114. In appropriate circumstances the peptides or conju gates of the invention may also include a “deactivating 0109. It is readily seen that, if the starting material is a group', which refers to a chemical group that is able to diolefin (16), the resulting product will be cyclic compound chemically react with, for example, the N terminal ( NH2) 17. The reaction has in fact been applied to creation of cycles or C-terminal ( COOH) group of the peptide unit, trans from olefin-functionalized peptides. See, e.g., Pernerstorfer forming it through a chemical reaction, into a suitable deriva et al., Chem. Commun., 20:1949-50 (1997); Covalent capture tive thereofthat maintains the specificity of the corresponding and stabilization of cylindrical B-sheet peptide assemblies, peptide moiety toward fibrin, but is unable to chemically react Clarket al., Chem. Eur: J., 5(2):782-792 (1999); Highly effi with, respectively, a carboxyl or an amino functionality on a cient synthesis of covalently cross-linked peptide helices by different moiety, and thus may not be involved in carboxa ring-closing metathesis, Blackwell et al., Angew. Chem., Int. mido reactions. Such groups may include acetyl (also referred Ed., 37(23):3281-3284 (1998); Synthesis of novel cyclic pro to as CH3(CO)— or Ac), amino groups and derivatives tease inhibitors using Grubbs olefin metathesis, Ripka et al., thereof such as , for example, —NH2, NH(CH3), Med. Chem. Lett., 8(4):357-360 (1998); Application of Ring H2NOC CH2-NH . Closing Metathesis to the Synthesis of Rigidified Amino 0115 Multimeric constructs including fibrin-binding pep Acids and Peptides, Miller et al., J. Am. Chem. Soc., 118(40): tides of the invention may be prepared using known linkers 9606-96.14 (1996); Supramolecular Design by Covalent Cap and techniques, such as, for example, those set forth in co ture, Design of a Peptide Cylinder via Hydrogen-Bond-Pro pending U.S.S.N. (US 2005/0147555), incorporated herein moted Intermolecular Olefin Metathesis, Clark et al., J. Am. by reference in its entirety. In a preferred embodiment Chem. Soc., 117(49): 12364-12365 (1995); Synthesis of Con homodimers including fibrin-binding peptides may be pre formationally Restricted Amino Acids and Peptides Employ pared. Such dimeric compounds have increased avidity and ing Olefin Metathesis, Miller et al., J. Am. Chem. Soc., 117 thus exhibit better binding to fibrin. US 2010/0158814 A1 Jun. 24, 2010 22

0116. In the practice of the present invention, a determi fibrin in vitro or in vivo, and particularly for detection and/or nation of the affinity of the fibrin-binding moiety for fibrin imaging of fibrin clots and pathological angiogenic pro relative to fibrinogen is a useful measure, and is referred to as cesses. Any suitable method of assaying or imaging fibrin specificity for fibrin. Standard assays for quantitating binding may be employed. and determining affinity include equilibrium dialysis, equi I0120 For detection of fibrin or fibrin-derived polypep librium binding, gel filtration, or the monitoring of numerous tides in Solution, a binding moiety according to the invention spectroscopic changes (such as a change in fluorescence can be detectably labeled, e.g., fluorescently labeled, radio polarization) that may result from the interaction of the bind labeled or enzymatically labeled, then contacted with the ing moiety and its target. These techniques measure the con solution, and thereafter formation of a complex between the centration of bound and free ligand as a function of ligand (or binding moiety and the fibrin target can be detected. As an protein) concentration. The concentration of bound polypep example, a fluorescently labeled fibrin binding peptide may tide (Bound) is related to the concentration of free polypep be used for in vitro fibrin detection assays, wherein the pep tide (Free) and the concentration of binding sites for the tide is added to a solution to be tested for fibrin under condi polypeptide, i.e., on fibrin, (N), as described in the following tions allowing binding to occur. The complex between the equation: fluorescently labeled fibrin-binding peptide and fibrin can be detected and quantified by measuring the increased fluores Bound=NXFree/((1/K)+Free). cence polarization arising from the fibrin-bound peptide rela 0117. A solution of the data to this equation yields the tive to that of the free peptide. association constant, K, a quantitative measure of the bind I0121 Alternatively, a sandwich-type ELISA assay may be ing affinity. The association constant, K is the reciprocal of used, wherein a fibrin binding moiety is immobilized on a the dissociation constant, K. The K is more frequently Solid Support such as a plastic tube or well, then the Solution reported in measurements of affinity. A peptide having a K. suspected of containing fibrin or a fibrin-derived polypeptide 1.5 times higher for fibrinogen than for fibrin would be con is contacted with the immobilized binding moiety, non-bind sidered low-specificity fibrin binder. A peptide having a K. ing materials are washed away, and complexed polypeptide is 10 times greater for fibrinogen than fibrin would be a moder detected using a suitable detection reagent, Such as a mono ate-specificity fibrin binder, and a peptide having a K, 100 clonal antibody recognizing fibrin. The monoclonal antibody times or more greater for fibrinogen than for fibrin would be is detectable by conventional means known in the art, includ termed highly specific for fibrin. Preferably the peptides and ing being detectably labeled, e.g., radiolabeled, conjugated agents of the present invention have a K at least 1.5 times with an enzyme such as horseradish peroxidase and the like, higher for fibrinogen than for fibrin, more preferably at least or fluorescently labeled. 10 times higher, even more preferably at least 100 times, and 0.122 For detection or purification of soluble fibrin or most preferably at least 1000 times higher. Preferred fibrin fibrin-derived polypeptides in or from a solution, a binding binding polypeptides have a K, for fibrin in the range of 1 moiety of the invention can be immobilized on a solid sub nanomolar (nM) to 100 micromolar (LM) and includes K, strate Such as a chromatographic Support or other porous values of at least 10 nM, at least 20 nM, at least 40 nM, at least material, then the immobilized binder can be loaded or con 60 nM, at least 80 nM, at least 1 uM, at least 5uM, at least 10 tacted with the solution under conditions suitable for forma uM, at least 20 uM, at least 40 uM, at least 60 uM, and at least tion of a binding moiety/fibrin complex. The non-binding 80 uM. portion of the Solution can be removed and the complex may 0118 Where fibrin binding moieties are employed as be detected, e.g., using an anti-fibrin or anti-binding moiety imaging agents, other aspects of binding specificity may antibody, or the fibrin target may be released from the binding become more important: Imaging agents operate in a moiety at appropriate elution conditions. dynamic system in that binding of the imaging agent to the I0123. A particularly preferred use for the polypeptides target is not in a stable equilibrium state throughout the imag according to the present invention is for creating visually ing procedure. For example, when the imaging agent is ini readable images of thrombi and pathologic angiogenic pro tially injected, the concentration of imaging agent and of cesses, to aid in the diagnosis, monitoring and treatment of agent-target complex rapidly increases. Shortly after injec such disorders. The fibrin binding polypeptides disclosed tion, however, the circulating (free) imaging agent starts to herein may be converted to imaging reagents for detecting clear through the kidneys or liver, and the plasma concentra thrombi by conjugating the polypeptides with a label appro tion of imaging agent begins to drop. This drop in the con priate for diagnostic detection. Such labels, referred to, for centration of free imaging agent in the plasma eventually example, as a detectable label or a diagnostically effective any causes the agent-target complex to dissociate. The usefulness moiety, include any moiety detectable by imaging proce of an imaging agent depends on the difference in rate of dures, that is to say any moiety able to provide, to improve or, agent-target dissociation relative to the clearing rate of the in any way, to advantageously modify the signal detected by agent. Ideally, the dissociation rate will be slow compared to a diagnostic imaging technique including, for instance, mag the clearing rate, resulting in a long imaging time during netic resonance imaging (MRI), radioimaging, X-ray imag which there is a high concentration of agent-target complex ing, light imaging, ultrasound imaging, thus enabling the and a low concentration of free imaging agent (background registration of diagnostically useful, preferably contrasted, signal) in the plasma. The dissociation rate of the complex is images when used in association with the said techniques. controlled by the dissociation rate constant, kff. Because Examples of detectable labels or diagnostically effective moi higher values of kff correspond to faster dissociation rates, it eties according to the invention include, for instance, chelated is preferable to obtain binding peptides which have a low kff gamma ray or positron emitting radionuclides; paramagnetic for use as imaging agents. metal ions in the form of chelated or polychelated complexes, 0119 The fibrin-binding moieties according to this inven X-ray absorbing agents including atoms having atomic num tion will be extremely useful for detection and/or imaging of ber higher than 20; an ultrasound contrastagent, including for US 2010/0158814 A1 Jun. 24, 2010 example gas-filled microvesicle, a dye molecule; a fluores as other active groups of detactable labels or therapeutic cent molecule; a phosphorescent molecule; a molecule agents, such, as for example, amino, thiol or carboxyl groups. absorbing in the UV spectrum; a quantum dot; a molecule I0128. The linker may also be a polyfunctional linking capable of absorption within near or far infrared radiations moiety or polyfunctional linker, which refers to a linear or and, in general, all the moieties which generate a detectable branched chain including at least 3 functional groups, one of Substance. them connecting the linking moiety with the peptide, and the 0.124 Preferably, a peptide of the invention exhibiting a remainder connecting the linking moiety with at least two strong ability to bind fibrin is conjugated or linked (directly or detectable labels and/or therapeutic agents. Such polyfunc via a linker) to a label appropriate for the detection method tional linking moieties may include, for example, N-branched ology to be employed. For example, the fibrin binder may be Lysine systems (see, f. i., Veprek, Pet al., J. Pept. Sci. 5, 5 conjugated with a paramagnetic chelate Suitable for magnetic (1999); 5, 203 (1999), polycarboxylic compounds and suit resonance imaging (MRI), with a radiolabel suitable for X-ray able derivative thereof in which the carboxylic group(s) are in imaging, with an ultrasound microsphere or liposome Suit a suitably activated or protected form, polyaminated com able for ultrasound detection, or with an optical imaging dye. pounds and suitable derivative thereof in which the amino 0.125. Alternatively, a peptide of the invention exhibiting a group(s) are in a suitably activated or protected form, and strong ability to bind fibrin is conjugated or linked (directly or amino acids and poly-amino acids Such as polyornithine, via a linker) to a therapeutic agent. Such compounds are polyarginine, polyglutamic acid, polyaspartic acid. useful for treatment oralleviation of diseases associated with I0129. Lipid molecules with linkers may be attached to fibrin. allow formulation of ultrasound bubbles, liposomes or other 0126. Additional modifications within the scope of the aggregation based constructs. Such constructs could be invention include introduction of linkers or spacers between employed as agents for targeting and delivery of a diagnostic the targeting sequence of the fibrin binding peptide and the reporter, a therapeutic agent (e.g., a chemical “warhead for detectable label or therapeutic agent. Use of such linkers/ spacers may improve the relevant properties of the binding therapy) or a combination of these. peptide (e.g., improve the binding ability, increase serum 0.130. In the present invention an especially preferred stability, adjust hydrophobicity or hydrophilicity, providing linker is GGGK. Additionally, the linkers GSAGSK (SEQID improved pharmacokinetic and pharmacodynamic proper NO. 137) and GAGSGK (SEQ ID NO. 138) are also pre ties, etc.). Indeed, use of an appropriate linker and/or spacer ferred. may provide an optimal distance between the fibrin-binding I0131. In general, the technique of using a detectably peptide and the detectable label or therapeutic agent, which labeled fibrin binding moiety is based on the premise that the may in turn improve the targeting capability of the com label generates a signal that is detectable outside the patient's pounds of the invention. These linkers may include, but are body. When the detectably labeled fibrin binding moiety is not restricted to, substituted or unsubstituted, saturated or administered to the patient suspected of having a fibrin related unsaturated, straight or branched alkyl chains, derivatized or disorder (such as a thrombus or pathological angiogenic pro underivatized polyethylene glycol, polyoxyethylene or poly cess), the high affinity of the fibrin binding moiety for fibrin vinylpyridine chains, one or more amino acids (and prefer causes the binding moiety to bind to fibrin and accumulate ably 3 or more amino acids and most preferably at least 4 or label at the site of interest. The signal generated by the labeled 6 amino acids, peptides from Straight, branched or cyclic peptide is detected by a scanning device which will vary amino acids, Sugars, oraliphatic or aromatic spacers common according to the type of label used, and the signal is then in the art, Substituted or unsubstituted polyamide chains; converted to an image of the area. derivatized or underivatized polyamine, polyester, polyethyl (0132 Magnetic Resonance Imaging enimine, polyacrylate, poly(vinylalcohol), polyglycerol, or I0133. The fibrin binding moieties of the present invention oligosaccharide (e.g., dextran) chains; glycosylated amino may advantageously be conjugated with a MRI detectable acid residues, alternating block copolymers; malonic, suc moiety, such as, for example, a paramagnetic metal chelator cinic, glutaric, adipic and pimelic acids; caproic acid; simple or iron particles (such as Superparamagnetic FeCparticles) in diamines and dialcohols; any of the other linkers disclosed order to form a contrast agent for use in MRI. Preferred herein as well as any other simple polymeric linker known in paramagnetic metal ions have atomic numbers 21-31, 39, 42. the art, for instance as described in WO 98/18497 and WO 43, 44, 49 or 57-83. This includes ions of the transition metal 98/18496. or lanthanide series which have one, and more preferably five 0127. Furthermore, linkers that are combinations of the or more, unpaired electrons and a magnetic moment of at least moieties described above, can also be employed to confer 1.7 Bohr magneton. The preferred paramagnetic metal is special advantage to the properties of the peptide. The linker selected from the group consisting of Fe(2+), Fe(3+), Cu(2+), may be a linear or branched and is preferably at least a Ni(2+), Rh(2+), Co(2+), Cr(3+), Gd(3+), Eu(3+), Dy(3+), divalent linking moiety. A "divalent linking moiety is a chain Tb(3+), Pm(3+), Nd(3+), Tm(3+), Ce(3+), Y(3+), Ho(3+), including two functional groups allowing for its conjugation Er(3+). La(3+), Yb(3+), Mn(3+), Mn(2+). Gd(3+) (also with a peptide on one side and a detectable label or therapeu referred to as Gd(III)) is particularly preferred for MRI due to tic agent on another. Preferably the divalent linking moiety its high relaxivity and low toxicity, and the availability of only permits conjugation with the terminal amino or carboxyl one biologically accessible oxidation state. Gd(III) chelates groups of the peptide and an appropriate functional group on have been used for clinical and radiologic MR applications the detectable label or therapeutic agent. A “functional since 1988, and approximately 30% of MR exams currently group' refers to specific groups of atoms within molecules or employ a gadolinium-based contrast agent. Additionally, moieties that are responsible for the characteristic chemical fibrin-binding moieties of the present invention also can be reaction of those molecules or moieties and may include, for conjugated with other MRI detectable moieties, such as, for example, the NH2 or —COOH groups of peptides, as well example, one or more Superparamagnetic particles. US 2010/0158814 A1 Jun. 24, 2010 24

0134. The practitioner will select a metal according to linear phosphonoalkyl derivatives disclosed in U.S. Pat. No. dose required to detect a thrombus and considering other 6,509.324; or of macrocyclic chelants such as texaphirines, factors such as toxicity of the metal to the subject. See, porphyrins, phthalocyanines. Tweedle et al., Magnetic Resonance Imaging (2nd ed.), Vol. 1, 0.136 Additional chelating ligands are ethylenebis-(2-hy Partain et al., eds. (W. B. Saunders Co. 1988), pp. 796-7. droxy-phenylglycine) (EHPG), and derivatives thereof, Generally, the desired dose for an individual metal will be including 5-C1-EHPG, 5Br-EHPG, 5-Me-EHPG, 5t-Bu proportional to its relaxivity, modified by the biodistribution, EHPG, and 5sec-Bu-EHPG: bis-2 (hydroxybenzyl)-ethyl pharmacokinetics and metabolism of the metal. The trivalent ene-diaminediacetic acid (HBED) and derivatives thereof, cation, Gd" is particularly preferred for MRI contrastagents, derivatives of 1,3-propylenediaminetetraacetic acid (PDTA) due to its high relaxivity and low toxicity, with the further and triethylenetetraaminehexaacetic acid (TTHA); deriva advantage that it exists in only one biologically accessible tives of 1,5,10-N,N',N'-tris(2,3-dihydroxybenzoyl)-tricat oxidation state, which minimizes undesired metabolization echolate (LICAM) and 1,3,5-N,N',N'-tris(2,3-dihydroxy of the metal by a patient. Another useful metal is Cr", which benzoyl) aminomethylbenzene (MECAM). Examples of is relatively inexpensive. representative chelators and chelating groups contemplated 0135 A chelator (also called a chelating ligand or chelat by the present invention are described in, for example, WO ing agent) is a chemical moiety, agent, compound or molecule 98/18496, WO 86/06605, WO 91/03200, WO95/28179, WO having one or more polar groups that act as a ligand for, and 96/23526, WO 97/36619, PCT/US98/01473, PCT/US98/ complex with, a paramagnetic metal. Suitable chelators are 20182, and U.S. Pat. No. 4,899,755, all of which are hereby known in the art and include acids with methylene phospho incorporated by reference. nic acid groups, methylene carbohydroxamine acid groups, 0.137 Preferred ligands according to the present invention carboxyethylidene groups, or carboxymethylene groups. In a are set forth in FIGS. 6a to 6c, together with suitable biblio preferred embodiment the chelators includes cyclic or linear graphic references concerning their preparation. polyamino polycarboxylic or polyphosphonic acids and con I0138 Particularly preferred are: DTPA, DTPA-GLU, tains at least one amino, thiol or carboxyl group. Examples of DTPA-Lys, DOTA, AAZTA, and the following AAZTA chelators include, but are not limited to, a polyaminopolycar derivatives: boxylic acid and the derivatives thereof, comprising, for example, diethylenetriamine pentaacetic acid (DTPA) and derivatives thereof such as benzo-DTPA, dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, dibenzyl DTPA; N,N-bis(2-(carboxymethyl)(methylcarbamoyl)me thylaminoethyl-glycine (DTPA-BMA); N-2-bis(car boxymethyl)amino-3-(4-ethoxyphenyl)propyl)-N-2-bis (carboxymethyl)aminoethylglycine (EOB-DTPA); Hoc1N N r 4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8, 11-triazatridecan-13-oic acid (BOPTA); N,N-Bis2-bis(2- HN (1,1-dimethylethoxy)-2-oxoethylaminoethyl-L-glutamic N acid 1-(1,1-dimethylethyl)ester N.N-bis(2-bis(carboxym ethyl)aminoethyl L-glutamic acid (DTPA-GLU); DTPA conjugated with Lys (DTPA-Lys); ethylenediaminetetraace tic acid (EDTA); 1,4,7,10-teraazacyclododecane 1,4,7,-tri acetic acid (DO3A) and derivatives thereof including, for example, 10-(2-hydroxypropyl)-1,4,7,10-teraazacy clododecane 1,4,7,-triacetic acid (HPDO3A): 1,4,7-triazacy clononane N,N',N'-triacetic acid (NOTA); 2-methyl-1,4,7, 10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (MCTA); 6-bis(carboxymethyl)aminoltetrahydro-6-me thyl-1H-1,4-diazepine-1,4(5H)-diacetic acid (AAZTA) pro vided by WO03008390 application, incorporated herein by reference, and derivatives thereof; 1,4,7,10-tetraazacyclotet radecane-1,4,7,10-tetraacetic acid (DOTA) and derivatives thereof, including for instance, benzo-DOTA, dibenzo DOTA, (C.O.'.C.".C.")-tetramethyl-1,4,7,10-tetraazacyclotet radecane-1,4,7,10-tetraacetic acid (DOTMA); or 14,8,11 tetraazacyclotetradecane-N,N',N',N"-tetraacetic acid (TETA); or corresponding compounds wherein one or more of the carboxylic groups is replaced by a phosphonic and/or phosphinic group, including, for instance, N,N'-bis-(pyri doxal-5-phosphate) ethylenediamine-N,N'-diacetic acid (DPDP): ethylenedinitrilotetrakis(methylphosphonic) acid (EDTP), 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra (methylenephosphonic) acid (DOTP), the phosphonoalkyl polyaza macrocyclic compounds for instance disclosed in U.S. Pat. No. 5,362.476 and U.S. Pat. No. 5,409,689 and the US 2010/0158814 A1 Jun. 24, 2010 25

the linker, such biodegradable functionalities can include -continued ester, double ester, amide, phosphoester, ether, acetal, and NH2 ketal functionalities. HOC HOC 0.141. In general, known methods can be used to couple the metal chelate and the fibrin binding moiety using Such link ers. See, e.g., WO95/28967, WO 98/18496, WO 98/18497 /-N and discussion therein. The fibrin binding moiety can be HOC -\- linked through its N- or C-terminus via an amide bond, for N example, to a metal coordinating backbone nitrogen of a metal chelate or to an acetate arm of the metal chelate itself. - The present invention contemplates linking of the chelate on HOC CO2H any position, provided the metal chelate retains the ability to bind the metal tightly in order to minimize toxicity. Similarly, \,7– CO2H the fibrin binding moiety may be modified or elongated in order to generate a locus for attachment to a metal chelate, HOC 1-4 7 provided such modification or elongation does not eliminate re its ability to bind fibrin. 0.142 MRI contrast reagents prepared according to the l CO2H disclosures herein may be used in the same manner as con ventional MRI contrast reagents. When imaging a thrombus, certain MR techniques and pulse sequences may be preferred 0.139. In accordance with the present invention, the chela to enhance the contrast of the thrombus to the background tor of the MRI contrast agent is coupled to the fibrin binding blood and tissues. These techniques include (but are not lim moiety directly or via a linker. The positioning of the chelator ited to), for example, black blood angiography sequences that should be selected so as not to interfere with the binding seek to make blood dark, Such as fast spin echo sequences affinity or specificity of the fibrin binding moiety. Preferably, (see, e.g., Alexander et al., Magnetic Resonance in Medicine, the chelate will be appended either to the N terminus or the C 40(2): 298-310 (1998)) and flow-spoiled gradient echo terminus, however the chelate may also be attached anywhere sequences (see, e.g., Edelman et al., Radiology, 177(1): 45-50 within the sequence. In a preferred embodiment, a chelator (1990)). These methods also include flow independent tech having a free central carboxylic acid group (e.g., DTPA-Asp niques that enhance the difference in contrast due to the T (B.-COOH)-OtBu) makes it easy to attach at the N-terminus difference of contrast-enhanced thrombus and blood and tis of the peptide by formation of an amide bond. The chelate Sue, such as inversion-recovery prepared or Saturation-recov could also be attached at the C-terminus with the aid of a ery prepared sequences that will increase the contrast linker. Alternatively, isothiocyanate conjugation chemistry between thrombus and background tissues. In addition, since could be employed as a way of linking the appropriate the present invention does not significantly alter T, methods isothiocyanto group bearing DTPA to a free amino group of T preparation may also prove useful (see, e.g., Gronas et anywhere within the peptide sequence. al., Journal of Magnetic Resonance Imaging, 7(4): 637-643 0140. In general, the fibrin binding moiety can be bound (1997)). Finally, magnetization transfer preparations may directly or covalently to the metal chelator (or other detect also improve contrast with these agents (see, e.g., Goodrich et able label), or it may be coupled or conjugated to the metal al., Investigative Radiology, 31(6): 323-32 (1996)). chelator using a linker, which may be, without limitation, 0143. The labeled reagent is administered to the patient in amide, urea, acetal, ketal, double ester, carbonyl, carbamate, the form of an injectable composition. The method of admin thiourea, Sulfone, thioester, ester, ether, disulfide, lactone, istering the MRI contrast agent is preferably parenterally, imine, phosphoryl, orphosphodiester linkages; Substituted or meaning intravenously, intraarterially, intrathecally, intersti unsubstituted Saturated or unsaturated alkyl chains; linear, tially, or intracavitarily. For imaging thrombi, intravenous or branched, or cyclic amino acid chains of a single amino acid intraarterial administration is preferred. For MRI, it is con or different amino acids (e.g., extensions of the N- or C-ter templated that the Subject will receive a dosage of contrast minus of the fibrin binding moiety); derivatized or underiva agent Sufficient to enhance the MR signal at the site of a tized polyethylene glycol, polyoxyethylene, or polyvinylpy thrombus at least 10%. After injection with the fibrin binding ridine chains; Substituted or unsubstituted polyamide chains; moiety-containing MRI reagent, the patient is scanned in the derivatized or underivatized polyamine, polyester, polyethyl MRI machine to determine the location of any thrombi. In enimine, polyacrylate, poly(vinyl alcohol), polyglycerol, or therapeutic settings, upon thrombus localization, a throm oligosaccharide (e.g., dextran) chains; alternating block bolytic can be immediately administered, if necessary, and copolymers; malonic, succinic, glutaric, adipic and pimelic the patient can be Subsequently scanned to visualize throm acids; caproic acid; simple diamines and dialcohols; and bus degradation. other simple polymeric linkers known in the art (see, e.g., WO 0144. Ultrasound Imaging 98/18497, WO 98/18496) or other linkers discussed herein. 0145 When ultrasound is transmitted through a sub Preferably the molecular weight of the linker can be tightly stance, the acoustic properties of the Substance will depend controlled. The molecular weights can range in size from less upon the velocity of the transmissions and the density of the than 100 to greater than 1000. Preferably the molecular Substance. Changes in the acoustic properties will be most weight of the linker is less than 100. In addition, it may be prominent at the interface of different substances (solids, desirable to utilize a linker that is biodegradable in vivo to liquids, gases). Ultrasound contrast agents are intense Sound provide efficient routes of excretion for the imaging reagents wave reflectors because of the acoustic differences between of the present invention. Depending on their location within liquid (e.g., blood) and gas-containing microvesicles, such as US 2010/0158814 A1 Jun. 24, 2010 26 microbubbles or microballoons, liposomes, or microspheres diethylaminocoumarin-3-yl)carbonyl)methyl-amino) dissolved therein. Because of their size, ultrasound octadecanoic acid; N-12-(((7'-diethylaminocoumarin-3-yl) microvesicles, liposomes, microspheres, and the like may carbonyl)methylamino)-octadecanoyl-2-aminopalmitic remain for a longer time in the blood stream after injection acid; N-Succinyldioleylphosphatidylethanolamine; 1,2-dio than other detectable moieties; a targeted fibrin-specific ultra leyl-sn-glycerol: 1,2-dipalmitoyl-sn-3-succinylglycerol: 1.3- Sound agent therefore may demonstrate Superior imaging of dipalmitoyl-2-Succinyl-glycerol: 1-hexadecyl-2-palmitoylg thrombi and sites of angiogenesis. lycerophosphoethanolamine or palmitoylhomocysteine; 0146 In this aspect of the invention, the fibrin binding alkylamines or alkylammonium salts, comprising at least one moiety may be linked to a material which is useful for ultra (Co-Co.), preferably (C-Cs), alkyl chain, Such as, for Sound imaging. The materials are employed to form instance, N-stearylamine, N,N'-distearylamine, N-hexadecy microvesicles (e.g., liposomes, microbubbles, microspheres, lamine, N,N'-dihexadecylamine, N-stearylammonium chlo or emulsions) containing a liquid or gas which functions as ride, N,N'-distearylammonium chloride, N-hexadecylammo the detectable label (e.g., an echogenic gas or material nium chloride, N,N'-dihexadecylammonium chloride, capable of generating an echogenic gas). Materials for the dimethyldioctadecylammonium bromide (DDAB), hexade preparation of Such microvesicles include amphiphilic com cyltrimethylammonium bromide (CTAB); tertiary or quater pounds such as Surfactants, lipids, sphingolipids, oligolipids, nary ammonium salts comprising one or preferably two (Co or phospholipids, proteins, polypeptides, carbohydrates, and Co), preferably (C-Cs), acyl chain linked to the N-atom synthetic or natural polymeric materials. See, for further through a (C-C) alkylene bridge, such as, for instance, description of suitable materials and methods, WO 98/53857, 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), WO 98/18498, WO 98/18495, WO 98/18497, WO 98/18496, 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and WO 98/18501, incorporated herein by reference in their 1.2-oleoyl-3-trimethylammonium-propane (DOTAP), 1,2- entirety. distearoyl-3-dimethylammonium-propane (DSDAP); and 0147 For contrast agents comprising Suspensions of sta mixtures or combinations thereof. bilized microbubbles (a preferred embodiment), amphiphilic 0.148. According to a preferred embodiment, at least one components are preferred. Suitable amphiphilic components of the compounds forming the microbubbles envelope is a include phospholipids; lysophospholipids; fatty acids, Such phospholipid, optionally in admixture with any of the other as palmitic acid, Stearic acid, arachidonic acid or oleic acid; above-cited materials. According to the present description, lipids bearing polymers, such as chitin, hyaluronic acid, poly the term "phospholipids” is intended to encompass any vinylpyrrollidone or polyethylene glycol (PEG), also referred amphiphilic phospholipid compound, the molecules of which as “pegylated lipids”; lipids bearing Sulfonated mono- di-, are capable of forming a stabilizing film of material (typically oligo- or polysaccharides; cholesterol, cholesterol Sulfate or in the form of a mono-molecular layer) at the gas-water cholesterol hemisuccinate; tocopherol hemisuccinate; lipids boundary interface in the final microbubbles suspension. with ether or ester-linked fatty acids; polymerized lipids; Accordingly, these materials are also referred to in the art as diacetyl phosphate; dicetyl phosphate; ceramides; polyoxy “film-forming phospholipids”. ethylene fatty acid esters (such as polyoxyethylene fatty acid 0149 Amphiphilic phospholipid compounds typically Stearates), polyoxyethylene fatty alcohols, polyoxyethylene contain at least one phosphate group and at least one, prefer fatty alcohol ethers, polyoxyethylated sorbitan fatty acid ably two, lipophilic long-chain hydrocarbon groups. esters, glycerol polyethylene glycol ricinoleate, ethoxylated 0150. Examples of suitable phospholipids include esters soybean sterols, ethoxylated castor oil or ethylene oxide (EO) of glycerol with one or preferably two residues of fatty acids and propylene oxide (PO) block copolymers; sterol aliphatic (the same or different) and phosphoric acid, wherein the acid esters including, cholesterol butyrate, cholesterol iso phosphoric acid residue is in turn bonded to a hydrophilic butyrate, cholesterol palmitate, cholesterol Stearate, lanos group, Such as, for example, choline (phosphatidylcholines— terol acetate, ergosterol palmitate, or phytosterol n-butyrate; PC), serine (phosphatidylserines—PS), inositol (phosphati sterol esters of Sugar acids including cholesterol glucu dylinositol), glycerol (phosphatidylglycerols—PG), ethano ronides, lanosterol glucoronides, 7-dehydrocholesterol glu lamine (phosphatidylethanolamines—PE), and the like coronide, ergosterol glucoronide, cholesterol gluconate, groups. Esters of phospholipids with only one residue offatty lanosterol gluconate, or ergosterol gluconate; esters of Sugar acid are generally referred to in the art as the “lyso' forms of acids and alcohols including lauryl glucoronide, Stearoylglu the phospholipids or lysophospholipids. Fatty acids present coronide, myristoyl glucoronide, lauryl gluconate, myristoyl in the phospholipids are in general long chain aliphatic acids, gluconate, or Stearoyl gluconate; esters of Sugars with ali typically containing from 12 to 24 carbon atoms, preferably phatic acids including Sucrose laurate, fructose laurate, from 14 to 22, the aliphatic chain mat contain one or more Sucrose palmitate. Sucrose Stearate, glucuronic acid, gluconic unsaturations or is preferably completely saturated. acid or polyuronic acid; Saponins including Sarsasapogenin, Examples of suitable fatty acids included in the phospholip Smilagenin, hederagenin, oleanolic acid, or digitoxigenin: ids include, for example, lauric acid, myristic acid, palmitic glycerol or glycerol esters including glycerol tripalmitate, acid, Stearic acid, arachidic acid, behenic acid, oleic acid, glycerol distearate, glycerol tristearate, glycerol dimyristate, linoleic acid, and linolenic acid. Preferably, saturated fatty glycerol trimyristate, glycerol dilaurate, glycerol trilaurate, acids such as myristic acid, pamitic acid, Stearic acid and glycerol dipalmitate, long chain alcohols including n-decyl arachidic acid are employed. alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, or 0151. Further examples of phospholipids are phosphatidic n-octadecyl alcohol; 6-(5-cholesten-3f6-yloxy)-1-thio-f-D- acids, i.e., the diesters of glycerol-phosphoric acid with fatty galactopyranoside; digalactosyldiglyceride; 6-(5-cholesten acids; sphingolipids such as Sphingomyelins, i.e., those phos 33-yloxy)hexyl-6-amino-6-deoxy-1-thio-B-D-galactopyra phatidylcholine analogs where the residue of glycerol diester noside; 6-(5-cholesten-3?-yloxy)hexyl-6-amino-6-deoxyl with fatty acids is replaced by a ceramide chain, cardiolipins, 1-thio-f-D-mannopyranoside; 12-(((7'- i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid, US 2010/0158814 A1 Jun. 24, 2010 27 glycolipids such as gangliosides, cerebrosides, etc., glucolip may contain other amphiphilic compounds including, for ids, Sulfatides and glycosphingolipids. As used herein, the instance, fatty acids, such as palmitic acid, Stearic acid, term "phospholipids' includes either naturally occurring, arachidonic acid or oleic acid; Sterols, such as cholesterol, or semisynthetic or synthetically prepared products that can be esters of sterols with fatty acids or with Sugar acids; glycerol employed either singularly or as mixtures. Examples of natu or glycerol esters including glycerol tripalmitate, glycerol rally occurring phospholipids are natural lecithins (phos distearate, glycerol tristearate, glycerol dimyristate, glycerol phatidylcholine (PC) derivatives) such as, typically, soya trimyristate, glycerol dilaurate, glycerol trilaurate, glycerol bean or egg yolk lecithins. dipalmitate; tertiary or quaternary alkyl-ammonium salts, 0152 Examples of semisynthetic phospholipids are the Such as 1,2-distearoyl-3-trimethylammonium-propane partially or fully hydrogenated derivatives of the naturally (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane occurring lecithins. (DPTAP), and mixtures or combinations thereof. 0153. Examples of synthetic phospholipids, which are a 0156 Preferably, the formulation (and particularly the preferred embodiment are e.g., dilauryloyl-phosphatidylcho microbubble envelope) includes at least on component bear line (“DLPC"), dimyristoylphosphatidylcholine (“DMPC), ing an overall net charge. Such as, for instance, a charged dipalmitoyl-phosphatidylcholine (“DPPC), diarachi amphiphilic material, preferably a lipid or a phospholipid. doylphosphatidylcholine (“DAPC), distearoyl-phosphati Examples of phospholipids bearing an overall negative dylcholine (“DSPC), 1-myristoyl-2-palmitoylphosphatidyl charge are derivatives, in particular fatty acid di-ester deriva choline (MPPC), 1-palmitoyl-2- tives, of phosphatidylserine, such as DMPS, DPPS, DSPS; of myristoylphosphatidylcholine (“PMPC), 1-palmitoyl-2- phosphatidic acid, such as DMPA, DPPA, DSPA; of phos stearoylphosphatidylcholine (“PSPC), 1-stearoyl-2- phatidylglycerol such as DMPG, DPPG and DSPG or of palmitoyl-phosphatidylcholine (SPPC), phosphatidylinositol, such as DMPI, DPPI or DPPI. Also dioleoylphosphatidylycholine (“DOPC), 1.2 Distearoyl-sn modified phospholipids, in particular PEG-modified phos glycero-3-Ethylphosphocholine (Ethyl-DSPC), dilauryloyl phatidylethanolamines, such as DPPE-PEG or DSPE-PEG, phosphatidylglycerol (“DLPG') and its alkali metal salts, can be used as negatively charged molecules. Also the lyso diarachidoylphosphatidylglycerol (“DAPG”) and its alkali form of the above cited phospholipids, such as lysophosphati metal salts, dimyristoylphosphatidylglycerol (“DMPG') and dylserine derivatives (e.g. lyso-DMPS, -DPPS or -DSPS), its alkali metal salts, dipalmitoyl-phosphatidylglycerol lysophosphatidic acid derivatives (e.g. lyso-DMPA, -DPPA (“DPPG') and its alkali metal salts, distearolyphosphatidylg or -DSPA) and lysophosphatidylglycerol derivatives (e.g. lycerol (“DSPG') and its alkali metal salts, dioleoylphos lyso-DMPG, -DPPG or -DSPG), can advantageously be used phatidylglycerol (“DOPG') and its alkali metal salts, as negatively charged compounds. Other examples of nega dimyristoyl phosphatidic acid (“DMPA) and its alkali metal tively charged compounds are bile acid salts such as cholic salts, dipalmitoyl phosphatidic acid (“DPPA) and its alkali acid salts, deoxycholic acid salts or glycocholic acid salts; metal salts, distearoyl phosphatidic acid (“DSPA), diarachi and (C-C), preferably (C-C) fatty acid salts such as, doyl phosphatidic acid (“DAPA) and its alkali metal salts, for instance, palmitic acid salts, Stearic acid salts, 1,2-di dimyristoyl phosphatidyl-ethanolamin-e (“DMPE), palmitoyl-sn-3-succinylglycerol salts or 1,3-dipalmitoyl-2- dipalmitoyl phosphatidylethanolamine (“DPPE), distearoyl Succinylglycerol salts. phosphatidyl-ethanolamine (“DSPE), dimyristoyl phos 0157 Preferably, the negatively charged compound is phatidylserine (“DMPS), diarachidoyl phosphatidylserine selected among DPPA, DPPS, DSPG, DSPE-PEG2000, (“DAPS), dipalmitoyl phosphatidylserine (“DPPS), dis DSPE-PEG5000 or mixtures thereof. tearoylphosphatidylserine (“DSPS), dioleoylphosphati 0158. The negatively charged component is typically dylserine (“DOPS), dipalmitoyl sphingomyelin (“DPSP), associated with a corresponding positive counter-ion, which and distearoyl sphingomyelin (“DSSP). can be mono- (e.g. an alkali metal or ammonium), di- (e.g. an 0154 Preferred phospholipids are fatty acid di-esters of alkaline earth metal) or tri-Valent (e.g. aluminium). Prefer phosphatidylcholine, ethylphosphatidylcholine, phosphati ably the counter-ion is selected among alkali metal cations, dylglycerol, phosphatidic acid, phosphatidylethanolamine, such as Li", Na', or K", more preferably Na. phosphatidylserine, phophatidylinositol or of sphingomy 0159. Examples of phospholipids bearing an overall posi elin. Particularly preferred phospholipids are DAPC, DSPC, tive charge are derivatives of ethylphosphatidylcholine, in DPPA, DSPA, DMPS, DPPS, DSPS and ethyl-DSPC. Most particular di-esters of ethylphosphatidylcholine with fatty preferred are DPPS or DSPC. Mixtures of phospholipids can acids, such as 1,2-distearoyl-sn-glycero-3-ethylphosphocho also be used, such as, for instance, mixtures of DSPE, DPPE, line (Ethyl-DSPC or DSEPC), 1,2-dipalmitoyl-sn-glycero-3- DPPC, DSPC and/or DAPC with DSPS, DPPS, DSPA, ethylphosphocholine (Ethyl-DPPC or DPEPC). The negative DPPA, DSPG, DPPG, Ethyl-DSPC and/or Ethyl-DPPC. counterion is preferably a halide ion, in particular chloride or 0155 Suitable phospholipids further include phospholip bromide ion. Examples of positively charged compounds that ids modified by linking a hydrophilic polymer, such as PEG can be incorporated into the envelope of microbubbles are or polypropyleneglycol (PPG) thereto. Phospholipids modi mono-, di- tri-, or tetra-alkylammonium salts with a halide fied by linking PEG thereto may be referred to herein as counterion (e.g. chloride or bromide) comprising at least one pegylated phospholipids. Examples of modified phospholip (Co-Co.), preferably (Ca-Cs), alkyl chain, Such as, for ids are phosphatidylethanolamines (PE) modified with poly instance mono- or di-stearylammonium chloride, mono or ethylenglycol (PEG), “PE-PEGs', i.e. phosphatidylethanola di-hexadecylammonium chloride, dimethyldioctadecylam mines where the hydrophilic ethanolamine moiety is linked to monium bromide (DDAB) or hexadecyltrimethylammonium a PEG molecule of variable molecular weight (e.g. from 300 bromide (CTAB). Further examples of positively charged to 5000 daltons), such as DPPE-PEG, DSPE-PEG, DMPE compounds that can be incorporated into the envelope of PEG or DAPE-PEG (where DAPE is 1,2-diarachidoyl-sn microbubbles are tertiary or quaternary ammonium salts with glycero-3-phosphoethanolamine.) The compositions also a halide counter ion (e.g. chloride or bromide) comprising US 2010/0158814 A1 Jun. 24, 2010 28 one or preferably two (Co-Co.), preferably (C-Cs), acyl polyethylene glycol (PEG), etc., malic acid, glycolic acid, chains linked to the N-atom through a (C-C) alkylene maltol, and the like. Such hydrophilic compounds also aid in bridge. Such as, for instance, 1,2-distearoyl-3-trimethylam homogenizing the microbubbles size distribution and monium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylam enhance stability under storage. Any suitable organic solvent monium-propane (DPTAP), 1,2-oleoyl-3-trimethylammo may be used as long as its boiling point is sufficiently low and nium-propane (DOTAP) O 1,2-distearoyl-3- its melting point is sufficiently high to facilitate Subsequent dimethylammonium-propane (DSDAP). drying. Typical organic solvents include, for example, diox (0160 DSEPC, DPEPC and/or DSTAP are preferably ane, cyclohexanol, tertiary butanol, tetrachlorodifluoro eth employed as positively charged compounds in the ylene (CCF) or 2-methyl-2-butanol. 2-methyl-2-butanol microbubble envelope. and C-ClaF are preferred. 0161 The positively charged component is typically asso 0.167 Prior to formation of the suspension of ciated with a corresponding negative counter-ion, which can microvesicles by dispersion in an aqueous carrier, the freeze be mono- (e.g. halide), di- (e.g. Sulphate) or tri-Valent (e.g. dried or spray dried phospholipid powders are contacted with phosphate). Preferably the counter-ion is selected from air or another gas. When contacted with the aqueous carrier among the halide ions, such as F (fluorine), Cl (chlorine) or the powdered phospholipids whose structure has been dis Br (bromine). rupted will form lamellarized or laminarized segments that 0162 Mixtures of neutral and charged compounds, in par will stabilize the microbubbles of the gas dispersed therein. ticular of phospholipids and/or lipids, can be satisfactorily This method permits production of Suspensions of employed to form the microbubble envelope. The amount of microbubbles that are stable even when stored for prolonged charged lipid orphospholipid may vary from about 95 mol% periods and are obtained by simple dissolution of the dried to about 1 mol %, with respect to the total amount of lipid and laminarized phospholipids (which have been stored under a phospholipid, preferably from 80 mol % to 20 mol%. desired gas) without shaking or any violent agitation. 0163 Preferred mixtures of neutral phospholipids and 0168 Alternatively, microvesicles, and particularly charged lipids or phospholipids are, for instance, DPPG/ microbubbles, can be prepared by Suspending a gas into an DSPC, DSTAP/DAPC, DPPS/DSPC, DPPS/DAPC, DPPE/ aqueous Solution at high agitation speed, as disclosed e.g. in DPPG, DSPA/DAPC, DSPA/DSPC and DSPG/DSPC. WO97/29783. Preferably, as disclosed in International patent 0164. In preferred embodiments, the phospholipid is the application WO 04/069284, a microemulsion can be prepared main component of the stabilizing envelope of the which contains the phospholipids (e.g., DSPC and/or DSPA) microbubbles, amounting to at least 50% (w/w) of the total in admixture with a lyoprotecting agent (Such as, for instance, amount of components forming the envelope of the gas-filled carbohydrates, Sugar alcohols, polyglycols and mixtures microbubbles. In some preferred embodiments, substantially thereof, as indicated in detail hereinafter) and optionally other the totality of the envelope (at least 90% and up to 100% by amphiphilic materials (such as Stearic acid), dispersed in an weight) can be formed of the phospholipid. emulsion of water and of a water immiscible organic solvent. 0.165 Any of the gases disclosed herein or known to the Preferred organic solvents are those having solubility in water skilled artisan may be employed; however, inert gases, such of 1.0 g/1 or lower, preferably lower about 0.01 g/l, and as SF or perfluorocarbons like CF, CFs and CF are include, for instance, pentane, hexane, heptane, octane, preferred, optionally in admixture with other gases Such as nonane, decane, 1-pentene, 2-pentene, 1-octene, cyclopen air, nitrogen, oxygen or carbon dioxide tane, cyclohexane, cyclooctane, 1-methyl-cyclohexane, ben 0166 The preferred microvesicle suspensions of the Zene, toluene, ethylbenzene, 1,2-dimethylbenzene, 1,3-dim present invention may be prepared from phospholipids using ethylbenzene, di-butyl ether and di-isopropylketone, known processes, such as, for example, freeze-drying or chloroform, carbon tetrachloride, 2-chloro-1-(difluo spray-drying solutions of the crude phospholipids in a Suit romethoxy)-1,1,2-trifluoroethane (enflurane), 2-chloro-2- able solvent or using the processes set forth in EP 554213; (difluoromethoxy)-1,1,1-trifluoroethane (isoflurane), tetra U.S. Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962: chloro-1,1-difluoroethane, perfluoropentane, EP 682530; U.S. Pat. No. 5,556,610; U.S. Pat. No. 5,846,518; perfluorohexane, perfluoroheptane, perfluorononane, per U.S. Pat. No. 6,183,725; EP 474833; U.S. Pat. No. 5,271,928; fluorobenzene, perfluorodecalin, methylperfluorobutylether, U.S. Pat. No. 5,380,519; U.S. Pat. No. 5,531,980; U.S. Pat. methylperfluoroisobutylether, ethylperfluorobutylether, eth No. 5,567,414; U.S. Pat. No. 5,658,551; U.S. Pat. No. 5,643, ylperfluoroisobutylether and mixtures thereof. The fibrin 553: U.S. Pat. No. 5,911,972; U.S. Pat. No. 6,110,443; U.S. binding peptide of the invention conjugated to a phospholipid Pat. No. 6,136,293; EP 619743: U.S. Pat. No. 5,445,813: U.S. (e.g. the lipopeptides discussed herein) can be admixed Pat. No. 5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat. No. together with the phospholipid forming the microvesicle's 6,187,288; and U.S. Pat. No. 5,908,610, which are incorpo envelope, in the microemulsion. Preferably, an aqueous Sus rated by reference herein in their entirety. Preferably, the pension of the fibrin binding peptide-phospholipid conjugate phospholipids are dissolved in an organic solvent and the and of a PE-PEG (e.g. DSPE-PEG2000) is first prepared, Solution is dried withoutgoing through a liposome formation which is then admixed together with an aqueous-organic stage. This can be done by dissolving the phospholipids in a emulsion comprising the phospholipid and the lyoprotecting suitable organic solvent together with a hydrophilic stabilizer agent. Preferably said mixing is effected under heating, e.g. Substance or a compound soluble both in the organic solvent form about 40° C. to 80° C. and water and freeze-drying or spray-drying the Solution. In 0169. Other excipients or additives may be present either this embodiment the criteria used for selection of the hydro in the dry formulation of the microbubbles or may be added philic stabilizer is its solubility in the organic solvent of together with the aqueous carrier used for the reconstitution choice. Examples of hydrophilic stabilizer compounds thereof, without necessarily being involved (or only partially soluble in water and the organic solvent are, e.g., a polymer, involved) in the formation of the stabilizing envelope of the like polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), microbubble. These include pH regulators, osmolality adjust US 2010/0158814 A1 Jun. 24, 2010 29 ers, viscosity enhancers, emulsifiers, bulking agents, etc. and polydioxanone; poly-beta.-aminoketones (A.S. Angeloni, P. may be used in conventional amounts. For instance com Ferruti, M. Tramontini and M. Casolaro. The Mannich bases pounds like polyoxypropylene glycol and polyoxyethylene in polymer synthesis: 3. Reduction of poly(beta-aminoketone glycol as well as copolymers thereof can be used. Examples (s)) to poly(gamma-aminoalcohol(s)) and their N-alkylation of viscosity enhancers or stabilizers are compounds selected to poly(gamma-hydroxyquaternary ammonium salt(s)), from linear and cross-linked poly- and oligo-saccharides, Polymer 23, pp 1693-1697, 1982.); polyphosphazenes (Al Sugars and hydrophilic polymers such as polyethylene glycol. lcock, Harry R. Polyphosphazenes: new polymers with inor 0170 As the preparation of gas-filled microbubbles may ganic backbone atoms (Science 193:1214-19 (1976)) and involve a freeze drying or spray drying step, it may be advan polyanhydrides. The microballoons of the present invention tageous to include in the formulation a lyophilization addi tive. Such as an agent with cryoprotective and/or lyoprotective can also be prepared according to the methods of WO-A-96/ effect and/or a bulking agent, for example an amino-acid Such 15815, incorporated herein by reference, where the microbal as glycine; a carbohydrate, e.g. a Sugar Such as Sucrose, man loons are made from a biodegradable membrane comprising nitol, maltose, trehalose, glucose, lactose or a cyclodextrin, or biodegradable lipids, preferably selected from mono- di-, a polysaccharide Such as dextran; or a polyoxyalkyleneglycol tri-glycerides, fatty acids, Sterols, waxes and mixtures Such as polyethylene glycol. thereof. Preferred lipids are di- or tri-glycerides, e.g., di- or 0171 In ultrasound applications the contrast agents tri-myristin, -palmityn or -Stearin, in particular tripalmitin or formed by phospholipid stabilized microbubbles can be tristearin. The microballoons may employ any of the gases administered, for example, in doses such that the amount of disclosed herein of known to the skilled artisan; however, phospholipid injected is in the range 0.1 to 200 ug/kg body inert gases such as fluorinated gases are preferred. The weight, preferably from about 0.1 to 30 g/kg. microballoons may be suspended in a pharmaceutically 0172 Other gas containing Suspensions include those dis acceptable liquid carrier with optional additives known to closed in, for example, U.S. Pat. No. 5,798,091, WO those of ordinary skill in the art and stabilizers. 97/29783, also EP 88.1915, incorporated herein by reference 0.175 Other gas-containing contrast agent formulations in their entirety. These agents may be prepared as described in include microparticles (especially aggregates of micropar U.S. Pat. No. 5,798,091 or WO97/29783. ticles) having gas contained therein or otherwise associated 0173 Another preferred ultrasound contrast agent com therewith (for example being adsorbed on the surface thereof prises ultrasound contrast agents in the form of “microbal and/or contained within Voids, cavities or pores therein). loons” of “microcapsules”. The terms “microballoon” or Methods for the preparation of these agents are as described “microcapsules' (here used interchangeably) refer to gas in EP 0122624; EP 0123235; EP 0365467; U.S. Pat. No. filled bodies with a material boundary or envelope. More on 5,558,857; U.S. Pat. No. 5,607,661; U.S. Pat. No. 5,637,289; microballoon formulations and methods of preparation may U.S. Pat. No. 5,558,856; U.S. Pat. No. 5,137,928: WO be found in EP 324938 (U.S. Pat. No. 4,844,882); U.S. Pat. 95/21631 or WO93/13809, incorporated herein by reference No. 5,711,933: U.S. Pat. No. 5,840,275; U.S. Pat. No. 5,863, in their entirety. 520; U.S. Pat. No. 6,123,922; U.S. Pat. No. 6,200,548; U.S. 0176 Any of these ultrasound compositions should also Pat. No. 4,900,540; U.S. Pat. No. 5,123,414: U.S. Pat. No. be, as far as possible, isotonic with blood. Hence, before 5,230,882: U.S. Pat. No. 5,469,854; U.S. Pat. No. 5,585, 112: injection, Small amounts of isotonic agents may be added to U.S. Pat. No. 4,718,433: U.S. Pat. No. 4,774,958; WO any of above ultrasound contrast agent Suspensions. The iso 95/01187; U.S. Pat. No. 5,529,766: U.S. Pat. No. 5,536,490; tonic agents are physiological Solutions commonly used in and U.S. Pat. No. 5,990.263, the contents of which are incor medicine and they comprise aqueous Saline solution (0.9% porated herein by reference. NaCl), 2.6% glycerol solution, 5% dextrose solution, etc. 0.174. The preferred microballoons have an envelope Additionally, the ultrasound compositions may include stan including a biodegradable physiologically compatible poly dard pharmaceutically acceptable additives, including, for mer or, a biodegradable solid lipid. The polymers useful for example, emulsifying agents, viscosity modifiers, cryopro the preparation of the microballoons of the present invention tectants, lyoprotectants, bulking agents etc. can be selected from the biodegradable physiologically com 0177. Any biocompatible gas may be used in the ultra patible polymers, such as any of those described in any of the Sound contrast agents of the invention. The term 'gas' as used following patents: EP 458745, U.S. Pat. No. 5,711,933, U.S. herein includes any Substances (including mixtures) Substan Pat. No. 5,840,275, EP 554213, U.S. Pat. No. 5,413,774 and tially in gaseous form at the normal human body temperature. U.S. Pat. No. 5,578,292, the entire contents of which are The gas may thus include, for example, air, nitrogen, oxygen, incorporated herein by reference. In particular, the polymer CO. hydrogen, nitrous oxide, a noble or inert gas such as can be selected from biodegradable physiologically compat helium, argon, Xenon or krypton, a radioactive gas Such as ible polymers, such as polysaccharides of low water solubil Xe' or Kr', a hyperpolarized noble gas such as hyperpo ity, polylactides and polyglycolides and their copolymers, larized helium, Xenon or neon, fluorinated gases (including copolymers of lactides and lactones Such as .epsilon.-capro for example, perfluorocarbons, SF SeF) a low molecular lactone, gamma.-Valerolactone and polypeptides. Other Suit weight hydrocarbon (e.g., containing from 1 to 7 carbon able polymers include poly(ortho)esters (see e.g., U.S. Pat. atoms), for example, an alkane Such as methane, ethane, a No. 4,093,709: U.S. Pat. No. 4,131,648; U.S. Pat. No. 4,138, propane, a butane, isobutene, isopentane or pentane, a 344; U.S. Pat. No. 4,180,646); polylactic and polyglycolic cycloalkane Such as cyclobutane or cyclopentane, an alkene acid and their copolymers, for instance DEXON (see J. Such as propene, propadiene or abutene, or an alkyne Such as Heller, Biomaterials 1 (1980), 51; poly(DL-lactide-co-epsi acetylene, an ether, a ketone, an ester, halogenated gases. Such lon.-caprolact-one), poly(DL-lactide-co-gamma.-Valerolac as halogenated, fluorinated or perfluorinated low molecular tone), poly(DL-lactide-co-gamma-...-butyrolactone), poly weight hydrocarbons (e.g. containing up to 7 carbon atoms) alkylcyanoacrylates; polyamides, polyhydroxybutyrate; and/or mixtures thereof. US 2010/0158814 A1 Jun. 24, 2010 30

0.178 Fluorinated gases are preferred, in particular perflu 0181 For the use in MRI the microvesicles will preferably orinated gases. Fluorinated gases include materials that con contain a hyperpolarized noble gas such as hyperpolarized tain at least one fluorine atom such as SF freons (organic neon, hyperpolarized helium, hyperpolarized Xenon, or mix compounds containing one or more carbon atoms and fluo tures thereof, optionally in admixture with air, CO, oxygen, rine, i.e., CF, CF, CFs, CFs, CF or CBrF3, CC1F. nitrogen, helium, Xenon, or any of the halogenated hydrocar C-ClFs, and CBrClF), fluorinated hydrocarbons, fluorinated bons as defined above. ketones such as perfluoroacetone, fluorinated ethers such as 0182 For use in scintigraphy, the microvesicle will pref perfluorodiethyl ether and perfluorocarbons. The term per erably contain radioactive gases such as Xe' or Kr' or fluorocarbon refers to compounds containing only carbon and mixtures thereof, optionally in admixture with air, CO, oxy fluorine atoms and includes, in particular, Saturated, unsatur gen, nitrogen, helium, krypton or any of the halogenated ated, and cyclic perfluorocarbons. The saturated perfluoro hydrocarbons as defined above. carbons, which are usually preferred, have the formula C.F.-- 0183 Since ultrasound vesicles may be larger than the 2, where n is from 1 to 12, preferably from 2 to 10, most other detectable labels described herein, in one preferred preferably from 3 to 8 and even more preferably from 3 to 6. embodiment they are be linked or conjugated to a plurality of Examples of biocompatible, physiologically acceptable per fibrin-binding polypeptides in order to increase the targeting fluorocarbons are: perfluoroalkanes, such as perfluo efficiency of the agent. Attachment to the ultrasound contrast romethane, perfluoroethane, perfluoropropanes, perfluo agents described above (or known to those skilled in the art) robutanes (e.g. perfluoro-n-butane, optionally in admixture may be via direct covalent bond between the fibrin-binding with other isomers such as perfluoro-isobutane), perfluoro polypeptide and the material used to make the vesicle or via a pentanes, perfluorohexanes or perfluoroheptanes; perfluoro linker, as described previously. For example, see WO alkenes, such as perfluoropropene, perfluorobutenes (e.g. 98/53857 generally for a description of the attachment of a perfluorobut-2ene) or perfluorobutadiene; perfluoroalkynes peptide to a bifunctional PEG linker, which is then reacted (e.g. perfluorobut-2-yne); and perfluorocycloalkanes (e.g. with a liposome composition. See also, Lanza et al., Ultra perfluorocyclobutane, perfluoromethylcyclobutane, perfluo sound in Med. & Bio., 23(6):863–870 (1997). rodimethylcyclobutanes, perfluorotrimethylcyclobutanes, 0184. A number of methods may be used to prepare sus perfluorocyclopentane, perfluoromethylcyclopentane, per pensions of microvesicles conjugated to fibrin-binding fluorodimethylcyclopentanes, perfluorocyclohexane, per polypeptides. For example, one may prepare maleimide-de fluoromethylcyclohexane and perfluorocycloheptane). Pre rivatized microbubbles by incorporating 5% (w/w) of ferred saturated perfluorocarbons include, for example, CF N-MPB—PE (1,2-dipalmitoyl-sn-glycero-3-phospho-etha C2F, CFs, CFs. CaFlo, CSF2 and C.F. Most preferably nolamine-4-(p-maleimido-phenyl butyramide), (Avanti the gas or gas mixture comprises SF or a perfluorocarbon Polar-Lipids, Inc) in the phospholipid formulation. Then, selected from the group consisting of CF, CFs CF, solutions of mercaptoacetylated fibrin-binding peptides (10 CSF2, CaF2, C7F1a, CsPs, with CFlo being particularly mg/mL in DMF), which have been incubated in deacetylation preferred. See also WO 97/29783, WO 98/53857, WO solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 M 98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO hydroxylamine.HCl, pH 7.5) are added to the maleimide 98/18501, WO 98/05364, WO 98/17324. activated microbubble suspension. After incubation in the 0179. In certain circumstances it may be desirable to dark, under gentle agitation, the peptide conjugated include a precursor to a gaseous Substance (e.g., a material microbubbles may be purified by centrifugation. that is capable of being converted to a gas in Vivo, often 0185. Compounds that can be used for derivatization of referred to as a “gas precursor). Preferably the gas precursor microvesicles and particularly microbubbles typically and the gas it produces are physiologically acceptable. The include the following components: (a) a hydrophobic portion, gas precursor may be pH-activated, photo-activated, tem compatible with the material forming the envelope of the perature activated, etc. For example, certain perfluorocarbons microbubble or of the microballoon, in order to allow an may be used as temperature activated gas precursors. These effective incorporation of the compound in the envelope of perfluorocarbons, such as perfluoropentane, have a liquid/gas the vesicle; said portion is represented typically by a lipid phase transition temperature above room temperature (or the moiety (dipalmitin, distearoyl); and (b) a spacer (typically temperature at which the agents are produced and/or stored) PEGs of different molecular weights, an amino acid chain, but below body temperature; thus they undergo a phase shift etc.), which may be optional in some cases (for example, and are converted to a gas within the human body. microbubbles may for instance present difficulties to be 0180. As discussed above, the gas can comprise a mixture freeze dried if the spacer is too long) or preferred in some of gases. The mixture may comprise any of the above gases in others (e.g., peptides may be less active when conjugated to a any ratio. In one preferred embodiment, the mixture may microballoon with short spacers); and (c) a reactive group include a conventional gas such as nitrogen, air or carbon capable of reacting with a corresponding reacting moiety on dioxide and a fluorinated as. Examples of Suitable gas mix the peptide to be conjugated (e.g., maleimido with the -SH tures can be found in WO94709829, which is herein incor group of cysteine). porated by reference. The following combinations are par 0186 Alternatively, fibrin-binding polypeptide conju ticularly preferred gas mixtures: a mixture of gases (A) and gated microbubbles may be prepared using biotin/avidin. For (B) in which, at least one of the gases (B), present in an example, avidin-conjugated microbubbles may be prepared amount of between 0.5-41% by vol., has a molecular weight using a maleimide-activated phospholipid microbubble Sus greater than 80 daltons and is a fluorinated gas and (A) is pension, prepared as described above, which is added to selected from the group consisting of air, oxygen, nitrogen, mercaptoacetylated-avidin (which has been incubated with carbon dioxide and mixtures thereof, the balance of the mix deacetylation solution). Biotinylated fibrin-binding peptides ture being gas A. are then added to the Suspension of avidin-conjugated US 2010/0158814 A1 Jun. 24, 2010 microbubbles, yielding a suspension of microbubbles conju Where a material necessary for forming the contrast agent is gated to fibrin-binding peptides. not already present in the container (e.g. a targeting ligand to 0187. Additionally fibrin binding peptides may be conju be linked to the phospholipid during reconstitution), it can be gated to phospholipids, these lipopeptides may then be used packaged with the other components of the kit, preferably in to prepare gas filled microvesicle ultrasound contrast agents. a form or container adapted to facilitate ready combination Preferably, the phospholipid may be selected from the group with the other components of the kit. consisting of phosphatidylethanolamines and modified 0191 No specific container, vial or connection system is phosphatidylethanolamines. The peptide and the phospho required; the present invention may use conventional contain lipid may be conjugated directly or via a linker, including for ers, vials and adapters. The only requirement is a good seal example a hydrophilic polymer, an amino acid chain, etc. between the stopper and the container. The quality of the seal, Particularly preferred phospholipids include phosphatidyle therefore, becomes a matter of primary concern; any degra thanolamines modified by linking a hydrophilic polymer dation of seal integrity could allow undesirable Substances to thereto. Examples of modified phosphatidylethanolamines enter the vial. In addition to assuring sterility, Vacuum reten are phosphatidylethanolamines (PE) modified with polyeth tion is essential for products stoppered at ambient or reduced ylenglycol (PEG), in brief"PE-PEGs', i.e. phosphatidyletha pressures to assure safe and proper reconstitution. The stop nolamines where the hydrophilic ethanolamine moiety is per may be a compound or multicomponent formulation linked to a PEG molecule of variable molecular weight (e.g. based on an elastomer, Such as poly(isobutylene) or butyl from 300 to 5000 daltons), such as DPPE-PEG, DSPE-PEG, rubber. DMPE-PEG or DAPE-PEG, DSPE-PEG2000, DSPE 0.192 Ultrasound imaging techniques that can be used in PEG3400, DPPE-PEG2000 and DPPE-PEG3400 are pre accordance with the present invention include known tech ferred, with DSPE-PEG2000 particularly preferred. Note that niques. Such as color Doppler, power Doppler, Doppler a salt form of the phospholipid may be used. Such as, for amplitude, stimulated acoustic imaging, and two- or three example, the trimethyl ammonium salt, the tetramethylam dimensional imaging techniques. Imaging may be done in monium salt, the triethylammonium salt, Sodium salt, etc. harmonic (resonant frequency) or fundamental modes, with Methods of preparing such lipopeptides are set forth in the the second harmonic preferred. examples. 0193 In ultrasound applications the contrast agents 0188 Some preferred methods of preparing targeted formed by phospholipid stabilized microbubbles may, for microvesicles with fibrin-binding polypeptides conjugated to example, be administered in doses such that the amount of phospholipids are included in the examples. phospholipid injected is in the range 0.1 to 200 g/kg body 0189 Unless it contains a hyperpolarized gas, known to weight, preferably from about 0.1 to 30 ug/kg. Microbal require special storage conditions, the lyophilized residue loons-containing contrastagents are typically administered in may be stored and transported without need of temperature doses such that the amount of wall-forming polymer or lipid control of its environment and in particular it may be supplied is from about 10 g/kg to about 20 mg/kg of body weight. to hospitals and physicians for on site formulation into a 0194 In a preferred embodiment, the ultrasound contrast ready-to-use administrable Suspension without requiring agents described herein are conjugated to one or more fibrin such users to have special storage facilities. Preferably in such binding moieties. As shown in the examples, these targeted a case it can be supplied in the form of a two-component kit, ultrasound contrast agents will localize at blood clots con which can include two separate containers or a dual-chamber taining fibrin, fibrin-containing tissue or sites of angiogenesis container. In the former case preferably the container is a and may be used to image clots, cancer orangiogenic tissue. conventional septum-sealed vial, wherein the vial containing 0.195 The ultrasound contrast agents of the present inven the lyophilized residue of step b) is sealed with a septum tion may further be used in a variety of therapeutic imaging through which the carrier liquid may be injected using an methods. The term therapeutic imaging includes within its optionally prefilled Syringe. In Such a case the Syringe used as meaning any method for the treatment of a disease in a patient the container of the second component is also used then for which comprises the use of a contrast imaging agent (e.g. for injecting the contrast agent. In the latter case, preferably the the delivery of a therapeutic agent to a selected receptor or dual-chamber container is a dual-chamber Syringe and once tissue), and which is capable of exerting or is responsible to the lyophilizate has been reconstituted and then suitably exert a biological effect in vitro and/or in vivo. Therapeutic mixed or gently shaken, the container can be used directly for imaging may advantageously be associated with the con injecting the contrast agent. In both cases means for directing trolled localized destruction of the gas-filled microvesicles, or permitting application of Sufficient bubble forming energy e.g. by means of an ultrasound burst at high acoustic pressure into the contents of the container are provided. However, as (typically higher than the one generally employed in non noted above, in the stabilized contrast agents according to the destructive diagnostic imaging methods). This controlled invention the size of the gas microbubbles is substantially destruction may be used, for instance, for the treatment of independent of the amount of agitation energy applied to the blood clots (a technique also known as Sonothrombolysis), reconstituted dried product. Accordingly, no more than gentle optionally in combination with the localized release of a hand shaking is generally required to give reproducible prod Suitable therapeutic agent. Alternatively, said therapeutic ucts with consistent microbubble size. imaging may include the delivery of a therapeutic agent into 0190. It can be appreciated by one of ordinary skilled in cells, as a result of a transient membrane permeabilization at the art that other two-chamber reconstitution systems capable the cellular level induced by the localized burst of the of combining the dried powder with the aqueous Solution in a microvesicles. This technique can be used, for instance, for an sterile manner are also within the scope of the present inven effective delivery of genetic material into the cells; optionally, tion. In such systems, it is particularly advantageous if the a drug can be locally delivered in combination with genetic aqueous phase can be interposed between the water-insoluble material, thus allowing a combined pharmaceutical/genetic gas and the environment, to increase shelflife of the product. therapy of the patient (e.g. in case of tumor treatment). US 2010/0158814 A1 Jun. 24, 2010 32

0196. The term “therapeutic agent” includes within its tion labels have large molar absorptivities, e.g. >10 cm'M meaning any Substance, composition or particle which may 1, while fluorescent optical dyes will have high quantum be used in any therapeutic application, Such as in methods for yields. Examples of optical dyes include, but are not limited the treatment of a disease inapatient, as well as any Substance to those described in WO 98/18497, WO 98/18496, WO which is capable of exerting or responsible to exert a biologi 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO cal effect in vitro and/or in vivo. Therapeutic agents thus 97/18841, WO 96/23524, WO 98/47538, and references cited include any compound or material capable of being used in therein. The photolabels may be covalently linked directly to the treatment (including diagnosis, prevention, alleviation, the fibrin binding moiety or linked to the fibrin binding moi pain relief or cure) of any pathological status in a patient ety via a linker, as described previously. (including malady, affliction, disease lesion or injury). 0202 After injection of the optically-labeled fibrin bind Examples of therapeutic agents include those discussed ing moiety, the patient is scanned with one or more light herein, Such as, for example, drugs, pharmaceuticals, bioac Sources (e.g., a laser) in the wavelength range appropriate for tive agents, cytotoxic agents, chemotherapy agents, radio the photolabel employed in the agent. The light used may be therapeutic agents, proteins, natural or synthetic peptides, monochromatic or polychromatic and continuous or pulsed. including oligopeptides and polypeptides, vitamins, steroids Transmitted, scattered, or reflected light is detected via a and genetic material, including nucleosides, nucleotides, oli photodetector tuned to one or multiple wavelengths to deter gonucleotides, polynucleotides and plasmids. In a preferred mine the location of fibrin in the subject. Changes in the embodiment the therapeutic agent is a drug useful in the optical parameter may be monitored over time to detect accu treatment of cancer, thrombotic disorders orangiogenic dis mulation of the optically-labeled reagent at the site of the orders. thrombus. Standard image processing and detecting devices 0.197 Optical Imaging, Sonoluminescence or Photoa may be used in conjunction with the optical imaging reagents coustic Imaging of the present invention. 0198 In another embodiment, the fibrin binding moieties 0203 The optical imaging reagents described above may of the invention may be conjugated (directly or via a linker) to also be used for acousto-optical or Sonoluminescent imaging an optical, Sonolumiscent or photoacoustic label. In a pre performed with optically-labeled imaging agents (see, U.S. ferred embodiment, the fibrin binding moieties of the inven Pat. No. 5,171,298, WO98/57666, and references therein). In tion are conjugated (directly or via a linker) to an optically acousto-optical imaging, ultrasound radiation is applied to active imaging moiety. Suitable examples of optically active the Subject and affects the optical parameters of the transmit imaging moieties include, for example, optical dyes, includ ted, emitted, or reflected light. In Sonoluminescent imaging, ing organic chromophores or fluorophores, having extensive the applied ultrasound actually generates the light detected. delocalized ring systems and having absorption or emission Suitable imaging methods using Such techniques are maxima in the range of 400-1500 nmi; a fluorescent molecule described in WO 98/57666. Such as, for example, fluorescein, a phosphorescent mol 0204 Additionally, the fibrin-binding moieties of the ecule; a molecule absorbing in the UV spectrum; a quantum invention may be attached to an enzyme Substrate that is dot; or a molecule capable of absorption of near or far infrared linked to both a light imaging reporter and a light imaging radiations. One preferred optically active moiety is 5-car quencher. The fibrin binding moiety serves to localize the boxyfluorescein (CF5). construct to the fibrin-bearing tissue of interest (e.g. a tumor), 0199. In accordance with the present invention, a number where an enzyme cleaves the enzyme Substrate, releasing the of optical parameters may be employed to determine the light imaging quencher and allowing light imaging of the location of fibrin with in Vivo light imaging after injection of fibrin-bearing tissue of interest. the subject with an optically-labeled fibrin binding moiety. 0205 Nuclear Imaging (Radionuclide Imaging) and Optical parameters to be detected in the preparation of an Radiotherapy. image may include transmitted radiation, absorption, fluores 0206 Fibrin-binding moieties also may be conjugated cent or phosphorescent emission, light reflection, changes in with a radionuclide reporter appropriate for Scintigraphy, absorbance amplitude or maxima, and elastically scattered SPECT, or PET imaging and/or with a radionuclide appro radiation. For example, biological tissue is relatively translu priate for radiotherapy. Constructs in which the fibrin binding cent to light in the near infrared (NIR) wavelength range of moieties are conjugated with both a chelator for a radionu 650-1000 nm. NIR radiation can penetrate tissue up to several clide useful for diagnostic imaging and a chelator useful for centimeters, permitting the use of the fibrin binding moieties radiotherapy are within the scope of the invention. of the present invention for optical imaging of fibrin in vivo. 0207 For use as a PET agent a peptide is complexed with 0200 Near infrared dye may include, cyanine or indocya one of the various positron emitting metalions, such as "Mn, nine derivatives such as, for example, Cy5.5, IRDye800. Fe, Cu, Ga, 'As, "Tc, or '''In. The binding moieties indocyanine green (ICG), indocyanine green derivatives of the invention can also be labeled by halogenation using including the tetrasulfonic acid substituted indocyanine green radionuclides such as 'F, I, I, I, I, 77Br, and 7.Br. (TS-ICG), and combinations thereof. Preferred metal radionuclides for scintigraphy or radio 0201 The fibrin binding moieties may be conjugated with therapy include "Tc, Cr, 7Ga, Ga, Sc, Cr, "7Tm, photolabels, such as optical dyes, including organic chro 14 Ce, In, 168 Yb, 175Yb. 140La, 90Y. 88y. 'Sim, 166Ho, mophores or fluorophores, having extensive delocalized ring 165 Dy 166Dy, 6.Cu, 62Cu, (Cu, 67Cu, 7Ru, 103Ru, 186Re, systems and having absorption or emission maxima in the 188Re, 208Pb, 2. Bi, 212Bi, 213Bi, 214Bi, 105Rh, 109Pd, 117 Sn, range of 400-1500 nm. The fibrin binding moiety may alter ''Prm, ''Tb, 77Lu, Au and Au. The choice of metal natively be derivatized with a bioluminescent molecule. The will be determined based on the desired therapeutic or diag preferred range of absorption or emission maxima for photo nostic application. For example, for diagnostic purposes the labels is between 600 and 1000 nm to minimize interference preferred radionuclides include Cu, "Ga, Ga, ''"Tc, and with the signal from hemoglobin. Preferably, photoabsorp ''' In. For therapeutic purposes, the preferred radionuclides US 2010/0158814 A1 Jun. 24, 2010

include Cu, 90Y. 105Rh, In, 117'Sn, 149Pm, 'Sim, 161Tb, Formula 5 is described in, for example, U.S. Pat. Nos. 5,627, 166Dy, 166Ho, 175Yb. 177Lu, 186-188Re, and 199Au.99mTc is 286 and 6,093,382, and the chelating group of Formula 6 is particularly preferred for diagnostic applications because of described in, for example, U.S. Pat. Nos. 5,662,885; 5,780, its low cost, availability, imaging properties, and high specific 006; and 5,976,495. activity. The nuclear and radioactive properties of Tc-99m make this isotope an ideal Scintigraphic imaging agent. This isotope has a single photon energy of 140 keV and a radioac tive half-life of about 6 hours, and is readily available from (1) a..'Mo-mTc generator. R R 0208. The metal radionuclides may be chelated by a chela tors. Suitable chelators include those discussed above, as well rooc-l / \ |- COOH as, for example, linear, macrocyclic, terpyridine, and NS, N N NS, or N. chelators, including for example, the ligands disclosed in U.S. Pat. No. 5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021,556, U.S. Pat. No. 5,075,099, and U.S. N N Pat. No. 5,886,142, and other chelators known in the art including, but not limited to, HYNIC, and bisamino bisthiol too- \ / COOH (BAT) chelators (see also U.S. Pat. No. 5,720,934). For R example, Na chelators are described in U.S. Pat. No. 6,143, 274; U.S. Pat. No. 6,093,382: U.S. Pat. No. 5,608,110; U.S. Pat. No. 5,665,329; U.S. Pat. No. 5,656,254; and U.S. Pat. | No. 5,688,487. Certain NS chelators are described in PCT/ CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. No. 5,662,885; U.S. Pat. No. 5,976,495; and U.S. (2) Pat. No. 5,780,006. The chelator may also include derivatives R R of the chelating ligand mercapto-acetyl-acetyl-glycyl-gly cine (MAG3), which contains an NS, and NS systems such rooc- / \ )-cool as MAMA (monoamidemonoaminedith-iols), DADS (NS diaminedithiols), CODADS and the like. These ligand sys tems and a variety of others are described in Liu and Edwards, Chem Rev. 99:2235-2268 (1999) and references therein. CN N) 0209. The chelator may also include complexes contain ing ligand atoms that are not donated to the metal in a tet too- \ / COOH radentate array. These include the boronic acid adducts of R technetium and rhenium dioximes, such as are described in U.S. Pat. No. 5,183,653: U.S. Pat. No. 5,387,409; and U.S. Pat. No. 5,118,797, the disclosures of which are incorporated by reference herein, in their entirety. 0210. In another embodiment, disulfide bonds of a fibrin

binding polypeptide of the invention are used as two ligands (3) for chelation of a radionuclide such as 'mTc. In this way the peptide loop is expanded by the introduction of Tc (peptide S S-peptide changed to peptide-S Tc—S-peptide). This has also been used in other disulfide containing peptides in the literature (Chen et al., J. Nucl. Med., 42:1847-1855 (2001)) while maintaining biological activity. The other chelating groups for Tc can be supplied by amide nitrogens of the backbone, another cystine amino acid or other modifications of amino acids. 0211 Particularly preferred metal chelators include those of Formula 1, 2, and 3 (for '''In and lanthanides such as paramagnetic Gd" and radioactive lanthanides, such as, for (4a) example '77Lu, Y. Sm, and 'Ho) and those of Formula 4, 5, and 6 (for radioactive’"Tc, Re, and Re) set forth below. 0212. These and other metal chelating groups are described in U.S. Pat. Nos. 6,093,382 and 5,608,110, which are incorporated by reference herein in their entirety. Addi tionally, the chelating group of Formula 3 is described in, for example, U.S. Pat. No. 6,143.274; the chelating group of US 2010/0158814 A1 Jun. 24, 2010 34

thetic or naturally occurring amino acids; all where n is 1-100. -continued Other variants of these structures are described, for example, (4b) in U.S. Pat. No. 6,093,382. The disclosures of each of the OH foregoing patents, applications and references are incorpo rated by reference herein, in their entirety. O 0214 Chelators may be covalently linked directly to the fibrin-binding moiety or linked to the fibrin-binding polypep tide via a linker, as described previously, and then directly labeled with the radioactive metal of choice (see, WO 98/52618, U.S. Pat. No. 5,879,658, and U.S. Pat. No. 5,849, HN O 261). 0215. The selection of a proper radionuclide for use in a particular radiotherapeutic application depends on many fac NH HN tors, including: 0216 a. Physical half-life This should belong enough to allow synthesis and purification of the radiotherapeu tic construct from radiometal and conjugate, and deliv ery of said construct to the site of injection, without HO OH significant radioactive decay prior to injection. Prefer (5a) ably, the radionuclide should have a physical half-life between about 0.5 and 8 days. i 0217 b. Energy of the emission(s) from the radionu NH HN clide—Radionuclides that are particle emitters (such as alpha emitters, beta emitters and Auger electron emit ters) are particularly useful, as they emit highly ener getic particles that deposit their energy over short dis HO OH Y . tances, thereby producing highly localized damage. (5b) Beta emitting radionuclides are particularly preferred, as the energy from beta particle emissions from these isotopes is deposited within 5 to about 150 cell diam eters. Radiotherapeutic agents prepared from these nuclides are capable of killing diseased cells that are relatively close to their site of localization, but cannot travel long distances to damage adjacent normal tissue iNH HN Such as bone marrow. 0218 c. Specific activity (i.e. radioactivity per mass of the radionuclide)—Radionuclides that have high spe cific activity (e.g., generator produced 'Y''' In, ''Lu) are particularly preferred. The specific activity of a HO OH radionuclide is determined by its method of production, (6) the particular target for which it is produce, and the OH properties of the isotope in question. 0219 Many of the lanthanides and lanthanoids include radioisotopes that have nuclear properties that make them Suitable for use as radiotherapeutic agents, as they emit beta particles. Some of these are listed in the table below.

TABLE 4 Approximate range Half-Life Max b- Gamma of b-particle (cell NHCOCH Isotope (days) energy (MeV) energy (keV) diameters 49-Pin 2.21 1.1 286 60 -Sm 1.93 O.69 103 30 0213. In the above Formulas 1 and 2, R is alkyl, preferably 66-Dy 340 O4O 82.5 15 methyl. In the above Formula 5. X is either CH or O, Y is 66-Ho 1.12 1.8 80.6 117 either C-C branched or unbranched alkyl: Y is aryl, ary 75-Yb 4.19 O.47 396 17 loxy, arylamino, arylaminoacyl: Y is arylkyl where the 77-Lu 6.71 OSO 208 2O alkyl group or groups attached to the aryl group are C-Co OY 2.67 2.28 150 branched or unbranched alkyl groups, C-C branched or Il-In 2.8.10 Auger electron 173, <5 * m unbranched hydroxy or polyhydroxyalkyl groups or poly emitter 247 alkoxyalkyl or polyhydroxy-polyalkoxyalkyl groups, J is C(=O)—, OC(=O) , SO. , NC(=O) , NC(=S) , wherein: Pm is Promethium, Sm is Samarium, Dy is Dyspro N(Y), NC(=NCH) , NC(-NH) , N=N sium, Ho is Holmium, Yb is Ytterbium, Luis Lutetium, Y is homopolyamides or heteropolyamines derived from Syn Yttrium, In is Indium. US 2010/0158814 A1 Jun. 24, 2010

0220. The use of radioactive rhenium isotope as an alter ReOCl(PPhs), and as ReO,(pyridine)", where Phis phenyl native to above lanthanides and lanthanoids is well known in and Bu is n-butyl. Other rhenium reagents capable of forming the art and is encompassed by the invention. a rhenium complex may also be used. 0221 Particularly 'Re isotopes have proved to be of 0228 Radioactively-labeled Scintigraphic imaging agents particular interest in nuclear medicine, having a large number provided by the present invention are provided having a Suit of applications in radiopharmaceutical therapy. able amount of radioactivity. In forming Tc-99m radioactive 0222. Thus, in a preferred embodiment, the invention complexes, it is generally preferred to form radioactive com includes novel radiotherapeutic agents in which fibrin-bind plexes in Solutions containing radioactivity at concentrations ing moieties of the inventions are conjugated to a suitably of from about 0.01 mCi to 100 mCiper mL. chelated radionuclide that emits ionizing radiations such as 0229 Generally, the unit dose to be administered has a beta particles, alpha particles and Auger or Coster-Kroning radioactivity of about 0.01 mCi to about 100 mCi, preferably electrons. More preferably, the fibrin binding moiety of the 1 mCi to 20 mCi. The solution to be injected at unit dosage is invention is labelled with a lanthanide or a lanthanoid radio from about 0.01 mL to about 10 mL. nuclide selected from Y, In, Pm, 'Sim, Dy, Ho, 0230 Typical doses of a radionuclide-labeled fibrin-bind ''Yb, and ''Lu. Examples of suitable chelating ligand may ing imaging agent according to the invention provide 10-20 be selected from those of FIGS. 6a to 6c. mCi for an adult human. After injection of the fibrin-specific 0223) The compounds of the invention labeled with thera radionuclide imaging agent into the patient, a gamma camera peutic radionuclides can find application either as a radiop calibrated for the gamma ray energy of the nuclide incorpo harmaceutical that will be used as a first line therapy in the rated in the imaging agent is used to image areas of uptake of treatment of a disease such as cancer, or in combination the agent and quantify the amount of radioactivity present in therapy, where the radiotherapeutic agents of the invention the site. Imaging of the site in vivo can take place in a matter could be utilized in conjunction with adjuvant chemotherapy of a few minutes. However, imaging can take place, if desired, (e.g., with one of the other therapeutic agents disclosed hours or even longer, after the radiolabeled peptide is injected herein), or as the therapeutic part of a matched pair therapeu into a patient. In most instances, a Sufficient amount of the tic agent. administered dose will accumulate in the area to be imaged 0224. In fact, the peptide moiety of the radiotherapeutic of within about 0.1 of an hour to permit the taking of scintipho the invention is able to localize the chelated radioactive iso tOS tope to the pathologic fibrin deposition, for instance, into 0231 Proper dose schedules for the radiotherapeutic com thrombi/clots, atherosclerois plaques and inflammation pounds of the present invention are known to those skilled in based damage involved in multiple Sclerosis and, especially the art. The compounds can be administered using many within Solid tumors. The cytotoxic amount of ionizing radia methods that include, but are not limited to, a single or mul tion emitted by the localized radioisotope is thus able to cause tiple IV or IP injections, using a quantity of radioactivity that the cell death of the pathologic tissue. is sufficient to cause damage or ablation of the targeted fibrin 0225 Complexes of radioactive technetium are particu containing tissue, but not so much that Substantive damage is larly useful for diagnostic imaging and complexes of radio caused to non-target (normal tissue). The quantity and dose active rhenium are particularly useful for radiotherapy. In required is different for different constructs, depending on the forming a complex of radioactive technetium with the energy and half-life of the isotope used, the degree of uptake reagents of this invention, the technetium complex, prefer and clearance of the agent from the body and the mass of the ably a salt of Tc-99m pertechnetate, is reacted with the tumor. In general, doses can range from a single dose of about reagent in the presence of a reducing agent. Preferred reduc 30-50 mCi to a cumulative dose of up to about 3 Curies for an ing agents are dithionite, Stannous and ferrous ions; the most adult human. preferred reducing agent is stannous chloride. Means for pre 0232. The radiotherapeutic compositions of the invention paring Such complexes are conveniently provided in a kit can include physiologically acceptable buffers, and can form comprising a sealed vial containing a predetermined require radiation stabilizers to prevent radiolytic damage to quantity of a reagent of the invention to be labeled and a the compound prior to injection. Radiation stabilizers are Sufficient amount of reducing agent to label the reagent with known to those skilled in the art, and may include, for Tc-99m. example, para-aminobenzoic acid, ascorbic acid, gentistic 0226. Alternatively, the complex may beformed by react acid and the like. ing a peptide of this invention conjugated with an appropriate 0233. A single, or multi-vial kit that contains all of the chelator with a pre-formed labile complex of technetium and components needed to prepare the complexes of this inven another compound known as a transfer ligand. This process is tion, other than the radionuclide, is an integral part of this known as ligand exchange and is well known to those skilled invention. in the art. The labile complex may be formed using such 0234. A single-vial kit preferably contains a chelating transfer ligands as tartrate, citrate, gluconate or mannitol, for ligand, a source of Stannous salt, or other pharmaceutically example. Among the Tc-99m pertechnetate salts useful with acceptable reducing agent, if required, and is appropriately the present invention are included the alkali metal salts such buffered with pharmaceutically acceptable acid or base to as the sodium salt, or ammonium salts or lower alkyl ammo adjust the pH to a value of about 3 to about 9. The quantity and nium salts. type of reducing agent used would depend highly on the 0227 Preparation of the complexes of the present inven nature of the exchange complex to be formed. The proper tion where the metal is radioactive rhenium may be accom conditions are well known to those that are skilled in the art. plished using rhenium starting materials in the +5 or +7 It is preferred that the kit contents be in lyophilized form. oxidation state. Examples of compounds in which rhenium is Such a single vial kit may optionally contain labile or in the Re(VII) state are NHReO or KReO. Re(V) is avail exchange ligands Such as glucoheptonate, gluconate, manni able as, for example, ReOC(NBu), ReOCl(ASPh4) tol, malate, citric or tartaric acid and can also contain reaction US 2010/0158814 A1 Jun. 24, 2010 36 modifiers such as diethylenetriamine-pentaacetic acid 0241. Such conjugates will be especially useful in treating (DPTA), ethylenediamine tetraacetic acid (EDTA), or C, B, or thrombus associated diseases, especially acute myocardial Y cyclodextrin that serve to improve the radiochemical purity infarction, stroke and pulmonary embolism in , and stability of the final product. The kit may also contain including humans, which method comprises administering to stabilizers, bulking agents such as mannitol, that are designed a in need thereof an effective amount of a fibrin to aid in the freeze-drying process, and other additives known binding moiety according to the invention conjugated with a to those skilled in the art. thrombolytic agent. The invention also provides the use of 0235 A multi-vial kit preferably contains the same gen Such conjugates in the manufacture of a medicament for the eral components but employs more than one vial in reconsti treatment of thrombus associated diseases in mammals, tuting the radiopharmaceutical. For example, one vial may including humans. Suitable thrombolytic agents for use in contain all of the ingredients that are required to form a labile this aspect of the invention include fibrinolytic enzymes, Tc(V) complex on addition of pertechnetate (e.g., the stan including plasminogen activators. The term plasminogen nous source or other reducing agent). Pertechnetate is added activator includes but is not limited to streptokinase, human to this vial, and after waiting an appropriate period of time, tissue plasminogen activator (tPA) and urokinase (both single the contents of this vial are added to a second vial that con and two-chain forms). Such enzymes are obtained from natu tains the ligand, as well as buffers appropriate to adjust the pH ral sources or tissues or by recombinant production. Other to its optimal value. After a reaction time of about 5 to 60 suitable thrombolytic agents include fibrinolytically active minutes, the complexes of the present invention are formed. It hybrid proteins (see, e.g., EP-A-155 387) which comprise is advantageous that the contents of both vials of this multi one chain of a 2-chain protease linked to a chain of a different vial kit be lyophilized. As above, reaction modifiers, 2-chain protease, at least one of the chains in the hybrid exchange ligands, stabilizers, bulking agents, etc. may be protein being derived from a fibrinolytically active protease: present in either or both vials. thrombolytic protein conjugates (see, e.g., EP-A-152 736), 0236. Therapeutic Applications Such as urokinase linked to reversibly blocked plasmin, 0237. In another embodiment of the invention, a fibrin derivatives of fibrinolytic enzymes in which the catalytic site binding moiety of the invention is conjugated to a therapeutic on the enzyme which is responsible for fibrinolytic activity is agent (also referred to as a therapeutically active agent or blocked by a human protein attached thereto by way of a moiety). reversible linking group, for example urokinase reversibly 0238. Unless otherwise provided, the term “therapeutic' linked to the active center of human plasmin, genetically as used herein includes at least partial alleviation of symp engineered derivatives including muteins of naturally occur toms of a given condition. The therapeutically active agents ring plasminogen activators; hybrid molecules (see, e.g., EP do not have to produce a complete alleviation of the Symp A-297 882); reversibly blocked in vivo fibrinolytic enzymes, toms to be useful. For example, treatment of an individual can Such as a binary complex between Streptokinase and plasmi result in a decrease in the size of a tumor or diseased area or nogen, most preferably a p-anisoyl Streptokinase/plasmino a blood clot, or even prevention of an increase in size of the gen complex without internal bond cleavage (anistreplase, tumor or diseased area, as well as partial alleviation of other described in U.S. Pat. No. 4,808.405); and the like. symptoms. Alternatively, treatment can also result in the 0242. The thrombolytic agents and the fibrin binding moi reduction in the number of blood vessels in an area of interest eties can be linked or fused in known ways, using the same or can prevent an increase in their number. Treatment can also type of linkers discussed above. Preferred linkers will be prevent or lessen the number or size of metastatic outgrowths Substituted or unsubstituted alkyl chains, amino acid chains, of the main tumor(s). polyethylene glycol chains, and other simple polymeric link 0239 Suitable examples of therapeutic agents according ers known in the art. More preferably, if the thrombolytic to the present invention include anticoagulant-thrombolytic agent is itself a protein, for which the encoding DNA or fibrinolytic agents capable of clots lysis, anti-angiogenic sequence is known, the thrombolytic protein and fibrin bind agents, cytotoxic agents including chemotherapeutic or ing polypeptide may be coexpressed from the same synthetic tumoricidal agents for selective killing and/or inhibiting the , created using recombinant DNA techniques. The cod growth of cancer cells and, especially, radiotherapeutic ing sequence for the fibrin binding polypeptide may be fused agents. in frame with that of the thrombolytic protein, such that the 0240. In one embodiment the therapeutic agent is a throm peptide is expressed at the amino- or carboxy-terminus of the bolytic or fibrinolytic agent. The fibrin-binding peptides of thrombolytic protein, or at a place between the termini, if it is the present invention can be used to improve the activity of determined that such placement would not destroy the thrombolytic and anti-angiogenic agents by improving their required biological function of either the thrombolytic pro affinity for fibrin and their residence time at a fibrin clot or at tein or fibrin binding polypeptide. A particular advantage of a site of pathological angiogenic activity. In this aspect of the this general approach is that concatamerization of multiple, invention, hybrid thrombolytic agents are provided by conju tandemly arranged fibrin binding polypeptides is possible, gating a fibrin binding polypeptide according to the invention thereby increasing the number and concentration of fibrin with a thrombolytic agent. Likewise, anti-angiogenic agents binding sites associated with each thrombolytic protein. In are provided by conjugating a fibrin-binding polypeptide this manner fibrin binding avidity is increased which would according to the invention with an anti-angiogenic agent. The be expected to improve the efficacy of the recombinant thera fibrin binding polypeptide portion of the conjugate causes the peutic protein. thrombolytic to “home' to the sites of fibrin clots or sites of 0243 In addition to thrombolytic agents, the fibrin bind angiogenesis, and to improve the affinity of the conjugate for ing peptides according to this invention can be used to deliver Such sites, so that the thrombolytic oranti-angiogenic activity other active agents to sites of fibrin in vivo or in vitro. For of the conjugate is more localized and concentrated at the example, Small molecule therapeutics or other therapeutic sites of interest. agents may be linked to one or more fibrin binding peptides US 2010/0158814 A1 Jun. 24, 2010 37 and the conjugate administered to a Subject or introduced to a 0247 The quantity of material administered will depend fibrin-containing Solution, and the fibrin-binding properties on the seriousness of the thromboembolic condition and posi of the conjugate will concentrate the Small molecule orthera tion and size of the clot. The precise dose to be employed and peutic agent at the sites of fibrin accumulation. In a particu mode of administration must perforce in view of the nature of larly preferred aspect, the fibrin binding peptides of the inven the complaint be decided according to the circumstances by tion may be used to deliver agents which are active in the the physician Supervising treatment. In general, dosages of presence of fibrin, such as angiogenesis promoters (e.g., the fibrin binder/thrombolytic agent conjugate will follow the fibroblast growth factor). The fibrin binding peptides may dosages that are routine for the thrombolytic agent alone, also be used to increase the blood clearance half-life of a although the improved affinity for fibrin added by the fibrin compound or drug, by causing accumulation of the com binder component may allow a decrease in the standard pound or drug in fibrin clots, from which it will be gradually thrombolytic dosage. Particular thrombolytics contemplated released. for use in this therapy (with examples of dose and method of 0244. The fibrin-binding polypeptides of the present administration) are as follows: invention may also be used to target genetic material to spe 0248. 1 streptokinase 1.0-3.0 megaunits over 30 min cific cells. For example, the binding peptides of the present utes to 3 hours anistreplase 30 units; 2-5 minute injec invention may be used to localize genetic material to cells or tion tPA (wild-type) 50-150 mg; infusion over up to 6 tissue containing fibrin. Thus such constructs may be useful hours two-chain urokinase 40-100 mg; infusion over up in gene therapy. The genetic material may include nucleic to 6 hours single-chain urokinase (3-12 megaunits) acids, such as RNA or DNA, of either natural or synthetic 30-100 mg; infusion over up to 5 hours hybrid plasmi origin, including recombinant RNA and DNA and antisense nogen 20-100 mg; injection or infusion activators and RNA and DNA. Types of genetic material that may be used derivatives muteins of plasminogen 10-100 mg; injec include, for example, carried on expression vectors tion or infusion activators Such as plasmids, phagemids, cosmids, yeast artificial chro 0249. In preferred features, the fibrin binding moiety is mosomes (YACs) and defective or “helper' viruses, antigene linked to the thrombolytic agent with a linker encompassing nucleic acids, both single and double stranded RNA and DNA an enzymatic cleavage site, e.g., an enzymatic cleavage site and analogs thereof. Such as phosphorothioate and phospho normally cleaved by enzymes in the coagulation cascade, rodithioate oligodeoxynucleotides. Additionally, the genetic Such as Factor Xa, thrombin, or plasmin cleavage sites, etc. material may be combined, for example, with lipids, proteins The thrombolytic agent preferably would not be activated or other polymers. Delivery vehicles for genetic material may until it is cleaved from the fibrin binding moiety at the site of include, for example, a virus particle, a retroviral or other the clot. Since cleavage of the thrombolytic agent would gene therapy vector, a liposome, a complex of lipids (espe occur at the site of the clot, the risk of unwanted bleeding cially cationic lipids) and genetic material, a complex of events at sites distant from the clot would be minimized. dextran derivatives and genetic material, etc. 0250 Alternatively, a therapeutic thrombolytic can be 0245. In a preferred embodiment the binding polypeptides loaded into an ultrasound vesicle that has been derivatized on of the invention are utilized in gene therapy for treatment of its surface with the fibrin binding moieties of the present diseases associated with angiogenesis. In this embodiment, invention. The vesicle may also be filled with an ultrasound genetic material, or one or more delivery vehicles containing efficient gas, such as, but not limited to, perfluoropropane or genetic material, e.g., useful in treating an angiogenesis-re perfluorobutane. Once the fibrin-specific vesicle has homed lated disease, may be conjugated to one or more fibrin-bind to the site of a thrombus, as monitored by ultrasound, the ing peptides of the invention and administered to a patient. frequency and energy of the ultrasound waves administered 0246. In the above treatment methods, the compounds can be altered to result in a controlled release of the throm may be administered by any convenient route customary for bolytic at the site of the thrombus (see, e.g., WO 93/25241). thrombolytic or therapeutic agents, for example parenterally, 0251. As discussed above fibrin-binding peptides of the enterally or intranasaly, and preferably by infusion or bolus present invention also can be used to improve the activity of injection, or by depot or slow release formulation. In a pre therapeutic agents such as anti-angiogenic or tumoricidal ferred embodiment, the composition may be formulated in agents against undesired angiogenesis Such as occurs in neo accordance with routine procedures as a pharmaceutical com plastic tumors, by homing in on areas undergoing angiogen position adapted for intravenous administration to human esis so that the therapeutic activity can be more localized and beings. Typically, compositions for intravenous administra concentrated at the sites of angiogenesis. tion are solutions in sterile isotonic aqueous buffer. Where 0252. In this aspect of the invention, hybrid agents are necessary, the composition may also include a solubilizing provided by conjugating a fibrin-binding polypeptide accord agent and a local anaesthetic Such as lignocaine to ease pain at ing to the invention with a therapeutic agent. The therapeutic the site of the injection. Generally, the ingredients will be agent may be a radiotherapeutic, discussed above, a drug, Supplied either separately or mixed together in unit dosage chemotherapeutic or tumoricidal agent, genetic material or a form, for example, as a dry lyophilized powder or water free gene delivery vehicle, etc. Such conjugates will be useful in concentrate in a hermetically sealed container Such as an treating angiogenesis-associated diseases, especially neo ampoule or Sachette indicating the quantity of active agent in plastic tumor growth and metastasis, in mammals, including activity units. Where the composition is to be administered by humans, which method comprises administering to a mam infusion, it can be dispensed with an infusion bottle contain mal in need thereof an effective amount of a fibrin-binding ing sterile pharmaceutical grade “water for injection' or polypeptide according to the invention conjugated with a saline. Where the composition is to be administered by injec therapeutic agent. The invention also provides the use of Such tion, an ampoule of sterile water for injection or saline may be conjugates in the manufacture of a medicament for the treat provided so that the ingredients may be mixed prior to admin ment of angiogenesis associated diseases in mammals, istration. including humans. US 2010/0158814 A1 Jun. 24, 2010

0253) Suitable therapeutic agents for use in this aspect of 0258 Pharmaceutically acceptable carriers, adjuvants and the invention include, but are not limited to: antineoplastic vehicles that may be used in the pharmaceutical compositions agents, such as platinum compounds (e.g., spiroplatin, cispl of this invention include, but are not limited to, ion exchang atin, and carboplatin), methotrexate, adriamycin, mitomycin, ers, alumina, aluminum Stearate, lecithin, serum proteins, ansamitocin, bleomycin, cytosine, arabinoside, arabinosyl Such as human serum albumin, buffer Substances such as adenine, mercaptopolylysine, Vincristine, buSulfan, chloram phosphates, glycine, Sorbic acid, potassium Sorbate, partial bucil, melphalan (e.g., PAM, L-PAM, or phenylalanine mus glyceride mixtures of Saturated vegetable fatty acids, water, tard), mercaptopurine, mitotane, procarbazine hydrochlo salts or electrolytes, such as protamine Sulfate, disodium ride, dactinomycin (actinomycin D), daunorubcin hydrogen phosphate, potassium hydrogen phosphate, Sodium hydrochloride, doxorubicin hydrochloride, taxol, mitomycin, chloride, Zinc salts, colloidal silica, magnesium trisilicate, plicamycin (mithramycin), aminoglutethimide, estramustine polyvinyl pyrrolidone, cellulose-based substances, polyeth phosphate Sodium, flutamide, leuprolide acetate, megestrol ylene glycol, Sodium carboxymethylcellulose, polyacrylates, acetate, tamoxifen citrate, testoiactone, triloStane, amsacrine waxes, polyethylene-polyoxypropylene-block polymers, (m-AMSA), aparaginase (L-aparaginase), Erwina aparagi polyethylene glycol and wool fat. nase, etoposide (VP-16), interferon cx-2a, Interferon cx-2b, 0259. The pharmaceutical compositions of this invention teniposide (VM-26, vinblastine sulfate (VLB), Vincristine may be administered by a variety of routes or modes. These Sulfate, bleomycin Sulfate, adriamycin, and arabinosyl; anti include, but not limited, to oral, intratracheal, Sublingual, angiogenic agents such as tyrosine kinase inhibitors with pulmonary, topical, rectal, nasal, buccal, vaginal, parenteral, activity toward signaling molecules important in angiogen or via an implanted reservoir. Implanted reservoirs may func esis and/or tumor growth such as SU5416 and SU6668 (Su tion by mechanical, osmotic, or other means. The term gen/Pharmacia & Upjohn), endostatin (EntreMed), angiosta parenteral as used herein includes intraperitoneal, paraverte tin (EntreMed), Combrestatin (Oxigene), cyclosporine, bral, periarticular, periostal, Subcutaneous, intracutaneous, 5-fluorouracil, vinblastine, doxorubicin, paclitaxel, intravenous, intra-arterial, intramuscular, intra-articular, daunorubcin, immunotoxins; coagulation factors; antivirals intrasynovial, intrasternal, intrathecal, intralesional and Such as acyclovir, amantadine azidothymidine (AZT or intracranial injection or infusion techniques. Zidovudine), ribavirin and vidarabine monohydrate (adenine 0260 Such compositions are preferably formulated for arahinoside, ara-A); antibiotics, antimalarials, antiprotoZo parenteral administration, and most preferably for intrave ans. Such as chloroquine, hydroxychloroquine, metroidazole, nous or intra-arterial administration. Generally, and particu quinine and meglumine antimonate; anti-inflammatories larly when administration is intravenous or intra-arterial, Such as diflunisal, ibuprofen, indomethacin, meclofenamate, pharmaceutical compositions may be given as a bolus, as two mefenamic acid, naproxen, oxyphenbutaZone, phenylbuta or more doses separated in time, or as a constant or non-linear Zone, piroxicam, Sulindac, tolmetin, aspirin and salicylates. flow infusion. 0254 As used herein the term “therapeutic’ includes at 0261) Details concerning dosages, dosage forms, modes least partial alleviation of symptoms of a given condition. The of administration, composition and the like are further dis fibrin-binding peptides and conjugates of the present inven cussed in a standard pharmaceutical text, such as Reming tion do not have to produce a complete alleviation of symp ton's Pharmaceutical Sciences, 18th ed., Alfonso R. Gennaro, toms to be useful. For example, treatment of an individual can ed. (Mack Publishing Co., Easton, Pa. 1990), which is hereby result in a decrease in the size of a tumor or diseased area, or incorporated by reference. a blood clot or prevention of an increase in size of the tumor 0262. As discussed supra, one embodiment of the present or diseased area or partial alleviation of other symptoms. invention relates to novel compounds comprising a fibrin Treatment can result in reduction in the number of blood binding moiety conjugated with at least one diagnostically or vessels in an area of interest or can prevent an increase in the therapeutically active moiety. number of blood vessels in an area of interest. Treatment can 0263. In particular, a preferred embodiment of the present also prevent or lessen the number or size of metastatic out invention includes compounds of general Formula (I) growths of the main tumor(s). 0255 Pharmaceutical Applications A-Y(-T). (I) 0256 Whether the fibrin binding moieties are to be used in 0264 wherein patients for detection and diagnosis or to facilitate the 0265 A is a fibrin-binding peptide moiety comprising an therapy. Such uses require that they be treated as pharmaceu amino acid sequence selected from the group consisting of tical agents. Pharmaceutical compositions of this invention the sequences provided in Table 1 or Table 2: comprise any of the compounds of the present invention, and 0266 Y is a suitable linking moiety connecting A with at pharmaceutically acceptable salts thereof, with any pharma least one T, when s is 2, the units Y may be the same or ceutically acceptable ingredient, excipient, carrier, adjuvant different from each other; or vehicle. 0267 T is, independently in each occurrence, a diagnos 0257 Pharmaceutical compositions of this invention can tically or therapeutically active moiety; be administered to mammals including humans in a manner 0268 s is 1 or 2, similar to other diagnostic or therapeutic agents. The dosage 0269 r is, independently in each occurrence, an integer to be administered, and the mode of administration will from 1 to 8: depend on a variety of factors including age, weight, sex, 0270 or a physiologically acceptable salt thereof. condition of the patient, and genetic factors, and will ulti 0271 Unless otherwise specified, the phrases “fibrin mately be decided by the attending physician or veterinarian. binding peptide moiety' or, simply, peptide moiety.” used In general, dosage required for diagnostic sensitivity orthera herein interchangeably, refer to a suitable derivative of the peutic efficacy will range from about 0.001 to 50,000 g/kg, corresponding fibrin-binding peptide of the sequences dis more usually 0.01 to 25.0 ug/kg of host body mass. closed herein, in which one or both of the N-terminal US 2010/0158814 A1 Jun. 24, 2010 39

( NH) and the C-terminal ( COOH) groups of the peptide cally active moiety or moieties T conjugated, through a Suit are functionalized through formation of a carboxamido bond able linking moiety Y, to the N-terminal ( NH) or, con with Y. If the C-terminal or N-terminal group is not function versely, to the C-terminal ( COOH) group of the peptide alized, it may be suitably protected or deactivated. Thus, moiety, thus resulting in a compound of Formula (I) in which “fibrin-binding peptide moiety” refers to that moiety result the peptide moiety A is functionalized at only one of its N- or ing from the original amino acid sequence of the fibrin C-terminal groups. binding peptide, following the said optional protection/deac tivation and the said carboxamido bond(s) formation. 0279 According to another preferred aspect of the inven 0272 Typically, for instance, in the case of the fibrin tion, within the compounds of Formula (I), S is 2. The com binding peptide having the amino acid sequence of Seq005 as pounds of the invention in which S-2 include, independently shown in Table 1, H.N. WQPCPAESWTFCWDP COOH in each occurrence, one or more diagnostically of therapeu (SEQ ID NO. 1), the corresponding fibrin-binding peptide tically active moiety or moieties T conjugated, through a suitable linking moiety Y. to each of the N-terminal ( NH) moieties include, for instance.: and the C-terminal ( COOH) groups of the peptide moiety A, thus resulting in compounds of Formula (I) in which the -HN-WOPCPAESWTFCWDP-CO peptide moiety is functionalized at both of the N- and C-ter minal groups. Pg-HN-WQPCPAESWTFCWDP-CO-, 0280 According to another preferred aspect of the inven and tion, within the compounds of Formula (I), ris an integer from 1 to 5. 0273 in which Pg is a suitable protecting/deactivating 0281. In one preferred embodiment of the invention Y is a group. linear or branched divalent linking moiety. The phrase “diva 0274. In the present invention, unless otherwise indicated, lent linking moiety' (or “divalent linker, used herein inter the phrase “protecting group', designates a protective group changeably) is intended to include a chain including a func adapted to preserving the function of the functional group to tional group which permits the conjugation of the linking which it is bound. Specifically, protective groups are used to moiety with the N- or the C-terminal group of A and, a second preserve amino function or carboxyl function. Appropriate functional group which permits conjugation with a diagnos protective groups may include, for example, benzyl, benzy tically or a therapeutically effective moiety T. loxycarbonyl, alkyl or benzyl esters, or other substituents 0282 Unless otherwise indicated, the term “functional commonly used for the protection of Such functions, which group' as used herein refers to specific groups of atoms are well known to those skilled in the art, for example Fmoc, within molecules or moieties that are responsible for the and protective groups described in conventional manuals characteristic chemical reactions of those molecules or moi such as T. W. Green, Protective Groups in Organic Synthesis eties. In the context of the invention, the functional groups (Wiley, N.Y. 1981). include the specific, suitably protected or suitably activated 0275 Unless otherwise indicated, the phrase “deactivat —NH terminal or —COOH terminal groups of the peptide ing group as used herein, refers to chemical groups that are moiety, of the linking moiety and of the diagnostically or able to react with the N-terminal ( NH) or the C-terminal therapeutically active moiety. In addition, these functional (—COOH) group of the peptide unit transforming it, through groups may include any other amine, thiol, carboxyl group a chemical reaction, into a suitable derivative thereof that present as a free group or as an optionally activated reactive maintains the specificity of the corresponding peptide moiety group on the said linking, diagnostically or therapeutically toward fibrin, but is unable to chemically react with, respec active moiety, thus allowing other cross-linking or coupling tively, a ( COOH) oran ( NH) functionality on a different reactions. In a preferred aspect of the present invention, the moiety, and thus may not be involved in carboxamido cross functional group are Suitably protected or Suitably activated linking reaction. —NH2 or —COOH groups allowing cross-linking reaction 0276 Suitable examples of deactivating groups comprise through carboxamido bonds ( NHCO ) and ( CONH ) Ac, where Ac is CH(CO)— when used, for instance, to formation. deactivate an ( NH) terminal group of the peptide chain to 0283 Preferably, the divalent linking moiety is a linear the corresponding, unreactive, AcHN— group. On the other C-Coo and, more preferably, a C-Cs, and, most preferably, side, NH or NH(CH), may be, for instance, used to a C-Cls alkyl chain, that is optionally interrupted by one or deactivate a terminal —COOH group by providing the cor more groups selected from —O—, —CONH-, -CO—, responding—CONH2 or —CONHCCH) unreactive amide. —NR—and—NHCO—, and optionally substituted by one 0277 According to a preferred aspect of the invention, or more —R group(s), wherein R is H or a C-C alkyl within the compounds of Formula (I), A is a fibrin-binding group, and R is a —CONH2 group or a (C-Cs)alkyl group, peptide moiety comprising the amino acid sequence of that is optionally substituted, in its turn, by a CONH2 group Seq005 as shown in Table 1, H.N. WQPCPAESWT or by an optionally Substituted benzene ring. As discussed FCWDP COOH (SEQID NO.1), in which each of the W, Q, Supra, the said divalent linker includes two functional groups P. C. A. E. S, T, F and D has the meaning conventionally connecting it, respectively, with A and with T. adopted when defining amino acids according to one letter 0284. Unless otherwise provided, the term “(C-C) alkyl code and in which the Camino acids in positions 4 and 12 are as used herein, designates a linear or branched, saturated or bonded to each other through a disulfide ( S S ) bond. unsaturated alkyl Substituent comprising from 1 to 5 carbon 0278. According to another preferred aspect of the inven atoms Such as, for example, methyl, ethyl, propyl, isopropyl. tion, within the compounds of Formula (I), S is 1. The com butyl, tert-butyl, penty1 and the like, wherein methyl, ethyl pounds of the invention having S-1 include, independently in and propyl are preferred. More preferably, the divalent link each occurrence, one or more diagnostically of therapeuti ing moiety Y includes one or more Sub-units selected, for US 2010/0158814 A1 Jun. 24, 2010 40 instance, from homobifunctional and heterobifunctional preferably, from 2 to 5 equal or different diagnostically or units and suitable combinations thereof. therapeutically effective moieties. 0285 Unless otherwise provided, the term “homobifunc 0314 Preferably, the said polyfunctional linker Y is a lin tional unit or moiety, refers to a unit or moiety having at least ear or branched C-Clso and, preferably, a C-C and, more two reactive functional groups which are the same. preferably, a C-C-7s alkyl chain, that is optionally interrupted 0286. Unless otherwise provided, the term "heterobifunc by one or more groups selected from —O—, —CONH . tional unit or moiety, refers to a unit or moiety having at least —CO——NHCO and —NR, and optionally substituted two different reactive groups. by one or more —R group(s), wherein R is H or a C-Cs 0287 Suitable examples of homobifunctional units alkyl group optionally substituted by a -COOH or NH include, for instance, dicarboxylic moieties and diamine moi group, and R may be a group selected from —NH . eties having formula, respectively, —COOH or a derivative thereof including, for instance, 0288 –OC Z CO , and lower alkyl esters or—CONHamide, a (C-Cs)alkyl option 0289 NH Z NH , ally substituted by a group selected from —COOH, where Z is a chain preferably selected from the following: —CONH and NHR or by an optionally substituted ben 0290 —CH2—(CH), , Zenering, the chain further including at least three functional 0291 —CH (CHO), , groups connecting the polyfunctional moiety with A and each 0292 -(CH2(CH)O), (CH), , of the said remaining functional groups with a diagnostically 0293 -(CH), NHCO-(CH), , or therapeutically effective moiety T. 0294) —(CH), NHCO CH-O-(CH2(CH)O), 0315 Suitable examples of the said polyfunctional linking (CH2) , moiety may include, for instance: 0295) -(CH2). CH(R)-(CH2), , 0316 (a) N-branched lysine systems (see, f. i., Veprek, 0296 (CH2). CH(R)-(CH2), NHCO CHO Petal., J. Pept. Sci. 5, 5 (1999); 5, 203 (1999), (CH-(CH2)O), (CH), , 0317 (b) Polycarboxylic compounds and suitable where n=1-10, m=1-5 and p-0-5,and derivatives thereof in derivative thereof in which the carboxylic group(s) are in which the carboxylic and the amino group(s) are in a Suitably a suitably activated or protected form, activated or protected form. 0318 (c) polyaminated compounds and suitable deriva 0297 Suitable examples of heterobifunctional units, for tive thereof in which the amino group(s) are in a Suitably instance, include: activated or protected form, 0298) - HN-(CH), CO , 0319 (d) amino acids and poly-amino acids such as 0299 HN-(CH), CH(R) CO , polyornithine, polyarginine, polyglutamic acid, polyas 0300 —HN-(CH2). O—(CH), O CH CO . partic acid. (0301 - HN (CH), CH(R)-(CH.), CO , 0320 In a preferred aspect of the invention, the polyfunc 0302) —OC CH(NR)-(CH), NH , tional linking moiety Y includes one ore more sub-unit(s) 0303 —OC (CH) —NHOC (CH)O—(CH), selected from the above homobifunctional and heterobifunc NH , tional units and one or more sub-unit(s) selected from the (0304) —HN-(CH.). CH(R)-(CH), NHCO following: CHO-(CH2(CH2)O), (CH), NH-, 0321 - HN-(CH), CH(NR) CO , wherein n, m and p are as above defined, and Suitable com 0322 –OC CH(NR)-(CH), NH-, binations thereof. 0323 —OC (CH), NR (CH), CO , 0305 More preferably, the divalent linking moiety Y of 0324) - HN CH(R)-(CH2)CO , the invention comprises one of the following units: 0325 —HN CH(R)-(CH), NH , (0306 —HN CH-CO , 10326) HN-(CH2) (CH) CH(R)) (CH), 0307 – OC (CH), CO , NH 0308 HN—(CH), O (CH), O—CH CO . (0327 - OC (CH,), ((CH.). CH(NR)-(CH), 0309 HN CH(CONH)–(CH), NH , NH 0310 NH CH(CONH) (CH), NHCO (CH) where n, m, p. R. and Ra have the above defined meaning CO—, 0328. In a particularly preferred aspect, the said polyfunc 0311 –CO CH-O-(CH), 13 O-(CH). NHCO tional Y moiety comprises one of the following sub-units: CHO (CH), O—(CH). NH , 0329 -Gly-Gly-Gly-Lys, or a suitable repetition and/or combination thereof 0312. In a particularly preferred aspect of the invention, (0330) —OC-(CH,), ((CH.). CH(NH))–(CH.) the divalent linking moiety Y is or comprises the unit-Gly NH-, Gly-Gly-Lys in which Gly is Glycine and Lys is Lysine, also 0331 —HN CH(CONH)–(CH), NHCO CH referred to as GGGK. O—(CH2) -O-(CH2). NH-, 0313. In a different embodiment of the invention, Y is a 0332 - OC CH(NH) (CH), NHCO CH linear or branched polyfunctional linking moiety. Unless oth (NH)–(CH), NH , erwise provided, “polyfunctional linking moiety', and “poly 0333 – OC (CH), CO , functional linker, used herein interchangeably, refer to a 0334 HN CH(COOH) (CH), NH , linear or branched chain including at least 3, preferably from 0335) - HN CH, CO , 3 to 8 and, more preferably, from 3 to 5 functional groups, one 0336 - OC (CH), CO , of them connecting the said polyfunctional moiety with the 0337 – HN-(CH), O-(CH), O CH-CO , N-terminal ( NH) or the C-terminal group (-COOH) 0338 –OC CH-O-(CH), O (CH). NH group of A and the remaining connecting the polyfunctional or a suitable repetition and/or combination thereof in which moiety with at least 2, preferably, from 2 to 7, and, more each of the HN and HOOC groups of the sub-unit US 2010/0158814 A1 Jun. 24, 2010 allows carboxamido cross-linking reactions resulting in the 44, 49 and between 57 and 83. More preferred are paramag possible elongation and/or ramification of the linking moiety. netic metal ions selected from the following: Fe(2+), Fe(3+), 0339. Any intermediate compounds according to the Cu(2+), Ni(2+), Rh(2+), Co(2+), Cr(3+), Gd(3+), Eu(3+), invention including, for instance, a peptide moiety A conju Dy(3+), Tb(3+), Pm(3+), Ndo 3+), Tm(3+), Ce(3+), Y(3+), gated with a suitable, optionally protected Y moiety or with a Ho(3+), Er(3+), La(3+), Yb(3+), Mn(3+), Mn(2+) wherein Suitable, optionally protected, Sub-unit of a Y moiety consti Gd(3+) is the most preferred. 0351. The phrase “contrast imaging agent' or “contrast tutes a further object of the present invention. These interme agent” refers to any detectable entity that can be used to in diates and their preparations are, for instance, detailed herein vitro visualize or detect fibrin units or fibrin deposition into or below, in the experimental section. on a biological element including cells, biological fluids and 0340. As a non limiting example, FIG. 1 includes the biological tissues originating from a live mammal patient, synthetic procedure for the preparation of a linker function and preferably, from human patient, as well as the in vivo alized peptide moiety of the invention (Seq005-P) in which identification and location offibrin and fibrin deposition in or the peptide moiety is the one comprising the amino acid on mammalian and, preferably, human body organs, regions sequence of Seq005 as shown in Table 1, and the conjugated or tissues when the said detectable entity is used in associa linking moiety is -GGGKJJ- (SEQID NO. 139) (where J is tion with a suitable diagnostic imaging technique. the Fmoc-8-amino-3,6-dioxaoctanoic acid; see Aldrich Neo 0352. The phrase “chelator”, “chelating ligand’ or system and Peptides International catalogue). “chelating agent, used herein interchangeably, refers to 0341 These novel intermediates including, the peptide chemical moieties, agents, compounds, or molecules charac moiety conjugated with a Suitable linking unit or a Suitable terized by the presence of polar groups able to a form a sub-unit thereof find application as intermediates for the complex containing more than one coordinate bond with a preparation of the compounds of Formula (I). transition metal or another metal entity. In a preferred aspect 0342. According to a preferred aspect of the invention, of the invention the chelating ligands include cyclic or linear within the compounds of Formula (I), T is a diagnostically polyamino polycarboxylic orphosphonic acids and contain at effective moiety or a radiotherapeutic moiety. least one amine, thiol, carboxyl group, present as free, option ally activated functionality that is suitable for use in the con 0343. The phrase “diagnostically effective moiety” or jugation reaction with a functional group of the spacer chain “imaging effective moiety', used herein interchangeably, Y. refers to any moiety detectable by imaging diagnostic proce 0353. The expression a “residue of a chelating agent', or a dures, that is to say any moiety able to provide, to improve or, “residue of a chelating ligand, used herein interchangeably, in any Way, to advantageously modify the signal detected by refers to that portion of the chelating ligand remaining after an imaging diagnostic technique including, for instance, the above conjugation. Preferably the conjugation is from an magnetic resonance imaging (MRI), radioimaging, X-ray acidic group on the chelating ligand or a Suitable derivative imaging, light imaging, thus enabling the registration of diag thereof with an amino group ( NH2) of the linking moiety Y. nostically useful, preferably contrasted, images when used in or, alternatively, between a Suitable reactive amino group of association with Such techniques. the chelating ligand and a terminal carboxy group (-COOH) 0344 Examples of diagnostically effective moieties of the Y moiety, or a suitable derivative thereof, so as to give according to the invention include, for instance, chelated rise to a carboxamido linkage. The acidic or the reactive gamma ray or positron emitting radionuclides; paramagnetic amino group of the chelating ligand involved in the crosslink metal ions in the form of chelated or polychelated complexes, ing reaction is suitably selected in order to not reduce or X-ray absorbing agents including atoms of atomic number modify the chelating capability of the ligand residue. 0354) The term “labelled” or “complexed' used in the higher than 20; a dye molecule; a fluorescent molecule; a context of a “chelating ligand labelled with a metal element'. phosphorescent molecule; a molecule absorbing in the UV refers to the formation of a chelate or coordinate complex spectrum; a quantum dot; a molecule capable of absorption between the metal and the chelating ligand. within near or far infrared radiations; moieties detectable by 0355 The term “metal entity” refers to a metalion that is ultrasound and, in general, all moieties which generate a detectable by an imaging technique. Such metal entities spe detectable substance. The skilled person in the art well know cifically include paramagnetic metal ions that are detectable that the imaging modality to be used have to be selected by imaging techniques such as Magnetic Resonance Imaging according to the imaging detectable moiety the diagnostic (MRI), or to a metal ion (e.g. radionuclide) that is detectable compounds of the invention include. by imaging techniques Such as Scintigraphic imaging, Single (0345 MRI Contrast Agents Photon Emission Computed Tomography (SPECT) and 0346. In a particularly preferred embodiment of the inven Positron Emission Tomography (PET) or even a radionuclide tion, within the compound of Formula (I), T is an MRI detect for therapy. able moiety. 0356 Suitable chelating ligands include those discussed (0347 Compounds of Formula (I) in which T is a MRI herein, particularly chelating ligands selected from the group detectable moiety are preferred for use as MRI contrast consisting of a polyaminopolycarboxylic acid and the agents. derivative thereof, comprising, for example, diethylenetri 0348 Accordingly, in one preferred aspect, the present amine pentaacetic acid (DTPA) and derivative thereof such as invention relates to novel MRI contrast agents of Formula (I) benzo-DTPA, dibenzo-DTPA, phenyl-DTPA, diphenyl in which T is a MRI detectable moiety. DTPA, benzyl-DTPA, dibenzyl DTPA; N,N-bis(2-(car (0349 Preferably, the said MRI detectable moiety com boxymethyl)(methylcarbamoyl)methylaminoethyl-gly prises the residue of a chelating ligand labelled with a para cine (DTPA-BMA); N-2-bis(carboxymethyl)amino-3-(4- magnetic metal element detectable by Magnetic Resonance ethoxyphenyl)propyl)-N-2-bis(carboxymethyl)amino Imaging (MRI) techniques. ethylglycine (EOB-DTPA); 4-carboxy-5,8,11-tris 0350 Preferred paramagnetic metal elements are those (carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13 having atomic number ranging between 20 and 31.39, 42, 43. oic acid (BOPTA); N,N-Bis 2-bis(2-(1,1-dimethylethoxy)- US 2010/0158814 A1 Jun. 24, 2010 42

2-oxoethylaminoethyl-L-glutamic acid 1-(1,1-dimethyl ethyl) ester N,N-bis(2-bis(carboxymethyl)aminoethyl L -continued glutamic acid (DTPA-GLU); DTPA conjugated with Lys CO2H (DTPA-Lys); ethylenediaminetetraacetic acid (EDTA): 14, 7,10-teraazacyclododecane 1,4,7-triacetic acid (DO3A); 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA); 10-(2-hydroxypropyl)-1,4,7,10- teraazacy clododecane 1,4,7,-triacetic acid (HPDO3A); 6-bis(car boxymethyl)aminoltetrahydro-6-methyl-1H-1,4-diazepine 1,4(5H)-diacetic acid (AAZTA) provided by WO03008390 application, incorporated herein by reference, and derivative thereof 2-methyl-1,4,7,10-tetraazacyclotetradecane-1,4,7, 10-tetraacetic acid (MCTA); (C.O.'.O.".C.")-tetramethyl-1,4,7, 10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTMA); or a polyaminophosphate acid ligand or deriva tive thereof, in particular, N,N'-bis-(pyridoxal-5-phosphate) ethylenediamine-N,N'-diacetic acid (DPDP), ethylenedini trilotetrakis(methylphosphonic) acid (EDTP) or a polyaminophosphonic acid ligand and derivative thereof, or polyaminophosphinic acid and derivative thereof, in particu lar 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetrakisme thylphosphonic) and 1,4,7,10-tetraazacyclotetradecane-1,4, 7,10-tetrakis methylene-(methylphosphinic) acid; or of NH2 macrocyclic chelators such as texaphyrins, porphyrins, phthalocyanines 0357 Preferred ligand according to the present invention include those of FIGS. 6a to 6c, which also include suitable bibliographic references concerning their preparation. 0358 Particularly preferred are: DTPA, DTPA-GLU, DTPA-Lys, DOTA, AAZTA, and the following derivatives thereof: HOC HOC- -COH COH N Hoc1N CO2H N 1-4 N HOC N 7

HN N N l Y CO2H CO2H 0359 Examples of particularly preferred MRI contrast agents of the invention comprise:

Chelated complex 1

S S H H N N CONH COONa h Kl HO HO CHCONH CONH CON C O 3 CONH CON CONH CONH CONH CONH CONH CONH CONH N H2NOCN, HNOCN, HNOCN, HNOCN, HNOC HNOC NH Gd3+\-COO OOC N 2Na" NaOOC /-COO N O \-COO NHoo1 No1 N1,N1 NH N 1n 1n 1n OCHIN, OCHIN, OCHN OCHN OCHN OCHN n-1-1-1 os-sO H

US 2010/0158814 A1 Jun. 24, 2010 44

-continued

Chelated complex 3

S S H H N N CONH2 COONa h Kl HO HO CONHl CONH CON CONH CONKl CONH CONH CONH CONH CONH CCONH CONH O NHCONHCONHCONHCOCH, H:NOCN-HNOCN-HNOCN-HNOCN-HNOC CS HNOC NH

NaOOC

N H

OOC COONaGd OOCN COO OOC N r COONaGd Jh-rOCHNN-COHN NHCO NHCO N7

ls COO OOC COONaGd OOC COONaGd o N- OCHN ls- OCHN Vy - Vy - NCOO NCOO

Chelated complex 4

H H N N CONH2 COONa Kl h HO HO CHCONH CONH CON CONH CONKl CONH CONH CONH CONH CONH CCONH CONH O H2NOCN, HNOCN, HNOCN, HNOCN, HNOC CS HNOC NH

NaOOC

N H US 2010/0158814 A1 Jun. 24, 2010 45

-continued OOCN- COONaGd OOCN COONaGd ors so A-roin-on-oil N COO OOC COONaGd lus NHCO1 OOC o COONaGd OOC ls - OCHN N COO

N so OOC Chelated complex 5

H H N N coona (1 | a CONH l Sn - S - S4-S-S 4S

O CH3CONH CONH CON NCONH1.cN CONH CONH CONH CONH CONH CONH CONH

"O-C-N /-CO.Na HNOC HNOC a/ HNOC YaY HNOC n1 HNOC NOC HNOC NH

OC-VN Gd's N C NaOOC OC * NHCO CONH 'O.C- /-CO, NCO H OC -v Gd+3 N N

QN H O NaOC Chelated complex 6 H N N CONH COONal ar

CHCONH CONH CON CONH Kl CONH CONH CONH CONH CONH CCONH CONH O HNOC HNOC a1 HNOC ne/ HNOC NY HNOC C2J HNOC NH s t Gd+3> N / NHCONu-n-CONH NH ... IN V-CO2 Cl2.5H161GdN29Na3O37S2 2952.16 2950.998.382

US 2010/0158814 A1 Jun. 24, 2010 47

-continued Chelated complex 9 H H N GOONaan-Nn 4n. CONH S Sn N HO HO

CHCONH CONH Kl CONH CONH CONH CONH CONH CONH CCONH CONH O

HNOC r HNOC NH NaOOC

NaOOC COO NHCO N N- COO Gd'3 / \- NHOC) N7

OOC u C23H159GdNoNa3O37S2 2926.12 2924.98.2732 Chelated complex 10 S

N CONH2 COOH CH3CONH CONH Kl CONH 6.CONH CONH CONH") CONH CONHin CONH H:NOCs-HNOCN-HNOCs-HNOCs-HNOCN-NOCs HNOCN-NH COO COO EX

NHoo1 No1 N1 O n1 NNH NaOOC-/N-N-N-on------COONa

Nuclear Imaging (Radionuclide Imaging) and graphic imaging, Single Photon Emission Computed Tomog Radiotherapy raphy (SPECT) and Positron Emission Tomography (PET). 0360. In another preferred embodiment of the invention, 0364 Preferably, the radioimaging detectable moiety within the compounds of Formula (I), T is a radioimaging comprises the residue of a chelating agent labelled with a detectable moiety or a radiotherapeutic moiety. radionuclide detectable by the said scintigraphic, SPECT or 0361 Compounds of Formula (I) in which T is a radio PET imaging techniques. imaging detectable moiety are preferred for use as radio 0365. When T is a radiotherapeutic moiety, it preferably graphic contrast agents. comprises a radionuclide which is therapeutically effective. 0362 Accordingly, in another preferred embodiment, the In a preferred embodiment, the radiotherapeutic moiety com present invention further relates to novel radiographic con prises the residue of a chelating ligand labelled with a thera trast agents of Formula (I) in which T is a radioimaging peutically active radionuclide. detectable moiety. 0366. Together with the chelating ligands discussed 0363 A “radioimaging detectable moiety' refers to a moi above, Suitable examples of chelating ligands for radionu ety that is detectable by imaging techniques such as Scinti clides may be selected from linear, macrocyclic, terpyridine, US 2010/0158814 A1 Jun. 24, 2010 48 and NS, NS, or N. chelators (see, moreover, ligand dis closed, for instance, in U.S. Pat. No. 5,367,080, U.S. Pat. No. -continued 5,364,613, U.S. Pat. No. 5,021,556, U.S. Pat. No. 5,075,099, U.S. Pat. No. 5,886,142), and other chelating ligands known (8) in the art including, but not limited to, HYNIC, TETA and R R bisamino bisthiol (BAT) chelators (see also U.S. Pat. No. 5,720,934). For example, N. chelating ligands are described hoc-l NI \N 1-cool in U.S. Pat. Nos. 6,143,274; 6,093,382; 5,608,110; 5,665, 329; 5,656,254; and 5,688,487. Certain NS chelators are N N described in PCT/CA94/00395, PCT/CA94/00479, PCT/ COOH CA95/00249 and in U.S. Pat. Nos. 5,662,885; 5,976,495; and too-r \ I 5,780.006. The chelator may also include derivatives of the R chelating ligand mercapto-acetyl-acetyl-glycyl-glycine N (MAG3), which contains an NS, and NS systems such as MAMA (monoamidemonoaminedithiols), DADS (NS diaminedithiols), CODADS and the like. These ligand sys tems and a variety of others are described in Liu and Edwards, Chem Rev. 1999...99, 2235-2268 and references therein. (9) 0367 The chelator may also include complexes contain HOOC-N | \ -COOH ing ligand atoms that are not donated to the metal in a tet radentate array. These include the boronic acid adducts of C, D technetium and rhenium dioximes, such as are described in U.S. Pat. Nos. 5,183,653: 5,387,409; and 5,118,797, the dis closures of which are incorporated by reference herein, in their entirety. 0368 Preferred radionuclides according to the present invention include 99. Tc, 5. Cr, 67'Ga, 68 Ga., 47“’Sc, 67 Tm, 0370. In the above Formulas 7 and 8, R is an alkyl, pref 14 Ce, In, 113In, 168 Yb, 175Yb. 140La, 90Y. 88y. 'Sim, erably methyl. For "Tc, Re, and Re radionuclides, 166Ho, 165Dy, 166Dy Cu, 62Cu, 64C s 67C s 7Ru, 10Ru, particularly preferred are the following ligands: 186Re, 188Re, 208Pb, 2. Bi, 212Bi, 213Bi, 214Bi, 105Rh, 109Pd, 117'Sn, 14°Pm, 161Tb, 77Lu, Au, '''Ag, Au, 51M s 52mm s 52Fe, 60Cu, 7°As, 94mTc, lion, 142Pr and Ga. 0369. The choice of a suitable ligand residue depends on (10) the radionuclide used for the ligand labelling. Thus, preferred residues of chelating ligands include those of FIGS. 6a to 6c (for 'In lanthanides and radioactive lanthanides, including, for instance 77Lu, Y, Sm, and Ho or for 7Ga, Ga., Cu, Cu, Cu, or 7Cu) and those of FIGS. 7a to 7b (for radioactive "Tc, Re, and 'Re). In particular, for metal entities including '''In, lanthanides and radioactive lan thanides, particularly preferred are the following ligand resi dues (11)

(7)

(12)

NH HN

n 2 COOH

OH OH US 2010/0158814 A1 Jun. 24, 2010 49

are incorporated by reference herein in their entirety. Addi -continued tionally, the above chelating group of Formula 9 is described (13) HO in, for example, U.S. Pat. No. 6,143,274; the chelating groups of the above Formula 14 and 15 are described in U.S. Pat. O Nos. 5,627.286 and 6,093,382, and the chelating group of Formula 16 is described in, for example, U.S. Pat. Nos. 5,662, 885; 5,780,006; and 5,976,495. 0372. In the Formula 14 and 15, X is either CH, or O, Y is either C-C branched or unbranched alkyl: Y is aryl, ary O loxy, arylamino, arylaminoacyl: Y is arylkyl where the alkyl HN group or groups attached to the aryl group are C-Co branched or unbranched alkyl groups, C-C branched or unbranched hydroxy or polyhydroxyalkyl groups or poly alkoxyalkyl or polyhydroxy-polyalkoxyalkyl groups, J is NH HN C(=O)—, OC(=O) , SO, NC(=O) , NC(=S) , N(Y), NC(=NCH) , NC(-NH) , N=N homopolyamides or heteropolyamines derived from Syn thetic or naturally occurring amino acids; all where n is 1-100. s r COOH Other variants of these structures are described, for example, OH OH in U.S. Pat. No. 6,093,382. The disclosures of each of the foregoing patents, applications and references are incorpo as well as the following ligand residues: rated by reference herein, in their entirety. 0373 The choice of the radionuclide will be determined based on the desired therapeutic or diagnostic application. (14) For uses in radiotherapy or radiodiagnostics preferred radio r nuclides are Pb, 7 Ga, Ga, 7 As, '''In, In, Y, 7Ru, NH HN 6|Cu, 62Cu, 6.Cu, 52Fe, 52Mn, 140La, 175Yb, S.Sm. 166Ho, 149Pm, 177Lu, 14Pr, 159Gd, 212Bi,47Sc, 149Pm,07Cu, Ag, Au, 161Tb, 5 Cr, 167Tm, 14 Ce, 168Yb, 88y. 165Dy, 166Dy 7Ru, 103Ru, 186Re, 188Re, 99mTc, 211 Bi, 212Bi, 213Bi, 214Bi, s re 'Rh, 'Pd, '7"Sn, "77Snand 'Au and oxides and nitrides OH OH thereof. For example, for therapeutic purposes (e.g., to pro (15) vide radiotherapy for primary tumors and metastasis), the preferred radionuclides include Cu, 'Y. 'Rh, '''In, 117 Sn, 149Pm, 153Sm, 161Tb, 166Dy, 166Ho, 175Yb. 177Lu, J ''Re, and 'Au, with 77Lu and Y being particularly (-)pi preferred. For diagnostic purposes (e.g., to diagnose and monitor therapeutic progress in e.g. primary tumors and metastases) the preferred radionuclides include Cu, ''Ga, i Ga, Tc, and '''In. NH HN 10374) "Tc is particularly preferred for diagnostic appli cations because of its low cost, availability, imaging proper ties, and high specific activity. The nuclear and radioactive properties of "Tc make this isotope an ideal scintigraphic OH OH imaging agent. This isotope has a single photon energy of 140 (16) keV and a radioactive half-life of about 6 hours, and is readily OH available from a Mo "Tc generator. O 0375 Preferred metal radionuclides for use in PET imag O ing are positron emitting metal ions, such as "Mn, Fe, 6O Cu, 68 Ga, 72'As, 94."Tc, or '''In.O 0376 Preferred for scintigraphic applications are radiodi r HN ' agnostic contrast agents of Formula (I) wherein T is the N s1 residue of a chelating ligand of FIGS. 7a to 7b, labelled with / V radionuclide selected from "Tc and ''Re. More pre H3C CH3 ferred are those in which T is the residue of a chelating ligand NHCOCH of formula from 22 to 33. Particularly preferred for scinti graphic applications are contrast agents of Formula (I) 0371. These and other metal chelating groups are wherein T is a residue of a ligands of formula from 22 to 33 described in U.S. Pat. Nos. 6,093,382 and 5,608,110, U.S. labelled with "Tc. Pat. No. 6,143,274; U.S. Pat. Nos. 5,627,286 and 6,093,382, 0377 Preferred radionuclides for use in radiotherapy U.S. Pat. Nos. 5,662,885; 5,780,006; and 5,976,495 which include Cu, 90Y. 105Rh, In, 7Sn, 14°Pm, 'Sim, 161Tb, US 2010/0158814 A1 Jun. 24, 2010 50

166Dy 166Ho, 175Yb. 90Y. 186/188Re, and Au, with 177Lu ery of said construct to the site of injection, without and 'Y being particularly preferred. significant radioactive decay prior to injection. Prefer ably, the radionuclide should have a physical half-life PET Imaging between about 0.5 and 8 days. 0393 b. Energy of the emission(s) from the radionu 0378. In a still further embodiment of the invention, within clide—Radionuclides that are particle emitters (such as the compounds of Formula (I), T is an optionally labelled alpha emitters, beta emitters and Auger electron emit Sugar moiety for use, when labelled, in PET Imaging. ters) are particularly useful, as they emit highly ener 0379 Accordingly, in another preferred aspect, the getic particles that deposit their energy over short dis present invention relates to compounds of Formula (I) in tances, thereby producing highly localized damage. which T is a suitably labelled sugar moiety. Beta emitting radionuclides are particularly preferred, 0380. In a preferred embodiment of the invention, T as the energy from beta particle emissions from these includes a Sugar moiety labeled by halogenation with radio isotopes is deposited within 5 to about 150 cell diam nuclides, such as, “I, II, I, II, 77Br 7Br and F, eters. Radiotherapeutic agents prepared from these wherein F is particularly preferred. nuclides are capable of killing diseased cells that are relatively close to their site of localization, but cannot Therapeutically Effective Agents travel long distances to damage adjacent normal tissue 0381. In another embodiment of the invention, within the Such as bone marrow. compounds of Formula (I), T is a therapeutic active moiety. 0394 c. Specific activity (i.e. radioactivity per mass of 0382 Compounds of Formula (I) in which T is a therapeu the radionuclide)—Radionuclides that have high spe tic active moiety are new and constitute a further object of the cific activity (e.g., generator produced 'Y''' In, ''Lu) present invention. are particularly preferred. The specific activity of a 0383 Suitable examples of therapeutic active moieties radionuclide is determined by its method of production, according to the present invention include thrombolytic or the particular target for which it is produce, and the fibrinolytic agents capable of lysis of clots, or cytotoxic properties of the isotope in question. agents for selective killing and/or inhibiting the growth of for 0395. Many of the lanthanides and lanthanoids include example, cancer cells, and radiotherapeutic agents. radioisotopes that have nuclear properties that make them 0384. In one embodiment of the invention T is one of the Suitable for use as radiotherapeutic agents, as they emit beta aforementioned thrombolytic or fibrinolytic agents and, pref particles. Some of these are listed in the table below. erably, Streptokinase or urokinase. Compounds of Formula (I) in which T includes a thrombolytic or a fibrinolytic agent TABLE NO. 4 are useful intreating thrombus associated diseases, especially Gamma Approximate range of acute myocardial infarction, in mammals, including humans. Half-Life Max b-energy energy b-particle (cell 0385 Thus, in a different aspect thereof, the invention Isotope (days) (MeV) (keV) diameters relates to the use of a compound of Formula (I) in which T 49-Pin 2.21 1.1 286 60 includes a thrombolytic or a fibrinolytic agent for the prepa -Sm 1.93 O.69 103 30 ration of a pharmaceutical formulation for treating thrombus 66-Dy 340 O4O 82.5 15 associated diseases in mammalian, including humans. 66-Ho 1.12 1.8 80.6 117 75-Yb 4.19 O.47 396 17 0386 Thus, in a still different aspect thereof, the present 77-Lu 6.71 OSO 208 2O invention relates to compounds of Formula (I) in which Tisan OY 2.67 2.28 150 antineoplastic agent. Il-In 2.8.10 Auger electron 173, <5 * m 0387 Suitable examples of the said agents include, for emitter 247 instance, the previously listed antineoplastic compounds as well as toxins. wherein: Pm is Promethium, Sm is Samarium, Dy is Dyspro 0388. In a preferred embodiment of the invention, T is a sium, Ho is Holmium, Yb is Ytterbium, Luis Lutetium, Y is radiotherapeutic agent comprising a therapeutic radionu Yttrium, In is Indium. clide. Preferably, T is residue of a chelating ligand that is 0396 The use of radioactive rhenium isotope as an alter labelled with a therapeutically active radionuclide. These native to above lanthanides and lanthanoids is well known in compounds are preferred for use as radiotherapeutic agents the art and is encompassed by the invention. according to the invention. 10397) Particularly ''Reisotopes have proved to be of 0389. Accordingly, in another embodiment, the invention particular interest in nuclear medicine, having a large number relates to novel radiotherapeutic agents of Formula (I) in of applications in radiopharmaceutical therapy. which T is the residue of a chelating ligand suitably labelled 0398. Thus, in a preferred embodiment, the invention with a therapeutically active radionuclide. relates to novel radiotherapeutic agents of Formula (I) 0390 Preferred radionuclides for use in radiotherapy wherein T is the residue of a suitably chelated radionuclide include Cu, 90Y. 105Rh, In, 117'Sn, 149Pm, 'Sim, 161Tb, that emits ionizing radiations such as beta particles, alpha 166Dy 16Ho, 175Yb, 90Y. 77Lu, 186/188Re, and Au, with particles and Auger or Coster-Kroning electrons. '77Lu and 'Y being particularly preferred. 0399. More preferably, T is the residue of a chelating 0391 The selection of a proper radionuclide for use in a ligand labelled with a lanthanide or a lanthanoid radionuclide particular radiotherapeutic application depends on many fac selected from 90Y, In, Pm, 15 Sm, Dy, Ho, 75Yb, tors, including: and ''Lu. Examples of suitable chelating ligand may be 0392 a. Physical half-life This should belong enough selected from those of FIGS. 6a to 6c. to allow synthesis and purification of the radiotherapeu 0400 Thus, in a particularly preferred embodiment, the tic construct from radiometal and conjugate, and deliv present invention relates to novel radiotherapeutic agent of US 2010/0158814 A1 Jun. 24, 2010

Formula (I) wherein T is the residue of a chelating ligand of 0412 Compounds of Formula (I) in which T is an optically FIGS. 6a to 6c. labelled with a therapeutically active nuclide active imaging moiety are new and constitute a further object 90Y. Ill In O 177Lu. of the present invention. These compounds are preferred for 04.01. In another preferred aspect, the invention relates to use as for optical imaging contrast agents. novel radiotherapeutic agents of Formula (I) wherein T is the 0413 Thus, in a still further embodiment, the invention residue of a chelating ligand of Formula 10 to 16 labelled with relates to novel contrast agents for optical imaging having 186Re or 188Re. Formula (I), in which T is an optically active imaging moiety. 0402. The compounds of the invention labeled with thera peutic radionuclides can find application either as radiophar 0414 Suitable examples of optically active imaging moi maceutical that will be used as a first line therapy in the eties include those discussed herein, for instance, a dye mol treatment of a disease such as cancer, or in combination ecule; a fluorescent molecule Such as, for example, fluores therapy, where the radiotherapeutic agents of the invention cein; a phosphorescent molecule; a molecule absorbing in the could be utilized in conjunction with adjuvant chemotherapy UV spectrum; a quantum dot; or a molecule capable of (e.g., with one of the other therapeutic agents disclosed absorption of near or far infrared radiations. herein), or as the therapeutic part of a matched pair therapeu 0415 Optical parameters to be detected in the preparation tic agent. of an image may include, e.g., transmitted radiation, absorp 0403. In fact, the peptide moiety of the radiotherapeutic of tion, fluorescent orphosphorescent emission, light reflection, the invention is able to localize the chelated radioactive iso changes in absorbance amplitude or maxima, and elastically tope to the pathologic fibrin deposition, for instance, into scattered radiation. For example, biological tissue is rela thrombi/clots, atherosclerois plaques and inflammation tively translucent to light in the near infrared (NIR) wave based damage involved in multiple Sclerosis and, especially length range of 650-1000 nm. NIR radiation can penetrate within Solid tumors. The cytotoxic amount of ionizing radia tissue up to several centimetres, permitting the use of the tion emitted by the localized radioisotope is thus able to cause diagnostic agents of the invention comprising a NIR moiety the cell death of the pathologic tissue. to image target-containing tissue in vivo. Salts 0416) Near infrared dye may include, cyanine or indocya nine derivatives, such as, for example, Cy5.5, IRDye800. 0404 Both the ligands and the paramagnetic or radionu indocyanine green (ICG), indocyanine green derivatives clide chelated compounds of Formula (I) can also be in the including the tetrasulfonic acid substituted indocyanine green form of a physiologically salt. (TS-ICG), and combination thereof. 04.05 The term “pharmaceutically acceptable salt”, as 0417. In another embodiment, the compounds of the used herein, refers to derivatives of the compounds of the invention may include photolabels, such as optical dyes, invention wherein the parent compound is modified by mak including organic chromophores or fluorophores, having ing the acid or basic groups not yet internally neutralized in extensively conjugated and hence delocalized ring systems the form of non-toxic, stable salts which does not destroy the and having absorption or emission maxima in the range of pharmacological activity of the parent compound. 400-1500 nm. The compounds of the invention may alterna 0406 Suitable example of the said salts include: mineral tively be derivatized with a bioluminescent molecule. The or organic acid salts, of basic residues such as amines; alkali preferred range of absorption maxima for photolabels is or organic salts of acidic residues such as carboxylic acids; between 600 and 1000 nm to minimize interference with the and the like. signal from hemoglobin. Preferably, photoabsorption labels 04.07 Preferred cations of inorganic bases which can be have large molar absorptivities, e.g. >10 cm'M', while Suitably used to prepare salts within the invention comprise fluorescent optical dyes will have high quantum yields. ions of alkali or alkaline-earth metals such as potassium, Examples of optical dyes include, but are not limited, to those Sodium, calcium or magnesium. Preferred cations of organic described in WO 98/18497, WO 98/18496, WO 98/18495, bases comprise, inter alia, those of primary, secondary and WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841, tertiary amines Such as ethanolamine, diethanolamine, mor WO 96/23524, WO 98/47538, and references cited therein. pholine, glucamine, N-methylglucamine, N,N-dimethylglu 0418 For example, the photolabels may be covalently camine. linked directly to the peptide moiety of the invention, or one 0408 Preferred anions of inorganic acids which can be or more of them may be linked thereto through a branching suitably used to salify the complexes of the invention com chain Y, as described previously. prise the ions of halo acids such as chlorides, bromides, 0419. After injection of the optically-labeled diagnostic iodides or other ions such as Sulfate. derivative according to the invention, the patient is scanned 04.09 Preferred anions of organic acids comprise those of with one or more light Sources (e.g., a laser) in the wavelength the acids routinely used in pharmaceutical techniques for the range appropriate for the photolabel employed in the agent. Salification of basic Substances such as, for instance, acetate, The light used may be monochromatic or polychromatic and Succinate, citrate, fumarate, maleate or oxalate. continuous or pulsed. Transmitted, Scattered, or reflected 0410 Preferred cations and anions of amino acids com light is detected via a photodetector tuned to one or multiple prise, for example, those of taurine, glycine, lysine, arginine, wavelengths to determine the location of target-containing ornithine or of aspartic and glutamic acids. tissue (e.g. angiogenic tissue) in the Subject. Changes in the optical parameter may be monitored over time to detect accu Optical Imaging mulation of the optically-labeled reagent at the target site. Standard image processing and detecting devices may be 0411. In one further preferred embodiment of the inven used in conjunction with the optical imaging reagents of the tion, T represents an optically active imaging moiety. present invention. US 2010/0158814 A1 Jun. 24, 2010 52

0420. In a preferred embodiment, the invention relates to maceutically acceptable salts thereof, in combination with novel optical imaging agents of Formula (I) wherein T is the one or more possible pharmaceutically acceptable carriers or residue of a 5-Carboxyfluorescein. excipients. 0427. In an even further aspect thereof, the invention relates to the use of the compounds of Formula (I) in whichT Use of Compounds of Formula I for Imaging and is a diagnostically active moiety for the diagnosis in vitro (of Treating Pathological Conditions Associated with ex vivo Samples) of pathological systems, including cells, Fibrin, Particularly Tumors biological fluids and biological tissues originating from a live mammal patient, and preferably, human patient. Additionally, 0421. As discussed above, the peptide moiety compounds the invention relates to the use of the compounds of Formula of the invention are able to selectively bind to fibrin and, in (I) in which T is a diagnostically active moiety for the prepa particular, to fibrin present in the extracellular matrix (EC) of ration of pharmaceutical compositions for use in the diagnos tumor or connective tissue of stroma thus acting as targeting tic imaging, in Vivo, of human body organ, regions or tissues, moiety able to localize an active moiety linked thereto to including tumorous or cancerous tissues and inflammations, fibrin depositions and, especially, to fibrin deposition inside wherein fibrin depositions occur. Solid tumors or metastatic tissues. 0428. In yet another aspect the invention provides a 0422 The compounds of the present invention may thus method for imaging Solid tumors or tumorous cells both in find advantageous application for the diagnosis, prevention vitro (ofex vivo Samples) and in Vivo, the method comprising and treatment of all pathological conditions associated with the use of a diagnostic imaging agent of the invention and an fibrin deposition, including clots and thromboembolic dis imaging technique. eases and, especially, Solid tumors and metastatic processes. 0429 Furthermore, the invention provides a method for Moreover, they may advantageously be used to follow up and treating and/or ameliorating Solid tumors or tumorous cells in monitor oncological therapy efficacy and tumor treatment Vivo, the method comprising administering a therapeutic results. agent of the invention. 0423. In particular, the compounds of Formula (I) in which T is a diagnostically active moiety according to the invention Preparations may find advantageous application for localizing and diag nostically visualizing fibrin deposition associated with ath 0430. The preparation of the compounds of Formula (I), in erosclerosis and plaque formation. In a further aspect com which T is the residue of a chelating agent labelled with a pounds of the invention allow diagnostic imaging of paramagnetic metalion or a radionuclide, either as such or in inflammatory processes, including demyelination processes the form of physiologically acceptable salts, represents a further object of the invention. and axonal damage involved in multiple Sclerosis and, in 0431 Isolation, conjugation and use offibrin binding moi general, of all inflammatory conditions associated with pro eties (and conjugates thereof with diagnostically ortherapeu cesses in which fibrin plays a role. In an especially preferred tically active moieties) in accordance with this invention will aspect, the compounds of Formula (I) in which T is a diag be further illustrated in the following examples. The specific nostically active moiety according to the invention may find parameters included in the following examples are intended advantageous application in localizing and visualizing fibrin to illustrate the practice of the invention, and they are not content inside Solid tumor and metastatic processes. presented to in any way limit the scope of the invention. 0424. In a further aspect, the compounds of Formula (I) in which T is a diagnostically active moiety may find application Examples in the non invasive histopathologic grading of solid tumors. A correlation, in fact, exists between the MRI derived measures 0432. The following materials were used in performing of the fibrin content inside the tumor mass the compounds of the Examples below: the invention provide and the histopathologic grade of the 0433) Materials: Fmoc-protected amino acids used were said Solid tumor. obtained from Nova-Biochem (San Diego, Calif., USA), 0425 The compounds of Formula (I) in which T is a Advanced ChemTech (Louisville, Ky., USA), Chem-Impex therapeutically active moiety may find advantageous appli International (Wood Dale Ill., USA), and Multiple Peptide cation for prevention, amelioration and/or treatment of patho Systems (San Diego, Calif., USA). DPPE, DSPE-PG4-NH. logical conditions associated with fibrin deposition, includ and DPPE-PG4-NH were obtained from Avanti Polar Lipids ing clots and thromboembolic diseases and atherosclerotic (Alabaster, Ala.). Fmoc-PEG3400-NHS was obtained from plaques and inflammatory damages associated with fibrin Shearwater Polymers (Huntsville, Ala.). Other reagents were deposition. In an especially preferred aspect, the compounds obtained from Aldrich Chemical Co. (Milwaukee, Wis.) and of Formula (I) in which T is a therapeutically active moiety VWR Scientific Products (Bridgeport, N.J.). Solvents for may find advantageous application for preventing, ameliorat peptide synthesis were obtained from Pharmco Co. (Brook ing and/or treating Solid tumors and metastatic processes field, Conn.). associated therewith. In an even more preferred embodiment, 0434 Examples 1-7 and 23 utilize and make reference to the compounds of Formula (I) in which T is a radiotherapeutic procedures A-L described below. moiety may be advantageously used to provide radiotherapy to a patient (particularly a human) in need thereof due to the Procedures for Peptide Synthesis presence of one or more solid tumors and metastatic pro 0435 Procedure A: Automated Peptide Solid Phase Pep cesses associated therewith. tide Synthesis 0426 In another embodiment, the invention concerns 0436 Individual peptides were prepared using an ABI pharmaceutical compositions containing, as an active ingre 433A instrument (Applied Biosystems, Foster City, Calif.). dient, at least one compound of Formula (I), including phar PAL-Peg-PS-Resin (1.2g, 0.18 mmol/g) or NovaSyn TGR US 2010/0158814 A1 Jun. 24, 2010

resin (1.25 g, 0.20 mmol/g) (NovaBiochem, Novato, Calif.) camine (10-100 mM in H2O). The mixture was stirred for 48 was used for all syntheses. The peptides were assembled on hand was then purified by preparative HPLC. resin using the FastMocTM protocol. After the synthesis, the 0451) Procedure I: Preparation of 5-Carboxyfluorescein resin was washed with DCM (2x) and dried. (CF5) Derivatives of Peptides 0437. Procedure B: Manual Coupling of Amino Acids 0438 DMF was used as the coupling solvent unless oth 0452. To a solution of a peptide in DMF (15 uL/mg) and erwise stated. The appropriate Fmoc-amino acid in DMF DIEA (20 equiv/equiv peptide) was added 5-carboxyfluores (0.25M solution, 3 equiv) was treated with HATU (0.5M in cein NHS ester (1.3-1.5 equiv.) in DMF (20 uL/mg). The NMP, 3.0 equiv) and DIEA (6.0 equiv). The mixture was mixture was stirred for 1-3 h. The reaction was monitored by shaken for -2 min and then was transferred to the synthesis mass spectroscopy and analytical HPLC. Upon completion of vessel containing the resin. The vessel was then shaken over the reaction, the crude was filtered and purified by preparative night at ambient temperature. The resin was filtered to remove HPLC. excess reagents and then washed (4x) with DMF. 0453 Procedure J: Preparation of Alloc-Gly-OH 0439 Procedure C. Manual Removal of the Fmoc Protect ing Group 0454 Gly-O-t-Bu. AcOH (1 g, 5.24 mmol) was dissolved 0440 The resin containing the Fmoc-protected amino acid in DCM (15 mL), and diallyl pyrocarbonate (1.1 g, 5.91 was treated with 20% piperidine in DMF (V/V, 15.0 mL/g mmol. 1.13 equiv) was added dropwise. The mixture was resin) for 10 min. The solution was drained from the resin. stirred at ambient temperature for 0.5h. Then DIEA (3.7g, 5 This procedure was repeated once and then followed by mL, 28.68 mmol. 5.47 equiv) was added. The mixture was washing the resin with DMF (4x). stirred at ambient temperature overnight. The volatiles were 0441) Procedure D: Removal of the iv.Dde Group (Solid removed and the crude residue was dissolved in EtOAc (100 Phase) mL/g of crude) and the organic layer was washed with 1N 0442. The resin containing the ivode-protected amino HCl (2x). The volatiles were removed and the crude was dried acid was treated with 10% (v/v) hydrazine in DMF (10 mL/g at high vacuum. NMR (500 MHz, CDC1) indicated a pure resin) for 10 min. The solution was drained from the resin. product and was consistent with the structure. The crude was This procedure was repeated once and then followed by then dissolved in a solution of TFA/DCM (1/1, V/v, 25 mL) washing the resin with DMF (4x). and the solution was stirred overnight. The volatiles were 0443) Procedure E: Removal of the iv.Dde Group from removed, EtOAc was added to wash any residue from the wall Peptides in Solution of the flask and then the volatiles were removed on the rotary 0444 The peptide (50 mg) was dissolved in DMF (2.0 evaporator. This was repeated. The resulting product was mL) and treated with neat hydrazine (40-200LL) for 10 min. dried overnight at high vacuum. NMR spectroscopy of the The mixture was diluted with water to a volume of 10 mL and material (CDC1,500 MHz) was consistent with the expected this was directly applied to a C18 reverse phase column and structure and the purity was found to be sufficient for use in purified by preparative HPLC as described in the general manual coupling protocols. procedures. 0455 Procedure K: Preparation of Alloc-Arg(Prmc)-OH 0445 Procedure F: Coupling of Fmoc-Adoa (Fmoc-J) 0446 Fmoc-Adoa (2 equiv) and HATU (2 equiv) were 0456 H-Arg(Prmc)-OH (5 g, 11.35 mmol) was dissolved dissolved in DMF and DIEA (4 equiv) was added to the in a mixture of HO and Dioxane (1/1, V/V, 125 mL), and mixture. The mixture was stirred for 1 min before transferring diallyl pyrocarbonate (6.34g, 34.05 mmol. 3.0 equiv) was the activated acid to the resin. The concentration of reagents added. The pH of the mixture was adjusted to >10.0 by adding was as discussed above for standard peptide couplings. The NaCO. The mixture was stirred and kept at reflux overnight. coupling was continued for 12 hat ambient temperature. The The volatiles were removed by rotary evaporation, the crude resin was drained of the reactants and washed with DMF (4x). was dissolved in EtOAc (100 mL/g of crude) and the solution In cases where two Adoa units were appended to the resin, the was washed with 1 NHCl (2x). The volatiles were removed by Fmoc group of the first appended Fmoc-Adoa unit was rotary evaporation, the crude was dissolved in CHCl and the removed (procedure C), the resin washed with DMF (4x) and Solution was loaded onto a silica gel column. The column was followed by coupling of the second Adoa moiety. eluted with two column volumes of CHC1 and then similarly 0447 Procedure G: Cleavage and Side-Chain Deprotec eluted with a 5% solution of MeOH in CHC1. Fractions tion of Resin Bound Peptides containing the desired compound were combined and the 0448 Reagent B (88.5:5:2 TFA:water:phenol:TIPS V/ Volatiles were removed by rotary evaporation and pumping at V/wt/v), 15 mL/g resin, was added to ~1.0 g of the resin and high vacuum to provide 4.2 g (70% yield) of Alloc-Arg(Prmc)- the vessel was shaken for 4.5 h at ambient temperature. The OH. The proton NMR spectrum (CDC1, 500 MHz) was resin was filtered and washed twice with TFA (5 mL/g resin). consistent with the expected structure and required purity. The filtrates were combined, concentrated to give a syrup which upon Trituration with 20 mL of EtO/g of resin gave a 0457 Procedure L: Removal of the Aloc Protecting Group solid residue which was stirred for 5-15 min and then centri from Peptides fuged. The Supernatant was decanted and the process was 0458. The Alloc-protected peptide was dissolved in 5-20 repeated three times. The resulting solid was dried under high mL/100 mg peptide of a solution of NMM:acetic acid:DMF vacuum or with a stream of dry nitrogen gas. (1:2:10). Pd(PPh) (1-10 equiv/equiv peptide) was added. 0449 Procedure H: Disulfide Cyclization The mixture was stirred for 0.5-4h. MS and analytical HPLC 0450. The precipitate obtained from trituration of the were used to check the reaction. After the reaction was com crude cleavage mixture with EtO was transferred to a beaker plete, the crude reaction mixture was diluted to twice its and DMSO (5-10 uL/mg crude peptide) was added. The pH of volume with 10%-25% CHCN in HO, filtered and purified the solution was adjusted to 8 by adding N-methyl-D-glu by preparative HPLC. US 2010/0158814 A1 Jun. 24, 2010 54

Methods for Analysis and Purification min, then linear gradient 15-70% B in 6 min: Flow rate: 3 0459 Analytical HPLC mL/min: Detection: UV, w=220 nm. 0460 Column: Waters Corp. X-Terra, MS Cs; 4.6 mm 0473 System E: Column: Waters XTerra MS-C18, 4.6 i.d.x50 mm; 5 um particle: Eluent A: Water (HPLC Grade mm i.d.x50mm; Particle size: 5 microns: Eluents: A: Water with 0.1%TFA by weight); Eluent B: Acetonitrile (0.1%TFA (0.1%TFA), B: Acetonitrile (0.1%TFA); Elution: linear gra by weight). Initial conditions and gradient elution profiles dient 15-60% B in 6 min; Flow rate: 3.0 mL/min: Detection: employed are described in the respective experimental pro UV, W220 nm. cedures for analysis of the title compounds. Elution rate: 3 0474 System F. Column: YMC C18, 4.6x250 mm; Elu mL/min: Detection: UV at 220 nm. ents: A: Water (0.1% TFA), B: Acetonitrile (0.1% TFA), 0461) Preparative HPLC Purification Initial condition: 20% B, Elution: lineargradient 20-80% B in 0462 Column: Waters Corp. X-Terra MS Cs; 50 mm 20 min; Flow rate: 1.0 mL/min: Detection: UV, W220 nm. i.d.x250 mm; 10 um particle: Eluents: Eluent A: Water 0475 System G: Column: Waters XTerra MS-C18, 4.6 (HPLC Grade with 0.1%TFA by weight); Eluent B: Aceto mm i.d.x50mm; Particle size: 5 microns: Eluents: A: Water nitrile (0.1% TFA by weight); Initial conditions and gradient (0.1%TFA), B: acetonitrile (0.1%TFA); Elution: Initial con elution profiles employed are described in the respective dition: 10% B, linear gradient 10-50% B over 8 min: Flow experimental procedures for analysis of the title compounds. rate: 3 mL/min: Detection: UV, w=220 nm. Elution rate: 100 mL/min: Detection: UV at 220 nm. 0476 System H. Column: Waters XTerra MS-C184.6x50 0463 Preparative HPLC Purification for Phospholipid mm, Particle size: 5 microns: Eluents: A: Water (0.1% TFA), Peptide Conjugates B: Acetonitrile (0.1%TFA); Elution: Initial condition: 5% B, 0464 Purification of Phospholipid Peptide Conjugates linear gradient 5-65% B in 8 min: Flow rate: 3 ml/min: Detec Employing the Kromasil Prep C4 HPLC Column tion: UV (a) 220 nm. 0465. The reaction mixture was diluted with distilled 0477 System I: Column: Waters XTerra C-4, 4.6x50mm: deionized water and purified on a reverse phase C4 prepara Eluents: A: Water (0.1% TFA), B: Acetonitrile:Methanol tive column (Kromasil R. Prep C, particle size 10 um, pore (1:1)(0.1% TFA); Elution: Initial condition: 80% B, linear size 300 A, 20x250 mm), using a gradient of 50-100% water gradient 80-100% B in 6 min.; Flow rate: 3.0 mL/min: Detec (0.1%TFA) into CH-OH:CHCN (1:1, viv, 0.1%TFA) at 100 tion: UV, W220 nm. mL/min over a period of 30 min. Fractions (15 mL) were 0478 System J. Column: YMC C-4, 4.6x250 mm; Elu analyzed by HPLC (column:YMCC-4, 5um,300 A, 4.6x250 ents: A: Water (0.1%TFA), B: Acetonitrile:Methanol (1:1)(0. mm) and the pure product-containing fractions were pooled. 1% TFA); Elution: Initial condition: 80% B, linear gradient Methanol was removed from the combined product-contain 80-100% B in 50 min.; Flow rate: 2.0 mL/min: Detection: UV, ing eluates by rotary evaporation; the resulting solution was =220 nm and ELSD: Sensitivity 10, Temp. 51 Deg C., Pres diluted with 10% aqueous acetonitrile, frozen and lyophilized sure 2.2 Torr. to provide the desired product. 0479. System K. Column:YMCC4; 250 mmx4.6 mmi.d.; 0466 Purification of Phospholipid Peptide Conjugates Particle size: 5.0 microns: Eluents: A:Water (0.1% TFA), B: acetonitrile (0.1% TFA); Elution: Initial condition: 80% B, Employing the Zorbax Prep C-3 HPLC Column linear gradient 80-90% Bover 100 min, then ramp to 100% B 0467. The diluted reaction mixture was loaded onto a Zor over 1 min, then hold at 100% B for 1 min: Flow rate: 2.0 bax C-3 column (21.2 mm i.d.x 150 mm) which was pre mL/min: Detection: UV, v=220 nm and ELSD: Sensitivity equilibrated with 25% B (CHCN with 0.1% TFA) at a flow 10, Temp. 51 Deg C., Pressure 2.2 Torr. rate of 30 mL/min. The column was eluted at 30 mL/min with 0480 System L: Column: YMC C-4, 4.6x250 mm; Elu the same eluent until the plug of DMF was eluted. The pro ents: A: Water (0.1% TFA), B: Acetonitrile/Methanol (1:1, portion of eluent B was then increased from 25% B to 30% B v/v)(0.1% TFA); Elution: Initial condition: 60% B, linear over 3 min and then ramped to 100% Bover 50 min. Fractions gradient 60-100% B in 20 min.; Flow rate: 2.0 mL/min: (15 mL) were collected and product-containing fractions Detection: UV, W220 nm and ELSD: Sensitivity 10, Temp. were pooled, frozen and lyophilized. 51 Deg C., Pressure 2.2 Torr. HPLC Methods Employed for Analysis of Compounds 0481 System M: Column: YMC C-4, 4.6x250 mm; Elu ents: A: Water (0.1% TFA), B: Acetonitrile/Methanol (1:1, 0468 HPLC Systems Employed for Analysis of Peptides v/v)(0.1% TFA); Elution: Initial condition: 50% B, linear and Phospholipid-Peptide Conjugates gradient 50-90% B in 10 min. Flow rate: 3.0 mL/min: Detec 0469 System A: Column: Waters XTerra MS-C184.6x50 tion: UV,220 nm and ELSD: Sensitivity 10, Temp. 51 Deg mm, Particle size: 5 microns: Eluents: A: Water (0.1% TFA), C., Pressure 2.2 Torr. B: Acetonitrile (0.1%TFA); Elution: linear gradient 5-55% B 0482 System N: Column: Zorbax 300SE C-3, 3 mm i.d.x in 7 min; Flow rate: 3 mL/min: Detection: UV, w=220 nm. 150 mm; 3.5 um particle: Eluent A : Water (0.1% TFA): 0470 System B: Column: Waters XTerra MS-C184.6x50 Eluent B: Acetonitrile (0.1%TFA). Initial condition: 50% B: mm, Particle size: 5 microns: Eluents: A: Water (0.1% TFA), Elution:linear gradient 50-90% B over 3 min, hold at 90% B B: Acetonitrile (0.1%TFA); Elution: linear gradient 5-65% B for 11 min; Elution rate: 0.5 mL/min: Detection: UV, w=220 in 7 min; Flow rate: 3 mL/min: Detection: UV, w=220 nm. nm and ELSD: Sensitivity 10, Temp. 51 Deg C., Pressure 2.2 0471) System C: Column: Waters XTerra MS-C184.6x50 Ton mm, Particle size: 5 microns: Eluents: A: Water (0.1% TFA), 0483 System O: Column: YMC C-4, 4.6x50 mm; Elu B: Acetonitrile (0.1%TFA); Elution: linear gradient 15-65% ents: A: Water (0.1% TFA), B: Acetonitrile-Methanol (1:1 B in 7 min; Flow rate: 3 mL/min: Detection: UV, W220 nm. v/v)(0.1% TFA); Elution: Initial condition: 75% B, linear 0472 System D: Column: Waters XTerra MS-C184.6x50 gradient 70-100% B in 10 min.; Flow rate: 3.0 mL/min: mm, Particle size: 5 microns: Eluents: A: Water (0.1% TFA), Detection: UV, W220 nm and ELSD: Sensitivity 10, Temp. B: Acetonitrile (0.1%TFA); Elution: Isocratic at 15% B for 1 51 deg C., Pressure 2.2 Torr. US 2010/0158814 A1 Jun. 24, 2010

0484 System P: Column: ES industries MacroSep C4. analysis is described in, for example, the following reference: 4.6x50mm; Particle size: 5 g; Eluents: A: Water (0.1%TFA), Fluorescence Polarization Technical Resource Guide Tech B: Acetonitrile/Methanol (1:1, v/v)(0.1%TFA); Elution: Ini nical Resource Guide 4" Edn. Invitrogen Corporation'501 tial condition: 25% B, linear gradient 25-100% over 7 min: Charmany Drive Madison, Wis. 53719 USA. Particularly, the Flow rate; 3 mL/min: Detection: UV (a) 220 mm mathematical analysis of the data and obtaining binding con stants is described in Chapter 8: Analysis of FP Binding Data Assay of Fibrin Binding Peptides by Direct Binding Fluores pp 8-2-8-7. cence Polarization (FP) Procedure Using 5-Carboxyfluore Assay of Fibrin Binding Peptides by Competition Binding sein Labeled Peptides Fluorescence Polarization (FP) Procedure using Competition 0485 Protocol of Unlabeled Test Peptides vs the Standard 5-Carboxyfluo 0486 1. Prepare 1 mL of 40 nM solution of 5-carboxy resein Labeled Peptide Seq000-CF5. fluorescein-labeled peptide in HEPES dilution buffer 0496 DDE at 10M concentration was aliquoted into a (HDB) with 0.01% Tween. 96-well plate. The standard 5CF-labeled peptide Seq000 0487. HDB (10 mM HEPES, 150 mM NaCl, 2 mM CF5 was added to DDE-containing wells to provide an initial CaCl) tracer concentration of 10M. An aliquot of competitor pep 0488 2. Dilute the 40 nM solution to obtain 1 ml of a 20 tide was added to each well in order to span a concentration nM solution of the test 5-carboxyfluorescein-labeled range of 10'M to 10M. The peptides were incubated with peptide. the DDE/Seq000-CF5 complex for 2 h. Then the anisotropy 0489. 3. Prepare a solution of DDE at a concentration value was read on the Tecan Polarion Plate Reader at 485 nm. that will be approximately 5-10 fold greater than the The competition curve was constructed using all of the con expected K. For the described assay an 8 LM concen centrations of the test peptide. The mathematical analysis of tration of DDE was prepared. the data and calculation of the ICso for the test peptides was 0490 4. Mix equal volumes of the DDE solution with accomplished using the regression routines in Prism Graph the 40 nM peptide solution. PadTM Software. The theoretical and mathematical basis for 0491 5. Prepare serial dilutions of DDE in a solution the experimental procedure and the data analysis is given in, consisting of the binding buffer with 0.01% Tween20 for example: “Practical Use of Fluorescence Polarization. In and 20 nM of the 5-carboxyfluorescein-labeled peptide. Competitive Receptor Binding Assays’-Section P of “Recep 0492 6. Make dilutions as shown in the following table tor Binding Assays’ http://www.Ncgc.Nih.Gov/Guidance/ in a Labsystems 384-well microplate and mix by Section5.Html#Practical-Fluor-Polar. CopyrightC) 2005, Eli repeated aspiration and dispensing of the solution into Lilly and Company and the National Institutes Of Health the wells. Chemical Genomics Center. The relative IC50 values for test 0493 7. Centrifuge the plate at 2000 RPM for 5 minutes peptides vs Seq000-CF5 were obtained by division of their to remove air pockets in the wells. IC50 by that obtained by titration of Seq005 into Seq005 0494 8. Read in Tecan Polarion Plate Reader at 485 nm. CF5/DDE complex. Thus lower relative IC50 indicates a to obtain the anisotropy value. stronger binding peptide. See Tables 1 and 2, Supra. 0497 Examples 1-3 below describe the preparation of exemplary peptides Seq016, Seq017, and Seq049. Target Protein 20 nM Peptide HDB Example 1 Row Solution (IL) Solution (IL) Solution (IL) O Preparation of O Ac-GWQPC*PWESWTFC*WDPGGGK NH, L. Row D L. Row E cyclic (5->13) peptide (Seq016) (SEQ ID NO. 7) L. Row F L. Row G 0498. The peptide sequence was prepared by SPSS from L. Row H Fmoc-PAL-PEG-PS resin (0.18 mmol/g, 1.38 g., 0.25 mmol) : L. Row I as described in procedure A using an ABI peptide synthesizer L. Row J L. Row K employing Fmoc chemistry which was implemented using L. Row L the FastMocTM protocol. Cleavage and side-chain deprotec L. Row M tion was conducted as described in procedure G and disulfide L. Row N cyclization was accomplished as described in procedure H. L. Row O L. Row P HPLC purification provided 105 mg (17.8% yield) of the 2 LL Target purified cyclic peptide. Protein Solution + 2 LL 40 nM Peptide Solution 0 Example 2 Preparation of 0495. The anisotropy (in mP millipolarization units) vs Ac SGSGJWQPC*PWESWTFC*WDPGGGK the logarithm of the concentration (micromoles/liter) of the NH. (cyclic 9->17) peptide (Seq017) (SEQ ID NO. receptor concentration is graphed. The dissociation constant 9) is obtained at the midpoint of the curve whose extrema are An and A, where A is the anisotropy of the free 0499 Procedures A, G and H were employed to prepare peptide and A is the anisotropy of the fully bound pep the peptide on a 0.266 mmol scale and HPLC purification tide. The theory, methods of operation and mathematical provided 130 mg (18.8% yield) of the pure product. US 2010/0158814 A1 Jun. 24, 2010 56

Example 3 0505 Examples 5 and 6 below describe the preparation of peptides bearing N-terminal Alloc-Arginine. Preparation of Ac RWQPC*PAESWT-Cha C*WDPGGGK NH cyclic (5->13) peptide Example 5 (Seq049) (SEQID NO. 69) Preparation of Alloc 0500. The peptide was prepared using the methods of pro RWQPC*PWESWTFC*WDPGGGK NH, cyclic cedures A, G and H. HPLC purification provided a 140 mg (5->13) peptide (Seq023-Aloc) (SEQID NO. 22) (27.5% yield) portion of the product as a fluffy white solid. 0501) Example 4 below and FIG. 1 describe and illustrate (0506. A 0.54 mmol (3 g) portion of W(N'-Boc)-Q(Trt)- the process used for the preparation of Adoa-Adoa linker P C(Trt)-P W(N'-Boc)-E(OtBu)-S(tEu)-W(N'-Boc)-T functionalized Seq005. (thBu)-F C(Trt)-W(N'-Boc)-D(OtBu)-P-GGG-K(Boc)- PAL-PEG-PS resin was prepared by automated SPSS Example 4 (procedure A). Aloc-Arg(Prmc) was appended to the N-termi nus using a modification of procedure B as follows: The resin Preparation of was added to a manual solid phase synthesis vessel and Sus Ac WQPC*PAESWTFC*WDPGGGK(JJ)-NH, pended in DMF (20 mL) by brief agitation. Aloc-Arg(Prmc)- cyclic (4-> 12) peptide, (Seq005-JJ) (SEQ ID NO. OH (524 mg, 1.00 mmol, 1.85 equiv), HATU (380 mg, 1.0 136) mmol. 1.85 equiv) and DIEA (257 mg, 347 uL. 1.98 mmol. 0502. The iv.Dde-protected peptide Ac W(N"-Boc)-Q 3.67 equiv) were added Successively with intervening agita (Trt)-P C(Trt)-P-A-E(OtBu)-Ser(tBu)-W(N'-Boc)-T tion of the vessel and the vessel was shaken overnight. The (thBu)-F C(Trt)-W(N"-Boc)-D(OtBu)-P-GGGK(ivDde)- coupling reaction was complete as indicated by a negative NH-TGR was assembled on a 130 umol scale (0.65 g resin) ninhydrin test. The resin was washed with DCM (3x20 mL) (procedure A). The ivode group was removed (procedure D) and dried. by treatment of the resin with 10% hydrazine in DMF (6.5 (0507 Reagent B (88:5:5:2-TFA: water: phenol: TIPS-V/v/ mL) for 10 min (2x). Then the resin was washed with DMF wit/v) (25 mL) was added to the vessel and the vessel was (4x). In a separate flask Fmoc-Adoa (100 mg, 0.26 mmol. 2.0 shaken at ambient temperature for 5 h. The resin was filtered equiv) in NMP (1 mL) was treated with HATU (99 mg, 0.26 and washed with TFA (2x5 mL). The combined filtrates were mmol. 2 equiv) in DMF (0.5 mL) and DIEA (67 mg, 91 LL. concentrated to a syrup which was triturated with EtO (20 0.52 mmol. 4 equiv) for 2 min after which the solution was mL) and the resulting solid was pelleted by centrifugation. transferred to the vessel containing the resin followed by The Supernatant liquid was decanted and the process was agitation of the vessel for 12 hat ambient temperature (pro repeated three times (procedure G). The resulting solid was cedure F). collected and cyclized (48 h) as described (procedure H) and 0503. The resin was washed with DMF (4x5 mL) and the purified by HPLC on a reverse phase C18 column. The prod Fmoc group was removed by treatment with 20% piperidine uct-containing fractions were pooled, frozen and lyophilized in DMF (10 mL, 2x10 min) followed by washing (4x10 mL) to provide 290 mg (21% yield) of the desired product. with DMF (procedure C). Then Fmoc-Adoa was coupled to the resinas described (vide supra) followed by removal of the Example 6 Fmoc protecting group (vide Supra) and washing of the resin. Preparation of Alloc-RWQPC*PAESWT-Cha Cleavage and side-chain deprotection (procedure G) was con C*WDPGGGK NH. (Seq057-Aloe) (SEQID NO. ducted for 4.5 h using Reagent B (10 mL). The resin was 86) drained and washed with TFA (5 mL) and the combined solutions were evaporated and triturated with ether to provide 0508. The peptide was prepared by the methods of proce the crude linear peptide as an off-white solid. The solid was dure A, B, G and H to give 230 mg (26.7% yield) portion of dissolved in DMSO (3 mL) after which the pH of the solution the product as a fluffy white solid. was adjusted to 8 by addition of 0.1M aqueous N-methylglu (0509. Example 7 below and FIG. 2 describe and illustrate camine. The mixture was stirred for 48 h (procedure H) dur the preparation of 5-carboxyfluorescein derivatives of pep ing which time the reaction was monitored by analytical tides. HPLC and mass spectroscopy. At the end of the reaction period the entire solution was diluted to 15 mL with 10% Example 7 CHCN in HO and the pH was adjusted to 2 by addition of Preparation of RWQPC*PAESWTFC*WDPGGGK aqueous TFA. (CF5)-NH cyclic (5->13) peptide (Seq056-CF5) 0504 The resulting solution was applied to a preparative reverse-phase C18 column and purified using a linear gradi (SEQID NO. 120) ent of 10% CHCN (0.1%TFA) into HO (0.1%TFA). Frac 0510. The peptide Alloc tions (15 mL) were collected and the pure product-containing RWQPC*PAESWTFC*WDPGGGK NH, cyclic (5->13) fractions were pooled, frozen and lyophilized to provide 42 peptide (Seq056-Aloc) (SEQID NO. 140) was prepared by mg (13% yield) of the peptide as a fluffy white solid which the methods of procedures A, B, G and H and purified by was characterized by HPLC and mass spectroscopy. HPLC: HPLC. The N-terminal Aloc N-CF5 derivative was pre to 3.83 min: Column: Waters XTerra MS-C18 4.6x50 mm: pared according to procedure I as follows: The peptide (70 Particle size: 5 microns: Eluents: A: Water (0.1% TFA), B: mg, 0.029 mmol) was dissolved in anhydrous DMF (1 mL) Acetonitrile (0.1%TFA); Elution: linear gradient 5-65% B in with stirring, after which DIEA (0.074 g, 100 uL, 0.572 7 min; Flow rate: 3 mL/min: Detection: UV, w=220 nm. Mass mmol. 19.7 equiv) was added followed by a solution of CF5 spectrum (API-ES). Neg. ion: M-H: 2480.6: M-2H/2: NHS (20 mg 0.042 mmol. 1.45 equiv) in anhydrous DMF. 1239.9 The mixture was stirred 1 h at ambient temperature. The

US 2010/0158814 A1 Jun. 24, 2010 58

Example 8 adjusted to 100 mL by addition of CH-OH (50% ) and CHCN water (1:1) (50%) and the resulting solution was Preparation of filtered to remove insoluble material. Ac WQPC*PAESWTFC*WDPGGGK(DSPE 0517. The filtered solution was loaded onto a C4 reverse PG4-Glut)-NH cyclic (4-> 12) peptide (Seq005 phase column (YMC, Prep C, 10 uM, 100 A, 30x250 mm) PL1) (SEQID NO. 123) which had been pre-equilibrated with 50% CH-OH and 0513. A Solution of the peptide CHCN (eluent A) water (eluent B) (1:1) at 30 mL/min. The Ac WQPC*PAESWTFC*WDPGGGK NH, cyclic column was washed with the same eluent until the plug of (4-> 12) peptide (Seq005) (SEQ ID NO.1) (150 mg, 0.069 DMF was eluted from the column. The mobile phase compo mmol) in DMF (1.0 mL) was added to a stirred solution of sition was then ramped to 70% B in 1 min and the elution was DSG (0.34 mmol. 112 mg, 5 equiv) and DIEA (15 mg, 20LL, continued at a linear gradient rate of 1% B/min to 100% Bat 0.12 mmol. 1.67 equiv) in DMF (1.0 mL). The mixture was which time the column was eluted with 100% B until the stirred for 0.5 h and the progress of the reaction was moni product was fully eluted from the column. Fractions (15 mL) tored by HPLC and MS. Upon completion of the reaction, the were collected and those containing the product in >98% volatiles were removed in vacuo and the residue was washed purity were collected and concentrated on a rotary evaporator with ethyl acetate (3x10 mL) to remove unreacted DSG. The to reduce the content of CHOH. The concentrated solution residue was dried, re-dissolved in anhydrous DMF (1.0 mL) was diluted with 10% CHCN in water, frozen and lyo and a solution of DSPE-PG4-NH. (134 mg., 0.048 mmol) in philized to provide 65 mg (60% yield) of the product as a DMF (1.0 mL) was added followed by DIEA (15 mg, 20 uL. colorless solid. 0.12 mmol, 1.67 equiv). The mixture was stirred for 16 h. The progress of the reaction was monitored by HPLC which indi Example 10 cated that the aminopegylated phospholipid was consumed at Preparation of 16 h. Ac-GWQPC*PWESWTFC*WDPGGGK(DSPE 0514. The reaction mixture was diluted with distilled, PG4-Glut)-NH cyclic (5->13) peptide (Seq016 deionized water and purified on a reverse phase preparative PL1) (SEQID NO. 126) column (YMC Prep C particle size 10 um, 30x250 mm), 0518. The peptide using a gradient of 50-75% water (0.1% TFA) into CH-OH: Ac-GWQPC*PWESWTFC*WDPGGGK NH, cyclic CHCN (1:1, v/v, 0.1%TFA) at a flow rate of 30 mL/min over (5->13) peptide (Seq016) (SEQ ID NO. 7) (90 mg, 0.038 a period of 5 min then ramping to 100% B over 50 min. mmol) was dissolved in anhydrous DMF (0.5 mL) and this Fractions (15 mL) were analyzed by HPLC and the pure solution was added to a solution of DSG (65 mg, 0.2 mmol. product-containing fractions were pooled. Methanol was 5.26 equiv) and DIEA (25 mg, 33.8LL, 0.2 mmol. 5.26 equiv) removed from the combined product-containing eluates by in anhydrous DMF (0.5 mL) with stirring. The mixture was rotary evaporation; the resulting solution was diluted with stirred 2 hand then EtOAc (20 mL) was added resulting in the 10% aqueous acetonitrile, frozen and lyophilized to provide formation of a solid which was pelleted by centrifugation. 108 mg (44% yield) of the desired product as a fluffy white The Supernatant liquid was decanted and this process was solid. repeated twice to remove remaining DSG. The solid was dried under vacuum (<0.1 mm) for 30 min followed by dis Example 9 solution by stirring in DMF (0.5 mL). Solid DSPE-PG4-NH Preparation of (53 mg, 0.19 mmol) was added portionwise and the resulting Ac SGSGSGSGWQPC*PWESWTFC*WDPGGG mixture was stirred overnight. The mixture was diluted with K(DSPE-PG4-Glut)-NH cyclic (12->20) peptide water (5 mL) and the crude mixture was purified by prepara (Seq024-PL1) (SEQID NO. 125) tive HPLC to give 60 mg (30% yield) of the desired productas a white lyophilizate. 0515. A solution of DSG (50 mg, 0.15 mmol, 4.41 equiv) and DIEA (20 mg, 0.15 mmol. 4.41 equiv) in anhydrous DMF Example 11 (2.0 mL) WaS stirred and the peptide Preparation of Ac SGSGSGSGWQPC*PWESWTFC*WDPGGGK Ac SGSGJWQPC*PWESWTFC*WDPGGGK NH cyclic (1220) peptide (Seq024) (SEQID NO. 23) (100 (DSPE-PG4-Glut)-NH cyclic (917) peptide mg, 0.034 mmol) was added in solid form portionwise to the (Seq017-PL1) (SEQID NO. 127) above solution. The mixture was stirred at room temperature for 30 min. The volume of the reaction mixture was adjusted 0519. The peptide to 50 mL by addition of EtOAc and the precipitated solid was Ac SGSGJWQPC*PWESWTFC*WDPGGGK NH, pelleted by centrifugation followed by decantation of the cyclic (9->17) peptide (Seq017) (SEQ ID NO. 9) (100 mg, Supernatant. The washing procedure was repeated (3x) to 0.039 mmol) was employed using the procedures described provide the glutaric acid monoamide mono-NHS ester of the for the preparation of SEQ005-PL1, HPLC purification pro peptide, whose identity was confirmed by mass spectral vided 53 mg (25.7% yield) of the target phospholipid-peptide analysis (M-2H)/2: 1546.1, (M-3H)/3: 1030.5), as a color conjugate. less solid. Example 12 0516. The glutaric acid monoamide mono-NHS ester of the peptide was dissolved in dry DMF-DCM (2.0 mL. 8:2. Preparation of RWQPC*PWESWTFC*WDPGGGK V/v). DIEA (40 mg, 54 uL, 0.31 mmol) was added and the (DSPE-PG4-Glut)-NH cyclic (5->13) peptide mixture was stirred. DSPE-PG4-NH. (45 mg 0.038 mmol, (Seq023-PL1) (SEQID NO. 128) 0.9 equiv) was added as a solid and the mixture was stirred for 0520 Alloc-RWQPC*PWESWTFC*WDPGGGK NH, 24h at ambient temperature. The volume of the mixture was cyclic (5->13) peptide (Seq023-Aloc) (SEQ ID NO. 142) US 2010/0158814 A1 Jun. 24, 2010 59

(100 mg, 0.04 mmol) in DMF (0.5 mL) was added to a were removed under high vacuum at 30°C. to give a viscous solution of DSG (50 mg, 0.153 mmol. 3.825 equiv) in DMF residue. The residue was triturated by addition of EtOAc (15 (0.5 mL). DIEA (7.4 mg, 10uL, 0.057 mmol. 1.43 equiv) was mL) to the vessel, resulting in the formation of a white solid added to the Solution and stirring was continued for 1 h at which was pelleted by centrifugation. The Supernatant was ambient temperature after which mass spectroscopy indi decanted and the Solid washed (3x) in the same manner and cated completion of the reaction. The volatiles were removed dried using a flow of dry nitrogen gas. under high vacuum to provide a semisolid residue. EtOAc (5 0524. The solid intermediate thus obtained was dissolved mL) was added to the residue resulting in the formation of a in anhydrous DMF (1 mL) with stirring and DIEA (110 mg. well-defined solid which was pelleted by centrifugation. The 149 uL, 0.86 mmol, 8.9 equiv relative to peptide) was added, Supernatant liquid was decanted and the washing process was followed by the dropwise addition of a solution of DSPE repeated (5x). This gave a 100 mg (92% yield) portion of a PG4-NH. (213 mg, 0.077 mmol. 0.8 equiv) in anhydrous white solid, the intermediate N'-glutaric acid monoamide DMF (1 mL). The mixture was stirred at ambient temperature mono-NHS ester of the peptide. A second run, conducted in overnight, after which HPLC analysis and mass spectroscopy exactly the same manner, provided an additional 100 mg of indicated the consumption of the lipid. white solid. Mass spectral analysis was consistent with the 0525. The volatiles were removed under high vacuum and presumed structure of the intermediate. The calculated the crude mixture was dissolved in a solution of 13 mL of monoisotopic molecular weight of the intermediate NHS NMM:HOAc:DMF (1:2:10, V/v/v) with stirring. Pd(PPh.) ester is 2713. Mass spectral analysis (API-ES negative ion) of (250 mg, 0.22 mmol. 2.25 equiv relative to peptide) was the white solid obtained by the above described procedure added to the stirred solution and stirring was continued for 4 gave peaks at 1356 (M-2H)/2) and 1431.6 (M+TFA-2H)/2). h. The solids were filtered and the resulting solution was 0521. The glutaric acid monoamide, mono-NHS ester of diluted to twice its volume with a solution of 25% CHCN in the peptide (200 mg, 0.074 mmol) was dissolved in DMF (1 HO. The resulting solution was loaded onto a Zorbax C-3 mL), and DIEA (11 mg, 15 uL, 0.085 mmol. 1.15 equiv) was column (21.2 mm i.d.x150 mm) which was pre-equilibrated added to the stirred solution. DSPE-PG4-NH. (160 mg, 0.8 with 25% B (CHCN with 0.1% TFA) at a flow rate of 30 equiv) in DMF (1 mL) was added to the mixture and stirring mL/min. The column was eluted at 30 mL/min with the same was continued overnight at ambient temperature. The Vola eluent until the plug of DMF was eluted. The proportion of tiles were removed at high vacuum. The residue was re eluent B was then increased from 25% B to 30% B over 3 min dissolved in 13 mL of NMM/HOAc/DMF (1:2:10, V/v/v). and then ramped to 100% B over 50 min. Fractions (15 mL) Pd(PPh) (300 mg, 3.0 equiv) was added in one portion. The were collected and product-containing fractions were pooled, mixture was stirred for 1 h. MS and analytical HPLC indi frozen and lyophilized. HPLCanalysis of the isolated product cated completion of the reaction. The crude reaction mixture indicated the need for additional purification. The purification was diluted with an equal volume of 20% CHCN in HO and was repeated and the product was isolated by freezing the insoluble material was filtered. The resulting solution was pooled pure product-containing fractions and lyophilization applied directly to a C4 reverse phase HPLC column and to provide 23 mg (4.5% yield) of the target phospholipid purified by preparative HPLC as described for Seq005-PL1. peptide conjugate. Fractions containing the pure product were pooled, frozen 0526 Example 15 below describes the preparation of and lyophilized to give 140 mg (29% yield based on the input lipopeptide DPPE-PG4-peptide conjugates. DSPE-PG4-NH) of the target compound as a fluffy solid. Example 15A Example 13 Preparation of Preparation of Ac RWQPC*PAESWT-Cha Ac WQPC*PAESWTFC*WDPGGGK(DPPE C*WDPGGGK(DSPE-PG4-Glut)-NH cyclic PG4-Glut)-NH cyclic (4-> 12) peptide (Seq005 (5->13) peptide (Seq049-PL1) (SEQ ID NO. 129) PL2) (SEQID NO.124) 0522) Ac RWQPC*PAESWT-Cha-C*WDPGGGK 0527 The process is adapted from that used for the prepa NH. (SEQ ID NO. 70) cyclic (5->13) peptide (94 mg., 0.04 ration of Seq005-PL1 except that DPPE-PG4-NH is mmol. 1.25 equiv) was employed to prepare a 57 mg (34% employed in place of DSPE-PG4-NH. Thus a solution of the yield) portion of the target phospholipid peptide conjugate in peptide Ac WQPC*PAESWTFC*WDPGGGK NH, the same manner as Seq005-PL1. cyclic (4-> 12) peptide (Seq005) (SEQ ID NO. 1) (66 mg, 0.03 mmol) and DIEA (11 mg, 15 uL, 0.085 mmol. 2.84 Example 14 equiv) in DMF (0.5 mL) was added to a stirred solution of DSG (33 mg, 0.10 mmol, 3.3 equiv) in DMF (0.5 mL). The Preparation of RWQPC*PAESWT-Cha mixture was stirred for 1 h and the progress of the reaction C*WDPGGGK(DSPE-PG4-Glut)-NH cyclic was monitored by HPLC and MS. Upon completion of the (5->13) peptide (Seq057-PL1) (SEQID NO. 130) reaction, the volatiles were removed in vacuo and the residue 0523) A solution of Alloc-RWQPC*PAESWT-Cha was washed with ethyl acetate (4x5 mL) to remove unreacted C*WDPGGGK NH cyclic (5->13) peptide (Seq057 DSG from the intermediate peptide glutaric acid monoamide Aloc)(SEQID NO. 143) (230 mg, 0.096 mmol) in anhydrous mono-NHS ester. DMF (1 mL) was added dropwise to a stirred solution of DSG 0528. The resulting solid was dried, re-dissolved in anhy (180 mg, 0.55 mmol. 5.75 equiv relative to peptide) in anhy drous DMF (1.00 mL) and stirred with a solution of DPPE drous DMF (0.5 mL) containing DIEA (110 mg. 0.86 mmol. PG4-NH. (95 mg, 0.035 mmol, 1.16 equiv) in DMF (1.0 mL) 8.9 equiv relative to peptide). The reaction mixture was for 24 hour. The progress of the reaction was monitored by stirred for 3 hand the progress of the reaction was monitored HPLC, which indicated complete consumption of the inter by analytical HPLC and mass spectroscopy. The solvents mediate peptide glutaric acid monoamide mono-NHS ester. US 2010/0158814 A1 Jun. 24, 2010 60

The solution was diluted with water and purified by reverse 0532. Example 16A below and FIG. 5 describe and illus phase C4 preparative column (Kromasil(R) Prep C, 10um, trate the preparation of DPPE-Glut-PG2-peptide conjugates. 300 A, 20x250 mm, flow rate 25 mL/min) using a gradient of 60-100% water (0.1% TFA) and a mixture of CH-OH and Example 1.6A CHCN (1:1, 0.1% TFA) over a period of 30 min. Fractions Preparation of were collected in 15 mL portions and analyzed by HPLC Ac WQPC*PAESWTFC*WDPGGGK(DPPE (Column: YMC C-4, 5 um, 300 A, 4.6x250 mm) using an Glut-PG2-JJ)-NH cyclic (4-> 12) peptide (Seq005 ELSD and a UV detector (0.220 nm). Pure product-contain PL3) (SEQID NO. 131) ing fractions were collected and concentrated on a rotary evaporator to remove the methanol from the eluate and the 0533. The peptide, resulting solution was diluted with 10% CHCN in H2O, Ac WQPC*PAESWTFC*WDPGGGK(JJ)-NH., cyclic frozen and lyophilized to afford 72 mg (48% yield) of the (4-> 12) peptide (Seq005-JJ) (SEQ ID NO. 136) (63 mg, required product as a fluffy white solid. 0.025 mmol) was dissolved in DMF (1.0 mL) and DIEA (9.8 mg, 13.3 uL, 0.076 mmol. 3 equiv) was added. The solution was stirred briefly and a solution of Fmoc-PG2-NHS (111 Example 15B mg, 0.032 mmol. 1.28 equiv) in DMF (1.0 mL) was added dropwise with stirring. The mixture was stirred for 16 hand Preparation of Comparative Peptide Seq000 conju HPLC analysis indicated the consumption of the starting gated to DSPE-PG4-Glut, referred to as Seq000-PL1 peptide. The reaction mixture was treated with piperidine (172 mg, 2004, 2.02 mmol. 80 equiv. final concentration -9% V/v in the reaction mixture) for 30 min after which the reac PG4-Glut)-NH cyclic (4-> 12) peptide Seq000-PL1 was pre tion mixture was diluted with HO and purified by preparative pared in a similar manner to the procedure of Example 15A. HPLC employing a reverse phase C4 column (Kromasil(R) Thus a Solution of the peptide Prep C, 10 um, 300 A, 20x250 mm) by the following Ac WQPC*PWESWTFC*WDPGGGK NH, cyclic method: elution: the plug of DMF was eluted at the initial (4-> 12) peptide (Seq000) (SEQID NO. 122) (115 mg, 0.05 condition of 20% CHCN in HO (0.1% TFA), then a linear mmol) and DIEA (130 mg, 176 uL. 1.0 mmol. 20 equiv) in gradient of 20% -80% CHCN (0.1% TFA) into HO (0.1% DMF (2.0 mL) was added to a stirred solution of DSG (100 TFA) over a period of 40 minata flow rate of 25 mL/min was mg, 0.30 mmol, 6 equiv) in DMF (0.5 mL). The mixture was employed to elute the product. Fractions (15 mL) were col stirred for 1 h and the progress of the reaction was monitored lected and those containing the pure product (HPLC analysis) by HPLC and MS. Upon completion of the reaction, the were pooled, frozen and lyophilized to provide 70 mg (47% volatiles were removed in vacuo and the residue was washed yield) of the PG2-derivatized peptide with ethyl acetate (4x20 mL) to remove unreacted DSG from Ac WQPC*PAESWTFC*WDPGGGK(NH-PG2-JJ) the intermediate peptide glutaric acid monoamide mono (SEQ ID NO. 143) - NH cyclic (4-> 12) peptide. NHS ester. 0534 A Solution of Ac WQPC*PAESWTFC*WDPGGGK(NH-PG2-JJ)- 0530. The resulting solid was dried, re-dissolved in anhy NH. (SEQID NO. 143) cyclic (4-> 12) peptide (50 mg, 0.009 drous DMF (1.0 mL) and stirred with a solution of DPPE mmol. 1.14 equiv) in DMF (1.0 mL) was treated with DIEA PG4-NH. (160 mg 0.0573 mmol. 1.11 equiv) in DMF (1.0 (14.8 mg, 20LL, 0.114 mmol. 14.6 equiv) followed by DPPE mL) for 24 hour. The progress of the reaction was monitored Glut-NHS (7 mg, 0.0078 mmol) in a 1:1 mixture of DMF: by HPLC, which indicated complete consumption of the CHCl (1.0 mL) and the mixture was stirred for 16hr at room intermediate peptide glutaric acid monoamide mono-NHS temperature. The solution was diluted with HO to 10 mL and ester. The solution was diluted with water and purified by purified by reverse phase C4 preparative column (Kromasil(R) reverse phase C4 preparative column (Kromasil(R) Prep C, 10 Prep C, 10um,300 A, 10x250 mm, flow rate, 10 mL/min) um, 300 A, 20x250 mm, flow rate 25 mL/min) using a gra using a gradient of 50-80% water (0.1% TFA)/acetonitrile: dient of 50-100% water (0.1%TFA) and a mixture of CHOH MeOH (1:1, 0.1% TFA) over a period of 40 min. The pure and CHCN (1:1, 0.1% TFA) over a period of 30 min. Frac product-containing fractions were pooled, frozen and lyo tions were collected in 15 mL portions and analyzed by HPLC philized to afford 26 mg (42% yield) of the target compound (Column: YMC C-4, 5 um, 300 A, 4.6x250 mm) using an as a fluffy white solid. ELSD and a UV detector (X=220 nm). Pure product-contain 0535 Examples 16B-16C below describe and illustrate ing fractions were collected and concentrated on a rotary the preparation of DPPE-Pro9-Glut-Ttda-Dga-peptide conju evaporator to remove the methanol from the eluate and the gates. Example 16D below describes and illustrates the resulting solution was diluted with 10% CHCN in H2O, preparation of a comparative lipopeptide, Seq000-PL2, from frozen and lyophilized to afford 138 mg (52% yield) of the required product as a fluffy white solid. Seq000(Adoa-Adoa) and DPPE. Example 16B Mass Spectrum: Method: MALDI, Mode: Positive Ion, IM+Na+2H): 5199 (Average) Preparation of Ac WQPCPAESWTFCWDPG SAGSK(DPPE-Pro9-Glut-Ttda-Dga)-NH cyclic 0531 HPLC: Ret. time: 5.88 min: Assay: >98% (area 96): (4-12) peptide (Seq005-PL4) (SEQ ID NO. 132) Column: YMCC-4, 5 uM, 300 A, 4.6x50mm; Eluents: A: Water (0.1%TFA), B: Acetonitrile:Methanol (1:1, v/v)(0.1% 0536 Preparation of Seq005-P2(Ttda-Dga) TFA); Elution: Initial condition: 80% B, linear gradient 0537 Chain elongation of the peptide was conducted on 80-100% B in 10 min.; Flow rate: 3.0 mL/min: Detection: UV 1.2 g of Fmoc-Pal-Peg-PS resin (0.17 mmol/g) on a 0.2 mmol (a) 220 nm & ELSD. scale (procedure A) to provide Ac W(N"-Boc)-Q(Trt)-P US 2010/0158814 A1 Jun. 24, 2010

C(Trt)-P-A-E(OtBu)-S(tBu)-W(N'-Boc)-T(tBu)-F-C(Trt)- (15 mL) for 15 h. The resin was washed with DMF (5x20 W(N'-Boc)-D(OtBu)-P-GS(tBu)-A-G-S(tEu)-K (iv.Dde)- mL). The peptide was cleaved from the resin using reagent B NH-Pal-Peg-PS resin. Resins from two runs were combined. (procedure G) and the crude solid peptide was subjected to The iv.Dde group was removed using 20 mL of 10% hydrazine disulfide cyclization (procedure H). The crude mixture was in DMF (2x10 min). Then diglycolic anhydride (0.464 g. 10 diluted with water to about five-fold its volume and applied to eq) and DIEA (0.516 g., 0.7 mL, 4.0 mmol) in DMF (20 mL) a Waters XTerra C-18 (250 mmx50 mm i.d.) column and were added to the resin and the resin was agitated for 15h. The purified using a linear gradient elution of ACN (0.1% TFA) resin was washed with DMF (5x20 mL) and then treated with into H2O (0.1%TFA) as described in the procedure titled. The N1-(tert-butoxycarbonyl)-1,3-diamino-4,7,10-trioxatride pure product-containing fractions were pooled, frozen and cane (0.513 g, 1.6 mmol. 4 eq), HATU (0.608 g, 1.6 mmol, 4 lyophilized to provide 227.7 mg (21.3% yield) of the peptide eq) and DIEA (0.413 g, 0.558 mL, 3.2 mmol, 8 eq) in DMF as a fluffy white solid. (20 mL) for 15 h. The resin was washed with DMF (5x20 (0542 Preparation of Ac WQPCPAESWTFCWDP mL). The peptide was cleaved from the resin using reagent B GAGSGK(DPPE-Pro9-Glut-Ttda-Dga)-NH cyclic (4-> 12) (procedure G) and the crude solid peptide was subjected to peptide (Seq005-PL5) from DPPE-Pro9-H and Seq005-P3 disulfide cyclization (procedure H). The crude mixture was (Ttda-Dga) diluted with water to about five-fold its volume and applied to (0543. DSG (75 mg, 0.230 mmol, 3.59 eq) in DMF (0.75 a Waters XTerra C-18 (250 mmx50 mm i.d.) column and mL) was stirred and to this mixture was added DPPE-(Pro) purified using a linear gradient elution of ACN (0.1% TFA) -H (100 mg 0.064 mmol) dissolved in DCM (0.5 mL). Then into H2O (0.1%TFA) as described in the procedure titled. The DIEA (0.03 g, 0.04 mL, 0.23 mmol. 3 equiv) was added and pure product-containing fractions were pooled, frozen and the mixture was stirred 4 h. Mass spectral analysis confirmed lyophilized to provide 175 mg (16.2% yield) of the peptide as the formation of the glutaric acid mono-amide-mono-NHS a fluffy white solid. ester of DPPE-(Pro). The volatiles were removed under high 0538 Preparation of Ac WQPCPAESWTFCWDPG vacuum and the crude residue kept for 2 hunder high vacuum. SAGSK(DPPE-Pro 9-Glut-Ttda-Dga)-NH cyclic (4->12) The resulting crude residue was triturated and washed with peptide (Seq005-PL4) from DPPE-Pro9-H and Seq005-P2 EtOAc to remove DSG and remaining traces of DMF and (Ttda-Dga) DIEA. The crude was redissolved in DCM (1 mL) and 0539 DPPE-(Pro)-H, 120 mg 0.077 mmol was added to Seq005-P3(Ttda-Dga) Ac WQPCPAESWTFCWDPG DSG (100 mg, 0.306 mmol. 4.0 eq) in DMF (2 mL), followed SAGSK(Ttda-Dga)-NH) (SEQID NO.145), (165 mg, 0.062 by DIEA (0.059 g, 0.08 mL, 0.46 mmol. 6.0 equiv); the mmol) in DMF (1 mL) was added, followed by DIEA (0.088 mixture was stirred 4 h. The volatiles were removed under g, 0.12 mL, 0.69 mmol. 11 equiv relative to the added pep high vacuum. The resulting crude residue was washed twice tide). The mixture was stirred at 40°C. for 15 h after which with EtOAc to remove DSG and remaining traces of DMF HPLC and MS analysis indicated formation of the desired and DIEA. The crude was redissolved in DMF (1 mL) and product. The resulting mixture was diluted with 35% MeOH Seq005-P2(Ttda-Dga) Ac-WQPCPAESWTFCWDPG in water (15 mL) and filtered using a 0.45 micron filter. The SAGSK(Dga-Ttda)-NH) (SEQID NO.145), (170 mg, 0.063 solution was purified by preparative HPLC on a C2 column. mmol) in DMF (1 mL) was added, followed by DIEA (0.088 The compound was applied to the column at 25% ACN g, 0.12 mL, 0.69 mmol. 11 equiv relative to the added pep MeOH 1:1, v/v (eluent B) in water (eluent A). After the tide). The mixture was stirred at 40°C. for 15h. The resulting compound was applied and the solvent plug eluted, the eluent mixture was diluted with 35% MeOH in water (25 mL) and composition was ramped to 50% B and then ramped from filtered using a 0.45 micron filter. The solution was purified 50-100% B over 30 min. The pure product containing frac by preparative HPLC. The pure product containing fractions tions were combined and most of the MeOH was removed by were combined, frozen and lyophilized to provide the product rotary evaporation. Tert-Butyl alcohol was added to the mix (90 mg. 32.8% yield) as a fluffy solid. ture and the mixture was frozen and lyophilized to give the product (125 mg, 46.5% yield) as a fluffy solid. Example 16C Example 16D Preparation of Ac WQPCPAESWTFCWDP GAGSGK(DPPE-Pro9-Glut-Ttda-Dga)-NH cyclic Preparation of Comparative Lipopeptide Seq000 (4-> 12) peptide (Seq005-PL5) (SEQ ID NO. 133) PL2 (0540 Preparation of Seq005-P3(Ttda-Dga) 0544 The fully side-chain protected peptide sequence 0541 Chain elongation of the peptide was conducted on Ac W(Boc)-Q(Trt)-P C(Trt)-P W(Boc)-E(O-t-Bu)-S 1.2 g of Fmoc-Pal-Peg-PS resin (0.17 mmol/g) on a 0.2 mmol scale (procedure A) to provide Ac W(N"-Boc)-Q(Trt)-P Bu)-P-GGGK(ivDde)-TGR was prepared on a 0.2 mmol C(Trt)-P-A-E(OtBu)-S(tBu)-W(N'-Boc)-T(tBu)-F C scale. The iv.Dde protecting group was removed from a 400 (Trt)-W(N"-Boc)-D(OtBu)-P-G-A-G-S(t-Bu)-G-K(ivDde)- mg (nominally 0.08 mmol) portion of the resin (procedure D). NH-Pal-Peg-PS resin. Resins from two runs were combined. The resin was washed with DMF (2x20 mL) and DCM (20 The iv.Dde group was removed using 20 mL of 10% hydrazine mL), re-suspended in DMF (10 mL) and treated with Fmoc in DMF (2x10 min). Then diglycolic anhydride (0.464 g. 10 Adoa (154 mg. 0.4 mmol), HOBt (54 mg. 0.4 mmol), DIC (51 eq) and DIEA (0.516 g., 0.7 mL, 4.0 mmol) in DMF (20 mL) mg, 62 uL, 0.4 mmol) and DIEA (139 uL, 0.8 mmol) for 4 h. were added to the resin and the resin was agitated for 15h. The The reagents were filtered off and the resin was washed with resin was washed with DMF (5x20 mL) and then treated with DMF (2x20 mL) and DCM (20 mL). The Fmoc group was N1-(tert-butoxycarbonyl)-1,3-diamino-4,7,10-trioxatride removed by treatment with 20% piperidine in DMF (2x20 cane (0.385 g, 1.2 mmol. 3 eq), HATU (0.456 g, 1.2 mmol. 3 mL) (modified procedure C) and the resin was washed with eq) and DIEA (0.310 g, 0.419 mL, 2.4 mmol. 6 eq) in DMF DMF (2x20 mL) and DCM (20 mL). Coupling with Fmoc US 2010/0158814 A1 Jun. 24, 2010 62

Adoa and Fmoc removal were repeated. The resin was re (0549 Separately, DPPE (15.8 mg-22.8 umoles) and suspended in DMF (7 mL) and treated with 3.6.9-trioxaun DPPG (4.2 mg-5.7 moles) were dispersed in a solution of decane-1,11-dioic acid anhydride solution prepared by the PEG4000 10% in distilled water (20mL) at 70° C. for 20 reaction of the corresponding acid (1.0 g, 0.45 mmol)) and minutes. The dispersion was then cooled to room tempera DIC (0.56 g. 0.45 mmol) in methylene chloride (5.0 mL) over ture. Perfluoroheptane (1.6 mL) was added to the aqueous a period of 12 hr., the solution was filtered and used directly phase using a high speed homogenizer (Polytron PT3000, for 16 hr. The reagents were filtered off and the resin was probe diameter of 3 cm) for 1 minute at 10000 rpm, to obtain washed with DCM (2x20 mL) and DMF (2x20 mL), re an emulsion. suspended in DCM (10 mL) and treated with a solution of dipalmitoyl phosphatidyl ethanolamine (690 mg, 1.0 mmol), 0550 The micellar suspension was mixed with the emul HATU (450 mg, 1.0 mmol) DIEA (400 mg) in DCM (5.0 mL) sion and the resulting mixture was heated at 80°C. for 1 hour and the mixture was allowed to shake for 26 hr (modified under agitation. After cooling at room temperature (1 hour), procedure B). The reagents were filtered off and the resin was the mixture was washed once by centrifugation (200 g/10 washed with DMF (2x20 mL) and DCM (2x20 mL) and min—Sigma centrifuge 3K10) to eliminate the excess of dried. The resin was then treated with Reagent B (30 mL) for phospholipids. The separated Supernatant (containing emul 4hr (procedure G). The resin was filtered off and the filtrate sified microdroplets of solvent) was recovered and re-sus was concentrated and treated with 200 mL of anhydrous EtO pended with the initial volume of a 10% PEG 4000 aqueous and the crude product was collected as a solid by filtration. Solution. This provided 400 mg of crude product which was dissolved 0551. The obtained suspension was sampled into DINER in DMSO (4.0 mL) after which the pH of the solution was vials (1 mL/vial). Then vials were cooled at -50° C. (Christ adjusted to 7.5 with an aqueous solution of N-methyl-D- Epsilon 2-12DS Freeze Dryer) and freeze-dried at -25°C. glucamine and stirred 48 h in air to effect formation of the and 0.2 mbar for 12 hours, with a final drying step at 30° C. cyclic peptide disulfide. The solution was diluted with water and 0.1 mbar for 7 hours. to a volume 40 mL and purified by reverse phase preparative 0552. The lyophilized product was then exposed to an HPLC (Kromasil R. Prep C, 10, 300 A, 20x250 mm, flow atmosphere containing CF/Nitrogen (50/50 by volume) rate 10 mL/min) using a gradient of 50-100% water (0.1% and the vials were sealed. TFA)/acetonitrile:MeOH (1:1, 0.1%TFA) over a period of 15 0553. The lyophilized product was dispersed in a volume min. The pure product-containing fractions were collected, of water twice the initial one by gentle hand shaking. combined and lyophilized to afford the target compound (28 mg, 10% yield) as a fluffy white solid. Example 18B (0545 Examples 17-20 below describe processes for pre paring targeted microbubbles with fibrin-binding polypep With Seq017-PL1 tides conjugated to phospholipids (lipopeptides). Such 0554 Example 18A was repeated by replacing Seq000 microbubbles are especially adapted for ultrasound imaging. PL1 with the same relative molar amount of Seq017-PL1. Example 17 Preparation of Targeted Microbubbles with DSPC/ Example 18C DPPG Envelope With Seq005-PL1 Example 17A 0555 Example 18A was repeated by replacing Seq000 With Comparative Lipopeptide Seq000-PL1 PL1 with the same relative molar amount of Seq005-PL1. 0546) 383 mg of a mixture DSPC/DPPG/Seq000-PL1 (molar ratio 47.5/47.5/5, corresponding to 157.5, 148.5 and Example 19 77.3 mg of the three components, respectively) and 22.6 g of PEG-4000 were solubilized in 120 g of t-butyl alcohol at 60° Preparation of Targeted Microbubbles with DSPC/ C., inawater bath. The solution was filled in vials with 0.8 mL DSPG Envelope of solution each. The samples were frozen at -45° C. and Example 19A lyophilized. The air in the headspace was replaced with a mixture of CF/Nitrogen (50/50) and vials capped and DSPC/DSPG Formulation with Comparative crimped. The lyophilized samples were reconstituted with 5 Lipopeptide Seq000-PL1 mL of HO per vial. 0556. An aqueous suspension of DSPE-PEG1000 (0.5 Example 17B mg-0.28 umole) and Seq000-PL1 (3.3 mg-0.63 umole) was With Seq005-PL1 prepared in 500 uL of distilled water at 60° C. to obtain a micellar Suspension. 0547 Example 17A was repeated, but replacing Seq000 0557 Separately, DSPC (18mg-22.75 umoles) and DSPG PL1 with the same relative molar amount of Seq005-PL1. (2 mg-2.53 umoles) were dissolved in cyclooctane (1.6 mL) Example 18 at 80°C. This organic phase was added to a PEG4000 10% Preparation of Targeted Microbubbles with DPPE/ Solution in water (20 mL) using a high speed homogenizer DPPG Envelope (Polytron T3000, probe diameter of 3 cm) for 1 minute at 9000 rpm, to obtain an emulsion Example 18A 0558. The micellar suspension was mixed with the emul With Comparative Lipopeptide Seq000-PL1 sion and the resulting mixture was heated at 80°C. for 1 hour (0548. An aqueous suspension of DSPE-PEG1000 (0.5 under agitation. After cooling to room temperature (1 hour), mg-0.28 umole) and Seq000-PL1 (3.3 mg-0.63 umole) was the obtained emulsion was washed once by centrifugation prepared in 500 uL of distilled water at 60° C. to obtain a (1500 g/10 min-Sigma centrifuge 3K10) to eliminate the micellar Suspension. excess of phospholipids. The separated Supernatant (contain US 2010/0158814 A1 Jun. 24, 2010

ing emulsified microdroplets of solvent) was recovered and under agitation. After cooling to room temperature (1 hour), re-suspended in the initial volume of a 10% PEG 4000 aque the obtained emulsion was washed once by centrifugation ous solution. (200 g/10 min-Sigma centrifuge 3K10) to eliminate the 0559 The obtained suspension was sampled into DINER excess of phospholipids. The separated Supernatant (contain vials (1 mL/vial). Then vials were cooled to -50° C. (Christ ing emulsified microdroplets of Solvent) was recovered and Epsilon 2-12DS Freeze Dryer) and freeze-dried at -25°C. re-suspended with the initial volume of a 10% PEG 4000 and 0.2 mbar for 12 hours, with a final drying step at 30° C. aqueous Solution. and 0.1 mbar for 7 hours. 0569. The obtained suspension was sampled into DINER 0560. The lyophilized product was exposed to an atmo vials (1 ml/vial). Then vials were cooled to -50° C. (Christ sphere containing CF/Nitrogen (35/65 by volume) and the Epsilon 2-12DS Freeze Dryer) and freeze-dried at -25°C. vials were sealed. and 0.2 mbar for 12 hours, with a final drying step at 30° C. 0561. The lyophilized product was then dispersed in a and 0.1 mbar for 7 hours. volume of water twice than the initial one by gentle hand 0570. The lyophilized product was then exposed to an shaking atmosphere containing CF/Nitrogen (35/65 by volume) and the vials were sealed. Example 19B 0571. The lyophilized product was dispersed in a volume DSPC/DSPG Formulation with Comparative Peptide of water twice than the initial one by gentle hand shaking Seq000-PL1 Example 20B 0562. Example 19A was repeated, but using 2.6 mg of DSPE-PEG1000 (1.44 umoles) and 1.9 mg of Seq000-PL1 With Seq017-PL1 (0.36 gmole) to prepare the micellar Suspension. 0572. Example 20A was repeated by replacing Seq000 PL1 with the same relative molar amount of Seq017-PL1. Example 19C DSPC/DSPG Formulation with Seq024-PL1 Example 200 0563 Example 19A was repeated, but using 2.6 mg With Seq005-PL1 Seq024-PL1 (0.45 umoles) and 0.8 mg DSPE-PEG1000 0573. Example 20 A was repeated by replacing Seq000 (0.45umoles) to prepare the micellar suspension. PL1 with the same relative molar amount of Seq005-PL1. Example 19D Example 20D DSPC/DSPG Formulation with Seq023-PL1 With Seq016-PL1 0564) Example 19A was repeated, but using 2.4 mg (0574) Example 20 A was repeated by replacing DSPG Seq023-PL1 (0.45 umoles) and 0.8 mg DSPE-PEG1000 with 5.8 umoles of stearate. (0.45umoles) to prepare the micellar suspension. Example 20E-20H Example 19E Preparation of Targeted Microbubbles with DSPC/ DSPC/DSPG Formulation with Seq016-PL1 DSPA Envelope 0565 Example 19A was repeated, but using 2.3 mg Example 20E Seq016-PL1 (0.45 umoles) and 0.8 mg DSPE-PEG1000 (0.45umoles) to prepare the micellar suspension. With Seq005-PL4 (0575 DSPC (16.3 mg-20.58 umoles), DSPA (3.7 mg-5.15 Example 20A-20D umoles) and Seq005-PL4(0.26 umoles, prepared as described above) were dissolved in cyclooctane (1.6 mL) at 80° C. Preparation of Targeted Microbubbles with DSPC/ 0576. The organic suspension was emulsified in a Stearate Envelope PEG4000 10% aqueous phase (20 mL) using a high speed Example 20A homogenizer (Polytron PT3000, probe diameter of 3 cm) for With Comparative Lipopeptide Seq000-PL1 1 minute at 8000 rpm to obtain the emulsion. (0577. The resulting emulsion was heated at 80° C. for 1 0566. An aqueous suspension of DSPE-PEG1000 (0.5 hour under stirring. After cooling at room temperature (1 mg-0.28 moles) and Seq000-PL1 12 (3.3 mg-0.63 umoles) hour), the emulsion was washed once by centrifugation (1500 was prepared in 500 uL of distilled water at 60° C. to obtain g/10 min-Sigma centrifuge 3K10) to eliminate the excess of the micellar Suspension. the phospholipid and the separated Supernatants (microdrop 0567 Separately, DSPC (18.2 mg-23.1 umoles) and stear lets) were recovered and re-suspended in twice the initial ate (1.8 mg-5.8 umoles) were dispersed in a solution of volume of a 10% PEG 4000 aqueous solution. PEG4000 10% in distilled water (20 mL) at 70° C. for 20 (0578. The emulsion was sampled in DINER vials (1 minutes. The dispersion was then cooled to room tempera mL/vial) and then lyophilized (laboratory freeze-dryer Lyo ture. Perfluoroheptane (1.6 mL) was added to the aqueous beta-35 TELSTAR) according the following sequence. phase using a high speed homogenizer (Polytron PT3000, 0579 Freezing: 2 hat-50° C. probe diameter of 3 cm) for 1 minute at 11000 rpm, to obtain 0580 Main Drying: 12 hat-25° C. and 0.2 mBar an emulsion. 0581 Final Drying: 6 hat 30° C. and 0.1 mBar 0568. The micellar solution was mixed with the emulsion 0582 Before redispersion, the lyophilisate was exposed to and the resulting mixture was heated at 60° C. for 4 hours an atmosphere containing CF/air (50/50 by volume). The US 2010/0158814 A1 Jun. 24, 2010 64 lyophilized product was then dispersed in a volume of water 0600 The organic suspension was emulsified in a twice the initial one by gentle hand shaking PEG4000 10% aqueous phase (20 mL) using a high speed homogenizer (Polytron PT3000, probe diameter of 3 cm) for Example 20F 1 minute at 8000 rpm to obtain the emulsion. 0601 The resulting emulsion was heated at 80° C. for 1 With Seq005-PL4 hour under stirring. After cooling at room temperature (1 0583 DSPC (16.3 mg-20.58 umoles), DSPA (3.7 mg-5.15 hour), the emulsion was washed once by centrifugation (1500 umoles) and Seq005-PL4(0.795 umoles, prepared as g/10 min-Sigma centrifuge 3K10) to eliminate the excess of described above) were dissolved in cyclooctane (1.6 mL) at the phospholipid and the separated Supernatants (microdrop 800 C. lets) were recovered and re-suspended in twice the initial 0584) The organic suspension was emulsified in a volume of a 10% PEG 4000 aqueous solution. 0602. The emulsion was sampled in DINER vials (1 PEG4000 10% aqueous phase (20 mL) using a high speed mL/vial) and then lyophilized (laboratory freeze-dryer Lyo homogenizer (Polytron PT3000, probe diameter of 3 cm) for beta-35 TELSTAR) according the following sequence. 1 minute at 8000 rpm to obtain the emulsion. 0603 Freezing: 2 hat-50° C. 0585. The resulting emulsion was heated at 80° C. for 1 0604 Main Drying: 12 hat-25° C. and 0.2 mBar hour under stirring. After cooling at room temperature (1 0605 Final Drying: 6 hat 30° C. and 0.1 mBar hour), the emulsion was washed once by centrifugation (1500 0606. Before redispersion, the lyophilisate was exposed to g/10 min-Sigma centrifuge 3K10) to eliminate the excess of an atmosphere containing CF/air (50/50 by volume). The the phospholipid and the separated Supernatants (microdrop lyophilized product was then dispersed in a volume of water lets) were recovered and re-suspended in twice the initial twice the initial one by gentle hand shaking volume of a 10% PEG 4000 aqueous solution. Example 21 0586. The emulsion was sampled in DINER vials (1 Dynamic Binding Test of Targeted Microvesicles mL/vial) and then lyophilized (laboratory freeze-dryer Lyo With Fibrin-Binding Peptides beta-35 TELSTAR) according the following sequence. Preparation of Fibrin-Coated Coverslips 0587 Freezing: 2 hat-50° C. 0607 Glass coverslips (40 mm in diameter, Bioptechs 0588 Main Drying: 12 hat-25° C. and 0.2 mBar Inc., Butler, Pa., USA) were coated with fibrin according the 0589 Final Drying: 6 hat 30° C. and 0.1 mBar following methodology. 0590. Before redispersion, the lyophilisate was exposed to 0608 Five mL of a solution of BSA 1% w/v in PBS pH 7.4 an atmosphere containing CF/air (50/50 by volume). The were added into a 60 mm Petri Dish containing one coverslip, lyophilized product was then dispersed in a volume of water and incubated at 37° C. for 15 min. Then the coverslip was twice the initial one by gentle hand shaking washed three times with 5 ml of Tween 80/PBS (0.1%, v:v). Twenty five gLof human thrombin solution at 5 U/mL were Example 20G added per mL of human fibrinogen solution (0.5 mg/mL in 50 mM sodium phosphate, NaCl280 mM, pH 7.4) and delicately With Seq005-PL5 mixed. Five mL of this solution were immediately distributed 0591 DSPC (16.3 mg-20.58 umoles), DSPA (3.7 mg-5.15 in each Petridish. The coverslips were incubated for one hour umoles) and Seq005-PL5(0.26 umoles, prepared as described at 37° C. and then dried overnight at 45° C. above) were dissolved in cyclooctane (1.6 mL) at 80° C. Binding Assay 0592. The organic suspension was emulsified in a 0609 Binding studies of targeted microvesicles were car PEG4000 10% aqueous phase (20 mL) using a high speed ried out using a parallel-plate flow chamber (FCS2. Bioptech homogenizer (Polytron PT3000, probe diameter of 3 cm) for Inc., Butler, Pa., USA) with a chamber gasket of 0.25 mm in 1 minute at 8000 rpm to obtain the emulsion. thickness, with a customized adapter for upside-down cham 0593. The resulting emulsion was heated at 80° C. for 1 ber inversion. The coated coverslip was inserted as a plate of hour under stirring. After cooling at room temperature (1 the flow chamber. Gas-filled Microvesicles (5x10 bubbles/ hour), the emulsion was washed once by centrifugation (1500 mL in 50% human plasma in PBS) were drawn through the g/10 min-Sigma centrifuge 3K10) to eliminate the excess of flow chamber using an adjustable infusion pump (Auto the phospholipid and the separated Supernatants (microdrop Syringe RAS50 Infusion Pump, Baxter, Deerfield, Ill., USA) lets) were recovered and re-suspended in twice the initial with a 60 mL syringe (Terumo). The pump flow rate was volume of a 10% PEG 4000 aqueous solution. adjusted to 1 mL/min to obtain the desired shear rate of about 0594. The emulsion was sampled in DINER vials (1 114s'. After 10 minutes, the flow was stopped and pictures mL/vial) and then lyophilized (laboratory freeze-dryer Lyo were taken randomly on different positions on the coverslip beta-35 TELSTAR) according the following sequence. (on surfaces of about 0.025 mm) using a 40x objective and a 0595 Freezing: 2 hat-50° C. CCD monochrome camera (F-View II, Soft Imaging Sys 0596) Main Drying: 12 hat-25° C. and 0.2 mBar tems, Germany) connected to an inverted Olympus IX 50 0597 Final Drying: 6 hat 30° C. and 0.1 mBar microscope. The number of microvesicles on each picture 0598. Before redispersion, the lyophilisate was exposed to was determined, averaged with respect to the total number of pictures and the obtained value was then divided by ten (to an atmosphere containing CF/air (50/50 by volume). The obtain the “slope', i.e. the average amount of bound lyophilized product was then dispersed in a volume of water microvesicles per minute). twice the initial one by gentle hand shaking 0610 For each preparation of Examples 17-20, the bind Example 20H ing assay was repeated four times thus obtaining an average value of the slope. The slope represents the microvesicle With Comparative Peptide Seq000-PL2 binding rate on the target Substrate. For instance, a slope value 0599 DSPC (16.3 mg-20.58 umoles), DSPA (3.7 mg-5.15 of8 indicates that an average of eighty (80) microvesicles was umoles) and Seq000-PL2 (0.26 umoles, prepared as bound on the coated coverslip in ten minutes. A higher slope described above) were dissolved in cyclooctane (1.6 mL) at indicates a better capacity of microvesicles to bind to the 800 C. target under flow conditions. US 2010/0158814 A1 Jun. 24, 2010 65

0611. In the following Tables 5-10, the binding activity of the microvesicles prepared according to Examples 17-20 TABLE 8-continued above is illustrated. As shown in these Tables, peptides Fibrin- Mi ic according to the present invention (particularly their respec- Bindingprin Preparationicrovesicle tive lipopeptides) show superior activities with respect to the Lipopeptide of Example Slope comparative peptide Seq000 (in particular, to corresponding SedO23-PL1 19D 8.5 lipopeptides Seq000-PL1 and Seq000-PL2). Seq016-PL1eqJ25 19E 11.5

TABLE 6 Fibrin- Microvesicle TABLE 9 Binding Preparation Lipopeptide of Example Slope Fibrin- Microvesicle Binding Preparation Seq000-PL1 17A 6.44 Lipopeptide of Example Slope Seq005-PL1 17B 8.54 Seq000-PL1 2OA 1.6 Seq017-PL1 2OB 3.1 Seq005-PL1 2OC 6.1 TABLE 7 Seq016-PL1 2OD 8.4

Fibrin- Microvesicle Binding Preparation Lipopeptide of Example Slope TABLE 10

s: Fibrin-Binding preparationMicrovesicle Seq005-PL1 18C 7.2 Lipopeptide of Example slope

Seq005-PL4 2OE 6.4 Seq005-PL4 2OF 8.O TABLE 8 Seq005-PL5 29OG 6.O Seq000-PL2 2OH 4.6 Fibrin- Microvesicle Binding Preparation Li popeptidetid offE Example SI ope Example 22 Seq000-PL1 19A 4.4 Preparation of the Chelated Complex 1 Seq000-PL1 19B 4.9 Seq024-PL1 19C 6.2 0612. The synthetic procedure for the preparation of the Chelated Complex 1 is set forth in Scheme 5.

Scheme 5 Poe H N N CONH(Trt) COOtBl Kl C STrt ) tBuO tBuO TrtS CH3CONH CONH CON NCONH Kl CONH CONH CONH CONH CONH CCONH Dr. GD-Oc HNOCHNOCHNOCSHNOCNNOC, HNOC NH

tBOOC

NHoo1 No1 N1 O n1NN N Fmen---o-so Boc A

1) 50% Morpholine in DMAC 7) 50% Morpholine in DMAC 2) Fmoc-GGG-OH, DIC, HOBt, DMAC 8) DTPA-Glu, DIC, HOBt, DMAC 3) 50%. Morpholine in DMAC 9) R. tBEtO 4) Fmoc-Lys(Fmoc)-OH, DIC, HOBt, DMAC 2. Asidiourification (prep. HPLC) 5)65 50%.Fmoc-Lys(Fmoc)-OH, Morpholine in DMAC DiC, HOBt, DMAC1) GdCl3, NaOH. pH 7 prep. 12) Desalting US 2010/0158814 A1 Jun. 24, 2010 66

-continued S S H N

CONH2 COONa

CHCONH CONH CON Kl C O ch, CONH CON CONH CONH CONH CONH CONH CONH CONH /-COO HNOC HNOC,N/ HNOC NHNOCNHNOCN-NOC, HNOC NH N

Gd3+\-COO NaOOC J OOCNN 2 Nat \- COO NA Nico1 No1 N1'nO 1YNH N \-COO 11NOCHN os-s H ÖCHNN-OCHNSOCANN-OCHN1NOCHN OCHN N-1N1\-1 O -COO E E N \-COO Gd3+ NHCO No-n i-coo OCHN COO YAN \-COO COO Neo COO ooc1YN1\1 N n1 N1)-coo ooc1N1\1 N n1N1\coo

OOC ! . .COO OOC - COO

0613 Chelated Complex 1 mL). DTPA-Glu (10.7 g; 14.4 mmol), HOBt (2.20 g; 14.4 0614 Fmoc-PAL-PEG-PS resin supported intermediate A mmol), DIC (2.26 mL, 14.4 mmol), DIEA (4.90 mL. 28.8 mmol) and DMAC (40 mL) were added to the resin. The prepared as schematized in FIG. 1 (5.00 g; 0.90 mmol) was Suspension was shaken for 24hat room temperature, filtered shaken in a SPPS vessel with DMAC (40 mL) for 1 h to swell and the resin washed with DMAC (5x40 mL), CHCl (5x40 the resin. After the solvent was filtered, Fmoc-GGG-OH mL) and then vacuum dried. The resin was shaken in a flask (1.48 g; 3.60 mmol), HOBt (0.55 g; 3.60 mmol), DIC (0.56 with “Reagent B” (150 mL) for 4.5 h. The resin was filtered mL: 3.60 mmol) and DMAC (40 mL) were added to the resin, and the Solution was evaporated under reduced pressure to the Suspension shaken for 6 hat room temperature, the mix afford an oily crude that, after treatment with EtO (40 mL), ture filtered and the resin washed with DMAC (5x40 mL). gave a precipitate. The precipitate was centrifuged, decanted The resin was then shaken with 50% morpholine in DMAC (7 and washed with EtO (4x40 mL) to give a white solid (2.10 mL) for 10 min, the mixture filtered and fresh 50% morpho g). This product (2.10 g) was dissolved in a mixture of DMSO (36 mL) and HO (4.0 mL) and the pH adjusted to 8 with line in DMAC (7 mL) was added. The suspension was stirred D-(-)-N-methylglucamine (1.23 g). The solution was stirred for 20 min then the mixture was filtered and the resin washed for 96 hours at room temperature and then purified by pre with DMAC (5x40 mL). Fmoc-Lys(Fmoc)-OH (2.13 g; 3.60 parative HPLC. The fractions containing the product were mmol), HOBt (0.55 g; 3.60 mmol), DIC (0.56 mL: 3.60 lyophilized to afford the desired chelating ligand (0.280 g; mmol) and DMAC (40 mL) were added to the resin, the 0.058 mmol) as a white solid. The ligand (0.240 g; 0.050 Suspension shaken for 6 hat room temperature, filtered and mmol) was suspended in HO (80 mL) and dissolved by the resin washed with DMAC (5x40 mL). The resin was then addiction of 0.1 NNaOH (8.50 mL: 0.85 mmol) up to pH 6.5. shaken with 50% morpholine in DMAC (7 mL) for 10 min, 5.187 mM aq. GdCl (38.3 mL: 0.202 mmol) was added the mixture filtered and fresh 50% morpholine in DMAC (7 maintaining pH 6.5 by means of 0.1 N NaOH (6.0 mL. 0.60 mmol). The solution was adjusted to pH 7.0 with 0.1 NNaOH mL) was added. The suspension was stirred for 20 min then and then loaded onto a XAD 1600 column and eluted with a the mixture was filtered and the resin washed with DMAC gradient HO/ACN (the desired product elutes with a percent (5x40 mL). Fmoc-Lys(Fmoc)-OH (4.26 g; 7.20 mmol), age of ACN-30) to give, after evaporation, compound the HOBt (1.10 g; 7.20 mmol), DIC (1.12 mL: 7.20 mmol) and Chelated complex 1, as sodium salt, (0.167 g; 0.029 mmol) as DMAC (40 mL) were added to the resin, the suspension a white solid. Overall yield 3.8%. shaken for 6 h at room temperature, filtered and the resin 0615. Analytical Data for Chelated Complex 1 washed with DMAC (5x40 mL). The resin was then shaken 0616) Mr (Molecular Weight): 5663.73 with 50% morpholine in DMAC (7 mL) for 10 min, the (C20s H276GdaNasNaoCss S2) mixture filtered and fresh 50% morpholine in DMAC (7 mL) 0617 CE (Capillary Electrophoresis): 88.5% (Area %) was added. The suspension was stirred for 20 min then the 0618 MS (Mass Spectroscopy): Obtained data consistent mixture was filtered and the resin washed with DMAC (5x40 with Chelated Complex 1 structure US 2010/0158814 A1 Jun. 24, 2010 67

Example 23 Preparation of the Chelated Complex 2 0619. The synthetic procedure for the preparation of the Chelated Complex 2 is set forth in Scheme 6.

Scheme 6

H N CONH(Trt) COOtBl Kl ? ) tBuO tBuO TrtS CHCONH3 CONH CON CONH CONKl CONH CONH CONH CONH CONH CCONH CONH O GO- OC HNOCN- HNOCN- HNOCN- HNOC 3) HNOC NH

NH

1) 10% Hydrazine in DMF 6) DTPA-Glu, DIC, HOBt, DMAC 2) Fmoc-Lys(Fmoc)-OH, DIC, HOBt, DMAC7) Reagent B, EtO 3) 50%. Morpholine IN DMAC 8) Air oxidati ificati HPLC 4) Fmoc-Lys(Fmoc-OH, DIC, HOBt, DMAC struction (prep. ) 5) 50%. Morpholine in DMAC 10) Desalting3:

S-S H N N CONH COONa Kl HO HO CH3CONH3 CONH CON CONH CONKl CONH CONH CONH CONH CONH CCONH CONH O HNOC HNOC n/ HNOC n/ HNOC n/ HNOC NOC, HNOC NH OOC Gd3+ r COO OOC- NS-SN-N-N-N-COO2 Na" NaOOC OOC 1-nois OCHN OCHN N n/ n/ y -COO N-C

OCHN C N COO Nico-to ooc1N1N1,N1,N1\cooN ooc1N1N1 Nin 1N1 Nooo

OOC COO OOC US 2010/0158814 A1 Jun. 24, 2010

0620 Chelated Complex 2 mL) and then vacuum dried. The resin was shaken in a flask with “Reagent B” (100 mL) for 4.5 h. The resin was filtered 0621. To Fmoc-PAL-PEG-PS resin supported intermedi and the Solution was evaporated under reduced pressure to ate B (3.00 g; 0.60 mmol) obtained according to Procedure A, afford an oily crude that, after treatment with EtO (40 mL), described above, was shaken in a SPPS vessel with DMAC gave a precipitate. The precipitate was centrifuged, decanted (25 mL) for 1 h to swell the resin. After the solvent was and washed with EtO (4x40 mL) to give a white solid (1.60 filtered, the resin was washed with DMF (5x25 mL). The g). This product (1.60 g) was dissolved in a mixture of DMSO resin was then shaken with 10% hydrazine in DMF (25 mL) (27 mL) and H2O (3.0 mL) and the pH adjusted to 8 with for 15 min, the solvent filtered and fresh 10% hydrazine in D-(-)-N-methylglucamine (0.94 g). The solution was stirred DMF (25 mL) was added. for 96 hours at room temperature and then purified by pre 0622. The suspension was stirred for more 20 min then the parative HPLC. The fractions containing the product were mixture was filtered and the resin washed with DMF (5x25 lyophilized to afford the desired chelating ligand (0.260 g; mL) and then DMAC (5x25 mL). Fmoc-Lys(Fmoc)-OH 0.060 mmol) as a white solid. The ligand (0.220 g; 0.050 (1.42 g; 2.40 mmol), HOBt (0.36 g.: 2.40 mmol), DIC (0.37 mmol) was suspended in HO (80 mL) and dissolved by mL: 2.40 mmol) and DMAC (25 mL) were added to the resin, addiction of 0.1 NNaOH (7.40 mL: 0.74 mmol) up to pH 6.5. the Suspension shaken for 24h at room temperature, filtered 6.21 mM aq. GdCl (32.60 mL: 0.202 mmol) was added and the resin washed with DMAC (5x25 mL). The resin was maintaining pH 6.5 by means of 0.1 N NaOH (6.10 mL. 0.61 then shaken with 50% morpholine in DMAC (25 mL) for 10 mmol). The solution was adjusted to pH 7.0 with 0.1 NNaOH min, the mixture filtered and fresh 50% morpholine in DMAC and then loaded onto a XAD 1600 column and eluted with a (25 mL) was added. The suspension was stirred for 20 min gradient HO/ACN (the desired product elutes with a percent then the mixture was filtered and the resin washed with age of ACN-30) to give, after evaporation, the Chelated com DMAC (5x25 mL). Fmoc-Lys(Fmoc)-OH (2.84 g; 4.80 plex 2, sodium salt, (0.174 g; 0.033 mmol) as a white solid. mmol), HOBt (0.73 g; 4.80 mmol), DIC (0.75 mL: 4.80 Yield 6.6% . mmol) and DMAC (25 mL) were added to the resin, the 0623 Analytical Data Suspension shaken for 24hat room temperature, filtered and 0624 Mr. 5202.26 (C7HaGdNNaOS) the resin washed with DMAC (5x25 mL). The resin was then 0625 CE: 87.8% (Area %) shaken with 50% morpholine in DMAC (25 mL) for 10 min, 0626 MS: Obtained data consistent with Chelated Com the mixture filtered and fresh 50% morpholine in DMAC (25 plex 2 structure mL) was added. The suspension was stirred for 20 min then the mixture was filtered and the resin washed with DMAC Example 24 (5x25 mL). DTPA-Glu (7.17 g; 9.60 mmol), HOBt (1.47 g; Preparation of the Chelated Complex 4 9.60 mmol), DIC (1.50 mL. 9.60 mmol), DIEA (3.26 mL: 0627 By following the synthetic procedure of Scheme 6, 9.60 mmol) and DMAC (25 mL) were added to the resin. The and by using the suitable AAZTA ligand instead of the cor Suspension was shaken for 24hat room temperature, filtered responding DTPA ligand the Chelated Complex 4 has been and the resin washed with DMAC (5x25 mL), CHC1 (5x25 also prepared. Chelated Complex 4 S S H N N COONa HO HO

ClCH3CONH CONHy C O Sl Slal CONH CONH CONH CONH CONH CONH HNOC HNOC NHNOCNHNOCNHNOCN-NOC,n/ HNOC NH OOC COONaGd J o o ls ! NaOOC OOC N N so V N OCHNN-OCHN n/ OCHN N N N E 7 Sco o H OOC COONaGd lu NHCO. NHCO OOC COONaGd ls ors N OCHN N- COO NHCO C VNN N l so OOC US 2010/0158814 A1 Jun. 24, 2010 69

(0628 Reference Complexes moiety to be conjugated to linker-functionalized peptide 0629. By starting from the previously known peptide intermediate, some chelated compounds have been prepared Ac-WQPC*PWESWTFC*WDGGGK NH, provided by as Reference Compounds for in vivo and in vitro tests. The WO02/055544 and by following the synthetic procedures of following reference compounds have, in particular, been pre Schemes 5 and 6, Suitably changing the chelating ligand pared:

Reference 1

H H H N N N

CONH2 -S-Frrn S-S4-S-S"Oil () 4s {l S-se CH3CONH CONH CON CONH IKl OOC CONH CONH CONH CONH CONH Gd3 Y HNOC HNOCHNOCall HNOC, HNOC ClaNOC HNOC NH ooc1\ oocOOC COONa Sooc NaOOC ) N or V N gr N N OCHN

Nooc-/-/2Clusus.COONa NHCO NH1 Yo O NHoo1 No1 N1 n1NNH H OCHN1N OCHN 1n OCHN 1n OCHNS-1a1n-O ~sO

--> NHCO NHCO COONa OCHN ~ Nooc1N1\1 N n1n N1,Nooo Noos-- ) Gd3 ls ) Gd -3 ls OOC COO OOC COO

Reference 2 H H H N N N CONH COONa 21 2 h S-S lHONS S SaS a S-SHO. Sa-S-S

CHCONH3 CONH Kl CONH (.KlCON CONH CONH)) CONH CONH l CONH) CONH l CONH O

HNOC, HNOC, HNOC, HNOC, HNOC NOC HNOC NH

NaOOC CO CO. OC ls ! NHoo1 No1 N1,N1O NH N cy---Gd'3 N 'N

ls CONa US 2010/0158814 A1 Jun. 24, 2010 70

-continued

Reference 3

COONa 21 CONH2 Kl' (, ) HO HO w S CHCONH CONH CON CONH CONH CONH CONHDC CONH CONH O HNOC HNOCHNOC HNOC HNOC CNOC HNOC NH

Reference 4

N N N

CONH2 OlS C. n HO n ClY S

CH3CONH CONH Cl (Kl CONH CONH CONH) CONH l CONH CONH CONH O HNO C HNOCS-HNOCN-HNOCN-HNOCNu-NOCNY a HNOC NH HOOC1

O NHCO 11a- n1NNH NaOOC OCHN n1no1n 1 os-sO H COO OOC-N-

COO US 2010/0158814 A1 Jun. 24, 2010 71

-continued Reference 5 S S H HN N N COONa CONH2 Sh HO HO CHCONH CONH Sl Kl CONH CONH CONH) CONH CONH CONH CONH O HNOC HNOCN-HNOCs-HNOCs-HNOCN-NOCHNOCN-NH

o N8. O O C s N O OOC -v NHoo1 No1 N1 n-1NNH HOOC-/ N N- --~en~~'s H

Example 25 TABLE 10-continued Fibrin MRI Imaging in Human/Mouse Clots Max signal 0630. In Vitro MRI Tests Enhancement at Comparison to 0631 Small plasma clots from different species (human, Compound 100 M Chelate Complex 1 guinea pig and mouse) ranging from 0.5-7 mm in diameter Reference Compound 5 41% -69% were formed by combining citrate plasma (1:3.V/v) phospho Reference Compound 4 23% -77% lipids (Reagent Pathromtin) and CaCl2 0.008 M (Dade Behring, Germany) into small 2 ml vials at 37°C. Clots were washed 3 times with TBS 1.x and incubated with contrast 0635. The results indicate that the compound of the inven agents at 100 uM for 30 minutes at 37°C. Two clots were tion, Chelated Complex 1, has the maximum signal enhance prepared for each solution of incubation. After the incubation ment. clots were washed 4 times with TBS 1x and placed into small Example 26 vials filled with TBS 1x for MRI analysis. T1-weighted 2D Spin-Echo (SE) images were acquired with the following Fibrin MRI Imaging in Tumor Models parameters: TR/TE=400/8 ms, spatial resolution=344 um and 0636. In Vivo MRI Tests: slice thickness-2 mm. Maximum signal intensity was mea 10637. Three groups of 5 mice each, inoculated with 2.10° Sured in each image containing clot and compared to the neuroblastoma cells with PBS, signal from a clots incubated in TBS. After MRI, clots were 0638 were formed and tested as follows: prepared for ICP analysis. 0639 Group a: Reference compound 2. 0632. The Chelated Complex 1 of the invention, Refer (0640 Group b: Chelate Complex 1 ence compound 1, Reference compound 2. Reference com (0641 Group c: ProHance(R) (see Protocol) pound 4, and Reference compound 5 were tested on human 0642 At day-10 post tumor cells inoculation, at least 4 clots by MRI at different concentrations in the incubation mice per group with similar tumor size (5-10mm in diameter) medium (25, 100 and 400 uM of complex). were selected for the in vivo MRI tests. After pre-contrast 0633 Results MRI acquisition (T1 and T2 high resolution 2D spin-echo and 0634. By comparing the signal enhancement in clots reg 3D gradient echo images), the test compounds were admin istered after incubation with the compounds at 100 uM con istered (25umol/kg iv.) and MRI was performed at 4 hand 24 centration, tested compounds could be classified in term of h post injection. Maximum signal intensity was measured in performance as follows: the entire tumor area and in each post contrast image covering the whole tumor and the signal was compared to the pre TABLE 10 contrast images. At the end of MRI exams, all animals were Max signal sacrificed and the tumor, blood, liver, kidneys and femoral Enhancement at Comparison to bone were removed and prepared for Gd assay by ICP-AES. Compound 100 M Chelate Complex 1 0643 (Inductively coupled plasma atomic emission spec troscopy). Chelated Complex 1 100% Reference Compound 2 68% -32% 0644. Results Reference Compound 1 44% -66% 0645. The size of the tumors in all groups was similar (volume of about 500 mm). US 2010/0158814 A1 Jun. 24, 2010 72

0646 A significant tumor signal enhancement was phospholipids, sodium chloride 2.4 g/L, Hepes 14.3 g/L, pH observed 4h post Chelated Complex 1 injection whereas with 7.6). The mixture was incubated for 2 minutes at 37°C., and the Reference Compound 2 the enhancement was low and did then 300 uL of calcium chloride solution, previously incu bated at 37°C., was added to obtain a new mixture that was not cover the whole tumor (<3 slices over 9, -30% of slices further incubated for 30 minutes at 37° C. The obtained clot covering the tumor). FIG.8 shows obtained T1 weighted MRI was transferred to a 5 mL tube, washed 3 times with 3 mL of images, registered at 2T, after injection of 25umol/kg of the TBS and incubated with the testarticle at the final concentra complex. For the ProHance(R) injection signal distribution tion ranging from 0.0 to 0.2 mM for 30 minutes at 37°C. At was only on the border and no signal enhancement was the end of incubation, clots were rinsed 4 times with 3 mL of observed inside the tumors. TBS to remove the unbound testarticle and then placed in 1.5 0647. In vitro and in vivo tests described in Examples 25 ml vials for MRI procedure. and 26 were performed by following the protocols below. 0669 High resolution MRI was then performed by using 0648. Protocols T1-weighted Spin-Echo (SE) sequences. T1 maps were also calculated from the acquisition of 2D gradient echo images or 0649 Screening of Fibrin Targeted Compounds of the inversion recovery spin echo images. Invention. 0670. At the end of experiment the clots were sent at the 0650. With the aim to test the specificity and efficiency of Analytical Laboratory for ICP-AES analysis. the compounds of the invention the following two protocols 0671 Assay of Gadolinium in Biological Samples have been developed and used. One is dedicated to the in vitro 0672 Apparatus MRI tests on clots and the other for the in vivo MRI tests on 0673. The assays were carried out on a Jobin-Yvon Mod neuroblastoma mouse model. 24 spectrometer operating with the following instrumental 0651. In Vitro Tests parameters: 0652 This protocol has been developed and used to test 0674) sample flow: 1 mL/min the specificity and efficiency of the fibrin targeted contrast 0675 plasma flame: 6000 to 10000° C. agents of the invention for sensitive detection of thrombus by 0676 wavelength: 342.247 nm. use of MRI. The study was performed on clots generated in 0677 Argon flow: nebulizer 0.3 L/min, transport gas vitro from different plasma species. Signal enhancement 0.2L/min, cooling gas 12 L/min. from each sample incubated with the fibrin targeted contrast 0678 Sample digestion was performed by a microwave agent was evaluated and compared to a standard contrast system (MDS-2000 CEM Corporation). 0679 Sample preparation and analytical conditions agent ProHance(R). 0680 Different preparation for each biological matrix 0653. Materials was adopted. 0654 Test Article 0681 Clot solutions were prepared by suspending the 0655 Compounds: The contrast agents tested were com clot in 1.5 mL of nitric acid (65% V/v). pounds of the present invention comprising a fibrin peptide, 0682) 1.5 mL of nitric acid (65% v/v) was added to an optional linker and at least one chelated complex of gado solutions of incubation (before and after incubation) and linium. to washing Solutions. 0656 Reagents 0683. The destruction of the organic matrix was per 0657 Compounds: Biological samples (species, biologi formed by Subjecting the samples to a wetashing process with cal fluid and strain): a microwave oven system. 0658) guinea pig plasma (Dunkin Hartley) 0684 Finally, the dried residues were dissolved with 3.0 0659 mouse plasma CDR-1 (ICR)BRIGS mL of HCL 5% (v/v) and then analysed by ICP-AES. 0660 rabbit plasma (New Zealand White) 0685. Data Processing. 0661 Storage: at -20° C. 0686 Briefly, linearity was evaluated for two standards, low and high, ranging from 0.00 to 20 mg (Gd)/L in HCl 5% 0662 Test System (v/v), respectively. The total content of gadolinium in the test 0663 The test system used was clots generated in-vitro sample was calculated by using the instrumental calibration from different plasma species (Human, guinea pig, mouse straight line and express as ug(Gd)/mL. and rabbit), chosenbecause they area suitable model and easy 0687 MRI Data Analysis to replicate for this study. 0688. The mean signal intensity was measured within a 0664) Equipment region of interest (ROI) including a clot, drawn by an operator 0665 All MRI experiments were performed on a 2T on each MR image using dedicated software (MR Research Oxford magnet (bore 45 cm i.d.), equipped with a self Systems Ltd, Surrey UK). Signal intensity enhancement shielded gradient set (16 cm i.d.) driven by six TECHRONR) (Enh96) was determined as: amplifiers (4:1:1 configuration) with a maximum gradient strength of 110 mT/m and slew rate of 75us, and interfaced to an MRRS console (MRResearch Systems Ltd, Surrey UK). A 0689 where SI and SI are the mean signal intensity of clots incubated without and with test articles, respec bird-cage resonator antenna (7.3 cm i.d.) was used. tively. 0666 Methods 0690 Protocol for In Vivo Tests 0667 In-Vitro Tests 0691. The study aim was to evaluate the specificity and 0668. The delivered Control Plasma Nwas obtained from efficiency offibrin targeted agents of the invention as contrast pooled plasma collected from healthy blood donors, stabi agents for sensitive detection of fibrin within solid tumor. lized with HEPES buffer solution (12 g/L) and lyophilized. 0692. The study was performed on a mouse model of Before use, Plasma Control N was reconstituted in distilled neuroblastoma induced by Neuro-2a cells subcutaneously water by shaking carefully the Suspension to dissolve the injected in the right flank of a A/Jmouse (see for instance: Y. lyophilized plasma. Clots were formed, in a 2 mL vial, to Chen. Effects of irradiated tumor vaccine and continuous which was added 300 ul of reconstituted plasma and 300 uL localized infusion of granulocyte-macrophage colony-stimu of Pathromtin SL (silicon dioxide particles, vegetable lating factor on neuroblastomas in mice. J. Pediatr Surg. US 2010/0158814 A1 Jun. 24, 2010

2003. 37(9), 1298–1304; Anthony D. Sandler, Hiroshi Chi 0722 High resolution T1-weighted Spin-Echo and Gradi hara and Gen Kobayashi. CpG Oligonucleotides Enhance the ent Echo sequences were used to achieve images with a Suit Tumor Antigen-specific Immune Response of a Granulocyte able contrast to detect tumors and to follow the contrasto Macrophage Colony-stimulating Factor-based Vaccine Strat graphic effects of test and reference articles. egy in Neuroblastoma Cancer Research 2003, 63, 394-399) 0723. At the end of the experiment, animals were sacri 0693 Signal enhancement kinetic within tumors was ficed. Tumor, blood, kidneys, liver and femoral bones were evaluated after injection of a fibrin targeted contrast agent of then taken, weighed and stored at about 4°C. until analyzed the invention and compared to a standard agent, ProHance(R). by ICP-AES. 0694. Materials 0695 Test Article Assay of Gadolinium in Biological Samples 0696 Compound: The contrast agents tested comprise a 0724 Apparatus fibrin targeted peptide, an optional linker and at least one 0725. The assays was carried out on a Jobin-Yvon Mod 24 chelated complex of gadolinium. spectrometer operating with the following instrumental 0697 Reference Article parameters: (0698 Compound: ProHance(R) 0726 sample flow: 1 mL/min 0699 Concentration: 0.5 M 0727 plasma flame: 6000 to 10000° C. 0700 Batch: T2059 0728) wavelength: 342.247 nm. 0701 Storage: at RT, protected from light 0729 Argon flow: nebulizer 0.3 L/min, transport gas (0702 Reagents 0.2L/min, cooling gas 12 L/min. (0703 Compound: Penicillin/Streptomycin (10000 0730 Sample digestion was performed by a microwave ug/mL). Supplier: Biochrom KG, Berlin, Germany system (MDS-2000 CEM Corporation). 0704 Compound: L-Glutamine (200 mM), supplier: 0731 Sample Preparation and Analytical Conditions SIGMA-ALDRICH, Steinheim, Germany 0732. Different preparation for each biological matrix was (0705 Compound: Foetal bovine serum, Supplier: adopted: HyClone(R), Logan, Utah, USA 0733. Tumor solutions were prepared by suspending 0706 Compound: Minimum Essential Medium Eagle, the tumor, accurately weighed, in 1.5 mL of nitric acid supplier: SIGMA-ALDRICH, Steinheim, Germany (65% v/v). (0707 Compound: Dulbecco's Phosphate Buffered Saline 0734) Liver was prepared by measuring the liver, accu (PBS, supplier: SIGMA Chemicals, St. Louis, Mo., U.S.A. rately weighed, in 1.5 mL of nitric acid (65% V/v). (0708 Compound: Zoletil 100, supplier: Virbac, Carros, 0735 Kidney solutions were prepared by suspending each France Compound: Rompun R, supplier: Bayer AG, kidney, accurately weighed, in 1.5 mL of nitric acid (65% Laverkusen, Germany v/v). (0709 Test System (0736 Blood solutions were prepared by mixing 1 mL of 0710 Animals blood in 1.5 mL of nitric acid (65% V/v). 0711 Species and strain: mouse A/J (chosen as it is a 0737 Bone solutions were prepared by suspending each Suitable model for pharmaco-toxicological and imaging stud femur, accurately weighed, in 1.5 mL of nitric acid (65% V/v). ies on neuroblastoma tumor). 0738. The destruction of the organic matrix was per 0712) Number and sex of animals: 10 males (5 males/ formed by Subjecting the samples to a wetashing process with group); 5 animals for possible replacements a microwave oven system. 0713 Weight and age at arrival: 20-25 g; 5-6 weeks old (0739 Finally the dried residues were dissolved with 3.0 0714 Supplier: Harlan Italy, S. Pietro al Natisone(UD), mL of HCL 5% (v/v) and then analysed by ICP-AES. Italy 0740 Data Processing. 0715 Equipment 0741 Briefly, linearity was evaluated for two standards, 0716 All the experiments were performed on a MRRS low and high, ranging from 0.00 to 20 mg (Gd)/L in HCl 5% console (MRResearch Systems Ltd, Surrey UK) interfaced to (v/v), respectively. The total content of gadolinium in the test a 2T Oxford magnet (bore 45 cm i.d.), equipped with a 16 cm sample was calculated by using the instrumental calibration i.d. self-shielded gradient set, driven by six TECHRONR) straight line and express as ug(Gd)/mL. amplifiers (4:1:1 configuration) with a maximum gradient 0742 MRI Data Analysis strength of 110 mT/m and slew rate of 75 us. As R.F. coil, a 0743. The mean signal intensity was measured within a quadrature resonator optimized to the mouse size was used. region of interest (ROI) including the entire tumor, drawn by 07.17 Methods an operator on each MR image using dedicated software (MR 07.18 Experimental Design Research Systems Ltd, Surrey UK). Signal intensity enhance 0719. Mouse neuroblastoma cell line (BS TCL 128 ment (Enh96) was determined as: Neuro-2a) was supplied by the Istituto Zooprofilattico Speri mentale della Lombardia e dell'Emilia, Brescia. The cells were grown in 90% MEM medium and 10% fetal bovine 0744 where SI and SI(t) are the mean signal intensity of serum, collected, washed two times with physiological saline tumor pre and post injection of the contrast agent (testarticles and resuspended in PBS (10° cells/0.2 mL). 10 cells were or reference), respectively. injected Subcutaneously in the right flank of each animal. 0745 Data collected (bodyweight, clinical signs, gross 0720. The tumor development was followed every other pathology examination) were subjected to qualitative analy day after inoculation by measuring the tumor diameter until S1S. the day of the MRI experiment. Animals with a tumor diam eter ranging from 300 to 700 mm were selected for in vivo Representative Embodiments imaging. Signal enhancement of the tumor was followed by 0746 The following non-limiting, enumerated embodi MRI up to 24 h after the administration of the test article and ments further describe the present invention: it was compared with that of the reference article. Animals 0747 1. An isolated fibrin-binding peptide having an were anaesthetized and during the experimental phase the amino acid sequence selected from the group consisting of anesthesia was maintained. the sequences provided in Table 1 or Table 2. 0721 The test articles and ProHance(R) (reference article) 0748 2. A diagnostic imaging agent comprising a fibrin were administered at doses in the range of 0.025-0.1 mmol/ binding peptide according to embodiment 1, wherein said kg. fibrin-binding peptide is linked to a detectable label. US 2010/0158814 A1 Jun. 24, 2010 74

0749. 3. The imaging agent of embodiment 2, wherein said fibrin-binding peptide is linked to at least one para- -continued magnetic metal atom Suitable for magnetic resonance CO2H imaging, optionally via a chelator. 0750 4. The imaging agent of embodiment 2, wherein HOC Nr CO2H said fibrin-binding peptide is linked to an echogenic label r Suitable for ultrasound imaging. N 0751) 5. The imaging agent of embodiment 2, wherein the fibrin binding peptide is linked to a diagnostic radionu --- No, clide, optionally via a chelator 0752 6. The imaging agent of embodiment 5, wherein the HOC diagnostic radionuclide is selected from the group consist- HOC CO2H ing of Cu, 7Ga, Ga., "Tc, and '''In. N /1 0753 7. The imaging agent of embodiment 2, wherein N HOC CO2H said fibrin-binding peptide is fluoresceinated. 5. 0754) 8. A therapeutic agent comprising a fibrin-binding N peptide according to embodiment 1, wherein said fibrin binding peptide is linked to a therapeutic agent. 0755 9. The therapeutic agent of embodiment 8, wherein N the fibrin-binding peptide is linked to a therapeutic radio nuclide, optionally via a chelator. HOC 0756 10. The therapeutic agent of embodiment 9, wherein HOC NH2 the therapeutic radionuclide is selected from the group HOC consisting of Cu, Y, Rh, '''In, ''7mSn, Pm, SSm, 161Tb, 166Dy, 166Ho, 175Yb, 177Lu, 186-188Re, and /-N l'Au. HOC N 0757 11. The imaging agent of embodiment 3, wherein N the chelator is selected from the group consisting of DTPA, N DTPA-GLU, DTPA-Lys, DOTA, AAZTA, and the follow ing derivatives thereof:

N /-coil Hoc1N r N HN N CO2H

CO2H 0758 12. The imaging agent of embodiment 3, selected from the group consisting of

Chelated complex 1

S S H H N N CONH COONa

CHCONH3 CONH CON CONH CONKl CONH CONH CONH) CONH CONH CCONH CONH O /-COO N H2NOCN, HNOCN, HNOCN, HNOCN, HNOC NOC HNOC NH Cnay/ (N-COON. CJNaOOC \ ,- COO O \-COO NHoo1 No1 N1,N1 NH N OCHN Yn OCHN 1n OCHN 1n OCHN ouls N-1-1-1 O H

US 2010/0158814 A1 Jun. 24, 2010 76

-continued

Chelated complex 3

S S H H N N CONH2 COONa h Kl HO HO CONHl CONH CON CONH CONKl CONH CONH CONH CONH CONH CCONH CONH O NHCONHCONHCONHCOCH, H:NOCN-HNOCN-HNOCN-HNOCN-HNOC CS HNOC NH

NaOOC

N H

OOC COONaGd OOCN COO OOC N r COONaGd Jh-rOCHNN-COHN NHCO NHCO N7

ls COO OOC COONaGd OOC COONaGd o N- OCHN ls- OCHN Vy - Vy - NCOO NCOO

Chelated complex 4

H H N N CONH2 COONa Kl h HO HO CHCONH CONH CON CONH CONKl CONH CONH CONH CONH CONH CCONH CONH O H2NOCN, HNOCN, HNOCN, HNOCN, HNOC CS HNOC NH

NaOOC

N H US 2010/0158814 A1 Jun. 24, 2010 77

-continued OOCN- COONaGd OOCN COONaGd ors so A-roin-on-oil N COO OOC COONaGd lus NHCO1 OOC o COONaGd OOC ls - OCHN N COO

N so OOC Chelated complex 5

H H N N coona (1 | a CONH l Sn - S - S4-S-S 4S

O CH3CONH CONH CON NCONH1.cN CONH CONH CONH CONH CONH CONH CONH

"O-C-N /-CO.Na HNOC HNOC a/ HNOC YaY HNOC n1 HNOC NOC HNOC NH

OC-VN Gd's N C NaOOC OC * NHCO CONH 'O.C- /-CO, NCO H OC -v Gd+3 N N

QN H O NaOC Chelated complex 6 H N N CONH COONal ar S-S-S-S-4-S S-S se 4S S-S

CHCONH CONH CON CONH Kl CONH CONH CONH CONH CONH CCONH CONH O HNOC HNOC a1 HNOC ne/ HNOC NY HNOC C2J HNOC NH s t Gd+3> N / NHCONu-n-CONH NH ... IN V-CO2 Cl2.5H161GdN29Na3O37S2 2952.16 2950.998.382

US 2010/0158814 A1 Jun. 24, 2010 79

-continued Chelated complex 9 H H N GOONaan-Nn 4n. CONH N

CHCONH CONH Kl O CONH CONH CONHhis CONH CONH CONH"r CONH CONH HNOC r HNOC NH NaOOC

NaOOC COO NHCO N N- COO ) H Gd'3 ) / \- NHOC N7

OOC l C23H159GdNoNa3O37S2 2926.12 2924.98.2732 Chelated complex 10 S

N CONH2 COOH CH3CONH CONH Kl CONH 6 CONH CONH CONH CONH CONH CONH H:NOCS-HNOCS-HNOCS-HNOCS-HNOCS-NOCShih, HNOCN-NH COO COO Y ls ! Hooc1 E N Gd3 O NaOOC-/N-N-on---o-soOOC- NHoo1 No1 N1,N1 NNH N COONa

0759) 13. The imaging agent of embodiment 4, wherein comprises a lipopeptide comprising a fibrin-binding pep the echogenic label Suitable for ultrasound imaging com tide of embodiment 1 linked to a phospholipid. 0763. 17. The ultrasound contrast agent of embodiment 16 prises a microballoon comprising a gas. comprising a lipopeptide selected from the group consist 0760 14. The imaging agent of embodiment 4, wherein ing of: Seq000-PL1, Seq005-PL1, Seq016-PL1, Seq017 the echogenic label Suitable for ultrasound imaging com PL1, Seq023-PL1 and Seq024-PL1. prises a microbubble comprising a gas. 0764. 18. The ultrasound contrast agent of embodiment 17, wherein the microbubble further comprises DSPC and 0761) 15. The imaging agent of embodiment 14, wherein DPPG. the microbubble comprises a mixture of two or more 0765. 19. The ultrasound contrast agent of embodiment selected form the group consisting of DSPC, DPPG, 17, wherein the microbubble further comprises DSPE DPPA, DSPE-PEG1000, DSPE-PEG2000, DSPE PEG1000, DPPE and DPPG. PEG3400, DPPE-PEG3400, palmitic acid and stearic acid. 0766. 20. The ultrasound contrast agent of embodiment 0762. 16. An ultrasound contrast agent comprising a 17, wherein the microbubble further comprises DSPE microbubble comprising a gas, wherein the microbubble PEG1000, DSPC and DSPG. US 2010/0158814 A1 Jun. 24, 2010 80

0767 21. The ultrasound contrast agent of embodiment 0781 s is 1 or 2, and 17, wherein the gas comprises SF or a perfluorocarbon, 0782 r is, independently in each occurrence, an integer optionally in admixture with air, nitrogen, oxygen or car from 1 to 8: bon dioxide. 0783 or a physiologically acceptable salt thereof. 0768 22. The ultrasound contrast agent of any one of 0784. 28. A compound of embodiment 27, wherein T is a embodiments 18 to 20, wherein the gas comprises CF, diagnostically active moiety. 0785 29. A compound of embodiment 28, wherein the optionally in admixture with air, nitrogen, oxygen or car diagnostically active moiety is selected from the group bon dioxide. consisting of a chelated gamma ray or positron emitting 0769 23. The ultrasound contrast agent of embodiment radionuclide, a paramagnetic metal ion in the form of a 16, further comprising a lyophilization additive and/or a chelated or polychelated complex, an X-ray absorbing bulking agent. agent including an atom of atomic number higher than 20, 0770 24. An isolated fibrin-binding peptide having an a dye molecule, a fluorescent molecule, a phosphorescent amino acid Sequence molecule, a molecule absorbing in the UV spectrum, a Ac-WQPC*PWESWTFC*WDPGGGK NH. (SEQ ID quantum dot, a molecule capable of absorption within near NO. 2), in which one or more of the following modifica or far infrared radiations, and a moiety detectable by ultra tions have been made: Sound. (0771 (a) replacement of Trp with Ala: 0786 30. A compound of embodiment 27, wherein T is a 0772 (b) replacement of Phe' with Cha; and therapeutically active moiety. 0787 31. A compound of embodiment 30, wherein the 0773 (c) addition of one or more polar amino acids at the therapeutically active moiety is selected from the group N terminus. consisting of a thrombolytic agent, a fibrinolytic agent, a 0774. 25. A diagnostic imaging agent comprising a fibrin cytotoxic agent, an agent for selective killing and/or inhib binding peptide of embodiment 24, wherein said fibrin iting the growth of tumor cells and a radiotherapeutic binding peptide is linked to a detectable label. agent. 0775. 26. A therapeutic agent comprising a fibrin-binding 0788 32. A compound of embodiment 27, wherein Y is peptide of embodiment 24, wherein said fibrin-binding selected from the group consisting of a linear or branched peptide is linked to a therapeutic agent. divalent linking moiety and a linear or branched polyfunc 0776 27. A compound of general Formula (I) tional linking moiety. A-Y(-T), (I) 0789) 33. A compound of embodiment 32, wherein the divalent linking moiety comprises—GGGK. 0777 wherein 0790 34. A compound of embodiment 27 wherein A com 0778 A is a fibrin-binding peptide moiety comprising an prises Seq005 as shown in Table 1. amino acid sequence selected from the group consisting of 0791 35. An intermediate compound comprising a pep the sequences provided in Table 1 or Table 2: tide moiety A conjugated with an optionally protected Y 0779 Y is a suitable linking moiety connecting A with at moiety or with an optionally protected, sub-unit of a Y least one T, when s is 2, the units Y may be the same or moiety, wherein A is a fibrin-binding peptide having an different from each other; amino acid sequence selected from the group consisting of 0780 T is, independently in each occurrence, a diagnos the sequences provided in Table 1 or Table 2 and Y is a tically or therapeutically active moiety; linking moiety.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 145

<21 Oc SEO ID NO 1 <211 LENGTH: 15 <212> TYPE PRT <213> ORGANISM: Artificial <22 Os FEATURE; OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs SEQUENCE: 1 Trp Gln Pro Cys Pro Ala Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15

SEO ID NO 2 LENGTH 19 TYPE PRT ORGANISM: Artificial FEATURE; OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide US 2010/0158814 A1 Jun. 24, 2010 81

- Continued

<4 OOs, SEQUENCE: 2 Trp Gln Pro Cys Pro Ala Glu Ser Trp Thr Phe Cys Trp Asp Pro Gly 1. 5 1O 15 Gly Gly Lys

<210s, SEQ ID NO 3 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide

<4 OOs, SEQUENCE: 3 Gly Pro Pro Gly Trp Glin Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys 1. 5 1O 15 Trp Asp Pro

<210s, SEQ ID NO 4 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs, SEQUENCE: 4 Gly Pro Pro Gly Trp Glin Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys 1. 5 1O 15 Trp Asp Pro Gly Gly Gly Lys 2O

<210s, SEQ ID NO 5 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide

<4 OOs, SEQUENCE: 5 Gly Gly Arg Gly Trp Glin Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys 1. 5 1O 15 Trp Asp Pro

<210s, SEQ ID NO 6 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs, SEQUENCE: 6 Gly Gly Arg Gly Trp Glin Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys 1. 5 1O 15 Trp Asp Pro Gly Gly Gly Lys 2O

<210s, SEQ ID NO 7 US 2010/0158814 A1 Jun. 24, 2010 82

- Continued

&211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide

<4 OO > SEQUENCE: 7 Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15

<210s, SEQ ID NO 8 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs, SEQUENCE: 8 Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15 Gly Gly Gly Lys 2O

<210s, SEQ ID NO 9 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-8-amino-3, 6-dioxaoctanoic acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid <4 OOs, SEQUENCE: 9 Ser Gly Ser Gly Xaa Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe 1. 5 1O 15 Cys Trp Asp Pro 2O

<210s, SEQ ID NO 10 &211s LENGTH: 24 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-8-amino-3, 6-dioxaoctanoic acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid

<4 OOs, SEQUENCE: 10 Ser Gly Ser Gly Xaa Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe 1. 5 1O 15 Cys Trp Asp Pro Gly Gly Gly Lys 2O US 2010/0158814 A1 Jun. 24, 2010 83

- Continued

<210s, SEQ ID NO 11 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-2- Cyclohexyl-L-alanine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid <4 OOs, SEQUENCE: 11 Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Xaa Cys Trp Asp Pro 1. 5 1O 15

<210s, SEQ ID NO 12 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-2- Cyclohexyl-L-alanine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid

<4 OOs, SEQUENCE: 12 Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Xaa Cys Trp Asp Pro Gly 1. 5 1O 15 Gly Gly Lys

<210s, SEQ ID NO 13 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-L-4-Fluorophenylalanine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid <4 OOs, SEQUENCE: 13 Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Xaa Cys Trp Asp Pro 1. 5 1O 15

<210s, SEQ ID NO 14 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature US 2010/0158814 A1 Jun. 24, 2010 84

- Continued <223> OTHER INFORMATION: Xaa-L-4-Fluorophenylalanine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid <4 OOs, SEQUENCE: 14 Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Xaa Cys Trp Asp Pro Gly 1. 5 1O 15 Gly Gly Lys

<210s, SEQ ID NO 15 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-L-3, 4-difluorophenylalanine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid <4 OOs, SEQUENCE: 15 Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Xaa Cys Trp Asp Pro 1. 5 1O 15

<210s, SEQ ID NO 16 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-L-3, 4-difluorophenylalanine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (11) . . (11) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid

<4 OOs, SEQUENCE: 16 Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Xaa Cys Trp Asp Pro Gly 1. 5 1O 15 Gly Gly Lys

<210s, SEQ ID NO 17 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide

<4 OOs, SEQUENCE: 17 Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15

<210s, SEQ ID NO 18 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial US 2010/0158814 A1 Jun. 24, 2010 85

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs, SEQUENCE: 18 Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15 Gly Gly Gly Lys 2O

<210s, SEQ ID NO 19 &211s LENGTH: 17 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide

<4 OOs, SEQUENCE: 19 Arg Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp 1. 5 1O 15

Pro

<210s, SEQ ID NO 2 O &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs, SEQUENCE: 2O Arg Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp 1. 5 1O 15 Pro Gly Gly Gly Lys 2O

<210s, SEQ ID NO 21 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide

<4 OOs, SEQUENCE: 21 Arg Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15

<210s, SEQ ID NO 22 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs, SEQUENCE: 22 Arg Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15 Gly Gly Gly Lys 2O US 2010/0158814 A1 Jun. 24, 2010 86

- Continued

<210s, SEQ ID NO 23 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide

<4 OOs, SEQUENCE: 23 Ser Gly Ser Gly Ser Gly Ser Gly Trp Glin Pro Cys Pro Trp Glu Ser 1. 5 1O 15 Trp. Thir Phe Cys Trp Asp Pro 2O

<210s, SEQ ID NO 24 &211s LENGTH: 27 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs, SEQUENCE: 24 Ser Gly Ser Gly Ser Gly Ser Gly Trp Glin Pro Cys Pro Trp Glu Ser 1. 5 1O 15 Trp. Thir Phe Cys Trp Asp Pro Gly Gly Gly Lys 2O 25

<210s, SEQ ID NO 25 &211s LENGTH: 18 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide

<4 OOs, SEQUENCE: 25 Lys Lys Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp 1. 5 1O 15 Asp Pro

<210s, SEQ ID NO 26 &211s LENGTH: 22 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs, SEQUENCE: 26 Lys Lys Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp 1. 5 1O 15 Asp Pro Gly Gly Gly Lys 2O

<210s, SEQ ID NO 27 &211s LENGTH: 21 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide US 2010/0158814 A1 Jun. 24, 2010 87

- Continued <4 OOs, SEQUENCE: 27 Lys Gly Lys Gly Lys Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr 1. 5 1O 15 Phe Cys Trp Asp Pro 2O

<210s, SEQ ID NO 28 &211s LENGTH: 25 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs, SEQUENCE: 28 Lys Gly Lys Gly Lys Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr 1. 5 1O 15 Phe Cys Trp Asp Pro Gly Gly Gly Lys 2O 25

<210s, SEQ ID NO 29 &211s LENGTH: 16 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-O-(2-Acetamido-2-deoxy -D-galactopyranosyl)-L-serine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid

<4 OOs, SEQUENCE: 29 Xaa Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15

<210s, SEQ ID NO 3 O &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-O-(2-Acetamido-2-deoxy -D-galactopyranosyl)-L-serine 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid <4 OOs, SEQUENCE: 30 Xaa Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15 Gly Gly Gly Lys 2O

<210s, SEQ ID NO 31 &211s LENGTH: 16 212. TYPE: PRT US 2010/0158814 A1 Jun. 24, 2010 88

- Continued

<213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-Tetrahydrofuran-2-carboxylic acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid

<4 OOs, SEQUENCE: 31 Xaa Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15

<210s, SEQ ID NO 32 &211s LENGTH: 2O 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Xaa-Tetrahydrofuran-2-carboxylic acid 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (1) . . (1) <223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid

<4 OOs, SEQUENCE: 32 Xaa Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys Trp Asp Pro 1. 5 1O 15 Gly Gly Gly Lys 2O

<210s, SEQ ID NO 33 &211s LENGTH: 19 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequnce: Fibrin-binding peptide

<4 OOs, SEQUENCE: 33 Arg Arg Gly Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys 1. 5 1O 15 Trp Asp Pro

<210s, SEQ ID NO 34 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence: Fibrin-binding peptide

<4 OOs, SEQUENCE: 34 Arg Arg Gly Gly Trp Gln Pro Cys Pro Trp Glu Ser Trp Thr Phe Cys 1. 5 1O 15 Trp Asp Pro Gly Gly Gly Lys 2O

<210s, SEQ ID NO 35